Engineering Bacterial Cellulose for Diverse Biomedical Applications
Sana Sandhu 1, Anindita Arpa 2, Xi Chen 2, Rahul Kumar 3, Justyn Jaworski 2, *
1. Department of Advanced Materials Science & Engineering, Sungkyunkwan University Advanced Institute of Nanotechnology, 300 Cheoncheon-dong, Jangan-gu, Suwon, S. Korea 440-746
2. Department of Bioengineering, University of Texas at Arlington, 500 UTA Blvd., Arlington, TX, United States of America 76010
3. Department of Chemistry, Sogang University, 35 Baekbeoreo Mapo-gu, Seoul, S. Korea 121-742
* Correspondence: Justyn Jaworski
Academic Editor: Hossein Hosseinkhani
Special Issue: Applications and Development of Biomaterials in Medicine
Received: September 15, 2019 | Accepted: November 20, 2019 | Published: November 25, 2019
Recent Progress in Materials 2019, Volume 1, Issue 4, doi:10.21926/rpm.1904006
Recommended citation: Sandhu S, Arpa A, Chen X, Kumar R, Jaworski J. Engineering Bacterial Cellulose for Diverse Biomedical Applications. Recent Progress in Materials 2019; 1(4): 006; doi:10.21926/rpm.1904006.
© 2019 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.
Increasing interest in bacterial cellulose due to the huge potential which exists for the development of this new biomaterial for medical applications has been met with recent growth in research in engineering this unique microbial manufactured material. The mechanical properties, porosity, and biocompatibility of bacterial cellulose derived biomaterials are particularly attractive for use in wound healing, tissue engineering, and drug delivery applications. Advances in synthetic biology and soft materials engineering are pushing the value of this biomaterial to new levels. This review provides and in-depth discussion of these most recent approaches in processing as well as physical, chemical, and genetic modification of bacterial cellulose toward further improvement and expansion of its functionality. In addition, we provide specific attention to the marketed applications of the resulting engineered materials for medical research and conclude with prospective areas of consideration for expanding the clinical utility of this biomaterial to new directions.
Bacterial cellulose; wound healing; tissue engineering; modification; composite
Bacterial cellulose (BC) is reported to have tremendous mechanical and chemical properties including high water holding capacity, tensile strength, and modulus of elasticity while being biocompatible . To relieve some of the environmental strain of utilizing wood derived cellulose, substantial developments have turned to the possibility of utilizing bacterial derived cellulose. A significant benefit of bacterial cellulose over plant-based cellulose is the lack of contaminants such as lignin and pectin . While the scale of bacterial cellulose production remains small, BC has far-reaching applications to various industries. Here we will focus primarily on how BC has significant value for medical products and biomedical applications (Figure 1). Throughout this review, we will offer a comprehensive look at the properties of BC and examine the recent advances in its modifications as well as provide a survey of the latest applications of bacterial cellulose in the area of biomedical materials. To begin understanding about BC and how this biomaterial can be engineered for medical applications, we must first consider how it forms from microbial cultures.
Figure 1 Biomedical applications of bacterial cellulose.
A number of bacterial have been reported as producers of extracellular cellulose including species from Gluconacetobacter, Agrobacterium, Aerobacter, Achromobacter, Azotobacter, Rhizobium, Sarcina, Salmonella and Escherichia . Certain species of Gluconacetobacter been found to be particularly efficient in their biological synthesis of cellulose including strains of G. hansenii . Biological synthesis of the interconnected cellulose that surrounds these cells (where this cellulose network is referred to as a pellicle) necessarily requires several genes whose products carry out the formation of the nanofibrous cellulose and its secretion . While the entire set of biosynthetic genes necessary to produce bacterial cellulose were previously not confirmed , recently it has been shown that a key set of genes could be transformed into other bacteria to confer production of cellulose pellicles . The genes include those within the bcs (bacterial cellulose synthesis) operon, specifically bcsA, bcsB, bcsC, and bcsD, as well as the genes cmcax and ccpAx. The roles of the respective genes have been reported, where bcsA yields the catalytic subunit of cellulose synthase while bcsB produces the regulatory subunit of the enzymes that binds to cyclic di-guanylic acid (cyclic di-GMP) . The cellulose synthase activity of the bcsA subunit can thus be allosterically regulated by cyclic di-GMP control of the bcsB switch. Production of bcsC is suggested to result in the formation of membrane channels for cellulose secretion while bcsD is believed to play a role in forming the cellulose into crystalline fibrils . Along with this are the downstream cmcax gene which encode for endo-beta-1,4glucanase that is secreted into the extracellular space and is believed to influence the assembly of cellulose ribbons when there is failure in arrangement by cleaving tangled chains of cellulose . The ccpAx product has proven to also be important in locating the bcs complex to the cell membrane and interacting with the bcsD subunits. Of course to provide the UDP-glucose necessary for the cellulose synthase to begin this process, the cell must have the common enzymes of glucose kinase to generate glucose-6-phosphate from glucose, phosphoglucomutase to isomerize glucose-6-phosphate to glucose-1-phospate, and UDP-glucose pyrophosphorylase to form UDP-glucose from UTP and glucose-1-phosphate.
