bone substitutes used for three-dimensional augmentation

original article

Bone substitutes used for three-dimensional augmentation 1

Abstract The study assessed the use of a new xenograft material. The aim of the present in vivo experiment was to obtain information on the clinical results and the rate of bone formation using two new experimental bone substitutes compared with an established bovine bone substitute and the autogenous graft, which is considered the gold standard for grafting. In the study miniature pigs were used as an animal model as they represent the highest similarities to human bone regarding anat-omy, morphology and remodelling and as a species are considered to be a close representation of human bone considering the process of bone formation. In a surgi-cal procedure the second premolars of 20 animals were extracted in all 4 quadrants and defects created which were filled with the different materials. After 30 days the first group was sacrificed and the second group after 60 days. To evaluate the rate of new bone formation sections were ground down for toluidine blue staining. Further-more, the technique of fluorochrome sequential label-ling was used to obtain information about the dynamics of new bone formation and remodelling processes in the loaded area at different times. A greater part of the de-fects loaded with the experimental blocks could not be integrated in the statistical analysis because the animals either lost the grafts a few weeks after transplantation or the healing was unsuccesssful. Histological examination revealed about 50% lower evidence of bone induction of the experimental blocks compared to the bovine ref-erence material. Fluorescence microscopy did not offer any significant differences between the bone biopsies.

The results of this study demonstrate that the use of the experimental blocks was less successful consider-ing healing and amount of new bone formation com-pared to the established bovine bone substitutes grafts. Nevertheless, the study showed that the combination of fluorescence polarization and histological analysis is an efficient method to obtain information about bone re-modelling and fibrous reactions on top of the basal bone by bone grafting experiments.

Keywords: Bone regeneration, Bone transplantation, Swine, Materials testing, Dental research

Introduction

When I was asked to contribute to a special edition of the International Journal of Stomatology and Occlusion Medicine in honor of Karl Donath I was proud and happy to fulfill this aim. Additionally I reflected on my perso-nal relationship to Karl, or should I say to the Donaths, as Karl was only part of the whole and the package is only complete with Erika. I have known Karl since the 1980s and started working and learning from him at the time I did my doctoral thesis in dentistry. Nevertheless, the rela-tionship over the years changed from a teacher-student relation to a godfather and son relation. At the beginning of his illness we had a special situation: he could not go to give a lecture on bone substitutes on a major congress in Vienna so he asked me if I would be willing to jump in as substitute. I felt honored and said yes without being clear about the things to come. When I prepared the lecture I found out that although we have been working so closely for ages both of us still had our own specific attitude to the topic, his from the perspective of an oral pathologist and mine from the perspective of a clinician. So I tried to change my perspective and to come from his side, which gave me a really hard time. But on the other hand the pre-

J. Stomat. Occ. Med.DOI 10.1007/s12548-011-0022-7

Bone substitutes used for three-dimensional augmentation

A technique revisited

Hendrik Döring, Christian Schmitt, Rainer Lutz, Stephan Eitner, Karl Andreas Schlegel

H. Döring () · C. Schmitt · R. Lutz · K. A. SchlegelDepartment of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glückstr. 11, 91054 Erlangen, Germanye-mail: [email protected]

S. EitnerDepartment of Prosthodontics, University of Erlangen-Nuremberg, Glückstr. 11, 91054 Erlangen, Germany

Received: 27 September 2011 / Accepted: 6 October 2011© Springer-Verlag 2011

2 Bone substitutes used for three-dimensional augmentation

original article

paration for this lecture gave me the opportunity to once again relocate my way of looking and to give the whole thing a more, maybe we could call it biological perspec-tive of things. To return back to our topic, one of our first papers together was a publication about non-resorbable bone substitutes and their behavior on incorporation [1]. The conclusions we had then are more or less still stand-ing and also the problems using bone substitutes for three dimensional defects. The search for an appropriate three-dimensional bone substitute material is still ongo-ing as it has to fulfill a number of criteria in order to be clinically successful. The list of criteria which should be fulfilled has already been described by Schweiberer and has been continuously added to over the past decades [2].

