Hagenmüller Henri, Hofmann Sandra, Kohler Thomas, Merkle Hans P, Kaplan David L, Vunjak-Novakovic Gordana, Müller Ralph, Meinel Lorenz
Institute for Biomechanics, ETH Zurich, 8093, Zurich, Switzerland.
Ann Biomed Eng. 2007 Oct;35(10):1657-67. doi: 10.1007/s10439-007-9338-2. Epub 2007 Jun 2.
The formation of bone-like tissue from human mesenchymal stem cells (hMSC) cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified over 44 days in culture using non-invasive time-lapsed micro-computed tomography (microCT). Each construct was imaged nine times in situ. From microCT imaging, detailed morphometrical data on bone volume density, surface-to-volume ratio, trabecular thickness, trabecular spacing, and the structure model index and tissue mineral density were obtained. microCT irradiation did not impact the osteogenic performance of hMSCs based on DNA content, alkaline phosphatase activity, and calcium deposition when compared to non-exposed control samples. Bone-like tissue formation initiated at day 10 of the culture with the deposition of small mineralized clusters. Tissue mineral density increased linearly over time. The surface-to-volume ratio of the bone-like tissues converged asymptotically to 26 mm(-1). Although in vitro formation of bone-like tissue started from clusters, the overall bone volume was not predictable from the time, number, and size of initially formed bone-like clusters. Based on microstructural analysis, the morphometry of the tissue-engineered constructs was found to be in the range of human trabecular bone. In future studies, non-invasive, time-lapsed monitoring may enable researchers to culture tissues in vitro, right until the development of a desired morphology is accomplished. Our data demonstrate the feasibility of qualitatively and quantitatively detailing the spatial and temporal mineralization of bone-like tissue formation in tissue engineering.
利用非侵入性延时微型计算机断层扫描（microCT），在44天的培养期内监测并量化了在丝素蛋白支架上于成骨培养基中培养的人间充质干细胞（hMSC）形成类骨组织的情况。每个构建体原位成像9次。通过microCT成像，获得了关于骨体积密度、表面积与体积比、小梁厚度、小梁间距、结构模型指数和组织矿物质密度的详细形态计量学数据。与未暴露的对照样品相比，基于DNA含量、碱性磷酸酶活性和钙沉积，microCT照射并未影响hMSC的成骨性能。类骨组织形成于培养第10天开始，伴有小矿化簇的沉积。组织矿物质密度随时间呈线性增加。类骨组织的表面积与体积比渐近收敛至26 mm(-1)。尽管类骨组织的体外形成始于簇状物，但无法根据最初形成的类骨簇的时间、数量和大小预测总体骨体积。基于微观结构分析，发现组织工程构建体的形态计量学处于人小梁骨范围内。在未来的研究中，非侵入性延时监测可能使研究人员能够在体外培养组织，直至实现所需的形态。我们的数据证明了在组织工程中定性和定量详细描述类骨组织形成的空间和时间矿化的可行性。
