Effect of titanium implants with strontium incorporation on bone apposition in animal models: A systematic review and meta-analysis

This systematic review aims to assess the efficacy of titanium (Ti) implant surfaces with or without strontium (Sr) incorporation on osseointegration in animal experimental studies. An electronic search was conducted using databases of PubMed and EMBASE up to November 2016 to identify studies focusing on osseointegration of strontium-modified titanium implants following PRISMA criteria. The primary outcome was the percentage of bone-to-implant contact (BIC) around the implants with or without strontium-modified surface. Of the 1320 studies, 17 studies fulfilling the inclusion criteria were finally included. A random effect meta-analysis was conducted based on BIC in 17 studies, and the results demonstrated considerable heterogeneity (I² = 79%). A sensitivity analysis found that three studies using the same surface modification method were the major source of the heterogeneity. Therefore, exploratory subgroup analysis was performed. Subgroup one including 14 studies showed a standard mean differences (SMD) of 1.42 (95% CI, 1.13–1.71) with no heterogeneity (I² = 0.0%), while subgroup two including the other three studies showed a SMD of 9.49.95% CI, 7.51–11.47) with low heterogeneity (I² = 0.1%). Sr-modified implants in both subgroups showed significantly higher BIC than unmodified implants (P < 0.01). The results showed a statistically significant effect of Sr-modified titanium implant surfaces on osseointegration and bone apposition in animal models.


Risk of bias and quality assessment of included studies.
The results of the risk of bias evaluation of included studies were shown in Fig. 2. For items 9 and 10, 60% of included studies reported the experiment was randomized at some level, while 29% reported blinding at any level during the study.
The ARRIVE criteria of included studies was shown in Table 2. The mean score of all studies was 17.2 (±1.98) out of a maximum of 24. All studies reported adequate information concerning title, abstract, introduction, ethical statement, species, surgical procedure, outcome evaluation, statistical analysis and results. Information Offermanns et al. 26

Bone to implant contact. All included studies measured the effect on BIC around implants with or without
Sr-enriched surface. A random effect meta-analysis was conducted based on BIC in 17 studies, and the overall results demonstrated considerable heterogeneity (I² = 79%). A sensitivity analysis found that three studies using the same surface modification method were the major source of the heterogeneity. Therefore, exploratory subgroup analysis was performed. The subgroup1 including 14 studies showed a standard mean differences (SMD) of 1.42 (95% CI, 1.13-1.71) with no heterogeneity (I² = 0.0%), while subgroup2 including the other three studies showed a SMD of 9.49 (95% CI, 7.51-11.47) with low heterogeneity (I² = 0.1%) (Fig. 3). The Sr-modified implants in both subgroups showed significantly higher BIC than unmodified implants (P < 0.01). However, high publication bias was found in the present study. (Begg, p = 0.039; Egger, p = 0.000) (Fig. 4.).
No meta-analysis could be conducted due to the considerable heterogeneity. 10 studies reported that significant higher bone area could be observed around strontium modified implants than those implants without strontium incorporation. However, no significant difference in bone area was reported in other four studies 28,30,37,42 .

Micro-CT evaluation and biomechanical test. Micro-computed tomography (micro-CT) evaluation
was performed in seven of included studies 29,31,32,[34][35][36] . In quantitative assessment, four studies 31,32,34,38 showed Sr-enhanced implants demonstrated stronger effect on all micro-CT parameters including bone volume per total volume (BV/TV), 3D bone distribution such as trabecular number (Tb.N), thickness (Tb.Th), and/or spacing (Tb. Sp) and/or the connective density (Conn.D) than control implants, while other three studies found no statistically significant changes in some parameters. Nine of included studies 28,29,31,32,[34][35][36][37][38] reported the results of biomechanical test including pull-out test or removal torque testing. All studies revealed a significant increase in implant fixation, with removal torque value, the maximal push-out force and/or the ultimate shear strength markedly raised compared to control groups.

