METHODS OF MANUFACTURING SUPERHYDROPHILIC IMPLANTS

The present invention relates to superhydrophilic implant materials; to methods by which such materials may be produced; and to the uses of such materials. For the purposes of the present invention, superhydrophilic materials should be taken as being those having a liquid contact angle at or close to zero.

Implant materials are used for a range of clinical purposes in modern medicine. These include the use of orthopaedic implants for use in the muscular or skeletal systems.

The early integration of implant materials within the body is an important factor for their clinical success. The surfaces of the implant represent the point of contact between the body and the implant, and exert a great deal of influence on how the implant interacts with the body. The response of biological cells, such as osteoblasts, to the implant may be dependent upon the topography, energy and chemical properties of the surface of the implant.

The challenge in the engineering surfaces of implant materials to be used in in contact with bones is to attract osteoblasts that produce a bone extracellular matrix, which ensures a high bone-implant contact. Cell attachment is one of the initial stages for subsequent proliferation and differentiation of osteoblastic cells. It is known that osteoblast adhesion, growth and differentiation are related to surface energy and roughness; however, the optimal surface properties for attachment, proliferation and differentiation of osteoblast are still not clearly defined.

Various techniques such as sand blasting and acid etching have been used in an attempt to roughen implant surfaces in order to improve their clinical performance and to encourage a stable mechanical interface between the implant and bone. However these techniques are subject to drawbacks. Sand blasting is reported to leave contaminations on the surface of implant materials, and both techniques produce surfaces with limited topography and chemical compositions.

Laser processing of titanium and stainless steels is a well-known process. Laser modified surfaces are advantageous due to their high degree of cleanliness and being free from contaminations. The process evaporates and melts the original surface layer and re-shapes a desirable micro- or nano-morphological structure with enhanced surface area and roughness to promote osseointegration.

Other methods for producing substructures on metallic implant material surfaces by a number of different techniques have been disclosed. Certain examples include shot blasting the surface with molten metal powders, laser processing, chemical etching, plasma assisted techniques or combination of these. Such techniques may employ coating with apatite by electro-chemical means in order to achieve a desired specific surface chemistry.

Methods in which pulsed laser beams are used to generate micro-grooves with a depth between 1-100 μηι on surfaces have been described previously, in the context of reducing and eliminating micro-cracks. Potential applications of this technology in medical implants have been suggested, since the micro-grooves have been found to enable directional cell growth.

It is an object of certain embodiments of the invention to provide methods of manufacturing superhydrophilic implant materials. It is an object of certain embodiments of the invention to provide alternative methods by which superhydrophilic implant materials may be manufactured. It is an object of certain embodiments of the invention to provide superhydrophilic implant materials. It is an object of certain embodiments of the invention to provide methods of treatment utilising superhydrophilic implant materials.

In a first aspect of the invention there is provided a method of manufacturing a superhydrophilic metal implant material, the method comprising laser processing, with a laser having a wavelength in the visible spectrum, the surface of a metal implant material in an atmosphere comprising nitrogen.

In a second aspect of the invention there is provided a superhydrophilic implant material manufactured by a method in accordance with the first aspect of the invention.

Within the context of the present disclosure superhydrophilic materials should be taken as being those having a liquid contact angle of 5° or less, and preferably of zero, or close to zero, as discussed further below. "An atmosphere", for the purposes of the present disclosure, may be taken as referring to the gaseous surroundings of the implant material the surface of which is being processed. Within this context, "an atmosphere comprising nitrogen", should be taken as relating to an atmosphere in which any detectable amount of nitrogen is present. Suitably such an atmosphere in accordance with these aspects of the invention may comprise at least 50% nitrogen by volume, at least 70% nitrogen by volume, at least 75% nitrogen by volume, at least 80% nitrogen by volume, at least 85% nitrogen by volume, at least 90% nitrogen by volume, or least 95% nitrogen by volume. Indeed, a suitable atmosphere may comprise 100% nitrogen by volume. Accordingly, it will be appreciated that air (which is generally taken as containing approximately 78% nitrogen by volume) may constitute an example of an atmosphere comprising nitrogen in accordance with certain embodiments of the methods of the invention. However, in certain embodiments, where an atmosphere comprising greater than 78% nitrogen by volume is preferred, air will not constitute an example of such an atmosphere.

The methods of the invention allow laser processing to modify both the surface topography and surface chemistry of the implant material. These modifications may occur simultaneously. The superhydrophilic surfaces of materials of the invention, in which surface chemistry and/or surface topography are modified, provide a significant improvement in cell adhesion compared to those known from the prior art. Laser processing of an implant material in an atmosphere comprising nitrogen may contribute to a change in surface chemistry of the material by leading to the production of metal nitrides. Laser processing may serve to modify the surface topography such that the surface area is increased.

Without wishing to be bound by any hypothesis, the inventors believe that the presence of nitrogen in the atmosphere at the time of processing influences surface chemistry by contributing to establishing the superhydrophilic surface of the material, and that increasing the proportion of nitrogen present in this atmosphere contributes to increased stability of the superhydrophilic properties obtained. This is consistent with the inventors' finding that methods of the invention in which laser processing is conducted in an atmosphere of substantially pure nitrogen produce materials of the invention that retain their superhydrophilic properties for significantly improved periods of time. The methods of the invention may be practiced in an atmosphere that comprises both nitrogen and oxygen. The presence of oxygen in the atmosphere surrounding the implant material may also contribute to a change in surface chemistry of the implant material, for example by increasing the concentration of metal oxides present. Air may represent a suitable atmosphere comprising both nitrogen and oxygen.

The presence of metal nitrides (and optionally metal oxides) may contribute to increased wettability of the surface of a material in accordance with the present invention. The presence of metal nitrides (and optionally metal oxides) may contribute to the ability of the surfaces of such materials to retain such wettability for increased duration compared to other known materials. Features of the surface topography (including size and shape of microfeatures and/or nanofeatures, as discussed elsewhere in the specification) may also contribute to increased wettability of materials in accordance with the present invention.

Suitable laser processing techniques for use in the methods of the invention may involve the selection of appropriate values in one or more of the following parameters: laser fluence; hatch distance; laser beam size and geometry, numbers of pulses/spots per location; number of scans; scanning speed; repetition rate in order to produce beneficial changes in the surface chemistry and/or topography of the implant material. Guidance as to how appropriate values may be selected or derived may be found elsewhere in specification, for example in the following paragraphs or in the Experimental Results section.

The changes to the surface topography caused by laser processing in the methods of the invention include the production of laser generated structures on the surface of the implant. These structures may have a height approximately twice that of the cells that are to be grown upon them. Examples of such structures include "micro cones" (with a height of between about 15 and 35 pm), or "micro grooves" The structures may optionally also comprise nanostructures (with a height of between about 10 nm and 800 nm), which may be formed upon the surface of such micro cones or micro grooves.

The changes to the surface topography increase surface area, and also roughness. This increase in roughness may serve to amplify the effects of the changes in surface chemistry, thus contributing to the superhydrophilic properties of the implant materials.

In a third aspect of the invention there is provided a superhydrophilic implant material comprising a surface enriched in metal nitrides. Such enrichment may be determined with reference to the concentration of metal nitrides in a sample of a suitable control material. In a fourth aspect of the invention there is provided a superhydrophilic implant material, a surface of which comprises at least 5% metal nitrides.

The surface of a superhydrophilic implant material in accordance with the invention (for example in accordance with the second, third or fourth aspects of the invention) may comprise between about 5% and 40% metal nitrides, such as between 10% and 30% metal nitrides. By way of example, a material of the invention may comprise about 10%, 15%, 20%, 25%, 30%, 35%, or 40% metal nitrides. In particular embodiments the surface of a superhydrophilic implant material in accordance with the invention may comprise at least 10% metal nitrides, and may comprise in the region of 14% metal nitrides. In suitable embodiments, the levels of metal nitrides in the surface of a superhydrophilic implant material in accordance with the invention may be enriched by at least 10% as compared to a relevant control material.

Superhydrophilic implant materials of the invention may additionally (or alternatively) possess one or more characterizing features of surface chemistry and/or surface topography independently selected from those considered below (which are discussed in more detail elsewhere in the specification).

Changed features of the surface chemistry of an implant material in accordance with the invention (such as an implant material produced by the methods of the invention) may include changes as compared to an untreated material. Such changes may include reducing carbon and hydro-carbon organic contaminations; and/or reducing carbon oxides; and/or increasing the oxygen to carbon ratio; as well as the impact upon metal nitrides and oxides.

Characteristic features of the surface topography may include the presence of micro structures on the surface of the material. Such micro structures (which may be between approximately 15 and 35 pm from peak to trough) may also have submicron features (nanostructures) on their surface. Suitable microstructures and nanostructures are described in further detail elsewhere in the specification, and these features are both able to increase the surface area of an implant material of the invention.

The surface topography of a material of the invention may comprise features (such as the microstructures referred to above) that have a period of around 30-50 pm. The surface topography may comprise structures that provide a balanced profile containing both "peaks" and "valleys".

The surface of a material of the invention may have a roughness (Ra) between 5 to 8 pm. The surface of a material of the invention may be substantially free from sharp features (such that the surface kurtosis is less than approximately 2). The absence of such sharp features may encourage a higher cell attachment.

The characteristic features of surface chemistry and/or surface topography outlined above may increase the ability of materials of the invention (or manufactured in accordance with the methods of the invention) to promote cell adhesion.

For the purposes of the present disclosure, references to "materials of the invention" should be taken as encompassing materials in accordance with either the second or third or fourth aspects of the invention, or materials used in a method in accordance with the first aspect of the invention.

