Optimization of dendrimer bridges in carbodiimide cross-linked amniotic membrane as a limbal epithelial stem cell niche

摘要

Introduction: In clinical ophthalmology, ex vivo expansion of epithelial stem cells derived from limbus is beneficial for establishment of regenerative medicine strategies. Although a variety of biomaterials have been studied as limbal epithelial cell (LEC) carriers, the amniotic membrane (AM) remains one of the most commonly used natural matrices due to excellent comeal tissue compatibility and stem cell preservation ability. In order to improve poor biological stability of collagenous tissue materials, a technique based on introduction of dendrimer bridges into carbodiimide cross-linked AM scaffolds is attempted here to overcome the limited cross-linking efficiency. We pay particular attention to the effects of dendrimer generation on nanoscale properties of carbodiimide cross-linked AM as a LEC niche. Materials and Methods: This study followed the tenets of the Declaration of Helsinki involving human subjects and was approved by Institutional Review Board of our institute. In the presence of the same amount of polypropylenimine (PPI) dendrimers of different generations (G-1, G-3, and G-5), the tissue specimens were cross-linked with 0.05 mmol carbodiimide and 0.01 mmol N-hydroxysuccinimide per mg AM. The PPI dendrimer-mediated chemical modification of AM was characterized by cross-linking density measurements, Fourier transform infrared spectroscopy, and electrokinetic analyses. Transmission electron microscopy and atomic force microscopy were performed to examine the matrix nanostructure, topography, and stiffness. Additionally, the degradability and compatibility of cross-linked AM were studied in vitro and in vivo. LEC-AM crosstalk was assessed to explore the relationship between nanoscale properties of biomaterial scaffolds and limbal epithelial progenitor cell behaviors. Results and Discussion: With increasing PPI dendrimer generation from G-1 to G-3, the cross-linking degree, mechanical strength, and weight remaining of test samples significantly increased, indicating that the dendrimers with high number of terminal amino groups can act as more effective cross-linking bridges to improve the stability of biological tissues. However, the AM treated with PPI G-5 dendrimers exhibited a relatively low cross-linking density than those of G-1 and G-3 groups, probably due to that the over existence of amino groups in the cross-linking reaction system is detrimental to cross-linkage formation between collagen molecular chains. The extent of aggregation of AM nanofibers was greatly affected by the PPI dendrimer-mediated cross-linking efficiency. Among the studied samples, the surface roughness and stiffness of G-3 groups were the highest. Additionally, these AM samples showed better ocular biocompatibility than those of G-5 groups, suggesting charge density-related cytotoxicity and tissue reaction. Our results demonstrated that the cross-linking of AM with PPI G-3 dendrimers can enhance the ability to promote LEC growth and sternness expression correlated with Wnt signaling pathway. Conclusion: The present study suggests that the generation number of PPI dendrimers may have a profound influence on the nanoscale properties of carbodiimide cross-linked AM biomaterial as a LEC niche.