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Optical densitometry study of the newly formed bone using rhBMP-2 in wistar rat mandibles/Estudio densitometrico del tejido oseo neoformado por el uso de rhBMP-2 en mandibulas de ratas wistar.

SUMMARY: The purpose of this study was to evaluate the efficacy of monoolein gel as a carrier for rhBMP-2 in the healing bone process of critical bone defects created in Wistar rats mandibles using digitalized radiographic method to analyze this process. In the group 1, the rhBMP-2 was dissolved in aqueous solution and in the group 2, the rhBMP-2 was combined with monoolein gel as a carrier. The results showed that in both of groups it was found efficient bone repair, with a great optical density in the group that the rhBMP-2 was combined with the monoolein gel, but without statistical difference between them.

KEY WORDS: Optical densitometry; rhBMP-2; Monoolein; Bone defects.

RESUMEN: El objetivo de este estudio fue evaluar la eficiencia del gel de monoolein como portador de la rhBMP-2 en el proceso de recuperacion de la osificacion en defectos osteos creados en mandibulas de ratos Wistar, por medio de radiografias digitalizadas como metodo de analisis. En el grupo 1, la rhBMP-2 fue disuelta en solucion acuosa y en el grupo 2, la rhBMP-2 fue combinada al gel de monoolein como portador. Los resultados mostraron que los dos grupos presentaron un eficiente reparo osteo, con mayor densidad optica en el grupo en que la rhBMP-2 fue combinada con el gel de monoolein, pero sin diferencia estadistica significante entre ellos.

PALABRAS CLAVE. Densitometria optica; rhBMP-2; Monoolein, Defectos oseos.

INTRODUCTION

Tissue development and regeneration are regulated by an interplay among various tissue inductive growth factors, formation of an appropriate vascular be to support the metabolic needs of the forming tissue mass, and a cell population capable of responding to the chemical cues and creating the new tissue. There has been considerable interest in understanding this signaling interplay in bone, because of its limited ability to heal on serious fracture or trauma and the limitations in common treatments for bone morbidity or replacement. BMPs are responsible for initiating cartilage and bone progenitor cell differentiation (Hanada et al., 2001) and sequencing new bone formation through endochondral ossification (Bonadio et al., 1998).

Bone morphogenetic protein (BMP) was first discovered by Urist in 1965. BMPs belong to the transforming growth factor-b superfamily. This family comprises a large number of growth and differentiation factors, which are related in terms of their primary amino acid sequences, and consists of the dimeric molecules BMP2 through BMP-8. Comparisons among the derived amino acid sequences of the BMPs found in the osteoinductive extracts of bone indicate that they fall into three subclasses. The first subclass contains BMP-2 and BMP-4, which are two highly related molecules that differ mainly in the amino terminal region, with BMP-2 containing a heparin-binding domain. In the second subclass are BMP-5, BMP-6, BMP7, which is also known as osteogenic protein-1 (OP-1), and BMP-8 (OP-2). These are slightly larger proteins than BMP2 and BMP-4, and there is 70% amino acid identity between these two subclasses. In the third subclass, and more distantly related to these factors, is BMP-3, which is also called osteogenin (Wozney 2002).

Successful carrier systems must enable vascular and cellular invasion, allowing the BMP to act as a differentiation factor. The carrier should be reproducible, non-immunogenic, moldable, and space-providing, so as to define the contours of the resulting bone. Moreover, the carrier should resorb completely following the initiation of bone induction, thus ensuring bone formation (Wikesjo et al., 2001). Various biomaterials have been tested as candidate carriers of BMPs. They include collagen, decalcified bone matrix, hyaluronan, hydroxyapatite, tricalcium phosphate, a hydroxyapatitecollagen composite, polylactic acid polymer, polylacticpolyglycolic polymer, gelatin, fibrin sealant, and composites of these materials. Each carrier material showed various advantages and disadvantages, and it has not yet been determined which of these materials constitutes the ideal carrier system for BMPs. The demands on the carrier materials may differ according to the indication and the site involved; therefore, the search for ideal carriers should be continued.

