FEDERAL UNIVERSITY OF SANTA CATARINA

DEPARTMENT OF CHEMICAL AND FOOD ENGINEERING

INSTITUTE OF BIOMATERIAL - WW7

ORGANIC-INORGANIC COMPOSITES FOR TISSUE ENGINEERING

CAMILA SCHROEDER

ERLANGEN- GERMANY

2014

FEDERAL UNIVERSITY OF SANTA CATARINA

DEPARTMENT OF CHEMICAL AND FOOD ENGINEERING

CAMILA SCHROEDER

INSTITUTE OF BIOMATERIAL - WW7

An internship report submitted to Federal University of Santa Catarina in partial fulfillment of the requirements of the degree in Food Engineering. Leader: Prof. Dr. Luismar Marques Porto Prof. Dr.-Ing. habil. Aldo R. Boccaccini Supervisor: Dr. Judith Bortuzzo

ERLANGEN- GERMANY

2014

ACKNOWLEDGMENTS

First of all, I would like to thank to Professors Boccaccini, Dachamir, and

Luismar, of making this dream possible. The experience has been an interesting and rewarding. I am especially grateful for their belief in this project and my abilities.

Thanks to Professor Boccaccini accept that I perform the project in the institute and has always demonstrated an interest and care in my research.

My special thank to my supervisor, Judith Bortuzzo, since the beginning was willing to help me. There were many hours of experiments, photos and more photos in the SEM.

Thanks to all my colleagues of the WW7 research group and from "Henkestraße" for the support.

Little farther, but not less important, InteLab group. Julia and Lya, you certainly were very important and encouraged me at times when I was most discouraged.

I must also thank João for his support, producing more and more samples to cross the seas, so I could continuous my project.

I am deeply grateful to my dear parents, Celso e Cleci that have always believed in me and gave me a lot of strength in all my decisions, plenty of support especially in difficult times. I love you so much!

Table of Contents

1. INTRODUCTION ........................................................................................................ 8 1.1 Introduction, Motivation and Background .................................................................... 8 1.2 Objectives ..................................................................................................................... 9

2. ENTERPRISE PRESENTATION ............................................................................ 10

3. LITERATURA REVIEW .......................................................................................... 11 3.1 Bacterial Cellulose ...................................................................................................... 11 3.2 Bioactive Glass ........................................................................................................... 13 3.3 Tissue Engineering ...................................................................................................... 13 3.3.1 Bone Tissue Engineering ......................................................................................... 14

4. MATERIAL AND METHODS ................................................................................. 16

5. RESULTS AND DISCUSSIONS ............................................................................... 18

6. CONCLUSIONS ......................................................................................................... 22

7. OUTLOOK ....................................................................... Error! Bookmark not defined.

8. REFERENCES ............................................................................................................ 24

ABSTRACT

Biomaterials for replacement and/or regeneration of bone tissue constitute important development in tissue engineering, due to its great demand and advantage to replace grafts. In the present study, the syntheses of a biodegradable composite and bioactive scaffolds were produced using bacterial cellulose (BC), a biodegradable polymer, incorporated with bioglass (BG) to stimulate scaffolds bioactivity. A bioactive scaffold encourages the surrounding tissue to regenerate. Bacterial cellulose synthesized by Gluconacetobacter hansenii is an excellent platform for tissue engineering and tiny particles can easily penetrate and become engrossed inside the porous BC matrix. BG has an interesting trait when immersed in body fluid, forms on its surface a layer of hydroxylcarbonate apatite, similar to the inorganic component of bone. BC membranes were coated with 1% and 10% v/v of 45S5 Bioglass® (SCHOTT Vitryxx ®, Germany) with distillated water, 25% ethanol and 50% ethanol solution. BC+BG composites illustrated that BG molecules penetrated the BC matrix and combined through a strong hydrogen bonding interaction. To understand the transformations occurred in BC membrane during scaffold syntheses, BC treated in water and ethanol, fluids involved in scaffold coating, are characterized by Fourier transform infrared (FTIR) and to investigate the morphology of the composite assessed by scanning electron microscopy (SEM). Bioactivity in simulated body fluid (SBF) was analyzed after 1, 3 and 7 days. The SEM investigation was repeated to evaluate the amount and morphology of the precipitated hydroxyapatite (HA). The chemical structure of the BC+BG favors the nucleation of hydroxyapatite on their surface, which indicates the potential for applications in tissue engineering for cartilaginous and bone tissue regeneration. In conclusion, the present results demonstrate most clearly the usefulness of the composite BC+BG as a novel and superior three-dimensional scaffold.

