PURION® processed human amnion chorion membrane allografts retain material and biological properties supportive of soft tissue repair.

The reparative properties of amniotic membrane allografts are well-suited for a broad spectrum of specialties. Further enhancement of their utility can be achieved by designing to the needs of each application through the development of novel processing techniques and tissue configurations. As such, this study evaluated the material characteristics and biological properties of two PURION® processed amniotic membrane products, a lyophilized human amnion, intermediate layer, and chorion membrane (LHACM) and a dehydrated human amnion, chorion membrane (DHACM). LHACM is thicker; therefore, its handling properties are ideal for deep, soft tissue deficits; whereas DHACM is more similar to a film-like overlay and may be used for shallow defects or surgical on-lays. Characterization of the similarities and differences between LHACM and DHACM was conducted through a series of in vitro and in vivo studies relevant to the healing cascade. Compositional analysis was performed through histological staining along with assessment of barrier membrane properties through equilibrium dialysis. In vitro cellular response was assessed in fibroblasts and endothelial cells using cell proliferation, migration, and metabolic assays. The in vivo cellular response was assessed in an athymic nude mouse subcutaneous implantation model. The results indicated the PURION® process preserved the native membrane structure, nonviable cells and collagen distributed in the individual layers of both products. Although, LHACM is thicker than DHACM, a similar composition of growth factors, cytokines, chemokines and proteases is retained and consequently elicit comparable in vitro and in vivo cellular responses. In culture, both treatments behaved as potent mitogens, chemoattractants and stimulants, which translated to the promotion of cellular infiltration, neocollagen deposition and angiogenesis in a murine model. PURION® processed LHACM and DHACM differ in physical properties but possess similar in vitro and in vivo activities highlighting the impact of processing method on the versatility of clinical use of amniotic membrane allografts.

Dehydrated human amniotic membrane regulates tenocyte expression and angiogenesis in vitro: Implications for a therapeutic treatment of tendinopathy.

Tendon injuries are among the most common ailments of the musculoskeletal system. Prolonged inflammation and persistent vasculature are common complications associated with poor healing. Damaged tendon, replaced with scar tissue, never completely regains the native structural or biomechanical properties. This study evaluated the effects of micronized dehydrated human amnion/chorion membrane (µdHACM) on the inflammatory environment and hypervascularity associated with tendinopathy. Stimulation of human tenocytes with interleukin-1 beta (IL1ß) induced the expression of inflammatory and catabolic markers, resulting in secretion of active MMPs and type 3 collagen that is associated with a degenerative phenotype. Treatment with µdHACM diminished the effects of IL1ß, reducing the expression of inflammatory genes, proteases, and extracellular matrix components, and decreasing the presence of active MMP and type 3 collagen. Additionally, a co-culture model was developed to evaluate the effects of µdHACM on angiogenesis associated with tendinopathy. Micronized dHACM differentially regulated angiogenesis depending upon the cellular environment in which it was placed. This phenomenon can be explained in part through the detection of both angiogenic protagonists and antagonists in µdHACM. Observations from this study identify a mechanism by which µdHACM regulates inflammatory processes and angiogenesis in vitro, two key pathways implicated in tendinopathic injuries.

Dehydrated Human Amniotic Membrane Inhibits Myofibroblast Contraction through the Regulation of the TGFß‒SMAD Pathway In Vitro.

Excessive fibrosis affects more than 100 million patients yearly, leading to the accumulation of extracellular matrix that compromises tissue architecture and impedes its function. Intrinsic properties of the amniotic membrane have alluded to its potential to inhibit excessive fibrosis; therefore, this study aimed to investigate the effects of dehydrated human amnion/chorion membrane (dHACM) on dermal fibroblasts and their role in fibrotic pathways. Human dermal fibroblasts were stimulated with TGFß1, triggering myofibroblast-like characteristics in vitro. Subsequent addition of dHACM in the continued presence of TGFß1 inhibited downstream signaling, leading to a reduction in the expression of known fibrotic and extracellular matrix genes. In addition, dHACM decreased alpha-smooth muscle actin, a stress filament responsible for contractile activity in scarring. The functional outcome of these effects was observed in an ex vivo model for cellular contraction. Hyperactivation of TGFß signaling increased the contractile capacity of myofibroblasts embedded within a collagen substrate. Simultaneous addition of dHACM treatment prevented the marked contraction, which is likely a direct result of the inhibition of TGFß signaling mentioned earlier. These observations may support the use of dHACM in the regulation of fibroblast activity as it relates to tissue fibrosis.

