The early fixation of bone screws after surgical implantation still remains a challenge in the field of traumatology. Whilst hydroxyapatite (HA) coatings are known to enhance the fixation of implants; their removal at a later time-point may be problematic. An HA coating has been developed to demonstrate that both implant fixation and safe removal are feasible in the same design. Accordingly the aim of this study was to compare the In-Vivo performance of thin biomimetic HA coated titanium screws to uncoated counterparts used as control after bilateral implantation in the femoral condyle of 36 New Zealand White Rabbits. The screws were analysed macroscopically, by histology, micro-CT and biomechanically at both two and six weeks post-implantation. The HA coated screws demonstrated excellent biocompatibility. At two weeks the HA coated screws demonstrated a significant increase in removal torque values as well as a strong trend towards higher pull-out forces. In addition histology confirmed a higher degree of osseointegration and direct bone to implant contact. At six weeks no difference in pull-out force and removal torque could be detected. SEM images confirmed the absence of any residual HA coating indicating a fast coating degradation <i>In-Vivo</i>. The low level of removal torque after full osseointegration at 6 weeks supports the feasibility of safe and easy removal of the implant. The HA coating under study appears to offer a unique characteristic of enhanced fixation with a minimal increase in removal torque after full osseointegration. This may be of value in clinical applications where it is necessary to assure both screw fixation and later removal.
As a sequel to the internal fixation of bony fractures implant loosening, migration and screw cut-out are major post-surgery complications that may require a second surgery to remediate [
After implantation in-vivo in bone there is typically an early resorption phase close to the implant [
A lapine animal model was used as it was thought to be sufficiently sensitive to demonstrate differences at two selected time points with respect to screw pull-out and screw removal torque. This was done at both two and six week time periods post implantation to represent early implant stabilization and subsequent full osseointegration.
The goal of the study was to determine if there would be improved implant fixation, at each time point, as measured by screw pull-out force: additionally that there would be a relatively modest screw removal torque indicating relatively low risk of damage to either the screw or the bone.
Ti6Al4V Ti cancellous screws (Ø 4.0 mm × 14 mm), obtained from Stryker AG (Selzach, Switzerland) were used as control samples. Thin HA coated (1 - 2 µm) cancellous bone screws (same type) served as test samples.
Untreated Ti cancellous bone screws were first sonicated in acetone for 5 minutes, then sonicated in ethanol for 5 minutes, and finally sonicated for 5 minutes in deionized water. An alkaline pretreatment was performed by placing the screws in a 5 M NaOH solution for 10 minutes at 70˚C. Thereafter the screws were placed in 1 liter of phosphate buffered saline (Dulbecco’s PBS, Sigma, Steinheim, Germany) for 72 h at 70˚C. A holder was devised that prevented coating of the screw heads and allowed for all screws to be simultaneously coated. Stirring of the PBS with a magnet stir bar was adjusted to minimize the formation of HA aggregates on the HA coating. After removal from the PBS solution, the screws were removed from the holder, rinsed in deionized water and dried in nitrogen atmosphere. HA coated Ti screws were denoted as Ti-HA followed by a characteristic suffix.
All animal work was approved by both an internal and an independent external ethical committee and performed according to the ethical guidelines of NAMSA Lyon, Chasse sur Rhône, France.
Thirty-two aged (>24 weeks) rabbits plus four reserve rabbits were randomly implanted with one test or one control article in each medial femoral condyle. At either two or six weeks after implantation, the rabbits were euthanized. The local tissue effects and bone healing performance were evaluated by macroscopic, radiographic, histopathology and micro-computer tomography analyses. The biomechanical properties of the bone/screw interface were evaluated with removal torque measurements. Alternatively, a pull-out test was performed at both time points. All investigations performed are summarized in
The reserve rabbits were operated on using the same surgical procedure as the other rabbits. They were followed in exactly the same manner up to the end of the study and were planned to potentially replace rabbits removed from the study in case of unforeseen rabbit loss or any adverse event. At the end of the study, no abnormal event had occurred requiring the inclusion of these rabbits. Therefore, all reserve animal sites were sampled (control and test) and used for biomechanical testing.
