A series of systematic reviews have illustrated the clinical and histological healing after maxillary sinus floor augmentation.1–5 Various biomaterial types have been used for this purpose, and the results have been analyzed.1,4 In a systematic review,1 clinical and histological results using synthetic bone substitutes were compared with those using autogenous bone or xenografts. Statistically significant differences in newly formed bone were seen between autogenous bone and synthetic bone. Nevertheless, the clinical outcomes were similar among groups using different grafting materials. Another systematic review confirmed the higher amount of new bone formation at sinuses augmented using autogenous bone compared with xenografts.4 In the same review, it was further concluded that a mixture of tricalcium phosphate and hydroxyapatite showed better results compared with bovine bone.
The healing at the antrostomies was described in animal6–8 and human studies.6–12 In animal studies,6–8 the healing at the antrostomies was often found incomplete, presenting connective tissue interposed in the central zones despite the use of collagen membranes to protect the access window. The use of membranes seems to improve the clinical outcomes5 and to increase vital bone formation.13 However, in a clinical study,9 the histological evaluation revealed no differences in bone formation between antrostomies covered or not covered by the collagen membrane. Nevertheless, a high content of connective tissue was reported.
Despite the high number of studies that reported histomorphometric outcomes from biopsies collected from the crest or from the lateral wall of the sinus after sinus floor augmentation, a direct comparison between the 2 outcomes is still missing. Hence, the aim of this study was to compare the histomorphometric outcomes of biopsies collected from the antrostomy and from the alveolar crest after a sinus-lift procedure.
Materials and Methods
Patient Selection
Twelve patients, 5 men and 7 women with a mean age of 55.3 ± 11.7 years, were recruited. Twelve sinus floor augmentation were performed, 7 in the left side and 5 in the right side of the maxilla.
This clinical study followed the Declaration of Helsinki on medical protocols and ethics. The protocol was presented to and approved by the ethical committee of the Corporación Universitaria Rafael Núñez, Cartagena de Indias, Colombia, before starting the study (protocol #01-2015; May 19, 2015). All treatments were performed at the Corporación Universitaria Rafael Núñez, Cartagena de Indias, Colombia, during the period from August 2015 to July 2016. Twelve volunteers were planned to be included in the study for biopsy collection. After 9 months from sinus floor elevation conducted using a lateral access, the biopsies were harvested from the alveolar crest and from the antrostomy. Surgical procedures, materials used, and possible complications were thoroughly illustrated to the patients. Written informed consent was signed by all participants for the full protocol procedures. The study was reported according to the STROBE guidelines.
According to a few articles reporting histomorphometric data on mineralized bone contained in biopsies harvested from either the alveolar crest or the antrostomy,12–15 a sample of 10 patients was calculated. The sample was increased to 12 patients, considering possible withdrawal or complications.
The following inclusion criteria were adopted: (i) presence of an edentulous atrophic zone in the posterior segment of the maxilla; (ii) height of the sinus floor ≤4 mm; (iii) need of a prosthetic fixed rehabilitation supported by implants; (iv) more than 21 years of age; (v) good general health; (vi) no contraindication for oral surgical procedures; and (vii) not being pregnant. The following exclusion criteria were as follows: (i) presence of systemic disorders; (ii) chemotherapy or radiotherapy; (iii) smokers >10 cigarettes per day; (iv) acute or a chronic sinusitis or other sinus pathologies; and (v) previous bone augmentation procedures in the region.
Clinical Procedures
The surgical procedures were performed by an expert surgeon (D.B.). Local anesthesia was provided, and crestal and releasing incisions were performed. Full-thickness flaps were elevated, and the alveolar bone was exposed. A lateral access window was prepared using a diamond insert (SFS 109 029; Komet-Brasseler-GmbH, Lemgo, Germany) mounted on a sonic-air surgical instrument (Sonosurgery TKD, Calenzano, Italy). The sinus mucosa was detached from the bone layer, and the elevated space was filled with a collagenated corticocancellous porcine bone (OsteoBiol Gen-Os, 250–1000 µm; Tecnoss, Giaveno, Italy). The antrostomy was covered with a collagen membrane (OsteoBiol Evolution, 0.3 mm; Tecnoss). A small nail in titanium was placed on the lateral wall as reference, and the flaps were sutured. Antibiotics (amoxicillin 875 mg/clavulanic acid 125 mg twice a day for 6 days), nonsteroidal anti-inflammatory drugs as needed, and mouth rinses with 0.12% chlorhexidine 3 times a day for 10 days were prescribed. The sutures were removed after 7 days, and the patients were included in a maintenance recalls for the full extent of the study.
