Implant functionalization with mesoporous silica: A promising antibacterial strategy, but does such an implant osseointegrate?

Abstract Objectives New strategies for implant surface functionalization in the prevention of peri‐implantitis while not compromising osseointegration are currently explored. The aim of this in vivo study was to assess the osseointegration of a titanium‐silica composite implant, previously shown to enable controlled release of therapeutic concentrations of chlorhexidine, in the Göttingen mini‐pig oral model. Material and Methods Three implant groups were designed: macroporous titanium implants (Ti‐Porous); macroporous titanium implants infiltrated with mesoporous silica (Ti‐Porous + SiO2); and conventional titanium implants (Ti‐control). Mandibular last premolar and first molar teeth were extracted bilaterally and implants were installed. After 1 month healing, the bone in contact with the implant and the bone regeneration in the peri‐implant gap was evaluated histomorphometrically. Results Bone‐to‐implant contact and peri‐implant bone volume for Ti‐Porous versus Ti‐Porous + SiO2 implants did not differ significantly, but were significantly higher in the Ti‐Control group compared with Ti‐Porous + SiO2 implants. Functionalization of titanium implants via infiltration of a SiO2 phase into the titanium macropores does not seem to inhibit implant osseointegration. Yet, the importance of the implant macro‐design, in particular the screw thread design in a marginal gap implant surgery set‐up, was emphasized by the outstanding results of the Ti‐Control implant. Conclusions Next‐generation implants made of macroporous Ti infiltrated with mesoporous SiO2 do not seem to compromise the osseointegration process. Such implant functionalization may be promising for the prevention and treatment of peri‐implantitis given the evidenced potential of mesoporous SiO2 for controlled drug release.


| INTRODUCTION
Alterations in manufacturing processes and surgical techniques in implant therapy together with decreasing costs have rendered implant therapy for oral rehabilitation in partially and fully edentulous patients common. Additionally, high success levels (>95%) and good predictability have motivated patients to choose treatment with implants (Buser et al., 2017;Esposito et al., 2014;Naert et al., 2002;Quirynen et al., 2014).
Developments in implant dentistry were mainly related to enhance the rate of implant osseointegration as well as to address conditions with impaired bone quality at the bone-implant interface (Joos et al., 2006;Nkenke & Fenner, 2006;Rupp et al., 2018). In order to accelerate the process of osseointegration and improve the strength of the established bone-implant interface, implant surface characteristics have been investigated extensively (Meirelles et al., 2008;Pellegrini et al., 2018;Smeets et al., 2016;Wennerberg & Albrektsson, 2010). Optimization of the boneimplant interface has been obtained by incorporating inorganic phases within or onto the titanium oxide layer, or by increasing the roughness Dohan Ehrenfest et al., 2011;Kim et al., 2010;Wennerberg & Albrektsson, 2010). Despite that, patients still lose implants as a result of mechanical or biological complications. Summarized research findings point out two aspects as main causes of implant failure: occlusal overload and periimplantitis (Naert et al., 2012;Renvert & Quirynen, 2015;Schwarz et al., 2018).
Research is currently focusing on the development of effective treatment protocols to prevent or to bring to a halt the periimplantitis disease in patients receiving dental implants Qin et al., 2018;Renvert & Polyzois, 2018;Sanz & Giannobile, 2018;Schwarz et al., 2015;Tonetti et al., 2015).
Whereas the surface characteristics of the implant components influence not only the biocompatibility, but also the bacterial adhesion and colonization, the mechanisms restricting the formation of dense layers of micro-organisms, so called biofilms, on these surfaces have been studied, with the rationale to decrease the initial bacterial adhesion and minimize the subsequent inflammation of the peri-implant tissues (Lang et al., 2000;Mabboux et al., 2004;Norowski & Bumgardner, 2009;Sardin et al., 2004;Sennhenn-Kirchner et al., 2009;Zhou et al., 2017). Among the possibilities to prevent or treat the increasing peri-implantitis incidence and at the same time reduce the risk of antibiotic resistance development related to systemic drug administration are implant surface modifications that prevent adhesion of pathogens at the implant surface (e.g., nanostructured TiO 2 ), the local administration of antimicrobial or antibiofilm drugs at the implant site (e.g., Zn-or Ag-modified TiO 2 , chlorhexidine-grafted titanium, chlorhexidine releasing hydroxyapatite coatings), or the physical removal (debridement) of biofilms (Campoccia et al., 2013;Hallström et al., 2012;Qian et al., 2012;Xu et al., 2017). In that context, we previously designed a dental implant composed of a porous titanium-silica (Ti/SiO 2 ) composite material and containing an internal reservoir that can be loaded with antimicrobial compounds. The antimicrobial compounds can diffuse in a controlled manner through the porous implant walls, thereby reducing microbial biofilm formation on the implant surface (Braem et al., 2015;De Cremer et al., 2017). Hence, these implants allow controlled release of compounds from the implant inside towards the tissues outside, favoring prevention and treatment of biofilm formation because of the local treatment while avoiding the occurrence or progression of peri-implantitis. Proof-of-concept for the drug delivery functionality and concomitant antimicrobial activity of chlorhexidine released from this composite material has been established in vitro, and this is in a biofilm preventive as well as curative setup using the oral bacterial pathogen Streptococcus mutans (Braem et al., 2015). Yet, to further prove the usefulness of this novel material in the clinic, its influence on osseointegration with and without a functional drug release needs to be addressed as well.
Therefore, the present experiment evaluated the osseointegration potential of such porous Ti/SiO 2 composite implant material in the absence of any drug release. Implant osseointegration was compared to the osseointegration of the macroporous Ti implant as such, as well as to a commercially pure dense Ti implant. The experiment was performed in the mandible of the Göttingen mini-pig and implant osseointegration was evaluated by histomorphometry.

