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International Journal of. Oral and Maxillofacial Surgery 36: 377–385. Chiapasco, M., Casentini, P. & Zaniboni, M. (2009). Bone augmentation procedures in ...

P.W. Ka¨mmerer* V. Palarie* E. Schiegnitz V. Nacu F.G. Draenert B. Al-Nawas

Influence of a collagen membrane and recombinant platelet-derived growth factor on vertical bone augmentation in implant-fixed deproteinized bovine bone – animal pilot study

Authors’ affiliations: P.W. Ka¨mmerer, E. Schiegnitz, F.G. Draenert, B. Al-Nawas, Department of Oral, Maxillofacial and Plastic Surgery, University Medical Centre of the Johannes Gutenberg-University Mainz, Mainz, Germany V. Palarie, Clinic for Oral & Maxillofacial Surgery, State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau, Moldova V. Nacu, Laboratory Tissue Engineering and Cellular Culture, State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau, Moldova

Key words: animal experiment, collagen membrane, deprotenized bovine bone, guided bone

Corresponding author: Peer W. Ka¨mmerer Assistant Professor, Department of Oral, Maxillofacial and Plastic Surgery University Medical Centre of the Johannes Gutenberg-University Mainz Augustusplatz 2, 55131 Mainz, Germany Tel.: +004906131 17 5459 Fax: +06131 17 6602 e-mail: [email protected]

Materials and methods: In 12 rabbits, a DBB-block was implant-fixed on the tibia in a split-

regeneration, nobel active dental implant, recombinant human platelet-derived growth factor-BB, tibia, vertical bone augmentation Abstract Objectives: Combinations of bone substitute block materials with membrane techniques as well as with growth factors are possible options to enhance the prognosis of vertical bone augmentation. Therefore, the aim of the pilot study was to compare the influence of a collagen membrane and a signal protein (rhPDGF-BB) on vertical bone augmentation with a stable fixed block material (deproteinized bovine bone [DBB]). leg-design. Included were: DBB only (control), DBB + collagen membrane (test), DBB + rhPDGF-BB (test) and DBB + rhPDGF-BB + collagen membrane (test). 24 samples were examined after 3 (n = 12) and 6 weeks (n = 12). Calculated parameters were new bone area (NBA;%), new vertical bone height (VBH; mm). Due to the pilot character of this study, single values are shown descriptively only. Results: After 3 weeks, there were constant higher NBA values in the rhPDGF-BB-group without membrane (NBA (%) DBB: 30/16/4; DBB + membrane: 25/17/7, DBB + rhPDGF-BB: 40/33/34, DBB + rhPDGF-BB + membrane: 0/30/16; VBH (mm) DBB: 1.2/1.2/1, DBB + membrane: 0.7/0.9/1, DBB + rhPDGF-BB: 0.7/0.9/1, DBB + rhPDGF-BB + membrane: 0/1.1/1). After 6 weeks, both membrane groups showed a constant higher NBA and VBH independent to the use of rhPDGF-BB (NBA DBB: 3/0/5, DBB + membrane: 20/35/31, DBB + rhPDGF-BB: 5/8/4, DBB + rhPDGFBB + membrane: 31/35/40; VBH DBB: 0.3/0.3/0.6, DBB + membrane: 1.6/2.4/2.1, DBB + rhPDGF-BB: 0.4/0.7/0.8, DBB + rhPDGF-BB + membrane: 1.8/2/1.8). Conclusions: For vertical augmentation, the addition of rhPDGF-BB to DBB-blocks may increase early bone growth. In the later phase, the use of a collagen membrane enhances new bone volume and height to a significant greater extend. Even if the results are higher than those in the non-membrane groups, the low gain of bone after the short time periods still needs improvement.

* These authors contributed equally to this work and are considered as joint first authors.

