Silicate Bioceramic Structure Control on Mesenchymal Stem Cell Proliferation and Differentiation

ID：MPS/DMR/BMAT(7623)0906817 PI：Sahai，Nita ORG：威斯康星大学题目：硅酸盐生物陶瓷结构对间充质干细胞增殖和分化的控制本奖项由2009年美国恢复和再投资法案(公共法律第111-5号)资助。研究价值：生物活性硅酸盐生物陶瓷或玻璃的化学成分以及所浸出的硅和钙浓度通过影响种植体表面形成的无定形二氧化硅和碳酸化羟基磷灰石(Ca5(PO4)3OH，HCA)层的织构，蛋白质的吸附，以及人类间充质细胞(HMSC)附着，强烈影响骨整合的复杂过程增殖和分化为能产生新骨的成骨细胞。与离子浓度不同，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以及由此产生的表面纹理的关键控制以前没有被研究过。PI假设，对于初始纹理相似的表面，硅酸盐结构对溶解机制起主要控制作用，影响离子释放的浓度和速率，影响沉淀的无定形二氧化硅和HCA层的织构，影响特定基因的激活，最终影响hMSC的增殖和向成骨细胞的分化。这一假设将通过进行批次和流过实验以及测定hMSC在伪硅灰石(β-CaSiO_3，PSW)和硅灰石(α-CaSiO_3，Wol)上的增殖和分化来验证。Psw晶型具有应变高能硅酸盐、三环？它在水中溶解迅速，而WOL中较坚固的硅酸链溶解较慢，而Bioglass®45S5提供了积极的对照。硅酸盐结构和初始表面纹理(粗糙度和颗粒大小)的影响将单独进行检查。溶解和析出的机制和速率将通过使用电感耦合等离子体光谱的周期性溶液分析(pH、Si、Ca、P)和使用扫描电子显微镜、能量色散X射线光谱和高分辨率透射电子显微镜的表面表征来确定。反应后的颗粒将作为hMSC培养的底物，在标准培养液中添加成骨分化因子。成骨细胞的增殖和分化将通过荧光和总DNA含量、骨钙素和碱性磷酸酶(成骨细胞产生的酶)分析以及实时聚合酶链式反应鉴定骨特异基因(CBFA-1、骨钙素、骨桥蛋白)来定期定量。定期的培养基取样将提供有关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体外实验，比典型的模拟体液中的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。BROADER影响：拟议工作的结果可能对第三代影响细胞和基因的生物陶瓷的开发产生重大影响，改善骨整合性能，为设计生物活性材料提供先验基础，以控制硅、钙和磷水平在优化水平上促进hMSC的增殖、分化和成骨细胞活性。因此，该结果有助于减少对昂贵的特殊材料合成和体外测试的依赖，缩短骨科植入物手术后的恢复期，并延长植入物在人体内的寿命。生物医学的影响对美国日益老龄化的人口尤其重要，预计未来30年髋关节和膝关节植入手术的需求将显著增长(~80%)。这项拟议的工作还为研究生和本科生提供了一个综合的跨学科培训计划，包括表面化学、晶体结构和细胞生物学。结果将在同行评议的期刊和会议上传播；纳入由国际和平协会和联合国际教授的研究生课程；并用于为威斯康星大学的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.