The aim of the present study was to evaluate histologically vertical bone regeneration outcomes after using bovine bone graft material in block and granular forms. The buccal bony plates of the outer mandibles of 10 New Zealand rabbits received Bio-Oss blocks that were immobilized using orthopedic mini-plates, and another 10 received granular forms that were gently packed and stabilized into the custom-made perforated metallic cubes. The mean graft area (GA), new bone area (NBA), bone-to-graft contact (BGC), and maximum vertical height reached by the new bone development (MVH) were histometrically evaluated and showed no significant differences between 2 graft types. The new bone was observed mostly close to the basal bone and developed penetrating the trabecular scaffold in the form of seams that covered the intralumen surfaces of the block type graft, while in the granular graft type the new bone was observed to grow between the graft particles usually interconnecting them. Either form of Bio-Oss was capable of providing considerable vertical bone augmentation.

Introduction

The presence of a sufficient volume of healthy bone at recipient sites is a major prerequisite for the success of dental osseointegrated implants.1  Vertical bone loss after tooth extraction can compromise oral rehabilitation with dental implants.2  Vertical augmentation of the residual alveolar bone can be achieved using guided bone regeneration, distraction osteogenesis, and fixation of onlay bone grafts.3  Although autologous onlay block-grafting techniques may provide an initial adequate vertical increase of the residual ridge height, subsequent graft resorption often ensues, compromising the ultimate results.4,5  In addition, the morbidity associated with intraoral autograft harvesting and the hospitalization needed to harvest material from extraoral sites has led to the use of bone-graft substitutes.68 

Deproteinized bovine bone mineral (Bio-Oss, Geistlich Pharma AG, Wolhusen, Switzerland) in granular form has been widely used as a bone-graft substitute in implant dentistry. Numerous experimental animal studies indicate that it may be incorporated into the bone tissue and that intimate contact will be established between the biomaterial and newly formed bone.912  In a number of studies, granular Bio-Oss has been suggested for vertical bone augmentation in deficient residual ridges in conjunction with stabilized rigid reinforced membranes13,14,31–33 or titanium mesh.15,16  Bio-Oss graft in block form has also been proposed for vertical augmentation of deficient residual ridges.3,7,1719,30  The need for membrane placement over the block graft to facilitate bone healing is controversial. A recent animal study by Zecha et al20  showed no additional bone in-growth after the application of biodegradable membranes over onlay block grafts. Similarly, no significant differences were found in sites treated with xenograft block material treated with and without collagen membranes.7  Findings in agreement with the previous studies were recorded after the application of nonresorbable barrier membranes on corticocancellous human block grafts.21 

The aim of the present study was to evaluate histologically and histomorphometrically vertical bone-regeneration outcomes in rabbit mandibles after using bovine bone graft (Bio-Oss) in block form without a membrane and in granular form covered by a customized metallic membrane.

Materials and Methods

Twenty male New Zealand rabbits weighing between 3 and 4 kg were separated into 2 groups. Ten animals, assigned to group A, received block-form grafts, and the remaining 10 animals (group B) received granular-form grafts in conjunction with custom-made perforated metallic chambers. The surgical protocol was approved by the responsible Animal Committee of the University of Thessaloniki (protocols 119 and 237).

Surgical protocol

The animals were preanesthetized with an intramuscular injection of diazepam (1–2 mg/kg Stedon 10 mg, Adelco, Chromatourgia Athinon, Athens, Greece). After 10 minutes, the animals were anesthetized with an intramuscular injection of ketamine (35 mg/kg Imalgene 1000, Merial, Lyon, France) and xylazine (5 mg/kg Rompun, Bayer AG, Leverkusen, Germany). The surgical area was shaved and disinfected using povidone iodine solution 10% (Betadine 10%, Mundipharma, GmbH, Germany). In addition, 1.8 mL of a local anesthetic (2% Xylocaine/epinephrine 1:80 000, Dentsply, Sankin, Tokyo, Japan) was used to enhance the anesthetic result.

