Bacterial cellulose modifications for biomedical applications

Universidade do Minho Escola de Engenharia Eugénia Cristina Queirós Teixeira October 2022 Eugénia Cristina Queirós Teixeira UMinho|2022 Bacterial cellulose modifications for biomedical applications Bacterial cellulose modifications for biomedical applications October 2022 Universidade do Minho Escola de Engenharia Eugénia Cristina Queirós Teixeira Work developed under the supervision of Doctor Francisco Miguel Portela da Gama and Doctor Pier Parpot Doctoral Thesis Doctorate in Biomedical Engineering Universidade do Minho Escola de Engenharia Bacterial cellulose modifications for biomedical applications DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii ACKNOWLEDGEMENTS After this long journey I would like to express my deepest gratitude to all the people who helped me during this work and make this thesis possible. I will start acknowledging my supervisors, Professor Miguel Gama and Prof. Pier Parpot for their help, support, patience and for being so understanding with me. A special thank to Prof. Miguel Gama for giving me the opportunity to develop this research project and, mainly, for never give up on me and encouraging me to keep going. To the University of Minho, particularly, to Centre of Biological Engineering and all the people that work there and were always available to helping me; a special thank to Joana Azevedo, Patrícia Dias and Carla Magalhães for their essential help on bioreactors. To all the people from LTEB lab for their partnership and continuous availability to listen, to help and to encourage me. All of you were crucial to finish this chapter, particularly, Ana Cristina Rodrigues, Fernando Dourado and also Sara Pinheiro; although she no longer belongs to LTEB group, her contribution was extremely important to part of this work. I also want to thank to Centre of Chemistry and to the excellent people that I met there: Marta Ferreira, Pedro Rocha, Daniela Carvalhal, Vânia Sousa, Luís Lema, Professor Isabel Neves: all of you are a good example to the good people that I met and, in some cases, I had the privilege to work with! To “my people” at UTAD: my “scientific father” Professor Pedro Tavares who started as my supervisor and stay in my life as my friend! Mariana Fernandes and Lisete Ferrnandes my friends and scientific advisors: thank you both for always supporting me! From UTAD, I cannot forget Professor José Ramiro which is always available for me and also the new good people that this work allowed me to meet: Professor Jose Eduardo Pereira, Professor Justina Prada and Professor Isabel Pires: all of you were an essential support on in vivo trials. I really want to acknowledge all my friends and family for all the support that they gave me in all moments of my life. A special thanks to my “sisters” Sofia Sousa and Cláudia Cardoso which are always there for me and who never let me gave up. To my parents and my brother for being the pillar of my life. Finally, to Nuno which is, simultaneously, my best friend, my husband, my inspiration and my strength. Without your love and support this thesis would never occur. You are the best gift that life has ever gave me. Lastly, I gratefuly acknowledge Fundação Portuguesa para Ciência e Tecnologia (FCT) for the PhD scholarship ref.: PhD Program in Biomedical Engineering" (ref: NORTE08-5369-FSE-000012 opens 4 PhD scholarship position with the financial support of ESF – European Social Fund, under Programa Operacional Regional do Norte - Norte2020. iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v BACTERIAL CELLULOSE MODIFICATIONS FOR BIOMEDICAL APPLICATIONS ABSTRACT The main goal of this doctoral project is to explore the huge potential of bacterial cellulose (BC) in distinct areas, with particular focus on biomedical field. Specifically, i) the impact of different strains, fermentation time and fermentation medium on the BC properties was assessed; ii) the potential of oxidized BC as a wound dressing was analyzed; iii) exploratory studies on the use of laser technology for the surface patterning of BC were developed. With regards to the study on the influence of different culture conditions on BC’s properties, two strains (ATCC 53582 and ATCC700718), two distinct culture media (HS and MOL medium) and different bioreactors (static and agitated) were used. The time of culture was different in the static (6,15 and 30 days) and agitated (8 days) fermenters. The obtained results allowed to conclude that only slight differences (with no statistical relevance) between static and agitated conditions were observed in what concerns to BC properties. Then, and intending to explore the applicability of BC on biomedical field, BC membranes with hemostatic activity were produced through electrochemical oxidation. After oxidation using tetramethylpiperidine-1-oxyl (TEMPO) radical, the samples were characterized through several techniques. The oxidation degree was evaluated by titration and the obtained values revealed that increased from 4% to 7% and up to 15%, corresponding to an applied charge of 400, 700 and 1200 Coulombs, respectively. In vitro and in vivo biodegradability of oxidized BC membranes were evaluated and compared with that of Surgicel®, a commercially used hemostatic dressing. The oxidized BC preserved the crystallinity and the 3D nano-fibrillar network, and demonstrated hemostatic activity, although not as effective as that of Surgicel®. In vivo assays demonstrated that the oxidized membranes did not induce an inflammatory response, revealing a good biocompatibility. Finally, the surface pattern of BC was achieved through direct design of the laser patterns on wet BC surface. The interaction of the texturized surfaces with fibroblasts (L929) was assessed. The obtained results using XPS and FTIR techniques showed that the use of laser did not chemically modify the BC surface. The biocompatibility of both pristine and modified BC membranes was evaluated using mouse skin fibroblasts cells. SEM results showed that fibroblasts were present in both BC membranes surfaces exhibiting its usual phenotype, and significant differences between both groups in terms of metabolic activity were not detected. These results showed the good biocompatibility of both pristine and modified BC membranes, further work being necessary to exploit the potential of this methodology. Keywords - Bacterial cellulose; biomedical applications; fermentation; hemostatic materials; surface patterning vi MODIFICAÇÕES NA CELULOSE BACTERIANA PARA APLICAÇÕES BIOMÉDICAS RESUMO Este projeto de doutoramento pretende explorar o enorme potencial da celulose bacteriana (CB) em variadas áreas, dando particular enfoque à área biomédica. Especificamente, i) foi analisado o impacto de diferentes estirpes, tempo de fermentação e meio de fermentação nas propriedades da CB; ii) foi analisado o potencial da CB oxidada no tratamento de feridas; iii) foram realizados estudos exploratórios com tecnologia laser para a padronização de superfícies de CB. Relativamente ao estudo da influência de diferentes condições de fermentação nas propriedades da CB, foram testadas duas estirpes (ATCC 53582 e ATCC700718), dois meios de cultura distintos (HS e MOL) e bioreatores (cultura estática e agitada). O tempo de cultura também foi distinto, na cultura estática (6,15 e 30 dias) e agitada (8 dias). Os resultados obtidos permitiram concluir que houve apenas pequenas diferenças (sem relevância estatística) entre a cultura estática e agitadas em termos de propriedades de CB. Seguidamente, e com o objetivo de explorar a aplicabilidade da CB na área biomédica, foram produzidas membranas de CB com atividade hemostática por oxidação eletroquímica. A oxidação foi realizada usando o radical tetrametilpiperidina-1-oxil (TEMPO). O grau de oxidação foi avaliado por titulação, tendo-se obtido valores de 4% para 7% e 15%, correspondendo a uma carga aplicada de 400, 700 e 1200 Coulombs, respetivamente. A biodegradabilidade in vitro e in vivo das membranas de CB oxidadas foi avaliada e comparada com o Surgicel®, uma compressa comercial usada no tratamento de feridas. A CB oxidada preservou sua a cristalinidade e estrutura nanofibrilar, demonstrando atividade hemostática, embora não tão eficaz quanto o Surgicel®. Os ensaios in vivo demonstraram que as membranas oxidadas não induzem resposta inflamatória, revelando boa biocompatibilidade.Finalmente, a modificação da superfície da membrana de CB foi efetuada através da padronização com laser. Os resultados obtidos pelas técnicas de XPS e FTIR mostraram que a utilização do laser não modificou quimicamente a superfície da CB. A biocompatibilidade das membranas de CB foi avaliada usando fibroblastos (L929). Os resultados da microscopia eletrónica de varrimento mostraram que os fibroblastos aderiram em ambas as superfícies exibindo o fenótipo característico, não sendo detetadas diferenças significativas entre os dois grupos em termos de atividade metabólica. Estes resultados mostraram a boa biocompatibilidade das membranas de CB com e sem padronização, sendo necessários estudos adicionais para explorar o potencial desta tecnologia. Palavras chave - Celulose bacteriana; aplicações biomédicas; fermentação; materiais hemostáticos; padronização da superfície. vii LIST OF CONTENTS Chapter 1 ............................................................................................................................ 18 Introduction ......................................................................................................................... 18 1.1 - Context and Motivation ............................................................................................ 19 1.2 - Objectives ................................................................................................................ 21 1.3 - Thesis Outline .......................................................................................................... 22 1.4 - Dissemination and Communications ........................................................................ 22 1.5 - References .............................................................................................................. 23 Chapter 2 ............................................................................................................................ 25 Literature Review .................................................................................................................. 25 2.1 – Bacterial cellulose: biosynthesis and properties ....................................................... 26 2.2 – Culture methods for BC production ......................................................................... 31 2.3 – Biomedical and cosmetic applications of BC ........................................................... 35 2.3.1 – Drug delivery systems ................................................................................ 35 2.3.2 – Tissue Engineering .................................................................................... 36 2.3.3 – Cosmetics ................................................................................................. 37 2.3.4 – Artificial vessels ......................................................................................... 38 2.3.5 – Wound healing dressings and artificial skin ................................................ 39 2.3.5.1 – Hemostasis ............................................................................................ 44 2.3.5.2 – Topical hemostatic agents ...................................................................... 47 2.3.5.3 – Polysaccharide-based hemostatic materials ............................................. 49 2.3.5.4 – Oxidation of BC ...................................................................................... 51 2.4 – Biomaterials and biocompatibility ............................................................................ 53 xiv Figure 5.1. The characteristics of the different patterns tested. .............................................. 138 Figure 5. 2. The surface of the wet BC membranes after to laser treatment. P1 and P2 differ only on the distance between lines which is 0.2 and 0.1 mm, respectively. ............................................. 141 Figure 5.3. The appearance of the patterns after dehydration using SEM equipment. ............. 142 Figure 5.4. XPS survey spectra (left) and the deconvolution of the carbon peak (right) for both BC and BCL samples. .......................................................................................................................... 143 Figure 5.5. FTIR spectra of BC and BCL membranes. ............................................................ 145 Figure 5.6. The deconvoluted absorption peaks attributed to the Iα (750 cm-1) and Iβ (719 cm-1) crystalline forms of cellulose obtained by ATR-FTIR. ......................................................................... 146 Figure 5.7. Cell viability quantified by MTT assay after 24h and 72h of incubation. Significant differences are indicated as follow: * P<0.05, **P<0.01 and *** P<0.001. ...................................... 147 Figure 5.8. L929 morphology after 24h and 72h of incubation (Magnification: 800x). ............ 149 xv LIST OF TABLES Table 2.1. Bacterial sources of BC with distinct structures and biological roles (4).................... 26 Table 2.2. Commercially available materials produced from BC [Adapted from (75)]. ............... 43 Table 2.3. Advantages and disadvantages of synthetic biomaterials [Adapted from (123)]. ....... 54 Table 3.1.The main components of HS and MOL medium. ...................................................... 81 Table 3.2. d-spacings, crystallite sizes (D(hkl)) and crystallinity (CrI) of bacterial cellulose in static conditions determined by XRD diffractograms. .................................................................................. 91 Table 3.3. d-spacings, crystallite sizes (D(hkl)) and crystallinity (CrI) of bacterial cellulose in agitated conditions determined by XRD diffractograms. .................................................................................. 92 Table 3.4. Degree of polymerization (DP) of bacterial cellulose for all tested conditions. ........... 98 Table 4.1. Oxidation degree (in %) corresponding to different applied charges and the duration of electrolysis. .................................................................................................................................... 116 Table 4.2. Width of the oxidized and non-oxidized BC fibers. .................................................. 119 Table 4.3. Crystallite size and crystallinity index of pristine and oxidized BC. .......................... 121 Table 4.4. Histological assessment scores of oxidized and non-oxidized BC membranes according to ISO standard 10993-6 (annex E). ................................................................................................ 125 Table 5. 1. Elemental composition of BC and BCL samples analyzed by XPS. ........................ 144 xvi LIST OF ABBREVIATIONS ATR-FTIR – Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy BAI – Biomedical-associated infections BC – Bacterial Cellulose BCL – Bacterial Cellulose laser CrI – Crystallinity Index CSL –Corn Steep Liquor CV – Cyclic Voltammetry DDS – Drug Delivey System DP – Degree of Polymerization EPS – Extracellular Polymeric Substances FBGC – Foreign Body Giant Cells FBR – Foreign Body Reaction/Response H&E – Hemotoxylin and Eosin stain HAs – Hemostatic Agents HS – Hestrin and Schramm medium HST – Hyperthophic Scar Tissue IL – Interleukin K. xylinus – Komagataeibacter xylinus LTS – Laser Texturing Surface MCP-1 – Monocyte Chemotactic Protein-1 MIP – Macrophage Inflammatory Protein NMR – Nuclear Magnetic Resonance Spectroscopy PBS – Phosphate Buffer Saline PDMS – Polydimethylsiloxane rcf – Relative Centrifugal Field RGD – Arginine-Glycine-Aspartic acid RGDS – Arginine-Glycine-Aspartic acid-Serine ROS – Reactive Oxygen Species SEM – Scanning Electron Microscope TE – Tissue Engineering xvii TEM – Transmission Electron Microscopy TEMPO – 2,2,6,6-Tetramethylpiperidine-1-oxyl radical TGF – Transforming Growth Factor vvm – volume of air per volume of reactor per minute XPS – X-ray Photoelectron Spectroscopy XRD –X-ray Diffraction Remarks: In general, the International System of Units (SI) was used in this work. Sometimes multiples and sub-multiples of the SI units were also used, as well as other non-SI units but allowed by SI, such as the use of liter to express volume. Some units not recognized by the SI were also used to express some variables, such as the volume percent (% v/v), and mass per volume percent (% m/v) to denote the composition of some solutions, the revolutions per minute (rpm) to indicate the agitation rates and the volume of air per volume of reactor per minute (vvm) to designate the aeration rates, due to the usual use in fermentation technology area. 18 Chapter 1 Introduction In this first chapter, the context and motivation that supported this thesis are explained; the objectives of the work are delineated, and the content of this manuscript is outlined for the reader. Finally, the scientific dissemination outcomes of the performed work (research papers and communications in science meetings) are reported. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 19 1.1 - CONTEXT AND MOTIVATION Given its superior technical properties, bacterial cellulose (BC) has a broad range of applications, such as in the food and cosmetic industries. Beyond this, and with particular interest to this work, the application of BC on biomedical field is also possible and widely explored. BC represents an alternative source of cellulose with good mechanical properties, high crystallinity, high degree of polymerization and high-water content. It is also biocompatible, hydrophilic and non-toxic, exhibiting high purity, since unlike plant cellulose it does not have any hemicelluloses, pectin and lignin (1–3). BC can be produced under static and agitated conditions. Depending on the culture method, the resulting macroscopic morphology, microstructure, and properties are different. Having this in consideration, the choice of the fermentative technique is related with the final application of BC. Comparing both methods, static culture is the most used process, due to its simplicity. Since the BC is produced at the air-medium interface, one of the main challenge on a bioreactor design is to ensure a higher air-medium surface (4,5). However, this fermentation technique is characterized by the high cost, low rate of production and some issues are described related to delivery of oxygen, being the reason why agitated fermentation has been proposed. With agitated fermentation it could be possible to produce BC with different features. Instead of a one-piece membrane obtained in static conditions, pellets or irregular masses and fibrous suspensions are obtained with different sizes that could vary from 10 µm to 10 nm in diameter, bearing several shapes, from spherical or ellipsoidal, to stellate. These new features are consequence of the rotation speed, culture time and the additives used in the culture medium (6). The appearance of agitated culture intended to overcome the issue related to the delivery of oxygen by increasing the oxygen transfer. However, this increase on oxygen transfer generates non-cellulose producing mutants that are often related with lower levels of BC production, limiting its up-scaling. Nevertheless, some bioreactor cultures seem to be able to produce high levels of BC. However, this implies higher operational complexity. Therefore, the simpler static culture has been frequently used to obtain BC despite the long cultivation time required. As previously mentioned, the fermentation method affects the resulting BC properties. Thus, it seems crucial to study that influence through the evaluation of different parameters, such as: distinct bacterial strains, different times of culture and, of course, different fermentation methods. BC and other natural polymers are extensively applied in biomedical field due to their special properties. In this field, BC has been explored for wound healing applications and also for tissue Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 20 engineering and drug delivery. Taking this into account, in this doctoral project two distinct applications on biomedical field are envisaged. The first one is related with the possible application of BC as a hemostatic dressing. Hemostasis is the first stage of wound healing process. It is ascribed to the ability of the body to control the flow of blood when a vascular injury occurs. Thus, hemostasis includes the production of blood clot and its consequent dissolution followed by the repair of the injured tissue. It has a complex interplay of four key elements: the vascular endothelium, platelets, the coagulation pathway and fibrinolysis (7). Different hemostatic approaches may be applied according to the type of injury. Surgicel®, made by Ethicon Inc. of Johnson & Johnson, is a bio-absorbable material widely applied for intraoperative hemostasis and prevent adhesion in surgery. This material act as a mesh for platelet adhesion and aggregation, helping the formation of an artificial clot, while its negative charge is likely to activate the secondary hemostasis as well (8,9). Nevertheless, some issues were reported associated with the use of Surgicel®. One example of these issues is the case in which a patient was diagnosed with intracranial hemorrhage (10). Aiming to overcome the reported Surgicel® issues, in this work the production of a new hemostatic material based on BC is aimed, using electrochemical methods. It is also hypothesized that surface oxidation may lead to a substantial improvement of the BC biodegradability. Finally, the surface modification of the BC topography, as a means to improve its biocompatibility as an implantable biomaterial, will be tested. When implanted in the human body, biomaterials contact with surrounding tissues and induce a host response. The nature of this reaction determines the biocompatibility of the biomaterial. The human immune system is composed by the innate and adaptive systems that plays an important role in reacting against any foreign materials. Indeed, the implantation of any biomaterial may trigger a reaction of the immune system called Foreign Body Reaction/Response (FBR) that induce the formation of a capsule of dense fibrous tissue surrounding the implant, which eventually cause its loss of functionality. Thus, host reactions will determine the success of integration and biological performance of the biomaterial (11,12). Related with FBR, the biomaterial-associated infections (BAI) is another critical issue to address when a biomaterial is implanted. BAI take advantage on the presence of a substrate for opportunistic pathogen colonization, which culminates into an inflammatory response. In fact, the combination between FBR and bacteria creates a dysregulated immune niche more susceptible to bacterial adhesion and infection (13). Thus, it is essential the development of new approaches targeting BAI in order to avoid the possible biomaterial failure. Considering this, the modification of the biomaterial surface could contribute to solve this problem since Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 21 after implantation, biomaterial surface is the stage where interactions between the material and cells occurs. Hence, topographical features at micro and nanometer scales offer a promising new methodology to control cell-surface interactions. One example of this new methodology is the laser surface texturing which is based on the direct treatment of a surface using a laser beam. It is characterized by its high reproducibility, and it can be performed through the production of a regular or irregular patterns of bumps, dimples, and (linear or non-linear) grooves. Furthermore, it modifies the surface roughness and chemistry in several materials without using toxic substances. Also, it is a high processing-speed methodology with a low risk of surface contamination due to the absence of direct contact during the process (14). Thus, in this work, it is also aimed to pattern wet BC membranes using a CO2 laser and evaluate the influence of this treatment not only on BC properties but also on fibroblasts (L929) adhesion. With this technique, here developed only at exploratory level, it is expected to overcome the above issues related with biomaterial implantation. 1.2 - OBJECTIVES The work developed in this doctoral project and presented in this manuscript aims not only to increase the existing knowledge related with BC but also to develop new and improved applications for BC, especially on biomedical field. The main objectives of this work are the following: • Understand the influence of fermentation conditions on BC properties in order to envision new applications for this material, with special focus on biomedical field. To achieve that different parameters will be tested: distinct strains, different times of culture and the influence of static and agitated fermentation; • In order to overcome some critical issues reported regarded to the use of Surgicel®, this work aim at producing BC membranes with hemostatic activity. For that, we carried out the electrochemical oxidation of BC membranes with the tetramethylpiperidine-1-oxyl (TEMPO) radical; • Finally, an exploratory study was performed intending to improve the interaction of BC with biological tissues through surface laser patterning. In this perspective, this project aims at presenting the ground basis for the development of innovative bacterial cellulose products with excellent technological properties that meet emerging needs. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 22 1.3 - THESIS OUTLINE This thesis is divided into 6 main chapters, in which chapter 4 was published as research paper in a scientific journal. Chapter 2 - Literature Review , is a scientific state-of-the-art review on the subject of the present thesis. The main BC properties that make it an astonishing material to be applied in different fields are discussed. Chapter 3 – The influence of different fermentation conditions on bacterial cellulose properties. reports the effect of using different bacterial stains, different fermentation methods, different culture media and times of culture on the BC properties. Chapter 4 – Hemostatic oxidized bacterial cellulose membranes: present the new properties acquired by BC after oxidation through electrochemical methods. Chapter 5 – Laser patterning of bacterial cellulose membranes surface and its influence on cell adhesion: a preliminary study on BC surface modification through laser pattering and its influence on fibroblasts adhesion. Chapter 6 – Conclusions and future work , a final overview of the thesis is performed, highlighting the main findings and conclusions, and adding some suggestions for the future work with BC modifications, especially for biomedical applications. 1.4 - DISSEMINATION AND COMMUNICATIONS The experimental work exhibited in this thesis was partially published in international journals, as well as communications in national and international science meetings. The scientific output originated from this work is listed below. Publications in peer-reviewed journals • Queirós, E.C., Pinheiro, S. P., Pereira, J. E., Prada, J., Pires, I., Dourado, F., Parpot, P., Gama, M., (2021). Hemostatic dressings made of oxidized bacterial nanocellulose membranes. Polysaccharides. 2(1), 80-99. https://doi.org/10.3390/polysaccharides2010006. • Queirós, E.C., Rodrigues A.C., Dourado, F., Gama, M., (2021). Effect of culturing conditions on the properties of the bacterial nanocellulose. In preparation. Conference Poster Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 23 • E. C. Queirós., S. P. Pinheiro., J. E. Pereira., J. Prada., I. Pires., P. Parpot., M. Gama (2019) " Synthesis and characterization of oxidized bacterial cellulose through electrochemical methods: its biodegradability and potential as hemostatic material", 4th International Symposium on Bacterial Nanocellulose, Porto - Portugal, October, 2019. • E. C. Queirós., S. P. Pinheiro., J. E. Pereira., J. Prada., I. Pires., P. Parpot., M. Gama (2019) " Synthesis and characterization of oxidized bacterial cellulose through electrochemical methods: its biodegradability and potential as hemostatic material",30th Annual Conference of the European Society for Biomaterials together with the 26th Annual Conference of the German Society for Biomaterials, Dresden - Germany, September, 2019. • E. C. Queirós., S. P. Pinheiro., P. Parpot., M. Gama (2018) "The potential of bacterial cellulose as hemostatic material", CHEMPOR2018, Aveiro - Portugal, October, 2018. • S. P. Pinheiro., E. C. Queirós., V. Carvalho., Gama, M., P. Parpot. (2017) "Development of hemostatic materials made of electrochemically oxidized bacterial cellulose", CEB Annual Meeting, Braga - Portugal, July, 2017. 1.5 - REFERENCES 1. Eslahi N, Mahmoodi A, Mahmoudi N, Zandi N, Simchi A. Processing and Properties of Nanofibrous Bacterial Cellulose-Containing Polymer Composites: A Review of Recent Advances for Biomedical Applications. 2019;60(1):144–70. 2. Gorgieva S, Trček J. Bacterial Cellulose: Production, Modification and Perspectives in Biomedical Applications. Nanomaterials. 2019;9(10). 3. R P, CR L, PL A, RG S. Bacterial cellulose: a versatile biopolymer for wound dressing applications. Microb Biotechnol. 2019;12(4):586–610. 4. Chawla PR, Bajaj IB, Survase SA, Singhal RS. Microbial cellulose: Fermentative production and applications. Food Technol Biotechnol. 2009;47(2):107–24. 5. Parte FGB, Santoso SP, Chou C-C, Verma V, Wang H-T, Ismadji S, et al. Current progress on the production, modification, and applications of bacterial cellulose. 2020;40(3):397–414. 6. Wang J, Tavakoli J, Tang Y. Bacterial cellulose production, properties and applications with Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 30 Figure 2.3. Cellulose crystal structure: a) Iα triclinic structure and b) Iβ monoclinic structure [Adapted from (16)]. The chains of BC are thus packed into cellulose Iα, cellulose Iβ and also amorphous domains. The main difference between Iα and Iβ regards their intra and inter-unit H-bonding network, being the interunit H-bonding also different for cellulose II. This type of polymorph is thermodynamically more stable than cellulose I, being obtained through the regeneration or mercerization of cellulose I. Indeed, cellulose I and II exhibited distinct conformation of the polysaccharide chain since the former is parallel and the later anti-parallel, which determines changes on the mechanical properties of the material, specifically a decrease on Young’s modulus from 27 GPa (cellulose I) to 21 GPa (cellulose II) (3). On the other hand, cellulose III can be obtained by swelling cellulose I or II with amines or liquid ammonia, while cellulose IV is classically formed by annealing of cellulose II or III in glycerol (18). BC has great mechanical properties (Young’s modulus around 15-35 GPa and tensile strength of 200-300 MPa), high degree of polymerization (up to 8000) and high water content - up to 200 times its dry weight (5). It is a biocompatible, hydrophilic and non-toxic material that exhibits high purity since unlike plant cellulose it does not have any hemicelluloses, pectin and lignin. Many studies demonstrate that BC does not trigger a foreign body reaction. Helenius et al. (2006) assessed the in vivo biocompatibility through subcutaneous implantation in rats for 1, 4 and 12 weeks. The chronic inflammation, foreign body responses, cell ingrowth and angiogenesis were evaluated through histology, immunohistochemistry and electron microscopy. The obtained results showed that there were no macroscopic and microscopic evidences of inflammation around the implants. Also, there were no signs Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 31 of fibrotic capsule or giant cells and BC was well integrated into the host tissue and did not induce any chronic inflammatory reactions (19). Pértile et al. (2013) also showed the good biocompatibility of BC in a long-term in vivo study. The BC samples were implanted in mice and it was showed that a mild and benign inflammatory reaction occurred, which decreased along time and did not induce a foreign body reaction. Furthermore, there were no signs of chronic inflammatory reaction or encapsulation, while formation of new blood vessels around and inside (in the periphery) the implants were observed (20). Zhou et al. (2019) showed that the in situ incorporation of a carboxymethyl group on BC surface increased the cell affinity and viability. Also, after implantation, the tissue reaction revealed that carboxymethylation considerably increased the biocompatibility, since a lower inflammatory reaction was observed (21). More recently, Zhang et al. (2020) evaluated the in vitro biocompatibility of a BC scaffold for corneal stroma replacement, through the cultured of rabbit corneal epithelial and stromal cells on the BC scaffold, during a 3-month follow-up. The obtained results showed that BC supported cell adhesion, proliferation and differentiation. On the other hand, in vivo tests further confirmed the good biocompatibility and stability of BC in rabbit cornea, making BC an attractive option for tissue engineering of corneal stroma (22). Summing up, BC has a huge potential to be applied in several fields. In biomedicine, BC has been suggested as wound dressing material, as a replacement for skin, cartilage, bone and blood vessels, performing as a scaffold in tissue engineering strategies and also as a drug delivery system (12,23). Nevertheless, and beyond biocompatibility, biodegradation is one other desirable requirement for many applications in biomedical field. The modification of BC in order to increase and improve its biodegradability has been attempted. BC may be chemically modified through its hydroxyl groups. Thus, an improved biodegradation may be achieved through the oxidation, making BC reabsorbable by the organism. Besides this, after oxidation BC displays other characteristics such as hemostatic features, thus being a suitable raw material for some applications as a medical device (24,25). The potential of BC in the food and cosmetic industries has also been extensively reported, in particular as a thickener and stabilizer of oil in water emulsions (26,27). 