Avaliação da interação biológica entre compósito de quitosana, colágeno e hidroxiapatita e tecido ósseo ovino / Investigation of biological interactions of chitosan-collagen-hydroxyapatite composite with bone tissue in sheep

As lesões em membros de grandes animais são um desafio para médicos veterinários, uma vez que somente a osteossíntese não garante resultados satisfatórios. Muitos pesquisadores vêm se dedicando ao desenvolvimento e estudo de substitutos ósseos produzidos de materiais naturais, como quitosana, colágeno e hidroxiapatita, que auxiliam na regeneração óssea. Seis ovinos fêmeas da raça Santa Inês foram submetidos a ostectomias unicorticais de sete milímetros de diâmetro na região proximal da superfície dorsomedial dos III/IV metacarpianos. Foi implantado compósito de quitosana, colágeno e hidroxiapatita em um membro torácico para avaliação da biocompatibilidade do material ao tecido ósseo ovino, e no membro contralateral foi reproduzida a mesma técnica, porém foi mantido sem preenchimento, como controle. Após 60 dias do procedimento cirúrgico, realizou-se biópsia óssea na área de interface entre biomaterial/osso (membro com compósito) e tecido neoformado/osso (membro controle), para realização de avaliação histológica do material não descalcificado, por meio de microscopia de luz e microscopia eletrônica de varredura. Na análise histomorfométrica, mediante microscopia de luz, foi possível identificar maior porcentagem de tecido neoformado em membro controle, quando comparado ao membro com compósito (80% e 63,5%, respectivamente; P<0,05). Por meio da microscopia eletrônica de varredura, observou-se invasão da estrutura interna do compósito por tecido ósseo neoformado. Não houve formação de tecido cicatricial, reação de corpo estranho ou resposta inflamatória crônica nas amostras analisadas. Conclui-se que o compósito de quitosana, colágeno e hidroxiapatita, quando implantado em tecido ósseo ovino, apresenta biocompatibilidade e perfil osteocondutor.(AU)

Fracture management poses a great challenge to large animal practitioners. Osteosynthesis alone is often insufficient to provide satisfactory outcomes in large animals; therefore, several research efforts have been made to investigate and develop bone substitutes capable of promoting bone regeneration. Chitosan-collagen-hydroxyapatite composites constitute a promising alternative given their similar composition to bone. Six Santa Inês ewes were submitted to the creation of experimental 7mm wide unicortical defects on the dorsomedial aspect of the proximal III/IV metacarpal bone diaphysis. Limbs were randomly selected for treatment with chitosan-collagen-hydroxyapatite composite or to serve as untreated controls. Biopsy fragments were collected from the bone/new bone or the bone/biomaterial interface (control and treated defects respectively) within 60 days of surgery; composite biocompatibility was assessed using light and scanning electron microscopy. Histomorphometric analysis under light microscopy revealed greater percentage of new bone tissue in control compared to treated defects (80% and 63.5% respectively; P<0.05). No scar tissue formation, foreign body or chronic inflammatory reactions were observed. Scanning electron microscopy revealed invasion of the composite by new bone tissue. The chitosan-collagen-hydroxyapatite composite studied is biocompatible with bone and shows osteoconductive properties in sheep.

The effect of the solute on the structure, selected mechanical properties, and biocompatibility of Ti-Zr system alloys for dental applications.

New titanium alloys have been developed with the aim of utilizing materials with better properties for application as biomaterials, and Ti-Zr system alloys are among the more promising of these. In this paper, the influence of zirconium concentrations on the structure, microstructure, and selected mechanical properties of Ti-Zr alloys is analyzed. After melting and swaging, the samples were characterized through chemical analysis, density measurements, X-ray diffraction, optical microscopy, Vickers microhardness, and elasticity modulus. In-vitro cytotoxicity tests were performed on cultured osteogenic cells. The results showed the formation essentially of the α' phase (with hcp structure) and microhardness values greater than cp-Ti. The elasticity modulus of the alloys was sensitive to the zirconium concentrations while remaining within the range of values of conventional titanium alloys. The alloys presented no cytotoxic effects on osteoblastic cells in the studied conditions.

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats.

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n=45) received daily injections of alendronate (n=27) or sterile saline solution as control (n=18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.

Micro-shear bond strength of different adhesives to human dental enamel.

