Abstract

Bone defects that cannot be healed completely are termed critical-sized defects and can be used to test bone grafts for medicine, dentistry, and periodontology. The aim of the present study was to detect the effects of a xenograft (Unilab Surgibone) on bone building in experimentally created parietal bone defects in rats. Standardized parietal bone defects were created in 16 rats, and each defect had a circular morphology 6 mm in diameter. The right defect sites were filled with porous particle material, and the left site was used as control. After the 3rd, 6th, and 12th months, rats were killed and tissue samples obtained from the related site of the cranium. Subsequently, histological sections were taken and stained with different stains for evaluation under light microscope. The rate of bone formation was assessed using a semiquantitative method. These results showed that dense collagenous tissue was observed in the control area during the third month, whereas xenograft particles were surrounded by a fibrous tissue layer at the implantation site. Osteoclast-like cells were also observed. There was also no significant bone repair at other observation periods. It can be concluded that the material used had no evidence of resorption and does not enhance bone formation. However, it seems biocompatible, osteoconductive, and could be used in a limited manner as a material for filling osseous defects in clinical practice.

Introduction

A significant number of investigations are currently being performed to elicit an ideal material to support bone repair or regeneration in defects formed for various reasons.13 In the experimental arena, it is important to test various biomaterials and transplants by locotypical tissues, which are expected to be involved in clinical applications in the future. From this point of view, bone defect models play a crucial role in the experimental testing of biomaterials.4 The application of natural materials such as allografts and xenografts seems to be a valid alternative to autografts.5,6 From this point of view, for a natural material to be suitable for application it must biocompatible, noninfectious, and nonantigenic.7,8 In addition, the material should not inhibit the normal activity of bone cells and the normal bone remodeling process.7 Xenografts are very well known and are generally derived from animal origins. They act as scaffolds for the ingrowth of osteoblasts and ensure space for new bone formation.9,10 Moreover, they are highly attractive because they carry minimal risk of contamination from infectious disease and do not compromise the patient's remaining tissues.11 In addition, because their physicochemical properties are similar to those of human bone, these natural biomaterials show great osteoconductive characteristics.12 Xenografts are very slowly resorbed.12,13 

A critical size bone defect can be defined as the smallest bone wound that does not heal without any osteopromotive material during the lifetime of an animal.14 These defects are suitable for testing biomaterials.15 One of these xenografts is Unilab Surgibone, which has been tested in previous investigations and reported as osteoconductive.16,17 

In the present study, we aimed to investigate the long-term effects of Unilab Surgibone on the bone-healing response in experimentally created parietal bone defects in rats.

Materials and Methods

This investigation was conducted on 16 Wistar Albino rats weighing 260 to 340 g. Prior to the operations, permission was obtained from the local ethical committee of the University of Cumhuriyet for Experiment Animals.

Materials

Unilab Surgibone (Missisauga, Ontario, Canada) is an extracellular composite matrix of hydroxyapatite and collagen of bovine bone. The chemical structure is similar to hydroxyapatite (Ca3[PO4]2Ca[OH]2) but contains 29% protein (collagen) and a few pores. The particular form (mean granular diameter between 600 and 800 µ) used in the present study is commercially available. Sterilization was made by ethylene oxide.

Surgical procedures

Anesthesia was induced with intramuscular injection (40 mg/kg) of tiletamine-zolazepam (Zoletil 100, Virbac, Carros, France). Before surgery, the dorsal part of the cranium was clipped free of fur and disinfected with povidone iodine. A 20-mm incision was made along the sagittal suture, and the skin, musculature, and periosteum were dissected to expose the parietal bones. Two identical, symmetrical, 6-mm-diameter, full-thickness critical-sized defects were created using a trephine under saline irrigation. Care was taken to avoid damage to the dura mater. The created experimental defects were filled with the test material, except for the control defects, which were left unfilled. After implantation of test material, the periosteum and muscles were sutured. Ten days after the operation, the sutures were removed and the implanted site was evaluated for signs of local intolerance (inflammation, necrosis, hemorrhage, exudate).

Histological analysis

Rats were killed by intravenous injection of an overdose of sodium pentobarbital a 3, 6, and 12 months after treatment. Blocks were removed from the related site of the cranium, which included the defects and normal bone. Specimens were fixed in 10% buffered neutral formalin for 24 hours and decalcified in a formic acid–hydrochloride acid combination for 24 hours. After rinsing with tap water, the specimens were dehydrated in increasing concentrations of ethanol and embedded in paraffin, and sections of different thicknesses were prepared in the transverse plane and stained with hematoxylin-eosin, Mallory azan, and Masson thrichrom. After these procedures, the specimens were evaluated under a light microscope (Jenamed 2, Carl Zeiss, Jena, Germany) for histological evaluation regarding the bone-healing response. Bone formation was classified according to a previously developed classification.18 None or minimal bone healing with fibrous tissue interposition was graded 0, partial bone healing with occasional fibrous tissue ingrowth was graded 1, and complete bone healing bridging the defect was graded 2. The Fisher exact test was used to analyze the data, and statistical significance was considered as P < .05.

