Laser surface modification of hydroxyapatite and glass-reinforced hydroxyapatite

Ana C. Queiroz^{, , a, b}, José D. Santos^{c, d}, Rui Vilar^e, Sónia Eugénio^{f, g} and F.J.Fernando J. Monteiro<sup>c, d

a</sup> INEB-Instituto de Engenharia Biomédica, Laboratório de Biomateriais, Rua do Campo Alegre 823, 4150-180, Porto, Portugal
^b Escola Superior de Tecnologia e Gestão, Apartado 574, 4901, Viana do Castelo Codex, Portugal
^c INEB-Instituto de Engenharia Biomédica, Laboratório de Biomateriais, Rua do Campo Alegre 823, 4150-180, Porto, Portugal
^d Faculdade de Engenharia da Universidade do Porto, Departamento de Engenharia Metalúrgica e de Materiais, Rua Roberto Frias, 4200-466, Porto, Portugal
^e IST-Instituto Superior Técnico, Av Rovisco Pais, 1049-001, Lisboa, Portugal
^f INEB-Instituto de Engenharia Biomédica, Laboratório de Biomateriais, Rua do Campo Alegre 823, 4150-180, Porto, Portugal
^g IST-Instituto Superior Técnico, Av Rovisco Pais, 1049-001, Lisboa, Portugal

Received 11 June 2003;  accepted 22 November 2003.  Available online 25 February 2004.
Biomaterials
Volume 25, Issue 19 , August 2004, Pages 4607-4614

1. Abstract

Surface treatment of materials with excimer laser radiation often results in the formation of a rough columnar or cone-shaped surface topography, which leads to a considerable increase in the surface area. As a result, the search for a non-porous bioactive material with adequate mechanical properties and a high surface to volume ratio, similar to porous materials, which could be used for drug delivery in the treatment of periodontitis, justified assessing excimer laser surface treatment to promote controlled roughning of hydroxyapatite (HA) and glass-reinforced hydroxyapatite (GR-HA). A KrF excimer laser with 248 nm radiation wavelength and 30 ns pulse duration was used for surface modification. The laser treatment was carried out in air, using wide ranges of radiation fluence and number of laser pulses. In order to identify the physico-chemical changes induced by the laser treatment and the column formation mechanisms in these materials, the treated surfaces were characterised by laser profilometry, scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Fourier transform infra-red spectroscopy (FTIR). Laser processing induced the formation of a surface topography consisting of cone-shaped features. The constitution of the surface layer was also modified, as revealed by FTIR, XPS and XRD.

This work has shown that laser surface modification increases the surface area of HA and GR-HA and is a promising technique to increase the reactivity and drug delivery capability of both materials.

Author Keywords: Laser; Hydroxyapatite; Surface modification; XPS; XRD; FTIR

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Materials preparation

2.2. Laser surface treatment

2.3. Surface characterisation

3. Results and discussion

3.1. Structural surface characterisation

3.2. Surface chemistry characterisation

4. Conclusions

Acknowledgements

References

3. 1. Introduction

Periodontitis is an oral disease that promotes periodontal bone defects, and, ultimately, alveolar bone loss [1 and 2]. The systemic approach to the treatment of this disease involves the administration of high antibiotic dosage for long periods of time. The increasing resistance of oral and media pathogens to common antibiotics has been dictating a restrictive use of systemic antibiotic therapy [3 and 4]. With a local delivery approach, the amount of antibiotic required should significantly decrease, as compared to a systemic treatment, and the period of time during which it would be administrated should also decrease [4 and 5]. In order to maximise local antibiotic delivery, the surface area of the drug releasing agent must be as high as possible, to accommodate large amounts of adsorbed drug. High surface areas may be obtained using porous materials and granules [6]. In this work a novel approach was used to induce high surface area on dense hydroxyapatite (HA) and glass-reinforced hydroxyapatite (GR-HA), by changing the surface topography via a KrF excimer laser treatment, using 248 nm wavelength radiation.