The surgical procedure was performed by an experienced veterinary surgeon using standard aseptic techniques. A skin incision was made on the medial side of the femur at the level of the epiphysis. The muscles were separated using blunt dissection to access the medial condyle and the periosteum was removed from the condyle surface. The entry point for drilling was localized between the distal part of the growth plate and the insertion of the collateral-lateral ligament. The direction of drilling was perpendicular to the bone surface. Bone defects were made using a cordless power tool. Drilling was initiated by introducing a 1.4 mm diameter guide wire to the depth of the opposite cortex. To avoid perforation of the lateral cortex, the guide wire was laser marked to indicate 10 mm insertion depth. A 2.7 mm diameter cannulated drill was then inserted with a stop at 10 mm depth. Continuous irrigation was applied to limit the thermal impact of drilling. After removal of the drill, the defect was extensively rinsed with saline to remove bone debris. The guide wire was removed together with the drill and the guide wire was not replaced and the tapping procedure was conducted without the guide wire. The drill
Time-period | 2 weeks | 6 weeks | |||
---|---|---|---|---|---|
Group | Test group | Control group | Test group | Control group | |
Number of sites | Histopathology analysis & micro-CT | 4 | 4 | 4 | 4 |
Pull-out test | 6 | 6 | 6 | 6 | |
Removal torque measurement | 6 | 6 | 6 | 6 | |
Reserve sites | 2 | 2 | 2 | 2 | |
Total | 18 | 18 | 18 | 18 |
hole was tapped using a 4 mm diameter screw tap. The guide wire was removed and the defect was rinsed again with saline to remove bone debris. The spacing device was used to create a standardized distance between bone surface and screw head. The screw was inserted until it was in contact with the spacer and was then unscrewed 1/4 turn to release the spacer. The standardized gap between screw head and bone was necessary to ensure that there was access for the pull-out test jig and additionally to prevent bone overgrowth covering the screw head. These steps were taken to minimize biological variance impact on the pull-out and removal torque measurements. The contralateral femur was similarly treated. The incisions were then closed by suturing the muscle and cutaneous layers separately with absorbable suture (Vicryl or PDS™ II, Ethicon). Medio-lateral and antero-pos- terior radiographs were taken to locate each screw (angle and insertion depth). Two and six weeks after implantation, the rabbits were sacrificed by an intravenous injection of pentobarbital (Dolethalâ, Vetoquinol) preparation.
The evaluation offixation strength by a pull-out test for each screw implanted in each distal femoral metaphysis was enabled by the preset 2.5 mm distance between the screw head and the bone surface allowing for the use of a special pull-out tool. The femurs were shortened and embedded in PMMA in containers as described above for the removal torque measurement. The containers were removed from the embedding tool and then each was placed in a materials testing machine (Z10, Zwick, Einsingen, Germany) in a special fixture, cf.
The crosshead of the material testing machine was moved upwards at a constant displacement rate of 5 mm/minute force and displacement were continuously registered until a clear drop in the pull-out force was observed.
A removal torque test was carried out to evaluate the torsional fixation of the screws. For this purpose a custom-made torsion apparatus was used. After explantation the femurs were shortened to 15 - 20 mm length, including the distal end, and were then embedded in polymethylmethacrylate (PMMA) (Technovit 3040, Heraeus Kutzer, Wehrheim, Germany). To achieve an exact alignment of the screw with the removal tool (standard surgical hex key) a special embedding tool was designed allowing for simultaneous embedding of 8 femurs, cf.
Subsequently the containers were mounted in the custom made test assembly, cf.
The effective torsion moment was the product of the registered force Freg, and the lever arm l (35 mm).
Selected coated and uncoated Ti bone screws were imaged with SEM (XL30 ESEM, FEI, Netherlands) using the secondary electron detector.
After complete fixation, implantation sites were dehydrated in alcohol solutions of increasing concentrations,
cleared in xylene and embedded in PMMA resin. After micro-CT analysis, one longitudinal central cross section of each site was obtained by a micro-cutting (thickness of each section between 20 to 30 µm) and grinding technique. Sections were stained with modified Paragon for qualitative, semi-quantitative and quantitative analysis.
Quantitative histomorphometric analyses were conducted by scanning and examining slides with a Zeiss Axioscope microscope equipped with a color images analyzing system (Samba, version 4.27, Samba Technologies, France). The regions of interest (ROI 1 and 2) were investigated as defined in
Quantitative analyses were further performed to assess the percentages of the contact and area density parameters within the ROIs 1 and 2. The contact parameters describe the bone to implant contact as well as the soft tissue contact and the bone marrow contact. The area density was divided into bone area density (percentage of the ROI occupied by bone tissue in terms of surface area), as well as soft tissue area, bone marrow area and implant area density. The quantitative results of ROI 1 and ROI 2 were summed for analysis and the resulting average values were evaluated.
