Processing and preservation of amniotic membranes as biological wound dressings

Amniotic membrane derived from the human placenta is a collagen-based extracellular matrix that contains various growth factors, which offers an excellent source of biological materials for tissue engineering applications. The aim of this study was to investigate different pretreatment conditions combined with gamma irradiation to establish the processing condition for long-term preservation of amniotic membranes as biological wound dressings. Human amniotic membranes (hAMs) were first isolated from donated placentas obtained by caseation, cleaned off blood remnants, and cut into 10 cm $\times 10 cm$ sheets. During the pretreatment step, 0.05 - 0.5% (w/v) hypochlorite solution or a combination of penicillin-streptomycin, neomycin, and amphotericin (PNA) solution at 50, 100, and 2.5 $\mu$ g/mL, respectively, was first performed to reduce bacterial contamination. After air drying under sterile environment, the amniotic sheets were individually packed and tightly sealed in double-layered polyethylene bags before exposed to gamma irradiation at 25 kGy under cold condition. Plate count methods were performed on as-received hAMs and after pretreatment and gamma irradiation steps to check the bacterial contamination on the samples. Results showed that pretreating the amniotic membranes in 0.5% (w/v) hypochlorite for 2 h or PNA for 18 h could sufficiently reduce the bacterial contamination below 103 colony-forming unit, and gamma irradiation under cold condition could maintain the membranes’ transparency as well as ensure the sterility of samples up to 6 months. Therefore, this combined pretreatment and irradiation steps could be applied to preserve dried hAMs for long-term use as wound dressings.