After 9 months from sinus floor elevation, biopsies were collected from the alveolar crest at the implant sites and from the antrostomy, using trephine burs.
Histological Preparation of the Biopsies
One biopsy was unavailable for histological evaluation so that the final sample was of 11 biopsies. The biopsies were removed from the trephines, washed in saline solution, and were immediately stored in 10% buffered formalin. Subsequently, the biopsies were dehydrated in an ascending series of alcohol and then embedded in a glycol-methacrylate resin (Technovit 7200 VLC; Kulzer, Wehrheim, Germany). After polymerization, the biopsies were sectioned following the longitudinal axis using a precision diamond disk to obtain specimens of about 150 μm followed by grounding to about 30 µm of width. The specimens were stained with acid fuchsin and toluidine blue.
Histomorphometric Evaluation
The histomorphometric evaluation was performed twice by a well-trained author (K.A.A.A.) blinded to the sites of biopsies harvesting. Mean values were used. Measurements were performed after a calibration with another author and K > 0.80. All histological analyses were performed in ARDEC Academy (Rimini, Italy) using an Eclipse Ci microscope (Nikon Corporation, Tokyo, Japan) connected to a digital video camera (Digital Sight DS-2Mv; Nikon Corporation, Tokyo, Japan). The software NIS-Elements D 4.10 (Laboratory Imaging; Nikon Corporation) was used for measurements. The percentages of mineralized bone, marrow spaces, xenograft residual particles, connective tissue, vessels, and inflammatory infiltrate were evaluated. The total bone was calculated as sum of mineralized bone and marrow spaces. To perform measurements, a point-counting procedure was used applying a lattice with squares of 75 μm superposed over the histological slides, using an objective of ×10.
Data Analysis
Mean values and SDs as well as 25th, 50th (median), and 75th percentiles were calculated for each outcome variable. Mean values, SDs, and 95% confidence intervals of the differences between alveolar crest and antrostomy biopsies were calculated for each analyzed variable.
The primary variables were newly formed bone and connective tissue. The other variables were considered as secondary ones. A Wilcoxon signed-rank test was used to analyze differences between biopsies from the alveolar crest and from the antrostomy. The level of significance was set at α 0.05.
Results
Mineralized bone was composed of newly formed bone (woven bone and parallel-fibered bone) and of lamellar bone, the latter mostly confined in the crestal region of the alveolar crest biopsies.
In the alveolar crest, the identification of the limits between the old pre-existing crestal bone and the newly formed bone was in most cases uncertain. The mineralized bone was composed of woven bone/parallel-fibered bone and of mature lamellar bone, and it was surrounding the particles of xenograft (Fig. 1). The content of mineralized bone (Table 1) was 40.1 ± 14.1% (P = 0.004 compared with the antrostomy; Table 2). Bone marrow was occupying the spaces within the trabeculae and was represented by 40.1 ± 18.0% (P = 0.059). The total amount of bone was 80.1 ± 23.2% (P = 0.003). Small amounts of connective tissue (3.9 ± 9.1%; P = 0.028) were observed, mainly around residues of xenograft particles that were still present at percentages of 14.7 ± 15.2 (P = 0.008). No evidence of osteoclastic activity was observed. Vessels were found in small amounts (1.2 ± 0.9%; P = 0.213), whereas inflammatory infiltrates (P = 0.102) were virtually absent.
;)
Fig. 1: A ground section illustrating the healing at the alveolar crest. Trabecular bone and bone marrow were occupying large part of the biopsies. Xenograft particles were still observed and surrounded by newly formed bone. Acid fuchsine and toluidine blue stain. Image grabbed at a magnification of ×20.
