ABSTRACT
The molecular complexity of the processes which lead to cell adhesion includes membrane and cytoskeletal proteins, involved in the focal adhesion formation, as well as signaling molecules tightly associated with the main intracellular regulatory cascades (Akt/PKB and MAPK/Erk). Dynamic environments, which create substrate deformations at determined frequencies and timing, have significant influences on adhesion mechanisms and in general in cellular behavior. In this work, we investigated the role of mechanical stretching (10% substrate deformation, 1 Hz frequency applied up to 60 min) on adhesion proteins (vinculin and focal adhesion kinase-FAK), related RhoGTPases (Rac1 and RhoA), and intracellular pathways (Akt/PKB and MAPK/Erk) in terms of activation and membrane recruitment in relation with cytoskeletal changes observed (membrane ruffling and filopodia formation). These changes are due to intracellular molecular rearrangements, acting with sequential concerted dynamics, able to modify the cytoskeletal conformation. The observed cellular response adds some important issues for better understanding the cellular behavior in environment which mimic as close as possible the physiological conditions.
Subject(s)
Focal Adhesion Kinase 1/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Stress, Mechanical , Vinculin/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Cell Adhesion/physiology , Cells, Cultured , Focal Adhesion Kinase 1/analysis , Mice , Mitogen-Activated Protein Kinase Kinases/analysis , Pseudopodia/physiology , Signal Transduction , Vinculin/analysis , rho GTP-Binding Proteins/analysisABSTRACT
The interest of scientists in the effects of mechanical stresses on cells is growing, in order to reproduce and understand cell behaviour in an environment closely reproducing physiological conditions. There have been many studies showing that mechanical stimulations are involved in regulating the proliferation, apoptosis and synthesis of proteins and cell morphology. In this study, we have considered the effects of a 20% stretching mechanical stress on MRC5 lung fibroblast cells in order to verify the role of survival/apoptotic pathways. As a survival pathway, the activation of Akt has been studied in association with pro-apoptotic or anti-apoptotic signals such as the Bax/Bcl-2 ratio and cleavage of caspases 3 and 9. Findings have shown the effects of overstressed cellular stretching to be a balance of a cause-and-effect reaction between survival and apoptosis.
Subject(s)
Apoptosis/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Signal Transduction/physiology , Stress, Mechanical , Androstadienes/pharmacology , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line , Cell Shape/drug effects , Cell Survival/drug effects , Cell Survival/physiology , Enzyme Inhibitors/pharmacology , Fibroblasts/drug effects , Humans , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , Wortmannin , bcl-2-Associated X Protein/metabolismABSTRACT
Two series of glasses of general formula (2-p) SiO2.1.1Na2O.CaO.pP2O5.xZnO (p=0.10, 0.20; x=0.0, 0.16, 0.35, and 0.78) have been analyzed for physico-chemical surface features before and after contact with simulated body fluid, morphological characteristics, and osteoblast-like cells behavior when cultured on them. The resulted good cell adhesion and growth, along with nonsignificant changes of the focal contacts, allow the authors to indicate HZ5 and HP5Z5 glasses as the ones having optimal ratio of Zn/P to maintain acceptable cell behavior, comparable to the bioactive glass (Bioglass) used as a control; results are also rationalized by means of three-dimensional models derived by molecular dynamic simulations, with decomposition and conversion rates optimized with respect to the parent Hench's Bioglass.
Subject(s)
Biocompatible Materials/chemistry , Glass/chemistry , Zinc/chemistry , Animals , Cell Adhesion , Cell Line , Cell Proliferation , Focal Adhesions , Materials Testing , Mice , Osteoblasts/cytology , Osteoblasts/physiology , Surface PropertiesABSTRACT
OBJECTIVE: To evaluate therapy and rheumatologic aspects of recurrent acute idiopathic pericarditis (RAIP). METHODS: We studied 46 patients. We used non-steroidal anti-inflammatory drugs (NSAIDs) at high dosage. We did not start corticosteroid: if already started, we planned a very slow tapering; 37 patients (80.4%) were treated with colchicine. We also assessed the frequency of ANA, anti-SSA and Rheumatoid factor. RESULTS: With our protocol recurrences dropped from 0.46 to 0.03 attacks/patient/month (p<0.00001) within 12 months and remained at the same level (0.024) till the end of the follow-up (mean 8 years). In the 37 patients treated with colchicine recurrences dropped from 0.5 to 0.03 (p<0.0001) within 12 months, and in 9 patients not given colchicine from 0.27 to 0.045 (p<0.005). When colchicine was used the decrease was significantly higher (0.47 vs 0.23) (p<0.001). In 27 (58.7%) patients ANA were positive at a titre >1/80, in 7 (15.2%) >1/160. Rheumatoid factor was positive in 7 (15.2%) and anti-SSA in 4 (8.7%). During the follow-up 4 (8.7%) new diagnosis of Sjogren and 1 (2.2%) of Rheumatoid Arthritis were made. CONCLUSION: NSAIDs at high dosage, slow tapering of corticosteroid and colchicine are very effective in RAIP. The improvement is more dramatic in colchicine treated patients, but also other patients can achieve good control of the disease. The finding of ANA, anti-SSA and the new rheumatological diagnoses support the involvement of autoimmunity.
