本文選題：電沉積技術(shù) + 仿生骨支架��； 參考：《東華大學(xué)》2015年博士論文

【摘要】：因創(chuàng)傷、腫瘤和疾病等原因造成的骨缺損是臨床醫(yī)學(xué)中常見的骨科病癥。目前臨床上治療骨缺損最常用的方法有自體骨移植和異體骨移植等,但這些方法均存在各自的局限性(如供體有限、易引發(fā)免疫排斥反應(yīng)等),從而限制了它們?cè)诠切迯?fù)中的應(yīng)用,骨組織工程為骨缺損的修復(fù)帶來了曙光�；谔烊还侵饕怯赡z原纖維、羥基磷灰石(HA)晶體組成的生物礦化體系和具有獨(dú)特的有機(jī)/無機(jī)分級(jí)結(jié)構(gòu),理想的仿生骨支架的開發(fā)就需要模擬天然骨的主要成分和特殊結(jié)構(gòu)。近年來,電沉積技術(shù)在生物醫(yī)學(xué)中的應(yīng)用日趨廣泛,特別是在構(gòu)建骨支架方面,從金屬涂層的制備逐漸向聚合物基底的沉積發(fā)展。電沉積能夠通過一步法在許多基底材料上沉積磷灰石礦物或其他粒子,與其他技術(shù)相比擁有很多優(yōu)點(diǎn),如設(shè)備簡(jiǎn)單、易操作,反應(yīng)條件溫和,生產(chǎn)成本低;沉積速度快;沉積物比較均勻等。如果能在聚合物基底上通過電沉積技術(shù)沉積骨鹽成分,制備可降解的有機(jī)/無機(jī)復(fù)合材料,在結(jié)構(gòu)、成分和功能上仿生天然骨,這將對(duì)仿生骨支架的研究具有重要的理論價(jià)值和應(yīng)用前景�；诖�,本課題以聚合物為沉積模板,通過電沉積技術(shù)制備有機(jī)/無機(jī)復(fù)合仿生骨支架,并對(duì)復(fù)合支架的理化性能和體內(nèi)外誘導(dǎo)成骨能力進(jìn)行研究。具體研究?jī)?nèi)容主要有:1.首先以聚合物為沉積模板,探究影響電沉積骨鹽成分的因素。選取了三種聚合物電紡膜為沉積模板:左旋聚乳酸(plla)、膠原蛋白和殼聚糖;四種金屬電極為工作電極:銅(cu)、鎳(ni)、鈦(ti)和不銹鋼(ss),研究了這三種聚合物材料和四種金屬電極對(duì)沉積磷酸鈣鹽形貌和結(jié)構(gòu)的影響。結(jié)果表明,基底材料能夠影響磷酸鈣鹽的形貌,而其組成差別不大,主要以ha為主。不同的金屬電極對(duì)磷酸鈣鹽的形貌和組成均有影響。隨后進(jìn)一步選擇plla膜為沉積模板和銅電極為工作電極,研究沉積時(shí)間、沉積電壓和沉積溫度對(duì)沉積磷酸鈣鹽形貌和結(jié)構(gòu)的影響。結(jié)果表明,不同的沉積參數(shù)使磷酸鈣鹽晶體呈現(xiàn)不同的形貌和組成,只有在適宜的沉積電壓、溫度和時(shí)間下,才能夠生成均一的磷酸鈣鹽涂層。通過對(duì)沉積機(jī)理的分析研究,發(fā)現(xiàn)涉及的因素主要對(duì)磷酸鈣鹽晶體的成核與生長(zhǎng)產(chǎn)生影響。2.進(jìn)一步研究了電沉積技術(shù)對(duì)三維支架上磷酸鈣鹽沉積的影響,并制備有機(jī)/無機(jī)復(fù)合三維支架。采用熱致相分離(tips)技術(shù)制備具有三維、納米纖維和多孔結(jié)構(gòu)的plla/聚己內(nèi)酯(pcl)支架,通過電沉積技術(shù)對(duì)三維支架進(jìn)行磷酸鈣鹽的沉積,獲得plla/pcl礦化支架。通過研究沉積電壓和時(shí)間對(duì)支架礦化的影響,得到了純度較高的ha礦物,且制得的礦化支架能保持多孔結(jié)構(gòu)。為研究礦化支架的體外成骨潛能,將骨髓間充質(zhì)干細(xì)胞(bmscs)種植到支架上并進(jìn)行培養(yǎng),發(fā)現(xiàn)礦化支架能夠促進(jìn)細(xì)胞粘附、提高堿性磷酸酶(alp)活性和促進(jìn)礦物基質(zhì)形成。將礦化前后的復(fù)合支架植入sd大鼠的顱骨缺損部位12w后,發(fā)現(xiàn)礦化支架能夠加速顱骨缺損的修復(fù)。綜上所述,礦化支架從成分上模擬天然骨的無機(jī)成分,并結(jié)合可降解的聚合物材料,制備的復(fù)合支架具有三維多孔納米纖維結(jié)構(gòu),能在很大程度上仿生細(xì)胞外基質(zhì)的結(jié)構(gòu)。因此,制備的三維礦化支架很有潛力用于骨組織工程中。3.為探討添加劑對(duì)磷酸鈣鹽晶體沉積的影響以及對(duì)干細(xì)胞成骨分化的影響,將鍶鹽加入到電解液中,探究其對(duì)磷酸鹽礦化過程的影響。制備plla電紡膜為沉積模板,將不同量的硝酸鍶加到電解液中,制備不同濃度的含鍶電解液,通過電沉積技術(shù)對(duì)plla膜進(jìn)行礦化,采用不同技術(shù)研究鍶的摻入對(duì)沉積的磷酸鈣鹽晶體的影響。將復(fù)合納米纖維膜與bmscs共培養(yǎng),研究鍶的摻入對(duì)細(xì)胞增殖和向成骨分化的影響。通過掃描電子顯微鏡(sem)觀察,鍶的摻入對(duì)沉積物的形貌產(chǎn)生了影響。隨著鍶濃度的增加,礦物晶體由花簇狀變?yōu)槠瑺?最后變?yōu)榍驙?且鍶的摻入增強(qiáng)了晶體的分散性、使晶型由多晶變?yōu)轭愃茊尉У男螒B(tài)。由離子釋放行為曲線可以看出,鍶的摻入加速了礦物晶體中鈣離子和磷酸根離子的釋放。細(xì)胞實(shí)驗(yàn)結(jié)果表明,一定濃度鍶的摻入能夠促進(jìn)bmscs的增殖,增強(qiáng)alp的活性,提高鈣結(jié)節(jié)的沉積以及骨鈣素(ocn)的表達(dá);在一定范圍內(nèi),摻入鍶的含量越高,對(duì)細(xì)胞的增殖和分化的促進(jìn)作用越明顯。4.進(jìn)一步研究了電沉積技術(shù)在材料表面引入成骨性微載體的可行性,以構(gòu)建功能性有機(jī)/無機(jī)骨修復(fù)材料。首先合成了氨基化的介孔硅(msns-nh2),用來負(fù)載具有成骨潛能的小分子藥物地塞米松(dex),制得dex@msns-nh2微載體;采用電沉積技術(shù)將其沉積到PLLA/PCL三維支架上,得到DEX@MSNs-NH2/PLLA/PCL復(fù)合支架。實(shí)驗(yàn)結(jié)果表明在沉積電壓為3 V時(shí),DEX@MSNs-NH2能夠均勻地沉積在PLLA/PCL支架上,獲得的復(fù)合支架仍能保持三維、多孔和納米纖維的結(jié)構(gòu),這將有利于細(xì)胞的粘附和向內(nèi)長(zhǎng)入。通過在復(fù)合支架上種植BMSCs,研究其對(duì)細(xì)胞的骨誘導(dǎo)性;并將復(fù)合支架植入SD大鼠顱骨缺損處,評(píng)估其體內(nèi)骨修復(fù)能力。細(xì)胞實(shí)驗(yàn)表明復(fù)合支架能夠支撐BMSCs的粘附、生長(zhǎng)和遷移。由ALP活性、茜素紅(ARS)染色和骨鈣素(OCN)染色結(jié)果表明,與PLLA/PCL和MSNs-NH2/PLLA/PCL支架相比,DEX@MSNs-NH2/PLLA/PCL復(fù)合支架對(duì)BMSCs的成骨分化具有更好的誘導(dǎo)作用。體內(nèi)顱骨缺損修復(fù)實(shí)驗(yàn)進(jìn)一步表明,制得的DEX@MSNs-NH2/PLLA/PCL復(fù)合支架能夠支撐顱骨缺損處細(xì)胞的粘附和遷移,進(jìn)而加速骨缺損的修復(fù)。因此,制備的功能性DEX@MSNs-NH2/PLLA/PCL復(fù)合支架有望開發(fā)成為有效的骨修復(fù)材料。
