新型一体化纤维环-髓核双相支架的制备与评估

FABRICATION AND ANALYSIS OF A NOVEL TISSUE ENGINEERED COMPOSITE BIPHASIC SCAFFOLD FOR ANNULUS FIBROSUS AND NUCLEUS PULPOSUS

目的用脱细胞脱钙骨基质和脱细胞髓核基质构建新型一体化纤维环-髓核双相支架,并进行理化检测,探讨其作为组织工程椎间盘支架的可行性。方法取猪股骨近端松质骨,塑形成外径10 mm、内径5 mm、厚3 mm的骨环,经脱脂、脱钙、脱细胞处理制备成纤维环相支架;取猪尾髓核组织,Triton X-100和脱氧胆酸联合脱细胞后粉碎、离心,制备脱细胞髓核基质;将制备的脱细胞髓核基质注入纤维环相支架中央,冷冻干燥,联合应用紫外照射和碳化二亚胺进行交联,制备一体化纤维环-髓核双相支架。通过大体观察、HE染色、扫描电镜观察支架结构;测定支架的孔隙率、吸水率、压缩弹性模量;MTT法检测25%、50%、100%支架浸提液对新西兰大白兔脂肪来源干细胞(adipose-derived stemcells,ADSCs)增殖的影响;将ADSCs接种到支架上,活/死细胞染色观察细胞在支架上的活性。结果大体观察支架呈乳白色;HE染色可见支架均质红染,无细胞成分残留;扫描电镜可见外层纤维环相支架孔径大小均匀一致,孔隙相互贯通,中央脱细胞髓核基质微丝相互连接,形成均匀网状结构,交界处连接紧密。纤维环相支架孔径为(343.00±88.25)μm,一体化纤维环-髓核双相支架孔隙率为82.98%±7.02%、吸水率为621.53%±53.31%,压缩弹性模量为(89.07±8.73)kPa。经MTT测定支架浸提液对ADSCs增殖无影响;活/死细胞染色可见细胞在支架上生长良好,无死细胞。结论用脱细胞脱钙骨基质和脱细胞髓核基质构建的一体化纤维环-髓核双相支架具有合适的孔径和孔隙率且无毒,力学性能与人正常椎间盘接近,是构建组织工程椎间盘的理想支架载体。

Objective To fabricate a novel composite scaffold with acellular demineralized bone matrirdacellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties. Methods Pig proximal femoral cancellous bone rings （10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness） were fabricated, and were dealed with degreasing, decalcification, and decdlularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-IO0 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells （ADSCs） were cultured with different concentrations of scaffold extract （25%, 50%, and 100%） to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining. Results The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with （343.00 ± 88.25） am oore diameter of the annulus fihrosus phase, 82.98% ± 7.02% porosityand 621.53% ＋ 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was （89.07 ± 8.73） kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell. Conclusion Novel composite scaffold made of acellular demineralized bone matrirdacellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering.