A systematic review on neutrophils interactions with titanium and zirconia surfaces: Evidence from in vitro studies

Abstract Objectives This systematic review aimed to assess in vitro studies that evaluated neutrophil interactions with different roughness levels in titanium and zirconia implant surfaces. Material and Methods An electronic search for literature was conducted on PubMed, Embase, Scopus, and Web of Science and a total of 14 studies were included. Neutrophil responses were assessed based on adhesion, cell number, surface coverage, cell structure, cytokine secretion, reactive oxygen species (ROS) production, neutrophil activation, receptor expression, and neutrophil extracellular traps (NETs) release. The method of assessing the risk of bias was done using the toxicological data reliability assessment tool (TOXRTOOL). Results Ten studies have identified a significant increase in neutrophil functions, such as surface coverage, cell adhesion, ROS production, and NETs released when interacting with rough titanium surfaces. Moreover, neutrophil interaction with rough–hydrophilic surfaces seems to produce less proinflammatory cytokines and ROS when compared to naive smooth and rough titanium surfaces. Regarding membrane receptor expression, two studies have reported that the FcγIII receptor (CD16) is responsible for initial neutrophil adhesion to hydrophilic titanium surfaces. Only one study compared neutrophil interaction with titanium alloy and zirconia toughened alumina surfaces and reported no significant differences in neutrophil cell count, activation, receptor expression, and death. Conclusions There are not enough studies to conclude neutrophil interactions with titanium and zirconia surfaces. However, different topographic modifications such as roughness and hydrophilicity might influence neutrophil interactions with titanium implant surfaces.


| INTRODUCTION
Ever since the ground-breaking work of Brånemark in the 1960s, different metals and their alloys have been used as dental implants due to their excellent biocompatibility, mechanical strength, and aesthetics (Brånemark et al., 1969). When an implant is inserted into the bone, an initial inflammatory response is triggered by the immune cells of myeloid origin predominated by neutrophils (Kolaczkowska & Kubes, 2013;Segal, 2005).
Neutrophils, the critical cellular player, activate an inflammatory cascade by producing cytokines, enzymes, and DNA fiber networks called neutrophil extracellular traps (NETs) (Brinkmann et al., 2004;Nauseef, 2016). The presumed neutrophil response to a dental implant is determined by several physical and chemical features of the implant surfaces, which include mechanical and physicochemical properties such as chemical composition, surface wettability, surface energy, and surface topography (Bowers et al., 1992;Galli et al., 2005;Ong et al., 1996).
Zirconia is considered a potential alternative to titanium implants due to its aesthetics, excellent biocompatibility, mechanical properties, and reduced bacterial biofilm formation (Christel et al., 1989;Langhoff et al., 2008;Piconi & Maccauro, 1999). However, due to its high hardness, surface roughening of zirconia has been technically challenging (Rottmar et al., 2019). Different surface topographies of zirconia were reported to increase osteoblast proliferation on a rough surface compared to a smooth surface (Bächle et al., 2007). A recent study showed a significantly increased cellular spreading and migration rate on rough zirconia surfaces (Sa = 3.36 μm) than on the rough titanium implant surfaces (Munro et al., 2020). From these studies mentioned above, it is assumed that the functional activity of neutrophils is determined by the implant surface characteristics raising questions regarding the exact nature of such interactions. Therefore, this systematic review aimed to assess in vitro studies that evaluated neutrophil interactions with different roughness levels in titanium and zirconia implant surfaces.

| MATERIALS AND METHODS
The reporting of this review complies with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines (Moher et al., 2009(Moher et al., , 2012. The PRISMA checklist is presented in Supporting Information Materials S1 and S2. Ethics approval was not required for this systematic review.

| Eligibility criteria
The eligibility criteria were based on the PICO (population, intervention, control, and outcomes) questions: How do neutrophils interact with titanium and zirconia surfaces? Studies   independently. Authors were contacted if any missing data or additional data were required from the eligible studies.

| Risk of bias in the included studies
The method of assessing the risk of bias was done using the toxicological data reliability assessment tool (TOXRTOOL) (Schneider et al., 2009). For in vitro studies, it uses a set of 18 questions or criteria. For each criterion, a score of "1" is provided when the response is "yes," or the criteria are addressed, while a score of "0" is given when the response is "no," that is, when the criteria are not addressed in the study. If the value is ≥15, Category 1 is assigned. For values >11, Category 2 is assigned, and for all values <11, Category 3 is assigned. Categories 1 and 2 represent that the data is reliable without and with restriction, respectively, while Category 3 indicates that the data reported from the study is unreliable.

