Extrusion of pyrophosphate into extracellular media by osteoarthritic cartilage incubates.

The distribution of calcium pyrophosphate mineral phase, almost exclusively confined to articular cartilage in chondrocalcinosis, and the high level of pyrophosphate (PPi) ion relative to serum in synovial fluid in patients with either chondrocalcinosis or advanced osteoarthritis led to an investigation of whether cartilage cells elaborate PPi ions. Incubates of articular cartilage from young rabbits but not from mature rabbits, as well as growth plates cartilage, released PPi into incubation media during a 4h period. Control rabbit ear cartilage and synovial membrane elaborated negligible amounts of PPi. The PPi was shown to be undialyzable but could be dissociated from the alkaline phosphatase by ultracentrifugation. In 16 patients with osteoarthritis, a substantial output of PPi by samples of articular cartilage from the knee was demonstrated. It is postulated that either rapid cell division and matrix synthesis found in the base of ulcerating osteoarthritic cartilage or remodeling calcified sites are the source of the PPi in such osteoarthritic cartilage. It is further hypothesized that this PPi output accounts at least in part for the elevated PPi levels found in synovial fluid of patients with osteoarthritis.

Demonstration of macromolecular inhibitors of calcification and nucleational factors in fluid from calcifying sites in cartilage.

An extracellular fluid phase (C(f1)), aspirated by micropuncture techniques from the hypertrophic cell zone of calcifying epiphyseal certilage, has been characterized in a calcifying system in vitro in respect to the behavior of sedimenting and supernatant fractions after high speed ultracentrifugation. To perform these tests on the starting samples of 20 nl of C(f1), macroscopic analytical methods were scaled down for the identification of relevant organic components, including hexuronic acid and proteinpolysaccharides (PPL). The mineral accretion system was designed to simulate physiologic conditions in the calcifying cartilage septa of normal rats, and the mineral used for seeding was an immature calcium phosphate similar to native cartilage mineral. Normal C(f1) or its dilutions in synthetic lymph up to 1:4 completely prevented mineral accretion in vitro. The inhibitory action was localized to the sedimented fractions after ultracentrifugation and could be destroyed by incubation with trypsin or hyaluronidase. The sediment of C(f1) contained 2 mg of hexuronic acid per ml of C(f1) and gave a strong reaction of identification for a light fraction of PPL by fluorescent antibodies to rat PPL. PPL fractions were tested in the same mineral accretion systems as C(f1) and exhibited responses similar to those of C(f1). Also, there was evidence of a mineral phase in C(f1) of normal rats, in C(f1) of rats with healing rickets, but not in C(f1) of untreated rachitic rats. These results are interpreted to indicate that certain PPLs function as an inhibitor of crystal growth at extracellular sites premonitory to calcification. Evidence for a low density inhibitor of mineral accretion was found in normal serum but not in C(f1).

Partition of calcium, phosphate, and protein in the fluid phase aspirated at calcifying sites in epiphyseal cartilage.

A reproducible method, adapted from renal micropuncture techniques, was developed for sampling 10-40 mmul of a clear fluid from epiphyseal cartilage of normal or rachitic rats in vivo, either from the hypertrophic cell zone (C(f1)) or surface resting cell cartilage (L(f1)). Characterization of this fluid depended upon quantitation of protein, total inorganic phosphate (P(it)), total calcium (Ca(t)), nucleotide, and hemoglobin in volumes of 20 mmul. Established methods for macroscale measurements of each of these parameters have been modified to permit direct spectrophotometric readings on samples of 10(-10)-10(-11) g. The fluid from hypertrophic and peripheral resting cell cartilage was of an extracellular nature as evidenced by a high chloride and sodium, as well as low potassium, protein, and nucleotide content. The pH of fluid isolated from endochrondral plates in vivo was measured under oil as a function of P(CO2) and the computed bicarbonate was elevated above concurrent serum levels. After ultracentrifugation of C(f1) of normal, rachitic, and healing rachitic animals, nonprotein-bound calcium (Ca(f)) and phosphate (P(if)) were determined on supernatant fluids. The hypertrophic cell cartilage fluid of rachitic rats was distinguished by a high ratio C(f1)/serum of P(if). This ratio returned to normal during treatment of rickets. The upper limit for ionic activity A(1) Ca(++) x A HPO(4) (=) was too low to initiate precipitation of brushite or dicalcium phosphate but was in a range of supersaturation in respect to crystalline apatites. Thus these data are consistent with initiation of calcification by heterogeneous nucleation of mineral in the septal matrix but can be reconciled alternately with a precipitation mechanism only if the site of initial mineral phase separation is outside the septal matrix.