UNLABELLED: The aim of this study was to evaluate the micro-shear bond strength of 5 adhesive systems to enamel, one single-bottle acid-etch adhesive (O), two self-etching primers (P) and two all-in-one self-etching adhesives (S). METHOD: Sixty premolar enamel surfaces (buccal or lingual) were ground flat with 400- and 600-grit SiC papers and randomly divided into 5 groups (n = 12), according to the adhesive system: SB2--Single Bond 2 (O); CSE--Clearfil SE Bond (P); ADS--AdheSE (P); PLP--Adper Prompt L-Pop (S); XE3--Xeno III (S). Tygon tubing (inner diameter of 0.8 mm) restricted the bonding area to obtain the resin composite (Z250) cylinders. After storage in distilled water at 37 degrees C for 24h and thermocycling, micro-shear testing was performed (crosshead speed of 0.5 mm/min). Data were submitted to one-way ANOVA and Tukey test (a = 5%). Samples were also subjected to stereomicroscopic and SEM evaluations after micro-shear testing. Mean bond strength values (MPa +/- SD) and the results of Tukey test were: SB2: 36.36 (+/- 3.34)a; ADS: 33.03 (17.83)a; XE3: 32.76 (+/- 5.61)a; CSE: 30.61 (+/- 6.68)a; PLP: 22.17 (+/- 6.05)b. Groups with the same letter were not statistically different. It can be concluded that no significant difference was there between SB2, ADS, XE3 and CSE, in spite of different etching patterns of these adhesives. Only PLP presented statistically lower bond strengths compared with others.

Effect of different enamel treatments on bond strength using resin dental adhesives.

OBJECTIVE: The aim of this investigation was to evaluate the effect different cleaning techniques used on enamel surfaces have on the bond strength of a composite resin to the dental enamel. METHOD AND MATERIALS: Eighty-eight bovine enamel fragments were mounted in acrylic resin bases. The enamel surfaces were sanded using 200-, 400-, and 600-grit sandpapers, thus creating a smear layer. These surfaces were then randomly divided in 2 groups: 1 of them received salivary contact for 10 minutes, and the other received it for 60 minutes. Then all surfaces were cleaned with a pumice and water paste applied with a rubber cup, followed by the application of biological detergent, sodium bicarbonate jet, or air/water spray. After cleansing, the enamel surfaces received the application of the Scotchbond Multi-Purpose adhesive system and Z100 composite resin, following the manufacturer's instructions, using an appropriate matrix. After storage at 37 degrees C for 8 days, traction tests were carried out using an Instron machine operating at 0.5 mm/minute. Some fractured specimens (randomly chosen) were analyzed under scanning electron microscopy. RESULTS: The statistical analysis using descriptive and inferential methods did not show significant differences between the different periods of time of salivary contact. The technique of pumice and water paste cleaning followed by the application of biological detergent was significantly more efficient than the others. Scanning electron micrographs of the fragments after traction tests confirmed these results. CONCLUSION: The technique of pumice and water paste cleaning followed by the application of biological detergent was the treatment that allowed the best results in terms of resin bonding to bovine enamel covered with acquired pellicle, and the sodium bicarbonate jet technique presented the lowest bond strength values and seemed to disturb the acid conditioning of enamel surfaces.

Protein-energy malnutrition alters histological and ultrastructural characteristics of the bone marrow and decreases haematopoiesis in adult mice.

Protein-energy malnutrition (PEM) decreases resistance to infection by impairing a number of physiological processes, including haematopoiesis. The aim of this study was to evaluate the microanatomical aspects of bone marrow (BM) in mice that were subjected to PEM, in particular, with respect to the components of the local extracellular matrix and the proliferative activity of haematopoietic cells. For this, histological, histochemical, immunohistochemical and ultrastructural techniques were used. Two-month old male Swiss mice were fed with a low-protein diet containing 4% protein and control mice fed a 20% protein diet. When the experimental group had attained a 25% loss of their original body weight, we collected the different biological samples. Malnourished mice had presented severe BM atrophy as well as a reduction in proliferating cell nuclear antigen and gelatinous degeneration. The malnourished mice had more fibronectin accretion in paratrabecular and endosteal regions and more laminin deposition in perisinusal sites than controls. Endosteal cell activation and hyperplasia were found, suggesting their participation in the process. Additionally, we have observed a decrease in the capacity of malnourished haematopoietic stroma to support the growth of haematopoietic stem cells (CD34+) in vitro. These findings point to a structural impairment of the haematopoietic microenvironments in mice with PEM, possibly hampering the interactions between cells and cellular signalling.

