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AIM: To investigate the potential of an ultrashort aromatic peptide hydrogelator integrated with hyaluronic acid (HA) to serve as a scaffold for bone regeneration. MATERIALS AND METHODS: Fluorenylmethyloxycarbonyl-diphenylalanine (FmocFF)/HA hydrogel was prepared and characterized using microscopy and rheology. Osteogenic differentiation of MC3T3-E1 preosteoblasts was investigated using Alizarin red, alkaline phosphatase and calcium deposition assays. In vivo, 5-mm-diameter calvarial critical-sized defects were prepared in 20 Sprague-Dawley rats and filled with either FmocFF/HA hydrogel, deproteinized bovine bone mineral, FmocFF/Alginate hydrogel or left unfilled. Eight weeks after implantation, histology and micro-computed tomography analyses were performed. Immunohistochemistry was performed in six rats to assess the hydrogel's immunomodulatory effect. RESULTS: A nanofibrous FmocFF/HA hydrogel with a high storage modulus of 46 KPa was prepared. It supported osteogenic differentiation of MC3T3-E1 preosteoblasts and facilitated calcium deposition. In vivo, the hydrogel implantation resulted in approximately 93% bone restoration. It induced bone deposition not only around the margins, but also generated bony islets along the defect. Elongated M2 macrophages lining at the periosteum-hydrogel interface were observed 1 week after implantation. After 3 weeks, these macrophages were dispersed through the regenerating tissue surrounding the newly formed bone. CONCLUSIONS: FmocFF/HA hydrogel can serve as a cell-free, biomimetic, immunomodulatory scaffold for bone regeneration.
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Ácido Hialurônico , Hidrogéis , Ratos , Animais , Bovinos , Hidrogéis/farmacologia , Hidrogéis/química , Ácido Hialurônico/farmacologia , Ácido Hialurônico/uso terapêutico , Osteogênese , Microtomografia por Raio-X , Cálcio/farmacologia , Ratos Sprague-Dawley , Regeneração Óssea , Periósteo , Alicerces Teciduais/químicaRESUMO
PURPOSE: Osteoporosis is characterized by a decrease in bone mineral density (BMD). A preliminary stage of the disease is progressive bone marrow adiposity, caused by imbalance between osteogenesis and adipogenesis in the marrow. Detection of osteoporosis relies on the quantification of BMD with techniques such as dual-energy X-ray absorptiometry. This work aimed to detect bone marrow changes in an experimental model of osteopenia using a low-field tabletop NMR scanner. METHODS: An experiment was performed on 32 female rats, 3 months old, 16 of which were ovariectomized (OVX) and 16 were sham-operated (sham). The femur and tibia from both hind limbs were isolated and underwent ex vivo NMR scans at four time points after the OVX and sham operations. NMR scans were complemented by BMD measurements and histology. RESULTS: Significant changes in the bone marrow of ovariectomized rats, relative to sham operated rats, were observed after 3.5 and 4.5 months. Bone marrow adiposity was detected by significant changes in T1 and T2 relaxation times, and in the diffusion coefficient. CONCLUSIONS: This study suggests a potential detection of changes to the bone marrow using a tabletop NMR device. Clinical translation may facilitate screening, early detection of bone weakening as a result of estrogen withdrawal, and monitoring of treatment efficacy. Magn Reson Med 78:860-870, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
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Medula Óssea/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Osteoporose/diagnóstico por imagem , Animais , Densidade Óssea , Medula Óssea/química , Feminino , Fêmur/diagnóstico por imagem , Ovariectomia , Ratos , Ratos Sprague-Dawley , Tíbia/diagnóstico por imagemRESUMO
Purpose: This pilot study aimed to explore the feasibility of scanning the human distal radius bone marrow in vivo to detect osteoporosis-related changes using magnetic resonance and evaluate whether the radius may serve as an accessible probing site for osteoporosis. This may lead in the future to the use of affordable means such as low-field MRI scanners for the monitoring of disease progression. Methods: A clinical trial was performed using a 3T MR scanner, including 26 women assigned into three study groups: healthy-premenopausal (n = 7; mean age 48.6 ± 3.5 years), healthy-postmenopausal (n = 10; mean age 54.5 ± 5.6 years), and osteoporotic-postmenopausal (n = 9; mean age 61.3 ± 5.6 years). Marrow fat composition was evaluated using T2 maps, a two-compartment model of T1, and a Dixon pulse sequence. Results: The osteoporotic group exhibited higher fat content than the other two groups and lower T2 values than the healthy-premenopausal group. Conclusions: Osteoporosis-related changes in the composition of the distal radius bone marrow may be detected in vivo using MRI protocols. The scanning protocols chosen here can later be repeated using low-field MRI scanners, thus offering the potential for early detection and treatment monitoring, using an accessible, affordable means that may be applied in small clinics. This trial is registered with MOH_2018-05-23_002247, NCT03742362.
