HA复合rhBMP-2转染的BMSCs对羊胫骨延长骨愈合的影响

THE EFFECT OF HA MIXED WITH ADENOVIRUS MEDIATED rhBMP-2 TRANSFERRED BMSCs OF GOATS ON DISTRACTION OSTEOGENESIS

目的评价HA复合rhBMP-2的腺病毒(adenovirus mediated rhBMP-2,Adv-rhBMP-2)转染的羊BMSCs对骨痂延长骨愈合的影响。方法成年山羊19只,雌雄不限,体重15～20kg。于每只羊髂嵴上穿刺抽取骨髓10mL,常规传代培养BMSCs。取第3代BMSCs,以感染复数200转染腺病毒。取0.25%胰蛋白酶消化转染后3d的细胞1×108个,与HA充分混匀制备Adv-rhBMP-2/BMSCs/HA。建立山羊右侧胫骨延长模型,术后立即于截骨部位注射自体细胞复合物。按术后注入物质不同随机分成4组:A组为Adv-rhBMP-2/BMSCs/HA组(n=6),B组为Adv-rhBMP-2/BMSCs组(n=5),C组为Adv-β-gal/BMSCs/HA组(n=4),D组为空白组(n=4)。术后第7天开始行胫骨延长,速度1mm/d,共延长4周。术后5、8、12周摄X线片观察骨痂生长情况,12周处死动物,取标本分别行骨密度检测、生物力学测定、组织学观察和骨形态计量学分析。结果X线片检查示,术后5、8周A、B组骨痂生成量明显多于C、D组,X线片定量评分A、B组明显高于C、D组,差异均有统计学意义(P<0.05);12周各组均在骨延长部位形成连续骨痂。骨密度测定:术后12周A、B、C、D组延长部位骨矿物质含量分别为(4.175±1.921)、(2.600±0.638)、(2.425±0.826)和(1.175±0.574)?g,骨矿物质密度分别为(0.612±0.196)、(0.630±0.159)、(0.450±0.166)和(0.266±0.113)g/cm2,A、B组显著高于C、D组(P<0.05)。生物力学测定:A、B、C、D组最大载荷分别为(490.20±155.08)、(350.59±80.48)、(221.95±68.79)和(150.65±92.29)N,弹性模量为(178.24±105.80)、(105.88±27.09)、(81.18±48.67)和(50.35±47.64)MPa,各指标A组显著高于C、D组(P<0.05)。组织学观察见A组骨延长处大量新生骨组织,骨小梁多为纵行网状排列。骨形态计量分析示A、B、C、D组新骨体积分别为72.35%±5.68%、67.58%±7.42%、49.63%±4.87%和38.87%±2.35%,A组新生骨生成量明显比D组多(P<0.05)。结论HA复合rhBMP-2修饰的BMSCs制备的组织工程骨可促进羊胫骨延长骨愈合。

Objective To evaluate the effectiveness of HA mixed with adenovirus mediated rhBMP-2 gene （AdvrhBMP-2） transferred BMSCs of goats on distraction osteogenesis. Methods Nineteen adult goats were used for the experiment, no matter they were male or female, and the weight of the goats were 15-20 kg. The 10 mL marrow was obtained from the iliac crest of each goat. The BMSCs was expanded and passaged conventionally. The 3th BMSCs was infected by Adv-rhBMP-2 at 200 multiplicity of infection （MOI）. The 1× 10^8 infected BMSCs were digested by 0.25% trypsin and collected, then mixed with HA. The right tibia lengthening models were developed, and mixture with BMSCs was injected in the osteotomy position. The goats were divided randomly into 4 groups according to the material injected in operation, group A： Adv-rhBMP-2/BMSCs/HA （n=6）; group B： Adv-rhBMP-2/BMSCs （n=5）; group C： Adv-β -gal/BMSCs/HA （n=4）; group D： sham without any injections （n=4）. After a seven-day latency period following ostectomy, distraction was carried out at a rate of 1 mm/day for 4 weeks. Roentgenography was practiced in 5, 8 and 12 weeks. After 12 weeks, the goats were sacrificed and dual-energy X-ray absorptiometry （DXA）, biomechanical test and histology results were studied. Results After five and eight weeks surgery, X-ray test showed the distraction callus was more in group A and B than group C and D, and the radiographic score was significantly higher in group A and B than in the other two groups（P 〈 0.05）; after 12 weeks surgery, the continued callus was formed in the distraction defects in all groups. DXA showed the mean bone mineral content of distraction callus in group A, B, C, D was （4.175 ± 1.921）, （2.600 ±0.638）, （2.425 ±0.826） and （1.175 ＋0.574） g, and the mean bone mineral density was （0.612 ±0.196）, （0.630 ± 0.159）, （0.450±0.166） and （0.266 ±0.113） g/cm2. The group A and B was significantly higher than group C and D （P 〈 0.05）. Biomechanical test showed the maximum loading of group A, B, C, D was （490.20± 155.08）, （350.59 ± 80.48）, （221.95± 68.79） and （150.65 ± 92.29） N, and elastic modulus was （178.24± 105.80）, （105.88 ± 27.09）, （81.18 ±48.67） and （50.35± 47.64） MPa. The group A was significantly higher than in group C and D （P 〈 0.05）. Histology observation revealed abundant bone formation in the distraction defects in group A, and the bone trabecula was arranged longitudinal and netlike. Histomorphology analysis revealed the bone volume in group A, B, C, D was 72.35%±5.68%, 67.58% ±7.42%, 49.63% ±4.87% and 38.87% ±2.35%, and the bone formation was significantly greater in group A compared with group D （P 〈 0.05）. Conclusion HA mixed with rhBMP-2 modified BMSCs can accelerate distraction osteogenesis in goats.