Ginseng used for bone tissue scaffold

The scaffold based tissue engineering materialized for bone tissue therapy. Gelatin-glutaraldehyde cross linked scaffold was prepared by solvent casting -porogen leaching method. It was characterized by FTIR and SEM microphotograph analysis. Absence of peak at waves no. 1625 cm?1 in ATR-FTIR indicated formation of cross-linking. FE-SEM micrograph showed honeycomb pad like structure with high porosity. Methanolic extract of Withania somnifera (Ashwagandha) root extract induced MC3T3 E1 osteoblast cell adhesion and proliferation on porous gelatin scaffold. GC-MS analysis pointed out presence of 4-amino- 2-ethyl-3-methylquinoline, an active phyto-chemicals having tissue regeneration potential. High anti-oxidant capacity down regulates cell death mechanism by scavenging free radical. The biocompatible gelatin scaffold has RGD moiety that attune the MC3T3 E1 osteoblast cell adhesion. Withania somnifera root extract may boost up cell proliferation on scaffold. Therefore treatment with Withania somnifera root extract may be the new approaches for designing bone tissue scaffold for bone tissue therapy.

Effect of mistletoe polysaccharose on mouse osteoblast cell line MC3T3-E1 promoting proliferation and inhibiting apoptosis and its possible mechanism / 解剖学报

Objective: To observe the effect of mistletoe polysaccharose on proliferation and apoptosis of osteoblast and to explore the possible mechanism. Methods: The mistletoe polysaccharose was extracted and made different concentrations (0.625 g/L, 1.25 g/L, 2.5 g/L, 5 g/L) to treat mouse skull bone cell line MC3T3-E1. Cell cycle and apoptosis were detected by flow cytometry. Cell proliferation and extracellular alkaline phosphatase secretion were detected by BrdU and alkaline phosphatase ELISA kits. The mRNA expression level of osteoblast related gene was detected by Realtime PCR. Results: Compared with the control group, the result of treatment groups with increasing mistletoe polysaccharose concentrations were as follows: the proportions of S phase and G2/M phase in cell cycle increased and the cell count Annexin V +/PI-significantly decreased, the number of cells increased, extracellular alkaline phosphatase activity obviously decreased. The increasing transcription levels of Runt-related transcription factor 2(Runtx2) and collagen type I alpha 1 (COL1A) were stabled over 2. 5 g/L and the up-regulation of mRNA expression of osteocalcin (OC) was steady at 1.25 g/L. Conclusion: Mistletoe polysaccharose can promote the proliferation and inhibit the apoptosis of MC3T3-E1 cells, and its possible mechanism might be related to down-regulating of extracellular alkaline phosphatase activity and the grow exhanced expression of OC, Runtx2 and COL1A.

Effects of rhPTH (1-34) on adhesion of MC3T3-E1 osteoblasts cultured on titanium surfaces of SLA / 安徽医科大学学报

Objective To evaluate the effects of the human recombinant parathyroid hormone [rhPTH (1 -34)] on the initial adhesion of MC3T3-E1 osteoblasts cultured on titanium surfaces of sand-blasted large grift acid-etched (SLA). Methods The optimal concentration of the rhPTH(1-34) action was determined by the CCK8 method. Then the MC3T3-E1 cells were divided into experimental groups treated with the optimal concentration of the rhPTH(1-34) medium and control groups treated with blank medium, and inoculated on SLA-treated titanium plates. DAPI immunocytochemistxy, scanning electron microscopy, and RT-PCR were used to count the cell adhesion of MC3T3-E1 cells on the surface of titanium plates. Morphological changes were observed and the expression of the integrin α1, α5, β1 mRNA in MC3T3-E1 cells was determined. Results Compared with the control group, the adherence of osteoblasts was more than that of the control group. The remodeling of actin filaments, the change of cell morphology, and the speed of adhesion and extension on the titanium plate were faster than that of the control group. The expression of the osteoblast integrin subunit α1, α5 and (31 was higher than that of the control group. Conclusion rhPTH (1-34) can promote the adhesion of MC3T3-E1 cells on pure titanium surface.

