Subchondral bone failure in overload arthrosis: a scanning electron microscopic study in horses

Mechanical overload leads to a common arthrosis in the metacarpal condyle of the fetlock joint of racehorses. This is usually asymptomatic but severe forms can cause lameness. Subchondral bone failure is often present and the predictability of the site provided an opportunity to study of the progression of bone failure from microcracks to actual collapse of subchondral bone. Twenty-five fetlock condyles from racehorses with various stages of disease were selected. Stages ranged from mild through severe subchondral bone sclerosis, to the collapse of bone and indentation or loss of cartilage known as 'traumatic osteochondrosis'. Parasagittal slices were radiographed and examined with scanning electron microscopy. Fine matrix cracks were seen in the subchondral bone layer above the calcified cartilage and suggested loss of water or other non-collagenous components. The earliest microcracks appeared to develop in the sclerotic bone within 1-3 mm of the calcified cartilage layer and extend parallel to it in irregular branching lines. Longer cracks or microfractures appeared to develop gaps as fragmentation occurred along the margins. Occasional osteoclastic resorption sites along the fracture lines indicated activated remodeling may have caused previous weakening. In one sample, smoothly ground fragments were found in a fracture gap. Bone collapse occurred when there was compaction of the fragmented matrix along the microfracture. Bone collapse and fracture lines through the calcified cartilage were associated with indentation of articular cartilage at the site.     

Baicalein alleviates osteoarthritis by protecting subchondral bone,inhibiting angiogenesis and synovial proliferation

Osteoarthritis (OA) is one of the most frequent chronic joint diseases with the increasing life expectancy. The main characteristics of the disease are loss of articular cartilage, subchondral bone sclerosis and synovium inflammation. Physical measures, drug therapy and surgery are the mainstay of treatments for OA, whereas drug therapies are mainly limited to analgesics, glucocorticoids, hyaluronic acids and some alternative therapies because of single therapeutic target of OA joints. Baicalein, a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi, has been widely used in anti-inflammatory therapies. Previous studies revealed that baicalein could alleviate cartilage degeneration effectively by acting on articular chondrocytes. However, the mechanisms involved in baicalein-mediated protection of the OA are not completely understood in consideration of integrality of arthrosis. In this study, we found that intra-articular injection of baicalein ameliorated subchondral bone remodelling. Further studies showed that baicalein could decrease the number of differentiated osteoblasts by inhibiting pre-osteoblasts proliferation and promoting pre-osteoblasts apoptosis. In addition, baicalein impaired angiogenesis of endothelial cells and inhibited proliferation of synovial cells. Taken together, these results implicated that baicalein might be an effective medicine for treating OA by regulating multiple targets.     

The influence of laser irradiation of low-power density on an experimental cartilage damage in rabbit knee-joints: an in vivo investigation considering macroscopic, histological and immunohistochemical changes]

In a total of 45 rabbits, knee-joint arthrosis was induced according to the Hulth & Telhag model. Depending on the post-operative survival time, the cartilage was investigated macroscopically, histologically and immunohistochemically (within a period of 10 days to 8 months). Thereafter, the influence of laser irradiation at a wavelength of 692.6 nm and energy densities of 1 and 4 J/cm2 on the cartilage morphology seven days following the exposure was examined. After joint instability surgery it was found out that the cartilage changes in the main stress area (MSA) and in regions outside the main stress area (ROMSA) progressed differently. Various qualitative and semi-quantitative changes were found for collagens I, II, IV and V, and for the glycoproteins fibronectin and tenascin. Immunohistochemically, there was a growing expression of collagen I in the apical layers, collagen II showed a stronger pericellular expression, and collagen IV showed, after an initial growth of the pericellular expression, a reduced territorial expression and a stronger apical-interterritorial expression in the osteoarthrotic cartilage. For fibronectin, the cellular expression turned out to grow in the ROMSA. In the MSA it decreased, but at the same time the interterritorial expression grew. For Tanascin, there was a decrease of the interterritorial expression in the radial zone while the pericellular and interterritorial expression of the apical layers of the osteoarthrotic cartilage grew. Lasing proved to significantly influence the osteoarthrotically changed cartilage when applied at an energy density of 1 J/cm2, i.e., the morphological changes had not yet progressed to the extent the control group had. Both the chondrocyte density and the glucosaminoglycan content turned out to be higher. When lasing was applied at higher energy densities, no significant difference among the control groups was found. Thus, it could be demonstrated in vivo that an arthrotic process decelerates through the influence of laser light of low-energy densities.     

