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Application of Bacterial Cellulose in Skin and Bone Tissue Engineering

Mujuan Pang, Yinghong Huang, Fansu Meng, Yong Zhuang, Hui Liu,
Manling Du, Qianqian Ma, Qi Wang, Zhen Chen, Lianyu Chen, Tiange Cai,
Yu Cai

PII: S0014-3057(19)31458-2
DOI: https://doi.org/10.1016/j.eurpolymj.2019.109365
Reference: EPJ 109365

To appear in: European Polymer Journal

Received Date: 19 July 2019

Revised Date: 4 November 2019
Accepted Date: 9 November 2019

Please cite this article as: Pang, M., Huang, Y., Meng, F., Zhuang, Y., Liu, H., Du, M., Ma, Q., Wang, Q., Chen,
Z., Chen, L., Cai, T., Cai, Y., Application of Bacterial Cellulose in Skin and Bone Tissue Engineering, European
Polymer Journal (2019), doi: https://doi.org/10.1016/j.eurpolymj.2019.109365

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 Application of Bacterial Cellulose in Skin and Bone

Tissue Engineering
Mujuan Pang 1*, Yinghong Huang 2*, Fansu Meng 3*, Yong Zhuang 1, Hui Liu 1,
Manling Du 1, Qianqian Ma 1, Qi Wang 4, Zhen Chen5, 6, Lianyu Chen5, 6, Tiange
Cai 7†, Yu Cai 1,8,9†
1 College of Pharmacy, Jinan University, Guangzhou 510632, China;

2 Guangxi International Zhuang Medicine Hospital, Nanning 530201, China;

3 Zhongshan Hospital of Traditional Chinese Medicine Affiliated to Guangzhou University of

TCM, Zhongshan, Guangdong 528400, China;

4 Guangzhou Jiayuan Pharmaceutical Technology Co., Ltd., Guangzhou 510663, China;

5 Department of Integrative Oncology, Cancer Center, Fudan University, Shanghai 200433, China

6 Department of Integrative Oncology, Shanghai Medical College, Fudan University, Shanghai

200433, China.

7 College of Life Sciences, Liaoning University, Shenyang 110036, China;

8 Cancer Research Institute of Jinan University, Guangzhou 510632, China;

9 International Cooperative Laboratory of Traditional Chinese Medicine Modernization and

Innovative Drug Development of Chinese Ministry of Education (MOE), School of Pharmacy,

Jinan University, Guangzhou 510632, China.

†Correspondence: Yu Cai:caiyu8@sohu.com;

Tiange Cai: caitiange@163.com.

* The authors contribute equally to the work.

Abstract
Bacterial cellulose (BC) produced by aerobic bacteria has the advantages of high
purity, high porosity, strong water absorption, high water holding capacity, high
mechanical strength and good biocompatibility. In this review, we briefly introduced
the characteristics of BC and the production and modification methods of BC, and
focused on the application of BC in skin tissue and bone tissue engineering. At the same
time, we summarized the role of BC as a wound dressing and bone repair material in
wound healing and bone repair, and prospects for the application of BC as a wound
dressing and bone repair material.
Graphical abstract

