Hindawi Publishing CorporationArthritisVolume 2013 Article ID 631959 7 pageshttpdxdoiorg1011552013631959

Research ArticleHow Different Methodologies of Harvesting andAnalysing the Samples Affect the Test Results in DeterminingJoint Mediators

Ibrahim Yilmaz1 Nevzat Selim Gokay2 Rifat Bircan3 Gamze V Saracoglu4

Sergulen Dervisoglu5 and Alper Gokce2

1 Tekirdag State Hospital Turkish Republic Ministry of Health 59100 Tekirdag Turkey2Department of Orthopaedics and Traumatology Namik Kemal University School of Medicine 59100 Tekirdag Turkey3 Department of Biology Namik Kemal University School of Science 59100 Tekirdag Turkey4Department of Public Health Namik Kemal University School of Medicine 59100 Tekirdag Turkey5 Department of Pathology Istanbul University Cerrahpasa School of Medicine 34099 Istanbul Turkey

Correspondence should be addressed to Alper Gokce agokceyahoocom

Received 9 August 2012 Revised 27 December 2012 Accepted 13 January 2013

Academic Editor Pierre Youinou

Copyright copy 2013 Ibrahim Yilmaz et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose This study has researched the affect of different methodologies of harvesting and analysing the samples in determiningthe mediators emerging after the rat articular cartilage injuryMaterials and Methods One hundred and forty-four male wistar ratswere divided into 2 groups Synovial fluid samples were taken from all of the rats We entered into the right knees of the rats ingroup I (119899 = 36) under anaesthesia and took cartilage tissue samples from their distal femur Samples were taken as reference valuesfor enzyme linked immunosorbent assay (ELISA) and histopathological evaluations We entered into the right knees of the rats ingroup II (119899 = 108) and formed complete layer of cartilage injury in their medial femoral condyles At the end of the 15th day therats were sacrificed after taking synovial fluid samples from their right knees creating defect in the rats in group II The molecularmarkers in the synovial fluid and cartilage tissue samples which were taken from the experimental and control groups (MMP-9MMP-13 TIMP-1 TNF-120572 and NO) were analysed by direct or indirect methodologies SPSS 180 Package program was used in thestatistical evaluation Students t-test where themeasurement variables between the experimental and control groupswere comparedwas applied Receiver Operating Characteristics (ROC) curves were used in the determination of the diagnostic sufficiency fromthe tissue Results No difference was found between TIMP-1 (119875 = 067) andMMP-9 (119875 = 028) levels in synovial fluid and cartilagetissue From the molecular markers when MMP-9 MMP-13 NO TIMP-1 TNF-1205721015840 the area under ROC curve and P values wereexamined MMP-13 (119875 lt 00001 95 CI 070ndash085) NO (119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 00001 95 CI091ndash098) results were found to be statistically significant Inferences The indirect ELISA protocol which we apply for the cartilagetissue as an alternative to synovial lavage fluid is a reliable method which can be used in the determination of articular cartilageinjury markers

1 Introduction

The molecular markers which emerge in the process afterthe articular cartilage injury are used in the monitoring ofdegenerative diseases prognosis determination monitoringof response to the treatment and identification of the diseasemechanism in molecular level [1 2] One important purposeof the measurement of molecular determinants is to examine

the disease quantitatively in the early stages when the carti-lage injury has not been radiologically determined yet

Chondrocytes have a directing role in the metabolismincluding the construction and destruction of matrix mole-cules in the lifetime [3 4] There are simultaneous changesin tissues in the articular cartilage injury and there is aneed for molecular markers related to each tissue for thecomprehensive evaluation of these changes However there

2 Arthritis

are no official study protocols for different tissues [5] As therecan be significant differences related to biological tissues theway of sampling of the markers which are obtained from dif-ferent tissues can influence the result should be predictablePharmaceutical researchers have published articles on thevalidity of the analytical method in the determination of themethod applied [5ndash10]

Molecular markers of the cartilage are molecules whichcan be in the structure of protein constituting collagen pro-teoglycan and extra cellular matrix (ECM) and they showincrease and decrease in several cartilage pathologies and inthe different stages of the same pathology [11]

Depending on the severity of the ongoing inflammationcartilage-basedmarkers in several intensities go out of the celland enter into the synovial fluid Parts of the cartilage ECMmacromolecules which are poured into the synovial fluid canpass to the systemic circulation [12]

This study aims to determine the biological markersoccurring in the articular cartilage injury directly by cartilagetissue biopsies and in synovial fluid and present the relation-ship between them

2 Materials and Methods

This study was conducted after the decision of the meetingdated June 1 2010 numbered 201004 and the permission ofthe Local Ethical Committee of Experimental Animals andthe Use of Live Mammal of Namik Kemal University Exper-imental analyses were repeated minimum 3 times In orderto minimize the differences in the technique anaesthesiainjury formation and analyses were performed by the sameresearchers

21 Materials Male wistar rats were supplied from Istan-bul University Experimental Medicine Research InstituteKetamine HCl (Ketalar 500mg) injectable 1 flacon Pfizerand xylazine (Rompun 2) injectable solution Parke-Daviswere used Protein extraction solution (78510) and proteaseinhibitor cocktail (87785) Thermo Scientific Pierce Biotech-nology Rockford IL amp 1105 were supplied from the UnitedStates of America NO kit was supplied from ELISA commer-cial kits IL-TIMP-1 (BMS2018BMS2018TEN) was suppliedfrom Cayman Chemical matrix metalloproteinase-9 (MMP-9) (BMS20162CE) and MMP-13 (BMS2022BMS2022TEN)platinum were supplied from ELISA eBioscience and TNF-120572 (KRC3011) kit was supplied from Invitrogen Mechanicaldisruption was provided with EpiSonic Multi-FunctionalBioprocessor 1100 Mindray MR 96 A Chinese brand devicewas used as EnzymeL120484nked-Immuno-SorbentAssay (ELISA)microplate reader The tissues which are obtained from 144wistar ratswere studied double-blind by the same researchers

22 Methods One hundred forty-four male wistar rats withan average weight of 300 grams were divided into 2 groupsSynovial fluid samples were taken from all of the ratsWhile taking synovial fluid the knee joint was entered bythe injector and the liquid which is given into the jointwas withdrawn 45 seconds later and taken into the tubes

Figure 1 Macro view of the actualization of the cartilage completelayer injury

containing EDTA Synovial fluid aspirates with PBS in therate of 75120583L 1 3were centrifuged at 24∘C in 3000RevolutionsPer Minute (rpm) for 15 minutes and cleared of cells andmaintained at minus 40∘C to be evaluated with ELISA Allof the samples were taken at 0500 in the morning

The rats in group I (control group 119899 = 36) were appliedarthrotomy under ketamine HCI and xylazine anaesthe-sia Cartilage tissue samples were taken from distal femurmedial condyles These samples were taken as reference val-ues for enzyme linked immunosorbent assay (ELISA) andhistopathological evaluations And then these rats weresacrificed

The rats in group II (119899 = 108) were applied arthrotomyunder ketamine HCl and xylazine anaesthesia and a com-plete layer of cartilage injury was formed in their medialfemoral condyles (Figure 1) [13 14] From the right kneesof the rats where injury was formed synovial fluid sampleswere taken by the same technique on the 15th day Thenarthrotomy was applied and cartilage tissue samples weretaken from their distal medial condyles These rats weresacrificed after this operation

The synovial lavage fluid and cartilage tissue samplestaken from the experimental and control groups were anal-ysed by direct or indirect methodologies by MMP-9 MMP-13 NO TIMP-1 TNF-120572 and commercial ELISA kits whichare cartilage injury molecular markers in line with companybulletins

23 Biochemical Analyses The samples were blindly studiedThe synovial fluid tissue samples which were taken from theexperimental and control groups were analysed by MMP-9MMP-13 NO TIMP-1 and TNF-120572 values ELISA kits and inline with the company bulletins

Before conducting analyses in the cartilage tissue the tis-sues weremade compatible for kit study procedure Porcelainmortar and pestle were subjected to 8 sodium hypochloritesolution They were dried after having been washed withbidistillated waterThey were wrapped with aluminium foliosand kept in the drying oven at 134∘C for 1 hour At the end ofthis time they were kept at minus20∘C for 30 minutes The carti-lage tissue sampleswhichwere broken upwith and pulverizedby a pestle in the presence of liquid nitrogen were takeninto cryo tubes and coded (Figure 2) They were weighed at

Arthritis 3

Figure 2 Mechanical disruption of the tissue which is frozen in thepresence of liquid nitrogen

the rate of 1 20 (wv) and transferred into eppendorf tubesby means of 15 no scalpel end Primarily 400120583L lysisbufferwas added on them Then a protease cocktail inhibitor tentimes the ready volume (making 1x the last concentration) isadded and vortexed for 1 minute and centrifuging processwas performed at +4∘C for 5minutes in 10000 (g) [15 16]Thesamples were kept at minus80∘C for one night For themechanicaldisruption of the cartilage tissue they were subjected to 2times vibration and ultrasonic sound waves operation for 10minutes under the same pressure at intervals of 20 minutesin sonicator by watering down with phosphate buffer saline(PBS pH = 7 4) in 15mLmicrotubes (max 400 120583L of samplecontent) And then in line with the company bulletins thesamples were placed in the wells MMP-9 MMP-13 TIMP-1and TNF-120572 450 nm NO was evaluated by reading at ELISAmicroplate reader at 540 nm wave length as a result of thecalculation of the data which was obtained over the nitrateand nitrite values Standards were studies as pairs

24 Histopathological Analyses In order to prove the car-tilage complete layer injury the cartilage tissue sampleswhich were taken from each of 6 rats from both groupswere also evaluated histopathologically The samples wereburied in paraffin-embedded blocks after the routine tissuemonitoring Five-micron sections were put to hematoxylin-eosin staining and examined in the light microscope (times100)Histological sections were examined in terms of 8 parametersand scored and evaluated [17]

25 Statistical Analysis Thedata was evaluated by using SPSS180 Package program Descriptive statistics were calculated(mean standard deviation) after performing data controlStudents t-test which is a test in the comparison of thequantitative data in the comparative analyses between theexperimental and control groups in which the measurementvariables between two groups are compared was appliedPartial correlation analysis was performed in order to explainthe relationship between thesemediators ReceiverOperatingCharacteristics (ROC) curves were used in the determinationof diagnostic sufficiency from the tissue

3 Results

31 Histopathological Evaluation The formation of the carti-lage complete layer injury was reported (Figure 3)

32 Statistical Evaluation All of the evaluations were eval-uated in 95 confidence interval and bilaterally Alphameaningfulness level was determined as le005 accordingto the specific activity results in synovial lavage fluid andcartilage tissue of the mediators (MMP-9 MMP-13 NOTIMP-1 and TNF-120572) emerging after complete layer articularcartilage injury

No meaningful difference was determined in statisticalterms between the TIMP-1 (119875 = 067) and MMP-9 (119875 =028) levels in synovial lavage fluid and cartilage tissue Ameaningful difference was found in statistical terms betweenthe MMP-13 (119875 lt 00001) TNF-120572 (119875 lt 00001) and NO(119875 lt 00001) levels (Table 1)

It was determined that the measurements in intragroupand intergroup TIMP-1 andMMP-9 mediators were variableand close measurements were obtained in NO TIMP-1 andTNF-120572 variables (Figure 4)

In the partial correlation analysis performed a negativelinear meaningful relationship was found between TIMP-1and MMP-13 (119875 lt 00001 119903 = minus038) a positive meaningfulrelationship was found between TIMP-1 and TNF-120572 (119875 lt00001 119903 = 033) A negative meaningful relationship wasfound between TIMP-1 and NO (119875 = 0001 119903 = minus026) Anda positive linear meaningful relationship was found betweenMMP-9 and TNF-120572 (119875 lt 00001 119903 = 034) and NO (119875 lt00001 119903 = 046) (Table 2)

When the area under ROC curve of TIMP-1 MMP-9MMP-13 NO and TNF-120572 markers and P values were exam-ined in the study groups MMP-13 (119875 lt 00001 95 CI070ndash085) NO (119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 00001 95 CI 091ndash098) results were found to bestatistically meaningful (Figure 5)

4 Discussion

Osteoartrit (OA) is a disease which emerges with the breakupof the balance between the construction and destruction ofthe cartilage in favour of destruction results in the loss ofthe cartilage and progresses slowly [18] Recently it has beenshown that the MMPs whose levels in the joint at the timeof injury increase play an important role in the destructionof the cartilage tissue [19] One of the tissue inhibitorswhich enable the balancing of these enzymes which aredestructive for the cartilage is identified as metalloproteinase[20] Particularly the metalloproteinase which increases inOA is produced by chondrocyte and synovial cells Sincethe number of these cells is higher than that in the cartilagetissue the main production source of MMP in the joint fluidis synovial tissue It is shown that metalloproteinase andTIMP increased in the synovial fluid in OA and collagenase-TIMP-1 complex level generally stayed under the level whichcan be determined by immunoassay [21] Lohmander et aldetermined that metalloproteinase and TIMP levels stayed

4 Arthritis

(a) (b)

(c) (d)

Figure 3 It is seen that complete layer of cartilage injury is histopathologically formed in the presence of HampE (times100) (a) Intact (b)fibrillation (c) fissure and (d) osteophyte formation

Table 1 Evaluating MMP-9 MMP-13 NO TIMP-1 and TNF-alpha levels in synovial lavage fluid and cartilage tissue types