The material should

• not interfere with the physiological healing process• not act or promote inflammatory reactions• should ideally be osteopromotive• should be easy to handle clinically• should provide a sufficient implant bed• should not include any biological hazards

In the search for this material animal research studies were carried out using artificial defects in miniature pigs and observing them at 2 time points, 30 days in order to obtain information on the early stage of wound healing and at 60 days to see the osseointegration of the different materials. In this study two new block materials of bovine origin as well as autogenous bone and Bio-Oss® block material (Geistlich, Wolhusen, Switzerland) were used.

The following questions should be answered by this study:

• How good is the clinical applicability, e.g. handling of the tested material?

• What findings can be observed clinically?• Are there differences comparing the form and stability

of the materials?• How is the biocompability of the materials to be rated?• How is histologically the fusion and the osseointegra-

tion of the materials always in comparison to the auto-genous block.

Material and methods

The miniature pig was the animal of choice for this expe-riment as the bone regeneration rate (1.2–1.5 μm/day) is comparable to that of man (1.0–1.5 μm/day; [3, 4]). A total of 6 bone defects were created in the forehead area of 18 adult female pigs (12 months old)1. The size of the defects created conformed to the definition of a critical size defect [5] and the defects were positioned at least 1 cm

1 The study was approved by the local animal committee of the government of Middle Frankonia, Ansbach, Germany.

apart to avoid biological interactions between the defects [5–7]. Ketamine hcl (5 mg/kgBW; tavet®, Ratiopharm, Ulm, Germany) was used as the intravenous anesthetic. Additionally, a local anesthetic (6 ml Ultracain-DS forte™, Hoechst, Frankfurt a. M., Germany) was applied. Initi-ally the second premolars in the upper and lower jaws of 20 miniature pigs were extracted, the buccal wall of the cavity was removed using a rotating burr and the bone substitutes were applied to the created defect situation which had a size of 1 × 0.5 × 1 cm (Fig. 2). For the auto-genous group the bone was harvested interforaminally from the lower jaw. In total four different groups were placed according to random to the defect: experimental blocks I and II, Bio-Oss® block and the autogenous block (Figs. 1, 3). All blocks were fixed to the defect using an osteosynthesis screw. Finally, the augmented area was covered by an individualized Bio-Gide® membrane, the flap repositioned and sutured using resorbable sutures (Vicryls® 3.0, Ethico, Norderstedt, Deutschland).

The animals were postoperatively treated with syste-mic antibiotics for 3 days, (Streptomycin™ 0.5 g/kgBW/day, Grünenthal, Stolberg, Germany).

After 30 and 60 days 10 animals each were sacrificed and evaluated and with this procedure 16 specimens of each group were obtained for each observation period. The bone samples were embedded in Technovit 9100™ (Heraeus/Kulzer, Werheim, Germany) by way of prepa-ring for the bone samples to be cut into thin sections [8] and subjected to microradiographic examination and light microscopy [9]. In each observation period at least 5 sections per sample were used for light microscopic eva-luation. For statistical purposes the Wilcoxon rank sum test with a significance level of 95% was used.

Microradiography

For the microradiographic examination the embed-ded bone samples were cut into thin sections of 180 μm thickness using a precision saw and a grinding machine (Exakt Gerätebau, Norderstedt, Germany). Finally, X-ray images of the samples were made in a Faxitron X-ray for 6 h at a tube voltage of 11 kV (Faxitron, Rohde and Schwarz, Köln, Germany; [9]). Quantitative image ana-lysis of microradiography was performed using the Bio-quant image analysis system (Bioquant Image Analysis Corporation, Nashville, TN). Attention was paid to select only the grafted area. The relationship between minera-lized and non-mineralized soft tissues in the defect was determined (Figs. 4, 5).

