Evaluation of ethylene oxide, gamma radiation, dry heat and autoclave sterilization processes on extracellular matrix of biomaterial dental scaffolds.

Scaffolds used to receive stem cells are a promising perspective of tissue regeneration research, and one of the most effective solutions to rebuild organs. In the near future will be possible to reconstruct a natural tooth using stems cells, but to avoid an immune-defensive response, sterilize the scaffold is not only desired, but also essential to be successful. A study confirmed stem cells extracted from rat's natural teeth, and implanted into the alveolar bone, could differentiate themselves in dental cells, but the scaffold's chemistry, geometry, density, morphology, adherence, biocompatibility and mechanical properties remained an issue. This study intended to produce a completely sterilized dental scaffold with preserved extracellular matrix. Fifty-one samples were collected, kept in formaldehyde, submitted to partial demineralization and decellularization processes and sterilized using four different methods: dry heating; autoclave; ethylene-oxide and gamma-radiation. They were characterized through optical images, micro-hardness, XRD, EDS, XRF, SEM, histology and sterility test. The results evidenced the four sterilization methods were fully effective with preservation of ECM molecular arrangements, variation on chemical composition (proportion of Ca/P) was compatible with Ca/P proportional variation between enamel and dentine regions. Gamma irradiation and ethylene oxide presents excellent results, but their viability are compromised by the costs and technology's accessibility (requires very expensive equipment and/or consumables). Excepted gamma irradiation, all the sterilization methods more than sterilizing also reduced the remaining pulp. Autoclave presents easy equipment accessibility, lower cost consumables, higher reduction of remaining pulp and complete sterilization, reason why was considered the most promising technique.

The effect of pores and connections geometries on bone ingrowth into titanium scaffolds: an assessment based on 3D microCT images.

In order to support bone tissue regeneration, porous biomaterial implants (scaffolds) must offer chemical and mechanical properties, besides favorable fluid transport. Titanium implants provide these requirements, and depending on their microstructural parameters, the osteointegration process can be stimulated. The pore structure of scaffolds plays an essential role in this process, guiding fluid transport for neo-bone regeneration. The objective of this work was to analyze geometric and morphologic parameters of the porous microstructure of implants and analyze their influences in the bone regeneration process, and then discuss which parameters are the most fundamental. Bone ingrowths into two different sorts of porous titanium implants were analyzed after 7, 14, 21, 28, and 35 incubation days in experimental animal models. Measurements were accomplished with x-ray microtomography image analysis from rabbit tibiae, applying a pore-network technique. Taking into account the most favorable pore sizes for neo-bone regeneration, a novel approach was employed to assess the influence of the pore structure on this process: the analyses were carried out considering minimum pore and connection sizes. With this technique, pores and connections were analyzed separately and the influence of connectivity was deeply evaluated. This investigation showed a considerable influence of the size of connections on the permeability parameter and consequently on the neo-bone regeneration. The results indicate that the processing of porous scaffolds must be focused on deliver pore connections that stimulate the transport of fluids throughout the implant to be applied as a bone replacer.

Cephalometric measures correlate with polysomnography parameters in individuals with midface deficiency.

To determine the association between cephalometric measurements and polysomnographic parameters in Brazilian patients with midface deficiency. This was a primary, clinical, observational, longitudinal, retrospective, analytical, and single-center study. Forty-eight patients with midface deficiency were divided into two groups as follows: those who underwent surgically assisted rapid palatal expansion (SARME) and those who received maxillary advancement (MA). Pre- and post-operative cephalometric and polysomnography measurements were obtained. Pearson's correlation was used to verify the presence of any significant associations between PSG scores and cephalometric measurements. Associations between BMI (Body Mass Index) and AHI (Apnea Hypopnea Index) as well as arousals were observed. In the SARME group, associations between AHI and SNA, UAS and MP-H, arousals and SNA, and Co-A and MP-H were noted. Associations between AHI and Co-A, PoOr-A and MP-H, arousals and UAS, and between minimum saturation of O2 and SNA, SNB, and Co-A were observed in the MA group. This study demonstrates the alterations in the middle third of the face that were related to sleep disturbance. In addition, it shows the associations between the polysomnographic parameters and the cephalometric representations corresponding to the analyzed deformities and transverse or anteroposterior maxillary deficiencies.

Chick Embryo Model for Homing and Host Interactions of Tissue Engineering-Purposed Human Dental Stem Cells.