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Some cases of asymptomatic traumatic cyst can be sizable; therefore, they require complete curettage and grafting with bone substitution materials. This case report presents a sizeable traumatic mandibular cyst in a young man treated by surgical exploration and filled with autologous dentin graft (ADG) prepared from an extracted impacted tooth 48 (FDI tooth-numbering system) and advanced platelet-rich fibrin (A-PRF). Initially, an A-PRF membrane was used to cover the apices of teeth 42 and 43, which were protruding into the defect to protect their periapical structures. Then, a grafting strategy was introduced to achieve two fronts of bone formation: one by stimulation of bone outgrowth from the periphery due to A-PRF cellular activity, and a second by bone deposition directly on dentin particles in the center of the defect. On CBCT scans performed 7 months postoperatively, arrays of trabeculae that were extending from bone boundaries of the cyst defect were merged with more condensed bone deposited on ADG residuals in the center, thus filling the defect. It was found that autologous dentin combined with cellular A-PRF activity is a powerful tool to restore even sizable bone defects in a relatively short time frame with adequate bone remodeling.
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Cistos , Fibrina Rica em Plaquetas , Dentina , Humanos , Masculino , Extração Dentária , Transplante AutólogoRESUMO
Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed.
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Diferenciação Celular/fisiologia , Proteínas da Matriz Extracelular/genética , Botões de Extremidades/citologia , Botões de Extremidades/metabolismo , Animais , Diferenciação Celular/genética , Embrião de Galinha , Galinhas , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. (45)Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of (45)Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.
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Calcificação Fisiológica/fisiologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Cálcio/metabolismo , Linhagem Celular , Meios de Cultura/química , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Selenito de Sódio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Transferrina/metabolismoRESUMO
PURPOSE: There is little knowledge about healing patterns for the socket with an intentionally retained root fragment: a socket shield. The clinical observation is soft tissue ingrowth next to the socket shield. The aim of this study was to evaluate the effectiveness of autologous grafting matrices in preventing soft tissue ingrowth. MATERIALS AND METHODS: Patient data from a private clinic were searched for sockets with a socket shield left to heal with blood clot or grafted with autologous materials: autologous platelet-rich fibrin (PRF), scraped particulate bone, cortical tuberosity bone plate, or particulate dentin and covered with PRF membranes. The included sites were exposed by the flap 4 months after the first surgery, and soft tissue ingrowth depth and width next to the root fragment were measured by a scaled probe and documented. RESULTS: Evaluation of 34 sites showed the greatest depth of soft tissue ingrowth in the nongrafted sockets (6.0 ± 0.0 mm). Grafting with PRF plugs (depth of 2.3 ± 0.2 mm) or particulate bone (depth of 2.7 ± 0.6 mm) decreased soft tissue ingrowth. Grafting with particulate dentin or cortical tuberosity bone plate resulted in a soft tissue ingrowth depth of only 1 mm, yielding the best clinical outcome. Radiography confirmed those findings. CONCLUSION: Autologous dentin particulate or tuberosity cortical bone plate is most effective for preventing soft tissue ingrowth.