Apart from the specialized enzymatic machinery needed to produce the bacterial cellulose into ribbons, the cell must also have the necessary means to generate the UDP-glucose from various carbon sources that are available in its environment. Figure 2 depicts a simplified biosynthesis pathway for bacterial cellulose in Acetobacter xylinum [7,8,9,10]. Different carbon sources may enter from the bacterial cellulose biosynthesis pathway in different ways as glucose, fructose, and galactose . Disaccharides like sucrose, lactose, or maltose and more complex sugars will of course first be converted to their respective monosaccharides via enzymatic hydrolysis, which are then fed directly into the cellulose biosynthesis pathway [8,11]. Different species of cellulose producing bacteria have been indicated to have preferential carbon sources, and the carbon source will itself affect the rate and yield of cellulose [11,12]. The rate of production in looking at the cellulose synthases function to polymerize UDP-glucose into β linked chains is also inherently linked to the regulation of this enzyme. As has been previously shown, cyclic-di-GMP is an allosteric activator of cellulose synthase implying that absence of the cyclic-di-GMP leads to inactive cellulose synthase . Indeed cyclic-di-GMP reversibly binds to cyclic-di-GMP binding protein (a membrane protein) and becomes unavailable; hence, controlling the equilibrium between bound and unbound cyclic-di-GMP which may be done via the intracellular potassium concentration may serve to push for enhanced bacterial cellulose production .
Figure 2 Bacterial cellulose biosynthesis pathway with metabolite denoted in black, metabolic pathways as black boxes, and enzymes involved in the respective reactions denoted in red.
As with most biomaterials of interest, it is not only the production of the cellulose but its hierarchical structure that lends itself to the resulting properties. Examination of the cell-directed assembly of cellulose has shown highly crystalline cellulose networks to result from secretion of the cellulose through the membrane embedded enzyme complexes discussed above. In contrast, it has been shown that transformation of only the bcsABC genes results in a non-crystalline material with no utility for manufacturing as a biomaterial . The organized self-assembly has been characterized in recent works revealing that van der Waals forces first facilitate the crystallization of cellulose chains into mini-sheets and hydrogen bonding of the mini-sheets into mini-crystals that emerge from the membrane bound pore complex as a single terminal complex (TC) subunit that are precisely spaced as to allow formation of crystalline cellulose I microfibrils . As illustrated in Figure 3, further organization of the microfibrils into bundles of microfibrils by sufficiently close proximity of neighboring TC subunits into a functional row of TCs and finally formation of ribbons have been shown that there is a significant hierarchical cellulose assembly process which is largely controlled by the bcsD driven arrangement of linear TC arrays and their orientation longitudinal to the axis of the cell . The highly organized nature of this resulting 3D bacterial cellulose network affords its superior strength to and stability for which it continues to find applications where limitations in its industrial use are predominantly attributed to its relatively low yield and higher cost than plant cellulose.
Production of the bacterial cellulose necessarily requires a culture environment, whether static or agitated bioreactor, for the cellulose producing strain of bacteria to be used along with considerations of the growth media (specifically the source of carbohydrates and other nutrients as well as the acidity). In general, the production rate of BC is directly dependent on the oxygen transfer coefficient of the culture where typically continuous cultures outperform batch cultures . Because this aerobic process occurs predominantly at the interface between the air and the medium for static cultures, the rate of BC production is relatively low in static batch cultures despite their yield of very uniform sheets of BC. Agitated fed-batch cultures with glucose supply and control over dissolved oxygen content have shown relative enhancement in yield up to 15g/L . The degree of agitation of cultures has benefits in generating homogenous distributions and enhancement of oxygenation but can also have drawback in requiring energy consumption and where issues of bacterial cellulose produced through agitation have weaker mechanical properties and are produced in small granules as opposed to larger pellicles depending on the degree of agitation . To reduce costs, air lift reactors have been found to require only 1/6th the energy of a stirred tank reactor for a given equivalent amount of BC production , where the use of air lift reactors become more beneficial as the degree of culture viscosity increases.
Figure 3 Hierarchical organization of bacterial cellulose (BC) formation into ribbons during biosynthesis of pellicles.
Aside from the culture method, the quantity of BC production is highly dependent on the composition of the media. Among the most common lab scale media is the Hestrin-Schramm formulation which has been reported to provide higher production than the Yamanaka media formulation . In comparison to Yamanaka formulations and Zhou formulations, the pH stability of the Hestrin-Schramm formulation was the most stable to buffering the gluconic acid by-product formation resulting from bacterial cellulose production . Because a decrease in the pH of the medium reduces the production of BC, studies have explored the use of additives including lignosulfonate to successfully inhibit gluconic acid oligomer formation. While lab scale media formulations are relatively expensive, to make bacterial cellulose economically feasible to a range of applications a significant amount of research has gone into identification of low-price culture medium carbon sources . For example, fruit juices have been examined for BC production by the strain G. persimmonis, and have shown muskmelon juice to provide over 8 grams of bacterial cellulose production per liter . Agricultural and industrial wastes have also been actively researched with cotton-based textile waste fabrics proving to be an interesting source of carbon feedstock after enzymatic treatment to produce 10.8 grams of BC per liter of culture . On par with this production level was the use of carbon-rich drainage water from rice wine production, resulting in BC production at yields of 10.38g/L . Surpassing this, the use of confectionery industry waste water as flour-rich hydrolysates allowed production of BC at 13g/L . It is worth noting that while the composition of the BC produced by these different means discussed above remains the same, the effects of their extent of polymerization, mechanical properties, water holding capacity, and degree of crystallinity are highly depending on the carbon sources as well as the production technique. Herein we’ll discuss more about the properties of bacterial cellulose as related to biomedical applications, how these properties and capabilities of bacterial cellulose have been altered and expanded in recent studies, and the biomedical products to which bacterial cellulose has been successfully implemented (Figure 4).
