DOI: Biomimetic Collagen Nanofibrous · DOI: 10.1002/adem.200980087 Biomimetic Collagen Nanofibrous Materials for Bone Tissue Engineering** By Wenfu Zheng,Wei Zhang* and Xingyu Jiang

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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.

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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

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W. Zheng et al./Biomimetic Collagen Nanofibrous Materials for Bone Tissue Engineering

techniques capable of generating collagen-based nanofibrous

structures: molecular self-assembly, electrospinning, and

phase separation; among these, electrospinning is the most

simple and efficient method and will be highlighted in this

review.[6–8] Electrospinning has recently emerged as a leading

technique for generating biomimetic scaffolds made of

synthetic or natural polymers. It is able to produce continuous

fibres with diameters of microns down to tens of nanometres.

Various types of electrospun collagen fibres have been

produced and characterized for tissue engineering.

In this review, we will first introduce the hierarchical

organization of mineralized collagen in bone, including the

HA crystal structure, collagen assembly, collagen–HA crystal

composite, and collagen–mineral interactions. Second, we will

briefly present biomimetic mineralization and bone grafts.

Then, we will introduce the three major techniques –

molecular self-assembly, electrospinning, and phase separa-

tion – that can generate collagen-based nanofibres. We will

focus on the electrospinning technique and the use of

electrospun collagen nanofibres in bone tissue engineering.

2. Hierarchical Organization of MineralizedCollagen in Bone Tissue

Bone is a highly complex and well-organized organ. Weiner

and Wagner identified seven discrete levels of hierarchical

organization in the bone:[1] major components, mineralized

collagen fibrils, fibril arrays, fibril array patterns, ostons,

spongy/compact bone and the whole bone. In this review, we

focus principally on the structure of HA crystals, collagens

and mineralized collagen composites because they can pro-

vide basis for better understanding of collagen–mineral inter-

action and implications with respect to designing biomimetic

materials for bone tissue engineering applications.

2.1. HA Crystals

The first level of hierarchy consists of molecular compo-

nents, including water, HA, collagen and non-collagenous

proteins (NCPs). HA, in a crystallized form, is the only

mineral constituent in mature bone. The dominant morphol-

ogy of HA crystals are plate-shaped and among the smallest

known biological crystals (30–50 nm long, 20–25 nm wide

and 1.5–4 nm thick).[9] The thicknesses of the crystals are

remarkably uniform and the surfaces are highly ordered.[10]

The reason for the plate shape of the crystal is not clear, a

plausible explanation is that the mineral formed initially

resembles octacalcium phosphate (OCP), which naturally

forms plate-shaped crystals.[11]

2.2. Collagen Assembly

The structural fibrous protein, type I collagen, comprises

approximately 85%–90% of the extracellular organic matrices

of bone. There are many classes of collagenous structures in

the ECM, including fibrils, networks and transmembrane

collagenous domains.[12] For brevity, we focus here on

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mineralized type I collagen fibrils, the basic building blocks

