Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism.@*METHODS@#MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells.@*RESULTS@#The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05).@*CONCLUSION@#Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

Regulation of osteoclast differentiation by CaMK Ⅱδ mediated through CREB and ERK1/2 signal pathways / 解放军医学杂志

Objective To investigate the function of cAMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK1/2) in osteoclast differentiation mediated by Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)δ,and elucidate the molecular mechanism thereof.Methods CaMK Ⅱδ RNA interference lentivirus vector was constructed and mouse RAW264.7 cells were transfected with the virus to determine the interference efficiency.After virus transfection,RAW264.7 cells were treated with 50ng/ml receptor activator of nuclear factor κB ligand (RANKL) and the phosphorylation levels of CREB and ERK1/2 were detected at different time points.The cells were also treated with PD98059,an ERK1/2 inhibitor,to determine the effect of ERK1/2 signal blocking on the expression of nuclear factor activated T-cells cytoplasmic 1 (NFATc1) and osteoclast differentiation.Results Interference efficiency of recombinant CaMK Ⅱδ virus vector was 77.2％ at mRNA level and 70.2％ at protein level.CaMK Ⅱδ RNA interference significantly suppressed phosphorylation of CREB and ERK1/2,and the levels ofp-CREB and p-ERK1/2 were down-regulated by 21％-55％ and 55％-64％,respectively.ERK1/2 inhibitor significantly down-regulated the protein expression of NFATc1,and the number of osteoclast,the number and size of bone resorption lacunae decreased by 39.3％,50.0％ and 52.3％,respectively.Conclusion CaMK Ⅱδ RNA interference may significantly suppress the phosphorylation of CREB and ERK1/2,and CREB and ERK1/2 have mediated the CaMK Ⅱδ-induced osteoclast differentiation.

Influence of zoledronate on osteoclast differentiation and gene expression of calmodulin and calmodulin-dependent protein kinase II / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of zoledronate acid on osteoclast differentiation and gene expression of calmodulin (CAM) and calmodulin-dependent protein kinase (CAMK)II.</p><p><b>METHODS</b>Receptor activation of nuclear factor κB ligand (RANKL) was used to induce differentiation of RAW264.7 cells into osteoclasts in vitro. The cells were divided into two groups, group A and group B. Both groups were treated with RANKL for 5 days, whereas group B was also treated with zoledronate for the last 2 days.Osteoclastogenesis and gene expression of CAM and CAMK II were examined.</p><p><b>RESULTS</b>In group B, the number of new-generated osteoclasts (≥3 nuclei), number and size of dentin resorption lacunaes were (23 ± 3) , (19 ± 2) and (4951 ± 223) µm(2) respectively, which were significantly lower than those [(44 ± 3) , (46 ± 1) and (13 331 ± 248) µm(2)] in group A (P < 0.01).mRNA and protein level of CAM and CAMK II were also significantly down-regulated in group B when compared with group A (P < 0.01) and the decrease was 26.7% and 37.2% respectively for CAM, 57.0% and 76.1% respectively for CAMK II.</p><p><b>CONCLUSIONS</b>Zoledronate acid could significantly inhibit formation and resorption function of osteoclasts. CAM and CAMKII may be involved in the inhibition process.</p>

Effect of glial cell derived neurotrophic factor on regeneration of facial nerve defects by autogenous vein conduit / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the effects of glial cell derived neurotrophic factor (GDNF) on regeneration of facial nerve defects by autogenous facial vein conduit.</p><p><b>METHODS</b>Thirty-six rabbits were used in this study and 10 mm-length facial nerve defects were made on both sides of all animals. The nerve gaps were bridged using autogenous posterior facial vein graft of the same side. The animals received injection of either saline (group A, n=16) or GDNF (group B, n=16) into the veins. Nerve function was evaluated by evoking nerve action potential immediately after operation and 4, 8 and 16 weeks after operation. Regenerated nerve samples were harvested at 4, 8, and 16 weeks after operation and processed for histology and transmitting electron microscopic examination (TEM).</p><p><b>RESULTS</b>Action potential did not exist immediately after operation but it was evoked at 4, 8, and 16 weeks in both groups. At 4 and 8 weeks after operation, the amplitude and width of action potential were significantly higher in group B than group A (P < 0.01), except wave width at 4 weeks, which showed no significant differences, while the latency period was significantly shorter in group B than that in group A (P < 0.01). At 16 weeks, action potential was similar between two groups, except wave amptitude, which was higher in group B than group A (P < 0.01). Morphologic and TEM examinations showed more matured myelinated nerve fibers and active Schwann's cells in group B when compared group A during the whole regeneration process.</p><p><b>CONCLUSION</b>GDNF can promote nerve regeneraat early stage during reconstruction of facial nerve defects by autogenous facial vein conduit and combination of GDNF and autogenous vein graft provides a valuable method for clinical reconstruction of facial nerve defects.</p>

