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Lubricin in human temporomandibular joint disc: An immunohistochemical study
archives of oral biology 57 (2012) 614–619
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Lubricin in human temporomandibular joint disc: An immunohistochemical study Rosalia Leonardi a,*, Giuseppe Musumeci b, Edoardo Sicurezza a, Carla Loreto b a b
Department of Dentistry, Faculty of Dentistry, University of Catania, Policlinico Universitario, Italy Department of Anatomy, Diagnostic Pathology, Forensic Medicine, Hygiene and Public Health, University of Catania, Italy
article info
abstract
Article history:
Aims: To evaluate, immunohistochemically, the presence and distribution of lubricin in
Accepted 12 December 2011
human temporomandibular joint (TMJ) discs without any degenerative changes, obtained from autopsies, in order to elucidate the TMJ lubrication system and disc tribology.
Keywords:
Methods: Immunohistochemistry for lubricin detection was carried out on 34 TMJ discs. Any
Temporomandibular joint
disc had signs of degenerative or inflammatory joint disease nor disc were displaced.
Disc
Sections were incubated with diluted rabbit polyclonal anti-lubricin antibody and scored
Chondrocyte
according to the percentage of lubricin immunopositive cells. Three different TMJ disc tissue
Lubricin
compartments taken from the intermediate zone were analysed, namely: the central region
Osteoarthritis
as well as the temporal (superior) and condylar (inferior) disc surfaces. The Friedman test,
Proteoglycan 4 (PRG4)
was used to compare lubricin at a protein level expression, amongst the regions of disc
Superficial zone protein (SZP)
specimens. Results: Staining was noted within the TMJ disc cell populations in every disc tissue sample, however, the number of disc cells immunolabelled varied according to disc tissue regions. The percentage of immunostained cells, was statistically significant lower in the central region than in each disc surface ( p < 0.0001), whilst any statistically significant difference was found when comparing the two surfaces one another. Conclusions: Lubricin is present in several location of TMJ disc being significantly more expressed at disc surfaces than in the central part. # 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Lubricin, also commonly referred to as superficial zone protein (SZP), is a large, multifaceted, water soluble glycoprotein encoded by the PRG4 gene. It has a molecular weight of 206 kDa and consists of approximately equal proportions of protein and glycosaminoglycans. Electron microscope measurements show that the lubricin molecule is a partially extended flexible rod and, in solution, occupies a smaller spatial domain than would be expected from structural predictions.1 This characteristic may aid the molecule’s boundary lubricating ability. Depletion of the lubricin function
has also been associated with camptodactyly-arthropathycoxa vara-pericarditis syndrome (CACP), an arthritis-like autosomal recessive disorder.2 Lubricin, which is present in synovial fluid, on the superficial layer of articular cartilage3–5 and tendons,6,7 contributes to the boundary lubrication properties facilitating low friction levels at the interfacing surfaces of articular cartilage.1 Lubricin biosynthesis and biodistribution is mostly regulated by cytokine and growth factor. In this respect, Exposure of synoviocytes, chondrocytes and cartilage explants to proinflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) results in a marked reduction in the expression and/or abundance of
* Corresponding author at: Department of Orthodontics, University of Catania, via S. Sofia n 78, Catania, Italy. Tel.: +39 3356692528. E-mail address: [email protected] (R. Leonardi). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.12.004
archives of oral biology 57 (2012) 614–619
secreted lubricin, with corresponding alterations in the amounts of cartilage-associated (boundary) lubricin. Conversely, treatment with transforming growth factor-beta (TGFbeta) significantly upregulates lubricin synthesis, secretion and cartilage boundary association.8 Lubricin is known to be involved in the joint lubrication system. This system consists of three parts: lubricin, surface active-phopholipids and hyaluronan (HA). As far as temporomandibular joint (TMJ) is concerned an increased friction between the moving parts has been considered one of the major factors in the aetiology of disc displacement.9 Accordingly, a collapsed lubrication system is considered a key factor in the chain of events leading up to internal derangements.9 However except for HA, no research has been carried out on lubricin in human TMJ. Recently a pivotal role of lubricin in joint lubrication has been pointed out in bovine meniscus of stifle joints10 as well as in caprine11 and human intervertebral discs.12 In fact, previous studies have demonstrated the presence of lubricin on the surface and within the tissue matrix of tendons6,7,13–18 and joint disc,11,12 suggesting that lubricin plays a role in lubricating the collagenous structures during sliding motion. Findings from these studies suggest that on one hand lubricin, in association with surface active phospholipids (SALP), acts as an extremely efficient boundary lubricant and protector of the articular surfaces,9,10,18 and on the other hand lubricin also plays a role on a smaller scale in lubricating the collagenous structures during motion. Accordingly, these findings prompted this immunohistochemical investigation, on the presence and distribution of lubricin in human TMJ discs without any degenerative changes, obtained from autopsies, in order to elucidate further aspects of the TMJ lubrication system and disc tribology.
2.
