Heparin-based microparticles for temporally controlled protein sequestration to modulate chondrocytic differentiation

摘要

Introduction: Cartilage injuries have limited healing capacity, and strategies for mesenchymal stem cell (MSC) therapies have multiple shortcomings, including difficulty In preventing hypertrophy during chondrogenesis. Thus, we employed heparin microparticles (MPs) as a means to potentially sequester endogenous growth factors away from cells and thereby reduce/inhibit cellular differentiation. Specifically, we hypothesized that heparin MPs could temporally delay differentiation in the model chondrocytic progenitor ATDC5 cell line. In addition, we hypothesized that core-shell heparin-poly(ethylene-glycol) (PEG) microparticles, in which heparin MPs are surrounded by a PEG shell, could further temporally modulate protein binding by delaying heparin mediated sequestration due to diffusion limitations through the PEG shell. Materials and Methods: Heparin methacrylamide MPs and PEG-diacrylate (DA) MPs were fabricated via water-in-oil or water-in-water emulsion. Core-shell heparin-PEG MPs were fabricated by suspending heparin MPs in a PEG-DA solution. 700 cell ATDC5 spheroids containing high and low levels of heparin or PEG MPs were formed via aggregation in agarose wells and cultured 18 days on orbital rotary culture. Core-shell MPs, heparin MPs, PEG MPs, and a blank control were incubated with human SDF-1 or BMP-2 and pull-down was assessed via ELISA after 2 and 24 hours on rotary at 4°C. Results and Discussion: In spheroid culture with heparin MPs, ATDC5 differentiation was delayed in dose-dependent manner. At days 12 and 18, glycosaminoglycan deposition was reduced as heparin MP dosage increased, as opposed to PEG MP controls (Fig 1 A). Chondrocytic gene expression markers were significantly down-regulated at early timepoints in high heparin MP group compared to no MP groups, a trend not observed in PEG MP groups (Fig 18). A core-shell MP technology was developed and fabrication was confirmed by confocal microscopy (Fig 2A). In protein pull-down studies, SDF-1 (7 kDa) was sequestered by core-shell MPs after 2 hours (Fig 2B), while BMP-2 (15 kDa) was only sequestered after 24 hours (Fig 2C), suggesting that core-shell MPs can temporally modulate sequestration. Conclusion: Heparin MPs delayed differentiation of ATDC5 cell spheroids in a dose-dependent manner, a trend not seen in the PEG MP groups, suggesting that growth factor sequestration is the mechanism behind this phenomenon. Furthermore, core-shell heparin-PEG MPs have the potential to temporally modulate growth factor sequestration. In core-shell MPs, BMP-2 (15 kDa) sequestration was temporally delayed while SDF-1 (7 kDa) sequestration was not, likely due to diffusion limitations of the larger BMP-2 protein through the PEG shell. Overall, these heparin-based MP technologies have the potential to temporally modulate chondrocytic differentiation and subsequent hypertrophy in therapeutic cell types such as MSCs by sequestering endogenous growth factors, a novel strategy for tissue regeneration.