a Departamento de Ortopedia, Santa Casa de São Paulo, São Paulo, SP, Brasil
b Serviço de Ortopedia e Traumatologia, Hospital Israelita Albert Einstein, Universidade Federal de São Paulo (Unifesp), São Paulo, SP,Brasil
c Instituto de Ortopedia, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, SP, Brasil
d Escola Paulista de Medicina, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brasil
Introduction
Primary osteoarthritis (OA) is a multifactorial disease charac-terized by irreversible joint degeneration, with formation ofosteophytes and reduction of the joint space. It presents thefollowing main symptoms: progressive increase in pain, loss offunction, limitations on day-to-day activities and restrictionson sports practice.
Some theories have correlated continual intense practiceof physical activity with development of primary OA in eliteathletes, caused by joint overloading. Moreover, there has beenspeculation regarding how the joint cartilage responds to thisoverloading. However, the relationship between joint cartilagedamage and intense physical activity still seems to be a matterof controversy in the literature.
Some studies have demonstrated that athletes whopractice sports that include rapid acceleration with instantdeceleration or continuous training with a high impact onjoints, or who compete at elite level for prolonged periods oftime, present greater likelihood of developing OA. However,the types and intensities of exercises that are harmful to jointcartilage remain unknown.
A presents an initial asymptomatic phase, which maybe influenced by the level of overloading to which the jointis exposed in a physical activity. However, identificationof this joint damage is difficult, given the limitations ofthe assessment instruments available. In this light, recentstudies have demonstrated that biochemical biomarkersare a potential option for early detection of asymptomaticOA.
Under physiological conditions, the metabolism of type IIcollagen is slow. Its fibrils have a half-life measured in years.In the initial stages of cartilage degeneration, degradation ofthese collagen fibrils is observed. Enzymes named metallo-proteinases are released, and these contribute toward thisdegradation, especially collagenases and aggrecanases. Col-lagenases are responsible for cleaving type II collagen andproduce fragments of this collagen. Specific antibodies forthese fragments can be detected in synovial fluid, blood orurine and have been studied as potential biomarkers for theonset of joint degradation.
When a joint component is degraded and ejected out of itsoriginal tissue, measuring this in the joint fluid would be themost precise method. One biomarker of joint degradation isthe C-telopeptide of type II collagen (CTX-II). Use of CTX-II asa marker for progression of cartilaginous lesions and its directrelationship with radiological grades and clinical scores forOA have been proven in the literature. Therefore, assaying of CTX-II levels seems to be an effective manner for ascertainingtype II collagen turnover.
Biomarkers are instruments for measuring the progressionof diseases or the effects of treatment on disease progres-sion. Thus, they can serve as tools for elucidating the effectsof exercise on joint cartilage and the possible developmentof primary OA.12,13In this light, the objective of the presentstudy was to analyze and compare blood serum assay levelsof the biomarker CTX-II in professional indoor soccer players,at three different times during one season.
Methods
This was a prospective longitudinal study that was approvedby the Research Ethics Committee of Santa Casa de Misericór-dia de São Paulo.
The study included 14 players in a professional indoor soc-cer team (under-21 category). The players were male, of meanage 19 years, all in the same team, and were subjected to thesame training and match load.
Players with previous knee surgery (two players) or whowere using chondroprotectant medications (three players)were excluded.
In addition, four players who were undergoing physio-therapy treatment for femoropatellar overload due to muscleimbalance in the pelvic belt were excluded because they pre-sented pain of patellar origin that limited their sports practiceat the beginning of the season.
CTX-II levels were assayed at three times: A - at the begin-ning of the pre-season; B - four months later (a time thatmarked the middle of the season); and C - at the end of theseason.
Blood samples (3 mL) were collected from each individualby means of simple puncture in the non-dominant arm, usingvacuum collection kits. The blood samples were centrifugedand stored at a temperature of -80 degrees, until all the sam-ples were ready to be tested.
Each sample was analyzed by means of an ELISA test fordetecting human CTX-II (Hu CTX-II kit, Cusabio Biotech, cata-log number CSB - E09323h, batch S20045731, produced in theUnited States). This kit presents 100% specificity for humanCTX-II alone, without cross-reactions, and with a minimumdetectable level of lower than 0.3 ng/mL. This test was per-formed in a private laboratory in the city of São Paulo, withcosts entirely borne by the researchers.
To compare the assayed levels between the three evalu-ation times, the nonparametric paired Wilcoxon statisticaltest and Bonferroni correction for multiple comparisons were
applied (95% confidence interval and p value < 0.05). This non-parametric test was the best option because it was impossibleto assume that the sample had normal distribution.
Results
Table 1 presents the data on each individual and the markerlevels observed for each of them at each time during the sea-son. It could be seen that two players (4 and 10) presenteddiscrepant data, with much higher assayed levels than those ofthe other players evaluated in this study, in all the evaluations.Nevertheless, these participants did not report any decline inphysical performance, or any presence of symptoms such aspain, edema or instability.
