Biochemical and Biophysical Analyses of Tissue-Engineered Bone Obtained from Three-Dimensional Culture of a Subset of Bone Marrow Mesenchymal Stem Cells
Biochemical and Biophysical Analyses of Tissue-Engineered Bone Obtained from Three-Dimensional Culture of a Subset of Bone Marrow Mesenchymal Stem Cells
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DOI：
10.1089/ten.tea.2009.0750
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发表时间：
2010-12-01
影响因子：
4.1
通讯作者：
Curcio, Francesco
中科院分区：
文献类型：
--
作者：
Ferro, Federico;Falini, Giuseppe;Curcio, Francesco
组织工程骨移植是治疗大小骨缺损的一种很有前途的方法。由于侵入性、伦理问题、文化和感染的可能性，目前的方法通常不能被患者耐受。自体移植已被指出是解决这些问题的一种方法。由于组织的可利用性，许多人已经提出使用来源于骨髓的培养细胞在培养物中扩增并诱导在骨组织中分化。文献中报告的数据表明，确实可以在体外生产组织替代品，但关于体外生产的骨质量，结果并不总是一致的。在本研究中，我们研究了诱导人骨髓间充质干细胞向骨分化的聚集体中的骨形成。骨诱导后，我们采用傅立叶变换红外光谱，扫描电子显微镜，透射电子显微镜和X-射线粉末衍射的矿物基质的特点。从骨髓中获得细胞，使用RosetteSep对CD3、CD11b、CD14、CD16、CD19、CD56、CD66b和血型糖蛋白A进行免疫耗竭，并在新培养基配方中培养4代，然后使其形成自发聚集体。在聚集前增殖结束时，通过荧光激活细胞分选（FACS）分析细胞中常规用于表征扩增的间充质干细胞的标志物，发现对于CD29非常均一（99%+/-1%）、CD73（99%+/-1%）、CD90（95%+/-4%）、CD105（96%+/-4%）和CD133（0%+/-1%）表达。我们的研究结果表明，不仅聚集的细胞表达成骨分化的主要标志物，如骨钙素，骨粘连蛋白，骨桥蛋白，骨唾液蛋白，而且无机基质是由磷灰石的结构和形态相似的天然骨，即使没有支架。
Grafts of tissue-engineered bone represent a promising alternative in the treatment of large and small bone defects. Current approaches are often badly tolerated by patients because of invasiveness, ethical problems, culture, and possibility of infection. Autologous grafts have been indicated as a solution to such problems. Because of tissue availability, many have proposed the use of cultured cells derived from bone marrow expanded in culture and induced to differentiate in bone tissue. Data reported in the literature show that it is possible to produce tissue substitutes in vitro indeed, but results are not always concordant regarding the in vitro produced bone quality. In the present work, we investigated bone formation in aggregates of human bone marrow-derived mesenchymal stem cells induced to differentiate in bone. After osteoinduction we characterized the mineral matrix produced using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray powder diffraction. Cells were obtained from bone marrow, subjected to immunodepletion for CD3, CD11b, CD14, CD16, CD19, CD56, CD66b, and glycophorin A using RosetteSep and cultured in a new formulation of medium for four passages and then were allowed to form spontaneous aggregates. At the end of proliferation before aggregation, cells were analyzed by fluorescent activated cell sorting (FACS) for markers routinely used to characterize expanded mesenchymal stem cells and were found to be remarkably homogeneous for CD29 (99% +/- 1%), CD73 (99% +/- 1%), CD90 (95% +/- 4%), CD105 (96% +/- 4%), and CD133 (0% +/- 1%) expression. Our results show that not only aggregated cells express the major markers of osteogenic differentiation, such as osteocalcin, osteonectin, osteopontin, and bone sialoprotein, but also the inorganic matrix is made of an apatite structurally and morphologically similar to native bone even without a scaffold.