ITBM-RBM 26 (2005) 212–217
http://france.elsevier.com/direct/RBMRET/
Article original
Polysaccharides as scaffolds for bone regeneration
Les polysaccharides comme matrices pour la régénération osseuse
M.A. Barbosa a,b,*, P.L. Granja a, C.C. Barrias a,b, I.F. Amaral a,b
a
INEB, Instituto de Engenharia Biomédica, Laboratório de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
b
Department Engenharia Metalúrgica e de Materiais, Faculdade de Engenharia, Univesidade do Porto, Porto, Portugal
Received 1 April 2005; accepted 15 April 2005
Available online 13 June 2005
Abstract
This review is concerned with the use of polysaccharides for bone regeneration. Cellulose, alginate and chitosan have been chosen as
examples of the formidable potential of this class of materials. Many others could be cited but our experience and research interests dictated
the choice. As in many areas of research we know how they started but we cannot see how and if they will end. Modification of cellulose was
our entrance to the world of polysaccharides and a lot of what we know we owe to Charles Baquey, to whom we would like to express our
immense gratitude. This chapter is dedicated to him, for his cooperation, unlimited enthusiasm and friendship.
© 2005 Elsevier SAS. All rights reserved.
Résumé
Cette revue s’inscrit dans l’utilisation de polysaccharides pour la régénération osseuse. La cellulose, l’alginate et le chitosan ont été choisis
comme exemple compte tenu du formidable potentiel de cette classe de matériaux. Beaucoup d’autres auraient pu être cités mais notre
expérience et nos intérêts en recherche ont guidé ce choix. La modification de cellulose a été notre introduction dans le monde des « polysaccharides » et la plupart de nos connaissances sont dues à Charles Baquey à qui nous voulons ici exprimer notre immense gratitude. Ce chapitre
lui est dédié pour son implication, son enthousiasme illimité et son amitié.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Polysaccharides; Bone regeneration; Cellulose; alginate; Chitosan
Mots clés : Polysaccharides ; Régénération osseuse ; Cellulose ; Alginate ; Chitosane
1. Benefits of polysaccharides for biomedical
applications
The use of natural polymers, or biopolymers, as structural
materials is not new. Nature itself has always used, for
instance, cellulose to provide the structure of higher plants,
chitin as the exoskeleton of several molluscs, keratin for thermoinsulation in hair, collagen for mechanical support in connective tissues, and silk in spiderwebs. At present, the socioeconomic situation of the modern world has raised the interest
* Corresponding author. Tel.: +351 22 607 4900; fax: +351 22 609 4567.
E-mail address: [email protected] (M.A. Barbosa).
1297-9562/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.rbmret.2005.04.006
in these materials. Oil embargos and high prices, associated
with the concerns of oil long-term availability, are favorable
to the replacement of oil-derived polymers by polymers
derived from renewable resources. Environmental concerns
are also playing an increasingly important role, contributing
to the growing interest in natural polymers due to their biodegradability, low toxicity and low disposal costs. The usually low manufacture costs of biopolymers, related to their
large agricultural availability and renewability, are additional
advantages. Furthermore, their versatility of chemical structures and their well-known chemistry allow the development
of advanced functionalized materials that can match several
varied requirements. In addition, the rapid advancement in
M.A. Barbosa et al. / ITBM-RBM 26 (2005) 212–217
understanding of fundamental biosynthetic pathways through
genetic manipulations will provide tailoring of biopolymer
structure and function, thus creating new opportunities for
these materials [1–3].
In the biomedical field, the degradation of natural polymers into physiological metabolites makes them excellent candidates for a wide range of applications, such as drug delivery. Polysaccharides, in particular, have some excellent
properties which make them the polymer group with the longest and widest medical applications experience: [4–7] nontoxicity (monomer residues are not hazardous to health), water
solubility or high swelling ability by simple chemical modification, stability to pH variations, and a broad variety of
chemical structures. This versatility makes these materials
able to overcome some disadvantages like low mechanical,
temperature and chemical stability, and proneness to microbial and enzymatic degradation, which, in some cases, can be
used as an advantage.