Evidence for a role of proteinpolysaccharides in regulation of mineral phase separation in calcifying cartilage.

Our previous studies have indicated the presence of a macromolecular inhibitor of in vitro mineral growth, as well as a mineral nucleational agent in extracellular matrix fluid aspirated by micropuncture methods from epiphyseal hypertrophic cell cartilage. In this report, new miniaturized methods were used to extract proteinpolysaccharide complexes (PPC) from cartilage, to isolate a light fraction (PPL-C), and further, to separate it into R1, R2, and SR2 subfractions. These methods were applied to PPL-C complexes separated from microdissected epiphyseal cartilages and to cetylpyridinium chloride (CPC) precipitates of extracellular matrix fluid aspirated from similar cartilages. Most of all of the inhibitory action on an in vitro system of mineral growth shown by whole cartilage PPL-C and by cartilage fluid PPC obtained from noncalcifying sites was contained in the R2 fraction which represented (1/4)-[unk] of the total hexuronate. The R2 fraction was diminished or absent from calcified cartilage fluids and from whole calcified epiphyseal septa. The ratio R1 + R2: SR2 ranged from 0.37 to 0.71 in the fluids and whole tissue samples of noncalcified cartilages. The R2 fraction was distinguished from SR2 by a 2- to 3-fold higher protein: hexuronate ratio. These data are interpreted to indicate that the inhibitory R2 fraction was degraded or otherwise inactivated at the zone of provisional calcification and that this inhibitor participates in the physiological mechanism that regulates endochondral calcification.

Localization of collagenase in the growth plate of rachitic rats.

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

Mechanism of interaction of digitalis with estradiol binding sites in rat uteri.

Digitalis preparations have a weak estrogenic effect in man. The data in animals are equivocal. We have studied.the biologic effect of both digitoxin and digoxin on the rat uterus in vivo and the interaction of these drugs with the rat uterus estrogen receptor in vitro. Digitoxin and estradiol significantly increased the uterine weight of immature rats, while digoxin did not. The interaction of digitoxin and digoxin with the rat uterus estrogen cytosol receptor was studied using protamine sulfate precipitation and dextran-coated charcoal (DCC) assays. Both methods gave a Kd for the estradiol-receptor interaction between 0.8-3.1 X 10(-9) M (n = 20). Digitoxin at concentrations of 0.5-2.0 X 10(-6) M significantly inhibited the binding of estradiol to the specific or saturable binding sites with minimal inhibition of hormonal binding to nonspecific sites. The binding was competitive with a calculated Ki for digitoxin of 5.2-7.8 X 10(-7) M (n = 18). Digoxin failed to inhibit estradiol binding to the receptor protein in vitro. We conclude that digitoxin probably acts directly as a weak estrogen and that this effect probably explains the estrogen-like side effects seen with digitoxin therapy in man.

Interaction of spironolactone and digitalis with the 5 alpha-dihydrotestosterone (DHT) receptor of rat ventral prostate.

