Biphasic calcium phosphate with or without hyaluronic acid vs. deproteinized bovine bone mineral for maxillary sinus augmentation. A randomized clinical trial

UNIVERSIDAD DE SEVILLA FACULTAD DE ODONTOLOGÍA DOCTORADO DE CIENCIAS DE LA SALUD BIPHASIC CALCIUM PHOSPHATE WITH OR WITHOUT HYALURONIC ACID VS. DEPROTEINIZED BOVINE BONE MINERAL FOR MAXILLARY SINUS AUGMENTATION. A RANDOMIZED CLINICAL TRIAL. Tesis Doctoral NICOLA ALBERTO VALENTE Sevilla, 2021 Tesis Doctoral – Nicola Alberto Valente 2 Sevilla, Enero de 2021 EUGENIO VELASCO ORTEGA, Profesor Titular de Odontología Integrada de Adultos y Gerodontología de la Facultad de Odontología y Director del Máster de Implantología Oral de la Universidad de Sevilla. ANTONIO BARONE, Profesor Titular del Departamento de Cirugía de la Facultad de Odontologia de la Universidad de Pisa. CERTIFICAN: Que D. NICOLA ALBERTO VALENTE, inscrito en el programa de Doctorado de Ciencias de la Salud de la Universidad de Sevilla, ha realizado bajo su tutela y dirección el trabajo de investigación titulado Biphasic calcium phosphate with or without hyaluronic acid vs. deproteinized bovine bone mineral for maxillary sinus augmentation. a randomized clinical trial, que consideramos satisfactorio para optar al título de Doctor en Odontología. Prof. Velasco Ortega Prof. Antonio Barone Table of contents 1. INTRODUCTION ................................................................................. 5 1.1. ANATOMY OF THE MAXILLARY SINUS ........................................................ 7 1.2. BIOMATERIALS ................................................................................................... 9 1.3. AIM ....................................................................................................................... 11 2. PATIENTS AND METHODS ............................................................ 13 2.1. STUDY DESIGN ................................................................................................. 14 2.2. RANDOMIZATION, ALLOCATION CONCEALMENT, BLINDING, CALIBRATION ............................................................................................................. 16 2.3. PRIMARY AND SECONDARY OUTCOMES .................................................. 16 2.4. INTERVENTIONS ............................................................................................... 16 2.4.1. SINUS AUGMENTATION ............................................................................................. 16 2.4.2. IMPLANT PLACEMENT ............................................................................................... 20 2.4.3. RESTORATIVE PHASE AND MAINTENANCE ........................................................... 20 2.5. PATIENT REPORTED OUTCOME MEASURES (PROMS) ............................ 20 2.6. COMPLICATIONS .............................................................................................. 21 2.7. HISTOLOGIC PROCEDURES ........................................................................... 21 2.8. STATISTICAL ANALYSIS ................................................................................ 22 3. RESULTS ............................................................................................. 24 3.1. PRIMARY OUTCOMES ..................................................................................... 25 3.1.1. HISTOLOGICAL RESULTS .......................................................................................... 25 Tesis Doctoral – Nicola Alberto Valente 4 3.1.1.1. CONTROL (ABBM) .................................................................................................... 25 3.1.1.2. TEST 1(TPC) AND 2 (TPC + HYALURONIC ACID) ................................................ 30 3.2. SECONDARY OUTCOMES ............................................................................... 32 3.2.1. MEAN BONE GAIN ....................................................................................................... 32 3.2.2. IMPLANT INSERTION TORQUE ................................................................................. 33 3.2.3. COMPLICATIONS AND IMPLANT FAILURES ........................................................... 33 3.2.4. PATIENT’S REPORTED OUTCOME MEASURES ...................................................... 34 4. DISCUSSION ....................................................................................... 36 5. CONCLUSIONS .................................................................................. 