Type X collagen synthesis by chick sternal cartilage and its relationship to endochondral development.

Our morphological studies have demonstrated that the appearance of localized, paired zones of primary calcification on either side of the midline of the 19-d embryonic chick sternum is heralded by the development of paired, translucent zones 2 d previously. Histological studies demonstrated that the majority of chondrocytes within these translucent zones are hypertrophic, and that the zones are surrounded by a margin of flattened nonhypertrophic cells. The discrete localization of these paired areas of hypertrophic chondrocytes and subsequent endochondral bone development allows for the direct correlation of the histological and biochemical characteristics of the zones sequentially during development and makes it possible to precisely match the synthetic activity to the cellular morphology, thereby eliminating possible minor but critical variations in developmental staging that could otherwise arise. Our studies have demonstrated that there is a direct spatial and temporal correlation between the degree of cellular maturation and the synthesis of type X collagen, and that the sudden and profound initiation of type X collagen synthesis on days 16-17 of development occurs concurrently with the attainment of hypertrophic characteristics by the majority of cells within the translucent zone. Before acquisition of these hypertrophic characteristics, the cells of this precalcification zone synthesize only type II and the minor cartilage collagens. Chondrocytes isolated from these regions in more immature sternae (i.e., 11+ d embryos) were found to synthesize high levels of type X collagen within 4 d of culture within collagen gels even though hypertrophic development and type X collagen synthesis by cells within this region would not normally have been apparent in ovo for several more days. These data indicate that there is a direct correlation between the development of hypertrophic characteristics and the synthesis of type X collagen, and that the maturation of chondrocytes in precalcification zones may be regulated by matrix components and/or stimulated by culture within collagen gels.

Synthesis of a low molecular weight collagen by chondrocytes from the presumptive calcification region of the embryonic chick sterna: the influence of culture with collagen gels.

The mature chick sternum is divisible almost equally into cephalic calcified and caudal cartilagenous regions. Isolation and culture of cells derived from embryonic precursors of these regions has revealed two discrete populations of cells with distinct morphological features and synthetic capabilities. Both cell populations grew well in culture within or upon collagen gels or upon plastic and maintained morphologies similar to those observed in the parent tissue. Polyacrylamide gel electrophoresis of radiolabeled proteins synthesized by the cells in culture demonstrated large differences in the types of collagens synthesized. Both chondrocyte populations synthesized type II and minor cartilage collagens but only chondrocytes isolated from the presumptive calcification region synthesized the previously identified, low molecular weight collagen, termed G collagen. Synthesis of G collagen was stimulated by culture within or upon collagen gels such that it represented an average of 65% of the total collagen synthesized by presumptive calcification region chondrocytes after 7 d of culture within collagen gels. Light and scanning electron microscopy demonstrated that the two chondrocyte types exhibited distinct morphological features and accumulated different extracellular matrices in culture.

The effect of charged synthetic polymers on proteoglycan synthesis and sequestration in chick embryo fibroblast cultures.

The polycation, poly(L-lysine), repressed the synthesis of glycosaminoglycans in secondary cultures of chick embryo skin fibroblasts and caused sequestration of glycosaminoglycans around the cells. The synthesis of chondroitin sulphate, dermatan sulphate, hyaluronic acid and a fourth component, thought to be heparan sulphate, were all inhibited to the same extent but the sequestration of the sulphated polymers were greater than that of the unsulphated. The sequestered material was retained around and not within the cells. Incubations with the polyanion, poly(L-glutamate), showed a slight stimulation of glycosaminoglycan synthesis and in these and control incubations (no additions to medium), most of the glycosaminoglycan synthesised appeared in the culture medium. The subsequent addition of poly(L-glutamate) to incubations containing poly(L-lysine) reversed the inhibitory and sequestering effect of the polycation. It was concluded that the inhibition of synthesis by poly(L-lysine) was either a direct effect of poly(L-lysine) on the cell membrane or a result of the high local pericellular concentration of sequestered proteoglycan.

A comparison of the glycosaminoglycans of weight-bearing and non-weight-bearing human dermis.

Twenty-six samples of human skin from three non-weight-bearing and 27 samples from three weight-bearing sites were obtained at autopsy from 11 subjects varying in age from 4 to 91 years. The dermis was idssected free of other tissues and after proteolytic digestion, the glycosaminoglycans were isolated quantitatively by ion-exchange chromatography. Analysis of the preparations showed that there was significantly more glycosaminoglycan in the dermis from weight-bearing sites (p less than 0.001). The major glycosaminoglycans were hyaluronic acid and dermatan sulfate with chondroitin sulfate as a minor component, but the relative proportions of these components were not significantly different in the two sets of samples (p greater than 0.1). It is suggested that the quantity of glycosaminoglycan in the dermis is influenced by the functional requirements of the dermis at that site.

A role for glycosaminoglycans in the development of collagen fibrils.

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.

Arterial connective tissue changes and distribution of 125I-labelled low density lipoprotein in hypertensive pigs.

Young pigs with hypertension of 10 weeks duration, resulting from cellophane perinephritis, were injected with 125I-labelled low-density lipoprotein ([125I]LDL) before being killed 24 h or 48 h later. Intimal thickening and increased acid mucopolysaccharide were demonstrated in the aortas and major arteries of the hypertensive animals. Increased accumulation of [125I]LDL was observed in the inner media beneath areas of intimal thickening. It is suggested that the primary effect of hypertension in atherosclerosis is to produce structural changes in arterial connective tissue which allow increased accumulation of LDL by altering the permeability and binding properties of the arterial wall.

The effect of centrifugal force on glycosaminoglycan production by aortic smooth muscle cells in culture.

Cultured smooth muscle cells from pig aorta subjected to centrifugation (48 h at 45 g over a 72-h period) increased their production of glycosaminoglycans by approximately 50%. The sulphated components, heparan sulphate, dermatan sulphate and chondroitin sulphate, showed a relatively greater increase than hyaluronic acid (66-34%). The results are consistent with the hypothesis that mechanical stress, such as hypertension, leads to increased accumulation of glycosaminoglycans in the aortic wall and that secondary trapping of lipoproteins by sulphated glycosaminoglycans produces atherosclerotic plaques.

Chondrons extracted from canine tibial cartilage: preliminary report on their isolation and structure.

We report on the morphology and structure of single and multiple chondrons isolated from homogenized samples of fresh and fixed canine tibial cartilage. Phase contrast, Nomarski, and scanning electron microscopy observations show each chondron to be composed of a chondrocyte and its pericellular matrix enclosed within a "felt-like" pericellular capsule. The extraction of intact chondrons from cartilage homogenates confirms the structural validity of the chondron concept and emphasizes the intrinsic mechanical strength of the capsule. Frayed collagen fibers radiate from multiple chondron columns suggesting a shear-resistant, structural interrelationship between capsular components and type II collagen fibers. Future development of chondron extraction procedures could provide a unique model with which to study the structure, biochemistry, and function of articular cartilage chondrocytes and their pericellular microenvironment.

Chondrons in cartilage: ultrastructural analysis of the pericellular microenvironment in adult human articular cartilages.

A combination of scanning and transmission electron microscopy was used to investigate the morphology and ultrastructure of normal human articular cartilage sampled from adult amputation specimens. This study confirms our previous observations on canine articular cartilage, which showed middle and deep layer chondrocytes surrounded by a pericellular matrix and enclosed within a pericellular capsule composed of filamentous and fine fibrillar materials. Pores in the "felt-like" organization of the capsular weave progressively decreased in size from the inner to the outer border of the capsule. Matrix vesicles were found embedded within the capsular weave and distributed throughout the territorial matrix. It is suggested that the chondrocyte, its pericellular matrix, and capsule together constitute the "chondron," a primary functional and metabolic unit of cartilage that acts hydrodynamically to protect the integrity of the chondrocyte and its pericellular microenvironment during compressive loading.

