Fibrous braided stents with antibacterial properties

Tese de Doutoramento (Programa Doutoral em Engenharia Biomédica)Nowadays, cardiovascular diseases are reported as a major cause of death and disability in developed countries. Therefore, stents have emerged as a solution to overcome this problem. However, common commercialize stents, are metallic, which present several disadvantages as corrosion, higher risk of restenosis and infection. In order to minimize the disadvantages associated with these stents, new materials, like fibrous materials, have begun to be used as well as surface modifications of biomaterials begun to be applied, in order to avoid those drawbacks, especially infection. Therefore, the main objective of this work was the development of a fibrous stent, able to compete with the mechanical properties of the commercial ones, with the advantaged of being coated with an antibacterial agent, able to avoid infections. For this purpose, braided stents were produced by varying materials, structural and process parameters, such as monofilament type and diameter, braiding angle and mandrel diameter. The influence of these design parameters on mechanical behavior, as well as stent's porosity, was thoroughly investigated, and suitable parameters were selected for developing a stent with mechanical characteristics and porosity matching with the commercial stents. According to the experimental results, suitable performance was achieved with a polyester stent designed with: monofilament diameter of 270 µm, braiding angle of 35°; and mandrel diameter of 6 mm, providing similar properties to commercial Nitinol stents (porosity above 70%, unchanged diameter during bending tests above 75%, force for longitudinal compression between [0.16-5.28] N and force for radial compression between [1.13-2.90] N). After the stent’s development, silver and silver oxide thin films were deposited by non-reactive and reactive pulsed dc magnetron sputtering. The coatings were characterized chemical, physical and structurally. This first approach in stent’s functionalization revealed that silver thin film formed a continuous layer, while silver oxide layer was composed of islands with hundreds of nanometers surrounded by small nanoparticles with tens of nanometers. In order to verify the antibacterial behavior of the coatings, halo inhibition zone tests were realized for Staphylococcus epidermidis and Staphylococcus aureus. Silver coatings did not show antibacterial behavior, contrarily to silver oxide coatings, which presented antibacterial properties against the studied bacteria. The presence of silver oxide phase along with the development of different morphology were pointed as the main factors in the origin of the antibacterial effect found in silver oxide thin film. With the purpose of understand the influence of oxygen species in the physical, chemical and structural properties of thin films new silver and silver oxide thin films were deposited, by nonreactive and reactive pulsed dc magnetron sputtering, with the variation of oxygen content. The resulted coatings revealed that silver coating forms a continuous layer. The incorporation of oxygen leads to the formation of a mixture of Ag2O + AgO phases. However, with the increase of the oxygen fraction, the mixture of oxides disappear and the resultant thin film became only AgO. Cytotoxicity tests were performed, demonstrating that in the silver oxide coating no cytotoxicity was found, making this coating able to be used in stents applications. The antibacterial behavior of coatings was, quantitatively, tested against Staphylococcus epidermis, showing that AgxO unlike Ag coating presented antibacterial behavior. The presence of silver oxide is the main reason for the antibacterial effect, probably due to the increased production of reactive oxygen species (ROS). Finally, and in order to evaluate the coating’s behavior when subjected to mechanical forces, coated silver and silver oxide stents were, again, mechanically tested, and no loss of adhesion or delamination were observed, indicating that coatings will support, adequately, the mechanical forces and the dangerous of delivering parts of coating in blood current will not occur. In conclusion, fibrous stents coated with silver oxide, present a good mechanical behavior, when compared with nitinol commercial stents, possess antibacterial properties and are no cytotoxic, which made this stent a promising candidate as a biomaterial and a viable substitute to metallic stents.Atualmente, as doenças cardiovasculares são consideradas a principal causa de morte e incapacidade nos países desenvolvidos. Os stents surgiram como uma solução para este problema, contudo, os