Surface chemistry of grafted expanded poly(tetrafluoroethylene) membranes modifies the in vitro proinflammatory response in macrophages.

A series of surface-modified expanded poly(tetrafluoroethylene) membranes showed varied levels of in vitro macrophage proinflammatory response. Membranes containing a mixture of phosphate and hydroxyl groups (as determined by X-ray photoelectron spectroscopy analysis) stimulate greater macrophage activation than samples containing a mixture of phosphate and carboxylic acid segments. The types of proteins that adsorbed irreversibly from serum onto the two samples with the highest and lowest cellular response were investigated using surface-matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Distinct differences in the number and type of proteins that adsorbed were observed between these samples. A correlation was found between the main protein components adsorbed onto the surfaces and the resulting in vitro proinflammatory response. This study strongly supports the hypothesis that the cellular response is not controlled directly by surface properties but is mediated by specific protein adsorption events. This in turn highlights the importance of better understanding and controlling the properties of intelligent surface-modified biomaterials.

Comprehensive characterization of grafted expanded poly(tetrafluoroethylene) for medical applications.

Successful implantation of any biomaterial depends on its mechanical, architectural, and surface properties. Materials with good bulk properties seldom possess the appropriate surface characteristics required for good biointegration. The present study investigates the results of surface modification of a highly porous, fully fluorinated polymeric substrate, expanded poly(tetrafluoroethylene) (ePTFE), with a view to improving the surface bioactivity and hence ultimately its biointegration. Modification involved gamma irradiation-induced graft copolymerization with the monomers monoacryloxyethyl phosphate (MAEP) and methacryloxyethyl phosphate (MOEP) in various solvent systems (water, methanol, methyl ethyl ketone, and mixtures thereof). In order to determine the penetration depth of the graft copolymer into the pores and/or the bulk of the ePTFE membranes, angle-dependent X-ray photoelectron spectroscopy (XPS) and magnetic resonance imaging (MRI) were used. It was found that the penetration depth was critically affected by the choice of monomer and solvent as well as by the technique used to remove dissolved oxygen from the grafting mixture: nitrogen degassing versus vacuum. Difficulties due to the porous nature of the membranes in establishing the lateral position of the graft copolymers were largely overcome by combining data from microattenuated total reflectance Fourier transfer infrared (µ-ATR-FTIR) mapping and time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. Results show that the large variation in graft heterogeneity found between different samples is largely an effect of the underlying substrate and choice of monomer. The results from this study provide the necessary knowledge and experimental data to control both the graft copolymer lateral position and depth of penetration in these porous ePTFE membranes.

Novel phosphate-grafted ePTFE copolymers for optimum in vitro mineralization.

Surface modification via graft copolymerization is an attractive method for optimizing polymers used in biomedical applications. We developed a novel method using a mixed solvent system (either water and dichloromethane (DCM) or water, methanol and DCM) consisting of two solvent phases for grafting 2-(methacryloyloxy)ethyl phosphate onto expanded polytetrafluoroethylene (ePTFE). This new method resulted in the fabrication of grafted membranes with greater grafting extents (GEs) (as evaluated from x-ray photoelectron spectroscopy (XPS)) in the organic phase than those obtained when grafting was carried out in a single phase. It also made it possible to graft in the aqueous phase, a process that is otherwise inhibited by the concomitant formation of large amounts of highly crystalline homopolymer. Thorough characterization of the grafted membranes using gravimetric, XPS and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) not only permitted evaluation of the grafting outcomes but also made it possible to analyze their dependence on monomer concentration and solvent composition. A selection of membranes was tested for their in vitro mineralization capacity using simulated body fluid. It was found that an 'ideal' mineralization outcome, i.e. a uniform coating of carbonated hydroxyapatite (cHAP) formed on the sample grafted in the aqueous phase of the water/DCM two-phase solvent system. A detailed discussion bringing together these results, as well as results from a series of earlier studies, allows conclusions regarding polymer chemistry and the topology necessary for cHAP mineralization.

Time-of-flight secondary ion mass spectrometry study of the orientation of a bifunctional diblock copolymer attached to a solid substrate.