Subject(s)
Autoantibodies/blood , Colchicine/therapeutic use , Pericarditis/drug therapy , Pericarditis/immunology , Tubulin Modulators/therapeutic use , Acute Disease , Adolescent , Adult , Aged , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antibodies, Antinuclear/blood , Drug Therapy, Combination , Female , Follow-Up Studies , Glucocorticoids/therapeutic use , Humans , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Pericarditis/diagnosis , Retrospective Studies , Rheumatoid Factor/blood , Secondary Prevention , Treatment OutcomeABSTRACT
A bioactive glass belonging to the system SiO(2)-CaO-Na(2)O was doped with silver ions by ion exchange in molten salts as well as in aqueous solution. The ion exchange in the solution was done to check if it is possible to prepare an antimicrobial material using a low silver content. The doped glass was characterized by means of X-ray diffraction, SEM observation, EDS analysis, bioactivity test (soaking in a simulated body fluid), leaching test (GFAAS analyses) and cytotoxicity test. It is demonstrated that these surface silver-doped glasses maintain, or even improve, the bioactivity of the starting glass. The measured quantity of released silver into simulated body fluid compares those reported in literature for the antibacterial activity and the non-cytotoxic effect of silver. Cytotoxicity tests were carried out to understand the effect of the doped surfaces on osteogenic cell adhesion and proliferation.
Subject(s)
Biocompatible Materials/chemistry , Glass/chemistry , Silver/chemistry , Anti-Bacterial Agents/chemistry , Body Fluids/chemistry , Calcium Carbonate/chemistry , Carbonates/chemistry , Cations, Monovalent , Cell Proliferation , Cell Survival , Durapatite/chemistry , Electron Probe Microanalysis , Humans , Ion Exchange , Microscopy, Electron, Scanning , Osteoblasts/cytology , Silicon Dioxide/chemistry , Spectrometry, X-Ray Emission , Spectrophotometry, Atomic , Surface Properties , X-Ray DiffractionABSTRACT
Biocompatibility evaluation is a fundamental step in developing new biomaterials. Implants derived from bovine tibial compact bone were analyzed with in vitro tests using fibroblast and osteoblast-like cells. Initially, cell attachment and proliferation were quantified. Results indicated that the pins did not interfere with normal cell adhesion and proliferation; more-over, cell morphology was observed using scanning electron microscopy (SEM) and confocal fluorescence microscopy. In vivo experiments to evaluate material osteointegration are currently in progress. (Journal of Applied Biomaterials and Biomechanics 2005; 3: 35-41).
ABSTRACT
The most significant complication in external fixation is pin tract infection causally related to the highly adaptive ability of bacteria to colonise the surfaces of "inert" biomaterials or of adjacent damaged tissue cells. The hypothesis that coating a pin with a silver-containing compound will decrease bacterial colonisation and/or pin tract infection has been confirmed in other studies in vitro and in vivo experiments. In this work, biocompatibility of silver-coated orthopaedic external fixation pins was compared with stainless steel controls in an in vitro study. Human peripheral blood lymphocytes were used to assess the possible genotoxic effect of silver, studying the frequency of sister-chromatid exchanges and micronuclei while fibroblasts (NIH 3T3) and osteoblast-like cells were used for cytotoxicity and cytocompatibility studies. These studies have shown that silver is neither genotoxic nor cytotoxic as compared to stainless steel, a material in wide use as a metal implant. At 4 days cells cultured on the silver-coated material evidenced good cell spreading and a higher cell count with respect to the uncoated material. It appears that the addition of silver onto implantable medical devices could be beneficial when specific biological properties, such as antibacterial behaviour, are required. Based on these and the previous bacterial studies it seems like the toxicity towards bacteria was quite a bit greater than that towards the human cells.
