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).

Methods
For this study, hBMSCs were isolated of three donors undergoing a bone marrow aspiration for non-union treatment. The hBMSCs were cultured in osteogenic medium, supplemented with citrulline or alanine in the following concentrations: 0, 5, 7.5, and 10 mM. 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. All measurements were performed at days 1, 3, 7, 14, and 21. 

Results
Citrulline supplementation showed increased cellular viability and proliferation while decreasing cytotoxicity. Citrulline supplemented hBMSCs showed increased expression of osteogenic marker genes collagen I, RUNX2, osteocalcin, and osteopontin, as well as enhanced mineralisation.

Discussion
This study revealed potential cellular mechanisms behind enhanced bone regeneration upon citrulline supplementation of specific amino acids and indicated a more advanced osteoblastogenesis, extracellular matrix formation and maturation, increased cellular proliferation, and decreased cytotoxicity. In conclusion, the supplementation of citrulline has shown to possess potential beneficial effects on the osteogenic capacities of hBMSCs and is therefore of interest in the field of bone regeneration.