Int. J. Oral Maxillofac. Surg. 2012; 41: 657666 doi:10.1016/j.ijom.2011.11.017, available online at http://www.sciencedirect.

com

Research Paper Biomaterials

Immunohistochemical characterization of wound healing at two different bone graft substitutes

M. Sager, D. Ferrari, M. Wieland, M. Dard, J. Becker, F. Schwarz: Immunohistochemical characterization of wound healing at two different bone graft substitutes. Int. J. Oral Maxillofac. Surg. 2012; 41: 657666. # 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The immunohistochemical characteristics of wound healing following application of a biphasic calcium phosphate or a collagen coated natural bone combined with a native collagen membrane in a dog model was assessed. Standardized buccal dehiscence-type defects were surgically created following implant bed preparation in 6 dogs. Following implant placement, defects were randomly lled with a collagen coated natural bone mineral (GBO), or a biphasic hydroxyapatite/beta tricalcium phosphate (SBC), and covered with a native collagen membrane. After 1, 4, and 9 weeks submerged healing, dissected blocks were processed for immunohistochemical (collagen type I (CI), osteocalcin (OC), angiogenesis (TG)) analysis. At 1 week, GBO and SBC granules were homogeneously surrounded by a well vascularized, non-mineralized tissue (NMT). CI and OC antigen reactivity was commonly observed adjacent to both bone graft substitutes. At 4 and 9 weeks, SBC and GBO granules were completely integrated into a secondly formed network of spongiosa. At 9 weeks, dissolution of some granules was observed in the SBC group. Adjacent to these granules, NMT was signicantly increased and revealed a pronounced CI, OC and TG antigen reactivity. The initial pattern of bone regeneration and graft integration was comparable in both groups; bone remodelling was more pronounced with SBC.

M. Sager2, D. Ferrari1, M. Wieland3, M. Dard4, J. Becker1, F. Schwarz1

1 Department of Oral Surgery, Heinrich Heine sseldorf, Germany; 2Animal University, Du Research Institute, Heinrich Heine University, sseldorf, Germany; 3MyoPowers Medical Du Technologies SA Chemin du Levant 98 1005, Lausanne, Switzerland; 4Department of Periodontology and Implant Dentistry, New York University, College of Dentistry, USA

Key words: guided bone regeneration; immunohistochemistry; animal study; biphasic calcium phosphate; collagen coated natural bone mineral; collagen membrane. Accepted for publication 8 November 2011 Available online 12 December 2011

Xenogenic derived type I and III collagen barrier membranes have been proven to predictably support guided bone regeneration (GBR) at dehisced implant sites3,15,26. Some of the potentially advantageous properties of collagen over synthetic
0901-5027/050657 + 010 $36.00/0

materials are related to: its hemostatic function, allowing early wound stabilization; chemotactic properties to attract broblasts; and semipermeability, facilitating nutritient transfer30,33,34. A major drawback, particularly of native collagen,

is fast biodegradation, resulting in a poor ability to maintain space thus compromising the secluded wound area33,41. Accordingly, native collagen is commonly used in combination with either bone grafts or bone graft substitutes to support the

# 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

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area compared with GBO35. Since there was no difference noted in BF at 24 weeks23, it could be suggested that initial mineralization varied between both types of bone graft substitutes. Conventional histology is not appropriate to further elucidate the complex biological pattern of wound healing during the initial stages of graft integration. A recent experimental animal study investigated the histological and immunohistochemical characteristics of a GBO and SBC supported GBR procedure in dehiscence-type defects at titanium implants5. Immunohistochemical labelling of nondecalcied tissue sections merely involved a qualitative assessment of the osteocalcin (OC) antigen reactivity within the area of newly formed mineralized tissue (MT) at 1, 4 and 9 weeks. Based on this evaluation, a distinctive difference between both groups could not be observed. The aim of the present study was to further elucidate the immunohistochemical characteristics of wound healing in both groups employing a quantitative assessment of angiogenesis, collagen type I (CI) and OC within the complete defect area including NMT and MT compartments.
Material and methods

membrane preserving its original position21,47. Owing to its osteogenic, osteoinductive and osteoconductive properties8, particulate autograft must still be considered to be a gold standard grafting material for the rehabilitation of dental implant-related defects7,25. Some potential drawbacks are related to its quantity at specic donor sites, an increased morbidity and patient discomfort, as well as its potential resorption7,25,27. In order to overcome some of these problems, various bone graft substitutes, ranging from alloplastic to coral, algae, or xenogenic derived materials have been introduced14. The ideal bone graft substitute requires at least some physicochemical properties such as biocompatibility, osteoconductivity, resorbability, and steadiness. The most common source of xenogenous hydroxyapatite (HA) is bovine bone, which has also been studied extensively for bone augmentation in combination with the GBR technique21,47,48. These grafting materials are also referred to as natural bone minerals, since proprietary processes are suggested to remove all cells and proteinaceous material, leaving behind an inert bone scaffold exhibiting a specic three-dimensional surface structure42. Recently, a natural bone mineral was coated with porcine derived collagen to render its surface even more attractive for cell adhesion BioOss Collagen1)35,40. (GBO, Amongst the alloplastic materials, HA and tricalcium phosphate (TCP) were most commonly used for bone regeneration in combination with GBR and well accepted due to lack of possible disease transmission5,16. Porous and non-porous HA materials reveal a high volume stability but are considered to be nonresorbable or slowly resorbable. In contrast, materials consisting of TCP were reported to be bioabsorbable but subsequently lack sufcient steadiness22,23. Recently, a new biphasic calcium phosphate (Straumann Bone Ceramic1; SBC) consisting of 60% HA and 40% b-TCP was introduced as an alternative bone graft substitute, combining the favourable properties of both components. Preliminary experimental animal studies have pointed to an intimate contact between newly formed bone and the remaining graft material1,10,23. After 8 weeks of healing, JENSEN et al.23 reported a signicantly higher amount of bone formation (BF) in defects treated with autografts or b-TCP. Similarly, at 9 weeks, SBC exhibited a signicantly higher amount of non-mineralized tissue (NMT) within the regenerated