que se encontram no mercado são metálicos, pelo que apresentam várias desvantagens como: corrosão, maior risco de reestenose e infeção. A fim de minimizar as desvantagens associadas a estes stents, novos materiais, como materiais fibrosos, começaram a ser utilizados, assim como, a modificação da superfície dos biomateriais, de forma a evitar a infeção. Consequentemente, o principal objetivo deste trabalho foi o desenvolvimento de um stent fibroso, capaz de competir com as propriedades mecânicas dos stents comerciais, com a vantagem de ser revestido com um agente antibacteriano, evitando, deste modo, a possibilidade de infeção. Para este efeito, foram produzidos diferentes stents entrançados, através da variação dos materiais, parâmetros estruturais e de processo, tais como: o tipo de monofilamento e o seu diâmetro, ângulo de entrançamento e diâmetro do mandril. A influência destes parâmetros no comportamento mecânico, bem como a porosidade dos stents foram estudadas detalhadamente. Para o desenvolvimento do stent foram selecionados os parâmetros adequados com o objetivo de obter caraterísticas mecânicas e porosidade compatíveis com os stents comerciais. De acordo com os resultados experimentais, a melhor performance foi obtida com um stent de poliéster com diâmetro de monofilamento de 270 µm, ângulo de entrançamento de 35° e o diâmetro do mandril de 6 mm, proporcionando propriedades semelhantes ao stents comerciais de nitinol (porosidade acima de70%, diâmetro inalterado durante os testes de dobragem acima de 75%, força de compressão longitudinal compreendida entre [0.16-5.28] N e força de compressão radial compreendida entre [1.13-2.90] N). Após a produção do stent, foram depositados filmes finos de prata e óxidos de prata, através da pulverização catódica pulsada em magnetrão, em regime não-reativo e reativo. Os revestimentos foram caracterizados química, física e estruturalmente. Esta primeira abordagem na funcionalização do stent revelou que o revestimento de prata formou um filme contínuo, enquanto o filme de óxido de prata era composto de ilhas, com centenas de nanómetros rodeadas por nanopartículas com dezenas de nanómetros. A fim de verificar o comportamento antibacteriano dos revestimentos, foram realizados testes de halo para Staphylococcus epidermidis e Staphylococcus aureus. Como resultado, o revestimento de prata não apresentou comportamento antibacteriano, ao contrário do revestimento de óxido de prata, que apresentou propriedades antibacterianas contra as bactérias estudadas. A presença da fase de óxido de prata juntamente com o desenvolvimento de morfologias diferentes foram apontados como os principais fatores na origem do efeito antibacteriano encontrado no revestimento de óxido de prata. Com o propósito de compreender a influência das espécies de oxigênio nas propriedades físicas, químicas e estruturais dos filmes finos, foram depositados novos revestimentos de prata e óxido de prata, por pulverização catódica em magnetrão, em regime não-reativo e reativo, com variação do teor de oxigénio. Os revestimentos resultantes revelaram que os revestimentos de prata formaram uma camada contínua. A incorporação do oxigénio levou à formação de uma mistura de fases de Ag2O + AgO. Contudo, com o aumento da fração de oxigénio, a mistura de óxidos desapareceu e os filmes finos resultantes apresentaram apenas a fase de AgO. Foram realizados ensaios de citotoxicidade, demonstrando que o revestimento de óxido de prata escolhido não é tóxico, tornando-o adequado para ser aplicado em stents. O comportamento antibacteriano dos revestimentos foi, quantitativamente, testado contra Staphylococcus epidermis, revelando que os revestimentos de AgxO, ao contrário dos revestimentos de Ag, apresentam um comportamento antibacteriano. A presença do óxido de prata é um dos principais fatores para o efeito antibacteriano, provavelmente, devido ao aumento da produção de espécies reativas de oxigénio (ROS). Finalmente, e de modo a avaliar o comportamento do revestimento quando submetido a forças mecânicas, os stents revestidos com prata e óxido de prata foram, novamente, testados mecanicamente, e não se observou perda de adesão ou a delaminação do filme. Estes resultados indicam que os revestimentos conseguem suportar, de forma adequada, as forças mecânicas e que não haverá risco de perda de revestimento para a corrente sanguínea. Em conclusão, os stents fibrosos revestidos com óxido de prata, apresentam um bom comportamento mecânico, quando comparado com os stents comerciais de nitinol, além de apresentarem propriedades antibacterianas e não serem citotóxicos, o que torna este stent um candidato promissor como substituto para os stents metálicos atuais

