See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/305716254 Comparative study of Bone tissue accelerated regeneration by latex membranes from Hevea brasiliensis and Hancornia speciosa Article · July 2016 DOI: 10.1088/2057-1976/2/4/045007 CITATIONS 7 READS 192 11 authors, including: Some of the authors of this publication are also working on these related projects: Leishmania View project NEW HETEROLOGOUS FIBRIN SEALANT AS BIOLOGICAL SCAFFOLD CANDIDATE FOR STEM CELLS View project Juliana Ferreira Floriano São Paulo State University 13 PUBLICATIONS   73 CITATIONS    SEE PROFILE Lígia Souza Lima Silveira da Mota São Paulo State University 49 PUBLICATIONS   196 CITATIONS    SEE PROFILE Edson Luiz Furtado São Paulo State University 193 PUBLICATIONS   936 CITATIONS    SEE PROFILE Rui Seabra Ferreira Jr São Paulo State University 109 PUBLICATIONS   732 CITATIONS    SEE PROFILE All content following this page was uploaded by Lígia Souza Lima Silveira da Mota on 19 June 2018. 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Please scroll down to see the full text. Download details: IP Address: 186.207.249.52 This content was downloaded on 28/07/2016 at 15:50 Please note that terms and conditions apply. Comparative study of bone tissue accelerated regeneration by latex membranes from Hevea brasiliensis and Hancornia speciosa View the table of contents for this issue, or go to the journal homepage for more 2016 Biomed. Phys. Eng. Express 2 045007 (http://iopscience.iop.org/2057-1976/2/4/045007) Home Search Collections Journals About Contact us My IOPscience iopscience.iop.org/page/terms http://iopscience.iop.org/2057-1976/2/4 http://iopscience.iop.org/2057-1976 http://iopscience.iop.org/ http://iopscience.iop.org/search http://iopscience.iop.org/collections http://iopscience.iop.org/journals http://iopscience.iop.org/page/aboutioppublishing http://iopscience.iop.org/contact http://iopscience.iop.org/myiopscience Biomed. Phys. Eng. Express 2 (2016) 045007 doi:10.1088/2057-1976/2/4/045007 PAPER Comparative study of bone tissue accelerated regeneration by latex membranes fromHevea brasiliensis andHancornia speciosa Juliana Ferreira Floriano1, FaustoCapuanoNeto2, Lígia Souza Lima Silveira daMota3, EdsonLuiz Furtado4,10, Rui Seabra Ferreira5,6, Benedito Barraviera5,6, Pablo JoséGonçalves7, LucianeMadureira deAlmeida8, FelipeAzevedoBorges9, Rondinelli DonizettiHerculano9 and Carlos Frederico deOliveiraGraeff1 1 Department of Physics, Júlio deMesquita Filho StateUniversity of São Paulo, Bauru (SP), Brazil 2 Department of Tropical Diseases, Júlio deMesquita Filho StateUniversity of São Paulo, Botucatu (SP), Brazil 3 Department ofGenetics, Júlio deMesquita Filho StateUniversity of São Paulo, Botucatu (SP), Brazil 4 Department of Plant Protection, Júlio deMesquita Filho StateUniversity of São Paulo, Botucatu (SP), Brazil 5 Department of Tropical Diseases andDiagnostic Imaging, Júlio deMesquita Filho StateUniversity of São Paulo, Faculty ofMedicine, Botucatu (SP), Brazil 6 Center for the Study of Venoms andVenomous Animals (CEVAP), Júlio deMesquita Filho StateUniversity of São Paulo, Botucatu (SP), Brazil 7 Institute of Physics, Federal University ofGoiás, Goiânia (GO), Brazil 8 StateUniversity ofGoiás, Ipameri (GO), Brazil 9 Biomaterials Group, Chemistry Institute Júlio deMesquita Filho StateUniversity of São Paulo, Araraquara (SP), Brazil 10 CNPq scholarship holder. E-mail: juli@ibb.unesp.br Keywords:Hevea brasiliensis,Hancornia speciosa, membrane, bioactivity, bone regeneration Supplementarymaterial for this article is available online Abstract Bone loss is a common problem after accidental traumas, cancers, congenital defects, and surgical procedures. The techniques normally used in large bone restoration involve complex and invasive procedures such as grafting. Thus, it is of interest to develop alternatives such as bioactivematerials to induce accelerated bone regeneration. Natural rubber (NR)membranes are potential candidates due to their characteristics such as biocompatibility, angiogenic potential, flexibility,mechanical stability, surface porosity, and permeability. The present study aims at assessing the osteogenic potential ofNR membranes of clones of high bioactivity ofHevea brasiliensis (RRIM600 and IAN873) and of Hancornia speciosa, as well as Physicochemical characterization at theNRmembranes by scanning electronmicroscopy, Fourier transform infrared spectroscopy, and tensile tests. Critical-size bone defects were surgicallymade in adultmale rabbit calvarium. Afibrin sealant (FS)was used tofix the membranes as a replacement for cyanoacrylate.We compared the respective osteogenic potentials of the testedmembranes against a control group in healthy animals. The newbone formedwas characterized using radiography, x-ray tomography, and histological andmorphometric studies. Our results show that bothmembranes have great potential for regenerating bone tissue, with higher bioactivity compared to the gold standard (PTFE), whichwas used as positive control. In bothNR membranes the stress–strain profile shows low stress at small strain, characteristic of elastomerwith a low degree of reticulation, followed by an increase in the stress at high deformation and twomain differences between bothNRLbiomembranes, related to its composition. The FS acted satisfactorily in the tests, being highly recommended as a substitute for cyanoacrylate in this type of application. 1. Introduction Large osseous defects resulting from traumas, cancers, congenital defects, or surgical procedures pose significant reconstructive problems. Generally, bone reconstruction is made through bone grafts or implants [1]. Artificial materials implanted into bone defects are generally encapsulated by fibrous tissue, RECEIVED 23May 2016 ACCEPTED FOR PUBLICATION 16 June 2016 PUBLISHED 27 July 2016 © 2016 IOPPublishing Ltd http://dx.doi.org/10.1088/2057-1976/2/4/045007 mailto:juli@ibb.unesp.br http://dx.doi.org/10.1088/2057-1976/2/4/045007 http://crossmark.crossref.org/dialog/?doi=10.1088/2057-1976/2/4/045007&domain=pdf&date_stamp=2016-07-27 http://crossmark.crossref.org/dialog/?doi=10.1088/2057-1976/2/4/045007&domain=pdf&date_stamp=2016-07-27 leading to their isolation from the surrounding bone. However, bioactive materials spontaneously bond to living bone without the formation of surrounding fibrous tissue [2]. In recent years, new techniques have been developed to stimulate bone reconstruction [3], which obtained excellent results, since bone tissue has a high regeneration potential with structural organiza- tion similar to native tissue. In particular, the use of latex or natural rubber (NR) membranes obtained from the sap of the rubber tree (Hevea brasiliensis) has shown high osteogenic potential [4]. This is due to its characteristics of biocompatibility and high angio- genic potential, as well as theflexibility andmechanical stability, surface porosity, and permeability of the biomembranes [5, 6]. These characteristics of NR membranes allow their performance as an occlusive mechanical barrier, hampering the migration of cells incompatible with the formation of new tissue, preventing competition with tissues of greater proliferative capacity and favor- ing osteogenesis [7]. Moreover, NR membranes can also act as a source of gradual release of substances with osteogenic potentials [8, 9]. Different studies have shown that these membranes integrate gradually into the bone, generate an acceleration of the bone for- mation, and improve the cicatrization process [10]. In this way, the use of NRmembrane provides great ben- efits for human health, preventing many difficulties such as bone transplants and grafts. Another great advantage of the use of these membranes is their low cost, since they can be produced on a large scale, being a biomaterial accessible to all [11]. The bioactivity of the latex origin is still unknown, since it has a large genetic variability. It is believed that there are clones of Hevea brasiliensis that produce latex with the greatest bioactive potential. In our previous work, we showed some preliminary evidence about this fact. In this paper, we compare two clones with the highest poten- tial, in in vivo tests [5]. Latex allergy from Hevea brasiliensis is a recurring scientific topic. It is well documented that NR can cause allergic reactions in humans; it may in part be related to the latex processing [12]. In our previous work [5], we used a method of collecting and proces- sing NR membranes without the use of any chemical additive that can cause allergy, where no negative responses were verified that indicate an allergic pro- cess. These