Original Research Article
Comparative ultrastructure evaluation of particle biomaterial of ovine origin
Joo Csar Zielak1
Allan Fernando Giovanini1
Tatiana Miranda Deliberador1
Ccero de Andrade Urban1
Corresponding author:Joo Csar Zielak5,300 Professor Pedro Viriato Parigot de Souza St. Campo CompridoZIP code 81280-330 Curitiba PRE-mail: firstname.lastname@example.org
1 Dental School, Positivo University Curitiba PR Brazil.
Received for publication: September 13, 2010. Accepted for publication: October 7, 2010.
Introduction: Xenogenic bone grafting biomaterials have been vastly studied. Easy clinical use and low cost have attracted professionals and patients. Alternatively to bovine biomaterial, ovine biomaterial can be developed. Objective: To compare by scanning electron microscopy the ultrastructure of a particle ovine biomaterial to equine and bovine commercially available biomaterials, besides to human bone from a tissue bank. Material and methods: Ovine mandibular bone, from a local sheep farm, was ground and submitted to a chemical and physical sterilization protocol to be compared to different biomaterial particles. Images were obtained and evaluated according to the quantity and area values of porosities (2), visual surface roughness, and Ca/P ratio. Results: There was no statistical difference among the sample images; however, the bovine particles presented marked porosities and roughness. Conclusions: In relation to porosity, experimental ovine bone presented closer characteristics to human bone than equine and bovine. Regarding to surface roughness, equine bone was the most similar to human bone, followed by ovine and bovine. Concerning to Ca/P ratio, ovine bone presented the lowest and most distant value from human bone, followed by equine and bovine.
Keywords: bone grafting; experimental; ultrastructure.
In Dentistry, in the event of great bone loss, grafting is essential for aesthetic and functional correction. Costa and Veinstein  reported the classical grafting division: 1) autogenous or self bone, i.e., when the tissue is transferred from one area (donor site) to another (receptor site) in the same subject, not provoking an immune reaction; 2) allogeneic, homogenous or homograft, i.e., tissues grafted among individuals of the same species with non-identical genes, such as fresh, frozen, lyophilized bone (FDBA), demineralized and lyophilized bone (DFDBA); 3) alloplastic, foreign body, inert, synthetically produced, used for tissue grafting, such as calcium phosphate, hydroxyapatite, bioceramic, among other types; and 4) xenografts, heterografts or heterologous, performed among individuals of different species, for example, from animal origin, such as the bovine bone, or from other animal vivarium, such as the equine bone. For each graft type, several materials are employed, generically named as biomaterial grafts.
Non autogenous materials of the aforementioned classification has evoked a great interest by the professionals, since the surgical procedure is faster, because the material to be grafted is already prepared, only requiring the receptor site. This reduces the risks due to the lack of donor site wound, being very advantageous for the patient [5, 14].
Biomaterials criteria selection and processing technique should be ef f icient and sa fe. By understanding the nature of the materia ls processing, particularly the inf luence of the several techniques of biological and biomechanical preservation and/or the processes of incorporation and association among materials, each biomaterial is selected according to the surgical reconstruction goal. Currently, the availability of different biomaterials have increased, and the agility in the acquisition of treatment techniques provided an opportunity of innovative ways of surpassing the countless reconstructive challenges besides offering to many people a more satisfactorily treatment option .
The use of bovine bone as a source of raw material for the production of grafts biomaterial has been studied for some time . After harvesting, the bone processing is carried out in order to remove the cellular rests, lipids and proteins. It is possible that these substances, of great potential for the receptors immune system activation, interfered in grafts biocompatibility. Bone treatment with peroxides for matrix decellularization helps to
maintain the collagen-apatite complex and produces a higher mechanical resistance of the obtained material .
