Osteoblast Proliferation and Morphology Analysis on Laser Modified Hydroxyapatite Surfaces: Preliminary Results

Biocompatibility has long been associated with surface microtopography, microtexture and microchemistry. The surface topography ultimately affects the nature and the strength of the interactions that occur at biomaterial-biological environment (cell adhesion, mobility, spreading and proliferation). Thus, it is necessary to produce and work with controlled microtopographical surfaces that present reproducible microdomains of a dimension similar to that of the biological elements of interest (for instance, cells). [1] There are a number of substrates that already have been studied (such as silicone, polystyrene, poly-L-lactic acid and titanium coated polystyrene) in terms of surface topography. [2] However, few studies are related to hydroxyapatite substrates. As it is well established, hydroxyapatite is a well known ceramic that is extremely used in medical applications, namely implants and coatings. In this work, the surface topography of dense hydroxyapatite substrates was altered by using KFr excimer laser. Excimer lasers produce high-intensity, pulsed ultraviolet radiation and are especially well suited for materials processing due to their large beam cross-section area, which permits using mask projection technologies to process relatively large areas in a single step.[3] Introduction Calcium phosphate ceramics are widely used as bone substitutes since they are biocompatible and bioactive. Having a chemical composition close to natural bone, calcium phosphate ceramics are promising bone substitute materials in orthopaedics, maxillofacial surgery and dentistry. Hydroxyapatite (HA) and tricalcium phosphate (TCP) are the most commonly used calcium phosphates, because their calcium/phosphorus (Ca/P) ratios are close to that of natural bone and they are relatively stable in physiological environment. HA is a major constituent of bone materials and is resorbed after a long time of residence in the body. In previous work [3], 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. With this treatment, it is possible to change the surface morphology, while maintaining the mechanical properties of the base material. Key Engineering Materials Vols. 309-311 (2006) pp. 105-108 online at http://www.scientific.net © (2006) Trans Tech Publications, Switzerland All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of the publisher: Trans Tech Publications Ltd, Switzerland, www.ttp.net. (ID: 195.23.32.141-20/09/06,12:09:06) Cone shaped formations can be obtained, leading to an increase in the actual surface area [3-9]. However, when developing such surface modified materials for biomedical applications, it is of outmost relevance to obtain a complete characterization of the biological interactions that occur at the bone tissue/material interface. For biomaterials thought for contact with bone tissue, osteoblastic cell lines are commonly used for biocompatibility tests and proliferation assays since they are efficient, reproducible and easy to culture. [4] Scanning Electron Microscopy and Confocal Laser Scanning Microscopy were used to assess cell adhesion, distribution and morphology on the substrate surface. Materials and Methods Samples preparation: Hydroxyapatite (commercially pure HA from Plasma Biotal, ref. P120) powder was sieved until a particle size less than 75 μm was achieved. Cylindrical hydroxyapatite discs with a diameter of 5.5 mm were then obtained by uniaxial compression at 157 MPa (Mestra Snow P3) and submitted to a sintering cycle as previously described [3]. For the laser surface treatment a KrF excimer laser with 248nm 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 a uniform fluence at the surface. The following laser processing parameters were selected: laser fluence of 1 J/cm 2 , pulse frequency of 10 Hz, and 1000 laser pulses per spot. All samples surface was irradiated. Cell culture procedures: The samples were seeded with MG 63 osteoblast like cells over a period of 5 days. They were cultured at 37 oC in a humidified atmosphere of 5% CO2 in air, in 75 cm 2 , flasks containing 10 ml of alphaminimum essential medium (α-MEM) (Gibco), 10% foetal calf serum (Gibco), 0,5% gentamicin (Gibco) and 1% fungizone (Gibco). The medium was changed every third day and, for sub-culture, the cell monolayer was washed with phosphate-bufered saline (PBS) (Sigma) and incubated with trypsin/EDTA solution (0.25% trypsin,1 mM EDTA; Sigma) for 5 min at 37oC to detach the cells. The effect of trypsin was then inhibited by adding the complete medium at room temperature. The passage of the cells was the 27 th . The discs were steam sterilized (120°C, 20 min), placed in non treated 96-well plates (14 mg/well, ca. 1 cm 2 surface area) to avoid cell adhesion to the bottom of the wells. The cells were seeded at 10 4 cells per sample and incubated at 37oC for three hours to allow cell adhesion. Afterwards, fresh medium was added until it reached a final volume of 150μL per well. After the culturing periods the samples were prepared for scanning electron microscopy and laser confocal scanning microscopy analyses. Results In previous work a homogeneous columnar structure onto the surface of the discs was obtained with this type of laser surface treatment. [3] Figure 1. SEM microphotograph of untreated (a) and laser treated hydroxyapatite discs (b). Bioceramics 18 106