温敏性bFGF/Dex脂质体-水凝胶复合支架的神经化骨再生评价

doi:10.19438/j.cjoms.2024.04.002

摘要/Abstract

摘要： 目的: 利用脂质体同时包载具有促进神经分化作用的碱性成纤维细胞生长因子（bFGF）和成骨诱导小分子（Dex）,与海藻酸钠溶液混合,构建一种具有温敏性和可注射性的bFGF/Dex脂质体-水凝胶复合支架,评价复合支架促进神经分化和新骨再生的作用。方法: 通过薄膜分散法制备bFGF/Dex脂质体,与RGD多肽活化后的海藻酸钠溶液混合,通过自由基聚合反应合成温敏性bFGF/Dex脂质体-水凝胶复合支架。使用Zeta粒径仪检测脂质体的粒径、多分散度和Zeta电位,扫描电镜观察支架的微观结构,利用万能材料试验机测定支架的机械强度,通过RT-PCR和免疫荧光染色检测bFGF促进hBMSCs成神经分化相关基因和蛋白的表达。构建兔颅骨缺损模型,通过micro-CT及免疫组织化学染色,评价复合支架在体内促进神经化骨再生的作用。采用SPSS 26.0软件包对数据进行统计学分析。结果: 成功构建温敏性bFGF/Dex脂质体-水凝胶复合支架,该支架在常温下（25 ℃）呈溶液状态且具有流动性,大于临界温度（32 ℃）时可迅速转变为凝胶状态。支架表面呈疏松多孔的蜂窝状,能够承担一定应力。体外成神经相关基因和蛋白表达提示,bFGF在诱导hBMSCs成神经分化中具有重要作用。动物实验结果表明,复合支架具有高效促进神经化骨再生的作用。结论: 具有温敏性、可注射性和突出生物功能的智能bFGF/Dex脂质体-水凝胶复合支架,为颌骨缺损修复提供了新的材料。

关键词: 颌骨缺损, 骨移植材料, 神经化骨再生, 温敏性复合支架

Abstract: PURPOSE: Liposomes were used to simultaneously encapsulate basic fibroblast growth factor (bFGF) that can promote nerve differentiation and osteogenic small molecule (Dex). Then, a temperature sensitive and injectable bFGF/Dex liposome-hydrogel composite sustained-release scaffold was constructed by mixing it with sodium alginate solution. METHODS: bFGF/Dex liposomes were prepared by film dispersion method, mixed with sodium alginate solution activated by RGD polypeptide, and thermosensitive bFGF/Dex liposome hydrogel composite scaffolds were synthesized by free radical polymerization. The particle size, polydispersity, and Zeta potential of liposomes were detected by Zeta particle size analyzer. Microstructure of the scaffold was observed under scanning electron microscopy, and mechanical strength of the scaffold was measured using a universal material testing machine. Real time fluorescence quantitative PCR (RT-PCR) and immunofluorescence staining were used to detect the expression of bFGF promoting neural differentiation related genes and proteins in hBMSCs. Rabbit skull defect models were constructed and the effect of the composite scaffold on promoting neurogenic bone regeneration in vivo was evaluated through micro-CT and immunohistochemical staining. SPSS 26.0 software package was used for statistical analysis. RESULTS: Thermosensitive bFGF/Dex liposome hydrogel composite scaffold was successfully constructed. It was in a solution state and had fluidity at normal temperature(25℃), and quickly changed to a gel state when higher than the critical temperature(32 ℃). The surface of scaffold was loose and porous, which could bear certain stress. The expression of genes and proteins related to neurogenesis suggested that bFGF had a strong promoting effect on neurogenesis in hBMSCs. The results of animal experiments showed that the composite had a good neurogenic bone regeneration effect. CONCLUSIONS: The intelligent bFGF /Dex liposome hydrogel composite scaffold with temperature sensitivity and outstanding biological function provides a new material for repair of jaw defects.

Key words: Jaw bone defect, Bone graft materials, Neurogenic bone regeneration, Temperature sensitivity composite scaffold

中图分类号:

R782

楚晨, 宗倩, 寻兴祥, 赵谦, 袁荣涛, 许晓. 温敏性bFGF/Dex脂质体-水凝胶复合支架的神经化骨再生评价[J]. 中国口腔颌面外科杂志, 2024, 22(4): 322-328.

CHU Chen, ZONG Qian, XUN Xing-xiang, ZHAO Qian, YUAN Rong-tao, XU Xiao. Evaluation of neurogenic bone regeneration of thermosensitive bFGF/Dex liposome hydrogel composite scaffold[J]. China J Oral Maxillofac Surg, 2024, 22(4): 322-328.

