Immunolocalization of matrix metalloproteinases in partial-thickness defects in pig articular cartilage. A preliminary report.

BACKGROUND: Partial-thickness defects in mature articular cartilage do not heal spontaneously. Attempts at repair often result in limited integration between the repair tissue and the surrounding cartilage, with formation of chondrocyte clusters adjacent to a zone of cartilage necrosis. In wound repair, spatially and temporally controlled expression of matrix metalloproteinases and their inhibitors have been implicated in proteolytic degradation of damaged extracellular matrix components, but the sequence of events following damage to cartilage is unknown. To determine this sequence, we studied the distribution of matrix metalloproteinases and their inhibitors during early in vivo repair of partial-thickness defects in pig articular cartilage. METHODS: With use of a model that elicits the ingrowth of mesenchymal cells into partial-thickness defects, partial-thickness defects were created in knee joint cartilage. The distributions of matrix metalloproteinase-1, 2, 3, 9, 13, and 14; tissue inhibitors of metalloproteinase-1 and 2; and the neoepitope DIPEN341 specifically generated following matrix metalloproteinase cleavage of aggrecan were determined by immunolocalization of repair tissue and surrounding cartilage excised from immature pigs during the first eight weeks of repair and from adult minipigs at eight days and three weeks. RESULTS: Synthesis of matrix metalloproteinase-13 was usually confined to hypertrophic chondrocytes in immature cartilage and to the radial zone in adult cartilage. Following injury, strong induction of matrix metalloproteinase-13 synthesis was observed in chondrocyte clusters surrounding lesions in all of the animals. The migration of macrophages into defects was prominent at two and eight days, with synthesis and deposition of matrix metalloproteinase-9 onto damaged cartilage matrix and newly synthesized matrix in the defect. The DIPEN341 neoepitope was localized to damaged cartilage matrix at eight days and six weeks, indicating partial degradation of aggrecan. Focal synthesis of matrix metalloproteinase-1, 3, and 14 and of tissue inhibitor of metalloproteinase-1 occurred at later times, suggesting continuous remodeling of the increasingly compact repair tissue. CONCLUSIONS: The expression of matrix metalloproteinase-13 by normal hypertrophic chondrocytes and the induction of synthesis in chondrocyte clusters adjacent to the zone of cartilage necrosis suggest that this enzyme participates in the pericellular proteolysis required for lacunar expansion. The localization of matrix metalloproteinase-9 to damaged cartilage matrix suggested matrix proteolysis, which was confirmed with DIPEN341 localization. Reduced matrix metachromasia persisted and was colocalized with DIPEN341 at six weeks. However, under the conditions investigated, there was only limited proteolytic degradation in the zone of cartilage necrosis. This may render the zone mechanically weakened, thereby contributing to subsequent instability of the region, and may form a barrier to integration of repair tissue with viable cartilage. CLINICAL RELEVANCE: Osteoarthritis initially involves the superficial layers of cartilage. The development of procedures to promote the healing or repair of early defects will have major advantages in terms of disease alleviation as well as economic importance. Identification of the enzymes involved in the early repair of partial-thickness defects in articular cartilage is clinically relevant because proteolysis of damaged matrix has to take place in order for repair tissue to integrate with surrounding healthy cartilage.

Measurement of localized cartilage volume and thickness of human knee joints by computer analysis of three-dimensional magnetic resonance images.

RATIONALE AND OBJECTIVES: This work demonstrates a new method for computerized measurement of the dimensions (thickness and volume) of articular cartilage for any specified region of the human knee joint. Three-dimensional magnetic resonance (MR) images optimized for cartilage contrast have been analyzed using computerized edge-detection techniques, and the reproducibility of articular cartilage thickness and volume measurements is assessed. METHODS: A fat-suppressed, three-dimensional SPoiled GRass MR sequence (45/7.5/30 degrees) with total scan time of approximately 12 minutes was used to acquire volume images of human knee joints at spatial resolution of 0.6 x 1.2 x 1.2 mm. Measurements were made using six repeated scans for three healthy volunteers over a period of 2 months. The subsequent semi-automated image processing to establish total cartilage volume and cartilage thickness maps for the femur required approximately 60 minutes of operator time. RESULTS: The mean coefficient of variation for total cartilage volume for the six repeated scans for the three volunteers was 3.8%, and the average coefficient of variation for the user-selected cartilage plugs was 2.0%. The cartilage thickness maps from the repeated scans of the same knee were similar. CONCLUSIONS: Standard resolution MR images with fat-suppressed contrast lead to an objective and reproducible measurement of spatial dimensions of articular cartilage when analyzed semi-automatically using computerized edge-detection methods.

