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D D Dean

Publications and source records attributed to D D Dean.

At least 127 records · Page 7Linked to original sources
PubMed1996

Vitamin D regulation of metalloproteinase activity in matrix vesicles.

Matrix vesicles (MVs) are enriched in matrix metalloproteinases (MMPs) capable of degrading proteoglycans. The aim of the present study was to identify which MMPs are present in MVs and determine whether these MMPs are regulated by 1,25-(OH)2D3 [1,25] and 24,25-(OH)2D3 [24,25]. To do this, growth zone (GC) and resting zone (RC) chondrocytes were isolated from rate costochondral cartilage and placed into culture. At confluence, GCs were treated with 1,25 and RCs with 24,25 for 24 hours. MVs, plasma membranes (PMs), and conditioned media were then collected from the cultures. RTPCR demonstrated the presence of mRNA for stromelysin-1 and 72 kDa gelatinase in both RCs and GCs, Casein zymography revealed activity at M(r) 48 and 28 kDa in MV, but not PM or conditioned media; Western analysis confirmed that this activity was stromelysin-1. Gelatinolytic activity, at low levels, was also found in MVs, but not PMs or conditioned media. When enzyme activity was measured using a proteoglycan bead assay, it was found that both GCs and RCs produced MVs and PMs containing neutral metalloproteinase. Both cells also produced MVs and PMs containing plasminogen activator. The addition of 1,25 to GCs caused a significant 4- to 5-fold increase in metalloproteinase activity in MVs, but not PMs. In contrast, MVs from cultures of RCs treated with 24,25 contained decreased metalloproteinase activity; enzyme activity in PMs was unaffected by 24,25. Plasminogen activator in MVs from RC was increased by treatment with 24,25, while MV enzyme activity was decreased after treatment of GC cultures with 1,25. This study shows that both RCs and GCs produce stromelysin-1 and 72 kDa gelatinase and that these enzymes are preferentially localized in MVs. Further, MMP and plasminogen activator activities in MVs and PMs are regulated by vitamin D metabolites.

24,25-Dihydroxyvitamin D 3↗
PubMed1995

Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63).

The effect of surface roughness on osteoblast proliferation, differentiation, and protein synthesis was examined. Human osteoblast-like cells (MG63) were cultured on titanium (Ti) disks that had been prepared by one of five different treatment regimens. All disks were pretreated with hydrofluroic acid-nitric acid and washed (PT). PT disks were also: washed, and then electropolished (EP); fine sandblasted, etched with HCl and H2SO4, and washed (FA); coarse sandblasted, etched with HCl and H2SO4, and washed (CA); or Ti plasma-sprayed (TPS). Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by brightfield and darkfield microscopy, cold field emission scanning electron microscopy, and laser confocal microscopy, while chemical composition was mapped using energy dispersion X-ray analysis and elemental distribution determined using Auger electron spectroscopy. The effect of surface roughness on the cells was evaluated by measuring cell number, [3H]thymidine incorporation into DNA, alkaline phosphatase specific activity, [3H]uridine incorporation into RNA, [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagenase-digestible protein (NCP), and [35S]sulfate incorporation into proteoglycan. Based on surface analysis, the five different Ti surfaces were ranked in order of smoothest to roughest: EP, PT, FA, CA, and TPS. A TiO2 layer was found on all surfaces that ranged in thickness from 100 A in the smoothest group to 300 A in the roughest. When compared to confluent cultures of cells on plastic, the number of cells was reduced on the TPS surfaces and increased on the EP surfaces, while the number of cells on the other surfaces was equivalent to plastic. [3H]Thymidine incorporation was inversely related to surface roughness. Alkaline phosphatase specific activity in isolated cells was found to decrease with increasing surface roughness, except for those cells cultured on CA. In contrast, enzyme activity in the cell layer was only decreased in cultures grown on FA- and TPS-treated surfaces. A direct correlation between surface roughness and RNA and CDP production was found. Surface roughness had no apparent effect on NCP production. Proteoglycan synthesis by the cells was inhibited on all the surfaces studied, with the largest inhibition observed in the CA and EP groups. These results demonstrate that surface roughness alters osteoblast proliferation, differentiation, and matrix production in vitro. The results also suggest that implant surface roughness may play a role in determining phenotypic expression of cells in vivo.