Anthraquinone Glycoside Aloin Induces Osteogenic Initiation of MC3T3-E1 Cells: Involvement of MAPK Mediated Wnt and Bmp Signaling

Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.

Effects of Scytosiphon lomentaria on osteoblastic proliferation and differentiation of MC3T3-E1 cells

BACKGROUND/OBJECTIVES: Bone formation and bone resorption continuously occur in bone tissue to prevent the accumulation of old bone, this being called bone remodeling. Osteoblasts especially play a crucial role in bone formation through the differentiation and proliferation. Therefore, in this study, we investigated the effects of Scytosiphon lomentaria extract (SLE) on osteoblastic proliferation and differentiation in MC3T3-E1 cells. MATERIALS/METHODS: A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and protein expression analysis of osteoblastic genes were carried out to assess the osteoblastic proliferation and differentiation. RESULTS: The results indicated that treatment of SLE promoted the proliferation of MC3T3-E1 cells and improved ALP activity. And, SLE treatment significantly promoted mineralized nodule formation compared with control. In addition, cells treated with SLE significantly upregulated protein expression of ALP, type 1 collagen, bone morphogenetic protein 2, runt-related transcription factor 2, osterix, and osteoprotegerin. CONCLUSIONS: The results demonstrate that SLE promote differentiation inducement and proliferation of osteoblasts and, therefore may help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.

The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells

OBJECTIVES: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. MATERIALS AND METHODS: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). RESULTS: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). CONCLUSIONS: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

Lentiviral-mediated RNAi targeting p38MAPK ameliorates high glucose-induced apoptosis in osteoblast MC3T3-E1 cell line.

The p38 mitogen activated protein kinase (p38MAPK) pathway is an important signaling cascade involved in cell growth, differentiation and apoptosis. High glucose activates p38MAPK pathway in different cells, including osteoblasts. In the present study, role of p38MAPK in high glucose induced osteoblast apoptosis and potential of RNA interference (RNAi) targeting p38MAPK as a therapy strategy have been reported. Lentiviral-mediated RNAi effectively reduced p38MAPK and p-p38MAPK expressions in osteoblastic cell line (MC3T3-E1) following high glucose (22 mM) induction. Inhibition of p38MAPK activity significantly suppressed high glucose induced apoptosis of MC3T3-E1 cell and was confirmed by flow cytometry and ultra-structural examination by transmission electronic microscope. Inhibition of p38MAPK also significantly attenuates caspase-3 and bax protein expressions, but increased significantly bcl-2 expression as determined by Western blot analysis. The results suggested that p38MAPK mediates high glucose induced osteoblast apoptosis, partly through modulating the expressions of caspase-3, bax and bcl-2. Inhibition of p38MAPK with lentiviral-mediated RNAi or its specific inhibitor provides a new strategy to treat high glucose induced osteoblast apoptosis.

Effect of RNAi targetingp38MAPK on high glucose-induced apoptosis of osteoblast MC3T3-E1 cell line / 中华内分泌代谢杂志