A method for estimating the Young's modulus of complete tracheal cartilage rings

Cartilage is primarily responsible for maintaining the stability of the large airways; yet very little is known about the mechanical properties of airway cartilage. This work establishes a technique whereby average values for the equilibrium modulus of excised tracheal cartilage rings can be obtained. An apparatus was designed to apply preset deformations to a tracheal segment and to monitor the deforming force. Segments of four human tracheae obtained postmortem and containing three rings were mounted in the apparatus after being stripped of posterior membrane. The load-deformation behavior was analyzed with a model on the basis of thin curved beam theory. Agreement between predicted deformed shapes and those observed was good in three of the four cases and in the case of a short length of longitudinally split rubber tube. The technique is suitable for comparing mechanical properties of cartilage before and after an intervention.     

LDH and LDH Isoenzymes of Synovial Fluid in the Horse

LDH is an intracellular enzyme, which when cells degenerate is released to the extracellular spaces and body fluids. Cells and organs in the mammalian body differ from each other with respect to their LDH isoenzyme patterns. These circumstances have led to the use of LDH isoenzyme determinations in laboratory diagnostic work. In the present investigation total LDH activity and LDH isoenzyme distribution in equine synovial fluid from healthy joints, joints with serous arthritis, osteochondrosis dissecans and arthrosis, were determined. The fluids from the diseased joints differed from normal synovial fluid with respect to total LDH activity, and the different joint diseases each seemed to give rise to a characteristic isoenzyme pattern. In order to examine possible sources of the increased LDH activity and altered isoenzyme patterns, blood plasma, red and white blood cells, synovial membrane and articular cartilage were also studied. It was found that LDH4 and LDH5 were present in high amounts in articular cartilage, and an increase in these isoenzymes was the most characteristic feature in synovial fluid from joints with arthrosis. The results were discussed in view of possible diagnostic value of isoenzyme determinations on synovial fluid.     

Basophilic line of the articular cartilage in normal and various pathological states

Epiphyses of long tubular bones in the man and animals of various age, as well as experimental material of the adjuvant arthritis, with special reference to the basal part of the articular cartilage have been studied by means of histological, histochemical and histometrical methods. The structural-chemical organization of the basophilic line (tidemark) of the articular cartilage ensures its barrier role and participation in regulating selective permeability. Reconstruction of the tidemark in the process of physiological ageing and in cases of the articular pathology is aimed to preserve its integrity and in this way a complete differentiation of the noncalcified and calcified structures is secured. Disturbance of the basophilic line results in changes of the articular selective permeability, in invasion of vessels and structural elements of the bone marrow, and in development of profound distrophic and destructive changes of the cartilage--in deforming artrosis. Deflations in the structural-chemical organization of the tidemark indicate certain disturbances in the state of the system articular cartilage--subchondral bone. These data can be of prognostic importance.     

