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G Lust

Publications and source records attributed to G Lust.

At least 55 records · Page 3Linked to original sources
PubMed1995

Self-diffusion monitors degraded cartilage.

This article demonstrates that both the bulk water self-diffusion coefficient (D) and the spatially resolved variation in D for lesion canine cartilage due to osteoarthritis is increased by about 25% over that of surrounding cartilage. This increase in D can be mimicked by enzymatic degradation of cartilage with trypsin, hyaluronidase, and collagenase, or by mechanical means. However, it is established here using excised disks of living cartilage whose proteoglycan and collagen contents were manipulated by biochemical intervention in tissue culture that the diffusion measurement is not sensitive to the proteoglycan content of cartilage. Instead, self-diffusion appears to monitor mesoscopic (nonspecific) tissue damage. These results show that D, measured in a spatially resolved manner by pulsed field gradient nuclear magnetic resonance imaging, can localize regions of cartilage degradation.

Animals↗
PubMed1995

Alternative splicing of ED-A and ED-B sequences of fibronectin pre-mRNA differs in chondrocytes from different cartilaginous tissues and can be modulated by biological factors.

The alternative splicing of the ED-A and ED-B segments of fibronectin pre-mRNA was examined in epiphyseal, costal, and meniscal cartilage from 3-week-old beagles and in nasal, tracheal, articular, and meniscal cartilage from 1- and 2-year-old Labrador retrievers. In contrast to the 100% expression of ED-B(+) mRNA that has been reported for embryonic chick cartilage (Bennett, V.D., Pallante, K.M., and Adams, S.K. (1991) J. Biol. Chem. 266, 5918-5924), all cartilages studied expressed both the ED-B(+) and ED-B(-) forms of fibronectin mRNA with the exception of the trachea, in which expression was 100% ED-B(-). Of all cartilages studied, only the meniscus had detectable levels of ED-A(+) mRNA. Placing articular cartilage chondrocytes in primary monolayer culture dramatically up-regulated the expression of ED-A(+) mRNA to 25% of the total, and this expression was further increased by the addition of transforming growth factor beta 1 or fucoidan to the culture medium. The expression of ED-B(+) mRNA remained at about 18% in the cultured chondrocytes and was not further affected by either transforming growth factor beta 1 or fucoidan. In contrast, dibutyryl cyclic adenosine monophosphate decreased the relative expression of both the ED-A(+) and ED-B(+) forms of fibronectin pre-mRNA. We concluded that the expression of ED-B(+) fibronectin remains relatively high in chondrocytes from cartilaginous canine tissues (15-35%) with the exception of the trachea, in contrast to the less than 10% expression of ED-B(+) fibronectin reported for other non-fetal tissues.

Alternative Splicing↗
PubMed1995

Accumulation of fibronectin in articular cartilage explants cultured with TGF beta 1 and fucoidan.

Fibronectin is a glycoprotein involved in cell matrix interactions. In osteoarthritis, fibronectin levels in the lesion cartilage are elevated up to 20-fold above control levels. In these experiments, explants of disease-free cartilage cultured in the presence of a combination of TGF beta 1 and the sulfated fucopolysaccharide, fucoidan, accumulated fibronectin at levels comparable to those found in osteoarthritic lesions. TGF beta 1 increased fibronectin synthesis, most of which was released to the medium. The addition of fucoidan favored retention of the newly synthesized fibronectin within the matrix. The fibronectin which accumulated as a result of these treatments was similar to the fibronectin in normal and osteoarthritic cartilage with respect to the ED-B+ alternative splice form. No change in the proteoglycan content of the cartilage explants with elevated fibronectin levels was detected.

Animals↗
PubMed1995

Antibody specific for extra domain B of fibronectin demonstrates elevated levels of both extra domain B(+) and B(-) fibronectin in osteoarthritic canine cartilage.