Material surface is capable of influencing the cellular colonization. Marins et al.  characterized the particles of an organic bovine biomaterial by scanning electronic microscopy and evaluated its application in vivo (rats) by light microscopy. The results demonstrated that the studied material, with its preserved micro-architecture, is slowly resorbed and can act as a space for cellular filling, enabling the angiogenesis, cellular migration, bone adhesion and neoformation at the lesions edge in which it was grafted.
Similar to bovine animals, other animals reared, on a regular basis and a large scale, are a source for graft biomaterials production. By comparing, for example, the bovine to ovine cattle, some factors should be taken into consideration: both can be fed only with pasture, because they are ruminant animals; sheep adapt better to climatic conditions and require a smaller physical space of pasture; besides that, the onset of their breeding and slaughtering is between 6 and 9 months , while bovine, generally, need to reach 24 months ; also ovines gestation period is only five months, while bovine demands 10 months [13, 17].
The possibility of disease transmission is other relevant factor that causes apprehension when animal origin biomaterials are an issue. Among the scariest diseases regarding to either bovine or ovine animals, is the transmissible spongiform encephalopathy (TSEs), which can be caused by the accumulation of modified glycoproteins on the individuals nervous tissues. The modified glycoproteins belong to a class of substances, so called prions. As it is known, TSEs occurring in humans have the same causative agent as bovine and ovine encephalopathies ; however, although prions can be resistant, there are methods to neutralize them .
Since study reports on ovine origin biomaterials are scarce, the aim of this study was to compare, through scanning electronic microscopy (SEM), the ultrastructure of an experimental particle biomaterial of ovine origin to commercially available equine and bovine bone and human bone particles coming from a muscle-skeletal tissue bank.
material and methodsThe ovine material was harvested from the
mandible bone of a sheep (Suffolk), 6-months old, coming from the Cabanha Kulik vivarium, located at Barreirinha neighborhood, Curitiba, Parana, Brazil.
The animal was slaughtered for feeding purposes by the vivariums employees, and its head was discarded. The same employee stored the sheeps head in a freezer (-12C). Next, at the laboratory, the mandible was separated and had its surface cleaned by the removal of soft tissue remnants (periosteum and muscular insertion) through knife and scalpel (Duflex, Curitiba, Parana, Brazil). Following, the sheeps mandible was washed in tap water and divided into fragments of about 2.0 x 1.0 x 0.5 cm. Some fragments were submitted to the following procedures: 1) immersion on 2% sodium hypochlorite, at 20C, for 12 hours, 2) washing in tap water (2 minutes), 3) immersion on 2% sodium metabisulphite solution, for 3 minutes, for hypochlorite inactivation, 4) washing in tap water (2 minutes), 5) drying through dry heat, at 20C, for 2 hours, 6) fragments grinding in electrical bone grinder (Kopp, Curitiba, Parana, Brazil), and 7) wet heat (autoclave), for 20 minutes, at 135C. By using the aforementioned procedures, a small bone fragment was grinded, coming from a surgical procedure and stored in the Muscle-skeletal tissue Bank of the Clinic Hospital of the Federal University of Parana.
Next, some ovine and human bone particles were stored in a desiccator with silica, mounted on a SEM support, and metalized (Baltec, Balzers, Germany). Other fragments of the commercially available biomaterials were also metalized and mounted on the support: equine (Spongy BIO-GEN BGS-05, Bioteck, Chieri, Torento, Italy) and bovine bone (Genox Inorg Spongy, Baumer, Mogi-Mirim, Sao Paulo, Brazil). The supports were taken into electronic microscopy for chemical and ultrastructural morphological evaluation of the surface (electrons dispersive energy spectroscopy) (SEM-EDS, JSM, 6360-LV, Jeol, Japan). The obtained images were analysed and compared by the aid of image analysis software (Image Tool 3.00, University of Texas Health Science Center, USA).
Through SEM image analysis, the visual aspect of the superficial roughness and porosity of the particles was observed at x 30, 150, 1,500, and 10,000 magnification (figure 1).
Human bone Ovine bone