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Investigation of collagen architecture in diseases of temporomandibular joint

Background: Temporomandibular joint (TMJ) is a stress bearing structure. Its resilience and functions are supported by collagen architecture. Visualization of this collagen networking can be done under polarizing microscope. This network gets modified in a variety of conditions affecting the TMJ likeankylosis, degenerative joint disorder, neoplastic conditions, trauma and ageing. The objective of this study is to analyze the histomorphological changes in TMJ conditions and correlate them with collagen architecture so as to project scoring criteria for distinguishing between osseous changes. Methods: Paraffin based H and E sections of TMJ conditions were obtained and observed under polarizing microscopy. Sample distribution was as follows: Degenerative joint disease (n=10), ankylosis (n=5) and osteochondroma (n=5). A scoring criterion was devised and cutoff value was determined. Results: Immature or formative tissue was found to have a score of less than and equal to five and mature tissue had a score of greater than and equal to six. Conclusion: Scoring criterion proposed in this study is a reliable technique and can be used for confirming the histological architectural changes within a diseased TMJ.


Introduction
Collagen fibrillar networking is the basic structure supporting the three-dimensional architecture of tissues. This provides tensile strength and volume maintenance [1]. Condylar pathologies may be developmental, traumatic, vascular, abnormal muscle pulling, nutritional, endocrinal, teratogenic and infectious in origin [2]. These disorders cause a change in structural integrity of TMJ histology and underlying collagen network. Most common etiology of TMJ ankylosis is after trauma or infection [3]. Few cases are congenital in origin [4,5]. Approximately 3% to 4% of population seek treatment for TMJ disorders, and roughly 70% of these patients suffer from disc displacement [6]. In this study, an attempt towards understanding architectural changes in collagen networking in various temporomandibular joint disorders has been made by means of polarizing microscopy.

Materials and methods
Hematoxylin and eosin stained paraffin-based sections were obtained from various temporomandibular joint conditions. Sample was distributed as follows: Degenerative joint disease (n=10), ankylosis (n=5) and osteochondroma (n=5). Polarizing light microscopy was used to delineate collagen fibril architecture in tissue sections. A qualitative PLM (Polarizing light microscopy) scoring system was designed ( Table 1). Institutional ethical committee clearance was obtained according to guidelines provided by Declaration of Helsinki. Radiographs were evaluated for all cases included in the study (Figure 9).

Results and observation
Diseased condylar tissue when observed under polarizing microscope exhibited a variety of patterns ( Table 2). Woven bone showed an irregular fishnet-like pattern suggestive of a forming tissue. Mature bone had parallel lamellations corresponding to collagen mineralizing at regular intervals. This was observed as thick parallel lines. Cartilage had no birefringence.
Overall scores observed were: a. Ankylosed condylar tissue (Figures 1 and 2               contrasted using Image J software and thickness measured (Supplementary figures S1 and S2).

Discussion
The normal biomechanical behaviour of a diarthrodial joint is dependent on the composition and ultrastructural organization of the articular cartilage. The collagen network of articular cartilage protects the chondrocytes, resists tensile forces produced by compression as well as interstitial swelling, provides attachment for proteoglycans, and anchors the doi: 10.7243/2052-7896-1-7 cartilage to the subchondral bone. Normally, articular cartilage that is regularly subjected to high levels of shear stress shows a high degree of collagen orientation and a thick superficial zone [1]. Collagen organization is critical factor responsible for load bearing and durability of TMJ. Many scoring systems have been proposed for studying the histomorphology and collagen orientation of the temporomandibular joint. These systems can be divided into a) semi-quantitative methods: OsScore and ICRS (International Cartilage Repair Society) I and II and b) qualitative technique like PLM (polarizing light microscopy) [7,8]. Polarized light microscopy is a traditional method for visualizing the collagen network architecture of condylar cartilage [9]. Polarized light microscopic technique can be used to measure collagen fibril orientation, parallelism, and birefringence [10,12]. To overcome the drawbacks associated with polarizing microscopy as quoted by Rieppo et al., two observers viewed the slides and scoring was done with unanimous decision. Linear polarizing microscopy can be performed on a light microscope with addition of two filters, polarizer and analyzer. The polarizer is placed after the light source and ensures only linear polarized light i.e., light in a single plane that is perpendicular to direction of light propagation, is transmitted to the specimen. Optically anisotropic materials change the direction of light through birefringence. The fibrillar structure of collagen splits the incident polarized light into two orthogonal rays depending on the direction of collagen at each point in the section. The analyzer filter positioned after the specimen is at right angle to the polarizer. The polarized specimen is observed through an eyepiece. The objective of using polarizing light microscopy is to assess the extent to which a sample demonstrates collagen organization. This microscopic technique can be used to visualize fibrillar collagen for describe its network, changes in collagen orientation during ageing, validation of collagen imaging modalities and for observing changes during loading and degradation. Use of polarizing microscope for scoring purposes offers a systematic assessment of collagen organization. In this study, this technique for microscopic visualization has been used to demarcate a cutoff scoring value for immature and mature osseous structures in temporomandibular joint disorders. Less than five score value is suggestive of an ongoing formative process whereas a score of greater than or equal to six is indicator of a stabilized structure (no remodeling process). Here, a new scoring criteria using histomorphology as well as polarizing light microscope has been proposed. Almeida et al., performed histologic and histomorphologic analysis of collagen using picrosirius red. They compared the light intensity between diseased TMJ and normal controls using image analysis software. No statistical difference was observed between both groups. However, this was a qualitative analysis and did not study collagen birefringence in terms of collagen remodeling in immature and mature bone [10]. In a contradictory study studying effects of distraction osteogenesis on temporomandibular joint, disorganization of collagen fibers was observed (H & E). However, no significant difference was found in histology and fiber thickness between the distracted and contralateral sides [11]. Panula et al., reported reduced birefringence and thinning of fibers in arthritic temporomandibular joints [1]. However, in this study, both thick and thin fibrils were found with varying intensities of birefringence. The authors have added few modifications in the PLM scoring proposed by Changoor et al., by including some additional groups so that the validity of the scoring system can be enhanced.
No other study has included other pathologies affecting temporomandibular joint for studying collagen architecture using any of the methodologies available. Hence, further validation of the method proposed in this study is required and perhaps can be verified with a larger sample size as well as by other investigators.

Conclusion
TMJ is a synovial joint that is affected by numerous changes that can be age-related or pathologic in nature. The underlying collagen architecture provides mechanical support and resilience to the joint. The changes in collagen structure are reflected upon the structural changes and defects in the joint itself [12].

Authors' contributions SC SJ
Research concept and design