A a H A O a b c d 2 a A R R A A C C h c c K s T p t K B B N W T T C h 0 Carbohydrate Polymers 153 (2016) 406–420 Contents lists available at ScienceDirect Carbohydrate Polymers j ourna l ho me pa g e: www.elsev ier .com/ locate /carbpol multipurpose natural and renewable polymer in medical pplications: Bacterial cellulose élida Gomes de Oliveira Baruda, Robson Rosa da Silvac, Hernane da Silva Barudb,c,∗, gnieszka Tercjakd, Junkal Gutierrezd, Wilton Rogério Lustrib, smir Batista de Oliveira Juniora, Sidney J.L. Ribeiroc School of Dentistry/Unesp, São Paulo State University – Unesp, Rua Humaitá, 1680, 14801-903, Araraquara, SP, Brazil University Center of Araraquara, UNIARA, Brazil Institute of Chemistry, São Paulo State University – Unesp, CP 355, Araraquara, SP, 14801-970, Brazil Group ‘Materials + Technologies’, Department of Chemical and Environmental Engineering, University of the Basque Country, UPV/EHU, Plaza Europa 1, 0018 Donostia-San Sebastián, Spain r t i c l e i n f o rticle history: eceived 7 April 2016 eceived in revised form 23 June 2016 ccepted 16 July 2016 vailable online 19 July 2016 hemical compounds studied in this article: ollagen (PubChem CID: 6913668) yaluronan (PubChem CID: 24759) hitosan (PubChem CID: 71853) elulose (PubChem CID: 71853) aolin (PubChem CID: 56841936) ilver (PubChem CID 104755) iO2 (PubChem CID: 162651) ropolis (PubChem CID: 10455788) etracycline (PubChem CID: 54675776) a b s t r a c t Bacterial cellulose (BC) produced by some bacteria, among them Gluconacetobacter xylinum, which secrets an abundant 3D networks fibrils, represents an interesting emerging biocompatible nanomaterial. Since its discovery BC has shown tremendous potential in a wide range of biomedical applications, such as artificial skin, artificial blood vessels and microvessels, wound dressing, among others. BC can be easily manipulated to improve its properties and/or functionalities resulting in several BC based nanocompos- ites. As example BC/collagen, BC/gelatin, BC/Fibroin, BC/Chitosan, etc. Thus, the aim of this review is to discuss about the applicability in biomedicine by demonstrating a variety of forms of this biopolymer highlighting in detail some qualities of bacterial cellulose. Therefore, various biomedical applications ranging from implants and scaffolds, carriers for drug delivery, wound-dressing materials, etc. that were reported until date will be presented. © 2016 Elsevier Ltd. All rights reserved. eywords: iopolymers acterial cellulose anocomposites ound healing issue scaffolds issue engineering ontents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 2. Wound dressings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408 3. Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 3.1. In vitro biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 3.2. In vivo biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Antimicrobial properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Surface modifications of BC for cell adhesion and growth . . . . . . . . . . . . . . . . . ∗ Corresponding author at: Institute of Chemistry, São Paulo State University – Unesp, C E-mail address: hsbarud@yahoo.com.br (H. da Silva Barud). ttp://dx.doi.org/10.1016/j.carbpol.2016.07.059 144-8617/© 2016 Elsevier Ltd. All rights reserved. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 P 355, Araraquara, SP, 14801-970, Brazil. dx.doi.org/10.1016/j.carbpol.2016.07.059 http://www.sciencedirect.com/science/journal/01448617 http://www.elsevier.com/locate/carbpol http://crossmark.crossref.org/dialog/?doi=10.1016/j.carbpol.2016.07.059&domain=pdf mailto:hsbarud@yahoo.com.br dx.doi.org/10.1016/j.carbpol.2016.07.059 H.G. de Oliveira Barud et al. / Carbohydrate Polymers 153 (2016) 406–420 407 6. Drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 7. Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412 8. Cardiovascular implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 9. Cartilage/meniscus implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 10. Bone and connective tissue repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 11. Dental/oral implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 12. Neural implants/dura máter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 13. Artificial cornea/contact lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 14. Urinary conduits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 15. Tympanic membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416 16. Other applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .416 17. Future opportunities and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 18. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .417 . . . . . . 1 d fi s m n i f g b ( o i p o t i a c s t G t Over the past decade, several BC based materials have been designed for a diversity of biomedical applications. There has been References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction Natural biopolymers in a variety of biocompatible materials and evices have been the main focus of intense research in medical eld and related areas. Consequently, continual efforts from many cientists surely led to the emergence of novel systems that closely imic the complex and hierarchical structures inherent to the ative tissue. The systems or medical devices include wound dress- ngs, medical implants, drug delivery, vascular grafts and scaffolds or tissue engineering (Reis et al., 2008). Naturally occurring biopolymers viz. collagen, hyaluronan, elatin, chitosan and cellulose have being explored in biomedicine ecause their properties are similar to those of the native tissue Rajwade, Paknikar, & Kumbhar, 2015; Silva et al., 2010). Particularly, cellulose is the most abundant natural biopolymer n earth, endowed with unique properties and being an ideal start- ng point for transforming it into useful materials. Cellulose is also resent in a wide variety of living species being harvested mainly btained from trees and cotton. The first report regarding the production of cellulose from bac- eria sources was done by Brown (Brown, 1886) in 1886. The author nvestigated the biosynthesis of cellulose by Acetobacter xylinum – n acetic acid bacteria, that secrets an abundant 3-D network of ellulose fibrils under aerobic conditions, using glucose as a carbon ource. The Acetobacter xylinum is the most efficient and inves- igated producer being reclassified thereafter within the genus luconacetobacter xylinus as G. xylinus. The schematic representa- ion of Fig. 1 illustrates the 3-D network of cellulose fibrils derived Fig. 1. Representative scheme of the 3-D network secreted by Acetobacter xylinum bac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .418 from bacteria. In terms of composition, bacterial cellulose (BC) is a polymer structurally similar to plant cellulose, however showing superior physicochemical properties (Ul-Islam, Khan, & Park, 2012). This feature is mainly addressed to the well-arranged 3-D network of fibers with diameter ranging from 3.0 to 3.5 �m, which in turn are assembled by bundles of thinner cellulosic fibers with diameter sizes down to micro- and nanoscale. In addition, compared to plant cellulose, BC fibers are free of lignin and hemicellulose (Barud et al., 2011; Klemm, Heublein, Fink, & Bohn, 2005; Svensson et al., 2005). Considering the strong interaction between hydroxyl groups, BC fibers express a tendency of self-assemble. An extended network is observed via both intramolecular and intermolecular hydrogen bonds (Capadona et al., 2009; Li, Lin, & Davenport, 2011) enabling the production of sheets with high surface area and porosity. Therefore, BC represents an exciting class of nanomaterial and since its discovery has shown tremendous potential as a useful biopolymer which offers a wide range of applications, especially the biomedical ones, including the use as biomaterial for artificial skin, artificial blood vessels and microvessels, wound dressing of second- or third-degree burn ulcers, and dental implants. Other studies with endothelial, smooth muscle cells and chondrocytes have shown that these cells presented good adhesion to BC (Ul- Islam et al., 2012). teria. In detail, hydroxyl groups of the highlighted sing nanofibril are evidenced. a prodigious increase in the number of scientific publication since 2000 as well as an astonishing growth in the number of citations 408 H.G. de Oliveira Barud et al. / Carbohydr r g t 2 R i m o w E F 2 m s u chronic wounds and found that the mean time for 75% epitheliza- S Fig. 2. Some of bacterial cellulose applications in biomedical field. eporting to BC biomedical materials. In recent years a number of ood review articles had highlighted the potential applications of his material (Fu et al., 2012; Fu, Zhang, & Yang, 2013; Jorfi & Foster, 015; Klemm et al., 2011; Kucinska-Lipka, Gubanska, & Janik, 2015; ajwade et al., 2015; Shah, Ha, & Park, 2010). The aim of this review is to discuss about the applicability