Effect of Cross-Linking Density on the Structures and Properties of Carbodiimide-Treated Gelatin Matrices as Limbal Stem Cell Niches

Abstract

Given that human amniotic membrane is a valuable biological material not readily available for corneal epithelial tissue engineering, gelatin is considered as a potential alternative to construct a cellular microenvironment. This study investigates, for the first time, the influence of cross-linking density of carbodiimide-treated gelatin matrices on the structures and properties of artificial limbal stem cell niches. Our results showed that an increase in the carbodiimide concentration from 1.5 to 15 mM leads to an upward trend in the structural and suture strength of biopolymers. Furthermore, increasing number of cross-linking bridges capable of linking protein molecules together may reduce their crystallinity. For the samples treated with 50 mM of cross-linker (i.e., the presence of excess N-substituted carbodiimide), abundant N-acylurea was detected, which was detrimental to the in vitro and in vivo ocular biocompatibility of gelatin matrices. Surface roughness and stiffness of biopolymer substrates were found to be positively correlated with carbodiimide-induced cross-link formation. Significant increases of integrin β1 expression, metabolic activity, and ABCG2 expression were noted as the cross-linker concentration increased, suggesting that the bulk crystalline structure and surface roughness/stiffness of niche attributed to the number of cross-linking bridges may have profound effects on a variety of limbal epithelial cell behaviors, including adhesion, proliferation, and stemness maintenance. In summary, taking the advantages of carbodiimide cross-linking-mediated development of gelatin matrices, new niches with tunable cross-linking densities can provide a significant boost to maintain the limbal stem cells during ex vivo expansion.

Hybrid silk fibroin–gelatin nanofibrous sheet for drug delivery and regenerative medicine: In‐vitro characterization and controlled release of simvastatin/protein

“…The XRD spectrum of in‐situ crosslinked G fibers exhibits one peak around 22.9°, and a small peek at 7.8° related to the triple helical structure 31 . The present results support the reported results by Manna et al and Lai et al about crosslinking G with immersing in carbodiimide (EDC) 32,33 . However, in the SF–G samples, due to the formation of the β‐sheet structure in SF fibers, a sharp peak appears around 20.5° indicates the β‐sheet crystal formation for treated SF 30 .…”

Physicochemical Characteristics of Chitosan-Based Hydrogels Modified with Equisetum arvense L. (Horsetail) Extract in View of Their Usefulness as Innovative Dressing Materials

“…It may be also stated that with an increasing amount of crosslinking agent, an increase in the tensile strength of the hydrogel was observed. This was also reported by Wong et al [42], Haryanto et al [43] and Lai et al [44].…”

Silica Release from Silane Cross-Linked Gelatin Based Hybrid Scaffold Affects Cell Proliferation

“…On the other hand, gelatin is a commercially available natural protein that is widely used as a tissue engineering scaffold due to its biodegradability and similarity to the more expensive collagen adhesive protein. To increase the stability of the gelatin based scaffolds, people have used various cross-linkers such as glutaraldehyde, , EDC·HCl, , and genipin . However, the use of those cross-linkers are limited because of their potential toxic effects and ineffective increase in mechanical strength.…”