Stimulation of osteoblastic differentiation and mineralization in MC3T3-E1 cells by antler and fermented antler using Cordyceps militaris

Ethnopharmacological relevance: For thousands of years antlers have been used in Asian countries to promote rapid healing, treat weight loss, slow growth in children, strengthen weak bones, and alleviate cold hands and feet. Aim of the study: The present study was performed to examine the effect of fermentation on the ability of antler to act as a stimulator of bone growth. Materials and methods: This study used pre-osteoblast MC3T3-E1 cells to examine factors related to bone growth, such as cell proliferation, production of alkaline phosphatase (ALP), and deposition of extracellular matrix proteins (e.g.; collagens, osteonectin, bone sialoprotein (BSP)), via the treatment of non-fermented and fermented antler. Results: Antler fermentation using Cordyceps militaris was carried out at 25 °C for seven days. The total content of sugar, sialic acid, and protein increased with fermentation time. Cell proliferation was greater in the fermented antler- (FA-) treated groups than in the NFA- (non-fermented antler-) treated groups, in which proliferation increased significantly up to 137% of the basal value. Significant increases in mRNA expression and ALP activity were found at FA concentrations of 50-100 μg/ml; at 100 μg/ml the activity had increased 119% compared to the control activity. For NFA and FA the expression levels of type I collagen mRNA significantly increased in a dose-dependent manner at all treatment doses. However, significant differences between the antler groups were not observed. Mineralization significantly increased by NFA and FA treatment to 183% and 241%, respectively, when compared to colostrum, as a positive control (165%). Conclusions: Antler treatment increased the proliferation of osteoblasts and bone matrix proteins, such as type I collagen and BSP. Antler fermented with Cordyceps militaris showed enhanced activity, and its stimulatory effects on cell proliferation and ALP production were greater than those of NFA. We surmise that these increases in activity were related to increased sialic acid content. Therefore, the results of this study suggest that the physiological effects of antler, including bone growth, may be increased through the fermentation process.