The effect of experimental osteoporosis on bone healing of autologous iliac crest graft around implants / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the influences of experimental osteoporosis (OP) on bone healing of autologous iliac crest graft around dental implants in rabbits.</p><p><b>METHODS</b>Twenty Japanese rabbits were randomly divided into two groups. Bilaterally ovariectomy was performed on experimental group and control group received sham-operation. Twelve weeks later, femoral bones were examined for bone mineral density (BMD) to verify OP status. Then bone defects were made in the proximal metaphysis of the tibiae and autologous iliac crest grafts with simultaneous implant placement were performed. The animals were killed at 8 and 12 weeks after bone graft surgery. Undecalcified sections were prepared and examined histologically and histomorphometrically.</p><p><b>RESULTS</b>Osteoporotic status caused by ovariectomy was verified by significantly decreased BMD in experimental group (P < 0.001). At 8 and 12 weeks after bone graft surgery, osseointegration was observed in both groups. However, thickness of cortical bone (TCB), bone volume in cancellous area (BVC), implant-bone contact rate (IBCR) at bone graft area all significantly decreased in experimental group when compared with control group (P < 0.01). Newly formed bone was also less in experimental group than that in control group.</p><p><b>CONCLUSION</b>Although experimental OP may not delay osseointegration of dental implants in autologous iliac crest graft, it certainly promotes resorption of bone grafts, decreases cancellous bone volume and implant-bone contact rate. Therefore it may be an important risk factor for patients receiving autologous bone graft with simultaneous implant placement.</p>

Expression of bone-related genes in bone marrow MSCs after cyclic mechanical strain: implications for distraction osteogenesis / 国际口腔科学杂志·英文版

<p><b>AIM</b>Understanding the response of mesenchymal stem cells (MSCs) to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis.</p><p><b>METHODOLOGY</b>In this study, a single period of cyclic mechanical stretch (0.5 Hz, 2,000 microepsilon) was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity was examined. The mRNA expression of six bone-related genes (Ets-1, bFGF, IGF-II, TGF-beta, Cbfa1 and ALP) was detected using real-time quantitative RT-PCR.</p><p><b>RESULTS</b>The results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfa1 expression became elevated later. The temporal expression pattern of ALP coincided perfectly with Cbfa1.</p><p><b>CONCLUSION</b>The results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.</p>

Response of bone marrow mesenchymal stem cells to mechanical stretch and gene expression of transforming growth factor-beta and insulin-like growth factor-II under mechanical strain / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the response of rat bone marrow mesenchymal stem cells (MSCs) to a single period of mechanical strain and expression patterns of transforming growth factor-beta (TGF-beta) and insulin-like growth factor-II (IGF-II) after mechanical stretch.</p><p><b>METHODS</b>Bone marrow MSCs were isolated from SD rats and cultured in vitro. A four-point bending apparatus were used to perform a single period of mechanical strain (2000 microepsilon, 40 min) on MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity of MSCs were examined and gene expression patterns of TGF-beta and IGF-II were detected by SYBR green quantitative real-time RT-PCR.</p><p><b>RESULTS</b>Cell proliferation, ALP activity and expression of TGF-beta and IGF-II were all significantly up-regulated in stretched MSCs when compared with their controls. The mRNA levels of TGF-beta and IGF-II got top increase immediately after mechanical loading and increased about 51.44 and 8.92 folds, respectively, when compared with control cells. Expression of TGF-beta and IGF-II decreased with time and returned to control level at 12 h after mechanical stimulus, despite of a small increase at 6 h.</p><p><b>CONCLUSION</b>The mechanical stretch can promote MSCs proliferation, up-regulate its ALP activity and induce a time-dependent expression increase of TGF-beta and IGF-II which in turn result in osteogenic differentiation of MSCs. Mechanical stimulus is a key stimulator for osteogenic differentiation of MSCs and vital for bone formation in distraction osteogenesis.</p>

Autogenous standard versus inside-out vein graft to repair facial nerve in rabbits / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate autogenous vein grafts and inside-out vein grafts as conduits for the defects repair in the rabbit facial nerves.</p><p><b>METHODS</b>The 10 mm segments of buccal division of facial nerve were transected for 48 rabbits in this study. Then the gaps were immediately repaired by autogenous vein grafts or inside-out vein grafts in different groups. All the animals underwent the whisker movement test and electrophysiologic test during the following 16 weeks at different time points postoperatively. Subsequently, the histological examination was performed to observe the facial nerve regeneration morphologically.</p><p><b>RESULTS</b>At 8 weeks after operation, the facial nerve regeneration has significant difference between the experimental group and the control group in electrophysiologic test and histological observation. However, at the end of this study, 16 weeks after operation, there was no significant difference between inside-out vein grafts and standard vein grafts in enhancing peripheral nerve regeneration.</p><p><b>CONCLUSION</b>This study suggest that both kinds of vein grafts play positive roles in facial nerve regeneration after being repaired immediately, but the autogenous inside-out vein grafts might accelerate and facilitate axonal regeneration as compared with control.</p>

