RESUMO
The use of two particulate bone graft substitute materials in experimental narrow marginal peri-implant bone defects was investigated with respect to early bone healing and implant stability. Porous titanium granules, oxidized white porous titanium granules (WPTG), and demineralized bovine bone mineral (DBBM) were characterized in vitro, after which the two latter materials were tested in experimental peri-implant bone defects in six minipigs, with empty defects as control. After mandibular premolar extraction, the top 5mm of the alveoli were widened to 6mm in diameter, followed by the placement of six implants, three on each side, in each pig. Six weeks of healing was allowed. The WPTG showed better mechanical properties. No significant differences in resonance frequency analysis were found directly after compacting or healing, and similar quantities of defect bone formation were observed on micro-computed tomography for all groups. Histomorphometric analysis demonstrated a more coronal bone-to-implant contact in the DBBM group, which also displayed more defect bone fill as compared to the WPTG group. The better mechanical properties observed for WPTG appear of negligible relevance for the early stability and osseointegration of implants.
Assuntos
Substitutos Ósseos/farmacologia , Implantação Dentária Endóssea/métodos , Implantes Dentários , Osseointegração/efeitos dos fármacos , Animais , Bovinos , Feminino , Microscopia Eletrônica de Varredura , Porosidade , Distribuição Aleatória , Suínos , Porco Miniatura , Titânio/farmacologia , Cicatrização/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
The aim of this study was to investigate the effect of TiO2 scaffold (SC) coated with an alginate hydrogel containing a proline-rich peptide (P2) on osteoblast proliferation and differentiation in vitro. Peptide release was evaluated and a burst release was observed during the first hours of incubation, and then progressively released overtime. No changes were observed in the cytotoxicity after 48 h of seeding MC3T3-E1 cells on the coated and uncoated TiO2 SC. The amount of cells after 7 days was higher on uncoated TiO2 SC than on alginate-coated TiO2 SC, measured by DNA content and scanning electron microscope imaging. In addition, while lower expression of integrin beta1 was detected for alginate-coated TiO2 SC at this time point, similar gene expression was observed for other integrins, fibronectin-1, and several osteoblast differentiation markers. After 21 days, gene expression of integrin beta3, fibronectin-1, osterix, and collagen-I was increased in alginate-coated compared to TiO2 SC. Moreover, increased gene expression of integrin alpha8, bone morphogenetic protein 2, interleukin-6, and collagen-I was found on P2 alginate-coated TiO2 SC compared to alginate-coated TiO2 SC. In conclusion, our results indicate that alginate-coated TiO2 SC can act as a matrix for delivery of proline-rich peptides increasing osteoblast differentiation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
Assuntos
Alginatos/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Osteoblastos/citologia , Peptídeos/farmacologia , Alicerces Teciduais/química , Titânio/farmacologia , Sequência de Aminoácidos , Animais , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/farmacologia , L-Lactato Desidrogenase/metabolismo , Camundongos , Dados de Sequência Molecular , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Peptídeos/química , Domínios Proteicos Ricos em Prolina , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Polyproline-rich synthetic peptides have previously been shown to induce bone formation and mineralization in vitro and to decrease bone resorption in vivo. Alginate hydrogel formulations containing these synthetic peptides (P2, P5, P6) or Emdogain® (EMD) were tested for surface coating of bone implants. In an aqueous environment, the alginate hydrogels disclosed a highly compact structure suitable for cell adhesion and proliferation. Lack of cytotoxicity of the alginate-gel coating containing peptides was tested in MC3T3-E1 cell cultures. In the present study, relative mRNA expression levels of integrin alpha 8 were induced by P5 compared to untreated alginate gel, and osteopontin mRNA levels were increased after 21 days of culture by treatment with synthetic peptides or EMD compared to control. Further, in agreement with previous results when the synthetic peptides were administered in the culture media, osteocalcin mRNA was significantly upregulated after long-term treatment with the formulated synthetic peptides compared to untreated and EMD alginate gel. These results indicate that the alginate gel is a suitable carrier for the delivery of synthetic peptides, and that the formulation is promising as biodegradable and biocompatible coating for bone implants.
