Influence of collagen-based integrin α1 and α2 mediated signaling on human mesenchymal stem cell osteogenesis in three dimensional contexts

Abstract

Collagen I interactions with integrins α₁ and α₂ are known to support human mesenchymal stem cell (hMSC) osteogenesis. Nonetheless, elucidating the relative impact of specific integrin interactions has proven challenging, in part due to the complexity of native collagen. In the present work, we employed two collagen-mimetic proteins-Scl2-2 and Scl2-3- to compare the osteogenic effects of integrin α₁ versus α₂ signaling. Scl2-2 and Scl2-3 were both derived from Scl2-1, a triple helical protein lacking known cell adhesion, cytokine binding, and matrix metalloproteinase sites. However, Scl2-2 and Scl2-3 were each engineered to display distinct collagen-based cell adhesion motifs: GFPGER (binding integrins α₁ and α₂ ) or GFPGEN (binding only integrin α₁ ), respectively. hMSCs were cultured within poly(ethylene glycol) (PEG) hydrogels containing either Scl2-2 or Scl2-3 for 2 weeks. PEG-Scl2-2 gels were associated with increased hMSC osterix expression, osteopontin production, and calcium deposition relative to PEG-Scl2-3 gels. These data indicate that integrin α₂ signaling may have an increased osteogenic effect relative to integrin α₁ . Since p38 is activated by integrin α₂ but not by integrin α₁ , hMSCs were further cultured in PEG-Scl2-2 hydrogels in the presence of a p38 inhibitor. Results suggest that p38 activity may play a key role in collagen-supported hMSC osteogenesis. This knowledge can be used toward the rational design of scaffolds which intrinsically promote hMSC osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2594-2604, 2018.

Keywords: collagen-mimetic proteins; human mesenchymal stem cells; integrin α1; integrin α2; osteogenesis.

Figure 1

Activation of MAPK pathways through integrin interactions. ERK, JNK and p38 MAPKs are differentially activated by integrin α₁β₁ and α₂β₁ signaling. Specifically, integrin α₂β₁ is uniquely associated with increased p38 activation. Figure adapted from Lal et al.

Figure 2

Flow cytometry characterization of the expression of the integrin α₁ and integrin α₂ in the hMSC population used in the PEG-Scl2-2 versus PEG-Scl2-3 comparison experiments as well as in the p38 inhibition study (the comparison and inhibition studies were conducted simultaneously to avoid differences in initial hMSC population).

Figure 3

Expression of phosphorylated ERK, JNK and p38 by hMSCs seeded on Scl2-1, Scl2-2 or Scl2-3 coated surfaces. For the purpose of comparison, results for the Scl2-2 and Scl2-3 groups were normalized to the basal expression levels associated with the Scl2-1 control. Results represent an average of n = 3-4 samples per treatment group. * indicates a significant difference from Scl2-1, p < 0.05. ^# indicates a significant difference from Scl2-2, p < 0.05.

Figure 4

Expression of osteogenic markers (A) runx2 and osterix and (B) OPN and calcium deposition in PEG-Scl2-2 and PEG-Scl2-3 hydrogels relative to PEG-Scl2-1 controls. Results represent n = 3-6 samples per formulation. * indicates a significant difference from the PEG-Scl2-1 gel, p < 0.05; ^# indicates a significant difference from the PEG-Scl2-2 gel, p < 0.05.

Figure 5

hMSC expression of osterix and OPN in PEG-Scl2-2 hydrogels exposed to p38 inhibition (SB203580 in DMSO) relative to DMSO-only controls (n=3-4 per formulation). For the purpose of comparison, measures for each marker have been normalized to the DMSO control gels. * the connected bar indicates a significant difference in overall osteogenic marker expression between treatment groups, p < 0.05.