Hydroxyapatite is a well-known bioceramic that is extensively used in medical applications [7 and 8]. Due to its reasonable mechanical behaviour under low-load conditions and excellent biocompatibility, combined with slow replacement by the host bone after implantation, this compound is commonly used, for example, as a coating for hip prostheses and dental implants [9 and 10]. The recently developed glass-reinforced hydroxyapatite [11 and 12] presents higher mechanical properties than HA and, when tested under in-vitro and in-vivo conditions, both as a coating [13 and 14] and a bulk material [15 and 16], leads to very interesting results.

Previous studies have shown that GR-HA may be a very promising material to be used as a scaffold for adsorption of specific antibiotics, like sodium ampicillin [17], due to the presence of controlled amounts of biodegradable phases, such as
-TCP, Ca₃(PO₄)₂, in the microstructure. These tricalcium phosphate (TCP) phases may initially desorb the drug at high concentration levels and initiate their degradation process in the physiological environment, faster than pure HA, leading to a faster response from the host bone.

Pulsed laser deposition (PLD) is being increasingly employed for the growth of calcium phosphate coatings on metallic substrates for implant applications [18, 19, 20 and 21]. The examination of the targets after PLD reveals that cone shaped formations appear at their surface as a result of the laser treatment, leading to an increase in the surface area [20, 22, 23, 24 and 25]. This is an obvious disadvantage in the case of PLD because the target damage makes coatings less homogeneous and causes droplet ejection, but it can be put to profit, instead of being applied to the target, the laser treatment is applied to the materials surface, aiming at changing its surface morphology [25, 26 and 27]. This treatment has the advantage of preserving the chemical and mechanical integrity of the material [28], something that does not happen when porous scaffolds are used as drug delivery agents. The present work is, therefore, a logical extension of previous work [6 and 17], since its purpose is enhancing the capacity of accommodating larger contents of drugs by increasing the surface area through laser treatment.

In the present work a KrF excimer laser was used for surface modification. The laser treatment was carried out using wide ranges of radiation fluence and number of laser pulses. The physico-chemical changes induced by the laser treatment were characterised by laser profilometry, scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR).

4. 2. Materials and methods

2.1. Materials preparation

Commercially pure HA (from Plasma Biotal, ref. P120) powder and GR-HA 16 mm diameter and 1.5 mm thickness cylinders were used. In order to obtain these samples, P₂O₅-CaO glass (75P₂O₅, 15CaO, 10CaF₂ mol%) was prepared using reagent grade chemicals. The chemicals were mixed and heated at 1350°C for 1 h. The mixture was then cast into water, dried for 24 h in a oven at 100°C, then ball milled and sieved until a particle size of <75 
m was achieved. HA was also sieved to approximately the same particle size. GR-HA was obtained with 7.5 wt% glass addition to HA powder. This mixture was wet milled for 8 h (with methanol as a suspending medium), dried for 24 h in a oven at 100°C and sieved to a particle size <75 
m, in order to obtain an homogeneous free flowing powder.

Cylindrical samples were obtained by uniaxial compression of the powders at 288 MPa, followed by sintering for 1 h at 1200°C, with a heating rate of 4°C/min and natural cooling inside the furnace. To obtain uniform surface roughness, all samples were grinded (280, 400, 600, 1000 mesh) and polished to 3 
m before laser irradiation. Roughness measurements were performed before and after the laser treatment, by laser profilometery.

2.2. Laser surface treatment

For the laser surface treatment a KrF excimer laser with 248 nm radiation wavelength and 30 ns pulse duration was applied. Processing was performed using a mask projection micromachining system. A square mask was used to define the laser spots projected onto the material surface, while a beam homogeniser allowed to obtain an uniform fluence at the surface. A preliminary series of laser treatments was performed using a wide range of processing parameters: fluences between 0.2 and 10 J/cm², pulse frequencies of 5, 50 and 100 Hz and number of pulses varying from 10 to 2000. This preliminary set of experiences enabled the most adequate laser processing conditions to be chosen on the basis of the surface modification induced in the material. The following laser processing parameters were selected for the systematic tests: laser fluences of 0.75 and 1 J/cm², pulse frequencies of 5 Hz, and 200, 500, 1000 and 2000 laser pulses.

