Background: Although caffeine generally offers benefits to human health, its impact on bone metabolism remains unclear. Aim and Methods: This study aimed to systematically evaluate the long-term effects of caffeine administration on osteoclasts, osteoblasts, and ovariectomy-induced postmenopausal osteoporosis (OP). Results: Our in vitro findings revealed that 3.125 and 12.5 μg/mL caffeine inhibited RANKL-mediated osteoclastogenesis in RAW 264.7 cells through the MAPK and NF-κB pathways, accompanied by the inactivation of nuclear translocation of nuclear factor NFATc1. Similarly, 3.125 and 12.5 μg/mL of caffeine modulated MC3T3-E1 osteogenesis via the AKT, MAPK, and NF-κB pathways. However, 50 μg/mL of caffeine promoted the phosphorylation of IκBα, P65, JNK, P38, and AKT, followed by the activation of NFATc1 and the inactivation of Runx2 and Osterix, ultimately disrupting the balance between osteoblastogenesis and osteoclastogenesis. In vivo studies showed that gavage with 55.44 mg/kg caffeine inhibited osteoclastogenesis, promoted osteogenesis, and ameliorated bone loss in ovariectomized mice. Conclusion: Conversely, long-term intake of high-dose caffeine (110.88 mg/kg) disrupted osteogenesis activity and promoted osteoclastogenesis, thereby disturbing bone homeostasis. Collectively, these findings suggest that a moderate caffeine intake (approximately 400 mg in humans) can regulate bone homeostasis by influencing both osteoclasts and osteoblasts. However, long-term high-dose caffeine consumption (approximately 800 mg in humans) could have detrimental effects on the skeletal system.

OBJECTIVE: Recent reports indicate that sclerostin is secreted by periodontal ligament tissue-derived (PDL) cells during orthodontic force loading and that the secreted sclerostin contributes to bone metabolism. However, the detailed mechanism is poorly understood. The aim of this study was to determine how PDL cells affect bone formation.
METHODS: Rat periodontal ligament tissue was immunohistochemically stained for sclerostin. Cultured primary PDL cells, osteoblasts, and skin fibroblasts (Sfbs) isolated from rat periodontal ligament tissue, calvaria, and skin, respectively, were examined. Osteoblasts were cultured with control conditioned medium (Cont-CDM) and PDL cell culture conditioned medium (PDL-CDM) for up to 21 days. Cultured osteoblasts were then stained with alkaline phosphatase and von Kossa stain. Osteoblasts cultured in each conditioned medium were analyzed by real-time quantitative PCR for bone Gla protein (Bgp), Axin2, and Ki67 expression. PDL cells used to obtain conditioned medium were analyzed for Sost, Ectodin and Wnt1 expression and compared with expression in Sfbs.
RESULTS: Expression of sclerostin was observed in periodontal ligament tissue by immunohistochemical staining. The formation of mineralization nodules was inhibited in PDL-CDM compared with Cont-CDM in osteoblast culture. In PDL-CDM, the expression levels of Bgp and Axin2 in osteoblasts were decreased compared with Cont-CDM. In PDL cells, expression levels of Sost and Ectodin were much higher than in Sfbs; however, expression of Wnt1 was lower in PDL cells compared with Sfbs.
CONCLUSIONS: PDL cells secrete various proteins, including sclerostin and suppress osteogenesis in osteoblasts through the canonical Wnt pathway.

Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis.

The surface morphology of titanium metal is an important factor affecting its hydrophilicity and biocompatibility, and exploring the surface treatment strategy of titanium metal is an important way to improve its biocompatibility . In this study , titanium (TA4) was firstly treated by large particle sand blasting and acid etching (SLA) technology, and then the obtained SLA-TA4 was treated by single surface treatments such as alkali-heat, ultraviolet light and plasma bombardment. According to the experimental results, alkali-heat treatment is the best treatment method to improve and maintain surface hydrophilicity of titanium. Then, the nanowire network morphology of titanium surface and its biological property, formed by further surface treatments on the basis of alkali-heat treatment, were investigated. Through the cell adhesion experiment of mouse embryonic osteoblast cells (MC3T3-E1), the ability of titanium material to support cell adhesion and cell spreading was investigated after different surface treatments. The mechanism of biological activity difference of titanium surface formed by different surface treatments was investigated according to the contact angle, pit depth and roughness of the titanium sheet surface. The results showed that the SLA-TA4 titanium sheet after a treatment of alkali heat for 10 h and ultraviolet irradiation for 1 h has the best biological activity and stability. From the perspective of improving surface bioactivity of medical devices, this study has important reference value for relevant researches on surface treatment of titanium implantable medical devices.
钛金属的表面形貌是影响其亲水性及生物相容性的重要因素，探究钛金属表面处理策略是提高其生物相容性的重要途径。本文先采用大颗粒喷砂酸蚀技术（SLA）处理钛金属A4（TA4），对得到的SLA-TA4进行碱热、紫外光照及等离子体轰击等单一方式表面处理。根据实验结果得出，碱热处理是提高并保持钛金属SLA-TA4亲水性的最佳单一处理方法。随后，在碱热处理的基础上，继续研究多种表面处理方式形成的钛金属表面纳米线网络结构及其生物性能。通过小鼠胚胎成骨前体细胞MC3T3-E1黏附实验，比较了不同方式表面处理后，钛金属材料支持细胞黏附、细胞铺展的能力，并根据不同表面处理方式形成的材料表面接触角、微坑深度及粗糙度等参数，分析探讨多种表面处理方式造成的生物活性差异的机制。结果表明，经碱热处理10 h及紫外照射1 h处理后的SLA-TA4 表现出最佳的生物活性及稳定性。从提高医疗器械表面生物活性的角度考虑，本文研究结果或对钛金属植入性器械的表面处理相关研究提供有价值的参考。.

