Ultrastructural and immunohistochemical study of the effect of sodium alendronate in the progression of experimental periodontitis in rats

The aim of the present research was to investigate the ultrastructural aspects and the immunoexpression of receptor activator of NFκB ligand (RANKL) and osteoprotegerin (OPG) on experimental periodontal disease of alendronate (ALN)‐treated rats. Male Wistar rats received daily injections of 2.5 mg/kg body weight of ALN during 7 days previously and 7, 14, and 21 days after the insertion of a 4.0 silk suture into the gingival sulcus around the right upper second molar. Specimens were fixed in 0.1% glutaraldehyde + 4% formaldehyde under microwave irradiation, decalcified in 4.13% EDTA and paraffin embedded for TRAP histochemistry and immunohistochemistry for RANKL and OPG, or embedded in Spurr epoxy resin for TEM analysis. ALN reduced the activity of osteoclasts and significantly decreased the resorption of the alveolar crest. In the control group the alveolar crest appeared resorbed by TRAP‐positive osteoclasts, which presented ultrastructural features of activated cells. The immunoexpression of RANKL was not inhibited by the drug; however, the expression of OPG was increased in the treated animals. The alveolar crest of ALN‐treated specimens at 21 days showed signs of osteonecrosis, like empty osteocyte lacunae, the exposed bone regions and bacterial infection. The results showed that ALN treatment in individuals with periodontal disease represents a risk of osteonecrosis because of the reduced activity of osteoclasts resultant of the increased immunoexpression of OPG. Microsc. Res. Tech. 77:902–909, 2014. © 2014 Wiley Periodicals, Inc.     

Effect of organic silicon,methylsulfonylmethane, and glucosamine sulfate in mandibular bone defects in rats

Organic silicon (OS), glucosamine sulfate (GS), and methylsulfonylmethane (MSM) have been related to bone and connective tissue health and have been considered as basic therapy for osteoarthrosis disorders. Therefore, the aim was to analyze the effect of the association of these three components in mandibular bone defects in rats. Nine rats were used for histocompatibility test. In each animal was implanted the composition (70% OS, 15% GS, 15% MSM) and gutta percha (control) under the dorsal subcutaneous tissue. The samples were collected at 7, 14, and 21 days post‐surgery and inflammatory events analyzed. In sequence, the composition was engrafted in mandibular bone defects of nine rats; bone defects without treatment were the control group. Analyses were performed at 7, 14, and 28 days post‐surgery and samples were evaluated by scanning electron microscopy (SEM). For the histocompatibility test, both groups had a moderate inflammatory process at 7 days post‐surgery and mild inflammatory process at 14 and 21 days. But in SEM analysis, the composition promotes an extensive reabsorption in cortical and crest alveolar bone, and great tooth root reabsorption. In conclusion, although the composition had positive result in the histocompatibility test, its direct application in mandibular bone defects caused intense resorption.     

Platelet‐rich fibrin and collagen membrane in the preservation of the alveolar bone: Feasibility of the elemental inorganic composition and scanning electron microscopy analysis

The success of dental implants is related to the amount, quality, and composition of the alveolar bone. The placement of platelet‐rich fibrin (PRF) clot associated with a resorbable collagen membrane (RCM) in a postextraction alveolus is a technique used for ridge preservation. This case report study analyzed the ultrastructural characteristics of cross‐sectioned alveolar bone that received PRF and RCM using scanning electron microscopy and the inorganic composition using “energy dispersive X‐ray spectrometry,” in order to explore the feasibility of this method to clinical studies. Three alveolar bone samples from two male patients (37 and 58 years old), obtained in the procedure of placing the dental implant, were analyzed. Two bone samples previously received PRF and RCM (M37 and M58), the third sample represented a physiological bone formation without treatment (M37‐control). The bone sample M37 showed irregularly shaped islets of calcified material intermingled with connective tissue. The other samples, from the 58‐year‐old patient with PRF and RCM (M58); and the other untreated bone sample from the same 37‐year‐old patient (M37‐control) showed similar ultrastructural morphology with trabecular conformation without islets agglomerations. The inorganic composition analysis showed higher concentrations of calcium and phosphorus in both samples treated with PRF and RCM in comparison to the untreated bone sample. The Ca/P ratio was higher in the M37 sample compared to the others samples. The results showed morphology and inorganic composition differences among the treatments used, suggesting that this method is feasible to analyze parameters of the alveolar bone tissue.     

