Expression of human bone morphogenic protein 7 in primary rabbit periosteal cells: potential utility in gene therapy for osteochondral repair.
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A commonly encountered problem in orthopedics is bone and cartilage tissue injury which heals incompletely or without full structural integrity. This necessitates development of improved methods for treatment of injuries which are not amenable to treatment using current therapies. An already large and growing number of growth factors which play significant roles in bone remodeling and repair have been identified in the past few years. It is well established that bone morphogenic proteins induce the production of new bone and cartilage. An efficient method of delivery of these growth factors by conventional pharmacological means has yet to be elucidated. We wished to evaluate the use of retroviral vector-mediated gene transfer to deliver genes of therapeutic relevance for bone and cartilage repair. To determine the feasibility of using amphotropically packaged retroviral vectors to transduce primary rabbit mesenchymal stem cells of periosteal origin, primary periosteal cells were isolated from New Zealand white rabbits, transduced in vitro with a retroviral vector bearing both the nuclear localized lacZ marker gene and the neo(r) gene, and selected in G418. We used a convenient model for analysis of in vivo stability of these cells which were seeded on to polymer scaffold grafts and implanted into rabbit femoral osteochondral defects. The nuclear localized beta-galactosidase protein was expressed in essentially 100% of selected cells in vitro and was observed in the experimental explants from animals after both 4 and 8 weeks in vivo, while cells transduced with a retroviral vector bearing only the neo(r) gene in negative control explants showed no blue staining. We extended our study by delivering a gene of therapeutic relevance, human bone morphogenic protein 7 (hBMP-7), to primary periosteal cells via retroviral vector. The hBMP-7 gene was cloned from human kidney 293 cell total RNA by RT-PCR into a retroviral vector under control of the CMV enhancer/promoter. Hydroxyapatite secretion, presumably caused by overexpression of hBMP-7, was observed on the surface of the transduced and selected periosteal cells, however, this level of expression was toxic to both PA317 producer and primary periosteal cells. Subsequently, the strong CMV enhancer/promoter driving the hBMP-7 gene was replaced in the retroviral vector by a weaker enhancer/promoter from the rat beta-actin gene. Nontoxic levels of expression of hBMP-7 were confirmed at both the RNA and protein levels in PA317 producer and primary periosteal cell lines and cell supernatants. This work demonstrates the feasibility of using a gene therapy approach in attempts to promote bone and cartilage tissue repair using gene-modified periosteal cells on grafts. (+info)
Arthroscopic findings in acute fractures of the ankle.
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We have evaluated prospectively the arthroscopic findings in acute fractures of the ankle in 288 consecutive patients (148 men and 140 women) with a mean age of 45.6 years. According to the AO-Danis-Weber classification there were 14 type-A fractures, 198 type B and 76 type C. Lesions of the cartilage were found in 228 ankles (79.2%), more often on the talus (69.4%) than on the distal tibia (45.8%), the fibula (45.1%), or the medial malleolus (41.3%). There were more lesions in men than in women and in general they were more severe in men (p < 0.05). They also tended to be worse in patients under 30 years and in those over 60 years of age. The frequency and severity of the lesions increased from type-B to type-C fractures (p < 0.05). Within each type of fracture the lesions increased from subgroups 1 to 3 (p < 0.05). The anterior tibiofibular ligament was injured with increased frequency from type-B.1 to type-C3 fractures (p < 0.05), but it was not torn in all cases. While lateral ligamentous injuries were seen more often in type-B than in type-C fractures (p < 0.05), no difference was noted in the frequency of deltoid ligamentous lesions. Our findings show that arthroscopy is useful in identifying associated intra-articular lesions in acute fractures of the ankle. (+info)
Mechanical behaviour of articular cartilage under tensile cyclic load.
