利用羟基磷灰石支架孔隙结构调控血管生长和异位骨形成

发布时间：2018-06-26 18:44

本文选题：多孔支架 + 孔隙结构可控　；参考：《西南交通大学》2016年博士论文

【摘要】：在骨组织工程支架中,多孔支架的物理结构特性是影响细胞行为、血管生长和骨形成的关键因素。它不但为细胞的生长和迁移提供模板,同时也为血管生长和骨形成提供充足的空间。虽然优化多孔支架结构特性可有效促进骨组织形成,但是单纯调控支架的物理结构促进骨再生的能力仍然是有限的。一些研究表面引入外源性生长因子和药物可显著增强骨组织再生,因此多孔支架与生物因子或药物有效结合协同调控血管生长和骨形成受到越来越多的关注。在本研究中,通过调控支架的结构参数(孔径、贯通性、孔径分布和表面形貌)探索多孔羟基磷灰石(HA)支架结构特性与血管生长和异位骨形成的关系,进一步优化支架的多孔结构。此外,通过将载药高分子微球均匀分布在涂覆有海藻酸的多孔HA支架孔壁表面构建多孔支架缓释体系,以增强多孔HA支架血管生长和骨形成能力。采用糖球造孔法通过去除糖球制备孔隙结构可控的3类多孔HA支架,控制糖球颗粒的粒径和热处理工艺精确调控多孔支架的宏孔孔径和贯通孔尺寸。在本研究中,通过热处理工艺将宏孔孔径分别为500-650、700-950和1100-1250 μm的多孔HA支架的贯通孔尺寸/宏孔孔径的比例(d/s比)统一调控约为0.26,确保3种孔径支架具有相似的贯通性,以精确研究宏孔孔径对异位骨形成和血管生长的影响。体内实验结果显示多孔支架的宏孔孔径不但影响血管生长和骨形成的速度,同时影响新形成骨的分布。宏孔孔径为700-950 μm的多孔HA支架比其他两种孔径多孔支架展现更高的血管数量、新骨生成量以及更为均匀的新骨分布。采用糖球造孔法和热处理工艺技术制备宏孔孔径为750-900μm、贯通孔径尺寸分别为87、228和367μm (d/s比分别为0.09、0.26和0.45)的3种多孔HA支架,通过体内植入实验研究多孔支架d/s比对异位诱导骨形成和血管生长的影响。结果显示多孔支架的d/s比不但影响血管的生长,同时也影响骨组织的形成。植入4周后结果显示新生血管的尺寸随着支架贯通孔径的增大而增大,体内植入12周后结果显示d/s比为0.45的支架由于抗压强度较低,在植入体内后发生脆裂导致新骨在支架内部不均匀的形成。d/s比为0.09的支架由于贯通孔径较低限制了新生血管的数量和尺寸,导致骨形成能力的降低。而d/s比为0.26的支架则展现了最高的新骨生成量以及更均匀的新骨分布。通过采用梯度糖球模板制备2种孔径分布相反的梯度分布多孔HA支架,即(1)内部孔径为500-650μm、外周孔径为1100-1250μm(HASL); (2)内部孔径为1100-1250μm、外周孔径为500-6500μm (HALS)。体内植入4周后结果显示虽然HASL外周和中心区域的新生血管数量无显著性差异,但是外周新生血管的尺寸明显大于中心区域的新生血管。而HALS外周和中心区域的新生血管数量几乎相同无显著性差异,但是中心区域的新生血管数量小于外周区域的新生血管。HASL内总的新生血管数量和尺寸大于HALS的新生血管数。体内植入12周后结果显示HASL在整个支架的外周及中心展现均匀的骨形成,而HALS只有外周有新骨的生成。HASL内总的新骨形成量明显高于HALS。因此表明梯度多孔支架的孔径分布不仅影响支架的血管化,还影响新形成骨的分布。外部大孔径的梯度多孔支架更有利于再生新骨组织在多孔支架中的整体生长。采用一种新型的糖球颗粒模板湿化处理技术制备宏孔孔壁表面具有沟槽结构的多孔HA支架。结果发现湿化处理可调控糖球模板表面的水分含量,而孔壁表面沟槽的宽度又由糖球模板表面上的水分的含量所控制。细胞实验显示沟槽结构可诱导细胞沿沟槽方向定向排列。基因表达结果显示沟槽结构有利于骨形成。通过一种新型的方法将载丹酚酸B (Sal B)的壳聚糖微球(CMs)均匀固定在涂覆有海藻酸的多孔HA支架表面构建支架缓释体系。为增强CMs和HA支架的结合力,选用海藻酸作为多孔HA支架的涂覆材料。结果显示通过静电吸附的作用,微球稳定地固定在涂有海藻酸的多孔HA支架表面。在吸附过程中,采用静置和震荡两种组装方式将Sal B/CMs组装到多孔支架表面。与静置组装方式相比,采用震荡组装方式的多孔支架表面上的Sal B/CMs分布更为均匀,通过对比不同浓度的海藻酸研究载Sal B的CMs (Sal B/CMs)在多孔支架表面上的分布情况。结果显示最优的海藻酸浓度为1%,因为采用1%海藻酸作为涂覆层保障微球在支架表面均匀地分布并且不会影响多孔HA支架宏孔孔壁表面的多孔结构。细胞实验结果显示采用1%海藻酸作为涂覆层组装Sal B/CMs的多孔HA支架在与细胞共培养3、7天后,明显促进细胞的增殖,并且细胞均匀地粘附在支架的表面。
[Abstract]:In bone tissue engineering scaffolds, the physical structure of porous scaffolds is a key factor affecting cell behavior, vascular growth and bone formation. It not only provides a template for cell growth and migration, but also provides sufficient space for vascular growth and bone formation. Although optimizing the structural characteristics of porous scaffolds can effectively promote bone formation, However, the ability to regulate the physical structure of scaffolds to promote bone regeneration is still limited. The introduction of exogenous growth factors and drugs on some surfaces can significantly enhance bone tissue regeneration. Therefore, more and more attention has been paid to the synergistic regulation of vascular growth and bone formation by porous scaffolds with biological factors or drugs. The structural properties of the porous hydroxyapatite (HA) scaffold were investigated by regulating the structural parameters of the scaffold (aperture, penetration, pore size distribution and surface morphology). The porous structure of the scaffold was further optimized. In addition, the high molecular weight microspheres were evenly distributed in the porous HA scaffold coated with alginate. The porous scaffold sustained-release system was constructed to enhance the vascular growth and bone formation ability of porous HA scaffolds. By using the sugar ball hole method, 3 kinds of porous HA scaffolds with controllable pore structure were prepared by removing sugar spheres. The size of the sugar sphere particles and the heat treatment process were controlled to accurately regulate the pore diameter and through hole size of porous scaffolds. In the heat treatment process, the penetration hole size / pore diameter ratio (d/s ratio) of the porous HA scaffold with the pore size of 500-650700-950 and 1100-1250 m, respectively, is regulated by about 0.26, ensuring the similar penetration of the 3 kinds of aperture scaffolds to accurately study the effect of the macropore diameter on the ectopic bone formation and the vascular growth. The results show that the pore diameter of the porous scaffold not only affects the speed of vascular growth and bone formation, but also affects the distribution of the newly formed bone. The porous HA scaffold with a macro pore diameter of 700-950 mu m exhibits higher blood vessel quantity, new bone formation and more uniform new bone distribution than the other two porous porous scaffolds. 