Alkaline Phosphatase↗
PubMed1995

Osteosarcoma hybrids can preferentially target alkaline phosphatase activity to matrix vesicles: evidence for independent membrane biogenesis.

Alkaline phosphatase is the marker enzyme for matrix vesicles, extracellular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma x fibrosarcoma hybrids in which alkaline phosphatase expression was greatly reduced, a phenomenon known as extinction. In the present study, we used to cell hybrids, LTA-1 and LTA-5, constructed from a human osteoblast-like osteosarcoma. TE85, and a mouse fibrosarcoma, La-t-, to examine the differential distribution of alkaline phosphatase between matrix vesicles and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasma membranes was extinguished, enzyme activity in matrix vesicles from LTA-1 hybrid cells was 34.2% of that present in matrix vesicles from the TE85 parent cells and 200 times that found in La-t- matrix vesicles. Matrix vesicles from LTA-5 had alkaline phosphatase levels similar to La-t-. When other membrane enzymes (phospholipase A2, 5'-nucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle and plasma membrane levels were similar to those of TE85 and significantly higher than in La-t- membrane fractions. Northern analysis detected mRNA for alkaline phosphatase in TE85 cells, but not in the hybrids or La-t- cells. In contrast, reverse transcription-polymerase chain reaction (RT-PCR) revealed alkaline phosphatase mRNA in the hybrid cells, but at very low levels. Taken together, the data indicate that regulation of plasma membrane and matrix vesicle alkaline phosphatase is independent and suggest that matrix vesicle biogenesis is independent and distinct from that of plasma membrane biogenesis. Analysis of 1B- and 1L-type alkaline phosphatase mRNA by RT-PCR showed that alternate promoter usage of the alkaline phosphatase gene was not responsible for the differential localization of this enzyme in matrix vesicle. Thus, it is likely that matrix vesicle and plasma membrane alkaline phosphatase are regulated differently at a post-transcriptional level.

5'-Nucleotidase↗
PubMed1995

Carbonic anhydrase is required for statoconia homeostasis in organ cultures of statocysts from Aplysia californica.

A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (L15) medium supplemented with salts and Aplysia haemolymph for four days at 17 degrees C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 x 10(-4) M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.

Animals↗
PubMed1995

Markers of primary mineralization are correlated with bone-bonding ability of titanium or stainless steel in vivo.

Critical events in the adaptation of osseous tissues to implant materials involve initial calcification of the newly synthesized bone. Previous studies indicated that bone-bonding but not nonbonding glass ceramics increase the matrix vesicle number, thereby compensating for delayed maturation of the extracellular organelles. The present study assessed whether this was also true for metal implants commonly used in orthopaedics and oral medicine. Bone-bonding titanium (Ti) or nonbonding stainless steel (SS) implants were placed in the right tibias of Sabra rats following ablation of the marrow. At 3, 6, 14, and 21 days postinjury, newly formed endosteal bone in the treated and contralateral limbs was removed and matrix vesicle-enriched membranes isolated. Alkaline phosphatase and phospholipase A2 specific activities and phosphatidylserine (PS) content were determined and compared with those of a nonsurgical control group. Results show that matrix vesicle alkaline phosphatase and phospholipase A2 activity and PS content was increased in the Ti-implanted limbs at 6 (peak), 14, and 21 days, although at levels less than observed in normal healing. Alkaline phosphatase activity remained elevated throughout the healing period. In contrast, these parameters were markedly inhibited in the SS-implanted limbs with respect to Ti or to normal healing. Both implants altered the systemic response associated with marrow ablation, but in an implant-specific manner. The results support the hypothesis that cells adjacent to bone-bonding materials can compensate for negative effects on primary mineralization during osteogenesis, whereas cells adjacent to nonbonding materials either do not compensate or are further depressed. The data support the use of the rat marrow ablation model as a tool for rapid, initial assessment of biomaterials in bone.

Alkaline Phosphatase↗
PubMed1995

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes.

Vitamin D3 metabolites affect the proliferation and differentiation of cartilage cells. Previous reports have shown that rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. These results indicate that 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness. These observations support the hypothesis that 24,25-(OH)2D3 plays a significant role in cartilage development.