[Abstract]:Bone defects caused by trauma, tumor and disease are common Department of orthopedics diseases in clinical medicine. The most commonly used methods in clinical treatment of bone defects are autogenous bone graft and allograft bone graft, but these methods have their own limitations (such as limited donor, easy to induce immune rejection, etc.), thus limiting their bone in bone. Bone tissue engineering has brought dawn to the repair of bone defects. Natural bone is mainly composed of collagen fiber, hydroxyapatite (HA) crystal and unique organic / inorganic hierarchical structure. The development of ideal biomimetic bone scaffold needs to simulate the main components and special structures of natural bone. In recent years, electrodeposition has been widely used in biomedicine, especially in the construction of bone scaffolding, the preparation of metal coating from the metal coating to the deposition of polymer substrate. Electrodeposition can deposit apatite or other particles on many base materials by one step method, which has many advantages compared with other technologies, such as set up. It is simple, easy to operate, mild reaction conditions, low production cost, rapid deposition rate, and more uniform sediment. If the bone and salt composition can be deposited on the polymer substrate by electrodeposition technology, biodegradable organic / inorganic composites are prepared, and the structure, composition and function are biomimetic of natural bone, which will be important for the study of biomimetic bone scaffold. Based on this, this paper uses polymer as a deposition template to prepare organic / inorganic composite biomimetic scaffolds by electrodeposition, and studies the physical and chemical properties of the composite scaffold and the ability to induce osteogenesis in vivo and in vivo. The main contents are as follows: 1. first, the polymer was used as a deposition template to explore the influence of electrodeposition. Three kinds of polymer electrospun membranes were selected as deposition templates: L-polylactic acid (PLLA), collagen and chitosan, and four metal electrodes as working electrodes: copper (Cu), nickel (Ni), titanium (TI) and stainless steel (SS). The effects of the three polymeric materials and four metal electrodes on the morphology and structure of the deposited calcium phosphate were investigated. The results show that the substrate can affect the morphology of calcium phosphate, but the difference is small, mainly ha. Different metal electrodes have influence on the morphology and composition of calcium phosphate. Then, the