Development of stepwise osteogenesis-mimicking matrices for the regulation of mesenchymal stem cell functions

Abstract

An extracellular microenvironment, including an extracellular matrix (ECM), is an important factor in regulating stem cell differentiation. During tissue development, the ECM is dynamically remodeled to regulate stem cell functions. Here, we developed matrices mimicking ECM remodeling during the osteogenesis of mesenchymal stem cells (MSCs). The matrices were prepared from cultured MSCs controlled at different stages of osteogenesis and referred to as "stepwise osteogenesis-mimicking matrices." The matrices supported the adhesion and proliferation of MSCs and showed different effects on the osteogenesis of MSCs. On the matrices mimicking the early stage of osteogenesis (early stage matrices), the osteogenesis occurred more rapidly than did that on the matrices mimicking undifferentiated stem cells (stem cell matrices) and the late stage of osteogenesis (late stage matrices). RUNX2 was similarly expressed when MSCs were cultured on both the early stage and late stage matrices but decreased on the stem cell matrices. PPARG expression in the MSCs cultured on the late stage matrices was higher than for those cultured on the stem cell and early stage matrices. This increase of PPARG expression was caused by the suppression of the amount of beta-catenin and downstream signal transduction. These results demonstrate that the osteogenesis-mimicking matrices had different effects on the osteogenesis of MSCs, and the early stage matrices provided a favorable microenvironment for the osteogenesis.

FIGURE 1.

Stepwise osteogenic differentiation of MSCs. A, MSCs were differentiated into osteoblasts on TCPS. ALP and alizarin red S stainings were performed after culture for the indicated period. Scale bar, 200 μm. B and C, expressions of ALP (B) and IBSP (C) during the osteogenesis of the MSCs were investigated by real-time PCR analysis. Data represent means ± S.D. (n = 3). *, p < 0.05.

FIGURE 2.

ECM alteration during the osteogenesis of MSCs in vitro. ECM proteins were investigated by immunocytochemical analysis. Cell nuclei were counterstained with methyl green. Stem cell, Early stage, and Late stage, undifferentiated MSCs and differentiating cells at the early and late stages of osteogenesis, respectively. The results of negative control are shown in supplemental Fig. 2. Scale bar, 200 μm.

FIGURE 3.

Preparation of the stepwise osteogenesis-mimicking matrices. A, the removal of cellular components from the stepwise osteogenesis-mimicking matrices was confirmed by cell nuclei and actin stainings. Cell nuclei and actin were stained in the samples before and after decellularization. Blue pseudocolor and green pseudocolor indicate cell nuclei and actin, respectively. B, remaining proteins after decellularization were confirmed by CBB staining. C, cell attachment activity on the stepwise osteogenesis-mimicking matrices was measured after 1 or 4 h of incubation. Data represent mean ± S.D. (n = 3). a, p < 0.01 versus TCPS (1 h); b, p < 0.01 versus BSA (1 h); c, p < 0.001 versus TCPS (4 h); d, p < 0.001 versus BSA (4 h). The differences among cell attachment activity of stepwise osteogenesis-mimicking matrices were not significant.

FIGURE 4.

Proliferation of MSCs on the stepwise osteogenesis-mimicking matrices. A, MSC proliferation was measured by a WST-1 assay. Data represent means ± S.D. (n = 3). *, p < 0.05 versus stem cell matrices (2 days). **, p < 0.01 versus early stage matrices (4 days). B, MSCs were cultured for 1 day and then were allowed to incorporate BrdUrd for 2 h. The results are expressed as a percentage of BrdUrd-incorporated cells to the total cell number. Data represent mean ± S.D. (n = 3). *, p < 0.05 versus stem cell matrices.

FIGURE 5.

Osteogenesis of MSCs on the stepwise osteogenesis-mimicking matrices. A, MSCs were cultured on the stepwise osteogenesis-mimicking matrices for 2 weeks in osteogenic medium. After the osteogenic culture, ALP staining of MSCs was done. B, positive areas of ALP staining on the stepwise osteogenesis-mimicking matrices measured after 2 weeks of osteogenic culture. The results are expressed as a percentage normalized to the positive area on TCPS. Data represent means ± S.D. (n = 3). *, p < 0.005; **, p < 0.01. C and D, MSCs were cultured on stepwise osteogenesis-mimicking matrices for 2 weeks in osteogenic medium. Expressions of ALP (C) and SPP1 (D) genes were investigated by real-time PCR analysis. The results are expressed as a percentage normalized to the expression level on TCPS. Data represent means ± S.D. (n = 3). *, p < 0.05.

FIGURE 6.

Expressions of transcription factors related to osteogenesis of MSCs. MSCs were cultured on stepwise osteogenesis-mimicking matrices for 2 weeks in osteogenic medium. The expression levels of RUNX2 (A), SP7 (B), HOXA2 (C), SOX9 (D), and PPARG (E) were measured by real-time PCR analysis. The results are expressed as a percentage normalized to the expression level on TCPS. Data represent means ± S.D. (n = 3). *, p < 0.05; **, p < 0.001; ***, p < 0.01.

FIGURE 7.

BMP-2 and Wnt signals regulated on the matrices. A, MSCs were cultured on stepwise osteogenesis-mimicking matrices for 2 weeks in osteogenic medium with or without exogenous BMP-2. The expression level of RUNX2 was measured by real-time PCR analysis. Data represent means ± S.D. (n = 3). *, p < 0.05. N.S., no significant difference. B, MSCs were cultured on stepwise osteogenesis-mimicking matrices for 2 weeks in osteogenic medium. The amount of total β-catenin was measured by Western blot analysis. The lower panel shows the control experiment. The sample from the MSCs cultured on TCPS with 25 mm of LiCl for 1 week was used as positive control of β-catenin. C, MSCs cultured on osteogenesis-mimicking matrices on stepwise osteogenesis-mimicking matrices for 2 weeks in osteogenic medium with 50 μm quercetin or DMSO. The expression level of PPARG was measured by real-time PCR analysis. Data represent means ± S.D. (n = 3). *, p < 0.05. N.S., no significant difference.