Effect of chitosan and Dysphania ambrosioides on the bone regeneration process: A randomized controlled trial in an animal model

The focus of this triple‐blind study was on evaluating the effect of chitosan combined with Dysphania ambrosioides (A) extract on the bone repair process in vivo. In total, 60 male Wistar rats (Rattus norvegicus albinus) weighing between 260 and 270 g were randomly selected for this study and distributed into four groups (n = 15). Group C (chitosan), Group CA5 (chitosan + 5% of D. ambrosioides), Group CA20 (chitosan + 20% of D. ambrosioides), and Group CO (Control‐Blood clot). In each animal, bone defects measuring 2 mm in diameter were performed in both tibias for placement of the substances. After 7, 15, and 30 days, the animals were sedated and sacrificed using the cervical dislocation technique and the tissues were analyzed under optical microscope relative to the following events: inflammatory infiltrate, necrosis, osteoclasts, osteoblasts, fibroblasts, periosteal, and endosteal bone formation. The data were evaluated to verify distribution using the Kolmogorov–Smirnov test, and variance, using the Levene test; as distribution was not normal, data were subjected to the Kruskal–Wallis and Dunn nonparametric tests (p < .05). A significant inflammatory infiltrate was observed in Group CA5 (p = .008) in the time interval of 7 days, and in Group C at 15 (p = .009) and 30 (p = .017) days. Osteoblastic activity was more significant in Group CA20 (p = .027) compared with CA5 in the time interval of 7 days. Group CA20 demonstrated a significantly higher endosteal and periosteal bone formation value in the time interval of 7 (p = .013), 15 (p = .004), and 30 days (p = .008) compared with the other groups. The null hypothesis was refuted, bone regeneration was faster in spheres with an association of chitosan and 20% extract, and complete bone repair occurred clinically at 15 days and histologically at 30 days. The spheres proved to be a promising method for the biostimulation of alveolar bone repair and bone fractures.     

Feasibility of high pressure freezing with freeze substitution after long‐term storage in chemical fixatives

Fixation of biological samples is an important process especially related to histological and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for transmission electron microscopy for many years, as it provides adequate preservation of the morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is a newer alternative method that rapidly freezes non‐cryoprotected samples that are then slowly heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This study addresses several issues related to tissue preservation for electron microscopy. Using mice liver tissue as model the difference between samples fixed chemically or with HPF immediately after excision, or stored before chemical or HPF fixation were tested with specific focus on the nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde (FA) is the best in preserving membrane morphology, 2.5% GA can be used as alternative for stored and then chemically processed samples, with 10% formalin being suitable as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural preservation, but HPF with FS can minimize these artifacts relative to other processing protocols. Microsc. Res. Tech. 76:942–946, 2013. © 2013 Wiley Periodicals, Inc.     

Morphometric,quantitative, and three‐dimensional analysis of the heart muscle fibers of old rats: Transmission electron microscopy and high‐resolution scanning electron microscopy methods

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high‐resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A‐, I‐, and H‐bands and their M‐ and Z‐lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm², a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Confocal laser scanning microscopy for the study of the morphological changes of the postextraction sites

