Functional adaptation of articular cartilage from birth to maturity under the influence of loading: a biomechanical analysis

REASONS FOR PERFORMING STUDY: The concept of functional adapatation of articular cartilage during maturation has emerged from earlier biochemical research. However, articular cartilage has principally a biomechanical function governed by joint loading. OBJECTIVES: To verify whether the concept of functional adaptation can be confirmed by direct measurement of biomechanical properties of cartilage. HYPOTHESIS: Fetuses have homogeneous (i.e. site-independent) cartilage with regard to biomechanical properties. During growth and development to maturity, the biomechanical characteristics adapt according to functional (loading) demands, leading to distinct, site-dependent biomechanical heterogeneity of articular cartilage. METHODS: Osteochondral plugs were drilled out of the surface at 2 differently loaded sites (Site 1: intermittent impact-loading during locomotion, Site 2: low-level constant loading during weightbearing) of the proximal articular cartilage surface of the proximal phalanx in the forelimb from stillborn foals (n = 8), horses of age 5 (n = 9) and 18 months (n = 9) and mature horses (n = 13). Cartilage thickness was measured using ultrasonic, optical and needle-probe techniques. The osteochondral samples were biomechanically tested in indentation geometry. Young's modulus at equilibrium, dynamic modulus at 1 Hz and the ratios of these moduli values between Sites 1 and 2 were calculated. Age and site effects were evaluated statistically using ANOVA tests. The level of significance was set at P<0.05. RESULTS: Fetal cartilage was significantly thicker compared to the other ages with no further age-dependent differences in cartilage thickness from age 5 months onwards. Young's modulus stayed constant at Site 1, whereas at Site 2 there was a gradual, statistically significant increase in modulus during maturation. Values of dynamic modulus at both Sites 1 and 2 were significantly higher in the fetus and decreased after birth. Values for both moduli were significantly different between Sites 1 and 2 from age 18 months onwards. The ratio of values between Sites 1 and 2 for Young's modulus and dynamic modulus showed a gradual decrease from approximately 1.0 at birth to 0.5-0.6 in the mature horse. At age 18 months, all values were comparable to those in the mature horse. CONCLUSIONS: In line with the concept of functional adaptation, the neonate is born with biomechanically 'blank' or homogeneous cartilage. Functional adaptation of biomechanical properties takes place early in life, resulting in cartilage with a distinct heterogeneity in functional characteristics. At age 18 months, functional adaptation, as assessed by the biomechanical characteristics, has progressed to a level comparable to the mature horse and, after this age, no major adaptations seem to occur. POTENTIAL RELEVANCE: Throughout life, different areas of articular cartilage are subjected to different types of loading. Differences in loading can adequately be met only when the tissue is biomechanically adapted to withstand these different loading conditions without injury. This process of functional adaptation starts immediately after birth and is completed well before maturity. This makes the factor of loading at a young age a crucial variable, and emphasises the necessity to optimise joint loading during early life in order to create an optimal biomechanical quality of articular cartilage, which may well turn out to be the best prevention for joint injury later in life.     

Differences in the topographical distribution of articular cartilage degeneration between equine metacarpo- and metatarsophalangeal joints

REASONS FOR PERFORMING STUDY: The equine metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joints, although having virtually the same geometrical appearance, differ in the prevalence of joint pathologies, such as osteochondral fragmentation, and in biomechanical behaviour. The recently developed cartilage degeneration index (CDI) technique offers a possibility to assess quantitatively differences in cartilage degeneration between these joints and to compare these with known differences in biomechanics and clinical observations. OBJECTIVES: To compare the topographical distribution of articular cartilage degeneration across the proximal articular surface of the proximal phalanx (P1) in the equine fore- and hindlimb. METHODS: In 24 distal hindlimbs from 24 horses, articular cartilage degeneration of the proximal articular surface of P1 was quantified using the CDI. Overall CDI value (CDI(P1)) and CDI values of 6 areas of interest were determined: the medial dorsal surface (mds), lateral dorsal surface (lds), medial central fovea (mcf), lateral central fovea (lcf), medial plantar surface (mps) and lateral plantar surface (lps). The joints were divided into 4 equally sized groups of increasing CDI(P1) values. From an existing CDI database of MCP joints, 24 joints were selected with matching CDI(P1) values to the MTP joints and CDI values for the same areas of interest were determined. RESULTS: In both the MCP and MTP joints, highest CDI values were determined at the dorsal articular surfaces. Values were not significantly different between fore- and hindlimbs. In contrast to the MCP joint, CDI values at the plantar joint margin were significantly higher compared to CDI values in the central sites in the MTP joint. CDI values for the plantar surfaces of P1 were significantly higher than those for the palmar surfaces in the forelimb in joints with advanced stages of OA; and values for the central regions of P1 were significantly lower in the hindlimb compared with the forelimb in joints with severe OA. CONCLUSIONS: In both fore- and hindlimbs, initial cartilage degeneration started at the dorsal articular margin of P1. There was a major difference in the spread of cartilage degeneration; in the forelimb both the central and palmar parts are about equally involved, whereas in the hindlimb the plantar parts were significantly more and the central parts significantly less involved. These differences can be linked to differences in biomechanical loading reported elsewhere. POTENTIAL RELEVANCE: This study supports the hypothesis that differences in biokinematics between fore- and hindlimbs are associated with differences in the development of cartilage degeneration and other joint pathologies such as osteochondral fragmentation in the MCP and MTP joints. This information is indispensable for a better understanding of the dynamic nature and progression of these joint disorders and may be of help when monitoring the effects of therapeutic interventions and preventative measures.     

