BMP-2 Levels Research Articles

Evaluation of the effects of smoking on the clinical and histological characteristics and levels of growth factors in autologous fibrin membranes.

Considering the increase in regenerative dental treatments, autologous fibrin membranes (ARMs) have been widely used in tissue engineering, favoring the regeneration of hard and soft tissues, accelerating angiogenesis and promoting cell differentiation and migration. This study proposed evaluate the differences in clinical characteristics and levels of the growth factors BMP-2, IGF, PDGF and VEGF between smokers and nonsmokers were evaluated according to the guidelines of the US Preventive Service Task Force (USPSTF). Fourteen smokers and 14 nonsmokers were selected. After processing, the samples were allowed to rest in tubes for 5min for the organization of the matrix and completion of the fibrin clot. Only the yellow portion and the buffy coat were removed and stored. Four clots were obtained from each donor and used to quantify the release of the growth factors BMP2, PDGF, IGF and VEGF. In addition, histological analyses were performed, and clinical characteristics were evaluated. The data were tabulated and subjected to statistical analysis with a significance level of 5%. In both groups, the membranes remained intact throughout the analysis period, indicating similar structural behavior. Histological evaluation of the membranes obtained from the participants revealed the presence of more homogeneous fibrin membranes in the nonsmoker group and many leukocytes bordering the entire fibrin clot. In the smoker group, heterogeneous fibrin clots, sometimes malformed, and fewer leukocytes in the region of the buffy coat and bordering the entire fibrin were observed. Smokers had significantly lower levels of VEGF, PDGF and BMP-2 (pg/ml in 1µg total protein) than smokers did (p < 0.05), and there was no significant difference in IGF levels (p > 0.05) between the groups. There was also no statistically significant difference in membrane size between smokers and nonsmokers (mm, p > 0.05). Smoking may interfere with the formation of the fibrin mesh and consequently affects the quality and regenerative capacity in in smoking patients.

Biodegradable ultrahigh-purity magnesium and its alloy ZX00 promote osteogenesis in the medullary cavity and glycogenolysis in the liver.

Magnesium (Mg)-based implants have become an attractive alternative to conventional permanent implants in the orthopedic field. While biocompatibility, degradation kinetics, and osseointegration of Mg-based implants have been mostly investigated, the impact of degradation products on bone remodeling and potential systemic effects remains unclear. The aim of this study was to evaluate the early and mid-term local and systemic tissue responses of degrading ultrahigh-purity ZX00 (Mg-Zn-Ca alloy) and ultrahigh-purity Mg (XHP-Mg) pins in a juvenile healthy rat model. The potential differences between implant types (degradable vs. permanent), implantation, and age-related changes were investigated using titanium (Ti), sham-operated, and control groups (non-intervention), respectively. Degradation products of ZX00 and XHP-Mg pins promote osteogenesis in the medullary cavity by upregulating the expression levels of Bmp2 and Opg within 14 days post-surgery. The higher degradation rate of XHP-Mg resulted in the accumulation of degradation products starting from day 3 and upregulation of different genes, particularly Ccl2 and Cepbp. Besides good osseointegration and new bone tissue formation, we found a more parallel hydroxyapatite/collagen orientation along Mg-based pins in the perimeter region compared to Ti pins. In the liver, reduced glycogen levels in Mg-based pins indicated that degradation products promote glycogenolysis, while only the ZX00 group showed a higher serum glucagon level on day 14. Results suggest that degrading ZX00 and XHP-Mg pins stimulate osteogenesis mainly via Bmp2 and Opg and promote glycogenolysis in the liver, while the higher degradation rate of XHP-Mg pins resulted in upregulation of different genes and metabolites. STATEMENT OF SIGNIFICANCE: Bioresorbable magnesium (Mg)-based implants are promising alternative candidates for orthopedic interventions. Until now, a few in vivo studies explored how Mg-based implants promote osteogenesis in the medullary cavity and modulate systemic tissue responses. Herein, results demonstrate i) the degradation rate of the Mg-based implants has a crucial effect on osteogenesis via regulating Bmp2 and Opg expression in the medullary cavity, ii) parallel HAp/collagen matrix pattern in ZX00 and XHP-Mg groups compared to the Ti group, iii) both Mg pins promote glycogenolysis in the liver. Our findings highlight dual role of Mg-based implants in bone remodeling and systemic metabolic modulation. Nevertheless, this is the first study to report the interaction between Mg-based implants and liver metabolism.

Mechanistic studies of chondroitin sulfate/dermatan sulfate isolated from freshwater fish discards on osteogenesis in MC3T3-E1 cells.

Glycosaminoglycans (GAGs) are essential bone extracellular matrix molecules that regulate osteoblast differentiation. Numerous studies have explored endogenous and exogenous GAG osteoanabolic activities using appropriate in vitro and in vivo models. However, GAGs' underlying the mechanism of action and structure-function relationships need to be elucidated in detail. Earlier, we showed that exogenous GAG can bring about osteogenesis in pre-osteoblast cells. In the present study, we have elucidated the mechanism of action of exogenous GAGs, especially of the chondroitin sulfate/dermatan sulfate (CS/DS) class on osteogenesis. GAGs were immobilized, and osteoblast differentiation was evaluated in MC3T3-E1 cells. Results indicated that GAGs supported osteoblast differentiation by promoting collagen production, extracellular matrix formation, and subsequent mineralization. We elucidated the mechanisms underlying these effects by assessing the key signaling molecules involved in osteogenesis in response to exogenous CS/DS with/without BMP2. CS/DS alone significantly increased pERK1/2 and ATF4 expression levels differentially in a time-dependent manner without significant effects on BMP2, RUNX2, and pSMAD5 protein expression. On the other hand, CS/DS, in the presence of BMP2, differentially increased BMP2, pSMAD5, pERK1/2, RUNX2, and ATF4 expression levels at various time points. Collectively, these results strongly suggest that CS/DS can promote osteogenesis, and in the presence of BMP2, it could promote SMAD-mediated ERK-dependent osteogenesis.