Diagnostic parameters in experimental renal infection.

Urinary tract infections are commonly encountered in medical practice but their laboratory diagnosis presents many difficulties. In this study we have used experimental models to assess the value of serum antibody, urinary antibody, rheumatoid factor, and the histochemical examination of renal tissue as diagnostic parameters. Under the conditions of the experiment the analyses did provide useful diagnostic information and may prove to be of value in the management of urinary tract infection in man.

The effect of dried mussel extract on an induced polyarthritis in rats.

A polyarthritis was induced in a number of rats by the intradermal injection of a modified Freund's adjuvant into the right foot pad. Powered extract of New Zealand mussel fed to the rats did not have any beneficial effect on either the development or the severity of the arthritis.

The development of the circle technique for determining the optimum line of tumour excision.

The healing of skin wounds is markedly influenced by their relationship to the tensional forces in the skin. Directional variations in skin extensibility which give rise to the cleavage line phenomenon and the skin tension lines are easily visualized by a recently developed skin marking technique. This technique makes it possible to plan accurately the optimum line of excision of skin tumours, including malignant melanomata, so as to allow primary linear wound closure without the necessity of undermining or grafting. The technique is particularly applicable to the excision of tumours of the back and limbs, and in addition, provides a warning if the local skin tensional state makes primary closure hazardous.

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.

Morphological and functional interrelationships of articular cartilage matrices.

The pericellular, territorial and interterritorial matrices of canine tibial cartilage have been identified ultrastructurally on the basis of their collagen fibre density and organisation, proteoglycan distribution and their structural response to experimentally applied compressive loads. In addition, a discrete pericellular capsule composed of fine, faintly banded fibrils is described which surrounds and encloses the pericellular matrix and chondrocytes of the middle and deep layers but not of the superficial layer. It is suggested that the fine fibrils which comprise this pericellular capsule represent some of the new minor collagen species recently localised in a similar position in hyaline cartilages. The densely compacted cupola which forms the articular pole of the capsule is frequently penetrated by a clearly defined pericellular channel, consistently orientated in the direction of the articular surface. Membrane-bound vesicles are observed in the pericellular matrix, within the lumen of the pericellular channel and accumulated in the territorial matrix immediately beyond the pericellular channel. The constancy of this distribution pattern strongly suggests a flow of material through the pericellular channel from the pericellular matrix to the territorial matrix and beyond, possibly in response to minute pressure gradients generated during compressive deformation of the non-distensible capsule. Furthermore, it is suggested that the random dispersal and subsequent rupture of matrix vesicles may represent a mechanism whereby chondrocytes, with limited mobility, could exercise homeostatic control over the cartilage matrix at some distance from the cell. Chondrocytes in the deeper layers of canine tibial cartilage are each surrounded by three distinct compartments, a pericellular matrix and capsule, a territorial matrix and an interterritorial matrix. The response of each of these concentric compartments to experimental load suggests that they function synergistically to produce an integrated, biological, hydro-elastic suspension system capable of resisting physiological compression.

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes.

Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 micron diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.

Analysis of the morphology and function of primary cilia in connective tissues: a cellular cybernetic probe?

More than 300 primary cilia have been identified electronmicroscopically in a variety of embryonic and mature connective tissue cells. To further define the enigmatic function of these cilia, we examined the interrelationships between the basal apparatus and cytoplasmic organelles and the ciliary shaft and the extracellular matrix. The basal diplosome was consistently associated with the secretory organelles including the maturing face of the Golgi complex, Golgi vacuoles and vesicles, the microtubular network, the plasma membrane, and coated pits and vesicles. Small vesicles and amorphous granules were also observed within the ciliary lumen and adjacent to the ciliary membrane. Microtubule-membrane bridges linked axonemal tubules to the ciliary membrane. The position, projection, and orientation of the axoneme were influenced by the structural organisation and mechanical properties of the matrix and frequently caused angulation of the ciliary shaft relative to the basal body. Located midway between the secretory apparatus and the extracellular matrix, primary cilia would appear ideally situated to mediate the necessary interaction between the cell and its surrounding environment prerequisite to the formation and maintenance of a functionally effective matrix. We propose that primary cilia in connective tissue cells could act as multifunctional, cellular cybernetic probes, receiving, transducing, and conducting a variety of extrinsic stimuli to the intracellular organelles responsible for effecting the appropriate homeostatic feedback response to changes in the extracellular micro-environment.