| RESULTS
The electronic search of the databases identified a total of 3147 papers: PubMed (n = 1110), Embase (n = 1090), Scopus (n = 666), and Web of Science (n = 281). After eliminating the duplicates and screening the titles and abstracts, 45 full texts were reviewed ( Figure 1). Finally, 14 articles were included in the qualitative analysis.   Table 1 summarizes the studies that evaluated immunological response, morphological changes, activation, and adhesion of neutrophils during interaction with titanium and zirconia implant surfaces.

| Risk of bias
In this review, 12 studies were found to have an overall score of ≥15 (Category 1), demonstrating that data from these studies are reliable without restrictions. One study had a score of 14 and belonged to Category 2, and one study had a score of 11 and belonged to Category 3. The scoring for all studies is shown in Table S2 and Figure S1 shows the number of studies belonging to the respective categories.

| Neutrophil behavior based on roughness
Ten studies assessed the interaction of neutrophils with titanium surfaces based on their roughness. One study identified intact cellular F I G U R E 1 PRISMA flowchart of the online databases searched and selection of studies for inclusion. morphology and reduced chemiluminescence activity (respiratory burst) by neutrophils when interacting with serum coated moderately rough hydrophilic titanium surfaces compared to moderately rough hydrophobic titanium surfaces (El Kholy et al., 2020). A second study identified neutrophils interacting with rough hydrophilic titanium surfaces to produce low levels of interleukin-1β (IL-1β), IL-6, IL-12, tumor necrosis factor-α, IL, neutrophils elastase, and myeloperoxidase with no NETs formation compared to neutrophils interacting with naive smooth and rough titanium surfaces (Abaricia et al., 2020).
However, activated neutrophils on smooth titanium and alloy surfaces released relatively low IL-ra and IL-8 fields (Schildhauer et al., 2009). Additionally, rapid neutrophil adhesion (80%-82%) and various stages of NETosis with completely spread NETs with swollen nuclei and chromatin alteration on SLA (Sandblasted, Large grit, Acidetched) titanium surfaces were observed. (Vitkov et al., 2015). In another study, neutrophils exposed to rough titanium surfaces had a fourfold higher surface attachment area showing a prominent shape and more cytoplasmic projections after 2 h compared to smooth titanium surfaces (Campos et al., 2014). However, CD11b and Lselectin expression in neutrophils were not influenced by titanium surface textures (Campos et al., 2014). Two studies also reported increased neutrophils adhesion, priming, ROS production and expression of CD 11b on rough titanium surfaces compared to smooth titanium surfaces (Eriksson et al., 2001;Nygren et al., 1997).
Significant production of ROS occurred earlier on smooth titanium surfaces compared to rough titanium surfaces (Eriksson et al., 2001).
No statistically significant differences in neutrophil cell count and production of ROS were observed on blasted Ti surfaces compared to other coated Ti surfaces (Arvidsson et al., 2011). Short-and longterm exposure of neutrophils to rough titanium surfaces showed increased adhesion and proliferation compared to nanostructural surfaces (Smith et al., 2013). Only one study investigated biocompatibility parameters of human bone marrow cells and reported that 12.2% of granulocytes adhered to naive titanium surfaces (Wilke et al., 1998). Based on these results, increased neutrophil adhesion, ROS production, and different stages of NETosis were observed on rough titanium surfaces. However, rough hydrophilic titanium surfaces seem to induce decreased levels of proinflammatory cytokines and ROS production and showed no NET formation from neutrophils. Eriksson and Nygren (2001b) investigated neutrophil functions based on adhesion receptors on hydrophilic and hydrophobic titanium surfaces. Eriksson and Nygren (2001a) found that neutrophils adhered to hydrophilic titanium surfaces in a FcγIII receptor (CD16). Expression of the FcγIII receptor on neutrophils was dominant during the initial hours, which gradually shifted towards CD11b expression later. Eriksson and Nygren (2001b) reported that neutrophil activation increased over time on hydrophilic titanium surfaces, which was evident from the decreased expression of CD62L. Additionally, the CD16 expression was higher during the initial hours at hydrophilic surfaces but only peaked after late hours at hydrophobic surfaces (Eriksson & Nygren, 2001b). The same study showed that neutrophils adhesion to hydrophilic and hydrophobic titanium surfaces was depressed by inhibiting hirudin (thrombin inhibition), reporting the expression of CD16 and CD11b to be thrombin dependent (Eriksson & Nygren, 2001b). A recent study also identified a significant reduction in L-selectin (CD62L) expression on titanium alloy surfaces by applying sheave r force, indicating an increased neutrophils activation compared to other highly polished medical implant surfaces such as stainless steel and sapphire crystal (Radley et al., 2019). These studies indicate that different adhesion receptors recognize hydrophilic and hydrophobic titanium surfaces.