Table 1: Histomorphometric Evaluation of Hard and Soft Tissue Components (n = 11)
Table 2: Mean Values, SDs, and Lower and Upper 95% Confidence Intervals (CI) of the Difference of the Mean values (n = 11)
The antrostomy was found partly corticalized in most biopsies, and the elevated space was occupied by marrow structures and trabecular mineralized bone that was surrounding the residual xenograft particles (Fig. 2, A and B). In few specimens, the antrostomy was mainly occupied by connective tissues that were embedding residual xenograft particles (Fig. 3). The mineralized bone represented 26.0 ± 10.8% of the total tissue contents, whereas marrow spaces occupied 23.4 ± 17.0%. The total amount of bone was 49.9 ± 25.6%. The soft tissue was 19.7 ± 19.4%. Residual xenograft particles represented 28.2 ± 15.7%, without evident signs of osteoclastic activity (Fig. 2, A and B). Vessels constituted 2.0 ± 2.3% while a low amount of inflammatory infiltrate was found (0.7 ± 1.3%) (Table 1).
Fig. 2: A and B, Ground sections illustrating the healing at the antrostomy. The antrostomy was found partly corticalized in most biopsies. Trabecular mineralized bone and bone marrow were occupying the elevated space. Newly formed bone was surrounding the xenograft particles. Acid fuchsine and toluidine blue stain. Original magnification (A) ×20), (B) ×100.
Fig. 3: Ground sections illustrating the healing at the antrostomy. In few specimens, the antrostomy was mainly occupied by connective tissues that were embedding residual xenograft particles. Original magnification ×100.
Discussion
The aim of this study was to compare the histomorphometric outcomes of biopsies collected from the antrostomy and from the alveolar crest of maxillary sinus after sinus lift augmentation. Higher percentages of mineralized bone and bone marrow were seen in the biopsies from the alveolar crest compared with those of the antrostomy. Conversely, higher residues of xenograft and of connective tissues were found in the antrostomy compared with the crestal biopsies.
In the alveolar crest region, the presence of about 40% of mineralized bone and 40% of marrow spaces within the area of interest for implant installation, together with a low content of residual grafted particles, pointed to a successful bone regeneration. The amount of mineralized bone reported in this study was in agreement with the outcomes of another similar clinical study in which autogenous bone mixed with either deproteinized bovine bone mineral or an allograft was used for sinus floor augmentation.14 Seven patients for groups were recruited, the sinus was filled with the respective biomaterial, and all antrostomies were covered using collagen membranes. After 6 months of healing, biopsies from the alveolar crest were retrieved. Mineral bone was represented by 41% and 35% at the xenograft and allograft groups, respectively. The results from this study are also in agreement with another clinical study in which sinus floor augmentation was performed bilaterally in 13 volunteers.15 Either biphasic calcium phosphate (BCP) or freeze-dried bone allograft (FDBA) was used for sinus floor augmentation, and collagen membranes were placed over the antrostomies. Biopsies from the alveolar crest were collected after 9 months of healing before implant placement. Mineralized bone was identified as 49% and 40% at BCP and at FDBA sites, respectively.
In the antrostomy region, a lower amount of mineralized bone (26%) was found compared with the alveolar crest biopsies. A total amount of 50% of mineralized bone and marrow spaces were found, and most biopsies seemed to be partly corticalized. The results from this study are in agreement with another clinical study in which 10 patients received a bilateral sinus floor augmentation using either an alloplast composed of 60% hydroxyapatite and 40% b-tricalcium phosphate or autogenous bone.12 A collagen membrane was placed to cover both sinuses. Biopsies were collected after 6 to 8 months. Mineralized bone was found at percentages of 28% at the alloplast sites and 37% at the autogenous bone sites. The histological healing at antrostomy was also described in a clinical study.9 Eighteen patients were recruited for sinus floor augmentation and randomly divided into 2 groups. A collagenated corticocancellous porcine bone similar to that used in this study was grafted into the elevated space. A collagen membrane was placed to cover the antrostomy in the control group, whereas, in the test group, no membranes were used. After 6 months of healing, biopsies were collected from the antrostomy and histologically analyzed. Newly formed bone was about 31% and 28% at the sites with or without collagen membrane, respectively.
High amounts of connective/soft tissues were reported in various studies that evaluated biopsies from the antrostomy region, ranging between 40% and 60%.9,10,12 However, marrow spaces were also included in these connective/soft tissues data. In this study, marrow spaces were evaluated separately, and a proportion of about 20% was assessed, providing a total amount of bone of about 50%. The amount of connective tissue that was isolating the biomaterial from bone structures and in which no vessels were found was present at percentage of 20%. These regions seemed to have very low chance to heal properly.