MR protocols for imaging the guinea pig knee.

Magnetic resonance images of the femorotibial joints of male Dunkin-Hartley guinea pigs were obtained in two and three dimensions at 2.35 T using a wide range of T1- and T2-weighted imaging sequences. The effect of slice position on visualisation of articular cartilage, bone and periarticular tissues in sagittal and coronal sections was investigated along with the resolution and signal/noise ratio achievable. Based on that survey, a two-dimensional spin echo sequence (repetition time = 1500 ms, echo time = 40 ms) was found to give optimum visualisation of the normal joint anatomy with in-plane resolution of 75 x 150 microns and a 1 mm slice thickness in an imaging time of 25 min. This protocol was also found to be highly effective in distinguishing many features of the spontaneous, osteoarthritic-like pathology found in the joints of older animals compared to juveniles and therefore provides a means of monitoring disease progression longitudinally. Three-dimensional spin echo imaging methods demonstrated focal changes in signal intensity in the articular cartilage of the medial tibial plateau in older animals. The resulting imaging times of several hours, however, precludes their routine use in vivo.

Magnetic resonance imaging, histology, and x-ray of three stages of damage to the knees of STR/ORT mice.

RATIONALE AND OBJECTIVES: The authors develop a scoring system for assessing those features of degeneration of the STR/ORT mouse knee visualizable by magnetic resonance (MR) imaging, and to validate those MR scores by comparison with x-ray and histology. METHOD: Magnetic resonance imaging, histology, and x-ray have been used in a cross-sectional study to visualize the anatomy and pathology of the knees of three pairs of male STR/ORT mice and their approximately age-matched female pairs. A scoring system was developed that distinguished the faster rate of damage of the males from the slower progressive changes seen in the females. RESULTS: Changes in the patellar tendon were observed in MR imaging of the 5-month-old male knee. Sagittal images showed other degenerative features such as sclerosis and loss of signal from synovial fluid after 9 months; osteophytes and degeneration of the tibial plateau were better visualized in the coronal plane. Cysts were poorly correlated to the progression of the disease. Similar trends were observed for four features scored in x-rays (sclerosis, joint space narrowing, cysts, and osteophytes) and cartilage degradation assessed using histology. In contrast, the age-matched females were less affected. CONCLUSIONS: Magnetic resonance imaging can identify joint degeneration in the knees of male mice, which develops more rapidly than in age-matched females. Those observations were validated by radiology and histology.

Cartilage swelling and loss in a spontaneous model of osteoarthritis visualized by magnetic resonance imaging.

The objective of this study was to investigate whether the rate of change in cartilage pathology could be effectively monitored by magnetic resonance imaging (MRI) as part of a longitudinal investigation of an osteoarthritis model in vivo, and to define the minimum requirements necessary to establish disease progression. Magnetic resonance images of the knee of eight male Dunkin-Hartley guinea-pigs were obtained at 8, 12, 18, 24, 30, 36 and 52 weeks of age using a two-dimension spin-echo sequence with a TR of 1500 ms and TE of 40 ms. The total thickness of the femoral and tibial cartilage was measured from those images. Over the same time course, sets of spin-spin relaxation-weighted images were acquired from two additional animals of the same age, from which the T2 relaxation times of water in the articular cartilage were estimated and compared with those of muscle and adipose tissue within the same joint. The cartilage thickness of all the animals increased during the first 6 months, then either stayed thicker (4/8) or became progressively thinner (4/8). Up to 18 weeks of age, the cartilage T2 value was between 23-24 ms but became elevated by 30 weeks and the mean value was more than 40 ms at the end of the study, T2 values for the muscle and adipose tissue remained within the range 30-33 ms, or 47 ms, respectively. We concluded that in this model, cartilage thickness measurements from an magnetic resonance image would not provide a reliable marker to stage osteoarthritis progression partly because the cartilage was so thin in a joint of this size, but also because the changes with time were not linear but biphasic. However, quantitation of the T2 relaxation values may provide a more predictable indicator of cartilage pathology for longitudinal studies because the changes were monotonic and independent of cartilage thickness.

