Matrix Depletion Research Articles

Influence of topologically closed packed phase formation on creep rupture life of directionally solidified nickel-base superalloys

Several experimental superalloy compositions based on the alloy IN792 were investigated. Creep rupture properties were measured and microstructures in the as-heat-treated condition and after creep rupture testing were evaluated. Two types of topologically closed packed (TCP) phases have been observed. Type 1 occurs in alloys with increased additions of the γ′-formers Al and Ta and shows detrimental effects on creep rupture life. Type 2 precipitates in Re-containing alloys are much smaller in size and volume fraction than type 1 and are found not to influence the creep rupture properties. The different effect of type 1 and 2 TCP phases can be rationalized in terms of the amount of precipitate and the resulting matrix depletion of solid solution strengtheners.

Histamine, histamine receptors (H1 and H2), and histidine decarboxylase expression by chondrocytes of osteoarthritic cartilage: an immunohistochemical study

Degeneration and loss of articular cartilage are the characteristic features of osteoarthritis (OA), with the appearance of fibrillations, cell clusters, matrix depletion, and changes in matrix composition all apparent. Histamine has a recognised role in allergic and inflammatory reactions, and is reported to affect several aspects of chondrocyte behaviour. The immunohistochemical (IHC) studies reported here have demonstrated histamine (H), both H1 and H2 receptors, and the histamine-producing enzyme histidine decarboxylase (HDC) in a variable proportion of human articular chondrocytes in OA cartilage specimens. Such observations were especially evident within the degenerative, superficial zone, and more so in late-stage disease. By contrast, "normal" age-matched cartilage specimens showed relatively little immunopositive staining for histamine and HDC. These findings strongly suggest that histamine and H-receptor expression by HAC in OA cartilage is potentially an important contributor to the atypical, aberrant phenotype of OA chondrocytes.

Gp49B1 deficiency is associated with increases in cytokine and chemokine production and severity of proliferative synovitis induced by anti‐type II collagen mAb

Mice with a disrupted gp49B gene, which encodes gp49B1 that is expressed on certain hematopoietic cells and has two immunoreceptor tyrosine-based inhibitory motifs (ITIM), exhibit augmented FcepsilonRI-initiated mast cell degranulation and resultant tissue edema. gp49B1-deficient (gp49B(-/-)) mice also exhibit exaggerated lipopolysaccharide (LPS)-induced intravascular neutrophil aggregation leading to cutaneous microangiopathy. To determine whether gp49B(-/-) mice exhibit elevated cytokine and chemokine levels leading to pathologic inflammation, we quantified clinical and morphologic parameters of arthritis and tissue levels of contributory mediators in gp49B(-/-) and gp49B1-sufficient (gp49B(+/+)) mice injected with anti-type II collagen monoclonal antibody (mAb) and LPS. Clinical scores for joint swelling and histological assessments of synovial thickness and cartilage matrix depletion at day 7 were significantly 2.3- to 2.5-fold greater and were more prolonged in gp49B(-/-) mice. At day 5, the amounts of IL-1beta, macrophage inflammatory protein (MIP)-1alpha, and MIP-2 were 2.1-, 2.5-, and 12-fold greater in joint extracts from gp49B(-/-) mice. A significant 2.7-fold more neutrophils infiltrated the synovium of gp49B(-/-) mice at day 7, and neutrophilia persisted with the delayed resolution of the synovitis. mAb-mediated depletion of neutrophils prevented the synovitis in both strains. Thus, gp49B1 counter-regulates the cytokine and chemokine induction and attendant neutrophilia that are all essential for synovitis and cartilage matrix depletion.

Long‐term in vivo stability of rabbit nasal septal cartilage following laser cartilage reshaping: A pilot investigation

