Enhanced bone healing around nanohydroxyapatite-coated polyetheretherketone implants: An experimental study in rabbit bone.

Journal of Biomaterials Applications http://jba.sagepub.com/

Enhanced bone healing around nanohydroxyapatite-coated polyetheretherketone implants: An experimental study in rabbit bone S Barkarmo, M Andersson, F Currie, P Kjellin, R Jimbo, CB Johansson and V Stenport J Biomater Appl published online 10 July 2014 DOI: 10.1177/0885328214542854 The online version of this article can be found at: http://jba.sagepub.com/content/early/2014/07/10/0885328214542854 A more recent version of this article was published on - Oct 3, 2014

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Hard Tissues and Materials

Enhanced bone healing around nanohydroxyapatite-coated polyetheretherketone implants: An experimental study in rabbit bone

Journal of Biomaterials Applications 0(0) 1–11 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214542854 jba.sagepub.com

S Barkarmo1, M Andersson2, F Currie3, P Kjellin3, R Jimbo4, CB Johansson1 and V Stenport1

Abstract Objective: To investigate the bone response to threaded polyetheretherketone (PEEK) implants coated with nanohydroxyapatite. Materials and methods: A total of 39 PEEK implants were coated with nanocrystalline hydroxyapatite and 39 uncoated implants were used as controls. The implant surface was characterized by optical interferometry and scanning electron microscope. The implants were inserted in the tibia and femur of 13 rabbits. After 6 weeks of healing, quantitative and qualitative analyses were performed. Results: The test implants showed significantly higher removal torque test values compared with the control group. Histomorphometric evaluation demonstrated higher bone-to-implant contact for the test implants; however, there were no differences in bone area between the groups. Qualitative histological analyses demonstrated inflammatory cellular reactions in close vicinity of both implant surfaces. A two-cell layer of foreign body giant cells was observed irrespective of sample type. Conclusion: Our findings demonstrate that implants with a threaded design render good stability to PEEK in both coated and uncoated implants. Nanohydroxyapatite-coated PEEK implants demonstrated improved bone formation compared with uncoated controls. Keywords Polyetheretherketone, nanotopography, hydroxyapatite, osseointegration, in vivo

Introduction Polyetheretherketone (PEEK), a non-resorbable polymer, has been commonly used in orthopedic surgery and spinal implant applications because of its excellent mechanical properties and biocompatibility.1 The PEEK material has similar elastic modulus to the cortical bone, which reduces the risk of stress shielding around the implant, and makes it a suitable material for orthopedic and spinal applications.2–4 Flexible manufacturing, the fact that it is easy to sterilize, and radiographic radiolucency are other advantages of the PEEK material. Moreover, PEEK is a bio-inert material resistant to chemical and thermal degradation due to its chemical structure. Its biocompatibility has been shown in both in vitro5,6 and in vivo studies.7 In one study, even though direct bone contact to PEEK

material was observed, the implants were also partially surrounded by fibrous tissue in interbody spinal fusion of sheep,8 suggesting that the PEEK alone has low bioactive properties. Therefore, researchers have focused 1

Department of Prosthodontics, Institute of Odontology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 2 Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg, Sweden 3 Promimic AB, Stena Center 1B, Gothenburg, Sweden 4 Department of Prosthodontics, Faculty of Odontology, Malmo¨ University, Malmo¨, Sweden Corresponding author: S Barkarmo, Department of Prosthodontics/Dental Material Sciences, The Sahlgrenska Academy, Institute of Odontology, University of Gothenburg, Box 450, 405 30 Gothenburg, Sweden. Email: [email protected]