A fusion protein containing the alternately spliced Extra Domain B [ED-B(+)] sequence of canine fibronectin was expressed in E. coli and the purified protein was used to produce an antibody specific for the ED-B(+) segment. This antibody recognized canine cartilage fibronectin but not canine plasma or synovial fluid fibronectin. Using this antibody, it was determined that ED-B(+) fibronectin increased proportionally with the total fibronectin in osteoarthritic cartilage. From this result, it was concluded that the chondrocyte is the source of much of the elevated fibronectin that appears in osteoarthritic cartilage.

Amidohydrolases↗
PubMed1995

Effects of stromelysin activity on proteoglycan degradation of canine articular cartilage explants.

We investigated whether stromelysin activity in the medium of canine articular cartilage explants is associated with proteoglycan degradation in these explants. Cartilage explants were treated with recombinant human interleukin 1 alpha (rh-IL-1 alpha), lipopolysaccharide, or canine monocyte-conditioned medium. Proteoglycan synthesis and degradation were measured. Metalloproteinase activity (inhibitable by tissue inhibitor of metalloproteinase 2) in the culture medium was measured by use of fluorimetry with a quenched fluorescent substrate. Western blots of the medium were probed with polyclonal antibodies to human stromelysin, collagenase, and gelatinase. Neither metalloproteinase activity nor proteoglycan degradation were inducible in canine cartilage explants treated with rh-IL-1 alpha. However, proteoglycan synthesis was significantly (P < 0.05) decreased by concentrations of 10 and 100 ng of rh-IL-1 alpha/ml. Metalloproteinase activity in the medium accompanied proteoglycan degradation of cartilage treated with lipopolysaccharide and monocyte-conditioned medium. The metalloproteinase released into the medium was identified as prostromelysin by results of western blotting.

Animals↗
PubMed1994

Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects.

Cartilage resurfacing by chondrocyte implantation, with fibrin used as a vehicle, was examined in large (12 mm) full-thickness articular cartilage defects in horses. Articular chondrocytes, isolated from a 9-day-old foal, were mixed with fibrinogen and injected with thrombin, in a 1:1 mixture, into 12 mm circular defects on the lateral trochlea of the distal femur of eight normal horses. The contralateral femoropatellar (knee) joint served as a control in which the defect was left empty. Synovial fluid from the femoropatellar joints was sampled on days 0, 4, 7, 30, 120, and 240 postoperatively. Groups of four horses were killed at 4 or 8 months postoperatively, and the repair tissue was evaluated by gross and histologic examination with use of hematoxylin and eosin and safranin O staining and by autoradiography. Biochemical analyses included quantitation of proteoglycan, total collagen, and type-II collagen in the repair tissue. Grossly, grafted defects had improved filling of the cartilage lesions; histologically, these areas consisted of differentiated chondrocytes in the deep and middle zones. The cellular arrangement in these zones resembled that of hyaline cartilage. The control defects contained poorly attached fibrous tissue throughout. Grafted tissue at 8 months had increased proteoglycan synthesis evident by both safranin O staining and autoradiography. Glycosaminoglycan quantitation by dye-binding assay confirmed a significantly elevated glycosaminoglycan content in grafted defects (58.8 micrograms/mg of dry weight) compared with control defects (27.4 micrograms/mg; p < 0.05). Similarly, the levels of chondroitin sulfate/dermatan sulfate was significantly elevated in the grafted defects, and this was the predominant glycosaminoglycan epitope present. There was a statistically significant (p < 0.05) increase in type-II collagen in the grafted tissue at 8 months (61.2% grafted; 25.1% control). This resurfacing attempt with use of allograft chondrocytes, secured in large full-thickness articular defects with polymerized fibrin, resulted in an improved cartilage surface in comparison with the control defects, a significantly greater aggrecan level, and a significantly higher proportion of type-II collagen.

Animals↗
PubMed1994

Cross-validation of cyanogen bromide-peptide ratios to measure the proportion of type II collagen in pepsin digests of equine articular cartilage, meniscus, and cartilage repair tissue.

