Materials and methods

Ethical considerations

This study was approved by the Human Research Ethics Committee of the School of Dentistry of Araraquara (CAAE: 32435614.5.0000.5416). In addition, the study protocol was registered in the Brazilian Clinical Trials Registry (ReBEC – RBR-7qhkrn - https://ensaiosclinicos.gov.br/rg/RBR-7qhkrn) on November 11, 2016, under the WHO Universal Trial Number (UTN) U1111-1173-6057. All participants read and signed an informed consent form prior to inclusion in the study. The fundamental ethical principles for research involving human subjects, as stated in the Declaration of Helsinki, were fully respected. The results from this clinical trial were reported according to the CONSORT guidelines. The patients were enrolled at the specialization clinic in implantology at the School of Dentistry of Araraquara.

Eligibility criteria

The inclusion criteria for this study were as follows: Age between 18 and 70 years; need for bilateral maxillary sinus floor elevation prior to the placement of osseointegrated dental implants; residual alveolar bone height adjacent to the maxillary sinus equal to or less than 4 mm; good systemic health; at least 4 months of post-extraction socket healing. Patients were excluded from the study if they met any of the following criteria: Current smokers or those who had stopped smoking within the previous 5 years; diabetic patients; use of medications or presence of systemic conditions that could alter bone metabolism; chronic upper airway infections; patients presenting asymptomatic sinus opacifications detected by the image exams; chronic use of anti-inflammatory drugs or antibiotics; presence of bruxism; alcohol abuse; drug dependence; pregnant women or those planning to become pregnant within the following year; a history of radiotherapy in the head or neck region.

Groups and study design

The maxillary sinuses of the patients were randomly assigned, in a 1:1 ratio, to be grafted with the following biomaterials: β-tricalcium phosphate (β-TCP; BETApro®, 99% purity and 20 a 600 μm of particle size, Procell, São Carlos, Brazil) or a combination of β-TCP and autogenous bone. Patients underwent the surgical procedure for the placement of the bone substitutes, and a 10-month healing period was allowed before implant placement surgery, during which biopsies were collected from the grafted sites. The implant prostheses were placed for rehabilitation six months after bone substitute placement. Randomization was carried out using a randomization table generated on the Random.org website (GJO). The operator only became aware of the side to be grafted with the different treatment combinations after performing the maxillary sinus lift (LCP).

Sample size

Regarding the sample size, the initial protocol estimated the inclusion of 20 participants. However, during the course of the study, we recalculated the sample size based on the variability observed in the early data and on the specific outcomes related to the maxillary sinus. This updated analysis indicated that 15 participants would provide adequate statistical power for the outcomes of interest. The sample size was calculated using a paired t-test based on tomographic data on bone formation from the study by Stiller et al., [9], which evaluated the volumetric stability of β-TCP grafts and β-TCP combined with hyaluronic acid for maxillary sinus floor elevation. The minimum difference between mean graft volume reductions was 13.90%, with a standard deviation of 5.00. Thus, considering a study power (β) of 0.9 and α of 0.05, a minimum sample size of 15 patients per group was determined. The sample size calculation was also based on the number of implants placed, with implant stability quotient (ISQ) defined as the primary outcome. Based on data from a previous study, a minimum clinically relevant difference of 10 ISQ units and a standard deviation of 8.45 units were considered [22]. Assuming a split-mouth design, a two-sided significance level of 5% (α = 0.05), and a statistical power of 80% (β = 0.20), the minimum required sample size was calculated to be 12 implants. Therefore, a minimum of 12 implants was required to detect a statistically significant difference between the treatment groups.

Surgical procedures: maxillary sinus floor elevation

Patients underwent maxillary sinus floor elevation surgery. Following local anesthesia, a full-thickness mucoperiosteal flap was raised to expose the lateral wall of the maxillary sinus. Using a round bur mounted on a contra-angle handpiece, an osteotomy was performed to access the sinus through the lateral wall. The sinus membrane was visualized by transparency, carefully elevated, and the lateral wall window was displaced into the sinus cavity. The grafts were inserted into the sinus in volumes of 2–5 g, depending on sinus anatomy and volume. One sinus was filled with a mixture of β-TCP and autogenous bone harvested from the patient’s mandibular retromolar region, while the contralateral sinus received only β-TCP. The mixture of autogenous bone and β-TCP was based on a visual assessment of the harvested autogenous bone volume. No weight-based measurements were performed. The graft materials were mixed intraoperatively to obtain an approximately 1:1 volume ratio before placement. After graft placement, the surgical site was covered with a resorbable collagen membrane (Genderm®, Baumer, Brazil) and sutured using 5 − 0 nylon sutures (Ethicon, Johnson & Johnson, Brazil). The donor site was closed in layers using 4 − 0 Vicryl sutures (Ethicon, Johnson & Johnson, Brazil) for internal closure and 5 − 0 nylon for external sutures. Postoperative medications were prescribed to all patients. Amoxicillin (500 mg) was administered orally three times daily for 7 days, ibuprofen (600 mg) three times daily for 5 days, and dipyrone (500 mg) four times daily for 3 days. Additionally, patients were instructed to rinse with a 0.12% chlorhexidine digluconate mouthwash three times daily for 14 days. Sutures were removed after 14 days.

