DataSheet1_Optimizing Barium Titanate Nanocomposite Bone Scaffolds for Biomineralization in Dynamic Compression Bioreactors Using Time-Lapsed Microstructural Imaging and Smart Thresholding.PDF

Bone scaffolds made of calcium phosphate polymer nanocomposites have limited osteoinductive properties. Piezoelectric materials have attracted considerable interest in bone tissue engineering due to their potential to promote osteogenesis through additional electrical stimulation. Time-lapsed micro-CT imaging is a time-effective tool for in vitro optimization of such scaffolds but is challenged by nanocomposites with a high attenuation coefficient, such as one containing high amounts of piezoelectric barium titanate. We used high-resolution end-point micro-CT scans combined with histology and Raman spectroscopy to screen polydopamine functionalized nanocomposites containing 3–27 vol% barium titanate for collagenous extracellular matrix formation and mineralization. All compositions showed well-connected extracellular matrix and birefringent matured collagen after seven weeks of static human mesenchymal stem cell cultures. Nevertheless, high-resolution micro-CT analysis combined with smart thresholding during image processing enabled us to observe modest differences in ECM mineralization between groups suggesting that a volume fraction of 9–21% barium titanate facilitated the formation of dense mineral clusters in the pores even in the absence of mechanical stimuli, further corroborated by Raman spectroscopy. The same image processing approach facilitated the analysis of time-lapsed micro-CT images of scaffold cultures in dynamic compression bioreactors where 9 vol% barium titanate was the best nanocomposite composition, resulting in a significant twofold increased maturation rate under dynamic conditions. On the other hand, barium titanate content of ≥15 vol% did not improve mineralization. At 27 vol%, the biomineralization of the collagenous extracellular matrix was even impeded in the nanocomposite scaffolds, as evidenced by histology stainings. Overall, our approach enables time-lapsed quantitative assessment of high X-ray absorbing nanocomposite scaffolds for biomineralization under dynamic compression, facilitating the optimization of such mechanically responsive scaffolds.以磷酸钙聚合物纳米复合材料制备的骨支架，其骨诱导性能较为有限。压电材料（piezoelectric materials）因可通过额外电刺激促进成骨，在骨组织工程领域受到广泛关注。延时显微CT（time-lapsed micro-CT）成像是体外优化此类支架的高效工具，但对于高衰减系数的纳米复合材料（如含大量压电钛酸钡（barium titanate）的复合材料）而言，该技术面临挑战。本研究采用高分辨率终点显微CT扫描结合组织学与拉曼光谱（Raman spectroscopy）技术，对含3~27体积百分比钛酸钡的聚多巴胺（polydopamine）功能化纳米复合材料进行筛选，以评估其胶原细胞外基质形成与矿化情况。在静态培养人骨髓间充质干细胞七周后，所有组分的支架均呈现出连通性良好的细胞外基质与具有双折射特性的成熟胶原。尽管如此，结合图像处理阶段的智能阈值分割进行的高分辨率显微CT分析，使我们能够观察到各组间细胞外基质矿化的细微差异：结果表明，钛酸钡体积分数为9%~21%时，即便无机械刺激，也可促进孔隙内形成致密的矿物团簇，这一结论进一步得到拉曼光谱的验证。采用相同的图像处理方法，可更便捷地分析动态压缩生物反应器（dynamic compression bioreactors）中支架培养的延时显微CT图像，其中钛酸钡体积分数为9%的纳米复合材料为最优组分，在动态培养条件下其成熟速率显著提升了一倍。另一方面，钛酸钡含量≥15体积百分比时，并未提升矿化效果。当钛酸钡体积分数为27%时，纳米复合支架的胶原细胞外基质生物矿化甚至受到抑制，这一点可通过组织学染色（histology stainings）结果得到证实。综上，本研究提出的方法可实现动态压缩条件下高X射线吸收纳米复合支架生物矿化的延时定量评估，有助于优化此类机械响应型骨支架。