Biocompatibility and osteogenesis of the castor bean polymer doped with silica ( SiO 2 ) or barium titanate ( BaTiO 3 ) nanoparticles 1

PURPOSE: To evaluate the biocompatibility and osteogenesis of castor oil polymer doped with SiO2 or BaTiO3 nanoparticles. METHODS: Twenty four male rats Wistar were submitted to bone defect filled with castor oil polymer. The animals were distributed in two experimental groups had been formed with 12 animals each: Group 1 Castor oil polymer doped with 0.30 grams of SiO2 replacing 0.30 grams of CaCO3. Group 2 Castor oil polymer doped with 0.30 grams of BaTiO3 replacing 0.30 grams of CaCO3. Euthanasia occurred 30 and 60 days after surgery and the femurs were sent to histological analysis and MEV. RESULTS: The implants were biocompatible and allowed for progressive osteogenesis through osteoconduction in both observation periods. There was significant bone neoformation at 30 and 60 days in both groups within the histomorphometric evaluation, but group 1’s osteogenesis was lesser in the 30 and 60-day periods observed when compared to the animals of group 2. The MEV morphometric evaluation evidenced a lesser percentage of osseous tissue filling within the BaTiO2-doped polymer. CONCLUSION: The castor oil polymer doped with SiO2 or BaTiO3 remained biocompatible and allowed for progressive osteogenesis in both observation periods.


Introduction
The history of tissue repair using biomaterials can be traced back to the Prehistoric Period and, during a long time, happened in a very empirical manner, expecting only they would be inert, non-toxic and well-tolerated 1 .The evolution in the biomaterials' employ involved the search of devices that stimulated the implant's integration with the nearest tissues through cellular activation, proliferation and differentiation in the implant's site, as well as the subsequent formation of the extracellular matrix intimately associated with the implanted material 2 .Among all the favorable aspects expected in a biomaterial, allowing for osteogenesis in the bone-implant interface, optimizing the osteoblasts' differentiation, is primordial from a clinical point of view 3 .In this sense, porous materials that work as support for bone growth, working as an outline that ensures the cellular infiltration to the implant's interior, have become noticed and are highly applied in experimental studies 4 .
Experimental studies have shown the applicability in vivo 5,6 and the benefits for castor oil polymer in vitro 7,8 , revealing the biocompatibility, osteointegration and, mainly, the diameter, conformation and intercommunication of the pores as the most important aspects to regulate vascular and cellular migration to the inside of these implants, allowing for bone neoformation.
However, despite the advantages presented by the castor oil polymer, an implant will hardly harbor all the ideal properties for bone replacement, especially when compared to autogenous grafts.In this context, the development of new composite materials that resemble natural bone structure, and capable of concentrating traits so as to ease osteogenesis promotion, has been the challenge of many researchers 9 .
Many studies pointed out promising results in the use of mesoporous silica (SiO 2 ) based biomaterials in osseous tissue repair processes 10,11 .According to Colilla et al. 12 , when these biomaterials are exposed to physiological environment, a series of chemical reactions happen in the tissue/material interface, leading to the material's incorporation to the living tissue.
Other studies have sought new ways to stimulate osteogenesis, through electrically-charged materials 13 .Studies involving piezoelectric ceramics for implantation were focused, for the most part, in materials containing barium titanate (BaTiO 3 ).BaTiO 3 is a ferroelectric ceramic characterized by the presence of spontaneous polarization and the ability to guide osteogenesis towards polarization.
Just like with the bone, the source of piezoelectric behavior in electric iron materials comes from the formation of an electric dipole due to an asymmetrical ion distribution 14 .
The study of the interaction of bioceramics with distinct osteogenic traits in the production of composite biomaterials, aiming to optimize the implant's properties, will allow for the development of an ideal framework for bone regeneration.As such, the aim of this study was to evaluate the biocompatibility and osteogenesis of castor oil polymer doped with SiO 2 or BaTiO 3 nanoparticles.