Date: Accepted 04 June 2012 To cite this article: Ka¨mmerer PW, Palarie V, Schiegnitz E, Nacu V, Draenert FG, Al-Nawas B. Influence of a collagen membrane and recombinant platelet-derived growth factor on vertical bone augmentation in implant-fixed deproteinized bovine bone – animal pilot study. Clin. Oral Impl. Res. , 2012, 1–9 doi: 10.1111/j.1600-0501.2012.02534.x

© 2012 John Wiley & Sons A/S

Atrophy of the alveolar ridges of the superior and inferior jawbone following tooth extraction, periodontal infections and trauma or operation defects is an often described and clinically important phenomenon (Araujo et al. 2002; Cawood et al. 2007; Nevins et al. 2009). Reconstruction methods for these vertical defects in human and animal trials have been studied extensively by evaluating healing events via histological, radiological and clinical outcomes. But the common results of these studies show that the vertical

regeneration of severe localized edentulous atrophic ridges remains a challenging procedure (Arau´jo & Lindhe 2005; Canullo et al. 2006; Simion et al. 2006; Tonetti & Hammerle 2008; Felice et al. 2009; Draenert et al. 2011; Schmitt et al. 2011). The available modalities for vertical reconstruction of the bone are compromised by different intra-operative and postoperative discomforts. “Gold standard” autogenous grafts require invasive techniques for the harvesting bone often from extra-oral regions. Despite the well-known

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advantages of autografts, such as the capacity for osteo-conduction as well as -induction and restricted immune reaction, there are also significant drawbacks, like induction of a secondary defect at the donor site, followed by possible infection and morbidity at the donor site (Schaaf et al. 2010; Blokhuis & Arts 2011). The resorption of such grafts has been reported to be up to 50% of the total volume of reconstructed site (Chiapasco et al. 2009). Vertical alveolar distraction is an alternative technique to harvesting operations (Misch 1997; Swennen et al. 2001). But these techniques have limitations due to deviceproblems, the additional scarring and the small amount of gained bone in proper direction, especially when vertical augmentation is indicated (Klesper et al. 2002). The alternative use of various particulated bone substitute materials with different membrane and growth factors is possible (Klein et al. 2010; Klein & Al-Nawas 2011; Zhou et al. 2011), but shows high limitations in the clinical outcome. Deproteinized bovine bone substitutes (DBB; Bio Oss, Geistlich Pharma, Wolhusen, Switzerland) show a resistance to resorption following placement into bony defects or as an onlay graft. This may provide long-term preservation of the vertical and interproximal bone height as well as of the corresponding aesthetics (Tarnow et al. 1992; Steigmann 2008). It has also been shown to induce periodontal and peri-implant bone regeneration, especially when used in conjunction with membranes. During bone regeneration by osteo conduction of the DBB-graft, pluripotent cells differentiate into osteoblasts, which can than produce osteocytes (Boyne et al. 1997). The implantation of DBB-blocks may provide additional volume stability (Chris Arts et al. 2006; Por et al. 2007; Schmitt et al. 2011). Although, DBB-block augmentation may be compromised by the block size, which may disable proper angiogenesis as well as migration of osteogenic cells (Avila et al. 2010). All together, in a current systematic review, no superior grafting approach for vertical augmentation could be identified (Klein & Al-Nawas 2011). The most common methods of ridge reconstructions include grafting procedures with coverage of a barrier membrane (Steinhauser & Obwegeser 1967; Buser et al. 1999; Urban et al. 2009; Friedmann et al. 2011). When using resorbable membranes together with an underlying, osteoconductive material, a gain in marginal bone was reported in several studies (Hammerle & Lang 2001; Zitzmann et al. 2001a, 2001b; Strietzel et al. 2006; Friedmann

