European Polymer Journal: Ning Lin, Alain Dufresne

European Polymer Journal 59 (2014) 302–325
Contents lists available at ScienceDirect
European Polymer Journal

journal homepage: www.elsevier.com/locate/europolj
Feature Article
Nanocellulose in biomedicine: Current status and future

prospect
Ning Lin, Alain Dufresne ⇑
Grenoble Institute of Technology (Grenoble INP), The International School of Paper, Print Media and Biomaterials (Pagora), CS10065, 38402 Saint Martin
d’Hères Cedex, France
a r t i c l e i n f o a b s t r a c t
Article history: Nanocellulose, a unique and promising natural material extracted from native cellulose,
Received 6 May 2014 has gained much attention for its use as biomedical material, because of its remarkable
Received in revised form 1 July 2014 physical properties, special surface chemistry and excellent biological properties (biocom-
Accepted 21 July 2014
patibility, biodegradability and low toxicity). Three different types of nanocellulose, viz.
Available online 1 August 2014
cellulose nanocrystals (CNC), cellulose nanofibrils (CNF) and bacterial cellulose (BC), are
introduced and compared in terms of production, properties and biomedical applications
Keywords:
in this article. The advancement of nanocellulose-based biomedical materials is summa-
Cellulose nanocrystals
Cellulose nanofibrils
rized and discussed on the analysis of latest studies (especially reports from the past five
Bacterial cellulose years). Selected studies with significant findings are emphasized, and focused topics for
Biomedical application nanocellulose in biomedicine research in this article include the discussion at the level
of molecule (e.g. tissue bioscaffolds for cellular culture; drug excipient and drug delivery;
and immobilization and recognition of enzyme/protein) as well as at the level of macro-
scopic biomaterials (e.g. blood vessel and soft tissue substitutes; skin and bone tissue
repair materials; and antimicrobial materials). Functional modification of nanocellulose
will determine the potential biomedical application for nanocellulose, which is also intro-
duced as a separated section in the article. Finally, future perspectives and possible
research points are proposed in Section 5.
Ó 2014 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Contents
1. What is nanocellulose? – Types and productions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

2. Why the choice of nanocellulose? – Unique properties in physics, chemistry and biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
2.1. Mechanical properties and potential nanoreinforcement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
2.2. Surface chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
2.3. Biological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
2.3.1. Biocompatibility and hemocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
2.3.2. Biodegradability in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
2.3.3. Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
3. Nanocellulose-based biomedical materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
3.1. Tissue bioscaffolds for cellular culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
3.2. Drug excipient and drug delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
⇑ Corresponding author. Tel.: +33 476826995; fax: +33 476826933.

E-mail address: [email protected] (A. Dufresne).
http://dx.doi.org/10.1016/j.eurpolymj.2014.07.025
0014-3057/Ó 2014 Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
N. Lin, A. Dufresne / European Polymer Journal 59 (2014) 302–325 303
3.3. Immobilization and recognition of enzyme/protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