In situ and ex vivo evaluation of an arthroscopic indentation instrument to estimate the health status of articular cartilage in the equine metacarpophalangeal joint

OBJECTIVE: To evaluate an arthroscopic indentation instrument (Artscan 200) for assessment of the health status of equine articular cartilage. STUDY DESIGN: In vitro experiment using equine isolated proximal phalanx (P1) specimens. SAMPLE POPULATION: P1 specimens from 39 horses (aged 1.5-22 years). METHODS: Reproducibility was tested by determination of the coefficient of variation (CV). Dynamic modulus and cartilage degeneration index (CDI) values were measured at 2 predefined sites (site 1, joint margin; site 2, joint center) to assess the accuracy and to evaluate the relation with surface integrity. RESULTS: CV was 9.0%. A significant decrease in indenter force was identified when dynamic modulus values decreased to <2.5 MPa (range of tested samples 0.9-8.1 MPa) and when CDI values at site 1 increased to >50% (range 5.4-72.8%). CONCLUSIONS: Technique reproducibility was adequate but accuracy was limited. The device identified degeneration-associated decreases in cartilage stiffness only when the mechanical properties of the cartilage were considerably changed. CLINICAL RELEVANCE: Usefulness of this indentation instrument during arthroscopic surgery would be limited in the initial phase of OA-like cartilage degeneration, but may yield important information in more advanced OA.     

Influence of age, site, and degenerative state on the speed of sound in equine articular cartilage

OBJECTIVE: To determine the speed of sound (SOS) in equine articular cartilage and investigate the influence of age, site in the joint, and cartilage degeneration on the SOS. SAMPLE POPULATION: Cartilage samples from 38 metacarpophalangeal joints of 38 horses (age range, 5 months to 22 years). PROCEDURE: Osteochondral plugs were collected from 2 articular sites of the proximal phalanx after the degenerative state was characterized by use of the cartilage degeneration index (CDI) technique. The SOS was calculated (ratio of needle-probe cartilage thickness to time of flight of the ultrasound pulse), and relationships between SOS value and age, site, and cartilage degeneration were evaluated. An analytical model of cartilage indentation was used to evaluate the effect of variation in true SOS on the determination of cartilage thickness and dynamic modulus with the ultrasound indentation technique. RESULTS: The mean SOS for all samples was 1,696 +/- 126 m/s. Age, site, and cartilage degeneration had no significant influence on the SOS in cartilage. The analytical model revealed that use of the mean SOS of 1,696 m/s was associated with maximum errors of 17.5% on cartilage thickness and 70% on dynamic modulus in an SOS range that covered 95% of the individual measurements. CONCLUSIONS AND CLINICAL RELEVANCE: In equine articular cartilage, use of mean SOS of 1,696 m/s in ultrasound indentation measurements introduces some inaccuracy on cartilage thickness determinations, but the dynamic modulus of cartilage can be estimated with acceptable accuracy in horses regardless of age, site in the joint, or stage of cartilage degeneration.     

Accuracy of diagnostic arthroscopy for the assessment of cartilage damage in the equine metacarpophalangeal joint

