Biomedical subjects

J C Pita

Publications and source records attributed to J C Pita.

At least 19 recordsLinked to original sources

PubMed1994

Mechanical properties of canine articular cartilage are significantly altered following transection of the anterior cruciate ligament.

The compressive, tensile, and swelling properties of articular cartilage were studied at two time periods following transection of the anterior cruciate ligament in the knee of greyhound dogs. An experimental protocol was designed to quantify the essential equilibrium and biphasic material properties of cartilage in tension, compression, and shear, as well as the parameters of isometric swelling behavior. All properties were measured at several sites to elicit differences between sites of frequent and less frequent contact. Hydration was determined at each site and was compared with the material properties of cartilage from corresponding sites. There were extensive changes in all compressive, tensile, and swelling properties of cartilage after transection of the anterior cruciate ligament. Twelve weeks after surgery, the intrinsic moduli were reduced significantly in compression (approximately 24% of control values), tension (approximately 64%), and shear (approximately 24%), and the hydraulic permeability was elevated significantly (approximately 48%). Significant increases in hydration (approximately 9%) also were observed, as well as a strong correlation of hydration with hydraulic permeability. The pattern of these changes was not found to differ with site in the joint, but significant differences were observed in the magnitude of change for cartilage from the femoral groove and the femoral condyle. The pattern and extent of changes in the material properties following transection of the anterior cruciate ligament indicate that altered loading of the joint severely compromises the overall mechanical behavior of articular cartilage. The observed loss of matrix stiffness in compression, tension, and shear is associated with increases in the deformation of the solid matrix, a diminished ability to resist swelling, and the increase in hydration observed in this study. The increased swelling and elevated water content were related directly to the increase in hydraulic permeability; this suggests an associated loss of fluid pressurization as the load support mechanism in the degenerated cartilage. Without a successful mechanism for repair, damage to the solid matrix may progress and lead to further degenerative changes in the biochemistry, morphology, and mechanical behavior of articular cartilage.

Animals↗

PubMed1994

Centrifugal and biochemical comparison of proteoglycan aggregates from articular cartilage in experimental joint disuse and joint instability.

Two models involving altered joint loading were compared with regard to their effects on the biochemical composition and proteoglycan aggregate structure of articular cartilage. Disuse atrophy was created in greyhound dogs by nonrigid immobilization of the right knee in 90 degrees of flexion, and joint instability was created by transection of the anterior cruciate ligament. Similarities and differences between the two experimental groups at two different time periods were examined to investigate why joint instability induces progressive and irreversible changes to the articular cartilage, whereas joint disuse induces changes that may be reversible when the joint is remobilized. The following studies were performed on the cartilage from all experimental and control groups: (a) compositional analyses to determine water, uronate, and hydroxyproline contents; (b) high performance liquid chromatography for detection of hyaluronan and chondroitin sulfates; and (c) centrifugation analyses of nondissociatively extracted and purified proteoglycans to isolate and quantify the populations of monomers and slow and fast-sedimenting families of aggregates. In general, all cartilage was found to have a decreased ratio of proteoglycan to collagen after 4 weeks of disuse, and this ratio returned to control values at 8 weeks. In contrast, cartilage had an elevated ratio of proteoglycan to collagen as well as increased hydration at 12 weeks after transection of the anterior cruciate ligament. The most striking contrast between the two models was the finding of an approximately 80% decrease in the content of hyaluronan at both time periods after transection of the anterior cruciate ligament, with no evidence of a change after disuse. The results of centrifugation analyses indicated a significant decrease in the quantity of proteoglycan aggregates in both models. However, this decrease was associated primarily with a loss of slow-sedimenting aggregates after disuse and a loss of both slow and fast-sedimenting aggregates after transection of the anterior cruciate ligament. Furthermore, the population of fast-sedimenting aggregates was depleted to a greater extent than that of the slow-sedimenting aggregates. The preservation of fast-sedimenting aggregates as well as hyaluronan after periods of joint disuse but not joint instability suggests a possible mechanism for the reversibility of cartilage changes. Although the proteoglycan aggregates were depleted after disuse atrophy, it is possible that an aggregate-depleted matrix could recover when normal proteoglycan synthesis is resumed. In contrast, although synthesis may be maintained or elevated after transection of the anterior cruciate ligament, the matrix may not be repopulated with aggregates because there is an insufficient amount of hyaluronan.

Analysis of Variance↗

PubMed1991

Proteoglycans nondissociatively extracted from different zones of canine normal articular cartilage: variations in the sedimentation profile of aggregates with degree of physiological stress.

