C. Anthony Poole

Chondrons From Articular Cartilage (II): Analysis of the Glycosaminoglycans in the Cellular Microenvironment of Isolated Canine Chondrons

A chondron rich preparation was isolated from mature canine tibial cartilage using low-speed homogenization techniques. Proteoglycans were extracted from this preparation by exhaustive treatment with 4M guanidine-HCl. A significant proportion of the total proteoglycan, measured as uronic acid, was resistant to extraction and represented 27.9% in intact cartilage chips and 18.6% in the chondron fraction. Histochemical examination of chondrons confirmed that extraction resistant proteoglycans remained within the capsule of the chondron after 4M guanidine-HCl treatment. Electrophoretic analysis of the glycosaminoglycans extracted from intact cartilage chips and the chondron fraction showed approximately equivalent amounts of chondroitin sulphate (79.3%), keratan sulphate (16.3%) and hyaluronic acid (4.3%) present. In contrast, the extraction resistant residue in the chondron fraction was significantly enriched for hyaluronic acid (10.5%, p less than 0.05) but was depleted of chondroitin sulphate (70.9%, p less than 0.05). The major chondroitin sulphate isomer in the resistant fraction was chondroitin 6-sulphate while in the soluble fraction, the quantities of the two isomers were approximately equivalent. Comparison with previously published data suggests a role for minor collagens in the retention of proteoglycans in the cellular microenvironment.

Immunolocalization of type IX collagen in normal and spontaneously osteoarthritic canine tibial cartilage and isolated chondrons

OBJECTIVE The pericellular localization of type IX collagen in avian and mammalian hyaline cartilages remains controversial, while its distribution during osteoarthritic degeneration is poorly understood. This study aimed to compare and contrast the immunohistochemical distribution of type IX collagen in normal mature and spontaneously osteoarthritic canine tibial cartilage. DESIGN Thick vibratome sectioning techniques were evaluated and compared with isolated chondrons using a range of streptavidin-linked probes in combination with light, confocal and transmission electron microscopy. RESULTS In normal intact samples, type IX collagen was concentrated in the pericellular microenvironment, while a weaker extracellular reaction around each chondron separated the territorial matrix from the unstained interterritorial matrix. Further differentiation was evident in isolated chondrons where the fibrous pericellular capsule stained more intensely than the tail and interconnecting segments between columnated chondrons. Two regions of type IX reactivity were identified in osteoarthritic tissue: an intensely stained superficial reactive region below the eroding margins, and normal deep layer cartilage where pericellular staining persists. The superficial reactive region was characterized by chondron swelling and chondrocyte cluster formation, a loss of pericellular type IX staining, and a significant increase in matrix staining between clusters. Disintegration and loss of fibrillar collagens was evident in both the swollen microenvironment and adjacent territorial matrices. CONCLUSIONS The results suggest that changes in type IX distribution, expansion of the pericellular microenvironment and chondrocyte proliferation represent key elements in the chondron remodeling and chondrocyte cluster formation associated with osteoarthritic degeneration.

Structural colocalisation of type VI collagen and fibronectin in agarose cultured chondrocytes and isolated chondrons extracted from adult canine tibial cartilage

Cell–matrix and matrix–matrix interactions are of critical importance in regulating the development, maintenance and repair of articular cartilage. In this study, we examined the structural colocalisation of type VI collagen and fibronectin in isolated chondrons and long‐term agarose cultured chondrocytes extracted from normal adult canine articular cartilage. Using double labelling immunohistochemistry in conjunction with dual channel confocal microscopy and digital image processing we demonstrate that type VI collagen and fibronectin are distributed in a similar staining pattern and are colocalised at the surface of cultured chondrocytes and isolated chondrons. The results suggest that type VI collagen and fibronectin may play a role in both cell–matrix adhesion and matrix–matrix cohesion in the pericellular microenvironment surrounding articular cartilage chondrocytes.

Confocal imaging of the human keratocyte network using the vital dye 5-chloromethylfluorescein diacetate.

Background: The human corneal stroma consists of intercalated layers of collagen and keratocytes. These cells are known to maintain the stroma and aid in repair but it is likely they have other crucial roles throughout the cornea. The complexity of their anatomy is revealed in this study by ex vivo in situ images of the human keratocyte covering a range of ages.

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

SummaryChick 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 μm 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.

The Efficacy of Subcutaneous Goretex Implants in Monitoring Wound Healing Response in Experimental Protein Deficiency

This combined biochemical and histological study demonstrated that subcutaneously implanted Goretex tubing can be used to monitor and detect variations in wound healing potential in rats subjected to experimental hypoproteinaemic and normal refeeding conditions. Induced hypoproteinaemia was observed to be associated with a marked diminution in cellular infiltration, collagen synthesis and fibrous deposition within the implant. All these effects were completely reversed by subsequent refeeding of normal diet. Although regional variations in fibroblastic response attributable to biologic variability, were observed within individual control implants, or between paired controls, they were relatively minor as compared to the marked differences observed at the macroscopic, microscopic and biochemical level between implants removed from normally fed and protein deficient animals.

Distribution of newly synthesized aggrecan in explant cultures of bovine cartilage treated with retinoic acid

This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when explant cultures of articular cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In cartilage explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.