(12) United States Patent (10) Patent No.: US 9,675,733 B2 Tseng Et Al

USOO9675733B2

(12) United States Patent (10) Patent No.: US 9,675,733 B2 Tseng et al. (45) Date of Patent: *Jun. 13, 2017

(54) METHODS OF MODULATING BONE 4,201,211 A 5/1980 Chandrasekaran et al. REMODELING 4,230,105 A 10, 1980 Harwood 4,292,299 A 9, 1981 Suzuki et al. 4,292.303 A 9, 1981 Keith et al. (71) Applicant: TissueTech, Inc., Doral, FL (US) 4,305.502 A 12/1981 Gregory et al. 4,327,725 A 5, 1982 Cortese et al. (72) Inventors: Scheffer Tseng, Pinecrest, FL (US); Ek 4,347,841 A 9/1982 Benyo et al. Kia Tan, Miami, FL (US); Hua He, 4476,116 A 10, 1984 Anik 4,624,848 A 11/1986 Lee Miami, FL (US) 4,871,549 A 10, 1989 Ueda et al. 4,968,509 A 11/1990 Radebaugh et al. (73) Assignee: TISSUETECH, INC., Doral, FL (US) 5,002,071 A 3, 1991 Harrell 5,011,692 A 4/1991 Fujioka et al. (*) Notice: Subject to any disclaimer, the term of this 5,017,381 A 5/1991 Maruyama et al. patent is extended or adjusted under 35 5,033,252 A 7, 1991 Carter 5,052,558 A 10, 1991 Carter U.S.C. 154(b) by 0 days. 5,093,487 A 3, 1992 Brown et al. This patent is Subject to a terminal dis 5,116,817 A 5, 1992 Anik claimer. 5,229,135 A 7/1993 Philippon et al. 5,260,068 A 11, 1993 Chen 5,260,069 A 11, 1993 Chen (21) Appl. No.: 15/348,736 5,323,907 A 6/1994 Kalvelage 5,336,168 A 8, 1994 Sibalis (22) Filed: Nov. 10, 2016 5,436,135 A 7/1995 Tayot et al. 5.437,287 A 8/1995 Phillips et al. Prior Publication Data 5,456,923 A 10, 1995 Nakamichi et al. (65) 5,461,140 A 10, 1995 Heller et al. US 2017/OO721 O2 A1 Mar. 16, 2017 5,508,040 A 4/1996 Chen (Continued) Related U.S. Application Data FOREIGN PATENT DOCUMENTS (63) Continuation of application No. 14/004,992, filed as application No. PCT/US2012/035678 on Apr. 27, CN 1193515 A 9, 1998 2012, now Pat. No. 9,526,770. CN 1203794. A 1, 1999 EP 1604695 A1 12/2005 (60) Provisional application No. 61/480.281, filed on Apr. KR 20010098.716 A 11 2001 28, 2011. WO WO-03077794 A2 9, 2003 WO WO-2004O26244 A2 4/2004 WO WO-2004060388 A1 T 2004 (51) Int. C. WO WO-2006094247 A2 9, 2006 A 6LX 35/50 (2015.01) WO WO-2007038686 A2 4/2007 A6 IK 35/54 (2015.01) WO WO-2011031489 A2 3, 2011 CI2N 5/00 (2006.01) CI2N 5/04 (2006.01) OTHER PUBLICATIONS A6IL 27/38 (2006.01) A6IL 27/36 (2006.01) Bae et al. Characterization of the Promoter Region of the Human (52) U.S. C. Transforming Growth Factor-B Type II Receptor Gene. J. Biol. CPC ...... A61L 27/3847 (2013.01); A61K 35/54 Chem. 270(49):29460-29468 (1995). (2013.01); A61L 27/3604 (2013.01); A61L (Continued) 27/365 (2013.01); A61 L 2430/02 (2013.01) (58) Field of Classification Search None Primary Examiner — Chris R Tate See application file for complete search history. Assistant Examiner — Douglas F White References Cited (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich (56) & Rosati U.S. PATENT DOCUMENTS 3,598,122 8, 1971 Zaffaroni (57) ABSTRACT 3,598,123 8, 1971 Zaffaroni 3,710.795 1, 1973 Higuchi et al. Disclosed herein, in certain instances, are methods of inhib 3,731,683 5, 1973 Zaffaroni 3,742.951 7, 1973 Zaffaroni iting osteoclast differentiation, bone resorption, bone forma 3,814,097 6, 1974 Ganderton et al. tion, and bone remodeling in an individual in need thereof, 3,921,636 11/1975 Zaffaroni comprising administering to the individual a composition 3,972,995 8, 1976 Tsuk et al. comprising Substantially fetal Support tissue product includ 3,993,072 11, 1976 Zaffaroni ing amniotic membrane and umbilical cord or an extract 3,993,073 11, 1976 Zaffaroni thereof, or a composition comprising Substantially isolated 3.996,934 12, 1976 Zaffaroni 4,031,894 6, 1977 Urquhart et al. HC-HA complex. 4,060,084 11, 1977 Chandrasekaran et al. 4,069,307 1, 1978 Higuchi et al. 4,077.407 3, 1978 Theeuwes et al. 10 Claims, 24 Drawing Sheets US 9,675,733 B2 Page 2

(56) References Cited with Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 45(5):1544-1552 (2004). U.S. PATENT DOCUMENTS Border et al. Transforming Growth Factor-B in Disease: The Dark 5,516,527 A 5, 1996 Curatolo Side of Tissue Repair. J. Clin. Invest. 90:1-7 (1992). 5,567,441 A 10, 1996 Chen Chen et al. Functions of hyaluronan in wound repair. Wound Rep 5,622,721 A 4, 1997 Dansereau et al. Reg 7:79-89 (1999). 5,665,378 A 9, 1997 Davis et al. Chen et al. Recombinant Adenovirus Coexpressing Covalent 5,686,105 A 11/1997 Kelmet al. Peptide/MHC Class II Complex and B7-1: In Vitro and In Vivo 5,700,410 A 12/1997 Nakamichi et al. Activation of Myelin Basic Protein-Specific T Cells. J. Immunol. 5,837,280 A 11/1998 Kenealy et al. 5,837,284 A 11/1998 Mehta et al. 167: 1297-1305 (2001). 5,840,329 A 11, 1998 Bai Colon et al. Transfer of Inter-O-inhibitor Heavy Chains to 5,869,090 A 2f1999 Rosenbaum Hyaluronan by Surface-linked Hyaluronan-TSG-6 Complexes. J. 5,932,545 A 8, 1999 Henkin et al. Biol. Chem. 2009. 284:2320-2331. 5.948,766 A 9, 1999 Milan et al. Cooke et al. Comparison of cryopreserved amniotic membrane and 5,977,175. A 11, 1999 Lin umbilical cord tissue with dehydrated amniotic membrane/chorion 6,152,142 A 11/2000 Tseng tissue. J Wound Care 23(10):465-474 (2014). 6,326,019 B1 12/2001 Tseng Day et al. Hyaluronan cross-linking: a protective mechanism in 6,328,765 B1 12/2001 Hardwicket al. 6,391,452 B1 5/2002 Antonsen et al. inflammation? Trends in Immunology 26(12): 637-643 (2005). 6,465,014 B1 10/2002 Moroni et al. Derynk et al. TGF-3 receptor signaling, Biochem. Biophys. Acta. 6,923,983 B2 8/2005 Morgan et al. 1333:F105-F150 (1997). 6,929,801 B2 8, 2005 Klose et al. Fortunato et al. Interleukin-10 and transforming growth factor-3 6,932,983 B1 8, 2005 Straub et al. inhibit amniochorion tumor necrosis factor-O production by con 6,946,144 B1 9, 2005 Jordan trasting mechanisms of action: Therapeutic implications in prema 7,494,802 B2 2/2009 Tseng et al. 8,071,135 B2 12/2011 Liu et al. turity. Am. J. Obstet. Gynecol. 177(4):803-809 (1997). 8, 105,634 B2 1/2012 Liu et al. Fortunato et al. Interleukin-10 inhibition of interleukin-6 in human 8,153,162 B2 4/2012 Tseng et al. amniochorionic membrane: Transcriptional regulation. Am. J. 8, 182,840 B2 5/2012 Tseng et al. Obstet. Gynecol. 175: 1057-1065 (1996). 8, 182,841 B2 5/2012 Tseng et al. Fortunato et al. The effect of transforming growth factor and 8, 187,639 B2 5/2012 Tseng et al. interleukin-10 on interleukin-8 release by human amniochorion 8.323,701 B2 12/2012 Daniel et al. may regulate histologic chorioamnionitis. Am. J. Obstet. Gynecol. 8,372.437 B2 2/2013 Daniel 8.420,126 B2 4/2013 Tseng et al. 17903):794-799 (1998). 8.440,235 B2 5/2013 Tseng et al. Fries et al. Inter-alpha-inhibitor, hyaluronan and inflammation. Acta 8,455,009 B2 6/2013 Tseng et al. Biochim Polonica 50(3):735-742 (2003). 8.460,714 B2 6/2013 Tseng et al. Gabbiani. The myofibroblast in wound healing and fibrocontractive 9,161,954 B2 10/2015 Tseng et al. diseases. J. Pathol. 200:500-503 (2003). 9,161.955 B2 10/2015 Tseng et al. Grande. Role of Transforming Growth Factor-B in Tissue Injury and 9,161,956 B2 10/2015 Tseng et al. Repair. Proc. Soc. Exp. Biol. Med. 214:27-40 (1997). 9,175,066 B2 11/2015 Tseng et al. Guo. CarbopolR Polymers for Pharmaceutical Drug Delivery 9,198.939 B2 12/2015 Tseng et al. Applications. Drug Delivery Technology 3(6):1-4 (2003). 2001/0041684 A1 11/2001 Lezdey et al. 2004/OO57938 A1 3, 2004 Ghinelli Hales et al. TGF-B-1 induces lens cells to accumulate C-Smooth 2004/O126323 A1 7/2004 Shevchuk et al. muscle actin, a marker for Subcapsular cataracts. Curr. Eye Res. 2007/0202189 A1* 8, 2007 Ahlfors ...... A61L 27/3604 13:885-890 (1994). 424,548 Hanada et al. Regulation of cytokine signaling and inflammation. 2008.0102135 A1 5, 2008 Olivier Cytokine & Growth Factor Reviews 13(4-5):413-421 (2002). 2008. O181935 A1* 7, 2008 Bhatia ...... A61K 38.39 Hao et al. Identification of Antiangiogenic and Antiinflammatory 424/443 Proteins in Human Amniotic Membrane. Cornea 19(3):348-352 2008.0193554 A1 8, 2008 Dua et al. (2000). 2008/0241211 A1 10/2008 Han et al. Hatano et al. Transplantation of amniotic membrane and limbal 2009, O130162 A2 5, 2009 Pathak et al. autograft in the treatment of recurrent pterygium. Clinical Ophthal 2009, 0226499 A1 9, 2009 Wisniewski et al. mology 50(6): 1101-1104 (1996) (English Abstract). 2010.0047214 A1 2/2010 Abramson et al. He et al. A simplified system for generating recombinant 2010/0272782 A1* 10, 2010 Owens ...... A61L 31,005 adenoviruses. PNAS USA95:25.09-2514 (1998). 424/443 He et al. Biochemical Characterization and Function of Complexes 2011/0212158 A1 9/2011 Tom et al. formed by Hyaluronan and the Heavy Chains of Inter-O-inhibitor 2011 O2562O2 A1 10/2011 Tom et al. (HC-HA) Purified from Extracts of Human Amniotic Membrane. J 2012fO141595 A1 6/2012 Tseng et al. Biol Chem 284(30):20136-20146 (Jul. 24, 2009). 2013, O156863 A1 6/2013 Tseng et al. Heiligenhaus et al. Improvement of HSV-1 Necrotizing Keratitis 2013,0344163 A1 12/2013 Tseng et al. with Amniotic Membrane Transplantation Invest Ophthalmol Vis 2014/O112998 A1 4/2014 Tseng et al. Sci 42(9): 1969-1974 (2001). 2016/0095931 A1 4/2016 Tseng et al. Hilmy et al. Physical and chemical properties of freeze-dried 2016/0106785 A1 4/2016 Tseng et al. 2016/0129049 A1 5/2016 Tseng et al. amnio-chorion membranes sterilized by y irradiation. Atom Indo 2016/0129050 A1 5/2016 Tseng et al. nesia 13(2): 1-3 (1987) Abstract only. 2016/0129051 A1 5/2016 Tseng et al. Hori. Amniotic Membrane Transplantation and Immune Reaction. 2016. O151424 A1 6/2016 Tseng et al. Folia Ophthalmologica Japonica 56(9):722-727 (2005) (English 2016,0184368 A1 Abstract). 6/2016 Tseng et al. Howes et al. Receptor for Advanced Glycation End Products and Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. OTHER PUBLICATIONS 45(10):37 13-3720 (2004). Jadin et al. Characterization of a Novel Recombinant Hyaluronan Bhutto et al. Localization of Collagen XVIII and the Endostatin Binding Protein for Tissue Hyaluronan Detection. Journal of Portion of Collagen XVIII in Ages Human Control Eyes and Eyes Histochemistry & Cytochemistry 62(9):672-683 (2014). US 9,675,733 B2 Page 3

(56) References Cited PCT/US2011/042679 International Preliminary Report on Patent ability dated Jan. 8, 2013. OTHER PUBLICATIONS PCT/US2011/042679 International Search Report and Written Opinion dated Mar. 9, 2012. Jester et al. Corneal Stromal Wound Healing in Refractive Surgery: PCT/US2012/035678 International Preliminary Report on Patent the Role of Myofibroblasts. Prog. Retin. Eye Res. 18(3):311-356 ability mailed Oct. 29, 2013. (1999). PCT/US2012/035678 International Search Report and Written Jester et al. Induction of C-Smooth Muscle Actin Expression and Opinion mailed Oct. 1, 2012. Myofibroblast Transformation in Cultured Cornea Keratocytes. Petraglia et al. Inhibin and Activin in Human Fetal Membranes: Cornea 15(5):505-516 (1996). Evidence of a Local Effect on Prostaglandin Release. J. Clin. Keelan et al. Activin A Exerts both Pro- and -Anti-inflammatory Endocrinol. Metab. 77(2):542-548 (1993). Effects on Human Term Gestational Tissues. Placenta 21:38-43 Riley et al. Production of inhibin forms by the fetal membranes, (2000). decidua, placenta and fetus at parturition. Hum. Reprod. 15:578-583 Kopp et al. Abrogation of Transforming Growth Factor-beta Sig (2000). Romero et al. The natural interleukin-1 receptor antagonist in the naling by SMAD7 Inhibits Collagen Gel Contraction of Human fetal, maternal, and amniotic fluid compartments: The effect of Dermal Fibroblasts. J. Biol. Chem. 280(22):21570-21576 (2005). gestational age, fetal gender, and intrauterine infection. Am. J. Lawrence. Transforming Growth Factor-f: a general review. Eur. Obstet. Gynecol. 171912-921 (1994). Cytokine Netw. 7:363-374 (1996). Ronnov-Jessen et al. Induction of C-Smooth Muscle Actin by Lee et al. An Agarose Gel Electrophoretic Method for Analysis of Transforming Growth Factor-B1 in Quiescent Human Breast Gland Hyaluronan Molecular Weight Distribution. Anal. Biochem. Fibroblasts. Lab. Invest. 68:696-707 (1993). 219:278-287 (1994). Rovere et al. The long pentraxin PTX3 binds to apoptotic cells and Li et al. An Experimental Study of the Effects of Human Amniotic regulates their clearance by antigen-presenting dendritic cells. Membrane on Human Retinal Pigment Epithelial Cell Proliferation Blood 96(13):4300-4306 (2000). in vitro. Acta Acadamiae Medicinae Militaris Tertia 25(5):407-409 Rugg et al. Characterization of complexes formed between TSG-6 (2003) (English Abstract). and inter-alpha-inhibitor that act as intermediates in the covalent Lieberman et al. Pharmaceutical Dosage Forms. 2 Ed. 1:209-214 transfer of heavy chains onto hyaluronan. J Biol Chem (1990). 280(27): 25674-25686 (2005). Liu et al. Biocompatibility and stability of disulfide-crosslinked Sanggaard et al. The transfer of heavy chains from bikunin proteins hyaluronan films. Biomaterials 26(23):4737-4746 (2005). to hyaluronan requires both TSG-6 and HC2. J Biol Chem Logan et al. Decorin Attenuates Gliotic Scar Formation in the Rat 283 (27): 18530-18537 (2008). Cerebral Hemisphere. Exp. Neurol. 159:504-510 (1999). Serini et al. The Fibronectin Domain ED-A Is Crucial for Marek et al. TGF-3-(transforming growth factor-B) in chronic Myofibroblastic Phenotype Induction by Transforming Growth Fac inflammatory conditions—a new diagnostic and prognostic marker? tor-f1. J. Cell. Biol. 142:873-881 (1998). Med. Sci. Monitl. 8(7):RA145-RA151 (2002). Shah et al. Control of Scarring in adult wounds by neutralising Massague et al. Controlling TGF-3 signaling. Genes and Develop antibody to transforming growth factor B. Lancet 339:213-214 ment 14:627-644 (2000). (1992). Milner et al. TSG-6: a multifunctional protein associated with Singh et al. Encyclopedia of Pharmaceutical Technology 2nd Ed. inflammation. J. Cell Sci. 116(10): 1863-1873 (2003). pp. 751-753 (2002). Moller-Pedersen et al. Neutralizing antibody to TGF-B modulates Solomon et al. Suppression of Interleukin la and interleukin 1b in stromal fibrosis but not regression of photoablative effect following human limbal epithelial cells cultured on the amniotic membrane PRK. Curr. Eye Res. 17:736-747 (1998). stromal matrix. Br. J. Ophthalmol 85:444-449 (2001). Monteleone et al. SMAD7 in TGF-B-mediated negative regulation Tan et al. Structural and Biological Comparison of Cryopreserved of gut inflammation. Trends in Immunology 25(10):513-517 (2004). and Fresh Amniotic membrane Tissues. Journal Biomaterial and Mukhopadhyay et al. Two distinct populations of tumor necrosis Tissue Engineering 4:379-388 (2014). factor-stimulated gene-6 protein in the extracellular matrix of Travis et al. Hyaluronan Enhances Contraction of Collagen by expanded mouse cumulus cell-oocyte complexes. Archives of Bio Smooth Muscle Cells and Adventitial Fibroblasts Role of CD44 and chemistry and Biophysics 394(2):173-181 (2001). Implications for Constrictive Remodeling. Cir. Res. 88:77-83 Na et al. Analysis of Human Amniotic Membrane Components as (2001). Proteinase Inhibitors for Development of Therapeutic Agent for Tseng et al. How Does Amniotic Membrane Work? Ocular Surface Recalcitrant Keratitis. Trophoblast Res. 13:453-466 (1999). J. 2(3): 177-187 (2004). Nakao et al. SMAD7: a new key player in TGF-b-associated Tseng et al. Suppression of Transforming Growth Factor-Beta disease. Trends in Molecular Medicine 8(8):361-363 (2002). Isoforms, TGF-B Receptor Type II, and Myofibroblast Differentia Neumann et al. High molecular weight hyaluronic acid inhibits tion in Cultured Human Corneal and Limbal Fibroblasts by Amni advanced glycation endproduct-induced NF-kB activation and otic Membrane Matrix. J. Cell Physiol. 179:325-335 (1999). cytokine expression. FEBS Ltrs. 453:283-287(1999). . Appl. No. 1 1/528,902 Office Action dated Apr. 2, 2009. Ochsner et al. Decreased expression of tumor necrosis factor-alpha . Appl. No. 1 1/528,902 Office Action dated Dec. 16, 2009. stimulated gene 6 in cumulus cells of the cyclooxygenase2 and EP2 . Appl. No. 1 1/528,902 Office Action dated Jan. 27, 2011. null mice. Endocrinology 144:1008-1019 (2003). . Appl. No. 1 1/528,902 Office Action dated Sep. 8, 2010. Oikawa et al. Inhibition of Angiogenesis by 15-Deoxyspergualin. J. . Appl. No. 1 1/528,980 Office Action dated Aug. 11, 2009. Antibiotics 44(9): 1033-1035. . Appl. No. 1 1/528,980 Office Action dated Jan. 10, 2011. PCT/US2006/37906 International Preliminary Report on Patent . Appl. No. 1 1/528,980 Office Action dated Nov. 13, 2008. ability dated Apr. 1, 2008. . Appl. No. 1 1/528,980 Office Action dated Oct. 15, 2010. PCT/US2006/37906 International Search Report and Written Opin . Appl. No. 1 1/529,658 Office Action dated Apr. 3, 2009. ion dated Jul. 11, 2007. . Appl. No. 1 1/529,658 Office Action dated Dec. 16, 2009. PCT/US2010/032452 International Preliminary Report on Patent . Appl. No. 1 1/529,658 Office Action dated Jan. 27, 2011. ability dated Oct. 25, 2011. . Appl. No. 1 1/529,658 Office Action dated Sep. 3, 2010. PCT/US2010/032452 International Search Report dated Dec. 27. . Appl. No. 1 1/535,924 Office Action dated Dec. 16, 2009. 2010. . Appl. No. 1 1/535,924 Office Action dated Jan. 31, 2011. PCT/US2010/46675 International Preliminary Report on Patent . Appl. No. 1 1/535,924 Office Action dated Mar. 31, 2009. ability dated Feb. 28, 2012. . Appl. No. 1 1/535,924 Office Action dated Sep. 8, 2010. PCT/US2010/46675 International Search Report and Written Opin . Appl. No. 13/262,725 Office Action dated Feb. 25, 2015. ion dated May 30, 2011. . Appl. No. 13/262,725 Office Action dated Jul. 17, 2014. US 9,675,733 B2 Page 4

(56) References Cited Yamaguchi et al. Negative regulation of transforming growth fac tor-B by the proteoglycan decorin. Nature 346(6281):281-284 OTHER PUBLICATIONS (1990). Ahmed et al. Expression and localization of alphavbeta6 integrin in . Appl. No. 13/322,896. Office Action dated Jan. 20, 2016. extraplacental fetal membranes: possible role in human parturition. . Appl. No. 13/322,896. Office Action dated Jul. 6, 2015. Mol Hum Reprod 10(3):173-179 (2004). Temma et al. Effects of 4-hydroxy-2-nonenal, a marker of oxidative . Appl. No. 13/322,896. Office Action dated Oct. 22, 2014. stress, on the cyclooxygenase-2 of human placenta in . Appl. No. 13/453,840 Office Action dated Aug. 21, 2012. chorioamnionitis. Mol Hum Reprod 10(3):167-171 (2004). . Appl. No. 13/704.231 Office Action dated Aug. 2, 2016. U.S. Appl. No. 13/704.231 Office Action dated Jan. 19, 2017. . Appl. No. 13/704.231 Office Action dated Feb. 11, 2016. U.S. Appl. No. 14/240,712 Office Action dated Nov. 28, 2016. . Appl. No. 13/704.231 Office Action dated Jun. 4, 2015. U.S. Appl. No. 14/880,135 Office Action dated Dec. 23, 2016. . Appl. No. 13/796,761 Office Action dated Dec. 9, 2014. English Translation of JP74043153B (App. S45-107284) (9 pgs.) . Appl. No. 13/802.204 Office Action dated Aug. 7, 2015. (Pub. Nov. 19, 1974). . Appl. No. 13/802.204 Office Action dated Feb. 26, 2015. Relucentietal. Cumulus oophorus extracellular matrix in the human . Appl. No. 13/802.204 Office Action dated Jan. 22, 2016. oocyte: a role for adhesive proteins. Ital J Anat Embryol 110(2 Supp 1):219-224 (2005). . Appl. No. 13/802.264 Office Action dated Jul. 16, 2015. Salustri et al. PTX3 plays a key role in the organization of the . Appl. No. 13/802.264 Office Action dated Nov. 28, 2014. cumulus oophorus extracellular matrix and in in vivo fertilization. . Appl. No. 13/802,359 Office Action dated Dec. 10, 2014. Development 131: 1577-1586 (2004). . Appl. No. 13/802.447 Office Action dated Dec. 15, 2014. U.S. Appl. No. 14/848,148 Office Action dated Mar. 20, 2017. . Appl. No. 14/004,992 Office Action dated Jun. 6, 2016. Wisniewski et al., Cytokine-induced gene expression at the cross . Appl. No. 14/004,992 Office Action dated Nov. 23, 2015. roads of innate immunity, inflammation and fertility: TSG-6 and .S. Appl. No. 14,886,946. Office Action dated Apr. 18, 2016. PTX3/TSG-14. Cytokine Growth Factor Rev 15(2-3): 129-146 Verbeek et al. Induction of alpha-Smooth muscle actin expression in (2004). cultured human brain pericytes by transforming growth factor-beta 1. Am. J. Pathol. 144:372-382 (1994). * cited by examiner U.S. Patent Jun. 13, 2017 Sheet 1 of 24 US 9,675,733 B2

F.G. 1

88.8 3.3: & a 88 s: :. -9-cars s: & ------

8 8x8 ties: sixties:

-: U.S. Patent Jun. 13, 2017 Sheet 2 of 24 US 9,675,733 B2

FG 2

::::::::::::::::::::::::::::: U.S. Patent Jun. 13, 2017 Sheet 3 of 24 US 9,675,733 B2

FG 3

U.S. Patent Jun. 13, 2017 Sheet 4 of 24 US 9,675,733 B2

FIG. 4

TRAP ESA

- - Neg CTL Pos CTL AME HCHA AM AMP (200ug/m (25ug/ml (200ug/ml (200ug/ml Protein) HA) Protein) Protein) U.S. Patent Jun. 13, 2017 Sheet S of 24 US 9,675,733 B2

FIG 5

U.S. Patent Jun. 13, 2017 Sheet 6 of 24 US 9,675,733 B2

F.G. 6

Pgs A&E HC.A. &: iais: 3A, Ai: yo g agi is: 88 is: Ai: 8:

U.S. Patent Jun. 13, 2017 Sheet 7 of 24 US 9,675,733 B2

U.S. Patent Jun. 13, 2017 Sheet 8 of 24 US 9,675,733 B2

FG, 8 U.S. Patent Jun. 13, 2017 Sheet 9 of 24 US 9,675,733 B2

FIG. 9A

RANK 50 ng frn awa --

3& 3. A U.S. Patent Jun. 13, 2017 Sheet 10 of 24 US 9,675,733 B2

-A {-A

U.S. Patent Jun. 13, 2017 Sheet 12 of 24 US 9,675,733 B2

FG. 9E

WW a. y: 7.637 Sirix} + 7,383 8: 0.33:38 s a. x & w W S.

hia Cocentration gift U.S. Patent Jun. 13, 2017 Sheet 13 of 24 US 9,675,733 B2

F.G. 10A RANKL (50 ng/ml)

PBS ES 25ugfri U.S. Patent Jun. 13, 2017 Sheet 14 of 24 US 9,675,733 B2

FG, OB RANKL Avi P-1

-- (ug/m

3.32.

S. U.S. Patent Jun. 13, 2017 Sheet 15 of 24 US 9,675,733 B2

F.G. 10C

U.S. Patent Jun. 13, 2017 Sheet 16 of 24 US 9,675,733 B2

F.G. 10D RANKL AMP-1 + (ug/ml)

O, C64

O.32

3. O

2CO

SCO

U.S. Patent Jun. 13, 2017 Sheet 18 of 24 US 9,675,733 B2

F.G. 10F

AMP DOSe Curve

70.00 C50: 20.1 g/ml

y = 9.065in(x) + 22.801. R at O. 94.73

U.S. Patent Jun. 13, 2017 Sheet 20 of 24 US 9,675,733 B2

FIG. 12

RAACY

:::::::::::::: × È?: & × ::::::::::::::::::::::::¿j

U.S. Patent US 9,675,733 B2

After &tatic Extract 38: s: is88 ta: essar is: U.S. Patent Jun. 13, 2017 Sheet 22 of 24 US 9,675,733 B2

F.G. 14

&fter St. ARSStaining Extractix:

s Cer

38& :::::8. 388 3:3:83. 38& 8:38. 33.38: 838:

