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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.25 n.3 Temuco sep. 2007

http://dx.doi.org/10.4067/S0717-95022007000300006 

 

Int. J. Morphol., 25(3):511-518, 2007.

 

Biochemical Analysis by Calcium Determination on the Newly Formed Bone in Critical Bone Defects Submitted to Different Treatments. A Pilot Study

Análisis Bioquímico para la Determinación de Calcio en Hueso Neoformado con Defectos Críticos Sometidos a Diferentes Tratamientos. Estudio Piloto

 

*Joao Paulo Mardegan Issa; **Sergio Olavo Petenusci; **Selma Siéssere;**Simone Cecilio Hallak Regalo; **Ruberval Armando Lopes; **Miguel Ángel Sala Di Matteo; **Mamie Mizusaki Iyomasa & ***Helton Luiz Aparecido Defino

* Graduate student, Faculty of Dentistry, University of Sao Paulo, Ribeiráo Preto, Sao Paulo, Brazil.
** Professor, Faculty of Dentistry, University of Sao Paulo, Ribeiráo Preto, Sao Paulo, Brazil.
*** Professor, Faculty of Medicine, University of Sao Paulo, Ribeiráo Preto, Sao Paulo, Brazil.

Dirección para correspondencia


SUMMARY: The aim of this study was to evaluate the calcium dosage of the newly formed bone, in critical bone defects created on the calvarial tissue and filled by different substances, in male Wistar rats. A cylindrical critical bone defect (6x2mm) was created in the posterior portion, left side, on the calvarial bone in nine animals and filled with: animals' coagulum, monoolein gel, poloxamer gel, monoolein gel + 1µg rhBMP-2, monoolein gel + 3µg rhBMP-2, monoolein gel + 7µg rhBMP-2, poloxamer gel + 1µg rhBMP-2, poloxamer gel + 3µg rhBMP-2, poloxamer gel + 7µg rhBMP-2. The results of this study showed a positive correlation between the calcium dosage analysis and the newly formed bone.

KEY WORDS: Bone repair; Biomaterials; rhBMP-2; Carriers; Calcium.

RESUMEN: El propósito de este estudio fue evaluar el contenido de calcio del hueso neoformado, en defectos óseos críticos provocados en la bóveda craneana y rellenados con diferentes substancias, en ratas Wistar machos. Un defecto crítico cilindrico (6x2mm) fue provocado en el lado izquierdo de la porción posterior del hueso de la bóveda craneana, en nueve animales y rellenado con coágulo sanguíneo del propio animal, gel de monoleina, gel de poloxamer, gel de monoleina + 1µg,g de rhBMP-2, gel de monoleina + 3µg rhBMP-2, gel de monoleina + 7µg rhBMP-2, gel de poloxamer + 1µg rhBMP-2, gel de poloxamer + 3µg rhBMP-2, gel de poloxamer + 7µg rhBMP-2. Los resultados de este estudio mostraron una correlación positiva entre el contenido de calcio y el hueso neoformado.

PALABRAS CLAVE: Reparación ósea; Biomateriales; rhBMP-2; Transportadores; Calcio.


INTRODUCTION

Osteogenic factors, and particularly members of the transforming growth factor (TGF-) superfamily, play a key role in bone formation and repair (Wozney and Rosen, 1998). BMP-2, as the prototype of the bone morphogenetic proteins, induces bone regeneration and ectopic bone formation in adult vertebrates (Reddi, 1997, 1998) and determines important steps during early embryogenesis and limb/wing development in animals (Hogan, 1996). Accordingly, BMP-2 and its close relative BMP-4 have been studied intensively in developmental biology, and these proteins are of interest for therapeutic applications. Althoµgh the protein is usually supplemented to the medium in a soluble form in vitro, there are constraints for sustained delivery due to the need to provide bioactive protein for an extended period of time in vivo. An ideal osteoinductive carrier for bone morphogenetic protein-2 (BMP-2) delivery should consist of a biomaterial that is able to deliver protein over the required physiological timeframe, without compromising the biological activity of the molecule or the surrounding tissue upon release. In addition, the scaffolds should biodegrade at a rate commensurate with new extracellular matrix production and be biocompatible.