Objective To examine the role of p38MAPK in high glucose-induced apoptosis of osteoblast MC3T3-E1 cell line, and to investigate its effect on the expressions of apoptosis-related molecules including caspase3, bax, and bcl-2. Methods The lentiviral vector containing short hairpin RNA targeting p38MAPK was constructed. The cultured osteoblast MC3T3-E1 cell were divided into 5 groups:normal control group(A group), high glucose group(B group), p38MAPK-shRNA transfection group(C group), signal transduction inhibitor group(D group), and transfection with negative control siRNA group(E group). RT-PCR was used to determine the p38MAPK mRNA expression levels in MC3T3-E1 cells. Flow cytometry(FCM)was employed to detect the cell apoptotic percentage. The protein levels of apoptosis-related molecules p38MAPK, p-p38MAPK, caspase-3, bax, and bcl-2 were assayed by Western blot. Ultrastructural alternation of MC3T3-E1 cell was observed under transmission electron microscopy(TEM). Results The lentiviral vector containing short hairp in RNA targeting p38MAPK was successfully constructed and transfected into MC3T3-E1 cells. RT-PCR result suggested that the siRNA targeting p38MAPK could effectively reduce the p38MAPK mRNA expression level induced by high glucose in MC3T3-E1 cell line. FCM showed siRNA significantly decreased high glucose-induced apoptosis percentage of MC3T3-E1 cells(P＜0.01). Meanwhile, we also found the siRNA significantly attenuated the proteins levels of p38MAPK, p-p38MAPK, caspase-3, and gene bax induced by high glucose in MC3T3-E1 cells, whereas the protein level of gene bcl-2 was enhanced remarkably when compared with high glucose group and negative control siRNA group(P＜0.01, P＜0.05).Conclusion The iRNA targeting p38MAPK suppressed high glucose-induced MC3T3-E1 cell apoptosis via inhibiting the activation of p38MAPK signaling pathway, thereby reducing the expressions levels of p-p38MAPK, caspase-3 and gene bax, and up-regulating the level of gene bcl-2.

The effect of rhBMP-2 on delta12-PGJ2 induced osteoblastic differentiation and mineralization / 대한치주과학회지

Prostaglandin plays a significant role in the local control of bone metabolism associated with periodontal disease. delta12-PGJ2 is a natural PGD2 metabolite that is formed in vivo in the presence of plasma. It is known for delta12-PGJ2 to stimulate calcification in osteoblastic cells. Bone morphogenetic protein(BMP) stimulated osteoblastic differentiation in various types of cells and greatly enhanced healing of bony defects. The purpose of this study was to evaluate the effect of rhBMP-2 on delta12-PGJ2 induced osteoblastic differentiation and mineralization in vitro. A human osteosarcoma cells line Saos-2 were cultured. In the test groups, 10-7M of delta12-PGJ2 or mixture of 10-8M of delta12-PGJ2 and 100ng/ml of rhBMP-2 or 100ng/ml of rhBMP-2 were added to culture media. After 1 day, 2 days and 4 days of culture period, the cell number was measured. Alkaline phosphatase activity was measure at 3 days. Reverse transcription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein at 8 hours, 1 day and 7 days. The ability to produce mineralized nodules in rat osteoblasts(MC3T3-E1) was evaluated at 21 days. The results were as follows : 1. rhBMP-2 or mixture of rhBMP-2 and delta12-PGJ2 inhibited cell proliferation of human osteosarcoma cells. 2. rhBMP-2 or mixture of rhBMP-2 and delta12-PGJ2 stimulated alkaline phosphatase activity significantly higher than delta12-PGJ2 alone. 3. rhBMP-2 or mixture of rhBMP-2 and delta12-PGJ2 stimulated mineralization compared to delta12-PGJ2 alone. 4. mRNA of alkaline phosphatase, BMP-2, cbfa 1, Type I collagen were detected in the group treated with delta12-PGJ2/rhBMP-2, rhBMP-2 alone, delta12-PGJ2 alone. These results show that mixture of delta12-PGJ2 and rhBMP-2 causes more bone formation than delta12-PGJ2 alone while the bone formation effects of mixture of delta12-PGJ2 and rhBMP-2 are less than those of rhBMP-2 alone. Further researches would be necessary to clarify the interactions of these agents.

On the Activity of MC3T3-E1 Cell in vitro

This study was performed to observe the effect of ultrasound(1.0MHz, 0.75W/cm2 and 1.0W/cm2) irradiation on cultured MC3T3-E1 cell, osteoblastic like cell with respect to the proliferation, protein synthesis, and alkaline phosphatase activity of the cells. The results were as follows: 1. The proliferation of MC3T3-E1 cells was increased on ultrasound irradiated group compared with control group. 2. The protein synthesis was not apparently increased on ultrasound irradiated group compared with control group. 3. The alkaline phosphatase activity level was not apparently increased on ultrasound irradiated group compared with control group. From the above results and other literatures, we could suggest that the ultrasound with the appropriate intensity and frequency may have important roles in stimulation of cell proliferation. Therefore the ultrasound may be used in the acceleration of the bone regeneration and bone fracture healing.

