Evaluation of Growth Factor Release and Stimulation of Proliferation of Wound Microenvironment Cells with Amnion/Chorion Grafts

Placental membranes, which have been utilized clinically to treat a range of complex and chronic wounds, contain numerous growth factors and cytokines. This study was designed to investigate which growth factors and cytokines are released from dehydrated amnion/chorion membranes (dACM), and how these growth factors interact with skin and vascular cells found within the wound microenvironment.

For 7 days, the release of 39 growth factors and cytokines from dACM relevant to wound healing was measured using a quantitative multiplex proteomics microarray with grafts from 7 different donors. To simulate the effect of these cytokines and their role in wound healing, proliferation assays with human dermal fibroblasts, human keratinocytes, and human umbilical vein endothelial cells (HUVECs) were conducted using conditioned media (CM) with releasate from dACM.

Cytokines were released in vitro from dACM for up to seven days at 37⁰C.  Fetuin A, Galectin-7, insulin-like growth factor binding protein 1 (IGFBP-1), insulin-like growth factor 1 (IGF-1), and transforming growth factor beta 1 (TGF-β1) were present at the highest concentrations per cm² within the dACM.  Releasate from dACM resulted in significantly increased fibroblast (14 days) and keratinocyte (7 days) proliferation relative to controls (28%±10% (p≤0.01, n=9) and 65%±22% (p≤0.01, n=6), respectively).  Additionally, dACM resulted in significantly increased proliferation of HUVECs, an important cell type for angiogenesis (212%±22% above controls (p≤0.01, n=6)).

These results describe the release of growth factors and cytokines from dACM over 7 days, which suggests these grafts may have a lasting impact at the application site over the course of multiple days. The mechanistic assays evaluating proliferation of fibroblasts, keratinocytes and endothelial cells confirm that the released growth factors and cytokines are active and stimulate proliferation of cells that play a role in the wound microenvironment.