Individuals 3, 5 and 10 presented pain at the origin ofthe patellar tendon in one knee, which became worse atthe end of the sports practice, improved with physiother-apy and did not limit their participation in training andmatches. Individual 13 presented a condition of non-limitingpain above the insertion of the pes anserinus. Also, individ-uals 6 and 8 complained of lateral pain in their knees, abovethe lateral epicondyle, during sports practice, which was alsonon-limiting.
Because of the tendinous origin of the pain in the individ-uals included in this study (patellar tendinopathy, anserinetendinopathy and friction of the iliotibial tract), it was decidednot to include them separately in statistical comparisonsregarding degradation of type II collagen exclusively of thecartilaginous tissue.
Comparison of CTX-II assay levels between different timesduring the season
Because of the limited size of the sample, which was selectedin a non-randomized manner because this was a closedindoor soccer team, the distribution of the individuals andresults from the sample cannot be considered to be normal.When this occurs, nonparametric statistical tests need to beused. In the graphic distribution of the data, two individu-als had values that were very different from those of the remainder of the team and which were considered to be out-liers.
To avoid the risk of statistical error, two separate analyseswere performed: 1 - comparisons using the complete data; and2 - comparisons using reduced data (without the informationon players 4 and 10) (Table 2).
In comparing the values from the first two analyses, i.e.beginning of the season versus middle of the season, no sta-tistically significant differences between the samples could beseen, in either of the comparisons (1 and 2).
In the analysis between the middle and the end of theseason, there was a significant difference in assayed levels,in the comparison among the 14 participants (p < 0.02). How-ever, when players 4 and 10 were taken out of the analysis,no significant difference could be seen (analysis with reduceddata).
In comparing the beginning and end of the season, bothanalyses (comparisons 1 and 2) indicated that there was a sta-tistically significant increase in the CTX-II biomarker (p < 0.003and p < 0.01, respectively). In other words, between the begin-ning and the end of the season, there was a significant increasein the level of the CTX-II joint degradation biomarker, irre-spective of whether the extreme results were taken intoconsideration.
Discussion
This study aimed to analyze and compare blood serum assaylevels for the biomarker CTX-II among professional indoor soc-cer players, at three different times during one season. Fromthe results observed, it could be seen that there was a statis-tically significant increase in CTX-II between the beginningand end of the season. It has been well established in theliterature that increased levels of the biomarker CTX-II are apredictive factor for joint degradation. Thus, it is believed thatearly identification of CTX-II levels would be a useful tool formaking equally early diagnoses of primary osteoarthritis (OA)and taking preventive action.
Corroborating the findings of the present study, O'Kaneet al.22evaluated different sports categories and demonstratedthrough urine analyses that samples from marathon run-ners presented higher CTX-II levels than those of swimmersor rowers. According to these authors, runners expose theirlower-limb joints to repetitive axial overloading, which maycause early damage to their joint cartilage.
The pure and simple increase in CTX-II in the playersstudied here does not necessarily mean that they are suf-fering or will suffer osteoarthrosis. It is known that patientswith arthrosis have high levels of this serum biomarker andincreased levels are directly related to radiographic worsening,according to the Kelgreen-Laurence classification.16What wecan be sure of from these data is that, at least during competi-tions, chondral degradation was higher in the patients studied,since the biomarker CTX-II comes from destruction of type IIcollagen, exclusively in joint cartilage.
It is possible that changes to training, intensification ofanaerobic exercises with muscle strengthening, compulsoryphysiotherapeutic follow-up and even use of chondroprotec-tant medications might be viable solutions for joint protection.In order to protect the joint health of soccer players, teamsshould start to think about this type of preventive strategy.
After all, the aim today should be prevention beforetreatment. Injury avoidance through practicing preventivemedicine should be the focus of exercise and sports medicine,since careers can certainly be prolonged in this manner.
The possible limitations of this study are the small samplesize (n = 14) and the absence of a control group for comparisonsbetween findings. The sample size is explained by the factthat this study investigated a single team within this category,whose members were all subjected to the same training andcompetition loads. It was decided not to use a control group,because our aim was only to ascertain whether there wouldbe any increase in joint collagen degradation over the courseof a single season of training and competition. In other words,the individuals became their own controls.
Other studies underway within the Sports Trauma Groupof Santa Casa de São Paulo are comparing types of sports andcontrol groups, including indoor soccer. Preliminary studiesindicate that there are large differences regarding type II col-lagen degradation.
The discrepancies encountered in our sample, in two ofthe individuals analyzed, can be explained by a variety oftheories. These include the notions that the markers mayhave been influenced by the individuals' hormonal state, diet or genetic factors,22given that neither of these individualsreported any reduction in physical performance, or any symp-toms such as pain, edema or instability. According to Damet al.,24the amount of cartilage degradation estimated fromthe biomarker CTX-II is related to the presence of pain.
Conclusion
There was a significant increase in the serum levels of CTX-II in the indoor soccer players, comparing the beginning andend of the season. These data suggest that increased degra-dation of type II joint collagen was occurring at the end ofthis period. It is clear that further studies are needed, withappropriate methodological rigor, so as to make effective con-tributions toward precise elucidation of the etiology of OA andits relationship with biomarkers as a tool for early diagnosis.
Conflicts of interest
The authors declare no conflicts of interest.
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