摘要
力生长因子(mechano-growth factor, MGF)是一种新发现的生长因子,它是胰岛素样生长因子-I(insulin like-growth factor-I, IGF-I)基因剪接变异的产物。该生长因子由力敏感细胞响应力学刺激而表达,已有研究表明MGF及其E肽(MGF- Ct24E)具有促进肌肉肥大、心肌保护及神经修复等功能。本实验室前期研究表明,MGF及MGF-Ct24E能够促进成骨细胞增殖,影响其分化、矿化过程,这提示MGF将会对骨折愈合过程产生影响。本研究围绕MGF-Ct24E在骨折愈合中的功能应用展开,包括以下两个方面的内容:一是MGF-Ct24E直接注射于骨缺损部位对骨折愈合的影响;二是MGF-Ct24E生物活性材料的制备与性能。主要研究内容和结果如下:
(1)MGF-Ct24E直接注射于骨缺损部位对骨折愈合的影响
通过手术在大白兔桡骨中段制造5mm的完全缺损骨折模型,术后第3天开始局部注射不同剂量的MGF-Ct24E(对照组注射PBS),连续注射5 d,1次/d。然后,进行如下检测:①术后第4,6,8周,X射线摄片观察骨折愈合情况;②术后第8周,通过组织学观察了解各组骨折部位细胞形态学变化。所有检测结果均表明,较高剂量的MGF-Ct24E(0.144mg/次)局部注射能够明显提高骨愈合质量,并且愈合时间提前2周以上。
(2)MGF-Ct24E生物活性材料的制备与性能研究
①首先,制备含有MGF-Ct24E的生物活性材料(MHPLA),即按照整体仿生思路,依次将马来酸酐、已二胺、MGF-Ct24E引入聚乳酸中,合成一种新型的仿生骨基质材料MHPLA,本研究中引入马来酸酐、己二胺为MGF-Ct24E引入聚乳酸中提供活性位点;
②采用分子量和分子量分布对引入MGF-Ct24E过程中对MHPLA分子量的影响进行表征,表征结果表明,采用依次引入改性单体的过程对本体聚合物本身有一定的影响,即相对于初始PDLLA的分子量,MHPLA分子量明显下降,但该分子量仍保持在骨折愈合材料的要求范围内;
③采用氨基酸分析、核磁氢谱对MHPLA中MGF-Ct24E的接入情况进行检测,为深入研究MGF-Ct24E生物活性材料在骨折愈合中的意义奠定基础,两种检测均表明,MGF-Ct24E被成功接入到聚乳酸上,通过整体仿生法制得一种新型骨基质材料。反应过程中MGF-Ct24E的转化率为25%,MHPLA中MGF-Ct24E的绝对含量为0.88 mmol/g;
④最后,通过体外细胞相容性研究,探讨了MGF-Ct24E生物活性材料对成骨细胞生长行为和功能行为的影响。将对照组、实验组材料与原代大鼠成骨细胞共培养,分别采用显微镜观察、MTT检测、ALP检测的方法对成骨细胞形态、成骨细胞的增殖、成骨细胞分化进行检测,可见成骨细胞在MHPLA材料上形态饱满,粘附及铺展状况良好,并且明显促进成骨细胞增殖的活性、抑制其分化,时间持续一周以上,所有结果均表明,MGF-Ct24E—生物活性材料(MHPLA)中的MGF-Ct24E保持其生物活性,促使MHPLA细胞相容性好,表现出明显地促进成骨细胞生长行为和功能行为的优势;另外,MGF-Ct24E—生物活性材料相对于游离的MGF-Ct24E具有作用时间延长的优势,因此将MGF-Ct24E—生物活性材料植入骨缺损部位有望起到更好的骨折愈合效果。
Mechano growth factor (MGF) is a novel growth factor from the alternative splicing of Insulin-like growth factor I (IGF-I) gene, which is expressed in response to the mechanic stimulation. Further studies have confirmed that MGF and its E peptide (MGF-Ct24E) could promote muscle hypertrophy, preserve cardiac muscle and repair the damage of nerves. Our previous studies concluded that MGF mRNA expressed highly in the cyclic stretched osteoblasts, suggested that MGF is a mechano-response molecule in bone and influences on the function of osteoblasts. these results indicated that MGF or MGF-Ct24E may play a major role in the process of bone healing. This research focus on the influence of MGF-Ct24E for fracture healing. Firstly, effects of the injected-MGF-Ct24E on radial fracture healing; Secondly, the preparation and property of the bioactive materials containing MGF-Ct24E. The main works and conclusions are included as follows:
(1) The effects of the injected-MGF-Ct24E on radial fracture healing
The animal models used in this study, with 5mm bone defect, were produced in the middle of the left radius in rabbits. After 3 days of the surgical operation, 0.2 mL MGF (0.36 and 0.72 g/L, control: PBS) was injected into the ends of fracture areas once per day for continuous 5 days. And then following detections were taken:①After 4, 6, 8 weeks, X-ray photography was used to evaluate the healing of fracture;②At 8th week, histological examinations were performed to observe the cell morphology. All results showed that MGF-Ct24E can accelerate fracture healing significantly in a rabbit model and shows a dose-dependent manner in a certain range.
(2) The preparation and property of the bioactive materials containing MGF-Ct24E.
①The bioactive materials containing MGF-Ct24E was prepared in the Research Center of Bioinspired Material Science & Engineering;
②GPC-MALLS was used to confirmed the influence of MGF-Ct24E introduction on molecular weight of PDLLA, which showed that the modified process reduced the molecular weight of PDLLA, however, did not weaken the function of PDLLA in fracture healing;
③Amino Acid Analysis (AAA) and 1H-NMR were used to determine the introduction and bioactivity of MGF-Ct24E, which is key to the study of influences of bioactive materials on fracture healing. The results of AAA exhibited that MGF-Ct24E were coupled to the MHPLA at a concentration of 0.88μmol/g and the coupling efficiency was found to be 25%.
④Examinations of cytocompatibity was used to probe the influences of MGF-Ct24E_bioactive materials on fracture healing. Firstly, osteoblasts were cultured on different materials films, with glass as control. The cytocompatibility of MHPLA with rats osteoblasts was estimated by means of cell morphology (by discrepancy microscope) and cell proliferation (by MTT assay) and ALP activity. The morphology observations revealed that the osteoblasts cultured on MHPLA spread wider than those on PDLLA and glass, and much more cells were confluent on MHPLA, comparing to the other two groups; The result of MTT showed osteoblasts grown faster on MHPLA than on HPLA and glass at 3rd, 5th, 7th; ALP showed that MHPLA delayed osteoblast differentiation. All results showed that the bioactivity of MGF-Ct24E on MHPLA is high. Moreover, effective time of MGF-Ct24E-bioactive materials on fracture healing is higher than MGF-Ct24E, which suggested potentially wide applications of MHPLA in biomedical areas, especially in tissue engineering.
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