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on techniques to apply biologically active agents to induce improved osteogenesis. Incorporation of bioactive materials into the polymer9 or modifications of the surface properties10 are two ways to achieve this. It has been proposed that a surface with low energy, such as PEEK, leads to decreased protein adsorption.11 Fibronectin, a protein important for cell adhesion, has shown to have better orientation on a hydrophilic surface. Consequently, alteration of the surface energy could lead to a surface better suited to osteoblasts creating faster bone integration.12–14 The chemical composition and the roughness of the surface are two factors that can be modified to create higher surface energy and thus promote bone healing.15 This can be achieved in a number of ways, for example by applying coating with osteoconductive properties on the PEEK surface, such as with hydroxyapatite (HA).10,16–18 Due to its similarity to the mineral phase of natural bone tissue, artificial HA has been used over several decades as a bioactive coating material19,20 and has been shown to have enhancing effects on osseointegration when coated onto implants.21 There are several techniques for coating HA onto PEEK surfaces. For example, surface modifications have been performed through electron beam deposition of titanium,22 plasma spray deposition of HA,10 and spin coat deposition of a thin layer of nanocrystalline HA.23 Some concern has been raised over the thickness of the HA coating and studies using thicker coatings of >10 mm have demonstrated complications with detachment of the coating from the bulk material.24,25 To avoid detachment, a thinner and more stable, nanometer-thick coating technique has been developed.26–28 Surface topography influences bone response not only at a micrometer level but also at the nanometer level of implants.29 Titanium implants coated with nanometer-sized HA particles have shown enhanced early bone formation in vivo.26 Even though the cellular effect on the osteogenesis has not been fully clarified, it has been suggested that the nanometer-sized particles facilitate adhesion of osteoblasts to the implant surface to accelerate bone formation.30–32 The beneficial influence on the bone healing around nanoHA-coated implants has been proposed to be caused by the surface nanotopography, along with altered chemistry, which together give a synergistic effect.33,34 In a previous study, cylinder-shaped PEEK implants were coated with nanoHA using a spin coating technique.23 Several of the implants were shown not to integrate, which was interpreted as a lack of primary

stability due to the cylinder shape. It is known that good initial stability is important for successful osseointegration35 and that implant design will influence the primary stability.36 A threaded, screw-shaped implant, compared to a cylinder-shaped implant, can offer better stability and an improved healing process without movement of the implant. The purpose of the present study was to investigate the bone response to threaded PEEK implants with nano-sized HA compared with uncoated implants.

Materials and methods Implants In total, 78 threaded PEEK implants were used in the present study. The implants had a diameter of 3.5 mm at the top and 3.2 mm at the bottom and a length of 4 mm. The upper part of the implants was straight and the lower part had a taper of 7.15 . The geometry of the implants is shown in Figure 1. The implants were divided into two groups. Half (39 implants) were used for nanoHA coating (test) and the other half were used as control implants (39 implants). All the implants were machined by PEEK-Optima (Invibio Ltd.) at Elos Medtech Pinol, Gørløse, Denmark.

Nanohydroxyapatite surface treatment The test implants were coated with nanocrystalline HA, according to the Promimic HAnanoTM method.37

Figure 1. Geometry of the threaded polyetheretherketone (PEEK) implants used. Measurements are given in mm.



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Prior to the coating procedure, the implants were ultrasonically cleaned with isopropanol and water to remove possible contamination from machining. The coating solution was a dispersion of nano-sized HA. The HA particles were produced in a liquid crystalline phase containing surfactants, organic solvent and an aqueous solution of Ca(NO3)2 and H3PO4.38 The liquid crystalline phase was diluted with organic solvent to reduce viscosity and create the coating dispersion. The dispersion was applied onto the implants by spin coating, performed at 2700 r/min for 5 s, and left to dry in air for 10 min. The implants were then heat-treated at a temperature of 325 C in an oxygen atmosphere to remove the surfactants and improve the adherence of the crystals to the polymer surface. The thickness of the resulting HA coating is inversely proportional to the spin coating rotational speed; the layer becomes thinner with increased rotating speed.39 The method resulted in an approximately 5–20 nm thin layer of crystalline HA on the implant surface.23 The implants were disinfected by immersion in 70% ethanol solution and dried at 120 C before insertion in the rabbits.