In a fifth aspect of the invention there is provided a method of treatment comprising providing a superhydrophilic implant material of the invention to a subject in need thereof.

The inventors have found that the methods and materials of the invention are of benefit in the manufacture of materials for use as medical implants. In particular the materials of the invention, such as those manufactured in accordance with the methods of the invention, may be suitable for use as orthopaedic implants. Examples of orthopaedic implants that may be produced using the materials and methods of the invention include knee, hip joints and teeth implants. Without detracting from the above, the methods and materials of the invention may also be of use in the manufacture of implant materials for other therapeutic uses. The methods of the invention may employ a diode pumped solid-state (DPSS) laser. A suitable DPSS laser may be based on Nd:YAG or Nd:YV0 ₄ . In particular embodiments the laser used may be a Nd:YV0 ₄ DPSS laser.

As set out above, the methods of the invention make use of laser processing utilising a laser having a wavelength in the visible spectrum. The visible spectrum may be considered to lie between approximately 380 nm and 740 nm. Merely by way of example, suitable wavelengths that may be used in the methods of the invention may be selected from the group consisting of: approximately 355 nm, and approximately 532 nm.

The use of a laser having a wavelength in the visible spectrum provides a number of advantages as compared to lasers having longer or shorter wavelengths. As compared to lasers having wavelengths in the infrared range, use of a laser having a wavelength in the visible spectrum enables a smaller diameter focal spot to be used, thus allowing higher resolution of the laser processing, and better accuracy control of the size of structures produced by such processing. The efficiency of beam absorption by metallic materials (such as metal implant materials) is also greater for lasers with wavelengths in the visible spectrum, as compared to those with wavelengths in the infrared spectrum.

The use of lasers having wavelengths in the visible light spectrum also has advantages when compared to lasers with ultraviolet wavelengths. The cost of optical components used in such visible light lasers is lower than those used in ultraviolet lasers (thus providing economic advantages), and the efficiency of energy conversion from electrical to optical forms is also improved.

Notwithstanding the above, methods of manufacturing a superhydrophilic implant material (such as a superhydrophilic metal implant material) involving laser processing the surface of the material in an atmosphere comprising nitrogen may alternatively be put into practice using a laser with a wavelength of between approximately 248 nm - 1064 nm (though such methods will not enjoy the benefits set out in the preceding paragraphs). Merely by way of example, a suitable wavelength that may be used in the context of such methods may be approximately 1064 nm. In certain embodiments of the methods of the invention the laser used is a scanning laser, though it is contemplated that the methods of the invention could also be put into practice by moving an implant material relative to a fixed laser.

In certain embodiments, the methods of the invention may employ a laser with a fluence of between approximately 1 J/cm ² and approximately 4 J/cm ² In a suitable embodiment the invention may employ a laser with a fluence of between approximately 1.51 J/cm ² and 2.47 J/cm ² . Suitable embodiments of the invention may employ a laser with a fluence of greater than 1.75 J/cm ² . For example, suitable embodiments may employ a laser with a fluence of greater than 2 J/cm ² , or greater than 2.2 J/cm ² , or greater than 2.4 J/cm ² .

In certain embodiments of the invention the laser processing may comprise patterning of the surface of the implant material with a pattern of substantially parallel scans. In certain embodiments the laser processing may employ overlapping parallel scans at different angles, such that the scans cross one another. The laser processing may comprise patterning of the surface of the implant material with a pattern of cross-hatched scans. In certain embodiments, such as those discussed in the preceding sentences, the laser processing may comprise patterning of the surface of the implant material with a pattern having a hatch distance (for these purposes the average distance between substantially parallel scans) of approximately 55 pm. Methods of the invention utilising cross scans may be capable of producing implant materials with preferred characteristics; however such methods may be slower than those in which only substantially parallel scans (i.e. without crossing scans) are employed.

The number of pulses and/or spots used in patterning the surface of the implant material may also be used in characterising the methods of the invention. For example, the number of pulses per mm ² may vary between 25,000 (e.g. 27,273) to approximately 165,000 (e.g. 163,636). Laser patterning in keeping with these embodiments may be used to provide a number of pulses per spot varies from about 80 (e.g. 82.5 in an embodiment where 27,273 pulses per mm ² are used) to about 500 (e.g. 495 in an embodiment where 163,636 pulses per mm ² are used).

Suitably, the methods of the invention may make use of a laser with a pulse duration of between approximately 1 ps and 100 ns. Merely by way of example, a "nanosecond laser" with a pulse duration of between approximately 1 ns and 100ns is suitable for use in the methods of the invention. In certain embodiments the methods of the invention may comprise patterning of the surface of the implant material with a laser with pulse duration of approximately 8 ns. Alternatively, a "picosecond laser" with a pulse duration of between approximately 1 ps and 100 ps may be used. The use of picosecond lasers of this sort may provide advantages in terms of increased speed with which surface patterning can be achieved.

In certain embodiments the methods of the invention may comprise patterning of the surface of the implant material through use of between 1 to 6 scans over an area to be patterned.

In certain embodiments the methods of the invention may comprise patterning of the surface of the implant material using a scanning speed of approximately 20 mm/s.

In certain embodiments the methods of the invention may comprise patterning of the surface of the implant material using a laser with a repetition rate of between around 15 kHz and around 35 kHz. In a suitable embodiment, a repetition rate of approximately 30 kHz may be used.

In certain embodiments the methods of the invention may comprise patterning of the surface of the implant material with a laser with a depth of focus of approximately 50 pm to approximately1500 pm). In a suitable embodiment, a laser may be used with a depth of focus of around 952 pm.

In certain embodiments of the methods of the invention laser with a spot diameter of approximately 10 pm to approximately 100 pm. In a suitable embodiment a laser with a spot diameter of 55 pm may be employed.

In certain embodiments the method of the invention is practiced in the absence of a shroud gas. Suitably a method of the invention may be practiced in atmospheric gases (i.e. a method in which atmospheric air constitutes the atmosphere comprising nitrogen).

Metal implant materials suitable for use in the methods or materials of the invention may be selected from those conventional in the field in which the implant material is to be used. Merely by way of example, implant materials may be selected from the group consisting of: titanium alloys; titanium; chromium alloys; stainless steel; magnesium alloys. In a particular embodiment, the implant material used in a method or material of the invention may comprise the titanium alloy Ti-6AI-4V.

The materials of the invention, such as those produced using the methods of the invention, are superhydrophilic. Suitably a superhydrophilic material in accordance with the present invention may have a water contact angle of 5° or less. For example, superhydrophilic material of the invention may have a water contact angle of approximately 0°.

Other superhydrophilic materials are known from the prior art such as Ti0 ₂ coatings, but such prior art superhydrophilic materials suffer from a number of drawbacks. For example the superhydrophilic properties of such materials in which laser texturing is performed in an Argon gas environment have a tendency to decrease rapidly with time. In contrast, the inventors have found that the materials of the invention are able to retain their superhydrophilic properties for extended periods. For example, materials in accordance with the invention where laser texturing is carried out in a N ₂ gas environment are able to retain their superhydrophilic properties for a month or longer when stored in a normal air atmosphere. This stability of the materials of the invention indicates that they have a "shelf life" that makes them suitable for manufacturing prior to their intended use. Furthermore, the superhydrophilicity of materials of the invention is not reduced if these materials are subjected to autoclaving. This is a standard technique by which implant materials may be sterilised prior to medical use, and so the ability of the materials of the invention to retain their beneficial properties during sterilisation is highly desirable.

In keeping with the above, it can be seen that the methods and materials of the invention provide notable advantages in terms of the stability of the superhydrophilicity that is achieved. Such materials may retain superhydrophilicity (illustrated by a contact angle at or around 0°) for at least one day, up to seven days, or even up to 15 days after laser processing. Indeed, the inventors have found that materials of the invention (produced by methods of the invention) may maintain a contact angle of less than 10 degrees (illustrative of superhydrophilicity) for at least a month after laser processing.

Materials of the invention, such as those manufactured by the methods of the invention, may be characterized with reference to one or more features of their surface topography. For example, materials of the invention may be characterized with reference to the height of the structures on their surfaces. The height of such structures may be measured from "peak to valley". In certain embodiments, structures on materials of the invention, such as those manufactured in a method of the invention, may have a mean height of between approximately 15 and 35 pm. These structures may have further submicron features on their surface. Such submicron features (or nanostructures) may have a height of between approximately 10 nm and 800 nm.

The materials may be characterised with reference to their shape. By way of example, suitable structures may have a generally conical form.

Materials of the invention, such as those manufactured by the methods of the invention, may be characterized with reference to the periodicity of structures on their surfaces. The period may be defined as the average distance between corresponding points of adjacent structures (for example the average distance between the tips of structures). The inventors have found that the period of structures on a material of the invention appear to correspond approximately to the hatch distance of the laser beam used in the laser patterning of the surface of the material. Thus the period of structures on the surface of a material of the invention may provide an indication as to the methods used to manufacture the material. In certain embodiments a material of the invention, such as a material manufactured by a method of the invention, may have a period between structures of approximately 30 to 60 pm, approximately 45 to 55 pm, or approximately 50 pm (and so methods of the invention may make use of cross hatching having a corresponding hatch distance of approximately 30 to 60 pm, approximately 45 to 55 pm, or approximately 50 or 55 pm). An preferred period may be around twice the maximum length of the cells that it is intended should be placed in contact with the surface.