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et al. 2011). Collagen membranes maintain a temporary barrier and space keeping function under provision of nutrient diffusion for cell proliferation and differentiation; it was furthermore proven that they are supporting an early transmembraneous angiogenesis (Schwarz et al. 2006, 2008a, 2008b). The degradation of those membranes starts shortly after implantation (von Arx et al. 2005). An additional bioactive optimization of DBB as a scaffold for the delivery of growth factors such as platelet-derived growth factor (PDGF) is an interesting option to induce further osteoinduction (Rocchietta et al. 2007; Simion et al. 2007; Schmitt et al. 2011; Stockmann et al. 2011). PDGF – a dimeric glycoprotein composed of either two A- (AA), B- (BB) or a combination of A- and B-chain (AB) – was discovered as a major mitogenic factor present in serum, secreted from the a-granules of platelets activated during the coagulation of blood (Klein et al. 2010). It works by means of angiogenesis and chemotaxis (Risau 1997; Moore et al. 2009; Ka¨mmerer et al. 2011; Nevins & Reynolds 2011). The results of animal studies as well as randomized controlled trials demonstrated the efficacy of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) for regeneration of cranial and ridges defects (Simion et al. 2006; Lynch et al. 2008; Moore et al. 2009; Nevins et al. 2009; Choo et al. 2011). Therefore, protein therapeutics with rhPDGF-BB have a significant potential to treat conditions affecting bone (Becker et al. 1992; Vikjaer et al. 1997). Because angiogenesis was observed to affect bone formation at ridge defects during the initial weeks, the evaluation of early stages of wound healing might be a particular interest for the assessment of the biologic activity of this factor (Jung et al. 2008; Schwarz et al. 2009). However, very little evidence exists regarding the influence of the DBB-rhPDGF-BB complex and DBB alone in combination with resorbable membranes in early healing of vertical bone defects. Also, the question of possible advantages by stabile fixation of alloplastic block materials in combination with growth factors and membrane techniques has not been answered. Therefore, the aim of the present study was to define the sequential healing events and the effects of the membrane with and without addition of rhPDGFBB – with stable, implant-fixed DBB-blocks as a vehicle carrier – that occur during initial stages at vertical bone augmentation in rabbits. The hypothesis is that there is a difference in histological formation of the new bone growth when using a collagen membrane and when using rhPDGF-BB as adjunct.

Materials and methods Study materials

Deproteinized bovine bone blocks (DBB; Bio-Oss®; Geistlich Pharma AG, Wolhusen, Switzerland), recombinant human plateletderived growth factor-BB (rhPDGF-BB; Sigma, St. Louis, MO, USA) as well as resorbable, noncross-linked collagen membranes (Bio-Gide®; Geistlich Pharma AG, Wolhusen, Schweiz) were used for bone augmentation. One titanium dental implant (3.5 9 11.5 mm NobelActive; Nobel Biocare, Zu¨rich, Switzerland) was used in each study site to stabilize bone blocks. Experimental animal model

The study was planned prospectively in accordance to the ARRIVE guidelines (Kilkenny et al. 2010). Twelve, 9 months old, 4 –5 kg, New Zealand white rabbits were used. After approval by the Ethics Committee of the State University of Medicine and Pharmacy (SMPhU) “N. Testemitanu”, Chisinau, Moldova, the surgical part of the project was performed at the SMPhU as well. All animals were treated in accordance with both policies and principles of laboratory animal care and with the European Union guidelines (86/609/ EEC). They were housed in individual cages in an animal room maintained at 22°C and 55% relative humidity with ventilation 18– 20 times/h and a 12-h light–dark cycle. They were allowed free access to diet and water. Adaptation and observation period was 1 week before surgery. The animals were operated under a general anaesthetic by intramuscular injections of a combination of a dose of 35 mg/kg ketamine and a dose of 5 mg/kg xylazine. The experiments were conducted using the tibia model via an anterior transdermal approach on both sides (n = 24). This was the model of choice as this is an accepted model for biomaterial research and technically favourable (Liljensten et al. 2000; Joosten et al. 2005; Calvo-Guirado et al. 2011; Draenert et al. 2011). In all animals, local bone of the proximal tibia was exposed after incision and elevation of the periosteum. The bone was carefully skimmed with a straight fissure carbide bur under copious irrigation with sterile 0.9% physiological saline to remove remaining soft tissue and to lay open fresh bone tissue. For each study site, in the middle of the DBB-block, a hole appropriate to the diameter of the implant (3.5 mm) was drilled and the implant was carefully inserted (Fig. 1). The block was cut into a size of 13.5 9 5 mm with a height of 5 mm and screwed down with the implant to immobilize it on the bone (Fig. 1). © 2012 John Wiley & Sons A/S

