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Rheumatology 05/2006; 45(4):488-90. · 4.06 Impact Factor
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ABSTRACT: The cow could be a suitable model for studies concerning functional changes of the cervix. However, as in many species, the bovine cervix becomes softer in texture during the follicular phase of the estrous cycle compared to the luteal phase. In the present study, we explored if changes in the collagen network take place that could be responsible for this phenomenon and if regional differences in water content, collagen content, and collagen degradation along the cross-sectional and longitudinal axes of the cervix were present. Two groups of nonpregnant animals with different progesterone status were studied. One group (n = 11) was under high progesterone influence, and the other group (n = 12) was under low progesterone influence. The water content was derived from the weight of the samples before and after lyophilization. The collagen content and the ratio of collagenous to noncollagenous proteins (hydroxyproline:proline ratio) were determined by performing amino acid analysis on hydrolyzed samples using high-performance liquid chromatography. Collagen denaturation was quantified with a colorimetric assay by determining the amount of hydroxyproline released from samples treated with alpha-chymotrypsine. The water content of the superficial layer of the submucosa was always significantly (P < 0.01) higher than the water content of the deep layer in the vaginal, mid, and uterine segments, but this was unrelated to the progesterone status of the animals. No effect of the tissue layers or of the progesterone status of the animals on the collagen content was observed, but an effect of segment was noted. The collagen content (mug/mg dry wt) in the vaginal segment of the cervix was significantly higher than in the mid (P < 0.05) and the uterine (P < 0.01) segments. The hydroxyproline:proline ratio showed the same pattern as the collagen content. The percentage of collagen denaturation in the superficial layer was always significantly (P < 0.01) higher than that in the deep layer, but no effect of the progesterone status or of the segment along the longitudinal axis was seen. It is concluded that regional differences in collagen biochemistry are present in the cervix of nonpregnant cows, which may account for the difference in firmness of different parts along the circular or the longitudinal axis of the cervix. However, differences in texture of the cervix between the two groups of cows could not be explained by differences in the collagen content, percentage of collagen denaturation, or water content.
Biology of Reproduction 12/2003; 69(5):1600-7. · 4.01 Impact Factor
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ABSTRACT: The objective of the present study was to assess if cervical ripeness could be quantified by measuring the percentage of denaturation of the collagen network of the stromal layer. Biopsy specimens from the caudal part of the cervix were obtained from nine pluriparous cows between Days 149 and 157 of gestation (second-trimester biopsy), at exactly Day 275 of gestation (term biopsy), and shortly after calving (calving biopsy). The samples were divided into a superficial stromal part and a deep stromal part. The water content was derived from the weight of the samples before and after lyophilization. A colorimetric assay was used to assess the percentage of collagen denaturation by determining the extinction at 570 nm of hydroxyproline released from alpha-chymotrypsine-treated samples. By incorporating a hydroxyproline standard series in the measurements, the insoluble collagen content (mug/mg dry wt) as well as the insoluble collagen concentration (mug/mg wet wt) could be derived. The water content of both layers of the cervix significantly increased between midpregnancy and parturition (P < 0.01). The insoluble collagen content and the insoluble collagen concentration were significantly increased at term (P < 0.01 and P < 0.05, respectively) but were significantly decreased at calving (P < 0.05 and P < 0.01, respectively). Both parameters showed no significant differences between the superficial and deep stromal layer, and they were significantly correlated with each other. A significant increase in the percentage denaturation of the deep stromal layer occurred between the second trimester and term pregnancy (P < 0.01), whereas at calving, the percentage denaturation had not significantly increased compared to term. The percentage of collagen denaturation of the superficial stromal layer did not significantly change with stage of gestation or at parturition. Our findings indicate that cervical ripening is a combination of increased collagen synthesis and increased percentage of collagen denaturation, whereas at calving, an increased digestion of the denatured collagen leads to increased collagen loss from the cervical connective tissue. The finding that cervical ripening mainly takes place in the deep stromal layer of the cervix emphasizes the importance of a detailed description of the tissue sampling sites for a proper interpretation of the results obtained from biochemical studies of the cervix.
