DOI: 10.1002/adem.200980087 Biomimetic Collagen Nanofibrous Materials for Bone Tissue Engineering** By Wenfu Zheng,Wei Zhang * and Xingyu Jiang 1. Introduction Bone – a highly complex and well-organized organ – refers to a family of remarkable hierarchical structures with different motifs that are all constructed of a basic building block, the mineralized collagen fibril. [1] The assembly includes an orderly deposition of hydroxyapatite (HA) minerals within a type I collagen matrix. The crystallographic c-axis of the HA is oriented parallel to the longitudinal axis of the collagen fibril. [2] Investigation and simulation of the hierarchical nano-fibril structure in nature can offer novel designs and methods of fabrication of functional materials, such as materials that can be used as tissue-engineering scaffolds and biomimetic materials. Collagen is a natural extracellular matrix (ECM) compo- nent of many tissues, such as bone, skin, tendon, ligament and other connective tissues. [2–4] One of the underlying hypoth- eses in collagen research, as related to biomaterials, is that evolutionary bioengineering has produced a material that has ideal properties for biological applications. An essential feature of this type of biomaterial is its excellent assembled structure, widespread occurrence in nature, and potential to complete degrade in biological environments, thus, collagen has been widely used as a practical biomaterial in tissue engineering. [5] The fibril structure of natural collagen offers great opportunities for fabricating artificial scaffolds to mimic autologous bone grafts. Currently, there are three basic REVIEW [*] W. Zheng, W. Zhang, and X. Jiang CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology 11 ZhongGuanCun Beiyitiao, Beijing 100190 (PR China) E-mail: [email protected][**] We thank the Human Frontier Science Program, the National Science Foundation of China (90813032, 20890020 and 50902025), the Ministry of Science and Technology (2006CB705600, 2007CB714502 and 2009CB930001) and the Chinese Academy of Sciences (KJCX2-YW-M15) for funding. Hierarchical assemblies of nanofibres are ubiquitous in nature. Mineralized type I collagen is the basic building block of hierarchically organized, highly complex structures of bone tissue. As a biomaterial, collagen is widely utilized in biomimetic nanofibrous matrix fabrication due to its inherent biocompat- ibility and widespread occurrence in nature. Nanotechnology has recently gained a new impetus due to the introduction of the concept of biomimetic nanofibres for tissue regeneration. The emergence of electrospinning techniques provides a new opportunity to fabricate nano-collagen fibres for bone tissue engineering. By orchestrating major parameters, collagen fibres with different components (pure or blended), sizes (nanometre to micrometre) and surface properties (mineralized or modified by functional bioactive molecules) have been developed and their effects on bone cell adhesion, prolifer- ation, migration and differentiation evaluated. This review briefly introduces natural mineralized collagen structures in bone, biomimetic mineralization and bone grafts, and in vitro mineralization of collagen nanofibres fabricated by using three major techniques – molecular self-assembly, electro- spinning, and phase separation. Their applications in bone tissue engineering are also discussed. We highlight the electrospinning technique in collagen nanofibre fabrication and its great potential for bone tissue regeneration. ADVANCED ENGINEERING MATERIALS 2010, 12, No. 9 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com B451
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DOI: 10.1002/adem.200980087
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Biomimetic Collagen NanofibrousMaterials for Bone TissueEngineering** By Wenfu Zheng,Wei Zhang* and Xingyu Jiang
Hierarchical assemblies of nanofibres are ubiquitous in nature. Mineralized type I collagen is the basicbuilding block of hierarchically organized, highly complex structures of bone tissue. As a biomaterial,collagen is widely utilized in biomimetic nanofibrous matrix fabrication due to its inherent biocompat-ibility and widespread occurrence in nature. Nanotechnology has recently gained a new impetus due tothe introduction of the concept of biomimetic nanofibres for tissue regeneration. The emergence ofelectrospinning techniques provides a new opportunity to fabricate nano-collagen fibres for bone tissueengineering. By orchestrating major parameters, collagen fibres with different components (pure orblended), sizes (nanometre to micrometre) and surface properties (mineralized or modified byfunctional bioactive molecules) have been developed and their effects on bone cell adhesion, prolifer-ation, migration and differentiation evaluated. This review briefly introduces natural mineralizedcollagen structures in bone, biomimetic mineralization and bone grafts, and in vitro mineralization ofcollagen nanofibres fabricated by using three major techniques – molecular self-assembly, electro-spinning, and phase separation. Their applications in bone tissue engineering are also discussed. Wehighlight the electrospinning technique in collagen nanofibre fabrication and its great potential for bonetissue regeneration.
