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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

Coculture of SSCs and me-CH increases GAG formation and collagen biosynthesis. (a) Morphology of me-CH at passage 0 and passage 2. (b) Multilineage differentiation assay shows that SSCs are able to differentiate into osteoblast (osteogenic), adipocyte (adipogenic), and chondrocytes (chondrogenic). One representative donor is shown. (c) Alcian blue staining was performed at week 4 to detect GAGs. Scale bar = 100 μm. (d) Quantitative GAG assay shows more GAGs deposited in coculture aggregates than monocultures of either SSC or me-CH (n = 5) at week 4. Error bar reflects standard deviation. (e) Total collagen contents were measured by hydroxyproline assay. The amount of hydroxyproline was expressed as μg/μg of DNA. *P < 0.05. **P < 0.01. P values were calculated with one-way ANOVA followed by Dunnett's test.
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fig1: Coculture of SSCs and me-CH increases GAG formation and collagen biosynthesis. (a) Morphology of me-CH at passage 0 and passage 2. (b) Multilineage differentiation assay shows that SSCs are able to differentiate into osteoblast (osteogenic), adipocyte (adipogenic), and chondrocytes (chondrogenic). One representative donor is shown. (c) Alcian blue staining was performed at week 4 to detect GAGs. Scale bar = 100 μm. (d) Quantitative GAG assay shows more GAGs deposited in coculture aggregates than monocultures of either SSC or me-CH (n = 5) at week 4. Error bar reflects standard deviation. (e) Total collagen contents were measured by hydroxyproline assay. The amount of hydroxyproline was expressed as μg/μg of DNA. *P < 0.05. **P < 0.01. P values were calculated with one-way ANOVA followed by Dunnett's test.

Mentions: Upon isolation from meniscus, fibrochondrocytes showed two types of morphology. Some of them are round-shaped, while others are fibroblast-like. After a few passages, a homogenous morphology appears (Figure 1(a)). SSCs were then cultured in differentiation medium to test their multilineage differentiation potentials (Figure 1(b)). Passage 2 of me-CH was used to coculture with SSC (passage 2). Monoculture of me-CH or SSC was used as control. All cell pellets were cultured in SF medium (serum free medium containing 1% L-glutamine, 0.2 mM ascorbic acid, 100 U/mL penicillin, and 10 μg/mL streptomycin). At week 4 after culture, pellets were harvested for histology, GAG quantification, and hydroxyproline assay. Alcian blue staining on paraffin sections showed evidence of sulfate GAG deposition in coculture and me-CH pellets (Figure 1(c)). Positively stained area retained typical morphology of fibrocartilage with chondrocytes emended long fibers. Meanwhile, barely any GAGs were detected in SSCs only pellets. As shown in Figure 1(d), GAGs in coculture pellets are about 5-fold more than the monoculture of SSC and 20% less than me-CH only pellets. This suggested that in coculture of two cells types, 50% of fibrochondrocytes produced 80% of GAGs compared to monoculture of me-CH in a 3-dimensional culture environment.


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)

Coculture of SSCs and me-CH increases GAG formation and collagen biosynthesis. (a) Morphology of me-CH at passage 0 and passage 2. (b) Multilineage differentiation assay shows that SSCs are able to differentiate into osteoblast (osteogenic), adipocyte (adipogenic), and chondrocytes (chondrogenic). One representative donor is shown. (c) Alcian blue staining was performed at week 4 to detect GAGs. Scale bar = 100 μm. (d) Quantitative GAG assay shows more GAGs deposited in coculture aggregates than monocultures of either SSC or me-CH (n = 5) at week 4. Error bar reflects standard deviation. (e) Total collagen contents were measured by hydroxyproline assay. The amount of hydroxyproline was expressed as μg/μg of DNA. *P < 0.05. **P < 0.01. P values were calculated with one-way ANOVA followed by Dunnett's test.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4379431&req=5

fig1: Coculture of SSCs and me-CH increases GAG formation and collagen biosynthesis. (a) Morphology of me-CH at passage 0 and passage 2. (b) Multilineage differentiation assay shows that SSCs are able to differentiate into osteoblast (osteogenic), adipocyte (adipogenic), and chondrocytes (chondrogenic). One representative donor is shown. (c) Alcian blue staining was performed at week 4 to detect GAGs. Scale bar = 100 μm. (d) Quantitative GAG assay shows more GAGs deposited in coculture aggregates than monocultures of either SSC or me-CH (n = 5) at week 4. Error bar reflects standard deviation. (e) Total collagen contents were measured by hydroxyproline assay. The amount of hydroxyproline was expressed as μg/μg of DNA. *P < 0.05. **P < 0.01. P values were calculated with one-way ANOVA followed by Dunnett's test.
Mentions: Upon isolation from meniscus, fibrochondrocytes showed two types of morphology. Some of them are round-shaped, while others are fibroblast-like. After a few passages, a homogenous morphology appears (Figure 1(a)). SSCs were then cultured in differentiation medium to test their multilineage differentiation potentials (Figure 1(b)). Passage 2 of me-CH was used to coculture with SSC (passage 2). Monoculture of me-CH or SSC was used as control. All cell pellets were cultured in SF medium (serum free medium containing 1% L-glutamine, 0.2 mM ascorbic acid, 100 U/mL penicillin, and 10 μg/mL streptomycin). At week 4 after culture, pellets were harvested for histology, GAG quantification, and hydroxyproline assay. Alcian blue staining on paraffin sections showed evidence of sulfate GAG deposition in coculture and me-CH pellets (Figure 1(c)). Positively stained area retained typical morphology of fibrocartilage with chondrocytes emended long fibers. Meanwhile, barely any GAGs were detected in SSCs only pellets. As shown in Figure 1(d), GAGs in coculture pellets are about 5-fold more than the monoculture of SSC and 20% less than me-CH only pellets. This suggested that in coculture of two cells types, 50% of fibrochondrocytes produced 80% of GAGs compared to monoculture of me-CH in a 3-dimensional culture environment.

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