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 [18].
Intervention description {11a}
Tomography
All participants must undergo CBCT tests before the surgical procedure (T1), in post-surgical follow-up at 1 week (T2) and 8 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® Guide (Plenum, Jundiaí, São Paulo, Brazil) will be used over 3 to 4 mm 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 [19] 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 [19]. Finally, the suture will be performed using Nylon thread, Nylon Blue 5.0 (Techsuture®, Bauru, São Paulo, Brazil).
Healing caps placement
After 6 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 [19].
Criteria for discontinuing or modifying allocated interventions {11b}
Criteria were 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. The patient will be discontinued from the study only if they experience a serious adverse event, which can be correlated with the device. So far, we have foreseen a serious allergic reaction to the device material and a serious infection.
Strategies to improve adherence to interventions {11c}
Adherence will be maximized by educating patients about post-operative care and the importance of this process to the treatment successful.
Relevant concomitant care permitted or prohibited during the trial {11d}
After the grafting procedure, the postoperative prescription will include Amoxicillin 875 mg + Potassium Clavulanate 125 mg every 12 h for 7 days, Nimesulide 100 mg every 12 h for 3 days, and Dipyrone 500 mg every 6 h for 3 days. Implant post-surgical medication will include Amoxicillin 500 mg every 8 h for 7 days, Nimesulide 100 mg every 12 h for 3 days, Dipyrone 500 mg every 6 h for 3 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, as described in the exclusion criteria.
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 as follows:
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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
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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 6 months
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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
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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
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Evaluate the influence of autogenous and personalized graft on collagen matrix production through alkaline phosphatase quantification
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Investigate the influence of the graft in the differentiation of endothelial cells through the quantification of vascular endothelial growth factor (VEGF)
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Evaluate the volume of mineralized new-formed bone tissue through micro-tomographic examinations
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Verify the adaptation of the bone graft to the host bone
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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 70° 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 (70°, 90°, absolute alcohol), allowing the piece to be finally cleared in xylene for 3 h 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, Kulzer 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 fourfold magnification objectives and 5-fold magnification eyepieces (Diastar - Leica Reichert & 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 digitalized 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’s blue associated with acid fuchsin, and analyzed under an optical microscope (DIASTAR – Leica Reichert & Jung products, Wetzlar, Germany) at × 100 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 min. 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, TX, EUA) are 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) will be used 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}
| Study period |
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Enrollment | Allocation | Post allocation |
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Timepoint | − T1 | T0 | T1 (intervention) | T2 (8-month follow up) | T3 (14-month follow up) |
---|
Patients visits | x | | | | |
Eligibility screen | x | | | | |
Informed consent | x | | | | |
Randomization: split-mouth | | x | | | |
Intervention
|
Tomography (T1, T2, and T3) | x | | x | x | |
Graft installation | | | x | | |
Implant placement and biopsies | | | | x | |
Healing caps placement | | | | | x |
Assessments
|
Surgery analysis | | | x | | |
Implant stability | | | | x | x |
Biopsies | | | | x | |
Biopsies analyses | | | | x | x |
Adverse events | | | x | x | x |
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 [19]. 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. To maintain the power of the study, the sample size should be maintained, thus if any participant dropout or is discontinued before the analysis of the primary outcome, a new participant will be recruited instead, until complete 20 participants.
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. Additionally, as the study will be carried out at a public university that serves patients free of charge in various undergraduate and graduate courses, the study will be disseminated to students and professors, allowing them to refer patients with the necessary inclusion criteria to be enrolled in this study.