2. Cellulose and its derivatives
Cellulose, a naturally occurring polymer produced by
plants, as well as by microorganisms, is the b (1 → 4) polymer of anhydroglucose (Fig. 1).
In the biomedical field, cellulose and its derivatives have
been extensively used for decades. The biocompatibility of
several cellulosics is well established [4–7.] Ikada has
described cellulose as a polymer usually exhibiting relatively
low protein adsorption and cell adhesion (particularly blood
cells), low immune response (low phagocytosis by macrophages and low interleukin-1 release), and inducing comparatively higher activation of the complement system [6]. Cellulose is poorly biodegradable in the body and is not digestible,
but it can be made hydrolysable by changing its higher order
structure [8].
Baquey and coworkers have pioneered and considerably
contributed to this field of study by firstly proposing the use
of regenerated cellulose hydrogels (RCH) for orthopedic
applications. Cellulose Regenerated by the Viscose process
(CRV®) was patented and thoroughly investigated in terms
of physico-chemical, mechanical and biological properties.
It was later chemically modified to enhance its bioactivity
through modifications such as phosphorylation, grafting of
adhesive peptides and oxidation.
CRV is obtained by the standard procedure for preparing
viscose cellulose [9–11]. Briefly, the starting cellulose material (most usually, refined wood pulp) is converted into alkali
Fig. 1. The cellulose molecule –(C6H10O5)n– in its chair configuration.
213
cellulose by steeping in sodium hydroxide, which is then aged.
Alkali cellulose is then converted into sodium cellulose xanthate with carbon disulfide, and finally the xanthate is dissolved in dilute alkali, and viscose is regenerated thereof. Filtration and purification determine the quality of the final
product and, in the case of CRV, is carried out according to
an original procedure [12]. The high swelling material
obtained by this method has been widely used for decades
for producing rayon fibers (textiles) and Cellophane® sheets
[9–11]. In the present case, CRV is highly purified, due to the
applications envisaged [12]. Sulfur content allowed is less
than 1000 ppm, according to regulations applied to Cellophane® for food applications [13,14]. CRV is highly stable to
gamma sterilization and ageing [14–16]. It was investigated
as implantable material in orthopedic surgery, as a sealing
material for the femoral component in hip prostheses, in place
of the acrylic cement, as well as dyaphyseal obturator [12–22].
It was envisaged to take advantage not only of its biostability
and good matching with mechanical properties of cortical
bone but also of its hydroexpansivity, allowing therefore a
satisfactory fixation to hard tissue [12–18]. The maximum
volume expansion of CRV is of approximately 60%. This
material has been shown to satisfy most of biocompatibility
requirements. It is cytocompatible although it seemed not to
allow proliferation of bone cells, after an initial attachment
of the cells. The biostability and osteoconduction of this material have been demonstrated in vivo, although a complete
osseointegration was not observed [19–22].
The applicability of cellulose phosphate (CP) as a biomaterial for orthopedic applications was then investigated to
improve the osseointegration of cellulose. CP has been used
for decades in the treatment of Ca metabolism-related diseases, such as renal stones, due to its high Ca binding capacity, associated with lack of toxicity and indigestibility [23–25].
It has also been widely used in affinity chromatography, owing
to its ability to specifically bind biologically active species,
such as enzymes and peptides [26,27].
Due to its capability of binding Ca and eventually growth
factors, CP can be envisaged as a promising alternative biomaterial, capable of promoting an adequate healing response
once implanted. CP was synthesized according to an optimization of the H3PO4/P2O5/Et3PO4/hexanol method [28,29].
Reaction parameters investigated were the swelling pretreatment, the sequence of addition of reagents, temperature
and time of reaction, and the relative quantities of reagents
[28]. The synthesis was optimized using microcrystalline cellulose. For the first time, cellulose triphosphate was reported,
which opened the possibility of using these materials more
efficiently in many applications where the functionality is
directly related to the phosphate content [28]. It was demonstrated that this technique promotes covalent binding of phosphate groups to the cellulose backbone, and only monoesters
are obtained. The reaction seemed to allow regioselective substitution in the C-6 carbon for low phosphate contents. CP
thus obtained was essentially amorphous, presented high
water swelling, and seemed resistant to gamma sterilization.