The effect of a static magnetic field of Nd-Fe-B magnet on alkaline phosphatase activity of MC3T3-E1 cells / 대한치과교정학회지

more efficient tooth movement. The purpose of this study was to evaluate the effect of different static magnetic fields of ND-Fe-B magnet on MC3T3-E1 cells by measuring the alkaline phosphatase activity and observing the amount of stained alkaline phosphatase. For measuring of alkaline phosphatase activity, MC3T3-E1 cells were seeded in first and third row of 12 well culture plate. And Nd-Fe-B magnets were positioned under the first column of first and third row to apply different static magnetic fields(first column:100mT ; second column:4.6mT ; third column:0.5mT ; forth column:0.0mT) to the cells for 7, 13, 19, and 25 days. For staining of alkaline phosphatase, MC3T3-E1 cells were seeded in 100mm culture plates. And Nd-Fe-B magnets were positioned under the corner of plates to apply different static magnetic fields(magnet side:100mT ; the opposite side:0.5mT) to the cells for 7, 13, 19 and 25 days. The results were as follows : 1. ALP activity was increased until day 19 in biochemical determination as well as in histochemical staining. 2. The application of higher magnetic field(100mT) suppressed ALP activity at day 13, 19, 25. On the contrary, the application of the lower magnetic field(4.6mT, 0.5mT) significantly enhanced the ALP activity. 3. Consistent with enzyme assay, histochemical staining of ALP also demonstrated that higher magnetic field(100mT) suppressed ALP activity, lower one(0.5mT) enhanced.

The effect of a static magnetic field on the bone nodule formation of MC3T3-E1 cells / 대한치과교정학회지

To evaluate the effect of a static magnetic field on the bone producing potential of MC3T3 El cells, the alkaline phosphatase activity was measured after the cells having been cultured under 76.4mT static magnetic field using a SmCos magnets for 5days, 7days, lldays, 15days and 2ldays for each cell culture group. Also, the amount of bone nodule stained with Alizarin red S was observed. The results were as follows. · The alkaline phosphatase activity of the 7, 11, and 15 days group among the experimental groups was decreased as compared with the control groups, and the decrease of alkaline phosphatase activity in the 11 days group was the most evident among them. · Any stained bone nodules of both groups had not been observed until the 11th day. The stained bone nodules in the control groups were found on the 15th day, but not in the experimental groups. The stained bone nodules were observed in both groups on the 21st day, but the control groups have more bone nodules than the experimental groups.

The effects of continuous and intermittent compressive pressure on alkaline phosphatase activity of MC3T3-E1 cells / 대한치과교정학회지

The propose of this study was to evaluate the difference of cellular activity dependent on intermittent compressive force by determining the alkaline phosphatase activity. Alkaline phosphatase activity was measured on control and experimental groups every 24, 48, 72hours. Experimental groups consisted of continous and intermittent compressive group which were compressed by 300g/cm2 of diaphram pump. The intermittent compressive group was connected by timer which was worked on 10 minutes an off 10 minutes. The results were as follows; 1. The alkaline phosphatase activity between control and experimental groups showed not significant difference at compressed 24 hours. 2. The alkaline phosphatase activity of experimental groups were more increased than control group at compressed 48 hours. 3. The alkaline phosphatase activity of intermittent compressive group showed significant increased to control group. Whereby continuous compressive group showed not significant difference to control at 72 hours. 4. The alkaline phosphatase activity of intermittent compressive group were stringly increased than continuous compressive groups. 5. Between experimental groups and control group no other morphologic changes were detected by microscopic findings.