ObjectiveTo study the effect of transforming growth factor β3 (TGF-β3), bone morphogenetic protein 2 (BMP-2), and dexamethasone (DEX) on the chondrogenic differentiation of rabbit synovial mesenchymal stem cells (SMSCs). MethodsSMSCs were isolated from the knee joints of 5 rabbits (weighing, 1.8-2.5 kg), and were identified by morphogenetic observation, flow cytometry detection for cell surface antigen, and adipogenic and osteogenic differentiations. The SMSCs were cultured in the PELLET system for chondrogenic differentiation. The cell pellets were divided into 8 groups: TGF-β3 was added in group A, BMP-2 in group B, DEX in group C, TGF-β3+BMP-2 in group C, TGF-β3+DEX in group E, BMP-2+DEX in group F, and TGF-β3+BMP-2+DEX in group G; group H served as control group. The diameter, weight, collagen type II (immuohistochemistry staining), proteoglycan (toluidine blue staining), and expression of cartilage related genes [real time quantitative PCR (RT-qPCR) technique] were compared to evaluate the effect of cytokines on the chondrogenic differentiation of SMSCs. Meanwhile, the DNA content of cell pellets was tested to assess the relationship between the increase weight of cell pellets and the cell proliferation. ResultsSMSCs were isolated from the knee joints of rabbits successfully and the findings indicated that the rabbit synovium-derived cells had characteristics of mesenchymal stem cells. The diameter, weight, collagen type II, proteoglycan, and expression of cartilage related genes of pellets in groups A-F were significantly lower than those of group G (P<0.05). RT-qPCR detection results showed that the relative expressions of cartilage related genes (SOX-9, Aggrecan, collagen type II, collagen type X, and BMP receptor II) in group G were significantly higher than those in the other groups (P<0.01). Meanwhile, with the increase of the volume of pellet, the DNA content reduced about 70% at 7 days, about 80% at 14 days, and about 88% at 21 days. ConclusionThe combination of TGF-β3, BMP-2, and DEX can make the capacity of chondrogenesis of SMSCs maximized. The increase of the pellet volume is caused by the extracellular matrix rather than by cell proliferation.
To study the method of isolating and culturing synovium-derived MSCs (SMSCs), and to investigate its multiple differentiation potential in vitro. Methods Three 2-month-old Changfeng hybrid swines weighing 8-10 kg (male and female) were used. SMSCs were harvested from the synovium of swine knee joints and cultured in vitro. When the SMSCs at passage 3 reached confluence, basic culture medium was removed, and the multi ple differentiationpotential of SMSCs was demonstrated in specific induction media (experimental group). The cells at passage 3 cultured with basic culture medium served as control group. After 21 days of chondrogenic differentiation, the cells underwent toluidine blue staining, immunohistochemistry staining and real-time fluorescence quantitative PCR detection. After 10 and 21 days of osteogenic differentiation, the cells underwent ALP staining and Al izarin red staining, respectively. After 21 days of adipogenic differentiation, the cells underwent Oil red O staining. Results SMSCs displayed long and thin or polygonal morphology 24 hours after culture. They prol iferated fast 48 hours after culture and presented large number of spindle-shaped cells with few globular cells 72 hours after culture. For the experimental group 21 days after chondrogenic induction, the cells were positive for toluidine blue staining with the formation of Aggrecan outside the cells; the immunohistochemistry staining revealed the expression of Col II; the real-time fluorescence quantitative PCR detection showed that the expressions of Col II A1, Aggrecan and SOX9 mRNA of the experimental group were greater than that of control group (P lt; 0.05). The cells were positive for ALP staining 10 days after osteogenic induction, and positive for Al izarin red staining 21 days after osteogenic induction, with the formation of calcium nodules. Oil red O staining displayed the formation of l i pid droplets inside the cells 21 days after adi pogenic induction. For the control group, the results of all the staining assays were negative except the ALP staining presenting with sl ight positive result. Conclusion SMSCs can be isolated from knee joint of swine and proliferate and differentiate into osteogenic, adi pogenic and chondrogenic cells in vitro. SMSCs may be a promising source of seed cells for tissue engineering.
ObjectivesTo investigate the ultrasound findings of the synovial hemangioma of knee (SHK) and to evaluate its value in clinical diagnosis.MethodsThe ultrasonographic manifestations and clinical data of 10 patients with SHK confirmed by surgery and pathology were retrospectively analyzed and compared with MRI findings, surgery and pathological results.ResultsSeven cases of SHK (6 cases of diffuse type, 1 case of limited type) were assessed by ultrasound, including 1 case of vascular origin, 1 case of supraorbital sac origin, 1 case with pigmented villonodular synovitis, 1 case with thrombosis, 2 cases accompanied with bone erosion and osteophyte formation, and 3 cases with joint cavity effusion. Ultrasonic findings of SHK were as followed: 7 cases of SHK were manifestate as diffuse mass with unclear boundary, irregular shape and uneven echo mass; 5 cases had mixed-echo mass with reticular structures inside, an increased volume in erect position and positive CDFI compression test; 1 case had heterogeneous hypoechoic mass with a nodular appearance and the positive compression test; 1 case as poorly-demarcated, irregular shape, heterogeneous hyperechoic mass without obvious blood flow signals under the compression test. There were no characteristic ultrasonic findings from other 3 cases of SHK.ConclusionsDiffuse SHKs have characteristic ultrasonograms. SHKs with localized and significant synovial hyperplasia have no specific ultrasonic manifestation and are easily misdiagnosed. Ultrasound is convenient, noninvasive and inexpensive. It can accurately evaluate the involvement of knee joint capsule and surrounding soft tissues. It can be used as the first line diagnostic modality for routine scanning of SHKs.