Material 119873 Mean Standard deviation 119875

TIMP-1 Synovial lavage fluid 84 301 261 067Cartilage Tissue 84 283 259

MMP-9 Synovial lavage fluid 90 592 163 028Cartilage Tissue 90 566 167

MMP-13 Synovial lavage fluid 90 068 023lt0001

Cartilage Tissue 90 047 024

TNF-120572 Synovial lavage fluid 84 032 009lt0001

Cartilage Tissue 84 014 007

NO Synovial lavage fluid 90 057 019lt0001

Cartilage Tissue 90 036 019

high for a long time in the knee joint after a traumatic injury[22]

In our study it was seen thatMMP-9 levels did not changeeven when different methodologies are used in synoviallavage fluid and cartilage tissue analyses in the samples whichwere examined after the formation of injury Likewise nochange was determined in TIMP-1 levels (Table 1)

The role of proinflammatory was shown in the articularcartilage injury of TNF-120572 which are among the cytokinesother than those aforementioned molecules [23 24] SerumTNF-120572 level is seen as a criterion of the synovial cellhyperactivity which becomes evident in the late stages of thecartilage injury [25] It is known that TNF-120572 in the jointwith OA mainly originates from the cells in the cartilage

tissue and these cells perform more cytokine expressionwhen compared to synovial cells [26]

In our study statistically meaningful differences weredetermined in terms of TNF-120572 and MMP-13 levels in thesamples which were obtained from synovial lavage fluid andcartilage tissue (Table 1) In the subsequent partial correla-tion analysis a negative meaningful relationship was foundbetween TIMP-1 and MMP-13 (119875 lt 00001 119903 = minus038)and a positive meaningful relationship was found betweenTIMP-1 and TNF-120572 (119875 lt 00001 119903 = 033) A positivelinear meaningful relationship was found between MMP-9 and TNF-120572 (119875 lt 00001 119903 = 034) (Table 2) It wasseen in ROC analysis that these two mediators can also beused in different methodologies in synovial lavage fluid and

Arthritis 5

Table 2 Partial Correlation analysis

Control variables TIMP-1 MMP-9 MMP-13 TNF-120572 NOMaterial

TIMP-1Correlation 100 minus014 minus038 033 minus026Significance (2-tailed) mdash 008 0000 0000 0001

MMP-9Correlation minus014 10 minus004 034 046Significance (2-tailed) 008 mdash 061 0000 0000

MMP-13Correlation minus038 minus004 1000 013 001Significance (2-tailed) 0000 061 mdash 009 088

TNF-120572Correlation 033 034 013 1000 minus004Significance (2-tailed) 0000 0000 009 mdash 059

NOCorrelation minus026 046 001 minus004 1000Significance (2-tailed) 0001 0000 088 059 mdash

Syno

vial

flui

dM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Cart

ilage

tiss

ueM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Figure 4 Distribution of the mediators in the synovial lavage fluidand cartilage tissue in all experimental groups (mean 95 CI)

cartilage tissue The molecular marker level results which areobtained from different areas in the literature and set forthwith different methodologies are also in conformity with thedata of our study [25ndash27]

In an experimental study it was it shown that NOsynthesis are stimulated by several cytokines such as TNF-120572 [28] Evidences regarding the fact that NO is a secondaryfundamental mediator after TNF-120572 in cartilage cells wereset forth in the same study [28] In our study on the otherhand NO level was determined to be meaningfully high inthe analysis results of the samples taken from synovial lavage

1080604020

1

08

06

04

02

0

Sens

itivi

ty

Specifity

ROC curve

Diagonal segments are produced by ties

Source of the curveTIMP-1TIMP-9MMP-13

TNF-120572NOReference line

Figure 5 ROC analysis where the validities of the mediators insynovial lavage fluid and cartilage tissue is evaluated

fluid in conformity with the TNF-120572 level in the experimentalgroups A positive linear relationship was found betweenTNF-120572 and NO (119875 lt 00001 119903 = 046) In the ROC analysisthe fact that these mediators which were taken from differentplaces and examined can also be analysed over a differentmethodology cartilage tissue was confirmed

6 Arthritis

The affect of MMP enzymes on cartilage is inhibited byTIMP [29 30] NO is overexpressed at synovial tissue ofinjured cartilage by the stimulus of inducible nitric oxide syn-thase andTNF-120572 [29ndash32] IL-1 andTNF-120572 release is increasedwith the affect of degraded matrix molecules and so NOexpression is also increased It is reported that MMP-13 isdischarged mostly via sinovial fluid [33] We think that thesedata explain why TNF-120572 MMP-13 and NOwere foundmorein synovial fluids in our results

We think the restriction of our study is that it cannot becrosschecked by a method other than ELISA method whichis used in the analysis of the samples

5 Conclusion

When among the molecular markers which are analyzedin the synovial lavage fluid MMP-9 MMP-13 NO TIMP-1and TNF-120572 the area under the ROC curve and P values areevaluated MMP-13 (119875 lt 00001 95 CI 070ndash085) NO(119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 0000195 CI 091ndash098) results are statistically significant As aresult as it can be analyzed from the synovial lavage fluid ofthe molecular injury markers such as MMP-9 MMP-13 NOTIMP-1 and TNF-120572 which emerge in the articular cartilageinjury we can say that it can also be analyzed in a reliablemanner by the indirect ELISAmethod described by us in thesamples which are obtained from the cartilage tissue

Acknowledgment

Theauthors thank theTurkishOrthopedic Society (TOTBID)for the grants in support of this study

References

[1] G Nagyeri M Radacs S Ghassemi-Nejad et al ldquoTSG-6protein a negative regulator of inflammatory arthritis forms aternary complex with murine mast cell tryptases and heparinrdquoThe Journal of Biological Chemistry vol 286 no 26 pp 23559ndash23569 2011

[2] K Tsuritani J Takeda J Sakagami et al ldquoCytokine receptor-like factor 1 is highly expressed in damaged human kneeosteoarthritic cartilage and involved in osteoarthritis down-stream of TGF-120573rdquo Calcified Tissue International vol 86 no 1pp 47ndash57 2010

[3] T A Stupina V D Makushin and M A Stepanov ldquoExperi-mental morphological study of the effects of subchondral tun-nelization and bone marrow stimulation on articular cartilageregenerationrdquo Bulletin Experimental Biology and Medicine vol153 no 2 pp 289ndash293 2012

[4] MA JordanG SVanThiel J Chahal and S JNho ldquoOperativetreatment of chondral defects in the hip joint a systematicreviewrdquo Current Reviews in Musculoskeletal Medicine vol 5 no3 pp 244ndash253 2012

[5] O S Ertas and A Kayali ldquoAn overview on analytical methodvalidationrdquo Journal of Faculty Pharmacy vol 34 no 1 pp 41ndash57 2005

[6] G Szepesi M Gazdag and K Mihalyfi ldquoSelection of high-performance liquid chromatographic methods in pharmaceuti-cal analysis III Method validationrdquo Journal of ChromatographyA vol 464 no 2 pp 265ndash278 1989

[7] A Kayal O Sogut and B Tuncel ldquoSome considerations on ana-lytical methods validations bioavailability bioequivalence andpharmacokinetic studiesrdquoEuropean Journal of DrugMetabolismand Pharmacokinetics vol 20 no 4 pp 271ndash274 1995

[8] A Marin A Lopez-Gonzalvez and C Barbas ldquoDevelopmentand validation of extraction methods for determination ofzinc and arsenic speciation in soils using focused ultrasoundapplication to heavy metal study in mud and soilsrdquo AnalyticaChimica Acta vol 442 no 2 pp 305ndash318 2001

[9] J G Morrison P White S McDougall et al ldquoValidation ofa highly sensitive ICP-MS method for the determination ofplatinum in biofluids application to clinical pharmacokineticstudies with oxaliplatinrdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 24 no 1 pp 1ndash10 2000

[10] V Grdinic and J Vukovic ldquoPrevalidation in pharmaceuticalanalysis part 1 fundamentals and critical discussionrdquo Journal ofPharmaceutical and Biomedical Analysis vol 35 no 1 pp 489ndash512 2004

[11] P Garnero J C Rousseau and P D Delmas ldquoMolecular basisand clinical use of biochemical markers of bone cartilage andsynovium in joint diseasesrdquoArthritis ampRheumatism vol 43 no1 pp 953ndash968 2000

[12] D A Walsh P Verghese G J Cook et al ldquoLymphatic vesselsin osteoarthritic human kneesrdquoOsteoarthritis Cartilage vol 20no 5 pp 405ndash412 2012

[13] Y Henrotin B Kurz and T Aigner ldquoOxygen and reactiveoxygen species in cartilage degradation friends or foesrdquoOsteoarthritis and Cartilage vol 13 no 8 pp 643ndash654 2005

[14] A Ostalowska E Birkner M Wiecha et al ldquoLipid peroxi-dation and antioxidant enzymes in synovial fluid of patientswith primary and secondary osteoarthritis of the knee jointrdquoOsteoarthritis and Cartilage vol 14 no 2 pp 139ndash145 2006

[15] K Haugbro J C Nossent T Winkler Y Figenschau and OP Rekvig ldquoAnti-dsDNA antibodies and disease classificationin antinuclear antibody positive patients the role of analyticaldiversityrdquo Annals of the Rheumatic Diseases vol 63 no 4 pp386ndash394 2004

[16] K J Jepsen F Wu J H Peragallo et al ldquoA syndrome ofjoint laxity and impaired tendon integrity in lumican- andfibromodulin-deficient micerdquo The Journal of Biological Chem-istry vol 277 no 38 pp 35532ndash35540 2002

[17] B E Gencosmanoglu M Eryavuz and S Dervisoglu ldquoEffectsof some nonsteroidal anti-inflammatory drugs on articularcartilage of rats in an experimental model of osteoarthritisrdquoResearch in Experimental Medicine vol 200 no 3 pp 215ndash2262001

[18] A J Hough ldquoPathology of osteoarthritisrdquo inArthritis and AlliedConditions W J Koopman Ed pp 1945ndash1968 Williams ampWilkins Baltimore Md USA 1997

[19] J D Sandy C R Flannery P J Neame and L S LohmanderldquoThe structure of aggrecan fragments in human synovial fluidEvidence for the involvement in osteoarthritis of a novelproteinase which cleaves the Glu 373-Ala 374 bond of theinterglobular domainrdquoThe Journal of Clinical Investigation vol89 no 5 pp 1512ndash1516 1992

[20] S Tseng A H Reddi and P E Di Cesare ldquoCartilage oligomericmatrix protein (COMP) a biomarker of arthritisrdquo BiomarkerInsights vol 4 pp 33ndash44 2009

[21] L S Lohmander L A Hoerrner and M W Lark ldquoMet-alloproteinases tissue inhibitor and proteoglycan fragmentsin knee synovial fluid in human osteoarthritisrdquo Arthritis ampRheumatism vol 36 no 2 pp 181ndash189 1993

[22] L S Lohmander H Roos L Dahlberg L A Hoerrnerand M W Lark ldquoTemporal patterns of stromelysin-1 tissue

Arthritis 7

inhibitor and proteoglycan fragments in human knee joint fluidafter injury to the cruciate ligament or meniscusrdquo Journal ofOrthopaedic Research vol 12 no 1 pp 21ndash28 1994

[23] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Immunol-ogy vol 14 pp 397ndash440 1996

[24] L Connell and I B McInnes ldquoNew cytokine targets in inflam-matory rheumatic diseasesrdquo Best Practice amp Research vol 20no 5 pp 865ndash878 2006

[25] M Sharif E George L Shepstone et al ldquoSerumhyaluronic acidlevel as a predictor of disease progression in osteoarthritis of thekneerdquo Arthritis amp Rheumatism vol 38 no 6 pp 760ndash767 1995

[26] C Melchiorri R Meliconi L Frizziero et al ldquoEnhancedand coordinated in vivo expression of inflammatory cytokinesand nitric oxide synthase by chondrocytes from patients withosteoarthritisrdquoArthritis amp Rheumatism vol 41 no 12 pp 2165ndash2174 1998

[27] R Hilf J L Wittliff W D Rector E D Savlov T C Halland R A Orlando ldquoStudies on certain cytoplasmic enzymesand specific estrogen receptors in human breast cancer and innonmalignant diseases of the breastrdquo Cancer Research vol 33no 9 pp 2054ndash2062 1973

[28] F A Van de Loo O J Arntz F H van Enckevort P L van Lentand W B van den Berg ldquoReduced cartilage proteoglycan lossduring zymosan-induced gonarthritis in NOS

2-deficient mice

and in anti-interleukin-1-treated wild-type mice with unabatedjoint inflammationrdquo Arthritis amp Rheumatism vol 41 no 4 pp634ndash646 1998

[29] R F Loeser ldquoIntegrins and cell signaling in chondrocytesrdquo Bi-orheology vol 39 no 1-2 pp 119ndash124 2002

[30] A R Poole F Guilak and S B Abramson ldquoEtiopathogenesisof osteoarthritisrdquo in Osteoarthritis Diagnosis and MEdicalSur-gical Management R WMoskowitz Ed pp 27ndash49 LippincottPhiladelphia Pa USA 4th edition 2007

[31] S R Goldring and M B Goldring ldquoThe role of cytokines incartilage matrix degeneration in osteoarthritisrdquo Clinical Ortho-paedics and Related Research no 427 supplement pp S27ndashS362004

[32] S Kamekura K Hoshi T Shimoaka et al ldquoOsteoarthritisdevelopment in novel experimental mouse models induced byknee joint instabilityrdquoOsteoarthritis and Cartilage vol 13 no 7pp 632ndash641 2005

[33] A R Poole M Kobayashi T Yasuda et al ldquoType II col-lagen degradation and its regulation in articular cartilage inosteoarthritisrdquo Annals of the Rheumatic Diseases vol 61 sup-plement 2 pp 78ndash81 2002