REASONS FOR PERFORMING STUDY: There are many noninvasive diagnostic methods used for evaluating chronic progressive joint disease, but each has severe limitations in the detection of early articular cartilage damage. OBJECTIVES: To evaluate the accuracy of arthroscopy as a diagnostic method for the assessment of the severity of cartilage surface damage on the proximal articular margin of the equine first phalanx (P1). HYPOTHESIS: That arthroscopic assessment of the visible cartilage provides 1) a good indication of the integrity of the cartilage surface and 2) a good estimation of the status of the cartilage surface of the entire articular area of P1. METHODS: Arthroscopic examination of the dorsal pouch of the metacarpophalangeal joint was performed in the left front limbs of 74 slaughter horses (age 5 months to 23 years). The appearance of the visible cartilage of P1 was scored by 2 independent arthroscopists, using the SFA arthroscopic grading system. The joints were dissected after completion of the arthroscopic procedure. The cartilage degeneration index (CDIP1) was determined and used as a quantitative measure for the overall degree of cartilage surface deterioration on the articular area of P1. Further, CDI values were determined for the dorsal articular margin of P1 (CDIdam), i.e. the area that can be visualised with arthroscopy. The CDIdam values were classified into 3 groups (CDIdam<25%, minor lesions; 25%45%, severe lesions). Differences between the 2 arthroscopists were evaluated statistically in a nonparametric test and Pearson correlation coefficients (r) with matching P values were determined for the correlations between SFA and CDIdam and between CDIP1 and CDIdam. The level of significance was set at P<0.05. RESULTS: Differences between SFA scores of the 2 arthroscopists were not significant (P = 0.22). In the group of joints with minor cartilage changes, there was no correlation between SFA and CDIdam (r = 0.12; P = 0.71), but there was a significant correlation between CDIP1 and CDIdam (r = 0.95; P<0.01). In the group with moderate cartilage damage, there was an increase in correlation between SFA and CDIdam (r = 0.27; P = 0.09) and a decrease in the correlation between CDIP1 and CDIdam (r = 0.48; P<0.01). In the group with severe cartilage changes, there was a significant correlation between SFA and CDIdam (r = 058; P<0.01), but no significant correlation between CDIP1 and CDIdam (r = 0.43; P = 0.06). CONCLUSIONS: Arthroscopic assessment of cartilage lesions on the proximal articular surface of P1 in joints with minor cartilage damage leads to an underestimation of the actual damage because proteoglycan depletion and light cartilage fibrillation cannot be detected arthroscopically. In cases with mild cartilage damage, the status of the cartilage surface of the visible area of P1 is a good representation of the status of the entire articular surface. In cases with severe cartilage lesions, there is an overestimation of real damage. In such joints, the arthroscopic scoring system provides reliable information, but the visible area is not representative of the entire articular surface. POTENTIAL RELEVANCE: From a practical viewpoint, it can be stated that the arthroscopic grading of visible lesions on the equine P1 gives the best impression of overall cartilage damage in joints with moderately severe cartilage lesions. It should be realised, however, that this is the result of an underestimation due to the shortcomings of the grading system, which is neutralised by an overestimation due to the fact that the severity of lesions on the visible area of P1 is not representative for the entire articular surface.     

Quantification and age-related distribution of articular cartilage degeneration in the equine fetlock joint

REASONS FOR PERFORMING STUDY: The equine fetlock joint has the largest number of traumatic and degenerative lesions of all joints of the appendicular skeleton. OBJECTIVE: To gain insight into the distribution of cartilage degeneration across the articular surface in relation to age in order better to understand the dynamic nature and progression of osteoarthritis (OA). HYPOTHESIS: That there would be a specific age-related distribution pattern of cartilage degeneration in the equine metacarpophalangeal joint. METHODS: The proximal articular cartilage surfaces of the first phalanges (P1) of 73 slaughter horses (age range 0.4-23 years) with different stages of osteoarthritis were scored semiquantitatively on a 0 to 5 scale and also assessed quantitatively using the cartilage degeneration index (CDI(P1)), which ranges from 0 to 100%. Furthermore, CDI values were determined for special areas of interest; medial dorsal surface (CDI(mds)), lateral dorsal surface (CDI(lds)), medial central fovea (CDI(mcf)) and lateral central fovea (CDI(lcf)). Correlations were calculated for CDI(P1) values and CDI values at the specific areas of interest with macroscopic scores and with age. RESULTS: There was a high correlation between the semiquantitative macroscopic score and the quantitative CDI(P1) values (r = 0.92; P < 0.001). A macroscopic score of 0 (i.e. no obvious cartilage degeneration) corresponded with a CDI(P1) mean +/- s.e. value of 25 +/- 2.8% and a macroscopic score of 5 (i.e. severe cartilage degeneration in localised areas) with a mean +/- s.e. value of 38.1 +/- 7.9%. There was a moderate but highly significant correlation between the CDI(P1) value and the age of the horses (r = 0.41; P < 0.001). Highest CDI values were calculated for the medial dorsal surface (from 10.6 +/- 2.8% at macroscopic Grade 0 to 63.1 +/- 8.4% at Grade 5). At the lateral dorsal surface, these values were 5.9 +/- 1.4% and 47.2 +/- 10.4%, respectively. The CDI(mcf) and CDI(lcf) were significantly lower (P < 0.05) than the CDI(mds) and CDI(lds) at all grades. The CDI(mcf) ranged from 1.0 +/- 2.9% at Grade 0 to 43.7 +/- 9.1% at Grade 5; laterally, these values were 1.5 +/- 2.6% and 15.2 +/- 6.2%, respectively. CONCLUSIONS: CDI grading increased from lateral to medial and from central to dorsal. This specific distribution pattern confirms the heterogeneous nature of the OA process and strongly supports an important role for biomechanical loading, superimposed on age-related changes, in the spread of the disorder over the joint. POTENTIAL RELEVANCE: Knowledge of the development of OA across the articular surface is essential for understanding the dynamic nature and progression of the disease and can form a basis for improvements in diagnostic and therapeutic approaches to degenerative joint disease.     