of BC n biomedicine by demonstrating a variety of forms of this biopoly- er either in pristine or as nanocomposites which expands its field f application. Some features of BC will also be highlighted in detail ith emphasis on reports that prove its utility in biomedicine. xamples of biomedical applications are schematically shown in ig. 2. . Wound dressings One of the first proposed and main direct applications of BC embranes in biomedical field is related to wound dressing as Fig. 3 hows. Fontana et al. (1990) were the pioneers in describing the se of BC to replace burned skin. Since then, literature has shown Fig. 3. Representative scheme of BC membrane as a wound dressing ource: Adapted from Czaja et al. (2007). ate Polymers 153 (2016) 406–420 an increasing number of papers related to wound dressing. Cellu- lose 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 (Kowalska-Ludwicka et al., 2013). Some BC based wound dressings are in fact available comer- cially: BioFill®, Bioprocess®, XCell®, and Gengiflex®, for periodontal diseases reconstruction (Farah, 1990). In terms of ideal wound dressing, the biomembrane BioFill® was one of the first commercial products that fulfils the main pre- requisites, including: low cost, good adherence to the wound, water vapor permeability, elasticity, transparency, durability, constitute a physical barrier for bacteria, is hemostatic, easy handling and it can be applicated with minimum exchanges. Additionally, the effectiveness of BioFill® in accelerating the healing process and pain relief has been proven in more than 300 cases (Czaja, Young, Kawecki, & Brown, 2007; Farah, 1990; Wouk et al., 1998). The analgesic mechanism of action of these wound dressings is not yet fully understood. However, some authors suggest that the healing mechanism involves the capture of ions by means of cellu- lose hydrogen bonds or the nano BC 3-D network mimics the skin surface creating optimal conditions for healing and regeneration is also proposed (Czaja et al., 2007; Wouk et al., 1998). It is important to point out that the utilization of BC as a wound dressing clearly shortened the healing time or wound closure over standard care when applied to non-healing lower extremity ulcers, as reported by many researchers (Czaja, Krystynowicz, Bielecki, & Brown, 2006; Czaja et al., 2007; Portal, Clark, & Levinson, 2009). Wet BC represents a novel class of wound dressing application in the treatment of partial thickness burns as proposed by Czaja et al. (2006, 2007). This type of dressing exhibited outstanding results since wet BC membranes are able to provide a favorable moist environment for a fast wound cleansing, and consequently a faster healing. Likewise, Portal and co-werkers (Portal et al., 2009) applied BC wound dressing (DermafillTM, AMD/Ritmed, Tonawanda, NY) for tion was reduced from 315 days without the application of BC to 81 days using a BC membrane. . In detail is exemplified the BC network covering the injured. ohydra i m a i 2 B 2 t r s t b B i w p m d c a ( d o t ( w o t s a n i t f i 3 b a ( r r t r p A m 2 t h 3 i o n H.G. de Oliveira Barud et al. / Carb Beyond the direct use above mentioned, improvement and mod- fications are required to enhance capabilities. BC can be easily anipulated forming nanocomposites with improved properties nd/or functionalities. In this way, several BC based nanocompos- tes have been fabricated. For example, BC/collagen (Albu et al., 014; Cai & Yang, 2011; Moraes et al., 2016; Saska et al., 2012) and C/gelatin (Nakayama et al., 2004; Wang, Wan, Luo, Gao, & Huang, 012) are nanocomposites with improved mechanical proper- ies that have been developed. The mechanical improvements are elated to an increase of thermal stability, elastic modulus and ten- ile strength. Saibuatong and Phisalaphong (2010) investigated the prepara- ion of BC fibrils and aloe vera nanocomposite films. They obtained io-polymer film by supplementing 30% (v/v) of aloe gel in the C culture medium which outcomes BC reinforced fibers with mproved properties in terms of mechanical strength, crystallinity, ater absorption capacity, and water vapor permeability in com- arison with unmodified BC films. Legeza et al. (2004) produced a BC wound dressing for the treat- ent of third degree burns that is impregnated with superoxide ismutase (an antioxidant) or poviargol (an antibiotic). Further, BC omposite with kaolin (a blood clotting agent) was proved to be wound healing material as much in a short term as long term Wanna, Alam, Toivola, & Alam, 2013). One of the utmost features on the design of wound healing ressing products is the enhancement of water holding capability f the final nanocomposite, in other words, increase the potential o retain water for a long time. Ul-Islam, Khan, Khattak, and Park 2011) found that BC/Chitosan (Ch) composite presented very slow ater release. Accordingly, BC-Ch could be applied to the treatment f hard to heal wounds, skin ulcers, bedsores, burns, and wounds hat needs frequent dressing changes (Ciechanska, 2004). Lin, Lien, Yeh, Yu, and Hsu (2013) recently reported the kin wound healing efficacy of BC-Ch composite in experiments ssessed with rat models. The authors found that the composite did ot produce any toxic effect on animal cells. Moreover, an exam- nation of the tissue regeneration process revealed that wounds reated with BC-Ch composites had epithelialized and regenerated aster than those treated with BC or commercially available dress- ng materials. . Biocompatibility ‘Biocompatibility’ refers to the ability of a given material to e non-toxic to the biological system, to perform satisfactorily nd elicit an appropriate host response upon specific application Torres, Commeaux, & Troncoso, 2012). Thus, biocompatibility is a esult of the complex interactions between an implant and the sur- ounding tissues. It is a fundamental property, configuring one of he required characteristics to a material be considered a biomate- ial. Due to structural similarities with extracellular matrix com- onents, such as collagen, BC is a highly biocompatible material. dditionally, unlike proteins, the polysaccharide nature of BC akes it less or even non-immunogenic (Petersen & Gatenholm, 011). There are several in vitro and in vivo studies in the litera- ure emphasizing the importance of structural characteristic of BC ydrogel and its biocompatibility. .1. In vitro biocompatibility Schwann cells were cultured on BC membranes and no signif- cant differences in the morphology and cellular functions were bserved on the basis of the results of microscopy (light and scan- ing electron), cell proliferation assay, flow cytometry and RT-PCR te Polymers 153 (2016) 406–420 409 (Zhu, Li, Zhou, Lin, & Zhang, 2014). Native BC allowed the prolifer- ation of L929 cells and human osteoblasts, according to Chen et al. (2009). Mendes et al. (2009) have assessed the biological response in the presence of a BC membrane after subcutaneous implan- tation in mice. They performed analysis of histological sections of the BC membrane and the surrounding tissue at 7, 15, 30, 60 and 90 days post-surgery. They found no evidence of foreign body reaction throughout the studied period. Polymorphonuclear cells and lymphocytes were observed at 7, 15 and 30 days post- surgery suggesting a mild inflammatory response. By contrast, at 60 and 90 days post-surgery, no inflammatory cell infiltration was observed. Human vein endothelial cells exhibited a great proliferation in a BC hydrogel which in turn displayed horizontal growth and occur- rence of interesting vertical migration of cells with regards the membrane. Due to the presence of different gradient of oxygen availability as a function of the depth of BC hydrogel, it was found that the cell penetration inside the BC hydrogel was limited up to a certain level of oxygen (Jeong et al., 2010; Recouvreux et al., 2011). It has been shown that human osteoblasts were able to attach and spread well on larger bacterial cellulose particles obtained in agitated cultures (Hu, Catchmark, & Vogler, 2013). Kim, Cai, and Chen (2010) prepared BC-gelatin composites to assess the biocompatibility. NIH3T3 fibroblast cells were seeded over pure BC and BC-gelatin composites that were incubated for 48 h. They found that the cells showed good adhesion and prolifer- ation although the biocompatibility was much better in BC-gelatin composites than that of pure BC. Accordingly, the prepared BC- gelatin scaffolds are bioactive, indicating that they can be used for wound dressing and as tissue engineering scaffolds (Kim et al., 2010). Similarly, Wang et al. (2012) have reported the synthe- sis of a BC/gelatin composite via crosslinking by procyanidin. The results showed that the proliferation, infiltration and adhesion of fibroblasts are improved with regard to native BC. Indeed, gelatin, a polypeptide derived from an extracellular matrix, and a denatured form of collagen, exhibits many properties such as good