Assessment of Bone Healing in Rabbit Calvaria Grafted with Three Different Biomaterials

Abstracts

This study evaluated the bone regeneration process in rabbit calvaria induced by three types of biomaterials: two xenogenous, consisting of deproteinized bovine bone, while the other was alloplastic, based on biphasic calcium phosphate. Five New Zealand white rabbits weighing between 2,900 and 3,500 g were submitted to four standard 8 mm-diameter perforations at the parietal bone. Three perforations were filled with three grafts and biomaterials, two of them received bovine Bio-Oss^(r) and Endobon^(r) Xenograft Granules, and the other consisted of fully alloplastic Straumann^(r) Bone Ceramic. The fourth remaining cavity was used as control with coagulum. After eight weeks, the animals were sacrificed, and the samples were prepared for morphometric and qualitative analysis. The cavities filled with alloplastic biomaterials showed higher percentages of newly formed bone (p<0.05), while the cavities with xenogenous biomaterials showed higher amount of residual graft (p<0.05). Although the results showed greater bone formation with Straumann^(r) Bone Ceramic, further studies are required to prove which is the more effective biomaterial for bone induction process.

biomaterials; bone regeneration; xenogen bovine graft; alloplastic graft.

Este estudo avaliou o processo de reparação óssea induzida por três biomateriais, dois de origem xenógena constituído de osso bovino desproteinizado e um aloplástico à base de fosfato de cálcio bifásico, em calota craniana de coelhos. Em cinco coelhos brancos da Nova Zelândia com peso entre 2.900 e 3.500 g, foram realizadas quatro perfurações padronizadas de 8 mm de diâmetro nos ossos parietais e enxertados dois biomateriais de origem bovina: Bio-Oss^(r) e Endobon^(r) Xenograft Granules e um totalmente aloplástico: Straumann^(r)Bone Ceramic. Uma cavidade permaneceu com coágulo e foi utilizado como controle. Após oito semanas os animais foram sacrificados e as amostras preparadas para análise morfométrica e qualitativa. Os resultados mostraram que as cavidades preenchidas com o biomaterial aloplástico apresentaram percentualmente maior quantidade de osso neoformado (p<0,05). Apesar dos resultados mostrarem maior neoformação óssea pelo Straumann(r)Bone Ceramic, há a necessidade de mais estudos para se comprovar qual biomaterial é mais efetivo no processo de indução óssea.

Introduction

The use of osseointegrated dental implants requires an adequate amount of bone tissue both in volume and in quality ⁽¹⁾. However, these conditions are not always available, requiring reconstruction with bone substitutes ⁽²⁾. Autogenous bone is regarded as the gold standard for these procedures because of its biological and physico-chemical properties, which are considered optimal ⁽³⁾. However, there is need for a second surgical procedure, increasing the risk of complications ⁽⁴⁾. The bone allograft presents less surgical procedures ⁽⁵⁾, although there are risks such as disease transmission ⁽⁶⁾. Because of these difficulties, many biomaterials of xenogenous or alloplastic origin are being researched for the purpose of bone reconstruction.

Xenogenous bovine bones used as bone substitutes are deproteinized and lyophilized. Biomaterials with these characteristics do not cause any immune response, so they are considered biocompatible ⁽⁷⁾. This category includes Bio-Oss^(r) (Geistlich-Pharma, Wolhusen, Switzerland), consisting of calcium carbonate apatite; it is osteoconductive, with porosity between 75% and 80% and can be used to raise the maxillary sinus membrane, resulting in proper osseointegration of the dental implant in the bone tissue ⁽⁸⁾. Bio-Oss^(r) is resorbed slowly, with residues found nine years after the initial graft ⁽⁹⁾.

Endobon^(r) Xenograft Granules (RegenerOss(tm); BIOMET 3i, Palm Beach Gardens, FL, USA) are another bone substitute consisting of bovine bone, both deproteinized and lyophilized. According to the manufacturer, it is granular in form, with porosity between 45% and 80% and consists of completely deproteinized bovine hydroxyapatite. Moreover, it is biocompatible, bioactive, osteoconductive and not resorbable ^{( 10 , 11 )}.