Early exercise advances the maturation of glycosaminoglycans and collagen in the extracellular matrix of articular cartilage in the horse

REASON FOR PERFORMING STUDY: Training at a very young age may influence the characteristics of the collagen network of articular cartilage extracellular matrix (ECM) in horses. OBJECTIVES: To investigate whether increasing workload of foals results in significant changes in the biochemical composition of articular cartilage ECM. METHODS: Thoroughbred foals (n = 33) were divided into 2 different exercise groups from age 10 days-18 months. One group (PASTEX; n = 15) was reared at pasture; the other (CONDEX; n = 18) underwent a specific additional training programme that increased workload by 30%. At mean age 18 months, 6 animals from each group were subjected to euthanasia. The proximal articular surface of the proximal phalanx of the right hindlimb was examined for the presence of damage using the cartilage degeneration index (CDI). Samples were taken from 2 sites with known different loading patterns. Slices were analysed for DNA, glycosaminoglycans (GAG), collagen and post translational modifications of collagen (formation of hydroxylysylpyridinoline [HP] and pentosidine crosslinks, and hydroxylysine [Hyl]), and exercise groups and different sites compared. RESULTS: There were no differences in CDI between PASTEX and CONDEX animals, indicating the absence of extra joint damage due to the exercise regimen. There were site-related differences for most biochemical variables, corroborating earlier reports. All biochemical variables showed differences between PASTEX and CONDEX groups at one of the sites, and some at both. GAG and collagen levels were lower in the CONDEX group whereas Hyl, HP crosslinks and pentosidine crosslinks were higher. CONCLUSIONS AND POTENTIAL RELEVANCE: A measurable effect of the conditioning exercise was demonstrated. The margin between too much and too little work when training foals may be narrower than intuitively presumed.     

Equine carpal articular cartilage fibronectin distribution associated with training, joint location and cartilage deterioration

Processes involved in equine carpal osteochondral injury have not been established. In other species, fibronectin appears important in chondrocyte-matrix interactions, and levels are increased in osteoarthritis. This investigation aimed to (a) describe fibronectin immunoreactivity in the middle carpal joint of 2-year-old Thoroughbreds, (b) determine topographical variations, (c) compare strenuously trained (Group 1) or gently exercised horses (Group 2) and (d) describe sites with early osteoarthritis. Group 1 (n = 6) underwent a 19 week high intensity treadmill training programme. Group 2 (n = 6) underwent 40 min walking until euthanasia. Dorsal and palmar sites on radial, intermediate and third carpal articular surfaces were prepared. Immunohistochemistry was performed using a biotin-streptavidin/peroxidase method. Cross-reactivity of rabbit antihuman fibronectin antiserum with equine fibronectin was confirmed using Western blotting. Results showed: (a) fibronectin was present primarily in pericellular and interterritorial matrix locations, (b) dorsal sites had zonal immunoreactivity compared to palmar sites, (c) Group 1 dorsal radial carpal cartilage had increased superficial staining compared to Group 2 and (d) fibrillated cartilage showed increased intracellular and local matrical immunoreactivity (superficial zone). These findings suggest topographical and exercise-related variations in fibronectin distribution, and indicate equine fibronectin is localised at sites of cartilage degeneration and released into the matrix by chondrocytes in the local area.     

New approach for quantitative assessment of articular cartilage degeneration in horses with osteoarthritis

OBJECTIVE: To evaluate a modified digital imaging technique for quantitative assessment of the grade of osteoarthritis across the proximal articular surface of the first phalanx in horses. SAMPLE POPULATION: 6 metacarpophalangeal (fet-lock) joint specimens from 6 horses with various stages of osteoarthritis. PROCEDURE: First phalanx specimens, together with 4 gray scale reference calibration targets, were positioned in a bath with the proximal articular cartilage surface submerged in saline (0.9% NaCl) solution. Digital images were obtained from the articular surface before and after staining with Indian ink. Computer-controlled gray level analysis of the nonstained and Indian ink-stained cartilage surfaces and gray scale reference calibration targets was performed by use of the mean pixel value (based on 255-gray scale). An increase in the mean pixel value after staining was used to calculate the cartilage degeneration index (CDI). RESULTS: The CDI of the proximal articular cartilage surface of the first phalanx specimens ranged from 9.2 +/- 5.7 (early stage osteoarthritis) to 41.5 +/- 3.6% (late stage osteoarthritis). The effect of repeating the measurement 6 times in nonstained (including repositioning) and stained specimens (including repositioning and restaining) was not significant. Up to 10 measurements of nonstained specimens could be made without refreshing the bath solution. In stained specimens, mean gray level increased significantly after the sixth measurement. CONCLUSIONS AND CLINICAL RELEVANCE: The modified digital imaging technique allowed quantitative assessment of cartilage degeneration across the articular cartilage surface. The CDI is the first quantitative measure for osteoarthritis-induced cartilage degeneration over an entire joint surface in horses.     

