With dental implant treatment becoming the gold standard, the need for effective bone augmentation prior to implantation has grown. This study aims to evaluate a bone augmentation strategy integrating three key growth factors: bone morphogenetic protein-2 (BMP-2), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF). Collagen scaffolds incorporating BMP-2, IGF-1, or VEGF were fabricated and categorized into five groups based on their content: scaffold alone; BMP-2 alone (BMP-2); BMP-2 and IGF-1 (BI); BMP-2, IGF-1, and VEGF (BIV); and BMP-2 and IGF-1 with an earlier release of VEGF (BI + V). The prepared scaffolds were surgically implanted into the calvarias of C57BL/6JJcl mice, and hard tissue formation was assessed after 10 and 28 days through histological, tomographic, and biochemical analyses. The combination of BMP-2 and IGF-1 induced a greater volume of hard tissue augmentation compared with that of BMP-2 alone, regardless of VEGF supplementation, and these groups had increased levels of cartilage compared with others. The volume of hard tissue formation was greatest in the BIV group. In contrast, the BI + V group exhibited a hard tissue volume similar to that of the BI group. While VEGF and CD31 levels were highest in the BIV group at 10 days, there was no correlation at the same time point between hard tissue formation and the quantity of M2 macrophages. In conclusion, the simultaneous release of BMP-2, IGF-1, and VEGF proved to be effective in promoting bone augmentation.

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-L-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via oxidative stress.

OBJECTIVE: To evaluate the 10-year influence of soft tissue height (STH) on crestal bone level changes (CBC) in bone-level implants with non-matching internal conical connections.
METHODS: From the initial 97 patients, 59 (19 men, 40 women, age 55.86 ± 9.5 years) returned for the recall visit. Based on baseline STH, they were categorized into T1 (thin STH ≤2 mm, n = 33), T2 (thin STH augmented with allogenic tissue matrix (ATM), n = 32), and C (thick STH >2 mm, n = 32). Implants were placed in the posterior mandible using a one-stage approach and received single screw-retained restorations. Clinical (PPD, BOP, PI) and radiographic examinations were conducted after 10 years, with CBC calculated mesial and distal to each implant.
RESULTS: After 10 years, implants in surgically thickened (T2) or naturally thick STH (C) showed bone gains of 0.57 ± 0.55 mm and 0.56 ± 0.40 mm, respectively (p < 0.0001) shifting from an initial CBC of -0.21 ± 0.33 mm to 0.36 ± 0.29 mm in the thick STH group and -0.2 ± 0.35 mm to 0.37 ± 0.29 mm in the surgically thickened STH group. Implants in naturally thin STH yielded a non-significant trend of bone loss (-0.12 ± 0.41 mm; p > 0.05).
CONCLUSIONS: Implants in thin STH (≤2 mm) exhibited greater CBC over the study period. Significant bone gains were observed in thick STH cases, indicating that naturally thick STH or STH augmentation with ATM may contribute to maintain CBC in long-term around implants.
CONCLUSIONS: This is the first long-term follow-up study suggesting that adequate soft tissue height around implants helps maintain stable peri‑implant bone levels. While tissue thickness plays a key role, other factors also interact with peri‑implant tissue height to sustain crestal bone stability over time.

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.

Alveolar bone defects caused by tooth loss often lead to challenges in implant dentistry, with a need for development of optimal bone biomaterials to predictably rebuild these tissues. To address this problem, we fabricated a novel bone block using platelet-rich fibrin (PRF) and Deproteinized Bovine Bone Mineral (DBBM), and characterized their mechanical and biological properties. The bone block was prepared by mixing DBBM, Liquid-PRF, and Solid-PRF fragments in various combinations as follows: (1) BLOCK-1 made with Solid-PRF fragments + DBBM, (2) BLOCK-2 made with Liquid-PRF + DBBM, (3) BLOCK-3 made with Solid-PRF fragments + Liquid-PRF + DBBM. The time for solidification and the degradation properties were subsequently recorded. Scanning electron microscopy (SEM) and tensile tests were carried out to investigate the microstructure and mechanical properties of each block. The bioactivity of the three groups towards osteoblast differentiation was also evaluated by culturing cells with the conditioned medium from each of the three groups including cell proliferation assay, cell migration assay, alkaline phosphatase (ALP) staining, and alizarin red staining (ARS), as well as by real-time PCR for genes encoding runt-related transcription factor 2 (RUNX2), ALP, collagen type I alpha1(COL1A1) and osteocalcin (OCN). BLOCK-3 made with Solid-PRF fragments + Liquid-PRF + DBBM had by far the fastest solidification period (over a 10-fold increase) as well as the most resistance to degradation. SEM and tensile tests also revealed that the mechanical properties of BLOCK-3 were superior in strength when compared to all other groups and further induced the highest osteoblast migration and osteogenic differentiation confirmed by ALP, ARS and real-time PCR. PRF bone blocks made through the combination of Solid-PRF fragments + Liquid-PRF + DBBM had the greatest mechanical and biological properties when compared to either used alone. Future clinical studies are warranted to further support the clinical application of PRF bone blocks in bone regeneration procedures.

