Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing Thomas J. Koob, Jeremy J. Lim, Nicole Zabek, Michelle Massee MiMedx Group, Inc., 1775 West Oak Commons Court NE, Marietta, Georgia Received 21 March 2014; revised 17 June 2014; accepted 8 August 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33265 Abstract: Human amniotic membrane allografts have proven grafts containing amnion and chorion are currently marketed effective at improving healing of cutaneous wounds. The for wound repair. To examine the role of tissue processing mechanism of action for these therapeutic effects is poorly technique in cytokine retention, cytokine contents in repre- understood but is thought to involve the resident growth fac- sentative dehydrated single layer wound care products were tors present in near term amniotic tissue. To determine the measured. The results demonstrated that cytokine content relative cytokine contribution of the amnion and chorion in varied significantly among the allografts tested, and that amniotic allografts, the content of 18 cytokines involved in PURIONVR Processed single layer amnion grafts contained wound healing were measured in samples of PURIONVR more cytokines than other single layer products. These Processed dehydrated amnion, chorion, and amnion/chorion results suggest that PURIONVR Processed dHACM contains membrane (dHACM) grafts by multiplex enzyme-linked substantially more cytokines than single layer amnion prod- immunosorbent assay array. Both amnion and chorion con- ucts, and therefore dHACM may be more effective at deliver- tained similar amounts of each factor when normalized per ing growth factors to a healing wound than amnion alone. dry weight; however, when calculated per surface area of tis- VC 2014 The Authors. Journal of Biomedical Materials Research Part B: sue applied to a wound, amnion contained on average only Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed 25% as much of each factor as the chorion. Therefore, an Mater Res Part B: Appl Biomater 00B: 000–000, 2014. allograft containing both amnion and chorion would contain four to five times more cytokine than a single layer amnion Key Words: wound healing, growth factor, amniotic mem- allograft alone. Both single layer amnion and multilayer allo- brane, dHACM How to cite this article: Koob TJ, Lim JJ, Zabek N, Massee M. 2014. Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing. J Biomed Mater Res Part B 2014:00B:000–000. INTRODUCTION brane that is underlain by a compact, collagen-rich tissue. Biological tissues from a variety of sources have been used The chorion is comprised primarily of dense collagen fibers to treat non-healing wounds. Skin autografts and human in an interfibrillar matrix containing proteoglycans and elas- skin allografts have been employed extensively for burns tic fibers. Cells are distributed throughout the amniotic and chronic wounds. A number of animal-derived xenograft membrane. Neither the amnion nor chorion is vascularized. tissues including porcine skin, urinary bladder, and small The amniotic membrane is a metabolically active tissue that intestinal submucosa (SIS) have been developed and mar- continually remodels and grows to accommodate the grow- keted for treatment of non-healing, topical wounds. Both ing conceptus. Remodeling of the tissue is governed by human allografts and xenografts require decellularization to growth factors, cytokines, chemokines, and related regula- remove immunoreactive cellular components, leaving an tory factors produced by the endogenous cells in the amni- acellular extracellular matrix scaffold for repair with varying otic membrane. amounts of biologically active factors. Recently human amni- Human amniotic membranes in the form of fresh, frozen, otic membrane allograft tissues have increased in popularity or minimally processed tissue allografts have proven effec- for treatment of non-healing wounds, partly due to their tive at improving healing of ophthalmic injuries, burns, and non-immunogenic properties; however, these tissues remain chronic wounds. Aside from the well-established barrier relatively poorly characterized. function, the mechanism of action for these therapeutic Human amniotic membrane is comprised of two distinct effects is poorly understood but is thought to involve the but conjoined tissues, amnion and chorion, both derived resident growth factors and cytokines normally present in from the inner layer of the placenta. The amnion faces the near term amniotic tissue. Little is known about what fetus and the chorion faces the uterus. The amnion consists growth factors and cytokines are present in amniotic mem- of a layer of epithelial cells anchored to a basement mem- brane; however, growth factors including but not limited to Correspondence to: T. J. Koob; e-mail: [email protected] Contract grant sponsor: MiMedx Group, Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. VC 2014 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. 