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Diabetic Synthesis

While repopulating wounded tissues through cell proliferation and migration, PURION Processed dHACM has also been shown to stimulate secretion of cytokines by cells, including secretion of immunomodulatory, angiogenic, and tissue growth promoting cytokines by fibroblasts, vascular endothelial cells, and stem cells. When treated with dHACM extracts, human dermal fibroblasts upregulated biosynthesis of bFGF, GCSF, and PlGF in vitro, three growth factors involved in healing. 0

Similarly, dHACM treatment induced production of over 30 angiogenic factors by human microvascular endothelial cells in vitro, including granulocyte macrophage colony-stimulating factor (GM-CSF), angiogenin, transforming growth factor ? 3 (TGF-? 3), and HB-EGF. 51 The role of stem cells in healing has also focused on the paracrine signaling properties of these cells, including their influence on the inflammatory status of injured tissues. 121 ADSCs, BM-MSCs, and HSCs were shown to modulate secretion of a number of cytokines involved in immunoregulation and mitogenesis in response to dHACM extracts.

Of these molecules, they can generally be classified into three unique functions: 1) chemokines and proteins related to leukocyte migration, 2) immunomodulatory cytokines, and 3) mitogenic growth factors and proteins related to tissue growth. 106 Upregulated chemokines, including I-309, IL-8, IL-16, MCP-1, MIP-1? , and MIP-1? , are known to direct chemotaxis of immune cells that are critical in healing, including monocytes and macrophages, neutrophils, T lymphocytes, dendritic cells, and eosinophils.

Similarly ICAM-1, which was upregulated in all three stem cell types, is an adhesion molecule that facilitates leukocyte endothelial transmigration,122 suggesting a potential role of ICAM-1 in the extravasation of circulating stem cells from the blood vessels toward the dHACM treated site. In wound settings, these chemokines are commonly expressed early following establishment of hemostasis, often peaking at day 1 with expression sustained through the first week of healing, and have also been shown to promote angiogenesis. 4 Immunomodulatory cytokines, including GDF-15, IL-1RA, and IL-6, were also upregulated in MSCs, ADSCs, and HSCs.

These cytokines are known to regulate various pro- and anti-inflammatory cues that are required for healing, including regulation of apoptosis, T cell activation, B cell differentiation, macrophage differentiation, hematopoiesis, and additional downstream immunomodulatory cues. 58, 123, 124 Finally, growth factors and growth factor regulatory molecules, including EGF, FGF-4, GH, IGFBP-1, IGFBP-2, and SCF, were secreted in response to dHACM. These growth cytokines are known to stimulate cell proliferation, migration, differentiation, cell survival, and protein synthesis, and have been shown to enhance healing of wounds. 25-128

Additionally, TIMP-1, which was also upregulated, is an inhibitor of MMPs and is actively involved in tissue remodeling processes. 129 These results indicate that in addition to the growth factors and cytokines released from dHACM tissue into the wound, dHACM continues to amplify these paracrine signals by inducing resident cells to produce additional regenerative growth factors, thus enhancing dHACM’s regenerative effect. Together this balance of regulatory signals modulates the healing response to provide an ideal balance of cues to promote healing. 8. 5 dHACM PROMOTES ANGIOGENESIS

Angiogenesis, or the creation of new blood vessels, is paramount during the late inflammatory and proliferative phases of wound healing since chronic wounds are commonly associated with poor circulation and vascularization. The following angiogenic cytokines were identified to be intrinsically present in PURION Processed dHACM: 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).

The dHACM had a direct effect on stimulating human dermal microvascular endothelial cells. dHACM caused HMVE cells to proliferate, recruited migration of endothelial cells, and induced production of over 30 angiogenic factors by these cells in vitro, including granulocyte macrophage colony-stimulating factor (GM-CSF), angiogenin, transforming growth factor ? 3 (TGF-? 3), and HB-EGF. 51

In an in vivo murine ischemic wound model, a steady increase in microvessels was observed in dHACM implants over a 4 week period to levels equivalent to healthy and healed skin, indicative of a dynamic intra-dHACM implant neovascular process. 1 Recruited bone marrow stem cells were also localized to sites of neovascularization within the implanted graft where they expressed angiogenic stromal derived factor 1? (SDF-1? ). 104 Taken together, these results demonstrate that MiMedx PURION Processed 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, as shown in Figure 41.

These results indicate that dHACM stimulates angiogenesis when applied to a wound through the presence of angiogenic factors coupled with dHACM’s effect on the production of angiogenic factors by endothelial cells. The effect of dHACM is amplified by increasing the number of endothelial cells in the wound via migration and proliferation and by inducing cells to produce additional angiogenic factors to produce a potent local angiogenic environment.

