RESUMO
Intensive work has been performed on the characterization of the mechanical properties of mineralised tissues formed in vivo. However, the mechanical properties of bone-like tissue formed in vitro have rarely been characterised. Most research has either focused on compact cortical bone or cancellous bone, whilst leaving woven bone unaddressed. In this study, bone-like mineralised matrix was produced by osteoblasts cultured in vitro on the surface of titanium alloys. The volume of this tissue-engineered bone is so small that the conventional tensile tests or bending tests are implausible. Therefore, nanoindentation techniques which allow the characterization of the test material from the nanoscale to the microscale were adopted. These reveal the apparent elastic modulus and hardness of the calcospherulite crystals (a representative element for woven bone) are 2.35 +/- 0.73 and 0.41 +/- 0.15 GPa, respectively. The nanoscale viscoelasticity of such woven bone was further assessed by dynamic indentation analysis.
Assuntos
Ligas/química , Desenvolvimento Ósseo/fisiologia , Alicerces Teciduais/química , Titânio/química , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Substitutos Ósseos/química , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Células Cultivadas , Dureza/fisiologia , Teste de Materiais , Nanoestruturas/química , Nanotecnologia/métodos , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Engenharia TecidualRESUMO
During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.
Assuntos
Desintegrinas/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Metaloendopeptidases/biossíntese , Osteoblastos/enzimologia , Osteogênese/fisiologia , Regulação para Cima/fisiologia , Proteínas ADAM , Proteína ADAMTS1 , Animais , Células Cultivadas , Desintegrinas/genética , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Metaloendopeptidases/genética , Proteoglicanas/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Extracellular ATP acting through purinoceptors may be an important factor in the modulation of bone turnover. In this study we cloned and sequenced the P2U purinoceptor from osteoclastoma, confirming the recently published human sequence. Furthermore, by the reverse transcriptase-linked polymerase chain reaction (RT-PCR) and Southern blotting we demonstrated expression of P2U receptor mRNA in bone, primary cultures of human bone-derived cells, and two osteosarcoma cell lines, Saos2 and Te85. P2U receptor transcripts were identified in alkaline phosphatase-positive human bone-derived cells isolated by flow cytometry providing strong evidence for the expression of the P2U purinoceptor in mature osteoblasts. P2U receptor transcripts were also detected in a purified giant cell population isolated from osteoclastoma, indicating that this receptor is also expressed by osteoclasts. These data suggest that purinergic agonists may play a role in the regulation of bone metabolism.
Assuntos
Neoplasias Ósseas/metabolismo , Osso e Ossos/metabolismo , Tumor de Células Gigantes do Osso/metabolismo , Osteoblastos/metabolismo , Receptores Purinérgicos P2/genética , Idoso , Fosfatase Alcalina/metabolismo , Sequência de Bases , Southern Blotting , Neoplasias Ósseas/patologia , Células Cultivadas , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Citometria de Fluxo , Tumor de Células Gigantes do Osso/patologia , Células Gigantes/metabolismo , Células Gigantes/patologia , Humanos , Masculino , Dados de Sequência Molecular , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteossarcoma/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/metabolismo , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
A simple technique has been developed to identify the resorption lacunae excavated by avian osteoclasts in vitro. Briefly, devitalized bovine bone wafers, with cells in situ, are fixed, stained with toluidine blue, and then examined by reflected light microscopy. Resorption pits are clearly visible using a x 10 objective, even when the resorbed areas are covered by a confluent layer of cells. The technique can be used to quantify the plan area, depth, and volume of the excavations. Depth and volume are assessed using the x 50 objective lens. This technique has been used to investigate the effects of retinyl acetate and EHDP on bone resorption.
Assuntos
Reabsorção Óssea/fisiopatologia , Microscopia/métodos , Osteoclastos/fisiologia , Animais , Bovinos , Embrião de Galinha , Diterpenos , Ácido Etidrônico/farmacologia , Técnicas Histológicas , Técnicas In Vitro , Osteoclastos/efeitos dos fármacos , Ésteres de Retinil , Vitamina A/análogos & derivados , Vitamina A/farmacologiaRESUMO
It has previously been reported that low extracellular pH stimulates the excavation of resorption lacunae by rodent osteoclasts in vitro. Using avian bone cells in a similar in vitro assay we have demonstrated that osteoclast activity is optimal at pH 7.20-7.40 and is inhibited at extremes of pH (less than 7.10 and greater than 7.60). Over the first 24 h of incubation at low pH there may be an increase in osteoclastic resorption but to a lesser extent than that reported for rodent cells. However, after 24-30 h in culture there is little or no further increase in bone resorption, presumably due to a cytotoxic effect of low pH acting either on the osteoclast directly or via nonosteoclastic bone cells. In contrast to a previous report, in which preincubation of wafers for 24 h had no effect on bone resorption, we found that preincubation of bone substrates at pH 6.50 for longer periods enhances subsequent resorption at pH 7.20.
