本文選題：無機小牛骨 + 原子力顯微鏡��； 參考：《南京大學》2015年博士論文

【摘要】：背景與目的骨量不足是口腔頜面外科手術中面臨的主要問題之一,其原因包括牙周病、外傷、腫瘤等。目前在臨床上主要通過自體骨、異體骨、異種骨、人工合成材料等材料進行植入,其中自體骨移植長期以來都被認為是植骨材料的“金標準”但自體骨也存在著明顯的缺點,如增加取骨術區(qū)、取骨區(qū)并發(fā)癥等。因此,研發(fā)接近自體骨植骨效果的組織工程骨一直是生物材料領域的熱點。目前國外組織工程骨領域的研究已經(jīng)使大量的材料在臨床使用和商品化,而國內(nèi)在相關科學研究和成果轉(zhuǎn)化之間的銜接上仍然存在一些不足。本研究立足于骨粉材料基礎研究,著眼于組織工程骨的構建和后期轉(zhuǎn)化的可行性操作,希望可以實現(xiàn)組織工程骨材料的自主研發(fā)。之前我們通過大量臨床經(jīng)驗積累,總結了目前使用的各種商品化骨粉,發(fā)現(xiàn)這些骨粉仍然存在一些不足。化學合成的骨替代材料由于與天然骨理化性質(zhì)的差異以及后期代謝成份的影響,其在臨床的使用并不廣泛,目前仍需要進一步完善研究。天然來源的骨替代材料如各種動物松質(zhì)骨來源的骨粉,由于具有和骨組織相近的多孔結構和近似的礦物質(zhì)組成,目前是臨床使用的主要材料,如小牛松質(zhì)骨來源的BioOss骨粉。該類型的煅燒骨主要成份為骨無機基質(zhì),并沒有骨誘導性,所以在臨床上存在體內(nèi)滯留時間過長,新生骨骨質(zhì)不理想等缺點。近年開發(fā)的組織工程骨如PepGen/P-15,其過于簡單的多肽表面修飾方法使所添加多肽P-15的活性和穩(wěn)定性不明確,也一定程度上限制了在臨床的應用。本課題將全面地對組織工程骨材料的研發(fā)和相關生物學評價進行系統(tǒng)性研究,旨在(1)研發(fā)具有可靠生物安全性和成骨性的胎牛松質(zhì)骨無機骨基質(zhì)；(2)對生產(chǎn)的無機骨基質(zhì)進行表面修飾,提高其成骨效率； (3)明確多肽P-15對于細胞粘附的作用。材料與方法選取胎牛松質(zhì)骨為原料,利用化學及煅燒法制備胎牛骨無機骨基質(zhì)(Fetal Anorganic Bovine-derived Matrix, FABM)。使用MTT法、SEM掃描電鏡、染色體畸變實驗等方法,檢測所制得FABM的生物安全性。使用球磨儀粉碎FABM,過篩制備納米級FABM后壓制成片。在制得的FABM片上培養(yǎng)人頜骨間充質(zhì)干細胞(Human jaw mesenchymal stem cells, HJMSC),觀察多肽P-15對細胞粘附形態(tài)的影響。通過常溫共沉積法在FABM片表面通過雙礦化法沉積磷酸鈣結晶,對FABM進行表面改性,提高FABM對成骨細胞的粘附及其成骨性。利用原子力顯微鏡(Atomic Force Microscope, AFM)的單細胞檢測技術,研究P-15對HJMSC彈性模量的影響,和P-15對于FABM顆粒與HJMSC細胞之間粘附力的影響。制備兔顱骨臨界骨缺損,植入所制得的骨粉,制備組織切片,觀察其遠期成骨性和骨材料的體內(nèi)代謝時間。結果1.所制備骨粉的物理化學性質(zhì)制得骨粉外觀為白色或類白色多孔顆粒,顆粒尺寸為0.5-2mm。其中超過粒度上限的顆粒重量不超過3%,小于粒度上限的顆粒重量不超過5%。X射線衍射譜中無明顯的其他磷酸鈣和結晶物質(zhì)峰,羥基磷灰石含量為96.9%。檢測pH值為：7.42,骨粉鈣磷比：1.76±0.27。2.所制備骨粉的生物安全性測試細胞毒試驗中,不同濃度的材料浸提液與細胞共培后,MTT檢測結果均未顯示明顯細胞毒性。熱源反應實驗中,三只動物(兔)體溫升高均小于0.6℃,判斷骨粉無熱源反應。急毒試驗中,試驗組和對照組動物活動無異常,未觀察到毒性反應,試驗組體重變化與對照組動物相比無明顯差異。皮膚刺激試驗中,所選用的三只家兔,注射浸提液后,試驗樣品與溶劑對照平均記分差為0.49,小于1.0,表明無明顯皮內(nèi)反應出現(xiàn)。染色體畸變試驗中顯微鏡下觀察染色體畸變,選取分散良好的中期分裂相(染色體數(shù)為2n±2)進行染色體畸變分析。結果得出骨粉材料未致染色體畸變率升高。3.多肽P-15對HJMSC的影響在20min和12h共培養(yǎng)時間條件下,通過SEM電鏡結果可見,有無P-15存在的FABM片表面附著的HJMSC,其粘附后的形態(tài)有差異。主要表現(xiàn)為細胞伸展狀態(tài)及絲狀板狀偽足的多少等形態(tài)學表現(xiàn)。AFM實驗中,有無P-15吸附的FABM顆粒,其對HJMSC的粘附力基本在30pN左右,無顯著差異,但在拉脫FABM顆粒與細胞時,有P-15參與的組別所消耗的能量為無P-15組的四倍。提示P-15能更有效地促進細胞和FABM之間的粘附。在P-15對于HJMSC的彈性模量研究中,細胞經(jīng)P-15共培養(yǎng)后,AFM試驗發(fā)現(xiàn)P-15改變了細胞膜彈性模量,提示P-15對細胞骨架有一定影響,并和P-15濃度有關(P0.05)4.骨粉植入動物實驗在兔顱骨制備1.8cm × 1.8cm的臨界骨缺損,植入制得的FABM,在不同時間點處死動物,得到組織切片。切片結果提示FABM生物相容性較好,未發(fā)現(xiàn)較明顯異物反應,在體內(nèi)的代謝時間為26周左右。結論(1)所制得FABM具有符合標注的理化性質(zhì)和良好的生物安全性,符合三類醫(yī)療器械的臨床前生物監(jiān)測標準。(2)多肽P-15對于HJMSC具有促粘附作用。P-15對細胞的彈性模量有影響,提示其可能改變細胞的細胞膜結構,從而影響細胞的粘附。多肽P-15可以作為有效的促成骨生長因子用于骨粉表面修飾。(3)共沉積法可以在FABM表面形成穩(wěn)定有序的結晶,通過沉積固定的生長因子可以起到緩釋長效的作用。(4)動物體內(nèi)實驗提示FABM有良好的生物安全性和成骨特性,其體內(nèi)代謝周期符合臨床需要。
[Abstract]:Background and objective bone shortage is one of the main problems in oral and maxillofacial surgery, which includes periodontitis, trauma, tumor and so on. It is now mainly implanted in the clinical materials such as autogenous bone, allograft bone, xenogeneic bone, artificial synthetic material and so on. Autogenous bone transplantation has long been considered as the "gold mark" of bone graft materials. However, there are obvious defects in the autogenous bone, such as increasing the area of osteotomy and the complication of bone taking. Therefore, the research and development of tissue engineering bone, which is close to the effect of autogenous bone graft, has always been a hot spot in the field of biological materials. There are still some shortcomings in the connection between scientific research and the transformation of results. This study is based on the basic research of bone powder materials, focusing on the construction of tissue engineering bone and the feasibility of the later transformation. We hope to realize the independent research and development of tissue engineering bone materials. We have accumulated a lot of clinical experience and summed up the current use. A variety of commercialized bone powder has been found that there are still some deficiencies in these bone powders. The chemical synthetic bone substitute is not widely used in clinical use because of its differences in physical and chemical properties of natural bone and the effect of later metabolic components. The natural source of bone substitute, such as various animal cancellous bones, is still needed. Bone meal is a major clinical material, such as BioOss bone powder derived from calf cancellous bone. The main component of this type of calcined bone is bone inorganic matrix and no bone inducibility, so there is a long time in the body and new bone bone in the body. The tissue engineering bone, such as PepGen/P-15, has been developed in recent years, such as the simple method of polypeptides surface modification, which makes the activity and stability of the added polypeptide P-15 not clear, and to some extent restricts the clinical application. This topic will make a comprehensive study on the research and development of tissue engineering bone material and the related biological evaluation system. The purpose of the study is (1) to develop an inorganic bone matrix with reliable biological safety and osteogenesis in fetal bovine cancellous bone; (2) surface modification of the inorganic bone matrix produced to improve its osteogenesis efficiency; (3) the