Prior to micro-CT scanning 14 screws were removed from the femurs (7 control and 7 test articles after 6 weeks in-vivo). The bones were placed with their embedding containers (including the PMMA) in a micro-CT (SkyScan 1172, Bruker-Mikroct, Kontich, Belgium) and scanned at a resolution of 30 µm. Subsequently, the cortical thickness was measured at 4 points (2 in 2 perpendicular planes) using an evaluation tool provided by the CT manufacturer (Skyscan) and averaged, cf.
After fixation and resin inclusion and before histological evaluation, the sites were analyzed via micro-CT (Bruettisellen, Switzerland). The samples were scanned by micro-computed tomography (CT 100, Scanco Medical AG, Switzerland) using an energy of 90 kVp at an intensity of 88 µA. The integration time was adjusted to 600 ms at a double fold frame average. Nominal isotropic resolution was set to 11.4 m. To segment bone around the implant, measured data was filtered using a three-dimensional constrained Gaussian filter with finite filter support (1 voxel) and filter width (sigma = 0.5). The outer border of the volume of interest (total volume = TV) for the bone region around the implant was defined by apredefined distance of 0.5 mm. from the implant thread surface cf.
Bone and implant were segmented as separate phases. As the interface of the implant to bone was blurred due to the partial volume effect, morphologic operators were used to identify the bone to implant contact area. The bone implant contact (BIC) was then analyzed slice by slice. As part of the implant head of the screw was outside the bone, the analysis region was limited to a height of 860 voxels starting 2 voxels above the tip of the implant.
After summarizing the valid data points, all relevant descriptive parameters were identified to assess the mean and variance of the data. Normality of all continuous variables was assessed with the Shapiro-Wilk Test [
The median of the maximum pull-out forces at two and six weeks post implantation are described in
Maximum removal torque values of both control and test articles at two and six weeks after implantation are summarized in
The SEM images shown in
In general, at two weeks a residue of the HA coating was found on the screw portion that was inserted into the bone. HA was visible below the screw head and down to just above the first thread and some proportion was
covered with HA growth, cf.
The HA coated test screws displayed clean areas without residual HA coating as well as spots clearly demonstrating residual HA at the screw interface. Cell attachment was observed within these areas.
At six weeks in-vivo the control screws showed areas of cell attachment directly at the titanium surface. The test articles did not show any evidence of residual HA at six weeks implantation in-vivo and only minor cell attachment, cf.
A total of 8 sections (4 control sites and 4 test sites) were analyzed for each time point. At two weeks implantation, the histopathologic analysis showed no local adverse effects in the control and test groups. Qualitatively a higher level of osseointegration was observed with the test article, cf.
At six weeks, the bone tissue matured in the two groups. Although a slightly higher amount of bone tissue was qualitatively observed with the test group, quantitatively no significant difference was seen between the test and control groups, cf.
In general at six weeks there was evidence of thickening of the bone trabeculae together with moderate signs of bone remodeling around the control and the test screws. A moderate grade of cancellous bone was seen to grow towards the surfaces of both the control and the test sites. The signs of osseointegration and osteoconduction slightly increased compared to two weeks for both groups. The osseointegrated bone debris (surgery-related) was remodeled after this time period. A significant increase of bone density in the test group was noted between two and six weeks, while bone density remained stable in the control group.
In conclusion, the HA coated test screw slightly outperformed the control article in terms of qualitative peri- implant bone healing, however, with the limitations of the study this difference did not achieve statistical significance.
The main finding during the micro-CT evaluation after removal torque testing was that the cortices were much thinner than the 1.5 mm. This was the threshold value above which Seebeck et al. [
The micro-CT for the ROI close to the interface showed no statistically detectable difference in bone volume
density (BV/TV) between test and control at two and six weeks. However, at six weeks a slight trend towards higher bone volume density was observable for the test articles, cf.
The present in-vivo HA performance study generated a number of findings. Whilst there was no detectable impact of the HA coating during surgical implantation, there were substantial differences in in-vivo behavior as demonstrated by the biomechanical and histological analyses. The measured removal torque significantly in-
creased for the HA coated test screws after two weeks compared to control screws. The same trend was seen in the pull-out forces measured at two weeks. These results, supported by histological results, demonstrate more rapid implant stabilization has been effected by the thin biomimetic HA coating. At six weeks no trends or statistical differences could be found within the biomechanical analyses. Whilst there was an increase in removal torque at 6 weeks the values measured were almost an order of magnitude below the torques that would damage the screwdriver or the screw itself. Therefore the removal of the screws was safe and straight forward even after full osseointegration had occurred. SEM analysis of the screw surface after removal torque measurements demonstrated signs of residual HA after two weeks, but no residual HA coating at six weeks, which confirms remodeling occurred during this time period. A positive effect of the coating was seen in the histological analyses where the HA coated screws induced a greater bone thickening and increased bone to implant contact.
