掺钆羟基磷灰石生物纳米复合材料的生物性能评价

Evaluation of biological properties of Gd-doped hydroxyapatite bio-nanocomposites

目的探讨新型掺钆羟基磷灰石(Gd-HA)复合支架的生物相容性,及其作为细胞培养材料和骨组织工程支架的可行性。方法化学合成Gd-HA复合支架并进行电子显微镜观察;实验分为3组:HA组、Gd-HA组和对照组;分离、培养和鉴定兔脂肪间充质干细胞(ADSCs),在ADSCs体外培养体系加入Gd-HA复合支架浸提液,通过活死细胞染色法评估细胞存活率及细胞毒性,CCK-8法评估支架内细胞增殖能力,茜素红染色评估支架对细胞成骨分化;通过皮肤刺激试验、全身急性毒性试验和支架肌内埋植部位肌肉组织及肝肾脏病理学观察支架材料毒性反应。结果Gd-HA复合支架电子显微镜下呈不规则空隙结构;细胞形态观察显示,ADSCs贴壁生长,呈长梭形;流式细胞术检测CD29阳性率为96.94%,CD44阳性率为97.90%,CD45阳性率为0.10%,CD34阳性率为0.46%;活死细胞染色显示,共培养5 d后Gd-HA组活细胞量明显优于羟基磷灰石(HA)组;CCK-8检测0~3 d内细胞增殖无明显差异;3 d后Gd-HA组明显优于HA组和对照组(P<0.05);Gd-HA组茜素红染色后钙结节沉积明显优于HA组和对照组,呈现更深的红色;浸提液接触皮肤后大体及皮肤组织HE染色观察均未见皮肤刺激现象;浸提液注入腹腔后各实验组一般状态良好,体质量稳步上升趋势(P>0.05);支架肌内埋植部位HE染色显示,Gd-HA组材料与肌肉组织的界面炎症反应明显高于对照组,随着植入时间延长炎性细胞浸润逐渐减轻,8周时材料周围组织形态接近正常肌肉组织,12周时肝肾HE染色观察未见病理性改变。结论Gd-HA复合支架具有良好的生物相容性,有利于细胞的增殖及成骨分化,有望成为组织工程中干细胞移植的良好载体。

Objective To investigate the biocompatibility of new gadolinium-doped hydroxyapatite(Gd-HA)composite scaffolds and to explore their feasibility as cell culture materials and bone tissue engineering scaffolds.Methods The Gd-HA composite scaffolds were chemically synthesized and placed under the electron microscope for observation.The experiment was divided into three groups,the HA group,the Gd-HA group,and the control group.Rabbit adipose-derived mesenchymal stem cells(ADSCs)were isolated,cultured and characterized,and the Gd-HA composite scaffold extract was added to the ADSCs in vitro culture system.Cell survival and cytotoxicity were assessed by live-dead cell staining,cell proliferation ability within the scaffolds was assessed by CCK-8 assay,and the scaffolds were assessed by alizarin red staining for cell osteogenic differentiation.The toxic reactions of the scaffold materials were observed by skin irritation test,systemic acute toxicity test and muscle tissue and liver and kidney pathology at the site of intramuscular implantation of the scaffolds.Results The Gd-HA composite scaffold showed irregular void structure under electron microscope.Cell morphology observation showed that ADSCs grew adherently to the wall and were long shuttle-shaped.The positivity rate of CD29 was 96.94%,CD44 was 97.90%,CD45 was 0.10%,and CD34 was 0.46%,which was obtained using flow cytometry.Live-dead cell staining showed that the amount of live cells in the Gd-HA group was significantly better than that in the hydroxyapatite(HA)group after 5 days of co-culture.CCK-8 assay showed no significant difference in cell proliferation within O-3 days.After 3 days,the Gd-HA group was significantly better than the HA group and the control group(P<0.05).Calcium nodule deposition after alizarin red staining was significantly better in the Gd-HA group than in the HA and control groups,showing a deeper red color.No skin irritation was observed in gross and skin tissue HE observations after the contact of the extract with the skin.The general condition of the experimental groups was good after the infusion of the extract into the abdominal cavity,and the body mass tended to increase steadily(P>0.05).HE staining showed that inflammatory reaction at the interface between the material and muscle tissue of the stent intramuscular implantation site in Gd-HA group was significantly higher than that of the control group,and the inflammatory cell infiltration was gradually reduced with the prolongation of implantation time.At the 8th weeks the morphology of the tissue around the material was close to normal muscle tissue,and no pathological changes were observed in the HE staining of liver and kidney at the 12th week.Conclusion Gd-HA composite scaffolds exhibit good biocompatibility and facilitate cell proliferation and osteogenic differentiation,and they are expected to serve as good carriers for stem cell transplantation in tissue engineering.