the temporomandibular joint ( tmj ) is a joint of the sinovial , double and bicondilar type .
it is situated between the condylar process and the mandibular fossa of the temporal bone .
it has an articular capsule , synovial membrane , ligaments and associated muscles . in comparison with other synovial joints ,
the tmj is unique because of the presence of the articular disc that separates it into two independent cavities , known as infra and supradiscal cavities , with distinct morphological features . at the end of the 12 gestational week
morphologically , the articular disc is a small elliptic fibrocartilaginous plate shaped like an s in sagittal cut and having its bigger axis in the posteromedial direction .
the articular disc is divided into four bands : anterior , intermediate , posterior and bilaminar zone .
the disc also plays an important functional role in translation and rotation movements , in a broad and passive manner .
the articular disc can be described as a concave lens with two faces ( superior and inferior ) , two margins ( medial and lateral ) and two extremities ( anterior e posterior ) . during the embryogenesis period ,
the articular disc of the tmj consists of a dense layer of mesenchymal cells that derived from the condylar blastema in the anterior region and from the glenoidal blastema in the posterior region . for sab
, the architecture of the articular disc is complex , with collagen fibres predominating in the longitudinal direction of the superior and inferior faces and cross - sectional and interposed oblique fibres predominating in the intermediate portion . for minarelli and liberti , the disc has compact collagen bands in the intermediate band , with fibres running in the anteroposterior direction .
takahashi and sato examined the distribution of types i and iii collagen , fibronectin and elastic fibres in the articular disc of 54 adult cadavers and 24 foetuses aged 12 , 16 , 20 , 24 , 28 and 32 weeks with light microscopy and scanning electron microscopy . according to the authors , at 12 weeks ,
the collagen fibres formed a thin layer on the entire disc ; at 16 weeks , the disc already had anteroposteriorly - oriented collagen fibres in the intermediate band and cross - sectional fibres were found in the most superficial layers of the disc ; finally , at 20 weeks , the authors found a disc consisting of a complex arrangement of collagen fibres , oriented in various directions . in terms of structural biology , the collagen fibres inside the articular disc of the tmj
are composed mainly of type i collagen and small amounts of type iii collagen . according to takahashi and sato , the concentration of type - i collagens in the articular discs of foetuses aged 20 to 24 weeks increased in all bands , especially in the anterior and intermediate bands .
meanwhile , the expression of type - three collagen in foetuses of the same gestational age increased only in the intermediate band .
confirmed that a large number of fibroblast - like cells in the human tmj disc was co - localized with lumican expression in their specimens .
in contrast , little lumican expression was observed in the vicinity of fibrochondrocytes and chondrocyte - like cells .
matsumoto et al . demonstrated the presence of has3 in the chondrocyte - like cells of both normal and deformed tmj discs in adults .
a study done at the institute of anatomy , university of catania , italy , used four articular discs from adult cadavers as controls .
a multi - directional preservation of the collagen bands was found in all samples , in addition to a predominance of fibrocyte - like cells , and in a smaller aggregated amount , chondrocyte - like cells .
the apparent presence of fibrocartilaginous areas also suggests the presence of type ii collagen fibers .
fibrous proteins ( collagen and elastin ) , responsible for the shape and structure of the lamina propria , are associated with resistance and elasticity , while interstitial proteins ( glycoproteins and proteoglycans ) control viscosity , hydration , and the tissue volume extracellular matrix . according to gray et al . , the characteristics of the layers of the lamina propria are intimately associated with the arrangement and distribution of the fibrous proteins in the extracellular matrix , especially collagen fibres , which support and absorb the physical stress caused on the articular disc by joint movement .
collagen belongs to a family of structural proteins with distinct chemical compositions , morphologies and functions .
collagen is a component of many organs and tissues and is the main extracellular matrix element of multicellular animals , representing from 2530% of the protein mass of the organism .
the different types of collagens have been organized into the following classes : fibrillar , associated with fibrils , non - fibrillar , filamentous , short- and long - chain .
it has also been demonstrated that some types of collagen are capable of aggregating molecules to form heterotypical fibrils .