A block copolymer consisting of a phosphate-containing moiety (poly[2-(methacryloyloxy)ethyl phosphate], PMOEP) and a keto-containing moiety (poly[2-(acetoacetoxy)ethyl methacrylate], PAAEMA) showed good stability after attachment to an APS amine-modified glass slide, as did both of the respective homopolymers. The PAAEMA homopolymer can attach to the APS amine groups via covalent linkages, while the PMOEP homopolymer most likely attaches through electrostatic interactions involving deprotonated phosphate and protonated amine groups. To elucidate the conformation of the block copolymer after attachment, particularly with respect to the PMOEP segment orientation, principal component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) spectra of the surface-attached polymer layers was performed. Comparison with the pure homopolymer spectra and interpretation after PCA indicate that the adsorbed conformation is not random. Rather, the copolymer is adsorbed in a conformation that preferentially exposes the PMOEP block toward the outer surface. We thus conclude that the most likely conformation of PMOEP-b-PAAEMA immobilized onto the APS-modified glass slide is via covalent interfacial linkages involving the PAAEMA block with the result that the surface is enriched in PMOEP tails. This in turn implies that under the conditions applied (dry DMF) the covalent coupling of keto groups to the amine groups of the aminated slide is more efficient than the proton transfer required for the generation of electrostatic attractions. This (partially) preferential orientation of the PMOEP-b-PAAEMA copolymer could have significant implications on interfacial interactions such as those involved in nucleation and the subsequent mineralization sequence of events in hydroxyapatite formation. The present study demonstrates that ToF-SIMS is a powerful tool not only for the investigation of the surface composition of adsorbed layers, but also for probing the molecular conformation of such adsorbed block copolymers, though care is required in the PCA analysis of multiple spectra.

Adsorption of well-defined fluorine-containing polymers onto poly(tetrafluoroethylene).

Adsorption of well-defined fluorinated polymers onto clinically relevant poly(tetrafluoroethylene) (PTFE) substrates offers an attractive method for modifying the surface properties of chemically inert PTFE. Reversible addition-fragmentation chain transfer (RAFT) was successfully used for synthesis of the polymers in this study: the homopolymers poly(2,3,4,5,6-pentafluorostyrene) (PFS), poly(2,2,3,3-tetrafluoropropyl acrylate) (PTFPA), and poly(2,2,3,3-tetrafluoropropyl methacrylate) (PTFPMA) as well as their block copolymers with tert-butyl acrylate ( (t)BA). Water-soluble blocks were synthesized through the hydrolysis of the t-butyl side groups of P( (t)BA) to the corresponding carboxylic acid. Adsorption of selected polymers onto PTFE from a series of solvents (methyl ethyl ketone (MEK), dimethylformamide (DMF), fluorobenzene (FB), dichloromethane (DCM)) was investigated using X-ray photoelectron spectroscopy (XPS) and sessile water drop measurements. The three homopolymers studied all adsorbed irreversibly (i.e., were not removed by washing) from organic solvents at ambient temperature. PFS displayed the highest adsorption, and was attributed to strong hydrophobic interactions. From angle-resolved XPS it was concluded that PFS became impregnated into the PTFE substrate down to depths of 100 A when using FB as a solvent. The carboxylic acid-containing block copolymers adsorbed more effectively from DMF (a good solvent for the poly(acrylic acid) block) compared to MEK. The resulting modified PTFE substrates displayed high stability with respect to desorption in aqueous solution, yet conformational changes of the adsorbed polymer resulted in a switchable hydrophobic-hydrophilic surface (in air or water, respectively). These results highlight the success of a facile and simple approach to irreversibly adsorb functional polymers to a nonfunctional fluorinated surface.

Influence of a diene impurity on the molecular structure of phosphate-containing polymers with medical applications.

We have demonstrated that the unacknowledged presence of almost 30% diene impurity in some commercial phosphate monomers had not only a significant effect on the molecular structure (topology) of a series of synthesized polymers but the instability of the ester functionalities during these polymerizations resulted in unexpectedly complex co-polymer chemistry.

FT-IR spectroscopy of fluoro-substituted hydroxyapatite: strengths and limitations.

Fluoro substituted hydroxyapatite (FHAp) samples were prepared by a cyclic pH method. Both calcined and uncalcined samples were subjected to elemental analysis (F, Ca, P) and X-ray diffraction (XRD) analysis to verify composition and phase purity. Good correlation between a-axis parameters and fluoride ion content was found for calcined samples, however, for uncalcined samples the fluoride ion content was higher than estimated from the a-axis values. Fourier transform infra red (FT-IR) spectroscopy analysis of the calcined samples showed OH band shifts and splitting in accordance with F-HO interactions affecting the OH vibration. We conclude that the OH libration (620-780 cm(-1) range) is more suited for estimation of fluoride ion content than the OH stretching. In contrast, uncalcined samples all displayed FT-IR spectra similar to that of hydroxyapatite (HAp) despite the presence of fluoride ions (18-73%). FT-IR emission spectroscopy was used to probe the changes occurring in the FT-IR spectra of HAp and FHAp samples upon heating. Interpretation of the spectral changes occurring during heating to 1,000 degrees C and subsequent cooling is given. Room temperature spectra of samples heated to various temperatures was used to determine the temperature necessary to produce FT-IR spectra displaying the expected OH bands. A model accounting for the combined observations is proposed.