Straumann^(r) Bone Ceramic (Biora AB, Malmoe, Sweden) is a fully alloplastic bone substitute based on biphasic calcium phosphate. It is granular, consists of 40% β-tricalcium phosphate and 60% hydroxyapatite, osteoconductive and presents 90% porosity ⁽¹²⁾. It is used to raise the sinus membrane ⁽¹³⁾ and results in trabecular bone structure with close contact with the biomaterial, thereby allowing the placement of dental implants after six months ⁽¹⁴⁾. It also produces fewer residual graft characteristics requiring additional studies ^{( 13 )}.

This study evaluated and compare bone healing in surgically prepared cavities in rabbit skulls using three different biomaterials, two of which are xenogenous, consisting of deproteinized and lyophilized bovine bone, and one alloplastic bone based on biphasic calcium phosphate.

Material and Methods

Experimental Design

Three bone substitutes were used, as follows: two xenogenous consisting of deproteinized and lyophilized bovine bone - Bio-Oss^(r) and Endobon^(r)Xenograft Granules, and a fully alloplastic one based on biphasic calcium phosphate in Straumann^(r) Bone Ceramic. They were surgically grafted into bone defects prepared in the skullcaps of five rabbits. After eight weeks, the samples were subjected to morphometric and qualitative analyses. For the statistical analysis, ANOVA and Tukey's test were conducted at a significance level of 0.05 using the statistical software SAS version 7.0 (SAS Institute, Cary, NC, USA).

Animal Study

The present study used five New Zealand male white rabbits, weighing between 2,900 g and 3,500 g. The study was approved by the ethics committee (protocol no. 011/12-CEP/ICS-UNIP) and was in accordance with the guidelines and rules for research involving animals according to State Law no. 11997 of August 25, 2005, which established the Code of Animal Protection of the State of São Paulo, Brazil.

The animals were kept in individual cages under the same environmental conditions before surgery and during the evaluation period. They were fed MP872 (Moinhos Primor S.A., São Paulo, SP, Brazil) and water ad libitum.

Surgical Procedures

Surgical procedures followed the previously proposed methodologies proposed by Aghaloo et al. ⁽¹⁵⁾ and Cavalcanti et al. ⁽¹⁶⁾. The animals were premedicated using a combination of 1 mg/kg of morphine (Cristália Produtos Químicos Farmacêuticos Ltda., Itapira, SP, Brazil), 1 mg/kg of midazolam (Cristália), 10 mg/kg of ketamine (Symtec do Brasil Ltda, Cotia, SP, Brazil) and 2 mg/kg of xylazine (Rompun^(r); Bayer S.A., São Paulo, SP, Brazil) administered intramuscularly. Anesthesia was induced with propofol (Cristália) at an intravenous dose of 2 mg/kg, as well as local anesthesia with 4% articaine hydrochloride with 1:100,000 epinephrine (DFL Industria e Comercio S.A., Rio de Janeiro, RJ, Brazil), via infiltration at the surgery site. Throughout the procedure, the animals had masks to provide 100% oxygen; they were monitored for heart rate, respiratory rate, oxygen saturation, hemoglobin, temperature and blood pressure (BP) (systolic BP, diastolic BP and PA average) in a non-invasive way with a veterinary multiparameter monitor (NeoVet^(r); Centaurus Medical, San Diego, CA, USA). Prophylactic antibiotic therapy was administered with enrofloxacin (Schering-Plough Saúde Animal Ind. e Com. Ltda, Cotia, SP, Brazil) at an intravenous dose of 5 mg/kg.

After obtaining anesthesia, the surgical region was shaved and applied a 2% iodine antiseptic solution. Next a sagittal incision was made approximately 10 cm in the midline of the skull, primarily in the skin, followed by the periosteum to expose the parietal bones. Four standardized bone defects were made; two on each bone, using a sterile 8-mm diameter trephine cutter drill (Implacil Material Odontológico Ltda., São Paulo, SP, Brazil) under copious irrigation with sterile saline solution.