Overload arthrosis: strain patterns in the equine metacarpal condyle

An overload arthrosis occurs consistently in the palmar region of the metacarpal condyle of the equine fetlock (metacarpophalangeal) joint characterized by subchondral bone sclerosis, devitalization and mechanical failure leading to collapse of the overlying articular cartilage. Samples were selected of joints with mild, moderate, and severe subchondral sclerosis, in which cartilage collapse had not yet occurred. An additional group that had severe sclerosis with focal rarefaction suggesting impending collapse was also studied (n=5/group). Parasagittal slices were milled to 2.0 mm thickness and subjected to palmar forces 50 to 200% of those applied by the sesamoid bone at angles corresponding to early, mid and late stance support phases of the gait cycle. From contact radiographs in the loaded and unloaded samples, strains were determined by recognizing displacements in the trabecular patterns using texture correlation analysis. Failure did not occur in any of the samples. Strains were generally proportional to the forces applied and greatest at midstance. Strain patterns varied between samples and with the different loading positions. With increased subchondral bone sclerosis there was greater shear strain in overlying trabeculae. Strain patterns were not consistently different within the sclerotic bone at the site of failure. Focally higher strains at the surface were sometimes related to the edge of the platen which was molded to mimic the sesamoid bone in vivo. These results indicate that sclerotic thickening of subchondral bone transmits stresses to overlying trabeculae. No consistent strain pattern was recognized where devitalization and mechanical failure occurs. Focally higher strains related to the edge of the opposing sesamoid bone may play a role.     

Characterization of cathepsins in cartilage

The presence of a cathepsin B-like enzyme in rabbit ear cartilage was established by the use of the synthetic substrates benzoyl-l-arginine amide and benzoyl-dl-arginine 2-naphthylamide. This was facilitated by using a technique that permits the incubation of a fixed weight of thin (18mu) cartilage sections with an appropriate exogenous substrate. The enzymic properties of cathepsin B in cartilage have been compared with an endogenous enzyme that liberates chondromucopeptide by degrading the cartilage matrix autocatalytically at pH5. Besides being maximally active at pH4.7, these cartilage enzymes are enhanced in activity by cysteine and inhibited by arginine analogues, iodoacetamide, chloroquine and mercuric chloride. They are not inhibited by EDTA, di-isopropyl phosphorofluoridate and diethyl p-nitrophenyl phosphate. When inhibiting the release of chondromucopeptide from cartilage at pH5, the arginine-containing synthetic substrates are hydrolysed simultaneously. These enzymes also share the same heat-inactivation characteristics at various pH values, being stable at acid pH and unstable at neutral and alkaline pH. The experimental evidence indicates that a cathepsin B-like enzyme may be partly responsible for the autolytic degradation of cartilage matrix at pH5.     

The hydrodynamic components in joints: proof of a membrane-cistern system within the calcified zone of the femur head cartilage

The theoretical postulate of a hydrodynamic system for the transmission of forces at the hip joint led to the discovery of a two-membrane system. Within the calcified cartilaginous zone of the femoral head there exists a system of small spaces and cisterns which can only be understood in the postulated sense as an overall acting hydrodynamic system. A correlation between the destruction of the calcified zone, i.e. the hydrodynamic system, and arthrosis is indicated. Ongoing studies and deliberations will help to discover the true model of the transmission of forces and impulses and to solve the problems of treating arthrosis.     

Studies on the cathepsins in elastic cartilage

1. The presence of several enzymes in rabbit ear cartilage was examined by a quantitative method that permits the incubation of a fixed weight of cartilage sections (18mum.) with an appropriate exogeneous substrate. 2. As the presence of cathepsins B and D in cartilage has already been established, evidence is now provided to show that cathepsins A and C are also present and are maximally active at pH5. 3. Cathepsin A was recognized by its hydrolysis of benzyloxycarbonyl-glutamyl-tyrosine and cathepsin C by its hydrolysis of glycyl-tyrosine amide; the cartilage also hydrolysed benzyloxycarbonyl-glutamyl-phenylalanine and benzoyl-dl-phenylalanine 2-naphthyl ester at pH5. 4. The acid phosphatase activity and the DNA content of cartilage have also been measured to provide a basis for comparison with the cathepsin activity of cartilage obtained from other sites and species.     

