Runx2 Expression Research Articles

P38 Signaling Mediates Naringin-Induced Osteogenic Differentiation of Porcine Metanephric Mesenchymal Cells.

To explore the potential of metanephric mesenchymal cells (MMCs) for osteogenesis and naringin's ability to enhance this process and its molecular mechanism. Porcine MMCs at 70 days of gestation were used as tool cells, cultured in osteogenic induction medium, identified by immunocytochemistry staining. Osteogenic potential of porcine MMCs and naringin's ability to enhance this process was tested by detecting changes in cell viability, alkaline phosphatase (ALP) activity, the expression of runt-related transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN), and the formation of mineralized nodules, and the application of the p38 signaling pathway inhibitor SB203580 vitiated the osteogenesis-promoting effect of naringin. Immunocytochemical staining showed that the cells were Vimentin and Six2(+), E-cadherin and CK-18(-). Naringin can activate the p38 signaling pathway to enhance the osteogenesis of porcine MMCs by increasing cell viability, ALP activity, the expressions of Runx2, OPN and OCN, and the formation of mineralized nodules (P<0.05). The application of p38 signaling pathway inhibitor SB203580 vitiated the osteogenesis-promoting effect of naringin, manifested by decreased ALP activity, the expressions of Runx2, OPN and OCN, and the formation of mineralized nodules (P<0.05). Naringin, the active ingredient of Chinese herbal medicine Rhizoma Drynariae for nourishing Shen (Kidney) and strengthening bone, enhances the osteogenic differentiation of renal MMCs through the p38 signaling pathway.

Relaxin-2 promotes osteoblastic differentiation mediated by epidermal growth factor and epidermal growth factor receptor signaling.

Loss of osteogenic differentiation potential of osteoblasts has been associated with the pathogenesis of osteoporosis. Thus, stimulation of osteoblastic differentiation is a therapeutic strategy for osteoporosis. Relaxin-2 is a peptide hormone with potent biological functions. However, the effects of Relaxin-2 in osteoblastic differentiation and osteoporosis have not been reported before. Here, we report a novel physiological role of Relaxin-2 in promoting osteoblastic differentiation and mineralization of MC3T3-E1 cells. Our results indicate that exposure to Relaxin-2 upregulated the expression, and elevated the activity of alkaline phosphatase (ALP) when MC3T3-E1 cells were cultured in osteogenic differentiation medium (OM). Additionally, Relaxin-2 upregulated the mRNA levels of osteocalcin (ocn), osteopontin (opn), and collagen type I alpha 1 (Col1a1). The alizarin red S staining assay revealed that Relaxin-2 promoted the mineralization of MC3T3-E1 cells. We also found that Relaxin-2 increased the expression of Runx-2 as well as the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). Importantly, silencing of EGF abolished the effects of Relaxin-2 in osteoblastic differentiation and related gene expression. These findings suggest that Relaxin-2 stimulates osteogenic differentiation through activating EGF/EGFR signaling.

Novel Chitosan-Gelatin Scaffold with Valproic Acid Augments In Vitro Osteoblast Differentiation of Mesenchymal Stem Cells.

The study aimed to develop a biodegradable scaffold incorporating valproic acid (VPA) for improved human bone marrow-derived mesenchymal stem cell (hBMSC) proliferation, differentiation, and bone mineral synthesis. A chitosan-gelatin (CH-G) scaffold was fabricated and loaded with varying concentrations of VPA (1, 3, 5 mM/L). In vitro studies assessed drug release, cell proliferation, morphology, mineralization, and gene expression. VPA was rapidly released from the scaffold, with over 90% cumulative release within seven days. Cells cultured on VPA-loaded scaffolds exhibited significantly enhanced proliferation and mineralization compared to the control. VPA treatment upregulated osteocalcin and runt-related transcription factor 2 (Runx-2) expression, key markers of osteogenic differentiation. The CH-G scaffold, particularly with 1 mM/L VPA, demonstrates excellent biocompatibility and promotes hBMSC-mediated bone regeneration. This novel approach holds promise for future applications in bone tissue engineering.