Biology of Reproduction 12/2003; 69(5):1608-14. · 4.01 Impact Factor
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ABSTRACT: The objective of this study was to investigate how molecular level changes in the collagen network affect its mechanical integrity. Our hypothesis is that the cleavage and unwinding of triple helices of collagen molecules significantly reduce the mechanical integrity of the collagen network in bone, whereas collagen crosslinks play a major role in sustaining the structural integrity of the collagen network. To test this hypothesis, the collagen molecular structure was altered in demineralized human cadaveric bone samples in the following two ways: heat induced unwinding and pancreas elastase induced cleavage of collagen molecules. Along with control specimens, the treated specimens were mechanically tested in tension to determine their strength, elastic modulus, toughness, and strain to failure. Also, the percentage of denatured collagen molecules and amounts of two major collagen crosslinks (hydroxylysylpyridinoline and lysylpyridinoline) were determined using high-performance liquid chromatography techniques. It was found that unwinding of collagen molecules may cause more reduction in stiffness (E) but less strain to failure (ef) than cleavage. Both collagen denaturation types cause similar changes in the strength (ss) and work to fracture (Wf) of the collagen network with no significant changes in hydroxylysylpyridinoline and lysylpyridinoline crosslinks. The results of this study indicate that the integrity of collagen molecules significantly affect the mechanical properties of the collagen network in bone, and that collagen crosslinks may play an important role in maintaining the mechanical integrity of the collagen network after collagen denaturation occurs.
Calcified Tissue International 09/2002; 71(2):186-92. · 2.38 Impact Factor
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ABSTRACT: The objective of this study was to document the development of biochemical heterogeneity from birth to maturity in equine articular cartilage, and to test the hypothesis that the amount of exercise during early life may influence this process. Neonatal foals showed no biochemical heterogeneity whatsoever, in contrast to a clear biochemical heterogeneity in mature horses. The process of formation of site differences was almost completed in exercised foals age 5 months, but was delayed in those deprived of exercise. For some collagen-related parameters, this delay was not compensated for after an additional 6 month period of moderate exercise. It is concluded that the functional adaptation of articular cartilage, as reflected in the formation of biochemical heterogeneity in the horse, occurs for the most part during the first 5 months postpartum. A certain level of exercise seems essential for this process and withholding exercise in early life, may result in a delay in the adaptation of the cartilage.
Equine Veterinary Journal 06/2002; 34(3):265-9. · 1.46 Impact Factor
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ABSTRACT: Subchondral bone provides structural support to the overlying articular cartilage, and plays an important role in osteochondral diseases. There is growing insight that the mechanical features of bone are related to the biochemistry of the collagen network and the mineral content. In the present study, part of the normal developmental process and the influence of physical activity on biochemical composition of subchondral bone was studied. Water content, calcium content and characteristics of the collagen network (collagen, hydroxylysine, lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) crosslinking) of subchondral bone were measured in newborn foals, 5-month-old foals (pasture-grown and box-confined) and 11-month-old foals at 2 differently loaded sites of the proximal articular surface of the first phalanx. During the first 5 months postpartum, water and hydroxylysine content decreased significantly while calcium and collagen content and the amount of HP and LP crosslinks increased significantly. The withholding of physical activity during this developmental phase affected the biochemical characteristics of subchondral bone only at the site that is loaded during physical exercise. At this site, calcium content and both HP and LP crosslink levels increased significantly less than in pasture-raised animals. During development from 5-11 months, measured parameters remained essentially constant, except for water content, which decreased further. It is concluded that substantial changes, presumed to be largely exercise-driven, take place during the normal process of development in the biochemical composition of equine subchondral bone. Normal development of subchondral bone is presumably important for the normal functional adaptation of this bone to the loading conditions it is subjected to and therefore essential to resist the future biomechanical challenges the horse will encounter during its athletic career. The findings from this study and the assumed important role of subchondral bone quality in the pathogenesis of osteochondral disease merit more attention to the role of the collagen network in subchondral bone.
Equine Veterinary Journal 04/2002; 34(2):143-9. · 1.46 Impact Factor
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ABSTRACT: The collagen network in human articular cartilage experiences a large number of stress cycles during life as it shows hardly any turnover after adolescence. We hypothesized that, to withstand fatigue failure, the physical condition of the collagen network laid down at adolescence is of crucial importance for the age of onset of osteoarthritis (OA).