Dependence of nanoscale friction and adhesion properties of articular cartilage on contact load

Boundary lubrication of articular cartilage by conformal, molecularly thin films reduces friction and adhesion between asperities at the cartilage-cartilage contact interface when the contact conditions are not conducive to fluid film lubrication. In this study, the nanoscale friction and adhesion properties of articular cartilage from typical load-bearing and non-load-bearing joint regions were studied in the boundary lubrication regime under a range of physiological contact pressures using an atomic force microscope (AFM). Adhesion of load-bearing cartilage was found to be much lower than that of non-load-bearing cartilage. In addition, load-bearing cartilage demonstrated steady and low friction coefficient through the entire load range examined, whereas non-load-bearing cartilage showed higher friction coefficient that decreased nonlinearly with increasing normal load. AFM imaging and roughness calculations indicated that the above trends in the nanotribological properties of cartilage are not due to topographical (roughness) differences. However, immunohistochemistry revealed consistently higher surface concentration of boundary lubricant at load-bearing joint regions. The results of this study suggest that under contact conditions leading to joint starvation from fluid lubrication, the higher content of boundary lubricant at load-bearing cartilage sites preserves synovial joint function by minimizing adhesion and wear at asperity microcontacts, which are precursors for tissue degeneration.     

Expression profile of carbonic anhydrases in articular cartilage

Carbonic anhydrases (CAs), which catalyze the reversible reaction of carbonate hydration, are important for cartilage homeostasis. The full spectrum of CA activity of all 13 isoenzymes in articular cartilage is unknown. This study quantified the mRNA profile of CAs in rat articular cartilage, using quantitative polymerase chain reactions. Among the 13 functional CAs, CAs II, III, Vb, IX, XII and XIII were significantly expressed at mRNA level by the chondrocytes in articular cartilage. To verify these significantly expressed CAs in articular cartilage at protein level, immunohistochemistry was performed. While CAs III, Vb and XII distributed in the full-thickness of cartilage, including the calcified zone of cartilage, CA II was mainly localized in the proliferative zone of cartilage. CA IX was limited in the superficial zone of cartilage and CA XIII expressed in the superficial and partially mid zone. These results provide a framework for understanding individual CAs as well as the integrated CA family in cartilage biology, including matrix mineralization.     

Mechanism of the reduction of Meckel's cartilage in man

The mechanism of reduction of the anterior end of Meckel's cartilage was studied in human embryos, with the following findings: 1. Meckel's cartilage is surrounded, from the outside and from below, by newly formed mandibular bone over the extent of the insertion of the musculus mylohyoideus. 2. Blood vessels from the newly formed bone penetrate Meckel's cartilage and break it down in the same way as in enchondral ossification of cartilaginous models of other bones. 3. The anlagen of the musculus mylohyoideus and musculus genioglossus are at first inserted on Meckel's cartilage; further muscle fibres, formed on the under surface of the two muscles, are inserted on the newly formed bone of the rudimentary mandible. Parallel to this process, the fibres on the upper surface of the muscles, which were originally inserted on Meckel's cartilage, disappear. The two processes combined lead to transposition of the insertions of the two muscles from Meckel's cartilage to the mandible. 4. In the area of the resorbed Meckel's cartilage, a minimum number of bone trabeculae are formed at the time of its resorption. The space left by Meckel's cartilage is taken over chiefly by the primitive medullary cavity of the rudimentary mandible, medially to the canal for the nerve and blood vessels.     

Holographic recording of the interference waves in muscle tissues

The method of interference holography is suggested for the investigation of biomechanical properties of human skeletal muscles. The method permitted the registration of a profile of deforming waves induced on the muscle surface by a special striking mechanism. Analytically and in model experiments it was demonstrated that deforming waves penetrated much deeper than skin and subcutaneous tissue layer. The experiments have confirmed that the skin has but little effect on interference pattern. The interferogram of deforming waves in the biceps muscle reflect distinctions in the muscle tissue elasticity along and transverse muscle fibers.     

Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G

The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.     

Morphofunctional peculiarities of the transverse processes of the axis, considerations on their pathogenetic significance.