Resveratrol alleviates thiram-induced tibial dyschondroplasia by regulating BMP-2, RUNX-2 and HIF-1α expressions in broiler chickens

Tetramethylthiuram disulfide (thiram) is a pesticide and fungicide, widely used in agriculture, and is a recognized cause of tibial dyschondroplasia (TD), a skeletal abnormality in fast-growing broiler chickens. This study aimed to investigate the effect of resveratrol (RSV) against TD induced by thiram in broilers. A total of 240 broilers were evenly divided into three groups (n = 80) i.e., Control, TD and RSV. All groups received feed and water ad libitum, whereas the TD and RSV groups received standard diet with an addition of thiram (50 mg/kg of feed) from 4–7 days to induce TD. After the cessation of thiram on day 8, the RSV group was administered RSV at 400 mg/kg body weight orally from 8–18 days. Histopathological changes in tibia were assessed using hematoxylin and eosin (H&E) staining. In addition, the mRNA and protein expressions of targeted genes were analyzed using RT-qPCR, immunohistochemistry, and western blotting. Thiram feeding resulted in an increased growth plate (GP) width, decreased vascularization, irregular shaped chondrocytes with pyknotic nuclei, lameness, poor production performance, reduced levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), alkaline phosphatase (ALP) and elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA). The mRNA and protein expressions of BMP-2 and RUNX-2 were downregulated, whereas HIF-1α was upregulated in TD affected broilers (p < 0.05). RSV administration resulted in a significant increase in mRNA and protein levels of BMP-2 and RUNX-2 (p < 0.05) and a decrease in the levels of HIF-1α. In addition, RSV improved vascularization, angiogenesis, alleviated lameness, and enhanced production efficiency. Furthermore, it restored the GP width, serum biochemical levels and liver antioxidant enzymes. In conclusion, this study indicated that resveratrol mitigated the adverse effects of thiram toxicity in broiler chickens.

Study on the Effects of Low-Intensity Pulsed Ultrasound and Iron Ions for Proliferation and Differentiation of Osteoblasts

ObjectiveThis study involved the proliferation and differentiation of osteoblasts treated with low-intensity pulsed ultrasound (LIPUS) and iron (Fe3+) ions, respectively. The biological effects of LIPUS and Fe3+ ions on the proliferation and differentiation of osteoblasts were also evaluated. MethodsMC3T3-E1 cells were seeded in six-well plates with the medium, which contained different concentrations of Fe3+ (0, 100, 200, 300, 400, 500, 600 and 700 μg L-1, respectively). LIPUS treatment was directed at the bottom of the plate for 20 min at an intensity of 80 mW cm-2 every day. ResultsViability results showed that a dose of 400 μg L-1 Fe3+ ions had the best effect at promoting osteogenic proliferation in cell culture. The results of alkaline phosphatase staining and mineralization indicated that the differentiation of osteoblasts was promoted by LIPUS and Fe3+ ions. Fluorescence staining results showed that the number of cell nuclei in the LIPUS, Fe3+ and LIPUS-Fe groups increased by 37.20%, 55.81% and 89.76%, respectively. Migration data indicated that migration and proliferation rates were increased by LIPUS and Fe3+, and the results of protein expression indicated that LIPUS and Fe3+ may increase the expression of Wnt, β-catenin, and Runx2, hence promoting normal bone regeneration and development. ConclusionThe combination of LIPUS (1.5 MHz, 80 mW cm-2) and Fe3+ accelerates the proliferation and differentiation of osteoblasts significantly compared with single-factor treatment (stimulated by LIPUS and Fe3+ ions, respectively). This study could establish a foundation for LIPUS-responsive biomaterials in the repair and regeneration of bone tissues.

