0003.‘996’) 7X OXOI-OilYSO 0

THE FINE STRUCTURE OF A SURFACE LAYER OVER THE FIBROUS ARTICULAR TISSUE OF THE RAT

MANDIBULAR CONDYLE

J. APPLETON

Electron Miscroscope Unit, School of Dental Surgery, Department of Dental Surgery. Pembroke Place, P.O. Box 147, Liverpool, L69 3BX, England

Summary-The collagenous articulating surface of the rat mandibular condyle was covered with a fibrillar-amorphous layer, the articular lamina, up to 1.3 pm in thickness. Beneath the articulating surface, only the fibrillar component was evident between the collagen fibres. .The articular lamina stained positively with colloidal iron together with the matrix between the collagen fibres for a depth of 0.3 pm. For a depth of 5 pm below the articulating surface, membrane-bound vesicles were densely stained. Washing with hyaluronidase or Pronase removed the articular lamina and abolished the staining reaction between the collagen fibres below the articulating surface, but the vesicles deep to the articulating surface were stained. The articular lamina appears to be a hyaluronate-protein layer and be important in boundary film lubrication.

INTRODUCTION

There is a thin but distinct surface layer or articular lamina covering the collagen fibres which form the articulating surface of the mandibular condyle in the rat (Appleton, 1975). A similar articular lamina is also present covering hyaline articular cartilage of the rat and other species. This lamina remains after being washed vigorously with normal saline, and is unlikely, therefore, to be formed as a result of the precipitation of synovial fluid during fixation and dehydration (Balaz, Bloom and Swann, 1966; Gardner, 1972, Weiss and Mirow, 1972; Finsterbush and Friedman, 1973). It is not analogous in any way to the thicker membrane observed coating hyaline articular carti- lage surfaces when viewed under polarized light and termed the ‘lamina splendens’ by McConaill (1951); this probably represents an interference fringe. It is possible that the articular lamina is an integral part of the articular tissue but it is not established whether it is continuous with and similar in composition to the interfibrillar matrix of the underlying fibrous ar- titular tissue or is a separate phase. My purpose was to investigate in more detail the structure and compo- sition of this articular lamina within the mandibular joint.

MATERIALS AND METHODS

Ten black and white rats of both sexes at 80 days of age were used to provide 20 mandibular condyles. Under Nembutal anaesthesia, the condylar process was divided beneath the condylar head and the con- dyles were removed with the overlying articular disc intact to avoid damage to the articulating surfaces. The articular disc was then quickly and carefully removed and the articular surface of the condyle was washed vigorously for several minutes in a stream of normal saline from a wash bottle. The mandibular condyles were processed according to one of the fol- lowing regimes:

1. Ten condyles were fixed in 6.25 per cent glutaral- dehyde in cacodylate buffer, pH 7.4, for 2 h and washed in buffer overnight followed by staining with the Mowry (1958) colloidal iron’ method by immer- sion of the whole cartilage in a freshly-made solution for 20min and then washing with 12 per cent acetic acid.

2. The remaining unfixed condyles were divided rinto two equal groups and one group was washed rfor 3 min with normal saline containing hyaluroni- dase (150 pg/ml) from bovine testis (35%500 i.u./mg) at 37°C and the other group with Pronase (Sigma Co. Ltd.) product p 150, type 6 from Streptococcus griseus in 0.2 M tris-HCI, 0.02 M CaCI, pH 7.5, at 55°C.

After fixation in glutaraldehyde, the enzyme- digested condyles were stained with Mowry colloidal iron solution. Controls were used in which the surface was washed with buffer, saline or enzyme solutions inactivated by boiling.

All tissues were dehydrated in a graded series of methanols and embedded in Araldite. Thin sections were prepared using a Beichert OMUII ultramicro- tome equipped with a diamond knife. After mounting on copper grids, sections were stained with a 25 per cent solution of uranyl acetate in methanol and exam- ined in a Philips E.M. 300 equipped with a liquid nitrogen anti-contamination device.

