Enhanced Osteogenesis Potential of MG-63 Cells through Sustained Delivery of VEGF via Liposomal Hydrogel

Abstract

The challenges of using VEGF to promote osteoblastic differentiation include a short half-life and a narrow therapeutic window. A carrier system combining hydrogel and liposomes may improve the therapeutic efficacy of VEGF for bone regeneration. This study aimed to investigate the effects of delivery of VEGF via liposomal hydrogel on the osteogenesis of MG-63 cells. Liposomal hydrogel scaffold was fabricated and then characterized in terms of the morphological and chemical properties using FESEM and FTIR. In 2.5D analysis, the MG-63 cells were cultured on liposomal hydrogel + VEGF as the test group. The osteogenic effects of VEGF were compared with the control groups, i.e., hydrogel without liposomes + VEGF, osteogenic medium (OM) supplemented with a bolus of VEGF, and OM without VEGF. Cell morphology, viability, and differentiation and mineralization potential were investigated using FESEM, MTT assay, ALP activity, and Alizarin red staining. The characterization of scaffold showed no significant differences in the morphological and chemical properties between hydrogel with and without liposomes (p > 0.05). The final 2.5D culture demonstrated that cell proliferation, differentiation, and mineralization were significantly enhanced in the liposomal hydrogel + VEGF group compared with the control groups (p < 0.05). In conclusion, liposomal hydrogel can be used to deliver VEGF in a sustained manner in order to enhance the osteogenesis of MG-63 cells.

Keywords: MG-63 osteoblast-like cells; hydrogel; liposomes; osteogenesis; vascular endothelial growth factor.

Figure 1

TEM images of (A) liposomes without VEGF and (B) liposomes with VEGF. (Scale bar: 200 nm.)

Figure 2

Photographs of the process of gelation for hydrogel without liposomes (a,b) and liposomal hydrogel (c,d). The tube inversion method was used to determine the gelation time (b,d).

Figure 3

Field emission scanning electron microscope images of hydrogel without liposomes (a–c) and liposomal hydrogel (d–f) at 100×, 300×, and 100k× magnifications. The white arrow (f) indicates the presence of liposomes in the liposomal hydrogel.

Figure 4

(A) The FTIR spectra of hydrogel without liposomes and liposomal hydrogel: (B) The cumulative release profile of VEGF from hydrogel with and without liposomes over 21 days. The cumulative release of VEGF in ng/mL was determined as a function of time by VEGF ELISA kit at 450 nm. Each value is expressed as mean ± SD (n = 3 per group). Chitosan, CS; β-glycerophosphate, BGP.

Figure 5

Surface cellular morphology viewed under FESEM at ×1000 magnification: Both spherical (black arrows) and spindle shaped cells (yellow arrows) were seen on day 0 (a,e). The presence of cytoplasmic extensions (white arrows, broken line) was observed on day 7 (b,f). Polygonal and flattened cells (black arrows, broken line) were seen on day 14 (c,g). On day 21, the liposomal hydrogel + VEGF group (h) showed denser cellular network and higher accumulation of mineralized nodules compared with the hydrogel without liposomes group (d). White arrows indicate deposition of calcium nodules.

Figure 6

The effects of sustained delivery of VEGF on cell viability was determined with MTT assay on days 0, 7, 14, and 21 of cultivation. Each value was expressed as mean ± SD (n = 3 per group). * denotes statistical significance between different culture time points of each group (p < 0.05). ** denotes significant differences between groups at each time point (p < 0.05).

Figure 7

The effects of sustained delivery of VEGF on ALP activity assayed on day 0, 7, 14, and 21 of cultivation, and the ALP activity is expressed as nmol/ng. Each value is expressed as mean ± SD (n = 3 per group). * denotes statistical significance between different culture time points of each group (p < 0.05). ** denotes significant differences between groups at each time point (p < 0.05).

Figure 8

The effects of sustained delivery of VEGF on mineralization: Alizarin Red S staining was quantitatively assayed at 570 nm on day 21. Each value is expressed as mean ± SD (n = 3 per group). ** denotes significant differences between groups at each time point (p < 0.05).