Bacterial cellulose: studies on biocompatibility, surface modification and interaction with cells

Tese doutoramento em Engenharia BiomédicaA wide variety of biomaterials and bioactive molecules have been applied in tissue engineering as scaffolds in order to provide an appropriate environment to the growth and differentiation of cells. However, creating devices for biological substitutes that enhance the regeneration of neural tissues is still a challenge, because of the difficulty in providing an active stimulation of nerve regeneration. Biological scaffolds can be composed of natural polymers combined with extracellular matrix molecules and have been shown to facilitate the constructive remodeling of many tissues by the establishment of an environment necessary for the regulation of cell processes. In this context, different biomaterials have been used as scaffolds to improve interactions between material/cells and repair neurological damages. In recent years, bacterial cellulose (BC) emerged as a promising biomaterial in tissue engineering due its properties: high crystalinity, wettability, high tensile strength, pure nanofibers network, moldability in situ and simple production. BC has been modified to further enhance cell adhesion and biocompatibility; as an alternative to peptide chemical grafts, BC allow the use of recombinant proteins containing carbohydrates binding domains (CBMs), such as the CBM3, which has affinity by cellulose, representig a attractive way to specifically adsorb bioactive peptides on cellulose surface. The goal of this work was to modify the bacterial cellulose improving the neuronal cell affinity and producing a scaffold with potential to be used in neural tissue engineering. For this purpose, two strategies were used: 1) adhesive peptides fused to a carbohydrate binding domain with affinity to cellulose and; 2) surface modification by nitrogen plasma treatment. Also, in this work, we analized the biocompatibility in a longterm approach of two different types of BC grafts and the effect of BC nanofibers subcutaneously implanted in mice. The recombinant proteins IKVAV-CBM3, exIKVAV-CBM3 and KHIFSDDSSECBM3, were successfully expressed in E. coli, purified and stably adsorbed to the BC membranes. The in vitro results showed that the exIKVAV-CBM3 was able to improve the adhesion of both neuronal and mesenchymal cells (MSC), while IKVAV-CBM3 and KHIFSDDSSE-CBM3 presented only a slight effect on mesenchymal cell adhesion, and no effect on the other cells. The MSCs neurotrophin expression by cells grown on BC membranes modified with the recombinant proteins was also verified. NGF was expressed and released by cells adhered on the BC membranes, creating a microenvironment that promotes neuronal regeneration. The nitrogen plasma treatment did not increase the wettability of the material, but increased the porosity and changed the surface chemistry, as noticed by the presence of nitrogen. XPS analysis revealed the stability of the modified material along time and autoclave sterilization. The cell adhesion and proliferation of HMEC-1 and N1E-115 cells was significantly improved in the plasma treated BC, in contrast with the 3T3 cells, revealing a cell-specific effect. Regarding in vivo studies, the BC implants caused a low inflammatory reaction that decreased along time and did not elicit a foreign body reaction. A tendency for calcification, which may be related to the porosity of the BC implants, was observed. However, this tendency was different depending on the BC tested. Regarding nanofibers implants, after 2 and 4 months post implantation, mostly of injected nanofibers remained in aggregates in the subcutaneous tissue. There was infiltration of cells in these aggregates of nanofibers, mostly macrophages, and there is evidence of phagocytosis of the material by these cells. Moreover, no differences were observed between the controls and implanted animals in thymocyte populations, B lymphocyte precursors and myeloid cells in the bone marrow.BC is a good material to be used as scaffold in tissue engineering applications. However, is still necessary to improve the interaction of cells with the material to obtain a matrix that supports the growth, differentiation and selectivity of cells. In our attempt to enhance and select neuronal attachment to BC, the recombinant proteins produced were able to improve cell adhesion and viability on BC membranes. Also, nitrogen plasma treatment proved to be an effective and economical surface treatment technique, which was also capable to improve the adhesion of endothelial and neuroblast cells to the material. Therefore, the surface modification leads to a better cell affinity with BC, probably contributing for a better biocompatibility in vivo. In the in vivo results, our work points to the necessity to further investigation to verify the tendency to BC to calcify in long-term circumstances. Meanwhile, the BC nanofibers seem to be an