the presence of heterotypical fibrils with different combinations in many tissues has already been reported .
evidence of these combinations obtained with immunohistochemical analysis has been observed between types i and v collagen in human skin , tendon and cornea and between types ii and xi , and between i and ii in cartilage . given the scarcity of studies that focus morphologically on the types of collagen in the tmj disc of human foetuses , we believe a better structural knowledge of the relationship of these proteins with the articular disc of the tmj is necessary .
thus , the objective of this study is to analyse the immunohistochemical expression of types i and iii collagen markers in the articular disc of the temporomandibular joint of human foetuses .
the protocol of this study was submitted to the unifesp research ethics committee and approved under number 0225/05 . our sample consisted of 10 human foetuses supplied by the universidade federal de uberaba , resulting in 20 tmj with gestational ages from 17 to 24 weeks .
the gestational age of the foetuses was determined by measuring the crown - rump ( cr ) length .
all foetuses were preserved in a 10% formalin solution and dissected by removing the skin , subcutaneous tissue and exposing the deep structures .
two sections of 3 m were taken from each paraffin block containing the collected material and were then spread on glass slides .
the streptavidin - biotin technique was used to analyse the immunohistochemistry reactions of types i and iii collagens .
sections were deparaffinised in three changes of xylene ( 10 min each ) and rehydrated in a graded series ( 100 , 80 and 70% ) of ethanol finishing in distilled water .
antigen sites were retrieved by pretreatment with 0.5 m acetic acid for 90 min at 37c and multiple rinses with distilled water .
endogenous peroxidases were quenched by incubating in 3% h2o2 ( 20 min , room temperature ) .
the slides were then washed 32 min in pbs and incubated in milk powder solution for 20 min to block nonspecific binding of antibody .
sections were incubated with 100 l of the primary antibody overnight at 4c.primary antibody was purified rabbit anti - human type i collagen , 1:800 ( novatec inc .
, baltimore , md , usa ) and rabbit monoclonal anti - human type iii collagen ( novatec inc . ) .
subsequently , slides were washed in pbs ( 32 min ) , and detection of the primary antibody was performed using the lsab kit ( dako cytomation , carpinteria , ca , usa ) .
sections were incubated with biotinylated secondary antibody for 30 min , washed ( 32 min in pbs ) , and incubated with streptavidin - peroxidase ( hrp ) conjugate for 30 min .
the sections were again washed ( 32 min in pbs ) before the antibody - hrp complex was visualized by incubation with diaminobenzidine ( dab lsab + system - hpr dako cytomation ) for 5 min .
slides were briefly counter - stained in harris hematoxylin and dehydrated , and cover slips added .
st louis , md , usa ) resin , analysed , and photographed with a light microscope ( olympus bx50 ) .
the tissues recommended by the manufacturer were used as positive antibody controls.the negative controls were the same cases used as positive controls , which were submitted to the immunohistochemical reaction described above , except for the incubation with the primary antibody , done with a buffer solution .
figure 1a shows numerous type i collagen fibres throughout the extension of the articular disc of the tmj
. however , these fibres are more abundant in the posterior band of articular disc ( 1b ) .
additionally , figure 1c shows a large concentration of type iii collagen fibres on the inferior face of the articular disc .
figure 1d shows type iii collagen fibres in the intermediate band of the articular disc .
figure 1immunohistochemical micrograph of type i collagen . a ) , foetus aged 19 weeks of intrauterine life ; there are collagen fibres in the entire disc ( a ) , mandibular head ( b ) and temporal bone ( c ) ; b ) , immunohistochemical photomicrograph of type i collagen in foetus at 22 weeks of intrauterine life ; the posterior band of the articular disc contains numerous complexly - arranged collagen fibres ; c )
, immunohistochemical micrograph of type iii collagen in foetus at 21 weeks of intrauterine life ; the inferior face ( a ) of the articular disc is delimited by numerous collagen fibres ; d ) , immunohistochemical micrograph of type iii collagen in foetus at 24 weeks of intrauterine life ; collagen fibres are arranged in many directions on the inferior face ( intermediate ) .
the heart tissues used as positive controls for collagen i and iii are represented in panels e ) and f ) , respectively . 