1. Introduction
Bone – a highly complex and well-organized organ – refers
to a family of remarkable hierarchical structures with different
motifs that are all constructed of a basic building block,
the mineralized collagen fibril.[1] The assembly includes an
orderly deposition of hydroxyapatite (HA) minerals within a
type I collagen matrix. The crystallographic c-axis of the HA is
[*] W. Zheng, W. Zhang, and X. JiangCAS Key Lab for Biological Effects of Nanomaterials andNanosafety, National Center for NanoScience and Technology11 ZhongGuanCun Beiyitiao, Beijing 100190 (PR China)E-mail: [email protected]
[**] We thank the Human Frontier Science Program, the NationalScience Foundation of China (90813032, 20890020 and50902025), the Ministry of Science and Technology(2006CB705600, 2007CB714502 and 2009CB930001) and theChinese Academy of Sciences (KJCX2-YW-M15) for funding.
site scaffolds. The initially precipitated amorphous calcium
phosphate, along with the collagen fibril, was transformed
into a crystalline apatite-like phase. Goissis and co-workers[61]
reported in vitro and in vivo biomimetic mineralization of
charged collagen assemblies with calcium phosphate depos-
ited in close resemblance to the D-periodicity of collagen fibril
assembly.[61] Pederson and co-workers[62] reported a strategy
for exploiting temperature driven self-assembly of collagen
and thermally triggered liposome mineralization to form a
mineralized collagen composite from an injectable precursor
fluid. Their results showed that heating of a liposome-
Fig. 1. a) High magnification of the mineralized collagen fibrils. The inset image is the sediffraction pattern of the mineralized collagen fibrils. The asterisk is the centre of the area, anarea is about 200 nm. b) HRTEM image of mineralized collagen fibrils. The long arrow inddirection of collagen fibril. Two short arrows indicate two HA nanocrystals. (Cited from Rewith permission from [63]. Copyright 2003, American Chemical Society
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containing suspension of acid-soluble collagen results in the
self-assembly of a mineralized collagen gel that may be
suitable as an injectable composite biomaterial.[62]
In 2003, our team, for the first time, verified the new
hierarchical self-assembly structure of nano-HA/collagen
(nHAC) composite in vitro using conventional and high-
resolution transmission electron microscopy.[63] We synthe-
tically prepared nano-fibrils of mineralized collagen as a
self-assembly model system to evaluate the possibility of
biomimetic materials with hierarchical structures similar to
those found in nature.[63] Collagen solutions of different pH,
temperature and ion strength were evaluated for the
formation of collagen fibrils. Transmission electron micro-
scopy (TEM) investigations revealed that the composites
formed consist of an intertwined assembly of collagen fibrils
bundles more than 1 mm long (Fig. 1). Each collagen fibril is
surrounded by a layer of HA nanocrystals grown on the
surface of the collagen fibrils. Each mineralized bundle of
collagen fibrils is much thicker than the self-assembled
collagen fibrils, implying that the self-assembled collagen
nanofibrils act as the template for HA precipitation.
Additionally, in order to discern the relative orientation of
the HA crystals with respect to collagen fibrils, electron
diffraction investigation have also been carried out. The
results demonstrated the preferential alignment of the HA
crystallographic c-axis with the collagen fibril longitudinal axis.
