Biocompatibility Studies of Nanoengineered Polycaprolactone and Nanohydroxyapatite Scaffold for Craniomaxillofacial Bone Regeneration.

Currently there is an increased demand for synthetic bone substitute materials (SBSMs) due to avoidance of donor-site surgery and morbidity. Attempts are done to create SBSM mimicking the bone microarchitecture for enhanced healing. In this study, the authors nanoengineered polycaprolactone (PCL) and nanohydroxyapatite (nHAp) composite scaffold by electrospinning. The nHAp is synthesized via hydrothermal process followed by microwave irradiation. In vitro biocompatibility evaluation with MG63 osteoblastic cell line showed enhanced cell proliferation in the PCL-nHAp scaffold than plain PCL by MTT assay and fluorescence microscopy. Increased osteogenesis in the PCL-nHAp scaffold was shown by the increased calcium load, alkaline phosphatase activity, and expression of osteogenic biomarkers namely osteocalcin, osteonectin, and osteopontin. In vivo studies conducted in rabbit femur bone defects showed increased bone regeneration in PCL-nHAp implanted defects. The results show that PCL-nHAp electrospun scaffold is biomimetic and highly osteogenic and thus a potential SBSM for critical size craniomaxillofacial bone defect applications.

Microarchitectural pattern of pristine maxillary bone.

PURPOSE: There is limited evidence available on the influence of location on bone density in the maxilla. Therefore, this study was aimed at comparing the microarchitecture of bone harvested from different nonatrophic maxillary locations. MATERIALS AND METHODS: A total of 37 partially edentulous subjects (aged 48.15 ± 15.85 years) were included in the study. A bone core biopsy specimen was obtained from one site per patient at the planned implant surgery location. Thirty-four specimens were used for microcomputed tomography (micro-CT) analysis. Mann-Whitney U tests (independent samples) were performed to determine whether the distributions of the six bone-related parameters showed significant differences between sexes and site locations. Study sites were categorized as either anterior (incisors and canines) or posterior (premolars and molars). The possible associations among variables (bone volume fraction [BV/TV], age, and five bone-related parameters) were examined using the Spearman rank correlation test. RESULTS: The mean BV/TV values showed no significant difference between the maxillary anterior (46.93 ± 26.2) and posterior (51.90 ± 28.42) locations. Statistically significant positive correlations were identified between BV/TV and trabecular thickness (Tb.Th) (r = 0.6, P < .001) and between BV/TV and trabecular number (Tb.N) (r = 0.49, P = .006). Statistically significant negative correlations were found between BV/TV and trabecular spacing (Tb.Sp) (r = -0.65, P < .001), between BV/TV and trabecular pattern factor (Tb.Pf) (r = -0.7, P <.001), and between BV/TV and the structural model index (SMI) (r = -0.68, P <.001). However, no correlations between BV/TV and age or sex were found. CONCLUSION: Bone density was independent of the anatomical location, assessed by micro-CT in the pristine nonatrophic maxillary bone. Studies with a larger sample size and different population should be conducted to validate the findings of the current project.

Segmental odontomaxillary dysplasia: report of a case emphasizing histopathological, immunohistochemical and scanning electron microscopic features.

Segmental odontomaxillary dysplasia (SOD) is a rare developmental disorder of the maxilla, and there is little information on its morphological features. Thus, the present article describes a case of SOD focusing on its histopathological, immunohistochemical and scanning electron microscopic features. Several dental abnormalities were present, including numerous dentin and pulp defects, altered composition of hard tissue, and proliferation of myofibroblasts in the pulp and the soft tissue surrounding affected teeth. This myofibroblastic proliferation was identified for the first time in SOD and may be involved in both bone and tooth resorption mechanisms.

Is micro-computed tomography reliable to determine the microstructure of the maxillary alveolar bone?