Assuntos
Reabsorção Óssea/fisiopatologia , Osteoclastos/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Concentração de Íons de HidrogênioRESUMO
We investigated the expression of cytokine transcripts in osteoblast-like cells derived from explants of pagetic and normal bone. A reverse transcription-linked PCR was used that allowed the simultaneous analysis of a range of cytokines. Normal osteoblast-like cells were found to contain the transcripts for IL-1 beta, IL-6, and TGF-beta 1. For the first time we detected in bone cells the two other mammalian isoforms of TGF-beta, beta 2, and beta 3. Furthermore, we have also identified mRNA for IL-3 and the novel chemotactic factor, IL-8. Using this sensitive technique it was not possible to detect mRNA for IL-1 alpha, IL-2, IL-4, IL-5, IL-7, TNF-alpha, or interferon-gamma. The human osteosarcoma cell line Saos-2 also showed a similar pattern of expression of these cytokines to primary osteoblast-like cells, with the exception that TNF-alpha was also identified. Cells isolated from pagetic bone showed essentially the same profile of cytokine expression as normal bone except that TNF-alpha was also detected in two of four samples. The cytokine profile of successive populations of cells harvested from one explant culture at 9, 22, and 57 days showed a consistent pattern of cytokine expression, demonstrating the phenotypic stability of the osteoblast-like cells in long-term cultures.
Assuntos
Citocinas/biossíntese , Osteíte Deformante/metabolismo , Osteoblastos/metabolismo , Sequência de Bases , Células Cultivadas , Citocinas/genética , DNA Complementar/análise , Humanos , Interleucinas/biossíntese , Interleucinas/genética , Dados de Sequência Molecular , Osteoblastos/citologia , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Transcrição Gênica , Fatores de Crescimento Transformadores/biossíntese , Fatores de Crescimento Transformadores/genética , Células Tumorais CultivadasRESUMO
It has been proposed that Paget's disease of bone is caused by the infection of bone cells with one or several paramyxoviruses. In this study we used the polymerase chain reaction (PCR), which allows the detection of very low levels of a target nucleic acid sequence, to study cultures of pagetic bone cells and samples of pagetic bone. Oligonucleotide primers were designed to flank a sequence of the nucleocapsid genome of measles virus and canine distemper virus (CDV). Within this fragment there were contrasting restriction endonuclease sites specific to measles or CDV that allowed identification of the original template. We were unable to detect paramyxovirus RNA in four strains of human bone cells outgrown from pagetic bone and one strain derived from an uninvolved site of a patient with Paget's disease. Furthermore, paramyxovirus sequences were not detected in cDNA prepared from six samples of pagetic bone biopsies. The work presented here further questions the role of measles and CDV in the abnormal remodeling observed in Paget's disease.
Assuntos
Osso e Ossos/microbiologia , Vírus da Cinomose Canina/isolamento & purificação , Vírus do Sarampo/isolamento & purificação , Osteíte Deformante/microbiologia , RNA Viral/análise , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Osso e Ossos/citologia , Células Cultivadas , Primers do DNA , DNA Complementar , Vírus da Cinomose Canina/genética , Feminino , Humanos , Masculino , Vírus do Sarampo/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Osteoblastos/microbiologia , Reação em Cadeia da PolimeraseRESUMO
We investigated the production of parathyroid hormone-related protein (PTHrP) by cells derived from explants of human bone. Using an immunoradiometric assay (IRMA), PTHrP was detected in conditioned medium from cultures of bone-derived cells from 6 of 7 patients investigated in this study. PTHrP mRNA was identified in human bone cells using reverse transcriptase-linked polymerase chain reaction (RT-PCR) and by Northern analysis. Transcripts for PTHrP were detected in a purified population of alkaline phosphatase positive cells isolated from human bone marrow cultures by flow cytometry, confirming the expression of PTHrP mRNA by cells of the osteoblastic lineage. Production of PTHrP was inhibited by 10(-6) M of the glucocorticoids, prednisolone and desacetylated deflazacort, in a dose-dependent manner. In addition, RT-PCR followed by Southern blot analysis detected a decrease in steady-state PTHrP mRNA in cultures of human bone-derived cells treated with 10(-6) M prednisolone.