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2 Arthritis

are no official study protocols for different tissues [5] As therecan be significant differences related to biological tissues theway of sampling of the markers which are obtained from dif-ferent tissues can influence the result should be predictablePharmaceutical researchers have published articles on thevalidity of the analytical method in the determination of themethod applied [5ndash10]

Molecular markers of the cartilage are molecules whichcan be in the structure of protein constituting collagen pro-teoglycan and extra cellular matrix (ECM) and they showincrease and decrease in several cartilage pathologies and inthe different stages of the same pathology [11]

Depending on the severity of the ongoing inflammationcartilage-basedmarkers in several intensities go out of the celland enter into the synovial fluid Parts of the cartilage ECMmacromolecules which are poured into the synovial fluid canpass to the systemic circulation [12]

This study aims to determine the biological markersoccurring in the articular cartilage injury directly by cartilagetissue biopsies and in synovial fluid and present the relation-ship between them

2 Materials and Methods

This study was conducted after the decision of the meetingdated June 1 2010 numbered 201004 and the permission ofthe Local Ethical Committee of Experimental Animals andthe Use of Live Mammal of Namik Kemal University Exper-imental analyses were repeated minimum 3 times In orderto minimize the differences in the technique anaesthesiainjury formation and analyses were performed by the sameresearchers

21 Materials Male wistar rats were supplied from Istan-bul University Experimental Medicine Research InstituteKetamine HCl (Ketalar 500mg) injectable 1 flacon Pfizerand xylazine (Rompun 2) injectable solution Parke-Daviswere used Protein extraction solution (78510) and proteaseinhibitor cocktail (87785) Thermo Scientific Pierce Biotech-nology Rockford IL amp 1105 were supplied from the UnitedStates of America NO kit was supplied from ELISA commer-cial kits IL-TIMP-1 (BMS2018BMS2018TEN) was suppliedfrom Cayman Chemical matrix metalloproteinase-9 (MMP-9) (BMS20162CE) and MMP-13 (BMS2022BMS2022TEN)platinum were supplied from ELISA eBioscience and TNF-120572 (KRC3011) kit was supplied from Invitrogen Mechanicaldisruption was provided with EpiSonic Multi-FunctionalBioprocessor 1100 Mindray MR 96 A Chinese brand devicewas used as EnzymeL120484nked-Immuno-SorbentAssay (ELISA)microplate reader The tissues which are obtained from 144wistar ratswere studied double-blind by the same researchers

22 Methods One hundred forty-four male wistar rats withan average weight of 300 grams were divided into 2 groupsSynovial fluid samples were taken from all of the ratsWhile taking synovial fluid the knee joint was entered bythe injector and the liquid which is given into the jointwas withdrawn 45 seconds later and taken into the tubes

Figure 1 Macro view of the actualization of the cartilage completelayer injury

containing EDTA Synovial fluid aspirates with PBS in therate of 75120583L 1 3were centrifuged at 24∘C in 3000RevolutionsPer Minute (rpm) for 15 minutes and cleared of cells andmaintained at minus 40∘C to be evaluated with ELISA Allof the samples were taken at 0500 in the morning

The rats in group I (control group 119899 = 36) were appliedarthrotomy under ketamine HCI and xylazine anaesthe-sia Cartilage tissue samples were taken from distal femurmedial condyles These samples were taken as reference val-ues for enzyme linked immunosorbent assay (ELISA) andhistopathological evaluations And then these rats weresacrificed

The rats in group II (119899 = 108) were applied arthrotomyunder ketamine HCl and xylazine anaesthesia and a com-plete layer of cartilage injury was formed in their medialfemoral condyles (Figure 1) [13 14] From the right kneesof the rats where injury was formed synovial fluid sampleswere taken by the same technique on the 15th day Thenarthrotomy was applied and cartilage tissue samples weretaken from their distal medial condyles These rats weresacrificed after this operation

The synovial lavage fluid and cartilage tissue samplestaken from the experimental and control groups were anal-ysed by direct or indirect methodologies by MMP-9 MMP-13 NO TIMP-1 TNF-120572 and commercial ELISA kits whichare cartilage injury molecular markers in line with companybulletins

23 Biochemical Analyses The samples were blindly studiedThe synovial fluid tissue samples which were taken from theexperimental and control groups were analysed by MMP-9MMP-13 NO TIMP-1 and TNF-120572 values ELISA kits and inline with the company bulletins

Before conducting analyses in the cartilage tissue the tis-sues weremade compatible for kit study procedure Porcelainmortar and pestle were subjected to 8 sodium hypochloritesolution They were dried after having been washed withbidistillated waterThey were wrapped with aluminium foliosand kept in the drying oven at 134∘C for 1 hour At the end ofthis time they were kept at minus20∘C for 30 minutes The carti-lage tissue sampleswhichwere broken upwith and pulverizedby a pestle in the presence of liquid nitrogen were takeninto cryo tubes and coded (Figure 2) They were weighed at

Arthritis 3

Figure 2 Mechanical disruption of the tissue which is frozen in thepresence of liquid nitrogen

the rate of 1 20 (wv) and transferred into eppendorf tubesby means of 15 no scalpel end Primarily 400120583L lysisbufferwas added on them Then a protease cocktail inhibitor tentimes the ready volume (making 1x the last concentration) isadded and vortexed for 1 minute and centrifuging processwas performed at +4∘C for 5minutes in 10000 (g) [15 16]Thesamples were kept at minus80∘C for one night For themechanicaldisruption of the cartilage tissue they were subjected to 2times vibration and ultrasonic sound waves operation for 10minutes under the same pressure at intervals of 20 minutesin sonicator by watering down with phosphate buffer saline(PBS pH = 7 4) in 15mLmicrotubes (max 400 120583L of samplecontent) And then in line with the company bulletins thesamples were placed in the wells MMP-9 MMP-13 TIMP-1and TNF-120572 450 nm NO was evaluated by reading at ELISAmicroplate reader at 540 nm wave length as a result of thecalculation of the data which was obtained over the nitrateand nitrite values Standards were studies as pairs

24 Histopathological Analyses In order to prove the car-tilage complete layer injury the cartilage tissue sampleswhich were taken from each of 6 rats from both groupswere also evaluated histopathologically The samples wereburied in paraffin-embedded blocks after the routine tissuemonitoring Five-micron sections were put to hematoxylin-eosin staining and examined in the light microscope (times100)Histological sections were examined in terms of 8 parametersand scored and evaluated [17]

25 Statistical Analysis Thedata was evaluated by using SPSS180 Package program Descriptive statistics were calculated(mean standard deviation) after performing data controlStudents t-test which is a test in the comparison of thequantitative data in the comparative analyses between theexperimental and control groups in which the measurementvariables between two groups are compared was appliedPartial correlation analysis was performed in order to explainthe relationship between thesemediators ReceiverOperatingCharacteristics (ROC) curves were used in the determinationof diagnostic sufficiency from the tissue

3 Results

31 Histopathological Evaluation The formation of the carti-lage complete layer injury was reported (Figure 3)

32 Statistical Evaluation All of the evaluations were eval-uated in 95 confidence interval and bilaterally Alphameaningfulness level was determined as le005 accordingto the specific activity results in synovial lavage fluid andcartilage tissue of the mediators (MMP-9 MMP-13 NOTIMP-1 and TNF-120572) emerging after complete layer articularcartilage injury

No meaningful difference was determined in statisticalterms between the TIMP-1 (119875 = 067) and MMP-9 (119875 =028) levels in synovial lavage fluid and cartilage tissue Ameaningful difference was found in statistical terms betweenthe MMP-13 (119875 lt 00001) TNF-120572 (119875 lt 00001) and NO(119875 lt 00001) levels (Table 1)

It was determined that the measurements in intragroupand intergroup TIMP-1 andMMP-9 mediators were variableand close measurements were obtained in NO TIMP-1 andTNF-120572 variables (Figure 4)

In the partial correlation analysis performed a negativelinear meaningful relationship was found between TIMP-1and MMP-13 (119875 lt 00001 119903 = minus038) a positive meaningfulrelationship was found between TIMP-1 and TNF-120572 (119875 lt00001 119903 = 033) A negative meaningful relationship wasfound between TIMP-1 and NO (119875 = 0001 119903 = minus026) Anda positive linear meaningful relationship was found betweenMMP-9 and TNF-120572 (119875 lt 00001 119903 = 034) and NO (119875 lt00001 119903 = 046) (Table 2)

When the area under ROC curve of TIMP-1 MMP-9MMP-13 NO and TNF-120572 markers and P values were exam-ined in the study groups MMP-13 (119875 lt 00001 95 CI070ndash085) NO (119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 00001 95 CI 091ndash098) results were found to bestatistically meaningful (Figure 5)

4 Discussion

Osteoartrit (OA) is a disease which emerges with the breakupof the balance between the construction and destruction ofthe cartilage in favour of destruction results in the loss ofthe cartilage and progresses slowly [18] Recently it has beenshown that the MMPs whose levels in the joint at the timeof injury increase play an important role in the destructionof the cartilage tissue [19] One of the tissue inhibitorswhich enable the balancing of these enzymes which aredestructive for the cartilage is identified as metalloproteinase[20] Particularly the metalloproteinase which increases inOA is produced by chondrocyte and synovial cells Sincethe number of these cells is higher than that in the cartilagetissue the main production source of MMP in the joint fluidis synovial tissue It is shown that metalloproteinase andTIMP increased in the synovial fluid in OA and collagenase-TIMP-1 complex level generally stayed under the level whichcan be determined by immunoassay [21] Lohmander et aldetermined that metalloproteinase and TIMP levels stayed

4 Arthritis

(a) (b)

(c) (d)

Figure 3 It is seen that complete layer of cartilage injury is histopathologically formed in the presence of HampE (times100) (a) Intact (b)fibrillation (c) fissure and (d) osteophyte formation

Table 1 Evaluating MMP-9 MMP-13 NO TIMP-1 and TNF-alpha levels in synovial lavage fluid and cartilage tissue types

Material 119873 Mean Standard deviation 119875

TIMP-1 Synovial lavage fluid 84 301 261 067Cartilage Tissue 84 283 259

MMP-9 Synovial lavage fluid 90 592 163 028Cartilage Tissue 90 566 167

MMP-13 Synovial lavage fluid 90 068 023lt0001

Cartilage Tissue 90 047 024

TNF-120572 Synovial lavage fluid 84 032 009lt0001

Cartilage Tissue 84 014 007

NO Synovial lavage fluid 90 057 019lt0001

Cartilage Tissue 90 036 019

high for a long time in the knee joint after a traumatic injury[22]

In our study it was seen thatMMP-9 levels did not changeeven when different methodologies are used in synoviallavage fluid and cartilage tissue analyses in the samples whichwere examined after the formation of injury Likewise nochange was determined in TIMP-1 levels (Table 1)

The role of proinflammatory was shown in the articularcartilage injury of TNF-120572 which are among the cytokinesother than those aforementioned molecules [23 24] SerumTNF-120572 level is seen as a criterion of the synovial cellhyperactivity which becomes evident in the late stages of thecartilage injury [25] It is known that TNF-120572 in the jointwith OA mainly originates from the cells in the cartilage

tissue and these cells perform more cytokine expressionwhen compared to synovial cells [26]

In our study statistically meaningful differences weredetermined in terms of TNF-120572 and MMP-13 levels in thesamples which were obtained from synovial lavage fluid andcartilage tissue (Table 1) In the subsequent partial correla-tion analysis a negative meaningful relationship was foundbetween TIMP-1 and MMP-13 (119875 lt 00001 119903 = minus038)and a positive meaningful relationship was found betweenTIMP-1 and TNF-120572 (119875 lt 00001 119903 = 033) A positivelinear meaningful relationship was found between MMP-9 and TNF-120572 (119875 lt 00001 119903 = 034) (Table 2) It wasseen in ROC analysis that these two mediators can also beused in different methodologies in synovial lavage fluid and

Arthritis 5

Table 2 Partial Correlation analysis

Control variables TIMP-1 MMP-9 MMP-13 TNF-120572 NOMaterial

TIMP-1Correlation 100 minus014 minus038 033 minus026Significance (2-tailed) mdash 008 0000 0000 0001

MMP-9Correlation minus014 10 minus004 034 046Significance (2-tailed) 008 mdash 061 0000 0000

MMP-13Correlation minus038 minus004 1000 013 001Significance (2-tailed) 0000 061 mdash 009 088

TNF-120572Correlation 033 034 013 1000 minus004Significance (2-tailed) 0000 0000 009 mdash 059

NOCorrelation minus026 046 001 minus004 1000Significance (2-tailed) 0001 0000 088 059 mdash

Syno

vial

flui

dM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Cart

ilage

tiss

ueM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Figure 4 Distribution of the mediators in the synovial lavage fluidand cartilage tissue in all experimental groups (mean 95 CI)

cartilage tissue The molecular marker level results which areobtained from different areas in the literature and set forthwith different methodologies are also in conformity with thedata of our study [25ndash27]

In an experimental study it was it shown that NOsynthesis are stimulated by several cytokines such as TNF-120572 [28] Evidences regarding the fact that NO is a secondaryfundamental mediator after TNF-120572 in cartilage cells wereset forth in the same study [28] In our study on the otherhand NO level was determined to be meaningfully high inthe analysis results of the samples taken from synovial lavage

1080604020

1

08

06

04

02

0

Sens

itivi

ty

Specifity

ROC curve

Diagonal segments are produced by ties

Source of the curveTIMP-1TIMP-9MMP-13

TNF-120572NOReference line

Figure 5 ROC analysis where the validities of the mediators insynovial lavage fluid and cartilage tissue is evaluated

fluid in conformity with the TNF-120572 level in the experimentalgroups A positive linear relationship was found betweenTNF-120572 and NO (119875 lt 00001 119903 = 046) In the ROC analysisthe fact that these mediators which were taken from differentplaces and examined can also be analysed over a differentmethodology cartilage tissue was confirmed

