混合附载内皮细胞与成骨细胞的nHA／RHLC／PLA复合支架材料修复犬股骨头缺损的观察

Experimental study of repairing femoral bone defects with nHA/RHLC/PLA scaffold composite withendothelial cells and osteoblasts in canines

目的探讨以nHA／RHk／PLA复合支架材料为载体混合附载BMSCs来源的内皮细胞与成骨细胞修复犬股骨头坏死的可行性。方法BMSCs向内皮细胞（ECs）、成骨细胞（OBs）诱导分化14d后，消化、收集细胞，调整细胞密度分别为1．0×10^6／ml，与nHA／RHLC／PLA支架材料共培养2-4h，制成支架-细胞复合体。选取6-8月龄杂种犬18只36后肢股骨头造模，根据骨缺损处植入物不同分为3组各12肢：A组（支架-ECs＋OBs）、B组（支架-OBs）、C组（单纯支架）。术后12、24周取材，行大体及组织学、免疫组化、形态学计量评价骨修复效果。结果组织学检测：A组3个月时见缺损区基底部有大量的类骨质和新生骨小梁形成，在骨小梁的边缘存在较多的成骨细胞，修复区血管丰富。成骨反应由基底向中心部推进。6个月时充填区骨小梁成熟明显，可见有编织骨形成。缺损区中央偶见碎裂的支架材料；新骨形成量和成骨细胞数由基底部向外周递减，骨小梁的成熟度也由基底向外周递减。B组3个月时缺损区基底部也可见明显成骨反应、类骨质形成，但骨小梁数目及成骨细胞、毛细血管均较A组少，纤维组织多。c组仅在缺损基底边缘可见少量新生骨组织，骨小梁数量少，骨小梁带窄；较多肉芽组织形成，见较多炎性细胞。缺损区中央绝大部分为碎裂的不成形的支架材料残留。24周时通过图像分析系统测量新生骨基质面积百分比均值，A组为16．7％±2．2％；B组为13．6％±1．2％，C组为9．6％±2．O％，三组间成骨量有差异统计学意义（F=23．217，P〈0．05）。结论骨髓基质干细胞来源的成骨细胞具有修复股骨头骨坏死的能力；内皮细胞和成骨细胞联合应用的成骨能力及血管化程度优于成骨细胞的单独修复，内皮细胞可以增强成骨细胞的成骨作用。

Objective To explore whether a tissue-engineered construct composed of autogenous endothelial cells, osteoblasts and a new bioresorbable nano-hydroxyapatite/recombinant human-like collagen/ polylaetic acid （nHA/RHLC/PLA） would enhance bone regeneration and repair femoral head defects in canine models. Methods The bone marrow stem cells （BMSCs） were isolated from bone marrow of canine ilium and cultured in Dulbeceo＇s modified eagle medium：nutrient mixture F-12 culture media for I week andthe second-generation BMSCs were further induced by osteogenic medium （1×10^8 mol/L dexamethasone, 10 mmol/L B-sodium glycerophosphate and 50 μg/ml vitamin C ） and by endothelial cell grow medium （ vascular endothelial growth factor and basic fibroblast growth factor） for 14 days in vitro. Thus BMSCs were induced into ECs and OBs. After the second passage, cells were digested and collected. And cell density was adjusted to 1.0×10^6/ml. The cells and nHA/RHLC/PLA scaffold were co-cultured for 2 - 4 hours then nHA/RHLC/PLA scaffold composites prepared. Cavity defects of 8 mm in diameter and 10 mm in height were made in femoral heads. The nHA/RHLC/PLA scaffold composited with ECs and osteoblasts （OBs） （group A） and composited with OBs （group B） were inserted into different defects while cell-free nHA/ RHLC/PLA scaffold served as controls （ group C）. New bone formation and defect repair were evaluated at 3 and 6 months by radiographic examination, histology and bone histomorphometry. Results New bone formation was evident as early as 3 months in groups A, B and C. At 6 months, abundant bone tissue within defects was observed in group A. The control animals with cell-free scaffold showed less bone formation at both timepoints. The scaffold of nHA/RHLC/PLA was degraded and absorbed gradually with the formation of new bone tissues. Histology and bone histomorphometry further revealed significantly increased trabecular bones in group A compared with groups B and C at 6 months postimplantation （ P 〈 0.01 ）. Conclusion More abundant new bone tissue may be found in the bone defect areas implanted with osteoblast-endotheliocyte composite than osteoblasts composite and scaffold materials only. ECs and osteoblasts derived from BMSC are ideal seed cells for repairing femoral head defects.