Dehydrated human amniotic membrane modulates canonical Wnt signaling in multiple cell types in vitro.

Canonical Wnt signaling is a major pathway known to regulate diverse physiological processes in multicellular organisms. Signaling is tightly regulated by feedback mechanisms; however, persistent dysregulation of this pathway is implicated in the progression of multiple disease states. In this study, proteomic analysis identified endogenous Wnt antagonists in micronized dehydrated human amnion/chorion membrane (µdHACM); thereby, prompting a study to further characterize the intrinsic properties of µdHACM as it relates to Wnt activity, in vitro. A TCF/LEF reporter cell line demonstrated the general ability of µdHACM to inhibit ß-catenin induced transcription activity. Furthermore, in vitro systems, modeling elevated Wnt signaling, were developed in relevant cell types including tenocytes, synoviocytes, and human dermal fibroblasts (HDFs). Stimulation of these cells with Wnt3A resulted in translocation of ß-catenin to the nucleus and increased expression of Wnt related genes. The subsequent addition of µdHACM, in the continued presence of Wnt-stimulus, mitigated the downstream effects of Wnt3A in tenocytes, synoviocytes, and HDFs. Nuclear localization of ß-catenin was abated with corresponding reduction of Wnt related gene expression. These data demonstrate the in vitro regulation of canonical Wnt signaling as an inherent property of µdHACM and a novel mechanism of action.

Human amniotic membrane modulates Wnt/ß-catenin and NF-κß signaling pathways in articular chondrocytes in vitro.

Objective: Inflammation, catabolism, and hypertrophy in chondrocytes play a central role in osteoarthritis (OA). The Wnt/ß-catenin and NF-κß pathways contribute to these degradative processes. This in vitro study evaluates the inhibitory effect of a novel therapeutic, micronized dehydrated human amnion/chorion membrane (µdHACM), as a potential treatment to offset elevated Wnt/ß-catenin and NF-κß signaling. Design: Three-dimensional human articular chondrocyte pellets were stimulated with an inflammatory cocktail to induce a degenerative phenotype. Treatments included varying doses of µdHACM. Protein and gene expression were analyzed using qRT-PCR, immunoblotting, and immunofluorescence to assess changes in the major constituents of Wnt/ß-catenin and NF-κß signaling. Regulation of catabolic activity was evaluated using enzymatic assays that detect MMP-13 and aggrecanase-mediated degradation products in conditioned media. Results: Confirmation of the model was established through the expression of specific markers and extracellular matrix genes, verifying a chondrogenic phenotype was maintained. Inflammatory stimulation elicited a change in the chondrocyte proteome and secretome, elevating Wnt/ß-catenin and NF-κß signaling and downstream expression of inflammatory, proteolytic, and hypertrophic markers, while decreasing primary cartilage matrix components, ACAN and COL2A1. µdHACM reversed these inflammatory-induced changes, suppressing phospho-GSK-3ß, ß-catenin expression/nuclear localization of the Wnt signaling axis and inhibiting IKKß, phospho-IκBα, and phospho-p65 in the NF-κß signaling cascade. Additionally, µdHACM altered expression of direct downstream targets, namely MCP1, MMP3, MMP13, ADAMTS4, ADAMTS5, RUNX2 and COL10A1. Moreover, µdHACM reduced MMP-13 and aggrecanase-mediated substrate degradation. Conclusion: µdHACM ameloriated the effects of inflammatory-induced degeneration in chondrocytes through Wnt/ß-catenin and NF-κß inhibition, subsequently downregulating key inflammatory, hypertrophic and catabolic mediators in vitro.