Proteoglycans were extracted and purified without dissociation (a-A1 preparations) from superficial and deeper layers of high weight-bearing (HWA) and low weight-bearing (LWA) areas of dog normal articular cartilage. These proteoglycans were then characterized by velocity gradient centrifugation. In each of the 4 different topographical regions, the weight average sedimentation coefficients related strongly with total hexuronate content of the tissue. In the superficial layers, almost all aggregates had low sedimentation coefficients: the aggregates were smaller and less abundant in LWA than in HWA. The deeper layers contained an additional population of faster sedimenting aggregates which appeared smaller and less abundant in LWA than in HWA. Quantification and functional characterization of aggregates as well as in vitro aggregating studies showed that the topographical differences in size and content of aggregates were related to differences in content of hyaluronate and link protein in the a-A1 preparations. Superficial a-A1 specimens contained twice as much hyaluronate as deeper a-A1 preparations and their hyaluronate content increased with degree of physiological stress. Deeper a-A1 specimens from weight-bearing areas did not differ in their hyaluronate content but experiments assessing the saturation with link protein of these different a-A1 preparations suggested that specimens from HWA contained more active link than those from LWA. In contrast, the capacity of aggregation of a-A1D1D1 proteoglycan monomers as well as the molecular weight (Mr = 5 x 10(5) and aggregating capacity of hyluronate molecules appeared very similar in all a-A1 preparations from areas of articular cartilage. It is hypothesized that the synthesis of the three constituents necessary for aggregate formation (i.e. proteoglycan monomers as well as hyaluronate and link protein molecules) increases with degree of physiological load and that aggregation helps to maintain within cartilage the high concentration of proteoglycans that are essential for its biomechanical functions. The reported topographical variations in the distribution of proteoglycan aggregates reflect probably a maximal adaptation of the physiologic and biomechanical properties of the matrix to meet the high stress levels experienced by the articular cartilage in vivo.

Animals↗

PubMed1991

Treatment of osteoarthritis with tiaprofenic acid: biochemical and histological protection against cartilage breakdown in the Pond-Nuki canine model.

Experimental and cage matched control animals were sacrificed 12 weeks after production of ligamentous instability in the right knee, and biochemical studies were performed on eroded OA and normal articular cartilage. Significant protection was afforded by tiaprofenic acid administered orally at 15 mg/kg body weight. Chondroprotection was manifested by reduction of fast sedimenting proteoglycan aggregates, as well as retention of hyaluronate content, and favorable proteoglycan aggregate S value levels. This agent showed significant chondroprotective action under the conditions of these studies.

Administration, Oral↗

PubMed1990

Kinetics of proteoglycans and cells in growth plate of normal, diabetic, and malnourished rats.

The metabolism of proteoglycans in normal growth plate and the changes in growth plate morphology induced by diabetes and malnutrition were studied in rats. The proteoglycans had a significantly faster turnover (half-life measured with [35S]sulfate labeling: 25-30 h) than the cells in the growth plate. Morphometric studies showed significant reductions of cell number, zone height, and [3H]thymidine incorporation in growth plates from rats with untreated streptozotocin-induced diabetes compared to normal rats. Similar, although less pronounced alterations were observed in malnourished, nondiabetic rats. Disaggregation and degradation of proteoglycans are probably necessary prerequisites for calcification. Our data indicate that the proteoglycans are in a dynamic state of rapid biosynthesis and degradation throughout the growth plate with a shift in the balance at the calcification front toward less synthesis and more degradation.

Animals↗

PubMed1989

Macro and micro rate zonal analytical centrifugation of polydisperse and slowly diffusing sedimenting systems in isovolumetric density gradients. Application to cartilage proteoglycans.

A method to study the polydispersity of zonally sedimenting and slowly diffusing macromolecules or particles in isokinetic or isovolumetric density gradients is presented. First, a brief theory is given for predicting the zonal profile after a "triangular" (or "inverse") zone is centrifuged. This type of zone is essential to preserve hydrodynamic stability of the very slowly diffusing polydisperse solutes. It is proven, both by semitheoretical considerations and by computer calculations, that the resulting concentration profile of macrosolute is almost identical with that obtainable with a rectangular zone coextensive with the triangular one and carrying the same total mass. Next, practical procedures are described for the convectionless layering of very small triangular zones (50 microL or less). The linearity and stability of the zones are experimentally tested and verified. Finally, the method is applied to cartilage proteoglycan preparations that included either the monomeric molecules only or both the monomeric and the aggregated ones. The zonal results are compared with those obtained by using conventional boundary sedimentation. The two sets of results are seen to coincide fairly well, thus proving that the present technique can add to preparative zonal centrifugation the analytical precision of boundary sedimentation. A multimodal polydisperse system is suggested to describe the aggregated proteoglycan macromolecules.

