BMP-2 gene-fibronectin-apatite composite layer enhances bone formation

Abstract

Background: Safe and efficient gene transfer systems are needed for tissue engineering. We have developed an apatite composite layer including the bone morphogenetic protein-2 (BMP-2) gene and fibronectin (FB), and we evaluated its ability to induce bone formation.

Methods: An apatite composite layer was evaluated to determine the efficiency of gene transfer to cells cultured on it. Cells were cultured on a composite layer including the BMP-2 gene and FB, and BMP-2 gene expression, BMP-2 protein concentrations, alkaline phosphatase (ALP) activity, and osteocalcin (OC) concentrations were measured. A bone defect on the cranium of rats was treated with hydroxyapatite (HAP)-coated ceramic buttons with the apatite composite layer including the BMP-2 gene and FB (HAP-BMP-FB). The tissue concentration of BMP-2, bone formation, and the expression levels of the BMP-2, ALP, and OC genes were all quantified.

Results: The apatite composite layer provided more efficient gene transfer for the cultured cells than an apatite composite layer without FB. The BMP-2 concentration was approximately 100-600 pg/mL in the cell-culture medium. Culturing the cells on the apatite composite layer for 27 days increased ALP activity and OC concentrations. In animal experiments, the tissue concentrations of BMP-2 were over 100 pg/mg in the HAP-BMP-FB group and approximately 50 pg/mg in the control groups. Eight weeks later, bone formation was more enhanced in the HAP-BMP-FB group than in the control groups. In the tissues surrounding the HAP button, the gene expression levels of ALP and OC increased.

Conclusion: The BMP-2 gene-FB-apatite composite layer might be useful for bone engineering.

Figure 1

Three-dimensional views of a hydroxyapatite ceramic button (HAP) and the implantation of HAP samples into bone defects (burr holes). A; HAPs were made for cranial repair (cranioplasty) in rats. Both sides of the HAP were cut in order for bone formation to extend into the space around the bone defect. B; The panel demonstrates how bone formation was measured. Bone formation was quantified by measuring the length of new bone extension into the inside of the bone defect and the thickness of the edges of the bone defect.

Figure 2

SEM photos of the EVOH-CP, EVOH-FB, EVOH-DNA and EVOH-DNA-FB substrates. Uniform layers were observed on the surfaces of all the samples. High magnification images (the lower micrographs) show that all these layers have a microflake-like architecture.

Figure 3

Relative luciferase assay. Relative luciferase light units (RLUs, normalized to the protein concentration) of extracts from MC3T3-E1 cells (empty columns) or C3H10T1/2 cells (solid columns) cultured on EVOH-DNA and EVOH-DNA-FB for 3 days. The values presented are the mean ± standard deviation. (n = 3, *p < 0.05, **p < 0.001).

Figure 4

BMP-2 gene expression levels and BMP-2 protein concentrations in in-vitro experiments. BMP2 gene expression levels in extracts from MC3T3-E1 cells (A) or C3H10T1/2 cells (B) cultured on EVOH-CP, EVOH-BMP, or EVOH-BMP-FB for 3 or 7 days. The empty columns indicate a 3-day culture and the solid columns a 7-day culture. (C) BMP-2 concentrations in the medium from cells cultured on EVOH-CP, EVOH-BMP or EVOH-BMP-FB for 7 days. The empty columns indicate the MC3T3-E1 cells and the solid columns the C3H10T1/2 cells. The values presented are the mean ± standard deviation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5

Development of MC3T3-E1 cells in in-vitro experiments. A; Alkaline phosphatase activity of cells cultured on EVOH-CP, EVOH-FB, EVOH-BMP or EVOH-BMP-FB for 9 or 27 days. B; Osteocalcin concentration of cells cultured on EVOH-CP, EVOH-FB, EVOH-BMP or EVOH-BMP-FB for 9 or 27 days. The empty columns indicate a 9-day culture and the solid columns a 27-day culture. The values presented are the mean ± standard deviation. (n = 3, *p < 0.01).

Figure 6

BMP-2 gene expression and BMP-2 protein concentrations in animal experiments. BMP-2 gene expression (A) and BMP-2 protein concentrations (B) were evaluated in bone defect tissue treated with HAP-CP, HAP-BMP or HAP-BMP-FB two weeks after the procedure. The values presented are the mean ± standard deviation (n = 3, *p < 0.05, **p < 0.01).

Figure 7

Evaluation of the animal experiments. A; Histological sections of the bone defects were stained with hematoxylin and eosin after demineralization. The bone defects were treated with HAP-CP, HAP-BMP or HAP-BMP-FB 8 weeks ago. The yellow dotted lines show the area of bone formation (indicated by new bone). Bone formation was observed between the cranium and the dural membrane, resulting in increased cranial thickness. Bone formation was also observed in the bone defect space as the extension of new bone. Bars indicate 100 μm. B; Bone formation was quantified in each group. The extension of new bone into the space left by the bone defect (open columns). The increased thickness of the cranium due to the bone formation (solid columns). The values presented are the mean ± standard deviation. (n = 5, *p < 0.05, **p < 0.01) C; ALP and OC gene expression in the bone defect tissue 8 weeks after the procedure. Open columns indicate ALP gene expression and solid columns indicate OC expression. The values presented are the mean ± standard deviation (n = 5, *p < 0.05, **p < 0.01).