A horizontal 2- to 3-cm long incision was then made to the buccal aspect of the mandible. The primary incision included the skin and subcutaneous layer, which were lifted using a pair of tweezers. A second incision followed and was extended deeply through the muscles to the periosteum. The buccal bone of the outer mandible was exposed using a mucoperiosteal elevator. The bony surface was flattened using a round number 2 carbide bur under copious irrigation with sterile saline solution. Then, with a drill, screw holes were made for the self-drilling screws used to immobilize the plates and metallic cubes. For the animals in group A, cubic form block grafts (Bio-Oss spongiosa block, Geistlich Pharma AG) were shaped using a scalpel blade to 4 × 4 × 4 mm dimensions.

The blocks were placed in direct contact with the host-bone surface (no holes in the basal bone were created) and immobilized using orthopedic mini-plates (Lorenz plating system, Biomet, Jacksonville, Fla) (Figure 1a). The plates were Π-shaped and matched the block dimensions; they were stabilized using screws (self-drilling screws, W Lorenz Surgical Inc, Biomet Microfixation Anticipate, Innovate) that were fixed in place using a specific hand screwdriver (Standard Handle and X-lock standard driver, Lorenz plating system, Biomet). For the animals in group B, the particulate bone graft material (Bio-Oss Particulate, Geistlich Pharma AG) was first irrigated with sterile saline solution and then gently packed into the custom-made perforated metallic cubes (4 × 4 × 4 mm internally). These were secured over the flattened lateral mandible region with the same screws used to immobilize the orthopedic plates of group A (Figure 1b).

Figures 1–3.

Figure 1. (a) The block-form bone graft was placed in direct contact with the bone surface and was immobilized using Π-shaped orthopedic mini-plates and bone screws. (b) The particulate-form bone graft was in the custom-made perforated metallic cubes secured with bone screws. Figure 2. Histologic samples of group A. General view of the new bone regeneration. It was derived from the basal native bone (a) and encountered mostly close to the basal bone as well as in the middle of the augmentation area (b) in the form of flowing patterns of new bone covering the walls of the lumens within the scaffold of Bio-Oss block. (Original magnification ×10 and stained with Sanderson's rapid bone stain [RBS].) Figure 3. (a) In this magnification of rectangle A in Figure 2a, new bone derived from the basal native bone and extending coronally within the framework of the block graft can be seen. (b) Magnification of the rectangle in Figure 2b. The current view is located in the middle region. New woven bone can be seen in close contact with the intra-lumen spaces of the Bio-Oss scaffold. (Original magnification ×40 and stained with Sanderson's RBS.)

Figures 1–3.

Figure 1. (a) The block-form bone graft was placed in direct contact with the bone surface and was immobilized using Π-shaped orthopedic mini-plates and bone screws. (b) The particulate-form bone graft was in the custom-made perforated metallic cubes secured with bone screws. Figure 2. Histologic samples of group A. General view of the new bone regeneration. It was derived from the basal native bone (a) and encountered mostly close to the basal bone as well as in the middle of the augmentation area (b) in the form of flowing patterns of new bone covering the walls of the lumens within the scaffold of Bio-Oss block. (Original magnification ×10 and stained with Sanderson's rapid bone stain [RBS].) Figure 3. (a) In this magnification of rectangle A in Figure 2a, new bone derived from the basal native bone and extending coronally within the framework of the block graft can be seen. (b) Magnification of the rectangle in Figure 2b. The current view is located in the middle region. New woven bone can be seen in close contact with the intra-lumen spaces of the Bio-Oss scaffold. (Original magnification ×40 and stained with Sanderson's RBS.)

