The effect of non-steroidal anti-inflammatory drugs on the osteogenic activity in osseointegration: a systematic review

Non-steroidal anti-inflammatory drugs are commonly used in implant dentistry for management of post-operative pain. The objective of this systematic review was to analyse the effect of non-steroidal anti-inflammatory drugs on the osteogenic activity of osteoblasts with an emphasis on its effect on osseointegration. A systematic literature search for in vitro, animal models, and clinical trials was conducted using Ovid, PubMed, Scopus, and Web of Science databases. Articles published since the introduction of selective COX-2 inhibitors, between January 1999 and July 2018, were selected. The integrated search followed the PRISMA statement with the following key terms: non-steroidal anti-inflammatory drug/s, titanium, osseointegration, and osteoblast. The review is registered at PROSPERO database: CRD42016051448. The titles and abstracts of each research article in the initial search (n = 875) were independently screened by two reviewers. A third independent reviewer reviewed the articles that were included by one but excluded by the other reviewer. This resulted in the cataloguing of 79 full-text manuscripts where the articles were assessed for the following criteria: the study investigates the effects of NSAIDs on osteoblasts, explores the COX pathway and its effect on osteogenic activity, and compares the effects of NSAIDs on osteoblasts with a control group. A total of 13 articles have been included for qualitative synthesis. There is a lack of consensus in the literature to explicitly conclude that there is a relationship between the use of post-operative NSAIDs and failed osseointegration; however, osseointegration does not appear to be negatively affected by NSAIDs in the human clinical studies.


Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are a group of drugs with anti-inflammatory, analgesic, and antipyretic effects. They are commonly used in dentistry for management of dental pain associated with inflammation. NSAIDs exert their effects through the inhibition of the cyclooxygenase (COX) enzyme, therefore interfering with the synthesis of prostaglandins (PG) and thromboxanes; PGs and thromboxanes are inflammatory mediators that are responsible for pain. COX has three isoforms: COX-1, COX-2, and COX-3. COX-1 exhibits characteristics of a constitutive enzyme, as its activity is associated with the involvement of PGs and thromboxanes in controlling normal physiological functions [1]. COX-2 exhibits characteristics of an inducible enzyme in inflammatory cells and is activated in response to pathological stimuli [2]. COX-3 is a variant of COX-1, though it shares the characteristics of both COX-1 and COX-2. The osseointegration process that is observed after implant insertion can be compared to bone fracture healing through the process of an inflammatory response in which the recruitment of osteoprogenitor cells occurs, followed by their downstream differentiation into osteoblasts that leads to bone deposition on the implant surface [3,4]. COX-1 is expressed in normal bone and at the site of bone fracture, whilst COX-2 is upregulated during inflammation and the initial stages of bone repair [5]. The effects of NSAIDs on altering bone growth, remodelling, and repair are generally not considered when prescribed for post-operative pain management after implant placement.
Hypoxia occurs locally in bone tissues when pathological conditions such as implant placement and bone fractures arise [6,7]. It has been established, through clinical studies of bone cultures, that hypoxia is directly responsible for directing the synthesis of prostaglandin E (PGE) by osteoblasts [6]. Therefore, the presence of the COX enzymes in bone healing is of importance [6]. Prostaglandins have the capacity to influence bone metabolism and can both induce and inhibit tissue repair mechanisms [6]. Local administration of PGE1 has been shown to stimulate bone formation, increase alveolar bone height, and induce formation of new cementum and periodontal ligament adjacent to the site of delivery in canine mandibles [8,9]. Furthermore, PGE2 has been shown to stimulate replication and differentiation of osteoblasts cultured on smooth titanium surfaces thereby increasing bone formation around titanium implants [10]. Additionally, PGs can also inhibit the formation and growth of osteoblasts [6]. Therefore, altered PG levels as a result of COX inhibition can have a negative impact on the role of PG in bone tissue, potentially causing a shift in precursor cell action towards bone resorption [6].
Cyclooxygenases have an important role in the production of PGs where these enzymes in bone tissues show increased activity under the influence of hypoxia-inducible factors [6,11]. Therefore, local activity of COX enzymes promotes bone formation and resorption through the production of PGs [12]. Non-selective NSAIDs are reported to inhibit the activity of COX-1 equally, if not more than COX-2 [2]. Therefore, NSAIDs inhibit the production of PGs at the site of implant placement or fracture, thereby influencing the bone healing cascade [13]. There is evidence from animal studies that indicate that COX-2 inhibitors delay done healing in diaphyseal fracture models in rats [13]. However, the exact roles of COX-1 and COX-2 in the PG production has not been ascertained in humans, and assumptions have been made suggesting a milder or non-significant inhibitory effect of selective COX-2 inhibitors on bone healing when compared to a non-selective COX inhibitor [2,13]. Furthermore, a systematic review conducted by Marquez-Lara et al. highlighted the great variability regarding the impact of NSAIDs on bone healing, and that there is no consensus regarding the impact of NSAIDs following orthopaedic procedures [14]. Therefore, the rationale of the present systematic review is to address the gaps in the literature by identifying if variables such as the dosage, duration of administration, and selectivity of post-operative NSAIDs negatively affect osseointegration.

