【摘要】 目的：筛选适宜尺寸的羟基磷灰石（HAP）颗粒负载骨形态发生蛋白2(BMP2)以构建HAP/BMP2活性骨植入物用于促骨缺损再生修复研究。方法：研磨过筛法制备HAP颗粒，取直径范围在0.50～1.00 mm、1.00～1.25 mm和1.25～1.50 mm 3种尺寸的颗粒，记为HAP-1、HAP-2和HAP-3。通过扫描电子显微镜（SEM）、CCK8法和模拟填充实验评估HAP颗粒的形貌、生物相容性和填充性。优选HAP颗粒负载BMP2构建HAP/BMP2活性骨植入物，并与人间充质干细胞（hBMSC）共培养，通过实时荧光定量聚合酶链式反应（qRT-PCR）、碱性磷酸酶（ALP）染色检测HAP/BMP2体外促成骨性能。选取15只8月龄、平均体重为（8.0±0.7）kg的比格犬采用随机数字表法分为3组，每组5只，构建颈椎大段缺损模型，研究活性骨植入物促骨修复性能。对照组骨缺损术后在C2～4间用接骨板及螺钉固定邻近椎体；HAP-2组骨缺损术后填充HAP-2骨植入物，行相同固定术；HAP-2/BMP2组术后填充HAP-2/BMP2活性骨植入物，行相同固定术。术后6、12个月，比格犬于侧卧位拍摄X线片后处死，取颈椎C1～4节段，并使用微型CT扫描C1～4节段样本，使用CT-An、CT-Vol进行3D重建。然后进行甲苯胺蓝染色，全景扫描并用ImageJ 2.1.0软件进行半定量分析，以评估植入物体内促进骨缺损修复再生的能力。结果：HAP-1(0.50～1.00 mm)、HAP-2(1.00～1.25 mm)和HAP-3(1.25～1.50 mm)3种尺寸HAP颗粒均保留了致密多孔晶体结构，HAP-1、HAP-2颗粒浸提液处理hBMSC 72 h无明显毒性（P>0.05），且与HAP-3比较，HAP-1、HAP-2颗粒显示出更好的填充性能。qRT-PCR结果证实HAP-1、HAP-2颗粒负载BMP2前后均能促进成骨相关基因Alp、Col1、Runx2的表达，且HAP-2/BMP2组效果最佳（P<0.001）。ALP染色显示HAP-2/BMP2组的ALP活性最强。体内促成骨实验中，X线片及微型CT结果显示，HAP-2/BMP2活性骨植入物在术后6、12个月显著促进比格犬颈椎缺损的新生骨组织再生和成熟，甲苯胺蓝染色结果证实HAP-2/BMP2组比格犬新生骨组织面积更大（P<0.001）。结论：不同尺寸HAP颗粒作为载体制备的HAP/BMP2活性骨植入物促成骨效果不同，其中HAP-2(1.00～1.25 mm)具有良好的结构、填充性、生物相容性和更优异的促成骨性能，具有临床价值。更多还原

【Abstract】 Objective: To screen suitable sizes of hydroxyapatite(HAP) particles loaded with bone morphogenetic protein-2(BMP-2) to construct HAP/BMP2 active bone implants for promoting regenerative repair of bone defects. Methods: HAP particles were prepared using grinding and sieving. Particles with diameter ranges of 0.5-1.0 mm, 1.0-1.25 mm, and 1.25-1.5 mm were selected and designated as HAP-1, HAP-2, and HAP-3, respectively. The morphology, biocompatibility, and filling properties of HAP particles were evaluated using scanning electron microscopy, CCK-8 assay, and simulated filling experiments. The optimal HAP particles were loaded with BMP-2 to construct HAP/BMP-2 active bone implants and co-cultured with human bone marrow mesenchymal stem cells(hBMSCs). The osteogenic potential of HAP/BMP-2 was assessed in vitro using quantitative real-time polymerase chain reaction(qRT-PCR) and alkaline phosphatase(ALP) staining. Fifteen 8-month-old beagle dogs, with an average weight of(8.0±0.7) kg, were randomly divided into three groups with five dogs in each group. A large cervical spine defect model was constructed to study the bone repair performance of active bone implants. Six and twelve months postoperatively, the Beagle dogs were euthanized after X-ray imaging in the lateral position. The C1-C4 cervical spine segments were collected and scanned using Micro-CT, and 3D reconstruction was performed using CT-An and CT-Vol. Toluidine blue staining was conducted, and panoramic scanning was performed for semiquantitative analysis using ImageJ 2.1.0 to evaluate the ability of the implants to promote regenerative repair of bone defects in vivo. Results: HAP particles of three different sizes—HAP-1(0.50-1.00 mm), HAP-2(1.00-1.25 mm), and HAP-3(1.25-1.50 mm) —all retained a dense porous crystal structure. The extracts from HAP-1 and HAP-2 particles showed no significant cytotoxicity to hBMSCs after incubating for 72 h(P>0.05), and both demonstrated better filling properties compared to HAP-3. qRT-PCR results confirmed that both HAP-1 and HAP-2 particles, before and after BMP2 loading, could promote the expression of osteogenic-related genes, Alp, Col1, and Runx2, with the HAP-2/BMP2 group showing the most significant effect(P<0.001). ALP staining revealed that the HAP-2/BMP2 group had the strongest ALP activity. In the in vivo osteogenesis experiments, X-ray and micro-CT results showed that HAP-2/BMP2 active bone implant significantly promoted the regeneration and maturation of new bone tissue in the cervical spine defects of Beagle dogs at 6 and 12 months post-surgery. Toluidine blue staining confirmed that the HAP-2/BMP2 group had a larger area of new bone tissue(P<0.001). Conclusions: The HAP/BMP2 implants prepared with HAP particles of different sizes exhibited varying osteogenic effects. Among them, HAP-2(1.00-1.25 mm) demonstrated excellent structure, filling properties, biocompatibility, and superior osteogenic performance, indicating significant clinical value.更多还原