Keywords
Bacterial cellulose; Skin wound healing; BC-based wound dressing; Bone tissue
engineering; Bone repair material
1. Introduce
1.1 Bacterial cellulose
At present, energy shortages and environmental pollution problems have become an
urgent problem to be solved. In order to meet the increasing demand for renewable
resources and environmentally compatible materials [1], the development of
biopolymers with abundance, renewability, low cost, environmental protection and
biodegradability has become a hot spot in materials science [2]. Natural biopolymers,
such as collagen, hyaluronic acid, gelatin, chitosan and cellulose, have been widely
used in medical research due to their similar properties to natural tissues. As the most
abundant polymer on the earth, cellulose can be produced by plants, fungi, algae and
bacteria. The annual output of cellulose pulp alone can reach 1014 tons [2, 3]. The
cellulose produced by bacteria was discovered in 1886 by British scientist A.J. Brown
when he was cultured Acetobacter under static conditions [4]. In order to distinguish it
from plant-derived cellulose, it is called microbial cellulose or bacterial cellulose.
Acetobacter is the earliest strain used to synthesize BC. Later, it was found that BC
can also be produced in bacteria such as Agrobacterium, Pseudomonas, Rhizobium, and
Escherichia [5]. Among them, Acetobacter xylinus (synonyms Gluconacetobacter
xylinus, Komagataeibacter xylinus) is currently considered to be the strain with the
strongest ability to synthesize cellulose. The biosynthesis of bacterial cellulose is the
polymerization of UDP-glucose into α-1, 4-glucan chains by the interaction of various
enzymes [6], and then the microfibers are formed by stripping the pores of the bacterial
cells in a strip form.There are three hydroxyl groups on each glucosyl ring in BC, so
the microfibers self-assemble into a fiber bundle having a diameter of 10-100 nm due
to the large number of hydrogen bonds [7], and then form a three-dimensional nanofiber
network with a large number of pores [8] to obtain a native BC.
1.2 Preparation of bacterial cellulose
The methods of BC culture can be roughly divided into static culture, agitation/shake
culture and bioreactor [9]. BC film is produced at the gas-liquid interface between
medium and air in static culture due to BC produced by aerobic bacteria. Therefore, the
overall yield of BC film in static culture is directly related to the surface area of the gas-
liquid interface [10]. The preparation and purification methods of static culture are
simple, namely BC gel sheets harvested at the gas-liquid interface were purified with
hot water and sodium hydroxide and then washed with a large amount of water to reach
a neutral pH to obtain pure BC film. But the high cost and the low productivity are two
main problems in static culture owing to its limited air contact area [11]. To improve
productivity, Agitation/shake culture has been developed to increase the rate of oxygen
transport in the medium [12]. However, it has been found that the BC produced by
agitation culture has a lower degree of polymerization, crystallinity and mechanical
properties versus static culture [13], and it is easy to produce a Cel mutant, resulting in
a decreased yield [14]. Therefore, agitation culture did not significantly increase the
productivity of BC [14, 15]. In order to overcome the drawbacks of static and agitated
cultures and achieve industrial production of BC, bioreactor culture has been studied to
increase the yield of BC. Although bioreactor culture has improved the yield of BC to
a certain extent, a bioreactor culture method that can simultaneously meet high yield
and good performance of BC has not appeared [9]. Therefore, even though the time of
static culture is long and its yield is low, it is still the most common technique for
preparing BC on a laboratory scale because it can produce BC with good property.
In addition, due to the importance of strains and culture conditions on BC yield, high-
yield mutants found by genetic techniques and optimized culture conditions are the
main research directions for increasing BC yield [10]. In order to reduce production
costs, some reports suggest that low-cost industrial by-products or industrial waste [16]
such as tobacco waste extract [17], agricultural by-products [18], toxic aromatic
hydrocarbons [19], sugar beet molasses and cheese whey [20], etc. can be used as
carbon or nitrogen sources, which possesses good prospects for the production of BC.
Furthermore, some studies have shown that the addition of certain additives to the
fermentation medium, such as ethanol [21], agar [22], sodium alginate [23], not only
increase BC production, but also have certain changes in BC crystallinity, mechanical
strength, particle size and so on. Even so, BC's production costs and productivity are
still difficult to overcome.
1.3 Characteristics of bacterial cellulose
Compared with plant cellulose, bacterial cellulose possesses more excellent
properties. For example, bacterial cellulose has high purity without lignin,
hemicellulose and other impurities. Therefore, its purification process is simple.
Moreover, it has almost no rejection and inflammatory reaction, and possesses excellent
biocompatibility, which is the main reason for its research in biomedicine [24]. BC has
a high degree of polymerization, such as the polymerization degree of Acetobacter
xylinus cellulose can be up to 16000. In addition, the crystallinity of BC is high, up to
95%, and it was reported that the Young's modulus of BC monofilament can be up to
114 GPa [8]. Thus, based on its excellent mechanical properties, it has been developed
into audio speaker diaphragm [25], food packaging film [26], shielding film [27], and
treating tympanic membrane perforation [28, 29] and so on. High porosity and three-
dimensional structure are the biggest superiority of BC; therefore, it is used in the fields
of sensing materials [30] and photocatalysts [31] due to its high porosity and large
specific surface area. Meanwhile, it is widely used in the cell culture [32] and wound
dressing [33] because of its favorable permeability, hygroscopicity, water holding
capacity and flexibility. In addition, BC possesses good controllability and
biodegradability during synthesis, which makes it favored in the medical field and
widely used in tissue engineering scaffolds [34], controlled release drugs [35] and other
research. Because of these characteristics, BC can be widely used in food, cosmetics
[36], capacitors [37], biomedical and tissue engineering, functional materials and other
fields. As shown in Figure 1, the number of BC publications has increased since 2000.
1200
Publication

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of

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Number

400 337
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18 Year of Publication

Figure 1. Annual number of publications on BC since 2000 (Web of Science™ search system,

search term “bacterial cellulose”)

1.4 Modification and application of bacterial cellulose

Although native BC possesses remarkable intrinsic properties, in order to fully
exploit the potential of BC and meet the requirements of different researches, various
methods to modify the physicochemical properties of BC have been used to improve
the added value of BC, which has been the focus of BC research [38]. For example,
changes in porosity, crystallinity, chemical structure and function. Depending on the
order of the modification, the BC modification can be divided into in situ modification
methods and ex situ modification methods [39]. In situ modification means that the
modification of BC occurs during the synthesis of BC by changing bacterial culture
conditions or adding other materials to the medium. Komagataeibacter
sucrofermentans is used to generate functional BC having unnatural characteristic
fluorescence with 6-carboxyfluorescein-modified glucose as a substrate [40]. Similarly,
BC films with antibacterial activity were produced from the medium containing the
hydrolyzed products of mulberry acid [41]. The addition of porogens such as paraffin
microspheres [42], gelatin microspheres [43], etc. to the culture medium can form pores
of a desired size on the BC. However, the ex situ modification is a method to modify
the surface of BC by physical or chemical methods after BC synthesis. For example,
by solution-dipping method, the antibacterial ingredients are loaded into BC to improve
the antibacterial activity of BC [44]. A series of BC derivatives with different structures
and properties were obtained by carboxymethylation [45], acetylation [46],
phosphorylation [47], esterification [48] and other graft copolymerization and
crosslinking reactions on the surface of BC. The in situ modification is simple in
operation and uniform in modification effect, but the application range is small because
of the strictness of bacterial growth environment. Although the ex situ modification
which always is used to modify the surface of the BC is complicated in operation, but
it is not limited by the bacterial growth environment, and has more modification
methods, which lead to a wide range of applications [49].

