The Mechanism of Cell Interaction and Response on Decellularized Human Amniotic Membrane: Implications in Wound Healing

Author(s): 
Mohit Bhatia, PhD; Marian Pereira, PhD; Hemlata Rana; Bhavani Stout; Craig Lewis, PhD; Sascha Abramson, PhD; Kristen Labazzo, PhD; Cynthia Ray; Qing Liu, PhD; Wolfgang Hofgartner, MD, DSc; Robert Hariri, MD, PhD

The human amniotic membrane has been used as a biological dressing for skin burns,wounds, and chronic leg ulcers. Its therapeutic effects have been attributed to its ability to promote epithelialization, inhibit fibrosis, and act as an antimicrobial agent.1 Human amniotic membrane products currently on the market, including Amniograft® and Prokera (Bio- Tissue Inc, Miami, Fla), are frozen with intact cellular structure, associated growth factors, and cytokines. ACELAGRAFT (Celgene Cellular Therapeutics, Cedar Knolls, NJ) is distinct in that it is a decellularized and dehydrated human amniotic membrane (DDHAM) with all cells and associated growth factors removed. As a result, DDHAM can be shipped and stored at room temperature. DDHAM has demonstrated potential as a wound healing product in ongoing studies for the treatment of acute and chronic ulcers. Even with all cells and related factors removed, DDHAM still possesses the necessary biological properties to augment wound healing processes. In this work, a detailed mechanistic analysis of the mode of interaction of cells with DDHAM is presented. The response of human dermal fibroblasts to DDHAM was evaluated to complement the current clinical studies with DDHAM for the treatment of skin wounds. The biochemical, cell biology, and gene array studies together provide a mechanistic understanding of the wound healing process as it relates to DDHAM.

Methods

Preparation of membranes. DDHAM was prepared using proprietary methods as previously described.2,3 In short, the amniotic membrane was excised from qualified term placentas and was washed and scraped to remove extraneous tissue and cells.This was followed by a decellularization of the tissue using deoxycholic acid and drying of the tissue using a gel dryer. An amniotic membrane treated as such is free of cells. The decellularized and dehydrated human amniotic membrane product is sold under the name ACELAGRAFT.

 

Biochemical analysis. The decellularized and dried membrane was completely solubilized using any of the 4 methods summarized in Scheme I. Tissue (1–3 mg) was solubilized in 1–3 mL of 10 mM HCl at 100°C for 1 h. Tissue was also digested using either collagenase (ratio of 1:100 at 37°C for 18 h) or pepsin (1 mg/mL) in 0.5 M acetic acid at 37°C for 18 h. To identify growth factors (cytokines and/or hormones), the amniotic membrane tissue was treated by methods previously described.4 The tissue was treated with a buffer (5 mL) consisting of 2 M guanidium HC1 with 100 mM tris buffer, (pH 7.2, 5 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1 mM β-mercaptoethanol) for 24 h at 4°C.The resulting supernatant was dialyzed against water for 24 h at 4°C and the sample was dried for biochemical analysis. Total collagen in DDHAM was quantitated using a Sircol assay kit (Accurate Chemical and Scientific Corp., Westbury, NY). Collagens I, III, and IV were quantitated using sandwich ELISAs with primary and secondary antibodies purchased from Rockland Immunochemicals, Inc. (Gilbertsville, Pa). Elastin and glycosaminoglycans (GAG) content were determined using the FASTIN and BLYSCAN dye based assay kits (Accurate Chemical and Scientific Corp., Westbury, NY). Fibronectin and laminin were quantitated using sandwich ELISA kits (Takara Bio Inc., Madison, Wis). Residual maternal hormones and growth factors were assessed using ELISAs from R&D Systems (Minneapolis, Minn).

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