Collagen crosslinks and their relationship to the thermal properties of calf tendons.

The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.

Age- and position-related heterogeneity of equine tendon extracellular matrix composition.

The digital flexor tendons of the neonate and adult horse have been compared with respect to variation in extracellular matrix composition along their length. Two pepsin-sensitive, acetic acid soluble proteins, molecular weight (Mr) 52 kD (np 52) and Mr 54 kD (np 54), were prominent throughout the length of neonatal tendons. In adult tendon, np 52 and np 54 were less abundant and restricted to the cannon (metacarpal) region. In contrast, a single pepsin- and collagenase-resistant protein of Mr 55 kD (fp 55) was exclusive to the fetlock (metacarpophalangeal joint) region regardless of age, although more distinct in the adult. Pepsin extracted fp 55 precipitated at 2.0 M sodium chloride: 0.5 M acetic acid and was further purified to homogeneity by bacterial collagenase digestion. Analysis of fp 55 amino acid composition revealed the presence of a large proportion of glycine residues (379 of 1001), suggesting a possible homology with the collagen family. These data demonstrate that the composition of equine digital flexor tendons varies with age, is heterogeneous along its length, and suggests that variation in tendon extracellular matrix composition is influenced by functional requirements.

Aggregational state and molecular order of tendons as a function of age.

Form or textural birefringence (FB) depends on the geometrical characteristics of rod-like molecules, like collagen. The degree of packing and ordered aggregation are factors that also play important roles in the FB. Since current information reports increases in the diameters of collagen fibrils according to age, a search to determine variations of intrinsic birefringence (IB) and FB of collagen in Achilles and tail tendons of rats as a function of age was carried out. The findings support the conclusion that there are increases on FB and IB as the animals age. These increases are a consequence of higher molecular order and aggregation of collagen in tendons of older animals. Furthermore, an accurate methodology was developed to detect and to control the effect of section thickness variation of the material used in this research.

Studies of compact hard tissues and collagen by means of Brillouin light scattering.

A measure of the elastic properties of tissue can be found from the propagation of sound in the tissue. Longitudinal sonic velocities were measured for mineralized turkey leg tendon (density 1.50 g/cc), deer antler (1.77 g/cc) and cow tibia (2.05 g/cc) in the 10 GHz frequency regime by means of Brillouin light scattering using a nine pass Fabry-Perot interferometer. Wet, air dried, mineralized and demineralized specimens were tested. Sonic velocity in each tissue increased with mineral content and decreased when the tissue was wet. All wet values are higher than for wet rat tail tendon collagen, axially and radially, but with considerably less anisotropy. The results are interpreted to indicate that bone matrix collagen is more highly crosslinked than tail tendon collagen. The loss of anisotropy is taken to correspond to a much higher crosslinking density between adjacent collagen molecules in mineralized tissue compared to rat tail tendon. The axial sonic velocity of dried rat tail tendon is almost that for low density dried mineralized tissue and greater than the radial sonic velocity of these tissues, but the radial sonic velocity for dried rat tail tendon is much lower, again corresponding to less crosslinking in this tissue. Longitudinal modulus, K, is defined as the tissue density times the square of the velocity. The compliance, 1/K, was found to be a linear function of density for each of the four conditions. It suggests that a Reuss formalism describes the elastic properties. Since the difference between the compliance for wet and dry tissue is also a linear function of density, the effect of water on the compliance is additive. The axial sonic velocity for cow bone is essentially constant over a frequency range spanning 10 orders. Presumably the axial sonic velocity is controlled by the continuity of the collagen fibers lying along the bone axis. The radial velocity decreases by 30% over this frequency range, probably due to the many levels of structure observed in long bone like osteons, Haversian canals and blood vessels, as well as internal surfaces like cement lines and between lamellae. The sonic anisotropy of hard tissues decreases considerably with increasing frequency. While rat tail tendon collagen is very anisotropic both sonically and optically, hard tissues whether wet, dry, mineralized or demineralized show much less anisotropy. The optical index of refraction, both axially and radially, was found by Brillouin scattering for the air dried demineralized tissues. A close match was found between optical and sonic anisotropy for all the demineralized tissues.

