丝蛋白复合支架对骨质疏松状态下骨缺损修复的比较性研究

发布时间：2018-05-14 11:45

本文选题：介孔生物玻璃(MBG)/丝蛋白支架 + 非介孔生物玻璃(BG)/丝蛋白支架　；参考：《武汉大学》2015年博士论文

【摘要】：骨质疏松症是一种因骨量低下,骨微结构破坏,导致骨脆性增加,易发骨折为特征的全身性骨病。[1]。该疾病在女性主要由绝经后雌激素下降引起,主要表现为骨形成的减少[2]。全球范围内约有2亿人正在经受骨质疏松的痛苦[3],同时,约50%的65岁以上绝经后高加索或亚洲女性经历骨质疏松引起的骨折[4]。近几年,多项研究显示,由于破骨细胞数目的增加及雌激素的减少,绝经后骨质疏松女性的骨折愈合时间延长。目前,药物治疗骨质疏松主要分为全身给药和局部药物缓释,应用的药物包括抗骨吸收类如雌激素替代疗法(HRT)、选择性雌激素受体调节剂(SEM)、双膦酸盐(Bisphosphates)和降钙素(Calcitonin),促骨形成类如甲状旁腺激素类(PTH1-3和PTH1-84)及氟化物(Fluoride),和兼有抗吸收及促形成作用的药物如雷尼酸锶(Strontium ranelate) [5]。尽管这些药物在临床上广泛应用于治疗骨质疏松及其伴发的骨折,对于骨质疏松状况下的局部骨缺损再生修复的研究更值得关注,特别是生物支架材料的植入对局部骨缺损愈合的疗效。丝素的主要由p-片层结构的纤维多肽组成,其主要成分包括甘氨酸、丙氨酸和丝胶[6]。天然丝素来源于蚕丝,通过基因工程技术或对丝素经过一系列的化学改性,形成可用于生物医学研究的丝蛋白[7]。丝蛋白因其诸多优点,例如质轻、可降解、高张力、高韧性、价格低廉和易于加工处理等,可制备成复合丝蛋白支架应用于韧带纤维、骨和软骨组织再生方面的研究[8,9]。体内和体外研究证明了丝素的各种生物和化学性能,包括低生物毒性、非炎性反应、非免疫原性、生物可降解性和生物吸收性等。相应地,研究者们将丝蛋白支架作为药物控释载体或种子细胞生长模板,广泛地应用于骨组织工程领域,然而,与陶瓷类支架相比,纯丝蛋白支架的骨引导性较差。相对而言,陶瓷类(例如磷酸钙和生物玻璃)支架作为生物骨替代材料广泛应用于口腔科和矫形骨科,这得力于它们与天然矿化骨有着极相似的无机成分、结构和机械性能[10]。磷酸钙(CaP)具有较高的蛋白亲和性,无机离子降解后形成再矿化层沉积于骨组织和支架材料之间,以增强骨与胞外基质界面的结合强度[11]。另外,生物玻璃(BGs)被称作原位组织再生修复的第三代生物材料,能够同时直接结合骨组织和软组织[12]。BGs能释放钠离子和钙离子,形成的羟基磷酸钙沉积层利用强化学键紧密地结合玻璃层和宿主骨组织,进而刺激新骨生成[13]。再者,生物玻璃类的新兴衍生物——介孔生物玻璃(MBGs),因其高度的表面改性和多孔性,相比传统BGs呈现出更优越的生物活性。体外细胞学实验发现,MBGs界面释放的硅、钙、磷和钠离子迅速提高胞外和胞内反应,极大提高其在生物医学科学领域的应用价值[14]。综上,我们设计和实施了如下三个实验来探讨以上问题：(1)首先实施大鼠卵巢摘除术建立骨质疏松动物模型,选用同月龄的正常大鼠为对照,研究和比较MBGs/丝蛋白支架和BGs/丝蛋白支架对骨髓问充质细胞的生物学影响,包括黏附、增殖和成骨向分化等；(2)进一步针对骨质疏松状况下的大鼠建立股骨缺损模型,比较MBGs/丝蛋白支架和BGs/丝蛋白支架对缺损局部区域的再生修复作用,包括骨形成和矿化、骨基质的成熟、再生骨形成过程中的软骨成骨和破骨活性等方面；(3)同时,运用相同的体内动物模型,研究CaP/丝蛋白支架对缺损局部区域的再生修复作用,包括骨形成和矿化、骨基质的成熟、再生骨形成过程中的软骨成骨和破骨活性等方面。第一部分比较介孔生物玻璃(MBG)与非介孔生物玻璃(BG)/丝蛋白支架对骨质疏松来源骨髓基质细胞的影响目的：研究MBG与BG/丝蛋白复合支架作为细胞载体,对于正常大鼠及骨质疏松诱导模型来源BMSCs增殖和成骨分化能力的影响材料和方法：选取3月龄Wistar大鼠行双侧卵巢摘除术,经2个月诱导期,行μCT重建分析、HE染色和Alizarin Red染色,鉴定骨质疏松模型的成功建立。选取同月龄正常大鼠为对照,分离、培养骨质疏松来源BMSCs并接种于三维丝蛋白支架,行SEM观察接种后细胞形态,用CCK-8试剂盒测定细胞增殖,PNPP法检测成骨诱导分化后细胞的ALP活性。结果：μCT结果显示,与假手术组相比,骨质疏松组股骨头局部松质骨BV/TV,Tb.N和Tb.Th均显著降低,Tb.Sp增高,股骨干皮质骨厚度降低。分离的骨质疏松大鼠原代MSCs (O-MSCs)在培养12天后细胞密度达到80%以上,而正常大鼠来源MSCs (S-MSCs)仅需9天达到同样生长密度。成骨诱导14天后,Alizarin Red染色显示O-MSCs组的结节数明显少于S-MSCs组,说明骨质疏松来源的MSCs的成骨能力下降。HE染色观察骨组织形态,骨质疏松大鼠松质骨的骨量下降、骨质结构破坏,骨内膜吸收引起皮质骨厚度减少,进而显现骨髓腔增大。结论：成功建立本课题所需的大鼠骨质疏松模型。MBG/丝蛋白支架有利于骨质疏松来源O-MSCs和正常大鼠S-MSCs的黏附、增殖和成骨诱导分化,可考虑用于体内骨缺损模型。第二部分比较介孔生物玻璃(MBG)与非介孔生物玻璃(BG)／丝蛋白支架对骨质疏松状态下骨缺损再生修复的影响目的：探讨与比较MBG与BG/丝蛋白复合支架在骨质疏松状态下股骨缺损的改建过程及再生修复效果。材料和方法：对Wistar大鼠行双侧卵巢摘除术,骨质疏松模型建立后,制备股骨远端2.5mm直径的穿通缺损,植入纯丝支架、BG/丝蛋白支架和MBG/丝蛋白支架,并设缺损无充填组为对照。术后14和28天,行μCT重建分析、HE染色、Safranin O染色、TRAP染色(N.Oc)和免疫组化(COL I, OPN, BSP and OCN)检测分析。结果：结合μCT结果、组织学观察和细胞学行为确立骨质疏松动物模型建立成功。术后第二周和第四周,缺损无充填组无法自行愈合,结合组织形态学和μCT定量分析,其余实验组再生骨量为MBG/丝蛋白组BG/丝蛋白组纯丝支架组。免疫组化检测成骨指标COL I, OPN, BSP和OCN,结果显示MBG/丝蛋白组能显著诱导胶原和非胶原基质的合成。结论：本研究结果提示,与纯丝蛋白支架和BG/丝蛋白支架相比,MBG/丝蛋白支架能加快骨质疏松状况下骨缺损的愈合进程,尽管该病理过程本身呈现显著的骨质破坏和骨量减少。同时,MBG/丝蛋白支架能更快地诱导成骨矿化并减少破骨活力。综合以上结论,MBG/丝蛋白支架可用于绝经后骨质疏松状况下局部骨质缺损得修复,是具有潜在治疗作用和临床应用价值的骨替代物。第三部分多孔磷酸钙(CaP)/丝蛋白支架对骨质疏松状态下骨缺损再生修复的影响目的：探讨多孔CaP/丝复合支架在骨质疏松状态下股骨缺损的改建过程及再生修复效果。材料和方法：对Wistar大鼠行双侧卵巢摘除术,骨质疏松模型建立后,制备股骨远端2.5mm直径的穿通缺损,植入多孔CaP/丝复合支架,并设空白组、缺损无充填组和纯丝支架组为对照。术后14和28天,行μCT重建分析、HE染色、Safranin O染色、TRAP染色(N.Oc)和免疫组化(Col I和OPN)检测分析。结果：结合μCT结果、组织学观察和体重变化确立骨质疏松动物模型建立成功。术后14和28天,缺损无充填组无法自行愈合。多孔CaP/丝支架组的BV/TV、骨再生指数、Col I和OPN的表达明显高于纯丝支架组,且支架残余量、N.Oc显著降低,愈合过程中可见纤维骨结构和软骨基质的形成。结论：基于组织工程途径的多孔CaP/丝复合支架对骨质疏松状态下的缺损修复治疗具有优越性和广阔的应用前景,为病理状态下的骨再生修复提出新的治疗思路。
