The present study analyzed the spatial organization of human superficial chondrocytes in the remaining intact cartilage of articular surfaces with focal, early and OA. Comparing the organizational patterns of chondrocytes distant to focal lesions with those observed in normal non-degenerative cartilage, the present study examined whether superficial chondrocytes experience destruction versus specific remodeling of their horizontal organization in early OA.
One of the important observations of the present study was that superficial chondrocytes, when distant to focal lesions in early OA, maintained a distinct spatial organization comparable to non-degenerative cartilage. In the intact cartilages of grade 2 joints, we observed similar horizontal patterns including chondrocyte strings, cluster, pairs and singles as we previously reported for intact cartilages (1
). Most importantly, the spatial organization of all intact condylar and some tibia but not patellofemoral groove cartilages of grade 2 joints was greatly modified in two ways. First, in early OA, we identified a novel spatial pattern in the superficial chondrocyte organization. This novel pattern consisted of chondrocytes that were aligned in two parallel lines in close proximity building double string patterns. In addition, the percentages, in which some of the ‘classical’ chondrocyte patterns occurred, had drastically changed in favor of the novel pattern. In each condylar and some tibia grade 2 samples, the novel spatial pattern co-existed with the organizational patterns of normal, non-degenerative cartilage. To the authors’ knowledge, the present study is the first to identify a distinct spatial re-organization of human superficial chondrocytes in response to early OA that results in significantly different organizational patterns compared to normal non-degenerative cartilage.
The present data unquestionably raises the question whether cellular proliferation may be responsible for the observed increase in the superficial chondrocyte numbers and the spatial remodeling of the cellular organization in early OA. Although proliferation of chondrocytes is controversial, many studies have shown that chondrocytes are in fact mitotically active (2
). In OA, the fibrillation- or lesion-associated cellular aggregation is thought to be related to chondrocyte proliferation (2
). Proliferative factors such as basic fibroblast growth factor (bFGF) from the synovial fluid (35
), cell shape changes, matrix depletion and disruption may induce proliferative cellular aggregation (25
). A study on advanced OA specimens doubted the existence of chondrocyte cell division and, consequently, the existence of cell proliferation in OA (33
). Observing mitoses as direct evidence of proliferation, they reported the absence of mitotic figures in advanced OA cartilage. Nevertheless, they concluded that the induction of cell division in adult and OA cartilage remains an open subject due to the existence of factors essential for cell division (33
). Most importantly, they considered chondrocyte proliferation to be likely in early OA (33
). Another study on cryoinjured cartilage analyzed the number of centrosomes as evidence for cells undergoing a cell cycle. They reported that 0.7 % of cryo-injured chondrocytes had two centrosomes and concluded that adult chondrocytes retain the ability to repopulate the matrix, possibly as an initial stage of cartilage regeneration (32
). In the present study, we showed that both the numbers of chondrocytes per cell group in the grade 2 condyles and the corresponding superficial zone cell density of the grade 2 distal femur were increased. Thus, we were able to demonstrate using two different methods that early OA-cartilages show an increase in superficial chondrocytes suggesting that proliferation in the articular surface had occurred.
The presence of a novel organizational pattern in the superficial zone may also be related to chondrocyte death. In the present study, we focused on the spatial location of vital superficial chondrocytes or those that have recently died by cartilage dissection or donor death. We excluded structurally disintegrated chondrocytes that underwent apoptosis or necrosis previous to donor death since we stained the DNA of nuclei. We noted that almost all depicted nuclei were represented by a clear and spatially defined DNA signal. However, it is possible that chondrocytes that underwent apoptosis or necrosis previous to donor death contributed to the modified organization by causing acellular areas without any fluorescent staining or by interrupting the existing nuclei signals with focal absence of fluorescent stain. In that case, one would expect an overall decreased number of nuclei signals or, for example, shorter or interrupted condylar strings. However, the strings that we observed in non-degenerate condyles were not shorter or interrupted in the intact cartilage of grade 2 joints. In contrast, they were modified to double strings. Consequently, most cell groups in the intact cartilage of grade 2 joints contained more chondrocytes per group than in non-degenerate cartilage. We also showed an increase in the cell density of the superficial zone in the grade 2 distal femur compared to grade 0–1. Both observations suggest that an increased number of chondrocytes was present in the grade 2 distal femur. Thus, it is unlikely that the death of chondrocytes that died previously was responsible for the depicted organizational modification.
