脂肪干细胞与纳米羟基磷灰石/胶原人工骨支架修复不规则骨创伤的基础研究

Tissue engineering and nanotechnology have enabled engineering of nanostructured materials to meet the current challenges in bone treatment owing to rising occurrence of bone diseases, accidental damages and defects. Nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering. The abundance and accessibility of adipose derived stem cells (ASCs) may prove to be novel cell therapeutics for bone repair and regeneration. ASCs were cultured on these scaffolds and were induced to undergo osteogenic differentiation in the presence of n-HA for BTE. Osteogenic differentiation of ASCs was confirmed using alkaline phosphatase activity (ALP), mineralization (ARS) and dual immunofluorescent staining using both ASCs marker protein and Osteocalcin, which is a bone specific protein. This was evident from the immunostaining and CMFDA images of ADSCs showing cuboidal morphology, characteristic of osteogenic lineage. . We are to develop a new kind of nano-hydroxyapatite collagen composite scaffold,combined with adipose-derived stem cells(ASCs),which will properly degradate and induce bone regeneration.It is crucial to synthesis the material and adjust the combination formula.After safy detection,we are to use this combined material to repair dogs' mandibular defection.One the other hand ,as we have already done some preparing work of ASCs researches,we are to carry on futher researches on the signal pathway and molecular conduction of the ossification of the ASCs,which will guide us to regulate the combination formula,and finally we might get a new ideal bone repair materical.All of these researches will provide instructive evidence for the bone defect repairation and regeneration.

本项目拟研制一种将脂肪干细胞（ASCs）与纳米羟基磷灰石/胶原（n－HA/C）支架材料相结合的新型骨修复材料；系统研究这种负载ASCs的骨修复材料的制备技术，实现脂肪干细胞与支架材料的复合；通过对狗下颌骨的节段性骨缺损的修复过程研究，探讨ASCs在成骨过程中的信号通路及传导机制，进而反馈指导ASCs和胶原蛋白复合纳米羟基磷灰石人工骨支架材料的配方调整，最终实现成骨过程与支架材料的降解过程的匹配，获得具有临床应用意义的人工骨修复材料。以上目标的实现，对用于不规则骨创伤修复的新材料的研究和应用均具有指导价值。

本课题组将纳米级羟基磷灰石与I型胶原复合，制备出具有良好物理及生物特性的仿生材料。脂肪干细胞（adipose-derived stem cells, ADSCs）可在支架材料表面及内部贴附生长，且在一定条件下于体内外均具有良好的成骨效应。本研究拟将ADSCs作为种子细胞，与所制备的纳米羟基磷灰石/胶原（nano-hydroxyapayite collagen，nHAC）支架复合，检测其对兔下颌骨缺损的修复效应。. 课题组曾对ADSCs抗衰老效应进行过初步探索，发现其分泌物可部分修复HDFs的内源性、外源性老化损伤；制备丝素-壳聚糖支架，发现其物理学特性初步符合生物材料要求，具有良好的生物相容性，认为其可以成为骨组织工程中较为理想的支架材料。正式实验阶段，本课题组成功制备nHAC支架材料，并测得其具有良好的机械性能、降解性及生物相容性，随后对兔下颌骨缺损标本进行检测发现：术后4周，对照组（A）3d-CT显示骨缺损腔未见缩小，大体观察腔内肉芽组织长入，镜下观察腔内散在脂肪组织及纤维结缔组织，未见骨小梁形成；nHAC组（B）及nHAC+ ADSCs组（C）3d-CT显示骨缺损腔无明显缩小，大体观察支架稍降解，有少量骨痂形成，但B组少于C组，镜下观察缺损边缘少量类骨质及大量成骨细胞分布，有骨小梁形成，B组均少于C组。术后8周，A组较前未见明显变化；B及C组3d-CT显示骨缺损腔缩小，大体观察支架部分降解，可见大量新生骨痂但B组少于C组，镜下可见缺损边缘大量类骨质和新生骨分布，大面积排列规整的骨小梁，材料内部有岛状新生骨散在分布和毛细血管长入，B组均少于C组。术后12周，A组较前未见明显变化；B及C组3d-CT显示骨缺损腔明显缩小，大体观察支架基本降解，腔内为新生骨痂填满，但B组强度弱于C组，镜下可见散在支架材料与新生骨组织交织，类骨质和新生骨较前明显增多，形态更为粗大致密且排列规则，可见编织骨及髓腔形成，B组均少于C组。. 本课题组制备的nHAC支架在物理及生物性能方面可满足骨组织工程对材料的要求，同ADSCs复合后对兔下颌骨缺损的修复作用更佳，为组织工程化骨在的进一步研究及应用奠定了基础。