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Fibrina Rica em Plaquetas , Alvéolo Dental , Tecido Conjuntivo , Humanos , Estudos Retrospectivos , Extração Dentária , Alvéolo Dental/diagnóstico por imagem , Alvéolo Dental/cirurgiaRESUMO
Chondrocyte apoptosis is thought to be an important step in the calcification of cartilage in vivo; however, there are conflicting reports as to whether or not this apoptosis is a necessary precursor to mineralization. The goal of this study was to determine whether or not apoptosis is necessary for mineralization in an in vitro murine micromass model of endochondral ossification. C3H10T1/2 murine mesenchymal stem cells were plated in micromass culture in the presence of 4 mM inorganic phosphate with the addition of the apoptogens, camptothecin, or staurosporine, to induce apoptosis. The rate and total accumulation of mineralization was measured with (45)Ca uptake. In these studies, both apoptogens increased the rate of mineralization, with staurosporine increasing (45)Ca accumulation by about 2.5 times that of controls and camptothecin increasing total amounts of mineralization about 1.5 times that of controls. Inhibiting cell apoptosis with the caspase inhibitor, ZVAD-fmk, to prevent apoptosis, caused slower rates of (45)Ca uptake; however, total amounts of (45)Ca accumulation reached the same values by day 30 of culture. FTIR data showed mineralization in all samples treated with 4 mM inorganic phosphate, with the highest mineral to matrix ratios in the camptothecin treated samples.
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Apoptose/fisiologia , Calcificação Fisiológica , Condrócitos/citologia , Animais , Aves , Cálcio/farmacocinética , Técnicas de Cultura de Células , Cinética , Células-Tronco Mesenquimais/citologia , CamundongosRESUMO
This study utilized radiographic comparative analysis in order to evaluate dimensional ridge changes four months after tooth extraction and immediate grafting with mineralized dentin particulate autograft and chopped plateletrich fibrin. Fiftyeight extraction sockets with up to 2 mm of missing buccal bone in the coronal aspect compared to the lingual bone were included. Graft material was covered with either a plateletrich fibrin membrane or collagen sponge with no effort to achieve primary closure. The dimensional changes of the ridge were assessed on conebeam computed tomography (CBCT) images acquired prior to extraction and four months later. The reduction in the buccal bone plate thickness 1 mm, 3 mm, and 5 mm below the buccal crest was -0.87 ± 0.84 mm, -0.60 ± 0.70 mm, and -0.41 ± 0.55 mm, respectively. The mean ridge width changes 1 mm, 3 mm, and 5 mm below the crest were -1.38 ± 1.24 mm, -0.82 ± 1.13 mm, and -0.43 ± 0.89 mm, respectively. The average midbuccal bone height gain was +1.1%, while the midlingual height gain was 5.6%. A mineralized dentin autograft with plateletrich fibrin is effective in preserving postextraction alveolar ridge dimensions.
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BACKGROUND: Periodontal disease is infectious in nature and leads to an inflammatory response. It arises from the accumulation of subgingival bacterial plaque and leads to the loss of attachment, increased probing depth, and bone loss. It is one of the world's most prevalent chronic diseases. In this study we developed and studied metronidazole-loaded 50/50 poly(DL-lactide-co-glycolide) (PDLGA), 75/25 PDLGA, and poly(DL-lactic acid) (PDLLA) films. These films are designed to be inserted into the periodontal pocket and treat infections with controlled-release metronidazole for >or=1 month. METHODS: The structured films were prepared using the solution-casting technique. Concentrated solutions and high solvent-evaporation rates were used to get most of the drug located in the bulk, i.e., in whole film's volume. The effects of copolymer composition and drug content on the release profile, cell growth, and bacterial inhibition were investigated. RESULTS: The PDLLA and 75/25 PDLGA films generally exhibited a low- or medium-burst release followed by a moderate release at an approximately constant rate, whereas the 50/50 PDLGA films exhibited a biphasic release profile. The drug released from films loaded with 10% weight/weight metronidazole resulted in a significant decrease in bacterial viability within several days. When exposed to human gingival fibroblasts in cell culture conditions, these films maintained their normal fibroblastic features. CONCLUSIONS: This study enabled the understanding of metronidazole-release kinetics from bioabsorbable polymeric films. The developed systems demonstrated good biocompatibility and the ability to inhibit Bacteroides fragilis growth; therefore, they may be useful in the treatment of periodontal diseases.
