Send Orders for Reprints to [email protected] 52 Recent Patents on Regenerative Medicine 2014, 4, 52-68 Recent Patents in Regeneration

Luminita Labusca1,5,*, Udo Greiser2, Viorel Nacu3, Florin Zugun-Eloae4 and Kaveh Mashayekhi5,6,7

1Department of Orthopedic Surgery, University Hospital Saint, Spiridon Iasi, 1st Independentei Boulevard Iasi, Ro- mania; 2Network of Excellence for Functional Biomaterials NFB, National University of Ireland, Galway, IDA Business Park, Dangan, Galway, Ireland; 3Laboratory of and Cell Culture, State Medical and Pharmaceuti- cal University “Nicolae Testemitanu”, Republic of Moldova Bd. Stefan Cel Mare 165, MD2004, Chisinau, Republic of Moldova; 4Department of Molecular Genetics and Immunology, “Gr.T.Popa: University of Medicine and Pharmacy Iasi, Romania, Universitatea de Medicina si Farmacie "Gr.T.Popa" Iasi Str. Universitatii Nr.16 700115 Iasi, Romania; 5SBIM Systems Bioinformatics and Modeling SBIM, GmbH, Basaltstr. 39, 60487 Frankfurt, Germany; 6BioTalentum, Godollo, Hungary Bio Talentum Ltd. Aulich Lajos Str. 26., 2100 Gödöll, Hungary; 7Faculty of Veterinary Medicine, Theoretical Epidemiology, Utrecht University, Theoretical Epidemiology Yalelaan 104, Room 2.052 3584 CM, Utrecht, Netherlands

Received: October 31, 2013; Accepted: January 1, 2014; Revised: January 3, 2014 Abstract: The limited capability of articular cartilage to heal after injury and the worldwide increase in the burden of diseases related to , are prompting numerous efforts in developing novel treatments. Methods of grafting, promoting tissue repair or regenerative medicine strategies are in different stages of investigation. However, in the last decades, inadequate few novel therapies have made their way to the clinic gaining limited acceptance. Some of the reasons of the relative lack of progress are the insufficient quality and stability of the repair tissue and the lack of serious evidence based medicine information to support the use and reimbursement of new procedures. This paper introduces the most recent patents which relate to cartilage regeneration. Grafting procedures and their conceptual development, regenera- tive medicine strategies as well as drug, physical and food supplements based methods are listed. Main issues encountered with every intervention type and the way new methods are addressing them, are also discussed. Keywords: Cartilage, defects, osteoarthritis, patents, regeneration, therapy.

I. INTRODUCTION II. BACKGROUND Articular cartilage has very limited capability to heal. II.1. Cartilage Traumatic and Degenerative Lesions Cartilage disruption results in progressive loss of joint mo- tion, pain and disability, decreasing the quality of life of the Traumatic lesions of the articular cartilage are disconti- nuities produced by acute disruption, or by repeated micro affected person. Globally, the number and severity of cases trauma. The subliminal damage results in superficial fibrilla- are on ascending trend, increasing the burden of disease and tion with cumulative effect which undermines the structural taking a high toll on healthcare resources [1]. Articular carti- and functional properties of the joint surface. lage covers surfaces within a joint, functioning as a shock absorber and facilitates motion. Its structure is adapted Two categories of posttraumatic cartilage lesions are to functioning under high compression, tensile and shear described. Superficial and chondral lesions are disruptions of . This ultra specialized tissue is aneural, avascular and joint surface, which do not penetrate the subchondral bone. alymphatic with low cellularity, high extracellular matrix With in vascularized structures, wound healing occurs through (ECM) content and a stratified cell-fiber distribution. The stages of necrosis, inflammation and repair. Chondral and nourishment of a relatively low cell population is provided superficial lesions do not disrupt the blood vessels. This type by nutrient diffusion from subchondral bone vessels and of injury induces tissue necrosis; however, the inflammatory synovial fluid. The same structural particularities, which are phase does not occur [3]. Consequently, the recruitment of the basis of the remarkable biomechanical endurance, are progenitor cells and the initiation of healing processes are considered to be the principal cause of the incapacity to heal absent [4]. have limited replicative potential after trauma and degeneration [2]. The purpose of this paper [5], therefore the repair of the defect can only be produced is to provide an overview of the latest developments of the by means of a so-called “extrinsic mechanism”, which mobi- increasing patent literature addressing the topic of cartilage lizes the periarticular-periosteal or intraarticular, synovial, regeneration. progenitors [6]. In the case of osteochondral lesions, the de- fect penetrates the subchondral bone. The local disruption of

the vascular network triggers the wound healing cascade. *Address correspondence to this author at the Department of Orthopedic Surgery, University Hospital, Saint Spiridon Iasi, 1st Independentei Boule- Nevertheless, the repair results in formation of a fibrous tis- vard Iasi, Romania; Tel: +40749162219; Fax: +40232272191; sue, unable to reproduce the complex biochemical and ultra- E-mail: [email protected] structural characteristics of the . Fibro carti-

2210-2973/14 $100.00+.00 © 2014 Bentham Science Publishers Recent Patents in Cartilage Regeneration Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 53 lage contains mainly type 1 fibers, has a low degree morphological deterioration followed by the generalized of collagen polymerization, as a result the newly formed joint failure, feature of advanced OA stages [19]. tissue has decreased endurance to compression and shear. Regenerative medicine (RM) is introducing the concept The mechanical failure of the repair tissue inevitably occurs of complete structural and functional restoration of tissues in variable amount of time after joint injury. The initial tear propagates within the normal surrounding cartilage inducing organs and systems made possible by the use of break- extended collagen fibers breakage and progressing to genera- through discoveries in the field of molecular cell and devel- lized joint destruction. opmental biology [20]. It is sought that using RM specific tools complete structural regeneration and functional reha- Osteoarthritis, the most common form of joint degenera- bilitation of affected joint would be possible. Biological joint tive disease, is considered to be the result of interaction be- restoration and complete re growth of hyaline cartilage could tween systemic and local, mechanical and metabolic factors address both traumatic defects as well as tissue deterioration [7]. The intimate mechanisms underlying OA occurrence and during various OA stages. progression are still to be elucidated. Age has been incrimi- nated in OA pathogeny as there are strong statistical correla- To this end, a wealth of basic and translational research is tions between advanced age and the prevalence of disease. currently going on. Specific RM strategies, cell therapy, tis- However age-related changes in metabolism sue engineering or are in different stages of and periarticular structures functioning do not obligatorly preclinical testing or clinical applicability. Introduced in the result in a degenerated joint surface. A distinct form of OA, early 1990’s by a Swedish group, autologus chondrocyte commonly known as posttraumatic arthritis (PTA) represents transplantation (ACT) uses a cell suspension of autologus the end stage in the natural evolution of a traumatized joint. adult cells to treat focal cartilage defects [21]. OA can be as well the consequence of disturbances in joint A development of ACT, matrix assisted chondrocyte im- anatomy, alignment and stability, in joint or muscle innerva- plantation/transplantation (MACI/MACT) can be considered a tions, or of inadequate muscle strength of various origins [8]. form of cartilage engineering. With this procedure, cells are Metabolic OA is a recently identified subtype of OA related delivered to the cartilage defect by means of a three- to the complex systemic disturbances associated with the dimensional matrix thereby improving their retention and en- metabolic syndrome. High blood pressure induces subchon- hancing their biological activity at implantation site [22]. Cur- dral bone ischemia which affects cartilage nutrient exchange and triggers bone remodeling. Ectopic lipid deposition rently several scaffolds and methods of cell seeding are com- within chondrocytes alters cellular metabolism. The en- mercially available [23, 24]. ChondroCelect™ was the first hanced level of adipokines associated with obesity induces advanced therapy medicinal product (ATMP) approved to be expression of proinflammatory factors and degradative en- marketed in Europe [25]. ACT has been reported as well to zymes [9]. Clinical research data support the fact that OA is improve mobility and reduce pain in patients with early os- a systemic disease having complex and intricate inflamma- teoarthritis [26]. However, the clinical advancement of adult tory, metabolic and proliferative signatures, not limited to cell based therapy is limited by technical inconveniences, cell and not initiated within the cartilage tissue itself [10, 11]. source related obstacles as well as by the availability and af- fordability of the procedure. Having an increased proliferative An abnormal tissue turnover with a balance in the favor and differentiation potential, mesnechymal stem cells (MSCs) of growth and reversion of mesenchymal cells to a develop- are looked upon as a suitable cell source for cartilage regen- mental-like phenotype could explain the aspect and the pro- eration. Case series of succesful use of MSC to treat cartilage gression of OA lesions [12]. Progenitor cells with migratory defects are reported [27]. Several clinical trials using autologus and proliferative potential could be demonstrated in OA or allogeneic MSCs for cartilage regeneration are going on cartilage [13], synovial membrane [14], subchondral bone [28]. In animal models, cell therapy was proven successful in and periarticular adipose tissue [15] of OA subjects. limiting OA progression [29] or in preventing PTA [30]. II.2. Cartilage Repair and Regeneration To date, no clinically available therapy could completely restore structure and function of normal adult articular carti- The attempt to cure the “troublesome cartilage ulcers” lage. Long term satisfactory clinical results with the use of remains still frustrating to modern orthopedic surgery [16]. ACI have been recently reported [31]. Regardless these en- Different methods of grafting make use of autologous or couraging results, to date, OA treatment still relies on symp- allogeneic cartilaginous or non-cartilaginous tissues to fill tomatic relief and on reconstructive joint surgery for the ad- joint surface defects [17]. Subchondral bone stimulating pro- vanced stages of cartilage deterioration and functional im- cedures such as subchondral drilling, microfracture, and pairment. abrasion arthroplasty are used to create controlled microle- sions within the osseous tissue underlying the defect. The blood clot produced by such procedures is rich in bone mar- II.3. Patent Search and Description row progenitors and is thought to induce cartilage repair In the following, the principal approaches in cartilage [18]. These interventions are capable to generate short term regeneration will be briefly outlined based on current thera- satisfactory results reducing pain and restoring mobility. peutic principles. Their major drawback is the poor quality of the repair tissue. The fibrous cartilage does not reproduce the structural orga- Patent search was performed in the following databases: nization and the mechanical endurance of the native hyaline “Patent Lens” of the organization, “Initiative for Open Inno- joint surface. Such repair is prone to progressive clinical and vation (IOI)”; free patents on line “FPO” and Google 54 Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 Labusca et al.