To evaluate the long-term effect of laser cartilage reshaping on rabbit nasal septal cartilage viability and mechanical integrity in an in vivo model. In vivo animal investigation. Rabbit septal cartilage specimens were laser (Nd:YAG, lambda = 1.32 mum, spot size 5.4-mm diameter, 10 W, 10 seconds, 50 Hz PPR) reshaped and subsequently reimplanted into an interscapular subcutaneous pocket. Specimens were harvested at 8 and 12 months and evaluated using photography, flow cytometry, and histology. Grossly, specimens showed alteration in the physical integrity with varying degrees of tissue resorption. The non-irradiated control specimens demonstrated significantly increased stiffness. Histologically, there was marked depletion of the extracellular matrix and an overall reduction in tissue mass in laser irradiated tissues. However, flow cytometry data identified viable chondrocytes in laser-irradiated specimens that were identical to those observed in controls. Study results demonstrate that the rabbit nasal septal cartilage model can be effectively used to study laser reshaping, however alternative recipient sites with perichondrial lining, such as the pinna, may provide a more realistic physiologic environment for reshaped graft tissue. The dosimetry used in this pilot study likely led to significant thermal injury. Study results underscore the importance of elucidating the optimal laser dosimetry required to initiate permanent shape change while minimizing thermal damage.

Hydrogen peroxide inhibits formation of cartilage in chicken micromass cultures and decreases the activity of calcineurin: implication of ERK1/2 and Sox9 pathways

Calcineurin was found as a positive regulator of chondrogenesis in chondrifying chicken micromass cultures (HDCs), as cyclosporine A (CsA) reduced both the amount of cartilage and the expression of mRNAs of aggrecan and the chondrogenic transcription factor Sox9. Cartilage formation was inhibited by H 2O 2 in a concentration-dependent manner without loss of cellular viability or severe decrease of cell number. Expression of both the mRNA and the unphosphorylated protein Sox9 was decreased, while its phosphorylation was stimulated by either H 2O 2 or CsA. Oxidative stress decreased the activity of calcineurin but the phosphorylation of the member of MAPK family ERK1/2 was extremely elevated either by 1 mM H 2O 2 or 2 μM CSA. The ERK inhibitor PD098059 attenuated the depletion of cartilage matrix as well as decreased the expression and phosphorylation of Sox9 in cultures treated with H 2O 2 or CsA. Our results suggest that the chondrogenesis-inhibiting effect of H 2O 2 is mediated, at least partly, by inhibition of calcineurin and by activation of ERK1/2. We also propose a regulatory role of calcineurin in the phosphorylation level of either ERK1/2 or Sox9 and a positive role of ERK1/2 in regulating both the expression level and the phosphorylation state of Sox9 in chicken HDCs.

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.

Effect of Proteoglycan Depletion on T2 Mapping in Rat Patellar Cartilage

To evaluate experimentally the sensitivity of T2 mapping with magnetic resonance (MR) imaging at 8.5 T in depicting variations in proteoglycan content and concurrent extracellular matrix of rat patellar cartilage. The study was performed in 36 immature (age, 5 weeks) and 36 mature (age, 10 weeks) Wistar rats. Maintenance and care of the rats were conducted in accordance with National Institutes of Health guidelines. Fifty-six rats underwent T2 mapping in 28 right patellae degraded with hyaluronidase for 1 and 6 hours and in 28 undegraded age-matched patellae that served as controls. After MR mapping, the rats were sacrificed, and the patellae were studied histologically to evaluate proteoglycan and collagen content and collagen network organization in cartilage. Biochemical analysis was performed in 88 patellae to quantify sulfated glycosaminoglycan and hydroxyproline content. Effects of age and/or degree of degradation were evaluated after rank transformation of continuous data by using rank analysis of variance (ANOVA). Associations between continuous variables were assessed with the Spearman rank correlation coefficient. Results of histologic analysis showed proteoglycan loss after hyaluronidase degradation without alteration of collagen network. No significant variation in hydroxyproline sulfate content was observed with depletion of proteoglycan. Proteoglycan losses of 19% and 13%, found after 1-hour degradation in immature and mature groups, respectively, were associated with significantly increased global T2 values (ANOVA, P < .001). Six-hour degradation resulted in more severe proteoglycan losses of 45% and 53% in immature and mature groups, respectively, inducing significant increases in global T2 values in immature and mature groups (ANOVA, P < .001). Variations in T2 values between superficial and deep cartilage zones were not affected by proteoglycan depletion. In rat patellar cartilage, T2 mapping permits detection of slight or severe proteoglycan depletion and concurrent changes of extracellular matrix when age-matched samples are compared.

