In Vivo and In Vitro Evaluation of a Hypothermically Stored Amniotic Membrane

Chronic wounds occur when the acute wound healing process becomes stalled and fails to progress through the subsequent phases of healing. Hypothetically stored amniotic membrane (HSAMº) has been shown to contain numerous growth factors, cytokines, and extracellular matrix proteins^1,2.

In the current study, we have evaluated the effects of HSAM both in vivo and in vitro. To determine the profile for released factors from HSAM in vitro, HSAM samples were cultured for 7 days and supernatants were analyzed using proteomic microarrays (RayBiotech). In vitro, human dermal fibroblasts and human keratinocytes were treated with assay media or media conditioned with HSAM (CM) and evaluated for proliferation and migration. CM was obtained by incubating HSAM grafts in assay media (AM) for 5 days at 4°C. Full-thickness skin defects in rats were treated with HSAM and assessed 9- and 21-days post-implantation for histological changes related to quality of wound repair, including evaluation of the epidermal layer, abnormal scar tissue formation, basket-weave matrix, and the presence of dermal appendages.  

When evaluating secreted factors in vitro, the total growth factors and cytokines released from HSAM over 7 days was approximately 70.6%±19.9%2. In vitro HSAM (25% CM) significantly promoted human fibroblast and human keratinocyte proliferation (1.17 and 1.53-fold change compared to AM, respectively) and migration (1.91 and 2.32-fold change compared to AM, respectively)2. Full-thickness wounds treated with HSAM resulted in epithelialization after 9 days, early epidermal formation, and increased basket-weave matrix compared to controls. By 21 days, HSAM-treated wounds displayed regenerate tissue that closely mimicked unwounded skin1. 

In summary, these results demonstrate the HSAM contains and releases cytokines and growth factors, and we found that HSAM released factors promoted in vitro cellular responses. Additionally, in vivo wounds treated with HSAM resulted in improved remodeling compared to controls.