The incision was then sutured in layers. The muscles and periosteum were sutured using resorbable Vicryl sutures (Ethicon Inc, Johnson & Johnson, Somerville, NJ), and the skin was sutured using nonresorbable silk sutures (Medipac, Kilkis, Greece). Subcutaneous injection with the antibiotic enrofloxacin (5 mg/kg Baytril 5%, Bayer Hellas AG, Athens, Greece) was administered to the animals for 3 days postoperatively. The animals were left to recover from the anesthesia in a warm and quiet location and kept for 2 months in separate cages to heal. During this period, the cages were cleaned every 3 days, and the animals were fed ad libitum.

After a 2-month healing period, the animals were anesthetized and humanely killed using intravenous injection of 10% sodium chloride (DEMO SA, Athens, Greece). The augmentation sites were retrieved from the mandibles using a mechanical microsaw. The stabilization plates were unscrewed and removed from the block-graft samples, while the metallic chambers from the group B animals were left in place during the histologic preparation procedures.

Histologic preparation

All samples were immersed in formaldehyde solution successively (in 30% for 1 hour and in 10% for 2 days) and prepared for nondecalcified histologic sections as described by Veis et al.22  The procedure included dehydration of the samples in ascending grades of alcohol (50%–100%) for 6 days and successive immersion in ascending series of resin solution (50%–100%) (Technovit 7200, Heraeus Kulzer GmbH, Wehrheim, Germany). The final 100% resin with the embedded specimens was polymerized for 12 hours under 430 nm light. A high-precision microtome (Accutom II, Struers, Copenhagen, Denmark) and grinding device (DAP-V, Struers) were used to obtain 50- to 80-μm thin sections glued on glass slides using a light-cure resin (3M ceramic primer and Kulzer Technovit 7210 VLC adhesive). Two central sections were taken from each specimen and stained with Sanderson's rapid bone stain and trichrome stain.

The histomorphometric evaluation was made using a light microscope (Axiostar Plus, Carl Zeiss, Göttingen, Germany). The resulting images were digitized using a digital camera (AxioCam ICc3, Carl Zeiss, Jena, Germany) and a frame grabber, and analyzed by the use of appropriate computer software (AxioVision v4.6.3, Carl Zeiss). In each specimen, the percentages of the new bone area (NBA), graft area (GA), bone-to-graft contact (BGC), and maximum vertical height of new bone (MVH) were measured and analyzed statistically. The MVH was expressed as the percentage of maximum coronal extent of new bone and the total vertical length of the augmentation area. The Mann-Whitney U test was used for BGC and NBA, where the values did not follow normal distribution, and the independent samples t test was used for GA and MVH with normal distribution values. The statistical level of significance was set to .05; the statistical analyses were made using the SPSS 12.0 software (SPSS Inc, Chicago, Ill).

Results

All surgical sites healed uneventfully without exhibiting any kind of dehiscence.

Histologic evaluation

Both the block and granular forms of Bio-Oss were integrated with the host bone. They maintained their original dimensions, and no signs of osteolysis or necrosis within the augmentation area were observed. New bone regeneration could be found in all histologic samples. It was encountered mostly close to the basal bone as well as in the middle of the augmentation area. Different bone-development patterns were observed in the 2 groups.

In group A (block form; Figure 2), the new bone derived from the basal native bone (Figure 3a: magnification of rectangle A in Figure 2a) and was developed by penetrating the trabecular scaffold in the form of seams that covered the intralumen surfaces of the graft (Figure 3b: magnification of rectangle in Figure 2b). In group B (granular form; Figure 4), the new bone was developed as rods and plates interspersed among the graft particles. Osteoblast layers in high activity were developed around the new bone bridges. Osteoid apposition could be seen as reddish layers in contact either with the newly formed bone or directly with the graft material (Figure 5a: magnification of Figure 4a, rectangle). In the specimens from group B, the new bone apposition interconnected the graft particles, as compared with the “flowing” pattern of new bone development on the walls of the lumens within the scaffold of Bio-Oss block (group A). However, it was not rare even in the group A specimens to observe a “bridging” effect by new bone growth within the graft spaces (Figure 6b, arrows). Generally, the closer the proximity to the basal bone, the higher the graft coverage was by new bone. This was notable in both groups.