Figure 4 Bacterial pellicle formed at the air liquid interface of a static batch culture. Adapted with permission from Wang et al. 
2. Properties of Bacterial Cellulose
2.1 Water Content
One of the most important properties of BC is its ability to intake and retain water. This makes BC a suitable material for biomedical applications, as the extracellular matrix of most living tissues largely consist of water . BC can hold from 60 to 700 times its dry weight in water, depending on how it’s manufactured or modified . Most of this water is chemically bound to the BC fibrils, and is not free. Specifically, 10% of this water that is chemically bound exists in the form of hydration shells around the cellulose microfibrils comprising the BC structure . The abundant hydrogen bonds that are present in between the cellulose microfibrils are what helps grants BC with its water retention capacity. There are several ways to modify BC in order to effect its swelling and water retention capacities. Chemical modification is the most common way, with the use of composites including silk fibroin being one such method . Silk fibroin has been successfully proven to substantially improve the swelling properties of BC  as have incorporation of other component into BC including chitosan and montmorillonite [27,28]. These approaches largely make use of altering the pore size, pore volume and surface area of the BC, which in turn affects its water retention and intake.
2.2 Mechanical Properties
BC is also known to have good mechanical properties, which makes it a good material for tissue engineering. In its hydrated form, it has a Young’s modulus of 10 MPa and a stress test value (at failure) of 1 MPa . In contrast, sheets prepared from dried bacterial cellulose have been reported to have a Young’s modulus of over 15 GPa along the plane of the sheet and tensile strengths of 260 MPa . By altering the water content of the BC, it is thereby possible to greatly affect the membrane stiffness, namely where decreasing water content will lead to increasing membrane stiffness . Other results have shown BC in its hydrated form to have a similar Young’s modulus of 11 MPa with percent elongation at breaking point of 35%; while, the dry BC exhibited a Young’s modulus of 1.3 GPa, and an almost zero percent elongation at break . Aside from hydration state, researchers have found several other ways to modulate the mechanical properties of BC for instance through the use of composites. One such example is to the use of cross-linked BC/collagen mixtures, which showed an increase in tensile strength by 57.9% over BC alone . By incorporating a layer of hydroxyapatite on the BC via biomimetic mineralization, research have even created a biomaterial with mechanical properties of interest for bone tissue scaffolds . In another method, the incorporation of paraffin beads during growth of the bacterial cellulose served as a porogen which after removal of the paraffin resulted in an interconnected porous network that would mimic the mechanical properties of extracellular matrix and this resulting BC scaffold promoted substantial regeneration of the human auricle . Yet a different approach is to generate a composite in situ by co-culture of G. hansenii and E. coli that produce a mannose-rich exopolysaccharide which incorporates into the BC microfibril network. The Young’s modulus for such BC increased from 2.6 GPa to 4.8 GPa by incorporating the co-cultured process; while the stress at breaking point increased 80 MPa as compared to 45 MPa for the monocultured BC .
BC is a biopolymer that is composed of ultrafine nanofibers, which give it a natural pellicle (hydrogel-like) structure . Specifically, it is composed of linear strands of ultrafine nanofibers, which assemble to form microfibrils that generate tight bundles giving rise to compact ribbon-like structures that form an interwoven network providing the pellicle . The structure is comprised of β-(1, 4)-linked D-glucose residues, with strong hydrogen bonds between the adjacent cellulose nanofibers [39,40]. This structure allows BC to have numerous hydrogen bonds, which is allows it to retain water and also form its hierarchical structure through inter and intra-molecular with neighboring hydroxyl groups . Because this nanofibrous network resembles aspects of the extracellular matrix of some tissues. This structure in combination with the ability of BC to be easily modified in situ and ex situ opens the potential for this material serving as an important biomaterial for wound healing and tissue regeneration applications. One such example is the use of silver nanoparticles (Ag-NPs), which form a robust BC-AgNP hybrid with excellent antimicrobial properties, making it ideal for wound healing . Another way is to utilize polyethylene glycol (PEG) to create scaffolds with improved viscoelasticity . There are also methods that can be used to template the BC to alter its surface as well as overall architecture. Agarose film scaffolds with honey-comb patterned grooves can be used to guide G. xylinus to produce honey-comb patterned BC . Polydimethylsiloxane (PDMS) substrates can be used to orientate BC fibers by controlling the ridged morphology of the PDMS . It is also possible to create microporous BC scaffolds, via the use of paraffin wax microspheres, to create ideal scaffolds (pore size: 300-500 μm) for bone regeneration , among other purposes.
Pore size is critical when developing scaffolds for tissue engineering. Depending on the type of tissue and the function of the scaffold, the pore size will vary anywhere from 100 nm (e.g. extracellular matrix) to 100 μm (e.g. neovascularization) . Figure 5 shows an example of the structure and porosity of bacterial cellulose. A good example of the effect of pore size was shown when researchers constructed a microporous BC scaffold with good interconnectivity of 300-500 μm size pores resulting in improved cell penetration and seeding within the scaffold . Other techniques that can be used to create porous BC scaffolds include freeze-drying BC-hydrocolloid mixtures, laser patterning, and 3D-printing [46,47,48].
