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Osteoarthritis is a highly prevalent disease, age being the main risk factor. The age-related accumulation of advanced-glycation-endproducts (AGEs) adversely affects the mechanical and biochemical properties of cartilage. The hypothesis that accumulation of cartilage AGEs in combination with surgically induced damage predisposes to the development of osteoarthritis was tested in vivo in a canine model. To artificially increase cartilage AGEs, right knee joints of eight dogs were repeatedly injected with ribose/threose (AGEd-joints). Left joints with vehicle alone served as control. Subsequently, minimal surgically applied cartilage damage was induced and loading restrained as much as possible. Thirty weeks after surgery, joint tissues of all dogs were analyzed for biochemical and histological features of OA. Cartilage pentosidine levels were ∼5-fold enhanced (p = 0.001 vs. control-joints). On average, no statistically significant differences in joint degeneration were found between AGEd and control-joints. Enhanced cartilage pentosidine levels did correlate with less cartilage proteoglycan release (R = −0.762 and R = −0.810 for total and newly-formed proteoglycans, respectively; p = 0.028 and 0.015 for both). The current data support the diminished cartilage turnover, but only a tendency towards enhanced cartilage damage in AGEd articular cartilage was observed. As such, elevated AGEs do not unambiguously accelerate the development of early canine OA upon minimal surgical damage. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1398–1404, 2012
Osteoarthritis (OA), with a high prevalence and increasing incidence due to the aging population, having a large impact on the patient's quality of life, is characterized by progressive cartilage damage, bone changes, and secondary synovial inflammation.1
As yet, the pathogenesis of OA is largely unknown. Several factors have been reported to predispose to the development of OA, such as genetic background, overweight, joint laxity, and muscle weakness.2 However, undisputedly, the most important risk factor for development of OA is age.3 The incidence of OA increases strongly with age: >50% of the population over 60 years of age is affected.4, 5 However, there are still many uncertainties how age contributes to the onset and progression of OA. Age-related changes in the articular cartilage are suggested to play an important role in the susceptibility of cartilage to OA.
One of the major age-related changes in articular cartilage is the spontaneous modification of proteins by non-enzymatic glycation resulting in the accumulation of advanced glycation endproducts (AGEs). Non-enzymatic glycation is a post-translational modification of proteins by reducing sugars. The spontaneous condensation of reducing sugars with free amino groups in lysine or arginine residues on proteins leads to the formation of AGEs.6 Pentosidine, a fluorescent AGE formed between lysine and arginine residues, is frequently used as marker for AGEs. AGEs are formed in all proteins, and can only be removed from the tissue when the protein is removed. The low turnover of proteins in articular cartilage results in an abundant accumulation of AGEs in this tissue with increasing age.7–9
AGEs are known to affect physical and chemical properties of proteins. Tissue strength is dependent upon the amount of crosslinks present10 and accumulation of AGEs is correlated with increased tissue stiffness of cartilage. Moreover, an increase in AGEs makes the cartilage more brittle,9, 11 making the tissue more prone to mechanical damage. In addition to mechanical changes AGEs also interfere with cellular processes. It has been demonstrated that increased AGE levels lead to decreased proteoglycan turnover (synthesis and release) of articular cartilage.12, 13 Altogether, these data suggest that increasing levels of AGEs in cartilage tissue, renders the tissue more prone to damage and limits repair activity, adding to development and progression of cartilage damage as seen in osteoarthritis.
Studying the effect of AGEs on cartilage damage in vivo, independent of age, necessitates animal models in which AGE levels of articular cartilage can artificially be enhanced by supplying high amounts of reducing sugars. This method was used in the canine anterior cruciate ligament transection (ACLT) model of OA.14 AGE levels in the knee cartilage of beagle dogs were artificially enhanced ∼5-fold by repeated intra-articular ribose injections. In this model, all joints developed OA due to joint instability but cartilage damage was more severe in the joints with artificially enhanced AGE levels than in the PBS-injected joints. As such this study supported the role of AGEs in progression of cartilage damage.
In the present study the role of AGEs in the development (initiation) of OA was studied. For this purpose minimal surgically applied chondral damage was applied. By use of minimal chondral damage and restraining joint loading it was anticipated that OA in normal joints would not develop spontaneously. Joints with artificially enhanced cartilage AGE levels were hypothesized to be more sensitive to the OA-inducing trigger and were expected to develop OA.
