Guided Bone Regeneration (GBR) uses biodegradable collagen membranes of animal origin tissues (dermis and pericardium). Their barrier effect prevents soft tissues to interfere with the regeneration of alveolar bone. However, their xenogeneic origin involves heavy chemical treatments which impact their bioactivity. Wharton's Jelly (WJ) from the umbilical cord is a recoverable surgery waste. WJ is mostly made from collagen fibers, proteoglycans, hyaluronic acid, and growth factors. WJ with immunologically privileged status and bioactive properties lends credence to its use as an allograft. Nevertheless, low mechanical properties limit its use in bone regenerative strategies. Herein, our objective is to develop a crosslinked WJ-based membrane to improve its strength and thus its potential use as a GBR membrane. The umbilical cords are collected after delivery and then stored at −20°C until use. The WJ membranes (1 × 5 × 12 mm) were obtained after the removal of blood vessels and amniotic tissue, washed, lyophilized, and stored at −20°C. WJ membranes were incubated in genipin solutions in decreasing concentrations (0.3 g / 100 mL − 0.03 g / 100 mL) for 24 hours at 37°C. The crosslinking degree was estimated by ninhydrin and confirmed by FTIR (Fourier-transform infrared spectroscopy) assays. The swelling rate was obtained after the rehydration of dry crosslinked WJ-membrane for 10 min in D-PBS. The mechanical properties were assessed in hydrated conditions on a tensile bench. The resistance to the degradation was evaluated by collagenase digestion (1 mg/mL for 60 hours) assay. The cytotoxicity of crosslinked WJ-membrane was evaluated in accordance with the standard ISO.10993-5 (i.e. Mitochondrial activity and Lactate Dehydrogenase release) against Mesenchymal Stem Cells (MSCs). Finally, the MSCs colonization and proliferation were followed after 21 days of culture on crosslinked WJ-membranes.Introduction and Objective
Materials and Methods
Alveolar bone resorption following tooth extraction or periodontal disease compromises the bone volume required to ensure the stability of an implant. Guided bone regeneration (GBR) is one of the most attractive technique for restoring oral bone defects, where an occlusive membrane is positioned over the bone graft material, providing space maintenance required to seclude soft tissue infiltration and to promote bone regeneration. However, bone regeneration is in many cases impeded by a lack of an adequate tissue vascularization and/or by bacterial contamination. Using simultaneous spray coating of interacting species (SSCIS) process, a bone inspired coating made of calcium phosphate-chitosan-hyaluronic acid was built on one side of a nanofibrous GBR collagen membrane in order to improve its biological properties. First, the physicochemical characterizations of the resulting hybrid coating were performed by scanning electron microscopy, X-ray photoelectron, infrared spectroscopies and high-resolution transmission electron microscopy. Then human mesenchymal stem cells (MSCs) and human monocytes were cultured on those membranes. Biocompatibility and bioactivity of the hybrid coated membrane were respectively evaluated through MSCs proliferation (WST-1 and DNA quantification) and visualization; and cytokine release by MSCs and monocytes (ELISA and endothelial cells recruitment). Antibacterial properties of the hybrid coating were then tested against S. aureus and P. aeruginosa, and through MSCs/bacteria interactions. Finally, a preclinical in vivo study was conducted on rat calvaria bone defect. The newly formed bone was characterized 8 weeks post implantation through μCT reconstructions, histological characterizations (Masson's Trichrome and Von Kossa stain), immunohistochemistry analysis and second harmonic generation. Biomechanical features of newly formed bone were determined.Introduction and Objective
Materials and Methods