88: U.S. Patent Jun. 13, 2017 Sheet 23 of 24 US 9,675,733 B2

F.G. 15

Ctrl Air U.S. Patent US 9,675,733 B2

US 9,675,733 B2 1. 2 METHODS OF MODULATING BONE tissue product comprises placental amniotic membrane REMODELING (PAM), umbilical cord amniotic membrane (UCAM), pla centa, umbilical cord, chorion, amnion-chorion, or any com CROSS-REFERENCE binations thereof. In some embodiments, the fetal support tissue product comprises frozen or previously frozen pla This application is a continuation of U.S. application Ser. cental amniotic membrane (PAM), frozen or previously No. 14/004,992, filed Dec. 31, 2013, which is the National frozen umbilical cord amniotic membrane (UCAM), frozen Phase entry of International Application No. PCT/US2012/ or previously frozen placenta, frozen or previously frozen 035678, filed Apr. 27, 2012, which claims priority to U.S. umbilical cord, frozen or previously frozen chorion, frozen Provisional Application No. 61/480.281, filed Apr. 28, 2011, 10 or previously frozen amnion-chorion, or any combinations all of which are incorporated herein by reference in their thereof. In some embodiments, the fetal support tissue entireties. product in the form of a pulverized powder or a homogenate. In some embodiments, the fetal Support tissue product is SUMMARY OF THE INVENTION cryopreserved, lyophilized, terminally sterilized, or a com 15 bination thereof. In some embodiments, the extract com Disclosed herein, in certain embodiments, are methods of prises HC-HA. In some embodiments, the extract is pre inhibiting bone resorption in an individual in need thereof, pared by ultracentrifugation. In some embodiments, the comprising administering to the individual a composition extract is prepared by at least 2 rounds of ultracentrifugation. comprising fetal Support tissue product or an extract thereof. In some embodiments, the extract is prepared by more than In some embodiments, the fetal Support tissue product 2 rounds of ultracentrifugation. In some embodiments, the comprises placental amniotic membrane (PAM), umbilical extract is prepared by at least 4 rounds of ultracentrifugation. cord amniotic membrane (UCAM), placenta, umbilical cord, In some embodiments, the composition further comprises a chorion, amnion-chorion, or any combinations thereof. In pharmaceutically-acceptable diluent, excipient or carrier. In Some embodiments, the fetal Support tissue product com Some embodiments, the composition is formulated as a prises frozen or previously frozen placental amniotic mem 25 Solution, Suspension, paste, ointment, oil, emulsion, micro brane (PAM), frozen or previously frozen umbilical cord emulsion, cream, lotion, gel, or combination thereof. In amniotic membrane (UCAM), frozen or previously frozen Some embodiments, the composition is formulated for con placenta, frozen or previously frozen umbilical cord, frozen trolled, Sustained, or delayed release. In some embodiments, or previously frozen chorion, frozen or previously frozen the composition is injected into an osteolytic joint. In some amnion-chorion, or any combinations thereof. In some 30 embodiments, the composition is administered by a patch. In embodiments, the fetal support tissue product in the form of Some embodiments, the patch is placed on an osteolytic bone a pulverized powder or a homogenate. In some embodi or an osteolytic joint. In some embodiments, the composi ments, the fetal Support tissue product is cryopreserved, tion is administered by an implant. In some embodiments, lyophilized, terminally sterilized, or a combination thereof. the implant is placed on an osteolytic bone or an osteolytic In some embodiments, the extract comprises HC-HA. In 35 joint. In some embodiments, the composition is adminis Some embodiments, the extract is prepared by ultracentrifu tered by an orthopaedic prosthesis. In some embodiments, gation. In some embodiments, the extract is prepared by at the composition is administered by a bone stent. In some least 2 rounds of ultracentrifugation. In some embodiments, embodiments, the composition is a sheet and is wrapped the extract is prepared by more than 2 rounds of ultracen around an osteolytic bone or an osteolytic joint. In some trifugation. In some embodiments, the extract is prepared by 40 embodiments, the methods further comprise administering at least 4 rounds of ultracentrifugation. In some embodi to the individual a calcium Supplement. In some embodi ments, the composition further comprises a pharmaceuti ments, the disease, disorder, or condition characterized by cally-acceptable diluent, excipient or carrier. In some excessive or undesired osteoclast differentiation is arthritis, embodiments, the composition is formulated as a solution, osteoporosis, a bone tumor, Paget’s Disease, alveolar bone Suspension, paste, ointment, oil, emulsion, microemulsion, 45 degradation, or any combination thereof. In some embodi cream, lotion, gel, or combination thereof. In some embodi ments, the arthritis is osteoarthritis, rheumatoid arthritis, ments, the composition is formulated for controlled, Sus psoriatic arthritis, or any combination thereof. tained, or delayed release. In some embodiments, the com Disclosed herein, in certain embodiments, are methods of position is injected into an osteolytic joint. In some promoting or inducing bone formation in an individual in embodiments, the composition is administered by a patch. In 50 need thereof in an individual in need thereof, comprising Some embodiments, the patch is placed on an osteolytic bone administering to the individual a composition comprising or an osteolytic joint. In some embodiments, the composi fetal Support tissue product or an extract thereof. In some tion is administered by an implant. In some embodiments, embodiments, the fetal Support tissue product comprises the implant is placed on an osteolytic bone or an osteolytic placental amniotic membrane (PAM), umbilical cord amni joint. In some embodiments, the composition is adminis 55 otic membrane (UCAM), placenta, umbilical cord, chorion, tered by an orthopaedic prosthesis. In some embodiments, amnion-chorion, or any combinations thereof. In some the composition is administered by a bone stent. In some embodiments, the fetal Support tissue product comprises embodiments, the composition is a sheet and is wrapped frozen or previously frozen placental amniotic membrane around an osteolytic bone or an osteolytic joint. In some (PAM), frozen or previously frozen umbilical cord amniotic embodiments, the methods further comprise administering 60 membrane (UCAM), frozen or previously frozen placenta, to the individual a calcium Supplement. frozen or previously frozen umbilical cord, frozen or pre Disclosed herein, in certain embodiments, are methods of viously frozen chorion, frozen or previously frozen amnion treating a disease, disorder, or condition characterized by chorion, or any combinations thereof. In some embodi excessive or undesired bone resorption, the method com ments, the fetal Support tissue product in the form of a prising administering to an individual in need thereof a 65 pulverized powder or a homogenate. In some embodiments, composition comprising fetal Support tissue product or an the fetal Support tissue product is cryopreserved, extract thereof. In some embodiments, the fetal support lyophilized, terminally sterilized, or a combination thereof. US 9,675,733 B2 3 4 In some embodiments, the extract comprises HC-HA. In an osteolytic bone or an osteolytic joint. In some embodi Some embodiments, the extract is prepared by ultracentrifu ments, the composition is administered by an orthopaedic gation. In some embodiments, the extract is prepared by at prosthesis. In some embodiments, the composition is admin least 2 rounds of ultracentrifugation. In some embodiments, istered by a bone stent. In some embodiments, the compo the extract is prepared by more than 2 rounds of ultracen sition is a sheet and is wrapped around an osteolytic bone or trifugation. In some embodiments, the extract is prepared by an osteolytic joint. In some embodiments, the methods at least 4 rounds of ultracentrifugation. In some embodi further comprise administering to the individual a calcium ments, the composition further comprises a pharmaceuti Supplement. In some embodiments, the disease, disorder, or cally-acceptable diluent, excipient or carrier. In some condition characterized by excessive or undesired osteoclast embodiments, the composition is formulated as a solution, 10 differentiation is arthritis, osteoporosis, a bone tumor, Pag Suspension, paste, ointment, oil, emulsion, microemulsion, et's Disease, alveolar bone degradation, or any combination cream, lotion, gel, or combination thereof. In some embodi thereof. In some embodiments, the arthritis is osteoarthritis, ments, the composition is formulated for controlled, Sus rheumatoid arthritis, psoriatic arthritis, or any combination tained, or delayed release. In some embodiments, the com thereof. position is injected into an osteolytic joint. In some 15 Disclosed herein, in certain embodiments, are methods of embodiments, the composition is administered by a patch. In inhibiting bone resorption in an individual in need thereof, Some embodiments, the patch is placed on an osteolytic bone comprising administering to the individual a composition or an osteolytic joint. In some embodiments, the composi comprising Substantially isolated HC-HA complex. In some tion is administered by an implant. In some embodiments, embodiments, the HC-HA complex is obtained by a process the implant is placed on an osteolytic bone or an osteolytic comprising: (a) providing a reaction mixture comprising: (i) joint. In some embodiments, the composition is adminis HA (e.g., HMWHA); (ii) ICI, wherein the ICI is optionally tered by an orthopaedic prosthesis. In some embodiments, in serum or isolated from serum; (iii) TSG-6, wherein the the composition is administered by a bone stent. In some TSG-6 is optionally recombinant; and (iv) PTX3, wherein embodiments, the composition is a sheet and is wrapped the PTX3 is optionally recombinant; wherein at least one of around an osteolytic bone or an osteolytic joint. In some 25 HA, ICI, TSG-6, or PTX3 is optionally generated by a embodiments, the methods further comprise administering plurality of cells present in the reaction mixture; (b) incu to the individual a calcium Supplement. bating the reaction mixture for a period of time sufficient to Disclosed herein, in certain embodiments, are methods of produce HC-HA complex; and (c) isolating and purifying treating a disease, disorder or condition characterized by a the HC-HA complex. In some embodiments, the substan deficiency of bone formation in an individual in need 30 tially isolated HC-HA complex is derived from placenta, thereof, the method comprising administering to an indi umbilical cord, chorion, amnion-chorion, placental amniotic vidual in need thereof a composition comprising fetal Sup membrane (PAM), umbilical cord amniotic membrane port tissue product or an extract thereof. In some embodi (UCAM), or any combinations thereof. In some embodi ments, the fetal Support tissue product comprises placental ments, the substantially isolated HC-HA complex is derived amniotic membrane (PAM), umbilical cord amniotic mem 35 from frozen or previously frozen placental amniotic mem brane (UCAM), placenta, umbilical cord, chorion, amnion brane (PAM), frozen or previously frozen umbilical cord chorion, or any combinations thereof. In some embodi amniotic membrane (UCAM), frozen or previously frozen ments, the fetal Support tissue product comprises frozen or placenta, frozen or previously frozen umbilical cord, frozen previously frozen placental amniotic membrane (PAM), or previously frozen chorion, frozen or previously frozen frozen or previously frozen umbilical cord amniotic mem 40 amnion-chorion, or any combinations thereof. In some brane (UCAM), frozen or previously frozen placenta, frozen embodiments, the composition further comprises a pharma or previously frozen umbilical cord, frozen or previously ceutically-acceptable diluents, excipient or carrier. In some frozen chorion, frozen or previously frozen amnion-chorion, embodiments, the composition is formulated as a solution, or any combinations thereof. In some embodiments, the fetal Suspension, paste, ointment, oil, emulsion, microemulsion, support tissue product in the form of a pulverized powder or 45 cream, lotion, gel, or combination thereof. In some embodi a homogenate. In some embodiments, the fetal Support ments, the composition is formulated for controlled, Sus tissue product is cryopreserved, lyophilized, terminally ster tained, or delayed release. In some embodiments, the com ilized, or a combination thereof. In some embodiments, the position is injected into an osteolytic joint. In some extract comprises HC-HA. In some embodiments, the embodiments, the composition is administered by a patch. In extract is prepared by ultracentrifugation. In some embodi 50 Some embodiments, the patch is placed on an osteolytic bone ments, the extract is prepared by at least 2 rounds of or an osteolytic joint. In some embodiments, the composi ultracentrifugation. In some embodiments, the extract is tion is administered by an implant. In some embodiments, prepared by more than 2 rounds of ultracentrifugation. In the implant is placed on an osteolytic bone or an osteolytic Some embodiments, the extract is prepared by at least 4 joint. In some embodiments, the composition is adminis rounds of ultracentrifugation. In some embodiments, the 55 tered by an orthopaedic prosthesis. In some embodiments, composition further comprises a pharmaceutically-accept the composition is administered by a bone stent. In some able diluent, excipient or carrier. In some embodiments, the embodiments, the methods further comprise administering composition is formulated as a Solution, Suspension, paste, to the individual a calcium Supplement. In some embodi ointment, oil, emulsion, microemulsion, cream, lotion, gel. ments, the disease, disorder, or condition characterized by or combination thereof. In some embodiments, the compo 60 excessive or undesired osteoclast differentiation is arthritis, sition is formulated for controlled, sustained, or delayed osteoporosis, alveolar bone degradation, Paget’s Disease, a release. In some embodiments, the composition is injected bone tumor, or any combination thereof. into an osteolytic joint. In some embodiments, the compo Disclosed herein, in certain embodiments, are methods of sition is administered by a patch. In some embodiments, the treating a disease, disorder, or condition characterized by patch is placed on an osteolytic bone or an osteolytic joint. 65 excessive or undesired bone resorption, the method com In some embodiments, the composition is administered by prising administering to an individual in need thereof a an implant. In some embodiments, the implant is placed on composition comprising Substantially isolated HC-HA com US 9,675,733 B2 5 6 plex. In some embodiments, the HC-HA complex is frozen umbilical cord amniotic membrane (UCAM), frozen obtained by a process comprising: (a) providing a reaction or previously frozen placenta, frozen or previously frozen mixture comprising: (i) HA (e.g., HMW HA); (ii) ICI, umbilical cord, frozen or previously frozen chorion, frozen wherein the ICI is optionally in serum or isolated from or previously frozen amnion-chorion, or any combinations serum; (iii) TSG-6, wherein the TSG-6 is optionally recom thereof. In some embodiments, the composition further binant; and (iv) PTX3, wherein the PTX3 is optionally comprises a pharmaceutically-acceptable diluents, excipient recombinant; wherein at least one of HA, ICI, TSG-6, or or carrier. In some embodiments, the composition is formu PTX3 is optionally generated by a plurality of cells present lated as a solution, Suspension, paste, ointment, oil, emul in the reaction mixture; (b) incubating the reaction mixture Sion, microemulsion, cream, lotion, gel, or combination for a period of time sufficient to produce HC-HA complex: 10 thereof. In some embodiments, the composition is formu and (c) isolating and purifying the HC-HA complex. In some lated for controlled, Sustained, or delayed release. In some embodiments, the substantially isolated HC-HA complex is embodiments, the composition is injected into an osteolytic derived from placenta, umbilical cord, chorion, amnion joint. In some embodiments, the composition is adminis chorion, placental amniotic membrane (PAM), umbilical tered by a patch. In some embodiments, the patch is placed cord amniotic membrane (UCAM), or any combinations 15 on an osteolytic bone oran osteolytic joint. In some embodi thereof. In some embodiments, the substantially isolated ments, the composition is administered by an implant. In HC-HA complex is derived from frozen or previously frozen Some embodiments, the implant is placed on an osteolytic placental amniotic membrane (PAM), frozen or previously bone or an osteolytic joint. In some embodiments, the frozen umbilical cord amniotic membrane (UCAM), frozen composition is administered by an orthopaedic prosthesis. In or previously frozen placenta, frozen or previously frozen Some embodiments, the composition is administered by a umbilical cord, frozen or previously frozen chorion, frozen bone stent. In some embodiments, the methods further or previously frozen amnion-chorion, or any combinations comprise administering to the individual a calcium Supple thereof. In some embodiments, the composition further ment. In some embodiments, the disease, disorder, or con comprises a pharmaceutically-acceptable diluents, excipient dition characterized by excessive or undesired osteoclast or carrier. In some embodiments, the composition is formu 25 differentiation is arthritis, osteoporosis, alveolar bone deg lated as a solution, Suspension, paste, ointment, oil, emul radation, Paget’s Disease, a bone tumor, or any combination Sion, microemulsion, cream, lotion, gel, or combination thereof. thereof. In some embodiments, the composition is formu Disclosed herein, in certain embodiments, are methods of lated for controlled, Sustained, or delayed release. In some treating a disease, disorder or condition characterized by a embodiments, the composition is injected into an osteolytic 30 deficiency of bone formation in an individual in need joint. In some embodiments, the composition is adminis thereof, the method comprising administering to an indi tered by a patch. In some embodiments, the patch is placed vidual in need thereof a composition comprising substan on an osteolytic bone oran osteolytic joint. In some embodi tially isolated HC-HA complex. In some embodiments, the ments, the composition is administered by an implant. In HC-HA complex is obtained by a process comprising: (a) Some embodiments, the implant is placed on an osteolytic 35 providing a reaction mixture comprising: (i) HA (e.g., bone or an osteolytic joint. In some embodiments, the HMWHA); (ii) ICI, wherein the ICI is optionally in serum composition is administered by an orthopaedic prosthesis. In or isolated from serum; (iii) TSG-6, wherein the TSG-6 is Some embodiments, the composition is administered by a optionally recombinant; and (iv) PTX3, wherein the PTX3 bone stent. In some embodiments, the methods further is optionally recombinant; wherein at least one of HA, ICI, comprise administering to the individual a calcium Supple 40 TSG-6, or PTX3 is optionally generated by a plurality of ment. In some embodiments, the disease, disorder, or con cells present in the reaction mixture; (b) incubating the dition characterized by excessive or undesired osteoclast reaction mixture for a period of time sufficient to produce differentiation is arthritis, osteoporosis, alveolar bone deg HC-HA complex; and (c) isolating and purifying the HC-HA radation, Paget’s Disease, a bone tumor, or any combination complex. In some embodiments, the Substantially isolated thereof. 45 HC-HA complex is derived from placenta, umbilical cord, Disclosed herein, in certain embodiments, are methods of chorion, amnion-chorion, placental amniotic membrane promoting or inducing bone formation in an individual in (PAM), umbilical cord amniotic membrane (UCAM), or any need thereof, comprising administering to the individual a combinations thereof. In some embodiments, the Substan composition comprising Substantially isolated HC-HA com tially isolated HC-HA complex is derived from frozen or plex. In some embodiments, the HC-HA complex is 50 previously frozen placental amniotic membrane (PAM), obtained by a process comprising: (a) providing a reaction frozen or previously frozen umbilical cord amniotic mem mixture comprising: (i) HA (e.g., HMW HA); (ii) ICI, brane (UCAM), frozen or previously frozen placenta, frozen wherein the ICI is optionally in serum or isolated from or previously frozen umbilical cord, frozen or previously serum; (iii) TSG-6, wherein the TSG-6 is optionally recom frozen chorion, frozen or previously frozen amnion-chorion, binant; and (iv) PTX3, wherein the PTX3 is optionally 55 or any combinations thereof. In some embodiments, the recombinant; wherein at least one of HA, ICI, TSG-6, or composition further comprises a pharmaceutically-accept PTX3 is optionally generated by a plurality of cells present able diluents, excipient or carrier. In some embodiments, the in the reaction mixture; (b) incubating the reaction mixture composition is formulated as a Solution, Suspension, paste, for a period of time sufficient to produce HC-HA complex: ointment, oil, emulsion, microemulsion, cream, lotion, gel. and (c) isolating and purifying the HC-HA complex. In some 60 or combination thereof. In some embodiments, the compo embodiments, the substantially isolated HC-HA complex is sition is formulated for controlled, sustained, or delayed derived from placenta, umbilical cord, chorion, amnion release. In some embodiments, the composition is injected chorion, placental amniotic membrane (PAM), umbilical into an osteolytic joint. In some embodiments, the compo cord amniotic membrane (UCAM), or any combinations sition is administered by a patch. In some embodiments, the thereof. In some embodiments, the substantially isolated 65 patch is placed on an osteolytic bone or an osteolytic joint. HC-HA complex is derived from frozen or previously frozen In some embodiments, the composition is administered by placental amniotic membrane (PAM), frozen or previously an implant. In some embodiments, the implant is placed on US 9,675,733 B2 7 8 an osteolytic bone or an osteolytic joint. In some embodi ously frozen placental amniotic membrane (PAM), frozen or ments, the composition is administered by an orthopaedic previously frozen umbilical cord amniotic membrane prosthesis. In some embodiments, the composition is admin (UCAM), frozen or previously frozen placenta, frozen or istered by a bone stent. In some embodiments, the methods previously frozen umbilical cord, frozen or previously fro further comprise administering to the individual a calcium Zen chorion, frozen or previously frozen amnion-chorion, or Supplement. In some embodiments, the disease, disorder, or any combinations thereof. In some embodiments, the fetal condition characterized by excessive or undesired osteoclast support tissue product in the form of a pulverized powder or differentiation is arthritis, osteoporosis, alveolar bone deg a homogenate. In some embodiments, the extract comprises radation, Paget’s Disease, a bone tumor, or any combination HC-HA. thereof. 10 Disclosed herein, in certain embodiments, are bone Disclosed herein, in certain embodiments, are patches implants comprising Substantially isolated HC-HA. In some comprising fetal Support tissue product or an extract thereof. embodiments, the bone implant is a bone stent or an osse In some embodiments, the fetal Support tissue product ointegrated implant. In some embodiments, the Substantially comprises placental amniotic membrane (PAM), umbilical isolated HC-HA is coated onto the outside of the bone cord amniotic membrane (UCAM), placenta, umbilical cord, 15 implant. In some embodiments, the Substantially isolated chorion, amnion-chorion, or any combinations thereof. In HC-HA elutes from the bone implant. In some embodi Some embodiments, the fetal Support tissue product com ments, the substantially isolated HC-HA complex is pro prises frozen or previously frozen placental amniotic mem vided in a composition comprising fetal Support tissue brane (PAM), frozen or previously frozen umbilical cord product or in an extract of fetal Support tissue product. In amniotic membrane (UCAM), frozen or previously frozen some embodiments, the substantially isolated HC-HA com placenta, frozen or previously frozen umbilical cord, frozen plex is obtained by a process comprising: (a) providing a or previously frozen chorion, frozen or previously frozen reaction mixture comprising: (i) HA; (ii) ICI, wherein the amnion-chorion, or any combinations thereof. In some ICI is optionally in serum or isolated from serum; (iii) embodiments, the fetal support tissue product in the form of TSG-6, wherein the TSG-6 is optionally recombinant; and a pulverized powder or a homogenate. In some embodi 25 (iv) PTX3, wherein the PTX3 is optionally recombinant: ments, the extract comprises HC-HA. wherein at least one of HA, ICI, TSG-6, or PTX3 is Disclosed herein, in certain embodiments, are patches optionally generated by a plurality of cells present in the comprising substantially isolated HC-HA. In some embodi reaction mixture; (b) incubating the reaction mixture for a ments, the substantially isolated HC-HA complex is pro period of time sufficient to produce HC-HA complex; and (c) vided in a composition comprising fetal Support tissue 30 isolating and purifying the HC-HA complex. In some product or in an extract of fetal Support tissue product. In embodiments, the substantially isolated HC-HA complex is some embodiments, the substantially isolated HC-HA com derived from placenta, umbilical cord, chorion, amnion plex is obtained by a process comprising: (a) providing a chorion, placental amniotic membrane (PAM), umbilical reaction mixture comprising: (i) HA; (ii) ICI, wherein the cord amniotic membrane (UCAM), or any combinations ICI is optionally in serum or isolated from serum; (iii) 35 thereof. In some embodiments, the substantially isolated TSG-6, wherein the TSG-6 is optionally recombinant; and HC-HA complex is derived from frozen or previously frozen (iv) PTX3, wherein the PTX3 is optionally recombinant; placental amniotic membrane (PAM), frozen or previously wherein at least one of HA, ICI, TSG-6, or PTX3 is frozen umbilical cord amniotic membrane (UCAM), frozen optionally generated by a plurality of cells present in the or previously frozen placenta, frozen or previously frozen reaction mixture; (b) incubating the reaction mixture for a 40 umbilical cord, frozen or previously frozen chorion, frozen period of time sufficient to produce HC-HA complex; and (c) or previously frozen amnion-chorion, or any combinations isolating and purifying the HC-HA complex. In some thereof. embodiments, the substantially isolated HC-HA complex is Disclosed herein, in certain embodiments, are composi derived from placenta, umbilical cord, chorion, amnion tions for administration to a bone comprising fetal Support chorion, placental amniotic membrane (PAM), umbilical 45 tissue product or an extract thereof. In some embodiments, cord amniotic membrane (UCAM), or any combinations the composition is administered by injection, a bone implant thereof. In some embodiments, the substantially isolated or a patch. In some embodiments, the bone implant is a bone HC-HA complex is derived from frozen or previously frozen stent. In some embodiments, the fetal Support tissue product placental amniotic membrane (PAM), frozen or previously or the extract thereof is coated onto the outside of a bone frozen umbilical cord amniotic membrane (UCAM), frozen 50 implant. In some embodiments, the fetal Support tissue or previously frozen placenta, frozen or previously frozen product or the extract thereof elutes from the bone implant. umbilical cord, frozen or previously frozen chorion, frozen In some embodiments, the fetal Support tissue product or previously frozen amnion-chorion, or any combinations comprises placental amniotic membrane (PAM), umbilical thereof. cord amniotic membrane (UCAM), placenta, umbilical cord, Disclosed herein, in certain embodiments, are bone 55 chorion, amnion-chorion, or any combinations thereof. In implants comprising fetal Support tissue product or an Some embodiments, the fetal Support tissue product com extract thereof. In some embodiments, the bone implant is a prises frozen or previously frozen placental amniotic mem bone stent or an osseointegrated implant. In some embodi brane (PAM), frozen or previously frozen umbilical cord ments, the fetal support tissue product or the extract thereof amniotic membrane (UCAM), frozen or previously frozen is coated onto the outside of the bone implant. In some 60 placenta, frozen or previously frozen umbilical cord, frozen embodiments, the fetal Support tissue product or the extract or previously frozen chorion, frozen or previously frozen thereof elutes from the bone implant. In some embodiments, amnion-chorion, or any combinations thereof. In some the fetal Support tissue product comprises placental amniotic embodiments, the fetal support tissue product in the form of membrane (PAM), umbilical cord amniotic membrane a pulverized powder or a homogenate. In some embodi (UCAM), placenta, umbilical cord, chorion, amnion-cho 65 ments, the extract comprises HC-HA. rion, or any combinations thereof. In some embodiments, Disclosed herein, in certain embodiments, are composi the fetal Support tissue product comprises frozen or previ tions for administration to a bone comprising Substantially US 9,675,733 B2 9 10 isolated HC-HA. In some embodiments, the composition is composition is formulated into PLGA copolymers. In some administered by injection, a bone implant or a patch. In some embodiments, the composition is formulated for controlled, embodiments, the bone implant is a bone stent. In some Sustained, or delayed release. In some embodiments, the embodiments, the fetal Support tissue product or the extract composition is injected into an osteolytic joint. In some thereof is coated onto the outside of a bone implant. In some embodiments, the composition is administered by a patch. In embodiments, the fetal Support tissue product or the extract Some embodiments, the patch is placed on an osteolytic bone thereof elutes from the bone implant. In some embodiments, or an osteolytic joint. In some embodiments, the composi the substantially isolated HC-HA complex is provided in a tion is administered by an implant. In some embodiments, composition comprising fetal Support tissue product or in an the composition is formulated into an orthopaedic prosthe extract of fetal Support tissue product. In some embodi 10 ments, the substantially isolated HC-HA complex is sis. In some embodiments, the fetal Support tissue product is obtained by a process comprising: (a) providing a reaction in the form of a sheet. In some embodiments, the compo mixture comprising: (i) HA; (ii) ICI, wherein the ICI is sition further comprises a backing. In some embodiments, optionally in serum or isolated from serum; (iii) TSG-6, the composition is wrapped around an osteolytic bone or an wherein the TSG-6 is optionally recombinant; and (iv) 15 osteolytic joint. In some embodiments, the methods further PTX3, wherein the PTX3 is optionally recombinant: comprise administering to the individual a calcium Supple wherein at least one of HA, ICI, TSG-6, or PTX3 is ment. optionally generated by a plurality of cells present in the Disclosed herein, in certain embodiments, are methods of reaction mixture; (b) incubating the reaction mixture for a promoting mineralization by osteoblasts in an individual in period of time sufficient to produce HC-HA complex; and (c) need thereof, comprising administering to the individual a isolating and purifying the HC-HA complex. In some composition comprising a fetal Support tissue product or an embodiments, the substantially isolated HC-HA complex is extract thereof. In some embodiments, the fetal support derived from placenta, umbilical cord, chorion, amnion tissue product is Substantially isolated placental amniotic chorion, placental amniotic membrane (PAM), umbilical membrane (PAM), umbilical cord amniotic membrane cord amniotic membrane (UCAM), or any combinations 25 (UCAM), placenta, umbilical cord, chorion, amnion-cho thereof. In some embodiments, the substantially isolated rion, or any combinations thereof. In some embodiments, HC-HA complex is derived from frozen or previously frozen the fetal support tissue product is frozen or previously frozen placental amniotic membrane (PAM), frozen or previously PAM, frozen or previously frozen UCAM, frozen or previ frozen umbilical cord amniotic membrane (UCAM), frozen ously frozen placenta, frozen or previously frozen umbilical or previously frozen placenta, frozen or previously frozen 30 cord, frozen or previously frozen chorion, frozen or previ umbilical cord, frozen or previously frozen chorion, frozen ously frozen amnion-chorion, or any combinations thereof. or previously frozen amnion-chorion, or any combinations In some embodiments, the fetal support tissue product is thereof. cryopreserved, lyophilized, terminally sterilized, or a com Disclosed herein, in certain embodiments, are methods of bination thereof. In some embodiments, the fetal support inhibiting osteoclast differentiation in an individual in need 35 tissue product is in the form of a pulverized powder or a thereof, comprising administering to the individual a com homogenate. In some embodiments, the fetal Support tissue position comprising product fetal Support tissue product or product does not comprise a vein or an artery, a cell with an extract thereof. In some embodiments, the fetal Support metabolic activity, active HIV-1, active HIV-2, active tissue product is Substantially isolated placental amniotic HTLV-1, active hepatitis B, active hepatitis C, active West membrane (PAM), umbilical cord amniotic membrane 40 Nile Virus, active cytomegalovirus, active human transmis (UCAM), placenta, umbilical cord, chorion, amnion-cho sible spongiform encephalopathy, or active Treponema pal rion, or any combinations thereof. In some embodiments, lidum. In some embodiments, the fetal Support tissue prod the fetal support tissue product is frozen or previously frozen uct is obtained from a human, non-primate human, cow or PAM, frozen or previously frozen UCAM, frozen or previ pig. In some embodiments, the composition further com ously frozen placenta, frozen or previously frozen umbilical 45 prises a pharmaceutically-acceptable diluents, excipient or cord, frozen or previously frozen chorion, frozen or previ carrier. In some embodiments, the composition is formulated ously frozen amnion-chorion, or any combinations thereof. as a solution, Suspension, paste, ointment, oil, emulsion, In some embodiments, the fetal Support tissue product is microemulsion, cream, lotion, gel, or combination thereof. cryopreserved, lyophilized, terminally sterilized, or a com In some embodiments, the composition comprises Substan bination thereof. In some embodiments, the fetal support 50 tially-isolated tissue product formulated into microspheres, tissue product is in the form of a pulverized powder or a microparticles, or liposomes. In some embodiments, the homogenate. In some embodiments, the fetal Support tissue composition is formulated into PLGA copolymers. In some product does not comprise a vein or an artery, a cell with embodiments, the composition is formulated for controlled, metabolic activity, active HIV-1, active HIV-2, active Sustained, or delayed release. In some embodiments, the HTLV-1, active hepatitis B, active hepatitis C, active West 55 composition is injected into an osteolytic joint. In some Nile Virus, active cytomegalovirus, active human transmis embodiments, the composition is administered by a patch. In sible spongiform encephalopathy, or active Treponema pal Some embodiments, the patch is placed on an osteolytic bone lidum. In some embodiments, the fetal Support tissue prod or an osteolytic joint. In some embodiments, the composi uct is obtained from a human, non-primate human, cow or tion is administered by an implant. In some embodiments, pig. In some embodiments, the composition further com 60 the composition is formulated into an orthopaedic prosthe prises a pharmaceutically-acceptable diluents, excipient or sis. In some embodiments, the fetal Support tissue product is carrier. In some embodiments, the composition is formulated in the form of a sheet. In some embodiments, the compo as a solution, Suspension, paste, ointment, oil, emulsion, sition further comprises a backing. In some embodiments, microemulsion, cream, lotion, gel, or combination thereof. the composition is wrapped around an osteolytic bone or an In some embodiments, the composition comprises Substan 65 osteolytic joint. In some embodiments, the methods further tially-isolated tissue product formulated into microspheres, comprise administering to the individual a calcium Supple microparticles, or liposomes. In some embodiments, the ment. US 9,675,733 B2 11 12 Disclosed herein, in certain embodiments, are methods of product is in the form of a pulverized powder or a homo inhibiting bone resorption in an individual in need thereof, genate. In some embodiments, the fetal Support tissue prod comprising administering to the individual a composition uct does not comprise a vein or an artery, a cell with comprising a fetal Support tissue product or an extract metabolic activity, active HIV-1, active HIV-2, active thereof. In some embodiments, the fetal support tissue HTLV-1, active hepatitis B, active hepatitis C, active West product is Substantially isolated placental amniotic mem Nile Virus, active cytomegalovirus, active human transmis brane (PAM), umbilical cord amniotic membrane (UCAM), sible spongiform encephalopathy, or active Treponema pal placenta, umbilical cord, chorion, amnion-chorion, or any lidum. In some embodiments, the fetal Support tissue prod combinations thereof. In some embodiments, the fetal Sup uct is obtained from a human, non-primate human, cow or port tissue product is frozen or previously frozen PAM, 10 pig. In some embodiments, the composition further com frozen or previously frozen UCAM, frozen or previously prises a pharmaceutically-acceptable diluents, excipient or frozen placenta, frozen or previously frozen umbilical cord, carrier. In some embodiments, the composition is formulated frozen or previously frozen chorion, frozen or previously as a solution, Suspension, paste, ointment, oil, emulsion, frozen amnion-chorion, or any combinations thereof. In microemulsion, cream, lotion, gel, or combination thereof. Some embodiments, the fetal Support tissue product is cryo 15 In some embodiments, the composition comprises Substan preserved, lyophilized, terminally sterilized, or a combina tially-isolated tissue product formulated into microspheres, tion thereof. In some embodiments, the fetal support tissue microparticles, or liposomes. In some embodiments, the product is in the form of a pulverized powder or a homo composition is formulated into PLGA copolymers. In some genate. In some embodiments, the fetal Support tissue prod embodiments, the composition is formulated for controlled, uct does not comprise a vein or an artery, a cell with Sustained, or delayed release. In some embodiments, the metabolic activity, active HIV-1, active HIV-2, active composition is injected into an osteolytic joint. In some HTLV-1, active hepatitis B, active hepatitis C, active West embodiments, the composition is administered by a patch. In Nile Virus, active cytomegalovirus, active human transmis Some embodiments, the patch is placed on an osteolytic bone sible spongiform encephalopathy, or active Treponema pal or an osteolytic joint. In some embodiments, the composi lidum. In some embodiments, the fetal Support tissue prod 25 tion is administered by an implant. In some embodiments, uct is obtained from a human, non-primate human, cow or the composition is formulated into an orthopaedic prosthe pig. In some embodiments, the composition further com sis. In some embodiments, the fetal Support tissue product is prises a pharmaceutically-acceptable diluents, excipient or in the form of a substantially-flattened sheet. In some carrier. In some embodiments, the composition is formulated embodiments, the composition further comprises a backing. as a solution, Suspension, paste, ointment, oil, emulsion, 30 In some embodiments, the composition is wrapped around microemulsion, cream, lotion, gel, or combination thereof. an osteolytic bone or an osteolytic joint. In some embodi In some embodiments, the composition comprises substan ments, the methods further comprise administering to the tially-isolated tissue product formulated into microspheres, individual a calcium Supplement. microparticles, or liposomes. In some embodiments, the Disclosed herein, in certain embodiments, are methods of composition is formulated into PLGA copolymers. In some 35 balancing bone resorption and bone formation, comprising embodiments, the composition is formulated for controlled, administering to an individual in need thereof a therapeuti Sustained, or delayed release. In some embodiments, the cally-effective amount of a composition comprising a fetal composition is injected into an osteolytic joint. In some Support tissue product or an extract thereof. In some embodi embodiments, the composition is administered by a patch. In ments, the fetal Support tissue product is Substantially iso Some embodiments, the patch is placed on an osteolytic bone 40 lated placental amniotic membrane (PAM), umbilical cord or an osteolytic joint. In some embodiments, the composi amniotic membrane (UCAM), placenta, umbilical cord, tion is administered by an implant. In some embodiments, chorion, amnion-chorion, or any combinations thereof. In the composition is formulated into an orthopaedic prosthe Some embodiments, the fetal Support tissue product is frozen sis. In some embodiments, the fetal Support tissue product is or previously frozen PAM, frozen or previously frozen in the form of a substantially-flattened sheet. In some 45 UCAM, frozen or previously frozen placenta, frozen or embodiments, the composition further comprises a backing. previously frozen umbilical cord, frozen or previously fro In some embodiments, the composition is wrapped around Zen chorion, frozen or previously frozen amnion-chorion, or an osteolytic bone or an osteolytic joint. In some embodi any combinations thereof. In some embodiments, the fetal ments, the methods further comprise administering to the Support tissue product is cryopreserved, lyophilized, termi individual a calcium Supplement. 