While previous studies have demonstrated the role of ubiquitin-conjugating enzyme 2C (UBE2C) in promoting β-cell proliferation and cancer cell lineage expansion, its specific function and mechanism in bone marrow mesenchymal stem/stromal cells (BMSCs) growth and differentiation remain poorly understood. Our findings indicate that mice with conditional Ube2c deletions in BMSCs and osteoblasts exhibit reduced skeletal bone mass and impaired bone repair. A significant reduction in the proliferative capacity of BMSCs was observed in conditional Ube2c knockout mice, with no effect on apoptosis. Additionally, conditional Ube2c knockout mice exhibited enhanced osteoclastic activity and reduced osteogenic differentiation. Furthermore, human BMSCs with stable UBE2C knockdown exhibited diminished capacity for osteogenic differentiation. Mechanistically, we discovered that UBE2C binds to and stabilizes SMAD1/5 protein expression levels. Interestingly, UBE2C\'s role in regulating osteogenic differentiation and SMAD1/5 expression levels appears to be independent of its enzymatic activity. Notably, UBE2C regulates osteogenic differentiation through SMAD1/5 signaling. In conclusion, our findings underscore the pivotal role of UBE2C in bone formation, emphasizing its contribution to enhanced osteogenic differentiation through the stabilization of SMAD1/5. These results propose UBE2C as a promising target for BMSC-based bone regeneration.

Gain-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) can cause high-bone-mass (HBM) phenotype, with 19 identified mutations so far. The A242T mutation in LRP5 has been found in 9 families, making it one of the most prevalent mutations. However, the correlation between the A242T mutation and HBM phenotype remains unverified in animal models. This study aimed to investigate the bone properties in a new transgenic mouse model carrying the LRP5 A241T missense mutation, equivalent to A242T in humans. Heterozygous Lrp5A241T mice were generated using CRISPR/Cas9 genome editing. Body weight increased with age from 4 to 16 weeks, higher in males than females, with no difference between Lrp5A241T mice and wild-type control. Micro-CT showed slightly longer femur and notably elevated trabecular bone mass of the femur and fifth lumbar spine with higher bone mineral density, bone volume fraction, and trabecular thickness in Lrp5A241T mice compared to wild-type mice. Additionally, increased cortical bone thickness and volume of the femur shaft and skull were observed in Lrp5A241T mice. Three-point bending tests of the tibia demonstrated enhanced bone strength properties in Lrp5A241T mice. Histomorphometry confirmed that the A241T mutation increased bone formation without affecting osteoblast number and reduced resorption activities in vivo. In vitro experiments indicated that the LRP5 A241T mutation enhanced osteogenic capacity of osteoblasts with upregulation of the Wnt signaling pathway, with no significant impact on the resorptive activity of osteoclasts. In summary, mice carrying the LRP5 A241T mutation displayed high bone mass and quality due to enhanced bone formation and reduced bone resorption in vivo, potentially mediated by the augmented osteogenic potential of osteoblasts. Continued investigation into the regulatory mechanisms of its bone metabolism and homeostasis may contribute to the advancement of novel therapeutic strategies for bone disorders.