Table 1. Patents Relating different Cartilage Regeneration Methods Issued between January 1, 2012 - October 3, 2013.

Chapter*/Patent Inventors /Asignees Status Title Definition (Summary) Number

III.1 Theodore, I. Malinin Univer- US granted patent Cartilage composition Particulate cartilage compositions for stimulating US8318212 sity of Miami, Miami, FL Nov. 27, 2012 and process for produc- chondrogenesis and producing cartilage regeneration (USA) ing the cartilage com- position

III.2. Chan, H.H., Kang, K.W., WO patent appli- Graft materials Graft material derived from mammalian cartilage with WO2012141454 Kim, J.Y., Ahn, J.H., Choi cation derived from significantly lowered risk by eliminating antigenicity, DM. October 18, 2012 mammalian cartilage pathogen and cytotoxicity through multi-step process- Hans Biomed Corp. ing treatments and inactivating all kinds of pathogens without affecting biomechanics

III.3. Kizer, N., Spiro, B., Bay, EU granted patent Cartilage particulate Cartilage treatment composition comprising: ca- EP2338441 H.M., Blanchard, CH. January 23, 2013 system daveric, allogeneic human cartilage particles from a Isto Technologies Inc. US granted patent human donor less than fifteen years of age at the time of donation including viable chondrocytes; and Zimmer Inc. September 3, 2013 a matrix, wherein the matrix is defined as a chon- droconductive media

III.4. Kizer, N., Spiro, B., Yao, J., US granted patent Cadaveric allogeneic Compositions having at least one neo cartilage parti- US8524268 Blanchard, C.H. September 3, 2013 human juvenile cle, juvenile cartilage particle or a combination thereof Zimmer Inc. cartilage implant and a matrix, and methods and devices that include the compositions

IV.A.1. Truncale, K.G., Gertzman, US granted patent Cartilage allograft Cartilage repair assembly comprising a shaped al- US8221500 A.A, Sunwoo, M.H., Tom- July 17, 2012 plug lograft structure of subchondral bone with an integral ford, W.W. overlying cartilage cap which is treated to remove Musculoskeletal cellular debris and and milled allograft transplant cartilage in a bioabsorbable carrier

IV.A.2. Juergen, F., Gassmeier, C., US granted patent Cartilage replacement Cartilage replacement implant for the biological US8231685 Aicher, W.K. July 31, 2013 Implant and method regeneration of a damaged cartilage area of articular Tetec Tissue Engineering for producing a cartilage in the human body, comprising a cell carrier Technologies AG, Reutlingen cartilage replacement which has a defect-contacting surface for placement (DE) implant on the damaged cartilage area and is formed and designed for colonization with human cell

IV.A.3. Mollenhauern, J. US patent applica- Implant and therapeutic Implant including a support material, cartilage cells US20120039961 Tetec Tissue Engineering tion composition for damage and/or precursor cells thereof, and a cartilage-specific Technologies AG February 16, 2012 and diseases related to collagen type. A method for preparing the implant, to the human animal a therapeutic composition, includes cartilage cells musculo skeletal system and/or precursor cells thereof and a cartilage-specific collagen type

IV.B.1. Archer, C.W., Haven, S.N., EU granted patent Method of isolation of Human isolated from the full depth of EP2111447 Dowthwaite, G. November 14, a human stem cell human cartilage tissue and/or isolated from aged University Cardiff 2012 from the full depth of human cartilage; and uses thereof cartilage

IV. B1. 2. Archer, C.W., Haven, S.N., US granted patent Isolation of human Method of isolation of human stem cells from all US8357534 Dowthwaite, G. January 22, 2013 articular stem cells depth of human cartilage isolating a human stem cell University Cardiff comprising:(a) obtaining human articular cartilage tissue from a full depth of the cartilage tissue; (b) digesting the tissue to release chondrocytes, by using ;(c) exposing the isolated chondrocytes to fibronectin and/or a fragment thereof containing an RGD sequence; and (d) isolating those cells that bind fibronectin or said fragment

Recent Patents in Cartilage Regeneration Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 55

Table (1) contd….

Chapter*/Patent Inventors /Assignees Status Title Definition (Summary) Number

IV.B.2 Katz, N., Pustilinik, F. US patent applica- Orthopedic applica- Orthopedic applications of mesenchymal stem cell US20120087983 Jointechlabs Inc. tion tions of encapsulated encapsulated and delivered for treatment of cartilage April 12, 2012 stem cells damage in joints, therapeutic composition prepared comprising a purified fraction of adipose-derived mesenchymal stem cells encapsulated in microbeads of a three-dimensional biocompatible matrix

IV.C1. Rueger, D.C., Kidley, R. US patent applica- Methods of treating Methods of repairing and regenerating cartilage tissue US20120077743 Stryker Corp. tion cartilage defects by administering into the cartilage or the area sur- March 21, 2012 rounding the cartilage a composition comprising a AU2011265308 therapeutically effective amount of a morphogenic January 19, 2012 protein

IV.C.2. Rosier, R.N., Zusick, M.J., US granted patent Protecting and repairing Compositions and methods related to promoting US8513193 Sampson, E., Bukata, S., August 20, 2013 cartilage and muscu- protection or repair of articular cartilage and/or Puzas, J.E., loskeletal soft tissues musculoskeletal soft tissue by contacting the carti- Awad, H. lage, tissues or cellular components thereof with a parathyroid hormone/parathyroid hormone-related University of Rochester protein (PTH/PTHrP) receptor agonist or releasing factor

IV.C.3. Zang, R., Peluso, D., Morris, EU granted patent Methods and compo- Methods and compositions for regenerating functional EP2174674 E.A. January 18, 2012 sitions for healing and and physiologically appropriate tissue repair for the Genetics Institute LLC. repair of the cartilage repair of articular cartilage injuries and defects, meth- ods of treating patients with articular cartilage injuries or defects, utilizing bone morphogenetic proteins (BMPs), which are known to have osteogenic and/or chondrogenic properties, and which may be produced via recombinant DNA technology

V.1. Iwasaki, N., Minami, A., US granted patent Compositions and Composition for regenerating cartilage or treating a US8372394, Kasahara, Y., Igarashi, T., February 2, 2013 methods for treating cartilage disease containing a monovalent metal salt of JP2008052999 Kawamura, K., Miyajima, C., AU2008217916 cartilage disease alginic acid that improves cartilage regenerative action Ohzawa, N., Imai, M. and ease of application to a cartilage injury lesion, and WO2008102855 March 1, 2013 Mochida Pharmaceutical Co., a composition for treating a cartilage disease, which JP granted Ltd., National University has the effects of protecting cartilage from mechanical Corporation Hokkaido Uni- March 03, 2009 irritation, inhibiting degenerative changes in cartilage versity WO granted caused by wear and inflammation, repairing a cartilage August 28, 2008 injury lesion, and inhibiting inflammation and pain of joint tissue

V.2. Suzuki, M., Ueno, K. US patent applica- Substrate for cartilage Substrate for cartilage cultivation, whith high safety US20120122791 Muto, N. tion cultivation using and growth promoting effect on cartilage cells, and a artificial collagen, and method for cartilage regeneration treatment containing JNC Corp. Tokyo May 17, 2012 method for cartilage artificial collagen, in particular, an artificial collagen regeneration treatment aqueous solution Regeneration effect is obtained by using the substrate the intraarticular of the substrate in and around the cartilage defect sit

V.3. Kato, M. US patent applica- Artificial cartilage and An artificial cartilage comprising 15-95% by mass of US20130116187 Hoya Corp. tion its production method collagen, 4.9-70% by mass of , and 0.1- May 09, 2019 20% by mass of

56 Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 Labusca et al.

Table (1) contd….

