Chitosan modified with PAP as a promising delivery system for melatonin in the treatment of osteoporosis: targeting the divalent metal transporter 1.

The demands for novel and efficient therapies have gradually increased with the rising concerns of osteoporosis (OP). The most popular method in promoting bone regeneration during osteoporotic conditions consists of loading bioactive materials with different drugs to treat osteoporotic bones by either promoting the process of osteogenesis, or by inhibiting the activity of osteoclasts. By analyzing single cell sequencing results, we found that divalent metal transporter 1 (DMT1) played a role in OP. Based on our previous results, we found that melatonin (MT) suppressed expression of DMT1 induced by high glucose during OP, so we determined the efficacy of MT for the treatment of OP. However, the clinical effects of MT on OP were unsatisfactory. To enhance its biological efficacy, we combined MT with porous gelatin chitosan (chitosan) and the conductive material, PLA-b-AP-b-PLA (PAP), then determined how MT incorporation in chitosan@PAP nanoparticles affected the ability to promote MC3T3-E1 osteogenesis and mineralization, both in vitro and in vivo. The results confirmed the effect of MT on DMT1. We then prepared and characterized composites prepared as nanofibers, and determined the efficacy of MT combined with chitosan-PAP modified hydrogels as a slow-release system in a femur model of osteoporosis mice, with associated properties suitable for bone tissue engineering. The results indicated that MT-loaded chitosan@PAP nanospheres showed favorable osteogenic functions, both in vivo and in vitro, providing a practical solution for bone regeneration for OP patients.

Comparing the effects of team-based and problem-based learning strategies in medical education: a systematic review.

BACKGROUND: Recently, there has been a concerted effort within medical schools to depart from conventional lecture-based learning approaches to alternative teaching methods such as team-based learning (TBL) and problem-based learning (PBL), with the aim of enhancing both student engagement and instructional efficacy. Despite this shift, a comprehensive review that directly compares the impacts of PBL and TBL methods in medical education is lacking. This study seeks to address this gap by conducting a meta-analysis that compares the effects of TBL and PBL in the context of medical education. METHODS: Studies from Embase, PubMed, Web of Science, China National Knowledge Infrastructure, and Chinese Wanfang Database were searched, from inception to July 11, 2023. A meta-analysis was performed using Stata 14.0, and a total of 10 studies (including 752 participants) were included. The standardized mean difference (SMD) was used to estimate pooled effects. Heterogeneity was detected using the I2 statistic and further explored using meta-regression analysis. RESULTS: Compared with PBL, TBL significantly increased the number of theoretical tests (SMD = 0.37, 95% CI: 0.02-0.73). Additionally, TBL greatly improved teamwork skills compared with PBL. However, there were no significant differences between the TBL and PBL groups concerning practical skill scores, learning interest, or understanding skills. CONCLUSION: TBL in the theoretical aspects of medical education appears to be more effective than PBL in improving theoretical test scores and teamwork skills, providing evidence for the implementation of TBL in medical education.

Poly-3-hydroxybutyrate-co-3-hydroxyvalerate(PHBV)-Polyethylene glycol 20k(PEG20k) as a promising delivery system for PT2399 in the treatment of disc degeneration.

Disc degeneration often leads to a highly prevalent symptom known as low back pain. Healthy nucleus pulposus tissue exhibited a hypoxic environment devoid of blood vessels, while degenerated nucleus pulposus experienced hypoxic deterioration and the formation of new blood vessels. In this study, the expression of important genes like HIF-2α was found to vary between normal and degenerated nucleus pulposus cells when compared to the hypoxic surroundings. The aim of this study was to examine how HIF-2α is controlled in nucleus pulposus cells under hypoxic conditions and its role in angiogenic mechanisms. To assess the impact of gradual inhibition of HIF-2α on disc degeneration, we utilized PHBV-based synthetic materials loaded with inhibitors of HIF-2α. Specifically, we employed LPS and PT2399 loaded PHBV-PEG20k (PP20) to intervene with human nucleus pulposus cells. Additionally, we treated APD rat models with PT2399 loaded PP20 to evaluate its effects. The expression levels of target markers in nucleus pulposus cells were detected using PCR, WB, and immunofluorescence. Additionally, the effect of drugs on disc degeneration was identified through HE staining. The findings indicated that HIF-2α, CAIX, PPP1R15A, VEGFA, and EGLN3 could potentially serve as new indicators of disc degeneration. Additionally, HIF-2α might contribute to the progression of disc degeneration through involvement in angiogenesis and the regulation of hypoxia. Furthermore, the utilization of PT2399 loaded PHBV-PEG20k (PP20) could potentially offer a fresh alternative for treating disc degeneration.

