Ratio Of Proteoglycan Research Articles

Changes in Matrix Components in the Developing Human Meniscus

Background: Treatment of meniscal tears is necessary to maintain the long-term health of the knee joint. Morphological elements, particularly vascularity, that play an important role in meniscal healing are known to change during skeletal development. Purpose: To quantitatively evaluate meniscal vascularity, cellularity, collagen, and proteoglycan content by age and location during skeletal development. Study Design: Descriptive laboratory study. Methods: Medial and lateral menisci from 14 male and 7 female cadavers aged 1 month to 11 years were collected and evaluated. For each meniscus, histologic and immunohistologic techniques were used to establish the ratio of the area of proteoglycan (safranin O) positivity to the total area (proteoglycan ratio), collagen type I and type II immunostaining positivity, number of blood vessels, and cell density. These features were evaluated over the entire meniscus and also separately in 5 circumferential segments: anterior root, anterior horn, body, posterior horn, and posterior root. Additionally, cell density and number of blood vessels were examined in 3 radial regions: inner, middle, and periphery. Results: Age was associated with a decrease in meniscal vessel count and cell density, while the proteoglycan ratio increased with skeletal maturity. Differences in vessel counts, cellular density, and proteoglycan ratio in different anatomic segments as well as in the inner, middle, and peripheral regions of the developing menisci were also observed. Collagen immunostaining results were inconsistent and not analyzed. Conclusion: The cellularity and vascularity of the developing meniscus decrease with age and the proteoglycan content increases with age. All of these parameters are influenced by location within the meniscus. Clinical Relevance: Age and location differences in meniscal morphology, particularly in the number of blood vessels, are expected to influence meniscal healing.

The Degree of Chondral Fragmentation Affects Extracellular Matrix Production in Cartilage Autograft Implantation: An In Vitro Study

To evaluate if the degree of chondral fragmentation affected extracellular matrix (ECM) production in cartilage fragment autograft implantation invitro. Cartilage was taken from 5 donors undergoing total hip replacement (mean age, 65.6years; standard deviation [SD], 3). The cartilage was minced to obtain 4 groups with different fragment sizes: (1) "fish scale" (diameter, 8mm; thickness, 0.3mm), (2) cubes with 2-mm sides, (3) cubes with 1-mm sides, and (4) cartilage paste (< 0.3mm). The cultures were maintained in chondrogenic medium for 6weeks. Biochemically, a proteoglycan (PG):DNA ratio was calculated as the best approximation of ECM production per cell. The ratio between PG released in the culture medium and the PG in the neocartilage (PGrel:PG) was used as a matrix stability index. Histologically, the slides were stained with safranin O fast green and collagen type II immunostaining. The titration of safranin O-positive cells and the Bern score were calculated. Regarding the PG:DNA ratio, group 4 performed significantly better than groups 1 (P= .001) and 3 (P= .02), whereas group 2 performed better than group 1 (P= .03). No significant difference was found regarding the PGrel:PG ratio and safranin O-positive cells. Regarding the Bern score, group 4 performed significantly better than groups 1 (P= .02), 2 (P= .04), and 3 (P= .03). We conclude that human cartilage fragmentation significantly affects ECM production invitro. Increased fragmentation enhances ECM production. Assuming a similar behavior invivo, we recommend mincing the cartilage into small pieces when performing the cartilage fragment autograft implantation technique in order to increase ECM production. Further invitro studies investigating cartilage of younger nonarthritic donors, as well as invivo studies, are needed.

