Influence of biomechanical and biochemical stimulation on the proliferation and differentiation of bone marrow stromal cells seeded on polyurethane scaffolds

Abstract

The aim of the present investigation was to compare the effects of cyclic compression, perfusion, dexamethasone (DEX) and bone morphogenetic protein-7 (BMP-7) on the proliferation and differentiation of human bone marrow stromal cells (hBMSCs) in polyurethane scaffolds in a perfusion bioreactor. Polyurethane scaffolds seeded with hBMSCs were cultured under six different conditions, as follows: 10% Cyclic compression at 0.5 and 5 Hz; 10 ml/min perfusion; 100 nM DEX; 100 ng/ml BMP-7; and 1 ml/min perfusion without mechanical and biochemical stimulation (control). On days 7 and 14, samples were tested for the following data: Cell proliferation; mRNA expression of Runx2, COL1A1 and osteocalcin; osteocalcin content; calcium deposition; and the equilibrium modulus of the tissue specimen. The results indicated that BMP-7 and 10 ml/min perfusion promoted cell proliferation, which was inhibited by 5 Hz cyclic compression and DEX. On day 7, the 5 Hz cyclic compression inhibited Runx2 expression, whereas the 0.5 Hz cyclic compression and BMP-7 upregulated the COL1A1 mRNA levels on day 7 and enhanced the osteocalcin expression on day 14. The DEX-treated hBMSCs exhibited downregulated osteocalcin expression. After 14 days, the BMP-7 group exhibited the highest calcium deposition, followed by the 0.5 Hz cyclic compression and the DEX groups. The equilibrium modulus of the engineered constructs significantly increased in the BMP-7, 0.5 Hz cyclic compression and DEX groups. In conclusion, the present results suggest that BMP-7 and perfusion enhance cell proliferation, whereas high frequency cyclic compression inhibits the proliferation and osteogenic differentiation of hBMSCs. Low frequency cyclic compression is more effective than DEX, but less effective compared with BMP-7 on the osteogenic differentiation of hBMSCs seeded on polyurethane scaffolds.

Keywords: biochemical stimulation; biomechanical stimulation; bone tissue engineering; polyurethane scaffold.

Figure 1.

(A) Porous polyurethane scaffold with 20-mm diameter and 5-mm thickness. (B) Scanning electron microscope (SEM) photograph of the polyurethane scaffold (scale bar, 100 µm). Star: macro pore; Arrow: micro pore. (C) SEM photograph of the human bone marrow stromal cell (hBMSC)-loaded scaffold 24 h after seeding (scale bar, 20 µm). Arrow: hBMSCs.

Figure 2.

MTS assay for the proliferation of human bone marrow stromal cells (hBMSCs) on the polyurethane scaffolds cultured under different conditions on days 7 and 14. *P<0.05, **P<0.01, n=3. Dex, dexamethasone; BMP-7, bone morphogenetic protein-7.

Figure 3.

Nuclear fast red staining of human bone marrow stromal cell-seeded scaffolds cultured under different conditions on (A) day 7 and (B) day 14 (scale bar, 100 µm). a, Control; b, Mechanical stimulation I group; c, Mechanical stimulation II group; d, Perfusion group; e, DEX group; f, BMP-7 group. DEX, dexamethasone; BMP-7, bone morphogenetic protein-7.

Figure 4.

Scanning electron microscopy images of human bone marrow stromal cells (hBMSCs) on the scaffolds cultured under different conditions on (A) day 7 and (B) day 14 (scale bar, 20 µm). Arrow: hBMSCs. a, Control; b, Mechanical stimulation I group; c, Mechanical stimulation II group; d, Perfusion group; e, DEX group; f, BMP-7 group. DEX, dexamethasone; BMP-7, bone morphogenetic protein-7.

Figure 5.

(A) mRNA expression of Runx2, COL1A1 and osteocalcin on days 7 and 14. Data are expressed as x-fold (2^−ΔΔCq) vs. mRNA of control on day 7. *P<0.05, **P<0.01, n=3. (B) ELISA assay for osteocalcin/protein estimated within the scaffolds on days 7 and 14. *P<0.05, n=3. DEX, dexamethasone; BMP-7, bone morphogenetic protein-7.

Figure 6.

Alizarin Red S staining of human bone marrow stromal cell-seeded scaffolds cultured under different conditions on (A) day 7 and (B) day 14 (scale bar, 100 µm). a, Control; b, Mechanical stimulation I group; c, Mechanical stimulation II group; d, Perfusion group; e, DEX group; f, BMP-7 group. DEX, dexamethasone; BMP-7, bone morphogenetic protein-7.

Figure 7.

Quantitative analyses of the mineralization from the Alizarin Red S staining images. *P<0.05, **P<0.01. DEX, dexamethasone; BMP-7, bone morphogenetic protein-7.

Figure 8.

Equilibrium modulus of human bone marrow stromal cell-seeded scaffolds cultured under different conditions on days 7 and 14 compared with empty scaffolds (baseline). *P<0.05, n=3. DEX, dexamethasone; BMP-7, bone morphogenetic protein-7.