Clin Oral Invest DOI 10.1007/s00784-017-2158-3

ORIGINAL ARTICLE

Sinus floor elevation using implants coated with recombinant human bone morphogenetic protein-2: micro-computed tomographic and histomorphometric analyses Daniel S. Thoma 1 & So-Ra Yoon 2 & Jae-Kook Cha 2 & Hyun-Chang Lim 3 & Jung-Seok Lee 2 & Seong-Ho Choi 2 & Ui-Won Jung 2

Received: 26 March 2017 / Accepted: 19 June 2017 # Springer-Verlag GmbH Germany 2017

Abstract Objectives The objective of this study was to determine the validity of a graft-free sinus floor elevation (SFE) procedure with simultaneous placement of recombinant morphogenetic protein-2 (rhBMP-2)-coated implants compared to uncoated control implants. Methods In 10 rabbits, SFE was performed on both sides. Dental implants were randomly placed in the sinus filled with a blood clot. Test implants were coated with rhBMP-2, whereas in the control group, implants were uncoated. Microcomputed tomographic and histomophometric analyses were performed at 4 and 8 weeks, including measurement for newly formed bone height (NBHm). Results Bone formation was evident along the implant surfaces up to the apex in test, but limited in control implants at 4 weeks. NBHm amounted to 5.1 mm (Q1 = 4.1; Q3 = 5.3) for test implants and to 3.4 mm (2.6; 3.7) for control implants at 4 weeks. NBHm then decreased to 8 weeks (3.4 mm (3.3; 3.7)) for test implants, whereas in control sites, NBHm increased slightly to 4.4 mm (4.1; 4.5) (p = 0.1250; p = 0.6250). Conclusions Implants coated with rhBMP-2 presented a strong osteogenic reaction at 4 weeks with more favorable

Daniel S. Thoma and So-Ra Yoon contributed equally to this study. * Ui-Won Jung [email protected] 1

Clinic for Fixed and Removable Prosthodontics and Dental Material Science, University of Zurich, Zurich, Switzerland

2

Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea

3

Department of Periodontology, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea

outcomes in terms of bone formation along the implant surface up to the apex compared to uncoated control implants. Remodeling and resorption process between 4 and 8 weeks did not further improve the outcomes in the test, but in the control group. Clinical relevance The use of rhBMP-2-coated implants in a graft-free SFE might show an advantage in early implant stability to prevent collapse of membrane. However, a potential clinical benefit still needs to be proven. Keywords Maxillary sinus augmentation . Bone morphogenetic protein-2 . Histology . Micro-computed tomography . Animal study . Dental implant

Introduction Atroph y of t he a l veola r proce ss and continu i ng pneumatization of the maxillary sinus after tooth extraction often result in a vertical deficiency for the placement of dental implants, thereby requiring a sinus floor elevation procedure (SFE) [1–3]. Traditionally, various graft materials from different sources were used to increase the vertical height in the posterior maxilla for a SFE and to maintain the bone volume around the implant [4–6]. The use of grafting materials such as bone substitutes as space filler is associated with some drawbacks. The grafting procedure itself increases the surgical time, the treatment cost, and the possibility of postoperative complications such as infection of the augmented sinus [7–9]. Moreover, bone substitute materials might delay the physiological ossification process at the early stage of healing [10]. Site-specifically, vital bone formation adjoining the implant surface may be more important for a biological and biomechanical support [11, 12]. Moreover, the quality of regenerated bone in a grafted sinus can be affected by the distance to the

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host bone. New bone formation predominantly originates from the existing alveolar bone at the base of the maxillary sinus. This results in a relatively long healing time until bone is formed along the implant surface, being far away from the osteogenic source [13]. In order to overcome these disadvantages of traditional SFE procedures using bone substitute materials, the use of a blood clot has been proposed [7–9, 14, 15]. Controversial data were obtained using this procedure with disadvantages observed that the membrane collapses leading to an unpredictable amount of bone formation [16]. Reasons might include a constant air pressure bearing on the Schneiderian membrane and/or an insufficient regenerative potential of the blood coagulum alone. More recently, a biologic mediator with a high osteogenic potential recombinant human bone morphogenetic protein-2 (rhBMP-2) coated onto the implant surface was considered to be a promising adjunct for SFE procedures without the use of bone substitute materials [17]. rhBMP-2 coated on dental implants might speed up the healing process by accelerating the ossification. Thereby, a mechanical stability preventing the membrane to collapse would be provided at an earlier stage. The aim of the present study was, therefore, to determine the validity of a graft-free SFE procedure with simultaneous placement of rhBMP-2-coated implants compared to uncoated control implants.