results are consistent with other studies in the literature that conclude that NR membranes do not causes an allergic reaction and induce inflamma- tory responses similar to a normal healing process [13]. Thus, it is of great interest to search for other lac- tiferous species that can produce bioactive latex with low allergenic potential. The latex obtained from Hancornia speciosa, a plant native to Brazil, typically found in the Amazon Rainforest and in the Caatinga and Cerrado vegeta- tion, may be an interesting alternative for biomem- brane production for medical application. The low protein content of Hancornia, when compared to Hevea, is one of the most remarkable findings of Mal- monge et al, indicating thatHancornia speciosa has the potential to have fewer allergic components. For this reason, in this work we use Hancornia speciosa latex membranes in order to compare their potential with rubber latex membranes, in in vivo studies of bone regeneration, in search for an alternative to atopic patients [14]. Among the existing methods to assess the bone regeneration potential of a new material, such as NR membranes, themodel of a calvarial critical-size defect (CSD) is widely accepted in the scientific community. It has the advantage, unlike other models, of being based on a defect size specifically large enough to heal bone tissue, thusminimizing the difficulties due to dif- ferences in age, species, and anatomical place [15, 16]. Moreover, the association of a CSD with image analy- sis techniques, such as radiography and computed tomography (CT), which permit themonitoring of the evolution of the process of newbone formation, allows a precise assessment of the newly formed bone stimu- lated by thematerial to be tested [17, 18]. The present study aims at assessing the osteogenic potential of the rubber tree latex membranes of the RRIM 600 and IAN 873 clones, and of Hancornia spe- ciosa latex membranes, using the CSD method. The RRIM600 and IAN 873 clones were selected to present high bioactivity, as reported in a previous study [5]. Hancornia speciosa latex, in turn, has good angiogenic activity [19] and may be an alternative to rubber tree latex for atopic patients. Another innovation of this study was to fix the membranes to the bone on the CSD by means of a fibrin sealant (FS), a kind of biolo- gical glue derived from snake venom, as a substitute for cyanoacrylate, a material commonly used, but pre- senting innumerable negative responses [20, 21]. 2.Materials andmethods 2.1.Membrane preparation NR membranes were produced from latex collected from two different clones, RRIM 600 and IAN 873. RRIM 600 trees are grown at Balsam Farm (HeveaTec group), located inNhandeara in the State of São Paulo. The membranes made of this clone were named NR- r1. The IAN 873 trees were grown on a farm belonging to the State University of São Paulo—UNESP, at Lageado Farm in Botucatu, the State of São Paulo. The membranes made from this clone were named NR-r2. The latex collection was carried out in the morning by making a half-spiral cut (½ S). The collected serum was stored at low temperatures to prevent sponta- neous coagulation. The collecting bowl was filled with ice, and a sterilized 200 ml glass bottle was placed in its center. The serum was centrifuged for 5 min at 1500 rpm to remove impurities gathered during the collection process. The membranes were produced 2 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al within a laminar flow hood by pouring about 6.5 ml of the serum onto a polystyrene 60×15 mm Petri dish and carried to an oven, where they were dried at 50 °C and remained for 48 h to complete the polymerization. The Hancornia speciosa NR membranes (NR-h) were produced with latex collected from a farm belonging to the State University of Goiás in Ipameri, the State of Goiás. The latex was collected in a ster- ilized container by a knife incision in the bark. The cut was approximately 10 cm long and 0.5 cm deep [19]. Distilled water was added in a ratio of about 1:1 (latex: water) to prevent coagulation. The latex was cen- trifuged for 5 min at 3500 rpm to remove impurities gathered during the collection process. The mem- branes were prepared by pouring about 10 ml of serum onto a Petri dish (10.00±0.05 cm in dia- meter). Typically, they were left for 3 days at 55 °C to complete the polymerization. The NRmembranes were sterilized at the Instituto de Pesquisas Energéticas Nucleares (IPEN-Brazil) by gamma radiation performed in aGammacell 220, with a cobalt source (Co60), and a dose of 25 kGy [22]. 2.2. Physicochemical characterization 2.2.1. Scanning electronmicroscopy (SEM) For SEM the membranes were fixed in 2.5% glutar- aldehyde for 24 h. After the initialfixation, thematerial was washed 3 times in 0.1 M phosphate buffer with a pH of 7.3, 5 min apart, and post-fixed in 1% osmium tetroxide in the same buffer for 30 min Dehydration was performed using ethyl alcohol and liquid CO2. The specimens were glued onto a suitable support and coated with a gold layer 20 nm thick using a thermal evaporator, Balzer model 010. The SEM images were formed in a Philipsmicroscopemodel 515. 2.2.2. Fourier transform infrared spectroscopy (FTIR) An attenuated total-reflectance Fourier transform infrared spectrometer (ATR-FTIR, Tensor 27, Bruker) equipped with a diamond-coated ATR crystal was used to characterize the chemical composition. ATR- FTIR was performed at a resolution of 4 cm−1, averaging 16 scans over a range of 4000–380 cm−1. 2.2.3.Mechanical tests Tensile tests on the specimens were carried out on a DL-2000 (EMIC) testing machine, with a 50 kgf load cell at a speed of 500 mmmin−1 and elongated until failure. The values were converted to stress–strain curves and the Young’s modulus was calculated from the initial linear part (0%–5% elongation). 2.3. In vivo experiments The critical-size calvarial defect (CSD) is commonly used to assess biomaterials promoting bone tissue growth [16]. A CSD is defined as the smallest size intra-osseous wound that will not heal spontaneously. It was originally developed as a model of non-ligated craniofacial fibrosis, and was conceived to standardize the testing of bone repairing materials that may be used as alternatives to bone implants [23–25]. To assess the osteogenic potential of the NRmem- branes, we used 60 New Zealand race adult male rab- bits, which were randomly distributed into 2 groups depending on the implantation period of 60 or 90 days, and subdivided into 5 treatment groups, each group containing 6 animals. 3 groups received NR membrane implants, and there was 1 positive control group and 1 negative control group during each test period. The animals were treated in accordance with the Ethical Principles for Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA). During this research, the use of animals fol- lowed all the current ethical norms, and it was approved by the Ethics Commission of the State Uni- versity of São Paulo—UNESP, on 11 January, 2009, protocol number 2612/46/01/08. The FS derived from snake venomwas kindly sup- plied by the Center for the Study of Venoms and Venomous Animals (CEVAP) of the UNESP; its con- stituents and instructions for use are stated in patents BR1020140114327 and BR1020140114360, and was used in this study for the fixation of the NR mem- branes in the CSD. At the time of use, the components had been previously thawed, reconstituted, mixed, and applied. Formore detail see [26]. For the surgical procedures, the animals were sedated with acepromazine (0.1 mg kg−1) and butor- phanol (0.1 mg kg−1), injected intravenously, and 15 min later, anesthetized with a combination of tile- tamine-zolazepam (10 mg kg−1) and xylazine (0.5 mg kg−1), administered intramuscularly. After trichotomy of the frontal bone region, the rabbits were placed in ventral decubitus, and the anti- sepsis of the surgical site was performed with povi- done-iodine and physiological solution. After placing the operative-field sheets, an incision was made in the skin and subcutaneous tissue, extending itself along the middle line from the external protuberance of the occiput to the eye level. The frontal, interscutular, and occipital muscles were incised, moved away with a rugine, and retracted to show the periosteum of the parietal bone. Then we created a circular, critical-sized defect about 2 cm in diameter with a trephine 1 cm in diameter, activated by a surgical micromotor and abundant irrigation with 0.9% saline solution. Two holes were made, which were joined with the help of a luer forceps, making one defect. All the procedures was carried out carefully to preserve the dura-mater membrane. The cortical and spongy bone was removed, showing the meningeal membrane. In the groups treated with theNRmembranes, the defect was occluded with amembrane fragmentmeasuring about 2 cm. On this membrane and also on the edge of the defect, we deposited about 1 ml of the FS to fix the sec- ond membrane fragment, which was placed in order 3 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al to cover the defect. As a negative control the defect was filled with fibrin clot [7], and as a positive control the defect was filledwith PTFEmembranes [27, 28]. The periosteum and muscles close to the defect were moved closer with a continuous suture, the sub- cutaneous tissue with an invaginating suture and the skin with stitches. The threads used were 3-0 and 4-0 nylon. Before anesthetic induction and 24 h after the sur- gical procedure, enrofloxacin at a dose of 5 mg kg−1 was injected intramuscularly. Flunixin meglumine (1 mg kg−1) was administered subcutaneously every 24 h during immediate post-surgery and for three more days. The surgical wounds were treated with povidone-iodine and the cutaneous stitches were removed on the 10th day after surgery. At the end of the period of 60 and 90 days, the ani- mals were euthanized with pentobarbital (>100 mg kg−1 intravenously), after anesthesia with ketamine and xylazine. After euthanasia, the animals were placed in the ventral decubitus position and a new trichotomy was performed; any alterations in the regions close to the defect were annotated. A vertical incision was made close to the defect with a margin of about 1 cm around it. After collection the pieces were fixed in 10% formaldehyde for analysis. 2.4. X-ray imaging After 30 days, radiographic images of the implanted animals were made using a GE OPTIMA XR 220AMX equipment set at 70 kVp, 8 mA, and 2 mAs. The x-ray sensorwas positioned 40 cm from the focus tube. After euthanasia (60 and 90 days), the collected pieces were also subjected to radiography. The CT images of the pieces collected after eutha- nasia were obtained using a Shimadzu SCT-7000 model, imagine planes were taken every 1 mm and 1 s (1:1), using a potential of 120 KV and a current of 50 mA. The bone density was estimated with E-Film Workstation 4.0.3 software (Merge Healthcare Incor- porated, Chicago, CA, USA) using a Hounsfield unit (HU) [15, 29]. 2.5. Tissue processing and histological analyses The collected pieces in 10% formaldehyde for 24 h were included in paraffin. The pieces were then cut and at least 4–6 laminas were examined per animal. The 5 μm thick histological sections were stained with Masson trichrome for histological analysis, which consists in the assessment of the osteogenic process, the evolution of the bone defects made in the calvaria of the rabbits, and the construction of a widescreen to display the defect repair process. 2.6.Histomorphometric analysis The histomorphometric analysis was assessed by the traditional Weibel method [30], in which one uses a test system of lines and points on a 168-point graticulate. The bone volume (N) of each lamina was calculated using the formula: = ´N Lm 168 100, where Lm matches the mean linear intercept (in μm). Therefore, this method allows the calculation of the bone volume found on the lamina analyzed as a percentage calculation. In this work, 10 fields of 5 histological laminas were analyzed per group, the analyzed places being randomly chosen within the region of the bone defects. The images of the sections were digitized for analysis [31]. 2.7. Statistical analysis Statistical analysis was performed using the Jandel Sigma Stat program (Jandel Corporation, San Rafael, CA, USA). The results of the CT bone density and morphometry were analyzed using ANOVA or Krus- kal-Wallis with a significance set atP<0.05. 3. Results 3.1. Physicochemical characterization A tensile test was performed to compare the mechan- ical behavior of both the NR membranes, and in all samples good reproducibility was obtained, indicating homogeneity. Figure 1 shows that both NRLs present elastomeric behavior, with high elongation and a low Young’s modulus. The NRL from Hevea brasiliensis appeared to be stiffer than the NRL from Hancornia speciosa due to a higher Young’s modulus, and more resistant with higher elongation and stress at rupture, as shown in table 1. Table 1 resumes the results of the tensile mechan- ical test. The low values for the Young’s modulus are due to the non-vulcanization usually employed for NR latex. Figure 2 shows that, in terms of molecular struc- ture, both NRLmembranes are composed of the poly- mer cis-1,4-polyisoprene. The band around 3040 cm−1 is assigned to =CH stretching of between 2963 cm−1 and 2845 cm−1, to CH3 and CH2 stretch- ing of 1658 cm−1, to C=C stretching of 1445 cm−1 and 1374 cm−1, to CH2 and CH3 deformation, respec- tively; 837 cm−1 to =CH out-of-plane bending, 567 cm−1 and 488 cm−1 to C–C–C deformation. The FTIR spectra agreed well with the literature and the theoretical calculation of the vibrations (vibrational spectra of cis-1,4-polyisoprene). The absence of the band in the 960–970 cm–1 range and the presence at 837 cm−1 confirmed the cis-configuration and not trans. The assignments are summarized in table 2. Figure 3 shows that all the NRmembranes present high surface porosity, as noted in the SEM micro- graphs, where it is also possible to observe cells that have adhered to the surface. The cells show to be adhered to the NR surface with a spread and flat morphology (figures 1(a) and (b)). The inset shows the 4 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al contact between the cell and surface in a higher magnification. 3.2. Radiographic analysis Figure 4 shows the radiography images of the implanted animals. Figures 4(a) and (b) shows the typical evolution of the surgical process after 30 days of operation. All animals with implantedNRmembranes showed good evolution during the post-operative period and, within 30 days, it was already possible to observe radiopaque regions at the edges of the defects, see for example the red arrow in figure 4(b). On the other hand, the negative and positive control groups showed no radiopaque region during this testing period (figure 4(a)). Figures 4(c)–(h) shows the typical radiography images. The groups treated with the NR- r1 membrane showed newly formed bone, as can be verified by the radiopaque regions in figures 4(c) and 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 2 4 6 8 10 12 14 Strain (mm/mm) S tre ss (M P a) 0.030 0.025 0.020 0.015 0.010 0.005 0 0.01 0.02 0.03 0.04 H. brasiliensis (RRIM 600) H. brasiliensis (IAN 873) H. specisa Figure 1. Stress–strain curve of theNRmembranes fromHevea brasiliensis (RRIM600),Hevea brasiliensis (IAN 873), andHancornia speciosa. The inset shows the initial linear part of the stress–strain curves. Table 1.Average value and standard deviation of the strain at break, ultimate tensile, andYoung’smodulus fromNRLs fromdifferent sources. Strain at break (%) Ultimate tensile strength (MPa) Young’smodulus (MPa) Hevea brasiliensis (RRIM600) 1278.82 1.28 0.61 Hevea brasiliensis (IAN873) 1031.38 0.68 0.63 Hancornia speciosa 1178.17 0.71 0.66 Figure 2. FTIR spectra ofNRmembranes fromHancornia speciosa andHevea brasiliensis. 5 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al (f). Similar results were found in the other NR groups, as can be seen in the supplementary data (S1). In figures 4(e) and (h), one can observe that the negative control during both testing periods showed little or no radiopaque area. The positive control showed some radiopaque region, but far less dense than the groups treated with NR, as can be observed in figures 4(d) and (g). In all the radiography, the arrows indicate the radiopaque regions in theCSD. 3.3. Bone densitymeasured byCT Figure 5 presents the CT images of the different animal groups for the period of 60 and 90 days. Figures 2(a) and (b) shows CT images of the group that was treated with NR-r1 membranes. The arrows represent the hyperdense regions observed. It is possible to observe that, during both testing periods, there is a hyperdense lamina in the basal region of the defect. The basal region is where the process of new bone formation starts. Moreover, it is possible to verify the excellent evolution of the process; the hyperdense regions are larger and more homogeneous in the 90 day group. Similar results were found for NR-r2 and NR-h, as shown in the supplementary data (S2). A small hyperdense laminamay be observed in the basal region of the defect in the positive control (figures 5(c) and (d)). If we compare this result with the results of the other tested groups, where the NR-r1,2 and NR-h membranes were used, it is evident that NR is more efficient in bone regeneration. On the other hand, as expected in the negative control group, there is no hyperdense region, showing that the CSD does not regenerate spontaneously. Figure 5(g) shows the calculated bone density assessed by CT. One can observe that the average HU of the defect in the groups treated with NR-r1,2 and NR-h is higher during both testing periods. During the 90 day period, the density increased significantly, in fact its value is close to the values of the native bone at the defect’s edge. In the positive control group, the bone density was not only lower than in the groups treated with NR, it also did not increase throughout the period. In the negative control, on the other hand, no newbonewas formed. 3.4.Histological andhistomorphometric analysis Figures 6(a)–(f) presents the results of the histological analyses of the collected pieces of the groups treated with NRmembranes, the positive control (PTFE), and the negative control (clot). In the NR membrane groups and positive control, when compared with the untreated or negative control group, it was possible to verify that, during the 60 day period, there is a large amount of new immature dense bone tissue; showing centripetal bone formation interspersed with bone marrow (indicated as M in the figure); connective tissue undergoing ossification in abundance (indicated as CT); an increase in the number of osteocytes in the new bonematrix; and the presence of osteoclasts. This is a clear indication of effective new bone formation and the good quality of the newly formed bone (figures 6(a)–(d)). In figure 6(a), which corresponds to the positive control group (treated with PTFE), one can observe some cells permeating the membrane due to its non-occlusive characteristics [32, 33]. It should be noted that all the treated groups’ NR membranes showed similar results; only one imagewas representa- tive of othermembranes due its resemblance. In the clot group bone formation (indicated with *) the features are restricted due to the adjacent com- petitive tissue. Figures 7(a)–(d) shows the same results as figure 6, but for the 90 day groups. In this case, for the groups treated with NR membranes figures (c) and (d) the new bone tissue is mature, its lamellar structure is well organized, it has an abundant presence of osteocytes, but small regions of immature primary bone and con- nective tissue undergoing ossification, filling a large amount of the lamellar bone trabeculae (blue arrow) interspersed with bone marrow, can be observed. In the positive control group—PTFE (figure 7(b)), on the other hand, there is an island of growth with mature new bone, composed of lamellar secondary new bone and a predominance of immature primary bone. Cells such as osteocytes and connective tissue undergoing ossification permeated the membrane. There are also osteocytes in abundance, and to a lesser extent the pre- sence of immature bone tissue areas with Haversian systems and the presence of mature lamellar bone in the group treated with NR membranes, as shown in figure 8, which presents the polarized-light photomicrograph of the groups tested except for the negative control. One can verify that in all the NR groups the bone organization improves with time. There is clear evidence of secondary bone present in the 90 day NR groups, which present parallel bone lamellae and concentric bone lamellae formingHaver- sian canals. On the other hand, in the positive control group (figures 6(b1) and (b2)), one can verify that there was no discernible difference in bone organiza- tion with the period of implantation. In this case there is an abundance of immature bone tissue during both the tested periods. It should be noted that the negative control group was not shown because similar results Table 2. FTIR assignments of theNRmembranes. Wavenumber (cm−1) Assignment 3040 =CH stretching 2963 Methyl CH3 asymmetric stretching 2904 Methyl CH3 symmetric stretching 2845 Methylene CH2 symmetric stretching 1658 C=C stretching 1445 Methylene CH2 deformation 1374 Methyl CH3 asymmetric deformation 837 =CHout-of-plane bending 567 C–C–Cdeformation 488 C–C–Cdeformation 6 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al were found during both periods, and due its resem- blance only the image is shown. According to table 3, one can see that, during the first testing period, there was no significant difference in bone volume in all the groups. During the second testing period, there were significant differences between the groups; those treated with latex mem- branes gaining higher bone volumes than those treated with PTFE. The bone volume of the group treated with PTFE during the second testing period was very simi- lar to the volume presented during the 60 day period. 4.Discussion TheNR fromHevea brasiliensis andHancornia speciosa has been very promising for biological applications and biomaterials because of its important features as a natural stimulation of angiogenesis and biocompat- ibility [5, 19]. Ferreira et al showed that the NR latex films produced by casting induce vessel growth in the chorioallantoic membrane CAM, and it can be con- sidered as a potential biomaterial [34]. Almeida et al indicated that the latex obtained from Hancornia speciosa and eluted in water presents significant angiogenic activity and does not present cytotoxic or genotoxic effects on the life system [19]. Besides the features mentioned, the NR shows good elasticity and lowmechanical hysteresis. A tensile test was performed to compare the mechanical beha- vior of both NR membranes, and in all the samples good reproducibility was obtained, indicating homo- geneity (figure 1). Table 1 resumes the results of the tensile mechanical test. The low values for the Young’s modulus are due to the non-vulcanization usually employed for the NR. Valadares et al and Rezende et al also obtained values below 1MPa with an elon- gation of 980%–1150% unvulcanized, even using higher temperatures to dry, which may explain their higher values for tensile strength [35, 36]. Both NRs showed elastomeric behavior, with high elongation and a low Young’s modulus. The NR from Hevea brasiliensis appeared to be stiffer than the NR fromHancornia speciosa due to a higher Young’smod- ulus, and to be more resistant with higher elongation and stress at rupture (table 1). In both biomembranes the stress–strain profile shows low stress at small strain, characteristic of elastomer with a low degree of reticulation, followed by an increase in the stress at high deformation, although Hancornia speciosa were less intense; this abrupt increase in stress is due the strain-induced crystallization. The small differences between theHevea Brasiliensismembranes may be due the latex clone compositions. Dall’Antonia et al observed differences between RRIM 600 and IAN 873, where IAN 873 presented higher Wallace plasticity and mooney viscosity, indicating a higher polymer chain or more crosslinking. RRIM 600, due its lower values, corroborated with the higher amount of nitro- genous compounds and acetonic extract, which enhances polymer chains’ mobility, acting as a plasti- cizer; it also had a higher percentage plasticity reten- tion index, related to better thermo-oxidative resistance. Thiols are one of those responsible for pro- tecting the latex organelle membrane, but they occupy the active sites for crosslinking, thereby preventing crosslinking, corroborating the differences in mechanical behavior in this work and in the other parameters (seasonal and clonal variations in the latex and raw) [37]. Figure 3. (a) SEMvisualization of theNR surface of the RRIM600 clone, representative of othermembranes due its resemblance. (b) The inset shows, with highermagnification, the cell surface contact. 7 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al The differences in tensile behavior may be due the different composition of both rubber particles. The NR extracted from Hevea brasiliensis have a higher protein content; Malmonge et al showed a higher percentage of nitrogen in Hevea brasiliensis (0.3%) than in Hancornia speciosa (0.06%), which were rela- ted to a higher protein content of 32 300 μg g−1 for Hevea brasiliensis and 1900 μg g−1 for Hancornia spe- ciosa. Almeida et al (2014) also observed differences in nitrogen content in both latexes [14, 19]. Malmonge et al also observed differences in acetone extract, which is related to a high lipid content; Hancornia speciosa presented 6.9%when compared toHevea bra- siliensis (2.59%). The rubber particles are stabilized by a phospholipid/protein layer that confers a colloidal stability to the liquid latex; in Hancornia speciosa a higher content of fatty acids may be needed to stabilize due its low amount of protein [14, 38]. The mechanical behavior of the NR fromHancor- nia speciosa is understudied, although the influence of proteins and fatty acids in the mechanical behavior of the NR from Hevea brasiliensis is well exploited from deproteinized NR (DPNR). Amnuaypornsri et al observed a reduction in elongation and tensile Figure 4.Radiographies of the animals’heads after 30 days of the CSDoperation: (a)negative control group (clot) and (b) group treatedwithNR-r1membrane. Radiographies of the defect area for the groups of 60 and 90 days: (c) and (f) treatedwithNR-r1 membranes, (d) and (g) positive control group (PTFE), (e) and (h)negative control group (clot). In all the radiographies the arrows indicate the radiopaque regions in theCSD. 8 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al strength [39]. Amnuaypornsri et al observed that, in general, the NR showed higher stress in all the ranges of deformation and higher values of anisotropic frac- tion than the DPNR, indicating that a naturally occur- ring network, originated by the interaction of proteins, plays a significant role in the orientation of isoprene molecules and the consequential strain-induced crys- tallization during deformation [40]. Amnuaypornsri et al observed that the DPNR showed lower strength, but higher strain at break than the NR. The decreases in tensile propertiesmay be attributed to the reduction in branch-points [41]. Other differences were observed in the NR com- position derived from each species, as discussed above. From the FTIR (figure 2) two main differences were observed between both NR membranes; the Hancornia speciosa NR is known to possess higher amount of fatty acids, while Hevea brasiliensis have a higher protein content [14]. In the Hancornia speciosa spectra a more broadened band of around 3500 cm−1 is observed, related to free fatty acids while the Hevea brasiliensis spectra have a band of around 3287 cm−1 and at 1547 cm−1, corresponding to N–H stretching and deformation from the amide group, respectively, which are related to its higher protein content [14]. Dall’Antonia et al in the mechanical and thermal characterization of the formulated and vulcanized nat- ural rubber clones: GT 1, IAN 873, PB 235 e RRIM 600 also obtained no differences in FTIR spectra regardless of the type clone studied (GT 1, IAN 873, PB 235 e RRIM 600) [37]. Almeida et al observed similar com- positions between both NR membranes from Hevea Figure 5.CT Images of theCSD region for the 60 and 90 day groups after implantation of themembranes: (a) and (b) group treated withNR-r1membranes, (c) and (d) positive control group (PTFE), (e) and (f)negative control group (clot). In (g) the average bone density of the center of theCSD in all the groups during the 60 and 90 day-periods after implantation, obtained byCT analysis. ** P<0.05 all theNR groups versus positive control during the 60 day testing period;#P<0.001 all theNR and positive control groups versus the negative control during the 60 day, and all theNRgroups versus the positive control and negative control during the 90 day period; therewas no significant difference in the comparison of theNR-r1, 2 groups versusNR-h during both testing periods. 9 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al brasiliensis and Hancornia speciosa, even when stabi- lized in ammonia or sterilized by gamma irradia- tion [19]. The NR membranes presented here were pro- duced from a non-ammoniated latex, which is essen- tial to the compatibility presented here. Moc et al observed better adhesion of the L929 fibroblast on the NR prevulcanized using gamma irradiation or peroxide than in prevulcanized sulfur or in non-vulca- nized high ammoniated latex [42]. Floriano et al stu- died the ammonia in different clones of Hevea brasiliensis and observed genotoxic and cytotoxic effects, as well as delays in wound healing in the ammoniated membranes, contrary to non-ammo- niated membranes, whose IAN 873 and RRIM 600 clones showed in vitro bioactivity [5]. Almeida et al Figure 6.Photomicrographs of the evolution of the bone defectmade in the calvaria of the rabbits treatedwith the positive control (PTFE), NRmembranes, and negative control (clot) (figures (a)–(f)) for 60 days. Figures (a), (c), and (e)widescreen aspect centripetal exhibiting bone formation interspersedwith bonemarrow (M) to areas of connective tissue (CT) located predominately in the central region of the defect. Note on the repair of the groups treatedwith PTFEmembranes (figure (a)) and Latex (figure (c)) the defective area of the insulation (green arrow), allowing the growth of bone tissue (blue arrow) towards the dural region-integument, while in the clot group (figure (e)) only a thin layer of newly formed bone (red arrow) on small areas of connective tissue (CT) can be observed. Figures (b), (d), and (f) details of the previous figures showing the areas of newbone on a trabecular arrangement (*) in the positive control (PTFE) (figure (b)) andNRmembranes (figure (d)). In the negative control (clot,figure (f)) the group bone formation (*) is restricted due to the adjacent competitor tissue (AT). Edge default=dotted line. Objective of 4× and 10×. TrichromeMasson. Figure 7.Photomicrographs of the evolution of the bone defectmade in the calvaria of the rabbits treatedwith PTFE and latex membrane (figures (a)–(d)) for 90 days. Figures (a) and (c)widescreen aspect showing the total defect repair. Note in the repair of the groups treatedwith the PTFEmembrane (figure (b)) and latex (figure (d)) to fill in for lots of lamellar bone trabeculae (blue arrow) interspersedwith bonemarrow (M). Figures (b), (d), and (f) showdetails of the previousfigures showing the areas of the trabecular bone tissue arrangement (*)PTFE (figure (B)) and Latex (figure (F)). Edge default=dotted line. Objective of 4× and 10×. Trichrome Masson. 10 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al also observed the detrimental effect in cell survival with ammoniated Hancornia speciosa mem- branes [19]. All the NR membranes presented high surface porosity, as shown in the SEMmicrographs (figure 3), where it is also possible to observe cells adhered to the surface. The cell adhesion in the NR membrane sur- face is shown as cells with a spread and flat morph- ology (figures 3(a) and (b)). Oh et al observed that membranes of poly(lactic-co-glycolic acid) PLGA/ F127 with a porosity of 40 μm facing the bone defect can improve adhesiveness with bone, while 50 nm at the top prevented fibrous connective tissue invasion but permeated nutrients; Cho et al also observed lower bone regeneration to the polycaprolactone PCL/Tween 80 nanofiber mesh with smaller pores compared to the PLGA/F127 or PCL/PLA, and that even 50 nm pores permit the permeability of BSA (bovine serumalbumin) [43, 44]. Following bone injury, a number of cellular and chemical events result in the regeneration of the bone tissue. Initially, the osteocytes produce an unminer- alized bonematrix, and later, bymeans of specific che- mical tracers, the mineralization of this tissue occurs [45]. The surface characteristics of the materials’ important aspects, whether their topography, poros- ity, roughness, chemistry, or others factors, play an essential part in osteoblast adhesion on biomaterials. Thus, attachment, adhesion, and spreading belong to the first phase of cell/material interactions and the quality of this first phase will influence the cell’s capa- city to proliferate and to differentiate itself on contact Figure 8.Histological images associatedwith polarized-light for: (a) theNR-r1 group; (b) the positive control group; (c)NR-r2; (d) NR-h. The images with (1) refer to the period of 60 days and (2) for 90 days after the defect creation. Table 3.Results of histomorphometric analysis showing the average bone volume in theNRmembranes groups and the positive control. Experimental group Average bone volume/field (±s.d.) NR-r1—60 days 23,9±8,07a NR-r1—90 days 45,83±20,4c NR-r2—60 days 27,12±15,9a NR-r2—90 days 43,1±24c NR-h—60 days 25,08±5,17a NR-h—90 days 45,23±10,26c PTFE—60 days 26,02±16,8a PTFE—90 days 30,95±21,1b a No significant difference comparing all the groups during the 60 day testing period. b P<0.05 Significantly different from comparing all the groups during the 90 day testing period. c P<0.001 Significantly different from comparing all the groups during the 60 day testing period versus the 90 day testing period. 11 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al with the implant [46]. All the NR membranes pre- sented in this study showed excellent superficial adhe- sion cell due to their porous surface, as in the set shown in the SEM micrographs (figure 3). This fact corroborates the results in the in vivo tests. The results from radiography show that, after the 30 day period of implantation, the groups treated with NR-r1, 2 and NR-h membranes already had radiopaque regions on the edge of the created CSD, when compared with the positive and negative control groups, as can be seen in figures 4(a) and (b), and figure S1 from the supple- mentary data. This fact indicates that the membranes can speed up the process of bone regeneration, speed- ing up the mineralization of the connective tissue dur- ing the stage of bone formation. During the 60 and 90 day period, the groups treated with NR-r1,2 andNR-h membranes showed larger and more homogeneous radiopaque regions than with the positive control, as can be observed in figures 1(c), (d), (f), and (g), and figure S1. This indicates that NR-r1,2 andNR-hmem- branes have greater potential for bone formation than PTFE membranes. When we compare the groups in which both NR-r1,2 and NR-h membranes were used with the negative control, it is possible to verify their great potential for osteogenesis induction, since dur- ing this testing period the negative control that received the fibrin clot showed no radiopaque area, indicating that there was no spontaneous bone regen- eration (figures 4(c), (f), (c), and (h)), and figure S1. Only during the 90 day period is it possible to observe the beginning of new bone formation on the edges of the defects in the negative control (figure 4(h)). The results obtained in this study are in accordance with studies found in the literature, which confirm that the use of latex membranes in surgical processes for bone regeneration is adequate, since the barriers formed prevent or hinder the migration of cells incompatible with the new tissue to be formed, and promote osteo- genesis. As expected from previous studies the animals treated with latex membranes showed better results than those treatedwith the PTFEmembrane [7]. The CT analyses carried out during the 60 and 90 day periods showed similar results to radiography, where the groups treated with NR-r1 (figure 5(a)), NR-r2 (figure 5(b)) and NR-h (figure S2 from the sup- plementary data) membranes showed hyperdense laminae on the edges of the defects, which grew larger and more homogeneous during the experiment. The CT analyses of the positive control group, during the available periods, showed very thin, hyperdense, and small homogeneous laminae (figures 5(c) and (d) and figure (S2)), different from those observed in the groups treated with NR-r1,2 and NR-h membranes. This fact shows that the osteogenic potential of the NR membranes is superior to the positive controls. How- ever, in the negative control group, the laminae are not observed in the CT images, since the CSDwas not cap- able of regenerating itself spontaneously. When we compare the groups treated with NR-r1,2 and NR-h with the negative control, it was possible to verify the potential of these membranes in this type of applica- tion, as can be verified in figures 5(a), (b), (e), (f), and figure S2. The analyses of bone density from the CT show that the bone density in the groups treated with NR- r1,2 and NR-h was higher than in the control groups during all the testing periods. Moreover, the groups treated with NR-r2 and NR-h presented smaller bone density when compared with the groups treated with the NR-r1 membrane, but this difference was not sta- tistically significant P>0.05 (figure 5(g)). Therefore, we can verify that there are no differences in bioactiv- ity between the samples tested. During the 60 day per- iod, the bone density corresponded to the D4 bone type formed by a thin layer of cortical bone covering themedullary bone with large trabeculae, that is to say, a more malleable bone. On the other hand, during the 90 day period, we verified in all the groups treated with NR-r1,2 and NR-h the presence of the D3 type bone, which is formed by a thin layer of cortical bone cover- ing the medullary bone with small trabeculae. This is a bone of better quality and resistance, closer to the native bone present in the regions to the left and right of the CSD [47, 48]. Therefore, bothHevea brasiliensis clones present high bone regeneration potential and are suitable for use as biomaterials for bone repair. The same results were verified forHancornia speciosa latex, indicating that it presents similar bone regeneration potential to that found in the Hevea brasiliensis samples. The results of the bone density found lower bone density in the groups treated with NR- r2 and NR-h, when compared to groups treated with the NR-r1 membrane, but the difference was not statistically sig- nificant P>0.05 (figure 5(g)). So there are no differ- ences in bioactivity between the samples tested. Both clones tested from Hevea brasiliensis have high poten- tial for bone regeneration, and are suitable for bone regeneration. The same thing is seen for Hancornia speciosa, which presents potential for bone regenera- tion similar to that found in the samples of Hevea brasiliensis. Our results confirm those found in the literature, verifying that the animals treated with NR showed sig- nificant improvement during the process of bone regeneration, and an increase in mature bone volume during all the periods of study up to 120 days. This result was strengthened by means of ESR analysis, which indicated a higher degree of bone mineraliza- tion in the group treatedwithNR [8]. From the histological analyses it was possible to verify that all the groups treated presented new bone during the first testing period, revealed by large areas marked in blue (Masson trichrome) (figures 6(a)–(f)). The use of polarized-lightmethods shows that the new bone is disorganized, which is a characteristic of pri- mary bone [49], but organized lamellar bone is also observed in smaller proportions, showing the 12 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al beginning of maturation of the newly formed bone (figures 8(a1), (b1), (c1), and (d1)). During the 90 day period, the groups treated with NR membranes have mature new bone (see the large red areas in figures 7(c) and (d)). These bones are remarkably organized; they have a lamellar structure with Haversian canals (figures 8(a2), (c2), and (d2)), contrary to what was verified in the group treated with PTFE membranes, where, during the second testing period, there was no evidence of large regions of mature bone, which only appeared in small fragments, confirming the CT results (figures 7(a)–(b) and 8(b2)). During this testing period, the PTFE group presented lower bone density than theNR-treated groups, showing that therewas no bone maturation. As shown in figures 6 and 7, all the NR-treated groups presented good osteoblastic activ- ity, regions with osteoclastic activities, and large amounts of ossifying connective tissue during both testing periods. On the other hand, in the negative control group (clot), there was no new bone, only a large number of fibroblastic cells and regions with dense connective tissue during both the testing peri- ods, as seen in figures 6(e) and (f), representative of the 2 testing period due its resemblance. These results are in accordance with the literature, proving that CSD cannot be repaired spontaneously [16]. Histomorphometric analyses showed that during the first testing period, all the treated groups showed similar bone volumes, although the group treated with NR-r1 showed a bone volume slightly lower than the other groups, but the difference was not statistically significant. During the second testing period, on the other hand, the groups treatedwithNR-r1,2 andNR-h presented a bone volume 67% higher than the group treatedwith PTFE, see table 3. This result indicates that the NR-r1,2 and NR-h membranes are more efficient in the process of bone regeneration, promoting a bone formation of good quality, where it was possible to verify a large amount of well organized mature new bone at the end of this study. Both Hevea clones present the same bioactivity potential in the bone regeneration, and are highly sui- table for this type of application, as can be seen in the excellent results presented in this paper. It was evident that the NR-h membranes are effective in the process of bone reparation, and their performance was very similar to the group treated with NR-r1,2. These results indicate that Hancornia speciosa latex is appro- priate for this type of application, and we thus believe it to be a promisingmaterial for the production of bio- materials aiming at bone repair, in particular for patients with atopy to rubber tree latex (Hevea brasi- liensis). Moreover, this latex is an easily accessible low- cost material, thus being a highly recommended mat- erial. Compared with other bone regeneration meth- ods as allografts, the NR membranes’ advantage of efficient bone regeneration and bone quality, do not transmit diseases and are biocompatible, which can- not occur with the allografts [50]. When compared with another methodology that utilizes Synthetic hydroxylapatite (tricalcium phosphate—TCP), the NR membranes present the advantage of promoting osteogenesis and bone regeneration through its bioac- tive components. On the other hand, TCP also fails to satisfy the standards set by those of autogenous bone because it does not have the organic, cellular comp- onent. To be effective in the process of bone regenera- tion, as with the synthetics, it must be placed in an osteogenic environment [51]. When compared with absorbable membranes, the NR membranes present numerous advantages such as occlusion, maintenance of adequate space for bone neoformation, osseointe- gration not requiring further surgery for removal of the implant site, unlike the absorbable membranes composed of polylactic acid, polygalactin 910, col- lagen, and dura mater. However, some membranes were associated with inflammatory reactions in the adjacent tissue or were quickly degraded by the enzy- matic activity of macrophages and neutrophils. Absorbable membranes that are available are not able to maintain the appropriate space unless they have a favorable defectmorphology [52]. 5. Conclusions The results of the physicochemical characterization show that both the NR membranes presented elasto- meric behavior, with high elongation and a low Young’s modulus, although the NR membrane from Hevea brasiliensis appeared to be stiffer than the NR membrane from Hancornia speciosa due to a higher Young’s modulus, and more resistant with higher elongation and stress at rupture. In both NR mem- branes the stress–strain profile showed low stress at small strain, characteristic of an elastomer with a low degree of reticulation, followed by an increase in the stress at high deformation. The FTIR results show two main differences between both NRL biomembranes, related to their composition. The Hancornia speciosa latex is known to possess higher amount of fatty acids, while Hevea brasiliensis have a higher protein content. All the NR membranes presented high surface porosity, as noted in the SEM micrographs and cells adhered to the surface. The results show that the NR-r1 and r2 mem- branes are effective in the process of bone repair, and prove superior to PTFE. No significant differences were found in the bioactivity of these two clones, both obtaining excellent results for this type of application. The same went for the NR-h membranes, presenting results very similar toNR-r1 and r2. The NR-r1,2 and NR-h membranes proved cap- able of promoting quality bone repair, evolving over time, and are thus highly recommended as biomater- ials for bone repair. Moreover, these membranes also 13 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al showed excellent applicability, elasticity, and resist- ance for application to bone defects. The NR-hmembrane can be used as an alternative to the other NR membranes, for possessing the same osteogenic potential, promoting satisfactory bone regeneration. The FS proved to be efficient for this type of appli- cation; all the membranes remained well adhered to the bone surface, there being no case of detachment of the membranes during the whole study, neither were there negative responses in the regions where the sea- lant was applied. Acknowledgments Our thanks go to Professor Dr Joaquin Coutinho Netto (in memoriam) for his great contribution to the study and his understanding of the bioactive proper- ties of latex; to Professor Dr Luis Fernando Barbisan, Professor Dr Sergio Luis Felisbino, and Msc. Ricardo Arantes for their assistance in the histologic, histomor- phometric analyses; and to Heveatec for ceding the latex samples. The authors acknowledge the funding received through the UEG for the translation of this article, as well as the funding received through Coordenação de aperfeiçoamento de pessoal de nível superior-CAPES INCTMN/Nanobiomed, CAPES AUX-PE Toxinology, and FAPESP for the develop- ment of this research. References [1] O’Brien F J 2001 Biomaterials & scaffolds for tissue engineeringMater. Today. 14 88–95 [2] KokuboT andTakadamaH2006Howuseful is SBF in predicting in vivo bone bioactivity?Biomaterials 2907 2915 [3] DimitriouR, Jones E,McGonagle D andGiannoudis P 2011 Bone regeneration: current concepts and future directions BMCMed. 9 66 [4] ErenoC,Catanzaro SAG, Pasetto S,Herculano RD, Silva CP, Graeff C FO, TavanoO, BaffaO andKinoshita A 2010 Latex use as an occlusivemembrane for guided bone regeneration J. Biomed.Mater. ResA 95A 932–9 [5] Floriano J F,Mota L S L S, Furtado E L, RossettoV JV and Graeff C FO2014 Biocompatibility studies of natural rubber latex fromdifferent tree clones and collectionmethods J.Mater. Sci.-Mater.Med. 25 461–70 [6] Neves-JuniorWFP,Graeff C FO, FerreiraM,MulatoM, BernardesMS andCoutinho-Netto J 2006 Elastic properties of natural rubber tubes produced by dip-coating J. Appl. Polym. Sci. 100 702–7 [7] Martins R, Kinoshita AMO,Nara TA andGuimarães SAC 2010Comparative study of bone response guided tissue regeneration technique—macroscopic evaluation Full Dent. Sci. 1 224–30 [8] HerculanoRD, Silva CP, ErenoC,Guimaraes SAC, Kinoshita AMOandGraeff C FO2009Natural rubber latex used as drug delivery system in guided bone regeneration (GBR)Mater. Res. 12 253–6 [9] SilvaGA, CoutinhoOP,Ducheyne P andReis R L 2007 Materials in particulate form for tissue engineering applications in bone J. Tissue Eng. Regen.Med. 1 97–109 [10] BalabanianCA,Coutinho JN, LamanoCTL, Lacerda SA and Brentegani LG2006 Biocompatibility of natural latex implanted into dental alveolus of rats J. Oral Sci. 48 201–5 [11] Ciapetti G, Stea S, Pizzoferrato A, Checchi L and Pelliccioni GA 1994A latexmembrane as an alternative device in theGTR technique: preliminary report on its biocompatibility J.Mate. Sci.-Mater.Med. 5 647–50 [12] Berthelot K, Lecomte S, Estevez I, Coulary-Salin B, BentalebA, CullinC,DeffieuxA and Peruch F 2012Rubber elongation factor (REF), amajor allergen component inHevea brasiliensis latex has amyloid properties PloSOne 7 1-12 [13] Thomazini J A,Mrué F, Lachat J J, Zaborowiski AC, Duran JHR,Ceneviva R andCoutinho-netto J 1997 Morpfological and biochemical characterization of a prosthesismanufactured from latex natural ofHevea brasiliensis formedical utilizationActaMicrosc. 6 798–9 [14] Malmonge J A, Camilo EC,MorenoRMB,Mattoso LHC and McMahanCM2009Comparative study on technological properties of latex and natural rubber fromHarconia speciosa gomes andHevea brasiliensis J. Appl. Polym. Sci. 111 2986–91 [15] MooneyMP and SiegelM I 2005Animalmodels for bone tissue engineering edGWnek andGBowlin Encyclopedia of Biomaterials and Biomedical Engineering (NewYork:Dekker) pp 1–19 [16] Hollinger JO andKleinschmidt C J 1990The critical size defect as an experimentalmodel to test bone repairmaterials J. Craniofac. Surg. 1 60–8 [17] PerkaC, SchultzbO, Spitzer R S, LindenhaynK, Burmester GR and SittingerM2000 Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece andfibrin scaffolds in rabbitsBiomaterials 21 1145–53 [18] RudinMandWeissleder R 2003Molecular imaging in drug discovery and developmentNat. Rev. Drug. Disc. 2 123–31 [19] Almeida LM, Floriano J F, Ribeiro T P,Magno LN, Mota L S L S,Mrué FNP,Melo-Reis P, LinoR S J, Graeff C FO andGonçalves P J 2014Hancornia speciosa latex for biomedical applications: physical and chemical properties, biocompatibility assessment and angiogenic activity J.Mater. Sci.-Mater.Med. 5 1–8 [20] Sampaio RL, Ranzani J J T, BrandaoCV S, Thomazini-Santos A I, Barraviera B, Barraviera S RCS and GinniniM J SM2007Use offibrin glue derived from snake venom in the repair of deep corneal ulcers experimental study in dogs (Canis familiaris, Linnaeus 1758) J. VenomAnim. Toxins Incl. Trop. Dis. 27 857–73 [21] Machado EG, Issa J PM, Figueiredo FAT, SantosGR, Galdeano EA, AlvesMC, Chacon E L, Ferreira Junior R S, Barraviera B andCunhaMR2015Anewheterologous fibrin sealant as scaffold to recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins for the repair of tibial bone defectsActaHistochemica 117 288–96 [22] ISO 11.137 2006Review: Sterilization ofHealth Care Products [23] Schmitz J P andHollingerO J 1986The critical size defect as an experimentalmodel for craniomandibulofacial nonunions Clin. Orthop. 205 299–308 [24] Wang J andGlimcherM J 1999Characterization ofmatrix- induced osteogenesis in rat calvarial bone defects: II. Origins of bone-forming cellsCalcif. Tissue Int. 65 486–93 [25] Gosain AK, Santoro TD, Song L S, Capel CC, Sudhakar PV andMatloubHS 2003Osteogenesis in calvarial defects: contribution of the dura, the pericranium, and the surrounding bone in adult versus infant animals Plast. Reconstr. Surg. 112 515–27 [26] Barros LC, Ferreira R S J R, Barraviera S RC S, StolfHO, Thomazini-Santos I A,Mendes-GianniniM J S, Toscano E and Barraviera B 2009Anewfibrin sealant fromCrotalus durissus terrificus venom: applications inmedicine J. Toxicol. Environ. HealthB 12 553–71 [27] BarderD, Lignelli J, Smith BMandBartee BK 2007Using a dense PTFEmembranewithout primary closure to achieve bone and tissue regeneration J. Oral.Maxillofac. 65 748–52 [28] Taguchi Y, AmizukaN,NakadateM,OhnishiH, Fujii N, OdaK,Nomura S andMaeda T 2005Ahistological evaluation for guided bone regeneration induced by a collagenous membraneBiomaterials 26 6158–66 14 Biomed. Phys. Eng. Express 2 (2016) 045007 J F Floriano et al http://dx.doi.org/10.1016/S1369-7021(11)70058-X http://dx.doi.org/10.1016/S1369-7021(11)70058-X http://dx.doi.org/10.1016/S1369-7021(11)70058-X http://dx.doi.org/10.1016/j.biomaterials.2006.01.017 http://dx.doi.org/10.1186/1741-7015-9-66 http://dx.doi.org/10.1002/jbm.a.32919 http://dx.doi.org/10.1002/jbm.a.32919 http://dx.doi.org/10.1002/jbm.a.32919 http://dx.doi.org/10.1007/s10856-013-5089-9 http://dx.doi.org/10.1007/s10856-013-5089-9 http://dx.doi.org/10.1007/s10856-013-5089-9 http://dx.doi.org/10.1002/app.23416 http://dx.doi.org/10.1002/app.23416 http://dx.doi.org/10.1002/app.23416 http://dx.doi.org/10.1590/S1516-14392009000200023 http://dx.doi.org/10.1590/S1516-14392009000200023 http://dx.doi.org/10.1590/S1516-14392009000200023 http://dx.doi.org/10.1002/term.1 http://dx.doi.org/10.1002/term.1 http://dx.doi.org/10.1002/term.1 http://dx.doi.org/10.2334/josnusd.48.201 http://dx.doi.org/10.2334/josnusd.48.201 http://dx.doi.org/10.2334/josnusd.48.201 http://dx.doi.org/10.1007/BF00120348 http://dx.doi.org/10.1007/BF00120348 http://dx.doi.org/10.1007/BF00120348 http://dx.doi.org/10.1371/journal.pone.0048065 http://dx.doi.org/10.1002/app.29316 http://dx.doi.org/10.1002/app.29316 http://dx.doi.org/10.1002/app.29316 http://dx.doi.org/10.1097/00001665-199001000-00011 http://dx.doi.org/10.1097/00001665-199001000-00011 http://dx.doi.org/10.1097/00001665-199001000-00011 http://dx.doi.org/10.1016/S0142-9612(99)00280-X http://dx.doi.org/10.1016/S0142-9612(99)00280-X http://dx.doi.org/10.1016/S0142-9612(99)00280-X http://dx.doi.org/10.1038/nrd1007 http://dx.doi.org/10.1038/nrd1007 http://dx.doi.org/10.1038/nrd1007 http://dx.doi.org/10.1007/s10856-014-5255-8 http://dx.doi.org/10.1007/s10856-014-5255-8 http://dx.doi.org/10.1007/s10856-014-5255-8 http://dx.doi.org/10.1590/S1678-91992007000400014 http://dx.doi.org/10.1590/S1678-91992007000400014 http://dx.doi.org/10.1590/S1678-91992007000400014 http://dx.doi.org/10.1016/j.acthis.2015.03.006 http://dx.doi.org/10.1016/j.acthis.2015.03.006 http://dx.doi.org/10.1016/j.acthis.2015.03.006 http://dx.doi.org/10.1097/00003086-198604000-00036 http://dx.doi.org/10.1097/00003086-198604000-00036 http://dx.doi.org/10.1097/00003086-198604000-00036 http://dx.doi.org/10.1007/s002239900737 http://dx.doi.org/10.1007/s002239900737 http://dx.doi.org/10.1007/s002239900737 http://dx.doi.org/10.1097/01.PRS.0000070728.56716.51 http://dx.doi.org/10.1097/01.PRS.0000070728.56716.51 http://dx.doi.org/10.1097/01.PRS.0000070728.56716.51 http://dx.doi.org/10.1080/10937400903442514 http://dx.doi.org/10.1080/10937400903442514 http://dx.doi.org/10.1080/10937400903442514 http://dx.doi.org/10.1016/j.joms.2006.10.042 http://dx.doi.org/10.1016/j.joms.2006.10.042 http://dx.doi.org/10.1016/j.joms.2006.10.042 http://dx.doi.org/10.1016/j.biomaterials.2005.03.023 http://dx.doi.org/10.1016/j.biomaterials.2005.03.023 http://dx.doi.org/10.1016/j.biomaterials.2005.03.023 [29] MadhuK,NairDMDand LiMS 2007Digital and advanced imaging in endodontics: a review J. Endodontics 33 1–6 [30] Weibel ER 1963 Principles andmethods for the morphometric study of the lung and other organs Lab. Investigation 12 131–55 [31] Brandt J Z, Silveira LT,Grassi T F, Anselmo-Franci J A, FávaroW J, Felisbino S L, Barbisan L F and ScaranoWR2014 Indole-3-carbinol attenuates the deleterious gestational effects of bisphenol A exposure on the prostate gland ofmale F1 rats Reprod. Toxicol. 43 56–66 [32] GelbDA1999 Immediate implant surgery: ten-year clinicai overviewCompendiumCont. Educ. Dent. 20 1185–92 [33] Lopez-Soler R I, BrennanMP,Goyal A,Wang Y, Fong P, TellidesG, Sinusas A,Dardik A andBreuer C 2007 Development of amousemodel for evaluation of small diameter vascular grafts J. Surg. Res. 139 1–6 [34] FerreiraM,MendonçaR J,CoutinhoNetto J andMulatoM2009 Angiogenic properties ofnatural rubber latex biomembranes and the serumfractionof heveaBraz. J. Phys.39564–9 [35] Valadares L F, Leite CAP andGalembeck F 2006 Preparation of natural rubber–montmorillonite nanocomposite in aqueousmedium: evidence for polymer–platelet adhesion Polymer 47 672–8 [36] RezendeCA, Bragança FC,Doi TR, Lee L T,Galembeck F and Boué F 2010Natural rubber-clay nanocomposites:mechanical and structural properties Polymer 51 3644–52 [37] Dall’Antonia AC,MartinsMA,MorenoRMB,MattosoHC, JobAE, Ferreira FC andGonçalves P S 2006 Evaluation of clones of the rawnatural rubber by standard test and dynamic mechanical properties Polímeros 16 239–45 [38] Sansatsadeekul J and Sakdapipanich J 2006 Origin of Colloidal Behavior ofNatural Rubber ParticlesMahidolUniversity (http://2mtecorth/th/seminar/msativ/pdf/P14pdf) [39] Amnuaypornsri S, NimpaiboonA and Sakdapipanich J 2009 Role of phospholipids and proteins on gel formation and physical properties ofNRduring accelerated storageKgk-Kaut GummiKunst. 62 88–92 [40] Amnuaypornsri S, Sakdapipanich J, Toki S,Hsiao B S, IchikawaN andTanaka Y 2008 Strain-induced crystallization of natural rubber: effect of proteins and phospholipidsRubber Chem. Technol. 81 753–66 [41] Amnuaypornsri S, Sakdapipanich J andTanaka Y 2010Highly purified natural rubber by saponificaion of latex: analysis of green and cured properties J. Appl. Polym. Sci. 118 3524–31 [42] MokK l, Evans SD and Evans SW2001Cell adhesion on natural rubber latexfilms: influence of surface properties Rubb. Res. 4 205–21 [43] ChoWJ, Kim JH,Oh SH,NamHH,Kim JMand Lee JH 2009Hydrophilized polycaprolactone nanofibermesh‐ embedded poly (glycolic‐co‐lactic acid)membrane for effective guided bone regeneration J. Biomed.Mater. Res.A 91 400–7 [44] Oh SH, Kim JH, Kim JM and Lee J H 2006 Asymmetrically porous PLGA/Pluronic F127membrane for effective guided bone regeneration J. Biomater. Sci. Polym. Ed. 17 1375–87 [45] Punnett L andWegmanDH2004Work-related musculoskeletal disorders: the epidemiologic evidence and the debate J. Electromyogr. Kinesiol. 14 13–23 [46] AnselmeK2000Osteoblast adhesion on biomaterials Biomaterials 21 667–81 [47] Cope J B and SamchukovML 2001Mineralization dynamics of regenerate bone duringmandibular osteodistraction J. Oral. Maxillofac. Surg. 30 234–42 [48] MischCE 2008Contemporary Implant Dentistry 3rd edn (St. Louis:Mosby) [49] Junqueira LC andCarneiro J 2013BasicHistology 13th edn (NewYork:McGraw-Hill) [50] Stevens B, Yang Y,Mohandas A, Stucker B andNguyenT 2008 A review ofmaterials, fabricationmethods, and strategies used to enhance bone regeneration in engineered bone tissues J. Biomed.Mater. Res.B 85 573–82 [51] HoexterDL 2002 Bone regeneration graftmaterials J. Oral Implantol. 28 290–4 [52] MonteiroA S,Macedo LG,MacedoNL andBalducci I 2010 Polyurethane and PTFEmembranes for guided bone regeneration: histopathological and ultrastructural evaluation Med.Oral. Patol. Oral Cir. Bucal. 15 401–6 15 Biomed. Phys. Eng. 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Introduction 2. Materials and methods 2.1. Membrane preparation 2.2. Physicochemical characterization 2.2.1. Scanning electron microscopy (SEM) 2.2.2. Fourier transform infrared spectroscopy (FTIR) 2.2.3. Mechanical tests 2.3. In vivo experiments 2.4. X-ray imaging 2.5. Tissue processing and histological analyses 2.6. Histomorphometric analysis 2.7. Statistical analysis 3. Results 3.1. Physicochemical characterization 3.2. Radiographic analysis 3.3. Bone density measured by CT 3.4. Histological and histomorphometric analysis 4. Discussion 5. Conclusions Acknowledgments References