Mechanically reinforced and bioactive core–shell nanofibrous membranes loaded with astragaloside IV for guided bone regeneration

The aim of this study was to determine the cytotoxicity properties of essential oil from Cinnamon (Cinnamomum burmannii) bark on rat bone marrow mesenchymal stem cells (rBMSCs). This research was an experimental laboratory with The Post Test Only Group as a research design. Essential oil from Cinnamon (Cinnamomum burmannii) bark obtained from steam distillation method and essential oil was made in a series dilution with a concentration of 0,5%, 0,25%, 0,125%, 0,0625% and 0,0312%. Furthermore, the cytotoxicity test on rBMSCs using MTT assay. Microplate containing rBMSCs that had been exposed to five concentrations was incubated in a 5% CO2 incubator with 37 °C for 24 hours. The result of MTT assay can be seen from the absorbent solution formazan crystal through ELISA reader with the specific wavelength of 595 nm which produces data in the form of Optical Density (OD). The result research showed that the rBMSCs cell life percentage in 0,5%, 0,25%, 0,125%, 0,0625% and 0,0312% of concentration respectively were 12,8%, 18,3%, 21,7%, 26,5% and 32,5%. Probit analysis is used to determine the LC50 of essential oils from Cinnamon (Cinnamomum burmannii) bark. It can be concluded that the smaller the concentration of essential oils given to rBMSCs, the higher the percentage of cell life. In this study, the concentration of essential oils which can kill 50% of rBMSCs was 0,004%. How to Cite this Article Pubmed Style , Lestari ND, MHE, Arimbi , Plumeriastuti aH. Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Study. SRP. 2020; 11(9): 378-383. doi:10.31838/srp.2020.9.54 Web Style , Lestari ND, MHE, Arimbi , Plumeriastuti aH. Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Study. http://www.sysrevpharm.org/?mno=8143 [Access: March 28, 2021]. doi:10.31838/srp.2020.9.54 AMA (American Medical Association) Style , Lestari ND, MHE, Arimbi , Plumeriastuti aH. Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Study. SRP. 2020; 11(9): 378-383. doi:10.31838/srp.2020.9.54 Vancouver/ICMJE Style , Lestari ND, MHE, Arimbi , Plumeriastuti aH. Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Study. SRP. (2020), [cited March 28, 2021]; 11(9): 378-383. doi:10.31838/srp.2020.9.54 Harvard Style , Lestari, . N. D., , . M. H. E., Arimbi, . & Plumeriastuti, . a. H. (2020) Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Study. SRP, 11 (9), 378-383. doi:10.31838/srp.2020.9.54 Turabian Style , Niken Dwi Lestari, Mustofa Helmi Effendi, Arimbi, and and Hani Plumeriastuti. 2020. Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Reviews in Pharmacy, 11 (9), 378-383. doi:10.31838/srp.2020.9.54 Chicago Style , Niken Dwi Lestari, Mustofa Helmi Effendi, Arimbi, and and Hani Plumeriastuti. Cytotoxic Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Study. Systematic Reviews in Pharmacy 11 (2020), 378-383. doi:10.31838/srp.2020.9.54 MLA (The Modern Language Association) Style , Niken Dwi Lestari, Mustofa Helmi Effendi, Arimbi, and and Hani Plumeriastuti. Cytotoxic Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Study. Systematic Reviews in Pharmacy 11.9 (2020), 378-383. Print. doi:10.31838/srp.2020.9.54 APA (American Psychological Association) Style , Lestari, . N. D., , . M. H. E., Arimbi, . & Plumeriastuti, . a. H. (2020) Effect of Essential Oil from Cinnamon (Cinnamomum burmannii) Bark on Rat Bone Marrow Mesenchymal Stem Cells: In Vitro Reviews in Pharmacy, 11 (9), 378-383. doi:10.31838/srp.2020.9.54

