Xenograft bone substitutes are commonly used to increase bone volume and height in the deficient posterior maxilla. The addition of enamel matrix derivate (Emdogain) could increase the efficiency of the bone healing process. The aim of this prospective randomized, controlled split-mouth design study was to compare the percentage of newly formed bone in sinus floor augmentation with deproteinized bovine bone mineral with or without the addition of enamel matrix derivative after 6 months of healing. Sixteen bilateral sinus floor augmentation procedures were performed. Deproteinized bovine bone mineral combined with enamel matrix derivative (test) and deproteinized bovine bone mineral alone (control) groups were randomly allocated within each patient. Six months after augmentation and concurrent to implant placement, bone biopsies were taken for histomorphometric analysis. Additionally, implant survival and peri-implant bone levels were radiographically assessed at baseline and 24 months after functional loading. Histomorphometric analysis revealed a significantly higher amount of newly formed bone in the test group compared with the control group (22.6% and 15.5%, respectively; P = .033). No significant differences in the amount of remaining graft or connective tissue was found. Enamel matrix derivative added to deproteinized bovine bone mineral particles significantly increased new bone formation in sinus lift procedures in edentulous or partially edentulous patients with deficient bilateral posterior alveolar ridges requiring augmentation for implant placement.
Rehabilitation by fixed prostheses of edentulous sinus areas is often complicated by the low residual bone. Sufficient bone availability is reported to be the key factor for successful implant placement.1 Although the use of short implants has been shown to be a suitable therapeutic option to overcome these obstacles in some cases,2 anatomical limitations can oblige the use of maxillary sinus augmentation procedures3 and have become a treatment option over the last 10 years. Lateral approach sinus floor elevation has been routinely performed in the last few years and is regarded as a predictable procedure.4,5 However, the choice of bone graft material for maxillary sinus augmentation remains controversial.6 Deproteinized bovine bone mineral (DBBM), such as autogenous bone, is a material that does not elicit an immune response and provides an osteoconductive platform for new bone growth. To reduce the donor site morbidity associated with harvesting autogenous bone while preserving the necessary graft volume, clinicians have begun to mix DBBM with autogenous bone.7,8 Nowadays, the material that shows the most favorable and reproducible clinical outcomes in sinus filling procedures is autogenous bone alone or mixed with DBBM. Moving 1 step further toward biomaterial-only sinus grafting, a Cochrane review showed that autogenous bone could be successfully replaced by alternative materials such as bone substitutes.9 Moreover, it was shown that implants placed in xenografts have statistically the same survival rate as those placed in autogenous bone particles.10 Therefore, although biomaterials are becoming more and more accepted as a treatment option for sinus grafting, clinicians and researchers are looking for additional options to increase the reliability of the healing process in the grafted site. One suggestion that has gained attention is to impart a more bioactive component to DBBM, through the addition of growth factors.11 Enamel matrix derivative (EMD; Emdogain, Straumann, Basel, Switzerland) is a widely used medical device whose main component is amelogenin protein. EMD was developed initially for the regeneration of periodontal ligament and root cementum and has been successfully used for a variety of periodontal applications, including the treatment of intrabony defects, recessions, and furcation defects. EMD is capable of enhancing periodontal wound healing and regeneration.12 It has also been found to stimulate microvascular primary endothelial cell proliferation and, as such, may promote periodontal regeneration by stimulating angiogenesis. This potential of EMD on wound-healing sites was confirmed in an in vivo study.13 In intrabony defects, regenerative surgery including application of EMD has been shown to improve clinical parameters such as probing depth reduction, clinical attachment level, and defect fill.11,14 There have been many clinical, histologic, and immunohistochemical studies evaluating the suitability of the association of a DBBM (BioOss, Geistlich Biomaterials, Wolhusen, Switzerland) with EMD in the treatment of intrabony defects around teeth.15–17 A Cochrane systematic review and meta-analysis showed that the combination of EMD and bone grafts in intrabony defects may result in additional clinical improvements compared with those obtained with EMD alone.18 Additionally, a recent study in rats suggested that EMD has the ability to enhance the speed of new bone formation when combined with natural bone mineral particles.19 Moreover, studies evaluating the mix between DBBM and EMD showed that this combination significantly improves cell attachment, proliferation, and differentiation of human primary osteoblasts, and periodontal ligament (PDL) cells compared with control particles in vitro.14 EMD was also shown to stimulate the release of growth factors, cytokines, and differentiation of various markers.20 All these results seem to support the adjunction of EMD with various grafting materials for regenerative treatment in intrabony defects.21,22 This association may potentiate the effect of each component, where the bone substitute may act as a space maintainer, while EMD may induce formation of bone tissue, which particularly interests us in this study.23,24 Taken together, all the aforementioned findings appear to provide the biologic rationale for the clinical use of this combination strategy. However, to date, there are no histologic and clinical studies on maxillary sinus grafting with Bio-Oss in combination with Emdogain. The aim of this study was to histomorphometrically and histologically analyze samples obtained 6 months after maxillary sinus augmentation procedures of grafted sinuses in human subjects using Bio-Oss in combination with Emdogain and to determine the clinical efficacy of this therapy.