Converting ocean nacre into bone mineral matrix composite for bone regeneration- in vitro and in vivo studies

Nacre of Pinctada maxima is a natural biomineralized matrix, appeared 7 million years before hominins. In this study, we converted nacre into a self-setting particle bound with multiple calcium orthophosphates that reassemble mammals’ bone mineral matrices for induction of bone regeneration. The nacre-based calcium orthophosphates composite (NCOC) exhibited a compression strength of 10 MPa, which is superior to human trabecular bone. In vitro bioactivity tests revealed the formation of apatite with nano-porous flake-like crystals on the composite surface that mimic HA structure of a human bone matrix. NCOC demonstrated efficient attachment and proliferation of osteoblast cells, promoting osteogenic differentiation by increasing expressions of RUNX2 and OPN. In vivo studies using rabbit back fascia demonstrated that NCOC displays better bone healing and biocompatibility than conventional bone substitute apatite in critical bone defect models. The degradation of calcium carbonate crystal in vivo does not compromise structural integrity of NCOC. Overall, our data showed that NCOC produced through self-setting reactions, presents advantages such as accelerated biodegradation and osteostimulative properties, making it a promising bone substitute for effective bone regeneration.

LMK-235 suppresses osteoclastogenesis and promotes osteoblastogenesis by inhibiting HDAC4

Osteoblasts and osteoclasts play an important role in maintaining the structural integrity of bone tissue, in which osteoclasts degrade bone structure and osteoblasts restore bone tissue. The imbalance of osteoblast and osteoclast function can lead to many bone-related diseases, such as osteoporosis and inflammatory osteolysis. The drug that can both promote bone formation and inhibit bone loss will be able to treat those diseases. In this study, it was found that LMK-235, an selective HDAC4/5 inhibitor, inhibited the differentiation and maturation of osteoclasts by regulating NF-κB and p-Smad2/3 signaling pathways via inhibition of HDAC4. At the same time, we found that LMK-235 promoted osteoblast mineralization by upregulating Runx2 expression via inhibition of HDAC4. In vivo, LMK-235 was able to alleviate lipopolysaccharide (LPS)-induced calvarial osteolysis and promote the repair of bone defects. Taken together, LMK-235 suppresses osteoclast differentiation and promotes osteoblast formation by inhibiting HDAC4. This may provide a valuable treatment for bone diseases caused by abnormal osteoclast bone resorption and osteoblast bone regeneration.

A natural agent, 5-deoxycajanin, mitigates estrogen-deficiency bone loss via modulating osteoclast-osteoblast homeostasis

Hyperactive osteoclasts and hypoactive osteoblasts usually result in osteolytic conditions such as estrogen-deficiency bone loss. Few natural compounds that both attenuating bone resorption and enhancing bone formation could exert effects on this imbalance. 5-Deoxycajanin (5-D), an isoflavonoid extracted from Cajan leaf with estrogen-like properties, were found to have beneficial pharmacological effects on rebalancing the activities of osteoclasts and osteoblasts. This study revealed that 5-D at the same concentration could inhibit osteoclastogenesis of BMMs and promoted osteoblast differentiation of BMSCs. 5-D not only attenuated the fluorescent formation of RANKL-induced F-actin belts and NFATc1, but also activated ALP and RUNX2 expressions. As to downstream factor expressions, 5-D could block osteoclast-specific genes and proteins including NFATc1 and CTSK, while increased osteogenic genes and proteins including OPG and OCN, as confirmed by Real-time PCR and Western Blotting. Additionally, the network pharmacology and molecular docking identified the involvement of 5-D in the MIF and MAPK signaling pathways and the stable binding between 5-D and MAPK2K1. Further Western blot studies showed that 5-D decreased the phosphorylation of p38 and ERK in osteoclasts, but promoted these phosphorylations in osteoblasts. In a female C57BL/6J mouse model of estrogen deficiency-induced bone loss, 5-D demonstrated efficacy in enhancing BMD through attenuating osteoclast activities and promoting osteogenesis. These results underscore the potential application of 5-D on treating osteolysis resulting from hyperactive osteoclasts and hypoactive osteoblasts, shedding light on modulating osteoclast-osteoblast homeostasis.

