Applications of bacterial cellulose and its composites in biomedicine.

Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6426-3

MINI-REVIEW

Applications of bacterial cellulose and its composites in biomedicine J. M. Rajwade & K. M. Paknikar & J. V. Kumbhar

Received: 8 November 2014 / Revised: 21 January 2015 / Accepted: 21 January 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Bacterial cellulose produced by few but specific microbial genera is an extremely pure natural exopolysaccharide. Besides providing adhesive properties and a competitive advantage to the cellulose over-producer, bacterial cellulose confers UV protection, ensures maintenance of an aerobic environment, retains moisture, protects against heavy metal stress, etc. This unique nanostructured matrix is being widely explored for various medical and nonmedical applications. It can be produced in various shapes and forms because of which it finds varied uses in biomedicine. The attributes of bacterial cellulose such as biocompatibility, haemocompatibility, mechanical strength, microporosity and biodegradability with its unique surface chemistry make it ideally suited for a plethora of biomedical applications. This review highlights these qualities of bacterial cellulose in detail with emphasis on reports that prove its utility in biomedicine. It also gives an in-depth account of various biomedical applications ranging from implants and scaffolds for tissue engineering, carriers for drug delivery, wound-dressing materials, etc. that are reported until date. Besides, perspectives on limitations of commercialisation of bacterial cellulose have been presented. This review is also an update on the variety of lowcost substrates used for production of bacterial cellulose and its nonmedical applications and includes patents and commercial products based on bacterial cellulose.

Keywords Bacterial cellulose . Nanocomposites . Biomedical applications . Biomaterials

J. M. Rajwade (*) : K. M. Paknikar : J. V. Kumbhar Centre for Nanobioscience, Agharkar Research Institute, G. G. Agarkar Road, Pune 411 004, India e-mail: [email protected]

Introduction According to Dorland’s medical dictionary, the word ‘biomedicine’ means ‘clinical medicine based on the principles of physiology and biochemistry’ (Quirke and Gaudillie`re 2008). Destructive processes such as fractures, infections and cancers are often a cause for pain and disfigurement and may lead to loss of function of various tissues that make up our body. Research in biomedicine can, in principle, provide new strategies for regeneration of such lost tissue using biomimetic structures and processes. This research is multidisciplinary, involving cell biologists, biomaterials scientists, chemists, physicists, etc. With the continual efforts from these researchers, novel systems that closely mimic the complex and hierarchical structures inherent to the native tissue are sure to emerge. These systems employ synthetic or manufactured polymers as well as natural biopolymers, which are often nanoscale materials. Naturally occurring biopolymers viz. collagen, hyaluronan, gelatin, chitosan and cellulose are being explored in biomedicine because their properties are similar to those of the native tissue. Bacterial cellulose was discovered during vinegar fermentation by A. J. Brown in 1886, but it is only in the recent past that its applicability in biomedicine is being realised. Bacterial cellulose represents a naturally occurring ‘nanomaterial’, and this quality has attracted researchers from all over the world as shown by the increasing numbers of annual publications that appear in ‘science direct’ containing ‘bacterial cellulose’, ‘biomedicine’, ‘biomedical applications’ or their combinations (Fig. 1) as the search terms. Bacterial cellulose (BC) is an unbranched polysaccharide, comprising of linear chains of β-1,4-glucopyranose residues and is produced extracellularly by microorganisms belonging to genera Gluconacetobacter (renamed as Komagataeibacter), Acanthamoeba, Achromobacter, Zooglea, Agrobacterium, Aerobacter, Azotobacter, Rhizobium, Sarcina, Salmonella, Escherichia, Pseudomonas, Alcaligenes, etc. (Ross et al.

Appl Microbiol Biotechnol Fig. 1 Publications based on bacterial cellulose (science direct search system; ‘bacterial cellulose’, ‘medical’ and ‘biomedical applications’ as search terms)

1991; Shoda and Sugano 2005; Jung et al. 2007; Yamada et al. 2012; Lee et al. 2014). Among all microorganisms reported for cellulose biosynthesis, Gluconacetobacter xylinum, an aerobic, non-pathogenic, rod shaped, Gram-negative bacterium, is the most efficient cellulose-producing species (Shoda and Sugano 2005). Traditionally, BC was obtained by fermenting agro residues and consumed as a dessert nata de coco by the natives of Philippines (Chawla et al. 2009).

Biosynthesis of BC Since its discovery, research efforts on bacterial cellulose were directed toward studying its biogenesis. According to the earliest report, a single A. xylinum cell could polymerise up to 200,000 glucose molecules per second (Schramm and Hestrin 1954). Assembly of cellulose microfibrils was visualised as a two-step process of polymerisation and crystallisation, in which polymerisation of glucose residues to form a glucan chain takes place in the membrane and final (second stage) crystallisation of the glucan chains into cellulose I occurs in the extracellular space (Benziman et al. 1980). Bacterial cellulose biosynthesis has been documented extensively by Ross et al. (1991). They have presented an account on bacterial cellulose synthesis machinery, genetics and its regulatory mechanism in a model organism Acetobacter xylinum. Uridine diphosphate glucose (UDP) forms the sugar nucleotide precursor for cellulose production. Four enzymatic steps have been characterised in cell extracts of Acetobacter xylinum that appear to make up the complete pathway from glucose to cellulose. These are phosphorylation of glucose by glucokinase, isomerisation of glucose-6-phosphate (Glc-6-P) to glucose-1-phosphate (Glc-1-P) by phosphoglucomutase, synthesis of UDP-glucose (UDPG) by UDPG-pyrophosphorylase, and the cellulose synthase reaction. Cellulose synthase comprising of three subunits (BcsA, BcsB and BcsC) is the only enzyme unique to cellulose biogenesis and is found to

be a cytoplasmic membrane protein (Ross et al. 1991). The product formed from UDP has been characterised enzymatically and chemically to be β-1,4-linked glucan. Cellulose producing cells have 50 to 80 pore-like sites in a row along its long axis. These are thought to be the sites where precellulosic polymers (10 to 15 chains) known as tactoidal aggregates are extruded. The tactoidal aggregates (1.5-nm fibrils) extruded out from pores on the cell envelope form a protofibril ca. 2–4-nm diameter, and these are further bundled in the form of ribbon-shaped microfibril of ca. 80×4 nm (Iguchi et al. 2000). In addition to these genes, CMCax (encoding for endo-β-1,4-glucanase) and CcpAx (encoding cellulose completing protein) are essential genes for cellulose biosynthesis (Kawano et al. 2002). Polymerisation of 1,4-β glucan chains is not very well understood as yet, and there are several hypotheses proposed to understand the mechanism. One hypothesis states that polymerisation contains a lipid intermediate, where glucose is first converted to a lipid molecule through glycosyltransferase in the plasma membrane (Deiannino et al. 1988; Lee et al. 2014). A second hypothesis suggests that glucose residues are attached onto the nonreducing end of the polysaccharide space during polymerisation of 1,4-β glucan (Brown and Saxena 2000; Lee et al. 2014). The crystal structure of complex of BcsA and BcsB containing a translocating polysaccharide indicates that they form a cellulose-conducting complex to extend the polysaccharide by one glucose unit (Morgan et al. 2013). According to a recent report, cellulose is secreted from the bacterium in the form of a ribbon at a rate of 2 μm/min and is composed of approximately 46 microfibrils (Lin et al. 2013).

Utility of BC to the microbe There are several theories on production of BC and its utility to the producer microbe. According to the oldest view, cellulose produced is believed to retain moisture and prevent the bacteria

Appl Microbiol Biotechnol

from dehydrating, it traps carbon dioxide produced during the tricarboxylic acid cycle and helps the bacteria to become floatable. This helps in maintaining the organism in an aerobic environment (Schramm and Hestrin 1954). The cellulose biofilm is known to enhance colonisation of rotting substrates by the bacterium and reduce the opportunity for organisms other than cellulose synthesising bacteria to compete successfully for a limited resource i.e. rotting fruit. Moreover, cellulose pellicle protects the bacterium from the hazardous effect of UV radiation because of its opaque nature (Williams et al. 1989). It is also known to confer mechanical, chemical and physiological protection to bacterium (Ross et al. 1991). Bacterial cellulose promotes cell adhesion during symbiotic or infectious interactions, protects the bacterium from heavy metal ions and improves nutrient transport by diffusion (Iguchi et al. 2000). In plant-associated bacteria, cellulose biofilm helps the bacteria anchor to the plant tissue, leading to a survival advantage under natural conditions (Romling 2002).

Fermentative production of BC Pioneering work of Schramm and Hestrin led to developing a defined medium for optimal production of BC by fermentation (Schramm and Hestrin 1954; Chawla et al. 2009). Bacterial cellulose production was at first studied in static cultures using several cellulose-producing bacteria (Schramm and Hestrin 1954). Although, traditionally, BC has been produced under static conditions, different applications demand fermentative production in either static, agitated or stirred conditions. However, the forms of cellulose produced under these conditions are completely distinct. Static cultures produce gelatinous pellicles; however, agitated cultures yield fibrous BC with a lower degree of polymerisation and crystallinity index and lower Young’s modulus which is as a result of shear stress produced during agitation (Watanabe et al. 1998). Further modifications in the stirred tank reactors led to developing airlift reactors that supply air or oxygen-fortified air to the culture medium, and circulation takes place by air pressure alone. BC produced in an airlift reactor was reported to be in the form of unique ellipsoidal pellets, which were larger and different from the fibrous BC produced in the stirred tank reactor (Shoda and Sugano 2005). Large pellets tend to reduce agitation of the culture broth, and therefore, attempts were made to reduce the pellet size by addition of water soluble polysaccharides (Chao et al. 2001). In shaking cultures, build-up of BC in the medium leads to an increase in the viscosity and decrease in the homogeneity of culture. This results in reduced oxygen levels in the medium thus affecting bacterial growth and cellulose production (Shoda and Sugano 2005). In the case of agitated cultures in the stirred tank reactors, vigorous mechanical agitation by an impeller is needed to increase the homogeneity of the culture medium (Kouda et al. 1997).

Another type of reactor, viz., rotating disk reactor, has been examined for producing BC. In a rotating disk reactor, the blades are partially submerged in the culture medium as well as exposed to the atmosphere. The use of plastic composite support (PCS) biofilm reactor for the improved production of BC has been demonstrated (Cheng et al. 2009). Although the production of BC was improved compared to suspended culture fermentation, most of the produced BC adhered to the agitator and formed large chunks resulting in difficulty in continuous production and real-time BC sampling. Recently, production of BC using a PCS biofilm reactor has been demonstrated in a semi-continuous manner (Lin et al. 2014). The results indicate that BC is produced in the form of pellicle with improved productivity compared to static cultures, suggesting that this method could be scaled up to meet the commercial needs. Production of BC using a trickling bed reactor was studied and compared with static and shaking fermentation methods. The results obtained showed that the degree of polymerisation, purity, water-holding capacity, porosity and thermal stability of trickling fermentation BC was higher than that of static fermentation BC and shaking fermentation BC. However, the crystallinity of BC obtained using trickling fermentation was lower than BC obtained in static and shaking fermentation (Lu and Jiang 2014). Various inert materials have been added to fermentative media to synthesise cellulose-based composites which have been explored for medical as well as non-medical applications. Several low-cost, easily available substrates including agro-wastes have been tested for fermentative production of bacterial cellulose (Table 1). Primarily, fruit juices, sugarcane molasses, corn steep liquor and wastes originating from wood processing industries have also been assessed as substrates for BC production. Especially wastes generated during fruit processing as well as discarded fruits contain high amounts of fructose and glucose which can be converted into value-added products. These waste substrates are low- or no-cost carbon feedstocks and have been evaluated with the primary aim of reducing the production costs of bacterial cellulose, which will in-turn help commercialisation of this versatile biopolymer.

Non-medical applications of BC and its composites According to reports, BC was consumed as a dessert by the natives of Philippines. Since then, applications of BC have been extended in diverse fields such as electronics, paper industry, packaging and biosensors. These applications have been realised using either BC in its native form or as a composite with other organic materials, biopolymers and inorganic materials including metal nanoparticles and nanowires. Composites of BC can be prepared because many hydroxyl groups are available on the surface. During fermentative

Appl Microbiol Biotechnol Table 1

Production of bacterial cellulose using various agrowastes

Substrate used

Organism

Reference

Pineapple waste Potato effluents, cheese whey permeate and sugar beet raffinate Molasses Sugarcane molasses Beet molasses

Acetobacter xylinum Acetobacter xylinum ATCC 10821

Ching and Muhammad 2007 Thompson and Hamilton 2001

Acetobacter xylinum subsp. sucrofermentans BPR2001 Acetobacter xylinum Gluconacetobacter xylinus ATCC 10245

Bae and Shoda 2005 Keshk and Sameshima 2006 Keshk et al. 2006

Saccharified food waste

Acetobacter xylinum KJ1

Li et al. 2011a

Kombucha tea

Gluconoacetobacter xylinus

Nguyen et al. 2008

Black strap molasses and corn steep liquor

Gluconoacetobacter xylinus

El-Saied et al. 2008

Orange, pineapple, apple, Japanese pear and pear juice Date syrup

Acetobacter xylinum NBRC 13693

Kurosumi et al. 2009

Gluconacetobacter. xylinus PTCC 1734

Moosavi-nasab et al. 2010

Pineapple peel juice and sugarcane juice

Gluconacetobacter swingsii sp

Castro et al. 2011

Grape skins aqueous extract, cheese whey, crude glycerol and sulphite pulping liquor Wheat straw

Gluconacetobacter sacchari

Carreira et al. 2011

Gluconacetobacter xylinus

Chen et al. 2013

Thin stillage (waste from rice wine distillery) Pineapple waste Citrus waste

Gluconacetobacter xylinus Acetobacter xylinum Gluconacetobacter intermedius CIs26

Wu and Liu 2012 Zakaria and Nazeri 2012 Yang et al. 2013

Coconut juice

Rhodococcus sp. MI 2

Tanskul et al. 2013

Citrus waste

Gluconacetobacter intermedius CIs26

Yang et al. 2013

Spruce hydrolysate

Gluconacetobacter xylinus

Guo et al. 2013

Waste beer yeast

Gluconacetobacter hansenii CGMCC 3917

Lin et al. 2014

Watermelon peels

Komagataeibacter kombuchae MCM B- 967

production, both organic and inorganic materials have been tested and these materials have been shown to integrate in the fibrillar network when BC is being produced by the bacteria. Therefore, the latter types of composites may be non-uniform. Nonmedical applications of BC and BC nanocomposites have been listed in Table 2. BC in its native form has also been explored for several biomedical applications because of its unique characteristics such as purity, crystallinity, high degree of polymerisation, water-holding capacity and high mechanical strength. However, BC in its native form is insoluble and lacks processibility. Therefore, to complement the characteristics that BC lacks, modifications of native BC and synthesis of its composites with other materials have been described. In the following section, the unique characteristics of bacterial cellulose that prove its superiority as a biomaterial for medical applications are discussed.

and biochemical properties such as ultrafine nanofibre network structure (1.5-nm width) (Patel and Suresh 2008). BC can be sterilised without adversely affecting its structure and properties, which makes it an implantable biomaterial (Czaja et al. 2007; Hu et al. 2009; Wan et al. 2009a). An ideal biomaterial (Davis 2003) should be as follows: & & & & &

Be of a biocompatible chemical composition thus capable of evading adverse tissue reactions Promote cellular interaction and tissue development Be biodegradable/bio-absorbable Possess an interconnected porous structure Show good mechanical properties to sustain loads and high wear resistance which would minimise generation of wear debris BC has been studied for the above properties.

Attributes of BC suited for applications in biomedicine Biocompatibility The purity and crystallinity of BC are superior in comparison to those of plant-derived cellulose, which makes it suitable for biomedical applications. Further, it displays unique structural

‘Biocompatibility’ refers to the ability of a given material to be non-toxic to the biological system, to perform satisfactorily


Non-medical applications of bacterial cellulose and its nanocomposites

Category

Application

References

Food

Dessert nata de coco Thickening, gelling, stabilizing, emulsifying and binding agent

Budhiono et al. 1999 Shi et al. 2014; Lin and Lin 2004

Packaging

Bacteriostatic sausage casing Active food packaging

Zhu et al. 2010 Dobre et al. 2011; Stoica-Guzun et al. 2012; Gao et al. 2014

Paper

High quality papers Flame retardant Sound and heat insulation, gas sorption, filtration, controlled release matrices Ultralight, Flexible, and Fire-Resistant Carbon Nanofiber Aerogels Data storage device Immobilization of denitrifying bacteria Urease immobilization Glucoamylase immobilization Yeast immobilization for ethanol fermentation

Surma-ślusarska and Presler 2008 Basta and El-Saied 2009 Haimer et al. 2010 Wu et al. 2013b

Rezaee et al. 2008 Akduman et al. 2013 Wu et al. 2013a Yao et al. 2011

Biosensors

Hydrogen peroxide bionsensor Formaldehyde sensors Humidity sensors

Zhang et al. 2010 Hu et al. 2011a Hu et al. 2011b

Electronics

Flexible displays Electronic paper displays Acoustic membranes Photocatalyst Flexible luminescent membranes Supercapacitors

Barud and Ribeiro 2013 Shah and Brown 2005 Indrarti et al. 1998 Yang et al. 2011 Yang et al. 2012 Wang et al. 2013

Reinforcing agent

Reinforcement for fine structures, such as fibers, polymer foams and the matrices of composites

Lee et al. 2009; Eichhorn et al. 2010

Aerogels

Magnetic aerogels Immobilization matrices

and elicit an appropriate host response upon specific application (Torres et al. 2012). Thus, biocompatibility is a result of the complex interactions between an implant and the surrounding tissues. This would mean that besides biocompatibility, any polymer to be used for biomedical application should have a low friction coefficient, suitable surface topography, chemistry and hydrophilicity (Wei et al. 2006; Gomathi et al. 2008). Structurally, because of similarities with extracellular matrix components, such as collagen, BC becomes a biocompatible material. Further, unlike proteins, the polysaccharide nature of BC makes it less or even non-immunogenic (Petersen and Gatenholm 2011).

In vitro biocompatibility Several in vitro studies assess the biocompatibility of BC, which mainly focus on cell attachment and proliferation. The results of the first documented study indicated that the murine fibrosarcoma cell line L929 did not proliferate well on native BC membranes, whereas modification of the ionic charge due to collagen adsorption promoted cellular adhesion. Further, tri-methyl ammonium β-hydroxy propyl BC (TMAHP-BC), a modified BC, promoted growth of L929

Olsson et al. 2010

cells (Watanabe et al. 1993). In contrast to this observation, it was reported that L929 cells and human osteoblasts proliferated very well on native BC (Chen 2009) and that mesenchymal stem cells grown on the cellulose membrane showed >95 % viability (Mendes et al. 2009). Human vein endothelial cells were found to proliferate and grow horizontally and migrate vertically into BC suggesting penetration inside the cellulose hydrogel up to a certain level of oxygen availability (Jeong et al. 2010; Recouvreux et al. 2011). In the case of Schwann cells cultured on BC membranes, no significant differences in the morphology and cellular functions were observed on the basis of the results of microscopy (light and scanning electron), cell proliferation assay, flow cytometry and RT-PCR (Zhu et al. 2014). The influence of different cellulose morphologies on cell proliferation was assessed in independent studies. ‘Densified BC’ produced by compressing BC to obtain a cellulose content of 17 % did not induce cytotoxic effects on L929 cells (Ávila et al. 2014). Human osteoblasts were able to attach and spread well on larger cellulose particles obtained in agitated cultures (Hu et al. 2013). Better spreading and uniform distribution of fibroblasts was obtained with RGD protein-modified BC, whereas cell aggregates were obtained on native BC membranes (Andrade et al. 2010).


Several BC composites have also been assessed for their in vitro biocompatibility. According to a recent study, cell ingrowth tendency of muscle-derived cells increased as the starch content of BC/potato starch composites increased, this being attributed to porous nature of the composite, while most of cells could only proliferate on the surface of native BC (Yang et al. 2014). Several composites viz. BC/poly(ethylene glycol), BC/chitosan, BC/gelatin and BC/collagen showed better NIH3T3 cell activity as compared to native BC (Cai and Kim 2010; Kim et al. 2010; Zhijiang and Guang 2011; Wang et al. 2012). However, human adipose-derived stem cells proliferated on BC/poly(2-hydroxyethyl methacrylate) to a lower extent in comparison to native BC membranes (Figueiredo et al. 2013). Thus, BC per se showed biocompatibility in vitro which could vary with the cell type tested. When cells are cultured in a 3D environment, a more in vivo-like phenotype can be achieved, and this is proved with several cell types including cardiac cells, lung cancer cells and fibroblasts. A scaffold provides a foundation for cell attachment, and several materials have been tested as scaffolds to support growth of cells. There is an increased interest in developing adipose tissue as an in vitro model for adipose biology and metabolic disease, and to this end, 2D and 3D porous scaffolds of bacterial nanocellulose and alginate were prepared recently (Krontiras et al. 2014). It was observed that mouse mesenchymal stem cells were scarcely distributed and showed limited formation of lipid droplets on 2D surfaces, whereas cells grown in macroporous 3D scaffolds contained more cells growing in clusters, containing large lipid droplets. Scaffolds with smaller pores contained larger cell clusters than scaffolds with bigger pores, with viable adipocytes present even 4 weeks after differentiation. Scaffolds with lower alginate fractions retained their pore integrity better. Thus, BC-based hydrogels were proved to be biocompatible when evaluated with established cell lines as well as stem cells and can be used for cell expansion in vitro followed by implantation in vivo.

In vivo biocompatibility As is the case with all biomaterials, although in vitro tests suggest non-cytotoxicity of BC, in vivo biocompatibility needs to be proved, as the material is intended for use in humans. BC was assessed as a substitute for dura mater in Mongrel dogs. Macroscopic examination of the grafts demonstrated good acceptance and adherence to the bone fragment (Mello et al. 1997). A detailed systematic evaluation of biocompatibility of BC was carried out by Helenius et al. (2006). Upon subcutaneous implantation in Wistar rats, the implants retained their shape without any macroscopic signs of inflammation up to 12 weeks. Expression of SM α-actin, fibrosis,

capsule formations or giant cells was not detected in and around the implants. Moreover, new blood vessels were formed around and growing into the implant. A similar study carried out in Swiss Albino mice reported a mild inflammatory response on day 7 of implantation, with no inflammatory response at 60 and 90 days post-surgery (Mendes et al. 2009). Subcutaneous implantation of native BC and BC modified with a chimeric protein Arg-Gly-Asp in sheep as a model system indicated slightly irritating nature of BC compared to the negative control (MAXIFLO™ ePTFE Vascular Prosthesis, Vascutek Ltd, Scotland) (Andrade et al. 2013). Further, densified BC did not show signs of inflammation around the implants over the implantation period (Ávila et al. 2014). Bionext® cellulose sponge (commercial cellulose produced from Acetobacter xylinum) used for nasal reconstruction in rabbits presented good biocompatibility and remained stable (Amorim et al. 2009). In another study, hollow tubes of BC synthesised by rolling method were implanted into the spatium intermusculare region, and data showed that BC did not exert toxic effects on nerve tissues in vivo up to 6 weeks post-implantation (Zhu et al. 2014). When tubular BC was used for the regeneration of peripheral nerves, the BC did not show any inflammatory tissue response and was susceptible to vascularisation (Kowalska-Ludwicka et al. 2013). Composites of BC tested in vivo also presented healing effects. In one such study, BC/hydroxyapatite (HA) composites were used to treat non-critical bone defects in rats (Saska et al. 2011). The results of their studies indicated that after 16 weeks, bone defects were completely repaired with some amount of BC/HA membrane present at implant site acted as a support material. Another study on biocompatibility showed that BC/potato starch composite was indeed biocompatible as evidenced by formation of new blood vessels in and around the composite (Yang et al. 2014). Several studies indicate good biocompatibility of BC, and hence, it can be inferred that the native membranes would not show genotoxicity and immunoreactivity. However, few studies use nanofibres that are obtained upon processing of BC membranes and hence genotoxicity and immunoreactivity have been examined. The in vitro genotoxicity of bacterial cellulose nanofibres was assessed using the single cell gel electrophoresis and the Salmonella reversion assays. The reversion assays showed that cellulose nanofibres were nonmutagenic, and the comet assay carried out using CHO cells indicated no or insignificant DNA damage (Moreira et al. 2009). Immunoreactivity studies of injected BC nanofibres in BALB/c mice showed that low and high dose of BC nanofibres did not significantly affect the lymph node and spleen weight as compared to positive control. BC nanofibres did not induce CD4+ and CD8+ T cells in both lymph node and spleen cells. These findings suggest that BC, owing to its non-toxicity and non-immunogenicity, is an ideal biomaterial for biomedical applications (Kim et al. 2013).