Relationship between synovial fluid levels of glycosaminoglycans, hydroxyproline and general MMP activity and the presence and severity of articular cartilage change on the proximal articular surface of P1

REASONS FOR PERFORMING STUDY: Osteoarthritis (OA) is one of the most prevalent and disabling chronic conditions affecting horses and leads to degeneration of articular cartilage. Diagnosis is based on clinical signs in combination with radiography, which is relatively insensitive and provides only an indication of accumulated damage. Alternative methods, such as molecular markers, are therefore needed that can quantitatively, reliably and sensitively detect osteoarthritic changes in the joints at an early stage of the disease. If such markers are to be used reliably, it is important to know the relationship between marker concentration and cartilage composition. OBJECTIVES: To study the relationship between cartilage composition, synovial fluid levels of glycosaminoglycans (GAGs), hydroxyproline (Hyp) and general matrix metalloproteinase (MMP) activity, and the presence and severity of articular cartilage damage on the articular surface of P1. METHODS: Synovial fluid (SF) was collected from the metacarpophalangeal joints of 60 mature horses, and levels of GAGs, Hyp and general MMP activity were determined. Further, GAG and denatured collagen content of the articular cartilage were determined at the dorsal articular margin of P1 (site 1) and central cavity (site 2). The presence and severity of cartilage change was quantified using the cartilage degeneration index (CDI), measured at the same 2 sites. Correlations between SF parameters, cartilage composition and degree of cartilage degeneration were sought using correlation analysis. RESULTS: There was no correlation between GAG or Hyp content of SF and the amount of GAGs or denatured collagen, respectively, in cartilage. In joints with moderate to severe cartilage damage, the GAG content of site 1 was significantly lower than in joints with no to minimal cartilage change (P = 0.005) and there was a negative correlation between the amount of denatured collagen and GAG content at site 1 in all joints (r = -039, P = 0.002). Further, in joints with moderate to severe cartilage damage, there was a significant positive correlation between MMP activity in SF and Hyp levels in SF (r = 0.72, P < 0.001) and CDI at sites 1 (r = 0.46, P = 0.03) and 2 (r = 0.43, P = 0.04). CONCLUSIONS: General MMP activity in joints with moderate to severe cartilage damage is related to the severity of those cartilage changes and to Hyp levels in SF. Glycosaminoglycan levels in SF are not directly related to MMP activity, GAG content of articular cartilage or severity of cartilage change. POTENTIAL RELEVANCE: Glycosaminoglycan levels in SF are not helpful for the early detection of cartilage lesions. In damaged joints, Hyp levels may give an indication of the severity of cartilage change as they are strongly related to MMP activity, but do not qualify as markers for the presence or absence of cartilage lesions.     

Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint

The aim of this study was to evaluate topographical differences in the biochemical composition of the extracellular matrix of articular cartilage of the normal equine fetlock joint. Water content, DNA content, glycosaminoglycan (GAG) content and a number of characteristics of the collagen network (total collagen content, levels of hydroxylysine- (Hyl) and the crosslink hydroxylysylpyridinoline, (HP) of articular cartilage in the proximal 1st phalanx (P1), distal 3rd metacarpal bone (MC), and proximal sesamoid bones (PSB) were determined in the left and right fetlock joint of 6 mature horses (age 5-9 years). Twenty-eight sites were sampled per joint, which included the clinically important areas often associated with pathology. Biochemical differences were evaluated between sampling sites and related with the predisposition for osteochondral injury and type of loading. Significant regional differences in the composition of the extracellular matrix existed within the joint. Furthermore, left and right joints exhibited biochemical differences. Typical topographic distribution patterns were observed for each parameter. In P1 the dorsal and palmar articular margin showed a significantly lower GAG content than the more centrally located sites. Collagen content and HP crosslinks were higher at the joint margins than in the central area. Also, in the MC, GAG content was significantly lower at the (dorsal) articular margin compared with the central area. Consistent with findings in P1, collagen and HP crosslinks were significantly lower in the central area compared to the (dorsal) articular margin. Biochemical and biomechanical heterogeneity of articular cartilage is supposed to reflect the different functional demands made at different sites. In the present study, GAG content was highest in the constantly loaded central areas of the joint surfaces. In contrast, collagen content and HP crosslinks were higher in areas intermittently subjected to peak loading which suggests that the response to a certain type of loading of the various components of the extracellular matrix of articular cartilage are different. The differences in biochemical characteristics between the various sites may help to explain the site specificity of osteochondral lesions commonly found in the equine fetlock joint. Finally, these findings emphasise that the choice of sampling sites may profoundly influence the outcome of biochemical studies of articular cartilage.     