3D-bioprinted poly(lactic acid)/β-TCP/mesoporous silica scaffolds: An investigation on in-vitro bioactivity and osteogenesis characteristics

The current work aims to formulate novel bioactive and biocompatible 3D printed poly (l-lactic acid)/Beta tri calcium phosphate composite scaffolds reinforced with different types of mesoporous silica materials [PLA/β-TCP/MSMs] for bone regeneration application, which is not envisaged earlier. The bioink encompassing 30 % organic content (PLA) and 70 % inorganic content (β-TCP and MSMs) is formulated and their rheological characteristics are evaluated. Optimization of process conditions for 3D printed PLA composite scaffolds was done and pneumatic extrusion is performed. The physico-chemical properties as well as in vitro biological characteristics were assessed for 3D printed PLA/β-TCP/MSMs based composite scaffolds. The existence of β-TCP and MSMs incorporated into PLA matrix was observed to fasten the formation of hydroxyapatite, as evidenced by in vitro bioactivity assessment. The in vitro cytocompatibility analysis revealed that the 3D printed PLA/β-TCP/MSMs composite scaffolds exhibit suitable biocompatible behaviour and osteogenic potential. The calcium mineralization and ALP expression were also noticed in higher levels for 3D printed PLA/β-TCP/MSMs composite scaffolds. Gene expression analysis confirmed the expression of COL1, OCN, BMP-2 and RUNX2 on 3D printed PLA/β-TCP/MSMs composite scaffolds. The results speculate that this novel formulation closely resembling the composition of natural bone might have promising applications in terms of in vivo bone tissue engineering.

Eucommia ulmoides extract regulates oxidative stress to maintain calcium homeostasis and improve diabetic osteoporosis.

Diabetic osteoporosis (DOP) is a secondary disease that severely affects the health and quality of life of patients with diabetes mellitus. This study aimed to explore the bone protective effect of aqueous extract of Eucommia ulmoides (EUL) in DOP mice. DOP mice were established using a high-sugar, high-fat diet and streptozotocin (STZ) (35 mg/kg for three consecutive days), and the EUL aqueous extract (2.5 g/kg/day) was orally administered for 6 weeks. The serum levels of oxidative stress-related factors, calcium, and phosphorus were assessed using biochemical assays. The osteoprotective effect of EUL was assessed using micro-computer tomography, three-point bending assay, histological analysis, and immunoblotting. Quantitative real-time polymerase chain reaction and western blotting were performed to detect the expression levels of calcium transport channel factors in the kidney and small intestine tissues. Furthermore, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the femur, kidney, and small intestine tissues were detected using western blotting and quantitative real-time polymerase chain reaction. EUL aqueous extract reduced blood glucose levels, increased body weight, and relieved symptoms in DOP mice (p < .05). It also increased bone mineral density, improved the bone microstructure, decreased the number of femoral osteoclasts, and increased the expression of femoral Runx2 and Bmp2 in DOP mice (p < .01). After 6 weeks of EUL aqueous extract administration, serum levels of SOD, CTA, calcium, and phosphorus were upregulated, whereas MDA levels were decreased (p < .01). The aqueous EUL extract also upregulated the expression of TRPV5, PMCA-1b, and CaBP-9 k in the kidney and small intestine of DOP mice (p < .01). Furthermore, the expression of Nrf2 and HO-1 in the kidney, small intestine, and femur tissues was increased (p < .01). EUL aqueous extract reduced blood glucose levels in DOP mice and regulated oxidative stress through the Nrf2/HO-1 pathway, thereby maintaining calcium homeostasis and ultimately improving bone quality. Our study suggested that EUL aqueous extract may be effective in the treatment of DOP.

Indoxyl Sulfate Inhibits Osteogenesis in Bone Marrow Mesenchymal Stem Cells through the AhR/Hes1 Pathway.