3.4. Substitutes/medical biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
3.4.1. Blood vessel replacement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
3.4.2. Soft tissue–ligament, meniscus, and cartilage replacements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
3.4.3. Nucleus pulposus replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
3.5. Advanced nanomaterials for tissue repair, regeneration and healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
3.5.1. Skin tissue repair and wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
3.5.2. Bone tissue regeneration and healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
3.5.3. Other tissue repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
3.6. Antimicrobial nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
3.7. Other biomedical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
4. Functional modification of nanocellulose for potential biomedical application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
5. Conclusions and remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Appendix A. Supplementary material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
1. What is nanocellulose? – Types and productions brils made of alternating crystalline and amorphous cellu-
lose domains, which attributes the morphology of CNF
With the emergence and development of nanotechnol- with soft and long chains. Due to the entanglement of long
ogy, cellulose, the most ancient and important natural cellulosic chains, it is not so easy to determine the length
polymer on earth revives and attracts more attention in of CNF (commonly regarded as higher than 1 lm) with
the new form of ‘‘nanocellulose’’ to be used as novel and microscopic techniques. Therefore, only the information of
advanced material. Nanocellulose is described as the prod- fibril width for CNF is generally provided in the studies,
ucts or extracts from native cellulose (found in plants, ani- which varies from 10 to 100 nm depending on the source
mals, and bacteria) composed of the nanoscaled structure of cellulose, defibrillation process and pretreatment [4].
material. Generally, the family of nanocellulose can be Contrary to the production of CNC and CNF, the biosyn-
divided in three types, (1) cellulose nanocrystals (CNC), thesis of BC is a process of construction from tiny unit (Å)
with other designations such as nanocrystalline cellulose, to small unit (nm). As shown in Fig. 1 (bottom images), BC
cellulose (nano) whiskers, rod-like cellulose microcrystals; is typically synthesized by bacteria (such as Acetobacter
(2) cellulose nanofibrils (CNF), with the synonyms of nano- xylinum) in a pure form which requires no intensive pro-
fibrillated cellulose (NFC), microfibrillated cellulose (MFC), cessing to remove unwanted impurities or contaminants
cellulose nanofibers; and (3) bacterial cellulose (BC), also such as lignin, pectin and hemicellulose. During the bio-
referred to as microbial cellulose [1,2]. synthesis of BC, the glucose chains are produced inside
The sources for CNC and CNF extraction are wood, cot- the bacterial body and extruded out through tiny pores
ton, hemp, flax, wheat straw, sugar beet, potato tuber, mul- present on the cell envelope. With the combination of glu-
berry bark, ramie, algae, and tunicin. As shown in Fig. 1 cose chains, microfibrils are formed and further aggregate
(top images), the production of CNC or CNF is a procedure as ribbons (nanofibers) [11]. These ribbons subsequently
consisting in converting the large unit (cm) to the small generate a web-shaped network structure with cellulosic
unit (nm). Chemically induced destructuring strategy, such fibers (BC), which has a diameter of 20–100 nm with dif-
as acid hydrolysis, is commonly performed for the extrac- ferent types of nanofiber networks.
tion of CNC from native cellulose, through the removal of It is crucial to discuss the issue of large-scale production
amorphous regions and preservation of highly-crystalline of nanocellulose, which determines the practical applica-
structure. Released nanoparticles (CNC) present a diameter tions of nanocellulose as available commercial products.
of 5–30 nm, and length of 100–500 nm (from plant cellu- According to the reports of ‘‘Future Markets Inc.’’ [12], a
lose), or length of 100 nm to several micrometers (from number of organizations have announced CNF and CNC
tunicate and algae celluloses). With microscopic observa- demonstration plants in Europe and North America. It
tions and light scattering techniques, the morphology seems that the countries in North America focus on the
and dimensions of CNC can be assessed as elongated rod- production of CNC, such as reported organizations of Bio
like (or needle-like) nanoparticles, and each rod can there- Vision (Canada), CelluForce (Canada) and US Forest Service
fore be considered as a rigid cellulosic crystal with no Forest Products Laboratory (USA); while European coun-
apparent defect [3]. tries are more interested in CNF, for instance reported
Regarding the preparation of CNF, mechanically induced organizations of Centre Technique du Papier (France), Stora
destructuring strategy is mainly applied, which involves Enso (Finland), UPM Fibril cellulose (Finland), Borregaard
high-pressure homogenization and/or grinding before and/ Chemcell (Noway), etc. In comparison with the large-scale
or after chemical or enzymatic treatment. Multiple mechan- production of CNC and CNF, the production of BC is rather
ical shearing actions can effectively delaminate individual limited, resulting from high cost to support the growth of
microfibrils from cellulosic fibers. Different from rigid bacteria and low yield. Despite numerous bioreactors that
CNC, CNF consists of both individual and aggregated nanofi- have been studied in the literature to produce BC on large
304 N. Lin, A. Dufresne / European Polymer Journal 59 (2014) 302–325
Fig. 1. Hierarchical structure of cellulose; top image (from large unit to small unit): cellulose nanocrystals (CNC), micro/nanofibrillated cellulose (MFC and
NFC); bottom image (from tiny unit to small unit): bacterial cellulose (BC). Transmission electron micrographs of sugar beet MFC (adapted with permission
from [5]), hardwood MFC (adapted with permission from [6]), ramie CNC (adapted with permission from [7]); and scanning electron micrographs of BC
ribbons (adapted with permission from [8]), nata-de-coco BC (adapted with permission from [9]), BC pellicle (adapted with permission from [10]).
scale, the highest BC productivity is only 0.38 g/(L h) with both ‘‘nano-enhanced’’ and completely new ‘‘nano-
the aerosol bioreactor [13]. The organization of Jenpoly- enabled’’ products have been envisioned ranging from bulk
mers (Germany) and Nutrasweet Kelco Company (USA, applications like rheological modifier, composite reinforce-
with the trade name of Primacel in the 1990s) were ever ment or paper additive, to high-end applications such as
reported to produce BC and related biomedical products. tissue engineering, drug delivery and functional material
[14]. All the properties of nanocellulose can be generally
classified in three parts, viz. physical properties, surface
2. Why the choice of nanocellulose? – Unique properties
chemistry, and biological properties. Associated with the
in physics, chemistry and biology
topic ‘‘biomedicine’’, the emphasis of this article is mainly
placed on the mechanical reinforcement, surface groups
As natural nanoscaled material, nanocellulose possesses
and charges, as well as various biological properties of
diverse characteristics different from traditional materials,
nanocellulose.
including special morphology and geometrical dimensions,
crystallinity, high specific surface area, rheological proper-
ties, liquid crystalline behavior, alignment and orientation, 2.1. Mechanical properties and potential nanoreinforcement
mechanical reinforcement, barrier properties, surface
chemical reactivity, biocompatibility, biodegradability, The mechanical properties of nanocellulose can be char-
lack of toxicity, etc. On the basis of these unique properties, acterized by its properties in both the ordered (crystalline)
Fig. 2. Schematic representation of the chemical structure and intra-, inter-molecular hydrogen bonds in crystalline cellulose.
and disordered (amorphous) regions of the nanoparticle. a Raman spectroscopic technique, which involved the
Cellulosic chains in disordered regions contribute to the determination of local molecular deformation of BC via a
flexibility and plasticity of the bulk material, while those shift in the central position of the 1095 cm–1 Raman band
in ordered regions contribute to the stiffness and elasticity [18].
of the material. The modulus of different types of nanocel- Originated from these impressive mechanical proper-
lulose is expected to result from a mixing rule between the ties, nanocellulose has been potentially used as a load-
modulus of the crystalline domains and the amorphous bearing element for various host materials. With the
fraction. Therefore, the stiffness and modulus of CNC with homogenous dispersion and strong interfacial adhesion,
more crystalline regions should be higher than those of the presence of high-modulus nanocellulose can exhibit
CNF and BC fibrils with both crystalline and amorphous the promising nanoreinforcement allowing proper stress
structures. transfer from host material (matrix) to the reinforcing
Since 1930s, the elastic modulus of crystalline cellulose phase (nanocellulose).
has been investigated either by theoretical evaluations or
by experimental measurements (wave propagation, X-ray 2.2. Surface chemistry
diffraction, Raman spectroscopy, and atomic force micros-
copy). A broad range of values was reported, and it is gen- From a structural point of view, cellulose is a high
erally accepted that the Young’s modulus of crystalline molecular weight homopolysaccharide composed of b-
cellulose (assimilated to the one of CNC) should be in the 1,4-anhydro-D-glucopyranose units (Fig. 2). These units
range 100–200 GPa, which gives specific values similar to do not lie exactly in the plane with the structure, but rather
Kevlar (60–125 GPa) and potentially stronger than steel they assume a chair conformation with successive glucose
(200–220 GPa). Recently, the elastic modulus of crystalline residues rotated through an angle of 180° about the molec-
cellulose was investigated from atomistic simulations ular axis and hydroxyl groups in an equatorial position
using both the standard uniform deformation approach [19]. The ability of these hydroxyl groups to form hydrogen
and a complementary approach based on nanoscale inden- bonds plays a major role in the formation of fibrillar and
tation, which was reported as 139.5 ± 3.5 GPa (similar to semicrystalline packing, which governs the important
Kevlar) [15]. In another study, Dri et al. performed the physical properties of this highly cohesive material [20].
atomic structure model of cellulose in tandem with quan- As indicated with blue dashed lines in Fig. 2, intramolecu-
tum mechanics to compute the Young’s modulus of crystal- lar hydrogen bonds occur primarily between the hydrogen
line cellulose, which predicted the modulus of crystalline borne by the OH group of the C3 carbon and ring oxygen of
cellulose as high as 206 GPa (similar to steel) [16]. the adjacent glucose unit (O5). The intermolecular hydro-
Again, a broad range of values for the longitudinal mod- gen bonds occur between the hydrogen of the OH–6 pri-
ulus of cellulose microfibrils (involving both CNF and BC) mary hydroxyl and oxygen in position O3 in a cycle of a
was reported based on different theoretical and experi- neighboring unit, as well as the hydrogen of OH–2 and
mental strategies. The accepted average value is around oxygen in position O6.
100 GPa for the modulus of cellulose microfibril. A three- It is well known that the unidirectional parallel orienta-
point bending experiment using atomic force microscopy tion of cellulose chains within the elementary fibrils,
tips was performed on cellulose microfibrils to calculate occurring during biosynthesis and deposition, induces the
the elastic modulus. The dimension of cellulose microfi- formation of crystals having hydroxyl functionality on
brils was found to significantly affect the mechanical prop- one end, known as the non-reducing end (shown in pink1
erties, and a value of 81 ± 12 GPa was reported to be the
longitudinal modulus of pulp CNF [17]. Recently, the mod- 1
For interpretation of color in Fig. 2, the reader is referred to the web
ulus of BC was reported as 114 GPa through the analysis of version of this article.
in Fig. 2), and hemiacetal functionality on the other, known CNF are rare. Some studies on CNC-based materials (such
as the reducing end (shown in green in Fig. 2). as hydrogels) only report experiments of cell cultivation,
One of the most specific characteristics of cellulose is through the growth, propagation and activity of cells to
that each of its glucose unit bears three hydroxyl groups, evaluate the conditions of material biocompatibility.
which endows nanocellulose a reactive surface covered Diverse CNC-based materials as bioscaffolds for cell culti-
with numerous active hydroxyl groups. For each anhydro- vation will be further discussed in following section.
glucose unit, the reactivity of hydroxyl groups on different Hemocompatibility (or blood compatibility) is another
positions is heterogeneous. The hydroxyl group at the 6 significant property of biocompatibility, especially for
position acts as a primary alcohol whereas the hydroxyl blood-contacting biomaterials and artificial organs, such
groups in the 2 and 3 positions behave as secondary alco- as artificial blood vessels, pumps, and artificial hearts.
hols. Indeed, the carbon atom which carries the hydroxyl Interestingly, recent study reported the regulation of blood
group in the 6 position is only attached to one alkyl group, metabolic variables by the presence of TEMPO-oxidized
while the carbons with the hydroxyl groups in the 2 and 3 cellulose nanofibers. The oral administration of TEMPO-
positions are joined directly to two alkyl groups, which will oxidized cellulose nanofibers to mice was proved to be
induce steric effects derived from the supramolecular effective for reducing the postprandial blood glucose,
structure of cellulose and the reacting agent [21]. It has plasma insulin, glucose-dependent insulinotropic polypep-
been reported that on the structure of cellulose, the hydro- tide, and triglyceride concentrations. It seems that TEMPO-
xyl group at the 6 position can react ten times faster than oxidized cellulose nanofibers have both promising
the other OH groups, while the reactivity of the hydroxyl hemocompatibility and unique biological activities [29].
group on the 2 position was found to be twice that of at Attributed to its biosynthesis procedure, BC is com-
the 3 position [22]. However, regarding the surface reactiv- monly regarded as a material possessing better biocompat-
ity of hydroxyl groups from nanocellulose (such as CNC), ibility than other types of nanocellulose. With an in vivo
the use of reactants or solvents may affect the reactivity study of subcutaneous BC implantation in rats for
of hydroxyl groups from different positions. Recent studies 12 weeks [30], no fibrotic capsule or giant cells were found,
reported the order of reactivity for hydroxyl groups on CNC indicating no foreign body reaction for the introduction of
as nucleophiles with OH-C6 = OH-C2 > OH-C3 by etherifi- BC in animals. Meanwhile, fibroblasts infiltrated BC, which
cation [23,24]. was well integrated into the host tissue, did not elicit any
Apart from reactive groups, another important issue for chronic inflammatory reactions [30]. Gama et al. investi-
the surface chemistry of nanocellulose is the surface gated the biocompatibility of small-diameter BC and
charges, which mainly refers to the negative sulfate esters peptide (Arg-Gly-Asp)-modified BC membranes subcuta-
ð—OSO 3 Þ on CNC. Surface sulfate esters are introduced on neously implanted in sheep for 1–32 weeks. Compared
CNC during sulfuric acid hydrolysis via condensation ester- with negative control samples [expanded polytetrafluoro-
ification (sulfation) between surface hydroxyls and a ethylene (ePTFE)], peptide-modified BC membranes were
H2SO4 molecule, using another H2SO4 molecule as a con- only mildly irritating to the tissue, with no significant dif-
densation agent. The H2SO4 hydrolyzed CNC, therefore, ferences in the inflammation degree [31]. In another study,
are highly negatively charged, and form a well-dispersed in vivo biocompatibility of the BC membrane was analyzed
aqueous colloidal suspension. Surface charge amount from through histological analysis of long-term subcutaneous
sulfate groups on CNC can be controlled through the dura- implants in mice. BC implants caused a mild and benign
tion and temperature of H2SO4 hydrolysis. Besides the inflammatory reaction that decreased with time and did
promotion of high stability of CNC in solvents, surface not elicit a foreign body reaction. Moreover, no differences
—OSO 3 groups with negative charges also provide CNC were observed between the controls and implanted ani-
the accessibility for biomedical application, such as elec- mals in thymocyte populations and in B lymphocyte pre-
trostatic adsorption of enzymes or proteins [25]. cursors and myeloid cells in the bone marrow [32]. With
the plasma recalcification time and whole blood clotting
2.3. Biological properties experiments, Gama et al. studied the hemocompatibility
of BC and BC-based biomaterials. It was reported that
2.3.1. Biocompatibility and hemocompatibility native BC and peptide (Arg-Gly-Asp)-modified BC mem-
Biocompatibility is referred to as the ability of a foreign branes both preserved original conformational structures
material implanted in the body to exist in harmony with and exhibited a favorable interaction (non-activation) with
tissue without causing deleterious changes, which is an platelets, which indicated BC and modified BC as promising
essential requirement for biomedical materials [26]. hemocompatible biomaterials [33]. Similar conclusions
Regarding the evaluation of cellulose biocompatibility, dif- were recently reported for the hemocompatibility study
ferent studies provide various results due to the range of of BC/polypyrrole [34] and BC/polyvinyl alcohol biocom-
methodologies and sample preparations. According to the posites [35].
early reports [27,28], cellulose can be generally considered
to be broadly biocompatible, invoking only moderate (if 2.3.2. Biodegradability in vivo
any) foreign body responses in vivo. However, it is well For some applications (e.g. artificial heart valves or
known that cellulose is not readily degraded by the human menisci), biocompatible, non-biodegradable materials
body because it lacks cellulolytic enzymes, which will may be acceptable whereas for other applications (e.g. arti-
inevitably cause some incompatibility. It is a pity that ficial bone grafts), the bioresorbable material enabling tis-
direct investigations on the biocompatibility of CNC and sue regeneration is preferable [36]. In terms of
biodegradation, cellulose may be considered as non- inflammation due to the easy self-aggregation and non-
biodegradable in vivo or, at best, slowly degradable, due degradation of nanocellulose in the body of animals.
to the lack of cellulase enzymes in animals. However, the Kovacs et al. initially studied the inherent eco-toxicol-
form (i.e. crystallinity, hydration and swelling) of cellulose ogy of cellulose nanocrystals with aquatic organisms
may affect the degree of degradation, absorption and (different species of fish) [42]. Rainbow trout hepatocytes
immune response. Nonenzymatic, spontaneous biodegrad- were selected as the model cells, and the toxicity monitor-
ability of cellulose chains may perhaps account for slow ing program as well as the in-depth toxicity assessment
breakdown of unaltered cellulose within the human body, component was included in the toxicity testing strategy.
though this is admittedly conjecture and has not been ade- With the eco-toxicological characterization, CNC was
quately studied [37]. In an early in vivo study, Miyamoto found to have low toxicity potential and environmental
et al. found that the degradation of cellulose and cellulose risk, but showed no harm to aquatic organisms at concen-
derivatives in canine specimens depended significantly on trations that could occur in receiving waters. In another
the cellulose crystalline form and chemical derivatization report, the cytotoxicity of CNC against nine different cell
[27]. Regenerated cellulose prepared by deacetylation of lines was determined both by 3-(4,5-dimethylthiazol-2-
cellulose acetate (presumably the highly crystalline cellu- yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and lac-
lose II polymorph) did not measurably degrade over the tate dehydrogenase (LDH) assay, and no cytotoxic effects
course of the 6-week experiment. Contrarily, however, up of CNC against any of these cell lines in the concentration
to 75% (w/w) of equivalent samples of amorphous regener- range and exposure time studied (0–50 lg/mL and 48 h)
ated cellulose were degraded and absorbed over the same were reported [46]. However, recently it was reported that
experimental period. Another study reported that CNC was CNC may induce some slight dose-dependent cytotoxic
actually more biodegradable than fullerenes and carbon and inflammatory effects on human lung cells, especially
nanotubes in aqueous environments, but without the the risk with inhalatory exposure under high concentra-
in vivo investigation of biodegradability [38]. Recently, oxi- tions of released CNC powders [43].
dized cellulose was rendered more vulnerable to hydroly- Regarding the toxicity of cellulose nanofibrils, no
sis and therefore potentially degradable by the human inflammatory effects or cytotoxicity on mouse and human
body. Based on this strategy, researchers attempted to macrophages, and only low acute environmental toxicity
enhance the biodegradability of nanocellulose through oxi- (assessed with kinetic luminescent bacteria test) have
dation, such as the report of improving BC degradability been reported [47]. VTT Technical Research Centre in
in vitro (in water, phosphate buffered saline, and simulated Finland proposed an evaluation report on the systemic
body fluid) through periodate oxidation [39,40]. With the study of CNF for in vitro cytotoxicity, genotoxicity, immu-
pre-c-irradiation and sodium periodate oxidation treat- notoxicity, and neurotoxicity, together with pharyngeal
ments on BC membranes, it was reported that in vitro deg- aspiration study on mice. The results revealed low cytotox-
radation of oxidized BC involved two major phases, (1) icity and no DNA or chromosome damage from CNF, but
initial rapid degradation of about 70–80% of the entire pulmonary inflammation for mouse experiment possibly
sample; (2) slower degradation of an additional 5–10% induced by the particulate/bacteria from CNF [49,50]. Pere-
which eventually levels off leaving a small amount of non- ira et al. evaluated the in vitro cytotoxicity and the effect on
resorbable material. Further experiments on in vivo degra- gene expression of CNF to fibroblasts cells. It was reported
dation (male New Zealand White rabbits) showed the that low concentrations of CNF (100 lg/mL) have no obvi-
marked degradation of oxidized BC membranes at all-time ous toxicity, whereas high concentrations of CNF (2000
points, with the most rapid degradation occurring in the and 5000 lg/mL) will cause the sharp decrease of cell via-
first 2–4 weeks [41]. bility and affect the expression of stress- and apoptosis-
associated molecular markers [52]. Alexandrescu et al.
2.3.3. Toxicology compared the cytotoxicity on fibroblast cells of pure CNF
Even though earlier studies have reported nanocellu- and surface modified-CNF with crosslinking agent polyeth-
lose to have no or low toxicity (comparable to that of table yleneimine (PEI) and surfactant cetyl trimethylammonium
salt), when used as biomedical materials, the issues of tox- bromide (CTAB). In comparison with no acute toxic phe-
icology and safety concerns for these natural nanomateri- nomena for pure CNF, both modified-CNF samples caused
als should be further emphasized. Since the beginning a significant reduction in cell viability and proliferation
over twenty years ago, the nanotoxicology research for [51]. Interestingly, in another recent study, cationic
nanoparticles has built a comprehensive assessment modified-CNF (trimethylammonium-CNF) was reported
system, such as for metallic nanoparticles (Au, Ag nanopar- to display a better cytocompatibility than unmodified
ticles, quantum dot, etc.) and carbon nanotubes. However, and anionic modified-CNF (carboxymethylated-CNF) [53].
the toxicology study of nanocellulose and nanocellulose- Attributed to biosynthesis procedure during the prepa-
based biocomposites is still restricted at a very preliminary ration, bacterial cellulose is commonly regarded as one of
stage (mainly on the level of cytotoxicity). Table 1 summa- the most biocompatible material in the family of nanocel-
rizes recent reports on toxicology experiments and conclu- lulose. As shown in Table 1, no cytotoxicity for BC was
sions for nanocellulose. On the whole, there is no evidence obtained according to the evaluation on osteoblast cells,
for serious influence or damage of nanocellulose on both endothelial cells, and mouse feeding experiment [54–56].
cellular and genetic level as well as in vivo organ and ani- Although studies conducted so far on nanocellulose
mal experiments. However, the inhalation of plentiful reported the absence of serious environmental and biolog-
nanocellulose (especially for CNC) may induce pulmonary ical concerns, research and systematic assessment of
Table 1
Toxicological evaluations of nanocellulose.
Type Toxicological experiment Conclusion Ref.