Osteogenic differentiation of rat bone marrow mesenchymal stem cell after transfection with recombinant pAd-bone morphogenetic protein-7 / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the effect of recombinant pAd-BMP-7 on osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSC).</p><p><b>METHODS</b>Recombinant pAd-bone morphogenetic protein (BMP) 7 was constructed and the titer of recombinant adenovirus was determined. pAd-BMP-7 and pAdTrack-CMV were used to transfect rat MSC. Transfection efficiency was measured by fluorescent microscope and BMP-7 expression was detected by RT-PCR and immunocytochemical analysis. The MSC were then randomly divided into 3 groups: group A received pAd-BMP-7 transfection, group B was transfected with pAdTrack-CMV, and group C received pAdTrack-CMV transfection plus bone supplements. Osteogenic differentiation of MSC was evaluated by examination of mineralization nodes formation.</p><p><b>RESULTS</b>The titer of pAd-BMP-7 reached about 2.0 x 10(15) pfu/L and transfection efficiency of exogenous gene was nearly 99% at day 2. The expression of exogenous gene sustained about 5 to 7 weeks, with a higher level during first 3 weeks. After transfection, transcription of BMP-7 and expression of BMP-7 protein were also verified in MSC. Compared with the negative results in group B, mineralization nodes were formed in both group A and group C. However, group A showed better formation of mineralization nodes than group C (P < 0.01).</p><p><b>CONCLUSIONS</b>The results of this study indicated that recombinant pAd-BMP-7 can successfully transfect rat MSC and accelerate their osteogenic differentiation. The technique explored in this study provides a unique and valuable gene engineering approach for reconstruction of craniofacial bone defects.</p>

The changes of cytoskeleton F-actin in rat bone marrow mesenchymal stem cells and calvarial osteoblasts under mechanical strain / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the response of rat bone marrow mesenchymal stem cells (MSCs and calvarial osteoblasts to mechanical strain and the consequent changes of cytoskeleton F-actin.</p><p><b>METHODS</b>Bone marrow MSCs and calvarial osteoblasts were isolated from SD rats and cultured in vitro. Mechanical stretch was performed on passage 3 cells at 2 000 microepsilon for 0, 2, 6 and 12 hours using four-point bending system. The response of cells and the distribution of F-actin were observed using fluorescent staining under laser scanning confocal microscope and the morphological parameters were quantified using image analysis software Laserpix.</p><p><b>RESULTS</b>Under mechanical stretch, the fluorescent staining decreased obviously at both MSCs and osteoblasts, and F-actin filaments were rearranged and became tenuous, thinner, and abnormally distributed. The outline of nucleus became unclear and apoptotic changes were observed at some of both cells. Cellular size decreased more significantly in MSCs than in osteoblasts. Quantity analysis showed that total area of cells, total fluorescent density and green fluorescent density (F-actin) were all significantly decreased in MSCs (P < 0.05 or P < 0.01), and total fluorescent density, green fluorescent density and red fluorescent density (nuclei) did also in osteoblasts (P < 0.05 or P < 0.01).</p><p><b>CONCLUSION</b>Mechanical stretch caused extensive response on both MSCs and osteoblasts which led to the rearrangement of F-actin filament and apoptosis in some of these cells. MSCs were more sensitive to mechanical strain than osteoblasts.</p>

Construction of recombinant plasmid pEGFP-BMP7 and its expression in rat bone marrow mesenchymal stem cells in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct recombinant plasmid pEGFP-BMP7 and determine its expression in rat bone marrow mesenchymal stem cells (MSCs) in vitro.</p><p><b>METHODS</b>cDNA of target gene was obtained from neonatal rat kidney by RT-PCR. After sequencing the target gene, the cDNA was subcloned into a eukaryote plasmid pEGFP-N1 by directed cloning and then digested with two restrictive endonucleases to verify the correctiveness of the recombinant plasmid pEGFP-BMP7. Rat bone marrow MSCs were transiently transfected with the pEGFP-BMP7 and transfection efficiency of the Green Fluorescent Protein (GFP) was determined. RT-PCR and immunocytochemical analysis were also performed to detect the expression of BMP7 in rat MSCs.</p><p><b>RESULTS</b>1 311 bp cDNA fragment was obtained by RT-PCR and sequence analysis showed it matched perfectly with that of rat BMP7 gene except a single nucleotide change at 756 bp from T to A. Digestion of the recombinant plasmid showed two 1.3 kb and 4.7 kb fragments and their size were same as those of BMP7 and pEGFP. This indicated that BMP7 cDNA was successfully subcloned into pEGFP. Transient transfection showed an efficiency of 33% at day 2 in rat MSCs. After transfection, transcription of BMP7 was detected in MSCs and expression of BMP7 protein was also verified.</p><p><b>CONCLUSION</b>Recombinant eukaryote plasmid pEGFP-BMP7 was successfully constructed and expressed in rat bone marrow MSCs. This procedure may provide a unique method for stimulation of callus formation in distraction osteogenesis and reconstruction of craniofacial bone defects.</p>