Ka¨mmerer et al  Vertical bone augmentation with deproteinized bovine bone

The new formed bone is woven and can be clearly distinguished from the cortical frontier. Statistics

Fig. 1. Schema of the DBB block together with the inserted implant.

The animals were randomly allocated to four groups with two time points of healing according to study design and observation periods (Table 1). DBB was randomly soak-loaded with 0.5 ml rhPDGF-BB (Schwarz et al. 2010) (concentration 1 mg/ml) or animal blood. In both groups, releasing incisions were made to achieve primary closure of the periosteum. To evaluate differences when using the collagen membrane, a randomized split-leg-design was employed: in one tibia, only the periosteum was closed over the block. In the other tibia, the additional collagen membrane was used under the periosteum (Table 1). The mucoperiosteal flaps, muscles, subcutaneous tissue and skin were advanced, repositioned anatomically and fixed via interrupted and mattress sutures with Vicryl 4–0 (Ethicon GmbH, Norderstedt, Germany). Biopsies and histological procedures

The animals were sacrificed 3 and 6 weeks after surgery with an excess dose of Pentobarbitone at 100 mg/kg. Samples were harvested and fixated with 4% paraformaldehyde. The specimens were cut in appropriate bony pieces after immersion fixation for 4 weeks and prepared for histological examination (Wagner et al. 1981; Donath & Breuner 1982). Briefly, all samples were cut down by a commercial water-cooled saw (Exakt, Hamburg, Germany) to a thickness of 5 mm perpendicular to the axis of the placed dental implants. The bone slices were

immediately embedded in PMMA (Technovit 7100; Heraeus Kulzer, Hanau, Germany) and then grinded to a thickness of 30– 50 lm. The specimens were stained with Toluidine Blue and then examined using a Leica DM8000 M microscope (Leica Microsystems, Heidelberg, Germany). For histomorphometrical calculations, all slides were digitalized. Histomorphometry

For histomorphometrical examinations, slides with the implant cut in the middle were used. The following parameter were assessed by two investigators (S. E., K. P. W.), blinded to the manner of augmentations: (1).

Area of newly formed bone at the augmented site (NBA). For this, the relation between the total area of the primary augmentation (2 9 5 9 5 mm) and the area of newly formed bone was evaluated on the left and the right side of the implant for each implant (%; red rectangle in Fig. 2). Total values in all specimens in each group were measured. Visible DBBparticles were not counted as new bone. (2). Newly mineralized bone as a marker for marginal vertical bone height (VBH; in mm) in the augmented area on five arbitrary chosen, equally distributed points at the left and five at the right side of every sample was measured (yellow vertical lines in Fig. 2).

Table 1. Schematical design of the animal experiments (total n = 24)

Group

Procedures

3 Weeks (Group size total n = 12)

Control Test 1 Test 2 Test 3

DBB DBB DBB DBB

3 3 3 3

alone + membrane + rhPDGF-BB + rhPDGF-BB + membrane

© 2012 John Wiley & Sons A/S

(1a) (1b) (2a) (2b)

6 Weeks (Group size total n = 12) 3 3 3 3

(3a) (3b) (4a) (4b)

The nature of this pilot experiment was seen to be explorative, therefore, neither prior sample size calculation nor power analysis was conducted. Due to the pilot character of the study and the small sample size per group – each group consisted of three implants under examination – all values were reported descriptively. For all parameters, total bone values (left + right; NBA, VBH) were examined. The 3- as well as the 6-week results were compared within the groups only. In addition, the dependent values within a rabbit in order to show the effect of the membrane were calculated. The analyses were conducted using SPSS version 20.0 for Mac (IBM, Armonk, NY, USA).