Figure 2. Application of bacterial cellulose in the biomedicine

Undoubtedly, functional BC has increased the added value of BC and expanded the
application of BC. Due to its excellent mechanical properties, outstanding in vivo and
in vitro biocompatibility, high pore diameter, strong moisture absorption and water
holding capacity, degradability and similar structure of extracellular matrix, BC is
widely used in sensors [50], optoelectronics [51], cosmetic [36], food [36], biomedicine
[53](as shown in figure 2) and other fields. In biomedicine, BC is widely used for tissue
engineering research because it has a structure similar to an extracellular matrix, which
is consistent with the characteristics of an ideal tissue engineering material [54]. This
paper reviews the recent research status of BC in skin and bone tissue engineering. It
will help readers understand the role of BC in skin and bone tissue-based repair
processes in recent years.
2. Application of BC in skin tissue engineering
Composed of epidermis, dermis and subcutaneous tissue, the skin is the body's
largest barrier organ with good plasticity and regenerative capacity, but skin integrity
may be compromised by surgery, burns, diabetes, tumors and other causes. When the
skin is damaged, the body will initiates complex reactions to repair the damaged skin
to prevent further damage or infection to restore the skin's shielding function [55].
Dressings have long been used to aid wound healing, and current moisturizing dressings
[56], including hydrogels, hydrocolloids, alginates and hydrated fibers, are increasingly
characterized by ideal dressings versus traditional wound dressings [57]. For example,
it can maintain a moist environment, absorb excess penetrant, provide mechanical
protection, free from microbial infection, avoid allergic reactions, reduce wound pain,
easy replacement, etc [56, 58]. Therefore, BC has been widely used in wound healing
dressings [38] to utilize its excellent biocompatibility, high tensile strength and
flexibility, high porosity and water holding capacity, and good gas and liquid
permeability.
2.1 Role in the hemostasis/inflammation phase of wound healing
Wound healing is a sophisticated and complex dynamic process that generally
involves three stages: hemostasis/inflammation, proliferation and remodeling [59].
Inflammation and hemostasis occur simultaneously in the early stages of wound healing.
Once the skin is damaged, collagen exposure triggers platelet aggregation and
vasoconstriction to reduce blood loss, while the activation of the innate immune system,
neutrophils and monocytes rapidly migrate to damaged skin to eliminate potentially
infected and necrotic tissue [60]. The expression level of COX-2 that is the final product
of thrombin will increases after skin damage, which will enhance cell migration and
proliferation [61]. When the BC film loaded with lidocaine was used to treat burn
wounds, researcher found that the COX-2 immune expression in the wounds was
enhanced and the MMP-9 content was low of the experimental group in comparison
with the control group. Skin appendages were found in the wound, with mild
inflammatory infiltration, which is conducive to promoting the healing of burn wounds
[62]. It is necessary to prevent fluid loss and infection when dealing with burn wounds.
Therefore, the use of hydrophilic moisturizing dressings to absorb the carrion on the
wound can promote wound healing [63]. Lin et al. compared the healing effects of BC,
BC/chitosan (Ch) film, TegadermTM hydrocolloid dressing and transparent film
dressing on wounds, and found that BC can maintain a suitable water environment for
exudative wounds without excessive dehydration due to its good water absorption
capacity and optimal water permeability. While the wound covered by BC-Ch has a
faster epithelial formation and regeneration and a better wound healing effect [64]. The
bacterial cellulose/acrylic acid (BC/AA) hydrogel synthesized by the 35kGy electron
beam showed excellent water absorption and water retention, and the bacterial cellulose
also increased the mechanical properties of the hydrogel. Furthermore, in the H35
hydrogel treatment group, neutrophils whose main function is to remove pathogens
from the clot at the time of injury were abundant in the subcutaneous tissues, and the
scab fell off earlier than other groups, which is considered that the wound is completely
healed once scab sheds and forms a new epidermal layer [65]. Additionally, Altun et
al. utilized the high mechanical properties of BC to prepare biodegradable and
biocompatible bandages by pressure swing and poly (methyl methacrylate) (PMMA),
which was also found to support wound healing on the skin [66].
2.2 Role in the proliferative phase of wound healing
Characteristics of the proliferative phase include the formation of granulation tissue,
proliferation and migration of keratinocytes, fibroblast proliferation, extracellular
matrix synthesis, resulting in regenerative epithelial and angiogenesis [67].
Additionally, there are many wound healing dressing studies based on the important
role of fibroblasts and endothelial cells in maintaining the continuity of skin tissue.
Volova et al. incorporated fibroblasts into BC/P (3HB/4HB) composites and found that
the BC/P (3HB/4HB) film prepared by solvent evaporation has good biocompatibility,
which is the best scaffold for fibroblast growth. While fibroblasts have been shown to
promote wound healing agents to promote epidermal epithelialization. Increased genes
expression levels of collagen I (Col-1) (a mark of connective tissue formation), keratin
10, and keratin 14 (marks of formation of the spinous and granular layers in the
epidermis during tissue regeneration) were detected by PCR, demonstrating the
effectiveness of adding wound-healing drugs or cells to accelerate skin regeneration
[68]. Similarly, keratin/BC composite dressings improve the attachment of dermal
fibroblasts and keratinocytes to the surface of the dressing [69]. In addition,
incorporation BC with vaccarin to prepare BC-Vac film has no cytotoxicity and shows
better biocompatibility. BC-vac film-covered wound appears the stratified squamous
epithelium, dense newborn subcutaneous tissue and fibrous connective tissue collagen
fibers. Moreover, it has also been observed that fibroblasts are more active, which may
contribute to epithelial formation and tissue remodeling, probably because flavonoid
glycosides in vaccarin can alleviate oxidative stress damage in endothelial cells and
promote endothelial cell proliferation and neovascularization [70]. Some dressings
designed and prepared by using Arg to promote collagen synthesis and directly
participate in wound repair can promote the migration and proliferation of fibroblasts
and keratinocytes, and the expression of Col-1 [71].
2.3 Role in the remodeling Phase of wound healing
Reconstruction of the extracellular matrix is the final stage of wound healing. At this
stage, whereas differentiation of myofibroblasts with fibroblasts results in smooth
muscle actin deposition and wound contraction. Simultaneously, collagen III is
replaced by collagen I in the extracellular matrix (ECM) [72], resulting in scar
formation and recovery of the barrier [73]. Since the porous and 3D structure of BC
supports cell proliferation and penetration, Mohamad et al. incorporated human
epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) into BC/AA
hydrogels to study the burn wound healing in the epithelium of athymic mice. The
expression of cytokeratin-14, Col-1 and α-smooth muscle actin showed that hydrogel
