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Background <p>Endochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin–polyethylene glycol (PEG) hydrogels loaded with transforming growth factor TGF-β instructed stable chondral MSC development in&#xa0;vivo. Here, we assessed this approach in&#xa0;vitro, utilizing heparin–PEG hydrogels or the pellet culture system with soluble heparin supplementation of chondrogenic medium.</p> Methods <p>Human MSCs were cultured in heparin–PEG hydrogels (22.4&#xa0;mg/mL crosslinked heparin, 120&#xa0;ng TGF-β1) or as pellet cultures treated with soluble heparin (0, 10, 100, 700&#xa0;μg/mL) in TGF-β1-containing (10&#xa0;ng/mL) chondrogenic medium. Chondral and endochondral signaling (1–3&#xa0;h, 4&#xa0;weeks) and cartilage matrix formation (4&#xa0;weeks) were analyzed using western blot, histology, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and enzyme activity.</p> Results <p>Unlike in&#xa0;vivo, human MSCs differentiated in heparin–PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in&#xa0;vitro. Interestingly, treating MSC pellets with soluble heparin (10–700&#xa0;µg/mL) revealed reduced TGF-β-SMAD3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation, which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/β-catenin pathway in&#xa0;vitro but insufficiently silenced TGF-β-SMAD1/5/9 activation and unfortunately reduced antihypertrophic prostaglandin PGE2 levels. Ultimately, treatment with 10&#xa0;µg/mL heparin reduced the upregulation of several hypertrophy markers (<i>MEF2C</i>, <i>IHH</i>, <i>IBSP</i> messenger RNAs [mRNAs], alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous antihypertrophic interventions (PTHrP pulses, WNT inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation.</p> Conclusions <p>Taken together, heparin-TGF-β showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic differentiation and context-dependent lineage-instructive properties—permitting endochondral commitment in&#xa0;vitro but chondral development in&#xa0;vivo. Thus, environmental contributions are mandatory to allow heparin–PEG-guided chondral versus endochondral lineage commitment of MSCs in&#xa0;vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources.</p> Graphical abstract <p></p>

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Dichotomous SMAD2/3 regulation and selective antihypertrophic activity of heparin during in vitro chondrogenesis of mesenchymal stromal cells

  • 期刊论文

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  • 发表日期：

    2026年3月17日

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  • Sven Schmidt,
  • Safak Chasan,
  • Helen F. Dietmar,
  • Felicia A. M. Klampfleuthner,
  • Eliane Hesse,
  • Tilman Walker,
  • Uwe Freudenberg,
  • Wiltrud Richter,
  • Solvig Diederichs

摘要

Background

Endochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin–polyethylene glycol (PEG) hydrogels loaded with transforming growth factor TGF-β instructed stable chondral MSC development in vivo. Here, we assessed this approach in vitro, utilizing heparin–PEG hydrogels or the pellet culture system with soluble heparin supplementation of chondrogenic medium.

Methods

Human MSCs were cultured in heparin–PEG hydrogels (22.4 mg/mL crosslinked heparin, 120 ng TGF-β1) or as pellet cultures treated with soluble heparin (0, 10, 100, 700 μg/mL) in TGF-β1-containing (10 ng/mL) chondrogenic medium. Chondral and endochondral signaling (1–3 h, 4 weeks) and cartilage matrix formation (4 weeks) were analyzed using western blot, histology, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and enzyme activity.

Results

Unlike in vivo, human MSCs differentiated in heparin–PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treating MSC pellets with soluble heparin (10–700 µg/mL) revealed reduced TGF-β-SMAD3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation, which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/β-catenin pathway in vitro but insufficiently silenced TGF-β-SMAD1/5/9 activation and unfortunately reduced antihypertrophic prostaglandin PGE2 levels. Ultimately, treatment with 10 µg/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP messenger RNAs [mRNAs], alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous antihypertrophic interventions (PTHrP pulses, WNT inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation.

Conclusions

Taken together, heparin-TGF-β showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic differentiation and context-dependent lineage-instructive properties—permitting endochondral commitment in vitro but chondral development in vivo. Thus, environmental contributions are mandatory to allow heparin–PEG-guided chondral versus endochondral lineage commitment of MSCs in vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources.

Graphical abstract