BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factor κB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen Ⅱ synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factor κB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen Ⅱ synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

BACKGROUND:Heterotopic ossification of skeletal muscle is a clinically serious complication.For heterotopic ossification of skeletal muscles,the cells involved in the process of heterotopic ossification remain unclear. OBJECTIVE:To investigate the involvement of myocytes,fascia cells,and endothelial cells in the process of heterotopic ossification in skeletal muscle and to observe the cell origin of heterotopic ossification in skeletal muscle induced by bone morphogenetic protein 4. METHODS:Both C2C12 cells and the myotubes formed by the C2C12 cells in the induction medium were cultured,and 500 ng/mL bone morphogenetic protein 4 was added to the medium respectively,and whether the C2C12 cells and myotubes continued to proliferate within 10 days under the treatment were observed under a microscope.Myogenic cells(L6,derived from rats)and fibroblast-derived cells(derived from human)were co-cultured.After treatment with 500 ng/mL bone morphogenetic protein 4 and 10 ng/mL transforming growth factor-β,osteogenic and chondrogenic differentiation potential within 21 days were observed using Safranine O staining and Alcian blue staining.Using transgenic animal FVB/N-TgN(TIE2-LacZ)182Sato mice,15 μL of adeno-associated virus-bone morphogenetic protein 4(5×1010 PFU/mL)were implanted in the thigh muscle space of genetic mice for 10 and 14 days.X-gal staining was used to observe the formation of new blood vessel endothelium in the differentiated bone. RESULTS AND CONCLUSION:(1)Bone morphogenetic protein 4 caused myotube breakdown and increased C2C12 cell proliferation.Compared with other groups,the pure fibroblast-derived cell group had a higher area of positive alcian blue and safarin O staining(P<0.05)and a lower area of alkaline phosphatase staining(P<0.05),while the pure L6 group had a bigger area of alkaline phosphatase staining(P<0.05)but a smaller area of positive alcian blue and safarin O staining(P<0.05).(2)Transplantation of adeno-associated virus-bone morphogenetic protein 4-adsorbed gelatin sponge into FVB/N-TgN(TIE2-LacZ)182Sato mice resulted in heterotopic ossification.(3)X-gal staining results demonstrated that there was no obvious staining in chondrocytes and differentiated bones and Tie2+ endothelial cells did not participate in the formation of the alienated bone.(4)These findings verify that fibroblasts are the primary source of osteoblasts during the adeno-associated virus-bone morphogenetic protein 4-induced ectopic endochondral ossification in skeletal muscle,but myogenic cells are the main source of osteoblasts.Tie2+ endothelial cells might not be the cell source for cartilage and bone.

Objective:To summarize and analyze the current application status of oral mucosal graft (OMG) technique in the repair of ureteral strictures in China, and clarify the feasibility, safety and effectiveness of this technique.Methods:The 175 patients who underwent repair of ureteral stricture using oral mucosal patches from June 2015 to February 2022 were etrospectively analyzed in 14 medical centers in China, including 49 cases in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 32 cases in Affiliated Seventh Medical Center of PLA General Hospital, 3 cases in The Second Hospital of Anhui Medical University, 6 cases in The First Affiliated Hospital of Zhengzhou University, 56 cases in Peking University First Hospital, 3 cases in Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 cases in Shanghai Sixth People' s Hospital, 4 cases in General Hospital of Estern Theater Command, 4 cases in Lanzhou University Second Hospital, 2 cases in Guizhou Province People 's Hospital, 2 cases in Peking University People' s Hospital, 5 cases in Jinzhou First People's Hospital, 5 cases in The First Affiliated Hospital of Wannan Medical College, 1 case in Shandong Provincial Hospital. In this study, 127 patients (72.6%) used lingual mucosal patches, 32(18.3%) labial mucosa, and 16(9.1%) buccal mucosa. The surgical approach for OMG ureteral reconstruction was mainly minimally invasive, with robot-assisted laparoscopy in 84 patients (48.0%), traditional laparoscopic surgery in 87 patients (49.7%), and open surgery in only 4 patients (2.3%). There were 133 males and 42 females with an average age of (35.0±17.2) years. The mean body mass index (BMI) and stenosis length were (23.1±4.1) kg/m 2 and (4.7±1.8) cm, respectively. The stricture was located in the left ureter in 116 patients, right ureter in 58 case and bilateral ureter in 1 case. The most common causes of ureteral stricture were endoscopic surgery in 88(50.3%)patients, congenital stricture in 55(31.4%)patients, failed ureteroplasty in 29(16.6%)patients, history