High-resolution transmission electron microscopy (HRTEM)
analysis of the parallel-aligned mineralized collagen fibrils has
revealed that crystal lattice is seen not only on the side area of
the collagen fibrils, but also in the middle area, and that the
electron density on the surface of the collagen fibrils is higher
than in the interior area. These findings indicate that HA
crystals grown on the surface of the collagen surround the
fibrils, giving the first direct evidence to support previous
lected area electrond the diameter of theicates the longitudef. [63]) Reproduced
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theories that this occurs.
Carbonated HA (CHA), another natural
component of bone, has excellent biocom-
patibility and osteoconductivity and
appears to be an excellent material for
bioresorbable bone substitutes. Liao and
co-workers[64] prepared nanocarbonated
hydroxyapatite–collagen composite via a
biomimetic self-assembly method. This
composite showed the same inorganic phase
of natural bone at the nanoscale level and a
low degree of crystallinity. TEM results
confirmed that the microstructure of this
composite is a mineralized collagen fibre
bundle, like the hierarchical structure of
natural bone.
4.2. Effect of Non-Collagenous Proteins
on Collagen Mineralization
Although collagen comprises about 90% of
total organic bone matrix, there are many
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other proteins present in small amounts in these tissues. These
so-called non-collagenous proteins are believed to play
essential roles in the formation of collagenous mineralized
tissues.[9] One of the common characteristics of these
non-collagenous proteins is the high content of acidic amino
acids, such as aspartate and glutamate. In a recent study,
Oslzta and co-workers[65] described the mineralization of
collagen fibrils in the presence of poly-Asp as an analogue of
noncollagenous acidic proteins. They propose a mechanism in
which poly-Asp stabilized amorphous calcium phosphate
initially formed in solution impregnates collagen fibrils and
transforms into crystalline mineral. In 2008, Deshpande and
co-workers[66] carried out bioinspired mineralization of
collagen fibrils in the presence of poly-Asp. The mineralized
collagen fibrils closely resemble structures in collagenous
mineralized tissues with respect to organization and crystal-
lography. Their results suggest that the presence of poly-Asp
in the mineralization solution triggered mineralization of
reconstituted collagen fibrils.
4.3. Calcium Phosphate as a Transfection Agent for Bone
Regeneration
Calcium phosphate, besides its role in bone mineralization,
has also been commonly used as a transfection agent in
non-viral gene delivery. This process relies on the fact that
Fig. 2. Atomic force micrographs of a) aligned collagen matrices, and, b) randomly oriented collagen matricesproduced by shear flow deposition and static fibril formation, respectively. Scale bar, 2mm. c) alignedfibrillar structures (open arrows) are visible, and, d) above this plane aligned mineralized nodules (closedarrows) are visible. Scale bars, 30mm, insets, 15mm. (Cited from Ref. [81]). Reproduced with permission from[81]. Copyright 2009, Elsevier
ning of collagen using fluoroalcohols has been reported to
yield collagen nanofibres that do not swell in aqueous
media,[106,107] but are readily soluble in water, tissue fluids
or blood.[91,100,108–111] Since gelatin is a water-soluble degra-
dation product of the originally water-insoluble collagen
fibril,[112] the water solubility of the electrospun collagen
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scaffolds means a possible conformational change of col-
lagen.[102] Further research that would essentially assure
protection of the triple-helical structure of collagen during
electrospinning should be emphasized. A possible alterna-
tive is the coating of collagen on electrospun nanofibres of
synthesized polymers. By doing this, the natural structure and
function of collagen could be essentially preserved and the
mechanical properties of nanofibres could be further tuned by
orchestrating the components of polymers. The coating of
collagen can also yield good biocompatibility of nanofibres