Osteoporosis, a prevalent chronic health issue among the elderly, is a global bone metabolic disease. Flavonoids, natural active compounds widely present in vegetables, fruits, beans, and cereals, have been reported for their anti-osteoporotic properties. Onion is a commonly consumed vegetable rich in flavonoids with diverse pharmacological activities. In this study, the trabecular structure was enhanced and bone mineral density (BMD) exhibited a twofold increase following oral administration of onion flavonoid extract (OFE). The levels of estradiol (E2), calcium (Ca), and phosphorus (P) in serum were significantly increased in ovariectomized (OVX) rats, with effects equal to alendronate sodium (ALN). Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) levels in rat serum were reduced by 35.7% and 36.9%, respectively, compared to the OVX group. In addition, the effects of OFE on bone health were assessed using human osteoblast-like cells MG-63 and osteoclast precursor RAW 264.7 cells in vitro as well. Proliferation and mineralization of MG-63 cells were promoted by OFE treatment, along with increased ALP activity and mRNA expression of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappaB ligand (RANKL). Additionally, RANKL-induced osteoclastogenesis and osteoclast activity were inhibited by OFE treatment through decreased TRAP activity and down-regulation of mRNA expression-related enzymes in RAW 264.7 cells. Overall findings suggest that OFE holds promise as a natural functional component for alleviating osteoporosis.

We fabricated a microfluidic chip (osteoblast [OB]-osteoclast [OC] chip) that could regulate the mixture amounts of OB and OC supernatants to investigate the effect of different supernatant distributions on osteogenesis or osteoclastogenesis. Computer-aided design was used to produce an OB-OC chip from polydimethylsiloxane. A pressure controller was assembled and different blends of OB and OC supernatants were correctly determined. OB and OC supernatants were placed on the upper panels of the OB-OC chip after differentiation for an in vitro evaluation. We then tested the changes in osteogenesis using MC3T3-E1 cells in the middle chambers. We observed that a 75:25 distribution of OB and OC supernatants was the most potent in osteogenesis. We then primed the osteogenic differentiation of MC3T3-E1 cells using an OB-OC mixed supernatant or an OB supernatant alone (supernatant ratios of 75:25 or 100:0, respectively). These cells were placed on the calvarial defect sites of rats. Microcomputed tomography and histological analyses determined a significantly higher bone formation in the group exposed to the OB-OC supernatant at a ratio of 75:25. In this study, we demonstrate the applicability of an OB-OC chip to evaluate the effect of different supernatant distributions of OB and OC. We observed that the highest bone-forming potential was in MC3T3-E1 cells treated with conditioned media, specifically the OB-OC supernatant at a ratio of 75:25.

OBJECTIVE: Tibial diaphysis fractures are common injuries resulting from high-to-low-energy traumas in patients of all age groups, but few reports currently provide complementary parameters for the assessment of bone healing processes in the postoperative period. Serum alkaline phosphatase (ALP) and the scores from the Radiographic Union Scale for Tibial Fractures (RUST) can promote new horizons in this context. Therefore, the aim of this study was to assess the behavior of ALP and RUST through within-subject comparisons from immediately post-surgery to 49 days after tibial diaphysis fracture repair.
METHODS: This article included four case studies where patients underwent the same procedures. Adults of both sexes aged 18 to 60 years with tibial fractures requiring surgery were included. After surgical intervention (T1), the patients were followed for 49 days after surgery, returning for follow-up appointments on the 21st (T2) and 49th (T3) days. At the follow-up appointments, new X-ray images were obtained, and blood samples were collected for ALP measurement.
RESULTS: Serum ALP levels increased by T2 following tibial reamed intramedullary nailing surgery. While this increase persisted into T3 for two patients, a decline was observed during the same period for the other two patients. Both events are indicative of the bone consolidation process, and RUST scores at the T3 corroborate this perspective for all patients included in this study. Considering that delta ALP (T3-T1 value) was lower in patients who exhibited the highest RUST score, we suggest that a synchronized analysis between ALP and RUST allows medics to diagnose bone consolidation.
CONCLUSIONS: Therefore, it can be concluded that the analysis of ALP alongside RUST may be complementary for evaluating bone consolidation following tibial reamed intramedullary nailing surgery, but future studies are needed to confirm this assertion.

