RESV preparation and in vivo experimental designs

RESV used in this study was purchased from Sigma-Aldrich (Sigma-Aldrich, USA) ⁸. For in vivo treatments, RESV was firstly dissolved in dimethyl sulfoxide (DMSO) ⁴³ at 200 mg/mL, and was then diluted with saline to 10 mg/mL (containing 5% DMSO). For in vitro treatments, RESV was firstly dissolved in DMSO ⁴³ at 1 M, and was then diluted with media to 1 mM (containing 0.1% DMSO). In vitro working solution was set at 10 μM according to previous dose-determining papers on osteogenic promotion in young MSCs ^44-46. Equal amount of DMSO was applied into culture media as the control at a final concentration of 0.001%.

RESV for in vivo application was injected at 100 mg/kg intraperitoneally via the right lower quadrant of the abdominal area with mice in a head-down position, once every other day for 2 months. Control groups were injected intraperitoneally with an equal amount of 5% DMSO in saline solvent. The dosage was chosen based on previous documents and our preliminary tests below the potential highest dose not to induce obvious systemic alterations, and reports applying this dosage to affect stem cell activity in response to chemical injury in vivo ^{47, 48}. The application route was selected considering the better control of applied dose by intraperitoneal injection over feeding for a proof-of-concept study (instead of a preclinical study), and the reported efficacy of mitochondrial intervention by intraperitoneal injection of RESV ⁴⁹. The experimental duration was determined given the time needed for chronic changes of bone mass by bone remodeling according to our observations ⁵⁰. The method of chronic intermittent application every other day was based on previous reports on feeding-based RESV delaying age-related deterioration in mice ¹⁶.

As the recipients of RESV administration, littermate SAMR1 and SAMP6 mice were randomly allocated into 8 groups, i.e., 4 groups of each strain: (1) the SAMR1 4-month control group (n = 5), (2) the SAMR1 6-month control group (n = 5), (3) the SAMR1+DMSO group (started at 4 months old and sacrificed at 6 months old) (n = 4), and (4) the SAMR1+RESV group (started at 4 months old and sacrificed at 6 months old) (n = 4), (5) the SAMP6 4-month control group (n = 5), (6) the SAMP6 6-month control group (n = 5), (7) the SAMP6+DMSO group (started at 4 months old and sacrificed at 6 months old) (n = 4), and (8) the SAMP6+RESV group (started at 4 months old and sacrificed at 6 months old) (n = 5).