RESUMEN
The topographical and chemical surface features of biomaterials are sensed by the cells, affecting their physiology at the interface. When placed on titanium, we recently discovered osteoblasts attempted caveolae-mediated phagocytosis of the sharp-edged microstructures. This active, energy-consuming process resulted in decreased osteoblastic cell functions (e.g. secretion of extracellular matrix proteins). However, chemical modification with plasma polymerized allylamine (PPAAm) was able to amplify osteoblast adhesion and spreading, resulting in better implant osseointegration in vivo In the present in vitro study, we analyzed whether this plasma polymer nanocoating is able to attenuate the microtopography-induced changes of osteoblast physiology. On PPAAm, we found cells showed a higher cell interaction with the geometrical micropillars by 30â min, and a less distinct reduction in the mRNA expression of collagen type I, osteocalcin and fibronectin after 24â h of cell growth. Interestingly, the cells were more active and sensitive on PPAAm-coated micropillars, and react with a substantial Ca2+ ion mobilization after stimulation with ATP. These results highlight that it is important for osteoblasts to establish cell surface contact for them to perform their functions.
Asunto(s)
Adhesión Celular , Materiales Biocompatibles Revestidos/química , Proteínas de la Matriz Extracelular/metabolismo , Expresión Génica , Osteoblastos/citología , Alilamina/química , Línea Celular , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Humanos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Polimerizacion , ARN Mensajero/análisis , Propiedades de Superficie , Titanio/químicaRESUMEN
Implantation of biomaterials can cause complications often associated with inflammatory reactions. However, repeated evaluation of the implant site would be burdening for patients. Alternatively, blood examinations with analysis of inflammatory serum markers could potentially be useful to reflect the local cellular response for detection and/or prediction of inflammation-related complications. Therefore, following intramuscular implantation of surface-modified Ti implants in rats, this study aimed at examining possible associations between the post-implantation time course of pro-inflammatory (INFγ, IL-2) and anti-inflammatory (IL-4, IL-10) cytokine serum concentrations and the local peri-implant tissue response after 56 days (pro-inflammatory CD68-positive monocytes/macrophages, anti-inflammatory CD163-positive macrophages, MHC class II-positive cells, activated natural killer cells and mast cells). Multivariate correlation analysis revealed a significant interaction between serum IFNγ and peri-implant tissue CD68-positive monocytes/macrophages (p = 0.001) while no interactions were found for other cytokines and cell types. Additional Pearson correlation analysis of IFNγ serum concentrations on each experimental day vs. the CD68-positive monocytes/macrophages response on day 56 demonstrated a consistently positive correlation that was strongest during the first three weeks. Thus, high early pro-inflammatory IFNγ serum concentration was associated with high late number of pro-inflammatory CD68-positive monocyte/macrophages and low early serum IFNγ with low late CD68-positive monocyte/macrophage numbers. Further studies aimed at examination of patient samples could establish the relevance of this association to predict clinical complications. After implantation of titanium samples, high early IFNγ serum concentrations were associated with a pronounced late pro-inflammatory CD68-positive monocyte/ macrophage (red circle) response, while no correlation was found for other investigated cytokines and inflammatory cells (green circle). In contrast, low early IFNγ serum concentrations were correlated with low late monocyte/ macrophage numbers.
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Implantes de Medicamentos , Interferón gamma/administración & dosificación , Macrófagos/inmunología , Animales , RatasRESUMEN
The long-term stability and γ-sterilisability of bioactive layers is the precondition for the application of implants. Thus, aging processes of a microwave deposited, plasma polymerized allylamine nanofilm (PPAAm) with positively charged amino groups were evaluated concerning physicochemical characteristics and cell adhesion capacity over the course of one year. XPS, FT-IR, surface free energy, and water contact angle measurements elucidated not only the oxidation of the PPAAm film due to atmospheric oxygen reacting with surface free radicals but also the influence of atmospheric moisture during sample storage in ambient air. Surprisingly, within 7 days 70% of the primary amino groups are lost and mostly converted into amides. A positive zeta-potential was verified for half a year and longer. Increasing polar surface groups and a water contact angle shift from 60° to 40° are further indications of altered surface properties. Nevertheless, MG-63 human osteoblastic cells adhered and spread out considerably on aged and additionally γ-sterilized PPAAm layers deposited on polished titanium alloys (Ti-6Al-4V_P). These cell-relevant characteristics were highly significant over the whole period of one year and may not be related to the existence of primary amino groups. Rather, the oxidation products, the chemical amide group, that is, seem to support the attachment of osteoblasts at all times up to one year.