Monoolein is a biodegradable polar lipid that has no marked toxic effects (Ganem-Quintanar et al., 2000). Depending on water content and temperature, several phases can be formed, including reverse cubic and hexagonal phases. The reverse cubic phase of monoolein and water is formed at room temperature, and has been shown to accommodate and sustain the release of drugs with varied physical chemical properties, including proteins and peptides (Lee & Kellaway 2000a, 2000b; Lara et al., 2005; Turchiello et al., 2003).

The purpose of this study was to evaluate the efficacy of monoolein gel as a carrier for rhBMP-2 in the healing bone process of critical bone defects created in Wistar rat mandibles using digitalized radiographic method to analyze this process.

MATERIAL AND METHOD

This study followed the requirements of the Ethics Committee on the Use of Animals in Experimentation at the University of Sao Paulo, Brazil.

Monoolein (Myverol 18-99, 98.1% monoglycerides, Naarden, The Netherlands) gel was obtained in a 7:3 (monoolein:water) proportion as described by Lara et al. 2005. Briefly, monoolein was weighed and heated to 45[degrees]C. Water in the same temperature was added and the mixture was left to rest until it became a transparent and viscous mass. BMP in water solution (1mg/mL) was added and the mixture homogenized. Amounts of the gel sufficient to carry 15[micro]g of BMP were applied in the created critical bone defects.

The recombinant human bone morphogenetic protein, type 2 (rhBMP-2) used in this study was obtained at TheodorBoveri-Institut fur Biowissenschaften, Am Hubland, Wurzburg, Germany.

The purity of rhBMP-2 was assessed by polyacrylamide gel electrophoresis followed by spectrophotometric determinations (Beckman-DU-70, USA) in the stained gel band. The spectrophotometer provides a standard graphic of the electrophoretic gel strain, supplying through the proteins locations and area measurement, the percentage of sample purity (Fig. 1A).

Surgical procedures. Thirty male Wistar rats (~350g) were selected and allowed to acclimatize for one week before the experiment. The animals were fed with commercial rat chow and had access to food and water ad libitum. The rats were anaesthetized using ketamine hydrochloride (60mg/Kg) associated with xylazine (5mg/ Kg), administered intraperitoneally. After randomly assigned into two equal groups, critical bone defects (5x5mm) were created in the middle region of the right hemi-mandible body of the animals (Fig. 1B) and filled with: 15 [micro]g rhBMP-2 in aqueous solution (group 1), 15 [micro]g rhBMP-2 combined with monoolein gel as a carrier (group 2).

Animals' perfusion. After two weeks, the animals were anaesthetized with urethane 37.5% (1.5g/Kg) and perfused with an intracardiac infusion of saline solution (100mL) followed by 10% formalin and paraformaldehyde 4% in phosphate buffer 0.2M (100mL). Thehemi-mandibles were removed for radiographic analysis (Fig. 1C).

Radiographic analysis. The right hemimandibles of both groups were separated from the skull and the soft tissues were carefully removed. These hemimandibles were placed over extrabuccal radiographic film (T-MAT, Kodak, Kodak do Brasil Ltda., Sao Jose dos Campos/SP, Brasil). A X-Ray apparel (Weber, type 11R, Weber Company, USA) was perpendicularly placed in relation to the film, 40cm of focus-film distance, and a single radiographic exposition was performed with 10mA, 64.5kVp e 0.16 seconds. All these factors were monitorated by apparel/system Victodreen NERO 6000B (Non-Invasive Evaluator Radiation Outputs, Modelo 6000B, Victoreen Inc-USA).