and better meet the requirement of tissue engineering
applications.[91,108,113–115] Another possible alternative is the
electrospinning of nanofibres using gelatin directly. Although
gelatin is a degradation product of collagen, electrospun
gelatin fibres have the same biocompatibility as does
collagen.[116–119]
It has been reported that continuous fibres could not
be spun from acidic aqueous solutions of pure collagen; the
addition of sodium chloride to the solution can promote the
formation of continuous fibres, perhaps due to the increase in
solution conductivity.[120] The low viscosity of the collagen
solution hinders the electrospinning process, leading to
the formation of beads or the failure of fibre formation. An
increase of the concentration of collagen solution[97] or
addition of polyethylene oxide (PEO) to the solution[120] can
increase the viscosity of the spinning solution and allow better
Fig. 3. a) A typical image of disorderly mats made of poly(vinyl alcohol) (PVA) fibres via conventionalelectrospinning. b–d) Images of arrays of PVA fibres fabricated via magnetic electrospinning: b) a digital cameraimage, and, c,d) scanning electron micrographs of the aligned fibers. (Cited from Ref. [86]) Reproduced withpermission from [86]. Copyright Wiley-VCH, 2007
control over fibre formation.[100] The strength
of the applied electric field controls the size of
electrospinning to develop biodegradable and biomimetic
scaffolds (Fig. 4). Optimizing conditions for type I collagen
produced a matrix composed of 100 nm fibres that exhibited
the 67 nm banding pattern characteristic of native collagen
(Fig. 4d).[97] The structural properties of electrospun collagen
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varied with the origin of tissue (type I from skin vs. type I from
placenta), the isotype (type I vs. type III) and the concentration
of the collagen solution used to spin the fibres. The final
diameters of electrospun collagen fibres varied in a concen-
tration-dependent manner – the higher the concentration of
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Fig. 4. a) SEM of calfskin type I collagen electrospun onto a static, cylindrical mandrel. Cut edges of thematrix illustrate the porous, three-dimensional nature of the scaffold. b) Detailed SEM of electrospun calfskintype I collagen. c) SEM of electrospun type I collagen isolated from human placenta. d) TEM of theelectrospun type I calfskin collagen. Electroprocessed fibres exhibit the 67 nm banding typical of nativecollagen (inserted scale bar, 100 nm). (Cited from Ref.[97]) Reproduced with permission from [91].Copyright 2002. American Chemical Society
collagen solution, the larger the fibre diameter. Other groups
evaluated the biocompatibility of single-component type I
collagen by seeding cells on it. Rho and co-workers[108]
investigated electrospinning of type I collagen for wound
healing. Cross-linked by glutaraldehyde, the collagen nanofi-
brous matrix showed good tensile strength, even in aqueous
solution. Collagen nanofibrous matrices treated with type I
collagen or laminin were functionally active in responses in
normal human keratinocytes and were very effective as
wound-healing accelerators in early-stage wound healing.[108]
Shih and co-workers[101] reported that MSCs grown on type I
collagen nanofibres had significantly higher cell viability than
a tissue culture polystyrene control. Single-cell reverse
transcription polymerase chain reaction (RT-PCR) of type I
collagen gene expression demonstrated higher expression on
cells seeded on the nanofibres. Therefore, type I collagen
nanofibres support the growth of MSCs and can be used as a
scaffold for bone tissue engineering.
In summary, electrospun pure collagen can provide a basic
matrix for in vitro cell culture, However, electrospun
nanofibres based on pure collagen protein still face many
problems, including low stability in water, poor resistance
to collagenase environments and poor thermal stability. The
pure electrospun collagen fibres are easily denatured during
the electrospinning process.[102,105] Thus, as an alternative,
electrospinning of the blends of collagen and synthetic
polymers were quickly developed.