Germany). Two animals were killed after 1, 4, and 9 weeks of submerged healing.
Surgical procedure

The animals, used in this experiment, have been described previously35. Briey, 6 beagle dogs (age 2024 months, mean weight 14.8 0.6 kg) exhibiting a fully erupted permanent dentition were used. During the experiment, the dogs were fed once per day with a soft food diet and water. Animal selection, management, and surgery protocol were approved by the Animal Care and Use Committee of the Heinrich Heine University and the Bezirksregierung Du sseldorf. The experimental segment of the study started after an adaptation period of 4 weeks. The study design has been described previously35. In brief, extraction of the mandibular and maxillary second, third and fourth premolars and the rst and second molars (P2M2) was performed bilaterally in all dogs. After a healing period of 4 months, standardized buccal dehiscencetype defects were bilaterally created following implant site preparation in the upper jaws (n = 4 defects per animal). Subsequently, 4 titanium implants were inserted per animal, and the defects randomly assigned to either a GBO or SBC supported GBR procedure. Accordingly, each animal received both types of bone graft substitutes in the upper jaws. Randomization was based on a computer generated list (RandList1, DatInf GmbH, Tu bingen,

The surgical procedure has been described Briey, intramuscular previously35. sedation was performed with 0.17 mg/kg acepromazine (Vetranquil 1%, CevaTiergesundheit, Du sseldorf, Germany), and anaesthesia was initiated using 21.5 mg/ kg thiopental sodium (Trapanal 2.5%, Altana GmbH, Konstanz, Germany). Inhalation anaesthesia for all surgical procedures was oxygen and nitrous oxide and isourane. To maintain hydration, all animals received a constant rate infusion of lactated Ringers solution whilst anaesthetized. For intraoperative analgesia, intravenous injection of 0.4 mg/kg piritramid (Dipidolor1, Janssen-Cilag GmbH, Neuss, Germany) and 4.5 mg/kg carprofene (Rimadyl1, PtzerPharma GmbH, Karlsruhe, Germany) was given. For postoperative analgesia, piritramid and carprofene were applied subcutaneously for 3 days in the same dose as described above. In addition, prophylactic clindamycine (Clerobe1, Pharmacia Tiergesundheit, Erlangen, Germany) (11.0 mg/kg body weight) was administered intra- and postoperatively for 3 days. P2M2 were carefully removed bilaterally in both jaws after reection of mucoperiosteal aps and tooth separation during the rst surgery. Wound closure was achieved using mattress sutures and the sites were allowed to heal for 4 months. In the second surgery, midcrestal incisions were performed to reect mucoperiosteal aps at the respective sites for implant insertion in the upper jaws. Preparation of surgical implant sites was performed bilaterally, at a distance of 20 mm apart, using a low-trauma surgical technique under copious irrigation with sterile 0.9% physiological saline. Standardized buccal dehiscence-type defects (4 mm in height from the crestal bone, 2 mm in depth from the surface of the buccal bone, and 3 mm in width mesiodistally) were created with a straight ssure carbide bur. All osteotomy procedures were performed under copious irrigation with sterile 0.9% physiological saline. A periodontal probe (PCP12, Hu-Friedy Co., Chicago, Illinois, USA) was used to ensure standardization of the defect size. Chemically modied sand-blasted, large grit and acid-etched titanium implants (SLActive1, Standard Plus Implant, Institut Straumann AG, Basel, Switzerland) (diameter 3.3 mm, length 8 mm) were inserted with good primary stability (lack of clinical implant

Wound healing at different bone graft substitutes

mobility) so that the borderline between the bony and transmucosal part of the implant coincided with the bone crest (BTB) (Fig. 1a). According to a split-mouth design, the defects were randomly augmented with either GBO (Geistlich BioOss1 Collagen, Geistlich, Wolhusen, Switzerland) (pore diameters 3001500 mm, particle size 0.251 mm, total porosity 7075%), or SBC (Straumann Bone Ceramic1, Institut Straumann AG, Basel, Switzerland) (pore diameters 100500 mm, particle size 0.5 1 mm, total porosity 90%). Both GBO and SBC particles were moistened in sterile saline for 5 min before placement into the defect. Following grafting, each defect site was covered by a non-cross-linked porcine derived type I and III bilayered collagen membrane (Geistlich BioGide1, Geistlich, Wolhusen, Switzerland) (BG). Each membrane was trimmed and adapted over the entire defect so as to cover 23 mm of the surrounding alveolar bone and to ensure stability of the graft materials. Neither sutures nor pins were used for membrane xation or stabilization (Fig. 1b). Following periosteal-releasing incisions, the mucoperiosteal aps were advanced, repositioned coronally and xed with consecutive as well as vertical or horizontal mattress sutures (Resorba1, Nu rnberg, Germany) to ensure submerged healing conditions (Fig. 1c). All surgical procedures were performed by the same experienced operator.
Retrieval of specimens