loaded with HEK and HDF can promote collagen deposition and improve the healing
rate of burn wounds. Therefore, they consider that BC/AA hydrogel gel is suitable as a
wound dressing and cell carrier [74].
2.4 Role in the chronic wounds and excessive healing
Compared with normal wound healing, chronic wounds are characterized by reduced
angiogenesis, stem cell recruitment and activation, and ECM remodeling [73]. The
main complication of diabetic peripheral neuropathy is ulceration of lower extremity
injuries that is a common chronic wound. Long-term inflammatory responses and
increased proinflammatory cytokines such as IL-1β and TNF-α are observed in diabetic
wounds [75]. Therefore, in order to understand the healing effect of BC film containing
propolis extract on diabetic wounds, it was found that the levels of neutrophils and pro-
inflammatory factors IL-1β and TNF-α were higher than those of the control group on
the seventh day of treatment, which can accelerate debridement process. On the 14th
day, the number of inflammatory cells and factors was lower than that of the control
group, which was consistent with the changes in the normal wound healing process,
possibly because the abundant flavonoids in the propolis extract exert anti-
inflammatory and antioxidant functions, thus accelerating the wound healing of
diabetes mellitus [76]. Therefore, the combination of bacterial cellulose with anti-
inflammatory or antioxidant ingredients is a feasible research direction for the treatment
of chronic wounds. In addition, Springer et al. assisted the measurement of three
different optical measurement procedures (MPT, OCT, CLSM) to diagnose chronic
wound healing by taking advantage of the biological compatibility, light transmittance,
mechanical strength, softness and physical shielding of BC [77].
Excessive healing, another abnormal condition of wound healing, will lead to the
formation of scars and the patient's appearance and limb dysfunction [78]. And all over-
healing wounds include the initialization of purulent inflammatory processes, up-
regulation of fibroblasts, and excessive ECM deposition [59]. Hu et al. established a
cross-slot micrograph on a porous BC by low-energy CO laser lithography, and the
tetrapeptide consisting of arginine-glycine-aspartate-serine (RGDS) was used as a
fibronectin to target micro-patterns immobilized on the surface of a BC cross-slot
column (RGDS-MPBC). Their animal experiments found that RGDS-MPBC can guide
the ordering of human skin fibroblasts (HSF) on the surface of the columnar platform;
a dense collagen network is gradually formed on the channel; the collagen fiber bundle
is thin and evenly distributed; and no HSF cells were found. These results indicate that
the tissue has fewer scars and a better healing effect [79]. In addition, Napavichayanun
et al. sequentially loaded sericin and polyhexamethylene biguanide (PHMB) into the
BC film by physical adsorption. In vivo experiments indicated that the wound treated
with this dressing has a lower inflammatory response, a higher degree of collagen
formation, and the potential to shrink scars. This phenomenon may be caused by the
fact that sericin can induce Col-1 in the wound to promote cell proliferation and
migration, and synergize with the action of the antimicrobial agent PHMB to accelerate
the healing process [80]. As the mechanism of scar formation remains controversial,
BC-based composites can alleviate the formation of scars, but extensive research is
needed to achieve the results of applying BC composites to cure scars.
2.5 Role in the antibacterial on wound healing
In addition, it is important to prevent bacterial infections during wound healing.
Because bacterial infections will exacerbate tissue necrosis, inhibit epithelialization and
fibroblast proliferation, which will cause a drop in wound strength [75]. The
antibacterial properties of BC are achieved through physical barriers, but they have no
antibacterial properties in themselves to prevent infection ingrowth [81]. The addition
of antibacterial active ingredients to BC to prepare a composite dressing is one of
research strategies of BC-based wound dressings. Other research strategies for BC-
based wound dressings are shown in Table 1. Utilizing the high-porosity of BC, the
small-molecule antibacterial agent is impregnated into the BC pores to achieve a long-
term antimicrobial effect on the wound slowly released [38], which is the most common
and simple method of preparing an antimicrobial bacterial cellulose dressing. For
example, the BC film was immersed in AgNO3 solution and then sterilized by UV
irradiation to prepare Ag0/BC film. Even after 7 days of soaking, the release
concentration of silver in the Ag/BC film was quite stable, and the antibacterial property
of the composite was confirmed by the disk diffusion method, indicating that the
composite film has good sustained release and antibacterial properties [82]. Wei [83]
and Wu [83] et al. use electrostatic interaction to combine Ag+ with the oxygen atoms
of BC. Therefore, the release rate of Ag+ decreases as the pH increases BC-Ag
nanocomposites exhibit excellent antibacterial properties against Escherichia coli,
Staphylococcus aureus, Bacillus subtilis and Candida albicans. Similarly, electrostatic
interaction is also one of common mechanism for loading macromolecular
antimicrobials into BC. For example, after modifying graphene oxide (GO) with
polyethyleneimine (PEI), the GO is loaded into BC due to the formation of a strong
hydrogen bond between the hydroxyl group of BC and the amine group, and the
electrostatic interaction makes GO-PEI / BC is easier to anchor on the surface of
microbial cells to enhance BC antibacterial activity [85]. In addition, the BC reactive
group can also be modified by a chemical reaction such as carboxylation, esterification
or alkylation to enhance the adsorption of BC on the antibacterial agent. For example,
the composite prepared by periodate oxidized bacterial cellulose (DABC) loaded with
chloramphenicol has the advantages of antibacterial property, biodegradability and
biocompatibility [86]. Moreover, Sajjad et al. designed a neotype of artificial substitute
for burns by combining the wound healing properties of BC with the antibacterial
activity of modified montmorillonite (MMTs). Animals wound treated with modified
MMTs-BC nanocomposites have enhanced wound healing activity, tissue regeneration,
regenerative epithelialization, healthy granulation tissue and angiogenesis. These
results indicate that the modified MMTs-BC nanocomposites can be used as a neotype
of artificial skin substitute for burn patients and as a scaffold material for skin tissue
engineering [87].
Beyond that, BC is also applied to local skin damage caused by parasites. Celes et
al. prepared BC-DETC by immersive BC in diethyl dithiocarbate (DETC) and DETC
slowly releases a controlled release system because of the BC hydroxyl and DETC's
sulfur non-bonding interactions, which significantly reduces lesion size, Leishmania
load and inflammatory response in infected sites by inhibiting superoxide dismutase I
[88].
Commented [l1]: Mark 1
Tabel 1. Research strategy of wound dressing based on bacterial cellulose
Strategy Modification Action principle Examples or
methods components added
 1. Increase the pore size
Improved cell Changing BC Better Good softness ,lower
distribution (such as
interaction physical properties, fiber density and higher porosity
porogen[89])
such as porosity, facilitates cell penetration
fiber diameter, 2. Artificially formed
material form [90]
wettability