Expression of stromelysins 2 and 3 in rabbit corneal epithelium upon UV radiation

AbstractPurpose Stromelysins belong to the group of matrix metalloproteinases (MMPs). These enzymes degrade many extracellular matrix proteins in the basement membrane, including proteoglycans and laminin, but are unable to cleave the fibrillar collagens. Activated stromelysin, via its capacity to catabolize proteoglycans, is likely to help reestablish transparency by returning the proteoglycan ratio to normal during remodeling. Purpose of this study was to investigate the effect of UVA and UVB rays on the expression of stromelysin 2 (MMP‐10) and 3 (MMP‐11) in the corneal epithelium.Methods In the first group of rabbits the corneas were irradiated with UVA lamp (365 nm, once a day during 4 days, and a dose per day 1.01 J/cm2). In the second group of rabbits the corneas were irradiated with UVB lamp (312 nm, once a day during 4 days, and a dose per day 1.01 J/cm2). Stromelysin 2 and 3 were examined on cryostat sections immunohistochemically by using sheep polyclonal anti‐MMP‐10 and anti‐MMP‐11 antibodies.Results Our immunohistochemical results showed that UVA rays did not change the expression of both stromelysins studied in the corneal epithelium. In contrast, UVB rays induced the increased expression of stromelysin 2 and 3 in corneal epithelial cells.Conclusion Even if further studies are necessary, our results point to the suggestions that increased stromelysins after UVB irradiation can participate in remodeling processes during corneal wound healing.

Overexpression of Transforming Growth Factor-β1 in the Valvular Fibrosis of Chronic Rheumatic Heart Disease

For the purpose of determining the pathogenic role of transforming growth factor-β1 (TGF-β1) in the mechanism of chronic rheumatic heart disease, we evaluated the expression of TGF-β1, proliferation of myofibroblasts, and changes in extracellular matrix components including collagen and proteoglycan in 30 rheumatic mitral valves and in 15 control valves. High TGF-β1 expression was identified in 21 cases (70%) of rheumatic mitral valves, whereas only 3 cases (20%) of the control group showed high TGF-β1 expression (p<0.001). Additionally, increased proliferation of myofibroblasts was observed in the rheumatic valves. High TGF-β1 expression positively correlated with the proliferation of myofibroblasts (p=0.004), valvular fibrosis (p<0.001), inflammatory cell infiltration (p=0.004), neovascularization (p=0.007), and calcification (p<0.001) in the valvular leaflets. The ratio of proteoglycan to collagen deposition inversely correlated with TGF-β1 expression in mitral valves (p=0.040). In conclusion, an ongoing inflammatory process, the expression of TGF-β1, and proliferation of myofibroblasts within the valves have a potential role in the valvular fibrosis, calcification, and changes in the extracellular matrix that lead to the scarring sequelae of rheumatic heart disease.

Aggrecan 1 expression in Dmm mice

Type II collagen is a major component of the cartilage extra-cellular matrix (ECM). The Disproportionate micromelia (Dmm) mouse contains a mutation in the C-terminal coding region of the type II collagen gene, Col2a1. This mutation causes severe skeletal abnormalities and is lethal in homozygotes (Dmm/Dmm). It also causes dwarfism in heterozygotes (Dmm/+). In Dmm/Dmm mice, type II collagen is retained in the endoplasmic reticulum (ER) of chondrocytes and is absent from the ECM. The structure of the ECM is severely disorganized. Dmm/+ mutants also show some accumulation of type II collagen in the ER, and the total amount of ECM is reduced, but the organization of collagen fibrils in the ECM appears relatively normal. This suggests that secretion of the proteoglycan components of the ECM in Dmm/+ mice is reduced in proportion to type II collagen. To identify a mechanism for this suppression, we used immunohistochemistry to determine whether aggrecan (a major component of cartilage proteoglycan) is retained in the ER of Dmm/+ fetal rib cartilage. We also used RT-PCR to determine whether aggrecan transcription is reduced. Aggrecan protein was not found to be abnormally retained in the ER. Transcription of the aggrecan gene, however, was reduced to one-third of wild-type in these cells. These results suggest that aggrecan levels in the cartilage of Dmm/+ mice are regulated by a feedback mechanism that acts at the level of transcription to maintain a correct ratio of proteoglycan to collagen. Funding through NIH AR048839.