Investigation of the molecular mechanism of quercetin in inhibiting ankylosing spondylitis ossification via the bone morphogenetic protein/smad signaling pathway.

Ankylosing spondylitis (AS) is a chronic inflammatory disease involving the spine and bone joints, which is characterized by hyperosteogeny, ossification of ligaments, and ankylosis. Quercetin is a natural polyphenolic compound with various biological activities such as antioxidant, anti-inflammatory, and anti-tumor. It was to explore the effect of quercetin on AS ossification and its molecular mechanism. In vitro culture of AS mesenchymal stem cells was conducted. Cells were treated with 0, 10, 30, 60, and 80μM quercetin, divided into control, 10μM, 30μM, 60μM, and 80μM groups. Alkaline phosphatase (ALP) staining, Alizarin Red staining, real-time quantitative polymerase chain reaction (qRT-PCR), and Western blot (WB) were employed to investigate the effect of quercetin on the expression of osteogenic-related genes and proteins. Additionally, bone morphogenetic protein (BMP) and Smad genes were knocked out to explore quercetin's regulation of BMP/Smad. In vivo experiments were conducted using 50 mice, including 10 in the normal group. An AS model was established in 36 mice, divided into negative control (n = 18, 0.9% saline) and quercetin groups (n = 18, quercetin). Safranin O-fast green (HE) staining and MicroCT scanning were performed before and 4weeks after injection. In the 60μM and 80μM quercetin groups, ALP activity, Ca2+ deposition area, and relative protein/mRNA levels of BMP-1, BMP-2, Smad1, Smad4, and Smad5 in AS mesenchymal stem cells were significantly lower compared to the control, 10μM, and 30μM groups (P < 0.05). The 80μM group exhibited lower levels than the 60μM group (P < 0.05). In the siRNA + 80μM group, the reduction in mRNA expression of BMP1, BMP2, Smad1, Smad4, and Smad5 was significantly greater compared to the siRNA group and the 80μM group (P < 0.05). At 4weeks post-injection, mice in the quercetin group showed significantly reduced severity of articular cartilage lesions, lymphocyte infiltration, and tissue edema, with no significant increase in sacroiliac joint fusion. Quercetin downregulates the expression of BMP and Smad-related proteins, inhibiting osteogenic differentiation of AS mesenchymal stem cells and effectively reducing ALP activity and Ca2+ deposition levels. These findings suggest that quercetin holds potential application value in the control and treatment of AS disease.

Stem Cells Within Three-Dimensional-Printed Scaffolds Facilitate Airway Mucosa and Bone Regeneration and Reconstruction of Maxillary Defects in Rabbits.

Background and Objectives: Current craniofacial reconstruction surgical methods have limitations because they involve facial deformation. The craniofacial region includes many areas where the mucosa, exposed to air, is closely adjacent to bone, with the maxilla being a prominent example of this structure. Therefore, this study explored whether human neural-crest-derived stem cells (hNTSCs) aid bone and airway mucosal regeneration during craniofacial reconstruction using a rabbit model. Materials and Methods: hNTSCs were induced to differentiate into either mucosal epithelial or osteogenic cells in vitro. hNTSCs were seeded into polycaprolactone scaffold (three-dimensionally printed) that were implanted into rabbits with maxillary defects. Four weeks later, tissue regeneration was analyzed via histological evaluation and immunofluorescence staining. Results: In vitro, hNTSCs differentiated into both mucosal epithelial and osteogenic cells. hNTSC differentiation into respiratory epithelial cells was confirmed by Alcian Blue staining, cilia in SEM, and increased expression levels of FOXJ1 and E-cadherin through quantitative RT-PCR. hNTSC differentiation into bone was confirmed by Alizarin Red staining, increased mRNA expression levels of BMP2 (6.1-fold) and RUNX2 (2.3-fold) in the hNTSC group compared to the control. Four weeks post-transplantation, the rabbit maxilla was harvested, and H&E, SEM, and immunohistofluorescence staining were performed. H&E staining and SEM showed that new tissue and cilia around the maxillary defect were more prominent in the hNTSC group. Also, the hNTSCs group showed positive immunohistofluorescence staining for acetylated α-tubulin and cytokerin-5 compared to the control group. Conclusions: hNTSCs combined with PCL scaffold enhanced the regeneration of mucosal tissue and bone in vitro and promoted mucosal tissue regeneration in the in vivo rabbit model.

