光固化水凝胶负载骨髓间充质干细胞修复大鼠颅骨缺损

Reconstruction of rat calvarial defects utilizing an ultraviolet-cured hydrogel loaded with bone marrow mesenchymal stem cells

摘要

目的 探讨新型甲基丙烯酸酐化明胶（gelatin methacryloyl，GelMA）/骨髓间充质干细胞（bone marrow mesenchymal stem cells ，BMSCs）复合水凝胶应用于大鼠颅骨缺损区的成骨性能，为骨再生生物材料的应用提供实验依据。方法 本研究经南京大学动物伦理委员会批准。通过组合光引发剂苯基（2，4，6-三甲基苯甲酰基）磷酸锂[lthium phenyl（2，4，6-trimethylbenzoyl）phosphinate，LAP]、GelMA、BMSCs构建光固化复合生物水凝胶GelMA/BMSCs，扫描电子显微镜（scanning electron microscope，SEM）和能量色散X射线光谱仪（energy dispersive X-ray spectroscopy，EDX)检测GelMA凝胶表面微观形态及元素构成，电子万能试验机测试凝胶压缩强度。将GelMA/BMSCs水凝胶在体外培养1、2、5 d后，通过CCK-8检测水凝胶包封的BMSCs细胞增殖活性，利用共聚焦显微成像技术观察其存活情况和细胞形态；在大鼠颅骨制备5 mm临界骨缺模型，经复合水凝胶治疗的为GelMA/BMSCs组，不做任何处理的为对照组。分别于术后第 4、8周拍摄Micro-CT测算骨缺损面积和新生骨指标，第8周处死大鼠取缺损区颅骨样本进行H&E染色、Van Gieson染色和Goldner染色评价新生骨的质量。结果 SEM观察到固化的GelMA内部呈现3D海绵状大孔凝胶网络，大孔形貌均匀，孔隙率为73.41%，孔径为（28.75 ± 7.13） μm；EDX结果显示C和O均匀分布在水凝胶的网状大孔结构上；水凝胶压缩强度为152 kPa，GelMA/BMSCs 培养第5 天，镜下细胞形态铺展，从卵圆形变为梭形，CCK-8检测结果显示细胞增殖159.4%。术后第4 周，Micro-CT三维重建图像显示对照组的骨缺损范围未见明显缩小，GelMA/BMSCs组骨缺损内部可见大量新骨生成；术后第8 周，对照组骨缺损仍无明显变化，仅可见少量新生骨，GelMA/BMSCs组颅骨缺损基本愈合；第4 周与第8 周的定量分析发现， GelMA/BMSCs组大鼠颅骨缺损处新生骨量（new bone volume，BV）、骨体积分数（new bone volume/total bone volume，BV/TV）、骨表面积（bone surface，BS）、骨表面积组织体积比（bone surface/total bone volume，BS/TV）均优于对照组（P<0.05）。第8 周组织学染色结果显示GelMA/BMSCs组骨缺损处形成连续且致密的骨组织，而对照组仅在缺损边缘可见少量不连续新骨生成，缺损处主要为纤维结缔组织。结论 光固化水凝胶的干细胞疗法具有良好生物安全性，在诱导大鼠颅骨缺损区新骨形成的同时促进骨成熟，具有潜在的临床转化前景。

Abstract

Objective To investigate the osteogenic properties of a methacrylated gelatin (GelMA) / bone marrow mesenchymal stem cells (BMSCs) composite hydrogel applied to the skull defect area of rats and to provide an experimental basis for the development of bone regeneration biomaterials. Methods This study was approved by the Animal Ethics Committee of Nanjing University. A novel photocurable composite biohydrogel was developed by constructing photoinitiators [lthium phenyl (2,4,6-trimethylbenzoyl) phosphinate, LAP], GelMA, and BMSCs. The surface morphology and elemental composition of the gel were examined using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The compressive strength of the gel was evaluated using an electronic universal testing machine. After in vitro culture for 1, 2, and 5 days, the proliferation of the BMSCs in the hydrogels was assessed using a CCK-8 assay, and their survival and morphology were examined through confocal microscopy. A 5 mm critical bone deficiency model was generated in a rat skull. The group receiving composite hydrogel treatment was referred to as the GelMA/BMSCs group, whereas the untreated group served as the control group. At the 4th and 8th weeks, micro-CT scans were taken to measure the bone defect area and new bone index, while at the 8th week, skull samples from the defect area were subjected to H&E staining, van Gieson staining, and Goldner staining to evaluate the quality of bone regeneration and new bone formation. Results SEM observed that the solidified GelMA showed a 3D spongy gel network with uniform morphology, the porosity of GelMA was 73.41% and the pore size of GelMA was (28.75 ± 7.13) μm. EDX results showed that C and O were evenly distributed in the network macroporous structure of hydrogel. The hydrogel compression strength was 152 kPa. On the 5th day of GelMA/BMSCs culture, the cellular morphology transitioned from oval to spindle shaped under microscopic observation, accompanied by a significant increase in cell proliferation (159.4%, as determined by the CCK-8 assay). At 4 weeks after surgery, a 3D reconstructed micro-CT image revealed a minimal reduction in bone defect size within the control group and abundant new bone formation in the GelMA/BMSCs group. At 8 weeks after surgery, no significant changes were observed in the control group's bone defect area, with only limited evidence of new bone growth; however, substantial healing of skull defects was evident in the GelMA/BMSCs group. Quantitative analysis at both the 4- and 8-week examinations indicated significant improvements in the new bone volume (BV), new bone volume/total bone volume (BV/TV), bone surface (BS), and bone surface/total bone volume (BS/TV) in the GelMA/BMSCs group compared to those in the control group (P<0.05). Histological staining showed continuous and dense formation of bone tissue within the defects in the GelMA/BMSCs group and only sporadic formation of new bone, primarily consisting of fibrous connective tissue, at the defect edge in the control group. Conclusion Photocuring hydrogel-based stem cell therapy exhibits favorable biosafety profiles and has potential for clinical application by inducing new bone formation and promoting maturation within rat skull defects.

关键词

骨缺损 / 骨组织工程 / 支架 / 水凝胶 / GelMA水凝胶 / 骨髓间充质干细胞 / 干细胞疗法 / 光固化 / 骨再生 / 骨修复

Key words

bone defect / bone tissue engineering / scaffold / hydrogel / GelMA hydrogel / bone marrow mesenchymal stem cells / stem cell therapy / photocuring / bone regeneration / bone repair

Author summay

丁梦，博士研究生，Email：[e-mail]

共同第一作者，李强，博士研究生，Email：[e-mail]