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Description
Introduction
Around 10% of long bone fractures display inadequate bone regeneration, leading to delayed- or non-unions. Amino acids have shown to play important roles in bone regeneration. In particular, citrulline has revealed itself as an enhancer of bone regeneration in murine bone defect healing models. Oral citrulline supplementation stimulated callus formation and lead to a balanced regulation of inflammation, resulting in a shorter inflammatory phase after fracture, thereby contributing to enhanced bone regeneration. However, the exact cellular mechanisms behind these enhanced regenerative capacities are unknown. The aim of this study was therefore to determine whether citrulline can enhance the osteogenic capacity of human Bone-marrow Mesenchymal Stem Cells (hBMSCs) as well as MG63 (osteosarcoma) cells.
Methods
For this study, hBMSCs of 5 healthy donors (mean age 59 ± 9; 4 males) and MG63 cells were cultured in osteogenic medium for a period of three weeks. Citrulline was supplemented in the following concentrations (mM): 0, 5, 7.5, 10. Alanine was administered in isocaloric control concentrations. Cellular viability was assessed through presto blue assay, proliferation by DNA quantification, and cytotoxicity through lactate-dehydrogenase assay. For the determination of the osteogenic capacity, ALP activity and alizarin red staining were performed, combined with qPCR for osteogenic marker genes. Measurements were performed at days 1, 3, 7, 14, and 21.
Results
For hBMSCs, the greatest effect of citrulline supplementation was observed at day 21, at a concentration of 7.5 mM. ALP activity and cellular viability were enhanced increasingly over time upon citrulline supplementation. Contrarily, cellular viability and ALP activity in the alanine supplemented and blank control groups remained constant per time point. Additionally, for all timepoints, citrulline supplemented hBMSCs showed enhanced cellular proliferation defined by DNA quantification as compared to the blank medium control. This effect was most evident for all time points at a concentration of 7.5 mM. Overall, alanine supplementation did not enhance cellular proliferation compared to the blank medium control.
For the MG63 cells, ALP activity was minimal, and no differences were observed between the two amino acid treated groups. Cellular proliferation and viability also did not show significant differences among the two amino acid treated groups.
Discussion
This study revealed potential cellular mechanisms behind enhanced bone regeneration upon supplementation of specific amino acids. Citrulline supplementation induced an increased expression of the early osteogenic differentiation marker ALP, and enhanced cellular proliferation in hBMSCs, suggesting that these cells contribute to the stimulatory effect of citrulline on bone regeneration. This study will further focus on the effect of citrulline on osteogenic capacities regarding mineralization and the expression of osteogenic marker genes runx2, osterix, osteocalcin, osteopontin, collagen type 1, and iNOS, and analysing the possible cytotoxic effect of citrulline supplementation.
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