Figure 5 Three dimensional network structure of bacterial cellulose showing fiber and pore structures. Adapted with permissions from Halib et al. 
Because bacteria like Komagataeibacter xylinus and G. hansenii, are used to produce bacterial cellulose in laboratories and industry, the lipopolysaccharide component of their outer membrane presents an endotoxin, which if not removed, would cause biocompatibility issues . One way to remove endotoxins and improve biocompatibility is to wash the bacterial cellulose (BC) with sodium hydroxide solution. Avila et al. carried out such a set of experiments where they washed BC hydrogels with sodium hydroxide, thereby reducing the endotoxin content from 2390 EU/ml to 0.1 EU/ml, thereby improving the biocompatibility considerably . In conjunction with this, an additional approach to modify the BC to even further improve biocompatibility is via in situ carboxymethylation . Such an approach can improve the biocompatibility by reducing the inflammatory response but even as a sterilized material BC has been shown to have good hemocompatibility and cytocompatibility .
Bacterial cellulose (BC) is highly resistant to degradation and can withstand high thermal, mechanical and chemical stress . This interesting property could inherently limit the in vivo use of this material in certain biomedical applications, as an ideal implantable tissue scaffold should degrade while it facilitates tissue growth. Nonetheless, cellulolytic enzymes can be used to degrade the material and this is often employed as are a number of other chemical processing strategies for native BC. One such group has shown that incorporating graphene oxide/hydroxyapatite with BC could help to create an osteoconductive scaffolds that have claimed to improve the biodegradability . A second group used sodium periodate and hyaluronic acid along with BC to synthesize a scaffolding material with improved degradability for bone tissue engineering applications . In general the in vivo use degradation approaches for bacterial cellulose are limited. BC composites with chitin have nonetheless been made which are enzymatically cleavable by metabolic engineering , and controlled oxidation of BC sheets that have been previously gamma irradiated offer a material that is more bioresorbable . In contrast, because microorganisms in the environment can readily cleave the beta glycosidic bonds of bacterial cellulose, BC has the ability to undergo relatively rapid biodegradation making it an attractive polymer with a low environmental footprint.
3. Modification of Bacterial Cellulose
3.1 In Situ Modification Techniques
Modification of bacterial cellulose can be conducted during the process of the bacterial cell culture (in situ modification) or in contrast be performed after the cellulose microfibers have been produced (ex situ modification). While ex situ modification are carried out after BC has been formed using chemical and physical methods, in situ modification often uses modulation of culture conditions include material additives or even simply changing the carbon source to ultimately impact the bacterial cellulose as it is being produced. Such modifications can be critical for tissue engineering application of bacterial cellulose for wound dressing and bone regeneration . Here we will discuss more about existing strategies to modulating the conditions for in situ modification including tailoring the culture conditions and degradation rates to alter porosity, fiber density, and crystallinity as well as incorporating functional groups through methods that modify the chemical structure.
During in situ modification, the mechanical properties as well as physiochemical and structural properties of the material can be altered by incorporating materials during the biosynthesis and release of the fibers from the bacteria into cellulose networks. Ultimately this affords a composite biomaterial with distinct properties. Collagen has been used as an in situ additive material to modify the color, thickness, roughness, stiffness, porosity, and crystallinity . Specifically, the by incorporating collagen into the nutrient medium during growth of BC, the porosity increased and the XRD patterns showed not to be a simple mixture but rather resulted in a new crystalline structure of the B fibrils. Other in situ methods include the use of polystyrene to control the crystallinity and porosity and in doing so the mechanical property and swelling behavior . By utilizing polystyrene pin templates with diameters of 60-300um during the static culture of BC, the researchers could control the pore formation after BC biosynthesis and removal of the template. Using this approach the pores exhibited no border failures which would otherwise facilitate crack propagation. The use of such microporous membranes has particular interest in tissue repair applications given the high rates of oxygenation.
The in situ modification of BC with mineral based components for the formation of biomaterials toward bone regeneration applications has also been widely employed. The use of magnesium calcite from the skeleton of sand dollars (Clypeaster subdepressus) has been used as an additive material, where the pore geometry of the sand dollar could be covered with the bacterial cellulose microfibril network during growth. The BC coated pores were particularly fitting for bone regeneration as the size is sufficient for cell migration and vascularization along with the BC surface coating providing an amenable surface for cell adhesion . Hydroxyapatite which is the natural mineral component of bone comprised of calcium, phosphorous, oxygen, and hydrogen has also been utilized for in situ growth of BC to affect the porosity and mechanical properties . By sequential incubating the BC in solutions of CaCl2 and followed by Na2HPO4, the BC nanocomposites could be prepared to show up to 50% of the total composite weight was the mineral phase of hydroxyapatite. The material has even been evaluated in noncritical bone defects in rat tibiae for up to 16 weeks where in vivo tests showed low inflammatory response. After 4 weeks defects were observed to be filled in by new bone tissue revealing these BC-HA membranes as effective for bone regeneration. Other groups have also explored the benefits of hydroxyapatite integrated bacterial cellulose for a variety of medical material implementations . The choice of the optimum conditions for the creation of the bone tissue precursor and the method of how the combined aggregation of hydroxyapatite and bacterial cellulose suspensions should be prepared is important. Techniques include varying the ratio of components during combined aggregation of hydroxyapatite and bacterial cellulose suspensions, introducing the hydroxyapatite suspension in the growth medium during BC biosynthesis, and controlled synthesis of the hydroxyapatite from precursor ions dispersed in the medium during bacterial cellulose formation. Depending on these different methods of formation of the hydroxyapatite/BC composite the resulting nanotexture and alignment of the hydroxyapatite relative to the BC were found to be altered . For example, the use of hydroxyapatite nanoparticles prepared from a wet chemical precipitation method from aqueous calcium nitrate and di-ammonium phosphate were pre-synthesized before incorporating into the culture media during biosynthesis of BC nanofibrils . From the compositional analysis of the final material, the amount of the hydroxyapatite mineral phase made up 23.7% of the total weight of the nanocomposite. The resulting composites show high cell viability when used for culture of human embryonic kidney cells. Related research has also shown that the formation and morphology of hydroxyapatite crystals is highly dependent on the extent of co-incubation time during BC scaffolds growth .