Diamines and aminoalcohols: neutral solvents for native collagen.

A substantial fraction of interstitial collagens (types I, III, V and VI) can be dissolved from finely divided, connective tissues by homogenization in 0.5-1 M ethylene diamine and certain other organic amines neutralized by hydrochloric or other acids to pH 7-8.5. The amount of collagen solubilized from the tendons and other tissues of young animals is similar to that extracted by dilute acetic acid. In ethylene diamine hydrochloride solutions, native type I collagen denatures at 38 degrees and it has a sedimentation coefficient similar to that measured in acidic citrate solutions. From these amine hydrochloride solutions native collagen fibrils can be regenerated simply by tenfold dilution with water or by dialysis. The form of these precipitates is determined by temperature, pH, enzyme pretreatment of the collagen, and the dialysate salt concentrations and composition. Weak gels can be formed with less than 0.1 mg collagen per ml. Regenerated matrices containing types V and VI collagens, and with different fibril sizes can be used to support cell growth or to form sheets or hydrated gels possibly suitable for prosthetic uses.

Tissue determinants of nuclear magnetic resonance relaxation times. Effect of water and collagen content in muscle and tendon.

Previous studies have suggested a possible relationship between tissue collagen content and nuclear magnetic resonance (NMR) relaxation times. To further investigate this relationship, we studied skeletal muscle, tendon, and the muscle/tendon transition area of normal gastrocnemius muscle from 10 dogs, and determined tissue water and collagen (hydroxyproline) content and NMR T1 and T2 relaxation times at 20 MHz. Water and hydroxyproline contents and T1 and T2 were significantly different among the three tissues. Both spin-lattice and spin-spin relaxation times were linearly related to tissue water content. A significant curvilinear inverse relationship between T1 and hydroxyproline (r2 = 0.93) and a significant inverse curvilinear relationship between T2 and hydroxyproline (r2 = 0.92) were found. Statistically controlling for hydroxy-proline concentration eliminated differences in T1 and T2 among the muscle, muscle/tendon transition, and tendon groups. Thus, NMR relaxation times of skeletal muscle and tendon appear to be influenced by both tissue water and collagen content.

The structure of avian type XII collagen. Alpha 1 (XII) chains contain 190-kDa non-triple helical amino-terminal domains and form homotrimeric molecules.

The monoclonal antibody 75d7, specific for type XII collagen (Sugrue, S.P., Gordon, M.K., Seyer, J., Dublet, B., van der Rest, M., and Olsen, B. R. (1989) J. Cell Biol., in press), was used to characterize the intact form of type XII collagen from chick embryo leg tendons. On an immunoblot of a 6% polyacrylamide gel of tendon extracts, one sharp band is recognized by the antibody at Mr = 220,000, while two fuzzy and poorly resolved bands are seen at Mr = 270,000 and Mr = 290,000. By immunoprecipitation of radiolabeled tendon culture media and electrophoresis of the precipitated material, bands with the same mobilities are observed, indicating that type XII collagen is not proteolytically processed in the extracellular space. Type XII collagen was extracted from tendons with 1 M NaCl in a Tris-HCl buffer and partially purified by concanavalin A-Sepharose and gel permeation chromatographies, using dot immunoblots to monitor the purification. Fractions highly enriched in bacterial collagenase-sensitive proteins with the same electrophoretic properties as type XII collagen were obtained. These fractions did not stain with Alcian blue and neither they nor the immunostained type XII collagen were affected by chondroitinase ABC digestion, indicating that type XII collagen is not a proteoglycan. A disulfide-bonded trimeric CNBr peptide was isolated by affinity chromatography on an antibody column and further purified by gel electrophoresis. Its NH2-terminal amino acid sequence was shown to be unique, demonstrating that type XII collagen is a homotrimer [alpha 1 (XII)]3. After bacterial collagenase digestion, both the immunopurified radiolabeled preparation and the purified tendon extract fraction showed by gel electrophoresis the presence of a large disulfide-bonded, 3 x 190-kDa, collagenase-resistant domain. Rotary shadowing and electron microscopy of the purified type XII fraction demonstrated that the molecule has the structure of a cross consisting of a 75 nm collagenase-sensitive tail, a central globule, and three 60 nm arms each ending in a small globule. After heat denaturation and renaturation, only a very large globule can be seen, attached to the triple helical tail. These results show that type XII collagen has a unique structure and is different from the other matrix constituents described so far.

