Chick Tendon Research Articles

An experimental study on incompletely cut chicken tendons—A comparison of two methods of management

One hundred and twenty tendons in the chicken were partially cut to a different extent either transversely or obliquely. In half of the tendons, the cuts were sutured and then immobilised for three weeks, while in the other half, they were left unsutured and started on free movement on the third day. From the gross examination and histological studies, the results were significantly better in the sutured and immobilised group. The results became significantly worse when the cut was more than 50%, and there was no difference between an oblique or a transverse cut.

腱の骨内固定に関する実験的研究

Intra-osseous fixation of transplanted flexor tendons has been used widely in clinical practice, but the anchoring process has not been elucidated well. In the present study, tendon fixation was observed histologically in chickens whose flexors are similar anatomically to human tendon. Right deep flexors of the third digits were cut at the insertion, and then reanchored to the distal phalanx by a pull-out method. Sixteen young chickens (2-3 months after hatcing) were used for the study and sacrificed 4 days to 12 weeks after the procedure. Histological changes at the re-anchored area were examined by light and scanning electron microscopy. A control study confirmed that deep flexor tendons of young chickens directly anchor to the bone tissue, where fine fibrils are dispersed in the bone matrix. Deep flexors re-anchored to the distal phalanx showed a healing process between the tendon and bone. Granulation tissue surrounding the tendon pulled through the anchor hole matured as newly formed connective tissue which connected the tendon and bone, and simultaneously ossification appeared from the bone marrow side. The point of insertion of the re-anchored tendon was made of three layers, consisting of tendon, fibrocartilage and bone, 8 weeks after the operation, similar to the normal anchorage. Ossification seemed to proceed with small granules, about 0.2 μ in size, which may correspond to matrix vesicles. It was indicated that firm fixation between tendon and bone is achieved at about 4 weeks when Sharpey's fibers appear histologically.

The effect of tensional load on isolated embryonic chick tendons in organ culture.

Digital flexor tendons isolated from 17-18 day embryonic chickens were cultured intact, either on steel mesh grids, or in an apparatus designed to apply a mechanical load to the tissue. Tendons cultured without an applied load continued to synthesize protein and glycosaminoglycans throughout a 7-day period, but DNA synthesis decreased during this time. Increases in both protein and DNA synthesis were observed in tendons experimentally loaded for 48-72 h. Glycosaminoglycan production by tendons isolated from 17-day embryos was also increased in loaded tendons, sulfated GAG being increased more than hyaluronic acid. The same loading regime applied to tendons from 18-day embryos produced a smaller, yet significant increase in sulfated glycosaminoglycans but hyaluronate production was reduced. These investigations demonstrate that embryonic chicken tendons can be maintained in a viable state in organ culture and may provide a useful model for studies of the effects of mechanical forces on the synthetic capability and structure of connective tissue cells.

Proteoglycan-collagen arrangements in developing rat, calf and chick tendons

Proteoglycan-collagen arrangements in developing rat, calf and chick tendons

Effect of Elevated Temperature on the Intracellular Degradation of Different Collagen Types

The intracellular degradation of newly synthesized collagen of types I, II and IV was studied under cell culture conditions at normal and elevated body temperatures. The degradation of type I procollagen was studied in both human skin and chicken tendon fibroblasts, type II procollagen in chicken sternal chondrocytes and type IV procollagen in a human tumor cell line (HT-1080). To avoid the effect of the different temperatures on isotope penetration and protein synthesis, the cells were pulsed at 37 °C. The degradation of the newly synthesized procollagen was then followed during the chase period at 37 °C and at different temperatures slightly above the melting temperature of type I procollagen. Degradation was clearly increased in the human skin fibroblasts when the temperature was raised to 41 °C, whereas the result in the chicken tendon fibroblasts at 43 °C was not as dramatic. The extent of degradation in chicken chondrocytes was resistant to changes in temperature, whereas that in the HT-1080 cells was clearly increased when the incubation temperature was raised to 43 °C.

Development of crimp morphology and cellular changes in chick tendons

Tendons from the tarsus metatarsus regions of chick embryos, ages 10, 14, 15, and 16 days, were examined by optical polarization, optical transmission, and electron transmission microscopy to study the development of crimps which are commonly observed in adult tendons, ligaments, and other collagenous tissues which support tension. It is shown that the development of crimp structure is accompanied by morphological changes in fibroblasts.