Evaluation of dehydrated human umbilical cord biological properties for wound care and soft tissue healing.

Chronic wounds are a significant health care problem with serious implications for quality of life because they do not properly heal and often require therapeutic intervention. Amniotic membrane allografts have been successfully used as a biologic therapy to promote soft tissue healing; however, the umbilical cord, another placental-derived tissue, has also recently garnered interest because of its unique composition but similar placental tissue origin. The aim of this study was to characterize PURION® PLUS Processed dehydrated human umbilical cord (dHUC) and evaluate the biological properties of this tissue that contribute to healing. This was performed through the characterization of the tissue composition, evaluation of in vitro cellular response to dHUC treatment, and in vivo bioresorption and tissue response in a rat model. It was observed that dHUC contains collagen I, hyaluronic acid, laminin, and fibronectin. Additionally, 461 proteins that consist of growth factors and cytokines, inflammatory modulators, chemokines, proteases and inhibitors, adhesion molecules, signaling receptors, membrane-bound proteins, and other soluble regulators were detected. Cell-based assays demonstrated an increase in adipose-derived stem cell and mesenchymal stem cell proliferation, fibroblast migration and endothelial progenitor cell vessel formation in a dose-dependent manner after dHUC treatment. Lastly, rat subcutaneous implantation demonstrated biocompatibility since dHUC allografts were resorbed without fibrous encapsulation. These findings establish that dHUC possesses biological properties that stimulate cellular responses important for soft tissue healing. © 2018 The Authors. Journal Of Biomedical Materials Research Part B: Applied Biomaterials Published By Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1035-1046, 2019.

Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane.

Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair.

Type I and II Diabetic Adipose-Derived Stem Cells Respond In Vitro to Dehydrated Human Amnion/Chorion Membrane Allograft Treatment by Increasing Proliferation, Migration, and Altering Cytokine Secretion.

Objective: Human amniotic membranes have been shown to be effective for healing diabetic foot ulcers clinically and to regulate stem cell activity in vitro and in vivo; however, diabetic stem cells may be impaired as a sequela of the disease. In this study, dehydrated human amnion/chorion membrane (dHACM) allografts (EpiFix®; MiMedx Group) were evaluated for their ability to regulate diabetic stem cells in vitro. Approach: Human adipose-derived stem cells (ADSCs) from normal, type I diabetic, and type II diabetic donors were treated with soluble extracts of dHACM and evaluated for proliferation after 3 days by DNA assay, chemotactic migration after 1 day by transwell assay, cytokine secretion after 3 days by multiplex ELISA, and gene expression after 5 days by reverse transcription-polymerase chain reaction. Results: Although diabetic ADSCs demonstrated decreased responses compared to normal ADSCs, dHACM treatment stimulated diabetic ADSCs to proliferate after 3 days and enhanced migration over 24 h, similar to normal ADSCs. dHACM-treated diabetic ADSCs modulated secretion of soluble signals, including regulators of inflammation, angiogenesis, and healing. All ADSCs evaluated also responded to dHACM treatment with altered expression of immunomodulatory genes, including interleukins (IL)-1α, IL-1ß, and IL-1RA. Innovation: This is the first reported case demonstrating that diabetic ADSCs respond to novel amniotic membrane therapies, specifically treatment with dHACM. Conclusion: dHACM stimulated diabetic ADSCs to migrate, proliferate, and alter cytokine expression suggesting that, despite their diabetic origin, ADSCs may respond to dHACM to accelerate diabetic wound healing.

Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro.

