Reducible Cross-links Research Articles

The effects of hyaluronan on tissue healing after meniscus injury and repair in a rabbit model.

To assess the effect of hyaluronan on meniscus injury and repair, we had 35 mature New Zealand White rabbits undergo bilateral meniscus injury and repair (19 in the peripheral region, and 16 in the inner region). A longitudinal tear was created in the medial meniscus and repaired with horizontally placed nylon sutures. The left knee joint received intraarticular injections of hyaluronan 1 week after surgery and once a week for 5 weeks. The right knees were injected with phosphate-buffered saline (the carrier vehicle of the hyaluronan). Twelve weeks after repair, tears in the peripheral region showed gross and histologic evidence of healing, with no difference between the vehicle- and hyaluronan-treated menisci. Biochemically, the ratio of reducible collagen cross-links in the hyaluronan-treated menisci was significantly higher than in the vehicle-treated menisci, indicating greater level of collagen remodeling. Biomechanically the vehicle- and hyaluronan-treated menisci demonstrated similarly high tearing load and fracture toughness. In the inner region, poor healing response was observed grossly and histologically in both treatment groups. Water content in the hyaluronan-treated menisci was significantly lower than in the vehicle-treated menisci, indicating a lower level of swelling. Hyaluronan treatment stimulated collagen remodeling in the peripheral region and inhibited swelling of the meniscus repaired in the inner region.

Changes in collagen cross-linking in degenerative disc disease and scoliosis.

Biochemical study of the changes in the collagen cross-link profile of human intervertebral discs collected at surgery from patients with either low back pain associated with disc degeneration or scoliosis. To determine whether changes occur in the collagen cross-link profile in the disc of patients with either low back pain associated with disc degeneration or scoliosis, which may well influence matrix integrity. Such changes in the cross-link profile of a tissue indicates increased matrix turnover and tissue remodeling and may have implications for the progression of these disorders. The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. The stability of the collagenous components and hence the mechanical integrity of connective tissues such as the disc is dependent on the degree and type of cross-links between the collagen molecules. This article reports results on the distribution of the different cross-links in the disc and the changes that occur with age, degenerative disc disease, and scoliosis. Thirty-three discs were obtained from patients with degenerative disc disease and 29 discs from patients with scoliosis. Samples were acid hydrolyzed and the collagen cross-links analyzed by either fractionation on an amino acid analyzer configured for cross-link analysis using ninhydrin postcolumn detection or fractionation by high-pressure liquid chromatography with fluorescence detection. The reducible cross-links and the mature cross-link all increased from the outer anulus fibrosus through into the nucleus pulposus. The highest levels of the mature cross-link were found in the cartilage end-plate. The nonenzymic derived cross-link, pentosidine, in contrast, showed little difference across the disc, but did show the expected age-related increase. In degenerative disc disease, no change in the levels of the reducible or mature cross-links was found, but a decrease was observed in the levels of the age-related cross-link pentosidine in the more severe disease samples. In scoliosis, significantly higher levels of the reducible cross-links were found on the convex than on the concave side of the scoliotic disc. These changes in the cross-link profile of the intervertebral disc in degenerative disc disease and scoliosis are indicative of increased matrix turnover and tissue remodeling and likely to have implications for the progression of these disorders.

Thermomechanical analysis of collagen crosslinking in the developing ovine thoracic aorta