43 6. BIBLIOGRAPHY ............................................................................... 45 1. Introduction Tesis Doctoral – Nicola Alberto Valente 6 The implant-prosthetic rehabilitation of the atrophic maxilla requires the creation of sufficient volumetric quantity of bone, through regeneration techniques, necessary to position the implants. Although the purpose of the regenerations is the same in the upper and lower jaws, the techniques, and above all their predictability, vary greatly1, 2. Within this wide variability, the balance probably hangs in favor of the upper posterior maxilla1. Maxillary sinus augmentation (MSA) is probably the most predictable and best performing technique.3-6 The maxillary sinus floor elevation technique with lateral approach has been used in implant surgery for almost 40 years by now with high predictability of success. It was first described by Tatum in 1976, but first published by Boyne and James in 1980.7, 8 In the first description of the technique present in the literature the sinus elevation was used not only to allow implant placement but, in 11 of the 14 cases reported, it was used in order to be able to reduce the posterior ridge, thus augmenting the inter-arch space and provide the patients with a conventional prosthesis.8 The technique has been further developed and modified during the years and its indications and directions have been refined by several authors. We distinguish now between a one stage procedure, with simultaneous insertion of the implants, or a two stage procedure, when the residual bone height is less then 4mm, but the basic procedure has essentially remained the same as was firstly described.9 Despite the wide spread use of this surgical technique and its relative safety there are several variables and possible complications that must be Tesis Doctoral – Nicola Alberto Valente 7 taken into consideration in the planning and during the surgical procedure itself. One of these is the blood supply and vascularization of the sinus cavity and Schneider membrane and, in particular of the lateral maxillary wall, which is of crucial importance as a source of blood supply for our graft material and because the accidental severing of a vessel during the antrostomy can be, in some case, a significant intraoperatory complication. This technique, however, requires that the clinician must have an absolute mastery of the maxillary sinus’ anatomy before performing a sinus lift to avoid inconveniences that can prolong times of realization or even postpone execution, in rare cases. A large-caliber antral alveolus artery, for example, can cause intense bleeding, while the presence of a septum can cause the tearing of a sinus membrane.10-12 1.1. Anatomy of the maxillary sinus The maxillary sinus is the largest of the paranasal sinuses, which also include the frontal, sphenoid and ethmoidal sinuses. It usually occupies a large part of the body of the maxillary bone; it develops between the second and third month of intrauterine life, with a volume of 0.1-0.2 cubic centimeters at the time of birth. In fact, its size and its degree of pneumatization increase with the eruption of the first permanent elements, until it matures at the end of the adolescence period. A further widening can come with the persistence, on the medium/long term, of an edentulism in correspondence of the area of the floor of the sinus. Tesis Doctoral – Nicola Alberto Valente 8 Classically, the maxillary sinus is described as a pyramidal pneumatic cavity. The base corresponds to the lateral wall of the nasal cavity, its upper part is where the opening of the maxillary sinus into the nasal meatus is located; There is also a mesio-buccal wall, depressed in correspondence with the canine fossa, a superior one which forms the floor of the orbit, a posterior one facing the anterior wall of the pterygomaxillary fossa. The last wall, as already mentioned, partly corresponds to the upper alveolar process and partly to the hard palate; in a dentate adult this is the most solid of the bony walls, even though it has recesses in correspondence with the roots of premolars and first molars; these will undergo a thinning with the progress of the individual's age. The blood supply of the maxillary sinus is provided by three branches of the Maxillary Artery (MA): the Greater Palatine Artery, the Infraorbital Artery (IOA), and the Posterior Superior Alveolar Artery (PSAA). The PSAA originated from the MA while it passes through the pterygopalatine fossa. It descends to penetrate the maxillary tuberosity to give terminal