Assuntos
Anti-Inflamatórios/farmacologia , Osteoblastos/metabolismo , Prednisolona/farmacologia , Pregnenodionas/farmacologia , Proteínas/genética , Adolescente , Adulto , Idoso , Fosfatase Alcalina/metabolismo , Sequência de Bases , Northern Blotting , Southern Blotting , Células da Medula Óssea , Células Cultivadas , Criança , Meios de Cultivo Condicionados , Primers do DNA/química , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Osteoblastos/citologia , Proteína Relacionada ao Hormônio Paratireóideo , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Bone cells are regulated by interactions with both growth factors and components of the extracellular matrix (ECM). Syndecans are cell-surface heparan sulfate proteoglycans known to play a role in cell adhesion and migration, and binding of growth factors. This study was performed to investigate the expression of syndecans by osteoblasts. Reverse transcription-linked polymerase chain reaction (RT-PCR) and Northern analysis detected syndecan transcripts in the human osteosarcoma cell lines MG-63, TE-85, SaOS-2, and U2OS; human osteoblast-like cells; rat calvarial osteoblasts; and in human bone. Western blot analysis of proteoglycans from MG-63 and TE-85 cells detected multiple heparan sulfate proteoglycan core proteins consistent with syndecan expression. Regulation of syndecan-1, -2, and -4 expression was investigated in TE-85, MG-63, and SaOS-2 cells, in response to interleukin (IL)-1beta, and IL-6, parathyroid hormone [PTH(1-34)], and 1,25(OH)2-vitamin D3. Northern analysis demonstrated that in the osteosarcoma cell lines there was no regulation of syndecan transcript levels in response to PTH(1-34) or 1,25(OH)2-vitamin D3 for 24 or 48 h. In contrast, when MG-63 and SaOS-2 cells were incubated with IL-1beta (0.01-10 ng/mL) and IL-6 (0.1-50 ng/mL) there was a dose-dependent decrease in mRNA levels for syndecan-1 and -2 at 24 and 48 h, but in response to IL-1beta upregulation in the levels of syndecan-4 transcripts. In addition, Northern analysis was performed on RNA isolated from neonatal rat calvarial osteoblasts cultured under conditions that promote osteogenesis for 0, 5, 13, 21, and 35 days. Syndecan-1 expression was observed to decrease during the culture period, syndecan-2 transcript levels increased, and there appeared to be no overall change in syndecan-4 levels. Controlled expression of syndecans by cells of the osteoblast lineage may be important in the regulation of osteoblastic proliferation and differentiation.
Assuntos
Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/farmacologia , Glicoproteínas de Membrana/genética , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proteoglicanas/genética , Animais , Sequência de Bases , Calcitriol/farmacologia , Linhagem Celular , Proteoglicanas de Heparan Sulfato/biossíntese , Humanos , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Sondas de Oligonucleotídeos/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , Hormônio Paratireóideo/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sindecana-1 , Sindecanas , Células Tumorais CultivadasRESUMO
The adhesive interactions that occur between bone cells and the developing matrix during bone formation help guide coupled remodeling and the maintenance of bone mass. Here, we provide evidence that acetylcholinesterase (AChE) is a novel osteoblast-derived mediator of cell-matrix interactions in bone. These findings complement an increasing body of evidence which suggests that AChE, in addition to its role in terminating cholinergic signaling, may be instrumental in regulating cellular differentiation and adhesion. We have shown, using RT-PCR, that osteosarcoma cell lines and primary cultures of osteoblasts express AChE mRNA. Expression appeared to be differentiation-dependent, and restricted to AChE splice variants containing the T subunit (exon 6). Immunofluorescent localization demonstrated that these osteoblastic cells expressed protein for AChE with an intracellular vesicular distribution. Immunohistochemistry on tissue sections confirmed AChE expression by osteoblasts in vivo, and revealed the presence of AChE along cement lines, also identified by enzyme histochemistry. In vitro functional studies indicated that osteoblast-like cells adhered specifically to and spread on AChE substrates, but did not interact with butyrylcholinesterase, a closely related protein. Our evidence strongly implicates AChE as a novel bone matrix protein, capable of mediating cell-matrix interactions, and as such may be a principal participant in organized bone formation and the regulation of remodeling.