Upper limb pyrophosphate tenosynovitis outside the carpal tunnel.

Three cases of calcium pyrophosphate dihydrate (CPPD) crystal deposits in tendon sheaths outside the carpal tunnel are reported. Crystals were shown by x ray diffraction analysis in one case and by compensated light microscopy in the other two. Surgical excision of the tendon synovial sheath had to be done in two cases (one case with CPPD crystal deposits).

Effects of short-term immobilization versus continuous passive motion on the biomechanical and biochemical properties of the rabbit tendon.

Little work has been done on the effect that continuous passive motion (CPM) may have on maintaining the mechanical and biochemical characteristics of tendons deprived of normal load-bearing stimuli. This study compared changes in mechanical properties and collagen concentrations of the tibialis anterior tendon between two groups of rabbits: control animals that received no treatment and experimental animals that received CPM to one ankle and immobilization to the other. Treatment duration was three weeks. Mechanical testing yielded values for elastic modulus, stiffness, stress, strain, and strain energy density at linear and maximum loads. Hydroxyproline concentrations were measured to determine tendon collagen content. Findings indicated that cyclic tensile loading of tendons by CPM lessened the harmful effects of immobilization. Linear load and stress for CPM and control tendons were similar, while the same measures for immobilized tendons were 16% and 25% less than control tendons. Ultimate strength of the control tendons, however, exceeded CPM tendons by 16% and immobilized tendons by 20%. Hydroxyproline levels were comparable for the CPM, control, and immobilized tissues. For a short duration (three weeks), CPM reduced the detrimental effects of immobilization in the tendon, primarily in the linear-load region.

A comparative electron microscopic study of apatite crystals in collagen fibrils of rat bone, dentin and calcified turkey leg tendons.

Crystal-collagen relationships in calcified turkey leg tendons and cortical bone and dentin of the rat were studied by bright field and selected-area dark field electron microscopy. The latter imaging technique enables the specific and direct visualization of apatite crystal sizes and their crystallographic orientations within collagen fibrils. Cortical bone possessed the longest mean c-axial length (170 +/- 50 A), then the tendon (142 +/- 43 A) and the smallest was dentin (110 +/- 30 A). Crystallographic orientations of apatite were found to alternate between a,b- and c-axial planes along the axial period of longitudinally sectioned collagen. This distribution of apatite may reflect a crystal alignment with collagen molecules as they spiral in superhelical fashion along the long axis of the collagen fibril. Apatite crystals were localized within both the gap and overlap zones of collagen fibrils even at very early stages of mineralization. The relative amounts of mineral within single collagen periods were determined as a function of electron absorbency. In the tendon at the onset of mineralization 80% of the mineral was located in the gap zone and 20% in the overlap zone; with further mineralization these relative amounts changed to 55% in the gap zone and 45% in the overlap zone. This 55/45% ratio observed in the heavily mineralized tendon was also observed in both cortical bone and dentin. The implications of these findings are discussed in view of collagen molecular ordering and the spread of apatite along collagen fibrils.

Ehlers-Danlos syndrome type IV (EDS IV) as model of a defective biopolymer composite material.

The connective tissue of a lethal EDS IV case was investigated for the reasons of the manifested disturbances of the arterial wall. This functional disorder was attributed to the mechanical decoupling of elastin and collagen, with the premise of a composite material consisting of cellular, fibrillar, lamellar and other matrix components. A conceivable relation between the manifested deficiency of type III collagen and a disturbed anchoring of elastin is shown. These findings are supported by biochemical, morphological, x-ray and mechanical data.

Effects of chondroitinase-ABC on proteoglycans and swelling properties of fibrocartilage in bovine flexor tendon.

