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

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.