Amino Acids Enhance the Effects of Dexamethasone on Osteogenesis by Rat Stem Cells

Abstract

Dental pulp tissue is a desirable cell source for tooth regeneration. However, the extirpation of dental pulp tissue from the tooth root canal causes a partial defect in a sound tooth, which is unacceptable, even for the purpose of tooth regeneration. Moreover, bone or dentine formation by mesenchymal stem cells (MSCs) from dental pulp tissue is slow because of the small number and low proliferative capacity of dental pulp cells containing MSCs. To promote the proliferation and differentiation of MSCs in vitro, a novel accelerator needs to be identified in addition to dexamethasone (Dex), β-glycerophosphate (β-GP), and ascorbic acid (Vc). Therefore, the present in vitro study investigated the effects of L(+)-arginine (Arg) and L(+)-lysine (Lys) as bioactive factors that promote mineralized nodule aggregate formation in MSC subcultures. Bone marrow cells obtained from the femur shafts of rats (rBMCs) were used. Mineralized nodule aggregates were formed by rBMCs in culture medium (MEM: Dulbecco’s modified Eagle’s medium) for subcultures containing Dex and additional Lys or Arg. Aggregates were decalcified in 10% formic acid to measure the level of Ca²⁺ as an indicator of osteo- or odontogenesis. The results obtained suggest that the addition of Arg to the medium for the rBMC subculture enhanced Dex-induced osteogenesis by rMSCs. The level of Ca²⁺ in calcified nodule aggregates obtained from the rBMC subculture was significantly smaller in MEM containing Dex (MEM-Dex (+)) than in that with 1.150 mmol of Arg (p < 0.001). No significant differences were observed in the level of Ca²⁺ in aggregates formed by rBMCs between MEM-Dex (+) containing 68.4 or 136.8 mmol of Lys or 0.575 mmol of Arg and that without these amino acids (p > 0.05). The level of Ca²⁺ measured following the addition of Arg at 1.150 mmol to 2 mL of MEM-Dex (+) was high. These results indicated that Dex in the medium supplemented with Arg as a cofactor actively promoted the osteogenic activity of MSCs.

Impact Statement

To rapidly induce osteogenesis in a subculture of stem cells, a novel factor as an accelerator needs to be obtained. Amino acids may promote the differentiation of stem cells and bone formation by mesenchymal stem cells. The present study examined the effects of arginine and lysine on bone formation in vitro using bone marrow cells from rats (rBMCs). The addition of arginine to culture medium with dexamethasone in a subculture significantly induced the formation of mineralized nodule aggregates by rBMCs. Therefore, arginine is a suitable cofactor of dexamethasone to promote osteogenesis by rBMCs.