RANKL Levels Research Articles

Melatonin supports nonsurgical periodontal treatment in patients with Type 2 diabetes mellitus and periodontitis: A randomized clinical trial.

Diabetes mellitus (DM)-associated hyperinflammatory host response significantly provokes periodontal tissue destruction. In this context, the support of nonsurgical periodontal therapy in diabetics with host modulation agents is a current field of study. This clinical study aims to investigate the clinical efficacy of melatonin supplementation and discuss its possible biological mechanisms in nonsurgical periodontal treatment in patients with DM and periodontitis through some fundamental markers. In this randomized controlled and single-blind study, 27 of 55 diabetic patients with periodontitis (stage III/IV and grade C) underwent full-mouth scaling and root planing (fmSRP) alone and 28 patients underwent melatonin administration (6mg daily, 30days) in addition to fmSRP (full-mouth scaling and root planing plus melatonin, fmSRP-mel). The potential therapeutic contribution of melatonin was evaluated clinically and biochemically (gingival crevicular fluid RANKL, OPG, MMP-8, and serum IL-1β levels) at 3rd and 6th months. Melatonin (tablet, 6mg daily, 30days) did not cause any local or systemic side effects. fmSRP alone resulted in significant reduction in serum IL-1β levels, pocket depths, gingival inflammation, and gingival crevicular fluid RANKL and MMP-8 levels (p<0.05). Moreover, melatonin supplementation resulted in a more significant decrease in bleeding and pocket depth scores at probing, especially at 3months (p<0.05). Furthermore, RANKL and MMP-8 levels were significantly lower at 3months and IL-1β levels at 6months compared to the control group (p<0.05). However, OPG levels were not affected significantly by the treatments (p>0.05). Melatonin, as a host modulation agent, significantly increases the clinical efficacy of fmSRP. The reduction in periodontal inflammation and pocket depths may be a result of marked suppression of RANKL-associated osteoclastogenesis and extracellular matrix damage by melatonin.

Soluble Receptor Activator of Nuclear Factor Ligand and Osteoprotegerin Levels in Gingival Crevicular Fluid among Cigarette Smokers and Non-smokers with and without Periodontitis.

This study aimed to measure and compare the levels of soluble receptor activator of nuclear factor ligand (RANKL) and osteoprotegerin (OPG) in the gingival crevicular fluid (GCF), as well as their ratio, in smokers and nonsmokers with periodontitis. Gingival crevicular fluid samples were collected using PerioPaper strips, from 150 individuals, who were categorized into three groups: current smokers with periodontitis stage III grades C and B (n = 50), nonsmokers with periodontitis stages I and II grade A (n = 50), and control healthy individuals (n = 50). The concentrations (pg/mL) of sRANKL and OPG in the GCF were measured by enzyme-linked immunesorbent assays (ELISA). The smokers' group exhibited the highest sRANKL (pg/mL) concentration as a subsequent lead to a higher sRANKL/OPG ratio. The healthy control group exhibited higher OPG and lower sRANKL concentration, subsequently, the sRANKL/OPG ratio was reduced compared with the other study groups. However, there was no statistical significance of sRANKL and its relative ratio between periodontitis stage III grades C and B, periodontitis stages I and II grade A, and healthy control individuals. There was a statistically significant positive moderate correlation between smoking duration (years) and the sRANKL (pg/mL) concentration and a statistically significant negative moderate correlation between OPG (pg/mL) concentration and cigarettes smoked per day. As a result, compared to the other research groups, smokers with periodontitis stage III grades C and B had greater GCF concentrations of sRANKL, lower OPG, and a higher sRANKL/OPG ratio. The difference in OPG (pg/mL) level was statistically significant. However, there was no statistically significant difference in sRANKL (pg/mL) or its relative ratio, sRANKL/OPG, across the groups. A characteristic that sets periodontitis apart is alveolar bone loss. Resorption is induced by RANKL and inhibited by OPG, resulting in a relative ratio. In light of this, the levels of RANKL and OPG may be helpful indicators for monitoring the activity of periodontal disease in both smokers and nonsmokers with and without periodontitis.

Screening for early rheumatoid arthritis using high-frequency ultrasound, serum RANKL, and OPG detection.

