中国实用口腔科杂志

中国实用口腔科杂志 ›› 2026, Vol. 19 ›› Issue (5): 580-589.DOI: 10.19538/j.kq.2026.05.009

• 论著 • 上一篇    下一篇

羟基磷灰石微管-壳聚糖支架诱导骨组织再生效果评价

张泽良，李维文   

  1. 长治医学院附属和平医院口腔科，山西 长治 046000
  • 出版日期:2026-09-30 发布日期:2026-09-30
  • 通讯作者: 张泽良
  • 基金资助:
    长治医学院附属和平医院青年基金（HPYJ202407）

  • Online:2026-09-30 Published:2026-09-30

摘要： 目的    评价羟基磷灰石微管（hydroxyapatite microtubules，HAMT）-壳聚糖（chitosan，CHS）支架诱导骨组织再生的效果。方法    研究于2022—2023年在长治医学院中心实验室进行。将HAMT、纳米羟基磷灰石（nano-hydroxyapatite，nano-HAP）分别与CHS混合，利用蜡球作为造孔剂制备HAMT-CHS支架和nano-HAP-CHS支架。通过扫描电镜、傅里叶变换红外光谱分析仪、X射线衍射仪、万能测试机、全自动压汞仪检测支架表征，并检测支架装载苯妥英钠的载药缓释性能。将支架与大鼠骨髓干细胞（bone marrow stem cells，BMSCs）共培养，通过CCK-8实验、碱性磷酸酶（alkaline phosphatase，ALP）染色检测支架的生物相容性，通过RT-qPCR和Western Blot检测支架上细胞的骨桥蛋白（osteopontin，OPN）、Runt相关转录因子2（Runt-related transcription factor 2，RUNX2）及成骨特异性锌指转录因子osterix（OSX）等成骨相关基因表达情况。选取9只8周龄SD大鼠构建颅骨缺损模型，根据缺损处不放置支架、放置HAMT-CHS支架及放置nano-HAP-CHS支架将所有大鼠分为空白组、T组和N组（每组3只），2个月后通过Micro-CT观察比较颅骨缺损部位的组织体积（tissue volume，TV）、骨体积（bone volume，BV）、骨表面积（bone surface，BS）、骨小梁厚度（trabecular thickness，Tb.Th）、骨小梁分离度（trabecular separation，Tb.Sp）、骨小梁数量（trabecular number，Tb.N）、骨体积分数（bone volume fraction，BV/TV）、骨表面积与骨体积比值（bone surface fraction，BS/BV）及骨表面积组织体积比（bone surface area density，BS/TV），并通过苏木精-伊红染色法染色观察新骨质形成情况。结果    ①支架表征：研究制备的HAMT-CHS支架纯度较高。HAMT-CHS支架和nano-HAP-CHS支架均呈梯度分布的孔隙结构，且孔隙间有裂孔相通；HAMT-CHS支架内壁可见管状结构排列，nano-HAP-CHS支架内壁较平整。HAMT-CHS支架的弹性模量［（166.717 ± 0.031）MPa］明显高于nano-HAP-CHS支架［（86.235 ± 0.025）MPa］，差异有统计学意义（t = 3523.727，P < 0.001）；其最大承受压力［（2.20 ± 0.05）N/mm2］明显高于nano-HAP-CHS支架［（1.70 ± 0.21）N/mm2］，差异有统计学意义（t = 4.131，P = 0.046）；而其孔隙率［（84.03 ± 4.35）%］与nano-HAP-CHS支架［（82.89 ± 3.53）%］比较，差异无统计学意义（t = 0.350，P = 0.745）。②载药缓释性能：nano-HAP-CHS支架的初始药物释放速度较快，7 d后2种支架均进入药物缓慢释放期，35 d时HAMT-CHS支架的药物累积释放量［（0.398 ± 0.021）mg］与nano-HAP-CHS支架［（0.407 ± 0.032）mg］比较，差异无统计学意义（t = 0.361，P = 0.737）。③生物相容性：HAMT-CHS支架和nano-HAP-CHS支架上的BMSCs增殖情况良好，共培养第1、3、5、7天HAMT-CHS支架上细胞在波长450 nm处的光密度（OD450）值均明显优于nano-HAP-CHS支架，差异均有统计学意义（均P < 0.05）。另外，共培养7 d后HAMT-CHS支架的ALP染色情况较nano-HAP-CHS支架更深。④成骨相关基因表达：共培养7 d时，HAMT-CHS支架细胞中OPN和RUNX2的mRNA相对表达量均明显高于nano-HAP-CHS支架，差异均有统计学意义（均P < 0.05）；而OSX的mRNA相对表达量比较差异无统计学意义（P > 0.05）。共培养14 d时，HAMT-CHS支架细胞中OPN和RUNX2的mRNA和蛋白相对表达量均明显高于nano-HAP-CHS支架，OSX的mRNA相对表达量明显低于nano-HAP-CHS支架，差异均有统计学意义（均P < 0.05），而OSX的蛋白相对表达量与nano-HAP-CHS支架比较差异无统计学意义（P > 0.05）。⑤动物实验：T组和N组颅骨缺损部位有明显新骨形成，空白组在缺损边缘有少量新骨形成。但3组TV、BV、BS、Tb.Th、Tb.Sp、Tb.N、BV/TV、BS/BV、BS/TV比较，差异均无统计学意义（均P > 0.05）。结论    研究成功制备了纯度较高、内壁微观结构粗糙的HAMT-CHS梯度孔隙支架，其机械性能、生物相容性及促进骨组织再生能力较佳，是具有广阔应用前景的骨组织替代材料。