The healing at the antrostomy was described in experimental studies in sheep6,7 and rabbits.8 In all studies, bone was seen forming from the margins of the antrostomy and proceeding toward the center over time. However, remaining defects of limited dimensions were found in 2 studies.6,8 In another study on an animal model, a BCP (60% hydroxyapatite, 40% beta-tricalcium phosphate) was used as the grafting material.7 After 12 weeks of healing, connective tissue and biomaterial were occupying the center of the antrostomy and no specimens showed a complete obliteration by bone.
The positive outcomes in the crestal region should partly due to the newly formed bone within the elevated space, but also to the pristine alveolar crest present at the time of sinus floor augmentation. It has to be considered that the base of the elevated space is the most suitable zone for bone regeneration due to the presence of the floor of the sinus and of the medial and lateral bone walls. Various experimental studies have described the patterns of healing within the elevated space and showed that bone is forming from the base and the walls of the sinus.6–8,16–20 The sinus mucosa seemed to be less involved in bone formation at least in the earliest phases of healing, despite its innate potential for bone formation.21–24
Particles of xenograft were still detectable in both biopsies, being about 15% at the crestal biopsies and about 28% at the antrostomy region. The higher content of biomaterial in the antrostomy region compared with the crestal region may be explained by the higher modeling and remodeling activities within the base of the sinus compared with the antrostomy region.
The main limitation of this study was the absence of clinical data that may support the long-term success of the implant treatment. The biopsies have to be retrieved paying attention not to compromise the implant sites.
Conclusion
In conclusion, higher amounts of mineralized bone and bone marrow were found in the alveolar crest compared with the antrostomy.
Roles/Contributions by Authors
K. Tanaka: Protocol design, statistical analysis, and data interpretation. G. Iezzi: Histological processing of the specimens, histological analysis, and images. A. Piattelli: Interpretation of the data and critical revision of the manuscript. M. Ferri: Patients recruitment and data collection. N. F. Mesa: Data collection and revision of the article. K. A. Apaza Alccayhuaman: Writing of the manuscript, histomorphometric evaluation, and data analyses. D. Botticelli: Protocol design, clinical sessions, writing of the manuscript, and the final approval.
Disclosure
This study was supported by ARDEC Academy, Ariminum Odontologica SRL, Rimini, Italia, and by the Ministry of Education, University and Research (M.I.U.R.), Rome, Italy.
Approval
The study was approved by the ethical committee of the Corporación Universitaria Rafael Núñez, Cartagena de Indias, Colombia (protocol #01-2015; May 19, 2015).
Disclosure
The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.
Acknowledgments
The biomaterials for sinus floor augmentation were supplied free of charge by Tecnoss Srl, Giaveno, Turin, Italy.
References
1. Starch-Jensen T, Mordenfeld A, Becktor JP, et al. Maxillary sinus floor augmentation with synthetic bone substitutes compared with other grafting materials: A systematic review and meta-analysis. Implant Dent. 2018;27:363–374.
2. Esposito M, Grusovin MG, Coulthard P, et al. The efficacy of various bone augmentation procedures for dental implants: A cochrane systematic review of randomized controlled clinical trials. Int J Oral Maxillofac Implants. 2006;21:696–710.
3. Jensen T, Schou S, Stavropoulos A, et al. Maxillary sinus floor augmentation with Bio-Oss or Bio-Oss mixed with autogenous bone as graft: A systematic review. Int J Oral Maxillofac Surg. 2012;41:114–120.
4. Corbella S, Taschieri S, Weinstein R, et al. Histomorphometric outcomes after lateral sinus floor elevation procedure: A systematic review of the literature and meta-analysis. Clin Oral Implants Res. 2016;27:1106–1122.
5. Pjetursson BE, Tan WC, Zwahlen M, et al. A systematic review of the success of sinus floor elevation and survival of implants inserted in combination with sinus floor elevation. J Clin Periodontol. 2008;35(8 suppl):216–240.
6. Scala A, Lang NP, Velez JU, et al. Effects of a collagen membrane positioned between augmentation material and the sinus mucosa in the elevation of the maxillary sinus floor. An experimental study in sheep. Clin Oral Implants Res. 2016;27:1454–1461.
7. Favero V, Lang NP, Canullo L, et al. Sinus floor elevation outcomes following perforation of the Schneiderian membrane. An experimental study in sheep. Clin Oral Implants Res. 2016;27:233–240.
