Design and setting of the study
The proposed study is designed as a pragmatic randomized controlled trial with a 5-year follow-up. Consolidated Standards of Reporting Trials (CONSORT) diagram is demonstrated in Table 1. We plan to enroll 36 edentulous patients (maxilla and/or mandible) in need of dental implant prothesis. The treatment protocol is to rehabilitate these patients with full-arch fixed implant-supported prosthesis with placement of four to eight implants in the maxilla and/or mandible. The restorative materials will be randomly divided into two groups: zirconia frameworks with ceramic veneering (Fig. 1) versus titanium frameworks with acrylic resin veneering (Fig. 2). The trial will last for five years and patients will be followed up per six months. During each visit, clinical examination, surface roughness test and submucosa plaque collection will be complete and biological and mechanical complications will be treated. X-ray assessment will be performed per year. Table 2 shows the participant timeline.
Table 1 Consolidated Standards of Reporting Trials (CONSORT) diagram
Table 2
TIMEPOINT | T0 | T1 | T2 | Month0 | Month6 | Month12 | Month18 | Month24 | Month30 | Month36 | Month 42 | Month 48 | Month 54 | Month 60 |
ENROLMENT: | | | | | | | | | | | | | | |
Eligibility screen | X | | | | | | | | | | | | | |
Informed consent | X | | | | | | | | | | | | | |
Implant surgery | | X | | | | | | | | | | | | |
Impression | | | X | | | | | | | | | | | |
Allocation | | | | X | | | | | | | | | | |
INTERVENTIONS: | | | | | | | | | | | | | | |
Final restoration: titanium | | | | X | | | | | | | | | | |
Final restoration: zirconia | | | | X | | | | | | | | | | |
ASSESSMENTS: | | | | | | | | | | | | | | |
Implant survival rate | | | | | | X | | | | X | | | | X |
Peri-implant plaque index | | | | X | X | X | X | X | X | X | X | X | X | X |
Bleeding on probing | | | | X | X | X | X | X | X | X | X | X | X | X |
Suppuration | | | | X | X | X | X | X | X | X | X | X | X | X |
Probing depth | | | | X | X | X | X | X | X | X | X | X | X | X |
Mechanical complication | | | | | X | X | X | X | X | X | X | X | X | X |
Paralleling X-ray | | | | X | | X | | X | | X | | X | | X |
Microbiotia sample collection | | | | X | X | X | X | X | X | X | X | X | X | X |
Surface roughness | | | | X | X | X | X | X | X | X | X | X | X | X |
The primary outcome variable is the difference between two groups for implant survival rate, peri-implant plaque index, peri-implant mucosal conditions, marginal bone resorption, peri-implant submucosal bacteria species. The secondary outcome variable is the difference of mechanical complication rates and surface roughnesses between two groups.
Ethical considerations
The proposed study is designed as a prospective single- center, randomized controlled trial. The trial has been approved by the Ethics Committee of Stomatology School and Hospital of Peking University, China (PKUSSIRB-202054027). In addition, the study has been registered in Clinical- Trials.gov and the identifier number is ChiCTR2000029470.
The systematic health condition of all the participants will be recorded. Before the implant surgery, all patients will receive clinical and radiographic assessment. All patients will also be required to receive oral hygiene instructions before implant surgery.
Recruitment
The recruitment of the participants will be initiated in May 2020 at the Stomatology School and Hospital of Peking University, China. Approximately 50 edentulous patients received full-arch implant-supported prosthesis every year in this hospital. By issuing announcements in the registered area and waiting area, it is expected that 36 eligible participants will be recruited within 2 years. Eligible patients will receive the study protocols and consent forms. Subjects will be included in the study until they sign the consent forms.