alveolar and dental branches that supply blood for the posterior superior teeth, gingiva, and lining of the antrum. The IOA courses along the infraorbital groove and canal after its origin from the MA, then it exits from the infraorbital foramen on the facial aspect of the maxilla. Along the course through the infraorbital canal, the IOA gives origin to superior anterior alveolar branches that provides blood supply to the anterior teeth and lining of the antrum.11, 13 Usually the PSAA and the IOA form anastomoses inside and outside the bony lateral antral wall that supplies the Schneiderian membrane and the epiperiosteal vestibular tissues. According to the literature, an intraosseous Tesis Doctoral – Nicola Alberto Valente 9 anastomosis is constantly present, while an extraosseous one is present in about 44% of the cases.14, 15 Of particular importance is the intraosseous anastomosis, which is also called Alveolar Antral Artery (AAA). It was first described in 1934 and it passes through the area where the bony window is most frequently opened during sinus elevation.16 1.2. Biomaterials In addition to being aimed at obtaining the quantity of bone necessary for implant positioning, the regenerative techniques should also allow gaining adequate bone quality, suitable not only for receiving the implant in its volume, but also for giving adequate stability immediately and over time. The biomaterial used can be a key factor in determining the quality of the regenerated bone. In general, there are two types of materials that can be used in MSA techniques: autogenous bone and bone substitutes, with the latter further distinguishable into allogeneic, xenogenic or synthetic. The exclusive use of autologous bone for MSA provides for the removal of large quantities of bone, with the need to open a second surgical site and a significantly increased morbidity and consequent discomfort of the patient. Bone substitutes avoid this inconvenience and, very often, are used in combination with limited quantities of autologous bone, taken in the proximity of the area to be regenerated, so as not to give up the osteogenic qualities of the latter. The bone substitute of choice in the MSA, perhaps because the most documented in the scientific literature, is the anorganic bovine bone mineral (ABBM). There is a large amount of studies in the literature analyzing different materials used in the MSA procedure, in several of these studies different Tesis Doctoral – Nicola Alberto Valente 16 2.2. Randomization, allocation concealment, blinding, calibration Randomization sequence was obtained using a random permutation sequence generator (Statistics Toolbox, MatLab 7.11, The MathWorks, Natick, MA) without any kind of minimization of confounding factors such as age, smoking habit, gender and augmentation area. 2.3. Primary and secondary outcomes Primary outcome: Histomorphometric parameters of the augmented bone. Secondary outcomes:  Mean bone gain measured at 9 months through CBCT evaluation  Intraoperative and post-operative complications  Implant insertion torque measured in Ncm  Early implant failure  Patient related outcome measures (PROMs) 2.4. Interventions 2.4.1. Sinus augmentation All the patients enrolled in the study received a session of oral hygiene and a periodontal examination before the surgical procedure to obtain a more favorable oral environment for wound healing. A CBCT was mandatory for all included cases to verify that the maxillary sinus was clear and that the residual bone height was 3 mm or less. A voxel size of 0.125 mm, with a set Tesis Doctoral – Nicola Alberto Valente 17 of the parameters to 8.0 mA, 80 kV and an exposure time of 12-18 s were applied. CBCT radiographic measurments were carried out by GD following the protocol described in a series of studies in order to standardize the procedure.31-34 All the enrolled patients received 2 gr. of amoxicillin and clavulanic acid (or 600 mg. of clindamycin for those who were allergic to penicillins) as a pre-medication one-hour before surgery. All patients rinsed for 1 minute with 0.2% chlorhexidine mouthwash (and twice a day for the following 3 weeks). Local anesthesia was administered, a mid-crestal incision with mesial and distal release were performed to access the lateral bone wall of the maxillary sinus, subsequently, ultrasound bone surgery (Piezosurgery ®, Carasco, Italy) with specific tips was used for the bone window osteotomy (Figure 1). The Schneiderian membrane was reflected and lifted up medially with flat sinus curettes. Once