Collagen type I and type II were purified from equine flexor tendon and articular cartilage, respectively. Equal amounts of these collagens were cleaved with cyanogen bromide, and 11 mixtures containing increasing proportions of type II collagen were separated in seven identical sodium dodecyl sulfate-polyacrylamide gels. The density of bands was measured in wet gels and the peak areas were used to form six ratios of peptide bands that had polynomial relationships with the known proportions of type I and type II collagen in the mixtures. Calibration curves for determining the proportion of type II collagen in the mixtures were constructed using ratios and combinations of ratios of peak areas. Cross-validation was used to identify calibration curves with the smallest squared prediction error or squared average prediction error for all combinations of ratios. Ratios of peak areas of each one of the seven gels were treated, in turn, as the "unknown," and a prediction was carried out using these unknowns and the ratios from the other six gels. Two ratios had the smallest squared average prediction error and calibration curves were computed for these ratios with all seven gels. These curves were used to estimate the proportion of type II collagen in the pepsin-soluble and the pepsin-resistant fractions of articular cartilage inner and outer meniscus, and cartilage repair tissue. Cross-validation enabled selection of the cyanogen bromide-peptide ratios for calibration curves that resulted in the most accurate estimation of the proportion of type II collagen in pepsin digests of tissues.

Animals↗
PubMed1994

Inhibition of relaxin-induced pubic symphyseal "relaxation" in guinea pigs by glycosaminoglycan polysulfates and pentosan polysulfate.

There are similarities between the actions of estrogen and relaxin on the connective tissues of the pubic symphysis and those of neutral proteases on cartilage in osteoarthritis, including cartilage hydration, proteoglycan loss, and dissolution of collagen fibers. We hypothesized that compounds known to inhibit cartilage breakdown in animal models of osteoarthritis, such as polysulfated GAGs, would also antagonize the actions of estrogen and relaxin that increase the laxity and mobility of the pubic symphyses of guinea pigs. Estrogen-primed guinea pigs were injected with relaxin or with relaxin and the test compound. The pubic symphyses were manually palpated 6 h later and the degree of mobility scored. Glycosaminoglycan polysulfates and pentosan polysulfate inhibited relaxin-induced pubic symphyseal relaxation, whereas other types of agents were without effect. The guinea pig pubic symphysis assay for relaxin may thus provide a novel rapid screening test for compounds with potential chondroprotective activity.

Animals↗
PubMed1994

Transmission of relaxin from lactating bitches to their offspring via suckling.

The 6-kDa polypeptide hormone relaxin (Rlx) has been identified in human and bovine milk, and we recently reported its presence in canine milk. We postulated that Rlx might be transferred via suckling to the newborn pups, where, by virtue of its known effects to increase the distensibility of the pelvic connective tissues, it could play a role in causing the excessive laxity of the capsule and ligaments of the coxofemoral joint that precedes the development of hip dysplasia in genetically predisposed animals. Rlx was found in the serum of dysplastic (HD+) bitches for up to 6 wk of lactation, whereas it was detected in the serum of nondysplastic (HD-) bitches for only 1-2 wk of lactation. Rlx concentrations in milk were up to 60-fold greater than in serum. Milk Rlx levels varied markedly, but were highest during the first week of lactation and decreased thereafter. There were no significant differences in milk Rlx concentrations between HD+ and HD- bitches. Although the source of Rlx in milk is unknown, it cannot be the ovary or uterus, since hystero-ovariectomy performed at the time of cesarean section did not eliminate Rlx from milk during subsequent lactation. In serum samples taken from newborn pups before suckling, there were significant quantities of Rlx, demonstrating that the hormone enters the fetus in utero. However, Rlx rapidly disappears from serum of pups prevented from suckling for five hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1994

Phenotype and biological activity of neonatal equine chondrocytes cultured in a three-dimensional fibrin matrix.