| Animals and surgery
Six 36-month-old female Göttingen mini-pigs (Ellegaard, Göttingen minipigs A/S, Dalmose, Denmark) were used as experimental animals, due to their known physiology and similarities with human physiology (Stadlinger et al., 2012). The Ethics Committee for animal research of KU Leuven approved the study (P074/2015), accordingly to the regulations and guidelines of the Belgian animal welfare.
Three implant groups were designed as follows: • Group 1 (n = 6): custom-made porous titanium screw-shaped implant  Because of the created defects around the implants, the implants did not have primary stability. Therefore, submersion underneath the gingiva of the implants was performed to allow proper healing. Given the height of the cover screws and the length of the implants of groups 1 and 2, and given the striving towards non-tension primary closure, the buccal periosteum was relieved using a curved blade in the apical-horizontal direction. Flaps and surgical wound margins were sutured. Furthermore, in order to avoid adverse mechanical forces affecting the implant osseointegration process because of mastication, a soft diet was applied from implant installation onwards.
After 1 month post-implantation, healing was evaluated under sedation through clinical evaluation and intraoral digital radiographs, similarly as described above (Figure 3). Subsequently, the animals were euthanized by an intravascular injection of an embutramidemebenzoniumjodide-tetracaine-HCL solution (1 ml/5 kg body weight, T61 ® , Intervet, Mechelen, Belgium) into the ear vein until cardiac arrest occurred.

| Sample processing and analysis
The implants with the surrounding jaw bone were harvested. The bone blocks were fixed by immersion in a CaCO 3− buffered formalin solution (4%), dehydrated in an ascending series of ethanol concentrations for 18 days and embedded separately by infiltration of a benzoylperoxide (0.018%)-methylmetacrylate solution.
X-rays of the bone samples with implants were taken to confirm the presence of the implants. These X-rays were also used as a reference to define the cutting plane for separating the bone block into two halves containing each one single implant. Subsequently, the embedded bone blocks were mounted on a precision diamond saw (Leica SP 1600, Leica Microsystems, Nussloch, Germany). The cutting orientation was defined parallel to the implant and perpendicular to the jaw. Slices of approximately 700 μm thickness were obtained.
Two or three sections per sample were selected for analysis. The sections were micro-ground under running tap water and polished to a final thickness of 120-130 μm (Exact 400 CS grinding device, Exact Technologies Inc., Norderstedt, Germany). Finally, sections were stained with a combination of Stevenel's blue and Von Gieson's picrofushin red that allowed the visualization of mineralized bone (red) and demineralized tissue (blue-green). Additionally, representative sections were analyzed by scanning electron microscopy (SEM), as described before (Braem et al., 2014). To this end and prior to histological staining, sections were ground with a 4000 grit SiC grinding

| Statistical analysis
Statistical analysis was performed using the software package Stat Plus, a statistical analysis program for MAC OS ® Version v6, for all the parameters. The F-test for variance, and t-test to compare the means, assuming equal (homoscedasticity) or unequal (heteroscedasticity) variances, were adopted. Means and standard deviations are given for each implant group. The results were verified using the one-tailed ttest (with 95% confidence intervals), with a significance level <0.05.

| Ethics statement
The Ethics Committee for animal research of KU Leuven approved the study (P074/2015), accordingly to the regulations and guidelines of the Belgian animal welfare.

