Hard Tissues and Materials

Fresh-frozen allografts combined with bovine bone mineral enhance bone formation in sinus augmentation

Journal of Biomaterials Applications 2015, Vol. 29(7) 1003–1013 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214552709 jba.sagepub.com

Felipe Perraro Sehn1, Rafael Rodrigues Dias1, Thiago de Santana Santos1, Erick Ricardo Silva1, Luiz Antonio Salata1, Gavriel Chaushu2 and Samuel Porfı´rio Xavier1

Abstract We evaluated histologically, histomorphometrically, and tomographically the effects of the association of fresh-frozen bone allograft (FFB) with bovine bone mineral (BBM) in maxillary sinus floor augmentation. In total, 34 maxillary sinuses from 29 patients, with a mean age of 51.32 (6.44) years, underwent sinus augmentation. Patients were divided into control and test groups (17 sinuses each). The controls were grafted with allograft bone, and the test group received a combination of FFB and BBM at a 2:1 ratio. After 6 months, bone samples were collected for histological and histomorphometric examinations. The implant survival rates were 93.02% (control group) and 100% (test group) at 6 months after functional loading. Median volumetric reductions of 28.32% (17.05–44.05) and 12.62% (5.65–16.87) were observed for the control and test groups, respectively. Statistically significant histomorphometric differences were found between the control and test groups regarding newly formed bone 12.54% (10.50–13.33) vs. 24.42% (17.62–35.92), p < 0.001, total bone 48.34% (39.03–54.42) vs. 61.32% (50.61–64.96), p ¼ 0.007, and connective tissue 51.66% (45.57–60.97) vs. 39.30% (35.03–49.37), p ¼ 0.007. The addition of BBM to allograft bone in maxillary sinus augmentation resulted in higher percentages of new bone formation and total bone, and permitted implant placement with a low rate of osseointegration failure at the 6-month follow-up. Keywords Fresh-frozen bone allograft, bovine bone mineral, maxillary sinus, histology, histomorphometry, bone volume, computed tomography

Introduction Autologous bone grafts are considered the ‘gold standard’ because of their osteogenic, osteoconductive, and osteoinductive properties.1–3 However, the disadvantages such as the inevitable donor site,4 bone availability limitations,5 use of general anesthesia in extensive cases, surgical morbidity at the donor region,6–10 and increased cost to the patient11,12 have lead to the search for other types of substitute graft materials. As an alternative to autologous bone, allograft bone (AB) has many advantages, such as the considerable decrease in surgical morbidity, in addition to greater availability and quantities.13,14 Fresh-frozen bone allograft (FFB) was compared to autologous intra-oral particulated bone grafts in sinus lift surgeries; they resulted in similar clinical results.

Furthermore, histology and histomorphometric findings demonstrated that the material had osteoconductive properties comparable with autologous bone.15 Other biomaterials have been proposed as bone graft substitutes in maxillary sinus lifting.16–25 Bovine bone mineral (BBM), an osteoconductive deproteinized particulated bone, is also indicated for maxillary sinus

1 Department of Oral and Maxillofacial Surgery and Periodontology. Ribeira˜o Preto Dental School, University of Sa˜o Paulo, Sa˜o Paulo, Brazil 2 Department of Oral and Maxillofacial Surgery, School of Dentistry, Tel Aviv University, Israel

Corresponding author: Samuel Porfı´rio Xavier, Department of Oral and Maxillofacial Surgery and Periodontology, Ribeira˜o Preto Dental School, University of Sa˜o Paulo, Avenida do Cafe´, S/N. 14040-904. Ribeira˜o Preto-SP, Brazil. Email: [email protected]

1004 surgery; it results in new bone formation and high rates of implant survival.26–32 It has been reported that the addition of BBM to autologous bone in different concentrations can be beneficial in graft bone formation33–35 and in maintaining stability.36 To date, no comparative information has been reported regarding the use of FFB and BBM in oral implantology. Thus, the purpose of this study was to assess a new surgical approach. The hypothesis was that a combination of particulated FFB combined with BBM in sinus floor augmentation will enhance new bone formation. The first aim was to assess new bone formation in a histological study and to conduct a histomorphometric evaluation. The second aim was to demonstrate that the combination of particulated FFM with BBM could maintain the bone volume of grafts placed in sinus floor augmentation. The third aim of the study was to evaluate the torque applied when the implants were placed in a sinus lift procedure 6 months after bone graft surgery.