OBJECTIVES: To analyze the reliability of micro-computed tomography (micro-CT) to assess bone density and the microstructure of the maxillary bones at the alveolar process in human clinics by direct comparison with conventional stereologic-based histomorphometry. MATERIALS AND METHODS: Analysis of osseous microstructural variables including bone volumetric density (BV/TV) of 39 biopsies from the maxillary alveolar bone was performed by micro-CT. Conventional stereologic-based histomorphometry of 10 bone biopsies was performed by optic microscopy (OM) and low-vacuum surface electronic microscopy (SEM). Percentages of bone between micro-CT and conventional stereologic-based histomorphometry were compared. RESULTS: Significant positive correlations were observed between BV/TV and the percentage of bone (%Bone) analyzed by SEM (r = 0.933, P < 0.001), by toluidine blue staining OM (r = 0.950, P < 0.001) and by dark field OM (r = 0.667, P = 0.05). The high positive correlation coefficient between BV/TV and trabecular thickness illustrates that a value of BV/TV upper than 50% squares with a bone presenting most of their trabecules thicker than 0.2 mm. The high negative correlation between BV/TV and trabecular separation shows that values of BV/TV upper than 50% squares with a bone presenting most of their trabecules separated less than 0.3 mm each other. CONCLUSION: BV/TV assessed by micro-CT correlates with the percentage of bone assessed by conventional stereologic-based histomorphometry. Micro-CT is a reliable technique to determine the bone density and the microstructure of the maxillary alveolar bone at the site of dental implant insertion.

Bone tissue microarchitectural characteristics at dental implant sites. Part 1: identification of clinical-related parameters.

OBJECTIVE: To identify the characteristics of bone tissue microarchitecture by microCT at dental implant bone sites, describing them in terms of clinical parameters. MATERIAL AND METHODS: Forty-six bone implant sites of the maxilla and mandible from 32 volunteers were evaluated by conventional radiographs and CT scans. During the installation of each implant, bone biopsies were removed using a trephine bur at the first drilling to prepare the socket implant. Each sample was evaluated by microCT and dimensional parameters measured. RESULTS: Factor analysis summarized the microparameters into four components, which accounted for 92.8% of the total variance. The identified factors were (1) architecture - variables affecting 3D trabecular bone configuration and organization, (2) density - variables relating to surface/volume ratios and volume/volume ratios, (3) bulk - variables relating to the amount of bone and (4) spacing - variable related to the distance between trabeculae and the quantity and organization of marrow spaces. CONCLUSION: These four summarized factors correspond to clinical and radiographically recognizable parameters used for routine bone quality evaluation for implant treatment planning, which can potentially influence the primary stability of dental implants. The understanding of factors related to bone microarchitecture might reveal important aspects of its mechanical properties, essential for implant success.

[Application of ultrasonography in diagnosis and treatment of acute inflammation of soft tissues in maxillofacial region and neck].

We investigated the value of ultrasonography (USG) and ultrasound-guided aspirations in diagnosis and treatment of acute inflammation of soft tissues in maxillofacial region and neck. We performed 174 USG in 136 patients (73 with abscesses and phlegmons, 27 with acute lymphadenitis, 16 with inflamed cysts, 12 with parotitis, 3 with sialolithiasis and 5 with diseases, simulated acute inflammation). Ultrasound-guided aspirations performed in 35 patients. We considered USG to be useful diagnostic tool in acute inflammation of soft tissues in maxillofacial region and neck. USG allowed to determine localization and stage of inflammation, discriminate it from non-inflammatory diseases and improve assessment of its dynamics. Ultrasound-guided aspirations was a valuable tool for diagnostic and treatment purposes in inflamed cysts.

Relationship between files that bind at the apical foramen and foramen openings in maxillary central incisors - a SEM study

Considering that instrumentation of the apical foramen has been suggested for root canal infection control, this study analyzed the relationship between the files that bind at the apical foramen and the foraminal openings in 50 maxillary central incisors. After preparation of the pulp chamber, access to the canal was obtained with #1 and 2 LA Axxess and K-files with tip cut were inserted up to the apical foramen until binding was felt. The files were fixed with methyl cyanoacrylate and the tooth-file sets were cross-sectioned 10 mm short of the apex. Scanning electron microscopic analysis was carried out and files and foraminal areas were measured using Image Tool software. Statistically significant difference (p<0.0001) was found between files and the apical foraminal areas. The mean foraminal area was 3.8 times larger than the mean file area. The results of this study suggest that it would require 4 files of greater size beyond the one that bound to the foramen in order to allow a better relationship between files and apical openings of maxillary central incisors.

Uma vez que a instrumentação do forame apical tem sido sugerida para o controle de infecção do canal radicular, este estudo analisou a relação entre as limas que se ajustam no forame apical e a abertura foraminal em cinquenta incisivos centrais superiores. Após o preparo da câmara pulpar, foi feito o acesso radicular com as brocas LA Axxess #1 e 2 e limas K com a ponta cortada foram inseridas até que oferecessem a sensação tátil de ajuste no forame apical. Foram fixadas com cianoacrilato de metila e o conjunto dente-lima foi seccionado a 10 mm aquém do ápice. Foi feita a microscopia eletrônica de varredura e as áreas das limas e dos forames foram medidas por meio do Image Tool software. A análise estatística demonstrou diferença significante entre as áreas das limas e dos forames (p<0,0001). A média da área dos forames apicais foi 3,8 vezes maior que a das limas. Os resultados deste estudo sugerem que seriam necessários 4 instrumentos de calibre maior além do que se ajustou para que haja melhor relação entre as limas e as aberturas foraminais nos incisivos centrais superiores.

Histomorphometric comparison of maxillary pristine bone and composite bone graft biopsies obtained after sinus augmentation.

INTRODUCTION: Sinus grafting is a technique oriented to facilitate implant placement in posterior atrophic maxillae. Several modifications of the original technique and a wide variety of materials have been proposed; most of them associated with implant survival rates. However, the quality of the bone obtained after the application of certain grafting materials has not been fully elucidated yet. The aims of this multicenter study were to analyse histomorphometrical samples obtained 6 months after sinus grafting using a composite graft consisting of anorganic bovine bone (ABB)+ autologous bone (AB), and to compare these samples with maxillary pristine bone biopsies. MATERIAL AND METHODS: Ninety maxillary sinus augmentations were performed for delayed implant placement (N = 90) in 45 consecutive patients (test group). Bone cores were harvested 6 months after grafting for histomorphometric and ultrastructural study. Control pristine bone biopsies were taken from the posterior maxilla of 10 patients (control). Bone radiographic changes were assessed up to 24 months after implant loading. RESULTS: The total mean values after analysis of test cores revealed a proportion of 46.08 + or - 16.6% of vital bone, 42.27 + or - 15.1% of non-mineralized connective tissue, and 37.02 + or - 25.1% of the remaining ABB particles. Significant bone remodeling activities were noticed in sinus grafting samples when compared with pristine bone. A statistically significant difference was observed in the number of osteoid lines between two groups, with higher values in the test one (15.1 + or - 11.48% vs. 2.5 + or - 2.2%, P = 0.0005). Ultrastructural study showed that vital trabecular bone was in intimal contact with ABB particles. Radiographic analysis revealed that the higher the proportion of remaining ABB, the lower the total vertical resorption of the graft. CONCLUSION: Sinus grafting constitutes an excellent model for the study of de novo bone formation patterns and graft consolidation, when a combination of different bone substitutes is applied. The combination of ABB+AB yields highly satisfactory outcomes from both a clinical and a histologic perspective.

[An ultrastructure study on the palatomaxillary suture of dog expanded by NiTi-SMA].

