Objective To study the roles of icariin and cyclic tensile strain (CTS) in promoting the osteogenic differentiation of adipose-derived stem cells (ASCs) and the molecular mechanisms involved.Methods ASCs were isolated from Sprague-Dawley rats and treated either with icariin (10-7 mol/L) or with 1000 μ,2000 μ or 3000 μ of CTS for 7 days,or with icariin plus CTS at 2000 μ for three days.Alkaline phosphatase (ALP) activity was detected after 3 and 7 days of intervention.Western blotting was performed to detect the expression of Runt-related transcriptional factor 2 (Runx2),Yes-associated protein (YAP) and connective tissue growth factor (CTGF) after the third day of the intervention.A reverse transcription polymerase chain reaction was performed at 7 days to detect the expression of osteopontin (OPN) and collagen la and after 3 days to detect the expression of the YAP target gene,CTGF and ankyrin repeating domain 1 (Ankrd1).Results Icariin and CTS at 1000 μ,2000 μ or 3000 μ could all significantly promote the expression of ALP protein.CTS at 2000 μ was the nost effective.The co-treatment with icariin and CTS significantly promoted ALP protein expression compared with icariin or CTS treatment alone.It also significantly promoted the expression of Runx2 and CTGF protein.Icariin or CTS (2000 μ) alone could not promote the expression of YAP protein,but icariin combined with CTS (2000 μ) promoted it significantly.Either icariin or CTS (2000 μ) could significantly promote ALP activity after 3 and 7 days,but icariin combined with CTS had the most obvious effect.Both icariin and CTS (2000 μ) could also significantly promote the expression of the osteogenesis-related genes OPN and collagen la,as well as the YAP targeted genes CTGF and Ankrdl.However,the combination of icariin and CTS had the greatest effect in promoting the expression of OPN mRNA,collagen la mRNA,CTGF mRNA and ankrdl mRNA.Conclusion Icariin and CTS co-treatment may promote osteogenic differentiation of ASCs via activating YAP expression.

Objective To compare the effects of sodium hyaluronate (SH) and celecoxib (CO) administration on the treatment of knee osteoarthritis (OA) and to investigate their influences on levels of uPA and MMP-3 in synovial fluid. Methods One hundred and thirty-six knee osteoarthritis (OA) patients from January 2010 to October 2011 were randomly enrolled into two groups: the SH group and the CO group. In the SH group, patients were injected with 2 mL sodium hyaluronate intra articulation once a week for 5 weeks. In the CO group , patients were given oral administration of celecoxib daily at a dosage of 200 mg for 5 weeks. Before and at 1 ,6 months after treatment, Lequesne′s index and VAS-pain were detected to assess the clinical results of these two drugs. The levels of uPA and MMP-3 in synovial fluid were measured by using ELISA assay. Results All patients were followed up for 6 months to 12 months. The Lequesne′s index and VAS-pain score were lowered at 1 and 6 moths after treatment in both the SH group and the CO group(P<0.05). In the CO group, however, higher Lequesne′s index and VAS-pain score were obtained at 6 months compare to that obtained at 1 month after treatment (P<0.05). In contrast, no significant difference of Lequesne′s index and VAS-pain score were shown at 6 months and at 1 month after treatment in the SH group (P>0.05). Dramatic reduction of the levels of uPA and MMP-3 in synovial fluid were observed in the SH group after treatment(P<0.01), while marginal changes were found in the CO group(P>0.05). Conclusion The redueced levels of uPA and MMP-3 in synovial fluid after treatment of sodium hyaluronate may contribute to its longer-lasting effect than that of celecoxib. Therefore , the combination of sodium hyaluronate with celecoxib may lead to better therapeutic effect on OA patients.