the sinus membrane was completely lifted a bioabsorbable pericardium membrane (Smartbrane, Regedent AG, Zurich, Switzerland) was applied to protect it (Figure 2). Figure 1: Bone window created with the aid of piezosurgical inserts Tesis Doctoral – Nicola Alberto Valente 18 Figure 2: Sinus membrane elevated and protected with a bioabsorbable membrane The randomization sealed envelopes were opened and the clinician allocated the patients to one of the three experimental groups: 1) Control group that received Demineralized Bovine Bone Mineral (Bio-Oss Cancellous, Geistlich, Wolhunsen, Switzerland); 2) Test group 1 that received TCP with particle size ranging from 250 to 1000 µm (Osopia, Regedent, Zurich, Switzerland); 3) Test group 2 that received TCP as in test group1 plus crosslinked Hyaluronic Acid (Hyadent BG, Regedent, Zurich, Switzerland) with a ratio 2 to 1. The sinus was grafted with the biomaterial corresponding to the group, the bony window was repositioned, stabilized and covered with a resorbable pericardium membrane (Figures 3, 4). Tesis Doctoral – Nicola Alberto Valente 19 Figure 3: Sinus grafted with biomaterial Figure 4: Bone window repositioned after completion of grafting All patients were prescribed 1gr amoxicillin + clavulanic acid twice daily for 7 days (for those who were allergic to penicillin 300mg clindamycin 3 times daily for 7 days) as post-operative prophylaxis. All patients received 8mg of dexamethasone immediately after surgery and 4 mg of dexamethasone per day were prescribed for the following 5 days. Analgesics Tesis Doctoral – Nicola Alberto Valente 20 were prescribed (1gr paracetamol 3 times daily) according to patients’ need. The use of removable temporary restorations was not recommended during the healing period. 2.4.2. Implant placement Nine months after the MSA a CBCT analysis was required to evaluate the total bone height gain. Depending on the treatment plan, between one and three implants, bone level (BL) or tissue level (TL), of two different manufacturers (Institut Straumann AG, Basel, Switzerland and Sweden & Martina, Due Carrare, Italy) were positioned. While preparing the osteotomy implant site a bone biopsy was harvested using a trephine. The implant insertion torque was measured in Ncm for each implant. 2.4.3. Restorative phase and maintenance After an osseointegration period of 3 months, the implants were restored. The prosthetic procedures were similar for all groups, impressions were taken with a polyether rubber material (Impregum, Espe Dental AG, Seefeld, Germany) and a customized impression tray. Final Zirconia screwretained restorations were delivered after a period of 2/3 weeks, peri-apical radiographs were taken and oral hygiene instructions were given to all participants in the study. 2.5. Patient reported outcome measures (PROMs) Tesis Doctoral – Nicola Alberto Valente 21 Two weeks after MSA all patients answered a questionnaire regarding the oral health-related quality of life using the short-form Oral Health Impact Profile (OHIP-14).35 This questionnaire consists of 14 questions each one formulated in the following way: "How often (impact item) because of problems with your teeth, mouth or dentures?" The impact items belonged to 7 different impact domains: 1. Functional limitations; 2. Physical pain; 3. Psychological discomfort; 4. Physical disability; 5. Psychological disability; 6. Social disability; 7. Handicap. Each question could be answered with a score from 0 to 4 corresponding to: 0 = never, 1 = hardly ever, 2 = occasionally, 3 = fairly often, and 4 = very often. 2.6. Complications Possible complications were recorded at different time points of the study. Failure of the implant was considered as: any mobility of the implant, any infection that required removal, or any implant fracture. Implant success criteria were chosen according to Buser et al. 1990.36 2.7. Histologic procedures Twenty-four bone biopsies, obtained from 24 maxillary sinuses, were fixed by immediate immersion in 10% buffered formalin and processed (Precise 1 Automated System; Assing, Rome, Italy) to obtain thin ground sections. The specimens were dehydrated in an ascending series of alcohol rinses and embedded in glycol-methacrylate resin (Technovit 7200 VLC; Kulzer, Wehrheim, Germany). The specimens were sectioned, along their longitudinal axis, with a high precision diamond disk at about 150 µm and ground down to about 30 µm Tesis Doctoral – Nicola Alberto Valente 22 with a specially designed grinding machine Precise 1 Automated System. Three