8. Omori Y, Ricardo Silva E, Botticelli D, et al. Reposition of the bone plate over the antrostomy in maxillary sinus augmentation: A histomorphometric study in rabbits. Clin Oral Implants Res. 2018;29:821–834.
9. Barone A, Ricci M, Grassi RF, et al. A 6-month histological analysis on maxillary sinus augmentation with and without use of collagen membranes over the osteotomy window: Randomized clinical trial. Clin Oral Implants Res. 2013;24:1–6.
10. Xavier SP, Santos Tde S, Sehn FP, et al. Maxillary sinus grafting with fresh frozen allograft versus bovine bone mineral: A tomographic and histological study. J Craniomaxillofac Surg. 2016;44:708–714.
11. Tawil G, Barbeck M, Unger R, et al. Sinus floor elevation using the lateral approach and window repositioning and a xenogeneic
bone substitute as a grafting material: A histologic, histomorphometric, and radiographic analysis. Int J Oral Maxillofac Implants. 2018;33:1089–1096.
12. Danesh-Sani SA, Wallace SS, Movahed A, et al. Maxillary sinus grafting with biphasic bone ceramic or autogenous bone: Clinical, histologic, and histomorphometric results from a randomized controlled clinical trial. Implant Dent. 2016;25:588–593.
13. Tarnow DP, Wallace SS, Froum SJ, et al. Histologic and clinical comparison of bilateral sinus floor elevations with and without barrier membrane placement in 12 patients: Part 3 of an ongoing prospective study. Int J Periodontics Restorative Dent. 2000;20:117–125.
14. Galindo-Moreno P, de Buitrago JG, Padial-Molina M, et al. Histopathological comparison of healing after maxillary sinus augmentation using
xenograft mixed with autogenous bone versus allograft mixed with autogenous bone. Clin Oral Implants Res. 2018;29:192–201.
15. Kolerman R, Nissan J, Rahmanov M, et al. Comparison between mineralized cancellous bone allograft and an alloplast material for sinus augmentation: A split mouth histomorphometric study. Clin Implant Dent Relat Res. 2017;19:812–820.
16. Scala A, Lang NP, de Carvalho Cardoso L, et al. Sequential healing of the elevated sinus floor after applying autologous bone grafting: An experimental study in minipigs. Clin Oral Implants Res. 2015;26:419–425.
17. Scala A, Botticelli D, Faeda RS, et al. Lack of influence of the schneiderian membrane in forming new bone apical to implants simultaneously installed with sinus floor elevation: An experimental study in monkeys. Clin Oral Implants Res. 2012;23:175–181.
18. Scala A, Botticelli D, Rangel IG Jr, et al. Early healing after elevation of the maxillary sinus floor applying a lateral access: A histological study in monkeys. Clin Oral Implants Res. 2010;21:1320–1326.
19. Iida T, Carneiro Martins Neto E, Botticelli D, et al. Influence of a collagen membrane positioned subjacent the sinus mucosa following the elevation of the maxillary sinus. A histomorphometric study in rabbits. Clin Oral Implants Res. 2017;28:1567–1576.
20. Caneva M, Lang NP, Garcia Rangel IJ, et al. Sinus mucosa elevation using Bio-Oss® or Gingistat® collagen sponge: An experimental study in rabbits. Clin Oral Implants Res. 2017;28:e21–e30.
21. Srouji S, Ben-David D, Funari A, et al. Evaluation of the osteoconductive potential of bone substitutes embedded with schneiderian membrane- or maxillary bone marrow-derived osteoprogenitor cells. Clin Oral Implants Res. 2013;24:1288–1294.
22. Srouji S, Ben-David D, Lotan R, et al. The innate osteogenic potential of the maxillary sinus (schneiderian) membrane: An ectopic tissue transplant model simulating sinus lifting. Int J Oral Maxillofac Surg. 2010;39:793–801.
23. Srouji S, Kizhner T, Ben David D, et al. The schneiderian membrane contains osteoprogenitor cells: In vivo and in vitro study. Calcif Tissue Int. 2009;84:138–145.
24. Gruber R, Kandler B, Fuerst G, et al. Porcine sinus mucosa holds cells that respond to bone morphogenetic protein (BMP)-6 and BMP-7 with increased osteogenic differentiation in vitro. Clin Oral Implants Res. 2004;15:575–580.