6 Arthritis

The affect of MMP enzymes on cartilage is inhibited byTIMP [29 30] NO is overexpressed at synovial tissue ofinjured cartilage by the stimulus of inducible nitric oxide syn-thase andTNF-120572 [29ndash32] IL-1 andTNF-120572 release is increasedwith the affect of degraded matrix molecules and so NOexpression is also increased It is reported that MMP-13 isdischarged mostly via sinovial fluid [33] We think that thesedata explain why TNF-120572 MMP-13 and NOwere foundmorein synovial fluids in our results

We think the restriction of our study is that it cannot becrosschecked by a method other than ELISA method whichis used in the analysis of the samples

5 Conclusion

When among the molecular markers which are analyzedin the synovial lavage fluid MMP-9 MMP-13 NO TIMP-1and TNF-120572 the area under the ROC curve and P values areevaluated MMP-13 (119875 lt 00001 95 CI 070ndash085) NO(119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 0000195 CI 091ndash098) results are statistically significant As aresult as it can be analyzed from the synovial lavage fluid ofthe molecular injury markers such as MMP-9 MMP-13 NOTIMP-1 and TNF-120572 which emerge in the articular cartilageinjury we can say that it can also be analyzed in a reliablemanner by the indirect ELISAmethod described by us in thesamples which are obtained from the cartilage tissue

Acknowledgment

Theauthors thank theTurkishOrthopedic Society (TOTBID)for the grants in support of this study

References

[1] G Nagyeri M Radacs S Ghassemi-Nejad et al ldquoTSG-6protein a negative regulator of inflammatory arthritis forms aternary complex with murine mast cell tryptases and heparinrdquoThe Journal of Biological Chemistry vol 286 no 26 pp 23559ndash23569 2011

[2] K Tsuritani J Takeda J Sakagami et al ldquoCytokine receptor-like factor 1 is highly expressed in damaged human kneeosteoarthritic cartilage and involved in osteoarthritis down-stream of TGF-120573rdquo Calcified Tissue International vol 86 no 1pp 47ndash57 2010

[3] T A Stupina V D Makushin and M A Stepanov ldquoExperi-mental morphological study of the effects of subchondral tun-nelization and bone marrow stimulation on articular cartilageregenerationrdquo Bulletin Experimental Biology and Medicine vol153 no 2 pp 289ndash293 2012

[4] MA JordanG SVanThiel J Chahal and S JNho ldquoOperativetreatment of chondral defects in the hip joint a systematicreviewrdquo Current Reviews in Musculoskeletal Medicine vol 5 no3 pp 244ndash253 2012

[5] O S Ertas and A Kayali ldquoAn overview on analytical methodvalidationrdquo Journal of Faculty Pharmacy vol 34 no 1 pp 41ndash57 2005

[6] G Szepesi M Gazdag and K Mihalyfi ldquoSelection of high-performance liquid chromatographic methods in pharmaceuti-cal analysis III Method validationrdquo Journal of ChromatographyA vol 464 no 2 pp 265ndash278 1989

[7] A Kayal O Sogut and B Tuncel ldquoSome considerations on ana-lytical methods validations bioavailability bioequivalence andpharmacokinetic studiesrdquoEuropean Journal of DrugMetabolismand Pharmacokinetics vol 20 no 4 pp 271ndash274 1995

[8] A Marin A Lopez-Gonzalvez and C Barbas ldquoDevelopmentand validation of extraction methods for determination ofzinc and arsenic speciation in soils using focused ultrasoundapplication to heavy metal study in mud and soilsrdquo AnalyticaChimica Acta vol 442 no 2 pp 305ndash318 2001

[9] J G Morrison P White S McDougall et al ldquoValidation ofa highly sensitive ICP-MS method for the determination ofplatinum in biofluids application to clinical pharmacokineticstudies with oxaliplatinrdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 24 no 1 pp 1ndash10 2000

[10] V Grdinic and J Vukovic ldquoPrevalidation in pharmaceuticalanalysis part 1 fundamentals and critical discussionrdquo Journal ofPharmaceutical and Biomedical Analysis vol 35 no 1 pp 489ndash512 2004

[11] P Garnero J C Rousseau and P D Delmas ldquoMolecular basisand clinical use of biochemical markers of bone cartilage andsynovium in joint diseasesrdquoArthritis ampRheumatism vol 43 no1 pp 953ndash968 2000

[12] D A Walsh P Verghese G J Cook et al ldquoLymphatic vesselsin osteoarthritic human kneesrdquoOsteoarthritis Cartilage vol 20no 5 pp 405ndash412 2012

[13] Y Henrotin B Kurz and T Aigner ldquoOxygen and reactiveoxygen species in cartilage degradation friends or foesrdquoOsteoarthritis and Cartilage vol 13 no 8 pp 643ndash654 2005

[14] A Ostalowska E Birkner M Wiecha et al ldquoLipid peroxi-dation and antioxidant enzymes in synovial fluid of patientswith primary and secondary osteoarthritis of the knee jointrdquoOsteoarthritis and Cartilage vol 14 no 2 pp 139ndash145 2006

[15] K Haugbro J C Nossent T Winkler Y Figenschau and OP Rekvig ldquoAnti-dsDNA antibodies and disease classificationin antinuclear antibody positive patients the role of analyticaldiversityrdquo Annals of the Rheumatic Diseases vol 63 no 4 pp386ndash394 2004

[16] K J Jepsen F Wu J H Peragallo et al ldquoA syndrome ofjoint laxity and impaired tendon integrity in lumican- andfibromodulin-deficient micerdquo The Journal of Biological Chem-istry vol 277 no 38 pp 35532ndash35540 2002

[17] B E Gencosmanoglu M Eryavuz and S Dervisoglu ldquoEffectsof some nonsteroidal anti-inflammatory drugs on articularcartilage of rats in an experimental model of osteoarthritisrdquoResearch in Experimental Medicine vol 200 no 3 pp 215ndash2262001

[18] A J Hough ldquoPathology of osteoarthritisrdquo inArthritis and AlliedConditions W J Koopman Ed pp 1945ndash1968 Williams ampWilkins Baltimore Md USA 1997

[19] J D Sandy C R Flannery P J Neame and L S LohmanderldquoThe structure of aggrecan fragments in human synovial fluidEvidence for the involvement in osteoarthritis of a novelproteinase which cleaves the Glu 373-Ala 374 bond of theinterglobular domainrdquoThe Journal of Clinical Investigation vol89 no 5 pp 1512ndash1516 1992

[20] S Tseng A H Reddi and P E Di Cesare ldquoCartilage oligomericmatrix protein (COMP) a biomarker of arthritisrdquo BiomarkerInsights vol 4 pp 33ndash44 2009

[21] L S Lohmander L A Hoerrner and M W Lark ldquoMet-alloproteinases tissue inhibitor and proteoglycan fragmentsin knee synovial fluid in human osteoarthritisrdquo Arthritis ampRheumatism vol 36 no 2 pp 181ndash189 1993

[22] L S Lohmander H Roos L Dahlberg L A Hoerrnerand M W Lark ldquoTemporal patterns of stromelysin-1 tissue

Arthritis 7

inhibitor and proteoglycan fragments in human knee joint fluidafter injury to the cruciate ligament or meniscusrdquo Journal ofOrthopaedic Research vol 12 no 1 pp 21ndash28 1994

[23] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Immunol-ogy vol 14 pp 397ndash440 1996

[24] L Connell and I B McInnes ldquoNew cytokine targets in inflam-matory rheumatic diseasesrdquo Best Practice amp Research vol 20no 5 pp 865ndash878 2006

[25] M Sharif E George L Shepstone et al ldquoSerumhyaluronic acidlevel as a predictor of disease progression in osteoarthritis of thekneerdquo Arthritis amp Rheumatism vol 38 no 6 pp 760ndash767 1995

[26] C Melchiorri R Meliconi L Frizziero et al ldquoEnhancedand coordinated in vivo expression of inflammatory cytokinesand nitric oxide synthase by chondrocytes from patients withosteoarthritisrdquoArthritis amp Rheumatism vol 41 no 12 pp 2165ndash2174 1998

[27] R Hilf J L Wittliff W D Rector E D Savlov T C Halland R A Orlando ldquoStudies on certain cytoplasmic enzymesand specific estrogen receptors in human breast cancer and innonmalignant diseases of the breastrdquo Cancer Research vol 33no 9 pp 2054ndash2062 1973

[28] F A Van de Loo O J Arntz F H van Enckevort P L van Lentand W B van den Berg ldquoReduced cartilage proteoglycan lossduring zymosan-induced gonarthritis in NOS

2-deficient mice

and in anti-interleukin-1-treated wild-type mice with unabatedjoint inflammationrdquo Arthritis amp Rheumatism vol 41 no 4 pp634ndash646 1998

[29] R F Loeser ldquoIntegrins and cell signaling in chondrocytesrdquo Bi-orheology vol 39 no 1-2 pp 119ndash124 2002

[30] A R Poole F Guilak and S B Abramson ldquoEtiopathogenesisof osteoarthritisrdquo in Osteoarthritis Diagnosis and MEdicalSur-gical Management R WMoskowitz Ed pp 27ndash49 LippincottPhiladelphia Pa USA 4th edition 2007

[31] S R Goldring and M B Goldring ldquoThe role of cytokines incartilage matrix degeneration in osteoarthritisrdquo Clinical Ortho-paedics and Related Research no 427 supplement pp S27ndashS362004

[32] S Kamekura K Hoshi T Shimoaka et al ldquoOsteoarthritisdevelopment in novel experimental mouse models induced byknee joint instabilityrdquoOsteoarthritis and Cartilage vol 13 no 7pp 632ndash641 2005

[33] A R Poole M Kobayashi T Yasuda et al ldquoType II col-lagen degradation and its regulation in articular cartilage inosteoarthritisrdquo Annals of the Rheumatic Diseases vol 61 sup-plement 2 pp 78ndash81 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Arthritis 3

Figure 2 Mechanical disruption of the tissue which is frozen in thepresence of liquid nitrogen

the rate of 1 20 (wv) and transferred into eppendorf tubesby means of 15 no scalpel end Primarily 400120583L lysisbufferwas added on them Then a protease cocktail inhibitor tentimes the ready volume (making 1x the last concentration) isadded and vortexed for 1 minute and centrifuging processwas performed at +4∘C for 5minutes in 10000 (g) [15 16]Thesamples were kept at minus80∘C for one night For themechanicaldisruption of the cartilage tissue they were subjected to 2times vibration and ultrasonic sound waves operation for 10minutes under the same pressure at intervals of 20 minutesin sonicator by watering down with phosphate buffer saline(PBS pH = 7 4) in 15mLmicrotubes (max 400 120583L of samplecontent) And then in line with the company bulletins thesamples were placed in the wells MMP-9 MMP-13 TIMP-1and TNF-120572 450 nm NO was evaluated by reading at ELISAmicroplate reader at 540 nm wave length as a result of thecalculation of the data which was obtained over the nitrateand nitrite values Standards were studies as pairs

24 Histopathological Analyses In order to prove the car-tilage complete layer injury the cartilage tissue sampleswhich were taken from each of 6 rats from both groupswere also evaluated histopathologically The samples wereburied in paraffin-embedded blocks after the routine tissuemonitoring Five-micron sections were put to hematoxylin-eosin staining and examined in the light microscope (times100)Histological sections were examined in terms of 8 parametersand scored and evaluated [17]

25 Statistical Analysis Thedata was evaluated by using SPSS180 Package program Descriptive statistics were calculated(mean standard deviation) after performing data controlStudents t-test which is a test in the comparison of thequantitative data in the comparative analyses between theexperimental and control groups in which the measurementvariables between two groups are compared was appliedPartial correlation analysis was performed in order to explainthe relationship between thesemediators ReceiverOperatingCharacteristics (ROC) curves were used in the determinationof diagnostic sufficiency from the tissue

3 Results

31 Histopathological Evaluation The formation of the carti-lage complete layer injury was reported (Figure 3)

32 Statistical Evaluation All of the evaluations were eval-uated in 95 confidence interval and bilaterally Alphameaningfulness level was determined as le005 accordingto the specific activity results in synovial lavage fluid andcartilage tissue of the mediators (MMP-9 MMP-13 NOTIMP-1 and TNF-120572) emerging after complete layer articularcartilage injury

No meaningful difference was determined in statisticalterms between the TIMP-1 (119875 = 067) and MMP-9 (119875 =028) levels in synovial lavage fluid and cartilage tissue Ameaningful difference was found in statistical terms betweenthe MMP-13 (119875 lt 00001) TNF-120572 (119875 lt 00001) and NO(119875 lt 00001) levels (Table 1)

It was determined that the measurements in intragroupand intergroup TIMP-1 andMMP-9 mediators were variableand close measurements were obtained in NO TIMP-1 andTNF-120572 variables (Figure 4)

In the partial correlation analysis performed a negativelinear meaningful relationship was found between TIMP-1and MMP-13 (119875 lt 00001 119903 = minus038) a positive meaningfulrelationship was found between TIMP-1 and TNF-120572 (119875 lt00001 119903 = 033) A negative meaningful relationship wasfound between TIMP-1 and NO (119875 = 0001 119903 = minus026) Anda positive linear meaningful relationship was found betweenMMP-9 and TNF-120572 (119875 lt 00001 119903 = 034) and NO (119875 lt00001 119903 = 046) (Table 2)