50 nally sterilized, or a combination thereof. In some embodi Disclosed herein, in certain embodiments, are methods of ments, the fetal Support tissue product is in the form of a inhibiting bone remodeling in an individual in need thereof, pulverized powder or a homogenate. In some embodiments, comprising administering to the individual a composition the fetal Support tissue product does not comprise a vein or comprising a fetal Support tissue product or an extract an artery, a cell with metabolic activity, active HIV-1, active thereof. In some embodiments, the fetal support tissue 55 HIV-2, active HTLV-1, active hepatitis B, active hepatitis C, product is Substantially isolated placental amniotic mem active West Nile Virus, active cytomegalovirus, active brane (PAM), umbilical cord amniotic membrane (UCAM), human transmissible spongiform encephalopathy, or active placenta, umbilical cord, chorion, amnion-chorion, or any Treponema pallidum. In some embodiments, the fetal Sup combinations thereof. In some embodiments, the fetal Sup port tissue product is obtained from a human, non-primate port tissue product is frozen or previously frozen PAM, 60 human, cow or pig. In some embodiments, the composition frozen or previously frozen UCAM, frozen or previously further comprises a pharmaceutically-acceptable diluents, frozen placenta, frozen or previously frozen umbilical cord, excipient or carrier. In some embodiments, the composition frozen or previously frozen chorion, frozen or previously is formulated as a solution, Suspension, paste, ointment, oil, frozen amnion-chorion, or any combinations thereof. In emulsion, microemulsion, cream, lotion, gel, or combination Some embodiments, the fetal Support tissue product is cryo 65 thereof. In some embodiments, the composition comprises preserved, lyophilized, terminally sterilized, or a combina Substantially-isolated tissue product formulated into micro tion thereof. In some embodiments, the fetal support tissue spheres, microparticles, or liposomes. In some embodi US 9,675,733 B2 13 14 ments, the composition is formulated into PLGA copoly ments, the methods further comprise administering to the mers. In some embodiments, the composition is formulated individual a calcium Supplement. for controlled, Sustained, or delayed release. In some Disclosed herein, in certain embodiments, are methods of embodiments, the composition is injected into an osteolytic treating a disease, disorder, or condition characterized by joint. In some embodiments, the composition is adminis excessive or undesired bone absorption by osteoclasts, the tered by a patch. In some embodiments, the patch is placed methods comprising administering to an individual in need on an osteolytic bone oran osteolytic joint. In some embodi thereof a therapeutically-effective amount of a composition ments, the composition is administered by an implant. In comprising a fetal Support tissue product or an extract Some embodiments, the composition is formulated into an thereof. In some embodiments, the fetal support tissue orthopaedic prosthesis. In some embodiments, the fetal 10 product is Substantially isolated placental amniotic mem Support tissue product is in the form of a Substantially brane (PAM), umbilical cord amniotic membrane (UCAM), flattened sheet. In some embodiments, the composition placenta, umbilical cord, chorion, amnion-chorion, or any further comprises a backing. In some embodiments, the combinations thereof. In some embodiments, the fetal Sup composition is wrapped around an osteolytic bone or an port tissue product is frozen or previously frozen PAM, osteolytic joint. In some embodiments, the methods further 15 frozen or previously frozen UCAM, frozen or previously comprise administering to the individual a calcium Supple frozen placenta, frozen or previously frozen umbilical cord, ment. frozen or previously frozen chorion, frozen or previously Disclosed herein, in certain embodiments, are methods of frozen amnion-chorion, or any combinations thereof. In treating a disease, disorder, or condition characterized by Some embodiments, the fetal Support tissue product is cryo excessive or undesired osteoclast differentiation, the meth preserved, lyophilized, terminally sterilized, or a combina ods comprising administering to an individual in need tion thereof. In some embodiments, the fetal support tissue thereof a therapeutically-effective amount of a composition product is in the form of a pulverized powder or a homo comprising a fetal Support tissue product or an extract genate. In some embodiments, the fetal Support tissue prod thereof. In some embodiments, the fetal support tissue uct does not comprise a vein or an artery, a cell with product is Substantially isolated placental amniotic mem 25 metabolic activity, active HIV-1, active HIV-2, active brane (PAM), umbilical cord amniotic membrane (UCAM), HTLV-1, active hepatitis B, active hepatitis C, active West placenta, umbilical cord, chorion, amnion-chorion, or any Nile Virus, active cytomegalovirus, active human transmis combinations thereof. In some embodiments, the fetal Sup sible spongiform encephalopathy, or active Treponema pal port tissue product is frozen or previously frozen PAM, lidum. In some embodiments, the fetal Support tissue prod frozen or previously frozen UCAM, frozen or previously 30 uct is obtained from a human, non-primate human, cow or frozen placenta, frozen or previously frozen umbilical cord, pig. In some embodiments, the composition further com frozen or previously frozen chorion, frozen or previously prises a pharmaceutically-acceptable diluents, excipient or frozen amnion-chorion, or any combinations thereof. In carrier. In some embodiments, the composition is formulated Some embodiments, the fetal Support tissue product is cryo as a solution, Suspension, paste, ointment, oil, emulsion, preserved, lyophilized, terminally sterilized, or a combina 35 microemulsion, cream, lotion, gel, or combination thereof. tion thereof. In some embodiments, the fetal support tissue In some embodiments, the composition comprises Substan product is in the form of a pulverized powder or a homo tially-isolated tissue product formulated into microspheres, genate. In some embodiments, the fetal Support tissue prod microparticles, or liposomes. In some embodiments, the uct does not comprise a vein or an artery, a cell with composition is formulated into PLGA copolymers. In some metabolic activity, active HIV-1, active HIV-2, active 40 embodiments, the composition is formulated for controlled, HTLV-1, active hepatitis B, active hepatitis C, active West Sustained, or delayed release. In some embodiments, the Nile Virus, active cytomegalovirus, active human transmis composition is injected into an osteolytic joint. In some sible spongiform encephalopathy, or active Treponema pal embodiments, the composition is administered by a patch. In lidum. In some embodiments, the fetal Support tissue prod Some embodiments, the patch is placed on an osteolytic bone uct is obtained from a human, non-primate human, cow or 45 or an osteolytic joint. In some embodiments, the composi pig. In some embodiments, the composition further com tion is administered by an implant. In some embodiments, prises a pharmaceutically-acceptable diluents, excipient or the composition is formulated into an orthopaedic prosthe carrier. In some embodiments, the composition is formulated sis. In some embodiments, the fetal Support tissue product is as a solution, Suspension, paste, ointment, oil, emulsion, in the form of a substantially-flattened sheet. In some microemulsion, cream, lotion, gel, or combination thereof. 50 embodiments, the composition further comprises a backing. In some embodiments, the composition comprises Substan In some embodiments, the composition is wrapped around tially-isolated tissue product formulated into microspheres, an osteolytic bone or an osteolytic joint. In some embodi microparticles, or liposomes. In some embodiments, the ments, the methods further comprise administering to the composition is formulated into PLGA copolymers. In some individual a calcium Supplement. embodiments, the composition is formulated for controlled, 55 Disclosed herein, in certain embodiments, are methods of Sustained, or delayed release. In some embodiments, the treating a disease, disorder, or condition characterized by composition is injected into an osteolytic joint. In some deficient or defective bone formation, the methods compris embodiments, the composition is administered by a patch. In ing administering to an individual in need thereof a thera Some embodiments, the patch is placed on an osteolytic bone peutically-effective amount of a composition comprising a or an osteolytic joint. In some embodiments, the composi 60 fetal Support tissue product or an extract thereof. In some tion is administered by an implant. In some embodiments, embodiments, the fetal Support tissue product is substan the composition is formulated into an orthopaedic prosthe tially isolated placental amniotic membrane (PAM), umbili sis. In some embodiments, the fetal Support tissue product is cal cord amniotic membrane (UCAM), placenta, umbilical in the form of a substantially-flattened sheet. In some cord, chorion, amnion-chorion, or any combinations thereof. embodiments, the composition further comprises a backing. 65 In some embodiments, the fetal Support tissue product is In some embodiments, the composition is wrapped around frozen or previously frozen PAM, frozen or previously an osteolytic bone or an osteolytic joint. In some embodi frozen UCAM, frozen or previously frozen placenta, frozen US 9,675,733 B2 15 16 or previously frozen umbilical cord, frozen or previously human, cow or pig. In some embodiments, the composition frozen chorion, frozen or previously frozen amnion-chorion, further comprises a pharmaceutically-acceptable diluents, or any combinations thereof. In some embodiments, the fetal excipient or carrier. In some embodiments, the composition Support tissue product is cryopreserved, lyophilized, termi is formulated as a solution, Suspension, paste, ointment, oil, nally sterilized, or a combination thereof. In some embodi emulsion, microemulsion, cream, lotion, gel, or combination ments, the fetal Support tissue product is in the form of a thereof. In some embodiments, the composition comprises pulverized powder or a homogenate. In some embodiments, Substantially-isolated tissue product formulated into micro the fetal Support tissue product does not comprise a vein or spheres, microparticles, or liposomes. In some embodi an artery, a cell with metabolic activity, active HIV-1, active ments, the composition is formulated into PLGA copoly HIV-2, active HTLV-1, active hepatitis B, active hepatitis C, 10 mers. In some embodiments, the composition is formulated active West Nile Virus, active cytomegalovirus, active for controlled, Sustained, or delayed release. In some human transmissible spongiform encephalopathy, or active embodiments, the composition is injected into an osteolytic Treponema pallidum. In some embodiments, the fetal Sup joint. In some embodiments, the composition is adminis port tissue product is obtained from a human, non-primate tered by a patch. In some embodiments, the patch is placed human, cow or pig. In some embodiments, the composition 15 on an osteolytic bone oran osteolytic joint. In some embodi further comprises a pharmaceutically-acceptable diluents, ments, the composition is administered by an implant. In excipient or carrier. In some embodiments, the composition Some embodiments, the implant is placed on an osteolytic is formulated as a solution, Suspension, paste, ointment, oil, bone or an osteolytic joint. In some embodiments, the emulsion, microemulsion, cream, lotion, gel, or combination composition is formulated into an orthopaedic prosthesis. In thereof. In some embodiments, the composition comprises Some embodiments, the fetal Support tissue product is in the Substantially-isolated tissue product formulated into micro form of a substantially-flattened sheet. In some embodi spheres, microparticles, or liposomes. In some embodi ments, the composition further comprises a backing. In some ments, the composition is formulated into PLGA copoly embodiments, the composition is wrapped around an mers. In some embodiments, the composition is formulated osteolytic bone or an osteolytic joint. In some embodiments, for controlled, Sustained, or delayed release. In some 25 the methods further comprise administering to the individual embodiments, the composition is injected into an osteolytic a calcium Supplement. In some embodiments, the methods joint. In some embodiments, the composition is adminis further comprise administering to the individual an NSAID, tered by a patch. In some embodiments, the patch is placed a corticosteroid, hyaluronan injections, a DMARD, an anal on an osteolytic bone oran osteolytic joint. In some embodi gesic, or any combination thereof. ments, the composition is administered by an implant. In 30 Disclosed herein, in certain embodiments, are methods of Some embodiments, the composition is formulated into an treating osteoporosis, the methods comprising administering orthopaedic prosthesis. In some embodiments, the fetal to an individual in need thereof a therapeutically-effective Support tissue product is in the form of a Substantially amount of a composition comprising a fetal Support tissue flattened sheet. In some embodiments, the composition product or an extract thereof. In some embodiments, the further comprises a backing. In some embodiments, the 35 fetal Support tissue product is substantially isolated placental composition is wrapped around an osteolytic bone or an amniotic membrane (PAM), umbilical cord amniotic mem osteolytic joint. In some embodiments, the methods further brane (UCAM), placenta, umbilical cord, chorion, amnion comprise administering to the individual a calcium Supple chorion, or any combinations thereof. In some embodi ment. ments, the fetal Support tissue product is frozen or Disclosed herein, in certain embodiments, are methods of 40 previously frozen PAM, frozen or previously frozen UCAM, treating arthritis, the methods comprising administering to frozen or previously frozen placenta, frozen or previously an individual in need thereof a therapeutically-effective frozen umbilical cord, frozen or previously frozen chorion, amount of a composition comprising a fetal Support tissue frozen or previously frozen amnion-chorion, or any combi product or an extract thereof. In some embodiments, the nations thereof. In some embodiments, the fetal Support arthritis is osteoarthritis, rheumatoid arthritis, psoriatic 45 tissue product is cryopreserved, lyophilized, terminally ster arthritis, or any combination thereof. In some embodiments, ilized, or a combination thereof. In some embodiments, the the fetal Support tissue product is Substantially isolated fetal support tissue product is in the form of a pulverized placental amniotic membrane (PAM), umbilical cord amni powder or a homogenate. In some embodiments, the fetal otic membrane (UCAM), placenta, umbilical cord, chorion, Support tissue product does not comprise a vein or an artery, amnion-chorion, or any combinations thereof. In some 50 a cell with metabolic activity, active HIV-1, active HIV-2, embodiments, the fetal Support tissue product is frozen or active HTLV-1, active hepatitis B, active hepatitis C, active previously frozen PAM, frozen or previously frozen UCAM, West Nile Virus, active cytomegalovirus, active human frozen or previously frozen placenta, frozen or previously transmissible spongiform encephalopathy, or active frozen umbilical cord, frozen or previously frozen chorion, Treponema pallidum. In some embodiments, the fetal Sup frozen or previously frozen amnion-chorion, or any combi 55 port tissue product is obtained from a human, non-primate nations thereof. In some embodiments, the fetal Support human, cow or pig. In some embodiments, the composition tissue product is cryopreserved, lyophilized, terminally ster further comprises a pharmaceutically-acceptable diluents, ilized, or a combination thereof. In some embodiments, the excipient or carrier. In some embodiments, the composition fetal support tissue product is in the form of a pulverized is formulated as a solution, Suspension, paste, ointment, oil, powder or a homogenate. In some embodiments, the fetal 60 emulsion, microemulsion, cream, lotion, gel, or combination Support tissue product does not comprise a vein or an artery, thereof. In some embodiments, the composition comprises a cell with metabolic activity, active HIV-1, active HIV-2, Substantially-isolated tissue product formulated into micro active HTLV-1, active hepatitis B, active hepatitis C, active spheres, microparticles, or liposomes. In some embodi West Nile Virus, active cytomegalovirus, active human ments, the composition is formulated into PLGA copoly transmissible spongiform encephalopathy, or active 65 mers. In some embodiments, the composition is formulated Treponema pallidum. In some embodiments, the fetal Sup for controlled, Sustained, or delayed release. In some port tissue product is obtained from a human, non-primate embodiments, the composition is injected into an osteolytic US 9,675,733 B2 17 18 joint. In some embodiments, the composition is adminis effective amount of a composition comprising a fetal Support tered by a patch. In some embodiments, the patch is placed tissue product or an extract thereof. In some embodiments, on an osteolytic bone oran osteolytic joint. In some embodi the fetal Support tissue product is Substantially isolated ments, the composition is administered by an implant. In placental amniotic membrane (PAM), umbilical cord amni Some embodiments, the implant is placed on an osteolytic otic membrane (UCAM), placenta, umbilical cord, chorion, bone or an osteolytic joint. In some embodiments, the amnion-chorion, or any combinations thereof. In some composition is formulated into an orthopaedic prosthesis. In embodiments, the fetal Support tissue product is frozen or Some embodiments, the fetal Support tissue product is in the previously frozen PAM, frozen or previously frozen UCAM, form of a substantially-flattened sheet. In some embodi frozen or previously frozen placenta, frozen or previously ments, the composition further comprises a backing. In some 10 frozen umbilical cord, frozen or previously frozen chorion, embodiments, the composition is wrapped around an frozen or previously frozen amnion-chorion, or any combi osteolytic bone or an osteolytic joint. In some embodiments, nations thereof. In some embodiments, the fetal Support the methods further comprise administering to the individual tissue product is cryopreserved, lyophilized, terminally ster a calcium Supplement. In some embodiments, the methods ilized, or a combination thereof. In some embodiments, the further comprise administering to the individual a bisphos 15 fetal support tissue product is in the form of a pulverized phonate, an estrogen analog, Raloxifene, Calcitonin, Teri powder or a homogenate. In some embodiments, the fetal paratide, calcium salts, sodium fluoride, RANKL inhibitors, Support tissue product does not comprise a vein or an artery, Strontium ranelate, or any combination thereof. a cell with metabolic activity, active HIV-1, active HIV-2, Disclosed herein, in certain embodiments, are methods of active HTLV-1, active hepatitis B, active hepatitis C, active treating alveolar bone degradation, the methods comprising West Nile Virus, active cytomegalovirus, active human administering to an individual in need thereof a therapeuti transmissible spongiform encephalopathy, or active cally-effective amount of a composition comprising a fetal Treponema pallidum. In some embodiments, the fetal Sup Support tissue product or an extract thereof. In some embodi port tissue product is obtained from a human, non-primate ments, the fetal Support tissue product is Substantially iso human, cow or pig. In some embodiments, the composition lated placental amniotic membrane (PAM), umbilical cord 25 further comprises a pharmaceutically-acceptable diluents, amniotic membrane (UCAM), placenta, umbilical cord, excipient or carrier. In some embodiments, the composition chorion, amnion-chorion, or any combinations thereof. In is formulated as a solution, Suspension, paste, ointment, oil, Some embodiments, the fetal Support tissue product is frozen emulsion, microemulsion, cream, lotion, gel, or combination or previously frozen PAM, frozen or previously frozen thereof. In some embodiments, the composition comprises UCAM, frozen or previously frozen placenta, frozen or 30 Substantially-isolated tissue product formulated into micro previously frozen umbilical cord, frozen or previously fro spheres, microparticles, or liposomes. In some embodi Zen chorion, frozen or previously frozen amnion-chorion, or ments, the composition is formulated into PLGA copoly any combinations thereof. In some embodiments, the fetal mers. In some embodiments, the composition is formulated Support tissue product is cryopreserved, lyophilized, termi for controlled, Sustained, or delayed release. In some nally sterilized, or a combination thereof. In some embodi 35 embodiments, the composition is administered by a patch. In ments, the fetal Support tissue product is in the form of a Some embodiments, the patch is placed on an osteolytic bone pulverized powder or a homogenate. In some embodiments, or an osteolytic joint. In some embodiments, the composi the fetal Support tissue product does not comprise a vein or tion is administered by an implant. In some embodiments, an artery, a cell with metabolic activity, active HIV-1, active the implant is placed on an osteolytic bone or an osteolytic HIV-2, active HTLV-1, active hepatitis B, active hepatitis C, 40 joint. In some embodiments, the composition is formulated active West Nile Virus, active cytomegalovirus, active into an orthopaedic prosthesis. In some embodiments, the human transmissible spongiform encephalopathy, or active fetal Support tissue product is in the form of a Substantially Treponema pallidum. In some embodiments, the fetal Sup flattened sheet. In some embodiments, the composition port tissue product is obtained from a human, non-primate further comprises a backing. In some embodiments, the human, cow or pig. In some embodiments, the composition 45 composition is wrapped around an osteolytic bone or an further comprises a pharmaceutically-acceptable diluents, osteolytic joint. In some embodiments, the methods further excipient or carrier. In some embodiments, the composition comprise administering to the individual a calcium Supple is formulated as a solution, Suspension, paste, ointment, oil, ment. In some embodiments, the methods further comprise emulsion, microemulsion, cream, lotion, gel, or combination administering to the individual a bisphosphonate. thereof. In some embodiments, the composition comprises 50 Disclosed herein, in certain embodiments, are methods of Substantially-isolated tissue product formulated into micro treating a bone tumor, the methods comprising administer spheres, microparticles, or liposomes. In some embodi ing to an individual in need thereof a therapeutically ments, the composition is formulated into PLGA copoly effective amount of a composition comprising a fetal Support mers. In some embodiments, the composition is formulated tissue product or an extract thereof. In some embodiments, for controlled, Sustained, or delayed release. In some 55 the fetal Support tissue product is Substantially isolated embodiments, the composition is administered by a patch. In placental amniotic membrane (PAM), umbilical cord amni Some embodiments, the patch is placed on the alveolar bone. otic membrane (UCAM), placenta, umbilical cord, chorion, In some embodiments, the composition is administered by amnion-chorion, or any combinations thereof. In some an implant. In some embodiments, the implant is placed on embodiments, the fetal Support tissue product is frozen or the alveolar bone. In some embodiments, the fetal support 60 previously frozen PAM, frozen or previously frozen UCAM, tissue product is in the form of a substantially-flattened frozen or previously frozen placenta, frozen or previously sheet. In some embodiments, the composition further com frozen umbilical cord, frozen or previously frozen chorion, prises a backing. In some embodiments, the composition is frozen or previously frozen amnion-chorion, or any combi placed on the alveolar bone. nations thereof. In some embodiments, the fetal Support Disclosed herein, in certain embodiments, are methods of 65 tissue product is cryopreserved, lyophilized, terminally ster treating Paget’s disease, the methods comprising adminis ilized, or a combination thereof. In some embodiments, the tering to an individual in need thereof a therapeutically fetal support tissue product is in the form of a pulverized US 9,675,733 B2 19 20 powder or a homogenate tissue product. In some embodi is purified by ultracentrifugation (e.g., four rounds of ultra ments, the fetal Support tissue product does not comprise a centrifugation). In some embodiments, the composition fur vein oran artery, a cell with metabolic activity, active HIV-1, ther comprises a pharmaceutically-acceptable diluents, active HIV-2, active HTLV-1, active hepatitis B, active excipient or carrier. In some embodiments, the composition hepatitis C, active West Nile Virus, active cytomegalovirus, is formulated as a solution, Suspension, paste, ointment, oil, active human transmissible spongiform encephalopathy, or emulsion, microemulsion, cream, lotion, gel, or combination active Treponema pallidum. In some embodiments, the fetal thereof. In some embodiments, the composition comprises Support tissue product is obtained from a human, non Substantially-isolated tissue product formulated into micro primate human, cow or pig. In some embodiments, the spheres, microparticles, or liposomes. In some embodi composition further comprises a pharmaceutically-accept 10 ments, the composition is formulated into PLGA copoly able diluents, excipient or carrier. In some embodiments, the mers. In some embodiments, the composition is formulated composition is formulated as a Solution, Suspension, paste, for controlled, Sustained, or delayed release. In some ointment, oil, emulsion, microemulsion, cream, lotion, gel. embodiments, the composition is injected into an osteolytic or combination thereof. In some embodiments, the compo joint. In some embodiments, the composition is adminis sition comprises Substantially-isolated tissue product formu 15 tered by a patch. In some embodiments, the patch is placed lated into microspheres, microparticles, or liposomes. In on an osteolytic bone oran osteolytic joint. In some embodi Some embodiments, the composition is formulated into ments, the composition is administered by an implant. In PLGA copolymers. In some embodiments, the composition Some embodiments, the implant is placed on an osteolytic is formulated for controlled, sustained, or delayed release. In bone or an osteolytic joint. In some embodiments, the Some embodiments, the composition is injected into an composition is formulated into an orthopaedic prosthesis. In osteolytic joint. In some embodiments, the composition is Some embodiments, the methods further comprise adminis administered by a patch. In some embodiments, the com tering to the individual a calcium Supplement. position is administered by a patch. In some embodiments, Disclosed herein, in certain embodiments, are methods of the patch is placed on an osteolytic bone or an osteolytic promoting mineralization by osteoblasts in an individual in joint. In some embodiments, the composition is adminis 25 need thereof, comprising administering to the individual a tered by an implant. In some embodiments, the implant is composition comprising Substantially isolated HC-HA com placed on an osteolytic bone or an osteolytic joint. In some plex. In some embodiments, the HC-HA complex is embodiments, the composition is formulated into an ortho obtained by a process comprising: (a) providing a reaction paedic prosthesis. In some embodiments, the fetal Support mixture comprising: (i) HA (e.g., HMW HA); (ii) ICI, tissue product is in the form of a substantially-flattened 30 wherein the ICI is optionally in serum or isolated from sheet. In some embodiments, the composition further com serum; (iii); (iii) TSG-6; wherein the TSG-6 is optionally prises a backing. In some embodiments, the composition is recombinant; and (iv) PTX3, wherein the PTX3 is optionally wrapped around an osteolytic bone or an osteolytic joint. In recombinant; wherein at least one of HA, ICI, TSG-6, Some embodiments, the methods further comprise adminis TSG-6 like protein is optionally generated by a plurality of tering to the individual a calcium Supplement. In some 35 cells present in the reaction mixture; (b) incubating the embodiments, the methods further comprise administering reaction mixture for a period of time sufficient to produce to the individual a chemotherapeutic agent, a bisphosphi HC-HA complex; and (c) isolating and purifying the HC-HA nate, Metastron, or any combination thereof. complex. In some embodiments, the Substantially isolated Disclosed herein, in certain embodiments, are methods of HC-HA complex is derived from placental amniotic mem inhibiting osteoclast differentiation in an individual in need 40 brane (PAM), umbilical cord amniotic membrane (UCAM), thereof, comprising administering to the individual a com placenta, umbilical cord, chorion or amnion-chorion, and is position comprising Substantially isolated HC-HA complex. optionally biochemically purified. In some embodiments, In some embodiments, the HC-HA complex is obtained by the substantially isolated HC-HA complex is derived from a process comprising: (a) providing a reaction mixture frozen or previously frozen placental amniotic membrane comprising: (i) HA (e.g., HMW HA); (ii) IOI, wherein the 45 (PAM), frozen or previously frozen umbilical cord amniotic ICI is optionally in serum or isolated from serum; (iii); (iii) membrane (UCAM), frozen or previously frozen placenta, TSG-6, wherein the TSG-6 is optionally recombinant; and frozen or previously frozen umbilical cord, frozen or pre (iv) PTX3, wherein the PTX3 is optionally recombinant; viously frozen chorion, or frozen or previously frozen wherein at least one of HA, ICI, TSG-6, or PTX3 is amnion-chorion, and is optionally biochemically purified. In optionally generated by a plurality of cells present in the 50 some embodiments, the HC-HA is purified by ultracentrifu reaction mixture; (b) incubating the reaction mixture for a gation (e.g., four rounds of ultracentrifugation). In some period of time sufficient to produce HC-HA complex; and (c) embodiments, the composition further comprises a pharma isolating and purifying the HC-HA complex. In some ceutically-acceptable diluents, excipient or carrier. In some embodiments, the substantially isolated HC-HA complex is embodiments, the composition is formulated as a solution, derived from placental amniotic membrane (PAM), umbili 55 Suspension, paste, ointment, oil, emulsion, microemulsion, cal cord amniotic membrane (UCAM), placenta, umbilical cream, lotion, gel, or combination thereof. In some embodi cord, chorion or amnion-chorion, and is optionally bio ments, the composition comprises Substantially-isolated tis chemically purified. In some embodiments, the HC-HA is Sue product formulated into microspheres, microparticles, or purified by ultracentrifugation (e.g., four rounds of ultracen liposomes. In some embodiments, the composition is for trifugation). In some embodiments, the Substantially isolated 60 mulated into PLGA copolymers. In some embodiments, the HC-HA complex is derived from frozen or previously frozen composition is formulated for controlled, Sustained, or placental amniotic membrane (PAM), frozen or previously delayed release. In some embodiments, the composition is frozen umbilical cord amniotic membrane (UCAM), frozen injected into an osteolytic joint. In some embodiments, the or previously frozen placenta, frozen or previously frozen composition is administered by a patch. In some embodi umbilical cord, frozen or previously frozen chorion or frozen 65 ments, the patch is placed on an osteolytic bone or an or previously frozen amnion-chorion, and is optionally osteolytic joint. In some embodiments, the composition is biochemically purified. In some embodiments, the HC-HA administered by an implant. In some embodiments, the US 9,675,733 B2 21 22 implant is placed on an osteolytic bone oran osteolytic joint. produce HC-HA complex; and (c) isolating and purifying In some embodiments, the composition is formulated into an the HC-HA complex. In some embodiments, the substan orthopaedic prosthesis. In some embodiments, the methods tially isolated HC-HA complex is derived from placental further comprise administering to the individual a calcium amniotic membrane (PAM), umbilical cord amniotic mem Supplement. brane (UCAM), placenta, umbilical cord, chorion or Disclosed herein, in certain embodiments, are methods of amnion-chorion. In some embodiments, the Substantially inhibiting bone resorption in an individual in need thereof, isolated HC-HA complex is derived from frozen or previ comprising administering to the individual a composition ously frozen placental amniotic membrane (PAM), frozen or comprising Substantially isolated HC-HA complex. In some previously frozen umbilical cord amniotic membrane embodiments, the HC-HA complex is obtained by a process 10 (UCAM), frozen or previously frozen placenta, frozen or comprising: (a) providing a reaction mixture comprising: (i) previously frozen umbilical cord, frozen or previously fro HA (e.g., HMWHA); (ii) ICI, wherein the ICI is optionally Zen chorion or frozen or previously frozen amnion-chorion. in serum or isolated from serum; (iii); (iii) TSG-6, wherein In some embodiments, the composition further comprises a the TSG-6 is optionally recombinant; and (iv) PTX3. pharmaceutically-acceptable diluents, excipient or carrier. In wherein the PTX3 is optionally recombinant; wherein at 15 Some embodiments, the composition is formulated as a least one of HA, ICI, TSG-6, or PTX3 is optionally gener Solution, Suspension, paste, ointment, oil, emulsion, micro ated by a plurality of cells present in the reaction mixture: emulsion, cream, lotion, gel, or combination thereof. In (b) incubating the reaction mixture for a period of time Some embodiments, the composition comprises Substan Sufficient to produce HC-HA complex; and (c) isolating and tially-isolated tissue product formulated into microspheres, purifying the HC-HA complex. In some embodiments, the microparticles, or liposomes. In some embodiments, the substantially isolated HC-HA complex is derived from pla composition is formulated into PLGA copolymers. In some cental amniotic membrane (PAM), umbilical cord amniotic embodiments, the composition is formulated for controlled, membrane (UCAM), placenta, umbilical cord, chorion or Sustained, or delayed release. In some embodiments, the amnion-chorion, and is optionally biochemically purified. In composition is injected into an osteolytic joint. In some some embodiments, the substantially isolated HC-HA com 25 embodiments, the composition is administered by a patch. In plex is derived from frozen or previously frozen placental Some embodiments, the patch is placed on an osteolytic bone amniotic membrane (PAM), frozen or previously frozen or an osteolytic joint. In some embodiments, the composi umbilical cord amniotic membrane (UCAM), frozen or tion is administered by an implant. In some embodiments, previously frozen placenta, frozen or previously frozen the implant is placed on an osteolytic bone or an osteolytic umbilical cord, frozen or previously frozen chorion or frozen 30 joint. In some embodiments, the composition is formulated or previously frozen amnion-chorion, and is optionally into an orthopaedic prosthesis. In some embodiments, the biochemically purified. In some embodiments, the HC-HA methods further comprise administering to the individual a is purified by ultracentrifugation (e.g., four rounds of ultra calcium Supplement. centrifugation). In some embodiments, the composition fur Disclosed herein, in certain embodiments, are methods of ther comprises a pharmaceutically-acceptable diluents, 35 balancing bone resorption and bone formation, comprising excipient or carrier. In some embodiments, the composition administering to an individual in need thereof a therapeuti is formulated as a solution, Suspension, paste, ointment, oil, cally-effective amount of a composition comprising Substan emulsion, microemulsion, cream, lotion, gel, or combination tially isolated HC-HA complex. In some embodiments, the thereof. In some embodiments, the composition comprises HC-HA complex is obtained by a process comprising: (a) Substantially-isolated tissue product formulated into micro 40 providing a reaction mixture comprising: (i) HA (e.g., HMW spheres, microparticles, or liposomes. In some embodi HA); (ii) ICI, wherein the ICI is optionally in serum or ments, the composition is formulated into PLGA copoly isolated from serum; (iii) TSG-6, wherein the TSG-6 is mers. In some embodiments, the composition is formulated optionally recombinant; and (iv) PTX3, wherein the PTX3 for controlled, Sustained, or delayed release. In some is optionally recombinant; wherein at least one of HA, ICI, embodiments, the composition is injected into an osteolytic 45 TSG-6, or PTX3 is optionally generated by a plurality of joint. In some embodiments, the composition is adminis cells present in the reaction mixture; (b) incubating the tered by a patch. In some embodiments, the patch is placed reaction mixture for a period of time sufficient to produce on an osteolytic bone oran osteolytic joint. In some embodi HC-HA complex; and (c) isolating and purifying the HC-HA ments, the composition is administered by an implant. In complex. In some embodiments, the Substantially isolated Some embodiments, the implant is placed on an osteolytic 50 HC-HA complex is derived from placental amniotic mem bone or an osteolytic joint. In some embodiments, the brane (PAM), umbilical cord amniotic membrane (UCAM), composition is formulated into an orthopaedic prosthesis. In placenta, umbilical cord, chorion or amnion-chorion, and is Some embodiments, the methods further comprise adminis optionally biochemically purified. In some embodiments, tering to the individual a calcium Supplement. the substantially isolated HC-HA complex is derived from Disclosed herein, in certain embodiments, are methods of 55 frozen or previously frozen placental amniotic membrane inhibiting bone remodeling in an individual in need thereof, (PAM), frozen or previously frozen umbilical cord amniotic comprising administering to the individual a composition membrane (UCAM), frozen or previously frozen placenta, comprising Substantially isolated HC-HA complex. In some frozen or previously frozen umbilical cord, frozen or pre embodiments, the HC-HA complex is obtained by a process viously frozen chorion or frozen or previously frozen comprising: (a) providing a reaction mixture comprising: (i) 60 amnion-chorion, and is optionally biochemically purified. In HA (e.g., HMWHA); (ii) ICI, wherein the ICI is optionally some embodiments, the HC-HA is purified by ultracentrifu in serum or isolated from serum; (iii) TSG-6, wherein the gation (e.g., four rounds of ultracentrifugation). In some TSG-6 is optionally recombinant; and (iv) PTX3, wherein embodiments, the composition further comprises a pharma the PTX3 is optionally recombinant; wherein at least one of ceutically-acceptable diluents, excipient or carrier. In some HA, ICI, TSG-6, or PTX3 is optionally generated by a 65 embodiments, the composition is formulated as a solution, plurality of cells present in the reaction mixture; (b) incu Suspension, paste, ointment, oil, emulsion, microemulsion, bating the reaction mixture for a period of time sufficient to cream, lotion, gel, or combination thereof. In some embodi US 9,675,733 B2 23 24 ments, the composition comprises Substantially-isolated tis Disclosed herein, in certain embodiments, are methods of Sue product formulated into microspheres, microparticles, or treating a disease, disorder, or condition characterized by liposomes. In some embodiments, the composition is for deficient or defective bone formation, the methods compris mulated into PLGA copolymers. In some embodiments, the ing administering to an individual in need thereof a thera composition is formulated for controlled, Sustained, or peutically-effective amount of a composition comprising delayed release. In some embodiments, the composition is substantially isolated HC-HA complex. In some embodi injected into an osteolytic joint. In some embodiments, the ments, the HC-HA complex is obtained by a process com composition is administered by a patch. In some embodi prising: (a) providing a reaction mixture comprising: (i) HA ments, the patch is placed on an osteolytic bone or an (e.g., HMW HA); (ii) ICI, wherein the ICI is optionally in osteolytic joint. In some embodiments, the composition is 10 serum or isolated from serum; (iii); (iii) TSG-6, wherein the administered by an implant. In some embodiments, the TSG-6 is optionally recombinant; and (iv) PTX3, wherein implant is placed on an osteolytic bone oran osteolytic joint. the PTX3 is optionally recombinant; wherein at least one of In some embodiments, the composition is formulated into an HA, ICI, TSG-6, or PTX3 is optionally generated by a orthopaedic prosthesis. In some embodiments, the methods plurality of cells