Inclusion criteria
1) Edentulous jaw
(2) General health,ASA I ~ II classification of American Society of Anesthesiologists
(3) Age over 22 years old
(4) Good oral hygiene and good compliance
(5) Signing an informed written consent form
Exclusion criteria
(1) Age under 22
(2) Poor oral hygiene and uncontrolled peridontitis
(3) Antibiotics use in the past 3 months
(4) Systemic disease: uncontrolled diabetes mellitus, cardiovascular disease, disease of immune system, blood disorders such as coagulation disorders, severe osteoporosis
(5) Long-term medication users: steroid, anti-epileptic drug, biophosphonates
(6) HIV infection, Hepatitis B, syphilis
(7) Bruxism
(8) Smoker
(9) Uncontrolled infection in the area intended for implant placement or other areas
(10) Maxillofacial tumor
(11) Face-neck radiotherapy
(12) Mental illness or high expectations
(13) Unable to sign informed consent
(14) Antibiotic use within the past 3 months
Interventions
The treatment protocol aims to rehabilitate these patients with full-arch fixed implant-supported prosthesis with placement of four to eight implants in the maxilla and/or mandible. The restorative materials will be randomly divided into two groups: zirconia ceramic frameworks with or without ceramic veneering versus titanium frameworks with acrylic resin veneering. Before prosthesis fitting, standard polishing procedure (GB/T 6060.2–2006) and examination will be done to ensure the same surface roughness between groups. The surface roughnesses of all specimens are measured with the help of a profilometer (Mitutoyo Surftest SJ-401; Mitutoyo Corp), according to a previous study [19].
Primary and secondary outcome variables
The primary outcome variable is the difference between two groups in implant survival rate, peri-implant plaque index, peri-implant mucosal tissue conditions, marginal bone resorption, peri-implant submucosal bacteria species. The secondary outcome variable is the difference of mechanical complication rates and surface roughnesses between two groups.
Clinical assessment
Clinical examination will be recorded at baseline (immediately after prosthesis placement) and every 6 months after final prosthesis.
The following parameters will be evaluated: peri-implant plaque index (PLI), Bleeding Index (BI), suppuration (0/1) and probing depth (PD). PLI, suppuration and PD is evaluated at four sites per implant (mesiobuccal, buccal, distobuccal and lingual/palatal) [20]. The PD will be measured to the nearest mm using a plastic graded probe (Hu-Friedy Manufacturing, Chicago, IL, USA). Bleeding Index (BI) is recorded at two sites per implant (buccal and lingual).
PLI: 0—no plaque in the gingival margin area; 1—thin plaque on the tooth surface of the gingival margin area, but it is not visible on inspection, if the side of the probe tip is used to scrape plaque; 2—Medium amount of plaque can be seen on the adjacent surface; 3—A large amount of soft dirt can be seen in the gingival sulcus or the gingival margin area and the adjacent surface.
BI: 0—absence of inflammation and absence of bleeding on probing; 1—mild marginal inflammation( slight change in color, little change in texture of any portion of but not the entire marginal or papillary gingival unit) and absence of bleeding on probing; 2—mild marginal inflammation (criteria as above but involving the entire marginal or papillary gingival unit) and spotting gingival sulcus bleeding on probing; 3—moderate inflammation (glazing, redness, edema, and/or hypertrophy of the marginal or papillary gingival unit) and linear gingival sulcus bleeding on probing; 4—severe inflammation(marked redness, edema, and/or hypertrophy of the marginal or papillary gingival unit) and bleeding on probing over the sulcus.
The two examiners will be trained and calibrated prior to and during the trial to achieve maximum reproducibility in the measurements [21]. For continuous periodontal clinical parameters (PD), the standard error of measurement will be evaluated. The average level of agreement between two examiners will be considered satisfactory when greater than 90% (Kappa test) for the other clinical variables.
To evaluate mechanical complication, an analysis variable will be used [22]. If no alterations are present, a “Alpha” classification will be attributed; if minor chipping occurs—not requiring any intervention besides polishing or recontouring without the need for prosthesis retrieval— the prosthesis will be recorded as “Bravo”; a “Charlie” classification will be attributed when the occurrence of major chipping, need for prosthesis retrieval and laboratory intervention; and finally, a “Delta” classification indicates fracture of the framework is present.
X-ray assessment
Marginal bone loss (MBL): immediately after final prosthesis, periapical radiograph will be taken for each implant. Periapical radiographs will also be taken per year after final prosthesis. For standardization, a paralleling technique will be conducted using an intramural digital system (Digora Toto, Soredex, Finland). A software program (Kodak Dental Imaging 6.1, Carestream Health®, Rochester, NY, USA) will be used to do radiographic analysis. The crestal bone level CBL will be measured as the vertical distance between 2 mm below the implant–abutment interface and the most crestal part of the alveolar bone [23, 24]. MBL will be measured mesially and distally for each implant. In each group, peri-implant MBL will be measured to the nearest mm.