2.3. Surface characterisation

Structural characterisation of the surface layer on all the laser treated samples was performed using scanning electron microscopy (SEM) at 15 KeV. Roughness measurements were carried out to calculate the ten-point average maximum peak-to-valley depth (R_z) and the average (R_a) roughness values for each test condition. For a number of selected samples, detailed characterisation was performed using energy dispersive X-ray analysis (EDS), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy with alternated total reflectance (FTIR-ATR), using the split-pea accessory, in order to evaluate possible chemical and structural changes induced by the laser treatment.

5. 3. Results and discussion

3.1. Structural surface characterisation

SEM observation of HA samples treated with 1 J/cm² and different numbers of laser pulses, shows that there is an increase in surface roughness with the number of pulses, due to the formation of cone-like surface artefacts (Fig. 1a-f). In a smaller scale, the same type of topography change is observed for the samples treated with 0.75 J/cm². GR-HA samples treated using the same processing conditions also shows a similar surface topography, though the surface roughness tends to be slightly larger in this case as compared to HA. This topography is potentially adequate for enhanced drug delivery (Fig. 1), since it presents a high surface area.

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Fig. 1. SEM microphotograph of (a) non-treated HA; (b) laser treated HA with 1 J/cm² intensity and 200 pulses; (c) laser treated HA with 1 J/cm² intensity and 1000 pulses; (d) non-treated GR-HA; (e) laser treated GRHA with 1 J/cm² intensity and 200 pulses; and (f) laser treated GR-HA with 1 J/cm² intensity and 1000 pulses.

SEM observations were corroborated by the roughness measurements. The roughness increases with increasing number of pulses. The surface roughness of HA tends to be lower than the roughness of GR-HA samples for a low number of pulses, but the opposite occurs for larger number of pulses (Fig. 2). The roughness differences between HA and GR-HA are, however, similar within the limits of experimental error, and the surface topography after laser processing is very homogeneous in both cases. Moreover, it seems to be almost independent of the samples composition. When samples processed with the two fluences used are compared, the roughness increases with increasing radiation fluence (Fig. 3). The same behaviour is observed for R_a and R_z parameters.

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Fig. 2. Average roughness of GR-HA (white) and HA (grey) samples obtained for 1 J/cm² pulse intensity.

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Fig. 3. Average roughness of GR-HA samples obtained for 1 J/cm² (zebra), 0.75 J/cm² (white).

To estimate the actual surface area generated by the laser surface treatment, after visualising the SEM images and obtaining the roughness results, a number of assumptions were made to conceive a simple model enabling the calculation of surface area values with an accuracy of >90%.

Since the treated areas were relatively small, the measurement of the actual surface area of processed samples could not be performed using macroscopic methods such as BET (Brunauer, Emmett, Teller) method and mercury porosimetry. Instead, a simple geometric model was used to estimate it.

The topography of the treated surface was modelled by an arrangement of similar cylinders. The number of cylinders per unit area and the average dimensions of the cylinders were experimentally determined on typical laser processed samples, using image analysis and stereology. The experimental measurements were performed on 40×40 
m (1600 
m²) areas. A number of test lines were drawn and the number of interceptions counted. Using the relation established by Saltykov [29], the cylinder perimeter length per unit area was calculated. Values of 0.78 and 0.69 
m/
m², were found for HA and GR-HA samples treated with 1000 laser pulses and 1 J/cm² fluence, respectively. The increase in surface due to the laser treatment is equal to the lateral area of the cylinders, which was calculated considering that the average cylinder height is equal to R_a (1.46 and 1.67 
m for HA and GR-HA, respectively). Values of 1620 
m² and 2080 
m² were obtained for HA and GR-HA samples, respectively, leading to actual surface areas of 3220 
m² and 3680 
m² for those samples (as compared to an initial area of 1600 
m²). The increase in surface area obtained is 101% for HA and 130% for GR-HA.

