BACKGROUND:Numerous studies have shown that mechanical stimulation is essential for the lineage-specific differentiation of bone marrow mesenchymal stem cells.However,osteogenic differentiation of bone marrow mesenchymal stem cells on surfaces with different roughnesses under mechanical stretching conditions is unknown. OBJECTIVE:To investigate the effects and action mechanisms of different roughness surfaces of polydimethylsiloxane(PDMS)on osteogenic differentiation of bone marrow mesenchymal stem cells under stretching conditions. METHODS:Three morphologies with different roughnesses(PDMS-120M,PDMS-1000M,and PDMS-10000M)were constructed on PDMS surfaces by means of different grits of sandpaper(120 grits,1 000 grits and 10 000 grits),and PDMS surfaces in contact with air served as a control group.With different amplitudes of 0%,2%,4%,and 6%,osteogenesis-related gene expression of bone marrow mesenchymal stem cells on different PDMS surfaces under static and stretching conditions was detected by RT-qPCR.RT-qPCR and western blot assay were used to detect the expression of SIRT1 gene and protein as well as osteogenesis-related genes and proteins in bone marrow mesenchymal stem cells on different roughness surfaces under 2%stretching conditions.Alkaline phosphatase staining and alizarin red staining were further used to observe the osteogenic differentiation ability of bone marrow mesenchymal stem cells on different PDMS surfaces under 2%stretching conditions. RESULTS AND CONCLUSION:(1)Bone marrow mesenchymal stem cells on the PDMS-1000M surface with a roughness of(13.51±2.11)μm had better osteogenic gene expression under static conditions.(2)Bone marrow mesenchymal stem cells on the PDMS surface in contact with air had better osteogenic differentiation under 4%stretching conditions,while bone marrow mesenchymal stem cells on the PDMS-1000M surface had better osteogenic differentiation under 2%stretching conditions.(3)Bone marrow mesenchymal stem cells on the PDMS-1000M surface with a roughness of(13.51±2.11)μm had better osteogenic differentiation under 2%stretching conditions,which may be related to activation of SIRT1 signaling pathway.

BACKGROUND:Many factors can cause disc degeneration,including aging,nutritional deficiency,and mechanical factors.The mechanical load is considered to be a very important factor,but its mechanism is still unclear. OBJECTIVE:To investigate the role of sclerostin(SOST)and Wnt/β-catenin signaling pathways in inducing degeneration of endplate cartilage. METHODS:Cartilage endplate cells were extracted from 4-week-old male Sprague-Dawley rats.Compressive stress was applied to endplate chondrocytes in vitro using a mechanical loading apparatus,and the cell viability was determined by the cell counting kit-8 assay at 1,3,5,and 7 days after compression.Western blot,reverse transcription quantitative PCR,and cellular immunofluorescence techniques were employed to examine intracellular cartilage markers(Aggrecan and type Ⅱ collagen)as well as calcification-related factors(Runx2 and osteocalcin).The expression of extracellular matrix degradation enzyme and genes related to the signaling pathway(SOST and β-catenin)was also analyzed. RESULTS AND CONCLUSION:Under compressive stress,the cell activity of endplate chondrocytes increased with both the duration and intensity of stress.Furthermore,the expression levels of Aggrecan and type Ⅱ collagen decreased in endplate cells under compressive stress,while those of calcification-related factors(Runx2 and osteocalcin)increased.Additionally,compressive stress promoted extracellular matrix degradation in endplate chondrocytes,leading to an increase in matrix metalloproteinase 3 and matrix metalloproteinase 13 expression.Abnormalities were observed in the Wnt/β-catenin signaling pathway within these cells under compressive stress,characterized by a decrease in specific inhibitory factor SOST expression accompanied by abnormal accumulation of β-catenin.To conclude,decreased SOST expression in endplate chondrocytes under compressive stress activates the Wnt/β-catenin signaling pathway,thereby promoting calcification,degeneration and extracellular matrix degradation in the cartilage endplate.