PLLA film is selected as the deposition template and copper electrode as the working electrode. The deposition time, the deposition voltage and the deposition temperature on the sedimentary phosphoric acid are studied. The effect of calcium salt morphology and structure shows that the different deposition parameters make the calcium phosphate crystals with different morphology and composition. Only the homogeneous calcium phosphate coating can be generated only at the suitable deposition voltage, temperature and time. The main factors involved in the calcium salt crystal are found by the analysis of the deposition mechanism. The effect of nucleation and growth on.2. further studies the effect of electrodeposition on the deposition of calcium phosphate on the three-dimensional scaffold and the preparation of an organic / inorganic composite three-dimensional scaffold. The plla/ polyhexyl (PCL) stents with three-dimensional, nanofibers and porous structures are prepared by thermal phase separation (TIPS). The plla/pcl mineralization scaffold was obtained by the deposition of calcium phosphate. By studying the effect of the deposition voltage and time on the mineralization of the scaffold, the high purity ha mineral was obtained, and the prepared mineralized scaffold could maintain the porous structure. In order to study the osteogenic potential of the scaffold in vitro, the bone marrow mesenchymal stem cells (BMSCs) were planted on the scaffold and cultured. It was found that the mineralized scaffold can promote cell adhesion, increase the activity of alkaline phosphatase (ALP) and promote the formation of mineral matrix. The mineralized scaffold can accelerate the repair of the skull defect after the mineralization of the mineralized scaffold into the skull defect site of SD rats, and the mineralized scaffold simulates the inorganic components of the natural bone from the composition. Combined with degradable polymer materials, the composite scaffolds prepared with a three-dimensional porous nanofiber structure can mimic the structure of the extracellular matrix to a large extent. Therefore, the prepared three-dimensional mineralized scaffold has the potential to use.3. in bone tissue engineering to explore the effect of additive on the deposition of calcium phosphate crystals and to the osteogenesis of stem cells. The influence of the differentiation is added to the electrolyte to explore the influence of the strontium salt on the process of phosphate mineralization. The PLLA electrospun membrane is prepared as a deposition template, and different amounts of strontium nitrate are added into the electrolyte, and different concentrations of strontium electrolyte are prepared. The PLLA membrane is mineralized by electrodeposition, and the incorporation of strontium to the deposition of strontium is studied by different techniques. Effects of calcium phosphate crystals. Co culture of