Surgical procedures: implant placement

Ten months after the sinus floor elevation procedure, implants were placed in the grafted areas. Following local anesthesia, a full-thickness mucoperiosteal flap was raised to expose the alveolar ridge for implant placement (Drive®, AQUA, HE connection, 4.3 × 10 mm or 4.3 × 11.5 mm, Neodent, Curitiba, Brazil). Implants were installed in the indicated regions, and for prosthetic convenience, some implants were also placed in native, non-grafted areas. During implant placement, biopsies were obtained using a 2-mm diameter trephine bur from the implant sites. After implant insertion, the surgical site was sutured using 5 − 0 nylon sutures (Ethicon, Johnson & Johnson, Brazil). Postoperative medication and care were identical to those after sinus graft surgery. Sutures were removed after 14 days. Implant prostheses were placed for rehabilitated six months after surgery. Of the 15 patients evaluated, 13 received full-arch implant-supported prostheses, while 2 patients received partial implant-supported prostheses for posterior segment rehabilitation. Neither the operator who placed the dental implants nor the patients were aware of the side assigned to each treatment combination after the maxillary sinus lift procedure (ASDSFCS).

Implant stability analysis

Implant stability was assessed at the time of placement using the Osstell® device (Osstell AB, Göteborg, Sweden), which determines implant stability through resonance frequency analysis by calculating the Implant Stability Quotient (ISQ). Results are displayed on the device on a scale from 1 to 100, with higher ISQ values indicating greater implant stability. Insertion torque values (Ncm) were also recorded during implant placement. Primary stability data were collected for all implants placed in the study participants, including implants inserted in grafted sites (β-TCP and β-TCP + AB), which presented ≤ 4 mm of residual bone height according to the eligibility criteria, as well as implants placed exclusively in native bone. Stability measurements obtained from implants placed in grafted sites and those placed exclusively in native bone were analyzed separately.

Histomorphometric analysis

Biopsies collected for histomorphometric analysis were fixed in 4% paraformaldehyde for 48 h. Samples were then decalcified in 7% EDTA for 60 days and subsequently processed for paraffin embedding. Histological sections of 5 μm thickness were obtained and stained with hematoxylin and eosin (H&E). Images of the sections were captured using a DIASTAR optical microscope (Leica Reichert & Jung Products, Germany) with a 4×/10× objective. The images were digitized with a DXC-107 A/107AP video camera (Sony Electronics Inc., Japan) attached to the microscope and transferred to a computer. The percentages of bone, soft tissues, and bone substitute were determined in two sections of each biopsy. Quantitative analysis was performed using image analysis software (ImageJ, Jandel Scientific, San Rafael, CA, USA). The analysis was performed by a trained and blinded evaluator (LLR).

Tomographic analysis

Radiographic images were acquired using a cone-beam computed tomography (CBCT) scanner (i-CAT Classic; Imaging Sciences International, Hatfield, PA, USA), generating DICOM-based datasets with a resolution of 96 dpi, 14-bit grayscale, and voxel size of 0.25 mm. The scanner was operated at 120 kVp, 5 mA, with an exposure time of 20 s. CBCT scans were performed preoperatively, 14 days after the maxillary sinus floor elevation procedure, and one week before implant placement. Using virtual planning software (DentalSlice Bioparts Software®, Brasília, Brazil), measurements of the maxillary sinuses were taken at the three time points, at precisely the same locations. The distal limit of the nasopalatine canal was adopted as a fixed reference point in all three scans. From this distal limit, slices were numbered to the central region of the grafted area, with two additional mesial and two additional distal slices, resulting in a total of five slices per maxillary sinus. This approach allowed measurement of the same region across all three CBCT scans. Linear measurements from the alveolar crest to the sinus floor (mm) were performed. Additionally, the percentage of residual bone in the grafted region after 10 months was compared to the bone height immediately after biomaterial insertion to evaluate the dimensional stability of the grafts. The analysis was performed by a trained and blinded evaluator (ACG).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 (San Diego, CA, USA). Normality of the data from resonance frequency analysis, histomorphometric analysis, and tomographic analysis was confirmed using the Shapiro-Wilk test (p > 0.05). Implant insertion torque data did not follow a normal distribution. Parametric one-way ANOVA followed by Tukey’s post-hoc test was used for comparisons between experimental groups for resonance frequency analysis, tomographic and histometric analysis. Non-parametric Kruskal-Wallis tests followed by Dunn’s post-hoc test were used for comparisons of implant insertion torque (βTCP vs. βTCP + autogenous vs. Native Bone). Linear and volumetric bone formation and the composition of the grafted areas were analyzed using paired t-tests. Additionally, a Pearson correlation test was performed to assess the relationship between graft stability and the residual bone in the posterior maxilla. All tests were conducted with a 95% confidence level.