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D S Howell

Publications and source records attributed to D S Howell.

At least 19 recordsLinked to original sources
PubMed1992

Enhancement of osteoinduction by vitamin D metabolites in rachitic host rats.

Diaphyseal bone from normal Sprague-Dawley rats was delipidated in chloroform-methanol and demineralized in 0.6 N HCl at 4 degrees C. The bones were then implanted for 7-28 days into rats made rachitic by a low-phosphate, vitamin D-deficient diet (VDP-) for 3 weeks. Bones from VDP- and normal rats were also implanted into normal hosts. When normal rats were used as the host environment, a consistent sequence of cartilage induction and bone formation was observed. Demineralized rachitic bone (RB) implanted into normal host rats resulted in cartilage and bone induction similar to that seen for normal bone (NB) implants. Transmission electron microscopy of RB in normal hosts revealed morphologically normal chondrocytes and cartilage matrix with normal mineralization. In contrast, implantation of NB in VDP- hosts resulted in delayed chondrogenesis and lack of calcification. Furthermore, similar results were observed when RB was implanted into VDP- hosts. Treatment of VDP- hosts with either 1 alpha-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 did not accelerate the sequential appearance of precartilage or cartilage. However, 24,25-(OH)2D3 administered alone or in combination with 1 alpha-OHD3 significantly increased the amount of calcified cartilage observed at 2 weeks postimplantation compared to implants from either untreated VDP-hosts or those treated only with 1 alpha-OHD3. New bone formation was observed at 4 weeks postimplantation in all vitamin D-treated groups as determined by von Kossa staining or direct electron microscope examination. There was no apparent difference in the quantitative or qualitative bone formed within the various vitamin D-treated groups. Serum calcium and phosphorus levels were lower and alkaline phosphatase levels were higher in VDP- hosts compared with normal animals or those treated with vitamin D metabolites. The results of this study show a reduction in the capacity of progenitor cells in VDP- rat hosts to respond to osteoinductive factor(s). This impaired response appears to be corrected by vitamin D metabolites.

24,25-Dihydroxyvitamin D 3↗
PubMed1992

Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans.

This study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and beta-glucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Animals↗
PubMed1992

Matrix vesicles contain metalloproteinases that degrade proteoglycans.

This study explored whether extracellular matrix processing enzymes are present in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. It was found that there was a differential distribution of enzyme activities related to the cartilage zone from which the cells were isolated. There was a 3-fold enrichment of total and active acid metalloproteinase in growth zone chondrocyte (GC) matrix vesicles whereas no enrichment in enzyme activity was observed in resting zone chondrocyte (RC) matrix vesicles. Total and active neutral metalloproteinase were similarly enriched 2-fold in GC matrix vesicles. TIMP, plasminogen activator and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found. The data indicate that matrix vesicles are selectively enriched in enzymes that degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Animals↗
PubMed1992

A mini review: proteoglycan aggregate profiles in the Pond-Nuki dog model of osteoarthritis and in canine disuse atrophy.

The Pond-Nuki dog model of osteoarthritis has characteristics which seem to mimic the human disease in early stages, particularly with respect to progressive changes in the cartilage matrix. Aggregating proteoglycans were studied using novel extraction and ultracentrifugation methods designed to separate very large macromolecules. With these methods two large peaks of proteoglycan (PG) aggregates (PGA-1 and PGA-2) were separated in preparative amounts and were shown to have unequivocal differences in composition in many respects. The profiles of these peaks have been studied as a function of joint location, topographic site, cartilage layer, presence of cartilage atrophy versus osteoarthritis, as well as treatment of the animals with various agents. Both link protein (essential for forming link-protein stabilized aggregates) and hyaluronate are required to regenerate normal aggregate profiles from the deficient aggregate fractions obtained from osteoarthritic cartilage. Canine proteoglycan link-stabilized aggregates (PGA-2) are confined to the middle and deep zone of cartilage. We believe that their reduction or elimination in the Pond-Nuki model results from a disturbance or loss of functional link protein (and hyaluronate), thereby weakening the middle and deep cartilage layers.