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Figure 1. Intra-operative lateral view of the lifted maxillary sinus wall, up to 13 mm high.

Materials and methods In this prospective, randomized, comparative study, 29 consecutive patients (21 females, 8 males) aged 18–65 years old (mean age, 51.32  6.44 years) treated between March 2008 and December 2013 were selected for sinus floor augmentation prior to implant placement in a single surgical center (Ribeira˜o Preto Dental School, University of Sa˜o Paulo, Brazil). All procedures were fully explained to the patients, who signed an informed consent form. The study protocol was approved by the Ribeira˜o Preto Dental School Ethical Committee. The inclusion criterion was a maximum 5 mm residual alveolar crest height in the posterior maxilla. Exclusion criteria were systemic diseases, use of bisphosphonate drugs, smokers, drinkers, any active infectious process, pregnancy, women with infants, inability to understand the purpose of the project, and any pathological disturbance at the maxillary sinus. Five patients were treated bilaterally in the test group, and the 24 other patients were treated in the test or control groups at only 1 sinus per patient. In total, 34 sinuses were included; all patients were operated on by the same experienced surgeon. Sinus lifting was based on a technique reported previously.15 The Schneiderian membranes were elevated to 13 mm (Figure 1) under local anesthesia (mepivacaine 2% with adrenalin 1:100,000) for grafting. Antibiotics prophylaxis was prescribed (amoxicillin, 1 g) before surgery. For the control group, 17 sinuses were augmented with fresh-frozen corticocancellous bone blocks from distal epiphyses (20  10  6 mm; Figure 2) from a

Figure 2. Corticocancellous FFB from femoral distal epiphysis before milling.

tissue bank intra-operatively particulated with a bone mill with ratchet (Neodent, Curitiba, Brazil). In the test group, 17 sinuses were grafted with a combination of fresh-frozen corticocancellous bone blocks from distal epiphyses (20  10  6 mm) and BBM (Bio-Oss particles, size 1–2 mm, Geistlich Biomaterials AG, Wolhusen, Switzerland) in a 2:1 ratio (Figure 3). All sinuses were grafted (Figure 4) with 3 cm3 of grafted bone measured with the aid of a syringe, and the lateral walls were covered with a collagenous membrane (Bio-Gide, Geistlich Biomaterials AG; Figure 5).

Volumetric evaluation Cone-beam computed tomography (CBCT) analysis was performed twice in both groups: one week after the grafting surgery (T1) and six months postsurgery (T2) using an iCat Classic (Imaging Sciences International, Hatfield, PA, USA) with exposure factors of 120 kV and 36.12 mAs with 0.25 mm reconstruction interval and slice thickness. The CT images were analyzed using Mimics software

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Figure 3. FFB particles beneath BBM (2:1 proportion) before homogenization.

Figure 6. Close-up view from a panoramic radiograph showing implant in the graft. Figure 4. FFB and BBM mixture filling the cavity.

from the middle of the lifted sinus. In this same surgical procedure, implants were installed (40 in the control group, 30 in the test group; Conexa˜o, Aruja´, Brazil). Then, six months after implant installation (Figure 6), the patients were rehabilitated prosthetically (Figure 7) and followed for six months.

Histomorphometric and histological evaluations

Figure 5. Collagen membrane covering the grafted site.

