Study setting {9}
The study will be performed with adult patients from the implantology course at the School of Dentistry, São Paulo State University (Unesp), Araraquara, Brazil, looking for implant-supported oral rehabilitation and needing graft in the atrophic anterior maxilla.
Eligibility criteria {10}
To be enrolled in this study, patients must present bone insufficiency in the anterior region of the maxilla, with a minimum height of 7 mm and a width of less than or equal to 5 mm (assessed by CBCT); be over 18 years old, and have voluntarily signed the consent form. The exclusion criteria are patients who have general contraindications to the surgical procedure. These include patients who: are undergoing radiation in the head and neck region; have immunosuppression or immunodepression; are being treated or undergoing treatment with anti-resorptive drugs or drugs that alter bone metabolism; have an untreated periodontal disease; have poor oral hygiene; have uncontrolled diabetes; have psychiatric issues or unrealistic expectations; are pregnant; are lactating; are smokers.
Who will take informed consent? {26a}
The principal investigator, his Ph.D. student (coordinating investigator), and other team members will obtain the informed consent.
Additional consent provisions for collection and use of participant data and biological specimens {26b}
Not applicable; no ancillary studies are foreseen.
Interventions
Explanation for the choice of comparators {6b}
To allow the installation of dental implants in atrophic alveolar ridges, bone grafts must be previously performed for bone volume and height reconstruction. The autogenous graft stands out for its osteoconduction, osteoinductive, and osteogenesis properties, being considered the gold standard for this type of intervention [17].
Intervention description {11a}
•Tomography:
All participants must undergo CBCT tests before the surgical procedure (T1), in post-surgical follow-up at one week (T2) and eight months (T3).
• Planning and production: patient-specific Plenum® Oss 3Dβ fit:
A trained professional will perform the patient-specific block planning (Mimics and 3-Matic, Materialise, Belgium) from the DICOM files, manufactured by additive manufacturing in a 3D ceramic printer (CeraFab 7500 LITHOZ). The patient-specific (personalized) graft will be produced using Lithography-based Ceramic Manufacturing technology. The process involves printing the virtual model (previously designed and exported in an STL file) with β-TCP slurry. After finishing the printing process, the grafts will be cleaned with a solvent to remove excess resin inside the pores. Then, it will be sintered in a muffle furnace at 1000°C and sent for sterilization by gamma irradiation.
• Preparation and installation of the autogenous bone graft and Plenum® Oss 3Dβ fit:
The intra- and extra-oral asepsis will be performed with 0.12% and 2% chlorhexidine digluconate. Next, the local anesthesia will be applied with Articaine 4% 1:100.000 (Nova DFL, Brazil), followed by a linear incision over the alveolar crest and two vertical incisions. Next, the mucoperiosteal flap will be detached, and the autogenous bone graft collection will be started. A linear incision will also be made in the donor site, followed by a mucoperiosteal flap detachment and a mandible branch exposure. Trunk-conical drills and discs mounted in a straight handpiece will be used for osteotomy. After the graft removal, it will incubate in saline solution while the receptor site is prepared. Grooves will be performed in the receptor site to stimulate the vascularization, and the autogenous bone graft will be fixed in the anterior region of the maxilla by fixing screws. On the opposite side of the maxilla (test group), grooves will also be performed, and the 3D printed block will also be fixed by fixing screws. Then, a resorbable polydioxanone membrane (Plenum, Jundiaí, São Paulo, Brazil) will be used over 3 to 4mm of the bone grafts, and the suture will be performed with Nylon 5.0 (Ethicon®, Jonhson & Jonhson, New Brunswick, Nova Jersey, EUA). All surgical procedures will be performed by the same professional.