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Alilamina , Membranas Artificiales , Nanoestructuras/química , Osteoblastos/metabolismo , Gases em Plasma , Titanio , Aleaciones , Alilamina/química , Alilamina/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Humanos , Ensayo de Materiales , Osteoblastos/citología , Oxidación-Reducción , Factores de Tiempo , Titanio/química , Titanio/farmacologíaRESUMEN
By means of plasma polymerization, positively charged, nanometre-thin coatings can be applied to implant surfaces. The aim of the present study was to quantify the adhesion of human bone cells in vitro and to evaluate the bone ongrowth in vivo, on titanium surfaces modified by plasma polymer coatings. Different implant surface configurations were examined: titanium alloy (Ti6Al4V) coated with plasma-polymerized allylamine (PPAAm) and plasma-polymerized ethylenediamine (PPEDA) versus uncoated. Shear stress on human osteoblast-like MG-63 cells was investigated in vitro using a spinning disc device. Furthermore, bone-to-implant contact (BIC) was evaluated in vivo. Custom-made conical titanium implants were inserted at the medial tibia of female Sprague-Dawley rats. After a follow-up of six weeks, the BIC was determined by means of histomorphometry. The quantification of cell adhesion showed a significantly higher shear stress for MG-63 cells on PPAAm and PPEDA compared to uncoated Ti6Al4V. Uncoated titanium alloyed implants showed the lowest BIC (40.4%). Implants with PPAAm coating revealed a clear but not significant increase of the BIC (58.5%) and implants with PPEDA a significantly increased BIC (63.7%). In conclusion, plasma polymer coatings demonstrate enhanced cell adhesion and bone ongrowth compared to uncoated titanium surfaces.
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Oseointegración , Polimerizacion , Prótesis e Implantes , Titanio , Aleaciones , Animales , Adhesión Celular , Línea Celular , Materiales Biocompatibles Revestidos , Femenino , Humanos , Osteoblastos/metabolismo , RatasRESUMEN
Copper (Cu) could serve as antibacterial coating for Ti6Al4V implants. An additional cell-adhesive layer might compensate Cu cytotoxicity. This study aimed at in vitro and in vivo evaluation of low-temperature plasma treatment of Ti6Al4V plates with Ti/Cu magnetron sputtering (Ti6Al4V-Ti/Cu), plasma-polymerized ethylenediamine (Ti6Al4V-PPEDA), or both (Ti6Al4V-Ti/Cu-PPEDA). Ti6Al4V-Ti/Cu and Ti6Al4V-Ti/Cu-PPEDA had comparable in vitro Cu release and antibacterial effectiveness. Following intramuscular implantation of Ti6Al4V-Ti/Cu, Ti6Al4V-PPEDA, Ti6Al4V-Ti/Cu-PPEDA and Ti6Al4V controls for 7, 14 and 56 days with 8 rats/day, peri-implant tissue was immunohistochemically examined for different inflammatory cells. Ti6Al4V-PPEDA had more mast cells and NK cells than Ti6Al4V, and more tissue macrophages, T lymphocytes, mast cells and NK cells than Ti6Al4V-Ti/Cu-PPEDA. Ti6Al4V-Ti/Cu had more mast cells than Ti6Al4V and Ti6Al4V-Ti/Cu-PPEDA. Results indicate that PPEDA-mediated cell adhesion counteracted Cu cytotoxicity. Ti6Al4V-Ti/Cu-PPEDA differed from Ti6Al4V only for mast cells on day 56. Altogether, implants with both plasma treatments had antibacterial properties and did not increase inflammatory reactions.