Solvent-cast direct-writing and electrospinning as a dual fabrication strategy for drug-eluting polymeric bioresorbable stents

Bioresorbable stents (BRS) are conceived to retain sufficient radial strength after implantation while releasing an antiproliferative drug in order to prevent vessel restenosis until complete resorption. Ongoing research trends involve the use of innovative manufacturing techniques to achieve thinner struts combined with optimized local drug delivery. This work presents a combination of solvent-cast direct-writing (SC-DW) and electrospinning (ES) using poly-l-lactic acid (PLLA) and poly(l-lactic-co-¿-caprolactone) (PLCL) as a new approach to generate everolimus-eluting BRS for cardiovascular applications. A Design of Experiment (DoE) was conducted to determine the optimal parameters to obtain a homogeneous coating with high specific surface. Manufactured stents were characterized by means of mechanical tests and scanning electron microscopy (SEM), with everolimus release in accelerated conditions quantified through High Performance Liquid Chromatography (HPLC). Drug loading was achieved either encapsulated in the struts of the stent or in an electrospun PLCL membrane covering the stent. In the former case, everolimus release was found to be insufficient, less than 3% of total drug loading after 8 weeks. In the latter, everolimus release considerably increased with respect to drug-loaded 3D-printed stents, with over 50% release in the first 6 hours of the test. In conclusion, everolimus release from PLCL-coated 3D-printed stents would match the dose and timeframe required for in vivo applications, while providing thinner struts than SC-DW drug-loaded stents.Peer ReviewedPostprint (published version

Materials and Textile Architecture Analyses for Mechanical Counter-Pressure Space Suits using Active Materials

Mechanical counter-pressure (MCP) space suits have the potential to improve the mobility of astronauts as they conduct planetary exploration activities. MCP suits differ from traditional gas-pressurized space suits by applying surface pressure to the wearer using tight-fitting materials rather than pressurized gas, and represent a fundamental change in space suit design. However, the underlying technologies required to provide uniform compression in a MCP garment at sufficient pressures for space exploration have not yet been perfected, and donning and doffing a MCP suit remains a significant challenge. This research effort focuses on the novel use of active material technologies to produce a garment with controllable compression capabilities (up to 30 kPa) to address these problems. We provide a comparative study of active materials and textile architectures for MCP applications; concept active material compression textiles to be developed and tested based on these analyses; and preliminary biaxial braid compression garment modeling results.United States. National Aeronautics and Space Administration (OCT Space Technology Research Fellowship Grant NNX11AM62H)MIT-Portugal Progra

Structural Design, Fabrication and Evaluation of Resorbable Fiber-Based Tissue Engineering Scaffolds

The use of tissue engineering to regenerate viable tissue relies on selecting the appropriate cell line, developing a resorbable scaffold and optimizing the culture conditions including the use of biomolecular cues and sometimes mechanical stimulation. This review of the literature focuses on the required scaffold properties, including the polymer material, the structural design, the total porosity, pore size distribution, mechanical performance, physical integrity in multiphase structures as well as surface morphology, rate of resorption and biocompatibility. The chapter will explain the unique advantages of using textile technologies for tissue engineering scaffold fabrication, and will delineate the differences in design, fabrication and performance of woven, warp and weft knitted, braided, nonwoven and electrospun scaffolds. In addition, it will explain how different types of tissues can be regenerated by each textile technology for a particular clinical application. The use of different synthetic and natural resorbable polymer fibers will be discussed, as well as the need for specialized finishing techniques such as heat setting, cross linking, coating and impregnation, depending on the tissue engineering application