Materials and methods Animals Ten male New Zealand white rabbits weighing 2.5–3.0 kg were used. The sample size was calculated with G*Power software v. 3.1 (University of Dusseldorf, Dusseldorf, Germany) at an alpha level of 0.05 and a statistical power of 95%. Based on the standard deviations observed in recent preclinical study [17], histological new bone height was set as a primary outcome variable, and a clinically relevant difference was set at 1 mm. The required sample size per group was 5; thus, 10 rabbits were prepared in consideration of two different healing periods (4 and 8 weeks). The animals were bred in separate cages under standard laboratory conditions, with ad libitum access to water and a standard laboratory pellet diet. Animal selection and care, the surgical protocol, and the preparation procedures were certified by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea (approval number:2013-0335).

roughened by a sandblasting and acid etching process. The coating of the ten test mini-implants with rhBMP-2 (Genoss, Suwon, Korea) was described previously [17]. In brief, under aseptic conditions, the rhBMP-2 solution was reconstituted to a 2.0 mg/mL liquid concentration with 1 ml hyaluronic acid. The implants were placed in sterile 0.5-mL wells (96 Stripwell plate, round well polypropylene, Sigma) that were filled with 0.1 mg/ea. (implant) rhBMP-2 (net volume of solution, 0.05 mL per implant) and incubated for 30 min. The implants were then air-dried for 16 h. All procedures were performed in a biological safety cabinet (Airstream, Class II, A2 type; Esco, Philadelphia, PA, USA) at room temperature. The coating of the implants encompassed a length of 3 mm to the apex. Surgical procedure The experimental surgical procedures were described in detail in previous publications [17, 18]. In brief, animals were sedated with general anesthesia by an intramuscular injection of a mixture of xylazine hydrochloride (Rompun, Bayer, Korea) and ketamine hydrochloride (Ketalar, Yuhan, Korea). The nasal dorsum of each of the rabbits was shaved, the surgical field disinfected using iodine solution, and, subsequently, local anesthesia applied by infiltration of 2% lidocaine. An incision was made on the skin along the midline of the nasal bone to expose the dorsal surface. Two circular windows (diameter of 5.5 mm) were prepared on both sides of the nasal bone using a trephine bur. The Schneiderian membrane of the sinus was carefully elevated and the implant sites were prepared 3 mm anterior to the windows. The mini-implants were then placed with the implant shoulder seated at the native bone and the apex protruding into the sinus cavity. Test and control implants were randomly (according to a computer-generated randomization list) allocated to either the left or right maxillary sinus. The periosteum and skin were sutured by layering with absorbable monofilament (4-0 Monosyn, B. Braun, Aesculap, PA, USA). The animals were carefully monitored during the further healing period for adverse tissue reactions at the surgical sites. The postoperative medication included antibiotics and analgesics for the first 7 days. After healing periods of 4 weeks (5 animals) and 8 weeks (5 animals), the animals were painlessly euthanized (Zoletil 50, Virbac S.A, Virbac Laboratories 06516, Carros, France). All surgical sites were macroscopically inspected, and any incidences were recorded. The sinus was block-resected including the surrounding soft tissues. Micro-computed tomography analysis

Experimental mini-implants A total of 20 mini-implants were specifically prepared for the experiment (6 mm in length and 3 mm in diameter; Dentium, Seoul, Korea). The mini-implant surface was moderately

All specimens were fixed in 10% formalin for 10 days and then scanned using a high-resolution (8.88 μm) microcomputed tomography (μCT) system (SkyScan 1173, Skyscan, Kontich, Belgium). Digital microradiographs were

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acquired at 130 kVp and 60 μA using a 1.0-mm-thick aluminum filter. The specimens were exposed to radiation for 500 ms after each 0.2° rotation. The pixel size was 14.91 μm. The volume of interest (VOI) was limited to 4 mm in a vertical direction and 10 mm in a mesial-distal direction. The following volumetric parameters were measured using a triangle meshing technique based on the marching-cubes method [19, 20]: 1. The total augmented volume (TV; mm3) within the volume of interest (VOI); the images obtained from the μCTs were reconstructed with axial, sagittal, and coronal planes. This allowed constituting one stereoscopic image in three-dimensional voxels. 2. The newly formed bone volume within TV (NBV; mm3); new bone was identified in the reconstructed images as voxels with gray scale values of 55–255 [17].