of extracorporeal shock wave lithotripsy in 13(7.4%)patients, radiotherapy history in 3(1.7%)patients and other causes in 6(3.4%)patients. Strictures were mainly located in the upper ureter, accounting for 61.7% (108/175 cases), followed by 36.0% (63/175) at the ureteropelvic junction and 2.3%(4/175)in the middle ureter. According to the surgical methods, the patients were divided into robot-assisted laparoscopic surgery group ( n=84), traditional laparoscopic surgery group ( n=87)and open surgery group ( n=4). Subgroup analysis of patients in robot-assisted laparoscopic and traditional laparoscopic surgery groups was performed. There were no significant difference in preoperative data between the two groups except for age (32.0±18.3) years vs.(37.0±15.9)years, P=0.040], BMI[(22.5±4.3)kg/m 2 vs. (23.7±3.6)kg/m 2, P=0.028], and etiology of stenosis [endoscopic injury, 34(40.5%) vs. 53(60.9%), P=0.012]. Preoperative hydronephrosis and stricture length were assessed by CTU and ureterography. Ureterography 7-9 weeks after surgery showed patency of the reconstructed segment, or no recurrence of hydronephrosis was judged as success. Evaluate the operation method, operation time, success rate, length of OMG in repairing ureteral stricture between laparoscopic and robot-assisted groups. Results:The overall success rate of oral mucosal graft repair surgery reached 97.7%(171/175). The success rate of ureteral reconstruction in the two groups were 96.4%(81/84)and 98.9%(86/87), respectively ( P=0.351), and the difference was not statistically significant. There was no significant difference for operation time, intraoperative blood loss, and mean oral mucosal length between the robotic and laparoscopic groups[(244.7±85.8) min and (222.7±83.5)min ( P=0.116), (58.9±38.6) ml and (68.4±45.5) ml ( P=0.217), (5.0±2.0) cm and (4.6±1.5) cm ( P=0.350)], respectively.Postoperative complications were reported in 23 (13.1%) patients, such as fever, urinary leakage, lymphatic leakage, infection, but only 2 (1.4%) cases patients had complications of Clavien-Dindo score ≥ Ⅲ. The two patients developed urinary stricture after surgery with failed conservative treatment, and no urinary stricture occurred following endoscopic treatment.The short-term (three months after surgery)incidence of complications in the site where the oral mucosa was taken, such as difficulty in opening mouth, pain, and swelling, was 12.0% (21/175), and there was no significant difference for oral complications between patients harvesting different length of mucosal graft. Conclusions:Ureteroplasty with oral mucosal graft is a safe, feasible and reliable technique for ureteral reconstruction. At present, minimally invasive technology is the main surgical approach for ureteroplasty, and there is no significant difference in operation time and success rate between robotic surgery and laparoscopic surgery.

Objective:To investigate the clinical treatment of large segmental humeral defects with unilateral external fixation and bone transport.Methods:A retrospective study was conducted of the 9 patients who had been treated at Department of Orthopedics, Shenzhen People's Hospital for large segmental humeral defects from September 2017 to June 2019. They were 5 males and 4 females with an average age of 29 years (from 21 to 41 years). Their defects were caused by trauma in 2 cases, by chronic osteomyelitis in 6 cases and by bone tumor in one case. The length of bone defect ranged from 4.2 to 9.0 cm, with an average of 5.9 cm. A unilateral external fixator was placed in operation, and adjusted regularly 7 to 10 days after operation for bone transport and bone lengthening to restore the length of humerus gradually. The external fixation bracket was removed after 3 to 4 layers of cortex were observed on X-ray films. Recorded were length and rate of humeral lengthening, fracture healing time, time for carrying external fixator and complications; the Disabilities of the Arm, Shoulder and Hand (DASH) scores were compared between preoperation and 15 months postoperation.Results:All the patients were followed up for 15 to 36 months (mean, 19 months). The length of lengthening averaged 5.9 cm (from 4.2 to 9.0 cm) with an average lengthening rate of 26%, the healing index 31 d/cm, the bone healing time 8.3 months, and the time for carrying external fixator 10.8 months(from 8.0 to 13.5 months). Their average DASH scores improved significantly from 25.0 ± 2.4 preoperation to 12.0 ± 1.8 at 15 months postoperation ( P<0.05). Good correction of large humeral defects was achieved in all but one case who reported temporary radial nerve paralysis. There were no such complications as neurovascular injury. The shoulder and elbow functions were basically normal after operation. Conclusions:In the treatment of large segmental humeral defects, unilateral external fixation plus bone transport can quickly repair the defects and recover the upper limb function of the patients.