Aging effect on functionalized silver-based nanocoating braided coronary stents

A previously developed fiber-based polyester (PES) stent, with mechanical properties comparable to commercial nitinol stents, was coated with metallic silver (Ag0) and silver oxides (AgxO) thin films through direct current (DC) magnetron sputtering. Ag0 and AgxO coatings provide antimicrobial properties to the stents to minimize the occurrence of coronary stent infections. Nevertheless, the stent interacts with the atmosphere and then with the biological fluids and may lead to the generation of silver species with diminished antimicrobial efficiency and/or prone to induce cytotoxicity. Therefore, stent coating nanostructures aged 3 months were thoroughly analyzed by X-ray photoelectron spectroscopy (XPS) and their antimicrobial and cytotoxicity properties were assessed. Aging led to the presence of silver carbonate and bicarbonate as well as chemisorbed oxygen species in Ag0 and AgxO coatings. Bactericidal efficacy was tested against an important nosocomial bacterium, particularly associated to indwelling devices: Staphylococcus epidermidis. Aged Ag0 and AgxO coating presented a Log reduction of 1 and 2 at their surface; respectively. However, aged stents were able to induce a Log reduction of 2 (Ag0) and 4 (AgxO) on the surrounding medium. Only aged AgxO stent was able to provide a mild reduction of the bacterium at its surface and a clear antimicrobial effect (Log reduction >3) within its vicinity. More importantly, both aged Ag0 and AgxO stents were shown to be compatible with fibroblasts cells indicating that they can be safely used as indwelling devices, despite the aging effectThis work was funded by FEDER funds through the Operational Competitiveness ProgramCOMPETE and by National Funds through Fundacao para a Ciencia e Tecnologia (FCT)-under the projects PTDC/CTM-TEX/28295/2017 and UID/CTM/00264/2019

Biodegradable polymeric fiber structures in tissue engineering

Tissue engineering offers a promising new approach to create biological alternatives to repair or restore function of damaged or diseased tissues. To obtain three-dimensional tissue constructs, stem or progenitor cells must be combined with a highly porous three-dimensional scaffold, but many of the structures purposed for tissue engineering cannot meet all the criteria required by an adequate scaffold because of lack of mechanical strength and interconnectivity, as well as poor surface characteristics. Fiber-based structures represent a wide range of morphological and geometric possibilities that can be tailored for each specific tissue-engineering application. The present article overviews the research data on tissue-engineering therapies based on the use of biodegradable fiber architectures as a scaffold

Bioresorbable Composite Stents for Enhanced Response of Vascular Smooth Muscle Cells

Formation of arterial plaque and stenosis is one of the main cardiovascular disease risk factors. Stenting is a popular approach to increase the inner diameter of the artery and provide an acceptable lumen gain. This is achieved by applying internal pressure to the arterial wall. Despite the desirable outcomes of this procedure, there are complexities and challenges that are being discussed among scholars in this area. Restenosis is one of these complications, in which smooth muscles cell start proliferation and remodeling in response of induced mechanical stresses. Another important issue is the placement of the stent and possible migration due to the continuous deformation and special contact state between tissue and stent struts. Finally, the mechanical properties of the stent and application of novel materials in order to improve its performance are the critical topics that also have been elaborated in the current research work. First of all, we developed a multi-scale model which is able to calculate load distribution in RVE scale and can be useful to assess the mechanical stresses experienced by smooth muscle cells. Moreover, stent migration has been simulated by using finite element modeling, and the effect of stent structure on this complication has been explained. Finally, the application of novel nano composite materials in stent design has been discussed. Developing 3D printed steel-PLLA and MgPLLA particle composites and the effect of added phases in micromechanical properties of composites has been evaluated. Advisor: Linxia G

