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. 2012 Feb;18(3-4):295-309.
doi: 10.1089/ten.TEA.2011.0136. Epub 2011 Oct 24.

Biomimetic extracellular matrix-incorporated scaffold induces osteogenic gene expression in human marrow stromal cells

Sriram Ravindran  1 , Qi Gao, Mrignayani Kotecha, Richard L Magin, Sachin Karol, Ana Bedran-Russo, Anne George
Affiliations

Biomimetic extracellular matrix-incorporated scaffold induces osteogenic gene expression in human marrow stromal cells

Sriram Ravindran et al. Tissue Eng Part A. 2012 Feb.

Abstract

Engineering biomaterials mimicking the biofunctionality of the extracellular matrix (ECM) is important in instructing and eliciting cell response. The native ECM is highly dynamic and has been shown to support cellular attachment, migration, and differentiation. The advantage of synthesizing an ECM-based biomaterial is that it mimics the native cellular environment. However, the ECM has tissue-specific composition and patterned arrangement. In this study, we have employed biomimetic strategies to develop a novel collagen/chitosan template that is embedded with the native ECM of differentiating human marrow stromal cells (HMSCs) to facilitate osteoblast differentiation. The scaffold was characterized for substrate stiffness by magnetic resonance imaging and nanoindentation and by immunohistochemical analysis for the presence of key ECM proteins. Gene expression analysis showed that the ECM scaffold supported osteogenic differentiation of undifferentiated HMSCs as significant changes were observed in the expression levels of growth factors, transcription factors, proteases, receptors, and ECM proteins. Finally, we demonstrate that the scaffold had the ability to nucleate calcium phosphate polymorphs to form a mineralized matrix. The results from this study suggest that the three-dimensional native ECM scaffold directly controls cell behavior and supports the osteogenic differentiation of mesenchymal stem cells.

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Figures

FIG. 1.
FIG. 1.
Model representing the experimental setup. It is a schematic representation that depicts the different steps involved in the generation of the ECM scaffold. ECM, extracellular matrix. Color images available online at www.liebertonline.com/tea
FIG. 2.
FIG. 2.
Immunohistochemical localization and SEM analysis of ECM components. (A–F) Representative images that show the presence of dentin matrix protein 1, bone sialoprotein, osteopontin, thrombospondin, fibronectin, and phosphoserine, respectively. (G, H) Rabbit and mouse secondary antibody controls, respectively. Scale bar: 50 μm for all images. (I) Representative image of DAPI-stained ECM scaffold showing absence of cellular DNA. Scale bar: 20 μm for this image. SEM, scanning electron microscopy. Color images available online at www.liebertonline.com/tea
FIG. 3.
FIG. 3.
Magnetic resonance imaging. (A1, A2) Representative apparent diffusion coefficient (ADC) slices of control and ECM-embedded scaffolds, respectively. Boxed regions mark areas for which diffusion coefficients were calculated. (A3) Graph showing ADCs for the control and ECM scaffold. Data represent mean±SD of the values from the five boxed regions. (B1, B2) Representative T1 relaxation slices for control and ECM-embedded scaffolds, respectively. Boxed regions mark areas for which T1 relaxation times were calculated. (B3) Graph showing T1 relaxation times for the control and ECM scaffold. Data represent mean±SD of the values from the five boxed regions. (C1, C2) Representative T2 relaxation slices for control and ECM-embedded scaffolds, respectively. Boxed regions mark areas for which T2 relaxation times were calculated. (C3) Graph showing T2 relaxation times for the control and ECM scaffold. Data represent mean±SD of the values from the five boxed regions.
FIG. 4.
FIG. 4.
SEM analysis of mineralized and nonmineralized ECM scaffolds and their mechanical properties. (A) SEM micrograph of the ECM-embedded scaffold before in vitro mineralization. (B1, B2) SEM micrograph of the ECM scaffold subjected to in vitro mineralization under physiological concentrations of calcium and phosphate ions and EDX analysis, respectively. (C1, C2) SEM micrograph of the ECM scaffold subjected to in vitro mineralization at high concentrations of calcium and phosphate ions and EDX analysis, respectively. (D1, D2) SEM micrograph of the control scaffold subjected to in vitro mineralization under physiological concentrations of calcium and phosphate ions and EDX analysis, respectively. (E1, E2) SEM micrograph of the control scaffold subjected to in vitro mineralization at high concentrations of calcium and phosphate ions and EDX analysis, respectively. (F) Graphical representation of the hardness data obtained by using a nanoindenter for the ECM scaffold before mineralization and after mineralization under high concentrations of calcium and phosphate ions. Data represent mean±s.e.m. EDX, energy-dispersive X-ray.
FIG. 4.
FIG. 4.
SEM analysis of mineralized and nonmineralized ECM scaffolds and their mechanical properties. (A) SEM micrograph of the ECM-embedded scaffold before in vitro mineralization. (B1, B2) SEM micrograph of the ECM scaffold subjected to in vitro mineralization under physiological concentrations of calcium and phosphate ions and EDX analysis, respectively. (C1, C2) SEM micrograph of the ECM scaffold subjected to in vitro mineralization at high concentrations of calcium and phosphate ions and EDX analysis, respectively. (D1, D2) SEM micrograph of the control scaffold subjected to in vitro mineralization under physiological concentrations of calcium and phosphate ions and EDX analysis, respectively. (E1, E2) SEM micrograph of the control scaffold subjected to in vitro mineralization at high concentrations of calcium and phosphate ions and EDX analysis, respectively. (F) Graphical representation of the hardness data obtained by using a nanoindenter for the ECM scaffold before mineralization and after mineralization under high concentrations of calcium and phosphate ions. Data represent mean±s.e.m. EDX, energy-dispersive X-ray.
FIG. 5.
FIG. 5.
Clustering of HMSCs on ECM scaffold. (A, A1) Representative z-stack projections of HMSCs cultured on control collagen/chitosan scaffold. (B, B1) Representative z-stack projections of HMSCs cultured on ECM-embedded collagen/chitosan scaffold. Circles depict clustering of HMSCs. HMSC, human marrow stromal cell. Color images available online at www.liebertonline.com/tea
FIG. 6.
FIG. 6.
ECM scaffold promotes cell–matrix and cell–cell interactions. (A, A1) Representative SEM images showing cell–matrix interactions. White arrows in A indicate cellular processes embedded in the scaffold. (A1) High-magnification image of the boxed area in A. (B, B1) Representative images showing cell–cell interactions on the ECM scaffold. Numbers 1 and 2 in B indicate two adjacent cells. (B1) Higher-magnification image of the boxed area in B. Arrows in B1 indicate points of cell–cell interaction.
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