Materials and Methods
All patients included in this prospective randomized and split-mouth study (n = 8) were treated for a bilateral sinus floor augmentation procedure (mean age, 59 ± 13 years; range, 47–72 years). Four patients were men, and 4 were women. They were referred to the Department of Periodontology at the University of Nice because of a deficient bilateral posterior alveolar ridge, preventing implant placement. Referred patients who fit the criteria for study inclusion were identified and approached for their willingness to take part, whereupon their informed consent was secured. The protocol of this prospective study (ClinicalTrials #NCT01870960) was submitted to and approved by the South Mediterranean II Ethical Research Committee (CCP Sud-Mediterranee II 13.018). For each case, the randomization was done by the clinical research director of our hospital, who faxed the surgeon which side to be used for the test sinus.
All patients were medically healthy and periodontally treated if necessary. Inclusion criteria were a maxillary partial or total edentulism involving the premolar/molar areas (Figure 1) and the presence of a Misch type 4 sinus situation.25 The preoperative diagnosis was made by carrying out orthopantomography combined with cone beam computerized tomography (CBCT), and analysis was performed with the software Planmeca Romexis (Planmeca, Inc, Hoffman Estates, Ill; Figure 2). Exclusion criteria were an acute myocardial infarction within the last 12 months, any medication that could interfere with bone healing, history of radiotherapy in the head and neck region, psychiatric problems, patients who smoke more than 10 cigarettes/d, and alcoholism or chronic drug abuse.
Maxillary sinus augmentation technique
Sixteen bilateral sinus lift procedures were prospectively performed by the same surgeon under local anesthesia. At each site, the crestal incision was prolonged by a vertical releasing incision, distal to the canine. After elevation of a mucoperiosteal flap, the buccal aspect of sinus wall was exposed to perform the lateral window using the Mectron Piezosurgery System (Genova, Italy). The height of the vertical osteotomy was approximately 10 mm. Then, the Schneiderian membrane was carefully elevated using manual sinus elevators. In 3 of the 16 cases, the sinus membrane was partially perforated and then sutured without any complications. The space created between the maxillary alveolar process and the elevated membrane was filled either with Bio-Oss mixed with Emdogain (test) or only with Bio-Oss (control). The flap was then repositioned and immobilized by means of 5-0 nonabsorbable polypropylene sutures to achieve closure of a tension-free flap. A second CBCT scan was taken immediately after sinus augmentation to verify that the material is well covered with an intact membrane. In all surgical procedures, antibiotic treatment (3 g amoxicillin + clavulanic acid per day) was prescribed for 7 days and analgesic treatment for 4 days (60 mg prednisolone + 3 g paracetamol per day). All patients were instructed to rinse their mouths with a 0.2% chlorhexidine solution twice a day for 2 minutes for 2 weeks. Patients were also advised not to blow their noses for 2 weeks and to cough or sneeze with an open mouth. The sutures were removed 2 weeks postoperatively. Dentures were not permitted for use until 2 weeks after surgery.