(ver. 8.13; Materialise, Leuven, Belgium) in DICOM files to assess differences between volumes of the grafts at different experimental times (T1–T2), according to Mazzocco et al.37 At six months after grafting, a single biopsy (n ¼ 34) was collected with a 2 mm trephine for histological and histomorphometric analyses

Biopsy samples were fixed in 10% formalin (pH 7) for 10 days. The specimens were dehydrated through an ascending series of alcohols. Then, the samples were embedded in LR White resin (London Resin Company, London, UK) and kept under stirring for 60 min. Subsequently, the specimens were stored and maintained for at least 12 h at 4 C. Next, they were kept in a vacuum for 1 h, agitated, and again stored in a refrigerator for 24 h. This routine was repeated for 15 days, with the resin being changed every 48 h. Resin polymerization was induced at 60 C. The specimens were bisected longitudinally using a precision cutting band (0.1 mm/D64; EXAKT, Norderstedt, Germany), and then sanded and polished with sandpaper and

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Journal of Biomaterials Applications 29(7) and Alizarin red or H&E were used to measure connective tissue (CT), new bone formation (NB: blue), residual AB (yellow), and residual BBM (red). From each biopsy, five sections were measured at 200  magnification and the values were averaged. The total fraction was calculated as a percentage of the total tissue volume, as previously described.39

Statistical analysis For statistical analyses, first a Shapiro-Wilk normality test showed a non-normal distribution of the data. Thus, non-parametric data are presented as medians (interquartile ranges, IQR). The Mann–Whitney U test was used to compare median values between groups tested. The Wilcoxon signed-rank test was used to compare median values of the volumetric data in the same group (initial and final). The SigmaPlot software (ver. 12.5; Systat Software, Witzenhausen, Germany) was used for all analyses. The level of significance was set at p  0.05.

Results Figure 7. Prosthetic crown over the implant.

polishing cloths (Hermes Abrasives Ltd., VA, USA). From each trephine, two slices were obtained for staining and observation under light microscopy with a thickness of 90 mm, and stained using a combination of Stevenel’s blue and Alizarin red.38 Bone biopsies that released passively from the trephines were fixed in 10% buffered formalin at pH 7.4 (>24 h). After fixation, the specimens were decalcified in 4% EDTA (ethylenediaminetetraacetic acid), and changed once per week. The bone pieces were then washed in running water for 1 h, followed by dehydration through an alcohol series. After dehydration, diaphanization was performed by placing the pieces in xylene. The fragments were then impregnated with paraffin wax in an oven at 60 C. The paraffin wax blocks were then cut on a microtome with a standardized spacing of 5 mm in thickness, which were divided in an interleaved manner for histological analyses with hematoxylin and eosin (H&E) staining. Two pieces of each biopsy were evaluated, and the one with the ‘best’ staining was chosen. Measurements were carried out at 200  magnification. A Leica DMLB Microsystems microscope (Leica Microsystems, Hesse, Germany) connected to a computer using a Leica DC300F digital camera (Leica Microsystems) was used for histomorphometric measurements. The computer software used to process and measure from the digitalized image was the Leica Application suite (ver. 4.1; Leica Microsystems). Sections stained with Stevenel’s blue

In the control group, 17 maxillary sinuses from 17 patients (13 females, 4 males) with a median age of 53 (48.5–58.5) years, augmented with fresh-frozen corticocancellous particulated bone block (FFB) were evaluated. In the test group, 17 maxillary sinuses from 12 patients (8 females, 4 males) with a median age of 52 (47–55) years were grafted with FFB and BBM in a 2:1 ratio. There was no statistical difference between the groups in age (p ¼ 0.351) or gender (p ¼ 0.472). All surgeries were well tolerated by the patients, with no complications during or after surgery. No sinus membrane perforation was apparent, nor was tissue dehiscence, infection or loss of local sensitivity. The mean surgical time was 50  15 min. The grafts were allowed to integrate for 6 months, at which time 70 implants were placed, 40 in the control group and 30 in the test group. The torque for the control group was 35 (32–40), whereas that for the test group was 45 (40–45) (p < 0.001). Each maxillary sinus received 1–3 implants, depending on the area to be rehabilitated. Three implants were lost in the control group due to non-osseointegration at the second surgery, 6 months after the insertion of implants, with survival rates for the control and test groups of 93.02% and 100%, respectively (p ¼ 0.862). Prosthetic loading was followed for 6 months in all patients.