CNC Acute lethal test Low toxicity potential [42]
Multi-trophic assays Low environmental risk
Animal experiments with fathead minnow and Zebrafish
reproduction tests
In vitro rainbow trout hepatocyte assay
Respiratory toxicity of aerosolized CNC on the human airway Low cytotoxicity [43]
with a co-culture of human monocyte-derived macrophages, Somewhat (pro-)inflammatory cytokines
dendritic cells and a bronchial epithelial cell line
In vitro gene mutations No evidence of high toxicity [44]
In vitro and in vivo chromosomal tests
Skin irritation and sensitization tests
Animal experiments with rat feeding study (28 d)
Cytotoxicity evaluation with L929 cells Low cytotoxicity at low CNC concentration [45]
Cytotoxicity evaluation with nine different cell lines No cytotoxic effects in the concentration range (0–50 lg/mL) [46]
and exposure time (48 h)
CNF Cytotoxicity evaluation with human monocyte and mouse No evidence of inflammatory effects or cytotoxicity [47]
macrophages
Kinetic luminescent bacteria test for acute environmental
toxicity
In vitro genotoxicity with enzyme comet assay No significant DNA damage [48]
Neurotoxicity and systemic effects with a nematode model Low or no cytotoxicity [49,50]
In vitro pharyngeal aspiration study for pulmonary No DNA and chromosome damage
immunotoxicity and genotoxicity with mice Pulmonary inflammation
Cytotoxicity evaluation with 3T3 fibroblast cells (including the No toxic phenomena for pure CNF [51]
test of cell membrane, cell mitochondrial activity and DNA Somewhat cytotoxicity for modified-CNF (with PEI or CTAB
proliferation) surface modification)
Cytotoxicity evaluation with bovine fibroblasts cells Low cytotoxicity at low CNF concentration (0.02–100 lg/mL) [52]
Effects of gene expression in vitro Reduction of cell viability and affection of the expression of
stress- and apoptosis-associated molecular markers at high CNF
concentration (2000–5000 lg/mL)
Cytotoxicity evaluation with human dermal fibroblasts No evidence of cytotoxicity for pure CNF [53]
Improved cytocompatibility of EPTMAC-modified CNF
BC Cytotoxicity evaluation with osteoblast cells and L929 fibroblast No evidence of cytotoxicity [54]
cells
Cytotoxicity evaluation with human umbilical vein endothelial No evidence of toxicity in vitro and in vivo [55]
cells
Animal experiment with C57/Bl6 male mouse
In vitro immunoreactivity with human umbilical vein Non-toxicity and non-immunogenicity [56]
endothelial cells
In vivo intraperitoneal injection study with BALB/c male mice
Abbreviations: PEI, polyethyleneimine; CTAB, cetyl trimethylammonium bromide; EPTMAC, glycidyltrimethylammonium chloride.
eco-toxicology of nanocellulose still need deeper investiga- is always a most concerned topic for biologists and
tions, especially aimed to the effects and mechanisms of material scientists. In some studies, information on nano-
nanoparticles aggregation in the body, and long-term cellulose and its application are mentioned as the term
in vivo toxicity evaluation of nanocellulose. Moreover, not ‘‘biocellulose,’’ which is attributed to the unique proper-
only the toxicity of nanocellulose itself, what the toxicity ties and potential of nanocellulose in the study of diverse
effects will be induced by the incorporation of nanocellu- biomedical materials. According to the report of ‘‘Future
lose is another important issue, which indicates the eco- Markets Inc.’’ in ‘‘The global market for nanocellulose
toxicology of nanocellulose-based materials. Despite no to 2017’’ published in October, 2012, there will be about
significant cytotoxicity of nanocellulose-based materials $ 97 billion estimates for medical and life sciences mar-
(generally hydrogels) in many studies [57–60], there was kets impacted by nanocellulose [12]. It is the aim of this
also a report of negative effect on biocompatibility for section to discuss the research in biomedical application
nanocellulose-based composites [61]. of nanocellulose with selected latest examples. From the
molecular level (cellular cultivation) to macroscopic
biomaterials (drug delivery, substitute implants, tissue
3. Nanocellulose-based biomedical materials repair, regeneration, etc.), diverse studies and new
frontiers together with future strategies on nanocellu-
The development of novel biomedical materials from lose-based biomedical materials are highlighted and
natural polymers for practical and clinical applications remarked.
3.1. Tissue bioscaffolds for cellular culture induced the spheroid formation of cells [69]. A novel scaf-
fold composed of natural polymers was reported to culture
Attributed to the properties of biocompatibility and NIH3T3 fibroblast, involving the components of pectin,
right mechanical properties similar to natural tissue, nano- carboxymethyl cellulose and CNF [70]. Recently, highly
cellulose-based biomaterials can provide a cell-friendly porous and biomimetic nanocomposites that allow for
environment to encourage cells attachment and prolifera- modulating the growth of L-929 fibroblasts were prepared
tion as a special tissue bioscaffold. Diverse cellular species by incorporating calcium peroxide (CaO2) and catalase into
cultured on nanocellulose-based biomaterials have been CNF matrix. Fig. 3(b and b0 ) shows CLSM images of L-929
reported, and the forms of these materials include hydro- fibroblasts cultured on pristine CNF and CNF modified with
gels, composites, electrospun nanofibers, sponges, and 15 wt% calcium peroxide/catalase composite scaffolds. The
membranes. BC seems to be the most prevalent choice addition of CaO2 and catalase induces the presence of
for the medium of cells culture because of its low cytotox- hydrogen peroxide (H2O2) or oxygen (O2), which affected
icity and high porosity. the survival of cells. Three-dimensional porous morphol-
Regarding CNC-based media for the culture of cells, ogy of CNF-based scaffolds both facilitated the diffusion
some studies applied conventional suspensions of unmod- of generated gases and provided great niches for cell
ified or fluorescent-modified nanocrystals as the environ- growth. It was reported that due to the generation of
ment for cells. From insect cells Sf9 and Hamster lung H2O2, cell attachment decreased, and cell proliferation
fibroblast V79, to human foreskin fibroblasts, human was delayed; while the generation of O2 played a useful
embryonic kidney cells HEK-293 and human lung cell, no role in supporting cell proliferation [71].
significant cytotoxicity to various cell models was found, Various BC-based bioscaffolds for the application of cel-
and promising cellular uptake and proliferation were lular culture can be mainly divided into three aspects,
reported in these studies [43,62–64]. Using a spin-coating which are BC pellicle/membrane scaffolds, BC/matrix
method, Dugan et al. prepared submonolayer film with ori- biocomposite scaffolds, and surface-modified BC pellicle
ented surfaces of adsorbed CNC. Due to the shape and scaffolds. Several latest studies focused on pure BC as scaf-
nanoscale dimensions of CNC, murine myoblasts cells folds for supporting cellular adhesion and proliferation.
C2C12 adopted increasingly oriented morphologies in Favi et al. investigated BC as a hydrogel scaffold for the cul-
response to more densely adsorbed and oriented nanocrys- ture of equine-derived bone marrow mesenchymal stem
tals surface. With a mean feature height of only 5–6 nm, cells (EqMSCs). As shown in Fig. 3(c and c0 ), BC hydrogels
CNC surface presented the smallest features to induce con- were cytocompatible, and significantly supported cellular
tact guidance in skeletal muscle myoblasts [65,66]. adhesion and proliferation. The cells seeded on the BC
Recently, electrospun nanofibers based on CNC, bearing hydrogel were observed to be viable and metabolically
suitable mechanical property, in vitro degradation and active [72]. Park et al. investigated the alteration of func-
basic cytocompatibility, were proved to be promising bio- tion of human umbilical vein endothelial cells treated with
nanocomposite scaffolds for cell culture. It was reported a,b-unsaturated aldehyde on BC scaffold. The study indi-
that electrospun maleic anhydride-grafted poly(lactic acid) cated that a,b-unsaturated aldehydes in cigarette smoke
nanofibers reinforced with CNC can be the supporting scaf- induce altered endothelial cell functions including mor-
folds to culture the human adult adipose derived mesen- phology, adhesion, proliferation, viability, and growth on
chymal stem cells (hASCs) and promote cell proliferation. BC [73]. Another work presented a cellular building unit
Low CNC concentration effectively improved the thermal made from microstrand-shaped BC covered with mamma-
stability and mechanical properties of scaffolds, but not lian cells. By folding or reeling the building unit, the multi-
significantly caused any cytotoxic effect on hASCs prolifer- ple shapes of millimeter-scale cellular constructs (coiled
ation within 7 days [67]. Another study reported the appli- and ball-of-yarn-shaped structures) were investigated.
cation of all-cellulose scaffold materials (CNC/cellulose) to Histological analysis of the cellular constructs indicated
culture cells, and the influence of CNC orientation in scaf- that the BC microstrand served as a pathway of nutrition
folds to cell growth was investigated. Fig. 3(a and a0 ) pre- and oxygen to feed the cells in the central region [74].
sents confocal laser scanning microscope (CLSM) images Recently, the laser patterning post-processing was used
of Human dental follicle cells (hDFCs) cultured on electro- on BC to solve the limitation of small and heterogeneous
spun CNC/cellulose nanofibers for 3 and 7 days. It was pore size for the ingrowth of cells. After laser perforation,
shown that CNC can well dispersed in electrospun scaffolds BC hydrogels displayed high biocompatibility and the
and achieve considerable orientation along the long axis resulting channels supported migration, matrix production
direction. Cultured cells can proliferate rapidly not only and phenotypic stabilization of bovine chondrocytes,
on the surface but also deep inside the scaffolds. More which qualified perforated-BC as a sustainable scaffold
interestingly, the aligned nanofibers of CNC/cellulose for cell ingrowth [75].
exhibited a strong effect on directing cellular organization Regarding the materials used for the BC/matrix scaffold
[68]. systems, chitosan, agarose, alginate, collagen, gelatin, poly-
Under controlled concentrations, CNF aqueous suspen- pyrrole, and poly(3-hydroxybutyrate-co-4-hydroxybuty-
sions can spontaneously form hydrogels to provide suit- rate) [P(3HB-co-4HB)] have been studied as matrices to
able environment with required mechanical support for culture cells. Polypyrrole was in situ polymerized onto
cell growth and differentiation. It was reported that CNF the surface of BC to produce the BC/polypyrrole membrane
hydrogels can promote the cellular differentiation of the scaffold, and performed the seeding of PC12 rat neuronal
human hepatic cell lines (HepG2 and HepaRG), and cells. Conductive polypyrrole coating on BC acted as an
Fig. 3. Confocal laser scanning microscope (CLSM) images of nanocellulose bioscaffolds for cell culture. Human dental follicle cells (hDFCs) cultured on
electrospun CNC/cellulose nanofibers for (a) 3 days, (a0 ) 7 days; scale bar = 50 lm (adapted with permission from [68]). L-929 fibroblasts cultured for 7 days
on (b) pristine CNF, (b0 ) CNF/15 wt% calcium peroxide/catalase composite; scale bar = 40 lm (adapted with permission from [71]). Equine-derived bone
marrow mesenchymal stem cell (EqMSCs) cultured on BC hydrogels for (c) 2 days, (c0 ) 14 days; scale bar = 50 lm (adapted with permission from [72]).
active interface for tissue engineering, which was benefi- coated with bone morphogenetic protein-2 (BMP-2). The
cial for the regulation of cell activity through electrical alkaline phosphatase activity assays indicated that BC
stimulations [76]. Incorporated with gelatin or hydroxyap- had a good biocompatibility and induced the differentia-
atite (Hap) to enhance the bioactivity, Wang et al. devel- tion of mouse fibroblast-like C2C12 cells into osteoblasts
oped a porous BC membrane with regular vertical pore in the presence of BMP-2 in vitro. Within a certain range
arrays via a laser patterning technique. Chondrogenic rat (0–3 mg/scaffold), the osteogenic activity of induced oste-
cells were cultured on these membrane scaffolds, and the oblasts was positively correlated to the concentration of
scaffolds well supported the attachment and proliferation BMP-2. In vivo subcutaneous implantation studies further
of cells together with the preservation of cellular viability showed that BC scaffolds modified with BMP-2 promoted
[77]. Another study prepared BC-biocomposite scaffolds more bone formation and higher calcium concentration
by freeze-drying using polymeric P(3HB-co-4HB) as matrix than the BC scaffolds alone at 2 and 4 weeks [79]. Another
and trifluoro-acetic acid as co-solvent. Chinese hamster surface modification strategy using plasma treatments (O2,