Histologic analysis After μCT scanning, the specimens were dehydrated and embedded in methylmethacrylate resin in a vacuum chamber system. Each block was cut using a diamond cutter (Exakt, Apparatebau, Norderstedt, Germany), and sections were sawed to a thickness of approximately 100 μm. These sections were then ground and polished on a diamond grinder to a thickness of 15 μm, mounted on microscope slides and stained with hematoxylin–eosin. The specimens were examined under a microscope equipped with a digital camera (BX50, Olympus, Tokyo, Japan). The acquired images of the slides were saved as digital files. Histomorphometric measurements were performed by a single experienced researcher (S.R.Y.) unaware of the treatment groups using an automated image analysis software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA) (Fig. 1):

Fig. 1 Schematic drawings illustrating the measured parameters. Region of interest (ROI) (mm2); The rectangle with the blue border indicates the area of interest (ROI). The subarea bordered by a dotted green line indicates the total augmented area (TA). Ant anterior, Post posterior, OH osseointegrated height, NOH non-osseointegrated height, PH protruding height, CBT cortical bone thickness, NBH new-bone height

thickness of the cortical (native) bone (CBT, mm) anterior (CBTa, mm) and posterior (CBTp, mm) (mean CBT for anterior and posterior = CBTm) length of the implant without contact to newly formed bone (PH-NBH) (OH, mm) anterior (OHa, mm) and posterior (OHp, mm) (mean OH for anterior and posterior = OHm) bone-to-implant contact (BIC, %); The percentage of newly formed bone in contact with the implant surface (not including CBT).

Linear measurements Primary outcome

mean (mean NBH, =NBHm, of anterior and posterior measurements of the newly formed bone height (NBH, mm) measured anterior (NBHa, mm) and posterior (NBHp, mm).

newly formed bone height (NBH, mm), measured anterior (NBHa, mm) and posterior (NBHp, mm) length of the implant protruding into the sinus cavity (PH, mm) anterior (PHa, mm) and posterior (PHp, mm) (mean PH for anterior and posterior = PHm)

Secondary outcomes

Surface measurements Two (anterior and posterior) rectangular regions of interest (ROI) were selected adjacent to the implant (width: 1.5 mm; height: the length of the implant protruding into the sinus cavity (PH)). The following outcomes measures were assessed: – –

Surface area of newly formed bone within ROI (NBA; mm2), anterior (NBAa, mm), and posterior (NBAp, mm) (mean NBA for anterior and posterior = NBAm) Augmented surface area within ROI (TA; mm2), anterior (TAa, mm), and posterior (TAp, mm) (mean TA for anterior and posterior = TAm)

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Statistical analysis

Histologic analysis

The median, minimum, and maximum values of radiographic and histometric parameters were calculated in all groups. Also, means and standard deviations are given in the tables. The paired data of the two groups were compared by using the Wilcoxon signed-rank tests because of the small sample sizes and the problematic normality of the data. For the different secondary outcomes, no correction for the multiple testing was applied. Because of the small sample size (n = 5) no p value can be below 0.05 for the Wilcoxon signed-rank test. Hence, the p values indicate only a possible fact for further studies. If p = 0.0625, it indicates in this case that the data of the two groups do not overlap w.r.t. location. For the primary endpoint, we derived a 90% nonparametric confidence interval because of the same reasons. We investigated the possible confounding factors Bsite,^ Banterior,^ and posterior^ by linear mixed models, but they did not have a significant influence on the treatment outcomes.