High resolution magnetic resonance imaging of the proximal interphalangeal joints. Correlation with histology and production of a three-dimensional data set.

The magnetic resonance imaging appearance of the proximal interphalangeal joints of cadavers was correlated with histology of the same specimen allowing many small-scale features to be identified that might otherwise have been misinterpreted. It enabled the magnetic resonance signal to be understood at a tissue and cellular level, allowing identification of synovial folds extending from the extensor tendon and volar plate, the entheses of the proper collateral ligament, the epitenon of the flexor tendons and the presence of osteophytes and sites of cartilage erosion. The main difficulties in matching two-dimensional magnetic resonance images with histology were the differing section thicknesses of the two methods and shrinkage of histological specimens. There are many advantages in producing high resolution three-dimensional datasets-the magnetic resonance section thickness is reduced and the individual components of the joint can be viewed simultaneously in two or more planes. A unique magnetic resonance atlas of three dimensional joint structure is presented.

Degenerative joint disease in the guinea pig. Use of magnetic resonance imaging to monitor progression of bone pathology.

OBJECTIVE: The suitability of magnetic resonance imaging (MRI) for serial monitoring of bone pathology in the guinea pig stifle joint, an in vivo model of osteoarthritis, was investigated. METHODS: MR images were compared with histologic features and radiographs of 1-mm-thick sections to determine the MR correlates of the bone changes. Ten guinea pigs were then imaged on 7 occasions over the first year of life, enabling serial measurements of subchondral bone thickness, subchondral pseudocysts, and osteophytes. RESULTS: The signal intensity of trabecular bone in MR images accurately reflected the degree of osteopenia and trabecular thinning noted around the cruciate ligament insertions. The extent of subchondral sclerosis and the development of marginal osteophytes were also accurately represented. Serial observations revealed that MRI can detect highly significant progression of lytic bone lesions, subchondral sclerosis, and osteophyte size over periods of 6 weeks. CONCLUSION: MRI is not only a reliable technique for the assessment of bone pathology but is also a useful tool for monitoring the progression of bone damage in osteoarthritis.

Visualization by magnetic resonance imaging of focal cartilage lesions in the excised mini-pig knee.

This study showed that magnetic resonance imaging can be used to visualize partial thickness cartilage lesions, 0.7 x 10 mm in area and 0.5 mm in depth, surgically induced in the femur (femoropatellar compartment) of a mini-pig knee joint. Formalin-fixed joints, intact as well as disarticulated, were studied by high resolution imaging using a 2.35 T, 31 cm horizontal-bore superconducting magnet. The two-dimensional and three-dimensional spatial resolutions achievable were as follows: 0.12 x 0.23 mm (two-dimensional) and 0.35 x 0.35 x 0.35 mm (three-dimensional) for the intact joint, and 0.08 x 0.08 mm (two-dimensional) and 0.14 x 0.14 x 0.27 mm (three-dimensional) for the disarticulated joint. These results demonstrate that magnetic resonance imaging, together with edge detection and volume rendering, can be used to visualize focal cartilage lesions.

Production of binding proteins and role of the insulin-like growth factor I binding protein 3 in human articular cartilage explants.

The aim of this study was to determine the production of insulin-like growth factor binding proteins (IGFBP) and the role of the IGFBP-3 in human normal (n = 2) and osteoarthritic (OA) articular cartilage (n = 14) explants. Binding proteins were studied in the medium by Western ligand blotting and Western blotting. Proteoglycan synthesis under insulin-like growth factor I (IGF-I) stimulation was studied after a pulse of 35SO4(2-) in the presence or absence of added IGFBP-3. Osteoarthritic explants released a doublet of IGFBPs with a 39/43 kDa Mr corresponding to the binding protein 3. Constitutive production from unstimulated OA cartilage was higher than from normal cartilage. IGF-I induced a 20-fold increase and IL-1 a 2-fold increase in IGFBP-3 release. A minor band around 30 kDa was also detectable. Studies of proteoglycan (PG) synthesis showed that the majority of OA cartilage explant samples responded weakly to IGF-I (100 ng/ml) stimulation (+33%), while the others were high responders (+180%). Co-incubation of IGF-I with recombinant (r) IGFBP-3 did not affect the rate of PG synthesis. However, while pre-incubation with rIGFBP3 for 72 h did not change the rate of PG synthesis in the high-responder group, it strongly increased PG synthesis in the low-responder group. This study demonstrates that the ability of IGF-I to enhance proteoglycan synthesis varied among the OA samples and may in part be dependent on the local level of IGFBP-3. This implies pathophysiological considerations in the limits of IGF-I action during the OA process.