When the area under ROC curve of TIMP-1 MMP-9MMP-13 NO and TNF-120572 markers and P values were exam-ined in the study groups MMP-13 (119875 lt 00001 95 CI070ndash085) NO (119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 00001 95 CI 091ndash098) results were found to bestatistically meaningful (Figure 5)

4 Discussion

Osteoartrit (OA) is a disease which emerges with the breakupof the balance between the construction and destruction ofthe cartilage in favour of destruction results in the loss ofthe cartilage and progresses slowly [18] Recently it has beenshown that the MMPs whose levels in the joint at the timeof injury increase play an important role in the destructionof the cartilage tissue [19] One of the tissue inhibitorswhich enable the balancing of these enzymes which aredestructive for the cartilage is identified as metalloproteinase[20] Particularly the metalloproteinase which increases inOA is produced by chondrocyte and synovial cells Sincethe number of these cells is higher than that in the cartilagetissue the main production source of MMP in the joint fluidis synovial tissue It is shown that metalloproteinase andTIMP increased in the synovial fluid in OA and collagenase-TIMP-1 complex level generally stayed under the level whichcan be determined by immunoassay [21] Lohmander et aldetermined that metalloproteinase and TIMP levels stayed

4 Arthritis

(a) (b)

(c) (d)

Figure 3 It is seen that complete layer of cartilage injury is histopathologically formed in the presence of HampE (times100) (a) Intact (b)fibrillation (c) fissure and (d) osteophyte formation

Table 1 Evaluating MMP-9 MMP-13 NO TIMP-1 and TNF-alpha levels in synovial lavage fluid and cartilage tissue types

Material 119873 Mean Standard deviation 119875

TIMP-1 Synovial lavage fluid 84 301 261 067Cartilage Tissue 84 283 259

MMP-9 Synovial lavage fluid 90 592 163 028Cartilage Tissue 90 566 167

MMP-13 Synovial lavage fluid 90 068 023lt0001

Cartilage Tissue 90 047 024

TNF-120572 Synovial lavage fluid 84 032 009lt0001

Cartilage Tissue 84 014 007

NO Synovial lavage fluid 90 057 019lt0001

Cartilage Tissue 90 036 019

high for a long time in the knee joint after a traumatic injury[22]

In our study it was seen thatMMP-9 levels did not changeeven when different methodologies are used in synoviallavage fluid and cartilage tissue analyses in the samples whichwere examined after the formation of injury Likewise nochange was determined in TIMP-1 levels (Table 1)

The role of proinflammatory was shown in the articularcartilage injury of TNF-120572 which are among the cytokinesother than those aforementioned molecules [23 24] SerumTNF-120572 level is seen as a criterion of the synovial cellhyperactivity which becomes evident in the late stages of thecartilage injury [25] It is known that TNF-120572 in the jointwith OA mainly originates from the cells in the cartilage

tissue and these cells perform more cytokine expressionwhen compared to synovial cells [26]

In our study statistically meaningful differences weredetermined in terms of TNF-120572 and MMP-13 levels in thesamples which were obtained from synovial lavage fluid andcartilage tissue (Table 1) In the subsequent partial correla-tion analysis a negative meaningful relationship was foundbetween TIMP-1 and MMP-13 (119875 lt 00001 119903 = minus038)and a positive meaningful relationship was found betweenTIMP-1 and TNF-120572 (119875 lt 00001 119903 = 033) A positivelinear meaningful relationship was found between MMP-9 and TNF-120572 (119875 lt 00001 119903 = 034) (Table 2) It wasseen in ROC analysis that these two mediators can also beused in different methodologies in synovial lavage fluid and

Arthritis 5

Table 2 Partial Correlation analysis

Control variables TIMP-1 MMP-9 MMP-13 TNF-120572 NOMaterial

TIMP-1Correlation 100 minus014 minus038 033 minus026Significance (2-tailed) mdash 008 0000 0000 0001

MMP-9Correlation minus014 10 minus004 034 046Significance (2-tailed) 008 mdash 061 0000 0000

MMP-13Correlation minus038 minus004 1000 013 001Significance (2-tailed) 0000 061 mdash 009 088

TNF-120572Correlation 033 034 013 1000 minus004Significance (2-tailed) 0000 0000 009 mdash 059

NOCorrelation minus026 046 001 minus004 1000Significance (2-tailed) 0001 0000 088 059 mdash

Syno

vial

flui

dM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Cart

ilage

tiss

ueM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Figure 4 Distribution of the mediators in the synovial lavage fluidand cartilage tissue in all experimental groups (mean 95 CI)

cartilage tissue The molecular marker level results which areobtained from different areas in the literature and set forthwith different methodologies are also in conformity with thedata of our study [25ndash27]

In an experimental study it was it shown that NOsynthesis are stimulated by several cytokines such as TNF-120572 [28] Evidences regarding the fact that NO is a secondaryfundamental mediator after TNF-120572 in cartilage cells wereset forth in the same study [28] In our study on the otherhand NO level was determined to be meaningfully high inthe analysis results of the samples taken from synovial lavage

1080604020

1

08

06

04

02

0

Sens

itivi

ty

Specifity

ROC curve

Diagonal segments are produced by ties

Source of the curveTIMP-1TIMP-9MMP-13

TNF-120572NOReference line

Figure 5 ROC analysis where the validities of the mediators insynovial lavage fluid and cartilage tissue is evaluated

fluid in conformity with the TNF-120572 level in the experimentalgroups A positive linear relationship was found betweenTNF-120572 and NO (119875 lt 00001 119903 = 046) In the ROC analysisthe fact that these mediators which were taken from differentplaces and examined can also be analysed over a differentmethodology cartilage tissue was confirmed

6 Arthritis

The affect of MMP enzymes on cartilage is inhibited byTIMP [29 30] NO is overexpressed at synovial tissue ofinjured cartilage by the stimulus of inducible nitric oxide syn-thase andTNF-120572 [29ndash32] IL-1 andTNF-120572 release is increasedwith the affect of degraded matrix molecules and so NOexpression is also increased It is reported that MMP-13 isdischarged mostly via sinovial fluid [33] We think that thesedata explain why TNF-120572 MMP-13 and NOwere foundmorein synovial fluids in our results

We think the restriction of our study is that it cannot becrosschecked by a method other than ELISA method whichis used in the analysis of the samples

5 Conclusion

When among the molecular markers which are analyzedin the synovial lavage fluid MMP-9 MMP-13 NO TIMP-1and TNF-120572 the area under the ROC curve and P values areevaluated MMP-13 (119875 lt 00001 95 CI 070ndash085) NO(119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 0000195 CI 091ndash098) results are statistically significant As aresult as it can be analyzed from the synovial lavage fluid ofthe molecular injury markers such as MMP-9 MMP-13 NOTIMP-1 and TNF-120572 which emerge in the articular cartilageinjury we can say that it can also be analyzed in a reliablemanner by the indirect ELISAmethod described by us in thesamples which are obtained from the cartilage tissue

Acknowledgment

Theauthors thank theTurkishOrthopedic Society (TOTBID)for the grants in support of this study

References

[1] G Nagyeri M Radacs S Ghassemi-Nejad et al ldquoTSG-6protein a negative regulator of inflammatory arthritis forms aternary complex with murine mast cell tryptases and heparinrdquoThe Journal of Biological Chemistry vol 286 no 26 pp 23559ndash23569 2011

[2] K Tsuritani J Takeda J Sakagami et al ldquoCytokine receptor-like factor 1 is highly expressed in damaged human kneeosteoarthritic cartilage and involved in osteoarthritis down-stream of TGF-120573rdquo Calcified Tissue International vol 86 no 1pp 47ndash57 2010

[3] T A Stupina V D Makushin and M A Stepanov ldquoExperi-mental morphological study of the effects of subchondral tun-nelization and bone marrow stimulation on articular cartilageregenerationrdquo Bulletin Experimental Biology and Medicine vol153 no 2 pp 289ndash293 2012

[4] MA JordanG SVanThiel J Chahal and S JNho ldquoOperativetreatment of chondral defects in the hip joint a systematicreviewrdquo Current Reviews in Musculoskeletal Medicine vol 5 no3 pp 244ndash253 2012

[5] O S Ertas and A Kayali ldquoAn overview on analytical methodvalidationrdquo Journal of Faculty Pharmacy vol 34 no 1 pp 41ndash57 2005

[6] G Szepesi M Gazdag and K Mihalyfi ldquoSelection of high-performance liquid chromatographic methods in pharmaceuti-cal analysis III Method validationrdquo Journal of ChromatographyA vol 464 no 2 pp 265ndash278 1989

[7] A Kayal O Sogut and B Tuncel ldquoSome considerations on ana-lytical methods validations bioavailability bioequivalence andpharmacokinetic studiesrdquoEuropean Journal of DrugMetabolismand Pharmacokinetics vol 20 no 4 pp 271ndash274 1995

[8] A Marin A Lopez-Gonzalvez and C Barbas ldquoDevelopmentand validation of extraction methods for determination ofzinc and arsenic speciation in soils using focused ultrasoundapplication to heavy metal study in mud and soilsrdquo AnalyticaChimica Acta vol 442 no 2 pp 305ndash318 2001

[9] J G Morrison P White S McDougall et al ldquoValidation ofa highly sensitive ICP-MS method for the determination ofplatinum in biofluids application to clinical pharmacokineticstudies with oxaliplatinrdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 24 no 1 pp 1ndash10 2000

[10] V Grdinic and J Vukovic ldquoPrevalidation in pharmaceuticalanalysis part 1 fundamentals and critical discussionrdquo Journal ofPharmaceutical and Biomedical Analysis vol 35 no 1 pp 489ndash512 2004

[11] P Garnero J C Rousseau and P D Delmas ldquoMolecular basisand clinical use of biochemical markers of bone cartilage andsynovium in joint diseasesrdquoArthritis ampRheumatism vol 43 no1 pp 953ndash968 2000

[12] D A Walsh P Verghese G J Cook et al ldquoLymphatic vesselsin osteoarthritic human kneesrdquoOsteoarthritis Cartilage vol 20no 5 pp 405ndash412 2012

[13] Y Henrotin B Kurz and T Aigner ldquoOxygen and reactiveoxygen species in cartilage degradation friends or foesrdquoOsteoarthritis and Cartilage vol 13 no 8 pp 643ndash654 2005

[14] A Ostalowska E Birkner M Wiecha et al ldquoLipid peroxi-dation and antioxidant enzymes in synovial fluid of patientswith primary and secondary osteoarthritis of the knee jointrdquoOsteoarthritis and Cartilage vol 14 no 2 pp 139ndash145 2006

[15] K Haugbro J C Nossent T Winkler Y Figenschau and OP Rekvig ldquoAnti-dsDNA antibodies and disease classificationin antinuclear antibody positive patients the role of analyticaldiversityrdquo Annals of the Rheumatic Diseases vol 63 no 4 pp386ndash394 2004

[16] K J Jepsen F Wu J H Peragallo et al ldquoA syndrome ofjoint laxity and impaired tendon integrity in lumican- andfibromodulin-deficient micerdquo The Journal of Biological Chem-istry vol 277 no 38 pp 35532ndash35540 2002

[17] B E Gencosmanoglu M Eryavuz and S Dervisoglu ldquoEffectsof some nonsteroidal anti-inflammatory drugs on articularcartilage of rats in an experimental model of osteoarthritisrdquoResearch in Experimental Medicine vol 200 no 3 pp 215ndash2262001

[18] A J Hough ldquoPathology of osteoarthritisrdquo inArthritis and AlliedConditions W J Koopman Ed pp 1945ndash1968 Williams ampWilkins Baltimore Md USA 1997

[19] J D Sandy C R Flannery P J Neame and L S LohmanderldquoThe structure of aggrecan fragments in human synovial fluidEvidence for the involvement in osteoarthritis of a novelproteinase which cleaves the Glu 373-Ala 374 bond of theinterglobular domainrdquoThe Journal of Clinical Investigation vol89 no 5 pp 1512ndash1516 1992

[20] S Tseng A H Reddi and P E Di Cesare ldquoCartilage oligomericmatrix protein (COMP) a biomarker of arthritisrdquo BiomarkerInsights vol 4 pp 33ndash44 2009

[21] L S Lohmander L A Hoerrner and M W Lark ldquoMet-alloproteinases tissue inhibitor and proteoglycan fragmentsin knee synovial fluid in human osteoarthritisrdquo Arthritis ampRheumatism vol 36 no 2 pp 181ndash189 1993

[22] L S Lohmander H Roos L Dahlberg L A Hoerrnerand M W Lark ldquoTemporal patterns of stromelysin-1 tissue

Arthritis 7

inhibitor and proteoglycan fragments in human knee joint fluidafter injury to the cruciate ligament or meniscusrdquo Journal ofOrthopaedic Research vol 12 no 1 pp 21ndash28 1994

[23] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Immunol-ogy vol 14 pp 397ndash440 1996

[24] L Connell and I B McInnes ldquoNew cytokine targets in inflam-matory rheumatic diseasesrdquo Best Practice amp Research vol 20no 5 pp 865ndash878 2006

[25] M Sharif E George L Shepstone et al ldquoSerumhyaluronic acidlevel as a predictor of disease progression in osteoarthritis of thekneerdquo Arthritis amp Rheumatism vol 38 no 6 pp 760ndash767 1995

[26] C Melchiorri R Meliconi L Frizziero et al ldquoEnhancedand coordinated in vivo expression of inflammatory cytokinesand nitric oxide synthase by chondrocytes from patients withosteoarthritisrdquoArthritis amp Rheumatism vol 41 no 12 pp 2165ndash2174 1998

