Influence of select extracellular matrix proteins on mesenchymal stem cell osteogenic commitment in three-dimensional contexts

Abstract

Growth factors have been shown to be powerful mediators of mesenchymal stem cell (MSC) osteogenic differentiation. However, their use in tissue engineered scaffolds not only can be costly but also can induce undesired responses in surrounding tissues. Thus, the ability to specifically promote MSC osteogenic differentiation in the absence of exogenous growth factors via the manipulation of scaffold material properties would be beneficial. The current work examines the influence of select extracellular matrix (ECM) proteins on MSC osteogenesis toward the goal of developing scaffolds with intrinsically osteoinductive properties. Fibrinogen (FG), fibronectin (FN) and laminin-1 (LN) were chosen for evaluation due to their known roles in bone morphogenesis or bone fracture healing. These proteins were conjugated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels and their effects on encapsulated 10T½ MSCs were evaluated. Specifically, following 1week of culture, mid-term markers of various MSC lineages were examined in order to assess the strength and specificity of the observed osteogenic responses. PEG-LN gels demonstrated increased levels of the osteogenic transcription factor osterix relative to day 0 levels. In addition, PEG-FG and PEG-LN gels were associated with increased deposition of bone ECM protein osteocalcin relative to PEG-FN gels and day 0. Importantly, the osteogenic response associated with FG and LN appeared to be specific in that markers for chondrocytic, smooth muscle cell and adipocytic lineages were not similarly elevated relative to day 0 in these gels. To gain insight into the integrin dynamics underlying the observed differentiation results, initial integrin adhesion and temporal alterations in cell integrin profiles were evaluated. The associated results suggest that α(2), α(v) and α(6) integrin subunits may play key roles in integrin-mediated osteogenesis.

Figure 1

Expression of osteogenic markers osterix, osteopontin, and osteocalcin by ELISA (osterix and osteocalcin) and cell counts (osteopontin). For ELISA assays, 6 samples per day 7 formulation were analyzed. The day 0 ELISA sample number was n = 4. For cell counts, sections from 4–8 separate samples of each formulation were evaluated. Validation of the cell counting assessment method is given in Supplementary Figure 3. For the purpose of comparison, ELISA and cell count measures for each protein have been normalized to the corresponding measure for PEG-FN gels. * indicates a significant difference, p < 0.05.

Figure 2

(A) Expression of myocardin and SM22α, as assessed by cell counting and ELISA methods, respectively. (B) Expression of PPAR and A-FABP, as assessed by cell counting and ELISA methods, respectively. (C) Expression of sox9 and collagen II by ELISA. For ELISA assays, 6–9 samples per day 7 formulation were analyzed. The day 0 ELISA sample number was n = 4. For cell counts, sections from 4 separate discs of each formulation were evaluated. For the purpose of comparison, ELISA and cell count measures for each protein have been normalized to the corresponding measure for PEG-FN gels.

Figure 3

Representative images of day 7 immunostaining for myocardin, PPAR, and osteopontin. Positive staining is indicated by brown (PPAR and osteopontin) or red (myocardin) coloration. Scale bar = 40 µm and applies to all images.

Figure 4

Day 7 and day 0 expression of various integrin alpha subunits as assessed by ELISA (n = 6–9 per day 7 formulation; n = 4 for day 0). For the purpose of comparison, ELISA measures for each protein have been normalized to the corresponding measure for PEG-FN gels. * indicates a significant difference, p < 0.05.