biocom- patibility, low immunogenicity, adhesiveness, promotion of cell adhesion and growth and low cost. Other composites such as BC-poly(ethylene glycol), BC-chitosan and BC-collagen showed better NIH3T3 cell activity as compared to native BC (Cai & Kim 2010; Zhijiang & Guang 2011). Human adipose-derived stem cells proliferated on BC-poly(2-hydroxyethyl methacrylate) to a lower extent in comparison to native BC mem- branes (Figueiredo et al., 2013). BC-collagen composites were synthesized for potential tissue engineering applications through an in situ synthesis strategy. It should be pointing out that high biodegradability, low antigenicity and cell-binding properties found for BC-collagen composites are distinguished characteristics of a suitable biomaterial with medical purposes (Luo et al., 2008). As shown throughout this manuscript several studies indi- cated good biocompatibility of BC, and consequently, it can be inferred that pristine BC membranes would not show genotoxi- city and immunoreactivity. Therefore, in vitro genotoxicity of BC nanofibers was assessed by single cell gel electrophoresis and the Salmonella reversion assays. The reversion assays showed that cel- lulose nanofibers were non mutagenic or genotoxic and the comet assay indicated no or insignificant DNA damage (Moreira et al., 2009). Regardless the systemic toxicity, Hagiwara et al., (2010) eval- uated the adverse effects of fermentation-derived BC when administered for both sex of F344 rats at dietary for 28 days. No macroscopic changes were observed neither adverse effects were manifested in hematology results. Additionally, no other treat- ment related changes were apparent observing blood biochemistry 4 ohydr r e 3 c t m w a f a c a d t i w m T w c i s 6 c b a 4 t s t a e a v t 2 t B b w c a n A a S m a D N d w t 10 H.G. de Oliveira Barud et al. / Carb esults. Thus, they concluded that BC does not cause any adverse ffect when fed to both sex of F344 rats at dietary for 28 days. .2. In vivo biocompatibility A detailed systematic evaluation of BC biocompatibility was arried out by Helenius et al. (2006). Upon subcutaneous implanta- ion in Wistar rats, the implants retained their shape without any acroscopic signs of inflammation up to 12 weeks. Using the improved multilayer fermentation method, BC films ere obtained. There were observed low cytotoxicity of the films nd good proliferation of human adipose-derived stem cells. Thus, ull-thickness skin wounds were made on the backs of BALB/c mice nd subsequent histological examinations demonstrated signifi- ant fresh tissue regeneration and capillary formation in the wound rea of BC groups on day 7 when compared with those commercial ressings and animal skins in other groups. These results indicate he high production efficiency of BC and its high clinical potential s due to the biocompatibility of the films (Fu et al., 2012). In a similar investigation conducted by Park et al. (2014), BC ound dressing materials were compared with two different com- ercial dressings, Vaseline gauze and Algisite M, in a rat model. his study showed that BC-dressed animals presented more rapid ound healing on day 14 without any evidence of toxicity when ompared to other groups. Hollow tubes of BC synthesized by rolling method were mplanted into the spatium intermusculare region, and data howed that BC did not revealed toxic effects on nerve tissues up to weeks post-implantation (Zhu et al., 2014). Another study on bio- ompatibility showed that BC/potato starch composite was indeed iocompatible as evidenced by formation of new blood vessels in nd around the composite (Yang, Chen, & Wang, 2014). . Antimicrobial properties BC provide a moist environment to a wound resulting in bet- er wound healing. However, in addition to biocompatibility, the econd major short coming associated with the medical applica- ion of BC is related to its non-bactericidal nature, presenting no ntimicrobial activity to prevent wound infection. As a result, sev- ral bactericidal elements have been attached to BC to enhance its ntimicrobial activities. It is reported that among the different antimicrobial agents, sil- er has been the most extensively studied and used since ancient imes to fight infections and prevent spoilage (Rai, Yadav, & Gade, 009). In this way BC-Ag nanocomposites have been prepared hrough a variety of routes for the same purpose. Remarkably, C-Ag nanocomposites were found to be effective against many acterial and fungal species, thereby reducing the chances of ound infection when utilized as dressing materials. Maneerung, Tokura, and Rujiravanit (2008) obtained a BC-Ag omposite by immersing BC in AgNO3 solution, while NaBH4 was pplied to reduce the Ag+ ions adsorbed on the surface of BC anofibers to produce metallic Ag nanoparticles. The freeze-dried g nanoparticle-impregnated BC nanocomposites exhibited strong ntimicrobial activity against Escherichia coli (Gram-negative) and taphylococcus aureus (Gram-positive). Sureshkumar, Siswanto, and Lee (2010) reported an easy ethod for preparing magnetic BC-Ag nanocomposites. Adding mixture of Fe3+ and Fe2+ ions, they first homogenized the 3- nanofibrous structure of BC and as the pH increased, magnetic Ps were precipitated and attached to the BC surface. A poly- opamine layer was then coated onto the magnetic BC nanofibers, hich reduced the Ag nanoparticles from AgNO3 solution onto he magnetic BC surface. The magnetization of the prepared BC-Ag ate Polymers 153 (2016) 406–420 nanocomposite was well maintained and impressive antimicrobial activity against the model microbial species have been shown. Some of us also prepared BC-Ag nanocomposites (Maria et al., 2009) by a simple method loading a large amount of Ag nanopar- ticles into BC. These composites showed large bactericidal effects, nearly 100% of antibacterial activities against Escherichia coli (Maria et al., 2010). Other nanocomposites were obtained by the associa- tion of Ag nanoparticles presenting antimicrobial activities (Barud et al., 2008, 2011). Wu et al. (2014) developed a novel method to synthesize and impregnate Ag nanoparticles onto BC nanofibres (AgNP-BC) to prevent Ag nanoparticles from dropping off 3-D nanofibrous BC structure and thus minimized the toxicity of these nanoparticles. They generated uniform spherical Ag nanoparticles (10–30 nm) and self-assembled on the surface of BC nanofibers forming a stable and evenly distributed hybrid nanostructure. Regardless the slow Ag+ release, AgNP-BC nanocomposites still exhibited significant antibacterial activities with more than 99% reductions in Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. More- over, AgNP-BC nanocomposites allowed attachment and growth of epidermal cells with no emerged cytotoxicity. The results demon- strated that AgNP-BC nanocomposites could reduce inflammation and promote wound healing. Still considering metals that exhibit antimicrobial activity, it has been reported that Cu nanoparticles have bactericidal effects comparable to Ag nanoparticles. Thus, Pinto, Neves, Pascoal, and Trindade (2012) reported the antibacterial activity of bionanocom- posites made of Cu and BC and plants cellulose as well. The authors reported striking 100% of antibacterial activity against S. aureus and K. pneumoniae. Composites of BC membrane impregnated with TiO2 also have been investigated. The improvement of antibacterial and con- ducting properties of BC showed that the introduction of TiO2 substantially promote the use of BC-TiO2 composites in biomedical applications (Gutierrez, Fernandes, Mondragon, & Tercjak, 2012; Gutierrez, Tercjak, Algar, Retegi, & Mondragon, 2012; Gutierrez, Fernandes, Mondragon, & Tercjak, 2013). Biomaterials prepared by the addition of different clays and never-dried BC hydrogel have been shown potential use in biomed- ical applications. Considering the ion exchange properties of montmorrilonite (MMT), researchers extended the work to the preparation of BC composites with modified MMTs (Ul-Islam, Khan, Khattak, & Park, 2013). BC hydrogels impregnated with Ca-MMT, Na-MMT and Cu-MMT were prepared and unveiled significant antibacterial activity (80%) of BC-clay composites. Propolis has meriting special attention. It is a natural substance with remarkable antifungal, antiviral, antioxidant, anti- inflammatory and antibacterial properties. Barud et al. (2013) produced propolis-BC membranes which were able to incorpo- rate propolis at the surface and interstices. It was evidenced that the polyphenolic compounds determination and the prominent antibacterial activity in the membrane were dose dependent, sup- porting the possibility of obtaining propolis-BC membranes at the desired concentrations, taking into consideration its application and its skin permanence time. In conclusion, the authors suggested that propolis-BC membrane may favor tissue repair in less time and more effectively in contaminated wounds. BC-chitosan composites were also found to have both bacterici- dal and