Fibroblasts inhibit osteogenesis by regulating nuclear-cytoplasmic shuttling of YAP in mesenchymal stem cells and secreting DKK1

Background Fibrous scars frequently form at the sites of bone nonunion when attempts to repair bone fractures have failed. However, the detailed mechanism by which fibroblasts, which are the main components of fibrous scars, impede osteogenesis remains largely unknown. Results In this study, we found that fibroblasts compete with osteogenesis in both human bone nonunion tissues and BMP2-induced ectopic osteogenesis in a mouse model. Fibroblasts could inhibit the osteoblastic differentiation of mesenchymal stem cells (MSCs) via direct and indirect cell competition. During this process, fibroblasts modulated the nuclear-cytoplasmic shuttling of YAP in MSCs. Knocking down YAP could inhibit osteoblast differentiation of MSCs, while overexpression of nuclear-localized YAP-5SA could reverse the inhibition of osteoblast differentiation of MSCs caused by fibroblasts. Furthermore, fibroblasts secreted DKK1, which further inhibited the formation of calcium nodules during the late stage of osteogenesis but did not affect the early stage of osteogenesis. Thus, fibroblasts could inhibit osteogenesis by regulating YAP localization in MSCs and secreting DKK1. Conclusions Our research revealed that fibroblasts could modulate the nuclear-cytoplasmic shuttling of YAP in MSCs, thereby inhibiting their osteoblast differentiation. Fibroblasts could also secrete DKK1, which inhibited calcium nodule formation at the late stage of osteogenesis.

Aqueous extract of freeze-dried Protaetia brevitarsis larvae promotes osteogenesis by activating β-catenin signaling

Objective: To investigate the effect of an aqueous extract of Protaetia brevitarsis (AEPB) on osteogenesis using preosteoblast MC3T3-E1 cells and zebrafish larvae. Methods: Flow cytometric analysis was used to measure the cytotoxicy. Alkaline phosphatase activity was detetmined using p-nitrophenyl phosphate as a substrate. Calcium deposition was detected using alizarin red staining along with osteogenic marker expression in preosteoblast MC3T3E1 cells. In addition, vertebral formation in zebrafish larvae was detected using calcein staining and osteogenic gene expression. Results: AEPB highly promoted the expression of osteogenic markers including runt-related transcription factor 2, osterix, and alkaline phosphatase, along with elevated levels of mineralization in MC3T3-E1 cells. Moreover, AEPB accelerated vertebral formation in zebrafish larvae accompanied by upregulated expression of osteogenic genes. FH535, an inhibitor of Wnt/β-catenin, suppressed AEPB-induced osteogenic gene expression and vertebral formation, indicating that AEPB stimulates osteogenesis by activating the Wnt/β-catenin signaling pathway. Conclusions: AEPB stimulates osteoblast differentiation and bone formation by activating β-catenin. Therefore, AEPB is a promising material that induces osteogenesis, and is useful for the treatment of bone resorption diseases.

Serum nesfatin-1 changes in osteonecrosis of the femoral head / 中国组织工程研究

BACKGROUND: Nesfatin-1, a novel feeding regulator, has been found to be closely linked to orthopedic diseases in recent years. The correlation between Nesfatin-1 and osteonecrosis of the femoral head (ONFH) remains unclear. OBJECTIVE: To determine the changes in plasma Nesfatin-1 level in patients with ONFH and to study its correlation with the development of ONFH. METHODS: From May to December 2016, we enrolled 86 patients with ONFH who were hospitalized in the Hip Preserving Ward, No. 3 Orthopedic Department, the First Affiliated Hospital of Guangzhou University of Chinese Medicine (including 13 ARCO stage II, 26 ARCO stage III, and 47 ARCO stage IV) and 86 healthy controls. In the ONFH group, there were 26 cases of steroid-induced ONFH, 40 cases of alcoholic ONFH, 12 cases of traumatic ONFH, and 8 cases of idiopathic ONFH. The levels of Nesfatin-1 in peripheral venous blood were measured by enzyme-linked immunosorbent assay (ELISA). Comparison was performed between different ARCO stages, different etiologies as well as before and after collapse. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (No. 20140307), and each patient had signed an informed consent form prior to inclusion in the study. RESULTS AND CONCLUSION: Plasma Nesfatin-1 level was significantly higher in ONFH patients than healthy controls (P=0.000). There were insignificant differences among different etiologies (P=0.956), but significant differences among different ARCO stages (P=0.011) as well as before and after collapse (P=0.003). There was a positive correlation between plasma Nesfatin-1 level and ARCO stages. Receiver operator characteristic curve analysis showed that Nesfatin-1 level had sensitivity (52.05%) and specificity (92.31%) in the diagnosis of ONFH. These findings indicate that Nesfatin-1 is closely related to the development of ONFH. It can reflect the degree of necrosis, which can provide reference for clinical diagnosis and treatment of ONFH.