Different biomaterials have been used as BMP-2 delivery systems in ectopic bone formation: poly(D,L-lactide) disks (Winn etal., 1999) poly(D,[SCAP]L-lactide-co-glycolide) (PLGA) porous scaffolds(Whang et al. 1998) and microparticles entrapped inside calcium-phosphate cement disks (Ruhe et al, 2003), collagen in the form of sponges (Winn et al; Uludag et al, 1999 and Uludag et al, 2001) and gels (Kim & Valentini, 2002), deorganified bovine bone particles (Winn et al.), honeycomb-shaped hydroxyapatite (Kuboki et al, 2001) and bioglass in the form of fibers (Mahmood et al, 2001). Additionally, hyaluronic acid scaffolds loaded with BMP-2 induced differentiation of the multipotent cell line C3H10T1/2 (Kim & Valentini). Delivery of BMP-2 has been extensively studied because of its importance in cellular differentiation. Delivery systems for other morphogens or cytokines in vitro and in vivo include poly(D,L-lactide), collagen sponges for BMP-4 and BMP-6 (Uludag et al.y 1999), collagen for BMP-7 (Rutherford & Gu, 2000), polyesterurethane for a cocktail containing bone-inducing BMPs (Saad et at, 2000), poly(propylene fumarate) for TGF-61 (Vehof et at, 2002). PLGA porous scaffolds for osteoblast stimulating factor-1 (Yang et at, 2003) and tricalcium phosphate and glass-ceramic and PLGA/glass-ceramic composites and human bone for BMP-4, basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF) (Ziegler et at, 2002). In all cases, a broader set of biomaterial functional features and control of release profiles of the morphogens or cytokines would be important attributes to expand the benefits of these systems.

Monoolein is a biodegradable polar lipid that has no marked toxic effects (Ganem-Quintanar et at, 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 drµgs with various physical chemical properties, including proteins and peptides (Lee & Kellaway, 2000 a, b; Lara et at, 2005 and Turchiello et at, 2003).

Chitosan is a linear cationic polysaccharide prepared from chitin, found in shells of shrimps, lobsters and crabs. Chitosan consists of N-acetyl glucosamine and is classified according to the degree of deacetylation (DDA) (Hejazi & Amiji, 2003). It presents biocompatibility and degradation in biological medium as well as interact with some mucosal membrane due to presents positive charges in physiological pH(Mieifl/., 2002).

Poloxamers are non-ionic polyoxyethylene-polyoxypropilene-polyoxiethylene (PEOn-PPOn-PEOn) tri-block copolymers with many pharmaceutical applications. Poloxamer 407 (P407), which has a molecular weight of 12,000 Daltons and a PEO/PPO ratio of 2:1 by weight, has been the most widely used of these copolymers. Solutions of P407 at concentrations of 20% show in situ thermoreversible gelation behavior (Schmolka, 1972; Bohorquez et at, 1999; Moore et at, 2000), which permit them to be administrated in cold liquid form by syringe. After administration, P407 cold solution forms a gel in situ at the site of the injection. The P407 gel is biocompatible with cells and body fluids (Park & Park, 1996). Used in pharmaceutical preparations, its low toxicity and weak immunogenic properties make it a suitable vehicle for drµg delivery (Johnston and Miller, 1985; Veyries et at, 1999 and Paavola et at).

The aim of this study was to evaluate the calcium dosage of the newly formed bone, in critical bone defects created on the calvarial tissue and filled by different substances, in male Wistar rats.

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.