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After healing periods of 1, 4, and 9 weeks, 2 animals each were killed by an overdose of sodium pentobarbital 3%. The oral tissues were xed by perfusion with 10% buffered formalin administered through the carotid arteries. Dissected blocks containing the experimental specimens were obtained and xed in 10% neutral buffered formalin solution for 47 days.
Histological preparation

Histological preparation of the tissue biopsies has been described previously35. In brief, dehydration was performed using ascending grades of alcohol and xylene. Tissue specimens were inltrated and embedded in methylmethacrylate (Technovit 9100 NEU, Heraeus Kulzer, Wehrheim, Germany) and prepared for non-decalcied sectioning. During this procedure, any negative inuence of polymerization heat was avoided by controlled polymerization in a cold atmosphere (4 8C). The specimens were completely polymerized

Fig. 1. (a) Standardized buccal dehiscence-type defects (4 mm in height from the crestal bone, 2 mm in depth from the surface of the buccal bone, and 3 mm in width mesio-distally) were bilaterally created in the upper jaws (4 per animal). Particular care was taken that the stabilized blood clot in defect area was preserved during augmentation. (b) Following defect augmentation using either GBO or SBC in a split-mouth design, all experimental sites were completely covered by BG. (c) All defect sites were left to heal in a submerged position.

after 20 h. Each titanium implant was cut in the bucco-oral direction along with its long axis using a diamond wire saw (Exakt1, Apparatebau, Norderstedt, Germany). Serial sections were obtained from the central aspect of each defect site, resulting in 36 sections of approximately 300 mm in thickness each11. All specimens

were glued with acrylic cement (Technovit 7210 VLC, Heraeus Kulzer, Wehrheim, Germany) to silanized glass slides (Super Frost, Menzel GmbH, Braunschweig, Germany) and ground to a nal thickness of approximately 40 mm. One part of the sections was stained with Masson Goldner Trichrome (MG)35, whilst the other part

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was prepared for immunohistochemical labelling.

Immunohistochemical labelling

For immunohistochemistry all tissue section were deplasted in xylol (2 30 min) followed by treatment with 2-methoxyethylacetate (2 20 min) and acetone (2 5 min). After rehydration in phosphate buffered saline (PBS), antigen unmasking was performed by incubating the slides for 15 min in trypsin (PAA Laboratories GmbH, Pasching, Austria) (0.05% in PBS) at 37 8C. After washing with PBS the activity of endogenous peroxidase was quenched with 0.9% hydrogen peroxide in PBS for 10 min at room temperature, the specimens were washed and non-specic binding sites were blocked with a blocking solution for 30 min (Dako Cytomation, Hamburg, Germany). The primary mouse monoclonal antibody to CI (1:60 dilution) (Acris Antibodies GmbH, Hiddenhausen, Germany), OC (1:40 dilution) (Acris Antibodies GmbH), transglutaminase II (TG) (1:40 dilution) (Labvision, Fremont CA, USA), and the corresponding unspecic antibody (mouse IgG1), respectively, as negative control were applied to tissue sections in a humidied chamber and incubated overnight at 8 8C. The crossreactivity of these antibodies with canine tissues has been extensively documented in previous studies35,37,38. The slides were washed in PBS and incubated with secondary biotinylated anti-mouse antibody (1:50 dilution) for 90 min at room temperature. After washing in PBS, the presence of antibody antigen complexes was visualized using a streptavidin-peroxidase solution (1:250 dilution) (Acris Antibodies GmbH) and AEC (3-amino-9-ethylcarbazole) as the chromogen (Acris Antibodies GmbH).
Histomorphometrical analysis

A colour CCD camera (Color View III, Olympus, Hamburg, Germany) was mounted on a binocular light microscope (Olympus BX50, Olympus, Hamburg, Germany) and used for the acquisition of digital images. Tissue sections were evaluated at an original magnication 100 using a software program (analySIS FIVE docu1, Soft Imaging System, Mu nster, Germany). A calibrated and masked examiner identied the augmented area (AA) (mm2) from the bottom of the bone defect (BD) to the most coronal level of the bone graft particles underneath the barrier membrane (Fig. 2a). The antigen reactivity of CI, OC, and TG was automatically

Fig. 2. (a) The augmented area (AA) (mm2) was measured from the bottom of the bone defect (BD) to the most coronal level of the bone graft particles underneath the collagen membrane (CM). Within AA, the area of non-mineralized tissue (NMT) (mm2) was assessed (GBO, 1 week, MG stain, original magnication 12.5). (b) The antigen reactivity of CI, OC, and TG was automatically estimated by the image analysis software as a percentage of NMT. This also included the amount of NMT within any area of newly formed mineralized tissue (MT) (GBO, 1 week, OC stain, original magnication 100).

