Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （...Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.展开更多
A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indi...A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF-βsignaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP-7). This enhancement of BMP-7 in the context of TGF-β in the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regulated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF-β signaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic setting.展开更多
AIM：To evaluate the effect of exogenous recombinant human bone morphogenic protein-7（rhBMP-7）on transforming growth factor-β（TGF-β）-induced epithelial mesenchymal cell transition（EMT）and assessed its antifibr...AIM：To evaluate the effect of exogenous recombinant human bone morphogenic protein-7（rhBMP-7）on transforming growth factor-β（TGF-β）-induced epithelial mesenchymal cell transition（EMT）and assessed its antifibrotic effect via topical application.METHODS：The cytotoxic effect of rhBMP-7 was evaluated and the EMT of human corneal epithelial cells（HECEs）was induced by TGF-β. HECEs were then cultured in the presence of rhBMP-7 and/or hyaluronic acid（HA）. EMT markers,fibronectin,E-cadherin,α-smooth muscle actin（α-SMA）,and matrix metaloproteinase-9（MMP-9）,were evaluated. The level of corneal fibrosis and the reepithelization rate were evaluated using a rabbit keratectomy model. Expression of α-SMA in keratocytes were quantified following treatment with different concentrations of rhBMP-7.RESULTS：Treatment with rhBMP-7 attenuated TGF-β-induced EMT in HECEs. It significantly attenuated fibronectin secretion（31.6%; P〈0.05）,the α-SMA protein level（72.2%; P〈0.01）,and MMP-9 expression（23.6%,P〈0.05）in HECEs compared with cells grown in the presence of TGF-β alone. E-cadherin expression was significantly enhanced（289.7%; P〈0.01）in the presence of rhBMP-7. Topical application of rhBMP-7 combined with 0.1% HA significantly reduced the amount of α-SMA~＋ cells by 43.18%（P〈0.05）at a concentration of 2.5 μg/mL and by 47.73%（P〈0.05）at 25 μg/mL,compared with the control group,without disturbing corneal reepithelization.CONCLUSION：rhBMP-7 attenuates TGF-β-induced EMT in vitro,and topical application of rhBMP-7 reduces keratocyte myodifferentiation during the early wound healing stages in vivo without hindering reepithelization. Topical rhBMP-7 application as biological eye drops seems to be feasible in diseases involving TGF-β-related corneal fibrosis with corneal reepithelization disorders.展开更多
BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate ...BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate during epiphyseal growth exist.AIM Verify the effect of alendronate on the growth epiphyseal plate,and compare its effect with the size of the femur during the double-staining of the immunolocalization of transforming growth factor-β1(TGF-β1)and bone morphogenetic protein-2(BMP2)in endochondral ossifing in specimens that have received alendronate.METHODS Forty newborn rats were randomly divided into two groups:a control group(were given applications of 1 mg/kg physiologic saline)and a group that received Alendronate(a dose of 2.5 mg/kg).These groups were then divided into two subgroups for euthanasia in two and 12 d of life.After euthanasia,the femurs were removed,and the femoral bones were measured linearly between the apex of the greater trochanter until the lower intercondylar midlle face to verify the probable bone growth between 3 and 12 d in control and alednroanto treated rats.Posteriorly,the surgical pieces were also sent to the histopathology laboratory to produce histological slides.The obtained slides were stained with hematoxylin and eosin to measure each of the cartilage zones in endochondral development.and other slides were immunohistochemically tested for anti-TGF-β1 and BMP-2 antibodies to investigate the immunolocalization of these proteins in the epiphyseal plaque area.RESULTS On the third day,some diferences between the control group and specimens treated with alendronate were verified.Macroscopiccaly,we found similarities in size between the femoral bones when we compared the control group with the specimens that received alendronate.On the 12^th day,the bone size of the mice receiving the drug was significantly smaller than those of the control group.These results coincide with changes in the TGF-β1 and BMP-2 expression.In the specimens that received alendronate,the TGF-β1 was expressed in some sites of trabecular bone that was neoformed,peripherally to the bone marrow area.The BMP-2 was also positive in proliferative chondrocytes and hypertrofic chondrocytes.On the 12^th day,all layers of chondrocytes exhibited positivity for BMP-2 in the specimens that received alendronate.In the interface between the trabecular bone and cartilage,an area of disorganized bone deposition was evident.Neoformed bone also appeared to be different at 12 d.In the control group,BMP-2 was positive in an intense area of bone trabeculae,whereas the alendronate-treated group showed TGF-β1 positive trabeculae and a greater bone area.CONCLUSION Alendronate alters the immunolocalization of TGF-β1 and BMP-2 simultaneously,a condition that changes the usual histological aspects of the cartilage zone and impairs epiphysis growth and femur growth.展开更多
Critical-sized bone defect repair in patients with diabetes mellitus remains a challenge in clinical treatment because of dysfunction of macrophage polarization and the inflammatory microenvironment in the bone defect...Critical-sized bone defect repair in patients with diabetes mellitus remains a challenge in clinical treatment because of dysfunction of macrophage polarization and the inflammatory microenvironment in the bone defect region.Three-dimensional(3D)bioprinted scaffolds loaded with live cells and bioactive factors can improve cell viability and the inflammatory microenvironment and further accelerating bone repair.Here,we used modified bioinks comprising gelatin,gelatin methacryloyl(GelMA),and 4-arm poly(ethylene glycol)acrylate(PEG)to fabricate 3D bioprinted scaffolds containing BMSCs,RAW264.7 macrophages,and BMP-4-loaded mesoporous silica nanoparticles(MSNs).Addition of MSNs effectively improved the mechanical strength of GelMA/gelatin/PEG scaffolds.Moreover,MSNs sustainably released BMP-4 for long-term effectiveness.In 3D bioprinted scaffolds,BMP-4 promoted the polarization of RAW264.7 to M2 macrophages,which secrete anti-inflammatory factors and thereby reduce the levels of pro-inflammatory factors.BMP-4 released from MSNs and BMP-2 secreted from M2 macrophages collectively stimulated the osteogenic differentiation of BMSCs in the 3D bioprinted scaffolds.Furthermore,in calvarial critical-size defect models of diabetic rats,3D bioprinted scaffolds loaded with MSNs/BMP-4 induced M2 macrophage polarization and improved the inflammatory microenvironment.And 3D bioprinted scaffolds with MSNs/BMP-4,BMSCs,and RAW264.7 cells significantly accelerated bone repair.In conclusion,our results indicated that implanting 3D bioprinted scaffolds containing MSNs/BMP-4,BMSCs,and RAW264.7 cells in bone defects may be an effective method for improving diabetic bone repair,owing to the direct effects of BMP-4 on promoting osteogenesis of BMSCs and regulating M2 type macrophage polarization to improve the inflammatory microenvironment and secrete BMP-2.展开更多
Objective:Calcific aortic valve disease(CAVD)affects millions of elderly people,and there is currently no effective way to stop or slow down its progression.Therefore,exploring the pathogenesis of CAVD is very importa...Objective:Calcific aortic valve disease(CAVD)affects millions of elderly people,and there is currently no effective way to stop or slow down its progression.Therefore,exploring the pathogenesis of CAVD is very important for prevention and treatment.Cartilage oligomeric matrix protein(COMP)have important role in cell phenotype change.This study is aimed to confirm whether COMP participate in CAVD and try to find the possible mechanisms.Methods:Human aortic valve tissues from Nanjing First Hospital(CAVD group,n=20;control group,n=11)were harvested.The expression level of COMP was tested by western blot and immunohistochemistry.Dual immunofluorescence staining was used for locating COMP.Bone morphogenetic protein-2(BMP2)signalling were tested by western blot.The animal model was also used to detect COMP level by immunohistochemistry.Results:The results showed that the expression level of COMP was significantly increased in the calcific valve samples when compared with that of the control valve(P<0.05);COMP was expressed near the calcific nodules and co-localized with a-smooth muscle actin(a-SMA).The protein levels of BMP2 and p-Smads 1/5/9 were markedly more highly expressed in the CAVD group than the control group(P<0.05).Furthermore,immunofluorescence detection showed that COMP and BMP2 were co-located in calcific valves.Conclusions:The above results suggested that upregulation of COMP and BMP2 may be associated with aortic valve calcification and that COMP may become a potential therapeutic target in human CAVD.展开更多
Bone diseases such as osteoporosis and periodontitis are induced by excessive osteoclastic activity,which is closely associated with inflammation.Benzydamine(BA)has been used as a cytokine-suppressive or non-steroidal...Bone diseases such as osteoporosis and periodontitis are induced by excessive osteoclastic activity,which is closely associated with inflammation.Benzydamine(BA)has been used as a cytokine-suppressive or non-steroidal anti-inflammatory drug that inhibits the production of proinflammatory cytokines or prostaglandins.However,its role in osteoclast differentiation and function remains unknown.Here,we explored the role of BA in regulating osteoclast differentiation and elucidated the underlying mechanism.BA inhibited osteoclast differentiation and strongly suppressed interleukin-1β(IL-1β)production.BA inhibited osteoclast formation and bone resorption when added to bone marrowderived macrophages and differentiated osteoclasts,and the inhibitory effect was reversed by IL-1βtreatment.The reporter assay and the inhibitor study of IL-1βtranscription suggested that BA inhibited nuclear factor-κB and activator protein-1 by regulating IκB kinase,extracellular signal regulated kinase and P38,resulting in the down-regulation of IL-1βexpression.BA also promoted osteoblast differentiation.Furthermore,BA protected lipopolysaccharide-and ovariectomy-induced bone loss in mice,suggesting therapeutic potential against inflammation-induced bone diseases and postmenopausal osteoporosis.展开更多
Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain un...Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain unclear.This study aimed to determine whether CKIP-1 affects osteogenic differentiation in MSCs and explore the relationship of CKIP-1 and inflammation.Methods:Bone marrow MSCs of CKIP-1 wild type(WT)and knockout(KO)mice were cultivated in vitro.Cell phenotype was analyzed by flow cytometry,colony formation was detected to study the proliferative ability.Osteogenic and adipogenic induction were performed.The osteogenic ability was explored by alizarin red staining,alkaline phosphatase(ALP)staining and ALP activity detection.Quantitative real-time polymerase chain reaction(qRT-PCR)was carried out to determine the mRNA expression levels of osteoblast marker genes.The adipogenic ability was detected by oil red O staining.Content of the bone was analyzed to observe the differences of bone imaging parameters including trabecular bone volume/tissue volume(BV/TV),bone surface area fraction/trabecular BV,trabecular number(Tb.N),and trabecular spacing(Tb.sp).Interleukin(IL)-1b was injected on WT mice of 2 months old and 18 months old,respectively.Difference in CKIP-1 expression was detected by RT-PCR and western blot.The relationship between CKIP-1 and inflammation was explored by RT-PCR and western blot.Results:ALP assays,alizarin red staining,and qRT-PCR showed that MSCs derived from CKIP-1 KO mice exhibited a stronger capability for osteogenesis.Micro-computed tomography detection showed that among 18-month-old mice,CKIP-1 KO mice presented significantly higher bone mass compared withWTmice(P=0.02).No significant difference was observed in 2-month-old mice.In vivo data showed that expression of CKIP-1 was higher in the bone marrow of aging mice than in young mice(4.3-fold increase at themRNA level,P=0.04).Finally,the expression levels of CKIP-1 in bone marrow(3.2-fold increase at themRNA level,P=0.03)and cultured MSCs were up-regulated on chronic inflammatory stimulation by IL-1b.Conclusions:CKIP-1 is responsible for negative regulation of MSC osteogenesis with age-dependent effects.Increasing levels of inflammation with aging may be the primary factor responsible for higher expression levels of CKIP-1 but may not necessarily affect MSC aging.展开更多
Objective： To study the mechanism of Huogu I Formula （活骨I方） in treating osteonecrosis of femoral head. Methods： Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group ...Objective： To study the mechanism of Huogu I Formula （活骨I方） in treating osteonecrosis of femoral head. Methods： Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 （BMP2）, transforming growth factor beta1 （TGFβ1）, Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand （OPG/RANKL） mRNA was detected by in situ hybridization. Results： Compared with the control group, serum levels of total cholesterol （TG）, triglyceride （TG） and low-density lipoprotein cholesterol （LDL-C） in the model group rose significantly. Positive cell counting of BMP2, TGF 131, Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ1, Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant. Conclusion： Huogu I Formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ1, Smads and OPG/RANKL of osteoclast in femoral head.展开更多
Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional(3 D)scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we de...Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional(3 D)scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone(PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 2^3 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2(BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells(BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase(ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.展开更多
基金supported by grants from the National Nature Science foundation of China(Grant Nos.30872912 and 30830108)
文摘Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.
文摘A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF-βsignaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP-7). This enhancement of BMP-7 in the context of TGF-β in the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regulated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF-β signaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic setting.
基金Supported by the Soonchunhyang University Research Fund,the WPM project,Ministry of trade,industry&energy(No.10037842)the National Research Foundation of Korea(No.NRF-2016R1C1B2015622)Recombinant human BMP-7 protein was kindly provided by Cellumed Co.,Ltd
文摘AIM：To evaluate the effect of exogenous recombinant human bone morphogenic protein-7（rhBMP-7）on transforming growth factor-β（TGF-β）-induced epithelial mesenchymal cell transition（EMT）and assessed its antifibrotic effect via topical application.METHODS：The cytotoxic effect of rhBMP-7 was evaluated and the EMT of human corneal epithelial cells（HECEs）was induced by TGF-β. HECEs were then cultured in the presence of rhBMP-7 and/or hyaluronic acid（HA）. EMT markers,fibronectin,E-cadherin,α-smooth muscle actin（α-SMA）,and matrix metaloproteinase-9（MMP-9）,were evaluated. The level of corneal fibrosis and the reepithelization rate were evaluated using a rabbit keratectomy model. Expression of α-SMA in keratocytes were quantified following treatment with different concentrations of rhBMP-7.RESULTS：Treatment with rhBMP-7 attenuated TGF-β-induced EMT in HECEs. It significantly attenuated fibronectin secretion（31.6%; P〈0.05）,the α-SMA protein level（72.2%; P〈0.01）,and MMP-9 expression（23.6%,P〈0.05）in HECEs compared with cells grown in the presence of TGF-β alone. E-cadherin expression was significantly enhanced（289.7%; P〈0.01）in the presence of rhBMP-7. Topical application of rhBMP-7 combined with 0.1% HA significantly reduced the amount of α-SMA~＋ cells by 43.18%（P〈0.05）at a concentration of 2.5 μg/mL and by 47.73%（P〈0.05）at 25 μg/mL,compared with the control group,without disturbing corneal reepithelization.CONCLUSION：rhBMP-7 attenuates TGF-β-induced EMT in vitro,and topical application of rhBMP-7 reduces keratocyte myodifferentiation during the early wound healing stages in vivo without hindering reepithelization. Topical rhBMP-7 application as biological eye drops seems to be feasible in diseases involving TGF-β-related corneal fibrosis with corneal reepithelization disorders.
文摘BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate during epiphyseal growth exist.AIM Verify the effect of alendronate on the growth epiphyseal plate,and compare its effect with the size of the femur during the double-staining of the immunolocalization of transforming growth factor-β1(TGF-β1)and bone morphogenetic protein-2(BMP2)in endochondral ossifing in specimens that have received alendronate.METHODS Forty newborn rats were randomly divided into two groups:a control group(were given applications of 1 mg/kg physiologic saline)and a group that received Alendronate(a dose of 2.5 mg/kg).These groups were then divided into two subgroups for euthanasia in two and 12 d of life.After euthanasia,the femurs were removed,and the femoral bones were measured linearly between the apex of the greater trochanter until the lower intercondylar midlle face to verify the probable bone growth between 3 and 12 d in control and alednroanto treated rats.Posteriorly,the surgical pieces were also sent to the histopathology laboratory to produce histological slides.The obtained slides were stained with hematoxylin and eosin to measure each of the cartilage zones in endochondral development.and other slides were immunohistochemically tested for anti-TGF-β1 and BMP-2 antibodies to investigate the immunolocalization of these proteins in the epiphyseal plaque area.RESULTS On the third day,some diferences between the control group and specimens treated with alendronate were verified.Macroscopiccaly,we found similarities in size between the femoral bones when we compared the control group with the specimens that received alendronate.On the 12^th day,the bone size of the mice receiving the drug was significantly smaller than those of the control group.These results coincide with changes in the TGF-β1 and BMP-2 expression.In the specimens that received alendronate,the TGF-β1 was expressed in some sites of trabecular bone that was neoformed,peripherally to the bone marrow area.The BMP-2 was also positive in proliferative chondrocytes and hypertrofic chondrocytes.On the 12^th day,all layers of chondrocytes exhibited positivity for BMP-2 in the specimens that received alendronate.In the interface between the trabecular bone and cartilage,an area of disorganized bone deposition was evident.Neoformed bone also appeared to be different at 12 d.In the control group,BMP-2 was positive in an intense area of bone trabeculae,whereas the alendronate-treated group showed TGF-β1 positive trabeculae and a greater bone area.CONCLUSION Alendronate alters the immunolocalization of TGF-β1 and BMP-2 simultaneously,a condition that changes the usual histological aspects of the cartilage zone and impairs epiphysis growth and femur growth.
基金supported by National Key R&D Program of China(2018YFB1105600/2018YFC2002300/2018YFA0703000)National Natural Science Foundation of China(81772326/81702124/81902195)+3 种基金Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai JiaoTong University School of Medicine(JYZZ070)Project of Shanghai Science and Technology Commission(18441903700/19XD1434200/18431903700/19441908700/19441917500)Translational Medicine Innovation Project of Shanghai Jiao Tong University School of Medicine(TM201613/TM201915)Project of Shanghai Jiading National Health and Family Planning Commission(KYXM 2018-KY-03).
文摘Critical-sized bone defect repair in patients with diabetes mellitus remains a challenge in clinical treatment because of dysfunction of macrophage polarization and the inflammatory microenvironment in the bone defect region.Three-dimensional(3D)bioprinted scaffolds loaded with live cells and bioactive factors can improve cell viability and the inflammatory microenvironment and further accelerating bone repair.Here,we used modified bioinks comprising gelatin,gelatin methacryloyl(GelMA),and 4-arm poly(ethylene glycol)acrylate(PEG)to fabricate 3D bioprinted scaffolds containing BMSCs,RAW264.7 macrophages,and BMP-4-loaded mesoporous silica nanoparticles(MSNs).Addition of MSNs effectively improved the mechanical strength of GelMA/gelatin/PEG scaffolds.Moreover,MSNs sustainably released BMP-4 for long-term effectiveness.In 3D bioprinted scaffolds,BMP-4 promoted the polarization of RAW264.7 to M2 macrophages,which secrete anti-inflammatory factors and thereby reduce the levels of pro-inflammatory factors.BMP-4 released from MSNs and BMP-2 secreted from M2 macrophages collectively stimulated the osteogenic differentiation of BMSCs in the 3D bioprinted scaffolds.Furthermore,in calvarial critical-size defect models of diabetic rats,3D bioprinted scaffolds loaded with MSNs/BMP-4 induced M2 macrophage polarization and improved the inflammatory microenvironment.And 3D bioprinted scaffolds with MSNs/BMP-4,BMSCs,and RAW264.7 cells significantly accelerated bone repair.In conclusion,our results indicated that implanting 3D bioprinted scaffolds containing MSNs/BMP-4,BMSCs,and RAW264.7 cells in bone defects may be an effective method for improving diabetic bone repair,owing to the direct effects of BMP-4 on promoting osteogenesis of BMSCs and regulating M2 type macrophage polarization to improve the inflammatory microenvironment and secrete BMP-2.
基金the general program of Science and Technology Development Foundation of Nanjing Medical University(No.NMUB2018314)Jiangsu Provincial Key Medical Discipline(Laboratory)(ZDXKA2016021).
文摘Objective:Calcific aortic valve disease(CAVD)affects millions of elderly people,and there is currently no effective way to stop or slow down its progression.Therefore,exploring the pathogenesis of CAVD is very important for prevention and treatment.Cartilage oligomeric matrix protein(COMP)have important role in cell phenotype change.This study is aimed to confirm whether COMP participate in CAVD and try to find the possible mechanisms.Methods:Human aortic valve tissues from Nanjing First Hospital(CAVD group,n=20;control group,n=11)were harvested.The expression level of COMP was tested by western blot and immunohistochemistry.Dual immunofluorescence staining was used for locating COMP.Bone morphogenetic protein-2(BMP2)signalling were tested by western blot.The animal model was also used to detect COMP level by immunohistochemistry.Results:The results showed that the expression level of COMP was significantly increased in the calcific valve samples when compared with that of the control valve(P<0.05);COMP was expressed near the calcific nodules and co-localized with a-smooth muscle actin(a-SMA).The protein levels of BMP2 and p-Smads 1/5/9 were markedly more highly expressed in the CAVD group than the control group(P<0.05).Furthermore,immunofluorescence detection showed that COMP and BMP2 were co-located in calcific valves.Conclusions:The above results suggested that upregulation of COMP and BMP2 may be associated with aortic valve calcification and that COMP may become a potential therapeutic target in human CAVD.
基金supported by the National Research Foundation(NRF)(Grant Nos.2017R1A2B2012435,2019R1C1C1011198 and 2019R1A5A6099645,Korea)funded by the Korean Ministry of Science,ICT and future Planning(MSIP).
文摘Bone diseases such as osteoporosis and periodontitis are induced by excessive osteoclastic activity,which is closely associated with inflammation.Benzydamine(BA)has been used as a cytokine-suppressive or non-steroidal anti-inflammatory drug that inhibits the production of proinflammatory cytokines or prostaglandins.However,its role in osteoclast differentiation and function remains unknown.Here,we explored the role of BA in regulating osteoclast differentiation and elucidated the underlying mechanism.BA inhibited osteoclast differentiation and strongly suppressed interleukin-1β(IL-1β)production.BA inhibited osteoclast formation and bone resorption when added to bone marrowderived macrophages and differentiated osteoclasts,and the inhibitory effect was reversed by IL-1βtreatment.The reporter assay and the inhibitor study of IL-1βtranscription suggested that BA inhibited nuclear factor-κB and activator protein-1 by regulating IκB kinase,extracellular signal regulated kinase and P38,resulting in the down-regulation of IL-1βexpression.BA also promoted osteoblast differentiation.Furthermore,BA protected lipopolysaccharide-and ovariectomy-induced bone loss in mice,suggesting therapeutic potential against inflammation-induced bone diseases and postmenopausal osteoporosis.
基金Supported by the grants from the National Key Research and Development Project(No.2017YFB1304300)Conversion Fund of PLA General Hospital(No.2017tm-018)the Clinical Research Support Fund of PLA General Hospital(No.2017fc-tsys-2013).
文摘Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain unclear.This study aimed to determine whether CKIP-1 affects osteogenic differentiation in MSCs and explore the relationship of CKIP-1 and inflammation.Methods:Bone marrow MSCs of CKIP-1 wild type(WT)and knockout(KO)mice were cultivated in vitro.Cell phenotype was analyzed by flow cytometry,colony formation was detected to study the proliferative ability.Osteogenic and adipogenic induction were performed.The osteogenic ability was explored by alizarin red staining,alkaline phosphatase(ALP)staining and ALP activity detection.Quantitative real-time polymerase chain reaction(qRT-PCR)was carried out to determine the mRNA expression levels of osteoblast marker genes.The adipogenic ability was detected by oil red O staining.Content of the bone was analyzed to observe the differences of bone imaging parameters including trabecular bone volume/tissue volume(BV/TV),bone surface area fraction/trabecular BV,trabecular number(Tb.N),and trabecular spacing(Tb.sp).Interleukin(IL)-1b was injected on WT mice of 2 months old and 18 months old,respectively.Difference in CKIP-1 expression was detected by RT-PCR and western blot.The relationship between CKIP-1 and inflammation was explored by RT-PCR and western blot.Results:ALP assays,alizarin red staining,and qRT-PCR showed that MSCs derived from CKIP-1 KO mice exhibited a stronger capability for osteogenesis.Micro-computed tomography detection showed that among 18-month-old mice,CKIP-1 KO mice presented significantly higher bone mass compared withWTmice(P=0.02).No significant difference was observed in 2-month-old mice.In vivo data showed that expression of CKIP-1 was higher in the bone marrow of aging mice than in young mice(4.3-fold increase at themRNA level,P=0.04).Finally,the expression levels of CKIP-1 in bone marrow(3.2-fold increase at themRNA level,P=0.03)and cultured MSCs were up-regulated on chronic inflammatory stimulation by IL-1b.Conclusions:CKIP-1 is responsible for negative regulation of MSC osteogenesis with age-dependent effects.Increasing levels of inflammation with aging may be the primary factor responsible for higher expression levels of CKIP-1 but may not necessarily affect MSC aging.
基金Supported by the National Natural Foundation of China(No. 81173417 and 30472131)
文摘Objective： To study the mechanism of Huogu I Formula （活骨I方） in treating osteonecrosis of femoral head. Methods： Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 （BMP2）, transforming growth factor beta1 （TGFβ1）, Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand （OPG/RANKL） mRNA was detected by in situ hybridization. Results： Compared with the control group, serum levels of total cholesterol （TG）, triglyceride （TG） and low-density lipoprotein cholesterol （LDL-C） in the model group rose significantly. Positive cell counting of BMP2, TGF 131, Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ1, Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant. Conclusion： Huogu I Formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ1, Smads and OPG/RANKL of osteoclast in femoral head.
基金supported by the National Natural Science Foundation of China (U1605225, 31570974, and 31470927)the Public Science and Technology Research Funds Projects of Ocean (201505029)+1 种基金the Promotion Program for Young and Middle-aged Teacher in Science and Technology Research of Huaqiao University (ZQN-PY107)the Program for Innovative Research Team in Science and Technology in Fujian Province University
文摘Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional(3 D)scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone(PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 2^3 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2(BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells(BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase(ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.