In diabetes, hyperglycemia caused by decreased insulin availability or increased resistance to insulin can negatively affect the cellular response to tissue injury, resulting in chronic wounds that are stalled in a state of chronic inflammation and unable to progress toward healing. This may be due in part to the impaired responsiveness of reparative stem cells at the wound.

The diabetes-induced state of hyperglycemia has been linked to having a deleterious effect on wound healing associated with changes in ADSC cellular morphology, decreased proliferation and migration and abnormal cellular differentiation. 130, 131 The altered pattern of growth factor and cytokine levels in diabetes may also contribute to the cellular complications seen in chronic wound tissues. 132 Reduced bioavailability of cytokines and growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGF-? and platelet derived growth factor (PDGF) have been implicated in the pathogenesis of chronic wounds,133 and abnormal expression of growth factors has been reported in studies of diabetic foot ulcer tissues. 134

Despite these impaired cellular responses in diabetic tissues, the eluted soluble growth factors and cytokines from dHACM were capable of stimulating ADSCs from type I and type II diabetic donors to proliferate, migrate, and modulate gene expression and secretion of immunomodulatory cytokines in vitro. 07 Type I and Type II diabetic ADSCs increased secretion of the immunomodulatory factors IL-6, IL-8, MCP-1, MIP-1b and RANTES and upregulated gene expression of IL-1? , IL-1? , and IL-1RA, which are responsible for the inflammatory regulation functionality of ADSCs in wound healing. 135 These immunomodulatory cytokines are essential for normal progression through wound healing. Thus, initial upregulation of certain pro-inflammatory factors through treatment with dHACM may be necessary for resetting the cascade of normal healing in a chronic wound.

Ultimately, regulation of, as opposed to elimination of, immunomodulatory cytokines by ADSCs is crucial re-establishing an acute wound healing trajectory. The results presented here demonstrate that while ADSCs from diabetic donors can have decreased capacities for proliferation and migration as well as altered cytokine secretion profiles compared to normal ADSCs, diabetic stem cells were capable of responding to dHACM treatment.

Type I and Type II diabetic ADSCs increased proliferation in response to dHACM, increased cellular migration in response to soluble factors eluted from dHACM tissue, and altered their immunomodulatory cytokine secretion and gene expression to produce a number of factors important to healing of chronic wounds, representing a first step in determining how stem cells in diabetic patients may respond to dHACM treatment to promote healing of chronic wounds. 8. 7

SCIENTIFIC DATA ARE SUPPORTED BY MIMEDX dHACM’S PROVEN CLINICAL EFFICACY Scientific experiments continue to generate new information in order to elucidate potential cellular and molecular mechanisms through which dHACM grafts promote healing. MiMedx has published several papers to date in peer reviewed journals that establish that MiMedx PURION Processed amniotic tissues (e. g. , EpiFix and AmnioFix) retain over 50 regulatory proteins, including cytokines, chemokines and growth factors that are naturally present in fresh amniotic membrane. 0-52 These studies demonstrate that PURION Processed amniotic tissues retain biologically active signals that directly cause responsive cells to proliferate, upregulate biosynthesis of important growth factors and immunomodulatory regulators, recruit mesenchymal stem cell migration towards the allograft, and enhance angiogenesis within healing wounds in vitro and in vivo. 50-52, 104, 106, 107

In contrast, competitive tissue allograft products have been shown to contain only a fraction of the soluble factors that are retained in dHACM through the PURION Process. 05 These wound care products also have not demonstrated the wealth of scientific data or clinical healing results established by MiMedx; however, due to the decreased growth factor and cytokine content in these grafts, competitive allograft tissues would not be expected to possess the same ability to modulate cell responses and stimulate healing as MiMedx’s PURION Processed tissues.

These in vitro and in vivo scientific results strongly suggest that PURION Processed dHACM is intimately involved with modulation of the cellular environments in wounds to elicit an improved healing response, and the reparative attributes of dHACM allografts are further supported by the numerous peer reviewed published clinical trials on their use on diabetic foot ulcers and venous leg ulcers, as well as initial investigations in plantar fasciitis, tympanoplasty (surgical eardrum repair), and osteoarthritis.

These trials clearly demonstrate a more rapid rate of healing in dHACM treated wounds, compared to standard of care, indicating that PURION Processed dHACM modulates inflammation and promotes improved tissue growth. 108, 109, 111, 112, 120 The wealth of scientific knowledge compiled by MiMedx suggests that PURION Processed dHACM releases soluble signals into the wound environment that promote cell proliferation, migration, and endogenous cytokine secretion and ultimately stimulates stem cell activity, angiogenesis, and modulation of inflammation to support healing.

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