To investigate the role of microRNA miR-488-5p, which showed increased expression after the disconnection of the inferior alveolar nerve, in promoting the osteogenic and neurogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), as well as its effect on promoting the neuralized tissue engineered bone regeneration. rBMSCs were subjected to in vitro neural or osteogenic differentiation induction cultures. The expression levels of miR-488-5p at different time points (days 0, 2, 4, and 7) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). miR-488-5p overexpression or low expression in rBMSCs was achieved by transfection with miR-488-5p mimics or inhibitors. Four groups, the miR-488-5p mimics, the miR-488-5p inhibitor, and their respective negative controls (NC), were established to investigate the effects of miR-488-5p on the neural differentiation and osteogenic differentiation of rBMSCs.A 5 mm diameter, full-thickness circular critical bone defect was created in the rat calvaria. The rats were treated with light-cured gelatin methacryloyl (GelMA) seeded with rBMSCs. The rats were divided into four groups: ①BLANK group: GelMA; ②BMSCs group: GelMA + rBMSCs; ③NC-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics NC; and ④miR-488-5p-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics. Specimens were obtained 4 and 8 weeks after surgery, and micro-CT was performed to measure and analyze bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface area/total volume (BS/TV) and trabecular number (Tb.N). The effects of neuralized tissue engineering bone formation in the defect area were assessed using Hematoxylin-Eosin (HE) staining, Masson staining, and tissue immunofluorescence staining of the nerve-specific protein soluble protein-100 (S100). As rBMSCs progressed toward neural or osteogenic differentiation, miR-488-5p expression increased significantly from day 0 to day 7. Regarding neural differentiation, the mimics group showed increased expression of neural-related genes and proteins compared with the mimics NC group, while the opposite result was observed in the inhibitor group. As for osteogenic differentiation, the mimics group showed increased expression of osteogenic genes and proteins, more intense alkaline phosphatase (ALP) and alizarin red staining (ARS) staining, and enhanced ALP activity compared with the mimics NC group, while the opposite result was observed in the inhibitor group. 4 and 8 weeks after critical calvarial defect construction in rats, the BLANK group had the least amount of new bone formation, while the BMSCs group and the NC-BMSCs group had similar and intermediate amounts of new bone formation. The miR-488-5p-BMSCs group had the most new bone formation. At 4 weeks, the BMD [(0.63±0.05) g/cm3 vs. (0.51±0.03) g/cm3], BV/TV (33.17%±6.43% vs. 18.11%±1.52%), BS/TV [(3.43±0.69) /mm vs. (2.46±0.20) /mm], and Tb.N [(0.92±0.21) /mm vs.(0.59±0.07) /mm] in the miR-488-5p-BMSCs group were significantly higher than those in the NC-BMSCs group. At 8 weeks, the BMD [(0.80±0.04) g/cm3 vs. (0.68±0.04) g/cm3], BV/TV (56.69%±6.22% vs. 42.36%±3.86%), and the number of S100-labeled nerve cells around the new bone (46.33±4.04 vs. 26.00±3.61) in the miR-488-5p-BMSCs group was also significantly higher than that in the NC-BMSCs group. miR-488-5p promoted the osteogenic and neurogenic differentiation of rBMSCs and promoted the formation of neuralized tissue-engineered bone in rat calvarial defects.

Bioinformatics analysis and validation of RNA methylation-related genes in osteogenic and adipogenic differentiation of rat bone marrow mesenchymal stem cells