24,25-Dihydroxyvitamin D 3↗
PubMed1994

Latent transforming growth factor-beta is produced by chondrocytes and activated by extracellular matrix vesicles upon exposure to 1,25-(OH)2D3.

Resting zone and growth zone (GC) costochondral chondrocytes constitutively release latent, but not active, transforming growth factor-beta (TGF-beta) into the culture medium. When exogenous TGF-beta is added to the culture medium, no autocrine effect is observed. However, when 1,25-(OH)2D3 is added, a dose-dependent inhibition of latent TGF-beta release is found. Messenger RNA levels for TGF-beta 1 are unchanged by treatment with either 1,25-(OH)2D3 or TGF-beta 1. Since active growth factor was not observed in the conditioned medium, we tested the hypothesis that latent TGF-beta could be activated in the matrix. GC matrix vesicles, extracellular organelles associated with matrix calcification, were able to activate latent TGF-beta 1 and TGF-beta 2 when preincubated with 1,25-(OH)2D3. In contrast, GC plasma membranes activated latent TGF-beta, and addition of 1,25-(OH)2D3 inhibited this activation. The 1,25-(OH)2D3-dependent decrease in latent TGF-beta in the medium, with no detectable change in mRNA level, and the inhibition of plasma membrane activation of latent TGF-beta by 1,25-(OH)2D3 suggest that 1,25-(OH)2D3 may act through post-transcriptional and/or nongenomic mechanisms. The results also suggest that latent TGF-beta is activated in the matrix and that 1,25-(OH)2D3 regulates this activation by a direct, nongenomic action on the matrix vesicle membrane.

24,25-Dihydroxyvitamin D 3↗
PubMed1994

2,3,7,8-Tetrachlorodibenzo-p-dioxin reduces the number, size, and organelle content of Leydig cells in adult rat testes.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters testicular steroidogenesis and reduces total Leydig cell volume in the testis. However, its effect on Leydig cell number, size, and organelle content had not been determined in adult rats. Adult male rats received a single intraperitoneal injection of TCDD at a dose of 0, 12.5, 25.0, or 50.0 micrograms/kg body weight. Testicular tissues were obtained from rats 4 weeks after treatment. Testes were vascularly perfused with glutaraldehyde, embedded in Epon 812, sectioned at 0.5 micron, stained with toluidine blue, and evaluated by stereology for number and size of Leydig cells. Specimens from control and high dose groups were prepared for electron microscopy to quantify Leydig cell organelle content. TCDD treatment reduced (P < 0.01) body weight in a dose-dependent fashion. Testicular weight was not significantly reduced by TCDD treatment. The volume of Leydig cell cytoplasm per testis was reduced (P < 0.01) four weeks after treatment. Reduction in total Leydig cell volume resulted from a reduced (P < 0.05) number of Leydig cells and a reduced (P < 0.01) size of individual Leydig cells. However, the volume density (percentage) of Leydig cells occupied by specific organelles was not influenced by TCDD treatment. As a result of reduced total Leydig cell volume with no change in volume density of organelles in Leydig cells, the volumes per testis of smooth endoplasmic reticulum and mitochondria were reduced (P < 0.01) by TCDD treatment. In conclusion, the TCDD-induced reduction in Leydig cell volume per testis is explained by reduced number and size of individual Leydig cells and resulted in a significant reduction in total volume of both Leydig cell smooth endoplasmic reticulum and mitochondria per testis. Reduction in content of organelles that are responsible for various key steps in steroidogenesis, could explain TCDD-reduced production of testosterone in rats.

Animals↗
PubMed1994

Nongenomic regulation of extracellular matrix events by vitamin D metabolites.

Vitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25-(OH)2D3 or 24,25-(OH)2D3 may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinase C activity. Chondrocytes metabolize 25-(OH)D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25-(OH)2D3 and 24,25-(OH)2D3 may interact directly with extracellular matrix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modification and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor-beta when incubated with exogenous 1,25-(OH)2D3. These observations suggest that nongenomic regulation of matrix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chondrocytes, may modulate events in the extracellular matrix at sites distant from the cell surface.

Animals↗
PubMed1994

Culture surfaces coated with various implant materials affect chondrocyte growth and metabolism.