Uremic toxins cause bone disorders in patients with chronic kidney disease (CKD). These disorders are characterized by low turnover osteodystrophy and impaired bone formation in the early stages of CKD. Evidence indicates that the aryl hydrocarbon receptor (AhR) mediates signals that suppress early osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, whether the AhR mediates the effects of indoxyl sulfate (IS), a uremic toxin, on BMSC osteogenesis remains unclear. We investigated whether IS affects osteogenesis through the AhR/Hes1 pathway. Expression levels of osteogenesis genes (Runx2, Bmp2, Alp, and Oc), AhR, and Hes1 were measured in mouse BMSCs (D1 cells). At concentrations of 2-50 μM, IS significantly reduced mineralization, particularly in the early stages of BMSC osteogenesis. Furthermore, IS significantly downregulated the expression of Runx2, Bmp2, Oc, and Alp. Notably, this downregulation could be prevented using an AhR antagonist and through Ahr knockdown. Mechanistically, IS induced the expression of Hes1 through AhR signaling, thereby suppressing the transcription of Runx2 and Bmp2. Our findings suggest that IS inhibits early osteogenesis of BMSCs through the AhR/Hes1 pathway, thus suppressing the transcription of Runx2 and Bmp2. Our findings may guide new therapeutic strategies against CKD-related bone disorders.

The role and mechanism of β-catenin-mediated skeletal muscle satellite cells in osteoporotic fractures by Jian-Pi-Bu-Shen formula.

Osteoporosis is a metabolic bone disease. β-Catenin is associated with fractures. Jian-Pi-Bu-Shen (JPBS) can promote the healing of osteoporotic fractures (OPF). However, the mechanism of β-catenin-mediated skeletal muscle satellite cells (SMSCs) in OPF by the JPBS is unclear. SMSCs were isolated and divided into five groups. The results showed that the survival rate of SMSCs was significantly higher in the low, medium, and high dose JPBS-containing serum groups after 7 days of incubation. The ALP activity and the number of SMSCs mineralized in the JPBS-containing serum intervention group were elevated. Axin, GSK-3β, β-catenin siRNAs were constructed and transfected into cells. Transfection of siRNAs reduced Axin, GSK-3β, and β-catenin expressions, respectively. β-Catenin-siRNA reversed ALP activity, the number of SMSCs mineralized, and the expression of β-catenin, BMP2, Runx2, COL-I, SP7/Ostrix, Osteocalcin, and BMP-7. Transcriptomic results suggested that the TNF signaling pathway associated with OPF was enriched. SD rats were subjected to the construction of OPF model by removing the ovaries. JPBS decreased the levels of PINP, ALP, CTX, and NTX through β-catenin in OPF rats, while increasing Runx2, β-catenin expressions through β-catenin at the broken end of fractures. Moreover, JPBS decreased BMC, BMD, and BV/TV and improved pathological damage through β-catenin in OPF rats. JPBS decreased the expression of Axin, GSK-3β mRNA, and protein, but increased the expressions of β-catenin, Pax7, COL-II, COL-II, BMP2, and Runx2 through β-catenin in OPF rats. In conclusion, JPBS inhibits Axin/GSK-3β expression, activates the β-catenin signaling, and promotes the osteogenic differentiation of SMSCs.

Effects of the dietary supplementation of manganese on growth, skeletal development, oxidative stress and bone metabolism related genes in gilthead seabream (Sparus aurata) larvae