Lysosomotropic agents ammonium chloride and chloroquine inhibit both the synthesis and secretion of procollagen by freshly isolated embryonic chick tendon cells

Freshly isolated embryonic chick tendon fibroblasts were incubated in suspension culture for six hrs in the presence or absence of ammonium chloride or chloroquine. Both agents caused a marked reduction in collagen production without inhibiting non-collagen protein synthesis. The small amounts of collagen synthesized in the presence of each agent were secreted into the culture medium at rates significantly lower than exhibited by control cells. Since ammonium chloride and chloroquine have been shown to inhibit lysosomal degradation of proteins, and interfere with the fusion of membranous vesicles and receptor recycling, these results suggest a requirement for at least one of these processes in the synthesis and secretion of procollagen.

The effect of cis-4-hydroxy-L-proline on intracellular degradation of newly synthesized collagen by freshly isolated chick tendon fibroblasts.

Freshly isolated embryonic chick tendon cells were incubated for 6 hrs in suspension culture in the presence of the proline analogue, cis-4-hydroxyproline to cause the cells to synthesize collagen which was incapable of becoming triple helical. The cells were evaluated for the percentage of total protein synthesis devoted to collagen and for the percentage of newly synthesized collagen which was rapidly degraded. Collagen production in the presence of cis-4-hydroxyproline was reduced from 25% to 7% of total protein synthesis. Under normal conditions the cells degraded 8% of their newly synthesized collagen but when the cells were incubated in the presence of cis-4-hydroxyproline, 25% of their total collagen synthesized was degraded to dialyzable peptides. The enhanced degradation of nonhelical, analogue containing collagen was inhibited by inhibitors of lysosome function. These observations provide support for the concept that fibroblasts are able to recognize and degrade a portion of their newly synthesized collagen, and that defective collagen may be selectively degraded by an intracellular lysosomal process. Enhanced intracellular degradation can in part explain the decrease in collagen production by freshly isolated tendon fibroblasts incubated with cis-4-hydroxyproline.

Biosynthesis of procollagen [(pro alpha 1 V)2 (pro alpha 2 V)] by chick tendon fibroblasts and procollagen (pro alpha 1 V)3 by hamster lung cell cultures.

Biosynthesis of procollagen [(pro alpha 1 V)2 (pro alpha 2 V)] by chick tendon fibroblasts and procollagen (pro alpha 1 V)3 by hamster lung cell cultures.

Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes.

Type II procollagen messenger ribonucleic acid (mRNA) was isolated from chick sternum and rat chondrosarcoma cells and translated in a reticulocyte lysate cell-free system. A high molecular weight band was identified as type II procollagen by gel electrophoresis, collagenase digestion, and specific immunoprecipitation. The translation of type II mRNA was specifically inhibited by addition of type I procollagen amino-terminal extension peptide. When this peptide was added to the media of cultured fetal calf chondrocytes, chick sternal chondrocytes, or chick tendon fibroblasts, no inhibition of collagen synthesis was evident. These data suggest a general regulation of collagen biosynthesis by these peptides in the cell-free translation system. However, as indicated by the cell culture experiments, cellular characteristics and evolutionary divergence of animal species seem to restrict the effect of the peptides.

Assembly and processing of procollagen type III in chick embryo blood vessels.

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.

Stress relaxation in tendons of chickens with scoliosis

The mechanical properties of the digital flexor and extensor tendons of chickens which develop idiopathic scoliosis were studied in stress relaxation experiments. The material from the chickens with scoliosis, when held at a fixed deformation, showed a more rapid and greater reduction in force required to maintain this deformation than did material from the control chickens. This mechanical abnormality could contribute to the spinal curvature.

Effect of the arginine analog, canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K m for arginine was 2.5 μ m and the K i for canavanine was 35 μ m. When fibroblasts from embryonic chick tendons were incubated with [ 3H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [ 3H]arginine so that at 3 m m the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [ 14C]proline or [ 3H]glycine and 3 m m canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.

The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized alpha-subunits and from preformed cross-reacting protein.

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.

Purification and characterization of the amino-terminal propeptide of Pro alpha 1(I) chains from embryonic chick tendon procollagen.

A peptide with an apparent molecular weight of 23,000 was isolated from the medium of cultured chick embryo tendons. Comparison of tryptic peptides derived from the medium peptide and from the amino-terminal, bacterial collagenase resistant portion of Type I procollagen indicated that the medium peptide represented the amino-terminal precursor-specific region of the pro alpha 1 (I) chain of procollagen. This conclusion was supported by the demonstration that antibodies against the medium peptide reacted with Type I procollagen in a radioimmune assay but did not react with a peptide derived from the carboxy-terminal propeptide of Type I procollagen. In addition, the reaction with Type I procollagen was inhibited with the purified amino-terminal, collagenase-resistant portion of pro alpha 1 (I) chains. Finally, amino acid sequencing demonstrated that the amino propeptide of dermatosparactic calf pN alpha 1 (I) chains and the medium peptide have similar amino-terminal sequences. Carbohydrate analysis established the presence of one residue of N-acetylglucosamine and a trace of mannose and galactose.

