發(fā)布時(shí)間：2018-05-14 11:45

  本文選題：介孔生物玻璃(MBG)/絲蛋白支架 + 非介孔生物玻璃(BG)/絲蛋白支架　； 參考：《武漢大學(xué)》2015年博士論文

【摘要】：骨質(zhì)疏松癥是一種因骨量低下,骨微結(jié)構(gòu)破壞,導(dǎo)致骨脆性增加,易發(fā)骨折為特征的全身性骨病。[1]。該疾病在女性主要由絕經(jīng)后雌激素下降引起,主要表現(xiàn)為骨形成的減少[2]。全球范圍內(nèi)約有2億人正在經(jīng)受骨質(zhì)疏松的痛苦[3],同時(shí),約50%的65歲以上絕經(jīng)后高加索或亞洲女性經(jīng)歷骨質(zhì)疏松引起的骨折[4]。近幾年,多項(xiàng)研究顯示,由于破骨細(xì)胞數(shù)目的增加及雌激素的減少,絕經(jīng)后骨質(zhì)疏松女性的骨折愈合時(shí)間延長(zhǎng)。目前,藥物治療骨質(zhì)疏松主要分為全身給藥和局部藥物緩釋,應(yīng)用的藥物包括抗骨吸收類如雌激素替代療法(HRT)、選擇性雌激素受體調(diào)節(jié)劑(SEM)、雙膦酸鹽(Bisphosphates)和降鈣素(Calcitonin),促骨形成類如甲狀旁腺激素類(PTH1-3和PTH1-84)及氟化物(Fluoride),和兼有抗吸收及促形成作用的藥物如雷尼酸鍶(Strontium ranelate) [5]。盡管這些藥物在臨床上廣泛應(yīng)用于治療骨質(zhì)疏松及其伴發(fā)的骨折,對(duì)于骨質(zhì)疏松狀況下的局部骨缺損再生修復(fù)的研究更值得關(guān)注,特別是生物支架材料的植入對(duì)局部骨缺損愈合的療效。絲素的主要由p-片層結(jié)構(gòu)的纖維多肽組成,其主要成分包括甘氨酸、丙氨酸和絲膠[6]。天然絲素來源于蠶絲,通過基因工程技術(shù)或?qū)z素經(jīng)過一系列的化學(xué)改性,形成可用于生物醫(yī)學(xué)研究的絲蛋白[7]。絲蛋白因其諸多優(yōu)點(diǎn),例如質(zhì)輕、可降解、高張力、高韌性、價(jià)格低廉和易于加工處理等,可制備成復(fù)合絲蛋白支架應(yīng)用于韌帶纖維、骨和軟骨組織再生方面的研究[8,9]。體內(nèi)和體外研究證明了絲素的各種生物和化學(xué)性能,包括低生物毒性、非炎性反應(yīng)、非免疫原性、生物可降解性和生物吸收性等。相應(yīng)地,研究者們將絲蛋白支架作為藥物控釋載體或種子細(xì)胞生長(zhǎng)模板,廣泛地應(yīng)用于骨組織工程領(lǐng)域,然而,與陶瓷類支架相比,純絲蛋白支架的骨引導(dǎo)性較差。相對(duì)而言,陶瓷類(例如磷酸鈣和生物玻璃)支架作為生物骨替代材料廣泛應(yīng)用于口腔科和矯形骨科,這得力于它們與天然礦化骨有著極相似的無機(jī)成分、結(jié)構(gòu)和機(jī)械性能[10]。磷酸鈣(CaP)具有較高的蛋白親和性,無機(jī)離子降解后形成再礦化層沉積于骨組織和支架材料之間,以增強(qiáng)骨與胞外基質(zhì)界面的結(jié)合強(qiáng)度[11]。另外,生物玻璃(BGs)被稱作原位組織再生修復(fù)的第三代生物材料,能夠同時(shí)直接結(jié)合骨組織和軟組織[12]。BGs能釋放鈉離子和鈣離子,形成的羥基磷酸鈣沉積層利用強(qiáng)化學(xué)鍵緊密地結(jié)合玻璃層和宿主骨組織,進(jìn)而刺激新骨生成[13]。再者,生物玻璃類的新興衍生物——介孔生物玻璃(MBGs),因其高度的表面改性和多孔性,相比傳統(tǒng)BGs呈現(xiàn)出更優(yōu)越的生物活性。