Objective To investigate the effect of hydrophilicity of titanium with micro/nanotopographical surface on the cell viability and osteogenic differentiation in rat bone marrow mesenchymal stem cells (rBMSCs). Methods Methods of anodic oxidation and sandblast-alkali heat were utilized to establish scale structures. rBMSCs were seeded on these two titanium discs, cell viability was detected at 1, 3, 5 and 7 days by CCK8 assay. Total protein values and alkaline phosphatase (ALP) activity were examined at 7 and 14 days. ALP, collagen-I(COL1) and runt related transcription factor 2 (RUNX2) mRNA expression at 7 days were detected by qRT-PCR. The t test was used to compare water static contact angle, CCK8, total protein concentration and PCR detection index of osteogenic differentiation. Results The titanium surface treated by anodic oxidation was regular ordered nanotube arrays, while that treated by sandblast-alkali heat exhibited a three dimensional mesh nano porous structure, which both groups presented a similar micro-nanotopographical feature. However, the static contact angle of water in anodic oxidation group was significantly enhanced than that of sandblast-alkali heat group (83.3±2.3 vs 47.7±2.0, t=11.54, P<0.001). Compared with anodic oxidation group, rBMSCs on the discs modified by sandblast-alkali heat showed enhanced cell viability at 3, 5 and 7 days (0.66 ±0.03 vs 0.52 ±0.03, 1.15 ±0.06 vs 0.85 ±0.05, 1.58 ±0.07 vs 1.26 ±0.07), and these differences were statistically significant (t=2.962、3.845、3.183, P=0.042、0.018、0.033). After 7 and 14 days, the total protein concentration of rBMSCs in sandblasting alkali heat group was higher than that of anodic oxidation group [(389±45) μg/ml vs (226±32) μg/ml, (1070±59) μg/ml vs (760±65) μg/ml], whose differences were statistically significant (t=3.319、3.518, P=0.029、0.025). Moreover, ALP activity of rBMSCs of sandblasting alkali heat group at 7 days and 14 days was higher than that of anodic oxidation group [(2.11±0.32) U/gprot vs (1.00±0.21) U/gprot, (6.13±0.57) U/gprot vs (3.92±0.51) U/gprot], which showed statistically significant difference (t=2.912、2.976, P=0.043、0.041). Finally, mRNA levels of ALP, COL1 and RUNX2 in rBMSCs of sandblasting alkali heat group is higher than that of anodic oxidation group at 7 days (1.86 ±0.24 vs 1.00 ±0.15; 2.05 ±0.16 vs 1.00 ±0.14; 2.28 ±0.18 vs 1.00 ±0.12), and these difference was statistically significant (t=3.383、5.012、5.710, P=0.028、0.007、0.005). Conclusion Micro-nanotopographical surface of titanium implant in sandblast-alkali heat group promotes cell viability and osteogenic differentiation of rBMSCs, which may be affected by its hydrophilicity. Key words: Rat bone marrow mesenchymal stem cells; Osteogenic differentiation; Micro-nanotopographical surface; Hydrophilicity