Differential MiRNA/mRNA Expression Profiling and Functional Network Analysis for MC3T3-E1 Cells with Microgravity Stimulation Based on RNA-seq / 医用生物力学

Objective To investigate the effect of microgravity on MC3T3-E1 osteoblast differentiation. Methods The differential miRNA and mRNA expression profiling of MC3T3-E1 cells during exposure to microgravity were established by RNA transcriptome sequencing technology (RNA-seq). The RNA sequencing results were validated using quantitative real-time polymerase chain reaction (q-PCR). Bioinformatic analyses were applied for further study of these differentially expressed miRNAs and mRNAs. Results Compared with control (CON) group, A total of 1 912 coding transcripts and 160 miRNAs were detected along with osteogenic differentiation in simulated microgravity (SMG) group. Bioinformatic analysis revealed 10 core regulatory genes including 7 mRNAs and 3 miRNAs. Based on the analysis and verification, one miRNA, miR-9_6666-5p, was identified, which might play an important role in osteogenic differentiation process under microgravity. Conclusions The process of osteoblast differentiation was repressed under microgravity which might be related to the changed expression profile of miRNA/mRNA. The research findings can contribute to the better understanding of the molecular mechanisms of mRNA and miRNAs in osteogenic differentiation and bone formation under the microgravity condition.

Research of simulated microgravity regulate MC3T3-E1 cells differentiation through the nuclear factor-kappa B signaling pathway / 生物医学工程学杂志

In this study, we aim to investigat the effect of microgravity on osteoblast differentiation in osteoblast-like cells (MC3T3-E1). In addition, we explored the response mechanism of nuclear factor-kappa B (NF-κB) signaling pathway to "zero- " in MC3T3-E1 cells under the simulated microgravity conditions. MC3T3-E1 were cultured in conventional (CON) and simulated microgravity (SMG), respectively. Then, the expression of the related osteoblastic genes and the specific molecules in NF-κB signaling pathway were measured. The results showed that the mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin (OCN) and type Ⅰ collagen (CoL-Ⅰ) were dramatically decreased under the simulated microgravity. Meanwhile, the NF-κB inhibitor α (IκB-α) protein level was decreased and the expressions of phosphorylation of IκB-α (p-IκB-α), p65 and phosphorylation of p65 (p-p65) were significantly up-regulated in SMG group. In addition, the IL-6 content in SMG group was increased compared to CON. These results indicated that simulated microgravity could activate the NF-κB pathway to regulate MC3T3-E1 cells differentiation.

(2R,3S)-Pinobanksin-3-cinnamate promotes osteoblast differentiation through cAMP and cGMP pathways

Abstract Flavones have the potential of being used as a dietary supplement for bone health promotion beyond calcium and vitamin D. Recent studies have showed that flavones enhanced bone formation and inhibited bone resorption by affecting osteoblast and osteoclast differentiation through various cell signaling pathways. In this study, we investigated the effects of a new flavone (2R,3S)-pinobanksin-3-cinnamate, isolated from the metabolites of the endophytic fungus Penicillium sp. FJ-1 of Acanthus ilicifolius L., Acanthaceae, on osteoblast differentiation by using MC3T3-E1 cells. It was observed that (2R,3S)-pinobanksin-3-cinnamate promoted osteoblast differentiation, as evidenced by increased mineralization process and alkaline phosphatase activity, as well as expression of genes encoding the bone differentiation. Moreover (2R,3S)-pinobanksin-3-cinnamate treatment upregulated the gene expression of wingless-type MMTV integration site family, bone morphogenetic protein and runt-related transcription factor 2, and protein expression of phosphor-Smad1/5/8, β-catenin and runt-related transcription factor 2 in MC3T3-E1 cells. The osteoblast differentiation effects induced by (2R,3S)-pinobanksin-3-cinnamate were attenuated by the bone morphogenetic protein antagonist Noggin, and wingless-type MMTV integration site family signaling pathway inhibitors Dickkopf-1. Co-treatment with adenosine 30,50-cyclic monophosphate and guanosine 30,50-cyclic monophosphate pathway inhibitors, H89 and KT5823, respectively, reversed the (2R,3S)-pinobanksin-3-cinnamate-induced activations of p-Smad1/5/8, β-catenin, and runt-related transcription factor 2. Our data demonstrated that (2R,3S)-pinobanksin-3-cinnamate promoted the osteoblast differentiation of MC3T3-E1 cells, at least partially through the adenosine 30,50-cyclic monophosphate and guanosine 30,50-cyclic monophosphate signaling pathways, providing the scientific rational to develop (2R,3S)-pinobanksin-3-cinnamate against bone loss-associated diseases.

Cellular zinc deficiency inhibits the mineralized nodule formation and downregulates bone-specific gene expression in osteoblastic MC3T3-E1 cells / 한국영양학회지

PURPOSE: Zinc (Zn) is an essential trace element for bone mineralization and osteoblast function. We examined the effects of Zn deficiency on osteoblast differentiation and mineralization in MC3T3-E1 cells. METHODS: Osteoblastic MC3T3-E1 cells were cultured at concentration of 1 to 15 µM ZnCl2 (Zn− or Zn+) for 5, 15 and 25 days up to the calcification period. Extracellular matrix mineralization was detected by staining Ca and P deposits using Alizarin Red and von Kossa stain respectively, and alkaline phosphatase (ALP) activity was detected by ALP staining and colorimetric method. RESULTS: Extracellular matrix mineralization was decreased in Zn deficiency over 5, 15, and 25 days. Similarly, staining of ALP activity as the sign of an osteoblast differentiation, was also decreased by Zn deficiency over the same period. Interestingly, the gene expression of bone-related markers (ALP, PTHR; parathyroid hormone receptor, OPN; osteopontin, OC; osteocalcin and COLI; collagen type I), and bone-specific transcription factor Runx2 were downregulated by Zn deficiency for 5 or 15 days, however, this was restored at 25 days. CONCLUSION: Our data suggests that Zn deficiency inhibits osteoblast differentiation by retarding bone marker gene expression and also inhibits bone mineralization by decreasing Ca/P deposition as well as ALP activity.

MicroRNA-221 promotes cell proliferation, migration, and differentiation by regulation of ZFPM2 in osteoblasts

Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.

The Experimental Study of Cyasterone on the Treatment of Osteoporosis through the Bidirectional Effect of Inhibiting Osteoclast Differetiation and Promoting Osteoblast Differentiation / 昆明医科大学学报

Objective To explore the influence of cyasterone on the osteoclast and osteoblast differentiation and then to investigate its effect on the bone quality in the osteoporosis mice. Methods CCK8 assay was firstly used to detect the toxic effect of cyasterone on the mouse bone marrow derived mononuclear macrophages (BMMs) and anterior osteoblast lines MC3T3E1. Cell apoptosis was measured by flow cytometry. Then TRAP staining and ALP staining were employed to detect osteoclast differentiation and osteoblast differentiation, respectively. Realtime PCR was carried out to test the expression of osteoclast special gene TRAP and osteogenesis crucial gene ALP. In vivo, 15 mice were divided into three groups: sham-operated group, OVX group and OVX+cyasterone treatment group. In treatment group, cyasterone was used as 5mg/kg every day. Sham-operated group and OVX group were treat with saline solution. After 4 weeks, the tibia was collected for Micro-CT detection to observe the bone quality and microstructure changes. Results Cyasterone with the concentration of less than 10 mg/L had no significant cytotoxicity nor influence on the apoptosis (P＞0.05) . Cyasterone could significantly inhibit the osteoclast differentiation of BMMs (P＜0.05), simultaneously, it also had the effect to promote the osteoblast differetiation of MC3T3E1. Real-time PCR indicated that cyasterone could block the expression of TRAP and increase the expression of ALP (P＜0.05) . In vivo, cyasterone was able to obviously improve the osteoporosis status caused by estrogen deficiency without general toxicity. Conclusion cyasterone could provide a good treatment for osteoporosis through the bidirectional effect of inhibiting osteoclast differetiation and promoting osteoblast differentiation.

LC/MS-based Analysis of Bioactive Compounds from the Bark of Betula platyphylla var. japonica and Their Effects on Regulation of Adipocyte and Osteoblast Differentiation

Betula platyphylla var. japonica (Betulaceae), also known as Asian white birch, is an endemic medicinal tree, the bark of which has been used in Chinese traditional medicine for the treatment of various inflammatory diseases. In our continuing search for bioactive compounds from Korean natural resources, a phytochemical investigation of the bark of B. platyphylla var. japonica led to the isolation of 7-oxo-β-sitosterol (1) and soyacerebroside I (2) from its ethanol extract as main components by liquid chromatography (LC)/mass spectrometry (MS)-based analysis. The structures of isolates were identified by comparison of ¹H and ¹³C nuclear magnetic resonance spectroscopic data and physical data with the previously reported values and LC/MS analyses. To the best of our knowledge, this is the first study to demonstrate that the isolated compounds, 7-oxo-β-sitosterol and soyacerebroside I, were isolated in B. platyphylla var. japonica. We examined the effects of the isolates on the regulation of adipocytes and osteoblast differentiation. These isolates (1 and 2) produced fewer lipid droplets compared to the untreated negative control in Oil Red O staining of the mouse mesenchymal stem cell line without altering the amount of alkaline phosphatase staining. The results demonstrated that both compounds showed marginal inhibitory effects on adipocyte differentiation but did not affect osteoblast differentiation.

Tridax procumbens flavonoids: a prospective bioactive compound increased osteoblast differentiation and trabecular bone formation

BACKGROUND: The Tridax procumbens extracts (TPE) are known for their ethno-medicinal properties to increase osteogenic functioning in mesenchymal stem cells. Recently, we found that the T. procumbens flavonoids (TPF) significantly suppressed the RANKL-induced osteoclasts differentiation and bone resorption. The TPF also promoted osteoblasts differentiation and bone formation demonstrated by increasing bone formation markers in cultured mouse primary osteoblasts. However, the effects of the TPF on in vivo bone formation remain unclear. In this study, we investigated the effects of the TPF on in vivo bone formation, injected the TPF (20 mg/kg) twice a day in the low calcium diet mice and killed them after 21 day. Radiographic and histomorphometric analyses were performed on the dissected bones to determine the anabolic effects of the TPF. RESULTS: Bone mineral density and bone mineral content of the TPF-treated mice were significantly increased compared to the control mice. Bone formation-related indices like osteoblast number, osteoblast surface, bone volume, mineralizing surface, mineral apposition rate and bone formation rate were significantly increased in the TPF-treated mice compared to the control mice. CONCLUSION: Our findings point towards the stimulation of bone formation by TPF, suggested that the TPF could be a potential natural anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Role of Notch1 signal pathway in osteogenic differentiation of human periodontal ligament stem cells / 局解手术学杂志

Objective To explore the effect of Notch1 signal pathway on the osteogenic differentiation in human periodontal ligament stem cells(PDLSCs) under dynamic strain.Methods PDLSCs were separated from freshly extracted teeth then identified and prolifed.Notch1 signal pathway was regulated by chemicals.Dynamic strains were applied to PDLSCs with the tension plus system.Then Notch1 signal pathway key factor Notch intracellular domain(NICD),osteoblastic related indexes alkaline phosphatase(ALP) and bone morphogenetic proteins 2 (BMP2) were detected by western blot expression.The deformation rate of stress parameters was 0 to 12%,and the frequency was 0.1 Hz.The loading time was 0 h,6 h,12 h and 24 h.Results As Notch1 signal pathway was activated,the expression of osteogenic markers ALP and BMP2 both reduced (P<0.05).On the contrary, the expression of osteogenic markers ALP and BMP2 both increased obviously (P<0.05) as Notch1 signal pathway was inhibited.Conclusion The activated Notch1 signal pathway will inhibit osteogenic differentiation of PDLSCs under dynamic tensile.

The effect of biomechanical stimulation on osteoblast differentiation of human jaw periosteum-derived stem cells / 대한악안면성형재건외과학회지

BACKGROUND: This study was to investigate the effect of biomechanical stimulation on osteoblast differentiation of human periosteal-derived stem cell using the newly developed bioreactor. METHODS: Human periosteal-derived stem cells were harvested from the mandible during the extraction of an impacted third molar. Using the new bioreactor, 4% cyclic equibiaxial tension force (0.5 Hz) was applied for 2 and 8 h on the stem cells and cultured for 3, 7, and 14 days on the osteogenic medium. Biochemical changes of the osteoblasts after the biomechanical stimulation were investigated. No treatment group was referred to as control group. RESULTS: Alkaline phosphatase (ALP) activity and ALP messenger RNA (mRNA) expression level were higher in the strain group than those in the control group. The osteocalcin and osteonectin mRNA expressions were higher in the strain group compared to those in the control group on days 7 and 14. The vascular endothelial growth factor (VEGF) mRNA expression was higher in the strain group in comparison to that in the control group. Concentration of alizarin red S corresponding to calcium content was higher in the strain group than in the control group. CONCLUSIONS: The study suggests that cyclic tension force could influence the osteoblast differentiation of periosteal-derived stem cells under optimal stimulation condition and the force could be applicable for tissue engineering.

Evaluation of osteogenic effects of BMP2 up-regulator E40071 in vitro / 药学学报

Bone morphogenetic protein 2 (BMP2) plays a key role in bone development and reestablishment. In the study, we screened up-regulators of BMP2 among 20 000 compounds through a cell-based high throughput screening model and a positive compound E40071[2-(4-(5-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-7-yl) piperazin-1-yl)ethan-1-ol] was found as the positive hit. The EC50 value of E40071 was 2.73 μmol·L-1. In vitro, E40071 upregulated the mRNA levels of BMP2 and the downstream transcription factors, Runx2 and Osx in MC3T3-E1 (subclone 14). Protein expression of Runx2 was up-regulated by E40071 through induction of Smad1/5/8 phosphorylation. The alkaline phosphatase (ALP) activity was increased by E40071. Moreover, E40071 promoted the mineralization of MC3T3-E1 (subclone 14) by Alizarin red S staining. In addition, E40071 markedly inhibited osteoclast differentiation of mice macrophage Raw264.7 induced by RANKL and reduced the expression of osteoclast differentiation markers, including MMP9 and NFATc1. The results suggest that E40071 is able to promote bone formation activity of osteoblasts and inhibit differentiation of osteoclasts.

Primary culture of murine spleen-derived mesenchymal stem cells by explant culture / 中国比较医学杂志

Objective This study aimed to establish a reliable primary culture protocol for preparing murine spleen-derived mesenchymal stem cells ( MSCs) by tissue explant culture.Methods Healthy mouse spleens were crushed by syringe handle to harvest spleen mesenchymal tissues.Then the tiny pieces of spleen tissue were digested by collagenase II before seeded into culture flasks.The morphological characteristics of spleen tissue-derived cells were observed under the inverted microscope.Further, the surface antigen profile of the cells was analyzed by flow cytometry (FACS).The cells were induced to differentiate into osteoblasts and adipocytes.Results The murine spleen-derived MSCs exhibited a spindle-shaped appearance.The FACS results showed that the spleen-derived MSCs highly expressed CD29, CD44, CD105 and Sca-1, but weakly expressed CD11b, CD34, CD45 and Ia. In addition, the spleen-derived MSCs steadily differentiated into osteoblasts and adipocytes in the induction medium.Conclusions A method of primary culture of murine spleen-derived MSCs by explant culture is successfully established.The harvested MSCs exhibit high purity and cell proliferation ability, and provide a reliable cell model for related researches.

Effect of spinal cord injury on signaling pathways in mesenchymal stem cell-derived osteoblasts / 中华创伤杂志

Objective To investigate the changes in Wnt/β-catenin,bone morphogenic protein (BMP),estrogen receptor (ER) and insulin-like growth factor (IGF) signaling pathways in bone marrow mesenchymal stem cells (BMSCs) differentiation to the osteoblasts after spinal cord injury (SCI) and understand the mechanism of osteoporosis after SCI.Methods Forty 6-week-old male rats were divided into SCI group (n =20) and control group (n =20) according to the random number table.Rats in SCI group were submitted to laminar osteotomy at T10-12 and given lower thoracic cord sharp transection.In control group,rat lower thoracic cord was only exposed without transaction.Femoral bone marrow density (BMD) of rat right side was determined at postoperative 3 months.Femoral bone marrow was harvested from rat left side.After BMSCs osteoblast differentiation,cells were harvested and used for examining expression of genes associated with the signaling pathways in the two groups using microarray technology and real-time PCR analysis.Results BMD in SCI group was significantly lower in the ephiphyses and metaphyses[(0.176 ± 0.017)g/cm2 and (0.170 ±0.016)g/cm2] compared to that in control group [(0.257 ± 0.023) g/cm2 and (0.196 ± 0.013) g/cm2,P ＜0.05].Microarray and PCR analysis revealed Wnt/β-catenin (eg.Wnt1,Wnt3a,Wnt5a,Lrp5,Ctnnb1,Lef1 and Axin),BMP (Tgfb1 and Bmpr1),IGF -1 (eg.IGF1 R,c-fos and c-Jun),and ER (eg.Esr1) signaling pathways in osteoblasts were significantly down-regulated in SCI group compared to these in control group (P ＜ 0.05).Conclusions The Wnt/β-catenin,BMP,ER,and IGF-1 signaling pathways in osteoblasts are significantly down-regulated after SCI,resulting in profound BMD loss.This indicates that these signaling pathways are implicated in the osteoporosis after SCI.

Synergistic Effects of Chios Gum Mastic Extract and Low Level Laser Therapy on Osteoblast Differentiation

In the present study, we evaluated the effect of CGM on osteogenic differentiation of cultured osteoblasts, and determined whether combination treatment with LLLT had synergistic effects on osteogenic differentiation. The results indicated that CGM promoted proliferation, differentiation, and mineralization of osteoblasts at the threshold concentration of 10 µg/ml; whereas, CGM showed cytotoxic properties at concentrations above 100 µg/ml. ALP activity and mineralization were increased at concentrations above 10 µg/ml. CGM in concentrations up to 10 µg/ml also increased the expression of osteoblast-activated factors including type I collagen, BMP-2, RUNX2, and Osterix. The CGM (50 µg/ml) and LLLT (80 mW for 15 sec) combination treatment group showed the highest proliferation levels, ALP activity, and mineralization ratios. The combination treatment also increased the levels of phosphorylated forms of p38, ATF2, PKD, ERK, and JNK. In addition, the osteoblast differentiation factors including type I collagen, BMP-2, RUNX2, and Osterix protein levels were clearly increased in the combination treatment group. These results suggested that the combination treatment of CGM and LLLT has synergistic effects on the differentiation and mineralization of osteoblastic cells.

Tridax procumbens flavonoids promote osteoblast differentiation and bone formation

BACKGROUND: Tridaxprocumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. RESULTS: TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. CONCLUSION: Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Effect of microgrooves and fibronectin conjugation on the osteoblast marker gene expression and differentiation

PURPOSE: To determine the effect of fibronectin (FN)-conjugated, microgrooved titanium (Ti) on osteoblast differentiation and gene expression in human bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Photolithography was used to fabricate the microgrooved Ti, and amine functionalization (silanization) was used to immobilize fibronectin on the titanium surfaces. Osteoblast differentiation and osteoblast marker gene expression were analyzed by means of alkaline phosphatase activity assay, extracellular calcium deposition assay, and quantitative real-time PCR. RESULTS: The conjugation of fibronectin on Ti significantly increased osteoblast differentiation in MSCs compared with non-conjugated Ti substrates. On the extracellular calcium deposition assays of MSCs at 21 days, an approximately two-fold increase in calcium concentration was observed on the etched 60-microm-wide/10-microm-deep microgrooved surface with fibronectin (E60/10FN) compared with the same surface without fibronectin (E60/10), and a more than four-fold increase in calcium concentration was observed on E60/10FN compared with the non-etched control (NE0) and etched control (E0) surfaces. Through a series of analyses to determine the expression of osteoblast marker genes, a significant increase in all the marker genes except type I collagen alpha1 mRNA was seen with E60/10FN more than with any of the other groups, as compared with NE0. CONCLUSION: The FN-conjugated, microgrooved Ti substrate can provide an effective surface to promote osteoblast differentiation and osteoblast marker gene expression in MSCs.

Estudo dos mecanismos envolvidos no processo de diferenciação em linhagem osteogênica de células-tronco mesenquimais da medula óssea de ratos Wistar e ratos espontaneamente hipertensos (SHR) / Study of the mechanisms involved in the process of differentiation into osteogenic lineage of mesenchymal stem cells from bone marrow of wistar rats and spontaneously hypertensive rats (SHR)

Células-tronco mesenquimais (CTMs) obtidas a partir da medula óssea são capazes de se diferenciarem, sobretudo, em condrócitos, adipócitos e osteoblastos. Durante a osteogênese in vitro, alguns parâmetros são utilizados para caracterizar este processo, tais como atividade da fosfatase alcalina (FAL), mineralização e expressão de proteínas associadas à osteoblastos. Ratos espontaneamente hipertensos (SHR) são um modelo animal de hipertensão essencial humana e desenvolvem hipertensão após 4 semanas de idade. Esta linhagem apresenta alterações significativas no metabolismo ósseo. O objetivo do presente estudo foi investigar se, o genótipo hipertensivo poderia interferir na diferenciação osteoblástica das CTMs de ratos SHR e qual mecanismo está alterado quando comparadas com a linhagem progenitora, ratos Wistar. Para isso, nós obtivemos CTMs da medula óssea de ratos Wistar e SHR com 4 semanas de idade, sem a hipertensão estabelecida, afim de avaliar somente o possível efeito do genótipo hipertensivo na diferenciação osteogênica in vitro. Nós induzimos, ou não, a diferenciação osteogênica in vitro por meio da utilização dos indutores osteogênicos: ácido ascórbico, β-glicerofosfato e dexametasona. Os resultados demonstraram que, CTMs indiferenciadas de SHR (SHRC) demonstraram taxa de proliferação aumentada em comparação a CTMs, na mesma condição, de Wistar (WC), e após a indução da osteogênica, a taxa de proliferação apresentou uma diminuição acentuada no grupo SHR (SHRMO) do que no grupo Wistar na mesma condição (WMO). Embora não fora observada diferença significativa na atividade da FAL entre SHRMO e WOM no 7° dia, a mineralização e a diferenciação osteoblástica foram menores no grupo SHRMO no mesmo período experimental. Os fatores de transcrição Osterix e β-catenina parecem estar envolvidos na diferenciação reduzida no grupo SHRMO, pois apresentaram menor expressão neste grupo experimental. Além disso, a expressão diminuída de proteínas associadas...

Mesenchymal stem cells (MSCs) from bone marrow are able to differentiate mainly into chondrocytes, adipocytes and osteoblasts. During in vitro osteogenesis, some parameters are used to characterize this process, such as the activity of alkaline phosphatase (ALP), mineralization and osteoblast-associated proteins expression. Spontaneously hypertensive rats (SHR) is an animal model of human essential hypertension. This animals developing hypertension after 4 weeks of age. This strain shows significant changes in bone metabolism. The aim of this study was to investigate whether the hypertensive genotype could influence the osteoblastic differentiation of MSCs from SHR and which mechanism are altered when compared to the parental strain, Wistar rats. For that, we have obtained bone marrow MSCs from Wistar and SHR rats at 4 weeks of age, without hypertension established in order to evaluate only the possible effect of hypertensive genotype on osteogenic differentiation in vitro. We induced or non-osteogenic differentiation in vitro using osteogenic inducers: ascorbic acid, dexamethasone and β-glycerophosphate. The results demonstrate that undifferentiated MSCs SHR (SHRC) showed increased proliferation rate compared to MSCs, in the same condition Wistar (WC) and after osteogenic induction, proliferation rate showed a marked decrease in SHR (SHRMO) than in Wistar group in the same condition (WMO). Although it was not observed significant difference in ALP activity between WMO and SHRMO on day 7, mineralization and osteoblast differentiation were lower on group SHRMO in the same experimental period. The transcription factors Osterix and β-catenin appear to be involved in reduced differentiation in SHRMO group because they showed lower expression in this experimental group. Furthermore, the decreased osteoblast-associated proteins such as OCN, BSP, OPN expression suggest that extracellular matrix SHRMO group has a lower quality in comparison to WMO group. Higher...