Human dental stem cells (hDSC) have a potential for regenerative therapies and could differentiate in vitro into many tissues, such as dentin, nerve, and vascular endothelium. Gallus gallus domesticus developing fertilized egg or chick embryo is an experimental model absent of xenografts rejection, largely employed in replacement of mammal species in scientific research and preclinical studies to evaluate angiogenesis and vasculogenesis, tissue differentiation, and embryonic development. This multiscale research deals with the homing and cell signaling effects of a standardized hDSC toward the receptor tissues of G. gallus domesticus in ovo. The hDSC were obtained from the explantation from third molars, characterized by cell cytometry, and employed without any further purification procedure. Four experimental groups were studied, according to the kind of cell tracing strategy, named: Control, mCherry-labeled hDSC, QTracker-labeled hDSC, and QTracker-exposed controls. The eggs were kept in an incubator temperature of 37.6°C and humidity 86%, and the embryos were euthanized after 10 days of incubation. In vivo fluorescence and histological analysis were conducted. The fluorescence of the embryos inoculated with mCherry hDSC or the QTracker hDSC was associated with the bones and the beak tooth, and labeled cell islands could be localized in part of the samples. The inoculation of the QTracker probe resulted in proliferating bone tissue labeling. The hDSC inoculated groups presented cartilage plate hypertrophy and atypical morphology, meanwhile Control eggs were negative. The results demonstrated that hDSC can migrate to the cartilaginous tissues of the chick embryos, survive in this environment, implant, and interfere with the growth of developing bone.

Osteoinductive effects of preoperative dexamethasone in human dental pulp stem cells primary culture.

AIM: The use of dexamethasone (DEX) in mesenchymal cell culture induces osteoblastic differentiation and, consequently, formation of mineralized tissues. Tissue engineering proposes the development of therapeutic strategies aimed at structural and functional regeneration of biological tissues. In this sense, cell characterization in vitro is critical to ensure the development of such techniques. Our objective was to evaluate the osteoinductive effect of DEX administered as a preoperative medication in primary cell culture of human dental pulp stem cell. METHODOLOGY: Cells from the third molar pulp were divided into two experimental groups, each with two preoperative medication protocols used in dental practice and differentiated by the intake of DEX in one of them. The assessment of proliferation, differentiation and viability through trypan blue, methylthiazol tetrazolium, and von Kossa and alizarin red assays, respectively, were held within fixed intervals: 7, 14, 21 and 28 days. CONCLUSION: This study has shown that DEX may influence in vitro human dental pulp stem cell behavior.

Tooth tissue engineering: tooth decellularization for natural scaffold.

AIM: Tissue engineering is a multidisciplinary science that aims to produce replacement organs and biological substitutes. One of the techniques involves decellularizing a biological organ without altering its structure. One challenge is how to demonstrate which method would be better for this process. METHODOLOGY: Fifty premolar teeth were divided into five groups: G1 (control): solution of 10% formaldehyde; G2: phosphate buffer saline (PBS), 28 g of tetrasodium ethylenediaminetetraacetic (EDTA), sodium hypochlorite 2.5% (SH); G3: PBS, EDTA and 40v hydrogen peroxide (HP); G4: PBS, EDTA, SH, enzymatic detergent (ED); and G5: PBS, EDTA, HP, ED. Each group was analyzed by scanning electron microscopy (SEM), x-ray, measured weights and color and received statistical analysis. CONCLUSION: This study demonstrated that G5 was the most appropriate method to obtain a natural scaffold.

Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

Effects of glucose and glutamine concentrations in human dental pulp stem cells viability. An approach for cell transplantation.

PURPOSE: To evaluate microscopic behavior and viability of dental pulp stem cells under glucose and glutamine deprivation. METHODS: Human tooth tissues were minced in isolated pieces and cultured until the desired cellular proliferation for experimental phases. Cells were cultured under variations of glucose and glutamine in both serum presence and absence, and then those cells were evaluated according to number and viability by MTT assay. The confocal microscopy analyzed cytoskeleton, nucleus, and mitochondria integrity. RESULTS: A low concentration of glucose favored cellular viability and microscopic behavior; the presence of glutamine in culture medium was favorable only when associated with glucose. The cellular biological potential in culture could be preserved in serum absence if nutritional requirements are adequate. CONCLUSION: Cell microscopic behavior and viability have demonstrated better patterns on serum-free low glucose culture medium with glutamine deprivation.

