Titanium and its alloys are frequently used as joint replacement materials. However, these materials eventually fail as the implant loosens due to a poor bone-implant interface. As the average life span increases, the need for better and longer lasting joint implants increases as well. The most successful metal implant materials currently have relatively smooth surfaces on the micron size scale. They are held in place with cements that have been largely successful, with most failures not occurring until after ten years [1]. However, to move beyond this limiting time scale researchers have been working on methods to modify the metal surface to enhance integration of the implant directly with surrounding bone.

A varied surface topography of both micron and submicron (>100 nm) sized features have been found to be of the largest benefit to osteoblast response and bone growth. Both size scales seem to be required for optimal cell attachment and differentiation into mature bone forming cells [2]. The ability to control the lateral spacing of these structures has also been shown to be important [3]. Certain textures have also been able to control cell growth, being more preferable to osteoblast adhesion than to other cell types [4].

A flexible, single step process has been developed that results in a surface texture that exhibits micron scale features with superimposed submicron scale ridges. The process uses femtosecond pulses of a laser to create a complex surface topography of micron scale peaks and troughs with submicron scale ridges across the entire surface. By varying the number of pulses and the overlap of the laser as it scans the surface, both the micron scale roughness and lateral spacing of features can be varied for optimization. This surface has been shown to have great promise as a biomaterial with future application in arthroplasty procedures.

Materials and Methods:

Titanium sheets, 50x50x25 mm, were purchased from Goodfellow (USA). Rabbit osteoblasts were isolated from the bone marrow of New Zealand White rabbits and human mesenchymal stem cells were purchased from Cambrex (USA). Promega MTS assay kits were purchased from Fisher Scientific (USA). All other cell culture materials were purchased from the Tissue Culture Core Facility at the University of Virginia.

Laser Texturing of Titanium: The titanium samples were placed on a stage inside a vacuum chamber (base pressure ~1 mTorr) and mounted on a high precision computer controlled X-Y stage. The samples were exposed to laser pulses of 800 nm wavelength and 130 fs pulse duration at a repletion rate of 1 kHz from a regeneratively amplified Spectra Physics Ti-sapphire laser system. The laser beam was focused along the normal onto the sample surface by a 0.5 m focal length coated lens and the laser fluence was adjusted by using a Glan calcite laser polarizer. The X-Y stage translation speed was adjusted to vary the average number of laser pulses impinging on a given surface area of the sample. The samples were then washed in acetone and methanol to remove metal debris and sterilized.

Contact Angle Analysis: The contact angle of deionized water and cell culture media was determined on the textured titanium surface and compared to the contact angle on control titanium samples with no surface modification. The analysis was performed with a Model 200 Standard Goniometer from ramé-hart instrument co. The contact angle was measured by image analysis using DROPimage software. Before contact angle analysis, the samples were sterlized by several different means to ensure consistent behavior across different protocols.

Cell Cultures and Proliferation Assay: Both textured and control samples of titanium were placed in the bottom of the wells in 24 well plates where they covered the majority of available surface area. The titanium was irradiated with UV light on each side for sterilization. Rabbit osteoblasts and human mesenchymal stem cells were seeded on the titanium samples at 10,000 cells/cm2 in 24 well plates and cultured for 3 days. The cell proliferation was analyzed at two time points, 24 hours and 72 hours. At these time points the titanium samples were washed with phosphtate buffered saline and transferred to new well plates. The MTS assay was performed at each time point to determine the number of viable cells on the titanium.

Results and Discussion:

The laser texturing process creates a hierarchical surface on metals. After treatment, the surface is textured with submicron scale ridges that measure approximately 250 nanometers in width superimposed on micron scale pillars and troughs. By changing the laser texturing conditions the pillars can be made with different height to diameter aspect ratios and cover the surface in varying density per unit area. This flexibility is key to creating an optimized surface in the single step process. The samples used in the discussed cell studies were textured with pillars that measured 10 microns in height and 15 microns at the base, tapering to approximately 1 micron at the peak.

The textured titanium surfaces remained very hydrophilic with no special treatment during the texturing process or during transfer from texturing chamber to cell culture. While carbon contamination can cause some chemically etched surfaces to become extremely hydrophobic [2], the titanium samples textured by this method remained hydrophilic with no measurable contact angle even after several weeks in normal atmosphere. The samples were sterilized by several methods, including steam autoclaving, UV irradiation, and liquid CO2 treatment. The same hydrophilic behavior was retained across all sterilization treatment methods.

Rabbit osteoblasts and human mesenchymal stem cells adhered well on the textured surface as evidenced by SEM images and the MTS assay. SEM qualitatively indicated an increase in cell number from day 1 to day 3 over that observed on an untreated control titanium surface. At the later time point, the cells spread well on the micron scale pillars and started forming bridges between neighboring pillars. This is corroborated by the MTS assay which indicated a larger increase in the number of viable cells from day 1 to day 3 on the textured surfaces than control titanium with no laser treatment.

Conclusions:

A novel method to create textured metals with a combination of micron and superimposed submicron scale features has been shown to enhance cell proliferation at early time points on titanium. This one step method of surface texturing has several advantages when compared to chemical treatment methods, including the retention of hydrophilic behavior without special processing and the ability to control lateral spacing without additional lithography. Both osteoblasts and mesenchymal stem cells respond favorably to the hierarchical structure of this unique surface with micron and submicron scale features. The synthesis method is flexible enough to continue optimization of surface features for further improvements in cell attachment and differentiation.

References:

1. Gioe TJ, Novak C, Sinner P, Ma W, Mehle S. Knee arthroplasty in the young patient. Clinical Orthopaedics and Related Research. 2007;464:83-87.

2. Zhao G, Raines AL, Wieland W, Schwartz Z, Boyan BD. Requirement for both micron- and submicron scale structure for synergistic responses of osteoblasts to substrate surface energy and topography. Biomaterials. 2007;28:2821-2839.

3. Khang D, Lu J, Yao C, Haberstroh KM, Webster TJ. The role of nanometer and sub-micron features on vascular and bone cell adhesion on titanium. Biomaterials. 2008;29:970-983.

4. Richert L, Vetrone F, Yi JH, Zalzal SF, Wuest JD, Rosei F, Nanci A. Surface nanopatterning to control cell growth. Advanced Materials. 2008;20:1488-1492.