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The present study could not demonstrate a clear difference in cartilage damage in AGEd-cartilage compared to control cartilage, upon minimal surgically applied damage. Compared to the positive control (clear OA features of the original Groove model) and negative control (healthy cartilage), artificially aging of the cartilage appeared to accelerate development of cartilage damage (OA), not reaching statistical significance. Additionally, high cartilage AGE levels demonstrated a low cartilage proteoglycan release, corroborating a diminished turnover of proteoglycans due to the AGEing of the tissue.
In this study, the pentosidine levels in the AGEd-joints were approximately fivefold increased compared to the PBS-injected joints. PBS-injected joints had AGE levels expected for dogs of this age. Only once before, artificial enhancement of AGEs was used in vivo in a canine model; also demonstrating a fivefold increase in cartilage AGE levels.14 It has been demonstrated that cartilage pentosidine in humans from young (about 20 years) to old (about 80 years) also increased fivefold.12 This means that the outcome of these experiments can be translated into the human situation so leading to more information about the development of OA and with that potential therapeutic strategies. As such it is concluded that the artificial aging was successful and of relevance to human conditions.
In the present study minor changes, characteristic of joint degeneration, were found in the PBS-injected joints, as compared to the negative controls (no treatment). The triggers for induction of joint degeneration were purposely kept to a minimum. The surgically induced cartilage damage was restricted to a maximum of four grooves on femoral condyles only, whereas in the regularly performed Groove model OA at least 10 grooves are made.18 Additionally, loading of the joints was minimized, knowing that loading adds to development of OA.23 In the present study, the animals were permanently housed individually in indoor pens (3.5 m2), the minimal reasonably acceptable space, to keep movement and with that loading of the joints to a minimum. This in clear contrast to active exercise in large groups on a large patio in case of the classical Groove model. Apparently, the minimal surgically applied damage of the femoral condyles and the remaining minimal loading is sufficient for minimal development of joint damage in the PBS-injected tibial joints surfaces. It should be kept in mind that the time for development of joint degeneration in the classic Groove model is 20 weeks,15 whereas it was 30 weeks for the AGEd animals in the present study. This prolonged follow-up was specifically chosen to provide an increased time window to enhance the chance for differences in the development of joint damage between the PBS-injected and AGEd-joints.
Most interesting, the minimal condylar surgical-induced damage and minimal joint loading did not result in spontaneous healing but actually resulted in development of damage on the untouched tibial plateau. Studies on the spontaneous healing or progression of damage of articular cartilage defects have been performed in several animal models, including dogs.17, 24, 25 Clearly even the minimal joint damage of the femoral condyles in the present study, with minimal loading is not healed in a period of 30 weeks but even slightly progressed to the tibial plateau.
However, the artificial aging of the cartilage only marginally accelerates the cartilage degeneration. No significant differences were observed although compared to the control group a tendency towards more damage was seen at a biochemical level, although less than in the classic Groove model without enhanced cartilage AGEing. It might well be that this minor difference was not related to development (initiation) of joint degeneration due to the artificial aging, but that it was due to slight progression of the degeneration already started. This corroborates the previous canine study where artificial AGEing was able to accelerate progression of OA study upon ACLT.14 As such in the present study the original approach to study development of OA was hampered by the minor development of joint degeneration in the control PBS-injected joints.
Interestingly, it appeared that within the group of AGEd-joints, proteoglycan release (turnover) was the lowest in those joints with the highest cartilage AGE levels. This corroborates previous findings that increased AGE levels in cartilage lead to increased cross-linking of proteins and with that to diminished release of these macromolecules.10 Also degradation of AGE-modified collagen by matrix metalloproteinases is impaired compared to unmodified collagen.26 This will impair repair activity of the cartilage. Despite this phenomenon, also observed in the artificially AGEd-joints in this study, changes were insufficient to clearly drive the development of joint degeneration in the present study set-up.
In conclusion, despite the fact that enhanced cross-linking of macromolecules by the AGEs restrains loss of proteoglycans, corroborating the diminished turnover of old cartilage, and a tendency toward enhanced cartilage damage in the artificially AGEd-joints, the present data do not support a predominant role for enhanced cartilage AGE levels in development of joint degeneration in early OA. As such the clinical relevance of AGEing of cartilage in development of OA remains obscure.