present in the reaction mixture; (b) incu further comprise administering to the individual a calcium 15 bating the reaction mixture for a period of time sufficient to Supplement. produce HC-HA complex; and (c) isolating and purifying Disclosed herein, in certain embodiments, are methods of the HC-HA complex. In some embodiments, the substan treating a disease, disorder, or condition characterized by tially isolated HC-HA complex is derived from placental excessive or undesired osteoclast differentiation, the meth amniotic membrane (PAM), umbilical cord amniotic mem ods comprising administering to an individual in need brane (UCAM), placenta, umbilical cord, chorion or thereof a therapeutically-effective amount of a composition amnion-chorion, and is optionally biochemically purified. In comprising Substantially isolated HC-HA complex. In some some embodiments, the substantially isolated HC-HA com embodiments, the HC-HA complex is obtained by a process plex is derived from frozen or previously frozen placental comprising: (a) providing a reaction mixture comprising: (i) amniotic membrane (PAM), frozen or previously frozen HA (e.g., HMWHA); (ii) ICI, wherein the ICI is optionally 25 umbilical cord amniotic membrane (UCAM), frozen or in serum or isolated from serum; (iii); (iii) TSG-6, wherein previously frozen placenta, frozen or previously frozen the TSG-6 is optionally recombinant; and (iv) PTX3. umbilical cord, frozen or previously frozen chorion or frozen wherein the PTX3 is optionally recombinant; wherein at or previously frozen amnion-chorion, and is optionally least one of HA, ICI, TSG-6, or PTX3 is optionally gener biochemically purified. In some embodiments, the HC-HA ated by a plurality of cells present in the reaction mixture: 30 is purified by ultracentrifugation (e.g., four rounds of ultra (b) incubating the reaction mixture for a period of time centrifugation). In some embodiments, the composition fur sufficient to produce HC-HA complex; and (c) isolating and ther comprises a pharmaceutically-acceptable diluents, purifying the HC-HA complex. In some embodiments, the excipient or carrier. In some embodiments, the composition substantially isolated HC-HA complex is derived from pla is formulated as a solution, Suspension, paste, ointment, oil, cental amniotic membrane (PAM), umbilical cord amniotic 35 emulsion, microemulsion, cream, lotion, gel, or combination membrane (UCAM), placenta, umbilical cord, chorion or thereof. In some embodiments, the composition comprises amnion-chorion, and is optionally biochemically purified. In Substantially-isolated tissue product formulated into micro some embodiments, the substantially isolated HC-HA com spheres, microparticles, or liposomes. In some embodi plex is derived from frozen or previously frozen placental ments, the composition is formulated into PLGA copoly amniotic membrane (PAM), frozen or previously frozen 40 mers. In some embodiments, the composition is formulated umbilical cord amniotic membrane (UCAM), frozen or for controlled, Sustained, or delayed release. In some previously frozen placenta, frozen or previously frozen embodiments, the composition is injected into an osteolytic umbilical cord, frozen or previously frozen chorion or frozen joint. In some embodiments, the composition is adminis or previously frozen amnion-chorion, and is optionally tered by a patch. In some embodiments, the patch is placed biochemically purified. In some embodiments, the HC-HA 45 on an osteolytic bone oran osteolytic joint. In some embodi is purified by ultracentrifugation (e.g., four rounds of ultra ments, the composition is administered by an implant. In centrifugation). In some embodiments, the composition fur Some embodiments, the implant is placed on an osteolytic ther comprises a pharmaceutically-acceptable diluents, bone or an osteolytic joint. In some embodiments, the excipient or carrier. In some embodiments, the composition composition is formulated into an orthopaedic prosthesis. In is formulated as a solution, Suspension, paste, ointment, oil, 50 Some embodiments, the methods further comprise adminis emulsion, microemulsion, cream, lotion, gel, or combination tering to the individual a calcium Supplement. thereof. In some embodiments, the composition comprises Disclosed herein, in certain embodiments, are methods of Substantially-isolated tissue product formulated into micro treating arthritis, the methods comprising administering to spheres, microparticles, or liposomes. In some embodi an individual in need thereof a therapeutically-effective ments, the composition is formulated into PLGA copoly 55 amount of a composition comprising Substantially isolated mers. In some embodiments, the composition is formulated HC-HA complex. In some embodiments, the arthritis is for controlled, Sustained, or delayed release. In some osteoarthritis, rheumatoid arthritis, psoriatic arthritis, or any embodiments, the composition is injected into an osteolytic combination thereof. In some embodiments, the HC-HA joint. In some embodiments, the composition is adminis complex is obtained by a process comprising: (a) providing tered by a patch. In some embodiments, the patch is placed 60 a reaction mixture comprising: (i) HA (e.g., HMW HA); (ii) on an osteolytic bone oran osteolytic joint. In some embodi ICI, wherein the ICI is optionally in serum or isolated from ments, the composition is administered by an implant. In serum; (iii); (iii) TSG-6, wherein the TSG-6 is optionally Some embodiments, the implant is placed on an osteolytic recombinant; and (iv) PTX3, wherein the PTX3 is optionally bone or an osteolytic joint. In some embodiments, the recombinant; wherein at least one of HA, ICI, TSG-6, or composition is formulated into an orthopaedic prosthesis. In 65 PTX3 is optionally generated by a plurality of cells present Some embodiments, the methods further comprise adminis in the reaction mixture; (b) incubating the reaction mixture tering to the individual a calcium Supplement. for a period of time sufficient to produce HC-HA complex: US 9,675,733 B2 25 26 and (c) isolating and purifying the HC-HA complex. In some four rounds of ultracentrifugation). In some embodiments, embodiments, the substantially isolated HC-HA complex is the composition further comprises a pharmaceutically-ac derived from placental amniotic membrane (PAM), umbili ceptable diluents, excipient or carrier. In some embodi cal cord amniotic membrane (UCAM), placenta, umbilical ments, the composition is formulated as a Solution, Suspen cord, chorion or amnion-chorion, and is optionally bio Sion, paste, ointment, oil, emulsion, microemulsion, cream, chemically purified. In some embodiments, the Substantially lotion, gel, or combination thereof. In some embodiments, isolated HC-HA complex is derived from frozen or previ the composition comprises Substantially-isolated tissue ously frozen placental amniotic membrane (PAM), frozen or product formulated into microspheres, microparticles, or previously frozen umbilical cord amniotic membrane liposomes. In some embodiments, the composition is for (UCAM), frozen or previously frozen placenta, frozen or 10 mulated into PLGA copolymers. In some embodiments, the previously frozen umbilical cord, frozen or previously fro composition is formulated for controlled, Sustained, or Zen chorion or frozen or previously frozen amnion-chorion, delayed release. In some embodiments, the composition is and is optionally biochemically purified. In some embodi injected into an osteolytic joint. In some embodiments, the ments, the HC-HA is purified by ultracentrifugation (e.g., composition is administered by a patch. In some embodi four rounds of ultracentrifugation). In some embodiments, 15 ments, the patch is placed on an osteolytic bone or an the composition further comprises a pharmaceutically-ac osteolytic joint. In some embodiments, the composition is ceptable diluents, excipient or carrier. In some embodi administered by an implant. In some embodiments, the ments, the composition is formulated as a solution, Suspen implant is placed on an osteolytic bone oran osteolytic joint. Sion, paste, ointment, oil, emulsion, microemulsion, cream, In some embodiments, the composition is formulated into an lotion, gel, or combination thereof. In some embodiments, orthopaedic prosthesis. In some embodiments, the methods the composition comprises Substantially-isolated tissue further comprise administering to the individual a calcium product formulated into microspheres, microparticles, or Supplement. In some embodiments, the methods further liposomes. In some embodiments, the composition is for comprise administering to the individual a bisphosphonate, mulated into PLGA copolymers. In some embodiments, the an estrogen analog, Raloxifene, Calcitonin, Teriparatide, composition is formulated for controlled, Sustained, or 25 calcium salts, sodium fluoride, RANKL inhibitors, Stron delayed release. In some embodiments, the composition is tium ranelate, or any combination thereof. injected into an osteolytic joint. In some embodiments, the Disclosed herein, in certain embodiments, are methods of composition is administered by a patch. In some embodi treating alveolar bone degradation, the methods comprising ments, the patch is placed on an osteolytic bone or an administering to an individual in need thereof a therapeuti osteolytic joint. In some embodiments, the composition is 30 cally-effective amount of a composition comprising Substan administered by an implant. In some embodiments, the tially isolated HC-HA complex. In some embodiments, the implant is placed on an osteolytic bone or an osteolytic joint. HC-HA complex is obtained by a process comprising: (a) In some embodiments, the composition is formulated into an providing a reaction mixture comprising: (i) HA (e.g., HMW orthopaedic prosthesis. In some embodiments, the methods HA); (ii) ICI, wherein the ICI is optionally in serum or further comprise administering to the individual a calcium 35 isolated from serum; (iii) TSG-6, wherein the TSG-6 is Supplement. In some embodiments, the methods further optionally recombinant; and (iv) PTX3, wherein the PTX3 comprise administering to the individual an NSAID, a is optionally recombinant; wherein at least one of HA, ICI, corticosteroid, hyaluronan injections, a DMARD, an anal TSG-6, or PTX3 is optionally generated by a plurality of gesic, or any combination thereof. cells present in the reaction mixture; (b) incubating the Disclosed herein, in certain embodiments, are methods of 40 reaction mixture for a period of time sufficient to produce treating osteoporosis, the methods comprising administering HC-HA complex; and (c) isolating and purifying the HC-HA to an individual in need thereof a therapeutically-effective complex. In some embodiments, the Substantially isolated amount of a composition comprising Substantially isolated HC-HA complex is derived from placental amniotic mem HC-HA complex. In some embodiments, the HC-HA com brane (PAM), umbilical cord amniotic membrane (UCAM), plex is obtained by a process comprising: (a) providing a 45 placenta, umbilical cord, chorion or amnion-chorion, and is reaction mixture comprising: (i) HA (e.g., HMW HA); (ii) optionally biochemically purified. In some embodiments, ICI, wherein the IOI is optionally in serum or isolated from the substantially isolated HC-HA complex is derived from serum; (iii); (iii) TSG-6, wherein the TSG-6 is optionally frozen or previously frozen placental amniotic membrane recombinant; and (iv) PTX3, wherein the PTX3 is optionally (PAM), frozen or previously frozen umbilical cord amniotic recombinant; wherein at least one of HA, ICI, TSG-6, or 50 membrane (UCAM), frozen or previously frozen placenta, PTX3 is optionally generated by a plurality of cells present frozen or previously frozen umbilical cord, frozen or pre in the reaction mixture; (b) incubating the reaction mixture viously frozen chorion or frozen or previously frozen for a period of time sufficient to produce HC-HA complex: amnion-chorion, and is optionally biochemically purified. In and (c) isolating and purifying the HC-HA complex. In some some embodiments, the HC-HA is purified by ultracentrifu embodiments, the substantially isolated HC-HA complex is 55 gation (e.g., four rounds of ultracentrifugation). In some derived from placental amniotic membrane (PAM), umbili embodiments, the composition further comprises a pharma cal cord amniotic membrane (UCAM), placenta, umbilical ceutically-acceptable diluents, excipient or carrier. In some cord, chorion or amnion-chorion, and is optionally bio embodiments, the composition is formulated as a solution, chemically purified. In some embodiments, the Substantially Suspension, paste, ointment, oil, emulsion, microemulsion, isolated HC-HA complex is derived from frozen or previ 60 cream, lotion, gel, or combination thereof. In some embodi ously frozen placental amniotic membrane (PAM), frozen or ments, the composition comprises Substantially-isolated tis previously frozen umbilical cord amniotic membrane Sue product formulated into microspheres, microparticles, or (UCAM), frozen or previously frozen placenta, frozen or liposomes. In some embodiments, the composition is for previously frozen umbilical cord, frozen or previously fro mulated into PLGA copolymers. In some embodiments, the Zen chorion or frozen or previously frozen amnion-chorion, 65 composition is formulated for controlled, Sustained, or and is optionally biochemically purified. In some embodi delayed release. In some embodiments, the composition is ments, the HC-HA is purified by ultracentrifugation (e.g., administered by a patch. In some embodiments, the patch is US 9,675,733 B2 27 28 placed on the alveolar bone. In some embodiments, the TSG-6, or PTX3 is optionally generated by a plurality of composition is administered by an implant. In some embodi cells present in the reaction mixture; (b) incubating the ments, the implant is placed on an alveolar bone. In some reaction mixture for a period of time sufficient to produce embodiments, the methods further comprise administering HC-HA complex; and (c) isolating and purifying the HC-HA to the individual a calcium Supplement. 5 complex. In some embodiments, the Substantially isolated Disclosed herein, in certain embodiments, are methods of HC-HA complex is derived from placental amniotic mem treating Paget’s disease, the methods comprising adminis brane (PAM), umbilical cord amniotic membrane (UCAM), tering to an individual in need thereof a therapeutically placenta, umbilical cord, chorion or amnion-chorion, and is effective amount of a composition comprising Substantially optionally biochemically purified. In some embodiments, isolated HC-HA complex. In some embodiments, the HC 10 the substantially isolated HC-HA complex is derived from HA complex is obtained by a process comprising: (a) frozen or previously frozen placental amniotic membrane providing a reaction mixture comprising: (i) HA (e.g., HMW (PAM), frozen or previously frozen umbilical cord amniotic HA); (ii) ICI, wherein the ICI is optionally in serum or membrane (UCAM), frozen or previously frozen placenta, isolated from serum; (iii) TSG-6, wherein the TSG-6 is frozen or previously frozen umbilical cord, frozen or pre optionally recombinant; and (iv) PTX3, wherein the PTX3 15 viously frozen chorion or frozen or previously frozen is optionally recombinant; wherein at least one of HA, ICI, amnion-chorion, and is optionally biochemically purified. In TSG-6, or PTX3 is optionally generated by a plurality of some embodiments, the HC-HA is purified by ultracentrifu cells present in the reaction mixture; (b) incubating the gation (e.g., four rounds of ultracentrifugation). In some reaction mixture for a period of time sufficient to produce embodiments, the composition further comprises a pharma HC-HA complex; and (c) isolating and purifying the HC-HA ceutically-acceptable diluents, excipient or carrier. In some complex. In some embodiments, the Substantially isolated embodiments, the composition is formulated as a solution, HC-HA complex is derived from placental amniotic mem Suspension, paste, ointment, oil, emulsion, microemulsion, brane (PAM), umbilical cord amniotic membrane (UCAM), cream, lotion, gel, or combination thereof. In some embodi placenta, umbilical cord, chorion or amnion-chorion, and is ments, the composition comprises Substantially-isolated tis optionally biochemically purified. In some embodiments, 25 Sue product formulated into microspheres, microparticles, or the substantially isolated HC-HA complex is derived from liposomes. In some embodiments, the composition is for frozen or previously frozen placental amniotic membrane mulated into PLGA copolymers. In some embodiments, the (PAM), frozen or previously frozen umbilical cord amniotic composition is formulated for controlled, Sustained, or membrane (UCAM), frozen or previously frozen placenta, delayed release. In some embodiments, the composition is frozen or previously frozen umbilical cord, frozen or pre 30 injected into an osteolytic joint. In some embodiments, the viously frozen chorion or frozen or previously frozen composition is administered by a patch. In some embodi amnion-chorion, and is optionally biochemically purified. In ments, the patch is placed on an osteolytic bone or an some embodiments, the HC-HA is purified by ultracentrifu osteolytic joint. In some embodiments, the composition is gation (e.g., four rounds of ultracentrifugation). In some administered by an implant. In some embodiments, the embodiments, the composition further comprises a pharma 35 implant is placed on an osteolytic bone oran osteolytic joint. ceutically-acceptable diluents, excipient or carrier. In some In some embodiments, the composition is formulated into an embodiments, the composition is formulated as a solution, orthopaedic prosthesis. In some embodiments, the methods Suspension, paste, ointment, oil, emulsion, microemulsion, further comprise administering to the individual a calcium cream, lotion, gel, or combination thereof. In some embodi Supplement. In some embodiments, the methods further ments, the composition comprises Substantially-isolated tis 40 comprise administering to the individual a chemotherapeutic Sue product formulated into microspheres, microparticles, or agent, a bisphosphinate, Metastron, or any combination liposomes. In some embodiments, the composition is for thereof. mulated into PLGA copolymers. In some embodiments, the composition is formulated for controlled, Sustained, or BRIEF DESCRIPTION OF THE DRAWINGS delayed release. In some embodiments, the composition is 45 injected into an osteolytic joint. In some embodiments, the The novel features of the invention are set forth with composition is administered by a patch. In some embodi particularity in the appended claims. A better understanding ments, the patch is placed on an osteolytic bone or an of the features and advantages of the present invention will osteolytic joint. In some embodiments, the composition is be obtained by reference to the following detailed descrip administered by an implant. In some embodiments, the 50 tion that sets forth illustrative embodiments, in which the implant is placed on an osteolytic bone oran osteolytic joint. principles of the invention are utilized, and the accompany In some embodiments, the composition is formulated into an ing drawings of which: orthopaedic prosthesis. In some embodiments, the methods FIG. 1 exemplifies the formation of osteoclast is regulated further comprise administering to the individual a calcium by RANK/RANKL and its downstream signaling pathway. Supplement. In some embodiments, the methods further 55 NFATcl is a critical transcriptional factor that regulates the comprise administering to the individual a bisphosphonate. expression of osteoclastogenesis-related genes including Disclosed herein, in certain embodiments, are methods of TRAP treating a bone tumor, the methods comprising administer FIG. 2 exemplifies macrophage RAW264.7 cells cultured ing to an individual in need thereof a therapeutically with (E-G, Pos CTL) and without (A-C, Neg CTL) 50 ng/ml effective amount of a composition comprising Substantially 60 RANKL stimulation. Osteoclasts began to form on Day 3 isolated HC-HA complex. In some embodiments, the HC (F) and complete differentiation was observed on Day 5 (G) HA complex is obtained by a process comprising: (a) as evidenced by positive TRAP staining. Bar=200 um. providing a reaction mixture comprising: (i) HA (e.g., HMW FIG. 3 exemplifies macrophage RAW264.7 cells stimu HA); (ii) ICI, wherein the ICI is optionally in serum or lated with 50 ng/ml RANKL and cultured with soluble isolated from serum; (iii) TSG-6, wherein the TSG-6 is 65 amniotic membrane extract (AME) (A), HC-HA complex optionally recombinant; and (iv) PTX3, wherein the PTX3 purified from AM (HC-HA)(B), AM lysate (AML) (C), AM is optionally recombinant; wherein at least one of HA, ICI, powder (AMP) (D), and on the epithelial side of intact US 9,675,733 B2 29 30 amniotic membrane (iAM)(E), the stromal side of iAM (F), (5 g/ml), AMP (200 ug/ml), ACP (100 g/ml), PLP (100 the basement membrane side of epithelially-denuded AM ug/ml), and WUC (100 ug/ml) significantly inhibit osteo (dAM) (G), the stromal side of dAM (H), the epithelial side clast formation. of lyophilized amniotic membrane 1AM (I), or the stromal FIG. 13 exemplifies the effects of AMP on osteogenesis. side of lyophilized amniotic membrane IAM (J). Cell mor After cultivation for 7 days, the cell morphology of groups phology on Day 3 (Column 1), Day 5 (A2-D2), and Day 6 with induction (Pos Ctrl, HC-HA, and AMP) is changed: a (E2-J2). TRAP staining (Column 3) revealed no large mul compact layer of cells appears in the periphery in Pos Ctrl tinucleated osteoclasts when cultured on AM and its deriva and HC-HA, but becomes clusters in AMP. Alizarin staining tives. Bar=200 um. (ARS) shows only the periphery is stained in Pos Ctrl and FIG. 4 exemplifies Neg CTL showing significantly lower 10 HC-HA, bur both the periphery and the center are stained in (p=0.001) TRAP reading compared to the Pos CTL. Inhibi AMP. FIG. 14 exemplifies the effects of AMP on bone matrix tory action for osteoclast formation was seen on all AM mineralization. When ARS is measured quantitatively, cell derivatives (200 lug/ml protein) with HC-HA (25 ug/ml HA) treated with AMP (125 g/ml) significantly promotes the being the most potent followed by AME, AMP, and AML. 15 mineralization (p=0.0001). FIG. 5 exemplifies mRNA expression of NFATcl (mea FIG. 15 exemplifies the effects of AMP on the prolifera sured by quantitative PCR on Day 5 as being significantly tion of osteoblasts. The cell proliferation in the control is downregulated by all AM derivatives (p<0.05). increased from Day 1 to Day 4. In contrast, with the FIG. 6 exemplifies Neg CTL showing significantly lower treatment of AMP (125 ug/ml), the cell proliferation is first (p=2.9E-12) TRAP reading compared to Pos CTL. Statisti decreased from Day 1 to Day 2, and then increased from Day cally significant inhibitory action for osteoclast formation 2 to Day 4. However, compared to that of the control, the cell (all p-0.05) was seen on all AM tissues including epithelial proliferation with AMP treatment is significantly lower at side of iAM, stromal side of iAM, basement membrane side Day 2 and Day 4 (p<0.05). of dAM and stromal side of dAM. Inhibitory action of FIG. 16 exemplifies the effects of AMP and mAMP on osteoclast was also preserved after gamma irradiation of all 25 osteoclastogenesis. mAMP is a powder of injectable Amino AM tissues. Fix, which is prepared by Mimedx Group (Kennesaw, Ga.) FIG. 7 exemplifies mRNA expression of RANK on Day using Purion' Process, which is different from what is 7 as being significantly downregulated for both HC-HA and disclosed in this invention based on heat drying and other stromal side of dAM. chemical processing. Both AMP and mAMP are suspended FIG. 8 exemplifies expression of 33-integrin, which is 30 into PBS and prepared at a series of concentrations (8, 40, required for normal osteoclast differentiation, as being sig and 200 ug protein/nal) and added to RANKL-induced nificantly downregulated by HC-HA, AML, AMP and RAW264.7 cells. After 5 days treatment, the TRAP activity stromal side of dAM. Suppression of B3-integrin by AME is in cells is measured. AMP significantly and dose-depend statistically significant on Day 5 (p=0.000001). ently inhibits TRAP activity (p<0.05). Additionally, AMP is FIGS. 9A-9E exemplify the effects of nHC-HA on osteo 35 significantly more potent than mAMP at the same concen clastogenesis. RAW264.7 cells is effectively induced into tration (p<0.05). osteoclasts at Day 6 with 50 ng/ml RANKL(a). The inhibi The abbreviation AME means amniotic membrane tion of osteoclast formation is dose dependent on nHC-HA extract. AME was made by extraction of amniotic mem purified from AM. (HC-HA) concentrations inhibit osteo brane with PBS followed by centrifugation to remove the clast formation while the highest concentration of high 40 pellet. molecular weight HA (100 ug/ml) does not inhibit the The abbreviation AML means amniotic membrane lysate. osteoclast formation (b). The color picture of TRAP colo AML was made by homogenating the wet amniotic mem rimetric assay is provided. The lighter of the color, the more brane without adding any buffers. The abbreviation AMP inhibition of the TRAP activity is (c). nPHC-HA as low as means amniotic membrane powder. AMP was made by 0.08 ug/ml significantly inhibits the osteoclast formation (d). 45 lyophilizing amniotic membrane followed by pulverizing it IC50 of HC-HA on the osteoclast formation is calculated to into powder. be about 0.1 g/ml (e). FIGS. 10A-10F exemplify the effects of AMP (amniotic DETAILED DESCRIPTION OF THE membrane powder) on osteoclastogenesis. Previous results INVENTION can be reliably reproduced (a). Like HC-HA, AMP dose 50 (0-500 ug/ml protein) dependently inhibits the osteoclast Certain Definitions formation (b). The color picture of TRAP colorimetric assay is provided (c and d). Osteoclast inhibition by AMP is As used herein, "placenta' means the organ that connects measured by TRAP Colorimetric assay (e). IC50 of AMP on a developing fetus to the maternal uterine wall to allow the osteoclast formation is calculated to be about 20.1 lug/ml 55 nutrient uptake, waste elimination, and gas exchange via the (f). maternal blood Supply. The placenta is composed of three FIG. 11 exemplifies the performance of several tissue layers. The innermost placental layer Surrounding the fetus products (AME, HC-HA, AML, AMP) in inhibiting osteo is called amnion. The allantois is the middle layer of the clast formation. All AM derivates significantly inhibit osteo placenta (derived from the embryonic hindgut); blood ves clast formation, and AMP prepared from different donors 60 sels originating from the umbilicus traverse this membrane. (AMP1-5) consistently showed inhibitory activity that were The outermost layer of the placenta, the chorion, comes into more potent than AML. contact with the endometrium. The chorion and allantois FIG. 12 exemplifies the performance of AMP in inhibiting fuse to form the chorioallantoic membrane. osteoclast formation. Powders were made from amniotic As used herein, “chorion' means the membrane formed membrane (AMP), chorion (CHP), amnion-chorion (ACP), 65 by extraembryonic mesoderm and the two layers of tropho whole placenta (PLP), whole umbilical cord (UCP), and blast. The chorionic villi emerge from the chorion, invade umbilical cord amniotic membrane (UAMP). Like HC-HA the endometrium, and allow transfer of nutrients from mater US 9,675,733 B2 31 32 nal blood to fetal blood. The chorion consists of two layers: (ICI) and hyaluronan (HA) by the catalytic action of tumor an outer formed by the trophoblast, and an inner formed by necrosis factor (TNF)-stimulated gene-6 (TSG-6). the Somatic mesoderm; the amnion is in contact with the As used herein, “sheet’ means any continuous expanse or latter. The trophoblast is made up of an internal layer of Surface. In some embodiments, a fetal Support tissue product cubical or prismatic cells, the cytotrophoblast or layer of 5 described herein is a flat sheet. The sheet can be any shape Langhans, and an external layer of richly nucleated proto and size. In some embodiments, the sheet is a square, circle, plasm devoid of cell boundaries, the syncytiotrophoblast. triangle, or rectangle. In some embodiments, the sheet The avascular amnion is adherent to the inner layer of the comprises multiple layers (e.g., of chorion, amnion-chorion, chorion. UCAM, PAM, placenta, umbilical cord or any combinations As used herein, "amnion-chorion' means a product com 10 thereof). prising amnion and chorion. In some embodiments, the As used herein, "homogenized' means (a fetal Support amnion and the chorion are not separated (i.e., the amnion tissue product that has been broken up into particles that are is naturally adherent to the inner layer of the chorion). In of substantially uniform size. Some embodiments, the amnion is initially separated from 15 “Substantially isolated' or "isolated” means that the fetal the chorion and later combined with the chorion during Support tissue product has been separate from undesired processing. materials (e.g., red blood cells, blood vessels, and arteries) As used herein, “umbilical cord means the organ that derived from the original source organism. Purity, or “iso connects a developing fetus to the placenta. The umbilical lation may be assayed by standard methods, and will cord is composed of Wharton's jelly, a gelatinous Substance ordinarily be at least about 10% pure, more ordinarily at made largely from mucopolysaccharides. It contains one least about 20% pure, generally at least about 30% pure, and vein, which carries oxygenated, nutrient-rich blood to the more generally at least about 40% pure; in further embodi fetus, and two arteries that carry deoxygenated, nutrient ments at least about 50% pure, or more often at least about depleted blood away. 60% pure; in still other embodiments, at least about 95% As used herein, "placental amniotic membrane (PAM) 25 pure. means amniotic membrane derived from the placenta. In As used herein, the Substantial preservation of biological some embodiments, the PAM is substantially isolated. activity or structural integrity means that when compared to As used herein, “umbilical cord amniotic membrane' the biological activity and structural integrity of non-pro (UCAM) means amniotic membrane derived from the cessed tissue, the biological activity and structural integrity umbilical cord. UCAM is a translucent membrane. The 30 of the fetal support tissue product has only decreased by UCAM has multiple layers an epithelial layer, a basement about 5%, about 10%, about 15%, about 20%, about 25%, membrane; a compact layer; a fibroblast layer; and a spongy about 30%, about 35%, about 40%, about 50%, about 60%. layer. It lacks blood vessels or a direct blood supply. In some The term “fresh' refers to tissue that is less than 10 days embodiments, the UCAM is substantially isolated. In some old following birth, and which is in substantially the same embodiments, the UCAM comprises Wharton's Jelly. In 35 form as it was following birth. some embodiments, the UCAM comprises blood vessels As used herein, “biological activity” means the activity of and/or arteries. In some embodiments, the UCAM comprises polypeptides and polysaccharides. In some embodiments, Wharton's Jelly and blood vessels and/or arteries. the activity of polypeptides and polysaccharides found in As used herein, "human cells, tissues, or cellular or umbilical cord (and substantially isolated umbilical cord), tissue-based products (HCT/Ps) means articles containing 40 UCAM (and substantially isolated UCAM), placenta (and or consisting of human cells or tissues that are intended for substantially isolated placenta), PAM (and substantially iso implantation, transplantation, infusion, or transfer into a lated PAM), chorion (and substantially isolated chorion), or human recipient. amnion-chorion (and Substantially isolated amnion-cho As used herein, "minimal manipulation” means (1) for rion). structural tissue, processing that does not alter the original 45 As used herein, “structural integrity' means the integrity relevant characteristics of the tissue relating to the tissue's of stroma and basement membrane that make up the UCAM, utility for reconstruction, repair, or replacement; and (2) for chorion, or amnion-chorion. In some embodiments, the cells or nonstructural tissues, processing that does not alter structural integrity of the UCAM results in suture pull out the relevant biological characteristics of cells or tissues. strength. As used herein, homologous use” means the repair, 50 As used herein, "hyaluronan' (or “HA) means a sub reconstruction, replacement, or Supplementation of a recipi stantially non-sulfated or non-sulfated glycosaminoglycan ent’s cells or tissues with an HCT/P that performs the same with linear repeating disaccharide units of glucuronosyl-N- basic function or functions in the recipient as in the donor. acetylglucosamine. In some embodiments, HA is obtained As used herein, “fetal Support tissue product” means any from a commercial Supplier (e.g., Sigma Aldrich or Abbott product comprising tissue that is Supports the development 55 Medical Optics, Irvine, Calif.). In some embodiments, HA is of a fetus. Examples of fetal support tissue include, but are obtained from a commercial Supplier as a powder. In some not limited to, (i) placental amniotic membrane (PAM), or embodiments, HA is obtained from a cell that expresses a substantially isolated PAM, (ii) umbilical cord amniotic hyaluronan synthases (e.g., HAS1, HAS2, and HAS3). In membrane (UCAM) or substantially isolated UCAM, (iii) certain instances, an HA synthase lengthens hyaluronan by chorion or Substantially isolated chorion, (iv) amnion-cho 60 repeatedly adding glucuronic acid and N-acetylglucosamine rion or Substantially isolated amnion-chorion, (v) placenta or to the nascent polysaccharide as it is extruded through the substantially isolated placenta, (vi) umbilical cord or sub cell membrane into the extracellular space. stantially isolated umbilical cord, or (vii) any combinations As used herein, "recombinant TSG-6' means a TSG-6 thereof. In some embodiments, the fetal support tissue protein that is produced by recombinant methods (i.e., the product is a sheet, a powder, or homogenate. 65 TSG-6 gene from a first source (e.g., a human TSG-6 gene) As used herein, “HC-HA’, means the covalent complex is cloned into a DNA molecule from a second source (e.g., formed between heavy chains (HCs) of inter-alpha-inhibitor a bacterial plasmid). US 9,675,733 B2 33 34 As used herein, “recombinant PTX3” means a PTX3 process. The number of osteoblasts tends to decrease with protein that is produced by recombinant methods (i.e., the age, affecting the balance of formation and resorption in the PTX3 gene from a first source (e.g., a human PTX3 gene) is bone tissue, and potentially leading to osteoporosis. cloned into a DNA molecule from a second source (e.g., a Disclosed herein, in certain embodiments, are methods of bacterial plasmid). inhibiting bone resorption in an individual in need thereof, As used herein, the “production bioreactor is the biore comprising administering to the individual a composition actor in which the final HC-HA complex disclosed herein is comprising fetal Support tissue or an extract thereof. In some made. embodiments, the bone resorption is undesired or excessive. The terms “subject' and “individual are used inter In some embodiments, inhibiting bone resorption promotes changeably. As used herein, both terms mean any animal, 10 mineralization by osteoblasts. In some embodiments, inhib preferably a mammal, including a human or non-human. iting bone resorption inhibits undesired or excessive bone The terms patient, Subject, and individual are used inter remodeling. In some embodiments, inhibiting bone resorp changeably. None of the terms are to be interpreted as tion balances bone resorption and bone formation. Further requiring the Supervision of a medical professional (e.g., a disclosed herein, in certain embodiments, are methods of doctor, nurse, physicians assistant, orderly, hospice 15 treating a disease, disorder, or condition characterized by worker). excessive or undesired bone resorption, the method com The terms “treat,” “treating or “treatment,” as used prising administering to an individual in need thereof a herein, include alleviating, abating or ameliorating a disease composition comprising Substantially isolated amniotic or condition symptoms, preventing additional symptoms, membrane or an extract thereof. Additionally disclosed ameliorating or preventing the underlying metabolic causes herein, in certain embodiments, are methods of inhibiting of symptoms, inhibiting the disease or condition, e.g., arrest osteoclast differentiation in an individual in need thereof, ing the development of the disease or condition, relieving comprising administering to the individual a composition the disease or condition, causing regression of the disease or comprising fetal Support tissue or an extract thereof. In some condition, relieving a condition caused by the disease or embodiments, the osteoclast differentiation is undesired or condition, or stopping the symptoms of the disease or 25 excessive. In some embodiments, inhibiting osteoclast dif condition either prophylactically and/or therapeutically. ferentiation promotes differentiation of osteoblasts and min Methods of Use eralization by osteoblasts. In some embodiments, inhibiting Bone Remodeling osteoclast differentiation inhibits undesired or excessive Bone remodeling (or bone metabolism) is the process by bone remodeling. In some embodiments, inhibiting osteo which mature bone tissue is removed from the skeleton 30 clast differentiation balances bone resorption and bone for (bone resorption) and new bone tissue is formed (ossifica mation. Also disclosed herein, in certain embodiments, are tion). methods of treating a disease, disorder, or condition char Bone resorption is the process by which osteoclasts break acterized by excessive or undesired osteoclast differentia down bone, releasing minerals from bone into the blood. tion, the method comprising administering to an individual Osteoclasts are large multinucleated cells formed by the 35 in need thereof a composition comprising Substantially fusion of cells from the monocyte/macrophage cell line. isolated amniotic membrane or an extract thereof. Osteoclasts are generally present on the outer layer of bone, In some embodiments, the methods comprise administer just beneath the periosteum. Attachment of the osteoclast to ing to an individual in need thereof a therapeutically the osteon begins the process. The osteoclast then induces an effective amount of a composition comprising a fetal Support infolding of its cell membrane and secretes collagenase. 40 tissue product or an extract thereof. Calcium, magnesium, phosphate and products of collagen In some embodiments, the fetal Support tissue product is are released into blood as the osteoclasts tunnel into the Substantially isolated amnion. In some embodiments, the mineralized bone. Osteoclasts have been indicated in skel fetal support tissue product is substantially isolated PAM. In etal diseases such as osteoporosis, rheumatoid arthritis and Some embodiments, the fetal Support tissue product is Sub Paget’s disease, and are responsible for osteolysis. 45 stantially isolated UCAM. In some embodiments, the fetal Osteoclasts have been indicated in skeletal diseases such Support tissue product is Substantially isolated placenta. In as osteoporosis, rheumatoid arthritis and Paget’s disease, Some embodiments, the fetal Support tissue product is Sub and are responsible for osteolysis. stantially isolated umbilical cord. In some embodiments, the As used herein, "osteolysis” means refers to the degen fetal Support tissue product is Substantially isolated chorion eration and/or dissolution of bone. In some embodiments, 50 or amnion-chorion. In some embodiments, the fetal Support osteolysis is the process of bone resorption, whereby the tissue product is frozen or previously frozen PAM. In some bone salts can be withdrawn by a humoral mechanism and embodiments, the fetal Support tissue product is frozen or returned to the tissue fluids, leaving behind a decalcified previously frozen UCAM. In some embodiments, the fetal bone matrix. Osteolysis can be caused, interalia, by disease, Support tissue product is frozen or previously frozen pla infection or ischemia. 