The radiographic image obtained was digitalized in a professional scanner (Expression 636, EPSON Ltda, USA) and cut accord to interested regions using the Adobe Photoshop 7.0.1 program (Adobe, Adobe System Corporation Inc., USA). The resulted images containing the newly formed bone were submitted to optical densitometry analysis using the Sidex program (S3DX, Sidex Company, New York, USA). For this analysis, it was traced two diagonal lines beginning from the vertices of a regular square, placed in the newly formed bone area. On each line it was measured 100 dots for optical density. The total value for optical density in mandible was the mean of the values found in these two lines (Fig. 1D).

Statistical analysis. Data analysis was performed using GLM ANOVA with Tukey test and 95% of significance level.

[FIGURE 1 OMITTED]

RESULTS

The data showed greatest values for optical density in the group that the rhBMP-2 was combined with monoolein gel (group 2), but without statistical difference in relation to the group that the rhBMP-2 was dissolved in aqueous solution (group 1) (p>0.05), (Table I). The greatest values for optical density in the group 2, indicated that in this group the bone repair process occurs more faster than the group 1, showing that the carrier used in this study, monoolein gel, permitted to bone morphogenetic protein stay in the place that it was inserted and promote the mesenchymal cells differentiation into osteoblastic and chondroblastic cells, osteoinduction process, that is responsible for bone repair.

DISCUSSION

The purpose of this study was to evaluate the efficacy of monoolein gel as a carrier for rhBMP-2 in the healing process of critical bone defects created in Wistar rats mandibles using the optical densitometry method.

Studies in medical and dentistry area are concentrated in the investigation of the new bone formation dynamic and in the mechanical characteristics of the newly formed bone (Jihua et al., 2002; Meyer et al., 2001; Novaes et al., 2004). In studies that it was used odontologic X-ray apparel (Landin et al., 2002; Looder et al., 2004; Mansini, 2000; Simoes et al., 2003), there is discussions about the possible variations in the exposition time, Kvp and Ma in different radiographic registers. These variations can affect the final results of the optical density.

The radiographic film (T-MAT, Kodak) and apparel Weber, type 11R, USA used in this work, permitted to obtain images of the thirty mandibles analyzed through a single radiographic exposition, removing these variables.

The Sidexis program for bone densitometry provides a numeric analysis of each dot contained in a linear trace made in a radiography using the program. These representative numbers of bone density established by the program were analyzed after a statistical study. The mean values obtained showed that there was not statistical difference between the groups 1 and 2.

The Wistar rat experimental model used in this study has several advantages such as allowing a rapid healing period, animals easily lodged and fed, resistance to climatic variations, low cost, besides being routinely used in other experimental conditions involving bone reconstruction. The mandible was chosen by being a representative bone of different signs and densities, and also for being important for the diagnosis of several diseases and anomalies (Garcia & Souza 1999; Mansini; Southard et al., 2000; Christgau et al., 1998; Ay et al., 2005; Jonasson 2005; Nackaerts et al., 2006). The inferior middle mandibular region was chosen in this model to evaluate rhBMP-2 osteoinduction capacity, since it shows trabecular and cortical bone, in addition, adequate volume for healing observation in sagittal, frontal and transversal planes.

The protein purity grade is an important factor directly influencing indexes of new bone formation. In this study, gel electrophoretic analysis followed by spectrophotometric determinations indicated that rhBPM-2 was highly pure, approximately 86%, thus explaining the large quantity of newly formed bone in the two groups of animals where it was applied. According to Wang et al., 1990, active principle purity affects not only indexes of new tissue formation but also the presence or absence of specific cellular types.