The defects were filled with different bovine bone substitutes. The first was filled with Bio-Oss^(r) with diameters between 250 and 1,000 μm. The second was filled with Endobon^(r) Xenograft Granules with diameters between 500 and 1,000 μm. The third was filled with Straumann^(r) Bone Ceramic, a completely alloplastic-based biphasic calcium phosphate consisting of 60% hydroxyapatite (HA) and 40% β-tricalcium phosphate (β-TCP), with diameters between 500 and 1,000 μm. One of the defects was maintained as control (Fig. 1).

Figure 1.
Defects in the parietal bone patterned and filled with graft materials. A: Control, B: Straumann^(r)Bone Ceramic, C: Bio-Oss^(r) and D: Endobon^(r)Xenograft Granules.

Grafting procedures were performed before the periosteum was repositioned with absorbable suture (Vicryl^(r) 4-0, Johnson & Johnson do Brasil Indústria e Comércio de Produtos para Saúde Ltda, São José dos Campos, SP, Brazil). Then, the skin was repositioned and sutured with nylon thread (5-0 Ethicon^(r) Johnson & Johnson). After suturing, the surgical region was washed with 2% iodine solution.

After completing the surgical procedures, xylazine reversal was induced by the antagonist yohimbine (PowerVet, São Paulo, SP, Brazil) at an intravenous dose of 0.1 mg/kg. Immediate postoperative analgesia was administered via intramuscular morphine at 1 mg/kg.

Postoperatively, the animals were administered the analgesic tramadol hydrochloride (Cristália) at 0.6 mL every 12 h, the anti-inflammatory drug dexamethasone (disodium phosphate) at 0.6 mL every 12 h for three days, and the antibiotic enrofloxacin 2.5% at 0.6 mL every 12 h for 1 week.

Sacrifice of Animals

The animals were sacrificed eight weeks after the surgical procedures, initfigially using a combination of xylazine (20 mg/kg) and ketamine (50 mg/kg) administered intramuscularly, followed by 25 mg/kg of sodium thiopental (Cristália) administered intravenously in the ear vein 15 min later.

Histological Analysis

The samples were demineralized in 20% formic acid for seven days, dehydrated in alcohol with increasing concentrations (70% -100%) for 1 h each, cleared in two changes of xylene for 1 h each and paraffin embedded at 60 °C for 1 h. Five micrometer slices were obtained, stained with hematoxylin and eosin (HE) and analyzed using light microscopy.

Morphometric Analysis

Five sections (5 μm) were obtained from the center of each sample from each animal (20x magnification). Histological slides were then obtained for histomorphometric analysis blind to the study treatment groups. A system of computerized image analysis was used consisting of a light microscope (Axioskop 2 plus^(r); Carl Zeiss, Oberkochen, Germany) coupled to a camera (Axio Cam HRc^(r); Carl Zeiss) connected to a microcomputer, which used the image analysis software Axion Vision^(r) rel 4.8 (Carl Zeiss). Percentages were calculated for newly formed bone, residual graft and connective tissue in the bone marrow.

Qualitative Analysis

Qualitative analysis of the samples was conducted using cone-beam computer tomography obtained with a scanner Gendex CB500^(r) (Gendex Dental Systems, Des Plaines, IL, USA) under the following settings: 120 kVp, voxel 0.125 mm and acquisition time of 23 s. For the three-dimensional reconstructions, the software INVIVO 5^(r) (Anatomage, San José, CA, USA) was used, with axial DICOM thickness of 1 mm.

Statistical Analysis

The histomorphometrIc data showed homoscedasticity of variances after application of the F test. Analysis of variance was performed, which showed differences between the treatments at a significance level of 0.05. Then was applied the Tukey's test, which compared the different treatments (coagulum, Bio-Oss^(r), Endobon^(r) Xenograft Granules and Straumann^(r)Bone Ceramic) according to different factors (newly formed bone, residual graft and connective tissue in the bone marrow). SAS 7.0 (SAS Institute, Cary, NC, USA) was used for the statistical analyses.

Results

Histological Analysis

After eight weeks, histological analysis showed the presence of new bone tissue in all cavities filled with different biomaterials. Presence of residual graft was also observed in the surgical defects filled with Straumann(r)Bone Ceramic and Bio-Oss(r) (Fig. 2).

