Articular cartilage is distinct from other tissues in that its cells, the articular chondrocytes, consume much less oxygen than do most other cell types (1). Chondrocytes derive energy mainly by the Embden-Meyerhof-Parnas pathway of glycolysis, which does not require oxygen (2, 3). How they use oxygen is still unclear, but the actual levels of oxygen that are present in cartilage affect cellular function. Although chondrocytes can survive for many days without oxygen (4), at low oxygen tensions (<5%), rates of matrix synthesis and energy production fall steeply in an oxygen tension–dependent manner (2, 4).
Little is known about oxygen tensions within articular cartilage. Articular cartilage is avascular, so all nutrients, including oxygen, are supplied to the chondrocytes by diffusion from surrounding sources, principally, from the synovial fluid. Oxygen tension falls with distance from the cartilage surface, and the gradient is dependent on the balance between the rate of oxygen transport through cartilage and the rate of consumption by the cells. The only reported measurements have been performed in vitro (5, 6), where it was found that oxygen tensions fell steeply with distance from the surface of cartilage plugs or constructs to levels that were 1–2.5% in the center. Based on these measurements, it is commonly asserted that cartilage is hypoxic and that oxygen tensions in the deep zone of articular cartilage are ∼1% (4, 7). However, in view of the inhibitory effect of low oxygen tensions on chondrocyte metabolism and matrix synthesis (2, 4), it would be surprising if such low oxygen tensions exist in vivo.
As a first step in determining oxygen levels in vivo, we calculated the oxygen levels through various cartilage samples. Since transport of small solutes such as oxygen through cartilage appears to occur mainly by diffusion (8, 9), we used Fick's law to evaluate oxygen tension gradients across cartilage; a similar approach has previously been used to estimate oxygen profiles across the growth plate (10, 11), the intervertebral disc (12, 13), and other avascular tissues, such as the cornea (14, 15). For this analysis, we first measured the effect of oxygen tension on the oxygen consumption rate, since such data are not available. We then examined how the synovial fluid concentration, cell density, cell density profile, cartilage thickness, and transport from the subchondral bone might affect oxygen levels in cartilage. The results of these calculations indicate that oxygen tensions in normal cartilage are unlikely to fall as low as 1%.
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- MATERIALS AND METHODS
The modeling analysis presented here shows that oxygen tension gradient between the synovial surface and the cartilage–bone interface is regulated by a number of factors that affect either the delivery of oxygen to cartilage or the rate of oxygen consumption. These factors include cell density and its distribution, cartilage thickness, oxygen tension in synovial fluid, oxygen supply from the subchondral surface, and oxygen consumption rate per cell. It is clear that oxygen tensions within cartilage will differ from joint to joint and from species to species. In all but the thinnest cartilage, oxygen tension falls steeply with distance from the surface (Figure 3A), but we estimate that even in the deepest zone, oxygen tension is unlikely to fall as low as 1% in normal joints.
Consumption of oxygen by the cartilage depends on the rate of oxygen consumption per cell. The available information for mammalian cartilage is summarized in Table 1. Published results for the same species vary considerably, possibly because of differences in sampling site, and hence, cell density, and possibly also because of age differences (21). Nevertheless, the average rate of all measurements available is about 11 nmoles/million cells/hour, which is close to the values we measured in bovine chondrocytes either in situ or isolated from the matrix. We therefore thought it reasonable to use our data on the oxygen consumption rate for most calculations, particularly since we also measured the oxygen tension–dependence of the oxygen consumption rate (Figure 1). Figure 3C shows that this latter factor must be considered; if oxygen consumption is taken as being constant and independent of oxygen tension, the depth of penetration of oxygen into cartilage is significantly lowered. It should be noted, however, that the oxygen consumption rate also varies with the concentration of other nutrients, such as glucose (3), although at present, there are insufficient data in this regard to use such features in a model.
Another factor that significantly affects the oxygen tension gradient is the cartilage thickness (Figure 3A), which determines the diffusion distance; solutions to the diffusion equation are very sensitive to the diffusional distance (22). The effective diffusivity (D) of oxygen in the matrix would also affect the oxygen transport. We used a value for D that is consistent with the finding that effective diffusivity of all small solutes in cartilage is ∼40–50% of their diffusivity in aqueous solution (16). We did not consider possible variations in diffusivity between cartilage types because of lack of data; D is likely to be higher in younger and more hydrated tissues (23) and, so, increase, rather than limit, the oxygen supply to these tissues.
The thickness of cartilage, apart from affecting delivery, also affects the total consumption, since together with cell density, it determines the total number of cells throughout the cartilage depth and, hence, the total amount of oxygen consumed. Stockwell (19) suggested that, indeed, the cell density was limited by nutritional constraints, since he found a relationship between cell density (ρ) and cartilage thickness (d; in mm), that is, ρ = 27.9 × d–0.88 million cells/ml (19), which predicts that the total cell number under a unit area of cartilage surface is relatively constant, independently of cartilage thickness. Our calculations (Figure 3A) indicated that oxygen profiles did not normalize according to the relationship described by Stockwell. Glucose, which unlike oxygen, is essential for the survival of the chondrocyte (4, 24), is also supplied to cartilage by diffusion (25); glucose gradients across cartilage are similar to those of oxygen, whereas the gradients of lactic acid, which is produced by the cells, are reversed, with concentrations lowest at the cartilage surface (12). Since chondrocytes consume both oxygen and glucose (and other metabolites, such as glutamine) and produce lactic acid (2, 3), gradients in these nutrients and metabolites cannot occur independently in vivo.