The aldosterone antagonist, spironolactone, has been shown to block the effects of exogenously administered androgen in rat. This suggests that interaction of the drug with androgen at the target tissues may occur. In this paper we have studied the possible interaction of spironolactone with the 5alpha-dihydrotestosterone (DHT)1 receptor of rat ventral prostate. The competitive receptor assay used involves precipitation of the 105,000 X g supernatant of the homogenized tissue with protamine sulfate, removal of the unprecipitated cytosol, and incubation of the precipitate in the presence of the appropriate [3H]DHT steroid solution at 0 C for 18 hours. Using this method the Kd (dissociation constant) for DHT in the rat prostate was in the range of 1.9-4.0 X 10(-9)M and the binding capacity was 0.21 pmol/mg protein. Spironolactone was found to interfere with the binding of DHT to the precipitated cytosol and displayed an estimated Kd of 1.3-4.6 X 10(-8)M. Several digitalis preparations were similarly studied. Digitoxin and digitoxigenin also interfered with the binding of [3H]DHT and had an estimated Kd of 0.8-3.6 X 10(-8)M. Digoxin interacted less strongly and its estimated Kd was 10(-6)M. We believe these results suggest an interaction of spironolactone and digitalis with the DHT receptor and may help explain some of their antiandrogenic actions in the rat and in man.

Evidence that testosterone can suppress pituitary gonadotropin secretion independently of peripheral aromatization.

Testosterone (T) was given to normal men with and without the concomitant administration of the aromatase inhibitor, delta 1-testolactone (Teslac), to examine the role of peripheral aromatization of T in gonadotropin regulation. When T was administered alone by continuous iv infusion (15 mg/day for 4 days), serum T increased 3-fold (P less than 0.01) and estradiol (E) increased by 50% (P less than 0.01). These changes were associated with a 50% decrease in serum LH and FSH concentrations (P less than 0.01). When T was infused into men taking Teslac (2000 mg/day), serum T levels doubled (P less than 0.01), but E levels did not change (13.4 +/- 1.5 vs. 13.5 +/- 1.0 pg/ml; P = NS). This pattern of plasma steroids, increased T and unchanged E, was also associated with significantly decreased serum LH and FSH concentrations (14.5 +/- 0.4 vs. 8.0 + 0.4 mIU/ml and 9.9 +/- 2.5 vs. 5.8 +/- 0.1 mIU/ml, respectively; P less than 0.01). These data support the hypothesis that T or one of its metabolites can modulate LH and FSH secretion independently of peripheral aromatization to E.

Interaction of digitalis and spironolactone with human sex steroid receptors.

Spironolactone and digitoxin have previously been shown to interact with cytosol androgen and estrogen receptors, respectively, in the rat. The interaction of digitoxin with human uterine cytosol estrogen binding protein and spironolactone with human prostate and newborn prepuce cytosol dihydrotestosterone (DHT) binding protein has been analyzed in this study. Specific estradiol binding was found only in premenopausal uteri. The dissociation constant for estradiol binding was 0.6--2.3 X 10(-9) M (n - 12). Digitoxin in concentrations varying between 0.5--2.0 X 10(-6) M inhibited specific estradiol binding with a Ki of 2.0--7.3 X 10(-7) M (n = 9). The dissociation constants for DHT and the human androgen cytosol binding protein in prostate and newborn prepuce were 0.27--3.0 X 10(-8) M (n = 12) and 0.6--2.0 X 10(-8) M (n = 5), respectively. Spironolactone at concentrations of 0.3--2.0 X 10(-6) M competitively inhibited this binding with an affinity about one order of magnitude less than that of DHT. Digitoxin and spironolactone did not displace estradiol and DHT, respectively, from testosterone-estrogen binding globulin in male or female plasma. The interaction of digitoxin with the human uterus estrogen binding protein and spironolactone with the human prostate and prepuce androgen binding protein is similar to our previous observations in the rat, and may explain the weak estrogenic effects of digitoxin and spironolactone in man.

SC 25152: A potent mineralocorticoid antagonist with reduced affinity for the 5 alpha-dihydrotestosterone receptor of human and rat prostate.