Subject(s)
Coated Materials, Biocompatible , External Fixators , Silver , 3T3 Cells , Adult , Animals , Cell Survival , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , In Vitro Techniques , Lymphocytes/cytology , Lymphocytes/drug effects , Male , Mice , Middle Aged , Silver/toxicity , Time FactorsABSTRACT
To understand the inflammatory potential of oxidised ultra high molecular weight polyethylene (ox-UHMWPE) compared with the virgin one (UHMWPE), we analysed in vitro the predisposition of their interaction with plasma proteins and cells involved in the inflammatory response. The adsorption on the surface of the two materials of adhesion proteins (Fibronectin and Albumin), and pro-inflammatory proteins (IgG and IgA) have been studied. Moreover, we have evaluated the materials effect on complement activation and on macrophages and monocytes-neutrophils behaviour. The two UHMWPE chemical forms adsorbed all the proteins studied; the only difference was in complement activation. Enzyme immunoassay results evidenced higher levels of factor Bb and iC3b in plasma after the contact with the oxidised form. Physico-chemical properties of the oxidised UHMWPE affected the attachment of the cells as demonstrated by macrophages adhesion experiments. UHMWPE favoured only a limited peritoneal macrophages (PMs) spreading (round-shaped cells); the cell spreading and presence of microvilli on the cell membranes was evident in the case of the oxidised form, suggesting the activation of these cells on this chemical form. Ox-UHMWPE evidenced a statistically significative increase in chemiluminescence values respect to human unstimulated peripheral blood mononuclear cells, an index of increased cell release of reactive oxygen metabolites. In conclusion, UHMWPE oxidative degradation with its chemical modification induces monocytes-neutrophils chemiluminescence activation and PMs morphology changes correlated with macrophage activation, data consistent with the complement activation results obtained in this study; such modifications, along with changes in mechanical properties, are related to implant failure.
Subject(s)
Biocompatible Materials/toxicity , Inflammation/chemically induced , Macrophages, Peritoneal/drug effects , Polyethylenes/toxicity , Adsorption , Animals , Biocompatible Materials/chemistry , Cell Adhesion/drug effects , Cells, Cultured , Complement Activation/drug effects , Fibronectins/metabolism , Humans , Immunoglobulin A/metabolism , Immunoglobulin G/metabolism , In Vitro Techniques , Inflammation/pathology , Inflammation/physiopathology , Inflammation Mediators/metabolism , Joint Prosthesis , Luminescent Measurements , Macrophage Activation/drug effects , Macrophages, Peritoneal/physiology , Macrophages, Peritoneal/ultrastructure , Materials Testing , Mice , Microscopy, Electron, Scanning , Oxidation-Reduction , Polyethylenes/chemistry , Reactive Oxygen Species/metabolism , Serum Albumin/metabolismABSTRACT
An in vitro evaluation of a biomedical device, which combines the mechanical properties of zirconia substrates with the bioactivity of two different glass layers (AP40 and RKKP), was performed. In this work, data on different kinds of analysis were reported both on as-sintered zirconia samples and on RKKP- and AP40-coated zirconia substrates. Structure, composition and morphology of the apatite layer growth on the coated samples after 30 days of soaking in an acellular simulated body fluid, serum protein adsorption, fibroblasts and human osteoblast-like cells adhesion, growth, morphology and biochemical aspects were studied. Results of soaking test in SBF, revealed the growth of an apatite layer on the surface of the glass-coated samples. Proteins adsorbed to the materials were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and results evidenced that the two glass-coated materials bound a higher amount of total protein than did the zirconia substrate. Fibroblasts and osteoblast-like cells cultured on RKKP- and AP40-coated zirconia showed a higher proliferation rate, leading to confluent cultures with higher cell density and a generally better expression of osteoblast alkaline phosphatase activity in comparison with zirconia substrate. In conclusion, our results indicate that the surface chemical characteristics of the two glass coatings AP40 and RKKP, with no great differences between them, substantially enhance zirconia integration with bone cells at least in vitro. This effect may be of significance in the stability of glass-coated zirconia orthopaedic and dental implants.