Equine neonatal chondrocytes were cultured in three-dimensional fibrin matrices under conditions of immediate implantation or implantation following monolayer culture for 6 days, and 3 cell concentrations (1 x 10(5), 1 x 10(6), and 5 x 10(6) chondrocytes/cm3). Equine fibrinogen was collected by cryoprecipitation and polymerized by use of activated bovine thrombin. The fibrin implants were harvested and analyzed histologically and biochemically at 3, 7, and 14 days after the chondrocytes were implanted in fibrin. The differentiation ratio (ratio of rounded, chondrocyte-like cells to stellate, fibroblast-like cells) was statistically higher for implants that received 5 x 10(6) precultured cells at all time periods than for implants that received 1 x 10(5) or 1 x 10(6) precultured cells. The differentiation ratio was statistically higher for implants that received 5 x 10(6) immediately implanted cells than for other implants at 7 days after implantation. At 14 days, implants that received 5 x 10(6) precultured chondrocytes had a higher differentiation ratio than did implants that received 5 x 10(6) chondrocytes that had not been precultured. Among implants that received precultured chondrocytes, total glycosaminoglycan and chondroitin sulfate content was lowest for implants that received only 1 x 10(5) cells. Among implants that received chondrocytes that had not been precultured, glycosaminoglycan content was not significantly different among the 3 cell concentrations, and chondroitin sulfate content was different only between implants that received 5 x 10(6) vs 1 x 10(6) cells. Only after the longest incubation period and at the highest cell concentration studied did preculturing of chondrocytes improve maintenance of phenotype. Preculturing did not appear to influence proteoglycan synthesis.

Animals↗
PubMed1994

Structure of equine type I and type II collagens.

Collagen type I was purified from equine skin and flexor tendon, and type II collagen was purified from equine articular cartilage. The proteoglycans in these tissues were extracted, using guanidine HCl; the collagens were solubilized, using pepsin digestion, then were selectively precipitated with NaCl. Gel electrophoresis indicated that the precipitates contained only type I or type II collagen. Amino acid analysis indicated that collagen constituted > 97% of the total protein in the precipitates. Hydroxylation of proline was 42.0 +/- 0.6% (mean +/- SEM) in alpha 1(I) and alpha 2(I), and was 48.1 +/- 1.3% in alpha 1(II) chains. The hydroxylation of lysine was 23.2 +/- 0.7% in alpha 1(I) and 34.1 +/- 0.9% in alpha 2(I) chains from tendon, and 49.6 +/- 4.3% in alpha 1(II) chains from cartilage. The cyanogen bromide (CB)-peptide patterns of chromatographically purified equine alpha 2(I) and alpha 1(II) chains were similar to those published previously for rat, bovine, and human alpha 2 and alpha 1 chains. However, the CB-peptide pattern of the equine alpha 1(I) chain resembled the guinea pig alpha 1(I) chain, which has no methionine between CB7 and CB6. Purified equine alpha 1(I)CB7,6 contained no methionine, methionine sulfoxide, or homoserine lactone. Mass of 42.26 kd was determined by use of mass spectrometry, and N-terminal sequence analysis established that the first 12 amino acids of this CB7,6 were identical to the sequence of human alpha 1(I)CB7.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1993

Evidence for a glycosaminoglycan chain on a portion of articular cartilage fibronectins.

Fibronectin heterogeneity is, in part, the result of post-translational modifications. In these experiments, cartilage fibronectins were purified by anion exchange chromatography, followed by gelatin affinity chromatography or immunoprecipitation, and, finally, sodium dodecyl sulfate--polyacrylamide gel electrophoresis (NaDodSO4 PAGE). A substantial, although variable, portion of the fibronectins from canine and equine cartilages of all ages required salt concentrations from 0.2 to 1.0 M for elution from DEAE-cellulose. This was in contrast to plasma fibronectin which eluted with 0.1 M NaCl, but these results were consistent with observations made on human cartilage by Brown and Jones (1990 J. Rheumatol. 17, 65-72). When cartilage explants were incubated with Na2 35SO4=, the cartilage fibronectins were sulfated and the fibronectins which eluted with high salt contained from 5- to 50-fold more radiosulfate than the fibronectins which eluted with 0.1 M NaCl. A fraction of the 35SO4= which copurified with the cartilage fibronectin and comigrated with it in NaDodSO4-PAGE could be removed by digestion with chondroitinase ABC. This suggested that a percentage of cartilage fibronectins are covalently linked to a chondroitin sulfate or dermatan sulfate chain and thus might also appropriately be called proteoglycans. Alternatively, there is a proteoglycan which binds so tightly to fibronectin that separation is not achieved even in the presence of urea, sodium dodecyl sulfate, and mercaptoethanol.