Derivatization Chemical introduction of cell-
reactions to alter responsive stimulating
surface properties functionalities to polymeric
substances to mimic natural cell
signaling unions and achieving
better cell adhesion
Impregnation with Cell adhesion molecules provide
specific cell biofunctionality to materials and
adhesion molecules improve the biocompatibility of
(e.g., proteins, desired cell lines.
3. Surface topography
during biosynthesis[91]
1. Introduction of cell
response stimulating
functional groups, (e.g.,
NH3-[92], COOH-[93]
etc.)
2. Surface
hydrophobization [81]
1. Growth factor[94]
2. Silk fibroin[95]
3. RGD peptides[79]

peptides)
1.Collagen cross[96]
Impregnation with combining the advantages of
secondary secondary components to 2.Gelatin[97]
components with prepare composite materials to
better cell affinity overcome some limitations of a 3.Lecithin[98]

single material
1. Antibiotics[99]
Introduction Inclusion of Slow release of antibiotic into
antibacterial antimicrobials into the wound to destroy bacteria 2. Inorganic
activity the BC matrix nanoparticles[81]

3. Antimicrobial
peptides[100]

4. Cationic antibacterial
agent [80]

5. A polymer with
antimicrobial activity
[101]

Surface Bacterial binding to the dressing
hydrophobization due to cell-surface
hydrophobicity effect and
eliminate them during dressing
1.Plasma treatment [81]
2. By chemical reactions
(e.g. grafting amphiphilic
block copolymers)[102]

changes
1. Introduction of amino
Fiber modification Covalently attached active sites
groups[92]
through covalent provide permanent antimicrobial
binding of cell activity and bacteria binding 2. Binding of RGDC
adhesion sites capacity peptides/gentamicin[103]