5.3.2. Collagen Blend Electrospinning. Blending collagen with
other natural and/or synthetic polymers can yield engineer-
linity apatite and enhanced transcript levels of bone-specific
markers than did the controls (without BMP-2), indicating that
these nanofibrous electrospun scaffolds are efficient delivery
systems for BMP-2. Besides, Zhong and co-workers[92]
developed collagen–glycaosaminoglycan (GAG) blended
nanofibrous scaffolds that showed excellent biocompatibility
with rabbit conjunctiva fibroblasts. In addition to coating or
blending for composite nanofibre fabrication, covalently
grafted protein on the nanofibre surface is proposed to be
another choice for functionalization, which has long been used
for surface modification for conventional biomaterials.
5.4. Mineralization of Electrospun Collagen Fibres
The incorporation of minerals into polymer nanofibres
may create more biomimetic constructions and improve the
mechanical properties of the composite. By initially miner-
alizing HA in the gelatin and then co-electrospinning
the mixed nanocomposite solution, Kim and co-workers[116]
obtained electrospun nonwoven membranes in which nano-
crystals of HA were well incorporated into electrospun gelatin
fibres. Up to 40% HA could be successfully incorporated using
this technique. The biocompatibility of the nanocomposite
was assessed by measuring the alkaline phosphate (ALP)
activity of MG 63 cells cultured on the nanocomposite. Cells
on the HA nanofibre (20% and 40% HA) expressed signifi-
cantly higher levels of ALP activities than those on pure
gelatin nanofibres. Thomas and co-workers[103] fabricated
nanostructured biocomposite scaffolds of type I collagen and
HA using electrostatic co-spinning. Structural characteriza-
tion confirmed the presence of well-dispersed nano-HA
mineral phase in the collagen matrix. The diameter and
surface roughness of the composite fibres increased with an
increase in nano-HA content compared with neat collagen
fibres. With the increase of the nano-HA content, the tensile
modulus of the nanofibres increased, perhaps due to an
increase in rigidity over the pure polymer when the HA is
added and/or the resulting strong adhesion between the two
materials.[103] The methods mentioned above are realized by
co-spinning of HA and collagen or gelatin, which finish the
mineralization and electrospinning at the same time. Another
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method is to coat HA onto the pre-fabricated electrospun
fibres. Liao and co-workers[158] have electrospun collagen and
PLGA into nanofibrous scaffolds with high porosity and
well-connected open pore network. In order to mimic the
chemical composition of native bone ECM, the electrospun
scaffolds were subjected to mineralization under optimal
conditions. The results showed that bone-like apatite forma-
tion is much abundant and uniform over collagen nanofibres
than PLGA under the same experimental conditions. They
found that, compared with PLGA, surface functional groups
of electrospun scaffolds strongly influence the mineral
formation and the active surface functionalities. For example,
carboxyl and carbonyl groups of collagen may be favourable
for apatite nucleation and crystal growth. Ngiam and
co-workers[133] mineralized electrospun nanofibres using a
calcium–phosphate dipping method. Mineralization of
nano-HA was achieved by subjecting the nanofibres in a
series of calcium and phosphate treatments, deemed the
alternate dipping method. PLGA and PLGA/collagen nano-
fibrous scaffolds were first immersed in CaCl2 solution,
followed by rinsing with deionised water. The scaffolds were
subsequently immersed in Na2HPO4 solution and rinsed with
deionised water. All nanofibres were subjected to 3 cycles of
this treatment to achieve mineralization. The functionalities of
osteoblastic cells, such as ALP activity and
protein expressions, were ameliorated on
mineralized nanofibres. Furthermore, they
found that the amount of nano-HA appeared
to have a greater effect on the early stages of
osteoblast behaviour (cell attachment and
proliferation) rather than the immediate/late
stages (proliferation and differentiation).
Fig. 5. SEM photomicrograph of cells on 500–1 000 nm nanofibres (a, b) and tissue culture polystyrene (c, d).Representative confocal microscopy of cell morphology of nanofibres with diameters of 50–200 nm (e),200–500 nm (f), and 500–1 000 nm (g). Nanofibres with diameters of 500–1 000 nmwere stained with rhodamine(red). (Cited from Ref. [101]) Reproduced with permission from [101]. Copyright 2006. Wiley