Animals↗
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↗
PubMed1979

Cartilage proteoglycan alterations in an experimentally induced model of rabbit osteoarthritis.

Size distribution of cartilage proteoglycans (PG) extracted from control and osteoarthritic rabbit knees after partial meniscectomy was analyzed. In normal control knees, about 30% of PG molecules were in aggregate form and average sedimentation constant was 60S. No aggregates were found in osteoarthritic cartilage, whether ulcer, rim about ulcer, or distant normal-appearing cartilage was examined. Weight average sedimentation constants for PG subunits were similar to controls, 15S. Up to 70% of guanidinium-extractable PG could be extracted from osteoarthritic cartilage by using 0.5M guanidine HC1 (GuHCl); sedimentation characteristics of extracted PG were similar to those using 4.0M GuHCl. Absence of aggregates is consistent with a disorder of link protein, hyaluronic acid, or PG subunit hyaluronic acid binding sites. Absence of subunit degradation was an enexpected finding. The demonstrated ability of 0.5 M GuHCl to extract large amounts of PG from osteoarthritic cartilage will allow study of cartilage proteoglycans in their native nondissociated state.

Animals↗
PubMed1979

Metal-dependent neutral proteoglycanase activity from monolayer-cultured lapine articular chondrocytes.

Neutral proteoglycanase and other protease activity from cellular and CM fractions of monolayer-cultured rabbit articular chondrocytes were studied. The cellular fraction comprising soluble cytoplasmic enzymes possessed concentration-dependent elastase-like esterase activity and activity against trypsin and chymotrypsin synthetic substrates but had little caseinase activity. The 20% ammonium sulfate precipitate of CM possessed more neutral caseinase activity than the 60% ammonium sulfate precipitate and the bulk of activity against the synthetic substrates. Activity against bovine nasal septum PG was present in these fractions. Both the 20% and 60% ammonium sulfate fractions reduced the viscosity and the S of the PG substrate. This activity was incompletely inhibited by preincubation with either 5 mM o-phenanthroline or 10 mM EDTA, indicating that it was paritally metal-dependent. The activity in the cellular fraction was also partially inhibited by o-phenanthroline but more so by EDTA. These data indicate that chondrocytes synthesize and secrete into the culture medium neutral proteoglycanase(s) capable of initiating degradation of PG derived from the neutral pH cartilage matrix. The inhibitory profiles, together with recent evidence of enzymes with similar activity extracted from cartilage suggested that the proteoglycanase enzyme(s) may occur in multiple forms.

Animals↗
PubMed1976

Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH.

Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromotography and characterized by disc electrophoresis, inhibition patterns, and action of proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, alpha-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathespins B1, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.

Cartilage, Articular↗
PubMed1976

Possible role of extracellular matrix vesicles in initial calcification of healing rachitic cartilage.

Fluid (20-30 nl) was aspirated by a modified renal micropuncture technique from vitamin D-, phosphate-deficient rats. The fluid revealed a mineral forming agent which could be sedimented at 140,000 X g for 8 hours, was resistant to acid demineralization, but was destroyed by heating, freezing and thawing as well as sonication, and blocked by phospholipase A at 10-5 M but not at 10-7 M. Electron microscopic study of the fluid sediment revealed images consistent with matrix vesicles. These data are consonant with the view that matrix vesicles, their remnants, or closely associated structures comprised the mineral forming agent.

Animals↗
PubMed1975

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.

Alkaline Phosphatase↗
PubMedDate not supplied

Pyrophosphate release by osteoarthritis cartilage incubates.

Incubates of articular cartilage from young but not mature rabbits, as well as growth plate cartilage, elaborated PPi into basel Eagle's medium during a 4-hour period. Control rabbit synovial membrane and ear cartilage elaborated negligible amounts of PPi. The PPi was shown to be nondialyzable 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. The present authors believe that either rapid cell division and matrix synthesis found in the base of ulcerating osteoarthritic cartilage or remodeling calcified sites comprise the origin of the PPi in such osteoarthritic cartilage. They further theorize that this PPi output accounts for a considerable fraction, if not all, of the elevated PPi levels found in synovial fluid of patients with osteoarthritis.

Aged↗