| RESULTS
Wound healing, both post-extraction and post-implantation was uneventful. However, out of the 23 installed implants, two, two and one implants were lost in implant groups 1, 2 and 3, respectively, resulting in an overall implant osseointegration success of 78%. Per group, the success rate was 66.66%, 71.42% and 90%, respectively.
Clinical inspection after 1 month of healing showed that some healing abutments had punctured the mucosa and were exposed to the oral cavity. This was observed ad random over the different groups. The gingiva and mucosa surrounding these exposed abutments was evaluated as healthy (Figure 3c).

| DISCUSSION
Implant dentistry research has focused on the optimization of dental implants, aiming to obtain faster and better results by modulating the biological process of bone healing. This can be obtained through different ways of alterations, such as the implant surface topography (macro-scale, micro-scale and nano-scale), the implant shape, and its cation is now gaining interest from another perspective, namely the perspective of preventing and tackling peri-implantitis (Bumgardner et al., 2011;Johnson & García, 2015;Karoussis et al., 2018;Shahi et al., 2017;Shi et al., 2015). Braem and co-workers established proof-of-concept for the drug delivery functionality of a mesoporous SiO 2 barrier incorporated into a high-strength macroporous Ti carrier (Braem et al., 2015;De Cremer et al., 2017). This novel and promising implant design possesses an internal reservoir which can be refilled, thereby ensuring controlled release of antimicrobial or antibiofilm compounds over sustained period of time.
Prior to evaluating the release of antibacterial compounds in a peri-implantitis set-up in vivo, evidence should first be provided that such an implant with specific surface characteristics osseointegrates as other implants do. Therefore, the present experiment was conducted for evaluation of the effect of SiO 2 functionalization of a macroporous implant on the peri-implant bone healing in vivo in the Göttingen mini-pig, by comparing to an identical macroporous implant without modification with SiO 2 , and to the well-documented c.p. titanium implant. It was hypothesized that the integration of SiO 2 in the implant does not hamper implant osseointegration.
Regarding the osteotomy defect, filling of it with newly formed bone was incomplete in all specimens in this stage of healing of 1 month post-implantation. These results are in agreement with previous work, investigating early healing periods in animal models (Botticelli et al., 2005;Rossi et al., 2012). Despite the absence of pri-  BV in the closer (BV 500 ) and broader (BV 800 ) implant vicinity was calculated. No significant differences were found for BIC and BV 500/800 for Ti-Porous + SiO 2 versus Ti-Porous, suggesting that the functionalization of the Ti-Porous implant with a mesoporous silica did not affect the peri-implant bone response, neither negatively nor positively. Likewise, Inzunza and co-workers illustrated that the viability and proliferation of the osteoblast-like cells is not altered in contact with a mesoporous SiO 2 coating on titanium (Inzunza et al., 2014).
Moreover, in combination with bioactive glass, which is known to improve osseointegration around titanium (Braem et al., 2014), such coatings can also accelerate the formation of bone tissue in the implant periphery (Covarrubias et al., 2016). As the mesoporous SiO 2 phase enables the controlled release of drugs into the implant surroundings (Braem et al., 2015) and as it was shown in the present study that SiO 2 does not affect the osseointegration of the implant, such "hybrid" implant designs are promising for further exploration and use in a peri-implantitis set-up.
It should be taken into account that cell behavior and the conse- interlocking and providing anti-rotational resistance to forces). These differences may have co-influenced the biological and mechanical micro-environment leading to a differential peri-implant bone healing response. In an attempt to exclude the implant macro-design differences between the control and experimental implants, a larger-thanclinically-advised osteotomy was created. is under consideration. Of note is that, in the classical surgical protocol of close bone-to-implant contact at installation, resident bone first resorbs prior to new bone formation (Rossi et al., 2014;Slaets et al., 2006;Slaets et al., 2007). A small gap around the implant during the early phases of healing may thus accelerate bone regeneration.

| CONCLUSION
Overall, based on the findings and the limitations of the present study, it can be concluded that the functionalization of a macroporous titanium implant with SiO 2 does not negatively affect the peri-implant bone healing response, as observed in the jaw bone of Göttingen mini-pigs. At the same time, the importance of the implant macrodesign, in particular the screw thread design in terms of number, width, depth and pitch, in a marginal gap implant surgery protocol was emphasized. Subsequent studies will explore the use of such Ti/SiO 2 implants in preventing and treating peri-implantitis given their potential for controlled release of antibiofilm compounds (Braem et al., 2015;De Cremer et al., 2017).

ACKNOWLEDGMENTS
The research leading to these results has received funding from the

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest in this study.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.