Implant surface analysis Scanning electron microscopy. The implant surfaces were analyzed with a scanning electron microscope (SEM) (LEO Ultra 55 FEG; Zeiss, Oberkochen, Germany) using an acceleration voltage of 4 kV. Prior to the SEM analysis, the implants were sputtered with a 40 nm thick layer of gold to eliminate any charging effect and improve the contrast. Three implants of each type were examined at a randomly chosen location using different magnifications. Topography. Three implants from each group were topographically characterized at micrometer level using an optical interferometer (MicroXamTM; PhaseShift, Tucson, AZ, USA). Each implant was measured at five thread peaks. A high-pass Gaussian filter of size 50 50 mm2 was used to separate roughness from errors of form and waviness, as recommended by Wennerberg and Albrektsson.40 The evaluation was performed with Surfascan software (Somicronic Instrument, Lyon, France). The following parameters were measured: Sa (mm) ¼ average roughness; average height deviation from a mean plane within the measuring area. Sds (1/mm2) ¼ summit density; the number of summits per unit area. Sdr (%) ¼ developed interfacial area ratio; additional surface area contributed by the roughness, compared with a totally flat plane.

Animals and surgical technique A total of 13 female New Zealand White rabbits were used in the experiment, which was approved by the animal ethics committee at Gothenburg University. The animals were adult (9 months of age) and weighed between 3.5 and 4.5 kg. The rabbits received one implant in each distal femoral metaphysis and two implants in each proximal tibia metaphysis. The animals were kept in separate cages during the complete healing time and had free access to tap water and standard diet. Antibiotics (Borgal; Intervet, Boxmeer, The Netherlands) were administered prophylactically at a dose of 0.5 mL per kg body weight at the time of surgery and for the following 3 days. At surgery, general anaesthesia was induced by intramuscular injections of fentanyl 0.3 mg/mL and fluanisone 10 mg/mL (Hypnorm; VetaPharma Ltd, Leeds, UK) at an initial dose of 0.5 mL per kg body weight and an intraperitoneal injection of diazepam (Stesolid Novum; Actavis, Hafnarfjordur, Iceland) at a dose of 2.5 mg per animal. Additional doses of Hypnorm at 0.1 mL per kg body weight were given during the surgical procedure. The rabbits’ hind legs were shaved and cleaned with clorhexidin. Local anaesthetic lidocain (Xylocainß; Astra Zeneca, So¨derta¨lje, Sweden) at a dose of 1 mL was injected at each insertion site in connection to the surgery. The skin and fascial layers were opened and closed separately. Both layers were sutured with resorbable sutures. The periosteum was gently pulled away and was not resutured. The implant sites were prepared using low speed bur with a graded series of drills with increasing diameter under saline irrigation and aseptic conditions. The same person inserted all implants. The animals were allowed to bear their full body weight immediately after surgery. Six weeks after implant insertion the animals were sacrificed with pentobarbital 60 mg/mL (Pentobarbitalnatrium vet; Apoteksbolaget AB, Stockholm, Sweden) after sedation with 1.0 mL Hypnorm.

Removal torque test After completed follow-up time, 32 of the implants in tibia were subjected to removal torque (RTQ) test. On three rabbits, both tibial implants were removal torquetested (n ¼ 12), while in ten rabbits the distal implants were tested (n ¼ 20). The proximal implants in these rabbits were retrieved for further analysis not reported in this study. The test was performed with a manual torque wrench with a strain gauge (BTG90CN-S; Tohnichi, Tokyo, Japan). The RTQ test itself is a destructive test that provides a direct reading of the implant



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stability in the bone bed. The torque necessary to loosen the implant from the bone bed is registered and the received value is recorded in Newton centimetre (Ncm).

Histomorphometric analysis All implants in femur were removed en bloc with the surrounding tissue and prepared as sections of cut and ground undecalcified tissue. The specimens were immersed in 4% neutral buffered formaldehyde, dehydrated in increasing grades of ethanol, pre-infiltrated in diluted and pure resin and subsequently imbedded in light-curing resin (Technovit 7200 VLC; Heraeus Kultzer GmbH & Co., Wehrheim, Germany). Preparation of undecalcified cut and ground sections with implants in situ was performed using the Donath technique and EXAKTÕ equipment (EXAKT Apparatebau GmbH & Co., Norderstedt, Germany) to a final thickness of approximately 10–20 mm.41,42 All sections were histologically stained in toluidine blue mixed with pyronin G and the most central section of each implant was used for quantitative and qualitative analysis. The histomorphometric quantifications involved the following:

(iii) in the ‘‘mirror image BAs’’ outside the three best consecutive threads.