In certain embodiments a material of the invention, such as a material manufactured by a method of the invention, may have a surface roughness that is at least 5 times, at least 10 times, or at least 30 times higher than the surface roughness of an untreated control material. In certain embodiments a material of the invention, such as a material manufactured by a method of the invention, may have a surface roughness, Ra, that is between approximately 4 and 8 pm as measured by MeX (described elsewhere in the specification), or that is between approximately 4 and 15 pm as measured by Wyko (described elsewhere in the specification). Materials of the invention, such as those manufactured by the methods of the invention, such as a material manufactured by a method of the invention, may have a surface area that is at least 50%, 100% or 150% greater than the surface area of an untreated control material.

Material of the invention may be characterised with reference to the skewness of their surface. While almost all manufacturing processes produce materials with surfaces that are positively skewed, in certain embodiments the methods of the invention are able to produce materials of the invention with surfaces that have both positive and negative skewness. Without wishing to be bound by any hypothesis, the inventors believe that this surface topography comprising both positive and negative skews may be of benefit in promoting cell adhesion.

Additionally or alternatively, materials of the invention, such as those produced by the methods of the invention, may be defined with reference to one or more features of the chemical composition of their surfaces. The chemical composition of the surfaces of implant materials is able to influence adsorption of extracellular matrix proteins to biomedical the material, and thus impact upon their interaction with biological cells. Chemical composition may be determined as an absolute value, or with reference to the composition of a control untreated material.

In certain embodiments a material of the invention, such as one made by a method of the invention, may have a surface oxygen content of greater than 30%. In certain embodiments a material of the invention may have a surface oxygen content that is 30% greater than a control untreated material.

In certain embodiments a material of the invention, such as one made by a method of the invention, may have a surface carbon content of less than 8%. In certain embodiments a material of the invention may have a surface carbon content that is 50% lower than a control untreated material.

The materials of the invention are also able to be characterised with reference to their relationships with biological cells with which they come into contact. Such characterisation may be additional or alternative to the other parameters discussed in the present application. The superhydrophilicity of materials of the invention contributes to their ability to promote adhesion of biological cells, such as osteoblasts. Indeed, the materials of the invention are able to promote adhesion of biological cells to a greater extent than implant materials known in the prior art. Without wishing to be bound by any hypothesis, the inventors believe that the promotion of adhesion to materials of the invention arises as a result of increased focal adhesion formation by cells in contact with the materials. This increase in focal adhesion formation may be demonstrated by an increase in number of focal adhesions, or an increase in the area of focal adhesions, or an increase in both the number and the area of focal adhesions, as compared to suitable controls (e.g. materials that have not been treated using a method of the invention). Protocols by which the ability of a material to bind cells can be investigated, as well as protocols by which the levels of cell adhesion on materials of interest can be investigated, are described elsewhere in the specification.

In certain embodiments a material of the invention, such as a material manufactured by a method of the invention, may promote binding of biological cells by at least 50%, or by at least 100%, or by at least 200% as compared to binding of said cells on an untreated control material. The biological cells binding of which is promoted may be osteoblasts. Binding of biological cells may be assessed in vitro as a proxy for activity occurring in vivo.

The invention will now be further described with reference to the accompanying Figures and Tables in which:

Figure 1 represents a schematic view of (a) parallel-scanning; (b) and cross-scanning laser patterns that may be used in the invention;

Figure 2 illustrates scanning electron micrographs showing the surface topography of textured implant materials produced using Ti-6AI-4V: (a) sample LC, laser fluence = 1.68 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, crossed scanning (3 scans in each direction); (b) sample LP, laser fluence = 1.68 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, parallel scanning (6 scans); (c) sample HC, laser fluence = 2.47 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, crossed scanning (3 scans in each direction); (d) sample HP, laser fluence = 2.47 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, parallel scanning (6 scans); Figure 3 illustrates three dimensional images of the surfaces of a material (Ti-6AI-4V) that has undergone parallel-scanning and a material that has undergone cross- scanning using lasers: (a) sample LC, laser fluence = 1.68 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, crossed scanning (3 scans in each direction); (b) sample LP, laser fluence = 1.68 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, parallel scanning (6 scans);

Figure 4 shows the results of surface roughness measurements (panel a) and surface area measurements, measured by Wyko, SPIP and MeX (panel b), conducted on a number of materials;

Figure 5 illustrates the surface skewness in X-direction (panel a) and in Y-direction (panel b) of materials;

Figure 6 shows the results of analysis of surface kurtosis of materials after laser treatment;

Figure 7 shows: (panel a) profile height measurement (Z, μ m); and (panel b) spacing between profile irregularities (d, μ m) in materials that have undergone laser processing;

Figure 8 shows the results of contact angle measurements on materials that have undergone laser processing (Ti-6AI-4V surface after regular contacts with deionised water);

Figure 9 illustrate changes in contact angle measurements from 0° to 132°. Laser treated ΤΊ-6ΑΙ-64 (sample LC created by using laser fluence = 1.68 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, crossed scanning (3 scans in each direction) with time and being exposed to deionised water (a) CA=0 ⁰ (b) CA=2 ⁰ (c) CA=8 ⁰ (d) CA=25 ⁰ (e) CA=78 ° (f) CA=132°;

Figure 10 illustrates the results of XPS analysis of: "a)" a blank Ti-6AI-7V material; and "b)" a material of the invention (HP);

Figure 11 illustrates calibration curves used in the MTT assays described. MTT calibration curve relating number of viable cells and MTT absorbance; (a) cell number range of 0 to 7x10 ⁵ , (b) cell number range of 1.2 to 7x10 ⁵ and the fitted line; Figure 12 compares the percentage of adherent osteoblasts after 22 hours culture on different laser treated surfaces: (a) laser treated (CA = 0 °) and blank (control) ΤΊ-6ΑΙ- 4V surfaces; (b) laser treated (CA = 0 °) and plastic tissue culture (control);

Figure 13 illustrates the results of cell attachment studies (percentage of adherent osteoblasts after 22 hours in culture) conducted on non-superhydrophilic materials on: (a) laser treated (CA > 100 °) and blank (control) Ti-6AI-4V (b) laser treated (CA > 100 °) and blank Ti-6AI-4V compared to plastic tissue culture (control);

Figure 14 sets out micrographs of cells attached to different surfaces. Coloured cells shown on: (a) plastic; (b) blank-1 ; (c) blank-2;

Figure 15 sets out confocal scanning microscopy images of phalloidin-labelled 2T3 osteoblasts on different laser treated Ti-6AI-4V surfaces (Panel "a)" LP; Panel "b)" LC; Panel "c)" HC; Panel "d)" HP) - magnification increases from left to right hand columns;

Figure 16 also shows confocal scanning microscopy images of phalloidin-labelled 2T3 osteoblasts on different laser treated surfaces (Panel "a)" HC; Panel "b)" HP);

Figure 17 shows fluorescence microscopy images of an osteoblast cultured for 22 hours on standard tissue culture plastic: panel "a" illustrates stress fibres, panel "b" illustrates focal contacts, while panel "c" shows the combination of the preceding panels;

Figure 18 shows fluorescence microscopy images of an osteoblast cultured for 22 hours on a "blank" titanium alloy substrate: panel "a" illustrates stress fibres, panel "b" illustrates focal contacts, while panel "c" shows the combination of the preceding panels;

Figure 19 shows a confocal scanning microscopy image of a phalloidin-labelled osteoblast on an "HP" sample (scale bar is 25 pm on all images);

Figure 20 also shows a confocal scanning microscopy image of a phalloidin-labelled osteoblast on an "LP" sample (scale bar is 10 pm on all images); Figure 21 is a confocal scanning microscopy image of a phalloidin-labelled osteoblast grown between two ridges (illustrated with arrows) on a laser treated surface ("LP");

Figure 22 compares deconvolved images of vinculin labelling of single osteoblasts grown on two different laser treated surfaces (Panel a: sample HP; Panel b: sample LP);

Figure 23 compares labelling for vinculin (panels a and c) and for focal contacts (panels b and d) in osteoblasts grown on two different surfaces ("a)" and "b)" - plastic substrate; "c)" and "d)" blank Ti-6AI-4V substrate). Scale bar is 10 pm on all images;

Figure 24 is a confocal microscopy image of an osteoblast labelled with phalloidin. The cell has been cultured on sample "LC". Panel "b)" (scale bar is 10 pm) is an enlargement of the indicated portion of Panel "a)" (scale bar is 50 pm). Cells extend along ridges on the surface of the material, as well as between ridges;

Figure 25 is also a confocal microscopy image of an osteoblast labelled with phalloidin. Panel "a)" (scale bar is 10 pm) is a cell grown on a sample of laser processed material "HP"; while Panel "b)" (scale bar is 25 pm) is a cell grown on a blank substrate. On the laser processed material the cell has grown along a ridge formed on the surface, and has an elongated shape. On the blank substrate the cell has extended in all directions and covers a larger area of the surface;

Figure 26 shows labelling for vinculin (panel a) and for focal contacts (panel b) in an osteoblast grown on a laser treated surface (sample HP);

Figure 27 also shows labelling for vinculin (panel a) and for focal contacts (panel b) in an osteoblast grown on a laser treated surface (sample LP);

Figure 28 shows changing water adhesion over time on a range of laser treated Ti- 6AI-4V surfaces;

Figure 29 shows XPS results comparing carbon peaks in: "a)" a control material; and "b)" a material of the invention (sample HP created by using laser fluence = 2.47 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, parallel scanning - 6 scans); Figure 30 shows XPS results comparing oxygen peaks in: "a)" a control material; and "b)" a material of the invention (sample HP created by using laser fluence = 2.47 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, parallel scanning - 6 scans);

Figure 31 shows XPS results comparing carbon peaks (panel a) and oxygen peaks (panel b) in samples of an aged material of the invention (sample HP created by using laser fluence = 2.47 J/cm ² , frequency = 30 kHz, speed = 20 mm/s, parallel scanning - 6 scans);

Figure 32 compares various surface properties (surface roughness, surface area increase, and cell attachment in panel a; surface skewness, surface kurtosis, and cell attachment in panel b) of a titanium alloy Ti-6AI-4V before and after treatment with the methods of the invention; and

Figure 33 compares cell attachment and the ratio of oxygen:carbon for different laser treated surfaces.

