Assistant Professor University of New Haven West Haven, Connecticut, United States
Introduction: The ability of human mesenchymal stem cells to differentiate into various cell lineages is crucial in regenerative medicine. Recent research has provided evidence that Chitosan/hydroxyapatite (CH-NPs), Gold (Au-NPs), Chitosan (C-NPs), Gold/hydroxyapatite (Au/HA-NPs), Chitosan/hydroxyapatite (CH-NPs), and Hydroxyapatite (HA-NPs) nanoparticles enhance osteogenesis in bone marrow-derived mesenchymal stem cells (BM-MSCs).1 However, the use of nanoparticles raises concerns about cytotoxicity and cell viability. Nanoparticles can disrupt the crucial cytoskeleton function when taken up by the cell, resulting in toxicity.2 The effect of bone-based nanoparticles (BNPs) on hMSCs is not entirely understood. BNPs take advantage of the proteins found in the extracellular matrix that are involved in osteoblast growth without the concern of cellular antigens3. We investigated the effect of BNPs on hMSC cell viability and osteogenic differentiation.
Materials and
Methods: Bone marrow-derived mesenchymal stem cells (BM-MSCs) were seeded in 18 wells of a 96-well plate at 2,000 cells per well. Cells were incubated in a humid incubator with 5% CO at 37°C overnight. BNPs were added to columns two, four, and six at a concentration of 20 ug/mL. On days one, three, and five, 8 uL of CCK-8 were added to one column of control and one column of experimental cells. Cells were incubated for four hours, and a microplate reader was used to record absorbance at 450 nm. An additional 15 wells were seeded in an 18-well plate and incubated overnight. Cells were treated with varying concentrations of BNPs (0, 5, 10, 20, 50 ug/ml) and incubated for one week. CCK-8 was added to each well and incubated for four hours before absorbance was read using a microplate reader. To examine the effects of BNPs on osteogenic differentiation, six wells of a 24-well plate were seeded with hMSCs in mesenchymal stem cell basal medium. When cells reached 80% confluency, BNPs were added to column three at a concentration of 20 ug/mL and incubated overnight. Osteogenic differentiation medium was added to columns two and three. Medium was changed every three days. After 14 days, cells were stained using Alizarin Red and imaged.
Results, Conclusions, and Discussions:
Results: Figure 1A shows the comparison of stem cell absorbance with and without BNPs on days one, three, and five after the addition of BNPs. Figure 1B shows the results of varying BNP concentrations on cell viability after seven days. The results indicate that BNPs do not disrupt regular cellular function. It is expected that the addition of BNPs will show more Alizarin red staining compared to the control wells.
Discussion: These results indicated that BNPs may positively affect BM-MSC viability after five days but have no significant effect before day five. When considering BNP concentration, 10 ug/mL appears to result in slightly higher cell viability after seven days. Based on these results, BNPs are not toxic to BM-MSCs. Increased Alizarin Red staining would indicate that there is greater calcium deposition when MSCs are treated with BNPs showing a positive effect on osteogenic differentiation.
Conclusion: The addition of BNPs affects the behavior of bone marrow-derived mesenchymal stem cells. (BM-MSCs) The presence of BNPs results in higher cell viability after five days, while the concentration of BNPs has a minor effect on the viability of BM-MSCs. The addition of BNPs to BM-MSCs may increase osteogenesis.
Acknowledgements (Optional): Members of the Wang Labq