Discussion
The primary finding of this meta-analysis was that Sr-modified implant surfaces significantly increased the percentage of BIC. Therefore, the null hypothesis should be rejected.
To our knowledge, this is the first systematic review to assess the effect of Sr-modified implants on enhancing osseointegration and bone apposition in animal experimental studies. To quantify the potential effect of Sr-containing surfaces on peri-implant bone apposition, a meta-analysis of BIC was performed. Overall, the results of subgroup meta-analysis revealed that titanium implants with strontium incorporation demonstrated significantly better BIC than unmodified implants in small (rats, rabbits) and large (dogs) animals. The result verified the expectation in vitro studies that the Sr-containing titanium surface is expected to shorten bone healing period and enhance implant osseointegration 43,44 . Similarly, this result was in agreement with studies for appraising the efficiency of Sr-modified magnesium (Mg) based implant 45,46 . In these studies, Sr-enriched implant showed significantly higher percentage of BIC than that of pure Mg or Mg alloy based implant.
It is worth mentioning that subgroup two reported Sr-modified surfaces had significant effect on BIC and the difference between implants with and without strontium incorporation was more significant than that in subgroup one. The identical electrochemical deposition process was applied to incorporate strontium-substituted hydroxyapatite (Sr-HA) into surface in these three studies indicating the heterogeneity may be caused by method of surface modification. It has been reported that an electrochemical process can produce a homogeneous 2-to approximately 3 μm HA coating and nano-hydroxyapatite (nano-HA) on the metallic substrate surface 47 . Yang et al. 48 reported that significant superiority of osseointegration and bone apposition was found when electrochemical deposition method was used. However, it is difficult to determine the best surface modification methods due to the limited information.
New bone area plays an important role in evaluating the osteoconductive property of biomaterials. The new mineralized bone tissue area inside all the implant threads was measured to evaluate the percentage of bone area. Several studies have demonstrated that Sr-containing biomaterials could increase new bone apposition. Studies for evaluating Sr-incorporated bioactive glass scaffolds and bone cement in impaired bone found that materials were covered by more new bone than unmodified groups 49,50 . In this review, ten studies using rat animal model reported significantly higher BA in Sr-modified implants than unmodified implants, while the other four studies used rabbit animal model reporting no significant difference in BA. The difference may be attributed to the different animal model, which implies different dynamics of bone formation especially in early healing intervals 51 . Therefore, additional preclinical and clinical studies should be performed to assess the effect of Sr-modified implant on bone apposition and osseointegration.   Table 2. Checklist of ARRIVE criteria reported by the included studies.
The 3-D micro-CT image clearly provides the information of bone-implant interface and trabecular microstructure of peri-implant bone tissue from both qualitative and quantitative perspectives. Seven included studies 29,31,32,[34][35][36]38 performed CT evaluation and four of them showed significantly improved BV/TV, Tb.N, Tb.Th, Tb.Sp and/or Conn.D. Other three studies found the parameters of CT evaluation were partially improved by Sr-modified titanium surface. Possible reasons that cause the disparity could be the different surface topography and Sr concentration of titanium implant surfaces. Furthermore, biomechanical testing further demonstrated a significant increase in implant fixation. It was apparent to be verified by the improved trabecular bone microarchitecture together with increased BIC and BA around the implant.
All the included studies reported that the improved implant osseointegration and bone apposition was attributed to the released Sr ions and modified surface topography. Actually, the exact mechanism regarding bone remodeling effect of Sr has not been clearly understood. Recently, it has been suggested that the possible mechanism of Sr relies on the calcium-sensing receptor (CaR) which is expressed in several types of pre-osteoblastic  cells and bone marrow stromal cells 52 . Through a CaR-mediated mechanism, Sr is reported to increase bone apposition by promoting pre-osteoblastic cell proliferation and differentiation, reducing osteoclast differentiation, enhancing matrix mineralization 52,53 . In addition, the surface topography changed by strontium incorporation process also contributed to bone-implant integration. Three of included studies revealed that micro/nanoscale topography enhanced new bone apposition and osseointegration of Sr-modified implants or had a synergistic effect with released Sr ions 28,29,35 . Hence, in-depth investigations are required to isolate the pure and independent effect of strontium or surface topography on improving bone-implant integration.
The present systematic review had several limitations. Firstly, the follow-up of included studies ranged from 1 week to 12 weeks, so it remained unclear whether the osseointegration of Sr-incorporated Ti implants would be a stable anchorage, which could contribute to their long-term survival. Secondly, high publication bias (Begg, p = 0.039; Egger, p = 0.000) was found in Begg's and Egger's test. Therefore, the results need to be interpreted with caution. Thirdly, none of the implants included in the present study were loaded. Thus, future studies should evaluate the effect of Sr-incorporated Ti implants under loading conditions.

