Patent Application
20080097618
a-1d are photographs of a retrieved prior art total hip arthroplasty implant showing the total assembly,
a and 2b are photomicrographs of representative images of topography of 2×2 μm2 section of a glass slide sample, (
a-3f are photomicrographs of scanning electron microscope (FE-SEM) images of CaP films possessing different surface functionalities, COOH (
a and 4b are photomicrographs of a FE-SEM micrograph of a CaP coating on COOH-terminated SAMs highlighting the detail of coating morphology using a Type I immersion technique;
a-6g are photomicrographs of a FE-SEM micrograph of CaP coatings biomimetically deposited on SAMs possessing different surface functionalities, COOH (
a-7d are photomicrographs of a FE-SEM at 30,000× magnification of images exhibiting different surface functionalities COOH(2):OH(1) (
a and 8b are photomicrographs of FE-SEM images of a CaP coating on COOH-terminated SAMs using Type IIB SCPS immersion technique;
a and 10b are photomicrographs of an atomic force microscope (AFM) image of the topographic (
a and 11b are photomicrographs of an AFM phase images of a polyethylene (PE) sample treated with UV irradiation for 60 minutes (
a-12c are photomicrographs at 30,000× magnification of SEM images of CaP particle morphologies on polyethylene (PE) substrates having undergone no pretreatment (
a-13c are photomicrographs at 15,000× magnification of SEM images of CaP coatings on polyethylene substrates having undergone no pretreatments (
The present invention overcomes various drawbacks in the prior art by proposing an improved method and apparatus of depositing calcium phosphate coatings for use in orthopaedic applications. These applications include, but are not limited to:
The current process overcomes drawbacks in prior art by focusing on surface chemical functionality as the primary factor in improving the deposition and substrate-coating adhesion characteristics of calcium phosphate-based films. Careful physiochemical manipulation of the surface functionality of metallic, polymeric, ceramic, or organic-based materials enhances the amount and density of potential CaP nucleation sites, without a prominent effect on surface topography.
Since surface chemistry, not topography is the primary factor effecting deposition and adhesion of CaP films, substrate materials can be used in their “as-machined” or raw state, without time-consuming, or capital intensive surface finish processes. This has the potential to dramatically reduce costs associated with the manufacturing of implantable medical devices. Compared to prior art, such as that put forth by Leitao et al., the current process decreases the number and duration of substrate pretreatments, which is another potential source of cost reduction. The current process also relies on the immersion of a substrate in a supersaturated calcium phosphate solution (SCPS) at ambient temperatures, in place of the more chemically complex simulated body fluid (SBF) at physiological temperatures (37° C.) used in prior art. Reducing the chemical complexity of the immersion solution eliminates the possibility of side-reactions that may occur when the process is scaled-up to industrial scale and places emphasis on the interaction between the substrate and the ions in solution. Ambient immersion temperatures eliminate the additional energy expenditures required to elevate and maintain solution temperatures at, or above physiological conditions.
Coating-substrate adhesion is a major problem facing the use of biomimetic CaP films with polymeric materials. Chemical etching, flame treatment, ultraviolet (UV) irradiation, corona treatment, and plasma treating are all methods currently employed in various non-medical industries to improve the adhesion of polymers with other materials. Over the years, similar treatments of polymers have been tested in laboratories for biomedical applications. Many reports suggest that surface polar groups are created during these processing steps and act as nucleation sites for the formation of CaP structures and increase the rate of CaP deposition.11-12,18-19 For example, exposing the substrate to UV radiation prior to immersion in a simulated body fluid solution has been shown to enhance substrate-apatite adhesion properties.19 The exposure of a substrate to UV radiation increased the density of polar groups at the substrate surface, which interact favorably with CaP nuclei.19 Alkali pretreatments and glow-discharge (GD) processing of substrates prior to biomimetic formation of CaP coatings also increased deposition rate and substrate-coating adhesion through a similar mechanism.11-12 It is interesting to note, however, that GD processing was the only method that reduced apatite formation induction time and enhanced substrate adhesion on polymers such as polyethylene terephthalate (PET), poly(methyl methacrylate) (PMMA), polyamide 6 (PA6), polyethersulfone (PESF), polyethylene (PE) and polytetrafluoroethylene (PTFE), while other treatment methods were only effective for certain polymers.11-12,19
It is therefore, rational to assume that the surface chemistry plays a key role in the formation and growth of biomimetic CaP films. To understand the effect of surface chemistry on nucleation, growth, and adhesion of CaP films, self-assembled monolayers (SAMs) of alkanethiols possessing differing surface functionalities were prepared on gold-coated glass and polystyrene substrates. The monolayers served as model organic substrates in the biomimetic deposition of CaP coatings from supersaturated calcium phosphate solutions. Methyl (CH3) terminated SAMs were meant to serve as a plausible model of a saturated hydrocarbon surface, such as polyethylene (common biomaterial). SAMs with oxygen-based functionality, either homogeneous (COOH, OH) or heterogeneous (OH+COOH, CH3+COOH, OH+CH3, OH+COOH+CH3), were selected to simulate organic polymer surfaces that had been modified by a generic oxidation pretreatment. Tanahashi and Matsuda also used SAMs in studying the formation of apatite structures from a simulated body fluid.20 They found that the growth rate of apatite coatings on SAMs with PO4H2 and COOH functional groups was substantially higher than for SAMs with CONH2, OH, and NH2, groups. There was practically no growing of the apatite structures on SAMs with methyl as the end functionality. In other studies on the formation of HA on Langmuir-Blodgett monolayers, Sato et al. reached a similar conclusion.21 The authors found nucleation and growth of HA on monolayers with carboxyl groups and practically no nucleation on the monolayers with amino groups.
In contrast to the above previous prior art endeavors, we investigate the influence of chemical functionality on the CaP coating morphology and substrate coverage when the biomimetic deposition process is carried out using supersaturated calcium phosphate solutions (SCPS) instead of simulated body fluids. Our previous research showed that the deposition rate for CaP coatings is accelerated from supersaturated solutions.10 To accomplish the stated objectives as set forth heretofore, it was necessary to first understand the influence of substrate surface chemistry and immersion techniques on the formation and morphology of CaP coatings in general. With this understanding, a specific method by which polyethylene components can bond directly to bone would be formulated.