Chondrolysis associated with cartilage canals of the epiphyseal cartilage of the distal humerus of growing pigs

The articular-epiphyseal (A-E) cartilage of the distal humeri of 7 pigs weighing 13.1 to 18.2 kg and of 3 pigs weighing 36.4 to 40.9 kg was studied. Frozen samples of A-E cartilage were stained for the presence of reduced nicotinamide adenine dinucleotide dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase, and uridine diphosphate galactose-4-epimerase. Additional frozen sections and paraffin-processed sections were stained using the Alcian blue-critical electrolyte concentration method, safranin O-fast green, and hematoxylin and eosin. An area of grossly visible, opaque A-E cartilage of the medial condyle corresponded to regions of chondrolysis of the epiphyseal cartilage. The chondrolytic regions contained chondrocytes that did not stain for enzymes, had reduced staining for proteoglycans in the matrix, and were located at the site where the A-E cartilage increased in thickness. Cartilage canals were associated with the chondrolytic areas. Cartilage canals in both groups of pigs were commonly in various stages of chondrification, some of which were associated with degenerative cartilage. The regions of chondrolysis may indicate sites of biomechanical weakness in the A-E cartilage during the transformation of the epiphyseal cartilage into bone.     

Mapping of donor and recipient site properties for osteochondral graft reconstruction of subchondral cystic lesions in the equine stifle joint

REASONS FOR PERFORMING STUDY: To improve osteochondral graft reconstruction of subchondral cystic lesions in the medial and lateral femoral condyles by matching the material properties of donor and recipient sites. OBJECTIVES: To measure biomechanical and biochemical parameters that influence the function and healing of osteochondral grafts used to reconstruct subchondral cystic lesions. HYPOTHESIS: Suitable donor sites are available within the stifle joint for reconstructing the femoral condyles, despite considerable regional property variation. METHODS: Fifty-six osteochondral cores were harvested from 6 distal femurs for initial studies that determined subchondral bone modulus of elasticity and ultimate stress. In a second study, 28 osteochondral cores were harvested from 6 distal femurs to measure cartilage aggregate modulus, thickness and sulphated glycosaminoglycan (sGAG) content. Using micro-CT imaging, subchondral bone mineral density and bone volume fraction were also measured. In both studies 2-dimensional contour plots using a bicubic interpolation method and normalised data were generated to allow visual comparison of joint surface characteristics. Statistical comparisons between donor and recipient site raw data were made using an ANOVA for repeated measures with a post hoc Tukey test. RESULTS: Material properties of cartilage and bone vary considerably over the surface of the stifle joint but the central region of the medial condyle, where subchondral cystic lesions freqdently occur, typically demonstrated bone strength and modulus values of the highest observed. Cartilage thickness and aggregate modulus were highest in the medial femoral condyle and axial aspect of the lateral condyle. CONCLUSIONS: Material properties of the grafts from the trochlear groove and axial aspect of the lateral trochlear ridge were the closest match for those found in the medial condyle, whereas properties of the lateral condyle were most similar to those found in the trochlear groove and axial aspect of the medial trochlear ridge.     

Evaluation of subchondral bone mineral density associated with articular cartilage structure and integrity in healthy equine joints with different functional demands

OBJECTIVE: To determine and correlate subchondral bone mineral density and overlying cartilage structure and tensile integrity in mature healthy equine stifle (low magnitude loading) and metacarpophalangeal (high magnitude loading) joints. ANIMALS: 8 healthy horses, 2 to 3 years of age. PROCEDURE: Osteochondral samples were acquired from the medial femoral condyle (FC) and medial trochlear ridge (TR) of the stifle joint and from the dorsal (MC3D) and palmar (MC3P) aspects of the distal medial third metacarpal condyles of the metacarpophalangeal joint. Articular cartilage surface fibrillation (evaluated via India ink staining) and tensile biomechanical properties were determined. The volumetric bone mineral density (vBMD) of the underlying subchondral plate was assessed via dual-energy x-ray absorptiometry. RESULTS: Cartilage staining (fibrillation), tensile moduli, tensile strength, and vBMD were greater in the MC3D and MC3P locations, compared with the FC and TR locations, whereas tensile strain at failure was less in MC3D and MC3P locations than FC and TR locations. Cartilage tensile moduli correlated positively with vBMD, whereas cartilage staining and tensile strain at failure correlated negatively with vBMD. CONCLUSIONS AND CLINICAL RELEVANCE: In areas of high joint loading, the subchondral bone had high vBMD and the articular cartilage surface layer had high tensile stiffness but signs of structural wear (fibrillation and low failure strain). The site-dependent variations and relationships in this study support the concept that articular cartilage and subchondral bone normally adapt to physiologic loading in a coordinated way.     