Nutrient regulation of cell density, as suggested by Stockwell (19), may depend on the total nutrient–metabolite environment rather than the fall in concentration of 1 essential nutrient, such as oxygen, alone. Indeed, recent data indicate that cell viability is governed by interactions between levels of nutrients and metabolites. Cartilaginous cells die more rapidly under glucose deprivation if lactic acid concentrations are high and pH is acidic, for instance (24).
As well as the total cell density, the cell density distribution also appears to be important. Cell density is highest in the superficial zone, decreases in the middle zone, and there is then a moderate increase in the deepest zone (20). When this distribution was incorporated into the model, the consequent oxygen tension was higher than that based on uniform cell density (Figure 3D). This result suggests that the cell distribution is optimized so that the limited nutritional supply can support a maximal number of chondrocytes.
In regard to delivery of oxygen and other nutrients to cartilage, there is no doubt that these diffuse into the cartilage from the synovial fluid, as has been shown both in vitro and in vivo (26–28). These nutrients are supplied to the synovial fluid by the synovial capillaries (25). Diffusion from the synovial capillaries through a thin film of synovial fluid to the center of the cartilage surface is, however, insufficient to adequately supply the cartilage, and without the “stirring” induced by joint movement, synovial fluid would be depleted of nutrients (25), which possibly explains in part why cartilage degrades in immobilized joints (29). Here, we only considered the situation of constant synovial fluid concentration. Our calculations find, as expected, that synovial fluid oxygen tension directly affects the oxygen tension across cartilage, with cartilage oxygen tension falling in proportion to the decrease in synovial fluid oxygen tension (Figure 2), if we assume that synovial fluid is the only source of supply.
But, a very important question is whether oxygen is also supplied from the subchondral bone. There is strong evidence that the subchondral bone is a source of nutrients to immature cartilage (30, 31), but for adult cartilage, the answer is somewhat controversial. Physically, the subchondral bone is not completely impermeable; it is highly vascularized, and some of its terminal vessels contact directly with the deepest hyaline cartilage layer (32). Studies of normal femoral heads from adult humans found vascular channels to be in contact with the undersurface of the calcified zone of cartilage (33), while some vessels from the subchondral bone pass through the subchondral plate and terminate in the basal cartilage layer (32, 34). Although some studies have suggested that in adult animals, nutrient supply to the cartilage from the bone is minimal (35), other experimental evidence indicates that nutrition from the subchondral bone is important for articular cartilage, even in humans and large animals. Autografts with subchondral supply implanted into baboons remained intact over 3 years, while interruption of contact between articular cartilage and the vascularized subchondral bone resulted in degeneration of the cartilage over the long term (36). Another convincing piece of evidence for a subchondral supply comes from an in vivo study which found that gadolinium(diethylenetriaminepentaacetic acid)2– penetrated adult knee cartilage from the articular surface after intraarticular injection, but from both the articular surface and the subchondral bone after intravenous injection (37).
It thus seems quite likely that the subchondral region is also a source of oxygen and other nutrients for the cartilage, but is it a significant source? Some have concluded that diffusion from the subchondral region is limited, despite the presence of penetrating vessels (38, 39). Others have suggested that up to 50% of nutrients are supplied via the subchondral bone (32). Our calculations show that both the value and position of the lowest oxygen tension are significantly modified by the subchondral supply (Figure 4). If indeed the cell density profile is optimized to the prevailing nutrient supply, the finding that the cell density is similar in the midzone and toward the subchondral plate (20) indicates that the subchondral supply should not be ignored when considering cartilage nutrition.
Since synovial fluid and subchondral bone are the only 2 sources of oxygen and other nutrients for cartilage, pathologic changes in these tissues may threaten normal cartilage maintenance. Synovial oxygen tensions may fall to very low levels in chronic inflammatory arthritis or other joint disorders (Table 2), leading to corresponding decreases in cartilage oxygen tension (Figure 2), possibly affecting matrix metabolism adversely (2, 4). In osteoarthritis, changes in the subchondral bone (40) may influence its possible role in nutrient transport. The first signs of osteoarthritic damage, in hip cartilage at least, have been reported to occur in the midzone (41), the region of lowest cell density and lowest nutrient concentration if subchondral transport is involved (Figure 4).
In conclusion, although the calculations shown here can only provide estimates of oxygen tension profiles across articular cartilage in vivo, our results indicate that only under the most unfavorable conditions—thick cartilage, low synovial fluid concentration, and no transport from the subchondral bone—does oxygen tension in cartilage fall to ∼1% in the deep zone (Figures 2 and 3A). Our calculations also suggest that a significant subchondral supply is required for the lowest oxygen tension in the normal knee joint to remain close to 5%, the tension found to be optimal for the maintenance of matrix metabolism (2, 42). Finally, it should be noted that although this report discusses only oxygen, the factors which regulate oxygen profiles also regulate gradients in other essential nutrients, such as glucose, and in metabolic products, such as lactic acid. Chondrocyte metabolism is likely to be regulated by the entire nutrient–metabolite milieu, rather than the concentration of oxygen alone.