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Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.

Suramin is an anti-neoplastic drug. Its actions include the inhibition of binding of urokinase-type plasminogen activator (uPA) to its receptor, an event which may prevent cartilage breakdown. The aim of this work was to determine the effect of suramin on cartilage resorption. Cartilage expiants, stimulated with interleukin-1alpha, tumour necrosis factor-alpha or retinoic acid were incubated with suramin. Release of incorporated (35)S-sulphate from pre-labelled expiants was used as a measure of proteoglycan breakdown and toluidine blue staining was used to visualise proteoglycan loss.Suramin inhibited the resorption of cytokine and retinoic acid-stimulated bovine nasal cartilage at concentrations between 100-1000 microM. These findings were confirmed by histochemistry. Though reversibility studies indicated that suramin toxicity could not be excluded above 100 muM, retention of suramin in the expiants may have contributed to this. There was no significant effect on lactate production up to 500 muM. The observed inhibition of cartilage resorption may reflect actions of suramin on the PA/plasmin system or on cytokine action.

The aim of this study was to investigate the effects of insulin-like growth factor-I, transforming growth factor-beta (TGF-beta), and interluekin-1 alpha (IL-1 alpha) on the deposition and degradation of a cartilage-like matrix in high-density pellet cultures of adult bovine chondrocytes. Proteoglycan was determined by toluidine blue staining and colorimetric assay. Type II collagen was determined by immunohistochemical staining and its unwinding in situ by a recently developed immunoassay. Bovine nasal chondrocytes cultured as pellets deposited a well-organized extracellular matrix of proteoglycan and type II collagen. Insulin-like growth factor-I (2-10 ng/ml) increased the synthesis and incorporation into the matrix of both these proteins. TGF-beta (2-10 ng/ml) also increased proteoglycan synthesis. However it inhibited proteoglycan deposition, presumably through increased degradation of the molecule, as shown by increased release of aggrecan fragments into the tissue culture medium. TGF-beta had no effect on type II collagen deposition. In pellet cultures of bovine nasal or articular chondrocytes, 20 ng/ml IL-1 alpha induced a significant degradation of both proteoglycan and type II collagen. The effect on collagen clearly involved proteolytic cleavage of its triple helix because there was an increase in the proportion of unwound type II collagen in the matrix, as well as a loss of total type II collagen. In explant cultures of intact bovine articular cartilage, incubation with 50 ng/ml IL-1 alpha stimulated significant degradation of the proteoglycan but no degradation of the type II collagen. These results demonstrate that although the articular chondrocytes are capable of degrading type II collagen when isolated, they do not do so in situ, presumably because of some inherent property of the mature extracellular matrix. This study demonstrates the utility of pellet cultures when investigating chondrocyte-mediated turnover of cartilage matrix and its modulation by cytokines and growth factors.

The annual regrowth of antlers in male deer is a unique example of complete bone regeneration occurring in an adult animal. Growth is initiated at the distal antler tip, which is similar to the epiphyseal growth plate in some respects. However, there is some debate as to whether this process represents “true” endochondral ossification. As part of the characterization of the developmental process in pre-osseus antler tissue, we have studied, by in situ hybridization, the spatial expression of mRNAs for types I, II, and X collagen. Viewed in a coronal plane, type I procollagen mRNA was observed in skin, the fibrous perichondrium, and the densely cellular area immediately adjacent to the perichondrium. Below this area, as cells began to assume a columnar arrangement and coincident with the appearance of a vasculature and synthesis of a cartilaginous matrix, transcripts for types I, IIA, IIB procollagen and X collagen were detected. Further down in the cartilage zone, the pattern of type I procollagen mRNA expression was altered. Here, the signal was detected only in a morphologically distinct subpopulation of small, flattened cells within the intercellular matrix at the periphery of the columns of chondrocytes. The alternative splice form of type II procollagen mRNA (IIA), characteristic of chondroprogenitor cells (Sandell et al. [1991] J. Cell Biol. 114:1307-1319), was expressed by a subset of cells in the upper region of the columns, indicating that this zone contains a population of prechondrocytic cells. Positive hybridization to type IIA was most abundant in these cells. In contrast, transcripts for the other procollagen splice form (IIB) and type X collagen were expressed by chondrocytes throughout the whole of the cartilage region studied. The translation and export of type II collagen and type X collagen were confirmed by detecting specific immunoreactivity for each. The spatial distribution of immunoreactivity for collagen types II and X was consistent with that of corresponding mRNAs. These data demonstrate for the first time the distinct pattern of expression of genes for major cartilage matrix macromolecules, the expression of the differentially spliced form of type II procollagen mRNA (IIA), and specifically the colocalization of types II and X collagen in the developing antler tip. Taken together, they strongly indicate that antler growth involves an endochondral process. © 1996 Wiley-Liss, Inc.

Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.

This is a study of the regulation of human articular chondrocyte proliferation by transforming growth factor beta (TGF beta) and interleukin-1 beta (IL-1 beta) in vitro. Human articular chondrocytes were cultured at different cell densities on plastic and on a collagen substratum, in the presence and absence of serum. The effects of TGF beta and IL-1 beta on proliferation of chondrocytes, as determined by [3H]thymidine incorporation, under these conditions of culture were examined. TGF beta was found to have both stimulatory and inhibitory effects on chondrocytes in vitro. Interactions between TGF beta and growth factors present in serum influence the modulation of chondrocyte proliferation by TGF beta. IL-1 beta caused a significant reduction of the TGF beta-stimulated increase in chondrocyte proliferation. The complex inter-relationships between TGF beta and IL-1 beta on chondrocytes have implications for cartilage repair.

The Transforming Growth Factor-beta (TGF beta) family of polypeptides elicits diverse biological actions on a wide range of cell types. There are known to be several isoforms of TGF beta coded for by different genes, with possibly differential expression and potencies. We have demonstrated that there is constitutive expression of three forms of transforming growth factor beta in adult human articular chondrocytes. The presence of 10% fetal calf serum in the culture medium may influence expression. The addition of transforming growth factor beta or interleukin 1 beta to the culture medium does not appear to consistently influence the expression of TGF beta by the cells.

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.

Together, the chondrocyte and its pericellular matrix have been collectively termed the chondron. Current opinion is that the pericellular matrix has both protective and signalling functions between chondrocyte and extracellular matrix. Formation of a native chondrocyte pericellular matrix or chondron structure might therefore be advantageous when tissue engineering a functional hyaline cartilage construct. The presence of chondrons has not been previously described in cartilage engineered on a scaffold. In this paper, we describe a modified immunochemical method to detect collagen VI, a key molecular marker for the pericellular matrix, and an investigation of type VI collagen distribution in engineered hyaline cartilage constructs. Cartilage constructs were engineered from adult human or bovine hyaline chondrocytes cultured on sponge or nonwoven fiber based HYAFF 11 scaffolds. Type VI collagen was detected in all constructs, but a distinctive, high-density, chondron-like distribution of collagen VI was present only in constructs exhibiting additional features of hyaline cartilage engineered using nonwoven HYAFF 11. Chondron structures were localized in areas of the extracellular matrix displaying strong collagen II and GAG staining of constructs where type II collagen composed a high percentage (over 65%) of the total collagen.

Serum alpha1 antitrypsin levels and phenotypes were examined in 1000 healthy adults in Northern Ireland. The Pi phenotype M accounted for 87-8% Pi MS for 7-3% and Pi MZ for 3-5%. The percentages of the rarer phenotypes were Pi FM 0-3, Pi IM 0-6, Pi SZ 0-4 and Pi Z--0-1. The range of alpha1 antitrypsin levels in different phenotypes showed considerable overlap. Pi MS, Pi MZ and Pi SZ sera gave mean levels of 81%, 61% and 40% respectively of the mean level obtained in Pi M sera and that of the Pi Z--minus serum, 5%. Two distinct types of anomalous pattern were encountered in sera from ten subjects.