Wound healing at different bone graft substitutes

estimated by the image analysis software as a percentage of NMT (mm2) within AA (Fig. 2b). This also included the amount of NMT within any area of newly formed MT. Prior to the start of the morphometrical analysis, a calibration procedure was initiated for both the image analysis software as well as the masked examiner and revealed that repeated measurements of 12 different sections were similar at >95% level.
Statistical analysis

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Table 1. Mean antigen reactivity (%) of CI, OC, and TG (SD) within the NMT (mm2 SD) of the augmented area after 1 week of healing (n = 6 animals). NMT SBC GBO P value*
*

CI 5.0 2.6 7.8 4.5 n.s.

OC 24.0 9.6 32.8 8.7 n.s.

TG 18.3 4.3 21.0 4.8 n.s.

3.3 0.6 3.6 0.5 n.s.

Between groups comparison (paired t test).

1 week

Statistical analysis was performed using a commercially available software program (SPSS1 16.0, SPSS Inc., Chicago, IL, USA). Mean values and standard deviations of CI, OC, and TG were calculated for each group in each dog. The data rows were examined with the Kolmogorow Smirnow test for normal distribution. For the statistical evaluation of the changes within groups over time, the paired t test was used. For the comparisons between groups, the unpaired t test was used. P values < 0.05 were considered to be signicant.
Results

Postoperative healing was considered generally uneventful in all dogs. No complications such as a premature exposure of the augmented sites, allergic reactions, abscesses or infections were observed throughout the study period. The results of the histomorphometrical analysis in both groups are presented in Tables 13.

Statistical analysis revealed no signicant differences with respect to mean NMT, CI, OC, and TG values between groups (P > 0.05, respectively; unpaired t test) (Table 1). Within AA, both types of bone graft particles were surrounded by a dense zone of NMT, exhibiting rst signs of CI antigen reactivity. These tiny collagen bres were oriented perpendicular to the graft particles. No differences with respect to bre orientation and structure were observed between groups (Fig. 3a and b). The newly formed NMT commonly revealed pronounced OC antigen reactivity. The signal appeared to be most intense adjacent to the bone graft particles (Fig. 3c and d). Immunohistochemical analysis of TG clearly indicated the formation of vascular-like tissue structures in both groups. This was particularly true for the NMT in the basal compartment of the wound area (Fig. 3e and f).
4 weeks

In both groups, wound healing was mainly characterized by newly formed trabeculae of woven bone, arising from open marrow spaces at BD. This zone of MT invaded the

defect area in coronal and lateral directions to the titanium implant surface as well as the particles of both bone graft substitutes (Fig. 4a). These areas of new BF were demarcated by pronounced OC antigen reactivity (Fig. 4b). Both groups revealed a signicantly decreased area of NMT (P < 0.05, respectively; paired t test), but the difference between groups was not signicant (P > 0.05; unpaired t test). The NMT adjacent to both bone graft substitutes revealed increased amounts of CI, OC, and TG antigen reactivity. Statistical signicance was only reached for CI and TG in the SBC groups (P < 0.05, respectively; paired t test). Whilst the differences between groups were not signicant for OC and TG (P > 0.05, respectively; unpaired t test), the mean amount of CI was signicantly higher in the GBO group (P < 0.05; unpaired t test) (Table 2). For both GBO and SBC particles, orientation and structure of the collagen bres was comparable to that noted after 1 week of healing (Fig. 4c and d). Close correlation was observed between angiogenesis and new BF. In particular, the dense network of blood vessels seemed to be surrounded by the newly formed trabeculae of woven bone. Most of these vessels resembled sinusoidal capillaries exhibiting frequent anastomoses

Table 2. Mean antigen reactivity (%) of CI, OC, and TG (SD) within the NMT (mm2 SD) of the augmented area after 4 weeks of healing (n = 6 animals). NMT SBC GBO P value*
* y

P valuey P < 0.05 P < 0.05

CI 11.3 2.5 18.3 3.0 P < 0.05

P valuey P < 0.05 n.s.

OC 28.0 7.0 34.8 6.5 n.s.

P valuey n.s. n.s.

TG 38.8 7.7 35.8 13.6 n.s.

P valuey P < 0.05 n.s.

2.7 0.7 2.9 0.6 n.s.

Between groups comparison (paired t test). Within groups comparison to 1 week (unpaired t test).

Table 3. Mean antigen reactivity (%) of CI, OC, and TG (SD) within the NMT (mm2 SD) of the augmented area after 9 weeks of healing (n = 6 animals). NMT SBC GBO P value*
* y

P valuey n.s. P < 0.05

CI 11.5 2.1 5.0 2.6 n.s.

P valuey n.s. n.s.

OC 33.5 9.7 18.0 5.5 P < 0.05

P valuey n.s. P < 0.05

TG 44.3 13.4 34.8 8.2 n.s.

P valuey P < 0.05 n.s.

2.6 0.5 1.7 0.5 P < 0.05

Between groups comparison (paired t test). Within groups comparison to 1 week (unpaired t test).