A novel poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-PEG-melatonin composite scaffold enhances for inhibiting bone tumor recurrence and enhancing bone regeneration.

Introduction: Postoperative comprehensive treatment has become increasingly important in recent years. This study was to repair tissue defects resulting from the removal of diseased tissue and to eliminate or inhibit the recurrence and metastasis of residual tumors under the condition of reducing the systemic side effects of chemotherapeutic drugs. To address these challenges, multifunctional scaffolds based local drug delivery systems will be a promising solution. Methods: An optimal drug-loaded scaffold material PHBV-mPEG5k (PP5) was prepared, which is biocompatible, hydrophilic and biodegradable. Furthermore, this material showed to promote bone healing, and could be conveniently prepared into porous scaffold by freeze-drying the solution. By means of introducing melatonin (MT) into the porous surfaces, the MT loaded PP5 scaffold with desirable sustained release ability was successfully prepared. The effectiveness of the MT loaded PP5 scaffold in promoting bone repair and anti-tumor properties was evaluated through both in vivo and in vitro experiments. Results and Discussion: The MT loaded PP5 scaffold is able to achieve the desired outcome of bone tissue repair and anti-bone tumor properties. Furthermore, our study demonstrates that the PP5 scaffold was able to enhance the anti-tumor effect of melatonin by improving cellular autophagy, which provided a therapeutic strategy for the comprehensive postoperative treatment of osteosarcoma.

GelMA Hydrogel as a Promising Delivery System for Osthole in the Treatment of Rheumatoid Arthritis: Targeting the miR-1224-3p/AGO1 Axis.

Rheumatoid arthritis (RA) is a multifaceted, chronic, progressive autoimmune disease. This study aims to explore the potential benefits of an enhanced drug delivery system utilizing optimized Gelatin Methacryloyl (GelMA) vectors in RA management. We evaluated the levels of miR-1124-3p and AGO1 in RA tissues and cell lines using qPCR, WB, and immunofluorescence. The effects of osthole on inflammatory response and joint morphology were determined by qPCR, H&E staining, and micro-CT. The data showed that miR-1224-3p was downregulated in RA tissues and HUM-iCell-s010RA cells, while the overexpression of miR-1224-3p in HUM-iCell-s010RA cells reduced the expression of IL-6 and IL-1ß. Luciferase assay demonstrated that AGO1 was a direct target gene of miR-1224-3p. Additionally, osthole treatment increased miR-1224-3p levels and decreased AGO1 expression. The release data showed that osthole loaded on GelMA was released at a slower rate than free osthole. Further studies in a mouse model of CIA confirmed that osthole-loaded GelMA was more effective in attenuating osteopenia in RA as well as alleviating autoimmune arthritis. These findings suggest that osthole can regulate the miR-1224-3p/AGO1 axis in RASFs cells and has the potential to be developed as a clinical anti-RA drug. GelMA could provide a new approach to long-term RA treatment.

Enhancer RNA SLIT2 Inhibits Bone Metastasis of Breast Cancer Through Regulating P38 MAPK/c-Fos Signaling Pathway.