effect of polypeptide P-15 on cell adhesion. Materials and methods were used to prepare fetal bovine cancellous bone as raw material and prepared by chemical and calcining methods. Fetal bovine bone mineral matrix (Fetal Anorganic Bovine-derived Matrix, FABM). Using MTT method, SEM scanning electron microscopy, chromosome aberration test and other methods to detect the biological safety of FABM. W mesenchymal stem cells, HJMSC), observe the effect of polypeptide P-15 on the cell adhesion morphology. By depositing calcium phosphate crystallization on the surface of FABM slices through the normal temperature co deposition method, the surface modification of FABM to improve the adhesion and osteogenesis of FABM to osteoblasts is improved. The single detail of the primary subforce microscope (Atomic Force Microscope) is used. The effect of P-15 on the modulus of elasticity of HJMSC and the effect of P-15 on the adhesion between FABM particles and HJMSC cells. The critical bone defect of the rabbit skull was prepared and the bone powder was implanted, and the tissue sections were prepared, and the long-term osteogenesis and the metabolism time in the body were observed. Results the physical and chemical properties of the 1. preparation of bone powder were made. The appearance of bone powder is white or white like white porous particles, the particle size of which is 0.5-2mm. above the upper limit of particle size is not more than 3%. The weight of particles less than the upper grain size is not more than other calcium phosphate and crystalline material peak in the 5%.X ray diffraction spectrum, and the content of hydroxyapatite is 96.9%. detection pH value: 7.42, bone meal calcium and phosphorus. In the biological safety test of bone powder prepared by 1.76 + 0.27.2., in the cytotoxicity test of bone powder, the results of MTT detection did not show obvious cytotoxicity. In the heat source reaction experiment, the body temperature of three animals (rabbits) was less than 0.6 degrees C, and the bone powder had no heat source reaction. There was no significant difference in animal activity and no observation of toxic reaction. There was no significant difference in weight change between the experimental group and the control group. In the skin irritation test, the average score of the test samples and the solvent was 0.49, less than 1 after the injection of the extract, which showed no apparent intradermal reaction appeared. In the chromosome aberration test, there was no obvious intradermal reaction in the three rabbits. Chromosome aberration was observed under the microscope, and the chromosomal aberration was analyzed with a well dispersed medium division phase (2n + 2). The results showed that the effect of.3. polypeptide P-15 on HJMSC was not caused by the increase of the chromosome aberration rate of bone powder material. The results of SEM electron microscopy showed that there was a FABM slice without P-15 under the co culture time of 20min and 12h. The morphology of the adherent HJMSC is different. The main expression is the morphology of the cell extension state and the number of filamentous pads. In the.AFM experiment, there is no P-15 adsorbed FABM particles, and the adhesion force to HJMSC is basically around 30pN, and there is no significant difference, but when the FABM particles and the cells are removed, there are P-15 participating groups. The energy consumption was four times that of the non P-15 group. Suggesting that P-15 could promote the adhesion between cells and FABM more effectively. In the study of the modulus of elasticity of HJMSC by P-15, after P-15 co culture, the AFM test found that P-15 changed the elastic modulus of the cell membrane, suggesting that P-15 has a certain effect on the cytoskeleton, and that the concentration of P-15 is related to the P-15 concentration (P0.05) 4. bone powder implantation In the animal experiment, the critical bone defect of 1.8cm x 1.8cm was prepared in the rabbit skull, and the FABM was implanted. The animals were killed at different time points and the tissue sections were sliced. The results showed that the biocompatibility of FABM was better, and the metabolic time in the body was 26 weeks in the body. Conclusion (1) the FABM has the physical and chemical properties. Quality and good biological safety conforms to the pre clinical biological monitoring standards of three kinds of medical instruments. (2) polypeptide P-15 has an effect on the elastic modulus of cells with the adhesion action of.P-15 to HJMSC, suggesting that it may change the cell membrane structure of the cell, thus affecting the cell adhesion. Polypeptide P-15 can be used as an effective factor to promote bone growth factor. The surface modification of bone powder. (3) co deposition can form a stable and orderly crystallization on the surface of FABM. By deposition of fixed growth factors, it can play a sustained and long-term effect. (4) in vivo experiments in animals suggest that FABM has good biological safety and osteogenic characteristics, and its metabolic cycle in vivo meets the clinical needs.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R318.08;R687