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Anti-Infecciosos Locais/administração & dosagem , Bacteroides fragilis/efeitos dos fármacos , Implantes de Medicamento , Metronidazol/administração & dosagem , Bolsa Periodontal/tratamento farmacológico , Implantes Absorvíveis , Infecções por Bacteroides/tratamento farmacológico , Células Cultivadas , Implantes de Medicamento/síntese química , Implantes de Medicamento/toxicidade , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Gengiva/efeitos dos fármacos , Humanos , Teste de Materiais , Testes de Sensibilidade Microbiana , Bolsa Periodontal/microbiologia , Poliésteres/síntese química , Poliésteres/toxicidade , Poliglactina 910/síntese química , Poliglactina 910/toxicidadeRESUMO
Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb-bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4 mM inorganic phosphate and no organic-phosphate esters; control cultures had 1 mM inorganic phosphate. Mineralization was monitored based on (45)Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis. Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin-LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating that the phosphatases were, in part, providing a source of inorganic phosphate. To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.
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Calcificação Fisiológica , Cartilagem , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/química , Células-Tronco Mesenquimais/fisiologia , Animais , Apigenina/metabolismo , Cartilagem/citologia , Cartilagem/fisiologia , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Embrião de Galinha , Inibidores Enzimáticos/metabolismo , Células-Tronco Mesenquimais/citologia , FosforilaçãoRESUMO
Periodontal diseases are initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response, degradation of dento gingival fibrous tissue and its detachment from root cementum. It is well accepted, that osteoclastic alveolar bone loss is governed exclusively through secretion of proinflammatory cytokines. Nevertheless, our findings suggest that once degradation of collagen fibers by MMPs occurs, a drop of cellular strains cause immediate release of ATP from marginal gingival fibroblasts, cell deformation and influx of Ca+2. Increased extracellular ATP (eATP) by interacting with P2×7 purinoreceptors, present on fibroblasts and osteoblasts, induces generation of receptor activator of nuclear factor kB ligand (RANKL) that further activates osteoclastic alveolar bone resorption and bone loss. In addition, increased eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3-inflammasome activation via P2×7. Then, the inflammatory cells secrete cytokines, interleukin IL-1, TNF and RANKL that further trigger alveolar bone resorption. Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. It seems therefore, that eATP is a key modulator that initiates the pathway of alveolar bone resorption and bone loss in patients with periodontal disease. In conclusion, we propose that strain release in gingival fibroblasts aligned on collagen fibers, due to activity of MMP, activates release of ATP that triggers the pathway of alveolar bone resorption in periodontitis. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis.
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Trifosfato de Adenosina/fisiologia , Perda do Osso Alveolar/fisiopatologia , Fibroblastos/citologia , Gengiva/citologia , Periodontite/fisiopatologia , Animais , Citocinas/metabolismo , Fibroblastos/metabolismo , Gengiva/metabolismo , Humanos , Inflamassomos/metabolismo , Leucócitos/metabolismo , Periodontite/microbiologiaRESUMO
Osteoporosis is characterized by reduction in trabecular bone in conjunction with increased marrow cell adiposity. While these changes occur within weeks, monitoring of treatment efficacy as performed by DEXA is sensitive only to long-term changes. MRI is sensitive to bone marrow changes but is less affordable. In a recent study, we have shown that a stray-field NMR can monitor bone marrow cellular changes that are related to osteoporosis. Objectives. To demonstrate sensitivity of a low-field tabletop NMR scanner to bone marrow dynamics following hormonal treatment in rats. Methods. Two-month-old female rats (n = 36) were ovariectomized (OVX) and dosed for the ensuing 3 or 5 weeks with 20 mg/kg of PTH(1-34). Hind limbs femurs and tibiae were isolated and underwent ex vivo microradiography and histology and NMR relaxometry at 6 weeks (preventive experiment) and 11 weeks (therapeutic treatment experiment) after OVX. Results. OVX rats developed osteoporotic changes including adipogenic marrow compared to Sham and PTH treated rats. T2 and ADC NMR relaxation coefficients were found to correlate with marrow composition. Conclusions. This study suggests that stray-field NMR, an affordable method that is sensitive to the rapid cellular changes in bone marrow, may have a clinical value in monitoring hormonal treatment for osteoporosis.