bacteriostatic activities against gram positive and negative bacteria (Ciechanska, 2004). When placed in contact with human fluid containing lysozyme, these composites are degraded resulting in glucosamine and N-acetylglucosamine units, which accelerate the wound healing process (Ciechanska, 2004). Finally, Wei, Yang, and Hong (2011) prepared BC films with ben- zalkonium chloride, an antimicrobial agent, in order to design a controlled release system to future acute wounds treatment. After ohydra p w t d 5 m s m p l o i c a b t t t P t p m p w t w f h T a i ( t f i ( r a b e B h d s l f A s b w t 2 B n m d w n H.G. de Oliveira Barud et al. / Carb erforming in vitro antimicrobial tests they concluded that the film as able to reduce the growth and proliferation of gram posi- ive bacteria demonstrating the potential controlled release system esigned. . Surface modifications of BC for cell adhesion and growth The surface of native BC generally offers poor cell attach- ent/adhesion because cellulose is biochemically very inert. It hould be pointed out that the interfacial characteristics of bio- aterials play a key role in cell adhesion. The surfaces should romote the specific absorption of proteins and subsequent cellu- ar interaction (Angelova, 1999). Several studies have been carried ut with the aim of modifying BC surface to optimize the BC-cells nteractions. These modifications stand to changes in the physical- hemical properties of BC structures, such as wettability, porosity nd surface. Thus, the modification of surfaces by plasma techniques are ecoming common in materials engineering. The most impor- ant advantage of this process is the ability to selectively change he surface properties, improving biocompatibility and mimicking he local tissue environment without altering the main attributes. lasma provides an effective mean to modify surfaces and optimize he biofunctionality (Chu, Chen, Wang, & Huang, 2002). Nitrogen lasma is frequently used to modify metals, polymers and poly- eric membranes, aiming the introduction of amino groups in the olymer surface and therefore, changing its polarity, reactivity and ettability (Charpentier, Maguire, & Wan, 2006). For example, Pertile, Andrade, Alves, and Gama (2010) increased he concentration of the functional amino groups of BC surface ith plasma. Thus, adhesion and proliferation studies were per- ormed on nitrogen plasma treated BC membranes by seeding uman endothelial cells (HMEC-1) and rat neuroblasts (N1E-115). he results showed a significant increase in the proliferation of cells pplied, demonstrating potential applications in tissue engineer- ng. Towards the improvement of cell adhesion, the same groups Pertile, Moreira, Andrade, Domingues, & Gama, 2012) also inves- igated the introduction in native BC of small signalling peptides ound in the proteins of the extra-cellular matrix (ECMs) such as the ntegrin-ligand sequences isoleucine-lysine-valine-alanine-valine IKVAV) mixed to a carbohydrate-binding module (CBM3). These ecombinant proteins were adsorbed to BC fibers surface and were ble to improve the adhesion of neuronal and mesenchymal cells, ut demonstrated no effect on other cell lines tested. To improve the biocompatibility of BC, basic fibroblast, human pidermal and keratinocyte growth factor were immobilized onto C surface with different ECMs such as collagen, elastin, and yaluronan (Lin, Chen, Ou, & Liu, 2011). As a result, human epi- ermal and collagen-modified BC supported the growth of human kin fibroblast. The attachment of cells to biomaterials can be improved by uti- izing adhesive amino acid sequences, such as Arg-Gly-Asp (RGD), ound in several ECMs proteins. Concerning this information, ndrade, Moreira, Domingues, and Gama (2010) conducted a study eeding fibroblasts in RGD protein-modified BC and native BC mem- ranes. Better spreading and uniform distribution of fibroblasts ere obtained with the surface modification whereas cell clus- ers were obtained on native BC membranes. Andrade et al. (2011, 012), also investigated the biocompatibility of small-diameter C and peptide (Arg-Gly-Asp)-modified BC membranes subcuta- eously implanted in sheep for 1–32 weeks. Peptide-modified BC embranes were mildly irritating to the tissue, with no significant ifferences in relation to the inflammation degree when compared ith expanded polytetrafluoroethylene (ePTFE) thereby used as a egative sample control. te Polymers 153 (2016) 406–420 411 6. Drug delivery Successful drug delivery systems are influenced by multiple fac- tors and one of which is the appropriate identification of materials for research and engineering of new drug delivery systems. BC is one such biopolymer that fulfils the criteria for consideration as a drug delivery material. In recent years, several drug-delivery systems based on nanocel- lulose material for various pharmaceutical applications have been proposed. Inumerous approaches for the preparation of BC-based nanocomposites by incorporating different guest substrates includ- ing small molecules, inorganic nanoparticles and polymers on the surfaces of BC nanofibers are exemplified. The delivery of the antibiotic tetracycline encapsulated on BC matrix was described by Stoica-Guzun, Stroescu, Tache, Zaharescu, and Grosu (2007). They compared irradiated (doses of 5 or 15 kGy) to non-irradiated BC membranes in an in vitro study and found that electron beam irradiated over BC-tetracycline system resulted into faster drug release rate. Lately, researchers applied BC membranes as systems for topi- cal release of lidocaine and ibuprofen (Trovatti et al., 2011, 2012). An in vitro drug release study using a phosphate buffer solution (pH 7.4) at 32 ◦ C showed a burst release profile in which more than 90% of the total drug was released in the first 20 min. The therapeutic applicability of different lidocaine delivery systems (BC membrane, a gel, and an aqueous solution) was evaluated in vitro with human epidermis. It was found that the permeation rate of lidocaine related to the BC membranes was significantly lower than those obtained with the other two conventional delivery systems (gels and aqueous solutions). In another study, Müller et al. (2013) investigated BC as potential drug-delivery system for proteins by investigating serum albumin as a model. They found that the freeze-dried BC samples showed a lower uptake of protein than the pristine BC and the biological stability of albumin was maintained during materials processing. Other models of studies were also described in literature inves- tigating possible effective delivery systems based on BC. Model tablets of Paracetamol (Mohd Amin, Abadi, Ahmad, Katas, & Jamal, 2012) were film coated with BC, using a spray coating technique, and in vitro drug release studies of these tablets were investigated. Physicochemical, morphological and thermal properties of BC films were studied. It was found that BC exhibited excellent ability to form soft, flexible and foldable films without the addition of any plasticizer, allowing the delivery of paracetamol, as in vitro assays indicates. BC-caffeine membranes (Silva et al., 2014) were prepared by a simple approach and the permeation of caffeine through human epidermis, from BC or from conventional formulation systems (solution and gel), was compared in vitro to assess their thera- peutic applicability. Diffusion studies with Franz cells showed that these materials are promising biosystems for topical delivery of caffeine, showing reproducibility and an extended and predictable caffeine release over time, leading to their potential use for cellulite attenuation. Bacterial cellulose (BC) membranes are used as the carrier for berberine hydrochloride and berberine sulphate to produce a new controlled release system (Huang et al., 2013). Release studies and transdermal experiments were carried out in vitro. Carrier BC can significantly extend the drug release time, in contrast to existing commercial carriers that were compared in the study. Freeze-dried BC membranes 10 mm thick were optimized for drug delivery. 