Measurement of articular cartilage stiffness of the femoropatellar, tarsocrural, and metatarsophalangeal joints in horses and comparison with biochemical data

OBJECTIVE: To determine normal cartilage stiffness values in different weight-bearing and non-weight-bearing areas of 3 different equine joints, and to evaluate the relationship between cartilage stiffness and glycosaminoglycan (GAG) and collagen content. STUDY DESIGN: Compressive stiffness of the articular cartilage was measured in 8 horse cadaver femoropatellar (FP), tarsocrural (TC), and metatarsophalangeal (MT) joints. Gross evaluation, collagen content, GAG content, and histologic appearance were assessed for each measurement location. ANIMALS: Eight equine cadavers (4 intact females, 4 castrated males; 7 Quarter Horse or Quarter Horse type, 1 Arabian; aged 4-12 years, weighing 400-550 kg). METHODS: The articular surfaces of 8 equine cadaver FP, TC, and MT joints were grossly evaluated for signs of articular cartilage pathology. Stiffness at preselected sites (FP joint-6 sites; TC joint-3 sites; MT joint-4 sites) was determined using an arthroscopic indentation instrument. Biochemical composition (collagen, GAG content) and histologic evaluation (modified Mankin score) were assessed for each measurement site. RESULTS: All cartilage from all sites evaluated was determined to be normal based on macroscopic and histologic assessments. No significant correlation between Mankin scores and cartilage stiffness values was observed. Site differences in cartilage stiffness were measured in all 3 joints (P<.001). GAG or collagen content had a significant positive correlation with stiffness values in 6 of 13 sites (P<.05, r>0.622, r2>0.387). CONCLUSION: Relative cartilage stiffness values measured in healthy equine joints are site dependent and can be measured using an indentation device intended for arthroscopic application. CLINICAL RELEVANCE: An indentation instrument provided an objective means of determining relative compressive stiffness of articular cartilage. Further research needs to be performed to confirm the site and joint differences observed in this study in clinically normal horses and to determine if the tester can be used clinically to predict articular cartilage pathology.     

Biomechanical characterisation of equine laryngeal cartilage

Reasons for performing the study: Upper airway obstruction is a common problem in the performance horse as the soft tissues of the larynx collapse into the airway, yet there is a paucity of information on biomechanical properties for the structural cartilage components. Objective: To measure the geometry and compressive mechanical properties of the hyaline cartilage to improve understanding of laryngeal function and morphology. Methods: A total of 11 larynges were harvested from Thoroughbred and Standardbred racehorses. During gross dissection, linear dimensions of the cricoid were obtained. From both the cricoid and arytenoid, specimens were cored to obtain 6 mm disc samples from 3 sites within the dorsal cricoid (caudal, middle and rostral) and 2 central sites in the arytenoids (inner, outer). The specimens were mechanically tested using radial confined compression to calculate the aggregate modulus and permeability of the tissue. The biomechanical data were analysed using a nested mixed effects model. Results: Geometrically, the cricoid has relatively straight walls compared to the morphology of human, ovine and canine larynges. There were significant observations of higher modulus with increasing age (0.13 MPa per year; P = 0.007) and stiffer cricoid cartilage (2.29 MPa) than the arytenoid cartilage (0.42 MPa; P<0.001), but no difference was observed between the left and right sides. Linear contrasts showed that the rostral aspect (2.51 MPa) of the cricoid was 20% stiffer than the caudal aspect (2.09 MPa; P = 0.025), with no difference between the arytenoid sites. Conclusions: The equine larynx is a well supported structure due to both the geometry and material properties of the cricoid cartilage. The hyaline structure is an order of magnitude higher in compressive modulus compared to the arytenoids and other hyaline‐composed tissues. Potential relevance: These characterisations are important to understand the biomechanics of laryngeal function and the mechanisms involved with surgical interventions.     

The distribution of cartilage oligomeric matrix protein (COMP) in equine carpal articular cartilage and its variation with exercise and cartilage deterioration

Based on previous studies where tendons receiving the most load have been shown to have the highest levels of cartilage oligomeric matrix protein (COMP), we hypothesized that COMP distribution in articular cartilage may be influenced by mechanical loading. This investigation aimed (a) to describe the pattern of COMP immunoreactivity in middle carpal joint cartilage of two-year-old Thoroughbred horses; (b) to determine topographical variations; (c) to compare high (group 1) and low (group 2) intensity training and (d) to describe COMP immunoreactivity at sites with early osteoarthritis.Group 1 (n =6) underwent a 19 week high-intensity treadmill training programme and group 2 (n =6) were given daily walking until euthanasia. Dorsal and palmar sites on radial and third carpal articular surfaces were prepared. Immunohistochemistry was performed with polyclonal rabbit anti-equine COMP antiserum using a biotin-streptavidin/peroxidase method. Results showed: (a) intracellular immunoreactivity was present in all cartilage zones, but the distribution of COMP staining within the matrix varied between cartilage zones; (b) differences in distribution between sites were not observed, but total COMP levels in exercised horses (n =2) did vary between sites with dorsal sites containing less COMP than palmar sites on the radial, intermediate and third carpal lateral facet; (c) group 1 cartilage showed marked interterritorial distribution in the deep layer compared to group 2 where staining was more generalized throughout the matrix and (d) fibrillated cartilage showed increased local immunoreactivity in the matrix. These findings demonstrate zonal variations in equine COMP distribution which may be influenced by loading.     

