预混磷酸钙骨水泥序贯缓释低剂量双因子及促老龄骨修复的研究

Previously, we have confirmed that nano-doped calcium phosphate cement delivery systems could provide a control release of the two growth factors, in which nano-doping significantly affected their release kinetics. The use of hydraulic calcium phosphate cements (CPCs) as bone substitute is impaired by their relatively poor handling due to the need to mix a powder and a liquid during surgery. To overcome these challenges, premixed CPC pastes have been developed and commercialised. Our further research shows that α-TCP nanoparticles can maintain the stability in the Sr solution, although with an unclear mechanism and degree efficacy. It provides scientific basis for construction of premixed calcium phosphate cements. .Guided by the previous studies，we intend to construct a two-phase biomaterial carrier made of β-TCP nanoparticles infused with premixed calcium phosphate cement (CPC), and this study would examine if the delivery of combined low doses of FGF-2 and BMP-2 from CPCs scaffold could enhance calvarial bone defect healing in old laboratory animals. The releationship between the controlled release performance, the morphological behavior and micro-structure will be investigated, according the basis of the two kinds of factors and final performance requirements. The effects of different sequential release patterns of FGF-2 and BMP-2 from CPC carriers on the osteogenic differentiation of low-population MSCs would also be investigated and the temporal organization of GFs release will be optimized. The project will explore the relationship between low-dose multiple growth factors delivery system and old bone regeneration in the same system, providing a basic study for bone healing in elderly.

前期我们已证实同质多相磷酸钙骨水泥（CPC）载体，能实现双因子初期的序贯缓释，但也发现传统CPC生长因子载体存在操作局限性。我们进一步的研究表明纳米α-TCP能够在锶离子溶液保持稳定性，虽然作用机制不明确，但为构建预混式CPC提供了研究基础。.本项目进一步利用同质多相CPC缓释双因子的方法，拟通过阳离子置换法设计预混式CPC，并掺入纳米β-TCP，创新地构建一种同质多相双因子（BMP-2/FGF-2）预混CPC载体。阐述预混CPC固化形成CPC的作用原理；模拟松质骨，调整原料组成、粒径等参数解决复合CPC相分离等关键技术；根据几种基质形态、降解速率的不同，调控双因子按需序贯释放。借此来探索低剂量双因子不同释放模式对模拟老龄骨微环境中低密度MSCs增殖、分化的影响。在材料、细胞水平和分子水平上多层次研究低剂量BMP-2主导的多因子缓释载体对老龄骨修复的作用。

随着人口老龄化，老年人骨质疏松性骨折难愈合问题也日显严峻。BMP-2在老年骨骼疾病治疗的作用已得到了充分的肯定，但基于高剂量BMP-2存在的安全隐患及BMP-2诱导成骨的作用机制，本课题通过两种低剂量生长因子（FGF-2、BMP-2）的序贯释放模式来研究其对老龄骨修复的作用。.首先，优化制备高分子改性微纳米γ-PGA/TCP，显著提高了β-TCP的亲水性、可降解性、生物相容性和吸附性。 .其次，优化构建预混磷酸钙骨水。通过调整α-TCP中无晶型含量以缩短激活水化反应时间，其中80%含量无晶型TCP可将激活时间缩短3天。定性和定量分析了TCP在Sr2+溶液中稳定性、Ca2+激活作用，从Sr→Ca交换动力学和反向交换动力学方面分析了预混磷酸钙骨水泥的形成机理。在水化反应激活过程中，67%-78%的Sr2+被Ca2+交换出来，在含80%无晶型TCP样品中约有34%的Sr2+残留与CDHA中，说明无晶型TCP对Sr2+亲和力更强，并且激活后膏剂中保持适量的Sr2+更有利于骨骼生长和血管再生。.然后，在以上基础上构建双因子预混磷酸钙骨水泥载体，分析双控载体的微结构、表/界面特性，解决了复合磷酸钙骨水泥抗溃散性和多相融合两个关键技术问题。研究了双因子释放动力学特征，处于外层因子的释放启动时间为18小时，而内层因子初始释放为48小时，释放速率也有各自的规律，能实现内外因子序贯缓释。研究表明影响生长因子释放动力学因素有很多，比如CPC性质微观结构，结晶度，密度和孔隙率的影响。 .最后，研究双因子不同释放模式对成骨细胞增殖和成骨分化的影响，体外模拟老龄骨微环境，测试不同低剂量的生长因子单独或联合使用对低密度MSCs的促增殖作用和成骨分化作用。另外，本项目还增加体内大鼠颅骨缺损模型，体内研究低剂量BMP-2主导的多因子缓释载体对骨修复的作用。研究表明，“先释放BMP-2再释放FCF-2”的释放模式更有利于低密度骨髓基质干细胞在载体材料表面增殖和成骨分化,以及体内骨修复作用，对诱导老龄骨缺损修复具有很好的应用潜力。