Objective: To investigate the effect and underlying molecular mechanism of interleukin-10 (IL-10) secreted by M2 macrophages on the osteogenic differentiation ability of rat bone marrow mesenchymal stem cells (BMMSCs) after irradiation. Methods: Between February 2024 and April 2025, eight healthy male SD rats aged 2 to 3 weeks were selected, primary BMMSCs and macrophages from SD rats were cultured in vitro. Macrophages were polarized to the M2 phenotype, and their surface markers were identified by flow cytometry and immunofluorescence. The experiment was divided into four groups: normal BMSC control group (CON), 4 Gy irradiation group (4Gy), 4 Gy irradiation+M2 macrophage co-culture group (4Gy+8 μm), and 4 Gy irradiation+M2 macrophage co-culture+IL-10 neutralizing antibody group(4Gy+8 μm+m/r IL-10). An in vitro cellular irradiation model was established by exposing BMMSCs to X-ray radiation at a dose of 4 Gy. A non-contact co-culture system between M2 macrophages and irradiated BMMSCs was established using Transwell chambers with an 8 μm pore size. After 48 hours of co-culture, ELISA was used to detect the IL-10 concentration in the supernatant of each group, and Western blot was used to measure the expression levels of key osteogenic differentiation markers [alkaline phosphatase(ALP),Runt-related transcription factor 2(RUNX2),osteocalcin(OCN)] and STAT3 signaling pathway-related proteins (p-STAT3, STAT3) in BMMSCs. The specific role of IL-10 was verified by adding an IL-10 neutralizing antibody. Results: Cell counting kit-8 assay results showed that on day 7 post-irradiation, the absorbance value of the 4 Gy Irradiation group (0.241±0.093) was significantly lower than that of the control group (1.794±0.083) (t=21.63, P<0.001). Western blotting analysis indicated that the expression levels of osteogenic markers ALP and RUNX2 in the irradiation group (0.819±0.074, 0.785±0.074) were significantly lower than those in the control group (1.000±0.067, 1.000±0.056) (t=3.16, P=0.034; t=4.01, P=0.016, respectively). Immunofluorescence analysis revealed that the fluorescence intensities of RUNX2 and ALP in the irradiation group (19.932±1.291, 7.316±0.089) were markedly weaker compared to the control group (31.154±3.352, 30.789±1.455). After co-culture with M2 macrophages, the proportion of viable cells in the co-culture group [(77.800±1.758)%] was significantly higher than that in the Irradiation group [(61.933±2.732)%] (P<0.001). Furthermore, the IL-10 concentration in the supernatant of the co-culture group [(46.39±1.879) pg/ml] was significantly higher than that in the irradiation group [(7.530±0.239) pg/ml] (t=36.74, P<0.001). Western blot results demonstrated that the expression levels of ALP, RUNX2, and OCN in the co-culture group were significantly higher than those in the Irradiation group (t=8.07, P=0.015; t=5.01, P=0.038; t=3.07, P=0.918, respectively). Adding 1 μg/ml IL-10 neutralizing antibody to the co-culture system significantly reduced the IL-10 level in the supernatant [(6.521±0.460) pg/ml] compared to the co-culture group [(26.270±6.486) pg/ml] (t=5.06, P=0.037). The p-STAT3/STAT3 ratio in the neutralizing antibody group (0.840±0.071) was significantly lower than that in the co-culture group (1.289±0.156) (t=4.27, P=0.051), and the osteogenic differentiation capacity of BMMSCs was also notably attenuated. Conclusions: M2 macrophages can improve the osteogenic differentiation ability of BMMSCs after irradiation damage by secreting IL-10 and activating the STAT3 signaling pathway.

Objective To investigate the effect of Irisin on osteogenic differentiation and Sost of rat bone marrow mesenchymal stem cells (BMSCs) . Methods Irisin was set to have different concentrations (0, 80, 100, 120 ng/mL) , and the optimal concentration of Irisin was determined by CCK-8 method. The BMSCs cultured with Irisin was the experimental group, while the control group was cultured without Irisin. The alizarin red and von Kossa were stained on the 3rd, 7th and 14th day to observe the positive region of osteogenic differentiation and the expression intensity. The mRNA and protein expression levels of the bone-related genes were detected by RT-PCR and Western bolt at 3rd and 10th days. The One-Way ANOVA and t-test were applied to perform the statistical analysis of the data. Results (1) Irisin at a concentration of 100 ng/mL promoted the proliferation of rat BMSCs significantly after cultured three days (OD=0.951; F=102.52, P<0.05) . (2) The depth of staining and the size as well as the number of calcium nodules in the experimental group were significantly higher than those in the control group. (3) Compared with the control group, the mRNA and protein expression levels of the bone-related genes: ALP, Lrp5, BMP2 and Smad1 were significantly increased, but the expression levels of Sost mRNA and protein were significantly decreased (P<0.001) . Conclusions (1) Irisin can inhibit the expression of the Sost gene (2) Irisin can promote the proliferation and osteogenic differentiation of rat BMSCs in vitro. (3) Wnt and BMP signaling pathway may play a role in Irisin promoting osteogenic differentiation of rat BMSCs. Key words: Bone marrow, mesenchymal stem cells; Signal transduction; Irisin; Sclerostin