Haemocompatibility In addition to biocompatibility, haemocompatibility is another important aspect to be studied with materials to be used as vascular grafts. When the haemocompatibility of BC was evaluated and compared with commercial grafts of expanded poly(tetrafluoroethylene) (ePTFE) and poly(ethyleneterephtalate) (PET), results indicated that BC-based grafts did not induce plasma coagulation. Upon biomaterial contact with blood, protein adsorption and denaturation are reported to trigger haemostasis (Fink et al. 2010). Adsorption of proteins on BC was reported, but haemolysis occurred in less than 2 % of the red cells, thus classifying BC as a non-haemolytic material (Andrade et al. 2011). It was reported that both BC and polyvinyl alcohol nanocomposites (used in cardiovascular applications) and native BC were haemocompatible (Leitão et al. 2013). Both the membranes appeared thrombogenic and induced less free Factor XII activation. BC/PVA exhibited a lower pro-coagulating activity than BC, but according to plasma recalcification profiles, both were haemocompatible.

Mechanical properties A notable feature of BC is its high aspect ratio (Huang and Gu 2011; Hu et al. 2014) and presence of a uniform, reticulated structure comprising ultrafine fibres (Patel and Suresh 2008; Wippermann et al. 2009), and hence BC exhibits desirable mechanical properties. In literature, bacterial cellulose has been produced in various forms, and depending on the form, variations in the mechanical properties have been reported. Mechanical properties of BC were first studied by Yamanaka et al. (1989). They used purified BC obtained from Acetobacter aceti AJ12368 cultivated under static conditions. The Young’s modulus of both air-dried and hot-pressed BC was found to be >18 GPa, and tensile strength of air-dried BC films was found to be as high as 260 MPa, whereas that for hot-pressed BC was found to be 216 MPa. The high Young’s modulus was attributed to the high density of the interfibrillar hydrogen bonds and large contact area due to very fine nature of fibrils (Yamanaka et al. 1989; Retegi et al. 2010). Further purification with alkaline and/or oxidative solutions significantly improved the mechanical properties, and the Young’s modulus of the resulting sheets was ~30 GPa (Nishi et al. 1990). The mechanical properties of single bacterial cellulose nanofibres with diameters ranging from 35 to 90 nm have been measured using atomic force microscopy. In this study, Young’s modulus of the nanofibres was found to be 78± 17 GPa, a value that is considerably high (Guhados et al. 2005).

The mechanical properties of bacterial cellulose make it an attractive material to be used for the regeneration of several types of tissue such as meniscus and blood vessels. In a study where the use of BC for the culture of smooth muscle cells was investigated, the mechanical properties of BC rings obtained from a 3-mm thick BC sheet were reported to be comparable to the porcine carotid artery and much higher than synthetic grafts of expanded polytetrafluoroethylene (Bäckdahl et al. 2006). BC has also been produced in the form of tubes with a Young’s modulus of approximately 5 MPa and sustains a blood pressure of 250 mmHg (Bodin et al. 2007a). Interestingly, the mechanical properties of BC were not adversely affected even after treatment with 0.1 M NaOH used for purification (McKenna et al. 2009). BC can also be produced in various shapes and forms to match the requirements of the desired tissue. In a study, BC was grown in crescent shape to mimic the menicus. In comparison to collagen meniscal implants and freshly harvested pig meniscus, the Young’s modulus of crescent-shaped bacterial cellulose was measured to be 1 MPa (0.01 MPa for collagen) (Bodin et al. 2007b). According to recent studies, BC with effective cellulose content of 13.7 % was found to have an equilibrium modulus of 2.4 MPa (comparable to 3.3 MPa of the native ear cartilage) (Nimeskern et al. 2013) and ranged from 2.2 MPa for native hydrogel (1 % cellulose) to 242 MPa for BC with 30 % cellulose according to Tanaka et al. (2014). Suture retention tests gave a load to break of 20 and 30 N for 10 and 20 % BC, respectively. This study showed promising results for the potential use of BC as a meniscus implant. In comparison to fibrillated pulp composites, BC-based composites [BC pellicles impregnated with phenolic resin and compressed at 100 MPa] were stronger with a Young’s modulus of 28 GPa attributed to the uniform, continuous and straight nanoscalar network of cellulosic elements oriented inplane (Nakagaito et al. 2005). Bacterial cellulose/poly(L-lactic) acid (PLLA) resin composites were used for understanding the fundamental stress-transfer processes in composites, and BC networks exhibited enhanced interaction with PLLA due to increased surface area (Quero et al. 2010). Bacterial cellulose gelatin double network hydrogels (elastic modulus of the composite was 1.7 MPa) with a very high water-holding capacity even under pressures as high as 3.7 MPa and showing complete recovery to its original shape even after repeated compression have been documented (Nakayama et al. 2004). In similar studies, bacterial cellulose/polyacrylamide (BC/PAAm) gels were able to sustain both high elongation and compression. A decrease in the water content of BC led to improvement in the tensile strength of BC/PAAm composite, where the tensile fracture stress of the composite was reported to be as high as 40±10 MPa, which was almost equal to that of the ligament (Hagiwara et al. 2010). Yet, another study described the preparation of bacterial cellulose-chitosan composites (BC-Ch) suitable for


wound dressing and other biomedical applications because of improvement in mechanical properties (Ul-Islam et al. 2011; Kim et al. 2010). Another important criterion to be considered in cardiovascular diseases is the mismatch of mechanical properties of the vascular graft with the surrounding native tissue that results in post-operative complications and ultimate graft failure. In this regard, composites of polyvinyl alcohol and bacterial cellulose have been proved to be an ideal substitute for soft tissue replacement (Millon et al. 2008).

Microporosity Especially for tissue engineering, a 3D microporous structure with retained hollow spaces is ideally suited. The porosities of native BC can be altered by varying the carbon source, culture time and inoculation volume; freeze-dried bacterial cellulose membranes had a more uniform pore size distribution and a higher porosity than hot air-dried BC (Tang et al. 2010). The development of microporous BC by using paraffin wax and starch particles during growth of Acetobacter xylinum subsp sucrofermentans BPR2001 was demonstrated (Bäckdahl et al. 2008; Bodin et al. 2010; Andersson et al. 2010; Zaborowska et al. 2010). A network of nanofibrils constituted the porous walls of the bacterial cellulose scaffolds, and spherical interconnected pores were observed. In another study, cylindrical nylon porogens were used during BC fermentation. The micro-channelled BC scaffolds thus formed consisted of a cellulose network more dense at the walls of the microchannels (600–700 μm) and an open fibrillar network at the top and bottom (Martínez et al. 2012). According to one more study, grinding BC and subsequent freezing and lyophilisation led to formation of porous bacterial cellulose sponges (Gao et al. 2011). Production of BC/ chitosan and BC/agarose composites by above process was also reported (Nge et al. 2010; Yang et al. 2011). The production of bacterial cellulose potato starch composites by addition of potato starch into the culture medium led to achievement a pore size of 40 μm (Yang et al. 2011). Pore sizes of up to 20 μm were obtained in BC with the surfactant-assisted foaming treatment (Yin et al. 2012).