OBJECTIVE: To explore the ultrastructure of the palate-maxillary sutures under tensile forces by transmission electron microscope (TEM). METHODS: The Suture expanders were made in NiTi-Shape memory alloy (NiTi-SMA). The maximum force was 3.5 N. Fourteen 8-month old mongrel dogs were used in the study. They were divided into three groups, (1) experimental group, (2) control group, (3) sham group. In the experiment and control groups, an 8 mm wide cleft was made by surgery. The suture expanders were fixed onto the palatine bones of the experimental group. The dogs of the experimental group were executed after 3, 7, 14, 28, 56 days of suture expansion. The change of suture tissue was examined by TEM. RESULTS: The cleft of the experiment group were closed at the ninth day of expansion. At the beginning, tissue rupture, exudation, death of fibroblasts, disruption of collagen and tear vessels were seen at the early stage of suture expansion. Then highly active functional manifestations were seen in both osteocytic and fibrocytic populations. At last, normal structure restored. CONCLUSIONS: Cell types and functional condition could be distinguished clearly by TEM. It suggests that the suture expansion should be the process of tissue repair and regeneration. The suture cells response, especially, the osteogenic response were the major factor of increasing suture width.

Back-scattered electron imaging and elemental microanalysis of retrieved bone tissue following maxillary sinus floor augmentation with calcium sulphate.

OBJECTIVES: To investigate the presence and composition of residual bone graft substitute material in bone biopsies from the maxillary sinus of human subjects, following augmentation with calcium sulphate (CaS). MATERIAL AND METHODS: Bone cores were harvested from the maxillary sinus of patients who had undergone a sinus lift procedure using CaS G170 granules 4 months after the initial surgery. Samples from seven patients, which contained residual biomaterial particles, were examined by field emission scanning electron microscopy and energy dispersive X-ray spectroscopy was used to determine the composition of the remaining bone graft substitute material. RESULTS: Residual graft material occurred in isolated areas surrounded by bone and consisted of individual particles up to 1 mm in length and smaller spherical granules. On the basis of 187 separate point analyses, the residual material was divided into three categories (A, B and C) consisting of: A, mainly CaS (S/P atomic% ratio > or =2.41); B, a heterogeneous mixture of CaS and calcium phosphate (S/P=0.11-2.4) and C, mainly calcium phosphate (S/P< or =0.11; C), which had a mean Ca : P ratio of 1.63+/-0.2, consistent with Ca-deficient hydroxyapatite. Linescans and elemental maps showed that type C material was present in areas which appeared dense and surrounded, or were adjacent to, more granular CaS-containing material, and also occurred as spherical particles. The latter could be disintegrating calcium phosphate in the final stages of the resorption process. CONCLUSIONS: CaS resorption in the human maxillary sinus is accompanied by CaP precipitation which may contribute to its biocompatibility and rapid replacement by bone.

[Evaluation of the efficacy of porous alumina ceramic use for defects elimination in maxillofacial region].

To study the biocompatibility of porous glass ceramics, experiments were conducted on: 75 white mice (Vistar); 11 rabbits (Shinshila) and 6 dogs. The porous alumina ceramic implants were embedded subperiostaly without additional fixation to bone surface. The osseointegration of implants, the size and form maintaining, changing of physical properties and long-term results in bone substitution were studied. The handling properties during surgery were also evaluated.

Guided bone regeneration at dehiscence-type defects using biphasic hydroxyapatite + beta tricalcium phosphate (Bone Ceramic) or a collagen-coated natural bone mineral (BioOss Collagen): an immunohistochemical study in dogs.

The aim of this study was to immunohistochemically investigate bone regeneration following application of either hydroxyapatite+beta tricalcium phosphate (BCG) or a collagen-coated natural bone mineral (BOC) in combination with a collagen membrane at dehiscence-type defects in dogs. Standardized buccal dehiscence defects were surgically created following implant bed preparation in six beagle dogs. Defects were randomly filled with either BOC (BioOss Collagen) or BCG (Bone Ceramic) according to a split-mouth design, and covered with a native porcine derived collagen membrane (BioGide). After 1, 4 and 9 weeks of submerged healing, dissected blocks were processed for immunohistochemical (osteocalcin) and histomorphometrical analysis (residual defect length, new bone-implant contact, area of new bone fill, percentage of osseointegrated bone-graft particles). Both groups revealed a significant decrease in mean residual defect length, and increases in mean new bone-implant contact, bone fill and percentage of osseointegrated bone-graft particles after 4 and 9 weeks of healing. Remaining BCG and BOC granules were completely integrated into a secondarily formed network of spongiosa, but there was no osteoclastic activity at the surface of either type of bone-graft particle. Both BCG and BOC may provide an osteoconductive scaffold to support guided bone regeneration procedures at dehiscence-type defects.