Animals↗

PubMed1989

Centrifugal characterization of proteoglycans from various depth layers and weight-bearing areas of normal and abnormal human articular cartilage.

Ultracentrifugal polydispersity differential [g(S)] distributions were determined for the proteoglycans of various postmortem human articular cartilage samples extracted from six lateral patellar grooves in nondissociative conditions after mild collagenase digestion of the tissue. The samples consisted of 53 slices (250 microns thick), from normal, mildly fibrillated, and extensively ulcerated knee joints. When statistically analyzed in various subgroupings, the obtained average sedimentation coefficients and polydispersity profiles supported the following conclusions: (a) loss of proteoglycan aggregation and sedimentability is confirmed to be a primary sign of cartilage matrix degradation; (b) higher S values for proteoglycans of the high weight (HW)-bearing areas and lower values for those of the low weight (LW)-bearing areas were a typical finding in normal cartilage samples; (c) inversion of this pattern was indicative of matrix degradation, suggesting that the HW regions are more affected than the LW-bearing areas; (d) the average S value distribution across cartilage thickness tended to resemble the corresponding proteoglycan content versus distance from articular surface; and (e) the deepest cartilage layer had, in most cases, the smallest amount of aggregates while the highest average sedimentability was observed at the middle zone of the normal samples. In the discussion, a role of proteoglycan aggregation for providing a means to "pack" more proteoglycans within the collagen meshwork and to control the generation of osmotic pressure gradients is suggested.

Adult↗

PubMed1989

Changes in the sedimentation profile of proteoglycan aggregates in early experimental canine osteoarthritis.

Osteoarthritis was induced in 12 normal dogs by severing of the anterior cruciate ligament of the right knees, the left knees serving as sham operated controls. The animals were killed at 7 and 14 weeks postsurgery. The total hexuronate, and thus proteoglycan, content of the articular cartilage of operated knees remained unaltered during the period of study. After pretreatment with a highly purified collagenase and in the presence of selected protease inhibitors, a higher proportion of the tissue hexuronate could be extracted from the different topographical areas of osteoarthritic joints under non dissociative conditions (70-75% versus 55-65% for control knees). The nondissociatively recovered osteoarthritic proteoglycans (a-A1 preparations) displayed progressive and consistent changes in their sedimentation profile. First, the size of the fast sedimenting or more saturated aggregates appeared to be reduced in the different regions of osteoarthritic joints at 7 weeks postoperatively. The disappearance of the faster sedimenting mode as well as a dramatic increase in the proportion of monomers were only detected in the topographical zones exhibiting the most severe surface damage and histologic abnormalities at 14 weeks postsurgery. The proteoglycan molecules present as "free" or "nonaggregated" monomers in a-A1 preparations recovered from normal and osteoarthritic cartilage at different time periods after surgery were separated from their corresponding aggregates by rate zonal centrifugation in isokinetic cesium sulfate gradient. Although they were severely depleted in keratan sulfate, the purified "free" and "aggregated" osteoarthritic monomers appeared to be normal in terms of aggregating capacity and size distribution, and were therefore not degraded. This progressive changes in size distribution of proteoglycan aggregates in the early stages of experimental canine osteoarthritis could contribute significantly to the biochemical and biomechanical alterations of osteoarthritic cartilage.

Animals↗

PubMed1988

Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans.

Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.

Animals↗

PubMed1988

EHDP-induced rachitic syndrome in rats is not reversed by vitamin D metabolites.

To examine whether either of the two known active vitamin D metabolites 1,25(OH)2D3 or 24,25(OH)2D3 could reverse the mineralization defect induced by 1-hydroxyethylidene-1,1-bis phosphonate (EHDP), a model of EHDP-induced rickets was used. Rats at the age of 31 days were injected for 10 consecutive days with EHDP (10 mg/kg). Other littermates were treated with a combination of EHDP and either 1,25(OH)2D3 or 24,25(OH)2D3 or were treated following 10 days of EHDP, with either of the vitamin D metabolites for an additional 72 hr. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscope studies of growth plate cartilage and bone. EHDP-treated rats revealed signs of typical rickets, manifested by widened growth plates and impaired bone mineralization. Transmission electron microscope (TEM) examination revealed matrix vesicles distributed throughout the growth plate; however, there appeared to be an arrest of the spread of the crystals at the provisional zone of calcification. Treatment with either 1,25(OH)2D3 or 24,25(OH)2D3 failed to reverse the rachitic condition of the animals. Serum calcium blood levels were elevated in the 1,25(OH)2D3 and EHDP-treated group. 1,25(OH)2D3 and 24,25(OH)2/D3 further increased the already elevated serum alkaline phosphatase levels observed in EHDP rats, although the increase observed with 1,25(OH)2D3 was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Progressive depletion of hyaluronic acid in early experimental osteoarthritis in dogs.