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Cobre/química , Etilenodiaminas/química , Inflamación/etiología , Gases em Plasma , Titanio/efectos adversos , Aleaciones , Animales , Biopelículas , Inmunohistoquímica , Inflamación/inmunología , Linfocitos/inmunología , Masculino , Pruebas de Sensibilidad Microbiana , Ratas , Ratas Endogámicas Lew , Staphylococcus aureus , TemperaturaRESUMEN
Surface modification of Titanium (Ti) by low-temperature plasma influences cell-material interactions. Therefore, this study aimed at examining serum cytokine levels and associations after intramuscular implantation (n = 8 rats/group) of Ti-plates with Plasma Polymerized Allyl Amine (Ti-PPAAm), Plasma Polymerized Acrylic Acid (Ti-PPAAc), and without such layers (Ti-Controls). Pro-inflammatory (IL-2, IFNγ, IL-6) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines were measured weekly for 56 days. Ti-PPAAm caused increased IL-2 (d7-14, d35), increased IFNγ (d35) and decreased IL-10 (d35, d49-56). Ti-PPAAc induced divergent anti-inflammatory cytokine changes with increased IL-4 (d28-56) and decreased IL-10 (d42-56). Ti-Controls elicited increased IL-2 (d42) and IFNγ (d35-42, d56). IL-6 was not detected and IL-13 only in three samples, thus they do not influence the response against these Ti implants. Correlation analysis revealed surface-dependent associations between cytokines indicating the involvement of different inflammatory cell populations. Concluding, different plasma modifications induce specific serum cytokine profiles and associations indicating distinct inflammatory responses.
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Antiinflamatorios/sangre , Citocinas/sangre , Mediadores de Inflamación/sangre , Implantación de Prótesis , Titanio/farmacología , Animales , Antiinflamatorios/metabolismo , Materiales Biocompatibles Revestidos/farmacología , Luces de Curación Dental , Citocinas/metabolismo , Equipos y Suministros/efectos adversos , Mediadores de Inflamación/metabolismo , Masculino , Metaboloma/efectos de los fármacos , Gases em Plasma/química , Gases em Plasma/farmacología , Implantación de Prótesis/efectos adversos , Implantación de Prótesis/rehabilitación , Ratas , Ratas Endogámicas Lew , Propiedades de Superficie , Titanio/químicaRESUMEN
New developments of antimicrobial implant surfaces doped with copper (Cu) ions may minimize the risk of implant-associated infections. However, experimental evaluation of the Cu release is influenced by various test parameters. The aim of our study was to evaluate the Cu release characteristics in vitro according to the storage fluid and surface roughness. Plasma immersion ion implantation of Cu (Cu-PIII) and pulsed magnetron sputtering process of a titanium copper film (Ti-Cu) were applied to titanium alloy (Ti6Al4V) samples with different surface finishing of the implant material (polished, hydroxyapatite and corundum blasted). The samples were submersed into either double-distilled water, human serum, or cell culture medium. Subsequently, the Cu concentration in the supernatant was measured using atomic absorption spectrometry. The test fluid as well as the surface roughness can alter the Cu release significantly, whereby the highest Cu release was determined for samples with corundum-blasted surfaces stored in cell medium.
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Implantation of biomaterials like titanium (Ti) causes inflammatory reactions possibly affecting implant functionality. Surface modifications could improve biocompatibility and functionality of implants. Biomembrane-derived phospholipids might be useful as implant coating due to their biomimetic properties. In vitro studies demonstrated beneficial effects for 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamin (POPE) as coating regarding interactions with cells and bacteria. Therefore, this in vivo study aimed at examining local inflammatory reactions after implantation of POPE-coated Ti plates. Ti implants with POPE attached non-covalently or covalent via octadecylphosphonic acid (OPA), with OPA alone and uncoated controls were simultaneously implanted intramuscularly in rats for 7, 14 and 56 days. The peri-implant tissue was quantitatively analyzed by immunohistochemistry for total macrophages, tissue macrophages, T cells, antigen-presenting cells and proliferating cells. Overall, both POPE-coated series were comparable to the controls. Furthermore, no differences were found between POPE coating on a covalently linked OPA monolayer and POPE coating dried from solution. Together with earlier in vitro results, this demonstrates the potential of phospholipids for implant surface modification.
Asunto(s)
Fosfolípidos/química , Titanio/química , Animales , Materiales Biocompatibles/química , Biomimética , Proliferación Celular , Humanos , Inmunohistoquímica/métodos , Inflamación , Macrófagos/citología , Ensayo de Materiales , Organofosfonatos/química , Fosfatidiletanolaminas/química , Ratas , Medicina Regenerativa/métodos , Factores de TiempoRESUMEN
Orthopaedic implants and temporary osteosynthesis devices are commonly based on Titanium (Ti). For short-term devices, cell-material contact should be restricted for easy removal after bone healing. This could be achieved with anti-adhesive plasma-fluorocarbon-polymer (PFP) films created by low-temperature plasma processes. Two different PFP thin film deposition techniques, microwave (MW) and radiofrequency (RF) discharge plasma, were applied to receive smooth, hydrophobic surfaces with octafluoropropane (C3F8) or hexafluorohexane (C6F6) as precursors. This study aimed at examining the immunological local tissue reactions after simultaneous intramuscular implantation of four different Ti samples, designated as MW-C3F8, MW-C6F6, RF-C3F8 and Ti-controls, in rats. A differentiated morphometric evaluation of the inflammatory reaction was conducted by immunohistochemical staining of CD68+ macrophages, CD163+ macrophages, MHC class II-positive cells, T lymphocytes, CD25+ regulatory T lymphocytes, NK cells and nestin-positive cells in cryosections of surrounding peri-implant tissue. Tissue samples were obtained on days 7, 14 and 56 for investigating the acute and chronical inflammation (n = 8 rats/group). Implants with a radiofrequency discharge plasma (RF-C3F8) coating exhibited a favorable short- and long-term immune/inflammatory response comparable to Ti-controls. This was also demonstrated by the significant decrease in pro-inflammatory CD68+ macrophages, possibly downregulated by significantly increasing regulatory T lymphocytes.
RESUMEN
Antimicrobial coating of implant material with poly(hexamethylene biguanide) hydrochloride (PHMB) may be an eligible method for preventing implant-associated infections. In the present study, an antibacterial effective amount of PHMB is adsorbed on the surface of titanium alloy after simple chemical pretreatment. Either oxidation with 5% H2 O2 for 24 hr or processing for 2 hr in 5 M NaOH provides the base for the subsequent formation of a relatively stable self-assembled PHMB layer. Compared with an untreated control group, adsorbed PHMB produces no adverse effects on SaOs-2 cells within 48 hr cell culture, but promotes the initial attachment and spreading of the osteoblasts within 15 min. Specimens were inoculated with slime-producing bacteria to simulate a perioperative infection. Adsorbed PHMB reacts bactericidally against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa after surface contact. Adhered SaOs-2 cells differentiate and produce alkaline phosphatase and deposit calcium within 4 days in a mineralization medium on PHMB-coated Ti6Al4V surfaces, which have been precontaminated with S. epidermidis. The presented procedures provide a simple method for generating biocompatibly and antimicrobially effective implant surfaces that may be clinically important.
Asunto(s)
Aleaciones/química , Antibacterianos/química , Biguanidas/química , Materiales Biocompatibles Revestidos/química , Titanio/química , Fosfatasa Alcalina/metabolismo , Antibacterianos/farmacología , Biguanidas/farmacología , Adhesión Celular , Diferenciación Celular , Línea Celular , Proliferación Celular , Materiales Biocompatibles Revestidos/metabolismo , Humanos , Osteoblastos/citología , Prótesis e Implantes , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus/efectos de los fármacos , Propiedades de SuperficieRESUMEN
Biomaterials should be bioactive in stimulating the surrounding tissue to accelerate the ingrowth of permanent implants. Chemical and topographical features of the biomaterial surface affect cell physiology at the interface. A frequently asked question is whether the chemistry or the topography dominates the cell-material interaction. Recently, we demonstrated that a plasma-chemical modification using allylamine as a precursor was able to boost not only cell attachment and cell migration, but also intracellular signaling in vital cells. This microwave plasma process generated a homogenous nanolayer with randomly distributed, positively charged amino groups. In contrast, the surface of the human osteoblast is negatively charged at -15 mV due to its hyaluronan coat. As a consequence, we assumed that positive charges at the material surface-provoking electrostatic interaction forces-are attractive for the first cell encounter. This plasma-chemical nanocoating can be used for several biomaterials in orthopedic and dental implantology like titanium, titanium alloys, calcium phosphate scaffolds, and polylactide fiber meshes produced by electrospinning. In this regard, we wanted to ascertain whether plasma polymerized allylamine (PPAAm) is also suitable for increasing the attractiveness of a ceramic surface for dental implants using Yttria-stabilized tetragonal zirconia.
RESUMEN
BACKGROUND: Biomaterial modifications-chemical and topographical-are of particular importance for the integration of materials in biosystems. Cells are known to sense these biomaterial characteristics, but it has remained unclear which physiological processes bio modifications trigger. Hence, the question arises of whether the dynamic of intracellular calcium ions is important for the characterization of the cell-material interaction. In our prior research we could demonstrate that a defined geometrical surface topography affects the cell physiology; this was finally detectable in a reduced intracellular calcium mobilization after the addition of adenosine triphosphate (ATP). RESULTS: This new contribution examines the cell physiology of human osteoblasts concerning the relative cell viability and the calcium ion dynamic on different chemical modifications of silicon-titanium (Ti) substrates. Chemical modifications comprising the coating of Ti surfaces with a plasma polymerized allylamine (PPAAm)-layer or with a thin layer of collagen type-I were compared with a bare Ti substrate as well as tissue culture plastic. For this purpose, the human osteoblasts (MG-63 and primary osteoblasts) were seeded onto the surfaces for 24 h. The relative cell viability was determined by colorimetric measurements of the cell metabolism and relativized to the density of cells quantified using crystal violet staining. The calcium ion dynamic of osteoblasts was evaluated by the calcium imaging analysis of fluo-3 stained vital cells using a confocal laser scanning microscope. The positively charged nano PPAAm-layer resulted in enhanced intracellular calcium ion mobilization after ATP-stimulus and cell viability. This study underlines the importance of the calcium signaling for the manifestation of the cell physiology. CONCLUSIONS: Our current work provides new insights into the intracellular calcium dynamic caused by diverse chemical surface compositions. The calcium ion dynamic appears to be a sensitive parameter for the cell physiology and, thus, may represent a useful approach for evaluating a new biomaterial. In this regard, reliable in vitro-tests of cell behavior at the interface to a material are crucial steps in securing the success of a new biomaterial in medicine.
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Biomaterials can cause a chronic local inflammation called foreign body reaction, with formation of foreign body giant cells (FBGC) by monocyte/macrophage fusion. However, FBGC appearance and role for biomaterials with different physicochemical properties are not yet fully understood. This study aimed at examining FBGC and inflammatory cells after intramuscular implantation of poly(l-lactide-co-d/l-lactide) (PLA) as membranes and uncoated electro-spun fiber meshes or meshes with a positively charged plasma-polymer coating into rats. After 7, 14 and 56 days, CD68+ and CD163+ macrophages, T lymphocytes, MHC-II+ cells, FBGC, and nestin-stained tissue area as regeneration marker were morphometrically analyzed. FBGC occurrence was primarily determined by material morphology, as their numbers for meshes were 10-fold higher during acute and 50-fold higher during chronic inflammation than for membranes but comparable between uncoated and coated meshes. CD68+ macrophages decreased around and within meshes, while CD163+ macrophages and MHC-II+ cells increased within meshes. T lymphocytes within meshes were higher for coated meshes, suggesting that the peri-implant tissue immunological response is also influenced by surface chemistry. FBGC were predominantly CD68+ and CD163- , and nestin-stained tissue area was negatively correlated with CD68+ monocytes/macrophages numbers and positively correlated with CD163+ macrophages numbers, highlighting differing roles in FBGC formation and tissue regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2726-2734, 2018.
Asunto(s)
Células Gigantes de Cuerpo Extraño/patología , Inflamación/etiología , Poliésteres/efectos adversos , Poliésteres/química , Prótesis e Implantes/efectos adversos , Animales , Células Presentadoras de Antígenos/metabolismo , Antígenos CD/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Masculino , Ratas Endogámicas Lew , Propiedades de Superficie , Linfocitos T/metabolismoRESUMEN
The crucial factor of metal implant ingrowth in the bone is the rapid cellular acceptance. Therefore, the knowledge about additionally used adhesion mechanisms of osteoblasts, like their negatively charged hyaluronan coat, generates new surface functionalization strategies. Here, titanium was coated with a very thin, adherent, cross-linked, pinhole- and additive-free allylamine plasma polymer layer (PPAAm) resistant to hydrolysis and delamination and equipped with a high density of positively charged amino groups. This plasma polymer-functionalization of titanium is advantageous concerning osteoblastic focal adhesion formation as vinculin and paxillin, actin cytoskeleton development and, in consequence in differentiated cell functions, compared to a pure titanium surface-but similar such as the collagen I bonded surface via a polyethylenglycol-diacid (PEG DA)-spacer.
Asunto(s)
Adhesión Celular , Materiales Biocompatibles Revestidos/química , Osteoblastos/fisiología , Plasma/metabolismo , Titanio/química , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Materiales Biocompatibles Revestidos/metabolismo , Colágeno/química , Colágeno/metabolismo , Medio de Cultivo Libre de Suero , Proteínas Fluorescentes Verdes/análisis , Humanos , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/farmacología , Osteoblastos/química , Osteoblastos/ultraestructura , Ratas , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Factores de Tiempo , Vinculina/análisisRESUMEN
Adhesion and spreading of cells on biomaterials are integrin-mediated processes. But recent findings indicate a key role of the cell membrane associated matrix substance hyaluronan (HA) in interface interactions. Because HA is a negatively charged molecule we assume that a biomaterial surface with an opposed charge could boost the first contact of the cell to the surface. Polished cp titanium (R(a)=0.19 microm) was coated with an amino-group containing plasma polymer (Ti PPA). For this purpose, a microwave excited, pulsed, low-pressure plasma was used. Additionally, collagen was immobilized on Ti PPA with polyethylene glycol diacid (PEG-DA), catalyzed by carbodiimide (CDI). The physico-chemical surface analytical techniques like XPS, FT-IR, water contact angle and zeta-potential verified the retention of the allylamine precursor structure. Human osteoblasts were cultured in serum-free Dulbecco's modified Eagle medium (DMEM). Adhesion and cell cycle phases were calculated by flow cytometry. Spreading and actin cytoskeleton were visualized by confocal microscopy. Gene expression of osteogenic markers was detected by real-time RT-PCR. Ti PPA is significantly advantageous concerning initial adhesion and spreading during the first hours of the cell contact to the surface. The proliferation of osteoblasts is positively influenced. Gene expression of the differentiation marker bone sialoprotein was upregulated after 24h. Our results demonstrate that functionalization of titanium with positively charged amino-groups is sufficiently enough to significantly improve initial steps of the cellular contact to the material surface.
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Alilamina/química , Osteoblastos/fisiología , Polímeros/química , Titanio/química , Actinas/química , Fosfatasa Alcalina/genética , Carbodiimidas/química , Catálisis , Adhesión Celular/fisiología , Ciclo Celular , Colágeno Tipo I/química , Colágeno Tipo I/genética , Colágeno Tipo I/efectos de la radiación , Citoesqueleto/química , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Microondas , Polietilenglicoles/química , Polietilenglicoles/efectos de la radiación , Polímeros/efectos de la radiación , Procolágeno/química , Procolágeno/genética , Procolágeno/efectos de la radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Propiedades de Superficie , Titanio/efectos de la radiación , Células Tumorales CultivadasRESUMEN
Topographical and chemical features of biomaterial surfaces affect the cell physiology at the interface and are promising tools for the improvement of implants. The dominance of the surface topography on cell behavior is often accentuated. Striated surfaces induce an alignment of cells and their intracellular adhesion-mediated components. Recently, it could be demonstrated that a chemical modification via plasma polymerized allylamine was not only able to boost osteoblast cell adhesion and spreading but also override the cell alignment on stochastically machined titanium. In order to discern what kind of chemical surface modifications let the cell forget the underlying surface structure, we used an approach on geometric microgrooves produced by deep reactive ion etching (DRIE). In this study, we systematically investigated the surface modification by (i) methyl-, carboxyl-, and amino functionalization created via plasma polymerization processes, (ii) coating with the extracellular matrix protein collagen-I or immobilization of the integrin adhesion peptide sequence Arg-Gly-Asp (RGD), and (iii) treatment with an atmospheric pressure plasma jet operating with argon/oxygen gas (Ar/O2). Interestingly, only the amino functionalization, which presented positive charges at the surface, was able to chemically disguise the microgrooves and therefore to interrupt the microtopography induced contact guidance of the osteoblastic cells MG-63. However, the RGD peptide coating revealed enhanced cell spreading as well, with fine, actin-containing protrusions. The Ar/O2-functionalization demonstrated the best topography handling, e.g. cells closely attached even to features such as the sidewalls of the groove steps. In the end, the amino functionalization is unique in abrogating the cell contact guidance.
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Adhesión Celular , Materiales Biocompatibles , Osteoblastos , Propiedades de Superficie , TitanioRESUMEN
Copper (Cu) could be suitable to create anti-infective implants based on Titanium (Ti), for example by incorporating Cu into the implant surface using plasma immersion ion implantation (Cu-PIII). The cytotoxicity of Cu might be circumvented by an additional cell-adhesive plasma polymerized allylamine film (PPAAm). Thus, this study aimed to examine in vivo local inflammatory reactions for Ti6Al4V implants treated with Cu-PIII (Ti-Cu), alone or with an additional PPAAm film (Ti-Cu-PPAAm), compared to untreated implants (Ti). Successful Cu-PIII and PPAAm treatment was confirmed with X-ray Photoelectron Spectroscopy. Storage of Ti-Cu and Ti-Cu-PPAAm samples in double-distilled water for five days revealed a reduction of Cu release by PPAAm. Subsequently, Ti, Ti-Cu and Ti-Cu-PPAAm samples were simultaneously implanted into the neck musculature of 24 rats. After 7, 14 and 56 days, peri-implant tissue was retrieved from 8 rats/day for morphometric immunohistochemistry of different inflammatory cells. On day 56, Ti-Cu induced significantly stronger reactions compared to Ti (tissue macrophages, antigen-presenting cells, T lymphocytes) and to Ti-Cu-PPAAm (tissue macrophages, T lymphocytes, mast cells). The response for Ti-Cu-PPAAm was comparable with Ti. In conclusion, PPAAm reduced the inflammatory reactions caused by Cu-PIII. Combining both plasma processes could be useful to create antibacterial and tissue compatible Ti-based implants.
