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Oral Maxillofac Surg8:2478-2483, 2010

Boundary-Lubricating Ability andLubricin in Synovial Fluid of Patients

With Temporomandibular Joint DisordersLili Wei, DDS, MD,* Haofei Xiong, DDS, MD,† Bo Li, DDS, MD,‡

Yong Cheng, DDS, PhD,§ and Xing Long, PhD�

Purpose: This study was conducted to measure the boundary-lubricating ability and lubricin concen-tration of synovial fluid (SF) from patients with different stages of temporomandibular joint disorders(TMDs) and establish relationships between them.

Patients and Methods: According to the imaging and clinical findings, TMD patients were dividedinto 3 subgroups: displaced disc with reduction, displaced disc without reduction, and osteoarthri-tis. The boundary-lubricating ability of SF was determined by the coefficient of friction (COF) of SFin vitro with a friction apparatus. The lubricin concentrations were quantified by enzyme-linkedimmunosorbent assays.

Results: The COF of SF in TMD patients was significantly higher than that of healthy control subjects,but no observed difference was found among patient subgroups. Furthermore, a significant decline inlubricin concentrations was found in the group with osteoarthritis, whereas there was no significantchange in the other groups. However, a significant correlation was not found between the COF and thelubricin concentrations in our study.

Conclusions: These findings showed that distinct changes in lubricin and boundary-lubricating abilityin the SF occurred with different stages of TMDs.© 2010 American Association of Oral and Maxillofacial Surgeons

J Oral Maxillofac Surg 68:2478-2483, 2010

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csiTds

eceived from The State Key Laboratory Breeding Base of Basic

cience of Stomatology (Hubei-Most) and Key Laboratory of Oral

iomedicine Ministry of Education, School and Hospital of Stoma-

ology, Wuhan University, Wuhan, China.

*PhD Student.

†PhD Student.

‡Senior Registrar, Department of Oral Radiology.

§Professor, Department of Oral Radiology.

�Professor and Head, Department of Oral and Maxillofacial

urgery.

Lili Wei and Haofei Xiong contributed equally to this article.

upported by a grant (No. 30811120100/H1407) from the National

atural Scientific Foundation of China.

Address correspondence and reprint requests to Dr Long:

epartment of Oral and Maxillofacial Surgery, The State Key

aboratory Breeding Base of Basic Science of Stomatology (Hu-

ei-Most) and Key Laboratory of Oral Biomedicine Ministry of

ducation, School and Hospital of Stomatology, Wuhan Univer-

ity, 237 Luo Yu, Wuhan, China; e-mail: xinglong1957@yahoo.

om.cn

2010 American Association of Oral and Maxillofacial Surgeons

278-2391/10/6810-0015$36.00/0

boi:10.1016/j.joms.2010.01.018

2478

oint lubrication responsible for maintaining the low-riction environment of the articular surfaces of synovialoints is a topic of continued interest. Synovial fluid (SF),n ultrafiltrate of blood with additives produced by theynovium, contributes to the lubrication and protectionf articular cartilage surfaces.1,2 Boundary lubricationefers to the ability of SF to reduce friction betweenpposed and pressurized cartilaginous surfaces indepen-ently of viscosity.3,4 SF lubricants such as hyaluronan,erum albumin, and lubricin have been believed to benvolved in the lubrication of joints. Lubricin, referred tos superficial zone protein, megakaryocyte-stimulatingactor precursor, and proteoglycan 4, is a chondropro-ective, mucinous glycoprotein and a product of theroteoglycan 4 gene.5-7 It is secreted by synovial fibro-lasts and chondrocytes, and it localizes in the SF, syno-ial tissue, and superficial zone of articular cartilage.3,8

Functions attributed to lubricin include protection ofartilage surfaces from protein deposition and cell adhe-ion, inhibition of synovial cell overgrowth, and partic-pation of boundary lubrication on cartilage surfaces.herefore abnormality in lubricin’s metabolism may in-uce pathologic changes in joints. Recent studies havehown that deficiency of lubricin could cause vulnera-

ility of the articular surfaces to mechanical and fric-

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WEI ET AL 2479

ional damage. This was further evidenced by the accel-rated progression of joint degeneration in individualsith camptodactyly arthropathy coxa vara pericarditis

yndrome, a disease linked to deficiency in the genencoding the lubricin protein, and by rapidly progres-ive joint degeneration observed in lubricin-null mice.9