[Abstract]:Osteoporosis is a kind of.[1]., which is characterized by low bone mass, bone microstructural damage, increased bone fragility and fracture - prone systemic osteopathy. The disease is caused mainly by estrogen decline after menopause, mainly manifested by the reduction of bone formation in [2]., about 200 million people are suffering from osteoporosis and [3], at the same time, about 50% A number of studies have shown that the fracture healing time of women with postmenopausal osteoporosis is prolonged due to the increased number of osteoclasts and decrease of estrogen, and the drug treatment of osteoporosis is mainly divided into systemic and local drug release in recent years. A number of studies have shown that [4]. and Asian women experienced osteoporotic fractures in the post menopause. The drugs used include anti bone resorption, such as estrogen replacement therapy (HRT), selective estrogen receptor modulator (SEM), bisphosphonates (Bisphosphates) and calcitonin (Calcitonin), bone forming groups such as parathyroid hormones (PTH1-3 and PTH1-84) and fluoride (Fluoride), and drugs such as strontium ranelate (strontium raniate), which have anti absorption and promoting effect. Strontium ranelate) [5]., although these drugs are widely used in the clinical treatment of osteoporotic and associated fractures, the study of regenerative repair of local bone defects in the condition of osteoporosis is worth more attention, especially the effect of the implantation of scaffold materials on the healing of local bone defects. Fibroin is mainly composed of p- lamellar structure. The main ingredients include glycine, alanine and sericin [6]. natural silk fibroin derived from silk. Through genetic engineering or a series of chemical modifications of silk fibroin, the silk protein [7]. silk protein, which can be used for biomedical research, has many advantages, such as light quality, degradation, high tension, high tenacity, low price and low price. [8,9]. and in vitro studies have demonstrated various biological and chemical properties of silk fibroin, including low biotoxicity, non inflammatory reaction, non immunogenicity, biodegradability and bioabsorptivity. The silk protein scaffold is widely used in the field of bone tissue engineering as a drug controlled release carrier or seed cell growth template. However, compared with the ceramic scaffold, the bone guide of the pure silk protein scaffold is poor. Relatively speaking, the ceramic (such as calcium phosphate and bioglass) scaffold is widely used in the Department of Stomatology as a biological substitute material in the Department of Stomatology. And orthopedic department of orthopedics, which is capable of having a very similar inorganic composition with the natural mineralized bone, structure and mechanical properties [10]. calcium phosphate (CaP) has high protein affinity. After the degradation of inorganic ions, the remineralized layer is deposited between the bone tissue and the scaffold materials to enhance the binding strength of the bone and extracellular matrix interface ([11].). Glass (BGs), a third generation biomaterial called in situ tissue regeneration, can simultaneously combine bone tissue and soft tissue directly with [12].BGs to release sodium and calcium ions. The formation of hydroxyl phosphate layer is tightly bonded to the glass layer and host bone tissue, and then stimulates the new bone to produce [13].. The new derivative, mesoporous biological glass (MBGs), has a better biological activity than the traditional BGs because of its high surface modification and porosity. In vitro cytological experiments found that the silicon, calcium, phosphorus and sodium ions released from the MBGs interface rapidly increased the extracellular and intracellular reactions, and greatly improved their application in the field of biomedical science. In [14]. comprehensive, we designed and implemented the following three experiments to