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Beneficial effects of coculturing synovial derived mesenchymal stem cells with meniscus fibrochondrocytes are mediated by fibroblast growth factor 1: increased proliferation and collagen synthesis.

Song X, Xie Y, Liu Y, Shao M, Wang W - Stem Cells Int (2015)

Bottom Line: Western blot showed that conditioned medium of SSCs can activate mitogen-activated protein kinase (MAPK) signaling pathways by increasing the phosphorylation of mitogen-activated regulated protein kinase 1/2 (MEK) and extracellular-signal-regulated kinases 1/2 (ERK).Overall, this study provided evidence that synovial MSCs can support proliferation and collagen synthesis of fibrochondrocytes, by secreting FGF1.Coimplantation of SSC and me-CH could be a useful strategy for reconstructing meniscus.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China.

ABSTRACT
Meniscus reconstruction is in great need for orthopedic surgeons. Meniscal fibrochondrocytes transplantation was proposed to regenerate functional meniscus, with limited donor supply. We hypothesized that coculture of synovial mesenchymal stem cells (SSC) with meniscal fibrochondrocytes (me-CH) can support matrix production of me-CH, thus reducing the number of me-CH needed for meniscus reconstruction. A pellet coculture system of human SSC and me-CH was used in this study. Enhanced glycosaminoglycans (GAG) in coculture pellets were demonstrated by Alcian blue staining and GAG quantification, when compared to monoculture. More collagen synthesis was shown in coculture pellets by hydroxyproline assay. Increased proliferation of me-CH was observed in coculture. Data from BrdU staining and ELISA demonstrated that conditioned medium of SSCs enhanced the proliferation and collagen synthesis of me-CH, and this effect was blocked by neutralizing antibody against fibroblast growth factor 1 (FGF1). Western blot showed that conditioned medium of SSCs can activate mitogen-activated protein kinase (MAPK) signaling pathways by increasing the phosphorylation of mitogen-activated regulated protein kinase 1/2 (MEK) and extracellular-signal-regulated kinases 1/2 (ERK). Overall, this study provided evidence that synovial MSCs can support proliferation and collagen synthesis of fibrochondrocytes, by secreting FGF1. Coimplantation of SSC and me-CH could be a useful strategy for reconstructing meniscus.

No MeSH data available.


Related in: MedlinePlus

Conditioned medium of SSCs increases proliferation of me-CH through FGF1 signaling pathway. (a) BrdU was stained for proliferating cells at day 3 after forming of aggregates. Pellets of me-CH were cultured in SF medium (serum free medium plus 5 μg/mL normal goat IgG), Con medium (conditioned medium plus 5 μg/mL normal goat IgG), or Con medium + anti-FGF1 (conditioned medium plus 5 μg/mL of goat antibody against FGF1). Positive cells are shown in red, indicated by white arrowheads. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (b) BrdU positive cells were quantified (N = 3). Data is shown as mean + standard deviation. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's test. *P < 0.05. **P < 0.01. (c) Immunofluorescent staining for FGF1 is performed on SSCs (passage 2). Red fluorescence shows positive staining of FGF1. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.
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fig3: Conditioned medium of SSCs increases proliferation of me-CH through FGF1 signaling pathway. (a) BrdU was stained for proliferating cells at day 3 after forming of aggregates. Pellets of me-CH were cultured in SF medium (serum free medium plus 5 μg/mL normal goat IgG), Con medium (conditioned medium plus 5 μg/mL normal goat IgG), or Con medium + anti-FGF1 (conditioned medium plus 5 μg/mL of goat antibody against FGF1). Positive cells are shown in red, indicated by white arrowheads. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (b) BrdU positive cells were quantified (N = 3). Data is shown as mean + standard deviation. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's test. *P < 0.05. **P < 0.01. (c) Immunofluorescent staining for FGF1 is performed on SSCs (passage 2). Red fluorescence shows positive staining of FGF1. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.

Mentions: To study the mechanism how SSCs increase proliferation of me-CH in coculture, conditioned medium of SSCs was collected and used to culture me-CH pellets. An FGF1 neutralizing antibody (5 μg/mL) was added to Con medium to neutralize active FGF1, since it is reported that FGF1 can be secreted by mesenchymal stem cells to stimulate the proliferation of chondrocytes [25]. Normal goat IgG (5 μg/mL) was added to SF medium and Con medium to eliminate the effects of unspecific binding of IgG. As shown in Figure 3(a), very few me-CH are BrdU positive, while much more positive cells were seen on the periphery of pellets cultured in Con medium. Stimulatory effects of Con medium on me-CH disappeared after neutralization of anti-FGF1 antibody. Quantification of BrdU positive cells in all conditions confirmed our impression on the proliferating cells (Figure 3(b)). Increase of BrdU positive cells in Con medium is significant when compared to SF medium and Con medium plus anti-FGF1. Expression of FGF1 by SSCs was evidenced by immunofluorescent staining on passage 2 cells (Figure 3(c)).