Assuntos
Acetilcolinesterase/metabolismo , Desenvolvimento Ósseo/fisiologia , Matriz Óssea/fisiologia , Osteoblastos/enzimologia , Animais , Adesão Celular/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Signaling between the various types of cells found in bone is responsible for controlling the activity of osteoblasts and osteoclasts, and therefore the regulation of bone mass. Our identification of a neuronal glutamate transporter in osteoblasts and osteocytes suggests the possibility that bone cells may use the excitatory amino acid glutamate as a signaling molecule. In these studies we report the expression of different subtypes of glutamate receptors in osteoblasts and osteoclasts in vitro and in vivo. We have identified expression in human and rat bone cells of N-methyl-D-aspartate receptor-1 (NMDAR-1) and 2D subunits and PSD-95, the NMDA receptor clustering protein associated with signaling in the central nervous system. In situ hybridization and immunohistochemistry localized NMDAR-1 expression to osteoblasts and osteoclasts in human tissue sections. These findings strengthen the suggestion that glutamate is involved in signaling between bone cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácido Glutâmico/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Receptores de N-Metil-D-Aspartato/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Sistema X-AG de Transporte de Aminoácidos , Animais , Composição de Bases , Transporte Biológico , Proteína 4 Homóloga a Disks-Large , Ácido Glutâmico/genética , Guanilato Quinases , Humanos , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/genéticaRESUMO
It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone-related peptide (PTHrP) gene and that N-terminal PTHrP immunoreactivity can be found in cell medium. Here we have firstly measured immunoreactive PTHrP in 8701-BC cell medium using antibodies raised against midregion and C-terminal fragments, and also demonstrated the expression of PTH/PTHrP receptor by 8701-BC cells. Secondly, we have examined the role, if any, elicited by diverse PTHrP domains on 8701-BC cell proliferation, and invasive behaviour in vitro related to production of extracellular proteolytic enzymes. Our data show that PTHrP [1-34], and, to a minor extent, [67-86] and [107-139], are anti-mitogenic but 'invadogenic' for 8701-BC cells, and suggest that diverse enzymatic activities may contribute to cell invasion in response to different PTHrP fragments. In light of the present data on a chemoattractive role for PTHrP in vitro, we hypothesize that this protein might intervene in local control of the invasive process in breast carcinoma.
Assuntos
Neoplasias da Mama/patologia , Proteínas/farmacologia , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Endopeptidases/metabolismo , Humanos , Dados de Sequência Molecular , Invasividade Neoplásica , Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo , Reação em Cadeia da Polimerase , Inibidores de Proteases/farmacologia , RNA Mensageiro/metabolismo , DNA Polimerase Dirigida por RNA , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/genética , Células Tumorais CultivadasRESUMO
A novel micro-cellular polymer with a well-defined and uniform micro-architecture has been developed as a three-dimensional support matrix for in vitro tissue engineering applications. This material is manufactured through a high internal phase emulsion (HIPE) polymerization route and may be modified with hydroxyapatite. The generic form of the support is known as PolyHIPE Polymer (PHP). By changing the chemical composition of the emulsion and the processing conditions, the pore size can be altered from sub-micron range to a few hundred microns and the porosity varied from 70% to 97%. Our work has investigated the use of this micro-porous polymer as a biomaterial to support the growth of osteoblasts, the bone forming cells in vitro. Three groups of polymers were used that had pore sizes of 40, 60 and 100 microm. Results demonstrated in vitro cell-polymer compatibility, with osteoblasts forming multicellular layers on the polymer surface and also migrating to a maximum depth of 1.4mm inside the scaffold after 35 days in culture. PHP was also able to support the differentiation of osteoblasts and the production of a bone-like matrix. The effect of modifying the polymer with hydroxyapatite was also studied and showed that there was a significant increase in osteoblast numbers penetrating into the polymer. There were few differences, between the pore sizes studied, on the overall penetration of osteoblasts into the polymer but the rate of movement into 100 microm PHP was significantly higher compared to the other sizes investigated. This study shows that osteoblasts seeded onto PHP demonstrate cellular attachment, proliferation and ingrowth leading to the support of an osteoblastic phenotype. Therefore this highly porous scaffold has a potential for bone tissue engineering.