Protocol and registration
The systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [15]. The review is registered at PROSPERO database, and the review protocol can be accessed at http://www.crd.york.ac.uk/PROSPERO/ display_record.php?ID=CRD42016051448.
The PROSPERO registration number is CRD4201605 1448.

Eligibility criteria
The review included in vitro, clinical and in vivo studies; animal models. Articles published since the introduction of selective COX-2 inhibitors in 1999 were included [3]. Studies published outside this time period, not in the English language, non-peer reviewed, and review studies were excluded.

Information sources
An electronic search into four databases: Ovid, Pubmed, Scopus, and Web of Science was performed to systematically identify the available literature. Articles published between January 1, 1999, and July 7, 2018, were considered.

Focus question
The focus question, used to guide the search strategy, according to the PICO schema is "Will variables such as the dosage, duration of administration, and selectivity of post-operative NSAIDs impair the bone healing around titanium implants?"

Search strategy
The search string comprised the combination of medical subject headings (MeSH) and free keywords. The linkage was conducted using the Boolean operator (AND, OR). The choice of keywords was intended to be broad to maximise the number of relevant studies considered. The following search strategy was applied to Ovid and PubMed: Furthermore, the following search strategy was applied to Scopus and Web of Sciences to supplement records identified through Ovid and PubMed: (non steroidal anti inflammatory agent OR non steroidal anti inflammatory agents OR non steroidal anti inflammatory drug OR non steroidal anti inflammatory drugs OR cyclooxygenase inhibition OR COX inhibition OR ibuprofen OR celecoxib) AND (osseointegration OR osteoblast OR osteoblasts OR titanium implant OR titanium implants OR dental implant OR dental implants)

Study selection
The titles and abstracts of each research article in the initial search were independently screened by the primary (JDL) and the second reviewer (TJ). A third independent reviewer (MN) reviewed the articles that were included by one but excluded by the other reviewer. The full-text manuscripts of the articles were catalogued in accordance to the "Eligibility criteria" section as mentioned above and were assessed for the following criteria: The study explored the COX pathway and its role in osseointegration. The effects of NSAIDs on osteoblasts attached to titanium are investigated (in vitro studies).

Data collection process
The full-text manuscripts of included studies were catalogued into in vitro, clinical, and in vivo studies. The data from the included studies were independently extracted by the primary (JDL) and the second reviewer (TJ) according to the "Data items" section as listed below. Disagreements or uncertainties were discussed with the third reviewer (MN) until an agreement was reached.

