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bioRxiv · 10.1101/2025.09.02.673657

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

Schmidt, S.·Chasan, S.·Dietmar, H. F.·Klampfleuthner, F. A. M.·Walker, T.·Freudenberg, U.·Richter, W.·Diederichs, S.
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Abstract

BackgroundEndochondral 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 beta (TGF-{beta}) instructed stable chondral MSC development in vivo. We here assessed this approach in vitro, utilizing heparin-PEG hydrogels or soluble heparin supplementation of chondrogenic medium. MethodsHuman MSCs were cultured in heparin-PEG hydrogels (22.4 mg/mL crosslinked heparin, 120ng TGF-{beta}1) or as hydrogel-free pellet cultures treated with soluble heparin (0, 10, 100, 700 g/mL) in TGF-{beta}1-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, qPCR, ELISA, and enzyme activity. ResultsUnlike in vivo, human MSCs differentiated in heparin-PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treatment with soluble heparin (10-700 {micro}g/mL) revealed reduced TGF-{beta}-small mother against decapentaplegic (SMAD)3 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/{beta}-catenin pathway in vitro but insufficiently silenced TGF-{beta}-SMAD1/5/9 activation and unfortunately reduced anti-hypertrophic prostaglandin E2 (PGE2) levels. Ultimately, treatment with 10 {micro}g/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP mRNAs, alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous anti-hypertrophic interventions (parathyroid-hormone related protein (PTHrP)-pulses, wingless-int (WNT)-inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation. ConclusionsTaken together, heparin-TGF-{beta} showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic power and context-dependent lineage-instructive properties: permitting endochondral differentiation 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/673657v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@140313aorg.highwire.dtl.DTLVardef@160049dorg.highwire.dtl.DTLVardef@cee22corg.highwire.dtl.DTLVardef@62e961_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

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Schmidt, S., Chasan, S., Dietmar, H. F., Klampfleuthner, F. A. M., Walker, T., Freudenberg, U., Richter, W., Diederichs, S.. 2025-09-02. Dichotomous SMAD2/3 regulation and selective anti-hypertrophic activity of heparin during in vitro chondrogenesis of mesenchymal stromal cells. https://doi.org/10.1101/2025.09.02.673657

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