Compression force promotes the osteogenic differentiation of periodontal ligament stem cells by regulating NAT10-mediated ac4C modification of BMP2

BackgroundOrthodontic treatment applies specific corrective forces to teeth, transmitting stress to periodontal tissue, thereby regulating the growth and development of periodontal ligament stem cells (PDLSCs). Recently, N-acetyltransferase 10 (NAT10) mediated N4-acetylcytidine (ac4C) modification is demonstrated to play a key role in the osteogenic differentiation of stem cells. Therefore, this study aimed explore the effects of Orthodontic treatment on the NAT10 mediated ac4C modification and osteogenic differentiation of PDLSCs.MethodsCompressive force was used to treat PDLSCs to simulate orthodontic force treatment. The ALP and ARS staining was performed to analyze the osteogenic differentiation of PDLSCs. Besides, ac4C dot blot and ac4C-RIP assays were performed to detect the global ac4C levels and BMP2 ac4C levels. The relationship between NAT10 and BMP2 was confirmed by RIP assay and immunofluorescence staining. The mRNA and protein levels of RUNX2, Oxterix and BMP2 were detected by RT-qPCR and western blot assays.ResultsCompressive force treatment promoted the osteogenic differentiation of PDLSCs, and enhanced the global ac4C levels and NAT10 levels in PDLSCs. NAT10 overexpression further promoted the osteogenic differentiation of compressive force treated PDLSCs. Besides, NAT10 overexpression increased ac4C levels of BMP2 and enhanced the mRNA stability of BMP2. Remodelin treatment significantly decreased the ac4C and mRNA levels of BMP2. Furthermore, BMP2 silencing reversed the role of NAT10 in the compressive force treated PDLSCs.ConclusionThis study demonstrated that compressive force promotes cell viability and osteogenic differentiation of PDLSCs by regulating BMP2 levels mediated by NAT10. NAT10 mediated ac4C levels of BMP2 is the key signaling axis of orthodontic stress in promoting cell growth and osteogenic differentiation of PDLSCs.Graphical abstract

Novel plasma cytokines identified and validated in children during lead exposure according to the new updated BLRV

Lead is a pervasive environmental contaminant with significant health risks, particularly to children. It is known for its neurotoxic and immunotoxic effects, causing developmental, cognitive, and behavioral impairments. Despite extensive research, the mechanisms of lead toxicity remain unclear. Cytokines, which are critical in immune response and inflammation, have emerged as potential biomarkers for lead toxicity. The recent Centers for Disease Control and Prevention (CDC) update to the blood lead reference value (BLRV) to 3.5 µg/dL emphasizes the need to explore novel biomarkers and mechanisms. The study involved 100 healthy children aged 1 to 5 years, divided into two groups based on BLRV: elevated (≥ 3.5 µg/dL) and low (< 3.5 µg/dL). The research consisted of two phases: discovery and validation. Plasma samples were analyzed using RayBio® Human Cytokine Antibody Arrays and Enzyme-linked immunosorbent assay (ELISA) for cytokine levels. Ethical approval was obtained, and statistical analyses included t-tests, chi-squared tests, pearson correlations, and multivariate logistic regression. Protein-protein interaction (PPI), Gene Ontology (GO) enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to explore the roles of significant differentially expressed proteins (DEPs). No significant differences in age, gender, or BMI between the two groups, but BLRV levels were significantly higher in the elevated BLRV group compared to the low BLRV group. In the discovery phase, significant changes in cytokine expression were identified, including increased levels of IL-6, IL-8, and IL-17, and decreased levels of BDNF, BMP-4, IGF-1, IL-7, IL-10, and Leptin. These findings were validated in the second phase using ELISA. Significant positive correlations were found between BLRV and IL-6, IL-8, and IL-17. Negative correlations were observed with BDNF, BMP-4, IGF-1, IL-7, IL-10, and Leptin. Multivariate regression confirmed that BLRV significantly affects these cytokine levels. PPI networks revealed that DEPs had strong interactions with multiple proteins, indicating their central role in lead toxicity. GO and KEGG analyses highlighted pathways related to neurotoxicity and inflammatory responses, including “negative regulation of myotube differentiation,” “neurotrophin signaling pathway,” and “alcoholism.” This study provides insights into the role of cytokines as biomarkers for lead toxicity and offers a comprehensive analysis of the mechanisms involved. The findings underscore the importance of early detection and intervention based on updated BLRV thresholds.

BMP-4 and fetuin A in systemic sclerosis patients with or without calcinosis.

Systemic sclerosis (SSc) is a connective tissue disease at the interface between inflammation and autoimmunity progressively leading to diffuse microvascular and fibrotic involvement of the skin and of multiple internal organs. Approximately, 20-40% of SSc patients suffer from cutaneous calcinosis, a debilitating manifestation due to calcium salt deposition in soft connective tissues, causing pain, ulceration, infection, and deformities, responsible of severe functional limitations. Pathomechanisms are poorly understood as well as markers/molecules capable to predict the risk of patients to develop calcinosis. An observational study was performed in 51 female patients, 25 with and 26 without calcinosis to compare clinical and laboratory parameters and to evaluate pro- and anti-calcifying circulating markers and the in vitro serum calcification potential (T50). Moreover, calcinosis samples were analyzed to characterize their mineral composition. Data demonstrate statistically significant differences in the prevalence of clinical manifestations and ACA and Scl70 autoantibodies in SSc patient with calcinosis compared to those without calcinosis. In SSc patients with calcinosis, serum levels of BMP-4 are higher, fetuin A might be regarded as a potential circulating prognostic marker and a negative correlation was observed between T50 and the global score of clinical manifestations, suggesting a potential predictive role of pro- and anti-calcifying molecules in SSc patients. Furthermore, calcinosis samples were characterized by the co-existence of phosphate and carbonate minerals with different stability and solubility. Further investigations on circulating markers in larger patient cohorts, especially at the early stages and throughout the natural course of the disease, may clarify their pathogenetic role in the SSc-related cutaneous calcinosis.

