Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

The Effect of the Plasma Treatment on PLGA Scaffold for Adhesion and Chondrogenic Differentiation of Human Adipose-derived Stromal Cells

High-density micromass culture was needed to take three dimensions culture with ASCs(adipose derived stromal cells) and chondrogenesis. However, the synthetic polymer has hydrophobic character and low affinity to cells and other biomolecules. Therefore, the surface modification without changes of physical and chemical properties is necessary for more suitable condition to cells and biomolecules. This study was performed to investigate the effect of surface modification of poly (lactic-co-glycolic acid)(PLGA) scaffold by plasma treatment (P(+)) on the adhesion, proliferation and chondrogenesis of ASCs, and not plasma treatment (P(-)). ASCs were isolated from human subcutaneous adipose tissue obtained by lipectomy and liposuction. At 1 hour 30 minutes and 3days after cell seeding onto the P(-) group and the P(+) group, total DNA amount of attached and proliferated ASCs markedly increased in the P(+) group (p < 0.05). The changes of the actin under confocal microscope were done for evaluation of cellular affinity, at 1 hour 30 minutes, the shape of the cells was spherical form in all group. At 3rd day, the shape of the cells was fiber network form and finely arranged in P(+) group rather than in P(-) group. RT-PCR analysis of cartilage-specific type II collagen and link protein were expressed in 1, 2 weeks of induction. Amount of Glycoaminoglycan (GAG) markedly increased in P(+) group(p < 0.05). In a week, extracellular matrix was not observed in the Alcian blue and Safranin O staining. However in 2 weeks, it was observed that sulfated proteoglycan increased in P(+) group rather than in P(-) group. In conclusion, we recognized that plasma treatment of PLGA scaffold could increase the hydrophilic property of cells, and provide suitable environment for high-density micromass culture to chondrogenesis.

The Effects of bFGF, VEGF and Micromass Culture on Proliferation and Differentiation of Human Chondrocytes

The acquisition of human chondrocytes for transplantation and cartilage coverage presents a major problem as these cells dedifferentiate rapidly during expansion in monolayer culture. Dedifferentiated chondrocytes change their shapes, metabolic states, and programs of matrix biosynthesis. We initiated this study on the basis of the hypothesis that bFGF, VEGF, and micromass culture can influence both the proliferation and their ability to express COL2A1 gene as a chondrogenic marker and Cbfa1 gene as an osteogenic marker. Chondrocytes in monolayer and micromass culture with or without bFGF and VEGF in vitro were collected and analyzed. In results, bFGF stimulated the proliferation of chondrocytes in monolayer culture. VEGF also stimulated the proliferation, but was less effective. The phenotype of chondrocytes was gradually changed in monolayer culture. Chondrocytes expanded in the presence of bFGF became dedifferentiated. However, dedifferentiated chondrocytes fully maintained their potential for redifferentiation in response to environmental changes. After transferring in micromass culture, chondrocytes which expanded with bFGF demonstrated high COL2A1 expression that was biochemically comparable to primary chondrocytes. Chondrocytes which expanded with VEGF demonstrated high Cbfa1 expression in both monolayer and micromass culture with passage times. This study provides that bFGF is needed to expand chondrocytes during tissue cultivation and additional three-dimensional environment is needed to maintain their differentiated phenotype. VEGF initiates the osteogenic potential during the chondrocyte expansion especially in micromass culture.

A study on chondrogenic potential in mandibular and limb bud mesenchymal cells of human embryos : A possible role of protein kinase C / 대한치과교정학회지

We have examined the in vitro stage-related chondrogenic potential of human mandibular and limb bud mesenchyme cells using micromass culture, Our results indicate that limb bud mesenchyme cells as early as stage 16 by Carnegie system (37 days), well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture, These results are correlated with stage-related chondrogenic potential of human limb bud in vivo as a result of Alcian blue staining. The proliferation of chondrogenic cells increased in the first 3 days after culture and then decreased. These results were correlated with the cell cycle analysis of which the number of G degrees/G1 phase increased markedly after 3 days of culture, while the percentage of cells in S phase was decreased, On the other hand, it was rarely differentiated in the mandible. We examined the effects of two PKC modulators such as phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, and staurosporine (STSN), an inhibitor of PKC. PMA inhibited the chondrogenesis, whereas STSN promoted the chondrogenesis in a dose dependent manner. In addition, PMA exerted no inhibitory effect when the cells were pretreated for 24 h with STSN, implying that the chondrogenic events might be settled at an early step in vitro and PKC may act as a negative modulator, Collectively, these results demonstrate, for the first time, the stage-related chondrogenic potential of human mandibular and limb bud mesenchyme cells and the role of PKC during chondrogenesis in vitro & in vivo.