High impact polystyrene (PS) (available from Dow Chemical) tensile bars were sectioned into 17×17 mm squares using a silicon carbide cutting wheel and smoothed using a surgical scalpel. Glass slides (GS) (obtained from Eerie Scientific) were cut into 37×25 mm rectangles using a diamond scalpel. Both PS and GS samples were subjected to ultrasonic cleaning in a diluted surfactant solution (Micro 90, manufactured by International Products Inc.) for five minutes. The samples were then rinsed with de-ionized water and ultrasonically cleaned in methanol (histological grade, obtained from Fisher Scientific) for an additional five minutes. After ultrasonic cleaning, the GS samples were rinsed once more with methanol and acetone (99.9%, Fisher Scientific) placed in a covered Pyrex dish and allowed to dry in an L-C oven for at least 1 hour at 100° C. The PS samples were rinsed with methanol (histological grade, Fisher Scientific) for 30 seconds following the ultrasonic cleaning and dried in the L-C oven at 70° C. for 15-20 minutes.
Both sides of the GS and PS samples were sputter-coated with a 2.5 nm thick gold film (Hummer 6.2, manufactured by Anatech LTD.). The average pressure of the vacuum was 0.075 Torr with a plasma discharge current of 0.015 amps during deposition.
Formation of SAMs on the sputtered gold surfaces took place from 1-10 mM solutions of alkanethiols in histological grade methanol at 22-25° C. Hexadecanethiol (HS(CH2)15CH3, MW=258.51 g/mol, 95.0%, available from Fluka Chemical), 16-mercaptohexadecanoic acid (HS(CH2)15COOH, MW=288.50 g/mol, 90.0%, (available from Aldrich) and 11-mercapto-1-undecanol (HS(CH2)11OH, MW=204.24 g/mol, 97.0%, (available from Aldrich) were used without further purification.
SAMs with CH3, OH, or COOH end functionality were prepared on gold-coated GS and PS substrates by immersion in 50 mL of respective thiol solutions in covered beakers for approximately 36 hours. Heterogeneous SAMs (CH3+COOH, OH+COOH, OH+CH3, or OH+COOH+CH3) were also prepared according to the same protocol using a mixture (1:1, 2:1, 1:2, 1:4 or 1:10) of respective solutions. Substrates with SAMs were rinsed with methanol to remove organic debris and physically adsorbed thiols. They were then placed in covered Pyrex dishes and dried in an L-C oven at 100° C. for at least 15 minutes. The substrates were stored in clean, covered Pyrex dishes for a maximum of three hours before they were immersed into supersaturated CaP solutions.
The self-assembled monolayers were characterized by measurements of advancing and receding (static) water contact angles using the sessile drop technique.22 A syringe equipped with a 0.5 mm Luer-tipped needle was filled with deionized water (18MΩ). A small drop was placed on the solid surface of interest and adding more water from the syringe increased its volume. When the size of the drop was increased, the three-phase contact line advanced over the dry solid surface creating conditions for the measurement of advancing contact angle. Measurements of advancing contact angles were carried out 10-15 seconds after enlargement of the drop size to allow the three phase contact line to stabilize its shape. Then, the needle tip was carefully lowered into the sessile drop and water was removed to reinforce the reduction in the length of the three-phase contact line and create conditions for receding contact angle measurements. The receding contact angles were measured 10-15 seconds after change in the drop volume was made. A Kruss-G10 goniometer with drop shape analysis software was used for measuring seven advancing and seven receding contact angles. Average values and their standard deviations are shown in Table I below.
Surfaces of selected substrates were imaged with the Dimension 3000 atomic force microscope (AFM, available from Digital Instruments). A Si3N4 cantilever tip (300 kHz, 40N/m) in intermittent contact mode was used. Scan sizes from 2×2 μm to 20×20 μm2 were used for characterization of surface roughness and asperity geometries as illustrated in
A supersaturated calcium-phosphate solution (SCPS) was prepared by combining 5.6 mM CaCl2.2H2O (MW=147.02 g/mol) (available from Fisher Scientific) and 3.34 mM NaH2PO4.H2O (MW=137.99 g/mol, 99.4%) (available from Fisher Scientific) in 1:1 volumetric ratio. The resting pH of the solution was approximately 3.2 and a tris-buffer (tris(hydroxymethyl)-aminomethane, C4H11NO3, MW=121.14 g/mol, 99+%, (available from Aldrich) was used to elevate and maintain the solution pH to approximately 7.35. Solution pH was monitored during preparation (pH-Meter, available from Metrohm Inc.).
Chemical immersion in the SCPS took place in one of three ways listed below.
Type I Immersion. This is a combined immersion technique, in which all samples were immersed in an 800 mL beaker containing 700 mL of SCPS. Beakers were covered during immersion by a sheet of paper to reduce dust and foreign objects in the solution. The samples were positioned by placing them in plastic microscope slide holders. Although orientation and position were held constant during the experiment, the samples were not perfectly vertical throughout immersion and faced the bottom of the beaker at an angle of 65-70 degrees. Using this technique sample surfaces were between 5 and 8 mm from each other. Surfaces facing downwards (not predisposed to sedimentation) were analyzed during characterization.
Type II Immersion. In the first set of experiments using the Type I immersion technique, it was found that the orientation of the sample has a profound effect on formation of CaP deposits and coatings. In order to avoid the accumulation of CaP precipitates on samples from supersaturated solutions, a vertical orientation was maintained throughout immersion. In this technique two different approaches were used when individual samples, or a set of a few samples were immersed into the SCPS solution.
Type IIA. Samples were immersed vertically in individual 50 mL beakers containing approximately 50 mL of SCPS. The beakers were covered with a thin polyolefin film throughout immersion. The SCPS was poured from a single batch that underwent constant stirring between dispersions into each beaker to ensure solution homogeneity. Vertical immersion was achieved either by implementing metal hangers attached to both the sample and to the parafilm covering the beaker, or by painted metal clips that held the samples vertical in solution. Distance of the samples from the sides of the glass beakers, which were being used as reaction cells, was held nearly constant throughout experimentation.
Type IIB. This is an improved Type IIA immersion technique that eliminates difficulties associated with hanging samples at perfect vertical orientation. Painted metal clips, that underwent a cleaning regime similar to that used for the actual samples, were attached to the bottom of PS samples with minimal obstruction of surface area. The clips were shaped in a manner such that they allowed the samples to be perfectly vertical while in solution. Immersion took place in a 100×50 mm Pyrex dish containing approx. 250 mL of SCPS with even spacing between samples that exceeded 3 cm. The dish was covered with a glass lens during experimentation. From 4 to 8 samples could be placed in the same solution during experimentation, securing the same deposition conditions for each sample.
