Background Osteoarthritis （OA） is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains ...Background Osteoarthritis （OA） is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 （IL-1） and tumor necrosis factor- α （TNF- α ） are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist （IL-1Ra） and soluble tumor necrosis factor- α receptor type Ⅰ （sTNF-RI） by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis. Methods Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 × 10^8 plaque-forming units （pfu） of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 × 10^8 pfu of adenovirus encoding either IL-1Ra （3 rabbits）, sTNF-RI （3 rabbits） or IL-1Ra and sTNF-RI in combination （3 rabbits）, into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 × 10^8 pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin （cartilage and synovium） and toluidine blue （cartilage）. The samples were examined by light microscopy and quantitatively evaluated. Results Intra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF- α blockade has little effect on joint destruction. Conclusion The enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.展开更多
Background Remodeling of the anterior cruciate ligament （ACL） graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal riboso...Background Remodeling of the anterior cruciate ligament （ACL） graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site （IRES） sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 （TGFβ1） and vascular endothelial growth factor 165 （VEGF165） genes （named Ad-VEGF165-IRES-TGFβ1）. We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts. Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR. Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβ1 and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers. Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.展开更多
Background Proprioception plays an important role in knee movements. Since there are controversies surrounding the overall recovery time of proprioception following surgery, it is necessary to define the factors affec...Background Proprioception plays an important role in knee movements. Since there are controversies surrounding the overall recovery time of proprioception following surgery, it is necessary to define the factors affecting proprioceptive recovery after anterior cruciate ligament （ACL） reconstruction and to investigate the relationship between proprioception and muscle strength. Methods A total of 36 patients who had their ACL reconstructed with a semitendinosus/gracilis graft （reconstructed group： 6 months post-surgery） and 13 healthy adults without any knee injury （control group） were included in the study. Knee proprioception was evaluated with a passive reproduction test. Isokinetic strength was measured using the Biodex System. Statistical analysis was used to compare proprioception of the reconstructed group versus the control group, and to define causal factors, including sex, hamstring/quadriceps ratio, and the course of injury before reconstruction, We also investigated the correlation between the passive reproduction error and quadriceps index. Results There was a significant difference in proprioception between the reconstructed and control groups （P 〈0.05）. When the course of injury before reconstruction was less than 4 months, there was a linear correlation with proprioception 6 months after the operation （r=0.713, P 〈0.05）. There was a positive correlation between post-surgery proprioception and the quadriceps index at 6 months post-surgery. Conclusions Impaired knee proprioception is observed 6 months after ACL reconstruction. Within 4 months of injury, early undertaking of reconstruction is associated with better proprioception outcome. Patients with enhanced proprioception have a better quadriceps index.展开更多
Background In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a n...Background In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Methods Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum （FBS） medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype （re-differentiation） and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining. Results Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium. Conclusion These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.展开更多
文摘Background Osteoarthritis （OA） is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 （IL-1） and tumor necrosis factor- α （TNF- α ） are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist （IL-1Ra） and soluble tumor necrosis factor- α receptor type Ⅰ （sTNF-RI） by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis. Methods Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 × 10^8 plaque-forming units （pfu） of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 × 10^8 pfu of adenovirus encoding either IL-1Ra （3 rabbits）, sTNF-RI （3 rabbits） or IL-1Ra and sTNF-RI in combination （3 rabbits）, into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 × 10^8 pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin （cartilage and synovium） and toluidine blue （cartilage）. The samples were examined by light microscopy and quantitatively evaluated. Results Intra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF- α blockade has little effect on joint destruction. Conclusion The enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.
文摘Background Remodeling of the anterior cruciate ligament （ACL） graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site （IRES） sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 （TGFβ1） and vascular endothelial growth factor 165 （VEGF165） genes （named Ad-VEGF165-IRES-TGFβ1）. We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts. Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR. Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβ1 and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers. Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.
文摘Background Proprioception plays an important role in knee movements. Since there are controversies surrounding the overall recovery time of proprioception following surgery, it is necessary to define the factors affecting proprioceptive recovery after anterior cruciate ligament （ACL） reconstruction and to investigate the relationship between proprioception and muscle strength. Methods A total of 36 patients who had their ACL reconstructed with a semitendinosus/gracilis graft （reconstructed group： 6 months post-surgery） and 13 healthy adults without any knee injury （control group） were included in the study. Knee proprioception was evaluated with a passive reproduction test. Isokinetic strength was measured using the Biodex System. Statistical analysis was used to compare proprioception of the reconstructed group versus the control group, and to define causal factors, including sex, hamstring/quadriceps ratio, and the course of injury before reconstruction, We also investigated the correlation between the passive reproduction error and quadriceps index. Results There was a significant difference in proprioception between the reconstructed and control groups （P 〈0.05）. When the course of injury before reconstruction was less than 4 months, there was a linear correlation with proprioception 6 months after the operation （r=0.713, P 〈0.05）. There was a positive correlation between post-surgery proprioception and the quadriceps index at 6 months post-surgery. Conclusions Impaired knee proprioception is observed 6 months after ACL reconstruction. Within 4 months of injury, early undertaking of reconstruction is associated with better proprioception outcome. Patients with enhanced proprioception have a better quadriceps index.
文摘Background In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Methods Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum （FBS） medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype （re-differentiation） and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining. Results Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium. Conclusion These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.