Subject(s)
Biocompatible Materials , Glass , Zirconium , Adsorption , Cell Line , Electrophoresis, Polyacrylamide Gel , Humans , In Vitro Techniques , Proteins/chemistryABSTRACT
The presence of apoptosis has been investigated in the interface membranes collected during revision surgery of loosened total hip joint arthroplasty (THAs). Terminal deoxyrobonucleotidyl transferase (TdT) assay for apoptotic DNA fragmentation quantification revealed a statistically significant presence of apoptosis in aseptic samples, obtained from both cementless (2.37+/-0.6%) and cemented (12.01+/-1%) prosthesis compared to septic samples where apoptosis was almost absent. Activated caspase-8 immunostaining was almost undetectable in septic samples, while in the aseptic samples active caspase-8 was present weakly in the cementless samples (1.35+/-0.22%) and strongly in the cemented ones (9.0+/-0.40%). The caspase-8 cytoplasmatic staining allowed the morphological recognition of positive cells both as fibroblast-like and immunocompetent cells. In aseptic cemented samples fibroblast-like cells were the most represented subpopulation in the caspase-8 positive population scored (76.6%) compared to the immunocompetent cells (23.4%). Caspase-8 activation is an upstream event in the apoptotic pathway triggered by the activation of cytokines receptors such as TNF-alpha receptor 1 (TNFR-1), and the presence of caspase-8 activation in fibroblast-like cells in the aseptic interface membranes of THAs suggests a possible TNF-alpha dependent apoptosis.
Subject(s)
Apoptosis , Biocompatible Materials , Caspases/metabolism , Fibroblasts/pathology , Prosthesis Failure , Arthroplasty, Replacement, Hip , Caspase 8 , DNA Fragmentation , Enzyme Activation , Fibroblasts/metabolism , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Time FactorsABSTRACT
The aim of this work was to realise bioactive coatings on full density alpha-alumina substrates. An SiO2-CaO-based glass (SC) and an SiO2-Al2O3-P2O5-K2O-CaO-F--based glass-ceramic (SAF) were used for this purpose. Specifically, SAF is a fluoroapatite containing glass-ceramic and previous studies have shown that it is a highly bioactive biomaterial. Furthermore, these fluoroapatite crystals possess a needle-shaped morphology which mimics that of hydroxylapatite found in human hard tissues, particularly in teeth. SAF is a very viscous glass-ceramic and for this reason an intermediate, less viscous, SC layer was interposed in direct contact with alumina aiming to obtain a good coating adhesion. Moreover, this intermediate layer strongly lowers the Al3+ diffusion and thus minimises both compositional changes in the SAF outer layer and the risk of detrimental modifications of the nature of the crystalline phases. A complete characterisation of the coated samples was performed by means of X-ray diffraction, optical and scanning microscopy. Coating adhesion on alumina was tested by comparative shear tests while biocompatibility was investigated on alumina. bulk SAF and on the realised coatings. For this purpose, cytotoxicity, adhesion and proliferation of human osteoblast-like cells were cultured onto the three materials. Results showed that the interposition of the SC layer was successful in allowing a good softening and spreading of the SAF outer layer and in avoiding the crystallisation of undesired crystalline phases maintaining the good bioactive properties of the bulk one. In vitro results on the coatings showed osteoblast-like cell behaviour similar to bulk fluoroapatite glass-ceramic and better respect to alumina substrates, being a promising index of bone material integration and of its in vivo possible applications.
Subject(s)
Aluminum Oxide , Apatites/chemistry , Bone Substitutes/chemistry , Ceramics , Animals , Apatites/pharmacology , Biocompatible Materials/chemistry , Cell Survival/drug effects , Cells/ultrastructure , Glass , Microscopy, Electron, Scanning , Osteoblasts/cytology , Osteoblasts/drug effectsABSTRACT
Biomaterials may initiate several and complex biological reactions in host tissues, and the cell-biomaterial interactions can determine the release of mediators including monocytes and lymphocytes chemotactic factors. The present work was aimed to investigate in vitro the macrophage biological reactions of a natural apatite obtained by heat treatment at 400 degrees C of bovine bone, compared to other ceramics usually used for dental and orthopedic applications, using synthetic apatite and three types of alumina as controls. Particles chemotactic activity and powders oxidative burst evidenced no monocyte macrophages sensitivity reaction for natural and synthetic hydroxyapatite powders at great granulometry (> 50 microm); data were confirmed by ultrastructural observations; electron microscopy analysis showed macrophages with the features of healthy cells in the presence of both natural and synthetic apatites while macrophages grown in the presence of alumina seemed to be negatively affected. In conclusion, among all ceramics tested, natural apatite displayed a good compatibility with living cells, being better tolerated than synthetic hydroxyapatite which in turn is better tolerated than alumina.