[27] R Hilf J L Wittliff W D Rector E D Savlov T C Halland R A Orlando ldquoStudies on certain cytoplasmic enzymesand specific estrogen receptors in human breast cancer and innonmalignant diseases of the breastrdquo Cancer Research vol 33no 9 pp 2054ndash2062 1973

[28] F A Van de Loo O J Arntz F H van Enckevort P L van Lentand W B van den Berg ldquoReduced cartilage proteoglycan lossduring zymosan-induced gonarthritis in NOS

2-deficient mice

and in anti-interleukin-1-treated wild-type mice with unabatedjoint inflammationrdquo Arthritis amp Rheumatism vol 41 no 4 pp634ndash646 1998

[29] R F Loeser ldquoIntegrins and cell signaling in chondrocytesrdquo Bi-orheology vol 39 no 1-2 pp 119ndash124 2002

[30] A R Poole F Guilak and S B Abramson ldquoEtiopathogenesisof osteoarthritisrdquo in Osteoarthritis Diagnosis and MEdicalSur-gical Management R WMoskowitz Ed pp 27ndash49 LippincottPhiladelphia Pa USA 4th edition 2007

[31] S R Goldring and M B Goldring ldquoThe role of cytokines incartilage matrix degeneration in osteoarthritisrdquo Clinical Ortho-paedics and Related Research no 427 supplement pp S27ndashS362004

[32] S Kamekura K Hoshi T Shimoaka et al ldquoOsteoarthritisdevelopment in novel experimental mouse models induced byknee joint instabilityrdquoOsteoarthritis and Cartilage vol 13 no 7pp 632ndash641 2005

[33] A R Poole M Kobayashi T Yasuda et al ldquoType II col-lagen degradation and its regulation in articular cartilage inosteoarthritisrdquo Annals of the Rheumatic Diseases vol 61 sup-plement 2 pp 78ndash81 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Arthritis

(a) (b)

(c) (d)

Figure 3 It is seen that complete layer of cartilage injury is histopathologically formed in the presence of HampE (times100) (a) Intact (b)fibrillation (c) fissure and (d) osteophyte formation

Table 1 Evaluating MMP-9 MMP-13 NO TIMP-1 and TNF-alpha levels in synovial lavage fluid and cartilage tissue types

Material 119873 Mean Standard deviation 119875

TIMP-1 Synovial lavage fluid 84 301 261 067Cartilage Tissue 84 283 259

MMP-9 Synovial lavage fluid 90 592 163 028Cartilage Tissue 90 566 167

MMP-13 Synovial lavage fluid 90 068 023lt0001

Cartilage Tissue 90 047 024

TNF-120572 Synovial lavage fluid 84 032 009lt0001

Cartilage Tissue 84 014 007

NO Synovial lavage fluid 90 057 019lt0001

Cartilage Tissue 90 036 019

high for a long time in the knee joint after a traumatic injury[22]

In our study it was seen thatMMP-9 levels did not changeeven when different methodologies are used in synoviallavage fluid and cartilage tissue analyses in the samples whichwere examined after the formation of injury Likewise nochange was determined in TIMP-1 levels (Table 1)

The role of proinflammatory was shown in the articularcartilage injury of TNF-120572 which are among the cytokinesother than those aforementioned molecules [23 24] SerumTNF-120572 level is seen as a criterion of the synovial cellhyperactivity which becomes evident in the late stages of thecartilage injury [25] It is known that TNF-120572 in the jointwith OA mainly originates from the cells in the cartilage

tissue and these cells perform more cytokine expressionwhen compared to synovial cells [26]

In our study statistically meaningful differences weredetermined in terms of TNF-120572 and MMP-13 levels in thesamples which were obtained from synovial lavage fluid andcartilage tissue (Table 1) In the subsequent partial correla-tion analysis a negative meaningful relationship was foundbetween TIMP-1 and MMP-13 (119875 lt 00001 119903 = minus038)and a positive meaningful relationship was found betweenTIMP-1 and TNF-120572 (119875 lt 00001 119903 = 033) A positivelinear meaningful relationship was found between MMP-9 and TNF-120572 (119875 lt 00001 119903 = 034) (Table 2) It wasseen in ROC analysis that these two mediators can also beused in different methodologies in synovial lavage fluid and

Arthritis 5

Table 2 Partial Correlation analysis

Control variables TIMP-1 MMP-9 MMP-13 TNF-120572 NOMaterial

TIMP-1Correlation 100 minus014 minus038 033 minus026Significance (2-tailed) mdash 008 0000 0000 0001

MMP-9Correlation minus014 10 minus004 034 046Significance (2-tailed) 008 mdash 061 0000 0000

MMP-13Correlation minus038 minus004 1000 013 001Significance (2-tailed) 0000 061 mdash 009 088

TNF-120572Correlation 033 034 013 1000 minus004Significance (2-tailed) 0000 0000 009 mdash 059

NOCorrelation minus026 046 001 minus004 1000Significance (2-tailed) 0001 0000 088 059 mdash

Syno

vial

flui

dM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Cart

ilage

tiss

ueM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Figure 4 Distribution of the mediators in the synovial lavage fluidand cartilage tissue in all experimental groups (mean 95 CI)

cartilage tissue The molecular marker level results which areobtained from different areas in the literature and set forthwith different methodologies are also in conformity with thedata of our study [25ndash27]

In an experimental study it was it shown that NOsynthesis are stimulated by several cytokines such as TNF-120572 [28] Evidences regarding the fact that NO is a secondaryfundamental mediator after TNF-120572 in cartilage cells wereset forth in the same study [28] In our study on the otherhand NO level was determined to be meaningfully high inthe analysis results of the samples taken from synovial lavage

1080604020

1

08

06

04

02

0

Sens

itivi

ty

Specifity

ROC curve

Diagonal segments are produced by ties

Source of the curveTIMP-1TIMP-9MMP-13

TNF-120572NOReference line

Figure 5 ROC analysis where the validities of the mediators insynovial lavage fluid and cartilage tissue is evaluated

fluid in conformity with the TNF-120572 level in the experimentalgroups A positive linear relationship was found betweenTNF-120572 and NO (119875 lt 00001 119903 = 046) In the ROC analysisthe fact that these mediators which were taken from differentplaces and examined can also be analysed over a differentmethodology cartilage tissue was confirmed

6 Arthritis

The affect of MMP enzymes on cartilage is inhibited byTIMP [29 30] NO is overexpressed at synovial tissue ofinjured cartilage by the stimulus of inducible nitric oxide syn-thase andTNF-120572 [29ndash32] IL-1 andTNF-120572 release is increasedwith the affect of degraded matrix molecules and so NOexpression is also increased It is reported that MMP-13 isdischarged mostly via sinovial fluid [33] We think that thesedata explain why TNF-120572 MMP-13 and NOwere foundmorein synovial fluids in our results

We think the restriction of our study is that it cannot becrosschecked by a method other than ELISA method whichis used in the analysis of the samples

5 Conclusion

When among the molecular markers which are analyzedin the synovial lavage fluid MMP-9 MMP-13 NO TIMP-1and TNF-120572 the area under the ROC curve and P values areevaluated MMP-13 (119875 lt 00001 95 CI 070ndash085) NO(119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 0000195 CI 091ndash098) results are statistically significant As aresult as it can be analyzed from the synovial lavage fluid ofthe molecular injury markers such as MMP-9 MMP-13 NOTIMP-1 and TNF-120572 which emerge in the articular cartilageinjury we can say that it can also be analyzed in a reliablemanner by the indirect ELISAmethod described by us in thesamples which are obtained from the cartilage tissue

Acknowledgment

Theauthors thank theTurkishOrthopedic Society (TOTBID)for the grants in support of this study

References

[1] G Nagyeri M Radacs S Ghassemi-Nejad et al ldquoTSG-6protein a negative regulator of inflammatory arthritis forms aternary complex with murine mast cell tryptases and heparinrdquoThe Journal of Biological Chemistry vol 286 no 26 pp 23559ndash23569 2011

[2] K Tsuritani J Takeda J Sakagami et al ldquoCytokine receptor-like factor 1 is highly expressed in damaged human kneeosteoarthritic cartilage and involved in osteoarthritis down-stream of TGF-120573rdquo Calcified Tissue International vol 86 no 1pp 47ndash57 2010

[3] T A Stupina V D Makushin and M A Stepanov ldquoExperi-mental morphological study of the effects of subchondral tun-nelization and bone marrow stimulation on articular cartilageregenerationrdquo Bulletin Experimental Biology and Medicine vol153 no 2 pp 289ndash293 2012

[4] MA JordanG SVanThiel J Chahal and S JNho ldquoOperativetreatment of chondral defects in the hip joint a systematicreviewrdquo Current Reviews in Musculoskeletal Medicine vol 5 no3 pp 244ndash253 2012

[5] O S Ertas and A Kayali ldquoAn overview on analytical methodvalidationrdquo Journal of Faculty Pharmacy vol 34 no 1 pp 41ndash57 2005

[6] G Szepesi M Gazdag and K Mihalyfi ldquoSelection of high-performance liquid chromatographic methods in pharmaceuti-cal analysis III Method validationrdquo Journal of ChromatographyA vol 464 no 2 pp 265ndash278 1989

[7] A Kayal O Sogut and B Tuncel ldquoSome considerations on ana-lytical methods validations bioavailability bioequivalence andpharmacokinetic studiesrdquoEuropean Journal of DrugMetabolismand Pharmacokinetics vol 20 no 4 pp 271ndash274 1995

[8] A Marin A Lopez-Gonzalvez and C Barbas ldquoDevelopmentand validation of extraction methods for determination ofzinc and arsenic speciation in soils using focused ultrasoundapplication to heavy metal study in mud and soilsrdquo AnalyticaChimica Acta vol 442 no 2 pp 305ndash318 2001

[9] J G Morrison P White S McDougall et al ldquoValidation ofa highly sensitive ICP-MS method for the determination ofplatinum in biofluids application to clinical pharmacokineticstudies with oxaliplatinrdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 24 no 1 pp 1ndash10 2000

[10] V Grdinic and J Vukovic ldquoPrevalidation in pharmaceuticalanalysis part 1 fundamentals and critical discussionrdquo Journal ofPharmaceutical and Biomedical Analysis vol 35 no 1 pp 489ndash512 2004

[11] P Garnero J C Rousseau and P D Delmas ldquoMolecular basisand clinical use of biochemical markers of bone cartilage andsynovium in joint diseasesrdquoArthritis ampRheumatism vol 43 no1 pp 953ndash968 2000

[12] D A Walsh P Verghese G J Cook et al ldquoLymphatic vesselsin osteoarthritic human kneesrdquoOsteoarthritis Cartilage vol 20no 5 pp 405ndash412 2012

[13] Y Henrotin B Kurz and T Aigner ldquoOxygen and reactiveoxygen species in cartilage degradation friends or foesrdquoOsteoarthritis and Cartilage vol 13 no 8 pp 643ndash654 2005

[14] A Ostalowska E Birkner M Wiecha et al ldquoLipid peroxi-dation and antioxidant enzymes in synovial fluid of patientswith primary and secondary osteoarthritis of the knee jointrdquoOsteoarthritis and Cartilage vol 14 no 2 pp 139ndash145 2006

[15] K Haugbro J C Nossent T Winkler Y Figenschau and OP Rekvig ldquoAnti-dsDNA antibodies and disease classificationin antinuclear antibody positive patients the role of analyticaldiversityrdquo Annals of the Rheumatic Diseases vol 63 no 4 pp386ndash394 2004

[16] K J Jepsen F Wu J H Peragallo et al ldquoA syndrome ofjoint laxity and impaired tendon integrity in lumican- andfibromodulin-deficient micerdquo The Journal of Biological Chem-istry vol 277 no 38 pp 35532ndash35540 2002

[17] B E Gencosmanoglu M Eryavuz and S Dervisoglu ldquoEffectsof some nonsteroidal anti-inflammatory drugs on articularcartilage of rats in an experimental model of osteoarthritisrdquoResearch in Experimental Medicine vol 200 no 3 pp 215ndash2262001

[18] A J Hough ldquoPathology of osteoarthritisrdquo inArthritis and AlliedConditions W J Koopman Ed pp 1945ndash1968 Williams ampWilkins Baltimore Md USA 1997

[19] J D Sandy C R Flannery P J Neame and L S LohmanderldquoThe structure of aggrecan fragments in human synovial fluidEvidence for the involvement in osteoarthritis of a novelproteinase which cleaves the Glu 373-Ala 374 bond of theinterglobular domainrdquoThe Journal of Clinical Investigation vol89 no 5 pp 1512ndash1516 1992

[20] S Tseng A H Reddi and P E Di Cesare ldquoCartilage oligomericmatrix protein (COMP) a biomarker of arthritisrdquo BiomarkerInsights vol 4 pp 33ndash44 2009

[21] L S Lohmander L A Hoerrner and M W Lark ldquoMet-alloproteinases tissue inhibitor and proteoglycan fragmentsin knee synovial fluid in human osteoarthritisrdquo Arthritis ampRheumatism vol 36 no 2 pp 181ndash189 1993

[22] L S Lohmander H Roos L Dahlberg L A Hoerrnerand M W Lark ldquoTemporal patterns of stromelysin-1 tissue

Arthritis 7

inhibitor and proteoglycan fragments in human knee joint fluidafter injury to the cruciate ligament or meniscusrdquo Journal ofOrthopaedic Research vol 12 no 1 pp 21ndash28 1994

[23] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Immunol-ogy vol 14 pp 397ndash440 1996

[24] L Connell and I B McInnes ldquoNew cytokine targets in inflam-matory rheumatic diseasesrdquo Best Practice amp Research vol 20no 5 pp 865ndash878 2006

