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 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 1 week 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 α2, αv, and α6 integrin subunits may play key roles in integrin-mediated osteogenesis.

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis

“…43 NH 2 -Arg-Gly-Asp-Ser-COOH (RGDS; American Peptide Company) and 3.4 kDa acryoyl-PEG succinimidyl valerate (ACRL-PEG-SVA; Laysan Bio, Inc.) were reacted a 1:1 molar ratio for 2 h in 50 mM sodium bicarbonate buffer (pH 8.5). The product (ACRL-PEG-RGDS) was purified by dialysis, lyophilized, and stored at -80°C until further use.…”

“…34,40,41 This latter property permits desired cell adhesion to be ''programmed'' into the PEGDA network through conjugation of bioactive moieties. 42,43 In the present work, we have tethered the peptide RGD to the PEGDA network to enable uniform levels of initial cell-matrix interactions across experimental groups.…”

Impact of secondary reactive species on the apparent decoupling of poly(ethylene glycol) diacrylate hydrogel average mesh size and modulus

“…Such adsorbed proteins are often major determinants of cell behavior, in addition to bioactive moieties deliberately conjugated to the material. 9–10 In contrast, the relatively inert nature of PEGDA hydrogels permits the controlled and defined investigation of biochemical cues on cell behavior. For controlled release applications, this “anti-fouling” property of PEGDA endows PEGDA hydrogel carriers with “stealth” properties.…”

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

“…PEG‐diacrylate was prepared as previously described by combining 0.1 mmol mL – dry PEG (3.4 kDa, Fluka), 0.4 mmol mL – acryloyl chloride, and 0.2 mmol ml – triethylamine in anhydrous dichloromethane, followed by stirring under argon overnight. The resulting solution was washed with 2 M K 2 CO 3 and separated into aqueous and dichloromethane phases to remove HCl.…”