Descriptive histology The specimen of each tissues presented bone regeneration without any enhanced inflammatory response. Newly formed bone predominantly regenerated along the implant surfaces and along the native cortical bone. This resulted in a pyramidal shape with the basis at the cortical native bone and the tip slightly more apical than the implant apex. A smooth transition between the native bone and the newly formed bone was observed. In the test group, the regenerated bone had a triangular shape leading up to the implant apex and appeared to cover a larger area than control implants. In control implant sites, the shape was more pyramidal and new bone formation did usually not reach the apex of the implants (Fig. 3).

Results Clinical observations Wound healing was uneventful in all animals. Neither postoperative infections nor exposure of implants were observed during the entire study period. In all sites, the Schneiderian membrane was intact as assessed by visual inspection.

Micro-computed tomography analysis In sagittal sections, de novo bone formation was evident exceeding in an apical direction the original cortical bone in both groups. However, the pattern of bone formation was not consistent. The newly formed bone was not evenly distributed among the spatial sections (anterior and posterior areas). In test sites, bone formation appeared to cover a wider area (further away from the implant surface), whereas in control sites, the area of bone formation was mostly limited to the implant surface (Fig. 2). The median augmented volume (TV) within the volume of interest (VOI) amounted to 60.3 mm3 (58.1; 73.8) at 4 weeks and to 61.3 mm3 (52.4; 80.7) at 8 weeks for test implants. The respective numbers for control implants were lower at both time-points amounting to 43.2 mm3 (36.1; 44.5) and to 30.0 mm3 (29.9; 64.4), respectively. The differences at 4 weeks (p = 0.0625) and 8 weeks (p = 0.3125) were not statistically significantly different. All data are summarized in Table 1.

Histomorphometric outcomes The median new bone height (NBHm) amounted to 5.1 mm (Q1 = 4.1; Q3 = 5.3) for test implants and to 3.4 mm (2.6; 3.7) for control implants at 4 weeks. NBHm then decreased up to 8 weeks resulting in median values of 3.4 mm (3.3; 3.7) for test implants, whereas in control sites, NBH increased slightly to 4.4 mm (4.1;4.5). The differences between the groups at both time-points were not statistically significant (p = 0.1250; p = 0.6250). The 90% nonparametric confidence intervals of the difference of the medians of the two groups were (−2.76, 1.00) at 4 weeks and (−0.92, 1.90) at 8 weeks. The median implant surface without contact to newly formed bone (OHm) was 4.7 mm (3.9; 4.7) for test implants at 4 weeks, then decreased at 8 weeks to 2.9 mm (2.6; 3.3). The respective numbers for control implants were 2.7 mm (2.5; 3.0) and 4.1 mm (2.8; 4.3). (p = 0.0625; p = 0.3125). The bone-to-implant contact (BIC) was higher at both time-points in the test group rendering 37.1% (22.1; 37.5) at 4 weeks and 33.1% (27.7; 35.3) at 8 weeks. The respective values in the control group were 8.7% (5.9; 27.6) and 25.4% (21.4; 26.4), respectively. The differences between two groups were significantly in favor of test implants at both time-points (p = 0.0625; p = 0.0625). Within the ROI, the augmented area surface (NBAm) was 0.66 mm2 (0.49; 0.66) and 0.63 mm2 (0.37; 0.69) at 4 and 8 weeks. In control sites, the respective numbers were 0.40 mm 2 (0.38; 0.52) and 0.59 mm 2 (0.41; 0.61). (p = 0.1250; p = 0.8125). All data are reported in Table 1. None of the intergroup comparisons revealed any statistically significant differences between the groups at any time-point (Table 2), since as mentioned, no p value of the Wilcoxon signed-rank test can be below 0.05 because of the small sample size (n = 5).

Clin Oral Invest Fig. 2 3D micro-computed tomography. Top: note the topography of the newly formed bone (red) around the protruding implants at 4 and 8 weeks post surgery. Red coding: augmented volume, green coding: miniimplant. Bottom: cross-sectional views in the sagittal plane. Test and control groups at 4 and 8 weeks of healing. Ant anterior, Post posterior

Discussion The present experimental trial evaluated implants with or without coating with rhBMP-2 for graft-free sinus elevation and demonstrated that (i) coating of implants with rhBMP-2 resulted in larger area of bone regeneration around the implant within the maxillary sinus based on descriptive μCT and histologic observations; (ii) the median new bone height along the implant surface protruding the maxillary sinus, BIC values and the augmented area of bone regeneration were more favorable for BMP-2-coated implants compared to control implants. Based on clinical observations, the maxillary sinus represents an ideal, contained defect area for bone regeneration [7,