We have used hydrothermal isometric tension (HIT) techniques in a sheep model to assess collagen crosslink stability and its contribution to the mechanical properties of the ovine thoracic aorta during perinatal and postnatal development. Aortic tissue was studied from fetal sheep, lambs, and adult sheep. Strips of tissue were loaded under isometric tension and heated to a 90 °C isotherm which was sustained for 3 hours. The decrease in load at this temperature is associated with collagen peptide bond hydrolysis and chain slippage, and the rate of this decrease is an inverse indicator of collagen crosslinking. The half-time of load decay ( t 1/2) was computed before and after tissue was treated with NaBH 4 which stabilizes immature, reducible crosslinks. We observed a two-fold increase in t 1/2 of untreated tissue from the lamb to the adult, indicating that aortic collagen crosslinking increased during postnatal development. Furthermore, the t 1/2 of NaBH 4-stabilized lamb tissue was similar to that of the untreated adult tissue, suggesting that much of the immature crosslinking in the lamb is stabilized during postnatal development. These observations suggest (a) increased crosslinking occurs during postnatal development and (b) that this increase is largely due to a conversion of immature crosslinks into their mature, heat stable form.

Analysis of Collagen and Elastic Fibers in Shoulder Capsule in Patients with Shoulder Instability

This study examined collagen cross-links, collagen fibril diameter and density, amino acid composition, and elastic fibers in shoulder capsule and skin in four patient groups: 1) unidirectional anterior instability (N = 8); 2) multidirectional instability/primary surgery (N = 6); 3) multidirectional instability/revision surgery (N = 6); and 4) no history of instability (N = 5). Compared with normal capsule, capsule from groups 1 and 2 had more stable and reducible collagen cross-links, significantly greater mean collagen fibril diameter, more cysteine, and a higher density of elastin staining. Compared with shoulder capsule in groups 1 and 2, shoulder capsule from group 3 contained significantly more reducible cross-links, smaller-diameter collagen fibrils, decreased collagen fibril density, and an increased density of elastin staining. There were no significant differences in any parameters between groups 1 and 2. We hypothesized that repeated capsular deformation in patients with shoulder instability results in changes in the capsule that increase its strength and resistance to stretching. Skin analyses demonstrated a significantly smaller mean collagen fibril diameter in skin from group 2 compared with group 1, suggesting the possibility of an underlying connective tissue abnormality.

Altered Posttranslational Modifications of Collagen in Keloid

Keloid is a tissue with an excessive accumulation of collagen. In this study, we have partially characterized post-translational modifications of type I collagen in human keloid in order to pursue their potential involvement in this pathology. The levels of lysyl hydroxylation of the helical portions of α1 and α2 chains of type I collagen in keloid were significantly higher than those of normal, while the levels of prolyl hydroxylation were identical between these two groups. The contents of the major reducible cross-links in dermal collagen, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymero-desmosine, were both significantly higher in keloids (up to sixfold) than those of normal. In addition, significant amounts of hydroxylysine-aldehyde derived cross-links that are characteristic of skeletal tissue collagens, dehydro-dihydroxylysinonorleucine (about 0.3 mole/mole of collagen) and pyridinoline (about 0.1 mole/mole of collagen), were found in keloids. These results indicate that keloid-forming cells are phenotypically different from those in normal dermis and that the collagen produced is highly cross-linked. The increased cross-linking provides the fibrils with more stability that may result in an accumulation of collagen.

Persistence of birefringence and specific collagen cross-links in dentine of teeth of the ‘Nuraghi’ population living in SardiniaBC 1500–1200

Well preserved bones of the Nuraghi population, discovered in 1980, are deposited in the Department of Biomedical Sciences, Division of Human Anatomy of the University of Sassari, in Sardinia, Italy. The Giant Tomb belongs to the megalithic monuments constructed in the period BC 1500–1200. The origin, history and the fate of Nuraghi population is not well known. Many cone-shaped fortresses are found in Sicily and megalithic monuments for burial purposes were built in their proximity. Due to the good macroscopic preservation of the mineralized parts of the Nuraghi skeletons we decided to compare the histological and biochemical structure of the recovered Nuraghi teeth with contemporary ones. Crown dentine of four Nuraghi molar and four premolar teeth were analysed and compared with contemporary molars and premolars removed for orthodontic reasons from 17–40-year-old patients in the Dental School, University of North Carolina at Chapel Hill USA. Evaluation of the collagen cross-links of dentine collagen was performed using a HPLC technique to analyze for hydrolysates. Nonreducible, stable cross-links pyridinoline and its lysyl analogue were quantified by fluorescence (excitation 330 nm, emission 390 nm). The concentration of reducible, labile cross-links referred to as DHLNL and HLNL (dehydro-dihydroxylisinorleucine) were identified and calculated by chromatography after the application of NaB3H4 with integration of respective peaks and converting the numbers into a residue per mole of collagen basis. The ultrastructure of collagen was estimated by its persistent birefringence in polarized light after staining with Picrosirus Red. It was found that dentine collagen of Nuraghi teeth resisted environmental factors sufficiently well to preserve the molecular ultrastructure of collagen fibres, proved by their birefringence and by the preservation of some of the stable and reducible collagen cross-links, characteristic for skeletal tissues. © 1998 John Wiley & Sons, Ltd.