3. Grafting amoxicillin
onto the surface[104]
1. Adding pore-forming
Changing the Changing surface Increasing available space and
agents[89] and foam
water holding area and aperture amount of trapping sites for
templates [105]in the
capacity distribution penetrating water molecules to process of biosynthesis
(WHC) and improve WHC; reducing the
2.Alkali treatment[106]
water release hydrophilic effect to improve
rate (WRR) WRR. 3.Laser piercing[107]

4.Gamma radiation[108]
1.Aloe vera gel[109]
Introduction of The influence of hydrophilic
hydrophilic components to WHC and WRR 2.Alginate[110]
components due to its nature
3.Chitosan[64]

4.Acrylic[65]

5.poly-AEM[111]
3. Application of bacterial cellulose in bone tissue engineering
3.1 Application of bacterial cellulose in bone repair
Bone is a mineralized connective tissue with unique wound healing capabilities, as
the most widely studied in tissue engineering. The primary bone tissue consists of two
parts, minerals consisting of calcium phosphate (70% of tissue) and organic proteins
(95% collagen I) [112]. When bone regeneration material is required to assist in
restoring the mechanical stability of the bone segment [113], and bone grafting, one of
the methods to repair bone damage, can promote bone healing and repair [114].
However, the application of bone grafts is limited by shortcomings such as appropriate
donor tissue, immune rejection, and disease transmission [115]. Due to the emergence
and development of regenerative medicine and tissue engineering technology, it has
become possible to prepare inexpensive, accessible, safe and effective synthetic bone
repair material. Ideal synthetic bone repair material need to stimulate specific cellular
responses, activate genes, and stimulate cell differentiation and production of ECM to
promote regeneration of damaged tissue [112]. The functions of synthetic bone repair
material currently studied are mainly manifested as providing structural support for cell
attachment, diffusion, migration, proliferation and differentiation [42]. Due to the
unique characteristics of BC, such as easy availability, cheapness, excellent
biocompatibility and slow degradation, it has attracted much attention from bone tissue
engineering researchers as a composite synthetic bone repair material [116]. In order to
improve the ability of BC composites to repair bone damage, some bone repair
components, such as hydroxyapatite, bone marrow mesenchymal stem cells (BMSC),
growth factors and extracellular matrix protein, and estrogen, are often impregnated
into BC structures.
3.1.1 Hydroxyapatite
Providing bone conduction is a basic function of bone graft materials, but we have
not found any report that BC replaces autologous bone graft as bone graft material
because of the poor rigidity of BC. At present, the use of BC for bone tissue engineering
research is mainly focused on promoting osteoinduction. Researchers have enhanced
the mechanical properties of BC stents by combining BC with materials with high
mechanical properties [117]. For example, dynamic treatment of BC film adsorbing
carboxymethyl cellulose triggers nucleation of calcium-deficient hydroxyapatite
(cdHAp), which provides rigidity to bone conduction materials. The results show that
cdHAp crystals cause increased cell adhesion and can be induced the differentiation of
osteoprogenitor cells [118]. In addition, Osteoblast adhesion that is the first step in
cell/material interaction can also be promoted by increasing the surface roughness of
the scaffold and the surface roughness will also affect the ability of cells to proliferate
and differentiate on the surface of the material [119]. For example, the cellulose-
gelatin/hydroxyapatite double network complex is based on higher thermal stability and
coarse super-surface morphology, which makes BMSC possess better adhesion and
greater potential of proliferation and differentiation [120].
3.1.2 Bone marrow mesenchymal stem cells
The study found that BMSC can secrete cytokines and play an important role in
regulating osteoclast differentiation, recruitment and proliferation of osteoblasts and
angiogenic cells [121]. Therefore, loading BMSC into BC is a research method for
preparing scaffolds or evaluating the performance of bone repair materials. For example,
horse-derived mesenchymal stem cells (EqMSC) were inoculated on BC scaffolds
whose pore provided support for EqMSC adhesion, ingrowth and migration. The results
of the study indicate that EqMSCs can differentiate into chondrocytes and bone cells
on BC [122]. Similarly, BC was used to culture BMSC. It also found that BC allowed
adhesion, expansion and biointegration of BMSC, and BC had low cytotoxicity and
slow degradation rate, making BC become an ideal biomaterial for slow healing
processes because the long process of reconstructing tissue requires a durable scaffold
[123]. Noh et al. inoculated human umbilical cord blood mesenchymal stem cells
(UCB-MSCs) on BC/Col scaffolds for culture. It was found that with the increase of
BC content, the stability of scaffold is stronger and calcium deposition is increased,
indicating that the composite scaffold with higher BC content is effective for late
osteogenic differentiation of UCB-MSC. Moreover, UCB-MSC-loaded BC/Col (5:1)
composite scaffolds on subcutaneous transplantation experiments showed more red
blood cells and α-SMA (angiogenic markers), suggesting neovascularization , which
may be because BC/Col (5:1) is able to maintain a stable porous microenvironment
allowing nutrient access and oxygen to diffuse better, while vascular formation helps
provide nutrients in the early stages of bone regeneration[124]. In order to increase bone
vessels, Zaborowska et al. incorporated vascular endothelial growth factor (VEGF) into
BC scaffolds, which can enhance the vascular formation, ossification and maturation
of new bone (callus) in femoral fractures in mice, and promote the bone bridging of