견종 경동맥을 이용한 자가 팽창성, 생분해성 폴리다이옥사논 스텐트의 생체적합성 연구

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의학과, 2021. 2. 오창완.Introduction: We report a preclinical study of polydioxanone (PDO) self-expanding, biodegradable, intracranial vascular stents for biocompatibility after experimental placement in canine carotid arteries and compare phosphorylcholine-coated and non-coated PDO stents. Materials and Methods: All experiments were approved by the committee of animal research. In vitro PDO filament degradation and platelet adhesion tests were performed. A total of 12 PDO stents and 12 phosphorylcholine-coated PDO stents were implanted in normal canine carotid arteries of 3.0- to 4.0-mm diameter in six dogs. The dogs were divided into three groups with 2 dogs per group. Each group was sacrificed at 4, 8, and 12 weeks after stent placement, respectively. Stent patency was assessed by angiography followed by a microscopic histological examination of the dissected carotid arteries. Results: Stent deployment was technically successful in all dogs without procedure-related complications. On angiographic analysis, in-stent stenosis (9.48 ± 7.13%) was documented in both stent groups at 4 weeks after implantation. Fifty percent of implanted carotid arteries were completely occluded with increased in-stent stenosis (15.80 ± 12.32%) in the other patent vessel at 8 weeks. Total occlusion at 12 weeks was observed in all implanted vessels. PDO filaments began to demonstrate a loss of 5% of their original mass by 4 weeks. PDO filaments started to degrade at 4 weeks and by 16 weeks reached about 50% of their original weight by using in vitro degradation examination. On histologic examination, the degradation of PDO stents started at 4-8 week and at 12 week periods, fragmentation of the braid was observed. According to the degradation of PDO polymer, PDO stents evoked extensive inflammatory responses at 8 and 12 week periods. Inflammatory scores (0-4) were 1.63 ± 0.71 at 4 weeks, 1.79 ± 0.83 at 8 weeks (p=0.360) and 2.33 ± 0.96 at 12 weeks (p=0.008). Although the phosphorylcholine-coated PDO stent showed statistical significance in platelet adhesion inhibition on in vitro platelet adhesions test compared with non-coated PDO stent, there was no different in vivo biocompatibility, such as luminal thrombosis formation and endothelialization between both stent groups. And, lower yet still severe inflammatory responses were observed for the phosphorylcholine-coated PDO stent group. Luminal thrombosis formation and histomorphometric stenosis also increased with increasing time intervals. Conclusion: The PDO stent induced an inflammatory reaction within the carotid artery with subsequent neo-intimal thickening. In 4-week follow-up period before polymer degradation, the vessel patency was preserved with mild in-stent stenosis. However, the degree of stenosis progressed according to the time interval and finally vessel occlusion was occurred. The observed tissue response may be attributable to the degradation of the polymer, which induced an inflammatory response, resulting in the occlusion of the arteries. The phosphorylcholine-coating did not show any difference compared with the non-coated PDO stent.