Hydroxyapatite thin films synthesized by pulsed laser deposition and magnetron sputtering on PMMA substrates for medical applications

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Abstract

Functionalized implants represent an advanced approaching in implantology, aiming to improve the biointegration and the long-term success of surgical procedures. We report on the synthesis of hydroxyapatite (HA) thin films on polymethylmetacrylate (PMMA) substrates – used as cranio-spinal implant-type structures – by two alternative methods: pulsed laser deposition (PLD) and radio-frequency magnetron sputtering (MS). The deposition parameters were optimized in order to avoid the substrate overheating. Stoichiometric HA structures were obtained by PLD with incident laser fluences of 1.4–2.75 J/cm2, pressures of 30–46.66 Pa and 10 Hz pulses repetition rate. The MS depositions were performed at constant pressure of 0.3 Pa in inert and reactive atmospheres. SEM-EDS, XRD, FTIR and pull-out measurements were performed assessing the apatitic-type structure of the prepared films along with their satisfactory mechanical adhesion. Cell viability, proliferation and adhesion tests in osteosarcoma SaOs2 cell cultures were performed to validate the bioactive behaviour of the structures and to select the most favourable deposition regimes. For PLD, this requires a low fluence of 1.4 J/cm2, reduced pressure of water vapours and a 100 °C/4 h thermal treatment. For MS, the best results were obtained for 80% Ar + 20% O2 reactive atmosphere at low RF power (∼75 W). Cells grown on these coatings exhibit behaviour similar to those grown on the standard borosilicate glass control: increased viability, good proliferation, and optimal cell adhesion. In vitro tests proved that HA/PMMA neurosurgical structures prepared by PLD and MS are compatible for the interaction with human bone cells.

Introduction

Our aim was to produce osteoinductive HA coatings for polymer polymethylmethacrylate (PMMA) cranial implants to be used in cranioplasty and vertebral stabilization.

Polymers such as ultra-high-molecular-weight polyethylene (UHMW PE) and polymethylmethacrylate (PMMA) found applications in acetabular cups, patellar prostheses, and as cements for fixing hip and joint prostheses and fillers in dentistry or vertebroplasty [1]. Between polymeric materials, PMMA is the aloplastic material the most used for the reconstruction of the cranial osseous defects. Unlike the other aloplastic implants, PMMA is a plastic both tough and easy to shape by heating. It is almost as tough as the bone. However, the PMMA plate has a feature superior to those of the bone, i.e. it does not fracture in the same manner, but in numerous fragments, avoiding this way the lesion of dura mater or of the cerebrum in case of the fracture [2].

Even if PMMA has the advantages of a low cost biomaterial and of the easy intraoperatory moulding and it is generally tolerated by human tissues, leading to neither necrosis nor adverse inflammatory reactions, it remains, however, an artificial prosthetic material, which inserted in the body could become encapsulated in a fibrous tissue matrix. This might generate a potential free space for infections, leading to the implant rejection. The risks of infection and contamination are even greater when the repair of the front-orbital defects involves the frontal sinus [2]. Therefore, the PMMA implants’ biological functionalization by other substances represents an actual trend in the field.

The main characteristic of artificial synthetic calcium phosphates (CaP), for which they are preferred in studies and applications concerning biomedical bone-substitute thin films deposition, is their innate similarity with the mineral compounds from bone and teeth, which represents the basis of osseoconductive behaviour [3], [4]. Hydroxyapatite (HA) Ca10(PO4)6(OH)2 presents the biggest stability of all calcium phosphates, being the less soluble in physiological conditions in the following series of relative solubility values (MCP > TTCP > α-TCP > DCPD > DCP > OCP > β-TCP > CDHA > HA) [1], [5].

The thin HA layers, herewith deposited by plasma and laser techniques, on medical polymer substrates, have the role to enhance the bioacceptance and to accelerate implant osseointegration in organism. The concept of composite HA coating/polymer systems, combining the biological and mechanical properties of the two components, aims to reduce the cytotoxicity or inflammatory reactions of surrounding tissues.

The HA–polymer composites mimic somehow the natural bone, formed by the inorganic phase (especially nanometric CHA) and organic compounds (mainly collagen type I). The nanometric dimension of the inorganic element, of high specific surface, similar to the one in the bony apatite is important from the point of view of the mechanical properties [6], [7]. It has been demonstrated that certain biochemical and physical properties of hydroxyapatites along with a certain surface structuration offer an intimate contact with bony tissue in vivo. It has also been demonstrated the possibility of stem cells differentiation [8], [9] in the presence of HA, result that allowed the production of stem cells inseminated foams designed to regenerate bone tissue.