• Dental implants installation and biopsy:
Eight months after the graft surgery, the patients will undergo implant placement surgery. The same aseptic care as described above will be performed. Following the local anesthesia, a linear incision will be made, a mucoperiosteal flap will be detached, and the fixing screws will be removed. Biopsies of the grafted areas will be obtained in the vestibular palatine sense direction [18] to involve the residual and new-formed bone without interfering with implant placement. The obtained biopsies will be immediately fixed in paraformaldehyde 4%. Then, the osteotomy sequence and implant placement will be performed, and the implants (Plenum®, Jundiaí, São Paulo, Brazil) will be placed. The resonance frequency analysis (RFA) will measure the primary implant stability using Osstell® (Integration Diagnostics AB, Goteborg, Switzerland). The measures will be performed on the mesial, distal, vestibular, and palatal regions. In the same surgical procedure, in the cases with high quantitative bone, mini-implants will also be used on the vestibule-palatine side for future histological analyses [18]. Finally, the suture will be performed using Nylon thread, Nylon Blue 5.0 (Techsuture®, Bauru, São Paulo, Brazil).
• Healing caps placement
After six months of implant placement, healing caps will be placed to begin the prosthetic phase. The cover screw will be removed, RFA will check the secondary implant stability, and the healing cap installed. In the cases where mini-implants were used, they will be retrieved by trephine drills for posterior analysis [18].
Criteria for discontinuing or modifying allocated interventions {11b}
Not applied because the study will evaluate a medical device in a split-mouth allocation; therefore, it is impossible to change the allocation once the device is installed.
Strategies to improve adherence to interventions {11c}
Not applied, no adherence to the intervention is required in this trial.
Relevant concomitant care permitted or prohibited during the trial {11d}
After the grafting procedure, the postoperative prescription will include Amoxicillin 875mg + Potassium Clavulanate 125mg every 12 hours for seven days, Nimesulide 100mg every 12h for three days, Dypirone 500mg every 6 hours for three days. Implant post-surgical medication will include Amoxicillin 500 mg every 8 hours for seven days, Nimesulid 100mg every 12 hours for three days, Dipirone 500mg every 6 hours for three days, and Chlorhexidine digluconate twice a day for 15 days.
Steroids and other drugs that interfere with calcium metabolism should not be administered during this clinical investigation.
Provisions for post-trial care {30}
According to the Brazilian law (CNS Resolution No. 466 of 2012, items II.21 and IV.3.g), this trial will assure the participant and the companion of the reimbursement of expenses arising from the participation and the presence for consultations. Additionally, if any damage occurs to the participant due to the interventions from this research, the participant will be compensated and will receive full assistance for the necessary time (CNS Resolution No. 466 of 2012, items II.3.1 and II.3.2).
Outcomes {12}
Primary endpoints
Evaluate the gain of increase/maintenance of the volume of the bone graft area and the quality of the newly formed bone tissue after eight months of the postoperative between the synthetic graft in a personalized block with the autogenous bone graft.
Secondary endpoints
The secondary endpoints are:
• Assess bone neoformation in the anterior region of the maxilla when the personalized block graft is used compared to the use of autogenous bone block graft. This will be done by evaluating the percentage of new-formed bone tissue, soft tissue, and the rate of residual material from the bone graft.
• Investigate whether the choice of bone graft interferes with the primary and secondary stability of implants installed in the region. Measures will be performed with RFA immediately after de implant placement and after six months.
• Evaluate the influence of the graft on the maturation of newly formed bone tissue through the quantification of osteocalcin (OCN) from the biopsies retrieved just before the implant placement.
• Assess the influence of the use of the graft type on the activity of osteoblastic cell differentiation through the quantification of morphogenetic protein -2 (BMP-2) from biopsies samples.
• Evaluate the influence of autogenous and personalized graft on collagen matrix production through alkaline phosphatase quantification.
• Investigate the influence of the graft in the differentiation of endothelial cells through the quantification of vascular endothelial growth factor (VEGF).
• Evaluate the volume of mineralized new-formed bone tissue through micro-tomographic examinations.
• Verify the adaptation of the bone graft to the host bone.
• Evaluate whether there is a difference in the operative time for performing the ceramic bone graft compared to the autogenous graft.
Study analysis:
• Volumetric analysis
Volume resorption will be evaluated by comparing the cone-beam computed tomography tests performed after (T2) the graft placement and eight months later (T3) with the software Horus Project®. In addition, from the CBCT T2 test, an analysis regarding the adaptation of the bone graft to the residual bone will be performed in both groups.