2.2 – CULTURE METHODS FOR BC PRODUCTION A previously mentioned, BC can be produced under static and agitated conditions. Depending on the culture method, the resulting macroscopic morphology, microstructure, and properties are different (4). In 1954, Hestrin and Schramm developed a culture medium to produce BC, commonly named as HS medium, that became widely used for researchers and producers. The medium is composed by 2% Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 32 glucose (main carbon source), 0.5% peptone, 0.5% yeast extract, 0.27% anhydrous disodium phosphate, 0.115% citric acid monohydrate and adjusted to pH 6 (28). The production costs are high, due to the low volumetric productivity, making critical the development of new approaches that solve this problem. In fact, long cultivation times requiring large amounts of culture medium and low production yields are some of the difficulties in the conventional production of BC. In order to answer these problems, different fermentation parameters have been studied such as pH control, carbon sources (sugarcane, molasses, sucrose and rotten fruit), the bacteria strain, oxygen delivery and the bioreactor design (29–31). Recently, another strategy applied by the researchers is related with the use of agro industrial wastes to create a culture media showing an increase of cellulose production with lower costs. Furthermore, the addition of inducers to activate the energy metabolism in the microorganism and/or reduce the formation of metabolic by-products is another tested cultivation strategy (6). The choice of the fermentative technique, i.e. static or agitated, is related with the final application of BC, since the physical, mechanical and morphological features are dependent of the culture process. Static culture for BC production is the most common technique due to its simplicity. This method is characterized by the cultivation in shallow bottles or trays containing the liquid growth medium. The culture can be performed for several days or weeks after inoculation and fermentation at 25ᵒ-30ᵒC and a pH of 3-7. During the cultivation, the formation of a floating layer of a gelatinous BC pellicle occurs on the interface, being the BC production directly related to the surface area of the air-liquid interface. The BC pellicle is visible at the surface of the liquid about 2 days after the beginning of the culture and its thickness increases with the culture time (4,31). In order to solve the high cost, low rate of production and the delivery of oxygen ascribed to the static culture, the agitated conditions have been proposed. In fact, in static conditions the delivery of oxygen occurs only at the interface; however, very high oxygen supply achieved in a stirred tank has also a negative influence, since it may lead to a decrease of BC production due to the selection of non-producing mutants. Indeed, in some works using agitated conditions a lower productivity as compared to that achieved in static ones was justified by the appearance of a non-cellulose producing mutant and the genetic instability of bacteria under dynamic conditions (32,33). Thus, the decrease on BC production is consequence of cellulose-negative mutants (Cel-), which become more frequent than the producing cells (Cel+). Furthermore, although not increasing the BC yield, dynamic conditions may allow the production of BC with different characteristics. Instead of a one-piece membrane obtained in static conditions, pellets or irregular masses and fibrous suspensions are obtained than can vary in size from 10 µm to 10 nm in diameter, bearing various shapes, from spherical or ellipsoidal, to stellate. These parameters are related with the rotating speed, culture time and also with the additives Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 33 used in the culture medium (4). According to Hu et al . (2010), in agitated culture, the rotation speed is a key parameter in the formation of sphere-like BC. The authors tested four rotational speeds obtaining spheres with distinct sizes. In fact, when the applied rotational speed was less than 100 rpm it was difficult to find any sphere-like BC particles and irregular shapes was obtained. In opposition, the spherical shape appeared at 125 rpm with a size around 8 mm. Moreover, the increase of the rotational speed to 150 rpm produced a different shape exhibiting a tail-like feature with a decrease around 2.5 mm on its size. For speeds above 200 rpm, the formation of sphere-like BC was inhibited and some interconnected BC particles were observed, with a diameter around 1 mm. Additionally, high rotational speed did not increase the amount of produced BC (34). A layered structure is observed in microstructure of the spherelike BC, where denser fibers and bacteria are present, while the interior region is hollow (4). The agitated culture was proposed aiming to increase not only the oxygen transfer rate but also the mass transfer rate through the induction of a low shear stress. Nevertheless, as mentioned above this type of culture is also responsible to produce non-cellulose mutant that are often related with lower levels of BC production limiting its up-scaling. However, some bioreactor cultures seem to be responsible to produce high levels of BC. Since the BC is produced at the air-medium interface, one of the main challenge on a bioreactor design is to ensure a higher air-medium surface. Figure 2.4 depicts the stirred tank, the rotating disk and airlift bioreactors which are some examples of different configurations used for BC production (4,31,32). Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 34 The stirred tank allows high levels of mass transfer but requires high energy consumption. The agitated conditions bring about operational issues related to the increased viscosity, difficulty in properly mixing the system as the concentration of BC increases, which makes the operation at large scale highly complex. On the other hand, airlift bioreactor not only reduce the energy demand but also is able to produce lower shear stress when compared with stirred tank bioreactors, as agitation is achieved through the supply of an appropriate amount of air or oxygen. In the case of rotating disk bioreactor, the disks are partially submerged in the culture medium while the other half is exposed to the atmosphere. As the disks rotate continuously, the bacteria adsorbed at the surface of the disks alternatively contacts the atmosphere and liquid media all over the time, supplying nutrients and oxygen. Thus, the BC film grows on the surface of the disks (32). Using this bioreactor design, several types of solids and fibers can be added directly to the medium being incorporated on the forming BC layer, improving its properties. The main goal of this bioreactor is to achieve BC with a more homogeneous structure. However, the yield is not considerably higher when compared with the one obtained from the static culture (4). Figure 2.4. The different bioreactors design and the correspondent obtained BC’s shape. A) stirred tank bioreactor producing BC pellets; b) rotating disk bioreactor producing BC sheets and c) airlift bioreactor producing thin layer BC pellets [Adapted from (31)]. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 35 To sum up, the main drawback in agitated cultures is the production of cellulose-negative mutants and operational complexity. Consequently, the simpler static culture has been frequently used to obtain BC despite the long cultivation time required. 2.3 – BIOMEDICAL AND COSMETIC APPLICATIONS OF BC Natural polymers are extensively applied in biomedical field due to their special properties. BC has mainly been studied for wound healing applications, as artificial skin and blood vessels and also for tissue engineering and drug delivery, as briefly reviewed ahead. 2.3.1 – Drug delivery systems Drug Delivery Systems (DDS) intents to achieve a targeted therapeutic action, minimizing the side effects and allowing the control on the effective drug concentration over long periods of time. They include several approaches, formulations, technologies and systems especially developed to transport medicines inside the body in a safely manner and efficiently achieve their desired therapeutic effects. Contrary to the conventional systems for the administration of drugs, which generally exhibited side effects due to a systemic, nonspecific bio-distribution and uncontrolled drug release characteristics, the modern DDS allow the reduction on the dosage frequency and maintain the drug concentration at target tissues for a longer period of time (35). The superior properties of BC in the design of DDS has been demonstrated. Specifically, on transdermal drug delivery, the use of BC prevents moisture evaporation and maintain the contact with the injured area, which helps the localized drug delivery to the target site (36). BC membranes for transdermal delivery using different drugs has been widely studied by the group of Freire and Silvestre, from the University of Aveiro. The studied drugs included lidocaine (37), ibuprofen (38), caffeine (39) and diclofenac (40). In all studies, the first step was the partial removal of water by squeezing the BC membrane, which is then loaded by absorption of a solution with the selected drug. Glycerol is also impregnated, yielding plasticizing effects and helping the penetration of the drugs across the stratum corneum . The authors showed SEM results demonstrating that the drugs were homogeneously distributed on the BC membranes. All the drugs were tested for in vitro skin permeation and compared with conventional formulations. The results demonstrated that the drugs had slower permeation rates when compared to conventional formulations, which represents a benefit when a long-term release is required. More recently, the same authors investigated the long-term storage stability of BC membranes loaded Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 36 with the same drugs, using accelerated testing conditions at different temperatures and relative humidity. All the systems were relatively stable with no significant structural and morphological changes or variations in the drug release profile. These results showed the potential of BC for dermal delivery systems (41). Given its 3D network, BC allows the encapsulation of other drugs and can act as drug carrier. Müller et al. (2013) studied the applicability of BC as DDS for proteins using serum albumin as model drug. Albumin loading on never-dried and freeze-dried BC was compared, both formulations showing a dependency of concentration, temperature, time and pre-swelling. It was shown that loading and release of the protein were governed by diffusion and swelling controlled mechanisms, similarly to the behavior of the most conventional hydrogels. Moreover, drug loading was higher for never-dried BC gels comparing with freeze-dried BC, which might be explained by the structural changes in the BC network during freezedrying. However, both formulations exhibited potential as suitable DDS (42). Another study performed by Moritz et al. (2014) showed the incorporation of octenidine in BC aiming to develop a ready-to-use system for wound treatment. The drug loading and release, mechanical characteristics, biocompatibility and antimicrobial efficacy were assessed. The resulting material exhibited improved healing and superior material properties, combined with an efficient infection control and minimized unwanted side effects. Moreover, octenidine loaded BC had comparable release profiles to already marketed products and was stable for up to 6 months storage without losing their physicochemical and biological features (43). Recently, Li et al. (2019) explored the potential of BC for oral delivery of melatonin. In order to prepare BC nanofiber suspensions, BC was previously hydrolyzed by sulfuric acid followed by the oxidation and the melatonin-loaded BC nanofiber suspension was prepared by emulsion solvent evaporation method. The obtained results showed that the new formulation had good thermodynamic stability and melatonin was uniformly distributed in the BC nanofibers suspension. In vitro release studies revealed that this formulation exhibited faster dissolution rate and much higher cumulative release rate and bioavailability than the commercially available melatonin formulation (44). 2.3.2 – Tissue Engineering Tissue engineering (TE) is another field where BC potential application has been widely studied. TE is an interdisciplinary field, merging knowledge from both engineering and life sciences, aiming at developing new bio-based implants to repair or replace and restore function of damaged tissues or organs (45,46). Several works describe the use of BC in TE. For instance, a novel BC-alginate composite scaffold obtained by freeze drying was tested by Kirdponpattara et al. (2015). In this work, BC-alginate scaffold showed a stable structure in both water and phosphate buffer saline (PBS), exhibiting highly Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 37 interconnected pores and good swelling ability demonstrating no cytotoxicity against L929 mouse fibroblast cells. Furthermore, for long-term culture, BC-alginate composite scaffolds were able to support cell attachment, spreading, and proliferation on the surface. However, under static conditions, the cell migration and growth inside the scaffolds were limited, due to insufficient porosity. All the obtained results indicated that this new scaffold facilitate diffusion, cell proliferation and tissue generation for TE applications (47). In other study, a highly porous and biocompatible regenerated BC-gelatin composite scaffold was produced. This scaffold exhibited interconnected pores, high porosity and rapid swelling, which ensure nutrient exchange ability during practical applications. Additionally, it allowed the adhesion and proliferation of fibroblasts and penetration up to a depth of 200 µm after 7 days of culture. It also enhanced the expression of metalloproteases which revealed that extended cell culture might lead to extracellular matrix production inside the scaffold (48). Osorio et al. (2019) modified the BC 3D surface in order to develop a novel approach to biomimic soft extracellular matrix chemistry using fibroblasts to immobilize adhesion proteins such as collagen and fibronectin. These proteins were responsible for activating the integrin adhesion pathways, generating a stronger cell adhesion to the biomaterial. Higher cell adhesion was observed, revealing that this approach was able to biomimic the chemical surface of soft extracellular matrix (49). BC membranes were also used by Klinthoopthamrong et al. (2020) to produce an active non-resorbable guided tissue regeneration membrane conjugated with plant-derived recombinant human osteopontin (p-rhOPN). Initially, BC was grafted with poly-acrylic acid brushes in order to introduce multiple carboxyl groups on BC surface providing an anchoring site for p-rhOPN conjugation. This process did not influence the mechanical strength and fibrous structure of BC and full coverage for p-rhOPN was obtained. It was also confirmed by different assays that the biological functions of p-rhOPN was preserved after surface immobilization and could enhance bone tissue regeneration. This new strategy potentially promote human periodontal ligament stem cells adhesion and osteogenic differentiation to a greater extent than BC alone (50). 