Background/Objectives: Guided bone regeneration (GBR) requires barrier membrane materials that balance biodegradation with mechanical stability. Magnesium (Mg)-based metals have good prospects for use as biodegradable barrier materials due to their elastic modulus, good biocompatibility, and osteogenic properties. In this study, gallium (Ga) was introduced into Mg to enhance the mechanical strength and optimize the degradation behavior of the alloy, addressing the limitations of conventional magnesium alloys in corrosion control and strength retention. Methods: Mg-xGa alloys (x = 1.0–3.0%, wt.%) were evaluated for biocompatibility, degradation, and osteogenic potential. Corrosion rates were calculated via weight loss, Mg2+ release, and pH changes. Osteogenic effects were assessed using rat bone marrow mesenchymal stem cells (rBMSCs) for alkaline phosphatase (ALP) activity, extracellular matrix (ECM) mineralization, and osteogenic-related gene expression. Optimal alloy was fabricated into barrier membranes with different pore sizes (0.85–1.70 mm) for the rabbit mandibular defect to evaluate the porosity effect on new bone formation. Results: Cytocompatibility tests established a biosafety threshold for Ga content below 3 wt.%. Mg-1Ga demonstrated uniform corrosion with a rate of 1.02 mm/year over 28 days. In vitro, Mg-1Ga enhanced ALP activity, ECM mineralization, and osteogenic gene expression. The 1.70 mm pore size group exhibited superior new bone formation and bone mineral density at 4 and 8 weeks. Conclusions: These results highlight Mg-1Ga’s biocompatibility, controlled degradation, and osteogenic properties. Its optimized pore design bridges the gap between collagen membranes’ poor strength and titanium meshes’ non-degradability, offering a promising solution for GBR applications.

BackgroundGlucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a progressive and disabling disease caused by long-term or high-dose glucocorticoid use. Decreased osteogenesis and proliferation of bone marrow mesenchymal stem cells (BMSCs) are the main pathogenesis of GIONFH. Platelet-rich plasma (PRP) has been shown to play a promising role in bone regeneration. However, the effects of PRP on glucocorticoid-induced BMSCs inhibition remains elusive. The objective of this study was to explore whether PRP could improve the in vitro biological activities of BMSCs inhibited by high-dose glucocorticoid in vitro.MethodsIn this study, a dexamethasone (Dex)-induced in vitro cell model was established. The effects of PRP on proliferation, migration, cell cycle and apoptosis of rat BMSCs induced with high-dose Dex compared to BMSCCTRL, using CCK-8 assay, transwell, flow cytometry and TUNEL assay, respectively. We further performed the alkaline phosphatase (ALP) and alizarin red (ALR) staining to explore the influence of PRP on osteogenic differentiation. Western Blot was used to detect the expression of Bcl-2, Caspase-3, RUNX2 apoptosis, and osteogenic-related proteins.ResultsWe observed increased apoptosis rate and Caspase-3 expression, and the decreased migration and osteogenic differentiation, and down-regulation of RUNX-2 and Bcl-2 expression in Dex-induced BMSCs. PRP could reverse these inhibitory effects of Dex, and enhance the BMSCs proliferation, migration, and osteogenic ability in vitro.ConclusionOur vitro study showed that PRP significantly protected BMSCs from Dex-induced apoptosis, and further promoted BMSCs proliferation, migration, and osteogenic differentiation. This study provides a scientific basis for the prevention and treatment of GIONFH with PRP. Meanwhile, it also lays the foundation for the application of PRP in other musculoskeletal diseases.

Developing scaffold materials with excellent biocompatibility, mechanical properties, and controlled drug release properties is vital to tissue engineering. In this study, we fabricated silk fibroin (SF)/poly(lactide-co-glycolide) (PLGA) nanofiber scaffolds containing recombinant human bone morphogenetic protein 2 (rhBMP2) and dexamethasone (DXM) via coaxial electrospinning, which were used in in vitro bone formation with rat bone marrow mesenchymal stem cells (rBMSCs). An in vitro drug release study was adopted to evaluate the sustained release potential of the core-shell structured nanofibers. Furthermore, we detected the potential of the SF/PLGA nanofiber membrane in vitro. In vitro studies showed that rhBMP2 still remained active on the nanofiber membrane. In addition, the dual-drug-loaded nanofiber membrane showed an early burst release of DXM and late sustained release of rhBMP2. rhBMP2 and DXM exhibited strong osteogenic differentiation potential when they acted on rBMSCs. Therefore, the SF/PLGA nanofiber membrane loaded with rhBMP2 and DXM has great potential for the enhancement of bone regeneration.