To conduct a comparative study of high-frequency ultrasound and magnetic resonance imaging (MRI) combined with serum RANKL and OPG detection, and assess the efficacy of high-frequency ultrasound with RANKL and OPG detection in screening early rheumatoid arthritis (RA). High-frequency ultrasound and MRI were performed on both carpal joints of 60 patients with early RA, and the frequencies of synovitis, joint effusion, tenosynovitis, and bone erosion detected by high-frequency ultrasound and MRI were observed. The serum levels of receptor activator for nuclear factor-κB ligand (RANKL) and osteoclastogenesis inhibitory factor (OPG) were also detected. The serum levels of RANKL and OPG were also detected in 80 normal healthy examinees. The data were recorded and statistically analyzed. The detection rates of carpal synovitis, joint effusion, tenosynovitis, and bone erosion in RA patients by high-frequency ultrasound were 81.66%, 69.16%, 63.33%, and 1.66%, respectively, while the detection rates by MRI were 80.00%, 71.66%, 65.00%, and 15.00%, respectively. There was no significant difference between high-frequency ultrasound and MRI in the detection rates of carpal synovitis, joint effusion, and tenosynovitis in RA patients (P>0.05), while the detection rate of bone erosion by high-frequency ultrasound was significantly lower than that by MRI. The serum levels of RANKL and OPG in RA patients were 231.47 and 68.71, respectively, while the serum levels of RANKL and OPG in normal healthy examinees were 123.51 and 385.05, respectively. The serum RANKL levels of RA patients were significantly higher than those of healthy examinees, while the serum OPG levels of RA patients were significantly lower than those of healthy examinees, which were statistically significant (P < 0.01). The AUC values of the ROC curves obtained by high-frequency ultrasound and MRI combined with serum RANKL and OPG detection in Synovitis modeling were 0.955 and 0.954, respectively. The AUC values of the ROC curves obtained from the joint fusion modeling using high-frequency ultrasound and MRI combined with serum RANKL and OPG detection were 0.949 and 0.950, respectively. The AUC values of the ROC curves obtained from modeling Tenosynovitis using high-frequency ultrasound and MRI combined with serum RANKL and OPG detection were 0.941 and 0.949, respectively. The AUC values of ROC curves obtained by combining high-frequency ultrasound and MRI with serum RANKL and OPG detection in Bone erosion modeling were 0.908 and 0.923, respectively. High-frequency ultrasound combined with serum RANKL and OPG detection has comparable effects to MRI on screening early RA, providing a safe, simple, and cost-effective screening method for the early detection of RA patients. Key Points • High-frequency ultrasound and MRI can effectively detect early lesions of the wrist joints in RA patients. • Ultrasound diagnosis has the advantages of being quick, inexpensive, and repeatable, making it the preferred choice of imaging examination for RA patients at an early stage.

Resolution of peri-implant mucositis following standard treatment: A prospective split-mouth study.

Peri-implant mucositis (PIM) is a pathological precursor of peri-implantitis, but its pattern of conversion to peri-implantitis is unclear and complicated to diagnose clinically, while none of the available protocols yield complete disease resolution. The aim of this study was the evaluation of PIM responsiveness to standard anti-infective mechanical treatment (AIMT) at clinical and biomarker levels, and estimation of the diagnostic capacity of bone markers as surrogate endpoints and predictors. Systemically healthy outpatients presenting one implant exhibiting clinical signs of inflammation confined within the soft tissue (PIM) and one healthy control (HC) implant at a non-adjacent position were included. Clinical parameters and peri-implant crevicular fluid samples were collected baseline and 6 months following mechanical therapy, to assess the levels of RANKL, OPG, and IGFBP2. PIM clustering was performed using machine learning algorithms. Overall, 38 patients met the inclusion criteria. Therapy resulted in the reduction of all clinical and biological indicators, but respective values remained significantly higher compared to HC. Clinical examination noted 30% disease resolution at the 6-month follow-up, while 43% showed no active bone resorption. OPG showed positive prognostic value for treatment outcome, while the clustering based on active bone resorption did not differ in terms of therapeutic effectiveness. AIMT is effective in reducing the clinical and biological indicators of PIM, but complete clinical resolution was achieved in only 30% of the cases. Around one third of PIM patients exhibited active bone resorption bellow clinical detectability that was not associated with disease progression and poor treatment responsiveness.