Materials and methods
One of five sections for each disc was stained with Haematoxylin & Eosin (H&E), in order to assign a histopatological degeneration score (HDS),24 the other sections were preserved for the immunohistochemical study. Briefly, this score takes into account pathological disc tissue transformation, i.e. collagen bundles, non-specific degenerative changes and the presence of blood vessels. This grading system results in a score ranging from 0 up to 8 for heavily degenerated disc tissue. Two investigators independently assigned the TMJ disc degeneration score. A score of more than 2 points lead to exclusion.
2.1.
Immunohistochemistry
Regarding immunohistochemical staining, at first endogenous peroxidase activity was quenched on TMJ disc specimens, with 3% H2O2 for 10 min. Non-specific binding of antibodies was blocked by normal horse/goat serum treatment [diluted 1:20 in phosphate buffered saline (PBS), 0.1% bovine serum albumin (BSA)]. The sections were irradiated (3 5 min) in capped polypropylene slide-holders with citrate buffer (pH 6), using a microwave oven (750 W) to unmask antigenic sites. Then, the sections were incubated with diluted rabbit polyclonal anti-lubricin antibody (Novus Biologicals, LLC, Littleton, CO, USA) (diluted 1:50 in PBS) overnight at +4 8C. The secondary antibody, biotinylated mouse/anti-rabbit IgG was applied (for 30 min, at RT), followed by the avidin–biotin– peroxidase complex (Vector Elite Kit Abbott, Chicago, IL, USA) for 30 min, at room temperature. The immunoreaction was visualized by incubating the sections for 4 min in a 0.1% 3,30 diaminobenzidine and 0.02% hydrogen peroxide solution (DAB substrate kit, Vector Laboratories, CA, USA). The sections were lightly counterstained with Mayer’s haematoxylin (Histolab Products AB, Goteborg, Sweden) and finally mounted in GVA mount (Zymed, Laboratories Inc., San Francisco, CA, USA).
2.2. Thirty-four TMJ discs, were obtained bilaterally from 17 cadavers (seven male and 10 female). The mean period (and standard deviation) between the death of the donor and tissue harvest was 14 3 h, with a maximum of 22 h. The cadaver donors ranged in age from 35 to 51 years, with a mean age of 46 7 years, at the time of death. The causes of death of the donors were cardiovascular disease (15 individuals) and cancer (two). Every donors’ clinical history revealed no generalized joint disease or TMJ arthropathy. Macroscopic examination of the discs showed no signs of degenerative or inflammatory joint disease nor disc displacement. The original sample collection, previously used in former studies,19–23 was from the Institute of Anatomy at the University of Catania, Italy. The discs were fixed overnight in 10% neutral buffered formalin (Bio-Optica, Milan, Italy). After fixation and overnight washing, each disc was sectioned through its centre along a parasagittal plane, perpendicular to its long axis. Each tissue block was dehydrated in graded ethanol, and embedded in paraffin preserving the longitudinal anatomical orientation. Specimens were sectioned at a thickness of 5 mm and placed on silanized glass slides (the anterior band being always on the right side of the slides).
615
Evaluation of immunohistochemistry
The analysis of immunostaining focused on the biconcave central part of the disc, which is commonly referred to, as the intermediate zone (i.e. the disc tissue between the anterior and posterior band). The lubricin-staining status was identified as either negative or positive. Positive staining was defined as the presence of a brown detection chromogen on the edge of the haematoxylin-stained cell nucleus, distributed within the cytoplasm, or in the immediate lacunar/pericellular space. The percentage of lubricin immunopositive cells (Extent Score) was independently evaluated by two investigators and scored as a percentage of the final number of 100 cells in five categories: 0 = <5%; 1 = 5–30%; 2 = 31–50%; 3 = 51–75%, and 4 = >75%. This score was assigned on an agreement basis following discussion. Counting was performed at 200 magnification. Three different TMJ disc tissue compartments were analysed, namely: the central region of the disc (thickness about 0.6 mm) and the temporal (superior) and condylar (inferior) disc surfaces, defined in this study as the tissue from the outer portion of the disc, i.e. upper and lower disc intra-articular space, that comes towards the central region of the disc for about 600 mm.
616
2.3.
archives of oral biology 57 (2012) 614–619
Positive and negative controls
Positive and negative controls were performed to test the specific reaction of primary antibodies used in this study at a protein level. For positive control testing the sections from bovine calf stifle joint underwent an immunoperoxidase process.10 The positive immunolabelling for lubricin was both perinuclear and cytoplasmic. For negative control testing sections of TMJ displaced discs were randomly drawn from degenerated disc samples. These were then treated with normal rabbit serum instead of the specific antibodies.
2.4.