Data items
The data collected from the included studies were arranged in the following fields: Author (year)-reveals the author/s and year of publication Sample (size)-describes the sample and sample size used in the study Treatment group (size)-describes the treatments used in study Methodology-describes the method of drug delivery Parameter-describes the parameter/s that are measured Outcome-describes the outcome/s of the experiments

Quality and risk of bias in individual studies
The quality and risk of bias assessments were performed independently by two reviewers (JDL and TJ) during the data extraction process. Any disagreements or uncertainties were discussed with the third reviewer (MN) until an agreement was reached. The quality and bias assessment for all studies addressed various bias domains. A Modified CONSORT checklist of items for reporting in vitro studies of dental materials outlined by Faggion (Tables 1 and 2) was used to assess quality and risk of bias of included in vitro studies [16]. The Cochrane Collaboration's Tool (Table 3) was used to assess quality and risk of bias of included human clinical studies [17]. A quality assessment of the methodology of the animal studies (Table 4) has been performed according to items (Table 5) of the ARRIVE guidelines [18].

Synthesis of results
The relevant data collected for qualitative synthesis are summarised in three critical analysis tables: in vitro studies (Table 6), clinical studies (Table 7), and in vivo studies (Table 8).

Statistical analysis
No meta-analyses could be performed due to the heterogeneity between the studies-different samples, experimental groups (drugs and concentrations), study models, and outcome measures.

Study selection
The electronic search in the databases of Web of Science, PubMed, Ovid, and Scopus resulted in the identification of 875 potential titles and abstracts. After removal of the duplicates, independent screening of the abstracts resulted in the selection of 79 studies for assessment of eligibility. A total of 13 studies were eligible and are included in the systematic review ( Fig. 1).

Exclusion of studies
The eligibility and study selection criteria as mentioned above were applied to the 79 full-text articles. A total of 66 studies were excluded after a full-text assessment for the following reasons: The study did not explore the role of COX pathway in osseointegration (n = 26). The effects of NSAIDs on osteoblasts were not investigated on titanium (n = 24). The study was a systematic review (n = 16).

Study characteristics
The included studies were catalogued into three groups characterised by the type of study: in vitro studies (Table 6), clinical studies (Table 7), and in vivo studies ( Table 8). The cataloguing provided a clearer understanding of the effects of NSAIDs in osseointegration in various study models, ultimately contributing to the sensitivity of the systematic review.

Quality and risk of bias assessment
The quality and risk of bias assessments of included studies are summarised in Tables 1, 2, 3, 4, and 5. The quality assessment revealed a high risk of bias (for one or more domain) for most of the included studies. The included in vitro studies had high risk of bias according to the Modified CONSORT checklist [16,19,20]. One clinical study was classified as unclear risk of bias (for one or more domain) according to the Cochrane Collaboration's Tool [17,21]. An in vivo animal study had an unclear risk of bias according to the ARRIVE guidelines [18,22].

Discussion
Non-steroidal anti-inflammatory drugs are widely used in clinical dentistry to manage post-operative inflammation and pain. Two systematic reviews have been performed to review the literature concerning the possible influence of NSAIDs on the osseointegration of titanium implants: a review conducted by Gomes et al. concluded that osseointegration is impaired in the presence of conventional NSAIDs, whilst the review conducted by Kalyvas et al. concluded that short-term post-operative NSAIDs do not appear to negatively impact osseointegration [3,4]. Despite these conflicting conclusions regarding post-operative use of NSAIDs, both Gomes et al. and Kalyvas et al. agreed that prolonged or long-term use of NSAIDs, particularly in patients with chronic diseases, impaired osseointegration and, therefore, reduced the success of implant surgery [3,4]. The current review extends on these existing reviews by identifying if dosage, duration of administration, and selectivity of post-operative NSAIDs impair osseointegration.