• Biopsies analyses
- Histological analysis
The obtained biopsies will be immediately stored in Paraformaldehyde 4% for 48h and after immersed in 70º alcohol solution for the micro-tomographic test. Then, the biopsies will be washed with running water for subsequent immersion in EDTA solution, which will be changed three times a week for 50 days. After evaluating the correct decalcification of the biopsies, the dehydration process will be carried out. Dehydration will be done in increasing alcohol baths (70º, 90º, absolute alcohol), allowing the piece to be finally cleared in xylene for 3 hours and embedded in paraffin.
The mini-implant biopsies will be washed with running water for 6 hours and then dehydrated in a growing series of ethanol (60 - 100%) and infiltrated and polymerized in light-curing resin (Technovit 7200 VLC, Kultzer Heraeus GmbH & CO, Wehrheim, Germany).
- Micro-tomographic analysis
Before starting the process for inclusion in paraffin, the biopsies will be scanned by a microtomography (Skyscan®, Aatselaar, Belgium. Camera Pixel: 12.45; X-ray tube power: 65 kVP, X-ray intensity: 385 µA, integration time: 300 ms, filter: Al-1 mm and voxel size: 18 µm3c). Then, the images will be reconstructed, spatially repositioned, and analyzed by specific software (NRecon, Data Viewer, CTAnalyser, Aatselaar, Belgium). The threshold used in the analysis will be 25-90 shades of gray, and the values of mineralized tissue volumes will be obtained as a percentage. A trained examiner blinded to the experimental groups will perform this analysis.
- Histomorphometric analysis
The paraffin blocks will be cut in serial cuts with 4μm thick along their entire length. To obtain the histological blades, the pattern of selecting the center cut of the block will be followed. Blades will be stained with hematoxylin and eosin (HE, Merck & Co. Inc., New Jersey, USA). Blades images will be captured and scanned using an optical microscope with four-fold magnification objectives and 5-fold magnification eyepieces (Diastar - Leica Eichert & Jung products, Germany), with a digital camera (DFC-300-FX, Leica Microsystems, Germany) with 1.3-megapixel resolution coupled to this microscope and connected to a microcomputer with a digitilized image analyzer software Image J 1,45 (Wayne Rasband National Institutes of Health, USA). The following parameters will be evaluated: newly formed bone percentage, soft tissue percentage, and residual bone graft percentage. Measurements will be performed by a single calibrated examiner blinded to experimental groups.
The mini-implant blocks with bone tissue will be cut at a central point using a cut and wear system (Exakt Apparatebeau, Hamburgo, Germany). The sections will be approximately 45 μm thick, stained with Stevenel blue associated with acid fuchsin, and analyzed under an optical microscope (DIASTAR – Leica Reichert & Jung products, Wetzlar, Germany) at 100X magnification. Histomorphometric evaluations will be performed with the image analysis software (Image J, San Rafael, CA, USA). In addition, the percentages of bone-implant contact (%BIC) and the bone area between turns (%BBT) will be evaluated separately in the region between bone tissue and the center of the mini-implant. A blind and trained examiner will perform these analyses.