9]. Various biomaterials ranging from autogenous bone to alloplastic, xenogeneic, and allogeneic bone substitute materials were successfully evaluated [4–6]. Moreover, recent clinical research demonstrated that even implants protruding the maxillary sinus and not being surrounded by biomaterials can obtain bone regeneration along the implant surfaces [7, 9, 14]. Data, however, are controversial and range extensively between different studies. One of the major drawbacks appears to be a potential collapse of the Schneiderian membrane, resulting in a limited bone formation at the apex of the dental implant and an unpredictable amount of the overall bone formation [16]. Therefore, the use of biologic mediators with a high osteogenic potential recombinant human bone morphogenetic protein-2 (rhBMP-2) coated onto the implant surface

Clin Oral Invest Table 1 Mean values, standard deviations, medians, and first and third quartiles for micro-computed tomography outcome measures at 4 and 8 weeks. p values calculated using the Wilcoxon signed-rank test Test Mean SD

Control Median Q1

Q3

Mean SD

Wilcoxon signed-rank-test Median Q1

Q3

p value

4 weeks Total volume (mm3; TV)

63.33 19.01 60.25

58.14 73.75 43.97 12.47 43.19

36.06 44.46 0.0625

Bone volume (mm3; bone V) New bone volume (mm3; NBV) Soft tissue volume (mm3; STV) 8 weeks Total volume (mm3; TV) Bone volume (mm3; bone V) New bone volume (mm3; NBV) Soft tissue volume (mm3; STV)

17.62 7.95 21.75 11.31 5.99 10.69 34.40 19.43 44.51 61.24 22.16 61.29 5.97 1.47 5.73 3.62 1.74 3.25 51.65 21.21 52.05

14.46 8.75 25.48 52.37 5.38 2.83 44.16

10.32 4.73 12.30 29.92 4.69 1.69 20.64

was proposed. This combination was suggested to speed up the healing process, improve osseointegration and bone formation at an earlier stage, while simultaneously preventing a collapse of the Schneiderian membrane [21, 22]. In a previous

23.04 13.01 48.59 80.74 6.80 3.51 68.35

14.04 5.81 13.28 6.03 2.66 4.79 23.90 15.37 18.00 43.53 19.20 29.98 7.26 5.18 5.87 2.67 1.31 2.14 33.60 23.32 21.08

14.84 6.64 31.23 64.39 7.13 3.03 56.67

0.4375 0.1250 0.6250 0.3125 1.0000 0.4375 0.4375

experiment, the effect of rhBMP-2 coated on the dental implants was evaluated using either blood or a collagen sponge as filler material within the sinus [17]. In that study, at 8 weeks of healing, only a limited effect of rhBMP-2 could be

Fig. 3 Histologic views of the test and control groups at 4 and 8 weeks. a Overall view (Scale bar = 2mm). b Higher magnification view of the new bone area (Scale bar = 200 μm). c Higher magnification view of the bone-implant contact (Scale bar = 200 μm). NB new bone, CB cortical bone

Clin Oral Invest Table 2 Mean values, standard deviations, medians, and first and third quartiles for histologic outcome measures at 4 and 8 weeks. p values calculated using the Wilcoxon signed-rank test Test Mean SD 4 weeks New bone height (mm; NBHm)

Control Median Q1

Q3

Mean SD

Median Q1

Q3

Wilcoxon signed-rank test p value

4.73 0.68

5.06

4.13

5.29

3.47

1.05

3.42

2.62

3.67 0.1250

Protruded height (mm; PHm)

5.30 0.34

5.49

5.34

5.49

5.38

0.21

5.37

5.36

5.46 0.5000

Cortical bone thickness (mm; CBTm) Osseointegrated height (mm; OHm)

0.29 0.10 4.39 0.48

0.25 4.70

0.25 3.89

0.27 4.70

0.26 2.68

0.08 0.80

0.22 2.69

0.20 2.49

0.33 1.000 3.05 0.0625

Non-osseointegrated height (mm; 0.95 0.76 0.80 NOHm) Bone to implant contact (%; BIC) 31.80 9.70 37.09 4.17 0.33 4.18 Total area (mm2; TAm) 0.68 0.29 0.66 New bone area (mm2; NBAm) Fibrous tissue anterior (mm2; FTAm) 3.48 0.22 3.41