Adherence of Actinobacillus actinomycetemcomitans on oral epithelial cells.

Actinobacillus actinomycetemcomitans is capable of colonizing mucosa and dental plaque and plays an important role in periodontal disease in young peoples and adult. Adherence mechanisms on epithelial cells, tooth or oral bacteria and gingival invasion probably are the initial steps in the pathogenesis of gingivitis or periodontitis. In this study, the adherence of A. actinomycetemcomitans on oral epithelial cells following subculturing were examined. The adherence on oral epithelial cells showed high in all the isolates values but with differences among them and at each time of subculturing. The adherence of A. actinomycetemcomitans FDC Y4 was stable in each of the subcultures. However, adhesion values of all the tested isolates were different except for strains #1, #38 and Y4, suggesting a heterogenicity within this microbial group. Morphologic variations were observed in extracellular structures of the A. actinomycetemcomitans tested. The adhesion process on oral epithelial cells of this organism can be influenced by subcultures, but additional studies are necessary to verify the influence of subculturing on adherence or other virulence factors.

SEM evaluation of the interaction between a three-step adhesive and dentin.

Interaction between resin tags and microtags of adhesive systems and dentinal collagen fibrils is a poorly understood aspect of adhesion. This study evaluated this interaction in 25 recently extracted human third molars. Each tooth was embedded in an epoxy resin and cross-sectioned to obtain two 1-mm-thick dentin disks. The outer dentin surfaces were polished with wet 600-grit sandpaper to create a uniform smear layer. After etching with 35% phosphoric acid for 15 seconds, the primer and adhesive of Scotchbond Multi-Purpose and the resin composite Z100 (3M Dental Products, St Paul, MN 55144, USA) were placed on the dentinal surfaces according to the manufacturer's instructions. The disks were left in distilled water at 37 degrees C for two weeks, then fractured perpendicular to the bonded surfaces in order to obtain two hemi-disks. The fractured surfaces were treated with 2N-chloridric acid and processed for scanning electron microscopy. Gold-coated specimens were examined with a JEOL 6100 scanning electron microscope. Results showed a hybrid layer with resin tags of approximately 100 microm in length and numerous and fine branching resin microtags. The tags and microtags created by this three-step adhesive system were observed in intimate contact with the collagen fibrils of dentin, even in deeper zones which were not affected by acid etching. It suggests that adhesion to dentin may include both micromechanical and chemical aspects.

Root resorptions in upper first premolars after application of continuous torque moment. Intra-individual study.

MATERIAL AND METHOD: With the purpose of investigating the occurrence, localization and extension of possible root resorptions after fixed appliance treatment with a continuous torque force, 28 upper first premolars orthodontically indicated for extraction from 14 patients were analyzed by scanning electron microscopy. Tooth movement was carried out with continuous moments of different magnitudes (300 cNmm, and 600 cNmm), using a biomechanical model with superelastic wires (stainless steel-NiTi-SE), which was specially designed and individually calibrated. The teeth were divided into one control group with four premolars (non-moved) from two patients, and two experimental groups (300 cNmm and 600 cNmm respectively) with six patients each. Each group was distributed intra-individually as follows: the right first premolar of six patients was extracted after 1 week of movement, the left first premolars were removed after 2, 3 and 4 weeks. After extraction, teeth were fixed, treated with 2% sodium hypochlorite solution for 6 hours in order to remove the organic tissue components, dehydrated, and metal-coated in a Balzers SCD 050 apparatus. RESULTS: The analysis in a scanning electron microscope (Jeol 6100, at 10-15 kV) revealed many resorption lacunae in the root surface, mainly on the lingual side in the apical third of the roots. Resorption processes were also observed on the buccal root surface in the cervical third. All experimental teeth showed resorption areas. Teeth which had been moved for a longer time period and with a higher magnitude of applied moments showed a higher degree of root resorption in width as well as in depth. Higher magnitude of moments produced exposure of root dentine, evidencing pronounced root resorption.

High-resolution immunocytochemistry of noncollagenous matrix proteins in rat mandibles processed with microwave irradiation.