Statistical analysis
Mean values and standard deviations were obtained for the Extent Score of the three disc portions examined in each sample. The Shapiro–Wilk test25 for normal data was used to detect the normality of distribution ( p > 0.05). Therefore, the data were analysed using the Friedman test, which allowed to compare lubricin protein expression amongst the regions of disc specimens. p-Values of less than 0.05 were considered statistically significant, Moreover, a post hoc Dunn’s Multiple Comparison test26 has been performed to find out between which regions the differences were statistically significant. Statistical computation was conducted using SPSS program (SPSS1 release 16.0, Chicago, IL, USA).
3.
Results
Degeneration score was assigned on disc section stained by HE (Fig. 1). Only one disc out of 34 obtained a degeneration score of more 2 points, thus it was excluded from the study, and the immunohistochemical investigation was carried out on 33 TMJ discs. Staining was noted within the TMJ disc cell populations in every disc tissue sample, but not every cell was immunolabelled by lubricin antibody (Figs. 2 and 3). Cellular staining was cytoplasmatic in close proximity to the nucleus. In some chondrocyte-like cells chromogen could also be appreciated in
Fig. 1 – TMJ disc stained by Haematoxylin & Eosin. Bar 50 mm.
Fig. 2 – Immunohistochemical staining of human TMJ disc. Positive intracellular staining for lubricin is indicated by brown chromogen. The single arrow indicate immunolabelled cells and the empty arrow not immunolabelled cells. Double arrow shows the chromogen in chondrocyte-like cell lacunar space. Bar 50 mm.
the lacunar space (i.e. the small cavity within the extracellular matrix, containing a chondrocyte) (Figs. 2 and 3). The number of disc cells immunolabelled varied according to the tissue regions (Fig. 4). Lubricin staining of the two disc surface specimens was significantly more extensive than that of the central region (Fig. 4). Accordingly, the median of the Extent Score of the disc central region was 1.39 whilst it was 3.61 on the temporal surface and 3.75 on the condylar surface, the differences being statistically significantly different ( p < 0.001) (Table 1). No statistically significant differences were obtained when comparing the two surfaces to each other ( p > 0.05). Lubricin staining within the disc cells was ubiquitous in regions of positive extracellular matrix staining (Fig. 5). Thus,
Fig. 3 – Immunohistochemical staining of human TMJ disc. Positive staining for lubricin is indicated by brown chromogen. Both TMJ disc cells and extracellular matrix are immunolabelled. Bar 100 mm.
617
archives of oral biology 57 (2012) 614–619
Fig. 4 – Lubricin staining on disc surface (right side) and at the inner portion of the TMJ disc (left side). Almost every disc cell is immunolabelled (arrow) at disc surface. The number of cells immunolabelled diminish at the inner portion (empty arrow). Bar 100 mm.
Fig. 5 – Scattered cells are immunolabelled by lubricin antibody. Around some immunolabelled cell also extracellular matrix is immunostained (arrow). Bar 150 mm.
there was no intracellular staining in cells located in unstained extracellular matrix (ECM). Sometimes the surrounding extracellular matrix around lubricin immunolabelled cells appeared also immunostained by lubricin antibody (Fig. 5). The extracellular matrix immunostaining also appeared more consistent at disc surface, and lubricin appeared present in a large band fashion and not as scattered staining.
has been established that HA affects the viscosity of synovial fluid but exhibits insignificant boundary lubrication.33,34 Lubricin was immunohistochemically detected both in TMJ disc cells and in extracellular matrix (ECM), however its distribution varied between the central region and the disc surface specimens. In fact, lubricin was more expressed at disc surfaces than in the central portion both in cells and in ECM. On the other hand in the inner portion of the central region, not every cell was immunolabelled, being lubricin staining detectable only in few scattered cells. Interestingly, the ECM surrounding these immunolabelled cells, was sometimes immunostained. This ECM moderate and scattered lubricin labelling found between collagenous fibres of our normal TMJ disc tissue specimens is in line with the literature. In fact, lubricin has been described in tendon and ligament tissues at the interface of collagen fibres. The detection of lubricin in this site of TMJ discs corroborates previous findings suggesting a lubrication role for lubricin within the collagen bundles, thus facilitating the relative motion of these fibrous structures.18 The fact that we detected a discrete lubricin layer on almost all disc surfaces, in almost all of the specimens, fall in line with previous findings on other joints.10–12 Although the mechanism underling this distribution was not assessed in this study, it is likely that it is related to variation in the function and structure of the disc as lubricin has been described as an important boundary lubricant on articulating surfaces, facilitating movement between opposing moving articular surfaces. Boundary lubricants are attached to and cover each articular surface, reducing the surface energy. SALP in
4.
