Introduction: Bone tissue engineering aims to induce new functional bone regeneration via the synergistic combination of biomaterials, cells, and factor therapy. The interaction between biomaterials and the surrounding tissue at the implant interface is essential for success or failure of implants. Biomaterial interacts with both osteoblasts and osteoclasts. Bone remodeling is controlled by two equal equal, but opposing, forces: bone formation by osteoblasts and bone destruction or resorption by osteoclasts. Successful bone remodeling should be balanced between bone formation and bone resorption. Ethyl-3, 4-dihydroxybenzoate (E-3, 4-DHB) is a component of the Rubus coreanus extract, and the extract has recently been shown to have a bone protecting-effect on postmenopausal osteoporosis in ovariectomized rats. However, the dual function of E-3, 4-DHB of inducing osteoblastic differentiation and inhibiting osteoclast differentiation has not been previously evaluated and warrants further evaluation.
Materials and methods: The osteoinductive ability of E-3, 4-DHB in preosteoblats and human mesenchymal stem cells (hMSCs) was examined. E-3, 4-DHB for future use in bone tissue engineering was evaluated by examination of early markers of differentiation (such as alkaline phosphatase [ALP], activity runt-related transcription factor 2 [Runx-2], and collagen type I expression) and late markers of osteoblast differentiation (bone nodule formation). Also, to evaluate the osteoinductive activity in a three-dimensional (3D) dynamic culture system, hMSCs were cultured in a HARV bioreactor for 21 days. In addition, differentiation of osteoclasts in response to E-3, 4-DHB was observed with TRAP activity and TRAP staining. Finally, in vivo study, alginate gel comprised of E-3, 4-DHB and cells was transplanted into the back subcutis of mice.
Results and discussion: Our results have shown that the ALP activity, Runx2 expression, collagen type 1 expression, and bone nodule formation of E-3, 4-DHB treated group have been significantly increased in comparison with the untreated group. hMSCs cultured in a bioreactor with osteogenic media containing E-3, 4-DHB showed more red or black color using ARS and von Kossa staining, compared with the hMSCs cultured in osteogenic media (control). In our in vitro study using RAW264.7 cells, we found that E-3, 4-DHB dose-dependently inhibited RANKL-induced osteoclast differentiation. These results suggest that E-3, 4-DHB has an inhibitory effect of osteoclast formation. Also, we verified the osteoinductive activity through animal testing.
Conclusion: Our results show that E-3, 4-DHB might have beneficial effects through regulation of both osteoblast and osteoclast differentiation. Therefore, we suggest that E-3, 4-DHB could be a strong candidate for dual regulation to increase osteoblast differentiation and decrease osteoclast differentiation.