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Background <p>Bone marrow–derived mesenchymal stem cells (MSCs) represent a promising source for cell-based regenerative cartilage therapies. However, in vitro chondrogenic differentiation of MSCs varies widely between donors, posing a significant challenge to the clinical translation of these therapies. Here, we explored the potential of pre-conditioning MSCs with TGF-β1 during monolayer expansion in order to enhance chondrogenic differentiation in poorly responsive MSCs and reduce donor-to-donor variability.</p> Methods <p>MSCs from six donors were expanded in monolayer using standard methods. For the final two days of expansion, half of the cells were pre-conditioned with 10 ng/mL TGF-β1, whereas the others were kept in standard expansion medium. MSCs were then detached for 28 days of chondrogenic differentiation in standard pellet culture in chondrogenic medium or in a 3D mechanically induced chondrogenesis model in polyurethane-fibrin scaffolds. After 28 days, samples were collected for gene expression analysis, sulphated glycosaminoglycan (sGAG) assay, DNA quantification and histological evaluations (Safranin-O/Fast green staining).</p> Results <p>Two days of TGF-β1 pre-conditioning during MSC expansion increased <i>COL2A1</i> expression after 28 days of pellet culture (<i>P</i> = 0.0217), but also elevated <i>COL10A1</i> (<i>P</i> = 0.0293), leaving the <i>COL2A1/COL10A1</i> ratio unchanged (<i>P</i> = 0.8522). There was a trend for an increased <i>COL2A1/COL1A1</i> ratio and an increased <i>ACAN</i> expression (<i>P</i> = 0.0837). Pre-conditioning had no effect on sGAGs retained in the pellets or released into the medium over the 28 days but slightly enhanced the Safranin-O staining on histological sections. When chondrogenesis was mechanically induced without exogenous TGF-β1, pre-conditioning had no effect on <i>COL2A1</i>, <i>COL10A1</i>, <i>COL1A1</i>, or <i>ACAN</i> gene expression. Similarly, no differences were observed in sGAG content (retained or released) or in Safranin-O staining.</p> Conclusions <p>TGF-β1 pre-conditioning enhanced some chondrogenic markers in static pellet culture, but these effects were small and did not translate to the more clinically relevant model of mechanically induced chondrogenesis. Furthermore, TGF-β1 pre-conditioning was associated with increased <i>COL10A1</i> expression, indicating a concurrent promotion of hypertrophic differentiation.</p>

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Modulating mesenchymal stromal cells chondrogenesis through TGF-β1 pre-conditioning during monolayer expansion in pellet culture and in a 3D mechanical load model

  • 期刊论文

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  • 发表日期：

    2026年7月30日

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  • Maja Schlittler,
  • Guoliang Chen,
  • Lauma Ievina,
  • Laura Mecchi,
  • Franziska L. Breulmann,
  • Martin J. Stoddart,
  • Elena Della Bella

摘要

Background

Bone marrow–derived mesenchymal stem cells (MSCs) represent a promising source for cell-based regenerative cartilage therapies. However, in vitro chondrogenic differentiation of MSCs varies widely between donors, posing a significant challenge to the clinical translation of these therapies. Here, we explored the potential of pre-conditioning MSCs with TGF-β1 during monolayer expansion in order to enhance chondrogenic differentiation in poorly responsive MSCs and reduce donor-to-donor variability.

Methods

MSCs from six donors were expanded in monolayer using standard methods. For the final two days of expansion, half of the cells were pre-conditioned with 10 ng/mL TGF-β1, whereas the others were kept in standard expansion medium. MSCs were then detached for 28 days of chondrogenic differentiation in standard pellet culture in chondrogenic medium or in a 3D mechanically induced chondrogenesis model in polyurethane-fibrin scaffolds. After 28 days, samples were collected for gene expression analysis, sulphated glycosaminoglycan (sGAG) assay, DNA quantification and histological evaluations (Safranin-O/Fast green staining).

Results

Two days of TGF-β1 pre-conditioning during MSC expansion increased COL2A1 expression after 28 days of pellet culture (P = 0.0217), but also elevated COL10A1 (P = 0.0293), leaving the COL2A1/COL10A1 ratio unchanged (P = 0.8522). There was a trend for an increased COL2A1/COL1A1 ratio and an increased ACAN expression (P = 0.0837). Pre-conditioning had no effect on sGAGs retained in the pellets or released into the medium over the 28 days but slightly enhanced the Safranin-O staining on histological sections. When chondrogenesis was mechanically induced without exogenous TGF-β1, pre-conditioning had no effect on COL2A1, COL10A1, COL1A1, or ACAN gene expression. Similarly, no differences were observed in sGAG content (retained or released) or in Safranin-O staining.

Conclusions

TGF-β1 pre-conditioning enhanced some chondrogenic markers in static pellet culture, but these effects were small and did not translate to the more clinically relevant model of mechanically induced chondrogenesis. Furthermore, TGF-β1 pre-conditioning was associated with increased COL10A1 expression, indicating a concurrent promotion of hypertrophic differentiation.