Mean values were calculated for the various regions that were involved in the measurements. These mean values were used for statistical comparisons of test and control samples.

Statistical analysis Statistical significance was evaluated using SPSS version 21.0 (SPSS IBM, Chicago, IL, USA). The histomorphometric data and the RTQ test results were analyzed with the Wilcoxon signed rank test. The significance level was considered as p 0.05.

Results Implant surface analysis Scanning electron microscope. SEM of the non-coated PEEK (control) and the nanoHA-coated (test) implants are shown in Figure 3. As can be seen in Figure 3(c) and (d), which were obtained at 60,000 magnification, the nanoHA-coated surface was completely covered by

Bone-to-implant contact The bone-to-implant contact (BIC) measurements were performed with a Leitz Metallux 3 light microscope (Leitz, Wetzlar, Germany) coupled to a Leitz Microvid unit connected to a PC. The measurements were performed directly in the eyepiece of the light microscope using both a 10 and a 16 objective. Measurements of the percentage of the BIC were performed along the entire length of both sides of the implant.

Bone area Microscopic images were acquired with a Nikon DSRi1 camera connected to a light microscope (Nikon Eclipse ME 600 L, Tokyo, Japan) with a 5 objective. The bone area (BA) measurements were conducted using semi-automatic image analysis software from Cuanto Implant (Uppsala, Sweden).43 The BA percentages inside the threads were calculated for both sides of the implant. Calculations were made in various regions of the implant (Figure 2): (i) in all threads around the entire implant, (ii) in the three best consecutive threads, and

Figure 2. Survey figure of the histological stained cut and ground section of a polyetheretherketone (PEEK) implant demonstrating the semi-automatic measurements of bone area (BA) both inside the threads and in the mirror image (MI) region outside the same threads. Bar ¼ 200 mm.



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elongated, nano-sized particles, which are absent on the non-coated surface. At lower magnification (20,000), comparing Figure 3(a) and (b), the HA particles on the test implant can be seen to have formed a uniform coating, covering the whole surface without alteration of the underlying microtopography. Topography. The surface topography measurements are presented in Table 1. Interferometer analysis revealed small differences between control and test implants with respect to all parameters tested (Sa, Sds, and Sdr), suggesting that application of nanoHA did not alter the microstructure.

Animals and surgical technique All 13 animals completed the 6 weeks’ follow-up. The healing was uneventful.

This resulted in RTQ results for 15 control and 15 test implants. The internal connection on the implant neck on three of the test implants was fractured at 14.5, 26, and 32.5 Ncm. These implants were therefore not loosening from the bone and higher values could not be received. The results of the RTQ test are presented in Figure 4. Mean values were significantly higher for the test implants compared with the control implants (p ¼ 0.001). The mean value for the test implants was

Table 1. Results of the surface roughness measurements using an interferometer. Implant

Sa (mm) SD Sds (/mm2) SD

Sdr (%) SD

Removal torque test

Control 0.85 0.29 Test 0.93 0.25

346,451.61 28,809.03 35.65 10.87 363,541.37 21,238.55 34.11 12.31

In one rabbit, we had technical difficulties to attach the torque peg into the implants. Consequently no removal torque values could be obtained for that animal.

Sa ¼ average roughness; Sds ¼ summit density; Sdr ¼ developed interfacial area ratio. The values are presented as means standard deviation (SD).

Figure 3. Scanning electron microscope (SEM) micrographs of the control and test polyetheretherketone (PEEK) implants. (a) Control implant at 20,000 . Bar ¼ 1 mm; (b) test implant at 20,000. Bar ¼ 1 mm; (c) control implant at 60,000. Bar ¼ 300 nm; and (d) test implant at 60,000 magnification. Bar ¼ 200 nm.