Implants placement and surgical re-entry
A CBCT was performed 6 months after sinus lift procedure to plan the implant placement. A total of 28 implants (Straumann Standard Plus SLActive implants, Institut Straumann AG, Basel, Switzerland) were placed in 1 surgery, in both control and test sites, by a 1-stage nonsubmerged process. Six patients received 4 implants (2 in the test side and 2 in the control side), and 2 patients received only 2 implants (1 per side). The diameter of 7 implants was 4.8 mm and their length was 8 mm, the diameter of 12 implants was 4.8 mm and their length was 10 mm, the diameter of 5 implants was 4.8 mm and their length was 12 mm, and the diameter of 4 implants was 4.1 mm and their length was 10 mm. For each side implants were systematically placed in the first molar area and in another site if other teeth were missing. Consequently, a bone core was taken from the molar site for each sinus. The biopsy was obtained using a standardized 3.5-mm trephine drill under sterile saline irrigation. The trephine was removed with the bone biopsy, immediately transferred into 10% eosinophilic paraformaldehyde, and further processed for histopathology, as described below. After an osseointegration period of 2 months, fixed implant-supported prostheses were performed in all the patients.
Histologic and histomorphometric evaluation
Biopsy-containing trephines were fixed for a total duration of 15 days, and the solution was changed twice during this period. After fixation, the trephine and the bone plug were immersed in 70% ethanol for 12 hours. Then the trephine was wrapped in a sterile gauze soaked in alcohol and sent to histologic processing and sectioning (AnaPath AG, Liestal, Switzerland). The fixed histologic specimens were embedded in methyl methacrylate as previously described.26 Briefly, nondecalcified sections from plastic-embedded tissue blocks were obtained according to a cutting-grinding technique. Sections were originally cut 500 μm thick and then ground to a final thickness of 30 to 50 μm and stained with paragon stain for microscopic evaluation. The most intact slide of biopsies from each sinus was chosen for the histomorphometric analysis. The new bone area (newly formed bone) to total area was calculated by measuring the tissue present inside the cannula of the trephine and then determining what percentage of this measured tissue was new bone (NB), remaining graft (RG), or connective tissue (CT). These experiments were performed using a Nikon Eclipse 90i microscope and the software Visiopharm Integrator System version 3.6.
Statistical analysis was done by an independent statistician. Means, standard deviations, and ranges were calculated to describe the patients' characteristics. Paired t-tests were performed to evaluate the outcomes differences between control and tests. An α level of significance was fixed to 0.05. The statistical and graphing software Prism version 8 (GraphPad Software Inc, San Diego, Calif) was used for all statistical analysis.
The radiographic analysis was performed with the software Planmeca Romexis. Initially, the mean vertical height of the alveolar bone on the computerized tomography scan between the most posterior aspect of the maxillary sinus and the oral cavity was not significant between tests sides and controls sites (2.6 and 2.9 mm, respectively). After grafting, the vertical bone heights were 12.5 ± 1.2 (tests sides) and 12.9 ± 0.7 mm (controls sides) (Figure 3).
All patients were successfully treated according to the same sinus lift procedure. Twenty-eight implants were placed in 1 surgery in both control and test sites by a 1-stage nonsubmerged process. No implants failed during osseointegration or after functional loading (Figure 4). At 24 months after placement, no implant showed any complication, and the survival rate was 100 %.
During the pilot drilling with the trephine, the grafted bone appeared to be of high density in both groups. The osteotomy sites bled freely, indicating good vascularization of the graft. Biopsies tended to be consistent in terms of the overall amount and situation of tissues within the cannula of the biopsy trephine. No inflammatory infiltrate was present in any of the sections. Granular size of the residual bone grafting material appeared consistent between the test and control sites. All areas of new bone formation were found to be associated with the presence of graft material. In fact, graft particles were completely surrounded by regenerated bone. Sometimes, trabeculae bony bridges were seen, linking islands of graft and bone with each other. Sometimes, graft particles were enveloped with soft tissue or were resorbed by multinucleated osteoclasts (Figure 5).