composite nanofibers with BMSCs to study the effect of strontium incorporation on cell proliferation and osteogenic differentiation. Through scanning electron microscopy (SEM), the doping of strontium has an effect on the morphology of the sediments. As the concentration of strontium increases, the mineral crystals change from flower clusters to flakes and eventually become spherical. The doping of strontium enhances the dispersivity of the crystal and makes the crystal form from polycrystalline to single crystal. It can be seen from the ion release behavior curve that the doping of strontium accelerates the release of calcium ions and phosphate ions in the mineral crystals. The experimental results show that a certain concentration of strontium can promote the proliferation of BMSCs and enhance the activity of ALP. To improve the deposition of calcium nodules and the expression of osteocalcin (OCN), the higher the content of strontium is, the more obvious the promoting effect on the proliferation and differentiation of the cells, the more.4. further studies the feasibility of the introduction of the osteogenic microcarrier on the surface of the material by electrodeposition technology, in order to construct functional organic / inorganic bone repair materials. Aminated mesoporous silicon (msns-nh2) is used to load the small molecular drug dexamethasone (DEX) with osteogenic potential to produce dex@msns-nh2 microcarrier. The DEX@MSNs-NH2/PLLA/PCL composite scaffold was deposited on the PLLA/PCL three-dimensional stent by electrodeposition technology. The experimental results show that DEX@MSNs-NH2 can sink evenly when the deposition voltage is 3 V. The composite scaffolds retained on the PLLA/PCL scaffold can still maintain three-dimensional, porous and nanofiber structures, which will benefit the adhesion and inward growth of the cells. By planting BMSCs on the composite scaffold, the bone inducibility of the cells is studied, and the composite scaffold is implanted in the skull defect of SD rats, and the bone repair ability in the body is evaluated. The results showed that the composite scaffold could support the adhesion, growth and migration of BMSCs. The results of ALP activity, alizarin red (ARS) staining and Osteocalcin (OCN) staining showed that the DEX@MSNs-NH2/PLLA/PCL composite scaffold had a better induction of osteogenic differentiation of BMSCs compared with PLLA/PCL and MSNs-NH2/PLLA/PCL stents. In vivo skull defect repair experiment entered into one. The results show that the prepared DEX@MSNs-NH2/PLLA/PCL composite scaffold can support the adhesion and migration of cells in the skull defect and accelerate the repair of bone defects. Therefore, the functional DEX@MSNs-NH2/PLLA/PCL composite scaffold is expected to be an effective bone repair material.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08;R687

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