innocuous material in mice subcutaneous tissue, and proved to be an eligible material to production of ECM-mimetic grafts.Actualmente, um grande número de materiais poliméricos com diferentes propriedades estão disponíveis para aplicações biomédicas. Têm sido exploradas várias abordagens com o objetivo de melhorar a interação entre os polímeros e as células, que por ser geralmente inadequada, provoca reações in vivo como inflamações, perdas de tecido local e encapsulamento dos implantes. Entre estas abordagens, a modificação das superfícies, como por exemplo a funcionalização dos materiais com peptídeos imobilizados ou grupos químicos incorporados, mostra vantagens na obtenção de interações específicas das células com os materiais resultando em uma melhoria na sua biocompatibilidade. A celulose bacteriana (CB) tornou-se um biomaterial em foco para aplicações biomédicas devido a sua alta resistência mecânica, hidrofilicidade, alta cristalinidade e pureza, baixo custo de produção e sua característica rede de nanofibras. Além disso, o uso de domínios de ligação à celulose é uma alternativa simples e específica de enxertar peptídeos bioativos à estrutura da celulose possibilitando uma maior afinidade celular. O objectivo deste trabalho foi modificar a CB para aumentar a afinidade de células neuronais, produzindo um scaffold com potencial para ser utilizado em engenharia de tecidos neuronal. Com este propósito, duas estratégias foram utilizadas: 1) o uso de peptídeos de adesão conjugados a um domínio de ligação a carbohidratos (CBM), com afinidade para a celulose e, 2) modificação da CB através do tratamento com plasma de nitrogênio. Também, dentro do âmbito deste trabalho, avaliouse a biocompatibilidade a longo prazo da CB, tanto de implantes como de nanofibras implantados subcutaneamente em camundongos. As proteínas recombinantes IKVAV-CBM3, exIKVAV-CBM3 and KHIFSDDSSECBM3 foram expressas em E.coli, purificadas e adsorvidas de maneira estável nas membranes de CB. Os resultados in vitro mostraram que o exIKVAV-CBM3 aumentou a adesão de células neuronais e mesenquimais, enquanto que o IKVAV-CBM3 e KHIFSDDSSE-CBM3 apresentaram apenas um pequeno efeito na adesão das células mesenquimais, e nenhum efeito nas outras células testadas. Também, a expressão de neurotrofinas pelas células mesenquimais nas membranas de CB modificadas com as proteínas recombinantes foi verificada, e verificou-se que o NGF é expresso e libertado por estas células aderidas na CB, criando um ambiente promotor da regeneração neuronal. O tratamento com o plasma de nitrogênio não aumentou a molhabilidade da CB, mas foi capaz de aumentar a porosidade e a química de superfície, evidenciado pela presença do grupo nitrogênio. As análises de XPS mostraram a estabilidade do material modificado 180 dias após o tratamento, e após a esterilização por autoclave. A adesão e a proliferação celular das linhagens endotelial (HMEC-1) e neuronal (N1E-115) foi aumentada significativamente na celulose tratada com plasma, em contraste com os fibroblastos 3T3, o que revelou um efeito célula-específico. Quanto aos estudos in vivo, os implantes de CB causaram apenas uma reação inflamatória de baixa intensidade, que decresceu ao longo do tempo, e não estimulou reação de corpo estranho. Foi observada uma tendência para calcificar nas membranas de CB menos porosas, indicando uma relação com a porosidade dos implantes. Quanto aos implantes de nanofibras, após 2 e 4 meses de implantação, verificou-se que a maior parte das nanofibras permaneceram em agregados no tecido subcutâneo. Houve infiltração de células nesses agregados de nanofibras, sendo a maioria macrófagos, e evidências de fagocitose do material por estas células. Também, não foram encontradas diferenças entre os controles e os animais implantados nas populações de timócitos, precursores de linfócitos B e células mielóides na medula óssea. A CB é um bom material para ser utilizado em aplicações de engenharia de tecidos. Entretanto, ainda é necessário a modificação deste material para aumentar sua interação com as células, obtendo assim uma matriz capaz de manter o crescimento, a diferenciação e a seletividade de células. Na nossa tentativa de aumentar e selecionar a adesão de células neuronais à CB, as proteínas recombinantes produzidas foram capazes de aumentar a adesão e a viabilidade celular neste material. Também, o tratamento por plasma de nitrogênio provou ser um tratamento de superfície econômico e efetivo, sendo capaz de aumentar a afinidade das células com a CB, o que poderá contribuir para um melhoramento da sua biocompatibilidade in vivo. Quanto aos testes in vivo, este trabalho aponta para a necessidade de investigação futura para verificar a tendência da CB em calcificar em circunstâncias a longo prazo. Entretanto, as nanofibras de CB parecem ser inócuas quando implantadas no tecido subcutâneo, sendo um material elegível para a produção de enxertos que mimetizem a matriz extracelular

Estudo in vitro da interação da linhagem de fibroblastos L929 com membranas de celulose bacteriana para aplicações em engenharia de tecidos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia Química.O estudo das interações entre células e substrato na engenharia de tecidos é de grande importância para a determinação das propriedades biológicas dos implantes. A adesão das células ao substrato influencia na morfologia, proliferação e viabilidade celular. Neste trabalho foram avaliadas a adesão, proliferação e viabilidade de fibroblastos de camundongo, linhagem L929, em suporte biopolimérico. Dois tipos de celulose bacteriana foram utilizados: uma produzida em laboratório e outra comercial (BIONEXT®). Os fibroblastos foram cultivados sobre as membranas em meio DMEM, suplementado com 10% de SBF a 37°C, contendo 5%CO2, e diferentes parâmetros celulares foram escolhidos com o objetivo de monitorar e avaliar o comportamento das células em diferentes tempos de cultivo nas membranas. Foram observadas diferenças morfológicas significativas nas células. Os fibroblastos permaneceram com morfologia arredondada. A celulose bacteriana permitiu a adesão, crescimento, proliferação e viabilidade das células. The study of the dynamic behavior and adhesion between cells and substrates in tissue engineering is of major importance to predict the final biological properties of tissue implants. The adhesion of cells on the substrate influences morphology, proliferation and cellular viability. In this work, adhesion, proliferation and viability of L929 mouse fibroblasts on bacterial cellulose (BC) membranes were evaluated in vitro. Two kinds of cellulose membranes were used; a produced in the laboratory and a commercially available (BIONEXT®). Fibroblasts were cultivated on the membranes in a DMEM medium supplemented with 10% of SBF at 37°C, containing 5% of CO2, and different cellular parameters were chosen to evaluate the cell behavior on the membranes in function of time. Significant morphologic differences were observed in the cells. Although the fibroblasts were well adhered to the membrane, they maintained a round-shape. Bacterial cellulose membranes allowed cell adhesion, growth, proliferation and viability

Bacterial cellulose production by Gluconacetobacter xylinus by employing alternative culture media

Bacterial cellulose (BC) is used in different fields as a biological material due to its unique properties. Despite there being many BC applications, there still remain many problems associated with bioprocess technology, such as increasing productivity and decreasing production cost. New technologies that use waste from the food industry as raw materials for culture media promote economic advantages because they reduce environmental pollution and stimulate new research for science sustainability. For this reason, BC production requires optimized conditions to increase its application. The main objective of this study was to evaluate BC production by Gluconacetobacter xylinus using industry waste, namely, rotten fruits and milk whey, as culture media. Furthermore, the structure of BC produced at different conditions was also determined. The culture media employed in this study were composed of rotten fruit collected from the disposal of free markets, milk whey from a local industrial disposal, and their combination, and Hestrin and Schramm media was used as standard culture media. Although all culture media studied produced BC, the highest BC yield60 mg/mLwas achieved with the rotten fruit culture. Thus, the results showed that rotten fruit can be used for BC production. This culture media can be considered as a profitable alternative to generate high-value products. In addition, it combines environmental concern with sustainable processes that can promote also the reduction of production cost.The authors would like to acknowledge the Brazil National Council of Technological and Scientific Development (CNPq, FAPESP, and CAPES), the financial support from FAPESP 2009/14897-7, and Fundacao para a Ciencia e a Tecnologia (FCT)/Portugal through the project PTDC/EBB-EBI/112170/2009 for the financial support and scholarship. Special thanks to Talita Almeida Vicentin for technical support