A better understanding of the remodeling process of postextraction sockets is essential in dental treatment planning. The aim of this study was to evaluate whether confocal laser scanning microscopy (CLSM) can be applied to imaging contour changes of postextraction sites, as well as to its quantification with image analysis of obtained three‐dimensional images. This work describes a new application of the CLSM technique. The system used was the OLS3100‐USS, LEXT model (Olympus®). CLSM was used for the surface analysis of the extraction site. The measurements taken with CLSM were: (1) mesio‐distal distance, (2) alveolar ridge thickness, and (3) vestibular and lingual alveolar ridge height. Results of study cast scanning at baseline, 1 and 3 months after tooth extraction, with CLSM are well‐detailed images of postextraction areas. The CLSM technique used in study casts is a valid method to measure the dimensional changes that happen in the edentulous area after tooth extraction. This technique allows the evaluation of changes in mesio‐distal distance, thickness of the alveolar ridge and alveolar ridge height based on the measurements on the alveolar contours. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Effect of Chenopodium ambrosioides on the healing process of the in vivo bone tissue

The focus of this double‐blind randomized study was on evaluating the effect of an aqueous extract of Mastruz (Chenopodium ambrosioides L.) on the bone repair process in vivo. In total, 36 male Wistar rats were randomly selected for this study, and divided into 3 groups (n = 12): Group HS (Hemostatic Sponge), Group SM (Hemostatic Sponge with Mastruz) and Group BC (Blood Clot). In each animal, bone defects measuring 2 mm in diameter were performed in both tibias for placement of the substances. After 3 and 10 days, the animals were sacrificed, and the tissues were analyzed under an optical microscope relative to the following events: inflammatory infiltrate; necrosis; young fibroblasts; osteoclastic and osteoblastic activity; endosteal and periosteal bone formation; and bone repair. The results were assessed by using Kruskal–Wallis and Mann–Whitney tests (p < .05). Inflammatory infiltrate demonstrated difference between Groups SM and BC in the time interval of 3 days (p = .004); an event related to the presence of the fibrin sponge and liquid of the extract, which induced a foreign body initial reaction. The presence of young fibroblasts (p = .003), osteoclastic (p = .003), and osteoblastic (p = .020) activity was statistically significant between Groups HS and BC in the time interval of 10 days; performance was related to the presence of the sponge within bone. As regards injured bone tissue repair, Group SM demonstrated a higher level of regenerative capacity (p = 0.004), due to a larger quantities of endosteal and periosteal bone formation, demonstrated in Group SM. The aqueous extract of mastruz stimulated bone neoformation, presenting wound closure with bone tissue at the end of 10 days.     

Fine Structure of Bacterial Adhesion to the Epithelial Cell Membranes of the Filiform Papillae of Tongue and Palatine Mucosa of Rodents: A Morphometric,TEM, and HRSEM Study

The palatine mucosa and filiform papillae of the dorsal tongue mucosae of rodents were examined using transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). In the HRSEM method, the samples were fixed in 2% osmium tetroxide, dehydrated in alcohol, critical point‐dried, and coated with gold‐palladium. In addition, the HRSEM technique was used for morphometric analysis (length, width, and length/width ratio of cocci and bacilli). For the TEM method, the tissues were fixed in modified Karnovsky solution (2.5% glutaraldehyde, 2% formalin in 0.1M sodium phosphate buffer, pH 7.4) and embedded in Spurr resin. The results demonstrated that there are thick polygonal keratinized epithelial cells where groups of bacteria are revealed in three‐dimensional images on the surface of filiform papillae in these animals. The bacterial membranes are randomly attached to the microplicae surface of epithelial cells. Morphometrics showed higher values of length and width of cocci in newborn (0 day) as compared to newborn (7 days) and adults animals, the bacilli showed no differences in these measurements. At high magnification, the TEM images revealed the presence of glycocalyx microfilaments that constitute a fine adhesion area between bacterial membranes and the membranes of epithelial microplicae cells. In conclusion, the present data revealed the fine fibrillar structures of bacteria that facilitate adhesion to the epithelial cell membranes of the oral cavity and morphometric changes in newborn (0 day) rats as compared with other periods. Microsc. Res. Tech. 76:1226–1233, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructural characteristics of ovine bone marrow‐derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM‐MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM‐MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM‐MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM‐MSCs were isolated from the iliac crest, cultured until they reached near‐confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT‐PCR analysis. RT‐PCR displayed that oBM‐MSCs express typical surface marker for MSCs. TEM revealed the presence of electron‐lucent cells and electron‐dense cells, both expressing the CD90 surface antigen. The prominent feature of electron‐lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM‐MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM‐MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM‐MSCs display electron‐dense and electron‐lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.     