We have compared the lysyl hydroxylation level and pyridinoline cross-link level of the collagen network of degenerated (DG) cartilage of the femoral knee condyle (representing a preclinical early stage of OA) with that of normal cartilage from the contralateral knee. The biological age of the collagen network was determined by means of pentosidine levels. For each donor, collagen modifications of normal cartilage were compared with DG cartilage that showed no significant remodeling of the collagen network (as evidenced by identical pentosidine levels).
DG cartilage contained significantly more hydroxylysine residues per collagen molecule in comparison with healthy cartilage from the same donor, both in the upper and lower half (the region near the articular surface and adjacent to bone, respectively). In addition, a significantly higher level of pyridinoline cross-linking was observed in the upper half of DG cartilage. Considering the biological age of the collagen network, the changes observed in DG cartilage must have been present several decades before cartilage became degenerated.
The data suggest that high levels of lysyl hydroxylation and pyridinoline cross-linking result in a collagen network that fails mechanically in long term loading. Areas containing collagen with low hydroxylysine and pyridinoline levels are less prone to degeneration. As such, this study indicates that post-translational modifications of collagen molecules synthesized during adolescence are causally involved in the pathogenesis of OA.
Osteoarthritis and Cartilage 03/2002; 10(2):127-34. · 3.90 Impact Factor
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ABSTRACT: The objective of this study was to investigate how molecular level changes in the collagen network affect its mechanical integrity. Our hypothesis is that the cleavage and unwinding of triple helices of collagen molecules significantly reduce the mechanical integrity of the collagen network in bone, whereas collagen crosslinks play a major role in sustaining the structural integrity of the collagen network. To test this hypothesis, the collagen molecular structure was altered in demineralized human cadaveric bone samples in the following two ways: heat induced unwinding and pancreas elastase induced cleavage of collagen molecules. Along with control specimens, the treated specimens were mechanically tested in tension to determine their strength, elastic modulus, toughness, and strain to failure. Also, the percentage of denatured collagen molecules and amounts of two major collagen crosslinks (hydroxylysylpyridinoline and lysylpyridinoline) were determined using high-performance liquid chromatography techniques. It was found that unwinding of collagen molecules may cause more reduction in stiffness (E) but less strain to failure (ef) than cleavage. Both collagen denaturation types cause similar changes in the strength (ss) and work to fracture (Wf) of the collagen network with no significant changes in hydroxylysylpyridinoline and lysylpyridinoline crosslinks. The results of this study indicate that the integrity of collagen molecules significantly affect the mechanical properties of the collagen network in bone, and that collagen crosslinks may play an important role in maintaining the mechanical integrity of the collagen network after collagen denaturation occurs.
Calcified Tissue International 01/2002; 71(2):186-192. · 2.38 Impact Factor
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ABSTRACT: The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37-200 degrees C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three-point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone, thereby supporting the hypothesis of this study.
Journal of Orthopaedic Research 12/2001; 19(6):1021-6. · 2.81 Impact Factor
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ABSTRACT: Progressive destruction of articular cartilage is a hallmark of osteoarthritis (OA) and rheumatoid arthritis (RA). Age-related changes in cartilage may influence tissue destruction and thus progression of the disease. Therefore, the effect of age-related accumulation of advanced glycation end products (AGEs) on cartilage susceptibility to proteolytic degradation by matrix metalloproteinases (MMPs) present in synovial fluid (SF) of OA and RA patients was studied.
Cartilage was incubated with APMA-activated SF obtained from OA or RA patients, and tissue degradation was assessed by colorimetric measurement of glycosaminoglycan (GAG) release. Cartilage degradation was related to the level of AGEs in cartilage from donors of different ages (33-83 years) and in cartilage with in vitro-enhanced AGE levels (by incubation with ribose). MMP activity in SF was measured using a fluorogenic substrate. AGE levels were assessed by high-performance liquid chromatography measurement of the glycation product pentosidine.
In cartilage from donors ages 33-83 years, a strong correlation was found between the age-related increase in pentosidine and the decrease in MMP-mediated tissue degradation (r = -0.74, P < 0.0005). Multiple regression analysis showed pentosidine to be the strongest predictor of the decreased GAG release (P < 0.0005); age did not contribute (P > 0.8). In addition, decreased MMP-mediated GAG release was proportional to increased pentosidine levels after in vitro enhancement of glycation (r = -0.27, P < 0.01). This was demonstrated for both OA and RA SF (for control versus glycated, P < 0.002 for all SF samples tested).