[25] M Sharif E George L Shepstone et al ldquoSerumhyaluronic acidlevel as a predictor of disease progression in osteoarthritis of thekneerdquo Arthritis amp Rheumatism vol 38 no 6 pp 760ndash767 1995

[26] C Melchiorri R Meliconi L Frizziero et al ldquoEnhancedand coordinated in vivo expression of inflammatory cytokinesand nitric oxide synthase by chondrocytes from patients withosteoarthritisrdquoArthritis amp Rheumatism vol 41 no 12 pp 2165ndash2174 1998

[27] R Hilf J L Wittliff W D Rector E D Savlov T C Halland R A Orlando ldquoStudies on certain cytoplasmic enzymesand specific estrogen receptors in human breast cancer and innonmalignant diseases of the breastrdquo Cancer Research vol 33no 9 pp 2054ndash2062 1973

[28] F A Van de Loo O J Arntz F H van Enckevort P L van Lentand W B van den Berg ldquoReduced cartilage proteoglycan lossduring zymosan-induced gonarthritis in NOS

2-deficient mice

and in anti-interleukin-1-treated wild-type mice with unabatedjoint inflammationrdquo Arthritis amp Rheumatism vol 41 no 4 pp634ndash646 1998

[29] R F Loeser ldquoIntegrins and cell signaling in chondrocytesrdquo Bi-orheology vol 39 no 1-2 pp 119ndash124 2002

[30] A R Poole F Guilak and S B Abramson ldquoEtiopathogenesisof osteoarthritisrdquo in Osteoarthritis Diagnosis and MEdicalSur-gical Management R WMoskowitz Ed pp 27ndash49 LippincottPhiladelphia Pa USA 4th edition 2007

[31] S R Goldring and M B Goldring ldquoThe role of cytokines incartilage matrix degeneration in osteoarthritisrdquo Clinical Ortho-paedics and Related Research no 427 supplement pp S27ndashS362004

[32] S Kamekura K Hoshi T Shimoaka et al ldquoOsteoarthritisdevelopment in novel experimental mouse models induced byknee joint instabilityrdquoOsteoarthritis and Cartilage vol 13 no 7pp 632ndash641 2005

[33] A R Poole M Kobayashi T Yasuda et al ldquoType II col-lagen degradation and its regulation in articular cartilage inosteoarthritisrdquo Annals of the Rheumatic Diseases vol 61 sup-plement 2 pp 78ndash81 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Arthritis 5

Table 2 Partial Correlation analysis

Control variables TIMP-1 MMP-9 MMP-13 TNF-120572 NOMaterial

TIMP-1Correlation 100 minus014 minus038 033 minus026Significance (2-tailed) mdash 008 0000 0000 0001

MMP-9Correlation minus014 10 minus004 034 046Significance (2-tailed) 008 mdash 061 0000 0000

MMP-13Correlation minus038 minus004 1000 013 001Significance (2-tailed) 0000 061 mdash 009 088

TNF-120572Correlation 033 034 013 1000 minus004Significance (2-tailed) 0000 0000 009 mdash 059

NOCorrelation minus026 046 001 minus004 1000Significance (2-tailed) 0001 0000 088 059 mdash

Syno

vial

flui

dM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Cart

ilage

tiss

ueM

ater

ial

95

CI

0

2

4

6

TIMP-1 MMP-9 MMP-13 NOTNF-120572

Figure 4 Distribution of the mediators in the synovial lavage fluidand cartilage tissue in all experimental groups (mean 95 CI)

cartilage tissue The molecular marker level results which areobtained from different areas in the literature and set forthwith different methodologies are also in conformity with thedata of our study [25ndash27]

In an experimental study it was it shown that NOsynthesis are stimulated by several cytokines such as TNF-120572 [28] Evidences regarding the fact that NO is a secondaryfundamental mediator after TNF-120572 in cartilage cells wereset forth in the same study [28] In our study on the otherhand NO level was determined to be meaningfully high inthe analysis results of the samples taken from synovial lavage

1080604020

1

08

06

04

02

0

Sens

itivi

ty

Specifity

ROC curve

Diagonal segments are produced by ties

Source of the curveTIMP-1TIMP-9MMP-13

TNF-120572NOReference line

Figure 5 ROC analysis where the validities of the mediators insynovial lavage fluid and cartilage tissue is evaluated

fluid in conformity with the TNF-120572 level in the experimentalgroups A positive linear relationship was found betweenTNF-120572 and NO (119875 lt 00001 119903 = 046) In the ROC analysisthe fact that these mediators which were taken from differentplaces and examined can also be analysed over a differentmethodology cartilage tissue was confirmed

6 Arthritis

The affect of MMP enzymes on cartilage is inhibited byTIMP [29 30] NO is overexpressed at synovial tissue ofinjured cartilage by the stimulus of inducible nitric oxide syn-thase andTNF-120572 [29ndash32] IL-1 andTNF-120572 release is increasedwith the affect of degraded matrix molecules and so NOexpression is also increased It is reported that MMP-13 isdischarged mostly via sinovial fluid [33] We think that thesedata explain why TNF-120572 MMP-13 and NOwere foundmorein synovial fluids in our results

We think the restriction of our study is that it cannot becrosschecked by a method other than ELISA method whichis used in the analysis of the samples

5 Conclusion

When among the molecular markers which are analyzedin the synovial lavage fluid MMP-9 MMP-13 NO TIMP-1and TNF-120572 the area under the ROC curve and P values areevaluated MMP-13 (119875 lt 00001 95 CI 070ndash085) NO(119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 0000195 CI 091ndash098) results are statistically significant As aresult as it can be analyzed from the synovial lavage fluid ofthe molecular injury markers such as MMP-9 MMP-13 NOTIMP-1 and TNF-120572 which emerge in the articular cartilageinjury we can say that it can also be analyzed in a reliablemanner by the indirect ELISAmethod described by us in thesamples which are obtained from the cartilage tissue

Acknowledgment

Theauthors thank theTurkishOrthopedic Society (TOTBID)for the grants in support of this study

References

[1] G Nagyeri M Radacs S Ghassemi-Nejad et al ldquoTSG-6protein a negative regulator of inflammatory arthritis forms aternary complex with murine mast cell tryptases and heparinrdquoThe Journal of Biological Chemistry vol 286 no 26 pp 23559ndash23569 2011

[2] K Tsuritani J Takeda J Sakagami et al ldquoCytokine receptor-like factor 1 is highly expressed in damaged human kneeosteoarthritic cartilage and involved in osteoarthritis down-stream of TGF-120573rdquo Calcified Tissue International vol 86 no 1pp 47ndash57 2010

[3] T A Stupina V D Makushin and M A Stepanov ldquoExperi-mental morphological study of the effects of subchondral tun-nelization and bone marrow stimulation on articular cartilageregenerationrdquo Bulletin Experimental Biology and Medicine vol153 no 2 pp 289ndash293 2012

[4] MA JordanG SVanThiel J Chahal and S JNho ldquoOperativetreatment of chondral defects in the hip joint a systematicreviewrdquo Current Reviews in Musculoskeletal Medicine vol 5 no3 pp 244ndash253 2012

[5] O S Ertas and A Kayali ldquoAn overview on analytical methodvalidationrdquo Journal of Faculty Pharmacy vol 34 no 1 pp 41ndash57 2005

[6] G Szepesi M Gazdag and K Mihalyfi ldquoSelection of high-performance liquid chromatographic methods in pharmaceuti-cal analysis III Method validationrdquo Journal of ChromatographyA vol 464 no 2 pp 265ndash278 1989

[7] A Kayal O Sogut and B Tuncel ldquoSome considerations on ana-lytical methods validations bioavailability bioequivalence andpharmacokinetic studiesrdquoEuropean Journal of DrugMetabolismand Pharmacokinetics vol 20 no 4 pp 271ndash274 1995

[8] A Marin A Lopez-Gonzalvez and C Barbas ldquoDevelopmentand validation of extraction methods for determination ofzinc and arsenic speciation in soils using focused ultrasoundapplication to heavy metal study in mud and soilsrdquo AnalyticaChimica Acta vol 442 no 2 pp 305ndash318 2001

[9] J G Morrison P White S McDougall et al ldquoValidation ofa highly sensitive ICP-MS method for the determination ofplatinum in biofluids application to clinical pharmacokineticstudies with oxaliplatinrdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 24 no 1 pp 1ndash10 2000

[10] V Grdinic and J Vukovic ldquoPrevalidation in pharmaceuticalanalysis part 1 fundamentals and critical discussionrdquo Journal ofPharmaceutical and Biomedical Analysis vol 35 no 1 pp 489ndash512 2004

[11] P Garnero J C Rousseau and P D Delmas ldquoMolecular basisand clinical use of biochemical markers of bone cartilage andsynovium in joint diseasesrdquoArthritis ampRheumatism vol 43 no1 pp 953ndash968 2000

[12] D A Walsh P Verghese G J Cook et al ldquoLymphatic vesselsin osteoarthritic human kneesrdquoOsteoarthritis Cartilage vol 20no 5 pp 405ndash412 2012

[13] Y Henrotin B Kurz and T Aigner ldquoOxygen and reactiveoxygen species in cartilage degradation friends or foesrdquoOsteoarthritis and Cartilage vol 13 no 8 pp 643ndash654 2005

[14] A Ostalowska E Birkner M Wiecha et al ldquoLipid peroxi-dation and antioxidant enzymes in synovial fluid of patientswith primary and secondary osteoarthritis of the knee jointrdquoOsteoarthritis and Cartilage vol 14 no 2 pp 139ndash145 2006

[15] K Haugbro J C Nossent T Winkler Y Figenschau and OP Rekvig ldquoAnti-dsDNA antibodies and disease classificationin antinuclear antibody positive patients the role of analyticaldiversityrdquo Annals of the Rheumatic Diseases vol 63 no 4 pp386ndash394 2004

[16] K J Jepsen F Wu J H Peragallo et al ldquoA syndrome ofjoint laxity and impaired tendon integrity in lumican- andfibromodulin-deficient micerdquo The Journal of Biological Chem-istry vol 277 no 38 pp 35532ndash35540 2002

[17] B E Gencosmanoglu M Eryavuz and S Dervisoglu ldquoEffectsof some nonsteroidal anti-inflammatory drugs on articularcartilage of rats in an experimental model of osteoarthritisrdquoResearch in Experimental Medicine vol 200 no 3 pp 215ndash2262001

[18] A J Hough ldquoPathology of osteoarthritisrdquo inArthritis and AlliedConditions W J Koopman Ed pp 1945ndash1968 Williams ampWilkins Baltimore Md USA 1997

[19] J D Sandy C R Flannery P J Neame and L S LohmanderldquoThe structure of aggrecan fragments in human synovial fluidEvidence for the involvement in osteoarthritis of a novelproteinase which cleaves the Glu 373-Ala 374 bond of theinterglobular domainrdquoThe Journal of Clinical Investigation vol89 no 5 pp 1512ndash1516 1992

[20] S Tseng A H Reddi and P E Di Cesare ldquoCartilage oligomericmatrix protein (COMP) a biomarker of arthritisrdquo BiomarkerInsights vol 4 pp 33ndash44 2009

[21] L S Lohmander L A Hoerrner and M W Lark ldquoMet-alloproteinases tissue inhibitor and proteoglycan fragmentsin knee synovial fluid in human osteoarthritisrdquo Arthritis ampRheumatism vol 36 no 2 pp 181ndash189 1993

[22] L S Lohmander H Roos L Dahlberg L A Hoerrnerand M W Lark ldquoTemporal patterns of stromelysin-1 tissue

Arthritis 7

inhibitor and proteoglycan fragments in human knee joint fluidafter injury to the cruciate ligament or meniscusrdquo Journal ofOrthopaedic Research vol 12 no 1 pp 21ndash28 1994

[23] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Immunol-ogy vol 14 pp 397ndash440 1996

[24] L Connell and I B McInnes ldquoNew cytokine targets in inflam-matory rheumatic diseasesrdquo Best Practice amp Research vol 20no 5 pp 865ndash878 2006

[25] M Sharif E George L Shepstone et al ldquoSerumhyaluronic acidlevel as a predictor of disease progression in osteoarthritis of thekneerdquo Arthritis amp Rheumatism vol 38 no 6 pp 760ndash767 1995

[26] C Melchiorri R Meliconi L Frizziero et al ldquoEnhancedand coordinated in vivo expression of inflammatory cytokinesand nitric oxide synthase by chondrocytes from patients withosteoarthritisrdquoArthritis amp Rheumatism vol 41 no 12 pp 2165ndash2174 1998

[27] R Hilf J L Wittliff W D Rector E D Savlov T C Halland R A Orlando ldquoStudies on certain cytoplasmic enzymesand specific estrogen receptors in human breast cancer and innonmalignant diseases of the breastrdquo Cancer Research vol 33no 9 pp 2054ndash2062 1973

[28] F A Van de Loo O J Arntz F H van Enckevort P L van Lentand W B van den Berg ldquoReduced cartilage proteoglycan lossduring zymosan-induced gonarthritis in NOS

2-deficient mice

and in anti-interleukin-1-treated wild-type mice with unabatedjoint inflammationrdquo Arthritis amp Rheumatism vol 41 no 4 pp634ndash646 1998

[29] R F Loeser ldquoIntegrins and cell signaling in chondrocytesrdquo Bi-orheology vol 39 no 1-2 pp 119ndash124 2002

[30] A R Poole F Guilak and S B Abramson ldquoEtiopathogenesisof osteoarthritisrdquo in Osteoarthritis Diagnosis and MEdicalSur-gical Management R WMoskowitz Ed pp 27ndash49 LippincottPhiladelphia Pa USA 4th edition 2007