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Fig. 3. Representative immunohistochemical views at 1 week. (a) First signs of positive CI antigen reactivity (arrows) within the newly formed dense NMT adjacent to SBC (original magnication 400). (b) These tiny bres were oriented perpendicular (arrows) to both bone graft particles (GBO, CI stain, original magnication 400). (c) The area of NMT revealed pronounced OC antigen reactivity (arrows) (SBC, original magnication 200). (d) The signal appeared to be more intense adjacent to the bone graft particles (arrows) (GBO, OC stain, original magnication 200). (e) Intense TG stain (arrows) adjacent to a SBC particle (original magnication 400). (f) Both groups commonly revealed the formation of vascular-like tissue structures (arrows) particularly in the basal compartment of the defect area (GBO, TG stain, original magnication 400). BV = blood vessel; BO = Geistlich BioOss1; MT = mineralized tissue; NMT = non-mineralized tissue; SBC = Straumann Bone Ceramic1.

(Fig. 4e). OC antigen reactivity appeared to be more intense within the NMT adjacent to newly formed blood vessels (Fig. 4f).
9 weeks

In both groups, newly formed MT had homogeneously organized the former defect area in coronal and lateral directions. Histological and immunohistochemical analysis revealed that both bone graft substitutes were completely integrated into this secondly formed network of spongiosa. Within this area, the mean amount of NMT was signicantly higher in the SBC

group (P < 0.05; unpaired t test) (Table 3). SBC particles revealed an increased contact with NMT, whilst GBO particles were frequently surrounded by newly formed MT. In general, histological observation failed to demonstrate any osteoclastic activity at the surface of both bone graft particles. Dissolution of SBC was commonly observed in areas where the bone graft particles were surrounded by NMT tissue (Fig. 5a and b). Whilst mean OC antigen reactivity remained unchanged in the SBC group, a signicant decrease was noted in the NMT tissue adjacent to GBO particles (P > 0.05, P < 0.05, respectively; paired t

test). The difference between both groups was statistically signicant (P < 0.05; unpaired t test) (Fig. 5c and d). Similarly, SBC particles revealed a signicant increase of mean TG antigen reactivity within the adjacent NMT (P < 0.05; paired t test). The difference between both groups did not reach statistical signicance (P > 0.05; unpaired t test) (Fig. 5e and f).
Discussion

The present study was designed to characterize wound healing immunohistochemically following application of two

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Fig. 4. Representative histological and immunohistochemical views at 4 weeks. (a) Newly formed trabecular bone, originating from open marrow spaces at BD, had started to invade the defect area in coronal direction (SBC, MG stain, original magnication 200). (b) Bone formation followed both bone graft materials and was demarcated by intense OC antigen reactivity (arrows) (GBO, original magnication 400). (c) The amount of CI (arrows) increased in both groups (SBC, original magnication 400). (d) Orientation and structure of the collagen bres (arrows) were comparable to that noted at 1 week (GBO, CI stain, original magnication 400). (e) Angiogenesis and new bone formation were tightly interconnected. Arrows indicate an intense TG antigen reactivity within a longitudinal sectioned blood vessel (SBC, original magnication 400). (f) OC stain was more intense within the NMT adjacent to newly formed blood vessels (arrows) (GBO, original magnication 400). BV, blood vessel. BV = blood vessel; BO = Geistlich BioOss1; MT = mineralized tissue; NMT = non-mineralized tissue; SBC = Straumann Bone Ceramic1.

different types of bone graft substitutes in combination with a native collagen membrane. Surgical creation of standardized buccal dehiscence-type defects in dogs is a commonly used model to evaluate bone regeneration at titanium implants9,28,36,38,43. Acute-type defects have a tendency to heal spontaneously and thus may have supported bone regeneration in all groups investigated. In general, the present results have indicated that both GBO and SBC revealed comparable osteoconductive properties and supported bone regeneration in coronal and lateral directions. The amount of BF in both groups has been reported previously35.

In brief, mean BF (mm2) in the SBC group increased from 0.03 0.01 at 1 week to 0.8 0.4 at 4 weeks, and to 1.6 0.4 at 9 weeks. In the GBO group, mean BF (mm2) increased from 0.02 0.01 at 1 week to 0.6 0.3 at 4 weeks, and to 1.8 0.5 at 9 weeks. The differences between both groups in terms of new BF were statistically not signicant. After 9 weeks of healing, the newly formed bone in the SBC group revealed a signicantly higher percentage of NMT compared with the GBO group35. This observation is in agreement with the present evaluation, since the area of NMT at 9 weeks was also signicantly higher in the SBC group. When

interpreting the results, one must keep in mind that NMT referred to the complete augmented defect area and was not limited to BF35. Immunohistochemical analysis of wound healing at 1 and 4 weeks revealed comparable characteristics in both groups. In particular, both bone graft substitutes were surrounded by a dense zone of NMT exhibiting comparable amounts of CI, OC and TG antigen reactivities. A signicant difference between groups in favour of GBO augmented sites was only observed for mean CI expression at 4 weeks. Since these values also tended to be higher in the GBO group at 1 week,

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Fig. 5. Representative histological and immunohistochemical views at 9 weeks. (a) SBC particles revealed an increased contact with NMT. In these areas, dissolution (arrows) of the graft material was commonly observed (MG stain, original magnication 400). (b) Both types of bone graft materials were well integrated into a secondly formed network of parallel bered bone. Osteoclastic activity was not observed (GBO, original magnication 400). (c) NMT areas within the newly formed bone adjacent to SBC particles revealed an intense OC antigen reactivity. Arrows indicate a supercial dissolution of the graft particles (original magnication 400). (d) GBO particles were most commonly surrounded by MT. The adjacent NMT was characterized by decrease OC antigen reactivity (arrows) (original magnication 400). (e) Cross- and longitudinally sectioned blood vessels within the NMT adjacent to SBC (TG stain, original magnication 400). (f) Unstained negative control (SBC, original magnication 400). BV = blood vessel; BO = Geistlich BioOss1; MT = mineralized tissue; NMT = non-mineralized tissue; SBC = Straumann Bone Ceramic1.