Early development of the lower deciduous dentition and oral vestibule in human embryos.

The aim of this work was to investigate the early development of the deciduous dentition and oral vestibule in the human embryonic lower jaw. Histological sections and three-dimensional reconstructions from prenatal weeks 6-9 were used. A continuous anlage for the oral vestibule did not exist in the mandible. In contrast to the upper jaw, where we previously observed that the dental and vestibular epithelia developed separately, two dento-vestibular bulges differentiated in the incisor region of the mandible. The lingual parts of each bulge were found to give rise to the respective central and lateral incisors, whereas the labial parts differentiated into the vestibular epithelium. In the canine and molar areas, the dental and vestibular epithelia originated separately. Later, the segments of the vestibular epithelium fused into the labial vestibular ridge, giving rise to the lower oral vestibule in the lip region. In the cheek region, the oral vestibule was found to originate in the mucosal inflection between the developing jaw and the cheek. A similar heterogeneous developmental base for the oral vestibule was also observed in the upper jaw. There is thus no general scheme for the early development of the dental and vestibular epithelia that applies to both the upper and lower jaws, and to both their anterior and posterior regions.

Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice.

Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior to palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G(1)/S progression of palatal mesenchymal cells through upregulation of p21(Cip1), leading to Rb hypophospholylation. Thus, RA appears to cause G(1) arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: an ultrastructural and cytochemical study

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4ºC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37ºC. In the first day, the specimens were irradiated 9 times and stored at 40ºC overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

A preservação da estrutura de ossos é dependente da qualidade e da velocidade em que ocorre o processo de desmineralização. Neste estudo foi observada a ultraestrutura de maxila de rato descalcificada utilizando microondas. Ratos Wistar sofreram perfusão com paraformaldeído e o segmento de maxila retirado e fixado em glutaraldeído. Após esta etapa algumas amostras foram descalcificadas por imersão em solução de Warshawsky durante 45 dias a 4(0)C. Outras amostras foram submetidas a irradiação por microondas (forno de microondas doméstico 700 Watts de potência), durante 20 s/350 W/ ± 37ºC. No primeiro dia foram realizadas um total de 9 irradiações e os espécimes foram deixadas posteriormente a 4ºC por 12 h na solução descalcificadora sem agitação. No segundo dia, os fragmentos foram submetidos à nova irradiação totalizando 20 banhos, trocando-se a solução e o gelo a cada banho. A seguir algumas amostras foram pós-fixadas com tetróxido de ósmio e outras com tetróxido de ósmio e piroantimonato de potássio. As amostras foram observadas em microscópio eletrônico de transmissão. Os resultados mostraram que o processo de descalcificação ativado por microondas reduziu para 48 h o período de descalcificação, o qual pelo método tradicional ocorre em 45 dias.

Element release from titanium devices used in oral and maxillofacial surgery.

Optical microscopy, scanning electron microscopy and X-ray microanalysis (EDS system) were used on c.p. titanium devices (21 grids and 10 plates) removed from 28 patients without signs of inflammation 6-24 months after surgery. Plates, grids and surrounding tissue were investigated to evaluate the titanium release and accumulation. Titanium was only present in the interfacial bone, probably due to fretting, and in all fibrous tissue surrounding the devices. Titanium content followed a decreasing gradient extending from the device surface and was not detected at a distance greater than 1mm. High titanium levels were found in blood cells in the connective tissue. In conclusion, titanium release from the devices stops only after bone is laid down on the titanium surfaces. Titanium release does not seem to interfere with the osteogenic process but perhaps may interact with it.