The mineral phase in calcified tissues represents an additional factor to be considered during their preservation for ultrastructural analyses. Microwave (MW) irradiation has been shown to facilitate fixative penetration and to improve structural preservation and immunolabeling in a variety of soft tissues. The aim of the present study was to determine whether MW processing could offer similar advantages for hard tissues. Rat hemimandibles were immersed in 4% formaldehyde + 0.1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2, and exposed to MWs for three periods of 5 min at temperatures not exceeding 37C. They were then decalcified in 4.13% EDTA, pH 7.2, for 15 hr, also under MW irradiation. Osmicated and non-osmicated samples were dehydrated in graded concentrations of ethanol and embedded in LR White resin. Sections of incisor, molars, and alveolar bone were processed for postembedding colloidal gold immunolabeling using antibodies against ameloblastin, amelogenin, bone sialoprotein, or osteopontin. Ultrastructural preservation of tissues was in most cases comparable to that obtained by perfusion-fixation, and there was no difference in distribution of labeling with those previously reported for the antibodies used. However, the immunoreactivities obtained were generally more intense, particularly at early stages of tooth formation. Amelogenin was abundant between differentiating ameloblasts and labeling for osteopontin appeared over the Golgi apparatus of odontoblasts after initiation of dentine mineralization. We conclude that MW irradiation represents a simple method that can accelerate the processing of calcified tissues while yielding good structural preservation and antigen retention. (J Histochem Cytochem 49:1099-1109, 2001)

Ultrastructure of cementum and periodontal ligament after continuous intrusion in humans: a transmission electron microscopy study.

An ultrastructural study of the cementum and periodontal ligament (PDL) changes after continuous intrusion with two different and controlled forces in humans was carried out. Twelve first upper premolars, at stage 10 of Nolla, orthodontically indicated for extraction from six patients (mean age 15.3) were used. They were divided into three experimental groups, distributed intra-individually as follows: control (not moved), continuously intruded for 4 weeks with 50 or 100 cN force, utilizing a precise biomechanical model with nickel titanium super-elastic wires (NiTi-SE), which were developed and calibrated individually. The teeth were extracted, fixed, decalcified, and conventionally processed for examination in a Jeol 100 CX II transmission electron microscope. Evident signs of degeneration of cell structures, vascular components, and extracellular matrix (EM) of cementum and PDL were observed in all the intruded teeth, with more severe changes towards an apical direction and in proportion to the magnitude of force applied. Resorptive areas and an irregular root surface of the intruded teeth were noticed, according to the same pattern described above. Concomitant, areas of repair were also revealed in the cementum and PDL although the magnitude of forces remained the same throughout the experimental period. Thus, a reduction of continuous force magnitude should be considered to preserve the integrity of tissues.

Ultrastructure of early mineral deposition during hyaline layer formation in rat molars.

There is no consensus on whether the first mineralized layer, the hyaline layer, that is juxtaposed to root dentine is a variety of dentine or cementum or even a tissue of epithelial origin. Some suggest that there is no intermediate tissue between the acellular extrinsic fibre cementum (AEFC) and the root dentine. Here, to study hyaline layer formation and mineralization we examined by transmission electron microscopy the early stages of root development in upper molars from 10 to 13 day old Wistar rats. In addition to conventionally processed material, undemineralized and unstained sections were examined, which showed the deposition of fine mineral crystals in contact with the mineralized surface of root dentine. Early mineralization of the hyaline layer occurred in the region of the inner basement membrane, which persisted between the inner cellular layer of Hertwig's epithelial root sheath and the outer mineralized root dentine. When the root sheath began its fragment, collagen fibrils from the developing periodontal ligament began to insert into the mineralising hyaline layer, which was 0.5-0.8 micron wide. As the fragmentation of the root sheath HERS increased, more collagen fibrils appeared intermingled with the mineralising hyaline layer. In more advanced stages, when the hyaline layer had become fully mineralized and the formation of the AEFC began, the hyaline layer could no longer be identified. Thus, the hyaline layer is clearly discernible at early stages of periodontal development. Subsequently, it is masked by intermingling of cementum and dentine and therefore it is not possible to detect it in the formed roots of rat molars.