55 centa. In some embodiments, the fetal Support tissue product Ossification (or, bone formation/mineralization) is the is frozen or previously frozen umbilical cord. In some process by which osteoblasts lay down bone. There are two embodiments, the fetal Support tissue product is frozen or processes resulting in the formation of normal, healthy bone previously frozen chorion or amnion-chorion. In some tissue: intramembranous ossification is the direct laying embodiments, the fetal Support tissue product does not down of bone into the primitive connective tissue (mesen 60 comprise a vein or an artery, a cell with metabolic activity, chyme), while endochondral ossification involves cartilage active HIV-1, active HIV-2, active HTLV-1, active hepatitis as a precursor. B, active hepatitis C, active West Nile Virus, active cyto Osteoblasts are fibroblasts that express bone sialoprotein megalovirus, active human transmissible spongiform and osteocalcin. Osteoblasts produce a matrix of osteoid, encephalopathy, or active Treponema pallidum. In some which is composed mainly of Type I collagen. Osteoblasts 65 embodiments, the fetal Support tissue product is obtained are also responsible for mineralization of this matrix. Zinc, from a human, non-primate human, cow or pig. In some copper and Sodium are some of the minerals required in this embodiments, the fetal Support tissue product is cryopre US 9,675,733 B2 35 36 served, lyophilized, terminally sterilized, or a combination some degree of osteoarthritis by age 65. Risk factors for thereof. In some embodiments, the fetal support tissue osteoarthritis include: prior joint trauma, obesity, and a product is in the form of a pulverized powder or a homo sedentary lifestyle. genate. Rheumatoid arthritis is a disorder in which the body’s In some embodiments, the methods comprise administer 5 immune system starts to attack body tissues. In rheumatoid ing to an individual in need thereof a therapeutically arthritis, most damage occurs to the joint lining and cartilage effective amount of a composition comprising Substantially which eventually results in erosion of two opposing bones. isolated HC-HA complex. In some embodiments, the sub Rheumatoid arthritis often affects joints in the fingers, stantially isolated HC-HA complex is derived from amnion wrists, knees and elbows. The disease is symmetrical (ap (PAM or UCAM), placenta, umbilical cord, or chorion. In 10 pears on both sides of the body) and can lead to severe some embodiments, the substantially isolated HC-HA com deformity. Rheumatoid arthritis occurs mostly in people plex is derived from frozen or previously frozen placental aged 20 and above. amniotic membrane (PAM), frozen or previously frozen Osteoclasts are often found in the affected joints of umbilical cord amniotic membrane (UCAM), frozen or individuals with arthritis and exacerbate bone and joint previously frozen placenta, frozen or previously frozen 15 destruction. Thus, disclosed herein, in certain embodiments, umbilical cord, frozen or previously frozen chorion, frozen are methods of treating arthritis. In some embodiments, the or previously frozen amnion-chorion, or any combinations methods comprise administering to an individual in need thereof. In some embodiments, the HC-HA complex is thereof a therapeutically-effective amount of a composition HC-HAHC-HA obtained by a process comprising: (a) pro comprising a fetal Support tissue product or an extract viding a reaction mixture comprising: (i) HA (e.g., HMW thereof. In some embodiments, the methods comprise HA); (ii) ICI, wherein the ICI is optionally in serum or administering to an individual in need thereof a therapeuti isolated from serum; (iii) TSG-6, wherein the TSG-6 is cally-effective amount of a composition comprising Substan optionally recombinant; and (iv) PTX3, wherein the PTX3 tially isolated HC-HA complex. In some embodiments, the is optionally recombinant; wherein at least one of HA, ICI, arthritis is osteoarthritis, rheumatoid arthritis, psoriatic TSG-6, or PTX3 is optionally generated by a plurality of 25 arthritis, or any combination thereof. cells present in the reaction mixture; (b) incubating the In some embodiments, the methods comprise administer reaction mixture for a period of time sufficient to produce ing to an individual in need thereof a therapeutically HC-HA complex; and (c) isolating and purifying the HC-HA effective amount of a composition comprising a fetal Support complex. tissue product or an extract thereof. In some embodiments, the composition further comprises 30 In some embodiments, the fetal Support tissue product is a pharmaceutically-acceptable diluents, excipient or carrier. Substantially isolated amnion. In some embodiments, the In some embodiments, the composition is formulated as a fetal support tissue product is substantially isolated PAM. In Solution, Suspension, paste, ointment, oil, emulsion, micro Some embodiments, the fetal Support tissue product is Sub emulsion, cream, lotion, gel, or combination thereof. In stantially isolated UCAM. In some embodiments, the fetal Some embodiments, the composition comprises Substan 35 Support tissue product is Substantially isolated placenta. In tially-isolated HC-HA formulated into microspheres, Some embodiments, the fetal Support tissue product is Sub microparticles, or liposomes. In some embodiments, the stantially isolated umbilical cord. In some embodiments, the composition is formulated into PLGA copolymers. In some fetal Support tissue product is Substantially isolated chorion embodiments, the composition is formulated for controlled, or amnion-chorion. In some embodiments, the fetal Support Sustained, or delayed release. In some embodiments, the 40 tissue product is frozen or previously frozen PAM. In some composition is injected into an osteolytic joint. In some embodiments, the fetal Support tissue product is frozen or embodiments, the composition is administered by a patch. In previously frozen UCAM. In some embodiments, the fetal Some embodiments, the patch is placed on an osteolytic bone Support tissue product is frozen or previously frozen pla or an osteolytic joint. In some embodiments, the composi centa. In some embodiments, the fetal Support tissue product tion is administered by an implant. In some embodiments, 45 is frozen or previously frozen umbilical cord. In some the implant is placed on an osteolytic bone or an osteolytic embodiments, the fetal Support tissue product is frozen or joint. In some embodiments, the composition is formulated previously frozen chorion or amnion-chorion. In some into an orthopaedic prosthesis. embodiments, the fetal Support tissue product does not Arthritis comprise a vein or an artery, a cell with metabolic activity, Arthritis is a joint disorder that involves inflammation of 50 active HIV-1, active HIV-2, active HTLV-1, active hepatitis one or more joints. There are over 100 different forms of B, active hepatitis C, active West Nile Virus, active cyto arthritis. The most common form, osteoarthritis (degenera megalovirus, active human transmissible spongiform tive joint disease), is a result of trauma to the joint, infection encephalopathy, or active Treponema pallidum. In some of the joint, or age. Other arthritis forms are rheumatoid embodiments, the fetal Support tissue product is obtained arthritis, psoriatic arthritis, and related autoimmune dis 55 from a human, non-primate human, cow or pig. In some eases. Septic arthritis is caused by joint infection. Symptoms embodiments, the fetal Support tissue product is cryopre common to all arthritic disorders include pain, Swelling, and served, lyophilized, terminally sterilized, or a combination joint stiffness, thereof. In some embodiments, the fetal support tissue Osteoarthritis is the most common form of arthritis. It product is in the form of a pulverized powder or a homo affects both the larger and the smaller joints of the body, for 60 genate. example the hands, feet, back, hip or knee. Osteoarthritis In some embodiments, the methods comprise administer develops as a result of wear on joints or injury to joints. ing to an individual in need thereof a therapeutically Osteoarthritis begins in the cartilage and eventually causes effective amount of a composition comprising Substantially the two opposing bones to erode into each other. Osteoar isolated HC-HA complex. In some embodiments, the sub thritis typically affects the weight bearing joints such as the 65 stantially isolated HC-HA complex is isolated from amnion back, spine, and pelvis. Osteoarthritis is most commonly a (PAM or UCAM), placenta, umbilical cord, or chorion. In disease of the elderly. More than 30 percent of females have some embodiments, the substantially isolated HC-HA com US 9,675,733 B2 37 38 plex is isolated from frozen or previously frozen placental therapeutically-effective amount of a composition compris amniotic membrane (PAM), frozen or previously frozen ing a fetal Support tissue product or an extract thereof. In umbilical cord amniotic membrane (UCAM), frozen or Some embodiments, the methods comprise administering to previously frozen placenta, frozen or previously frozen an individual in need thereof a therapeutically-effective umbilical cord, frozen or previously frozen chorion, frozen amount of a composition comprising Substantially isolated or previously frozen amnion-chorion, or any combinations HC-HA complex. thereof. In some embodiments, the methods comprise administer In some embodiments, the HC-HA complex is obtained ing to an individual in need thereof a therapeutically by a process comprising: (a) providing a reaction mixture effective amount of a composition comprising a fetal Support comprising: (i) HA (e.g., HMW HA); (ii) IOI, wherein the 10 ICI is optionally in serum or isolated from serum; (iii) tissue product or an extract thereof. TSG-6, wherein the TSG-6 is optionally recombinant; and In some embodiments, the fetal Support tissue product is (iv) PTX3, wherein the PTX3 is optionally recombinant; Substantially isolated amnion. In some embodiments, the wherein at least one of HA, ICI, TSG-6, or PTX3 is fetal support tissue product is substantially isolated PAM. In optionally generated by a plurality of cells present in the 15 Some embodiments, the fetal Support tissue product is Sub reaction mixture; (b) incubating the reaction mixture for a stantially isolated UCAM. In some embodiments, the fetal period of time sufficient to produce HC-HA complex; and (c) Support tissue product is Substantially isolated placenta. In isolating and purifying the HC-HA complex. Some embodiments, the fetal Support tissue product is Sub In some embodiments, the composition further comprises stantially isolated umbilical cord. In some embodiments, the a pharmaceutically-acceptable diluents, excipient or carrier. fetal Support tissue product is Substantially isolated chorion In some embodiments, the composition is formulated as a or amnion-chorion. In some embodiments, the fetal Support Solution, Suspension, paste, ointment, oil, emulsion, micro tissue product is frozen or previously frozen PAM. In some emulsion, cream, lotion, gel, or combination thereof. In embodiments, the fetal Support tissue product is frozen or Some embodiments, the composition comprises Substan previously frozen UCAM. In some embodiments, the fetal tially-isolated HC-HA formulated into microspheres, 25 Support tissue product is frozen or previously frozen pla microparticles, or liposomes. In some embodiments, the centa. In some embodiments, the fetal Support tissue product composition is formulated into PLGA copolymers. In some is frozen or previously frozen umbilical cord. In some embodiments, the composition is formulated for controlled, embodiments, the fetal Support tissue product is frozen or Sustained, or delayed release. In some embodiments, the previously frozen chorion or amnion-chorion. In some composition is injected into an osteolytic joint. In some 30 embodiments, the fetal Support tissue product does not embodiments, the composition is administered by a patch. In comprise a vein or an artery, a cell with metabolic activity, some embodiments, the patch is placed on an osteolytic bone active HIV-1, active HIV-2, active HTLV-1, active hepatitis or an osteolytic joint. In some embodiments, the composi B, active hepatitis C, active West Nile Virus, active cyto tion is administered by an implant. In some embodiments, megalovirus, active human transmissible spongiform the implant is placed on an osteolytic bone or an osteolytic 35 encephalopathy, or active Treponema pallidum. In some joint. In some embodiments, the composition is formulated embodiments, the fetal Support tissue product is obtained into an orthopaedic prosthesis. from a human, non-primate human, cow or pig. In some In some embodiments, the methods further comprise embodiments, the fetal Support tissue product is cryopre administering to the individual a calcium Supplement. In served, lyophilized, terminally sterilized, or a combination Some embodiments, the methods further comprise adminis 40 thereof. In some embodiments, the fetal support tissue tering to the individual an NSAID, a corticosteroid, hyaluro product is in the form of a pulverized powder or a homo nan injections, a DMARD, an analgesic, or any combination genate. thereof. In some embodiments, the methods comprise administer Osteoporosis ing to an individual in need thereof a therapeutically Osteoporosis is a bone disease characterized by reduction 45 effective amount of a composition comprising Substantially of bone mineral density (BMD), deterioration of bone isolated HC-HA complex. In some embodiments, the sub microarchitecture, and alteration of the amount and variety stantially isolated HC-HA complex is isolated from amnion of proteins in bone. Osteoporosis is defined as a bone (PAM or UCAM), placenta, umbilical cord, or chorion. In mineral density that is 2.5 standard deviations or more below some embodiments, the substantially isolated HC-HA com the mean peak bone mass (average of young, healthy adults) 50 plex is isolated from frozen or previously frozen placental as measured by DXA. Osteoporosis is classified as primary amniotic membrane (PAM), frozen or previously frozen type 1, primary type 2, or secondary. The form of osteopo umbilical cord amniotic membrane (UCAM), frozen or rosis most common in women after menopause is referred to previously frozen placenta, frozen or previously frozen as primary type 1 or postmenopausal osteoporosis. Primary umbilical cord, frozen or previously frozen chorion, frozen type 2 osteoporosis or senile osteoporosis occurs after age 55 or previously frozen amnion-chorion, or any combinations 75 and is seen in both females and males at a ratio of 2:1. thereof. Finally, secondary osteoporosis may arise at any age and In some embodiments, the HC-HA complex is obtained affect men and women equally. This form of osteoporosis by a process comprising: (a) providing a reaction mixture results from chronic predisposing medical problems or dis comprising: (i) HA (e.g., HMW HA); (ii) IOI, wherein the ease, or prolonged use of medications such as glucocorti 60 ICI is optionally in serum or isolated from serum; (iii) coids, when the disease is called steroid- or glucocorticoid TSG-6, wherein the TSG-6 is optionally recombinant; and induced osteoporosis (SIOP or GIOP). (iv) PTX3, wherein the PTX3 is optionally recombinant: Osteoporosis results from excessive bone resorption by wherein at least one of HA, ICI, TSG-6, or PTX3 is osteoclasts and insufficient bone formation by osteoblasts. optionally generated by a plurality of cells present in the Thus, disclosed herein, in certain embodiments, are methods 65 reaction mixture; (b) incubating the reaction mixture for a of treating osteoporosis. In some embodiments, the methods period of time sufficient to produce HC-HA complex; and (c) comprising administering to an individual in need thereof a isolating and purifying the HC-HA complex. US 9,675,733 B2 39 40 In some embodiments, the composition further comprises Support tissue product is frozen or previously frozen pla a pharmaceutically-acceptable diluents, excipient or carrier. centa. In some embodiments, the fetal Support tissue product In some embodiments, the composition is formulated as a is frozen or previously frozen umbilical cord. In some Solution, Suspension, paste, ointment, oil, emulsion, micro embodiments, the fetal Support tissue product is frozen or emulsion, cream, lotion, gel, or combination thereof. In previously frozen chorion or amnion-chorion. In some Some embodiments, the composition comprises Substan embodiments, the fetal Support tissue product does not tially-isolated tissue product or HC-HA formulated into comprise a vein or an artery, a cell with metabolic activity, microspheres, microparticles, or liposomes. In some active HIV-1, active HIV-2, active HTLV-1, active hepatitis embodiments, the composition is formulated into PLGA B, active hepatitis C, active West Nile Virus, active cyto copolymers. In some embodiments, the composition is for 10 mulated for controlled, Sustained, or delayed release. In megalovirus, active human transmissible spongiform Some embodiments, the composition is injected into an encephalopathy, or active Treponema pallidum. In some osteolytic joint. In some embodiments, the composition is embodiments, the fetal Support tissue product is obtained administered by a patch. In some embodiments, the patch is from a human, non-primate human, cow or pig. In some placed on an osteolytic bone or an osteolytic joint. In some 15 embodiments, the fetal Support tissue product is cryopre embodiments, the composition is administered by an served, lyophilized, terminally sterilized, or a combination implant. In some embodiments, the implant is placed on an thereof. In some embodiments, the fetal support tissue osteolytic bone or an osteolytic joint. In some embodiments, product is in the form of a pulverized powder or a homo the composition is formulated into an orthopaedic prosthe genate. sis. In some embodiments, the fetal Support tissue product is In some embodiments, the methods comprise administer in the form of a substantially-flattened sheet. In some ing to an individual in need thereof a therapeutically embodiments, the composition further comprises a backing. effective amount of a composition comprising Substantially In some embodiments, the composition is wrapped around isolated HC-HA complex. In some embodiments, the sub an osteolytic bone or an osteolytic joint. In some embodi stantially isolated HC-HA complex is isolated from amnion ments, the methods further comprise administering to the 25 (PAM or UCAM), placenta, umbilical cord, or chorion. In individual a calcium Supplement. In some embodiments, the some embodiments, the substantially isolated HC-HA com methods further comprise administering to the individual a plex is isolated from frozen or previously frozen placental bisphosphonate, an estrogen analog, Raloxifene, Calcitonin, amniotic membrane (PAM), frozen or previously frozen Teriparatide, calcium salts, sodium fluoride, RANKL inhibi umbilical cord amniotic membrane (UCAM), frozen or tors, Strontium ranelate, or any combination thereof. 30 previously frozen placenta, frozen or previously frozen Alveolar Bone Degradation umbilical cord, frozen or previously frozen chorion, frozen Degradation of the alveolar bone around the teeth often or previously frozen amnion-chorion, or any combinations results from periodontitis. If left untreated, alveolar bone thereof. degradation may result in loss of teeth. A diagnosis of In some embodiments, the HC-HA complex is obtained alveolar bone degradation is established by inspecting the 35 by a process comprising: (a) providing a reaction mixture Soft gum tissues around the teeth with a probe and evaluating comprising: (i) HA (e.g., HMW HA); (ii) IOI, wherein the X-ray films to determine the amount of bone loss around the ICI is optionally in serum or isolated from serum; (iii) teeth. TSG-6, wherein the TSG-6 is optionally recombinant; and Degradation of the alveolar bone is often exacerbated by (iv) PTX3, wherein the PTX3 is optionally recombinant: excess bone resorption by osteoclasts and insufficient bone 40 wherein at least one of HA, ICI, TSG-6, or PTX3 is formation by osteoblasts. Thus, disclosed herein, in certain optionally generated by a plurality of cells present in the embodiments, are methods of treating alveolar bone degra reaction mixture; (b) incubating the reaction mixture for a dation. In some embodiments, the methods comprising period of time sufficient to produce HC-HA complex; and (c) administering to an individual in need thereof a therapeuti isolating and purifying the HC-HA complex. cally-effective amount of a composition comprising a fetal 45 In some embodiments, the composition further comprises Support tissue product or an extract thereof. In some embodi a pharmaceutically-acceptable diluents, excipient or carrier. ments, the methods comprise administering to an individual In some embodiments, the composition is formulated as a in need thereof a therapeutically-effective amount of a Solution, Suspension, paste, ointment, oil, emulsion, micro composition comprising Substantially isolated HC-HA com emulsion, cream, lotion, gel, or combination thereof. In plex. 50 Some embodiments, the composition comprises Substan In some embodiments, the methods comprise administer tially-isolated tissue product or HC-HA formulated into ing to an individual in need thereof a therapeutically microspheres, microparticles, or liposomes. In some effective amount of a composition comprising a fetal Support embodiments, the composition is formulated into PLGA tissue product or an extract thereof. copolymers. In some embodiments, the composition is for In some embodiments, the fetal Support tissue product is 55 mulated for controlled, Sustained, or delayed release. In Substantially isolated amnion. In some embodiments, the Some embodiments, the composition is applied to the alveo fetal support tissue product is substantially isolated PAM. In lar bone. In some embodiments, the composition is admin Some embodiments, the fetal Support tissue product is Sub istered by a patch. In some embodiments, the patch is placed stantially isolated UCAM. In some embodiments, the fetal on the alveolar bone. In some embodiments, the composition Support tissue product is Substantially isolated placenta. In 60 is administered by an implant. In some embodiments, the Some embodiments, the fetal Support tissue product is Sub implant is placed on the alveolar bone. In some embodi stantially isolated umbilical cord. In some embodiments, the ments, the fetal Support tissue product is in the form of a fetal Support tissue product is substantially isolated chorion substantially-flattened sheet. In some embodiments, the or amnion-chorion. In some embodiments, the fetal Support composition further comprises a backing. In some embodi tissue product is frozen or previously frozen PAM. In some 65 ments, the composition is wrapped around the alveolar bone. embodiments, the fetal Support tissue product is frozen or In some embodiments, the methods further comprise admin previously frozen UCAM. In some embodiments, the fetal istering to the individual a calcium Supplement. US 9,675,733 B2 41 42 Paget’s Disease thereof. In some embodiments, the fetal support tissue Paget’s disease is a chronic bone disorder resulting is product is in the form of a pulverized powder or a homo enlarged and misshapen bones. Bones in individual with genate. Paget’s Disease are often weakened due to an imbalance in In some embodiments, the methods comprise administer bone resorption and bone formation, resulting in pain, ing to an individual in need thereof a therapeutically misshapen bones, fractures, and arthritis in the joints near effective amount of a composition comprising Substantially the affected bones. Paget’s disease typically is localized to isolated HC-HA complex. In some embodiments, the sub one or a few bones. Paget’s Disease may result from a viral stantially isolated HC-HA complex is isolated from amnion infection or genetic factors. (PAM or UCAM), placenta, umbilical cord, or chorion. In Paget’s disease proceeds in three stages: osteoclastic 10 some embodiments, the substantially isolated HC-HA com activity, mixed osteoclastic-osteoblastic activity, and plex is isolated from frozen or previously frozen placental exhaustive stage. The first stage is characterized by an amniotic membrane (PAM), frozen or previously frozen increase in the rate of bone resorption at localized areas. umbilical cord amniotic membrane (UCAM), frozen or These localized areas of osteolysis are seen radiologically as previously frozen placenta, frozen or previously frozen an advancing lytic wedge in long bones or osteoporosis 15 umbilical cord, frozen or previously frozen chorion, frozen circumscripta in the skull. The osteolysis is followed by a or previously frozen amnion-chorion, or any combinations compensatory increase in bone formation induced by osteo thereof. blasts recruited to the area. Bone is laid down in a disorga In some embodiments, the HC-HA complex is obtained nized and chaotic fashion rather than the normal linear by a process comprising: (a) providing a reaction mixture lamellar pattern. The resorbed bone is replaced and the comprising: (i) HA (e.g., HMW HA); (ii) IOI, wherein the marrow spaces are filled by an excess of fibrous connective ICI is optionally in serum or isolated from serum; (iii) tissue with a marked increase in blood vessels, causing the TSG-6, wherein the TSG-6 is optionally recombinant; and bone to become hypervascular. The bone hypercellularity (iv) PTX3, wherein the PTX3 is optionally recombinant: may then diminish, leaving a dense pagetic bone, also wherein at least one of HA, ICI, TSG-6, or PTX3 is known as burned-out Paget’s disease. 25 optionally generated by a plurality of cells present in the Paget’s disease results from excessive bone resorption reaction mixture; (b) incubating the reaction mixture for a and insufficient bone formation. Thus, disclosed herein, in period of time sufficient to produce HC-HA complex; and (c) certain embodiments, are methods of treating Paget’s Dis isolating and purifying the HC-HA complex. ease. In some embodiments, the methods comprising admin In some embodiments, the composition further comprises istering to an individual in need thereof a therapeutically 30 a pharmaceutically-acceptable diluents, excipient or carrier. effective amount of a composition comprising a fetal Support In some embodiments, the composition is formulated as a tissue product or an extract thereof. In some embodiments, Solution, Suspension, paste, ointment, oil, emulsion, micro the methods comprise administering to an individual in need emulsion, cream, lotion, gel, or combination thereof. In thereof a therapeutically-effective amount of a composition Some embodiments, the composition comprises Substan comprising Substantially isolated HC-HA complex. 35 tially-isolated tissue product or HC-HA formulated into In some embodiments, the methods comprise administer microspheres, microparticles, or liposomes. In some ing to an individual in need thereof a therapeutically embodiments, the composition is formulated into PLGA effective amount of a composition comprising a fetal Support copolymers. In some embodiments, the composition is for tissue product or an extract thereof. mulated for controlled, Sustained, or delayed release. In In some embodiments, the fetal Support tissue product is 40 Some embodiments, the composition is injected into an Substantially isolated amnion. In some embodiments, the osteolytic joint. In some embodiments, the composition is fetal support tissue product is substantially isolated PAM. In administered by a patch. In some embodiments, the patch is Some embodiments, the fetal Support tissue product is Sub placed on an osteolytic bone or an osteolytic joint. In some stantially isolated UCAM. In some embodiments, the fetal embodiments, the composition is administered by an Support tissue product is Substantially isolated placenta. In 45 implant. In some embodiments, the implant is placed on an Some embodiments, the fetal Support tissue product is Sub osteolytic bone or an osteolytic joint. In some embodiments, stantially isolated umbilical cord. In some embodiments, the the composition is formulated into an orthopaedic prosthe fetal Support tissue product is substantially isolated chorion sis. In some embodiments, the fetal Support tissue product is or amnion-chorion. In some embodiments, the fetal Support in the form of a substantially-flattened sheet. In some tissue product is frozen or previously frozen PAM. In some 50 embodiments, the composition further comprises a backing. embodiments, the fetal Support tissue product is frozen or In some embodiments, the composition is wrapped around previously frozen UCAM. In some embodiments, the fetal an osteolytic bone or an osteolytic joint. In some embodi Support tissue product is frozen or previously frozen pla ments, the methods further comprise administering to the centa. In some embodiments, the fetal Support tissue product individual a calcium Supplement. In some embodiments, the is frozen or previously frozen umbilical cord. In some 55 methods further comprise administering to the individual a embodiments, the fetal Support tissue product is frozen or bisphosphonate or calcitonin. previously frozen chorion or amnion-chorion. In some Bone Tumors embodiments, the fetal Support tissue product does not As used herein, “bone tumor” means the uncontrolled comprise a vein or an artery, a cell with metabolic activity, and/or undesired growth of tissue in bone. Bone tumors are active HIV-1, active HIV-2, active HTLV-1, active hepatitis 60 either benign or malignant. They are further classified as B, active hepatitis C, active West Nile Virus, active cyto “primary tumors”, which originate in bone or from bone megalovirus, active human transmissible spongiform derived cells and tissues, and “secondary tumors” which encephalopathy, or active Treponema pallidum. In some originate in other sites and metastasize to the bone. embodiments, the fetal Support tissue product is obtained Primary bone tumors may be neoplastic, developmental, from a human, non-primate human, cow or pig. In some 65 traumatic, infectious, or inflammatory. Examples of benign embodiments, the fetal Support tissue product is cryopre bone tumors include osteoma, osteoid osteoma, osteochon served, lyophilized, terminally sterilized, or a combination droma, osteoblastoma, enchondroma, giant cell tumor of US 9,675,733 B2 43 44 bone, aneurysmal bone cyst, and fibrous dysplasia of bone. In some embodiments, the HC-HA complex is obtained Malignant primary bone tumors include osteosarcoma, by a process comprising: (a) providing a reaction mixture chondrosarcoma, Ewing's sarcoma, fibrosarcoma, and other comprising: (i) HA (e.g., HMW HA); (ii) IOI, wherein the types. ICI is optionally in serum or isolated from serum; (iii) Bone tumors often result in undesired bone degradation. TSG-6, wherein the TSG-6 is optionally recombinant; and Undesired bone degradation may be caused by the presence (iv) PTX3, wherein the PTX3 is optionally recombinant: of metastatic tumor cells that stimulate formation and acti wherein at least one of HA, ICI, TSG-6, or PTX3 is Vation of osteoclasts. Undesired bone degradation may also optionally generated by a plurality of cells present in the arise from the formation of osteoblastic metastases. Thus, reaction mixture; (b) incubating the reaction mixture for a disclosed herein, in certain embodiments, are methods of 10 period of time sufficient to produce HC-HA complex; and (c) treating bone tumors. In some embodiments, the methods isolating and purifying the HC-HA complex. comprising administering to an individual in need thereof a In some embodiments, the composition further comprises therapeutically-effective amount of a composition compris a pharmaceutically-acceptable diluents, excipient or carrier. ing a fetal Support tissue product or an extract thereof. In In some embodiments, the composition is formulated as a Some embodiments, the methods comprise administering to 15 Solution, Suspension, paste, ointment, oil, emulsion, micro an individual in need thereof a therapeutically-effective emulsion, cream, lotion, gel, or combination thereof. In amount of a composition comprising Substantially isolated Some embodiments, the composition comprises Substan HC-HA complex. tially-isolated tissue product or HC-HA formulated into In some embodiments, the methods comprise administer microspheres, microparticles, or liposomes. In some ing to an individual in need thereof a therapeutically embodiments, the composition is formulated into PLGA effective amount of a composition comprising a fetal Support copolymers. In some embodiments, the composition is for tissue product or an extract thereof. mulated for controlled, Sustained, or delayed release. In In some embodiments, the fetal Support tissue product is Some embodiments, the composition is injected into an Substantially isolated amnion. In some embodiments, the osteolytic joint. In some embodiments, the composition is fetal support tissue product is substantially isolated PAM. In 25 administered by a patch. In some embodiments, the patch is Some embodiments, the fetal Support tissue product is Sub placed on an osteolytic bone or an osteolytic joint. In some stantially isolated UCAM. In some embodiments, the fetal embodiments, the composition is administered by an Support tissue product is Substantially isolated placenta. In implant. In some embodiments, the implant is placed on an Some embodiments, the fetal Support tissue product is Sub osteolytic bone or an osteolytic joint. In some embodiments, stantially isolated umbilical cord. In some embodiments, the 30 the composition is formulated into an orthopaedic prosthe fetal Support tissue product is substantially isolated chorion sis. In some embodiments, the fetal Support tissue product is or amnion-chorion. In some embodiments, the fetal support in the form of a substantially-flattened sheet. In some tissue product is frozen or previously frozen PAM. In some embodiments, the composition further comprises a backing. embodiments, the fetal Support tissue product is frozen or In some embodiments, the composition is wrapped around previously frozen UCAM. In some embodiments, the fetal 35 an osteolytic bone or an osteolytic joint. In some embodi Support tissue product is frozen or previously frozen pla ments, the methods further comprise administering to the centa. In some embodiments, the fetal Support tissue product individual a calcium Supplement. In some embodiments, the is frozen or previously frozen umbilical cord. In some methods further comprise administering to the individual a embodiments, the fetal Support tissue product is frozen or chemotherapeutic agent, a bisphosphinate, Metastron, or any previously frozen chorion or amnion-chorion. In some 40 combination thereof. embodiments, the fetal Support tissue product does not Fetal Support Tissue Products comprise a vein or an artery, a cell with metabolic activity, Disclosed herein, in certain embodiments, are methods of active HIV-1, active HIV-2, active HTLV-1, active hepatitis inhibiting osteoclast differentiation in an individual in need B, active hepatitis C, active West Nile Virus, active cyto thereof, comprising administering to the individual a com megalovirus, active human transmissible spongiform 45 position comprising product fetal Support tissue product or encephalopathy, or active Treponema pallidum. In some an extract thereof. Disclosed herein, in certain embodiments, embodiments, the fetal Support tissue product is obtained are methods of promoting mineralization by osteoblasts in from a human, non-primate human, cow or pig. In some an individual in need thereof, comprising administering to embodiments, the fetal Support tissue product is cryopre the individual a composition comprising a fetal Support served, lyophilized, terminally sterilized, or a combination 50 tissue product or an extract thereof. In some embodiments, thereof. In some embodiments, the fetal support tissue the fetal Support tissue product is Substantially isolated product is in the form of a pulverized powder or a homo placental amniotic membrane (PAM), umbilical cord amni genate. otic membrane (UCAM), placenta, umbilical cord, chorion, In some embodiments, the methods comprise administer amnion-chorion, or any combinations thereof. Disclosed ing to an individual in need thereof a therapeutically 55 herein, in certain embodiments, are methods of inhibiting effective amount of a composition comprising Substantially bone resorption in an individual in need thereof, comprising isolated HC-HA complex. In some embodiments, the sub administering to the individual a composition comprising a stantially isolated HC-HA complex is isolated from amnion fetal support tissue product or an extract thereof. Disclosed (PAM or UCAM), placenta, umbilical cord, or chorion. In herein, in certain embodiments, are methods of inhibiting some embodiments, the substantially isolated HC-HA com 60 bone remodeling in an individual in need thereof, compris plex is isolated from frozen or previously frozen placental ing administering to the individual a composition compris amniotic membrane (PAM), frozen or previously frozen ing a fetal Support tissue product or an extract thereof. umbilical cord amniotic membrane (UCAM), frozen or Disclosed herein, in certain embodiments, are methods of previously frozen placenta, frozen or previously frozen balancing bone resorption and bone formation, comprising umbilical cord, frozen or previously frozen chorion, frozen 65 administering to an individual in need thereof a therapeuti or previously frozen amnion-chorion, or any combinations cally-effective amount of a composition comprising a fetal thereof. Support tissue product or an extract thereof. Disclosed US 9,675,733 B2 45 46 herein, in certain embodiments, are methods of treating a methods, the fetal support tissue product is in the form of a disease, disorder, or condition characterized by excessive or sheet, pulverized powder or a homogenate. undesired osteoclast differentiation, the methods comprising Umbilical Cord and UCAM administering to an individual in need thereof a therapeuti Umbilical cord is recovered from any suitable source cally-effective amount of a composition comprising a fetal (e.g., a hospital or tissue bank). Umbilical cord can be Support tissue product or an extract thereof. Disclosed obtained from any mammal. Such as a human, non-human herein, in certain embodiments, are methods of treating a primate, cow or pig. disease, disorder, or condition characterized by excessive or The umbilical cord is kept frozen (e.g., at or below 0°C.) undesired bone absorption by osteoclasts, the methods com until donor and specimen eligibility has been determined. In prising administering to an individual in need thereof a 10 therapeutically-effective amount of a composition compris some embodiments, freezing the umbilical cord kills sub ing a fetal Support tissue product or an extract thereof. stantially all cells found in the umbilical cord. In some Disclosed herein, in certain embodiments, are methods of embodiments, freezing the umbilical cord kills substantially treating a disease, disorder, or condition characterized by all cells found in the umbilical cord while maintaining or deficient or defective bone formation, the methods compris 15 increasing the biological activity of the umbilical cord ing administering to an individual in need thereof a thera relative to fresh (i.e., non-frozen) umbilical cord. In some peutically-effective amount of a composition comprising a embodiments, freezing the umbilical cord results in the loss fetal support tissue product or an extract thereof. Disclosed of metabolic activity in substantially all cells found in the herein, in certain embodiments, are methods of treating umbilical cord. In some embodiments, freezing the umbili arthritis, the methods comprising administering to an indi cal cord results in the loss of metabolic activity in substan vidual in need thereof a therapeutically-effective amount of tially all cells found in the umbilical cord while maintaining a composition comprising a fetal Support tissue product or or increasing the biological activity of the umbilical cord an extract thereof. Disclosed herein, in certain embodiments, (e.g., its anti-inflammatory, anti-scarring, anti-antigenic, and are methods of treating osteoporosis, the methods compris anti-adhesion properties) relative to fresh (i.e., non-frozen) ing administering to an individual in need thereof a thera 25 umbilical cord. In some embodiments, the umbilical cord is peutically-effective amount of a composition comprising a not frozen. If the umbilical cord is not frozen, it is processed fetal support tissue product or an extract thereof. Disclosed as described below immediately. In some embodiments, the herein, in certain embodiments, are methods of treating umbilical cord is dried (e.g., by lyophilization). In some alveolar bone degradation, the methods comprising admin embodiments, drying the umbilical cord kills substantially istering to an individual in need thereof a therapeutically 30 all cells found in the umbilical cord. In some embodiments, effective amount of a composition comprising a fetal Support drying the umbilical cord results in the loss of metabolic tissue product or an extract thereof. Disclosed herein, in activity in substantially all cells found in the umbilical cord. certain embodiments, are methods of treating Paget’s dis All processing is done following Good Tissue Practices ease, the methods comprising administering to an individual (GTP) to ensure that no contaminants are introduced into the in need thereof a therapeutically-effective amount of a 35 UCAM products. composition comprising a fetal Support tissue product or an The umbilical cord is tested for HIV-1, HIV-2, HTLV-1, extract thereof. Disclosed herein, in certain embodiments, hepatitis B and C, West Nile virus, cytomegalovirus, human are methods of treating a bone tumor, the methods compris transmissible spongiform encephalopathy (e.g., Creutzfeldt ing administering to an individual in need thereof a thera Jakob disease) and Treponema pallidum using FDA licensed peutically-effective amount of a composition comprising a 40 screening test. Any indication that the tissue is contaminated fetal Support tissue product or an extract thereof. In some with HIV-1, HIV-2, HTLV-1, hepatitis B and C, West Nile embodiments of any of the aforementioned methods, the virus, or cytomegalovirus results in the immediate quaran fetal Support tissue product is Substantially isolated amnion. tine and Subsequent destruction of the tissue specimen. In some embodiments of any of the aforementioned meth Further, the donor's medical records are examined for risk ods, the fetal Support tissue product is Substantially isolated 45 factors for and clinical evidence of hepatitis B, hepatitis C, PAM. In some embodiments of any of the aforementioned or HIV infection. Any indication that the donor has risk methods, the fetal Support tissue product is substantially factors for, and/or clinical evidence of infection with HIV-1, isolated UCAM. In some embodiments of any of the afore HIV-2, HTLV-1, hepatitis B and C, West Nile virus, cyto mentioned methods, the fetal Support tissue product is megalovirus, human transmissible spongiform encephalopa Substantially isolated placenta. In some embodiments of any 50 thy (e.g., Creutzfeldt-Jakob disease) and Treponema palli of the aforementioned methods, the fetal support tissue dum results in the immediate quarantine and Subsequent product is substantially isolated umbilical cord. In some destruction of the tissue specimen. embodiments of any of the aforementioned methods, the In some embodiments, substantially all of the blood is fetal Support tissue product is substantially isolated chorion