Chapter*/Patent Inventors /Assignees Status Title Definition (Summary) Number

VI.1. Kandel, R., Pilliar, R., Gryn- WO patent appli- Methods for repairing Compositions, dosage forms and methods for stimu- WO2012094739 pas, M., St-Pierre JPH. Mount cation cartilage lating cartilage growth, regeneration or repair, and Sinai Hospital Corp. July 19, 2012 treating cartilage defects and diseases using short chain polyphosphates having a chain length of greater than 5 phosphate units with particular application in the treatment of osteoarthritis

VI.2. Bolshakova, A.Y., Borishpol- WO patent appli- Pharmaceutical com- Pharmaceutical composition in the form of a suspen- WO2012082013 skiy, A.L., Knyazkin, G.J., cation position for stimulating sion for the prevention and treatment of problems in Melnikova, N.B., Polukhin, June 21, 2012 the regeneration of the physiological and reparative regeneration of tis- I.V., Polukhin, O.V., supporting tissue and sues of the musculoskeletal system, namely bone Pyanzina I.S. articular cartilage tissue and articular cartilage. The pharmaceutical (variants) compositions contain a substance called “Mitsellat of calcium carbonate and magnesium”

VI.3. Vukicevic, S., Jelic, M. US granted patent Methods and compo- Compositions are described to regenerate cartilage in a US8119591 Genera Istrazivanja d.o.o., February 21, 2012 sition for regenerating partial thickness defect or area of reduced volume of Kalinovica (HR) articular cartilage articular cartilage comprising an infiltration suppressor agent and a columnar growth promoting agent

VII.1. Slayton, M.H., Barthe, P.G. US patent applica- Slayton MH, Barthe Methods of treating damaged cartilage targeting the US20120165848 Guided therapy systems tion PG/ Guided therapy damaged cartilage in region of interest, directing LLC. June 20, 2012 Systems LLC therapeutic ultrasound energy to the damaged cartilage WO patent appli- cation February 9, 2012

VIII.1. Raederstorff, D., Schwager, US Patent applica- Slayton MH, Barthe The use of hydroxyltyrosol for inducing or enhancing US20130005682 J., Wertz, K. tion PG/ Guided therapy cartilage repair or cartilage regeneration. nutraceutical DSM IP Assets B.V., January 1, 2013 Systems LLC and pharmaceutical compositions comprising hy- droxyltyrosol for regeneration and repair of cartilage Heerlen, TE. (NL) injuries in joints, in particular of traumatic cartilage injuries

* reference to the description within the text. patents. Key words were: “cartilage” AND “regeneration” saicplasty™” (Smith& Nephew) or “OATS™” (Arthrex). filtering for granted patents and patent applications recorded The method is technically challenging due to concern over during January 01, 2012 and October 03, 2013 across all the remaining gaps between plugs, the questionable chon- available jurisdictions and languages/ Results are presented drocyte viability at the margins of the graft and the uneven- in the following text and summarized in Table 1. ness of the reconstructed joint surface. Synthetic osteochon- dral plugs are designed to simplify the procedure and de- III. AUTO-, ALLO- OR XENO- PARTICULATE CAR- crease operating time eliminating the issues with donor site TILAGE GRAFTS pathology [34]. Large “dome” osteochondral plugs have been used as salvage method for reconstructing large joint Soft tissues such as autologus perichondrum or pe- defects resulting after posttraumatic or post tumor removal riosteum have been used in grafting cartilage defects. Pe- especially in young subjects. The main problems encoun- riosteum has been more largely used, especially in pediatric tered with grafting procedures are the low amount of patients [32]. Autologus or allogeneic osteocondral grafts are autologus tissue availability, the risk for disease transmission particularly used to treat full-thickness joint defects. Cryo- in case of allonegeic or xenografts, the quality of the biome- preserved grafts from cadaver donors can be used as well, chanical properties as well as the stability of the transplant. however fresh tissue is preferred for reasons of cells viability The use of particulate graft tissue is sought to increase the and mechanical stability of the implant [33]. Autologus os- mechanical stability of the graft and the active surface for teochondral transfer (OATS) consists in the removal of cy- delivery. An efficient method for obtaining lindrical plugs of ostheochondral tissue from a non-bearing cartilage grafts for facial augmentation or for the treatment portion of joint cartilage such as trochelar groove in the knee of cartilage defects in human joint is the use of a mixture of joint followed by impactation of the grafts within the de- morcelized cartilage matrix, fibrin sealant and growth factors fect(s). Osteochondral transfer is referred to as “Mo- (such as Fibroblast Growth Factor, FGF) [35]. Recent Patents in Cartilage Regeneration Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 57