In vitro studies
The effects of NSAIDs on the osteogenic activity of osteoblasts have been extensively studied at the molecular pharmacological level [23]. However, only two studies have been identified that investigated the effect of NSAIDs on osteoblasts attached to titanium surfaces ( Table 6). In the study conducted by Boyan et al., their results demonstrated that a non-selective COX inhibitor (indomethacin, 0.1 μM), a selective COX-1 inhibitor (resveratrol, 1 and 10 μM), and a selective COX-2 inhibitor (NS-398, 1 and 10 μM) did not have a significant effect on the number of cells derived from human osteosarcomas [20]. Furthermore, Boyan et al. demonstrated that the NSAIDs reduced prostaglandin E 2 (PGE2) production of cells attached to a rough titanium surface. Their results indicated that both COX-1 and COX-2 are involved in the production of osteocalcin, PGE2, and TGF-β1 by osteoblasts [20]. They also demonstrated that osteoblasts produced increased levels of PGE2 on rough titanium surfaces and that this was correlated with increased alkaline phosphatase activity and osteocalcin production [20]. This suggests that PGE2 may have a role in osteoblast proliferation and differentiation on titanium surfaces, and that this favourable effect of PGE2 Boyan et al. [20] Key: (+) = low risk of bias, (?) = unclear risk of bias, (−) = high risk of bias  [19,20].

Clinical studies
The clinical evidence demonstrating the effects of NSAIDs on the osseointegration of titanium dental implants is limited with only two clinical trials and one retrospective study identified in the database searches (Table 7). In the clinical trial conducted by Alissa et al., the effect of a 7-day post-operative course of ibuprofen (600 mg, taken four times daily) on the marginal bone level around dental implants was investigated [21]. They found that there were no statistically significant differences between the treated and placebo groups in the mean marginal bone level around dental implants at 3 and 6 months after implant placement [21]. In a similar clinical trial conducted by Sakka et al., the effect of a 7-day course of ibuprofen (600 mg, taken four times daily) on the marginal bone level around dental implants was also investigated [24]. They found that there were no significant differences between the ibuprofen and non-ibuprofen groups, consistent with the findings of Alissa et al., when comparing the changes in marginal bone level around dental implants [24]. However, a retrospective study conducted by Winnett et al. postulated that the adverse biological events following dental implant placement were associated with perioperative use of NSAIDs [25]. Winnett [25].

Animal studies
The influence of NSAIDS on bone healing in animal models has been shown to be related to the duration of treatment and drug selectivity [5]. A total of seven studies were identified that investigated the effect of NSAIDs on the osseointegration of titanium implants in animals: mice, rabbits, and rats ( Table 8).
The duration of treatment is a factor to consider when using NSAIDs, and a study conducted by Goodman et al. investigated the effect of a selective COX-2 inhibitor   Key: (+) = low risk of bias, (?) = unclear risk of bias, (−) = high risk of bias (rofecoxib, 12.5 mg/kg/day) administered for 6 weeks on bone growth in a bone harvest chamber during three different time periods: initial 2 of the 6 weeks, final 2 of the 6 weeks, and continuously for 6 weeks [26]. The bone harvest chamber was a titanium device that was implanted into the tibia of rabbits and had an inner removable core with canals that allowed for bone ingrowth into the inner chamber. Their results revealed that rofecoxib given continuously for 6 weeks significantly reduced bone ingrowth and osteoblasts per area compared with the control (no treatment), whilst rofecoxib given for 2 weeks did not appear to interfere with bone ingrowth and number of osteoblasts [26]. Furthermore, the studies conducted by Ribeiro et al. investigated the effect of long-term administration (60 days) of a selective COX-2 inhibitor (meloxicam, 3 mg/kg/day) on the bone growth on a titanium implant [27,28]. Their results also indicated that long-term use of a selective COX-2 NSAID significantly reduces bone-to-implant contact, bone area, and bone density, ultimately leading to failed osseointegration [27]. The data gathered in both studies suggest that duration of treatment is an important factor in the use of selective COX-2 NSAIDs, as short periods of rofecoxib and meloxicam did not adversely affect osseointegration [26,27]. The COX selectivity of NSAIDs and their interference with prostaglandin synthesis have been shown to inhibit bone healing [23]. Goodman et al. performed a follow-up study where the titanium bone harvest chamber was again implanted into the tibia of rabbits and the rabbits were treated with water (control), a non-selective COX inhibitor (naproxen, 110 mg/kg/day), or a selective COX-2 inhibitor (rofecoxib, 12.5 mg/kg/day) for a 4-week period [29]. Their results again demonstrated that COX-2 inhibition significantly decreased bone ingrowth, where rofecoxib also decreased the number of osteoblasts per area [29]. Furthermore, the conclusions by Goodman et al. were supported by the study by Chikazu et al. where titanium implants were inserted in wild-type and COX-2-knockout mice [30]. Their results revealed that the expression of COX-2 was induced in bone surrounding the implants in wild-type mice, but not in COX-2-knockout mice and that the bone-to-implant  The following parameters were assessed after 5 days: osteocalcin content, PGE2 content, and TGF-β1 content.
Indomethacin, resveratrol, and NS-398 had no effect on osteocalcin content. Indomethacin and resveratrol blocked PGE2 production. NS-398 had no effect on PGE2 production on smooth surfaces but caused a reduction on rough surfaces. Indomethacin blocked TGF-β1 production on rough surfaces. Resveratrol blocked TGF-β1 on TPS. NS-398 did not cause TGF-β1 inhibition.  Ibuprofen was the most commonly prescribed, 600 mg q.i.d.