Discussion
As far as we know, this is the first report regarding the presence of lubricin in human TMJ discs. As a matter of fact, every previous investigation on human TMJ has dealt only with another component of the joint lubricating system: the HA. The question whether HA could be used for treatment of TMJ disorders remains without any clear answer as findings from these studies have revealed contradictory results. Some studies reported that HA injections into affected joints improved their mobility and reduced pain and inflammation.27–30 On the opposite, an other study have questioned31 the effect of HA injections into affected joints for the routine treatment of TMJ disfunction, as they showed a limited therapeutic effect.32 In this respect, systematic reviews reported that there is insufficient, consistent evidence to either support or refute the use of hyaluronate for treating patients with TMD.33,34 For the sake of clarity it should be underlined that at the beginning HA was considered the predominant articular boundary lubricant, whilst nowadays it
Table 1 – Median and range plus values of the Extent Scores in the different sections. Disc region Temporal surface Central region Condylar surface
25% Percentile
Median
75% Percentile
Lower 95% CI of mean
Upper 95% CI of mean
3.40 1.14 3.51
3.61 1.39 3.75
3.75 1.47 3.86
3.50 1.31 3.60
3.65 1.45 3.78
Friedman test ( p value) p < 0.0001
618
archives of oral biology 57 (2012) 614–619
association with lubricin act as extremely efficient boundary lubricant and protector of articular surfaces.9 The increased friction between the articulating surfaces when lubrication is lacking has been hypothesized as major causative factor in TMJ disc displacement.9 Therefore, in the near future, it could be interesting to evaluate the expression using an animal model in which TMJ damage is induced in order to evaluate lubricin expression during the progression of disease. Last but not least, our findings may also have implications for bioengineering strategies, as lubricin secreting cells should be provided at disc surfaces as well as inside the central part.
11.
Funding
14.
The study was supported by the University of Catania, Grant ex-60% PRA.
10.
12.
13.
15.
Competing interests 16.
None declared.
Ethical approval
17.
All experimental protocols were in compliance and under the regulations of the university of Catania.
18.
references
19.
1. Swann DA, Slayter HS, Silver FH. The molecular structure of lubricating glycoprotein-I, the boundary lubricant for articular cartilage. J Biol Chem 1981;256:5921–5. 2. Rhee DK, Marcelino J, Al-Mayouf S, Schelling DK, Bartels CF, Cui Y, et al. Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing. J Biol Chem 2005;280:31325–32. 3. Lee SY, Nakagawa T, Reddi AH. Induction of chondrogenesis and expression of superficial zone protein (SZP)/lubricin by mesenchymal progenitors in the infrapatellar fat pad of the knee joint treated with TGF-beta1 and BMP-7. Biochem Biophys Res Commun 2008;376:148–53. 4. Nugent-Derfus GE, Chan AH, Schumacher BL, Sah RL. PRG4 exchange between the articular cartilage surface and synovial fluid. J Orthop Res 2007;25:1269–76. 5. Ohno S, Schmid T, Tanne Y, Kamiya T, Honda K, OhnoNakahara M, et al. Expression of superficial zone protein in mandibular condyle cartilage. Osteoarthritis Cartilage 2006;14:807–13. 6. Funakoshi T, Schmid T, Hsu HP, Spector M. Lubricin distribution in the goat infraspinatus tendon: a basis for interfascicular lubrication. J Bone Joint Surg Am 2008;90:803–14. 7. Funakoshi T, Spector M. Chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells. J Orthop Res 2010;28:716–25. 8. Jones AR, Flannery CR. Bioregulation of lubricin expression by growth factors and cytokines. Eur Cell Mater 2007;13:40–5. discussion 45. 9. Nitzan DW, Nitzan U, Dan P, Yedgar S. The role of hyaluronic acid in protecting surface-active phospholipids
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from lysis by exogenous phospholipase A(2). Rheumatology (Oxford) 2001;40:336–40. Schumacher BL, Schmidt TA, Voegtline MS, Chen AC, Sah RL. Proteoglycan 4 (PRG4) synthesis and immunolocalization in bovine meniscus. J Orthop Res 2005;23:562–8. Shine KM, Spector M. The presence and distribution of lubricin in the caprine intervertebral disc. J Orthop Res 2008;26:1398–406. Shine KM, Simson JA, Spector M. Lubricin distribution in the human intervertebral disc. J Bone Joint Surg Am 2009;91: 2205–12. Sun Y, Berger EJ, Zhao C, Jay GD, An KN, Amadio PC. Expression and mapping of lubricin in canine flexor tendon. J Orthop Res 2006;24:1861–8. Sun Y, Chen MY, Zhao C, An KN, Amadio PC. The effect of hyaluronidase, phospholipase, lipid solvent and trypsin on the lubrication of canine flexor digitorum profundus tendon. J