Bilateral Crosslinking with Glutaraldehyde and 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide: An Optimization Strategy for the Application of Decellularized Human Amniotic Membrane in Tissue Engineering.

Introduction: The decellularized human amniotic membrane (dHAM) emerges as a viable 3D scaffold for organ repair and replacement using a tissue engineering strategy. Glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) can increase the biomechanical properties of dHAM. However, the crosslinking process is associated with biochemical changes and residual toxic materials, dampening the biocompatibility of the dHAM. From a histologic point of view, each side of the amniotic membrane is biologically different. While the dHAM basement membrane side is rich in growth factors, the stromal side of the dHAM contains more connective tissue matrix (e.g., collagen fibers) which supports its biomechanical properties. Biocompatibility and biomechanical properties are two important challenges in the field of materials science. In this study, for the first time, the stromal and basement membrane side are cross-linked with GTA and EDC, respectively, to optimize the biocompatibility of the treated dHAM while sparing the GTA-mediated biomechanical improvements. Methods: Crosslinking was carried out on dHAM in three groups: EDC, GTA and bilateral treatment with EDC&GTA. Mechanical resistance, degradability, and crosslinking measurements were performed on treated dHAM. The viability of mesenchymal stem cells (MSCs) on the scaffolds was evaluated by the MTT assay. The expression levels of surface markers and images of the MSCs were thoroughly studied. Results: The results obtained showed that bilateral treatment of dHAM with EDC and GTA increased mechanical resistance. Similarly, the evaluation of surface markers revealed that bilaterally treated dHAM sustains the stemness and viability of MSCs at a level equal to that achieved with EDC alone. The SEM images indicated that the MSCs maintained adhesion on EDC&GTA-cross-linked dHAM. Conclusion: The current study explores a pioneering treatment of dHAM, a material long recognized for its regenerative properties, in a novel context. This research delves into the utilization of dHAM cross-linked with EDC&GTA, demonstrating its optimized efficacy in tissue engineering. The enhanced crosslinking technique significantly alters the membrane's properties, amplifying its durability and therapeutic potential. In this novel bilateral treatment strategy (EDC and GTA), improving mechanical properties by GTA on the stromal surface and maintaining the biocompatibility of EDC on the side of the basement membrane of dHAM had been attained together. By investigating the handling and impact of this cross-linked membrane, this study unveils a new approach in leveraging a well-known material through an innovative process, revolutionizing its application in wound care.