Study Of Construction Of Integrated Tissue Engineering Intervertebral Disc Based On Natural Bone Matrix Gelatin/Cartilage Extracellular Matrix Compounding With Disc Cells Of Goat

Objective:To explore the feasibility of construction of tissue engineered intervertebral disc based on natural bone matrix gelatin/cartilage extracellular matrix compounding with disc cells of goat.Methods:1. A hollow bone matrix gelatin ring with an outer diameter of10mm, internal diameter of5mm and height of3mm was prepared by decalcification and decellularization of pig’s proximal femoral cancellous bone. Acellular cartilage homogenate, obtained by grinding pig’s proximal femoral cartilage, was injected into the centre of hollow bone matrix gelatin ring and the integrated scaffold was prepared after freeze-drying and cross-linking. The scaffold was evaluated using Hoechst33258staining, Sirius red staining and, scanning electron microscopy. The scaffold porosity, water adsorption and compressive property were assessed under wet condition.2. The AF and NP cells, isolated from sheep, were observed for cell morphology under inverted microscope. Disc cell phenotypes were identified by staining the1st passage disc cells with toluidine blue, safranin O, and type Ⅰ, and Ⅱ collagen Immunocytochemistry staining. The1st generation AF cells and NP cells were seeded onto the AF phase and NP phase respectively and the cell-scaffold hybrids cultured in vitro for two days were evaluated through inverted microscope, histological staining and scanning electron microscopy, and LIVE/DEAD viability staining. Detecting the gene expression of type Ⅰ, Ⅱ collagen and aggrecan in disc cells of native disc and cell-scaffold hybrids by Real-time fluorescence quantitative PCR technique.3. The1st generation disc cells were labeled with PKH26dye and seeded onto the corresponding parts of the scaffold. The hybrids were cultured in vitro for two days, and then implanted subcutaneously into nude mice. After6weeks culture in vivo, evaluation of the hybrids were earried out with in vivo small animal imaging system and fluorescence microscope, Safranin O, and type I and II collagen immunohistochemical staining. Results:1. The scaffold has smooth surface and clear boundaries. Microscopic study observed transparent nucleus phase and milky white annulus phase. Hoechst33258staining showed no residual cell remnant and Sirius red staining showed closely linked deep red annulus phase and yellow and green nucleus phase. Pores in the scaffold were evenly distributed and connected under SEM with an average pore size of (372.3±139.8)μm in AF phase and (112.4±21.8) μm in NP phase. Water absorption was655.7%±78.6%, average compressive elastic modulus of integrated scaffold was49.06±15.57kpa, and the modulus of elasticity in normal intervertebral disc compression was135.9±28.9kPa providing a statistically significant difference P<0.05.2. AF cells cultured in vitro were long spindle shaped whereas NP cells were short spindle shaped. Senescence of disc cells began to emerge from generation5. Toluidine blue and Safranin O staining were positive for both AF and NP cells. Type Ⅰ collagen immunohistochemical staining was positive for AF cells and negative for NP cells whereas type Ⅱ collagen immunohistochemical staining was weakly positive for AF cells and positive for NP cells. Scanning electron microscopy displayed good cell adhesion on the scaffold. HE and LIVE/DEAD staining respectively, show good cell adhesion and viability on the scaffold. Levels of gene expression of type Ⅰ, Ⅱ collagen and aggrecan of cells in hybrids were higher than that in native disc.3. PKH26labeled intervertebral disc cells had evenly distributed red fluorescence. A lot of cells were viable even after6weeks in vivo culture as seen by in vivo small animal imaging system and fluorescence microscope. Hybrids were positive in type Ⅰ collagen immunohistochemistry and Safranin O staining but negative in type Ⅱ collagen immunohistochemistry staining.Conclusion:Construction of tissue engineered intervertebral disc based on natural bone matrix gelatin/cartilage extracellular matrix compounding with disc cells of goat is feasible.