Objective To detect the expression levels of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-3 (MMP-3),MMP-9,MMP-13 and MMP-14 in the patients with osteoarthritis(OA)before and after arthroscopic debridement,and to explore the influence of arthroscopic debridement in the expressions of uPA, MMP-3,MMP-9,MMP-13, and MMP-14.Methods 420 cases of synovial fluid from knee OA patients undergoing arthroscopic debridement were obtained before operation. After six months follow-up, 350 cases of synovial fluid samples were obtained and according to inclusion and exclusion criteria, 228 synovial fluid were selected to analyze.The expression levels of uPA,MMP-3,MMP-9,MMP-13,and MMP-14 were measured by ELISA assay.Pain intensity of these patients before operation and six months after operation were recorded using the Visual Analogue Scale/Score(VAS).The differences of the expression levels of uPA,MMP-3,MMP-9, MMP-13,and MMP-14 between before operation and after operation were compared.The relationship between the expression levels of uPA, and MMP-3, MMP-9, MMP-13, MMP-14 and VAS was analyzed with Spearman analysis.Results All the patients were followed up for 36.5 months. Compared with before operation, the expression levels of uPA and MMP-3 in the synovial fluid of the patients after arthroscopic debridement were significantly decreased(P<0.01),the expression levels of MMP-9 and MMP-13 were also decreased (P<0.05), but the MMP-14 expression level showed no significant change.The expression levels of uPA,MMP-3,MMP-9, MMP-13,MMP-14 were positively associated with VAS before arthroscopic debridement (r=0.361,r=0.417, r=0.136,r=0.514,r=0.156,P<0.05 );uPA and MMP-3 were positively correlated with VAS after arthroscopic debridement(r=0.981,r=0.831,P<0.01),as well as the expression level of MMP-13 and VAS, but there were no significant differences between the expression levels of MMP-9, MMP-14 and VAS. Conclusion The decreased levels of uPA,MMP-3 and MMP-13 in synovial fluid may contribute to the pain-relief effects of arthroscopic debridement.

Objective To investigate the curative effects of damage control surgery in treatment of borderline severe multiple fractures.Methods The study involved 37 patients with severe multiple fractures(ISS of 16-42,Pape classification of borderline type)surgically treated from January 2009 to June 2010.All patients underwent staged surgeries according to damage control principles:hemostasis,debridement and provisional fixation were given in the first stage;antishock,anti-coagulation dysfunction,anti-hypothermy,anti-infection were performed in the second stage;definitive operations for fractures were performed as soon as possible in the third stage,provided that their physiological condition was permitted.Results All the patients received effective treatments such as debridement,provisional fixation and antishock in the first and second stages,with no iatrogenic secondary injury.The patients had significant recovery in the 6-14 months of follow-up,with total disability rate of 16％.Conclusion The staged operations according to damage control principles are effective and safe in treatment of borderline severe multiple fractures.

The osteogenic in vitro effect of low intensity pulsed ultrasound (LIPUS) on SD rat adipose-derived stem cells (ADSCs) was investigated.Rat ADSCs underwent LIPUS (intensity=100 mW/cm2) or sham exposure for 8 min per treatment once everyday in vitro,and then the alkaline phosphatase (ALP) activity and mineralized nodule formation were assessed to evaluate the osteogenic effect of LIPUS on ADSCs.To further explore the underlying mechanism,the osteogenic-related gene mRNA expression was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR) at 1st,3rd,5th,7th day after exposure repectively.Westen blot was used to evaluate the protein expression levels of two osteogenic differentiation associated genes at 7th and 14th day repectively.It was found that ALP activity was increased after LIPUS exposure and LIPUS resulted in mineralized nodule formation of ADSCs in vitro.LIPUS-treated ADSCs displayed higher mRNA expression levels of runt-related transcription factor 2 (Runx2),osteocalcin (OCN),ALP and bone sialoprotein (BSP) genes than controls,and the protein levels of Runx2 and BSP were also increased.The results suggested that LIPUS may induce the osteogenic differentiation of ADSCs in vitro.

Matrix metalloproteinase-2 (MMP-2) level and the ERK1/2 signal pathway are dependent factors for the growth and metastasis of cancer.However,the impact of MMP-2 in combination with ERK1/2 in tumor patients with drug resistance is unknown.To determine the relationship between MMP-2 and the ERK1/2 signal pathway,we established an adriamycin (ADM)-induced MG-63 (ADM-MG-63) cell line.With the increase of the ERK1/2 pathway blocker PD98059,we detected the expression levels of MMP-2 and p-ERK1/2 by Western blot in ADM-MG-63 cells.In ADM-MG-63 cells transfected with MMP-2-siRNA,the expression of ERK1/2 was detected for understanding the function of the ERK1/2 signal pathway.Three siRNAs for MMP-2 (MMP-2-siRNA) were designed,and the optimal one was selected and tested at different time points of 24,48 and 72 h.Under an ADM-induced condition,ADM-MG-63 cells were finally stable living in the medium of ADM (200 ng/mL).PD98059 could effectively suppress the expression levels of p-ERK1/2 and MMP-2.When the MMP-2 was silenced by using MMP-2-siRNA,the expression of p-ERK1/2 was enhanced.It is concluded that MMP-2 may be involved in ADM resistance dependent on ERK1/2 signal pathway,suggesting interference in ERK1/2 may be a new method of targeted therapy for tumor resistance.

Matrix metalloproteinase-2 (MMP-2) level and the ERK1/2 signal pathway are dependent factors for the growth and metastasis of cancer. However, the impact of MMP-2 in combination with ERK1/2 in tumor patients with drug resistance is unknown. To determine the relationship between MMP-2 and the ERK1/2 signal pathway, we established an adriamycin (ADM)-induced MG-63 (ADM-MG-63) cell line. With the increase of the ERK1/2 pathway blocker PD98059, we detected the expression levels of MMP-2 and p-ERK1/2 by Western blot in ADM-MG-63 cells. In ADM-MG-63 cells transfected with MMP-2-siRNA, the expression of ERK1/2 was detected for understanding the function of the ERK1/2 signal pathway. Three siRNAs for MMP-2 (MMP-2-siRNA) were designed, and the optimal one was selected and tested at different time points of 24, 48 and 72 h. Under an ADM-induced condition, ADM-MG-63 cells were finally stable living in the medium of ADM (200 ng/mL). PD98059 could effectively suppress the expression levels of p-ERK1/2 and MMP-2. When the MMP-2 was silenced by using MMP-2-siRNA, the expression of p-ERK1/2 was enhanced. It is concluded that MMP-2 may be involved in ADM resistance dependent on ERK1/2 signal pathway, suggesting interference in ERK1/2 may be a new method of targeted therapy for tumor resistance.

Objective To investigate the biological effects of low intensity pulsed ultrasound (LIPUS) on the proliferation and osteogenic differentiation of adipose tissue-derived stem cells (ADSCs) in vitro.Methods Primary ADSCs were harvested from the inguinal fat pads of 4-week-old female Sprague-Dawley rats,cultured in vitro and purified by magnetic-activated cell sorting.Surface ADSC markers were identified by flow cytometry.LIPUS at 100 mW/cm2 was used to stimulate the cultured cells.Flow cytometry was performed for cell cycle analysis.Cellular proliferation was evaluated via CCK8 chromatometry,and a proliferation index was calculated.ADSCs were assigned to 4 groups:a negative control group,a LIPUS group,an osteoinduction group and a LIPUS plus osteoinduction group,and treated accordingly.Alkaline phosphatase (ALP) activity was determined at the 7th and 14th day in each group,and calcium nodes were marked by Von Kossa staining.The levels of osteogenic differentiation in the different groups were evaluated.Results The ADSCs of passage 3 expressed CD 34low,and CD29high CD44high,which was consistent with the characteristics of ADSC surface markers.Proliferation was upregulated significantly in the LIPUS group compared with the negative control group.ALP activity was also elevated significantly and it resulted in mine-ralization.The highest mineralization rate was observed in the LIPUS plus osteoinduction group.Conclusions LIPUS not only can stimulate the proliferation of rat ADSCs,it also promotes their osteogenic differentiation.

This study examined the effect of small interfering RNA-mediated β-catenin knockdown on the survival,invasion and chemosensitivity of human osteosarcoma cells (U2-OS cells).The siRNA against β-catenin was constructed and transfected into U2-OS cells.The expression of β-catenin was detected by qRT-PCR and Western blotting.Cell growth and apoptosis was detected in the presence or absence of doxorubicin by MTT and flow cytometry,respectively.Cell invasion ability was measured by transwell assay.The results showed that the transfection of β-catenin siRNA resulted in decreased expression of β-catenin,suppression of invasion and motility of U2-OS cells,reduced chemosensitivity to doxorubicin in vitro,and little change in cell growth and apoptosis.Additionally,down-regulated MT1-MMP expression was found after transfection.It was concluded that knockdown of β-catenin gene may decrease the invasive ability of human osteosarcoma cells through down-regulated MT1-MMP expression,and the chemosensitivity of osteosarcoma cells against doxorubicin.

This study examined the effect of small interfering RNA-mediated β-catenin knockdown on the survival, invasion and chemosensitivity of human osteosarcoma cells (U2-OS cells). The siRNA against β-catenin was constructed and transfected into U2-OS cells. The expression of β-catenin was detected by qRT-PCR and Western blotting. Cell growth and apoptosis was detected in the presence or absence of doxorubicin by MTT and flow cytometry, respectively. Cell invasion ability was measured by transwell assay. The results showed that the transfection of β-catenin siRNA resulted in decreased expression of β-catenin, suppression of invasion and motility of U2-OS cells, reduced chemosensitivity to doxorubicin in vitro, and little change in cell growth and apoptosis. Additionally, down-regulated MT1-MMP expression was found after transfection. It was concluded that knockdown of β-catenin gene may decrease the invasive ability of human osteosarcoma cells through down-regulated MT1-MMP expression, and the chemosensitivity of osteosarcoma cells against doxorubicin.