Wollenweber Marcus, Domaschke Hagen, Hanke Thomas, Boxberger Sabine, Schmack Gerhilt, Gliesche Konrad, Scharnweber Dieter, Worch Hartmut
Max Bergmann Center of Biomaterials, Dresden, Germany.
Tissue Eng. 2006 Feb;12(2):345-59. doi: 10.1089/ten.2006.12.345.
Controlling the differentiation of human mesenchymal stem cells (hMSC) and providing tissue functions in engineered constructs before implantation are major challenges. Beside the additives in culture media, the artificial niches inside a scaffold can serve this purpose. To prepare niches favoring the osteoblastic differentiation of hMSCs, components of the extracellular matrix of bone were immobilized on fabrics of poly(3-hydroxybutyrate). Aqueous gels of fibrillar bovine collagen I, with or without addition of chondroitin sulphate (CS), were immobilized on the textile scaffold, sub-structured in a freeze-drying process, and cross-linked. hMSCs of four donors were isolated from bone marrow. After expansion, the cells were seeded dynamically onto the scaffolds. From thereon, the culture was transferred into perfused vessels and partly submitted to dexamethasone to promote osteogenic differentiation. During their 4 weeks of culture, the cells' distribution and morphology throughout the scaffolds were characterized by laser scanning microscopy (LSM) and scanning electron microscopy (SEM). Photospectrometrically the cells' viability (MTT) and alkaline phosphatase (ALP) production were assessed. The transcription of osteoblast-specific markers was elucidated with polymerase chain reaction (PCR) tests. Cells on CS-containing scaffolds in the presence of dexamethasone showed the highest ALP production. PCR monitored an increase of osteoblastic markers. All scaffolds showed higher calcium deposition than cell-free controls. These results lead to the conclusion that a niche containing CS renders the differentiation of hMSCs toward osteoblastic cells more specific.
控制人间充质干细胞(hMSC)的分化并在植入前的工程构建物中提供组织功能是主要挑战。除了培养基中的添加剂外,支架内部的人工微环境也可起到这一作用。为了制备有利于hMSC成骨分化的微环境,将骨细胞外基质的成分固定在聚(3-羟基丁酸酯)织物上。将含有或不含有硫酸软骨素(CS)的纤维状牛I型胶原水凝胶固定在纺织支架上,该支架在冷冻干燥过程中进行亚结构构建并交联。从四名供体的骨髓中分离出hMSC。扩增后,将细胞动态接种到支架上。此后,将培养物转移到灌注血管中,并部分给予地塞米松以促进成骨分化。在为期4周的培养过程中,通过激光扫描显微镜(LSM)和扫描电子显微镜(SEM)对支架内细胞的分布和形态进行表征。通过分光光度法评估细胞活力(MTT)和碱性磷酸酶(ALP)的产生。用聚合酶链反应(PCR)试验阐明成骨细胞特异性标志物的转录情况。在存在地塞米松的情况下,含CS支架上的细胞显示出最高的ALP产生量。PCR监测到成骨标志物增加。所有支架的钙沉积均高于无细胞对照。这些结果得出结论,含有CS的微环境使hMSC向成骨细胞的分化更具特异性。