III.1. Granted patent US8318212 describes a method of mammalian (human, horse, pig, cow and mouse) by means stimulating chondrogenesis and producing cartilage regen- of successive treatments claimed of consistently lower toxic- eration using non demineralized autologus, allogeneic or ity and contamination risk without altering native tissue xenogeneic hyaline of cartilage particles with properties or the reconstructive capabilities. The method diameter of 60 - 500microns. The cartilage is harvested with offers a possibility for obtaining safe virus free allo- and a scalpel from articular cartilage of major joints (such as xenografts which are easily accepted by the host and can femoral heads, acetabulum, distal femoral surfaces, or hume- eliminate concerns such as foreign body sensation, infection, ruses) up to the subchondral without being exposed to high pain, escape and mobility of grafts observed using the con- temperatures or to any physical or chemical treatment (such ventional grafting methods. The procedure is presented as as demineralization agents), which might alter their natural having minimal side effects and significant reduced cost. The properties. The cartilage can be further prepared by grinding steps to be undertaken are: The harvest (preparation of the in any suitable apparatus after drying, subsequently freeze- mammalian cartilage); the treatment with hypertonic sodium drying in liquid nitrogen. The cartilage mixture can be used chloride solution; the exposure to a virus inactivating solu- to filling a defect by impactation and seizing to adjust the tion; treatment with an alkali solution; extraction of or joint surface. The method is sought to induce regeneration of freezing agent; the final step consisting in gamma irradiation the surface layer by means of the innovative distribution of of the graft. Gamma irradiation with 25kGy is the efficient the multiple faceted microparticles. The method is presented dose for complete chondrocyte removal, therefore eliminat- to have been successful in filling the defects created in the ing the risk of graft antigenicity. The quality of ECM in condylar cartilage of non-human primates. Quality of the samples of cartilage processed using this protocol was- filling tissue was assessed by histology (Hematoxilin Eosin, demonstrated histologically. Masson's Trichrome (MT) and PAS, Romanowski-Giemsa and Safranin-O staining). This Verhoeff Van Gieson (VVG) staining revealed the presence method is claimed to being preferrable by the clinicians due of substantial amount of collagen and elastic fibbers. Sa- to the easiness in handling the morcelized graft and for being franin-O staining demonstrated the presence of proteogly- suitable for all defect types, however, information about the cans and Alcian blue that of glucose amino glycans (GAGs). type of the regenerated cartilage (fibrous or hyaline) has still Three dimensional (3D) porous structure assessed by elec- to be provided [36]. tron microscopy was found to be conserved. The biological stability of grafts was tested by intramuscular implantation in III.2. European granted patent EP2338441 and US patent rats, biopsy at three months of the implanted pouch showed US8524268 are introducing methods of using compositions of allogenic human cartilage particles from a human cadaver “very little inflammation” with no signs of foreign body re- action [39]. This method of obtaining affordable cartilage less than fifteen years of age at the time of donation includ- graft material is appealing, however further studies about ing viable chondrocytes within the minced cartilage. The quality and stability of the implant in larger animal models, mixture is denominated as chondro-inductive and chondro- as well as the safety of the xeno derived grafts, will be conductive matrix. Donor areas containing collagen type-II needed. (COL2A1) cartilage such as costal cartilage, nasal cartilage, trachea cartilage, sternum cartilage are the preferred sites for III.4. US patent US8221500 relates to a method of ob- graft harvesting. Condro-inductivity is presented in the de- taining cartilage allograft plugs consisting of a shaped por- scription as the capability of the transplanted graft to induc- tion of allograft subchondral bone and the overlying carti- ing proliferation, growth differentiation and/or other matura- lage, treated to remove the cell debris and proteoglycans, it is tion of chondrocytes or chondroprogenitors in vivo. Chon- designed to be implanted together with milled allograft carti- dro-conductivity is described as being the property of the lage tissue included in a bioabsorbable carrier. The structure graft to offering a milieu for proliferation, differentiation, can be used as an implant for fitting cartilage defects in vari- growth, ingrowth and/or orientation of cartilage from sur- ous joints such as shoulder or knee. In order to address the rounding tissue toward the defect, after implantation. Carti- common problem of graft retention, cartilage defects can be lage particles can be encapsulated in fibrinogen, thrombin or previously drilled to accommodate the pegs of the implant. hyaluronate or within other patented or commercially avail- The mixture of milled cartilage tissue and bioresorbable car- able synthetic or mixed matrices, further cultivated in vitro rier forming a paste or a gel added to the osteochondral plug to obtaining a cartilage graft. The particulate cartilage grafts is claimed to increase the cellular transfer towards the can be tailored to accommodate the clinical defect. The con- grafted area. The allograft constructs can be used in combi- dro-inductive/chondro-coinductive matrix has to be secured nation with growth factors (such as FGF-2, FGF-5, IGF-1, in place with the use of a retainer. This retainer may be a TGF-, BMP-2, BMP-7, PDGF and VEGF) or with alloge- flap, plug, disc, sheet or patch of living cells, such as pe- neic cells such as chondrocytes, MSCs or with demineral- riosteum cells, other natural tissue membrane or a synthetic ized bone matrix, insulin, insulin- growth factor -1 (IGF-1), membrane. The method has been proved efficient in filling IHH, PTH or a commercially available bioactive glue. The experimental osteochondral femoral defects in a goat model preferable method of implantation is via arthroscopic sur- [37, 38]. This patent introduces an interesting source of vi- gery, alternatively by open surgery. Plug diameter can be 4- able cartilage; however donor availability as well as ethical 10mm, small enough to fit the endoscopic cannula but large concerns could limit the clinical applicability of the proce- enough to reduce to the minimum the number of implants, dure. the subchondral bone part can be cut to accommodate the III.3. World Intellectual Property Organization joint contour and to obtain an even joint surface. Both (WIPO/WO) patent application WO2012141454 relates to plugs and morcelized cartilage material can be frozen or obtaining hyaline, elastic or fibro cartilage grafts from freeze dried. The minced cartilage with particle size of 1 58 Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 Labusca et al. mm can be lyophilized to a water content of 0.1-8% and developments in the field of cartilage engineering includes mixed with the carrier (e.g., HA) in a ratio of 15-30% to formulation of new cell scaffolds/cell carriers, the identifica- 85-70% Autologus cells of choice cultivated in vitro can be tion of novel cell sources beneficial for in vitro or in situ injected within the cartilage matrix before or during or after regeneration, identification of growth factors/bioactive plug implantation [40]. molecules mixtures and formulations proven successful in cartilage protection and/or regeneration additionally use of IV. CARTILAGE TISSUE ENGINEERING cell free scaffolds designed to stimulate in situ cartilage re- generation. RM proposes ex vivo engineering of implantable tissues or organs in order to substitute the missing, diseased or con- IV.A. Cell Carriers genitally abnormal body parts. Three principal compounds cells, biomaterials and bioactive molecules are described as Natural derived or synthetic scaffolds are used as cell “the triad of tissue engineering” (TE) [41]. Cells are the main delivery substrates for the purpose of cartilage regeneration. actors of regeneration, sought to recompose or to repair Several recent patents are introducing novel approaches in missing or damaged structures. Biomaterials are used as biomaterial formulation or processing. scaffolds aiming to host, direct and sustain growth, differen- IV.A.1. US patent US8231685 describes a method for tiation and organization of cells in the process of “re”- producing cartilage implant aimed to fit the damaged area forming functional tissues. Bioactive molecules (growth fac- and to act as a cell carrier and a defect filling material. The tors, , and ) are used to modulate cell biomaterial can be either biocompatible synthetic or differentiation, protein expression, cell-cell and cell- a natural collagen consisting of non-woven material with a extracellular matrix (ECM) interactions. Cartilage engineer- porous structure such as a chitin, or hyaluronic acid ing has proven to be a challenge. Despite the apparent struc- derivates with pore width of 80-150μm and carrier layer tural simplicity of the tissue, reproducing the remarkable thickens of 0.3-0.4mm. The implant is designed to be an- biomechanical endurance characteristics of the native tissue chored within the defect either by resorbable suture materi- the stable integration of the engineered graft within the func- als, pins or adhesive materials. It is envisaged that it can ac- tional context of the joint are problems still to be solved. In commodate different type of cells, adult i.e. chondrocytes or order to produce the best functional graft, several choices mesenchymal stem cells or used cell free and implanted so as have to be made: Identification of the best cell source i.e. bleeding occurs from the cartilage defect within the implant. adult or stem cells, procurement ease, specific expansion and It is preferable that cell inoculation to be administered before differentiation potential; supportive structure engineering material implantation to ascertain a sufficient number of ma- methods, biodegradability of scaffold as to accomplish the trix producing elements capable of ECM substitution until dual role of cell carrier and temporary tissue substitute; iden- the complete degradation of the biomaterial. The material tification of growth factors or cytokines as well as the best can be split to accommodate the joint surface profile in order method of delivery capable to ascertain maintenance of cell to reduce the gap formation at the interface with the host. phenotype over time [42]. Currently, various combinations Traumatic, degenerative or inflammatory lesions in any of of cell, scaffolds and bioactive molecules are in different the human joints (such as knee, ) can be stages of product development for cartilage engineering. treated using method [52]. This patent provides an interest- Reports of successful grafting cartilage defects with en- ing solution for accommodating different size and shape of gineered cartilage in animal models are abundant in the re- cartilage defects, however, the procedure of anchoring of the cent literature [43]. Several products are already in clinical graft might prove technically demanding in clinical settings. ® use. Autologus induced matrix chondrogenesis, AMIC has IV.A.2. US patent application US20120039961 relates to been reported to reduce pain and improve function in me- an implant consisting of support material, cartilage cells or dium follow up [44]. Improved VAS, IKDC, KOOS, Ly- progenitors and cartilage specific collagen type to be used sholm, and Tegner scores were reported at 12, 24 and 36 for treating damaged or diseased human or animal muscu- months at 3 years follow up in patients with cartilage defects loskeletal damage. Cell types suitable for the intervention are up to 8.6cm2 after “biological arthroplasty” using bone mar- autologus or allogeneic chondrocytes or chondroblasts origi- row aspirate covered with a collagen type-I/-III matrix [45]. nating from MSCs. The cells are expanded in culture for two Engineered cartilage composed of autologus bone marrow or more passages. A “cartilage specific collagen type” such mesenchymal stem cells (BMMSCs) seeded on collagen as collagen type-VI is reported to be retained by the cells scaffold was reported to heal full thickness medial femoral condyle defects in OA patients [46] Different scaffolds such during in vitro cultivation through self-microencapsulation. as agarose [47], alginate, gelatin [48], collagen types I and II With the deposition of ECM around the cells, released pro- [49], and photopolymerizedpoliethylene oxide teins can be retained and more efficiently contribute to tissue [50] are reported to induce MSCs chondrogenesis in vitro or repair. The cartilage specific collagen can be biological, hu- in animal models. Growth factors commonly used for modu- man or animal origin, microbiologicaly produced or recom- lating chondrogenesis are the members of TGF- super fam- binant. Collagen types-II, VI, IX, XI and combinations ily, Insulin Growth Factor (IGF, FGF and PDGF) and Wnt thereof can be used, however collagen type-VI, as the natural proteins enhance chondrogenic differentiation is shown to constituent of chondrocyte pericellular matrix is preferred. cross-talks with TGF- pathway [51]. Continuous efforts are Non cartilage specific such as collagen type-I can made to finding the most efficient combination of cells, scaf- be used as well. The implant can include bio-active mole- folds and growth factors to ensure the best graft properties, cules, such as growth factors (such as TFG- family, BMPs, endurance, cell survival and ECM protein production. Recent IGF) adhesion factors, receptor agonists or