for construction of the hierarchical structure of bone. Type I

collagen is characterized by its fibrous nature and each fibril

is a triple helix consisting of three covalently cross-

linked,[13,14] left-handed polyproline peptide chains inter-

twined in a right-handed fashion.[2] Two of the three chains,

a1 chain, have an identical amino acid sequence and are

distinct from the a2 chain, which consist of a highly

conserved composition of amino acids. The triple helix of

type I collagen is approximately 300 nm long and 1.5 nm wide

and consists of about 1 000 amino acids per chain. Moreover,

the specific packing arrangement of the triple helix – of

alternating overlap and gap zones – results in the formation

of collagen fibrils possessing a high degree of axial alignment

and the exhibition of a characteristic D-banding (the

fingerprint of fibrous collagens).[15–19] In 1963, a simplified

structural model for collagen self-assembly in two dimen-

sions was proposed by Hodge and Petruska.[20] According to

this model, type I collagen self-assembles by forming

molecules staggered by approximately 22% of their length

(67 nm) with respect to their nearest neighbour. The hole or

gap (47 nm in length) and overlap (20 nm in length) zone

comprised the periodic staggered distance D (67 nm). Based

on high voltage electron microscopic tomography, collagen

was found to be packed in three dimensions through a strict

and contiguous alignment of its composite hole and overlap

zones. In 2001, Orgel and co-workers[21,22] reported the first

electron-density map of a type I collagen fibre at molecular

anisotropic resolution (axial: 0.516 nm; lateral: 1.11 nm) using

synchrotron radiation. Their data confirmed that collagen

microfibrils have a quasi-hexagonal unit cell, on which other

researchers had generally agreed. The quasi-hexagonal unit

cell contains five triple-helical molecule monomers as the

basis for an accurate model of the collagen fibril. The

molecular packing of the triple-helix monomers in this model

results in triple-helix neighbours arranged to form super-

twisted, right-handed microfibrils that interdigitate with

neighbouring microfibrils – leading to a spiral-like structure

for the mature collagen fibril. Their model advances the

provocative idea that the collagen fibril is a networked

nanoscale rope.

2.3. Collagen–HA Crystal Composite

The bone can be considered as an apatite-reinforced

collagen composite at the molecular level, where HA crystals

are intimately associated with the collagen framework in

which they form, resulting in a highly complex but ordered

mineral–organic composite material.[1] The basic structure of

the collagen–crystal composite is that HA crystals are

embedded in the holes or gaps inside the collagen fibril to

form intrafibrillar mineralization, all other crystals are located

either between triple-helical molecules or outside the

fibrils.[23,24] The characteristic of the intrafibrillar mineraliza-

tion is that platelets of HA arranged in the direction along the

long-axis of the fibril. In addition to the uniaxial orientation,

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platelets are often illustrated as being coherently aligned in the

plane perpendicular to the long axis of the fibril, stacked in

parallel arrays like a ‘‘deck of cards’’, as described by Traub

and co-workers.[25] The nano-scale crystal–collagen composite

can further organize into layers or lamellae a few microns

thick, and these in turn are arranged in a variety of ways into

higher-order structures – osteons, the most common high-

er-order structures in bone.[1]

2.4. Collagen–Mineral Interactions

The nucleation of HA crystals on collagen fibres has been

extensively investigated in previous studies.[26,27] It has been

demonstrated that carboxyl groups (�COOH) are the major

nucleation sites for collagen fibrils and the binding of calcium

ions on the negatively charged carboxylate groups of collagen

is one of the key factors for the first-step nucleation of HA

crystals.[28] The carboxyl groups are present in about 11% of

the amino acid residues of collagen molecules. In a neutral

solution, more than 99% of the carboxyl groups of aspartyl and

glutamyl ionize to carboxyl groups, which favours chelation

of calcium ions. The carboxyl groups on the outside of the

collagen threefold spiral are one kind of site for collagen

mineralization. Many researchers focused on the relationship

between the fine structure of the periodic banding pattern of

type I collagen, the onset and progression of mineralization

and elastic strain energy storage during tensile deformation

of the protein.[29–31] Their works have led to biomechanical

considerations of elastic energy storage in collagen and the

molecular basis for elastic and viscous deformation, as well

as for energy storage of collagen.[32] It is proposed that tensile

deformation of collagen involves stretching of the flexible

regions in its hole and overlap zones that are opened to

provide possible binding sites for calcium and phosphate ions

involved in mineralization.[29,32]

3. Biomimetic Mineralization and Bone TissueEngineering

3.1. Biomineralization and Biomorphic Mineralization

The study of biomineralization in nature is particularly

interesting because it provide a great source of inspiration

for the design of advanced materials and could offer new

strategies to regenerate human mineralized tissues.[33–36]

Nature’s mineralizers use small amounts of organic macro-

molecules to manipulate nucleation, growth, microstructure

formation and, consequently, the properties of their miner-

al-based materials. For example, Suzuki and co-workers

recently reported that an acidic matrix protein, Pif, in the

pearl oyster Pinctada fucata, which specifically binds to

aragonite crystals, is a key macromolecule for nacre

formation.[37] Inspired by natural biomineralization, bio-

morphic mineralization – a technique that produces materi-

als with morphologies and structures resembling those

of nature living things through the employment of

bio-structures as templates for mineralization – is an

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emerging field for developing advanced materials. For

example, Garcı́a-Ruiz and co-workers recently revealed that

crystallization in purely inorganic systems can also yield

so-called ‘‘biomorphs’’ that resemble those of biomater-

ials.[38,39]

In this review, we focus on biomineralization of bone tissue

and bone tissue engineering. Mineralization of bone –

essential for its hardness and strength – involves a well-

orchestrated process in which crystals of calcium phosphate

are produced by bone-forming cells and laid down in precise

amounts within the bone’s fibrous matrix or scaffolding.