Fibrocartilaginous regions of bovine deep flexor tendon were treated with chondroitinase-ABC and trypsin in order to extract proteoglycans from the extracellular matrix and thereby investigate the contribution of proteoglycan and collagen organization to tissue material properties. Chondroitinase-ABC digestion of tendon specimens for 24 h resulted in extraction of 60% of tissue glycosaminoglycan and leaching of the degraded large proteoglycan from the tissue residue. The totally degraded core protein of the small dermatan sulfate proteoglycan remained with the tissue residue, indicating that it is specifically associated with the tissue residue and that this association is not dependent on the glycosaminoglycan chains. Treatment of residues with trypsin after chondroitinase-ABC digestion depleted the specimens of proteoglycan. Bulk swelling tests on enzyme-extracted specimens showed that the distinct swelling properties of the fibrocartilaginous regions of the distal flexor tendon could be partially accounted for by elevated levels of proteoglycan. Swelling tests also showed that the distinct collagen organization of this region contributes significantly to the tissue's material properties. These results suggest that the fibrocartilaginous organization and composition of the articulating layer of distal tendon are adapted for mechanical requirements unique to this site, which receives compressive and frictional loads in addition to tensile loads.

Collagen crosslinking and cartilage glycosaminoglycan composition in normal and scoliotic chickens.

The amounts of lysine-derived crosslinks in collagens from tendon, cartilage, intervertebral disc, and bone and changes in the composition of sternal cartilage glycosaminoglycans were estimated in two lines of chickens, a control-isogenic line and a line that develops scoliosis. In the scoliotic line, scoliosis first appears at 3-4 weeks and progressively increases in severity and incidence so that 90% of the birds express the lesion by week 10. We have reported previously that cartilage, tendon, and bone collagens from scoliotic birds are more soluble than corresponding collagens from normal birds. Herein, collagen crosslinking and altered proteoglycan metabolism are examined as possible mechanisms for the differences in collagen solubility. At 1 week of age there were fewer reducible crosslinking amino acids (hydroxylysinonorleucine, dihydroxylysinonorleucine, and lysinonorleucine) in collagens from sternal cartilage and tendon in the scoliotic line than in the isogenic line. However, by week 3 and at weeks 5 or 7 values were similar in both groups. The amounts of hydroxypyridinium in vertebral bone and intervertebral disc collagen were also similar in both groups of birds. Consequently, differences in collagen crosslinking do not appear to be a persistent developmental defect underlying the expression of scoliosis in the model. However, differences were observed in cartilage proteoglycans and glycosaminoglycans from the scoliotic line that were not present in cartilage from the isogenic line. The average molecular weight of the uronide-containing glycosaminoglycans was 30% less in the scoliotic line than in the isogenic line, i.e., 12,000 compared to 18,000. The size distribution of cartilage proteoglycans from the scoliotic line also differed from that of proteoglycans from the isogenic line.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal-collagen relationships in calcified turkey leg tendons visualized by selected-area dark field electron microscopy.

The distribution and orientation of biological apatite crystals in calcified turkey leg tendons were studied by selected-area dark field electron microscopy. This imaging technique enables the direct visualization of apatite and the specific determination of the crystallographic axes (a, b-axes or c-axis) within calcified collagen fibrils. This study shows that at early stages of mineralization, rod-shaped apatite crystals (5-20 nm in length) were localized and dispersed within gap zones bordering both the collagen molecule C- and N-terminal regions. At later stages of mineral deposition the crystals were more extensive, occupying greater areas of the gap zone and, in addition, apatite crystals were found to occur in the overlap zones. The orientation of apatite crystals was observed to be an alternating and interlocking distribution of a, b-axes and c-axis along the axial period of collagen fibrils. This distribution is interpreted as representing a continuous rotation of apatite axial orientation along the collagen period.

Adaptation of bone and tendon to prolonged hindlimb suspension in rats.

The rat hindlimb suspension model was used to ascertain the importance of ground reaction forces in maintaining bone and tendon homeostasis. Young female Sprague-Dawley rats were randomly assigned to either a suspended or a nonsuspended group. After 28 days, femur bones and patellar tendons were obtained for morphological and biochemical analyses. Prolonged suspension induced a significant change in the geometric configuration of the femur middiaphysis by increasing the minimum diameter (12%) without any significant alterations in cortical area, density, mineral, and collagen concentrations. Femur wet weight, length, DNA, and uronic acid concentrations of suspended animals were not significantly different from bones of nonsuspended rats. However, the collagen and proteoglycan concentrations in patellar tendons of suspended rats were 28% lower than the concentrations of matrix proteins in tissues obtained from nonsuspended animals. These data suggest that elimination of ground reaction forces induces alterations in tendon composition and femur diaphyseal shape by changing regional rates in bone remodeling and localized tendon strain. Therefore it appears that ground reaction forces are an important factor in the maintenance of cortical bone and patellar tendon homeostasis during weight-bearing conditions.