1H high-resolution magic angle spinning nuclear magnetic reso- nance (1diffusion-ordered spectroscopy, DOSY) analysis showed that there was an interaction between the drugs and BC. Despite the structure of BC, the media and the solubility of the drug that can influence the sustained-release behavior, the results indicate 4 ohydr t l o 2 i g s B e r n i ( c o c T m c 7 t c t s n b p a 2 g n p i c ( t a e G w p t s n p w i i f s e e t ( ( 12 H.G. de Oliveira Barud et al. / Carb hat BC can be a useful material for drug delivery significantly pro- onging the release time of the drugs, either for oral administration r transdermally. Other group of researchers prepared BC-glycerin (Almeida et al., 014) membranes as supports for drug topical delivery which skin rritation potential of BC was evaluated in human subjects. The ood skin tolerance found after a single application under occlu- ion reinforces the putative interest of BC-glycerin membranes. esides modifying the mechanical properties, the inclusion of glyc- rin resulted in a skin moisturizing effect which could be clinically elevant for the treatment for skin diseases characterized by dry- ess, such as psoriasis and atopic dermatitis. Mono and multilayer materials from PVA and BC incorporat- ng vanillin as natural antimicrobial ingredient were prepared Stroescu, Stoica-Guzun, & Jipa, 2013). The composite films were haracterized by means of SEM and FTIR. The release mechanism f vanillin from composites films was investigated which diffusion oefficients are ranging from 1.69 × 10−12 to 3.84 × 10−12 m2 s−1. he vanillin release is influenced by films composition and the ultilayer films were found to be promising in order to achieve ontrolled release of vanillin. . Scaffolds In tissue engineering area a 3D cell-culture system is required o provide the geometrical basis and building blocks to provide ell attachment. Scaffolds are novel systems that closely mimic he complex and hierarchical structures inherent to the native tis- ue being designed to provide the microenvironment that cells eed to proliferate, migrate and differentiate. In addition, some iomechanical properties such as stiffness and elasticity that lay important roles in controlling terminal differentiation are lso improved (Bäckdahl, Esguerra, Delbro, Risberg, & Gatenholm, 008). Several materials have been tested as scaffolds to support rowth of cells. Nanocellulose produced by Gluconacetobacter xyli- us is an emerging biomaterial. As mentioned in Section 1, a ure nanocellulose fibril network is synthesized by the bacteria n any desired shape which microarchitecture and porosity that an be designed by controlling the bacteria fermentation process Bäckdahl et al., 2008; Rambo et al., 2008). It is also important o point out that the bacterial nanocellulose (BNC) network has very high affinity for water which results in hydrogel-like prop- rties promoting an ideal environment to host cells (Petersen & atenholm, 2011). Therefore, Bäckdahl et al. (2006) also developed BC scaffolds ith controlled microporosity by using paraffin wax and starch articles during culture and removing these particles once the cul- ivation process finished. The BC scaffolds were then seeded with mooth muscle cells for investigating the potential tissue engi- eered blood vessel application. Freeze-drying techniques allowed the preparation BC- oly(ethylene glycol) (PEG) scaffold composites by immersing et BC pellicle in PEG aqueous solution (Cai & Kim, 2010). Strong nteractions between BC and PEG were observed with a decrease n the crystallinity and improvement in thermal stability. Gao et al. (2011) prepared BC sponge scaffolds using emulsion reeze-drying technique in order to obtain high porosity and con- equently, high surface area. The prepared sponges also exhibited xcellent cell compatibility and fibrous synovium-derived mes- nchymal stem cells (MSCs) could proliferate well on and inside he matrix. Freeze-drying technique was applied by some of us as well Oliveira Barud et al., 2015) in the preparation of BC/silk fibroin SF) sponge scaffolds. In vitro tests proved non-cytotoxic or geno- ate Polymers 153 (2016) 406–420 toxic character of these nanobiocomposites. SEM images revealed a greater number of fibroblast cells (L929 cell line) attached at the BC/SF:50% scaffold surface if compared with the surface of pure BC, suggesting that the presence of fibroin improved cell attach- ment as is possible to see in Fig. 4. This could be related to the SF amino acid sequence that act as cell receptors facilitating cell adhe- sion and growth. Consequently, BC/SF:50% scaffolds configured an excellent option in bioengineering, depicting its potential for tissue regeneration and cultivation of cells on nanobiocomposites. There is an increased interest in developing adipose tissue as an in vitro model for adipose biology and metabolic disease. Krontiras, Gatenholm, and Hagg (2015) recently prepared 2D and 3D porous scaffolds of BC and alginate. The 3D scaffolds were engi- neered by crosslinking homogenized cellulose fibrils using alginate and freeze-drying the mixture to obtain a porous structure. They found that on 2D surfaces, the cells were scarcely distributed and showed limited formation of lipid droplets, whereas cells grown in macroporous 3D scaffolds contained more cells growing in clus- ters, containing large lipid droplets. Scaffolds with lower alginate relative content retained their pore integrity better. The authors concluded that 3D culturing of adipocytes in BC macroporous scaf- folds is a promising method for fabrication of adipose tissue as an in vitro model for adipose biology and metabolic disease. They also suggest that BC-alginate can be used as injectable gel which will be enable to deliver adipose or progenitor cells directly in the defects which is aimed to be repaired. Other studies have confirmed that different cells, such as human embryonic kidney cells (HEK) (Grande, Torres, Gomez, & Bañó, 2009), bone forming osteoblasts (OB) and fibroblasts (Chen et al., 2009), and human smooth muscle cells (SMC) (Petersen & Gatenholm, 2011) etc., can grow in the presence of BC scaffolds. Hutmacher (2001) has identified several requirements that tissue engineering scaffolds should fulfill. Among them, biodegrad- ability seems to be the most difficult requirement to meet for BC-based biomaterials, as cellulase enzymes capable of perform- ing cellulose hydrolysis are not present in animals. Li, Wan, Li, Liang, and Wang (2009) have reported the enhancement of the biodegradation of BC in vitro (in water, phosphate-buffered saline and simulated body fluid) through periodate oxidation. This chem- ical treatment preserved the original network structure of BC intact enabling to prepare a BC-based scaffold that could degrade in water, phosphate buffered saline (PBS) and the simulated body fluid (SBF). In terms of scaffolds, Si et al. (2014) and Luo et al. (2014) prepared Graphene oxide–bacterial cellulose (GO/BC) nanocom- posite hydrogels with well-dispersed GO in the network of BC. The in situ biosynthesis was developed by adding GO suspension into the culture medium of BC. The experimental results indicate that GO nanosheets are uniformly dispersed and well-bound to the BC matrix and that the 3D porous structure of BC is sustained. This is responsible for efficient load transfer between the GO rein- forcement and BC matrix. Compared with the pure BC, the tensile strength and Young’s modulus of the GO/BC nanocomposite hydro- gel containing 0.48 wt% GO are significantly improved by about 38 and 120%, respectively. The GO/BC nanocomposite hydrogels are promising as a new material for tissue engineering scaffolds. A BC-alginate scaffold composite (N-BCA) was fabricated by sequential steps of freeze-drying and crosslinking with Ca2+ (Kirdponpattara, Khamkeaw, Sanchavanakit, Pavasant, & Phisalaphong, 2015). A mechanically stable structure of N-BCA with open and highly interconnected pores in the range of 90–160 �m was constructed. For long-term culture, the scaffold supported attachment, spreading and proliferation of human gingival fibro- blast (GF) on the surface. Because of its biocompatibility and open macroporous structure, N-BCA could potentially be used as a scaf- fold for tissue engineering. H.G. de Oliveira Barud et al. / Carbohydrate Polymers 153 (2016) 406–420 413 Fig. 4. To test the hypothesis that the addition of silk fibroin to cellulose scaffolds increases cell adhesion (48 h), L-929 cells were seeded in MC and MC/SF scaffolds. SEM i n SEM s S a b 1 b ( s a a p h p s t g B t ( T d m f b ( s b o c c fi s mages of the cells attached to MC (a) and MC/SF (b) scaffolds surface; cross-sectio caffolds. ource: Reprinted with permission from Oliveira Barud et al. (2015). Lecithin is a natural mixture of phospholipids and neutral lipids nd the existence of the hydrocarbon groups on the surface was elieved to lead to improved blood compatibility (Nakaya & Li, 999). In an attempt to improve the biological behavior of pristine acterial cellulose (BC), Zhang et al. (2015) immobilized lecithin LEC) on the surface of BC nanofibers by solution immersion and ubsequent chemical crosslinking with proanthocyanidin (PA). The s-prepared LEC-immobilized BCs (denoted as BC/LECs) were char- cterized by SEM, FTIR, and XRD, and their dynamic mechanical roperties, thermal stability, and hydrophilicity were assessed. It as been found that BC/LECs retain the three-dimensional (3D) orous network structure of pristine BC. The BC/LECs still demon- trate favorable mechanical properties, surface hydrophilicity, and hermal stability. More importantly, preliminary cell studies sug- est that the BC/LECs show improved cell behavior over pristine C. BC-synthesizing bacteria in medium containing carbon nano- ubes (CNTs) coated with an amphiphilic comb-like polymer APCLP) formed an hybrid scaffold (CNT-BC-Syn) (Park