antagonists, anti- Recent Patents in Cartilage Regeneration Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 59 biotics, chemokines i.e. cytotactin, laminin, fibronectin, STRO-1, MSX1 or Notch-1. The clonally derived whole type-IV, V and VII collagens or synthetic peptides consisting depth cartilage stem cell population were shown, by in ovo of various bioactive sequences. The implant requires to be assays, to be able to participate into various chick connective structured as 3D matrix by various processing methods (e.g., tissues such as bone, cartilage, and tendon producing human cross linking). The supportive structure can contain rein- collagen type-I The human aged cartilage was demonstrated forcements such as proteins, polysaccharides, silk or cotton to display a positive intracellular labeling for stem cell fibbers containing open pores ranging 50-500μ, capable of marker transcription factor MSX1 [56, 57]. The being designed as a multilayered structure. The support ma- patent describes a novel cell source opening the possibility of terial can be loaded with the selected cell population, culti- using local progenitor populations for different strategies of vated for 1-2days allowing cell migration within the mate- RM Further development of such procedures will contribute rial. The therapeutic composition consisting of an aqueous to clinical applicability of local progenitor cell recruitment. solution of the cartilage specific collagen type, which is IV.B.2. US patent application US20120087983 reports added to the culture incubated for 48hours. The implant can details on a method of using micro-beads encapsulated adi- be used as regeneration approach particularly in tissue engi- pose derived stem cells ADSCs to be used in diverse ortho- neering for the treatment of posttraumatic defects of muscu- pedic applications for example treating articular cartilage loskeletal system or damaged areas such as cartilage, in- defects (ACDs). The invention relates to a 3D platform for tervertebral disc and wounds in human and animal subjects. stem cell delivery, specifically ADSCs included in biocom- As an example, a clinical application of an implant con- patible hydrogels formed into microbeads. The formulation structed using autologus chondrocytes originating from the is claimed to offering protection and support for the cells radial head of a female patient is given. Cells were pre- compared to cell suspension method. The therapeutic factors incubated for 48hours in an aqueous solution of collagen produced by the cells migrate after implantation through type-VI, than loaded on a porous layered membrane consist- layer towards the targeted orthopedic tissues, con- ing of allogeneic pericardial membrane and cross linked sequently enhance contact with tissue and improve bioactive gelatin aimed as to be re implanted in the cartilage defect of effects. The platform is claimed to promote healing in carti- the same patient [53]. The microencapsulation method offers lage damage induced by OA, ageing or trauma and to reju- a novel approach for enhancing engineered cartilage biologi- venate tissues adjacent to the diseased cartilage. Any of cal and mechanical properties. Further animal models studies adult, embryonic or induced pluripotent stem (IPS) cells can would be needed to confirm histological and mechanical be used with this formulation. This includes endothelial tis- stability of the grafts. sue from umbilical cord vein, endothelial tissue from fore- skin, endometrial tissue, human embryonic stem cells IV.B. Cell Sources for Cartilage Regeneration (HESCs), adipose tissue or patient specific IPS. The pre- ferred source is stromal vascular fraction derived from non- Progenitor cell populations exists within different articu- pathological post natal adipose tissue. Human adipose tissue lar zones of normal cartilage i.e. superficial zone, Ranvier is obtained from living donors during surgical or cosmetic groove and periarticular structures i.e. fat pad, synovial tis- liposuction procedures. Cell isolation can be done enzymati- sue and subchondral bone [54, 55]. caly i.e. trypsine digestion or using mechanical procedures IV.B.1. Patents EP2111447 and US8357534 relate to a such as agitation, sonic or thermal energy. Pluripotent cells method of obtaining human stem cells from the full depth of can be separated from other cell population using size frac- human cartilage includingly aged cartilage. The procedure tionation, density gradient, granularity, morphologically or relays on harvesting full depth human cartilage, enzymatic by positive or negative immunosorting procedure using digested with a mixture of pronase and collagenase, isolated magnetic activated cell sorting (MACS), fluorescence acti- cells purified by filtering or centrifugation, cultivated on vated cell sorting (FACS) or affinity chromatography. Cells fibronectin, RDG senquence or combination containing are further expanded in vitro in a defined culture media such substrate. The patent claims the isolation of human stem as Dulbecco’s modified Eagle’s Medium (DMEM), with or cells from the whole depth of normal cartilage tissue as well without antibiotics, glutamine and/or fetal bovine serum as from aged cartilage, previouslly conssidered less (FBS). Such cell population can be used for orthopedic ap- metabolically active. In contrast with bovine progenitors plication in “therapeutically effective amount” further de- isolated using a similar protocol, human cells appear to be fined as the amount of cells considered effective for a given immortal in culture, and overcoming the Hayflick limit. The application. The pluripotent cells described can be driven cells could produce cartilage when cultured in pellets after towards chondrogenic differentiation and administrated by 70 population doublings and therefore can undergo massive means of pharmaceutically acceptable solutions or stored i.e. expansion practicaly useful for cartilage engineering. The frozen, until needed. To obtain supportive 3-D platforms, isolated cells of the claimed exhibites phenotype plasticity cells are dispersed within biocompatible/biodegradable gel and can be used to engineer various connective tissues. The or polymeric matrix, which protect, maintain and cultivate method of isolation base on cell pupulations from full depth cells for a period of 5-14days after implantation. This ex- of human cartilage, both mature and aged (i.e. tends the duration for release of therapeutic proteins to the macroscopically thinner) cartilage, which can be isolated by healing tissue. are manually or automatically formed the high affinity to fibronectin and RDG sequence. Such into microbeads of 10-50μL in size, each bead including cells can undergo more than 52 population doublings, 2,000-10,000 stem cells, cells can furthermore continue to capable of differentiattion into several connective tissue increase the release of bioactive factors. The concentration of phenotypes expressing stem cells surface markers such as 2000 cells per μl is preferred, claimed to enable cell mainte- 60 Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 Labusca et al. nance and viability up to 14days after implantation. The fects proved efficient in overcoming limited in vivo life time composition of microbeads including viable autologus of GFs. The neo tissue produced by this method stained posi- ADSCs can be applied superficially to augment autolgous tive for collagen type-II [65]. Cartilage engineering strate- adipose tissue transplant procedures or injected within the gies involve the use of one or several GFs and different mo- subchondral bone adjacent to a cartilage lesion. The inven- dalities of delivery [66]. Transient application of TGF-3 tion further discloses a disposable chip-device consisting of increased Glycosaminoglycans (GAGs) content and com- two micro channels, a pump and a droplet formation system pressive properties of hydrogels seeded with bovine chon- for the automatic formulation of gel microbeads. Keeping drocytes [67] or MSCs when compared to continuous appli- stem cells encapsulated maintains cell-cell interaction, pro- cation, possibly due its role in initial phases of chondrogene- liferation and protein expression activity as compared to cell sis. Sequential administrating of FGF-2/TGF-1 induced suspension furthermore enhancing storage, cryopreservation chondrogenesis while IGF-1 enhanced matrix protein pro- i.e. freezing or vitrification, facilitates cell maintenance, duction [67]. transport and delivery. One of the possible orthopedic appli- IV.C.1. US patent application US20120077743 reports a cations of encapsulated stem cell delivery aims treating carti- method of repairing and regenerating cartilage tissue by local lage defects produced by OA or trauma. Encapsulated cells administration of a morphogenetic protein. Delivery can be can be administrate within the subchondral bone of the carti- made within the cartilage, menisci or intervertebral disc tis- lage defect(s) allowing the bioactive factors produced by the sue or in the areas surrounding cartilage such as synovial cells to migrate within surrounding tissue. The administra- fluid. The invention claims the potential use of a large panel tion can be performed via arthroscopy or percutaneously of bioactive molecules, to cite only a few OP-1, BMP-5, using an image intensifier, repeated as necessary until ob- BMP-6, GDF-5, GDF-6, GDF-7, CDMP-1, CDMP-2 and taining the desired effects [58]. CDMP-3. In a more preferred embodiment, the morphogenic This method of cell encapsulation could prove appealing protein is OP-1 or any aminoacid sequence having at least to extending the otherwise limited efficiency of ADSCs in 70% homology with the C-terminal 102-106 amino acids as cartilage regeneration. well as with the seven domain, of human OP-1. Morphogenetic protein of natural origin can be used besides IV.C. Bio-active Molecules for Cartilage Regeneration; molecules obtained by biosynthesis and chimerical proteins. Growth Factors Synthetic native or nonnative morphogenes can also be used. Consensus osteogenic proteins (COPs) can be expressed as The application of growth factors (GFs) is a promising fusion proteins in prokaryote organisms reported haveingos- field for cartilage repair within regenerative medicine. In vitro teogenic or chondrogenic role in animal models. The pre- and animal studies have proven the role of transforming ferred pharmaceutical composition is in a liquid form to be growth factor- super family (TGF-), fibroblast growth administered directly into the site of the damaged tissue factor family (FGF) [59] insulin-like growth factor-I (IL-1), within conventional carriers. Microparticulate drug carriers and platelet-derived growth factor (PDGF) in modulating the such as liposomes or microspheres can be prepared to con- local microenvironment for stimulating chondrogenesis and tain the morphogenes of the invention to be released in the ECM protein synthesis [60]. Growth factors exercise ana- fluids bathing the treated tissue (synovial fluid or blood bolic effect on chondrocytes and stimultate ECM protein stream) or included within a biodegradable scaffold com- production. Their proliferative and chondrogenic effect on posed of, for example, Polylactic acid (PLA), polyglycolic MSCs can be used to modulate cartilage regeneration. Major acid (PG) or alginate cross linked hydrogel. The method is issues with the use of GF for RM strategies are the biological reported successful in treating dog model of osteochondral availability, the limited in vivo life time as well as their com- knee defects using “time release” intra-articular delivery of plex and intricate, species dependent mechanism of action. OP-1, 16 weeks after surgery and in sheep model of chondral Platelet rich plasma (PRP) has been identified as a rich defects treated with microsphere-releasing OP-1 [68]. Local source of autologus GFs. PDGF originating from autologus administration of different formulations containing growth platelet lysate was efficient in MSCs expansion designed to factors has remarkable potential in cartilage regeneration. treat various orthopedic conditions [61]. TGF-1 has a Precise control of timing and duration of release of bioactive stimulatory role on chondrocyte ECM protein production, molecules will be needed while attempting clinical applica- increase synovial cells and MSCs chondrogenesis. However, tions. when delivered locally within the joints of animal models IV.C.2. US patent US8513193 relates to a method of they were reported to increase synovial proliferation, in- promoting protection and repair of the cartilage and other flammation and fibrosis and to induce osteophyte formation. musculoskeletal tissue such as meniscus, intervertebral disc, Such reports are rising safety concerns in the eventuality of muscle, tendon or ligament using a parathyroid hormone or a clinical applications of GF based regeneration strategies parathyroid hormone-related protein (PTH/PTHrP) receptor [62]. TGF-3stimulates ECM deposition and proved effi- agonist or releasing factor. Type 1 PTH receptor (PTHR-1) cient in the repair of rabbit models of cartilage injury [63]. is only expressed by chondrocytes in early stages of degen- Evidence exists referring to interspecies variability regarding erative processes. It is as well normally expressed in a par- the effect of TGF isoforms in MSCs-based chondrogenesis; ticular stage of cartilage development during endochondral therefore results have to be considered carefully when trans- bone formation as well as in the epiphyseal cartilaginous lating towards human application [64]. Liquid osteoprote- growth plate. PTHrP stimulates growth plate chondrocyte gerin -1, OP-1/BMP-7 infused continuously via mini osmotic proliferation and proteoglycan synthesis. PTH and PTHrP pump device in sheep models of chondral knee cartilage de- inhibits the chondrocyte hypertrophy, expression of matrix- Recent Patents