Electron microscopy of human articular cartilage]

Scanning and transmission microscopy of the articular cartilage was performed in femoral condyles of persons at the age of 30-50 years. It was demonstrated that hyaline cartilage is covered with a protective fibrillar layer consisting of tightly pressed collagenous fibrillae with an underlying layer of fibroblastic cells. In the intracellular substance of the hyaline cartilage fibrillar structures form a complex reticular web with vertical arrangement of the main collagenous fasiculi. In the superficial layer of the hyaline cartilage the collagenous fibrillae and their fasciculi form arcade-like structures. Lacunar chondrocytes have a rough villose surface, cellular secrete is discharged as round granules through cytoplasmic membrane. Ultrastructural changes in chondrocytes are observed simultaneously with their degenerative-dystrophic changes.     

Matrix vesicles in aging cartilage.

The calcification process that occurs in aging has been studied with the electron microscope in costal and tracheal cartilage of rats and in human costal cartilage. In these tissues, the early stage of the calcification process is induced and regulated by matrix vesicles in the same way as it occurs in epiphyseal cartilage, bone, and dentine. However, the spreading of inorganic substance from vesicles into the surrounding matrix is frequently impaired in aged cartilage, either because of a too low concentration of calcium ions, or because the structure of the cartilage matrix is not suitable for inorganic substance deposition. This shows that matrix vesicles have a calcium affinity and calcium-binding potentiality greater than that of other components of the cartilage matrix. Most matrix vesicles are produced by "Verd?mmerung der Zellen." This degenerative process of the chondrocytes leads also to the formation of pericellular halos consisting of aggregates of amorphous substance and thin filaments. Part of the material that forms these aggregates seems to be produced by disruption of matrix vesicles. Within this disruptive material, thick collagen fibrils can be formed. Moreover, this material seems capable of inducing calcification. These findings suggest that matrix vesicles, by releasing their content into the matrix, can be involved in some way in collagen formation, and that the released material maintains the calcium affinity and calcium-binding property it has within the vesicles.     

Collagen-polysaccharide gel as a model of intercellular substance of the connective tissue]

Gel samples forming at 37 degrees C in the solutions containing tropocollagen and various polysaccharides were examined by electron microscopy. Contracting gel clots formed in the solutions containing chondroitin sulfate, proteoglycine from the tracheal cartilage, gum arabic. Electron microscopy showed such clots to be permeated with collagen fibrillae with transverse striations and a period of 640 A. An association between the density of the forming gel and the nature of the polysaccharide component is discussed. Gel forming in the solutions containing tropocollagen and various polysaccharides is regarded as a model of the connective tissue intercellular substance.     

Cortisone induced alterations of costal cartilage in single and in parabiosed rats

Summary Cortisone-treated Buffalo rats have been parabiosed with untreated controls of the same age. The optical and electron microscopy, including histochemistry, of costal cartilage of these rats has been compared with that in single cortisone treated rats, single controls, and control parabiosed with control rats, at 14 and 28 days after parabiosis.Single cortisone-treated rats, in comparison to controls, have shown the greatest alteration in cellular morphology and in the extracellular matrix both at 14 and at 28 days. Cortisone-treated parabiosed rats demonstrate a gradation of these alterations. Cellular alterations include enhancement of lipid and glycogen deposition concurrently with the presence of numerous large cytoplasmic vacuoles containing beaded irregularly-shaped filaments, banded or unbanded collagen-like fibrils, and/or electron dense lamellar bodies. In the extracellular matrix, matrix vesicles, amianthoid fibers, randomly oriented unbanded fibrillar materials, and filament-like materials are most prominent in the single cortisone-treated rats and they are progressively less prominent in the cortisone-treated parabiosed rats, and in the parabiosed and single controls. Calcification of the extracellular matrix follows a similar pattern and is observed initially in pericellular halos of the single cortisone and in cortisonetreated rats parabiosed with controls.Histochemical techniques have shown that chondroitin sulfate is less demonstrable in the single cortisone and in the cortisone-treated parabiosed rats than it is in the single or parabiosed controls at 14 days but, at 28 days, all untreated or treated rats, single or parabiosed are basically comparable. Glycoproteins are prominent in the single cortisone-treated rats both at 14 and at 28 days and, at these same times, they are progressively less prominent in the cortisone-treated parabiosed rats and in the single or parabiosed controls.Many of the cortisone induced alterations in costal cartilage are suggestive of enhancement of the aging process.Supported in part by NIH HD 07074     

Nasal reconstruction with autologous rib cartilage: a 43-year follow-up.