2.3.3 – Cosmetics The application of BC in skin treatments, essentially as facial masks, is known for a long time. Amnuaikit et al. (2011) evaluated the effects of BC masks in humans and their satisfaction with the product. The obtained results allowed to conclude that a single application of the BC masks enhanced moisture uptake by facial skin and the patient expressed satisfaction with the product (51). Pacheco et al. (2018) used cosmetic masks based on BC membranes to incorporate two different active cosmetics. One of them was composed by oat, rosemary, calendula extracts and hydroviton. The other one was Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 38 composed by propolis extract at 30% of polypropylene glycol. The results revealed that the loading of the BC membranes with both active cosmetics did not modify its pristine nanofibrilar structure and could be used as support for sustained release. The sensory tests indicated high scores for skin adhesion and mask handling and the release of formulations from BC membranes demonstrated altogether, an appropriated system for cosmetic application (52). Another work driven by Perugini et al. (2020) studied the effect of different cosmetics (anti-aging, lifting, and cell renewal) on skin moisturizing, color, viscoelastic properties, skin surface smoothness, wrinkle reduction, dermal homogeneity and stratum corneum renewal. A significant decrease on skin roughness and wrinkle breadth was observed, as well as a significant improvement on dermis homogeneity and firmness after two months of treatment with “anti-aging” BC-based masks. Moreover, after one month of treatment it was observed that: i) when BCbased “lifting” masks were used there was a substantial improvement on skin firmness and elasticity and, ii) the use of “cell-renewal” purifying and regenerating BC-based masks promoted the production of new skin cells through a mild exfoliating action, which improved the stratum corneum cohesion. These findings showed the effective tailored system to release into the skin different active components (53). Amorim et. al. (2020) also presented a biomaterial entitled BioMask based on BC and enriched with natural propolis, aiming to help in the healing of inflammations caused by acne. The incorporation of the natural propolis did not change the nanofibrilar structure of BC and enabled superior mechanical properties, suggesting that it could be applied as a vehicle for releasing active components in a more efficient way. This work showed that the combination of natural biodegradable polymers with natural active extracts might provide new biotechnological products that address the needs of the world market, which pursues safer and environmentally friendly alternatives (54). 2.3.4 – Artificial vessels BC can be synthesized with different shapes, generating an ideal substrate for cell attachment and proliferation, thus making it a good candidate to develop artificial vessels, as shown by Klemm et al . (2001;2009). In these works, the authors designed a material called BActerial SYnthesized Cellulose (BASYC®). This new tubular product was shaped directly during the cultivation and applied as artificial blood vessel, since it exhibited enough moldability and similar mechanical properties. It was implanted in the carotid arteries of rats and pigs, revealing long-term stability while maintaining the bypass unobstructed for 3 months. In fact, in a first work, the BC implants were placed on an artificial defect of the carotid artery of rats for 1 year. The results revealed the formation of neointima and ingrowth of active fibroblasts. In a second work, the grafts were implanted into carotid arteries of pigs and remove after 3 Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 39 months. The obtained results indicated that novel BC engineering technique produced stable vascular tubes, confirming the highly attractive approach to in vivo tissue engineered blood vessels (55,56). Andrade et al. (2010) functionalized BC with chimeric peptides containing a cellulose-binding module and adhesion peptides in order to improve the adhesion of human microvascular endothelial cells (HMEC) to BC. The study revealed that the presence of adhesion sequences significantly increased the HMEC attachment to BC surface and also stimulated angiogenesis. Therefore, the use of recombinant peptides containing the adhesion sequences allowed to control the interaction of this material with cells through its high affinity and specificity for cellulose surfaces (57). Several different materials were used to functionalized and improve BC properties. One example is poly (vinyl alcohol) (PVA). This material was responsible for enhanced tensile strength and improved water permeability of BC, which is important to maintain their long-term integrity (58). Similar results were obtained by Leitão et al. (2016) which also proved the good properties of BC for this application. The novel approach developed by these authors also exhibited a dense, malleable, and mechanically strong tubular BC prosthesis. Preliminary in vivo studies revealed the presence of neo-vessels and endothelial cells on the luminal surface of the graft (59). In other work, BC was modified through the deposition of chitosan into the fibril network. Furthermore, heparin was also chemically grafted into the BC tubes aiming to improve the anticoagulation and endothelialization performance. The strength at break increased but burst pressure slightly decreased with the novel methodology. However, and as aimed, the anticoagulation and endothelialization performance was improved remarkably with the presence of both chitosan and heparin, revealing the great potential to be applied as small-diameter vascular prosthesis (60). 2.3.5 – Wound healing dressings and artificial skin Wound healing is a dynamic biological process that leads to the tissue regeneration of wounded tissue. It includes the participation of several cellular and matrix components encompassing four different stages: i) coagulation (immediately after injury), ii) inflammation (starting shortly after injury) during which swelling occurs, iii) cell proliferation, where new tissue and blood vessels are formed and, finally, iv) the maturation phase, where the remodeling of new tissue occurs (Fig. 2.5). Actually, there is a significant overlap between all these phases and the entire healing process could take months to be complete (61). Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 46 two different mechanisms: the tissue factor pathway and the contact pathway (or extrinsic and intrinsic, respectively). Figure 2.7. Illustration of blood vessel hemostatic mechanism [Adapted from (84)]. Both pathways converge resulting in the formation of a fibrin clot which strengthens the primary platelet plug. In the intrinsic pathway, exogenous material with negative charge activates factor XII (FXII). This induces a downstream proteolytic activation of other coagulation factors until the factor X (FX) is activated. In more detail, the intrinsic pathway occurs ultimately through thrombin activation by FXII. It is activated when FXII, PK (proteins prekallikrein) and HK (high-molecular weight kininogen) assemble on a suitable surface or polymer. Activated FXI will activated FIX, which then acts with its cofactor (FVIII) to form tenase complex on a phospholipid surface to activate FX. In turn, the extrinsic pathway initiates when trauma of the vasculature expose tissue factor. This event will activate coagulation factor VII (FVIII) in the presence of calcium ions, promoting the conversion of FX to FXa. Thus, both pathways converge at the production of FXa. In the final common pathway FX with FV transforms the prothrombin (FII) into thrombin, which converts fibrinogen (FI) to fibrin. Fibrin is stabilized by FXIIIa (activated by thrombin), which cross-links the fibrin monomers producing a stable clot (79,82–84). Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 47 Finally, the fibrinolytic system intents to dissolve blood clots along the process of wound healing and also prevents the formation of blood clots in healthy blood vessels. This is a concurrently running system whose activation occurs during the coagulation cascade, aiming to limit the size of the clot. It is composed by three serine proteases existing as zymogens (i.e., proenzymes in the blood). Thus, fibrinolysis is an enzymatic process whereby plasmin dissolves the fibrin clot into fibrin degradation products. This enzyme is produced by the protease tissue-type plasminogen activator (tPA) and urokinasetype plasminogen activator (uPA) released from vascular endothelium (77,79). 2.3.5.2 – Topical hemostatic agents Hemorrhagic episodes are inevitable during a surgery procedure. Massive bleeding is controlled using standard surgical techniques like stitches, ligatures or clips. However, when diffuse bleeding occurs or the natural physiologic hemostasis is not enough to control hemorrhagic episodes, it is pivotal the use of hemostatic materials. Topical hemostatic agents (HAs) are a useful tool to supplement the coagulation system when the conventional procedures do not effectively control bleeding. Furthermore, the ideal HAs should exhibit high hemostatic action, low tissue reactivity, biodegradability, low cost, and specificity for different situations. There is no single ideal hemostatic agent meeting all these requirements, being imperative the careful choice of the suitable product for a given operative environment (85,86). HAs can be broadly divided into active and non-active (Fig. 2.8), distinguished by having or not physiologically active substances that may interfere on the blood clotting process. However, there are some HAs that reveal characteristics of both groups and they are called as flowable HAs (87). Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 48 Figure 2.8. Available topical hemostatic agents [Adapted from (87)]. Active HAs include thrombin, alone or in combination with other mechanical HAs, such as gelatin sponge. They provide high concentrations of thrombin, converting fibrinogen into fibrin at the bleeding site, forming a clot. On the other hand, and as previously mentioned, the non-active HAs do not intrinsically contain thrombin or other biologically active components, being composed only by the mechanical hemostatic agents and synthetic sealants. The former is characterized by the ability to produce a physical matrix that provides a barrier over the bleeding site and include oxidized cellulose, gelatins, and polysaccharides spheres. This type of HAs should be left in place until clot formation and then gently removed to avoid disruption and re-bleeding. The synthetic sealants are essentially applied to prevent suture hole bleeding in cardiac and vascular procedures, acting as adhesives and forming a watertight barrier over the bleeding sites. Finally, combining features of the mechanical and active HAs, arise the flowable HAs. This kind of HAs are able to block blood flow and actively convert fibrinogen in blood into fibrin at the bleeding site. They are divided into bovine collagen combined with human pooled- Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 49 plasma thrombin, and animal source gelatin combined with recombinant human thrombin, bovine, or human pooled-plasma thrombin. After application, flowable agents required the reconstitution of thrombin, which is mixed with a gelatin matrix. The final product displays a foam-like consistency being delivered through a blunt-nosed or blending applicator to the target site, expanding up to 20% over 10 minutes after application. In postponed applications, the product might be stored during 3 hours after the mixing process is completed. The excess of the product must be clean through saline irrigation without any risk of clot disruption. Both clot and the remaining product could stay in situ, both being resorbed in 6-8 weeks (87–89). 2.3.5.3 – Polysaccharide-based hemostatic materials The previous section introduced the different available types of topical HAs. There are several different hemostatic strategies that may be selected according to the injury type (90). Polysaccharides are natural polymers composed of sugar building blocks that allow chemical and physical modification. There are several works describing the use of polysaccharide-based materials as topical HAs, tissue adhesives and sealants. Beyond others polysaccharides, chitosan and cellulose are widely applied in this field (83). Chitosan based hemostatic agents have been investigated due to its positive charge which is, in fact, a double-edged sword in this area. Usually, it accelerates red blood cell adhesion, platelet adhesion and activation, while, at the same time, it inhibits the activation of the contact system. Thus, and in order to improve its hemostatic efficacy, its modification is required. HemCon®, Celox® and Colgel® are some examples of chitosan-based HAs (91–93). Cellulose, specially, oxidized cellulose (OxCell)-based HAs are also commonly used. When applied into the bleeding site, OxCell is able to absorb the blood and entrap different components such as blood proteins, platelets and red blood cells. This leads to an increase of the blood coagulation factors concentration accelerating the blood coagulation process until the clot formation. This phenomenon produces a gel-like “pseudo-clot” that can act as a barrier to block the blood flow. Furthermore, the coagulation process is induced by the negatively charged carboxylic groups on OxCell surface through the activation of the coagulation factor XII (94–96). Oxycel® and Surgicel® are some examples of OC-based HAs available as woven and nonwoven, respectively (86,92,97). Surgicel® made by Ethicon Inc. of Johnson & Johnson Medical Limited is a bio-absorbable material widely applied for intraoperative hemostasis and adhesion prevention in surgery. Surgicel® seems to act as a mesh for platelets adhesion and aggregation, helping on the formation of an artificial clot, while its negative charge is likely to activate Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 50 the secondary hemostasis as well. It is advisable to remove Surgicel® when the hemostasis is achieved. However, the most common procedure is to left it in situ to reabsorb spontaneously, usually without any secondary effects. If left in situ , the material become gelatinous in 24 to 48 hours, while the complete degradation should occur between 4 and 6 weeks (86,92,98). Nevertheless, there are different reported cases describing the presence of Surgicel® residues and several problems are ascribed to its use. One of such cases was the presence of a recurrent gastrointestinal stromal tumor four months after surgical resection, that was ascribed to the intra-abdominal foreign-body granuloma caused by the presence of Surgicel® residues (99). Another case reported the formation of a foreign body reaction, contributing to the development of an intracranial giant-cell granuloma. In this case, the patient was diagnosed with intracranial hemorrhage which was assigned to the use of Surgicel® (100,101). Other reports concerned the use of Surgicel® in thoracotomy in order to control hemorrhage. However, it passed through the intervertebral foramen causing spinal cord compression (102,103). Considering all the problems associated to the available materials, it is important to improve and/or develop new hemostatic dressings. In this context, other cellulose-based materials have been broadly explored and several modifications have been tested to increase its hemostatic potential. Vosmanska et al. (2014) modified OxCell with inert argon plasma which caused significant changes on chemical composition of the surface layers, as well as changes in morphology of those layers. The plasma treatment yielded a more acidic material owing to higher content of hydroxyl and carboxyl groups, which caused larger inhibition zones against E. coli and S. epidermidis , thus showing an improvement on antibacterial properties. The modification using inert argon plasma also improved the required properties for hemostatic function ascribed to oxidized cellulose (104). On the other hand, the functionalization of OxCell with chitosan is also widely described in the literature. Oxidized cellulose-chitosan sponges may be prepared by lyophilization, showing better hemostatic effect on hepatic trauma when compared to oxidized cellulose alone. Besides this, the proposed material revealed greater biodegradability and biocompatibility, suggesting its potential as a surgical hemostat (105). Cheng et al. (2019) developed a N, O -carboxymethyl chitosan/oxidized regenerated cellulose ( N, O -CS/ORC) composite gauze. This material was degradable and had excellent antimicrobial activity against S. aureus and E. coli , which is important to prevent wound infection. Moreover, when tested in a rat model with abdominal wall defect and cecum abrasion, it revealed to be effective to prevent the formation of peritoneal adhesion (106). Another study developed by Demirekin et al. (2015) proposed a novel oxidized regenerated cellulose powder using two different metal ions (sodium and potassium). The materials showed bactericidal activity against S. aureus and in vivo studies with rats Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 51 indicated that powder materials performed a quick blood coagulation and did not damage the tissue at the application area (96). 