Objective:To explore the efficacy of different unipolar electrocoagulation power on pathological injury of porcine kidney suffering suture-free partial nephrectomy (SFPN).Methods:From April 2018 to July 2018, nine Guizhou pigs were selected, with an average age of 3 years and an average weight of 48 kg. According to different hemostatic power of unipolar electrocoagulation during open partial nephrectomy, they were divided into three groups(60W group, 80W group, and 100W group), with 3 in each group. The left kidney was exposed with a surgical incision, parallel to the lumbosacral muscle.The left renal artery was clamped and about 2 cm renal tissue was excised at the middle pole of the left kidney. 60W, 80W and 100W were used by unipolar electrocoagulation for hemostasis until no bleeding occurred after the artery clamp was released. The total ischemia time was controlled within 20 min. Temperature was measured by a multi-channel thermometer probe which was inserted into the healthy kidney tissue at a distance of 2 mm, 5 mm, and 10 mm away from the unipolar electrocoagulation hook, and the upper pole of the kidney far away from the operation area. The time of operation, the volume of renal bleeding, the time of hemostasis and the temperature were recorded. On the 7th day after operation, the left kidneys were taken and the pathological changes were observed by toluidine blue staining.Results:All operations were completed safely and successfully. The operation time in 60W group, 80W group, and 100W group was (41.2±5.5)min, (35.1±3.7)min, (31.3±2.2)min , respectively. There was no significant difference of operation time among those group ( P>0.05). The blood loss of renal was (35.3±4.1)ml, (21.4±4.7)ml, (15.3±4.1)ml, respectively. The blood loss in the 100W group and 80W group was less than that in the 60W group ( P<0.05). And the blood loss in the 100W group was less than that in the 80W group ( P<0.05). The hemostasis time was (15.2±1.9)min, (10.1±1.4)min, (6.4±0.8)min. The hemostasis time in the 100W and 80W groups was less than that in the 60W group ( P<0.05). And the hemostasis time in the 100W group was less than that in the 80W group ( P<0.05). At the place of 10 mm away from the electrocoagulation hook, the temperature in the three groups were (33.1±1.1)℃, (34.0±1.0)℃, (34.3±0.6)℃, which was not significantly different from that of the respective upper poles. And there was no significant difference between the three groups( P>0.05). At the place of 5 mm and 2 mm away from the electrocoagulation hook, the temperature in the 100W group (41.7±1.3)℃, (61.4±6.4)℃ and the 80W group (38.6±2.4)℃, (50.3±6.0)℃ was higher than that in the 60W group (36.9±4.1)℃, (42.0±4.7)℃, and the temperature in 100W group is higher than that in 80W group ( P<0.05). When the power was 60W, 80W or 100W, the temperature in the place 10 mm away from the electrocoagulation hook was less than that in the place 5 mm away from the electrocoagulation hook ( P<0.05), and the temperature of the place 5 mm away from the electrocoagulation hook was lower than that of the place 2 mm away from the electrocoagulation hook ( P<0.05). The total pathological injury depth of wounds in 60W, 80W, 100W group was (7 323±50)μm, (8 119±100)μm, (8 896±40)μm, respectively. The depth in 100W group and 80W group was deeper than that in 60W group ( P<0.05), and the depth in 100W group was deeper than that in 80W group ( P<0.05). Conclusions:In SFPN, the hemostatic effect of three different monopolar electrocoagulation output power is satisfactory. With the increase of power, the hemostasis speed is faster. However, the temperature of surrounding healthy renal tissue would be higher, and the total pathological injury depth would be deeper.