radius segment gap defects in rabbits [42].
3.1.3 Growth factor and extracellular matrix protein
 In the bone repair process, the BC film impregnated with growth factor and
extracellular matrix protein molecules can also promote cell attachment, proliferation
and induce osteogenesis. For example, BC-HA nanocomposite impregnated with
osteogenic growth peptide (OGP) or pentapeptide OGP can enhance osteoblast
differentiation activity in early bone regeneration [125]. Collagen, the most abundant
protein in ECM and the main organic component of bone tissue, is always combined
with BC as a composite material, because collagen is a bioactive molecule that can
stimulate a better regeneration process of bone [126]. Many studies have shown that a
variety of growth factors can induce pluripotent cells to differentiate into osteoblasts in
a non-bone environment to form new bone [127]. Among them, BMP-2 differentiated
from mesenchymal stem cells has strong osteoinductive activity and is widely used in
bone tissue engineering. For example, Shi et al. implanted a BMP-2-loaded BC
scaffolds into an ectopic bone model in SD rats, performed tissue staining, measured
alkaline phosphatase (ALP) activity and calcium deposition to evaluate bone formation.
They observed higher ALP activity and greater calcium deposition compared to the
control group. After 2 weeks of implantation, the BMP-2/BC scaffolds group was
almost filled with new bone tissue, probably because the pore size of BC achieved local
delivery of BMP-2, increasing the concentration of BMP-2 at the site of injury, and
better inducing osteogenesis [94].
3.1.4 Estrogen
In addition to growth factors, estrogen in bone has a role in mediating osteoblast
formation and bone resorption of osteoclasts through nuclear and film-associated
estrogen receptors [128]. Phytoestrogens were reported that it can also affect the
balance between osteogenesis and adipogenic differentiation of MSCs, thereby
stimulating osteogenic differentiation. Kheiry et al. incorporated phytohormone fisetin
into BC scaffolds to inoculate BMSCs for cell culture, and determined ALP activity to
determine BMSC differentiation. The researchers observed calcium precipitation on the
BC stent and found the expression of osteocalcin and osteopontin in real-time PCR
experiments. Osteocalcin, the late osteoblast-specific protein produced only by mature
osteoblasts, is responsible for the expression of various bone cell types. In addition,
ALP activity was most pronounced on day 10, indicating that the fisetin/BC scaffold
supports BMSC attachment, proliferation, and bone-specific matrix biosynthesis [129].
3.2 Application of bacterial cellulose in cartilage tissue engineering
In vivo cartilage mainly includes hyaline cartilage, elastic cartilage and fibrocartilage.
The most abundant cartilage is hyaline cartilage covering the joint surface; elastic
cartilage is present in the outer ear, epiglottis and larynx; and fibrous cartilage is present
in the intervertebral disc, joint capsule and ligament [130]. Chondrocytes are the main
cells in cartilage, and cartilage has a very limited ability to self-repair and regenerate
because mature chondrocytes do not divide, have low levels of metabolism, and have
no blood vessels and nerves [131] .
Autografting of chondrocytes or osteochondral plugs is currently the only surgical
treatment for repairing damaged cartilage tissue. However, autografting requires two
operations and the tissue recovery time is long [132]. Therefore, the promotion of new
cartilage tissue formation by implanting natural ECM or biopolymer scaffolds can
avoid the drawbacks of autografting, which is a new strategy for the treatment of
cartilage defects. Svensson et al. evaluated BC secreted by A. oxysporum with bovine
chondrocytes, which has good biocompatibility, mechanical properties and porosity,
and similar Young's modulus with articular cartilage, so that unmodified BC supports
chondrocyte proliferation and growth into the scaffold, indicating that BC has the
potential to serve as a scaffold for cartilage tissue engineering [34]. Native BC pores
are usually 0.02-10 micron in size, which is smaller than mammalian cells and limits
the inward growth of cells. It was found that materials with pores of 300-500nm size
have better effect of promoting cell inward growth. This was demonstrated by the BC
scaffolds prepared by laser perforation [133], or by adding pore-causing agents such as
paraffin microspheres [42] and agarosaccharide particles [89]. In addition, the rate of
degradation of the ideal scaffold should be comparable to the rate of formation of new
tissue, while the degradation rate of BC in the human body is slow because the human
body lacks enzymes to degrade BC. In order to prepare BC materials with suitable
degradation rate, Yadav et al. prepared BC scaffolds with N-acetylglucosamine
residues as carbon source, and combined with adult bone marrow-derived
mesenchymal stem cells, demonstrating that porous MBC scaffolds that are easily
degraded by lyme can support the adhesion, proliferation and synthesis of hMSCs rich
in cartilage ECM dense structure [134]. Based on the characteristics of cartilage tissue,
the avascular cartilage layer composed of chondrocytes and collagen II and
glycosaminoglycan (GAG) while Osteoblasts existing in the vascular pedicled
subchondral bone layer composed of HA and collagen I, Kumbhar et al. implanted a
bilayer support consisting of BC-HA and BC-GAG into the knee joint of the model
animal. No signs of cartilage regeneration were observed in the control group at the
third month of surgery. However, in the bilayer scaffold group, intact new cells and
new proteins was observed in the defect site, and the new tissue was fully fused with
the surrounding healthy tissue, which accelerated the regeneration of articular cartilage
and subchondral bone in the rat osteochondral defect (OCD) model, indicating its
