Biophysical characteristics of genipin-crosslinked amniotic membrane bio-scaffold

Abstract

Objective To investigate the characteristics and feasibility of genipin-crosslinked amniotic membrane(AM) as bio-scaffold. Methods Human umbilical cord mesenchymal stem cells (hUCMSCs) were isolated from fresh umbilical cord and cultured by adherent method.The expressions of PE-CD34, PE-CD45, PE-CD90, FITC-105 and FITC-Oct-4, the markers of hUCMSCs, were detected by flow cytometry.Alizarin red and oil red O staining were performed to identify the cells after adipogenesis and osteogenesis induction on the third-generation cells.Human AMs were treated at 37 ℃ and 45 ℃ by 0.5% and 1% genipin solution for 24, 36 and 48 hours respectively, and the mechanical properties of AM in each group were measured and compared.The hUCMSCs were divided into only hUCMSCs culture group, fresh AM group, crosslinked AM group, gelatin group and crosslinked AM+ gelatin group, and the cells were cultured in the corresponding medium.The content of hydroxyproline among the groups was detected with hydroxyproline kit, and proliferation of the cells (absorbance) was assayed by MTT method to evaluate the biological compatibility of crosslinked AM. Results The maximum tensile displacement of the crosslinked-AM by 0.5% and 1% genipin was (8.31±0.43)mm and (4.49±0.37)mm respectively, and those after crosslinked with 0.5% genipin under the 37 ℃ and 45 ℃ for 24 hours was (9.89±1.09)mm and (5.39±0.59)mm, respectively, showing a significant difference between them (t=6.389, P 0.05). The loading force of the crosslinked-AM was significantly higher in the 1% genipin treated group than that in the 0.5% genipin treated group (P 0.05). Conclusions Different crosslinked temprature, crosslinking period and concentration of genipin impact the mechanical properties of AM.Crosslinked AM with genipin is feasible as a carrier scaffold of artificial cornea because of less tissue toxicity and better plasticity. Key words: Amniotic membrane; Cross-linking reagents/pharmacology; Stress, mechanical; Tensile strength; Umbilical cord mesenchymal stem cells; Biological compatibility; Genipin