To investigate the characteristics and feasibility of genipin-crosslinked amniotic membrane(AM) as bio-scaffold.

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.

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 maximum tensile displacement of the crosslinked-AM was gradually decreased as the lapse of crosslinking time, and an insignificant difference was found among 24, 36 and 48 hours after 0.5% genipin treatment under the 37 ℃ (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), and the loading force of the AM was significantly increased in 45 ℃, 0.5% genipin, 24 hours crosslinked group compared with the 37 ℃, 0.5% genipin, 24 hours crosslinked group (t=5.528, P<0.05). The content of hydroxyproline in the AM was (1.28±0.36), (2.03±0.49) and (2.11±0.10)mg/g in the 1% genipin crosslinked AM group, 0.5% genipin crosslinked AM group and fresh AM group, respectively, and the content of hydroxyproline in the AM in the 1 % genipin group was significantly lower than that in the 0.5% genipin group in the fresh AM group (both at P<0.05). The proliferative values of the hUCMSCs were significantly lower in the only hUCMSCs culture group, fresh AM group and gelatin group were significantly reduced in comparison with the crosslinked AM group and crosslinked AM+ gelatin group (all at P<0.05). There was no significant difference in the proliferative values of the hUCMSCs between crosslinked AM group and crosslinked AM+ gelatin group (P>0.05).

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.

Amniotic membrane; Cross-linking reagents/pharmacology; Stress, mechanical; Tensile strength; Umbilical cord mesenchymal stem cells; Biological compatibility; Genipin