To investigate the role of miR-144-3p in regulating osteogenic differentiation of bone marrow mesenchymal stem cells and predict its target genes. Rat bone marrow mesenchymal stem cells (BMSCs) with induced osteogenic differentiation were examined for the expressions of Runx2, OCN and miR-144-3p. The effects of transfection with a miR-144-3p mimic or a miR-144-3p inhibitor were tested on the osteogenic differentiation of the BMSCs. The changes in the expressions of the predicted target of miR-144-3p in the BMSCs during induced osteogenic differentiation were examined using Western blotting and qRT-PCR. Rat BMSCs with induced differentiation into osteoblasts exhibited a progressive increase in the expressions of Runx2 and OCN (two markers of osteogenic differentiation), while the expression of miR-144-3p gradually decreased during the differentiation till reaching the lowest level at 21 days of induction. In rat BMSCs, transfection with the miR-144-3p mimic significantly decreased ALP activity (P &lt; 0.05) wile transfection with the miR-144-3p inhibitor significantly increased ALP activity (P &lt; 0.05) in rat BMSCs. Analysis based on miRanda, microRNA.org database and TargetScan suggested that Smad4 was the most likely target gene of miR-144-3p. The results of qRT-PCR showed no significant differences in expression levels of Smad4 among the cells with different treatments (P &gt; 0.05), while Western blotting revealed a significantly decreased expression of Smad4 in the cells transfected with miR-144-3p mimics and an increased Smad4 expression in the cells transfected with the miR-144-3p inhibitor as compared with the control cells (P &lt; 0.05). miR-144-3p participates in the regulation of osteogenic differentiation of rat BMSCs, and its inhibitory effect on osteogenic differentiation is achieved probably by decreasing the expression level of Smad4.

Objectives Spinal cord injury (SCI) is caused by external direct or indirect factors with high disability rate, which may even endanger the life of patients. To explore the role of bone morphogenetic protein 7 (BMP-7) in the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into neurons in vitro. Methods BMSCs were isolated and cultured by whole bone marrow adherence method. Adipogenic induction and osteogenic differentiation were used to test the multi⁃directional differentiation ability of BMSCs. Results After 28 days of adipogenic induction, BMSCs showed lipid droplets in the cytoplasm. After osteogenic induction, there were opaque lumps of mineral nodules in BMSCs. There were also orange-red or red mineral nodules in the extracellular matrix. The BMSCs in the 75 ng/ml BMP-7 group were morphologically similar to the neurons. After induction with BMP-7 for 2 h, the NF200 mRNA expression was higher, mRNA expression levels of SYN1, MAP2 and GFAP were higher. Positive rate of immunofluorescence staining in the BMP-7 group was notably increased. The positive rate of NSE immunofluorescence staining in the BMP-7 group was higher. Conclusion BMP-7 can induce rat BMSCs to differentiate into neurons in vitro.

Tissue engineering envisions functional substitute creation for damaged tissues. Insulin-like growth factor-1 (IGF-1) plays roles in bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation (OD), and we investigated its specific mechanism. BMSCs were cultured and OD was induced. Surface antigens (CD105, CD90, CD44, CD45, CD34) were identified by flow cytometry. Adipogenic, chondrogenic, and osteogenic differentiation abilities of BMSCs were observed. BMSCs were cultured in osteogenic medium containing 80 ng/mL IGF-1 for 3 weeks. Alkaline phosphatase activity, calcification level, osteogenic factor (runt related protein 2 [RUNX2], osteocalcin [OCN], osterix [OSX]), total (t-) ERK1/2 and phosphorylated- (p-) ERK1/2 levels, and SRY-related high-mobility-group box 4 (SOX4) levels were assessed by alkaline phosphatase staining and Alizarin Red staining, Western blot, and reverse transcription-quantitative polymerase chain reaction. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway inhibitor (PD98059) was used to inhibit the MAPK/ERK pathway in IGF-1-treated BMSCs. Small interfering-SOX4 was transfected into BMSCs to down-regulate SOX4. IGF-1 increased alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels in BMSCs, indicating that IGF-1 induced rat BMSC OD. SOX4, and p-ERK1/2 and t-ERK1/2 levels were elevated in IGF-1-induced BMSCs, which were annulled by PD98059. PD98059 partly averted IGF-1-induced rat BMSC OD. SOX4 levels, alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels were reduced after SOX4 down-regulation, showing that downregulation of SOX4 averted the effect of IGF-1 on inducing rat BMSC OD. IGF-1 induced rat BMSC OD by stimulating SOX4 via the MAPK/ERK pathway.