discuss the above problems: (1) first, the rat model of osteoporosis was established by ovariectomy, and the normal rats of the same month age were selected as the control. The biological effects of MBGs/ silk protein scaffold and BGs/ silk protein scaffold on bone marrow mesenchymal cells were studied and compared, including adhesion, Proliferation and osteogenic differentiation, and so on; (2) to further establish the model of femur defect in rats with osteoporosis and compare the regenerative repair effect of MBGs/ silk protein scaffold and BGs/ silk protein scaffold on the local area of the defect, including bone formation and mineralization, bone matrix maturity, and cartilage osteogenesis and osteoclast activity during the process of reproducing bone. (3) (3) at the same time, using the same animal model in vivo to study the regeneration and repair effect of the silk protein scaffold on the partial region of the defect, including bone formation and mineralization, the maturation of bone matrix, the osteogenesis and osteoclast activity of the cartilage in the process of regenerative bone formation. The first part is compared with the mesoporous Bioglass (MBG) and non mesoporous Bioglass (BG) / The effect of silk protein scaffold on bone marrow stromal cells derived from osteoporosis Objective: To study the effects of MBG and BG/ silk protein composite scaffold as cell carriers on the proliferation and osteogenic differentiation of normal rats and osteoporosis induced models from BMSCs and bone differentiation: 3 month old Wistar rats were selected for bilateral ovariectomy, after 2 months. In the induction period, CT reconstruction analysis, HE staining and Alizarin Red staining were used to identify the successful establishment of the osteoporosis model. The normal rats of the same month age were selected as the control, separated, the osteoporosis source was cultured and inoculated with the three-dimensional silk protein scaffold. The morphology of the cells after the inoculation was observed by SEM, the cell proliferation was measured by the CCK-8 kit and the PNPP method was used to detect the osteogenesis. ALP activity of the cells after differentiation. Results: the results of Mu CT showed that, compared with the sham group, the BV/TV, Tb.N and Tb.Th of the femoral head in the osteoporotic group decreased significantly, the Tb.Sp increased and the femoral shaft cortical bone thickness decreased. The cell density of the primary MSCs (O-MSCs) in the isolated osteoporotic rats was more than 80% after 12 days of culture, and the normal size was normal. The rat source MSCs (S-MSCs) only needed the same growth density in 9 days. 14 days after osteogenesis induction, Alizarin Red staining showed that the number of nodules in group O-MSCs was significantly less than that of group S-MSCs, indicating that the osteogenesis of MSCs in osteoporotic MSCs decreased.HE staining to observe bone tissue morphology, bone mass decreased in osteoporotic rats, bone structure destruction, and endintima Absorption caused cortical bone thickness to decrease and then show bone marrow cavity enlargement. Conclusion: the successful establishment of rat osteoporosis model.MBG/ silk protein scaffold is beneficial to osteoporosis source O-MSCs and normal rat S-MSCs adhesion, proliferation and osteogenesis induced differentiation, which can be used in bone defect model in vivo. The second part compares mesopore Effects of Bioglass (MBG) and non mesoporous Bioglass (BG) / silk protein scaffold on the regeneration of bone defects in osteoporotic state: To explore and compare the remodeling process and regenerative effect of MBG and BG/ silk protein composite scaffold under osteoporosis. Materials and methods: bilateral ovariectomy for Wistar rats After the establishment of the osteoporosis model, the perforating defect of the 2.5mm diameter of the distal femur was prepared, the pure silk stent, the BG/ silk protein scaffold and the MBG/ silk protein scaffold were implanted, and the defect free group was set as the control. 