one might speculate that the specic collagen coating of this bone graft substitute has contributed to these results. In this context, however, it must be emphasized that type I collagen is mainly expressed by osteoblasts and represents a major organic component of the alveolar bone matrix2,20. OC is the most abundant bone-specic non-collagenous protein that is exclusively synthesized by osteoblasts, odontoblasts and hypertrophic chondrocytes4,17,19. All these data taken together with the present results seem to indicate that osteoblastic differentiation within NMT was initiated after 1 week of healing in both groups. The observation

that OC antigen reactivity appeared to be more intense adjacent to both types of bone graft particles is in agreement with recent experimental data. In this study immunohistochemical analysis was limited to a qualitative assessment of OC35. The present evaluation revealed a pronounced proliferation of vascular-like tissue structures at 1 week in both groups. Angiogenesis was assessed by the use of primary mouse monoclonal antibodies to TG, an enzyme that stabilizes the structures of tissues by covalently ligating extracellular matrix molecules. Previous experimental studies provide clear evidence that TG is directly involved in

the process of angiogenesis6,18. The observation that new blood vessel formation may be induced within 1 week of healing corroborates the results of recent animal studies employing the same defect model38,39. When interpreting the present evaluation, it was also observed that mean antigen reactivity of CI, OC, and TG was reduced in the GBO group at 9 weeks. This was particularly noted for OC, reaching statistical signicance compared to the values obtained at 1 week. In contrast, the NMT adjacent to SBC particles revealed a stable CI and OC signal, and even a signicant increase in the mean amount of TG. There might be several

Wound healing at different bone graft substitutes

reasons to explain this difference between both groups. It must be emphasized that OC also seems to be involved in the bone remodelling process and may act via a negative feed back mechanism13. The observation that GBO particles were most commonly embedded in MT at 9 weeks coupled with the lack of graft resorption may explain, at least in part, the reduced OC antigen reactivity in this group. Even though the percentage of osseointegrated particles within the area of newly formed bone has been reported to be comparable for both bone graft substitutes, SBC particles revealed a signicantly higher amount of NMT35. In these areas, histological observation has pointed to a dissolution of the remaining graft material, which was commonly associated with a pronounced OC and TG antigen reactivity. Since OC is incorporated into the bone matrix, it was suggested that OC fragments may even be released during bone resorption29. Osteogenic cells have also been observed to arise from pericytes adjacent to small blood vessels in connective tissue24,31,32. These ndings might explain, at least in part, the more intense OC antigen reactivity adjacent to SBC particles after 9 weeks of healing. An initial dissolution of SBC was also observed after 8 weeks of healing in the mandible of mini pigs23. Histological observation revealed the presence of multinucleated giant cells on the surface of particles that were not covered by MT. Even though there were no signs of any cell-mediated resorption lacunae, these surfaces revealed a higher penetration of the staining agent, thus pointing to an initial dissolution of the graft material23. In these areas, dissolved biphasic CaP apatite crystals might improve the osteoconductive properties of the grafting material due to a reprecipitation of a biological apatite layer46. In this context, one must also realize that the turnover rate of bone remodelling in dogs has been reported to be approximately four times faster than the human turnover rate12. Whilst resorption of b-TCP was observed after 12 weeks in dogs, it took about 68 months when implanted in humans44,45. In conclusion, the present study has indicated that the initial pattern of bone regeneration and graft integration was comparable in both groups. Bone remodelling appeared to be more pronounced with SBC. For future studies, it might be hypothesized that localized ridge augmentation procedures employing SBC as a bone graft substitute may generate a more reactive and thus favourable biological environment to support the osseointegration of titanium implants.
6.

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Competing interests

The authors F. Schwarz, M. Sager, D. Ferrari, B. Hartig and J. Becker declare that they have no conict of interests related to this study. M. Wieland was and M. Dard is an employee of Institut Straumann AG, Basel, Switzerland, the manufacturer of one of the tested bone graft substitutes (Straumann Bone Ceramic1).
Funding

7.

8.

The study materials were kindly provided by Geistlich Biomaterials, Wolhusen Switzerland, and Institut Straumann AG, Basel, Switzerland.
Ethical approval

9.