Figure 34: SEM micrograph of laser textured surfaces of 316L stainless steel in nitrogen by using different laser parameters. Percentage of laser power, the hatch distance and the scanning pattern is mentioned for each micrograph. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all samples.

Figure 35: SEM micrograph of laser textured surfaces of pure titanium in nitrogen by using different laser parameters. Percentage of laser power, the hatch distance and the scanning pattern is mentioned for each micrograph. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all samples.

Figure 36: SEM micrograph of laser textured surfaces of Ti-6AI-4V in nitrogen by using different laser parameters. Percentage of laser power, the hatch distance and the scanning pattern is mentioned for each micrograph. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all samples.

Figure 37: SEM micrograph of laser textured surfaces of Ti-6AI-4V in argon by using different laser parameters. Percentage of laser power, the hatch distance and the scanning pattern is mentioned for each micrograph. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all samples. Figure 38: SEM micrograph of laser textured surfaces chosen for further analysis (a) Ti-6AI-4V structured with 70%P, 20 pm hatch, parallel pattern in nitrogen (b) pure Ti structured with 70%P, 20 pm hatch, crossed pattern in nitrogen (c) 316L stainless steel structured with 70%P, 20 pm hatch, crossed pattern in nitrogen (d) 316L stainless steel structured with 70%P, 30 pm hatch, crossed pattern in nitrogen, (e) 316L stainless steel structured with 70%P, 20 pm hatch, crossed pattern in air (f) Ti- 6AI-4V structured with 70%P, 35 pm hatch, parallel pattern in argon. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all samples.

Figure 39: Contact angle measurement (a) untreated stainless steel, contact angle θ=79 ⁰ (b) An example of laser textured stainless steel surface, contact angle θ=0 ⁰

Figure 40: EDX results after laser texturing of stainless steel in nitrogen and air atmospheres. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all textured samples.

Figure 41 : EDX results after laser texturing of Ti-6AI-4V in nitrogen and air atmospheres. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all textured samples.

Figure 42: EDX results after laser texturing of Ti-6AI-4V in argon atmosphere (to produce a comparator implant material). A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all textured samples.

Figure 43: 2T3 osteoblast adhesion on plastic tissue culture (control), untreated (Blank) and laser textured Ti-6AI-4V substrates

Figure 44: 2T3 osteoblast adhesion on plastic tissue culture (control), untreated (Blank) and laser textured Ti substrates

Figure 45: 2T3 osteoblast adhesion on plastic tissue culture (control), untreated (Blank) and laser textured stainless steel substrates

Figure 46: Comparing 2T3 osteoblast adhesion on different laser textured substrates

Figure 47: 2T3 osteoblast adhesion on plastic tissue culture (control), untreated (Blank) and Ti-6AI-4V substrate in different gas atmospheres with variying laser parameters. Laser- argon was treated with 70% power, 35 pm hatch distance, parallel patter, 30 kHz and 20 mm/s. Laser- N2 was treated with 70% power, 20 pm hatch distance, parallel patter, 30 kHz and 20 mm/s. Laser- air was treated with 70% power, 20 pm hatch distance, crossed patter, 30 kHz and 20 mm/s. HA is hydroxyapatite coated Ti-6AI-4V sample. The last column is grit blasted Ti-6AI-4V.

Figure 48: HOb adhesion on plastic tissue culture (control), untreated (Blank) and laser textured Ti-6AI-4V (optimum) substrate

Figure 49: Nd: YV0 ₄ (DPSS) laser was used to texture pin-shape implants (a) the laser system (b) a close-up picture.

Figure 50: Graph comparing the strength of integration of experimental implants implanted in sheep. Increasing strength (shown on the Y-axis) indicates increasing extent of osseointegration between host cells and the implant surface. Implant materials of the invention (manufactured by a method of the invention) exhibited favourable characteristics as compared to control implant materials (using prior art surface treatments such as machine finishing, grit blasting, or hydroxyapatite coating).

STUDY 1

Use of a method of the invention to prepare a material of the invention

Implant material samples of industrial aerospace grade Ti-6AI-4V were cleaned in ethanol in an ultrasonic bath for 5 minutes.

A diode pumped Nd:YV04 laser with a wavelength of 532 nm (in the visible spectrum: green), a pulse duration of 8 ns and a repetition rate of 30 kHz was used to process samples in atmospheric conditions without a shroud gas. The laser beam with a near Gaussian intensity distribution (M2 ~ 1.5) was focused onto the surface of the implant material with a spot diameter of 55 pm. The depth of focus (defined for the purposes of the present specification as the distance either side of the beam waist over which the beam diameter grows by 5%) was calculated to be 952 pm. This depth of focus is sufficiently large to result in a negligible change of the spot size on the flat target throughout the experiment.

Laser fluence was varied between 1.51 J/cm ² and 2.47 J/cm ² . The damage threshold of titanium (which is to say the fluence threshold that makes some sort of modification on the surface of titanium) has previously been reported to be 0.6 J/cm ² .

The laser beam was raster scanned over the surface of targets using a computer controlled galvo-scanning system equipped with a flat field lens. The number of laser scans and the hatch distance (the distance between adjacent laser scans) were varied to study the effect of laser scanning parameters on the textures produced on surface of the implant for each value of the laser fluence. The latter was necessary because the laser repetition rate and the scan velocity multiple passes were required in order that the desired numbers of pulses at each spot on the target surfaces were achieved. Both parallel (laser scanning on X-direction, Figure 1a) and cross-hatched (laser scanning on X and Y directions, Figure 1b) patterns were performed in each case.

Initially four materials of the invention (produced from the same starting material using the above method of the invention) were selected for further surface characterisation and biocompatibility tests. Details of these selected materials of the invention and the methods by which they were prepared are set out in Table 1. The hatch distance of 55 pm was used for all structures. Samples are referred to as LP (Low laser power, Parallel pattern), LC (Low laser power, Crossed pattern), HP (High laser power, Parallel pattern) and HC (High laser power, Crossed pattern).

The number of pulses per spot (npps, = pulse repetition rate x beam diameter / scanning speed) and number of pulses per unit area were calculated as 495 and 163636 / mm ² respectively and were kept constant for all samples. Table 1 shows the laser parameters and the pattern in each case.

Characterization of materials of the invention prepared using a method of the invention

Characterization of surface topography

Surface metrology was analysed by performing scanning electron microscopy on the materials of the invention, and using MeX (Alicona) software as well as Wyko NT1100 optical profiling and SPIP (Scanning Probe Image Processor).

Characterisation of hydrophilicity

Contact angle measurement was performed by using both deionised water and osteblast cell medium in a contact angle and surface tension analyzer (FTA188). Osteblast cell medium comprised alpha MEM (Minimum Essential Medium) supplemented with 10% FCS (Fetal Calf Serum), penicillin and streptomycin, and 2mM glutamine.

Characterisation of surface chemical composition

X-ray photoelectron spectroscopy (XPS) was used to examine the surface chemical compositions of the materials of the invention. XPS was carried out on a Kratos Axis Ultra instrument using monochromated Al Ka radiation. The X-ray source was run at a power of 150 W; the survey scans were acquired with a pass energy of 80 eV; high resolution core-level spectra were acquired at a pass energy of 20 eV. The angle between the X-ray beam and the analyser plane was 60° and photoelectrons were collected normal to the sample surface.

Binding energies were referenced to the C1s level of the adventitious carbon at 285.0 eV. Data processing was undertaken using CASA XPS. Cell culture and cell attachment assay

Cell attachment to the materials of the invention was investigated by assaying attachment of 2T3 mouse osteoblasts to the samples. 2T3 osteoblasts were cultured on the samples and then the cell number was analysed using an MTT assay.

Prior to use, the 2T3 osteoblasts were cultured in 75 cm ² flasks and maintained in an incubator at a temperature of 37°C regulated with 5%C0 ₂ , 95% air, and saturated humidity. aMEM supplemented with 10% foetal calf serum (FCS), penicillin/streptomycin and 2mM glutamine was used as the cell culture medium. Prior to confluence, the cells were sub-cultured by splitting.

Before cell seeding, the samples of the materials of the invention (comprising ΤΊ-6ΑΙ- 4V treated with a method of the invention) were rinsed in 100% ethanol and sterilized in an autoclave (120°C for 20 minutes) and then placed in 24-well plates (Corning, Fisher Scientific). Trypsinized cells were seeded onto the specimens at a concentration of 1x10 ⁵ per well as determined by a cell counter machine (Scharfe system, Casy 1).

The cells were incubated for 22 hours in a water saturated atmosphere of 95% air/5% C0 ₂ on either tissue culture plastic, control samples of Ti-6AI-4V that had not been treated in accordance with the invention (referred to as "blank" in the accompanying results) or the materials of the invention. Following incubation cell attachment was quantified using a MTT assay.