In each experiment, Type I and II, samples remained immersed in the SCPS for three days with daily solution refreshments. During solution refreshments, the old solution was carefully pipetted out of the beaker(s). New SCPS was carefully poured back in making sure not to disturb the samples. After three days of immersion, the samples were removed and gently washed with de-ionized water, placed in a covered Pyrex dish and dried in an L-C oven at 75° C. for 15 minutes.
Morphology and coverage of calcium phosphate coatings were examined under scanning electron microscopy, using a JEOL6400 SEM instrument. Approximately 12 mm wide sections were cut from the GS samples by a diamond scalpel. The size of the PS samples did not dictate that they be sectioned for observation. Samples were then mounted to aluminum pucks and sputter-coated with 2.5 nm of Au—Pd film to enhance resolution and reduce charging effects during SEM. SEM imaging was performed at an accelerating voltage of 20 kV and a working distance of 8 mm. Images were gathered via the digital image acquisition system in place with 1000 pixels per line and a dwell time of 0.1 msec for optimal image quality.
Field-emission scanning electron microscopy (FE-SEM, Hitachi S-4700, available from Hitachi Inc.) was employed to more closely examine structures of deposited CaP films. An accelerating voltage of 5 kV and working distance of either 5.6 or 10.5 mm was used to resolve nanometer-scale features within the films.
The SEM and FE-SEM images were analyzed using Scion Image 4.02 (manufactured by Scion Corporation) to gather information about the size of CaP particles/structures in the coatings.
Energy dispersive x-ray spectrometer (EDS) installed on the JEOL6400 SEM instrument was used to gather information about the calcium to phosphorus ratio of the deposited coatings. Carbon sputtering was used instead of Au—Pd sputtering for EDS analysis as the M5-peak for gold (2.2057 keV) interfered with the Kα-peak of phosphorus (2.1455 keV). EDS analysis was performed at 20 kV of accelerating voltage with a working distance of 39 mm and a magnification of 100,000×. A live time of 100 seconds and dead time setting of approximately 22% was used for all compositional characterization with the EDS. Measurements were made in five separate locations on each sample using the same settings and average value and standard deviation are shown in
A 31×41 transparent grid was super-imposed over SEM micrographs representative of the morphology of CaP films produced by Type I and Type IIA immersion techniques on the monolayers. “Void fraction” in the porous coating was determined as the ratio of grid points that fell on pores and the total number of grid points. These measurements are presented as percentage of surface area of the images.
CaP particles were separated from one of the supersaturated solutions for evaluation of their size and morphology. An SCPS solution was prepared and poured through a glass micro-fiber filter (0.5 μm pore size, Whatman) under suction. The filter was then washed with acetone to remove water and placed in an L-C oven at 75° C. for 48 hours prior to observation by FE-SEM. Imaging of particles was carried out with FE-SEM at accelerating voltage of 5 kV and working distance of 5.6 and 10.5 mm. The particle size was determined from collected images using Scion Image 4.02 software.
a and 2b show representative AFM images of GS and PS samples at different resolution. Both types of samples possessed nano-scale roughness such as shown in the AFM image in
Grainy texture of the GS sample surface shown in
Microscopic irregularities and characteristic “pitting” sites with a size of a few micrometers and variation in height of up to about 1 μm, characteristic of extruded amorphous-crystalline polymers, are evident on the surface of the PS samples (
Contact angles measured for water drops on SAMs prepared in this investigation are shown in Table 1. Receding contact angle measurements were difficult to obtain on carboxyl and hydroxyl-terminated SAMs, as these values were near zero. The small profile of the receding drops challenged the ability of the drop shape analysis software to discern between the liquid and solid surface. Contact angles near zero are reported as smaller than 5 degrees.
The hydrophobic character of CH3-terminated SAM is revealed by high advancing water contact angles, approximately 111 and 96 degrees for GS and PS samples, respectively. Typical advancing contact angle values measured for such SAMs are in the range of 110-112 degrees.22 Contact angles several degrees smaller recorded for CH3-terminated SAM formed on the PS sample suggests less dense packing of thiol molecules in the monolayer structure and/or submicroscopic imperfections of this particular sample. Receding water contact angles of 84-88 degrees were measured and they are also indicative of certain imperfections of the samples used in this investigation. Because both GS and PS samples covered with CH3-terminated SAM had different roughness characteristics, but still showed similar receding contact angles, we expect that the samples had some degree of heterogeneity. In our previous research, we speculated that this heterogeneity might result from the intergranular area of the gold coating that is uncovered with thiols.23
COOH and OH-terminated SAMs served as hydrophilic surfaces in this investigation. Similar advancing water contact angles were recorded for both polar SAMs, COOH-terminated SAMs being more hydrophilic as revealed by smaller contact angles, particularly when prepared on gold-coated PS substrates. A value close to zero degrees for advancing water contact angles was reported for freshly prepared COOH and OH-terminated SAMs,24 whereas as high as 84 and 65 degrees were reported in our previous paper.23 Aging of the SAMs and its sensitivity to air-born contaminants are expected to be the major reasons for such a broad variation in measured contact angles for SAMs with COOH and OH functional groups. These relatively high-energy surfaces have a tendency to reduce their surface energy by adsorbing organics from laboratory air.
The low standard deviation for most of the measured contact angles and reasonable contact angle hysteresis values (difference between advancing and receding contact angles) are also indicative of a relatively good quality samples prepared in this investigation.
Any of the heterogeneous SAMs containing the methyl functionality produced large water contact angles suggesting that the thiols with methyl groups dominated the molecular structure of SAMs. The SAMs with mixed OH and COOH functionality demonstrated contact angles that are larger than contact angles measured on homogeneous COOH or OH-terminated SAMs. This result is of no surprise as 16-mercaptohexadecanoic acid is longer by four methylene units than 11-mercapto-1-undecanol and surfaces composed of these two thiols have triple surface functionality, two polar OH and COOH, and nonpolar CH2.25
Deposition characteristics of CaP on SAMs differed greatly depending on the conditions of immersion. Type I immersion experiments yielded CaP films with a spherical morphology, while Type IIa and IIb immersion methods produced films with a plate-like structure. The variance in morphology is indicative of a difference in the underlying deposition mechanism between the two immersion techniques and is discussed hereinafter.
Type I Immersion Experiment. The SEM images of CaP coatings obtained according to Type I SCPS immersion are shown in
The particles on the homogeneous SAMs were quite spherical in nature and differed between functionalities in their densities and packing structures. CaP particles on heterogeneous functionality SAMs deviated from sphericity showing an elongated structure with the OH+CH3 SAM having the largest aspect ratio. Type II immersion techniques failed to produce coatings of similar structures as discussed below.
