Fabrication And Evaluation Of A Novel Annulus Fibrosus-Nucleus Pulposus Biphasic Scaffold For Intervertebral Disc Tissue Engineering

Objective The objective of this study is to fabricate and characterize a natural acellular demineralized bone matrix gelatin (DBMG) and cartilage extracellular matrix (CECM) scaffold for spinal intervertebral disc tissue engineering.Methods1. Annulus Fibrous scaffold:Pig proximal femoral cancellous bone rings (external diameter10mm, internal diameter5mm, and thickness3mm) were fabricated, then dealed with high-pressure water washing, degreasing, decalcification and other related treatment. Make the bone rings as the fibrous scaffold SEM observation and porosity measurement, water absorption rate and biomechanical analysis. MTT test was also done to assess cytotoxicity of the scaffolds.2. Annulus fibrosus-Nucleus pulposus Biphasic Scaffold: Pig cartilage micro filaments about5μm-100μm were prepared after pulverization and gradient centrifugation and made into3.5%suspension after acellularization treatment. Placing the suspension into the middle of pig proximal femoral cancellous bone rings, The scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Hydrochloride and N-hydroxysucinimide. Then freeze-dry the scaffold. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate and biomechanical analysis. ADSCs were seeded into the scaffold. The proliferation of the ADSCs was analyzed using inverted microscope and SEM.3. Annulus fibrous biphasic scaffold: Cortical bone and cancellous bone rings (thickness5mm) were fabricated. Then dealed with high-pressure water washing, degreasing, decalcification and other related treatment. Place the suspension into the middle of pig proximal femoral cancellous bone rings, freeze-drying and cross-linked. The porosity measurement, water absorption rate and biomechanical were analysis.Results1. Annulus Fibrous scaffold:The scaffold shows white, flexible in moist state, porous cancellous ring. The histological staining showed that there are no cell fragments in the scaffolds. The scaffold has good pore interconnectivity with pore diameter401.4±13.1μm,62.12±1.52%porosity and409.77±11.34%water absorption rate. MTT test showed that the scaffolds do not exert any cytotoxic effect on adipose-derived stem cells. 2. Annulus fibrosus-Nucleus pulposus Biphasic Scaffold:The scaffold shows yellowish-white, flexible in moist state. The scaffold has good pore interconnectivity with pore diameter middle101.3±22.5μm,83.24±4.31%porosity and421.45±8.43%water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs test indicated that cells covered the scaffolds uniformly and the cell growing well.3. Annulus fibrous biphasic scaffold:The scaffold shows light yellow. Witch has good mechanical property51.41±2.71%porosity and350.15±3.21%water absorption rate.Conclusion1. Novel DBMG scaffold had good pore diameter and porosity, appropriate biomechanical character, non-toxicity and good biocompatibility, which make it a suitable candidate for fibrous scaffold tissue engineering.2. Novel DBMG and CECM scaffold had good porosity, appropriate biomechanical character, non-toxicity and good biocompatibility, The matrix components similar to spinal intervertebral disc, which make it a suitable candidate as an spinal intervertebral disc tissue engineering.3. Cortical bone and cancellous bone complex scaffold has broad prospect.