The bones of the human body support the structures of the body and provide protection for a person’s internal organs. Bone metabolic diseases are on the rise due to a significant increase in life expectancy over a short period of time. Therefore, we investigated the osteoblast differentiation promoting and osteoclastogenesis inhibitory activities of fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf). We evaluated the alkaline phosphatase (ALP) activity of MC3T3-E1 mouse calvarial-derived osteoblasts. We also evaluated expression of ALP, osteocalcin (OCN), and runt-related transcription factor 2 (Runx2), which regulate osteoblast differentiation. To assess effects on osteoclast formation, tartrate-resistant acid phosphatase (TRAP) activity in RAW264.7 cells was analyzed. ALP activity increased by 121-136% and 140-156%, respectively in the presence of HR1901-BS and HR1901- BSaf. Expression of osteoblast differentiation factor also increased significantly. We also confirmed that HR1901-BS and HR1901-BSaf decreased TRAP activity in osteoclasts by 35-47% and 23-39%, respectively. Our results showed that fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf) increase bone mineralization and osteoblast differentiation activity in MC3T3-E1 cells, and inhibit bone resorption activity in RAW264.7 cells. In conclusion, fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf) can be used as an effective natural resource for preventing and treating bone-related diseases.

본 연구는 방사선 육종 차조기와 백출 복합물의 조골세포 분화 활성 및 파골세포 형성 억제를 조사하였다. 차조기와 백출 복합물은 MG-63 세포에서 ALP 활성 및 arlizarin red 염색을 확인하였고 조골세포 형성의 영향은 RAW 264.7 세포에서 TRAP 활성과 TRAP 염색을 진행하였다. 세포 독성 시험에서 차조기와 백출 복합물은 50 ㎍/㎖ 농도 이하에서 안전한 것으로 확인되었다. ALP 활성 및 골석회화 형성 능력은 대조군보다 활성이 낮았으나, 파골세포에서 TRAP 활성을 유의적으로 감소시켰으며, 효과적으로 TRAP(+) 다핵세포를 억제하였다. 따라서 차조기와 백출 복합물은 골 흡수 억제 활성을 향상시켜 뼈 관련 질환의 예방 및 치료에 효과적인 것으로 보여진다.

Calcium is the most abundant stored mineral in the human body and is especially vital for bone health; thus, calcium deficiency can cause bone-related diseases, such as osteopenia and osteoporosis. However, a high concentration of serum calcium, which is commonly known as hypercalcemia, can also lead to weakened bones and, in severe cases, osteosarcoma. Therefore, it is necessary to maintain the concentration of calcium that is appropriate for bone biology. In the present study, we aimed to elucidate the effects of high concentration of calcium, approximately 2 folds the normal calcium level, on osteoblast differentiation. The CaCl2 treatment showed dose-dependent suppression of the alkaline phosphatase activity and mineralized nodule formation. Calcium showed cytotoxicity at an extremely high concentration, but a moderately high concentration of calcium that results in inhibitory effects to osteoblast differentiation showed no signs of cytotoxicity. We also confirmed that the CaCl2 treatment repressed the mRNA expression and protein abundance of various osteogenic genes and transcriptional factors. Considered together, these results indicate that a high concentration of calcium negatively regulates the osteoblast differentiation of C2C12 cells.

Berberine has been used clinically for more than a decade to treat various diseases, has been shown to be effective in osteoblast differentiation, and is a potential treatment option for osteoporosis. However, compared with existing osteoporosis drugs, berberine is somewhat less effective. This study aimed to identify a new compound with efficacy superior to that of berberine. The osteogenic activities of various berberine derivatives were evaluated via cell differentiation in C2C12 preosteoblast cell lines. Alkaline phosphatase (ALP) staining assay and structure– activity relationship demonstrated that compound 2b had a potent osteogenic effect. Furthermore, compound 2b dose dependently increased ALP activity and showed no toxicity at the effective concentration, indicating its efficacy. Additionally, compound 2b upregulated BMP2-induced transcriptional activity in a promoter activity assay using ALP, BSP, and OC promoters.

