藻酸盐凝胶微球复合双相磷酸钙陶瓷构建程序化双重3D培养体系

Low cell viability, cell survival rate and unsatisfactory vascularization ability within the engineered constructs have always been great challenges for tissue engineered bone application. Building 3D culture system to simulate the micro-environment of seed cells may be a potential method to improve these. Thus, based on our previous study we plan to build a dual 3D programmed culture system. This system includes three steps: 1: Seeding epithelial progenitor cells with biophasic calcium phosphate scaffold to improve the vascularization of the tissue engineered bone, which completes the first level of 3D culture system. 2. Using placenta derived mesenchymal stem cells alginate beads to increase cell viability and survival rates, that is the second level of 3D culture system. 3. Finally, we seed stem cells alginate beads into epithelial progenitor cells composite calcium phosphate bioceramic scaffold to form a dual 3D programmed culture system. The content of the culture medium and the growth factors are adjusted according to the expression level of osteogenesis and angiopoietic related mRNA and growth factors. We will investigate this 3D culture system systemically ranging from its in vitro and in vivo bio-characterization, and compare the repair effectiveness of our culture system in rabbit large bone defect with the conventional tissue engineered boon establishment method through histology, imageology and molecular biology investigation. Our research will provide new experimental evidence and theoretical basis for clinic applying.

种子细胞存活率低，生物学功能降低以及较差的体内血管化仍是组织工程骨应用的主要问题。建立3D培养模型模拟种子细胞体内生物学微环境有望解决这些问题。基于课题组的前期研究，我们设计构建一套组织工程骨程序化双重3D培养体系。此体系的构建分为三步，1：将人脐带血内皮祖细胞与双相磷酸钙陶瓷支架复合以改善血管化问题，实现第一重3D培养。2：构建藻酸盐凝胶包裹人胎盘来源间充质干细胞微球进行第二重3D培养，以提高干细胞生物活性和存活率。3：将以上两套3D体系按程序进行复合，进行双重3D诱导培养。通过检测成骨、成血管相关基因表达水平及因子分泌水平，调整各阶段培养基、因子的配比优化支架材料的成骨和成血管效果。本研究将对该3D培养体系进行系统的生物学研究，并通过组织学、影像学以及分子生物学等手段对此体系在兔大段骨缺损模型中的骨修复效果与常规组织工程骨构建方法进行对比，为组织工程骨的构建提供新思路和理论依据

低细胞活力，低细胞存活以及差强人意的血管生成能力一直以来是组织工程骨应用中的巨大挑战。构建3D培养体系以模拟组织工程骨中种子细胞的微环境或许是一种极具潜力的提高细胞活力，生存率以及血管生成能力的方法。因此基于我们前期的研究工作，我们意在于构建一种双重3D程序化培养系统应用于组织工程骨领域。在研究方法中，我们首先将干细胞与藻酸盐凝胶进行微球制备实现第一重3D构建，之后将微球复合于我们制备的双相磷酸钙陶瓷支架材料中以形成第二重的3D程序化培养体系。并通过骨生成以及血管生成相关因子的释放程度对培养基成分以及生长因子进行优化。在构建完这一套体系后，我们对其进行了一系列的体内外的特性和功能学研究。在所获得的实验结果中，我们发现藻酸盐凝胶细胞微球复合双向磷酸钙陶瓷材料具有优于其他对照组（无微球直接复合细胞组，单一支架材料组，无材料修复处理组）在骨新生和血管新生方面的能力。因此，我们研究项目所构建的这一套双重3D培养体系为未来的临床骨修复，组织工程骨构建领域提供了新的研究证据和理论。