서론: 저자는 자가 팽창성, 생분해성 폴리다이옥사논 스텐트의 생체적합성을 확인하기 위하여, 견종 경동맥에 스텐트 삽입 후 영상의학적, 조직학적 검사를 시행하였다. 또한, 추가적으로 포스폴린콜린 코팅한 스텐트와 비교하고자 하였다. 대상 및 방법: 모든 동물 실험은 동물 실험 위원회의 승인 하에 진행하였다. 총 여섯 마리의 개(비글)의 3mm에서 4mm 크기의 양측 경동맥에 총 12개의 폴리다이옥사논 스텐트와 12개의 포스폴린콜린 코팅한 폴리다이옥사논 스텐트를 삽입하였다. 개는 각각 두 마리씩 스텐트 삽입 후 4주, 8주, 12주에 혈관조영검사 및 조직검사를 시행하였다. 결과: 24개의 스텐트는 모두 합병증 없이 삽입하였다. 혈관 조영 검사 상에서 스텐트 삽입 후 4주 경과 후, 스텐트 내 협착은 9.48 ± 7.13% 보였으며, 8주 경과 시에는 15.80 ± 12.32%의 협착 소견이 보였다. 하지만, 8주 경과한 경동맥의 50%와 12주 경과한 경동맥은 완전 폐색된 상태였다. 체외에서 시행한 폴리다이옥사논 섬유 분해 실험에서, 4주에 분해되기 시작하여 16주에는 초기 질량의 50%의 감소를 보였다. 조직학적 검사 상에서 폴리다이옥사논 스텐트는 4에서 8주 사이에 분해하기 시작하였으며, 12주에는 작은 분절로 분열된 소견이 관찰되었다. 또한, 폴리다이옥사논 고분자의 분열에 따라, 8주에서 12주 추적 검사에서 스텐트 주변의 심한 염증성 반응을 동반하였다. 염증 수치 (0-4)는 4주에 1.63 ± 0.71, 8주에 1.79 ± 0.83, 12주에 2.33 ± 0.96으로 점차 증가하였으며, 12주에는 4주에 비해 통계적으로 유의하게 증가하였다. 체외 혈소판 응집 실험에서는 포스폴린콜린 코팅한 스텐트에서 혈소판 응집이 통계적으로 유의하게 감소하였으나, 생체 실험에서는 혈관내 혈전 생성이나 혈관 내피 생성 등에서 코팅하지 않은 스텐트와 차이점을 보이지 않았다. 또한, 포스폴린콜린 코팅한 스텐트에서는 염증 수치가 낮게 관찰되기는 하였으나, 통계적 의의는 보이지 않았다. 혈관내 혈전 생성 및 조직학적 협착도 염증 수치와 비슷하게 시간 경과에 따라 증가하는 소견을 보였다. 결론: 이 동물 실험에서 폴리다이옥사논 스텐트는 분해됨에 따라 혈관 내에서 심한 염증 반응과 혈관 내막의 과증식을 유발하였다. 생분해가 활발하게 일어나기 전의 4주 경과에서는 경도의 스텐트내 협착은 관찰되나 혈관 개방성은 유지되었다. 하지만, 시간 경과에 따라 혈관 협착이 증가하였으며, 이러한 염증 반응 및 혈관 내막의 증식으로 최종적으로 혈관의 완전 폐색이 유발되었다. 포스폴린콜린 코팅한 스텐트도 같은 반응을 보였다.Abstract i Contents iv List of tables v List of figures vi Introduction 1 Material and Methods 4 Results 12 Discussion 30 Conclusion 36 References 37 Abstract in Korean 46Docto

Bioresorbable Composite Stents for Enhanced Response of Vascular Smooth Muscle Cells

Formation of arterial plaque and stenosis is one of the main cardiovascular disease risk factors. Stenting is a popular approach to increase the inner diameter of the artery and provide an acceptable lumen gain. This is achieved by applying internal pressure to the arterial wall. Despite the desirable outcomes of this procedure, there are complexities and challenges that are being discussed among scholars in this area. Restenosis is one of these complications, in which smooth muscles cell start proliferation and remodeling in response of induced mechanical stresses. Another important issue is the placement of the stent and possible migration due to the continuous deformation and special contact state between tissue and stent struts. Finally, the mechanical properties of the stent and application of novel materials in order to improve its performance are the critical topics that also have been elaborated in the current research work. First of all, we developed a multi-scale model which is able to calculate load distribution in RVE scale and can be useful to assess the mechanical stresses experienced by smooth muscle cells. Moreover, stent migration has been simulated by using finite element modeling, and the effect of stent structure on this complication has been explained. Finally, the application of novel nano composite materials in stent design has been discussed. Developing 3D printed steel-PLLA and MgPLLA particle composites and the effect of added phases in micromechanical properties of composites has been evaluated. Advisor: Linxia G