Figures 4–6.

Figure 4. Histologic samples of group B, which used the granular form of Bio-Oss bone graft. New bone was developed as rods and plates among the interspersed graft particles. (Original magnification ×10. Trichrome stain on the left and Sanderson's rapid bone stain [RBS] on the right.) Figure 5. (a) Magnification of group B samples. This magnification of the rectangle in the middle region of Figure 4b reveals new bone growth interconnecting the graft particles. Osteoblast layers in high activity were developed around the new bone bridges (black arrows). Osteoid apposition can be seen as reddish layers in contact either with the newly formed bone or directly with the graft material. (Original magnification ×40, trichrome stain left.) (b) Slender spongiosa with large size osteocytes characterize woven bone in the early maturation stage. (Original magnification ×40, Sanderson's RBS.) Figure 6. (a) In histologic samples from group A (block form bone graft), a “bridging” effect of the new bone growth could be seen within the graft trabeculae. A clear bridge consisting of composite new bone was developed interconnecting 2 proximal edges of the graft scaffold. (b) Woven bone with numerous large size osteocytes in early development stage can be seen among proximal graft trabeculae. (Original magnification ×40, stained with Sanderson's RBS.)

Figures 4–6.

Figure 4. Histologic samples of group B, which used the granular form of Bio-Oss bone graft. New bone was developed as rods and plates among the interspersed graft particles. (Original magnification ×10. Trichrome stain on the left and Sanderson's rapid bone stain [RBS] on the right.) Figure 5. (a) Magnification of group B samples. This magnification of the rectangle in the middle region of Figure 4b reveals new bone growth interconnecting the graft particles. Osteoblast layers in high activity were developed around the new bone bridges (black arrows). Osteoid apposition can be seen as reddish layers in contact either with the newly formed bone or directly with the graft material. (Original magnification ×40, trichrome stain left.) (b) Slender spongiosa with large size osteocytes characterize woven bone in the early maturation stage. (Original magnification ×40, Sanderson's RBS.) Figure 6. (a) In histologic samples from group A (block form bone graft), a “bridging” effect of the new bone growth could be seen within the graft trabeculae. A clear bridge consisting of composite new bone was developed interconnecting 2 proximal edges of the graft scaffold. (b) Woven bone with numerous large size osteocytes in early development stage can be seen among proximal graft trabeculae. (Original magnification ×40, stained with Sanderson's RBS.)

The new bone patterns developed in various maturation stages. In the early stages, it was found as primary slender spongiosa with large osteocytes (Figure 5b, arrows; Figure 6a, arrows) and as woven bone with osteoblasts, osteoclasts, and osteons in the active remodeling phases. In some areas in both groups, the trabecular bridges consisted of woven bone in the center with lamellar development at the periphery, resembling composite bone and revealing the tendency for early maturation of the newly formed bone (Figure 6b, from group A).

New bone growth was not evident only in the space within the grafted area. In both groups, bony projections were observed starting from the basal bone laterally to the seating borders of the block graft and climbing up to the middle of the block height (Figure 2b [arrows]; Figure 4b [arrows]). In some specimens from group B, these bony projections invaded the augmentation area through the lateral holes of the metallic chambers, sometimes in close contact with the metallic walls (Figure 4b [arrows]). The basal host bone appeared to have a tendency to incorporate the block graft and/or the metallic chamber.

The coronal part of both graft forms was invaded by fibrovascular connective tissue. Although no signs of inflammation were observed, new bone formation in this area was either slender or nonexistent. Only in 2 samples from group A was new bone formation observed in the coronal region (Figure 7: magnification of Figure 2a, rectangle C). In a few samples from group B, new bone was observed in the coronal region, but interestingly, it was in contact with the walls of the metallic membrane rather than around the proximal graft particles (Figure 2b, arrowheads).

Figure 7.