Nerve injury induced protein 1 (Ninjurin1) was originally described in neuroscience in which the expression of Ninjurin1 was regulated by Schwann cells and dorsal root ganglion neuronal cells of damaged nerve tissues. After the first discovery of Ninjurin1, the widespread expression of Ninjurin1 in adult and embryonic tissues have been observed including bone marrow, peripheral blood lymphocytes, thymus, and heart. Currently, the Ninjurin1 mediated positive regulation of pre-osteoclasts fusion and osteoclast development was reported. The bone homeostasis is dynamically balanced by bone-resorbing activity of osteoclast and bone-forming activity of osteoblast. Until now, the role of Ninjurin1 was never been described in osteoblastogenesis. Therefore, in this study, we have evaluated the expression and function of Ninjurin1 in osteoblast. The ample expression of Ninjurin1 was observed in bone marrow of mouse tibia sections but it was barely expressed in osteocytes. And also the expression levels of Ninjurin1 were gradually increased during osteoblast differentiation of calvarial pre-osteoblast, C2C12, and MC3T3-E1 cells. Importantly, the expression of Ninjurin1 was increased in the absence of osteogenic stimulus, BMP2, which suggests the cell density-dependent regulation of Ninjurin1. The controlled expression of Ninjurin1 by cell-density was evidently shown in not only pre-osteogenic osteoblast lineage cells but also in non-osteogenic cancer cells such as HeLa and A549 cells. In addition, the isoform-specific knockdown of Ninjurin1 remarkably reduced the alkaline phosphatase (ALP)-positive osteoblast differentiation. Thus, our results suggest a previously unappreciated mechanism of Ninjurin1 expression and also suggest its role on osteoblastogenesis.

Osteoporosis is a common disease characterized by bone mass reduction, leading to an increased risk of bone fracture, and it is caused by an imbalance of osteoblastic bone formation and osteoclastic bone resorption. Current osteoporosis drugs aim to reduce the risk of bone fracture, either by increasing osteoblastic bone formation or decreasing osteoclastic bone resorption. However, osteoblasts and osteoclasts are closely coupled, such that any reagent altering the differentiation or activity of one eventually affects the other. This tight coupling between osteoblasts and osteoclasts not only limits the therapeutic efficacy but also threatens the safety of osteoporosis drugs. This review will discuss the biological mechanisms of action of currently approved medications for osteoporosis treatment, focusing on the osteoblast–osteoclast coupling.

Endoplasmic reticulum (ER) stress is well known as a suppressor in osteoblast differentiation and activating transcription factor 3 (ATF3) could be induced by a various extracellular signals including cytokines, hormones, DNA damage. Up to date, although the role of ATF3 have been studied, the function of ATF3 in osteoblast differentiation is still not clear yet. Our study showed that expression level of ATF3 could be incresed by tunicamycin which is ER stress inducer in preosteoblasts. BMPs, which are secreted by osteoblasts, can be important regulators in osteogenic differentiation. The stress-responsive transcription factor ATF3 is a negative regulator of osteoblast differentiation in MC3T3-E1 cells. In this study, we verified that BMP2-stimulated osteoblast differentiation could be inhibited by over-expressed ATF3 through regulating alkaline phosphatase (ALP) expression and activation.

Nicotine of tobacco component has a controversial impact in the clinical outcome of dental implants. Although numerous nicotine effects on bone healing around implants have been presented, it is rarely reported in vitro study about normal human osteoblast(NHost) from oral and maxillofacial area at the surface of implants. The purpose of the present study was to evaluate the effect of nicotine on the proliferation and differentiation response of NHost to plasmatic and salivary levels of nicotine reported in smokers at the surface of screw-type plasma-sprayed titanium implants. NHosts were seeded on the surface of titanium implants and cultured for 21 days in α-MEM supplemented with 10% FBS, 50mg/ml ascorbic acid, 5mM β-glycerophosphate and 100nM dexamethasone. Seeded implants were exposed to various nicotine concentration(0.05-0.5mg/ml) from 1 to 21 days, and characterized for cell morphology, proliferation, differentiation, alkaline phosphatase(ALP) activity and ionized calcium concentration(Cai) of medium. Continuous exposure to higher nicotine concentration(above 0.3mg/ml) induced a dose- and time-dependent vacuolation of the cytoplasm, and a tendency to detach from the implant surface. 0.05mg/ml(lower nicotine concentration) did not cause significant effects in the cell proliferation and ALP activity. 0.1-0.2mg/ml caused evident dose-dependent effects in increased cell proliferation, ALP activity and earlier onset of matrix mineralization at levels up to 0.2mg/ml, while a dose-dependent inhibitory effect at 0.3-0.5mg/ml. Cai concentration of control group was decreased at 14 days. Increased Cai concentration at 0.1-0.2mg/ml, decreased Cai concentration at 0.3mg/ml and no change at 0.5mg/ml during the culture period were seen. It suggested that nicotine concentration could paly an role in modulating NHost activity as a contributing factor associated with proliferation and differentiation of NHost at the surface of implants.

