本文關(guān)鍵詞： 血管化 水凝膠 1-磷酸鞘氨醇 羧甲基殼聚糖 醛基化透明質(zhì)酸　出處：《東華大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：大段骨的缺損修復(fù)一直是臨床治療的難題。近年來,骨組織工程制備的仿生骨支架能夠從結(jié)構(gòu)和功能上模仿天然骨,為大段骨缺損的修復(fù)帶來了希望。骨再生是一個復(fù)雜過程,包括成骨細胞的長入和增殖、營養(yǎng)物質(zhì)和氧氣的輸送等,這些過程都需要血管參與,因此骨移植支架的前期血管化具有重要意義。目前,骨組織工程中常用的有效促血管化的方法有:血管誘導(dǎo)因子引入、成骨細胞和血管內(nèi)皮細胞與支架材料共培養(yǎng)、相關(guān)基因的引入以及顯微外科體外重建等。其中,在支架材料內(nèi)負載促血管化因子是一種簡單而有效的方法。本課題以介孔二氧化硅納米顆粒(MSNs)作為血管化功能性藥物1-磷酸鞘氨醇(S1P)的微載體,再將其與透明質(zhì)酸/殼聚糖基天然高分子制備的水凝膠復(fù)合,構(gòu)建可用于骨支架前期血管化的有效控釋載體。首先,利用不同濃度羧甲基化殼聚糖(4,6和8 wt%)與醛基化透明質(zhì)酸通過席夫堿反應(yīng)制備三種純水凝膠樣品,通過對純水凝膠樣品的物化性能表征和生物相容性研究篩選出一種可作為后續(xù)研究的高強度水凝膠。然后,制備聚電解質(zhì)藻酸鈉(ALG)和殼聚糖(CHI)表面修飾的MSNs,作為藥物的微載體,通過聚電解質(zhì)層修飾提升粒子的生物相容性和增強粒子對藥物的裝載性能。進一步制備了復(fù)合微載體的納米復(fù)合水凝膠,研究納米粒子的加入量對水凝膠理化性能的影響,對體外藥物釋放行為以及體外降解行為進行了研究。研究了細胞在納米復(fù)合水凝膠表面和內(nèi)部的生長情況,并評價了載藥納米復(fù)合水凝膠對內(nèi)皮細胞的募集和體內(nèi)促血管化的能力。實驗結(jié)果表明,純水凝膠的力學(xué)性能和交聯(lián)度隨著羧甲基化殼聚糖濃度的增加而增強,但細胞增殖卻相應(yīng)降低。因此,綜合考慮水凝膠的力學(xué)性能和細胞增殖情況,選取6 wt%羧甲基化殼聚糖用于后續(xù)研究。力學(xué)性能表征結(jié)果顯示,納米粒子的加入明顯增強納米復(fù)合水凝膠的力學(xué)性能(11.06-36.83 kPa),有效提升了天然水凝膠的力學(xué)性能。細胞實驗結(jié)果表明,納米復(fù)合水凝膠更利于細胞的增殖,少量的牛血清蛋白(BSA)的引入能夠改善細胞的鋪展。Transwell細胞遷移實驗結(jié)果顯示,載有S1P的納米復(fù)合水凝膠表現(xiàn)對內(nèi)皮細胞募集的作用。雞胚尿囊絨毛膜實驗和小鼠皮下包埋實驗結(jié)果進一步表明,載有S1P的納米復(fù)合水凝膠能夠有效促進周圍組織血管再生。而且,當載藥納米復(fù)合水凝膠與大孔支架結(jié)合,在小鼠體內(nèi)植入3周后,大孔支架淺層觀察到新生血管的長入。因此,制備的載有S1P的納米復(fù)合水凝膠藥物控釋載體在骨組織工程血管化領(lǐng)域具有潛在的應(yīng)用前景。
[Abstract]:In recent years, biomimetic bone scaffolds prepared by bone tissue engineering can mimic natural bone in structure and function, which brings hope to repair large segment bone defect. Bone regeneration is a complex process. These processes, including the growth and proliferation of osteoblasts, the transport of nutrients and oxygen, all require the participation of blood vessels, so the pre-vascularization of bone graft scaffolds is of great significance. The effective vascularization methods used in bone tissue engineering include the introduction of angiogenic factors, co-culture of osteoblasts and vascular endothelial cells with scaffold materials, the introduction of related genes and the reconstruction of microsurgery in vitro. It is a simple and effective method to load vascularization factor into scaffold materials. In this study, mesoporous silica nanoparticles (MSNs) were used as microcarriers of vascularized functional drug, sphingosine 1-phosphate (S1P). Then it was combined with hydrogel prepared by hyaluronic acid / chitosan based natural polymer to construct an effective controlled release carrier for early vascularization of bone scaffold. Three kinds of pure hydrogels were prepared by the reaction of carboxymethylated chitosan with aldohyaluronic acid at different concentrations for 6 and 8 wts through Schiff base reaction. A kind of high strength hydrogel which can be used as a follow-up study was selected by studying the physicochemical properties and biocompatibility of pure hydrogel samples. Then, the surface modified MSNs of polyelectrolyte sodium alginate (ALG) and chitosan (CHI) were prepared and used as microcarriers of the drug. Through the modification of polyelectrolyte layer to improve the biocompatibility of particles and enhance the loading properties of the particles, the nanocomposite hydrogels with composite microcarriers were further prepared, and the effects of the amount of nanoparticles on the physical and chemical properties of hydrogels were studied. The drug release behavior and degradation behavior in vitro were studied. The growth of cells on the surface and inside of nano-composite hydrogels was studied. The ability of drug loaded nano-composite hydrogels to recruit endothelial cells and promote vascularization in vivo was evaluated. The results showed that the mechanical properties and crosslinking degree of pure hydrogels increased with the increase of carboxymethylated chitosan concentration. However, cell proliferation was decreased. Therefore, considering the mechanical properties of hydrogel and cell proliferation, 6 wt% carboxymethylated chitosan was selected for further study. The addition of nano-particles significantly enhanced the mechanical properties of nano-composite hydrogels 11.06-36.83 KPA, and effectively enhanced the mechanical properties of natural hydrogels. The results of cell experiments showed that nano-composite hydrogels were more conducive to cell proliferation. The introduction of a small amount of bovine serum protein (BSA) can improve the proliferation of cells. Transwell cell migration results show that, The effect of S1P loaded nano-composite hydrogel on endothelial cell recruitment. Chicken embryo chorionic chorioallantoic test and mouse subcutaneous embedding test further showed that, Nano-composite hydrogels containing S1P could effectively promote vascular regeneration in peripheral tissues. Moreover, when drug-loaded nano-composite hydrogels were combined with macroporous scaffolds, they were implanted in mice for 3 weeks. The growth of neovascularization was observed in the superficial layer of the macroporous scaffold. Therefore, the S1P loaded nano-composite hydrogel drug controlled release carrier has a potential application prospect in the field of bone tissue engineering vascularization.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08;R68

【引證文獻】

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1 梅建國;莊夕棟;張穎;莊金秋;苗立中;吳信明;劉吉山;謝金文;丁壯;沈志強;;細胞培養(yǎng)微載體及其在生物醫(yī)藥領(lǐng)域的應(yīng)用[J];生物技術(shù);2017年05期

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本文編號：1550687