BACKGROUND: Breast cancer (BRCA) is the most common cancer in women, while the bones are one of the most common sites of metastasis. Although new diagnostic methods or radiation or chemotherapies and targeted therapies have made huge advances, the occurrence of bone metastasis is also linked with poorer survival. Enhancer RNAs (eRNAs) have been demonstrated to participate in the progression of tumorigenesis and metastasis. However, the role of eRNAs in BRCA bone metastasis remains largely unclear. METHOD: Gene expression profiling of 1,211 primary BRCA and 17 bone metastases samples were retrieved from The Cancer Genome Atlas (TCGA) database, and the significant prognostic eRNAs were identified by Cox regression and least absolute shrinkage and selection operator (LASSO) regression. The acceptable accuracy and discrimination of the nomogram were indicated by the receiver operating characteristic (ROC) and the calibration curves. Then target genes of eRNA, immune cell percentage by CIBERSORT analysis, immune genes by single-sample gene set enrichment analysis (ssGSEA), hallmark of cancer signaling pathway by gene set variation analysis (GSVA), and reverse phase protein array (RPPA) protein chip were used to build a co-expression regulation network and identified the key eRNAs in bone metastasis of BRCA. Finally, Cell Counting Kit-8 (CCK8) assay, cell cycle assay, and transwell assay were used to study changes in cell proliferation, migration, and invasiveness. Immunoprecipitation assay and Western blotting were used to test the interaction and the regulation signaling pathways. RESULTS: The 27 hub eRNAs were selected, and a survival-related linear risk assessment model with a relatively high accuracy (area under curve (AUC): 0.726) was constructed. In addition, seven immune-related eRNAs (SLIT2, CLEC3B, LBPL1, FRY, RASGEF1B, DST, and ITIH5) as prognostic signatures for bone metastasis of BRCA were further confirmed by LASSO and multivariate Cox regression and CIBERSORT analysis. Finally, in vitro assay demonstrated that overexpression of SLIT2 reduced proliferation and metastasis in BRCA cells. Using high-throughput co-expression regulation network, we identified that SLIT2 may regulating P38 MAPK/c-Fos signaling pathway to promote the effects of metastasis. CONCLUSION: Based on the co-expression network for bone metastasis of BRCA, we screened key eRNAs to explore a prognostic model in predicting the bone metastasis by bioinformatics analysis. Besides, we identified the potential regulatory signaling pathway of SLIT2 in BRCA bone metastasis, which provides a promising therapeutic strategy for metastasis of BRCA.

The Na+/K+ ATPase Inhibitor Ouabain Attenuates Stemness and Chemoresistance of Osteosarcoma Cells.

BACKGROUND The purpose of this study was to explore the effects of the Na+/K+ ATPase inhibitor ouabain in regulating osteosarcoma (OS) cell stemness. MATERIAL AND METHODS Western blot, qPCR, sphere-forming analysis, DNA methylation analysis, and Ca²âº concentration detection were performed to evaluate the stem-like traits of cells and ouabain-induced effects and related mechanisms on OS cell stemness. Cell viability assessment was performed to evaluate the effect of ouabain on OS cell chemosensitivity. RESULTS Ouabain reduced the ALDH1 activity, the expression of critical stemness regulators, sphere size and number, and migration, invasion, and adhesion ability, but had little effects on cell viability. Additionally, the intracellular Ca²âº concentration and methylation level of the critical stemness regulators were higher in OS cells than in spheres formed by OS cells. Mechanistic studies revealed that ouabain leads to DNA methylation of stemness markers through increasing intracellular Ca²âº concentration. Notably, inhibition of Ca²âº channel or DNA methylation rescued the inhibition of ouabain on OS cell stemness. Additionally, ouabain enhances cisplatin sensitivity of OS cells, which is involved in Ca²âº channel and DNA methylation. CONCLUSIONS This work provides a potential compound for treating OS patients, especially OS patients with chemoresistance.

MicroRNA­195­5p inhibitor prevents the development of osteoarthritis by targeting REGγ.