potential for treating OCD [132] .
BC is not only used in the repair of hyaline cartilage, but also in the repair of elastic
cartilage, such as auricular deformity and nose skew. Ávila et al. found that BC with
17% increase in cellulose content was similar to auricular cartilage in terms of
mechanical strength and host tissue response. After one week of retention in the body,
the implants were tightly bound to the surrounding soft tissue, while blood and tissue
fluid were adsorbed into the implant material. It indicated that the material can be used
as a non-absorbable auricular cartilage tissue engineering biomaterial [135]. In addition,
human nasal septal chondrocytes were inoculated into BC/alginate double-layer
scaffolds. After 6 weeks of in vitro culture, a rich and uniform distribution of cells and
new cartilage was observed throughout the porous layer of the scaffold. At 8 weeks
post-implantation, the presence of proteoglycan and collagen II demonstrated the
formation of new cartilage in the porous layer, suggesting that the double-layer BNC
stent has the potential to treat severe auricle defects [136]. Sevim et al. placed the
bacterial cellulose and fascia containing the chopped cartilage in the subcutaneous
region of the back of the rat to compare its health and integrity with the degree of
vascularization, fibrosis and chronic inflammation. There were no statistically
significant differences in cartilage health and integrity between the two groups,
suggesting that bacterial cellulose transplantation can be used as a surrogate for fascial
transplantation in rhinoplasty [137]. Combined with 3D bio-printing technology, BC,
as a bio-ink, is expected to replace autologous costal cartilage and nasal cartilage,
becoming a biomaterial for auricle reconstruction and rhinoplasty because of its unique
properties.
3.3 Application of bacterial cellulose in dentistry
Guided bone regeneration (GBR) requires a shielding film to shield the rapidly
proliferating epithelium and connective tissue, providing space for slow-growing cells
to form bone [138]. GBR is often used in oral implant therapy, and the barrier
membrane (DBM) is used to provide a space away from the surrounding connective
tissue for tissue growth and bone regeneration of the periodontal ligament. Therefore,
DBM should have good biocompatibility, be able to be degraded after the formation of
new tissue, and have sufficient mechanical and physical properties to play the barrier
function [139]. Because BC meets the requirements of DBM, it is used for DBM
research. BC/collagen material carrying osteogenic growth peptide (OGP (10-14)) acts
as a shielding membrane to maintain bone defect space during bone healing and
promote bone hyperplasia [140]. However, the biodegradation rate of BC is slow.
Studies have found that the biodegradation rate of native BC can be increased by
electron beam irradiation, which is expected to replace the cytotoxic collagen
membrane as an important re-absorption shielding membrane [141].
When caries develop into pulp infections, root canal therapy is often used as a way
to completely purify the root canal system. Removal of root residue, dentin filling,
lavage fluid and other factors will affect the efficacy of root canal. Therefore, the highly
adsorbable material has a greater advantage in removing residual liquid, which will help
improve the efficacy of root canal treatment. Utilizing the crystallinity, mechanical
strength and adsorption capacity of BC, Yoshino et al. rolled the BC sheets into a point
form standardized according to ISO 45. Compared with commercially available paper
points (PP), BC materials have higher tensile strength, higher average drug release rate
and 10 times higher water absorption than PP, indicating that it has good potential as a
material for root canal treatment [142]. In addition, the synthesized composite material
by combining the mineral binder powder and the biocellulose membrane possesses
smaller average grain size and specific surface area, faster interaction with water, and
shorter coagulation time, thus, it has high application potential in root canal filling,
channel filling or dentin mineralization [143].
4. Concluding remarks
Based on its hydrophilicity, biocompatibility, mechanical properties, moisture
retention and porosity, BC composites are involved in hemostasis/inflammation,
proliferation and remodeling of skin wound healing, which improves the speed and
effectiveness of skin wound healing by promoting cell adhesion, migration,
proliferation, differentiation and avoiding microbial infection. In addition, BC
composites can induce the generation of bone cells to repair bone tissue defects, which
has development potential in both plastic surgery and dentistry. At present, the
application research of BC in skin and bone tissue engineering is mainly focused on
improving the BC added value, and it is mainly in the laboratory stage. There are fewer
BC products that have been used in clinical practice, mainly as wounds and burn
dressings such as Dermafill, Bioprocess, Biofill and Suprasorb. Furthermore, Gengiflex
can be used to restore root tissue and SyntheCel can be used as a dura substitute. In
addition, Basyc is used as a vascular implant for coronary bypass surgery. Now a large
number of BC-based facial mask products have emerged, but the price of BC mask is
more expensive than other mask products due to its low yield and high cost. Although
there are few commercial products for biomedical applications in BC, with the joint
efforts of scientists, the potential of BC as an excellent nano-renewable material will
be well developed and applied in clinical treatment.

Acknowledgements
This study was financially supported by the Science and Technology Program of
Zhongshan (2016B1005) and the Program of Traditional Chinese Medicine Bureau of
Commented [l2]: Mark 2
Guangdong Province (20161264).

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