Single-Column High-Performance Liquid Chromatographic–Fluorescence Detection of Immature, Mature, and Senescent Cross-Links of Collagen

A high-performance liquid chromatographic–fluorescence detection method of reducible (immature) and nonreducible (mature and senescent) cross-links of collagen was established without the use of a radioisotope and preliminary fractionation step. This method used a gradient elution procedure of sodium citrate buffer containing 7% ethanol. The reducible cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible cross-link (histidinohydroxylysinonorleucine) were detected byO-phthalaldehyde derivatization with the postcolumn method, whereas other nonreducible cross-links (pyridinoline, deoxypyridinoline, and pentosidine) were detected by natural fluorescence. The linear ranges of contents of theO-phthalaldehyde derivative cross-links and the natural fluorescent nonreducible cross-links were 20–600, 5–500 (pyridinoline, deoxypyridinoline), and 0.2–20 pmol (pentosidine), respectively. Tissue containing 1–2 mg dry wt of collagen was adequate for duplicate analyses of the reducible and nonreducible cross-links. An equivalent of 0.25 mg of hydrolyzed collagen could be analyzed by this HPLC system. Using this system, age-related changes in the cross-links of collagen from human connective tissues were also investigated.

The effects of hyaluronan on the meniscus and on the articular cartilage after partial meniscectomy.

The effect of hyaluronan (molecular weight = 8 x 10(5)) on the meniscus and on the articular cartilage was assessed after partial meniscectomy in a rabbit model. On gross examination, remodeled meniscus appeared as newly synthesized translucent tissue, and was seen in both vehicle- and hyaluronan-treated menisci. Histologically, safranin O staining revealed the strong presence of glycosaminoglycans in the newly remodeled tissue, and polarized light demonstrated the absence of mature collagen architecture. Hydration of the hyaluronan-treated menisci was significantly less than that of the vehicle-treated menisci, and the reducible collagen cross-link dihydroxylysinonorleucine was significantly increased in the hyaluronan-treated menisci compared with the vehicle-treated menisci, indicative of a greater degree of collagen remodeling. In situ hybridization of vehicle- and hyaluronan-treated menisci revealed a high level of type I procollagen mRNA expression and minor expressions of types II and III mRNA. Expression of the type I collagen gene appeared to be more pronounced in the hyaluronan-treated menisci than in the vehicle-treated menisci. The tibial plateaus revealed mild cartilage fibrillation after partial meniscectomy. A statistically significant difference between vehicle- and hyaluronan-treated cartilage was not demonstrated in the present study because of the slow development (i.e., 12 weeks) of osteoarthritis after partial meniscectomy in the rabbit model. These results suggest that in the rabbit model, hyaluronan enhances collagen remodeling and inhibits meniscal swelling after partial meniscectomy in the avascular region.