Osteogenic protein-1 promotes the formation of tissue-engineered cartilage using the alginate-recovered-chondrocyte method

This study examined the effects of a growth factor, recombinant human osteogenic protein-1 (rhOP-1), on the formation of tissue-engineered cartilaginous tissue by adult bovine articular chondrocytes using the alginate-recovered-chondrocyte (ARC) method. To ascertain if rhOP-1 enhances the formation of the cell-associated matrix (CM) and the characteristics of CM formation, bovine articular chondrocytes were first cultured for up to 14 days in alginate beads in medium supplemented with serum, with or without rhOP-1. Then, the recovered chondrocytes and their associated CM were resuspended in medium, with or without OP-1, seeded onto culture inserts, and incubated for an additional 14 days. The fabricated ARC tissues were subjected to biochemical and histological analyses. The addition of rhOP-1 to the medium in the alginate bead culture step resulted in an increased accumulation of both proteoglycan (PG) and collagen, with a ratio of PG to collagen that was higher than that found in native adult cartilage. The addition of rhOP-1 in the second step had a similar stimulatory effect during 14 days of culture. Histological examination of the tissue formed under all conditions revealed a cartilage-like matrix, stained strongly by toluidine blue. The thickness of the tissues obtained from culture conditions that included the addition of rhOP-1 was four times greater than that of the tissues cultured without rhOP-1. Using the ARC method, rhOP-1 enhanced the formation of matrix and generated a voluminous tissue-engineered cartilaginous construct. These characteristics may be beneficial in generating constructs that can cover large defects.

The effect of a 5-day space flight on the immature rat spine

Background context: Spaceflight has many reported effects upon the musculoskeletal system structure and function. This study was designed to determine the effect of a 5-day flight on the rat spine.Methods: In September 1991, 8 neonatal rats were flown aboard the Space Shuttle Columbia flight STS-48 during a 5-day mission. Upon return to earth, the spines were dissected, frozen and shipped to our laboratory. Matched ground-based rats were used as controls. The spines were radiographed and then slowly thawed. Individual vertebrae were subjected to compressive biomechanical testing using an Instron tester (Instron Corp, Canton, MA, USA) and then processed for determination of calcium and phosphorus content. The intervertebral discs were placed in physiological saline and the stress-relaxation characteristics measured. The discs were then lyophilized and assayed for collagen and proteoglycan content. Disc height on radiographs was measured by image analysis.Results: After space flight, the heights of the discs were found to be 150 to 200 microns greater, although the values were not statistically significant. There was no difference in the resiliency of the thoracic discs as determined by stress-relaxation. However, in the lumbar discs, space flight increased the resiliency (p<.01). There was no difference in water content. In both the thoracic and lumbar discs there was a 3.3-fold increase in hydroxyproline–proteoglycan ratio after space flight. However, because of the small sample size, these values were not statistically significant. In the vertebrae, there was no difference in calcium–phosphate ratio or compressive strength.Conclusions: These data suggest that even after a short 5-day flight, the spine begins to undergo biomechanical and biochemical changes. In addition, the weightless environment in space may provide a good model to study the effects of immobilization on earth.

Mechanical behavior and biochemical composition of canine knee cartilage following periods of joint disuse and disuse with remobilization