Modulating the porosity of BC scaffolding for bone regeneration applications has also implemented the use of paraffin beads as sacrificial porogens. Such processes are discussed elsewhere in this review, but findings in developing chondrogenic scaffolds for auricular and nasal reconstruction revealed this enhance porosity of the BC provided in situ facilitated 150-500um pore sizes that were capable of uptake, adhesion, and differentiation of chondrocytes, where after 2 and 3 weeks were found to produce cartilaginous matrix proteins of aggrecan and collagen type II, respectively . Similar approaches have shown that the microporous BC generated with the use of paraffin porogens could yield a scaffold in which seeded osteoprogenitor cells could distribute themselves into clusters and form dense mineral deposits that could serve promising in bone tissue engineering applications . The use of paraffin templated BC scaffolds have been utilized in other tissue engineering applications as well including reconstruction of urinary conduits which proved successful after seeding with human stem cells to differentiate into urothelial and smooth muscle cells and formation of a multilayered urothelium .
In looking to other in situ methods for BC modification, adding potato starch into the media during culture was found to result in increased porosity of the structure (with 40um pore sizes) . When the concentration was greater than one percent, a local compact orientation in the surface morphology was observed while the lower layer remained transparent. Interestingly, when used in a cell culture environment, the ingrowth of cells increased for scaffolds with increased starch content. Implantation of the BS composite with potato starch showed that the lower layer was capable of neovascularization. Bacterial cellulose has even been used in a wound dressing formulation when coated on cotton gauze samples during its biosynthesis in a static culture medium . After sterilization, the composite had high water absorbency and large capability of wicking which are important characteristics of wound dressings. Further in situ modification methods during biosynthesis of BC utilized 30% aloe gel supplemented to the culture media to result in a material with higher water absorption capacity and water vapor permeability than native BC. This may be attributable to the change in the average pore size of the aloe supplemented BC as compared to the unmodified BC . Incorporating BC nanocrystalline chitin powder has also been carried out, where the chitin had undergone partial deacetylation as to provide antibacterial activity to the composite . The nanocomposites were prepared in situ by the addition of deacetylated chitin nanofibers to the culture medium during BC biosynthesis and were also prepared ex situ by post-modified mixing chitin with disintegrated BC in an aqueous suspension. Interestingly, they found the bactericidal activity of the nanocomposites to increase as a function of deacetylated chitin content. Further uses of natural in situ composites with BC included chitosan/heparin co-synthesis mixtures which are being developed for vascular tissue engineering applications as to prevent the formation of blood clots . Researchers have fabricated these structures into tubular shapes as scaffolds and have confirmed the presence of chitosan and heparin on the surface of the BC fibrils comprising the tubes but have also shown the composites to facilitate ingrowth and proliferation after cell seeding experiments.
For the prospect of using bacterial cellulose for target-specific biofiltration applications, researchers have looked to utilizing BC as a support material for immobilize antibodies capable of target selecting reception. Specifically, anti-human serum albumin affibodies have been covalently immobilized onto specially modified BC which have been carboxylated. By incorporating carboxymethylcellulose into the BC culture media, the resulting BC fibers grown in situ exhibited carboxylation to allow a chemical functional handle for covalent coupling by EDC-NHS cross-linking with amines present on the affibodies . As an alternative to in situ BC carboxylation, this could also be carried out by alkaline TEMPO oxidation. The resulting material was formed into a tubular network that was used as a potential biological filter.
3.2 Ex Situ Modification Techniques
Among the ex situ modification techniques which are implicitly occurring after the initial biosynthesis of the BC pellicle, there are those which proceed by physical approaches vs. chemical methods. Here we will first discuss several of these approaches to BC modification ex situ. In one approach, the use of electrostatic interaction between the electronegative bacterial cellulose and the positive nitrogen cations of nephelauxetic gel . The composite has been found to be functional at 0.25% of gel for generating a scaffold with good adhesion and proliferation in cultures of NIH 3T3fibroblasts. Examining other techniques based on non-covalent surface modification of BC membranes, researchers have exploited a protein unit referred to as carbohydrate binding module CBM3 capable of binding to the bacterial cellulose . In a specific implementation of this technique, research have make a fusion peptide of the laminin mimetic domain IKVAV onto the CBM3 domain for binding and coating the bacterial cellulose. Utilizing this approach, it has been shown that PC12 and mesenchymal stem cells could better adhere to the IKVAV displaying BC membranes and more importantly facilitated the cells release of NGF to show potential in neuronal tissue engineering applications. Other peptides of interest such as the integrin binding domain RGD have also been functionalized onto bacterial cellulose by grafting to promote cell adhesion . By also incorporating within the scaffolds enriched antimicrobial agents like gentamicin the functionalized scaffolds proved bactericidal activity while allowing human fibroblast growth. Osteoinductive BC-based materials have also been developed using a multi-component composite . By utilizing graphene oxide, hydroxyapatite, and BC matrix, a compact scaffold network had been developed showing biocompatibility and osteoinductive potential as well as promoting cell adhesion and cell growth.