Another contributing factor relevant to the described organizational changes may be structural loss of the superficial zone in early OA. In that case, translucent nuclei fluorescent signals from the deeper zones may have mistakenly been analyzed as superficial zones. However, we verified the presence of intact superficial zones by histological analyses. Thus, structural loss of the superficial zone unlikely contributed to the grade 2-specific modification of the horizontal chondrocyte organization. Furthermore, we showed that OA-typical cellular aggregates were never observed distant to lesions where the quantified spatial and numerical organizational changes occurred. Because we were unable to establish organizational patterns within these OA-typical cellular aggregates, we termed them ‘unorganized’. The ‘unorganized’ cellular aggregation did not contribute to the modification of the organizational patterns of superficial chondrocytes in early OA.
Often, stereological methods are applied to assess cell organization and density of articular cartilage (21
). However, these studies did not identify the spatial and density-related cellular characteristics, which the present and our previous study unraveled (1
). Therefore, we believe that the presently applied non-stereological methods were justified. Because it has been suggested that the yield of released cells from human cartilage is relatively low and perhaps depends on the amount of damage from slicing cartilage, we calculated the ratio between the densities of the superficial zone and the deeper zones and found that it was comparable to those of other studies (21
Furthermore, comparisons of percentages of groups with different denominators must be interpreted cautiously because the percentage of cells in certain organizational patterns may be skewed by different total numbers of cells. Other metrics or a true 3-D study may be useful because a 3-D analysis showed recently that the nearest neighbor distance did not vary significantly with growth from the fetal to calf to adult stage of cartilage whereas the angle to the nearest neighboring cell was sensitive to changes in bovine chondrocyte organization (39
). In contrast, the present and our previous study (1
) showed that the metric “nearest neighbor distance” was sensitive enough to elucidate differences in the spatial organization of human superficial zone chondrocytes. Whereas the present study examined human cartilage, the previously mentioned study analyzed bovine tissue. The difference in the sensitivity of the metric “nearest neighbor distance” may partially be caused by species-derived differences. Nevertheless, it may have been advantageous for the present study to also assess the angle from a cell to its nearest neighbor.
In the current study, we demonstrated that human superficial chondrocytes underwent a remodeling of their organizational structure distant to local fibrillations or lesions. The remodeling process led to the formation of a novel organizational pattern across the intact articular surface suggesting a generalized and coordinated chondrocyte response to distant early OA. That chondrocytes are capable of remodeling their organizational structure was recently shown by a study, which assessed the organizational structure by the angle to the nearest neighboring cell. It was shown that the cell organization changed dramatically during growth, ultimately attaining the classical zonal organization of adulthood (39
). The capacity of a generalized response is, however, not limited to chondrocytes during growth. A comparable ability of OA-chondrocytes was recently revealed by our laboratory when we demonstrated that the up-regulation of matrix turnover in ankle cartilage remote from OA lesions was similar to that adjacent to OA lesions (14
). This up-regulation of matrix turnover was evident in ankle but not knee cartilage. However, the generalized upregulation of matrix-turnover suggested a coordinated response of the articular surface to repair the focally damaged matrix (14
). Thus, superficial chondrocytes seem capable of coordinated responses.
The question as to the physiological and clinical relevance arises from the reported remodeling of the cellular organization in early OA. Our laboratory showed strong correlations between the spatial organizational chondrocyte patterns and the anatomical joint type in which they occurred (1
). Those findings may suggest an association of the organizational surface structure with local biomechanical or metabolic environments. Thus, changed local conditions in early OA may require the chondrocytes to adapt their spatial organization. However, it is not known how chondrocytes could benefit from an organizational remodeling, or whether it is actively sought out or a passive consequence of proliferation or synthesis. We believe that the increase in chondrocyte numbers may serve as recruitment of metabolically active units as an ultimately failing attempt to repair focal damage. A number of studies has shown that enhanced synthesis of extracellular matrix components is present in OA (40
). The increase in cell numbers may suggest a not well understood regenerative potential of the remaining intact cartilage in early OA.
Studies on early OA are rare, partially because only a few laboratories have access to intact human donor tissues, whereas an abundance of studies on surgical end-stage OA specimens dominate the literature. However, in advanced OA, two studies analyzed the regenerative cellular potential; they showed that OA chondrocytes can generate type II collagen and proteoglycan-rich cartilage transplants (49
). Although their bioactivity seemed lower (49
), these advanced OA chondrocytes were considered suitable for autologous chondrocyte implantation (ACI) based on their mRNA expression patterns (49
). Taken together, the present study implies clinical relevance because a regenerative potential may undoubtedly be desirable in tissue engineering and justifies further investigations of the capabilities of early OA chondrocytes. Further animal and human studies are currently being undertaken in our laboratory to elucidate the functional significance of the elucidated remodeling of the spatial organization of human superficial chondrocytes with early OA.