Beneficial effects of coculturing synovial derived mesenchymal stem cells with meniscus fibrochondrocytes are mediated by fibroblast growth factor 1: increased proliferation and collagen synthesis.

Song X, Xie Y, Liu Y, Shao M, Wang W - Stem Cells Int (2015)

Conditioned medium of SSCs increases proliferation of me-CH through FGF1 signaling pathway. (a) BrdU was stained for proliferating cells at day 3 after forming of aggregates. Pellets of me-CH were cultured in SF medium (serum free medium plus 5 μg/mL normal goat IgG), Con medium (conditioned medium plus 5 μg/mL normal goat IgG), or Con medium + anti-FGF1 (conditioned medium plus 5 μg/mL of goat antibody against FGF1). Positive cells are shown in red, indicated by white arrowheads. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (b) BrdU positive cells were quantified (N = 3). Data is shown as mean + standard deviation. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's test. *P < 0.05. **P < 0.01. (c) Immunofluorescent staining for FGF1 is performed on SSCs (passage 2). Red fluorescence shows positive staining of FGF1. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4379431&req=5

fig3: Conditioned medium of SSCs increases proliferation of me-CH through FGF1 signaling pathway. (a) BrdU was stained for proliferating cells at day 3 after forming of aggregates. Pellets of me-CH were cultured in SF medium (serum free medium plus 5 μg/mL normal goat IgG), Con medium (conditioned medium plus 5 μg/mL normal goat IgG), or Con medium + anti-FGF1 (conditioned medium plus 5 μg/mL of goat antibody against FGF1). Positive cells are shown in red, indicated by white arrowheads. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (b) BrdU positive cells were quantified (N = 3). Data is shown as mean + standard deviation. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's test. *P < 0.05. **P < 0.01. (c) Immunofluorescent staining for FGF1 is performed on SSCs (passage 2). Red fluorescence shows positive staining of FGF1. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.
Mentions: To study the mechanism how SSCs increase proliferation of me-CH in coculture, conditioned medium of SSCs was collected and used to culture me-CH pellets. An FGF1 neutralizing antibody (5 μg/mL) was added to Con medium to neutralize active FGF1, since it is reported that FGF1 can be secreted by mesenchymal stem cells to stimulate the proliferation of chondrocytes [25]. Normal goat IgG (5 μg/mL) was added to SF medium and Con medium to eliminate the effects of unspecific binding of IgG. As shown in Figure 3(a), very few me-CH are BrdU positive, while much more positive cells were seen on the periphery of pellets cultured in Con medium. Stimulatory effects of Con medium on me-CH disappeared after neutralization of anti-FGF1 antibody. Quantification of BrdU positive cells in all conditions confirmed our impression on the proliferating cells (Figure 3(b)). Increase of BrdU positive cells in Con medium is significant when compared to SF medium and Con medium plus anti-FGF1. Expression of FGF1 by SSCs was evidenced by immunofluorescent staining on passage 2 cells (Figure 3(c)).

Bottom Line: Western blot showed that conditioned medium of SSCs can activate mitogen-activated protein kinase (MAPK) signaling pathways by increasing the phosphorylation of mitogen-activated regulated protein kinase 1/2 (MEK) and extracellular-signal-regulated kinases 1/2 (ERK).Overall, this study provided evidence that synovial MSCs can support proliferation and collagen synthesis of fibrochondrocytes, by secreting FGF1.Coimplantation of SSC and me-CH could be a useful strategy for reconstructing meniscus.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China.

ABSTRACT
Meniscus reconstruction is in great need for orthopedic surgeons. Meniscal fibrochondrocytes transplantation was proposed to regenerate functional meniscus, with limited donor supply. We hypothesized that coculture of synovial mesenchymal stem cells (SSC) with meniscal fibrochondrocytes (me-CH) can support matrix production of me-CH, thus reducing the number of me-CH needed for meniscus reconstruction. A pellet coculture system of human SSC and me-CH was used in this study. Enhanced glycosaminoglycans (GAG) in coculture pellets were demonstrated by Alcian blue staining and GAG quantification, when compared to monoculture. More collagen synthesis was shown in coculture pellets by hydroxyproline assay. Increased proliferation of me-CH was observed in coculture. Data from BrdU staining and ELISA demonstrated that conditioned medium of SSCs enhanced the proliferation and collagen synthesis of me-CH, and this effect was blocked by neutralizing antibody against fibroblast growth factor 1 (FGF1). Western blot showed that conditioned medium of SSCs can activate mitogen-activated protein kinase (MAPK) signaling pathways by increasing the phosphorylation of mitogen-activated regulated protein kinase 1/2 (MEK) and extracellular-signal-regulated kinases 1/2 (ERK). Overall, this study provided evidence that synovial MSCs can support proliferation and collagen synthesis of fibrochondrocytes, by secreting FGF1. Coimplantation of SSC and me-CH could be a useful strategy for reconstructing meniscus.

No MeSH data available.


Related in: MedlinePlus