Zhou Yefang, Chen Fulin, Ho Saey Tuan, Woodruff Maria Ann, Lim Tit Meng, Hutmacher Dietmar W
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 119260, Singapore.
Biomaterials. 2007 Feb;28(5):814-24. doi: 10.1016/j.biomaterials.2006.09.032. Epub 2006 Oct 11.
In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone-calcium phosphate (mPCL-CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5 x 5 x 4mm(3)) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10 x 10 x 4mm(3)) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.
在本研究中，将包含多层猪骨髓基质细胞（BMSC）的细胞片与由医用级聚己内酯 - 磷酸钙（mPCL - CaP）制成的完全相互连通的支架组装在一起，用于构建结构和功能骨移植体。从培养瓶中收获BMSC片，并将其包裹在预先接种的复合支架周围。分层的细胞片与支架/细胞构建体良好整合并保持活力，在长达8周的培养过程中，支架内外均可见矿化结节。构建体内的细胞经历了经典的体外成骨分化，伴随着碱性磷酸酶活性和骨相关蛋白表达的升高。在体内，将两组细胞片 - 支架/细胞构建体移植到裸鼠皮下。第一组装构建体（5×5×4mm³）由BMSC片组装而成，在植入前培养8周。第二组装构建体（10×10×4mm³）在与BMSC片组装后立即植入，无需进一步体外培养。对于两组，构建体内均形成了新皮质且血管化良好的松质骨，骨体积高达40%。组织学和免疫组织化学检查显示，新生骨组织由接种的BMSC形成，骨形成主要遵循软骨内途径，编织骨基质随后成熟为完全矿化的致密骨；呈现出天然骨的组织学特征。这些发现表明，利用BMSC片技术结合从机械、营养物质运输和血管化角度优化结构的复合支架，可以再生出与天然骨相似的大骨组织。