Ex situ physical composites of bacterial cellulose with chitosan have been recently explored for instance through a one-step solution based strategy or infusion of chitosan into the BC matrix . In general, post-processing techniques such as freeze-drying are used to enhance porosity and retain the interconnected network of the bacterial cellulose. BC sponges with characteristic shapes as determined by the shape of appropriate molds were used during emulsion freeze drying . The ability of such sponges to provide a scaffold for cell proliferation was demonstrated with mesenchymal stem cell that penetrated 150um into the scaffolds after 7 days of culture. Othersponge like BC derived scaffolds have been produced using with a recent example being a nanocomposite with silk fibroin . Like the pure BC sponges, BC/silk-fibroin composites proved to have good interconnections in the porous network but through the use of 50% silk fibroin provided biocompatibility and cell adhesion. While structural proteins like silk fibroin and collagen have been widely used as an ex situ physical mixture with B, there have also been prior reports of the use of signaling proteins being incorporated into BC scaffolds. An interesting example is that of the use of a bacterial cellulose scaffold loaded with BMP-2 (bone morphogenetic protein 2) for osteogenesis . In using such a biomaterial for implantation in mice, the scaffold could effectively increase the local concentration of signaling molecules to induce differentiation of fibroblasts into osteoblasts where the osteogenic activity was found to be related to the amount of BMP-2 coating the BC surface. A much higher amount of bone formation was observed in vivo for the BMP-2 coated BC in comparison to the BC alone. This exemplifies not only the prospect of using BC as a component for tissue engineering but also by virtue of the loading capacity and release we can see that BC may serve as a good localized delivery system. In another example of the delivery capabilities of BC, a zinc oxide nanocomposite preparation with wet BC pellicles was produced to provide antimicrobial activity. The researchers confirmed good dispersion and release properties of the ZnO nanoparticles from the BC matrix . Tailoring such properties as controlled release could provide a means for the delivery of antimicrobial agents. As the BC scaffolds alone are not known for being antibacterial, loading of the BC structures and films with antimicrobials such as benzalkonium chloride can be achieved by simple soaking in solution . Such a simple preparation procedure could then be used to prepare a dry film for use as a wound dressing with sustained antimicrobial activity for at least 24 hours and is adaptable to other antimicrobial agents including antibiotics, silver, and surfactants.
In looking at not only the direct modification strategies but also the prospects of manipulating the physical properties by the processing techniques used, we see that various techniques exists which can be implemented to fabricated bacterial cellulose including gas forming, 3D printing, phase separation approaches, as well as the use of salt leaching or paraffin embedding to alter porosity. For example a recent fabrication method for generating bacterial cellulose fibers with unprecedented tensile strengths of 826 MPa and Young’s modulus of 65.7 GPa have been successfully achieved by a wet‐drawing and twisting process (Figure 6) . Other important works have shown that changes in the pore size and porosity can plays a significant role in the applications of the bacterial cellulose. There continue to be ongoing efforts in developing more effective methods for the fabrication of bacterial cellulose [81,82]. The rationale in developing novel methods by which to vary the pore size and porosity is the inherent result this has in changing the final properties and applications of the bacterial cellulose. Among the processing methods to modulate the porosity, salt leaching is a very common method in use with bacterial cellulose [83,84,85]. In general, this method utilizes salt crystals placed within the material to serve as a mold for future pores where later after biosynthesis of the bacterial cellulose the material can then be hardened and the salt removed by dissolution with the appropriate solvent. A major advantage of this process is that the final pores can be easily formed and are of a tunable desired size dependent on the different salts particle size. In a related and widely implemented approach, paraffin wax microspheres are used as sacrificial porogens that are used to take up space during biosynthesis or hardening of the BC material and then removed to generated an empty space of the desired pore size [44,86].
Figure 6 Processing of bacterial cellulose by a wet-drawing and twisting approach provided high strength macrofibers. Adapted with permission from Wang et al. 