Synthesis of soluble phosphate polymers by RAFT and their in vitro mineralization.

Soluble linear (non-cross-linked) poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-(methacryloyloxy)ethyl phosphate) (PMOEP) were successfully synthesized through reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization and by keeping the molecular weight below 20 K. Above this molecular weight, insoluble (cross-linked) polymers were observed, postulated to be due to residual diene (cross-linkable) monomers formed during purification of the monomers, MOEP and MAEP. Block copolymers consisting of PMAEP or PMOEP and poly(2-(acetoacetoxy)ethyl methacrylate) (PAAEMA) were successfully prepared and were immobilized on aminated slides. Simulated body fluid studies revealed that calcium phosphate (CaP) minerals formed on both the soluble polymers and the cross-linked gels were very similar. Both the PMAEP polymers and the PMOEP gel showed a CaP layer most probably brushite or monetite based on the Ca/P ratios. A secondary CaP mineral growth with a typical hydroxyapatite (HAP) globular morphology was found on the PMOEP gel. The soluble PMOEP film formed carbonated HAP according to Fourier transform infrared (FTIR) spectroscopy. Block copolymers attached to aminated slides showed only patchy mineralization, possibly due to the ionic interaction of negatively charged phosphate groups and protonated amines.

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications.

The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of CaP minerals, whereas all the grafted membranes revealed the growth of CaP minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity (theta(adv) = 120 degrees, theta(rec) = 80 degrees) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces (theta(adv) = 60 degrees and 80 degrees, theta(rec) = 25 degrees and 15 degrees, respectively) exhibited irregular growth of non-apatitic CaP minerals. Irreversibly adsorbed protein measured after a 1h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3-6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample. The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro.

Investigation into the diffusion of water into HEMA-co-MOEP hydrogels.

Cross-linked homopolymers and copolymers of 2-hydroxyethyl methacrylate, HEMA, and ethylene glycol methacrylate phosphate, MOEP, have been synthesized, and the diffusion of water into these systems has been investigated. Only polymers with 0-20 mol % MOEP exhibited ideal swelling behavior as extensive fracturing occurred in the systems with greater than 20 mol % MOEP as the polymers began to swell during water sorption. Gravimetric studies were used in conjunction with magnetic resonance imaging of the diffusion front to elucidate the diffusion mechanism for these systems. In the case of the cross-linked HEMA homopolymer gels, the water transport mechanism was determined to be concentration-independent Fickian diffusion. However, as the fraction of MOEP in the network increased, the transport mechanism became increasingly exponentially concentration-dependent but remained Fickian until the polymer consisted of 30 mol % MOEP where the water transport could no longer been described by Fickian diffusion.

Calcium phosphate nucleation on surface-modified PTFE membranes.

Highly porous PTFE membranes are currently being used in facial reconstructive surgery. The present study aims at improving this biomaterial through creating a more bioactive surface by introducing ionic groups onto the surface. The unmodified PTFE membrane does not induce inorganic growth after immersion in simulated body fluid (SBF) for up to 4 weeks. Copolymeric grafting with acrylic acid (AAc) by means of gamma irradiation and subsequent in vitro testing in SBF reveals that this copolymer initially acts as an ion-exchange material and subsequently induces growth of a calcium phosphate phase (Ca/P=2.7) when large amounts (15%) of pAAc are introduced onto the membrane surface. This copolymer is not expected to function well from a biomaterials perspective since SEM showed the pores on the surface to be partly blocked. In contrast, the surface of monoacryloxyethyl phosphate (MAEP)-modified samples is altered at a molecular level only. Yet the modified materials are able to induce calcium phosphate nucleation when the external surface coverage is 44% or above. The initial inorganic growth on these membranes in SBF has a (Ca+Mg)/P ratio of 1.1 (presumably Brushite or Monetite). The secondary growth, possibly calcium-deficient apatite or tricalcium phosphate, has a (Ca+Mg)/P ratio of 1.5. This result is a promising indicator of a bioactive biomaterial.