Increased cartilage AGEs resulted in decreased cartilage degradation by MMPs from SF, indicating that aged cartilage is less sensitive than young cartilage to MMP-mediated cartilage degradation, such as occurs in OA and RA. Therefore, the level of cartilage glycation may influence the progression of these diseases.
Arthritis & Rheumatism 11/2001; 44(11):2562-71. · 7.87 Impact Factor
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ABSTRACT: The prevalence of osteoarthritis (OAs) increases with age and coincides with the accumulation of advanced glycation endproducts (AGEs) in articular cartilage, suggesting that accumulation of glycation products may be involved in the development of OA. This study was designed to examine the effects of accumulation of AGEs on the turnover of the extracellular matrix of human articular cartilage.
Chondrocyte mediated cartilage degradation (GAG release, colorimetric) was measured in human articular cartilage of donors aged 19-82 years (N=30, 4-day culture). In addition, to mimic the age-related increase in AGE levels in vitro, cartilage was cultured in the absence or presence of glucose, ribose or threose. Cartilage degradation and proteoglycan synthesis ((35)SO(2)(-4) incorporation) were measured and related to the degree of cartilage AGE levels (fluorescence at 360/460 nm).
Chondrocyte-mediated degradation of articular cartilage (i.e. GAG release) decreased with increasing age of the cartilage donor (r=-0.43, P< 0.02). In vitro incubation of cartilage with glucose, ribose or threose resulted in a range of AGE levels that was highly correlated to the chondrocyte-mediated cartilage degradation (r=-0.77, P< 0.001, N=26). In addition, in these in vitro glycated cartilage samples, a decrease in proteoglycan synthesis was observed at increasing AGE levels (r=-0.54, P< 0.005, N=25).
This study shows that an increase in AGE levels negatively affects the proteoglycan synthesis and degradation of articular cartilage. In combination, these two effects reduce the turnover of the cartilage and thereby the maintenance and repair capacity of the tissue. By this mechanism, the age-related increase in cartilage AGE levels may contribute to the development of OA.
Osteoarthritis and Cartilage 11/2001; 9(8):720-6. · 3.90 Impact Factor
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ABSTRACT: Subchondral bone provides structural support to the overlying articular cartilage and plays an important role in osteochondral diseases. There is growing insight that the mechanical features of bone are related to the biochemistry of the collagen network. In this study the effect of exercise on water, calcium and the collagen network (total collagen, lysyl-hydroxylation, hydroxylysylpyridinoline, and lysylpyridinoline crosslinks) of subchondral bone at two differently loaded sites (site 1: intermittently loaded; site 2: constantly loaded) is investigated in foals. Exercise influenced calcium content and levels of both types of crosslinks at site 1, but had no influence on site 2. There was no concomitant increase in lysyl-hydroxylation level with the rise in crosslinks. Levels of lysyl-hydroxylation and lysylpyridinoline crosslinking were lower at site 1 than at site 2. It is concluded that exercise affects the post-translational modifications of the collagen component of subchondral bone. Loading also appears to play a role in site-related topographical differences. The lack of any relation between the sum of pyridinoline crosslinks and the amount of triple helical hydroxylysine gives support to a recent hypothesis that lysyl-hydroxylation of the triple helix and the telopeptides are under separate control.
The Veterinary Journal 08/2001; 162(1):24-32. · 2.24 Impact Factor
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H K Ronday,
W H van der Laan,
P P Tak,
J A de Roos, R A Bank,
J M TeKoppele,
C J Froelich,
C E Hack,
P C Hogendoorn,
F C Breedveld,
J H Verheijen
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ABSTRACT: To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-positive cells at sites of erosion in the rheumatoid synovium.
Granzyme B was added to [(3)H]proline/[(35)S]sulphate-labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of (35)S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of (3)H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry.
Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B-positive cells were present throughout the synovium and at the invasive front.
The presence of granzyme B-positive cells at the invasive front of the synovium together with its ability to degrade articular proteoglycans supports the view that granzyme B may contribute to joint destruction in rheumatoid arthritis.
Rheumatology 02/2001; 40(1):55-61. · 4.06 Impact Factor
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ABSTRACT: Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p < 0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p < 0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p < 0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p < 0.0005). Interestingly, the slopes of the curves of AGEs versus % d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens.
