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

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.


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).


1. Dua HS,Azuara-Blanco A. Amniotic membrane transplantation. Br J Opthalmol. 1999;83(6):748–752.

2. Hariri RJ, Kaplunovsky AM, Murphy PA. Collagen biofabric and methods of preparing and using the collagen biofabric. US Patent Application Publication; 2003: No. 20030187515.

3. Hariri RJ, Kaplunovsky AM, Murphy PA. Collagen biofabric and methods of preparing and using the collagen biofabric. US Patent Application Publication; 2004: No. 20040048796.

4. McDevitt CA, Wildey GM, Cutrone RM. Transforming growth factor-beta1 in a sterilized tissue derived from the pig small intestine submucosa. J Biomed Mater Res A. 2003;67(2):637–640.

5. Miranti CK, Brugge JS. Sensing the environment: a historical perspective on integrin signal transduction. Nat Cell Biol. 2002;4(4):E83–90.

6. Toy LW. Matrix metalloproteinases: their function in tissue repair. J Wound Care. 2005;14(1):20–22.

7. Reunanen N, Westermarck J, Häkkinen L, et al. Enhancement of fibroblast collagenase (matrix metalloproteinase- 1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways. J Biol Chem. 1998;273(9):5137–5145.

8. Gharaee-Kermani M, Phan SH. Role of cytokines and cytokine therapy in wound healing and fibrotic diseases. Curr Pharm Des. 2001;7(11):1083–1103.

9. McDonald JA, Kelley DG, Broekelmann TJ. Role of fibronectin in collagen deposition: Fab’ to the gelatinbinding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix. J Cell Biol. 1982;92(2):485–492.

10. Velling T, Risteli J,Wennerberg K, Mosher DF, Johansson S. Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta 1 and alpha 2beta1. J Biol Chem. 2002;277(40):37377–37381.

11. Chung CY, Erickson HP. Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix assembly and are essential for matrix incorporation of tenascin-C. J Cell Sci. 1997;110(Pt 12):1413–1419.

12. Roman J, McDonald JA. Fibulin’s organization into the extracellular matrix of fetal lung fibroblasts is dependent on fibronectin matrix assembly. Am J Respir Cell Mol Biol. 1993;8(5):538–545.

13. Pereira M, Rybarczyk BJ, Odrljin TM, Hocking DC, Sottile J, Simpson-Haidaris PJ. The incorporation of fibrinogen into extracellular matrix is dependent on active assembly of a fibronectin matrix. J Cell Sci. 2002;115(Pt 3):609–617.

14. Sottile J, Hocking DC. Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions. Mol Biol Cell. 2002;13(10):3546–3559.

15. Low QE, Drugea IA, Duffner LA, et al. Wound healing in MIP-1alpha(-/-) and MCP-1(-/-) mice. Am J Pathol. 2001;159(2):457–463.

16. Feugate JE, Wong L, Li QJ, Martins-Green M. The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue. BMC Cell Biol. 2002;3:13.

17. Blumenfeld I, Ullmann Y, Laufer D, Livne E. Enhancement of burn healing by growth factors and IL-8. Ann Burns Fire Disasters. 2000;13(4):220–230.

18. Gallucci RM, Simeonova PP, Matheson JM, et al. Impaired cutaneous wound healing in interleukin-6 deficient and immunosuppressed mice. FASEB J. 2000;14(15):2525–2531.

19. Schmidt JA, Mizel SB, Cohen D, Green I. Interleukin-1, a potential regulator of fibroblast proliferation. J Immunol. 1982;128(5):2177–2182.

Post new comment

  • Lines and paragraphs break automatically.
  • Web page addresses and e-mail addresses turn into links automatically.
  • Use to create page breaks.

More information about formatting options

Enter the characters shown in the image.