Tumour necrosis factor alpha, interferon gamma and dexamethasone regulate IGF-I-maintained collagen production in cultured human fibroblasts.

We examined the effects of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the production of collagen by human infant foreskin fibroblasts. Collagen synthesis was maintained in the presence of IGF-I so that cytokine effects could be examined in the absence of serum. TNF alpha inhibited IGF-I-maintained collagen production in a dose-dependent manner. Maximal suppression of 50% was attained at a concentration of 7.5 ng/ml. IFN gamma also suppressed collagen accumulation in IGF-I-maintained cells, with a maximal inhibition to 55% at 375 U/ml. The rate of collagen formation relative to total protein production for secreted proteins was calculated. This value decreased from 10.3% for IGF-I-cultured cells to 6.2% and 8.4% with the inclusion of TNF alpha and IFN gamma respectively, indicating that inhibition was selective for collagen. TNF alpha (5 ng/ml) and IFN gamma (250 U/ml) together suppressed IGF-I-maintained collagen production to approximately 35%, with a decrease from 10.3% to 2.9% in collagen production relative to total protein. The inclusion of a serum-free period prior to the addition of TNF alpha to the cultures resulted in a further inhibition to 15% of control. This increase in inhibition was not seen if dexamethasone was present in the serum-free period prior to cytokine addition. These data showed that IGF-I-maintained collagen formation is suppressed by the proinflammatory cytokines TNF alpha and IFN gamma, and that these interactions are influenced by dexamethasone. Proinflammatory cytokines interact in a complex manner with other serum factors to modulate IGF-I-stimulated extracellular matrix production and may have important roles in regulating tissue repair.

Magnetic resonance imaging protocol optimization for evaluation of hyaline cartilage in the distal interphalangeal joint of fingers.

RATIONALE AND OBJECTIVES: To identify a single magnetic resonance imaging (MRI) protocol that will provide optimal signal-to-noise ratio, resolution, and image contrast with minimal susceptibility artifacts and that will allow clear delineation and visualization of cartilage, fluid, bone, tendons, and ligaments within the distal interphalangeal (DIP) joint of the human hand. METHODS: A highly optimized 2.4 T MRI system was constructed from a 31-cm horizontal bore magnet, using a solenoid radiofrequency coil. This was used to study the DIP joints of 16 healthy, asymptomatic volunteers. RESULTS: A range of image contrast protocols were explored, including spin-echo T1 and T2, field echo, chemical shift suppression to give water only images, and magnetization transfer. Susceptibility variations were explored by changing the field strength from 0.6 to 2.4 T. A spin-echo protocol with TR = 1500 msec and TE = 30 msec can routinely produce images with resolution 0.075 x 0.150 for a slice thickness of 1 mm in 13 minutes. That protocol can visualize simultaneously compact and trabecular bone, two layers of cartilage, synovial fluid, and synovium within the joint, tendons and ligaments, and the volar plate. CONCLUSIONS: Although the contrast is not fully optimized for any one tissue, the spin echo protocol (TR = 1500, TE = 30) provides sagittal MR images, which clearly delineate the major structures of interest within the DIP joint, and which will be used in future studies to compare changes in the DIP joint because of aging or osteoarthritis. Experience gained by applying the above methods to a total of 16 healthy, asymptomatic volunteers has enabled a single sequence to be identified, which although not optimized for any one tissue, nevertheless visualized simultaneously and clearly delineated compact and trabecular bone, two layers of cartilage, synovial fluid, and synovium within the joint.

High resolution MR imaging of joint degeneration in the knee of the STR/ORT mouse.