In this study, we investigated the effect of bisphosphonate on the osteoblastic differentiation of human dental stem cells (hDPSCs). In the first experiment, we evaluated the effect of bisphosphonate on the differentiation of hDPSCs into osteoblasts by alkaline phosphatase staining after culturing hDPSCs. As a result, on day 13, the osteogenic differentiation of hDPSC was suppressed at 5 μM in clodronate and 2 μM in zolendronate. In NBP, osteogenic differentiation is more suppressed. In second experiment, cytotoxicity and proliferation test, the cell proliferation (examined by MTT assay) was more suppressed as the concentrations of zolendronate were larger than those of alendronate and clodronate. Western blotting, a third experiment, was found that AKT phosphorylation was inhibited in cell signaling proteins involved in cell proliferation inhibition and death by bisphosphonate concentration. In human dental stem cells, bisphosphonates inhibit osteoblast differentiation, and this phenomenon is clearly observed in NBPs (zolendronate), and it has been found that it is related to AKT phosphorylation of cell signaling proteins.

To determine the medicinal properties of Pleurotus cornucopiae and Safflower Oil (PS), the osteoblast effect was investigated. PS can prevent estrogen deficiency-induced bone loss. It was taken the measurements of biochemical factors such as serum osteocalcin, alkaline phosphatase, IGF-1, and bone status. Forty adult female Sprague-Dawley rats were randomly assigned to four equal groups for 12 weeks of treatment: (1) sham operation (SHAM) + vehicle; (2) ovariectomy (OVX) + vehicle; (3) OVX + 17β-estradiol (25 μg/kg); (4) OVX + PS (300 mg/kg/d, PO). At the end of the experiment, bone turnover, and trabecular microarchitecture were assessed by micro-computed tomography (micro-CT), and histopathology, respectively. Ovariectomized rats experienced remarkable increases in global volumetric bone density, and trabecular microarchitecture deterioration. These OVX-induced pathological changes are reversible in that most of them could be mostly corrected upon 17β-estradiol treatment. PNS treatment significantly prevented bone mass loss and microarchitecture deterioration. Moreover, PS enhanced osteoblast activity but suppressed osteoclast turnover, as evidenced by decreased levels of serum C-terminal telopeptides of type I collagen and elevated levels of alkaline phosphatase. PS mitigates estrogen deficiency-induced deterioration of trabecular microarchitecture.

In this paper, the in vitro biocompatibility of graphene film (GF) with osteoblasts was evaluated through cell adhesion, viability, alkaline phosphatase activity, F-actin and vinculin expressions, versus graphite paper as a reference material. The results showed that MG-63 cells exhibited stronger cell adhesion, better proliferation and viability on GF, and osteoblasts cultured on GF exhibited vinculin expression throughout the cell body. The rougher and wrinkled surface morphology, higher elastic modulus and easy out-of-plane deformation associated with GF were considered to promote cell adhesion. Also, the biomineralization of GF was assessed by soaking in simulated body fluid, and the GF exhibited enhanced mineralization ability in terms of mineral deposition, which almost pervaded the entire GF surface. Our results suggest that graphene promotes cell adhesion, activity and the formation of bonelike apatite. This research is expected to facilitate a better understanding of graphene-cell interactions and potential applications of graphene as a promising toughening nanofiller in bioceramics used in load-bearing implants.

Pleurotus cornucopiae (PC) mushrooms is found in the field and commonly known in Japan as Tamogidake mushrooms. Recently it has been reported that PC also alleviating the toxicity of heavy metals. However little is known about mechanism of the action of PC on osteoblast differentiation, especially in transcription factor. Inhibitor of DNA binding-1 (Id-1) function has been linked to the proliferation, migration, and senescence of cells, and studies about relationship between Id-1 and biological function. Therefore, this study was aimed to investigate the effect of PC on osteoblast differentiation and expression of Id-1 and Id-2. PC treatment increased ALP, Col 1 and OCN. PC treatment up-regulated the mRNA levels of Id-1 and Id-2 genes. This PC–induced osteoblast differentiation is more effective in lower doses rather than high doses. This study shows that expression of Id-1 and Id-2 was increased in a dose-dependent manner during PC-induced osteoblast differentiation.

In the present study, we evaluated the effect of CGM on osteogenic differentiation of cultured osteoblasts, and determined whether combination treatment with LLLT had synergistic effects on osteogenic differentiation. The results indicated that CGM promoted proliferation, differentiation, and mineralization of osteoblasts at the threshold concentration of 10 μg/ml; whereas, CGM showed cytotoxic properties at concentrations above 100 μg/ml. ALP activity and mineralization were increased at concentrations above 10 μg/ml . CGM in concentrations up to 10 μg/ml also increased the expression of osteoblast-activated factors including type I collagen, BMP-2, RUNX2, and Osterix. The CGM (50 μg/ml) and LLLT (80 mW for 15 sec) combination treatment group showed the highest proliferation levels, ALP activity, and mineralization ratios. The combination treatment also increased the levels of phosphorylated forms of p38, ATF2, PKD, ERK, and JNK. In addition, the osteoblast differentiation factors including type I collagen, BMP-2, RUNX2, and Osterix protein levels were clearly increased in the combination treatment group. These results suggested that the combination treatment of CGM and LLLT has synergistic effects on the differentiation and mineralization of osteoblastic cells.