Tissue integrity,costs and time associated with different agents for histological bone preparation

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin‐fixed and paraffin‐embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morsés solution (Group V). Serial, 3‐μm‐thick sections were obtained and stained with hematoxylin‐eosin to calculate the percentage of osteocyte‐occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi‐quantitative analysis to verify the adjacent muscle‐to‐bone integrity and preservation of collagen fibres. The highest percentage of osteocyte‐occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8–5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p < .001). Group IV (EDTA) had the highest integrity of muscle and collagen type I and III (P < 0.01). Demineralization with 10% acetic acid was the most effective at preserving bone tissue, while 5% EDTA was the best at maintaining collagen and adjacent muscle to bone. In conclusion, nitric acid at 5% showed the most efficient result as it balanced both time and cost as a demineralizing solution.     

MicroCT examination of human bone specimens: effects of polymethylmethacrylate embedding on structural parameters

X‐ray microtomography permits the nondestructive investigation of trabecular and cortical bone specimens without special preparation of the sample. To do a quantitative characterization, the cross‐section images have to be binarized, separating bone from nonbone. For this purpose, a widely used method is uniform thresholding. However, for commonly available microtomography scanners which use a polychromatic X‐ray source, it is unclear what effect the surrounding medium (e.g. air, saline solution, polymethylmethacrylate) has on the threshold value used for the binarization. In the literature an easy procedure to find the optimal uniform threshold value for a given acquisition condition is reported. By applying this procedure, the present work investigated whether a microtomography scan of trabecular bone samples in air or embedded in polymethylmethacrylate gave the same results in terms of structural parameters. The gold standard, that is, histological sections, was used as a reference. Two fixed threshold values were found, one for the microtomography scans performed in air and one for the scans with the same samples embedded in polymethylmethacrylate. These were applied on the correspondent microtomography images for the estimation of structural parameters, such as bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index. Paired comparisons were made in bone volume fraction between histological sections and microtomography cross‐sections for the same bone samples scanned first in air and then embedded in polymethylmethacrylate, by which no significant differences were found. Paired comparisons were also made in bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index for the same samples over volumes of interest of 4 × 4 × 4 mm³ between microtomography scans in air and scans with the samples embedded in polymethylmethacrylate. Neither these comparisons showed significant differences. This leads to the conclusion that structural parameters estimated by microtomography for human trabecular bone samples scanned either in air or embedded in polymethylmethacrylate are not affected by the surrounding medium (i.e. presence or absence of polymethylmethacrylate), provided that the corresponding optimal threshold value is applied for each acquisition condition.     

Osseointegration of hydroxyapatite and remodeling‐resorption of tricalciumphosphate ceramics

Background: Cancellous bone defects surrounded by still intact bone structures never heal. Ceramics offer a solution providing osteoconductive scaffolds. Purpose: The purpose of the study is to evaluate whether structured β‐TCP and HA implants can reconstruct cancellous bone defects, which role micro‐ and macro‐porosity, stiffness and surface area play; finally the indication for both materials based on its resorbability. Material & Methods: 10 German Shepard dogs were operated on both tibial heads implanting shell‐like fully interconnected ceramic cylinders, using a wet grinding hollow drill coated with diamonds. β‐TCP was compared with HA. A polychromatic sequential labelling with 4 different fluorochromes controlled bone formation dynamics. Non‐decalcifying histology after perfusion fixation and vessel casting was performed. μ‐CT was combined with high resolution microradiography and histology on thin ground crossections. The stages after 6 weeks, 2, 3, 4 months and 15 months were evaluated. Results: In spite of osseointegration of HA and β‐TCP, the osseointegration of both materials was completely different. Both shell‐like bone void fillers were osseointegrated in a sandwich‐like manner. HA yielded primarily a reinforcement of the recipient's cancellous‐bone bed and full osseointegration after 4 months, whereas β‐TCP‐implants were fully osseointegrated after 6 weeks. HA did not show signs of resorption. The resorption of the β‐TCP resulted during remodelling. The final stage showed restitution “ad integrum” of the β‐TCP defects with a physiological architecture, whereas HA was integrated in the cancellous bone construction providing 600 μm measuring macropores showing osteoinductive properties. Microsc. Res. Tech. 76:370–380, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of TGF‐β1 on mineralization mediated by rat calvaria‐derived osteogenic cells

In this study, we have analyzed the viability and cell growth, as well as, the mineralization of extracellular matrix (ECM) by alizarin red and von Kossa staining of calvaria‐derived osteogenic cultures, treated with TGF‐β1 alone or associated with Dex comparing with acid ascorbic (AA) + β‐glicerophosphate (βGP) (positive mineralization control). The expression of the noncollagenous proteins bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN) were evaluated by indirect immunofluorescence. In addition, the main ultrastructural morphological findings were assessed by transmission electron microscopy. Osteogenic cells were isolated of calvaria bone from newborn (2‐day‐old) Wistar rats were treated with TGF‐β1 alone or with dexamethasone for 7, 10, and 14 days. As positive mineralization control, the cells were supplemented only with AA+ βGP. As negative control, the cells were cultured with basal medium (α‐MEM + 10%FBS + 1%gentamicin). The treatment with TGF‐β1, even when combined with Dex, decreased the viability and cell growth when compared with the positive control. Osteoblastic cell cultures were positive to alizarin red and von Kossa stainings after AA + βGP and Dex alone treatments. Positive immunoreaction was found for BSP, OPN and FN in all studied treatments. Otherwise, when the cell cultures were supplemented with TGF‐β1 and TGF‐β1 + Dex, no mineralization was observed in any of the studied periods. These present findings suggest that TGF‐β1, in the studied in vitro doses, inhibits the proliferation and differentiation of osteoblastic cells by impairment of nodule formation.     

Aged garlic extract ameliorates the oxidative stress,histomorphological, and ultrastructural changes of cisplatin‐induced nephrotoxicity in adult male rats

Aim: Aged garlic extract (AGE) is a natural dietary substance having different antioxidant free‐radical‐scavenger compounds that ameliorates the toxicity of the oxidative stress. This study aimed to investigate the effect of AGE on cisplatin (CP)‐induced nephrotoxicity in rats. Materials and Methods: Twenty‐four, adult male Wistar albino rats were randomly divided into four groups namely control, AGE‐treated (a single oral dose of 250 mg/kg/day for 21 days), CP‐treated (a single intraperitoneal dose of 7.5 mg/kg on Day 16), and AGE + CP‐treated (AGE at a dose of 250 mg/kg/once daily for 21 days and a single dose of CP of 7.5 mg/kg intraperitoneally on Day 16). Body weight and absolute and relative kidney weights of each rat were calculated. Serum creatinine, uric acid, and urea levels were determined. Level of malondialdehyde and reduced glutathione and activity of superoxide dismutase and catalase of renal tissues were measured. Renal specimens from each rat were prepared for both light and electron microscopic examinations. Results: Interstitial cell infiltration, hemorrhage, glomerular atrophy, necrosis, and tubular degeneration were observed after CP treatment. Superoxide dismutase and catalase activities and glutathione level were significantly decreased and malondialdehyde level was significantly increased in CP‐treated rats compared with AGE + CP‐treated animals. A remarkable improvement in the histopathological and ultrastructural changes induced by CP in renal tissues was observed in AGE + CP‐treated rats. Conclusion: AGE exhibited antioxidant effect that could ameliorate the nephrotoxic effects of CP. Microsc. Res. Tech. 78:452–461, 2015. © 2015 Wiley Periodicals, Inc.     