Architectural remodeling of fresh meniscal allograft transplantation in a rabbit joint: Its long-term protective effect on articular cartilage

This study assessed a fresh medial meniscal allograft transplantation model in the rabbit knee. Biological characterization included assessment of collagen remodeling of the allograft and the potential protection against cartilage degeneration. Allograft transplantation was performed on the left knee, and total meniscectomy on the right knee. Forty-seven rabbits were operated on and assessed at 9, 12, and 26 weeks. Fresh medial meniscal allografts showed collagen remodeling that paralleled the revascularization and cellular proliferation of the allografts. Revascularization was shown as early as 9 weeks from the periphery, extending to the inner one-third of the allograft by 26 weeks. Viability assessment of the meniscal allograft cells showed live cells at the periphery of the allograft at 9 and 12 weeks. At time 0, i.e., the time of the transplant, few viable cells were observed within the donor tissues. Biochemically, collagen remodeling, in terms of increased reducible collagen crosslinks, i.e., dihydroxylysino-norleucine, and the percentage of collagen present, was seen throughout the 26-week observation period. At 26 weeks, the meniscal allografts inhibited degenerative changes of the femoral and tibial cartilage compared to results with total meniscectomy.

Ibuprofen protects alpha-crystallin against posttranslational modification by preventing protein cross-linking.

Posttranslational modification of bovine alpha-crystallin by D-erythrose-4-phosphate, fructose-6-phosphate, D-ribose-5-phosphate and carbamylation using potassium cyanate induced the loss of chaperone-like activity, as assessed by gamma-crystallin aggregation. The presence of high-molecular-weight aggregates indicated that erythrosylated, fructosylated and carbamylated alpha-crystallins were modified by non-reducible cross-linking. In contrast, ribosylation of alpha-crystallin induced the formation of reducible cross-links. Analysis of ribosylated, erythrosylated and carbamylated alpha-crystallin using non-denaturing acrylamide gels showed that the cross-linking did not sterically inhibit the normal aggregate formation or alter the oligomerisation of the aggregate. Co-incubation of ibuprofen in the presence of alpha-crystallin and the modifying agents protected the chaperone-like activity of alpha-crystallin, enabling the inhibition of gamma-crystallin aggregation. In addition, ibuprofen inhibited the formation of both reducible and non-reducible cross-linked high-molecular-weight alpha-crystallin aggregates. We show in this paper that ibuprofen can inhibit in vitro cross-linking events responsible for the loss of chaperone-like activity of alpha-crystallin and suggest that the protective effect of ibuprofen may be exerted by the binding of ibuprofen breakdown products to alpha-crystallin lysine groups, preventing posttranslational modification responsible for the loss of chaperone-like activity.

Reduced concentration of collagen reducible cross links in human trabecular bone with respect to age and osteoporosis

The decrease of bone strength in relation to age and osteoporosis is more pronounced than would be expected from the relative deficit in the amount of bone. Besides bone mass, the mechanical properties of cancellous bone also depend on the microarchitecture and possibly on the molecular structure of inorganic and organic components. The present study examines the bone collagen, especially the collagen cross links, in relation to age and osteoporosis. Samples of vertebral trabecular bone were taken at autopsy from 43 normal individuals, aged 15–90 years. Eleven of these served as sex- and age-matched controls for similar samples from 11 osteoporotic individuals, 70–90 years. The volume of each trabecular bone sample was estimated. After removal of the marrow, the trabeculae were ground to powder and decalcified. The extractability of the bone collagen was studied by repeated extractions with acetic acid and pepsin. The divalent reducible collagen cross-links, dehydro-dihydroxylysinonorleucine (DHLNL) and dehydro-hydroxylysinonorleucine (HLNL), were determined by reducing the bone collagen with tritiated potassium borohydride followed by ion-exchange chromatography. The mature trivalent pyridinium cross links were determined by reverse-phase HPLC with fluorescence detection. The extractability of collagen prepared from the vertebral trabecular bone of control individuals was increased with age. Bone collagen of osteoporotic individuals showed increased extractability and a substantial decrease in the concentration of the divalent reducible collagen cross links (DHLNL reduced by 30% and HLNL by 24%) compared with the sex- and age-matched controls. No alterations were observed in the concentration of the pyridinolines. The divalent reducible cross-links are the most frequent known cross links in bone (2–4 times the concentration of the pyridinium cross links). These changes would therefore be expected to reduce the strength of the bone trabeculae and could explain why the osteoporotic individuals had bone fractures even though the collagen density (mg/cm 3) did not differ from that of the sex- and age-matched controls. The microarchitecture of the cancellous bone was not assessed. The osteoporotic and control individuals seemed to have the same amount of trabecular bone, but the quality of the osteoporotic bone collagen was reduced.

