Proliferative and Osteogenic Supportive Effect of VEGF-Loaded Collagen-Chitosan Hydrogel System in Bone Marrow Derived Mesenchymal Stem Cells

Abstract

The use of hydrogel (HG) in regenerative medicine is an emerging field and thus several approaches have been proposed recently to find an appropriate hydrogel system. In this sense, this study developed a novel HG system using collagen, chitosan, and VEGF composites for culturing mesenchymal stem cells (MSCs), and investigated their ability for osteogenic differentiation and mineral deposition. Our results showed that the HG loaded with 100 ng/mL VEGF (HG-100) significantly supported the proliferation of undifferentiated MSCs, the fibrillary filament structure (HE stain), mineralization (alizarin red S and von Kossa stain), alkaline phosphatase, and the osteogenesis of differentiated MSCs compared to other hydrogels (loaded with 25 and 50 ng/mL VEGF) and control (without hydrogel). HG-100 showed a higher VEGF releasing rate from day 3 to day 7 than other HGs, which substantially supports the proliferative and osteogenic properties of HG-100. However, the HGs did not increase the cell growth in differentiated MSCs on days 14 and 21 due to the confluence state (reach stationary phase) and cell loading ability, regardless of the VEGF content. Similarly, the HGs alone did not stimulate the osteogenesis of MSCs; however, they increased the osteogenic ability of MSCs in presence of osteogenic supplements. Accordingly, a fabricated HG with VEGF could be used as an appropriate system to culture stem cells for bone and dental regeneration.

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

“…In the current study, the proliferation stimulative effect of VEGF was no longer significant after the cell differentiation was initiated on around day 14. This current observation is consistent with the results in other studies also reporting that cell proliferation declines after two weeks of the osteogenic induction [46,52,53]. Both the hydrogels, with and without liposomes groups, exhibited higher ALP activities than the OM and OM + VEGF groups, consistent with the findings by Takagishi et al (2006) [54], in which the promotion of osteogenic differentiation was reported in MG-63 cells cultured on gelatin hydrogels.…”

“…The findings of the current study showed that the effect of sustained delivery of VEGF from liposomal hydrogel was the most superior compared with the hydrogel without liposomes carrier system reported by Wu et al (2019) [45] and Elango (2023) [46], as well as the bolus delivery of VEGF without carrier [47]. This is explained by the sustained delivery of VEGF over the 21-day culture period.…”

“…The study by Wu et al (2019) on dental pulp stem cells (DPSCs) reported that the odontogenic effect of 100 ng/mL VEGF loaded in a hydrogel delivery system was significantly greater than 100 ng/mL VEGF supplementation without carriers [45]. This finding was supported by the most recent study by Elango (2023), in which the differentiation and mineralization were accelerated when human bone mesenchymal stem cells were cultured on a collagen-hydrogel carrier system loaded with 100 ng/mL VEGF [46]. On the other hand, the studies by Behr et al (2011) [60] and Aksel et al (2017) [57] showed that different concentrations of VEGF (5-200 ng/mL) did not appear to induce significantly different osteoinductive effects on human adipose-derived stem cells and human dental pulp stem cells, respectively.…”

“…The heterogeneity between studies may be influenced by the cell type, culturing conditions, and duration of time exposure of cells to VEGF. A recent study showed enhanced osteogenesis in human bone mesenchymal stem cells cultured for 21 days on a collagen-hydrogel scaffold [46]. However, the study by Aksel et al (2017) on dental pulp stem cells showed that the highest levels of mineralization and expression of the osteogenic gene were seen when VEGF was given only during the first week of incubation, instead of continuous supplementation over 21 days [57].…”

“…Throughout these phases, expressions of specific osteogenic markers are temporally and sequentially organized [43,44]. Previous studies only compared the effects of VEGF-containing hydrogel without any secondary nanocarrier with negative controls over 14-21 days [45,46]. Therefore, in order to further investigate the effects of VEGF on osteogenesis in this study, three methods of delivery of VEGF, namely bolus delivery (OM + VEGF group), burst release (hydrogel without liposomes + VEGF), and sustained delivery (liposomal hydrogel + VEGF), were conducted, and the effects on cell proliferation, differentiation, and mineralization were evaluated for 21 days.…”