Acute effects of ovariectomy on wound healing of alveolar bone after maxillary molar extraction in aged rats.

Acute effects of ovariectomy on the bone wound healing processes after maxillary molar extraction in aged rats were examined by means of quantitative scanning electron microscopy (SEM), backscattered electron image (BSE) analysis and energy-dispersive X-ray (EDX) microanalysis. Six-month-old female rats underwent either sham operation or bilateral ovariectomy, and 7 days postoperatively, the maxillary first molars were extracted. On post-extraction days 7, 30 and 60, the dissected maxillary bone surfaces were examined by SEM to reveal the bone formative and resorptive areas around the extracted alveolar sockets. In addition, the resin-embedded maxillae were micromilled in the transverse direction through the extracted alveolar sockets, and the newly-formed bone mass on the buccal bone surfaces and within the extracted sockets was examined by BSE analysis. Compared with sham-operated controls, the extent of newly-formed bone mass on the buccal bone surfaces in OVX rats was significantly decreased, due to increased bone resorption. On the other hand, new bone formation within the extracted sockets was similar in the experimental groups. In EDX microanalysis of these newly-formed bone matrices, both Ca and P weight % and Ca/P molar ratio were similar in the experimental groups. Our results suggest that 1) acute estrogen deficiency induced by ovariectomy stimulates sustained bone resorption, but has less effect on bone formation, and 2) bone wound healing after maxillary molar extraction within extracted alveolar sockets is not significantly delayed by ovariectomy, but bony support by newly-formed bone mass on the maxillary bone surfaces at the buccal side of the extracted sockets is significantly decreased, due to increased bone resorption.

Effects of ovariectomy on bone morphology in maxillae of mature rats.

Postmenopausal oestrogen deficiency results in bone loss (osteoporosis) in humans and experimental animals. The loss of trabecular bone in the ovariectomized (OVX) rat provides a useful experimental model of post-menopausal osteoporosis. At 5 months after ovariectomy of 3-month-old female rats, the mid and distal femurs and maxillae were dissected and processed for quantitative backscattered electron microscopic examinations. Histomorphometric analysis of femurs in OVX rats showed significant loss in metaphyseal trabecular bone areas compared with sham-operated controls; no significant bone loss was observed in the cortical bone areas of mid-diaphyses in OVX rats. Net bone areas in the maxillae of OVX rats was similar to that of sham-operated controls. Bone structure of maxillae in OVX rats was also similar to that in controls. Our results suggest that, in this animal model of osteoporosis, prominent bone loss occurs mainly in the bone areas formed by endochondral ossification such as distal femurs, but those areas formed by intramembranous ossification such as mid-femurs and maxillae sustained less effects by OVX.

Cell death during tooth eruption in the rat: surrounding tissues of the crown.

We investigated the occurrence of apoptosis and other types of cell death around the crown during tooth eruption of the rat upper molar. The TdT-mediated-dUTP-biotin nick end labeling (TUNEL) method and transmission electron microscopy (TEM) were employed. Apoptosis was detected by both TUNEL and TEM in part of the reduced enamel epithelium and connective tissue in the resorbing bony crypt of the pre-erupted tooth. In TEM, a large number of cells showed condensed chromatin and membrane-bound small bodies (apoptotic bodies). Macrophages that phagocytosed apoptotic bodies could be detected. Based upon the distance between bone surface and these apoptotic cells, and the characteristics of their organelles, we suggested that the apoptotic cells might be osteocytes, bone-lining cells (osteoblasts), and macrophages. We surmised that the osteoclasts had also died. Cells which contained autophagic vacuoles and autophagosomes, and others whose cytoplasm had dissolved, were also frequently observed. No progressive cell death was found in the oral epithelium or the fibrous connective tissue over the crown. These results suggest that apoptosis gives rise to some cell death during tooth eruption, but that other types of cell death also occur in various cells.