Orthop Res 2008;26:1225–9. Taguchi M, Sun YL, Zhao C, Zobitz ME, Cha CJ, Jay GD, et al. Lubricin surface modification improves extrasynovial tendon gliding in a canine model in vitro. J Bone Joint Surg Am 2008;90:129–35. Taguchi M, Sun YL, Zhao C, Zobitz ME, Cha CJ, Jay GD, et al. Lubricin surface modification improves tendon gliding after tendon repair in a canine model in vitro. J Orthop Res 2009;27:257–63. Taguchi M, Zhao C, Sun YL, Jay GD, An KN, Amadio PC. The effect of surface treatment using hyaluronic acid and lubricin on the gliding resistance of human extrasynovial tendons in vitro. J Hand Surg Am 2009;34:1276–81. Schmidt TA, Gastelum NS, Nguyen QT, Schumacher BL, Sah RL. Boundary lubrication of articular cartilage: role of synovial fluid constituents. Arthritis Rheum 2007;56:882–91. Leonardi R, Lo Muzio L, Bernasconi G, Caltabiano C, Piacentini C, Caltabiano M. Expression of vascular endothelial growth factor in human dysfunctional temporomandibular joint discs. Arch Oral Biol 2003;48: 185–92. Leonardi R, Loreto C, Barbato E, Caltabiano R, Lombardo C, Musumeci G, et al. MMP-13 (collagenase 3) localization in human temporomandibular joint discs with internal derangement. Acta Histochem 2008;110:314–8. Leonardi R, Villari L, Bernasconi G, Piacentini C, Baciliero U, Travali S. Cellular S-100 protein immunostaining in human dysfunctional temporomandibular joint discs. Arch Oral Biol 2000;45:411–8. Leonardi R, Villari L, Piacentini C, Bernasconi G, Baciliero U, Travali S. CD44 standard form (CD44H) expression and distribution in dysfunctional human temporomandibular joint discs. Int J Oral Maxillofac Surg 2000;29:296–300. Loreto C, Musumeci G, Leonardi R. Chondrocyte-like apoptosis in temporomandibular joint disc internal derangement as a repair-limiting mechanism. An in vivo study. Histol Histopathol 2009;24:293–8. Leonardi R, Rusu MC, Loreto C. Temporomandibular joint disc: a proposed histopathological degeneration grading score system. Histol Histopathol 2010;25:1117–22. Shapiro SS, Wilk MB. An analysis of variance test for normality. Biometrika 1965;52:9. Dunn. Multiple comparisons using rank sums. Technometrics 1964;6:12. Bertolami CN, Gay T, Clark GT, Rendell J, Shetty V, Liu C, et al. Use of sodium hyaluronate in treating temporomandibular joint disorders: a randomized, doubleblind, placebo-controlled clinical trial. J Oral Maxillofac Surg 1993;51:232–42. Hepguler S, Akkoc YS, Pehlivan M, Ozturk C, Celebi G, Saracoglu A, et al. The efficacy of intra-articular sodium
archives of oral biology 57 (2012) 614–619
hyaluronate in patients with reducing displaced disc of the temporomandibular joint. J Oral Rehabil 2002;29:80–6. 29. Long X, Chen G, Cheng AH, Cheng Y, Deng M, Cai H, et al. A randomized controlled trial of superior and inferior temporomandibular joint space injection with hyaluronic acid in treatment of anterior disc displacement without reduction. J Oral Maxillofac Surg 2009;67:357–61. 30. Obara T, Mabuchi K, Iso T, Yamaguchi T. Increased friction of animal joints by experimental degeneration and recovery by addition of hyaluronic acid. Clin Biomech (Bristol Avon) 1997;12:246–52.
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31. Meng QG, Long X. A hypothetical biological synovial fluid for treatment of temporomandibular joint disease. Med Hypotheses 2008;70:835–7. 32. Manfredini D, Piccotti F, Guarda-Nardini L. Hyaluronic acid in the treatment of TMJ disorders: a systematic review of the literature. Cranio 2010;28:166–76. 33. Shi Z, Guo C, Awad M. Hyaluronate for temporomandibular joint disorders. Cochrane Database Syst Rev 2003:CD002970. 34. Tadmor R, Chen N, Israelachvili JN. Thin film rheology and lubricity of hyaluronic acid solutions at a normal physiological concentration. J Biomed Mater Res 2002;61:514–23.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Lubricin in human temporomandibular joint disc: An immunohistochemical study Rosalia Leonardi a,*, Giuseppe Musumeci b, Edoardo Sicurezza a, Carla Loreto b a b
Department of Dentistry, Faculty of Dentistry, University of Catania, Policlinico Universitario, Italy Department of Anatomy, Diagnostic Pathology, Forensic Medicine, Hygiene and Public Health, University of Catania, Italy
article info
abstract
Article history:
Aims: To evaluate, immunohistochemically, the presence and distribution of lubricin in
Accepted 12 December 2011
human temporomandibular joint (TMJ) discs without any degenerative changes, obtained from autopsies, in order to elucidate the TMJ lubrication system and disc tribology.
Keywords:
Methods: Immunohistochemistry for lubricin detection was carried out on 34 TMJ discs. Any
Temporomandibular joint
disc had signs of degenerative or inflammatory joint disease nor disc were displaced.