The greatest values for optical densitometry were found in the group that the growth factor rhBMP-2 was combined with monoolein gel. Although the rhBMP-2 seemed to be able to induce formation of new bone tissue by itself, its association with an immobilizing carrier probably potentiates this effect, what is in agreement with previous reports (Wozney et al., 1990). The increased osteoinductive capacity of the rhBPM-2/monoolein association observed in this study can be explained by other investigators who considered that there is a direct relationship between the osteoinductive potential of the morphogenetic protein and the local carrier retention capacity (Winn et al., 1998; Uludag et al., 2000, 2001; Kamakura et al., 2004; Maire et al., 2005). It is widely known that recombinant human morphogenetic protein type 2 (rhBMP-2), chosen for this study, is an osteoinductor protein that acts chemotactically in the differentiation of mesenchymal cells into bone synthezed cells, like osteoblasts and chondroblasts (Ducy & Karsenty 2000; Schilephake 2002; Arosarena & Collins 2005). Although the morphogenetic protein is able to promote osteoinduction by itself (Desilets et al., 1990), many studies on bone repair have shown that the process is optimized by association of the protein to a sustained release carrier.

The sustained release system used in this study, monoolein gel, is already being used as sustained release material of several drugs and medicines including proteins and peptides (Geraghty et al., 1997; Lee et al., 2005). The gels physical and chemical properties such as consistency, viscosity, biocompatibility to neighboring tissues are adequate for implantation, allowing rhBPM-2 to remain in situ and to adhere to the bone defect walls. Furthermore, they are of low cost and easily synthesized from carbohydrates and fats (Geraghty et al.; Lee et al. 2000a,b).

The results of this study showed that in both of groups it was found efficient bone repair, with a great optical density in the group that the rhBMP-2 was combined with the monoolein gel, but without statistical difference between them.

ACKNOWLEDGEMENTS. We are grateful to FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo) for financial support (04/12013-0).

Received: 11-12-2006 Accepted: 11-04-2007

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ISSA, M. J. P.; NASCIMENTO, C.; SATO, S.; BARBOSA, S. R. E.; BENETTI, T. E.; ALBUQUERQUE JR., F. R. Optical densitometry study of the newly formed bone using rhBMP-2 in Wistar rat mandibles. Int. J. Morphol., 25(2):347-352, 2007.

ISSA, M. J. P.; NASCIMENTO, C.; SATO, S.; BARBOSA, S. R. E.; BENETTI, T. E.; ALBUQUERQUE JR., F. R. Estudio densitometrico del tejido oseo neoformado por el uso de rhBMP-2 en mandiculas de ratas Wistar. Int. J. Morphol., 25(2):347-352, 2007.

Correspondence to:

Joao Paulo Mardegan Issa

Faculdade de Odontologia de Ribeirao Preto - USP

Departamento de Morfologia, Estomatologia e Fisiologia

Av. Cafe S/N, Ribeirao Preto, SP, Brazil

CEP: 14040-904

E-mail: jpmissa@forp.usp.br

* Joao Paulo Mardegan Issa; * Cassio do Nascimento; * Sandra Sato; * Rodrigo Edson Santos Barbosa; * Emerson Toffanello Benetti & ** Rubens Ferreira de Albuquerque Junior

* Graduate student, Faculty of Dentistry, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil.

** Professor, Faculty of Dentistry, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil. Grant 04/12013-0. Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil.
Table I. Means (pixels) and standard deviations of the group 1 (rhBMP-2
in aqueous solution) and group 2 (rhBMP-2 combined with monoolein gel)
in 15 animals

      Group 1                Group 2

Mean     [+ or -] SD   Mean     [+ or -] SD

180.91      10.82      190.08      10.93
209.79       8.55      229.71      14.96
214.79      19.86      208.79      13.14
179.04      6,51       199.09      11.18
17,198      19.02      192.91      11.19
2t0.01      10.76      218.09      14.17
199.24      18.91      189.26      12.26
225.59      22.50      255.60      17.14
169.64      9,13       169.74      10.08
184.41      7.97       210.96      13.96
170.43      8.81       197.34      11.36
190.28      28.73      198.30      11.18
183.93      14.35      215.35      16.75
184.00      14.31      205.07      12,54
l79.04      6.51       214.93      15.03
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Author:Issa, Joao Paulo Mardegan; do Nascimento, Cassio; Sato, Sandra; Barbosa, Rodrigo Edson Santos; Benet
Publication:International Journal of Morphology
Date:Jun 1, 2007
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