0.53

1.65

2.67

0.97

2.94

2.32

2.97 0.0625

22.10 37.50 15.16 12.25 4.08 4.33 3.14 0.77 0.49 0.66 0.43 0.10 3.41 3.66 2.71 0.70

8.65 3.03 0.40 2.65

5.88 27.59 0.0625 2.76 3.75 0.0625 0.38 0.52 0.1250 2.45 3.22 0.1250

8 weeks New bone height (mm; NBHm) Protruded height (mm; PHm)

3.61 0.82 5.50 0.11

3.37 5.53

3.26 5.41

3.67 5.53

4.04 5.57

0.92 0.17

4.42 5.59

4.10 5.46

4.52 0.6250 5.68 0.8125

0.27 0.05

0.26

0.24

0.27

0.32

0.06

0.32

0.28

0.33 0.1875

Osseointegrated height (mm; OHm) 2.86 0.55 2.92 Non-osseointegrated height (mm; 2.34 0.38 2.19 NOHm) Bone to implant contact (%; BIC) 32.18 4.97 33.09 2.44 0.72 2.40 Total area (mm2; TAm) 0.59 0.24 0.63 New bone area (mm2; NBAm) Fibrous tissue anterior (mm2; FTAm) 1.85 0.53 1.71

2.65 2.02

3.35 2.73

3.61 1.96

0.96 0.91

4.11 1.45

2.82 1.26

4.33 0.3125 2.77 0.0625

Cortical bone thickness (mm; CBTm)

27.74 35.34 24.50 2.00 3.01 2.65 0.37 0.69 0.50 1.65 2.38 2.15

observed. This, to some extent, is in line with the results of the present study focusing on the later time-point, demonstrating a limited positive effect of rhBMP-2 coating compared to uncoated implants. It was speculated that an earlier time-point would be more appropriate to detect differences since rhBMP2 results in a strong osteoinductive activity predominantly at early time-points. Moreover, no advantage of using an absorbable collagen sponge was observed. In order to further evaluate coating of dental implants using rhBMP-2 without using a filler material, the present study was designed. The present study indicates that rhBMP-2-coated implants were more favorable for a number of outcome measures compared to uncoated implants based on μCT and histologic analyses. Predominantly, the median new bone height was 50% higher at the early time-point of 4 weeks compared to control implants without coating, demonstrating that the coating effect of rhBMP-2 fulfilled the expectations and did speed up the healing process as well as bone regeneration along the implant surface. Moreover, descriptive histology revealed bone formation up to the apex of the implant. Based on these observations, one might conclude that, following an initial swelling associated with the use of rhBMP-2 and the surgical procedures, a collapse of the Schneiderian membrane at later follow-up time-points appears to happen less likely and might improve longer term implant survival rates. Interestingly, between 4 and 8 weeks, bone formation along the implant

4.24 25.41 1.41 2.12 0.14 0.59 1.36 1.51

21.38 26.43 0.0625 2.06 2.20 0.8125 0.41 0.61 0.8125 1.45 1.80 0.8125

surfaces slightly increased at control, but decreased at coated implants. Previous studies indicated that when rhBMP-2 was used for localized bone regeneration and consecutive healing time-points were analyzed following a burst and a strong initial bone formation, remodeling processes are initiated [13, 23]. Obviously, the effect of rhBMP-2 is strongly related to the early healing phase with a burst release and subsequent remodeling processes that may lead to a decreasing bone formation [24]. These data, however, are based on preclinical experiments and have not been observed in clinical studies. A possible explanation for such remodeling processes might include that hard tissues, once augmented and not being subjected to a loading, may undergo resorption processes. The bone-to-implant contact is considered a key outcome measure when assessing implant osseointegration. In the test group, maximal BIC values were obtained at 4 weeks and did not improve over the course of the next 4 weeks. In the control group, BIC values were minimal at the early time-point, but then increased without reaching the level of the rhBMP-2coated implants. Even though bone formation around the implants based on μCT and histologic outcome measures did not improve between 4 and 8 weeks, but rather presented decreasing values, BIC values were not affected by these remodeling processes. From a clinical point of view and in order to ensure long-term successful outcomes, implant osseointegration needs to be stable. The extent and amount of osseointegration needed