OBJECTIVE: To investigate the effect of sinusoidal alternating electromagnetic field (SEMF) on fracture healing and its mechanism.
METHODS: Femoral fracture model was established using SPF male Wistar rats, 30 model rats were randomly divided into model control (MC) and SEMF groups with 15 rats in each group. The SEMF group was given 50 Hz 1.8 mT for 90 min every day, and the MC group was not treated. X-ray examinations were performed every two weeks to determine the formation of bone scabs in each group of rats. Three rats were sacrificed after 2 and 4 weeks of treatment in both groups. Protein was extracted from the fractured femurs, and the expression of type Ⅰ collagen (COL-1), Osterix (OSX), Runt-related transcription factor 2 (RUNX2), and vascular endothelial growth factor (VEGF) protein level was detected by immunoblotting. After 8 weeks, the femur on the operated side was taken for micro-CT scanning to observe fracture healing, angiography to observe blood vessel growth, and organs such as hearts, livers, spleens, lungs, and kidneys were taken for safety evaluation by hematoxylin-eosin staining (HE staining).
RESULTS: The bone scab scores of the SEMF group were significantly higher than those of the MC group after 2, 4, 6, and 8 weeks of treatment (all P<0.01); the fracture healing of the SEMF group was better than that of the MC group after 8 weeks, and the bone volume scores of the two groups were 0.243±0.012 and 0.186±0.008, respectively, with statistically significant differences (P<0.01); and the number of blood vessels in the SEMF group was also more than that of the MC group after 8 weeks. The results of protein blotting method showed that the protein expression of VEGF, COL-1, RUNX2, and OSX was higher in the SEMF group than in the MC group after 2 and 4 weeks of treatment (all P<0.05), and the HE staining showed that there was no abnormality in histopathological observation of examined organs in both groups.
CONCLUSIONS: SEMF can accelerate fracture healing by promoting the expression of osteogenic factors and vascular proliferation without significant adverse effects.
目的: 观察正弦交变电磁场（SEMF）对骨折愈合的影响并探讨其影响骨折愈合的作用机制。方法: 30只无特定病原体级雄性Wistar大鼠建立骨折模型后按随机分组法分为模型对照组和SEMF组，每组15只。SEMF组每天给予50 Hz 1.8 mT的SEMF 90 min，模型对照组不做任何处理。每两周进行X射线检查判断各组大鼠骨痂形成情况，在电磁场干预后的第二周和第四周各组分别处死3只大鼠并取术侧骨折处骨组织提取蛋白，蛋白质印迹法检测Ⅰ型胶原蛋白（COL-1）、Osterix蛋白（OSX）、Runt相关转录因子2（RUNX2）、血管内皮生长因子（VEGF）蛋白的表达。8周后取术侧股骨行显微CT扫描观察骨折愈合、行血管造影观察血管生长情况，并取心、肝、脾、肺、肾等器官采用苏木精-伊红染色（HE染色）进行安全性评价。结果: SEMF组治疗2、4、6、8周后骨痂评分均显著高于模型对照组（均P<0.01）；8周后SEMF组骨折愈合情况优于模型对照组，两组骨体积分数分别为0.243±0.012和0.186±0.008，差异有统计学意义（P<0.01）；8周后SEMF组血管数也均优于模型对照组。蛋白质印迹法结果显示，SEMF组治疗2、4周后VEGF、COL-1、RUNX2、OSX蛋白表达均高于模型对照组（均P<0.05）。HE染色结果显示，SEMF组和模型对照组各器官组织病理学观察均无异常。结论: SEMF能够通过促进不同时期成骨因子的蛋白表达和血管增生从而加速骨折的愈合，并且无明显不良反应。.

OBJECTIVE: The aim of this study was to understand the temporal and spatial distribution of canonical endochondral ossification (CEO) and non-canonical endochondral ossification (NCEO) of the normal growing rat condyle, and to evaluate their histomorphological changes following the simultaneous hypotrophy of the unilateral masticatory closing muscles with botulinum toxin (BTX).
METHODS: 46 rats at postnatal 4 weeks were used for the experiment and euthanized at postnatal 4, 8, and 16 weeks. The right masticatory muscles of rats in experimental group were injected with BTX, the left being injected with saline as a control. The samples were evaluated using 3D morphometric, histological, and immunohistochemical analysis with three-dimensional regional mapping of endochondral ossifications.
RESULTS: The results showed that condylar endochondral ossification changed from CEO to NCEO at the main articulating surface during the experimental period and that the BTX-treated condyle presented a retroclined smaller condyle with an anteriorly-shifted narrower articulating surface. This articulating region showed a thinner layer of the endochondral cells, and a compact distribution of flattened cells. These were related to the load concentration, decreased cellular proliferation with thin cellular layers, reduced extracellular matrix, increased cellular differentiation toward the osteoblastic bone formation, and accelerated transition of the ossification types from CEO to NCEO.
CONCLUSIONS: The results suggest that endochondral ossification under loading tended to show more NCEO, and that masticatory muscular hypofunction by BTX had deleterious effects on endochondral bone formation and changed the condylar growth vector, resulting in a retroclined, smaller, asymmetrical, and deformed condyle with thin cartilage.

The silent information regulator T1 (SIRT1) is linked to longevity and is a crucial mediator of osteoblast function. We investigated the direct role of Sirt1 during bone modeling and remodeling stages in vivo using Tamoxifen-inducible osteoblast-specific Sirt1 conditional knockout (cKO) mice. cKO mice exhibited lower trabecular and cortical bone mass in the distal femur. These phenotypes were coupled with lower bone formation and bone resorption. Metabolomics analysis revealed that the metabolites involved in glycolysis were significantly decreased in cKO mice. Further analysis of the quantitative acetylome revealed 11 proteins with upregulated acetylation levels in both the femur and calvaria of cKO mice. Cross-analysis identified four proteins with the same upregulated lysine acetylation site in both the femur and calvaria of cKO mice. A combined analysis of the metabolome and acetylome, as well as immunoprecipitation, gene knockout, and site-mutation experiments, revealed that Sirt1 deletion inhibited glycolysis by directly binding to and increasing the acetylation level of Glutamine oxaloacetic transaminase 1 (GOT1). In conclusion, our study suggested that Sirt1 played a crucial role in regulating osteoblast metabolism to maintain bone homeostasis through its deacetylase activity on GOT1. These findings provided a novel insight into the potential targeting of osteoblast metabolism for the treatment of bone-related diseases.