CaP particle sizes were analyzed from the SEM images and found to be 230±21 nm, 243±19 nm, and 210±14 nm for COOH, OH, and CH3 terminated SAMs. The difference in aspect ratio between CaP particles deposited on heterogeneous functionality surfaces is noticeable. CaP particles were 152±8 nm by 130±11 nm on the OH+COOH surface, 260±33 nm by 96±6 nm for the CH3+COOH surface, and 248±37 nm by 88±5 nm for the OH+CH3 surface.
The reason for coatings of different morphologies formed on molecularly heterogeneous surfaces in this experiment is not clear to us. We are also unaware of any report that presents structures of CaP coatings similar to those shown in
Further examination of CaP films on COOH-terminated substrates by FE-SEM highlighted the details of coating morphology as shown in
Type II Immersion Experiment. When Type II CaP deposition was used the CaP coating morphology was different as illustrated in
It is hypothesized that the differences in coating morphology of CaP film formation between the immersion techniques are due mainly to differences in conditions during immersion of substrates in the SCPS. As discussed earlier, in the case of Type I SCPS immersion experiments, the spherical morphology of the particles as illustrated in
Void fraction of the coatings was examined in regions representative of particle morphologies. This parameter was determined from 2-dimensional SEM images and should not be mistaken with porosity, although is meant to characterize the overall morphology of the CaP films. It should also not be confused with surface area coverage that is described hereinafter. Void fraction of the coatings varied greatly between the surface functionalities. Evenly dispersed voids and 12.2% void fraction was noted on the carboxyl-terminated monolayer. Concentrated open spacing comprising 13.9% of surface area shown in the image was observed on methyl-terminated monolayers. Evenly dispersed openings and a limited 8.8% void fraction was calculated for the hydroxyl-terminated monolayer.
CaP films on heterogeneous functionality SAMs showed characteristics exhibited by each of the homogeneous functionality components used to produce the surface. For instance, dispersion of pores on the heterogeneous OH(1)+COOH(1) functional surface was less concentrated than those observed on homogeneous OH surfaces, but also less evenly distributed than those observed on homogeneous COOH surface. Open space was greatest on CH3+COOH (12.9%) and OH+COOH (12.1%) surfaces. Limited void fraction (7.1%) was observed for the OH+CH3 sample.
In implementing Type IIA immersion for heterogeneous OH+COOH monolayers with varying OH:COOH ratio, no clear relationship was found between increasing amounts of hydroxyl-terminated thiols in solution and porosity of the deposited CaP films. OH(4)+COOH(1) surfaces produced CaP films with void fraction accounting for 22.7% of the surface area shown in
A uniform coating was produced on COOH and OH(1)+COOH(1) as illustrated in
Despite the differences in coating morphology, trends in substrate coverage were similar regardless of the immersion technique implemented. Substrate coverage of 100% was achieved on COOH, OH+COOH and OH+COOH+CH3-terminated SAMs. As stated previously though, the coatings on the OH+COOH+CH3 terminated SAM were extremely thin. CH3+COOH terminated SAM substrates were approximately 80-85% covered with a CaP film. Heterogeneous functionality OH+CH3 substrates elicited an approximate surface area coverage of 35-40%. Homogeneous CH3 and OH substrates had the least amount of substrate coverage with 15% and 10%, respectively.
Varying the ratio of OH to COOH terminal sites on the SAMs had little effect on particle size and morphology as shown in
Calcium to phosphorus ratios of CaP films formed on self-assembled monolayers of differing functionalities varied for coatings produced in different experiments (as shown in Table 2).
When Type I immersion was employed, the coatings were very calcium-rich. However, when Type IIB immersion was used, the films were calcium-deficient, with the overall Ca:P ratio lower than that of stoichiometric hydroxyapatite. This difference in coating compositions may highlight the difference in the underlying mechanism of deposition between the two immersion methods. The calcium-rich nature of the films, similarity in morphology to CaP particles separated from solution and the charge number of calcium ions (2+) compared to phosphate complexes (1−) imply that Type I immersion relies on an electrostatic interaction for the deposition of CaP films.
It is also worth noting that hydroxyl-dominated SAMs and SAMs with an equal ratio of hydroxyl and other groups elicited the most calcium-rich coatings when Type I and Type IIA immersion was employed. When Type IIB immersion was used, hydroxyl containing SAMs had the lowest calcium to phosphorus ratios.
From this investigation of the effects of surface chemistry on nucleation, growth and adhesion of CaP films, it was found that the self-assembled monolayers (SAMs) of alkanethiols served as model organic substrates in the biomimetic deposition of CaP coatings from supersaturated calcium phosphate solutions. It was further found that homogeneous COOH-terminated SAMs and heterogeneous OH+COOH SAMs produced CaP coatings that covered 100% of the substrate surface area within three days of chemical immersion. SAMs containing a predominantly methyl, or hydroxyl character failed to elicit continuous CaP films covering all of the surface area. This trend of surface area coverage was observed regardless of the immersion method employed, which suggests that the deposition process is strongly dependent on surface functionality.
Both deposition methodology and substrate surface functionality had also influenced the chemical composition of the formed calcium phosphate coatings. Calcium to phosphorus ratios of CaP films varied from about 1.3 to 2.7 for coatings produced on SAMs of differing functionalities. OH-terminated SAMs induced formation of deposits with enriched calcium content whereas coatings with stoichiometry close to that for hydroxyapatite were preferentially formed on SAMs having COOH groups.
Having gained a clearer insight of the surface chemistry variables associated with the adhesion of CaP films in a biomimetic deposition process it now became necessary to apply this learned knowledge to a substrate of choice.