Autogenous costal cartilage has long been a popular material for nasal augmentation. The history of autogenous cartilage transplantation is reviewed. Two patients are presented who underwent nasal augmentation with autologous costal cartilage with a 43-year follow-up on each patient.     

Effect of neonatal head X-irradiation on growth of costal and tibial cartilage in rats: a histochemical and electron microscopic study

Long-Evans rats were exposed to a single dose of head X-irradiation (600 rads) at 2 days of age. Experimental and sham irradiated rats were sacrificed at 14, 20-21, 23, 41-45, and 70-71 days. Tibial epiphyseal width and the number of cells in the epiphyseal plate were determined. Histochemical and electron microscopic studies were carried out on both costal and epiphyseal cartilage. Histochemical techniques revealed a reduction in chondroitin sulfate at 14 days in both costal and epiphyseal cartilage of X-irradiated rats. Epiphyseal cartilage demonstrated recovery subsequently, and this was followed by a normal decrease of chondroitin sulfate with increasing age, but costal cartilage did not recover. Collagen synthesis was also reduced in both costal and epiphyseal cartilage, but not as dramatically as chondroitin sulfate. Except for some electron dense cells and reduced scalloping of the cell membrane, costal chondrocytes from irradiated rats did not show major ultrastructural alterations. In contrast, epiphyseal chondrocytes demonstrated radiation induced alterations in organelles, in enhanced glycogen deposition, and in retardation of chondrocyte maturation. Extracellularly in both costal and epiphyseal cartilage of irradiated rats, collagen density and matrix granules were reduced, while calcification of the matrix was enhanced. Beyond 45 days, the effects of irradiation were markedly reduced. Comparisons of the histochemical results with metabolic studies carried out previously in cartilage from the same animals indicated a more direct concordance of the histochemical results with the pattern of physical growth and supported the usefulness of morphologic and histochemical techniques in the analysis of the growth disorder in the head-irradiated rat.     

Elastostatic analysis of the human thoracic skeleton

An elastostatic, finite element model (designated THORAX I) of the human thoracic skeleton has been developed. The model includes the primary load-carrying members of the thorax; namely, the sternum, costal cartilage, ribs, and vertebral column. The soft tissue has been neglected.

Using gross geometric data measured from a skeleton with an apparent ‘small’ frame and approximate cross-sectional properties, the THORAX I model has been subjected to three loading distribution applied to the anterior chest wall in the anterior-posterior direction. Calculations were carried out on the IBM 7094 computer, and primary attention was focused upon the displacement fields of the sternum, costal cartilage and ribs and stresses in costal cartilage and ribs. The sternum and rib nodal point displacement fields are reported in detail, and a simple 2-degree-of-freedom model for the sternum, which correlates well with the analytic results, is also presented. Maximum normal stresses in the cartilage and bony regions of the individual ribs for one loading condition are also given.     

