Osteogenic differentiation of human mesenchymal stem cells synergistically enhanced by biomimetic peptide amphiphiles combined with conditioned medium

Abstract

An attractive strategy for bone tissue engineering is the use of extracellular matrix (ECM) analogous biomaterials capable of governing biological response based on synthetic cell-ECM interactions. In this study, peptide amphiphiles (PAs) were investigated as an ECM-mimicking biomaterial to provide an instructive microenvironment for human mesenchymal stem cells (hMSCs) in an effort to guide osteogenic differentiation. PAs were biologically functionalized with ECM isolated ligand sequences (i.e. RGDS, DGEA), and the osteoinductive potential was studied with or without conditioned medium, containing the supplemental factors of dexamethasone, β-glycerol phosphate and ascorbic acid. It was hypothesized that the ligand-functionalized PAs would synergistically enhance osteogenic differentiation in combination with conditioned medium. Concurrently, comparative evaluations independent of osteogenic supplements investigated the differentiating potential of the functionalized PA scaffolds as promoted exclusively by the inscribed ligand signals, thus offering the potential for therapeutic effectiveness under physiological conditions. Osteoinductivity was assessed by histochemical staining for alkaline phosphatase (ALP) and quantitative real-time polymerase chain reaction analysis of key osteogenic markers. Both of the ligand-functionalized PAs were found to synergistically enhance the level of visualized ALP activity and osteogenic gene expression compared to the control surfaces lacking biofunctionality. Guided osteoinduction was also observed without supplemental aid on the PA scaffolds, but at a delayed response and not to the same phenotypic levels. Thus, the biomimetic PAs foster a symbiotic enhancement of osteogenic differentiation, demonstrating the potential of ligand-functionalized biomaterials for future bone tissue repair.

Figure 1

Cell proliferation of hMSCs over 28 days. All samples were cultured in conditioned media (GM + Osteogenic Supplements) with the exception of PA-RGDS+GM, which used normal GM only. *PA-RGDS+GM promoted significantly greater cell attachment than PA-DGEA, PA-S, and TCP after 28 days (p < 0.05).

Figure 2

ALP histochemical staining to qualitatively evaluate osteogenic differentiation. hMSCs were cultured on all coating conditions for up to 28 days in both normal GM and conditioned media (GM + Osteogenic Supplements). Representative images depicting the ALP activity for all samples in the tissue culture plate wells are shown.

Figure 3

Gene expression profile for Runx2 over 28 days. All samples were cultured in conditioned media (GM + Osteogenic Supplements) with the exception of PA-RGDS+GM, which used normal GM only. Values are expressed as a mean ± standard deviation relative to PA-S (dashed line) for all incubation periods. For each time point, *PA-RGDS promoted significantly more expression than all other coating conditions, **PA-DGEA expressed more than PA-RGDS+GM, and §indicates samples greater than the normalization control of PA-S (p < 0.05).

Figure 4

Gene expression profile for ALP over 28 days. All samples were cultured in conditioned media (GM + Osteogenic Supplements) with the exception of PA-RGDS+GM, which used normal GM only. Values are expressed as a mean ± standard deviation relative to PA-S (dashed line) for all incubation periods. For each time point, significantly more expression was observed in comparison to *PA-RGDS+GM and TCP, **PA-RGDS+GM only, #TCP only, and §indicates samples greater than the normalization control of PA-S (p < 0.05).

Figure 5

Gene expression profile for OCN over 28 days. All samples were cultured in conditioned media (GM + Osteogenic Supplements) with the exception of PA-RGDS+GM, which used normal GM only. Values are expressed as a mean ± standard deviation relative to PA-S (dashed line) for all incubation periods. For each time point, significantly greater expression was found compared to *all other coating conditions, **both PA-RGDS and TCP, #TCP only, and §indicates samples greater than the normalization control of PA-S (p < 0.05).