Response of a preosteoblastic cell line to cyclic tensile stress conditioning and growth factors for bone tissue engineering

Abstract

Bone regeneration can be accelerated by utilizing mechanical stress and growth factors (GFs). However, a limited understanding exists regarding the response of preosteoblasts to tensile stress alone or with GFs. We measured cell proliferation and expression of heat-shock proteins (HSPs) and other bone-related proteins by preosteoblasts following cyclic tensile stress (1%-10% magnitude) alone or in combination with bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1). Tensile stress (3%) with GFs induced greater gene upregulation of osteoprotegerin (3.3 relative fold induction [RFI] compared to sham-treated samples), prostaglandin E synthase 2 (2.1 RFI), and vascular endothelial growth factor (VEGF) (11.5 RFI), compared with samples treated with stimuli alone or sham-treated samples. The most significant increases in messenger RNA expression occurred with GF addition to either static-cultured or tensile-loaded (1% elongation) cells for the following genes: HSP47 (RFI=2.53), cyclooxygenase-2 (RFI=72.52), bone sialoprotein (RFI=11.56), and TGF-β1 (RFI=8.05). Following 5% strain with GFs, VEGF secretion increased 64% (days 3-6) compared with GF alone and cell proliferation increased 23% compared with the sham-treated group. GF addition increased osteocalcin secretion but decreased matrix metalloproteinase-9 significantly (days 3-6). Tensile stress and GFs in combination may enhance bone regeneration by initiating angiogenic and anti-osteoclastic effects and promote cell growth.

FIG. 1.

Illustration of a computer-controlled Flexcell^® tension bioreactor. Color images available online at www.liebertonline.com/tea

FIG. 2.

Cell morphology measured following 6 days of cyclic tension (1% elongation) for cells exposed to static conditions (A) and tension located at the edge (B) and center (C) of a six-well BioFlex^® culture plate with a flexible culture substrate coated with type I collagen (scale bar=100 μm). The arrow in (B) indicates the direction of actin according to the shape of the loading post. The images in B show different cell morphology depending on the area of the BioFlex^® culture plate. Color images available online at www.liebertonline.com/tea

FIG. 3.

Expression of messenger RNA (mRNA) for heat-shock proteins (HSPs) and bone-related proteins on days 3 and 6 following cyclic tension alone (1% elongation). HSP27 (A); HSP47 (B); HSP70 (C); osteopontin (OPN) (D); cyclooxygenase-2 (COX-2) (E); type I collagen (F); alkaline phosphate (ALP) (G); bone sialoprotein (BSP) (H); matrix metalloproteinase-13 (MMP-13) (I). *Statistically significant difference between the tension-treated groups and control (p<0.05).

FIG. 4.

Expression of mRNA for HSPs following 24 h cyclic tension (1%, 3%, 5%, and 10% elongation) and growth factors (GFs). HSP27 (A); HSP47 (B); HSP70 (C). *Statistical significance between the control group and the treated groups (GF alone; tension alone for all protocols; GF and tension protocols in combination). **Statistically significant difference between groups experiencing static with GFs and tension with GFs. ^#Statistically significant difference between groups exposed to tension alone and with GFs (p<0.05).

FIG. 5.

Expression of mRNA for bone matrix proteins following 24 h conditioning with cyclic tension (1%, 3%, 5%, and 10% elongation) and GFs. OPN (A); osteocalcin (OCN) (B); BSP (C); type I collagen (D). *Statistical significance between the control group and the treated groups (GF alone; tension alone for all protocols; GF and tension protocols in combination). **Statistically significant difference between groups experiencing static with GFs and tension with GFs. ^#Statistically significant difference between groups exposed to tension alone and with GFs (p<0.05).

FIG. 6.

Expression of mRNA for osteoprotegerin (OPG) (A), MMP-9 (B), TGF-β1 (C), and bone morphogenetic protein receptor type II (BMPR-2) (D) following 24 h conditioning with cyclic tension (1%, 3%, 5%, and 10% elongation) and GFs. *Statistical significance between the control group and the treated groups (GF alone;, tension alone for all protocols; GF and tension protocols in combination). **Statistically significant difference between groups experiencing static with GFs and tension with GFs. ^#Statistically significant difference between groups exposed to tension alone and with GFs (p<0.05). TGF-β1, transforming growth factor β1.

FIG. 7.

Expression of mRNA for COX-2 (A) and prostaglandin E synthase 2 (PGES-2) (B) following 24 h conditioning with cyclic tension (1%, 3%, 5%, and 10% elongation) and GFs. *Statistical significance between the control group and the treated groups (GF alone; tension alone for all protocols; GF and tension protocols in combination). **Statistically significant difference between groups experiencing static with GFs and tension with GFs. ^#Statistically significant difference between groups exposed to tension alone and with GFs (p<0.05).

FIG. 8.

Vascular endothelial growth factor (VEGF) mRNA and protein secretion following conditioning with cyclic tension and GFs. PCR (A) was performed after 24 h cyclic tension (1%, 3%, 5%, and 10% elongation) with GFs and secreted concentration (B) was acquired from the culture media collected for two durations: days 0–3 and 3–6 of tensile stress (5% elongation) with GFs. (A) *Statistical significance between the control group and the treated groups (GF alone; tension alone for all protocols; GF and tension protocols in combination). (B) *Statistically significant difference between each control group and the treated groups (GF alone; tension alone for all protocols; GF and tension protocols in combination) measured on days 3 and 6; ^&statistically significant difference between tension for 3 and 6 days. N.D. denotes no detection level by measuring the protein using the ELISA kit (p<0.05). (A, B) **Statistically significant difference between groups experiencing static conditions with GFs and tension with GFs. ^#Statistically significant difference between groups experiencing tension alone and tension with GFs.

FIG. 9.

Secretion of bone-related proteins following cyclic tension (5% elongation) in combination with GFs for two cultivation durations: days 0–3 and 3–6. OPN (A); OPG (B); OCN (C); MMP-9 (D) secretion. *Statistically significant difference between each control group and groups following tension measured on days 3 and 6. **Statistically significant difference between groups exposed to static conditions with GFs and those treated with tension and GFs. ^&Statistically significant difference between response for 3 and 6 days of tension (p<0.05). ^#Statistically significant difference between groups experiencing tension alone and tension with GFs. N.D. denotes no detection level by measuring the protein using ELISA.

FIG. 10.

MC3T3-E1 cell proliferation following combinatorial stress conditioning with cyclic tension (5% elongation) and GFs measured with MTS assay following 3 and 6 days of tension. *Statistically significant difference between the control group and the treated groups after 3 and 6 days of tension. ^&Statistically significant difference between cellular response for 3 and 6 days of tension. **Statistically significant difference between groups treated with tension and GFs and groups exposed to static conditions with GFs (p<0.05).