The enhancement of chondrogenic differentiation of human mesenchymal stem cells by enzymatically regulated RGD functionalities

Abstract

A thiol-acrylate photopolymerization was used to incorporate enzymatically cleavable peptide sequences into PEG hydrogels to induce chondrogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs). An adhesive sequence, RGD, was designed with an MMP-13 specific cleavable linker. RGD promotes survival of hMSCs encapsulated in PEG gels and has shown to induce early stages of chondrogenesis, while its persistence can limit complete differentiation. Therefore, an MMP-13 cleavage site was incorporated into the peptide sequence to release RGD mimicking the native differentiation timeline. Active MMP-13 production of encapsulated hMSCs was seen to increase from day 9 to 14 and only in chondrogenic differentiating cultures. Seeded hMSCs attached to the material prior to enzymatic cleavage, but a significant population of the cells detach after cleavage and release of RGD. Finally, hMSCs encapsulated in RGD-releasing gels produce 10 times as much glycosaminoglycan as cells with uncleavable RGD functionalities, by day 21 of culture. Furthermore, 75% of the cells stain positive for collagen type II deposition where RGD is cleavable, as compared to 19% for cultures where RGD persists. Collectively, these data provide evidence that temporal regulation of integrin-binding peptides is important in the design of niches in differentiating hMSCs to chondrocytes.

Figure 1

Chemical structures for PEGDA (A), CPENFFGRGDSG (B) and CRGDSG (C).

Figure 2

Measurement of active MMP-13 produced by hMSCs encapsulated in plain PEG gels and cultured in either control stem cell media (□) or chondrogenic differentiation media (■). MMP-13 production was normalized to DNA content to better quantify MMP production per cell basis.

Figure 3

Characterization of peptide release from PEG gels. Varying concentrations of tryptophan containing peptide sequence were copolymerized with PEG and treated with 0, 10, 50, 100 and 500ng/ml exogenously delivered MMP-13. PEG-peptide concentrations were as follows; 10mM (■), 5mM (●), 0mM (□), 10mM scramble sequence (▲). UV-Vis measurements for released tryptophan residue (A) were normalized to loaded concentrations to determine a release profile. (B) RGD ELISA was performed on 10mM peptide containing PEG gels treated with either 0 or 500ng/ml exogenously delivered MMP-13. Surface concentration determining RGD release was fit to a standard curve of known RGD concentrations.

Figure 4

hMSCs were entrapped in PEG-peptide gels in either control (▲) or chondrogenic (■) media. The composition of the gels was either PEG with 10mM CRGDSG (open symbols) or PEG with 10mM CPENFFGRGDSG (closed symbols). The cell constructs were analyzed for viability as measured via DNA amounts and normalized to the initial time point (A). These cell/gel materials were also analyzed for glycosaminoglycan deposition (B). Finally the cell surface integrin (α5β1) was stained in the cleavable RGD chondrogenic culture on day 3 (C) and day 24 (D) to determine the regulation of this adhesion binding complex. Heavy red staining on day 3 is indicative of integrin surface markers, where little to no red staining on day 24 indicates that this marker has been downregulated by the cell.