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M.A. Barbosa et al. / ITBM-RBM 26 (2005) 212–217
This synthesis route was effectively applied to the surface
modification of RCH, resulting in highly phosphorylated
products, with increased surface roughness and hydrophilicity [29].
The assessment of the mineralization of CP in simulated
physiological conditions, i.e. the ability to induce the formation of an apatite (the mineral of bone) layer, showed that
only the Ca salt of CP promoted formation of apatite. The
interface between the polymer and the mineral layer was continuous [30]. The Ca salt of surfaces phosphorylated for 8 h
promoted higher mineralization extents than the Ca salt of
surfaces treated for shorter or longer periods. The preincubation of unmodified RCH in Ca also resulted in the
homogeneous formation of an apatite layer, although in this
case the interface between the polymer and the mineral layer
was not continuous [31]. In vitro biocompatibility studies in
cultured bone cells showed that CP is not cytotoxic, independently of the phosphate content. However, CP promoted poor
rates of cell attachment, proliferation and differentiation,
which were attributed to the negative charge, associated with
the high hydrophilicity of the cellulose derivative [32]. On
the contrary, unmodified and slightly oxidized RCH promoted high rates of cell attachment, proliferation and differentiation. In this case, an apatite layer was observed between
the cellulose surface and the cell layer, which was attributed
to the synthesis of extracellular matrix by the osteoblastic
cells [33]. Finally, animal implantation studies in rabbits
revealed the biocompatibility of both unmodified and phosphorylated cellulose, as well as their osteoconductive properties [34]. A full osseointegration could not be observed,
although some remodeling activity of the bone tissue was
observed when CP was used.
The promising results obtained with slightly oxidized
regenerated cellulose encouraged pursuing this modification
as a way of promoting cellulose degradability coupled with
increased bone tissue compatibility. Oxidized regenerated cellulose is used as an absorbable wound dressing, under the
trade name of Surgicel® (Johnson and Johnson), as a gauze
which becomes gelatinous after contact with blood, thus
adhering to the surrounding tissue [35–39]. Skoog demonstrated that Surgicel® promotes osseous and soft tissue regeneration in maxillary reconstructive surgery, when used as superiosteal graft before flap closure [36]. It was described as a
good hemostatic agent, which is completely absorbable [36].
Degenshein et al. [37] also observed the biodegradability of
Surgicel®, after clinical use in several different types of surgical procedures. Finn et al. reported good osseous regeneration using Surgicel® as hemostatic agent [38]. Galgut [39]
has reported an enhancement of post-operative healing of periodontal defects in human patients treated surgically through
the use of oxidized cellulose as a hemostatic material. Healing was characterized as satisfactory, with no inflammation,
resorption or necrosis and the materials showed antibacterial
properties. Some studies also suggest no effect on bone induction by oxidized cellulose [40,41]. Granja et al. [33] have
demonstrated that when slightly oxidized, RCH induced high
rates of bone cells colonization in vitro, and promoted the
formation of an apatite layer in bone cells culture, which is
an unusual behavior. These findings suggest that they can be
good candidates as scaffolds for bone tissue engineering, by
pre-colonizing them with bone cells before implantation. Further studies are currently ongoing to explore new oxidation
procedures [42,43].
Martson et al. [44] reported regenerated cellulose viscose
sponges as compatible implantable tissue matrices for bone
tissue regeneration. They described this material as slowly
degradable [45]. Cellulose viscose sponges have also been
proposed as connective tissue regeneration matrices [45,46].
de Taillac et al. [42,43] are presently investigating the applicability of oxidized and macroporous regenerated cellulose
structures for promoting cell colonization of 3-D structures.
In an alternative attempt to provide cellulose with enhanced
bioactivity a new method to immobilize bioactive molecules
onto cellulose, namely with peptides bearing the RGD (ArgGly-Asp) sequence, was proposed by de Taillac et al. [47–49].