Subject(s)
Biocompatible Materials/pharmacology , Ceramics/pharmacology , Hydroxyapatites/pharmacology , Macrophages, Peritoneal/drug effects , Aluminum Oxide/pharmacology , Animals , Apatites/chemical synthesis , Apatites/pharmacology , Cattle , Ceramics/chemistry , Chemotaxis, Leukocyte/drug effects , Humans , Luminescent Measurements , Macrophages, Peritoneal/physiology , Macrophages, Peritoneal/ultrastructure , Mice , Mice, Inbred BALB C , Monocytes/drug effects , Monocytes/physiology , Monocytes/ultrastructureABSTRACT
Ultra-high molecular weight polyethylene (UHMWPE) sterilization with gamma rays induced high oxidation levels both on the surface and in the bulk that alter its structure and mechanical properties. The oxidation process of gamma-radiated UHMWPE induces a reduction of molecular weight and, consequently, a less abrasive resistance that has been related, among others, to the failure of UHMWPE in vivo. To explain the role of cells in such events, human osteoblast-like cells were seeded onto UHMWPE and oxidized UHMWPE discs. Cellular viability and morphology were evaluated along with matrix metalloproteinases (MMPs) production and activity. Oxidized UHMWPE did not induce any significant cytotoxic effects as observed by lactate dehydrogenase activity compared to the nonoxidized form; no changes in the cell morphology after 4 and 8 days proliferation were observed. In growth medium metalloproteinase 2 (gelatinase-A, MMP-2) was produced and released by osteoblast-like cells. We observed that cells grown onto oxidized UHMWPE discs decreased the release and activity of MMP-2 after 4 and 8 days culture compared to cells grown on control and non-oxidized UHMWPE discs; metalloproteinase 9 (gelatinase-B, MMP-9) release was not significantly influenced. The absence of cytotoxic and morphological effects in the presence of a down-regulation of MMP-2 release and activity suggest that oxidized polyethylene surfaces may modulate matrix remodeling and, consequently, bone formation.
Subject(s)
Extracellular Matrix/metabolism , Matrix Metalloproteinase 2/metabolism , Osteoblasts/metabolism , Polyethylenes/chemistry , Polyethylenes/metabolism , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Cell Size , Cell Survival , Cells, Cultured , Gamma Rays , Humans , Materials Testing , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinases , Osteoblasts/cytology , Oxidation-Reduction , Surface PropertiesABSTRACT
The results of a surface analysis performed on a fluoroapatite-based glass ceramic (SAF) also coating a full-density alpha-alumina substrate (SAF-alumina coating) are presented. These two materials have also been evaluated after soaking in simulated body fluid to understand their ability to induce hydroxyapatite growth on them. Aiming to understand the fluoroapatite glass-ceramic interaction with some plasma proteins, in the second part of this study, fibronectin, albumin, immunoglobulin G, IgA, and complement factor C3c SAF binding have been evaluated; surface activity on complement activation has also been quantified. SAF-alumina coating provides good sites for the nucleation and growth of an apatite layer, equivalent to the mineral component of bone and binds preferentially plasma fibronectin, which is well known to enhance cell adhesion and spreading. Moreover, SAF-alumina coating reduces alumina complement activation directly or via reduced IgA binding. Alumina was shown to bind the same C3 fragments as Zymosan, used as complement activating control, and to induce increased levels of serum soluble iC3b and Bb. A mechanical resistant material with enhanced bioactivity, bone integration, and reduced inflammatory potential respect to alumina has been obtained.