Cell behaviour assay and immunofluorescence microscopy

To perform microscopy samples were cleaned and sterilized as for the MTT assay. 1x10 ⁵ cells per well were seeded in a 24-well plate. After 22 hours culture samples were moved to a new 24-well plate and prepared for microscopy at room temperature. Cells were briefly washed with complete Hanks' solution (Gibco, UK) at 37 °C and fixed in 2% (w/v) paraformaldehyde (Sigma, UK) in PBS for 15 minutes. Cells were then permeabilized for 15 minutes in 0.5% Triton X-100 (Sigma, UK) in PBS, followed by incubation in 0.02M glycine (Sigma, UK) in phosphate buffered saline (PBS) for 15 minutes. Finally in order to block non-specific binding, cells were incubated in 5% goat serum with 1% bovine serum albumen (BSA) in PBS for 15 minutes. To detect cytoskeletal actin filaments, cells were incubated with 0.5 pg/ml phalloidin (Alexa Fluor 488 phalloidin, Invitrogen, UK) for 45 minutes. Samples were mounted onto glass coverslips and imaged by using a Nikon C1 confocal on an upright 90i microscope with a 60x/ 1.40 Plan Apo objective and 3x confocal zoom. Due to the complexity of structures, actin stress fibres were not clearly observed. Therefore to get a closer look at how cells attach to each substrate, microscopy was repeated with a lower cell density of 1x10 ⁴ cells per well. Double staining was performed by using anti-vinculin antibody (mouse a-vinculin hVIN-1 , Sigma) (1 : 100) followed by Alexa Fluor-conjugated goat anti-mouse antibody (1 :200) (Alexa Fluor 594, goat anti-mouse IgG (H+L), Invitrogen, USA) for localization of focal contacts.

All antibody incubations were performed in a humidified environment for 45 minutes at room temperature. Before mounting for microscopic observation, cells were stained with 0.5 Mg/ml phalloidin to label the actin cytoskeleton. Between each incubation step, samples were washed twice in PBS. Samples were mounted onto glass coverslips and imaged using a widefield (Olympus BX51) microscope, a confocal laser scanning microscope (Leica TCS SP5 AOBS) and an inverted microscope (IX70; Olympus) controlled by a Deltavision system (Applied Precision). Cells were also cultured and labelled as above on a plastic tissue culture surface (cell culture dish, Corning, obtained from Fisher Scientific, UK).

Images on plastic and blank metal substrate were collected on a Olympus BX51 upright microscope using a 60x/0.65-1.25 Plan Fin objective and captured using a Coolsnap HQ camera (Photometries) through MetaVue Software (Molecular Devices).

Due to the complex surface structure of the samples there was the possibility of cells attaching in irregular shapes. To investigate this, 1x10 ⁵ cells were seeded in each well of a 24-well plate containing laser treated samples followed by labelling with phalloidin (as explained before).

Images were collected on a Leica TCS SP5 AOBS upright confocal using a 63x/1.4 HCX PL Apo objective and 3x confocal zoom.

Confocal microscopy confirmed the necessity of a system capable of imaging the focal contacts on different heights (Z-slices) on laser treated samples. For this purpose a Delta Vision RT (Applied Precision) restoration microscope using a 60x/ 1.42 Plan Apo objective and the Sedat filter set (Chroma 89000). The system imaged each sample on different Z4evels (slices were 0.5 μηι apart) and images on every slice were analysed. A density of 1x10 ⁴ cells were seeded in each well of a 24-well plate containing laser treated samples followed by labelling with phalloidin and anti- vinculin.

Results

Analysis of surface topography

A method of the invention in which the surface Ti-6AI-4V substrates was processed using a 532 nm Nd:YV04 laser beam produced materials of the invention, the surfaces of which (illustrating the structures present as part of the surface topography) can be seen in the scanning electron micrographs of Figure 2. The height of the structures formed was in the range of 15 to 35 μηη and the structures included both regular and irregular features. The structures were formed using a method in which 495 pulses were applied per spot. The period of structures (calculated as the average distance between tips of structures) was measured as ~ 50 μητ This corresponded closely to the 55 μηη beam hatch distance used in the method of the invention. The period of structures was almost constant between all four materials of the invention produced, since the same laser hatch distance and the same number of pulses per spot were used in each manufacturing method.

While the different materials of the invention, produced using different methods of the invention, shared many properties in common, differences between the materials (consistent with the differences in the methods by which they were manufactured) were observed.

SEM micrographs (Figure 2) show that the lower laser fluence created smaller structures on the surface of the material. Parallel scans created an apparent line pattern on the surface, while the cross-hatched scan resulted in a mesh pattern.

Figure 3 shows three dimensional images of two surface types (obtained by MeX). The texture on a parallel scanned sample (Figure 3b) appeared more directionalised whereas the crossed scanning created a less regular texture (Figure 3a). Surface roughness measurements were obtained by using both Wyko and MeX, and the result of these can be seen in Figure 4. Using both systems, the general trend was the same although Wyko generated more noise especially on crossed scanned samples as a result of its data collection method.

The total surface area created on each sample was determined by calculating Developed Interfacial Area Ratio (Sdr). Sdr was obtained by using Wyko, SPIP and MeX, and it can be seen that the methods of the invention result in an increase of surface area. All Sdr measurements (Figure 4) follow the same pattern. Sdr graph also followed the same trend as surface roughness, showing a higher surface area increase on crossed scans compared to parallel ones and also a rise on Sdr in materials produced by a method in which a higher laser fluence was used. The largest area on the substrate surface was created by using higher laser power in crossed scans (sample HC).

Skewness (Rsk) is another parameter that describes the shape of a profile by measuring the symmetry of the deviation of a profile about its mean line. Almost all common machining and fabrication methods produce positively skewed surfaces. Skewness is defined as:

Where y(x) is the surface profile sampled by the set of n points yi.

Figure 5 shows the result of skewness analysis on materials of the invention in both X and Y directions. The results reveal that methods using a higher laser fluence and a parallel scanning (HP) created the only surface which showed a combination of positive and negative skewness (Figure 5). This surface also has the minimum Rsk values in both directions.

The sharpness of the profile peaks is measured as the surface kurtosis (Sku) which is defined as:

1 ^m " r ί w

mnS _q Figure 6 shows the result of kurtosis analysis on samples of materials of the invention. It can be seen that the sharpness of peaks is decreased in methods where the laser power is increased. As a result the sample of a material of the invention produced using a method employing a high power laser and parallel scanning patterning has the lowest Sku value.

Figure 7 (a) shows the mean peak to valley height of a profile in X direction (against laser scanning) on each sample. Figure 7 shows mean spacing of profile irregularities on X direction. Laser hatch distance was 55 pm in all cases. Using different powers and multiple scans changed the spacing between features on each sample

Analysis of hydrophilicity

Wettability measurements indicated that the methods of the invention dramatically decreased the contact angle of the materials the invention produced, as compared to untreated control samples of Ti-6AI-4v. Using both deionised water and osteblast cell medium on samples immediately after laser irradiation, revealed no contact angle (almost zero degrees) in all materials of the invention produced by the methods of the invention. This means that a droplet of these liquids immediately spread and covered the whole surface of the materials of the invention. These contact angle measurement results indicate that the surfaces of the materials of the invention are superhydrophilic.

Superhydrophilicity could be maintained on the surface for a few weeks (up to 6 weeks) when the substrate was kept in atmospheric conditions. If materials of the invention came into regular contact with deionised water (for example every other day) and were kept in soft plastic bags (resealable polyethylene plastic bag, Fisher Scientific, UK). Without wishing to be bound by any hypothesis, the inventors believe that this loss of superhydrophilicity may be caused by contamination with hydrocarbons present in the material of the plastic bags.

Contact angle started to increase after a week. Figure 8 shows the contact angle measurement changes over time for samples kept in such conditions. Contact angle value on a blank sample raised slightly (from 70 ⁰ to 76 °) following regular contact with deionised water over 21 days. Table 2 shows examples of contact angle measurements on samples LC, LP, HC and HP.

The changes in contact angle measurement are illustrated in Figure 9, in which the panels illustrate contact angle values increasing from 0° to 132°.

XPS was also carried out on freshly laser treated (superhydrophilic) and aged (high contact angle) samples with the same type of structures, result of which are presented in Figure 10.

Surface chemistry

The results revealed the presence of Ti, Al, V as well as O, C and N on all Ti-6AI-4V samples before and after laser treatment in a method of the invention. Results of the survey scan showed that laser irradiation could eliminate some impurities on the substrate. Traces of Fe, Zn, Ca, CI and K appeared on the blank untreated control sample, which were removed after laser irradiation in a method in accordance with the invention. Tables 3 and 4 present the atomic percentage of elements before and after laser irradiation, showing that using high laser fluence reduced the carbon contamination and increased the oxygen concentration.

Cell attachment

Cell attachment was investigated by assaying 2T3 osteoblast attachment on the materials of the invention and untreated control samples. 2T3 osteoblasts were cultured on the materials of the invention and untreated control samples and then the cell number was analysed using MTT assay.

A calibration curve was first obtained which relates the MTT values to the number of attached cells. Cultures were maintained until near confluence. Trypsinized cells were seeded in two 24-well plate (Corning, Fisher Scientific) at different densities. Both plates were seeded with the same cell densities. Incubation was carried out in a humidified atmosphere of 95% air and 5% C0 ₂ at 37°C for 22 hours.

Cells in one plate were trypsinized and counted (Scharfe system, Casy 1 machine) to find the total number of cells present after 22 hours. Cells in the second plate were incubated with the MTT reagent for 2 hours and finally acidified isopropanol (isopropanol containing 0.04M HCI) was added to lyse the cells and solubilize the coloured crystals.