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on the long run, is, however, unknown. Previous studies, analyzing BIC values at implant sites with or without rhBMP-2 presence indicated similar outcomes, with high values at early healing time-points and no further improvements thereafter. In the clinical point of view, the costs associated with procedure of rhBMP-2 could be a limitation to use in everyday clinic. However, the Escherichia coli-derived rhBMP-2 used in the present study is much more economical to produce than the CHO-cell-derived rhBMP-2. Also, the costs for coating of implant would be much less than the cost for soaking of bone material, because the amount of rhBMP-2 for coating is relatively small. Moreover, one might take into consideration that by using coated implants, bone substitute materials would be obsolete. As such, treatment costs per site would be comparable to traditional procedures. The outcomes of the present study are limited in terms of a relatively low number of animals/sites per time-point, probably preventing significant outcomes for the majority of the assessed parameters. One might speculate that an increased number of sites would result in potentially stronger conclusions.

References 1.

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Conclusions 7.

Dental implants coated with rhBMP-2 rendered a number of benefits in the present sinus elevation model. This predominantly included a strong osteogenic reaction at the 4-week time-point resulting in more favorable outcomes in terms of bone formation along the implant surface up to the apex compared to uncoated control implants. Remodeling and resorption process between 4 and 8 weeks did not further improve the outcomes in the test, but in the control group. A potential clinical benefit might still need to be proven. Acknowledgements The support of Prof. Jürg Hüsler for performing the statistical analysis is highly acknowledged.

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Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT & Future Planning) (No. NRF2017R1A2B2002537).

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13. Ethical approval This article does not contain any studies with human participants performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Informed consent required.

For this type of study, formal consent is not

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Esposito M, Felice P and Worthington HV (2014) Interventions for replacing missing teeth: augmentation procedures of the maxillary sinus. The Cochrane database of systematic reviews:CD008397. doi: 10.1002/14651858.CD008397.pub2 Jensen J, Simonsen EK, Sindet-Pedersen S (1990) Reconstruction of the severely resorbed maxilla with bone grafting and osseointegrated implants: a preliminary report. Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons 48:27–32 discussion 33 Raja SV (2009) Management of the posterior maxilla with sinus lift: review of techniques. Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons 67:1730–1734. doi:10.1016/j.joms.2009. 03.042 Nkenke E, Stelzle F (2009) Clinical outcomes of sinus floor augmentation for implant placement using autogenous bone or bone substitutes: a systematic review. Clin Oral Implants Res 20(Suppl 4):124–133. doi:10.1111/j.1600-0501.2009.01776.x Pjetursson BE, Tan WC, Zwahlen M, Lang NP (2008) A systematic review of the success of sinus floor elevation and survival of implants inserted in combination with sinus floor elevation. J Clin Periodontol 35:216–240. doi:10.1111/j.1600-051X.2008.01272.x Shanbhag S, Shanbhag V, Stavropoulos A (2014) Volume changes of maxillary sinus augmentations over time: a systematic review. The International journal of oral & maxillofacial implants 29:881– 892. doi:10.11607/jomi.3472 Balleri P, Veltri M, Nuti N, Ferrari M (2012) Implant placement in combination with sinus membrane elevation without biomaterials: a 1-year study on 15 patients. Clin Implant Dent Relat Res 14:682– 689. doi:10.1111/j.1708-8208.2010.00318.x Moon JW, Sohn DS, Heo JU, Shin HI, Jung JK (2011) New bone formation in the maxillary sinus using peripheral venous blood alone. Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons 69:2357–2367. doi:10.1016/j.joms.2011.02.092 Thor A, Sennerby L, Hirsch JM, Rasmusson L (2007) Bone formation at the maxillary sinus floor following simultaneous elevation of the mucosal lining and implant installation without graft material: an evaluation of 20 patients treated with 44 Astra Tech implants. Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons 65:64– 72. doi:10.1016/j.joms.2006.10.047 Araujo M, Linder E, Lindhe J (2009) Effect of a xenograft on early bone formation in extraction sockets: an experimental study in dog. Clin Oral Implants Res 20:1–6. doi:10.1111/j.1600-0501.2008. 01606.x Junker R, Dimakis A, Thoneick M, Jansen JA (2009) Effects of implant surface coatings and composition on bone integration: a systematic review. Clin Oral Implants Res 20(Suppl 4):185–206. doi:10.1111/j.1600-0501.2009.01777.x Wennerberg A, Albrektsson T (2009) Effects of titanium surface topography on bone integration: a systematic review. Clin Oral Implants Res 20(Suppl 4):172–184. doi:10.1111/j.1600-0501. 2009.01775.x Kim JS, Cha JK, Cho AR, Kim MS, Lee JS, Hong JY, Choi SH, Jung UW (2015) Acceleration of bone regeneration by BMP-2loaded collagenated biphasic calcium phosphate in rabbit sinus. Clin Implant Dent Relat Res 17:1103–1113. doi:10.1111/cid.12223 Hatano N, Sennerby L, Lundgren S (2007) Maxillary sinus augmentation using sinus membrane elevation and peripheral venous blood for implant-supported rehabilitation of the atrophic posterior maxilla: case series. Clin Implant Dent Relat Res 9:150–155. doi: 10.1111/j.1708-8208.2007.00043.x