Surface modification of bacterial cellulose by nitrogen-containing plasma for improved interaction with cells

Bacterial cellulose (BC) membranes were modified with nitrogen plasma in order to enhance cell affinity. The surface properties of the untreated and plasma modified BC (BCP) were analyzed through contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The effect of the plasma treatment on the adhesion of microvascular (HMEC-1), neuroblast (N1E-115) and fibroblast (3T3) cell lines was analyzed. The nitrogen plasma treatment did not increase the wettability of the material, but increased the porosity and surface chemistry, as noticed by the presence of nitrogen. XPS analysis revealed the stability of the modified material along time and autoclave sterilization. The cell adhesion and proliferation of HMEC-1 and N1E-115 cells was significantly improved in the BCP, in contrast with the 3T3 cells, revealing a cell-specific effect. This work highlights the potential of plasma treatment for the modification of the BC surface properties, enhancing its potential for biomedical applications.Programme Alβan, the European Union Programme of High Level Scholarships for Latin America.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasil.Fundação para a Ciência e a Tecnologia (FCT) – POCTI, Portugal

Melatoninergic System in Parkinson’s Disease: From Neuroprotection to the Management of Motor and Nonmotor Symptoms

Melatonin is synthesized by several tissues besides the pineal gland, and beyond its regulatory effects in light-dark cycle, melatonin is a hormone with neuroprotective, anti-inflammatory, and antioxidant properties. Melatonin acts as a free-radical scavenger, reducing reactive species and improving mitochondrial homeostasis. Melatonin also regulates the expression of neurotrophins that are involved in the survival of dopaminergic neurons and reduces α-synuclein aggregation, thus protecting the dopaminergic system against damage. The unbalance of pineal melatonin synthesis can predispose the organism to inflammatory and neurodegenerative diseases such as Parkinson’s disease (PD). The aim of this review is to summarize the knowledge about the potential role of the melatoninergic system in the pathogenesis and treatment of PD. The literature reviewed here indicates that PD is associated with impaired brain expression of melatonin and its receptors MT1 and MT2. Exogenous melatonin treatment presented an outstanding neuroprotective effect in animal models of PD induced by different toxins, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat, and maneb. Despite the neuroprotective effects and the improvement of motor impairments, melatonin also presents the potential to improve nonmotor symptoms commonly experienced by PD patients such as sleep and anxiety disorders, depression, and memory dysfunction

Bacterial cellulose: long-term biocompatibility studies

The bacterial cellulose (BC) secreted by G. xylinus is a network of pure cellulose nanofibers, which has high crystallinity, wettability and mechanical strength. These characteristics make BC an excellent material for tissue engineering constructs, noteworthy for artificial vascular grafts. In this work, the in vivo biocompatibility of BC membranes produced by two G. xylinus strains was analyzed through histological analysis of long-term subcutaneous implants in the mice. The BC implants caused a mild and benign inflammatory reaction that decreased along time and did not elicit a foreign body reaction. A tendency to calcify over time, which may be related to the porosity of the BC implants, was observed, especially amo the less poro s BC-1 implants. In addition, the potential toxicity of BC nanofibers - obtained by chemical-mechanical treatment of BC membranes - subcutaneously implanted in mice was analysed through bone marrow flow cytometry, blood and histological analyses. After 2 and 4 months post implantation, the nanofibers implants were found to accumulate cytoplasmically, in subcutaneous foamy macrophages aggregates. Moreover, no differences were observed between the controls and implanted animals in thymocyte populations and in B lymphocyte precursors and myeloid cells in the bone marrow.R. A. N. P. gratefully acknowledges support by the Programme Alssan, the European Union Programme of High Level Scholarships for Latin America (Scholarship No. E07D401931BR). S. M. is recipient of a SFRH/BPD/64726/2009 fellowship from Fundacao para a Ciencia e a Tecnologia (FCT, Portugal). R. M. G. da C. (SFRH/BD/37565/2007) and A. C. (SFRH/BD/31354/2006) are recipients of a PhD fellowship from Fundacao para a Ciencia e a Tecnologia (FCT, Portugal). Funding by FCT and the program COMPETE of the project PTDC/EBB-EBI/112170/2009 is acknowledged