RESULTS

The articulating surface formed by the collagen fibres of the articular tissue was covered by a distinct layer or articular lamina which often appeared to be up to 1.3 pm in thickness (Fig. l), but this may be exaggerated by the oblique plane of section. Closer examination showed that this articular lamina con- sisted of a fibrillar component and an amorphous

719

720 J. Appleton

component forming aggregates which were widely separated except when immediately adjacent to the collagen fibres. where they formed a more continuous layer (Fig. 2). Between the parallel collagen fibres deep TV the articulating surface, there was material which was similar in structure to the fibrillar com- ponent of the articular lamina. although the amor- phous component was not evident (Fig. 2).

Staining with colloidal Iron produced a dense depo- sit of iron particles both on and below the articulating surface (Fig. 3). The most dense deposit of iron was above and immediately adjacent to the articulating surface and consisted of aggregates of iron particles about 30nm in diameter. The most superficial layer of iron was less dense, consisting of particles about 3 nm in diameter (Fig. 4). For a depth of approxi- mately 0.3 pm below the articulating surface. there were some aggregates of iron particles 30nm in dia- meter. It was not possible to determine if the particles were specifically associated with the inter-fibrillar matrix component (Fig. 4). There was little inter- tibrillar staining below a depth of 0.3pm. However, the membranes of the numerous vesicles present in the fibrous articular tissue were heavily stained for a depth of up to 5pm below the articulating surface (Fig. 3).

Washing the condyle surface briefly with testicular hyaluronidase produced complete disintegration of the articular lamina with consequent exposure of the underlying collagen fibres (Fig. 5). ,The enzyme Pro- nase removed the articular lamina equally well, but also produced some loosening of the surface collagen fibres at the articulating surface and clearly removed the librillar component from between the collagen fibres (Fig. 6). If the condyles washed with the enzyme solutions were then stained with colloidal iron there was almost complete abolition of the staining above and immediately below the articulating surface formed by the collagen fibres. However, there were a few scattered iron particles in the deeper inter-fibril- lar matrix and the cell membranes of the numerous vesicles scattered throughout the matrix were still in- tensely stained (Fig. 6).

The structure of the articular tissue in the mandibu- lar joint of the rat is different from hyaline cartilage present in other synovial joints (Appleton, 1975). However, for depths between 3 and 5pm. the articu- Iar tissue was essentially similar in structure to hya- line cartilage in consisting chiefly of bundles of CO]- lagen fibres 2G-25 nm in diameter lying parallel to the articulating surface and separated by inter-fibrillar matrix. My findings do not support the suggestion (Gardner. 1972) that the articular lamina is lost dur- ing specimen preparation in the absence of Ruth- enium Red or cetyl pyridinium chloride.

The articular lamina stained positively with col- loidal iron particles showing more clearly its distribu- tion in relation to the collagen fibres of the articular surface. The articulating surface was completely covered with stained material and there was also some staining of the matrix between the collagen fibres for up to a depth of 0.3 pm below the articulat-

ing surface. The dense staining of the articular lamina and the limitation of inter-fibrillar matrix staining to a maximum depth of 0.3 pm is unlikely to be an arti- fact. In view of the persistence of staining of the deeper inter-fibrillar matrix after enzyme digestion, though that in the more superficial layers was abol- ished, it is improbable that staining of the articular lamina results from simple surface accumulation of iron particles. There appears to be no problem of penetration of the 3 nm iron particles because cell membranes and vesicle membranes were stained well below the limits of the inter-fibrillar matrix staining. Therefore, it seems likely that in rice there is a layer of colloidal iron-positive staining material on the art!- culating surface of the fibrous articular tissue. f

The degree to which colloidal iron is a specific his- tochemical stain for acid polysaccharides is uncertain but it has been widely used as such (Mowry. 1958: Curran and Clark. 1964: Curran, Clark and Lovell. 1965: Wetzel, Wetzel and Spicer. 1966). As the articu- lar lamina is stained with colloidal iron and rapidly removed with hyaluronidase, it may contain hyalur- onate. a major component of synovial fluid (Wright, Dowson and Kerr, 1973). However. the proteolytic enzyme Pronase is equally. if not more. effective in removing the articular lamina and. therefore. it prob- ably also contains a protein component.

The articular lamina may be important in the lubri- cation mechanisms within the mandibular joint. There are two basic types of joint lubrication: one in which the articulating surfaces are separated by a fluid film and the other in which there is surface-to- surface contact but molecular protection is afforded by layers attached to the surfaces. This latter type IS referred to as boundary lubrication (Wright and Dowson. 1976).