With elastic properties similar to biological tissue found in joint capsules and the ability to achieve a smooth surface through common machining techniques, polyethylene has gained wide-spread support as a material of choice for bearing surfaces in total shoulder, knee and hip arthroplasty.32 Most polyethylene components used in joint replacements are equipped with a metal component such as the tibial tray in total knee arthroplasty, or the acetabular cup in total hip arthroplasty shown in
As stated above, bioactive calcium-phosphate (CaP) coatings have been employed in several orthopedic applications to provide an environment conducive to bone growth at the surface of implants.10-16,19,37 Plasma sprayed hydroxyapatite (HA) is the most common form of bioactive coating used to enhance hard tissue integration with orthopedic implants. Since the plasma spray deposition method is a line of sight process, coatings produced on implants with complex geometries (screws, hip stems) are often non-uniform in terms of substrate coverage and coating thickness.10 The plasma spray deposition process also entails the use of high temperatures which can lead to heterogeneous coating properties and increased crystal sizes. Increased crystal sizes effectively reduce nano-scale surface roughness, which has been shown to negatively impact the state of differentiation of osteoblast cells.38 Chemical immersion biomimetic processes have therefore gained favor with the use of polyethylene substrates. Coating the non-bearing surface of polyethylene components with a bone-like, biomimetic CaP film by a similar method would increase the integration of the device with the patient's surrounding hard tissue (osseointegration).10,28,30,37,39-44 Through the process of osseointegration, the polyethylene would be integrated into the structure of the hard tissue thereby imparting long-term stability of the device in vivo. A direct benefit of this could be the elimination of devices commonly used in conjunction with the polyethylene bearing components, thereby reducing the mass and number of components to be implanted. As mentioned previously, this would also reduce the generation of wear debris in the joint capsule.
A major challenge facing the deposition of biomimetic CaP films on polyethylene substrates is the adhesion characteristics between the film and substrate. Adhesion between metallic substrates and CaP films have been shown to be much higher when compared to polymeric substrates.11-12,19 Kokubo and others have been successful in applying substrate pretreatments to polymeric materials to improve the adhesion characteristics of biomimetic CaP films, but details of the effects of these substrate pretreatments are not deeply understood.11-12,19
In this investigation, extruded polyethylene tensile bars were sectioned, cleaned and characterized using contact angle analysis and atomic force microscopy (AFM). Samples were then subjected to UV irradiation, or glow discharge processing for varying amounts of time. The effects of these pretreatments on the surface properties of the polyethylene samples were investigated using contact angle analysis and AFM. Samples were then immersed in a supersaturated calcium phosphate solution for deposition of a biomimetic CaP film. Nano-scratch testing was performed on the CaP-coated polyethylene samples to examine the effects of the substrate pretreatments on substrate-coating adhesion characteristics. Scanning electron microscopy was used to evaluate the morphology of the resulting coating. Fourier transform infrared spectroscopy (FTIR), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) were used to evaluate the composition and structure of the coatings.
Tensile specimens of commercial high density polyethylene (available from Dow Chemical Co., PE) were sectioned along the neck with a silicon carbide cutting wheel to yield 12.5×12.5 mm samples. The edges were smoothed and extraneous material was removed using a surgical scalpel. Samples then underwent 5 minutes of ultrasonic cleaning in methanol (histological grade), surfactant solution (Micro 90, from International Products Inc.) and deionized water (18MΩ). The PE samples were then dried in an L-C oven for 10 minutes at 75° C.
Three cleaned PE samples were placed in a UV irradiation chamber (available from BioForce Laboratories Inc.) for 10, 30 or 60 minutes. Samples were placed approximately 50 mm away from the light source and were subjected to UV irradiation with main wavelengths of 184.9 and 253.9 nm.
PE samples were subjected to radio-frequency driven glow discharge processing (Jupiter II Reactive Ion Etcher, March Instruments Inc.) for 10, 30, 60 or 300 seconds under an O2 environment with a gas flow of 25 cm3/s at a pressure of 186 mTorr. A forward power of 50 Watts and a frequency of 13.56 MHz were used to process the samples for the varying time.
Atomic force microscopy was used to image untreated PE substrates and to investigate any topographical and structural changes brought about by the surface pretreatments. Silicon cantilevers (˜300 kHz, ˜40N/m) were used for sample surface scanning in Tapping Mode. Scan sizes of 1 μm2 were used for characterization of surface roughness and phase differences. At least three scans were performed on each untreated and treated PE surface to gather information representative of each of the surfaces.
The polyethylene substrates were characterized by measurements of advancing and receding contact angles performed by implementation of the sessile drop technique.22 A Kruss-G10 goniometer with drop shape analysis software was used for measuring seven advancing and seven receding contact angles of de-ionized water, ethylene glycol (HOCH2CH2OH, MW=62.07 g/mol, 99+%, Fisher Scientific), glycerine (C3H8O3, MW=92.09 g/mol, Fisher Scientific) and diiodomethane (CH2I2, MW=267.84 g/mol, J.T. Baker Inc.).
The surface tension and surface tension components of the substrates were calculated using the semi-empirical Lifshitz-van der Waals Lewis acid-base interaction model developed by van Oss, Good, and Chaudhury.45 This model asserts that the surface tension of a material is the sum of the Lifshitz-van der Waals components of surface energy and the geometric mean of the electron acceptor and donor parameter of the material.
A supersaturated calcium-phosphate solution (SCPS) was prepared in accordance with previously described methods by combining 5.6 mM CaCl2.2H2O (MW=147.02 g/mol, from Fisher Scientific) and 3.34 mM NaH2PO4.H2O (MW=137.99 g/mol, 99.4%, also available from Fisher Scientific) in 1:1 volumetric ratio.10 The resting pH of the solution was approximately 3.2 and a tris-buffer (tris(hydroxymethyl)-aminomethane, C4H11NO3, MW=121.14 g/mol, 99+%, Aldrich) was used to elevate and maintain the solution pH to approximately 7.35. Solution pH was monitored during preparation (pH-Meter, available from Metrohm Inc.).
After substrate pretreatment, samples were immersed vertically in the SCPS. Deposition took place in a Pyrex dish filled with approximately 250 mL of SCPS, while the samples were held in a vertical position by painted metal clips. SCPS immersion lasted for three days with daily solution refreshments. Solution refreshments were performed by pipetting the old solution from the container and gently pouring in fresh solution, so as to not disturb the samples. On the final day of immersion, samples were removed from the SCPS, rinsed with DI water and allowed to dry in an L-C oven at 75° C. for 10 minutes.
CaP morphology was examined using the JEOL JSM-6400 scanning electron microscope (SEM) with an accelerating voltage of 20 kV. Cap-coated PE samples were sputtered with 2.5 nm of carbon to improve SEM image quality and reduce charging effects.
The SEM images were analyzed using Scion Image 4.02 (available from Scion Corporation) to gather information about the size of CaP particles/structures in the coatings.
Energy dispersive x-ray spectrometry (EDS; JEOL JSM-6400) was used to elicit information regarding the calcium to phosphorus ratio of the CaP coatings. Spectra were gathered from five different locations on the coating and quantitative analysis was performed using Spirit software.