The effect on chondrocyte metabolism of culture surfaces sputter-coated with various materials used for orthopaedic implants was studied and correlated with the stage of cartilage cell maturation. Confluent, fourth-passage chondrocytes from the costochondral resting zone and growth zone of rats were cultured for 6 or 9 days on 24-well plates sputter-coated with ultrathin films of titanium, titanium dioxide, aluminum oxide, zirconium oxide, and calcium phosphate (1.67:1). Corona-discharged tissue culture plastic served as the control. The effect of surface material was examined with regard to cell morphology; cell proliferation (cell number) and DNA synthesis ([3H]thymidine incorporation); RNA synthesis ([3H]uridine incorporation); collagenase-digestible protein, noncollagenase-digestible protein, and percentage of collagen production; and alkaline phosphatase-specific activity, both in the cell layer and in trypsinized chondrocytes. Cell morphology was dependent on surface material; only cells cultured on titanium had an appearance similar to that of cells cultured on plastic. While titanium or titanium dioxide surfaces had no effect on cell number or [3H]thymidine incorporation, aluminum oxide, calcium phosphate, and zirconium oxide surfaces inhibited both parameters. Cells cultured on aluminum oxide, calcium phosphate, zirconium oxide, and titanium dioxide exhibited decreased collagenase-digestible protein, noncollagenase-digestible protein, and percentage of collagen production, but [3H]uridine incorporation was decreased only in those chondrocytes cultured on aluminum oxide, calcium phosphate, or zirconium oxide. Chondrocytes cultured on titanium had greater alkaline phosphatase-specific activity than did cells cultured on plastic, but the incorporation of [3H]uridine and production of collagenase-digestible protein, noncollagenase-digestible protein, and percentage of collagen was comparable. The response of chondrocytes from the growth zone and resting zone to culture surface was comparable, differing primarily in magnitude. Cell maturation-dependent effects were evident when enzyme activity in trypsinized and scraped cells was compared. These results indicate that different surface materials affect chondrocyte metabolism and phenotypic expression in vitro and suggest that implant materials may modulate the phenotypic expression of cells in vivo.

Alkaline Phosphatase↗
PubMed1994

Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of beta-glycerophosphate and ascorbic acid.

Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 micrograms/ml), beta-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or beta-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.

3T3 Cells↗
PubMed1994

Nodule formation and calcification of mandibular condylar cartilage cell cultures mimic in vivo ultrastructure.

Mandibular condylar cartilage (MCC) of growing mammals contains four layers of cells which display a series of increasingly differentiated phenotypes and which culminate in a terminally differentiated cell that produces a calcified matrix. In this study, MCC cells were placed into culture in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 micrograms/ml ascorbic acid. After 12 h in culture, transmission electron microscopy revealed the presence of multiple cell types that underwent differentiation with additional time in culture. By 7 days, fusiform-shaped cells were seen that contained numerous actin-like cytofilaments and micropinocytotic vesicles characteristic of myofibroblasts. Chondroblast-like cells were also observed. By 10 days, without addition of beta-glycerophosphate or dexamethasone, these cellular events culminated in the formation of mineralized nodules containing matrix vesicles. The nodular surface at day 13 consisted of two or more layers of myofibroblast-like cells, while the deeper zones of the nodule contained cells displaying a morphology typical of calcified hypertrophic chondroblasts. These ultrastructural observations are consistent with the hypothesis that cells from the MCC are capable of recapitulating in culture the maturational events seen in vivo. This cell culture model may be useful for investigating cell-mediated cartilage calcification without the addition of exogenous phosphate or other regulatory factors.

Animals↗
PubMed1994

Evidence for receptors specific for 17 beta-estradiol and testosterone in chondrocyte cultures.

