Combined effects of TGFbeta1 and BMP2 in serum-free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation.
This study investigated the effects of TGFbeta1, BMP2 or a combination of both on the chondrogenic differentiation of mesenchymal stem cells (MSCs) in a serum-free micromass culture system in vitro. Putative MSCs harvested from the iliac crest of 4-5 month old New Zealand White Rabbits were expanded and cultured in three-dimensional high density micromass aggregate cultures containing TGFbeta1, BMP2 or a combination of both, in the absence of serum. After 14-20 days of culture, chondrogenic differentiation of the MSCs was assayed by toluidine blue staining, immunohistochemistry and semi-quantitative RT-RCR of type I collagen (CI) and type II collagen (CII). Quantitative measurements of cell proliferation and sulfated glycosaminoglycan (s-GAG) were also carried out to assess the growth rate and matrix deposition of the cultured aggregates. Both immunohistochemical staining and semi-quantitative RT-PCR showed that the combination of BMP2 and TGFbeta1 resulted in a marked enhancement of collagen II synthesis, with minimal collagen I expression, which would suggest hyaline-like cartilage formation. Additionally, BMP2 and TGFbeta1 had a synergistic effect on matrix proteoglycan deposition, as assessed by metachromatic toluidine blue staining. This is consistent with the quantitative measurement of glycosaminoglycans, whereby a significant increase in GAG/DNA was noted in the co-treatment group. Hence, it can be concluded that the combination of BMP2 and TGFbeta1 has a synergistic effect on the differentiation of MSC into hyaline-like cartilage tissue.
Toh
Wei Seong
WS
Faculty of Dentistry, National University of Singapore, Stem Cell Laboratory, Singapore.
Liu
Hua
H
Heng
Boon Chin
BC
Rufaihah
Abdul Jalil
AJ
Ye
Chao Peng
CP
Cao
Tong
T
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D020219:Chondrogenesis / Q000187:drug effects*; D004357:Drug Synergism; D006025:Glycosaminoglycans / Q000096:biosynthesis; D051457:Hyaline Cartilage / Q000166:cytology*; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D006025:Glycosaminoglycans; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C013786:A73025