Si-CaP/自体微小颗粒骨支架载体复合BMSCs促进脊柱融合的实验研究

Spinal fusion has been widely used in the treatment of spinal surgical diseases.Autogenous bone graft is generally recognized as the gold standard to spinal fusion. However , the nonunion incidence rate in sigle level spinal fusion remains 5%～35% in clinical cases .For this reason, seeking a better way to improve spinal fusion rate and enhance successful spinal fusion outcome has been an essential topic to the orthopaedic surgeon..In the past few years, with the development of tissue engineering technology , finding new solutions to avoid nonunion became feasible. This study will focus on the construction of silicate substituted calcium phosphate（Si-CaP）/autogenous tiny morselized bone scaffold , which possesses osteoinduction,osteoconduction and osteogenesis. The scaffold will be combined with the cultivative bone marrow stromal cells （BMSCs）into New Zealand rabbit. The adhesive and proliferative effectiveness of Si-CaP/autogenous tiny morselized bone scaffold on BMSCs and it's inductive influence on BMSCs to osteoblast transformation will be observed as well. .Using New Zealand rabbit spinal fusion model , the scaffold with BMSCs will be placed bilaterally in the region spanning L5-L6 transverse processes. Gross observation , histomorphological examination, biomechanics detection, radiology examination and molecular biology method will be applied to investigate the outcome of spinal fusion and the fate of the BMSCs in vivo. This study will definitely provide a new idea to improve the successful rate of spinal fusion in clinical practice.

脊柱融合广泛应用于脊柱外科疾病的治疗过程中。虽然自体骨是公认的促进脊柱融合的金标准，但仍会导致5%～35%单节段脊柱融合的患者发生不融合。因此,寻求提高脊柱融合率和促进脊柱融合的方法成为骨科医生研究的重要课题。近年来，随着组织工程技术的发展，为解决脊柱不融合的问题提供了新的思路。本实验通过构建具有骨诱导性、骨传导性和骨生成作用的硅磷酸钙（Si-CaP）/自体微小颗粒骨支架载体，并与新西兰兔骨髓基质细胞（BMSCs）复合培养，观察Si-CaP/自体微小颗粒骨支架载体对BMSCs的粘附和增殖的影响以及其诱导BMSCs向成骨细胞转化的作用。将BMSCs与Si-CaP/自体微小颗粒骨支架载体复合植入新西兰兔脊柱融合的动物模型中，通过大体观察、组织学观察、生物力学检测、影像学检测、分子生物学等方法研究脊柱融合情况和BMSCs在体内的转归情况。从而为临床上提高脊柱融合率提供一种新的有效的方法。

目的：构建Si-CaP/微小颗粒骨支架载体，探讨Si-CaP/微小颗粒骨降解性能、力学性能、空间结构及生物学性能。方法：实验分为：Si-CaP组、微小颗粒骨组及Si-CaP/微小颗粒骨组，将三者置于DMEM培养液中进行体外降解，在培养3d、7d、14d和28d时，取出各组样本在体外降解过程中培养液的pH值，及各组失重率、吸水率、压缩应力、弹性模量、微观形态、孔隙率及孔隙之间连通性的变化，微小颗粒骨内骨细胞的转归情况及基因表达规律。结果：随着降解时间的延长，各组降解液的pH值均升高；各组支架载体的失重率不断增加；各组支架载体的吸水率在不同时间段呈现不同的变化趋势；各组支架载体的压缩应力和弹性模量均缓慢下降，至降解28d时达到最小值。扫描电镜和Micro-CT观察，可见Si-CaP/微小颗粒骨表面及孔壁发生降解，孔隙率增大，孔与孔之间的连通性增加，并在Si-CaP/微小颗粒骨的表面及孔壁上形成类骨磷灰石。HE观察见Si-CaP/微小颗粒骨内Si-CaP和微小颗粒骨紧密结合，并且随着降解时间的延长，充填于微小颗粒骨间的Si-CaP结构逐渐松散。微小颗粒骨内骨陷窝逐渐空虚，体外降解14d后骨细胞溶解消失。降解前后Si-CaP组未见COL-I mRNA和DMP1mRNA的表达；Si-CaP/微小颗粒骨和微小颗粒骨的COL-I mRNA和DMP1mRNA的表达量随着时间的变化呈现不同的变化趋势。结论：Si-CaP/微小颗粒骨支架载体具有有利于细胞粘附、增殖及分化的空间结构，良好的体外降解性能、力学强度及生物学活性，可作为自体骨的替代材料修复骨缺损。成果及计划：综述《骨组织工程中硅磷酸钙材料研究进展》发表在《国际骨科学杂志》。在《Journal of Biomedical Materials Research Part A》投稿2篇。下一步在体外将BMSCs与Si-CaP/微小颗粒骨复合培养，进一步建立新西兰兔脊柱融合的动物模型，观察Si-CaP/BMSCs/微小颗粒骨支架载体在体内促进脊柱融合情况。