The ability of this peptide sequence to bind a variety of cells
makes it and interesting approach to modify biomaterials surfaces. In this approach silane-derivatized spacer arms were
first attached to the surface of cellulose as an intermediate for
the covalent linkage of RGD containing peptides. Peptides
were found to be covalently bound to cellulose. Bone cells
attachment and proliferation were considerably increased in
cellulose modified with the RGD peptide sequence.
3. Alginates
Alginates are naturally occurring polysaccharides that have
been finding increasing applications in the biotechnology
field. They belong to a family of linear copolymers of b-Dmannuronic acid and a-L-guluronic acid residues, which can
be arranged in different proportions and sequences along the
polymer chain [50,51]. Sodium alginate and most other alginates from monovalent metals are soluble in water, forming
solutions of considerable viscosity. Due to their suitable rheological properties, alginates have long been used in the pharmaceutical industry as thickening or gelling agents, as colloidal stabilizers and as blood expanders [51].
Sodium alginate forms relatively stable hydrogels through
ionotropic gelation in the presence of many multivalent ions,
being Ca2+ the most widely used. The crosslinking process
can be carried out under very mild conditions, at low temperature and in the absence of organic solvents, and hydrogels of different shapes can be prepared. Several therapeutic
agents, including antibiotics, enzymes, growth factors and
DNA, have already been successfully incorporated in alginate gels, retaining a high percentage of biological activity
[50,51]. Moreover, alginate hydrogels have been widely studied for cartilage and bone regeneration applications as scaffolds and vehicles for biologically active molecules or cells
[52,53].
In our laboratory, alginate has been employed in the development of microspheres with a uniform size, to be used as
M.A. Barbosa et al. / ITBM-RBM 26 (2005) 212–217
fillers of bone defects and as carriers for enzymes. A specific
application is the use of the system as an adjuvant therapeutic strategy to treat bone lesions associated with a genetic
disturbance called Gaucher disease (GD). Microspheres were
developed as vehicles for the recombinant enzyme glucocerebrosidase (GCR), currently employed in GD treatment.
GCR-loaded alginate microspheres were prepared [54] by
droplet formation under a coaxial air flow, followed by ionotropic gelation of the polymeric matrix in the presence of Ca2+.
The enzyme was successfully entrapped in alginate microspheres, retaining full activity and exhibiting improved stability at physiological pH. Studies using GCR-deficient fibroblasts from a GD patient showed that released GCR was
internalized by cells, significantly enhancing their residual
enzymatic activity, with only half of the dose required using
free-GCR. The in vitro GCR release profile was characterized by an initial high burst effect followed by a stage of very
slow release.
Whether or not this release-kinetics is adequate for the
therapeutic efficiency of GCR in the in vivo targeting of bone
resident Gaucher cells needs to be evaluated. However, the
possibility of obtaining different patterns of enzyme release
increases the range of applications of the system. Moreover,
it has been previously reported that attachment-dependent
cells are unable to specifically interact with alginate hydrogels, which promote minimal protein adsorption, presumably due to their high hydrophilic nature [55].
Enhanced cell adhesion in polymer–ceramic composites
in comparison with their polymeric counterparts has been previously demonstrated by other authors [56]. Therefore, as a
strategy to modulate GCR release-kinetics and, at the same
time, improve cell adhesion to alginate microspheres, calcium phosphate particles were added to the polymeric matrix
and calcium phosphate-alginate microspheres were prepared
[57,58]. Two different types of calcium phosphates were used:
hydroxyapatite (HAp), a ceramic widely used in orthopedic
applications, and calcium titanium phosphate (CTP), a
ceramic that is currently under investigation in our laboratory and which may be used as an immobilization matrix for
several enzymes. The ceramic powders were mixed with alginate and microspheres were prepared by the technique already
described, resulting in the formation of an alginate hydrogel
network with entrapped ceramic particles [57,58]. In this case,
the enzyme was incorporated in the microspheres before gel
formation in two different ways: pre-adsorbed onto the
ceramic particles or dispersed in the polymeric/ceramic paste.
The two strategies used for GCR entrapment resulted in distinct release profiles. When GCR was pre-adsorbed onto the
ceramic powders prior to microspheres formation the initial
burst was significantly reduced, compared to the one of alginate microspheres, and a more gradual release was obtained,
showing that modulation of release-kinetics was achieved.
The adhesion of osteoblastic-like cells to calcium
phosphate-alginate microspheres of different compositions
was analyzed [58]. The ceramic-to-polymer ratio strongly
influenced the ability of cells to adhere and spread on the
215
microspheres surface. No adherent cells could be found on
the surface of control alginate microspheres and only dispersed round cells and/or multicellular aggregates could be
observed on microspheres with a low percentage of calcium
phosphates. However, on microspheres prepared with a high
ceramic-to-polymer ratio (0.4 w/w ceramic, using a 1.5% w/v
alginate solution) cells were able to attach, spread and adopt
a spindle-shaped morphology.
4. Chitosan
Chitosan is a biocompatible and bioresorbable biopolymer that may be used for a wide number of biomedical applications, such as sutures, wound dressings, bone substitutes,
tissue engineering and drug and gene delivery vehicles
[59,60]. Chitosan is a linear copolymer of glucosamine and
N-acetyl glucosamine in a b1 → 4 linkage, usually obtained
by N-deacetylation of chitin under alkaline conditions [61].
The degree of N-acetylation (DA) together with the molecular weight are the most important parameters for its characterization. The DA, which is by definition the molar fraction
of N-acetylated units, is a structural parameter influencing
charge density, crystallinity and solubility, including the propensity to enzymatic degradation, with higher DAs leading
to faster biodegradation rates [62,63]. Similarly to most
polysaccharides, chitosan has the ability to elicit specific cellular functions. N-acetyl glucosamine attracts polymorphonuclear leucocytes, inducing the release of cytokines, which
favor the histoarchitectural organization of connective tissues [64].
In orthopedics, its enzymatic degradability associated to
its structural similarity to extracellular matrix glycosaminoglycans makes it an attractive biopolymer for bone tissue
repair. Numerous bone filling materials have been developed
in which chitosan is used in combination with calcium phosphates, essentially as a binding agent, or associated to biological signaling molecules [65,66]. In addition, its versatility to be processed into injectable, porous and membranar
forms without use of toxic solvents makes chitosan an interesting material to be used as a non-protein temporary scaffold, for bone regeneration [67]. Presently, an increasing number of anchorage-dependent cells, including bone cells, are
being cultured on 2-D and 3-D chitosan-based matrices, envisaging cell-based regenerative therapies [66,68–70].
Cell adhesion, migration and cell growth kinetics of a number of cells, including keratinocytes and fibroblasts, are known
to depend on the DA of chitosan, lower DAs favoring cell
adhesion [71–73]. However, no osteoblast adhesion studies
have been reported. Therefore, our group has decided to investigate this aspect and the results are due to appear shortly.
The ability of phosphorylated chitosan (P-chitosan) membranes to nucleate calcium phosphates under simulated physiologic conditions, was investigated by our group [74].
P-chitosan membranes presenting a surface P at.% of about
2.87 as shown by XPS studies, were used. The membranes
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were immersed in Ca(OH)2 or NaOH solutions, in order to
obtain the Ca or the Na salts, respectively. SEM-EDS studies
revealed the presence of a calcium phosphate mineral layer
all over the surface of P-chitosan membranes, after incubation in Ca(OH)2 solution. The release of ionically bound phosphate functionalities, under alkaline conditions, possibly contributed to the formation of calcium phosphate precursor sites,
due to the chelation of calcium ions from solution. During
the immersion in simulated body fluid (SBF), a multilayered
porous mineral structure composed of a partially carbonated
and poorly crystalline apatite was formed on the surface of
these membranes, as shown by EDS, ATR-FTIR and XRD
analysis. In addition, Ca was also found in the bulk of the
membrane, as expected, since the inner of the membrane is
also phosphorylated. No mineral formation could be observed
on unmodified chitosan membranes, nor on NaOH-treated
P-chitosan membranes, suggesting that Na ions were not
exchanged by Ca ions from SBF.
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