Clin Oral Invest 15.

16.

17.

18.

19.

Lundgren S, Andersson S, Gualini F, Sennerby L (2004) Bone reformation with sinus membrane elevation: a new surgical technique for maxillary sinus floor augmentation. Clin Implant Dent Relat Res 6:165–173 Kim HR, Choi BH, Xuan F, Jeong SM (2010) The use of autologous venous blood for maxillary sinus floor augmentation in conjunction with sinus membrane elevation: an experimental study. Clin Oral Implants Res 21:346–349. doi:10.1111/j.1600-0501. 2009.01855.x Baek WS, Yoon SR, Lim HC, Lee JS, Choi SH, Jung UW (2015) Bone formation around rhBMP-2-coated implants in rabbit sinuses with or without absorbable collagen sponge grafting. Journal of periodontal & implant science 45:238–246. doi:10.5051/jpis. 2015.45.6.238 Choi Y, Yun JH, Kim CS, Choi SH, Chai JK, Jung UW (2012) Sinus augmentation using absorbable collagen sponge loaded with Escherichia coli-expressed recombinant human bone morphogenetic protein 2 in a standardized rabbit sinus model: a radiographic and histologic analysis. Clin Oral Implants Res 23:682–689. doi:10. 1111/j.1600-0501.2011.02222.x Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R (2010) Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 25:1468–1486. doi:10.1002/jbmr. 141

20.

Jung UW, Unursaikhan O, Park JY, Lee JS, Otgonbold J, Choi SH (2015) Tenting effect of the elevated sinus membrane over an implant with adjunctive use of a hydroxyapatite-powdered collagen membrane in rabbits. Clin Oral Implants Res 26:663–670. doi:10. 1111/clr.12362 21. Boyne PJ, Marx RE, Nevins M, Triplett G, Lazaro E, Lilly LC, Alder M, Nummikoski P (1997) A feasibility study evaluating rhBMP-2/absorbable collagen sponge for maxillary sinus floor augmentation. The International journal of periodontics & restorative dentistry 17:11–25 22. Nevins M, Kirker-Head C, Nevins M, Wozney JA, Palmer R, Graham D (1996) Bone formation in the goat maxillary sinus induced by absorbable collagen sponge implants impregnated with recombinant human bone morphogenetic protein-2. The International journal of periodontics & restorative dentistry 16:8– 19 23. Lee EU, Lim HC, Hong JY, Lee JS, Jung UW, Choi SH (2016) Bone regenerative efficacy of biphasic calcium phosphate collagen composite as a carrier of rhBMP-2. Clin Oral Implants Res 27:e91– e99. doi:10.1111/clr.12568 24. Thoma DS, Cha JK, Sapata VM, Jung RE, Husler J, Jung UW (2016) Localized bone regeneration around dental implants using recombinant bone morphogenetic protein-2 and platelet-derived growth factor-BB in the canine. Clinical oral implants research in press. doi:10.1111/clr.12989