RANKL and MMP8 levels in Gingival Crevicular Fluid (GCF) as oral biomarker for periodontal condition

Objective: We assessed the diagnostic potential of RANKL and MMP8 in gingivitis and periodontitis patients. Material and Methods: Sixty Seven otherwise gingivitis and periodontitis patient were enrolled at Faculty of Dentistry Universitas Brawijaya and Janti Health Service Malang. Gingival crevicular fluid was sampled modified gingival index include normal (n= 26), mild inflammation (n= 25), moderate (n=10), and severe inflammation (n=6). For periodontitis parameter, GCF was sampled pocket depth such as pocket depth 0-3,5 mm (n= 49), pocket depth 3,6-5,5 mm (n=11), pocket depth &gt;5,5 mm (n=7). RANKL and MMP8 were determined by ELISA (Enzyme-linked immunosorbent assay). Then, One Way ANOVA analysis was used. Results: RANKL showed significantly difference in gingivitis patient (p&lt;0.05), but not periodontitis patient (p&gt;0.05). However MMP8 did not show any significant difference (p&gt;0.05) between gingivitis and periodontitis. RANKL showed a good discrimination performance. RANKL and MMP8 present in the identification of periodontal disease. Conclusion: RANKL presented more accurate diagnostic marker for periodontitis than gingivitis.

Assessment of osteoprotegerin and RANKL levels and several cardiovascular risk scoring systems in acromegaly.

The OPG/RANKL (osteoprotegerin/receptor activator of nuclear factor kappa-B) system, which plays a crucial role in bone metabolism, is also associated with vascular calcification. Acromegaly is characterized by excessive secretion of growth hormone and insulin-like growth factor, and studies have demonstrated an elevated risk of cardiovascular disease in individuals with acromegaly. In this study, our objective was to investigate the relationship between OPG/RANKL and various cardiovascular risk scoring systems. We recruited 44 consecutive acromegaly patients and 41 healthy controls with a similar age and gender distribution for this study. While RANKL levels were significantly higher in the acromegaly group compared to the controls, OPG levels were not found to be significantly different between the two groups. Furthermore, within the acromegaly group, RANKL levels were significantly higher in patients with active acromegaly compared to those with controlled acromegaly. Osteoprotegerin levels showed a positive correlation with the Framingham risk score (FRS) in the acromegaly group. Linear regression analysis revealed an association of OPG with FRS (adjusted R2 value of 21.7%). OPG and RANKL may serve as potential markers for assessment of cardiovascular calcification and prediction of the cardiovascular risk status in acromegalic patients.

O-Linked N-Acetylglucosamine Transferase Regulates Bone Homeostasis Through Alkaline Phosphatase Pathway in Diabetic Periodontitis.

Periodontitis is one of the most common complications of diabetes, which seriously affects patients' life quality. It is important to find the key factors and mechanisms to improve the treatment of periodontitis. In our study, high glucose (HG) and lipopolysaccharide (LPS) treated human periodontal ligament cells (hPDLCs) and LPS treated diabetic mice was used to establish the diabetic periodontitis model in vitro and in vivo. O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) and O-linked N-acetylglucosamine transferase (OGT) protein levels were detected by western blot assay. Cell counting kit-8, alkaline phosphatase (ALP), and alizarin red staining (ARS) assays were used to observe the O-GlcNAcylation and OGT effects on cell viability and osteoblast differentiation. Co-immunoprecipitation (Co-IP) assay was used to detect the relationship between OGT and ALP. The results showed that the levels of OGT and O-GlcNAcylation were significantly increased in both cell and mouse models. ALP and ARS staining results showed that silencing of OGT or inhibition of O-glycosylation notably improved osteogenic differentiation, increased the osteoprotegerin (OPG) protein levels and decreased the receptor activator for nuclear factor-κB Ligand (RANKL) protein levels of the HG and LPS treated hPDLCs. In diabetic periodontitis mice, knockdown of OGT relieved the injury of gingival tissue, increased the ALP and OPG levels and decreased the RANKL levels. Besides, ALP interacted with OGT protein, and OGT protein was found to act on ALP serine 513 glycosylation. In conclusion, our study demonstrated that excessive O-GlcNAcylation could restrain osteoblast differentiation by O-glycosylation in ALP.

Verbascoside Inhibits/Repairs the Damage of LPS-Induced Inflammation by Regulating Apoptosis, Oxidative Stress, and Bone Remodeling.

