ARTÍCULOS ORIGINALES

Bone substitute in the repair of the post-extraction alveolus

 

Sebastián Fontana ¹, Luis M. Plavnik ¹, Sandra J. Renou ², Marta E. González de Crosa ³

¹ Department of Histology & Embriology (“A”). School of Dentistry, National University of Córdoba, Argentina.
² Department of Oral Pathology, School of Dentistry, University of Buenos Aires, Argentina.
³ Scientific & Technologic area of the “CREO Foundation”. Córdoba, Argentina.

CORRESPONDING AUTHOR Dr. Sebastian Fontana Boulevard Chacabuco 770, 2o P CP 5000; Cordoba Argentina. e-mail: sfontana@odo.unc.edu.ar

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ABSTRACT

In recent years there has been increasing interest in the choice of the best material for bone substitutes. Experimental models enable estimation of biological potential, efficacy and safety of a biomaterial before its clinical application. The aim of this study was to evaluate the response of a bone substitute, UNC bone matrix powder (MOeP-UNC), for repairing the postextraction alveolus in Wistar rats. Rats’ first lower molars were extracted. The right alveoli were filled with MOeP-UNC hydrated with physiological saline (Experimental Group, EG), and the left alveoli were used as Control Group (CG). Thirty days after extraction, the animals were killed and the samples processed. Histological sections were made in vestibular- lingual direction at the level of the mesial alveolus of the first inferior molar (Guglielmotti et al. J. Oral Maxillofac. Surg. 1985;43(5):359-364). Repair of the alveoli at 30 days after extraction was evaluated histologically. Repair of the alveolus was optimum in the control group at 30 days, and the EG showed presence of MOeP-UNC particles in close contact with newly formed bone tissue (osseointegration). In the experimental model used, at 30 days post-surgery, the MOeP-UNC particles integrate compatibly with newly formed bone tissue.

Key words: Bone healing; Osteoinduction; Osteogenesis; Freeze dried bone.

RESUMEN

Substituto óseo en la reparación de alvéolos post-extracción

En los ultimos anos se ha incrementado el interes por la eleccion del material mas adecuado como sustituto oseo. Los modelos experimentales permiten estimar el potencial biologico, la eficacia y seguridad de un biomaterial, previo a su aplicacion clinica. El objetivo del presente estudio fue evaluar la respuesta de un sustituto oseo, matriz osea-UNC en polvo (MOeP-UNC), en la reparacion alveolar post-exodoncia en ratas Wistar. Se realizo la exodoncia de los primeros molares inferiores. En los alveolos derechos se coloco MOeP-UNC hidratada con solucion fisiologica (Grupo Experimental, GE). Los alveolos izquierdos, fueron utilizados como Grupo Control (GC). A los 30 dias post-exodoncia los animales fueron sacrificados y las muestras obtenidas se procesaron, se realizaron cortes histologicos en sentido vestibulo-lingual a la altura del alveolo mesial del primer molar inferior (Guglielmotti et al. J Oral Maxillofac Surg. 1985;43(5):359-364). Se realizo la evaluacion histologica de la reparacion de los alveolos a los 30 dias post cirugia. El grupo control presento una optima reparacion alveolar a los 30 dias y en el GE se evidencio la presencia de las particulas de MOeP-UNC en intimo contacto con el tejido oseo neoformado (oseointegracion). En el modelo experimental utilizado, a los 30 dias post-cirugia las particulas MOePUNC se integran de manera compatible con el tejido oseo neoformado.

Palabras clave: Cicatrizacion osea; Osteoinduccion-osteogenesis; Hueso desecado y congelado.

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INTRODUCTION

In recent years there has been increasing interest in the choice of the best material for substitutes used for repairing bone defects. The main aims of a biomaterial for bone tissue are that it should: a) restore form and function, b) significantly shorten the physiological process of ossification and c) be easy to manipulate¹. Some of the alternatives for bone substitutes are the following:

Autologous or autogenous grafts are taken from the patient him/herself and may be harvested from different intra-oral donor sites (chin, maxillary tuberosity, ascending branch) or extra-oral donor sites (iliac crest, tibia, calvaria). This is the material of choice because of its high osteogenic capacity and low antigenicity, e.g.autologous bone.