It is reported that in the literature that HA is able to increase the osseointegrative ability of screws and fixation pins and results in increased fixation strength of up to 2.5 times that of non-coated implants as measured by removal torque [
The six week time period was selected as an appropriate time point to look at the histological and biomechanical behavior after full osseointegration of the implant. Slaets et al. [
After HA remodelling and full osseointegration, the implant provided a good in growth and attachment to the bone, which was seen from histology. The values measured at six weeks for removal torques and pull-out forces comparing test samples and control articles resulted in no statistically observable difference in contrast to what was demonstrated at two weeks. This may be explained by the remodelling of the HA coating in-vivo. During SEM evaluation of the surface after screw removal only minor residual HA coating could be found on the HA coated screws after two and practically none at six weeks. The wear off or delamination of the HA coating during insertion can be excluded as there was evidence of significant differences in behaviour between HA coated and non-coated screws. Therefore, it could be postulated that the thin HA coating remodelled very rapidly in-vivo and that the absence of the coating at six weeks enabled easy and safe removal of the HA coated screws. Another potential explanation for the absence of coating could be that it disconnected during screw removal from the bone. This would be supported by the fact that only minor areas of HA coating could be seen even at two weeks in-vivo. The microstructure of the titanium surface underlying the HA-coating was not changed by the NaOH pretreatment prior to the coating process, as confirmed by inspection with SEM (not shown). Thus, the implant surface showed the same removal characteristics as seen for the non-coated reference implant after HA remodelling and the screw was removed without damage to the bone or to the surrounding tissue. The implant did not show increased pull-out forces as observed for plasma-sprayed HA coatings, which have very slow degradation rates [
To summarize, the two weeks histological evaluation, pullout and torsion tests showed effects and suggest that the coating had a measureable influence. In contrast the micro-CT showed no significant detectable difference for the defined ROI. Therefore the HA coating seems to have an early, very local, effect and there are some signs, although non-significant, that this early effect directly at―or close to―the implant interface is seen at the later time point in the form of increased bone formation. The HA coating does not act like an active pharmaceutical ingredient diffusing from the implant and additionally affecting the biological structures in the vicinity of the implant. A distance effect from the implant was reported earlier both by Peter et al. and Wermelin et al. [
Micro-CT measurements performed after removal torque evaluation gave information on the cortical thickness of the bones. The cortices were measured to be far below 1.5 mm. Seebeck et al. [
The in-vivo performance of the HA coating on titanium screws confirmed stabilization of the implant which was still safely removable even after osseointegration. Significantly higher removal torques and a trend towards higher pull-out forces were measured for the HA coated implant compared to the non-coated implant after two weeks in-vivo. This supported the hypothesis of more early and more rapid stabilization in the presence of the HA coating. Nevertheless, after full osseointegration of the screw implants, the biomechanical tests demonstrated safe and relatively easy implant removal, which is an absolutely essential requirement for such implants if they fail or require revision surgery. SEM images confirmed disconnection of the HA coating and/or HA remodeling in-vivo. Histology confirmed that the HA coating was not visible at both time points and had been replaced with newly formed bone that was intimate with the implant surface, whereas controls showed greater fibrous tissue at the implant surface. This unique “disconnection” characteristic has never been demonstrated before. Metallic implants with bioactive HA coatings are expected to enable the implant to bear load at earlier time points. This was confirmed in the present study by the significantly higher biomechanical removal torques for the HA coated screws after osseointegration in-vivo. Thus the HA coating design goal of enablingmore rapid fixation post-surgery whilst being still safely removable after full osseointegration was achieved. Additionally the structure of the coating provides the ability to load it with active pharmaceutical ingredients and further focus on anti-infective and/or anti-osteoporotic topics.
The European Commission as part of the 7th Framework programme―BIODESIGN as well as the Swedish Science Council is gratefully acknowledged for financial support. Claudia Beimel is acknowledged for performing the statistical analysis. NAMSA is acknowledged for performing the animal study.