Mouse calvarial defect Model: An approach for the micro‐tomographic evaluation of polymer scaffolds

The ability of bone repair scaffolds to form bone is traditionally evaluated using cell culture and animal experiments. Mouse calvarial organ culture maintains the natural cell‐to‐cell and cell‐to‐matrix relationships as well as the anatomical order, and this model has been used to study the biological behavior of intramembranous bones. The aim of this study was to evaluate the potential of mouse calvarial organ culture to be used as an in vitro model to study the bone regenerative ability of bone repair polymer scaffolds. Critical size defects (CSD) were created in the parietal bones. Electrospun poly(ε‐caprolactone) scaffolds were placed into one group of defects. The remaining defects served as a control. The bones were cultured for 38 days and analyzed with μCT, phase‐contrast microscopy, dissecting microscopy, scanning electron microscopy, and energy‐dispersive X‐ray analyses. This organ culture technique is easily available and could permit researchers to quickly establish a valuable database of candidate bone repair scaffolds. Microsc. Res. Tech. 77:1037–1043, 2014. © 2014 Wiley Periodicals, Inc.     

Histological evaluation on bone regeneration of dental implant placement sites grafted with a self-setting alpha-tricalcium phosphate cement

This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R. Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology.     

Microwave exposure increases bone demineralization rate independent of temperature

There is a long‐standing controversy regarding an effect of microwaves, independent of increasing temperature, on the rate of bone demineralization. In this study, we exposed standardized samples of gerbil femur to constant microwave exposure while maintaining the demineralizing solution (ethylenediamine tetraacetic acid, EDTA) at 20 °C. Random samples were selected at 3 h intervals, embedded in plastic and sectioned for histological evaluation to determine the extent of demineralization. The time to complete demineralization was significantly faster with microwave exposure (33 h) compared to non‐exposure on a tissue rotator (45 h) in a limited amount (5 mL/24 h) of EDTA. The presence of bone marrow was a significant barrier to the rate of demineralization and resulted in an asymmetrical pattern of mineral extraction. Samples without bone marrow were completely demineralized after 21 h of exposure to microwaves and EDTA. Additional comparisons were made between samples exposed to an effectively infinite supply of demineralizing agent (bone marrow intact). There was a significant increase in rate with unlimited demineralizing agent with (24 h) or without (30 h) microwaves when compared to tissue demineralized on the rotator. Our results establish a positive effect of microwaves on the rate of bone demineralization which is independent of temperature.     

Oleic acid‐added embedding medium for histological analysis of hard tissue

For the histological analysis of hard tissue such as bone, various acrylate‐based materials have been used as an embedding medium. However, commercial embedding media are expensive, and cutting the embedded block takes a long time. In this study, mixtures of methyl methacrylate (MMA), di‐butyl‐phthalate (DBP), and oleic acid (OA) were tested for possible application as an embedding medium for large and small undecalcified bone specimens. Mechanical properties were tested in a compressive mode. We investigated the change of hydrophilicity in the sectioned surface by measuring the contact angle depending on the OA. Crystallinity was analyzed using a X‐ray diffractometer (XRD). Surface analysis was performed using a confocal laser scanning microscope. To determine the staining efficiency of staining dyes, hamatoxylin‐eosin (H&E) and Masson's trichrome (MT) staining methods were performed for the histological analysis of bone‐implant complex. We confirmed that the investigated embedding media showed good properties such as optimal mechanical strength appropriate for cutting the embedded block and proper staining efficiency for histological analysis. Therefore, the MMA/DBP/OA mixtures can be used as an embedding media appropriate for various hard tissues and bone‐implant complex. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

运用模块化方法建立口腔修复有限元模型

以工程领域中的模块化方法为核心建立了牙颌组织的有限元模型库。对标本进行CT扫描、数据提取、和三维重建技术建立牙颌组织的实体模型。对实体模型切分模块 ,得到一套正常牙列的模块化模型库 ,并在其基础上派生一系列反映上下颌牙生理、病理情况的模型库。上述方法可建成正常牙、牙槽骨不同程度吸收、牙周不同程度松动、缺牙区牙槽骨、固定桥修复体、可摘局部义齿修复体等有限元模型。将模块化设计与有限元方法相结合 ,可高效地生成适用于口腔修复学设计和分析的有限元模型库。