Assuntos
Materiais Biocompatíveis/química , Substitutos Ósseos/química , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Polímeros/química , Estirenos/química , Engenharia Tecidual/métodos , Animais , Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Teste de Materiais , Osseointegração/fisiologia , Porosidade , Ratos , Propriedades de SuperfícieRESUMO
The cellular reaction to wear debris may result in the failure of an artificial joint's fixation to the skeleton. The influence of debris opsinization on cell activity has received little attention. This study seeks to establish whether different proteinaceous culture environments may invoke variant cellular responses to debris challenge. Consideration of the zeta potential of a low density polyethylene particle group and an ex vitro ultrahigh molecular weight polyethylene particle group revealed that the nature of the protein adsorbants is related to the concentration of the proteins in solution. Furthermore, the composition of the adsorbed layer was shown to vary with the spectra of proteins in solution. In standard cell culture conditions zeta potential approached zero, indicating the high probability of particle agglomeration. Cell challenge studies with U937 macrophages showed that BSA and FCS protein adsorption mediated increased cell adhesion, while bovine IgG showed little change over control values. No changes in behavior of osteoblastic cells were observed in similar experiments.
Assuntos
Materiais Biocompatíveis/farmacologia , Macrófagos/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Polietilenos/farmacologia , Adsorção , Algoritmos , Animais , Linhagem Celular Tumoral , Coloides/farmacologia , Meios de Cultura , Campos Eletromagnéticos , Humanos , Tamanho da Partícula , Proteínas/química , Ratos , Soroalbumina Bovina/química , Células U937RESUMO
Buprenorphine is a potent partial opioid agonist that is analyzed in urine to (i) monitor adherence to maintenance or detoxification therapy and (ii) detect illicit use. Buprenorphine analysis is commonly conducted on urine by immunoassay, but is subject to cross-reactivity from other drugs/drug metabolites, including morphine, codeine and dihydrocodeine. This study reports false-positive buprenorphine analysis [Thermo Fisher Scientific cloned enzyme donor immunoassay (CEDIA)] in patients who denied unauthorized buprenorphine use prior to sampling, but who had been prescribed amisulpride. In two cases, confirmatory analysis by liquid chromatography-tandem mass spectrometry was negative (<0.5 µg/L) for buprenorphine and metabolites and positive for amisulpride. Although the cross-reactivity of amisulpride and sulpiride in the CEDIA buprenorphine assay is low (estimated at 0.003 and 0.002%, respectively), it remains a significant consideration given the likely high concentrations of these compounds in urine relative to the low cutoff of the buprenorphine assay. Neither amisulpride nor sulpiride was listed as potential sources of interference on the CEDIA data sheet when this work was performed. These findings highlight the importance of confirming immunoassay-positive buprenorphine results using a more selective analytical technique.
Assuntos
Buprenorfina/urina , Técnicas Imunoenzimáticas/métodos , Sulpirida/análogos & derivados , Sulpirida/urina , Adulto , Amissulprida , Buprenorfina/administração & dosagem , Cromatografia Líquida de Alta Pressão , Reações Falso-Positivas , Humanos , Imunoensaio/métodos , Masculino , Detecção do Abuso de Substâncias/métodos , Sulpirida/uso terapêuticoRESUMO
Topography and surface chemistry have a profound effect on the way in which cells interact with an implant, which in turn impacts on clinical use and performance. In this paper we examine an electrochemical polishing approach in H2SO4/methanol that can be applied to the widely used orthopaedic/dentistry implant material, Ti6Al4V, to produce structured surfaces. The surface roughness, as characterized by R(a), was found to be dependent on the time of electropolishing but not on the voltage parameters used here. The surface chemistry, however, was dependent on the applied electrochemical potential. It was found that the chemical composition of the surface layer was modified during the electrochemical process, and at high potentials (9.0 V) a pure TiO2 layer of at least 10 nm was created on top of the bulk alloy. Characterization of these surfaces with rat cells from the osteoblast lineage provided further evidence of contact guidance by microscale topography with morphology analysis correlating with surface roughness (R(a) 300550 nm). Formation of a bone-like matrix after long-term culture on these surfaces was not strongly dependent upon R(a) values but followed the voltage parameter. These findings suggest that the surfaces created by treatment at higher voltages (9.0 V) produced a nanoscale layer of pure TiO2 on the Ti6Al4V surface that influenced the programme of cellular differentiation culminating in osteogenesis.
Assuntos
Materiais Biocompatíveis/química , Osteoblastos/citologia , Osteoblastos/fisiologia , Titânio/química , Ligas , Animais , Sobrevivência Celular , Células Cultivadas , Galvanoplastia , Teste de Materiais , Ratos , Propriedades de SuperfícieRESUMO
The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of protein trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 microm were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as 10 microm and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that protein could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the morphogenetic properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (bone) regeneration in dental and orthopaedic applications using osteoinductive PEG hydrogel micropatterns.