Objective To investigate the method and the effectiveness of arthroscopy and/or arthrotomy combinedwith postoperative radiotherapy for diffuse pigmented villonodular synovitis (PVNS) of the knee. Methods BetweenSeptember 2000 and August 2010, 97 patients with diffuse PVNS of the knee were treated. There were 38 males and 59 femaleswith a median age of 33 years (range, 8-75 years). The disease duration ranged from 1 week to 30 years, including 52 left kneesand 45 right knees. There were 10 recurrent cases. The extention and flexion of the knee joint were (1.9 ± 2.3)° and (122.9 ± 5.6)°,respectively; the Lysholm score was 43.2 ± 6.7; and the International Knee Documentation Committee (IKDC) score was53.2 ± 5.7, preoperatively. According to the scope and degree of the knee joint lesions, simultaneous anterior and posteriorsynovectomy was performed under arthroscopy in 82 cases, synovectomy under arthroscopy and removal of posterior extraarticularlesion by arthrotomy in 3 cases, synovectomy and the soft tissue lesions resection under arthroscopy in 9 cases, andstaging resection and bone graft in 3 cases. After operation, 76 patients received postoperative radiotherapy. Results Poplitealartery was injuryed in 1 case and the branch of popl iteal veins were injuryed in 3 cases during operation. Intra-articularhemorrhage occurred in 1 case at 3 days after operation. The other patients achieved heal ing of incision by first intentionwithout nerve damage and other complications. All patients were followed up 1 year and 3 months to 11 years and 2 months(median, 61 months) postoperatively. During follow-up, 89 cases had no relapse. At 15 months after operation, the extentionand flexion of the knee joint were (0.2 ± 1.3)° and (135.9 ± 6.6)°, respectively; the Lysholm score was 89.8 ± 5.8; and the IKDCscore was 87.8 ± 5.8. All indexes were significantly improved when compared with the preoperative ones (P lt; 0.05). At 6 monthsto 8 years postoperatively, 8 cases had occurrence, and they had sl ight limitation of the range of motion but had no pain andswelling of the knees after reoperation. Conclusion According to the scope and degree of the knee joint lesions, arthroscopyand/or arthrotomy combined with postoperative radiotherapy should be chosen for diffuse PVNS of the knee so as to obtain good effectiveness. Radiotherapy and enough total radiation dose are important factors to insure no recurrence.
ObjectiveTo investigate the role of CXCL13 in the onset and development of knee osteoarthritis by observing and comparing the expression of CXCL13 between osteoarthritis and normal synovium. MethodsThe synovium samples were collected from 30 patients with osteoarthritis who received total knee replacement (osteoarthritis group), including 11 males and 19 females with an average age of 66.7 years (range, 62-76 years). The synovium samples were collected from 22 patients without osteoarthritis who underwent traumatic amputation (control group), including 15 males and 7 females with an average age of 51.3 years (range, 48-56 years). The NimbleGen microarray detection was used to defect differentially expressed genes; the immunohistochemistry staining, Western blot, and real-time quantitative PCR (qRT-PCR) were used to detect the expressions of CXCL13 mRNA and protein. ResultsThere were 451 up-regulated genes and 810 down-regulated genes in the 22 885 genes which contained by mRNA gene chip, and CXCL13 gene expression was down-regulated. Immunohistochemistry staining and Western blot assay showed that the expression of CXCL13 protein was significantly lower in osteoarthritis group (0.408 0±0.101 8) than in control group (0.785 9±0.057 9) (t=15.630, P=0.000). qRT-PCR results showed that the expression of CXCL13 mRNA was significantly lower in osteoarthritis group (0.011 7±0.003 2) than in control group (1.041 4±0.129 7) (t=43.634, P=0.000). ConclusionLow expression of CXCL13 in the knee osteoarthritis synovium tissue may be associated with the onset and development of knee osteoarthritis.
Objective To explore the technique of arthroscopic treatment of synovial chondromatosis of the hip and to evaluate its effectiveness. Methods Between July 2009 and June 2011, 15 patients with synovial chondromatosis of the hip underwent arthroscopic synovectomy and removal of loose bodier. Of 15 patients, 11 were male and 4 were female, aged from 21 to 45 years with an average of 33.1 years. The location was the left side in 6 cases and the right side in 9 cases. The disease duration was 12-43 months (mean, 23 months) Pain and functional motion limitation were the main clinical symptoms. The visual analogue scale (VAS) score was 5.8 ± 1.1; the range of motion (ROM) of the hip was (149.8 ± 27.5)°; the Harris hip score was 54.5 ± 13.3. Results All incisions healed by first intention. All the patients were followed up 6 months to 2 years (mean, 17.4 months). At last follow-up, the VAS score was 2.0 ± 1.2; the ROM of the hip was (258.3 ± 35.4)°; the Harris hip score was 93.0 ± 18.7; and the above indexes were significantly improved when compared with preoperative values (P lt; 0.05). No recurrence was found on postoperative MRI. Conclusion Arthroscopic treatment of synovial chondromatosis of the hip has the advantages of minimal invasion, quick recovery, and best recovery of hip function and ROM.