Establishment and characterization of chondrocyte cell lines from the costal cartilage of SV40 large T antigen transgenic mice

Complete understanding of the physiology and pathology of the cartilage is essential to establish treatments for a variety of cartilage disorders and defects such as rheumatoid arthritis, congenital malformations, and tumors of cartilage. Although synthetic materials have been used in many cases, they possess inherent problems including wear of the materials and low mechanical strength. Autograft has been considered very effective to overcome these problems. However, the limitation of the transplant volume is a major problem in autograft to be overcome. The costal cartilage is the most serious candidate for donor site transplantation, since it is the largest permanent hyaline cartilage in the body. To investigate the possibility using the costal cartilage as a transplant source, we have established and characterized three mouse chondrocyte cell lines (MCC-2, MCC-5, and MCC-35) derived from the costal cartilage of 8-week-old male SV40 large T-antigen transgenic mice. At confluence, all the cell lines formed nodules that could be positively stained with alcian blue (pH 2.5). The size of nodules gradually increased during culturing time. After 2 and 6 weeks of culture, RT-PCR analysis demonstrated that all three cell lines expressed mRNA from the cartilage-specific genes for type II collagen, type XI collagen, aggrecan, and link protein. Furthermore, type X collagen expression was detected in MCC-5 and MCC-35 but not in MCC-2. Any phenotypic changes were not observed over 31 cell divisions. Immunocytochemistry showed further that MCC-2, MCC-5, and MCC-35 produced cartilage-specific proteins type II collagen and type XI collagen, while in addition MCC-5 and MCC-35 produced type X collagen. Treatment with 1alpha, 25-dihydroxyvitamin D(3) inhibited cell proliferation and differentiation of the three cell lines in a dose-dependent manner. These phenotypic characteristics have been found consistent with chondrocyte cell lines established from cartilage tissues other than costal cartilage. In conclusion, costal cartilage shows phenotypic similarities to other cartilages, i.e., articular cartilage and embryonic limbs, suggesting that costal cartilage may be very useful as the donor transplantation site for the treatment of cartilage disorders. Furthermore, the cell lines established in this study are also beneficial in basic research of cartilage physiology and pathology.     

Helical structure of P pili from Escherichia coli. Evidence from X-ray fiber diffraction and scanning transmission electron microscopy.

The structure of the P pili from Escherichia coli has been studied using X-ray fiber diffraction and scanning transmission electron microscopy (STEM). Analysis of the fiber diffraction data indicates that the pili are constituted largely of structural subunits arranged helically with approximately 33 subunits in 10 turns in an axial repeat of 244.5 +/- 1.8 A. Radial electron density distributions calculated from equatorial diffraction data and STEM data indicate that the pili are about 65 A in diameter with a small central cavity roughly 15 A across. The principal protein component of the pili is PapA, which has a molecular weight of 16.5 kDa. Assuming that each subunit consists of a single PapA molecule, the mass-per-unit-length of the pili predicted from the X-ray data is 2.23 kDa/A. Measurements of mass-per-unit-length were also made through the analysis of STEM images. These measurements indicate a value of 2.13 +/- 0.14 kDa/A. STEM images demonstrated the presence of thin, thread-like structures emerging from the ends of pili and spanning breaks in the pili structure. These structures, which have been observed under other conditions, have been termed fibrillae. In the STEM images the fibrillae appear about 20 A in diameter. The mass-per-unit-length of the fibrillae was estimated using the STEM data to be 0.4 kDa/A. These data are consistent with the fibrillae representing an unwound or unraveled form of the pili proteins overstretched to about five times the length they would have in the intact pili.     

The effect of calcification on the structural mechanics of the costal cartilage

The costal cartilage often undergoes progressive calcification with age. This study sought to investigate the effects of calcification on the structural mechanics of whole costal cartilage segments. Models were developed for five costal cartilage specimens, including representations of the cartilage, the perichondrium, calcification, and segments of the rib and sternum. The material properties of the cartilage were determined through indentation testing; the properties of the perichondrium were determined through optimisation against structural experiments. The calcified regions were then expanded or shrunk to develop five different sensitivity analysis models for each. Increasing the relative volume of calcification from 0% to 24% of the cartilage volume increased the stiffness of the costal cartilage segments by a factor of 2.3–3.8. These results suggest that calcification may have a substantial effect on the stiffness of the costal cartilage which should be considered when modelling the chest, especially if age is a factor.     