Substances. The recombinant human bone morphogenetic protein, type 2 (rhBMP-2) used in this study was obtained at Theodor-Boveri-Institut fiir Biowissenschaften, Am Hubland, Wiirzburg, Germany.

Monoolein (Myverol 18-99, 98.1% monoglycerides, Naarden, The Netherlands) gel was obtained in a 7:3 (MonooleimWater) proportion as described before (Lara et at). Briefly, monoolein was weighed and heated to 45°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 (lmg/mL) was added and the mixture homogenized.

Chitosan (Hidagem HCMF, Cognis, Spain) gel was prepared at dispersing 8.3 mg of chitosan in 1.0 mL of rhBMP-2 water solution followed of acidifying with acetic acid (1.0%).

Poloxamer 407 (Pluronicx F-127®) was purchased from BASF, Brazil.

Amounts of the gels sufficient to carry 1, 3 and 7mg of rhBMP-2 were applied in the critical bone defects.

Animals. Nine male Wistar rats (n=9), 350g, were selected and allowed to acclimatize for one week prior to surgery. The animals were fed with commercial rat chow and had access to food and water ad libitum. A cylindrical critical bone defect (6x2mm) was created in the posterior portion, left side, on the calvarial bone and filled with animals' coagulum (Group 1), monoolein gel (Group 2), poloxamer gel (Group 3), monoolein gel + 1µg rhBMP-2 (Group 4), monoolein gel + 3µg rhBMP-2 (Group 5), monoolein gel + 7µg rhBMP-2 (Group 6), poloxamer gel + 1µg rhBMP-2 (Group 7), poloxamer gel + 3µg rhBMP-2 (Group 8), poloxamer gel + 7µg rhBMP-2 (Group 9).

Surgical procedure. The rats were anaesthetized with standard anesthetic cocktail consisting of ketamine hydrochloride (60mg/Kg) and xylazine (5mg/Kg), administered intraperitoneally. Surgery was performed using aseptic techniques. Alinear incision was made throµgh the skin, and the osteotomy was performed using a high-speed drill, adjusted to 3000rpm, and abundant irrigation.

Animals sacrifice. After two weeks, the animals were anaesthetized with urethane 37.5% (0.4mL/ lOOg) and the calvarial bone samples were removed for biochemical analysis, calcium dosage analysis. The samples were inserted into individual beckers and weighed with the objective to determine the fresh weight of the samples (organic component + mineral component) and heated until 700°C, for 15 hours to determine only the mineral compound. After balance determination, the mineral component was diluted in chloridric acid IN and the calcium dosage was determined by colorimetric method, using a photocolorimeter (Micronau, Brazil) and a comercial kit (Labtest Sistemas Diagnósticos Ltda, Belo Horizonte, Brazil).

RESULTS

Clinical observations. The postoperative process was considered normal, without infection or other complications.

Calcium dosage analysis. Considering the weight of the fresh bone samples, mineral plus organic components, it was obtained the following values (mg) for the nine groups, in sequence: 18.50mg; 2.60mg; 2.70mg; 5.70mg; 10.60; 19.80mg; 10.60mg; 9.80mg; 5.90mg (Fig. 1).


Considering the calcined weight of the bone samples, mineral component, it was obtained the following values (mg) for the nine groups, in sequence: 6.50mg; 1.40mg; 5.40mg; 4.30mg; 7.10mg; 13.30mg; 8.10mg; 11.20mg; 16.50mg (Fig. 2).


Considering the calcium dosage in the calcined bone samples, it was obtained the following values (µg) for the nine groups, in sequence: 128.42big; 60.29µg; 149.56µg; 21.28(µg; 328.89µg; 822.23µg; 328.11µg; 399.37mg; 743.93µg (Fig. 3).


Considering the calcium dosage (µg/mg) of the calcined bone samples, it was obtained the following values (µg) for the nine groups, in sequence: 7.78mg; 43.06µg; 27.69µg; 4.95µg; 46;32µg; 61.82µg; 40.50µg; 61.44µg; 66.42µg (Fig. 4).