1 epidermal growth factor (EGF), basic fibroblast growth fac- Human placentas were donated under informed consent, tor (bFGF), keratinocyte growth factor (KGF), transforming following Caesarean sections, as regulated by the Food and growth factor (TGF) alpha and beta, hepatocyte growth fac- Drug Administration’s (FDA) Good Tissue Practice and tor (HGF), and nerve growth factor (NGF) have been identi- American Association of Tissue Banks (AATB). All donors fied within fresh and preserved amniotic tissues.1–3 were tested to be free of infectious diseases, including HIV, In order to preserve the bioactivity of fresh tissues, amni- HTLV, Hepatitis B and C, syphilis, and CMV. Amnion and cho- otic membranes have been frozen or dehydrated to prevent rion were isolated from placenta and processed with a pro- denaturation of the growth factors within the tissue. Once prietary PURIONVR Process that involves gentle cleansing of dehydrated, membranes can be stored at room temperature the layers. The amnion and chorion were either processed without risk of hydrolysis and degradation. Dehydrated separately, or laminated to form a two layer graft, and the human amnion/chorion membranes (dHACM) contain a tissues were dehydrated under controlled drying condi- number of growth factors that are known to play a role in tions.21 EpiFixVR (MiMedx Group, Marietta, GA) was used as normal wound healing, including cell recruitment and prolif- the bilayer dHACM in this study. eration, modulation of inflammation, and regulation of ECM Human amnion allografts sold in a dry configuration degradation/synthesis.4 Previous analyses have determined were obtained commercially and directly prepared for that PURIONVR Processed dHACM contains over 50 growth enzyme-linked immunosorbent assays (ELISAs) and histol- factors, cytokines, chemokines, and regulatory factors.5 ogy. The products tested were BioD DryFlex (n 5 5), BioD Human amniotic membranes have been used extensively AmnioExCel (n 5 5), Bone Bank SteriShield Single Layer to promote healing in ophthalmic injuries,6–9 and recent (n 5 1), and Bone Bank SteriShield II Dual Layer (n 5 2). study has established that amniotic membranes are an effec- tive therapy for healing of chronic cutaneous wounds.10,11 ELISAs Human amniotic membrane allografts have been shown to Samples of amnion and chorion were PURIONVR Processed reduce inflammation, pain, and scarring while promoting separately, sterilized, weighed, and prepared for growth fac- accelerated wound healing.10–18 Amniotic membrane allo- tor analyses. Weighed, minced samples were placed in lysis grafts also provide a biological barrier for the wound, as buffer containing protease inhibitors for 24 hours at 4C. well as a matrix for cell proliferation and tissue growth. Tissues were then homogenized, centrifuged to remove tis- In order to ensure that safe amniotic tissue allografts can sue residue, and standard ELISAs were used to measure the be obtained while also preserving bioactivity for clinical effec- content of each growth factor/cytokine (RayBiotech, Inc., tiveness and stability for long-term storage and off the shelf Norcross, GA). Fifteen growth factors/cytokines were meas- availability, MiMedx Group, Inc. (Marietta, GA) developed a ured in each sample. Three metalloproteinase inhibitors gentle cleansing and dehydration process (PURIONVR Process) were also measured. Growth factor content was normalized to preserve and maintain the biological activities inherent in to the dry mass of starting tissue or to the surface area of native amniotic tissue.19–21 Both single layer amnion allografts the tissue. (AmbioDry2VR ) and amnion/chorion composite grafts (EpiFixVR and AmnioFixVR ) are produced using the PURIONVR Process. Multiplex ELISA arrays Previous reports have established that PURIONVR Proc- Content of growth factors in EpiFix, DryFlex, AmnioExCel, essed dHACM grafts contain a large cohort of regulatory fac- SteriShield, and Sterishield II was measured with multiplex tors involved in inflammation and tissue regeneration, and ELISA arrays (RayBiotech, Inc.), which are capable of