Objective To observe the effect of different concentrations of parathyroid hormone 1-34 on the adipogenic potential of rat bone marrow mesenchymal stem cells (BMSCs). Methods ① Rat bone marrow mesenchymal cells were separated and expanded by adherent culture. The morphology of cells was observed and cell surface markers were examined by flow cytometry.② The multi-lineage differentiation capability of cells was examined by culturing cells under conditions favorable for adipogenic and osteogenic differentiation. ③ Taken P3 of BMSCs for test, different concentrations of PTH1-34 (0, 10-10, 10-9, 10-8 mol/L) were used to stimulate BMSCs respectively, 14 days later, lipoprotein lipase (LPL) activity were measured by enzyme linked immuno-sorbent assay (ELISA), mRNA expression of alkaline phosphatase (ALP) and PPARγ-2 were measured by realtime polymerase chain reaction (PCR).④Statistical analysis: data were presented as ±s. All statistical analysis was performed with windows Statistical Praduct and Serice Solutions (SPSS) 13.0. One-way analysis of variance (ANOVA) was applied to determine the difference between groups. Least signisicant difference (LSD) was used to determine the difference between the two randomized groups. Differences were considered significant at a value of P<0.05. Results The cells expressed CD44, CD29 but without expression of CD45. By culturing in adipogenic medium for 3 weeks and in osteogenic medium for 4 weeks respectively, and then identified by oil red O and Alizarin red, the cells were successfully induced to adipocytes and osteogenesis. Expressions of LPL were 11.20±0.16, 7.62±0.48, 5.84±0.57, 5.32±0.52, mRNA expressions of PPAR7-2 were 2.80±0.05, 1.36±0.23, 0.94±0.11, 0.78±0.04, ALP activity were 0.191 ±0.016, 0.333±0.024, 0.549±0.025, 0.684±0.021 respectively. Compared with the control group, different concentrations of PTH1-34 groups could decrease mRNA expression of LPL and PPARγ-2. ALP activity were increased(P<0.05). Conclusion PTH1-34 inhibits BMSCs of adipogenic differentiation and promotes osteogenic differentiation in a dose-dependent manner. Key words: Teriparatide; Mesenchymal stem cells; Bone marrow; Adipogenic differentiation; Osteogenic differentiation

To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. The BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined，and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. The isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant ( P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B ( P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B ( P>0.05). VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.

Osteogenesis is a complex process of bone formation regulated by various factors, yet its underlying molecular mechanisms remain incompletely understood. The present study aimed to investigate the role of S100A16, a novel member of the S100 protein family, in the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) and uncover a novel Smad4-mitogen-activated protein kinase (MAPK)/Jun N-terminal kinase (JNK) signaling axis. In the present study, the expression level of S100A16 in bone tissues and BMSCs from ovariectomized rats was evaluated and then the impact of S100A16 silencing on osteogenic differentiation was examined. Increased S100A16 expression was observed in bone tissues and BMSCs from ovariectomized rats, and S100A16 silencing promoted osteogenic differentiation. Further transcriptomic sequencing revealed that the Smad4 pathway was involved in S100A16 silencing-induced osteogenesis. The results of western blot analysis revealed that S100A16 overexpression not only downregulated Smad4 but also activated MAPK/JNK signaling, which was validated by treatment with MAPK and JNK inhibitors U0126 and SP600125. Overall, in the present study, the novel regulatory factors influencing osteogenic differentiation were elucidated and mechanistic insights that could aid in the development of targeted therapeutic strategies for patients with osteoporosis were provided.