Results The postoperative healing was uneventful in all animals. No complication such as swelling, fracture, infection or allergic reaction was observed within the study period. No premature exposure of the augmented bone was seen. All animals (n = 12; 24 samples) could be included in the descriptive statistical analysis (Table 1). Area of newly formed bone (NBA) at the augmented site

After 3 weeks, in group 1a, a total mean NBA of 20% (single values for the animals (SV): 30.3%; 15.9%; 13.8%) was seen. NBA in group 1b was 16.3% (SV: 24.8%; 16.6%; 7.4%) in group 2a, 35.28% (SV: 39.6%; 32.6%; 33.6%) and in group 2b 15.4% (SV: 0%; 30.3%; 15.9%) respectively. Values are given in Table 2 and Fig. 3. Group 2a showed a constant higher NBA. After 6 weeks, in group 3a, only 2.47% NBA was seen (SV: 2.5%; 0%; 4.9%). In group 3b, the calculated mean NBA was 28.5% (SV: 19.8%; 34.9%; 30.7%), in group 4a 5.3% (SV: 5.1%; 7.5%; 4.4%) and in group 4b 35.54% (SV: 30.9%; 35.3%; 40.4%) respectively. All values are given in Table 2 and Fig. 3. Both membrane groups (3b and 4b) showed a higher NBA than the non-membrane groups (Fig. 4a and b). There was no manifest difference between groups 3b and 4b. Newly mineralized, vertical bone growth (VBH)

After 3 weeks, a mean VBH of 1.1 mm (SV: 1.2 mm; 1.2 mm; 1 mm) was measured in group 1a. A VBH of 0.87 mm (SV: 0.7 mm;

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Ka¨mmerer et al  Vertical bone augmentation with deproteinized bovine bone

0.9 mm; 1 mm) was seen in group 1b, a VBH of 0.88 mm (SV: 0.7 mm; 0.9 mm: 1 mm) in group 2a and a VBH of 0.7 mm (SV: 0 mm; 1.1 mm; 1 mm) in group 2b. Values for both are given in Table 3 and Fig. 5. There were no obvious differences between the groups. After6 weeks, in group 3a, the mean VBH was 0.4 mm (SV: 0.3 mm; 0.3 mm; 0.6 mm). In group 3b, 2.02 mm (1.6 mm; 2.4 mm; 2.1 mm), in group 4a, 0.62 mm (SV: 0.4 mm; 0.7 mm; 0.8 mm) and in group 4b, 1.87 mm (SV: 1.8 mm; 2 mm; 1.8 mm) were calculated (Table 3 and Fig. 5). Group 3b as well as group 4b (membrane groups) had a constant higher mean VBH than the non-membrane groups (Fig. 6a and b). The difference between group 3b and 4b was not evident.

Fig. 2. Histological specimen (toluidine blue, original magnification 920) of a sample from the DBB + rhPDGF-BB group after 3 weeks.

Table 2. Absolute values of mean area of new formed bone (%) after 3 and 6 weeks. Single values for each animal (SV) are given Group Total 3 weeks DBB alone DBB + membrane DBB + rhPDGF-BB DBB + rhPDGF-BB + membrane Total 6 weeks DBB alone DBB + membrane DBB + rhPDGF-BB DBB + rhPDGF-BB + membrane

Area of new formed bone (%)

SV (%)