Human-derived placental tissues have been shown in randomized clinical trials to be effective for healing chronic wounds, and have also demonstrated the ability to recruit stem cells to the wound site in vitro and in vivo. In this study, PURION(®) Processed dehydrated human amnion/chorion membrane allografts (dHACM, EpiFix(®) , MiMedx Group, Marietta, GA) were evaluated for their ability to alter stem cell activity in vitro. Human bone marrow mesenchymal stem cells (BM-MSCs), adipose derived stem cells (ADSCs), and hematopoietic stem cells (HSCs) were treated with soluble extracts of dHACM tissue, and were evaluated for cellular proliferation, migration, and cytokine secretion. Stem cells were analyzed for cell number by DNA assay after 24 h, closure of an acellular zone using microscopy over 3 days, and soluble cytokine production in the medium of treated stem cells was analyzed after 3 days using a multiplex ELISA array. Treatment with soluble extracts of dHACM tissue stimulated BM-MSCs, ADSCs, and HSCs to proliferate with a significant increase in cell number after 24 h. dHACM treatment accelerated closure of an acellular zone by ADSCs and BM-MSCs after 3 days, compared to basal medium. BM-MSCs, ADSCs, and HSCs also modulated endogenous production of a number of various soluble signals, including regulators of inflammation, mitogenesis, and wound healing. dHACM treatment promoted increased proliferation and migration of ADSCs, BM-MSCs, and HSCs, along with modulation of secreted proteins from those cells. Therefore, dHACM may impact wound healing by amplifying host stem cell populations and modulating their responses in treated wound tissues. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1495-1503, 2016.

Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing.

Human amniotic membrane allografts have proven effective at improving healing of cutaneous wounds. The mechanism of action for these therapeutic effects is poorly understood but is thought to involve the resident growth factors present in near term amniotic tissue. To determine the relative cytokine contribution of the amnion and chorion in amniotic allografts, the content of 18 cytokines involved in wound healing were measured in samples of PURION® Processed dehydrated amnion, chorion, and amnion/chorion membrane (dHACM) grafts by multiplex enzyme-linked immunosorbent assay array. Both amnion and chorion contained similar amounts of each factor when normalized per dry weight; however, when calculated per surface area of tissue applied to a wound, amnion contained on average only 25% as much of each factor as the chorion. Therefore, an allograft containing both amnion and chorion would contain four to five times more cytokine than a single layer amnion allograft alone. Both single layer amnion and multilayer allografts containing amnion and chorion are currently marketed for wound repair. To examine the role of tissue processing technique in cytokine retention, cytokine contents in representative dehydrated single layer wound care products were measured. The results demonstrated that cytokine content varied significantly among the allografts tested, and that PURION® Processed single layer amnion grafts contained more cytokines than other single layer products. These results suggest that PURION® Processed dHACM contains substantially more cytokines than single layer amnion products, and therefore dHACM may be more effective at delivering growth factors to a healing wound than amnion alone.

Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration.

BACKGROUND: Chronic wounds are associated with a number of deficiencies in critical wound healing processes, including growth factor signaling and neovascularization. Human-derived placental tissues are rich in regenerative cytokines and have been shown in randomized clinical trials to be effective for healing chronic wounds. In this study, PURION® Processed (MiMedx Group, Marietta, GA) dehydrated human amnion/chorion membrane tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for properties to support wound angiogenesis. METHODS: Angiogenic growth factors were identified in dHACM tissues using enzyme-linked immunosorbent assays (ELISAs), and the effects of dHACM extract on human microvascular endothelial cell (HMVEC) proliferation and production of angiogenic growth factors was determined in vitro. Chemotactic migration of human umbilical vein endothelial cells (HUVECs) toward pieces of dHACM tissue was determined using a standard in vitro transwell assay. Neovascularization of dHACM in vivo was determined utilizing a murine subcutaneous implant model. RESULTS: Quantifiable levels of the angiogenic cytokines angiogenin, angiopoietin-2 (ANG-2), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), platelet derived growth factor BB (PDGF-BB), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) were measured in dHACM. Soluble cues promoted HMVEC proliferation in vitro and increased endogenous production of over 30 angiogenic factors by HMVECs, including granulocyte macrophage colony-stimulating factor (GM-CSF), angiogenin, transforming growth factor ß3 (TGF-ß3), and HB-EGF. 6.0 mm disks of dHACM tissue were also found to recruit migration of HUVECs in vitro. Moreover, subcutaneous dHACM implants displayed a steady increase in microvessels over a period of 4 weeks, indicative of a dynamic intra-implant neovascular process. CONCLUSIONS: TAKEN TOGETHER, THESE RESULTS DEMONSTRATE THAT DHACM GRAFTS: 1) contain angiogenic growth factors retaining biological activity; 2) promote amplification of angiogenic cues by inducing endothelial cell proliferation and migration and by upregulating production of endogenous angiogenic growth factors by endothelial cells; and 3) support the formation of blood vessels in vivo. dHACM grafts are a promising wound care therapy with the potential to promote revascularization and tissue healing within poorly vascularized, non-healing wounds.