- Immunohistochemical analysis
Histological sections from the same paraffin blocks used for the histomorphometric analysis will be mounted on blades with an appropriate glass surface (Fisher Superfrost Plus; Thermo Fisher Scientific - Waltham MA, USA). The immunohistochemical reaction will be performed using the indirect immunoperoxidase technique with an amplifier. Therefore, 3% hydrogen peroxide will be used (Merck Laboratories, Kenilworth, Nova Jersey, USA) to inhibit endogenous peroxidase. Antigen recovery will be performed by immersing the slides in citrate phosphate buffer, pH=6, kept in a steamer (moist heat) for 20 minutes. Blocks of nonspecific reactions will be performed with bovine albumin (Sigma, San Luis, Missouri, USA) and skim milk. The primary antibodies against osteocalcin, alkaline phosphatase, VEGF, and BMP2 (Santa Cruz Biotechnology, Dallas, Texas, EUA), and bone metabolism markers. BMP2 is one of the main proteins related to bone neoformation and development, while alkaline phosphatase and osteocalcin allow the evaluation of collagen matrix production and bone mineralization. Vascular endothelial growth factor (VEGF) enables the assessment of local angiogenesis. As a secondary antibody, the biotinylated anti-goat antibody produced in rabbits will be used (Pierce Biotechnology, Waltham, Massachusetts, USA), the amplifier will be Avidin and Biotin (Vector Laboratories, Burlingame, California, USA), and diaminobenzidine (Dakocytomation, Carpinteria, California, USA) as the chromogen. At the end of the revelation by Diaminobenzidine, the counterstaining of the histological sections will be performed with Harris Hematoxylin. The sections will be analyzed under an optical microscope (LeicaR® DMLB, Heerbrugg, Switzerland) as well as the expression of proteins, coupled to an image capture camera (Leica® DFC 300FX, Leica microsystems, Heerbrugg, Switzerland) and connected to a microcomputer with scanned image analyzer software (Leica Camera Software Box®, Leica Imaging Manager -IM50 Demo Software).
• Implant stability analysis
The primary and secondary implant stability will be analyzed using the Osstell® device (Osstell AB, Gothenburg, Sweden). This device determines the implant stability by the resonance frequency analysis. The system includes using a SmartPeg fixed to the implant through an integrated screw. The SmartPeg is excited by a magnetic impulse from the measuring probe of the portable instrument, and the implant stability coefficient (ISQ) is calculated from the response signal. Results are displayed on the instrument ranging from 1 to 100. The higher the ISQ number, the greater the stability of the implant.
Stability measurements will be obtained in two directions: buccal-palatal and mesiodistal. Values will be compared within groups about primary and secondary stability. They will also be compared between groups to assess whether there is a difference between implant stability in the test and control groups.
Participant timeline {13}
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Enrollment
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Allocation
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Post Allocation
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TIMEPOINT
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-T1
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T0
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T1 (Intervention)
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T2 (8-months follow up)
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T3 (14-months follow up)
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Patients visits |
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Randomization: split-mouth |
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Tomography (T1, T2, and T3) |
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Implant placement and biopsies |
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ASSESSMENTS
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Surgery analysis
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| Sample size {14}
Sample calculations were performed based on tomographic bone formation data from the study, which evaluated the volumetric stability of autogenous and allogeneic corticomedullary block grafts used for volume increase associated with edentulous alveolar ridges [18]. This choice was due to the lack of studies comparing β-tricalcium phosphate blocks and autogenous bone and the similarity of the methodology for evaluating the stability of block grafts. As a result, it was verified that the minimum difference between the means of treatments concerning the percentage of volumetric reduction of the graft was 11.90, with a standard deviation of 12.09. Therefore, considering a study power (1-β) of 0.8 and an α power of 0.05, a minimum sample of 20 participants was determined, each of whom will receive two grafts, one synthetic in a personalized block and another in an autogenous block.
Recruitment {15}
Recruitment will be performed by local radio and social media (Facebook). The advertisement will invite patients who need implant rehabilitation on both sides of the maxilla, and after the patients attend a screening visit, the inclusion criteria will be assessed.
Assignment of interventions: allocation
Sequence generation {16a}
The treatments will be randomized using 20 binary sets (0- control treatment: autogenous graft and 1- test treatment: Plenum® Oss 3Dβ fit). The sets, 1 to 20, will correspond to each patient according to the enrolment order. The binaries numbers 0 and 1 will be randomized, corresponding to the treatment that will be applied. From left to right, the first number will define the treatment of the right side of the maxilla, and the second number will determine the treatment which will be applied on the left side.
Concealment mechanism {16b}
Randomization of treatments will be performed using the ResearchRandomizer software (https://www.randomizer.org/).
Implementation {16c}
The trial sponsor and the Principal investigator team will generate randomization with the allocation sequence.
Assignment of interventions: Blinding
Who will be blinded {17a}
Not applicable; this is an open trial.
Procedure for unblinding if needed {17b}
Not applicable; this is an open trial.