Volumetric evaluation In the control group, the median volume of grafted bone was 2.86 (2.08–3.33) cm3 and the difference was

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Table 1. Volumetric values in the control group.

Table 2. Volumetric values in the test group.

Control group

Test group 3

Bone volume (cm3)

Bone volume (cm ) Patient

Initial

Final

Difference

Resorption %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Mean SD

2.48 4.28 1.09 2.86 3.92 1.82 1.61 2.97 2.45 3.22 1.28 2.92 2.68 2.91 2.35 3.45 4.45 2.75 0.73

1.09 3.32 1.03 2.05 1.85 0.83 1.35 2.22 1.85 2.88 1.05 2.67 1.78 1.97 1.52 1.39 2.95 1.87 0.58

1.39 0.96 0.06 0.81 2.07 0.99 0.26 0.75 0.6 0.34 0.23 0.25 0.9 0.94 0.83 2.06 1.5 0.88 0.44

56.04 22.42 5.5 28.32 52.8 54.39 16.14 25.25 24.48 10.55 17.96 8.56 33.58 32.3 35.31 59.71 33.7 30.41 13.47

Patient

Initial

Final

Difference

Resorption %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Mean SD

2.21 2.61 1.32 1.9 1.96 2.61 2.14 3.88 3.17 2.87 5.77 3.92 5.86 1.89 2.3 1.93 1.61 2.88 0.98

1.97 2.57 1.08 1.86 1.62 2.25 1.55 3.34 2.76 2.63 5.6 3.89 5.12 1.58 1.83 1.73 1.45 2.60 0.97

0.24 0.04 0.24 0.04 0.34 0.36 0.59 0.54 0.41 0.24 0.17 0.03 0.74 0.31 0.47 0.2 0.16 0.30 0.16

10.85 1.53 18.18 2.10 17.34 13.79 27.57 13.91 12.93 8.36 2.94 0.76 12.62 16.40 20.43 10.36 9.93 10.58 0.05

statistically significant (p < 0.001) between T1 and T2, with a median volume of 0.830 (0.30–1.19) cm3. The median resorption rate for the control group was 28.32% (17.05–44.05) (Table 1). In the test group, the median volume of grafted bone was 2.30 (1.91–3.52) cm3 and the difference was statistically significant (p < 0.001) between T1 and T2, with a median of 0.24 (0.16–0.44) cm3. The median resorption rate for the test group was 12.62% (5.65–16.87) (Table 2). Comparing the groups, the differences in the initial and final bone volumes were statistically significant (p < 0.001), but the rate of resorption was not significant (p ¼ 0.314).

In the test group, BBM particles were detected in close contact with new bone and FFB (Figure 8c) with visible osteoid matrix bridges (Figure 8d) and osteoblastics cells surrounding it. It was not possible to recognize protein staining inside the BBM particles. New bone areas were present with osteocytic lacunae filled with osteocytes. Howship’s lacunae were detected with osteoclasts in close contact with grafted biomaterial (Figure 9). There was no sign of acute or chronic inflammatory infiltrate.

Histology

Histomorphometrically, there was no statistically significant differences (p ¼ 0.818) between the groups in terms of the amount of bone graft materials. In the control group, the median value was 38.20% (28.38– 41.80), ranging from 23.26% to 44.6%, and the sum of the FFB and BBM material in the test group was a median value of 35.41% (21.42–45.45), with a range from 15.55% to 58.6% (Table 3). In the histomorphometric analyses, FFB, BBM, and new bone were stained in different colors (Figure 10). Residual FFB was statistically significantly higher in the control group (38.20%, 28.38–41.80) than in the test group (16.04%, 10.46–22.42) (p ¼ 0.001).