Bone morphogenetic protein levels relate to osteoarthritis and neurobehavioral outcomes in cognitively unimpaired older adults

AbstractBackgroundOsteoarthritis (OA) is a degenerative disorder of bone aging and risk factor for cognitive decline. Bone morphogenetic proteins (BMPs) are growth factor proteins that regulate skeletal and neural development, and circulating BMPs may mediate molecular cross‐talk between bone and brain. The present study examined plasma BMP levels in relation to OA and neurobehavioral outcomes in cognitively unimpaired (CU) older adults.MethodsParticipants included 65 CU adults (mean age= 76.45, 60% female) enrolled in the UCSF Brain Aging Network for Cognitive Health (BrANCH) study who underwent cross‐sectional neurological examination, neuropsychological testing, and blood draw. Participants were classified as OA (n=31) or control (CTL; n=34) based on clinician exam (UDS Medical Conditions). Plasma was assayed for 11 different BMPs via SomaScan v4.1. Composite z‐scores were calculated for cognitive domains of memory and executive functioning. Mood symptoms were quantified with the 30‐item Geriatric Depression Scale (GDS). ANCOVA compared BMP concentrations between OA and CTL, adjusting for age and sex. Separate multiple linear regressions examined the relationship between OA‐associated BMPs and neurobehavioral outcomes across the entire sample (cognitive z‐scores, GDS total score), adjusting for age, sex, and education.ResultsOA and CTL groups did not significantly differ on demographics or neurobehavioral outcomes (ps&gt;0.10). Five BMPs were differentially abundant (ps&lt;.05) between OA and CTL (lower in OA: BMP5, BMP7, BMP10, BMP6; higher in OA: BMP4). In neurobehavioral models, lower BMP6 and higher BMP4 levels related to worse executive functioning (BMP6: β=0.27, p=.03; BMP4: β=‐0.28, p=.02) and higher GDS scores (BMP6: β=‐0.33, p=.02; BMP4: β=0.34, p=.01). Higher BMP10 also related to lower GDS scores (β=‐0.31, p=.02). No associations were observed between BMPs and memory z‐scores (ps&gt;0.05).ConclusionIn a cohort of CU older adults, OA related to differential plasma abundance of BMPs. In turn, circulating BMP levels, specifically BMP4, BMP6, and BMP10, were associated with executive function and depressive symptoms. These data help provide a molecular link connecting skeletal disorders of aging (e.g., OA) to adverse neurobehavioral outcomes. Given the diverse immunovascular, metabolic, and growth regulatory functions of BMPs, additional mechanistic work is needed to identify the precise pathways by which systemic BMPs interact with the central nervous system.

Elabela alleviates cuproptosis and vascular calcification in vitaminD3- overloaded mice via regulation of the PPAR-γ /FDX1 signaling

BackgroundVascular calcification is a crucial pathophysiological process associated with age-related cardiovascular diseases. Elabela, a recently identified peptide, has emerged as a significant player in the regulation of cardiovascular function and homeostasis. However, the effects and underlying mechanisms of Elabela on age-related vascular calcification remain largely unexplored.MethodsIn-vivo vascular calcifications of C57BL/6J mice (8-week-old) and young (8-week-old) or aged (72-week-old) SD rats were injected with vitamin D3 (VitD3) or saline, respectively. Furthermore, the VitD3-overloaded mice received Elabela (1 mg/kg/d), peroxisome proliferators-activated receptor-γ (PPAR-γ) activator Rosiglitazone (5 mg/kg/d) or copper-ionophore Elesclomol (20 mg/kg/d), respectively. As for in-vitro studies, primary rat vascular smooth muscle cells (VSMCs) were isolated from aortas and cultured for explore the role and underlying mechanism of Elabela in vascular calcification.ResultsThere were marked increases in FDX1 and Slc31a1 levels in both aortas and VSMCs during vascular calcification, coinciding with a rise in copper levels and a decrease in Elabela levels. Alizarin red and von-Kossa staining indicated that the administration of Elabela effectively hindered the progression of vascular cuproptosis and arterial calcification in VitD3-overloaded mice and rat arterial rings models. Moreover, Elabela significantly suppressed osteogenic differentiation and calcium deposition in VSMCs and strikingly reversed high phosphate-induced augmentation of FDX1 expression, DLAT aggregation as well as intracellular copper ion levels. More importantly, Elabela exhibited remarkable abilities to prevent mitochondrial dysfunctions in primary rat VSMCs by maintaining mitochondrial membrane potential, inhibiting mitochondrial division, reducing mitochondrial ROS production and increasing ATP levels. Interestingly, Elabela mitigated cellular senescence and production of pro-inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-18 and TNF-α, respectively. Furthermore, Elabela upregulated the protein levels of PPAR-γ in VitD3-overloaded mice. Administrating PPAR-γ inhibitor GW9662 or blocking the efflux of intracellular copper abolished the protective effect of Elabela on vascular calcification by enhancing levels of FDX1, Slc31a1, Runx2, and BMP2.ConclusionElabela plays a crucial role in protecting against vascular cuproptosis and arterial calcification by activating the PPAR-γ /FDX1 signaling. Elabela supplementation and cuproptosis suppression serve as effective therapeutic approaches for managing vascular calcification and related cardiovascular disorders.

The Expression Level of Inflammation-Related Genes in Patients With Bone Nonunion and the Effect of BMP-2 Infected Mesenchymal Stem Cells Combined With nHA/PA66 on the Inflammation Level of Femoral Bone Nonunion Rats.