Manganese (Mn) is an essential element necessary for normal growth, antioxidant defense and maintaining bone health. Knowledge about the effects of dietary Mn levels in gilthead seabream (Sparus aurata) larvae performance is still scarce. To determine the effects of dietary Mn levels on growth, survival, skeletal development and expression of antioxidant and bone metabolism related genes, gilthead seabream larvae were fed six experimental microdiets supplemented with manganese sulfate monohydrate (MnSO4.H2O) (contained 6.3, 39, 75, 79, 200 and 270 mg Mn/kg diet). After 26 days of feeding, dietary Mn levels had no effect on larval survival. However, larvae fed the non-supplemented diet (6.3 mg Mn/kg) showed signs of Mn deficiency, including growth reduction, low whole body Mn content, lower expression of mnsod, runx2, oc, and reduced bone mineralization. The poor vertebral body mineralization and low bone relative gene expression in seabream larvae was related to a marked increase in the number of fish with total skeletal anomalies and anomalies of the caudal vertebrae region. On the contrary, larvae fed with Mn supplemented diet up to 270 mg Mn/kg showed increased growth and whole body Mn content, up-regulated mnsod, runx2, alp, opn and oc expression, as well as increased bone mineralization, and reduced number of fish with skeletal anomalies. Therefore, the findings suggest that adequate dietary Mn supplementation positively influences the growth, skeletal development, and antioxidant defense of gilthead seabream larvae, preventing the manifestation of Mn deficiency-related issues.

New Insights on the Therapeutic Potential of Runt-Related Transcription Factor2 for Osteoarthritis: Evidence from Mendelian Randomization.

Research has highlighted the role of runt-related transcription factor2 (Runx2) in the development of osteoarthritis (OA); however, its causal association remains unclear. This study aimed to explore whether Runx2 expression is causally associated with OA and assess its therapeutic potential for OA. Genetic proxy instruments for Runx2 expression were obtained from gene expression quantitative trait locus (eQTLs) study of eQTLGen Consortium (n = 31,684). Aggregated genome-wide association study (GWAS) data for OA (including all OA [177,517 cases and 649,173 controls], knee OA (KOA) [62,497 cases and 333,557 controls], and hip OA (HOA) [36,445 cases and 316,943 controls]) were extracted from the Genetics of Osteoarthritis Consortium. We integrated eQTLs data with OA GWAS data to estimate their causal association and to estimate the potential of Runx2 as a drug target in the treatment of OA using summary data-based Mendelian randomization (SMR) analysis. Furthermore, different OA GWAS data (including all OA [77,052 cases and 378,169 controls], KOA [24,955 cases and 378,169 controls], and HOA [15,704 cases and 378,169 controls]) derived from the GWAS Catalog database were used for replication study. SMR analysis showed that high expression levels of Runx2 were associated with an increased risk of all OA [odds ratio (OR) 1.044, 95% confidence interval (CI) 1.023-1.067; P = 5.03 × 10-5], KOA (OR1.040, 95%CI 1.006-1.075; P = 0.021), and HOA (OR1.067, 95%CI 1.022-1.113; P = 0.003). This suggests that Runx2 inhibitors may have promising potential for the treatment of OA. Notably, the causal effects of Runx2 with all OA (OR1.053, 95%CI 1.027-1.079; P = 3.95 × 10-5) and KOA (OR1.043, 95%CI 1.001-1.087; P = 0.045) were repeated in the replication study, but limited evidence supported the association of Runx2 expression levels with HOA (OR1.045, 95%CI 0.993-1.101; P = 0.094). Our analyses indicate a positive correlation between Runx2 expression and OA risk across all three phenotypes, suggesting the potential of Runx2 inhibitors in the treatment of OA and providing evidence from a genetic perspective.

Reversal of heavy arterial calcification in a rat model of chronic kidney disease using targeted ethylene diamine tetraacetic acid-loaded albumin nanoparticles.

Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy. Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification in vitro, and then they were treated with EDTA. Human calcified femoral arteries were also treated ex vivo with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification. EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs vs. 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 vs. 29.815±24.169 mm3, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of BMP2 and RUNX2 (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (BMP2, RUNX2) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show ex vivo reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 vs. day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy. This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.

Osteogenic Differential Expression of Rabbit Adipose-Derived Mesenchymal Stem Cells by PPARγ Transfection Coated with Nanoparticles

Adipose-derived stem cells (ADSCs) have advantages in utilization. Nanoparticles (NP) are degradable, nontoxic and have good biocompatibility, besides, they can encapsulate and transport water-soluble, fat-soluble and deoxyribonucleic acid. This study explored the influence of NP-coated PPARγ transfection on osteogenic differentiation of rabbit ADSCs. After preparing PPARγ transfection system coated with nanoparticles, Petri dishes containing the same amount of cells were divided at the beginning of cell culture into blank group (not transfected), control group and observation group (transfected with PPARγ coated NP). Alkaline phosphatase (ALP) staining and Western Blot were done to detect the contents of lipoprteinlipase (LPL), Runx2 and serum osteocalcin (BGP) protein. The levels of Runx2, BGP and PPARγ mRNA were measured by RT-PCR. After ADSCs cells were stained, a small number of cells adhered to each other, and the cells were mainly fibroblast-like and spindle-shaped, and proliferated in the form of colonies. The levels of Runx2 and BGP in the observation group were significantly lower than those in the control group (p &lt; 0.05) after the ADSCs cells were cultured for 7 and 14 days. The expression of LPL and PPARγ mRNA in the observation group was higher control group (p &lt; 0.05). Transfection of PPARγ coated with nanoparticles inhibited the osteogenic differentiation of rabbit ADSCs by inhibiting the expression of Runx2 and BGP and promoting the expression of LPL and PPARγ.

3D-printed titanium scaffolds loaded with gelatin hydrogel containing strontium-doped silver nanoparticles promote osteoblast differentiation and antibacterial activity for bone tissue engineering.

Bone tissue engineering offers a promising alternative to stimulate the regeneration of damaged tissue, overcoming the limitations of conventional autografts and allografts. Recently, titanium alloy (Ti) implants have garnered significant attention for treating critical-sized bone defects, especially with the advancement of 3D printing technology. Although Ti alloys have impressive versatility, their lack of cellular adhesion, osteogenic and antibacterial properties are significant factors that contribute to their failure. Hence, to overcome these obstacles, this study aimed to incorporate osteoinductive and antibacterial cue-loaded hydrogels into 3D-printed Ti (3D-Ti) scaffolds. 3D-Ti scaffolds were synthesized using the direct metal laser sintering method and loaded with a gelatin (Gel) hydrogel containing strontium-doped silver nanoparticles (Sr-Ag NPs). Compared with Ag NPs, Sr-doped Ag NPs increased the expression of Runx2 mRNA, which is a key bone transcription factor. We subjected the bioactive 3D-hybrid scaffolds (3D-Ti/Gel/Sr-Ag NPs) to physicochemical and material characterization, followed by cytocompatibility and osteogenic evaluation. The microporous and macroporous topographies of the scaffolds with Sr-Ag NPs showed increased Runx2 expression and matrix mineralization, with potent antibacterial properties. Therefore, the 3D-Ti scaffolds incorporated with Sr-Ag NP-loaded Gel hydrogels favored osteoblast differentiation and antibacterial activity, indicating their potential for orthopedic applications.

Development of polyvinyl alcohol nanofiber scaffolds loaded with flaxseed extract for bone regeneration: phytochemicals, cell proliferation, adhesion, and osteogenic gene expression.

Introduction: Bone tissue engineering seeks innovative materials that support cell growth and regeneration. Electrospun nanofibers, with their high surface area and tunable properties, serve as promising scaffolds. This study explores the incorporation of flaxseed extract, rich in polyphenolic compounds, into polyvinyl alcohol (PVA) nanofibers to improve their application in bone tissue engineering. Methods: High-performance liquid chromatography (HPLC) identified ten key compounds in flaxseed extract, including polyphenolic acids and flavonoids. PVA nanofibers were fabricated with 30 wt.% flaxseed extract (P70/E30) via electrospinning. We optimized characteristics like diameter, hydrophilicity, swelling behavior, and hydrolytic degradation. MG-63 osteoblast cultures were used to assess scaffold efficacy through cell adhesion, proliferation, viability (MTT assay), and differentiation. RT-qPCR measured expression of osteogenic genes RUNX2, COL1A1, and OCN. Results: Flaxseed extract increased nanofiber diameter from 252nm (pure PVA) to 435nm (P70/E30). P70/E30 nanofibers showed higher cell viability (102.6% vs. 74.5% for pure PVA), although adhesion decreased (151 vs. 206 cells/section). Notably, P70/E30 enhanced osteoblast differentiation, significantly upregulating RUNX2, COL1A1, and OCN genes. Discussion: Flaxseed extract incorporation into PVA nanofibers enhances bone tissue engineering by boosting osteoblast proliferation and differentiation, despite reduced adhesion. These properties suggest P70/E30's potential for regenerative medicine, emphasizing scaffold optimization for biomedical applications.

Target analysis and identification of curcumin against vascular calcification

To investigate the mechanism of curcumin (CUR) on vascular calcification (VC), we screen for common targets of CUR and atherosclerosis and verify the targets genes in vivo and in vitro experiments. The common targets of CUR and AS were screened and obtained using different databases. These target genes were analyzed by GO and KEGG pathway enrichment analysis. PPI network analysis was performed and to analyze the key targets. A rat VC model was constructed and CUR was fed for three weeks. The changes of vascular structure and calcium salt deposition were observed in H&E and Von Kossa staining. Further, the expression of these target proteins was detected in the primary VSMCs of VC. The 31 common targets were obtained. GO functional enrichment analysis obtained 1284 terms and KEGG pathway enriched 66 pathways. The key genes were identified in the cytoHubba plugin. The molecular docking analysis showed that CUR bound strongly to EGFR, STAT3 and BCL2. The animal experiments showed the deposition calcium salt reduced by the CUR administration. These proteins BMP2, RUNX2, EGFR, STAT3 and BAX expression were upregulated in VC group and CUR attenuated the upregulated expression. The signal protein Akt and p65 expression increased in VC group and decreased in CUR group. We identified some common target genes of CUR and AS and identified these key genes. The anti-VC effect of CUR was associated with the inhibition of upregulation of EGFR, STAT3 and RUNX2 expression in VSMCs.

Effects of Etanercept on Experimental Osteoarthritis in Rats: Role of Histone Deacetylases.

Mounting evidence suggests that histone deacetylases (HDAC) inhibitors reduce cartilage destruction in animal models of osteoarthritis (OA). Tumor necrosis factor (TNF)-α-blocking treatment for OA may provide effective joint protection by slowing joint damage. To investigate the effects of intraperitoneal administration of etanercept (a TNF-α inhibitor) on OA development in rats and changes in the nociceptive behavior of rats and expression of HDACs, RUNX2, and MMP13 in cartilage. Induction of OA in Wistar rats was accomplished through anterior cruciate ligament transection (ACLT). One or five milligrams (mg) of etanercept was administered intraperitoneally for 5 consecutive weeks after ACLT to the ACLT + etanercept (1 and 5 mg/kg) groups. Nociceptive behavior and changes in knee joint width were analyzed. Cartilage was evaluated histologically and immunohistochemically. ACLT + etanercept significantly improved mechanical allodynia and weight-bearing distribution compared to ACLT alone. In OA rats treated with etanercept, cartilage degeneration and synovitis were significantly less pronounced than those in ACLT rats. OA-affected cartilage also showed reduced expression of HDAC 6, 7, RUNX-2, and MMP-13 in response to etanercept but increased expression of HDAC4. Our study demonstrated that etanercept therapy (1) attenuated the development of OA and synovitis in rats, (2) reduced nociception, and (3) regulated chondrocyte metabolism, possibly by inhibiting cell HDAC6 and HDAC7, RUNX2, and MMP13 and increasing HDAC4 expression. Based on new evidence, etanercept may have therapeutic potential in OA.