in Cartilage Regeneration Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 61 degrading enzymes (MMPs), expression of Type-X collagen, a cell population consisting in autologus chondrocytes or which are characteristics of hypertrophic phenotype of chon- mixture of chondrocytes and stem cells, which have been drocytes having as a result cartilage mineralization and bone previously cultivated in vitro with or without the addition of formation. PTH regulation is dependent on Indian Hedgehog a growth factor. Various members of BMP family produced (IHH), their concerted action is necessary for bone growth by DNA recombinant technology such as rhBMP-2 -18 or control; however, its activation in adult cartilage is a con- GDF family such as rhGDF1 -12 can be used. The growth tributor to joint degeneration. PTH/PTHrP, IHH as well as factor can be applied to the tissue source in a buffer solution other regulatory molecules such as matrix metalloproteinase In a rabbit model of cartilage defect, allogeneic osteochon- MMP-9, MMP-13 and BMP6 are present in the growth plate dral grafts bathed in rhBMP-2 (0.5mg/ml) showed signifi- is normally absent in adult cartilage and detectable in early cant healing of the defect after 4 weeks with no sign of joint stages of cartilage degeneration. PTHrP is expressed by pro- inflammation, reduced focal degeneration and improved os- liferating chondrocytes in OA cartilage. Enhanced proteo- teochondral integration of the graft as well as reduced fi- glycan synthesis is present in cartilage and other muscu- brous tissue repair in the subchondral bone compartment loskeletal tissues early after injury, interpreted as an attempt compared to controls. In a rabbit model of cartilage defect to produce tissue repair. The intermittent activation of treated with rhBMP, autografts harvested from the same PTH/PTHrP receptor in skeletal tissues results in stimulation knee joint showed improved graft integration, reduced graft of matrix synthesis, usually collagen and proteoglycans, and cartilage degeneration and an increase in cartilage formation cellular proliferation. The method is able to induce repair at the edge of the graft. rhBMP treated autologus osteochon- preventing degradation of articular and non-articular carti- dral grafts in non-human primates knee cartilage defects dis- lage and other musculoskeletal tissues such as skeletal mus- played better graft integration, with no sign of osteophyte cle, intervertebral disc, tendon and ligaments. Intermittent formation, increased amount of new cartilage formation be- administration of PTH/PTHrP receptor agonist or PTH re- tween the graft and host cartilage, and better integration with leasing factor can be performed alone or combined with the surrounding cartilage. Less fibrous tissue appeared at the various other treatments such as chondrocyte or stem cell subchondral bone interface, compared to saline treated con- implantation, with or without a scaffold material, or com- trols, assessed by histology-histochemistry methods [70] and bined with other medication such as anti-inflammatory peripheral quantitative computered tomography (pQCT) for drugs. Intermittent PTHR1 stimulation of chick chondrocyte the bone compartment. Retention of rhBMP within the os- in culture was shown to induce cell proliferation and proteo- teochondral grafts was measured in in vivo rabbit and non- glycan synthesis without turning on the hypertrophic pheno- human primate models. Radioactively labeled rhBMP was type as opposed to continuous stimulation induced progres- detectable up to 22days compared to 11days in the control sion to hypertrophic phenotype. [71]. The method of modulating repair/regeneration by means of recombinant GF has been already approved as a Intermittent exposure to PTH fragments 1-34 stimulated method for enhancing bone formation during spinal fusion cellular repair and suppression of hypertrophy in a mice procedures. Dosage and timing of treatment will need to be model of severe joint injury (cruciate and menisci trans sec- tion, a condition which induce severe posttraumatic OA in improved in order to increase safety and efficiency of such formulations for cartilage repair. both mice and humans). Chondrocyte maturation markers (MMP-13, ADAMTS-5, and PTHR-1) were immunohis- tologically detected and proven significantly increased in V. ACELLULAR SCAFFOLDS FOR CARTILAGE meniscal and cartilage tissue immediately after injury in REPAIR mice models and in human subjects as compared to normal The use of cell free biomaterials represents an appealing controls. PTH treatment of rabbit meniscal cells induced up- approach in cartilage repair. Such devices are intended to regulation of mRNA for cyclin D1 (CCND1), marker of me- repair the joint surface, restoring the cartilage integrity and niscal cell proliferation. In a rabbit model of meniscal injury, overall tissue mechanical properties acting as well as cell local administration of PTH for 4 weeks narrowed the defect attractant. Surrounding cell population from the subchondral and increased proteoglycan content and cellularity compared bone, synovial tissue or cartilage layer could be mobilized by to saline treated controls [69]. If proven successful in large the intrinsic properties of the material. The use of “smart animal models, the method of modulating PTH axis could be biomaterials’ incorporating slow release cytokines or GFs translated to preventing cartilage degeneration after trauma are proven to increase their efficiency [72]. The “in situ car- or in very early stages of OA. tilage regeneration” would avoid extracorporeal cell manipu- IV.C.3. EU granted patent EU2174674 relates to a lation facilitating regulatory approval and clinical accessibil- method of treating joint defects using recombinant BMPs, ity. Natural, synthetic and composite materials such as poly- recombinant human protein (rhBMP-2) aiming to regenerate glycolic acid (PGA), hyaluronan scaffold nanostructured 3D functional cartilage with good strength and stability. rhBMP- chitosan and chondroitin sulfate multilayers [73] or poly lac- 2 as a method for augmentation of cartilage repair is pre- tic-co-glycolid acid (PLGA) microspheres releasing collagen sented to heaving potential in treating OA, delaying or re- type-I and BMP-7 [74] are tested for such purposes. The use ducing the need for joint reconstructive surgery. It can be of hydrogels could be particularly beneficial as the cell free used to treat full thickness acute cartilage defects caused by scaffold could be delivered via miminal invasive procedures trauma or in combination with other therapies that result in and photoplymerized in situ reducing patient morbidity, inva- formation of an osteochondral defect within a joint. The re- siveness of the surgery and reducing costs [75]. combinant growth factor is added to a tissue source which V.1. US patent US8372394 presents a method of regen- can be autologus, allogeneic or synthetic osteochondral graft, erating cartilage defects using a monovalent salt of alginic 62 Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 Labusca et al. acid. The composition has the structure of a gel, which can proportionally amount. This results in a structure which has be contracted in the treated area with addition of calcium good mechanical strength and affinity for the living tissue. ions. It has the viscosity of 400-20000millipascal per second This structure is claimed to being suitable to be used as arti- (mPa/s) and sufficient fluidity to allow the minimally inva- ficial cartilage. The method of fabrication comprises three sive delivery within the joint. The use of a solution of CaCl2 steps; first a mixture of hyaluronic acid and collagen is pre- in the same time with the alginate salt increased local viscos- pared, then a second composition comprising proteoglycan ity up to 2000mPas or more. The authors claim in this condi- and collagen. First and second compositions, are mixed to- tions the method can be used for treating cartilage defects gether, freeze-dried and cross linked. anti gravitationally. MSCs can be embedded within the com- The resultant composition is pulverized, dispersed under position, however, the use of cell free scaffold is able to in- water and freeze dried for the second time. The artificial duce cartilage regeneration and prevents further degradation cartilage obtained by this method can be further sterilized by of the joint surface. The compound acts as a mechanical pro- irradiation. The collagen used can be of animal origin ob- tector, efficient in reducing pain and stiffness related to the tained from skins, , , tendons, internal organs joint injury. It is claimed that the composition has an effect of mammals such as cow, pig, horse, rabbit and rat, or birds in lowering joint inflammation and pain related to aging, such as chicken or various species of fish. Proteoglycans trauma, osteoarthritis, disc injury, meniscus injury or osteo- may be aggrecan, versican, neurocan, brevican, decorin, chondritis dissecans, inhibiting degenerative disease, and biglycan or others, can be mammalian, bird or fish sources. protecting the cartilage tissue [76]. Among the possible use of glycosaminoglicans such as V.2. US patent application US20120122791relates to a chondroitin sulfate, dermatan sulfate, heparan sulfate, ker- formulation of artificial collagen used as such or with differ- atan sulfate, heparin, nevertheless hyaluronic acid is pre- ent additives for cartilage regeneration. In the form of aque- ferred. The structure can be cross linked by a physical or ous solution it can be used for promotion of cartilage regen- chemical method, porosity is preferable within 60-99% with eration or as substrate for cartilage cultivation. The formula- pore diameter of 50-800m. The method of fabrication re- tion can be used even for treating a cartilage defect by means sults in formation of structures having high similarity to hu- of open or arthroscopic surgery, either as such, placed man cartilage in terms of composition, biomechanical and around a grafted cartilage defect, or as a vehicle for cell de- self-organization properties [78]. Further tests will be needed livery. By changing the concentration of artificial collagen to ascertain the safety, efficiency and properties of such used, different degrees of consistency of the solution can be structures when used in living organisms. obtained, accommodating the clinical needs. Cartilage cells can be of autologus or allogeneic source, adult chondrocytes VI. DRUGS FOR CARTILAGE REGENERATION or MSCs, cultivated prior to implantation to the desired cell Pharmaceutical intervention for cartilage regeneration volume. Arginine-glycine-aspartic acid sequence (RDG) can purposes stands as an area of incoming research. To date the be contained by the formulation to enhancing proliferation, only clinical available substance having chondroprotective adhesion and matrix production capability of the cells. The effect is sought to be hyaluronic acid. Hyaluronic acid salts artificial collagen aqueous solution has joint protective effect in different formulation are used primarily for pain relief in which is comparable with clinically available hyaluronic acid mild to severe stages of OA. Its use was clinically approved salts currently used for treating symptomatic knee and hip as a method for synovial fluid replacement by means of in- joints. Conditioned as a artificial collagen sponge for culti- tra-articular injections referred to as viscosupplementation vating rabbit chondrocytes, the formulation increased pro- [79]. Sodium hyaluronate-based commercially available vis- teoglycan, DNA as well as aggrecan, collagen type-II and cosupplementation devices have been reported to express a sox-9 gene expression compared to control cells cultivated chondroprotective effect by promoting the restoration of on bovine collagen matrix. The formulation of artificial col- proteoglycan (PG) depletion induced by chymopapain in lagen has the advantage of not having antigenicity and pri- rabbits [80]. Promotion of cartilage matrix synthesis and onic disease transmission risk. The formulation can be used proteoglycan reaggregation could be obtained with the use of as a scaffold for delivery of a cartilage growth factor. It was viscosupplemetation devices [81]. proven efficient in treating cartilage defects when injected intraarticularly within rabbit knees. Seven weeks after ad- VI.1. US granted patent US8119591 refers to a method for regenerating articular cartilage using an infiltration sup- ministration of artificial collagen solution, joint surface was pressor agent and a columnar growth promoting agent. The macroscopically smooth, showing significant International developmental characteristic of articular cartilage consists in Cartilage Repair Society score (ICRS) compared to the sa- columnar pattern of cell growth, which is not reproduced by line treated controls, the defects were reported to be filled any current available method of treating cartilage defects. with tissue positive for collagen type-II, based on Safranin-O Moreover, superficial and chondral joint surface defects are staining and immunohistochemistry [77]. Further medium usually either left untreated or converted to an osteochondral and long term studies will support the usefulness of artificial lesion in order to promote subchondral bone bleeding and the collagen formulations for clinical applications. promotion of a repair tissue. The invention presents a V.3. US patent application US20130116187 relates to a method for the combined use of one substance to inhibit method of manufacturing artificial cartilage composed of synoviocytes and fibroblasts infiltration to the defect site and cross linked collagen (15-95%), proteoglycan (4.9-70%) and of another to promote cartilage cell columnar growth. The hyaluronic acid 0.1-20%. The invention is based on freeze inhibitory agent is described to be either a corticoid