體外細(xì)胞學(xué)實(shí)驗(yàn)發(fā)現(xiàn),MBGs界面釋放的硅、鈣、磷和鈉離子迅速提高胞外和胞內(nèi)反應(yīng),極大提高其在生物醫(yī)學(xué)科學(xué)領(lǐng)域的應(yīng)用價(jià)值[14]。綜上,我們?cè)O(shè)計(jì)和實(shí)施了如下三個(gè)實(shí)驗(yàn)來探討以上問題：(1)首先實(shí)施大鼠卵巢摘除術(shù)建立骨質(zhì)疏松動(dòng)物模型,選用同月齡的正常大鼠為對(duì)照,研究和比較MBGs/絲蛋白支架和BGs/絲蛋白支架對(duì)骨髓問充質(zhì)細(xì)胞的生物學(xué)影響,包括黏附、增殖和成骨向分化等；(2)進(jìn)一步針對(duì)骨質(zhì)疏松狀況下的大鼠建立股骨缺損模型,比較MBGs/絲蛋白支架和BGs/絲蛋白支架對(duì)缺損局部區(qū)域的再生修復(fù)作用,包括骨形成和礦化、骨基質(zhì)的成熟、再生骨形成過程中的軟骨成骨和破骨活性等方面；(3)同時(shí),運(yùn)用相同的體內(nèi)動(dòng)物模型,研究CaP/絲蛋白支架對(duì)缺損局部區(qū)域的再生修復(fù)作用,包括骨形成和礦化、骨基質(zhì)的成熟、再生骨形成過程中的軟骨成骨和破骨活性等方面。第一部分 比較介孔生物玻璃(MBG)與非介孔生物玻璃(BG)/絲蛋白支架對(duì)骨質(zhì)疏松來源骨髓基質(zhì)細(xì)胞的影響目的：研究MBG與BG/絲蛋白復(fù)合支架作為細(xì)胞載體,對(duì)于正常大鼠及骨質(zhì)疏松誘導(dǎo)模型來源BMSCs增殖和成骨分化能力的影響材料和方法：選取3月齡Wistar大鼠行雙側(cè)卵巢摘除術(shù),經(jīng)2個(gè)月誘導(dǎo)期,行μCT重建分析、HE染色和Alizarin Red染色,鑒定骨質(zhì)疏松模型的成功建立。選取同月齡正常大鼠為對(duì)照,分離、培養(yǎng)骨質(zhì)疏松來源BMSCs并接種于三維絲蛋白支架,行SEM觀察接種后細(xì)胞形態(tài),用CCK-8試劑盒測(cè)定細(xì)胞增殖,PNPP法檢測(cè)成骨誘導(dǎo)分化后細(xì)胞的ALP活性。結(jié)果：μCT結(jié)果顯示,與假手術(shù)組相比,骨質(zhì)疏松組股骨頭局部松質(zhì)骨BV/TV,Tb.N和Tb.Th均顯著降低,Tb.Sp增高,股骨干皮質(zhì)骨厚度降低。分離的骨質(zhì)疏松大鼠原代MSCs (O-MSCs)在培養(yǎng)12天后細(xì)胞密度達(dá)到80%以上,而正常大鼠來源MSCs (S-MSCs)僅需9天達(dá)到同樣生長(zhǎng)密度。成骨誘導(dǎo)14天后,Alizarin Red染色顯示O-MSCs組的結(jié)節(jié)數(shù)明顯少于S-MSCs組,說明骨質(zhì)疏松來源的MSCs的成骨能力下降。HE染色觀察骨組織形態(tài),骨質(zhì)疏松大鼠松質(zhì)骨的骨量下降、骨質(zhì)結(jié)構(gòu)破壞,骨內(nèi)膜吸收引起皮質(zhì)骨厚度減少,進(jìn)而顯現(xiàn)骨髓腔增大。結(jié)論：成功建立本課題所需的大鼠骨質(zhì)疏松模型。MBG/絲蛋白支架有利于骨質(zhì)疏松來源O-MSCs和正常大鼠S-MSCs的黏附、增殖和成骨誘導(dǎo)分化,可考慮用于體內(nèi)骨缺損模型。第二部分比較介孔生物玻璃(MBG)與非介孔生物玻璃(BG)／絲蛋白支架對(duì)骨質(zhì)疏松狀態(tài)下骨缺損再生修復(fù)的影響目的：探討與比較MBG與BG/絲蛋白復(fù)合支架在骨質(zhì)疏松狀態(tài)下股骨缺損的改建過程及再生修復(fù)效果。