In order to confirm these results, the surface area was also evaluated using laser profilometry data and Underwood equation [30]. This method lead to surface area estimations of 3520 
m² and 4240 
m² for HA and GR-HA, respectively, representing increases of 120 and 165%, in fair agreement with the previous results.

3.2. Surface chemistry characterisation

Since the aim of this work was to enhance the surface area of the bulk material, only the samples that presented large surface specific area were chosen for detailed surface characterisation, namely HA and GR-HA samples treated with a fluence of 1 J/cm² and 200 and 1000 pulses, i.e. HA200, HA1000, GR-HA200 and GR-HA1000, respectively.

The FTIR spectrum of un-treated HA (Fig. 4) shows phosphate peaks (at 1100, 1003 and 555 cm⁻¹) and hydroxyl group peaks (at 3571 and 632 cm⁻¹). A small shift in the phosphate peaks position as compared to their standard position is noticed, indicating a deficiency of calcium in the apatite [31 and 32]. The spectra of laser treated samples are similar, but the peaks increasingly lose definition when the number of pulses increases, revealing a loss of crystallinity of the material. Simultaneously, a shoulder develops in the 750 cm⁻¹ region with an amplitude that increases as the number of pulses increases. This peak can be attributed to
-TCP [33], and its presence shows that the high temperatures developed during the laser treatment induced the formation of this phase, due to the transformation of HA.

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Fig. 4. FTIR-ATR analysis of HA treated and untreated samples.

The FTIR spectrum for the GR-HA sample before laser treatment (Fig. 5) shows
-TCP peaks (543, 550, 570 and 578 cm⁻¹), together with the typical HA peaks. After laser processing some peaks are considerably spread and broadened, indicating a loss of crystallinity, while the shoulder at 750 cm⁻¹, which appeared in HA samples, here it is even more evident. Already clearly visible in the sample treated with 200 pulses, it transforms into an independent peak for the sample treated with 1000 pulses. This fact indicates a change in surface composition, that, in this case, consists, not only on the formation of
-TCP [33], but also on the probable appearance of poorly crystallised calcium pyrophosphates [34].

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Fig. 5. FTIR-ATR analysis of GR-HA treated and untreated samples.

XPS results are in agreement with those obtained by FTIR. Phosphorus (Fig. 6a), calcium (Fig. 6b) and oxygen ( Fig. 6c) peaks are observed. The phosphorus peaks in HA and GR-HA spectra are increasingly shifted towards higher energy with increasing number of pulses, indicating the presence of other phosphate phases besides hydroxyapatite. The phosphorus peak of GR-HA could be resolved into a doublet characteristic of hydroxyapatite and TCP, the shift towards higher binding energy for the GR-HA200 sample indicating that the amount of TCP increases. For the GR-HA1000 sample a new doublet appears at 135.6 eV, which is characteristic of pyrophosphate [35]. The oxygen peaks in GR-HA and GR-HA200 spectra consist of two peaks, corresponding to hydroxyapatite and TCP respectively. After 1000 laser pulses, the spectrum presents a new peak that may be attributed to the pyrophosphate phase [34]. The evolution of the calcium peaks corroborates the behaviour observed in phosphorus and oxygen peaks. In conclusion, the phases identified by XPS are in agreement with those observed by FTIR, and confirm the presence of pyrophosphate in the GR-HA sample treated with 1000 pulses.

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Fig. 6. XPS lines for (a) phosphorous; (b) calcium; and (c) oxygen.

The X-ray diffractograms of untreated samples reveal the presence of hydroxyapatite and a very small proportion of calcium oxide for HA samples, and hydroxyapatite and
-TCP for GR-HA [17]. The diffractograms of the laser treated samples may contain information deriving not only from the laser-processed area, but also from the unprocessed surface. For this reason only the XRD spectra of GR-HA samples are considered, because they are the only ones where a change in the phase composition was noticed: the hydroxyapatite peaks lose relative importance and
-TCP peaks develop when the number of pulses increases.