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Qualitative description

20 p=0.001 15 10 5 0 nanoHA

Ctr

Figure 4. Graph showing the results of the removal torque (RTQ) test. The bars represent mean values and standard deviation (SD) in Newton centimetre (Ncm). Differences between groups were tested using Wilcoxon signed rank test with significance level p 0.05.

Table 2. Mean values of the percentage of bone implant contact (BIC) along the length of both sides of the implant. Bone area (BA) and mirror image (MI) BA are presented in all threads and in the three best consecutive threads. Standard deviation is given within parenthesis. Results were analyzed with Wilcoxon signedrank test with significance level p 0.05. Parameter

Test implants

Control implants

Statistics

BIC BA, all threads BA, three best MI BA, three best

39 (14) 68 (12) 90 (3) 96 (2)

33 (12) 68 (9) 87 (4) 98 (1)

p ¼ 0.02 n.s. p ¼ 0.05 n.s.

n.s. ¼ not significant.

15.4 8.8 (6.0–32.5) and for the control implants 8.5 5.7 (2.5–21.0).

Histomorphometric analysis The quantitative results (13 test and 13 control implants) for BIC and BA (standard deviation, SD) are shown in Table 2. In general the results of the histomorphometric evaluations indicated higher mean values for the test implants. The mean BIC values were 39% 14 (13.8–60.8) for the test implants and 33% 12 (13.8–59.6) for the controls (p ¼ 0.02). The mean BA in all threads was 68% 12 (44.3– 89.0) and 68% 9 (52.5–81.0) for the test and control implants, respectively. Mean BA in the three best consecutive threads was 90% 3 (84.0–95.0) for the test and 87% 4 (79.6–94.0) for the control implants (p ¼ 0.05). Finally, mean values for mirror image BA in the three best consecutive threads were 96% 2 (90.1–99.5) for the test and 98% 1 (94.3–99.4) for the control implants.

All implants were integrated in the bone tissue without capsula formation. Irrespective of whether they were test or control implants, the inspection of the toluidine blue-stained cut and ground sections using the light microscope revealed that the implants were surrounded by various amounts of mostly newly formed bone tissue, i.e. along the entire surface as well as in the apical region (Figure 5(a)). At higher magnification, demarcation lines were clearly seen between the darker stained new bone and the lighter stained old bone. Various degrees of woven bone in contact with the implant were evident (Figure 5(b)). In some samples, non-mineralized tissue could be seen. They appeared to be osteoid-like structures in close contact with the implant. Osteoblasts were difficult to detect. At the same time bone-forming regions, i.e. bone surfaces with osteoid and visible osteoblasts entrapped in the osteoid layer, could be observed (Figure 5(c) and (d)). However, a distinct osteoblast rim on the osteoid was difficult to detect in these regions being present at some distance away from the implant. The implant surface in soft tissue regions was often covered or coated with an elongated formation of foreign body giant cells (FBGCs) appearing like a collar on the implant surface. This formation was often a two-cell layer collar having darker stained cells in close apposition to the implant surface, and lighter cells towards the tissue (Figure 5(e)). In other areas, where soft tissue regions were present, small blood vessels, fibroblasts, some plasma cells, irregular-shaped giant cells, and several macrophages including foam cells could be observed. The cytoplasm in the latter cells was dark stained (Figure 5(f)). Often cells, when seen inside soft tissue regions, were ovoid or circular and light stained, with a distinct darker surrounding rim (Figure 5(g)). Some, possibly detached, implant material could be observed in phagocytic cells (Figure 5(h)). These findings were similar for both test and control implants.