3.2. Bone Grafts and Bone Tissue Engineering

Bone tissue engineering is critical for healing of large

defects. Autografts, allografts, and xenografts can be used for

bone healing. The use of autogenous bone grafts is widely

accepted in the treatment of bone defects due to their oste-

oconductive,[40,41] osteogenic[42] and osteoinductive[43] pro-

perties and the lack of immunogenicity[44] or possible disease

transmission. However, chronic donor site pain and

morbidity,[45] limited availability, variable quality, potential

donor site infection and longer operation times[46] have

limited its use.[47,48] Allograft (human cadaver bone) is a

successful alternative to autograft bone in the clinical

setting,[49,50] but is associated with additional disadvantages,

including potential host rejection,[51] limited supply in some

locations, excessive resorption, potential disease transmis-

sion, toxicity associated with sterilization[48,52] and ethical

concerns. Xenograft (animal bone) finds rather infrequent

application in bone grafting owing to concerns about

immunogenicity and the risk of species-to-species transmis-

sible diseases.[53,54] There is a clear and urgent need to

provide alternatives to these bone grafts. Tissue engineering

of bone by combining scaffold materials with tissue cells and

biological cues is considered to be a promising alternative to

traditional bone graft strategies and has become a rapidly

expanding research area for bone repair and regeneration. A

range of biomaterials have been employed as bone tissue

engineering scaffolds, which capable of promoting the

differentiation of immature progenitor cells down an

osteoblastic lineage (osteoinduction), encouraging the

in-growth of surrounding bone (osteoconduction) and

integrating into the surrounding tissue (osseointegration)

such that the implant is anchored into the defect site in a

manner to prevent micromotion and allow the implant to fuse

the defect and surrounding bone.[36] Since the hierarchical

self-assembly of collagen–HA composite is the naturally

occurring process during bone generation and growth, as a

key ingredient of natural bone, collagen shows excellent

biological properties in bone regeneration. It shows excellent

osteoconduction and osteoinduction, and has the capability

to form direct biochemical bonding with the host tissue,

which will in turn hasten the tissue regeneration at the

defective site. Therefore, collagen–HA composite is an

excellent scaffold for bone tissue engineering.

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4. Self-assembly of Collagen Nanofibres forBone Tissue Engineering

4.1. Self-assembly of Nano-HA/Collagen (nHAC)

Composite

Since the mineralized collagen fibril is the basic structural

and functional unit of the collagenous mineralized tissues, the

ability to recreate it in vitro is essential for the development of

nanostructured bioinspired materials for mineralized tissue

repair. In addition to this, the fibril structure of natural

collagen offers great opportunities to mimic autologous bone

grafts. Thus, the in vitro self-assembly and mineralization of

collagen have attracted considerable attention.[55,56] As early

as the 1950s, the ability of extracted collagen monomers to

self-assemble into native-like fibrils was being investigated

extensively.[15,18,19,57,58] Early studies to mimic the composi-

tion and structure of bone focused on using simulated body

fluid (SBF) with reconstituted type I collagen. Glimcher and

co-workers reported that HA was nucleated in the hole

zones of self-assembled collagen fibres.[59] By combining the

collagen fibril assembly and the calcium phosphate formation

in one process step, Bradt and co-workers[60] obtained

homogeneously three-dimensional apatite–collagen compo-

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


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

calcium ions are known to form ionic

complexes with the helical phosphates of

DNA and these complexes have easy trans-

portability across the cell membrane via ion

channel-mediated endocytosis.[67,68] A recent

study evaluated the potential of a collagen/

calcium phosphate scaffold as a delivery

system for naked plasmid DNA.[69] The

results showed that the delivery of a naked

therapeutic plasmid encoding VEGF 165

from the collagen/calcium phosphate scaf-

fold in a bone defect resulted in increased

bone formation.