Objective To study the effects of the periosteum，synovium andcartilage tissues on the gene expressions of proteoglycan, collagen Ⅱ, andnuclear factor kappa B (NF-κB) and to investigate the different effects of these tissues on cartilage regeneration. Methods In 20 New Zealand white rabbits, 20 cartilage explants were taken from the knee joints in each rabbit, the sizeof which was 4 mm×4 mm×4 mm. All the cartilages were divided into the following 4 groups and cultured for 7 days: Group A, with 5 pieces (2 mm×2 mm) of the synovium of theknee joints in each dish; Group B, with 5 pieces (2 mm×2 mm) of the periosteum ineach dish; Group C, with 5 pieces (2 mm×2 mm×2 mm) of the cartilage in each dish; and Group D, with no addition of other tissues (control group). RNA was extracted from the cells of the cartilage explants (4 mm×4 mm×4 mm) in all the dishes. Thegene expressions of proteoglycan, collagen Ⅱ and NF-κB were defected by a reversetranscription-polymerase chain reaction (RT-PCR).Results In group A, the gene expression of proteoglycan was significantly decreased. The relative density of this gene expression had a significant difference when compared with that in group D (1.09±0.21 vs. 1.25±0.25, Plt;0.05); the gene expressions of collagen Ⅱ and NF-κB were also decreased, but they had no significant differences when compared with those in group D (Pgt;0.05). In groupB, the gene expressions of proteoglycan, collagen Ⅱ, and NF-κB were significantly increased. The relative densities of these gene expressions were 1.60±0.26, 1.57±0.24, and 4.20±2.22, respectively, which had significant differences when compared with those in group D (Plt;0.05). In group C, the relative density of the gene expression of collagen Ⅱ was 1.43±0.28, which had a significant difference when compared with that in group D (Plt;0.05), but therelative densities of the gene expressions of proteoglycan and NF-κB had no significant differences when compared with those in group D (Pgt;0.05). Conclusion The results indicate that the periosteum can up-regulate the gene expressions of proteoglycan, collagen Ⅱ and NF-κB. The NF-κB is likely to be an important nuclear transcription factor related to cartilage regeneration. The results also suggest that the periosteum maybe better in facilitating the cartilage repair and regeneration in clinical practice.
ObjectiveTo explore the pathological role of high mobility group box chromosomal protein 1 (HMGB1) in osteoarthritis (OA) by comparing the difference of HMGB1 in the synoviocytes between OA and normal knees. MethodsSynoviocyte lines from OA and normal knees were collected and cultured. Immunohistochemistry and Western blot were applied to identify the difference of HMGB1 between the OA and normal synoviocyte lines. The eukaryotic expression vector containing human Pgenesil-1/HMGB1 small interfering RNA (siRNA) were constructed and identified. The synoviocyte lines were transfected with the eukaryotic expression vector of Pgenesil-1/HMGB1 siRNA (Pgenesil-1/HMGB1 siRNA group) and with Pgenesil-1 plasmid (Pgenesil-1 group) and were not transfected as a control (untransfected group). Western blot was applied to identify the difference of HMGB1 among groups, and the levels of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) protein synthesis in the supernatants were measured by ELISA. ResultsPrimary knee synoviocytes cultured in vitro were fibroblast-like cells with longspindle shape. The immunohistochemistry and immunofluorescence results showed positive staining for HMGB1 in cytoplasm and weak positive staining in the nucleus in the OA synoviocyte line, but positive staining for HMGB1 in the nucleus and weak positive staining in the cytoplasm in the synoviocyte line of normal knee. The level of HMGB1 in the OA synoviocytes (0.687±0.025) was significantly higher than that of normal synoviocytes (0.172±0.030) (t=32.159, P=0.000) by Western blot. The recombinant plasmid Pgenesil-1/HMGB1 siRNA was successfully constructed. The expression of HMGB1 protein in Pgenesil-1/HMGB1 siRNA group (0.134±0.048) was significantly lower than that of Pgenesil-1 group (0.581±0.032) and untransfected group (0.514±0.069) (P<0.05). ELISA results showed that IL-1β and TNF-α in supernatants of Pgenesil-1/HMGB1 siRNA group were significantly lower than those of Pgenesil-1 group and untransfected group (P<0.05). ConclusionThe up-regulated expression and expressed location (from nucleus to cytoplasm) of HMGB1 in the synoviocyte are closely related to OA. The siRNA targeting inhibition of HMGB1 gene expression can obviously inhibit IL-1β and TNF-α in supernatants of the OA synoviocyte line and delayed the inflammation of OA.