ARTÍCULOS ORIGINALES

Use of anorganic bovine bone matrix in an experimental model of bone healing

 

Marcela A Redondo¹, Sandra J Renou², Sebastián A Puia³, Osvaldo R Costa¹, María B Guglielmotti ²

¹ Department of Periodontics, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina
² Department of Oral Pathology, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina
³ Department of Oral and Maxillofacial Surgery II, School of Dentistry, University of Buenos Aires, Argentina.

CORRESPONDENCE Dr Sandra Judith Renou, Department of Oral Pathology, School of Dentistry, University of Buenos Aires, MT Alvear 2142 2A, (C1122AAH), Buenos Aires. Argentina. e-mail: sandrarenou@gmail.com

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ABSTRACT

The dimensions of the alveolar bone surrounding the tooth are not maintained post tooth-extraction probably as a consequence of the bone remodeling process and the biomechanical demands on bone. The use of biomaterials as bone substitutes in the post-tooth-extraction socket promotes bone repair, regardless of damage to bone structures during the surgical procedures. The aim of the present study was to evaluate the effectiveness of anorganic bovine bone matrix (ABBM) as a bone substitute, in an experimental model of post-tooth extraction bone healing in the rat. Radiographic follow-up was performed at 7, 14, and 30 days, and showed persistence of the biomaterial inside the experimental alveoli. At 14 and 30 days post-tooth extraction, particles surrounded by bone tissue were observed in the middle sector of the alveoli. The osteoconductive property of ABBM was demonstrated using the present experimental model of active osteogenesis, thus showing its usefulness as a bone substitute. Persistence of the particles at the studied experimental time points did not affect post-tooth extraction bone healing.

Key words: Bone substitutes; Bone healing; Tooth extraction; Rat.

RESUMEN

Utilización de matriz ósea esponjosa anorgánica bovina en un modelo experimental de reparación ósea

Las dimensiones del hueso alveolar que rodea a la pieza dentaria, no se mantienen después de la exodoncia. Este hecho seria consecuencia del proceso de remodelado óseo y del requerimiento biomecánico. La utilización de biomateriales como sustitutos óseos en los alveolos, facilitan o promueven la reparación ósea, independientemente que se haya producido traumatismo de las estructuras óseas durante la maniobra quirúrgica. El objetivo del presente estudio fue evaluar la efectividad de una matriz ósea esponjosa anorgánica (MOEA) como sustituto óseo, en un modelo experimental de reparación ósea en el alveolo post-extracción en ratas. Se realizo el estudio radiográfico en los distintos tiempos experimentales: 7, 14 y 30 dias, evidenciando la persistencia del biomaterial. A los 14 y 30 días post-exodoncia se evidenciaron las partículas rodeadas de tejido óseo en el sector medio del alveolo. Es importante destacar que la utilización de (MOEA), como sustituto óseo en el alveolo post-exodoncia de rata, evidencio su capacidad osteoconductiva. La persistencia de las partículas del biomaterial en los tiempos estudiados no interfirió en la reparación ósea.

Palabras clave: Sustituto óseo; Reparación ósea; Exodoncia; Ratas.

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INTRODUCTION

Post tooth-extraction socket bone healing requires approximately two months before complete bone repair can be observed, after which bone remodeling takes place^1,4. Although it holds true that the reported descriptions of the stages of bone repair from the moment the tooth is extracted are based on histologic and histomorphometric studies performed in experimental models in rats and dogs, among other experimental animals^5-9, it is also well documented that observations in animal models apply to humans, rendering post-tooth extraction socket healing in animals and humans comparable^10-12. It is also well documented that bone tissue metabolism in each of the stages and the mechanisms involved in socket bone healing are species specific, differing between animals and humans. For example, post tooth-extraction socket healing is slower in humans than in dogs¹³, and the metabolic activity index is faster in rats than in humans¹⁴. The dimensions of the alveolar bone surrounding the tooth are not maintained post tooth-extraction^1-4, probably as a consequence of the bone remodeling process and the biomechanical demands on bone¹⁵. The use of biomaterials as bone substitutes in the post-tooth extraction socket promotes bone repair, regardless of damage to bone structures associated with the surgical procedures^3,16-17. There are different types of bone substitutes, each of which exhibits different properties:
1) Osteoinduction: It implies chemotaxis, mitosis, and differentiation of mesenchymal cells to osteoblasts or chondroblasts; eg, demineralized bone powder^18-22 .
2) Osteoconduction: The implanted biomaterial serves as a scaffold for osteoblasts; such is the case of bank-bone grafts and hydroxyapatite^{23- 26}.
3) Osteopromotion: The implanted biomaterial stimulates viable osteoblasts; for example: bioactive glass ceramic materials^27-31.