Identification of lysine-derived crosslinks in porcine collagen type X from growth plate and newly mineralized bone.

Intact collagen type X cannot readily be extracted from the growth plate. Both the use of pepsin to release this molecule from tissue and the relative solubility of collagen type X following treatment of chick embryos with beta-aminopropionitrile (Chen et al., 1992) suggest that the insolubility may by brought about by the formation of lysine-derived crosslinks. By immunocytochemical labelling using antibodies specific for collagen type X, we have shown that this collagen type persists in the cartilaginous spicules present in metaphyseal bone and appears to be colocalized with collagen type II. The combined concentration of the reducible bifunctional crosslinks, dihydroxylysinonorleucine and monohydroxylysinonorleucine, in collagen type X isolated from the premineralized and newly mineralized growth plate was about 0.6 residues/ molecule, a level which might explain the relative intractability of collagen type X. Pyridinoline and deoxypyridinoline were present in very small amounts in collagen type X; this suggests that, unlike the situation in other types of collagen, few of the bifunctional crosslinks undergo maturation to pyridinium compounds. Although it is clear that collagen type X contains lysinederived crosslinks, work is in progress to establish which molecule also participates in the formation of these crosslinks.

Alteration of extracellular matrix in dilated cardiomyopathic hamster heart.

The purpose of this study was to characterize the collagen in hereditary dilated cardiomyopathic hamster hearts, and to examine the participation of the collagen in the occurrence and progression of cardiomyopathy. BIO 53.58 hamsters (5, 10, 20 weeks old) were used as the model of dilated cardiomyopathy. Flb hamsters were used as controls. The collagen content was almost constant at any age in the Flb hamsters, but increased with age in BIO 53.58 hamsters. Type III collagen increased significantly in BIO 53.58 hamsters at 10 weeks. The acetic acid solubility of collagen decreased in BIO 53.58 hamsters as the fibrosis progressed, but was unchanged in controls. Reducible crosslinks showed a tendency to decrease progressively in BIO 53.58 hamsters. There were no differences between Flb and BIO 53.58 hamsters at 5 weeks, but its expression in BIO 53.58 hamsters at 10 and 20 weeks of age increased compared to Flb controls. These findings indicate that in the early phase of cardiomyopathy the extracellular matrix of the myocardium is rich in type III collagen. In the later phase, the matrix resembles that of hard tissues, whose collagen is mainly of type I collagen and is insoluble. These data suggest that the increased collagen synthesis may impair the cardiac function in the development of cardiomyopathy.

Cross-Linking and the Molecular Packing of Corneal Collagen

We have quantitatively characterized, for the first time, the cross-linking in bovine cornea collagen as a function of age. The major iminium, reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL) and dehydro-histidinohydroxymerodesmosine (deH-HHMD). The former rapidly diminished after birth; however, the latter persisted in mature animals at a level of 0.3–0.4 moles/mole of collagen. A nonreducible cross-link, histidinohydroxylysinonorleucine (HHL), previously found only in skin, was also found to be a major mature cross-link in cornea. The presence of HHL indicates that cornea fibrils have a molecular packing similar to skin collagen. However, like deH-HHMD, the HHL content in corneal fibrils only reaches a maximum value with time about half that of skin. These data suggest that the corneal fibrils are comprised of discrete filaments that are internally stabilized by HHL and deH-HHMD cross-links. This pattern of intermolecular cross-linking would facilitate the special collagen swelling property required for corneal transparency.