BACKGROUND: Osteoblastic responses play a crucial role in the success of oral implants. Enhanced proliferation of osteoblast cells is associated with reduced cell mortality and an increase in bone regeneration. This study aims to evaluate the osteoblastic responses following oral implantation.
METHODS: Osteoblast stem cells were harvested and subsequently cultivated using cell culture techniques. The osteoblastic phenotype of the extracted cells was confirmed by examining the extracellular matrix. Cell morphogenesis on functionalized biomaterial surfaces was assessed through indirect immunofluorescence staining. The cellular response was investigated in the presence of two types of implant materials: titanium (Ti) and alumina-toughened zirconia (ATZ). Cell viability and apoptosis were quantitatively assessed using MTT assays and flow cytometry, respectively.
RESULTS: The survival of osteoblastic lineage cells was moderately reduced post-implantation. Viability in the Ti implant group remained at approximately 86%, while in the ATZ group, it was observed at 75%, which is considered acceptable. Moreover, there was a significant disparity in cell survival between the two implant groups (p < 0.05). Analysis of apoptosis levels at various concentrations revealed that the rate of apoptosis was 3.6% in the control group and 18.5% in the ATZ group, indicating that apoptosis or programmed cell death in the ATZ-treated group had increased nearly four-fold (p < 0.05).
CONCLUSIONS: The findings of this study indicate a reduction in osteoblastic cell line survival following implant treatment, with titanium implants exhibiting superior performance in terms of cell survival. However, it was also noted that the incidence of apoptosis in osteoblast cells was significantly higher in the presence of zirconium-based implants.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a principal regulator of calcium homeostasis through activation of the vitamin D receptor (VDR). Previous studies have shown that 2α-(3-hydroxypropyl)-1,25D3 (O1C3) and 2α-(3-hydroxypropoxy)-1,25D3 (O2C3), vitamin D derivatives resistant to inactivation enzymes, can activate VDR, induce leukemic cell differentiation, and increase blood calcium levels in rats more effectively than 1,25(OH)2D3. In this study, to further investigate the usefulness of 2α-substituted vitamin D derivatives, we examined the effects of O2C3, O1C3, and their derivatives on VDR activity in cells and mouse tissues and on osteoblast differentiation of dedifferentiated fat (DFAT) cells, a cell type with potential therapeutic application in regenerative medicine. In cell culture experiments using kidney-derived HEK293 cells, intestinal mucosa-derived CaCO2 cells, and osteoblast-derived MG63 cells, and in mouse experiments, O2C2, O2C3, O1C3, and O1C4 had a weaker effect than or equivalent effect to 1,25(OH)2D3 in VDR transactivation and induction of the VDR target gene CYP24A1, but they enhanced osteoblast differentiation in DFAT cells equally to or more effectively than 1,25(OH)2D3. In long-term treatment with the compound without the medium change (7 days), the derivatives enhanced osteoblast differentiation more effectively than 1,25(OH)2D3. O2C3 and O1C3 were more stable than 1,25(OH)2D3 in DFAT cell culture. These results indicate that 2α-substituted vitamin D derivatives, such as inactivation-resistant O2C3 and O1C3, are more effective than 1,25(OH)2D3 in osteoblast differentiation of DFAT cells, suggesting potential roles in regenerative medicine with DFAT cells and other multipotent cells.

The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and intrinsic transcriptional machinery. While rodent osteoblastic differentiation has been extensively studied, research on human osteogenesis has been limited by cell sources and existing models. Here, we systematically dissect hPSC-derived osteoblasts to identify functional membrane proteins and their downstream transcriptional networks involved in human osteogenesis. Our results reveal an enrichment of type II transmembrane serine protease CORIN in humans but not rodent osteoblasts. Functional analyses demonstrated that CORIN depletion significantly impairs osteogenesis. Genome-wide ChIP enrichment and mechanistic studies show that p38 MAPK-mediated CEBPD upregulation is required for CORIN-modulated osteogenesis. Contrastingly, the type I transmembrane heparan sulfate proteoglycan SDC1 enriched in MSCs exerts a negative regulatory effect on osteogenesis through a similar mechanism. ChIP-seq, bulk and single-cell transcriptomes, and functional validations indicated that CEBPD plays a critical role in controlling osteogenesis. In summary, our findings uncover previously unrecognized CORIN-mediated CEBPD transcriptomic networks in driving human osteoblast lineage commitment.