Osteoarthritis (OA) is a common inflammatory joint disease. MicroRNAs (miRNAs/miRs) have been reported to be involved in the pathogenesis of OA; however, the role of miRNAs in OA remains largely unexplained. The purpose of the present study was to investigate the expression and role of miR­195­5p in OA, and to further explore the mechanism. The expression level of miR­195­5p was measured using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). TargetScan and a luciferase reporter assay were used to reveal the associations between miR­195­5p and REGγ (also known as PSME3). To investigate the role of miR­195­5p in OA, a cell model of OA was established by treating ATDC5 cells with lipopolysaccharide (LPS). Then an MTT assay was conducted to detect cell proliferation ability, and an Annexin V­fluorescein isothiocyanate/propidium iodide apoptosis detection kit was used to measure cell apoptosis. In addition, the levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α were determined using ELISA. Furthermore, gene and protein expression was measured via RT­qPCR and western blot assay, respectively. The results revealed that miR­195­5p was significantly upregulated in the articular cartilage tissues of patients with OA and in LPS stimulated ATDC5 cells. REGγ was a direct target of miR­195­5p. The repressed cell proliferation ability and enhanced cell apoptosis of ATDC5 cells induced by LPS were reversed by miR­195­5p downregulation. Furthermore, LPS stimulation significantly upregulated the levels of IL­1ß, IL­6 and TNF­α, while miR­195­5p downregulation markedly reduced the expression of inflammatory factors induced by LPS. The results also revealed that a miR­195­5p inhibitor inhibited the LPS induced repression of the Wnt/ß­catenin signaling pathway and activation of nuclear factor (NF)­κB signaling pathway in ATDC5 cells. Notably, the results of the present study also indicated that all of the effects of the miR­195­5p inhibitor on ATDC5 cells were reversed by REGγ silencing. In conclusion, the results indicated that the miR­195­5p inhibitor served a protective role in OA by inhibiting chondrocyte apoptosis and inflammatory responses by regulating the Wnt/ß­catenin and NF­κB signaling pathways.

Suppressive oligodeoxynucleotide-induced dendritic cells rein the aggravation of osteoarthritis in mice.

OBJECTIVE: To explore the effect of suppressive oligodeoxynucleotide-induced dendritic cells (S-DCs) in the osteoarthritis (OA) therapy. METHODS: S-DCs were prepared from splenic CD11c + cells by in vitro culture with suppressive oligodeoxynucleotide. The function and phenotypes of S-DCs were measured by ELISA and flow cytometry. The innate immune signaling pathways were detected by western blotting in the non-treated DCs and S-DCs upon stimulation. In vivo, we employed an iodoacetate-induced OA mice model. S-DCs were transferred by intravenous route. The weight bearing of mice was evaluated and pro-inflammatory factors in OA joint were measured by real-time PCR. Treg cell ratio and CD4 + IL10+ cells in spleen were detected by flow cytometry at day 5 post OA induction. RESULTS: The S-DCs showed less inflammatory phenotypes upon stimulation. The expression of pro-inflammatory cytokines and mature makers in the S-DCs were blunt, due to the impaired innate immune signal transduction. In an iodoacetate-induced OA model, transfer of S-DCs significantly controlled the process of OA. Restricted inflammatory responses were observed in the joint of S-DC recipients. Moreover, after S-DC transfer, Tregs and CD4 + IL10+ cells were mounted in the spleen. CONCLUSION: Transfer of suppressive oligodeoxynucleotides-induced autologous DCs may represent a potential agent to control the aggravation of OA in patients.

Leukotriene D4 induces cellular senescence in osteoblasts.

Aging is associated with the development of osteoporosis, in which cellular senescence in osteoblasts plays a key role. Leukotriene D4 (LTD4), an important cysteinyl leukotriene (cysLT), is a powerful pro-inflammatory mediator formed from arachidonic acid. However, little information regarding the effects of LTD4 on the pathogenesis of osteoporosis has been reported before. In the present study, we defined the physiological roles of LTD4 in cellular senescence in osteoblasts. Our results indicate that LTD4 treatment decreased the expression of SIRT1 in a dose-dependent manner in MC3T3-E1 osteoblastic cells. Additionally, LTD4 significantly increased the expression of p53, p21 and plasminogen activator inhibitor-1 (PAI-1). LTD4 was also found to elevate the activity of ß-galactosidase (SA-ß-Gal) but to prevent BrdU incorporation. Our results indicate that cysteinyl leukotriene receptor 1 (cysLT1R) could be detected in MC3T3-E1 osteoblastic cells at both the mRNA and protein levels. However, cysLT2R was not expressed in these cells. Interestingly, we found that knockdown of cysLT1R or use of the selective cysLT1R antagonist montelukast abolished the LTD4-induced reduction in SIRT1 and increase in p53, p21, and PAI-1. Notably, knockdown of cysLT1R by transfection with cysLT1R siRNA or treatment with montelukast attenuated the LTD4-induced increase in SA-ß-Gal activity. Our study shows for the first time that LTD4 has a significant impact on cellular senescence in osteoblasts.