steroid drying a dispersed composition of the three substances at such as dexamethasone either cartilage derived morphoge- Recent Patents in Cartilage Regeneration Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 63 netic proteins (CDMPs 1, 2) or combinations. The columnar sion arthroplasty, with graft procedures like osteochondral growth promoting agent can be a growth factor such as transfer or with cell therapy like ACT or mesenchymal stem BMPs -2, -4, -6, -7, FGFs such as FGF-8, IGFs such as IGF- cell therapy. The invention offers as well a method for en- 1, and combinations thereof. The inhibitory and stimulatory hancing ECM of a bioengineered cartilage. Chondrocytes agents can be delivered separately or mixed together in the harvested from different cartilage zones can be cultured on a area of cartilage damage during arthroscopic or open surgery substrate in the presence of short chain polyphosphate to procedures. Antibiotics, anti-inflammatory compounds, anti- increase thickness, dry weight, proteoglycan and collagen viral agents, anti-cancer agents can be added to the formula- content of the engineered tissue. Short chain polyphosphates tion. Inhibitory cocktail of 10μmol of dexamethasone and can be administered continuously during in vitro chondro- 40μg of CDMP-2 was administered three days after induc- cyte culture (1-5weeks) or periodically (e.g., at 3, 4, 5, 6 tion of chondral defects in sheep knee, followed by columnar days after initiation of the culture. The method has been effi- growth stimulatory cocktail of BMP-7 (50μg), dexametha- cient in stimulating cartilage matrix deposition in cell culture sone (10μmol) and CDMP-2 (40μg), at 10, 40, 70, and 120 of deep zone calves chondrocytes cultivated on membrane days after initial surgery Eight months (240 days) after initial inserts and in punch biopsy-sized tissue culture. Cultures surgery, histology revealed that chondral defects were totally exposed to different concentration of inorganic short chain or partially filled by formation of new articular chondrocytes polyphosphates had higher dry weight, DNA, collagen and organized into columns within chondrocytic lacunae as well proteoglycan content. Immunofluorescence showed accumu- as newly deposited extracellular matrix [82]. This approach lation of administered polyphosphate within new formed of reproducing developmental cartilage growth could result cartilage matrix. Intraarticular injection of polyphosphate in the regeneration of endurant adult cartilage. (PP) stopped OA progression in guinea pig model after 2 times treatment per week intraarticular administration, as VI.2. World patent application WO2012082013 refers to assessed by histology of cartilage samples, with significantly a pharmaceutical composition in form of a suspension con- taining a substance called "Mitsellat of calcium carbonate increased in collagen and proteoglycan normalized to DNA content compared to untreated controls [84]. If proven suc- and magnesium" claimed to induce bone and cartilage regen- cessful in large animal models of OA and in longer follow eration claimed successful in animal models applied for the up, the method could make an easy to use form of cartilage prevention and treatment of musculoskeletal tissue regenera- regeneration procedure. tion which is particularly efficient in treatment of diabetic patients [83]. If proven successful in animal models, such approach will contribute to the design of specific therapies VII. PHYSICAL INTERVENTION FOR CARTILAGE for cartilage damage in the context of metabolic diseases. REGENERATION VI.3. WO patent applicationWO2012094739 refers to a Physical interventions such as bracing [85], magnetic method of stimulating cartilage growth and regeneration for therapy [86], or electrotherapy are commonly applied in the the treatment of cartilage defects or diseases. The method treatment of cartilage pathology as conservative methods for involves intraarticular administration of a pharmaceutical symptomatic relief. More evidence is accumulating regard- composition containing therapeutically effective amount of a ing their role in improving range of motion (ROM) in reduc- short chain polyphosphate containing more than 5 phosphate ing joint pain and in improving the quality of life and the units. Inorganic phosphate of various amounts are claimed to self-perceived health and physical functions [87]. Electro- stimulate cartilage growth, regeneration reducing cartilage therapeutic control of cell function is under investigation for mineralization when introduced at the site requiring treat- their role in regenerating connective tissues. The hypothesis ment by intraarticular delivery within ankle, knee, hip, fin- is based on the assumption that trans-membrane molecular ger, wrist, elbow, shoulder joints. ECM restoration is flux modulated via externally applied electric fields can im- achieved by the increase of collagen and proteoglycan con- prove the quality of the cartilage and bone regeneration and tent at the site of cartilage regeneration. It is claimed to pre- accelerate the integration of engineered grafts after in vivo vent the progression of cartilage degradation at sites where implantation [88]. Microcurrent application was reported as traumatic or degenerative changes have occurred by restor- an efficient application in accelerating repair of non-articular ing and/or increasing cartilage matrix accumulation. The hyaline cartilage in prepuberal rats [89]. invention could have particular application in osteoarthritis VII.1. US patent application US20120165848 relates to a treatment. Short chain polyphosphates can be delivered lo- method for treating injured or degenerated cartilage using cally in a slow releasing manner incorporated within a car- ultrasound energy. It is intended to be used as a modality to rier, excipient or vehicle, a biodegradable polymer or mole- target the cartilage defect or OA lesion via an arthroscopic cule, or by oral administration. When intended for stopping probe, which has a housing component containing the ultra- progression of osteoarthritis, the polyphosphates can be sound transducer on a distal end of the probe and a control- combined for intraarticular delivery with corticosteroids such ler, which controlling the probe. The housing device can as methylprednisolone acetate 1-25mg/ml or a viscosupple- contain a position sensor, an interface for wireless communi- mentation agent. The method can be used in combination cation and a rechargeable power supply. The cartilage defect with symptomatic treatment of OA such as non-steroidal can be accessed and the injured or deteriorated portion of the anti-inflammatory drugs (NSAIDS), analgesics, viscosup- cartilage can be ablated via the ultrasound probe. The probe plementation, and nutriceuticals. It can be used as well in is used to induce micro fractures at the site of the injury in combination with known surgical methods for cartilage re- order to initiate re-growth of cartilage. The method can be pair, which induce fibrocartilage formation e.g. bone marrow used as well for welding a portion of cartilage with the con- stimulation techniques, microfracture or Pridie drilling, abra- formal distribution of ultrasound aiming to initiate re-growth 64 Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 Labusca et al. of cartilage to the subchondral bone. The therapeutic ultra- chondrocytes [93]. Curcumin (diferuloylmethane), the major sound may be streamed for different purposes. One ultra- constituent of turmeric has been widelly investigated based sound energy effect can be applied for the ablative and hae- on the antiinflamatory, anti-oxidant as well as analgesic mostatic and the second for the hydrodynamic, diathermic, properties. In human cartilage explants or chondrocyte cul- and resonance induced tissue effects. The method claims to ture. It was reported inhibiting the the catabolic and degrada- be protective for the tissue layers above the cartilage defect tive effects after IL-1, LPS, and TNF- exposure inhibiting i.e. skin, subcutaneous tissue and muscle and other surround- the production of MMP-3, MMP-9, and MMP-13 [94] re- ing joint structures. Application of ultrasound energy in- storing glucosaminoglycans (GAG) production and type-II creases the heat shock proteins and promote white blood collagen synthesis [95]. Food and synthetic compounds are cells mediated healing of the targeted areas of cartilage in the proposed to having beneficial effect on regenerating cartilage muscle and connective tissue layer, increase local [96]. release, causing partial shrinking of the collagen and alters the protein configuration in the region of interest (ROI). The VIII.1. US patent application US20130005682 referes to biological effects on the treated tissues are claimed to be the use of hydroxytyrosol [4-(2-hydroxylethyl)-1,2- immediate or delayed cell apoptosis at the site of injury, col- benzenediol, CAS: 10597-60-1] for inducing cartilage repair lagen remodelling, modification of biochemical cascades at and regeneration. The invention claims that hydroxytyrosol the site of injury (e.g., wound healing), increasing the blood induce or enhance chondrocyte proliferation, increase ECM perfusion, stimulation of the collagen production, cell protein production and consequently promotes cartilage re- growth, angiogenesis and cell permeability modification. generation. The substance is proposed to be useful for the The method is claimed to be useful in order to remove por- treatment of cartilage injury induced by trauma, promoting tions of degenerated cartilage without damaging the adjacent joint health, prevent stiffness and increase mobility in the structures, during micro fracture surgery or for the treatment affected joints. The compound can be produced by several of degenerative disc disease in the spine [90]. This could steps of 3,4-dihydroxymandelic acid hydrogenation or from represent an easy to use method for cartilage stimulation, natural sources e.g., products and by-products derived from further histological and biomechanical analysis of the tissue the olive tree. It is claimed that the product can be used to will be needed to ascertain the quality of repair. manufacture different forms of dietary supplements such as food bars or meal replacement formulations along with ga- VIII. FOOD SUPPLEMENTS IN CARTILAGE RE- lenic formulation for oral or topical administration. The ef- GENERATION fective amount of hydroxytyrosol in the blood plasma after Food supplements are extensively used as adjuvant ther- administration is estimated between 10-15μM [97]. This apy for the treatments of symptoms associated with different approach could overcome the issues with standardization of forms of cartilage deterioration. Glucosamine and chon- bioactive compounds encountered with the use of food ex- droitine sulphate (CS) preparations have been reported to tracts. Further studies are needed to analyse the quality of the successful in the treatment of the knee, finger and interverte- repair tissue. bral disc symptomatic amelioration, due to stimulatory effect on chondrocyte, ECM protein production and inhibition of SUMMARY matrix breakdown. Major concerns exist about the quality of Scrutiny of the latest patent literature in cartilage regen- the studies and reports of clinical results followed by the eration allows for several general remarks. Some of the cur- potential bias introduced by the company sponsorship and rent granted patents or patent applications are developing the quality of the available food supplements preparation. It already existent methods in cartilage repair such as cartilage has been proposed that glucosamine and CS can pass the defect grafting aiming to improve known shortcomings of gastrointestinal tract reaching cartilage tissue, potentially existent procedures such as graft availability or stability after having a chondroprotective effect and, presumably a regen- implantation. A large cathegory of patents are reffering to erative effect. However the biochemical mechanism uderly- implementation of cartilage engineering methods focusing ing the hypothesised effects needs further clarification [91]. on one or another of the compounds of the so called tissue Biochemical mechanisms underlying some of the tradition- engineering triad (cells, biomaterials and bioactive mole- ally used food supplements, fruits and herbs extracts such as cules). Identification of new cell source in the full depths of pomegranate, green tea, cat’s claw, devil’s claw, ginger, In- mature cartilage opens to the possibility of using local pro- dian olibaum, turmeric and ananas have been reported. genitor populations for regenerating cartilage. Interesting Green tea catechins, especially epigallocatechin-3-gallate developments are provided by the patents focusing on (EGCG) accounting for 59% of the dry weight extract are growth factor or growth modulators delivery. Due to their reported to induce inhibitory effect on proteolitic enzymes simplicity in application such methods could develop into (MMP-1, MMP-13, ADAMT-1, 4) in OA chondrocytes, efficient therapies. However, further studies to certify their supressing IL-1, IL-6 TNF- induced inflammatory re- safety and efficiency in clinical settings are needed. sponse in human chondrocytes [92]. EGCG can regulate the expression of cytokines, chemokines, MMPs, aggrecanase, Acellular scaffolds designed to attract surrounding cell ROS, NO and COX-2. It has been shown to inhibiting ad- populations aiming in situ cartilage regeneration composed vanced glycated end products (AGE) -stimulated expression of biomaterials which are both endurant and easy to handle of TNF- and MMP-13 effect mediated via suppression of offers the perspective of simplifyed regulatory approval for p38-MAPK, JNK, and NF-B activation in human OA chon- product development. Recent Patents in Cartilage Regeneration Recent Patents on Regenerative Medicine 2014, Vol. 4, No. 1 65