Regeneration of cartilage tissue by autologous chondrocytes transplantation for cartilage defects in a experimental bovine model

To evaluate the effects of chondrocytes transplantation on the regeneration of cartilage by intraarticular injection or injection into blood clots at cartilage defects, eight full-thickness cartilage defects were created surgically on the articular surface of each femoral trochlea of two calves. Autologous chondrocytes were isolated individually from the cartilage pieces collected at the creation of defects. And isolated cells were cultured in monolayers for proliferation. Cells were injected into synovial fluid (Group 2, n=11) or into the blood clots at the cartilage defects (Group 3, n=5) of the left femoropatellar joint on weeks 2 and 3, respectively after the operation. The defects (Group 1, n=16) of right femoropatellar joint were left untreated in the control group. After 14 weeks, repaired tissues were evaluated based on gross and histological examinations. In Group 3, more repaired tissues and a better interface between the repaired tissue and host cartilage were observed compared with the results for Groups 1 and 2. Moreover, cartilaginous tissue were observed more in defects of Group 3 than in defects of other groups. In conclusion, the present study suggests that the injection of cells into the blood clot at a cartilage defect might be applicable for the regeneration of damaged cartilage.     

Influence of site and age on biochemical characteristics of the collagen network of equine articular cartilage

OBJECTIVE: To determine variations in biochemical characteristics of equine articular cartilage in relation to age and the degree of predisposition for osteochondral disease at a specific site. SAMPLE POPULATION: Articular cartilage specimens from 53 horses 4 to 30 years old. PROCEDURE: Healthy specimens were obtained from 2 locations on the proximal articular surface of the first phalanx that had different disease prevalences (site 1 at the mediodorsal margin and site 2 at the center of the medial cavity). Water, total collagen, and hydroxylysine contents and enzymatic (hydroxylysylpyridinoline [HP]) and nonenzymatic (pentosidine) crosslinking were determined at both sites. Differences between sites were analyzed by ANOVA (factors, site, and age), and age correlation was tested by Pearson's product-moment correlation analysis. Significance was set at P< 0.01. RESULTS: Correlation with age was not found for water, collagen, hydroxylysine contents, and enzymatic cross-linking. Nonenzymatic crosslinking was higher in older horses and was linearly related to age (r = 0.94). Water and collagen contents and HP and pentosidine crosslinks were significantly higher at site 1. Hydroxylysine content was significantly lower at site 1. CONCLUSIONS: Except for nonenzymatic glycation, the composition of articular cartilage collagen does not change significantly in adult horses. A significant topographic variation exists in biochemical characteristics of the articular cartilage collagen network in equine metacarpophalangeal joints. These differences may influence local biomechanical properties and, hence, susceptibility to osteochondral disease, as will greater pentosidine crosslinks in older horses that are likely to cause stiffer and more brittle cartilage.     

Effects of intramuscular polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage.

The effect of intramuscular polysulfated glycosaminoglycan (PSG) on repair of cartilage injury was evaluated in eight horses. In each horse, one middle carpal joint had both a partial-thickness and a full-thickness articular cartilage defect created. In the contralateral middle carpal joint, chemical articular cartilage injury was created by intra-articular injection of 50 mg sodium monoiodoacetate (MIA). Horses were divided into two groups for treatment. Group 1 horses (control) received an intramuscular injection of normal saline every four days for a total of seven injections starting seven days after cartilage injury. Group 2 horses received 500 mg of PSG intramuscularly every four days for seven treatments starting seven days after cartilage injury. Horses were maintained for 12 weeks. Horses were evaluated clinically, and their middle carpal joints were evaluated radiographically and arthroscopically at the end of the study. Joint tissues were also collected and examined microscopically. The only significant difference between groups was slightly greater matrix staining intensity for glycosaminoglycans in the radiate articular cartilage layer in MIA injected and PSG treated joints. Partial-thickness defects had not healed and the predominant repair tissue in full-thickness defects was fibrous tissue. It was concluded that using this joint injury model, 500 mg PSG administered intramuscularly had no effect on the healing of articular cartilage lesions, and minimal chondroprotective effect from chemically induced articular cartilage degeneration.