Hypertrophy in Mesenchymal Stem Cell Chondrogenesis: Effect of TGF-β Isoforms and Chondrogenic Conditioning

Abstract

Induction of chondrogenesis in mesenchymal stem cells (MSCs) with TGF-β leads to a hypertrophic phenotype. The hypertrophic maturation of the chondrocytes is dependent on the timed removal of TGF-β and sensitive to hypertrophy-promoting agents in vitro. In this study, we have investigated whether TGF-β3, which has been shown to be more prochondrogenic compared to TGF-β1, similarly enhances terminal differentiation in an in vitro hypertrophy model of chondrogenically differentiating MSCs. In addition, we tested the impact of the time of chondrogenic conditioning on the enhancement of hypertrophy. MSCs were chondrogenically differentiated in pellet culture in medium containing TGF-β1 or TGF-β3. After 2 or 4 weeks, chondrogenic medium was switched to hypertrophy-inducing medium for 2 weeks. Aggregates were analyzed histologically and biochemically on days 14, 28 and 42. The switch to hypertrophy medium after 14 days induced hypertrophic cell morphology and significant increase in alkaline phosphatase activity compared to the chondrogenesis only control using both TGF-β1 and TGF-β3. After 28 days predifferentiation, differences between hypertrophic and control groups diminished compared to 14 days predifferentiation. In conclusion, chondrogenic conditioning with both TGF-β isoforms similarly induced hypertrophy in our experiment and allowed the enhancement of the hypertrophic chondrocyte phenotype by hypertrophic medium. Enhancement of hypertrophy was seen more clearly after the shorter chondrogenic conditioning. Therefore, to utilize this experimental model as a tool to study hypertrophy in MSC chondrogenesis, a predifferentiation period of 14 days is recommended.

Effect of parathyroid hormone-related protein in an in vitro hypertrophy model for mesenchymal stem cell chondrogenesis

“…These cells are not hypertrophic chondrogenic cells because they do not express type II or type X collagen. The extracellular matrix in this area of the aggregates is known to contain type I collagen [2] and we have seen this in previous experiments [6]. We speculate that these cells express a more bone-like than a hypertrophic chondrocyte-like phenotype.…”

“…Pellets were digested in Sigma papain digestion solution [150 μg/ml in phosphate-buffered saline (PBS), 6 mM cysteine HCl, 6 mM ethylenediaminetetraacetic acid (EDTA), pH 6.0] at 60°C overnight. Four samples per condition were prepared.…”

“…During chondrogenesis, MSCs express markers of hypertrophic chondrocytes like type X collagen, alkaline phosphatase (ALP) and others [4][5][6]. A wide array of additional genes is regulated, stage specifically, in a manner very similar to the profiles seen during embryonic limb development [5].…”

Cartilage-Bone Interface Features, Scaffold and Cell Options for Regeneration

“…In the category of stem/progenitor cells, bone marrow derived stem cells, adipose derived stem cells, synovial stem cells and embryonic stem cells were demonstrated to be appropriate cell choices for osteochondral regeneration [24,25]. Specific differentiation of these cells into proper lineages is achieved by incorporating relevant biomolecules into scaffolds in a specially controlled manner.…”

The role of growth factors in stem cell-directed chondrogenesis: a real hope for damaged cartilage regeneration

“…The TGF-β superfamily members regulate cell proliferation, differentiation and apoptosis during embryogenesis. They include the secretory isoforms: TGF-β1, -β2 and -β3, with TGF-β3 having the highest chondrogenic potential of all isoforms, and their action results in rapid cell differentiation [21]. The TGF-βs and BMPs bind to the extracellular domains of specific receptors containing serine/threonine kinase activity in their intracellular domains, and require Sma and Mad related (SMAD) proteins for signal transduction within the cells (Fig.…”