Self-Assembling Peptide RADA16 Nanofiber Scaffold Hydrogel-Wrapped Concentrated Growth Factors in Osteogenesis of MC3T3

Abstract

Concentrated growth factors (CGFs) are widely used in surgery with bone grafting, but the release of growth factors from CGFs is rapid. RADA16, a self-assembling peptide, can form a scaffold that is similar to the extracellular matrix. Based on the properties of RADA16 and CGF, we hypothesized that the RADA16 nanofiber scaffold hydrogel could enhance the function of CGFs and that the RADA16 nanofiber scaffold hydrogel-wrapped CGFs (RADA16-CGFs) would perform a good osteoinductive function. This study aimed to investigate the osteoinductive function of RADA16-CGFs. Scanning electron microscopy, rheometry, and ELISA were performed, and MC3T3-E1 cells were used to test cell adhesion, cytotoxicity, and mineralization after administration with RADA16-CGFs. We found that RADA16 endowed with the sustained release of growth factors from CGFs, which can help maximize the function of CGFs in osteoinduction. The application of the atoxic RADA16 nanofiber scaffold hydrogel with CGFs can be a new therapeutic strategy for the treatment of alveolar bone loss and other problems that require bone regeneration.

Keywords: CGFs; RADA16; alveolar bone cleft; alveolar bone grafting; osteogenesis.

Figure 1

CGF preparation. Blood was drawn from the arm vein, and the tubes were centrifuged under the CGF preparation procedure. Three blood fractions were found at the end of the process. The middle fraction was extracted as the CGF composition and squashed to form the CGF membrane.

Figure 2

Properties of CGFs. (a) Sample preparation for ELISA quantification. Shredded CGF membrane and 2% w/v RADA16 solution (500 µL) were softly mixed in a 1.5 mL EP tub. Then, 500 μL PBS was added to the tube and put into a 37 °C water bath. The buffer was extracted at 1 h, 6 h, 12 h, 1 d, 2 d, 3 d, 5 d, 7 d, and 10 d, and another 1 mL PBS was added into the tubes for 1 h, and extracted for ELISA quantification. (b,c) SEM image of CGFs. Bars, 500 μm and 20 μm, respectively.

Figure 3

Properties of RADA16. (a) Mass spectroscopy of the synthetic peptide. (b) High-performance liquid chromatography of synthetic peptides. (c,d) SEM images of RADA16 (1%). Bars, 5 μm and 1 μm, respectively. (e) Rheometry performance of RADA16 (1%).

Figure 4

Growth factors released from RADA-CGFs. (a) Concentration of PDGF-BB at each time point. *, p < 0.05. (b) Concentration of VEGF at each time point.

Figure 5

Cell proliferation of MC3T3 with RADA16-CGF. (a) MC3T3 cell morphology co-cultured with different composites after 3 days. Scale bars, 100 μm. (b) Comparison of the number of adherent cells between the control, CGFs, RADA16, and RADA16-CGF groups at different timepoints. *, p < 0.05; **, p < 0.005; ***, p < 0.001; ****, p < 0.0001. (c) Comparison and tendency chart of the CCK-8 assays for the proliferation of MC3T3 cells co-cultured with different composites. *, p < 0.05; ***, p < 0.001.

Figure 6

Effect of RADA16-CGFs on mineralization capability. (a) ALP staining images after culturing on the different composites for 7 days. (b) Optical microscopy images of alizarin red S staining after culturing on the different composites for 21 days. Scale bars, 100 μm. (c) Quantitative analysis of calcium nodules on the composites at a wavelength of 562 nm. *, p < 0.05; **, p < 0.005; ***, p < 0.001. (d) The gene expression of osteogenic differentiation-related proteins (Alp) of MC3T3 cells co-cultured with different composites. *, p < 0.05; **, p < 0.005.