Morphology of the pericellular capsule in articular cartilage revealed by hyaluronidase digestion.

To allow a more valid comparison between our previous ultrastructural data and the immunolocalization of type IX and other minor collagen species in cryosectioned cartilage, we examined both normal and testicular hyaluronidase-digested canine tibial cartilage by electron microscopy. Removal of matrix proteoglycans caused the pericellular capsule to collapse against the cell surface, suggesting that its normal anatomical position is mediated by pericellular matrix hydration. Detailed examination of the pericellular capsule and pericellular channel revealed fine, faintly banded fibrils and an amorphous component somewhat similar in structure to basement membrane collagens. Matrix vesicles and the electron-dense material of the interterritorial matrix were only partially digested by hyaluronidase. We propose that the pericellular capsule is composed of a "felt-like" network of minor collagen species which act synergistically to maintain both the composition of the pericellular matrix and the integrity of the chondrocyte/pericellular matrix complex during compressive loading.

The immunoperoxidase localization of type X collagen in chick cartilage and lung.

We have localized type X collagen in chick cartilage and lung by use of affinity-purified antibodies raised against the purified protein. Examination of the centers of primary endochondral development in the embryonic sternum, developing cartilage in the embryonic tubular bones and growth plate cartilages demonstrated the specific association of type X collagen with regions of hypertrophic chondrocytes in these tissues. Furthermore these studies revealed a tendency for type X collagen to accumulate adjacent to regions of active vascular invasion and an apparent lag between the initial intracellular accumulation and the matrix deposition of type X collagen. A lag in the matrix accumulation of type X collagen was also shown biochemically and immunohistochemically in chondrocytes in culture. In chick lung type X collagen was localized to the smooth muscle of the blood vessels and to smooth muscle surrounding alveolar ducts.

Glycosaminoglycan synthesis by Dupuytren's cells in culture.

Glycosaminoglycan synthesis by cells cultured from nodule or band tissues of patients with Dupuytren's disease was compared with that of skin fibroblasts cultured from uninvolved areas of the palm. No differences were observed in culture which could account either for the abnormal glycosaminoglycan content or the increased cellularity of the diseased tissues. It is suggested that the behavior of Dupuytren's cells in vivo may result from a response to local conditions within the palm rather than from the expression of an irreversible change in their glycosaminoglycan metabolism.

The glycosaminoglycans of Dupuytren's disease.

The total and individual glycosaminoglycan (GAG) content at various stages of the Dupuytren disease process and in samples of normal palmar connective tissue (palmar dermis, palmar fascia and digital flexor tendon) from the hands of uninvolved age-matched controls have been assayed and compared. Morphological comparisons between the different tissues were made by histological examination of sections stained to demonstrate collagen fiber patterns and glycosaminoglycan distribution. Significant differences in the type and amount of GAG were found between the various manifestations of the disease process, i.e., nodules, cellular and fibrous bands, and between these and the normal palmar connective tissues. In the most actively proliferating cellular regions chondroitin sulfate levels were 11 times greater than those of the normal palmar connective tissues, whereas dermatan sulfate tissue levels showed a fourfold increase. On the other hand, tissue concentrations of hyaluronate were similar to those of normal palmar connective tissue. The relationship of these differences in GAG levels to the development and maturation of the normal palmar connective tissues and the Dupuytren's process is discussed.