It has previously been shown that spironolactone possesses antiandrogenic activity in the rat and interacts with rat prostate 5 alpha-dihydrotestosterone cytoplasmic receptors to block the nuclear uptake of this hormone. Current evidence suggests that this androgen receptor interaction may be an important mechanism through which spironolactone causes endocrine side effects in rat and man. We have analyzed the interactions of several spirolactone analogs with the androgen receptor of human and rat prostate and the mineralocorticoid receptor of human and rat kidney. One analog, SC 25152, was found to have considerably reduced affinity for the prostate 5 alpha-dihydrotestosterone receptor [Ka = 24 +/- 1% and 19 +/- 6% (mean +/- SE) in the human and rat, respectively, of the Ka for spironolactone] while maintaining similar affinity for the mineralocorticoid receptors of human and rat kidney [Ka = 113 +/- 37% and 86 +/- 7% (mean +/- SE), respectively, of the Ka for spironolactone]. These findings would predict this analog to have reduced antiandrogenicity at equivalent therapeutic doses.

Diminution of large pituitary tumor after replacement therapy for primary hypothyroidism.

Thyroid-stimulating, hormone-producing tumors of the pituitary may be associated with primary hypothyroidism. The case presented here illustrates the rapid resolution of a large pituitary tumor after thyroid hormone replacement in a patient with primary hypothyroidism.

Effects of vitamin D metabolites on healing of low phosphate, vitamin D-deficient induced rickets in rats.

A model of low-phosphate, vitamin D-deficient rachitic rats was used to compare the effects of 1 alpha(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3 on cartilage and bone. The rats were maintained for 3 weeks on a high-calcium, low-phosphate, vitamin D-deficient diet, during which period they developed severe rickets. The rachitic rats were injected for 2 or 3 consecutive days with a physiologic dose of either metabolite. Other littermates were given a single dose of 50,000 IU of cholecalciferol in combination with a normal diet. 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 microscopic studies of the growth plate cartilage and bone. Treatment with 1 alpha (OH)D3 or with 1,25(OH)2D3, in spite of increasing Ca and P levels in the Cfl, induced only partial healing of the rickets. In contrast, 24,25(OH)2D3 or vitamin D with a normal diet resulted in complete morphologic and biochemical healing of the rickets. Transmission electron microscopic (TEM) studies have shown partial mineralization of the wide hypertrophic zone of the growth plate following treatment with 1 alpha(OH)D3 or with 1,25(OH)2D3. Mineralization was more complete with 24,25(OH)2D3 treatment. The results of this study emphasize the importance of 24,25(OH)2D3 for normal endochondral bone formation and mineralization.

Structural differences between two populations of articular cartilage proteoglycan aggregates.

To determine if articular cartilage contains structurally distinct populations of proteoglycan aggregates, we extracted and purified proteoglycans from canine knee cartilage under associative conditions. Equilibrium density gradient centrifugation separated three proteoglycan populations, on the basis of differences in sedimentation velocity, into groups of 21, 106, and 270 S. Electron microscopic examination showed that the 21 S samples contained free aggrecan molecules and clusters of aggrecan molecules, with a mean of five aggrecan molecules per cluster. The 106 and 270 S samples contained proteoglycan aggregates consisting of central hyaluronan filaments with multiple attached aggrecan molecules. The two populations of aggregates did not differ in mean aggrecan length or in the spacing of aggrecan molecules along the hyaluronan filaments, but the slower sedimenting aggregates (106 S) had significantly shorter hyaluronan filaments as measured by electron microscopy (mean hyaluronan length, 400 compared with 1,162 nm) and one-third as many aggrecan molecules per aggregate (mean number of aggrecan molecules per aggregate, 15 compared with 44). This study shows that articular cartilage contains aggrecan clusters and two structurally distinct populations of proteoglycan aggregates. The differences between the two types of aggregate, in particular the number of aggrecan molecules per aggregate, may reflect differences in their assembly, stability, or turnover and give them different mechanical and biological properties.

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.

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.

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.

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.

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.

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.

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.