The cysteinyl leukotriene receptor 1 (CysLT1R) antagonist montelukast suppresses matrix metalloproteinase-13 expression induced by lipopolysaccharide.

Bacterial products such as LPS are critical factors responsible for bone destruction. MMP-13, a member of the matrix metalloproteinase family, plays a critical role in the proteolytic degradation of extracellular matrix components, which includes collagen fibrils in the bone matrix. Montelukast is a selective cysteinyl leukotrienes receptor 1 (cysLT1R) antagonist used clinically for the treatment of asthma, as it reduces eosinophilic inflammation in airways. This study aims to explore the role of montelukast in regulating MMP-13 expression induced by LPS in osteoblasts. Our results indicate that LPS stimulated cysLT1R expression in mouse MC3T3-E1 osteoblasts in a dose- and time-dependent manner. Notably, LPS-induced up-regulation of MMP-13 was ameliorated by treatment with montelukast in a dose-dependent manner. Furthermore, treatment with montelukast stimulated the expression of SOCS3, an inhibitor of MMP-13. Silencing of SOCS3 abolished the inhibitory effects of montelukast on MMP-13 expression. Mechanistically, we found that montelukast suppressed LPS-induced nuclear translocation of NF-κB p65 as well as NF-κB transcriptional activity by inhibiting the phosphorylation and degradation of IκBα. These data suggest that montelukast can modulate inflammatory events in bone diseases.

Antagonism of cysteinyl leukot-riene receptor 1 (cysLT1R) by montelukast regulates differentiation of MC3T3-E1 cells under overloaded mechanical environment.

Long-term exposure to overloaded mechanical environment induces bone fatigue damage symptoms and osteoblast damages. Montelukast is a selective cysteinyl leukot-riene receptor 1 (cysLT1R) antagonist, which has been used for the treatment of bronchial asthma in clinics. In the current study, we have identified a novel pharmacological role of montelukast by finding that it has protective properties against overload damage in osteoblastic MC3T3-E1 cells. Firstly, our results show that CysLT1R is expressed in MC3T3-E1 cells. Mechanical tensile strain of 5000-7000 µÎµ resulted in a significant upregulation of CysLT1R in osteoblastic MC3T3-E1 cells in an intensity dependent manner. Secondly, MTT assay indicates that loading with 5000 µÎµ mechanical strain inhibited cell proliferation, which was suppressed by montelukast treatment. Furthermore, montelukast promotes cell differentiation by increasing the expression of ALP and RUNX2. Alizarin Red S staining assay showed that montelukast abolished the inhibitory effects of overload mechanics on osteoblast mineralization. Mechanistically, the effect of montelukast on osteoblastic differentiation acted by activating the extracellular regulated protein kinases (ERK) pathway. The obtained results suggested that montelukast promotes proliferation and differentiation in osteoblasts exposed to overload mechanics.

Corticosterone suppresses IL-1ß-induced mPGE2 expression through regulation of the 11ß-HSD1 bioactivity of synovial fibroblasts in vitro.

The aim of the present study was to investigate the correlation between glucocorticoid activity regulation, prostaglandin E2 (PGE2) synthesis, and synovial inflammation inhibition activity, through microsomal prostaglandin E synthase-1 (mPGES-1) expression regulated by the glucocorticoid pre-receptor regulator, 11ß-hydroxysteroid dehydrogenase-1 (11ß-HSD1). In the present study, fibroblast-like synovial cells of rats were studied as a cell model. Cells were stimulated with 10 ng/ml interleukin (IL)-1ß for 24 h, and were subsequently, within the next 24 h, treated with or without 10-6 mmol/l corticosterone alone or with 100 nmol/l PF915275. At the end of the second 24 h, PGE2 levels in culture supernatants were assayed. Cells were harvested for mRNA evaluation of 11ß-HSD1, mPGES-1, IL-1ß and tumor necrosis factor (TNF)-α, and protein detection of 11ß-HSD1 and mPGES-1 using reverse transcription-qualitative polymerase chain reaction and western blot analysis, respectively. Corticosterone was demonstrated to suppress the mRNA expression levels of inflammatory factors, such as TNF-α and PGE2, induced by IL-1ß in vitro. Simultaneously, expression levels of 11ß-HSD1 decreased significantly at the mRNA and protein levels (P<0.05). Cortisol concentration in the medium of the group treated with corticosterone was significantly increased (P<0.05) compared with that of the control group; however, the cortisol concentration was decreased in the medium when the conversion bioactivity of 11ß-HSD1 was inhibited by PF915275, while the changes in 11ß-HSD1 and mPGES-1 mRNA expression levels and PGE2 content were reversed in the medium. These results indicated that a significant positive correlation (P<0.01) may exist between mRNA and protein expression levels. To conclude, 11ß-HSD1 is a key regulator for the synthesis of mPGES-1 and PGE2 in the inflammatory synovial cells in vitro, suggesting a potential interference target for osteoarthritis.