Artificial cartilage substitute could find their place bridg- stages or of particular joint environments after injury. Like- ing cartilage repair and joint replacement procedures. Con- wise, the direct effect of stem cell therapy could be applied trolled production of bioactive compounds in food extracts after biomarker based identification of preclinical and pre- and/or natural sources could generate quantitative and quali- imagistic stages of OA or PTA. such as the anabolic effect in tative studies on their effect on cartilage metabolism setting inducing regenerative processes and capability of modulating up their use based on scientific principles. inflammation, which can have significant impact in disease management [101]. Going to the next level, the molecular CURRENT & FUTURE DEVELOPMENTS mechanisms involved in pathological responses as well as in tissue repair could be clearly mapped and organized. Com- Today as couple of centuries ago, cartilage defects and/or plex intricate determinants such as metabolic context, expo- degeneration remain problems difficult to manage and still a sure to environment and seasonal factors, dietary influence potential disabling factor for an increasing fraction of popu- and physical activity could be correctly evaluated in order to lation. Different attempts are made to meet the challenge of producing a functionally well integrated structure with as improve the treatment choice. Computer modeling of the close as possible similarity to the native cartilage. The large joint microenvironment and its functioning at macro scale number of papers being published yearly and the impressive could predict the immediate and long term effects of a cer- number of patents issued on the topic demonstrate that an tain therapy, waiving the need for animal studies and facili- ultimate solution is still to be found. Every each approach tating the marketing approval of truly regenerative proce- presented has its advantages and disadvantages; however dures. none would have the potential to offer a solution suitable for every type of cartilage disease. Any attempt to regenerate the CONFLICT OF INTEREST joint surface will need to address the specific molecular, biomechanical and metabolic characteristic of the joint envi- The authors confirm that this article content has no con- ronment as well as patient’s expectation for functional re- flict of interest. covery. There is a necessity to redefine all the mentioned particularities of joint functioning by approaching cartilage ACKNOWLEDGEMENTS pathology in a descriptive as well as an integrative manner. The authors are thankful to the reviewers of this manu- Thus, a classification of cartilage degenerative diseases as script for their significant contribution in improving the con- well as of individual response after trauma at a molecular tent and the aspect of the manuscript. level would define target(s) for existent and emerging thera- pies leading to the discovery and patenting of a new genera- tion of molecules to be used in the prevention and treatment ABBREVIATIONS of cartilage tissue deterioration. This could be further inte- ACI/ACT = Autologus chondrocyte implanta- grated within the complex micro and macro scale systems in tion/transplantation which the tissue is functioning. Particularities related to joint [99] as well as individual differences in tissue metabolism BMP = Bone marrow protein could be differentially treated for improving results. ECM = Extracellular matrix It is obvious that the biomechanical, metabolic and func- EP = European Patent tional context of a limited cartilage defect is different from any of the OA stages; however there is no precise description GDF = Growth differentiation factor and no parameters to define this. Posttraumatic OA is the end GF = Growth factor stage of several years of evolution of a traumatized joint hav- ing symptomatic, histological and imagistic similarity with ICRS = International cartilage repair society primary OA. However, large individual variability exists MACI/MACT = Matrix assisted chondrocyte implanta- regarding the joint response to injury as there are in integrat- tion/transplantation ing the effect treatments such as open reduction and internal fixation (ORIF) procedure or arthroscopic repair. Moreover OA = Osteoarthritis the degree of professional or recreational use of the affected PTA = Posttraumatic arthritis joint is ultimately individual. Perceiving cartilage structure as a dynamic result of complex intricate determinants at a TGF = Transforming growth factor beta micro- and macro scale would enable the choice of the most WO = Short from suitable repair, regeneration or joint reconstruction strategy. Assessment of quality of the repair (fibrous or hyaline, fetal WIPO = World intellectual property organization or adult mature cartilage) would increase predictability of the results enabling correct evaluation of recovery time and pa- REFERENCES tient rehabilitation [99]. [1] Bitton R. The economic burden of osteoarthritis. Am J Manag Care The use of advanced genomic technologies and systems 2009;15(8 Suppl): 230-5. biology approach is sought to identify OA specific biomark- [2] Wang Y, Wei L, Zeng L, He D, Wei X. Nutrition and degeneration of articular cartilage. 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