Sambucus sieboldiana (SS) is a member of the family Caprifoliaceae and has been recommended as a functional material because of its several bioactivities. Although numerous literatures are available on the pharmacological and biological activities, the biological activity of SS in bone regeneration process has not yet been well-defined. Therefore, in this study, the effect of SS was investigated in the proliferation and differentiation of MC3T3-E1 osteoblastic cell line. The treatment of SS did not significantly affect the cell proliferation in MC3T3-E1 cells. SS significantly accelerated the mineralization and significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (OC) mRNAs, compared to the control, in the differentiation of MC3T3-E1 cells. SS significantly accelerated the decrease of osteonectin (ON) mRNA expression as compared with the control in a time-dependent manner in the differentiation of MC3T3-E1 cells. These results suggest that the SS facilitate the osteoblast differentiation and mineralization in MC3T3-E1 osteoblastic cells. Therefore, there may be potential properties for development and clinical application of bone regeneration materials.

Bioactive peptides function effectively with a minimal amount compared to proteins. Recently SPARC related modular calcium binding 1 (SMOC1) has been implicated in regulating osteoblast differentiation and limb and eye development. In this study we synthesized a peptide covering 16 amino acids derived from the extracellular calcium binding (EC) domain of SMOC1, and its effects on proliferation and osteoblast differentiation of human bone marrow mesenchymal stem cells were examined. Treatment of SMOC1 peptide did not modulate proliferation of BMSCs. However, mineralization of BMSCs was significantly increased with a dose dependent manner. Consistently expression of osteoblast differentiation marker genes including type 1 collagen and osteocalcin was also dose dependently increased. Taken together, these results suggest that peptide derived from the EC domain of SMOC1 recapitulates at least partially osteogenic function of SMOC1.

Recently, extensive research has been performed in the field of orthopedic medicine to develop cell-based therapies for the restoration of injured bone tissue. But there has been rarely reported about rehabilitaton of oral and maxillofacial bone defect using self-derived osteoblasts. Normal human osteoblast cell(NHost) was previously established into marrow-derived human mesenchymal stem cells for their capacity to proliferate and differentiate into osteoblasts under various culture conditions. The purpose of this study was to examine proliferation and differentiation of NHosts effected by growth factors with ALP activity and RT-PCR. After NHosts were cultured under basal and osteogenic medium at 37℃ and 5% CO2, they were analyzed by ALP activity and RT-PCR. BMP-2 under osteogenic medium decreased growth rate of NHosts compared to under osteogenic medium. BMP-2 under osteogenic medium induced osteoblastic differentiation in NHosts by increased ALP activity. The differentiating capacity of NHosts under osteogenic medium showed that NHosts expressed higher mRNA expression levels of OSX and OCN, while that of RUNX2 decreased after BMP-2 treatment. It suggested that NHosts having characteristics of osteoprecursor cells might be more advanced in their osteogenesis development by BMP-2, making NHosts an interesting biological tool for treatment of skeletal defects and diseases of oral and maxillofacial bone.

Diabetic patients tend to exhibit delayed bone formation and osteoblast differentiation, which results in osteopenia. Recently, numerous clinical reports suggest that 635-nm light irradiation improves bone regeneration and wound healing, and reduces pain in patients suffering from diabetes. The purpose of the present study was to test the hypothesis that 635-nm irradiation can influence bone formation by MC3T3-E1 osteoblasts cultured on high concentrations of glucose(25mmol/L D-glucose) in the presence or absence of phorbol 12-myristate 13-acetate(PMA), and to establish an in vitro pathological model of bone formation. The effect of 635-nm irradiation on bone formation was investigated using Alizarin Red S staining, and alkaline phosphatase enzyme activ ity and calcium deposition assays. In addition, gene expression of the o steogenic markers BMP-2, osterix and osteocalcin were assayed by RT-PCR. Calcium deposition by MC3T3-E1 cells was reduced in the presence of high concentrations of glucose or by PMA supplementation. However, 635-nm irradiation led to an increase in calcium deposition by MC3T3 cells, followed by increased bone mineralization. mRNA expression of BMP-2 and osterix at an early stage and of osteocalcin at a late stage was significantly upregulated by 635-nm irradiation in MC3T3-E1 cells supplemented with high concentrations of glucose. Irradiation at 635 nm increases bone mineralization in MC3T3-E1 cells cultured in vitro on high concentrations of glucose and alters osteogenic gene expression, which accelerates bone formation in hyperglycemic conditions.