Silicate Bioceramic Structure Control on Mesenchymal Stem Cell Proliferation and Differentiation

ID：MPS/DMR/BMAT（7623）0906817 PI：Sahai，Nita ORG：威斯康星大学标题：硅酸盐生物陶瓷结构对间充质干细胞增殖和分化的控制该奖项由2009年美国复苏和再投资法案资助（公法111-5）.知识产权：生物活性硅酸盐生物陶瓷或玻璃的化学组成以及浸出的Si和Ca浓度通过影响无定形二氧化硅和碳酸盐的质地而强烈影响骨整合的复杂过程。羟基磷灰石（Ca 5（PO 4）3OH，HCA）层在植入物表面形成，蛋白质的吸附，以及人间充质干细胞（hMSC）附着、增殖和分化成产生新骨的成骨细胞。不同于离子浓度，离子释放速率对细胞活性的影响，以及硅酸盐结构和溶解机制对离子释放速率和总浓度的关键控制，以及对所得表面纹理的关键控制，以前没有研究过。PI假设，对于类似初始纹理的表面，硅酸盐结构对溶解机制发挥主要控制作用，影响离子释放的浓度和速率、沉淀无定形二氧化硅和HCA层的纹理、特定基因的激活以及最终hMSC增殖和分化成骨细胞。将通过进行分批和流通实验并确定hMSC在假硅灰石（β-CaSiO 3，psW）和硅灰石（α-CaSiO 3，Wol）上的增殖和分化来测试该假设。PSW多晶型物具有应变高能硅酸盐？三环？与Wol中溶解较慢的较强硅酸盐链相比，Bioglass® 45 S5在水中快速溶解，而Bioglass® 45 S5提供了阳性对照。硅酸盐结构和初始表面纹理（粗糙度和粒度）的影响将被独立检查。将通过使用电感耦合等离子体发射光谱法进行定期溶液分析（pH、Si、Ca、P），并通过使用扫描电子显微镜、能量色散X射线光谱法和高分辨率透射电子显微镜进行表面表征，确定溶解和沉淀机制和速率。反应的沉淀将用作在补充有成骨分化因子的标准培养基中培养hMSC的底物。通过荧光和总DNA含量、骨钙素和碱性磷酸酶分析（成骨细胞产生的酶）以及使用真实的时间聚合酶链反应鉴定骨特异性基因（Cbfa-1、骨钙素、骨桥蛋白），定期定量增殖和分化成骨细胞。定期的培养基取样将提供细胞存在时离子释放速率的信息。概念方法和实验设计提供了一组全面的离体实验，这些实验比模拟体液中的典型体外批量实验更接近体内条件，同时分别检查每个参数的影响。拟议工作的结果可能会对第三代细胞和基因影响生物陶瓷的开发产生重大影响，这些生物陶瓷具有改善的骨整合特性，通过为设计生物活性材料提供先验基础，所述生物活性材料将Si、Ca和P水平控制在用于hMSC增殖、分化和成骨细胞活性的最佳水平。因此，这些结果有助于减少对昂贵的特设材料合成和体外测试的依赖，减少骨科植入手术后的术后恢复期，并增加植入物在人体内的寿命。对于美国日益老龄化的人口来说，生物医学意义尤其重要，预计未来三十年对髋关节和膝关节植入手术的需求将显著增加（约80%）。拟议的工作还提供了一个综合的，跨学科的培训计划，包括表面化学，晶体结构和细胞生物学的研究生和本科生。结果将在同行评审的期刊和会议上传播;纳入PI和co-PI教授的研究生课程;并用于为威斯康星州大学的K-12学生开发博物馆展览。

ID: MPS/DMR/BMAT(7623) 0906817 PI: Sahai, Nita ORG: University of WisconsinTitle: Silicate Bioceramic Structure Control on Mesenchymal Stem Cell Proliferation and DifferentiationThis award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).INTELLECTUAL MERIT: The chemical composition of bioactive silicate bioceramics or glasses and the leached Si and Ca concentrations strongly influence the complex process of osteointegration by affecting the texture of the amorphous silica and carbonated hydroxyapatite (Ca5(PO4)3OH, HCA) layers formed at the implant surface, the adsorption of proteins, and human mesenchymal stem cell (hMSC) attachment, proliferation and differentiation into osteoblasts that produce new bone. Distinct from ion concentrations, the effect of ion release rates on cellular activity, and the critical control exerted by silicate structure and dissolution mechanisms on ion release rates and overall concentrations, and on resulting surface texture has not been examined previously. The PIs hypothesize that, for surfaces of similar initial texture, silicate structure exerts a major control on the dissolution mechanism, affecting the concentration and rate of ions released, the texture of the precipitated amorphous silica and HCA layers, the activation of specific genes and, ultimately, hMSC proliferation and differentiation into osteoblasts. This hypothesis will be tested by performing batch and flow-through experiments and determining hMSC proliferation and differentiation on pseudowollastonite (beta-CaSiO3, psW) and wollastonite (alpha- CaSiO3, Wol). The psW polymorph possesses strained high-energy silicate ?three-rings? that dissolve rapidly in water versus the stronger silicate chains in Wol that dissolve slower, while Bioglass® 45S5 provides a positive control. Silicate structure and initial surface texture (roughness and particle size) effects will be examined independently. The dissolution and precipitation mechanisms and rates will be established by periodic solution analysis (pH, Si, Ca, P) using Inductively-Coupled Plasma Optical Emission Spectroscopy and by surface characterization using Scanning Electron Microscopy with Energy-Dispersive X-Ray Spectroscopy and High Resolution Transmission Electron Microscopy. Reacted pellets will be used as substrates for hMSC cultures in standard culture media supplemented with osteogenic differentiation factors. Proliferation and differentiation into osteoblasts will be quantified periodically by fluorescence and total DNA content, osteocalcin and alkaline phosphatase analyses (enzymes produced by osteoblasts), and identification of bone-specific genes (Cbfa-1, osteocalcin, osteopontin) using real time polymerase chain reaction. Periodic culture medium sampling will provide information on ion release rates in the presence of cells. The conceptual approach and experimental design provides a comprehensive group of ex vivo experiments that are closer to in vivo conditions than typical in vitro batch experiments in simulated body fluids, while examination of the effects of each parameter separately.BROADER IMPACTS: The results of the proposed work could have a significant impact on the development of third-generation cell-and gene-affecting bioceramics with improved osteointegration properties, by providing an a priori basis for designing bioactive materials that control Si, Ca and P levels at optimized levels for hMSC proliferation, differentiation and osteoblast activity. The results could thus help to reduce dependence on expensive ad hoc materials synthesis and in vitro testing, decrease the post-operative recovery period following orthopaedic implant surgery and increase the life-span of the implant in the human body. The biomedical implications are especially critical for an increasingly aging population in the USA, where a significant rise (~80%) is projected in the demand for hip and knee implant surgeries over the next three decades. The proposed work also provides an integrated, interdisciplinary training program including surface-chemistry, crystal structure and cell biology to graduate and undergraduate students. Results will be disseminated in peer-reviewed journals and at conferences; incorporated into graduate courses taught by the PI and co-PI; and used to develop a Museum Exhibit for K-12 students at University of Wisconsin.