Emission rates for electron tunneling from InAs quantum dots to GaAs substrate

Tunneling emission rates of electrons from InAs quantum dots (QD) in a GaAs matrix depletion region have been calculated for application in the analysis of experimental data from deep-level transient spectroscopy (DLTS). The hybridization among metastable states localized in the InAs QD and continuum states in the GaAs substrate in the DLTS measurement is evaluated from the local densities of states. Two physical quantities have been calculated, the broadening of the metastable state localized in the InAs QD, i.e., the charge-transfer rates from the quantum dot to the substrate, and the corresponding hybridization coefficients. The metastable state is broadened significantly only when the bias is large enough, whereas the hybridization coefficients increase almost linearly with the bias. The effects of the direct Coulomb interactions and exchange energies among electrons initially confined in the QD have been included and found to be very significant. Increasing the number of electrons initially confined in the QD from 1 to 4, the charge-transfer rates increase by a factor of 1.8.

Osteogenic Protein-1 inhibits matrix depletion in a hyaluronan hexasaccharide-induced model of osteoarthritis

Objective: To examine the capacity of recombinant osteogenic protein-1 (OP-1) to inhibit the cartilage extracellular matrix damage that follows treatment with hyaluronan hexasaccharides (HA6). Design: The effects of OP-1 were examined on isolated human chondrocytes grown in alginate beads as well as articular cartilage slices treated with hyaluronan hexasaccharides. Changes in the relative expression of messenger RNA for hyaluronan synthase- 2, aggrecan and CD44 were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Cartilage proteoglycan biosynthesis was examined by a 35S-sulfate incorporation assay. Cell-associated matrix of human chondrocytes was visualized by the use of particle exclusion assay, and alcian blue staining. Cartilage slices were examined for accumulation of proteoglycan by Safranin-O, and hyaluronan by a specific biotinylated probe. Results: Combined OP-1 and HA6 treatment resulted in enhanced expression of mRNA for aggrecan and HAS-2, compared to the treatment with HA6 only. This increased expression of aggrecan mRNA was paralleled by an increased synthesis of cartilage proteoglycan especially retained in the cell-associated matrix. Co-treatment with OP-1 inhibited the HA6-induced depletion of cell-associated matrices as well as HA6-induced depletion of hyaluronan and proteoglycan within cartilage tissue slices. Conclusions: These results demonstrate that OP-1 can abrogate the catabolic events associated with a HA6-induced matrix depletion model of osteoarthritis. The mRNA levels of two major cartilage extracellular matrix components, aggrecan and hyaluronan synthase-2 are enhanced above values obtained by either OP-1 or HA6 treatments alone.

A dose titration of triamcinolone acetonide on insulin-like growth factor-1 and interleukin-1-conditioned equine cartilage explants.

Previous in vitro pilot studies have defined a potentially beneficial effect of insulin-like growth factor-1 (IGF-1) and triamcinolone acetonide (TA) on interleukin-1 (IL-1)-conditioned equine cartilage. Furthermore, an optimal dose for IGF-1 treatment alone has been documented previously using the same test system as in the current project. To perform a dose titration of TA on IL-1-conditioned equine articular cartilage explants in the presence of an optimised IGF-1 dose, in order to optimise a triamcinolone concentration for use in combination with IGF-1 for future investigations. Cartilage explants were harvested from the distal femur of a normal horse. The effect of a clinically relevant TA dose range was evaluated in the presence of IL-1 and IGF-1 through measurement of proteoglycan (PG) matrix metabolism (synthesis and degradation). TA and IGF-1 in combination inhibited the IL-1-induced release of PG matrix components (glycosaminoglycan or GAG) from the articular cartilage, as well as producing a significant increase in GAG synthesis. This experiment provided proof of principle that a combination treatment appears to be able to combat the IL-1-induced matrix depletion, while enhancing anabolic metabolism within the articular cartilage. The use of IGF-1 in conjunction with TA in vivo has the potential to provide beneficial anabolic effects not seen with TA alone.

MR imaging of normal and matrix-depleted cartilage: correlation with biomechanical function and biochemical composition.