Magnification of rectangle C in the coronal region of Figure 2a. A rare observation of new bone growth at the coronal region of a block form graft consisting of composite bone (ie, areas of lamellar and woven bone in close contact with the trabeculae walls of the block graft framework). (Original magnification ×40, stained with Sanderson's rapid bone stain.)

Figure 7.

Magnification of rectangle C in the coronal region of Figure 2a. A rare observation of new bone growth at the coronal region of a block form graft consisting of composite bone (ie, areas of lamellar and woven bone in close contact with the trabeculae walls of the block graft framework). (Original magnification ×40, stained with Sanderson's rapid bone stain.)

Histomorphometric analysis

The mean GA in group A was 37.08% ± 12.96%. It was relatively lower but not statistically significant in group B: 31.74% ± 6.26% (P = .212; >.05). The mean NBA in group A was 9.68% ± 6.22%, whereas in group B the mean NBA was lower, 5.71% ± 2.83%. The difference was marginally not statistically significant (Mann-Whitney U test P = .065; >.05). Following the same pattern, the mean BGC in group A was 35.13% ± 28.12%, while in group B it was slightly higher (39.22% ± 22.79%) but still without a statistically significant difference (Mann-Whitney U test P = .525; >.05). The last measurement was the mean value of the maximum vertical height reached by the MVH. In group A, it was 78.78% ± 13.28%. It was slightly higher in group B (83.22% ± 9.28%). This difference was not statistically significant (P = .353; >.05).

Discussion

Rabbit tibia22  and calvaria21,23  have been used as experimental models in numerous studies for testing biomaterials and surgical techniques. However, to approximate conditions in the oral cavity, an experimental rabbit mandible model was used in the present study. A similar experimental model was used in another recent study.8  Although surgical access to the lateral mandible is not as convenient as it is to the tibia and calvaria, after the cortical surface was flattened, it presented a beneficial base for stabilizing materials for further evaluation of the vertical bone augmentation outcomes. In the present study, the surgical area was covered by the periosteum, muscle layers, and skin, and despite the relative functional pressure during mastication, all surgical sites healed uneventfully. The instability of the particulate grafting material limits its use for vertical bone augmentation; it requires protection by biological barriers or the presence of bone walls.24  A solid metallic chamber thus was used in this study to achieve both accommodation of the particulate material and comparable dimensions with the block grafts. The block grafts were stabilized using Π-shaped orthopedic plates instead of placing screws in small size block grafts. The brittle consistency of the block grafts has been shown to compromise stability when bone screws are used.3,6 

Standard parameters (NBA, GA, BGC, and MVH) were used to assess the augmentation outcomes and differences between the 2 graft forms. The experimental protocols were not similar, and no studies were found using the protocol employed in the present study. Thus, the results of the present study were compared with other studies in which the 2 Bio-Oss graft forms or other bone grafts were used for vertical or lateral bone augmentation without the immediate placement of dental implants.

Comparison with studies using block type grafts

Schwarz et al25  compared similar Bio-Oss graft forms in dogs either alone or in conjunction with rhBMP-2 and rhPDGF-5 growth factors; however, the authors attempted lateral instead of vertical bone augmentation, and their measurements were reported in absolute mm2 instead of percentages. Their results nonetheless agree with the present study in that comparable osteogenic outcomes were found for the 2 graft forms. As in the present study, they found trabeculae of woven bone deriving from open marrow spaces of the adjacent alveolar basal bone, but no obvious differences were reported with respect to the bone-regeneration patterns for the 2 graft forms. In contrast, the present study clearly showed that new bone development interconnected the interspersed granular graft particles, as compared with the flowing pattern of new bone covering the walls of the lumens and resembling a seam within the scaffold of Bio-Oss block graft.

Kim et al21  used 4-mm high Bio-Oss collagen block graft stabilized with bone screws for vertical bone augmentation in rabbit calvaria. They found lower values for the vertical bone gain (1.88 mm) and percentage of new bone fill (4.89%), as compared with the present study where the maximum gain in height was about 3 mm and the percentage of new bone fill was 9.68% ± 6.22% in group A. However, the 2 studies differed in the region augmented (calvaria instead of mandible), the graft stabilization method (screws instead of plates), and the consistency of the block grafts (Bio-Oss collagen instead of cancellous Bio-Oss alone).