The hyaluronic acid (HA) content of articular cartilage was studied in early experimental osteoarthritis (OA) in 16 normal dogs. The anterior cruciate ligament in the right knees of the dogs was transected; their left knees served as sham operated controls. The animals were killed at 7 and 14 weeks postsurgery. Although their total hexuronate, and thus proteoglycan, content remained unaltered during the period of study, the different weight-bearing areas of the OA knees displayed a progressive and significant decrease in HA content. We found no differences in the molecular weight and in vitro aggregating capacity of the HA molecules from OA cartilage versus those from control cartilage. This early relative depletion of HA could contribute significantly to the biochemical alterations of OA cartilage. Furthermore, it appears to be a good parameter for the differentiation of changes related to OA and changes related to aging.

Animals↗

PubMed1988

Quantification and characterization of hyaluronic acid in different topographical areas of normal articular cartilage from dogs.

Normal articular cartilage from adult dogs was analyzed for hyaluronate, hexuronate and hydroxyproline. The low weight-bearing areas of both tibial plateaus and femoral condyles displayed a higher collagen content and a lower proteoglycan content than the regions of maximum contact. Both superficial and deeper layers contained more hyaluronate in areas of maximum than of minimum contact. On the other hand, in each weight-bearing area, the proportion of hyaluronate relative to total proteoglycan content appeared twice as much in the superficial layers than in their corresponding underlying zones. The molecular weight and in vitro aggregating capacity of the hyaluronate molecules were however quite similar in the different topographical areas of the articular tissue.

Animals↗

PubMed1988

Aspiration and characterization of predentin fluid in developing rat teeth by means of a micropuncture and micro-analytical technique.

A fluid phase was aspirated in vivo and in vitro from predentin or pulp of developing rat teeth by means of a micropuncture technique. Pooled aspirates (approx. 2 nL) were analyzed for P, Na, K, Ca, Mg, and S by electron probe microtechniques (Lechene and Warner, 1979). Compared with pulp fluid, currently and previously studied cartilage fluids, as well as serum, predentin fluid showed elevated K, depressed Na, Cl, and Ca, as well as increased P. Statistical analysis was possible for only a few groups of comparisons among the elemental profiles. Ultrastructural examination of the aspiration site and of the aspirates showed no evidence of contamination with cell organelles or other formed elements. The micropuncture technique used was a critically precise and laborious procedure; possible contamination with intracellular fluid could not be avoided. The consistently low Mg concentration found in the aspirates, however, supports our view that the samples were primarily extracellular.

Animals↗

PubMed1987

Biochemical characterization of fracture callus proteoglycans.

The changes in proteoglycan molecules during the initial stages of fracture healing in rats were characterized. Following extraction of callus proteoglycan components with dissociative solvents, the components were purified in a cesium chloride density gradient. The recovered proteoglycans were characterized with respect to their molecular size distribution using gel filtration chromatography and a centrifugal transport methodology. During this early healing period, a decrease was observed in the relative proportion of the aggregate and in the hydrodynamic size and sedimentation coefficients of these molecules. While some molecular degradation could have occurred during the early stages of fracture healing, the dominant change of the proteoglycan molecules seemed to be disaggregation. No significant difference was observed in the proportion of aggregates reformed when exogenous hyaluronate and link glycoproteins were allowed to interact with the two corresponding monomer preparations. The molecular changes of the proteoglycan molecules seem to parallel those occurring during endochondral calcification of rat epiphyseal cartilage.

Animals↗

PubMed1987

Tensile properties of human knee joint cartilage. II. Correlations between weight bearing and tissue pathology and the kinetics of swelling.