lung fibroblast cells were incubated on this composite scaf- N2, or CF4 plasmas) on BC reported altered changes of sur-
fold for 48 h, which exhibited the capability of cell adhe- face property involving more hydrophilic BC with O2 or N2
sion and proliferation, as well as better biocompatibility plasma treatment, and hydrophobic BC with CF4 plasma
than pure P(3HB-co-4HB) scaffold [78]. treatment. Furthermore, different surface plasma treat-
To further enhance cell attachment on BC-based bio- ments on BC scaffolds will provide distinct effects for the
scaffolds, surface modifications can be performed, such as adhesion of L-929 fibroblast and Chinese hamster ovary
protein or peptide coatings, plasma treatment, surface sul- cell line. It was reported that the cell adhesion and prolif-
fation or phosphorylation. Shi et al. prepared a BC scaffold eration of both cells was significantly improved on
CF4-modified BC, while unremarkable increase of cells pro- system for drug delivery. The presence of CNC in algi-
liferation for O2 or N2-modified BC scaffolds was reported nate-based microspheres showed more consistent swelling
in comparison with pristine BC scaffold [80]. Kuzmenko patterns, higher encapsulation efficiency, and promising
et al. reported a universal method of protein bioconjuga- sustained release profiles of the drug [87]. Regarding the
tion on BC scaffolds in order to increase cell adhesion. application of nanocellulose in the fabrication of hydrogels,
The surface of BC scaffolds was modified with two pro- CNC was chemically grafted with cyclodextrin and partici-
teins, fibronectin and collagen type I, through the biocon- pated in the architecture of hydrogels via in situ inclusion
jugation applying 1-cyano-4-dimethylaminopyridinium interactions. The drug release study revealed the perfor-
(CDAP) tetrafluoroborate as the intermediate catalytic mance of hydrogels as drug carriers for the in vitro release
agent. Effective promotion of cell attachment by CDAP of doxorubicin and exhibited the behavior of prolonged
treatment to BC scaffolds was shown for human umbilical drug release with special release kinetics and mechanisms,
vein endothelial cells and the mouse mesenchymal stem which were the ‘‘obstruction effect’’ and ‘‘locking effect’’
cells [81]. Recently, the surface oxidized modification of attributed to the good dispersion of the nanoparticles
BC with TEMPO-C6 or dialdehyde-C2, 3 was also reported and the formation of a rigid network of CNC [91]. Kolakovic
to promote the adhesion and proliferation of cells [53,82]. et al. reported the application of CNF films for long-lasting
sustained drug delivery by a filtration processing. The drug
3.2. Drug excipient and drug delivery release studies showed generally sustained drug release
over periods of three months for model drugs. Interest-
Possessing excellent compaction property, cellulose ingly, with the same CNF drug carriers, the release of indo-
has a long history of application in the pharmaceutical methacin showed diffusion limited release, while
industry, in particular as pharmaceutical excipients to itraconazole and beclomethasone showed almost zero-
condense drug-loaded matrices as suitable tablets for oral order release kinetics. The dependence of model drug used
administration. Despite an extended history of use in tab- for release kinetics was attributed to the different drug sol-
leting, there is still continuing research into the use of ubilities in the dissolution medium, and the varied effects
cellulose with new types of cellulose (viz. nanocellulose) of drug binding to the CNF chains [100]. In another study,
in advanced drug-loaded systems whereby the rate of Valo et al. coupled a genetically engineered hydrophobin
tablet disintegration as special excipients, or prolonged fusion protein with cellulose binding domains (CBD) and
drug release as novel drug carriers. As a drug delivery coated itraconazole drug nanoparticles for subsequent
excipient, Burt et al. investigated the capability of pure binding to CNF. Hydrophobin or hydrophobin-double CBD
CNC to bind water soluble antibiotics (tetracycline and was selected to facilitate drug molecules binding to CNF
doxorubicin), and the potential of cationic-CNC to bind matrix. The presence of CNF provided protection for drug
nonionized hydrophobic anticancer agents (docetaxel, nanoparticles and notably increased formulation storage
paclitaxel, and etoposide) [83]. Besides direct use as stability during the formulation process and storage. It
excipient, CNC can also be used as co-stabilizer to was reported that in the carrier system containing
improve the physicochemical and flow properties of poly- hydrophobin-coated CNF, drug nanoparticles around
meric excipients. Acrylic beads prepared via emulsion 100 nm could be stored for more than ten months [89].
polymerization using CNC as co-stabilizer were proved Regarding the studies of BC in drug delivery, Huang et al.
to be a suitable excipient. The presence of CNC affected recently investigated the effects of BC membranes for the
positively the size and size distribution of the bead excip- delivery of berberine hydrochloride and berberine sulfate
ient, which formed a stable structure together with low in comparison with commercial tablet. It was reported that
flow time and reduced cotangent of angle [84]. In another BC is a promising drug carrier to significantly extend the
work, investigating spray-drying treatment on tablets, release duration of model drugs [104]. Müller et al. per-
CNF exhibited a better ability to pack with lower powder formed BC as a hydrogel carrier for bovine serum albumin
porosity than commercial microcrystalline cellulose, as the model drug. It was shown that freeze-dried BC sam-
which indicated novel spray-dried CNF excipient for tab- ples exhibited lower uptake of albumin than native, never-
let production [85]. With the same technique of spray- dried BC and that release of the model drug was a result of
drying, BC was film-coated on tablets, and provided soft, both diffusion- and swelling-controlled processes. Further
flexible, and foldable nanocellulose films, which exhibited studies using luciferase as the model protein indicated that
better mechanical properties in comparison with tradi- the three-dimensional structure and activity of this protein
tional Aquacoat ECD materials (polymeric materials com- can be preserved during the binding and release from BC
posed of 30 wt% aqueous ethylcellulose dispersion) hydrogels [94].
materials [86]. Unlike traditional trapping strategy, some researchers
Common forms of nanocellulose-based drug carriers recently attempted to directly attach drug molecules on
can be mainly divided into three aspects, viz. microspheres nanocellulose, which was performed using covalent cou-
(or microparticles), hydrogels (or gels), and membranes (or pling between modified nanocellulose and drug molecules.
films). Table 2 summarizes various drug carrier systems With a series of oxidation, reductive-amination, and ester-
based on nanocellulose. It was reported that solid carriers ification reactions in aqueous media, a novel CNC-based
formed from nanocellulose and different matrices spatially delivery system attached to the syringyl alcohol linker
trapped drug molecules, and imparted the regulation of through a c-aminobutyric acid spacer molecule can be pro-
drug release. Lin et al. developed a pH-sensitive CNC/ duced, on which small model amine drugs (e.g., phenylpro-
sodium alginate microsphere-based controlled release panolamine) can be covalently connected [110]. Similarly,
Table 2
Drug carrier systems based on nanocellulose.
Carrier form Material component Model drug Release time and medium Mechanism model Ref.
Nanocellulose Matrix
Microsphere CNC EA; MMA; BMA Propranolol hydrochloride 12 h in pH 6.8 PBS – [84]
or bead Sodium Theophylline 16 h in pH 7.4, pH 6.8, pH 1.0 Ritger-Peppas [87]
alginate PBS equation
CNF – Indomethacin; nadolol; atenolol; 10–14 d in pH 7.4 PBS Baker-Lonsdale [88]
metoprolol tartrate; verapamil; mathematical
ibuprofen model
Hydrophobin Itraconazole 90 min in pH 1.2 NaCl/HCl – [89]
solution
Suspension CNC CTAB Paclitaxel; docetaxel; DOX; TET; 1–4 d in PBS – [83]
etoposide
Chitosan Procaine hydrochloride 100 min in pH 8 NaCl solution – [90]
oligosaccharide
Hydrogel or CNC Cyclodextrin/ DOX 6.5 d in water Ritger-Peppas [91]
gel Pluronic equation
Cyclodextrin/ Bovine serum albumin 20 h in pH 7.4 PBS – [92]
Pluronic
Regenerated Bovine serum albumin 48 h in simulated body fluid Fickian diffusion [93]
cellulose law
BC – Bovine serum albumin 48 h in pH 7.4 PBS Ritger-Peppas [94]
equation
– Collagen; hyaluronan; growth 36–96 h in PBS – [95]
factors
Acrylic acid Bovine serum albumin 8 h in simulated intestinal Ritger-Peppas [96]
fluid equation
Polyacrylamide Theophylline 24 h in pH 7.4 PBS Ritger-Peppas [97]
equation
Membrane or CNF – Paracetamol 5–10 min in water – [85]
coating for – Lysozyme 10 h in pure water or water/ Fick’s second law [98]
tablet ethanol solution
– Caffeine 9 h in water Higuchi equation [99]
– Indomethacin 30 d in pH 5.0 phosphate Higuchi equation [100]
buffer
Itraconazole 90 d in pH 1.2 NaCl/HCl
solution
Beclomethasone dipropionate 90 d in water
BC – Paracetamol 2 h in pH 5.8 PBS – [86]
– Lidocaine 7 h in pH 7.4 PBS – [101]
– Lidocaine; ibuprofen 8 h in pH 7.4 PBS Fickian diffusion [102]
law
– Caffeine 15 h in pH 7.4 PBS – [103]
– Berberine hydrochloride, 24 h in pH 2.1 HCl or H2SO4 Ritger-Peppas [104]
berberine sulfate solution; pH 6.8 PBS; pH 12.0 equation
NaOH solution
– Glycerin 24 h in in vivo evaluation – [105]
(skin)
Poly(vinyl Vanillin 1 h in water Ritger-Peppas [106]
alcohol) equation; Fickian
diffusion law
Nanofiber CNC Poly(lactic Columbia blue 48 h in water Higuchi equation [107]
acid)
Hordein/zein Riboflavin 24 d in pH 7.4 PBS – [108]
Aerogel CNF – Beclomethasone dipropionate 700 min in pH 8.0 SDS solution – [109]
Abbreviations: BMA, butylmetacrylate; CTAB, cetyl trimethylammonium bromide; DOX, doxorubicin hydrochloride; EA, ethyl acrylate; MMA, methyl-
methacrylate; TET, tetracycline hydrochloride; PBS, phosphate-buffered solution; SDS, sodium dodecyl sulfate.
with the binding of bi-functional fusion protein on CNF and drug release, interactions between drug molecules and
CNC, hydrophobic solid drug nanoparticles can be nanocellulose [112], as well as possible reduction or
adsorbed by the packed protein film on nanocellulose destruction of drug activity and structure. Recently, it
and can improve the long-term stability of drugs under was reported that the surface charges on nanocellulose
physiological condition [111]. (TEMPO-oxidized negatively charged CNF) presented the
The use of natural nanocellulose to deliver drugs is an adverse impact on the chemical stability of drug molecules
attractive idea, but many issues are still under investiga- (aspirin), which will accelerate the decomposition of drugs
tion, especially regarding the influence and regulation of [113].
3.3. Immobilization and recognition of enzyme/protein for the immobilization of enzyme and protein. Another
key point in enzyme/protein immobilization is the selec-
To overcome the problem of instability and rapid loss tion of the immobilization method, such as adsorption,
of catalytic activity during the operational and storage entrapment, or covalent binding on carrier material.
periods resulting from autolysis, unfolding, and aggrega- Available hydroxyl groups and possible negative charges
tion of enzyme and protein, the immobilization of (CNC and CNF) on the surface of nanocellulose provide
enzyme/protein on carriers has been considered as a the possibility of enzyme/protein immobilization on the
powerful technique in diverse fields ranging from food basis of chemical conjunction and electrostatic adsorp-
technology to biomedical and biosensor engineering. An tion. Table 3 summarizes recent studies on surface immo-
ideal carrier material for enzyme/protein immobilization bilization of enzyme or protein on nanocellulose on the
should be biocompatible without compromising the pro- basis of different strategies (chemical binding or physical
tein structure and biological activity. Furthermore, this adsorption). Covalent immobilization of enzyme/protein
carrier material should be easily processed to enhance on nanocellulose can provide significantly high enzyme/
the enzyme/protein loading and activity as well as the protein loading and excellent stability, but always treated
stability in both operation and storage. As a nontoxic, with complicated chemistry procedure. Physical approach
noncarcinogenic, biocompatible, and in no way injurious is simple, cheap, and allows better preservation of the ori-
in the biological environment material, nanocellulose ginal structure of enzyme/protein, but with limited load-
meets the rigid medical requirements of suitable carrier ing and efficiency of immobilization.
Table 3
Surface immobilization of enzyme or protein on nanocellulose.
Strategy Type Enzyme or protein model Specific procedure Ref.

