复合构建组织工程骨对犬缺损颌骨的诱导再生及修复

Repair of dog jaw defect with compounded tissue-engineered bone

目的:利用骨髓基质干细胞、人重组骨形成蛋白2及聚乳酸复合构建组织工程骨,诱导颌骨再生,修复颌骨缺损,并对其成骨效能进行评价。方法:实验于2003-04/2004-04在青岛大学附属医学院中心实验室完成。选取普通级杂种犬12只,随机分为4组,即骨髓基质干细胞+人重组骨形成蛋白2+聚乳酸组、人重组骨形成蛋白2+聚乳酸组、聚乳酸+骨髓基质干细胞组、聚乳酸组,3只/组。全部动物双侧下颌骨体部分别造成30mm×12mm椭圆形缺损,用自身对照法,右侧骨缺损为实验侧,左侧为空白对照侧。取犬骨髓基质干细胞诱导为成骨细胞,给予不同材料复合后植入颌骨缺损区:骨髓基质干细胞+人重组骨形成蛋白2+聚乳酸组植入骨髓基质干细胞、人重组骨形成蛋白2与聚乳酸;人重组骨形成蛋白2+聚乳酸组植入人重组骨形成蛋白2与聚乳酸;聚乳酸+骨髓基质干细胞组植入聚乳酸、骨髓基质干细胞;聚乳酸组植入聚乳酸。术后第2,4,8周行X线、组织病理及扫描电镜检查,观察成骨情况。结果:实验纳入12只犬,全部进入结果分析。①培养过程中细胞形态观察:原代培养24h后部分细胞贴壁,48h后贴壁细胞向四周伸展,边缘的细胞呈梭形,散在生长的细胞呈椭圆形或梭形;10～12d后细胞集落间相互融合成单层,形态多为梭形。细胞传代培养后4h开始贴壁,8～10h基本贴壁完全,细胞大部分向梭形、多角形细胞转化,带有数个突起。②传代细胞密集区VonKossa染色结果:细胞密集区出现大面积黑染区域,即钙结节,而细胞密度较低的区域并无着色。③各组第8周时两侧成骨情况大体观察:骨髓基质干细胞+人重组骨形成蛋白2+聚乳酸组实验侧缺损为板层骨修复,质硬,部分区域呈骨性突起;人重组骨形成蛋白2+聚乳酸组实验侧缺损为不完全修复;聚乳酸+骨髓基质干细胞组实验侧缺损可见散在骨岛形成;聚乳酸组实验侧缺损区仅在边缘有新骨形成。各组空白对照侧均为纤维组织充填。④各组第8周两侧成骨情况X线检查结果:实验侧:骨髓基质干细胞+人重组骨形成蛋白2+聚乳酸组植入区可见较规则骨小梁像;人重组骨形成蛋白2+聚乳酸组植入区可见少量密度高的骨痂影形成;聚乳酸+骨髓基质干细胞组骨床边界较模糊,植入区可见密度高的骨痂影形成;聚乳酸组植入区点片状密度较高的影像。各组空白对照侧8周时植入区均无阻射影像。⑤各组第8周两侧成骨情况组织学检查:实验侧:骨髓基质干细胞+人重组骨形成蛋白2+聚乳酸组新生骨形成大片状结构,植入区内骨髓腔开始形成,聚乳酸大部分降解,新生血管数目增多,有的穿过降解的聚乳酸内部;人重组骨形成蛋白2+聚乳酸组以纤维成骨方式为主,也可见软骨化骨过程,骨小梁增多;聚乳酸+骨髓基质干细胞组缺损区有较多散在骨岛形成,可见软骨化骨;聚乳酸组骨缺损不完全修复,纤维组织嵌于缺损区,骨床边缘成骨细胞较活跃;骨髓基质干细胞+人重组骨形成蛋白2+聚乳酸组实验侧的成骨量明显优于其余3组;各组空白对照侧均未见有新骨形成,缺损为纤维组织充填。⑥骨髓基质干细胞+人重组骨形成蛋白2+聚乳酸组第8周两侧成骨情况扫描电镜检查结果:实验侧聚乳酸大部分吸收,被骨组织替代,可见小血管穿入聚乳酸内,新骨内骨细胞分布均匀,骨细胞位于陷窝内,孔隙内可见骨质沉积;空白对照侧见大量纤维蛋白,未见骨组织形成。结论:骨髓基质干细胞在体外可定向诱导为成骨细胞,并能钙化形成新骨。外源性骨形成蛋白可促进骨髓基质干细胞增殖,与降解速率及骨再生速率相匹配的聚乳酸复合,具有快速成骨的效能,可作为良好的骨缺损修复材料。