参考文献

[1] Guo K, Zhao HM, Chen GK, et al.PAP polypeptide promotes osteogenesis in jaw bone defect repair by inhibiting inflammatory reactions[J]. Front Bioeng Biotechnol, 2022, 10: 916330.
[2] Zhao J, Zhou YH, Zhao YQ, et al.Oral cavity-derived stem cells and preclinical models of jaw-bone defects for bone tissue engineering[J]. Stem Cell Res Ther, 2023, 14(1): 39-58.
[3] Zhang WB, Saxena S, Fakhrzadeh A, et al.Use of Human dental pulp and endothelial cell seeded tyrosine-derived polycarbonate scaffolds for robust in vivo alveolar jaw bone regeneration[J]. Front Bioeng Biotechnol, 2020, 8: 796.
[4] Al Maruf DSA, Ghosh YA, Xin H, et al.Hydrogel: a potential material for bone tissue engineering repairing the segmental mandibular defect[J]. Polymers (Basel), 2022, 14(19): 4186.
[5] Xie C, Ye J, Liang R, et al.Advanced strategies of biomimetic tissue-engineered grafts for bone regeneration[J]. Adv Healthc Mater, 2021, 10(14): e2100408.
[6] Cao SJ, Zhao Y, Hu YM, et al.New perspectives: in-situ tissue engineering for bone repair scaffold[J]. Compos B Eng, 2020, 202: 108445.
[7] Sun X, Yang S, Tong S, et al.Study on exosomes promoting the osteogenic differentiation of adscs in graphene porous titanium alloy scaffolds[J]. Front Bioeng Biotechnol, 2022, 10: 905511.
[8] Zhang M, Li Y, Feng T, et al.Bone engineering scaffolds with exosomes: a promising strategy for bone defects repair[J]. Front Bioeng Biotechnol, 2022, 10: 920378.
[9] Mai TP, Park JB, Nguyen HD, et al.Current application of dexamethasone-incorporated drug delivery systems for enhancing bone formation[J]. J Pharmaceut Investigat, 2023, 53(5): 643-665.
[10] Wang XD, Li SY, Zhang SJ, et al.The neural system regulates bone homeostasis via mesenchymal stem cells: a translational approach[J]. Theranostics, 2020, 10(11): 4839-4850.
[11] 王旭东, 张成瑶, 张士剑, 等. 同期神经化增强髂骨瓣术后骨髓间充质干细胞的活性[J]. 上海口腔医学, 2020, 29(6): 561-566.
Wang XD, Zhang CY, Zhang SJ, et al.Simultaneous innervation revitalizes bone marrow mesenchymal stem cells from postoperative iliac grafts[J]. Shanghai Journal of Stomatology, 2020, 29(6): 561-566.
[12] Cho Y, Baek J, Lee E, et al.Heparin-mediated electrostatic immobilization of bFGF via functional polymer films for enhanced self-renewal of human neural stem cells[J]. J Mater Chem B, 2021, 9(8):2084-2091.
[13] Son B, Kim M, Won H, et al.Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration[J]. J Nanobiotechnology, 2023, 21(1): 310.
[14] Ergin AD, Uner B.Characterization, optimization, and in vitro evaluation of cholesterol-free liposomes[J]. J Drug Deliv Sci Technol, 2023, 84: 104468.
[15] Moody CT, Brown AE, Massaro NP, et al.Restoring carboxylates on highly modified alginates improves gelation, tissue retention and systemic capture[J]. Acta Biomater, 2022, 138: 208-217.
[16] Ouyang Y, Zhao J, Wang S.Multifunctional hydrogels based on chitosan, hyaluronic acid and other biological macromolecules for the treatment of inflammatory bowel disease: a review[J]. Int J Biol Macromol, 2023, 227: 505-523.
[17] Pentlavalli S, Chambers P, Sathy BN, et al.Simple radical polymerization of poly(alginate-graft-N-isopropylacrylamide) injectable thermoresponsive hydrogel with the potential for localized and sustained delivery of stem cells and bioactive molecules[J]. Macromol Biosci, 2017, 17(11): 1700118.
[18] Xun XX, Qiu JZ, Zhang J, et al.Triple-functional injectable liposome-hydrogel composite enhances bacteriostasis and osteo/angio-genesis for advanced maxillary sinus floor augmentation[J]. Colloids Surf B Biointerfaces, 2022, 217: 112706.
[19] Lencina MMS, Ciolino AE, Andreucetti NA, et al.Thermoresponsive hydrogels based on alginate-g-poly (N-isopropylacrylamide) copolymers obtained by low doses of gamma radiation[J]. Eur Polym J, 2015, 68: 641-649.
[20] Ivakhnitskaia E, Chin MR, Siegel D, et al.Vinaxanthone inhibits Semaphorin3A induced axonal growth cone collapse in embryonic neurons but fails to block its growth promoting effects on adult neurons[J]. Sci Rep, 2021, 11(1): 13019.
[21] Yang F, Liu Y, Tu J, et al.Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF[J]. Nat Commun, 2014, 5: 5627.
[22] Xing SG, Zhou YL, Yang QQ, et al.Effects of nanoparticle-mediated growth factor gene transfer to the injured microenvironment on the tendon-to-bone healing strength[J]. Biomater Sci, 2020, 8(23): 6611-6624.
[23] Bochicchio S, Dalmoro A, Lamberti G, et al.Advances in nanoliposomes production for ferrous sulfate delivery[J]. Pharmaceutics, 2020, 12(5): 445-470.
[24] Patrick PS, Bear JC, Fitzke HE, et al.Radio-metal cross-linking of alginate hydrogels for non-invasive in vivo imaging[J]. Biomaterials, 2020, 243: 119930.