X-ray diffraction (XRD, Scintag XDS 2000 Diffractometer) was employed to gather information about the structure and composition of the deposited CaP film. An untreated PE sample underwent immersion in SCPS for five days with daily solution refreshments to deposit enough material for this test. The sample was then allowed to dry in an L-C oven for 1 hour at 75° C. The CaP-coated PE sample was then subjected to an 18-hour XRD scan utilizing Cu—Kα radiation (λ=1.54 Å). The Bragg-Brentano diffractometer was used in the θ-2θ configuration.
The 5-day coating composition was also analyzed by Fourier transform infrared spectroscopy (FTIR, Spectrum AssureID, obtained from Perkin-Elmer Inc.). An attenuated total reflectance (ATR) attachment was added to the FTIR to permit analysis of the coating.
A nano-indenter system (Nanoindenter XPS, from MTS Inc.) equipped with a Berkovich-style scratch tip was used for interrogation of coating-substrate adhesion parameters via scratch testing. The Berkovich tip was ramped through the coating with an applied normal load range of 0-20 mN and a scratch length of 500 μm. A single batch of ten scratches was performed on each sample. Scratches were spaced 100 μm apart to sample a representative area of the coating.
Scratches on each of the samples were examined under SEM. Images of all ten scratches, and groups of three scratches within the batch were gathered at an accelerating voltage of 20 kV, and at a magnification of 55× and 250× respectively. Digital image analysis software (Scion Image, from Scion Corporation) was used to digitally measure the distance from the point of initial load application to the point of coating delamination for each scratch. The length was then used to determine the applied normal load and lateral force (applied lateral force perpendicular to the scratch path) by correlating the position at which delamination occurred with the position of the scratch tip. After determining the position of the scratch tip, the force parameters were located in the data file generated and recorded during the scratch tests. All scratch lengths were measured to ensure that only the 500 μm prescribed length of the scratch was taken into account.
As a basis for comparison, 1 μm2 AFM scans were performed on a cleaned, untreated polyethylene substrate.
AFM phase images illustrated in
UV irradiation and glow discharge processing yielded block-shaped domains at the surface of the PE samples. The block-shaped regions were larger and more pronounced on the glow discharge treated samples when compared to the UV treated samples.
Analysis of the roughness parameters ascertained by AFM and summarized in Table 3, below, suggest little, if any, effect of substrate pretreatment on surface topography. There is a slight drop in the roughness parameter (ratio between the actual surface area and the projected surface area of the scan) noted for samples having undergone glow discharge pretreatments. Geometric roughness (RMS) and average roughness (Ra) remain unchanged, for the most part, throughout the different oxidation pretreatments.
Table 4 summarizes the advancing and receding contact angles, along with the contact angle hysteresis (difference between advancing and receding contact angles) for untreated and treated PE surfaces.
Contact angle hysteresis values and low standard deviations for most samples imply that the surfaces are of good quality and the results are reproducible. Non-uniform etching and oxidation of polymer are expected to produce irreproducible surfaces of different heterogeneity pattern.
Advancing water contact angles of nearly 90° for untreated PE samples were observed and are indicative of a hydrophobic material. The values are lower by 5-15 degrees than what is usually reported for polyethylene in literature.22,48 Exposure of the samples to UV irradiation or glow discharge changed the hydrophobic character of the polymer to a more hydrophilic character, as evidenced by a reduction in the advancing contact angle values measured for water drops. The production of surface polar groups (carboxyl, hydroxyl, carbonyl, esters, etc.) upon physico-chemical pretreatment is well-documented.11-12,19
As can be seen in Table 4, above, glow-discharge processing provided the most dramatic reduction in advancing water contact angles. As is the case for all treatments, a saturation point in the reduction of contact angles was observed. This saturation in contact angle reduction may be related to the achievable number of polar groups at the sample surface by particular treatment technique. Overall, UV irradiation had the least severe impact on advancing water contact angles.
Samples having undergone a glow discharge pretreatment with a forward power of 50 W were shown to have the highest total surface energy and the highest electron donor (γ−) parameter as shown in Table 5, below. The γ− parameter is defined as a quantification of the active sites that attract electrons from another material, in terms of surface energy. This attraction and sharing of electrons may not only aid in improving substrate coverage by biomimetic CaP films, but it may also contribute to the enhancement of adhesion of these films to the polymeric substrate.
The appearance of spherical CaP particle morphologies first noted during the self-assembled monolayer (SAM) study, Type I immersion experiment shown in
Particle and cluster morphologies were similar within each type of substrate pretreatment. Overall coating morphologies did vary between the different substrate pretreatment groups however. CaP clusters were evenly dispersed on the control, UV irradiated and glow discharge treated samples.
AFM analysis of the PE substrates before and after the prescribed pretreatments indicate little or no change in topography. This fact suggests that surface functionality plays a role in determining the deposition characteristics of CaP films on PE substrata. Similarities in the coating morphologies and deposition characteristics also suggest that the oxidation pretreatments aid in the production of chemically similar surface of the PE samples.
Substrate coverage was near 100% for all treated and untreated substrates. CaP films on the PE substrate treated with UV irradiation for 60 minutes showed an 80-85% coverage of the free surface area.
Near 100% substrate coverage of the untreated (control) PE sample suggests that some sort of surface functionality was preexisting. Results from the surface chemistry on nucleation, growth and adhesion on CaP films indicated that CaP nucleation and growth should be weak on surfaces possessing a dominant “methyl” (CH3) character, such as would be the case for untreated PE substrates. Stearates had been incorporated into the polymer blend as mold release agents during PE processing at the Dow Corporation49 and they are probably responsible for deposition of CaP films on untreated polyethylene samples.
Substrate pretreatments were found to have little effect on the calcium to phosphorus ratios of the bone-like CaP films when this parameter was investigated by EDS as shown in Table 6, below. Irradiation of the PE substrates with UV light tends to produce a film with a lower Ca:P ratio. Increased time of exposure to the UV light intensified this effect.
The infrared spectrum of the CaP coating on a PE substrate illustrated in
Table 7, above, provides a summary of the critical normal loads obtained from nano-scratch tests of CaP coatings on untreated and treated PE substrata. The critical normal loads, while they provide little information regarding the substrate-coating adhesive strength in shear loading, do show a fairly clear relationship based on substrate pretreatments. These normal loads can provide a basis for comparison of the relative adhesion of biomimetic CaP films on PE substrata.