DISCUSSION

The aim of this study was to evaluate the calcium dosage by biochemical analysis of the newly formed bone, in critical bone defects created on the calvarial tissue and filled with biomaterials combined or not with rhBMP-2 is different concentrations, in male Wistar rats. It was found in this study a straightly relationship between the newly formed bone, more expressed in the groups that the rhBMP-2 was applied in more quantities, and calcium dosage. Thus, the greatest values for calcium dosage were found when it was inserted 7|µg rhBMP-2, independently of the gel carrier used. These results are interesting because they show that the newly formed bone is accompanied by biochemical alteration, represented by the mineralization of this tissue, calcium dosage analysis. Thus, it is possible to relate that besides the rhBMP-2 action to convert mesenchymal cells into osteoblastic and chondroblastic cells, osteoinduction process, the resultant tissue after its action is an organized bone, which it is interesting for clinical application in many fields of the Medical Sciences.

The Wistar rat experimental model used in this study is an excellent animal experimental model with some positive characteristics, 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 calvarial bone tissue was chosen to evaluate the bone repair because it shows favorable bone structure, trabecular and cortical bone, in addition, adequate volume to observe the healing bone process in sagittal, frontal and transversal planes.

The biomaterials used in this study, monoolein, chitosan and poloxamer gels, are already being used as sustained release media of several drµgs and medicines including proteins and peptides (Geraghty et al., 1997; Nielsen et al., 1998; Lee and Kellaway, 2000b; Paavola et al,2000;Leeetal, 2001;BonacucinaeiaZ., 2005;Bhattarai et al, 2005; Kofuji et al, 2005 and Kao et al, 2006). The gels physical and chemical properties such as consistency, viscosity, biocompatibility to neighboring tissues are adequate for implantation, allowing rhBMP-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 (Nylander et al, 1996; Geraghty et al; Lee and Kellaway, 2000b; Shah et al, 2001; Lee et al, 2005 and Boyd et al, 2006).

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 rhBMP-2 was highly pure, 86% of purity, explaining the large quantity of newly formed bone found in animal groups where it was applied and a positive correlation between these newly formed bone and calcium dosage analysis.

The effects of growth factor dose are intensely investigated not only in terms of efficiency but also cost. In this study bone defects with 6x2mm were treated with 1, 3 and 7Mg of rhBMP-2 during 2 weeks before animals sacrifice and analysis. Other authors, like Yasko et al, 1992, reported bone regeneration after creation a critical bone defect with 5mm of extension in rat femurs and insertion of 11 µg rhBMP-2, by mechanical, radiographic and histological evidence while a much smaller dose, lAµg rhBMP-2, produced new bone tissue, but not sufficient to close the defect. Higher dosages, as 20mg of rhBMP-2 applied to supra-alveolar bone defects, 5mm of height, in Beagle dogs showed substantial bone regeneration after 8 weeks (Sigurdsson et al, 1995). On the other hand, extremely high dosages as 50 \ig rhBMP-2 combined with collagen matrix and applied to 4mm defects in rat skulls were capable to produce new bone formation (Inoda et al, 2004), but did not show advantages in relation to smaller and sustained release dosages as shown by Wang et al. (1990) who tested 0.5 to 115 |LLg dosages of rhBMP-2.

The results of this study showed a positive correlation between the calcium dosage analysis and the newly formed bone.

 

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Correspondence to:

Dr. Joao Paulo Mardegan Issa
Faculdade de Odontología de Ribeirao Preto - USP
Departamento de Morfología, Estomatología e Fisiología
Av. Café S/N
CEP: 14040-904
Ribeirao Preto, SP, BRAZIL

Phone: +55-16-36024095 Fax: +55-16-36330999

E-mail: jpmissa@forp.usp.br

Received: 15-03-2007 Accepted: 22-06-2007

 

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