Recently, sex hormones were shown to stimulate chondrocyte differentiation and matrix protein synthesis in vitro in a sex-specific and maturation-dependent manner. The aim of the present study was to determine whether cytosolic receptors in these cells would specifically bind 17 beta-estradiol and testosterone, and if so, whether binding was gender- and maturation-dependent. Confluent, fourth passage cultures of cells derived from male or female rat costochondral growth zone and resting zone cartilage were homogenized and specific binding of 17 beta-estradiol or testosterone measured in the cytosolic fraction. Scatchard analysis indicated the presence of a high-affinity 17 beta-estradiol receptor (Kd = 4.5 to 8.7 x 10(-11) M), with low binding capacity (3.9 to 11.2 fmol/mg protein). Chondrocytes from female rats were found to have a significantly greater binding capacity for 17 beta-estradiol than chondrocytes from male rats. However, cells from both sexes had binding capacities that were independent of cell maturation. A high-affinity testosterone receptor (Kd = 4.3 to 6.3 x 10(-11) M) with low binding capacity (4.1 to 5.9 fmol/mg protein) was found in both males and females, but no difference in binding capacity was noted, either as a function of gender or stage of cell maturation. Immunohistochemistry using antibodies against 17 beta-estradiol and testosterone and the 17 beta-estradiol nuclear receptor (D-75) confirmed that 17 beta-estradiol and testosterone receptors were present in chondrocytes from both male and female rats. These data demonstrate that chondrocytes from growth zone and resting zone cartilage are capable of binding both 17 beta-estradiol and testosterone. This suggests that these hormones mediate their direct effects on chondrocytes via receptors specific for their appropriate ligand. The sex-specific effects of 17 beta-estradiol may be due to differences in receptor number between chondrocytes derived from female and male rats. In contrast, the sex-specific effects of testosterone may be regulated at the post receptor level since no differences in binding capacity were found between males and females.

Animals↗
PubMed1993

Treatment of canine osteoarthritis with insulin-like growth factor-1 (IGF-1) and sodium pentosan polysulfate.

The potential therapeutic effects of insulin-like growth factor-1 (IGF-1) and sodium pentosan polysulfate (PPS) were evaluated in an anterior cruciate ligament-deficient canine model of osteoarthritis (OA). A control group of animals received no treatment or surgery (N). The remaining four groups of animals received anterior cruciate transection and either no treatment (OA), intra-articular IGF-1 (IGF-1), intra-muscular PPS (PPS), or a combination of intra-articular IGF-1 and intra-muscular PPS (IGF-1/PPS). All therapy was begun 3 weeks after surgery and continued for 3 weeks. At 6 weeks, articular cartilage from the femoral condyle was evaluated for anatomy, histology (Mankin grade) and biochemistry. Anatomically, only cartilage from dogs in the IGF-1/PPS group approximated that found in N. Mankin scores indicated less severe disease in both PPS and IGF-1/PPS groups compared with the OA group. Consistent with histology, the level of active neutral metalloproteinase was lower in cartilage from the PPS group compared with the OA group. Active and total neutral metalloproteinase, tissue inhibitor of metalloproteinases (TIMP), total collagenase, uronate and hydroxyproline contents were all near normal in the IGF-1/PPS group. In a model of mild OA, therapeutic intervention with IGF-1 and PPS appeared to successfully maintain cartilage structure and biochemistry. From these data, it is hypothesized that proteinase activity was successfully blocked by PPS, and that this allowed the observed growth factor induced effects. As we unravel the various factors that regulate cartilage metabolism, it is becoming apparent that combinations of agents will be needed to effectively control cartilage repair in OA. The addition of PPS to IGF-1 shows promise as a therapeutic intervention and introduces a new rational approach to therapy of OA.

Animals↗
PubMed1993

The effects of methotrexate on normal and osteoarthritic lapine articular cartilage.

OBJECTIVE: The effects of methotrexate (MTX) on articular cartilage and its influence on the development of osteoarthritis (OA) lesions were tested in a lapine partial medial meniscectomy model. METHOD: Animals were divided into groups consisting of unoperated and operated rabbits that either received or did not receive MTX treatment. After 8 weeks knee articular condylar cartilage was examined for gross and histologic anatomy, active and total neutral metalloproteinases, tissue inhibitor of metalloproteinase (TIMP), DNA, uronic acid and hydroxyproline content. RESULTS: Carbon black retention and histologic scores revealed moderately severe changes in the OA animals with a tendency to less severe changes in OA animals receiving MTX. Unoperated animals receiving MTX had abnormal cartilage that displayed pitting and elevations in histologic score. Active and total neutral metalloproteinase and TIMP were elevated in both untreated and treated OA animals when compared to either unoperated or unoperated and treated animals. CONCLUSION: Articular cartilage with lesser amounts of neutral metalloproteinase and high amounts of TIMP levels often seen with other therapeutic modalities for OA, were not observed with MTX therapy. Our data suggest that MTX may have limited value in the treatment of OA.

Animals↗
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↗