These studies clearly demonstrate that dehydrated human amnion/chorion membranes (dHACM) contain a large number of pro-angiogenic growth factors, including angiogenin, angiopoietin-2, EGF, bFGF, HB-EGF, HGF, PDGF-BB, PlGF, and VEGF. This partial list of growth factors does not encompass the entire array of physiologically important and biologically active molecules present in dHACM . These particular growth factors, however, are likely to be responsible for the clinical benefits of this dHACM allograft, in relation to neovascularization and healing within chronic wounds. These soluble signals in dHACM also stimulated human microvascular endothelial cells to proliferate in vitro, and further, to increase production of a number of endogenous growth factors, cytokines, and receptors related to angiogenesis. Furthermore, dHACM tissue promoted chemotactic migration of human endothelial cells in vitro, suggesting that these soluble factors are capable of recruiting endothelial cells to promote wound re-vascularization. Our findings strongly support dHACM exerting therapeutic actions both directly and indirectly by activating multiple signaling pathways that promote angiogenesis within healing wounds.
Our previous work demonstrated that PURION® Processed dHACM tissue also retains a collection of growth factors, anti-inflammatory molecules, and tissue inhibitors of metalloproteinases that play other important roles in wound healing. Specifically, this material or its extracts, promoted the proliferation of dermal fibroblasts and the recruitment of progenitor cells in vitro and in vivo . Combined with the present results, dHACM allografts contain a wide array of soluble signals, only a fraction of which have been identified to date, which may stimulate healing through a variety of signaling pathways and physiological mechanisms.
These multifunctional mechanisms offer advantages for dHACM as a treatment of difficult to heal chronic wounds. In normal wounds, the initial fibrin clot acts as both a matrix for repair and as a reservoir for growth and cell recruitment factors. Inflammatory cells, including neutrophils, monocytes, and lymphocytes then invade the wound and release further growth factors and cytokines that initiate wound repair mechanisms, including keratinocyte and fibroblast migration/proliferation, microvascular endothelial cell recruitment, proliferation, and angiogenesis, and nerve sprouting within the granulation tissue . In chronic wounds, however, including diabetic and venous ulcers characterized by deficiencies in vascularization and cytokine signaling, the exogenous delivery of key cytokines and growth factors may be necessary to restore a molecular balance and achieve healing .
Single growth factors may be insufficient to overcome to multiple deficiencies in most chronic wounds, as demonstrated by the modest efficacy and limited utility of recombinant human PDGF (becaplermin) as a single agent as shown in a meta-analysis of well-designed clinical trials . By contrast, dHACM contains additional angiogenic factors, specifically VEGF and bFGF (FGF2), both potent angiogenic cytokines that promote endothelial cell proliferation and migration . We also identified PlGF which not only has direct angiogenic effects, but acts synergistically with VEGF to stimulate wound angiogenesis . The growth factors angiopoietin-1 and 2 are critical for regulating the stabilization and remodeling of blood vessels [23, 26]. Our finding that dHACM also stimulates human microvascular endothelial cells to increase production of a variety of angiogenic cytokines and growth factors suggests this material’s ability to amplify the initial signals provided by the allograft itself, potentially even beyond the lifespan of the allograft.
Previous studies of amniotic membranes in regulating angiogenesis have reported conflicting data, with some describing enhanced vascularization while others have observed inhibition of angiogenesis. Anti-angiogenic effects have been widely reported in cases of ocular/corneal surgery , while improved vascularization and healing have been reported when used to promote healing of cutaneous wounds [29, 30]. This dichotomy may be explained in part by the presence of both anti-angiogenic (including thrombospondin-1, endostatin, and anti-angiogenic TIMPs)  and pro-angiogenic factors (including VEGF and PDGF) [32, 33] in amniotic tissues. Combined with the previously described role of the local environment in regulating angiogenesis, the specific response to the amniotic membrane graft cytokine milieu may thus be largely dependent on the target tissue and the location of the implantation.
In this study, subcutaneous implantation of dHACM tissues in a murine model demonstrated a steady increase in vascularization through day 28. More specifically, dHACM implants went from being completely avascular following implantation, to having a vascular density similar to both normal and healed skin within 4 weeks. This dynamic vascular remodeling is in line with our in vitro findings, and consistent with the time course of clinical trial data , supporting the retention of biological activity by growth factors present in PURION® Processed dHACM grafts. The prolonged pro-angiogenic effects from a single implant may offer potential practical benefits compared to advanced wound interventions that require more frequent (daily or biweekly) applications.
The angiogenic properties of amniotic membrane grafts demonstrated here apply only to PURION® Processed, dehydrated amnion/chorion laminated grafts produced by MiMedx. The PURION® Process has been optimized to preserve the bioactivity of native amniotic growth factors and cytokines, while other amniotic membrane grafts processed by different methods may not retain biologically active angiogenic factors and, therefore, may lack the ability to stimulate angiogenesis. This is particularly true for single layer amnion products, which are inherently much thinner and therefore contain far fewer growth factors, as well as decellularized amniotic membrane grafts, which have been processed to remove cells and soluble factors from the tissue matrix.
The clinical value of dHACM grafts for use as therapy in non-healing wounds has been demonstrated by clinical research, even as further translational studies are underway. Treatment with dHACM allografts was reported to improve healing in patients with a variety of wound types for which traditional therapies were ineffective . Additionally, refractory wounds that healed after dHACM treatment were reported not to recur with long-term follow-up . Finally, in a small, prospective, randomized clinical trial, Zelen et al. demonstrated a significant increase in the healing rate of diabetic foot ulcers treated with dHACM compared to those treated with a standard therapeutic regimen, with 77% and 92% of dHACM wounds healed at weeks 4 and 6, respectively, compared to only 0% and 8% of controls . The aggregate data of dHACM suggests this material could be studied for comparative effectiveness with other available wound healing products.