2.3.5.4 – Oxidation of BC Different methods have been tested to chemically modify BC membranes, being the surface acetylation, alkali, sulfuric acid and phosphorylation treatments some of the applied methods (25). The chemical modification at the C1 and C4 positions of the glucopyranose ring usually promotes degradation, resulting in a considerable decrease of crystallinity through the reduction of microfibril length and the loss of the exclusive mechanical behavior of BC (107). Beyond the previously mentioned methods, chemical modification of BC can be also achieved through oxidation. Generally, this method is accompanied by degradation and a concomitant decrease in the degree of polymerization which may be explained by the oxidation reaction per se, the β-elimination from carbonyls being responsible to the shortening of the chain. BC may be oxidized through different methods: radiation, energy impact or also by the application of oxidizing reagents (108), where the chemical oxidants can be selective or non-selective. Nitrogen oxides, ozone, alkali metal nitrites and nitrates and permanganates are examples of non-selective oxidants, while periodates and nitroxyl radicals are selective (109). Periodates open the pyranose ring leading to the oxidation of C2 and C3 to aldehydes (108,110). Furthermore, chemical modification at C1 and C4 regions usually promotes degradation resulting in a considerable decrease in crystallinity through the reduction of microfibril length and also a loss mechanical properties (107). Alternatively, the 2,2,6,6Tetramethylpiperidine-1-oxyl (TEMPO) nitroxyl radical has been widely applied to oxidize cellulose since it is able to convert polysaccharides into the corresponding polyuronic acids through the selective oxidation of the primary hydroxyl groups at C6 to carboxyl. In fact, TEMPO is able to promote a selective conversion of alcoholic hydroxyl groups to aldehydes, ketones and, as mentioned, to carboxyl groups (111). In this process (Fig. 2.9), TEMPO is oxidized through a one-electron transfer reaction to the corresponding oxoammonium. This oxoammonium is the active oxidant in the primary alcohol oxidation. Then, the electrooxidation of the hydroxylamine regenerates TEMPO+, in situ . Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 52 Figure 2.9. Electrochemical oxidation mediated by TEMPO radical [Adapted from (112)]. This electrochemical approach is a suitable alternative to the chemical co-oxidants like NaClO-NaBr and water-acetonitrile-NaClO-NaClO2. The anodic regeneration of the oxidizing species instead of the primary oxidants is considered cleaner (112). Furthermore, TEMPO is also characterized by its stability, non-toxicity and non-mutagenic features (113), high reaction rate and yield, allowing a controlled modification of the polysaccharides (114). However, the selective oxidation of C6 primary hydroxyl only occurs on BC microfibrils surfaces or in cellulose I crystallites without taking place inside the crystallites (115,116) (Fig. 2.10). After oxidation, the carboxylate groups formed on microfibrils surface exhibit negative charges causing repulsive effects between the microfibrils, helping on the individual microfibrils disintegration through mild mechanical treatment in water (117,118). Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 53 Figure 2.10. Representation of C6 primary hydroxyl oxidation on cellulose microfibril surface mediated by TEMPO [Adapted from (117)]. Additionally, the presence of carboxylate groups on the microfibril surface may affect the surface properties such as topography, composition, roughness and surface free energy (107). Due to all these effects, oxidized BC exhibit new features such as in vivo degradability and hemostatic properties (119) which in association with all other BC properties make it an excellent material for biomedical applications. 2.4 – BIOMATERIALS AND BIOCOMPATIBILITY 2.4.1 – Cell-biomaterial interactions Although considered highly biocompatible, BC may be further improved as a biomaterial. In the last decades, the use of biomaterials in biomedical field, namely as implantable devices or to promote functional tissue regeneration, increased exponentially. According to Williams (2009), a biomaterial is “a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of ant therapeutic or diagnostic procedure” (120). After implantation, biomaterials contact with surrounding cells eliciting a host response that corresponds to the first steps of tissue repair. In fact, the immune response to biomaterials depends of different factors such as the method of implantation, the source of biomaterials Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 54 and their physicochemical properties, molecular weight, chemical composition, mechanical properties and degradation rate. Modern implant designs intend to use this immune response to improve implant integration while avoiding chronic inflammation, foreign body reactions and the risk of losing the proposed function (121,122). Biomaterials can be classified as natural or synthetic. In the former case, they are derived from animals, microbes, or plants and are similar to materials familiar to the body. Also, they are biodegradable and the natural degradation might occur in the body through enzymes. On the other hand, synthetic biomaterials can be divided into four groups: metals, polymers, ceramics and composites and the main advantages and disadvantages of all the groups are present on table 2.3. Table 2.3. Advantages and disadvantages of synthetic biomaterials [Adapted from (123)]. Biomaterial Advantages Disadvantages Metal Strong, tough, and ductile Corrodible, dense, and hard fabrication Polymers Resilient and easy fabrication Fragile, deformable, and degradable Ceramics High biocompatibility, inert, and strong in compression Hard fabrication, brittle, and not resilient Composites Strong in compression Hard fabrication Metals are essentially used on artificial joints for hips and knees and the most commonly used are stainless steel, pure titanium and, titanium alloys. Polymeric biomaterials are applied in facial prosthesis, tracheal tubes and in medical adhesives and sealants. Polyesters, polytetrafluoroethylenes, and polyurethanes are examples of polymeric biomaterials. On the other hand, due to its poor fracture toughness, the application of ceramics is almost limited to restorative material in dentistry. Finally, Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 55 composites are broadly used in prosthetic limbs since their low density/weight and high strength make them suitable for this application (123). As previous mentioned, after implantation biomaterials will contact with surrounding cells and induce a host response. The human immune system is composed by the innate and adaptive systems that plays an important role in reacting against any foreign materials. Both systems involve several humoral and cellular factors that are crucial to ensure an effective immune response. Figure 2.11 present the cells and proteins that mediate this immune response. The implantation of any biomaterial into the organism may cause a reaction of the immune system called by Foreign Body Reaction/Response (FBR). This reaction is responsible for the formation of a capsule of dense fibrous tissue surrounding the implant, which eventually cause its loss of functionality. In fact, after implantation, host reactions will determine the success of integration and biological performance of the biomaterial (124–127). These reactions involve different steps following the injury associated to the implantation procedure, blood-material interactions, provisional matrix formation, acute and chronic inflammation, granulation tissue development, FBR, and fibrosis/fibrous capsule formation (128). Figure 2.11. Cells and proteins involved in mediating effective immune response [Adapted from (124)]. FBR is a result of a complex interaction between the innate and adaptive immune system which is not yet completely understood. The current understanding of this intricate process encompassed five phases: i) protein adsorption, ii) acute inflammation, iii) chronic inflammations, iv) foreign body giant cell Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 62 also of the spatial arrangement on BC substrates was performed, allowing the control of the adhesion and differentiation of the cellular mediators and actuators of fibrosis (154,155). Figure 2.14. Cell morphology on surface structured BC using hexagonal and squared patterns [Adapted from (155)]. Based on these works, Robotti et al. (2020), developed a micro-engineered BC coating for cardiac implantable electronic devices. A set of micro-wells arranged in a hexagonal pattern were created on a BC substrate which was used to coat a cardiac implant. This strategy was proven to reduce by 66% the thickness of the fibrotic tissue around the implant, both the generator and the proximal parts of the leads remaining fully free from fibrotic tissue (156). Other works have been developed based on BC patterning with laser (157–159). Although BC is considered a promising nanoscaffold for tissue engineering, the absence of large pores for cell ingrowth is a limitation. Thus, laser patterning was used to create regular vertical pore arrays in order to obtain novel porous BC membranes with large pores. The biological properties were also improved through the BC membranes modification with gelatin and hydroxyapatite, making this novel material a suitable alternative in bone tissue engineering fields (157). In the same field, Ahrem et al. (2014) used 3D laser perforation of never-dried BC hydrogels to increase the size and heterogeneity of the pores. Laser perforation slightly performed structural modifications (i.e., fiber or globular aggregates), but did not induce any chemical modifications. Furthermore, the resulting channels supported cell migration into the BC hydrogels, the matrix production and phenotypic stabilization of chondrocytes (158). More recently, Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 63 Hu et al. (2019) used low-energy CO2 laser photolithography to construct a crossed groove/column micropattern on the BC surface, where the tetrapeptide Arginine-Glycine-Aspartic acid-Serine (RGDS) was immobilized, improving the affinity to fibroblasts. After hydrating, the new formulation did not damage the integrity of the micropattern, suggesting its potential to be applied in a highly hydrated environment. Moreover, the crossed groove/column micropattern structure on the BC showed dual affinities to human skin fibroblasts cells and collagen which allowed not only the cell migration but also the manipulation of collagen distribution (159). To sum up, surface topography may be used to control cell behavior on surfaces, specifically cell adhesion, spreading, proliferation and differentiation. 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Demirekin ZB, Sezer UA, Karatopuk DU, Sezer S. Development of Metal Ion Binded Oxidized 78 Chapter 3 The influence of different fermentation conditions on bacterial cellulose properties The influence of fermentation conditions on BC properties was evaluated. Different parameters were tested: distinct bacterial strains, different times of culture and the influence of static versus agitated fermentation. Two bacterial strains were evaluated (ATCC 53582 and ATCC700718) and two distinct culture media were used (HS and MOL). On static conditions, three times of culture were tested (6,15 and 30 days) while in agitated culture, BC was only cultivated for 8 days. The samples obtained were characterized using different techniques, such as: XRD, ATR-FTIR and the degree of polymerization was also determined. The obtained results revealed only slight differences (with no statistical relevance) between static and agitated conditions in what concerns to BC properties. A slight decrease on degree of polymerization and BC’s crystallinity on agitated culture seems to occur, when compared to static fermentation. Although, the chemical structure of BC was preserved since in both fermentation methods and all the time-points, the crystallinity and the fraction of Iα was high for all the samples. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 79 3.1 - INTRODUCTION Bacterial cellulose is a biopolymer widely present in nature and produced by different organisms, ranging from plant to algae, fungi and bacteria (1). Even though plant and bacterial cellulose are chemically identical, substantial differences have been found between them in what concerns macromolecular properties and also in purity since, contrarily to plant cellulose, BC is secreted without hemicelluloses and lignin (2). The synthesis of BC is an intricate process involving several enzymes and complexes of catalytic and regulatory proteins. The process includes the formation of the cellulose precursor Uridine diphosphate glucose (UDP glucose) followed by glucose polymerization into the (1,4) glucan chain. Then, the secreted glucan chains aggregate and assemble, creating cellulose ribbons at nanoscale size, which in turns produced a network structure with high porosity. After that, a 3D structure is generated through crosslinking with other elementary fibrils and development pf microfibrils (3). Overall, BC may be produced using two different fermentation methods, e.g., static or agitated. Static culture is the most common technique due to its simplicity as it involves low, or no shear power being extensively applied to produce BC pellicles. The culture can be performed for several days or weeks after inoculation. The production of BC occurs at the air-liquid interface. Nevertheless, the commercial application of BC produced this way is still limited due to its low productivity and increased incubation time-scale (1,4). On the other hand, the BC produced through agitated culture exhibit several different forms such as fibrous suspensions, pellets, spheres or even irregular masses, due to the agitation and availability of oxygen and nutrients in adequate amounts in the bulk phase. Moreover, features like size, shape and amount of BC are associated to the rotating speed, oxygen supply, agitator configuration, the duration of the culture and the shear force in culture medium (5,6). The main drawback of agitated culture is the possibility of converting some cells into non-cellulose producing mutants decreasing the productivity (6). Beyond this, strains and culture conditions have a considerable effect on the microstructure and physicochemical properties of the produced BC, namely on properties such as crystallinity and fraction of the Iα allomorph (7). In this work, we intended to study the influence of culture medium, the time of culture and the used strain on the BC’s properties. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 80 3.2 – MATERIALS AND METHODS 3.2.1 – Fermentation conditions 3.2.1.1 – Bacterial strains Komagataeibacter xylinus (ATCC 700718 and ATCC 53582), from the American Type Culture Collection were used to produce BC under static and agitated conditions. The strains were kept in HestrinSchramm culture medium (HS medium) (8), in solid state with 2% (m/v) agar (Acros Organics). For agitated fermentation only ATCC 700718 strain was used. 3.2.1.2 – Inoculum preparation The inoculum preparation procedure was the same for both ATCC 700178 and ATCC 53582, according the previous work developed by the group (9). In short, Komagataeibacter xylinus cells were grown in 1 L conical flasks with 100 mL of HS medium, comprising (in % m/v): 2.0 glucose (Fisher Chemical), 0.5 peptone (OXOID), 0.5 yeast extract (OXOID), 0.27 disodium phosphate di-hydrated (Na2HPO4.2H2O) (Panreac) and 0.115 citric acid (Panreac). The initial pH was set at 5.5 using 18% (v/v) HCl (Fisher-Chemical). Before inoculation, the cultivation medium was autoclaved at 121°C, 1 bar for 20 min. The medium was incubated for 48 h at 30ᵒC under static conditions. The inoculum preparation was the same for both static and agitated fermentation. 