3 kinds of porous HA scaffolds with macropore diameter of 750-900 m and 87228 and 367 mu m respectively (d/s ratio 0.09,0.26 and 0.45 respectively) were prepared by processing technology. The effect of d/s ratio of porous scaffold on ectopic bone formation and vascular growth was investigated by implantation in vivo. The results showed that the d/s ratio of porous scaffolds not only affected blood vessels. Growth, but also the formation of bone tissue. 4 weeks after implantation, the results showed that the size of the neovascularization increased with the increase of the perforation diameter of the stent. After 12 weeks in the body, the results showed that the d/s ratio 0.45 was low in compressive strength, and the embrittlement after implantation resulted in the uneven.D/s ratio of the new bone in the stent. The scaffold limited the number and size of the new blood vessels and reduced the bone formation ability. The d/s ratio of 0.26 to the scaffold showed the highest new bone formation and more uniform new bone distribution. The gradient distribution of porous HA scaffolds, that is, (1) internal pores, were prepared by using a gradient sugar sphere template. The diameter was 500-650 mu m, the circumference aperture was 1100-1250 mu m (HASL), and the inner aperture was 1100-1250 m and the peripheral aperture was 500-6500 mu m (HALS). The results showed that there was no significant difference in the number of neovascularization in the peripheral and central regions of the peripheral blood, but the size of the neovascularization in the peripheral blood was obviously greater than that of the neovascularization in the central region. And HALS There was no significant difference in the number of neovascularization in the peripheral and central regions, but the number of neovascularization in the central region was less than that of the new vascular.HASL in the peripheral region. The number and size of the new blood vessels in the new vascular.HASL were larger than that of the HALS. The results showed that HASL was uniform in the peripheral and center of the whole stent after the body implantation. Bone formation, and the formation of new bone in the HALS only with new bone in.HASL, is obviously higher than that of HALS.. Therefore, the pore size distribution of the gradient porous scaffold not only affects the vascularization of the scaffold, but also affects the distribution of the newly formed bone. The gradient porous scaffold with large external aperture is more beneficial to the overall growth of the new bone tissue in the porous scaffold. A porous HA scaffold with groove structure on the surface of the macroporous wall was prepared by a new type of sugar ball particle formwork. The results showed that the wetting treatment could regulate the water content of the surface of the sugar sphere template, and the width of the groove on the surface of the hole was controlled by the content of water on the surface of the sugar sphere template. Cell experiments showed the groove junction. The gene expression results show that the groove structure is beneficial to the formation of bone. The chitosan microsphere (CMs) of B (Sal B) is uniformly fixed to the porous HA scaffold coated with alginate on the surface of the scaffold to construct a scaffold release system. The binding force of CMs and HA scaffolds is selected. As the coating material of the porous HA scaffold, the alginic acid shows that the microspheres are immobilized on the surface of the porous HA scaffold coated with alginate by electrostatic adsorption. In the process of adsorption, the Sal B/CMs is assembled on the surface of the porous scaffold by two assembly methods of static and concussion. The distribution of Sal B/CMs on the surface of the porous scaffold is more uniform. The distribution of CMs (Sal B/CMs) on the porous scaffold on the surface of Sal B is studied by contrasting alginate with different concentrations. The results show that the optimal alginate concentration is 1%, because the use of 1% alginate as coating layer is evenly distributed on the surface of the scaffold and will not be used. The porous structure of the macroporous surface of the porous HA stent was affected. The results of cell experiment showed that the porous HA scaffold which used 1% alginate as the coating layer to assemble Sal B/CMs obviously promoted the cell proliferation after co culture with the cells, and the cells adhered to the surface of the scaffold evenly.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;TB383.4

【参考文献】