Assuntos
Materiais Biocompatíveis/química , Osteoblastos/fisiologia , Osteogênese/fisiologia , Polietilenoglicóis/química , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Crescimento Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Portadores de Fármacos/química , Hidrogéis/química , Teste de Materiais , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos , Propriedades de Superfície , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
Osteoclasts are derived from haematopoietic stem cell precursors of the monocyte/macrophage cell lineage, through interaction with factors that are believed to include M-CSF and RANKL. VEGF is a proangiogenic cytokine that has been shown to promote osteoclast differentiation and survival. In this study, we assessed the role of VEGF and its receptors in osteoclastogenesis, in vitro, by culturing osteoclast precursors in the presence of VEGF, VEGF receptor-specific ligands, and blocking antibodies to VEGF receptors. Activation of VEGFR1 in the presence of RANKL induces osteoclast differentiation. Stimulating the receptors individually induced increased resorption by osteoclasts compared to controls but not to the level observed when stimulating both receptors simultaneously. We have shown that VEGF induces osteoclast differentiation through its action on VEGFR1. The way in which VEGF mediates its effect on mature osteoclast activity, however, may be through its interaction with both receptor subtypes.
Assuntos
Diferenciação Celular/fisiologia , Osteoclastos/citologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Reabsorção Óssea , Células Cultivadas , Humanos , Osteoclastos/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Vascular endothelial growth factor (VEGF) is a proangiogenic cytokine that is expressed highly in many solid tumours often correlating with a poor prognosis. In this study, we investigated the expression of VEGF and its receptors in bone metastases from primary human breast tumours and further characterised its effects on osteoclasts in vitro. Breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2 (vascular endothelial growth factor receptor 1 and 2), demonstrating that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 and VEGFR2. RAW 264.7 cells (mouse monocyte cell line) and human peripheral blood mononuclear cells (PBMCs) were cultured with VEGF, RANKL and M-CSF. VEGF and RANKL together induced differentiation of multinucleated, tartrate-resistant acid phophatase (TRAP)-positive cells in similar numbers to M-CSF and RANKL. The PBMCs were also able to significantly stimulate resorption of mineralised matrix after treatment with M-CSF with RANKL and VEGF with RANKL. We have shown that VEGF in the presence of RANKL supports PBMC differentiation into osteoclast-like cells, able to resorb substrate. Vascular endothelial growth factor may therefore play a role in physiological bone resorption and in pathological situations. Consequently, VEGF signalling may be a therapeutic target for osteoclast inhibition in conditions such as tumour osteolysis.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Osteólise/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Animais , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/farmacologia , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor (PTH/PTHrP-R) genes, that N-terminal, mid-regional and C-terminal immunoreactive PTHrP can be found in cell conditioned medium and, furthermore, that exogenously added PTHrP (1-34), (67-86) and, to a minor extent, (107-139) are anti-mitogenic but promote Matrigel invasion by this cell line. It has also been reported that PTHrP gene expression is selectively switched on in those 8701-BC clonal lines endowed with a higher proliferation rate and invasive ability in vitro. Here we have first examined the presence of PTH/PTHrP-R transcript in the different 8701-BC clones by PCR and Southern blot analysis. Second, we have studied the growth and invasive response in vitro to PTHrP fragments by some of these clones, i.e. BC-3A, BC-61 and BC-66, selected on the basis of their lower (BC-3A) or higher (BC-1 and BC-66) Matrigel invasion ability and their expression of PTHrP (positive for BC-61 and BC-66) and PTH/PTHrP-R (positive for BC-61). Our data show the existence of clonal heterogeneity for PTH/PTHrP-R mRNA and for the proliferative and invasive responses elicited by treatment with diverse PTHrP fragments. In particular: (i) the sensitivity to PTHrP (1-34) is restricted due to the uneven expression of PTH/PTHrP-R; (ii) BC-3A cells (the less 'aggressive' clone) are resistant to the anti-mitogenic effect of the PTHrP domains and, most noticeably, exhibit a growth-potentiating response to PTHrP (67-86) opposite to that found for both the parental 8701-BC cells and the two other clones; (iii) all PTHrP fragments tested induced the expression of a growth-restraining and invasion-promoting phenotype by BC-61 cells (one of the more 'aggressive' clones). Present data in vitro support the hypothesis that in vivo PTHrP may be a key element in local control of the invasive process during breast carcinoma development and that its role may be, in turn, dependent upon the biological characteristics and the level of malignancy of the target cells within the multiclonal population of a primary tumour.