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INTRODUCTION: Although fatigue has been implicated in cartilage failure, there are only two published studies in this area, by the same author. However, in these previous studies cartilage was tested in the direction parallel to that of collagen orientation in the superficial layer, where it possesses greater tensile strength. In the present work, articular cartilage was also tested along the direction perpendicular to that of the collagen. Furthermore, the study investigated topographic and zonal variations in the fatigue behaviour of cartilage from the human knee. METHODS: Specimens were tested in a specially constructed apparatus that allowed the number of cycles at specimen failure, as well as the load and elongation of the specimen, to be monitored for each specimen. To date, some 72 specimens have been tested, all from the same knee joint, though from different sites and at different depths within the cartilage layer. RESULTS AND CONCLUSIONS: The most impressive of the outcomes of this study is the scatter of the data. Considering all the specimens used, the range of number of load cycles to failure was between 2 and 1.5 million. The zonal variation in fatigue behaviour was similar to that in tensile modulus reported previously; the surface and deep layers seemed to have better fatigue properties whether tested in the direction parallel or perpendicular to that of the collagen in the superficial layer. The middle layer was far weaker, suggesting that highly packed and ordered fibres in the surface and deep zones have better mechanical properties than the more random and loose fibres in the middle zone. The variation in fibre organization through the cartilage thickness was also reflected in the differences observed in the elongation of the specimen during the test. The surface and deep zones had a higher stiffness than the middle zone. Cartilage had better fatigue resistance when the specimen was loaded in a direction parallel rather than perpendicular to the collagen within the surface layer. This was true whether specimens were harvested from the superficial, intermediate or deep layer. There were many factors that confounded attempts to estimate the likely fatigue life from the data obtained in such a study. (+info)
Altered fracture repair in the absence of MMP9.
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The regeneration of adult skeletal tissues requires the timely recruitment of skeletal progenitor cells to an injury site, the differentiation of these cells into bone or cartilage, and the re-establishment of a vascular network to maintain cell viability. Disturbances in any of these cellular events can have a detrimental effect on the process of skeletal repair. Although fracture repair has been compared with fetal skeletal development, the extent to which the reparative process actually recapitulates the fetal program remains uncertain. Here, we provide the first genetic evidence that matrix metalloproteinase 9 (MMP9) regulates crucial events during adult fracture repair. We demonstrate that MMP9 mediates vascular invasion of the hypertrophic cartilage callus, and that Mmp9(-/-) mice have non-unions and delayed unions of their fractures caused by persistent cartilage at the injury site. This MMP9- dependent delay in skeletal healing is not due to a lack of vascular endothelial growth factor (VEGF) or VEGF receptor expression, but may instead be due to the lack of VEGF bioavailability in the mutant because recombinant VEGF can rescue Mmp9(-/-) non-unions. We also found that Mmp9(-/-) mice generate a large cartilage callus even when fractured bones are stabilized, which implicates MMP9 in the regulation of chondrogenic and osteogenic cell differentiation during early stages of repair. In conclusion, the resemblance between Mmp9(-/-) fetal skeletal defects and those that emerge during Mmp9(-/-) adult repair offer the strongest evidence to date that similar mechanisms are employed to achieve bone formation, regardless of age. (+info)
The value of sonography with micro convex probes in diagnosing meniscal tears compared with arthroscopy.
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OBJECTIVE: Meniscal tears are common in knee injuries. Sonography has been used in the knee to evaluate meniscal tears. Linear probes with high resolution have been used, and the overall accuracy of sonography has been more than 70% in many studies. In this study, we used a 6.5-MHz micro convex probe to evaluate meniscal tears, and the results were compared with arthroscopic findings. METHODS: Four hundred six knee joints with knee pain and a clinical indication for arthroscopy were examined from the popliteal fossa with the 6.5-MHz micro convex probe. Those patients with positive sonographic findings who had an arthroscopic examination (100 patients) were included in our study, and the results were compared. The results were statistically analyzed by the Fisher exact test. RESULTS: One hundred knees with sonographic examinations underwent arthroscopic evaluation. Three age groups were included in the study (20-30, 30-40, and >40 years). Comparison of the results between the two methods showed sensitivity of 100% and specificity of 95% for sonography in detecting meniscal tears. The positive predictive value for the medial meniscus was 95%, and the negative predictive value was 100%; these values for the lateral meniscus were 93% and 100%, respectively. CONCLUSIONS: Meniscal tears are common in all age groups. The use of sonography allows rapid, low-cost, and noninvasive exploration of meniscal tears as a first-line diagnostic method. We recommend high-resolution micro convex probes, which better fit the anatomic concavity of the popliteal fossa, as efficient investigation tools. (+info)
Stem cells in orthopedics: current concepts and possible future applications.