Osteocytes play an important role as regulators of both osteoclasts and osteoblasts, and some proteins that are secreted from them play a role in bone remodeling and modeling. LPS affects bone structure because it is an inflammatory factor, despite verbascoside's potential for bone preservation and healing. Osteocytes may also be involved in the control of the bone's response to immunological changes in inflammatory situations. MLO-Y4 cells were cultured in either supplemented -MEM alone with a low serum to inhibit cell growth or media with LPS (10 ng/mL) and/or verbascoside (50 g/mL) to show the LPS effect. In our research, LPS treatment increased RANKL levels while decreasing OPG and RUNX2 expression. Treatment with verbascoside reduced RANKL expression. In our work, verbascoside increased the expression of OPG and RUNX2. In MLO-Y4 cells exposed to verbascoside, SOD, CAT, and GSH activities as well as the expression levels of bone mineralization proteins like PHEX, RUNX2, and OPG were all elevated.

Hyperbaric oxygen therapy exerts anti-osteoporotic effects in obese and lean D-galactose-induced aged rats.

Obesity accelerates the aging processes, resulting in an aggravation of aging-induced osteoporosis. We investigated the anti-osteoporotic effect of hyperbaric oxygen therapy (HBOT) in obese- and lean-aged rats through measurement of cellular senescence, hypoxia, inflammation, antioxidants, and bone microarchitecture. Obese and lean male Wistar rats were injected with 150 mg/kg/day of D-galactose for 8 weeks to induce aging. Then, all rats were randomly given either sham or HBOT for 14 days. Metabolic parameters were determined. Expression by bone mRNA for cellular senescence, hypoxia, inflammation, antioxidative capacity, and bone remodeling were examined. Micro-computed tomography and atomic absorption spectroscopy were performed to evaluate bone microarchitecture and bone mineral profiles, respectively. We found that HBOT restored the alterations in the mRNA expression level of p16, p21, HIF-1α, TNF-α, IL-6, RANKL, RANK, NFATc1, DC-STAMP, Osx, ALP, and Col1a1 in the bone in obese-and lean- aging rats. In obese-aging rats, HBOT increased the level of expression of Sirt1 and CuZnSOD mRNA and diminished the expression level of HIF-2α and ctsk mRNA to the same levels as the control group. However, HBOT failed to alter catalase and OCN mRNA expression in obese-aged rats. HBOT partially improved the bone microarchitecture in obese-aged rats, but completely restored it in lean-aged rats. Interestingly, HBOT protected against obesity-induced demineralization in obese-aged rats. In summary, HBOT exerts an anti-osteoporotic effect in lean-aged rats and prevents some, but not all the negative effects of obese-aged conditions on bone health. Therefore, HBOT is considered as a potential therapy for aging-induced osteoporosis, regardless of obese status.

Periodontal Disease, Local and Systemic Inflammation in Puerto Ricans with Type 2 Diabetes Mellitus.

We assessed the associations between serum or gingival crevicular fluid (GCF) endothelial and inflammatory mediators and chronic PD among T2DM Hispanic adults. We enrolled 248 Puerto Rican residents with T2DM aged 40-65 years. The exposures included serum inflammatory mediators (IL-1b, IL-6, IL-10, and TNF-α), endothelial adhesion molecules, RANKL levels, and the GCF content of these analytes from a subset of 158 samples. The outcomes included the percent of sites with a probing pocket depth (PPD) ≥ 4 mm and clinical attachment loss ≥ 4 mm. Adjusted logistic regression models were fit to the categorized outcomes. Increased serum IL-10 (Adj. OR: 1.10, p = 0.04), sICAM-1 (Adj. OR: 1.01; p = 0.06), and elevated serum IL-1β (Adj. OR: 1.93; p = 0.06) were statistically significant or close to being significantly associated with a percent of sites with PPD ≥ 4 mm. An increase in GCF IL-1α (Adj. OR: 1.16; p < 0.01) and IL-1β (Adj: 2.40; p = 0.02) was associated with periodontal parameters. Our findings suggested that oral and systemic endothelial and inflammatory mediators are associated with periodontal clinical parameters among Hispanic adults with T2DM.

Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells.

This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients. HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay. 1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05). 1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

The unique role of bone marrow adipose tissue in ovariectomy-induced bone loss in mice.