Biodegradability Bacterial cellulose does not rapidly degrade in the human body due to high degree of crystallinity and absence of enzymes that break the β(1-4) glycosidic linkage of cellulose (Helenius et al. 2006; Zaborowska et al. 2010). Bacterial cellulose is a slow/non-degrading material in vivo and in vitro which makes it suitable for use as a scaffold providing a longterm support. Reabsorption of the BC membranes implanted

as a substitute for the dura mater in dogs, as indicated by decrease in membrane thickness, was reported (Mello et al. 1997). BC implanted in the nasal dorsum of rabbits showed slight fragmentation at the end of 6 months with no signs of pathological inflammation (Amorim et al. 2009). Other in vivo studies have shown that BC implanted subcutaneously in rats retains its size and shape even after 12 weeks of implantation indicating non-biodegradability of BC (Helenius et al. 2006; Mendes et al. 2009; Ávila et al. 2014). In vitro degradation studies have shown fragmentation of BC fibrils and formation of fuzzy aggregates after being immersed for 8–12 weeks in phosphate-buffered saline at pH 7.25 and temperature 37 °C (Chen 2009; Chen et al. 2011). The in vivo study carried out with composite membranes of BC/hydroxyapatite (HA) proved that structural components of BC-HA, viz., the HA particles and BC nanofibres because of their size promote the reabsorption of this biomaterial (Saska et al. 2011). In view of the slow degradation of BC, several attempts have been made to enhance the degradability of BC. In one such study, amorphous regions of BC were oxidised chemically using periodate to form a biodegradable 2,3-dialdehyde bacterial cellulose (Li et al. 2009). In a second study, BC membranes were irradiated with γ-radiation and showed rapid degradation in the first 2–4 weeks in vivo (Czaja et al. 2014). According to yet another study, incorporation of enzyme cellulase in BC imparted bioabsorbable characteristic to BC (Hu and Catchmark 2011).

Alterations in surface chemistry BC has abundant active functional hydroxyl groups, which makes it suitable for combination with different nanostructures by providing powerful interaction of BC with surrounding species. It also acts as a promising template in the synthesis of a great variety of nanostructures with desired properties and functionalities (Huang and Gu 2011; Hu et al. 2014). BC can play the role of reducing agent, structure-directing agent and stabiliser in the formation of BC/AgNP nanocomposites (Yang et al. 2012). There have been several attempts of enhancing the biomedical properties of BC by surface modification. BC membranes coated with calcium-deficient hydroxyapatite have been reported as analogues to bone to be used in bone tissue engineering (Wan et al. 2007; Grande et al. 2009). BC has also been modified with adhesion sequences [Arg-Gly-Asp (RGD) peptide] (Andrade et al. 2010). Phosphorylated bacterial cellulose (PBC) adsorbed transition metal ions and lanthanide metal ions (Oshima et al. 2008) and favoured protein adsorption (Oshima et al. 2011). BC covalently attached with Cibacron Blue (CB) F3GA has been tested for the rapid analysis of proteins with high efficiency and resolution due to the


nanoporous structure (Tamahkar et al. 2010). Recently, synthesis of gentamicin-activated BC membranes which were bactericidal against Streptococcus mutans but exhibited no toxicity to human dermal fibroblasts was reported (Rouabhia et al. 2014). The experimental findings demonstrated that nitrogen plasma treatment of BC membranes led to reduction in hydrophilicity and induced a change in the surface topography. Modification by nitrogen plasma improved adhesion of human microvascular endothelial cells by 2-fold and by 25 % in the case of neuroblasts (Pertile et al. 2010). In a separate study, extracellular matrices such as collagen, elastin and hyaluronan as well as growth factors were immobilised onto macroporous BC, and these modifications led to high cell proliferation suggestive of biocompatibility (Lin et al. 2011a). TEMPO-mediated oxidation of BC produced more degradable BC networks and increased water retention making it a smart hydrogel, which could be used for several biomedical applications including self-assembling drug delivery devices (Li et al. 2009; Spaic et al. 2014). Apart from the above properties, unique features of BC such as bioadaptibility (Hong and Qiu 2008), inertness, hypoallergenity, bioconsistency and chemical stability have been described (Moreira et al. 2009; Mohd Amin et al. 2012). Additionally, water-holding capacity and water release rate of BC/Chitosan composite and native BC have also been assessed (Ul-Islam et al. 2011; Kim et al. 2010). From the foregoing section, it is clear that properties such as biocompatibility, haemocompatibility, mechanical properties and a unique surface chemistry make bacterial cellulose an attractive material for biomedical applications. Although more research into obtaining a structure that exhibits uniform porosity is required, the fact that the chemical nature permits modification makes it indeed a promising biomaterial. Thus, BC in its native form as well as a nanocomposite shows versatile properties making it a biomaterial suited for a variety of applications in biomedicine that range from implants to drug delivery which have been dealt with in detail in the following section.

Applications of bacterial cellulose and its nanocomposites in biomedicine Wound dressings/artificial skin Bacterial cellulose has been used as natural polymeric wound care material since the 1980s. BC has many characteristics of an ideal wound dressing. It is known to promote autolytic debridement, reduce pain and accelerate granulation, ensuring proper wound healing (De Olyveira et al. 2011). Further, BC helps in creation of a moist environment at the wound site and absorption of exudates and can readily conform to the contour

of the wound forming a tight physical barrier between the wound and the surrounding environment, preventing microbial infections. Furthermore, when BC is brought in contact with blood, it attenuates thrombogenicity (Fink et al. 2010). In a clinical study, the effectiveness of a commercial BC-based wound dressing (XCell®) for the treatment of venous leg ulcers was assessed (Alvarez et al. 2004). XCell® created a protective, moist, hypoxic environment similar to an undisturbed wound. In a separate study, wound healing and epithelisation with BC-based wound dressings was demonstrated (Solway et al. 2010, 2011). The other investigations also show that BC protects the wound tissue creating suitable conditions for healing and tissue regeneration (Fu et al. 2012). All this is suggestive of a high clinical potential of BC. Cellulose dressings are recommended as a temporary covering for the treatment of wounds, including pressure sores, skin tears, venous stasis, ischemic and diabetic wounds, second-degree burns, skin graft donor sites, traumatic abrasions and lacerations, and biopsy sites by the manufacturers (KowalskaLudwicka et al. 2013). There are several successful attempts at imparting antimicrobial properties to BC. According to the earliest report, direct impregnation of bacterial cellulose pellicles with silver nanoparticles imparted antimicrobial characteristic to BC (Maneerung et al. 2008). Similarly, impregnation of BC membranes with silver salts and subsequent reduction of ions using various reducing agents has been described by several researchers (Pinto et al. 2009; Jung et al. 2009; Luiz et al. 2010; Barud et al. 2011). Further antimicrobial potential of these materials was assessed. The process of photoassisted synthesis of silver nanoparticles and their stabilisation using variety of biopolymers including BC has been optimised and patented [USA (Patent No.7514600), Eurasia (Patent No. 010338), China (Patent No. 1950142), South Africa (Patent No. 2006/08551), Sri Lanka (Patent No. 14287), Singapore (Patent No. 127299)] (Paknikar 2009). These materials were then assessed for their antimicrobial activity against both Gram-positive and Gram-negative bacteria (Jain et al. 2009) and for their toxicity in vitro (Arora et al. 2008; Jain et al. 2009). Silver loading capacity and antimicrobial activity of bacterial cellulose produced using various carbon sources (glucose, maltose and sucrose) were studied, and it was observed that content and size of silver nanoparticles varied on different BC membranes, due to differences in microstructure, porosity and crystalline nature (Yang et al. 2012). A new type of BC-based wound dressing impregnated with superoxide dismutase and poviargol stimulated the healing of thermal burns of the skin in acute radiation disease (Legeza et al. 2004) and that native BC was beneficial for wound healing (Schönfelder et al. 2005). Interestingly, bacterial cellulose/collagen type I composite was able to achieve in vitro reduction of protease, interleukins and ROS activity (Wiegand et al. 2006). In another study, the potential of freezedried bacterial cellulose membranes coated with


benzalkonium chloride solution for healing of wounds caused by acute traumas was proved (Wei et al. 2011). A wide variety of BC composites has also been explored as potential wound dressing materials. The synthesis of bacterial cellulose/chitosan composites by in situ fermentation method was reported (Ciechańska 2004). Poly(3-hydroxubutyrate-co4-hydroxubutyrate)/BC composite scaffold was bioactive and suitable for cell adhesion/attachment suggesting that these composites can be used as wound dressings or in tissueengineering applications (Zhijiang et al. 2012). Further, BC composite with kaolin was proved as short-term and longterm wound healing materials (Wanna et al. 2013). In a study on dermal toxicity of biopolymer-stabilised silver nanoparticles, it was proved that supplementary materials such as hyaluronic acid, cerium oxide and gentamicin enhanced healing of superficial wounds by suppressing bacterial infections and promoting epithelisation (Paknikar et al. 2013). In a recent study, BC in combination with a synthetic polymer, viz., poly(2-hydroxyethyl methacrylate) (PHEMA), was used for the preparation of dry wound dressings (Figueiredo et al. 2013). Unique type of sponges prepared by incorporating alginates in BC have been described with compatibility with human keratinocytes and gingival fibroblasts, good tear resistance during sewing procedures and a good potential to be used in the oral cavity to cover the surgical wounds (Chiaoprakobkij et al. 2011). Cardiovascular implants Apart from its use as a wound dressing material, BC has also been explored as an artificial blood vessel. Taking advantage of the moldability of bacterial cellulose, it was synthesised in the form of very regular tubes using a patented matrix technique during fermentation. This product was patented under the name ‘BASYC’ and could be stored up to 6 weeks at 4 °C. The inner surface roughness ranged between 7 and 14 nm, which was similar to that of blood vessels. The tensile testing of transverse sections of BC and rat blood vessels showed that the load capacities were comparable (mean value ~800 mN), and the BASYC tubes resisted the blood pressure of 0.02 MPa. In vivo implantation studies showed that, after 4 weeks the tubes were surrounded with connective tissue, pervaded with small vessels, the tubes had a patency rate of 100 % and no signs of rejection. Moreover, tube was covered with well-oriented endogenous cells and was identical to the control vessel (Klemm et al. 2001). Materials that are often used for replacement as vascular grafts are not suitable in small calibre blood vessels primarily due to thrombosis and occlusion. The search for newer nonthrombogenic materials with mechanical properties that mimic the native vessel has led to the exploration of BC. The mechanical properties of bacterial cellulose were comparable to porcine carotid artery and better than expanded

polytetraflourethylene (Bäckdahl et al. 2006). The utility of PVA/BC nanocomposites for the replacement of cardiovascular tissues was reported, as it would mimic the role of collagen and elastin (Millon and Wan 2006). Later on, it was proved that by controlling material and processing parameters, the PVA-BC nanocomposite could exhibit a broad range of mechanical properties, particularly anisotropy, which is important when the material is to be used as a vascular graft (Millon et al. 2008; Mohammadi 2011). An in vivo study was conducted in pigs and results proved that BC hollow tubes could indeed serve as potential vascular conduits (Wippermann et al. 2009). There were no apparent changes such as dilatation, dehiscence, or aneurysm formation in any of the grafts. The data obtained in a different study also indicates that BC can be grafted as stable vascular conduits thus confirming an attractive approach to in vivo tissue engineered blood vessels (Schumann et al. 2009). Cartilage-meniscus implants Repair of cartilage defects is a major clinical need due to the limited regeneration capacity of the cartilage tissue. Materials for artificial cartilage are required to be not only tough but also resistant to biodegradation, which is a phenomenon, whereby a material implanted in a living body deteriorates over time by biological mechanisms. The need for bio-mimicking scaffolds has led to the exploration of BC as a scaffold material. A study showed that BC did not induce activation of pro-inflammatory cytokines during in vitro macrophage screening protocol. Chondrocytes seeded on the BC membranes showed proliferation and collagen type II production, indicating suitability as a bio-mimicking scaffold (Svensson et al. 2005). In a recent study, a biodegradable form of lysozyme susceptible BC was synthesised using metabolically engineered Gluconacetobacter xylinus. Modified bacterial cellulose (MBC) was produced in HS medium supplemented with Nacetylglucosamine (GlcNAc) residues in static conditions, which yielded scattered cellulosic fibres. This type of BC was explored for cartilage repair, in which human mesenchymal stem cells proliferated at a higher level on MBC as compared to native BC. The material was reported to be a novel in vivo degradable scaffold for chondrogenesis (Andersson et al. 2010; Yadav et al. 2013). Recently, BC with cellulose content of 15 % has been proposed as a non-resorbable implant material for auricular cartilage replacement, since it matches the mechanical strength and most importantly the host tissue response of human auricular cartilage (Ávila et al. 2014). Bone tissue implants The synthesis of a composite based on BC as a template for biomimetic apatite formation was reported (Hutchens et al.


2006). To form these, purified cellulose obtained from A. xylinum X-2 was sequentially incubated in calcium chloride followed by dibasic sodium phosphate solution. Calcium phosphate particles (50–90 %) of size range of 20–50 nm were precipitated in BC hydrogel, which was more favourable as a bone implant. The results of another study indicated that HABC nanocomposites could be formed by using phosphorylated BC as the starting material (Fang et al. 2009). The composites were biocompatible and useful as a biomaterial for bone tissue engineering. A different approach for the synthesis of bacterial cellulose hydroxyapatite nanocomposites was described (Zimmermann et al. 2011). In this study, native BC obtained using A. xylinum subsp. sucrofermentas BPR2001 was modified using carboxymethyl cellulose (CMC). The latter was used for the introduction of negative charges, and apatite particles were deposited under dynamic conditions. Osteoprogenitor cells were found to attach and spread on mineralised BC probably due to rough surface, whereas they occurred in clusters on native BC. It is known that the apatite formed from animal bone shows faster repair than synthesised apatite. Hence, BC goat bone apatite (GBA) nanocomposites were synthesised (Fan et al. 2012). Proliferation and promotion of cell differentiation was observed using L929 cells indicating that BC-GBA nanocomposite could be a promising bone filler material for treating bone defects and reconstruction. Similar results have been obtained with SaOS-2 cells which used BC produced by A. xylinum ATCC 52582 (Tazi et al. 2012). In an attempt to further improve the biological properties of BC and use it as a scaffold for bone tissue engineering, BC collagen composites have been developed upon surface modifications of BC. Osteogenic cells could be successfully cultured attesting BC collagen as an alternative material for bone tissue engineering applications (Saska et al. 2012). Another study confirmed that poly-lysine (PLL) can be introduced on BC obtained using A. xylinum M-12 which makes it structurally similar and molecularly different from natural ECM. These PLL-coated BC nanofibres proved to act as nanotemplates and induced the formation of nanosized platelet-like, calciumdeficient, B-type carbonated HAp (Gao et al. 2011). It was established that BC could be used as a local delivery system for the growth factor BMP-2 that promotes bone formation (Shi et al. 2012). The effect of incorporation of growth peptides onto BC and its effect on bone regeneration was also assessed (Saska et al. 2012). Osteogenic growth peptide (OGP) and the C-terminal penta-peptide OGP was immobilised on the surface of BC via hydrogen bonding, and highest development of the osteoblastic phenotype was demonstrated with CHO-K1 cells in vitro. The study used BC pellicles produced by Glucoacetobacter xylinus. Stem cells are an ideal source for tissue engineering applications as they can differentiate into various cell types. The differentiation potential of human adipose-derived

mesenchymal stem (HASCs) cells into osteoblasts on BC was evaluated. The HASCs grew very well on BC obtained by culturing Acetobacter xylinum ATCC 53582, forming a coherent layer. Furthermore, in the presence of osteogenic medium, significant mineralisation was observed proving that BC could be used as scaffold for seeding cells in bone tissue engineering (Zang et al. 2014). Neural implants The problem of nervous tissue reconstruction is challenging. In this context, BC was assessed as a scaffold for nerve tissue regeneration, where BC fibres maintained a continuous path that promoted infiltration of cells. The study showed that mesenchymal stem cells adhered to BC proliferated and expressed nerve growth factor neurotrophin thus creating a microenvironment that promotes neuronal regeneration (Pértile et al. 2012). Recently, BC was explored for the preparation of nerve conduits for repairing peripheral nerve injuries. In this study, a consortium comprising five bacterial strains and four kinds of yeasts was used to obtain BC membranes and conduits. The in vitro data indicated that BC possessed good biocompatibility with Schwann cells and exerted no adverse hematological and histological effects upon in vivo implantation in SpragueDawley rats (Zhu et al. 2014). In one of the studies, BCderived tubes were also tested for their regenerative potential effect in Wistar rats showing damaged peripheral nerves. Biocompatibility of implants was affirmed by low-level tissue response and susceptibility to vascularisation. Implanted tubes did not change their original size and shape and allowed the accumulation of neurotrophic factors inside, thus facilitating the process of nerve regeneration (Kowalska-Ludwicka et al. 2013). In a recent study, BC obtained using Acetobacter xylinum was assessed in repair of dural defects in rabbits. BC exhibited a decreased inflammatory response compared to traditional materials. The long-term effect of this new dural material, however, needs to be validated in larger animals (Xu et al. 2014). Other biomedical implants Artificial cornea Corneal disease is a leading cause of blindness, and an estimated 10 million people worldwide have lost their eyesight due to corneal disease or illness. Hence, the need for corneal transplants has led to the search of a wide variety of biomaterials to be used as bioengineered corneas. The properties of BC such as its nanoporous structure, excellent mechanical properties that help to maintain the intraocular pressure and definite pellucidness make it a favourable material to be used as an artificial cornea. A BC-polyvinyl alcohol hydrogel with water content and light transmittance comparable to that of


natural cornea was synthesised. The hydroxyl groups in both the polymers provided the necessary interfacial interactions in the composite (Wang et al. 2010). Water-holding capacity, light transmission, improved mechanical and excellent thermal properties indicated the hydrogel composite to be a very promising optically functional material. Further studies on such aspects would be extremely helpful for achieving success in engineered corneas. Urinary conduits A rise in the number of bladder cancer patients has led to development of biocompatible, functional urinary conduits. A 3D porous BC scaffold produced in a tubular fermentation system containing paraffin as a porogen was used. Under static as well as 3D dynamic conditions, BC conduits were seeded with human urine-derived stem cells (USC). Cultured urothelial and smooth muscle cells were found to express higher urothelial markers. Similar results were obtained with in vivo studies when the scaffolds were implanted in athymic mice indicating that BC does not elicit any fibrotic capsule formation. The study indicated that a porous bacterial cellulose scaffold provides favourable conditions for urine-derived stem cell differentiation assisting in the development of a tissue-engineered urinary conduit (Bodin et al. 2010). These structures hold promise for use in patients with end-stage bladder diseases requiring bladder reconstruction. Dental implants The potential of BC as a dental canal treatment material for intracanal asepsis was evaluated in a recent study (Yoshino et al. 2013). This unique study used pointed cellulose prepared using BC membranes produced by two strains of Acetobacter hansenii (ATCC 700178 and ATCC 35059). Pointed BC when compared to commercially available plant-cellulosebased paper points (PP) showed a higher liquid absorption capacity and expansion capacity. The tensile strength and drug release efficiency of wet BC was greater under simulated ambient conditions proving that BC has a great potential for use in dental root canal treatment.

Drug delivery applications Achieving reproducibility of the drug dose in topical formulation and the loss of material because of contact with garments or surfaces is a major drawback in topical drug delivery. BC membrane hydrogels also have been assessed for applications in drug delivery. The diffusion potential of BC membranes in electron beam irradiated and non-irradiated samples was investigated by loading tetracycline. The results showed that non-irradiated BC allowed faster drug movement

compared with that afforded by the irradiated BC suggesting the potential of BC membranes for transport and adsorption of drugs onto BC membranes (Stoica-Guzun et al. 2007). Another study was conducted for the preparation of BC membranes loaded with lidocaine, an anesthetic drug (Trovatti et al. 2011). BC membranes have the advantage of precise drug delivery and holding capacity of the drug molecule to avoid loss. This achieved modulation of the bioavailability of lidocaine, easy application being another advantage. As an extension to this study, the therapeutic feasibility of BC membranes as drug delivery systems was assessed in which the permeation of two model drugs (lidocaine hydrochloride and ibuprofen) through human epidermis was studied in vitro. BC membranes loaded with drugs could be used to modulate the bioavailability of drugs for percutaneous administration. This would be advantageous in the design of unique drug delivery systems that have the ability to absorb exudates as well as to adhere to irregular skin surfaces (Trovatti et al. 2012). BC has been explored as transdermal drug delivery membranes using diclofenac sodium salt as a model drug and glycerol as a plasticiser. The drug release was governed by its diffusion through the porous 3D network providing sustained release. This property can be successfully combined with its good biocompatibility and absorption properties (Silva et al. 2014). Recently, BC membranes were tested as supports for topical or transdermal drug delivery with good tolerance during the 24-h patch tests (Almeida et al. 2014). BC hydrogels were synthesised using lyophilised BC powder and acrylic acid (AA) which were thermo- and pH-responsive. The pH-responsive behaviour resulted in a lower in vitro drug-release rate in simulated gastric fluid suggesting its usefulness in gastric environments (Mohd Amin et al. 2012). On experimentation, the results indicated that hydrogels prepared using solubilised BC were useful and even superior for oral drug delivery (Pandey et al. 2013). In situ fermentation of BC together with hydroxypropyl methylcellulose (HPMC, a well-established tablet excipient) produced HPMC-BC (HBC). The latter, as expected, improved rehydration and small-molecule absorption and hence could be used for delivery of small drug molecules (Guimard et al. 2007; Abeer et al. 2014). In one more study on the permeation rates of caffeine from BC membranes, the rate of permeation was observed to be lower than conventional formulations suggesting that these membranes could be used for predictable caffeine release over time proving their potential for cellulite attenuation (Silva et al. 2014). Applications of BC (native form as well as composites) remain primarily in the area of topical drug delivery or oral drug delivery agent (Silvestre et al. 2014). However, a more detailed systematic research is indeed required in the future to develop new materials based on BC and to tailor such materials into responsive materials and prepare BC-drug conjugates for drug delivery applications.


BC [in the form of nanocrystals prepared by acid hydrolysis] has been explored for the preparation of emulsions. Using BC nanocrystals, the stabilisation of an oil-in-water emulsion was reported (Kalashnikova et al. 2011). The emulsion was stable for several months. The emulsion stability was attributed to the particle irreversible adsorption associated with the formation of a 2D network. Further studies on these aspects can be used to prepare emulsions containing drugs and can be assessed for oral as well as dermal drug delivery agents. Extensive studies for proving the utility of BC in oral drug delivery are essential.

Commercialisation The properties such as high purity, hydrophilicity and mechanical strength have led to numerous applications of BC. At an industrial scale, BC is currently being produced in the form of pellicles obtained after static cultivation. Spherical morphology and aggregated forms are obtained under stirred cultivation conditions. To suit applications, various reactor configurations involving revolving wires, silicone tubes and packed bead reactors containing spherical beads have been reported. A greater understanding of cellulose biosynthesis in the specialised bacterial groups, formulating new media and optimisation of process parameters, can help in obtaining cellulose on a large scale. Few commercial products based on bacterial cellulose are enlisted in Table 3 and patents on bacterial cellulose are listed in Table 4. Products based on the ‘nanoforms’ of cellulose such as nanocrystals and nanowhiskers are being explored in various applications, and once the practical applications are realised, the demand of cellulose will increase leading to commercial production of BC. A ‘demand’ for cellulose arising from researchers exploring newer applications of bacterial cellulose will necessitate its ‘supply’ which will be realised only if fermentative production of cellulose is carried out on a commercial scale. In the case of products intended for non-medical applications, BC can be obtained from a variety of low-cost, easily available waste materials; however, medical applications require Table 3

production under highly defined conditions often employing synthetic medium. This probably resulted in lesser attention towards this excellent versatile material in the context of medicine. Concerted efforts from researchers across various scientific disciplines in designing media and novel reactor configurations would lead to increase in efficiency of production leading to its commercialisation.

Concluding remarks Bacterial cellulose is indeed a unique biomaterial known to mankind. Several properties of this biological polymer can be attributed to the structure of bacterial cellulose. The nanofibre network formed during synthesis is a distinct hierarchical structure as far as carbohydrate polymers are concerned. Not only the parent material in the form of fibrils but also the nanoscale in the form of cellulose is attracting attention of researchers all over the world. In the case of bacterial cellulose, due to its crystalline nature and high purity, it becomes particularly useful for production of nanofibres, nanocrystals, nanowhiskers, etc. Such nanoscale materials have not been fully explored for practical applications. Large-scale cultivation of cellulose overproducing bacteria may seemingly help meet the future requirement of nanoscale cellulose. A greater and in-depth understanding of cellulose biosynthesis in the specialised bacterial groups will give more clues at obtaining cellulose on a large-scale. Specific improvements in the design of fermentors/reactors and optimisation of media can complement these studies. To meet the demands of bacterial cellulose and to ensure high productivity, the choice of fermentor/reactor vessel design is very critical. The stirred tank systems are energy intensive because of power required for mechanical agitators. The cellulose produced in such systems has a reticulated structure and can be used in preparation of nanocomposites. Static cultures produce cellulose with lamella structures and less branching and these become more suited for biomedical applications. Reactors that allow continuous cultivation (which also achieves removal of toxic byproducts) employing genetically stable cultures would be the

Commercial products based on bacterial cellulose for biomedical applications

Product

Application

Reference

Biofill®

Temporary wound dressing

Fontana et al. 1990; Klemm et al. 2006; Keshk 2014

Gengiflex®

Regeneration of periodontal tissues, guided bone tissue regeneration

Chawla et al. 2009; Klemm et al. 2006; Keshk 2014

Prima Cel™

Wound dressing for treating ulcers

Chawla et al. 2009

Bioprocess™

Wound dressing

Torres et al. 2012

XCell®

Wound dressing to treat chronic ulcers

Alvarez et al. 2004; Czaja et al. 2007

Dermafill™ BASYC®

Wound dressing Artificial blood vessel, cuff for nerve suturing

Solway et al. 2010 Klemm et al. 2001; Klemm et al. 2006; Schumann et al. 2009


Patents based on bacterial cellulose

Application

Product

Patent number

Artificial dura mater

Bacterial cellulose

CN 201010563139.9 Liu et al. 2011

Poly (vinyl alcohol)-bacterial cellulose

ZL 200710015537.5 Ma et al. 2010

Carboxymethyl cellulose-bacterial cellulose composite membrane

CN 200910126692.3 Cao et al. 2009

Bacterial cellulose hollow tube

European Patent No. 0396344 Yamanaka et al. 1990

Artificial skin graft

Bacterial cellulose film

Skin tissue repair

Bacterial cellulose film

U.S. patent 4912049 Farah 1990 ZL 200810047793.7 Yang et al. 2010

Artificial blood vessel

Wound dressing material

Bacterial cellulose collagen composite

CN 201110300494.1 Zheng et al. 2012

Silver-Bacterial cellulose composite membrane

CN 201110192110.9 Wang et al. 2011

Bacterial cellulose

US 20110286948 A1 Lin et al. 2011b

Photocatalytic particles loaded bacterial cellulose membranes

US 2009/0209897 A1 Limaye et al. 2009

Bacterial cellulose-hyaluronic acid- nano silver composites

CN 201010139908.2 Wang et al. 2011

Liquid loaded BC

US Patent 4,655,758 Johnson and Johnson 1980 WO 2011150482 Saska et al. 2011

Bone and connective tissue repair

Resorbable composites of bacterial cellulose, collagen and hydroxyapatite

Bone tissue repair

Hydroxyapatite modified bacterial cellulose

Scaffold matrix

Bacterial cellulose membrane Regenerated cellulose and oxidized cellulose membranes

CN 200910036754.1 Lin and Zhang 2009 CN 201110191767.3 Yin et al. 2011 US 6,800,753 Kumar 2004 CN 200910067684.6 Wan et al. 2009b

Anticoagulant membrane

Bacterial cellulose -heparin composite

In vivo implants

Solvent dehydrated microbial cellulose

U.S. Patent 6,599,518 Oster et al. 2003

Soft tissue replacement, medical devices

Poly (vinyl alcohol)-bacterial cellulose nanocomposites

Personal cleansing applications

Bacterial cellulose network, cationic polymer

U.S. 2005 / 0037082 A1 Wan and Millon 2005 US 2011/0039744 A1 Heath et al. 2011

Air filtering face mask

Bacterial cellulose-nano silver

Antibacterial mask

Bacterial cellulose and silver compounds

Antivirus mask

PVA and bacterial cellulose

Blood purification

Carboxymethyl bacterial cellulose composite

CN Patent 200910126692.3 Cao et al. 2009

Therapeutic cosmetic preparation

Bacterial cellulose hydrogel loaded with powdered cosmetic Bacterial cellulose microfibrils

US Patent 2009/0041815 Legendre 2009 US. Patent 6534071 Tournilhac and Lorant 2000 ZL 201020239963.4 Li et al. 2011b

Oil in water emulsion in cosmetic preparation Cold pack

Bacterial cellulose hydrogel

ZL 200910149665.8 Zhong 2011 JP 2011167226 Nakamura and Nakamura 2011 CN 201110078333.2 Zhang et al. 2012


need of the hour. Researchers have attempted the use of genetically modified strains for increasing the cellulose yield and reducing the generation time, and it is hoped that the cellulose production can be commercialised after seeking requisite additional regulatory permissions for medical applications. The production of bacterial cellulose can be undertaken using lowcost, indigenously available materials as carbon sources in fermentative processes. So also, many studies indicate use of food processing wastes as raw materials required for bacterial cellulose production. The latter are expected to drastically reduce the production costs. From the foregoing review of literature, the wide range of applications of bacterial cellulose particularly in the field of medicine has been realised which encompasses applications in tissue engineering, wound dressings, functional grafts, drug delivery, etc. The material is also being studied with reference to development of new biomedical devices and for replacement of degenerated/lost/impaired tissue. Its utilisation and applicability in various forms ranging from never dried, freeze dried and densified nanocrystalline powder, other forms such as films, spheres and tubes, and the production of 3D implant prototype of the human ear suggest that it could be possible to produce bacterial cellulose into any shape and form especially in the context of repair of tissue defects. The biocompatibility and durable nature of the biopolymer have probably given it an edge over synthetic polymers thus arousing great interest amongst researchers all over the world. The importance of interdisciplinary research in the domain of microbial cellulose is being realised and will soon lead to development of ‘ideal’ and ‘tailor-made’ materials for biomedicine and may motivate industrialists to venture into setting up of large-scale systems devoted for production of this versatile biopolymer. The non-medical applications of cellulose in electronics to catalysts require surface modification, and the chemical composition permits functionalisation and even incorporation of non-metals, metals and polymeric materials in the porous matrix. Achieving good porosity in the 3D cellulose structures during synthesis is still a challenge, and few porogens have been tested. More research is required which would eventually lead to creation of microbial cellulose-based structures with uniform porosity suited for both medical and non-medical applications. With the recent interest among researchers, several nanostructured matrices with improved functionality and structural features will soon be available. Many publications report the use of native and modified bacterial cellulose for repair of bone, connective tissue, blood vessels, skin, etc. This is primarily due to its several advantages such as biocompatibility, mechanical strength, transparency, hydrophilicity, moldability, etc. However, all these can be realised when bacterial cellulose can be prepared by manufacturers commercially at low-medium scale level in wet/ moist form which is easily available for use. Efforts that focus on large-scale production of BC-composites are essential with

more emphasis on composites containing nanocellulose, cellulose whiskers and microfibrillated cellulose of bacterial origin. These would be useful for the expanding scope of applications of bacterial cellulose—the naturally occurring pure polymer. Commercial production of bacterial cellulose at industrial scale is indeed the greatest obstacle, but with concerted efforts of chemical engineers, biologists and materials scientists, this material will continue to be a biomaterial of choice. Various applications would motivate more and more people to set up factories producing native bacterial cellulose as well as cellulose-based composites. This will also lessen the requirement of plant-derived cellulose proving to be an eco-friendly approach.

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