 
a ) , foetus aged 19 weeks of intrauterine life ; there are collagen fibres in the entire disc ( a ) , mandibular head ( b ) and temporal bone ( c ) ; b ) , immunohistochemical photomicrograph of type i collagen in foetus at 22 weeks of intrauterine life ; the posterior band of the articular disc contains numerous complexly - arranged collagen fibres ; c ) , immunohistochemical micrograph of type iii collagen in foetus at 21 weeks of intrauterine life ; the inferior face ( a ) of the articular disc is delimited by numerous collagen fibres ; d ) , immunohistochemical micrograph of type iii collagen in foetus at 24 weeks of intrauterine life ; collagen fibres are arranged in many directions on the inferior face ( intermediate ) .
the heart tissues used as positive controls for collagen i and iii are represented in panels e ) and f ) , respectively .
minarelli and liberti have a stated that the anterior band contains 85% of type i collagen fibres and 15% of type iii collagen fibres , while the posterior contain 94% of type i collagen fibres and 6% of type iii collagen fibres .
consider that the fibres inside the disc are composed mainly of type i collagen and are responsible for roughly 80% of the dry mass of the disc .
figures 1a and 1b show numerous type i collagen fibres throughout the tmj disc , especially in the posterior band .
figure 1c shows a great concentration of type iii collagen fibres located on the inferior face of the disc , while figure 1d shows complexly arranged type iii collagen fibres running in many different directions .
this does not mean that the anterior , intermediate and posterior bands of the disc were not marked by the antibody , but when type i collagen fibres are compared with those of type iii , there is a prevalence of type i in the posterior band of the disc and a prevalence of type iii on the entire inferior face of the disc . in foetuses and children ,
type i collagen fibres have a stratified arrangement , organized in fibre bundles with an anteroposterior , laterolateral and oblique orientation forming mesh . in the posterior band of the disc , in agreement with our findings ,
the laterolateral fibre bundles were marked and , in agreement with minarelli and liberti , they formed a ring that surrounded the disc .
in contrast , in the intermediate band of the disc , there was a prevalence of longitudinally oriented fibres in the anteroposterior direction .
descriptions of the arrangement that constitute the architecture of the articular disc have been made in humans of different ages and also in primates . again regarding the composition of collagen fibres , it is important to point out that the aggregation of fibrillar collagen molecules to form heterotypical fibrils has already been described in many structures such as skin , tendon , meniscus , cartilage , human cornea and heart . in our study
we observed that these heterotypical fibrils had probably formed , in agreement with takahashi and sato who demonstrated that the disc of foetuses of various gestational ages contains types i and iii collagens , and probably type ii , as judged berkovitz et al . by the presence of fibrocartilaginous areas in the articular disc .
the distribution pattern of the fibres throughout the foetuses articular discs is different from that observed in adults articular discs .
the following hypothesis may be considered : for the distribution patterns of types i and iii collagens to change as the foetus develops and in adults , some events , such as sucking , small mandibular movements and load , begin during the maturation stage , after the twelfth week of development , and are lifelong .
thus , findings from this study may lead us to conclude that immunohistochemical expression of types i and iii antibodies is positive for the presence for these collagen fibres and heterotypical fibril meshes form between those two types of collagen .
 the objective was to study the morphology of the articular disc and analyse the immunohistochemical expression of types i and iii collagen markers in the temporomandibular joint ( tmj ) disc of human foetuses of different gestational ages . 
 twenty tmj from human foetuses supplied by universidade federal de uberaba with gestational ages from 17 to 24 weeks were studied . 
 the gestational age of the foetuses was determined by measuring the crown - rump ( cr ) length . 
 macroscopically , the foetuses were fixed in 10% formalin solution and dissected by removing the skin and subcutaneous tissue and exposing the deep structures . 
 immunohistochemical markers of type i and iii were used to characterize the existence of collagen fibres . 
 analysis of the immunohistochemical markers of types i and iii collagen revealed the presence of heterotypical fibril networks . 
Introduction
Materials and Methods
Results
Discussion