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Stem cells are the cells that have the ability to divide for indefinite periods in culture and to give rise to specialized cells. Sources of these cells include embryo, umbilical cord and certain sites in adults such as the central nervous system [CNS] and bone marrow. Its use hold promise of wide spread applications particularly in areas of spinal cord injury, difficult non-unions, critical bone defects, spinal fusions, augmentation of ligament reconstructions, cartilage repair and degenerative disc disorders. This review article contains current information derived from Medline searches on the use in various orthopedic subspecialties. Some issues remain at the forefront of the controversy involving stem cell research - legislation, ethics and public opinion, cost and concentration methods. As is true with any new technology, the enthusiasm for this technology that has potential to influence virtually every orthopedic case management, must be balanced by subjecting it to stringent clinical and basic research investigations. (+info)
Microfracture and bone morphogenetic protein 7 (BMP-7) synergistically stimulate articular cartilage repair.
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OBJECTIVE: Microfracture is used to treat articular cartilage injuries, but leads to the formation of fibrocartilage rather than native hyaline articular cartilage. Since bone morphogenetic protein 7 (BMP-7) induces cartilage differentiation, we hypothesized that the addition of the morphogen would improve the repair tissue generated by microfracture. We determined the effects of these two treatments alone and in combination on the quality and quantity of repair tissue formed in a model of full-thickness articular cartilage injury in adolescent rabbits. DESIGN: Full-thickness defects were made in the articular cartilage of the patellar grooves of forty, 15-week-old rabbits. Eight animals were then assigned to (1) no further treatment (control), (2) microfracture, (3) BMP-7, (4) microfracture with BMP-7 in a collagen sponge (combination treatment), and (5) microfracture with a collagen sponge. Animals were sacrificed after 24 weeks at 39 weeks of age. The extent of healing was quantitated by determining the thickness and the surface area of the repair tissue. The quality of the repair tissue was determined by grading specimens using the International Cartilage Repair Society Visual Histological Assessment Scale. RESULTS: Compared to controls, BMP-7 alone increased the amount of repair tissue without affecting the quality of repair tissue. Microfracture improved both the quantity and surface smoothness of repair tissue. Compared to either single treatment, the combination of microfracture and BMP-7 increased both the quality and quantity of repair tissue. CONCLUSIONS: Microfracture and BMP-7 act synergistically to stimulate cartilage repair, leading to larger amounts of repair tissue that more closely resembles native hyaline articular cartilage. (+info)
Results after microfracture of full-thickness chondral defects in different compartments in the knee.
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OBJECTIVE: To determine if the clinical results after microfracture of full-thickness cartilage lesions deteriorate over a period of 36 months. METHODS: Between 1999 and 2002 85 patients (mean age 39.5 years) with full-thickness cartilage lesions underwent the microfracture procedure and were evaluated preoperatively and 6, 18 and 36 months after surgery. Exclusion criteria were meniscal pathologies, axial malpositioning and ligament instabilities. Baseline clinical scores were compared with follow-up data by paired Wilcoxon-tests for the modified Cincinnati knee and the International Cartilage Repair Society (ICRS)-score. The effects of the lesion localization and Magnetic resonance imaging (MRI) parameters were evaluated using the Pearson correlation and independent samples tests. RESULTS: Both scores revealed significant improvement 18 months after microfracture (P<0.0001). Within the second 18 months after surgery there was a significant deterioration in the ICRS-score (P<0.0001). The best results could be observed in chondral lesions of the femoral condyles. Defects in other areas of the knee deteriorated between 18 and 36 months after microfracture. MRI 36 months after surgery revealed best defect filling in lesions on the femoral condyles with significant difference in the other areas (P<0.02). The Pearson coefficient of correlation between defect filling and ICRS-score was 0.84 and significant at the 0.01 level. CONCLUSIONS: Microfracture is a minimal invasive method with good short-term results in the treatment of small cartilage defects. A deterioration of the results starts 18 months after surgery and is most evident in the ICRS-score. The best prognostic factors have young patients with defects on the femoral condyles. (+info)