Histochemical aspects of the differentiation of adventitious cartilage on the membrane bones of the embryo chick

Summary The histochemistry of the adventitious cartilage of the chick has been studied and compared with both primary cartilage and the bone on which the adventitious cartilage develops. The distribution of DNA, RNA, collagen, acid mucopolysaccharide, mucoprotein, glycogen, lipid, alkaline phosphatase and inorganic phosphate has been studied. Adventitious cartilage was found to have the histochemistry of primary hypertrophic cartilage and to calcify. The appearance of lipid and alkaline phosphatase activity coincided with the onset of calcification.The proliferating osteogenic and chondrogenic cells of the chick embryo have been classified and compared histochemically. Collagen synthesis was found to be high in the osteogenic cells and acid mucopolysaccharide and mucoprotein synthesis high in the chondrogenic cells.It has been postulated that the morphogenetic switch from osteogenesis to adventitious chondrogenesis most probably involves a change in the rate of collagen and acid mucopolysaccharide synthesis by the germinal cells of the membrane bones.     

In situ split costal cartilage graft harvesting through a small incision using a gouge

A costal cartilage graft is one of the most useful materials in reconstructive plastic surgery. In this article, a technique of in situ split costal cartilage graft harvesting through a small incision (2 to 3 cm) using a gouge is described. The technique used has many advantages: it is a simple technique, is easy to learn, and can be performed quickly through a small incision. By avoiding complete costal cartilage graft harvesting, the associated potential complications such as pleural perforation, chest wall deformities, long-lasting postoperative pain, and incisional scar length are reduced. This technique will be useful in selected cases for which a complete block of costal cartilage graft is not needed.     

Demonstration of neurosecretory substance in previously scanned specimens: adaptation of the Gomori aldehyde-fuchsin method for correlative SEM/TEM/LM histochemistry.

The Gomori aldehyde-fuchsin (AF) method for selective staining of neurosecretory substance (NSS) has been adapted to tissue previously prepared for both scanning and transmission electron microscopy (SEM/TEM). The procedure results in precise correlation of light microscopic (LM) histochemistry with SEM/TEM of the same tissue.     

Modeling costal cartilage using local material properties with consideration for gross heterogeneities

Contemporary computer models of the thorax designed to predict injury in automobile collisions model the costal cartilage as a homogeneous material using properties derived from local material characterization tests. No studies have validated the accuracy of these models in predicting the structural mechanics of costal cartilage. Two heterogeneities - the perichondrium and calcified regions - may affect the behavior of costal cartilage in a manner not accounted for by current models. This study sought to investigate the predictive ability of subject-specific models of whole costal cartilage segments, with the calcified regions modeled distinctly and with the perichondrium removed (from the physical specimens as well as from the simulations). Finite element models were developed in the case of five cadaveric costal cartilage segments. The properties of the cartilage were derived from indentation testing of each specimen, where the characteristic average instantaneous elastic moduli ranged from 8.7 to 12.6 MPa. Matched simulations and experiments were then performed, subjecting each specimen to cantilever-like loading with a dynamic posterior displacement of the sternal boundary (all other boundary degrees-of-freedom fixed). The models predicted the resulting peak anterior-posterior forces generated on the costal boundary with a minimum error of 1% and a maximum error of 36%. These results provide support to the previous implicit assumption that insight can be gained into the structural behavior of costal cartilage by observing the local material properties (when calcified regions are included and the perichondrium is removed). Future work includes the addition of the perichondrium, so as to model the whole costal cartilage composite structure.     

Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.     

Adherence to epithelial cells and ultrastructure of fosfomycin-resistant mutants of group A streptococci

Adherence of three strains of group A streptococci and their fosfomycin-resistant mutants to HEp-2 tissue culture cells was compared with some cell-surface characteristics, i.e. ultrastructure and hydrophobicity. Among Fosr mutants, both well-adhering and weakly adhering mutants were found. Clonal analysis of the mutants proved their greater stability in the adherence. Well-adhering parent strains of streptococci and Fosr mutants exhibited surface fibrillae in contrast to weakly adhering Fosr mutants which were devoid of fibrillae or contined fibrillae of lower electron density. Decrease of adherence of Fosr mutants of two strains was accompanied by a decrease of their hydrophobicity.     