Figure 2.
Bone formation in defects in the parietal bone of a rabbit filled with different biomaterials. A: Control, B: Straumann^(r)Bone Ceramic, C: Bio-Oss^(r), and D: Endobon^(r)Xenograft Granules (black asterisk = new bone, blue asterisk = residual graft, Original magnification, 100x; HE).

Morphometric Analysis

Morphometric analysis showed the following amounts of newly formed bone: Bio-Oss(r) (57.00±7.76), Endobon(r)Xenograft Granules (58.42±6.29) and Straumann(r)Bone Ceramic (78.06±8.95). The percentage of new bone formation was greater (p<0.05) in the bone defects filled with Straumann(r)Bone Ceramic than in those grafted with Bio-Oss(r) or Endobon(r) Xenograft Granules (Table 1). Residual graft material displayed the following levels: BioOss(r) with 27.58±6.16, Endobon(r)Xenograft Granules with 25.42±8.82 and Straumann(r)Bone Ceramic with 6.28±1.79, with the last one showing significantly lower levels (p<0.05) (Table 1). The presence of connective tissue in the bone marrow was not significantly different (p>0.05) among the biomaterials used for filling bone defects (Table 1).

Table 1.
Mean and SD values in percentages (%) of bone neoformation, residual graft and connective tissue in the bone defects, using different biomaterials after 8 weeks.

• Columns with different letters indicate statistical differences (Tukey's - 0.05) N/A= not applicable

Qualitative Analysis

The qualitative analysis was based on images of three-dimensional reconstructions obtained by cone-beam computer tomography. They showed that the cavities filled with Bio-Oss^(r) and Endobon^(r) Xenograft Granules presented higher hyperdensity and better homogeneity. Cavities grafted with Straumann^(r) Bone Ceramic showed greater qualitative irregularities. The control (coagulum) showed hypodense images of the defects (Fig. 3).

Figure 3.
Three-dimensional reconstruction obtained from cone-beam computer tomography showing repaired cavities. A: control, B: Straumann^(r)Bone Ceramic, C: Bio-Oss^(r) and D: Endobon^(r) Xenograft Granules.

Discussion

When the study objective is to evaluate bone healing in an animal model using bone substitutes, it is essential to verify whether the substitute material complies with the concept of "critical size defects". This concept has different thresholds according to the animal species and site of the defect. Use of standard defects with 8 mm diameter in the parietal bones of rabbit calvaria allowed large increases in their interface with bone graft materials used in this study. This was a reasonable choice in view of previously shown experimental results ⁽¹⁷⁾. This region was also important because it has similar embryological origin and morphology to the maxilla and has limited anatomic area of mechanical stress and relative stability of the neighboring structures, both making it ideal for evaluating osteogenesis induced by biomaterials ⁽¹⁸⁾. A period of eight weeks was appropriate to assess late repair, including new bone tissue resorption of the graft material, bone remodeling and bone regeneration ⁽¹⁹⁾.

Analysis of bone regeneration after eight weeks showed that alloplastic biomaterials showed greater levels of newly formed bone when compared with biomaterials of deproteinized bovine bone. This result is similar to that obtained when Bio-Oss(r) was compared over the same observation period, which is based on biphasic calcium phosphate with the same composition (HA/βTCP in a proportion of 60/40) in shapes of "donut" granules at the submicron level (i.e., from 300-400 μm pores for central particles 0.8 mm) (MegaGen Implant, Kyungsan, Korea) ⁽²⁰⁾. However, different results were observed in studies assessing maxillary sinus membrane elevation ^{( 13 , 21 )} and in those studying rabbit calvaria after eight weeks involving standard defects with 6.5 mm diameter ⁽⁶⁾. These studies found no statistically significant differences in the levels of newly formed bone tissue induced by these biomaterials

Xenogenous biomaterials showed similar behavior, both in the amount of newly formed bone, as in the amount of residual graft. The results may be explained by the origin of the tested materials, as both were of bovine origin, deproteinized and lyophilized, had similar granule diameters (Bio-Oss^(r), 250-1,000 μm and Endobon^(r)Xenograft Granules, 500-1,000 μm) and had similar porosity (Bio-Oss^(r) between 75% and 80% and Endobon^(r)Xenograft Granules between 45% and 85% (BIOMET 3i (tm)). The results of the present study were similar to those reported by Tovar et al. ⁽²²⁾, who compared the Bio-Oss^(r), OsteoGraf N-300^(r) (Dentsply International, York, PA, USA), and two variations of a newly developed biomaterial (Dentsply). After eight weeks of evaluation, no statistical difference in the amount of newly formed bone was observed.