The work of Radin, Swann and Weisser (1970) and Wright and Dowson (1976) suggests that hyaluronate alone is not important in boundary lubrication. If the viscosity of synovial fluid is reduced by the destruc- tion of the hyaluronate molecule with hyaluronidase, the lubricating properties of the synovial fluid remains the same. However. the destruction of the protein factor in the synovial fluid with trypsin does not affect its viscosity but considerably reduces its lubricating properties. My findings suggest that within the mandibular joint there may be a hyaluronate- protein layer bound to the articular surfaces which could be important in boundary lubrication.

3cktlo~.lrdgrn1enrs~I gratefully acknowledge the technical assistance of Mrs. V. R. Bradley.

REFERENCES

Appleton J. 1975. The ultrastructure of the articular tissue of the mandibular condyle in the rat. Arch ore/ &o/.

20, 823-826. Balaz E. A.. Bloom G. D. and Swann D. A. 1966. Fine

structure and glycosaminoglycan content of the surface layer of artiCU]ar cartilage. Frdn Pi-or. Fedn Am. S~C,S rxp. Eiol. 25, 1813-1816.

Curran R. C. and Clark A. E. 1964. The use of the colloida] iron method for acid polysaccharides in electron micro- scopy. B&hem. J. 90. 2P.

Curran R. C., Clark A. E. and Lovell D. 1965. Acid muco- polysaccharides in electron microscopy. J. Anar. 99, 427434.

A surface articulating layer in the mandibular joint 721

Finsterbush A. and Friedman B. 1973. Early changes in immobilized rabbits knee joints. A light and electron microscope study. C/in. Orfhop. 92, 305-319.

Gardner D. L. 1972. The influence of microscopic tech- nology on knowledge of cartilage surface structure. Ann. rheum. Dis. 31, 235-258.

McConaill M. A. 1951. The movement of bones and joints--IV. The mechanical structure of articular carti- lage. J. Bone Jr Sury. 339, 251-259.

Mowry R. W. 1958. Improved procedure for the staining of acidic polysaccharides by Mtillers colloidal (hydrous) ferric oxide and its combination with the Feulgen and the periodic acid Schiff reactions. Lab. Inrest. 7, 566576.

Radin E. C.. Swarm D. A. and Weisser P. A. 1970. Separ-

ation of hyaluronate free lubricating fraction from syno- vial fluid. Nature 288, 377-378.

Weiss C. and Mirow S. 1972. An ultrastructural study of osteoarthritic changes in the articular cartilage of human knees. J. Bone Jr Sury. 54A. 954972.

Wetzel M. G., Wetzel B. K. and Spicer S. S. 1966. Ultra- structural localization of acid mucosubstances in the mouse colon with iron containing stains. J. Cdl Biol. 30, 299-3 15.

Wright W.. Dowson D. and Kerr J. 1973. The structure of joints. Inr. Rev. Connect. Tissue Res. 6, 1055125.

Wright W. and Dowson D. 1976. Lubrication and carti- lage. J. Anar. 121. 107-118.

Plate I overleaf.

722 J. Appleton

Plate 1.

Fig. I. The articular tissue of the rat mandibular condyle showing the distinct layer of fibrillarramor- phous material covering the articulating surface. x 11.000

Fig. 2. The tibrillar-amorphous material forms a layer on the collagen fibres but between the fibres only the fibrillar component is evident. x 79,000

Fig. 3. Staining with colloidal iron has produced an electron-dense deposit covering the articulating surface together with some sub-surface staining for a depth of 0.3 pm. The membranes of vesicles

are densely stained. x 17,000

Fig. 4. Aggregates of iron particles about 30 mm in diameter on the articulating surface and immediately below the articulating surface. More superficially, individual iron particles about 3 nm in diameter

can be seen. x 158.000

Fig. 5. The surface fibrillar-amorphous material almost completely removed by treatment with hyalur- onidase. x 17,000

Fig. 6. Absence of staining with colloidal iron in the surface layer after digestion with Pronase. There is some sub-surface staining and vesicular membranes are stained with iron. x 28.000

A surface articulating layer in the mandibular joint 123

Plate 1