UV irradiated samples show an overall decline in critical normal load values with increasing treatment time. However, the critical load is slightly higher than that for the control sample when the PE is treated for shorter times. Samples that had undergone glow-discharge processing with a forward power of 50 W showed a dramatic increase in critical normal load values when compared with the rest of the samples. The greatest critical normal load was achieved with a glow discharge treatment time of 30 seconds. Samples treated for 60 and 300 seconds showed a decrease in the critical normal load values, which still exceeded the values attained by the control sample and samples treated by UV irradiation.
Reductions in adhesion on samples that have undergone prolonged exposure to UV irradiation and/or glow discharge processing have been observed in previous work.11,19 Kokubo et al. hypothesized that extensive exposure of polymers to oxidizing environments actually compromises the structural integrity of the polymer surface.11-12,19 Degradation of the surface structure would invariably have a negative impact on the coatings adhered to it.
An alternative hypothesis to the structural degradation of the polymer is that the oxidation pretreatments actually remove the additives that act as nucleation sites inherent in the polymer. Once the removal of the surfactant reaches equilibrium, nucleation-enhancing polar groups begin to form in crystalline domains of the polymer. This hypothesis is further supported by the fact that glow discharge treatments increase the distribution of crystalline regions at the polymer surface since the amorphous phase is preferentially etched by the process.
The result that glow discharge processing enhances the substrate-coating adhesion characteristics of biomimetic CaP films on polyethylene substrates is in accordance with the integral work performed by Kokubo et al. They reported enhanced adhesion characteristics on a number of polymeric substrates after glow discharge processing due to the increased density of polar groups at the sample surface.11-12,19 Polar groups (carboxyls, carbonyls, hydroxyls, esters, etc.) produced by the glow discharge process act as strong nucleation sites for CaP formation. It follows that the increase in nucleation sites should improve the adhesion of the CaP films. The severe increase in the electron donor parameter (γ−) may be the underlying mechanism by which adhesion is improved by the production of these polar functional groups.
From these two studies the following conclusions can be formulated and used in the formation of an implant coating process for the delivery of biologically active substances, i.e. growth factors, bone morphogenic proteins (BMP) stem cells (osteoprogenitor cells, progenitor cells, bone marrow stromal cells, etc.)
As the conclusions of the previous studies clearly suggest the ability to utilize substrate materials in their raw (as machined, as received) state and not requiring treatments to attain a specific level of contact surface roughness (as is clearly taught in prior art coating methods) has resulted in drastic reduction of production time, labor costs and material costs in the delivery system of the invention. However, this does not suggest that a good cleaning regime is not implemented. Failure to implement the specified cleaning regime in previous research led to the inability to deposit CaP films on metallic substrates. The presence of dust, organic contaminants from the laboratory environment may impart an unfavorable charge to the substrates thereby negating deposition of CaP on the substrates.
Dust particles and other organic contaminants may favor heterogeneous nucleation due to the reduction in free energy associated with the binding of CaP articles to the contaminants, rather than with the substrate itself. Accordingly, the process of the invention proposes the following substrate cleaning regime:
Substrate Cleaning Regime for Metals:
Substrate Cleaning Regime for Polymers:
The cleaning regime is followed by a substrate pretreatment, which also differs for metals than for polymers. For metallic substrates, the invention proposes the use of a precalcification step (immersion in boiling CaOH2) to aid in the formation of a calcium-titanate film, which acts to further complex with calcium ions when the substrate is subsequently immersed in a supersaturated calcium phosphate solution (SCPS). It is thought that the Ca2+ ions from the precalcification step will actually penetrate into the oxide film present on the metal substrate prior to treatment. These Ca2+ ions attract calcium phosphate clusters and may actually further supersaturate the SCPS in proximity to the substrate, thereby enhancing deposition of the CaP films.
Polymer substrates pretreatment according to the invention, proposes the use of glow-discharge processing. The use of glow-discharge (a.k.a. corona discharge, or plasma treating) processing has been used in several other industries to enhance the wettability of polymers in applications such as the improvement of ink printability and adhesion. In the application of the invention, it was found that glow discharge processing actually enhances a specific surface energy component, known as the electron donor parameter. The electron donor parameter is a quantification of the number of sites on a surface that will actively share electrons with another material. To the knowledge of the inventors, the electron donor parameter has not been addressed in any body of literature to date regarding the deposition of any type of coating on any substrate. This knowledge was gained only by experimentation (empirical evidence). Scission reactions occurring during glow-discharge processing are known to produce surface polar groups (carbonyl, carboxyl, esters, etc.), which impart specific effects to the surface energy and surface energy components of a particular material. Previous to our research, the effect of glow discharge processing on the deposition and adhesion of CaP films to polymeric substrates was investigated (Kokubo et al.). However, there was a failure to elucidate the exact mechanism by which glow discharge processing enhances deposition and adhesion of CaP films.
The above substrate pretreatments are summarized as follows:
Metallic Substrate Pretreatment:
Polymeric Substrate Pretreatment:
Most prior art publications concerning the study of deposition of CaP films on various substances centers around the use of simulated body fluids (solution to mimic the composition of human blood plasma). Simulated body fluids (SBF) are principally used to study how bone growth will occur in vivo, by doing in vitro experiments. Since the objective of the invention is simply to prove a CaP coated substrate to use as a delivery vehicle, rather than understand how bone growth will occur, the invention proposes the use of a supersaturated calcium phosphate solution (SCPS) that contains only calcium and phosphate ions. It is felt that by reducing the number of different ionic species in solution, the industrial viability of the process is enhanced.
The composition of the SCPS is most important. In particular, the ratio of calcium to phosphorus in the SCPS plays a major role in the final composition of the coating. A ratio of 1.67 calcium to phosphorus is preferred to achieve the final composition of the coating. This ratio is equal to that of so-called stoichiometric hydroxyapatite. As set forth below, the SCPS is composed of 5.6 mM CaCl2.2H2O and 3.34 mM NaH2PO4.H2O. Changing the calcium to phosphorus ratio of the solution may lead to changes in supersaturation of one ion with respect to another and change the final composition of the coating. Changes in this ratio may also affect the rate of speed of the deposition process, thereby leading to undesired changes in coating coverage, morphology and composition.
It is preferred that the SCPS is buffered to a pH of 7.40±0.05 for metallic and polymeric substrates. Theoretical work has shown that the thermodynamics and kinetics of CaP precipitation from a supersaturated solution are largely pH dependent. Slight changes in pH may yield the precipitation of specific phases of CaP that have different compositions, morphologies, crystal structures and solubility characteristics. Tris(hydroxymethyl)aminomethane is used in place of other types of buffers due to its ability to elevate and maintain the solution pH to specific levels with only small amounts of buffer.