In the control group, residual FFB was evident (Figure 8a) with empty osteocytic lacunae and a lamellar pattern area, as was new bone formation with osteocytic lacunae filled with viable osteocytes and woven bone, and osteoblastic cells in close contact with osteoid matrix connected through bridges between AB particles and new bone formation (Figure 8b). Multinucleated giant cells, similar to osteoclasts, and phagocytic projections in close contact with the remodeling bone area were seen. There was no sign of an acute or chronic inflammatory infiltrate.

Histomorphometry

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Figure 8. (a) Residual allogeneic bone (AB) in close contact with the new bone (NB) area and osteoid matrix (green arrows). Osteoblastics cells (white arrows) in close contact with osteoid matrix; filled osteocytic lacunae (black arrows) with osteocytes; empty osteocytic lacunae (yellow arrows) in AB (staining with Stevenel’s blue and Alizarin red). (b) Osteoid matrix bridge (green arrows) between new bone (NB) area, and allograft bone particles. Osteoblastic cells (white arrows) in close contact with osteoid matrix; filled osteocytic lacunae with osteoids (black arrows) at the NB area (staining with Stevenel’s blue and Alizarin red). (c) Close contact between bovine bone mineral particles (BBM), NB, and AB. Filled osteocytic lacunae with osteoids (black arrows) and osteoclast cells (red arrow) are presented (staining with Stevenel’s blue and Alizarin red). (d) An osteoid matrix bridge (black arrows) is seen between new bone (NB) formation, allograft bone particles, and BBM (staining with Stevenel’s blue and Alizarin red).

12.54% (10.50–13.33), and ranging from 7.72% to 15.32% for the control group, and a median value of 24.42% (17.62–35.92), ranging from 10.76% to 45.16% in the test group. Total bone was significantly higher (p ¼ 0.007; Graph 2) in the test group (61.32%, 50.61–64.96), ranging from 40.6% to 79.7%, than in the control group (48.34%, 39.03–54.42), ranging from 37.55% to 58.03%. For connective tissue, a statistically significant difference was found between the control group (51.66%, 45.57–60.97), ranging from 41.97% to 62.45%, and the test group (39.30%, 35.03–49.37), ranging from 20.29% to 59.39% (p ¼ 0.007; Table 4).

Figure 9. Bovine bone mineral (BBM) in close contact with new bone (NB) formation and allograft bone (AB). Visible bone formation (white arrows) inside BBM and the presence of osteoclastic cells (black arrows) are evident (staining with hematoxylin and eosin, H&E, 200  magnification).

Remaining BBM was found in the test group (12.47%, 6.31–21.64). In terms of new bone formation, statistically significant differences were found between the groups (p < 0.001; Graph 1) with a median value of

Discussion In this prospective, randomized, and comparative study, we evaluated the effects of BBM and FFB in 34 maxillary sinuses grafted for sinus floor augmentation. Few reports have described the use of FFB in sinus floor elevation. Recently, Xavier et al.15 observed similar rates of implant survival and new bone formation for sinus augmentation whether FFB or autologous bone was used for grafting. Stacchi et al.40 used allografts in sinus lifting and detected new bone

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formation in close contact with preexisting bone, with no signs of inflammatory infiltrate. Other studies have shown that FFB is osteoconductive, with active bony remodeling and no sign of inflammatory tissue, suggesting that FFB is appropriate for sinus floor augmentation.41 Similar to previous reports, our results showed

Table 3. Histomorphometric values in the control group. Control group

Gender

Age

Total bone %

F F F F F M M F M F F F F F M F F Mean SD

62 72 51 48 56 47 52 49 50 61 44 56 58 53 44 59 55 53.94 5.58

50.75 55.48 38.28 38.45 53.99 38.58 47.44 47.72 55.69 49.07 50.95 54.86 58.03 37.55 39.48 46.65 48.34 47.72 5.60

New bone %

Residual allograft bone %

Connective tissue %

9.65 13.23 12.7 10.55 11.34 15.32 14.87 8.8 12.54 7.72 12.75 14.36 13.43 11.85 10.62 12.86 10.44 11.94 1.71

41.1 42.25 25.58 27.9 42.65 23.26 32.57 38.92 43.15 41.35 38.2 40.5 44.6 25.7 28.86 33.79 37.9 35.78 6.21