The human temporomandibular joint (TMJ) maye one of the most sophisticated and complex tri-ological systems. Temporomandibular joint disor-ers (TMDs) are common disorders of the mandibularotion system with distinct clinicopathologic charac-

eristics. Classical features of TMDs include displacedisc with reduction (DDR), displaced disc withouteduction (DDNR), and osteoarthritis (OA). Disc dis-lacement is considered as an initial change in TMJathology, frequently leading to secondary tissueamage, and it precedes the onset of TMJ OA andven disc perforation.10,11 The collapse of lubricationunction is assumed to be an important cause in theathogenesis of TMDs because lubrication plays a criti-al role in maintaining low-friction and low-wear prop-rties of condyle cartilage during various jaw move-ents.12 Furthermore, it is very likely that pathologic

hange in lubrication function is accompanied by differ-nt stages in the pathogenesis of TMDs.However, few reports have explored lubrication

unction, which is primarily investigated through theeasure of lubricin contents and boundary-lubricat-

ng ability, in different subgroups of TMD patients.herefore this study was performed to examine lubri-in and boundary-lubricating ability in the SF aspi-ated from patients with different stages of TMDs andstablish a relationship between them.

atients and Methods

SUBJECTS

The subjects included in this study were recruitedrom the clinic at the School and Hospital of Stoma-ology of Wuhan University, Wuhan, China. Sevenealthy volunteers and 34 patients with TMDs partic-

pated in the study. Clinical examination was per-ormed, and signs and symptoms were recorded dur-ng the patient’s initial visit. The same 2 trainednvestigators performed the assessments throughoth clinical examinations and radiography. Subjec-ive pain during mouth opening and eating was ratedy use of a visual analog scale ranging from 0 (noain) to 100 (maximum pain imaginable). Maximalouth opening was measured with a millimeter ruler

nd defined as the distance between the maxillary andandibular central incisors. Radiographic examina-

ion included transcranial views (open and closedouth) and tomography of the TMJ for determination

f bony changes such as osteophytes and erosion. o

uperior joint space arthrography was used to deter-ine the anterior disc displacement. Clinical diag-oses of TMDs were made according to the clinicaliagnostic criteria for TMD.13 The patients were di-ided into 3 subgroups according to the imaging andlinical findings: DDR, DDNR, and OA. All the dataere collected during the patient’s first visit, andone of the patients had any treatment previously.

COLLECTION OF SF SAMPLES

Volunteers and patients were given no medicationor at least 2 weeks before the SF sampling was per-ormed. During collection of samples, a local anes-hetic (2% lidocaine) was injected into the TMJ extra-apsular region, and then a total of 1 mL of salineolution was injected into the superior compartmentf the joint. The patient was asked to open and closeis or her mouth to mix the saline solution with SF.he SF and saline solution mixture was aspirated andeinjected 5 times. Then, the sample was aspiratednd collected. After being centrifuged at 4°C (3,000 gor 10 minutes) for cell and tissue debris removal, allhe samples were stored at �70°C until the assaysere performed.14 For all experiments, informed con-

ent was obtained, and the protocol was approved byhe Human Research Ethics Committee, School andospital of Stomatology, Wuhan University.

BOUNDARY-LUBRICATING ABILITY ANALYSIS OF SF

Boundary-lubricating ability was tested with a fric-ion apparatus, as described previously by Davis etl15 and Jay et al.16 This test system involves a ballupper specimen) that slides against a rotating disclower specimen) under a prescribed set of condi-ions. The load is applied vertically downward with aotor-driven carriage that uses the force or load sen-

or for feedback to maintain a constant load. Theearing system was axially loaded within a gimbalsystem free to rotate around 2 perpendicular horizon-al axes. A steel ball and ceramic as bearing materialsere chosen because they offer a steady surface with

eadily reproducible conditions from test to test. Aontact pressure of 3.1 MPa was generated when theearing was loaded with 1 kg with a sliding velocity of.17 mm/s. The friction apparatus recorded displace-ents of the gimbals system around the vertical load-

ng axis through a linear displacement voltage trans-ucer, the output voltage of which was directlyroportional to the magnitude of the frictionalorque. The signal was related to the coefficient ofriction (COF [�]) by a calibration with known fric-ional torque. Every measurement of the COF wasarried out at 25°C and was preceded by a measure-ent of the COF with 0.9% sodium chloride (NaCl).hus all measurements of COF are relative to the COF

f 0.9% NaCl. The addition of 200 mL of 0.9% NaCl

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2480 SYNOVIAL FLUID IN TMD

nd, later, 200 �L of test lubricant was followed byringing the bearing surfaces close enough so that theolution wet both surfaces. After 5 minutes for equili-ration, the bearing was brought to rest on the ceramics it was oscillating, and the voltages were automaticallyecorded. Typically, COF values were reproducible toithin 10%, and at least 3 values were recorded and

veraged. Values of COF stabilized after 2 minutes andere recorded every 30 seconds. The final value was the

verage of values during 300 seconds.16,17

ENZYME-LINKED IMMUNOSORBENT ASSAYANALYSIS OF LUBRICIN IN SF

Protein-containing solutions were quantified byse of the Micro bicinchoninic acid protein assayPierce Biotechnology, Rockford, IL), according tonstructions provided by the manufacturer, excepthat the assay was downscaled to a total volume of00 �L. An enzyme-linked immunosorbent assayELISA) kit for human lubricin (Yope Biotechnol-gy, Shanghai, China) was used to measure the

ubricin concentrations in the SF samples accordingo the manufacturer’s instructions.

