Experimental Study On Osteochondral Differentiation Of ADSCs And Novel Tissue Engineered Scaffolds For Intervertebral Disc

Objective:1. The osteochondral differentiation ability of ADSCs were investigated to provide ideal seed cells for osteochondral tissue engineering; The chondrogenic ability of ADSCs induced by co-culturing with chondrocytes or culturing with induction medium were compared to providing a more effective induction method.2. To fabricate a novel composite scaffold with CFDBM/acellular nucleus pulposus matrix and verify the feasibility of it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties; To investigate effects of different decellularized methods on structure and biomechanical properties of AF to provide evidence for AF tissue engineering.Methods:1. ADSCs were isolated from rabbits. ADSCs at passage3were cultured with osteogenic or chondrogenic medium. The morphology of induced ADSCs was observed by microscope. After14days, induced ADSCs were detected with histological staining and RT-PCR to investigate osteochondral differentiation ability.2. ADSCs were induced to chondrocytes by co-culturing with chondrocytes or culturing with induction medium. The morphology of the induced ADSCs was observed by microscope. After14days, induced ADSCs were detected with toluidine blue, immunohistochemistry and PCR to compare induction effect of two methods.3. Pig proximal femoral cancellous bone rings were fabricated and dealed with degreasing, decalcification, decellularization to prepare AF phase of scaffold. NP taken from pig tails were decellularized, crushed and centrifugalized to prepare NP ECM which was injected into the center of AF phase. Then the composite scaffold was freeze-dryed and cross-linked. The scaffold was investigated by general observation, HE and SEM, as well as porosity, water absorption rate and compressive elastic modulus. Effect of scaffold extract on ADSCs proliferation was examined by MTT and viability of ADSCs seeded on scaffold was detected by Live/Dead staining.4. Fresh pig AF were dissected and decellularized with Triton X-100, SDS or trypsin. After decellularization process, general observation, histological staining and SEM were carried out to examine cell removal efficacy and ultrastructure. Besides, collagen content, GAG content and biomechanical properties were detected.Results:1. ADSCs cultured in vitro had a spindle shape, rapid proliferation and good stability. Induced ADSCs became osteoblasts or chondrocytes in morphology.14days later, alkaline phosphatase, alizarin red and von Kossa staining of ADSCs cultured with osteogenic medium were all positive. Toluidine blue and type Ⅱ collagen immunohistochemistry staining of ADSCs cultured with chondrogenic medium were positive and gene expressions of collagen II, aggrecan and SOX9increased greatly.2. ADSCs induced by two methods gradually became chondrocytes in morphology.14days later, toluidine blue and type II collagen immunohistochemistry staining were all positive in two groups, ADSCs culturing with induction medium staining deeper and having higher transcriptional levels of collagen Ⅱ, aggrecan and SOX9.3. The scaffold looked white. HE staining revealed there were no cell fragments. SEM showed the pores of AF phase were uniform and interconnected, acellular NP ECM forming an even network and the junction of scaffold closely connected. The scaffold had a porosity of82.98%±7.02%with (343.00±88.25)μm pore diameter of AF phase and621.53%±53.31%water absorption rate. Compressive elastic modulus was (89.07±8.73)kPa. MTT indicated scaffold extract had no influence on cell proliferation. Live/Dead showed ADSCs had a good proliferation on the scaffold.4. Histological and SEM showed there were no residual cells in three groups with the structure of AF in Triton X-100group not disturbed, trypsin group slightly damaged and SDS group severely damaged. There was no statistical difference on collagen content between each group and natural AF, but GAG content all decreased. Mechanical properties deterioration occurred in no group except SDS group.Conclusion:1. ADSCs can differentiate into osteoblasts or chondrocytes under certain conditions; ADSCs can differentiate into chondrocytes by co-culturing with chondrocytes or culturing with induction medium, and the induction effect of latter method is better.2. Composite AF-NP scaffold made of CFDBM/acellular NP matrix had good pore diameters and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, which make it a suitable scaffold for intervertebral disc.3. Triton X-100-treated AF retained major ECM after thoroughly cell removal, preserved major structure and adequate mechanical strength, which make it a suitable candidate as an alternative scaffold for AF tissue engineering.