To correlate articular cartilage function, as reflected in biomechanical properties and biochemical composition, with magnetic resonance (MR) imaging parameters of normal articular cartilage and cartilage partially depleted of matrix components. Normal articular cartilage from 12 porcine patellae was evaluated biomechanically, biochemically, and with MR imaging (with and without gadolinium enhancement). The patellae were then enzymatically treated to deplete the matrix of either collagen or proteoglycan and then reevaluated biomechanically, biochemically, and with MR imaging. Correlations between cartilaginous tissue function and MR imaging parameters were made. Analysis of variance was performed to assess the effect of enzymatic treatment on measured parameters. Linear correlations among the MR imaging, biochemical, and biomechanical parameters were performed to determine the strengths of the relationships. P <.05 indicated statistically significant differences. Biochemical, biomechanical, and MR analyses enabled detection of changes caused by matrix depletion (P <.05). T2 was the most useful MR imaging parameter for distinguishing proteoglycan loss from collagen loss. T2 correlated significantly with both biomechanical modulus (indicative of cartilage stiffness; P <.001, R2 = 0.51) and biochemical proteoglycan content (P <.001, R2 = 0.44). Differentiation between proteoglycan loss and collagen loss in terms of T1 improved with gadolinium enhancement. With gadolinium enhancement, proteoglycan depletion was associated with a greater decrease in T1 than collagen depletion (P <.05). An association between biochemical and biomechanical functional status and MR imaging parameters of articular cartilage was demonstrated. Linear correlations existed between modulus and proteoglycan content in terms of T2. Additionally, proteoglycan loss and collagen loss had differing effects on gadolinium-enhanced T1 when it was expressed as the ratio of T1 after gadolinium enhancement/T1 before gadolinium enhancement.

Electrodialysis as a Sample Preparation Tool for Capillary Electrophoresis of Small Anions – A Proof of Principle

The use of capillary electrophoresis (CE) for biomedical applications or control of waste water effluents is often restricted, due to the complex matrix embedding trace concentrations of the analyte. Selective removal of interfering ionic species is challenging when analyte and matrix are similar in size and charge. An electrodialysis method applying a home-made module for sample desalting is described. Chloride, sulfate and nitrate ions were employed at 50 or 100 mM (referring to concentrations in real samples from clinical or environmental origin) to simulate matrix compounds. Sulfanilic acid was selected as a model analyte ion at millimolar or sub-millimolar concentrations. Process analysis was performed with ion chromatography coupled on-line. For a simple monitoring of the process in application, conductivity measurement in the sample compartment could be successfully established. Within 45 minutes, the matrix concentration was decreased by a factor of about 10, whilst the analyte was only diluted by a factor of about 3. The rate of the matrix depletion was constant over the whole process and controlled by the constant current generated from the voltage source. Speeding up the process by applying higher current densities failed, due to resulting thermal stress at the membranes. Electrodialysis results were independent of both the matrix anion and matrix cation identity, indicating a good robustness against variations of the sample matrix.

A mechanism underlying the movement requirement for synovial joint cavitation.

Many studies have highlighted the importance of movement-induced mechanical stimuli in the development of functional synovial joints. However, such phenomenological results have failed to provide a full explanation of the mechanism essential for the morphogenesis of fluid-filled joint cavities. We have previously demonstrated that the large glycosaminoglycan hyaluronan (HA), in association with its principal cell surface receptor CD44, plays a major role during the morphogenesis of chick joints. We have taken cells from the surface of recently cavitated joints and subjected them to a brief period of dynamic mechanical strain (3800 microE for 10 min) and measured changes in HA synthesis/release, CD44 expression and HA synthase gene expression. In addition, we subjected cells to matrix depletion prior to the application of mechanical strain in order to examine any potential modulatory function of the ECM during the cell response to strain. Removal of the cell-associated HA-containing matrix with hyaluronidase significantly increased the release of HA into tissue culture media over 24 h and is associated with increased CD44 expression, alterations in HA synthase gene expression and enhanced binding of HA to the cell surface. Such changes in HA release were shown to be blocked by addition of exogenous HA and synergistically enhanced by the application of dynamic mechanical strain. These results show that cell-matrix interactions modify the response of embryonic cells to mechanical strain and provide further insight into the mechano-dependent mechanism of joint cavity morphogenesis.

Glucosamine and chondroitin sulfate: biological response modifiers of chondrocytes under simulated conditions of joint stress

Objective: To test the hypothesis that chondrocytes are more responsive to the chondroprotective agents, glucosamine (glcN) and chondroitin sulfate (CS), under in vitro conditions simulating in vivo joint stress.Design: Synthetic and anticatabolic activities of bovine articular cartilage were assayed using 35-sulfate labeling and assaying the specific activity of glycosaminoglycans (GAGs) under the conditions of enzyme-induced matrix depletion, heat stress, mechanical compression and cytokine stress.Results: The response of cartilage to simulated conditions of in vivo stress varies, depending on the type stress and age of the animal. Cartilage from aged animals was more responsive to stress and to glcN and CS. Pronase-induced matrix depletion and mechanical stress increased proteoglycan synthetic activity. Exposure to glcN and CS significantly enhanced this stress response from 85 to 191% and from 40 to 1000%, respectively. Heat stress and stromelysin digestion decreased synthetic activity, which was reversed or normalized on exposure to glcN and CS. Cartilage from young joints was somewhat refractory to the level of stress imposed and to treatment with glcN and CS.Conclusion: The metabolic response of cartilage from aged animals to glcN and CS under simulated conditions of in vivo stress is significantly greater than that seen in nonstressed or young tissue. By enhancing the ‘protective’ metabolic response of chondrocytes to stress, glcN and CS may improve its ability for repair and regeneration. These observations suggest that these compounds function as biological response modifiers (BRMs), agents which boost natural protective responses of tissues under adverse environmental conditions.

Differential recovery of glycosaminoglycan after IL-1-induced degradation of bovine articular cartilage depends on degree of degradation.

In the present study we examined cartilage matrix repair following IL-1-induced matrix depletion. Previous data indicated that, in some cases, chondrocytes can synthesize macromolecules to establish a functional extracellular matrix in response to a matrix-damaging insult or when placed in a three-dimensional environment with inadequate matrix. However, the conditions under which such 'repair' can occur are not entirely clear. Prior studies have shown that chondrocytes in trypsin-depleted young bovine articular cartilage can replenish tissue glycosaminoglycan (GAG) and that the rate of replenishment is relatively uniform throughout the tissue, suggesting that all chondrocytes have similar capacity for repair. In the present study we used the characteristic heterogeneous distribution of matrix depletion in response to IL-1 exposure in order to investigate whether the severity of depletion influenced the rate of GAG replenishment. We used the delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) method to monitor the spatial and temporal evolution of tissue GAG concentration ([GAG]). For both mild (n=4) and moderate (n=10) IL-1-induced GAG depletion, we observed partial recovery of GAG (80% and 50% of baseline values, respectively) over a 3-week recovery period. During the first 2 weeks of recovery, [GAG] increased homogeneously at 10-15 mg/ml per week. However, during the third week the regions most severely depleted following IL-1 exposure showed negligible [GAG] accumulation, whereas those regions affected the least by IL-1 demonstrated the greatest accumulation. This finding could suggest that the most severely degraded regions do not recover fully, possibly because of more severe collagen damage; this possibility requires further examination.

Regulation of cell polarity, radial intercalation and epiboly in Xenopus: novel roles for integrin and fibronectin.

Fibronectin (FN) is reported to be important for early morphogenetic movements in a variety of vertebrate embryos, but the cellular basis for this requirement is unclear. We have used confocal and digital time-lapse microscopy to analyze cell behaviors in Xenopus gastrulae injected with monoclonal antibodies directed against the central cell-binding domain of fibronectin. Among the defects observed is a disruption of fibronectin matrix assembly, resulting in a failure of radial intercalation movements, which are required for blastocoel roof thinning and epiboly. We identified two phases of FN-dependent cellular rearrangements in the blastocoel roof. The first involves maintenance of early roof thinning in the animal cap, and the second is required for the initiation of radial intercalation movements in the marginal zone. A novel explant system was used to establish that radial intercalation in the blastocoel roof requires integrin-dependent contact of deep cells with fibronectin. Deep cell adhesion to fibronectin is sufficient to initiate intercalation behavior in cell layers some distance from the substrate. Expression of a dominant-negative beta1 integrin construct in embryos results in localized depletion of the fibronectin matrix and thickening of the blastocoel roof. Lack of fibronectin fibrils in vivo is correlated with blastocoel roof thickening and a loss of deep cell polarity. The integrin-dependent binding of deep cells to fibronectin is sufficient to drive membrane localization of Dishevelled-GFP, suggesting that a convergence of integrin and Wnt signaling pathways acts to regulate radial intercalation in Xenopus embryos.