slices were obtained from each specimen, subsequently stained with acid fuchsin and toluidine blue before the analysis. Histological analysis was carried out using a light microscope (Laborlux S, Leitz, Wetzlar, Germany) connected to a high-resolution video camera (3CCD, JVCKY-F55B, JVC, Yokohama, Japan) and interfaced with a monitor connected to a computer. This optical system was associated with a digitizing pad (Matrix Vision GmbH, Oppenweiler, Germany) and a histomorphometry software package with image capturing capabilities (Image-Pro Plus 4.5, Media Cybernetics Inc., Immagini & Computer Snc, Milano, Italy). One single well-trained examiner, who was not involved in the surgical treatment, evaluated the histological results. Percentages of newly formed bone, marrow spaces and residual graft particles were reported. 2.8. Statistical analysis Power analysis was employed to determine the sample size by using a 0.05 significance level and a power of 90%, based on the results reported in previous review paper concerning histomorphometric outcomes.37 Sample size from 3 to 5 per treatment arm was required to detect significant difference between the bone substitute material groups by using secondary outcomes, that is, percentage of residual biomaterial and connective tissues. Sample size was increased with a factor 1.25 for possible drop-out of subjects. Final sample size per group ranged from 4 to 6 subjects. Descriptive statistics were calculated, including means, standard deviations, medians, and confidence intervals. Age, baseline bone height, insertion torque, complications, bone gain and PROMs scores were analyzed Tesis Doctoral – Nicola Alberto Valente 23 using the one-way analysis of variance (ANOVA) test, while the statistical analysis of the histomorphometric values for new bone, marrow spaces and residual graft particles was made using one-way multivariate analysis of variance (MANOVA). The level of significance was established at 5% (P = .05), and the analysis was carried out using statistic software (SPSS version 26, IBM). 3. Results Tesis Doctoral – Nicola Alberto Valente 25 Thirty-six patients were considered eligible, 12 patients were excluded from the study for the following reasons: 5 patients refused to give their consent to participate in a randomized clinical trial, 3 patients had a thickening of the sinus mucosa that required additional examinations, 2 patients were under treatment with Direct Oral Anticoagulants and 2 patients after being enrolled and treated had to move to another city due to their job. Baseline values for age, gender, initial bone height and number of implants placed are summarized in table 1. Table 1 – Baseline parameters Parameter Test 1 (BCP) Test 2 (BCP + Hyaluronic acid) Control (ABBM) Group comparison Age (mean ± SD) 57.63 ± 13.97 60.63 ± 11.21 49.5 ± 11.28 P = 0.194 Gender ratio (m/f) 3/5 3/5 3/5 Initial bone height (mm) ± SD 2.5 ± 0.38 2.63 ± 0.23 2.69 ± 0.46 P = 0.591 Total implants 16 13 15 3.1. Primary Outcomes 3.1.1. Histological results Three biopsy samples were not analyzed (one for each group) because the biopsies were damaged during their removal from the trephine. A total of 21 biopsies were examined, specifically 7 for each group. All biopsy samples at low magnification showed a certain amount of new bone formation. 3.1.1.1. Control (ABBM) Tesis Doctoral – Nicola Alberto Valente 32 Considering the different histological components observed in biopsies, based on the different grafting materials, there was a statistically significant difference, F (6, 32) = 16.83, P < 0.0005; Wilk’s Λ = 0.058, partial η2 = 0.759. When analyzing the between-subject effects, the graft material used had a statistically significant effect on both non-mineralized tissue (F [2, 18] = 72.003; P < .0005; partial η2 = 0.889), and residual graft particles (F [2, 18] = 126.268; P < .0005; partial η2 = 0.933), but not on new bone (F [2, 18] = 1.819; P = 0.191; partial η2 = 0.168), so the percentage of new bone was not statistically different between the three groups. The Tukey HSD post hoc test revealed that non-mineralized tissues were statistically significantly different between test 1 and control (P < 0.0005), test 2 and control (P < 0.0005), but not between test 1 and test 2 (P = 0.948). Similarly, RGP were statistically significantly different between test 1 and control (P < 0.0005), test 2 and control (P < .0005), but not between and test 1 and test 2 (P = 0.985). 