Homologous or allogeneic grafts, or allografts come from individuals of the same species, but who are genetically different. The types most often used are freeze dried bone allograft (FDBA) and demineralized freeze dried bone allograft (DFDBA), also called demineralized bone matrix.

Heterologous grafts or xenografts come from an animal of a different species, eg deproteinized bovine bone mineral (Bio-Oss, Giestlich Pharma).

Alloplastic or synthetic grafts are synthetically manufactured materials². Among the most often used of these materials are bioactive glass (Bioglass) and tricalcium phosphate (TCP)2. The new bone that will form in a bone defect treated with particulate grafts is mediated by processes of osteogenesis, osteoconduction and/or osteoinduction. These processes could take place individually or simultaneously, depending on the nature of the material used^3-6.
According to the literature, the ideal filling material is autologous bone graft, since there is the possibility to retain cell viability, graft revascularization and there is no possibility of disease transmission^1,7-9. However, obtaining an autologous bone graft requires a surgical procedure at the donor site, with the consequent risk of postoperative morbidity (infection, pain, hemorrhage, muscular weakness and neurological injury, among others). Surgical time also increases considerably; and in some cases the amount of graft harvested may be insufficient ^10,11. Thus, the use of alternative filling materials has increased. Biomedical publications^1,6,7 reflect the search for an ideal substitute for autologous bone, nevertheless, there is some controversy regarding methodology and interpretation of the results^7,11,12. The aim of this study is to evaluate the response of a bone substitute, UNC bone matrix powder (MOeP-UNC), for repair of the post-extraction alveolus in Wistar rats.

MATERIALS AND METHODS

Filling Material
UNC bone matrix powder (MOeP-UNC) is human bone tissue from the Bone Bank at Cordoba Hospital (Cordoba, Argentina), which meets the requirements established by Argentina’s National Institute for Organ Donation and Transplantation (Instituto Nacional Central Unico Coordinador de Ablacion e Implante, INCUCAI). Bone from a single donor is processed, lyophilized and sterilized by gamma radiation. The Human Tissue Processing Plant of the Blood Derivative Laboratory at Cordoba National University is authorized by INCUCAI and the National Medication, Food and Medical Technology Administration (Administracion Nacional de Medicamentos Alimentos y Tecnologia Medica, ANMAT). The UNC Bone Matrix is registered as Medical Technology (ANMAT) under medical product register number 1007-1/2).

Histomorphometric Measurement of Particles
Sixty bone substitutes particles were measured using image analysis software (Image Pro-Plus 4.5) to determine their equivalent diameter. Equivalent diameter is obtained by considering the irregular area of the particle under observation to be a perfect circle¹³ (Fig. 1).

Fig. 1: Macroscopic appearance of MOeP-UNC bone substitute (Original Magnification x 45).

Surgical Procedure
Sixteen male Wistar rats weighing 80 g (} 10 g) were anesthetized with Ketamine solution (8 mg/100 g body weight; Ketamina Zoovet, Lab. Zoovet, Argentine) and Xilazine (1.28 mg/100 g body weight; Sedomin, Lab Konig, Argentine). The methodology described by Guglielmotti MB et al.14 was followed for the extractions. In order to achieve the approach and perform surgical procedures in the rat buccal cavity, a special examination table was designed to keep the animals in dorsal decubitus position with the mouth held open by means of a system of fasteners. The first lower molars were extracted using instruments that were proportional to the size and shape of the teeth. After extraction, the right alveoli were filled with MOePUNC hydrated with physiological saline (Experimental Group, EG) and the left alveoli were left unfilled (Control Group, CG). NIH standards for the use and care of laboratory animals were followed (NIH publication No 85-23, revised 1985). The experimental protocol was approved by the Committee of Bioethics of the School of Medical Science at Cordoba National University. Thirty days after the extraction the rats were killed, and the hemimaxillaries were dried, radiographed, demineralized and embedded in paraffin. Vestibulo-lingual sections were made at the level of the mesial alveolus of the first inferior molar for histological study. A descriptive analysis was made of the presence of MOeP particles implanted in the alveoli after extraction and newly for med bone tissue directly related to them (osseo integration).

RESULTS

Histomorphometric Analysis
The image analysis software determined that the equivalent diameter of the particles was 535.42 μm (} 233.90), ranging from 125.85 μm to 1069.86 μm. (Fig. 2).