removed from the umbilical cord. In some embodiments, or amnion-chorion. In some embodiments of any of the 55 substantially all of the blood is removed from the umbilical aforementioned methods, the fetal Support tissue product is cord before the umbilical cord is frozen. In some embodi frozen or previously frozen PAM. In some embodiments of ments, blood is not removed from the UC. In some embodi any of the aforementioned methods, the fetal Support tissue ments, blood is not removed from the umbilical cord before product is frozen or previously frozen UCAM. In some the umbilical cord is frozen. embodiments of any of the aforementioned methods, the 60 In some embodiments, the umbilical cord tissue is washed fetal Support tissue product is frozen or previously frozen with buffer with agitation to remove excess blood and tissue. placenta. In some embodiments of any of the aforemen In some embodiments, washing with agitation reduces the tioned methods, the fetal Support tissue product is frozen or wash time. In some embodiments, the umbilical cord is previously frozen umbilical cord. In some embodiments of contacted with an isotonic buffer. In some embodiments, the any of the aforementioned methods, the fetal Support tissue 65 umbilical cord is contacted with saline, PBS, PBS 1X, product is frozen or previously frozen chorion or amnion Ringer's solution, Hartmann's solution, TRIS-buffered chorion. In some embodiments of any of the aforementioned saline, HEPES-buffered saline, EBSS, HBSS, Tyrode's salt US 9,675,733 B2 47 48 Solution, Gey’s Balanced Salt Solution, DMEM, EMEM, UCAM) is maintained in the sheet. In some embodiments, GMEM, RPMI, or any combinations thereof. the natural biological activity of the umbilical cord (or, any In some embodiments, the umbilical cord is cut into isolated parts thereof, e.g., UCAM) is maintained in the multiple sheets. The size of the sheets depends on the desired sheet. In some embodiments, the natural structural integrity use of the product derived from the umbilical cord. and the natural biological activity of the umbilical cord (or, In some embodiments, the section of the umbilical cord is any isolated parts thereof, e.g., UCAM) is maintained in the not cut further. In some embodiments, the section of the sheet. umbilical cord is cut into Smaller portions. Optionally, in In some embodiments, a powder or homogenate is made some embodiments, additional cuts are made in the Whar by: obtaining umbilical cord, processing the umbilical cord ton's Jelly to help shape the UC. 10 (see above for processing methods), and grinding (or pull In some embodiments, the cut umbilical cord tissue is Verizing) or homogenizing the umbilical cord. In some optionally washed again with buffer to further remove embodiments, the powder or homogenate is made with excess blood and tissue. whole umbilical cord. Optionally, the umbilical cord is In some embodiments, part or all of the Wharton's Jelly further processed by removing Wharton's Jelly and/or the is removed from the umbilical cord. The desired thickness of 15 umbilical arteries and veins. Alternatively, the Wharton's the fetal Support tissue product determines how much, if any, Jelly and/or the umbilical arteries and veins are note of the Wharton's Jelly is removed. In some embodiments, removed. In some embodiments, the powder or homogenate the Wharton's Jelly is not removed. is made by isolating the UCAM from the rest of the The umbilical cord comprises two arteries (the umbilical umbilical cord. In some embodiments, the natural biological arteries) and one vein (the umbilical vein). In some embodi activity of the umbilical cord (or, any isolated parts thereof) ments, the vein and arteries are removed from the umbilical is substantially maintained. cord. In certain instances, the vein and arteries are Sur In Some embodiments, the powder or homogenate is rounded (or suspended or buried) within the Wharton's Jelly. prepared by any Suitable method. In some embodiments, the In some embodiments, the vein and arteries are removed powder or homogenate is prepared by use of a homogenizer concurrently with the removal of the Wharton's Jelly. In 25 (e.g., an ultrasonic homogenizer), Sonicator, pulverizer, War Some embodiments, the veins and arteries are not removed. ring blender, grinding mill/jar, bead beater, or any combi If a UCAM sheet is the desired fetal support tissue nation thereof. product, then generation of the UCAM sheet comprises In Some embodiments, the powder or homogenate is substantially isolating the UCAM from the umbilical cord. prepared by use of a grinding jar. In some embodiments, the After substantially pure UCAM has been obtained, the 30 umbilical cord (or, any isolated parts thereof) is lyophilized UCAM is optionally washed with buffer to remove excess prior to being placed in the grinding jar. A grinding ball is blood and tissue. dropped in the grinding jar and the grinding jar is sealed. The In some embodiments, isolated UCAM is flattened fol grinding jar is immersed into liquid nitrogen for 5 min and lowing separation from the umbilical cord, generating a flat then placed in a mill and ground at a 30 HZ grinding cycle UCAM sheet comprising isolated UCAM. In some embodi 35 for 4 min. ments, the isolated UCAM is rolled following separation In some embodiments, the umbilical cord (or, any isolated from the umbilical cord, generating a tubular UCAM sheet. parts thereof) is lyophilized by any suitable method (e.g., In some embodiments, the UCAM sheets are in any suitable exposure to a liquid gas, placement in a freezer). In some shape (e.g., a square, a circle, a triangle, a rectangle) and into embodiments, the umbilical cord (or, any isolated parts any Suitable size. 40 thereof) is placed in the vacuum chamber of a lyophilization In some embodiments, the UCAM sheets are contacted device until all or substantially all fluid (e.g., water) has been with a buffer to remove substantially all remaining red blood removed. In some embodiments, the umbilical cord (or, any cells. In some embodiments, the chorion or amnion-chorion isolated parts thereof) is lyophilized following freezing (e.g., sheets are contacted with an isotonic buffer. In some exposure to a temperature below 0°C., -20°C., -40°C., embodiments, the chorion or amnion-chorion sheets are 45 -50° C., -60° C., -70° C., -75° C., -80° C., -90° C., -100° contacted with saline, PBS, PBS 1X, Ringer's solution, C.). Hartmann's solution, TRIS-buffered saline, HEPES-buff In some embodiments, the natural biological activity of ered saline, EBSS, HBSS, Tyrode's salt Solution, Gey’s the umbilical cord (or, any isolated parts thereof) is main Balanced Salt Solution, DMEM, EMEM, GMEM, RPMI, or tained in the powder or homogenate. any combinations thereof. 50 In some embodiments, an extract is made from the In some embodiments, multiple layers of isolated UCAM umbilical cord or any isolated parts thereof. In some are combined to generate a layered UCAM sheet. The embodiments, the natural biological activity of the umbilical layered UCAM sheet is any suitable thickness. In some cord (or, any isolated parts thereof) is maintained in the embodiments, the layered UCAM sheet comprises two, extract. In some embodiments, a homogenate or powder is three, four, five, six, seven, eight, nine, or ten layers of 55 made as described above. In some embodiments, the homo isolated UCAM. In some embodiments, the layered UCAM genate or powder is centrifuged to generate an extract (i.e., sheet comprises more than ten layers of isolated UCAM. a chorion extract or an amnion-chorion extract). Any Suit In some embodiments, the UCAM sheet is optionally able method of centrifugation may be used. In some embodi contacted with a Substrate (i.e., a Supportive backing). In ments, the extract comprises the Supernatant. In some some embodiments, UCAM sheet is not contacted with a 60 embodiments, the extract comprises the precipitant. In some substrate. In some embodiments, the UCAM sheet does not embodiments, the extract is subject to additional extraction require a particular orientation relative to the Substrate (i.e., methods (e.g., HABP affinity chromatography, or immuno any side of the UCAM may be in contact with the substrate). affinity chromatography). In some embodiments, the UCAM sheet is orientated such In some embodiments, the method of making the extract that the epithelial layer is in contact with the substrate. 65 comprises: (a) mixing the homogenate or powder with cold In some embodiments, the natural structural integrity of PBS buffer without protease inhibitors, to generate a PBS the umbilical cord (or, any isolated parts thereof, e.g., mixture, (b) centrifuging the PBS mixture, and (c) isolating US 9,675,733 B2 49 50 the extract, to generate an isolated extract. In some embodi embodiments, drying the placenta kills Substantially all cells ments, the cold PBS buffer and tissue product are combined found in the placenta. In some embodiments, drying the in a 1:1 ratio. In some embodiments, the PBS mixture is placenta results in the loss of metabolic activity in Substan centrifuged at 48,000xg 4°C. for 30 min. tially all cells found in the placenta. In some embodiments, the method of making the extract All processing is done following Good Tissue Practices further comprises purifying the extract. The number of (GTP) to ensure that no contaminants are introduced into the purification steps depends on the desired purity. In some tissue grafts or PAM. embodiments, the purification comprises at least 2 rounds of The placenta is tested for HIV-1, HIV-2, HTLV-1, hepa ultracentrifugation. In some embodiments, the purification titis B and C, West Nile virus, cytomegalovirus, human comprises more 2 rounds of ultracentrifugation. In some 10 embodiments, the purification comprises at least 4 rounds of transmissible spongiform encephalopathy (e.g., Creutzfeldt ultracentrifugation. In some embodiments, the method of Jakob disease) and Treponema pallidum using FDA licensed purifying the isolated extract comprises: (d) dissolving the screening test. Any indication that the tissue is contaminated isolated extract in CsCl/4M guanidine HCl at the initial with HIV-1, HIV-2, HTLV-1, hepatitis B and C, West Nile density of 1.35 g/ml, to generate a CsCl mixture, (e) 15 virus, or cytomegalovirus results in the immediate quaran centrifuging the CsCl mixture at 125,000xg for 48 h at 15° tine and Subsequent destruction of the tissue specimen. C., to generate a first purified extract, (f) extracting the first Further, the donor's medical records are examined for risk purified extract and dialyzing it against distilled water to factors for and clinical evidence of hepatitis B, hepatitis C, remove CsCl and guanidine HCl, to generate a dialysate. In or HIV infection. Any indication that the donor has risk Some embodiments, the method of purifying the isolated factors for, and/or clinical evidence of infection with HIV-1, extract further comprises (g) mixing the dialysate with 3 HIV-2, HTLV-1, hepatitis B and C, West Nile virus, cyto volumes of 95% (v/v) ethanol containing 1.3% (w/v) potas megalovirus, human transmissible spongiform encephalopa sium acetate at 0°C. for 1 h, to generate a first dialysate/ thy (e.g., Creutzfeldt-Jakob disease) and Treponema palli ethanol mixture, (h) centrifuging the first dialysate/ethanol dum results in the immediate quarantine and Subsequent mixture at 15,000xg, to generate a second purified extract, 25 destruction of the tissue specimen. and (i) extracting the second purified extract. In some In some embodiments, the PAM is not isolated from the embodiments, the method of purifying the isolated extract placenta before further processing begins. In some embodi further comprises: (i) washing the second purified extract ments, the PAM is isolated from the placenta (generating with ethanol (e.g., 70% ethanol), to generate a second isolated PAM) before further processing begins, purified extract/ethanol mixture; (k) centrifuging the second 30 purified extract/ethanol mixture, to generate a third purified In some embodiments, substantially all of the blood is extract; and (1) extracting the third purified extract. In some removed from the placenta. In some embodiments, some embodiments, the method of purifying the isolated extract blood is removed from placenta. In some embodiments, the further comprises: (m) washing the third purified extract blood is not removed from the placenta. with ethanol (e.g., 70% ethanol), to generate a third purified 35 In some embodiments, the placenta is washed with buffer extract/ethanol mixture; (n) centrifuging the third purified with agitation to remove excess blood and tissue. In some extract/ethanol mixture, to generate a forth purified extract; embodiments, washing with agitation reduces the wash time. and (o) extracting the forth purified extract. In some embodi In some embodiments, the placenta is contacted with a ments, the purified extract comprises HC-HA complex. buffer. In some embodiments, the placenta is contacted with Placenta and Placental Amniotic Membrane (PAM) 40 an isotonic buffer. In some embodiments, the placenta is Placenta is recovered from any Suitable source (e.g., a contacted with saline, PBS, PBS 1X, Ringer's solution, hospital or tissue bank). Placenta can be obtained from any Hartmann's solution, TRIS-buffered saline, HEPES-buff mammal. Such as a human, non-human primate, cow or pig. ered saline, EBSS, HBSS, Tyrode's salt Solution, Gey’s The placenta may be frozen, previously frozen, or fresh (i.e., Balanced Salt Solution, DMEM, EMEM, GMEM, RPMI, or not frozen). 45 any combinations thereof. Where the placenta is not processed into a pulverized In some embodiments, the placenta is washed with an immediately after it has been obtained, it is processed for isotonic buffer or tissue culture media, and any Suitable storage (e.g., it is frozen or dried). In some embodiments, the antibiotic. In some embodiments, the antibiotic is cipro placenta is frozen for storage. In some embodiments, the floxacin, amphotericin B, penicillin, streptomycin, neomy placenta is frozen at or below 0°C. In some embodiments, 50 cin or a combination thereof. In some embodiments, the the placenta is frozen until donor and specimen eligibility antibiotic is ciprofloxacin and amphotericin B. In some has been determined. In some embodiments, the placenta is embodiments, the antibiotic is penicillin, streptomycin, neo placed in a cryo-preservative before being frozen. In some mycin, and amphotericin B. embodiments, freezing the placenta kills Substantially all In some embodiments, the placenta is cut into multiple cells found in the placenta. In some embodiments, freezing 55 sections (e.g., using a scalpel). The size of the sections the placenta kills substantially all cells found in the placenta depends on the desired use of the product derived from the while maintaining or increasing the biological activity of the placenta. placenta relative to fresh (i.e., non-frozen) placenta. In some In some embodiments, the cut placentatissue is optionally embodiments, freezing the placenta results in the loss of washed again with buffer to further remove excess blood and metabolic activity in substantially all cells found in the 60 tissue. placenta. In some embodiments, freezing the placenta results If a PAM sheet is the desired fetal support tissue product, in the loss of metabolic activity in substantially all cells then generation of the PAM sheet comprises any or all of the found in the placenta while maintaining or increasing the following steps in addition to the preceding steps. biological activity of the placenta relative to fresh (i.e., In some embodiments, PAM is substantially isolated from non-frozen) placenta. If the placenta is not frozen, it is 65 the placenta. After substantially isolated PAM has been processed as described below immediately. In some embodi obtained, the PAM is optionally washed with buffer to ments, the placenta is dried (e.g., by lyophilization). In some remove excess blood and tissue. US 9,675,733 B2 51 52 In some embodiments, isolated PAM is cut into in any exposure to a temperature below 0°C., -20°C., -40°C., Suitable shape (e.g., a square, a circle, a triangle, a rectangle) -50° C., -60° C., -70° C., -75° C., -80° C., -90° C., -100° and into any Suitable sizes. C.). In some embodiments, the PAM sheets are contacted with In some embodiments, the natural biological activity of a buffer to remove substantially all remaining red blood 5 the placenta or any isolated parts thereof is maintained in a cells. In some embodiments, the chorion or amnion-chorion powder or homogenate. sheets are contacted with an isotonic buffer. In some In some embodiments, an extract is made from the embodiments, the chorion or amnion-chorion sheets are placenta or any isolated parts thereof. In some embodiments, contacted with saline, PBS, PBS1x, Ringer's solution, Hart the natural biological activity of the placenta or any isolated mann's solution, TRIS-buffered saline, HEPES-buffered 10 parts thereof is maintained in an extract. In some embodi saline, EBSS, HBSS, Tyrode's salt Solution, Gey’s Bal ments, a homogenate or powder is made as described above. anced Salt Solution, DMEM, EMEM, GMEM, RPMI, or In some embodiments, the homogenate or powder is cen any combinations thereof. trifuged to generate an extract (i.e., a chorion extract or an In some embodiments, multiple layers of isolated PAM amnion-chorion extract). Any suitable method of centrifu are combined to generate a layered PAM sheet. The layered 15 gation may be used. In some embodiments, the extract PAM sheet is any suitable thickness. In some embodiments, comprises the Supernatant. In some embodiments, the the layered PAM sheet comprises two, three, four, five, six, extract comprises the precipitant. In some embodiments, the seven, eight, nine, or ten layers of isolated PAM. In some extract is subject to additional extraction methods (e.g., embodiments, the layered PAM sheet comprises more than HABP affinity chromatography, or immunoaffinity chroma ten layers of isolated PAM. tography). In some embodiments, the PAM sheet is optionally con In some embodiments, the method of making the extract tacted with a substrate (i.e., a Supportive backing). In some comprises: (a) mixing the homogenate or powder with cold embodiments, PAM sheet is not contacted with a substrate. PBS buffer without protease inhibitors, to generate a PBS In some embodiments, the PAM sheet does not require a mixture, (b) centrifuging the PBS mixture, and (c) isolating particular orientation relative to the Substrate (i.e., any side 25 the extract, to generate an isolated extract. In some embodi of the PAM may be in contact with the substrate). In some ments, the cold PBS buffer and tissue product are combined embodiments, the PAM sheet is orientated such that the in a 1:1 ratio. In some embodiments, the PBS mixture is epithelial layer is in contact with the substrate. centrifuged at 48,000xg 4°C. for 30 min. In some embodiments, the natural structural integrity of In some embodiments, the method of making the extract the placenta (or any isolated parts thereof; e.g., PAM) is 30 further comprises purifying the extract. The number of maintained in the sheet. In some embodiments, the natural purification steps depends on the desired purity. In some biological activity of the placenta (or any isolated parts embodiments, the purification comprises at least 2 rounds of thereof) is maintained in the sheet. In some embodiments, ultracentrifugation. In some embodiments, the purification the natural structural integrity and natural biological activity comprises more 2 rounds of ultracentrifugation. In some of the placenta (or any isolated parts thereof; e.g., PAM) is 35 embodiments, the purification comprises at least 4 rounds of maintained in the sheet. ultracentrifugation. In some embodiments, the method of In some embodiments, a powder or homogenate is made purifying the isolated extract comprises: (d) dissolving the by: obtaining placenta, processing the placenta (see above isolated extract in CsCl/4M guanidine HCl at the initial for processing methods), and grinding (or pulverizing) or density of 1.35 g/ml, to generate a CsCl mixture, (e) homogenizing the placenta. In some embodiments, the pow 40 centrifuging the CsCl mixture at 125,000xg for 48 h at 15° der or homogenate is made with whole placenta. In some C., to generate a first purified extract, (f) extracting the first embodiments, the powder or homogenate is made by iso purified extract and dialyzing it against distilled water to lating the PAM from the rest of the placenta. In some remove CsCl and guanidine HCl, to generate a dialysate. In embodiments, the natural biological activity of the placenta Some embodiments, the method of purifying the isolated (or, any isolated parts thereof) is Substantially maintained. 45 extract further comprises (g) mixing the dialysate with 3 In some embodiments, the powder or homogenate is volumes of 95% (v/v) ethanol containing 1.3% (w/v) potas prepared by any suitable method. In some embodiments, the sium acetate at 0°C. for 1 h, to generate a first dialysate/ powder or homogenate is prepared by use of a homogenizer ethanol mixture, (h) centrifuging the first dialysate/ethanol (e.g., an ultrasonic homogenizer), Sonicator, pulverizer, War mixture at 15,000xg, to generate a second purified extract, ring blender, grinding mill/jar, bead beater, or any combi 50 and (i) extracting the second purified extract. In some nation thereof. embodiments, the method of purifying the isolated extract In some embodiments, the powder or homogenate is further comprises: (i) washing the second purified extract prepared by use of a grinding jar. In some embodiments, the with ethanol (e.g., 70% ethanol), to generate a second placenta (or, any isolated parts thereof) is lyophilized prior purified extract/ethanol mixture; (k) centrifuging the second to being placed in the grinding jar. A grinding ball is dropped 55 purified extract/ethanol mixture, to generate a third purified in the grinding jar and the grinding jar is sealed. The extract; and (1) extracting the third purified extract. In some grinding jar is immersed into liquid nitrogen for 5 min and embodiments, the method of purifying the isolated extract then placed in a mill and ground at a 30 HZ grinding cycle further comprises: (m) washing the third purified extract for 4 min. with ethanol (e.g., 70% ethanol), to generate a third purified In some embodiments, the placenta (or, any isolated parts 60 extract/ethanol mixture; (n) centrifuging the third purified thereof) is lyophilized by any suitable method (e.g., expo extract/ethanol mixture, to generate a forth purified extract; Sure to a liquid gas, placement in a freezer). In some and (o) extracting the forth purified extract. In some embodi embodiments, the placenta (or, any isolated parts thereof) is ments, the purified extract comprises HC-HA complex. placed in the vacuum chamber of a lyophilization device Chorion and Amnion-Chorion until all or substantially all fluid (e.g., water) has been 65 Placenta comprising chorion is recovered from any Suit removed. In some embodiments, the placenta (or, any iso able source (e.g., a hospital or tissue bank). Placenta can be lated parts thereof) is lyophilized following freezing (e.g., obtained from any mammal. Such as a human, non-human US 9,675,733 B2 53 54 primate, cow or pig. The placenta may be frozen, previously begins. In some embodiments, the chorion or amnion frozen, or fresh (i.e., not frozen). chorion is isolated from the placenta before further process In some embodiments, the chorion is immediately iso ing begins. lated from the placenta. In some embodiments, the chorion In some embodiments, substantially all of the blood is is not immediately isolated from the placenta. In some 5 removed from the placenta, chorion, or amnion-chorion. In embodiments, the chorion or amnion-chorion (or, placenta Some embodiments, some blood is removed from the pla comprising chorion) is frozen for storage. In some embodi centa, chorion, or amnion-chorion. In some embodiments, ments, the chorion or amnion-chorion (or, placenta compris the blood is not removed from the placenta, chorion, or ing chorion) is frozen at or below 0°C. In some embodi amnion-chorion. 10 In some embodiments, the placenta, chorion, or amnion ments, the chorion or amnion-chorion (or, placenta chorion is contacted with a buffer. In some embodiments, the comprising chorion) is frozen until donor and specimen placenta, chorion, or amnion-chorion is contacted with an eligibility has been determined. In some embodiments, the isotonic buffer. In some embodiments, the placenta, chorion, chorion or amnion-chorion (or, placenta comprising cho or amnion-chorion is contacted with saline, PBS, PBS1x, rion) is placed in a cryo-preservative before being frozen. In 15 Ringer's solution, Hartmann's solution, TRIS-buffered Some embodiments, freezing the chorion or amnion-chorion saline, HEPES-buffered saline, EBSS, HBSS, Tyrode's salt (or, placenta comprising chorion) kills Substantially all cells Solution, Gey’s Balanced Salt Solution, DMEM, EMEM, found in the chorion or amnion-chorion. In some embodi GMEM, RPMI, or any combinations thereof. ments, freezing the chorion or amnion-chorion (or, placenta In some embodiments, the placenta, chorion, or amnion comprising chorion) kills Substantially all cells found in the 20 chorion is contacted with a Suitable antibiotic. In some chorion or amnion-chorion while maintaining or increasing embodiments, the antibiotic is ciprofloxacin, amphotericin the biological activity of the chorion or amnion-chorion B. penicillin, Streptomycin, neomycin or a combination relative to fresh (i.e., non-frozen) chorion or amnion-cho thereof. In some embodiments, the antibiotic is ciprofloxacin rion. In some embodiments, freezing the chorion or amnion and amphotericin B. In some embodiments, the antibiotic is chorion (or, placenta comprising chorion) results in the loss 25 penicillin, Streptomycin, neomycin, and amphotericin B. of metabolic activity in substantially all cells found in the In some embodiments, the chorion or amnion-chorion is chorion or amnion-chorion. In some embodiments, freezing cut into multiple sheets (e.g., using a scalpel). The size of the the chorion or amnion-chorion (or, placenta comprising sheets depends on the desired use of the product derived chorion) results in the loss of metabolic activity in substan from the chorion or amnion-chorion. In some embodiments, 30 chorion or amnion-chorion is cut into in any suitable shape tially all cells found in the chorion or amnion-chorion while (e.g., a square, a circle, a triangle, a rectangle) and into any maintaining or increasing the biological activity of the suitable sizes. chorion or amnion-chorion relative to fresh (i.e., non-frozen) In Some embodiments, the chorion or amnion-chorion chorion or amnion-chorion. If the chorion or amnion-cho sheet is contacted with a buffer to remove substantially all rion is not frozen, it is processed as described below 35 remaining red blood cells. In some embodiments, the cho immediately. In some embodiments, the chorion or amnion rion or amnion-chorion sheet is contacted with an isotonic chorion (or, placenta comprising chorion) is dried (e.g., by buffer. In some embodiments, the chorion or amnion-cho lyophilization). In some embodiments, drying the chorion or rion sheet is contacted with saline, PBS, PBS1x, Ringer's amnion-chorion (or, placenta comprising chorion) kills Sub solution, Hartmann's solution, TRIS-buffered saline, stantially all cells found in the chorion or amnion-chorion. 40 HEPES-buffered saline, EBSS, HBSS, Tyrode's salt Solu In some embodiments, drying the chorion or amnion-cho tion, Gey’s Balanced Salt Solution, DMEM, EMEM, rion (or, placenta comprising chorion) results in the loss of GMEM, RPMI, or any combinations thereof. metabolic activity in substantially all cells found in the In some embodiments, multiple layers of isolated chorion chorion or amnion-chorion. or amnion-chorion are combined to generate a layered All processing is done following Good Tissue Practices 45 chorion or amnion-chorion sheet. In some embodiments, a (GTP) to ensure that no contaminants are introduced into the layered chorion or amnion-chorion sheet comprises at least tissue grafts or PAM. one layer of chorion and at least one layer of amnion. The The chorion or amnion-chorion (or, placenta comprising layered chorion or amnion-chorion sheet is any Suitable chorion) is tested for HIV-1, HIV-2, HTLV-1, hepatitis Band thickness. In some embodiments, the layered chorion or C, West Nile virus, cytomegalovirus, human transmissible 50 amnion-chorion sheet comprises two, three, four, five, six, spongiform encephalopathy (e.g., Creutzfeldt-Jakob dis seven, eight, nine, or ten layers of isolated chorion or ease) and Treponema pallidum using FDA licensed screen amnion-chorion. In some embodiments, the layered chorion ing test. Any indication that the tissue is contaminated with or amnion-chorion sheet comprises more than ten layers of HIV-1, HIV-2, HTLV-1, hepatitis B and C, West Nile virus, isolated chorion or amnion-chorion. or cytomegalovirus results in the immediate quarantine and 55 In Some embodiments, the chorion or amnion-chorion Subsequent destruction of the tissue specimen. sheet is optionally contacted with a Substrate (i.e., a Sup Further, the donor's medical records are examined for risk portive backing). In some embodiments, chorion or amnion factors for and clinical evidence of hepatitis B, hepatitis C, chorion sheet is not contacted with a substrate. In some or HIV infection. Any indication that the donor has risk embodiments, the chorion or amnion-chorion sheet does not factors for, and/or clinical evidence of infection with HIV-1, 60 require a particular orientation relative to the Substrate (i.e., HIV-2, HTLV-1, hepatitis B and C, West Nile virus, cyto any side of the chorion or amnion-chorion may be in contact megalovirus, human transmissible spongiform encephalopa with the substrate). In some embodiments, the chorion or thy (e.g., Creutzfeldt-Jakob disease) and Treponema palli amnion-chorion sheet is orientated Such that the epithelial dum results in the immediate quarantine and Subsequent layer is in contact with the substrate. destruction of the tissue specimen. 65 In some embodiments, the natural structural integrity of In some embodiments, the chorion or amnion-chorion is the chorion or amnion-chorion is maintained in the sheet. In not isolated from the placenta before further processing Some embodiments, the natural biological activity of the US 9,675,733 B2 55 56 chorion or amnion-chorion is maintained in the sheet. In embodiments, the purification comprises at least 4 rounds of Some embodiments, the natural structural integrity and the ultracentrifugation. In some embodiments, the method of natural biological activity of the chorion or amnion-chorion purifying the isolated extract comprises: (d) dissolving the is maintained in the sheet. isolated extract in CsCl/4M guanidine HCl at the initial In some embodiments, a powder or homogenate is made density of 1.35 g/ml, to generate a CsCl mixture, (e) by: obtaining placenta comprising chorion, processing the centrifuging the CsCl mixture at 125,000xg for 48 h at 15° placenta and isolating the chorion (see above for processing C., to generate a first purified extract, (f) extracting the first and isolation methods), and grinding (or pulverizing) or purified extract and dialyzing it against distilled water to homogenizing the chorion or amnion-chorion. In some remove CsCl and guanidine HCl, to generate a dialysate. In embodiments, the powder or homogenate is made with 10 Some embodiments, the method of purifying the isolated chorion. In some embodiments, the powder or homogenate extract further comprises (g) mixing the dialysate with 3 is made with amnion-chorion. In some embodiments, the volumes of 95% (v/v) ethanol containing 1.3% (w/v) potas natural biological activity of the chorion or chorion-amnion sium acetate at 0°C. for 1 h, to generate a first dialysate/ (or, any isolated parts thereof) is Substantially maintained. ethanol mixture, (h) centrifuging the first dialysate/ethanol In some embodiments, the powder or homogenate is 15 mixture at 15,000xg, to generate a second purified extract, prepared by any suitable method. In some embodiments, the and (i) extracting the second purified extract. In some powder or homogenate is prepared by use of a homogenizer embodiments, the method of purifying the isolated extract (e.g., an ultrasonic homogenizer), Sonicator, pulverizer, War further comprises: (i) washing the second purified extract ring blender, grinding mill/jar, bead beater, or any combi with ethanol (e.g., 70% ethanol), to generate a second nation thereof. purified extract/ethanol mixture; (k) centrifuging the second In some embodiments, the powder or homogenate is purified extract/ethanol mixture, to generate a third purified prepared by use of a grinding jar. In some embodiments, the extract; and (1) extracting the third purified extract. In some chorion or amnion-chorion is lyophilized prior to being embodiments, the method of purifying the isolated extract placed in the grinding jar. A grinding ball is dropped in the further comprises: (m) washing the third purified extract grinding jar and the grinding jar is sealed. The grinding jar 25 with ethanol (e.g., 70% ethanol), to generate a third purified is immersed into liquid nitrogen for 5 min and then placed extract/ethanol mixture; (n) centrifuging the third purified in a mill and ground at a 30 HZ grinding cycle for 4 min. extract/ethanol mixture, to generate a forth purified extract; In some embodiments, the chorion or amnion-chorion is and (o) extracting the forth purified extract. In some embodi lyophilized by any suitable method (e.g., exposure to a ments, the purified extract comprises HC-HA complex. liquid gas, placement in a freezer). In some embodiments, 30 Storage the chorion or amnion-chorion (or, any isolated parts In some embodiments, a fetal Support tissue product thereof) is placed in the vacuum chamber of a lyophilization described herein is stored for later use. In some embodi device until all or substantially all fluid (e.g., water) has been ments, storing a fetal Support tissue product described herein removed. In some embodiments, the placenta (or, any iso does not destroy the natural biological activity of the fetal lated parts thereof) is lyophilized following freezing (e.g., 35 Support tissue product. In some embodiments, storing a fetal exposure to a temperature below 0°C., -20°C., -40°C., Support tissue product described herein does not destroy the -50° C., -60° C., -70° C., -75° C., -80° C., -90° C., -100° natural structural integrity of the fetal Support tissue product. C.). Sterilization In some embodiments, the natural biological activity of In some embodiments, a fetal Support tissue product the chorion or amnion-chorion is maintained in the powder 40 described herein is subject to terminal sterilization by any or homogenate. Suitable (e.g., medically acceptable) method. In some In some embodiments, an extract is made from the embodiments, a fetal Support tissue product described herein chorion or amnion-chorion. In some embodiments, the natu is exposed to gamma radiation for a period of time Sufficient ral biological activity of the chorion or amnion-chorion is to sterilize the fetal support tissue product. In some embodi maintained in the extract. In some embodiments, a homo 45 ments, a fetal Support tissue product described herein is genate or powder is made as described above. In some exposed to gamma radiation at 25 kGy for a period of time embodiments, the homogenate or powder is centrifuged to sufficient to sterilize the fetal support tissue product. In some generate an extract. Any Suitable method of centrifugation embodiments, a fetal Support tissue product described herein may be used. In some embodiments, the extract comprises is exposed to gamma radiation at 17-30 kGy for a period of the Supernatant. In some embodiments, the extract com 50 time sufficient to sterilize the fetal support tissue product. In prises the precipitant. In some embodiments, the extract is Some embodiments, a fetal Support tissue product described subject to additional extraction methods (e.g., HABP affinity herein is exposed to an electron beam for a period of time chromatography, or immunoaffinity chromatography). sufficient to sterilize the fetal support tissue product. In some In some embodiments, the method of making the extract embodiments, a fetal Support tissue product described herein comprises: (a) mixing the homogenate or powder with cold 55 is exposed to X-ray radiation for a period of time sufficient PBS buffer without protease inhibitors, to generate a PBS to sterilize the fetal support tissue product. In some embodi mixture, (b) centrifuging the PBS mixture, and (c) isolating ments, a fetal Support tissue product described herein is the extract, to generate an isolated extract. In some embodi exposed to UV radiation for a period of time sufficient to ments, the cold PBS buffer and tissue product are combined sterilize the fetal support tissue product. in a 1:1 ratio. In some embodiments, the PBS mixture is 60 In some embodiments, the dose range of the gamma centrifuged at 48,000xg 4°C. for 30 min. radiation is between 10 kGy and 60 kGy, e.g., between 17 In some embodiments, the method of making the extract kGy and 30 kGy, between 17 kGy and 20 kGy, between 20 further comprises purifying the extract. The number of kGy and 24 kGy, 25 kGy and 30 kGy. In some embodiments, purification steps depends on the desired purity. In some the placental product is radioprotected by exposure to a embodiments, the purification comprises at least 2 rounds of 65 radioprotectant. In some embodiments, the radioprotectant ultracentrifugation. In some embodiments, the purification comprises glycerol, propylene glycol, trehalose, manitol, comprises more 2 rounds of ultracentrifugation. In some DMSO, or a combination thereof. US 9,675,733 B2 57 58 In some embodiments, a fetal Support tissue product In some embodiments, a fetal Support tissue product described herein is placed in a suitable radio-protectant. In described herein is placed in a suitable cryo-preservative. In Some embodiments, the radio-protectant comprises Glyc Some embodiments, the cryo-preservative comprises Glyc erol, Propylene Glycol, DMSO, Trehalose, Mannitol, or a erol, Propylene Glycol, DMSO, Trehalose, Mannitol, or a combination thereof. In some embodiments, the radio-pro combination thereof. In some embodiments, the cryo-pre tectant comprises Glycerol, Propylene Glycol, or a combi servative comprises Glycerol, Propylene Glycol, or a com nation thereof. bination. Storage Medium Lyophilization In some embodiments, the fetal Support tissue product is In some embodiments, the fetal Support tissue product 10 disclosed herein is lyophilized. In some embodiments, stored in any suitable storage medium. In some embodi lyophilizing a fetal Support tissue product described herein ments, the storage medium is DMEM, Liebowitz’s medium, does not destroy the natural biological activity of the fetal MEM, NCTC, or any combination thereof. Support tissue product. In some embodiments, lyophilizing a In some embodiments, the storage medium comprises a fetal support tissue product described herein does not high oncotic or hyperosmotic agent (also referred to as a 15 destroy the natural structural integrity of the fetal support "plasma expander”). In some embodiments, the hyperos tissue product. motic agent is propylene glycol, glycerol: Sugars, such as In some embodiments, the fetal Support tissue product glucose, Sucrose, maltose, dextrose, and the like; dimethyl disclosed herein is lyophilized following freezing. In some sulfoxide (DMSO); dimethylamine (DMA); polyvinylpyr embodiments, the fetal Support tissue product disclosed rolidone (PVP); sorbitol, glutathione (GSH); ascorbic acid; herein is lyophilized following freezing by any suitable rosmarinic acid (RO); riboflavin; dextran; albumin; treha method (e.g., exposure to a liquid gas, placement in a lose; mannitol; or any combination thereof. The hyperos freezer). motic agent maintains an optimal hydration state of the fetal In some embodiments, a frozen tissue product disclosed support tissue product. Preferably, the hydration of the fetal herein is placed in the vacuum chamber of a lyophilization support tissue product is maintained between about 60% and 25 device until all or substantially all fluid (e.g., water) has been 90% hydration for the intended purpose. In some embodi removed. ments, the hyperosmotic agent makes up about 10% to 90% Rehydration of the storage medium, preferably 10% to about 50%, and In some embodiments, a dehydrated or lyophilized prod more preferably about 30% to about 50%. In some embodi uct tissue product disclosed herein is partially or fully ments, the fetal support tissue product is stored in 10%, 30 rehydrated. In some embodiments, dehydrated or 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lyophilized product tissue product disclosed herein rehy glycerol. In some embodiments, the fetal support tissue drated by contacting the fetal support tissue product with a product is stored in 10%, 20%, 30%, 40%, 50%, 60%, 70%, buffer or with water. In some embodiments, the fetal support 80%, 90% or 100% propylene glycol. tissue product is contacted with an isotonic buffer. In some In some embodiments, the fetal Support tissue product is 35 embodiments, the fetal Support tissue product is contacted stored in 50% DMEM+50% Glycerol. with Saline. In some embodiments, the fetal Support tissue Cryopreservation product is contacted with PBS. In some embodiments, the In some embodiments, a fetal Support tissue product fetal Support tissue product is contacted with Ringer's described herein is frozen for cryopreservation by any Solution. In some embodiments, the fetal Support tissue Suitable method (e.g., exposure to a liquid gas, placement in 40 product is contacted with Hartmann's solution. In some a freezer, graduated cryopreservation). In some embodi embodiments, the fetal Support tissue product is contacted ments, cryopreserving a fetal Support tissue product with a TRIS-buffered saline. In some embodiments, the fetal described herein does not destroy the natural biological support tissue product is contacted with a HEPES-buffered activity of the fetal support tissue product. In some embodi saline; 50% DMEM+50% Glycerol; 10%, 20%, 30%, 40%, ments, cryopreserving a fetal Support tissue product 45 50%. 