3.2. Secondary Outcomes 3.2.1. Mean bone gain Mean bone gain values, measured on CBCT, are shown in table 3 was 11.05 ± 1.5 mm for the control group, 12.25 ± 2.3 mm for the test 1 group, 11.04 ± 2.3 mm for the test 2 group, the difference observed between the groups was not statistically significant (F [2.20] = 0.881, P = 0.43) (Table 3). Tesis Doctoral – Nicola Alberto Valente 33 Table 3 – Mean bone gain per group Test 1 (BCP) Test 2 (BCP + Hyaluronic acid) Control (ABBM) Group comparison Mean bone gain (mm) ± SD 12.25 ± 2.33 11.04 ± 2.29 11.05 ± 1.53 P = 0.403 3.2.2. Implant insertion torque A total of 44 implants, 15 in the control group, 16 in the test 1 group and 13 in the test 2 group, were placed. Precisely, 10 Bone Level and 34 Tissue Level implants were used, 16 Straumann(Bone and Tissue Level Implants, Straumann, Basel Switzerland)) and 28 Sweden & Martina (Premium/Prama, Sweden & Martina, Due Carrare, Italy) respectively. The mean insertion torque value was 41.9 ± 6.5 Ncm for the control group, 22.5 ± 5.3 N/cm for the test group 1 and 25.7 ± 6 N/cm for the test group 2. The difference between the groups was statistically significant (F [2.20] = 23.745, P <0.0005). The post hoc Tukey test revealed that the difference was statistically significant between the control group and the two test groups (P <0.0005), but not between the two test groups (P = 0.563). 3.2.3. Complications and implant failures The number of complications detected, in the various phases of the study, was on average 1.33 ± 0.95 in total, and for the control, test 1 and test 2 groups respectively 0.88 ± 0.85, 1.38 ± 1.06 and 1.13 ± 0.99. No statistically significant difference was found between the groups in terms of Tesis Doctoral – Nicola Alberto Valente 34 complications (F [2.21] = 0.535, P = 0.593). No implant failures were recorded at any stage. 3.2.4. Patient’s reported outcome measures Mean scores for the OHIP-14 questionnaire are reported in table 4, overall scores for control, test 1 and test 2 groups were respectively 8.63 ± 6.41, 12.13 ± 5.7 and 12.88 ± 6.23. The difference between the total OHIP- 14 scores of the 3 groups was not statistically significant (F [2.21] = 1.098, P = 0.352). The only statistically significant differences between the three test groups were found for question 2 (P = 0.001), 3 (P = 0.038), and 12 (P = 0.002). The Tukey post hoc test shows the differences between the groups. For question 2, the control group showed a statistically significant difference compared to both the test group 1 (P = 0.001) and 2 (P = 0.021), while the test groups were not significantly different from each other (P = 0.329). The only statistically significant difference for question 3 was between the control group and the test 2 group (P = 0.044), albeit only slight. For question 12 the test 2 group showed a statistically significant difference from both the control group (P = 0.004) and the test group 1 (P = 0.004). Test group 2 and control group did not show statistically significantly differences from each other (P = 1). Tesis Doctoral – Nicola Alberto Valente 35 Table 4 – OHIP-14 scores per group Impact item of the question Test 1 (BCP) Test 2 (BCP + Hyaluronic acid) Control (ABBM) Group comparison Had trouble in pronouncing words 0.38 ± 0.52 0.50 ± 0.53 0.50 ± 0.93 P = 0.916 Felt that sense of taste had worsened 2.38 ± 0.52 1.75 ± 0.89 0.50 ± 1.07 P = 0.001 Had painful aching in your mouth 1.25 ± 1.03 2.63 ± 1.30 1.00 ± 1.41 P = 0.038 Found it uncomfortable when eating food 1.38 ± 1.06 0.88 ± 1.13 0.50 ± 0.76 P = 0.233 Been feeling selfconscious 1.88 ± 1.64 1.38 ± 1.06 2.63 ± 1.68 P = 0.263 Have you felt tense 0 ± 0 0 ± 0 0 ± 0 Diet has been unsatisfactory 0 ± 0 0 ± 0 0 ± 0 Had to interrupt meals 0.63 ± 0.92 0.50 ± 0.93 0.25 ± 0.46 P = 0.639 Found it difficult to relax 0.50 ± 0.93 0.25 ± 0.70 0.38 ± 1.06 P = 0.861 Been a bit embarrassed 0 ± 0 0 ± 0 0 ± 0 Been irritable with other people 0 ± 0 0 ± 0 0 ± 0 Had difficulty during usual jobs 0 ± 0 0.88 ± 0.83 0 ± 0 P = 0.002 Felt that life less satisfying 0.63 ± 0.91 0.63 ± 0.74 0.75 ± 1.38 P = 0.963 Been totally unable to function 0.25 ± 0.46 0.50 ± 0.93 0.88 ± 1.25 P = 0.420 Total 12.12 ± 5.67 12.87 ± 6.27 8.62 ± 6.41 P = 0.352 4. Discussion Tesis Doctoral – Nicola Alberto Valente 37 While the percentage of new augmented bone in the three groups at histological exam wasn’t statistically significant, the difference in the percentage of non-mineralized tissue and residual graft material was statistically significant. It is not easy to analyze the results of a study on MSA, especially the histological ones, in fact it is