[31] S R Goldring and M B Goldring ldquoThe role of cytokines incartilage matrix degeneration in osteoarthritisrdquo Clinical Ortho-paedics and Related Research no 427 supplement pp S27ndashS362004

[32] S Kamekura K Hoshi T Shimoaka et al ldquoOsteoarthritisdevelopment in novel experimental mouse models induced byknee joint instabilityrdquoOsteoarthritis and Cartilage vol 13 no 7pp 632ndash641 2005

[33] A R Poole M Kobayashi T Yasuda et al ldquoType II col-lagen degradation and its regulation in articular cartilage inosteoarthritisrdquo Annals of the Rheumatic Diseases vol 61 sup-plement 2 pp 78ndash81 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Arthritis

The affect of MMP enzymes on cartilage is inhibited byTIMP [29 30] NO is overexpressed at synovial tissue ofinjured cartilage by the stimulus of inducible nitric oxide syn-thase andTNF-120572 [29ndash32] IL-1 andTNF-120572 release is increasedwith the affect of degraded matrix molecules and so NOexpression is also increased It is reported that MMP-13 isdischarged mostly via sinovial fluid [33] We think that thesedata explain why TNF-120572 MMP-13 and NOwere foundmorein synovial fluids in our results

We think the restriction of our study is that it cannot becrosschecked by a method other than ELISA method whichis used in the analysis of the samples

5 Conclusion

When among the molecular markers which are analyzedin the synovial lavage fluid MMP-9 MMP-13 NO TIMP-1and TNF-120572 the area under the ROC curve and P values areevaluated MMP-13 (119875 lt 00001 95 CI 070ndash085) NO(119875 lt 00001 95 CI 072ndash086) and TNF-120572 (119875 lt 0000195 CI 091ndash098) results are statistically significant As aresult as it can be analyzed from the synovial lavage fluid ofthe molecular injury markers such as MMP-9 MMP-13 NOTIMP-1 and TNF-120572 which emerge in the articular cartilageinjury we can say that it can also be analyzed in a reliablemanner by the indirect ELISAmethod described by us in thesamples which are obtained from the cartilage tissue

Acknowledgment

Theauthors thank theTurkishOrthopedic Society (TOTBID)for the grants in support of this study

References

[1] G Nagyeri M Radacs S Ghassemi-Nejad et al ldquoTSG-6protein a negative regulator of inflammatory arthritis forms aternary complex with murine mast cell tryptases and heparinrdquoThe Journal of Biological Chemistry vol 286 no 26 pp 23559ndash23569 2011

[2] K Tsuritani J Takeda J Sakagami et al ldquoCytokine receptor-like factor 1 is highly expressed in damaged human kneeosteoarthritic cartilage and involved in osteoarthritis down-stream of TGF-120573rdquo Calcified Tissue International vol 86 no 1pp 47ndash57 2010

[3] T A Stupina V D Makushin and M A Stepanov ldquoExperi-mental morphological study of the effects of subchondral tun-nelization and bone marrow stimulation on articular cartilageregenerationrdquo Bulletin Experimental Biology and Medicine vol153 no 2 pp 289ndash293 2012

[4] MA JordanG SVanThiel J Chahal and S JNho ldquoOperativetreatment of chondral defects in the hip joint a systematicreviewrdquo Current Reviews in Musculoskeletal Medicine vol 5 no3 pp 244ndash253 2012

[5] O S Ertas and A Kayali ldquoAn overview on analytical methodvalidationrdquo Journal of Faculty Pharmacy vol 34 no 1 pp 41ndash57 2005

[6] G Szepesi M Gazdag and K Mihalyfi ldquoSelection of high-performance liquid chromatographic methods in pharmaceuti-cal analysis III Method validationrdquo Journal of ChromatographyA vol 464 no 2 pp 265ndash278 1989

[7] A Kayal O Sogut and B Tuncel ldquoSome considerations on ana-lytical methods validations bioavailability bioequivalence andpharmacokinetic studiesrdquoEuropean Journal of DrugMetabolismand Pharmacokinetics vol 20 no 4 pp 271ndash274 1995

[8] A Marin A Lopez-Gonzalvez and C Barbas ldquoDevelopmentand validation of extraction methods for determination ofzinc and arsenic speciation in soils using focused ultrasoundapplication to heavy metal study in mud and soilsrdquo AnalyticaChimica Acta vol 442 no 2 pp 305ndash318 2001

[9] J G Morrison P White S McDougall et al ldquoValidation ofa highly sensitive ICP-MS method for the determination ofplatinum in biofluids application to clinical pharmacokineticstudies with oxaliplatinrdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 24 no 1 pp 1ndash10 2000

[10] V Grdinic and J Vukovic ldquoPrevalidation in pharmaceuticalanalysis part 1 fundamentals and critical discussionrdquo Journal ofPharmaceutical and Biomedical Analysis vol 35 no 1 pp 489ndash512 2004

[11] P Garnero J C Rousseau and P D Delmas ldquoMolecular basisand clinical use of biochemical markers of bone cartilage andsynovium in joint diseasesrdquoArthritis ampRheumatism vol 43 no1 pp 953ndash968 2000

[12] D A Walsh P Verghese G J Cook et al ldquoLymphatic vesselsin osteoarthritic human kneesrdquoOsteoarthritis Cartilage vol 20no 5 pp 405ndash412 2012

[13] Y Henrotin B Kurz and T Aigner ldquoOxygen and reactiveoxygen species in cartilage degradation friends or foesrdquoOsteoarthritis and Cartilage vol 13 no 8 pp 643ndash654 2005

[14] A Ostalowska E Birkner M Wiecha et al ldquoLipid peroxi-dation and antioxidant enzymes in synovial fluid of patientswith primary and secondary osteoarthritis of the knee jointrdquoOsteoarthritis and Cartilage vol 14 no 2 pp 139ndash145 2006

[15] K Haugbro J C Nossent T Winkler Y Figenschau and OP Rekvig ldquoAnti-dsDNA antibodies and disease classificationin antinuclear antibody positive patients the role of analyticaldiversityrdquo Annals of the Rheumatic Diseases vol 63 no 4 pp386ndash394 2004

[16] K J Jepsen F Wu J H Peragallo et al ldquoA syndrome ofjoint laxity and impaired tendon integrity in lumican- andfibromodulin-deficient micerdquo The Journal of Biological Chem-istry vol 277 no 38 pp 35532ndash35540 2002

[17] B E Gencosmanoglu M Eryavuz and S Dervisoglu ldquoEffectsof some nonsteroidal anti-inflammatory drugs on articularcartilage of rats in an experimental model of osteoarthritisrdquoResearch in Experimental Medicine vol 200 no 3 pp 215ndash2262001

[18] A J Hough ldquoPathology of osteoarthritisrdquo inArthritis and AlliedConditions W J Koopman Ed pp 1945ndash1968 Williams ampWilkins Baltimore Md USA 1997

[19] J D Sandy C R Flannery P J Neame and L S LohmanderldquoThe structure of aggrecan fragments in human synovial fluidEvidence for the involvement in osteoarthritis of a novelproteinase which cleaves the Glu 373-Ala 374 bond of theinterglobular domainrdquoThe Journal of Clinical Investigation vol89 no 5 pp 1512ndash1516 1992

[20] S Tseng A H Reddi and P E Di Cesare ldquoCartilage oligomericmatrix protein (COMP) a biomarker of arthritisrdquo BiomarkerInsights vol 4 pp 33ndash44 2009

[21] L S Lohmander L A Hoerrner and M W Lark ldquoMet-alloproteinases tissue inhibitor and proteoglycan fragmentsin knee synovial fluid in human osteoarthritisrdquo Arthritis ampRheumatism vol 36 no 2 pp 181ndash189 1993

[22] L S Lohmander H Roos L Dahlberg L A Hoerrnerand M W Lark ldquoTemporal patterns of stromelysin-1 tissue

Arthritis 7

inhibitor and proteoglycan fragments in human knee joint fluidafter injury to the cruciate ligament or meniscusrdquo Journal ofOrthopaedic Research vol 12 no 1 pp 21ndash28 1994

[23] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Immunol-ogy vol 14 pp 397ndash440 1996

[24] L Connell and I B McInnes ldquoNew cytokine targets in inflam-matory rheumatic diseasesrdquo Best Practice amp Research vol 20no 5 pp 865ndash878 2006

[25] M Sharif E George L Shepstone et al ldquoSerumhyaluronic acidlevel as a predictor of disease progression in osteoarthritis of thekneerdquo Arthritis amp Rheumatism vol 38 no 6 pp 760ndash767 1995

[26] C Melchiorri R Meliconi L Frizziero et al ldquoEnhancedand coordinated in vivo expression of inflammatory cytokinesand nitric oxide synthase by chondrocytes from patients withosteoarthritisrdquoArthritis amp Rheumatism vol 41 no 12 pp 2165ndash2174 1998

[27] R Hilf J L Wittliff W D Rector E D Savlov T C Halland R A Orlando ldquoStudies on certain cytoplasmic enzymesand specific estrogen receptors in human breast cancer and innonmalignant diseases of the breastrdquo Cancer Research vol 33no 9 pp 2054ndash2062 1973

[28] F A Van de Loo O J Arntz F H van Enckevort P L van Lentand W B van den Berg ldquoReduced cartilage proteoglycan lossduring zymosan-induced gonarthritis in NOS

2-deficient mice

and in anti-interleukin-1-treated wild-type mice with unabatedjoint inflammationrdquo Arthritis amp Rheumatism vol 41 no 4 pp634ndash646 1998

[29] R F Loeser ldquoIntegrins and cell signaling in chondrocytesrdquo Bi-orheology vol 39 no 1-2 pp 119ndash124 2002

[30] A R Poole F Guilak and S B Abramson ldquoEtiopathogenesisof osteoarthritisrdquo in Osteoarthritis Diagnosis and MEdicalSur-gical Management R WMoskowitz Ed pp 27ndash49 LippincottPhiladelphia Pa USA 4th edition 2007

[31] S R Goldring and M B Goldring ldquoThe role of cytokines incartilage matrix degeneration in osteoarthritisrdquo Clinical Ortho-paedics and Related Research no 427 supplement pp S27ndashS362004

[32] S Kamekura K Hoshi T Shimoaka et al ldquoOsteoarthritisdevelopment in novel experimental mouse models induced byknee joint instabilityrdquoOsteoarthritis and Cartilage vol 13 no 7pp 632ndash641 2005

[33] A R Poole M Kobayashi T Yasuda et al ldquoType II col-lagen degradation and its regulation in articular cartilage inosteoarthritisrdquo Annals of the Rheumatic Diseases vol 61 sup-plement 2 pp 78ndash81 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Arthritis 7

inhibitor and proteoglycan fragments in human knee joint fluidafter injury to the cruciate ligament or meniscusrdquo Journal ofOrthopaedic Research vol 12 no 1 pp 21ndash28 1994

[23] M Feldmann F M Brennan and R N Maini ldquoRole ofcytokines in rheumatoid arthritisrdquo Annual Review of Immunol-ogy vol 14 pp 397ndash440 1996

[24] L Connell and I B McInnes ldquoNew cytokine targets in inflam-matory rheumatic diseasesrdquo Best Practice amp Research vol 20no 5 pp 865ndash878 2006

[25] M Sharif E George L Shepstone et al ldquoSerumhyaluronic acidlevel as a predictor of disease progression in osteoarthritis of thekneerdquo Arthritis amp Rheumatism vol 38 no 6 pp 760ndash767 1995

[26] C Melchiorri R Meliconi L Frizziero et al ldquoEnhancedand coordinated in vivo expression of inflammatory cytokinesand nitric oxide synthase by chondrocytes from patients withosteoarthritisrdquoArthritis amp Rheumatism vol 41 no 12 pp 2165ndash2174 1998

[27] R Hilf J L Wittliff W D Rector E D Savlov T C Halland R A Orlando ldquoStudies on certain cytoplasmic enzymesand specific estrogen receptors in human breast cancer and innonmalignant diseases of the breastrdquo Cancer Research vol 33no 9 pp 2054ndash2062 1973

[28] F A Van de Loo O J Arntz F H van Enckevort P L van Lentand W B van den Berg ldquoReduced cartilage proteoglycan lossduring zymosan-induced gonarthritis in NOS

2-deficient mice

and in anti-interleukin-1-treated wild-type mice with unabatedjoint inflammationrdquo Arthritis amp Rheumatism vol 41 no 4 pp634ndash646 1998

[29] R F Loeser ldquoIntegrins and cell signaling in chondrocytesrdquo Bi-orheology vol 39 no 1-2 pp 119ndash124 2002

[30] A R Poole F Guilak and S B Abramson ldquoEtiopathogenesisof osteoarthritisrdquo in Osteoarthritis Diagnosis and MEdicalSur-gical Management R WMoskowitz Ed pp 27ndash49 LippincottPhiladelphia Pa USA 4th edition 2007

[31] S R Goldring and M B Goldring ldquoThe role of cytokines incartilage matrix degeneration in osteoarthritisrdquo Clinical Ortho-paedics and Related Research no 427 supplement pp S27ndashS362004

[32] S Kamekura K Hoshi T Shimoaka et al ldquoOsteoarthritisdevelopment in novel experimental mouse models induced byknee joint instabilityrdquoOsteoarthritis and Cartilage vol 13 no 7pp 632ndash641 2005

[33] A R Poole M Kobayashi T Yasuda et al ldquoType II col-lagen degradation and its regulation in articular cartilage inosteoarthritisrdquo Annals of the Rheumatic Diseases vol 61 sup-plement 2 pp 78ndash81 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom