RESUMEN
The modification of titanium (Ti) surfaces by ion-implantation has previously been reported to enhance osseointegration in vivo. However, the mechanisms underlying the apparently improved biocompatibility of these novel implant materials are unknown. The aim of this study is, therefore, to determine the precise effects of calcium ion-implanted Ti on the functional activity of bone cells in vitro. Flow cytometry (FCM) and the reverse transcriptase polymerase chain reaction (RT-PCR) were used to measure the response of bone-derived cells to key bone-associated components, including alkaline phosphatase (ALP), bone morphogenetic protein receptor-1B (BMPR-1B), bone sialoprotein (BSP), osteonectin (ON), and osteopontin (OPN). FCM analysis showed that BMPR-1B, BSP and particularly OPN were significantly up-regulated in MG-63 cells cultured on Ca-implanted Ti compared with control nonimplanted Ti. Moreover, the effects of this novel Ca-Ti surface were found to be mediated, at least partly, via gene activation, since RT-PCR demonstrated the presence of notably elevated levels of OPN mRNA transcripts in the MG-63 cells. These findings thus show that Ti surfaces implanted with Ca ions can enhance the expression of certain bone-associated components in vitro, and suggest that this effect could be the cause of the potential benefit of this material on bone in vivo.
Asunto(s)
Huesos/citología , Huesos/efectos de los fármacos , Calcio/química , Calcio/farmacología , Ensayo de Materiales/métodos , Prótesis e Implantes , Titanio/metabolismo , Adolescente , Antígenos de Neoplasias/metabolismo , Huesos/metabolismo , Línea Celular Tumoral , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Masculino , Osteopontina/genética , Osteopontina/metabolismo , Activación TranscripcionalRESUMEN
Ca ion implantation of Ti surfaces has previously been reported to enhances osseointegration in vivo. Although the mechanisms underlying the response of bone cells to these novel surfaces still remain unclear, it is possible that Ca ion-implanted Ti (Ca-Ti) may influence the growth of new bone by modulating the progression of the cell cycle. In the present study we have, therefore, examined the precise effects of Ca ion-implantation of Ti on the bone-like MG-63 cell line in vitro. The results of flow cytometry analysis showed that this surface markedly enhanced the proportion of cells which expressed Ki-67, a cell proliferation-associated nuclear antigen, compared with cells grown on the non-implanted Ti (control) surface. In addition, cultures grown on Ca-Ti and synchronized at the G1/S boundary by hydroxyurea more rapidly re-entered and progressed through the S and G2/M phases of the cell cycle than their counterparts on Ti. Ca ion-implantation also significantly increased the numbers of mitotic cells. These results thus show that alteration of the surface chemistry of Ti by high-energy implantation with Ca ion was able to substantially modulate the progression of the bone cell cycle, and suggest a possible means of enhancing the response of bone cells to implant materials.
Asunto(s)
Calcio/química , Calcio/farmacología , Ciclo Celular/efectos de los fármacos , Osteosarcoma/patología , Titanio/química , Adolescente , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Demecolcina/farmacología , Humanos , Hidroxiurea/farmacología , Masculino , Ensayo de MaterialesRESUMEN
This study reports the relationship between the biocompatibility and surface properties of experimental bone cements. The effect of hydroxyapatite (HA) or alpha-tri-calcium phosphate (alpha-TCP) incorporated into bone cements prepared with methyl methacrylate as base monomer and either methacrylic acid or diethyl amino ethyl methacrylate (DEAEMA) as comonomers was investigated. The in vitro biocompatibility of these composite cements was assessed in terms of the interaction of primary human osteoblasts grown on the materials over a period of 5 days and compared with a control surface. These results were related to the surface properties investigated through a number of techniques, namely Fourier transform infrared, contact angle measurements, X-ray photoelectron spectroscopy and energy dispersive analysis of X-rays. Complementary techniques of thermal analysis and ion chromatography were also performed. Biocompatibility results showed that the addition of alpha-TCP improves biocompatibility regardless of comonomer type. This is in contrast to HA-based cements where cell proliferation was significantly lower. Surface characterisations showed that structural integrity of the materials was maintained in the presence of the acid and base comonomers, and water contact angles were reduced particularly in DEAEMA containing materials. Furthermore, ion chromatography confirmed higher Ca2+ and PO4(3-) ion release by both types of ceramics, particularly for those containing DEAEMA. In conclusion, the incorporation of acidic and basic comonomers to either HA or alpha-TCP ceramics containing bone cements can have differential effects upon the attachment and proliferation of bone cells in vitro. Moreover, those cements consisting of alpha-TCP and containing DEAEMA comonomer indicated the most favourable biocompatibility.
Asunto(s)
Cementos para Huesos/química , Osteoblastos/citología , Materiales Biocompatibles , Fosfatos de Calcio , División Celular , Cerámica , Durapatita , Humanos , Metacrilatos , Microscopía Electrónica de Rastreo , Osteoblastos/ultraestructura , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Rayos XRESUMEN
The use of calcium ion (Ca) implantation of titanium (Ti), previously reported to encourage osseointegration in vivo, has been investigated using an in vitro model in order to understand the basic mechanisms involved in the response of target cells to such surfaces. Polished Ti discs were implanted with high, medium and low (1x10(17), 1x10(16), 1x10(15)ionscm-2) doses of Ca ions at 40 keV. The effects of different levels of Ca implantation on morphology, attachment and spreading of MG-63 cells seeded on the surface of control (non-implanted) Ti and Ca-Ti discs were assessed. Further, to understand cell-material interactions at a molecular level, the expression of beta1 and alpha5beta1 integrins and the formation of vinculin-positive focal adhesion plaques were examined. In addition, the effects of pre-immersion of the Ca (high)-Ti in tissue culture medium on cell attachment were measured and correlated with specific chemical changes at the Ti surface. Our findings suggest that Ca implantation can affect the adhesion of MG-63 cells both qualitatively and quantitatively. However, this effect appears to depend on the level at which Ca ions are implanted. Results showed that although cell adhesion on Ca (high)-Ti was initially reduced, it nevertheless was not only restored but substantially increased with progressing culture times. In addition, a significantly enhanced cell spreading, formation of focal adhesion plaques and expression of integrins were measured on this particular surface. In contrast, no marked differences were observed in cell behaviour on Ca-Ti (low and medium). Pre-immersion studies indicated that the decrease in cell attachment to Ca (high)-Ti at early time periods may be linked to the presence of Ca- and P-rich particles on the surface. The absence of these particles at 24 h was consistent with a significant increase in cell attachment.
Asunto(s)
Calcio/química , Materiales Biocompatibles Revestidos/química , Oseointegración/fisiología , Osteoblastos/citología , Osteoblastos/fisiología , Titanio/química , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Tamaño de la Célula , Supervivencia Celular , Humanos , Integrinas/metabolismo , Iones , Ensayo de Materiales , Propiedades de SuperficieRESUMEN
This study investigated the surface chemistry of urban fine particles (PM(2.5)), and quantified the adsorbed and desorbed species after exposure to bronchoalveolar lavage fluid (BALF). Urban background and roadside PM(2.5) samples of different mass concentration and total weight were collected in triplicate in the South Bronx region of New York City. Simultaneously, the concentrations of other atmospheric pollutants (CO, NO(x), SO(2), O(3), elemental carbon) were measured, and weather conditions were recorded. The collected PM(2.5) samples underwent one of three treatments: no treatment, treatment in vitro with BALF, or treatment in a saline solution (control). The surfaces of untreated, saline-treated, and BALF-treated PM(2.5) samples were analyzed using x-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). These results were then compared with ambient air pollutant concentrations, weather variables, selected BALF characteristics, and results from a previous London study conducted using identical preparation methods by XPS analysis only. Both XPS and ToF-SIMS detected PM(2.5) surface species and observed changes in surface concentrations after treatment. XPS analysis showed the surface of untreated urban PM(2.5) consisted of 79 to 87% carbon and 10 to 16% oxygen with smaller contributions of N, S, Si, and P in the samples from both background and roadside locations. A wider variety of other inorganic and organic species (including metals, aliphatic and aromatic hydrocarbons, and nitrogen-containing molecules) was detected with ToF-SIMS. Surface characteristics of particles from the roadside and background sites were very similar, except for higher (p <.05) nitrate concentrations at the roadside, which were attributable to higher roadside NO(x) concentrations. Comparable species and quantities were identified in a previous study of London PM(2.5), where PM(2.5) surface chemistry differed considerably depending on the source, particularly in surface concentrations of oxygen and trace species. After treatment with BALF the N-C signal detected by XPS analysis increased in the average by 372 +/- 203%, indicating significant surface adsorption of protein or other N-containing biomolecules. Lower (nonsignificant) N-C signals were observed for smoker BALF, compared to nonsmoker BALF. ToF-SIMS data confirmed protein adsorption after BALF treatment--smoker BALF resulted in lower levels of adsorbed proteins compared to nonsmoker BALF. ToF-SIMS also indicated an adsorption of phospholipid on the treated PM(2.5) surfaces. The primary phospholipid in BALF is dipalmitoylphospatidylcholine (DPPC), although positive identification was not possible due to low concentrations at the PM(2.5) surface. Oxygen content of PM(2.5) surfaces was the most significant determinant of both N-C and phospholipid adsorption. The XPS signal of the soluble species NH(+)(4), NO(2-)(3), Si, and S decreased in both saline- and BALF-treated samples, showing that these species may be bioavailable in the lung. Similarly, ToF-SIMS analysis suggests the bioavailability of Na(+) and Al(+) as well as NH(+)(4) and Si(+).