Keywords: Bacterial Cellulose, bioglass, bone tissue engineering, hydroxyapatite, bioactivity.

1. INTRODUCTION

1.1 Introduction, Motivation and Background

According to World Health Organizations (WHO), a survey compares data from 1990 and 2012 showing that life expectancy has increased an average of 6 years in the world. As the global population continues to age, the number of patients with bone diseases and hence the demand for bone replacement is increasing dramatically.

The loss of an organ or tissue due to cancer, disease or trauma is a critical problem in human health care. An attractive and promising approach to address such issue is to create biological or hybrid substitutes for implantation into the body. (Langer and Vacanti, 1993; Lanza et.al., 2007).

In order to reduce health care costs, public incentive are focusing on developing new devices and sustainable technologies that can improve the quality of life population. The advantage of this approach is the reduced number of operations needed and resulting in a shorter recovery time for the patient.

Tissue engineering is a considerable option as this interdisciplinary field aims on the development of adequate replacements for nonfunctional tissue to maintain, restore, or improve its function (Langer and Vacanti, 1993), using different approaches such as the development of medical devices and artificial organs (Palsson and Bhatia, 2004; Tsuda et.al., 2007).

Numerous materials show promise options for medical applications. These materials used for scaffolds can be natural or synthetic such as ceramic, metals or polymers (Ifkovits and Burdick, 2007).

A fascinating and renewable natural material is bacterial cellulose, a polysaccharide, three-dimensional fibrous web that is secreted by bacteria such as Gluconacetobacter hansenii (Klemm et.al., 2001; Rambo et.al., 2008). BC demonstrates a serious of distinguished structural features and properties such high purity and crystallinity, biocompatibility, good mechanical properties and durability make it a material with a high potential for the development of scaffolds and products with high biotechnological value (Czaja et.al., 2007; Barud et.al., 2011; Yamanaka et.al., 1998).

An ideal bone tissue engineering scaffold needs to follow specific criteria like ability to deliver cells, excellent osteoconductivity and bioactivity, good biodegradability, appropriate mechanical properties, highly porous structure and commercialization potential. (Temenoff and Mikos, 2000; Jones and Boccaccini, 2005; Freyman et.al., 2001).

Bioactive glasses show excellent osteoconductivity and bioactivity, ability to deliver cells, and controllable biodegradability (Wilson et.al., 1981; Hench et.al., 1971, ,1997). Larry Hench discovered biomedical applications of glass by developing the well-known 45S5 Bioglass®. Thereafter, 45S5 Bioglass®, was successfully marketed worldwide as a bone replacement material (Hench and Mater, 2006).

The composite of bacterial cellulose membranes and bioglass is an attractive alternative for development or regeneration of tissue. The new physicochemical and biological properties of the scaffold create great potential for applications in bone tissue engineering.

1.2 Objectives

The objectives of this research project involves the design, development and characterization of novel multifunctional scaffolds for hard and soft tissue engineering applications based on combination of biopolymers and inorganic fillers and coatings.

The effect of an inorganic coating on bioactivity and mechanical properties is being investigated of the newly developed scaffolds. The final goal is to create an ideal scaffold for tissue engineering.

2. INSTITUTE PRESENTATION

The Institute of Biomaterials of Friedrich-Alexander Universität (WW7) is developing a range of nanostructured biomaterials for medical implants, tissue engineering and drug delivery.

New biomaterials based on bioactive glasses and polymer/nanoparticle composites are being investigated specially, with electric field assisted methods being developed for the fabrication of biomedical nanostructures. Specific tissues being considered in current research projects are bone, cartilage and cardiac tissue.

Biomaterials research in our group is being carried in a very interdisciplinary environment and in close collaboration with a number of researchers and academics with expertise in cell biology, biochemistry, biotechnology and medical sciences in Erlangen, Germany and worldwide.