20 16.3 35.27 15.43

30.3; 15.9; 13.8 24.8; 16.6; 7.4 39.6; 32.6; 33.6 0; 30.3; 15.9

2.47 28.49 5.31 35.544

2.5; 0; 4.9 19.8; 34.9; 30.7 5.1; 7.5; 4.4 30.9; 35.3; 40.4

Membrane effect

After 3 weeks, the membrane had a negative effect on the area of new formed bone (group 1a vs. 1b: mean value 3.7% [SV 5.5%; +0.7%; 6.4%]; group 2a vs. 2b: mean value 19.84% [SV 39.6%; 2.3%; 17.7%]). There was also a slightly negative effect on the VBH (group 1a vs. 1b: mean value 0.27 mm [ 0.5 mm; 0.3 mm; 0 mm]; group 2a vs. 2b: mean value 0.18 mm [ 0.7 mm; +0.2 mm; 0 mm] Tables 4 and 5). After 6 weeks, there was a persistent additional new bone area formation in the membrane group (group 3a vs. 3b: mean value + 26.02 [SV + 17.3; +34.9; +25.8]; group 4a vs. 4b: mean value + 30.23 [SV + 25.8; +27.8; +36]). The same positive effect was seen for the VBH (group 3a vs. 3b: mean value + 1.62 mm [+1.3 mm; +2.1 mm; +1.5 mm]; group 4a vs. 4b: mean value + 1.25 mm [+1.4 mm; +1.4 mm; +1 mm] Tables 4 and 5).

Discussion

Fig. 3. Scatterplots showing the area of new formed bone (%) in the different samples after 3 (black rectangle) and 6 weeks (grey circle).

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The bony vertical augmentation for functional as well as aesthetic reconstruction is a widespread, though critical method as longterm stability is desirable (Rocchietta et al. 2008). Therefore, the histological study aimed to determine the effect of membrane usage as well as of rhPDGF-loaded DBB on early bony healing after vertical augmentation in the rabbit tibia. The animal model used is well established for vertical augmentation and implant examination purposes (Casap et al. 2011; de Macedo et al. 2009; Khojasteh et al. 2011). Although, to the best of our knowledge, this is the first experimental rabbit study reporting on early bone growth for vertical augmentation using membrane procedures in © 2012 John Wiley & Sons A/S

Ka¨mmerer et al  Vertical bone augmentation with deproteinized bovine bone

(b)

(a)

Fig. 4. (a and b) Representative histological specimen (toluidine blue, original magnification 920) of a sample from the DBB + rhPDGF-BB + membrane-group (a) as well as from the DBB-group (b) after 6 weeks.

Table 3. Mean vertical bone height (mm) after 3 and 6 weeks. Single values for each animal (SV) are given Group Total 3 weeks DBB alone DBB + membrane DBB + rhPDGF-BB DBB + rhPDGF-BB + membrane Total 6 weeks DBB alone DBB + membrane DBB + rhPDGF-BB DBB + rhPDGF-BB + membrane

combination with rhPDGF-soaked and implant-fixed DBB-blocks. The tension on the soft tissue covering the defect after intraoral augmentation is similar to the tension on the overlying soft tissue in the tibia model. Schwarz et al. conducted a similar study at chronic-type lateral ridge defects in the dog mandible. Although, it has to be kept

Vertical bone growth (mm)

SV (%)

1.14 0.87 0.88 0.7

1.2; 1.2; 1 0.7; 0.9; 1 0.7; 0.9; 1 0; 1.1; 1

0.4 2.02 0.62 1.87

0.3; 1.6; 0.4; 1.8;

0.3; 0.6 2.4; 2.1 0.7; 0.8 2; 1.8

in mind, that horizontal augmentation cannot be compared to vertical augmentation techniques (Schwarz et al. 2010). Deproteinized bovine bone is a xenogenic bone material with a high degree of biocompatibility; its structure is similar to cancellous bone (Jensen et al. 1996). After slow remodelling over time, incorporation into

Table 4. Effect of the membrane by means of area of new formed bone (mean values as well as single values dependent within an animal) after 3 and 6 weeks. All values are given in % Area of new formed bone (%) Without membrane 3 weeks DBB DBB + PDGF 6 weeks DBB DBB + PDGF

