Yuan Wei, Zheng Jun, Qian Jinyu, Zhou Xiaoxiao, Wang Minghui, Wang Xiuhui
Department of Orthopedic Surgery, Affiliated Zhoupu Hospital, Shanghai University of Medicine & Health Sciences, Shanghai, 201318, P.R.China.
Department of Orthopedics and Traumatology, Yueyang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 200437,
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2017 Jul 15;31(7):862-869. doi: 10.7507/1002-1892.201703043.
To observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model.
SVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson's trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining.
The cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C ( <0.05), and in group B than groups A and C ( <0.05). HE staining, Masson's trichrome staining, and immunohistochemistry staining for OCN staining exhibited a large number of cartilage cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and there was no effective new bone formation in groups A and C.
The combination of sustained release of rhBMP-2 and freshly SVFs can significantly promote spinal fusion in rat model, providing a theoretical basis for further clinical applications.
观察大鼠脂肪组织来源的基质血管成分细胞（SVFs）联合重组人骨形态发生蛋白-2（rhBMP-2）缓释在大鼠腰椎融合模型中的作用效果。
通过Ⅰ型胶原酶消化法从4月龄大鼠双侧腹股沟区皮下脂肪中获取SVFs。采用仿生磷灰石涂层工艺通过rhBMP-2与β-磷酸三钙（β-TCP）共价结合制备缓释载体。采用BCA法检测缓释效果。选取32只大鼠建立腰椎后外侧融合模型，分为4组，每组8只。分别将脱钙骨基质（DBX）支架+PBS、DBX支架+rhBMP-2/β-TCP缓释载体、DBX支架+SVFs、DBX支架+rhBMP-2/β-TCP缓释载体+SVFs植入A、B、C、D组。术后8周采用X线片、手动触诊脊柱及高分辨率显微CT评估脊柱融合情况；分析骨密度（BMD）和骨体积分数；通过HE染色和Masson三色染色评估新骨形成情况，采用免疫组织化学染色检测骨钙素（OCN）。
rhBMP-2在2周时累计释放量约为40%，表明rhBMP-2具有缓释效果；而对照组在2周内几乎释放完毕。术后8周，手动触诊脊柱、X线及显微CT联合评估显示，D组骨形成最强（100%，8/8），其次为B组（75%，6/8）、C组（37.5%，3/8）、A组（12.5%，1/8）。显微CT分析显示，D组的BMD和骨体积分数显著高于A、B、C组（<0.05），B组高于A、C组（<0.05）。HE染色、Masson三色染色及OCN免疫组织化学染色显示，D组有大量软骨细胞及骨基质沉积，呈现出与长骨矿化相似的活跃成骨过程。B组骨形成弱于D组，A、C组无有效新骨形成。
rhBMP-2缓释与新鲜SVFs联合应用可显著促进大鼠模型中的脊柱融合，为进一步临床应用提供理论依据。