The transverse process C2 plays an important role in the stabilization of the vertebral unit C2/C3 and the protection of the vertebral artery and the cervical spinal cord. The anatomical disposition of the transverse processes C2 and especially the lateral processes may be different from one subject to another. Certain anatomical variants of the transverse process can exert a negative effect on the stabilizing and protective function of the same. Our observations suggest that a maldevelopment or a lesion of the transverse process of the axis may induce extra stresses in the vertebral unit C2/C3 with subsequent development of a zygapophyseal arthrosis at this level. Any zygapophyseal arthrosis (uni- or bilaterally) should lead us to think of this possibility too.     

Contact analysis of biphasic transversely isotropic cartilage layers and correlations with tissue failure.

Failure of articular cartilage has been investigated experimentally and theoretically, but there is only partial agreement between observed failure and predicted regions of peak stresses. Since trauma and repetitive stress are implicated in the etiopathogenesis of osteoarthritis, it is important to develop cartilage models which correctly predict sites of high stresses. Cartilage is anisotropic and inhomogeneous, though it has been difficult to incorporate these complexities into engineering analyses. The objectives of this study are to demonstrate that a transversely isotropic, biphasic model of cartilage can provide agreement between predicted regions of high stresses and observed regions of cartilage failure and that with transverse isotropy cartilage stresses are more sensitive to convexity and concavity of the surfaces than with isotropy. These objectives are achieved by solving problems of diarthrodial joint contact by the finite-element method. Results demonstrate that transversely isotropic models predict peak stresses at the cartilage surface and the cartilage-bone interface, in agreement with sites of fissures following impact loading; isotropic models predict peak stresses only at the cartilage-bone interface. Also, when convex cartilage layers contacted concave layers in this study, the highest tensile stresses occur in the convex layer for transversely isotropic models; no such differences are found with isotropic models. The significance of this study is that it establishes a threshold of modeling complexity for articular cartilage that provides good agreement with experimental observations under impact loading and that surface curvatures significantly affect stress and strain within cartilage when using a biphasic transversely isotropic model.     

COMPENSATORY GROWTH IN THE RAT TIBIA

When the proximal cartilage plate of one tibia of a rat is sterilized by radiation, compensatory growth occurs at the distal plate of the same bone. This growth is marked by small changes in the cell kinetic parameters and histology of the growth cartilage when compared with the distal cartilage plate in the unirradiated tibia. The changes are consistent with a delay in the maturation of the plate showing compensatory growth. Some possible mechanisms are considered, but the evidence available at present does not give decisive support to any particular theory.     

Normal contact of elastic spheres with two elastic layers as a model of joint articulation.

An analytical model of two elastic spheres with two elastic layers in normal, frictionless contact is developed which simulates contact of articulating joints, and allows for the calculation of stresses and displacements in the layered region of contact. Using various layer/layer/substrate combinations, the effects of variations in layer and substrate properties are determined in relation to the occurrence of tensile and shear stresses as the source of crack initiation in joint cartilage and bone. Vertical cracking at the cartilage surface and horizontal splitting at the tidemark have been observed in joints with primary osteoarthritis. Deep vertical cracks in the calcified cartilage and underlying bone have been observed in blunt trauma experiments. The current model shows that cartilage stresses for a particular system are a function of the ratio of contact radius to total layer thickness (a/h). Surface tension, which is observed for a/h small, is alleviated as a/h is increased due to increased load, softening and/or thinning of the cartilage layer. Decreases in a/h due to cartilage stiffening lead to increased global compressive stresses and increased incidence of surface tension, consistent with impact-induced surface cracks. Cartilage stresses are not significantly affected by variations in stiffness of the underlying material. Tensile radial strains in the cartilage layer approach one-third of the normal compressive strains, and increase significantly with cartilage softening. For cases where the middle layer stiffness exceeds that of the underlying substrate, tensile stresses occur at the base of the middle layer, consistent with impact induced cracks in the zone of calcified cartilage and subchondral bone. The presence of the superficial tangential zone appears to have little effect on underlying cartilage stresses.