Animal selection, management, and surgery protocol were approved by the Animal Care and Use Committee of the Heinrich Heine University and the Bezirksregierung Du sseldorf.
References
1. Artzi Z, Weinreb M, Carmeli G, Lev-Dor R, Dard M, Nemcovsky CE. Histomorphometric assessment of bone formation in sinus augmentation utilizing a combination of autogenous and hydroxyapatite/biphasic tricalcium phosphate graft materials: at 6 and 9 months in humans. Clin Oral Implants Res 2008;19:68692. 2. Becker J, Schuppan D, Rabanus JP, Rauch R, Niechoy U, Gelderblom HR. Immunoelectron microscopic localization of collagens type I, V, VI and of procollagen type III in human periodontal ligament and cementum. J Histochem Cytochem 1991;39:10310. 3. Becker J, Al-Nawas B, Klein MO, Schliephake H, Terheyden H, Schwarz F. Use of a new cross-linked collagen membrane for the treatment of dehiscence-type defects at titanium implants. A prospective randomized controlled double blinded clinical multicenter study. Clin Oral Implants Res 2009; 20:7429. 4. Boivin G, Morel G, Lian JB, Anthoine-Terrier C, Dubois PM, Meunier PJ. Localization of endogenous osteocalcin in neonatal rat bone and its absence in articular cartilage: effect of warfarin treatment. Virchows Arch A Pathol Anat Histopathol 1990;417:505 12. 5. Brunel G, Brocard D, Duffort JF, Jacquet E, Justumus P, Simonet T, Benque E. Bioabsorbable materials for guided bone regeneration prior to implant placement and 7-year

10.

11.

12.

13.

14.

15.

16.

follow-up: report of 14 cases. J Periodontol 2001;72:25764. Buemi M, Galeano M, Sturiale A, Ientile R, Crisafulli C, Parisi A, Catania M, Calapai G, Impala P, Aloisi C, Squadrito F, Altavilla D, Bitto A, Tuccari G, Frisina N. Recombinant human erythropoietin stimulates angiogenesis and healing of ischemic skin wounds. Shock 2004;22:16973. Chiapasco M, Zaniboni M, Boisco M. Augmentation procedures for the rehabilitation of decient edentulous ridges with oral implants. Clin Oral Implants Res 2006;17:13659. Chiriac G, Herten M, Schwarz F, Rothamel D, Becker J. Autogenous bone chips: inuence of a new piezoelectric device (Piezosurgery) on chip morphology, cell viability and differentiation. J Clin Periodontol 2005;32:9949. Cho KS, Choi SH, Han KH, Chai JK, UM, Kim CK. Alveolar bone forWikesjo mation at dental implant dehiscence defects following guided bone regeneration and xenogeneic freeze-dried demineralized bone matrix. Clin Oral Implants Res 1998;9:419 28. Cordaro L, Bosshardt DD, Palattella P, Rao W, Serino G, Chiapasco M. Maxillary sinus grafting with Bio-Oss or Straumann Bone Ceramic: histomorphometric results from a randomized controlled multicenter clinical trial. Clin Oral Implants Res 2008;19:796 803. Donath K. The diagnostic value of the new method for the study of undecalcied bones and teeth with attached soft tissue (SageSchliff (sawing and grinding) technique). Pathol Res Pract 1985;179:6313. Draper HH. Bone loss in animals. In: Draper HH, editor. Advances in Nutritional Research. New York: Plenum Press; 1994. p. 5371. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G. Increased bone formation in osteocalcin-decient mice. Nature 1996; 382:44852. Esposito M, Grusovin MG, Coulthard P, Worthington HV. The efcacy 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. Friedmann A, Strietzel FP, Maretzki B, Pitaru S, Bernimoulin JP. Histological assessment of augmented jaw bone utilizing a new collagen barrier membrane compared to a standard barrier membrane to protect a granular bone substitute material. Clin Oral Implants Res 2002;13:58794. Fugazzotto PA. Success and failure rates of osseointegrated implants in function in regenerated bone for 72 to 133 months. Int J Oral Maxillofac Implants 2005;20:7783.