4.4. Cells Response to Mineralized

Aligned Collagen Fibres

In vivo, collagen fibrils are arranged in

complex three-dimensional arrays, often in

an aligned manner, to fulfil certain biome-

chanical functions. Collagen is found as

parallel fibre bundles in tendons and liga-

ments,[70] as concentric waves in bone[9] and

as oriented fibrils in the superficial zone of

articular cartilage[71] or as orthogonal lattices

in the cornea.[72] This spatial organization

imparts mechanical strength to the tissue

and impacts cellular functions.[3] It is thus

important to create aligned collagen fibril

matrices in vitro and investigate its influence

on cell behaviour. Several approaches


have been introduced to reconstruct aligned collagen matrices

in vitro. Elsdale and Bard presented a technique that involved

unidirectional draining of a supporting coverslip during

gelation of a collagen solution.[73] Additionally, exposing a

gelling collagen solution to a strong magnetic field aligns

collagen fibrils due to the diamagnetic properties of collagen

molecules.[74,75] Guo and Kaufman[76] also made use of

magnetic fields, but did so by adding magnetic beads to the

collagen solution and then aligning the gelling collagen

solution by moving the beads towards their poles. Aligned

collagen nanofibre matrices have also been produced by

electrospinning[77] or use of a mica surface in combination

with hydrodynamic flow.[78] Lastly, collagen fibril alignment

has been observed in microfluidic channels (<100mm width)

as result of a short initial pressure-driven flow and subsequent

static gelation of a collagen solution.[79] Lanfer and co-

workers[80] introduced a microfluidic system in fabricating

aligned fibrillar collagen matrices (Fig. 2a). They carried out

two variants of the streaming experiments: one was the

deposition of collagen fibrils during fibril formation, in which

the number and size of the fibres were concentration-

dependent; the other was the deposition of fully developed

collagen fibrils, in which matrices consisting of long, highly

aligned and individual collagen fibrils were produced by

streaming a collagen solution containing ‘‘ready-made’’


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collagen fibrils. Other factors, such as flow rate and substrate

properties, all influence the collagen alignment. Then, they

cultured mesenchymal stem cells (MSCs) on the aligned type I

collagen structures to assess their impact on MSC growth

and differentiation.[81] In addition, they refined the aligned

collagen matrices by incorporating glycosaminoglycan (GAG)

heparin to demonstrate the versatility of the applied method

to study multiple ECM components in a single system. The

reconstituted and aligned ECM structures maintained and

allowed multilineage (osteogenic/adipogenic/chondrogenic)

differentiation of MSCs. Most noticeable was the observation

that, during osteogenesis, ordered matrix mineralization was

deposited on the aligned collagen substrates (Fig. 2c and d).

The results shed light on the regulation of MSCs through

directional ECM structures and demonstrate the versatility of

these cell culture platforms for guiding the morphogenesis of

tissue types with anisotropic structures.[81]

In summary, the development of novel self-assembled

HA/collagen composite structures should improve our

understanding of collagen-mediated mineralization in bone

tissues, and provide the basic theoretical support for the

fabrication of HA/collagen composites and their application

in bone regeneration.[82,83]

5. Biomimetic Electrospun CollagenNanofibres for Bone Tissue Regeneration

In the body, the majority of human tissues and organs,

such as bone, tendon and skin, are attached on hierarchically

organized fibrous structures with the fibre size realigning

from the nanometre to the millimetre scale.[1] The nano-

scale structure of the ECM provides a natural web of intricate

nanofibres to support cells and present an instructive

background to guide their behaviour.[84] As such, scaffolds

consisting of nanofibres have now been extensively used to

mimic these natural tissue matrixes. Scaffolds with nanofibre

architectures have bigger surface areas for absorbing proteins

and present more binding sites to cell-membrane receptors.

Using nanofibres, the engineering of a number of tissues,

including cartilage, bone, arterial blood vessel, heart and

nerve, has been attempted.[7,85] Collagen – the natural fibrous

constituent of native tissues – is also widely utilized to

fabricate scaffolds serving as an active analogue of native

ECM.[5] Conventional polymer processing techniques have

difficulty in producing fibres smaller than 10mm in diameter,

which are several orders of magnitude larger than native ECM

(50–500 nm). For this reason, there has been a concerted effort

to develop methods of producing nanofibres to more

adequately simulate the ECM geometry.