Accumulation of bone marrow adipose tissue (BMAT) is always seen in osteoporosis induced by estrogen deficiency. Herein, we aimed to investigate the mechanisms and consequences of this phenomenon by establishing a mouse model of osteoporosis caused by ovariectomy (OVX)-mimicked estrogen deficiency. Micro-CT, osmium tetroxide staining, and histological analyses were performed to examine the changes in bone microstructure, BMAT and white adipose tissue (WAT) in OVX mice compared to sham mice. The osteogenesis and adipogenesis of primary bone marrow stromal cells (BMSCs) isolated from sham and OVX mice were compared in vitro. The molecular phenotypes of BMAT and WAT were determined and compared by quantitative PCR (qPCR). Bone marrow adipocyte-conditioned medium (BMA CM) was prepared from sham or OVX mice for coculture assays, and BMSCs or bone marrow monocytes/macrophages (BMMs) were isolated and subjected to osteoblast and osteoclast differentiation, respectively. Cell staining and qPCR were used to assess the effects of BMAT on bone metabolism. OVX-induced estrogen deficiency induced reductions in both cortical and trabecular bone mass along with an expansion of BMAT volume. At the cellular level, loss of estrogen inhibited BMSC osteogenesis and promoted BMSC adipogenesis, whereas addition of estradiol exerted the opposite effects. In response to estrogen deficiency, despite the common proinflammatory molecular phenotype observed in both fat depots, BMAT, unlike WAT, unexpectedly exhibited an increase in adipocyte differentiation and lipolytic activity as well as the maintenance of insulin sensitivity. Importantly, BMAT, but not WAT, presented increased mRNA levels of both BMP receptor inhibitors (Grem1, Chrdl1) and Rankl following OVX. In addition, treatment with BMA CM, especially from OVX mice, suppressed the osteoblast differentiation of BMSCs while favoring the osteoclast differentiation of BMMs. Our study illustrates that OVX-induced estrogen deficiency results in bone loss and BMAT expansion by triggering imbalance between the osteogenesis and adipogenesis of BMSCs. Furthermore, expanded BMAT, unlike typical WAT, may negatively regulate bone homeostasis through paracrine inhibition of osteoblast-mediated bone formation and promotion of osteoclast-mediated bone resorption.

Bone remodeling in experimental diabetes mellitus and surgical menopause in Wistar rats

BACKGROUND: Osteoporosis is metabolic skeletal disease characterized with low bone mass, bone microarchitecture disturbance that together lead to high prevalence of fragility fractures. Postmenopausal osteoporosis accounts for about 80% of the osteoporosis structure in women over 50 years. Diabetes mellitus (DM) is an independent risk factor for low-traumatic fractures. The incidence of both type 2 DM and osteoporosis increases during menopause. Therefore, the study of bone metabolism in experimental diabetes and surgical menopause seems important.THE AIM of the study was to investigate bone metabolism parameters during menopause and experimental type 2 DM.MATERIALS AND METHODS: The half of female Wistar rats had been subjected to bilateral ovariectomy at the beginning of the experiment. Diabetes mellitus (DM) was modelled using a high-fat diet and streptozotocin+nicotinamide. Four weeks after the following groups were formed: «Сontrol» (females without any interventions receiving standard chew, n=5) «OE» (females after ovariectomy n=5), «DM» (females with DM, n=4), «OE+DM» (females after ovariectomy with DM, n=4). The observation period lasted 8 weeks. Bone turnover and calcium-phosphorus metabolism markers (osteocalcin, osteoprotegerin (OPG), nuclear factor-kappa-beta receptor activator ligand (RANKL), sclerostin, fibroblast growth factor-23 (FGF-23), calcium, phosphorus) were measured in the end of experiment. Bone histomorphometry was performed after euthanasia.RESULTS: Phosphorus level was significantly lower both in the «OE» group (1.63 [1.58; 1.65] mmol/L) and in the «DM» group (2.81 [2.57; 2.83] mmol /l) compared to the «Control» group (3.12 [2.55; 3.24] mmol/l) (p&lt;0.001). This marker was significantly higher in the «OE+DM» group (2.79 [2.46; 2.81] mmol/l) in comparison to the «OE» group (2.79 [2.46; 2.81] mmol /l), p=0.025. Osteocalcin level was significantly lower in the «DM» group (8.1 [7.8; 9.2] ng/ml) compared to the «Control» group (16.97 [14.07; 17.07] ng/ml ), p=0.005. A weak negative correlation (r= -0.5, p&lt;0,05) was found between glucose and osteocalcin levels (p=0.03). RANKL level was significantly lower in the «OE+DM» group (278,1 [273.1; 289.7] pg/mL) compared to the «OE» group (400.6 [394.5; 415.1] pg/mL), besides the OPG/RANKL ratio was higher in this group (0.03 [0.02; 0.035] and 0.01 [0.004; 0.014], respectively), p=0.05. In the «OE» group lower OPG level (5.1 [1.5; 5.6] pmol/L) and OPG/RANKL ratio (0.01 [0.003; 0.014]) were obtained in comparison to the «Control» group (12.3 [8.8; 14.2] pmol/l and (0.34[0.33; 0.4], p=0.025 and p=0.07, respectively. The area of bone trabeculae in the epiphyseal zone was the largest in the «Control» group (42 [39; 45]) %; the difference was significant compared to the «OE» group (29 [25; 33] %, p=0.011) and the «OE+DM» group (30 [23; 25] %, p=0.016). The area of bone trabeculae in the metaepiphyseal zone was also the largest in the «Control» group (49 [46; 52] %) compared to the «OE» (35 [25; 39] %), «DM» (31 [26; 34] %), «OE+DM» (35 [33; 38] %), p&lt;0.001. There was no difference in the thickness of the bone trabeculae among the groups.CONCLUSION: DM induction can significantly inhibit bone remodeling in animals without menopause, which is reflected in a lower osteocalcin level. Bone turnover during DM and surgical menopause is characterized by lower RANKL levels and higher OPG/RANKL ratio. The effect of ovariectomy on bone metabolism was manifested in more extensive decrease in bone trabeculae area than in DM.