Regulation of renal proximal tubular epithelial cell fibroblast growth factor-2 generation by heparin

Progression of renal disease is closely correlated to the degree of renal interstitial fibrosis, and evidence is increasing that epithelial cells of the renal proximal tubule (PTCs) may contribute to its pathogenesis. Such cytokines as basic fibroblast growth factor (FGF-2) have been implicated in progressive renal injury, and we previously showed that PTCs are a source of this cytokine. FGF-2 is characterized by its high affinity for heparin, and numerous studies have suggested that heparin may modify the progression of renal disease. The current study examined how heparin influenced FGF-2 generation and bioactivity in the human renal epithelial PTC line, HK-2. Incubation of HK-2 cells with heparin led to a dose- and time-dependent increase in FGF-2 concentration in the culture supernatant that was not accompanied by alterations in FGF-2 messenger RNA expression, assessed by reverse-transcriptase polymerase chain reaction and Northern analysis. The heparin-induced increase in FGF-2 concentration was accompanied by a decrease in the amount of FGF-2 bound to the extracellular matrix, although this accounted for only a small proportion of the total FGF-2 generated. Induction of FGF-2 by 2-O-desulfated heparin, together with a reduction in total cell-associated FGF-2 and anti-FGF-2 antibody binding to fixed permeabilized cells after the addition of heparin, suggested that the FGF-2 released was mainly derived from a preformed intracellular source. That FGF-2 was predominantly derived from an intracellular pool was also confirmed by pulse chase experiments. The addition of heparin resulted in the generation of bioinactive FGF-2, judged by in vitro fibroblast proliferation. Conversely, heparitinase treatment of supernatant samples from heparin-treated cells and the addition of 2-O-desulfated heparin resulted in the generation of active FGF-2, suggesting that the generation of bioinactive FGF-2 was related to binding of FGF-2 by extracellular heparin after its release from cells. These data show that heparin depletes both the cell and surrounding matrix of FGF-2 and suggest that FGF-2 released from cells was mainly derived from a preformed intracellular source. Furthermore, FGF- 2 released from epithelial PTCs after the application of heparin was bioinactive. This likely resulted from released FGF-2 binding to an excess of extracellular heparin. Results presented here therefore suggest a mechanism by which heparin, through its effect on depletion of matrix and cells of FGF-2 and its generation in an inactive form, may influence progressive renal interstitial fibrosis. © 2001 by the National Kidney Foundation, Inc.

Expression of the transcriptional factor Egr-1/BTEB2 in cardiac xenograft vascular remodeling

IT IS important to consider that phenotypic change of smooth muscle cells (SMCs) is one of the important factors involved in vascular remodeling in cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty. Phenotypic change of SMCs induces fibrosis of vessels by collagen synthesis, degradation of vessels by depletion of extracellular matrix, or intimal thickening by proliferation and migration of SMCs. In xenograft rejection, phenotypic change of SMCs plays an important role in the process of forming vascular lesion formation, as it does in common other cardiovascular diseases. However, little is known about this mechanisms. Early growth response factor-1 (Egr-1) and basic transcriptional element binding protein-2 (BTEB2) regulate multiple genes involved in the remodeling of vessels in various vascular diseases. Egr-1 is a transcriptional factor of BTEB2, which is itself a transcriptional factor regulating nonmuscle type myosin heavy chain (SMemb), which is expressed in embryonic SMCs of fetal aorta and proliferating SMCs. Therefore, Egr-1 and BTEB2 play an important role in the dedifferentiation of SMCs. It would be useful to consider expression of Egr-1 and BTEB2 in cardiac xenografts to investigate the mechanisms of xenotransplant rejection.

Effect of transforming growth factor-beta on proteoglycan synthesis by chondrocytes in relation to differentiation stage and the presence of pericellular matrix.