Tendon echogenicity: ex vivo study.

Recent publications discussing the echogenicity of normal tendon have described it variously as hyperechoic or hypoechoic. Since the echogenicity of tendon has been used to define normality and abnormality, certain knowledge of the normal echogenicity of tendon is crucial. Fresh tendon and muscle from beef hock was scanned with sector- and linear-array-transducer imaging at multiple angles and distances. The echogenicity of tendon was found to be very angle-dependent, a characteristic known as anisotropy. Scanned perpendicular to its long axis with a linear-array transducer, tendon was significantly more echogenic than muscle. With a change in angle, echogenicity of tendon decreased relative to that of muscle (the echogenicity of muscle remained the same), becoming isoechoic at angles of 2 degrees -7 degrees and hypoechoic at greater angles. Tendon studied with a sector transducer exhibited varying echogenicity. If echogenicity is used as a diagnostic criterion, the angle of the interrogating ultrasound beam must be very specifically defined.

Fibril-forming collagens in lamprey.

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.

Mechanical properties of rat tail tendon in relation to proximal-distal sampling position and age.

The mechanical properties of 3, 15 and 25 month-old rat tail tendons were investigated in relation to proximal-distal sampling location along the fibre length. For the 15 and 25 month-old tendons maximum load as well as collagen content per mm fibre length (unit collagen) increased markedly from the proximal to the distal location. A linear regression analysis of the collagen content and mechanical parameters (maximum load, maximum slope of the load-strain curve and energy absorption) showed that these parameters were linearly correlated to the collagen content. However, normalization of the mechanical parameters with regard to the collagen content did not cancel the dependency of the parameters on proximal-distal sampling location. Normalized load and energy values for the 3 month-old tendons and normalized slope values for the 15 and 25 month-old tendons were found to decrease from proximal to distal location. These findings showed that tail tendons are heterogeneous along their length in respect to mechanical strength. The regression analysis also indicated the existence of an inverse relationship between unit collagen and mechanical quality of the collagen. Alternatively, the mechanical properties of tendon fibres might be influenced by other components than collagen.

Keratan sulfate is a component of proteoglycans in the compressed region of adult bovine flexor tendon.

A monoclonal antibody (ET-4-A-4) was used to identify keratan sulfate (KS) as a constituent of bovine flexor tendon. KS was present in at least 500-fold higher amounts in the fibrocartilaginous region of the tendon that is subjected to compressive forces in vivo (mean = 0.03% of tissue dry weight) than in the more proximal regions subjected only to tensional forces. The KS was associated with proteoglycans of three size categories that could be separated by Sepharose CL-4B chromatography. The largest proteoglycan (Vo) contained approximately 4% KS and was predominant in the surface region of the tendon subjected directly to compressive forces. A population of somewhat smaller molecules (Kav approximately equal to 0.3) contained less than or equal to 20% KS and proportionately less chondroitin sulfate (CS) or dermatan sulfate (DS). The KS chains of this population were not digested by keratanase. This population was dominant in tissue from the middle layer of the fibrocartilaginous region. A third population of small KS proteoglycans or fragments (Kav approximately equal to 0.6) comprised 40% of the KS found in the deepest layer of the compressed tendon region. This smaller component was independent of the small DS proteoglycans.

MZ15, a monoclonal antibody recognizing keratan sulphate, stains chick tendon.

Biochemical, morphometric and electron histochemical methods have failed to demonstrate the presence in tendon of keratan sulphate, a common component of connective tissue proteoglycans. Using a monoclonal antibody specific for keratan sulphate, a positive localization of this molecule was made in the gastrocnemius tendon of stage 44 chicken embryos both at the light and electron microscopical levels.