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Design of an osteoinductive extracellular fibronectin matrix protein for bone tissue engineering

International Journal of Molecular Sciences (2015) 16(4) 7672-7681
DOI: 10.3390/ijms16047672
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Abstract

Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05).

Figures

  • Figure 1. Cloning, expression, and purification of osteoinductive and integrin-binding fibronectin (oFN and iFN, respectively). The cDNA coding for bone morphogenetic protein-2 (BMP-2) and the ninth and tenth type-III fibronectin domains (FNIII9-10) was cloned into the pBAD-HisB expression vector. Schematic representations of the fusion proteins and western blotting analysis of iFN (FNIII9-10) and oFN (BMP-2-FNIII9-10) are shown at 37 and 50 kDa, respectively.
  • Figure 2. Cell adhesion activity of osteoinductive fibronectin on MC3T3-E1 cells. Cells were seeded at a density of 1 × 105 cells/well on plates coated with iFN, oFN, or BMP-2 and incubated for 30 min at 37 °C. Adhesion of MC3T3-E1 cells was measured as described in Experimental Section. The cell adhesion activity is expressed as absorbance and represented as mean ± standard deviation (n = 3).
  • Figure 3. Alkaline phosphatase (ALP) activity of osteoinductive fibronectin (oFN, 5 μg·mL−1) on MC3T3-E1 cells, at 3 and 10 days. Cells were seeded at a density of 1 × 103 cells/well on plates coated with integrin-binding fibronectin (iFN) or oFN and incubated. Non-treated cells were used as control. ALP activity was normalized versus control and is represented as mean ± standard deviation (n = 3). * p < 0.05 compared with iFN-loaded groups or control.
  • Figure 4. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells seeded on fibronectin-loaded collagen matrix, at 10 days. Collagen matrices were placed in 24-well plates, and MC3T3-E1 cells were cultured on collagen matrices loaded with integrin-binding fibronectin (iFN) or osteoinductive fibronectin (oFN, 5 μg·mL−1). Inset: image of a collagen matrix. The results are reported as mean ± standard deviation (n = 3). * p < 0.05 compared with iFN-loaded groups or control.
  • Figure 5. Osteogenic differentiation activity of osteoinductive fibronectin (oFN) matrix protein as determined by real-time PCR. MC3T3-E1 cells were cultured on collagen matrices loaded with integrin-binding fibronectin (iFN) or oFN (5 μg·mL−1) in 24-well plates. The mRNA levels of OPN, RUNX2, and Col I were measured, and the relative mRNA level of each gene was normalized to that of GAPDH. The results are reported as mean ± standard deviation (n = 3). * p < 0.05 compared with iFN-loaded groups or control.
  • Table 1. Sequences of the primers used in the real-time polymerase chain reaction (PCR).

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CITATION STYLE

APA

Lee, S., Lee, D. S., Choi, I., Pham, L. B. H., & Jang, J. H. (2015). Design of an osteoinductive extracellular fibronectin matrix protein for bone tissue engineering. International Journal of Molecular Sciences, 16(4), 7672–7681. https://doi.org/10.3390/ijms16047672

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