Assuntos
Alginatos , Substitutos Ósseos/química , Osteoblastos/citologia , Peptídeos/química , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Adesão Celular , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Materiais Revestidos Biocompatíveis/química , Proteínas do Esmalte Dentário/química , Proteínas do Esmalte Dentário/farmacologia , Ácido Glucurônico , Ácidos Hexurônicos , Hidrogéis , Cadeias alfa de Integrinas/genética , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteopontina/genética , Peptídeos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.
Assuntos
Amelogenina/uso terapêutico , Proteínas do Esmalte Dentário/uso terapêutico , Doenças Periodontais/cirurgia , Amelogenina/fisiologia , Calcificação Fisiológica/fisiologia , Sequência Conservada , Proteínas do Esmalte Dentário/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Humanos , Metaloproteinases da Matriz/fisiologia , Osteogênese/fisiologia , Regeneração/efeitos dos fármacos , Reabsorção da Raiz/fisiopatologia , Cicatrização/fisiologiaRESUMO
BACKGROUND: Trauma induces local and subsequent systemic inflammatory reactions. Aberrant reactions can lead to a systemic inflammatory response syndrome, with a potentially lethal outcome. Our aim was to investigate the early local cytokine kinetic compared to systemic changes in a standardized surgical trauma. METHODS: In 7 patients with total hip replacement, drained blood samples and venous blood samples were taken 3 times within the first day after the operation. Interleukin (IL) release was assessed by a multiplex antibody bead kit and compared to pre-operative values. RESULTS: The major findings were significant increases in systemic levels of IL-6 and in local levels of IL-6, IL-8 and IL-1 receptor antagonist (IL-1Ra), and that the local levels of these cytokines were significantly higher than the systemic ones after surgery. Besides, there were only modest changes in local and systemic levels of tumour necrosis factor alpha, IL-1 beta, IL-2, IL-2Ra, IL-4, IL-5, IL-7, IL-10, IL-12, IL-13, IL-15 and IL-17. CONCLUSIONS: The acute sterile inflammation after major orthopaedic surgery is principally characterized by significantly increased local and systemic levels of IL-6 and significantly increased local levels of IL-8 and IL-1Ra. Furthermore, the concentrations are higher at the local site of injury. Hence, we conclude that systemic cytokine levels might not reflect local reactions.
Assuntos
Artroplastia de Quadril , Citocinas/sangue , Adulto , Idoso , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Interleucina-8/sangue , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: The innate immune system is suppressed after major orthopaedic surgery, implicating a risk of septic complications. Whole-blood ex vivo testing with lipopolysaccharide (LPS) has shown a depression of the tumour necrosis factor alpha (TNF-alpha) production until 12 days postoperatively. As part of the innate immune system, the Toll-like receptors TLR2 and TLR4 recognize antigens from Gram-positive and Gram-negative bacteria, respectively. The receptors CD14 and CD11b are involved in the LPS receptor complex, whereas human lymphocyte antigen (HLA)-DR binds endotoxin peptides. It is still uncertain whether the expression of all these receptors changes after major surgery. METHODS: In 6 patients undergoing hip arthroplasty, we investigated three times the display of TLR4, TLR2, CD14, CD11b, and HLA-DR on monocytes by fluorescence-activated cell sorting and white blood cell counts during 12 days postoperatively. At the same time, the plasma levels of interleukin (IL)-1beta, IL-4, IL-6, IL-10, IL-13, and TNF-alpha were measured. RESULTS: There was no significant change in the expression of TLR4, CD14, CD11b, HLA-DR, and TLR2. Monocyte count and cytokine analysis did not differ from the ones pre-operatively taken. CONCLUSIONS: After aseptic orthopaedic surgery, there is no change in the display of the LPS receptor complex on monocytes. Other mechanisms have to be investigated to gain insight into the decrease of the TNF-alpha production capacity postoperatively.