A wide variety of biomaterials has been used in experimental^{20,22,25,32-34} and clinical studies^{17, 27}. Although most grafts are capable of preserving bone tissue volume and contour at the extraction site, there is controversy regarding the quality of the bone that forms around the graft. This issue gains further significance when treatment involves placing an implant, for when bone repair around the filling is not adequate, the physical and biological properties of the newly formed bone tissue are not suitable to meet the biomechanical requirements of the implant. In addition, Irinakis T. emphasized the importance of monitoring peri-implant mucosa, since preserving the bone structures before and after surgical procedures ultimately facilitates reaching the mucosa surrounding the implant-supported prosthesis and maintaining adequate hygiene³⁵. Thus, adequate maintenance of peri-implant mucosa has significant clinical implications, given that oral hygiene is an important determinant of long-term treatment success.
The bone graft of choice is human demineralized freeze-dried allografts of cortical and cancellous bone. They are obtained from human bone-banks, and are subjected to a number of treatments, such as fragmentation, saponification, lyophilization, and decalcification, among others. Based on the above, the aim of the present study was to evaluate the effectiveness of Anorganic Bovine Bone Matrix as a bone substitute, in an experimental model of post-tooth extraction bone healing in the rat.

MATERIALS AND METHODS

Thirty male Wistar rats, 70 ± 10 g body weight (b.w.), were used. The animals were anesthetized by intraperitoneal injection of 8 mg of Ketamine (KetalarR, Parke-Davis, Morris Plains, NJ) and 1.28 mg Xylaxine (RompumR, Bayer, Leverkusen, Germany) per 100g / b.w. The right and left lower first molars were extracted following the technique described by Guglielmotti et al5. Anorganic Bovine Bone Matrix (OsteodensR-Pharmatrix, Argentina) particles ranging in size from 250 to 1000 mm, were placed in the fresh extraction socket of the extracted right mandibular first molar. No filling material was placed in the left post-tooth extraction socket, which served as control.
The guidelines for the care and use of laboratory animals were observed³⁶. The experimental protocol was approved by the Ethics Committee of the School of Dentistry of the University of Buenos Aries. The rats were fed regular chow and water ad libitum; no antibiotic therapy was administered. The animals were euthanized 7, 14, and 30 days post-tooth extraction respectively, and weighed; the mandibles were resected and fixed in 10% formalin solution. All hemimandibles were radiographed and decalcified in 25% formic acid for 48 hours, and processed for embedding in paraffin. The samples were sectioned in a bucco-lingual orientation at the level of the mesial alveolus of the lower first molar to obtain 5μm to 7μm thick sections. Both experimental and control sections were stained with hematoxylin-eosin.

RESULTS

Radiographic study
A time-dependent increase in radiopacity was observed in control alveoli (Fig. 1a). The presence of the bone substitute, which was more opaque than the newly formed bone tissue, was detected in the experimental group at all experimental time points (Fig. 1b).

Fig. 1: a: Control group: 30 days post-tooth extraction. Note the presence of radiopaque tissue filling the post-tooth extraction socket of the first molar. b: Experimental group: 30 days post-tooth extraction. Note the presence of bone substitute particles surrounded by the newly formed bone tissue in the post-tooth extraction socket of the first molar.