Light microscopy

The samples used for microradiography were reduced as described by Donath and Breuner (Microsaw, Exakt Gerätebau) to 20–30 µm and stained in toluidine blue-O [8]. Subsequently, the specimens were morphologically evaluated by means of light microscopy (Figs. 4, 5).

original article


The microradiography and toluidine blue-O stained preparations were evaluated by two investigators and for each sample a minimum, maximum and average was specified. With the Mann and Whitney U test the measu-red values were compared between two groups. To com-

pare the results quantitatively, p-values were calculated and a p-value < 0.05 was designated as significant. Due to the small sample size the non-parametric-specific Wil-coxon rank sum test with a significance level of 0.05 was

Fig. 1 Schematic drawing of the critical size defect for the left and right sides (blue Bio-Oss® block, red experimental block I, brown experimental block II, white = autologous block)

Fig. 2 Preparation of the critical size defectFig. 3 Implantation of the bone substitute material

Fig. 4 Direct comparisson of Toluidine Blue-O staining (5x) and corresponding microradiographic images (5x) for Bio-Oss® Block (left) and Experimental Block II (right)


original article

chosen (Axum 7, Math Software, Cambridge, MA; SPSS 12.0.2, SPSS ., Chicago, IL).

Clinical results

The use and application of the tested materials was simple and uneventful but even in the healing period in 13 animals the tested materials showed some healing or inflammatory problems. These problems led to a total or partial loss of the block materials. Finally at 30 days there were only 4 experimental blocks I and 1 experimental block II left for observation. At 60 days only 2 experimen-tal blocks I and 5 experimental blocks II could be evalua-ted (Table 1).

Light microcopy

At 30 days

The autogenous and conventional Bio-Oss block groups showed a de novo bone formation in the defect margins as well as signs of fusion in the autogenous group. In the test groups the limited number of specimens available made it difficult to draw clear conclusions but on the information gained it can be stated that the de novo bone formation was limited as numerous multinucleated giant cells could be seen. The pictures made under polarized light confirmed the de novo bone formation on the Bio-Oss blocks whereas the test blocks only exhibited a high number of collagenous fibers without signs of minerali-zation, showing a fibrous tissue reaction (Fig. 6).

At 60 days

Autogenous and conventional Bio-Oss® block groups both showed a good bony incorporation. In the test groups complete exfoliation of the material or chronic inflammation was sometimes observed. On a close look no real enhancement of de novo bone formation could be seen in the two test groups whereas the two chosen control groups, autogenous and Bio-Oss® block showed the same good results on a histological basis as they exhi-bited clinically (Fig. 6).

Fig. 5 Direct comparisson of Toluidine Blue-O staining (5x) and corresponding microradiographic images (5x) for Bio-Oss® Block (left) and Experimental Block I (right)

Table 1 Block samples available for further processing in this study

Experi-mental block I

Experi-mental block II

Bio-Oss® block

Sacrificed after 30 days

Samples with inflammatory processes

4 6 6

Samples without inflammatory processes

3 1 1

Sacrificed after 60 days

Samples with inflammatory processes

2 3 1

Samples without inflammatory processes

4 4 4

original article


Discussion

Studies on bone healing and bone restructuring require an animal model that allows the transfer of the results to humans. With the chosen model this demand could be fulfilled [10–12]. The clinical results achieved in this study are in accordance with what can be found in the literature [13–15]. The use of bone substitutes in implan-tology is limited to cases which provide so-called spa-ce-providing defects. This means that the defects are suitable for the placement of particulate material. The use of block material needed for the augmentation of so-called non-space providing defects is still the domain of the autogenous transplant [16–18]. In comparison to the results of a prior study using blocks of a bovine sub-stitute material in an animal model [19] similar de novo bone formation rates could not be detected in the test materials neither by means of microradiographical nor light microscopic examination. The histological exami-nation showed particles of bone augmentation material enclosed in the newly formed bone which corresponds with the results of Schlegel and Donath in a study on the resorption of xenogenous hydroxylapatites [1]. The histological analysis of the experimental blocks 1 and 2 show significantly less bony development in the defect area compared to the Bio-Oss® group. The assessment of the histology showed that for the most part of the expe-rimental blocks they were surrounded by a connective tissue layer separating the bone substitute material and the local bone. This collagen capsule, which enclosed the inserted material, was noticeably thicker than it was for the Bio-Oss® blocks. Newly formed bone could be, if ever displayed, almost exclusively found in the transitional

zone between bone substitute material and local bone. The bovine reference material showed a good integrity which could also be demonstrated by Carmagnola et al. [20]. In particular, after 60 days, in the defect edges and circular around the Bio-Oss® body, a higher proportion of newly formed bone was evident. In the microradiogra-phic representation bone replacement material and less mineralized bone could easily be differentiated. Such structures could be found only sporadically or not at all on the substitute experimental blocks, if they were not already prematurely exfoliated.