Assuntos
Artroplastia de Quadril , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Adulto , Idoso , Antígeno CD11b/metabolismo , Citocinas/sangue , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.
Assuntos
Amelogênese/fisiologia , Amelogenina/metabolismo , Antígenos CD/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Primers do DNA , Cães , Imunofluorescência , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraspanina 30RESUMO
BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD), extracted from porcine tooth buds, has been shown to promote periodontal healing in patients with severe periodontitis. This involves modulation of the inflammatory response followed by the onset of periodontal regeneration. Based on these observations, we examined the ability of EMD to modulate the release of a pro-inflammatory cytokine [tumor necrosis factor (TNF)-alpha], an anti-inflammatory cytokine (interleukin-10) and a chemokine (interleukin- 8) in whole human blood challenged by bacterial cell wall components. MATERIAL AND METHODS: Whole blood from healthy donors was challenged by lipopolysaccharide or peptidoglycan and incubated with different concentrations of EMD or a cAMP analogue 8-(4-chlorophenyl)thio-cAMP (8-CPT-cAMP). TNF-alpha, interleukin-8 and interleukin-10 were analysed from plasma by enzyme-linked immunosorbent assay (ELISA) while cAMP levels of peripheral blood mononuclear cell lysates were analysed by enzyme immunoassay (EIA). RESULTS: We found that EMD attenuated the release of TNF-alpha and interleukin-8 in whole blood from healthy donors challenged by lipopolysaccharide or peptidoglycan, while the release of interleukin-10 was unchanged. Enamel matrix derivative also produced a four-fold increase in the cAMP levels of peripheral blood mononuclear cell lysates. Like EMD, 8-CPT-cAMP attenuated the formation of TNF-alpha, but not of interleukin-10, in blood challenged by lipopolysaccharide. CONCLUSION: Enamel matrix derivative limits the release of pro-inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it has anti-inflammatory properties. We propose that this effect of EMD is, at least partly, secondary to an increase in the intracellular levels of cAMP in peripheral blood mononuclear cells.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas do Esmalte Dentário/farmacologia , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/sangue , AMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli , Humanos , Interleucina-10/sangue , Interleucina-8/sangue , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Staphylococcus aureus , Suínos , Tionucleotídeos/farmacologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
According to recent studies, amelin (ameloblastin, sheathlin) is expressed in young odontoblasts at the initiation of dentin formation during odontogenesis. The purpose of the present investigation was to study whether amelin is also expressed at the onset of trauma-induced reparative dentin formation. The mandibular developing first molars of 5-day-old rats were surgically taken out, and their pulp tissue briefly separated from the inner dentin surface and immediately repositioned. Then the teeth were re-implanted in their alveoli. At 0, 2, 4, 6, 8, 12 or 14 days after surgery, the animals were sacrificed and the experimental teeth evaluated by histology and immunohistochemistry for amelin. At 2, 4, 6 and 8 days after surgery, the detached and traumatized odontoblasts in the experimental teeth exhibited increasing signs of degeneration and loss of intracellular structures. At days 6 and 8 after surgery, immunohistochemistry revealed a strong staining for amelin in the traumatized odontoblastic layer. Twelve and 14 days after replantation, only necrotic cell remnants of the traumatized odontoblasts were discernible. At this stage, no amelin could be detected by immunostaining. A wide zone of an unorganized mineralized tissue surrounded the odontoblastic cell remnants. On the pulpal side of the unorganized tissue, a new, highly organized tubular reparative dentin layer was observed, bordered by columnar odontoblast-like cells abutting on newly formed predentin. The results indicate that the initiation of trauma-induced reparative dentin formation mimics that of primary dentin formation and that amelin seems to be involved in both processes, possibly as a signaling molecule.
Assuntos
Proteínas do Esmalte Dentário/análise , Polpa Dentária/lesões , Dentina Secundária/fisiopatologia , Animais , Anticorpos , Colágeno , Corantes , Tecido Conjuntivo/patologia , Proteínas do Esmalte Dentário/genética , Polpa Dentária/patologia , Dentina Secundária/metabolismo , Dentina Secundária/patologia , Dentinogênese/fisiologia , Corantes Fluorescentes , Seguimentos , Expressão Gênica , Imuno-Histoquímica , Dente Molar , Necrose , Odontoblastos/metabolismo , Odontoblastos/patologia , Odontoblastos/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.
Assuntos
Adesão Celular/efeitos dos fármacos , Proteínas do Esmalte Dentário/farmacologia , Substâncias de Crescimento/biossíntese , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , AMP Cíclico/biossíntese , Fibroblastos/metabolismo , Células HeLa/metabolismo , Humanos , Técnicas Imunoenzimáticas , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Ligamento Periodontal/citologia , Fator de Crescimento Derivado de Plaquetas/biossíntese , Regeneração/efeitos dos fármacos , Fator de Crescimento Transformador beta/biossínteseRESUMO
Familial severe hypodontia of the permanent dentition is a rare condition. The genetics of this entity remains unclear and several modes of inheritance have been suggested. We report here an increase in the number of congenitally missing teeth after the mating of affected subjects from two unrelated Norwegian families. This condition may be the result of allelic mutations at a single gene locus. Alternatively, incompletely penetrant non-allelic genes may show a synergistic effect as expected for a multifactorial trait with interacting gene products. This and similar kindreds may allow identification of genes involved in growth and differentiation of dental tissues by linkage and haplotype association analysis. Brittle nails, delayed growth of the hair, and delayed teething in the probands support the grouping of these conditions among the ectodermal dysplasias.
Assuntos
Anormalidades Múltiplas/genética , Alelos , Displasia Ectodérmica/genética , Heterogeneidade Genética , Anormalidades Dentárias/genética , Adulto , Anodontia/genética , Criança , Displasia Ectodérmica/classificação , Epistasia Genética , Feminino , Genes Dominantes , Cabelo/anormalidades , Cabelo/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Varredura , Modelos Genéticos , Unhas Malformadas , LinhagemRESUMO
Tumour viruses are thought to contribute to the development of one fifth of all human cancers, although the mechanisms involved are still obscure. Human papilloma virus (HPV) is a DNA virus associated with oral carcinomas. It has been shown that virus DNA has to become integrated into cellular DNA in order to transform normal to malignant cells. Cellular oncogenes and tumour suppressor genes are potential cancer genes. They are involved in the control of growth and differentiation of normal cells. It is known that structural or regulatory changes (activation) of these genes will lead to malignant transformation. Virus integration will sometimes take place in close relation to cellular oncogenes. Such incorporation may result in oncogene activation. Other cellular factors that may contribute to the development of oral squamous cell carcinoma are also discussed.
Assuntos
Carcinoma de Células Escamosas/microbiologia , Vírus de DNA Tumorais/patogenicidade , Neoplasias Bucais/microbiologia , Oncogenes , Papillomaviridae/patogenicidadeRESUMO
Mouse amelogenin cDNA was used in hybridization assays with genomic DNA, cut with the restriction enzyme Eco RI, from the edentulous chicken (Gallus domesticus), the monophyodont mouse (as control), diphyodont man, and the polyphyodont fishes Atlantic salmon (Salmo salar) and seawolf (Anarrhichas lupus). The hybridization assay was performed under stringent conditions with non-radioactive probes. Hybridization was obtained with mouse (6.4-kb band), man (9-kb and 13-kb bands), and seawolf (18-kb band) genomic DNA. This demonstrates DNA sequence similarities between these species, and supports the theory that DNA sequences encoding enamel proteins appear to be highly conserved during the evolution of vertebrates. Lack of hybridization in salmon and chicken may be due to sequence divergences or structural differences in an amelogenin gene analog, or it may be that no amelogenin gene is present in these animals.