Conclusion
Based on available evidence so far, it can be concluded that Sr-modified titanium implants could enhance osseointegration and new bone formation of peri-implant area in animal models. Nonetheless, future clinical investigations are needed to verify the safety and effectiveness of Sr-modified implants. Search strategy and study selection. Databases of PubMed and EMBASE were searched up to November 2016 for relevant articles published, using search terms "strontium", in combination with "osseointegration", "bone apposition", "osteogenic", "osteogenesis", "new bone formation", "bone to implant contact" and "bone regeneration". Additionally, bibliographies of the selected studies and relevant review articles were also scrutinized for cross-references. Titles and abstracts of searches were initially screened by two authors (SJY, LY). Uncertainty in the determination of eligibility was resolved by discussion. Two authors reviewed full-text articles independently and final inclusion was based on the inclusion criteria.

Methods
Inclusion and exclusion criteria. The inclusion criteria for the study selection were: 1. Studies regarding titanium implants modified with strontium; 2. Studies reporting the percentage of bone-to-implant contact of Sr-modified and unmodified implants; 3. Studies with a minimum of 3 implants/group; The exclusion criteria for the study selection were: 1. In vitro studies; 2. Studies assessing the combined effect of Sr and other inorganic elements (e.g. Ag, phosphate) modified surface without strontium-only test group.
Risk of bias and quality assessment. The risk of bias (RoB) of included studies was assessed using the SYRCLE RoB tool for animal studies 55 . The tool, which aims to assess methodological quality, was adapted to appraise bias in animal studies. RoB was evaluated by providing a response of "high", "low" or "unclear" in each of the 10 items. As reported in a previous review, a modified RoB tool was used in which items 9 and 10 were adjusted to include information on whether the experiment was randomized or blinded at any level (Fig. 2.). Reporting quality of the included studies was assessed based on a modified ARRIVE guidelines in which a checklist of 24 items was included 56 . Each item was judged as "0" (not reported) or "1" (reported). The total score of each of included studies was also recorded ( Table 2). Data extraction. Two independent reviewers (SJY, LY) extracted data from the full-texts of selected articles.
General information, animal parameters (total number, species), methods of strontium incorporation, evaluation time points, analysis methods and outcomes and implant parameters (total number, material, length, diameter, shape, location and surface characteristics of test and control implants) were retrieved. The primary outcome and secondary outcomes were extracted (Tables 3 and 4). If data were only expressed graphically, numerical values were requested from the authors, and if a response was not received, digital ruler software was used to measure graphical data (ImageJ, National Institutes of Health, Bethesda, MD).
Scientific RepoRts | 7: 15563 | DOI:10.1038/s41598-017-15488-1 Statistical analysis. The primary and secondary outcomes were present in descriptive statistics. The standardized mean differences, together with 95% confidence intervals, were analyzed using random-effect model. Heterogeneity was tested using the I 2 statistic to describe the proportion of total variation. Values of 25, 50 and 75% were regarded as low, moderate and considerable heterogeneity, respectively 57 . When the value was >50%, qualitative analysis was conducted. A forest plot was generated, and heterogeneity was calculated by use of the statistical software package STATA (v11.0; StataCorp, College Station, TX). A p value < 0.05 was considered to indicate statistical significance, unless specified otherwise.
Potential publication bias was assessed using Begg's funnel plots and Egger's test at the p < 0.10 level of significance.