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BACKGROUND: Several studies have shown that sectioning bundles of collagen fibers in the marginal gingiva during surgical procedures in animals is a distinct stimulus for alveolar bone resorption. Normally, gingival and periodontal fibroblasts, which reside on these collagen fibers, create physiological traction forces generated by the cytoskeleton. By splitting the fibers, traction forces are released, inducing changes in the cytoskeleton and cell shape. In this study, four drugs were selected, including cytochalasin D, EDTA, sodium orthovanadate, and H-7, all influencing the cytoskeleton-integrin-extracellular matrix (ECM) pathway, for their ability to reduce alveolar bone loss by local application. METHODS: The drugs were applied locally only once at the site of mucoperiosteal flap surgery in a rat model. Cytochalasin D (1 microl/microl), EDTA (0.24 mg/microl), sodium orthovanadate (0.02 mg/microl), and H-7 (0.10 microl/microl), each separately, were carried by a protective paste and placed immediately after elevating the flap. The analysis of alveolar bone loss was performed 3 weeks after surgery by scanning the microradiographic films of the mandible cross-sections. The percentages of cross sections with no, moderate, or severe bone loss in treated in comparison to non-treated rats are presented. RESULTS: EDTA, sodium orthovanadate, and H-7 were significantly effective in reducing alveolar bone loss. They were effective in reducing the amount of severe bone loss by 53%, 20%, and 58% while increasing the number of sections with no bone loss by 25%, 23%, and 35%, respectively. Cytochalasin D reduced alveolar bone loss insignificantly. CONCLUSION: EDTA, sodium orthovanadate, and H-7 are effective in reducing alveolar bone loss in rats following mucoperiosteum surgery.
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Perda do Osso Alveolar/tratamento farmacológico , Citoesqueleto/efeitos dos fármacos , Doenças Mandibulares/tratamento farmacológico , Doenças Maxilares/tratamento farmacológico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Perda do Osso Alveolar/prevenção & controle , Análise de Variância , Animais , Quelantes/uso terapêutico , Citocalasina D/uso terapêutico , Ácido Edético/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Masculino , Doenças Mandibulares/prevenção & controle , Doenças Maxilares/prevenção & controle , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Ratos , Ratos Wistar , Vanadatos/uso terapêuticoRESUMO
Osteomalacia is a pathological bone condition in which there is deficient primary mineralization of the matrix, leading to an accumulation of osteoid tissue and reduced bone mechanical strength. The hypothesis that there are no qualitative or quantitative differences in osteomalacic bone mineral or matrix compared to disease-free bones was tested by examining unstained sections of polymethyl methacrylate (PMMA) embedded iliac crest biopsies using Fourier transform infrared imaging (FTIRI) at approximately 6-microm spatial resolution. Controls were seven female subjects, aged 36-57, without apparent bone disease. The experimental group consisted of 11 patients aged 22-72, diagnosed with osteomalacia. The spectroscopic parameters analyzed in each data set were previously established as sensitive to bone quality: phosphate/amide I band area ratio (mineral content), 1660/1690 cm(-1) peak ratio (collagen cross-links), and the 1030/1020 cm(-1) peak ratio (mineral crystallinity). The correspondence between spectroscopic mineral content (phosphate/amide I ratio) and ash weight was validated for apatite crystals of different composition and crystallite size. The FTIRI results from the biopsies expressed as color-coded images and pixel population means were compared with the nonparametric Mann-Whitney U test. There were no significant differences in the cortical parameters. Significant difference was found in the mineral content of the trabecular regions with a lower mean value in osteomalacia (P = 0.01) than in controls. Mineral crystallinity tended to be decreased in the trabecular bone (P = 0.09). This study supports the hypothesis that, in osteomalacia, the quality of the organic matrix and of mineral in the center of bone does not change, while less-than-optimal mineralization occurs at the bone surface. This study provides the first spectroscopic evaluation of whole bone mineral and matrix properties in osteomalacia, demonstrating that there are few differences in collagen cross-links between biopsies from patients with osteomalacia and from individuals without histological evidence of bone disease.