The present results show that laser processing is a worthy alternative to other processes of surface roughening because it is relatively fast, it is a non-contact process, and it is easily automated. Surface roughening is due to the development of columnar or cone-shaped artefacts [25, 36, 37, 38, 39 and 40]. Knowledge of the factors that control the formation of these artefacts is important since it may allow one to understand how to control their development in order to achieve particular properties. The formation of cones or columns during pulsed laser processing has been the subject of considerable scientific discussion and several mechanisms have been proposed to explain it. A shadowing effect has been suggested to explain column growth in polymers and YbaCuO-type ceramics [37]. In this mechanism, vaporisation-resistant regions on the processed surface are assumed to shield the underlying region from incident radiation, leading to column growth. As a result, the top of the columns must be at the level or below the level of the initial surface. Recently, Sánchez et al. [41] reported that when Si is processed in air using ArF laser radiation (
=193 nm) columns often grow considerably above the original Si surface. This behaviour is incompatible with a shadowing mechanism and the authors suggested that columns growth is due to liquid flow and material expansion upon solidification. A similar result was described by Pedraza et al. [42] in Si processed with KrF laser radiation (
=248 nm) in air, O₂, and SiF₆. The columns presented a resolidified droplet at their tip, leading the authors to conclude that the columns tips melt at each laser pulse, while the columns bases remain solid, an observation that rules out upwards-fluid flows along the columns as the mechanism of material transport for column growth, in this case. Instead, Pedraza et al. [42] suggested that mass transport should occur in the gas phase. More recently, Usoskin et al. [43] showed, on the basis of theoretical considerations, that, once nucleated, columns and cones may grow in the vertical direction because radiation is reflected on the lateral walls of these artefacts and concentrates in the intercolumnar regions, leading to enhanced ablation in these regions. Simultaneously, ablation from lateral walls decreases because these walls are tilted towards the incoming radiation. In the present work, the development of cone-shaped features is observed, leading to increasing roughening of the surface. The SEM examination reveals melting of a surface layer of material, but no evidence of extensive fluid flow. Therefore, the cones could not be formed by a hydrodynamic mechanism [41]. Conversely, the observed morphology and the results of surface structural and chemical characterisation are consistent with the shadowing mechanism of column growth, suggested by Foltyn et al. [37] (shadowing being due to the chemical inhomogeneity and structural changes of the materials surface). It is also consistent with the column growth mechanism proposed by Usoskin et al. [43].

6. 4. Conclusions

The surface roughness increases with the number of pulses both for HA and GR-HA samples, as observed by SEM and laser profilometry, resulting in doubling the actual surface area for samples treated with 1000 pulses and, 1 J/cm². XPS, FTIR and XRD results indicate changes in the surface layer constitution. XPS analysis of HA laser treated samples show shifts of the Ca and P peaks, suggesting the formation of other phosphates. The changes induced in HA tend to displace the peaks towards values similar to untreated GR-HA. For GR-HA, a small shift is also noticed for the sample treated with 200 laser pulses. For the sample treated with 1000 pulses, the peak deviation noticed may be due to loss of crystallinity and also to the presence of a non-crystalline pyrophosphate phase.

FTIR results confirm this loss of crystallinity by a spreading and broadening of the peaks. For treated samples a shoulder at approximately 750 cm⁻¹ is observed, characteristic of
-TCP and/or pyrophosphate. The same peak is noticed for the GR-HA sample treated with 1000 laser pulses. The progressive transformation of HA to
-TCP is confirmed by XRD of GR-HA samples.

The formation of a columnar or cone shaped surface topography as a result of laser processing can be explained by a combination of shadowing and progressive reduction of the radiation fluence due to the increasing inclination of the surface towards incident radiation. This topography seems very promising as a means to increase the surface roughness and the reactivity of both HA and GR-HA.

7. Acknowledgements

The authors would like to acknowledge Dr. Iain Gibson for technical support on XRD analysis and Project "TEXMED", ref. POCTI/FCB/41402/2001 for financial support.

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Corresponding author. INEB-Instituto de Engenharia Biomédica, Laboratório de Biomateriais, Rua do Campo Alegre 823, 4150-180, , Porto, , Portugal

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