Effects of glucose and glutamine concentrations in human dental pulp stem cells viability. An approach for cell transplantation

PURPOSE: To evaluate microscopic behavior and viability of dental pulp stem cells under glucose and glutamine deprivation. METHODS: Human tooth tissues were minced in isolated pieces and cultured until the desired cellular proliferation for experimental phases. Cells were cultured under variations of glucose and glutamine in both serum presence and absence, and then those cells were evaluated according to number and viability by MTT assay. The confocal microscopy analyzed cytoskeleton, nucleus, and mitochondria integrity. RESULTS: A low concentration of glucose favored cellular viability and microscopic behavior; the presence of glutamine in culture medium was favorable only when associated with glucose. The cellular biological potential in culture could be preserved in serum absence if nutritional requirements are adequate. CONCLUSION: Cell microscopic behavior and viability have demonstrated better patterns on serum-free low glucose culture medium with glutamine deprivation. .

TGF-ß1 on induced osteogenic differentiation of human dermal fibroblast.

PURPOSE: To evaluate the role of transforming growth factor beta 1 (TGF-ß1) on the induced osteogenic differentiation of human dermal fibroblasts. METHODS: We performed four groups with cultured dermal fibroblasts according to the culture medium: CONTROL (DMEM culture medium); TGF-ß1 (DMEM culture medium with 10 ng/ml of TGF-ß1); OSTEOG (DMEM culture medium with 0.5 µg/ml of ascorbic acid, 10 mmol/l of ß-glycerophosphate and 10 nmol/L of dexamethasone); and OSTEOG/TGF-ß1 (osteogenic medium with 10 ng/ml of TGF-ß1). Alkaline phosphatase (ALP) activity and the amount of osteocalcin (OC) in the supernatant, as well as the capability to form calcium phosphate deposits, were analysed for 28 days RESULTS: There were significant differences (p<0.05) between CONTROL and TGF-ß1 groups in comparison with OSTEOG and OSTEOG/TGF-ß1 groups in the ALP activity and OC amount. Although, both osteogenic groups had the same behavior with regard the expression curve during the experimental time, the OSTEOG/TGF-ß1 group achieved significantly higher ALP and OC levels and showed no significant difference in the levels of mineralized deposits and in comparison with the levels found in the OSTEOG group. CONCLUSION: The addition of transforming growth factor beta 1 to the osteogenic culture medium increased the activity of alkaline phosphatase and the amount of osteocalcin, but TGF-ß1 did not alter the presence of mineralized calcium phosphate deposits.

Piezoelectric osteotomy for the placement of titanium implants in rabbits: histomorphometry study.

OBJECTIVE: To evaluate bone remodeling around dental implants inserted into recipient sites prepared using either the piezoelectric or the conventional drilling technique. MATERIAL AND METHODS: Twenty-four male New Zealand white rabbits (4 months, 2.70 kg) received dental implants (3.3 mm diameter and 6 mm length) on the medial surface of the tibia and were divided into 3 groups (n = 8). Group I was euthanized at 7 days; group II, at 14; and group III, at 28 days. Each animal received four implants, two in the right and two in the left tibia (96 implants were installed). Each tibia was operated by the same technique, and there are therefore neighbor's implants installed by different techniques. Histomorphometric parameters were used: the volume occupied by trabecular bone around the implants (BV/TV), media thickness, separation and number of trabeculae around the loops, and the contact area (interface) directly between the bone and implant (BIC). RESULTS: BV/TV was similar for both techniques (P = 0.291). Reduction in trabecular thickness was observed for both techniques (P < 0.05), but then returned to prior levels, with no significant difference between techniques (P = 0.217). Trabecular number increased from day 7 to day 14 (P < 0.001) and remained constant afterward for both techniques. No difference in BIC was observed between techniques on day 28 (P = 0.961). CONCLUSIONS: Piezoelectric osteotomy allowed bone formation for osseointegration of titanium implants, was not associated with bone necrosis, and provided results similar to those of the conventional technique. The piezoelectric technique can be considered a viable alternative in dental implantology.