60%, 70%, 80%, 90% or 100% glycerol; and/or 10%, described herein does not destroy the natural structural 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% integrity of the fetal Support tissue product. propylene glycol. In some embodiments, a fetal Support tissue product HC-HA Complex described herein is exposed to a liquid gas (e.g., liquid Disclosed herein, in certain embodiments, are methods of nitrogen or liquid hydrogen). In some embodiments, the 50 inhibiting osteoclast differentiation in an individual in need tissue product disclosed herein is exposed to liquid nitrogen. thereof, comprising administering to the individual a com In some embodiments, the fetal Support tissue product position comprising HC-HA complex. Disclosed herein, in disclosed herein does not contact the liquid gas. In some certain embodiments, are methods of promoting mineraliza embodiments, the fetal Support tissue product disclosed tion by osteoblasts in an individual in need thereof, com herein is placed in a container and the container is contacted 55 prising administering to the individual a composition com with liquid gas. In some embodiments, the fetal Support prising HC-HA complex. Disclosed herein, in certain tissue product disclosed herein is exposed to the liquid gas embodiments, are methods of inhibiting bone resorption in until the fetal support tissue product is frozen. an individual in need thereof, comprising administering to In some embodiments, the fetal Support tissue product the individual a composition comprising HC-HA complex. disclosed herein is frozen by exposure to a temperature 60 Disclosed herein, in certain embodiments, are methods of below about 0°C. In some embodiments, a fetal support inhibiting bone remodeling in an individual in need thereof, tissue product described herein is frozen by exposure to a comprising administering to the individual a composition liquid gas. comprising HC-HA complex. Disclosed herein, in certain In some embodiments, the fetal Support tissue product embodiments, are methods of balancing bone resorption and disclosed herein is frozen by graduated cryopreservation 65 bone formation, comprising administering to an individual (e.g., by lowering down the temperature in a computer in need thereof a therapeutically-effective amount of a generated program). composition comprising HC-HA complex. Disclosed herein, US 9,675,733 B2 59 60 in certain embodiments, are methods of treating a disease, contacting occurs for at least 6 hours, at least 12 hours, at disorder, or condition characterized by excessive or unde least 24 hours, at least 36 hours, at least 48 hours, at least 60 sired osteoclast differentiation, the methods comprising hours, or at least 72 hours. administering to an individual in need thereof a therapeuti In some embodiments, the method further comprises cally-effective amount of a composition comprising HC-HA immobilizing HA (e.g., HMW HA) to a stationary support complex. Disclosed herein, in certain embodiments, are (e.g., by cross-linking). In some embodiments, the stationary methods of treating a disease, disorder, or condition char support comprising HA (e.g., HMW HA) is contacted with acterized by excessive or undesired bone absorption by ICI (e.g., ICI purified from serum, IOI in serum), TSG-6 (or, osteoclasts, the methods comprising administering to an recombinant TSG-6), and PTX3 (or, recombinant PTX3). In individual in need thereofatherapeutically-effective amount 10 Some embodiments, the contacting occurs for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, of a composition comprising HC-HA complex. Disclosed at least 48 hours, at least 60 hours, or at least 72 hours. In herein, in certain embodiments, are methods of treating a Some embodiments, the stationary Support is washed to disease, disorder, or condition characterized by deficient or remove any unbound components. defective bone formation, the methods comprising admin 15 In some embodiments, the method further comprises HA istering to an individual in need thereof a therapeutically binding protein (HABP). In some embodiments, HABP is effective amount of a composition comprising HC-HA com affixed to a stationary Support (e.g., by cross-linking). In plex. Disclosed herein, in certain embodiments, are methods Some embodiments, the stationary Support comprising of treating arthritis, the methods comprising administering HABP is contacted with HA (e.g., HMW HA), ICI, TSG-6 to an individual in need thereof a therapeutically-effective (or, recombinant TSG-6), and PTX3 (or, recombinant amount of a composition comprising HC-HA complex. PTX3). In some embodiments, the contacting occurs for at Disclosed herein, in certain embodiments, are methods of least 6 hours, at least 12 hours, at least 24 hours, at least 36 treating osteoporosis, the methods comprising administering hours, at least 48 hours, at least 60 hours, or at least 72 hours. to an individual in need thereof a therapeutically-effective In some embodiments, the stationary Support is washed to amount of a composition comprising HC-HA complex. 25 remove any unbound components. Disclosed herein, in certain embodiments, are methods of In Some embodiments, ICI is isolated from serum. In treating alveolar bone degradation, the methods comprising Some embodiments, ICI is in serum. In some embodiments, administering to an individual in need thereof a therapeuti ICI is isolated from a cell or plurality of cells. cally-effective amount of a composition comprising HC-HA In some embodiments, TSG6 is isolated from a cell or a 30 plurality of cells (e.g., a tissue extract). In some embodi complex. Disclosed herein, in certain embodiments, are ments, TSG6 is not isolated from a cell or a plurality of cells methods of treating Paget’s disease, the methods comprising (e.g., a tissue extract). In some embodiments, TSG6 is administering to an individual in need thereof a therapeuti prepared by recombinant technology. cally-effective amount of a composition comprising HC-HA In some embodiments, PTX3 is isolated from a cell or a complex. Disclosed herein, in certain embodiments, are 35 plurality of cells (e.g., a tissue extract). In some embodi methods of treating a bone tumor, the methods comprising ments, PTX3 is not isolated from a cell or a plurality of cells administering to an individual in need thereof a therapeuti (e.g., a tissue extract). In some embodiments, PTX3 is cally-effective amount of a composition comprising HC-HA prepared by recombinant technology. complex. In some embodiments of any of the aforemen Purification of HC-HA tioned methods, the HC-HA complex is isolated from a fetal 40 In some embodiments, the HC-HA is purified by any support tissue (e.g., UCAM, PAM, umbilical cord, placenta, suitable method. In some embodiments, the HC-HA com chorion, amnion-chorion, or any combinations thereof) and plex is purified by centrifugation (e.g., ultracentrifugation, purified. In some embodiments of any of the aforementioned gradient centrifugation), chromatography (e.g., ion methods, the HC-HA complex is obtained by a process exchange, affinity, size exclusion, and hydroxyapatite chro comprising (a) providing a reaction mixture comprising: (i) 45 matography), gel filtration, or differential Solubility, ethanol HA (e.g., HMWHA); (ii) ICI, wherein the ICI is optionally precipitation or by any other available technique for the in serum or isolated from serum; (iii); (iii) TSG-6, wherein purification of proteins (See, e.g., Scopes, Protein Purifica the TSG-6 is optionally recombinant; and (iv) PTX3. tion Principles and Practice 2nd Edition, Springer-Verlag, wherein the PTX3 is optionally recombinant; wherein at New York, 1987; Higgins, S.J. and Hames, B. D. (eds.), least one of HA, ICI, TSG-6, or PTX3 is optionally gener 50 Protein Expression: A Practical Approach, Oxford Univ ated by a plurality of cells present in the reaction mixture: Press, 1999; and Deutscher, M. P., Simon, M.I., Abelson, J. (b) incubating the reaction mixture for a period of time N. (eds.), Guide to Protein Purification: Methods in Enzy Sufficient to produce HC-HA complex; and (c) isolating and mology (Methods in Enzymology Series, Vol 182), Aca purifying the HC-HA complex. demic Press, 1997, all incorporated herein by reference). Manufacturing HC-HA 55 In some embodiments, the HC-HA complex is purified by In some embodiments of any of the aforementioned ultracentrifugation. The number of purification steps methods, the HC-HA complex is obtained by a process depends on the desired purity. In some embodiments, the comprising (a) providing a reaction mixture comprising: (i) purification comprises at least 2 rounds of ultracentrifuga HA (e.g., HMWHA); (ii) ICI, wherein the ICI is optionally tion. In some embodiments, the purification comprises more in serum or isolated from serum; (iii); (iii) TSG-6, wherein 60 2 rounds of ultracentrifugation. In some embodiments, the the TSG-6 is optionally recombinant; and (iv) PTX3. purification comprises at least 4 rounds of ultracentrifuga wherein the PTX3 is optionally recombinant; wherein at tion. In some embodiments, the method of purifying the least one of HA, ICI, TSG-6, or PTX3 is optionally gener isolated extract comprises: (d) dissolving the isolated extract ated by a plurality of cells present in the reaction mixture: in CsCl/4M guanidine HCl at the initial density of 1.35 g/ml, (b) incubating the reaction mixture for a period of time 65 to generate a CsCl mixture, (e) centrifuging the CsCl mix Sufficient to produce HC-HA complex; and (c) isolating and ture at 125,000xg for 48 h at 15° C., to generate a first purifying the HC-HA complex. In some embodiments, the purified extract, (f) extracting the first purified extract and US 9,675,733 B2 61 62 dialyzing it against distilled water to remove CsCl and In some embodiments, the method comprises ICI isolated guanidine HCl, to generate a dialysate. In some embodi from serum. In some embodiments, the method comprises ments, the method of purifying the isolated extract further ICI that is not isolated from serum. In some embodiments, comprises (g) mixing the dialysate with 3 volumes of 95% the method comprises ICI that is expressed by a cell or a (v/v) ethanol containing 1.3% (w/v) potassium acetate at 0° 5 plurality of cells in a bioreactor. In some embodiments, the C. for 1 h, to generate a first dialysate/ethanol mixture, (h) plurality of cells constitutively expresses ICI. centrifuging the first dialysate/ethanol mixture at 15,000xg, In some embodiments, the method comprises TSG-6 that to generate a second purified extract, and (i) extracting the is isolated from a cell or a plurality of cells (e.g., a tissue extract). In some embodiments, the method comprises second purified extract. In some embodiments, the method O TSG-6 that is not isolated from a cell or a plurality of cells of purifying the isolated extract further comprises: (i) wash- 1 (e.g., a tissue extract). In some embodiments, the method ing the second purified extract with ethanol (e.g., 70% comprises TSG-6 that is expressed by a cell or a plurality of ethanol), to generate a second purified extract/ethanol mix cells in a bioreactor. In some embodiments, the cell or ture; (k) centrifuging the second purified extract/ethanol plurality of cells constitutively generates TSG-6. In some mixture, to generate a third purified extract; and (1) extract 15 embodiments, the cell or plurality of cells constitutively ing the third purified extract. In some embodiments, the expresses TSG-6. In some embodiments, the cell or plurality method of purifying the isolated extract further comprises: of cells is contacted with at least one factor known to (m) washing the third purified extract with ethanol (e.g., upregulate TSG-6. 70% ethanol), to generate a third purified extract/ethanol In some embodiments, the method comprises PTX3 that mixture; (n) centrifuging the third purified extract/ethanol is isolated from a cell or a plurality of cells (e.g., a tissue mixture, to generate a forth purified extract; and (o) extract extract). In some embodiments, the method comprises PTX3 ing the forth purified extract. The preferred purification that is not isolated from a cell or a plurality of cells (e.g., a method comprises four rounds of ultracentrifugation. tissue extract). In some embodiments, the method comprises In some embodiments, the HC-HA complex is purified by PTX3 that is expressed by a cell or a plurality of cells in a immunoaffinity chromatography. In some embodiments, anti 25 bioreactor. In some embodiments, the cell or plurality of HC1 antibodies, anti-HC2 antibodies, or both are generated cells constitutively generates PTX3. In some embodiments, and affixed to a stationary Support. In some embodiments, the cell or plurality of cells constitutively expresses PTX3. the unpurified HC-HA complex (i.e., the mobile phase) is In some embodiments, the cell or plurality of cells is passed over the Support. In certain instances, the HC-HA contacted with at least one factor known to upregulate complex binds to the antibodies (e.g., via interaction of (a) 30 expression of PTX3. an HC1 antibody and HC1, (b) an HC2 antibody and HC2, In some embodiments, a cell that constitutively (a) or (c) both). In some embodiments the support is washed expresses ICI; (b) expresses TSG-6; or (c) PTX3, is gener (e.g., with PBS) to remove any unbound or loosely bound ated by any Suitable method. In some embodiments, a cell molecules. In some embodiments, the Support is then that constitutively (a) expresses IC.I; (b) expresses TSG-6; or washed with a solution that enables elution of the HC-HA 35 (c) expresses PTX3 is generated by introducing point muta complex from the support (e.g., 1% SDS, 6M guanidine tions into a gene encoding (a); (b) TSG-6; or (c) PTX3. In HCl, or 8M urea). Some embodiments, the mutations are substitution muta In some embodiments, the HC-HA complex is purified by tions, deletion mutations, or insertion mutations. affinity chromatography. In some embodiments, HABP is In some embodiments, a cell that constitutively generates generated and affixed to a stationary Support. In some 40 HA is produced by any suitable method. In some embodi embodiments, the unpurified HC-HA complex (i.e., the ments, a cell that constitutively generates HA is produced by mobile phase) is passed over the Support. In certain introducing point mutations into a gene encoding HAS1, instances, the HC-HA complex binds to the HABP. In some HAS2, HAS3, or a combination thereof. In some embodi embodiments the support is washed (e.g., with PBS) to ments, the mutations are Substitution mutations, deletion remove any unbound or loosely bound molecules. In some 45 mutations, or insertion mutations. In some embodiments, a embodiments, the support is then washed with a solution that cell that constitutively generates HA is produced by con enables elution of the HC-HA complex from the support. tacting the cell with at least one factor known to upregulate In some embodiments, the HC-HA complex is purified by HAS1, HAS2, HAS3, or a combination thereof. a combination of HABP affinity chromatography, and immu Pharmaceutical Compositions noaffinity chromatography using anti HC1 antibodies, anti 50 Disclosed herein, in certain embodiments, are methods of HC2 antibodies, or both. inhibiting osteoclast differentiation in an individual in need Bioreactor thereof, comprising administering to the individual a com In some embodiments, the HC-HA complex is made by position comprising product fetal Support tissue product or use of live cells. In some embodiments, the method com an extract thereof. Disclosed herein, in certain embodiments, prises contacting (a) hyaluronan (HA); (b) IC.I; (c) TSG-6: 55 are methods of promoting mineralization by osteoblasts in and (d) PTX3; wherein one or more components is gener an individual in need thereof, comprising administering to ated or expressed by a plurality of cells in a bioreactor. the individual a composition comprising a fetal Support In some embodiments, the method comprises HA that is tissue product or an extract thereof. In some embodiments, obtained from a commercial Supplier. In some embodiments, the fetal Support tissue product is Substantially isolated the method comprises HA that is generated by a plurality of 60 placental amniotic membrane (PAM), umbilical cord amni cells in a bioreactor. In some embodiments, the plurality of otic membrane (UCAM), placenta, umbilical cord, chorion, cells constitutively generate HA. In some embodiments, the amnion-chorion, or any combinations thereof. Disclosed plurality of cells constitutively expresses HAS1, HAS2, herein, in certain embodiments, are methods of inhibiting HAS3, or a combination thereof. In some embodiments, the bone resorption in an individual in need thereof, comprising plurality of cells are contacted with at least one factor known 65 administering to the individual a composition comprising a to upregulate HAS1, HAS2. HAS3, or a combination fetal support tissue product or an extract thereof. Disclosed thereof. herein, in certain embodiments, are methods of inhibiting US 9,675,733 B2 63 64 bone remodeling in an individual in need thereof, compris Solid dosage forms, powders, immediate release formula ing administering to the individual a composition compris tions, controlled release formulations, fast melt formula ing a fetal Support tissue product or an extract thereof. tions, tablets, capsules, pills, delayed release formulations, Disclosed herein, in certain embodiments, are methods of extended release formulations, pulsatile release formula balancing bone resorption and bone formation, comprising tions, multiparticulate formulations, and mixed immediate administering to an individual in need thereof a therapeuti and controlled release formulations. cally-effective amount of a composition comprising a fetal In some embodiments, the compositions described herein Support tissue product or an extract thereof. Disclosed are formulated as solutions. In some embodiments, the herein, in certain embodiments, are methods of treating a compositions are formulated as gels. In some embodiments, disease, disorder, or condition characterized by excessive or 10 the compositions described herein are formulated as lipo undesired osteoclast differentiation, the methods comprising Somes. In some embodiments, the compositions described administering to an individual in need thereof a therapeuti herein are formulated as foams. In some embodiments, the cally-effective amount of a composition comprising a fetal compositions described herein are formulated as paints. In Support tissue product or an extract thereof. Disclosed Some embodiments, the compositions described herein are herein, in certain embodiments, are methods of treating a 15 formulated as in situ forming spongy materials. disease, disorder, or condition characterized by excessive or In some embodiments, the compositions disclosed herein undesired bone absorption by osteoclasts, the methods com are formulated for immediate release. In some embodiments, prising administering to an individual in need thereof a the compositions are formulated for controlled release (e.g., therapeutically-effective amount of a composition compris delayed release or Sustained release). In some embodiments, ing a fetal Support tissue product or an extract thereof. the compositions are formulated for immediate release and Disclosed herein, in certain embodiments, are methods of controlled release. As used herein, "controlled release' treating a disease, disorder, or condition characterized by includes delayed release, Sustained release, extended deficient or defective bone formation, the methods compris release, variable release, pulsatile release and bi-modal ing administering to an individual in need thereof a thera release. The controlled-release aspect of the compositions peutically-effective amount of a composition comprising a 25 described herein is imparted by any suitable excipients. By fetal support tissue product or an extract thereof. Disclosed way of example only, such excipients include polymers and, herein, in certain embodiments, are methods of treating Viscosity enhancing agents. arthritis, the methods comprising administering to an indi In some embodiments, the compositions are biodegrad vidual in need thereof a therapeutically-effective amount of able. a composition comprising a fetal Support tissue product or 30 Pharmaceutical compositions may be formulated in a an extract thereof. Disclosed herein, in certain embodiments, conventional manner using one or more physiologically are methods of treating osteoporosis, the methods compris acceptable carriers including excipients and auxiliaries ing administering to an individual in need thereof a thera which facilitate processing of the active compounds into peutically-effective amount of a composition comprising a preparations which can be used pharmaceutically. Proper fetal support tissue product or an extract thereof. Disclosed 35 formulation is dependent upon the route of administration herein, in certain embodiments, are methods of treating chosen. Any of the well-known techniques, carriers, and alveolar bone degradation, the methods comprising admin excipients may be used as Suitable and as understood in the istering to an individual in need thereof a therapeutically art. A Summary of pharmaceutical compositions described effective amount of a composition comprising a fetal Support herein may be found, for example, in Remington. The tissue product or an extract thereof. Disclosed herein, in 40 Science and Practice of Pharmacy, Nineteenth Ed (Easton, certain embodiments, are methods of treating Paget’s dis Pa.: Mack Publishing Company, 1995); Hoover, John E. ease, the methods comprising administering to an individual Remington's Pharmaceutical Sciences, Mack Publishing in need thereof a therapeutically-effective amount of a Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., composition comprising a fetal Support tissue product or an Eds. Pharmaceutical Dosage Forms, Marcel Decker, New extract thereof. Disclosed herein, in certain embodiments, 45 York, N.Y., 1980; and Pharmaceutical Dosage Forms and are methods of treating a bone tumor, the methods compris Drug Delivery Systems, Seventh Ed. (Lippincott Williams & ing administering to an individual in need thereof a thera Wilkins 1999), herein incorporated by reference in their peutically-effective amount of a composition comprising a entirety. fetal Support tissue product or an extract thereof. In some Pharmaceutical compositions including a compound embodiments, the compositions further comprise pharma 50 described herein may be manufactured in a conventional ceutically acceptable diluent(s), excipient(s), or carrier(s). In manner, Such as, by way of example only, by means of Some embodiments, the compositions further comprise an conventional mixing, dissolving, granulating, dragee-mak adhesive. In yet other embodiments, the compositions fur ing, levigating, emulsifying, encapsulating, entrapping or ther comprise a penetration enhancer. In some embodiments, compression processes. the compositions comprise a viscosity enhancing agent. 55 Excipients In other embodiments, the pharmaceutical compositions In some embodiments, the compositions further comprise further comprise a further therapeutic agent. For a non pharmaceutically acceptable diluent(s), excipient(s), or car limiting disclosure of further therapeutic agents see the rier(s). Combination Therapies section herein. In some embodiments, the compositions further comprise The pharmaceutical compositions described herein can be 60 an antifoaming agent. "Antifoaming agents' reduce foaming administered to a Subject by multiple administration routes, during processing which can result in coagulation of aque including but not limited to, oral, parenteral (e.g., intrave ous dispersions, bubbles in the finished film, or generally nous, Subcutaneous, intramuscular), intranasal, buccal, topi impair processing. Exemplary anti-foaming agents include cal, rectal, or transdermal administration routes. The phar silicon emulsions or Sorbitan sesquoleate. maceutical formulations described herein include, but are 65 In some embodiments, the compositions further comprise not limited to, aqueous liquid dispersions, self-emulsifying an antioxidant. "Antioxidants' include, for example, buty dispersions, Solid solutions, liposomal dispersions, aerosols, lated hydroxytoluene (BHT), sodium ascorbate, ascorbic US 9,675,733 B2 65 66 acid, Sodium metabisulfite and tocopherol. In certain e.g., hydrophilic polymers, electrolytes, Tween R. 60 or 80, embodiments, antioxidants enhance chemical stability PEG, polyvinylpyrrolidone (PVP; commercially known as where required. Plasdone(R), and the carbohydrate-based dispersing agents In some embodiments, the compositions further comprise Such as, for example, hydroxypropyl celluloses (e.g., HPC. a binder. “Binders' impart cohesive qualities and include, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses e.g., alginic acid and salts thereof; cellulose derivatives Such (e.g., HPMC K100, HPMC K4M, HPMC K15M, and as carboxymethylcellulose, methylcellulose (e.g., Metho HPMC K10OM), carboxymethylcellulose sodium, methyl cel(R), hydroxypropylmethylcellulose, hydroxyethylcellu cellulose, hydroxyethylcellulose, hydroxypropylcellulose, lose, hydroxypropylcellulose (e.g.) Klucel(R), ethylcellulose hydroxypropylmethylcellulose phthalate, hydroxypropylm (e.g., Ethocel(R), and microcrystalline cellulose (e.g., Avi 10 ethylcellulose acetate stearate (HPMCAS), noncrystalline cel(R); microcrystalline dextrose; amylose; magnesium alu cellulose, magnesium aluminum silicate, triethanolamine, minum silicate; polysaccharide acids; bentonites; gelatin: polyvinyl alcohol (PVA), vinyl pyrrolidone/vinyl acetate polyvinylpyrrolidone/vinyl acetate copolymer, crosspovi copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol done; poVidone; starch; pregelatinized Starch; tragacanth, polymer with ethylene oxide and formaldehyde (also known dextrin, a Sugar, such as Sucrose (e.g., Dipac.R.), glucose, 15 as tyloxapol), poloxamers (e.g., Pluronics F68(R), F88(R), and dextrose, molasses, mannitol, Sorbitol. Xylitol (e.g., Xyl F108(R), which are block copolymers of ethylene oxide and itabR), and lactose; a natural or synthetic gum Such as propylene oxide); and poloxamines (e.g., Tetronic 908R, acacia, tragacanth, ghatti gum, mucilage of isapol husks, also known as Poloxamine 908R, which is a tetrafunctional polyvinylpyrrolidone (e.g., Poly Vidone(R). CL, Kollidon(R) block copolymer derived from sequential addition of pro CL, Polyplasdone RXL-10), larch arabogalactan, Veegum(R), pylene oxide and ethylene oxide to ethylenediamine (BASF polyethylene glycol, waxes, sodium alginate, and the like. Corporation, Parsippany, N.J.)), polyvinylpyrrolidone K12, In some embodiments, the compositions described herein polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or further comprise buffering agents, for example to impart a polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl physiological acceptable pH. Suitable buffering agent acetate copolymer (S-630), polyethylene glycol, e.g., the include acids Such as acetic, boric, citric, lactic, phosphoric 25 polyethylene glycol can have a molecular weight of about and hydrochloric acids; bases Such as Sodium hydroxide, 300 to about 6000, or about 3350 to about 4000, or about Sodium phosphate, Sodium borate, Sodium citrate, sodium 7000 to about 5400, sodium carboxymethylcellulose, meth acetate, Sodium lactate and tris-hydroxymethylaminometh ylcellulose, polysorbate-80, Sodium alginate, gums, such as, ane; and buffers such as citrate/dextrose, Sodium bicarbonate e.g., gum tragacanth and gum acacia, guar gum, Xanthans, and ammonium chloride. Such acids, bases and buffers are 30 including Xanthan gum, Sugars, cellulosics, such as, e.g., included in an amount required to maintain pH of the Sodium carboxymethylcellulose, methylcellulose, sodium composition in an acceptable range. carboxymethylcellulose, polysorbate-80, sodium alginate, In some embodiments, the compositions further comprise polyethoxylated Sorbitan monolaurate, polyethoxylated Sor a carrier. A “carrier' or “carrier materials” include any bitan monolaurate, povidone, carbomers, polyvinyl alcohol commonly used excipients in pharmaceutics and should be 35 (PVA), alginates, chitosans and combinations thereof. Plas selected on the basis of compatibility with compounds ticizers such as cellulose or triethyl cellulose can also be disclosed herein, such as, compounds of any of Formula D used as dispersing agents. Dispersing agents particularly and the second agent, and the release profile properties of the useful in liposomal dispersions and self-emulsifying disper desired dosage form. Exemplary carrier materials include, sions are dimyristoyl phosphatidylcholine, natural phospha e.g., binders, Suspending agents, disintegration agents, fill 40 tidylcholine from eggs, natural phosphatidylglycerol from ing agents, Surfactants, Solubilizers, stabilizers, lubricants, eggs, cholesterol and isopropyl myristate. wetting agents, diluents, and the like. "Pharmaceutically In some embodiments, the compositions further comprise compatible carrier materials' may include, but are not a diluent. The term "diluent refers to chemical compounds limited to, acacia, gelatin, colloidal silicon dioxide, calcium that are used to dilute the compound of interest prior to glycerophosphate, calcium lactate, maltodextrin, glycerine, 45 delivery. Diluents can also be used to stabilize compounds magnesium silicate, polyvinylpyrrollidone (PVP), choles because they can provide a more stable environment. Salts terol, cholesterol esters, sodium caseinate, Soy lecithin, dissolved in buffered solutions (which also can provide pH taurocholic acid, phosphotidylcholine, Sodium chloride, tri control or maintenance) are utilized as diluents in the art, calcium phosphate, dipotassium phosphate, cellulose and including, but not limited to a phosphate buffered saline cellulose conjugates, Sugars sodium Stearoyl lactylate, car 50 Solution. In certain embodiments, diluents increase bulk of rageenan, monoglyceride, diglyceride, pregelatinized starch, the composition to facilitate compression or create Sufficient and the like. See, e.g., Remington. The Science and Practice bulk for homogenous blend for capsule filling. Such com of Pharmacy, Nineteenth Ed (Easton, Pa.; Mack Publishing pounds include e.g., lactose, starch, mannitol, Sorbitol, dex Company, 1995); Hoover, John E., Remington's Pharma trose, microcrystalline cellulose such as Avicel(R: dibasic ceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; 55 calcium phosphate, dicalcium phosphate dihydrate; trical Liberman, H. A. and Lachman, L., Eds. Pharmaceutical cium phosphate, calcium phosphate; anhydrous lactose, Dosage Forms, Marcel Decker, New York, N.Y., 1980; and spray-dried lactose; pregelatinized Starch, compressible Pharmaceutical Dosage Forms and Drug Delivery Systems, Sugar, Such as Di-Pac(R) (Amstar); mannitol, hydroxypropy Seventh Ed. (Lippincott Williams & Wilkins 1999). lmethylcellulose, hydroxypropylmethylcellulose acetate In some embodiments, the compositions further comprise 60 Stearate. Sucrose-based diluents, confectioner's Sugar, a dispersing agent and/or viscosity modulating agents. “Dis monobasic calcium Sulfate monohydrate, calcium sulfate persing agents.” and/or "viscosity modulating agents' dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed include materials that control the diffusion and homogeneity cereal Solids, amylose; powdered cellulose, calcium carbon of a drug through liquid media or a granulation method or ate; glycine, kaolin; mannitol, Sodium chloride; inositol, blend method. In some embodiments, these agents also 65 bentonite, and the like. facilitate the effectiveness of a coating or eroding matrix. In some embodiments, the compositions further comprise Exemplary diffusion facilitators/dispersing agents include, a disintegrant. The term “disintegrate” includes both the US 9,675,733 B2 67 68 dissolution and dispersion of the dosage form when con lulose, hydroxypropylmethylcellulose, hydroxyethylcellu tacted with gastrointestinal fluid. "Disintegration agents or lose, polysorbate-80, Sodium alginate, polyethoxylated disintegrants' facilitate the breakup or disintegration of a Sorbitan monolaurate, polyethoxylated Sorbitan monolau Substance. Examples of disintegration agents include a rate, povidone and the like. starch, e.g., a natural starch Such as corn starch or potato 5 In some embodiments, the compositions further comprise starch, a pregelatinized starch such as National 1551 or a surfactant. "Surfactants' include compounds Such as Amijel R., or sodium starch glycolate such as Promogel(R) or sodium lauryl sulfate, sodium docusate, Tween 60 or 80, ExplotabR), a cellulose such as a wood product, methylcrys triacetin, vitamin E TPGS, sorbitan monooleate, polyoxy talline cellulose, e.g., Avicel(R), Avicel(R) PH101, Avicel(R) ethylene Sorbitan monooleate, polysorbates, polaxomers, PH102, Avice1(R) PH105, Elcema(R) P100, Emcocel(R), Viva 10 bile salts, glyceryl monostearate, copolymers of ethylene cel(R), Ming TiaR), and Solka-FlocR, methylcellulose, cros oxide and propylene oxide, e.g., Pluronic R (BASF), and the carmellose, or a cross-linked cellulose, such as cross-linked like. Some other surfactants include polyoxyethylene fatty sodium carboxymethylcellulose (Ac-Di-SolR), cross-linked acid glycerides and vegetable oils, e.g., polyoxyethylene carboxymethylcellulose, or cross-linked croscarmellose, a (60) hydrogenated castor oil; and polyoxyethylene alkyle cross-linked Starch Such as sodium starch glycolate, a cross 15 thers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol linked polymer Such as crosspoVidone, a cross-linked poly 40. In some embodiments, Surfactants may be included to vinylpyrrolidone, alginate Such as alginic acid or a salt of enhance physical stability or for other purposes. alginic acid Such as Sodium alginate, a clay Such as In some embodiments, the compositions further comprise Veegum R. HV (magnesium aluminum silicate), a gum Such a viscosity enhancing agent. 'Viscosity enhancing agents' as agar, guar, locust bean, Karaya, pectin, or tragacanth, include, e.g., methyl cellulose, Xanthan gum, carboxymethyl Sodium starch glycolate, bentonite, a natural sponge, a cellulose, hydroxypropyl cellulose, hydroxypropylmethyl Surfactant, a resin Such as a cation-exchange resin, citrus cellulose, hydroxypropylmethyl cellulose acetate Stearate, pulp, sodium lauryl Sulfate, Sodium lauryl Sulfate in com hydroxypropylmethyl cellulose phthalate, carbomer, poly bination starch, and the like. vinyl alcohol, alginates, acacia, chitosans and combinations In some embodiments, the compositions further comprise 25 thereof. a preservative. Suitable preservatives include mercury-con In some embodiments, the compositions further comprise taining Substances such as merfen and thiomersal; stabilized a wetting agent. “Wetting agents' include compounds Such chlorine dioxide; and quaternary ammonium compounds as oleic acid, glyceryl monostearate, Sorbitan monooleate, Such as benzalkonium chloride, cetyltrimethylammonium Sorbitan monolaurate, triethanolamine oleate, polyoxyethyl bromide and cetylpyridinium chloride. 30 ene Sorbitan monooleate, polyoxyethylene Sorbitan mono In some embodiments, the compositions described herein laurate, Sodium docusate, Sodium oleate, sodium lauryl further comprise salts. In other embodiments, compositions sulfate, sodium doccusate, triacetin, Tween 80, vitamin E described herein further comprise one or more salts in an TPGS, ammonium salts and the like. amount Sufficient to impart a physiologically acceptable Dosage Forms osmolarity. Such salts include those having Sodium, potas 35 The compositions described herein e.g., HC-HA, fetal sium or ammonium cations and chloride, citrate, ascorbate, Support tissue extracts, fetal Support powders and homoge borate, phosphate, bicarbonate, sulfate, thiosulfate or nates) can be formulated for administration to a subject via bisulfite anions; suitable salts include sodium chloride, any conventional means including, but not limited to, oral, potassium chloride, sodium thiosulfate, sodium bisulfite and parenteral (e.g., intravenous, Subcutaneous, or intramuscu ammonium sulfate. 40 lar), buccal, intranasal, rectal or transdermal administration In some embodiments, the compositions further comprise routes. As used herein, the term "subject' is used to mean an a solubilizer. “Solubilizers” include compounds such as animal, preferably a mammal, including a human or non triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium human. The terms patient and Subject may be used inter lauryl sulfate, sodium doccusate, vitamin ETPGS, dimethy changeably. lacetamide, N-methylpyrrolidone, N-hydroxyethylpyrroli 45 Moreover, the pharmaceutical compositions (e.g., HC done, polyvinylpyrrolidone, hydroxypropylmethyl cellu HA, fetal Support tissue extracts, fetal Support powders and lose, hydroxypropyl cyclodextrins, ethanol, n-butanol, homogenates) disclosed herein can be formulated into any isopropyl alcohol, cholesterol, bile salts, polyethylene glycol Suitable dosage form, including but not limited to, aqueous 200-600, glycofurol, transcutol, propylene glycol, and dim oral dispersions, liquids, gels, syrups, elixirs, slurries, Sus ethyl isosorbide and the like. 50 pensions and the like, for oral ingestion by a patient to be "Stabilizers' include compounds such as any antioxida treated, Solid oral dosage forms, aerosols, controlled release tion agents, buffers, acids, preservatives and the like. formulations, fast melt formulations, effervescent formula In some embodiments, the compositions further comprise tions, lyophilized formulations, tablets, powders, pills, dra a Suspending agent. "Suspending agents' include com gees, capsules, delayed release formulations, extended pounds such as polyvinylpyrrolidone, e.g., polyvinylpyrroli 55 release formulations, pulsatile release formulations, multi done K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone particulate formulations, and mixed immediate release and K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl controlled release formulations. acetate copolymer (S630), polyethylene glycol, e.g., the Solutions polyethylene glycol can have a molecular weight of about In some embodiments, the compositions (e.g., HC-HA, 300 to about 6000, or about 3350 to about 4000, or about 60 fetal Support tissue extracts, fetal Support powders and 7000 to about 5400, sodium carboxymethylcellulose, meth homogenates) disclosed herein re formulated as Solutions. ylcellulose, hydroxypropylmethylcellulose, hydroxymethyl Solutions include aqueous solutions, dispersions, and emul cellulose acetate stearate, polysorbate-80, hydroxyethylcel sions. See, e.g., Singh et al., Encyclopedia of Pharmaceu lulose, sodium alginate, gums, such as, e.g., gum tragacanth tical Technology, 2" Ed., pp. 754-757 (2002). and gum acacia, guar gum, Xanthans, including Xanthan 65 The aqueous Suspensions and dispersions described gum, Sugars, cellulosics, such as, e.g., Sodium carboxym herein can remain in a homogenous state, as defined in The ethylcellulose, methylcellulose, sodium carboxymethylcel USP Pharmacists Pharmacopeia (2005 edition, chapter US 9,675,733 B2 69 70 905), for at least 4 hours. The homogeneity should be coat(R) USP 2910 (Shin-Etsu)); carboxymethylcellulose determined by a sampling method consistent with regard to sodium; methylcellulose; hydroxyethylcellulose; hydroxy determining homogeneity of the entire composition. In one propylmethyl-cellulose phthalate: hydroxypropylmethyl embodiment, an aqueous Suspension can be re-suspended cellulose acetate Stearate; non-crystalline cellulose; magne into a homogenous Suspension by physical agitation lasting 5 sium aluminum silicate; triethanolamine; polyvinyl alcohol less than 1 minute. In another embodiment, an aqueous (PVA): 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with Suspension can be re-suspended into a homogenous Suspen ethylene oxide and formaldehyde; poloxamers (e.g., Pluron sion by physical agitation lasting less than 45 seconds. In yet ics F68(R), F88(R), and F108R, which are block copolymers of another embodiment, an aqueous Suspension can be re ethylene oxide and propylene oxide); or poloxamines (e.g., Suspended into a homogenous Suspension by physical agi 10 Tetronic 908 R, also known as Poloxamine 908 R). tation lasting less than 30 seconds. In still another embodi Wetting agents suitable for the aqueous Suspensions and ment, no agitation is necessary to maintain a homogeneous dispersions described herein are known in the art and aqueous dispersion. include, but are not limited to, cetyl alcohol, glycerol Examples of disintegrating agents for use in the aqueous monostearate, polyoxyethylene Sorbitan fatty acid esters Suspensions and dispersions include, but are not limited to, 15 (e.g., the commercially available Tweens R. Such as e.g., a starch, e.g., a natural starch Such as corn starch or potato Tween 20R) and Tween 80R (ICI Specialty Chemicals)), and starch, a pregelatinized starch such as National 1551 or polyethylene glycols (e.g., Carbowaxs 3350R) and 1450R), Amijel R., or sodium starch glycolate such as Promogel(R) or and Carbopol 934(R) (Union Carbide)), oleic acid, glyceryl Explotab(R); a cellulose such as a wood product, methylcrys monostearate, Sorbitan monooleate, Sorbitan monolaurate, talline cellulose, e.g., Avicel(R), Avicel(R) PH101, Avicel(R) triethanolamine oleate, polyoxyethylene Sorbitan PH102, Avice1(R) PH105, Elcema(R) P100, Emcocel(R), Viva monooleate, polyoxyethylene Sorbitan monolaurate, sodium cel(R), Ming TiaR), and Solka-FlocR, methylcellulose, cros oleate, sodium lauryl Sulfate, Sodium docusate, triacetin, carmellose, or a cross-linked cellulose, such as cross-linked vitamin E TPGS, sodium taurocholate, simethicone, phos sodium carboxymethylcellulose (Ac-Di-SolR), cross-linked photidylcholine and the like carboxymethylcellulose, or cross-linked croScarmellose; a 25 Suitable preservatives for the aqueous suspensions or cross-linked Starch Such as Sodium starch glycolate; a cross dispersions described herein include, for example, potas linked polymer Such as crospovidone; a cross-linked poly sium Sorbate, parabens (e.g., methylparaben and propylpa vinylpyrrolidone; alginate Such as alginic acid or a salt of raben), benzoic acid and its salts, other esters of parahy alginic acid such as sodium alginate; a clay Such as droxybenzoic acid such as butylparaben, alcohols such as Veegum R. HV (magnesium aluminum silicate); a gum Such 30 ethyl alcohol or benzyl alcohol, phenolic compounds such as as agar, guar, locust bean, Karaya, pectin, or tragacanth; phenol, or quaternary compounds such as benzalkonium sodium starch glycolate; bentonite; a natural sponge; a chloride. Preservatives, as used herein, are incorporated into Surfactant; a resin Such as a cation-exchange resin; citrus the dosage form at a concentration Sufficient to inhibit pulp; Sodium lauryl Sulfate; sodium lauryl Sulfate in com microbial growth. bination starch; and the like. 35 Suitable viscosity enhancing agents for the aqueous Sus In some embodiments, the dispersing agents suitable for pensions or dispersions described herein include, but are not the aqueous Suspensions and dispersions described herein limited to, methyl cellulose, Xanthan gum, carboxymethyl are known in the art and include, for example, hydrophilic cellulose, hydroxypropyl cellulose, hydroxypropylmethyl polymers, electrolytes, Tween R. 60 or 80, PEG, polyvi cellulose, Plasdon R S-630, carbomer, polyvinyl alcohol, nylpyrrolidone (PVP; commercially known as Plasdone(R). 