Discussion The results of the present study demonstrate that nanoHA-coated, threaded implants presented increased osseointegration compared with the uncoated control implants. The results were confirmed by significantly higher removal torque values and percentage of BIC. In an earlier published pilot study, we reported data indicating the enhancing effect of cylindrically shaped, nanoHA-coated PEEK implants.23 This was an interesting model as the implant was cylindrical for the sole purpose of evaluating the effect of the nanotopography. However, there were difficulties in obtaining initial



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Figure 5. Images of undecalcified cut and ground sections from both test and control polyetheretherketone (PEEK) implants inserted in rabbit femur for 6 weeks. The sections were histologically stained with toluidine blue mixed with pyronin G. (a) Survey picture of a test implant demonstrating new bone formation around the entire implant. This particular sample demonstrated 51% bone-to-implant contact (BIC). Bar ¼ 500 mm. (b) Quite extensive, newly formed bone in contact with the implant (test implant) can be observed inside the threads. Arrows show demarcation lines between old cortical bone (OB) and new bone (NB). Bar ¼ 100 mm. (c) Image showing bone-forming regions close to the implant (control implant). The asterisk marks osteoid with a layer of light stained osteoblast on the surface. Such bone-forming regions were more often observed facing the marrow cavity. Bar ¼ 50 mm. (d) At higher magnification an osteoid-like layer can be observed close to the implant surface while in other regions osteoblasts were entrapped in the osteoid layer (arrows). Bar ¼ 20 mm. (e) Implant surface (test implant) in a non-mineralized region covered with an elongated formation of foreign body giant cells (FBGCs) appearing like a two-layer collar on the implant surface (asterisk). Bar ¼ 20 mm. (f) Clusters of multinucleated cells (arrow) and a giant foam cell (arrowheads) including macrophages can be observed inside soft tissue regions close to the implant (control implant). Bar ¼ 10 mm. (g) Large macrophages, both round and ovoid-shaped, and plasma cells (arrowheads) were often localized to soft tissue regions close to the implant surface. Note the irregular shaped surface of the implant (test implant). Some material, which has possibly detached from the implant, can be seen in cells (arrows). Bar ¼ 10 mm. (h) Soft tissue cavity, with ongoing bone formation (osteoblasts can be observed entrapped/embedded in osteoid) and possibly implant material in phagocytic cells (arrow). Bar ¼ 10 mm.



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stability. Therefore in the present study, threaded implants were used for both control and test groups in order to warrant initial stability. The PEEK material has favorable biomechanical properties for various applications in bone; however, improved bone conductive capacity may be beneficial to improve quality and quantity of the surrounding bone. The HA used in the present study was synthesized according to a soft-template method creating nano-sized apatite on the surface that resembles the apatite in bone tissue.38 It has been suggested that the bioactive mechanism of HA is due to reactions that occur, in which ion exchange on the bone–implant interface creates a biologically equivalent apatite layer on the implant surface.44 Nano-sized HA has been applied on titanium implants showing improved bone response;21 however, another study using the same coating does not report any enhanced osseointegration.27 In another study, plasma sprayed HA on CRF-PEEK inserted in rabbit bone showed enhanced bone response after 6 weeks compared to uncoated implants.45 The enhanced bone healing has been attributed not only to the surface chemistry that is received from the HA but also to the nanometer-scaled structures. Meirelles et al.26 showed improved bone healing on electropolished surfaces where the microstructure was removed to investigate the effects on nanoHA particles. It has been proposed that nanometer-sized particles accelerate bone formation by facilitating adhesion of osteoblasts to the implant surface.46 The surface topography at the microlevel, confirmed by the interferometer, presented no significant differences between the control and test groups, which indicates that there were no alterations in the surface microlevel. This is one of the important aspects of the study since our hypothesis was that the nano-sized HA coating would enhance osseointegration. Although the micro roughness should preferably be in the suggested moderately rough range, the objective of the study was to evaluate the effect of nanotopography, and for this reason, we utilized a base substrate with smooth microtopography. A potential risk of using threaded implants is that the coating agent may be sheared off at insertion. Since the coating is nanothin, the latter is difficult to control with the evaluation techniques used in the present study. Even though the surface topography did not show differences on a micrometer scale there were significantly higher RTQ values in favor of the test implants. The higher RTQ values indicate stronger biomechanical bonding between the nanoHA-coated PEEK implants and the bone tissue, resulting in improved osseointegration. We interpret this bone response to