3.2.1.3 – Static fermentation In the static fermentation both ATCC 700178 and ATCC 53582 strains were used, and different culture media were evaluated. In the case of ATCC 700178, two distinct culture media were tested: HS medium supplemented with absolute ethanol (referred to as ATCC 700178_HS) and a culture media prepared using molasses (referred to as ATCC 700178_MOL). On the other hand, in the case of ATCC 53582 only HS medium was used without ethanol supply (ATCC 53582_HS), since according to previous studies ethanol does not increase the production of BC by this strain. For both strains, after inoculum preparation, the formed cellulose pellicle was shaken to release the bacteria entrapped within the cellulose matrix into the residual medium and culture in fresh culture medium. Furthermore, only for ATCC 700178 and prior to culture, the cells were transferred to sterile conical flasks with new culture medium, making up 10% (v/v) of the final volume of fermentation Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 81 containing a culture media, prepared using (in % m/v): 4.0 molasses (a gift from RAR - Refinarias de Açúcar Reunidas, S.A; Portugal), 0.7 Corn Steep Liquor (a gift from COPAM Companhia Portuguesa de Amidos, S.A; Portugal), 0.15 citric acid (Panreac), and 0.26 disodium phosphate di-hydrated (Panreac). In order to simplify the distinguish between HS and MOL medium, on table 3.1 are present the main components of both culture media: Table 3.1.The main components of HS and MOL medium. Culture media Components HS • 2.0 of glucose (% m/v); • 0.5 of peptone (m/v); • 0.5 of yeast extract (% m/v); • 0.27 of disodium phosphate di-hydrated (% m/v); • 0.115 of citric acid (% m/v). MOL • 4.0 of molasses (% m/v); • 0.7 of Corn Steep Liquor (% m/v); • 0.15 of citric acid (% m/v) • 0.26 of ammonium sulphate (%m/v). ATCC 53582_HS cells were incubated with a ratio of 1% (v/v) of inoculum to 99% (v/v) of fresh HS medium while both ATCC 700178_HS and ATCC 700178_MOL cells were incubated with a ratio of 0.5% (v/v) of inoculum to 1.5% (v/v) of absolute ethanol and to 98% (v/v) of fresh HS medium supplemented with absolute ethanol for ATCC 700178_HS and fresh culture media prepared using molasses for ATCC 700178_MOL. In all tested conditions cells were incubated at 30°C for 6, 15 and 30 days under static conditions. 3.2.1.4 – Agitated fermentation Cells were grown in 1L conical flasks, as described on the “Inoculum preparation” section. Agitated fermentation was performed in an Eppendorf DASGIP® Parallel Bioreactor System for Microbial Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 82 Applications (Switzerland) with 2L vessels, with a working volume of 1L. Since for this fermentation method only ATCC 700178_HS was used, the experiments were performed by mixing 0.5% (v/v) of the inoculum with 1.5% (v/v) of absolute ethanol and 98% (v/v) of HS medium for a final volume of 1 L, using a stirring speed of 150 rpm. The temperature was kept at 30 ᵒC and the air flow rate was controlled at 1.5 vvm. Initial pH was 5.5 and the initial dissolved oxygen was 30 % of saturation. The experiments were performed for 8 days in quadruplicate. In order to summarize the tested parameters, in the follow diagram the tested conditions are present: Figure 3.1. Diagram of all tested conditions on BC production. 3.2.2 – BC purification After incubation, the culture medium was discharged and BC was collected, crushed and washed thoroughly to remove the remaining medium components and eliminate bacterial cells. First of all, BC was treated with 0.1M sodium hydroxide solution (NaOH) during 72h, with changes of the washing solution every 24h. After that, NaOH was rejected, and BC was washed three times (30 minutes each) with 2% (v/v) Divosan Hypochlorite and then with distilled water. Subsequently, BC was treated with 4% (v/v) acetic acid to decrease the pH and then with distilled water until the pH of the supernatant became Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 83 neutral. The purified BC was autoclaved at 121°C, 1 bar, for 20 min and stored at 4°C until used. Prior to use, BC was frozen at -80°C and freeze-dried at -100°C (Scanvac Coolsafe) during 5 days. This procedure was applied for both static and agitated conditions. 3.2.3 – BC characterization 3.2.3.1 – SEM The lyophilized BC samples were placed onto the sample stage and sputter-coated with an Au 5nm film. Observations were performed using a Scanning Electron Microscope, SU8010, Hitachi, Japan, at an accelerating voltage of 5.0 kV. For each sample, the nanofibers diameters were measured by analyzing two different SEM micrograph with Fiji ImageJ, performing an average of 100 readings per sample. 3.2.3.2 – TEM The lyophilized BC samples obtained by static fermentation. For ATCC 700178 the high-resolution morphologies were performed using a Field Emission Transmission Electron Microscope (FETEM, Talos F200X, FEI Company, Hillsboro, USA). To obtain individual BC fibers for FETEM characterization, 0.2-0.3 mg BC was dispersed in 2 ml ethanol, followed by ultrasound dispersion for 4 hours. For ATCC 53582 the high-resolution morphologies were performed using a Field Emission Transmission Electron Microscope JEOL JEM 2100. To obtain individual BC fibers for FETEM characterization, a suspension of 0.02 % of BC fibers was dispersed in Mili-Q water followed by homogenization vortex and ultrasound dispersion for 2 hours and 3-5 drops of sample on a TEM grid placed on filter paper. 3.2.3.3 – XRD The crystallinity was assessed using a PANalytical X’Pert Pro MPD diffractometer equipped with X’Celerator detector and secondary monochromator. All BC samples were analyzed at room temperature using a CuKα radiation and Bragg-Bentano geometry, 0.017°/step and 100 s/step. The crystallinity index (CrI) (Equation 1) was calculated using the Segal, Creely, Martin & Conrad (1959) (10) equation, i.e., it was calculated as a function of the maximum intensity of the diffraction peak Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 84 from the crystalline region (I200), at an angle of 2θ ~ 22.5°, and the minimum intensity from the amorphous region (Iam), at an angle of 2θ ~ 18°. 𝐶𝑟𝐼 (%)=(𝐼200 − 𝐼𝑎𝑚) 𝐼200 (1) The apparent crystallite size (CS) in the crystallographic planes (1–10), (110) and (200) were calculated assuming that the crystals exhibited uniform size and shape and according to Scherrer’s equation (Equation 2): 𝐶𝑆 =𝐾𝜆 𝐹𝑊𝐻𝑀 cos 𝜃 (2) where K is a dimensionless factor dependent on the method used to calculate the amplitude (K = 0.9), λ is the wavelength of the incident X-ray (λ = 0.15 nm), FWHM is the width of the diffraction peak at half-maximal height (in radians) and θ is the angle of the diffraction peak of the crystalline phase (Bragg’s angle). The diffractograms were analyzed by Rietveld refinement with Powder Cell software. 3.2.3.4 – ATR-FTIR All BC samples were analyzed using an ALPHA IIBruker spectrometer (Ettlingen, Germany) with a diamond-composite attenuated total reflectance (ATR) cell. Data were collected using OPUS software, which is integrated in the FTIR equipment. The measurements were recorded with a wavenumber range from 4000 cm-1 to 400 cm−1, with a resolution of 4 cm−1 and 64 scans per sample. The ATR clamp and platform were cleaned with a cotton swab dampened with isopropyl alcohol and then allowed to dry between the analysis of each sample. All assays were done in duplicate for each sample. 3.2.3.5 – Determination of % Iα mass fraction Through FTIR spectra, hydrogen-bonding vibrational bands are present in the 700-800 cm-1 range (11). Allomorphs Iα and Iβ have their bands around 750 cm-1 and 710 cm-1, respectively, and they have been used to estimate the ratio of these two allomorphs. The Gaussian function was fitted under the peaks around 750 cm-1 and 710 cm-1. The mass fraction of the Iα allomorph was calculated using the following equation (12) (Equation 3): Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 85 % 𝐼𝛼 = 2.55 × 𝐴𝐼𝛼 (𝐴𝐼𝛼 − 𝐴𝐼𝛽)− 0.32 (3) where AIα and AIβ are ascribed to the integrated intensities of the contributions from celluloses Iβ and Iα at 710 cm-1 and 750 cm-1, respectively. The Iα mass fractions in the celluloses was determined by the Gaussian deconvolution of the peaks, subtracted from a local linear background, and their integration using OriginPro software. 3.2.3.6 – Degree of polymerization All BC samples were cut into small pieces and mixed with 5 mL of distilled water under agitation for 30 min. Then, 5 mL of cupri-ethylenediamine (CED, Sigma-Aldrich) saturated with copper (II) hydroxide was added; the mixture was agitated for 60 min and then centrifuged for 5 min at 7000 rcf. Finally, the intrinsic viscosities of the BC samples were obtained using an Ubbelohde capillary-tube viscometer from Rheotek, placed in a water bath at 25°C. The viscosity values were obtained according to the standard ES ISO 5351:2012 (Pulps—Determination of limiting viscosity number in cupri-ethylenediamine (CED) solution). Thus, the viscosity ratio ηratio (dimensionless unit) was calculated using the following equation (Equation 4): 𝜂𝑟𝑎𝑡𝑖𝑜 =𝜂 𝜂0 = ℎ × 𝑡 (4) where h is the viscometer constant that was determined to be 0.132 s−1, and t is the efflux time (in seconds) of the test solution. The viscosity-averaged degree of polymerization (DPv) was calculated using the Mark–Houwink–Sakurada equation (13): [𝜂]= 𝐾 × 𝐷𝑃𝑣𝛼 (5) where, for BC samples, K = 0.0002 and α = 1.9705 (14). Finally, the molecular mass of BC was calculated from the relationship: 𝐷𝑃 𝑣=𝑀 162 (6) Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 86 where M is the molecular mass of the BC and 162 (g/mol) equals the molecular mass of an anhydroglucose unit (15). All the samples were analyzed in triplicate. 3.3 – RESULTS AND DISCUSSION The results presented on SEM and TEM sections (3.3.1 and 3.3.2, respectively) were obtained only for static fermentation and only for 6 days of culture. Furthermore, in the case of ATCC 700178 only one media was used: ATCC 700178_MOL. Besides the high costs ascribed to agitated conditions, on this type of fermentation, the produced BC stayed attached on the blades of the bioreactor which causes some losses on the obtained material. Thus, and aiming to reduce the costs associated to this process, both techniques were applied for both strains but only for static fermentation. Despite using only one type of fermentation, these results were very important since it gave some insights about the influence of the strain on some BC properties. In Fig. 3.2 are illustrated the BC appearance obtained for both static and agitated fermentations. Static Fermentation Stirred bioreactor Figure 3.2. The appearance of obtained BC after static and agitated fermentation. 3.3.1 – SEM Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 87 In order to understand the influence of producing BC using ATCC 53582 and ATCC 700178 strains on its morphology, it was performed a SEM analysis and the results are presented on Fig. 3.3. Fiber diameter ATCC 53582 ATCC 700178_MOL Figure 3.3. SEM images and mean fiber diameter of BC produced by different strains under static conditions. Scale bar: 5 µm. According to Fig. 3.3, for both strains, the complex structure of BC composed by a 3D randomly network with interconnected fibers was presented in both strains. Furthermore, the calculated mean fiber diameter of the BC samples was around 96 nm for ATCC 58532 (16,17) and around 43 nm for ATCC 700178_MOL. The higher values obtained for ATCC 53582 might be explained by the assembling of several fibers, as shown on the figure 3.3. On the other hand, the values obtained for ATCC 700178_MOL are in the range of the mean values obtained by Chen et al. (2017) (18). 3.3.2 – TEM TEM analysis was also applied on BC samples produced by both strains under static conditions, and the obtained results are presented on Fig. 3.4. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 94 Static bioreactor - 30 days of culture Stirred Bioreator – 8 days Figure 3.6. ATR-FTIR spectra for all tested conditions. (–) ATCC 53582_HS; (–) ATCC 700178_HS; (–) ATCC 700178_MOL and (–) ATCC 700178_HS under agitated conditions. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 95 All the samples exhibited the typical bands ascribed to the chemical structure of BC: (O–H symmetrical stretching around 3350 cm-1; C–H stretching around 2900 cm-1; C–O–C stretching around 1162 cm-1; C–O stretching at 1030 cm-1; vibration of amorphous cellulose (stretching of the glucose ring) around 899 cm-1 (30–33). Moreover, there were no other significant differences between the spectra since in all of them, all peaks appeared in the same position and at comparable intensities. 3.3.5 – Determination of % Iα mass fraction Figure 3.7 show the typical bands ascribed to both allomorphs, Iα and Iβ, at 750 cm-1 and 710 cm-1 respectively, demonstrating the ability of the bacteria to produce both allomorphs simultaneously. According to Imai et al.(1998) the absorbance bands at 3240 cm-1 and 3270 cm-1 are ascribed to Iα, and Iβ, respectively (34). The Iα mass fractions in the celluloses was determined by the Gaussian deconvolution of the peaks and the obtained results are presented in the following figure. Static bioreactor - 6 days Static bioreactor - 15 days Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 96 Static bioreactor - 30 days Stirred bioreactor – 8 days Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 97 Figure 3.7. The deconvoluted absorption peaks attributed to the Iα (750 cm-1) and Iβ (719 cm-1) crystalline forms of cellulose obtained by ATR-FTIR. Figure 3.7 show that the Iα values vary between 43 and 67%. These values were in agreement with the literature (35–38). Moreover, the mass fraction of Iα slight increased all over the time for all the samples that were tested in static conditions, since for 6 days of culture the values were between 43% and 48%, while for 30 days these values varied from 50% to 67%. The strain ATCC 700178 seems to have a slightly higher amount of Iα allomorph, both in static (50-67%) and agitated (63%) conditions. However, the differences observed are not dramatic. The results suggest that fermentation method does not affect significantly the mass fraction of Iα, although slight differences were observed for both strains and also for the culture medium. 3.3.6 – Degree of polymerization The degree of polymerization characterizes the number of repeating units present in a polymer. According Isogai et al. (1998) the degree of polymerization of cellulose may have some variations related to its source (39). The influence of fermentation conditions - culture medium, time of culture and the strain – on the degree of polymerization was evaluated and the obtained results are summarized on table 3.4. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 98 Table 3.4. Degree of polymerization (DP) of bacterial cellulose for all tested conditions. ATCC 53582_HS ATCC 700178_HS ATCC 700178_MOL DP 6 days 3070 ± 116 3010 ± 131 2866 ± 97 15 days 3004 ± 55 2976 ± 86 2565 ± 8 30 days 2942 ± 48 2852 ± 47 2436 ± 42 Stirred bioreactor 8 days 2659 ± 75 According to table 3.4, the degree of polymerization exhibited high values (2500-3000), which is expectable for BC samples (40–42). The value observed decreased over the cultivation time for all strains. Moreover, and despite the different fermentation times (6 versus 8 days), it seems possible to conclude that under agitated conditions the degree of polymerization tend to be smaller, comparing to static culture. The value obtained for 8 days of agitated culture the DP was 2659, while for the same strain in static culture a value of 2866 was obtained after 6 days. A similar reduction of DP in agitated samples was also observed by Xiang et al. (2017) (41) and might be explained by the shear stress present under stirred conditions (43). 3.4 – CONCLUSIONS This work aimed to study the influence of using different fermentations conditions, as well different strains, culture media and times of culture, on the BC properties. In general, the obtained results revealed only slight differences (with no statistical relevance) between static and agitated conditions in what concerns to BC properties. In fact, agitated culture seems to induce a slight decrease on degree of polymerization and on BC’s crystallinity. Beside this, the obtained ATR-FTIR results confirmed that the chemical structure of BC was preserved since in both culture methods and all the time-points, the mass fraction of Iα was higher for all the samples. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 99 3.5 – REFERENCES 1. Wang J, Tavakoli J, Tang Y. 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(2021), Bacterial cellulose modifications for biomedical applications 110 amplitude using 100 points), with a recycle delay of 5 s, a contact time of 2.0 ms and 1H 90° excitation pulse of 3.10 µs. All chemical shifts are quoted in parts per million from tetramethylsilane (TMS). 4.2.6 – ATR-FTIR Freeze-dried oxidized and non-oxidized BC membranes were analyzed using an ALPHA IIBruker spectrometer (Ettlingen, Germany) with a diamond-composite attenuated total reflectance (ATR) cell. Data was collected using OPUS software which is integrated on the FTIR equipment. The measurements were recorded with a wavenumber range from 4000 to 400 cm−1, with a resolution of 4 cm−1 and 64 scans per sample. The ATR clamp and platform were cleaned with a cotton swab dampened with isopropyl alcohol and allowed to dry between analysis of each coupon. All assays were done in duplicate for each sample. 4.2.7 – SEM Freeze-dried oxidized and non-oxidized BC membranes morphology was assessed by SEM (FEI Quanta 650 FEG; acceleration voltage from 3-10 kV). Both oxidized and non-oxidized BC membranes were added to aluminum pin stubs with electrically conductive carbon adhesive tape (PELCO Tabs™), with the excess removed using compressed air. Samples were coated with 2,5 nm of Au for improved conductivity. The analysis was conducted at (3-10 kV) with intensity point. For the quantitative analysis of the length of the fibers, at the least five different fibers were selected using the software ImageJ. 4.2.8 – XRD The crystalline structure was assessed using a PANalytical X’Pert Pro MPD diffractometer equipped with X’Celerator detector and secondary monochromator. Freeze-dried oxidized and non-oxidized BC membranes were analyzed at room temperature using a CuKα radiation and Bragg-Bentano geometry, 0.017°/step and 100 s/step. The crystallinity index (CrI) (Equation 1) was calculated using Segal, Creely, Martin & Conrad (1959) (33) equation, i.e., it was calculated as a function of the maximum intensity of the diffraction peak from the crystalline region (I200), at an angle of 2 ∼ 22.5°, and the minimum intensity from the amorphous region (Iam), at an angle of 2  ∼ 18° Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 111 𝐶𝑟𝐼 (%)=(𝐼200 − 𝐼𝑎𝑚) 𝐼200 (1) The apparent crystallite size (CS) in the crystallographic planes (1-10), (110) and (200) were calculated assuming that the crystals exhibit uniform size and shape and using Scherrer’s equation: 𝐶𝑆 =𝐾𝜆 𝐹𝑊𝐻𝑀 cos 𝜃 (2) where K is a dimensionless factor dependent upon the method used to calculate the amplitude (K = 0.9), λ is the wavelength of the incident X-ray (λ = 0.15 nm), FWHM is the width of the diffraction peak at halfmaximal height (in radians), and  is the angle of the diffraction peak of the crystalline phase (Bragg’s angle). The diffractograms were analyzed by Rietveld refinement with Powder Cell software. 4.2.9 – In vitro degradation of oxidized BC membranes The degradation of oxidized and non-oxidized BC membranes was tested in vitro using ultra-pure water at 37°C. The freeze-dried BC membranes were cut into pieces weighting between 8 to 12 milligrams (the exact mass was recorded). Then, they were immersed in 2 mL of ultra-pure water in centrifuge tubes. After incubation during 3, 7, 14 and 63 days the samples were taken out, dried in a woven at 50°C overnight, and weighed. The original mass of each sample was designated as mi while the mass after degradation was designated as mf . Thus, the mass loss rate was calculated using the following equation (29): 𝑀𝑎𝑠𝑠 𝑙𝑜𝑠𝑠 = 𝑚𝑖− 𝑚𝑓 𝑚𝑖 ×100 (8) 4.2.10 – Biological assays and hemostatic behaviour of BC membranes 4.2.10.1 – Preparation of blood samples Whole blood was collected from healthy bovine animals from on Izicar – Fábrica do Produtos Porcinos, Lda (Vila Nova de Famalicão, Portugal) using citrated (3.2%) 3 mL vacuum blood-collection tubes (Vacuette, Portugal) and transported on ice. 4.2.10.2 – Whole blood clotting samples Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 112 Three mL of whole blood were placed in each well of a 24-well plate. Then, 300 µL of 0.1 M CaCl2 (Riedel-de Haën) were added to induce clotting and the mixture was incubated for 0, 5, 10, 15 and 25 minutes at room temperature, with i) oxidized; ii) non-oxidized BC membranes; iii) Surgicel® (reference material); iv) glass microspheres (Ø0.40-0.60mm, Startorius) (positive control) and v) bare polystyrene (empty well, negative control). A volume of 100 µL of activated whole blood were collected for each time point and 2.5 mL of distilled water were added to each well followed by incubation for 5 minutes, to lyse the red blood cells which were not trapped in the thrombus, and release hemoglobin. Afterwards, the concentration of the released hemoglobin was measured by transferring 200 µL of the supernatant to a 96-well plate and analyzed in a spectrophotometer at λ=540 nm. In this method, a faster clot formation leads to a faster reduction of the detected absorbance value. All the samples were analyzed in triplicate (34). 4.2.11 – In vivo biocompatibility tests of BC membranes All the procedures were carried out in strict accordance with the recommendations for care and use of laboratory animals of the EU directive (2010/63/EU) and National (Decreto-Lei 113/2013) legislation for animal experimentation and welfare. The experimental procedures were carried out with the approval of the Portuguese competent authority, Direção Geral de Alimentação e Veterinária (DGAV, Lisboa, Portugal) with animal ethic approval number 0421/000/000/2017. Eighteen Wistar female rats (Charles River Laboratories, Les Oncins, France) weighing approximately 250 g were used in this study. All animals were kept in ventilation, humidity and temperature-controlled rooms with a 12/12-hour light/dark cycle. The animals were housed on cages (3 animals/cage) with corn kernels and received food pellets and water ad libitum. All animals were implanted subcutaneously with 3 untreated BC disks with 13 mm in diameter on the left side and 3 oxidized BC disks with 13 mm in diameter in the right side on the animal’s back. Animals were randomly assigned and blindly divided into 3 groups. The first group (n=6) in which the implants were removed after 3 days. A second group (n=6) in which the implants were removed after 14 days. The third group (n=6) in which the implants were removed after 56 days. 4.2.11.1 – Surgical procedures Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 113 Rats were anaesthetized with ketamine (80 mg/kg) and dexmedetomidine (0.2 mg/kg) by intraperitoneal injection, and then prepared for surgery as follows: the back of the animal, form the neck trough the tail, was shaved, antisepticised with chlorhexidine, and dried. Bodies were covered with sterile sheets except for the incision line. Three skin incisions (length, 2–3 cm) were made on the midline of the back of the animal. On each side three subcutaneous pockets (with approximately 30 mm) were carefully created using mosquito forceps. The implants were introduced into the pockets without fixation, located 20 mm from the line of incision without touching one another. The soft tissues and the skin were reapproximated with monofilament absorbable synthetic sutures (glyconate USP 5/0). Implant site location was performed by placing a subcutaneous stitch with non-absorbable suture. An ophthalmologic gel was applied to prevent drying of the eyes. Additionally, buprenorphine (0.05 mg/kg) was administered at the end of the surgical procedure immediately before the wound closure was completed. Postoperative pain control was also carried out with buprenorphine (0.05 mg/kg subcutaneous, twice daily) for 2 days. The animals were sacrificed under general anesthesia with a lethal dose of pentobarbital (80 mg/kg), delivered by intracardiac injection. Implants were then carefully removed in conjunction with all surrounding tissues and fixed with 4% neutral buffered formalin. After that, both oxidized and non-oxidized BC membranes were fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin. Cross sections (5 µm) were cut and stained with Hematoxylin and Eosin (H&E) for cellular infiltration and inflammatory response (35), as described below. 4.2.11.2 – Histological evaluations The implants and the surrounding tissue were collected at 3, 14and 56-days post implantation. Samples were fixed in 10% formalin, paraffin embedded, cut in 5 μm and stained with H&E for histological evaluation. For each sample, the biological response parameters were evaluated at the implant–tissue interface with three high power fields (×400) by two pathologists and recorded in an appropriate formulary. All the biological response parameters were evaluated according to the ISO standard 109936 which included the extent of fibrosis/ fibrous capsule (layer in micrometers) and inflammation; degeneration, as determined by changes in tissue morphology; the number and distribution from the material–tissue interface of the inflammatory cell types, namely polymorphonuclear neutrophilic leucocytes (PMN), lymphocytes, plasma cells, macrophages and multinucleated cells; the presence, extent and type of necrosis; other tissue alterations such as vascularization and fatty infiltration. Based Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 114 on the scoring system for the parameters, a total value was obtained for each animal in each group (35). The collected data were submitted to statistical analyses. The data were analyzed with one-way analysis of variance followed by the Tukey multiple comparisons test was used to evaluate the statistical differences between the groups using GraphPad Prism 5.0 software. Differences were considered statistically significant at P < 0.05. 4.3 – RESULTS AND DISCUSSION BC with three different oxidation degrees i.e., 4%, 7% and 15%, corresponding to the generation of 400, 700 and 1200 Coulombs (samples BC 400C, BC 700C and BC 1200C, respectively), were obtained and analyzed by FTIR, SEM, XRD and for in vitro degradation. However, the more oxidized membranes were very fragile, making their handling quite difficult after preparation. Hence, BC 1200C samples were excluded from degree of polymerization measurements, NMR analysis and hemostatic assays. Finally, and in order to reduce the number of animals, the in vivo biocompatibility tests were performed using only the BC 700C membranes. 4.3.1 – CV Before each electrolysis, a CV study of the BC oxidation using TEMPO mediator and carbonate buffer electrolyte was performed. Taking into account the preliminary voltammetric study carried out, the applied potential values were set between 0.7 and 0.8 V vs. SCE. Three electrolysis were carried out on the carbon Toray electrode in the presence of BC and TEMPO. The voltammetric study was performed also with TEMPO, in the absence of the BC membrane, as well as at the beginning and at end of the BC electrolysis (Fig. 4.1). In addition, CV was also used to evaluate the electroreactivity of TEMPO and its chemical stability. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 115 (a) (b) (c) Figure 4.1. Cyclic voltammograms of Toray Carbon electrode in presence of TEMPO (___) and of TEMPO with BC membrane at the beginning (___) and at the end of the electrolysis (___). The voltammograms corresponding to (a) BC 400C, (b) BC 700C and (c) BC 1200C membranes were acquired at room temperature and at scan rate of 50 mV s-1. The successively obtained voltammograms remained unchanged, showing the stability of the electrocatalytic materials. Furthermore, they were similar to those obtained by other authors (36–38) and all of them exhibiting a characteristic profile and good reversibility, featuring the anodic peak ascribed to the reversible oxidation of TEMPO corresponding to the active oxidant, the ion oxoammonium (TEMPO+), which was reduced into its hydroxylamine form during the cathodic sweep (28). The current intensities remained almost unchanged after the introduction of BC, accompanied by a slight shift in the oxidation potential towards more anodic values. On the other hand, at the end of electrolysis, a decrease on the current intensity was observed in the anodic peak, revealing the oxidation of BC membranes. This decrease in the current intensity also was directly related to an increase in the oxidation degree of the BC membranes. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 116 4.3.2 – Oxidation degree The oxidation degree of BC membranes was determined by titration. The values obtained using Equation 1 are shown on table 4.1. Table 4.1. Oxidation degree (in %) corresponding to different applied charges and the duration of electrolysis. Sample % Oxidation degree Duration of electrolysis (h) BC 400C 3.68 ±1.03 2.39 ± 0.52 BC 700C 6.45 ± 1.13 6.07 ± 1.25 BC 1200C 14.92 ± 0.31 14.5 ± 4.94 The results showed that the duration of the electrolysis was proportional to the applied charged and that roughly, the oxidation degree increased directly with the applied charge, with the values increasing from 4 to 15% of oxidation depending on the conditions used. These relatively small oxidation degrees were expected, since the reaction occurs only at the surface of the fibers, and the mediator is not able to penetrate the crystalline domains of cellulose. Moreover, the obtained values were similar to those obtained by Sezer et al. (39) where for 6h of reaction, the oxidation degree was around 7, and for 12h was approximately 9. 4.3.3 – Degree of polymerization The obtained results show that the degree of polymerization decrease with an increase on the oxidation degree, as follow: 1959 ± 63 for non-oxidized BC > 1841 ± 36 for BC 400C > 1401 ± 65 for BC 700C. These results are in accordance with other works (31,40–43). Considering that the inner part of the crystallites is not expected to undergo depolymerization, this must occur to significant extent at the surface level as to lead to the observed average reduction in DP, which justifies the increasing fragility of the membranes as the oxidation increases. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 117 4.3.4 – NMR NMR was used to confirm the specific oxidation of C6 of the BC membranes. The obtained spectra are presented on Fig. 4.2. (a) (b) Figure 4.2. NMR spectra of (a) 400C and (b) 700C membranes compared with non-oxidized BC membranes. The signal at 104.5, 84.5 and 61.7 ppm were assigned to C1, C4 and C6, respectively. However, the peaks between 75.3-70 ppm, corresponding to C2, C3 and C5, were not easily assessed since the resolution was not good enough (29,44,45). The peak at 61.7 ppm, related to C6 primary hydroxyl groups, presented a lower intensity in the oxidized membranes. On the other hand, the peak at 64.9 ppm corresponding to the C6 primary hydroxyl groups inside the crystalline fibrils and the C2/C3 peaks remained unchanged, as expected, since the hydroxyl groups located in the inner structure of the crystallites hardly suffer oxidation (46). Finally, the peak around 174.6 ppm, that corresponds to the carboxylate groups produced during the electrolysis, further confirm the oxidation of hydroxyl groups (21). As expected, the intensity of this peak was found to be directly related with the oxidation degree. 4.3.5 – ATR-FTIR ATR-FTIR spectra of the non-oxidized BC and different degrees of oxidized BC are shown in Fig. 4.3. The usual bands ascribed to the chemical structure of BC were observed: (O–H symmetrical stretching around 3350 cm-1; C–H stretching around 2900 cm-1; C–O–C stretching around 1162 cm-1; C– O stretching at 1030 cm-1; vibration of amorphous cellulose (stretching of the glucose ring) around 899 Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 118 cm-1. Regarding the oxidized BC, the most relevant modifications were the appearance of a C=O stretching band at around 1628 cm-1 that confirmed the formation of carboxylic groups (–COOH) (23,47). This peak also confirmed that the hydroxyl groups at the C6 position in the BC had been successfully converted into carboxyl groups. Figure 4.3. FTIR spectra of oxidized and non-oxidized BC membranes. There were no other relevant differences between the spectra of the BC and oxidized BC membranes; thus, as it was observed from the NMR analysis, it may be concluded that highly selective oxidation was achieved. 4.3.6 – SEM The influence of oxidation on BC membranes morphology was assessed by SEM. The obtained images are present on Fig. 4.4. The width of the fibers was also measured, and the values are presented on table 4.2. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 119 (a) b) (c) d) Figure 4.4. SEM images of (a) non-oxidized and (b) 400C, (c) 700C and (d) 1200C BC membranes. (Magnification: 50000 x). Table 4.2. Width of the oxidized and non-oxidized BC fibers. Sample Width of the fibers (nm) Non-oxidized BC 44.3 ± 2.14 BC 400C 39.13 ± 2.04 BC 700C 38.75 ± 1.15 BC 1200C 29.56 ± 1.83 SEM images revealed that oxidation did not affect the morphology of the fibers. The BC exhibited the usual compact 3D network structure of the nanofibrils, which remained almost similar after oxidation, showing that the electrochemical oxidation did not considerably affect their structure (48). According to Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 126 14 days Non-oxidized BC 3 2 1 2 1 0 1 1 0 BC 700C 2 2 0 2 1 0 0 3 0 56 days Non-oxidized BC 1 2 0 2 1 0 0 3 0 BC 700C 1 2 0 2 0 0 0 4 0 0= Same as when implanted; 1= Minimal; 2= Mild; 3= Moderate; 4 = Severe According to Fig. 4.9 and table 4.4, 3 days post implantation, there was a predominance of neutrophils, lymphocytes, polymorphonuclear cells (PMN) and macrophages infiltrated at the implant-tissue interface in both experimental groups. However, the tissue reaction was more evident on non-oxidized samples comparing to oxidized ones revealing that the acute response was more expressive in the former case. This was also confirmed by the presence of necrosis only in this experimental group. At days 14 and 56, the inflammatory reaction was still more expressive in the non-oxidized samples, with mononuclear cells being the predominant ones. As expected, fibrosis only occurred after 14 days of implantation, being more expressive after 56 days around the oxidized BC membranes, which induced a higher fibroplasia. For all the time-points, the presence of giant cells was minimal, suggesting that the possible foreign body reaction (FBR) would be also minimal. This was confirmed macroscopically since BC membranes were well integrated with the rat connective tissue (35). Even after 56 days of implantation, the oxidized membranes where still in place. Although the in vitro results showed that these samples were degrade to limited extension in 63 days, more extensive degradation was expected to occur in vivo . This cannot be quantified, but a significant fractions of material remained at the end of the trials. Although the in vivo biocompatibility of BC has been characterized by several authors (17,18,35,69–73), no studies have been performed to assessed the behavior of oxidized BC in vivo . Interestingly, the oxidized BC seemed to attract fewer inflammatory cells than the pristine material, which is generally considered highly biocompatible, although it gives rise to a thicker fibrosis comparing to non-oxidized samples. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 127 4.4 – CONCLUSIONS Bacterial cellulose membranes were oxidized using electrochemical methods, acquiring hemostatic features and became partially degradable. The obtained results using FTIR and NMR techniques allowed us to conclude that the oxidation reaction occurred mainly at the surface layer of the cellulose fibers. Furthermore, both morphology and crystallinity of the oxidized membranes were preserved, although a slight reduction in crystallinity occurred after oxidation. This decrease in crystallinity was only around 4%, even for the samples with higher oxidation degree, e.g., the pristine BC had approximately 92%, while the oxidized sample had around 88%. The in vitro degradability of oxidized membranes and their hemostatic potential were evaluated using Surgicel® as a control. The obtained results demonstrated that the oxidized BC exhibited higher hemostatic activity than the pristine material, although not as effective as that of Surgicel®. The in vivo biodegradability and biocompatibility of oxidized membranes were assessed through subcutaneous implantation of the membranes in the rat, and the results showed a highly biocompatible behavior, triggering only a mild inflammation process. 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(2021), Bacterial cellulose modifications for biomedical applications 134 135 Chapter 5 Laser patterning of bacterial cellulose membranes surface An exploratory study was performed intending not only to explore another biomedical application of BC membranes, but also to understand the influence of surface laser patterning on fibroblast adhesion. A CO2 laser was used, and two different patterns were tested: parallel lines (=) and perpendicular lines (#). When parallel lines were used, the distances between lines were also studied, namely 0.1 and 0.2 mm. The pristine (BC) and laser treated BC (BCL) were analysed by XPS, ATR-FTIR and SEM. The obtained results allowed to conclude that the use of laser did not chemically modify the BC surface and that it is indeed possible to carve the surface of BC using this approach. The effect of surface patterning on cell adhesion was assessed. SEM results showed that fibroblasts were present both surfaces exhibiting its usual phenotype. Furthermore, the metabolic activity did not reveal significant differences between the pristine BC and the patterned surfaces. In general, these preliminary results showed the potential of the laser technology to modify the surface topography opening the room for further studies aiming at improving the performance of BC as an implantable biomaterial. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 142 Figure 5.3. The appearance of the patterns after dehydration using SEM equipment. Shrinkage of the sample upon dehydration is probably responsible for the poor perception of 3D patterning on the SEM images. Because of that, it was hard to measured and confirmed the distance between lines, which should be 0.2 mm and 0.1 mm, for P1 and P2 patterns, respectively. Actually, and despite the freeze-drying process could maintain the morphology of nanoscale materials, this process also weakens the contrast and clarity at the micron scale. Consequently, it was problematic to obtain good and clear SEM images of the produced patterns. In order to circumvent these issues, in forthcoming work the samples will be frozen using liquid nitrogen, which has been shown to better preserve the 3D architecture of the BC membrane through the freeze-drying process. On the other hand, on electronic microscope image (on the right) it was also possible to visualize the lines/patterns on the surface of the BC membranes, bearing the expected in between space. Despite the presence of the patterns, they were not regular and homogeneous as in other works in the literature (7,14,15). In fact, Jin et al. (2018) created patterned BC membranes using PDMS templates at the culture medium-air interface, during the fermentation process, with different size of grooves (50, 30, 10 µm). With this strategy, they were able to produce regular grooves on PDMS templates which were suitably transferred onto BC surface, which had almost the same regular grooves and stripes structure as the PDMS. The authors also tested the effect of pattern surface on fibroblasts (L929) proliferation. They conclude that the patterned surfaces had an inhibitory effect on L929 cells proliferation, especially those with 10 µm stripes, dimensions close to those of the cells. In this work, the produced patterns were bigger compared to the ones in the mentioned reference. Here, the distance between lines were 0.1 and 0.2 mm, one order of magnitude larger. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 143 Nevertheless, the laser technology is rather flexible and can be used to produce different patterning effects, further work being necessary to fully exploit the potential of the technology (7). 5.3.1 – XPS XPS analysis provides valuable information not only about surface elemental composition but also on its functional groups. BC and BCL membranes were analyzed by XPS in order to study the possible influence of the laser treatment on chemical composition. The laser treatment leads to evaporation of water and combustion of the organic matter (cellulose in this case), residues of the combustion may remain attached to the membrane following the treatment, a possibility that was verified by XPS. The obtained results are present in Fig. 5.4. Also, the relative atomic concentration of C, O and N on the BC and BCL surface is shown on table 5.1. BC BC L Figure 5.4. XPS survey spectra (left) and the deconvolution of the carbon peak (right) for both BC and BCL samples. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 144 Table 5. 1. Elemental composition of BC and BCL samples analyzed by XPS. Samples Carbon (%) Oxygen (%) Nitrogen (%) BC 57.8 41.8 0.4 BCL 59.2 39.9 0.9 As expected, general XPS spectra for both BC and BCL samples revealed that the main elements detected were carbon and oxygen. Beyond these main elements, nitrogen was also detected, in very small amounts, being its presence ascribed to remaining’s of the BC biosynthesis process, possibly to some contaminating proteins (16). According to table 5.1, there was a slight decrease on the oxygen concentration on the surface after laser treatment, while a slight increase on carbon and nitrogen concentration was observed. The relative fraction of each element is in agreement with Wang et al. (2020) work and with the survey scan spectra where the most intense peaks are ascribed to carbon (around 283.95 eV) and oxygen (around 531.05 eV) (17). Further information related with surface chemistry can be assessed through deconvolution of the XPS spectra, as depicted on Fig. 5.4. The C 1s peak can be deconvoluted into four components: C1 which corresponds to C-C bonds (around 284 eV); C2, assigned to carbon with one oxygen bond C-O (around 287 eV); C3, where carbon binds with one oxygen through a double bond C=O (around 288 eV). Finally, and although hard to detect in present work, for both BC and BCL samples, C4 is recognized as the carbon linked to two oxygen bonds O-C=O (around 289 eV) (16–20). Since the obtained results for both BC and BCL were very similar, it may be concluded that laser treatment did not induce chemical modifications on the surface of the membranes. Nevertheless, a larger number of samples must be processed in order to confirm whether the difference in the carbon and oxygen content is significant. 5.3.2 – ATR-FTIR Figure 5.5 shows the ATR-FTIR spectra of bacterial cellulose membranes before and after laser treatment. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 145 Figure 5.5. FTIR spectra of BC and BCL membranes. The typical bands ascribed to the structure of cellulosic substrates are observed: O–H symmetrical stretching around 3350 cm-1; C–H stretching around 2900 cm-1; C–O–C stretching around 1162 cm-1; C– O stretching at 1030 cm-1 and vibration of amorphous cellulose (stretching of the glucose ring) around 899 cm-1 (21–23). As it is possible to observe, the spectra before and after the treatment were indistinguishable which confirms that the samples preserve their chemical structure, being these results consistent with XPS ones. Beyond this, it was also calculated the Iα mass fraction through Gaussian deconvolution of the peaks and the obtained results are present in the following figure. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 146 Figure 5.6. The deconvoluted absorption peaks attributed to the Iα (750 cm-1) and Iβ (719 cm-1) crystalline forms of cellulose obtained by ATR-FTIR. Figure 5.6 exhibits the typical bands attributed to both allomorphs Iα (at 750 cm-1) and Iβ (at 710 cm-1 ) of the celluloses in which the absorbance bands at 3240 cm-1 and 3270 cm-1 are ascribed to Iα and Iβ, respectively (24). Despite a slight increase on the Iα values from 61% at ~65% after laser treatment, in general, the obtained values were very similar. Moreover, these high values on Iα mass fraction is in accordance to the literature (25–27). 5.3.3 – Cell viability To assess the in vitro differential cell affinity, mouse fibroblasts L929 cells were cultured in direct contact with BC and BCL samples and the cell viability was measured using MTT assay. Figure 5.7 shows the relative viability of L929 cells after an incubation period of 24h and 72h. Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 147 Figure 5.7. Cell viability quantified by MTT assay after 24h and 72h of incubation. Significant differences are indicated as follow: * P<0.05, **P<0.01 and *** P<0.001. It was shown that there were no significant differences between BC-based surfaces and TCPS demonstrating that they were not cytotoxic. Furthermore, metabolic activity increased from 24h to 72h of culture in all groups, more expressively on Tissue Culture Polystyrene (TCPS). Besides, the obtained cell density on TCPS act as reference to evaluate if the cells are proliferating normally (2). Cell viability was similar on BC and BCL surfaces, suggesting that the surface patterning did not affect significantly the cell affinity of the material, but it suggests a trend towards a lower cell proliferation. According to Robotti et al. (2018) a decrease on cell density along of the culture time was expected. In fact, the authors observed a maximal reduction of 65% of cell density on patterned surfaces when compared to the flat control, after 72h of incubation. The authors tested hexagonal and square patterns featuring pits with 3 μm < d < 10 μm and 6 μm < i < 20 μm which are the ones imparting PDMS surfaces showing the most efficient antiadhesive properties (15). Furthermore, the decrease on cell density was also observed by Jin et al. (2018) where the patterned surfaces having an inhibitory effect on L929 cells proliferation in vitro . The in vivo results showed that the modified surface also inhibited inflammatory response, decreased the accumulation of fibroblasts with consequent decreasing on HST effect (7). In contrast, the results presented by Kurniawan et al. (2012) revealed that the treatment of BC membranes with different plasmas was able to enhance cell affinity. Their results indicated that fibroblast adhesion and proliferation was significantly enhanced by the plasma treatment highlighting the great potential of this surface treatment on the improvement of BC biocompatibility. However, when using plasma treatment both the Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 148 chemistry and the topography of the material are modified, thus these results are not comparable to those obtained using laser technology (2). 5.3.4 – SEM The surface morphology of the BC and BCL membranes after cell culture and the morphology of the cells were examined by SEM and the results are presented on Fig. 5.8. 24h 72h B C P1 Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 149 P2 P3 Figure 5.8. L929 morphology after 24h and 72h of incubation (Magnification: 800x). In all the conditions, membranes exhibited a dense network where it was very hard to observe the individual cellulose fibers. Furthermore, it was possible to observe the presence of cells showing their adhesion on the biomaterial surface. The BC surface thus supports the adhesion of fibroblasts and enabled their proliferation. The in vitro adhesion of cells involves different phases: i) sedimentation where the initial attachment is related with the adherence of the round cell body to the surface; ii) cell attachment, where cells acquire flattened shape and spread onto the surface, increasing the contact area and iii) cell spreading and stable adhesion, where cells fully spread onto the surface through reorganization and distribution of the actin skeleton, to increase adhesion strength (28). Figure 5.8 showed that L929 cells were able to attach and spread on BC surfaces with and without surface Queirós, E.C. (2021), Bacterial cellulose modifications for biomedical applications 150 patterning. There was an increase on cell number in every surfaces along the time, being this increase difficult to compare in quantitative terms. Furthermore, the growing fibroblasts cells onto BC and BCL samples exhibited the typical cell morphology with usual spindle shape and spread covering the material surface (15,28–30). This behavior was more evident after 72h of culture since a higher number of elongated cells was present demonstrating that at 24h of culture, the cells were on the first stage of adhesion. 5.4 – CONCLUSIONS This exploratory work intended to enhanced the properties of BC membranes for biomedical applications through its surface patterning using a CO2 laser. Contrarily to other works where the BC producing bacteria were cultured on microstructured PDMS molds, yielding BC membranes with the desired topographical shape, in this work the patterns were designed directly on the BC membranes. In fact, it was possible to draw the pattern after BC production, but we had some difficulties on keeping those patterns upon drying. However, it is important to pattern the surface on wet membranes since the cell culture must be performed in this environment. This issue may be overcome by freezing the BC in liquid nitrogen. 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