et al., 2015). hese scaffolds showed excellent osteoconductivity and osteoin- uctivity that led to high bone regeneration efficacy whose strategy ay open a new avenue for development of 3D biofunctional scaf- olds for regenerative medicine. A stretchable BC nanofiber pellicle was successfully produced y using dissolved oxygen in a conventional cultured medium Nagashima, Tsuji, & Kondo, 2016). The resulting pellicle could be tretched by up to 1.5 times to provide oriented crystalline nanofi- rous films which function was evidenced by direct video imaging f the motion of the bacteria. In conclusion, stressed environment ould offer a promising nanofibrous film rich in the cellulose I� rystalline phase, which opens up the potential of this nanofibrous lm for application as a scaffold, reinforcement material, or other tructural material. images of MC (c) and MC/SF (d) evidenced that the cells did not migrate into the 8. Cardiovascular implants Processing properties of BC has lead to important applications as artificial blood vessels. Klemm et al. (Klemm, Schumann, Udhardt, & Marsch, 2001; Klemm et al., 2005, 2006; Schumann et al., 2009; Wippermann et al., 2009) have developed prototypes of BC tubes (patented BASYC®-tubes) synthesized in the form of regular tubes with different inner diameter, wall thickness and length, using a patented matrix technique during fermentation. The initial studies showed that the BC tubes have very good surgical handling and can be sterilized in standard ways. In a follow-up in vivo study with rats, pigs, and sheep, the BC tubes were successfully used to replace carotid arteries. (Klemm et al., 2001; Schumann et al., 2009; Wippermann et al., 2009). Similarly, Putra, Kakugo, Furukawa, Gong, and Osada (2008) found that culturing BC in oxygen-permeable silicone tubes with inner diameter <8 mm yields, a tubular BC gel of the desired length, inner diameter, and thickness with uniaxially oriented fibrils were obtained. The tubes presented excellent mechanical properties and holds promise for use as a microvessel or soft tissue material in medical and pharmaceutical applications. Due to clinical conditions of thrombosis and occlusion, materials that are often used for replacement as vascular grafts are not pri- marily suitable in small caliber of blood vessels. There is a pursuit for newer non thrombogenic materials with mechanical proper- ties that mimic native vessel which has led to the exploration of BC. The mechanical properties of BC were comparable to porcine carotid artery and were better than expanded polytetrafluorethy- lene (Bäckdahl et al., 2006). Heparin (Hep) was also hybridized with the BC network to build Hep–BNC nanofibrous scaffolds with anticoagulant properties for potential use in vascular tissue engi- neering (Wan et al., 2011). The potential use of BC-based composites for the produc- tion of heart valve replacements of cardiovascular tissues was 4 ohydr a W p b P p i a p d o r 2 9 t i o p c e b 2 m c Y fi p t f l d u b c t ( ( i o m n t s a s t m o m l a t r 14 H.G. de Oliveira Barud et al. / Carb lso reported by Millon and Wan, (2006); Millon, Guhados, and an (2008) and Mohammadi (2011). The authors prepared a BC- oly(vinyl alcohol) (PVA) composite that mimics the mechanical ehavior of native porcine heart valve leaflets. Furthermore, BC- VA nanocomposite could exhibit a broad range of mechanical roperties aiming at mimicking not only the non-linear mechan- cal properties displayed by porcine heart valves, but also their nisotropic behavior. This property is related to the ability of the repared material in withstanding tensile forces depending on the irection of the fibers. This peculiarity emphasizes the importance f this material when used as a vascular graft by controlling mate- ial and processing parameters (Millon et al., 2008; Mohammadi, 011). . Cartilage/meniscus implants Due to the limited regeneration capacity of the cartilage tissue, he repair of cartilage defects configures a challenge and a clin- cal need. Materials for artificial cartilage are required to be not nly tough and resistant but also proof of biodegradation. As BC resents excellent mechanical properties and low biodegradability, hondrocytes were seeded on BC membranes and showed prolif- ration and collagen type II production, indicating suitability as a io-mimicking scaffold for cartilage replacement (Svensson et al., 005). Bodin, Concaro, Brittberg, and Gatenholm (2007) compared the echanical properties of a BC gel with traditional collagen menis- al implant material and real pig meniscus. It was found that the oung’s modulus of BC gel is similar to the one of pig meniscus, and ve times higher than the one of collagen material. The results of romising cell migration and controlled meniscus shape indicated hat BC can be an attractive material as meniscus implant. Another study conducted by Lopes et al. (2011) investigated the riction and wear behaviors of BC pellicles against bovine articu- ar cartilage. Due to the wear mechanism involving high plastic eformation, BC biomaterials possess low friction coefficient val- es (about 0.05) and preservation of the mating surfaces. This BC iomaterial was reported to be a potential replacement of artificial artilage for articular joints. In order to mimic the ultrastructure of the central region of he knee meniscus, Martínez, Brackmann, Enejder, and Gatenholm 2012) fabricated BC devices together with micro-channels Ø ∼ 500 �m). Results showed that the micro-channels could facil- tate the alignment of cells and collagen fibers, and the parallel rientation of collagen fibers in contrast strengthen the tissues, aking it suitable for knee meniscus and tendons replacement. Articular chondrocytes from young adult patients as well as eonatal articular chondrocytes were seeded with various seeding echniques onto the porous BC scaffolds. Furthermore, DNA analy- is implied that the chondrocytes proliferated within the porous BC nd with some further development, this novel biomaterial can be a uitable candidate for cartilage regeneration applications according o Andersson, Stenhamre, Bäckdahl, and Gatenholm (2010). Recently, Ávila et al. (2014) proposed a non-resorbable implant aterial for auricular cartilage replacement based on BC with 15% f cellulose content, since it matches the mechanical strength and ainly the host tissue response of human auricular cartilage. In terms of BC composites that presents applications in carti- age tissues, Azuma et al. (2007) concluded that BC-poly(dimethyl crylamide) double network gel has mechanical properties similar o the mechanical properties of cartilage and that may meet the equirements of artificial cartilage. ate Polymers 153 (2016) 406–420 10. Bone and connective tissue repair Bone is a composite material comprising basically an organic phase (collagen and noncollagenous proteins) and an inorganic mineral phase (calcium hydroxyapatite). Nanocellulose and its bio- composites have been proved to be promising materials for the culture of various cells, including osteoblast and chondroblast, indi- cating that nanocellulose based materials have the potential for bone tissue regeneration and healing. BC can be a good matrix for obtaining different types of calcium carbonate (CaCO3) crystals with improved biocompatibility. Stoica- Guzun et al. (2012) have used calcium chloride (CaCl2) and sodium carbonate (Na2CO3) as starting reactants to promote calcium car- bonate deposition on BC membranes. A membrane composed of BC and hydroxyapatite (Hap) was developed as biomaterial for potential bone regeneration, which delivered prone growth of osteoblast cells, high level of alkaline phosphatase activity, and greater bone nodule formation (Tazi et al., 2012). The better osteoblasts adhesion, proliferate and mineraliza- tion from BC-Hap biomaterials were expected to facilitate quick regeneration of bone tissue. Similarly, Grande et al. (2009) pro- duced BC-Hap scaffolds for biomedical applications and obtained excellent results in terms of regeneration of bone and connective tissues. Researchers also prepared and characterized BC-Hap compos- ites (Hong et al., 2006; Wan et al., 2006). They found that the HAp crystals are partially substituted with carbonate, resembling nat- ural bones. The nanocomposites containing HAp with structural features close to those of biological apatites are attractive for appli- cations as artificial bones and scaffolds for tissue engineering. Saska et al. (2011) prepared BC-Hap nanocomposites. They eval- uated the biological properties and performance of the material with respect to bone regeneration in defects of rat tibiae (Saska et al., 2011). The BC-Hap membranes were effective for bone regeneration and accelerated new bone formation. In addition, reabsorption of the membranes was slow, suggesting that it takes longer to this composite to be completely reabsorbed. Other authors (Wan et al., 2009; Yin et al., 2011) have induced a negative charge on