Electrospinning is a well-known and ubiquitous technique

to produce nanofibres and has been extensively used in

various applications including immunoassays,[86,87] nano-

catalysis[88] and molecular sensors.[89] In particular, electro-

spinning is an extremely promising method for generating

nanofibrous scaffolds from either natural or synthetic

biodegradable polymers to simulate the cellular microenvir-


onment. By using this technique, people can rapidly produce

fibres of nanoscale and conveniently tailor the physical,

chemical, mechanical and biological properties of a material

for cellular environments and specific applications, such as

tissue engineering.[88,90] Many biodegradable synthetic poly-

mers have been electrospun into fibrous meshes and

successfully used in cell-culture systems for tissue engineer-

ing.[91–93] In recent years, novel nanofibrous scaffolds of native

polymers, such as collagen, gelatin and elastin, fabricated by

electrospinning have been reported for tissue-engineering

constructs. Such scaffolds are found to closely mimic native

ECM in term of components and physical structures, because

of their natural origin and nanofibrous dimension. Here, we

focus on the electrospinning processes of collagen fibres and

their use in bone-tissue engineering.

5.1. A Brief Introduction to Basic Principles of

Electrospinning

In a typical electrospinning experiment, a polymer solution

or melt is pumped through a thin nozzle. The nozzle simu-

ltaneously serves as an electrode, to which a high electric field

of 100–500 kV m�1 is applied. The distance to the counter

electrode is 10–25 cm in laboratory systems. When a high

voltage is applied to the solution, a jet is formed as soon as the

applied electric field strength overcomes the surface tension of

the solution. When travelling towards the grounded collecting

plate, the jet becomes thinner as a consequence of solvent

evaporation and fibres are formed.[94]

5.2. Optimized Conditions for Collagen Electrospinning

Continuous, uniform collagen fibres with suitable mechan-

ical property are desirable in electrospinning. However, there

are several parameters that influence the quality of the

electrospun fibres. These parameters include the solvent used

(solution viscosity, solution conductivity and solvent volati-

lity), the strength of the electric field applied, the flow rate and

the collecting distance.[95,96] An appropriate solvent system is

a crucial factor for the successful electrospinning of nano-

fibres. In general, 1,1,1,3,3,3-hexafluoropropan-2-ol (HFP) is a

widely used solvent for the electrospinning of collagen

fibres.[97–101] 2,2,2-trifluoroethanol (TFE) has also been used

in some experiments.[92,102,103] However, it has been reported

that fluoroalcohols can lead to conformational change of

native proteins.[104] Although electrospinning fibres exhibit

the 67 nm banding typical of native collagen in early

publication,[97] some recent reports showed that most of

the triple-helical collagen was apparently lost when it

is electrospun out of fluoroalcohols, such as HFP or

2,2,2-trifluoroethanol (TFE).[102–105] Additionally, electrospin-

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

the fibres formed, from several microns in

diameter to tens of nanometres. A subopti-

mal field strength could lead to bead defects

in the spun fibres or even failure in jet

formation. Matthews and co-workers opti-

mized the voltage input parameters for type I

collagen electrospinning.[97] By setting the

concentration of collagen at 0.083 g mL�1 in

HFP and varying voltages from 15 to 30 kV,

they found that the most prominent forma-

tion of fibres took place at 25 kV with an

optimal air gap distance of approximately

125 mm.[97] Other groups reported that an

applied voltage of 10 kV, distance of 15 cm

and flow rate of 5mL min�1 is a suitable

condition for pure collagen electrospin-

ning.[121]

It is well known that well-ordered fibres

may be suitable for many applications in

tissue engineering.[122,123] There have been a

few approaches to improving the orderliness

of electrospun fibres. Matthews and co-

workers[97] used a rotating mandrel as a

ground target to collect collagen fibres. By

controlling the rotation speed of the mandrel,

they obtained collagen fibres aligned along

the axis of rotation. Katta and co-workers[124]

employed a macroscopic copper wire-framed


rotating drum as the collector, and the electrospun fibres

collected on the drum as it rotated were parallel to each other.

Theron and co-workers[125] described an electrostatic fiel-

d-assisted assembly technique using a tapered and grounded

wheel-like bobbin to position and align individual nanofibres

into parallel arrays. These methods can fabricate more or less

aligned fibres; however, they still have some drawbacks.