Bone nonunion delays fracture end repair and is associated with inflammation. Although bone nonunion can be effectively repaired in clinical practice, many cases of failure. Studies have confirmed that BMP-2 and nHA/PA66 repaired bone defects successfully. There are few studies on the effects of the combined application of BMP-2 and NHA/PA66 on bone nonunion osteogenesis and inflammation. We aimed to investigate the expression level of inflammation-related genes in patients with bone nonunion and the effect of BMP-2-infected mesenchymal stem cells combined with nHA/PA66 on the level of inflammation in femur nonunion rats. We searched for a gene expression profile related to bone nonunion inflammation (GSE93138) in the GEO public database. Bone marrow mesenchymal stem cells (MSCs) of SD rats were cultured and passed through. We infected the third generation of MSCs with lentivirus carrying BMP-2 and induced the infected MSCs to bone orientation. We detected the expression level of BMP-2 by RT-PCR and the cell viability and alkaline phosphatase (ALP) activity by CCK8 and then analyzed the cell adhesion ability. Finally, the levels of related inflammatory factors, including C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and Erythrocyte Sedimentation Rate (ESR), were detected in nonunion rats. Our findings: The patients with nonunion had up-regulated expression of 26 differentially inflammatory genes. These genes are mainly enriched in innate immune response, extracellular region, calcium ion binding, Pantothenate and CoA biosynthesis pathways. The expression level of BMP-2 in the Lenti-BMP-2 group was higher (vs. empty lentivirus vector group: t=5.699; vs. uninfected group t=3.996). The cell activity of the MSCs + BMP-2 + nHA/PA66 group increased gradually. After being combined with nHA/PA66, MSCs transfected with BMP-2 spread all over the surface of nHA/PA66 and grew into the material pores. MSCs + BMP-2 + nHA/PA66 cells showed positive ALP staining, and the OD value of ALP was the highest. The levels of CRP, IL-6, TNF-alpha, and ESR in the MSCs + BMP-2 + nHA/PA66 group were lower than those in the MSCs and MSCs + nHA/PA66 group but higher than those in MSCs + BMP-2 group. The above comparisons were all P<0.05. The findings demonstrated that the expression level of inflammation-related genes increased in the patients with bone nonunion. The infection of MSCs by BMP-2 could promote the directed differentiation of MSCs into osteoblasts in the bone marrow of rats, enhance the cell adhesion ability and ALP activity, and reduce inflammation in rats with bone nonunion.

Modeling of Healing of Intra-Articular Fractures of Long Tubular Bones in Rats

Background: We present a new original method of modeling an intra-articular fracture of the long tubular bone in laboratory animals.Objective: To create a new experimental model of an intra-articular fracture of the long tubular bone and to approve it for comprehensive evaluation of morphofunctional parameters of cartilage and bone healing.Materials and methods: The experiment was performed on 30 white sexually mature Wistar rats. Modeling involved layer-by-layer dissection of tissues along the anteromedial surface and formation of an incomplete fracture of the femoral medial condyle using a sharp L-shaped working end of an 0.2 × 0.2 сm instrument perpendicular to the medial condyle with a slight single muscle force. The rats were euthanized in groups of 10 animals on days 7, 14, and 30. We performed knee radiography and determined blood osteocalcin and BMP-7 levels. The Mankin score was used in the histological evaluation of the articular cartilage changes. The groups were compared using the Kruskal-Wallis test. P &lt; .05 was considered statistically significant.Results: We noted statistically significant differences (P &lt; .0001) when comparing the groups using the Mankin score. The post hoc test revealed statistically significant differences in the Mankin scores between days 7 and 14 (P = .014) and between days 7 and 30 (P &lt; .0001). Group comparisons showed no statistically significant differences in blood osteocalcin levels (P = .518). However, there were statistically significant differences (P = .0028) between the groups in blood BMP-7 levels. The post hoc test revealed statistically significant differences in blood BMP-7 levels between days 7 and 14 (P = .048) and between days 14 and 30 (P = .0195).Conclusions: The developed experimental model of intra-articular fractures of long tubular bones allows for a comprehensive assessment of reparative processes of cartilage and bone tissue. The model is close to the real mechanism of pathology development.

Circulating BMP-7 Level is Independent of Sarcopenia in Older Asian Adults.

In vitro and animal studies have demonstrated that BMP-7, renowned for its osteogenic properties, also exerts beneficial effects on muscle metabolism by enhancing myogenesis and reversing muscle atrophy. Despite being proposed as a common regulatory factor for both muscle and bone, the impact of BMP-7 on human muscle health has not been thoroughly investigated. This cross-sectional study involved 182 community-dwelling older adults who underwent a comprehensive geriatric assessment in South Korea. Sarcopenia was diagnosed using Asian-specific cutoffs, and serum BMP-7 levels were quantified via enzyme immunoassay. The mean age of the participants was 72.2 ± 7.3 years, with 62.6% being female. After adjustments for confounders, serum BMP-7 levels were not significantly different between individuals with and without sarcopenia, nor were there differences based on skeletal muscle mass, strength, or physical performance levels (P = 0.423 to 0.681). Likewise, no correlations were detected between circulating BMP-7 levels and any sarcopenia assessment metrics such as skeletal muscle index, grip strength, gait speed, or chair stand completion times (P = 0.127 to 0.577). No significant associations were observed between increases in serum BMP-7 concentrations and the risk of sarcopenia or poor muscle phenotypes (P = 0.431 to 0.712). Stratifying participants into quartiles based on serum BMP-7 levels also indicated no differences in sarcopenia-related parameters (P = 0.663 to 0.996). Despite experimental evidence supporting BMP-7's role in muscle metabolism, this study found no significant association between serum BMP-7 levels and clinical indicators of muscle health in older adults. These findings challenge the utility of serum BMP-7 as a biomarker for sarcopenia in this demographic.