Previous studies reported HA coatings synthesized on metallic substrates by pulsed laser deposition [5], [10], [11], [12] and magnetron sputtering [5], [13], [14], [15].

Last couple of years witnessed a gradual replacement of traditional, plasma spray thick-coated implants with adherent thin films produced by magnetron deployment. From our knowledge no reliable methods to produce bioactive coatings on PMMA substrates are reported.

Pulsed laser deposition, a highly performant technique as far as the chemical and physical properties are concerned (mechanical, structural, stoichiometric) it is not released for commercial use yet but it is constantly improved, tested, and attested by many research studies. Animal experiments have shown the compatibility of nanostructured HA–PLD films with clinical implantation, evidenced by an almost twofold implant resistance due to the improved adherence to the bone tissue, compared to the bare titanium implants [16]. Although the statistical data on their in vivo behaviour is still limited we could predict a better stability on long-term, based on an almost natural compatibility between the bone and the implant at a chemical and structural level with a resulting lower rate of revisions.

On the other part, magnetron sputtering (MS) is a method which is known to provide good control over thickness and composition of hydroxyapatite coatings. Uniform and high-purity adherent films on large areas can be easily deposited by this technique [13], [14], [17]. The target material is sputtered by the bombardment of high energy ions accelerated over the cathode sheath potential. The ion bombardment produces the emission and acceleration of the secondary electrons, which play an important role in maintaining the plasma around the cathode. A magnetic field confines the ionizing energetic electrons near the cathode allowing operation at high plasma densities and low pressures. Target-generated secondary electrons do not bombard substrates because they are trapped in cycloid trajectories near the target, and thus do not contribute to increased substrate temperature and radiation damage. This should allow the use of substrates that are temperature-sensitive such as PMMA with minimal adverse effects. In addition, this class of sputtering sources produces higher deposition rates than conventional sources and lends itself to a future application in biomedical industry [18].

We tried to obtain an improvement of PMMA implant-type substrates in terms of bio-functionalization by HA coating by two methods: PLD and MS at temperature below the softening temperature of PMMA (115 °C).

Section snippets

Materials and methods

Thin films of HA were deposited on PMMA substrates, using two different techniques: magnetron sputtering (MS), for PMMA–HA.1, PMMA–HA.2, PMMA–HA.3, PMMA–HA.4 samples, and pulsed laser deposition (PLD), for PMMA–HA.A1, PMMA–HA.A2, PMMA–HA.B and PMMA–HA.C.

In case of FTIR and EDS measurements we preferred flat silicon wafers (1 0 0) as substrates in order to have reliable results. Thus it was possible to avoid potential noise caused by the non-uniformity of PMMA implant-type substrates, and also the

Thickness determination

The deposition conditions of the MS–HA films are displayed in Table 2. In case of MS films no change in deposition rate was noticed when oxygen was added to the working atmosphere. A 1.2 nm/min sputtering rate was estimated. In order to obtain uniformity, in case of rotating substrates, the deposition duration was increased three times.

The tested PLD films had a thickness of 100–120 nm, appropriate for thin coatings for osteointegrating implants.

SEM

The HA/PMMA surface morphology was investigated by

Discussions

We analyzed the viability, proliferation and adhesion of osteoblasts to HA films deposited on PMMA by PLD and MS deposition techniques.

SEM and XRD results confirmed the amorphous nature of the PLD and MS films, revealing uniform and smooth surface morphologies in case of MS, and rougher for PLD. FTIR measurements exhibited the presence of characteristic phosphate bands of apatite. The EDS results confirmed the apatitic nature of the prepared coatings: a stoichiometric quality of PLD films (Ca/P =

Conclusions

In this study we demonstrated the versatility of two deposition techniques, pulsed laser deposition and magnetron sputtering, for obtaining bio-functionalized PMMA neurosurgical implants. The viability, proliferation and adhesion tests performed on HA/PMMA structures were analysed.

From biological point of view, the optimal regime established in the case of PLD samples deposited at room temperature required a low fluence of 1.4 J/cm2, reduced pressure of water vapours and a thermal

Acknowledgements

This work was supported under the Contract PN2 71-110/2007 (BIOSTIMP) financed by the Romanian Ministry of Education, Research and Youth. We thank Dr. Karine Anselme for the kind gift of providing the SaOs2 cell line and to Mrs. Livia Sima for her valuable support.

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