Chemical CNC Lysozyme Esterification [114]
conjunction Coupling with enzyme
Peroxidase Surface activation [115]
Coupling with protein
Alcohol oxidase; CGTase Surface deposited gold nanoparticles [116]
Coupling with enzyme
Glucose oxidase Polyetherimide surface cationization [117]
Deposited gold nanoparticles
Tryptophan-based peptides Surface TEMPO-oxidation [118]
Coupling with peptides
Human neutrophil elastase Esterification with glycine [119]
Coupling with elastase
Papain enzyme Embedment of Fe3O4 and Au on the surface [120]
Surface activation
b-Casein Azide modification on CNC reducing ends [121]
Acetylene modification on b-Casein
Click reaction for the coupling of protein
CNF Alkaline phosphatase; anti-hydrocortisone antibody Amine/epoxy/carboxylic acid modification [122]
Avidins Surface TEMPO-oxidation [123]
Physical adsorption CNC Heptapeptide Adsorption and identification of peptide [124]
Diblock protein (Elastin-co-Cartilage oligomeric Copolymerization of diblock proteins [125]
matrix) Adsorption of proteins
CNF Pancreatic serine protease trypsin Synthesis of nanocellulose-based hydrogel [126]
Entrapment of protein
Antihuman IgG antibody Oxidation and activation [127]
Adsorption of protein
Human immunoglobulin G (IgG) Surface grafting from poly(AMA-co-HEMA) [128]
Adsorption of modified peptide (acetylated-
HWRGWVA)
Laccase Adsorption of enzyme [129]
Lipase Adsorption of enzyme [130]
BC Hemoglobin; myoglobin; albumin; lysozyme Surface phosphorylation [131]
Hemoglobin Surface quaternary ammonium [132]
Glutamate decarboxylase Preactivation [133]
Adsorption of enzyme
Urease Surface modification with dye molecule [134]
Adsorption of enzyme
Abbreviations: Poly(AMA-co-HEMA), poly(2-aminoethyl methacrylate hydrochloride-co-2-hydroxyethylmethacrylate).

