A standardised ‘off the shelf’ substrate for enhanced tissue engineering

Purpose Amniotic membrane (AM) is a popular ophthalmic tissue engineering adjunct. We previously developed a highly effective thermolysin-based denuding technique that preserves basement membrane integrity This technique has been validated clinically using fresh frozen AM (FAM). We now propose a standardised dry-prepared ‘off the shelf’ AM tissue engineering substrate. Methods Dry preserved thermolysin denuded AM (DAM), and FAM denuded with ethylenediaminetetraacetic acid (EDTA), and dispase -based methodologies were prepared. Denuding efficiencies were compared using electron microscopy. The effect of denuding on AM molecular composition was investigated and characterised using proteomics. The propensity of DAM to support stem cells was explored using fluorescent immunohistochemistry for defined markers. Results Electron microscopy demonstrated thermolysin denuding efficiency was comparable in FAM and DAM. Proteomic analyses showed effective removal of epithelial cell proteins in DAM, but not EDTA–based denuding techniques. Whilst similar enzymatic activity to thermolysin, mechanical scraping reduces the efficacy of dispase denuding. Collagens IV, VI, periostin, βig-h3 and VLA-6 are targets of thermolysin activity. DAM maintains stem cell characteristics and is most effective in preventing differentiation. Conclusion Conventional EDTA and dispase procedures for preparing AM for tissue engineering are ineffective at removing cells whilst preserving the basement membrane. Combining our novel thermolysin denuding and dry preservation techniques improves the overall quality of AM for tissue-engineered constructs. Therefore, we propose a fist of its kind, stable and dry-stored ‘off the shelf’ construct for enhanced ocular surface tissue engineering.