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

Abstract

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 β3 (TGF-β3), 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.

Keywords: Amnion; Amnion/chorion grafts; Angiogenesis; Chorion; Chronic wounds; Endothelial cells; Growth factors; Soft tissue regeneration; VEGF; Wound healing; dHACM.

Figures

Figure 1

Effects of extracts of dHACM on microvascular endothelial cell proliferation in vitro. dHACM extracts promoted proliferation of HMVECs over controls; however, no dose response was observed at these concentrations. The p values shown indicate statistical significance from their respective controls. Values shown are means ± standard deviation (n = 5).

Figure 2

Change in growth factor production by human microvascular endothelial cells when cultured in the presence of varying concentrations of dHACM extract. Endothelial cells increased production of a number of angiogenic factors when cultured in the presence of dHACM extract, compared to untreated cells cultured without extract (green/blue). A few regulatory factors were also down regulated in the presence of extract (red/yellow), while many factors remained unchanged (black), relative to untreated controls.

Figure 3

Average number of migrated human umbilical vein endothelial cells (HUVECs) per field of view in response to dehydrated human amnion/chorion tissue allografts (dHACM). Representative micrographs and cell counts indicated that greater migration was observed in response to larger samples, relative to their smaller counterparts. HUVEC migration in complete medium was significantly greater than all other samples (p ≤ 0.05). * indicates significantly greater migration than basal medium and 2.0 mm groups (p ≤ 0.05). Scale bar – 200 μm.

Figure 4

Neovascular response following in vivo dHACM implantation. An increasing number of CD31 positive microvessels were seen within the implanted dHACM over time, ultimately reaching the level of normal and previously injured skin by day 28. (A) CD31 positive microvessels in dHACM over 28 days were stained green, while cell nuclei were counterstained blue with DAPI. (B) Vessel counts demonstrated an increase in vascularization approaching that of healthy and healing skin after 28 days. Scale bar – 50 μm.