Implants' presentation and preparation
The implants were prepared following the instructions recommended by the maker (BIOMECÂNICA Ind. e Com. de Prod.Ortopédicos Ltda).Castor oil polymer is obtained by mixing the pre-polymer and poliol (Liquid fraction), and the calcium carbonate (CaCO 3 ) (Powder fraction) is added to the polyurethane's basic components aiming to give it porosity and improve the base resistance and elasticity 9,10 .
The SiO 2 nanoparticles were prepared by the UFMS Materials group through Microemulsion mediated sol-gel and the particle size control was taken through surfactant usage.The BaTiO3 nanoparticles were prepared by the University Center of Grande Dourados (UFGD)'s Physics Department and synthesized through High-Energy Ball Milling (HEBM).Both nanostructured particles had a clear, dry powder state.
To prepare the biomaterial blocks, the components were inserted into a Becker and weighed individually, in preset proportions per group, and with each component insertion, the weighs were zeroed for the next insertion.After all components were inserted, they were mixed until they gained an aspect of mass with great adherence.Afterwards, the biomaterial was placed in a Teflon plate and pressed to create blocks 1.00mm thick with a diameter 2.00mm wide.The blocks obtained were sterilized individually through ethylene oxide to be used in the operatory procedure.

Animal characteristics and formation of experimental groups
Twenty-four Wistar rats, adult males, weighing between 250 and 300 grams, aged three months, were obtained from UNIGRAN's Central Animal Laboratory.Throughout the entire experiment the animals were kept in 12 hour photoperiods, with minimal noise, solid rations and water ad libitum, staying under watch for a period of two days, before their use in the experiment.
The animals were spread randomly in two distinct experimental groups, according to the implanted material.In group 1's animals, the bone defect was filled with castor oil polymer doped with 0.30 grams of SiO 2 , equal to 10% the powder fraction, replacing 0.30 grams of CaCO 3 .In group 2, the animals had their bone defect filled with castor oil polymer implants with 0.30 grams of BaTiO 3 (10% of the Powder fraction) added, replacing 0,30 grams of CaCO 3 .

Surgical procedure
After a semiologic exam realized by the veterinarian, the animals received pre-anesthetic medication with Ketamine at 5% (50mg/kg) (Sepso Ind. e Com.Ltda, Jacareí, SP) plus Xilasina a 2% (10mg/kg) (Sepso Ind. e Com.Ltda, Jacareí, SP), through intra-peritoneal and for anesthetic maintenance Isoflurane was administered via inhalation, through a facial cone with oxygen and universal vaporizer with a 1 L.min -1 flux.
Having stated the anesthetic plan, the animals were placed for fur rasping on the side of their lower right leg.With aseptic technique, access to the diaphysis of the femur in the proximal (cranium-lateral) side was performed for confection of the bone defect.After bone exposure, a defect of 2mm diameter was performed using a spherictype #1016 (KG Sorensen) diamond-tipped drill under constant irrigation with physiological serum perforating the bone cortical until reaching the medullar canal.After implanting the composite, synthesis through plans with polyamide nylon 0.30 needlepoint was performed.

Euthanasia
After the 30 and 60-day observation periods, the animals were submitted to euthanasia with intraperitoneal infusion of lethal dose of sodium Thiopental (120mg/ml).

Histomorphometric and morphometric through Scanning Electron Microscopy (SEM)
The total areas of the implant and bone neoformation, both in the histological blade images, as in the images taken through SEM, were calculated through the manual delimitation of areas and the measuring was made using the ImageJ 1.0 for Mac program.Initial measurements for the areas (pixels) were converted to µm, at a 1 to 6.25 proportion (1 pixel = 6.25 µm).For statistical analysis the proportions of bone neoformation were considered, through the quotient between the neoformation area and the total defect area (proportion of neoformed bone: neoformed bone area/ total defect area), being expressed as a percentage.
Images of the blades destined to histomorphometric scans were captured through an Olympus ® Japan -BX41 light microscope, tinted with Hematoxylin and Eosin and the standardized 10X increase to allow for general observation of the implanted area and the nearby tissue.
To obtain the SEM images a JEOL-brand JSM-6380LV-model scanning electron microscope from UFMS' Multiuser Laboratory of Materials Analysis (MULTILAM) was used.The images of the transversally-sectioned pieces, sent for morphometric analysis in the SEM, were captured using Backscattering Electrons (BSE), which allow for a contrast increase between structures of different compositions and densities, favoring the differentiation between the implant and osseous tissue.

Statistical analysis
Data was organized so as to compare the percentage values of bone neoformation in relation to the implanted polymer area.Intergroup comparisons were performed through the Mann-Whitney Test.The significance level considered was 95% (p<0.05).
Biocompatibility, traits in the process of tissue neoformation, osteogenesis and the morphological and structural aspects of the biomaterials were analyzed and the results are expressed in a descriptive manner.

All animals had an uneventful postoperative course
and exhibited normal behavior.The presence of SiO 2 or BaTiO 3 in the castor oil polymer did not affect the material's properties, which remained biocompatible, retained the internal porosity, and revealed itself to be capable of promoting osteoconduction.

Descriptive histopathological evaluation
In Group 1's animals, after 30 days, small areas of bone neoformation in the bone-implant interface were noticed.
Aside from the apposition of neoformed osseous tissue in the edges of the bone defect, discreet osteogenic activity towards the implanted material's interior was noticed.After 60 days, increase of bone growth around and inside the implant was observed, with abundant presence of mature osseous tissue and intense fibroblastic activity producing the collagenous matrix that precedes bone mineralization.The bone occupation within the biomaterial reinforces the osteointegration trait and suggests that the castor oil polymer doped with SiO 2 can be reabsorbed (Figure 1).G2's animals' histological evaluation at 30 days revealed discreet bone neoformation around the implant, however, with intense fibroblastic activity towards the defect's interior, characterizing the formation of the immature bone matrix that will replace the mature osseous tissue.As well, the presence of osteoclasts promoting material degradation for posterior osteogenesis is noticed.At 60 days, the bone defect was partially occupied by lamellar bone rich in osteocytes, few inflammatory mononuclear cells, and reduction in osteoblastic activity.Great proximity of mature osseous tissue with the biomaterial was noticed as well, suggesting osteointegration (Figure 2).

Scanning electron microscopy: descriptive morphological evaluation
Images obtained through secondary electrons (SE) and through backscattering electrons (BSE) of the animals belonging to group 1, with 30 days of observation, displayed endosteum bone growth, well-integrated interface between the neoformed bone and the implant and the biomaterial's pores displaying abundant osteoprogenitor cell activity secreting the initial bone matrix.After 60 days of observation, the implants matching group 1's animals were wrapped by osseous tissue.The bone neoformation happened through the periosteum and endosteum, small pores next to the interface were filled by mature osseous tissue and the implant remains integrated to the bone through an immature, still forming bone matrix (Figure 3).Group 2's animals, after 30 days of implanting, displayed discreet osteogenesis and little cell activity around the implant.The material remained well coapted with the neoformed bone around it, even with alterations of the biomaterial's surface in relation to the bone.At 60 days, there was increase of osteogenesis in the interface, with bone growth towards the inside of the biomaterial, however, there was little bone neoformation happening through the periosteum and endosteum.A few pores of varying diameters were noticed next to the interface, filled in with mature osseous tissue, and osteogenic cells promoting bone formation over the implant (Figure 4).

Histomorphometric analysis
The results of the histomorphometric analysis of the groups and periods studied are represented on Tables 1 and 2

Discussion
Many studies 5,7,10 point out the advantages of using castor oil polymer as a framework for bone growth, however, castor oil polymer integrates itself to the receiving bone in a slower and more incomplete manner when compared to autogenous bone graft 15 , especially for not displaying specific biological traits.
In the present study, the animals of group 1 received castor oil polymer implants doped with SiO 2 and the animals of group 2 with BaTiO 3 , both replacing the equivalent to 10% of CaCO 3 in their respective groups, with the aim of optimizing the material's characteristics.Morphometric intragroup analysis of the bone neoformation percentage in relation to the polymer implant area showed there was significant increase of osteogenesis around and within the polymer in both groups.
Saran et al. 9 also stated the osteogenesis progression after implanting castor oil polymer in the medullar canal of rabbit tibia.According to the authors, at the 90-day post-implant period, deposition of immature osseous tissue in contact with the implant could still be observed.The neoformed bone around the biomaterial took on a lamellar aspect as time went on, after 120 and 150 days of observation.
Del Carlo et al. 16 observed the migration of osteoprogenitor cells invading the surface porous structure of castor oil polymer, followed by the direct bone deposition and concluded that castor oil polymer behaves like a passive framework that allows for progressive bone neoformation and osteoconduction, however, the biomaterial was not capable of promoting osteoinduction.
According to Leonel et al. 17 , castor oil polymer revealed itself to be a supporting material to the regenerative process of bone defects created experimentally in the zygomatic arc of rats.To the authors, bone neoformation, which was greater in the final periods of examination, happened through osteoconduction, given that castor oil polymer allowed for tissue growth among its pores and over its external surface since the starting periods of observation.
In this study, at the end of the observation period (60 days), there was no meaningful difference with regards to the percentage of bone neoformation around and within the implants during intergroup comparison, which suggests similarity in the nanoparticles' osteogenetic potential.
The biological behavior of the SiO 2 and BaTiO 3 nanoparticles, as well as the manner which each ceramic stimulates osteogenesis, is quite divergent.While SiO 2 has an important role in living tissue biomineralization and its ability to induce osteoprogenitory cells to the lesion's location 18 , BaTiO3 stimulates bone neoformation due to its dielectric and piezoelectric properties 14 .
Nacer et al. 10 evaluated the castor oil polymer doped with silica nanoparticles' in vivo behavior, and confirmed an increase in number of osteoprogenitory cells around and within the composite when compared to the isolated castor oil polymer implant.
According to the authors, the success of castor oil polymer with 10% silica can be attributed to SiO 2 's osteoinducing nature, along with the nanoparticles altering the support's morphology, granting great coarseness to the external surface.
According to Henson and Getgood 19 , the presence of silica nanoparticles added in scaffolds has an important role in osteogenesis, for they can simulate the extracellular matrix's internal architecture, fundamental tissue-material interaction.
Terriza et al. 20 , upon evaluating SiO 2 's biocompatibility and bioactivity in poly (D,L-lactic acid lactic-co-glycolic acid) (PLGA), concluded that bioactive inorganic materials, like SiO 2 granulates, improve synthetic biopolymers' osteogenic performance due to their excellent chemical stability and the tight relationship between silicate and calcium deposition on its surface.
Studies centered in utilizing electrically-charged materials have shown promising results in stimulating osteogenesis.Feng et al. 21implanted hydroxyapatite and BaTiO 3 ceramics in dog mandibles and confirmed that BaTiO 3 implants promoted greater growth and bone repair.According to the authors, tissue growth around the implant happened in an orderly manner, which increased the efficacy of osteogenesis in the BaTiO 3 ceramic's surface.Yu et al. 22 concluded that BaTiO3's piezoelectric capacity is greater when implanted, when compared to synthetic hydroxyapatite.
Ciofani et al. 23 evaluated the effects of barium titanate nanoparticles over proliferation and differentiation of the mesenquimal cells of rats, and concluded that the BaTiO 3 nanoparticles promoted a meaningful increase in the formation of hydroxyapatite deposits during bone neoformation.

Conclusion
Castor oil polymer doped with BaTiO 3 or SiO 2 nanoparticles behaved like a biocompatible framework, capable of allowing for progressive bone neoformation within its communicative porous structure, promoting induction and aggregation of bone cells over its surface and, subsequently, speed up the osteogenesis process.
the experimental procedures were analyzed and approved by the Ethic Committee in the Use of Animals of UFMS with protocol number 494/2013 and complied with the Council for International Organization of Medical Sciences (CIOMS)'s ethical code for animal experimentation.

FIGURE 3 -
FIGURE 3 -Group 1's animals' MEV-taken photomicrograph montage after 30 (A and B) and 60 (C and D) days of observation.A -Implant's transversal dimensions.BSE, x40.B -Biomaterial's pores containing osteoprogenitor cells in activity.SE, x5000.C -General view of implant and the transversal dimensions of the biomaterial and neoformed bone tissue.BSE, x30.D -Immature bone matrix being formed in the bone-implant interface.SE, x1000.

FIGURE 5 -
FIGURE 5 -Comparison of the value averages of bone neoformation in relation to implanted polymer area to the MEV reading after 30-day experiment period (Mann-Whitney, p = 0.0209).

FIGURE 6 -
FIGURE 6 -Comparison of the value averages of bone neoformation in relation to implanted polymer area to the MEV reading after 60-day experiment period (Mann-Whitney, p = 0.2482).

FIGURE 7 -
FIGURE 7 -Comparison of the value averages of bone neoformation in relation to implanted polymer area to the histological analysis after 30day experiment period (Mann-Whitney, p = 0.0433).

FIGURE 8 -
FIGURE 8 -Comparison of the value averages of bone neoformation in relation to implanted polymer area to the histological analysis after 60day experiment period (Mann-Whitney, p = 0.3367).

TABLE 1 -
Intergroup comparison of average values ± standard deviation of bone neoformation percentage in relation to implanted polymer area.

TABLE 2 -
Intragroup comparison of average values ± standard deviation of bone neoformation percentage in relation to implanted polymer area.