Animals↗
PubMed1993

Effect of compressive loading and unloading on the synthesis of total protein, proteoglycan, and fibronectin by canine cartilage explants.

Full-thickness canine articular cartilage explants were subjected to compressive loads equivalent to a uniaxial stress of 0.025-1.2 MPa. A single cycle (18 h) of unconfined compression resulted in inhibition of total protein, proteoglycan, and fibronectin synthesis. The inhibition of fibronectin synthesis followed that of total protein synthesis. The magnitude of inhibition increased nonlinearly with increasing load levels. The signal that depressed synthesis remained effective for several hours after removal of load, but by 24 h proteoglycan synthesis had partially recovered and fibronectin and protein synthesis had fully recovered and sometimes exceeded the rate of synthesis in free-swelling controls. Forty-eight hours after five cycles of intermittent unconfined compression with similar loads, proteoglycan content and synthesis did not differ in loaded disks and in disks that were never loaded in vitro. Interestingly, the percentage of water in disks that had never been loaded in vitro increased significantly after 10 days in culture, relative to the percentage of water in free-swelling disks on the day of harvest. Intermittent compressive loading in the range of 0.5-1.2 MPa partially prevented this increase. Our results confirmed the previously reported inhibition of biosynthesis with static loading but also suggested that exposure to intermittent compressive loading may help to maintain the normal ratio of dry to wet weight in the explant.

Animals↗
PubMed1993

Effects of exercise and polysulfated glycosaminoglycan on repair of articular cartilage defects in the equine carpus.

Our aim was to determine if mild to moderate postoperative exercise and intra-articular polysulfated glycosaminoglycan result in improved repair of large, experimentally induced osteochondral defects in a weight-bearing surface of equine joints. Arthroscopic debridement was used to produce full-thickness defects in a weight-bearing area of the radial carpal bones in 18 ponies. The ponies were randomly assigned to two groups balanced for age: nine animals in the exercise and nine in the no exercise group. Six ponies in each group were medicated weekly for 5 weeks with an intra-articular injection of polysulfated glycosaminoglycan in one middle carpal joint beginning at the time of operation. Walking (twice daily) was begun 6 days postoperatively, and by the twelfth week postoperatively the ponies were trotting for 25 min and walking for 15 min twice daily. At the time of the ponies' death, 17 weeks postoperatively, each defect had an average of 50-75% coverage with repair tissue. Exercised, medicated joints had a significantly smaller area of coverage with repair tissue than exercised, nonmedicated joints. Cartilaginous repair tissue from exercised ponies contained significantly more glycosaminoglycan and type-II collagen (r = 0.53, p < 0.05). The ratio of hydroxylysine to hydroxyproline was significantly lower and the ratio of collagen content to total protein was significantly higher in the repair tissue of medicated joints than in the repair tissue of nonmedicated joints; this is consistent with the presence of less type-II collagen in the repair tissue in medicated joints. We concluded that postoperative exercise was beneficial and that the immediate postoperative use of intra-articular polysulfated glycosaminoglycan was detrimental to the development of cartilaginous repair tissue in large osteochondral defects of equine joints.

Amino Acids↗
PubMed1993

A new pressure chamber to study the biosynthetic response of articular cartilage to mechanical loading.