The absorbance of each well was then measured by using a plate reader (Dynex Technologies MRXII, using "revelation" software) at a wavelength of 570 nm. The number of viable cells is directly proportional to the amount of colour produced. This measurement determined the relationship between 2T3 osteoblast cell number and absorbance (calibration curve, Figure 1 1). A linear regression was used to relate the number of cells to an absorbance value in the range of cell numbers that was used in this study.

The MTT assay was performed on materials of the invention prepared as described above, and untreated control samples, to compare the number of adherent cells. After cleaning and sterilizing each sample was located in a well in a 24-well plate. To reduce the number of cells attaching to the plastic surface around each sample (samples were square and the wells circular), the wells which would hold metal samples were pre-treated with 3% BSA in PBS over night before performing the assay. This method was first tested on plastic wells and visual inspection showed that it effectively prevented the cell attachment.

2T3 osteoblasts were cultured on plastic and blank Ti-6AI-4V (control) as well as laser treated materials of the invention. Equal numbers of cells (5x10 ⁵ ) were seeded on the sterilized samples and plastic wells. Samples were incubated at 37 °C for 22 hours. After that samples were transferred to a new 24-well plate to enable accurate counting of the cells attached to metal surface, followed by adding acidified isopropanol. The final solution was mixed thoroughly to provide a homogenous colour and was read using a plate reader at a wavelength of 570 nm. Finally the number of viable cells on each surface was calculated by using the calibration curve (Figure 11).

Data on cell attachment are presented in Figure 12. Cell numbers were first normalised against the plastic tissue culture surface since this is a surface optimised for cell adhesion. Figure 12 compares cell attachment on blank Ti-6AI-4V and superhydrophilic laser treated materials of the invention. Samples were assayed immediately after laser treatment to ensure that contact angle was zero. Results show that Nd:YV0 ₄ laser treatment of Ti-6AI-4V in methods of the invention (producing materials of the invention) increased cell attachment dramatically compared to untreated metal. The highest cell attachment was observed on sample HP (created with high laser fluence, parallel scanning). On the other hand, sample LC (created with low laser fluence, crossed scanning) showed the lowest number of cells among laser treated substrates. Statistical analysis indicated that all four types of samples were statistically different from an untreated surface, illustrating that each of these implant materials that had undergone laser processing provided advantages (in terms of cell adhesion) as compared to untreated materials.

Figure 12 (b) compares the number of adherent cells between a standard tissue culture plastic surface (24-well plate from Corning, Fisher Scientific) and materials of the invention or untreated metal substrates. Result revealed that cell attachment on both HP and HC samples were higher than a plastic tissue culture surface with sample HP being significantly different from the plastic. Sample HC was found statistically equal to the plastic substrate. Although improved as compared to untreated materials (as discussed above) samples treated by low laser fluence (LH and LP) were found to be statistically lower in cell attachment when compared to the plastic surface (which is known to be well suited cell culture), as were the untreated blank metal.

Cell attachment was also observed on laser treated surfaces with a higher contact angle (non-superhydrophilic) (Figure 13). For this purpose, samples were kept in plastic bags and had a number of incidences of contact with deionised water. Wettability was measured to ensure a contact angle formed on the surface (CA>100°). Results revealed that laser treatment improved cell attachment compared to a blank substrate. However, the difference was not statistically significant when using a low fluence laser beam (LH and LP). This was unlike the result found on superhydrophilic surfaces where cell adherence appeared statistically significantly different on laser textured and blank samples for all four types of laser treatments (Figure 12).

Comparing Ti-6AI-4V and plastic tissue culture surfaces showed sample HP having the highest cell adherence and therefore not different from a plastic surface. The rest of Ti-6AI-4V substrates (HC, LC, LP and blank) revealed significantly lower cell attachment compared to a plastic surface (Figure 13 b). This was also dissimilar from cell attachment on zero contact angle (Figure 12 b). While performing the MTT assay, it was also observed that osteoblasts did not attach uniformly on blank samples. Figure 14 shows osteoblasts completely covering a plastic well and partially covering the blank samples. Laser treated samples could not be imaged as the natural colour of the surface is dark and therefore the blue coloured cells were not visible. However laser treated samples all revealed a compact uniform layer of bone cells attached to the surface. It will be appreciated that this indicates that the materials of the invention, but not the blanks, are able to stimulate a uniform distribution and attachment of the initial bone cells which improves the function of the implant.

Cell behaviour

The result of confocal scanning microscopy can be seen in Figure 15. Sample LP (fig 15a) (previously shown the lowest cell attachment by MTT) showed the lowest number of cells followed by sample LC (fig 15b), HC (fig 15c) and finally sample HP (fig 15d) showing the highest number of cells. It can also be seen that cells on sample HP, strongly modified their shape and location according to the underlying structures and therefore expanded along the laser path (grooves) (Figure 15d). Panels a and b of Figure 16 shows phalloidin labelling of 2T3 osteoblasts and the underlying structures on samples HC and HP.

Figure 17 and Figure 18 show the result of double staining of 2T3 osteoblasts with both phalloidin and anti-vinculin. This was performed to provide detailed information of how cells attached to different substrates by labelling both actin stress fibres in green (Figure 17a and Figure 18a) and focal contacts in red (Figure 17b and Figure18b ).

To find out if the cells were attaching 3-dimensionally into the laser created structures another confocal microscopy was performed. Figure 19 shows an osteoblast at different depths of a HP sample. The distance between each step is 1 pm and the cell was observed over 26 pm depth. Figure 19 shows the first 8 pm down the structures tip. The size of scale bar is 25 pm in all pictures.

Confocal microscopy also showed the interesting result that cells not only attach along the walls of ridges but in some cases they were able to form a "bridge" between two ridges. To show this a single cell was viewed over 68 pm depth in steps of 1.84 pm on a sample LP. Figure 20 shows the top 9 images. This can be seen in a closer look in Figure 21 which shows one of the z slides form Figure 20. Ridges on the structure are indicated by arrows.

Confocal microscopy confirmed the necessity of a system capable of imaging the focal contacts on different heights (Z-slices) on laser treated samples. For this purpose a Delta Vision RT (Applied Precision) restoration microscope with a 60x/ 1.42 Plan Apo objective and the Sedat filter set (Chroma 89000) was used. The images were collected using a Coolsnap HQ (Photometries) camera with a Z optical spacing of 0.5μιη. Raw images were then deconvolved using the Softworx software and maximum intensity projections of these deconvolved images are shown in the results.

Figure 22 shows the result of vinculin labelling (maximum intensity projection) images of a single osteoblast on two different laser textures.

Comparing the number of focal contacts and their areas between cells grown on plastic and cells grown on blank metal provides important information about the cell attachment on each substrate. Ten individual cells were analysed on both plastic and blank substrates, result of which can be seen in Table 5.

The results indicated that although the total cell area for each individual cell on plastic and blank substrates was not statistically different, the number and the area of focal contacts were dramatically reduced on the blank (sample) compared to the plastic (substrate). The minimum focal contacts that cells developed in 22 hours culture appeared the same on both substrates but the largest focal contact area were again different. Thus the ratio of total focal contact area to the total cell area was considerably greater on plastic than on the blank substrate.

Based on above findings it could be suggested that one of the reasons for a dramatic decrease in cell attachment to a blank Ti-6AI-4V compared to standard plastic tissue culture surface is the number and area of focal contacts in each cell is reduced. Although the reason is still unclear, it was found that osteoblasts were unable to develop focal contacts as well as they do on a plastic substrate (Table 5).

The result of a second confocal microscopy on the laser treated sample with highest cell attachment (sample HP) is presented in Figure 19. It could be observed that cells extended along the ridges of the structures and formed an elongated shape in respond to the underlying structure. Here the cell was attached more towards the tip of the structure rather than of the bottom of the valley. The deepest distance downwards that the cell extended to, was around 19 pm. This could be as a result of a smaller gap in the depth of the structure as well as the possibility of not enough nutrition available in that depth.

Other images showed that cells not only attach along the walls of ridges but in some cases they were able to form a "bridge" between two ridges (Figure 20). More detailed pictures supported this effect. Figure 24 shows how osteoblasts attach both to the walls of a structure (Figure 24a and Figure 25a) and across two walls (Figure 24b) creating a "bridge". Cells were extended on blank metal as there were no irregularities on the substratum to guide or restrict their shape (Figure 25b).

To be able to identify the focal contact areas developed by cells on laser structured samples a Delta Vision RT (Applied Precision) restoration microscope was used, enabling imaging of focal contacts at different heights (Z-slices) on the structures. These images were then deconvolved. The same method of "Threshold" adjustment was applied on every slice of each image by using Image J software to enable analysis of focal contacts.

Figure 26 shows the result of vinculin labelling of an osteoblast on sample HP and the mask of all focal contacts formed on every slice, which is a merged picture of all Z-level slices. Figure 27 shows the result of vinculin labelling on sample LP and the merged mask.

Table 6 compares the two cells described above. The cell on sample HP extended to 9 pm in the Z direction and formed focal contacts with a total area of 71 pm ² . However, the one observed on sample LP was just extended to 5.5 pm in the Z direction and formed focal contacts with a total area of 10 pm ² .

The result of focal contact comparison on sample HP and LP which previously showed the highest and lowest cell attachment respectively is presented in Table 7. The analysis is based on the average data from ten individual cells on each sample.