4. Medical Application and Marketed Products from Bacterial Cellulose
Bacterial cellulose has been considered a fitting biomaterial for a number of medical application (Table 1) including wound healing, tissue regeneration, and drug delivery among others due to its biocompatibility, low cytotoxicity and versatile modification options . In addition to these properties, novel formulations, implementations, and modification of bacterial cellulose have been identified which has yielded a substantial amount of new intellectual property (Table 2) In recent years, utilizing modified BC for surface wound dressing has gained significant attention as afforded by advancements in both bioengineering and material science. For example, the use of coniferyl alcohol as a composite hydrogel with BC has helped in providing substantial anti-microbial properties to BC where this area was previously problematic. The consistent release of coniferyl alcohol can maintain up to 72 hours of antimicrobial activity, along with the high porous nature of BC itself, serves as promising alternative to chronic wound healing . The incorporation of BC with vaccarin, a flavonoid glycoside extracted from vaccaria seed, has been used in wound treatment experiments to promote vascularization and has shown the ability to retain up to 73.6% cell viability after 14 days incubation. In this study, the skin tissue healed with minimal complications and was found to promote cell proliferation during wound healing . Suspension of BC with TiO2 solution to enhance its anti-microbial capability has also been used to accelerate wound healing rate in vivo  as has resveratrol which has been shown to promote anti-inflammatory responses and enhance revascularization when incorporated with BC proving to accelerate skin regeneration . The integration of stem cells with combinations of BC and acrylic acid composite scaffolds have proven to provide a moderately successful solution for full thickness dermal tissue wound healing .
Table 1 Biomedical applications of bacterial cellulose and key features in choosing that material.
Table 2 Intellectual property filings for bacterial cellulose technologies with medical applications.
Though BC application weighed heavily on their capabilities for wound dressing applications, there are a number of findings for its use in a variety of medical implementations. Due to the high porosity, the ability for BC to promote revascularization and angiogenesis on targeted sites has been exploited. An implanted BC scaffold on mice (Figure 7) has for instance shown to harbor favorable condition for angiogenesis as well as trigger no inflammatory response, and from these implantation studies, the onset of angiogenesis was observed as soon as 1 week after implantation . In a different approach, heparinized BC provided a means for controlled release of VEGF to promote vascularization, and in recent studies have shown detectible angiogenesis in vivo as soon as 2 weeks after implantation with blood vessel density ranging from 1.35% to 2.61% . In a more direct approach, artificial blood vessels have been fabricated from BC and have shown promising results in animal studies where artificial vessels possessed acceptable in vitro hemolytic rates (0.097%-0.42%) with no apparent triggering of immune cells providing a strong indication of its biocompatibility . Other than artificial vessels, BC has also contributed to the advancement of ophthalmic tissue regeneration. Research in examining the optical properties of BC have shown through thickness-controlled studies of BC cellulose that respectable level of transparency of 88% to 95% . A specially designed BC contact lens has even been developed to serve as a bandage for corneal damage . In the case of osteo-regenerative properties, BC scaffolds have shown comparable cell proliferation rates to collagen scaffolds but were capable of sustaining a higher porosity . The use of bacterial cellulose with increasing pore sizes has been shown to enhance the cell proliferation (Figure 8) . Nanoskin®, a bacterial cellulose skin graft, has also recently been demonstrated as a means for regeneration of large scale dermal tissue. During implantation studies conducted on guinea pigs, the deep dermal defects showed full recover without scar formation up to a 2×4cm area . The combination of BC and silk have also been implemented as skin grafts showing less inflammatory response compared to other marketed skin graft products as well as inducing relative improvements in comfort based on test subject surveys .
Figure 7 Surgical procedure for suturing of Nanoskin® in the caudal position on a wound. Reproduced with permissions from Kaminagakura et al. 
Figure 8 Bacterial Cellulose derived artificial extracellular scaffold for cartilage formation. Reproduced with permissions from Akaraonye et al. 
In addition to its extensive applications in wound healing and tissue regeneration, BC has shown to possess properties amenable for implementation in drug delivery technologies. In one example, a BC has been utilized in a nanocomposite with graphene oxide to enhance the carrying capacity of ibuprofen and has shown its sustained release under even highly acidic environments . Halogenation of bacterial cellulose capsules have also been utilized, where development through nanostabilized Pickering emulsions have revealed the BC capsules to have prolonged release profiles of therapeutic drugs . Another promising route for drug-delivery systems is through the use of BC undergoing gelatin hybridization which allows for significant swelling of up to 500% for drug encapsulation .