49.25 44.52 61.72 61.55 46.01 61.42 52.56 52.28 44.31 50.93 49.05 45.14 41.97 62.45 60.52 53.35 51.66 52.27 5.60

areas of new bone formation near resorption sites in both groups, indicating bone remodeling due to the presence of new bone formation and osteoclastic giant cells in contact with both BBM and FFB. Many authors have assessed the survival rate of implants in the maxillary posterior area. When installed in a conventional manner and without the need for bone grafts, a survival rate of 99.7% resulted.42 Johansson et al.43 reported a survival rate of 87.4% with implants placed in maxillary sinus grafted with particulated autologous iliac crest bone. Sbordone et al.44 evaluated maxillary sinus lifted with autologous bone, and found a rate of 93.3% after the 6-year follow-up, whereas Sbordone et al.45 reported 95.8% after the 3-year follow-up using autologous bone. Pieri et al.46 studied success rates of 90 implants placed in maxillary sinus grafted with autologous bone and BBM (1:1) at the 1-year follow-up, with

Graph 1. New bone formation in both groups, showing a statistical difference (p < 0.001).

Figure 10. Histomorphometric analysis of the test group. (a) Staining with Stevenel’s blue and Alizarin red, 200  magnification. (b) Selection of histomorphometric constituents, showing new bone (NB) formation (blue) in close contact with bovine bone mineral (BBM, red). Union of allograft bone (AB) particles (yellow) and BBM (red) with NB formation (blue).

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Graph 2. Total bone formation in both groups, showing a statistical difference (p < 0.001).

Table 4. Histomorphometric values in the test group. Test group

Gender

Age

Total bone %

F F F M F F F M M F F F F F F M F Mean SD

55 55 54 50 25 54 59 28 28 51 52 52 56 56 61 47 47 48.82 8.13

41.02 65.27 61.32 70 60.71 63.3 40.6 61.52 60.09 79.7 62.25 51.66 43.04 49.57 64.65 59.83 67.86 58.96 8.10

New bone %

Residual allograft bone %

BBM %

Connective tissue %

21.05 28.92 22.4 38.28 45.16 44.9 24.57 17.99 12.2 21.09 37.63 34.21 11.71 25.94 17.26 24.42 10.76 25.79 8.76

16.04 23.01 21.83 5.01 6.38 34.62 9.6 18.61 11.78 56.21 12.14 10.11 13.83 16.68 10.82 17.05 51.61 19.72 10.42

3.91 13.34 17.08 26.71 9.17 7.35 6.42 24.92 36.1 2.39 12.47 7.34 17.49 6.21 36.56 18.36 5.49 14.78 8.67

58.97 34.72 46.11 29.99 39.28 36.69 59.39 38.47 39.9 20.29 37.74 48.33 56.95 50.42 35.34 39.98 32.14 41.45 8.40

a survival rate of 98.7%. Viscioni et al.13 analyzed the effectiveness of FFB, reporting a 96.4% implant survival rate. Yoon et al.47 investigated biomaterials (autologous bone, AB, BBM) in sinus lifting, and achieved a mean implant survival rate of 90.9% at the 1-year follow-up. Similarly, in our 6-month