14 and 28 days after the operation, CT reconstruction was performed, HE staining, Safranin O staining, TRAP staining (N.Oc) and immunohistochemistry (COL I, OPN, and OPN) were used. Results: combined with the results of micron CT, the animal model of osteoporosis was established successfully by histological observation and cytological behavior. Second weeks and four weeks after operation, the defect without filling group could not heal itself, combined with histomorphology and quantitative analysis of CT, the remainder of the other experimental group was the pure silk scaffold group of the BG/ silk protein group of MBG/ silk protein group. The results of COL I, OPN, BSP and OCN showed that the MBG/ silk protein group could significantly induce the synthesis of collagen and non collagen matrix. Conclusion: the results of this study suggest that the MBG/ silk protein scaffold can accelerate the healing process of bone defect in osteoporotic condition compared with pure silk protein scaffold and BG/ silk protein scaffold, although this pathological process is in this process. MBG/ silk protein scaffold can induce bone mineralization and reduce osteoclast faster. The MBG/ silk protein scaffold can be used to repair local bone defects in postmenopausal osteoporosis, and it is a potential therapeutic and clinical value of bone substitute. Third The effect of partial porous calcium phosphate (CaP) / silk protein scaffold on the regeneration of bone defects in osteoporotic state objective: To explore the remodeling process and regenerative effect of porous CaP/ filament composite scaffold under osteoporosis. Materials and methods: bilateral ovariectomy for Wistar rats and the establishment of osteoporosis model The perforating defect of 2.5mm diameter of the distal femur was prepared, and the porous CaP/ silk composite scaffold was implanted, and the blank group was set up, the defect free group and the pure silk stent group were compared. 14 and 28 days after the operation, the reconstruction analysis of CT, HE staining, Safranin O staining, TRAP staining (N.Oc) and immunohistochemistry (Col I and OPN) were detected. Results: histological observation combined with the results of micron CT and immunohistochemical staining (Col I and OPN). The animal model of osteoporosis was established successfully. 14 and 28 days after operation, the defect without filling group could not heal itself. The expression of BV/TV, bone regeneration index, Col I and OPN in the porous CaP/ silk stent group was significantly higher than that in the pure silk stents, and the residual volume of the scaffold and the N.Oc decreased significantly. The fibrous bone structure and the cartilage matrix were visible during the healing process. Conclusion: the porous CaP/ filament composite scaffold based on the tissue engineering approach has the advantages and broad application prospects for the repair and treatment of the defects in the osteoporosis state, and puts forward new treatment ideas for the repair of bone regeneration under the pathological condition.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R580

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