The peri-implantitis lesions (PI) are defined as having PPD ≥ 5 mm, with positive suppuration or BI ≥ 1 and radiographic evidence of bone loss (> 2 mm) or according to consensus [25]. The peri-implant mucositis lesions (PM) are defined as being positive for suppuration or BI ≥ 1 and at the same time, no radiographic evidence of bone loss. The healthy implant sites (HI) are determined as having probing depths ≤ 4 mm, BI = 0, with no radiographic evidence for bone loss.
The rates of peri-implantitis and peri-implant mucositis will be calculated 1year, 3 years and 5 years after final restoration.
Laboratory assessment
1.Microbiological monitoring
1.1 Sample collection
Sulcus sampling will be performed immediately before prosthetic treatment and every 6 months after final prostheses. Before sampling, antimicrobial mouth wash should not be used within the past 48 hours, and the patient should not take food within the past 1 hr. Prior to sampling, clinical sites will be isolated and dried. If there is any supragingival/supramucosal plaque and calculus, they will be carefully removed. Submucosal plaque around one single implant will be sampled by inserting 4 sterile paper points (No. 30) into the base of the sulcus or pocket for 20 s. The paper points will be placed in labeled Eppendorf tubes and frozen, and transport to the laboratory for the subsequent extraction of DNA. 4 paper points around one single implant will be analyzed together as a unit.
1.2 Processing of microbiological samples
Bacterial identification and classification using 16S rRNA(ribosomal Ribonucleic Acid) gene sequencing technology-Polymerase Chain Reaction (PCR) samples (Sequencing of V1 and V3), library preparations, library quality inspections, and quantifications will be performed on qualified DeoxyriboNucleic Acid (DNA) samples of the oral cavity microorganisms, and the identified TAG sequences will be used for sample differentiation. Qualified libraries will be sequenced by the Illumina Hiseq 2500 high-throughput sequencing platform. Paired-End (PE) reads obtained by Hiseq / Miseq sequencing are spliced into one sequence, and the target sequence is subjected to quality control filtering. The filtered sequence is compared with a reference database, and the chimeric sequence is removed to obtain the final optimized sequence. Operational taxonomic units (OTU) cluster analysis and species classification annotations are based on optimized sequences, diversity index analysis is based on OTU clustering results, species structure analysis and species difference analysis are based on taxonomic information. Beta diversity analysis, principal co-ordinates analysis (PCoA analysis), and Linear Discriminant Analysis (LDA) Effect Size analysis will be used to compare differences in microbial diversity and differences in significant microbial species between two different groups.
2. Surface roughness assessment
The surface roughness will be measured using a profilometer (Mitutoyo Surftest SJ-401; Mitutoyo Corp) [19]. The tests will be performed before prosthetic delivery and every 6 months after final prostheses. To standardize the surface roughness measurements, 6 points are tested, which are located at mesial buccal, buccal, distal buccal, distal lingual, lingual, and mesial lingual around each abutment. For each point, measurements and analysis will be repeated twice. The reproducibility will be assessed by calculating the intraclass correlation coefficient (ICC) with a confidence interval of 95%.
Randomization, allocation and blinding
The subjects will be randomly divided into two groups according to the prosthetic materials: group 1: titanium framework with acrylic resin veneering; group 2: zirconia framework with /without ceramic veneering. The allocation of patients will be randomized using computer-generated permuted block randomization, and the allocation ratio will be 1:1. Randomization will be performed by sealed envelopes that will be opened after final impression being taken.
Sample analysis of microbiota in laboratory will be blinded after assignment to interventions. Each sample has a number associated with an allocation sequence, dental position information, and acquisition time information. The analyst of the PCR laboratory does not know the source of the sample. Interim statistical analysis will be done 1 year and 3 years after final prostheses. Final statistical analysis will be done at the end of this trial. The analyst is blinded to both the allocation of the patients and the interim analysis results.