666

Sager et al.
of bone matrix proteins by osteoclast cathepsins. Int J Biochem 1993;25:54550. Postlethwaite AE, Seyer JM, Kang AH. Chemotactic attraction of human broblasts to type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci U S A 1978;75:8715. Reilly TM, Seldes R, Luchetti W, Brighton CT. Similarities in the phenotypic expression of pericytes and bone cells. Clin Orthop 1998:95103. Rickard DJ, Kassem M, Hefferan TE, Sarkar G, Spelsberg TC, Riggs BL. Isolation and characterization of osteoblast precursor cells from human bone marrow. J Bone Miner Res 1996;11:31224. Rothamel D, Schwarz F, Sager M, Herten M, Sculean A, Becker J. Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat. Clin Oral Implants Res 2005;16:36978. Schwarz F, Rothamel D, Herten M, Sager M, Becker J. Angiogenesis pattern of native and cross-linked collagen membranes: an immunohistochemical study in the rat. Clin Oral Implants Res 2006;17:4039. Schwarz F, Herten M, Ferrari D, Wieland M, Schmitz L, Engelhardt E, Becker J. Guided bone regeneration at dehiscence-type defects using biphasic hydroxyapatite + beta tricalcium phosphate (Bone Ceramic) or a collagen-coated natural bone mineral (BioOss Collagen): an immunohistochemical study in dogs. Int J Oral Maxillofac Surg 2007;36:1198206. Schwarz F, Herten M, Sager M, Wieland M, Dard M, Becker J. Bone regeneration in dehiscence-type defects at chemically modied (SLActive) and conventional SLA titanium implants: a pilot study in dogs. J Clin Periodontol 2007;34:7886. Schwarz F, Jepsen S, Herten M, Aoki A, Sculean A, Becker J. Immunohistochemical characterization of periodontal wound healing following nonsurgical treatment with uorescence controlled Er:YAG laser radiation in dogs. Lasers Surg Med 2007;39:428 40. steSchwarz F, Rothamel D, Herten M, Wu feld M, Sager M, Ferrari D, Becker J. Immunohistochemical characterization of guided bone regeneration at a dehiscence-type defect using different barrier membranes: an experimental study in dogs. Clin Oral Implants Res 2008;19:40215. Schwarz F, Sager M, Ferrari D, Herten M, Wieland M, Becker J. Bone regeneration in dehiscence-type defects at non-submerged and submerged chemically modied (SLActive) and conventional SLA titanium implants: an immunohistochemical study in dogs. J Clin Periodontol 2008;35:6475. 40. Sculean A, Chiantella GC, Windisch P, Arweiler NB, Brecx M, Gera I. Healing of intra-bony defects following treatment with a composite bovine-derived xenograft (Bio-Oss Collagen) in combination with a collagen membrane (Bio-Gide PERIO). J Clin Periodontol 2005;32:7204. 41. Sela MN, Kohavi D, Krausz E, Steinberg D, Rosen G. Enzymatic degradation of collagen-guided tissue regeneration membranes by periodontal bacteria. Clin Oral Implants Res 2003;14:2638. 42. Spector M. Anorganic bovine bone and ceramic analogs of bone mineral as implants to facilitate bone regeneration. Clin Plast Surg 1994;21:43744. 43. Stentz WC, Mealey BL, Gunsolley JC, Waldrop TC. Effects of guided bone regeneration around commercially pure titanium and hydroxyapatite-coated dental implants II. Histologic analysis. J Periodontol 1997; 68:93349. 44. Suba Z, Takacs D, Gyulai-Gaal S, Kovacs K. Facilitation of beta-tricalcium phosphateinduced alveolar bone regeneration by platelet-rich plasma in beagle dogs: a histologic and histomorphometric study. Int J Oral Maxillofac Implants 2004;19:8328. 45. Wiltfang J, Schlegel KA, Schultze-Mosgau S, Nkenke E, Zimmermann R, Kessler P. Sinus oor augmentation with beta-tricalciumphosphate (beta-TCP): does plateletrich plasma promote its osseous integration and degradation? Clin Oral Implants Res 2003;14:2138. 46. Yamada S, Heymann D, Bouler JM, Daculsi G. Osteoclastic resorption of biphasic calcium phosphate ceramic in vitro. J Biomed Mater Res 1997;37:34652. 47. Zitzmann NU, Naef R, Scharer P. Resorbable versus nonresorbable membranes in combination with Bio-Oss for guided bone regeneration. Int J Oral Maxillofac Implants 1997;12:84452. 48. Zitzmann NU, Scharer P, Marinello CP, Schupbach P, Berglundh T. Alveolar ridge augmentation with Bio-Oss: a histologic study in humans. Int J Periodontics Restorative Dent 2001;21:28895.

17. Gallop PM, Lian JB, Hauschka PV. Carboxylated calcium-binding proteins and vitamin K. N Engl J Med 1980;302:14606. 18. Haroon ZA, Hettasch JM, Lai TS, Dewhirst MW, Greenberg CS. Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis. FASEB J 1999;13:178795. 19. Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol Rev 1989;69:9901047. 20. Helder MN, Bronckers AL, Woltgens JH. Dissimilar expression patterns for the extracellular matrix proteins osteopontin (OPN) and collagen type I in dental tissues and alveolar bone of the neonatal rat. Matrix 1993;13:41525. 21. Hockers T, Abensur D, Valentini P, Legrand mmerle CH. The combined use of R, Ha bioresorbable membranes and xenografts or autografts in the treatment of bone defects around implants. A study in beagle dogs. Clin Oral Implants Res 1999;10:48798. 22. Jensen SS, Broggini N, Hjorting-Hansen E, Schenk R, Buser D. Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res 2006;17:23743. 23. Jensen SS, Yeo A, Dard M, Hunziker E, Schenk R, Buser D. Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res 2007;18:75260. 24. Long MW, Robinson JA, Ashcraft EA, Mann KG. Regulation of human bone marrowderived osteoprogenitor cells by osteogenic growth factors. J Clin Invest 1995;95:8817. 25. McAllister BS, Haghighat K. Bone augmentation techniques. J Periodontol 2007;78: 8817. 26. Moses O, Pitaru S, Artzi Z, Nemcovsky CE. Healing of dehiscence-type defects in implants placed together with different barrier membranes: a comparative clinical study. Clin Oral Implants Res 2005;16: 2109. 27. Nkenke E, Schultze-Mosgau S, RadespielTroger M, Kloss F, Neukam FW. Morbidity of harvesting of chin grafts: a prospective study. Clin Oral Implants Res 2001;12:495 502. 28. Oh TJ, Meraw SJ, Lee EJ, Giannobile WV, Wang HL. Comparative analysis of collagen membranes for the treatment of implant dehiscence defects. Clin Oral Implants Res 2003;14:8090. 29. Page AE, Hayman AR, Andersson LM, Chambers TJ, Warburton MJ. Degradation

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

Address: Frank Schwarz Department of Oral Surgery Westdeutsche Kieferklinik Heinrich Heine University sseldorf D-40225 Du Germany Tel: +49 211 8118149 Fax: +49 211 1713542 E-mail: Frank.Schwarz@med.uni-duesseldorf.de