The specific preparation of SCPS according to the invention is as follows:
Preparation of the Supersaturated Calcium Phosphate Solution (SCPS):
f. Allow SCPS to reach 25° C.
Change in solution temperature effects the supersaturation of the solution, thereby influencing the rate of deposition of CaP films on the substrate. The use of a solution temperature of 25° C. was found to be ideal for the specific concentration of reagents used. Most prior art research have proposed the use of a physiological temperature of 37° C. during deposition experiments. Use of this elevated temperature with the above described invention procedure of the SCPS would reduce the supersaturation and retard the deposition process. Elevated temperatures also necessitate the expenditure of energy, which increases the processing costs associated with this biomimetic technique.
During early attempts to develop the process of the invention, substrates were simply laid flat on the bottom of a glass beaker filed with SCPS. Upon removal from the SCPS beaker, it was found that deposition preferentially occurred on the surface in contact with the glass beaker. Interactions between the solution, substrate and silicate ions in the glass skewed the results of experimentations. The proximity of the substrate to a flat surface may also predispose the system to intense local changes in solution chemistry (pH, ion concentration, etc.). These earlier mishaps resulted in the implementation of the use of a vertical immersion technique, as set forth below in detail, in which no part of the substrate is in contact with, nor is in any significant proximity to the sides of a container during immersion.
Immersion of Samples in SCPS
The final Ca:P ratio of the CaP films is approximately 1.34 which is significantly less than that of hydroxyapatite. From the result of the invention process, it can be confidently concluded that the invention process films are in the form of octacalcium phosphate (OCP) with some amorphous phases present. Most bioactive coatings used and reported in the prior art are in the form of hydroxyapatite. Prior art indicates that OCP is a precursor to hydroxyapatite. If hydroxyapatite is the desired phase of calcium phosphate for a particular application, extra time in the SCPS without solution refreshments will facilitate a phase transformation from OCP to hydroxyapatite.
By itself, the CaP films deposited via this biomimetic process act as a scaffold for bone growth on orthopedic implants. Thus, the biomimetic coatings add the attribute of osteoconductivity to the implant. To maximize bone growth a system must also induce bone growth, or possess the attribute of osteoinductivity. The inventors have shown that osteoinductive and therapeutic agents can be incorporated within the calcium phosphate structure during the coatings process.
In order to combine the attributes of osteoconductivity and osteoinductivity to the overall process by the use of biological active substances (growth factors, bone morphogenic proteins (BMP), stem cells-osteoprogenitor cells, progenitor cells, bone marrow cells, etc) it is necessary to propose minor additional considerations to the above set forth inventive process. For example, deposition experiments have shown a strong bonding affinity between CaP particles and osteogenic proteins in solution. This can be explained by electrostatic interactions between the proteins, Ca2+ and PO4− ions in the aqueous environment. It is hypothesized that the system free energy is reduced by complexation between the proteins and CaP particles, as described in classical equation of heterogeneous nucleation.
Maintaining the biological activity of therapeutic agents during their incorporation within the CaP matrix is of primary importance. The therapeutic agents are denatured by extremely acidic, or moderately basic environments. It has been observed that the range of pH values used in the described process does not reduce the biological activity of the incorporated agents. In fact, recent research suggests that the application of a BMP+CaP coating to resorbable polymeric substances may actually help preserve the potency of the BMPs, since the breakdown products produce a slightly acidic environment. For the addition of BMPs to the process, the substrate cleaning regime and pretreatment procedure as set forth hereinabove remains the same. The preparation of the supersaturated calcium phosphate solution (SCPS) adds an additional step as follows:
The immersion process set forth above remains the same. However, the invention proposes an additional step after the immersion process is completed. Release profiles of BMPs from CaP films are characteristically bimodal, with an initially high diffusion rate that tapers off to a more constant rate over time.8,10,12 The second phase of release would sustain cellular activity for an extended period of time, leading to a higher probability of successful bone fusion. Secondary release would also enhance the rate of bone growth and confine the BMPs to a more local region of application. To support this bimodal release profile the invention proposes, as a final step after the immersion process is completed and immediately before implantation, that the implant materials having the BMP+CaP coating thereon is dipped in BMPs just prior to implantation. Once implanted, the BMPs coated on the implant materials as a result of the dipping step will provide an initial burst of cellular activity at the proposed fusion site due to its water-soluble nature of the site. Due to its characteristic bimodal nature rate of the release profile, after the initial burst of cellular activity the BMP's will begin to defuse away from the CaP coating and continue cellular activity at the fusion site. This cellular activity will be more localized and sustained for a longer period of time than the prior art practice of using a BMP soaked collagen sponge. The CaP film will begin to dissolve allowing for the usage of the calcium phosphate ions in the mineralization process. The CaP film will also act as a scaffold for new bone formation at the proposed fusion site. Degradation of the interbody device will allow for greater load-sharing to take place between the device and the biological system, which will enhance fusion quality and rate.
It is envisioned that the implant with a CaP+BMP coating thereon is also intended to be used as a delivery vehicle for stem and/or progenitor cells. Differentiation and adhesion of progenitor cells is enhanced by the nano-scale topography and favorable surface chemistry of the BMP+CaP films. While the exact mechanism is not clearly understood, it is hypothesized that the combination and frequency of micro- and nano-scale roughness of the coating enhances the adhesion of the cells. Surface energy parameters, such as the electron acceptor parameter may also explain the increase in differentiation and adhesion. For this application the same procedure as set forth above is used. The only additional step is that after the implant with the BMP+CaP co-precipitated coating thereon has been dipped in a BMP solution the coated device is allowed to dry and after the material has dried, the cells are cultured on the surface of the implant. The culturing process is not specific. Nothing that occurs during the culturing process will detrimentally affect the coatings or implant materials.
While the present invention has been described in terms of a preferred embodiment, it is apparent that other forms could be adopted by one skilled in the art. For example the teachings of the present invention encompass any reasonable substitutions or equivalents of claim limitations. Those skilled in the art will appreciate that other applications, including those outside of the biomedical industry, are possible with this invention. Accordingly, the present invention is not limited to only the disclosed preferred embodiments and its equivalents. Accordingly, the scope of the present invention is to be limited only by the following claims.
The application claims the benefit of Unites States Provisional Application Ser. No. 60/852,545, filed on Oct. 18, 2006.
| Number | Date | Country | |
|---|---|---|---|
| 60852545 | Oct 2006 | US |
