However, we didnt detect the percentage of the cells administered remain at the subsequent time points. matured extracellular matrix. Stromal cell-derived factor-1 (SDF-1) enhanced the migration of hMeSPCs, whereas AMD3100 abolished the chemotactic effects of SDF-1 on hMeSPCs, both in vitro and in vivo. In an experimental OA model, transplantation of hMeSPCs effectively protected articular cartilage, as evidenced by reduced expression of OA markers such as collagen I, collagen X, and hypoxia-inducible factor 2 but increased expression of collagen II. Our study demonstrated for the first time that intra-articular injection of hMeSPCs enhanced meniscus regeneration through the SDF-1/CXCR4 axis. Our study highlights a new strategy of intra-articular injection of hMeSPCs for meniscus regeneration. = 3 in each time point) at 1, 2, and 3 weeks postsurgery. The mRNA expression levels of SDF-1 within injured meniscus were then analyzed, as previously described . The primer sequences used in this study are listed in Table 1. In Vivo Chemotaxis and Loss-of-Function Assay One week after meniscectomy, 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-stained hMeSPCs (6 106 cells in 50 L phosphate-buffered saline [PBS], pretreated with 10 g/ml AMD3100 for 2 hours at 37C) were injected into the right knee. As control, the left knee was injected with normal hMeSPCs in PBS alone. Four weeks after operation, the Kodak in vivo FX small animal imaging system was used to evaluate the migration of injected hMeSPCs within the meniscus defect. This experiment was repeated three times. Meniscectomy and hMeSPC Injection Six female rats weighing 200C220 g were used in this Clidinium Bromide study. The rats were treated with cyclophosphamide (150 mg/kg) 24 hours before the meniscectomy. The anterior half of medial meniscus was removed at the level of the medial collateral to create a defect . hMeSPCs (6 106 in 50 l PBS) were injected into the right knee at 1 week and 2 weeks after meniscectomy, whereas the same volume of PBS was injected into the left knee as control. After euthanasia, three meniscuses of rats from each experimental group were subjected to histological evaluation at Clidinium Bromide the 4-week and 12-week time points. Treatment Rabbit Polyclonal to MAP4K3 of animals was in accordance with standard guidelines approved by the Zhejiang University Ethics Committee. Cell Labeling and Detection The hMeSPCs used for in situ repair of meniscus were prestained with DiI/6-carboxyfluorescein diacetate (CFDA). To evaluate the survival of implanted hMeSPCs in the meniscus defect, a noninvasive Kodak in vivo FX small animal imaging system was used Clidinium Bromide to analyze the samples at 4 and 12 weeks postmeniscectomy . The positively stained cells within histological sections of the hMeSPC-treated group were observed under fluorescence microscopy, whereas DAPI was used to stain the cell nuclei. Histology Hematoxylin and eosin and safranin O staining were performed, as described previously . Macroscopically, regeneration of the injured meniscus was evaluated by area assay, and the degeneration of femoral and tibial articular cartilage was evaluated directly after ink staining . Histological scoring was performed, as described previously . Briefly, four sections from each sample were graded blindly by three observers. Histology evaluation was performed using the International Cartilage Repair Society visual histological assessment scale, including surface, matrix, cell distribution, and depth. Transmission Electron Microscopy At 4 and 12 weeks postsurgery, tissue specimens from the hMeSPC-treated and control groups were fixed according to standard procedures for transmission electron microscopy (TEM) to assess the cell morphology of the regenerated meniscus . Immunostaining A series of 8-m-thick sections were used for immunohistochemical staining. Rabbit anti-Col1 (Anbo Biotechnology Co., San Francisco, CA, http://www.anbobio.com), mouse anti-Col2 (Calbiochem, San Diego, CA, http://www.emdbiosciences.com), rabbit anti-Col10 (Abcam, Cambridge, U.K. , http://www.abcam.com), and rabbit anti-Hif-2 (Abcam), together with goat anti-mouse (Beyotime) or goat anti-rabbit (Beyotime) secondary antibodies, were used to detect the expression of these proteins within the degenerated articular cartilage . Statistical Analysis All quantitative data sets are expressed as mean SD. Students test was performed to assess whether there were statistically significant differences in the results of different data sets, with a value of < .05 being considered significantly different. Results Characterization of hMeSPCs A subpopulation of meniscus cells attached and formed colonies 10C12 days after initial seeding (Fig. 1A). The colonies were heterogeneous in morphology at P0, possibly reflecting differences in cell origin from the meniscus tissue. A multipotent homogeneous population of MSC-like cells became apparent after further culture (Fig. 1B). These results confirmed the clonogenicity and multipotency of meniscus-derived cells in vitro, as previously reported . Flow cytometry results showed that the cells expressed high Clidinium Bromide levels of MSC markers, including CD44 (89.39%), CD90 (99.36%), CD105 (95.58%), and CD166 (96.23%), but not the hematopoietic markers CD34 (1.47%).