A prototype chamber was used to apply a precise cyclic or static load on articular cartilage explants under sterile conditions. A variable pressure, pneumatic controller was constructed to power the chamber's air cylinder, capable of applying, with a porous load platen, loads of up to 10 MPa at cycles ranging from 0 to 10 Hz. Pig articular cartilage explants were maintained successfully in this chamber for 2 days under cyclic mechanical loading of 0.5 Hz, 0.5 MPa. Explants remained sterile, viable and metabolically active. Cartilage responded to this load with a decreased synthesis of fibronectin and a small but statistically significant elevation in proteoglycan content. Similar but less extensive effects on fibronectin synthesis were observed with the small static load (0.016 MPa) inherent in the design of the chamber.

Animals↗
PubMed1993

Joint laxity and its association with hip dysplasia in Labrador retrievers.

A study was done to determine whether radiographic-distraction measurement of coxofemoral joint (hip) laxity at 4 and 8 months of age can serve as a predictor of hip dysplasia in older Labrador Retrievers. The method of Smith, Biery, and Gregor was used for radiologic examination of hips and for evaluation of radiographs. Mean (+/- SEM) distraction laxity (ie, distraction index) for 10 adult disease-free dogs was 0.29 +/- 0.05, whereas a group of 8 dogs with dysplastic hips had mean distraction index of 0.60 +/- 0.10 (P < 0.05). Mean distraction index at 4 months of age for 11 pups of 4 litters from matings between dogs with normal hips was 0.39 +/- 0.07, and was 0.54 +/- 0.04 for 31 pups of 7 litters from matings between dogs with hip dysplasia. The distraction index and, thus, joint laxity at that age was significantly (P = 0.0351) different for the 2 groups. The distraction index at 4 months correlated positively with the distraction index at a later age at necropsy (r = 0.43; P = 0.0289). Distraction index < 0.4 at 4 months of age predicted normal hips in 88% of cases and distraction index > or = 0.4 predicted hip dysplasia in 57% of the dogs. Logistic regression modeling indicated that the odds of a hip being normal decreased with increasing distraction index, and thus, with increasing joint laxity. The logistic regression models provided a reasonable mathematical description of the data. Based on the logistic model of the data, distraction indexes between 0.4 and 0.7 at either 4 or 8 months of age were not associated strongly enough with evidence of disease to be clinically reliable in predicting, on an individual basis, the outcome for dysplastic hip conformation when dogs were older. Index > 0.7 was associated with high probability for developing dysplastic joints and distraction index < 0.4 predicted normal hips with high probability.

Animals↗
PubMed1993

Effect of intramuscularly administered polysulfated glycosaminoglycan on articular cartilage from equine joints injected with methylprednisolone acetate.

Intra-articularly administered, long-acting corticosteroids are a beneficial treatment for many equine joint disorders because they alleviate inflammation and signs of pain, but they also exert detrimental effects on the biochemical composition and morphologic features of articular cartilage. Chondroprotective drugs have been shown to mitigate some of the deleterious effects of intra-articularly administered corticosteroids on articular cartilage of laboratory animals. Twenty-one ponies were assigned at random to receive 1 of 3 treatments in the right middle carpal joint. Group-1 ponies (n = 8) had methylprednisolone acetate (MPA; 0.2 mg/kg of body weight) and saline solution administered intra-articularly and IM, respectively. Group-2 ponies (n = 9) received MPA (0.2 mg/kg) and polysulfated glycosaminoglycan (GAG; 2 mg/kg). Group-3 ponies (control; n = 4) had saline solution administered intra-articularly and IM. The corticosteroid or saline solution was injected into the right middle carpal joint on day 1. The IM administered polysulfated GAG or saline solution was administered at the same time, then was repeated every 3 days for 20 days. Ponies were euthanatized 21 days after initial injection by overdose of pentobarbital sodium. The cartilage of younger ponies was significantly (P < 0.05) more responsive to the proteoglycan-depleting effects of MPA. Ponies < 10 years old of groups 1 and 2 had significantly (P < 0.05) lower GAG content in the articular cartilage than did control ponies. Systemic treatment with polysulfated GAG did not result in a protective effect against proteoglycan loss from the articular cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