Protein translation elongation factor-1 alpha from Trypanosoma brucei binds calmodulin.

The following study examines the calmodulin (CaM) branch of the calcium signal pathway in the protozoan parasite Trypanosoma brucei. To accomplish this goal, a subset of cytosolic CaM-binding proteins (CaMBPs) was partially purified by a combination of DE52 and CaM-Sepharose affinity chromatography. Monoclonal antibodies (CBP-KK1) were used to clone the cDNA for a 53-kDa CaMBP from a lambda ZAP expression library of the metacyclic stage of T. brucei. The deduced amino acid sequence of clone CaMBP-12B had 81% overall amino acid identity to the translation elongation factor-1 alpha (EF-1 alpha) from Euglena gracilis and 76% identity to the rabbit EF-1 alpha. Rabbit EF-1 alpha was recognized by CBP-KK1 and was shown to bind to CaM-Sepharose in a calcium-dependent manner. By contrast, the complex of EF-1 alpha beta gamma did not bind CaM-Sepharose. A heterobifunctional sulfhydryl derivative of CaM (N-succinimidyl 3-(2-pyridyldithio)propionate-CaM) formed reducible cross-links with EF-1 alpha in solution but not with the complex of EF-1 alpha beta gamma. Biotinylated CaM bound weakly to trypanosome and rabbit EF-1 alpha in a gel overlay assay. This report demonstrates the direct interaction between CaM and the translation elongation factor EF-1 alpha.

P28. Phylloquinone (vitamin K 1) content of bone in young adults

Low-intensity pulsed ultrasound (LIPUS) has distinct effects on biologic mineralization at intensities of <100 mW/cm2. Intensity-dependent differences in the pattern of accelerated mineralization may be due to different alterations in regulation of collagenous matrix formation. However, little is known about the influence of LIPUS on collagen metabolism in the context of mineralization processes. Therefore, we attempted to evaluate differential effects of two intensities of pulsed ultrasound (30 vs. 120 mW/cm2) on collagen post-translational modification and mineralization in osteoblastic MC3T3-E1 cells. Murine osteoblastic MC3T3-E1 cells were exposed to pulsed ultrasound (1.5-MHz, 200-ms burst sine wave at 1.0-kHz frequency, either 30 or 120 mW/cm2 SATA, for 20 min/day from Day 14 to Day 35 postconfluence). Expression patterns of lysyl oxidase (LO), procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 1 (PLOD1, LH1), and 2 (PLOD2, LH2) was examined using quantitative PCR. Quantitative analysis of reducible immature cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature cross-links (pyridinoline and deoxypyridinoline) as well as analysis of the maturation of immature to mature cross-links were performed. Exposure to 30 mW/cm2 LIPUS upregulated LH2 mRNA expression and enzyme activity compared to controls. It was associated with increased relative amounts of telopeptidyl hydroxylysine (Hyl)-derived cross-links beginning on Day 14, upregulated LO mRNA expression, increased total reducible and nonreducible cross-links, and increased ratios of newly formed nonreducible to reducible cross-links. Similarities in the pattern of cross-link formation and calcium deposition in matrices between 30 mW/cm2 LIPUS-treated MC3T3-E1 cultures and bone suggest that 30 mW/cm2 LIPUS may promote the maturation of collagenous matrix as a scaffold for calcification. In contrast, exposure to 120 mW/cm2 ultrasound increased calcium accumulation compared to control at Day 35, but increases were delayed until Day 25. No differences in the extent and pattern of cross-links were observed compared to controls. These results suggest that the promotion of mineralization induced by 120 mW/cm2 may be attributed to other factors involved in mineralization process rather than cross-link pattern. Our results demonstrated the existence of differential effects of lower versus higher intensities of ultrasound on mineralization processes in vitro.