While there are many properties of bacterial cellulose that are attractive from the perspective of biomedical applications, one critically unfavorable element is the poor antibacterial and antifungal nature of native bacterial cellulose. Fortunately, there has been a number of methodologies including surface functionalization, embedding of nanoparticles, and impregnation with plant extracts which have proven successful in enhancing the antimicrobial properties of BC. Among the functionalization strategies, researchers have shown sialylation treatment of BC to generate amine functional groups could result in significantly reduced growth of S. aureus (a gram positive) but not E. coli (gram negative) . Interestingly, a similar sialylation treatment of BC to generate amine functionalized BC previously showed a greater increase in antibacterial activity with proportion to the extent of amine grafting which resulted in reduced bacterial adhesion for E. coli, S. aureus, B. subtilis, and even fungal C. albicans . This reduced adhesion was believed to be attributed to the increased hydrophobicity of the surface thereby decreasing the surface wettability to enhance antimicrobial performance by minimizing water contact; however, additional rationale for the antimicrobial was attributed to the positively charged surface interacting strongly with the negatively charged cell membranes leading to cell disruption but this did not seem to have the same effect on human HEK293 cells. In looking at bacterial cellulose composites with nanoparticles, groups have used CuO nanoparticle formation in a BC films by ex situ (ultrasonication of CuO dispersion with pellicle) and in situ (precipitation of CuO nanoparticles) synthesis methods and have shown the ex situ nanoparticle composite BC method to produce a more effective bactericidal product than the in situ method in reducing S. aureus and E. coli . The related approaches were used by one of the same researchers from the aforementioned to form a BC pellicle with MgO nanoparticles, but this MgO composite with BC was found to have much higher bactericidal effects that for the case with the CuO nanoparticles . Cellulose composites with silver nanoparticles have also been examined for their antibacterial effects and have shown that silver nanoparticles less than 20nm in diameter showed better zones of inhibition than composites with silver nanoparticles greater than 20nm in diameter . Bacterial cellulose has also been impregnated with ZnO nanoparticles (grown to 70-100nm in diameter) by immersing the BC pellicle in zinc nitrate solution followed by a NaOH solution . The release of Zn2+ ions from the BC films was believed to the mechanism for the antibacterial activity which showed reduced growth of S. aureus and B. subtilis gram positive bacteria as well as E. coli and P. aeruginosa gram negative bacteria. As ion release can be a critical factor for producing antibacterial effect, BC composited have also been formed by immersion in suspensions of montmorillonite (a hydrous alumina-silicate clay) modified to containing Cu, Na, or Ca . In this case, the zones of inhibition for the montmorillonite-BC composites with release of Cu were found to inhibit growth of E. coli and S. aureus, while release of Na inhibited MRSA, S. aureus, and E. coli, and the release of Ca inhibited E.coli, S. aureus, and C. fruendii. More complex composites of graphene oxide with TiO2 nanoparticles have also been filled into porous BC and when irradiated with near UV (365nm) was able to generate reactive oxygen species to significantly reduce the viability of S. aureus . Nanoparticle formulation of natural compounds like curcumin have also been used successfully for improving the antimicrobial properties of bacterial cellulose as confirmed by live/dead cell staining assays . Natural antibacterial plant extracts like silymarin isolated from milk thistle have been used to make nanoparticles with zein, and immersing BC hydrogels in the silymarin-zein nanoparticle suspensions has been used to produce films which inhibited growth E. coli, S. aureus, and P. aeruginosa though more so for the air dried films as compared to the lyophilized films . Other extracts, like scrophulariastriata boiss extract as well as mulberry leaf acid hydrolysate have been added to the growth media during production of BC films with the latter showing reduction in the growth of both E. coli and S. aureus which they attributed to antibacterial flavonoids of the mulberry leaf extract that were embedded in the bacterial cellulose for sustained release [141,142].
Bacterial cellulose hydrogel composites containing lignin dehydrogenative polymer of coniferyl alcohol have shown prolonged periods of release (72 hours) to provide antibacterial activity against P. aeruginosa, S. aureus, Serratia sp., L. monocytogenes, and S. typhimurium . Other natural products like thymol (the monoterpenoid phenol derivative of cymene extracted from thyme) when used for soaking of BC films allowed absorption and when used to examine for antibacterial effect resulted in release of the thymol to facilitated large zones of inhibition against S. aureus, E. coli, P. aeruginosa, and K. penumoniae . Finally, we would like to make mention of a bacterial cellulose composite made with Brazilian propolis extract. In using the propolis with C. cassia oil, the researchers were able to generate a self-microemulsifying formation that when loaded into BC hydrogel showed significant zones of inhibition for S. aureus, S. aureus methicillin resistant, S. epidermidis, K. pneumonia, P. aeruginosa, and E. coli .
5. Conclusions and Outlook
BC manufacturing happens at a smaller scale than conventional plant cellulose but holds higher interest from a medical and ecological perspective due to providing a non-toxic, biocompatible, fully biodegradable, and renewable material source. While significant advances have been made in BC processing, identifying low cost substrates, and development of efficient BC producing bacterial strains, there remains to be commercial scale bioprocessing of BC that is economically feasible to compete with traditional cellulose for low cost applications as opposed to higher value biomedical applications. Room for future investigations to lower the costs of BC continue to exist in finding culturing conditions that may utilize waste materials for feed-stock. For overcoming areas of difficulty for clinical progress, future work in providing better quality control over the porosity and consistency throughout the material, where the culture environment creates non-uniformities not only batch to batch but even for internal vs external regions of the same batch. The issue of requiring consistency for biomedical applications can be complicated from the property of bacterial cellulose to be an easily tunable material and can inherently be affected by production processes intended for enhancing yield or reducing costs. In addition to the challenges of establishment of consistent manufacturing for bringing BC based biomedical products to market, there is also the important aspects of assuring non-inferiority over current technologies. The considerable efforts to improve its processing ability for construction of tailored devices and improving its antimicrobial properties have similarly pushed forward the feasibility of new and exciting biomedical products. A promising future for new biomedical technologies based on BC will certainly stem from their adaptability in bestowing new functions through their easy of modification and capability of formulating composites. Perhaps with this natural fibrous network capable of facilitating cell adhesion, we will continue to see improvements in synthetic composites to provide antimicrobial properties and further manipulate the porosity to satisfy control of cell uptake while providing a barrier with high water content for wound healing and skin regeneration applications. While commercial products based on BC are already available in these areas, additional progress in providing encapsulation of drugs are expected to expand its use to practical transdermal and drug delivery applications. It is anticipated that as continued interest in the genetic engineering of these BC producing organisms finds ways for incorporating additional monosaccharides as we have recently seen, we’ll continue to find new biomedical application areas than originally imagined.
All authors contributed to this work by research of relevant literature, writing, figure preparation, and editing of this work.
The authors have declared that no competing interests exist.
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