The role of lysine-132 and arginine-136 in the receptor-binding domain of the K99 fibrillar subunit.

The gene encoding the K99 fibrillar adhesin of Escherichia coli has been modified by oligonucleotide-directed, site-specific, mutagenesis. The tryptophan-67, lysine-132, lysine-133 or arginine-136 were replaced by leucine, threonine, threonine and serine, respectively. The threonine-133 mutant fibrillae were indistinguishable from wild-type fibrillae. In contrast, replacement of lysine-132 or arginine-136 by threonine or serine, respectively, resulted in mutant fibrillae which had completely lost adhesive capacity, suggesting that the positive charges of these residues are essential for the interaction with the negatively charged sialic acid residue of the receptor molecules. After the replacement of tryptophan-67 with leucine neither fibrillae nor subunits were detectable, indicating that the mutant product is unstable and that tryptophan-67 has an essential structural role in the K99 subunit.     

Reconstruction of the nose damaged by cocaine

Nasal snorting of cocaine crystals causes destruction of the septal and nasal mucosa, which eventually provides exposure of the septal cartilage and nasal bones. This exposure eventually leads to septal chrondritis and nasal bone osteomyelitis. As this process continues, the severe loss of cartilage and bone allows gradual to total collapse of the nose. Correction of this deformity is best achieved by supplying new lining; this is possible by turning nasolabial flaps into the nasal vestibule to replace the lost and released lining. Once this has been accomplished, costal cartilage grafts can be inserted along the bridge and alae to maintain the structural integrity of the reconstruction.     

Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli

The role of specific amino acid residues of the K88ab and K99 fibrillar adhesins in the binding to erythrocytes and antibodies has been studied by chemical modification. It appeared that: (1) The integrity of the single disulfide bridge in the K99 subunits is essential for the binding of the fibrillae to the glycolipid receptors, but not for the recognition and binding of specific anti-K99 antibodies. (2) Modification of one lysine residue per subunit with 4-chloro-3,5-dinitrobenzoate results in the loss of the adhesive capacity of K99 fibrillae. Lysine residue are not important for the adhesive activity of K88ab fibrillae. Three or five lysine residues per subunit, respectively, can be modified without an effect on the immunological properties of the K99 and K88ab fibrillae. (3) Limited reaction of K99 and K88ab fibrillae with 2,3-butanedione destroys the adhesive activity of both fibrillae. This inactivation corresponds with the loss of one (K99) or two (K88ab) arginine residues per subunit. Ultimately, in K99 three, and in K88ab four, arginine residues per subunit can be modified without affecting the binding of specific antibodies. (4) Modification of five out of the nine carboxyl groups contained in the K99 subunit suppresses the recognition of specific anti-K99 antibodies, but carboxylates are not important for the adhesive activity of K99 fibrillae. Modification of two additional carboxylates in K99 results in an insoluble product. (5) Tyrosine residues are most probably not present in the adhesive or antigenic sites of K99 fibrillae. Modification of six out of the ten tyrosine residues in the K88ab subunit results in a decrease in adhesive activity but has no effect on the reaction with anti-K88ab antibodies.     

FORMATION,STRUCTURE AND FUNCTION OF CARTILAGE

Improved investigative techniques including electron microscopy, isotope tracings and improved histochemistry have greatly increased knowledge of the function of cartilage as a body tissue. Highly complex and delicate enzyme systems contained in the cartilage cell are involved in cartilage matrix formation and in the processes of calcification and cartilage repair. Heat, various drugs, freezing, and changes in the chemical environment damage or destroy these enzyme systems and interfere with the growth and function of cartilage. Hyaline cartilage to be transplanted must be handled with great care to preserve the cellular enzyme systems—otherwise the graft will be resorbed and clinical failure will result.