be the effect of the bioactive properties of the coating agent. Other studies have demonstrated positive correlations to RTQ values and bone-contact measurements that also could explain the findings in this study.47 The histomorphometric analysis showed significantly more BIC between the test and control implants after 6 weeks of healing. There were no differences in BA in all threads between the groups but when comparing the three best consecutive threads there was significantly more BA in the test implant group. The quantification of the BA was done using a novel semi-automatic software (Cuanto Implant, Uppsala, Sweden).43 This software categorizes the pixels of the histological images as bone tissue, non-bone tissue or implant. Subsequently, BA in regions of interest is quantified objectively, which can eliminate the variation between different observers. However, it should be noted that measurements with this software can be miscalculated due to both artifacts and differences in staining quality, even if it is possible to make manual corrections after the measurement is done. The most striking qualitative observation in both the test and the control samples was the numerous boneforming regions in close vicinity to the implants although osteoblasts on the osteoid were difficult to detect. However, bone-forming areas located on the opposite side, i.e. facing the marrow cavity, demonstrated osteoid with rims of both dark and light stained osteoblasts of various shapes. The reason for such differences in osteoblast occurrence cannot be explained based on the methods used in this study. In some cases osteoid-like tissue could be observed in direct apposition to the surfaces. Whether these less mineralized regions are related to disturbed bone formation, delayed bone formation or enhanced bone formation can only be speculated. Such regions are rarely observed on metal implants while in the present study they were prominent (C Johansson, personal communication, August 2013). Moreover, the observation of elongated and stretched-out FBGCs is another finding that is not often observed on metal implants such as machined Ti, Nb, and CoCr.47 Machined metal implants are fairly rough and the FBGCs formed are completely different to the ones observed in the present study.48 Moreover, on rough HA-coated surfaces, for example plasma-sprayed HA coatings used for orthopaedic devices and oral implants, the FBGCs that have been observed on the implant surfaces were quite large and round-shaped.49,50 The implants in the present study were ‘‘smooth’’ compared to machined metal implants, for example. This formation of long lines of two-layer cell coating on the implant surfaces may be ‘‘biomaterial-specific’’ and has been observed by others.51,52 In vitro and in vivo studies have shown that ‘‘flat surfaces’’, surface chemistry and surface



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morphology affect macrophage adhesion and the development of FBGC formation.53 The presence of FBGCs is related to chronic inflammation. In order to observe interfacial FBGCs in histological sections from various biomaterials, proper tissue handling and sectioning is of outmost importance. Proper fixation and preservation of the tissue must be done, sections must be thin, and appropriate staining protocols must be followed.54 The observation of ‘‘occasional FBGCs’’ in the paper by Toth et al.8 is most likely due to thick, 150–400 mm sections. The sections in the present study were of 15 mm thickness. Moreover, Toth et al.’s samples were first biomechanically tested followed by preparation of histological sections.8 Biomechanical testing can render interfacial artifacts—another reason for not observing elongated, surface-coated FBGCs. In conclusion, in this study the effect of nanoHAcoated PEEK implants was evaluated using a biomechanical test, as well as histomorphometry and qualitative histology after 6 weeks’ follow-up. Our findings show that the threaded, screw-shaped design gives good bone stability of the PEEK implant, both coated and uncoated. NanoHA-coated PEEK implants demonstrated improved bone formation compared with uncoated controls. Acknowledgements We would like to thank research technician Petra Hammarstro¨m Johansson for preparing the histological sections, and Dr Hamid Sarve for his valuable guidance on the histological evaluation.

Funding This study was supported by grants from the Hjalmar Svensson Research Foundation, the Wilhelm and Martina Lundgren Science Foundation, Sigge Perssons & Alice Nybergs stiftelse (Gothenburg Dental Society), the Royal Society of Arts and Sciences in Gothenburg, and the Nanoscience and Nanotechnology Area of Advance at Chalmers University of Technology.

Declaration of conflicting interests None declared.

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Tissue Healing Around Dental Implants With Marginal Bone Defects With and Without Flap Elevation: An Experimental Study in Dogs.

The combined effects of undersized drilling and implant macrogeometry on bone healing around dental implants: an experimental study.

Microscopy of bone cells, bone tissue, and bone healing around implants.

An unusual bone loss around implants.

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