Identification of an intronic enhancer regulating RANKL expression in osteocytic cells

The bony skeleton is continuously renewed throughout adult life by the bone remodeling process, in which old or damaged bone is removed by osteoclasts via largely unknown mechanisms. Osteocytes regulate bone remodeling by producing the osteoclast differentiation factor RANKL (encoded by the TNFSF11 gene). However, the precise mechanisms underlying RANKL expression in osteocytes are still elusive. Here, we explored the epigenomic landscape of osteocytic cells and identified a hitherto-undescribed osteocytic cell-specific intronic enhancer in the TNFSF11 gene locus. Bioinformatics analyses showed that transcription factors involved in cell death and senescence act on this intronic enhancer region. Single-cell transcriptomic data analysis demonstrated that cell death signaling increased RANKL expression in osteocytic cells. Genetic deletion of the intronic enhancer led to a high-bone-mass phenotype with decreased levels of RANKL in osteocytic cells and osteoclastogenesis in the adult stage, while RANKL expression was not affected in osteoblasts or lymphocytes. These data suggest that osteocytes may utilize a specialized regulatory element to facilitate osteoclast formation at the bone surface to be resorbed by linking signals from cellular senescence/death and RANKL expression.

Anti-Osteoclastogenesis effects of gambir extract gel in periodontitis

Objective: RANK-RANKL-OPG signaling is being considered as the pathway periodontitis induces osteoclastogenesis. Alveolar bone resorption and increased osteoclast activity are both triggered by a greater RANKL/OPG ratio in periodontitis. Gambir contains catechins and quercetin, which have antimicrobial, antioxidant, and anti-inflammatory properties. This study aimed to analyze the anti-osteoclastogenesis effect of gambir extract gel through RANKL and OPG serum levels, the number of osteoblasts, and osteoclasts in alveolar bone tissue of Wistar rats. Material and Methods: Twenty-four male Wistar rats were divided into four equal groups, each receiving a different treatment: Group I received a placebo gel, Group II received Tetracycline gel, Group III received 8% gambir extract gel, and Group IV received 10% gambir extract gel. After 21 days, the serum levels of RANKL and OPG were assessed using ELISA. Hematoxylin-eosin staining of the bone tissue preparation was followed by microscopic counting of osteoblasts and osteoclasts. Results: Significant differences in RANKL serum levels were seen in Group I (0.70 + 0.020) with Group III (0.031 + 0.014) (p=0.018) and Group I with Group IV (0.038 + 0.012) (p=0.041). Serum levels of OPG differed significantly between Groups I (0.165+0,125) and IV (0.536+0.182) p=(0.015). The number of Osteoblast in Group I (1.16+ 0.40) and IV (1.33+1.03) were significantly different (p=0.023). The number of Osteoclast in Group I (4.33+1.50) and IV (1.5+0.83) were significantly different (p=0.008). Conclusion: Gambir Extract Gel 10% inhibits osteoclastogenesis by increasing osteoblast and decreasing osteoclast numbers by more than 8%, decreasing RANKL, and elevating OPG serum levels.&#x0D; &#x0D; Keywords: Gambir Extract Gel, Osteoclastogenesis, Periodontitis