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Osso e Ossos/patologia , Osteomalacia/patologia , Adulto , Biópsia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Porous polymeric scaffolds play a key role in most tissue-engineering strategies. A series of non-degrading porous scaffolds was prepared, based on bulk-copolymerisation of 1-vinyl-2-pyrrolidinone (NVP) and n-butyl methacrylate (BMA), followed by a particulate-leaching step to generate porosity. Biocompatibility of these scaffolds was evaluated in vitro and in vivo. Furthermore, the scaffold materials were studied using the so-called demineralised bone matrix (DBM) as an evaluation system in vivo. The DBM, which is essentially a part of a rat femoral bone after processing with mineral acid, provides a suitable environment for ectopic bone formation, provided that the cavity of the DBM is filled with bone marrow prior to subcutaneous implantation in the thoracic region of rats. Various scaffold materials, differing with respect to composition and, hence, hydrophilicity, were introduced into the centre of DBMs. The ends were closed with rat bone marrow, and ectopic bone formation was monitored after 4, 6, and 8 weeks, both through X-ray microradiography and histology. The 50:50 scaffold particles were found to readily accommodate formation of bone tissue within their pores, whereas this was much less the case for the more hydrophilic 70:30 counterpart scaffolds. New healthy bone tissue was encountered inside the pores of the 50:50 scaffold material, not only at the periphery of the constructs but also in the center. Active osteoblast cells were found at the bone-biomaterial interfaces. These data indicate that the hydrophobicity of the biomaterial is, most likely, an important design criterion for polymeric scaffolds which should promote the healing of bone defects. Furthermore, it is argued that stable, non-degrading porous biomaterials, like those used in this study, provide an important tool to expand our comprehension of the role of biomaterials in scaffold-based tissue engineering approaches.
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Células da Medula Óssea/citologia , Substitutos Ósseos/química , Regeneração Tecidual Guiada/métodos , Osteogênese/fisiologia , Ácidos Polimetacrílicos/química , Povidona/análogos & derivados , Povidona/química , Crânio/citologia , Engenharia Tecidual/métodos , Células 3T3 , Animais , Células da Medula Óssea/fisiologia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Interações Hidrofóbicas e Hidrofílicas , Teste de Materiais , Camundongos , Polímeros/química , Ratos , Ratos Endogâmicos Lew , Crânio/fisiologia , Propriedades de SuperfícieRESUMO
Microarray gene expression analysis was utilized to identify genes upregulated in primary rat calvaria cultures in response to mechanical force. One of the identified genes designated CMF608 appeared to be novel. The corresponding full-length cDNA was cloned and characterized in more details. It encodes a putative 2597 amino acid protein containing N-terminal signal peptide, six leucine-rich repeats (LRRs), and 12 immunoglobulin-like repeats, 10 of which are clustered within the C-terminus. Expression of CMF608 is bone-specific and the main type of CMF608-positive cells is mesenchymal osteochondroprogenitors with fibroblast-like morphology. These cells reside in the perichondral fibrous ring of La Croix, periosteum, endosteum of normal bone as well as in the activated periosteum and early fibrous callus generated postfracture. Expression of CMF608 is notably absent from the regions of endochondral ossification. Mature bone cell types do not produce CMF608 with the exception of chondrocytes of the tangential layer of the articular cartilage, which are thought to be under constant mechanical loading. Ectopic expression of CMF608 in HEK293T cells shows that the protein is subjected to post-translational processing and its N-terminal approximately 90 kDa polypeptide can be found in the conditioned medium. Ectopic expression of either the full-length cDNA of CMF608 or of its N-terminal region in CMF608-negative ROS17/2.8 rat osteosarcoma cells results in transfected clones displaying increased proliferation rate and the characteristics of less-differentiated osteoblasts compared to the control cells. Our data indicate that CMF608 is a unique marker of early osteochondroprogenitor cells. We propose that it could be functionally involved in maintenance of the osteochondroprogenitor cells pool and its down-regulation precedes terminal differentiation.