Data collection and management
Plans for assessment and collection of outcomes {18a}
For security, CT scans will be saved on the M3 Health’s server, which already has a security protocol. The biopsies analysis will be performed with positive controls for the antibody reactions.
Data management will follow the same procedures for verification, validation, and security of electronic systems used by the sponsoring company, M3 Health.
Plans to promote participant retention and complete follow-up {18b}
Data from participants which have already been collected will be used in the analysis referring to the completed steps, even if they do not conduct the clinical investigation.
Data management {19}
Data will be maintained at Electronic File Management System (SharePoint platform, Microsoft 365) and organized by the study sponsor. The hardcopy source documents will be stored at M3 Health in an appropriate room with controlled access. Patients’ identity will be determined by a letter/number code composed of the first and last letter of the name, the first and last letter of the last name, and the birth year.
Confidentiality {27}
According to the current legislation regarding General Law of Data Protection (LGPD), number: 13.709/2018.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
Not applicable, the study will not collect biological specimens for genetics or molecular analyses.
Statistical methods
Statistical methods for primary and secondary outcomes {20a}
Data from this study will be analyzed for their distribution by the Shapiro-Wilk normality test. If the data are distributed according to normality, parametric tests will be used for the inferential analysis of the data. The comparison between groups in each experimental period will be performed using the unpaired t-test. The longitudinal evaluation of these data within each group will be evaluated using the ANOVA test for repeated samples complemented by the Tukey test. If the data are not distributed according to normality and the qualitative parameters, they will be analyzed inferentially using non-parametric statistical tests. The comparison between groups in each experimental period will be performed using the Mann-Whitney test. The longitudinal evaluation of these data within each group will be evaluated using the Friedman test complemented by the Dunn test. The Biostat software version 5.3 will be used to perform the statistical analysis of this project.
Interim analyses {21b}
The Principal investigator will decide to suspend the study if any serious adverse event is reported until the cause is identified. If it is verified that the event was not related to the medical device, the study will restart under sponsor permission. However, if the cause of the serious adverse event is related to the investigational device and the investigation is terminated, the interim results will be accessed by the Principal investigator, that will perform the correspondent analysis.
Methods for additional analyses (e.g. subgroup analyses) {20b}
Not applicable; all the analyses are already described in the study protocol.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}
This study needs 20 participants to conclude the statical analysis of the primary endpoint. Then, a new participant will be recruited whenever there is any dropout or loss of follow-up until the collection of the biopsy of 20 participants.
Plans to give access to the full protocol, participant level-data and statistical code {31c}
Not applicable; only the principal investigator, the research team, the sponsor, and the Brazilian National Health Surveillance Agency (ANVISA) will access participant-level datasets and statistical code.
Oversight and monitoring
Composition of the coordinating center and trial steering committee {5d}
Not applicable; this is a single-center trial. The communication will be directly between the sponsor team, the principal investigator, and the coordinating investigator via telephone, e-mails, and internet meetings, following sponsor security protocol.
Composition of the data monitoring committee, its role and reporting structure {21a}
The sponsor monitoring team will perform all monitoring processes. The protocol describes the visit timing according to the research phases.
Adverse event reporting and harms {22}
Adverse effects will be registered in the case report form. According to the severity level of the event, the necessary actions are detailed, described in the protocol, following ANVISA obligations and recommendations.
Frequency and plans for auditing trial conduct {23}
The audit monitoring visit will be conducted by the sponsor at least once during the clinical investigation, in the stitch removal phase after graft installation (3rd visit of the participants) or whenever the sponsor understands that there are severe or repeated Protocol deviations, or when there is suspicion of fraud when requested or suggested by a regulatory authority.
Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}
Protocol amendments will be communicated to ANVISA. If they are substantial and directly reflect on subjects’ participation, they will be resubmitted to the same ethics committee that approved the study and then submitted to ANVISA for protocol amendment.
Dissemination plans {31a}
An annual report will be elaborated and submitted to ANVISA. After data analysis, the research will publish the results in an appropriate indexed life science journal. Participants will be communicated the results via e-mail or WhatsApp.