BACKGROUND: Osteoarthritis is a common cause of pain and disability in the elderly. The underlying cause is the combination of inappropriate mechanical stress, inflammatory mediators and biochemical factors. Curcumin has been shown to have anti-oxidant, anti-inflammatory, anti-bacterial, anti-tumor, neuroprotective, and cardioprotective effects, radiation protection and therapeutic effects on osteoarthritis. Similarly, magnesium sulfate can relieve joint pain and inhibit joint destruction. OBJECTIVE: To investigate the mechanism by which the combined use of curcumin and magnesium sulfate exerts synergistic effect to promote inflammatory chondrocyte reverse differentiation.METHODS: Primary cultured inflammatory chondrocytes were subjected to monolayer culture in vivo. Cell proliferation assay (MTS) was used to detect the proliferation of inflammatory chondrocytes under in vitro monolayer culture conditions. The experimental cells were divided into four groups and underwent 3D induced reverse differentiation culture for 18 days: single culture of chondrocytes (chondrocyte group) , inflammatory chondrocyte cultured with curcumin (curcumin group) , inflammatory chondrocytes cultured with magnesium sulfate (magnesium sulfate group) , and inflammatory chondrocytes cultured with curcumin and magnesium sulfate (combination group). RESULTS AND CONCLUSION: MTS proliferation experiments showed that inflammatory chondrocytes at passage 3 had a higher rate of early proliferation and a lower degree of differentiation. Quantitative PCR results showed that the mRNA levels of type II collagen, proteoglycan and SOX9 were significantly higher in the combination group than in the curcumin group or magnesium sulfate group (P < 0.01). The size of the gross specimens and the positive area of chondrocyte reverse differentiation for alcian blue staining and safranin O staining in the combination group were significantly larger than those in the other three groups (P < 0.01). TUNEL staining results indicated that the positive area of apoptosis-specific staining in the combination group and magnesium sulfate group was significantly smaller than that in the other two groups (P < 0.01). Therefore, the combined use of curcumin and magnesium sulfate has the synergistic effect to promote the reverse differentiation of inflammatory chondrocytes.

Objective To establish a prostate urethral re-epithelialization model with Chinese rural canine by 2 μm laser vaporization resection.Methods We used 2 μm laser to vaporiz prostate of 15 uncastrated male Chinese rural canines from March to April in 2016.These canines mean age was (6.3 ± 0.6) years(ranging 5-7 years),and weight was (20.5 ± 1.3) kg(ranging 18-22 kg).We began to surgery in which we saw the protruding part of the prostate in urethra,and narrow prostate urethra after a successful anesthesia by intraperitoneal injection of chloral hydrate.The operation time,anesthesia time,survival rate,first time to drink water,first time to feed,first time to stand,first time to defecate,the time when canine bladders rinse became clear,wound healing time were recorded.After 3 days,1 week,2 weeks,3 weeks and 4 weeks,we randomly select 3 canines to observe regeneration of prostate urethra wound under cystoscope.After surgery,the bladder,prostate and prostate distale urethra were removed to make specimen and measure the diameter size of prostate.The HE staining and immunohistochemistry was performed in each sample.Results The experimental operation time was (70.5 ± 18.3) min (ramging 50-90 min).The average anesthesia time was (120.1 ± 21.1) min (ranging 95-145 min).The survival rate was 100％.In post surgery duration first standing time,first eating time,first drinking water time,first defecation time were (6.5 ± 1.8) h,(10.3 ± 2.1) h,(23.7 ± 5.6) h,(26.3 ± 3.1) h,respectively.The time when canine bladders rinse became clear and wound healing time were (5.2 ± 1.6) d,(8.7 ± 1.5) d respectively.Cystoscopy observated that the wound was covered by pale necrotic tissue 3 d and 1 week after operation,covered by epithelium 2 weeks after operation,covered by more thicker epithelium 3 weeks after operation,covered by epithelium which color was close to normal urothelium 4 weeks after operation.HE staining observated that the wound wasn't covered by epithelium 3 d after operation,partial wound was covered by flaky single or 2-3 cubic regenerated epithelial 1 week after operation,all wound was covered by epithelial which was lack of polar 2 weeks after operation,wound was covered by polarity epithelium which was thicken to 5-6 layer and observated a little umbrella cells on the surface 3 weeks after operation,wound was covered by polarity epithelium which was thicken to 5-6 layer and observated much umbrella cells on the surface 4 weeks after operation.Immunohistochemical staining observated that urinary spot protein from the wound or epithelium was negative 3 d,1 and 2 weeks after operation,urinary spot protein from the part of epithelium was positive 3 weeks after operation,and urinary spot protein from all epithelium was positive 4 weeks after operation.Conclusion It is feasible to establish prostate urethral re-epithelialization model in the Chinese rural canine by 2 μm laser vaporization resection of the prostate.