Fig. 2: Particle size in UNC bone matrix powder (n: 60).

 

Histological Analysis
Thirty days post-extraction, the alveolus in the control group was completely filled with lamellar bone. In all the experimental cases, the MOeP-UNC particles were found to be surrounded by newly formed bone (osseointegration). Osteocytic cells in their lacunae, peripheral osteo blasts and presence of osteoid matrix were found in the newly formed bone tissue (Figs. 3, 4 and 5).

Fig. 3: Bone particles (*) in alveolus 30 days after extraction. (H/E – Original Magnification x 45).

Fig. 4: Newly formed bone tissue (B) in direct relation (↑) to the bone substitute (*). (H/E Original Magnification x 100).

Fig. 5: Bone particle (*) and newly formed bone tissue (B) closely related to its surface (↑). (H-E Original Magnification x 400).

The presence of filler material did not interfere with repair of the post-extraction alveolus in this experimental model. During the study time (30 days) the MOeP-UNC particles integrated compatibly with the newly formed bone tissue.

DISCUSSION

Autologous bone is considered to be the gold standard for bone substitution, but some disadvantages have been reported, as mentioned above^10,11. Ochandiano Caicoya suggests that the cells in this type of graft die when they are more than 100 mm away from a vascular source (it is estimated that 95% of the graft osteoblasts undergo necrosis)¹². Under these circumstances, the advantages of this type of filling would be lost and it would behave as a nonliving graft. Regarding allografts, those of human origin are most often used, such as freeze-dried bone allograft (FDBA), which was the first to be used, and Demineralized Bone Matrix (DFDBA), which was described by Urist, showing that its extra-skeletal implantation in experimental animals resulted in heterotopic ossification by means of an osteoinductive mechanism¹⁵.
To date, clinical and experimental studies do not agree on whether FDBA or DFDBA-based fillings trigger osteoinduction and/or osteoconduction processes, probably due to controversies regarding methodology and interpretation of results, which are systematically repeated in the literature8,16. In this regard, the post-extraction repair of the alveolus model in rats¹⁴ was selected for our study with the aim of obtaining transferable results on biocompatibility and osteoconduction of MOePUNC. The results showed that the implanted particles did not interfere with post-extraction repair of the alveolus in rats and that over the study period (30 days) the MOeP-UNC particles integrated compatibly with the newly formed bone tissue (osteoconduction). These results agree with those of Glowacki¹⁷, who states that if non-critical bone defects are filled with biomaterials (e.g., in the case of post-extraction alveoli where only the crest wall is missing), only osteocompatibilty and osteoconductivity are being demonstrated, but not osteoinduction.
Although it has been proposed by means of different experimental models that demineralized bone particles (DFDBA) have an osteoinductive effect^15,18, other studies have questioned the benefits of this type of filling^19,20. A possible cause of this variability in the results may be that the age of bone donors was not considered in tissue banks²⁰. It is also very important to monitor closely all the steps required for demineralization and sterilization (whether with ethylene oxide, high temperatures or gamma radiation) because they might reduce bone induction by up to 40%²¹ or cause protein denaturation and/or inactivation, particularly of BMP, which is determinant in osteoinduction^22,23.
Particle size seems to be another key variable regarding the success of bone substitutes used as fillers²⁴, which is the reason why this study measured MOeP-UNC particle size using Image Pro-Plus 4.5 image analysis software. Mean diameter was found to be 535.42 μm. These data place MOeP-UNC particles within parameters described in previous studies, which suggest that particles ranging from 125 to 1000 mm produce greater osteogenic effect than those smaller than 125 mm or larger than 1000 mm^18,24. We therefore recommend the use of MOeP-UNC particles for bone fillings, since, in contrast to autografts, they do not require a donor site for harvesting; they have high mineral content, providing better physical properties than DFDBA particles, and they trigger osteoconductive effects.

ACKNOWLEDGMENTS

We are grateful to UNC Biotecnia, Blood Derivative Laboratory at Cordoba National University, for providing Bone Matrix Powder (MOeP-UNC). This Project was approved by the Secretariat of Science and Technology at Cordoba National University (SeCyT, UNC - No: 05-J060).

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