The mechanical behavior and biochemical composition of articular cartilage were studied in an experimental model of joint disuse, in which the canine knee was immobilized in a sling at 90 degrees of flexion. Articular cartilage from the surface zone of the femur was tested in an isometric tensile test and full-thickness cartilage on the tibial plateau was tested in a compressive indentation test. Water, proteoglycan and collagen contents were measured in site-matched samples. Site-specific increases in the tensile moduli (approximately 88% above control values in distal femoral groove) were observed in cartilage after 8 weeks of joint disuse, and after 3 weeks of remobilization following either 4 (approximately 140%, distal and proximal femoral groove) or 8 weeks (approximately 140%, distal femoral groove) of joint disuse. In contrast, the compressive properties of cartilage determined in the indentation test exhibited no change from control values with joint disuse or disuse followed by remobilization. Water contents increased at some sites on the tibia after 8 weeks of joint disuse (approximately 6% of tissue wet weight, posterior site), but not in the surface zone tissue of the femur. Proteoglycan/collagen and cartilage thickness were not found to change with disuse or disuse followed by remobilization. Reduced values for the ratio of proteoglycan:water were observed in the surface zone tissue of the femur (approximately 23%, distal femoral groove) and in the full-thickness tissue of the tibia (approximately 21%, anterior and posterior sites) after periods of joint disuse. In this study, the measured material properties suggest that the articular surface remains intact following periods of disuse or disuse with remobilization. This finding suggests one important difference between this model of joint disuse and other experimental models in which cartilage changes are both progressive and degenerative, such as surgically-induced joint instability.

Regulation of Rat Mast Cell Protease 1 Activity

Rat mast cell protease 1 (RMCP-1) is a chymotrypsin-like serine protease (chymase) that is specifically expressed by connective-tissue-type mast cells. It is stored in the secretory granules of the cells in a complex with heparin proteoglycan, and the chymase/heparin proteoglycan complexes are released following mast cell activation. The present study was undertaken to examine if the association with heparin proteoglycan influenced the regulation of RMCP-1 by various macromolecular protease inhibitors. Endogenous mast cell heparin proteoglycan was shown to significantly block the inhibition of RMCP-1 by the serpins alpha 1-protease inhibitor and alpha 1-antichymotrypsin, as well as the inhibition by alpha 2-macroglobulin, soybean trypsin inhibitor and plasma. The blocking of protease inhibition showed an optimum at a RMCP-1/proteoglycan ratio of 5:1 (by mass), corresponding to approximately 80 RMCP-1 molecules bound/proteoglycan molecule. Chymase activity present on intact peritoneal mast cells, i.e. present in its native complex with heparin proteoglycan, was also shown to be largely resistant to inhibition by alpha 1-antichymotrypsin and alpha 1-protease inhibitor. Heparin 10-saccharides and 20-saccharides were inefficient in preventing the interaction of RMCP-1 with alpha 1-antichymotrypsin, whereas pig mucosal heparin (approximately 50 monosaccharide units) blocked protease inhibition. We have previously shown that heparin potentiates the catalytic activity of RMCP-1 and, in the present study, we show that the mechanism for chymase activation involves a sixfold reduction of the Km,app value of RMCP-1 for the chromogenic substrate S-2586. Thus, the association of mast cell chymase with heparin proteoglycan may serve both to potentiate the catalytic activity of the enzyme and to increase the life-span of the chymases by preventing their inhibition after exocytosis.

The effect of proteoglycans on the formation of fibrils from collagen solutions

The precipitation of collagen fibrils from solutions at 37 °C and approximately physiological pH and ionic strength is retarded markedly in the presence of small amounts of proteoglycan monomer (PGS), proteoglycan aggregate (PGC), reduced and alkylated PGS, or cyanogen bromide-cleavage products of PGS. The polysaccharide-peptide fragments produced from PGS with papain, trypsin, cathepsin D, or the protein core obtained by the digestion of PGS with chondroitinase ABC are ineffective in this regard. In the presence of the materials which affected the rate of precipitation of the collagen fibrils, the specific absorbance, ΔA sp, of the collagen gels was directly related to specific retardation, R sp, when the ratio of proteoglycan to collagen was less than 25 μg/mg, suggesting that the size and/or organization of the fibrils in the gels was dependent on the presence of proteoglycans. PGS binds to collagen if it is present in the solution before the collagen fibrils form and at a maximum of about 1 molecule of PGS for every 25–30 molecules of collagen. Although the protein core of PGS does not retard fibrillogenesis, it does bind to collagen and does so even in the presence of PGS. The data support the thesis that the organization of collagen fibrils in tissues may be related to amounts and kinds of proteoglycans in the tissues.