Ultrastructure of cementum and periodontal ligament after continuous intrusion in humanas: a transmission electron microscopy study / Ultraestrutura de cemento e ligamento periodontal depois da intrusão contínua em humanos: um estudo de microscopia eletrônica de transmissão

An ultrastructural study of the cementum and periodontal ligament (PDL) changes after continuous intrusion with two different and controlled forces in humans was carried out. Twelve first upper premolars, at stage 10 of Nolla, orthodontically indicated for extraction from six patients (mean age 15.3) were used. They were divided into three experimental groups, distributed intra-individually as follows: control (not moved), continuously intruded for 4 weeks with 50 or 100 cN force, utilizing a precise biomechanical model with nickel titanium super-elastic wires (Niti-SE), which were developed and calibrated individually. The teeth were extracted, fixed, descalcified, and conventionally processed for examination in a Jeol 100 CX II transmission electron microscope. Evident signs of degeneration of cell structures, vascular components, and extracellular matrix (EM) of cementum and PDL were observed in all the intruded teeth, with more severe changes towars an apical direction and in proportion to magnitude of force applied. Resorptive areas and an irregular root surface of the intruded teeth were noticed, according to the same pattern described above. Concomitant, areas of repair were also revealed in the cementum and PDL although the magnitude of forces remained the same throughout the experimental period. Thus, a reduction of continuous force magnitude should be considered to preserve the integrity of tissues

SEM study of different treatments of the smear layer on different cavity walls.

The present study evaluated the effects of slightly demineralizing treatments on dentinal cavity walls, since some recently developed adhesive procedures are applied over the smear layer. Ten experimental treatments--mechanical, chemical or mechanical/chemical--were applied on MOD cavity walls prepared in vitro with diamond burs. The dentinal surface of a lateral and of a pulpal wall of each cavity was evaluated through scanning electron microscopy, and the effects of the treatments were compared. All treatments except air/water spray removed some of the smear layer, and slight differences were observed regarding the studied cavity walls: on dentin from pulpal walls the enamel hatchet associated with tannic acid produced a better effect than the other treatments, and on dentin from lateral walls the biological detergent rubbed with cotton pellets was a little more effective than the other treatments. The effect of the smear layer treatment on dentin is different according to the wall and to the applied treatment.

Macrophages and apoptosis in the stellate reticulum of the rat enamel organ.

Tooth germs of upper first molars of 1, 3, and 5-d-old rats, fixed in formaldehyde, were stained for the detection of apoptosis by the TUNEL method, and by the azo-dye method for the demonstration of acid phosphatase. For conventional light and electron microscopy the specimens were fixed in glutaraldehyde-formaldehyde and embedded in glycol methacrylate and Araldite. Results showed that macrophages, present in the stellate reticulum, contained basophilic bodies and TUNEL-positive globules, i.e. apoptotic bodies, in their interior. Macrophages also possessed strong acid phosphatase activity. Electron microscopy showed the presence of large vacuoles inside the macrophages containing dense fragmented material. Taken together these results suggest that the intra-epithelial macrophages of the stellate reticulum engulf apoptotic bodies.

Ultrastructural preservation of rat embryonic dental tissues after rapid fixation and dehydration under microwave irradiation.

Adequate preservation of the cells and matrix of mineralising tissues remains difficult, as organic components and initial mineral deposits may be lost during conventional processing for electron microscopy. In this study, we have reduced significantly the processing time using microwave irradiation. Rat molar tooth germs were fixed in 4% glutaraldehyde + 4% formaldehyde with 0.1 M sodium cacodylate in a laboratory microwave oven for two periods of 20 s with a maximal temperature of 37 degrees C. After conventional washing and post-fixation, specimens were dehydrated in graded ethanols under microwave irradiation for a total of 7 min 20 s. For comparison, some specimens were processed by conventional methods. After embedding, ultrathin sections were examined by electron microscopy. In differentiating ameloblasts and odontoblasts, plasma membranes, mitochondria, rough endoplasmic reticulum, the Golgi complex, together with all other cytoplasmic organelles exhibited excellent preservation. Microtubules, microfilaments and coated vesicles were particularly evident. Crystal-like mineral deposits were conspicuously present in relation to dentine matrix vesicles and collagen fibrils as well as in enamel matrix. The matrix of forming enamel had a globular electron-lucent appearance. It is concluded that this is a rapid method which provides a preserved or even improved morphology.

Apoptosis in the early involuting stellate reticulum of rat molar tooth germs.