[CONDITIONED MEDIUM OF BONE MARROW MESENCHYMAL STEM CELLS ALLEVIATE INHIBITING EFFECT OF DEXAMETHASONE ON OSTEOGENETIC CAPABILITY OF OSTEOBLAST].

OBJECTIVE: To explore the paracrine effect of bone marrow mesenchymal stem cells (BMSCs) on dexamethasone-induced inhibition of osteoblast function in vitro. METHODS: The serum free conditioned medium of mouse BMSCs cultured for 24 hours was prepared for spare use. The 3rd passage of MC3T3-E1 cells were divided into 4 groups: the control group (group A), dexamethasone group (group B), dexamethasone+BMSCs conditioned medium (1:1) group (group C), and BMSCs conditioned medium group (group D). After 24 hours of culture, the alkaline phosphatase (ALP) content was determined; the protein expressions of RUNX2 and Osteocalcin were detected by Western blot; and the gene expressions of collagen type I-α 1 (COL1A1), RUNX2, ALP, and Osteocalcin were detected by real-time fluorescence quantitative PCR (RT-qPCR); alizarin red staining was used to observe calcium nodules formation at 21 days. RESULTS: After cultured for 24 hours, ALP content was significantly lower in groups B, C, and D than group A, and in group B than groups C and D (P < 0.05), but no significant difference was found between groups C and D (P > 0.05). The relative protein expression of RUNX2 of group B was significantly lower than that of groups A, C, and D (P < 0.05), but difference was not significant between groups A, C, and D (P > 0.05). The relative protein expression of Osteocalcin was significantly lower in group B than groups A, C, and D, in groups A and C than group D (P < 0.05), but difference had no significance between groups A and C (P > 0.05). The relative gene expressions of RUNX2, Osteocalcin, COL1A1, and ALP of groups B, C, and D were significantly lower than those of group A (P < 0.05); the relative gene expressions of RUNX2, Osteocalcin, and ALP were significantly higher in group D than groups B and C, in group C than group B (P < 0.05). The gene expression of COL1A1 was significantly higher in group D than group B (P < 0.05), but difference was not significant between groups B and C, and between groups C and D (P > 0.05). The cells of group A all died at 6 days after culture; at 21 days, the calcium no dule staining was positive by alizarin red in groups B, C and D, and the degree of the staining gradually increased from groups B to D. CONCLUSIONS: BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.

A novel process for obtaining phenylpropanoic acid precursor using Escherichia coli with a constitutive expression system.

Phenylpropanoids are widely used in food supplements, pharmaceuticals, and cosmetics with diverse benefits to human health. Trans-cinnamic acid or p-coumaric acid is usually used as the starting precursor to produce phenylpropanoids. Synthetic bioengineering of microbial cell factories offers a sustainable and flexible alternative method for obtaining these compounds. In this study, a constitutive expression system consisting of Rhodotorula glutinis phenylalanine/tyrosine ammonia lyase was developed to produce a phenylpropanoic acid precursor in Escherichia coli. To improve trans-cinnamic acid and p-coumaric acid production, BioBrick optimization was investigated, causing a 7.2- and 14.2-fold increase in the yield of these compounds, respectively. The optimum strain was capable of de novo producing 78.81 mg/L of trans-cinnamic acid and 34.67 mg/L of p-coumaric acid in a shake flask culture. The work presented here paves the way for the development of a sustainable and economical process for microbial production of a phenylpropanoic acid precursor.