骨髓基质干细胞与软骨脱细胞基质多孔支架异位构建组织工程化软骨的实验研究

Fabrication a novel cartilage ECM-derived 3-D porous acellular matrix scaffold and in vivo cartilage tissue engineering with PKH26-1abeled chondrogenic bone marrow-derived mesenchymal stem cells

目的探讨骨髓基质于细胞（bone marrow mesenchymal stem cells，BMSCs）与软骨脱细胞基质多孔支架（cartilage ECM—derived porous scaffold，CEDPS）在裸鼠体内异位构建软骨的可行性，并建立利用PKH26荧光和分子荧光活体成像系统无创评估组织工程化细胞-支架复合体在体内生长情况的新方法。方法PKH26荧光标记成软骨诱导的BMSCs，接种入CEDPS支架，体外进行电镜、Dead／Live荧光染色观察，然后植入裸鼠背部，4周后利用分子荧光活体成像系统无创伤性评估组织工程化组织在裸鼠体内生长情况，取材进行组织学以及Ⅱ型胶原免疫荧光检测，与荧光图像比较。结果体外培养的BMSCs—CEDPS复合体电镜检查结果表明随着培养时间的增加，细胞在支架中增殖显著，细胞基质分泌增加，Dead／Live染色表明BMSCs在支架内部活性良好。4周后活体荧光示踪显示BMSCs在支架内生长良好，无扩散趋势，BMSCs—CEDPs复合体在裸鼠体内生成软骨样组织，番红“O”、甲苯胺蓝染色、Ⅱ型胶原免疫组化染色阳性，免疫荧光检查表明构建的软骨样组织内的细胞来源为接种的PKH26标记的BM—SCs。结论利用BMSCs和CEDPS支架能够在裸鼠皮下异位构建类软骨样组织。PKH26标记与分子荧光活体成像系统结合，能够无创伤性评估组织工程化组织的种子细胞在动物体内生长情况与转归。

Objective The objective of this study is non-destructive evaluation of tissue engineering constructs in vivo using PKH26 and molecular light imaging system, and exploring the feasibility for tissue engineering cartilage using canine chondrocytes and porous cartilage acellular matrix scaffold. Methods After induced by chondrogenic medium, BMSCs were seeded into the CEDPS scaffolds, cell attachment was confirmed by SEM and the viability of attached cells on the scaffold was confirmed by a live/dead assessment. Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds in vitro and implanted subcutaneously into nude mice. Then in vivo fluorescent imaging system was used for evaluating the cell-scaffold constructs. After 4 weeks, the constructs was analyzed by histology, immunohistochemistry and immunofluorescence examnation. Results SEM showed a large mount of extracellular matrix around the cells as time grown. Dead/Live staining in the confocal microscopy of cell-scaffold constructs revealed cells with green fluorescence （live cell）. Four weeks later, cartilage-like tissue formed in nude mice, with positive staining for Safranin O, tuoluidine blue and collagen II. Cells in the samples seemed to confirm that they originated from the labeled BMSCs, as confirmed by in vivo fluorescent imaging and immunofluorescence examination. Conclusion Cartilage ECM-derived scaffolds can be used for effective cartilage tissue engineering both in vitro and in vivo. As well, PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo.