A nonobstructive condition: Medial arterial calcification / 中南大学学报(医学版)

Vascular calcification, including intimal and medial calcification, is closely associated with a significant increase in cardiovascular diseases. Although increased understandings were achieved, people still know much more about intimal calcification than medial calcification because the latter doesn't obstruct the arterial lumen, commonly considered as a non-significant finding. We clarified the pathologic characteristic of medial calcification, its difference from intimal calcification, principally focused on its clinical relevance, such as diagnosis, nosogenesis, and hemodynamics. We underline the importance of identifying and distinguishing medial calcification, understanding its effect to local/systematic arterial compliance, and relationship to diabetic neuropathy. Recent studies emphasize do not ignore its predictive role in cardiovascular mortality. It is of great clinical significance to summarize the mechanisms of occurrence, lesion characteristics, diagnostic methods, pathogenic mechanisms, hemodynamic changes, and the distinction as well as association of intimal calcification with intimal calcification.

Bioactivity of the peptide P17-bone morphogenetic protein-2/nano-hydroxyapatite-mineralized collagen composite / 中国组织工程研究

BACKGROUND: A mineralized collagen composite, i.e. nano-hydroxyapatite/collagen (nHAC) has biomimetic three-dimensional structure and good bioactive properties. As a bone tissue engineering material, it is widely used in bone defect repair. A newly designed P17-bone morphogenetic protein-2 (P17-BMP2) has good biocompatibility and osteogenic capacity. Therefore, the composite scaffold material was prepared by combining the new P17-BMP-2 and nHAC, which might be used for the enhancement of osteogenic capacity in the treatment of bone defects. OBJECTIVE: To investigate the bioactivity of the P17-BMP-2/nHAC composite. METHODS: Rabbit bone marrow mesenchymal stem cells were seed on the P17-BMP-2/nHAC composite and nHAC. After 3 and 7 days of culture, the relative expression level of alkaline phosphatase was detected by RT-PCR. The subcutaneous implantation of P17-BMP-2/nHAC (experimental group) and nHAC (control group) into Sprague-Dawley rats was performed. Masson staining was performed for histological analysis at 12 and 35 days of implantation. P17-BMP-2/nHAC (experimental group) and nHAC (control group) were implanted into the white rabbit mandibular box-shaped bone defect, respectively. At 5 and 15 weeks, gross observation and X-ray were performed. The study was approved by the Medical Ethics Committee of China Medical University School & Hospital of Stomatology. RESULTS AND CONCLUSION: (1) The relative expression level of alkaline phosphatase in the P17-BMP-2/nHAC group was significantly higher than that in the nHAC group (P < 0.05). (2) The result of subcutaneous implantation showed that the acute inflammatory response initiated by the P17-BMP-2/nHAC or nHAC was not found. More activated fibroblasts growing into the implants could be found on the sections of P17-BMP-2/nHAC compared to that of nHAC at 35 days after implantation. (3) In the bone defect repair test, gross observation showed that both materials held good defect repair ability, the defect area began to reduce at 5 weeks after implantation, and the defect surface became flat at 15 weeks after implantation. X-ray examination showed that compared with the control group, the defect area was more significantly reduced in the experimental group. (4) These results indicate that P17-BMP-2/nHAC composite scaffold has higher bioactivity and a stronger ability to repair bone defect.

Effects of zinc gluconate carbon dots in cell imaging and inducing osteoblastic differentiation in vitro / 吉林大学学报(医学版)

Objective: To synthesize the zinc gluconate carbon dots (Zn-CDs) by one-step hydrothermal method, and to investigate their effects on the cell imaging and inducing osteoblastic differentiation of preosteoblasts in the mice. Methods: The Zn-CDs were synthesized by one-step hydrothermal method, and the characteristics were observed and detected by transmission electron microscope (TEM), Fourier transform-infrared spectrum (FT-IR) and fluorescence spectrometer. The MC3T3-E1 cells were divided into blank control group and experimental groups; different concentrations (0.01, 0.10, 1.00, 10.00, 100.00, 1 000 mg middot; L-1) of Zn-CDs were added into the cells in experimental groups, and nothing was added into the cells in blank control group. MTT assay was used to determin the relative growth rate (RGR) of MC3T3-E1 cells in various groups; the imaging characteristics of MC3T3-E1 cells were observed under confocal microscope; the relative expression levels of Runt-relateed transcription factor-2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC) mRNA in the MC3T3-E1 cells in various groups were detected by qRT-PCR; the number of calcified nodules was detected by alizarin red staining. Results: The TEM results showed that the particle size of Zn-CDs was about 5. 25 nm. The fluorescence spectrometer results showed that the Zn-CDs had the fluorescence properties of 360 nm ultraviolet excited light and 450 nm blue emission light, and the Zn-CDs showed the characteristics of excitation wavelength dependence. The FT-IR results showed that the surface of Zn-CDs was mainly composed of carboxyl groups and hydroxyl groups. Compared with blank control group, the RGR of MC3T3-E1 cells in 1 000. 00 mg middot; L-1 Zn-CDs group was decreased significantly at 24 h after co-culture (P<0. 01). The results of fluorescence imaging showed that the blue, green and red fluorescence in the MC3T3-E1 cells, the outline was clear, and the fluorescence intensity of the cytoplasm was stronger than that of the nucleus after co-cultured with Zn-CDs. The qRT-PCR results showed that the relative expression levels of Runx2, ALP and OC mRNA were significantly increased with the increasing of Zn-CDs concentration. The alizarin red staining results showed that the number of calcium deposits in the MC3T3-E1 cells in different concentrations of Zn-CDs groups were more than that in blank control group after induced for 21 d. Conclusion: Zn-CDs can effectively perform the fluorescence imaging in the MC3T3-E1 cells, and Zn-CDs have a certain ability to promote the osteoblastic differentiation of the MC3T3-E1 cells.

Down-regulation of osteocytic TGF-β/Smad4inhibits the osteoblastic and osteoclastic differentiation in mouse BMSCs / 基础医学与临床

Objective To determine the effect of ostecytic TGF-β/Smad4 signaling on osteoblastic and osteoclastic differentiation in bone marrow stromal cells (BMSCs).Methods Mice with osteocytic TGF-β/Smad4 conditional knock down (Smad4ot CKD) were generated as previously by crossing DMP1-8kb-Cre mice with Smad4lox(ex8)/lox(ex8) mice.The osteocytes were isolated from tibial and femoral diaphysis and co-cultured with wild-type BMSCs.ALP staining, Alizarin red staining and TRAP staining were performed to show osteoblastic and osteoclastic differentiation.Then, their marker genes were detected by qPCR and proteins measured by Western blot.ResultsThe expression of Runx2 and Osterix were reduced in smad4 CKDot co-cultured with BMSCs compared with controls(P<0.01).Similarly, the specific markers of osteoblastic differentiation were decreased (P<0.01).Additionally, the expression of RANKL was not significantly changed in with BMSCs.However, OPG was highly expressed incontrol group compared with smad4 CKD in co-cultured group (P<0.05).Thus, the radio of RANKL/OPG was significantly reduced (P<0.05).Furthermore, the expression of RANK was inhibited.Conclusions The terminally-differentiated osteocytes are the cells regulating bone metabolism, while down-regulation of osteocytic-TGF-β/Smad4 inhibits BMSC osteoblastic and osteoclastic differentiation.

Role of TAZ in genistein induced osteoblastogenic differentiation of mouse bone marrow-derived mesenchymal stem cells / 中华内分泌代谢杂志

Objective To investigate the role of transcriptional-coactivator with PDZ-binding motif( TAZ) in genistein-induced osteoblastogenic differentiation of mouse bone marrow-derived mesenchymal stem cells ( BMSCs) .Methods Mouse BMSCs were cultured in phenol red-freeα-MEM containing osteogenic supplements for inducing osteogenic differentiation.BMSCs were transfected with siRNA-TAZ and treated with genistein.The temporal sequence of osteoblastic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity (ALP) and calcium deposition.The mRNA expression of bone sialoprotein ( BSP) and osteocalcin ( OC) were detected by reverse transcription-polymerase chain reaction(RT-PCR).The binding interaction between TAZ and cbfa1 was identified by co-immunoprecipitation.Results TAZ expression was detected during the induction of osteogenic differentiation, the ALP activity and calcium deposition were significantly decreased in BMSCs which were transfected with siRNA-TAZ.Genistein(0.01-1 μmol/L) exhibited a dose-dependent effect on TAZ expression in mouse BMSCs cultures.Treatment with genistein ( 1 μmol/L ) resulted in increased ALP avtivity and calcium deposition of BMSC cultures as function of time.Genistein(1μmol/L) also promoted the nuclear localization of TAZ and augmented the interaction between TAZ and cbfa1, and by which upregulated cbfa1-mediated gene expression such as BSP and OC.However, the ALP avtivity and calcium deposition, as well as the expression of BSP and OC were not promoted by genistein in BMSCs transfected with siRNA-TAZ.Conclusion These data suggest that the TAZ plays an important role in genistein-induced osteoblastic differentiation of mouse BMSCs cultures.

Hypoxia inhibits the expression of semaphorin 3A in breast cancer cells and regulates the differentiation of preosteoblast cells / 肿瘤

Objective: To compare the effect of MCF-7 conditioned medium under normoxia or hypoxia condition on the differentiation of osteoblastic precursor MC3T3-E1 cells after exposing breast cancer MCF-7 cells to hypoxia, and to investigate the role of semaphorin 3A (Sema3A) on osteogenic differentiation regulated by hypoxia in breast cancer with bone metastasis. Methods: The conditioned medium was collected from breast cancer MCF-7 cells cultured under the normoxia or hypoxia condition for 48 h, and used to incubate osteoblastic precursor MC3T3-E1 cells. The content and activity of alkaline phosphatase (ALP) in MC3T3-E1 cells were evaluated by BCIP/NBT ALP color development kit and ALP activity assay kit, respectively. The mineralization of MC3T3-E1 cells was detected by alizarin red S (ARS) staining. The expressions of Sema3A mRNA and protein in MCF-7 cells under normoxia or hypoxia condition for 0, 12, 24 and 48 h were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The recombinant human Sema3A (rhSema3A) was added into MC3T3-E1 cells incubated with MCF-7 hypoxia conditioned medium, then the mRNA expression levels of osteogenic differentiation-related proteins ALP, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and Osterix (Osx) in MC3T3-E1 cells were measured by real-time fluorescent quantitative PCR. Results: Compared with normoxia cultured MCF-7 conditioned medium (NorCM) group, the activity of ALP in MC3T3-E1 cells was significantly decreased in hypoxia cultured MCF-7 conditioned medium (HyCM) group (P<0.05), and the area of mineralization nodes stained with ARS was also markedly reduced in HyCM group (P<0.05). The mRNA and protein expressions of Sema3A in MCF-7 cells after hypoxia culture for 24 and 48 h were significantly lower than those after normoxia culture for the same time (all P<0.05). After rhSema3A was used to treat MC3T3-E1 cells which were incubated with HyCM, the mRNA expression levels of ALP, RUNX2, OCN and Osx were significantly increased as compared with the rhSema3A untreated group (all P<0.05). Conclusion: Hypoxia down-regulates the expression of Sema3A in MCF-7 cells in vitro, and inhibits the differentiation of osteoblast, which indicates that Sema3A is probably an important factor for the inhibition of osteoblastic differentiation by breast cancer conditioned medium under hypoxic environment.

Effect of titanium surface microgrooves and thermal oxidation on in vitro osteoblast responses / 대한치과보철학회지

PURPOSE: We aimed to investigate the effect of combined various microgrooves and thermal oxidation on the titanium (Ti) and to evaluate various in vitro responses of human periodontal ligament cells (PLCs). MATERIALS AND METHODS: Grade II titanium disks were fabricated. Microgrooves were applied on titanium discs to have 0/0 microm, 15/3.5 microm, 30/10 microm, and 60/10 microm of respective width/depth by photolithography. Thermal oxidation was performed on the microgrooves of Ti substrata for 3 h at 700degrees C in air. The experiments were divided into 3 groups: control group (ST), thermal oxidation group (ST/TO), and combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO). Surface characterization was performed by field-emission scanning microscopy. Cell adhesion, osteoblastic differentiation, and mineralization were analyzed using the bromodeoxyurdine (BrdU), Alkaline phosphatase (ALP) activity, and extracellular calcium deposition assays, respectively. Statistical analysis was performed using the oneway analysis of variance and Pearson's bivariate correlation analysis (SPSS Version 17.0). RESULTS: In general, the combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO) showed significantly higher levels compared with the control (ST) or thermal oxidation (ST-TO) groups in the BrdU expression, ALP activity, and extracellular calcium deposition. Gr60-TO group induced highest levels of cell adhesion and osteoblastic differentiation. CONCLUSION: Within the limitation of this study, we conclude that the Ti surface treatment using combined microgrooves and thermal oxidation is highly effective in inducing the cell adhesion andosteoblastic differentiation. The propose surface is also expected to be effective in inducing rapid and strong osseointegration of Ti oral implants.

Gene expression profile of continuous mechanical stress-induced osteoblastic differentiation of rat bone marrow stromal cells / 医用生物力学

Objective To evaluate differences in genes expression of rat bone marrow stromal cells (rBMSCs) under continuous mechanical strain by gene microarray technology．Methods rBMSCs were isolated and cultured in vitro. Continuous stresses with amplitude of 10% and frequency of 1 Hz were applied on rBMSCs for 6 hours by Flexercell mechanical loading system to investigate rBMSC gene expression profiles, and quantitative PCR was used to verify gene expression changes related to osteoblastic differentiation. Results Compared with the control group, 1 244 differentially expressed genes were found in mechanical loading group, among which 793 genes were up-regulated, while 451 genes were down-regulated．GO (gene ontology) analysis suggested that differentially expressed genes were mainly involved in multicellular organismal development, cell differentiation, chemotaxis, cell adhesion and so on. Four signaling pathways as Notch, Wnt, FGF and IGF might participate in the regulation of stress-induced osteoblastic differentiation. PCR validation results were consistent with the gene chip results. Conclusions Mechanical stress could induce osteoblastic differentiation of the BMSCs, while several differentially expressed genes screened by gene microarray may attribute to this process.

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin, and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Effect of perfusion flow rate on proliferation and osteoblastic differentiation of human mesenchymal stem cells / 医用生物力学

Objective To investigate the effect of different perfusion flow rates on proliferation and osteoblastic differentiation of human mesenchymal stem cells (hMSCs) in large scale β-TCP (tricalcium phosphate) scaffold at perfusion bioreactor. Methods hMSCs isolated from iliac bone marrow aspiration were loaded into large scale β-TCP scaffold and cultured in perfusion bioreactor at the perfusion flow rate of 3, 6 or 9 mL/min for 15 days. The culture media were collected for D-glucose consumption assay every 3 days. After perfusion culture for 15 days, the cell-scaffold composites were harvested for assessment of cell viability by MTT colorimetric method, SEM observation and osteogenic gene expression by real-time PCR. Results The proliferation of hMSCs assayed by daily glucose consumption showed that at early stage of culture, cells proliferated faster at flow rate of 9 mL/min than at 3 or 6 mL/min (P<0.001); while at late stage of culture, cells proliferated faster at flow rate of 6 mL/min (P<0.05). The cell viability indicated that the cell-scaffold composites at flow rate of 6 mL/min exhibited the most viable cells (P<0.001). SEM indicated that all the macropores of the scaffold at different flow rates were filled with cellular layers. All cellular layers at flow rate of 3 mL/min were incompact, but that at 9 mL/min were compact; at flow rate of 6 mL/min, the cellular layers were either compact or incompact. Real-time PCR revealed that after perfusion culture for 15 days, the mRNA expression of osteobalstic genes including ALP and OP, were enhanced significantly at flow rate of 6 and 9 mL/min as compared to that at 3 mL/min (P<0.01); however, the 9 mL/min group presented the higher OC expression than 3 and 6 mL/min group (P<0.001). Conclusions At early stage of perfusion culture, the proliferation of hMSCs was promoted at flow rate of 9 mL/min, while at late stage, there was more viable cells in scaffolds at flow rate of 6 mL/min. The osteoblastic differentiation of hMSCs was facilitated with the increase of perfusion flow rate, which was attributed to the increased flow shear stress.

Effects from mechanical stimulation and osteogenic chemical inductor on osteoblastic differentiation of rat bone mesenchymal stem cells / 医用生物力学

Objective To investigate the effect from mechanical stimulation and osteogenic chemical inductor on osteoblastic differentiation markers and formation of calcified nodules in rat bone mesenchymal stem cells (rBMSCs). Method The rBMSC were cultured in medium contained with or without osteogenic chemical inductor. The cyclic biaxial mechanical strain (2%), at a frequency of 1 Hz, was applied to the rBMSCs for periods of 2 hours each time, at intervals of 2 hours, 3 times every day, lasting 3 days and 6 days, respectively. The mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteocalcin (OCN) were analyzed with real time fluorescent quantitation reverse transcription polymerase chain reaction(qRT-PCR) and formation of calcified nodules were detected with alizarin red staining method. Results The mRNA expression of ALP, COL I and OCN were significantly increased in induced group compared with that in the corresponding uninduced group and calcified nodule was observed in the osteogenic chemical inductor group after 6 days with mechanical stimulation. Conclusions Osteogenic chemical inductor and mechanical stimulation can promote the osteoblastic differentiation of rBMSCs.

Disturbed Osteoblastic Differentiation of Fibrous Hamartoma Cell from Congenital Pseudarthrosis of the Tibia Associated with Neurofibromatosis Type I

BACKGROUND: Fibrous hamartoma is the key pathology of congenital pseudarthrosis of the tibia (CPT), which was shown to have low osteogenicity and high osteoclastogenicity. This study further investigated the mechanism of impaired osteoblastic differentiation of fibrous hamartoma cells. METHODS: Fibroblast-like cells were obtained from enzymatically dissociated fibrous hamartomas of 11 patients with CPT associated with neurofibromatosis type I (NF1). Periosteal cells were also obtained from the distal tibial periosteum of 3 patients without CPT or NF1 as control. The mRNA levels of Wnt ligands and their canonical receptors, such as Lrp5 and beta-catenin, were assayed using reverse transcriptase PCR (RT-PCR). Changes in mRNA expression of osteoblast marker genes by rhBMP2 treatment were assayed using quantitative real time RT-PCR. Changes in mRNA expression of transcription factors specifically involved in osteoblastic differentiation by rhBMP2 treatment was also assayed using quantitative real-time RT-PCR. RESULTS: Wnt1 and Wnt3a mRNA expression was lower in fibrous hamartoma than in tibial periosteal cells, but their canonical receptors did not show significant difference. Response of osteoblastic marker gene expression to rhBMP2 treatment showed patient-to-patient variability. Col1a1 mRNA expression was up-regulated in most fibrous hamartoma tissues, osteocalcin was up-regulated in a small number of patients, and ALP expression was down-regulated in most fibrous hamartoma tissues. Changes in mRNA expression of the transcription factors in response to rhBMP2 also showed factor-to-factor and patient-to-patient variability. Dlx5 was consistently up-regulated by rhBMP2 treatment in all fibrous hamartoma tissues tested. Msx2 expression was down-regulated by rhBMP2 in most cases but by lesser extent than control tissue. Runx2 expression was up-regulated in 8 out of 18 fibrous hamartoma tissues tested. Osterix expression was up-regulated in 2 and down-regulated in 3 fibrous hamartoma tissues. CONCLUSIONS: Congenital pseudarthrosis of the tibia appears to be caused by fibrous hamartoma originating from aberrant growth of Nf1 haploinsufficient periosteal cells, which failed in terminal osteoblastic differentiation and arrested at a certain stage of this process. This pathomechanism of CPT should be targeted in the development of novel therapeutic biologic intervention.

Dikkopf-2, -3 and -4 Enhance Early Osteoblastic Differentiation in Mouse Mesenchymal Progenitor Cells and Stimulate Apoptosis in Osteoblastic Cells

OBJECTIVES: The inhibitory effect of Dickkopf (Dkk)-1 on osteoblastic differentiation through blocking Wnt signaling has been well studied. However, the role of other members of the subfamily of Dkks remains unclear. We have examined the role of different Dkks on osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. METHODS: Osteoblastic differentiation was induced by treatment of Wnt-3a with Dkks or vehicle in C3H10T1/2 cells and alkaline phosphatase (ALP) activity was measured. Serum deprivation induced apoptosis was performed with pre-treatment of Dkks or vehicle in MC3T3-E1 cells and methyl thiazolyl tetrazolium (MTT) assay was done. RESULTS: Dkk-2 at low concentrations (5 and 20 nM) and Dkk-3, -4 at any concentrations (5 to 100 nM) significantly increased Wnt-3a-induced ALP activity, whereas Dkk-2 at high concentration (100 nM) significantly reduced. Treatment of Dkk-2, -3 and -4 at high concentration (100 nM) showed significant decreases of Wnt/beta-catenin transcriptional activity, whereas no effects were seen at low concentration (20 nM). In parallel experiments, treatment of Dkk-1 showed robust dose dependent inhibition not only in ALP activity but also in Wnt/beta-catenin transcriptional activity. Dkk-2, -3 and -4 increased serum deprivation-induced apoptosis in MC3T3-E1 mouse osteoblasts, while Dkk-1 had no effect. CONCLUSIONS: We found that unlike Dkk-1, Dkk-3 and -4 stimulated early osteoblastic differentiation at various concentrations regardless of their inhibitory effects on Wnt/beta-catenin transcriptional activity at high concentration. Dkk-2 had a biphasic effect where the lower doses significantly increased ALP activity while the high dose was inhibitory. Dkk-2, -3 and -4 stimulated osteoblast apoptosis whereas Dkk-1 had no effect.

Evaluation of osteogenic activity and mineralization of cultured human dental papilla-derived cells

Correlation between vascular endothelial grwoth factor signaling and mineralization during osteoblastic differentiation of cultured human periosteal-derived cells

Synergistic Effect of Isoflavones on the BMP-4 MediatedOsteoblastic Differentiation of C2C12 Myoblast / 대한정형외과학회잡지

PURPOSE: Isoflavones are rich in soybean and are known to affect bone formation. This study examined the effects and modes of action of isoflavones on the differentiation of C2C12 myoblasts in the presence of the bone morphogenetic protein (BMP)-4. MATERIALS AND METHODS: The isoflavones, daidzein, genistein or equol, and/or BMP-4 were added alone or in combination to C2C12 myoblasts. After 72 hours culture, the cells were stained for the early osteoblastic differentiation marker, alkaline phosphatase (ALP). The ALP activity was determined by comparing the color of the stained images as well as by spectrophotometry. The expression profiles of the extracellular matrix (ECM) genes responsible for the extensive remodeling at the cell surface were analyzed using agene expression microarray after treating thesamples with daidzein. RESULTS: ALP staining of BMP-4 or the isoflavones-treated cells showed that BMP-4 increased the activity of ALP in a dose dependent manner, whereas the isoflavones alone did not induced any remarkable increase. However, the ALP activity increased when the cells were treated with BMP-4 and any of the three isoflavones. The macrogen mouse MAC array data showed that the ECM genes, Mmp13 and Mmp3, were up-regulated by daidzein, whereas Col4a2, Col5a1 and Mmp9 were down-regulated. CONCLUSION: Isoflavones induce osteoblastic differentiation when combined with BMP-4, which is possibly achieved by modulating the expressional levels of various ECM genes.

Evaluation of osteogenic activity and mineralization of cultured human periosteal-derived cells

The effects of Clomiphene on the proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells / 中国药理学通报

Aim: To investigate the effects of Clomiphene (CLO) on proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. Methods: Mouse BMSCs, cultured in phenol red-free, α-MEM supplemented with 10% charcoal-stripped fetal bovine serum, were added β-glycerolphosphate and Vitamin C for in-ducing osteoblastic differentiation, and treated with 0.1 nmol·L-1 to 10 nmol·L-1 CLO. The cell proliferation was measured by cell number count. The temporal sequence of osteoblastic differentiation in BMSCs was assessed by measuring alkaline phosphatase (ALP) activity and calcium deposition. Nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium using commercial NO kit. Results: CLO (0.1-10 nmol·L-1) resulted in a dose-dependent increase in cell number, ALP activity and calcium deposition in the BMSCs cultures. CLO treatment was associated with elevated nitrite formation in conditioned media of BMCSs cultures. Concurrent treatment with ICI 182, 780 (0.1 μmol·L-1), an estrogen receptor antagonist, or N ω-nitro-L-arginine methylester (L-NAME, 6 mmol·L -1), a NOS inhibitor eliminated the stimulation of CLO on the proliferation and osteoblastic differentiation of BMSCs cultures, and abolished the CLO (1 nmol·L-1)-induced increase in NO production. Conclusion: Our results indicated that CLO at the range of 0.1-10 nmol·L-1 might be a estrogen-like receptor (ER) agonist, and stimulate the proliferation and osteoblastic differentiation of mouse BMSCs through ER/NO pathway.

Effect of Extracts from Safflower Seeds on Osteoblastic Differentiation and Intracellular Free Calcium Concentration in MC3T3-E1 Cells

Very little research has been carried out on safflower seed for the prevention and treatment of the bone deficiency diseases, including osteoporosis, which are supported by scientific evidences. In the present study, 3microliter of 0.1% dried crude extract or 2microliter of 0.1% dried aqueous fraction were shown to significantly accelerate the rate of differentiation of osteoblast. Also, the crude extract and aqueous fraction increased the [Ca2+]i of the cultured osteoblast cells: 3microliter of 0.1% dried crude extract and 2microliter of 0.1% dried aqueous fraction significantly increased the [Ca2+]i of the cultured osteoblast cells (8x104) to the extent that it deserves a considerable attention. Furthermore, the crude extract and aqueous fraction increased the [Ca2+]i of the cultured osteoblast cells, and 300microM Cd2+, specific calcium channel blocker, completely blocked the increase. Therefore, the increased [Ca2+]i of the cultured osteoblast cells by safflower seed component continued to activate calcium channel.

Osteoblastic differentiation of adult stem cells by Biphasic Calcium Phosphate / 대한치주과학회지

The present study was to determine the influence of micro-macro biphasic calcium phosphate(MBCP) on proliferation and differentiation of human marrow-derived mesenchymal stem cells. Primary stem cells were cultured from bone marrow and 3-4 passaged cells were used. This study tested the proliferative effects by cell counting. Collagen sythensis, alkaline phosphatase activity, expression of osteocalcin and bone sialoprotein by Western blot analysis were evaluated. The cellular proliferation of ASC was not influenced by MBCP. Collagen synthesis of ASC cultured on MBCP significantly increased at 5th and 7th days(p<0.05). The ALP activity in ASC cultured on MBCP significantly increased at 5th and 7th days(p<0.05). The expression of OC and BSP incresaed in ASC cultured on MBCP. These results suggest that MBCP may stimulates the osteoblastic activity of ASC.