Radiographic bone loss.
Vertical bone height of remaining implants. NSAID cohort experienced more implant failures than the non-NSAID cohort.
The NSAID cohort experienced more cases of radiographic bone loss greater than 30% of the vertical height of their remaining implants.   Their results revealed no statistically significant differences between the experimental groups and the control [22]. Furthermore, a recent study conducted by Salduz et al. that investigated the effect of a non-selective COX inhibitor (diclofenac, 5 mg/kg/day) and a selective COX-2 inhibitor (celecoxib, 3 mg/kg/day) administered for 8 weeks on bone growth and osseointegration on two different alternative titanium surfaces revealed no statistically significant differences in the biomechanical and histological results between the experimental groups and the control, suggesting that long-term use of NSAIDs does not affect osseointegration [32]. The data collected in animal studies regarding duration of treatment and drug selectivity is inconsistent, and there is a lack of consensus on the influence of NSAIDs on osseointegration in animal models.

Limitations
The majority of the included studies revealed a high risk of bias, and conclusions from studies that have a high risk of bias are sufficient to affect interpretation of data [16][17][18]. Publication and selection bias is apparent in several included studies, as the negative effects of NSAIDs on osseointegration can be expected in the studies that administered NSAID at a high concentration and/or for a prolonged period of time. The conclusions of this systematic review were largely based on animal studies, as there are very few published in vitro and clinical studies relating to the effect of NSAIDs on osseointegration. The effects of NSAIDs on osseointegration in animals cannot be

Conclusions
The analgesic and therapeutic effects of NSAIDs are achieved by COX-2 inhibition [4]. It is likely that COX inhibition by NSAIDs is detrimental to the bone healing process, given the favourable actions of PG on this process [4]. Osteoblasts have the capacity to produce PGs, where PGE2 is most abundant, through the COX pathway though the evidence asserting that PGs have a direct role in bone healing is inconclusive [1,23]. Furthermore, there is insufficient evidence in the current literature to explicitly conclude that there is a relationship between the use of NSAIDs and early implant failure. However, osseointegration does not appear to be negatively affected by NSAIDs in the human clinical studies, which contrasts with the experimental in vitro and in vivo animal studies. Furthermore, there are no human clinical studies that have investigated the effect of a selective COX-2 NSAID on osseointegration. Therefore, further research with an emphasis in human clinical studies comparing the effect of the COX selectivity of NSAIDs on osseointegration is required.