The effects of transforming growth factor-beta (TGF-beta) on proteoglycan synthesis of chondrocytes are controversial. The hypothesis that the differential effect of TGF-beta is related to the differentiation stage of the chondrocytes is investigated in this study. Rabbit auricular chondrocytes were cultured in alginate. When seeded in alginate immediately after isolation, cells keep their cartilaginous phenotype. When cells are first cultured in monolayer, they lose their cartilaginous phenotype and become dedifferentiated. We used three different cell populations: (1) Differentiated cells (P0: immediately after isolation); (2) partially (de)differentiated cells (P1: after one passage in monolayer); (3) dedifferentiated cells (P4: after four passages in monolayer). Cells were characterized by morphology using electron microscopy, amount of proteoglycans using the Farndale assay and type of collagen produced using immunohistochemistry. The effects of addition of 10 ng/ml TGF-beta2 for 7 days to P0, P1 and P4 cells were compared. TGF-beta was added either directly from the start of the alginate culture, or after a preculture period of three weeks in alginate. The amount of proteoglycans was increased in all chondrocyte populations when TGF-beta was added immediately after seeding in alginate, indicating that the effect of TGF-beta on proteoglycan synthesis does not depend on the differentiation stage of cells. After preculture in alginate, stimulation of proteoglycan synthesis (as measured by amount of proteoglycans and 35S-sulfate incorporation) had vanished. This effect was independent of differentiation stage . A dose-response experiment with TGF-beta (1, 10, 50 ng/ml) confirmed this differentiation-stage-independent effect of TGF-beta on proteoglycan synthesis. Stimulation by TGF-beta can be retained after enzymatic digestion of the pericellular matrix and reseeding of the cells in alginate, indicating the importance of pericellular matrix for the effect of TGF-beta on matrix synthesis. Alkaline phosphatase (ALP) activity was largely inhibited by TGF-beta in P0 chondrocytes, either with or without preculture in alginate. After culturing in monolayer, ALP activity was not substantially changed by TGF-beta. This indicates that the effect of TGF-beta on ALP activity, in contrast to the effect on proteoglycan synthesis, does depend on the differentiation stage of the cells. Furthermore, the fact that ALP synthesis in P0 cells is still inhibited by TGF-beta after preculture indicates that these cells remain responsive to TGF-beta. This provides additional evidence for the importance of the pericellular matrix for regulation of the effect of TGF-beta on proteoglycan synthesis. The results indicate that, in pathological cartilage, matrix depletion might be the trigger for increased matrix synthesis in reaction to TGF-beta, suggesting an important role for TGF-beta in cartilage repair.

Presence of cartilage-derived morphogenetic proteins in articular cartilage and enhancement of matrix replacement in vitro.

To investigate the effects of the cartilage-derived morphogenetic proteins (CDMPs) in an in vitro cartilage explant model that mimics the chondrocytic response to matrix depletion, and to demonstrate their presence in articular cartilage. Adult bovine articular cartilage and postmortem specimens from adult human donors with and without osteoarthritic (OA) lesions were stained by immunohistochemistry using polyclonal antibodies specific for CDMP-1 and CDMP-2. Extracts of bovine articular cartilage were analyzed by Western blotting for the presence of the CDMPs. Bovine articular cartilage explants were depleted of their matrix by trypsin digestion, followed by a 7-day culture period in a chemically defined serum-free basal medium (BM), with or without recombinant CDMPs 1 and 2. The metabolic activity of chondrocytes was measured by 35S-sulfate incorporation into macromolecules. Newly synthesized proteoglycans (PGs) were analyzed using Sephacryl S-500 HR gel chromatography. The expression levels of the messenger RNA (mRNA) for chondrogenic markers were investigated by Northern analysis. CDMP-1 and CDMP-2 were detected in both bovine and human healthy and OA articular cartilage. Treatment of matrix-depleted cartilage explants with CDMPs 1 and 2 increased equally the incorporation of 35S-sulfate into PGs compared with tissue maintained in BM. Gel chromatography analysis indicated that aggrecan was the predominant PG species. Northern blot analysis showed that the expression of link protein, type II collagen, and aggrecan mRNA transcripts was not modulated by CDMP treatment. This study shows the presence of CDMP-1 and CDMP-2 in adult bovine and human articular cartilage. In addition, our in vitro data indicate that CDMPs 1 and 2 stimulate the metabolic activity of articular chondrocytes. Therefore, these signaling molecules may be contributing to the maintenance of the integrity of the joint surface.