The biomaterial-based biphasic calcium phosphate showed a lower amount of residual grafting (p<0.05) compared with biomaterials consisting of deproteinized bovine bone. The result was similar to the one found in a study that evaluated the behavior of the Straumann^(r)Bone Ceramic and Bio-Oss^(r) for floor elevation of the maxillary sinus surgery ⁽¹³⁾. Several studies have shown that the presence of residual graft using biomaterials based on deproteinized bovine bone is due to its slow resorption ^{( 9 , 11 , 23 , 24 )}. Traini et al. ⁽²³⁾ suggested that the difficulty of Bio Oss^(r) resorption is related to its high content of calcium and absence of proteins. In the study by Ramirez-Fernandez et al. ⁽²⁴⁾, performed in tibiae of rabbits, Endobon^(r)Xenograft Granules was less absorbable than OsteoBiol^(r)mp3 (TECNOSS srl, Giaveno, Italy) from swine. In the alloplastic biomaterial-based biphasic calcium phosphate (HA/βTCP in a proportion of 60/40), the βTCP constituent is quickly resorbed and replaced with natural bone providing faster remodeling, while the HA constituent resorbed slower, allowing preservation of the bone volume ^{( 13 , 14 )}.

Cavities filled with blood clot had morphometrically greater amount of newly formed bone tissue than the cavities filled with biomaterial-based bovine bone (p<0.05) and similar to the amount found in cavities filled with biomaterial-based phosphate biphasic calcium (p>0.05). This result was also observed by Rokn et al. ⁽⁶⁾, who suggested that this may be related to the repositioning and suturing of the periosteum during surgical procedures that act as a natural membrane, inducing guided tissue regeneration. Other studies in rabbit's calvaria, also showed newly formed bone tissue similar between the clot and the platelet-rich plasma ⁽¹⁵⁾, and between the clot and biodegradable hydrogel gelatin ⁽²⁵⁾. Borie et al. ⁽¹⁸⁾ showed that the clot had lower bone formation compared to autogenous bone and allogenic human bone in rabbit calvaria.

Despite the differences with the control group, the coagulum alone is not effective in the clinical setting. In augmentation procedures involving implant placement, thickness or height of the alveolar bone is important; so, the coagulum is less than ideal because it lacks resistance to compression from the soft tissue. For such procedures, it is essential that the graft material has osteoconductive properties (i.e., it should be able to create structural support for new bone formation), emphasizing the importance of evaluating biomaterials used for this purpose.

Three-dimensional reconstructions by cone-beam tomography showed that the cavities with bovine bone presented higher hyperdensity, more homogeneous and smaller amount of new bone, while cavities with biphasic calcium phosphate showed greater irregularities, but larger amounts of new bone. These results may be related to the greater amount of residual graft present in areas that received xenogenous biomaterials. Presence of more such biomaterials, which have slow resorption ⁽²³⁾, could provide images with greater regularity than in areas with greater amount of new bone. These reconstructions have also shown that cavities with blood clot showed no filling at the center of the surgical defect. These results suggest that the newly formed bone is concentrated at the edges of the surgical defect, reinforcing the clinical argument that in cases of bone reconstruction a bone substitute with osteoconductive feature is necessary.

The results of this study showed that there is need for further studies to assess biomaterials that are more effective in the process of bone induction than those used in this study. Both qualitative and quantitative techniques are warranted to better appreciate the results as they relate to clinical applicability.

The results suggest that all biomaterials induced bone formation after eight weeks. Although the alloplastic biomaterial induced more bone formation, further studies are essential to prove which types of biomaterials are more effective for bone induction.

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Publication Dates

• Publication in this collection
  Sep-Oct 2014

History

• Received
  19 Dec 2013
• Accepted
  28 July 2014