Furthermore, fluorescence labelling microscopy sho-wed that reossification of the defects filled with the bone substitute material only took place locally. In contrast, the reossification of the defects that were filled with auto-logous bone was found simultaneously all over the defect area. As expected, the bone substitute materials had only osteoconductive potential. The autologous bone block confirmed its standing as the gold standard in this study. Both qualitatively and quantitatively it was able to achieve the highest rates of bone formation which is consistent with other studies [21, 22]. Even after 60 days in consideration of the microradiograpic distinction of transplanted autologous bone block and local bone a clear difference could no longer be made. Besides the already mentioned characteristics of osteointegration, osteoconduction, osteoinduction and osteogenesis it still has angiogenic capabilities with associated vascu-larization [23] which allows a rapid revitalization of the graft and its cells. The consolidation of the bony defects followed the principles of primary angiogenous reos-sification. Progression of the bone formation from the peripheral areas to the central region was found. In this

Fig. 6 Box-and-whisker plot showing the amount of de novo bone formation in the region of interest with the different blocks after 30 and 60 days

80,00

60,00

40,00

20,00

0,00

Bone

postoperative day 30 postoperative day 60

Bio-Oss® Block

Exp. Block IExp. Block IIAutolog. Block


original article

process the bone substitute materials serve as a guide rail for the formation of new bone trabeculae. The results of the Bio-Oss® material also reaffirmed their clinically widespread and largely complication-free use. The resorption stability and osteoinductive properties make it a popular bone substitute in transplant surgery. Thus the results found in the autogenous and Bio-Oss® groups could not be reconfirmed in the two test groups.

The data obtained in this study demonstrated that healing of experimental blocks I and II showed sufficient mechanical stability over a longer period and sufficient recoverability to cellular exhibits. With respect to the per-sistence of the introduced, non-exfoliated experimental blocks and the Bio-Oss® augmentations, no significant differences were found. The absorbability is a crucial parameter for the success of a bone substitute. If resorp-tion takes place too fast this would put the integrity in the reconstruction phase at risk [24]. However, the primary aim remains to set up a bone substitute as a guide rail so that bone tissue can fill the spaces [25, 26]. Studies on the absent biodegradability of the bovine bone sub-stitute material have already been conducted [1, 27, 28]. After the investigation period of 60 days, no evidence of absorption could be determined in the area of the Bio-Oss® blocks. Also in the border areas of the experimen-tal blocks I and II no resorption lacunes could be found. Particularly through the combination of autologous bone with non-resorbable bone substitute, atrophy and resorption of the grafts can be prevented. However, the desired osseous integration was not achieved and the new block material showed significantly worse values in all points compared to the bovine reference material. Furthermore, there was absent evidence for the onset of collagen synthesis and proliferation. To concrete state-ments regarding the foreign body reaction, interaction with the surrounding tissue and osseointegration, furt-her studies have to be set up with different composition and porosity of the experimental block material.

Conclusion

In this study it was concluded that the intense global ambition over the past two decades have not yet produ-ced a block bone substitute material suitable for use in defects of the oral cavity on all premises initially stated. Even if in some published case reports xenogenous block materials “work” [15, 29] in the oral cavity the histologi-cal evidence as well as the predictability in the sense of guided bone regeneration is still lacking available data. It has to be concluded that for the non-space providing model autogenous bone still remains the state of the art for the time being.

Conflict of interest The authors declare that there is no current or potential conflict of interest in relation to this article.

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bone substitutes used for three-dimensional augmentation

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