Preliminary Study on Effect of Lactiplantibacillus plantarum on Osteoporosis in the Ovariectomized Rat.

Osteoporosis is a growing global health concern primarily associated with decreased estrogen in postmenopausal women. Recently, some strains of probiotics were examined for potential anti-osteoporotic effects. This study intended to evaluate the impacts of Lactiplantibacillus plantarum MGE 3038 strain (MGE 3038) in ovariectomized rats. For this purpose, twelve weeks old female Wistar rats (n=21; 250-300 g) were divided into 3 groups; ovariectomy (OVX) group, OVX/MGE 3038 group and Sham group (control). In these groups; two went through respective OVX and one had daily MGE 3038 administration through oral gavage. Prior to 16 weeks after OVX, we collected blood samples and extracted the tibiae. We scanned the extracted tibiae by in-vivo micro-computed tomography (micro-CT) and evaluated pathology by hematoxylin and eosin (H&E) and Masson's trichrome staining. The serum levels of C-telopeptide of type I collagen (CTX), osteocalcin (OC), and the receptor activator of nuclear factor-ĸB ligand (RANKL) were examined. The OVX/MGE 3038 group showed increases in bone mineral density, trabecular bone volume, trabecular number, and trabecular thickness (Tb.Th), and a decrease in trabecular spacing than the OVX group. However, OVX/MGE 3038 group and control group were measurably comparable in Tb.Th. Micro-CT, H&E, and Masson's trichrome findings exhibited increased preservation and maintenance of trabecular bone structure in the OVX/MGE 3038 group in comparison to the OVX group. In serum, the levels of CTX, OC and RANKL were significantly different between the OVX and OVX/MGE 3038 groups. Taken together, L. plantarum MGE 3038 could be helpful for the treatment of osteoporosis.

Changes in Serum Bone Metabolism Markers after Living Donor Liver Transplantation (LDLT) and Their Association with Fracture Occurrences.

Living donor liver transplantation (LDLT) is lifesaving, but can lead to osteoporosis and fractures. In our 3-year study of 25 LDLT recipients, we observed significant reductions in lumbar spine and femoral neck T scores, along with bone resorption marker reductions and liver regeneration marker increases. Serum calcium levels increased, while osteoprotegerin (OPG) decreased and Dickkopf-related protein 1 (DKK-1) increased. Patients who suffered fractures within 3 years of LDLT had higher serum OPG, lower serum nuclear factor kappa B ligand (RANKL), a higher OPG/RANKL ratio and higher serum DKK-1 levels. OPG, RANKL, OPG/RANKL ratio and DKK-1 levels before LDLT predicted hip or spine fractures within three years after LDLT. Further research is necessary to determine the optimal level of osteoclastic activity for preventing fracture onset.

Bone remodelling marker expression in grafted and ungrafted sheep tooth extraction sockets: A comparative randomised study

ObjectiveTo compare key markers of bone remodelling in a sheep tooth extraction model for sockets left to heal naturally or grafted with the bovine-derived xenograft Bio-Oss® covered with a collagen Bio-Gide® membrane. DesignRight side premolar teeth were removed from thirty Romney-cross ewes. Standardised sockets in each sheep were randomly allocated treatments, a grafted test and an empty control. At 4-, 8- and 16-weeks sheep were euthanized and tissue collected (N = 10/group). RANK, RANKL and OPG immunohistochemical analysis was performed (n = 3). RANK, RANKL, OPG, COL1A1, TIMP3, SP7 and MSX2 mRNA expression levels were determined using RT2-qPCR assays (n = 3). ResultsHistologically, more new woven bone was observed in the test group at all time points. Strong RANK and RANKL expression was found in both groups; at all time points with stronger RANK staining in the test group at 8 and 16 weeks. Strong OPG staining was localized to both osteoblasts and connective tissues. RANK receptor mRNA was expressed at a lower level in the test group (−4.26-fold; p = 0.02) at 4 weeks and SP7 at 16 weeks (−2.89-fold; p = 0.04). COL1A1 and TIMP3 mRNA expression increased significantly over time in the control group (p = 0.045, F = 5.4 and p = 0.003, F = 42.2 respectively). ConclusionSocket healing over time was comparable. The sheep tooth extraction model was found to be suitable for the evaluation of changes in the alveolar bone at the molecular level.