Available hydroxyl groups and negative charges on the CNF [122]. More studies reported the physical adsorption
surface of CNC provide the possibility of enzyme/protein to immobilize enzyme or protein on CNF. In order to
immobilization on the basis of chemical conjunction and enhance the interactions of enzyme/protein immobiliza-
electrostatic adsorption. Regarding the chemical strategy, tion on CNF, some studies attempted to modify the surface
some studies directly immobilized enzyme/protein on of CNF before the entrapment of enzyme/protein mole-
CNC with chemical grafting, such as immobilization of cules, such as oxidation and activation pretreatments
lysozyme on amino-glycine-CNC with carbodiimide-acti- [127] and surface polymeric grafting [128].
vation coupling reaction [114]; or peroxidase on CNC with The immobilization of enzyme/protein on BC is mainly
the activation of cyanogen bromide treatment [115]. achieved by physical interactions between BC and original
Another approach consists in functionalizing with smaller enzyme/protein molecules, such as electrostatic adsorp-
nanoparticles (generally gold nanoparticles, Au), and then tion of proteins on modified BC with surface phosphoryla-
realizing the immobilization of enzyme/protein on CNC tion or quaternary ammonium [131,132]. Recently, the
with the aid of inorganic nanoparticles. Luong et al. inves- properties and feasibility of BC membrane for the immobi-
tigated CNC/Au as a catalytic platform for enzyme immobilization of glutamate decarboxylase was reported. With a
lization, which exhibited significant biocatalytic activity pre-activation treatment followed by protein adsorption,
and preservation of original activity. The recovered specific immobilized glutamate decarboxylase on BC membrane
activities were about 70% and 95% for CGTase and alcohol exhibited good retention of protein activity (89.17%), least
oxidase enzymes, respectively [116]. More complicated leakage, and high stability (5% loss), which was associated
carrier based on CNC/PEI/Au was developed to immobilize to the porosity of the carrier material [133].
glucose oxidase enzyme [117]. Mahmoud et al. developed
a special nanocomposite consisting of magnetite nanopar- 3.4. Substitutes/medical biomaterials
ticles (Fe3O4) and Au nanoparticles embedded on CNC as a
magnetic support for covalent conjugation of papain and Promising mechanical properties and good biocompati-
facilitated the recovery of immobilized papain [120]. The bility of nanocellulose promote its research and develop-
conjugated material retained high enzyme activity and ment as substitute/medical biomaterial, such as the
good stability and reusability. Based on the similar strategy replacement of blood vessel (vascular graft), soft tissue,
of enzyme/protein immobilization, labeled DNA or enzyme and nucleus pulposus. The studies of nanocellulose as
was immobilized on CNC as a bioprobe, and used for the blood vessel replacement are most attracting and fruitful,
identification or recognition of target DNA sequence, as reported from the effects in various animal experiments
enzyme molecules, or as the platform for immunoassays before clinical research. Regarding the studies on nanocel-
and diagnostics [135,136]. Edwards et al. reported a color- lulose as soft tissue and nucleus pulposus, most reports are
imetric approach for the detection of human neutrophil still in the fundamental stage, and mainly focus on the
elastase (HNE) using peptide conjugated CNC [119]. comparison of different properties between nanocellu-
Recently, the immobilizing effects of CNC and the diblock lose-based materials and real organs.
proteins bearing two different self-assembling domains
[elastin (E) and the coiled-coil region of cartilage oligo- 3.4.1. Blood vessel replacement
meric matrix protein (C)] were investigated. It was One of the most common treatments to cardiovascular
reported that the protein CE with prevalent displaying of disease is the coronary bypass graft surgery, which is per-
the E domain interacted more with CNC leading to a stron- formed to supply blood to the heart tissue with a suitable
ger network, while the protein EC, which is predominantly blood vessel replacement. Possessing good mechanical
C-rich on its surface, did not interact as much with CNC. strength (a burst pressure of up to 880 mmHg) and blood
This study suggested that the surface characteristics of biocompatibility, it is possible to develop nanocellulose
the protein polymers, due to folding and self-assembly, (especially for BC) as material for artificial tubes used as
were important factors for the interactions with CNC, and potential replacement of small (<4 mm) or large (>6 mm)
therefore of significant influence on the overall immobili- size vascular grafts. The team of Dieter Klemm (University
zation efficiency [125]. With the purpose of identifying Jena and Polymer Jena, Germany) was the first research
specific crystalline region of CNC for the immobilization organization to investigate and apply artificial vascular sub-
of enzyme or protein, Guo et al. reported the phage display stitute obtained with biomaterials from BC. They have dis-
technology involving biopanning assays and enzyme- cussed the application of BC as blood vessel replacement
linked immunosorbent assay to investigate this binding in some publications [137–139], and especially described
property. A model of consensus peptide was efficiently a clinical product named BActerial SYnthesized Cellulose
immobilized on CNC, and the analysis indicated that pep- (BASYCÒ) with high mechanical strength in wet state, enor-
tide exhibited a bent structure when bound, allowing the mous water retention property, and low roughness of inner
Y5 amino acid to form a CH/p stacking interaction and H- tube surface. It is reported that BASYCÒ from BC has been
bond with the glucose ring of cellulose [124]. successfully used as the artificial blood vessel in rats and
Regarding chemical conjunction of enzyme or protein pigs for microsurgery [140,141]. In comparison with con-
on CNF, some studies directly immobilized enzyme/protein ventional synthetic vascular graft materials, e.g. polyester
macromolecules on CNF with grafting reactions, such as (Dacron) and ePTFE, biosynthetic BC tubes can be suitable
avidins binding on oxidized CNF [123], protein immobili- for small diameter (<4 mm) vascular conduits, and restrain
zation of alkaline phosphatase and anti-hydrocortisone the phenomena of thrombus induction and stenosis. BASYC,
antibody on amine, epoxy, and carboxylic acid modified BActerial SYnthesized Cellulose (BC tubes) with different
Fig. 4. Examples of substitutes from nanocellulose. (a) BASYC, BActerial SYnthesized Cellulose (BC tubes) with different dimensions (adapted with
permission from [145]). (b) Vascular prostheses made of CNF-polyurethane placed between the brachiocephalic trunk and the right common carotid artery
in male patient (adapted with permission from [146]). (c) Comparison between pig meniscus (left) and BC hydrogel (right) (adapted with permission from
[147]). (d) Negative silicone mold used to guide the bacteria during the bacterial culture to reproduce the large-scale features of the outer ear (left); and 3D
BC implant prototype (1% effective cellulose content) produced in the shape of the whole outer ear according to the 3T MRI scanning technique (right)
(adapted with permission from [148]).
dimensions are shown in Fig. 4(a). Recently, various proper- the performance of these nanocellulose implants (in large
ties and biology evaluation of BC tubes as blood vessel animal and even human body studies) are still required,
replacement have been investigated, involving the issues addressing in particular their long-term stability and suit-
of BC biomaterial-induced coagulation [142], cell attach- ability to replace small-caliber blood vessels without sig-
ment, proliferation, viability and invasion [143], hemody- nificant thrombogenicity, study of compliance between
namic analysis and microcirculatory evaluation [144], etc. the graft and the surrounding native vessels, together with
Different from BC biosynthetic procedure, it is impossi- the postoperative complications
ble to directly fabricate the tubes from CNC and CNF.
Therefore, the development of CNC or CNF-based blood 3.4.2. Soft tissue–ligament, meniscus, and cartilage
vessel replacement commonly includes the use of a matrix replacements
material. Recently, Brown et al. reported the synthesis of The design and fabrication of materials suitable for soft
CNC/fibrin biocomposites for the potential application of tissue replacement are important aspects of the biomedical
small-diameter replacement vascular graft. CNC was cova- application. The demand for biomaterials to be used for
lently grafted on fibrin matrix, and provided nanorein- soft tissue replacement should not only provide similar
forcement in terms of strength and elasticity to the mechanical properties as the tissue it replaces, but also
composites [149]. However, this material has not been improve lifespan, biocompatibility, durability, and low
tested in vivo. Novel biomaterials from polyurethane rein- degree of calcification. Using a double-network method,
forced with CNF have been reported to be potentially used BC/polyacrylamide (PAAm) gels can be synthesized by
as vascular replacement. The presence of CNF in polyure- combining BC gel as the first network, and PAAm as the
thane improved the elastic properties of the material, cou- second network in the presence of a crosslinker. The BC/
pled with low thrombogenicity and exceptional physical PAAm gels presented high elongation and high tensile frac-
and mechanical properties. CNF/polyurethane biomateri- ture stress (40 ± 10 MPa), which was similar to the tensile
als, with a wall thickness of 0.7–1.0 mm, were applied as fracture stress of ligament (38 ± 10 MPa), and could be
vascular prostheses between the brachiocephalic trunk potentially used as ligament replacement [150]. Mathew
and the right common carotid artery in a 26-year-old male et al. also reported the preparation of CNF and CNF/colla-
patient with multiple endocrine neoplasia 2B (Fig. 4(b)). It gen composites for potential ligament. Both composites
is a pity that no further effect of this CNF/polyurethane bio- exhibited mechanical properties and stress relaxation
material in clinical study was reported. behavior comparable to those of natural ligaments and
Based on decades of research, it is successful to produce tendons. Further in vitro biocompatibility study on these
nanocellulose-based materials for blood vessel composites showed a positive response concerning
replacement under proper control of the biotechnological adhesion/proliferation and differentiation for both human
formation. However, detailed in vivo characterization of ligament and endothelial cells [151,152].
Gatenholm et al. compared the mechanical properties a nontoxic and biocompatible platform to cover growth fac-
of BC gel with traditional collagen meniscal implant mate- tors or cells, which will activate and accelerate the process
rial and real pig meniscus. It was reported that the Young’s of tissue repair and regeneration. Most studied applications
modulus of BC gel is similar to the one of pig meniscus, and of nanocellulose-based biomaterials for tissue repair,
five times higher than the one of collagen material. The regeneration, and healing are skin tissue repair (wound
results of promising cell migration and controlled menis- dressings) and bone tissue regeneration and healing.
cus shape (as shown in Fig. 4(c)) indicated that BC can be
an attractive material as meniscus implant [147]. Another
study investigated the friction and wear behaviors of BC 3.5.1. Skin tissue repair and wound healing
pellicles against bovine articular cartilage. The tribological Regarding skin repair materials (also called wound
assessment of the sliding pairs for BC was performed using dressings), an important characteristic is their ability to
reciprocating pin-on-flat tests. Due to the wear mechanism absorb exudate during the dressing process, and its
involving high plastic deformation combined with the for- removal from a wound surface after recovery. The draw-
mation of tribological rolls at the contact interface, BC bio- backs of traditional skin tissue repair materials, e.g. gauze,
materials possessing low friction coefficient values (about are their strong permeability, which will cause the tight
0.05) and preservation of the mating surfaces can be adhesion of repair materials on the desiccated wound
obtained. This BC biomaterial was reported to be a poten- surface and thus induce new trauma on removal [156].
tial replacement of artificial cartilage for articular joints Considering its significant biological properties, interest
[153]. Recently, based on the 3T MRI scanning technique, on nanocellulose (especially BC) for novel wound care
an ear-shaped BC prototype material was produced from has steadily increased. BC skin tissue repair biomaterials
a negative ear mold, as shown in Fig. 4(d). Meanwhile, it can be fabricated by a multilayer fermentation method,
was reported that the mechanical properties of BC bioma- which showed low cytotoxicity and good proliferation of
terials can be regulated by the effective cellulose contents. human adipose derived stem cells. According to the animal
This study proved that BC is a promising material to reach experiments and histological examinations, more rapidly
mechanical properties of ear cartilage replacement, and fresh tissue regeneration and significant capillary forma-
can be produced in patient-specific ear shapes [148]. tion in the wound area with BC-based biomaterials were
reported compared with commercial dressings [157,158].
3.4.3. Nucleus pulposus replacement In another study, the effects of BC as wound dressing
Nucleus pulposus is a gelatinous core inside two verte- material were evaluated on animal experiments (male 6-
bral bodies for intervertebral disks, which is important to week-old Sprague-Dawley rats), which proved that the
provide flexibility and dissipate the stresses acting on the presence of BC can promote wound healing by accelerating
spine. It is reported that about 80% of the world population contractions through the accumulation of extracellular
suffers from back pain, and in 75% of cases this is a direct matrix [159]. Some studies also attempted to combine
consequence of degenerative processes of the disc, in par- nanocellulose with various natural matrices in order to
ticular nucleus pulposus degeneration. In recent studies, a develop enhanced biocomposites for potential skin tissue
biocomposite hydrogel with carboxymethylated CNF was repair materials, such as collagen [160], gelatin [161], algi-
prepared by UV polymerization of N-vinyl-2-pyrrolidone nate [162], chitosan [163], cotton gauze [164], poly(ethyl-
for the replacement of native human nucleus pulposus. ene glycol) [165], and poly(vinyl alcohol) [166].
The biocomposite hydrogel containing 0.4% v/v of carbo- BC-based biomaterials have been reported to be applied
xymethylated CNF with DS of 0.17 presented a close in clinical practice. Non-healing lower extremity ulcers
behavior to native nucleus pulposus, such as low strain were treated with a BC wound dressing. The time required
values after cyclic compression tests, and similar relaxa- for 75% reduction in wound size was compared for 11
tion properties [154]. Further study demonstrated that this chronic wounds with and without the presence of BC.
biocomposite hydrogel can act as a potential nucleus pul- The mean period of wound healing without the addition
posus implant attributed to its adequate swelling ratio of BC was 315 days (95% confidence interval (CI): 239–
and improved mechanical properties, which may be bene- 392 days), while with the incorporation of BC to these
ficial to restore the annulus fibrosus loading and the height chronic wounds, the mean time of wound healing reduced
of the intervertebral discs [155]. to 81 days (95% CI: 50–111 days). In this case, the use of BC
wound dressing was reported to significantly shorten the
3.5. Advanced nanomaterials for tissue repair, regeneration time for the tissue repair of non-healing lower extremity
and healing ulcers compared with standard care [167]. In the studies
of clinical effects of BC for skin tissue repair, clinical trials
Tissue repair and regeneration is the process of renewal, were conducted on 34 patients suffering from severe ther-