almost impossible to find studies that make a comparative analysis between the exact same materials, different biomaterials are often used in combination with others. For example, ABBM is often used with autologous bone (AB) or other biomaterials, calcium phosphate, in addition to being often used in combination with other materials, is sometimes used in its form of beta tricalcium phosphate alone or, as in our study, mixed with HA. A systematic review with meta-analysis of Danesh-Sani SA et al.17 shows how, in studies using xenografts as grafting material, the histomorphometric results relating to new bone, residual biomaterial and non-mineralized tissue (26.82 ± 26.54, 29.33 ± 46.74, 44.86 ± 21.26), are very similar to the results that we obtained with ABBM, while the same cannot be said for the reported mean values relating to alloplastic materials. However, in the latter category the authors insert a very diverse variety of materials (TCP, BCP, bio-glass, corals, calcium carbonate, calcium sulphate, etc.), therefore, a real comparison with our results obtained with BCP is not possible. A series of RCTs conducted by the same group of authors reported histological values obtained from biopsies collected with the same timing of our study, 9 months. The results obtained differ from ours but also in this Tesis Doctoral – Nicola Alberto Valente 38 case the comparability is limited by the fact that xenograft was used, while in our study we also used TCP and HA.38-40 In the above-mentioned systematic review a comparative analysis was performed, based on histomorphometric outcomes between several graft biomaterials and consistent with our findings no statistically significant difference for newly formed bone was found when comparing xenografts with alloplastic biomaterials.17 On the other hand, the authors of the systematic review did not find significant differences for residual graft materials and non-mineralized tissues; this was in contrast with our study, where the residual graft particles were significantly higher in the control group than in the 2 test groups, and the non-mineralized tissues were significantly lower in the control group when compared to the test groups. As already mentioned, however, a varied amount of different types of materials falls under the categories of xenografts and alloplastic materials. Calcium phosphate has a greater tendency to reabsorb and leave less biomaterial residues as shown in our study and confirmed by a study from Boëck-Neto41 in which the percentage of residual biomaterial, in the maxillary sinus, at histological control was 6.19%, although in this cited study new bone percentages were found higher than in ours, probably because Calcium phosphate was mixed with autologous bone in a 2:1 ratio. A more recent study by Wagner et al.42, similarly to our study, compares the use of BCP mixed with fibrin sealant with the use of autologous bone mixed with ABBM. In this latter study, the histological results relating to the ABBM + AB graft are very similar to ours obtained with ABBM only, conversely the results obtained with BCP + fibrin sealant show percentages Tesis Doctoral – Nicola Alberto Valente 39 of new bone and non-mineralized tissue (20.6% and 54 %) similar to our study albeit slightly lower, and a percentage of residual biomaterial much higher than our data (25.4%). In general, CaP-based grafting materials have a rather unpredictable rate of resorption, thus are less able to maintain the grafted volumes, and have a greater structural fragility, probably, as our results show, due to the greater presence of residual non-mineralized tissues43. The advantage of using these biomaterials is their ability to stimulate osteoblastic proliferation and differentiation thanks to their composition, structure and crystallinity which is similar to that of calcium hydroxyapatite, the main inorganic component of the bone.44-46 The other variable included in the second test group of this study was the addition of HA to BCP. The use of HA for bone regeneration still has a limited amount of evidence in the literature, however some results are promising. In vitro, Asparuhova et al.21 showed that HA, when added to preosteoblastic mesenchymal stromal cells, strongly induces the growth of osteoprogenitors and the expression of genes encoding for bone matrix proteins. Mendes et al.47, in an animal study on rats showed that HA alone in post-extraction sockets showed accelerated bone healing compared to the control at 21 days. Kim et al.48 showed how HA in infected