Morphological properties of articular chondrocytes in various experimental and clinical conditions

Abstract

Previous work has suggested that there exists a relationship between chondrocyte morphology and matrix metabolism. Changes to chondrocyte morphology have been reported in human cartilage however it is unclear if these are involved in the degenerative process associated with osteoarthritis (OA). In this work, the morphology of human and bovine chondrocytes has been characterised under a range of conditions. Bovine chondrocytes have been utilised in these experiments as bovine cartilage is non-degenerate and the chondrocytes have ‘normal’ morphology. However, if human cartilage have been used instead then there is possibility of having chondrocytes of mixed shapes i.e. both ‘normal’ and ‘abnormal’ cells. The thesis aimed at experimentally inducing morphological changes to chondrocytes to determine whether these changes resemble those observed in human cartilage. The ultimate aim is to model these changes to clarify the link between morphology and matrix metabolism by determining how morphological changes influence matrix metabolism. A classification system was developed for chondrocyte morphology allowing the quantification of chondrocyte shapes under different conditions permitting statistical comparisons. The different conditions utilised were (1) non-degenerate and mildly-degenerate human articular cartilage and (2) two in vitro models (a) weak 3D agarose gels to study the effect of gel strength and increasing concentrations of foetal calf serum (FCS) on morphology of bovine chondrocytes and (b) scalpel induced mechanically-injured bovine cartilage model to study in situ chondrocyte viability and morphology at the injured site in various culture conditions. Additionally, the effect of raised medium osmolarity on the response of chondrocytes to injury was studied to determine if the abnormal morphology could be reversed. Using fluorescence-mode confocal laser scanning microscopy (CLSM), chondrocyte viability, volume and morphology were determined and quantified by using VolocityTM 3D image analysis software. Histological evaluation of matrix by using Haematoxylin and eosin, Alcian blue and Masson’s trichrome staining of matrix produced by chondrocytes cultured in strong or weak agarose gels and in injured cartilage was determined. Additionally, immunohistochemical evaluation of matrix (collagen Types I & II) produced by chondrocytes was also performed. Results demonstrated that in non-degenerate human femoral head cartilage, ~83% chondrocytes were normal in morphology and 17±2% chondrocytes had cytoplasmic processes as compared to mildly-degenerate cartilage where 35±5% abnormal chondrocytes with cytoplasmic processes were present. In non-degenerate cartilage, 11±3% chondrocytes formed small sized clusters however clustering was quite evident in the superficial zone of mildly-degenerate human femoral head cartilage where 43±16% chondrocytes had formed large clusters. In mildly-degenerate cartilage the number of abnormal chondrocytes with processes, length of processes and number of processes per cell were greater in the superficial as compared to mid and deep zones. A model was developed to study the effect of external supporting agarose gel on chondrocyte morphology and also to determine the influence of FCS. Bovine chondrocytes cultured in weak gels after 7 days developed similar morphological changes as those observed in degenerate human cartilage. However, in the strong gels only few chondrocytes with morphological changes were present i.e. similar to non-degenerate cartilage. These morphological changes (development of clusters and processes) occurred more rapidly with increasing concentrations of FCS. Histology revealed less Alcian blue staining intensity around chondrocytes cultured in weak gels as compared to strong gels suggesting altered matrix produced by abnormal chondrocytes. FCS and gel strength were therefore proposed as related factors in regulating chondrocyte morphology. In the bovine injured cartilage explant model, after 14 days chondrocytes at the injury in the presence of FCS or synovial fluid (SF) produced morphological changes. These changes comprised cell enlargement, flattening, elongation and production of cytoplasmic processes. In the absence of FCS or SF, chondrocytes at the injury remained unaffected and were morphologically ‘normal’. Throughout the cartilage and even in the absence of subchondral bone, chondrocytes displayed morphological abnormalities in the presence of FCS or SF. These findings suggested that this is not the property of chondrocytes in the superficial layers alone rather it is due to the extent of penetration of the ‘factors’ into the matrix and there is no possibility of interference of injured site with osteocytes or bone factors. Histology revealed that these abnormal chondrocytes showed less staining with Alcian blue at the injury suggesting that these morphological changes might play a role in the changes to matrix metabolism. By raising the osmolarity of the culture medium these changes were inhibited and chondrocytes maintained their normal morphology. The results suggest that morphogenic/proliferative factors in FCS or SF and strength/damage to the matrix may be inter-related and act as potent controllers of chondrocyte morphology. Raised osmolarity was found to inhibit the morphological changes suggesting the possibly that hyperosmolarity can antagonise the effects of these factors. The key conclusions from the thesis were (a) in non-degenerate human femoral cartilage a large percentage of chondrocytes ~83% were normal in morphology and the rest were abnormal however in mildly-degenerate cartilage 35±5% abnormal chondrocytes with processes were present (b) the changes to chondrocyte morphology (development of clusters and processes) were exacerbated with cartilage degeneration (c) chondrocytes cultured in the weak gels produced morphological changes as compared to strong gels (d) chondrocytes at the injury displayed marked morphological changes in the presence of FCS or SF (e) by raising the medium osmolarity these morphological changes to chondrocytes at the injury were inhibited. These results show that chondrocyte morphology is complex and strongly dependent on the environmental settings. Experimental conditions were therefore identified which showed increased chondrocyte volume, abnormal morphology with cytoplasmic processes, enhanced proliferation/cluster formation and matrix changes. These changes to volume and morphology of chondrocytes in the models studied in this work had certain similarities to the changes observed in human cartilage suggesting that these shape changes may play a role in the changes to matrix metabolism occurring in OA. These findings may be of translational relevance in clinical and experimental research into cartilage injury and degeneration by providing new insights in understanding the role played by chondrocyte morphology in cartilage degeneration and injury.