The new bone formation in human maxillary sinuses using two bone substitutes with different resorption types associated or not with autogenous bone graft: a comparative histomorphometric, immunohistochemical and randomized clinical study

Abstract Objective The aim of this study is to evaluate the new bone and connective tissue formation and the biomaterial remaining after maxillary sinus bone augmentation using 5 different bone substitutes. The osteocalcin immunolabeling was performed to demonstrate their calcification and the possibility of receiving dental implants. Methodology 40 patients underwent maxillary sinus bone augmentation and were divided in 5 groups: Group 1 with 8 maxillary sinuses were grafted with autogenous bone graft (AB); Group 2 with 8 maxillary sinuses grafted with bioactive glass (BG); Group 3 with 8 maxillary sinuses grafted with bioactive glass added to autogenous bone graft (BG + AB) 1:1; Group 4 with 8 maxillary sinuses grafted with Bio-Oss (BO) and Group 5 with 8 maxillary sinuses grafted with Bio-Oss added to autogenous bone graft (BO + AB) 1:1. Results In group AB, 37.8% of bone was formed in the pristine bone region, 38.1% in the intermediate and 44.5% in the apical region. In group BG, 43.6% was formed in the pristine bone, 37% in the intermediate and 49.3% in the apical region. In group BG + AB 1:1, 39.0% was formed in the pristine bone region, 34.8% in the intermediate and 36.8% in apical region. In group BO, 33.4% was formed in the pristine bone, 32.5% in the intermediate and 34.3% in the apical region. In group BO + AB 1:1, 32.8% was formed in the pristine bone, 36.1% in intermediate and 27.8% in the apical regions. The immunolabeling for osteocalcin showed an intensive staining for all groups, which could demonstrate the calcification of the bone formed. Conclusion This study showed that the groups evaluated formed a suitable lamellar bone in the maxillary sinus reconstruction after six months of bone healing, thus being indicated to receive dental implants.


Introduction
Physiological phenomena such as bone resorption and maxillary sinus pneumatization occur when the posterior maxillary teeth are lost. 1 Since oral rehabilitation using dental implants is impossible at this point, bone reconstruction is the procedure of choice.
Different surgical techniques that depend on the residual bone on the maxillary sinus floor have been developed. 2 The posterior maxillary reconstruction is then considered a predictable and reliable procedure.
Since its creation by Boyne and James, different bone substitutes have been used as autogenous bone grafts, xenografts and allografts, among others. [3][4][5] However, an ideal bone substitute is yet to be described in the literature. 6 Among the numerous biomaterials created and researched over the years, the autogenous bone graft (AB) is considered the "gold standard" due to its specific properties, namely osteoconduction, osteoinduction, and osteogenesis.  Quality assessment was conducted according to the CONSORT Statement's RCT checklist 11 (Figure 1).

Sample size
Based on previous studies, the number of maxillary sinuses to be grafted were determined by performing a power test on the website www.lee.dante.br 12 . For this test, the difference of the mean was 15.1, with a standard deviation of 9.9. The test was conducted in a 1-tailed hypothesis with a 5% level of significance and a 80% power. The results indicated a minimum of 5 samples for each group.

Randomization
Drawing lots was performed to randomize and decide the sites to be grafted with each material by a clinical assistant.

Inclusion & exclusion criteria
All patients were subjected to facial cone beam computed tomography (CBCT) to evaluate the maxillary sinuses, as well as the proximity of mandibular canal and inferior dental roots where the autogenous bone graft were harvested. The inclusion criteria were: patients with maxillary sinuses with less than 5 mm of bone remaining; those who decided to be rehabilitated with dental implants; and those with bone in the mandibular symphysis or in the retromolar region. Exclusion criteria were: patients that reported uncontrolled systemic disease, untreated periodontal disease, or sinus pathologies; smokers; patients without enough bone at the mandibular regions to be harvested for the autogenous bone graft; patients who was also osteotomized using a 701 drill and removed using a chisel. 9,12,13 The bone blocks were grounded with a bone crusher (Neodent; Curitiba, PR, Brazil).
The maxillary sinuses were treated according to Boyne and James 3 (1980) using a posterior maxillary approach to expose the lateral wall of maxillary bone followed by a osteotomy using a nº 6 sphere drill to access the Schneiderian membrane and its elevation.  indicating the absence of staining and scores "1," "2," or "3" indicating low, moderate, or intense staining, respectively.

Statistical analysis
The Kolmogorov-Smirnov test was performed to indicate the parametric or non-parametric distribution of the samples. In case of normal distribution, a comparison among the groups was made using the ANOVA test followed by Tukey's multiple comparison test. In case of non-parametric distribution, a Kruskal-Wallis test was performed. A priori, p-value<0.05 was considered significant for all tests.

Results
Forty patients (22 men and 18 women) aged between 32 and 65 years old were subjected to unilateral maxillary sinus bone augmentation using the 5 types of bone grafts purposed in our study.  Besides this, statistically significant differences were    apical region (p=0.011) of group BG. (Figure 3 A, B) Therefore, our results were in accordance with hypothesis H 0 .

Immunohistochemistry evaluation
Group AB presented high level "3" immunolabeling results for osteocalcin, which points to a mature bone that is able to receive dental implants for group BG; for group BG + AB 1:1; for group BO and group BO + AB 1:1 (Figure 2 Osteocalcin immunolabeling sections).

Discussion
In our study, we evaluated the bone formation Biogran has a peculiar way of resorption: body fluids react with these bone substitute due to its unstable structure. A silica gel and calcium phosphate layer are formed around the particle surface, which allow a combined process of chemical dissolution and macrophage action. 10 With simultaneous colonization of osteoblasts, the particle disintegrates. A rate of bioactive glass particles remaining from 0% to 4.2% was reported after 6 months of bone healing in maxillary sinus bone augmentation, similar to our  in direct contact with the biomaterial remaining with lamellar formation. In our study, we also found low rates of new bone formation; however, the histological findings presented lamellar bone and a connective tissue well cellularized. Thus, the H 0 hypothesis was accepted.
The osteocalcin outcomes demonstrate that all bone substitutes evaluated in this study were calcified and able to receive dental implants, similar to previous studies. 8,15 The limitation of our study was the evaluation of dental implants in a long-term. According to literature, however, high rates of success are observed when Bio-Oss is used to reconstruct the bone height in maxillary sinuses. 30 Despite the outcomes showed in this study, further studies are required to evaluate other periods and demonstrate if the biomaterial particle rates influence bone healing.
In conclusion, our study showed the formation of a suitable lamellar bone in the patients that underwent maxillary sinus reconstruction after six months of bone healing in all groups, thus being indicated to receive dental implants.