Subject(s)
Aluminum Oxide/chemistry , Ceramics , Glass , Adsorption , Apatites , Complement Activation , Microscopy, Electron, Scanning , Proteins/chemistry , Surface PropertiesABSTRACT
A prospective multicentric study was carried out in patients having metal-on-metal METASUL components (Sulzer Medica, Winterthur, Switzerland) in order to check the following null hypotheses: H1: The concentration of Co, Cr, Ni, and Mb in blood and urine is not modified by the implant of a hip prosthesis with METASUL components at 6 months. H2: The incidence of markers of chromosomal damage [sister chromatid exchanges (SCEs) and micronuclei (Mni)] in lymphocytes is not modified by the implant of METASUL components at 6 months. H3: The concentrations of Co, Cr, Ni, and Mb in blood and urine did not correlate with the incidence of the markers of chromosomal damage. The measurements showed a 2-fold increase of Co in blood, a 10-fold increase of Co in urine, a 1.5-fold increase of Cr in the blood, and a 3-fold increase of Cr in the urine at a follow-up of 6 months from the operation; there was also a significant increase in the Ni blood concentration at the 7 day checkup. The study cohort did not show any modification in the frequency of markers of chromosomal damage in the peripheral lymphocytes at any of the observation times. The amount of the SCEs and Mni recorded at all the observation times did not correlate with each other or with any of the ion levels measured in the blood and in the urine.
Subject(s)
Chromosome Aberrations , Hip Prosthesis/adverse effects , Metals, Heavy/blood , Metals, Heavy/urine , Adult , Arthroplasty, Replacement, Hip/adverse effects , Female , Humans , Ions/blood , Lymphocytes , Male , Micronuclei, Chromosome-Defective , Middle Aged , Mutagenicity Tests , Sister Chromatid ExchangeABSTRACT
The advantages of transmucosal healing implants with a bioactive zirconia collar as a support for partially fixed prosthodontic restorations are optimal peri-implant marginal tissue sealing, reduction in plaque accumulation and satisfactory aesthetic results. The zirconia used in this study evidenced not only optimal clinical performances, but also good biocompatibility. The results from this study demonstrated that zirconia coating enhances fibroblasts and osteoblast-like cell adhesion, spreading and proliferation, favoring microscopic tissue/cell in-growth and clinical implant fixation improvement. From clinical analysis, it emerged that the treatment group obtained better scores in every peri-implant parameter. This evidence attests faster stabilization of soft and hard tissues around both the transmucosal zirconia collar and at the crestal level of the implant. A reduced plaque accumulation around the implant with zirconia collar could provide a better peri-implant microbiological en-vironment by allowing the soft tissues expression of optimal sealing and good bone adaptation to loading. From these clinical and radiographic comparative analyzes, it emerged that in the treatment group the mean values were always similarly low. A rapid stabilization of both hard and soft peri-implant tissues was documented in the 1st yr. In the treatment group, there was the formation of stable tissue sealing the zirconia collar, which could preserve mucosal and bone levels. In conclusion, 2-yr clin-ical results demonstrated that implants supporting fixed restorations using transmucosal healing implants with a zirconia collar appeared a valid method, reporting 100% implant survival rates. Moreover, in vivo results obtained using strict parame-ters to assess the peri-implant status affirmed that a zirconia collar offers excellent biological acceptance. Our preliminary in vitro results statistically evidenced increased fibroblast and osteoblast adhesion and proliferation to zirconia compared to tita-nium, and an index of enhanced material integration with bone and soft tissue cells. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 143-50).
ABSTRACT
Poly-L-lactide acid (PLLA) scaffold has been modified to enhance its osteoconductive and osteoinductive properties in view of a bone tissue engineering application. Two approaches have been followed: (i) coating with laminin or fibronectin and (ii) grafting with arginine-glycine-aspatic acid (RGD) or SIKVAV peptides. Moreover we have added a bioactive molecule 1,25-(OH)2 D3 into the scaffold that shows better cellular interaction to implement osteoinduction and osteogenesis. The two coatings promoted only cell adhesion in the very short term while even if grafted scaffolds had cell seeding efficiency similar to ungrafted PLLA, the grafted ones supported better the proliferation of seeded human osteoblast (hOB) and human mesenchymal stem cells (hMSCs) over 1 week of culture. Our data showed that in view of bone integration and bone regeneration, PLLA grafting with RGD can be considered a good substrate to induce hOB adhesion and proliferation but having no significant effect on the osteogenic induction, the scaffold has to be reinforced with osteoinductive molecules. It can be concluded from reverse transcriptase polymerase chain reaction results, alkaline phosphatase activity and mineralization assays that 1,25(OH)2 D3 reinforced RGD-PLLA keeps increased cell proliferation supported by an upregulation of the studied osteogenic markers and induced hMSCs differentiation into osteoblasts demonstrating osteoinductivity and osteoconductivity of the new formulated scaffold. These results can lead to a future application of RGD-D3-PLLA as an osteogenic material for bone replacement..