Objective To investigate the protective effect of thyroid hormone on spinal cord injury neurons and its molecular mechanism.Methods A DMEM culture medium with a volume fraction of 10％ fetal bovine serum was cultured with the dorsal ridge neurons of RN-dsc rats.The neurons were inoculated in the culture plate after the digestion of trypsin and treated differently as follows:(1) control group:DMEM treatment with no drugs or serum;(2) H2O2 group:serum-free DMEMtreatment containing 100 μmol/L H2O2;(3) H2O2 + 10-6 mol/L triiodothyronine (T3) group:serumfree DMEM treatment containing 100 μnol/L H2O2 and 10-6mol/L T3;(4) H2O2 + 10-5 mol/L T3 group:serum-free DMEM treatment containing 100 μ mol/L H2O2 and 10-5 mol/L T3;(5) negativecontrol group:transfection of negative control mimics with LipofectamineTM2000 reagent;(6) miR-210 group:transfection of miR-210 mimics with LipofectamineTM 2000 reagent.Cell viability,apoptosis number,and expressions of nuclear factor E2 correlation factor 2 (Nrf-2) antioxidant pathway molecules and miR-210 were determined.After transfection of miR-210 mimics and negative control mimics,expressions of Nrf-2 antioxidant pathway molecules were determined.Results The cell proliferation activity and protein expressions of Nrf-2,antioxidant reaction elements (ARE),superoxide dismutase 2 (SOD2),and heme oxygenase (HO-1) in H2O2 group (0.39 ±0.06,0.52 ±0.08,0.31 ±0.08,0.25 ± 0.05,respectively) were significantly lower than those in control group (1.00 ± 0.15,1.00 ± 0.17,1.00 ± 0.13,1.00 ± 0.11,respectively) (P ＜ 0.05),while the apoptosis numbers and the expressions of miR-210 were significantly higher than those in control group (P ＜ 0.05).The cell proliferation activity and protein expressions of Nff-2,ARE,SOD2,HO-1 in H2O2 + 10-6mol/L T3 group and H2O2 +10-5 mol/L T3 group were significantly higher than those in the H2O2 group (P ＜ 0.05),while apoptosis numbers and expressions of miR-210 were significantly lower than those in H2O2 group (P ＜ 0.05).The cell proliferation activity and protein expressions of Nrf-2,ARE,SOD2,HO-1 in H2O2 + 10-5mol/L T3 group (0.88 ±0.14,0.84 ±0.12,0.72 ±0.09,0.69 ±0.09) were significantly higher than those in H2O2 + 10-6mol/L T3 group (0.73 ±0.09,0.71 ±0.08,0.58 ±0.09,0.52 ±0.08) (P＜0.05),while apoptosis numbers and expressions of miR-210 were significantly lower than those in H2O2 + 10-6mol/L T3 group.The protein expressions of Nrf-2,ARE,SOD2,and HO-1 in miR-210 group (0.37 ±0.06,0.24 ±0.05,0.45 ± 0.08,0.49 ± 0.07,respectively) were significantly lower than those in negative control group (1.00±0.13,1.00±0.19,1.00±0.15,1.00±0.14,respectively) (P＜0.05).Conclusion Thyroid hormone can inhibit the expression of Nrf-2 in oxidative stress injury process of neurons by inhibiting the expression of miR-210,and hence reduce the oxidative stress injury of spinal cord neurons.

BACKGROUND:Mesenchymal stem cells from human skeletal muscle exhibit multi-directional differentiation potential under the influence of osteogenic proteins such as bone morphogenetic protein 4 (BMP4). But the differentiation of a specific cell subpopulation is not yet clear.OBJECTIVE:To characterize the multi-directional differentiation potential of mesenchymal stem cells from human skeletal muscle based on the expression of different surface markers.METHODS:Four different subpopulations were isolated from the human skeletal muscle by fluorescence-activated cell sorting based on their expression of the myogenic-specific marker CD56 and the endothelial-specific markers CD34 and CD144, including CD56+, CD56+CD34+CD144+, CD34+CD144+, and unsorted groups. Osteogenic differentiation of the four groups of the cells was displayed by Von Kossa staining after the treatment with BMP4 alone or BMP4 plus transforming growth factor β3. Chondrogenic differentiation of these cells was displayed by Alcian blue staining. Bone metabolism was assessed by alkaline phosphatase staining.RESULTS AND CONCLUSION:No significant difference in the bone metabolism was found among four groups after the treatment with BMP4 (P > 0.05). Osteogenic and chondrogenic potentials of the four cell subpopulations were significantly different. Under the same osteogenic induction, the CD56+ cells exhibited strongest potential for osteogenic differentiation; and under the same chondrogenic induction, the CD56+CD34+CD144+ cells exhibited better potential for chondrogenic differentiation than the CD56+ cells. These findings indicate that the osteogenic and chondrogenic potentials are intimately associated with the type of mesenchymal stem cells from human skeletal muscle:the CD56+ cells are closely related to the osteogenic potential, while the CD56+CD34+CD144+ cells have stronger chondrogenic potential.