Focal contact analysis on samples HP and LP showed that cell morphology and attachment were dramatically different between the two structures. The result revealed that on sample HP (the sample with highest cell attachment observed by MTT) a single osteoblast on average was extended to 16 slices in the Z direction and formed focal contacts in 14 of those slices. Therefore on this type of structure, cells formed focal contacts on nearly every slice analysed on Z level. However, on sample LP (the sample with lowest cell attachment observed by MTT) the total number of slices a cell could extended to was decreased to 10 and the number of slices with a focal contacts decreased even further to and average of 3 slices, showing that focal contacts formed in less than a third of Z-slices.

On a HP substrate, osteoblasts were extended to a larger area both on the X-Y plane (Total cell area projected on horizontal plane, Table 7) and in the Z-direction (Z dimension, Table 7) compared to a LP substrate. All measurements between the two structures are statically different.

Discussion

The first step of cell to surface interactions in an anchorage dependent cell is "cell attachment". Focal contacts are one of the means by which a cell attaches to the underlying substratum. One way to test the cell attachment is by measuring the number and the area of focal contacts. Considering that the bond density is constant in focal contacts and that focal contacts are the only significant areas of cell attachment, then the area of focal contacts can be used as an indicator of the strength of cell adhesion.

Biocompatibility results confirmed that Nd:YV0 ₄ laser surface texturing of Ti-6AI-4V samples encouraged osteoblast attachment. It was also revealed that using superhydrophilic substrates improved cell attachment. This showed that the surface composition also plays an important role in cell adherence. Although the topography was the same on both zero and non-zero contact angle surfaces, superhydrophilicity increased cell attachment to above that on standard tissue culture plastic.

This can be explained by the topography and the chemical compositions of different structures. Analysing the surface structures on different samples revealed sample HP having the second highest surface roughness and surface area increase (higher than LP and LC and lower than HC ) among all four analysed structures. Studying the surface skewness showed that this type of structure obtained the lowest Rsk value, making it a balanced surface containing both peaks and valleys. It also showed that sample HP was the only type of structure with both positive and negative skewness (in X and Y direction respectively). Surface kurtosis analysis revealed sample HP with the lowest Sku value among all surfaces, showing that features contained the lowest "sharpness".

Figure 32 show that cell attachment follows the same trend as roughness and area increase except for sample HC and HP where the symmetry of the texture and sharpness of the peaks came into account and resulted in sample HP obtaining the highest cell attachment.

From these result, the inventors suggest that cell attachment is promoted by increasing the surface area of a material of the invention (as long as the following specification is maintained: period of structures around 50 pm, depth of structures does not increase 30 urn, roughness between 5 to 8 pm, a balanced profile containing both peaks and valleys and avoiding sharp features - by maintaining surface kurtosis less than 2).

Surface composition analysis (XRD, EDX) showed the increased amount of oxygen and oxide compounds on the Ti-6AI-4V samples after laser irradiation, which caused a dramatic increase in surface energy. It was also revealed that the carbon contamination reduced dramatically after laser treatment.

Previous studies reported that cells favour a higher surface oxygen level and a higher surface energy. It is important to note that all four types of laser created samples (LC, LP, HC and HP) had a zero contact angle and therefore the same wettability characteristic. However, cell attachment was significantly different between them. This result indicated that, there are a number of elements capable of influencing biocompatibility.

One of these parameters is the surface topography (which was discussed above) and the other is chemical composition of the surface. Figure 33 indicates that cell attachment is closely associated with the oxygen to carbon ratio on different samples. Using a higher laser fluence increased the amount of oxygen and decreased the carbon contamination, resulting in a higher cell attachment. Results of this study indicated that higher amount of oxygen on the surface, lower carbon contamination, higher surface area and finally desired topography features on the surface which all were created in one single laser processing could increase the cell attachment by 154% compared to untreated sample and by 12% compared to tissue culture plastic.

It is interesting to question how cell attachment on some laser treated surfaces could exceed that on the plastic substrate, because cells can cover 100% of the plastic surface. This probably does not indicate that laser-treated titanium is a more adhesive surface than tissue culture plastic. Rather it could be because the total surface area of the titanium was increased after laser surface structuring. Since the cells were able to extend down into the structure they had more surface area for adhesion and so more cells were able to adhere. It was also observed later (Microscopy results) that cells alter their shape and adapt an elongated form on laser created structures while they extend fully on a plastic substrate as there were no features on the surface.

To find out the effect of wettability changes on cell attachment which occurred when samples aged, MTT was performed on both freshly made (zero contact angle, superhydrophilic) and aged (high contact angle) laser textures. Results indicated that cell attachment decreased as a result of contact angle increase and the subsequent drop in the wettability property which is in agreement with previous studies.

Table 8 compares untreated, freshly laser treated (HP-fresh) and aged laser treated (HP-aged) samples. It shows that laser treatment increased the oxygen to carbon ratio on the surface by 8% and increased the adhesion tension by 66% compared to untreated surface. This chemical change as well as the new topography, resulted in decreasing the contact angle dramatically (from 70 ° to 0 °) and finally increasing the cell attachment by 154% compared to the untreated surface. If a freshly laser treated sample (HP-fresh) and an aged laser treated (HP-aged) sample were compared when the topography is the same, therefore the only parameter affecting the cell adhesion is chemical composition. In this case decreasing oxygen to carbon ratio by 47% resulted in a significant increase in the contact angle (from 0° to 135°), a significant decrease in adhesion tension (from 72.8 to -50 mJ/m2) and therefore 28% decrease in cell attachment. The decrease in cell attachment in this case was as a result of the chemical composition (and the subsequent contact angle change) only.

Overall, the results showed that when comparing different laser treatments, a 22% difference in oxygen to carbon ratio between the samples, did not change the contact angle (θ= 0 ° for all sample) but did change the cell attachment. However, a 47% difference in oxygen to carbon ratio between the fresh and aged laser treated samples (sample HP) resulted in a significant increase in contact angle (decrease in wettability) and reduction in cell attachment (Table 9).

Cell shape and morphology have been shown to be noticeably different on different materials. Variation in cell morphology can be observed by light microscopy assisted with actin and vinculin stains. Early studies also revealed a strong dependence between the cell adhesion/proliferation and the substrate chemistry. Morphological changes then can be analysed and quantified by performing image analysis that report changes in cell shape and area and focal contact areas.

Result of this study showed that 2T3 osteoblasts on laser textured Ti-6AI-4V samples in accordance with the invention adopted different shapes according to the topography of underlying substratum by extending along laser path especially on sample HP (Figure 15d) which is in agreement with previous studies. This effect can be seen more clearly in Figure 15b, where cells and the underlying structure are both captured in a confocal microscope image. Higher magnifications in Figure 15c and Figure 15d (sample HC and HP) also showed both focused and de-focused areas, indicating that cells were extending into 3-dimensinal features of the structures.

As explained in the introduction, assuming that focal contacts are the only areas of significant cell attachment then the focal contact analysis can be used to identify the cell adhesion strength as it has been used in previous studies.

Double staining of 2T3 osteoblast by using phalloidin and vinculin provided detailed information of stress fibres and focal contact areas (Figure 17and Figure 18). It was also observed that both actin and vinculin staining were concentrated on focal contacts (high colour intensities, green and red in Figure 17 and Figure 18). Image analysis was then performed by comparing cell morphology on a plastic substrate and a blank sample by using WCIF Image J 1.37c software. To do this, each image was spatially calibrated after which a "Threshold" adjustment was applied on the vinculin labelled (red) image to restrict the analysis to focal contacts. This allows the software to select the higher colour intensity areas (focal contacts). The software was directed to distinguish these areas, measure them and create a mask (Figure 23).

The Experimental Results discussed in this section illustrate the effectiveness of methods of the invention, and also beneficial properties of materials of the invention. Some of these findings suggest that the use of lasers with a fluence of greater than 1.75 J/cm ² provides advantages in the methods of the invention.

STUDY 2

Abstract

The osseointegration of implants is directly related to the early interactions between osteoblastic cells and the implant surface. In this study the behaviour of 2T3 osteoblasts was compared on the following metal implant material surfaces before and after laser texturing:

• 316L stainless steel,

• pure titanium and

• Ti-6AI-4V.

These implant materials underwent laser processing in a nitrogen atmosphere (methods and materials of the invention), and in argon shroud gas (comparator methods and materials). Scanning electron microscopy showed distinct micro/nano topographies. Contact angle of deionised water on all samples was measured immediately after laser texturing and then over time.

EDX was performed to reveal the changes of surface chemistry before and after laser texturing in different gas atmospheres.

The optimum sample (sample with the highest cell attachment) was then compared to hydroxylapatite coated and grit blasted substrates. The optimum sample was then further tested with human osteoblast cells to verify that mouse and human osteoblasts both exhibited improved attachment to the implant materials of the invention.

Finally the optimum texture was transferred on pin-shaped implants which were implanted in sheep for 3 months. The results of a comparison between untreated and optimum laser treated samples 12 weeks post implantation is briefly described, and illustrates the increased strength (resulting from improved osseointegration) associated with in vivo integration of implant materials of the invention. Introduction

It is well-known that the surface topography and chemistry have profound impact on cell adhesion and growth. As described in the previous example, the inventors have created novel surfaces on titanium alloy (medical grade) which resulted in significant improvement of osteoblast cell adhesion indicating enhanced biocompatibility of osteoimplants.