AIM： To construct tissue-engineered bone by compounding bone marrow stromal cells （BMSCs）, recombinant human morphogenetic bone protein-2 （rhBMP2） and polylactic acid（PLA） to induce jaw regeneration and repair jaw defects, and evaluate osteogenic activities of the tissue-engineered bone. METHODS： Our experiment was conducted in the central laboratory of Qingdao University Medical College from April 2003 to April 2004. Twelve hybrid dogs of common grade were randomly divided into 4 groups： BMSC＋rhBMP2＋PLA group, rhBMP2＋PLA group, BMSC＋PLA group and PLA group, 3 dogs in each group. Bone defects with size of 30 mm×12 mm were made at the bilateral mandibular bone of dogs. By self-control method, the right mandibular bones were taken as experimental sides and the left ones were as blank controls. The dog BMSCs were cultured and induced to differentiate into osteoblasts. Different compounded tissueengineered bones were transplanted into the mandibular defect： BMSC ＋rhBMP2＋PLA group was implanted with BMSCs, rhBMP2 and PLA, rhBMP2＋PLA group with rhBMP2 and PLA, BMSC ＋PLA group with BMSCs and PLA, and PLA group with PLA. At 2, 4, 8 weeks after implantation, X-ray, histopathological and scanning electron microscope observation were performed to evaluate the bone formation. RESULTS： The selected 12 dogs were all involved in the result analysis. ①Form of BMSCs during culture： Some BMSCs adhered to the wall 24 hours after primary culture, but at 48 hours, the adhesive cells diffused around, among which those in the margin were in fusiform shape, and the dispersed cells were elliptic or fusiform. At 10 to 12 days, the cell colonies were confluent each other and formed a monolayer, most of which were in fusiform shape. The cells began to adhere to the wall 4 hours after subculture, and all of them adhered to the wall 8 to 10 hours after subculture, most of which were differentiated into fusiform or polygonal cells with numerous processes.②The Von Kossa staining results of the subcultured cells in dense region： A large-area region was stained dark in cells, and the dark region was the calcified nodule, whereas the cells in the low dense region were not stained. ③Gross observation of bilateral bone formation at 8 weeks in all the groups： In the BMSC＋rhBMP2＋PLA group, the lamellar bone was repaired to be hard in the defects of experimental sides, and bony processes were found in partial region; In the rhBMP2 ＋PLA group, the defects of the experimental sides were not repaired completely; In the BMSC＋PLA group, dispersed bone islands formed in the defects of the experimental sides; In the PLA group, formation of new bone was only found in the margin of defects of the experimental sides. The defects of the blank control sides were filled with fibrous tissues in all the groups. ④X-ray results of both sides in all the groups at 8 weeks： For the defects of the experimental sides： Image of regular bone trabeculae were observed in the BMSC＋rhBMP2＋PLA group; A small quantity.of callus images in high density formed in the implanted region in the rhBMP2＋PLA group; Margin of bone bed was unclear, and high-density callus formed in the implanted region in the BMSC ＋PLA group; Image in spot and piece shape was found in high density in the PLA group. No X-ray images were found in the blank control sides in all the groups. ⑤Histological examination results of both experimental and blank control sides in all the groups at 8 weeks： For the defects of the experimental sides： In the BMSC＋rhBMP2＋PLA group, new bone formed a big piece, bone medullary cavity was found in implanted region, most of PLA was degraded, and new blood vessels became more, some of which passed through the degraded PLA; In the rhBMP2＋PLA group, fibrous bone formed mainly, cartilaginous bone was also found, and bone trabecula became more; In the BMSC＋PLA group, lots of dispersed bone islands formed and cartilaginous bone was found in the defects; In the PLA group, the bone defects were repaired incompletely, fibrous tissues were found in the defects, and many osteoblasts were active in the margin of bone bed. The quantity of new-formed bone was remarkably more in the BMSC ＋rhBMP2＋PLA group than in the other three groups. Fibrous tissues filled the defects instead of new bone in the blank control sides in all the groups. ⑥Results of scanning electron microscopy of osteogenesis of both sides in the BMSC＋rhBMP2＋PLA group at 8 weeks： In the experimental sides, the PLA was almost absorbed and substituted by bone tissues, small vessels entered into the PLA, osteocytes of new bone distributed evenly in the lacuna, and bone matrix deposited in the pore; In the blank control sides, abundant fibrins were found, however no bone tissues formed. CONCLUSION： The BMSCs can be induced to differentiate into the osteoblasts in vitro, which then form new bone after calcification. Exogenous bone morphogenic proteins can not only improve the proliferation of BMSCs, but also compound with the PLA, which matches BMSCs in degradation velocity and bone regeneration velocity, to accelerate bone formation, so the compound is a satisfactory material to repair bone defect.