Peptide mapping of cornea collagens from chick embryos by dodecylsulfate/polyacrylamide gel electrophoresis.

Using differential salt fractionation, different collagen types were obtained from corneas of 17-day-old chick embryos. The collagen precipitated by 1.7 M and 2.5 M NaCl consisted of type I collagen, having an alpha 1: alpha 2 ratio of 2. The collagen precipitated by 5 M NaCl contained alpha A, alpha B, alpha 1 and alpha 2 chains having an alpha A: alpha B ratio of 1 and an alpha 1: alpha 2 ratio of 10. The alpha 1 and alpha 2 chains isolated from corneas were found to have higher mobility in polyacrylamide gel electrophoresis in sodium dodecylsulfate as compared with that of their counterparts isolated from embryonic chick tendons. When the alpha 1 and alpha 2 chains isolated by polyacrylamide gel electrophoresis in sodium dodecylsulfate were subjected to limited protease degradations, the peptide maps obtained from corneal alpha chains were not identical to those of tendon alpha chains. It is suggested that the type I collagen in cornea is different from type I collagen in tendon. Also the results indicated that the alpha 1 chain present in the 5 M NaCl precipitate is a type I alpha 1 chain.

Synthesis of types I, III and AB2 collagen by chick tendon fibroblasts in vitro.

Tendons from 14--17-day-old chick embryos contain predominantly type I collagen and about 5% AB2 collagen; type III collagen is not detectable by biochemical methods, such as sodium dodecyl sulfate/polyacrylamide gel electrophoresis or cyanogen bromide pattern, but can be visualized by immunofluorescence staining with collagen-type-specific antibodies. Similarly, freshly dissociated tendon cells secrete only type I collagen into the culture medium but no significant amounts of type III collagen [Uitto, J., Lichtenstein, J. R., and Bauer, E. A. (1976) Biochemistry, 15, 4935--4942]. Transfer of tendon cells from chick embryos to monolayer conditions, however, initiated synthesis of type III collagen in about 10% of the cells within three days, as visualized by immunofluorescence staining. Secretion of type III collagen into the culture medium can also be detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. With increasing number of passages the number of cells producing type III collagen reached levels of about 80% after the third passage, while 90% of all cells stained positively for type I collagen. This is reflected by an increase of production of type III collagen as determined by CM-cellulose chromatography. Using velocity sedimentation, the secretion of type III procollagen and of pN-collagen (carrying the amino-terminal extension only), into the culture medium of a second-passage tendon cell culture was detected. This study provides new evidence that the phenotype of cells may alter during transfer from the environment in vivo to conditions in vitro and that additional changes may occur with time in culture.

Effect of various aminoacid analogues on chick tendon procollagen synthesis and secretion: Selective inhibition by S-2-aminoethyl cysteine

Matrix-free chick embryo tendon cells were incubated with [ 14C]-proline in the presence of various aminoacid analogues and the effects of the analogues on [ 14C]-proline incorporation, [ 14C]-hydroxyproline synthesis and secretion of labeled molecules were examined. It was found that the structural lysine analogue S-2-aminoethylcysteine was a potent inhibitor of procollagen synthesis and secretion. At a concentration of 1 mM it produced a 75% decrease in [ 14C]-hydroxyproline synthesis and a 90% decrease in the secretion of [ 14C]-hydroxyproline-containing macromolecules.

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions. The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10 −6 M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.

Procollagen processing. Limited proteolysis of COOH-terminal extension peptides by a cathepsin-like protease secreted by tendon fibroblasts.

An enzymatic activity, capable of removing the COOH-terminal extensions of type I chick procollagen, has been demonstrated in embryonic chick tendons and in cultured tendon fibroblasts utilizing two new methods of analysis. The protease was purified by a combination of ultrafiltration concanavalin A affinity chromatography and gel filtration. The isolated protein has an apparent Mr of 43,000 by gel filtration and sodium dodecyl sulfate gel electrophoresis. The enzyme shows a major pH optimum at 4.2 and is susceptible to inhibitors such as pepstatin and leupeptin; it therefore seems related to the cathepsins. The possibility that this enzyme plays a role in the limited proteolytic processing of procollagen is discussed.