Histologic Results
The control group showed the typical features of post-tooth extraction bone repair: granulation tissue filling the alveolus and newly formed woven bone in the apical third of the alveolus at 7days; woven bone filling a large portion of the alveolus at 14 days; and lamellar bone replacing the woven bone at 30 days. The histological study of the experimental group showed the presence of bone substitute particles at all the experimental time points. Granulation tissue and woven bone were observed around and close to the particles at 7 days post-tooth extraction, and laminar bone tissue surrounding and aggregating the particles could be seen at 14 and 30 days (Fig.2 a-b, Fig. 3 a-b).

Fig. 2: a: Experimental group: 14 days post-tooth extraction. Note the negative histologic image corresponding to the particles, which were surrounded by bone and granulation tissue. (H-E- Orig. Mag. X40). b: Experimental group: 14 days post-tooth extraction. Note the negative histologic image corresponding to the particles, and the surrounding bone tissue (↑). (H-E – Orig. Mag. X40).

Fig. 3: a:Experimental group: 30 days post-tooth extraction. Note the lamellar bone tissue surrounding the particles, which differ in shape and size. (H-E – Orig. Mag. X10). b: Experimental group: 30 days post-tooth extraction. Higher magnification allows observing the presence of lamellar bone tissue surrounding a particle and covered by osteoblasts (↑). (H-E – Orig. Mag. X40).

DISCUSSION

Artzi et al³⁷ studied the influence of porous bovine bone mineral on human extraction socket healing 9 months post-extraction. The authors found newly formed bone characterized by abundance of cellular woven-type bone in the coronal area, while lamellar arrangements could be identified only in the more apical region. They concluded that the biomaterial was "an appropriate biocompatible bone derivative in fresh extraction sockets for ridge preservation". In agreement with the above, an experimental study in dogs showed the filling biomaterial to be in contact with woven bone first, and with lamellar bone at a later stage³⁸. The study also analyzed particle density at different experimental time points until particle resorption and replacement by lamellar bone was complete³⁹.
The aforementioned studies demonstrate the osteoconductive capacity of anorganic bovine bone, which has also been reported in experimental studies in rats and dogs under different experimental conditions, as well as in clinical studies^{3, 20-22}. It must be pointed out that the experimental works cited above were performed using the biomaterial alone or in combination with guided tissue regeneration membranes. The results of the present study demonstrate the osteoconductive capacity of anorganic bovine bone matrix, and show its usefulness as a post-tooth extraction filling biomaterial. The experimental model used herein could serve to further evaluate the effect of systemic and local factors, which previous works by our research group have shown to affect bone healing^40-47.
Other aspects that must be taken into account are the time and mechanisms involved in the resorption of the filling biomaterial. Ideally, this process ends when the material is resorbed or biodegraded and fully replaced by lamellar bone, which is able to withstand biomechanical loads⁴⁸. This process can take from five months to over a year in humans, as shown by Skoglund et al⁴⁹ and Avera et al⁵⁰, who encountered particles upon surgical reentry 44 months post-placement, and by Paolantonio et al⁵¹, who found that particles persisted 4 years post-placement. The mechanism involved in filling biomaterial resorption is not yet fully understood. Some authors have suggested that osteoclasts are involved, whereas others posit that enzymes play a role in this process^52,53.
Zitzmann et al observed lacunar type resorption, both in areas with bone filling and in those with newly formed bone. It is therefore evident that bone remodeling takes place normally in both areas⁵³. Another advantage to this biomaterial is its radiopacity, since it allows performing radiographic follow-up in both animal models and in humans. In fact, Schlegel and Donath⁵⁴ performed a 7-year radiographic follow-up of patients receiving a bone substitute. In the present study, radiographic follow-up was performed at each of the studied time points (7, 14, and 30 days), and confirmed the presence of the filling biomaterial in the experimental alveoli.

CONCLUSION

The experimental model used in the present study demonstrates the osteoconductive properties of locally manufactured anorganic bovine bone matrix, and confirms that it does not affect post-tooth extraction bone healing. Further studies should be conducted to analyze the bone substitute in combination with a Guided Bone Regeneration membrane.

ACKNOWLEDGEMENTS

This work was supported by research Grants UBACYT 20020100100657 from the University of Buenos Aires, Argentina and CONICET PIP 11220090100117 from National Research Council (CONICET), Argentina

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