Laser surface engineering utilizes the high power density available from focused and localized laser sources to modify the material on and near the surface. Depending on the material and process parameters, it involves modification of microstructure, grain refinement, phase transformations, alloying and mixing of multiple materials, and formation of composite system on the surface without affecting the bulk material. Laser surface modification methods are advantageous as they create a surface with a high degree of cleanliness and free from contaminations. Laser surface structuring, increases the surface area and surface roughness. It is capable of generating a contaminant free surface resulted from the evaporation of the original surface layer followed by fast re-solidification. A number of researchers studied laser surface texturing but there are still great potentials associated with this method due to its capability of creating variety of features with different shapes, sizes and depths. Different laser parameters can generate regular or irregular surface textures with different geometries. The process modifies both the surface topography and surface chemistry although it could be directed in a way to minimize one effect which means being capable of generating unique geometries with specific chemical compositions.

This study illustrates that osteoblast attachment may be even further improved by using different substrates, changing the shroud gas while texturing the surface and modifying the storage conditions after texturing.

A range of laser textured surfaces were created on different substrates

Using a Nd: YV0 ₄ (DPSS) laser and varying laser parameters, a number of different textures were created on the surface of three different substrates, 316L stainless steel, pure titanium and titanium alloy (Ti-6AI-4V). The laser beam was raster scanned over the surface of targets using a computer controlled galvo-scanning system equipped with a flat field lens. Both parallel (laser scanning on X-direction, Figure 1a) and crossed (laser scanning on X and Y directions, Figure 1 b) patterns were performed. Texturing was also performed in different gas atmospheres (nitrogen in accordance with the invention, and argon for comparator purposes) for various controlled surface chemistries. Representative examples are shown in Figure 34, Figure 35, Figure 36 and 37. Among these structures a few were selected for contact angle measurement, chemistry analysis and the osteoblast attachment. The selection was based on the success of previous textures in encouraging osteoblast attachment.

Table 10 shows the percentage of laser power used and the resulting fluence. Contact angle measurement

Contact angle is an indication of the surface wettability which is directly related to the biocompatibility of the surface. All surfaces created by the methods of the invention (or the comparator methods conducted in an argon atmosphere) are superhydrophilic having a zero contact angle (deionised water) as shown in Figure 39. However the contact angle increased with time depending on the storage conditions.

Table 1 1 shows the changes in contact angle of deionised water on different samples when samples were stored in air. In this case, some samples stayed superhydrophilic up to 15 days. Table 12 shows the contact angle of a particular laser textured (in nitrogen) Ti-6AI-4V when stored in aluminium foil. It shows minor increases in contact angle (can still be considered zero) after three and half months, indicating a better storage method. In case of texturing in argon, although the initial surface was superhydrophilic but the contact angle increased rapidly from the second day after texturing. The contact angle was also measured on hydroxyapatite coated and grit blasted Ti-6AI-4V samples (Table 13).

Surface chemistry analysis

Laser texturing modifies the surface chemistry which has a profound effect on cell adhesion. EDX was performed to indicate the changes in surface chemistry after laser texturing in different gas atmospheres on 316L stainless steel and Ti-6AI-4V (Figure 39, Figure 40 and 41 and Table 14, Table 15 and 16).

Osteoblast attachment was tested on laser textured surfaces

Laser textured surfaces created above were screened for optimum cell-surface interactions by growing 2T3 osteoblsts on the substrate as can be seen in Figure 43 to Figure 47. The optimum texture with highest cell attachment (laser textured Ti-6AI- 4V in nitrogen - a material of the invention) was then tested against grit blasted and hydroxyapatite coated Ti-6AI-4V samples and Ti-6AI-4V textured in argon atmosphere (Figure 46)

Human osteoblast attachment was tested on optimum laser textured surface

The optimum texture (Ti-6AI-4V textured with 70% laser power, 20 μιη hatch distance, parallel patter, 30 kHz frequency and 20 mm/s scanning speed) was tested for human osteoblast (HOb) attachment by means of a MTT test (Figure 48). The percentage of cells attached to the textured sample and the blank sample are significantly different (PO.0205).

The optimum texture was translated on pin-shaped Ti-6AI-4V implants

The final step was translating the optimum texture (Table 12) on pin-shaped Ti-6AI- 4V implants. To do this a combination of a rotation stage (to rotate the sample) and a X-Y stage (to move the sample along its length) was used to direct the laser beam (Figure 49b). The texture was successfully created on pins which were implanted in sheep for a three-month follow up including a torque test and histology to find out the bone attachment to implants.

The results of this study are shown in Figure 49, which compares the strength of integration of experimental implants surgically placed in sheep. Increasing strength indicates increasing extent of osseointegration between host cells and the implant surface. Implant materials of the invention (manufactured by a method of the invention) exhibited favourable characteristics as compared to control implant materials (using prior art surface treatments such as machine finishing, grit blasting, or hydroxyapatite coating).

Summary

This project illustrated the ability to enhance osteoblast attachment by using different substrates and changing the shroud gas while texturing the surface and modifying the storage conditions after texturing. 316L stainless steel, pure titanium and titanium alloy (Ti-6AI-4V) were textured by using a Nd: YV0 ₄ (DPSS) laser in nitrogen, argon and air atmospheres. Textured samples were stored in air and in aluminium foil to test the effect of the storage method on the surface contact angle and therefore the surface biocompatibility. The results indicated that methods of the invention in which a Ti-6AI-4V substrate underwent laser texturing (70% laser power, 20 pm hatch distance, parallel patter, 30 kHz and 20 mm/s) in nitrogen atmosphere created a surface with highest 2T3 osteoblast attachment. Cell attachment on this sample was higher than that of grit blasted and hydroxyapatite coated Ti-6AI-4V. Human osteoblast attachment was tested on this optimum sample (an implant material of the invention, manufactured by a method of the invention) and was significantly higher compared to a blank sample.

The optimum sample was translated onto pin-shaped implants. The result of a three- month implantation of these implant materials of the invention showed that they exhibited favourable osseointegration characteristics in vivo when compared to control materials representative of the prior art.

Table 1 : Laser parameters and pattern

Table 2: Contact angle measurement of different laser created structures after two months being kept in atmospheric conditions with a few contacts with deionised water

Table 3: XPS analysis, atomic percentage of Ti-6AI-4V surface layer before and after laser irradiation and contact angle of each sample

Blank LC LP HC HP

C (%) 44.06 45.91 45.4 40.07 40.55

N (%) 1.89 0.58 0.66 0.52 0.93

Ti (%) 7.31 12.16 1 1.89 12.6 12.73

V (%) 0.36 0.51 0.6 0.88 0.69

O (%) 40.72 37.5 38.12 41.07 40.69

Al (%) 2.56 3.34 3.33 4.86 4.41

Traces of other 3.1 0 0 0 0

elements (%)

Contact angle 70 0 0 0 0

(Degree) Table 4: XPS analysis, atomic percentage of Ti-6AI-4V surface layer on blank, superhydrophilic (HP-fresh) and high contact angle (HP-aged) samples

Table 5: Focal contact comparison of ten osteoblasts on blank Ti-6AI-4V and plastic substrates after 22 hours in culture

Table 6: Focal contact comparison of two individual cells on samples HP and LP

Table 7: Focal contact comparison of ten osteoblasts on laser treated substrates after 22 hours in culture

Total Number Total area Total cell area z dimension (pm) number of of slices of focal projected on

slices with focal contacts horizontal

contacts (pm ² ) plane (pm ² )

HP 16 14 82.5 799.1 8.2

LP 10 3 6 554.3 5.2

P value 6.4x10 ^"b 5.3x10-5 6.6x10-12 0.02 6.4x10 ^"b Table 8: Comparing untreated, freshly laser treated and aged laser treated Ti-6AI-

4V samples

Table 9: Cell attachment comparison between different laser textures (fresh) and between fresh and aged laser textures

Table 10: Nd: YV0 ₄ Laser power percentage and the resulting fluence

Nd: YV0 Laser power percentage Fluence (J/cm ² )

60% 0.674

65% 1.39

70% 2.21

100% 9.17 Table 11 : Contact angle before and after laser texturing on different substrates when samples stored in air. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all textured samples.

Table 12: Contact angle of one particular Ti-6AI-4V textured sample (optimum sample) in nitrogen when stored aluminium foil.

Table 13: Contact angle of hydroxyapatite coated and grit blasted Ti-6AI-4V

CA (degree)

Hydroxyapatite coated Ti64 38

Grit blasted ΤΊ64 ^iiiiiiiiiiiiiiiii^

Table 14: Atomic percentage of elements based on EDX results before and after laser texturing of stainless steel in nitrogen and air atmospheres. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all textured samples.

Table 15: Atomic percentage of elements based on EDX results before and after laser texturing of Ti-6AI-4V in nitrogen and air atmospheres. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all textured samples.

Ti64-Crossed- Ti64-Crossed- Ti64-Parallel- Ti64-Parallel-

Atomic % Blank

20 μηι-Ν2 20 μιη-air 20 μηι-Ν2 20 μιη-air

C 5.00 5.11 5.09 3.42 -

N 11.22 - 4.21 - -

O I 31.91 60.80 33.22 51.19 -

Al 3.66 2.40 2.62 3.02 9.16

Ti 46.19 30.33 52.84 40.67 87.75

V I ²⁰² 1.35 2.02 1.69 3.09 Table 16: Atomic percentage of elements based on EDX results before and after laser texturing of Ti-6AI-4V in argon atmosphere. A laser frequency of 30 kHz and scanning speed of 20 mm/s were used for all textured samples.

Ti 64-60% power- Ti 64-65% power- Ti64-70% power-

Atomic % Blank Crossed- 20 μηι- Crossed- 20 pm- Crossed- 35 pm- argon argon argon

Al 9.16 9.43 4.16 4.73

Ti 87.75 87.36 I 92.01 91.69

V 3.09 3.21 3.84 3.58

C - -

N - - -

0 - - -