TGF-β1 on induced osteogenic differentiation of human dermal fibroblast

PURPOSE: To evaluate the role of transforming growth factor beta 1 (TGF-β1) on the induced osteogenic differentiation of human dermal fibroblasts. METHODS: We performed four groups with cultured dermal fibroblasts according to the culture medium: CONTROL (DMEM culture medium); TGF-β1 (DMEM culture medium with 10 ng/ml of TGF-β1); OSTEOG (DMEM culture medium with 0.5 µg/ml of ascorbic acid, 10 mmol/l of β-glycerophosphate and 10 nmol/L of dexamethasone); and OSTEOG/TGF-β1 (osteogenic medium with 10 ng/ml of TGF-β1). Alkaline phosphatase (ALP) activity and the amount of osteocalcin (OC) in the supernatant, as well as the capability to form calcium phosphate deposits, were analysed for 28 days RESULTS: There were significant differences (p<0.05) between CONTROL and TGF-β1 groups in comparison with OSTEOG and OSTEOG/TGF-β1 groups in the ALP activity and OC amount. Although, both osteogenic groups had the same behavior with regard the expression curve during the experimental time, the OSTEOG/TGF-β1 group achieved significantly higher ALP and OC levels and showed no significant difference in the levels of mineralized deposits and in comparison with the levels found in the OSTEOG group. CONCLUSION: The addition of transforming growth factor beta 1 to the osteogenic culture medium increased the activity of alkaline phosphatase and the amount of osteocalcin, but TGF-β1 did not alter the presence of mineralized calcium phosphate deposits. .

Tooth tissue engineering: the influence of hydrophilic surface on nanocrystalline diamond films for human dental stem cells.

New techniques for tissue engineering (TE) are rapidly emerging. The basic concept of autologous TE is to isolate cells from small biopsy specimens, and to expand these cells in culture for subsequent seeding onto biodegradable scaffolds. Nanocrystalline diamond films have attracted the attention of researchers from a variety of different areas in recent years, due to their unique and exceptional properties. In this approach, human dental stem cells (hDSCs) were characterized by flow cytometry and grown on diamond films with hydrogen (H)-terminated and oxygen (O)-terminated surfaces for 28 days, and then removed by lysis and washing with distilled water. Energy dispersive spectroscopy analysis was performed, showing that the regions with O-terminated surfaces contained much higher levels of deposited calcium, oxygen, and phosphorus. These results suggest that the extracellular matrix was considerably more developed in the O-terminated regions, as compared with the H-terminated regions. In addition, optical microscopy of hDSCs cultured on the diamond substrate with H- and O-terminated surfaces, before washing with distilled water, showed preferential directions of the cells arrangement, where orthogonal lines suggest that the cells appeared to be following the O-terminated regions or hydrophilic surface. These findings suggest that O-terminated diamond surfaces prepared on biodegradable scaffolds can be useful for mineralized dental tissue formation.

Cytogenetic instability of dental pulp stem cell lines.

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

Tooth tissue engineering: optimal dental stem cell harvest based on tooth development.

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

Exodontia em paciente valvopata sob uso de medicaçäo anticoagulante: relato de caso clínico / Exodontics in valvopath patients using anticoagulation therapy: a case report

O tratamento odontológico em pacientes portadores de cardiopatias que utilizam anticoagulantes orais tornou-se uma rotina constante no consultório odontológico nos últimos anos, exigindo do cirugiäo-dentista uma abordagem específica e uma atuaçäo interdisciplinar com as várias equipes de saúde que acompanham o paciente. Os autores apresentam alguns aspectos relativos ao tratamento odontológico de um paciente valvopata, em especial aos que recebem medicaçäo anticoagulante oral, por meio de revisäo de literatura dos últimos 10 anos e do relato de um caso clínico

Desenvolvimento de tecido dental na mandíbula de ratos Lewis por meio de técnicas de engenharia de tecidos / Development of dental tissues in lewis rat's jaw by tissue engineering techniques