Subject(s)
Osseointegration/drug effects , Polyesters/pharmacology , Tissue Scaffolds/chemistry , Animals , Biomarkers/metabolism , Cattle , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Gene Expression Regulation/drug effects , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteogenesis/drug effects , Serum Albumin, Bovine/metabolism , Spectrophotometry, Ultraviolet , Tensile Strength/drug effectsABSTRACT
The interactions between the surface of synthetic scaffolds and cells play an important role in tissue engineering applications. To improve these interactions, two strategies are generally followed: surface coating with large proteins and surface grafting with small peptides. The proteins and peptides more often used and derived from the extracellular matrix, are fibronectin, laminin, and their active peptides, RGD and SIKVAV, respectively. The aim of this work was to compare the effects of coating and grafting of poly(L-lactide) (PLLA) films on MRC5 fibroblast cells. Grafting reactions were verified by X-ray photoelectron spectroscopy. Cell adhesion and proliferation on coated and grafted PLLA surfaces were measured by cell counting. Vinculin localization and distribution were performed on cell cultured on PLLA samples using a fluorescence microscopy technique. Finally, western blot was performed to compare signals of cell adhesion proteins, such as vinculin, Rac1, and RhoA, as well as cell proliferation, such as PCNA. These tests showed similar results for fibronectin and laminin coated PLLA, while RGD grafting is more effective compared with SIKVAV grafting. Considering the overall view of these results, although coating and grafting can both be regarded as effective methods for surface modification to enhance cell adhesion and proliferation on a biomaterial, RGD grafted PLLA show better cell adhesion and proliferation than coated PLLA, while SIKVAV grafted PLLA show similar adhesion but worse proliferation. These data verified different biological effects depending on the surface modification method used.
Subject(s)
Coated Materials, Biocompatible/metabolism , Fibroblasts/cytology , Oligopeptides/metabolism , Polyesters/metabolism , Cell Adhesion , Cell Line , Coated Materials, Biocompatible/chemistry , Fibroblasts/metabolism , Humans , Lung/cytology , Oligopeptides/chemistry , Polyesters/chemistryABSTRACT
The transmission of mechanical forces to cells is followed among all by biological signals related to changes in the assembly or disassembly of integrins associated linker proteins, such as vinculin. We applied for 3 hours 2% cyclic mechanical strain at the frequency of 1 Hz to human fibroblasts cultured on a deformable substrate; substrate deformation resulted to modify the number, length and area of vinculin positive focal adhesion contacts when compared to not stretched cells. The mechanism behind these morphological changes is related to Akt and RhoA roles in focal adhesion assembly. In the case of Akt and Rho inhibition, focal contacts disassembled only in presence of stretching mechanical stress, highlighting the role of mechanical stress on focal adhesion maturation in terms of multimolecolar assembly which from focal complexes leads to fibrillar adhesion.
Subject(s)
Cell Shape , Stress, Mechanical , Cell Adhesion , Cell Membrane/metabolism , Cells, Cultured , Focal Adhesions/metabolism , Humans , Microscopy, Fluorescence , Phosphatidylinositol 3-Kinases/metabolism , Vinculin/biosynthesis , Vinculin/geneticsABSTRACT
The aim of this study was to design a functional bio-engineered material to be used as scaffold for autologous mesenchymal stem cells in ligament tissue engineering. Polyelectrolyte modified HEMA hydrogel (HEMA-co-METAC), applied as coating on silk fibroin fibres, has been formulated in order to take advantage of the biocompatibility of the polyelectrolyte by increasing its mechanical properties with silk fibres. Human bone marrow mesenchymal stem cells behaviour on such reinforced polyelectrolyte has been studied by evaluating cell morphology, cell number, attachment, spreading and proliferation together with collagen matrix production and its mRNA expression. Silk fibroin fibres matrices with HEMA-co-METAC coating exhibited acceptable mechanical behaviour compared to the natural ligament, good human mesenchymal stem cell adhesion and with mRNA expression studies higher levels of collagen types I and III expression when compared to control cells on polystyrene. These data indicate high expression of mRNA for proteins responsible for the functional characteristics of the ligaments and suggest a potential for use of this biomaterial in ligament tissue-engineering applications.