Assuntos
Osso e Ossos/fisiologia , Condrócitos/fisiologia , Osteócitos/fisiologia , Biossíntese de Proteínas , Células-Tronco/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Fraturas Ósseas/genética , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Crânio/fisiologia , Estresse Mecânico , Regulação para CimaRESUMO
Mesenchymal cells isolated from chick limb-buds, when plated in micromass culture, differentiate into chondrocytes, forming a mineralizable matrix. Because these cells produce prostanoids during differentiation, this system was used to test the hypothesis that E-series prostanoids are involved in chondrocyte-mediated calcification. Prostaglandins E(2) (PGE(2)) and E(1) (PGE(1)), and the E-series analog, misoprostol (MP), increased chondrocyte cAMP content in the presence of the phosphodiesterase inhibitor IBMX. The increases for PGE(1) and PGE(2) at their saturation concentrations were twofold to threefold greater than for MP at its saturation concentration. At culture day 7, 9, or 11 (the day that mineralization commenced), the maximal cyclic adenosine monophosphate (cAMP) production was at a concentration of 250--500 ng/ml PGE(2), 500--1000 ng/ml PGE(1), and less-than-or-equal5000 ng/ml MP. At these concentrations, PGE(1) and PGE(2), but not MP, stimulated chondrocyte differentiation. (45)Ca accumulation in mineralizing, as compared to nonmineralizing, similarly treated control cultures was not altered by the addition of indomethacin and/or prostanoid when the phosphate source was inorganic phosphate. Because the prostanoids decrease alkaline phosphatase activity, initial beta-glycerophosphate-mediated mineralization was inhibited by each of the prostanoids.
RESUMO
In summary, the present commentary proposes a hypothesis that alveolar bone remodeling and bone loss in periodontitis, periodontal surgery, and in orthodontic tooth movement is triggered by a common "strain relaxation" signaling pathway of gingival and periodontal fibroblasts. The abrupt splitting, degradation, or relaxation of collagen fibers in the marginal periodontium produces a "strain relaxation" signal in the local fibroblasts which reside on these fibers, activating an ECM-integrin-cytoskeleton pathway. A cascade of cellular reactions which lead to osteoclastic bone resorption starting on the inner aspect (periodontal) of the alveolar bone then persists. A novel therapeutic approach is suggested here by using locally delivered drugs intervening in the cell contractile apparatus.
Assuntos
Perda do Osso Alveolar/fisiopatologia , Fibroblastos/fisiologia , Periodontite/fisiopatologia , Periodonto/citologia , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/prevenção & controle , Animais , Antibacterianos/farmacologia , Colágeno/química , Análise do Estresse Dentário , Fibroblastos/efeitos dos fármacos , Humanos , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Transdução de Sinais , Estresse Mecânico , Linfócitos T/fisiologia , Resistência à Tração , Tetraciclina/farmacologiaRESUMO
BACKGROUND: Bone graft substitutes are currently used individually or in various combinations in reconstructing bone defects. Nacre, marine mineralized structure, was recently proposed as a very biocompatible and osteoinductive material for use in periodontal and implant surgery. Our aim was to investigate the interaction between natural nacre and fresh bone marrow, during bone development, in an ectopic site of DA rats. Surface modifications of nacre were tested. METHODS: Demineralized bone matrix (DBM) cylinders (demineralized cortex of diaphysis) prepared from rat femurs were filled with fresh marrow, which was removed from other 2-month old DA rat femurs. Natural nacre particle or nacre which was treated with HCl, phosphate buffer saline (PBS), and Ca(OH)2 to modify its surface was placed into the DBM cylinders. The cylinders were implanted subcutaneously at the thoracic region of growing DA rats. After 4 weeks the cylinders were surgically removed, fixed in buffered formalin, and x-rayed. Scans of the microradiographs and histological evaluation of the DBM cylinders including bone developed at the interface of nacre and its surface modifications were compared to marrow controls. RESULTS: The results show that natural nacre is a poor conductive biomaterial in a bone developmental environment. Nacre surface treated with Ca(OH)2 and PBS was found to be most biocompatible. In this group, new bone was apposed directly on the nacre surface and the total amount of bone was highest in comparison to other treatment groups. CONCLUSIONS: This study does not support previous observations that nacre is osseoinductive. Our model system seems to be very sensitive and capable of testing interaction between surface modifications of biomaterials and fresh marrow in the process of new bone development.