材料和方法：對(duì)Wistar大鼠行雙側(cè)卵巢摘除術(shù),骨質(zhì)疏松模型建立后,制備股骨遠(yuǎn)端2.5mm直徑的穿通缺損,植入純絲支架、BG/絲蛋白支架和MBG/絲蛋白支架,并設(shè)缺損無充填組為對(duì)照。術(shù)后14和28天,行μCT重建分析、HE染色、Safranin O染色、TRAP染色(N.Oc)和免疫組化(COL I, OPN, BSP and OCN)檢測(cè)分析。結(jié)果：結(jié)合μCT結(jié)果、組織學(xué)觀察和細(xì)胞學(xué)行為確立骨質(zhì)疏松動(dòng)物模型建立成功。術(shù)后第二周和第四周,缺損無充填組無法自行愈合,結(jié)合組織形態(tài)學(xué)和μCT定量分析,其余實(shí)驗(yàn)組再生骨量為MBG/絲蛋白組BG/絲蛋白組純絲支架組。免疫組化檢測(cè)成骨指標(biāo)COL I, OPN, BSP和OCN,結(jié)果顯示MBG/絲蛋白組能顯著誘導(dǎo)膠原和非膠原基質(zhì)的合成。結(jié)論：本研究結(jié)果提示,與純絲蛋白支架和BG/絲蛋白支架相比,MBG/絲蛋白支架能加快骨質(zhì)疏松狀況下骨缺損的愈合進(jìn)程,盡管該病理過程本身呈現(xiàn)顯著的骨質(zhì)破壞和骨量減少。同時(shí),MBG/絲蛋白支架能更快地誘導(dǎo)成骨礦化并減少破骨活力。綜合以上結(jié)論,MBG/絲蛋白支架可用于絕經(jīng)后骨質(zhì)疏松狀況下局部骨質(zhì)缺損得修復(fù),是具有潛在治療作用和臨床應(yīng)用價(jià)值的骨替代物。第三部分多孔磷酸鈣(CaP)/絲蛋白支架對(duì)骨質(zhì)疏松狀態(tài)下骨缺損再生修復(fù)的影響目的：探討多孔CaP/絲復(fù)合支架在骨質(zhì)疏松狀態(tài)下股骨缺損的改建過程及再生修復(fù)效果。材料和方法：對(duì)Wistar大鼠行雙側(cè)卵巢摘除術(shù),骨質(zhì)疏松模型建立后,制備股骨遠(yuǎn)端2.5mm直徑的穿通缺損,植入多孔CaP/絲復(fù)合支架,并設(shè)空白組、缺損無充填組和純絲支架組為對(duì)照。術(shù)后14和28天,行μCT重建分析、HE染色、Safranin O染色、TRAP染色(N.Oc)和免疫組化(Col I和OPN)檢測(cè)分析。結(jié)果：結(jié)合μCT結(jié)果、組織學(xué)觀察和體重變化確立骨質(zhì)疏松動(dòng)物模型建立成功。術(shù)后14和28天,缺損無充填組無法自行愈合。多孔CaP/絲支架組的BV/TV、骨再生指數(shù)、Col I和OPN的表達(dá)明顯高于純絲支架組,且支架殘余量、N.Oc顯著降低,愈合過程中可見纖維骨結(jié)構(gòu)和軟骨基質(zhì)的形成。結(jié)論：基于組織工程途徑的多孔CaP/絲復(fù)合支架對(duì)骨質(zhì)疏松狀態(tài)下的缺損修復(fù)治療具有優(yōu)越性和廣闊的應(yīng)用前景,為病理狀態(tài)下的骨再生修復(fù)提出新的治療思路。
[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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R580

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