【摘要】：由于創(chuàng)傷、感染以及發(fā)育異常等原因?qū)е碌拇竺娣e骨缺損的修復在臨床治療上仍面臨巨大的挑戰(zhàn)。目前常用的自體和異體移植治療方法都存在不同程度的問題。組織工程的出現(xiàn)為骨缺損修復的治療提供了新的選擇方法。構(gòu)建生物特性與正常骨相似的組織工程骨是目前骨組織工程研究的熱點。羥基磷灰石的組成與人體骨的無機成分相似。尤其是納米級羥基磷灰石,因具有良好的生物相容性、骨傳導性和骨再生性,被廣泛應用于骨組織工程領(lǐng)域。但目前的納米級羥基磷灰石存在尺寸均一性和水分散性較差的問題,限制了其在骨組織工程中的應用。從仿生礦化的角度出發(fā),利用納米技術(shù),制備水分散性優(yōu)良的納米羥基磷灰石,構(gòu)建新型實用的骨組織修復材料,有望推動骨組織工程的發(fā)展。絲素蛋白由于其獨特的機械性能、生物相容性和緩慢的降解性,受到越來越多的關(guān)注,成為骨組織工程的基質(zhì)材料。本文首先以尺寸均一的絲素蛋白納米顆粒為模板仿生礦化制備納米級羥基磷灰石,通過調(diào)控工藝參數(shù),獲得尺寸均一,能在水中穩(wěn)定分散、具有核殼結(jié)構(gòu)的羥基磷灰石/絲素蛋白納米顆粒,并研究發(fā)現(xiàn)了絲素蛋白包覆層對穩(wěn)定納米顆粒的關(guān)鍵作用。隨后,將此納米顆粒直接分散在絲素蛋白溶液中形成均一的絲素蛋白與羥基磷灰石混合物,經(jīng)過冷凍干燥制備絲素蛋白/羥基磷灰石復合支架。與已有報道的絲素蛋白/羥基磷灰石復合支架相比,本研究所制備的復合支架不僅具有更高的羥基磷灰石含量,同時實現(xiàn)了羥基磷灰石納米顆粒在納米尺度的均勻分布,以及力學強度的顯著提高。體外細胞實驗結(jié)果表明,在不添加成骨生長因子的情況下,復合支架中羥基磷灰石的含量和分布影響骨髓間充質(zhì)干細胞的生長和成骨分化能力。納米顆粒含量高且分布均勻的絲素蛋白復合支架能夠提供更適宜的環(huán)境,顯著提高間充質(zhì)干細胞的生長和成骨分化能力。與此同時,利用上述具有核殼結(jié)構(gòu)的羥基磷灰石/絲素蛋白納米顆粒作為載體,成功加載骨生長因子骨形態(tài)蛋白2(BMP-2),并實現(xiàn)了因子的可控釋放。BMP-2加載率高達99.6%,可穩(wěn)定釋放21天以上,沒有出現(xiàn)“突釋”現(xiàn)象。細胞實驗結(jié)果表明同原有體系相比,現(xiàn)有的控釋體系能夠更好地促進間充質(zhì)干細胞的生長和向成骨細胞的分化。隨后,將BMP-2控釋體系同絲素蛋白/羥基磷灰石復合支架制備技術(shù)結(jié)合,構(gòu)建能夠可控釋放BMP-2,且羥基磷灰石均勻分布的復合支架體系,以構(gòu)建適合骨修復的微環(huán)境。同不含BMP-2的復合支架相比,上述體系不僅獲得了更好的成骨分化效果,同時還通過調(diào)控BMP-2的釋放行為實現(xiàn)了干細胞向成骨細胞分化能力的優(yōu)化。動物實驗結(jié)果進一步表明,同原有加載BMP-2的絲素蛋白基支架相比,該復合支架顯示出更好地誘導骨再生的能力�？傊�,本文以尺寸均一的絲素蛋白納米顆粒為模板,調(diào)控羥基磷灰石納米顆粒的生成,不僅優(yōu)化了絲素蛋白/羥基磷灰石骨材料的設計,同時實現(xiàn)了更為有效控釋BMP-2的載體的制備以及同絲素蛋白/羥基磷灰石骨材料的復合,最終構(gòu)建出適合骨再生的微環(huán)境。更重要的是,基于對上述不同因素的調(diào)控,研究了不同關(guān)鍵因素影響干細胞分化以及骨修復的作用,為新型骨材料的設計提供了理論參考。
[Abstract]:The repair of large-area bone defects due to trauma, infection, and developmental abnormalities still faces great challenges in clinical treatment. There are different degrees of problems in the methods of autograft and allograft treatment. The appearance of tissue engineering provides a new method for the treatment of bone defect repair. The construction of tissue engineering bone with similar biological characteristics and normal bone is a hot spot in the research of bone tissue engineering at present. The composition of the hydroxyapatite is similar to that of the human bone. in particular, that nano-level hydroxyapatite is widely used in the field of bone tissue engineering because of good biocompatibility, bone conductivity and bone regeneration. However, the present nano-grade hydroxyapatite has the problems of poor size and poor water dispersibility, and the application of the nano-hydroxyapatite in the bone tissue engineering is limited. Starting from the angle of biomimetic mineralization, nano-hydroxyapatite with excellent water dispersibility is prepared by using nano-technology, and a novel practical bone tissue repair material is constructed, which is expected to promote the development of bone tissue engineering. The silk fibroin is a matrix material for bone tissue engineering due to its unique mechanical properties, biocompatibility and slow degradation. The preparation method comprises the following steps of: firstly, performing bionic mineralization on the silk fibroin nano-particles with uniform size as a template to prepare the nano-scale hydroxyapatite, and obtaining the hydroxyapatite/ silk fibroin nano-particles with uniform size, stable dispersion in water, The key role of the coating of silk fibroin in stabilizing nanoparticles was also studied. then, the nano particles are directly dispersed in the silk fibroin solution to form a uniform silk fibroin and a hydroxyapatite mixture, and the fibroin/ hydroxyapatite composite stent is prepared by freeze drying. Compared with the existing reported silk fibroin/ hydroxyapatite composite stent, the composite stent prepared by the research institute not only has higher hydroxyapatite content, but also realizes the uniform distribution of the hydroxyapatite nano-particles in the nano-scale and the remarkable improvement of the mechanical strength. The results of in vitro cell experiment show that in the absence of bone growth factor, the content and distribution of the hydroxyapatite in the composite scaffold can affect the growth of the mesenchymal stem cells and the ability of bone differentiation. The silk fibroin composite stent with high nanometer particle content and uniform distribution can provide a more suitable environment, and can obviously improve the growth and the bone differentiation capacity of the mesenchymal stem cells. At the same time, the bone morphogenetic protein 2 (BMP-2) was loaded successfully by using the hydroxyapatite/ silk fibroin nanoparticles with the core shell structure as a carrier, and the controllable release of the factor was realized. The loading rate of BMP-2 is up to 99.6%, and can be stably released for more than 21 days without any 鈥渂urst鈥，

本文編號：2316222