© 2012 John Wiley & Sons A/S

20 (30.3; 15.9; 13.8) 35.27 (39.6; 32.6; 33.6) 2.47 (2.5; 0; 4.9) 5.31 (5.1; 7.5; 4.4)

Membrane effect 3.7 ( 5.5; +0.7; 6.4) 19.84 ( 39.6; 2.3; 17.7) +26.02 (+17.3; +34.9; +25.8) +30.23 (+25.8; +27.8; +36)

native bone has been described (Jensen et al. 1996); this can be supported by the findings of our study. It has been widely discussed, that the high regenerative potential of autologous bone transplants is due to the transfer of several vital cell type (mesenchymal stem cells, osteoblast as well as their precursor cells) and local autologous growth factors (Blokhuis & Arts 2011; Schmitt et al. 2011). Therefore, an additional bio-functionalization of DBB, for example with rhPDGF-BB, is a nearby option. Details about the absorption and the probably uncontrolled release kinetics of rhPDGF in combination with DBB-blocks are still unknown. Bateman et al. examined that the in vitro absorption of PDGF to b-TCP-carriers occurs in a concentration- as well as timedependent manner. The in vitro release was – with a release of approximately 45% after 10 days – slower than the in vivo release (Bateman et al. 2005). Accordingly, the use of rhPDGF-BB soaked DBB seemed reasonable. To see an effect, a comparable high dose of PDGF was chosen in the study. After 3 weeks of healing, histomorphometrical analysis revealed that rhPDGF-BB tends to increase the total area of new bone at the augmented site. Compared to the DBB-only group (mean NBA: 20%), the addition of PDGF-BB nearly doubled this value (mean NBA: 35.27%). The membrane rather prohibited this positive effect (mean NBA: 15.4%). Although, as the differing single values show, this effect cannot be seen as being statistically significant. The early positive effect of rhPDGF-soaked DBB on formation of new bone is in accordance to previous studies (Simion et al. 2006; Schwarz et al. 2010). After 6 weeks, the effect of the membranes on new bone volume as well as on new vertical bone height seems to outperform the PDGF effect. Simion and co-workers observed a beneficial effect of a natural bone mineral with addition of rhPDGF-BB 4 months after vertical ridge augmentation in dogs. In their study, the best effect was achieved without coverage of a collagen barrier membrane. The authors concluded that membrane exclude osteogenic cells derived from the periosteum (Simion et al. 2006, 2007). These findings are in contrast to the results of our study as well as to the current findings regarding early transmembraneous angiogenesis (Schwarz et al. 2006, 2008a, 2008b) as well as reported successful guided bone regeneration techniques (Kim et al. 2010). Schwarz et al. could show that the collagen membrane did not interfere with bone induction by a growth factor (rhBMP-2) (Schwarz et al. 2008a, 2008b).

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Ka¨mmerer et al  Vertical bone augmentation with deproteinized bovine bone

Fig. 5. Scatterplots showing the new vertical bone growth (mm) in the different samples after 3 (black rectangle) and 6 weeks (grey circle).

(a)

(b)

Fig. 6. (a and b) Representative histological specimen (toluidine blue, original magnification 920) of a sample from the DBB + rhPDGF-BB + membrane group (a) as well as from the DBB-group (b) after 6 weeks.