Journal of Biological Chemistry 01/2001; 275(50):39027-31. · 4.77 Impact Factor
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ABSTRACT: In order to assess the influence of strenuous exercise on collagen characteristics of articular cartilage, the response of the collagen network was studied in seven 2-year-old Thoroughbreds subjected to strenuous exercise compared to 7 nontrained individuals. After 13 weeks, the animals were subjected to euthanasia, fetlock joints of the forelimbs were scored macroscopically after Indian Ink staining, and articular cartilage from different locations of the articular surface of the proximal first phalanx was sampled and analysed for water content, collagen content, hydroxylysine content and amount of hydroxylysylpyridinoline (HP) crosslinks. Gross lesions were significantly more severe in the exercised than in the nonexercised group. In the control animals, the characteristic site-specific differences in collagen parameters were found as described earlier, but in the strenuously exercised animals this physiological biochemical heterogeneity had disappeared. In the exercised animals, an increase in water content and a sharp decrease in HP crosslinking was found that was correlated with the presence of wear lines. It is concluded that the strenuous exercise provoked significant alterations in the characteristics of the collagen network of the articular cartilage of the fetlock joint which were suggestive of microdamage and loosening of the collagen network. The collagen component of cartilage, in contrast to the proteoglycan component, is known to have a very limited capacity for repair and remodelling due to an extremely low turnover rate. Therefore, alterations within the articular collagen network might be expected to play an important role in the pathophysiology of degenerative joint disorders.
Equine Veterinary Journal 12/2000; 32(6):551-4. · 1.46 Impact Factor
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ABSTRACT: To provide evidence for the hypothesis that the loss of tensile strength of osteoarthritic (OA) cartilage (resulting in swelling-the hallmark of OA) is due to an impaired collagen network and not to loss or degradation of proteoglycans.
The amount of degraded collagen molecules, the fixed charge density (FCD) on a dry-weight basis, the degree of swelling in saline, and the instantaneous deformation (ID; a test reflecting the tensile stiffness of the collagen network) were measured in full-depth OA femoral condyle samples. In addition, levels of the crosslink hydroxylysylpyridinoline (HP), the amount of degraded collagen molecules, and the degree of swelling were determined in the 3 zones (surface, middle, and deep) of OA cartilage. We also compared the ID of normal and OA cartilage.
In full-depth OA cartilage, a close relationship was found between swelling and ID. Swelling and ID correlated strongly with the amount of degraded collagen molecules, and were not related to FCD. OA cartilage showed the same zonal pattern in HP levels as normal cartilage (i.e., an increase with depth). No relationship was found between collagen crosslinking and swelling of the surface, middle, and deep zones. In all 3 zones, swelling was proportional to the amount of degraded collagen molecules. Compared with that of normal cartilage, the change in ID of OA cartilage was most pronounced at the surface in a direction parallel to the direction of the collagen fibrils.
The decreased stiffness of the OA collagen network (as measured by swelling and ID) is strongly related to the amount of degraded collagen molecules. The anisotropy in ID parallel and perpendicular to the direction of the fibrils revealed that the impairment of strength resides mainly in, and not between, the fibrils. Proteoglycans play only a minor role in the degeneration of the tensile stiffness of OA cartilage.
Arthritis & Rheumatism 11/2000; 43(10):2202-10. · 7.87 Impact Factor
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ABSTRACT: Although >80% of the mineral in mammalian bone is present in the collagen fibrils, limited information is available about factors that determine a proper deposition of mineral. This study investigates whether a specific collagen matrix is required for fibril mineralization. Calcifying callus from dog tibias was obtained at various times (3-21 weeks) after fracturing. At 3 weeks, hydroxylysine (Hyl) levels were almost twice as high as in control bone, gradually reaching normal levels at 21 weeks. The decrease in Hyl levels can only be the result of the formation of a new collagen network at the expense of the old one. The sum of the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) in callus matched that of bone at all stages of maturation. However, the ratio HP/LP was 2.5-4.5 times higher in callus at 3-7 weeks than in normal bone and was normalized at 21 weeks. Some 40% of the collagen was nonmineralized at the early stages of healing, reaching control bone values (approximately 10%) at 21 weeks. In contrast, only a small increase in callus mineral content from 20.0 to 22.6 (% of dry tissue weight) from week 3 to 21 was seen, indicating that initially a large proportion of the mineral was deposited between, and not within, the fibrils. A strong relationship (r = 0.80) was found between the ratio HP/LP and fibril mineralization; the lower the HP/LP ratio, the more mineralized the fibrils were. Because the HP/LP ratio is believed to be the result of a specific packing of intrafibrillar collagen molecules, this study implies that mineralization of fibrils is facilitated by a specific orientation of collagen molecules in the fibrils.