follow-up results, high survival rates were found, ranging from 93.02% to 100%, in the control and test groups, respectively. For each grafting material, there are different values for torque in the literature. Johansson et al.43 evaluated implant insertion torque in sinus lifting surgery with particulated autologous bone from the hip, and showed a mean of 41.1  26 N. Pieri et al.46 compared the behavior of 90 implants inserted in sinus lifts, grafted with autologous bone and BBM (1:1) at the 1-year follow-up, reporting a mean insertion torque of 29.18  6.4 N. Degidi et al.48 measured the initial torque from implants installed in maxillary sinus lifts with autologous bone and BBM (1:1), and reported a torque of 26.48  20.8 N after 6 months. In this study, a statistically significant difference (p < 0.001) between the control group (35, 32–40) and the test group (45, 40–45) was evident, showing better primary stability in the test group. Various results have been reported regarding the volumetric and density behavior of autologous bone, particulated or in blocks. According to Sbordone et al.,49 although the volumetric changes with particulated bone were higher than with bone blocks, their use promoted higher values of density over 6 years of evaluation. When comparing autologous and allogeneic bone blocks 6 months after sinus floor augmentation, Sbordone et al.50 demonstrated a volumetric reduction of 14% related to the freeze-dried allogeneic bone compared to autologous bone, both from the hip. In the present study, the median volumetric change in the test group was 12.62%. The authors of the present study report two reasons for this volumetric maintenance in the grafts. The first is the true volumetric maintenance of the bone graft, with decreased resorption of the biomaterial formed at the union of the AB and the BBM. Other authors, like Mazzocco et al.,37 evaluated the volumetric behavior of grafts in sinus floor augmentation with BBM material at 100%, 8–9 months after grafting surgery, with a mean of bone resorption of 10%. Jensen et al.51 studied volumetric changes in bone grafts with BBM and autologous bone. Their results showed that the volumetric reduction was significantly influenced by BBM levels, and that the reduction was not influenced by the embryological origin of the bone. A second reason for the decrease in volumetric resorption is that it is not possible to differentiate newly formed bone from grafted bone via CT analysis, so assessing volume is influenced by both grafted bone and newly formed bone. In the present study, residual AB was found in both groups with empty osteocytic lacunae and a lamellar pattern area in close contact with new bone formation, with characteristically filled osteocytic lacunae with viable osteoids and woven bone, which was in close

Sehn et al. contact with osteoid matrix, creating bone bridges between allograft particles and the new bone area. Similar results have been reports by others.40,41,52,53 The result of residual AB at 6 months in our control group was similar to the 34.9% reported by Xavier et al.15 Moreover, bridges of new bone connecting residual allograft particles and BBM in the test group were found, showing the osteoconductive characteristics of the biomaterials. No inflammatory infiltrate was detected in any of the bone biopsies, consistent with the results by Acocella et al.41 Data regarding new bone in maxillary sinus grafted with allogeneic bone are limited and conflicting in the literature. While Acocella et al.41 showed 40.57% new bone formation, Chaushu et al.54 reported 26.1%, and Sohn et al.55 reported 17.88% and 8.26%. These discrepancies may be explained by different evaluation methods, variation between evaluators’ histomorphometry, number of patients, evaluation periods, and even the amount of bone remaining in the residual ridge in the area of posterior maxillary atrophy. In our research, new bone formation in the control group was 12.54% (10.50–13.33), whereas it was 24.42% (17.62–35.92) in the test group (p < 0.001). The higher amount of new bone in the test group can likely be explained by the presence of BBM (14.78  8.67%), which probably enhanced bone formation, as reported by Pettinicchio et al.56 They analyzed the histological behavior between three biomaterials for bone grafts, and observed higher amounts of new bone formation when BBM was used. Consistent with our results, Bassil et al.57 observed connection by bridges between newly formed bone and BBM particles. They concluded that BBM was a biocompatible and osteoconductive material. These results suggest that the presence of BBM favored a greater amount of bone formation in the test group. Moreover, the higher percentage of total bone in the test group, i.e. 61.32% (50.61–64.96) compared to the control group, i.e. 48.34% (39.03–54.42) (p ¼ 0.007) can be considered a consequence of the sum of residual AB, new bone, and BBM material. Both materials behaved as osteoconductive scaffolds and their use can be encouraged as alternative grafts in sinus lift surgeries. The association of BBM with FFB resulted in higher rates of osseointegration, amount of new bone formation, and total bone. Acknowledgments We thank Adriana Luisa Gonc¸alves Almeida, Denny Marcos Garcia, Dimitrius Leonardo Pitol and Sebastia˜o Carlos Bianco for laboratory and statistical technical support and Professor Paulo Tambasco de Oliveira for histological and histomorphometric assistance. The English in this document

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Declaration of conflicting interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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