After 6 months, the average value of newly formed bone was 22.6 ± 5.2% in the Bio-Oss + Emdogain grafted sinuses. Comparatively, the control group (Bio-Oss alone) resulted in a significantly lower amount of new bone, with a mean value of 15.5 ± 6.9% (P < .05). Among the 8 patients who were bilaterally grafted, 7 of them had better results in test side. However, the test and control groups showed comparative levels of remaining graft (20.1 ± 10% and 20 ± 11.7%, respectively) and CT (43.1 ± 9.4% and 37.2 ± 16.5%, respectively; Table 1).
Study power after calculation
With a proposed sample size of 8 patients and based on the assumptions of a mean difference of 11.1% in new bone and a standard deviation of 8.2% (SD = 5.2 for test, SD = 6.9 for control, and a correlation of 0.10), the study would have a power of 90.4%. If the new bone mean difference would be only 10.0%, the power would decrease to 83.8%, if 9.0% to 75.7%, and for 8.0% to 65.8%. The considered α level was 0.05 and the test was 2 tailed.
This study aimed to compare the sinus lift procedure with Bio-Oss alone or in combination with Emdogain. As expected, the clinical findings demonstrated a good regenerative capacity for both groups in this clinical situation at 6 months after surgery, as confirmed by histomorphometric analysis. However, we observed a significantly higher percentage of newly formed bone in test sides grafted with Bio-Oss plus Emdogain compared with control sides grafted with Bio-Oss alone (22.6% vs 15.5%, respectively). No significant difference in percentage of residual graft and connective tissue was found for either group.
The result of 15.5% of new bone in the control group grafted with Bio-Oss alone is in accordance with a recent study that reported an average percentage of 17.6% newly formed bone after 8 months. Nevertheless, we found a smaller proportion of residual bone substitute material compared with this study (our study, 20% vs 29.9%).4 Our results also corroborate those of another study using Bio-Oss alone, with percentages of regenerated bone and residual graft material of 19% and 40%, respectively.27 More recently, a histomorphometric study showed new bone formation of 24.6%, mean value of the connective tissue of 42.6%, and remaining biomaterial of 25.4%28 after 6 months of healing, additionally supporting the results presented herein with the control group.
Biopsies were done 6 months after sinus lift procedure in our study. Recent analyses showed the ratio of mineralized newly formed bone increased slightly from 5 to 11 months. In fact, histomorphometric and microcomputerized tomography assessments highlighted the higher proportion of newly formed bone at 8 and 11 months (later stages) compared with 5 months (early stages). Moreover, residual particles are fewer when the duration of healing period is increased.29 In our study, the bone cores were taken at 6 months, suggesting that higher percentages of newly formed bone could have been found at 8 or 11 months after sinus lift.
No inflammatory infiltrate was observed in any of the sections. However, because of the later healing point used in this study, it is not possible to determine if Emdogain had an effect on the inflammatory status associated with early healing of the defect site. It appears that Emdogain does not result in a chronic inflammatory state, as witnessed by the lack of inflammatory infiltrate at 6 months. Moreover, granular size of the residual bone grafting material appeared consistent between the test and control sites, demonstrating Emdogain does not contribute to any dissolution of the bone graft material. All areas of new bone formation were found to be associated with the presence of graft material, which suggests that Emdogain indeed precipitates onto the surface of the graft material and directly contributes to an osteopromotive environment on the DBBM material. Considering the increase in new bone formation in the presence of Emdogain, the mechanism appears to be based on an increase of the osteoconductivity of the bone graft material rather than imparting an osteogenic signal.
The use of autologous bone is considered the gold standard in bone grafting techniques. Nevertheless, it presents some drawbacks, especially regarding operative morbidity, namely, the need for two or more surgical sites in cases of greater amount of donor tissue, including extraoral sources. Additionally, fast resorption/remodeling times, resulting in larger defect collapse, have been reported when autologous bone grafts were used. Indeed, groups have more recently investigated the effect of mixing DBBM with autologous bone using varying mixing percentages in an attempt to conserve the attractive osteogenic component of the autogenous graft but also to preserve the defect volume for a longer time period because of the nonresorptive properties of the DBBM.30 Obviously, a DBBM-based bone graft with osteogenic properties would offer an attractive alternative to autologous grafts. Bio-Oss has become one of the most widely used materials for sinus floor elevation.31 It is often assimilated as a reference for other grafting biomaterials,32 which is why it was chosen as the control for our study.