BC by the adsorption of polyvinylpyrroli- done (PVP) to initiate the nucleation of Hap via dynamic simulated body fluid treatment. Shi et al. (2009) introduced an alkaline treat- ment before the biomimetic mineralization process in order to improve the mineralization efficiency. On the other hand, Zhang et al. (2009) have used a phosphorylation reaction to introduce phosphate groups to the surface of BC and promote the growth of calcium phosphate. Wan et al., (2009) have also shown that phos- phorylation effectively triggers Hap formation on BC which allowed BC-Hap composites with a third phase. Recently, Pigossi et al. (2015) evaluated the potential of BC- Hap composites associated with osteogenic growth peptide (OGP) or pentapeptide OGP (10–14) in bone regeneration in critical-size calvarial defects in mice. OGP is proteolytically cleaved, thus gen- erating the osteogenic growth peptide C-terminal pentapeptide (NH2-YGFGG-OH), named OGP(10–14). OGP (10–14) may be the physiologically active form of OGP because it is this C-terminal pen- tapeptide that activates the cytoplasmic OGP signalling pathway (Gabarin et al., 2001). Therefore, this suggests that OGP (10–14) is the bioactive form of OGP (Chen et al., 2002; Greenberg et al., 1993). In this study, the BC-Hap, BC-Hap-OGP, and BC-Hap-OGP (10–14) membranes were analyzed at 3, 7, 15, 30, 60, and 90 days. In each period, the specimens were evaluated by micro-computed tomography (�CT), descriptive histology, gene expression of bone biomarkers by qPCR and VEGFR-2 (vascular endothelial growth fac- tor) quantification by ELISA. The researchers found that at 60 and 90 days, a high percentage of bone formation was observed by �CT for BC-Hap and BC-Hap-OGP (10–14) membranes. High expres- ohydra s w m b p p w i c 1 r e a a i P c w t d i i t c v a a t p t f s f u t A c M c 1 S h m d m r a i s f m r a n H.G. de Oliveira Barud et al. / Carb ion of some bone biomarkers, such as Alpl, Spp1, and Tnfrsf11b, as also observed. They concluded that the BC-HA membrane pro- oted a better bone formation in critical-size mice calvarial defects. Fan et al. (2013) reported the fabrication of novel bone repair iomaterials with the introduction of goat bone apatite in BC. The roduced biomaterial can stimulate bone cells proliferation and romote cell differentiation as demonstrated in vitro assays. Note- orthy, Lee, Kim, Lee, and Park (2013) evaluated in vivo assays by mplanting silk fibroin-BC membranes to successfully promote the omplete healing of segmental defects of zygomatic arch of rats. 1. Dental/oral implants Some of the first applications in dental field were related to the egeneration of periodontal disease through guided tissue regen- ration technique (GTR). Novaes and Novaes (1992, 1993) reported dequate GTR results in periodontal defects in humans as well s in GTR for bone formation in association with osseointegrated mplants using the commercial BC membrane, Gengiflex®. Chiaoprakobkij, Sanchavanakit, Subbalekha, Pavasant, and hisalaphong (2011) developed a composite based on bacterial ellulose/alginate to be used as a temporary dressing of surgical ounds of the oral mucosa. The material features a unique design: he outer layer is thicker to prevent bacterial contamination and ehydration of the wound while the inner layer is porous and ntended to drain exudates. Preliminary in vitro tests of biocompat- bility showed a good performance of the material which enabled he proliferation of keratinocytes and gingival fibroblasts. BC was also reported as an innovative material for dental root anal treatment in animal experiments. In comparison with con- entional paper point materials, BC exhibited greater compatibility nd biological characteristics for dental root canal treatment. The bsorption rate of BC-based biomaterials was about 10-fold greater han that of paper point materials, and BC-based biomaterials can reserve better tensile strength under wet condition and, in addi- ion, it showed maintenance of physical integrity and only a small oreign body reaction (Yoshino et al., 2013). Cellulose whiskers may be also obtained from BC. Nanometric cale whiskers (nanowhiskers) can be used in materials rein- orcement. Jinga et al. (2014) prepared BC nanowhiskers and sed commercial MTA (Mineral Trioxide Aggregates) cement for he preparation of some composites: MTA-E (Mineral Trioxide ggregates-experimental), MTA-10% biocell and MTA-33% biocell ements. BC was observed to accelerate the hardening processes of TA cement, decreasing in the same time the relative quantity of alcium hydroxide crystals. 2. Neural implants/dura máter Nervous tissue reconstruction is a really challenging problem. elf-regeneration may be difficult depending on the extent of the arm. In this context, BC was assessed as a substitute for dura ater in Mongrel dogs. Macroscopic examination of the grafts emonstrated good acceptance and adherence to the bone frag- ent (Mello, Feltrin, Neto, & Ferraz, 1997). Pertile et al. (2012) eported the application of BC as a scaffold for nerve tissue regener- tion, where BC fibers maintained a continuous path that promoted nfiltration of cells. The researchers showed that mesenchymal tem cells adhered to BC proliferated and expressed nerve growth actor neurotrophin thus creating a microenvironment that pro- otes neuronal regeneration. BC was also reported to be developed as biomaterial for the econstruction of damaged peripheral nerves via cellulosic guid- nce channels. In vivo experiments were conducted on the femoral erve of Wistar rats for three months. Results evaluation from his- te Polymers 153 (2016) 406–420 415 tological analysis and postoperative observation of motor recovery showed that BC neurotubes can effectively prevent the formation of neuromas, while allowing the accumulation of neurotrophic factors inside, and facilitating the process of nerve regeneration (Kowalska-Ludwicka et al., 2013). The preparation of nerve conduits for repairing peripheral nerve injuries was explored using BC for it. In vitro data indicated that BC is biocompatible with Schwann cells and presented no adverse hematological and histological effects upon in vivo implantation in rats (Zhu et al., 2014). Xu et al. (2014) applied BC to repair dural defects in rabbits. Despite the long-term effect of this new dural material needs to be validated in larger animals, results showed that BC exhibited a decreased inflammatory response compared to traditional materi- als. 13. Artificial cornea/contact lens This is another interesting applicability of BC that is also few documented in literature. A Brazilian research group developed and patented (Messaddeq, Ribeiro, & Thomazini, 2008) a special mechanism to conform BC into correct angles and shape to pro- duce contact lenses for cornea regeneration. Wet BC membranes are cut in a round shape and then are compressed with the stick that presents a semi-spherical end onto the base, under a constant 150 ◦C heat. Thus, there can be obtained contact lens shaped inter- nally by the compression stick and externally by the mold, as shown in Fig. 5. In an effort to extend this work, Cavicchioli et al. (2015) impreg- nated ciprofloxacin (CPX) with and without 2-hydroxypropyl-�- cyclodextrin (�CD) into BC membrane in the shape of a contact lens by using aforementioned method in order to improve their therapeutic potential. Pure and impregnated membranes did not exhibit cytotoxicity, genotoxicity or mutagenicity effects. Other- wise, the BC-CPX membrane was only cytotoxic. They concluded that, except for BC-CPX, the investigated materials are promising for biomedical applications, especially as a contact lens used for regeneration or protection against bacteria. 14. Urinary conduits According to the American Cancer Society, bladder cancer is the second most common urologic malignancy in the United States, after prostate cancer. The chance of developing bladder cancer is about 1 in 27 for men and 1 in 85 for women. In order to treat malig- nancies that have invaded the bladder muscle, surgical resection of the tumor, followed by the creation of a continent urinary reservoir using segments of the small or large intestine is often necessary. Urinary diversion after radical cystectomy in patients with bladder cancer normally takes the form of an ileal conduit or neobladder. However, such diversions are associated with a number of compli- cations including increased risk of infection. A plausible alternative is the construction of a neobladder (or bladder tissue) in vitro using autologous cells harvested from the patient. Biomaterials can be used as a scaffold for naturally occurring regenerative stem cells to latch onto to regrow the bladder smooth muscle and epithe- lium. Such engineered tissues show great promise in urologic tissue regeneration, but are faced with a number of challenges. Thus, Bodin et al. (2010) produced microporous BC scaffolds seeded with human urine-derived stem cells to form a tissue- engineered conduit for use in urinary diversion. The cells were also induced to differentiate into urothelial and smooth muscle cells. The effects of urethral reconstruction with a three-dimensional (3D) porous BC scaffold prepared by gelatin sponge interfering in the BC fermentation process and seeded with lingual keratinocytes 416 H.G. de Oliveira Barud et al. / Carbohydrate Polymers 153 (2016) 406–420 F to ma B k mad M f rege w p s m u w 1 i p s C o f f t e e o a r p i t m p i t p t 2 ( o e ( ig. 5. (a, b) BC contact lens shaped by the designed device and c) device designed C membrane in a round form; a compression piece in the shape of a cylindrical stic ESSADDEQ, Y., RIBEIRO, S. J. L., THOMAZINI, W. Contact lens for therapy in cases o as evaluated in a rabbit model. Results demonstrated that 3D orous BC seeded with lingual keratinocytes enhanced urethral tis- ue regeneration without inducing inflammatory reactions and, at 3 onths postoperatively, macroscopic examinations and retrograde rethrograms of urethras revealed that all urethras maintained ide calibers (Huang et al., 2015). 5. Tympanic membrane Tympanic membrane (TM) perforation is a very common clin- cal problem resulting into conductive hearing loss and chronic erforations. Acute persistent or chronic TM perforations require urgical interventions such as myringoplasty or tympanoplasty. urrent strategies of tissue engineering are focused on treatment f regeneration of TM perforation will probably eliminate the need or conventional surgery. However, it is critical to understand the actors that contribute to the success or failure of TM perforations reatment. As such, several scaffolds and biomolecules have been valuated for TM tissue engineering. TM regeneration by tissue ngineering approach may be considered the greatest advance in tology. BC is presented as an alternative that is safe, biocompatible, nd has low toxicity. Recently, Kim et al. (2013) reported the fabrication of a nanofib- illar patch by using BC as a wound-healing scaffold for TM erforation. BC is endowed with the expected properties of an deal material for traumatic eardrum perforation repair: a nanos- ructured surface, biocompatibility, transparency, and appropriate echanical. In vitro, BC nanofibrillar patch promoted the adhesion, roliferation and migration of tympanic membrane cells. Next, n vivo assays applying Sprague-Dawley rats demonstrated that he presence of BC patch materials significantly increased the tym- anic membrane healing rate as well as recovered the function of ympanic membrane better than spontaneous healing (Kim et al., 013). A randomized controlled trial was conducted by Silveira et al. 2016). Forty patients with TM perforation secondary to chronic titis media were included and randomly assigned in two groups: xperimental group (20), treated with BC graft and control group 20) treated with autologous temporal fascia (fascia). The surgi- nufacture contact BC lens. The device is constituted by a snip piece which cuts the e of metal and a base with a semi-spherical undercut at the center made by Teflon. neration of cornea, has bacterial cellulose base. Patent number: PI0603704-6. cal time, hospital stay, time of epithelialization and the rate of TM perforation closure were evaluated and also hospital costs were compared. Despite the closure of perforations were similar in both groups, the average operation time in the fascia group was 76.50 min versus 14.06 min for BC. Finally, a 92% remarkable cost reduction in Brazilian public health system was detected when the hospital costs of fascia group were compared to BC group. 16. Other applications Regardless many BC applications in the biomedical field, there is still plenty to explore. Noteworthy, Recouvreux et al. (2011) synthesized a large organ-like 3D BC hydrogels with the poten- tial applications in implantable tissue and organ scaffolds such as kidney or liver. Tests in structural characteristics, mechanical properties and biocompatibility are all carried out and a superior performance could be totally expected. De Souza, Olival-Costa, Da Silva, Pontes, and Lancellotti (2011) conducted an in vivo study to evaluate the medialization, inflam- matory response, and healing of vocal folds after implantation of a membrane of microbial cellulose in 32 rabbits. The animals received a 0.25 mm2 membrane of BC in one side of vocal folds and 0.3 cc of distilled water at the other side as represented in the schematic illustration in Fig. 6. The results showed that after 120 days of implantation the material is relatively stable with no major modifications suggesting that BC is a useful material in medial displacement procedures of the vocal folds, as it causes minimal inflammatory reaction and does not extrude. There is a vast array of literature on the topic of BC nanocom- posites matrices and devices for tissue regeneration demonstrating a promising future on new research related to tissue engineering including bioprinting 3D field. Lately, Nimeskern et al. (2013) designed a BC-based ear-shaped prototype material from the reconstruction of gradient–echo mag- netic resonance imaging (MRI). The BC was bioprinted by using a negative silicone mold where the bacteria was guided to reproduce the large-scale features of the outer ear. This study was extremely important to confirm that BC is a promising tissue engineering material with appropriate mechanical properties for ear cartilage H.G. de Oliveira Barud et al. / Carbohydrate Polymers 153 (2016) 406–420 417 Fig. 6. Schematic drawing of the steps of cellulose implantation showing (A) the scissors opening the thyroid cartilage under direct endoscopic view; (B) the forceps grabbing the cellulose through the thyroid window; and (C) the cellulose positioned lateral to the thyroarytenoid muscle). Source: Adapted from De Souza et al. (2011). Fig. 7. Biofabrication of a patient-specific ear-shaped BC implant. (i) The bioprinter consists of a high-precision motion system and a microdispensing system. (ii) Transverse s 3D BC S r s 1 m t a e t a b f t i n i p s 1 f lice isolated from a spoiled gradient–echo MRI scan of the volunteer’s left ear. The ource: Reprinted with permission from Nimeskern et al. (2013). eplacement. Thereby, it may be used to create patient-specific ear hapes as displayed in Fig. 7. 7. Future opportunities and challenges It is expected that BC and BC based nanocomposites will be ore widely applied in biomedical fields due to the unique struc- ure of BC such as high moldability, excellent biocompatibility nd exceptional mechanical properties. Herein, we outlined sev- ral BC application in different areas of biomedicine. However, here were still some challenges to be overcome with respect to all reas mentioned in this work and many more, such as controlled iodegradation, porosity control, quality consistency, structure dif- erence between surface layer and core area of BC. It is important o point out that more work is expected on BC based nanocompos- tes due to the high-value-added of functional materials and many ovel applications can be expected as current trends. Wherefore, mproving properties, reducing production costs and designing a roper industrial fabrication line for BC based nanocomposites are ome great examples of main future goals for researchers. 8. Conclusions Bacterial cellulose is a natural renewable polymer synthesized rom the bacterium Gluconacetobacter xylinus that is the only implant prototype was fabricated in the shape of the whole outer ear. known species capable to produce cellulose on an industrial scale. Owning the uniform structure and morphology, BC is free of lignin and hemicellulose and it is endowed with unique characteristics such as high purity, high crystallinity, remarkable mechanical prop- erties, good chemical stability and high water-holding capacity. BC is featured as a completely biocompatible polymer that can be produced in almost any shape due to its high moldability during formation. BC is a really interesting emerging biomaterial being distinctive in several aspects and since its discovery has shown tremendous potential as an effective biopolymer in various fields, because the structural aspect of BC is far superior to those of plant cellulose. Generally, bacterial cellulose has attracted con- siderable interests in various application fields no matter acting as a matrix or using it in its modified state. Therefore, litera- ture exhibits several biomaterials that were associated with BC, such as collagen, gelatin, fibroin, propolis, chitosan, silver, alginate, hydroxyapatite, BC nanowiskers to reinforce materials, and others, resulting in composites and nanocomposites that were described in detail throughout this article review. Thus, this manuscript revised and presented a great number of different BC-based materials that were designed for biomedical applications (dressings, scaffolds, drug delivery systems), among others. 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