RESUMO
Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.
Assuntos
Bradicinina/metabolismo , Fator IX/metabolismo , Fator XII/metabolismo , Fibrina/metabolismo , Calicreínas/metabolismo , Trombina/metabolismo , Coagulação Sanguínea/fisiologia , Bradicinina/química , Cálcio/química , Cálcio/metabolismo , Cátions Bivalentes , Fator IX/química , Fator XI/química , Fator XI/metabolismo , Fator XII/química , Fibrina/química , Humanos , Calicreínas/química , Cinética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Ligação Proteica , Trombina/químicaRESUMO
Adverse tissue responses to prostheses wear particles and released ions are important contributors to hip implant failure. In implant-related adverse reactions T-lymphocytes play a prominent role in sustaining the chronic inflammatory response. To further understand the involvement of lymphocytes in metal-on-metal (MoM) implant failure, primary human lymphocytes were isolated and treated with cobalt-chromium (Co-Cr) wear debris and Co ions, individually, and in combination, for 24, 48 and 120 h. There was a significant increase in cell number where debris was present, as measured by the Neutral Red assay. Interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) secretion levels significantly decreased in the presence of metal particles, as measured by ELISA. Interleukin-2 (IL-2) secretion levels were significantly decreased by both debris and Co ions. Flow cytometry analysis showed that the metal nanoparticles induced a significant increase in apoptosis after 48-h exposure. This investigation showed that prolonged exposure (120 h) to metal debris induces lymphocyte proliferation, suggesting that activation of resting lymphocytes may have occurred. Although cytokine production was affected mainly by metal debris, cobalt toxicity may also modulate IL-2 secretion, and even Co ion concentrations below the MHRA guideline levels (7 ppb) may contribute to the impairment of immune regulation in vivo in patients with MoM implants.
Assuntos
Ligas de Cromo/toxicidade , Cobalto/toxicidade , Prótese de Quadril/efeitos adversos , Linfócitos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Técnicas In Vitro , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Espectrofotometria Atômica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with both FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p<0.05) amounts of Co and Cr ions into the culture medium, and significant (p<0.05) cellular uptake of both ions. There was also an increase (p<0.05) in apoptosis after a 48h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p<0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions+debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one.
Assuntos
Apoptose/fisiologia , Cromo/metabolismo , Cobalto/metabolismo , Próteses Articulares Metal-Metal/efeitos adversos , Monócitos/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cromo/toxicidade , Ligas de Cromo/metabolismo , Ligas de Cromo/toxicidade , Cobalto/toxicidade , Relação Dose-Resposta a Droga , Humanos , Monócitos/efeitos dos fármacos , Células U937RESUMO
Hip resurfacing with cobalt-chromium (CoCr) alloy was developed as a surgical alternative to total hip replacement. However, the biological effects of nanoparticles generated by wear at the metal-on-metal articulating surfaces has limited the success of such implants. The aim of this study was to investigate the effects of the combined exposure to CoCr nanoparticles and cobalt ions released from a resurfacing implant on monocytes (U937 cells) and whether these resulted in morphology changes, proliferation alterations, toxicity and cytokine release. The interaction between prior exposure to Co ions and the cellular response to nanoparticulate debris was determined to simulate the situation in patients with metal-on-metal implants receiving a second implant. Effects on U937 cells were mainly seen after 120h of treatment. Prior exposure to Co ions increased the toxic effects induced by the debris, and by Co ions themselves, suggesting the potential for interaction in vivo. Increased TNF-α secretion by resting cells exposed to nanoparticles could contribute to osteolysis processes in vivo, while increased IFN-γ production by activated cells could represent cellular protection against tissue damage. Data suggest that interactions between Co ions and CoCr nanoparticles would occur in vivo, and could threaten the survival of a CoCr metal implant.
Assuntos
Ligas de Cromo/toxicidade , Cobalto/toxicidade , Nanopartículas Metálicas/toxicidade , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Prótese de Quadril , Humanos , Interferon gama/metabolismo , Interleucina-6/metabolismo , Monócitos , Fator de Necrose Tumoral alfa/metabolismo , Células U937RESUMO
Modified Portland cement porous scaffolds with suitable characteristics for load-bearing bone tissue engineering applications were manufactured by combining the particulate leaching and foaming methods. Non-crosslinked polydimethylsiloxane was evaluated as a potential reinforcing material. The scaffolds presented average porosities between 70 and 80% with mean pore sizes ranging from 300 µm up to 5.0 mm. Non-reinforced scaffolds presented compressive strengths and elastic modulus values of 2.6 and 245 MPa, respectively, whereas reinforced scaffolds exhibited 4.2 and 443 MPa, respectively, an increase of â¼62 and 80%. Portland cement scaffolds supported human osteoblast-like cell adhesion, spreading, and propagation (t = 1-28 days). Cell metabolism and alkaline phosphatase activity were found to be enhanced at longer culture intervals (t ≥ 14 days). These results suggest the possibility of obtaining strong and biocompatible scaffolds for bone repair applications from inexpensive, yet technologically advanced materials such as Portland cement.
Assuntos
Cimentos Ósseos/química , Dimetilpolisiloxanos/química , Teste de Materiais , Osteoblastos/metabolismo , Engenharia Tecidual , Alicerces Teciduais/química , Linhagem Celular , Humanos , Osteoblastos/citologia , Porosidade , Suporte de CargaRESUMO
The need for a suitable scaffolding material for load bearing bone tissue engineering still has yet to be met satisfactorily. In this study, Portland cement and Portland cement/metakaolin (MK) blends were processed to render them biologically and mechanically suitable for such application. Portland cement was mixed with MK at different ratios. The slurries were hydrated under atmospheric (noncarbonated samples) and high-CO2 conditions (carbonated samples). The mechanical properties were characterized via compressive tests. The bioactivity was analyzed in a simulated body fluid solution. Scanning electron microscopy and energy dispersive spectroscopy were used to evaluate sample morphology and chemistry. The cytocompatibility (direct contact assay, MTT test, and alkaline phosphatase activity) was tested using human osteoblast-like cells. Cell responses were observed via conventional and electron microscopy. The results showed that the implementation of MK did not significantly influence the mechanical properties. All the samples evidenced bioactive behavior. Cell experiments confirmed a highly cytotoxic response to the noncarbonated specimens. The introduction of MK as well as the CO2 pretreatment significantly improved the cytocompatibility of the specimens. These results show that properly processed Portland cement and Portland cement/MK blends could present suitable properties for the development of load-bearing scaffolding structures in bone tissue-engineering applications.
Assuntos
Osso e Ossos , Materiais de Construção , Caulim , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Cimentos Ósseos , Humanos , Teste de Materiais , Osteoblastos/citologiaRESUMO
Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of beta-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC < 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC = 2.2), which resulted in expression patterns of the osteogenic markers more consistent with the observed differentiation process. The results suggest that beta-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR.