Additive effect of pulp pequi oil (Caryocar brasiliense Camb. ) on the biocompatibility of collagen and gelatin membranes in subcutaneous implants / Efeito aditivo do óleo de polpa de pequi (Caryocar brasiliense Camb. ) sobre a biocompatibilidade de membranas de colágeno e gelatina implantadas no subcutâneo

Trauma or disease inflicted by tissue injuries may cause tissue degeneration. The use of biomaterials for direct or indirect repair has emerged as a promising alternative, and has become an important research topic. The pequi fruit (Caryocar brasiliense Camb.) has shown antifungal, antibacterial, anti-inflammatory, healing, antitumor, and antioxidant properties. The objective of this study was to develop a new biomaterial using a combination of collagen, gelatin, and pulp pequi oil, and to evaluate its biocompatibility in comparison with that of biomaterials produced without pulp pequi oil. Membranes were prepared from a mixture of bovine tendon collagen, commercial gelatin, and pulp pequi oil. The inflammatory and cicatricial processes were assessed via histopathology of the tissue interface/implants in the subcutaneous tissues and quantitative evaluation of leukocyte and collagen production in Wistar rats. It was observed that the presence of pequi oil reduced the amount of foreign-body giant cells and favored the recruitment of fibroblasts (P< 0.01), thereby promoting greater production of collagen membrane than that in the membranes of control samples. Therefore, it can be concluded that the addition of pequi oil improved the biocompatibility of collagen and accelerated the healing process.(AU)

Trauma ou lesões causadas por doenças podem enfraquecer e degenerar os tecidos humanos e animais. O uso de biomateriais para reparação direta ou indireta surgiu como uma alternativa promissora e tornou-se um importante tema de pesquisa. O óleo de pequi (Caryocar brasiliense Camb.) mostrou propriedades antifúngicas, antibacterianas, anti-inflamatórias, curativas, antitumorais e antioxidantes. O objetivo deste estudo foi obter um novo biomaterial, produzido pela combinação de óleo de pequi, colágeno e gelatina, para avaliar sua biocompatibilidade em comparação às membranas produzidas sem o óleo. As membranas foram preparadas por meio da mistura de colágeno de tendão bovino, gelatina comercial e óleo de pequi. Os processos inflamatórios e cicatriciais foram avaliados por histopatologia da interface / implantes de tecido subcutâneo de ratos Wistar para avaliação quantitativa da produção de leucócitos e colágeno. Observou-se que a presença de óleo de pequi reduziu a quantidade de células gigantes de corpo estranho e favoreceu o recrutamento de fibroblastos (P<0,01), promovendo, assim, maior produção da membrana de colágeno em comparação com a membrana de controle. Portanto, pode-se concluir que a adição de óleo de pequi melhorou a biocompatibilidade do colágeno e acelerou o processo de cicatrização.(AU)

Use of collagen/chitosan sponges mineralized with hydroxyapatite for the repair of cranial defects in rats.

In traumatology, we encounter several clinical challenges that involve extensive bone loss primarily related to trauma, conditions that can be treated with autologous grafts. A good alternative is the use of synthetic biomaterials as substitutes. These polymers provide a suitable environment for the growth of new bone and vascular tissue, which are essential for repair. Collagen/hydroxyapatite composites have proven to be biocompatible and to behave mechanically. Furthermore, the addition of chitosan contributes to the formation of a three-dimensional structure that permits cell adhesion and proliferation, thus improving osteogenesis. The aim of this study was to evaluate bone formation during the repair of bone defects experimentally induced in the skull of rats and grafted with a polymer blend consisting of bovine tendon collagen and chitosan combined with hydroxyapatite. Thirty animals were used for the creation of a defect in the left parietal bone and were divided into three groups of 10 animals each: a control group without biomaterial implantation, a group receiving the blend of collagen and chitosan, and a group receiving this blend combined with hydroxyapatite. Each group was subdivided and the animals were sacrificed 3 or 8 weeks after surgery. After sacrifice, the skulls were removed for macroscopic photodocumentation and radiographic examination. The samples were processed for histological evaluation of new bone formation at the surgical site. Macroscopic and radiographic analysis demonstrated the biocompatibility of the blends. Histologically, the formation of new bone occurred in continuity with the edges of the defect, with the observation of higher volumes in the grafted groups compared to control. Mineralization of sponges did not stimulate bone neoformation, with bone repair being incomplete over the experimental period. In conclusion, mineralization by the addition of hydroxyapatite should be better studied. However, the collagen/chitosan sponges used in this study are suitable to stimulate osteogenesis in cranial defects, although this process is slow and not sufficient to achieve complete bone regeneration over a short period of time.

Zinc phthalocyanines attached to gold nanorods for simultaneous hyperthermic and photodynamic therapies against melanoma in vitro.

Studies indicate that hyperthermic therapy using gold nanorods and photodynamic activity with many photosensitizers can present a synergistic effect, and offer a great therapeutic potential, although more investigation needs to be performed before such approach could be implemented. We proposed to investigate the effect of the attachment of phthalocyanines on the surface of gold nanorods (well-characterized devices for hyperthermia generation) for the elimination of melanoma, one of the most important skin cancers due to its high lethality. Following the synthesis of nanorods through a seed-mediated method, the efficacy of photodynamic therapy (PDT) and hyperthermia was assessed separately. We chose to coat the nanorods with two tetracarboxylated zinc phthalocyanines - with or without methyl-glucamine groups. After the coating process, the phthalocyanines formed ionic complexes with the cetyltrimethylammonium bromide (CTAB) that was previously covering the nanoparticles. The nanorod-phthalocyanines complexes were analyzed by transmission electron microscopy (TEM), and their singlet oxygen and hydroxyl radical generation yields were assessed. Furthermore, they were tested in vitro with melanotic B16F10 and amelanotic B16G4F melanoma cells. The cells with nanoparticles were irradiated with laser (at 635nm), and the cell viability was assessed. The results indicate that the photodynamic properties of the phthalocyanines tested are enhanced when they are attached on the nanorods surface, and the combination of PDT and hyperthermia was able to eliminate over 90% of melanoma cells. This is a novel study because two tetracarboxylated phthalocyanines were used and because the same wavelength was irradiated to activate both the nanorods and the photosensitizers.

Biosilicate/PLGA osteogenic effects modulated by laser therapy: In vitro and in vivo studies.

The main purpose of the present work was to evaluate if low laser level therapy (LLLT) can improve the effects of Biosilicate®/PLGA (BS/PLGA) composites on cell viability and bone consolidation using a tibial defects of rats. The composites were characterized by scanning electron microscope (SEM) and reflection Fourier transform infrared spectrometer (FTIR). For the in vitro study, fibroblast and osteoblast cells were seeded in the extract of the composites irradiated or not with LLLT (Ga-Al-As, 808nm, 10J/cm2) to assess cell viability after 24, 48 and 72h. For the in vivo study, 80 Wistar rats with tibial bone defects were distributed into 4 groups (BS; BS+LLLT; BS/PLGA and BS/PLGA+LLLT) and euthanized after 2 and 6weeks. Laser irradiation Ga-Al-As (808nm, 30J/cm2) in the rats was performed 3 times a week. The SEM and FTIR results revealed that PLGA were successfully inserted into BS and the microparticles degraded over time. The in vitro findings demonstrated higher fibroblast viability in both BS/PLGA groups after 24h and higher osteoblast viability in BS/PLGA+LLLT in all periods. As a conclusion, animals treated with BS/PLGA+LLLT demonstrated an improved material degradation and an increased amount of granulation tissue and newly formed bone.

Characterization and biological evaluation of the introduction of PLGA into biosilicate®.

The aims of this study were to characterize different BS/PLGA composites for their physicochemical and morphological characteristics and evaluate the in vitro and in vivo biological performance. The physicochemical and morphological modifications were analyzed by pH, mass loss, XRD, setting time, and SEM. For in vitro analysis, the osteoblast and fibroblast viability was evaluated. For in vivo evaluations, histopathology and immunohistochemistry were performed in a tibial defect in rats. After incubation, all composites presented lower values in pH and mass loss over time. Moreover, XRD and SEM analysis confirmed that the composites degraded over time. Additionally, pore formation was observed by SEM analysis after incubation mainly in BS/PLGA groups. BS/PLGA showed significantly increased in osteoblast viability 24 h. Moreover, BS/PLGA composites demonstrated an increase in fibroblast viability in all periods analyzed when compared to BS. In the in vivo study, after 2 and 6 weeks of implantation of biomaterials, histopathological findings revealed that the BS/PLGA composites degrades over time, mainly at periphery. Moreover, can be observed the presence of granulation tissue, bone formation, Runx-2, and RANKL immunoexpression in all groups. In conclusion, BS/PLGA composites present appropriate physicochemical characteristics, stimulate the cellular viability, and enhance the bone repair in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1063-1074, 2017.

Avaliação da interação biológica entre compósito de quitosana, colágeno e hidroxiapatita e tecido ósseo ovino / Investigation of biological interactions of chitosan-collagen-hydroxyapatite composite with bone tissue in sheep

As lesões em membros de grandes animais são um desafio para médicos veterinários, uma vez que somente a osteossíntese não garante resultados satisfatórios. Muitos pesquisadores vêm se dedicando ao desenvolvimento e estudo de substitutos ósseos produzidos de materiais naturais, como quitosana, colágeno e hidroxiapatita, que auxiliam na regeneração óssea. Seis ovinos fêmeas da raça Santa Inês foram submetidos a ostectomias unicorticais de sete milímetros de diâmetro na região proximal da superfície dorsomedial dos III/IV metacarpianos. Foi implantado compósito de quitosana, colágeno e hidroxiapatita em um membro torácico para avaliação da biocompatibilidade do material ao tecido ósseo ovino, e no membro contralateral foi reproduzida a mesma técnica, porém foi mantido sem preenchimento, como controle. Após 60 dias do procedimento cirúrgico, realizou-se biópsia óssea na área de interface entre biomaterial/osso (membro com compósito) e tecido neoformado/osso (membro controle), para realização de avaliação histológica do material não descalcificado, por meio de microscopia de luz e microscopia eletrônica de varredura. Na análise histomorfométrica, mediante microscopia de luz, foi possível identificar maior porcentagem de tecido neoformado em membro controle, quando comparado ao membro com compósito (80% e 63,5%, respectivamente; P<0,05). Por meio da microscopia eletrônica de varredura, observou-se invasão da estrutura interna do compósito por tecido ósseo neoformado. Não houve formação de tecido cicatricial, reação de corpo estranho ou resposta inflamatória crônica nas amostras analisadas. Conclui-se que o compósito de quitosana, colágeno e hidroxiapatita, quando implantado em tecido ósseo ovino, apresenta biocompatibilidade e perfil osteocondutor.(AU)

Fracture management poses a great challenge to large animal practitioners. Osteosynthesis alone is often insufficient to provide satisfactory outcomes in large animals; therefore, several research efforts have been made to investigate and develop bone substitutes capable of promoting bone regeneration. Chitosan-collagen-hydroxyapatite composites constitute a promising alternative given their similar composition to bone. Six Santa Inês ewes were submitted to the creation of experimental 7mm wide unicortical defects on the dorsomedial aspect of the proximal III/IV metacarpal bone diaphysis. Limbs were randomly selected for treatment with chitosan-collagen-hydroxyapatite composite or to serve as untreated controls. Biopsy fragments were collected from the bone/new bone or the bone/biomaterial interface (control and treated defects respectively) within 60 days of surgery; composite biocompatibility was assessed using light and scanning electron microscopy. Histomorphometric analysis under light microscopy revealed greater percentage of new bone tissue in control compared to treated defects (80% and 63.5% respectively; P<0.05). No scar tissue formation, foreign body or chronic inflammatory reactions were observed. Scanning electron microscopy revealed invasion of the composite by new bone tissue. The chitosan-collagen-hydroxyapatite composite studied is biocompatible with bone and shows osteoconductive properties in sheep.

Feasibility study of collagen membranes derived from bovine pericardium and intestinal serosa for the repair of cranial defects in ovariectomised rats.

The indication of biomaterials has increased substantially in the regenerative therapy of bone defects. However, in addition to evaluating the physicochemical properties of biomaterials, the quality of the recipient tissue is also essential for the osseointegration of implants, as abnormalities in bone metabolism, such as gonadal hormone deficiency, can influence bone healing. This study evaluated the osteoregenerative capacity of collagen membranes derived from bovine pericardium and intestinal serosa in the repair of cranial defects in ovariectomised rats. Thirty female Wistar rats were submitted to surgical creation of a 5-mm cranial bone defect. The rats were divided into a control group (not ovariectomised) and an ovariectomised group. The non-ovariectomised group was divided into three subgroups: control (G1) in which the defect was not filled with the biomaterial, and two subgroups (G2 and G3) that received the bovine pericardium- and serosa-derived collagen membranes, respectively. The ovariectomised group was divided into the same subgroups (G4, G5, and G6). The animals were sacrificed 8 weeks after surgery. The calvaria were removed for macroscopic and radiographic photodocumentation and processed for histomorphometric analysis of bone healing at the surgical site. Macroscopic, radiological, and microscopic analyses demonstrated the biocompatibility of the implanted collagen membranes, as indicated by the absence of infiltration and signs of inflammation at the surgical site. Histologically, discrete immature bone neoformation projecting from the margins of the defect was observed at the surgical site in ovariectomised groups when compared to the non-ovariectomised groups. The volume of newly formed bone was significantly higher in the non-ovariectomised groups (G1: 7.83%±1.32; G2: 21.33%±1.96; and G3: 22.83%±0.98) compared to the respective ovariectomised subgroups (G4: 3.16%±0.75; G5: 16.83%±0.98; and G6: 16.16%±0.75), thus demonstrating the deleterious effects of ovariectomy on bone homeostasis. Higher volumes of newly formed bone were observed in the groups receiving the membrane grafts (G2, G3, G5, and G6) compared to the control groups (G1 and G4). In conclusion, the bilateral ovariectomy compromises the ability to repair bone lesions grafted with osteoconductive biomaterials as in the case of collagen membranes derived from both bovine pericardium and intestinal serosa.

A bacteriophage detection tool for viability assessment of Salmonella cells.

Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

Spintronic platforms for biomedical applications.

Since the fundamental discovery of the giant magnetoresistance many spintronic devices have been developed and implemented in our daily life (e.g. information storage and automotive industry). Lately, advances in the sensors technology (higher sensitivity, smaller size) have potentiated other applications, namely in the biological area, leading to the emergence of novel biomedical platforms. In particular the investigation of spintronics and its application to the development of magnetoresistive (MR) biomolecular and biomedical platforms are giving rise to a new class of biomedical diagnostic devices, suitable for bench top bioassays as well as point-of-care and point-of-use devices. Herein, integrated spintronic biochip platforms for diagnostic and cytometric applications, hybrid systems incorporating magnetoresistive sensors applied to neuroelectronic studies and biomedical imaging, namely magneto-encephalography and magneto-cardiography, are reviewed. Also lab-on-a-chip MR-based platforms to perform biological studies at the single molecule level are discussed. Overall the potential and main characteristics of such MR-based biomedical devices, comparing to the existing technologies while giving particular examples of targeted applications, are addressed.

Picomolar detection limit on a magnetoresistive biochip after optimization of a thiol-gold based surface chemistry.

The surface biochemistry plays a crucial role in the development of stable and reproducible bioanalytical devices. Very often, it represents the bottleneck of a successful integration of magnetoelectronic transducers with the biological receptors on its interface. Here is discussed how a thiolgold surface chemistry can be tailored and optimized in order to allow the biofunctionalization of a magnetoresistive biochip, preventing loss of viability by corrosion while improving its sensitivity. Two important parameters, type of buffer solution and salt concentration (globally ionic strength), were evaluated in the effectiveness of the sulfur-gold linkage and further influence on the biomolecular recognition between single stranded DNA molecules. A third, not less important variable under investigation was the blocking solution. Non-specific adsorption of magnetic labels to the sensing surface still is a major problem to be addressed. The effect of two well known blocking molecules (bovine serum albumin (BSA)) and thiolated polyethylene-glycol (SH-PEG)) on the prevention of non-specific adsorption of targets and labels are compared. The best conditions were selected using an optical microscopic characterization method. Optical images were analyzed for magnetic particles quantification and results presented as a percentage of surface coverage. The optimized protocol was further implemented on real magnetoresistive devices to assess its electric compatibility and bioassay performance. A good reproducibility (about 9% error) among different devices measuring the same target concentration was achieved. Also a reduced non-specific binding signal of 43 microV for non-complementary targets (30% complementarity) compares with a 500 microV for fully complementarity. A linear range on the biological detection of magnetically labeled target ssDNA oligonucleotides is demonstrated. Consequently, the limit of detection at the standard operational conditions of the device is situated at the picomolar range.

Femtomolar limit of detection with a magnetoresistive biochip.

In this paper the biological limit of detection of a spin-valve-based magnetoresistive biochip applied to the detection of 20 mer ssDNA hybridization events is presented. Two reactional variables and their impact on the biomolecular recognition efficiency are discussed. Both the influence of a 250 nm diameter magnetic particle attached to the target molecule during the hybridization event and the effect of a magnetic focusing system in the hybridization of pre-labeled target DNA (assisted hybridization) are addressed. The particles carrying the target molecules are attracted to the probe active sensor sites by applying a 40 mA DC current on U-shaped aluminium current lines. Experiments comparing pre-hybridization versus post-hybridization magnetic labeling and passive versus magnetically assisted hybridization were conducted. The efficiency of a passive hybridization is reduced by about 50% when constrained to the operational conditions (sample volume, reaction time, temperature and magnetic label) of an on-chip real-time hybridization assay. This reduction has shown to be constant and independent from the initial target concentration. Conversely, the presence of the magnetic label improved the limit of detection when a magnetically assisted hybridization was performed. The use of a labeled target focusing system has permitted a gain of three orders of magnitude (from 1 pM down to 1 fM) in the sensitivity of the device, as compared with passive, diffusion-controlled hybridization.

Notes on a plant parasite fungus in Portugal: Gymnosporangium cornutum

A rust fungus identified as Gymnosporangium cornutum was found on Sorbus aucuparia in Serra da Estrela (Manteigas), and the disease was severe at that location. Despite the abundance and worldwide occurrence of the genus Gymnosporangium, studies in Portugal are still limited(AU)

Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil.

Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation (deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3 percent, respectively. In 74.3 percent of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation ( deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

Nonstoichiometric hydroxyapatite-anionic collagen composite as support for the double sustained release of gentamicin and norfloxacin/ciprofloxacin.

This work studied the sustained release of ciprofloxacin or norfloxacin and gentamicin from nonstoichiometric hydroxyapatite (nHA) and anionic collagen composite. Within the first 24 and 48 h, the total antibiotic supply was significantly higher than the minimal inhibitory concentration required for the majority of the gram-negative bacteria. Although gentamicin was completely released from the matrix after 48 h by a normal diffusion mechanism, ciprofloxacin or norfloxacin release was characterized by a 2-phase release mechanism due to binding to nHA by complexation with calcium ion. Under the conditions studied, most of the norfloxacin or ciprofloxacin only will be disposable due to bioresorption or dissociation of the complexes. In conclusion, due to its biocompatibility nHA-anionic collagen composite may be a convenient support for the double sustained release of the antibiotics gentamicin and ciprofloxacin/norfloxacin for the control of bone infection while promoting bone tissue growth.

The controlled release of antibiotic by hydroxyapatite: anionic collagen composites.

Major problems with the treatment of osteomyelitis are associated with poor antibiotic distribution at the site of infection due to limited blood circulation to the skeletal tissue. Improved treatment procedures have been used in drug delivery systems that include bioceramics and natural and synthetic polymers. This work reports the development of anionic collagen:hydroxyapatite composite paste for sustained antibiotic release. Antibiotic release by the composite was characterized by two steps. In the first, 15.0+/-4.9% was released in the first 5 h (n = 53) by a normal Fick diffusion mechanism. In the second step, only 16.8+/-2.2% was released after 7 days. In conclusion, hydroxyapatite:anionic collagen composite can be an efficient support for sustained antibiotic release in the treatment of osteomyelitis because most of the antibiotic release may be associated with composite bioresorption, thus permitting antibiotic release throughout the healing process. Hydroxyapatite:anionic collagen paste showed good biocompatibility associated with bone tissue growth with material still being observed after 60 days from the time of implants.