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Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40 yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Osteoclasts with elevated bone resorption are commonly present in postmenopausal osteoporosis, and other osteolytic pathologies. Therefore, suppressing osteoclast generation and function has been the main focus of osteoporosis treatment. Betulinic acid (BA) represents a triterpenoid mainly purified from the bark of Betulaceae. BA shows multiple biological activities, including antitumor and anti-HIV properties, but its effect on osteolytic conditions is unknown. Here, BA suppressed receptor activator of nuclear factor-kB ligand (RANKL)-associated osteoclastogenesis and bone resorptive function, as assessed by tartrate-resistant acid phosphatase (TRAP) staining, fibrous actin ring generation, and hydroxyapatite resorption assays. Mechanistically, BA downregulated the expression of osteoclastic-specific genes. Western blot analysis revealed that BA significantly interrupted ERK, JNK and p38 MAPK activation as well as intracellular reactive oxygen species (ROS) production, thus altering c-Fos and NFATc1 activation. Corroborating the above findings in cell-based assays, BA prevented ovariectomyassociated bone loss in an animal model. In conclusion, these findings suggest that BA can inhibit osteoclast generation and function as well as the RANKL signaling pathway, and might be used for treating osteoclast-related osteoporosis.

We aim to compare the effects of simvastatin and combination of simvastatin and nylestriol on bone metabolism in ovariectomized (OVX) rats. Fifty healthy Wistar female rats were randomly allocated into 5 groups: sham + saline group (group A), OVX + saline group (group B), OVX + simvastatin (5 mg·kg·d) (group C), OVX + nylestriol (0.01 mg·kg·d) (group D), and OVX + simvastatin (3 mg·kg·d) + nylestriol (0.005 mg·kg·d) (group E). All mice were orally administrated with saline or medicine dissolved in saline for 10 weeks. Body weight of rats before and after the experiment was measured. Twenty-four hours after the experiment, calcium (Ca), creatinine (Cr), and hydroxyproline in urine were detected. Serum levels of osteocalcin (bone Gla-protein, BGP) and alkaline phosphatase (ALP) were measured. Bone mineral density was detected and trabecular bone was observed after the isolation of femur and tibia. Remarkably decreased serum BGP and increased serum ALP levels were detected in group B compared with those in group A. However, notably increased serum BGP and decreased serum ALP levels were found in groups C, D, and E compared with those in group B; femoral and tibial bone mineral density decreased in group B compared with that in group A, but increased in groups C, D, and E compared with that in group B. Simvastatin and combination of simvastatin and nylestriol promote formation of new bone, increase bone density, and improve bone microstructure damage in OVX rats.

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