The Expression Level of Inflammation-Related Genes in Patients With Bone Nonunion and the Effect of BMP-2 Infected Mesenchymal Stem Cells Combined With nHA/PA66 on the Inflammation Level of Femoral Bone Nonunion Rats

Bone nonunion delays fracture end repair and is associated with inflammation. Although bone nonunion can be effectively repaired in clinical practice, many cases of failure. Studies have confirmed that BMP-2 and nHA/PA66 repaired bone defects successfully. There are few studies on the effects of the combined application of BMP-2 and NHA/PA66 on bone nonunion osteogenesis and inflammation. We aimed to investigate the expression level of inflammation-related genes in patients with bone nonunion and the effect of BMP-2-infected mesenchymal stem cells combined with nHA/PA66 on the level of inflammation in femur nonunion rats. We searched for a gene expression profile related to bone nonunion inflammation (GSE93138) in the GEO public database. Bone marrow mesenchymal stem cells (MSCs) of SD rats were cultured and passed through. We infected the third generation of MSCs with lentivirus carrying BMP-2 and induced the infected MSCs to bone orientation. We detected the expression level of BMP-2 by RT-PCR and the cell viability and alkaline phosphatase (ALP) activity by CCK8 and then analyzed the cell adhesion ability. Finally, the levels of related inflammatory factors, including C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and Erythrocyte Sedimentation Rate (ESR), were detected in nonunion rats. Our findings: The patients with nonunion had up-regulated expression of 26 differentially inflammatory genes. These genes are mainly enriched in innate immune response, extracellular region, calcium ion binding, Pantothenate and CoA biosynthesis pathways. The expression level of BMP-2 in the Lenti-BMP-2 group was higher (vs. empty lentivirus vector group: t=5.699; vs. uninfected group t=3.996). The cell activity of the MSCs + BMP-2 + nHA/PA66 group increased gradually. After being combined with nHA/PA66, MSCs transfected with BMP-2 spread all over the surface of nHA/PA66 and grew into the material pores. MSCs + BMP-2 + nHA/PA66 cells showed positive ALP staining, and the OD value of ALP was the highest. The levels of CRP, IL-6, TNF-α, and ESR in the MSCs + BMP-2 + nHA/PA66 group were lower than those in the MSCs and MSCs + nHA/PA66 group but higher than those in MSCs + BMP-2 group. The above comparisons were all P˂0.05. The findings demonstrated that the expression level of inflammation-related genes increased in the patients with bone nonunion. The infection of MSCs by BMP-2 could promote the directed differentiation of MSCs into osteoblasts in the bone marrow of rats, enhance the cell adhesion ability and ALP activity, and reduce inflammation in rats with bone nonunion.

Integrin α2β1 deficiency enhances osteogenesis via BMP-2 signaling for accelerated fracture repair

Previous studies have shown that the absence of the collagen-binding integrin α2β1 confers protection against osteoporosis, primarily by enhancing osteoblast-mediated matrix formation, with a particular increase in collagen type I production. This study aimed to elucidate the mechanism underlying this increased matrix production. Our findings demonstrate that osteoblasts lacking integrin α2 secrete a pro-osteogenic factor that activates both TGF-β and BMP signaling pathways. Among these, BMP-2 was identified as the key signaling protein responsible for this effect, as its expression was significantly upregulated during osteoblast differentiation. Moreover, integrin α2 deficiency led to earlier and elevated BMP-2 secretion at the cell surface during osteogenesis, which promoted accelerated osteoblast differentiation. This phenomenon likely contributes to enhanced matrix production in aging animals, providing a protective effect against osteoporosis.To explore the broader implications of this phenotype, we utilized a fracture healing model. In integrin α2-deficient 12 weeks old female mice, elevated serum levels of BMP-2 were detected during the early stages of fracture repair. This upregulation of BMP signaling within the fracture callus accelerated the healing process, resulting in faster formation and mineralization of the cartilaginous callus. Additionally, the elevated BMP-2 levels facilitated earlier differentiation of chondrocytic cells, evidenced by the premature appearance of collagen type II- and type X-positive cells during endochondral ossification. Despite the accelerated healing, the overall biomechanical integrity of the repaired fractures remained uncompromised.Thus, the modulation of integrin α2β1 presents a promising therapeutic target for enhancing fracture repair by regulating BMP-2 signaling in a physiologically relevant manner.

Assessment of experimental circulating biomarkers in volume and pressure overload: insights from a cohort study