Disc
Sections were incubated with diluted rabbit polyclonal anti-lubricin antibody and scored
Chondrocyte
according to the percentage of lubricin immunopositive cells. Three different TMJ disc tissue
Lubricin
compartments taken from the intermediate zone were analysed, namely: the central region
Osteoarthritis
as well as the temporal (superior) and condylar (inferior) disc surfaces. The Friedman test,
Proteoglycan 4 (PRG4)
was used to compare lubricin at a protein level expression, amongst the regions of disc
Superficial zone protein (SZP)
specimens. Results: Staining was noted within the TMJ disc cell populations in every disc tissue sample, however, the number of disc cells immunolabelled varied according to disc tissue regions. The percentage of immunostained cells, was statistically significant lower in the central region than in each disc surface ( p < 0.0001), whilst any statistically significant difference was found when comparing the two surfaces one another. Conclusions: Lubricin is present in several location of TMJ disc being significantly more expressed at disc surfaces than in the central part. # 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Lubricin, also commonly referred to as superficial zone protein (SZP), is a large, multifaceted, water soluble glycoprotein encoded by the PRG4 gene. It has a molecular weight of 206 kDa and consists of approximately equal proportions of protein and glycosaminoglycans. Electron microscope measurements show that the lubricin molecule is a partially extended flexible rod and, in solution, occupies a smaller spatial domain than would be expected from structural predictions.1 This characteristic may aid the molecule’s boundary lubricating ability. Depletion of the lubricin function
has also been associated with camptodactyly-arthropathycoxa vara-pericarditis syndrome (CACP), an arthritis-like autosomal recessive disorder.2 Lubricin, which is present in synovial fluid, on the superficial layer of articular cartilage3–5 and tendons,6,7 contributes to the boundary lubrication properties facilitating low friction levels at the interfacing surfaces of articular cartilage.1 Lubricin biosynthesis and biodistribution is mostly regulated by cytokine and growth factor. In this respect, Exposure of synoviocytes, chondrocytes and cartilage explants to proinflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) results in a marked reduction in the expression and/or abundance of
* Corresponding author at: Department of Orthodontics, University of Catania, via S. Sofia n 78, Catania, Italy. Tel.: +39 3356692528. E-mail address: [email protected] (R. Leonardi). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.12.004
archives of oral biology 57 (2012) 614–619
secreted lubricin, with corresponding alterations in the amounts of cartilage-associated (boundary) lubricin. Conversely, treatment with transforming growth factor-beta (TGFbeta) significantly upregulates lubricin synthesis, secretion and cartilage boundary association.8 Lubricin is known to be involved in the joint lubrication system. This system consists of three parts: lubricin, surface active-phopholipids and hyaluronan (HA). As far as temporomandibular joint (TMJ) is concerned an increased friction between the moving parts has been considered one of the major factors in the aetiology of disc displacement.9 Accordingly, a collapsed lubrication system is considered a key factor in the chain of events leading up to internal derangements.9 However except for HA, no research has been carried out on lubricin in human TMJ. Recently a pivotal role of lubricin in joint lubrication has been pointed out in bovine meniscus of stifle joints10 as well as in caprine11 and human intervertebral discs.12 In fact, previous studies have demonstrated the presence of lubricin on the surface and within the tissue matrix of tendons6,7,13–18 and joint disc,11,12 suggesting that lubricin plays a role in lubricating the collagenous structures during sliding motion. Findings from these studies suggest that on one hand lubricin, in association with surface active phospholipids (SALP), acts as an extremely efficient boundary lubricant and protector of the articular surfaces,9,10,18 and on the other hand lubricin also plays a role on a smaller scale in lubricating the collagenous structures during motion. Accordingly, these findings prompted this immunohistochemical investigation, on the presence and distribution of lubricin in human TMJ discs without any degenerative changes, obtained from autopsies, in order to elucidate further aspects of the TMJ lubrication system and disc tribology.
2.
Materials and methods
One of five sections for each disc was stained with Haematoxylin & Eosin (H&E), in order to assign a histopatological degeneration score (HDS),24 the other sections were preserved for the immunohistochemical study. Briefly, this score takes into account pathological disc tissue transformation, i.e. collagen bundles, non-specific degenerative changes and the presence of blood vessels. This grading system results in a score ranging from 0 up to 8 for heavily degenerated disc tissue. Two investigators independently assigned the TMJ disc degeneration score. A score of more than 2 points lead to exclusion.
2.1.
Immunohistochemistry
Regarding immunohistochemical staining, at first endogenous peroxidase activity was quenched on TMJ disc specimens, with 3% H2O2 for 10 min. Non-specific binding of antibodies was blocked by normal horse/goat serum treatment [diluted 1:20 in phosphate buffered saline (PBS), 0.1% bovine serum albumin (BSA)]. The sections were irradiated (3 5 min) in capped polypropylene slide-holders with citrate buffer (pH 6), using a microwave oven (750 W) to unmask antigenic sites. Then, the sections were incubated with diluted rabbit polyclonal anti-lubricin antibody (Novus Biologicals, LLC, Littleton, CO, USA) (diluted 1:50 in PBS) overnight at +4 8C. The secondary antibody, biotinylated mouse/anti-rabbit IgG was applied (for 30 min, at RT), followed by the avidin–biotin– peroxidase complex (Vector Elite Kit Abbott, Chicago, IL, USA) for 30 min, at room temperature. The immunoreaction was visualized by incubating the sections for 4 min in a 0.1% 3,30 diaminobenzidine and 0.02% hydrogen peroxide solution (DAB substrate kit, Vector Laboratories, CA, USA). The sections were lightly counterstained with Mayer’s haematoxylin (Histolab Products AB, Goteborg, Sweden) and finally mounted in GVA mount (Zymed, Laboratories Inc., San Francisco, CA, USA).