MR imaging of the knee joints of the mouse at 2.35 T produces an in-plane resolution of 35 x 70 microns and a slice thickness of 600 microns from the sagittal and coronal planes, in less than 60 min. In normal, live DBA mice, which have no known pathology, the images clearly resolve many joint structures, such as the cruciate ligaments, menisci, and articulating surfaces of the femur and tibia. Gross pathological changes were identified first in excised knees from cadaver mice of the STR/ORT strain. Males of this strain spontaneously exhibit rapid joint degeneration with increasing age compared to females, in which much milder symptoms develop more slowly. Thickening of the patellar tendon, displacement of the patella, deformity, and sclerosis were clearly distinguishable in males compared with females older than 7 mo of age. The same degenerative features were evident in MR images of the knees of the live male but not female STR/ORT mice of this age, indicating that it would be possible to monitor longitudinally by MR imaging progressive development of joint changes in this osteoarthritic model.

A combined analysis and magnetic resonance imaging technique for computerised automatic measurement of cartilage thickness in the distal interphalangeal joint.

It is well known that magnetic resonance imaging (MRI) contrast can be controlled, albeit sometimes at the expense of image resolution and signal-to-noise ratio, and most studies of articular joints have used a single MRI protocol, which is optimised for subjective image analysis. Inevitably that single protocol frequently compromises the detection of one or another of the boundaries between which any measurement must be made. This paper describes an alternative approach in which the criteria for computerised edge detection necessary for fully automated measurement of cartilage thickness are used to define the MRI acquisition parameters. This necessitates the combined use of two MRI sequences, one optimised for the cartilage-bone boundary, and the other for cartilage-synovial fluid. This provides a highly effective combination and its efficacy is demonstrated for the distal interphalangeal joint of a range of asymptomatic adults.

Visualisation of mass transport of small organic molecules and metal ions through articular cartilage by magnetic resonance imaging.

Magnetic resonance imaging of water has been used to visualise the migration of three paramagnetic species, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO, 1), Cu2+ ions, and copper ethylenediamine-tetraacetate (CuEDTA, 2) through cartilage on the femoral condyle of the chicken knee. The migration of copper ions is dominated by strong binding with the cartilage. In contrast, both 1 and 2 bind weakly, and their diffusion can be followed as a progressive wave through the cartilage and subsequently into the trabecular bone structure.

Dexamethasone potentiates the stimulatory effect of insulin-like growth factor-I on collagen production in cultured human fibroblasts.

We examined the effects of insulin-like growth factor-I (IGF-I) and dexamethasone on the production of collagen by cultures of human infant foreskin fibroblasts, and the interaction between these two factors. IGF-I at 500 ng/ml maximally increased collagen accumulation fourfold. Collagen was increased twofold relative to total protein production. Dexamethasone at a concentration of 1 mumol/l reduced collagen production by between 25% and 40% in unstimulated cells and those cultured with up to 100 ng IGF-I/ml. However, dexamethasone did potentiate collagen production in cells stimulated with 250 ng IGF-I/ml. This potentiation was independent of any effects of IGF-I or dexamethasone on prostaglandin (PG)E2 production. Transforming growth factor-beta (TGF-beta) is also a potent stimulator of collagen formation. However, no potentiation of TGF-beta-stimulated collagen production by dexamethasone was apparent. The mechanism by which dexamethasone potentiates IGF-I-stimulated collagen production was investigated. Dexamethasone treatment increased IGF-I binding to the type 1 IGF receptor without altering the binding affinity. Dexamethasone also attenuated the secretion of IGF-binding proteins by IGF-I-maintained cells.

Upregulation of insulin-like growth factor I gene expression in the lesions of osteoarthritic human articular cartilage.

Expression of insulin-like growth factor I (IGF-I)mRNA and IGF-I protein was studied in human osteoarthritic and young articular cartilage by in situ hybridisation and immunohistochemistry. In situ hybridisation showed that relatively low amounts of IGF-I mRNA signal were present in anatomically normal regions of osteoarthritic and young cartilage. In fibrillated osteoarthritic cartilage, however, the signal intensity was significantly higher than in non-fibrillated cartilage. Particularly high levels of IGF-I mRNA were present in the surface cell clones of more advanced lesions, the amount of signal being about four to five times greater than in anatomically normal cartilage. The amount of message varied with cartilage depth. In young cartilage there was less IGF-I mRNA in the superficial zone than in the middle and deep zones. In fibrillated regions of osteoarthritic joints the amount of message in surface cells was greater than in deeper regions. A specific human IGF-I antibody was used to show the presence intracellularly of IGF-I protein in osteoarthritic and young cartilage. Raised levels of IGF-I message in osteoarthritic chondrocytes may represent an attempt at increased matrix repair, operating by an autocrine/paracrine mechanism.