Abstract Introduction Experimental biomarkers including Angiopoietin-2 (ANG2), bone morphogenetic protein 10 (BMP10), fibroblast growth factor 23 (FGF23), and insulin-like growth factor-binding protein 7 (IGFBP7) are hypothesized to reflect cardiac pathophysiological processes that are potentially present in adverse cardiac remodeling in volume or pressure overload. This study aims to elucidate the relationship between these circulating biomarkers and the presence of hemodynamically significant pressure or volume overload and adverse outcomes and compare it to N-terminal pro B-type natriuretic peptide (NT-proBNP). Methods We studied the association between the four experimental circulating biomarkers in patients with pressure or volume overload with reference to none/mild valvular disease in N=1302 of an observational outpatient cardiology cohort. The median age was 63.0 (25th/75th quartile 51.5, 71.4) years, 445 (34.2%) were women. Circulating biomarkers were quantified using a novel antibody-based method. Logistic regression models with cox regression plots were employed to assess the association of each biomarker with the severity of valve disease concerning volume or pressure overload, adjusted for age and sex. Results In the overall population, elevated levels of ANG2, BMP10, FGF23 and IGFB7 were positively associated with the presence of significant volume overload (ANG2: Odds ratio (OR) 1.26 (95% confidence interval (CI) 1.17-1.35), p&amp;lt;0.001; BMP10: OR 2.57 (CI 1.89-3.48), p&amp;lt;0.001; FGF23: OR 1.51 (CI 1.14-1.20), p=0.004; IGFBP7: OR 1.39 (CI 1.14-1.69), p=0.001, NT-proBNP: OR 1.69 (CI 1.47-1.95), p&amp;lt;0.001). No statistically significant association between circulating biomarkers and pressure overload was observed. Over a median follow-up of 4 years, 31 (20.1%) participants died in the group of volume overload and 9 (40.9%) individuals in the group of pressure overload. After multivariable-adjustment, elevated levels of BMP 10 showed a borderline association with an increased risk of all-cause mortality in individuals with volume overload compared to none/mild valvular disease: hazard ratio (HR) 1.51 (CI 0.96- 2.38), p=0.075. Higher concentrations of all biomarkers were predictive of all-cause mortality for both individuals with volume overload and those with none/mild valvular disease (ANG2: HR 3.72 (CI 1.67-8.33), p=0.001; BMP10: HR 1.65 (CI 1.20-2.26), p=0.002; FGF23: HR 2.08 (CI 1.65-2.62), p&amp;lt;0.001; IGFBP7: HR 1.55 (CI 1.25-1.91), p&amp;lt;0.001; NT-proBNP: HR 1.72 (CI 1.47-2.00), p&amp;lt;0.001). Conclusions This study demonstrates that circulating biomarkers involved in distinct pathophysiological pathways of inflammation, fibrosis and calcification are elevated in patients with hemodynamically significant volume overload. Since they are also related to mortality, their clinical role needs to be explored further, also in context with the routine biomarker NT-proBNP.

Alleviating vascular calcification with Bushen Huoxue formula in rats with chronic kidney disease by inhibiting the PTEN/PI3K/AKT signaling pathway through exosomal microRNA-32.

Vascular calcification (VC) significantly raises cardiovascular mortality in chronic kidney disease (CKD) patients. VC is characterized by the phenotypic transformation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells, mediated by exosomes derived from calcified VSMCs and the exosomal microRNAs (miRNA) which may trigger some signals to recipient VSMCs. Bushen Huoxue (BSHX) formula has demonstrated its clinical efficacy in CKD and its protective role in CKD-VC rats has also been observed. However, little is known about its underlying mechanism. To establish a VC model, aortic VSMCs from rats were induced to osteogenic differentiation by high-level phosphate (HP) in vitro. The expression of exosome and calcification makers were analyzed by western blot, including CD9, CD63, α-SMA, BMP-2, and Runx2, respectively. Differential expression of exosomal miRNAs in normal and HP-induced VSMCs were identified by using whole miRNA microarray technology. GO and KEGG analyses were performed to determine the significant enrichment of functions and signaling pathways in the target genes. In vivo, the CKD-VC rat model was established by administering adenine gavage combined with a high phosphorus diet. The rats were divided into normal control, model, low-dose BSHX, medium-dose BSHX, high-dose BSHX groups, and sevelamer groups. The blood biochemical parameters were measured. Renal histopathology and aortic calcification were observed. Western blot detected the levels of the calcification markers. Quantitative real-time PCR (qPCR) assay detected exosomal microRNA-32 (miR-32) mRNA expression in the aorta, the most differentially expressed exosomal miRNA previously identified. Phosphatase and tensin homolog located on chromosome ten (PTEN)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway components were also tested by western blot. Exosomal miRNA-32 and PI3K/AKT signaling pathways were highly differentially expressed between normal and HP-induced VSMCs. In vivo, BSHX improved blood biochemical parameters, renal histopathology, and aortic calcification in CKD-VC rats. BSHX increased the expression level of α-SMA and decreased the level of BMP-2 and Runx2. BSHX also lowered the expression level of exosomal miR-32 mRNA, enhanced PTEN expression, therefore, reduced p-PI3K and p-AKT levels in the aorta. BSHX alleviated VC in CKD rats by downregulating exosomal miR-32 expression in the aorta, thereby promoting PTEN expression and inhibiting the PI3K/AKT signaling pathway.

Comparative Study of Photobiomodulation Effects on Alveolar Socket Hard Tissue Healing in Rats: Application of 980 nm Versus 810 nm Lasers.

Background: This study aimed to explore the differential effects of photobiomodulation (PBM) via 980 nm and 810 nm lasers on the hard tissue healing of rat alveolar sockets, with a focus on a comparative analysis of hard tissue regeneration and osteogenic gene expression. Objective: This study aimed to explore the effects of PBM using 980 nm and 810 nm lasers on hard tissue healing of rat alveolar sockets, focusing on hard tissue regeneration and osteogenic gene expression. Materials and Methods: Thirty-six male Wistar rats (5 weeks old) had both right and left maxillary first molars extracted. Post extraction, the right alveolar sockets received PBM treatment with either 980 nm (0.3 W, 18 J/cm2) or 810 nm (0.1 W, 6 J/cm2) lasers for seven days, whereas the left sockets served as controls. Rats were euthanized on days 3, 7, 14, and 28 for histopathological, immunohistochemical, micro computed tomography (micro-CT), and quantitative polymerase chain reactionanalyses. Results: On day 3, early granulation tissue, neovascularization, and inflammatory cell aggregates were observed in all groups. By day 7, active osteoclasts and osteoblasts were noted, with a significant increase in CD31-positive cells in the 980 nm group (p < 0.05). Day 14 showed new bone formation, and by day 28, increased cancellous bone and collagen content were present in all groups, with no significant differences between them (p > 0.05). Gene expression analysis revealed higher BMP-2 and Runx-2 levels in laser-treated groups on day 14 (p < 0.05), with the 980 nm group having higher BMP-2 levels than the 810 nm group (p < 0.05). Bone sialoprotein expression was higher in laser-treated groups on days 14 and 28 (p < 0.05), and osteocalcin expression was highest in the 980 nm group on both days (p < 0.05). Micro-CT analysis showed no significant differences among groups in bone mineral density, bone surface (BS)/bone volume (BV), or bone volume (BV)/TV (total volume) indices. Conclusion: PBM with 980 nm and 810 nm lasers promotes early-stage hard tissue healing in extraction sockets, with the 980 nm laser more effectively enhancing osteogenic gene expression, suggesting its potential as an adjunctive therapy in dental and oral surgery.