Results

In all specimens, minimal inflammation was detected. On the other hand, there was no necrosis or tumor formation in the implantation and control regions (Figures 1 through 10). The defect area in the control group was filled with fibrous tissue in the 3rd, 6th, and 12th months. There was no callus formation (Figures 1, 4, and 7).

Dense collagen fiber accumulation was evident in connective tissue surrounding the xenograft residues in the experimental group in the third month. In some areas, bone matrix was found to be synthesized, and although osteocytes were localized in the matrix, osteoblasts were seen in the surrounding region of the matrix (Figures 2 and 3). While many xenograft residues were observed in the third month, their amounts were decreased by the 6th and 12th months (Figures 2, 5, and 8). The bone matrix was found to be increased in connective tissue surrounding the xenograft in the 6th and 12th months (Figures 6, 9, and 10). A small number of osteoclast-like cells were evident in the implantation area throughout the experimental process. However, no inflammation, no necrosis, and no tumorigenesis were evident (Figure 9). There were no statistically significant differences in bone-healing scores for the different observation periods (P > .05) (Tables 1 through 3).

Discussion

The long-term results of biomaterials have additional and valuable contributions to the literature concerning their fate and bone repair. Therefore, in this study, the long-term effect of Unilab was evaluated in critical-sized defects in rat cranium within different observation periods. Critical-sized defects are important for testing the biomaterials on bone-healing response. A 6-mm critical-sized defect was selected,18 but some studies do not accept this defect size as a critical-sized defect.19 Indeed, such a designed study can contribute additional information to the literature with regard to clinical situations.

The impact of different materials on bone building has been studied using very different experimental designs. Various models, animal types, species, anatomical regions, sizes of the bone defect, functional implications, surgical conditions, evaluation periods, and assessment methods and criteria have been employed. This diversity of experimental designs makes it difficult to compare the outcomes evaluated in different studies. From this point of view, a consistent and standardized testing model is desirable to provide a suitable assessment and comparison among the different materials and approaches used for bone building.20 

The current literature shows that xenografts have been well investigated in some experimental21,22 and clinical17 studies or in combination with mine matrix proteins.23 The material tested in the present study was previously evaluated experimentally16 and clinically.17 Generally, the results of research have shown that xenografts are biocompatible and osteoconductive. However, some investigations have revealed their poor and negative effects.17 

In 2009, Develioglu et al24 were the first to report the early bone-healing results of Unilab Surgibone in rat cranial defects. The short-term results of Unilab Surgibone were evaluated in 5-mm critical-sized defects in rats. No adverse effects or material resorption were seen, and osteoconductive properties were accentuated. In contrast to the previous study, we detected resorption of biomaterial and minimal bone building in some specimens in the present study. These results confirm the significance of long-term observation outcomes of this biomaterial.

In the present study, 3-, 6-, and 12-month results were observed and used for assessing the fate of the material. At 6 and 12 months, minimal material resorption and bone building were observed. This revealed that the material used has slowly resorbable properties. Although in some specimens we observed resorption of the material and bone-building areas, the material cannot be classified as a promising material that has a great effect on bone formation.

Generally, an ideal bone substitute material should be osteoinductive, osteoconductive, and remain in the body only as long as necessary to change the defect by newly formed bone.25 In addition, the resorption rate of the material should be known before application. Thus, research on a biomaterial's resorbability has been predominantly performed on animal models, a practice that presents limitations such as ethical concerns, high costs, and limited transferability to humans.26 With regard to resorbability, various factors have been considered. One of these factors is porosity and pore size. Previous investigations have shown that the optimal pore size is 200–400 µm. This allows maximal bone ingrowth, but it is poorly understood whether this pore size would be optimal for osteoclastic development and activity.27 Slowly resorbing or nonresorbable bone substitutes may have advantages in bone regeneration because of their ability to maintain augmented tissue volume over the long term.28,29 Our findings, with the exception of some specimens, revealed that this material shows evidence of resorption and minimal bone building. Particle size and chemical composition could have played a role in slowly resorbing of the material used in this study.

Conclusion

Experimentally testing materials before clinical use is very important; critical-sized defects fulfill this necessity. This study confirms the biocompatibility and osteoconductivity of the present material. Long-term results are useful for better understanding the biomaterial and recipient bone interaction, as well as the fate of the material. In this way, the resorbability and bone-building effects can be explained more accurately. Briefly, Unilab Surgibone has shown minimal evidence of resorption and minimal bone formation in only some specimens. Therefore, it may be considered for limited application in various defects confronted in dental and periodontal fields. Further research should be conducted with regard to its benefit in clinical practice.

Acknowledgments

The authors thank Dr Laurent Dupoirieux for his valuable help in editing the article.

Note

The study was presented as a poster at Europerio 6, Stockholm, Sweden, 2009.

References

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Author notes

1

Department of Periodontology, Faculty of Dentistry, Cumhuriyet University, Sivas, Turkey

2

Department of Histology and Embryology, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey

3

Department of Periodontology, Faculty of Dentistry, Gazi University, Ankara, Turkey