40 alginates, acacia, chitosans and combinations thereof. The and the carbohydrate-based dispersing agents such as, for concentration of the Viscosity enhancing agent will depend example, hydroxypropylcellulose and hydroxypropyl cellu upon the agent selected and the viscosity desired. lose ethers (e.g., HPC, HPC-SL, and HPC-L), hydroxypro In addition to the additives listed above, the liquid for pyl methylcellulose and hydroxypropyl methylcellulose mulations can also include inert diluents commonly used in ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, and 45 the art, Such as water or other solvents, solubilizing agents, HPMC K10OM), carboxymethylcellulose sodium, methyl and emulsifiers. Exemplary emulsifiers are ethyl alcohol, cellulose, hydroxyethylcellulose, hydroxypropylmethyl-cel isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl lulose phthalate, hydroxypropylmethyl-cellulose acetate alcohol, benzylbenzoate, propyleneglycol, 1,3-butylenegly Stearate, noncrystalline cellulose, magnesium aluminum sili col, dimethylformamide, sodium lauryl Sulfate, Sodium doc cate, triethanolamine, polyvinyl alcohol (PVA), polyvi 50 cusate, cholesterol, cholesterol esters, taurocholic acid, nylpyrrolidone/vinyl acetate copolymer (Plasdone R, e.g., phosphotidylcholine, oils, such as cottonseed oil, groundnut S-630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with oil, corn germ oil, olive oil, castor oil, and Sesame oil, ethylene oxide and formaldehyde (also known as tyloxapol), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, poloxamers (e.g., Pluronics F68(R), F88(R), and F108(R), which fatty acid esters of Sorbitan, or mixtures of these Substances, are block copolymers of ethylene oxide and propylene 55 and the like. oxide); and poloxamines (e.g., Tetronic 908R, also known as In some embodiments, the pharmaceutical compositions Poloxamine 908R, which is a tetrafunctional block copoly (e.g., HC-HA, fetal Support tissue extracts, fetal Support mer derived from sequential addition of propylene oxide and powders and homogenates) described herein can be self ethylene oxide to ethylenediamine (BASF Corporation, Par emulsifying drug delivery systems (SEDDS). Emulsions are Sippany, N.J.)). In other embodiments, the dispersing agent 60 dispersions of one immiscible phase in another, usually in is selected from a group not comprising one of the following the form of droplets. Generally, emulsions are created by agents: hydrophilic polymers; electrolytes; Tween R. 60 or vigorous mechanical dispersion. SEDDS, as opposed to 80; PEG, polyvinylpyrrolidone (PVP); hydroxypropylcellu emulsions or microemulsions, spontaneously form emul lose and hydroxypropyl cellulose ethers (e.g., HPC, HPC sions when added to an excess of water without any external SL, and HPC-L): hydroxypropyl methylcellulose and 65 mechanical dispersion or agitation. An advantage of SEDDS hydroxypropyl methylcellulose ethers (e.g. HPMC K100, is that only gentle mixing is required to distribute the HPMC K4M, HPMC K15M, HPMC K10OM, and Pharma droplets throughout the solution. Additionally, water or the US 9,675,733 B2 71 72 aqueous phase can be added just prior to administration, Subject. By way of non-limiting example, paints include which ensures stability of an unstable or hydrophobic active collodions, and solutions comprising saccharide siloxane ingredient. Thus, the SEDDS provides an effective delivery copolymers and a cross-linking agent. Collodions are ethyl system for oral and parenteral delivery of hydrophobic ether/ethanol solutions containing pyroxylin (a nitrocellu active ingredients. SEDDS may provide improvements in lose). After application, the ethyl ether?ethanol solution the bioavailability of hydrophobic active ingredients. Meth evaporates leaving behind a thin film of pyroxylin. In ods of producing self-emulsifying dosage forms are known Solutions comprising saccharide siloxane copolymers, the in the art and include, but are not limited to, for example, saccharide siloxane copolymers form the coating after U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563, each of evaporation of the solvent initiates the cross-linking of the which is specifically incorporated by reference. 10 Gels saccharide siloxane copolymers. The paints contemplated In some embodiments, the compositions (e.g., HC-HA, for use herein, are flexible such that they do not interfere fetal Support tissue extracts, fetal Support powders and with the growth or movement of a bone or joint. In some homogenates) disclosed herein are formulated as a gel. In embodiments, the paints are applied as a liquid (i.e. solution, Some embodiments, the compositions (e.g., HC-HA, fetal 15 Suspension, or emulsion), a semisolid (i.e. a gel, foam, paste, Support tissue extracts, fetal Support powders and homoge or jelly) or an aerosol. nates) disclosed herein are formulated as solvent release Foams gels. In some embodiments, the compositions (e.g., HC-HA, In some embodiments, the compositions (e.g., HC-HA, fetal Support tissue extracts, fetal Support powders and fetal Support tissue extracts, fetal Support powders and homogenates) disclosed herein are formulated as actinic homogenates) disclosed herein is formulated as a foam. radiation curable gels. In some embodiments, the composi Examples of suitable foamable carriers for use in the com tions (e.g., HC-HA, fetal Support tissue extracts, fetal Sup positions e.g., HC-HA, fetal Support tissue extracts, fetal port powders and homogenates) disclosed herein are formu Support powders and homogenates) disclosed herein lated as thermoreversible gels. In other embodiments, the include, but are not limited to, alginate and derivatives compositions (e.g., HC-HA, fetal Support tissue extracts, 25 thereof, carboxymethylcellulose and derivatives thereof, fetal Support powders and homogenates) disclosed herein collagen, polysaccharides, including, for example, dextran, are formulated hydrogels. dextran derivatives, pectin, starch, modified starches Such as As used herein, 'gels'. Sometimes referred to as jellies, starches having additional carboxyl and/or carboxamide are semisolid systems consisting of either Suspensions made groups and/or having hydrophilic side-chains, cellulose and up of Small inorganic particles or large organic molecules 30 interpenetrated by a liquid. Gels include a single-phase or a derivatives thereof, agar and derivatives thereof. Such as two-phase system. A single-phase gel consists of organic agar stabilized with polyacrylamide, polyethylene oxides, macromolecules distributed uniformly throughout a liquid in glycol methacrylates, gelatin, gums such as Xanthum, guar, Such a manner that no apparent boundaries exist between the karaya, gellan, arabic, tragacanth and locust bean gum, salts dispersed macromolecules and the liquid. Some single 35 thereof (e.g., Sodium alginate), or combinations thereof. phase gels are prepared from synthetic macromolecules Liposomes (e.g., carbomer) or from natural gums, (e.g., tragacanth). In In some embodiments, the compositions (e.g., HC-HA, Some embodiments, single-phase gels are generally aque fetal Support tissue extracts, fetal Support powders and ous, but will also be made using alcohols and oils. Two homogenates) disclosed herein are formulated as liposomes phase gels consist of a network of Small discrete particles. 40 or lipid particles. Phospholipids that are gently dispersed in Gels are hydrophobic or hydrophilic. The base of a an aqueous medium form multilayer vesicles with areas of hydrophobic gel may be liquid paraffin with polyethylene or entrapped aqueous media separating the lipid layers. Soni fatty oils gelled with colloidal silica, or aluminum or Zinc cation, or turbulent agitation, of these multilayer vesicles Soaps. The base of hydrophobic gels usually consists of results in the formation of single layer vesicles, commonly water, glycerol, or propylene glycol gelled with a suitable 45 referred to as liposomes. Suitable phospholipids for use in gelling agent (e.g., tragacanth, starch, cellulose derivatives, the formation of liposomes, include for example, phospha carboxyvinylpolymers, and magnesium-aluminum sili tidylcholines, ethanolamines and serines, sphingomyelins, cates). cardiolipins, plasmalogens, phosphatidic acids and cerebro Polymers composed of polyoxypropylene and polyoxy sides. Preferred phospholipids are, for example, phosphati ethylene form thermoreversible gels when incorporated into 50 dylcholine, phosphatidyl ethanolmine, phosphatidyl serine, aqueous Solutions. These polymers have the ability to phosphatidyl inositol, lysophosphatidylcholine, phosphati change from the liquid state to the gel State attemperatures dylglycerol. close to body temperature. In some embodiments, a liposome formulation may fur Paints ther comprise a lipophilic additive. Examples of such addi In some embodiments, the compositions (e.g., HC-HA, 55 tives include by way of example only, Stearylamine, phos fetal Support tissue extracts, fetal Support powders and phatidic acid, tocopherol, cholesterol, cholesterol homogenates) disclosed herein are formulated as paints. As hemisuccinate and lanolin extracts. used herein, a “paint', also known as film formers, means Creams and Lotions Solutions comprised of a solvent, a monomer or polymer, an In some embodiments, the compositions (e.g., HC-HA, tissue product or extract thereof, and optionally one or more 60 fetal Support tissue extracts, fetal Support powders and pharmaceutically-acceptable excipients. After application to homogenates) disclosed herein are formulated as creams. In a target bone, the solvent evaporates leaving behind a thin certain instances, creams are semisolid (e.g., soft Solid or coating comprised of the monomers or polymers, and the thick liquid) formulations that include a fetal Support tissue active agent. The coating protects active agents and main product described herein (e.g., a flat tissue product sheet, a tains them in an immobilized state at the site of application. 65 pulverized tissue product, or a homogenized tissue product) This decreases the amount of active agent that may be lost dispersed in an oil-in-water emulsion or a water-in-oil and correspondingly increases the amount delivered to the emulsion. US 9,675,733 B2 73 74 Ointments Miscellaneous In some embodiments, the compositions (e.g., HC-HA, In some embodiments, the pharmaceutical composition fetal Support tissue extracts, fetal Support powders and described herein comprise microencapsulated tissue prod homogenates) disclosed herein are formulated as ointments. ucts and/or extracts thereof (e.g., HC-HA). In some embodi In certain instances, ointments are semisolid preparations ments, one or more other compatible materials are present in that soften or melt at body temperature. the microencapsulation material. Exemplary materials Pastes include, but are not limited to, pH modifiers, erosion facili In some embodiments, the compositions (e.g., HC-HA, tators, anti-foaming agents, antioxidants, flavoring agents, fetal Support tissue extracts, fetal Support powders and and carrier materials such as binders, Suspending agents, homogenates) disclosed herein are formulated as pastes. In 10 disintegration agents, filling agents, Surfactants, solubilizers, certain instances, pastes contain at least 20% solids. In stabilizers, lubricants, wetting agents, and diluents. certain instances, pastes are ointments that do not flow at Exemplary microencapsulation materials useful for delay body temperature. ing the release of the formulations including compounds Patches described herein, include, but are not limited to, hydroxy In some embodiments, the compositions (e.g., HC-HA, 15 propyl cellulose ethers (HPC) such as Klucel(R) or Nisso fetal Support tissue extracts, fetal Support powders and HPC, low-substituted hydroxypropyl cellulose ethers homogenates) disclosed herein are administered via a patch. (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) In some embodiments, the compositions described herein such as Seppifilm-LC, Pharmacoat(R), Metolose SR, Metho are dissolved and/or dispersed in a polymer or an adhesive. cel(R)-E, Opadry YS, PrimaFlo, Benecel MP824, and Bene In some embodiments, a film, a patch disclosed herein is cel MP843, methylcellulose polymers such as Methocel(R)- constructed for continuous, pulsatile, or on demand delivery A, hydroxypropylmethylcellulose acetate Stearate Aqoat of a composition described herein. (HF-LS, HF-LG, HF-MS) and MetoloseR), Ethylcelluloses Wound Dressings (EC) and mixtures thereof such as E461, Ethocel(R), Aqua In some embodiments, the compositions (e.g., HC-HA, lon R-EC, Surelease.R., Polyvinyl alcohol (PVA) such as fetal Support tissue extracts, fetal Support powders and 25 Opadry AMB, hydroxyethylcelluloses such as Natrosol(R), homogenates) disclosed herein are administered via a wound carboxymethylcelluloses and salts of carboxymethylcellu dressing. Wound dressings include, but are not limited to loses (CMC) such as Aqualon(R)-CMC, polyvinyl alcohol gauzes, transparent film dressings, hydrogels, polyurethane and polyethylene glycol co-polymers such as Kollicoat IRR, foam dressings, hydrocolloids and alginates. monoglycerides (Myverol), triglycerides (KLX), polyethyl Implants/Prosthesis 30 ene glycols, modified food starch, acrylic polymers and In some embodiments, the compositions (e.g., HC-HA, mixtures of acrylic polymers with cellulose ethers such as fetal support tissue extracts, fetal support powders and EudragitR) EPO, Eudragit R. L30D-55, Eudragit R FS 30D homogenates) disclosed herein are administered via Eudragit R L100-55, EudragitR) L100, EudragitR) S100, implants and prostheses. In some embodiments, the com Eudragit R. RD100, EudragitR) E100, Eudragit R L12.5, positions (e.g., HC-HA, fetal Support tissue extracts, fetal 35 Eudragit R S12.5, Eudragit R NE30D, and Eudragit R NE Support powders and homogenates) are administered by 40D, cellulose acetate phthalate, sepifilms such as mixtures bone implants or bone stents. of HPMC and stearic acid, cyclodextrins, and mixtures of In some embodiments, the compositions (e.g., HC-HA, these materials. fetal Support tissue extracts, fetal Support powders and In still other embodiments, plasticizers such as polyeth homogenates) disclosed herein are administered via a bone 40 ylene glycols, e.g., PEG 300, PEG 400, PEG 600, PEG implant. In some embodiments, the bone implant is an 1450, PEG 3350, and PEG 800, stearic acid, propylene osseointegrated implant. As used herein, an "osseointegrated glycol, oleic acid, and triacetin are incorporated into the implant’ means a three dimensional implant containing microencapsulation material. In other embodiments, the pores into which osteoblasts and Supporting connective microencapsulating material useful for delaying the release tissue can migrate. In some embodiments, the bone implant 45 of the pharmaceutical compositions is from the USP or the comprises a composition described herein. In some embodi National Formulary (NF). In yet other embodiments, the ments, the bone implant is a dental implant. In some microencapsulation material is Klucel. In still other embodi embodiments, the bone implant is used for knee or joint ments, the microencapsulation material is methocel. replacement. In some embodiments, the bone implant is a In some embodiments, the fetal Support tissue products or craniofacial prosthesis (e.g., an artificial ear, orbital pros 50 extracts thereof (e.g., HC-HA) are incorporated within nano thesis, nose prosthesis). particles, microparticles, or microspheres. In some embodiments, the compositions (e.g., HC-HA, Microspheres usually have a spherical shape, although fetal Support tissue extracts, fetal Support powders and irregularly-shaped microparticles are possible. Micro homogenates) disclosed herein are administered via a bone spheres may vary in size, ranging from Submicron to 1000 stent. In some embodiments, the compositions (e.g., HC 55 micron diameters. The fetal Support tissue products and HA, fetal Support tissue extracts, fetal Support powders and extracts thereof are encapsulated in microspheres by any homogenates) disclosed herein are coated onto the outside of Suitable methods. Generally, the agent is dispersed or emul the stent. In some embodiments, the compositions (e.g., sified, using stirrers, agitators, or other dynamic mixing HC-HA, fetal support tissue extracts, fetal support powders techniques, in a solvent containing a wall-forming material. and homogenates) disclosed herein elute from the stent into 60 Solvent is then removed from the microspheres, and there the Surrounding bone. In some embodiments, the bone stents after the microsphere product is obtained. are inserted into the intramedullary canal of a bone. In some Nanoparticles are also contemplated for use with the fetal embodiments, the bone stent is placed in the sinus tarsi. In Support tissue products and extracts described herein. Nano Some embodiments, the bone stent in placed in a knee or particles are material structures of about 100 nm or less in joint. In some embodiments, the bone stent is placed in a 65 size. One use of nanoparticles in pharmaceutical composi bone fracture. In some embodiments, the bone stent is tions is the formation of Suspensions as the interaction of the expandable or contractable. particle Surface with solvent is strong enough to overcome US 9,675,733 B2 75 76 differences in density. Nanoparticle Suspensions are steril administering to an individual in need thereof a therapeuti ized as the nanoparticles are small enough to be subjected to cally-effective amount of a composition comprising a fetal sterilizing filtration (see, e.g., U.S. Pat. No. 6,139,870, Support tissue product or an extract thereof. Disclosed herein incorporated by reference for such disclosure). Nano herein, in certain embodiments, are methods of treating a particles comprise at least one hydrophobic, water-insoluble bone tumor, the methods comprising administering to an and water-indispersible polymer or copolymer emulsified in individual in need thereofatherapeutically-effective amount a solution or aqueous dispersion of Surfactants, phospholip of a composition comprising a fetal Support tissue product or ids or fatty acids. The nanoparticles may be obtained by any an extract thereof. In some embodiments of any of the suitable methods. These methods include vaporization meth aforementioned methods, the methods further comprise ods, such as free jet expansion, laser vaporization, Spark 10 administering a further therapeutic agent. erosion, electro explosion and chemical vapor deposition; In some embodiments, the pharmaceutical compositions physical methods involving mechanical attrition (e.g., disclosed herein result from the mixing or combining of "pearlmilling technology, Elan NanoSystems), Super criti more than one active ingredient and includes both fixed and cal CO2 and interfacial deposition following solvent dis non-fixed combinations of the active ingredients. The term placement. In one embodiment, the solvent displacement 15 “fixed combination” means that the active ingredients, e.g. method is used. The size of nanoparticles produced by this a compound described herein and a co-agent, are both method is sensitive to the concentration of polymer in the administered to a patient simultaneously in the form of a organic solvent; the rate of mixing; and to the Surfactant single entity or dosage. The term “non-fixed combination' employed in the process. Continuous flow mixers provide means that the active ingredients, e.g. a compound described the necessary turbulence to ensure Small particle size. One herein and a co-agent, are administered to a patient as type of continuous flow mixing device that is optionally separate entities either simultaneously, concurrently or used to prepare nanoparticles has been described (Hansen et sequentially with no specific intervening time limits, al J. Phys Chem 92, 2189-96, 1988). In other embodiments, wherein such administration provides effective levels of the ultrasonic devices, flow through homogenizers or Supercriti two compounds in the body of the patient. The latter also cal CO2 devices may be used to prepare nanoparticles. 25 applies to cocktail therapy, e.g. the administration of three or Combination Therapies more active ingredients. Disclosed herein, in certain embodiments, are methods of In some embodiments, the further therapeutic agent is a inhibiting osteoclast differentiation in an individual in need calcium Supplement, a calcium salt, or a combination thereof, comprising administering to the individual a com thereof. In some embodiments, the further therapeutic agent position comprising a fetal Support tissue product or an 30 is calcium carbonate, coral calcium, calcium citrate, calcium extract thereof. Disclosed herein, in certain embodiments, phosphate, calcium lactate, a calcium chelate (e.g., calcium are methods of inhibiting bone resorption in an individual in malate, calcium aspartate, calcium fumarate), or any com need thereof, comprising administering to the individual a binations thereof. composition comprising a fetal Support tissue product or an In some embodiments, the further therapeutic agent is a extract thereof. Disclosed herein, in certain embodiments, 35 NSAID. In some embodiments, the further therapeutic agent are methods of inhibiting bone remodeling in an individual is a salicylate, a propionic acid derivative, an acetic acid in need thereof, comprising administering to the individual derivative, and enolic acid derivative, a fenamic acid deriva a composition comprising a fetal Support tissue product or tive, a selective COX-2 inhibitor, a sulphonanilide, or any an extract thereof. Disclosed herein, in certain embodiments, combinations thereof. In some embodiments, the further are methods of balancing bone resorption and bone forma 40 agent is aspirin, diflunisal, Salsalate, ibuprofen, naproxen, tion, comprising administering to an individual in need fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, thereof a therapeutically-effective amount of a composition oxaprozin, loxoprofen, indomethacin, Sulindac, etodolac, comprising a fetal Support tissue product or an extract ketorolac, diclofenac, nabumetone, piroXicam, meloxicam, thereof. Disclosed herein, in certain embodiments, are meth tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic ods of treating a disease, disorder, or condition characterized 45 acid, meclofenamic acid, flufenamic acid, tolfenamic acid, by excessive or undesired osteoclast differentiation, the celecoxib, rofecoxib, Valdecoxib, parecoxib, lumiracoxib, methods comprising administering to an individual in need etoricoxib, firocoxibm, nimeSulide, licofelone, or any com thereof a therapeutically-effective amount of a composition binations thereof. comprising a fetal Support tissue product or an extract In some embodiments, the further therapeutic agent is a thereof. Disclosed herein, in certain embodiments, are meth 50 corticosteroid. In some embodiments, the further therapeutic ods of treating arthritis, the methods comprising adminis agent is a hydrocortisone type corticosteroid, an acetonide, tering to an individual in need thereof a therapeutically a betamethasone type corticosteroid, a corticosteroid ester effective amount of a composition comprising a fetal Support (e.g., a halogenated corticosteroid or a labile prodrug ester). tissue product or an extract thereof. In some embodiments, In some embodiments, the further therapeutic agent is the arthritis is osteoarthritis, rheumatoid arthritis, psoriatic 55 hydrocortisone, hydrocortisone acetate, cortisone acetate, arthritis, or any combination thereof. Disclosed herein, in tiXocortol pivalate, prednisolone, methylprednisolone, pred certain embodiments, are methods of treating osteoporosis, nisone, triamcinolone acetonide, triamcinolone alcohol, the methods comprising administering to an individual in mometasone, amcinonide, budesonide, desonide, fluocino need thereof a therapeutically-effective amount of a com nide, fluocinolone acetonide, halcinonide, betamethasone, position comprising a fetal Support tissue product or an 60 betamethasone sodium phosphate, dexamethasone, dexam extract thereof. Disclosed herein, in certain embodiments, ethasone sodium phosphate, fluocortolone, hydrocortisone are methods of treating alveolar bone degradation, the 17-valerate, aclometasone dipropionate, betamethasone val methods comprising administering to an individual in need erate, betamethasone dipropionate, prednicarbate, thereof a therapeutically-effective amount of a composition clobetasone-17-butyrate, clobetasol-17-propionate, fluocor comprising a fetal Support tissue product or an extract 65 tolone caproate, fluocortolone pivalate, and fluprednidene thereof. Disclosed herein, in certain embodiments, are meth acetate, hydrocortisone-17-butyrate, 17-aceponate, 17-bute ods of treating Paget’s disease, the methods comprising prate, Prednicarbate, or any combinations thereof. US 9,675,733 B2 77 78 In some embodiments, the further therapeutic agent is a dissociation buffer, and seeded at 169 cells/mm with hyaluronan or Sodium hyaluronate. C-MEM/10% FBS differentiation media without phenol red. In some embodiments, the further therapeutic agent is a After 24 h, seeded macrophage RAW 264.7 cells were DMARD (disease-modifying antirheumatic drug). In some treated with 50 ng/ml RANKL (except Neg CTL) and embodiments, the further therapeutic agent is adalimumab, 5 simultaneously treated with either PBS1x (Pos CTL), AME azathioprine, chloroquine, hydroxychloroquine, (200 lug/ml Protein), HC-HA (25 ug/ml HA), or on either cyclosporine, D-penicillamine, etanerecpt, golimumab, side (epithelium, stroma or basement membrane) of intact infliximab, leeflunomide, methotrexatem minocycline, AM (iAM), denuded AM (dAM), and lyophilized AM rituximab, Sulfasalazine, Sodium aurothiomalate, auranofin, (1AM). Cells were observed for cell death and formation of or any combinations thereof. 10 multinucleated cell (osteoclast) for a period of 5-7 days. In some embodiments, the further therapeutic agent is a TRAP Assay bisphosphonate. In some embodiments, the further thera After termination, cells were fixed with acetone and peutic agent is a N-containing bisphosphonate. In some stained with tartrate resistant acid phosphatase leukocyte kit embodiments, the further therapeutic agent is a non-N- (TRAP). Cell lysate was also collected withl%TritonX-100 containing bisphosphonate. In some embodiments, the fur- 15 extraction buffer for analysis with TRAP Colorimetric ther therapeutic agent is etidronate, clodronate, tiludronate, Assay. pamidronate, neridronate, olpadronate, alendronate, iban Formation of large multinucleated osteoclast cells was dronate, risedronate, Zoledronate, or any combination observed for the Pos CTL (FIG. 2 E-G) but not Neg CTL thereof. (FIG. 2 A-C). TRAP staining (FIG. 3) revealed no large In some embodiments, the further therapeutic agent is an 20 multinucleated osteoclast cells when RAW264.7 cells were estrogen analog. In some embodiments, the further thera cultured on the epithelial side of intact amniotic membrane peutic agent is estrodiol, phytoestrogen, diethylstilbestrol, or (iAM), the stromal side of iAM (F), the basement membrane any combinations thereof. side of epithelially-denuded AM (dAM) (G), the stromal In some embodiments, the further therapeutic agent is a side of dAM (H), the epithelial side of lyophilized amniotic selective estrogen receptor modulator (SERM). In some 25 membrane IAM (I), or the stromal side of lyophilized embodiments, the further therapeutic agent is raloxifene, amniotic membrane IAM (J). clomifene, femarelle, ormeloxifene, tamoxifen, toremifene, Following TRAP Colorimeteric analysis, the Neg CTL lasofoxifene, or any combinations thereof. showed significantly lower (p=2.9E-12) TRAP reading com In some embodiments, the further therapeutic agent is a pared to the Pos CTL. Inhibitory action for osteoclast member of the calcitonin-like protein family. In some 30 formation was seen for all AM derivatives (FIG. 4) with or embodiments, the further therapeutic agent is Calcitonin. without gamma irradiation. In some embodiments, the further therapeutic agent is qPCR parathyroid hormone. In some embodiments, the further Total RNAs were also collected for quantitative measure therapeutic agent is a recombinant parathyroid hormone. In ment of RANK, B3-Integrin, and NFATcl transcripts. some embodiments, the further therapeutic agent is Teri- 35 Quantitative RT-PCR revealed that levels of NFATcl paratide. mRNA were significantly downregulated (p<0.05) in In some embodiments, the further therapeutic agent is a response to all AM derivatives (FIG. 5). Expression of RANKL inhibitor. In some embodiments, the further thera RANK was found to be significantly downregulated for both peutic agent is denosumab. HC-HA and stromal side of dAM (FIG. 7). Expression of In some embodiments, the further therapeutic agent is a 40 B3-integrin (FIG. 8), which is required for normal osteoclast chemotherapeutic. Any Suitable chemotherapeutic agent is differentiation, was found to be significantly downregulated contemplated for use with the fetal support tissue product by HC-HA, AML, AMP, and stromal side of dAM. Sup compositions disclosed herein. Suitable chemotherapeutic pression off33-integrin by AME is statistically significant on agents include those that are efficacious in the treatment of Day 5 (p=0.000001). osteosarcoma, Ewing's sarcoma, chondrosarcoma, and 45 fibrosarcoma. In some embodiments, the further therapeutic Example 2: AME, AML, and AMP Inhibit agent is ifosfamide, etopside, carboplatin, cisplatin, cyclo Osteoclast Formation from RANKL Stimulated phosphamide, doxorubicin, epirubicin, methotrexate, PEG RAW 264.7 Macrophage Cells interferon alfa-2b, or any combinations thereof. In some embodiments, the further therapeutic agent is 50 Murine RAW 264.7 macrophage cells were seeded at a radiation therapy, proton therapy, or a combination thereof. density of 4.0x10 cells/96 well (30-40% confluent), cul In some embodiments, the further therapeutic agent is tured in C-MEM media without Phenol Red and supple sodium fluoride. mented with 10% FBS, 100 lug/ml penicillin & streptomy In some embodiments, the further therapeutic agent is cin. 24 hours after seeding, cells were treated with or without Strontium ranelate. 55 50 ng/ml RANKL stimulation. Experimental groups were In some embodiments, the further therapeutic agent is simultaneously treated with AMP, AML or AME with pro Metastron (i.e., strontium-89 chloride). tein concentration of 200 lug/ml. On Day 5, the culture was terminated and analyzed by TRAP staining and TRAP EXAMPLES Colorimetric Assay. 60 The result from TRAP staining (FIG. 2) shows that Example 1: AM Tissue and its Derivatives Prevent osteoclasts (multi-nucleated cells) were not found on the Differentiation of Osteoclasts in Response to Neg CTL while large multi-nucleated cells were found on RANKL the Pos CTL. AME inhibited osteoclast formation but did not inhibit RAW macrophage cell proliferation (FIG. 2a). Cell Culture and Treatment 65 Osteoclast formation and RAW macrophage cell prolifera Macrophage RAW264.7 cells were cultivated in DMEM/ tion were also inhibited by AML-1 and AML-2 from 2 10% FBS until 80% confluence, harvested with enzyme-free different donors (FIG. 11), but such inhibition was not US 9,675,733 B2 79 80 complete because Small multi-nucleated cells could be seen UCAP with protein concentration of 100 ug/ml. On Day 5, after TRAP staining. Osteoclast formation and RAW mac the culture was terminated and analyzed by TRAP Colori rophage cell proliferation were also inhibited by AMP from metric Assay. 5 different donors (FIG. 11) at the same protein concentra Osteoclast formation was inhibited by all powder derived tion of 200 ug/ml as AML. from amniotic membrane (AMP), chorion (CHP), amnion The result from TRAP staining shows that the inhibitory chorion (ACP), placenta (PCP), whole umbilical cord action for osteoclast formation was seen on all AM deriva (UCP), and umbilical cord AM (UCAP) (FIG. 12). tives. There was no significant difference between the TRAP Colorimetric Assay reading between AMP and AME (Table Example 4: HC-HA Inhibits the Formation of 1). For two donors, the TRAP ELISA reading for AMP is 10 Osteoclasts from RANKL. Stimulated RAW 264.7 significantly lower than AML (Table 1) from the same donor Macrophage Cells (p=0.04 and p=0.02) at the same protein concentration 200 ug protein/ml (FIG. 11). Murine RAW 264.7 macrophage cells were seeded at a 15 density of 4.0x10 cells/96 well (30-40% confluent), cul TABLE 1. tured in C-MEM media without Phenol Red and supple mented with 10% FBS, 100 lug/ml penicillin & streptomy p-values cin. 24 hours after seeding, cells were treated with or without Comparison p = value 50 ng/ml RANKL stimulation. Experimental groups were simultaneously treated with AMP 1-5 AME O.24 AMP 1-2 AML 1-2 1.02E-OS high molecular weight HA or HC-HA prepared at a series of HA concentrations (0, 0.008, 0.04, 0.2, 1, 5, and 25ug/ml). On Day 5, the culture was terminated and analyzed by TRAP staining and TRAP ELISA. TABLE 2 25 The result from TRAP staining shows that osteoclasts OD readings and p-values (multi-nucleated cells) are not found on the negative control while large multi-nucleated cells are found on the positive OD+ standard p-value (compared control (FIG. 9A). Conditions deviation to Pos CTL) Even at the highest concentration (25 g/ml), HAdoes not Neg CTL O.O37 O.OO3S O.OO13 30 inhibits the formation of multinucleated cells and the expres Pos CTL O.53 - 0.11 sion of TRAP. In contrast, the inhibition by HC-HA is AME O.O73 O.O15 O.OO28 HC.HA (25 g/ml HA) O.O23 O.OO81 O.OO23 detected as low as at 0.08 ug/ml, and dose-dependently AML-1 O.18 O.OO97 O.OO67 increased until the multinucleated cells are completely AMP-1 O.046 O.O18 O.OO22 eliminated at 25 g/ml (FIG. 9B). AML-2 O.19 O.OS2 O.OO34 35 These observations are also confirmed by quantitatively AMP-2 O.093 O.OOSS O.OO36 measured TRAP activity (FIGS. 9C and 9D). From the Mean AMP 1-5 O.087 - O.O41 O.OO17 TRAP Colorimetric assay, the IC50 of HC-HA's inhibition on the osteoclast formation induced by RANKL is 0.1 lug/ml (FIG.9E). TABLE 3 40 Example 5: AMP Promotes the Mineralization of Protein Content of AMP from 5 different donors Osteoblasts BCA Protein(Ig). Total Total Protein Powder Powder Powder Protein(mg) Osteoblast precursor MC3T3-E1 cells were maintained in Reading usediml Weight Weight from 1 45 DMEM/10% FBS but were re-suspended into C-MEM/10% (ig/ml) (mg/ml) (mg) (mg) whole AM FBS and seeded at 1x10/ml on 24 well plastic (2 ml per AMP-1 13933 44.6 312 1340 419 well, and designated as Day 1 from here onwards) for 2 AMP-2 8777 30 293 880 257 days. AMP-3 14527 42 346 12SO 432 AMP-4 16516 49 337 1836 619 The culture medium was replaced with either PBS (Neg. 50 Ctrl), osteoblast-inducing reagents (0.2 mMascorbic acid AMP-5 22,727 51.2 444 1408 625 2-phosphate and 10 mM glycerol 2-phosphate, Pos. Ctrl), or osteoblast-inducing reagents plus 0.1 g/ml HC-HA or 125 ug/ml AMP. The cell culture medium was changed every 3 Example 3: Powder Derived from Amniotic Mem days until on Day 18. From Day 8 onwards, the osteoblast brane (AMP), Chorion (CHP), Amnion-Chorion 55 inducing reagents was additionally supplemented with mela (ACP), Placenta (PCP), Whole Umbilical Cord tonin (50 ng/ml). (UCP), or Umbilical Cord AM (UCAP) Inhibits At Day 18, cells were assayed with alizarin red staining Osteoclast Formation from RANKL Stimulated (ARS) to measure the mineralization of differentiated osteo RAW 264.7 Macrophage Cells blasts. Negative control was not stained by ARS but the 60 positive control was stained in red (FIG. 13). Murine RAW 264.7 macrophage cells were seeded at a Treatment with HC-HA (0.1 g/ml) has a similar staining density of 4.0x10 cells/96 well (30-40% confluent), cul as that of the positive control (FIG. 13). However, treatment tured in C-MEM media without Phenol Red and supple with AMP (125 ug/ml) yields much darker staining (AMP), mented with 10% FBS, 100 lug/ml penicillin & streptomy indicating that more minerals are generated (FIG. 14). cin. 24 hours after seeding, cells were treated with or without 65 The differences were also observed after ARS stained 50 ng/ml RANKL stimulation. Experimental groups were cells were removed by 4 M GnCl, showing less cells were simultaneously treated with AMP. CHP, ACP, PLP, UCP and left in AMP treated wells (FIG. 14). Some cells migrate from US 9,675,733 B2 81 82 the monolayer into the AMP particles and use it as a scaffold The orthopedic prosthesis is Surgically placed to replace for differentiation and mineralization. the individual’s damaged knee. Quantitative measurement of ARS (FIG. 14) shows HC While preferred embodiments of the present invention HA (0.1 ug/ml) does not significantly increase or decrease have been shown and described herein, it will be obvious to the mineralization when compared to the positive control 5 those skilled in the art that such embodiments are provided (p>0.05). In contrast, cells treated with AMP (125 ug/ml) by way of example only. Numerous variations, changes, and significantly promotes the mineralization compared to either substitutions will now occur to those skilled in the art the positive control or HC-HA (p=0.0001). The color without departing from the invention. It should be under changes related to the concentrations of alizarin red or in stood that various alternatives to the embodiments of the samples are shown in FIG. 14. 10 invention described herein may be employed in practicing Example 6: Use for the Treatment of an Arthritic the invention. It is intended that the following claims define Joint the scope of the invention and that methods and structures within the scope of these claims and their equivalents be An individual with rheumatoid arthritis is identified. A covered thereby. pharmaceutical composition for injection comprising HC 15 What is claimed is: HA is prepared. 1. A method of promoting or inducing osteogenesis in an The composition is injected at the arthritic joints of the individual in need thereof, comprising contacting a bone or individual. joint in the individual with a therapeutically-effective amount of a fetal Support tissue that comprises: (a) cells, Example 7: Use in the Treatment of Osteolysis 2O substantially all of which are dead, and (b) HC-HA; thereby promoting or inducing osteogenesis in the individual. An individual with an area of decalcified bone is identi 2. The method of claim 1, wherein the fetal support tissue fied. A wound dressing comprising placental powder is is placental amniotic membrane (PAM), umbilical cord prepared. amniotic membrane (UCAM), placenta, umbilical cord, The tubular tissue graft is surgically placed around the 25 chorion, amnion-chorion, or combinations thereof. area of decalcified bone. 3. The method of claim 1, wherein the fetal support tissue is frozen or previously frozen. Example 8: Use of an Implant in the Treatment of 4. The method of claim 1, wherein the fetal support tissue Osteolysis is a sheet, a powder, or a homogenate. 30 5. The method of claim 1, wherein the fetal support tissue An individual with an area of decalcified bone is identi is cryopreserved, lyophilized, terminally sterilized, or a fied. An implant coated in HC-HA is prepared. combination thereof. The implant is Surgically placed near the area of decal 6. The method of claim 1, wherein contacting the joint cified bone. with the fetal Support tissue comprises injecting the fetal 35 Support tissue into the joint. Example 9: Use of an Patch in the Treatment of 7. The method of claim 1, wherein contacting the bone or Alveolar Bone Degradation joint with the fetal Support tissue comprises wrapping a fetal An individual with alveolar bone degradation is identi Support tissue sheet around the bone or joint. fied. A sheet of substantially-flat amnion-chorion patch is 40 8. The method of claim 1, wherein the individual in need prepared. thereof has arthritis, osteoporosis, a bone tumor, Paget’s Disease, alveolar bone degradation, or any combination The patch is placed on an alveolar bone at the site of thereof. alveolar bone degradation. 9. The method of claim 1, wherein contacting the bone or joint with the fetal Support tissue comprises contacting the Example 10: Use of an Orthopaedic Prosthesis in 45 bone or joint with a medical device comprising the fetal the Treatment of Osteoarthritis Support tissue. An individual with a damaged knee due to osteoarthritis 10. The method of claim 9, wherein the medical device is is identified. An orthopedic prosthesis comprising chorion a patch, implant, orthopedic prosthesis, or bone stent. homogenate derivative is prepared. k k k k k