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2 宋擇眾;通過檢測堿性磷酸酶和骨鈣素2的表達水平定量分析組織工程骨成骨影響因素[D];山東大學;2015年

3 張敏;脈沖電磁場促進大鼠骨髓間充質(zhì)干細胞活性的實驗研究[D];第四軍醫(yī)大學;2015年

4 周權;同種異體胎兔BMSCs為種子細胞構建組織工程骨的實驗研究[D];南華大學;2015年

5 蔡升勇;基于形函數(shù)控制的組織工程骨架孔隙分布結構實體建模技術研究[D];上海交通大學;2010年

6 張弓;多基因修飾組織工程骨中其基因表達時限的檢測[D];重慶醫(yī)科大學;2009年

7 劉勇;組織工程骨血管神經(jīng)化構建的早期觀察[D];第一軍醫(yī)大學;2007年

8 穆天旺;血管束、感覺神經(jīng)束植入構建組織工程骨血管神經(jīng)化的早期形態(tài)學觀察[D];南方醫(yī)科大學;2009年

9 拓振合;應用組織工程骨膜及其脫蛋白骨輔助支架修復兔橈骨大段骨缺損[D];蘭州大學;2014年

10 吳治林;個體化組織工程骨修復人長骨缺損的回顧性對比研究[D];第三軍醫(yī)大學;2014年

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