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Excellent osseointegration of permanent implants is crucial for the long lasting success of the implantation. To improve the osseointegrative potential, bio-inert titanium alloy surfaces (Ti6Al4V) are modified by plasma chemical oxidation (PCO®) of the titanium-oxide layer to a non-stoichiometric, amorphous calcium phosphate layer. The native titanium-oxide film measuring only a few nanometers is converted by PCO® to a thick porous calcium phosphate layer of about 10µm. In a second step the PCO surface is combined with a cell adhesive plasma-polymerized allylamine (PPAAm) nano film (5 and 50nm). Independent of the PPAAm coating homogeneity, the human osteoblast-like MG-63 cells show a remarkable increase in cell size and well-developed filopodia. Analyses of the actin cytoskeleton reveal that the cells mold to the pore shape of the PPAAm-covered PCO, thereby establishing a strong attachment to the surface. Interestingly, we could demonstrate that even though our untreated PCO shows excellent hydrophilicity, this alone is not sufficient to facilitate fast cell spreading, but the positive surface charges mediated by PPAAm. This multilayer composite material guarantees enhanced interlocking of the cells with the porous surface.
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Cerámica/química , Materiales Biocompatibles Revestidos/química , Polímeros/química , Citoesqueleto de Actina/efectos de los fármacos , Aleaciones , Alilamina/química , Fosfatos de Calcio/química , Adhesión Celular/efectos de los fármacos , Línea Celular , Materiales Biocompatibles Revestidos/farmacología , Humanos , Microscopía Electrónica de Rastreo , Oxidación-Reducción , Espectroscopía de Fotoelectrones , Porosidad , Propiedades de Superficie , Titanio/químicaRESUMEN
A common method to derive both qualitative and quantitative data to evaluate osseointegration of implants is histomorphometry. The present study describes a new image reconstruction algorithm comparing the results of bone-to-implant contact (BIC) evaluated by means of µCT with histomorphometry data. Custom-made conical titanium alloyed (Ti6Al4V) implants were inserted in the distal tibial bone of female Sprague-Dawley rats. Different surface configurations were examined: Ti6Al4V implants with plasma-polymerized allylamine (PPAAm) coating and plasma-polymerized ethylenediamine (PPEDA) coating as well as implants without surface coating. After six weeks postoperatively, tibiae were explanted and BIC was determined by µCT (3D) and afterwards by histomorphometry (2D). In comparison to uncoated Ti6Al4V implants demonstrating low BIC of 32.4% (histomorphometry) and 51.3% (µCT), PPAAm and PPEDA coated implants showed a nonsignificant increase in BIC (histomorphometry: 45.7% and 53.5% and µCT: 51.8% and 62.0%, resp.). Mean BIC calculated by µCT was higher for all surface configurations compared to BIC detected by histomorphometry. Overall, a high correlation coefficient of 0.70 (p < 0.002) was found between 3D and 2D quantification of BIC. The µCT analysis seems to be suitable as a nondestructive and accurate 3D imaging method for the evaluation of the bone-implant interface.
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Materiales Biocompatibles Revestidos , Oseointegración , Prótesis e Implantes , Titanio , Algoritmos , Aleaciones , Animales , Etilenodiaminas , Femenino , Imagenología Tridimensional , Ensayo de Materiales , Poliaminas , Ratas , Ratas Sprague-Dawley , Tibia/diagnóstico por imagen , Tibia/patología , Tibia/cirugía , Microtomografía por Rayos XRESUMEN
An effective amount of the antiseptic agent PHMB cannot simply be placed on the surface of titanium alloys where hydrocarbons were removed by different purification procedures. Pre-treatment of Ti6Al4V specimen with 5% H2O2 in 24 h results in extra introduced -OH and -COOH groups as well as an adsorbed water film on the surface, which provide the base for the subsequent formation of a relatively stable and multi-layered PHMB film. The superficially adhering PHMB film produces no adverse effects on MG63 cells within a 48 h-cell culture, but promotes the initial attachment and spreading of the osteoblasts on the modified Ti6Al4V surface within 15 min. After direct bacterial inoculation of the active sample, the PHMB film reacts antimicrobially against Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative strains (Pseudomonas aeruginosa, Escherichia coli) after surface contact. The bactericidal efficacy is only slightly reduced after using of the same specimen for re-testing the antibacterial activity. MG63 cells adhere and proliferate within 48 h on a PHMB film-containing Ti6Al4V surface, which has been pre-contaminated with S. aureus. Bacterial biofilms were only revealed in controls without PHMB.