The nonequilibrium or kinetic swelling behavior of normal, fibrillated, and osteoarthritic (OA) (removed from total knee joint replacements) human knee joint cartilage has been measured using our isometric tensile apparatus (ITA). We found that large local variations exist in the manner with which human knee joint cartilage swells, including anisotropic effects, inhomogeneities, and dependence on local biochemical composition and pathological condition. The ITA provides three convenient biomechanical parameters--peak stress (sigma p), stress relaxation (sigma R), and diffusion coefficient (D)--to quantify the kinetics of swelling. We used these parameters to quantify and differentiate the kinetic swelling behavior of normal, fibrillated, and osteoarthritic cartilage, as well as the swelling behavior of cartilage from high and low weight-bearing areas. Also, these kinetic swelling parameters correlated very well, though by varying degrees, with such biochemical measures as collagen/proteoglycan ratio, hexosamine content/wet weight, and hydroxyproline content/dry weight, providing important insight into the mechanisms and processes involved during the course of swelling. Hence, the kinetic swelling behavior of cartilage should be used to provide important information not obtainable from equilibrium swelling studies.

Body Water↗

PubMed1986

Characterization of the proteoglycans recovered under nondissociative conditions from normal articular cartilage of rabbits and dogs.

Pretreatment of articular cartilage with a highly purified collagenase in the presence of selected protease inhibitors allowed the extraction under nondissociative conditions of 65% of the tissue hexuronate. Extracted proteoglycans were purified by two successive equilibrium centrifugations in Cs2SO4 and CsCl, respectively, and then characterized by their sedimentation properties. The use of labeled proteoglycan preparations demonstrated that no detectable degradation was introduced by the new extraction procedure. When applied to growth cartilage of rachitic rats the sedimentation profile of the purified proteoglycans was practically identical to that of the proteoglycan molecules recovered by micropuncture-aspiration. Proteoglycans were extracted from normal articular cartilage of rabbits and dogs with either the new procedure or 4.0 M guanidine HCl. The purified aA1 and A1 preparations were characterized by their sedimentation properties. The aA1 contained a higher proportion of aggregates which sedimented as two distinctive populations of molecules. This bimodal distribution of the aggregates was never observed in the A1 preparations even when the dissociative extraction was performed after collagenase pretreatment of cartilages. The two extraction procedures, however, extracted the same proteoglycan monomers since the aA1-D1 and A1-D1 preparations had similar biochemical composition and g(s) distribution functions. These observations and additional in vitro aggregation studies suggested that the differences in the size and proportion of aggregates between the aA1 and A1 preparations result from a more efficient recovery of link glycoproteins in nondissociative extractions that could have determined two structurally different hyaluronate molecules.

Animals↗

PubMed1986

Tensile properties of human knee joint cartilage: I. Influence of ionic conditions, weight bearing, and fibrillation on the tensile modulus.

The flow-independent (intrinsic) tensile modulus of the extracellular matrix of human knee joint cartilage has been measured for normal, fibrillated, and osteoarthritic (removed from total knee joint replacements) cartilage. The modulus was determined in our isometric tensile apparatus and measured at equilibrium. We found a linear equilibrium stress-strain behavior up to approximately 15% strain. The modulus was measured for tissues from the high and low weight-bearing areas of the joint surfaces, the medial femoral condyle and lateral patello femoral groove, and from different zones (surface, subsurface, middle, and middle-deep) within the tissue. For all specimens, the intrinsic tensile modulus was always less than 30 MPa. Tissues from low weight-bearing areas (LWA) are stiffer than those from high weight-bearing areas (HWA). The tensile modulus of the ECM correlates strongly with the collagen/proteoglycan ratio; it is higher for LWA than for HWA. Osteoarthritic cartilage from total knee replacement procedures has a tensile stiffness less than 2 MPa.

Adult↗

PubMed1985

Boundary centrifugation in isovolumetric and isokinetic cesium sulfate density gradients: application to cartilage proteoglycans and other macromolecules.

A boundary sedimentation methodology is described that avoids plateau dilution and simplifies the calculation of centrifugal parameters. The technique is designed for the preparative ultracentrifuge and uses a newly developed sectorial cell. It is based on previous developments of the transport method and depends on isokinetic or isovolumetric Cs2SO4 density and viscosity gradients. These gradients are prepared with a single-chamber mixing device, and the only two parameters required for their calculations are presented in a tabulated form for general use with most available rotors and cell sizes. Conditions are specified (1) to assure that the density and shape of the sedimenting molecules remain invariant through the selected electrolytic gradient, (2) to monitor the gradient profiles, and (3) to verify attainment of isokinetic or isovolumetric sedimentations. A set of equations is presented to calculate the average and transport sedimentation coefficients and the differential sedimentation coefficient distribution for both the isokinetic and isovolumetric centrifugal regimes. The method was applied to slowly diffusing polydisperse proteoglycan monomers, to a paucidisperse DNA from bacteriophage PM2, and to a diffusible monodisperse system (purified bovine serum albumin). In all cases, the expected results were obtained.

Animals↗