Sample size
Sample size has been calculated by NCSS-PASS software according to previous studies. At 5 years, peri-implant bone loss in metal-resin/metal-ceramic IFCDP was 0.9 mm (standard deviation [SD] 0.4 mm), peri-implant bone loss in ceramic IFCDP was 0.6 mm, (standard deviation [SD] 0.1 mm) [26]. In our study, the criterion for significance is set at α = 0.05 (type I error) and at β = 0.10 (type II error). The analysis is two-tailed. Assuming the dropout rate at 15%, in order to determine if there is a difference in the amount of bone loss between the two groups, 18 cases per group and 36 cases in total are finally determined. Another study included twenty edentulous subjects who received two mandibular implants. The abutments were either Titanium or zirconium dioxide (non-submerged implant placement, within-subject comparison, left-right randomization). After 3 months, mean absolute counts (mean) for P. gingivalis from titanium abutment versus zirconia abutment were 1000000(SD:0) versus 64000(SD:36770). Mean absolute counts (mean) for P. Intermedia from titanium abutment versus zirconia abutment were 600088(SD:952117) versus 3600089(SD:804935) [8]. In this study, the criterion for significance will be set at α = 0.05 (type I error) and at β = 0.10 (type II error). The analysis will be two-tailed. Assuming the dropout rate at 15%, in order to determine if there is a difference in the amount of P. gingivalis and P. Intermedia between the two groups, 3 patients per group and 6 patients in total will be required.
In sum, the sample size is determined to be 36 in total and 18 per group.
Statistical analysis
All the statistical computations will be handled by the Statistical Package for Social Sciences software (SPSS, version 19.0 for Mac, SPSS Inc., Chicago, IL, USA).
1.Clinical monitoring and X-ray assessment
Continuous variables will be described by mean+/- standard deviation or median. Grade and quantitative data will be described by percentage. Age and other basic information between group 1 and group 2 will be compared by independent t test. Gender, implant survival rates, peri-implantitis rates and peri-implant mucositis rates between test group and control group will be compared by Chi-square test. PD and X-ray indices between group 1 and group 2 will be compared by independent t test. Generalized estimating equation (GEE) tests will be used to evaluate the differences of PLI and BI between two groups and among in follow-ups. Actual p-values are reported with significant differences accepted at 0.05.
2.Surface roughness assessment
Roughnesses between group 1 and group 2 will be compared by independent t test. Actual p-values are reported with significant differences accepted at 0.05.
3.Microbiological monitoring
The mean counts (× 105) of individual bacterial species and the percentage of the total DNA probe will be calculated initially in each implant, then per subject and averaged across patients between groups. Periodontal pathogens include P. gingivalis, F. nucleatum subspecies, P. intermedia and etc. The proportions for the species will be distributed into the six complexes and the “other” group, as proposed by Socransky et al [27]. Differences between group1 and group 2 for microbiologic parameters will be sought using the Wilcoxon signed rank test. Adjustments for multiple comparisons [28] will be performed when the bacterial species are evaluated simultaneously. The level of significance will be set at 5%.
Alpha and beta diversity analyses will be performed using Primer7 and QIIME2 [29, 30]. Alpha diversity, Shannon's diversity index of both species number and their distribution, Margalef's index of numbers, and Pielou's index of evenness of distribution [31, 32, 33] will be analyzed, and the significance of the differences between groups will be derived using an unpaired Student's t test. Beta diversity analysis includes visualization of data at multiple taxonomic levels, with unweighted and weighted UniFrac distance metrics so to generate PCoA plots [34]. Analysis of similarity (ANOSIM) tests will be performed to determine whether microbial communities are significantly different between groups. White's non-parametric test will be applied to test the differences in specific microbiota taxonomic abundance between the groups, with a cutoff of 0.005 false discovery rate using the software package STAMP [35].
Dissemination of results
The results of the trial will be published in international peer-reviewed journals. A summary of the study results will also be recorded at ClinicalTrials.gov to allow general access to obtain findings.
Interim analyses
Interim statistical analysis will be done 1 year and 3 years after final prostheses. The analyst will be blinded to the allocation of the patients and will submit the analysis results to data and safety monitoring board DSMB. DSMB will announce an early close as long as DSMB find the drop-out implants or patients exceed 20% of the enrolled implants or patients.