post-extraction sockets caused greater bone formation thanks to its osteoinductive but also bacteriostatic and anti-inflammatory properties. Three interesting clinical studies show the results of the use of HA in bone regeneration. Alcantara et al.49, in a cone beam analysis, describe how HA, in post-extractive sockets, shows more bone volume at 30 days Tesis Doctoral – Nicola Alberto Valente 40 compared to the control, but an insignificant difference at 90 days. Lorenz et al.50 showed good bone formation induction ability of BCP+HA in postextraction sockets at 4 months, however the study did not have a control group. Finally, in a study on lateral sinus augmentation, Knabe et al.51, described good results, in terms of bone volumes obtained in regenerated sinuses, both for TCP alone and for TCP + HA at 6 months, but greater bone formation with TCP alone. However, in the two groups the TCP was not in the same form, being in granules in the TCP+HA group, as a putty scaffold in the TCP alone group, thus representing a major confounding factor. As mentioned, the results are promising, but larger and better designed clinical studies are needed to draw definitive conclusions. From in vitro and animal studies we could say that HA probably plays an important role in accelerating bone formation in the initial phases, so in our study the 9-month follow-up is not able to highlight its beneficial effects. If this role were confirmed through clinical studies, shorter waiting times after bone grafting could be hypothesized for implant placement. Comparing the bone height gain obtained after MSA with other studies present in the literature has a very relative value, in fact it is not only the material used that determines this outcome, but also and above all the technique used and other anatomical factors such as the width of the sinus. This was illustrated in a study by Stacchi et al.52 which shows how the width of the maxillary sinus affects not only the final bone height gain, but also the quality, measured histomorphometrically, of the regenerated bone. Certainly the xenogenic grafts, having a greater persistence of residual material, guarantee a greater medium and long-term persistence of the volumes with greater increase in height, as demonstrated also by recent studies53, 54, and Tesis Doctoral – Nicola Alberto Valente 41 also have improved mechanical strength properties as demonstrated in this study by the insertion torque values43. Surely this data on the highest insertion torque in the ABBM group reconnects to the histological data that shows values on the total mineralized tissue (new bone and graft residues) that are significantly higher in the control group. Overall, the incidence of complications was very low in all groups, with no complications in the implant and restorative phase and the few complications related to episodes of hematoma or swelling in the postoperative period after MSA and 6 perforations of the sinus membrane during MSA, all successfully repaired. This data is supported by similar evidence in the literature, in particular a systematic review with meta-analysis of Raghoebar et al.55 which shows data similar to ours as seen in the studies included in the analysis. The last of the parameters that this study proposed to evaluate was that related to the influence of oral health, in this case after an MSA procedure such, on the quality of life. We decided to do it with a questionnaire, adopted by many other studies, handed to the patient at a specific time point, but with questions that related to the entire previous period35, 56-58. The total scores of the OHIP-14 fpr the three groups were very low, showing that, despite its invasiveness, an MSA procedure does not have a negative impact on the quality of life of our patients. There was a statistically significant difference between the three groups only for three questions, however the scores were so low that reading this data in favor or against one of the three groups would represent, in our opinion, an interpretative distortion. In general, no question, for any group, scored higher than 2, which corresponds to "occasionally" and four questions, the numbers 6, 7, 10 and 11, received an average score of 0. Tesis Doctoral – Nicola Alberto Valente 48 18. Miron RJ, Zhang Q, Sculean A, et al. Osteoinductive potential of 4 commonly employed bone grafts. Clin Oral Investig. Nov 2016;20(8):2259- 2265. doi:10.1007/s00784-016-1724-4 19. Schmidt JR, Vogel S, Moeller S, et al. 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