Araújo et al18  compared Bio-Oss block grafts with similar autologous bone blocks for lateral ridge augmentation in dogs. The blocks were cylinder-shaped, 8 mm in diameter and 3 mm thick. Their observations agreed with those in the present study in that the dimensions of the Bio-Oss block graft remained unchanged, and the coronal region was filled only with small amounts of spots of new bone and basically connective tissue mentioning that the major quantity of new bone was observed close to the basal host bone. In addition, they found higher new bone development (23%) within the graft framework, but this difference can be attributed to the higher observation period (6 months), the standardized defects, the location where the blocks were placed, and the use of dogs instead of rabbits as experimental animals.

In agreement with the present study were the results published recently by Schmitt et al26  who tested vertical bone augmentation in sheep using Bio-Oss block type grafts alone or in combination with specific growth factors. They found 10.02% ± 5.43 new bone formation in Bio-Oss alone group as compared to 9.68% ± 6.22% in the present study, and the interesting point was the addition of growth factors had no any promotional effect in new bone formation.

Comparison with studies using granular type grafts

The coverage of a membrane is normally needed to contain granular form Bio-Oss or other granular bone grafts27  in clinical studies.13,15,16  However, in animal studies, metallic cylinders, chambers, domes or other rigid materials were used as membranes to accommodate particulate bone grafts instead of membranes.23,28,29  Torres et al23  used metallic cylinders 4 mm in height in rabbit calvarias to evaluate vertical bone augmentation with particulate anorganic bovine bone (ABB) alone or in combination with platelet-rich plasma. Their findings for the ABB-only group were similar to those of the present study. In a recent study, Dung and Tu28  used caps to cover granular alloplastic HA in rabbit calvaria and found lower mean vertical new bone gain (from 75% to 50%) in comparison to the mean 83.22% ± 9.28% of relative measurement found in the present study indicating the higher osteogenic capability of Bio-Oss bone graft. Another similar research by Zigdon et al29  using gold domes secured on rat calvaria showed different results presenting the Bio-Oss collagen being significantly less osteogenic as compared either with β-TCP or the granular Bio-Oss in the present study. It is interesting to notice that in 2 studies both block21  and granular29  types of collagen Bio-Oss present lower osteogenic capability as compared to normal Bio-Oss graft types evaluated in the present study.

As in the previous studies, this study used a specific cribriform metallic cube-shaped chamber as a membrane to accommodate the particulate graft. On the contrary, the block-form bone grafts were placed without membrane coverage since its use is controversial. It has been claimed that suturing of the thin mucoperiosteal flap on top of the membrane over the block graft may compromise the vascular supply development both into the graft mass and in the tissue flaps, preventing them from attaching to the underlying bone graft during healing.30,31  Evaluation of bone-augmentation outcomes after the use of the same block graft in conjunction with a membrane presents a topic for further research.

Conclusions

Within the limits of this study, the use of both block and particulate deproteinized bovine bone mineral graft resulted in considerable vertical bone augmentation outcomes. The mean values of GA and NBA were slightly higher in the block-form group, while the mean values for the MVH and BGC were slightly higher in the particulate group. However, no statistically significant differences were found between the 2 groups.

Abbreviations

     
  • ABB

    anorganic bovine bone

  •  
  • BGC

    bone-to-graft contact

  •  
  • GA

    graft area

  •  
  • MVH

    maximum vertical height reached by the new bone development

  •  
  • NBA

    new bone area

Acknowledgments

The study was self-supported, but Arriani Pharmaceuticals SA provided materials (Bio-Oss block and granular grafting material and straight plate 4-hole Biomet Microfixation Anticipate, Innovate) that were used in the study.

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