Although we believe our study shows the benefit of EMD and BioOss in patients requiring sinus floor augmentation for dental implant placement, we recognize that the study does have certain limitations. For example, age-related health factors were not controlled in this study; however, the augmentation procedure in question is generally performed in older patients who have been edentulous in the posterior region for some time, hence the necessity for sinus grafting to allow sufficient bone for implant placement. The authors also recognize that 8 patients, giving 8 sets of data each for BioOss alone and BioOss plus EMD could be considered a relatively low number in terms of clinical investigations. The authors consider that the data acquired are sufficient to see a positive effect, but we also recognize that it may be difficult to extrapolate the results to a wider population (ie, beyond patients who are fully or partially edentulous in the premolar and/or molar region in the maxilla and with insufficient bone for implant placement). It may also be difficult to apply to other challenging clinical situations (ie, other situations that may require bone augmentation for implant placement, such as narrow crestal ridges in the mandible or in anterior situations). We therefore urge a note of caution in interpreting these results to other situations.
Another limitation is that, as with many clinical studies in the dental field, surgery was performed by a single, very experienced surgeon in a specialized university clinic setting. Patients were referred to the Department of Periodontology at the University of Nice because of their challenging clinical situation. The same surgery performed in a private practice setting by dental practitioners who may be less experienced or less specialized may therefore have different results. In addition, although the healing time of 6 months is sufficient to assess the formation of new bone histologically, many dental practitioners may be more cautious and wait longer before attempting to place implants after such a sinus augmentation procedure. This partly has ethical implications, because waiting longer may not be desirable for the patients but is partly because of the greater experience of the clinician in this and similar studies, who can therefore attempt implant rehabilitation in a shorter time frame. Finally, the only measured parameter that showed a significant difference was the amount of new bone after 6 months. The authors recognize that, although this may not be the most important parameter, it is not the only consideration (ie, remaining graft may be important if another practitioner was to use a different type of bone graft material). The amount of connective tissue may be the most important factor depending on the type of implant and restoration used, which may be different from that used by the department in this study.
Possible future research could include an investigation in the use of the combination of BioOss and EMD in other situations where bone augmentation may be required, whether for implant placement or not. This could include, for example, narrow ridges in the edentulous or partially edentulous posterior mandible, or in the case of bone deficiency, in the anterior region. The possibility to evaluate new bone formation at different time points using biopsies could also be considered, although there are, of course, ethical considerations with this because it would require well-informed volunteers and would need to be approved by a stringent ethics committee. A consideration of age-related factors could also be made by evaluating the results in patients in different age groups.
Within the constraints of this study, it appeared that EMD loaded onto DBBM bone grafts results in a positive regenerative outcome in terms of new bone formation in sinus floor augmentation in edentulous or partially edentulous patients with deficient bilateral posterior alveolar ridges. Although the mechanisms for such a phenomenon are difficult to elucidate in a clinical setting, preclinical models would allow a more concerted histologic analysis of the healing process.33
The present findings indicate that the use of Bio-Oss plus Emdogain for sinus floor augmentation to allow implant placement in patients with deficient bilateral posterior alveolar ridges represents an interesting alternative procedure. Future studies, not only clinical studies with a higher patient recruitment, but also translational preclinical studies are recommended to confirm the promising results.
cone beam computerized tomography
deproteinized bovine bone mineral
enamel matrix derivative
The authors thank Ms Jane Fenner-Magnaldo for writing assistance and manuscript review in English and Prof Laurence Lupi, Université Côte d'Azur, for statistical analysis.
Institut Straumann provided materials and histology services for this study. Benjamin Pippenger and Michel Dard are employed by Institut Straumann.