restoration, and growth that makes the function of diseased mal burns covering 9–18% of the total body surface area, in
and damaged cells, organs, and tissues resilient to natural which 22 of the patients received BC as testing group. It
fluctuations. From bacteria to humans, all species have spe- was reported that the adherence of BC membrane to the
cific ability of tissue repair and regeneration. Different from wound surface was excellent in avoiding dead spaces,
the effects of substitute implants, the behavior of tissue which indicated that the application of BC dressing in the
repair and regeneration for organism inherently originates treatment of partial thickness burns to promote a favorable
from the individual self. Although no property of tissue environment for fast wound cleansing and rapid healing
regeneration or repair for nanocellulose itself, it can provide [168]. Similar conclusions on BC improving skin tissue
repair in clinical research were also reported in recent pub- were expected to meet the requirements of an ideal
lications [169,170]. wound-healing platform for tympanic membrane perfora-
On the basis of fundamental researches on the develop- tion. The tympanic membrane cells were found to well
ment of BC-based skin repair materials, some companies adhere and proliferate on the BC nanofibrillar patch, and
have launched several commercial products in wound in vitro the growth and migration of cells were promoted
healing system. BioFill Produtos Bioetecnologicos (Curitiba, under the guidance of BC patch. Specific effects of BC patch
PR Brazil) developed a series of products based on BC, materials on the regeneration and healing of tympanic
including BiofillÒ and BioprocessÒ (used in the therapy of membrane tissues were investigated through in vivo ani-
burns, ulcers as temporary artificial skin), and GengiflexÒ mal study (12 weeks Sprague-Dawley rats). It was reported
(applied in treatment of periodontal diseases). Another that the presence of BC patch materials significantly
company, Xylos Corporation in the US, has developed increased the tympanic membrane healing rate as well as
several medical devices using BC since 1996. The XCellÒ recovered the function of tympanic membrane better than
family offered by Xylos Corporation has been marketed spontaneous healing [175].
in the US since 2003. Unlike BC dressings manufactured BC was reported to be developed as biomaterial for the
by BiofillÒ, the XcellÒ product is claimed to have a dual- reconstruction of damaged peripheral nerves via cellulosic
function of both hydration and absorption to maintain guidance channels. In vivo experiments were conducted on
the ideal moisture balance [171]. the femoral nerve of Wistar rats for three months. Results
evaluation from histological analysis and postoperative
3.5.2. Bone tissue regeneration and healing observation of motor recovery showed that BC neurotubes
Developing effective bone regeneration therapy is a can effectively prevent the formation of neuromas, while
long-term attracting clinical topic. Bone loss caused by allowing the accumulation of neurotrophic factors inside,
trauma, neoplasia, reconstructive surgery, congenital and facilitating the process of nerve regeneration [176].
defects, or periodontal disease is a major health problem
worldwide. As mentioned in the Section 3.1, nanocellulose 3.6. Antimicrobial nanomaterials
and its biocomposites have been proved to be the promis-
ing scaffolds for the culture of various cells, including Wound infection caused by high bacterial levels, espe-
osteoblast and chondroblast, which indicates that nanocel- cially the burn wounds, traumatic injuries, and surgical
lulose-based materials have the potential for bone tissue procedures, is a significant reason for delayed or prolonged
regeneration and healing. However, studies on nanocellu- wound healing. Adherence and survival of pathogenic bac-
lose for bone tissue regeneration and healing applications teria on the surface of wounds leading to concomitant
are still at the fundamental stage, and only few publica- transmission to new hosts significantly contribute to the
tions report the practical effects on animal experiments. proliferation of pathogens, which considerably increases
A membrane composed of BC and hydroxyapatite was the threat to human health. With increasing awareness of
developed as biomaterial for potential bone regeneration, infectious diseases and antibiotic resistance, many studies
which showed the promotion of growth of osteoblast cells, were dedicated to the development of effective surface dis-
high level of alkaline phosphatase activity, and greater infection and alternative materials bearing antimicrobial
bone nodule formation. The better osteoblasts adhesion, and other bioactive characteristics, viz. antimicrobial
proliferate and mineralization from BC/hydroxyapatite wound dressing. Nanocellulose can provide a porous net-
biomaterials were expected to facilitate quick regeneration work structure in the architecture of biomaterials, which
of bone tissue [172]. Saska et al. further evaluated the bio- is beneficial for potential transfer of antibiotics or other
logical properties and practical effects of BC/hydroxyapa- medicines into the wound, meanwhile serving as an effi-
tite membranes for bone regeneration with in vivo cient physical barrier against any external infection
animal experiments. The biomaterials were embedded to [177]. It is also reported that antimicrobial nanomaterials
improve noncritical bone defects in rat tibiae at 1, 4, and from nanocellulose (carbohydrate nature) commonly
16 weeks. Low crystallinity hydroxyapatite crystals pre- exhibited compatibility with biological tissue as well as
sented a Ca/P molar ratio of 1.5, similar to physiological significant bioavailability and biodegradability [178,179].
bone. The BC/hydroxyapatite membranes were proved to However, because of the fact that nanocellulose itself has
accelerate new bone formation at the defect sites for bone no antimicrobial activity and cannot prevent wound infec-
regeneration in rat tibiae according to in vivo tests for tion, nanocellulose-based antimicrobial biomaterials are
4 weeks [173]. Recently, goat bone apatite was reported generally achieved by the conjunction of antimicrobial
to be introduced in BC for the fabrication of novel bone agents and nanocellulose using physical or chemical
repair biomaterials, which can stimulate bone cell prolifer- approaches. According to different types of antimicrobial
ation and promote the cell differentiation. However, no agents, nanocellulose-based antimicrobial biomaterials
in vivo experiment was reported in this study [174]. can be divided in two parts, including nanomaterials incor-
porated with inorganic antimicrobial agents (mainly
3.5.3. Other tissue repair involving silver particles (Ag) and its derivatives), and
Recently, the fabrication of a nanofibrillar patch by organic antimicrobial agents (e.g. lysozyme).
using BC and its application as a wound-healing platform It is reported that among the different antimicrobial
for traumatic tympanic membrane (ear drum) perforation agents, silver has been most extensively studied and used
was reported. The nanostructured surface, biocompatibil- since ancient times to fight infections and prevent spoil-
ity, transparency, and appropriate mechanical properties age. The silver nanoparticles with effective antibacterial,
antifungal and antiviral properties are proved as a promis- acid; C: cysteine) with the crosslinking followed by cova-
ing antibacterial agent [180]. Chemical reduction (AgNO3/ lent attachment of gentamicin onto the surface of the BC
reducing agent) and simple impregnation are common membrane network. It was reported that these gentami-
approach to introduce a silver antimicrobial agent into cin–RGDC-grafted BC membranes were bactericidal
nanocellulose-based materials. The antimicrobial efficacy against Streptococcus mutans but nontoxic to human der-
of Ag nanoparticle in nanocellulose-based biomaterials mal fibroblasts, which showed potential application in
depends on the size and shape of synthesized nanoparti- wound healing or drug delivery systems [207].
cles. It was reported that CNC nanohybrid materials con- Recently, some researchers studied the antibacterial
taining dendritic nanostructured Ag showed better property of nanocellulose with surface derivatization,
antibacterial activity than that of sphere nanostructured which means the development of nanocellulose antibacte-
Ag [181]. Recent studies attempted to incorporate both rial materials without the use of antibacterial agents. Via a
Ag nanoparticles and CNC in polymeric matrices to offer nucleophilic displacement reaction starting from cellu-
the synergy effects of antibacterial and mechanical rein- lose-p-toluenesulfonic acid ester, the surface of CNF can
forcement, such as poly(lactic acid)/CNC/Ag [182,183], be amino-functionalized. Interestingly, it was reported that
and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/CNC/ electrospun PVA nanofibers containing this amino-modi-
Ag materials [184] and waterborne polyurethane/CNC/Ag fied CNF (with 6-deoxy-6-trisaminoethyl-amino agent at a
[185]. Regarding the antibacterial nanomaterials from degree of substitution of 0.67) exhibited a high antimicro-
CNF and Ag nanoparticles, it was reported that composites bial activity against S. aureus and Klebsiella pneumonia
composed of CNF and Ag nanoparticles can be fabricated [208]. However, it was a pity that the antimicrobial mecha-
by an electrostatic assembly approach via polyelectrolytes nism from this amino-modified CNF was not investigated.
as macromolecular linkers between CNF and Ag nanoparti- Similar study was also reported on chemical grafting of ami-
cles [186]. Fluorescent silver nanoclusters were dipped noalkyl groups onto the surface of BC nanofibrillar network
into CNF materials with the mediation by poly(methacrylic to provide its antimicrobial activity. In this study, the chem-
acid). The presence of fluorescent silver nanoclusters pro- ical structure of amino-modified cellulose was compared
vided both fluorescence and antibacterial activities for similarly with chitosan, which was regarded as the origin
the composites [187]. The studies of Ag nanoparticle or and mimicking of antimicrobial property for modified BC
its derivatives (AgCl, silver sulfadiazine) introduced in BC [209]. Butchosa et al. reported the use of partially deacety-
to develop BC/Ag antibacterial nanomaterials were most lated chitin nanocrystals in BC materials to develop all-
intensively studied, which exhibited a high antimicrobial polysaccharide antimicrobial composites. It was reported
activity [188–193,179] and good biocompatibility. Besides that this ‘‘green’’ composite with all natural components
Ag nanoparticles, recent studies reported that zinc-oxide showed strong antibacterial activity with 99 ± 1% inhibition
nanoparticles (ZnO) on CNC [194], CNF [195] or BC [196] of bacterial growth [210]. Recently, Ul-Islam et al. reported
materials also showed some antibacterial effects. that BC composites incorporated into small concentrations
With the aim of avoiding toxicity and unsustainable of Cu-montmorillonite exhibited some antimicrobial activ-
effect of inorganic Ag nanoparticles, some novel organic ity, but without any mechanism investigation [211].
antibacterial agents, were incorporated in nanocellulose Most reported materials with nanocellulose with or
to develop novel antibacterial materials. The reported without antimicrobial agents in the forms of suspension,
organic antibacterial agents used in nanocellulose-based composite, porous membrane/film, and electrospun nano-
materials include CNC: porphyrin; CNF: octadecyldimethyl fiber, all present promising antimicrobial effects against,
(3-trimethoxysilylpropyl) ammonium chloride [197], alli- e.g. Gram-positive bacterium (S. aureus), and Gram-
cin and lysozyme [198], chitosan–benzalkonium chloride, negative bacterium (Escherichia coli). However, many
chitosan–methylisothiazolinone; and BC: gentamicin, e- interesting properties and pivotal issues on nanocellu-
polylysine [199], benzalkonium chloride [200], sorbic acid lose-based antimicrobial biomaterials are still unknown,
[201,202]. CNC was reported to be covalently grafted on especially regarding the reasonable balance between the
cationic porphyrin groups, which showed excellent effi- improvement of antimicrobial activity, duration of antimi-
ciency of photodynamic inactivation towards bacteria. This crobial effect, and control of normal human cell damage.
strategy was expected as the development of potential
photobactericidal nanomaterials [203,204]. Liu et al. 3.7. Other biomedical applications
reported the preparation of sodium alginate/CNF antibac-
terial composites with the addition of chitosan–benzalko- Besides the traditional biomedical applications dis-
nium chloride or chitosan–methylisothiazolinone as cussed before, nanocellulose has been attempted to be used
antibacterial agents. Both antibacterial agents were nano- in some new fields with special functions. A highly porous
spherical shape (30 nm and 10 nm) and adsorbed on the CNF/polypyrrole composite was developed as an electro-
surface of CNF during several min, under the driving forces chemically controlled solid phase extraction biomaterial
of hydrogen bonds and electrostatic interactions. Further- for the capture of DNA oligomers. This biocomposite pos-
more, these composites were reported to display promis- sessed a total anion exchange capacity of about 1.1 mol/
ing mechanical strength and excellent antibacterial kg, and was reported to extract and release the negatively
activity against Staphylococcus aureus [205,206]. In order charged fluorophore-tagged DNA oligomers through the
to enhance the antibacterial activity of BC, gentamicin- galvanostatic oxidation and reduction of conformal poly-
activated BC membranes were prepared by chemically pyrrole layer (30–50 nm) on CNF substrate. Resulting from
grafting RGDC peptides (R: arginine; G: glycine; D: aspartic the high surface area of porous structure, the ion exchange
capacity of CNF/polypyrrole composite bore two orders of DNA sizes (10 bp–15 kbp) as well as a high resolution of
magnitude higher than traditional ion exchange material, single-nucleotide polymorphisms even though the viscos-
and showed faster and better control of the polypyrrole ity of BC medium was less than 5 cP [214].
charge for the capture of DNA oligomers [212]. In another BC was also reported as an innovative material for den-
study, this CNF/polypyrrole composite prepared with the tal root canal treatment. In comparison with conventional
same strategy and source of CNF, was applied as the hemod- paper point materials, BC showed greater compatibility
ialysis membrane to purify blood. It was reported that this and biological characteristics for dental root canal treat-
biomaterial exhibited an effective removal of small uremic ment. The absorption rate of BC-based biomaterials was
toxins in blood and an improvement in thrombogenic prop- about 10-fold greater than that of paper point materials,
erties with the coating of heparin, which were attributed to and BC-based biomaterials can preserve better tensile
superior ion exchange capability and large surface area of strength under wet condition meeting the requirement of
the membrane. It should be also noted that due to the intro- high-expansion of dental root canal biomaterials. In addi-
duction of natural CNF, the hemocompatibility of this com- tion, it was reported that when used for dental root canal
posite biomaterial was much better than commercial treatment in animal experiments, BC-based biomaterials
synthetic membranes (such as polysulfone) [34,213]. showed maintenance of physical integrity, and only a small
Tabuchi et al. reported the ability of BC medium to foreign body reaction [215].
separate DNA fragments due to a double-mesh concept
combined with a stereo effect from BC-intrinsic three- 4. Functional modification of nanocellulose for
dimensional micrometer- to a nanometer-network potential biomedical application
structure. It was shown that a solution of 0.49% hydroxy-
propylmethyl cellulose polymer containing 0.3% BC frag- Diverse biomedical applications of nanocellulose
ments allowed excellent separation for a wide range of discussed in Section 3 are exciting, but modification of
Fig. 5. Chemical structure of some fluorescent labeling molecules grafted on CNC.