6421 Imaging Phenotype May Predict Genotype In Human Pheochromocytoma: Relationships Between Genetic Mutation Clusters, Metabolite Transporter Expression, And Radiotracer Uptake

Abstract Disclosure: I. Sakuma: None. M. Fujimoto: None. D.F. Vatner: None. K. Yokote: None. T. Tanaka: None. Context [1]23I-metaiodobenzylguanidine scintigraphy (MIBG) and [1]8F-fluoro-2-doxy-D-glucose positron emission tomography (FDG-PET) are helpful for the localization of pheochromocytoma. The norepinephrine transporter (NET) and the glucose transporter 1 (GLUT1) are thought to be critical for the uptake of these radioisotopes. NET expression is activated by Paired Like Homeobox (PHOX2) and glucocorticoid receptor (GR) in neuroblastoma cells. Around 70% of pheochromocytomas carry well-known mutations. Pheochromocytoma-associated genes are divided into two clusters: the pseudohypoxia-related cluster 1 and the kinase signaling-related cluster 2. Cluster 1 mutation are associated with noradrenergic pheochromocytomas with an elevated metastatic risk. Cluster 2 tumors demonstrate an adrenergic phenotype with a less aggressive course. How mutations of cluster 1 and cluster 2 impact the expression of NET/GLUT1 and on radiotracer uptake remains unclear. Hypothesis Cluster 1 and cluster 2 will have different effects on the expression of transcription factors associated with chromaffin cell differentiation with resultant differential effects on NET/GLUT1 expression. Methods 42 patients with pheochromocytoma were evaluated by MIBG and FDG-PET. Tumor gene expression was evaluated by real-time qPCR, and mutation analyses were performed by Sanger or next-generation sequencing. Results 88% of tumors were MIBG-avid, and 67% were FDG-avid. The expression of NET in MIBG-negative tumors was 70% lower than in MIBG-positive tumors. The expression of GLUT1 in FDG-PET-negative tumors was 75% lower than in FDG-PET-positive tumors. The expression of NET positively correlated with transcription factors involved in chromaffin cell maturation, including PHOX2A, PHOX2B, GATA binding protein 3 (GATA3), GR, catecholamine synthases, and insulinoma associated 1 (INSM1). The JASPAR database for promoter analysis revealed putative responsive regions of the transcription factors PHOX2A, PHOX2B, GATA3, GR, and INSM1 on the NET gene. Seven patients (16.7%) had mutations in cluster 1, and fourteen patients (33.3%) had mutations in cluster 2. Cluster 2 exhibited elevated NET, PHOX2A, PHOX2B, GATA3, GR, and INSM1 expression levels compared to cluster 1. Cluster 1 showed a higher expression level of GLUT1 and BMP4 that is associated with early chromaffin cell differentiation. Conclusions In pheochromocytomas, MIBG and FDG uptake correlated with the expression levels of NET and GLUT1, respectively. NET expression is associated with chromaffin cell differentiation transcription factor expression. We propose that pheochromocytomas that carry Cluster 1 mutations are more likely to be missed in MIBG due to low NET expression, while pheochromocytomas that carry Cluster 2 mutations are more likely to be missed on FDG-PET due to low GLUT1 expression with a more mature chromaffin cell phenotype. Presentation: 6/2/2024

Soybean Isoflavones Alleviate Osteoarthritis Through Modulation of the TSC1/mTORC1 Signaling Pathway to Reduce Intrachondral Angiogenesis

ABSTRACT Background The incidence of osteoarthritis (OA) is increasing, yet its pathogenesis remains largely unknown. Recent studies suggest that abnormal subchondral bone remodeling plays a crucial role in OA development, highlighting a gap in clinical treatments targeting this aspect. Soybean Isoflavone (SI) has shown potential in treating OA, although its mechanisms are not fully understood. Methods This research investigated the effects of SI on subchondral bone remodeling in an OA rat model, assessing joint damage, OARSI scores, and type H vessel formation (CD31hiEmcnhi expression). Additionally, the expression of ALP, OCN, BMP, and TSC1 was evaluated to determine involvement of the mTORC1 pathway. In vitro studies on IL-1β-induced osteoblasts further examined the impact of SI on TSC1/mTORC1 signaling and related markers. Results SI treatment reduced joint damage and OARSI scores in the rat OA model, significantly decreasing CD31hiEmcnhi expression, indicating a reduction in type H vessel formation. SI also downregulated ALP, OCN, and BMP expression while upregulating TSC1, suggesting inhibition of the mTORC1 signaling pathway and VEGF release. In vitro, SI increased TSC1 expression and decreased mTORC1 signaling, VEGF, ALP, OCN, and BMP levels in IL-1β-induced osteoblasts. Conclusion SI targets the TSC1/mTORC1 signaling pathway to suppress osteoblast activation and VEGF release, inhibiting type H vessel formation and slowing abnormal subchondral bone remodeling. These findings provide a novel therapeutic approach for OA by focusing on subchondral bone remodeling mechanisms.