Tendon hypertrophy is associated with increased hydroxylation of nonhelical lysine residues at two specific cross-linking sites in type I collagen.

This study was designed to investigate whether the changes in lysine hydroxylation known to occur in hypertrophic tendon occur randomly or at specific lysine residues in the type I collagen molecule. Peptides corresponding to the two known major cross-linking sites of type I collagen (a lysine (or hydroxylysine) at position 9N cross-linked to a hydroxylysine at 930 and a lysine (or hydroxylysine) at position 16C cross-linked to a hydroxylysine at position 87) were prepared by collagenase digestion, size fractionation, and separation by high performance liquid chromatography from normal chicken tendon and from chicken tendon subjected to increased tensile load as a result of muscle hypertrophy. The ratio of the difunctional cross-links dihydroxylysinonorleucine to hydroxylysinonorleucine in normal tendon is 0.75:1; this ratio is increased to 6:1 in hypertrophic tendon. The dihydroxylysinonorleucine to hydroxylysinonorleucine ratio is increased to the same extent in samples of the purified cross-linked peptides derived from both the N-terminal and C-terminal lysine aldehyde residues. On the other hand, the relative hydroxylysine content of preparations of the pooled larger helical peptides obtained by cyanogen bromide digestion of normal and hypertrophic tendons was essentially identical. These results demonstrate that there is a specific increase in hydroxylation of only the N- and C-terminal non-helical lysine residues that participate in the formation of the reducible difunctional cross-links of type I collagen in hypertrophic tendon, while the extent of hydroxylation of lysine residues in the helical regions is not affected. The specific mechanism by which the enzyme lysyl hydroxylase acting on its substrate can distinguish between lysine residues destined to be in non-helical versus helical regions in a nascent collagenous peptide that has not yet attained its final secondary structure remains to be defined.

Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage. Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine. Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.

A Mouse 3T6 Fibroblast Cell Culture Model for the Study of Normal and Protein-Engineered Collagen Synthesis and Deposition into the Extracellular Matrix

Mouse 3T6 i ro asts deposited an organize co agenous extrace u ar matrix during longterm culture in the presence of ascorbic acid. The matrix produced by the cells had a similar comprising distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III aand V collagens. By day more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction. Incorporation of NaB3H4 into reducible crosslinks and aldehydes directly demonstrated the involvement of the α1(I)CB6 an α2(I)CB3.5 in crosslinks. The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization. Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation. The existence of a preformed matrix did however, increase the efficiency with which newly synthesized collagen was incorporated into the pericellular matrix. At day 0, when there was no measurable a matrix present, 29% of the collagen synthesised wad deposited, while by 15,88% of the collagen was laid down in the matrix. The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix.

Site comparisons of dentine collagen cross-links from extracted human teeth

Covalent intermolecular cross-links in collagen provide the dentine matrix with stability and tensile strength. The density of collagen cross-links varies depending on the site within the same tissue. This variation is probably due to factors such as the different amounts of stress and different turnover rates at the respective sites. The aim was to quantify the collagen cross-links in different tooth groups as an adaptation to functional requirements. For this purpose, the types and content of major cross-links in dentine collagen of human teeth from three different sites were measured and compared: incisors, premolar-canines and molars. After removal of cementum and pulp, 23 extracted teeth at different ages (27–69 yr) were individually pulverized and demineralized with EDTA. Collagen was reduced with standardized NaB 3H 4, hydrolysed, and subjected to amino acid and cross-link analyses. Each cross-link was quantified on the basis of mole per mole of collagen. The results indicated (1) all teeth contained labile, reducible (dehydro-dihydroxylysinonorleucine and dehydro-hydroxylysinonorleucine) and stable, non-reducible (pyridinoline and its lysyl analogue) cross-links, and (2) the content of both reducible and non-reducible cross-links was least in incisors and greatest in molars. This suggests that dentine collagen matrix maybe functionally adaptive.