本文關鍵詞： 納米磷酸鈣 殼聚糖 可注射 成骨分化 組織工程　出處：《浙江理工大學》2013年碩士論文　論文類型：學位論文

【摘要】：隨著人口老齡化現(xiàn)象的加劇和交通/運動創(chuàng)傷的增加，人們對骨移植材料的需求也日益增多。利用組織工程骨來實現(xiàn)硬組織的修復替代正成為本領域一種最為理想的治療手段。從臨床角度來看，可注射組織工程骨的使用減少了患者的痛苦，避免了手術(shù)感染的風險，不易留疤且縮減了治療的費用，因而受到越來越多地重視。 本文以溫敏性殼聚糖水凝膠為流動相，以納米磷酸鈣顆粒(nCP)為藥物載體，以骨髓間充質(zhì)干細胞(MSCs)為種子細胞，構(gòu)建可注射組織工程骨，利用納米磷酸鈣顆粒優(yōu)異的吸附和載藥性能實現(xiàn)對藥物和生長因子的可控釋放，進而促進可注射組織工程骨對骨缺損的治療和修復。具體研究內(nèi)容和結(jié)果如下： 1.利用化學共沉淀方法制備了納米磷酸鈣顆粒，并對其形貌、結(jié)晶度和孔隙率等性能進行表征。通過原核表達制備了人重組骨形態(tài)發(fā)生蛋白2（rhBMP-2），并利用所制備的顆粒為載體負載了rhBMP-2和地塞米松（Dex），研究了藥物負載量、釋放速度及周期。結(jié)果表明：所制備的nCP顆粒呈均勻球狀，結(jié)晶性較好，平均粒徑為56.2±5.8nm，平均孔徑為11.96nm，孔隙率和比表面積分別達到47.2%和89.337m~2/g，有利于后續(xù)實驗藥物的負載及釋放；rhBMP-2基因可在大腸桿菌BL21中成功表達，純化后蛋白產(chǎn)量為27mg每升培養(yǎng)基，尿素梯度透析復性產(chǎn)率為35.9%；每50mg顆粒上rhBMP-2的負載率為56.1±0.493%，Dex的負載率為12.8±0.372%，負載的Dex和rhBMP-2可以較緩慢的速度持續(xù)釋放半個月左右，證明nCP是較理想的藥物載體，滿足骨組織工程的應用要求。 2.制備了殼聚糖/β-甘油磷酸鈉/納米磷酸鈣（CS/GP/nCP）水凝膠，對水凝膠的凝膠時間、形貌、低溫下的儲存性能進行了表征。結(jié)果表明，隨著GP濃度的提高，CS/GP/nCP體系的pH隨之增高，凝膠時間逐漸變短，4℃下保持液態(tài)的時間更長。CS/GP/nCP水凝膠呈連續(xù)多孔的海綿狀結(jié)構(gòu)，孔徑在100μm左右，適宜細胞的增殖，以及營養(yǎng)物質(zhì)和生長因子的傳遞和運輸。 3.成功地提取大鼠的骨髓間充質(zhì)干細胞，，并與CS/GP/nCP水凝膠和載藥nCP顆粒復合構(gòu)建可注射組織工程骨，對其生物相容性進行表征。結(jié)果表明制備的可注射組織工程骨材料具有良好的生物相容性，MSCs能夠在材料中正常增殖。當CS/GP/nCP體系中的Dex濃度為10~(-9)M左右時，對MSCs的增殖有一定的促進作用；當Dex濃度過高時，會抑制MSCs的增殖。當rhBMP-2的濃度達到30μg/mL時，對體系中MSCs的成骨分化有顯著的促進作用。Dex和rhBMP-2共同作用時，相對于不含藥物或僅含單種藥物的體系，CS/GP/nCP凝膠中的MSCs的增殖速度更快，且促成骨分化的效果更顯著。
[Abstract]:As population ageing intensifies and traffic / sports trauma increases. The demand for bone graft materials is also increasing. The use of tissue engineering bone to achieve the replacement of hard tissue is becoming the most ideal treatment in this field. From the clinical point of view. The use of injectable tissue engineered bone reduces the pain of patients, avoids the risk of surgical infection, does not scar easily and reduces the cost of treatment, so more and more attention is paid to it. In this paper, the injectable tissue engineered bone was constructed using thermo-sensitive chitosan hydrogel as mobile phase, nano-calcium phosphate (CPN) as drug carrier and bone marrow mesenchymal stem cells (MSCs) as seed cells. The controllable release of drugs and growth factors can be realized by the excellent adsorption and drug loading of nano-calcium phosphate particles. In order to promote the treatment and repair of bone defects with injectable tissue engineered bone, the specific research contents and results are as follows: 1. Nanometer calcium phosphate particles were prepared by chemical coprecipitation method and their morphology was analyzed. The crystallinity and porosity were characterized. The recombinant human bone morphogenetic protein 2rhBMP-2 was prepared by prokaryotic expression. The rhBMP-2 and dexamethasone Dexo were loaded with the prepared particles as the carrier. The drug loading, release rate and cycle were studied. The results showed that the prepared nCP particles were spherical in shape. The average particle size is 56.2 鹵5.8 nm, the average pore size is 11.96 nm, and the porosity and specific surface area are 47.2% and 89.337 mg / g, respectively. It is beneficial to the loading and release of the following experimental drugs; RhBMP-2 gene was successfully expressed in Escherichia coli BL21. The protein yield was 27mg / L and urea gradient dialysis renaturation yield was 35.9mg / l. The loading rate of rhBMP-2 on 50mg granules was 56.1 鹵0.493% and 12.8 鹵0.372% respectively. The sustained release of Dex and rhBMP-2 at a slower rate for about half a month proved that nCP was an ideal drug carrier and could meet the requirements of bone tissue engineering. 2. Chitosan / 尾 -glycerophosphate / nano-calcium phosphate / CS / GP / nCP-based hydrogels were prepared. The gel time, morphology and storage performance at low temperature were characterized. With the increase of GP concentration, the pH of CS / GP / nCP system increases and the gel time becomes shorter. At 4 鈩

本文編號：1493093