Objetivo: Verificar a viabilidade do desenvolvimento de tecidos dentais, organizados ou não na mandíbula, utilizando técnicas de Engenharia de Tecidos, usando diferentes polímeros biodegradáveis, em dois tempos de aderência celular na semeadura dos arcabouços, antes da cirurgia de implantação. Método: A amostra foi dividida em quatro diferentes grupos. O grupo controle C I, composto de receptores de implantes de germes dentais de molares; os gruposcontrole C II e C III, com receptores de implantes de arcabouço de PGA e PLGA respectivamente, sem células; e o grupo experimental IV, com receptores de implantes de arcabouço de PGA ou PLGA, com células dentais semeadas, obtidas por dissociação enzimática de germes de molares, de doadores de quatro dias de idade. Neste grupo, 207 doadores de germes dentais de molares foram utilizados para a cultura primária de células e 21 receptores para a implantação dos arcabouços semeados com células dentais distribuídos em: 10 receptores para o PGA, dos quais cinco receberam os implantes com 1 hora e os outros cinco receberam com 12 horas de espera para aderência celular. Após 12 semanas de implantação, os ratos receptores dos três grupos-controle e do experimental foram sacrificados e as mandíbulas removidas para as análises macroscópica, radiográfica e histológica. Resultados: Nossos resultados consideraram os critérios de semelhança ou não dos tecidos obtidos pela técnica de Engenharia de Tecidos, dos implantes dos arcabouços de PGA ou PLGA semeado com células dos doadores na mandíbula dos receptores com os grupos-controle de : implantes de germes dentais (C I) e arcabouço de PGA e PLGA sem células semeadas (C II e C III), além da avaliação do tempo de espera de 1 ou 12 horas para aderência das células dentais antes da cirurgia de implantação. No grupo controle C I, observa-se discreta erupção dos germes implantados na mandíbula, enquanto que nos outros dois, C II com PGA e C 111 com PLGA, ambos sem ...(au)

Avaliaçäo da variaçäo da pressäo arterial em pacientes especiais devida à administração de anestésico local sem vasoconstritor / Avaliation above the arterial pression variation among special patients according to the anesthesic loco administration without constrictor vases

Os autores realizaram a avaliaçäo de variaçäo da pressäo arterial em 53 pacientes, em tratamento na Disciplina de Odontologia para Pacientes Especiais, após a aplicaçäo de anestesia local à base de hidroclorido de mepivacaína 3 por cento sem vasoconstritor "Safety - Plus" (Septodont). Os resultados revelaram alteraçöes na pressäo diastólica, com discreta queda

Diagnóstico, planejamento e proservaçäo de um paciente portador de síndrome de Marfan - relato de caso clínico / Diagnosis, planning and cares after the treatment of patient carrier of Marfan's syndrome - report of clinical case

A síndrome de Marfan é uma anomalia genética, autossômica dominante cujos primeiros relatos foram descritos por Willians (1876). De acordo com ECHEVERRIA e CASTRO(4) (1982), et al. (15) (1982) e CHAVEZ DIAZ et al.² (1983) säo sinônimos desta síndrome: dolicostenomielia, aracnodactillia e distrofia mesodérmica congênita. A tríade que caracteriza a síndrome é composta pelos comprometimentos esqueléticos (deformidades toráxicas, escápula alada...), cardíacos (aneurisma e insuficiência aórtica, prolapso de válvula mitral...) e oculares (ectopia lentis, glaucoma...). Os achados clínicos mais freqüentes desta síndrome säo: a aracnodactilia, a insuficiência aórtica e a ectopia do cristalino. Como manifestaçöes bucais, encontram-se na literatura investigada, palato ogival, retrognatismo por hipoplasia mandibular, implantaçäo irregular com apinhamento dos elementos dentários e aumento dos seios paranasais. Neste relato, os autores apresentam as características clínicas e manifestaçöes bucais desta síndrome, com finalidade de proporcionar ao cirurgiäo-dentista informaçöes necessárias para o diagnóstico precoce, estabelecendo plano de tratamento adequado às limitaçöes do paciente

Cuidado no tratamento odontológico em paciente com anemia aplástica: relato de dois casos clínicos / Dental care in a aplastic anemia patient: report of two cases

Relato de casos de dois pacientes com anemia aplástica (ou aplasia de medula) que receberam cuidados odontológicos antes de ir para transplante de medula óssea (TMO). Ao início do tratamento a paciente do caso 1 apresentava sangramento gengival espontâneo devido a trombocitopenia associado à doença periodontal, grande acúmulo de placa bacteriana, agravada pela ausência de higiene bucal. Foi realizado a orientaçäo de higiene bucal, procedimentos periodontais básicos e exodontia do dente 45. Ao final do tratamento a condiçäo bucal satisfatória, näo havendo episódios hemorrágicos. No caso 2 o paciente apresentava raízes residuais que foram extraídas com anestesia local e posterior sutura, não havendo também nenhuma intercorrência hemorrágica. Os pacientes foram submetidos ao TMO em condiçöes bucais satisfatórias e com controle de higiene bucal