2.2. Thirty-four TMJ discs, were obtained bilaterally from 17 cadavers (seven male and 10 female). The mean period (and standard deviation) between the death of the donor and tissue harvest was 14 3 h, with a maximum of 22 h. The cadaver donors ranged in age from 35 to 51 years, with a mean age of 46 7 years, at the time of death. The causes of death of the donors were cardiovascular disease (15 individuals) and cancer (two). Every donors’ clinical history revealed no generalized joint disease or TMJ arthropathy. Macroscopic examination of the discs showed no signs of degenerative or inflammatory joint disease nor disc displacement. The original sample collection, previously used in former studies,19–23 was from the Institute of Anatomy at the University of Catania, Italy. The discs were fixed overnight in 10% neutral buffered formalin (Bio-Optica, Milan, Italy). After fixation and overnight washing, each disc was sectioned through its centre along a parasagittal plane, perpendicular to its long axis. Each tissue block was dehydrated in graded ethanol, and embedded in paraffin preserving the longitudinal anatomical orientation. Specimens were sectioned at a thickness of 5 mm and placed on silanized glass slides (the anterior band being always on the right side of the slides).
615
Evaluation of immunohistochemistry
The analysis of immunostaining focused on the biconcave central part of the disc, which is commonly referred to, as the intermediate zone (i.e. the disc tissue between the anterior and posterior band). The lubricin-staining status was identified as either negative or positive. Positive staining was defined as the presence of a brown detection chromogen on the edge of the haematoxylin-stained cell nucleus, distributed within the cytoplasm, or in the immediate lacunar/pericellular space. The percentage of lubricin immunopositive cells (Extent Score) was independently evaluated by two investigators and scored as a percentage of the final number of 100 cells in five categories: 0 = <5%; 1 = 5–30%; 2 = 31–50%; 3 = 51–75%, and 4 = >75%. This score was assigned on an agreement basis following discussion. Counting was performed at 200 magnification. Three different TMJ disc tissue compartments were analysed, namely: the central region of the disc (thickness about 0.6 mm) and the temporal (superior) and condylar (inferior) disc surfaces, defined in this study as the tissue from the outer portion of the disc, i.e. upper and lower disc intra-articular space, that comes towards the central region of the disc for about 600 mm.
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2.3.
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Positive and negative controls
Positive and negative controls were performed to test the specific reaction of primary antibodies used in this study at a protein level. For positive control testing the sections from bovine calf stifle joint underwent an immunoperoxidase process.10 The positive immunolabelling for lubricin was both perinuclear and cytoplasmic. For negative control testing sections of TMJ displaced discs were randomly drawn from degenerated disc samples. These were then treated with normal rabbit serum instead of the specific antibodies.
2.4.
Statistical analysis
Mean values and standard deviations were obtained for the Extent Score of the three disc portions examined in each sample. The Shapiro–Wilk test25 for normal data was used to detect the normality of distribution ( p > 0.05). Therefore, the data were analysed using the Friedman test, which allowed to compare lubricin protein expression amongst the regions of disc specimens. p-Values of less than 0.05 were considered statistically significant, Moreover, a post hoc Dunn’s Multiple Comparison test26 has been performed to find out between which regions the differences were statistically significant. Statistical computation was conducted using SPSS program (SPSS1 release 16.0, Chicago, IL, USA).
3.
Results
Degeneration score was assigned on disc section stained by HE (Fig. 1). Only one disc out of 34 obtained a degeneration score of more 2 points, thus it was excluded from the study, and the immunohistochemical investigation was carried out on 33 TMJ discs. Staining was noted within the TMJ disc cell populations in every disc tissue sample, but not every cell was immunolabelled by lubricin antibody (Figs. 2 and 3). Cellular staining was cytoplasmatic in close proximity to the nucleus. In some chondrocyte-like cells chromogen could also be appreciated in
Fig. 1 – TMJ disc stained by Haematoxylin & Eosin. Bar 50 mm.