nanocellulose before the development of practical materi- led to significant advancement with the promise of even
als is also important, which will determine its potential greater advances likely to come in the future. Overall,
biological applications. creating controlled properties, reliable and reproducible
By the means of introducing fluorescent molecules on production techniques for biocompatible nanocellulose
the surface, nanocellulose can be converted to functional (not only for BC) will be essential and beneficial to pave
nanoparticles with fluorescent labeling ability. It is the way for greater acceptance of nanocellulose as a com-
expected that fluorescent modification on nanocellulose mercially available material in biomedical applications.
enables the potential use in biomedical fields, such as opti- Further comparison and investigation on different effects
cal bioimaging, biosensor, and photodynamic therapy. On of the three types of nanocellulose (CNC, CNF and BC) will
the other hand, characterized by various fluorescence tech- determine their respective applications in biomedical
niques, fluorescent labeled nanocellulose is easier to be materials.
traced and evaluated for toxicity and bioactivity in materi- Specifically, regarding cellular bioscaffold, the mecha-
als. Since the first report of fluorescent labeling on CNC nisms for cells and nanocellulose interaction remain enig-
with fluorescein-50 -isothiocyanate (FITC) molecule [216], matical and require intensive in vivo study. Furthermore,
more and more studies focus on this topic. To date, diverse on the basis of mechanism analysis, it is possible for future
fluorescent molecules have been attempted to covalently study to regulate the interactions between cells and nano-
attach on the surface of CNC, including FITC, Rhodamine cellulose through controlling the macro- and microstruc-
B isothiocyanate [62], pyrene dyes [217], terpyridine and ture of nanocellulose. Different pharmaceutical
its derivatives [218], 1-pyrenebu-tyric acid N-hydroxy suc- molecules, together with growth factors, or antigenic
cinimide ester [219], 5-(and-6)-carboxyfluorescein succin- factors will be combinatorially organized in nanocellu-
imidyl ester, 5-(and-6)-carboxytetramethylrhod-amine lose-based drug carriers, and used for synergically medical
succinimidyl ester, Oregon Green 488 carboxylic acid, therapy purposes. The studies on the development of tis-
succinimidyl ester [220], PEI-chlorin p6 derivatives [221], sue substitutes and repair biomaterials have made positive
5-(4, 6-dichlorotriazinyl) aminofluorescein [222], and 7- progress (especially with BC), which promotes the launch
amino-4-methylcoumarin [223]. Fig. 5 shows the chemical of several commercial products and practical usage in
structure of some fluorescent molecules that have been clinic. On the one hand, novel nanocellulose-based tissue
grafted on CNC. It is sure that there is still a long way for substitutes and repair biomaterials will be more versatile
practical application of fluorescent CNC in biology, but it with the possible incorporation of biocompatible factors
is also undoubted that the fluorescently modified CNC is or functional factors (such as anticoagulant factors). On
so attracting that the breakthrough in this topic may bring the other hand, covalent attachment of biologically active
the revolution of biomedical materials. ligand molecules to the nanocellulose framework can
Surface grafting of amino acid molecules can offer bio- enhance and alter its characteristics for specific applica-
logically active building blocks on nanocellulose, which tions, which may improve interactions between materials
may contribute to the potential of nanocellulose to be used and human tissues.
as a nanocarrier for DNA delivery. Chemical conjunction From both scientific and economic viewpoints, nanocel-
between amino acid and CNC or CNF can be achieved with lulose, the resource and gift provided by Nature, is on the
two strategies: (1) esterification reaction between Fmoc- threshold of a breakthrough driven by recent extraordinary
amino acid and CNC, and removal of Fmoc-protecting group activities in the field of biomedical applications.
[224]; (2) activation of oxidized CNF to form a stable active
ester, and grafting of amino acid with the formation of
Acknowledgement
novel amide bond [225]. Recently, using the molecular rec-
ognition ability of DNA oligomeric base pairs, duplexing
The authors are grateful to the support of China Schol-
complementary DNA oligonucleotides have been grafted
arship Council (CSC) under Grant No. 2011695007.
onto CNC to produce DNA-based biocompatible nanomate-
rials, which may be used as special biomaterials
for enzyme/protein immobilization [226]. Ferrocene- Appendix A. Supplementary material
decorated CNC can be prepared by grafting ethynylferro-
cene onto azide functionalized CNC using azide–alkyne Supplementary data associated with this article can be
cycloaddition reaction, which can be assembled in found, in the online version, at http://dx.doi.org/10.1016/
three-dimensional structures for potential application in j.eurpolymj.2014.07.025.
biosensors and bioelectrochemical assemblies [227].
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sodium alginate films. J Agric Food Chem 2013;61:6562–7. doctoral research at Université Joseph Fourier
[206] Liu K, Lin X, Chen L, Huang L, Cao S. Dual-functional chitosan– and Grenoble Institute of Technology in
methylisothiazolinone/microfibrillated cellulose biocomposites for France. He received his PhD in 2014 at the
enhancing antibacterial and mechanical properties of agar films. International School of Paper, Print Media and
Cellulose 2014;21:519–28. Biomaterials (Pagora) in Grenoble Institute of
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Production of biocompatible and antimicrobial bacterial cellulose Alain Dufresne. His research interests involve
polymers functionalized by RGDC grafting groups and gentamicin. surface modification, design, development
ACS Appl Mater Interfaces 2014;6:1439–46. and application of nanomaterials based on
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Interfaces 2013;5:3290–7. the Department of Electronic at the Toulouse
[210] Butchosa N, Brown C, Larsson PT, Berglund LA, Bulone V, Zhou Q. National Institute of Applied Science. He is
Nanocomposites of bacterial cellulose nanofibers and chitin professor at the International School of Paper,
nanocrystals: fabrication, characterization and bactericidal Print Media and Biomaterials (Pagora) in
activity. Green Chem 2013;15:3404–13. Grenoble Institute of Technology. His main
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nanoreinforced composite films: novel wound dressing material characterization of polymer nanocomposites
with antibacterial properties. Cellulose 2013;20:589–96.
reinforced with nanoparticles extracted from
[212] Razaq A, Nyström G, Strømme M, Mihranyan A, Nyholm L. High-
renewable resources.
capacity conductive nanocellulose paper sheets for
electrochemically controlled extraction of DNA oligomers. PLoS
One 2011;6:e29243 [p. 9].
[213] Ferraz N, Leschinskaya A, Toomadj F, Fellström B, Strømme M,
Mihranyan A. Membrane characterization and solute diffusion in

European Polymer Journal: Ning Lin, Alain Dufresne

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European Polymer Journal 59 (2014) 302–325

Contents lists available at ScienceDirect

European Polymer Journal

Nanocellulose in biomedicine: Current status and future

1. What is nanocellulose? – Types and productions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

⇑ Corresponding author. Tel.: +33 476826995; fax: +33 476826933.

3.3. Immobilization and recognition of enzyme/protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

Type Toxicological experiment Conclusion Ref.

Strategy Type Enzyme or protein model Speciﬁc procedure Ref.

Abbreviations: Poly(AMA-co-HEMA), poly(2-aminoethyl methacrylate hydrochloride-co-2-hydroxyethylmethacrylate).

Fig. 5. Chemical structure of some ﬂuorescent labeling molecules grafted on CNC.

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