When the enamel organ of the rat tooth germ is fully developed at the tip of the prospective cusp, amelogenesis begins, and at this site the overlaying stellate reticulum begins its involution. During the involution process, there is a gradual decrease in intercellular spaces, invasion by blood vessels, appearance of macrophage-like cells and reduction in the number of stellate reticulum cells. Since reduction or disappearance of cells during embryonic development in organs and tissues has been shown to occur by apoptosis, we decided to examine early involuting regions of the stellate reticulum in the hope of detecting apoptosis. For this purpose, upper first molars of Wistar newborn rats aged 1 and 3 days were fixed in formaldehyde for the TUNEL method and in glutaraldehyde-formaldehyde for light and electron microscopy. Paraffin sections revealed TUNEL-positive structures, i.e. brown-yellow-stained bodies, in the central portion of the stellate reticulum, and next to the outer enamel epithelium and stratum intermedium. Examination of ultrathin sections confirmed the TUNEL findings: some stellate reticulum cells showed nuclei containing crescent-like electron-opaque condensed masses of peripheral chromatin, typical of apoptosis. Also, apoptotic bodies of various sizes and appearances were frequently observed within stellate reticulum cells. We should like to suggest that apoptosis is associated with the reduction in the number of cells during regression of the reticulum.

Root resorptions in upper first premolars after application of continuous intrusive forces. Intra-individual study.

A scanning electron microscopy study of possible root resorptions and their localization after application of continuous forces of different magnitudes was conducted. Twelve upper first premolars, indicated for extraction, were previously intruded with constant forces. The teeth were divided into 3 groups: 1. non-moved control teeth, 2. continuous force application of 50 cN for 4 weeks, 3. continuous force application of 100 cN for 4 weeks. Specially designed NiTi-SE-stainless steel springs were utilized to exert the actual forces. After experimental tooth movement, the extracted teeth were dehydrated, metal-coated and examined by scanning electron microscopy. The intruded teeth showed resorptive areas consisting of lacunae (concavities) in the mineralized root surface. The teeth moved with 50 cN showed in the apical third several, in the medial third few, and in the cervical third no resorptive areas. In the case of the teeth moved with 100 cN, we observed resorptive areas in most of the apical third--including the apex contour-, several in the medial third, and none in the cervical third. In the control group no resorptions were observed. Thus, our results suggest that intrusion of human teeth with continuous forces induces root resorption, depending on the magnitude of force applied.

Scanning electron microscopy examination of resorbing enamel surfaces in unexfoliated primary molar teeth.

We examined unexfoliated primary molar teeth by scanning electron microscopy to study the resorbing internal enamel surfaces. They were fixed in a cacodylate buffered glutaraldehyde-formaldehyde mixture, treated with 2 percent sodium hypochlorite, washed, dehydrated with ethanol and air-dried, before sputter-coating with a 40 nm layer of gold. The examination with a Jeol 6100 scanning electron microscope revealed large areas of enamel resorption, characterized by closely adjoining honeycomb-like lacunae (concavities). In higher magnification, lacunae show enamel with various patterns of resorption: whereas in some regions, only the central portions of prisms are removed; in others, the prism sheath regions are seen below the other enamel structures. Also, regions showing a random pattern of resorption are observed. In addition, lacunae showing resorbing enamel prisms in longitudinal and other orientations are also observed. The results of the present study reveal, therefore, that in unexfoliated primary teeth, the odontoclasts first remove the coronal dentin and then reach and resorb large areas of enamel. Furthermore, the various patterns of resorption support the idea that removal of enamel by odontoclasts depends upon the orientation of enamel structures, rather than of the different degrees of mineralization suggesting that enamel structures do not possess different inorganic/organic composition.

Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarisation.

We have examined freeze-fracture replicas of maxillary first molar tooth germs of newborn rats at early stages of dentinogenesis to study the development of tight junctions in the distal plasma membrane of differentiating odontoblasts. In addition, freeze-fracture was combined with filipin to observe the distribution of cholesterol on the distal plasma membrane of odontoblasts during differentiation. Only gap junctions were present in early differentiating odontoblasts. The distal plasma membrane exhibited low cholesterol content, which might indicate high fluidity. With the beginning of mineral deposition in matrix-vesicles, the first signs of tight junction formation were observed. Further development revealed increasingly complex focal tight junctions. In later stages, when mineralisation is observed progressing to the fibrillar and non-fibrillar constituents of the matrix, well developed focal tight junctions were detected. Concomitantly, cholesterol in distal portions of the odontoblast plasma membrane increased, indicating, probably, a higher rigidity. Thus, a distal plasma membrane domain is established, odontoblasts become fully differentiated, and partial compartmentalisation of matrix occurs. At this stage, odontoblasts may be able to secrete specific matrix molecules to ensure the progression of mineralisation.