Fig. 2 – Immunohistochemical staining of human TMJ disc. Positive intracellular staining for lubricin is indicated by brown chromogen. The single arrow indicate immunolabelled cells and the empty arrow not immunolabelled cells. Double arrow shows the chromogen in chondrocyte-like cell lacunar space. Bar 50 mm.
the lacunar space (i.e. the small cavity within the extracellular matrix, containing a chondrocyte) (Figs. 2 and 3). The number of disc cells immunolabelled varied according to the tissue regions (Fig. 4). Lubricin staining of the two disc surface specimens was significantly more extensive than that of the central region (Fig. 4). Accordingly, the median of the Extent Score of the disc central region was 1.39 whilst it was 3.61 on the temporal surface and 3.75 on the condylar surface, the differences being statistically significantly different ( p < 0.001) (Table 1). No statistically significant differences were obtained when comparing the two surfaces to each other ( p > 0.05). Lubricin staining within the disc cells was ubiquitous in regions of positive extracellular matrix staining (Fig. 5). Thus,
Fig. 3 – Immunohistochemical staining of human TMJ disc. Positive staining for lubricin is indicated by brown chromogen. Both TMJ disc cells and extracellular matrix are immunolabelled. Bar 100 mm.
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Fig. 4 – Lubricin staining on disc surface (right side) and at the inner portion of the TMJ disc (left side). Almost every disc cell is immunolabelled (arrow) at disc surface. The number of cells immunolabelled diminish at the inner portion (empty arrow). Bar 100 mm.
Fig. 5 – Scattered cells are immunolabelled by lubricin antibody. Around some immunolabelled cell also extracellular matrix is immunostained (arrow). Bar 150 mm.
there was no intracellular staining in cells located in unstained extracellular matrix (ECM). Sometimes the surrounding extracellular matrix around lubricin immunolabelled cells appeared also immunostained by lubricin antibody (Fig. 5). The extracellular matrix immunostaining also appeared more consistent at disc surface, and lubricin appeared present in a large band fashion and not as scattered staining.
has been established that HA affects the viscosity of synovial fluid but exhibits insignificant boundary lubrication.33,34 Lubricin was immunohistochemically detected both in TMJ disc cells and in extracellular matrix (ECM), however its distribution varied between the central region and the disc surface specimens. In fact, lubricin was more expressed at disc surfaces than in the central portion both in cells and in ECM. On the other hand in the inner portion of the central region, not every cell was immunolabelled, being lubricin staining detectable only in few scattered cells. Interestingly, the ECM surrounding these immunolabelled cells, was sometimes immunostained. This ECM moderate and scattered lubricin labelling found between collagenous fibres of our normal TMJ disc tissue specimens is in line with the literature. In fact, lubricin has been described in tendon and ligament tissues at the interface of collagen fibres. The detection of lubricin in this site of TMJ discs corroborates previous findings suggesting a lubrication role for lubricin within the collagen bundles, thus facilitating the relative motion of these fibrous structures.18 The fact that we detected a discrete lubricin layer on almost all disc surfaces, in almost all of the specimens, fall in line with previous findings on other joints.10–12 Although the mechanism underling this distribution was not assessed in this study, it is likely that it is related to variation in the function and structure of the disc as lubricin has been described as an important boundary lubricant on articulating surfaces, facilitating movement between opposing moving articular surfaces. Boundary lubricants are attached to and cover each articular surface, reducing the surface energy. SALP in
4.
Discussion
As far as we know, this is the first report regarding the presence of lubricin in human TMJ discs. As a matter of fact, every previous investigation on human TMJ has dealt only with another component of the joint lubricating system: the HA. The question whether HA could be used for treatment of TMJ disorders remains without any clear answer as findings from these studies have revealed contradictory results. Some studies reported that HA injections into affected joints improved their mobility and reduced pain and inflammation.27–30 On the opposite, an other study have questioned31 the effect of HA injections into affected joints for the routine treatment of TMJ disfunction, as they showed a limited therapeutic effect.32 In this respect, systematic reviews reported that there is insufficient, consistent evidence to either support or refute the use of hyaluronate for treating patients with TMD.33,34 For the sake of clarity it should be underlined that at the beginning HA was considered the predominant articular boundary lubricant, whilst nowadays it
Table 1 – Median and range plus values of the Extent Scores in the different sections. Disc region Temporal surface Central region Condylar surface
25% Percentile
Median
75% Percentile
Lower 95% CI of mean
Upper 95% CI of mean
3.40 1.14 3.51
3.61 1.39 3.75
3.75 1.47 3.86
3.50 1.31 3.60
3.65 1.45 3.78
Friedman test ( p value) p < 0.0001
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association with lubricin act as extremely efficient boundary lubricant and protector of articular surfaces.9 The increased friction between the articulating surfaces when lubrication is lacking has been hypothesized as major causative factor in TMJ disc displacement.9 Therefore, in the near future, it could be interesting to evaluate the expression using an animal model in which TMJ damage is induced in order to evaluate lubricin expression during the progression of disease. Last but not least, our findings may also have implications for bioengineering strategies, as lubricin secreting cells should be provided at disc surfaces as well as inside the central part.
11.
Funding
14.
The study was supported by the University of Catania, Grant ex-60% PRA.
10.
12.
13.
15.
Competing interests 16.
None declared.
Ethical approval
17.
All experimental protocols were in compliance and under the regulations of the university of Catania.
18.
references
19.
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