In summary, the separation of the periosteum (providing essential cell resources for rhPDGF mediated bone formation (Schwarz et al. 2010)) did not influence early new bone formation in a negative way. The results rather lead to the hypothesis, that the collagen membrane might only primarily exclude the vascularization as well as the ingrowth of osteoprogenitor cells from the periosteum (Eyre-Brook 1984; Skawina & Gorczyca 1984; Schwarz et al. 2010). The assumed additional

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membrane functions such as stabilization of the blood coagulum and to keep away unwanted soft tissue cells (Zitzmann et al. 2001a, 2001b; Friedmann et al. 2002; Hammerle et al. 2008) did not seem to enhance early bone growth. Therefore, because of the closure-function of the membrane, the early effect of rhPDGF + DBB + membrane on bone formation is not different to those in the non-rhPDGF-group. After 6 weeks, a transmembraneous neo-vasculari-

zation with higher mineralization of new bone and a biodegradation of the respective collagen membrane might have taken place. In addition, the collagen membrane may stabilize particulated bone graft materials at non-self-contained defects (Schwarz et al. 2010) such as before vertical augmentation. Mechanical immobility is needed to achieve biological healing (Hausamen & Neukam 1992). As solid, implant-immobilized DBBblocks were used, this stabilization may have a smaller impact as for DBB granules. Schwarz et al. could show that the collagen membrane degrades after 4–6 weeks of healing in dogs (Schwarz et al. 2008a, 2008b). Accordingly, after 6 weeks, the positive membrane effect was evident. This is in contrast to the results of Rothamel et al., although this group used a cross-linked collagen membrane with long-barrier function compared to the non-cross-linked membrane with resorption after shorter time in our study (Rothamel et al. 2009). It can be considered as a drawback for membrane use that an early exposure of collagen membranes to the oral environment may jeopardize the outcome due to infection or rapid disintegration (De Sanctis et al. 1996; Sela et al. 2003). This complication is still common (Simion et al. 2007). Jensen and Terheyden stated in a review, that the incidence of soft tissue dehiscences was higher for non-resorbable than for resorbable membranes (Jensen & Terheyden 2009). Previous studies could not see a difference between VBH in DBB-blocks or DBB-blocks pre-treated with either BMP or VEGF (Kim et al. 2010; Schmitt et al. 2011). This supports the findings of the present study as, after 6 weeks as no significant differences between the DBB and the DBB + rhPDGF-BBgroup were seen. New VBH was shown to be from the contact area of the inserted material. This points out again the need of direct bone-transplantcontact for successful augmentation (Schmitt et al. 2011) and emphasizes the good value of implant-fixed bone substitutes. Furthermore, an additive effect of the implant surfaces on initial ossification was measured. This is in accordance to prior studies (Rothamel et al. 2008).

Conclusion and clinical relevance The present study indicates that the addition of rhPDGF-BB to DBB-blocks has a potential to enhance early bone formation for vertical augmentation and the use of a membrane in © 2012 John Wiley & Sons A/S

Ka¨mmerer et al  Vertical bone augmentation with deproteinized bovine bone

Table 5. Effect of the membrane by means of area of vertical bone growth (mean values as well as single values dependent within an animal) after 3 and 6 weeks. All values are given in mm Vertical bone growth (mm) Without membrane 3 weeks DBB DBB + PDGF 6 weeks DBB DBB + PDGF

1.14 (1.2; 1.2; 1) 0.88 (0.7; 0.9; 1)

0.27 ( 0.5; 0.3; 0) 0.18 ( 0.7; +0.2; 0)

findings illustrate even more, that after 6 weeks, the usage of a collagen membrane is the key to maximize the new bone height.

Acknowledgements: The study was funded by a grant of the Osteology Foundation (No. 09-095). Nobel Biocare, Zu¨rich, Switzerland, supported the dental implants as well as insertion kits. The authors declare that they have no conflict of interest.

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0.4 (0.3; 0.3; 0.6) 0.62 (0.4; 0.7; 0.8)

this early phase has no benefit or can diminish bone formation, perhaps by reducing the necessary vascular supply. Furthermore, the

Membrane effect

At this time, rhPDGF-BB has no or a very limited additional effect. Accordingly, the animal experiments are giving hints that the additional collagen membrane may enhance the clinical prognosis of vertical augmentation procedures.

+1.62 (+1.3; +2.1; +1.5) +1.25 (+1.4; +1.4; +1)

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