可控释氧型组织工程材料促进种子细胞存活与成骨分化并用于修复极限股骨缺损的实验研究

The osteo-differentiation and osteogenesis of seeding cells has important significance on bone tissue engineering. However, majority of seeding cells cannot survive in the transplanted bioscaffold which mainly duo to hypoxia. Using oxygen releasing agent to supply oxygen has been accepted as a potential strategy to solve this problem. To now, the suitable and safe range of oxygen concentration for cell survival and osteo-differentiation is unknown. The existing oxygen releasing materials are not controllable which releasing oxygen only in short term. Above issues strictly limited the application of the oxygen releasing technique. Therefore, the present study planed to use the peripheral blood mesenchymal stem cells (PBMSCs) as the seeding cell model to study that the effects of different oxygen concentration on the survival and osteo-differentiation of seeding cells. Using CaO2 and PLGA to fabricate a novel oxygen generating microspheres which can release oxygen long-term and release oxygen is quantitatively controllable. Then, the microspheres and PBMSCs will be added into a basic bone tissue engineering materials for repairing critical size femur bony defect. This study is not only designed to study of controllable oxygen generating microspheres’ effects on the survival and osteo-differentiation of PBMSCs, but also to explore the mechanism of it by Western blot, RT-PCR as well as to test bone regeneration effect by histological,micro CT and biomechanical. The core objectives of this study are fabricating a novel controlled oxygen releasing material and providing an efficient strategy for the treatment of large bone defects.

种子细胞成骨分化并形成新骨质对组织工程修复骨缺损具有重要意义，移植后因缺氧而存活困难却制约了种子细胞的应用。用释氧剂供氧被公认可解决此问题，但适宜于细胞存活与成骨分化的氧浓度范围尚未知。已往的释氧材料均有释氧时间短、释氧量不可控的缺陷，故实际应用效果不佳。为此，本课题拟用外周血间充质干细胞（PBMSCs）为种子细胞模型研究不同氧浓度对其存活与分化的影响,以得出安全有效的浓度范围。用CaO2和PLGA构建释氧时程长、释氧量可控的缓释微囊，并将其与PBMSCs共同加入基础骨组织工程材料中，经体外检测后用于移植修复大鼠股骨极限缺陷模型。本课题除了研究可控释氧微囊对PBMSCs存活与成骨分化效率外，还将通过Western Blot, RT-PCR等探讨其作用机制，通过组织学、micro-CT和生物力学检测骨再生的效果。核心目的是构建全新的可控释氧型骨组织工程材料，为有效修复大段骨缺损提供治疗策略。

背景：种子细胞成骨分化并形成新骨质对组织工程修复骨缺损具有重要意义，移植后因缺氧而存活困难却制约了种子细胞的应用。用释氧剂供氧被公认可解决此问题，但适宜于细胞存活与成骨分化的氧浓度范围尚未知。已往的释氧材料均有释氧时间短、释氧量不可控的缺陷，故实际应用效果不佳。为此，本课题拟用外周血间充质干细胞（PBMSCs）为种子细胞模型研究不同氧浓度对其存活与分化的影响,以得出安全有效的浓度范围。用CaO2和PLGA构建释氧时程长、释氧量可控的缓释微囊，并将其与PBMSCs共同加入基础骨组织工程材料中，经体外检测后用于移植修复大鼠股骨极限缺陷模型。本课题除了研究可控释氧微囊对PBMSCs存活与成骨分化效率外，还将通过Western Blot, RT-PCR等探讨其作用机制，通过组织学、micro-CT和生物力学检测骨再生的效果。核心目的是构建全新的可控释氧型骨组织工程材料，为有效修复大段骨缺损提供治疗策略。.研究内容：1.探讨适合干细胞存活与成骨分化的氧浓度范围。2.可控释氧材料的构建与检测。3.体外检测释氧微囊对支架材料中种子细胞的存活与分化的影响。4.体内实验检测种子细胞的存活、分化及复合材料对骨修复的影响。5.机制研究。.重要结果及关键数据：1、种子细胞增殖及成骨分化的最适氧浓度分别为6%和9%左右。2、成功用CaO2和gelatin为原料，利用乳化-化学交联法构建可控释氧微囊，该释氧微囊能够长期缓慢释氧28天。3、该释氧微球能够改善极低氧条件下间充质干细胞的存活与成骨分化。4、该释氧微球联合自聚合肽（SAP）及间充质干细胞能够促进骨缺损修复及种子细胞存活。.科学意义：1.研究不同氧浓度对干细胞存活与分化的影响，为构建可控释氧材料提供基础；2.构建能可控释氧的复合材料，既尽可能延长释氧时程，又控制释氧率防止短时间内释放过高浓度氧导致种子细胞氧中毒；3.检测可控释氧材料在促进干细胞存活、成骨分化及其对骨修复等方面的效果；4.探讨可控释氧材料对干细胞存活及成骨分化的可能机制。本研究有望为组织工程修复大段骨缺损提供更加有效且切实可行的治疗策略。