STATISTICAL ANALYSIS

Results of all measurements were presented asean � SD. Data were analyzed with SPSS software,

ersion 13.0 (SPSS, Chicago, IL), and statistical differ-nces in each group were subjected to a 1-way analysisf variance test (� � 0.05). Pair-wise multiple compar-sons were carried out by a Bonferroni test (� � 0.05)n the cases where the analysis of variance testhowed significant differences. The correlation coef-cient was calculated to establish the relationshipetween lubricin concentration and COF.

Table 1. CHARACTERISTICS AND CLINICAL EXAMINATIO

Group No. of Subjects Gender (M/F)Age [Mean

(yr

ontrol 7 3/4 27 (18DR 11 2/9 24 (14DNR 12 3/9 31 (20A 11 0/11 38 (22

ei et al. Synovial Fluid in TMD. J Oral Maxillofac Surg 2010.

Table 2. COF AND LUBRICIN CONCENTRATION OF SF A

Control

OF (�) 0.247 � 0.006ubricin concentration (�g/mL) 7.496 � 0.468

P � .05 versus control.P � .05 versus all other groups.

ei et al. Synovial Fluid in TMD. J Oral Maxillofac Surg 2010.

esults

CLINICAL FINDINGS

Table 1 shows characteristics and clinical examina-ion findings of the TMD patients and control subjects.

COF OF SF

Compared with the healthy control subjects (0.247 �.006), the mean COF (�) was significantly increasedn the DDR group (0.266 � 0.011), DDNR group0.268 � 0.010), and OA group (0.275 � 0.009) (P �05). Nevertheless, no difference was found amongubgroups of TMD patients (P � .05). The data andtatistical analysis are shown in Table 2 and Figure 1.

LUBRICIN CONCENTRATION IN SF

The lubricin concentrations were 7.496 � 0.468g/mL in the SF of healthy control subjects, 7.160 �.249 �g/mL in DDR patients, 7.215 � 1.117 �g/mL

n DDNR patients, and 5.689 � 1.313 �g/mL in OAatients (Fig 2). No significant difference was foundetween the healthy control subjects and DDR orDNR patients (P � .05), whereas OA patients pre-

ented a much lower lubricin concentration than allther groups (P � .05). The data and statistical anal-sis are shown in Table 2 and Figure 2x

CORRELATION ANALYSES

Correlation analysis of COF and lubricin concentra-ions was performed for all SF samples (r � �0.291,2 � 0.085, P � .05) (Fig 3). However, no significantorrelation was found between the COF and the lu-ricin concentrations in our study.

DINGS OF TMD PATIENTS AND CONTROL SUBJECTS

e)] Pain on Visual AnalogScale [Mean (Range)]

Maximal Mouth Opening(mm)

0 (0) 38.87 � 6.7938 (0-75) 41.56 � 4.8950 (20-85) 32.64 � 6.7355 (30-85) 38.38 � 4.21

TED FROM CONTROL SUBJECTS AND TMD PATIENTS

DDR DDNR OA

66 � 0.011* 0.268 � 0.010* 0.275 � 0.009*60 � 1.249 7.215 � 1.117 5.689 � 1.313†

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iscussion

TMDs are some of the most common diseases of theMJ and have a broad spectrum of subgroups. Al-

hough the pathogenesis of TMDs remains obscure,he impaired joint lubrication due to the alteration ofroperties and components of SF may profoundly

mpact TMJ function and development of TMDs.18

nder physiologic bearing, the liquid membraneormed by the SF plays a major role in joint lubrica-ion, and under the condition of high overloading, 2ides of the surfaces of the articular cartilage attach toach other, and boundary lubrication of SF may play aole in low-friction articulation in the microenviron-ent.19,20 Given the important protective properties

IGURE 2. Lubricin concentrations in SF samples from healthyontrol subjects and patients with DDR, DDNR, and OA. No sig-ificant difference was found between the healthy control subjectsnd DDR/DDNR patients (P � .05). There were significant differ-nces when OA patients were compared with the 3 other groupsP � .05 [asterisk]).

IGURE 1. COFs of SF from healthy control subjects and patientsith DDR, DDNR, and OA. There were significant differencesetween the healthy control subjects and each patient group (P �05 [asterisks]), but no differences were found among the patientubgroups (DDR, DDNR, and OA) (P � .05).

ei et al. Synovial Fluid in TMD. J Oral Maxillofac Surg 2010.

Sei et al. Synovial Fluid in TMD. J Oral Maxillofac Surg 2010.

hat SF confers on articular cartilage, it is essential tonderstand the effect of boundary lubrication in theF of different stages of TMDs. Thus in this study theF boundary-lubricating ability in different subgroupsf TMDs and lubricin concentrations of these SF sam-les were tested. The results showed that, comparedith healthy control subjects, the boundary-lubricat-

ng ability of SF weakened in the patient groups ofDR, DDNR, and OA, because the COF (� value) wasigher than that of healthy control subjects in all 3atient groups. Meanwhile, lubricin concentrations ofF were shown to be near normal levels in the DDRnd/or DDNR group but decreased in the OA group.owever, a remarkable correlation between COF and

ubricin was not found in this study.The COF in this study showed a higher range of

alues when compared with other studies.17,21,22 Theiversity may be explained by a dissimilar surface

nteracting with SF or a different method for collect-ng SF.23 Compared with a cartilage-on-cartilage bear-ng system, a bearing apparatus that isolated boundaryubricant conditions was used in our study. This sys-em has the advantage of reproducibility, and it mim-cs the results of this bearing, although COF values

ere higher overall.4,21 Because it is difficult to obtainative SF from the human TMJ, SF was collectedhrough a dilution method in this study. This maynevitably affect the COF to some extent because theoundary-lubricating ability of diluted SF behaves dif-erently compared with the native SF under physio-ogic conditions.

Lubricin is a key mediator of boundary lubricationnd plays an important role in the functional integrityf the diarthrodial joint. In this study an ELISA assayas used to measure the lubricin concentrations of

IGURE 3. Correlation analysis of COF and lubricin concentra-ions. No significant correlation was found between them (r2 �.085, P � .05).

ei et al. Synovial Fluid in TMD. J Oral Maxillofac Surg 2010.

F. When one is collecting SF samples from the TMJ,

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2482 SYNOVIAL FLUID IN TMD

here is the concern that different volumes of salineolution aspirate may potentially influence the out-omes. Thus in this study the lubricin concentrationsere calculated against their total protein concentra-

ions before the comparison was made. The results ofhe ELISA assay showed that there was almost nohange in the lubricin concentrations in the earlytages of TMDs. In contrast, a significant decrease wasbserved in the late stage of TMD (ie, OA). Theseesults may broaden the concept that a chemo-me-hanical process and complex microenvironmentsovern lubricin metabolism within synovium and ar-icular cartilage in the TMJ. Lower lubricin concentra-ions may be part of a feedback mechanism withynovium and articular cartilage, which are both tar-ets of the milieu and the active participants whenlteration of the biochemo-mechanical milieu resultedn changes in the microenvironments of the SF. There-ore these findings indicate that the damage to theynovium and articular cartilage may not be serious inhe early stages of TMDs whereas the secreting andepairing function of synovium and articular cartilageill diminish, ultimately resulting in a decrease in

ubricin concentrations in SF with the exacerbation ofhe TMD.

Although many investigators only singled out pa-ients with internal derangement and/or OA,24,25 DDRnd DDNR were investigated in this study consideringistinct differences in patients’ symptoms and clinicaligns. However, no significant difference in the lubri-in concentration was found when these 2 groupsere compared with healthy control subjects individ-ally.OA is a degenerative joint disease with the destruc-

ion of articular cartilage, and it frequently results inisability and dysfunction of the TMJ. Researchersave shown that many cytokines, such as interleukin�, tumor necrosis factor �, and matrix metallopro-einase—which were regarded as the major patho-ogic mediators associated with arthritis—were de-ected in SF in patients with OA.26,27 These cytokinesay stimulate the secretion of latent cysteine pro-

eases (eg, cathepsin B) and/or neutrophil elastase,hich can reduce the synthesis but increase the deg-

adation of lubricin.4,28,29 Such findings might be usedo explain the decline in lubricin concentrations ob-erved in the OA group in our study.

Interestingly, our results showed no significant cor-elation between boundary-lubricating ability and lu-ricin concentrations. Of note, although the depositsf lubricin within the SF and the articular surface are

ikely responsible for key functional determinants ofubricin boundary-lubricating ability, it could be spec-lated that in practice, the lubrication of SF constitu-nts is complex, and any lubricant alone cannot rep-

icate it fully. Many molecules present in SF, with a

ariety of interactions and structure-function relation-hips, and may provide the framework for the poten-ially complete recapitulation of the boundary-lubri-ating ability.In conclusion, this study showed that distinct

hanges in lubricin and boundary-lubricating ability inhe SF occurred with different stages of TMDs. Fur-her studies should elucidate the role of lubricin,oundary lubrication, and complex, intrinsic mecha-isms implicated in TMDs.

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