Objective To establish two osteoarthritis models of destabilization of the medial meniscus(DMM)and chronic ankle instability(CAI)in mice,and compare the effects of knee osteoarthritis with varus deformity on ipsilateral ankle cartilage degeneration.Methods Thirty 6-week-old C57BL/6J male mice were randomly divided into a control group and two surgical groups(DMM group and CAI group),respectively.The progression of ankle joint degeneration was quantitatively evaluated through behavioral observation,imaging techniques and histopathology analysis in each group of mice over a 12-week period.Results A decline in gait stability and balance was observed in two surgical groups.Compared to the control group,the time required to cross the balance beam was increased by 23.20％,and the number of slips was increased by 43.26％at 12th week postoperatively in the DMM group.The bone volume fraction and bone mineral density of ankle joints also increased.Meanwhile,wear and tear of the ankle cartilage were found,with the formation of osteophytes,and OARSI score was increased by 88.89％.These changes in ankle joint were more pronounced in the CAI group.Conclusions This mouse model-based study revealed a coupling relationship between the knee and ankle motion.Knee osteoarthritis with varus deformity could lead to a significant ankle joint degeneration,while the damage was less severe than that observed in CAI.

BACKGROUND:TD-198946 is a small molecule drug known to induce stem cells to form cartilage;however,its effect on osteogenic differentiation remains unclear.OBJECTIVE:To investigate the effects of the small molecule drug TD-198946 on promoting osteoblastic differentiation of rat bone marrow mesenchymal stem cells and its mechanism of action.METHODS:Bone marrow mesenchymal stem cells (BMSCs) were extracted from SD rats.CCK-8 assay was used to evaluate the effect of different concentrations of TD-198946 on the proliferation of bone marrow mesenchymal stem cells to determine the optimal concentration of TD-198946.Then,the optimal concentration of TD-198946 and osteogenic medium were added to induce osteogenic differentiation of bone marrow mesenchymal stem cells.On day 3,qRT-PCR was used to detect the expression of alkaline phosphatase,Runt-related transcription factor 2,osteopontin,osteocalcin,and type Ⅰ collagen genes.On day 7 of osteogenic induction,alkaline phosphatase staining and western blot assay were performed to detect the expression of Runt-related transcription factor 2,type Ⅰ collagen,AKT,p-AKT,PI3K,and p-PI3K proteins.On day 21 of osteogenic induction,Alizarin red staining was conducted.RESULTS AND CONCLUSION:(1) CCK-8 assay results revealed that 100 nmol/L TD-198946 could promote the proliferation of bone marrow mesenchymal stem cells.(2) The results of alkaline phosphatase staining and alizarin red staining showed that TD-198946 could promote osteogenic differentiation of rat bone marrow mesenchymal stem cells.(3) qRT-PCR results showed that TD-198946 could promote the expression of osteogenic genes alkaline phosphatase,Runt-related transcription factor 2,osteopontin,osteocalcin,and type Ⅰ collagen.(4) Western blot assay results showed that TD-198946 could enhance the expression of Runt-related transcription factor 2,type Ⅰ collagen,p-PI3K,and p-AKT.The results indicate that the small molecule drug TD-198946 may induce osteogenic differentiation of rat bone marrow mesenchymal stem cells by activating the PI3K/AKT signaling pathway.

Objective To study the characteristics of gait behavior in a mouse model of chronic ankle instability and provide a reference for the study of the mechanism of chronic ankle instability as well as drug screening and evaluation.Methods Thirty C57BL/6J male mice were randomly divided into a control group(n=15)and an injury group(n=15).In the control group,the ankle joint underwent sham operation,and in the injury group,the anterior talofibular ligament and calcaneofibular ligament of the left ankle joint were transected.Gait parameters were analyzed in each group using TreadScan passive gait analysis system.Results Compared with the control group,the injury group showed a 28.43％increase(P＜0.05)in average standing time and a 23.07％increase(P＜0.05)in the percentage of standing time,whereas the average swing time and the percentage of swing time were shortened by 50.63％(P＜0.001)and 19.75％(P＜0.01),respectively.The average braking time and average stride time in the injury group were also shortened by 18.37％(P＜0.01)and 37.86％(P＜0.001),respectively.The injury group exhibited a decrease in step length,anterior-posterior step width,and mediolateral step width by 36.96％,13.66％,and 8.10％,respectively.The total movement speed and instantaneous speed decreased by 8.05％and 11.12％,respectively,while the stride frequency increased by 51.41％.The average footprint area and average maximum standing area decreased by 8.8％and 13.24％,respectively,and foot pressure decreased by only 3％.The plantar pressure distribution in the injury group was uneven,with a more obvious decrease in plantar pressure in the hindfoot,especially a 13.92％decrease in plantar pressure in the right posterior quadrant.Conclusions Mice with chronic ankle instability adopt a more conservative walking pattern during the motion,reducing movement volume and amplitude to improve coordination and stability during walking.

Objective To study the characteristics of gait behavior in a mouse model of chronic ankle instability and provide a reference for the study of the mechanism of chronic ankle instability as well as drug screening and evaluation.Methods Thirty C57BL/6J male mice were randomly divided into a control group(n=15)and an injury group(n=15).In the control group,the ankle joint underwent sham operation,and in the injury group,the anterior talofibular ligament and calcaneofibular ligament of the left ankle joint were transected.Gait parameters were analyzed in each group using TreadScan passive gait analysis system.Results Compared with the control group,the injury group showed a 28.43％increase(P＜0.05)in average standing time and a 23.07％increase(P＜0.05)in the percentage of standing time,whereas the average swing time and the percentage of swing time were shortened by 50.63％(P＜0.001)and 19.75％(P＜0.01),respectively.The average braking time and average stride time in the injury group were also shortened by 18.37％(P＜0.01)and 37.86％(P＜0.001),respectively.The injury group exhibited a decrease in step length,anterior-posterior step width,and mediolateral step width by 36.96％,13.66％,and 8.10％,respectively.The total movement speed and instantaneous speed decreased by 8.05％and 11.12％,respectively,while the stride frequency increased by 51.41％.The average footprint area and average maximum standing area decreased by 8.8％and 13.24％,respectively,and foot pressure decreased by only 3％.The plantar pressure distribution in the injury group was uneven,with a more obvious decrease in plantar pressure in the hindfoot,especially a 13.92％decrease in plantar pressure in the right posterior quadrant.Conclusions Mice with chronic ankle instability adopt a more conservative walking pattern during the motion,reducing movement volume and amplitude to improve coordination and stability during walking.

Objective To establish two osteoarthritis models of destabilization of the medial meniscus(DMM)and chronic ankle instability(CAI)in mice,and compare the effects of knee osteoarthritis with varus deformity on ipsilateral ankle cartilage degeneration.Methods Thirty 6-week-old C57BL/6J male mice were randomly divided into a control group and two surgical groups(DMM group and CAI group),respectively.The progression of ankle joint degeneration was quantitatively evaluated through behavioral observation,imaging techniques and histopathology analysis in each group of mice over a 12-week period.Results A decline in gait stability and balance was observed in two surgical groups.Compared to the control group,the time required to cross the balance beam was increased by 23.20％,and the number of slips was increased by 43.26％at 12th week postoperatively in the DMM group.The bone volume fraction and bone mineral density of ankle joints also increased.Meanwhile,wear and tear of the ankle cartilage were found,with the formation of osteophytes,and OARSI score was increased by 88.89％.These changes in ankle joint were more pronounced in the CAI group.Conclusions This mouse model-based study revealed a coupling relationship between the knee and ankle motion.Knee osteoarthritis with varus deformity could lead to a significant ankle joint degeneration,while the damage was less severe than that observed in CAI.

BACKGROUND:TD-198946 is a small molecule drug known to induce stem cells to form cartilage;however,its effect on osteogenic differentiation remains unclear.OBJECTIVE:To investigate the effects of the small molecule drug TD-198946 on promoting osteoblastic differentiation of rat bone marrow mesenchymal stem cells and its mechanism of action.METHODS:Bone marrow mesenchymal stem cells (BMSCs) were extracted from SD rats.CCK-8 assay was used to evaluate the effect of different concentrations of TD-198946 on the proliferation of bone marrow mesenchymal stem cells to determine the optimal concentration of TD-198946.Then,the optimal concentration of TD-198946 and osteogenic medium were added to induce osteogenic differentiation of bone marrow mesenchymal stem cells.On day 3,qRT-PCR was used to detect the expression of alkaline phosphatase,Runt-related transcription factor 2,osteopontin,osteocalcin,and type Ⅰ collagen genes.On day 7 of osteogenic induction,alkaline phosphatase staining and western blot assay were performed to detect the expression of Runt-related transcription factor 2,type Ⅰ collagen,AKT,p-AKT,PI3K,and p-PI3K proteins.On day 21 of osteogenic induction,Alizarin red staining was conducted.RESULTS AND CONCLUSION:(1) CCK-8 assay results revealed that 100 nmol/L TD-198946 could promote the proliferation of bone marrow mesenchymal stem cells.(2) The results of alkaline phosphatase staining and alizarin red staining showed that TD-198946 could promote osteogenic differentiation of rat bone marrow mesenchymal stem cells.(3) qRT-PCR results showed that TD-198946 could promote the expression of osteogenic genes alkaline phosphatase,Runt-related transcription factor 2,osteopontin,osteocalcin,and type Ⅰ collagen.(4) Western blot assay results showed that TD-198946 could enhance the expression of Runt-related transcription factor 2,type Ⅰ collagen,p-PI3K,and p-AKT.The results indicate that the small molecule drug TD-198946 may induce osteogenic differentiation of rat bone marrow mesenchymal stem cells by activating the PI3K/AKT signaling pathway.

BACKGROUND:Traction is clinically used for the early treatment of intervertebral disc degeneration,but its effect on the normal intervertebral disc remains unknown.Whether it directly causes intervertebral disc degeneration or has a positive effect is the key point of this study. OBJECTIVE:To design a static traction model and observe the effect of static traction on the nucleus pulposus and annulus fibrosus of the intervertebral disc METHODS:Twenty Sprague-Dawley rats,3 months of age,were included in the study.The intervertebral disc spaces between 7/8,8/9 and 9/10 were stretched by 1 mm,and the intervertebral disc spaces between 6/7 and 10/11 were used as control.Five rats were randomly selected at 2,4,6,and 8 weeks of traction to perform MRI T2-weighted scans of the caudal vertebra,tissue section staining,and RT-PCR gene assays for anabolic metabolism to observe the effects of static traction on the nucleus pulposus and annulus fibrosus of the intervertebral disc. RESULTS AND CONCLUSION:After short-term static traction of the rat caudal vertebra,the T2-weighted image signal in the nucleus pulposus region was enhanced.During the traction period,nucleus pulposus cells grew well,the intercellular matrix was abundant,and the annulus fibrosus arranged regularly.The RT-PCR results showed that after traction,the mRNA expression of proteoglycan increased,the expression of matrix metalloproteinase-3 decreased,the expression of type Ⅰ and Ⅱ collagen decreased,and the expression of matrix metalloproteinase-13 increased and tissue inhibitor of matrix metalloproteinase 1 increased.These gene results also indicated that traction made proteoglycan more inclined to an anabolic state,and type Ⅰ and Ⅱ collagen more inclined to a catabolic state.To conclude,static traction promotes proteoglycan anabolism making the nucleus pulposus moist.

Objective To study the closure time of neonatal ductus venosus and the Z score regression equation, and to explore the variation of closure time of neonatal ductus venosus with gestationalage.Methods Color doppler ultrasound was applied to detect the closure time of neonatal ductus venosus in normal newborns, Z score regression equation ( lnY =a +bX +cX2 ) for the closure time of neonatal ductus venosus ( Y ) was developed by regression analysis which used gestational age ( X) as an independent variable. The Z scores of the closure time of neonatal ductus venosus in different gestational age were calculated by the formula [ Z = ( M - Y )/S x , M for observation value, Y for predictivevalue].Results There were 432 cases in our study.The closure time of neonatal ductus venosus was negatively related to gestational age ( r = -0. 938 , P <0. 001 ) . The nonlinear regression equation was lnY= -5. 228+0. 089X-0. 000228X2, R2 =0. 854, Sx =0. 214(P<0. 001). Based on the predicted mean of the closure time and Sx related to different gestational age, Z scores for specific closure time of neonatal ductus venosus could be calculated by the formula [ Z =( M -Y)/Sx ] . The Z scores were normally distributed,and did not change with gestational age.Conclusions Theclosure & nbsp;time of neonatal ductus venosus is negatively related to gestational age. The Z scores obtained by the predicted nonlinear regression equation are normally distributed.