Lyophilization of dehydrated human amniotic membrane: a proactive approach to preserve growth factors for enhanced wound healing

The preservation of key growth factors in the human amniotic membrane (hAM) that are involved in tissue regeneration and wound healing is the primary focus of this research work. Here, we quantified the total protein content and the major growth factors present in different sample preparations of hAM. The membrane is initially processed, dehydrated, and gamma-irradiated and subsequently subjected to histological staining, cytotoxicity assays, and total protein estimation. The ELISA method was used to quantify TGF b1, bFGF, PDGF-BB, VEGF-A, and EGF in three distinct preservation samples: tissue homogenate (AC-H), ball milled powder (AC-P), and lyophilized powder (AC-L). An in-vitro scratch assay was performed to analyse cell migration and wound healing. Higher TGF-b1 and FGF-b concentrations indicate the potential impact of HAM on re-epithelialization and granular tissue formation. For major growth factors, the quantification shows no significant differences between the samples. On treating the wound area with concentrations of 0.4 mg/ml and 0.6 mg/ml, the remaining wound area for AC-H, AC-L, and AC-P are 39.71%, 40.31%, 55.99% and 25.48%, 62.8%, and 29.65%, respectively. This indicates the presence of growth factors in the membrane promotes wound healing and facilitates cell migration and proliferation. This study provides insights into the quantity of key growth factors within the human amniotic membrane, thereby presenting the approach as a viable option for treating chronic wounds. Additionally, as lyophilization preserves more growth factors and offers greater stability and shelf life than other preservation techniques, it may be an appropriate substitute for ball milling.