Efficacy of a Thermally Triggered Injectable Hydrogel CS/Col /PLA/GP and BMSCs for Cartilage Tissue Engineering
Authors: Li Mingjing , Li Fan 
Published: October 2020

Abstract
BackgroundArticular cartilage has limited self-repair ability. Tissue engineering is considered to be one of the most promising therapeutic approaches. Chitosan (CS) based hydrogels are the most widely used scaffolds which still need improvement. The purpose of this study was to investigate the efficacy of a thermally triggered injectable chitosan / type II collagen / polylactic acid / sodium β-glycerophosphate (CS/Col/PLA/GP) hydrogel and bone marrow mesenchymal stem cells (BMSCs) for the treatment of cartilage defects in rabbit knee joints. Material/MethodsThe CS-based hydrogels consisting of CS, Col II, PLA and GP were fabricated by chemical cross-linking method. The gel forming time and elastic modulus of these hydrogels were measured. We tested the viability, proliferation and differentiation of rabbit BMSCs cultured in the hydrogels by fluorescence staining, CCK-8 and PCR method. The hydrogels combined with or without BMSCs were injected into cartilage defects in rabbit knee joints and the materials were collected at 8 weeks after surgery. The repair effect of cartilage defects was evaluated based on gross observation, HE, safranin O and immunohistochemical staining. ResultsThe CS/Col/PLA/GP hydrogel was liquid at room temperature and gelled after 7.5±0.41min at 37°C. CS/Col /PLA/GP hydrogel had a modulus of 8.90 ± 0.12 kPa while CS/GP and CS/Col/GP hydrogels had the modulus of 4.07 ± 0.24 kPa and 4.93 ± 0.09 kPa. The results of Live/Dead cell viability assay reveal that most of BMSCs remained alive in the hydrogels. CCK-8 assay shows that the number of cells in CS/Col /PLA/GP hydrogel was significantly higher in comparison to the other groups on days 2 and 3 of cell culture (p<0.05). Aggrecan mRNA expression in the CS/Col /PLA/GP gel was the highest (p<0.05). Sox9 mRNA expression in the CS/Col /GP group was the highest, in which CS/Col /PLA/GP hydrogel was higher than the CS/GP hydrogel(p<0.05). Furthermore, CS/Col/PLA/GP and CS/Col /GP hydrogels showed higher COL2A1 mRNA expression in comparison to CS/GP constructs (p<0.05). In vivo studies showed that approximately 90% of the cartilage defects of rabbits treated by the hydrogel and BMSCs were repaired with hyaline-like tissue without obvious inflammation response. HE, safranin O, and immunohistochemical staining showed that the hyaline like cartilage was formed in cartilage defects, and the collagen content in the new generated cartilage was similar to the normal cartilage. The neocartilage was thinner than the surrounding normal cartilage, but it exhibited integration with adjacent healthy tissue. The abundant well-defined chondrocytes were aligned in several apparent chondrocyte clusters in the new generated cartilage.ConclusionsThe thermo-sensitive injectable CS/Col/PLA/GP composite hydrogel has better ability to promote survive, proliferation and chondrogenic differentiation of seeded BMSCs as compared against CS/Col/GP and CS/GP hydrogels. Combined with BMSCs to repair cartilage defects of rabbit knee joints, they can effectively reduce the cartilage defect area, and the new generated cartilage is comparable to normal cartilage structure. In addition, abundant availability and simple fabrication process also make CS/Col/PLA/GP composite hydrogel a suitable candidate scaffold in cartilage tissue engineering.