Properties of dehydrated human amnion/chorion composite grafts: Implications for wound repair and soft tissue regeneration.

PURION(®) processed dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Marietta, GA) tissue products were analyzed for the effectiveness of the PURION(®) process in retaining the native composition of the amniotic membrane and preserving bioactivity in the resulting products. dHACM was analyzed for extracellular matrix (ECM) composition through histological staining and for growth factor content via multiplex ELISA arrays. Bioactivity was assessed by evaluating endogenous growth factor production by human dermal fibroblasts in response to dHACM and for thermal stability by mechanical tests and in vitro cell proliferation assays. Histology of dHACM demonstrated preservation of the native amnion and chorion layers with intact, nonviable cells, collagen, proteoglycan, and elastic fibers distributed in the individual layers. An array of 36 cytokines known to regulate processes involved in inflammation and wound healing were identified in dHACM. When treated with dHACM extracts, bioactivity was demonstrated through an upregulation of basic fibroblast growth factor, granulocyte colony-stimulating factor, and placental growth factor biosynthesis, three growth factors involved in wound healing, by dermal fibroblasts in vitro. After conditioning at temperatures ranging from -78.7 to +73.5°C, dHACM retained its tensile strength and ability to promote proliferation of dermal fibroblasts in vitro. Elution experiments demonstrated a soluble fraction of growth factors that eluted from the tissue and another fraction sequestered within the matrix. The PURION(®) process retains the native composition of ECM and signaling molecules and preserves bioactivity. The array of cytokines preserved in dHACM are in part responsible for its therapeutic efficacy in treating chronic wounds by orchestrating a "symphony of signals" to promote healing.

Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing.

Human amnion/chorion tissue derived from the placenta is rich in cytokines and growth factors known to promote wound healing; however, preservation of the biological activities of therapeutic allografts during processing remains a challenge. In this study, PURION® (MiMedx, Marietta, GA) processed dehydrated human amnion/chorion tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for the presence of growth factors, interleukins (ILs) and tissue inhibitors of metalloproteinases (TIMPs). Enzyme-linked immunosorbent assays (ELISA) were performed on samples of dHACM and showed quantifiable levels of the following growth factors: platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, transforming growth factor α (TGFα), TGFß1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PLGF) and granulocyte colony-stimulating factor (GCSF). The ELISA assays also confirmed the presence of IL-4, 6, 8 and 10, and TIMP 1, 2 and 4. Moreover, the relative elution of growth factors into saline from the allograft ranged from 4% to 62%, indicating that there are bound and unbound fractions of these compounds within the allograft. dHACM retained biological activities that cause human dermal fibroblast proliferation and migration of human mesenchymal stem cells (MSCs) in vitro. An in vivo mouse model showed that dHACM when tested in a skin flap model caused mesenchymal progenitor cell recruitment to the site of implantation. The results from both the in vitro and in vivo experiments clearly established that dHACM contains one or more soluble factors capable of stimulating MSC migration and recruitment. In summary, PURION® processed dHACM retains its biological activities related to wound healing, including the potential to positively affect four distinct and pivotal physiological processes intimately involved in wound healing: cell proliferation, inflammation, metalloproteinase activity and recruitment of progenitor cells. This suggests a paracrine mechanism of action for dHACM when used for wound healing applications.