Journal of Bone and Mineral Research 10/2000; 15(9):1776-85. · 6.37 Impact Factor
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ABSTRACT: Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated with a stiffer collagen network in cartilage. However, even in cartilage, pentosidine levels themselves represent <1 cross-link per 20 collagen molecules, and as such cannot be expected to contribute substantially to the increase in collagen network stiffness. In the present study, we investigated a broad range of Maillard reaction products in cartilage collagen in order to determine whether pentosidine serves as an adequate marker for AGE levels. Not only did the well-characterized AGEs pentosidine, N(epsilon)-(carboxymethyl)lysine, and N(epsilon)-(carboxyethyl)lysine increase with age in cartilage collagen (all P<0.0001), but also general measures of AGE cross-linking, such as browning and fluorescence (both P<0.0001), increased. The levels of these AGEs are all higher in cartilage collagen than in skin collagen. As a functional measure of glycation the digestibility of articular collagen by bacterial collagenase was investigated; digestibility decreased linearly with age, proportional to the extent of glycation. Furthermore, the arginine content and the sum of the hydroxylysine and lysine content of cartilage collagen decrease significantly with age (P<0.0001 and P<0. 01 respectively), possibly due to modification by the Maillard reaction. The observed relationship between glycation and amino acid modification has not been reported previously in vivo. Our present results indicate that extensive accumulation of a variety of Maillard reaction products occurs in cartilage collagen with age. Altogether our results support the hypothesis that glycation contributes to stiffer and more brittle cartilage with advancing age.
Biochemical Journal 09/2000; 350 Pt 2:381-7. · 4.90 Impact Factor
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ABSTRACT: The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.
Journal of Bone and Mineral Research 08/2000; 15(7):1330-6. · 6.37 Impact Factor
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ABSTRACT: Biochemical heterogeneity of cartilage within a joint is well known in mature individuals. It has recently been reported that heterogeneity for proteoglycan content and chondrocyte metabolism in sheep develops postnatally under the influence of loading. No data exist on the collagen network in general or on the specific situation in the horse. The objective of this study was to investigate the alterations in equine articular cartilage biochemistry that occur from birth up to age one year, testing the hypothesis that the molecular composition of equine cartilage matrix is uniform at birth and biochemical heterogeneity is formed postnatally. Water content, DNA content, glycosaminoglycan content (GAG) and biochemical characteristics of the collagen network (collagen content, hydroxylysine content and hydroxylysylpyridinoline [HP] crosslinks) were measured in immature articular cartilage of neonatal (n = 16), 5-month-old foals (n = 16) and yearlings (n = 16) at 2 predefined differently loaded sites within the metacarpophalangeal joint. Statistical differences between sites were analysed by ANOVA (P<0.01), and age correlation was tested by Pearson's product moment correlation analysis (P<0.01). In neonatal cartilage no significant site differences were found for any of the measured biochemical parameters. This revealed that the horse has a biochemically uniform joint (i.e. the cartilage) at birth. In the 5-month-old foals and yearlings, significant site differences, comparable to those in the mature horse, were found for DNA, GAG, collagen content and hydroxylysine content. This indicates that functional adaptation of articular cartilage to weight bearing for these biochemical parameters takes place during the first months postpartum. Water content and HP crosslinks showed no difference between the 2 sites from neonatal horses, 5-month-old animals and yearlings. At both sites water, DNA and GAG decreased during maturation while collagen content, hydroxylysine content and HP crosslinks increased. We propose that a foal is born with a uniform biochemical composition of cartilage in which the functional adaptation to weight bearing takes place early in life. This adaptation results in biochemical and therefore biomechanical heterogeneity and is thought to be essential to resist the different loading conditions to which articular cartilage is subjected during later life. As collagen turnover is extremely low at mature age, an undisturbed functional adaptation of the collagen network of articular cartilage at a young age may be of significant importance for future strength and resistance to injury.
Equine Veterinary Journal 06/2000; 32(3):217-21. · 1.46 Impact Factor