My work at the WW7 research group was at the field of hard and soft tissue engineering. The first idea was a design and fabrication of a composite between a biomaterial of bacterial cellulose, that was produced and provided from The Integrated Technologies Laboratory – InteLab, (Federal University of Santa Catarina, Brazil) and bioactive glass, based in tissue engineering constructs. Characterizations of the new scaffold were carried out including mechanical properties, bioactivity in simulated body fluid and analysis of the structure.

3. LITERATURE REVIEW

3.1 Bacterial Cellulose

Cellulose, frequently derived from plants, is the most abundant organic composite in nature; however, microorganisms such as bacteria, algae and fungi can synthesize cellulose. Recently, bacterial cellulose has aroused interest such as a biomaterial to tissue engineering. Gram-negative bacterium Gluconacetobacter xylinus and Gluconacetobacter hansenii produce cellulose, these species are recognized as capable of producing cellulose on a commercial scale, and have the ability to use a variety of carbonaceous substrates for the biosynthesis. (Czaja et.al., 2006; Kurosumi et.al., 2009).

Formed by union of two D-glucose molecules by β-(1→4) glycosidic bonds. Hydroxyl groups establish interactions of hydrogen bonds type, those are responsible for the rigidity, nanofibers formation and render the cellulose insoluble in water and most organic solvents. (Kennedy et.al., 1982 ; Klemm et.al., 2009).

Bacterial cellulose has superior properties compared to plant cellulose, shows unique physicochemical properties such as great stability, low toxicity, nonallergenicity, high tensile strength, crystallinity and can be safely sterilized (Cai, Hou, and Yang, 2011; Maneerung, Tokura, and Rujiravanit, 2008; Klemm et.al., 2001). BC has been investigated and utilized as food products, biomedical applications, drug delivery systems, and biocomposites. In medicine such as engineering of bone tissue (Zaborowska et.al., 2010) or artificial knee menisci (Bodin, 2007), scaffolds for tissue engineering (Dugan, Gough, and Eichhorn, 2013; Wang et al., 2013) and manufacturing artificial blood vessels for microsurgery (Klemm et.al., 2001).

 Fig. 1 - Biomedical applications based materials bacterial cellulose. (Fu et.al., 2011).

The greatest advantage of BC is the high porosity that allows other materials to be incorporated and form reinforced composites. (Huang and Gu, 2011). BC-based composites are being investigated, such as alginate, silica, collagen, chitosan, polyaniline composites, silver, gold, palladium, platinum and titanium oxide have been utilized. (Serafica, Mormino and Bungay, 2002; Zhang et al., 2010; Ashori et.al., 2012; Shi et.al., 2012).

Then a variety of inorganic nanoparticles or nanowires can be formed through precipitation, oxidation–reduction and sol–gel reaction as shown in Fig.2.

 Fig. 2 - Schematic diagram of the in situ preparation of particles/BC composites. (Hu et al, 2014).

The Integrated Technologies Laboratory - InteLab (www.intelab.ufsc.br) has developed the production process of BC in several forms as films, composites and 3D macrostructures.( Recouvreux et.al., 2008;. Recouvreux et.al., 2011.)

 Fig. 3 - Applications of bacterial cellulose in biomedical area. (a) and (b) skin substitute (Czaja et.al., 2007).; (c) and (d) blood vessels (Klemm et.al., 2001)

3.2 Bioactive Glass

Bioceramics are ceramics used for the repair and reconstruction of diseased or damaged parts of the musculo-skeletal system (Hench, 1998), in which bioglass is a type of glass having the potential to be used as a medical implant material.

Biomedical applications of bioglass were discovered in 1969, when Hench developed the 45S5 Bioglass®, witch had excellent biocompatibility as the ability of bone bonding, excellent osteoconductivity, ability to deliver cells and controllable biodegradability, render promising scaffold material for tissue engineering (Wilson et al. 1981; Hench et al, 1971,1973, 1997). 45S5 Bioglass®, whose proportions are 45%SiO

2,

24.5% Na2O, 24.5% CaO, 6% P2O5, in weight percent, is the most bioactive glass (Hench et.al., 1971; 2006).

Bioactivity is defined and characterized by different behaviors of biomaterials when under different environments. The formation of hydroxyapatite layers in the in vitro test indicate bioactivity of BG, while in the in vivo test is indicated by the response of an organism, especially the cell proliferation and ingrowth (Hench and Mater, 2006).

Three dimensional BG scaffold is one of the most attractive premises for the future because of its excellent bioactivity and potential for stimulating osteogenesis (Xynos et.al., 2000) and angiogenesis (Leu et.al., 2009; Gorustovich et.al., 2010).

Has an interesting trait when immersed in a body fluid, a layer of hydroxyl carbonate apatite (HCA), similar to the inorganic component of bone, forms on its surface. The stages of reactivity of BG when in body fluid, have been proposed by L.L. Hench :

- Na+ ions leach out - SiOH formation - Formation of a silica-rich layer - Formation of a Ca-P rich layer - Crystallization of hydroxyl carbonate apatite

The HCA phase that forms on bioactive implants are chemically and structurally equivalent to the mineral phase in bone, providing interfacial bonding.

3.3 Tissue Engineering

Is an interdisciplinary field applying the methods of engineering and life science to create artificial constructs that restore, maintain or improve tissue functions (Williams, 2004). An alternative approach is to implant scaffolds for tissue ingrowth directly in vivo with the purpose to stimulate and to direct tissue formation in situ (Mano et.al., 2004;

Suchanek and Yoshimura, 1998).

One of the major themes in tissue engineering is scaffold fabrication. A scaffold is an artificial extra cellular matrix (ECM), which serves as temporary support where isolated cells are introduced to form tissue. (Ma, 2004)

In basis, TE attempts to mimic the function of natural tissue, being biocompatible, biodegradable, promote cell attachment and mechanical stable. Therefore, to optimize the development of functional biological substitutes, the natural circumstances have to be fundamentally understood. (Lanza et.al., 2000).

 Fig. 4 - Schematic illustration of the tissue engineering principle.(Papenburg, 2009)

3.3.1 Bone Tissue Engineering

Ideal bone scaffolds needs ability to deliver cells, excellent osteoconductivity, good biodegradability, appropriate mechanical properties, highly porous structure and commercialization potential. (Temenoff and Mikos, 2000; Jones and Boccaccini, 2005; Freyman et.al., 2001).

Bone tissue engineering systems have included demineralized bone matrix, collagen composites, calcium phosphate, polylactide, polylactide-polyethylene glycol, hydroxyapatite, dental plaster and titanium (Croteau, 1999; Tsuruga et.al., 1997).

First, biocompatibility of the substrate materials is imperative; the mechanical properties of the scaffold must be sufficient and not collapse during the patient’s normal activities. As with all materials in contact with the human body, must be easily sterilizable to prevent infection (Chaikof et.al., 2002).

 Fig. 5 - A schematic showing the idea of bioscaffold.( Wang, 2013)

Advantageous properties of two or more types of materials can be combined to suit better the mechanical and physiological demands of the host tissue.

4. MATERIAL AND METHODS

 Bacterial cellulose membranes were kindly provided by The Integrated

Technologies Laboratory – InteLab, (Federal University of Santa Catarina, Brazil). The material was produced by a static cultivation of Gluconacetobacter Hansenii on Manitol

growth medium for 7 days at 30 ◦C. BC membranes were sterilized by an autoclave, and used in this study.

 Fig. 6 - BC membrane produced in static condition (Recouvreux, 2008).

 The bioactive glass material was melt derived 45S5 Bioglass® powder (particle

size 2µm) by SCHOTT Vitryxx ®, Germany.

Composites syntheses were done in situ between BC membrane and 45S5 Bioglass® powder. The interaction might be physical or occur through definite hydrogen bonding. Tiny particles can easily penetrate and become engrossed inside the porous matrix of the BC.

BC-BG composite were prepare in bottles with 50 mL in each, the solution used were (1) distillated water, (2) 25% ethanol and (3) 50% ethanol in water. Bacterial cellulose membranes were coated in solutions with 1 and 10% v/v 45S5 Bioglass®.

 Fig. 7 - Scaffolds synthesis at 360 rpm (IKAMAG® RT15power) and room temperature.

After 6 and 24 hours, at a 360 rpm and room temperature, the composites were sterilized by autoclaving (121°C for 20 min). The BC-BG scaffolds formed were identified as BC-BG-water, BC-BG-25% and BC-BG-50%.

After the composite synthesis the samples were rinsed using deionized water, and dehydrated with ethanol solutions (30%, 50%, 70%, 80%, 90%, 95% and 99,8%) for 15 minutes each and dried in liquid CO2 (Leica, EM CDP300, Germany).

Scanning electron microscopy (SEM) was performed using the ZEISS AURIGA to investigate the morphology of precipitated bioglass particles. Dried samples were coated with a gold coating, and were examined at 1.20 kV.

Fourier transform infrared (FTIR) spectroscopy is a surface sensitive technique used to analyze the chemical transformations occurring on the surface of the composite of BC+BG. The results are then compared with the BC membrane without any treatment.

Bioactivity in simulated body fluid was analyzed. The SBF solution was prepared according to the protocol developed by Kokubo (Kokubo 1990, 2006) with ion concentrations approximately equal to those of human blood plasma. Composites of bacterial cellulose and bioglass were immersed in 40 ml of SBF in sterilized falcons and

placed inside an incubator at controlled temperature of 37 ◦C. Samples were extracted from the SBF solution after 1, 3 and 7 days. The SBF was not replaced in these times. Once removed from the incubation and dried, the SEM investigation was repeated to evaluate the amount and morphology of the precipitated HA.

5. RESULTS AND DISCUSSIONS

A major part analysis involves analyzing whether and how bioglass, reacts with BC membrane after water and ethanol treatment. Therefore, to begin with, the behavior of BG particles in the fluids with which it comes into contact with the scaffold processing monitored and selected the most suitable mold material for scaffold design.

To understand the size and structure of the material, images from SEM showed the interaction of nanowires of the bacterial cellulose and real size and formats of bioglass particles.

After 24h of distillated water treatment, was possible to see a homogeneous deposit of bioglass inside the BC membrane, as show Fig.9, however, with ethanol solution coating this deposition and interaction was lower (Fig. 10). For a more direct evidence of interaction of our BC+BG scaffolds, we investigated SEM images and chemical composition of the material.

 Fig. 8 - Morphological observation of BC structure and BG by SEM. a) and b) bacterial cellulose fibers; c) and d) bioglass particles.

 

a) b)

d) c)

 Fig. 9 – BC+BG composite after 24h coated 1% v/v bioglass in water solution

 Fig. 10 – BC+BG composites after 24h coated in a) distillated water; b) 24% ethanol in water; c)

50% ethanol.

Scaffold morphology was assessed with SEM. BG appears integrated within the polymeric matrix, which is an open-cell matrix and interconnected.

 Fig. 11 - SEM micrographs of the morphological interaction 1% v/v BG with the bacterial cellulose fibers after 24h in: a) destillated water, b) 25% ethanol and c) 50% ethanol solution.

a) b) c)

BC-BG composites illustrated that BG molecules penetrated the BC matrix and combined through a strong hydrogen bonding interaction. The cross section image (Fig.13) shows that bioglass particles were attached and deposited around the fibers and inside the BC membrane.

 Fig. 12 - Cross section of BC+BG composite.

After 7 days of treatment in simulated body fluid, increase precipitate HA layers formed on the surface of 45S5 bioglass, As showed Fig. 19.

a)  

c)  

b)  

d)  

e)   f  )  

Fig. 13 – Membranes after 7 days immersion in SBF. Composites BC+BG 1% v/v in coated solutions: a) and b) destilated water; c) and d) 25% ethanol; e) and f) 50% ethanol solution.

The energy dispersive X-ray (EDX) analysis of a BC-BG composite samples after incubation time of 7 days in SBF, confirming HA formation.

 Fig. 14 - EDX analysis of BC+BG 1% coated in 50% ethanol after incubation time of 7 days in

SBF.

 

6. CONCLUSIONS

Biomaterials based on bacterial cellulose were developed combined with bioactive glass. A versatile and facile method for scaffold and is applicable for a wide range of materials.

Composite BC-BG scaffolds are made using the selected scaffold processing technique of solvent casting. This composite appears to be a promising candidate for applications in soft and hard tissue engineering. It would be suitable also other applications such as filtration, drug delivery.

The microscopy images obtained from the different solution showed the presence homogeneous distribution of bioglass inside the membranes for all the cases, althought in 50%EtOh solution the quantity is lower.

The preliminary BG study demonstrates that higher concentration of ethanol; lower will be the bonds and deposition on BG in the BC membrane.

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