| Neutrophil behavior based on receptor expression
Moreover, the activation and adhesion were increased in hydrophilic surfaces compared to hydrophobic surfaces.

| Neutrophil interaction with titanium and zirconia implant surfaces
Only one study compared neutrophils interaction between titanium alloy and zirconia toughened alumina surfaces (Radley et al., 2018).
The neutrophils count was not significantly different between titanium and zirconia surfaces, and the neutrophil expression of CD62L did not differ between these surfaces.

| DISCUSSION
This systematic review indicated that neutrophils functions, such as adhesion, surface coverage, attachment, and NET release, are influenced by topographic modifications on titanium surfaces. It has been demonstrated that a rapid surface coverage and stronger neutrophil adhesion can be seen on rough titanium surfaces compared to smooth titanium surfaces (Campos et al., 2014;Eriksson et al., 2001;Vitkov et al., 2015). Additionally, SEM analysis has shown different morphological features of neutrophils, such as flat cells, cytoplasmic projections, and surface attachment on rough titanium surfaces (Campos et al., 2014). Like neutrophils, macrophage adhesion, morphology, and phenotype can be modulated by implant surface roughness (Chen et al., 2010;Geiger et al., 2009;Soskolne et al., 2002). Also, previous studies have shown increased osteoblast adhesion on rough titanium surfaces compared to smooth one's Fields (Bowers et al., 1992;Michaels et al., 1991). Additionally, studies have shown that osteoblast morphology can vary between rough and smooth titanium surfaces (Bowers et al., 1992;Boyan et al., 2003). Therefore, early biological events such as cell behavior and functions seem to be influenced by surface roughness.
Neutrophils seem to produce low levels of proinflammatory cytokines and enzymes and high anti-inflammatory cytokines when interacting with rough hydrophilic Ti surfaces (Abaricia et al., 2020).
Likewise, studies on macrophages also showed low levels of ELANGOVAN ET AL. proinflammatory cytokines in response to micro-rough hydrophilic titanium surfaces (Alfarsi et al., 2014;Hamlet et al., 2012). Studies on osteoblast have also found that increased hydrophilic surfaces improved osteogenic differentiation (Olivares-Navarrete et al., 2011;Vlacic-Zischke et al., 2011). In addition, micro-rough hydrophilic surfaces seem to be positively involved in the earlier onset of the osseointegration (Lang et al., 2011). These findings suggest that surface hydrophilicity seems to attenuate the production of proinflammatory cytokines and may promote faster wound healing. (Vitkov et al., 2015) showed that rough Ti surfaces triggered the NET release and histone citrullination. In contrast, Abaricia et al. (2020) demonstrated no NET formation on rough hydrophilic Ti surfaces compared to naive rough and smooth Ti surfaces.
Therefore, it is plausible to believe that implant surface roughness/ chemistry can affect the NETotic response.
It has been shown that neutrophils interaction with rough Ti surfaces can induce ROS to release (Eriksson et al., 2001;El Kholy et al., 2020), which might lead to local tissue damage, delayed wound healing and even loosening of implants (Hwang et al., 2019;Segal, 2005). Studies have also reported that Ti ions released from implant surfaces trigger macrophages and osteoblast to produce increased ROS levels (Vermes et al., 2001;Żukowski et al., 2018 clinical and in vivo studies are needed to confirm the relevance of in vitro findings. Finally, the lack of homogeneous quantitative data for meta-analysis and methodological heterogeneity to assess the interaction/behavior of neutrophils can also be a drawback of the present systematic review.

| CONCLUSION
There are not enough studies to draw any conclusion about neutrophil interactions with titanium and zirconia surfaces. However, different topographic modifications such as roughness and hydrophilicity might influence neutrophil interactions with titanium implant surfaces.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable-no new data was generated, or the article describes entirely theoretical research.