关键词: 羟基磷灰石微管, 壳聚糖, 骨再生, 组织工程

Abstract: Objective    To evaluate the efficacy of hydroxyapatite microtubule（HAMT）-chitosan（CHS）scaffolds in inducing bone tissue regeneration. Methods    The study was conducted at the Central Laboratory of Changzhi Medical College from 2022 to 2023. HAMT and nano-hydroxyapatite（nano-HAP）were separately blended with CHS to fabricate HAMT-CHS and nano-HAP-CHS scaffolds using paraffin spheres as the pore forming substance. The characteristics of scaffolds were evaluated via scanning electron microscopy，Fourier transform infrared spectroscopy，X-ray diffraction，a universal testing machine，and automated mercury intrusion porosimetry. The drug-loading sustained-release profiles of the scaffolds loaded with phenytoin sodium were also evaluated. Bone marrow stem cells（BMSCs）isolated from rats were co-cultured with the scaffolds. Biocompatibility was assessed using CCK-8 assays and alkaline phosphatase（ALP）staining. The expression of osteogenesis-related genes，including osteopontin（OPN），Runt-related transcription factor 2（RUNX2），and osterix（OSX），was quantified via RT-qPCR and Western Blot. Nine 8-week-old Sprague-Dawley rats were utilized to establish cranial defect models. The rats were randomly allocated into three groups（n = 3 per group）：the blank group（no scaffold placement），Group T（HAMT-CHS scaffold implantation），and Group N（nano-HAP-CHS scaffold implantation）. Two months after implantation，Micro-CT was performed to observe and compare the tissue volume（TV），bone volume（BV），bone surface（BS），trabecular thickness（Tb.Th），trabecular separation（Tb.Sp），trabecular number（Tb.N），bone volume fraction（BV/TV），bone surface fraction（BS/BV），and bone surface area density（BS/TV）at the defect sites. Additionally，hematoxylin and eosin staining was performed to observe new bone formation. Results    ①Scaffold characteristics：the fabricated HAMT-CHS scaffolds exhibited high purity. Both HAMT-CHS and nano-HAP-CHS scaffolds displayed a gradient porous architecture with interconnected pores. Microtubular structures were aligned along the inner walls of the HAMT-CHS scaffolds，whereas the inner walls of the nano-HAP-CHS scaffolds were relatively smooth. The elastic modulus of the HAMT-CHS scaffolds ［（166.717 ± 0.031）MPa］ was significantly higher than that of the nano-HAP-CHS scaffolds ［（86.235 ± 0.025）MPa］（t = 3523.727，P < 0.001）. The maximum compressive strength of the HAMT-CHS scaffolds ［（2.20 ± 0.05）N/mm2］ was also significantly greater than that of the nano-HAP-CHS scaffolds ［（1.70 ± 0.21）N/mm2］（t = 4.131，P = 0.046）. However，no statistically significant difference in porosity was observed between the HAMT-CHS［（84.03 ± 4.35）%］and nano-HAP-CHS scaffolds ［（82.89 ± 3.53）%］（t = 0.350，P = 0.745）. ②Drug-loading sustained-release performance：the nano-HAP-CHS scaffolds exhibited a faster initial drug release rate. After 7 days，both scaffolds entered a phase of sustained，slow drug release. At day 35，the cumulative drug release of the HAMT-CHS scaffolds［（0.398 ± 0.021）mg］showed no significant difference compared to that of the nano-HAP-CHS scaffolds［（0.407 ± 0.032）mg］（t = 0.361，P = 0.737）. ③Biocompatibility：BMSCs proliferated robustly on both HAMT-CHS and nano-HAP-CHS scaffolds. On days 1，3，5，and 7 of co-culture，the optical density values at wavelength 450 nm（OD450）of the cells on the HAMT-CHS scaffolds were significantly better than those on the nano-HAP-CHS scaffolds（all P < 0.05）. Furthermore，after 7 days of co-culture，ALP staining was noticeably more intense in the HAMT-CHS scaffolds than in the nano-HAP-CHS scaffolds. ④Osteogenesis-related gene expression：at day 7 of co-culture，the relative mRNA expression levels of OPN and RUNX2 in the HAMT-CHS scaffolds were significantly higher than those in the nano-HAP-CHS scaffolds（both P < 0.05），while no significant difference was found for OSX mRNA expression（P > 0.05）. At day 14 of co-culture，both the relative mRNA and protein expression levels of OPN and RUNX2 were significantly higher in the HAMT-CHS scaffolds than in the nano-HAP-CHS scaffolds（P < 0.05）. In contrast，the relative mRNA expression of OSX in the HAMT-CHS scaffolds was significantly lower than that in the nano-HAP-CHS scaffolds（P < 0.05），while its protein expression levels showed no statistically significant difference between the two scaffolds（P > 0.05）. ⑤In vivo animal study：distinct new bone formation was observed at the cranial defect sites in both Group T and Group N，whereas the blank group showed only a small amount of new bone at the defect margins. However，no statistically significant differences were observed among the three groups regarding TV，BV，BS，Tb.Th，Tb.Sp，Tb.N，BV/TV，BS/BV，and BS/TV（all P > 0.05）. Conclusion    In this study，HAMT-CHS gradient porous scaffolds with high purity and a rough inner-wall microstructure are successfully fabricated. The scaffolds exhibit superior mechanical properties，excellent biocompatibility，and enhanced capacity to promote bone tissue regeneration，representing a highly promising bone substitute material.

Key words: hydroxyapatite microtubule, chitosan, bone regeneration, tissue engineering

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