Clinical value of RANKL, OPG, IL-6 and sclerostin as biomarkers for fibrous dysplasia/McCune-Albright syndrome.

Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare genetic bone disease caused by a somatic mutation in the GNAS gene. Currently used bone turnover markers (BTMs) do not correlate with the clinical picture and are not useful to predict or monitor therapy success. This study assessed the correlation of RANKL, OPG, RANKL/OPG ratio, IL-6 and sclerostin with the classic BTMs alkaline phosphatase (ALP), procollagen type 1 propeptide (P1NP) and beta crosslaps (CTX), with pain, skeletal burden score (SBS) and response to bisphosphonate or denosumab treatment. Ninety-six serum samples of adult patients >18years of age with any subtype of FD/MAS were included from the biobank facility of the Leiden University Medical Center, Center for Bone Quality between 2015 and 2021. Standard laboratory assessments were assessed as part of usual care. The concentrations of potential biomarkers RANKL, OPG, sclerostin, IL-6 were analyzed. Data on FD/MAS subtype, age, pain, treatment history and treatment response were retrieved from the electronic patient files. Baseline characteristics were summarized by descriptive statistics. Correlations of the concentrations of the potential biomarkers with classic bone turnover markers, SBS and pain scores were cross-sectionally assessed by Spearman rank order correlation. Correction for multiple testing was performed by Benjamini and Hochberg False Discovery Rate. A sensitivity analyses was performed by excluding patients with SBS below 15 and patients using antiresorptive medication at the time of blood withdrawal or within the wash-out period. In patients treated with bisphosphonates or denosumab after blood withdrawal, pre-treatment concentrations were compared in patients with and without therapy response by Mann Whitney U test. The median age of the patients was 41.2 (Q1-Q3 25.9-52.2) years, 62.5% was female. Median SBS was 2.5 (Q1-Q3 0.5-7.8). RANKL level correlated weakly with ALP (Spearman rho 0.309, p=0.004, n=84), but not with P1NP or CTX. The RANKL/OPG ratio, OPG, IL-6 and sclerostin did not correlate with ALP, P1NP or CTX. None of the potential biomarkers correlated with SBS or pain. Results of the sensitivity analyses were comparable. Pre-treatment biomarker levels were similar in patients with and without improvement in pain scores following bisphosphonate therapy. Pre-treatment RANKL and sclerostin were comparable between patients with and without improvement in pain scores after denosumab therapy. Pre-treatment IL-6 level and the RANKL/OPG ratio seemed to be higher in patients with response to denosumab (IL-6: median 0.64 (Q1-Q3 0.53-0.74) pg/mL, n=6, RANKL/OPG: median 0.062 (Q1-Q3 0.016-0.331), n=5) compared to patients without response (IL-6: median 0.35 (0.20-0.54) pg/mL, n=5, RANKL/OPG: 0.027 (0.024-0.046), n=4). Pre-treatment IL-6 correlated with the improvement in maximum pain scores (rho 0.962, p<0.001, n=9) and average pain scores (rho 0.895, p=0.001, n=9) reported during denosumab therapy. Increased concentrations of RANKL, IL-6, sclerostin and of the RANKL/OPG ratio do not indicate severity of FD/MAS, as no correlation was observed of these potential biomarkers with the classic BTMs and SBS. Biomarker levels did not correlate with pain and had no value in predicting bisphosphonate treatment response. These biomarkers are not superior over the currently used methods of assessing ALP, P1NP and CTX or evaluating SBS to establish disease extent or activity and provide no reliable results. Yet, possibly pre-treatment IL-6 and the RANKL/OPG ratio may have some predictive value for clinical response to denosumab. Therefore, studies investigating disease activity and treatment response should include lesional imaging and patient-reported outcome measures.