Recently, our team reported a facile and effective approach to

fabricating well-aligned arrays and multilayer grids by a

technique called magnetic electrospinning, where magnetized

fibres are stretched into essentially parallel fibres over large

areas (more than 5 cm� 5 cm) in a magnetic field (Fig. 3). It is

suitable for fabricating aligned fibrous matrix for biomimetic

use.[86]

Generally speaking, by orchestrating various conditions,

we can get desired structures we need. Table 1 summarizes

the electrospinning conditions from recently reported studies.

5.3. Strategies for Collagen Electrospinning

5.3.1. Pure Collagen Electrospinning. The electrospinning of

single-component fibres was tried in early studies. As a

natural ECM component, the electrospun collagen fibres

display similar biochemical and mechanical properties as

native collagen. In 2002, Matthews and co-workers[97]

produced a nanofibrous matrix of type I collagen via


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Table 1. Reported conditions for electrospinning of collagen fibres.

Material Solvent and concentration Parameter Diameter [nm]Cross-linking agent

and time Reference

Collagen type I HFP, 0.083 g mL�1 Voltage¼ 25 kV 100–730 Glutaraldehyde (GTA)

vapour, 24 h

[97]

Distance¼ 125 mm

Feed rate¼ 5 mL h�1

Target mandrel rotating¼ 4 500 rpm

Collagen type I HFP, 8% wt.-% Voltage¼ 13 kV 300–375 – [126]

Distance¼ 130 mm

Feed rate¼ 1.2 mL h�1

Collagen type I HFP, 6.7 wt.-% Voltage¼ 12 kV 350� 250 – [127]

Distance¼ 120 mm


Collagen type I HFP, 50 mg mL�1; HFP,

TFE 180 mg mL�1Voltage¼ 10–15 kV ca. 100 – [102]

Distance¼ 120–150 mm

Feed rate¼ 0.75–1.2 mL h�1

Collagen type I HFP, 80 mg mL�1 Voltage¼ 15 kV ca. 250 30% GTA vapour, 48 h [128]

Distance¼ 150 mm


Target mandrel rotating¼ 15 m s�1

(linear velocity)

Collagen type I TFE, 100 mg mL�1 Voltage¼ 25 kV 500–700 GTA vapour, 24 h [103]

Distance¼ 125 mm


Collagen type I TFE, 55 mg mL�1 Voltage¼ 22 kV ca. 1 000 Different concentration of

GTA solution, 12 h

[129]


Feed rate¼ 8–12 mL h�1

Target mandrel rotating¼ 200 rpm

Collagen type I HFP, 8% v/v Voltage¼ 40 kV 100–1 200 25% GTA vapour,

different time

[108]

Distance¼ 215 mm


Collagen type 1 HFP, 8% w/v Voltage¼ 19–21 kV 100–600 25% GTA vapour, 24 h [105]



Collagen type I HFP, 4–12 wt.- % Voltage¼ 30 kV 50–1 000 EDC [101]


Collagen type I HFP, 4–12 wt.-% Voltage¼ 30 kV 50–1 000 EDC [130]


Collagen type II HFP, 40 mg mL�1 Voltage¼ 22 kV ca. 496 25% GTA solution, 24 h [99]


Collagen type III HFP, 40 mg mL�1 Voltage¼ 25 kV 250� 150 GTA vapour, 24 h [97]

Distance¼ 125 mm



collagen type I /

collagen type III

HFP, 60 mg mL�1 Voltage¼ 25 kV 390� 290 GTA vapour, 24 h [97]

Distance¼ 125 mm



Collagen type I/elastin 0.010 M HCl, 1%–5% w/v Voltage¼ 22 kV 220–600 EDC/NHS [100]



Collagen type 1/PLGA HFP, 4.5%, 5%, 6%, 8%,

10% w/v

Voltage¼ 18 kV 382� 125 – [131]

Distance¼ 120 mm


Collagen type I/gelatin HFP, 8.3% w/v Voltage¼ 10 kV 77� 41 to 485� 187 HMDI

(1,6-diisocyanatohexane)

[117]

Distance¼ 150 mm

Feed rate¼ 1–10 mL h�1

Collagen type I/GAG Water and TFE.10–20 wt-% Voltage¼ 10–20 kV 100–600 15% GTA vapour, 3 d [92]


Feed rate¼ 0.5–1.5 mL h�1

Collagen type I/PLLA HFP, 5% w/v Voltage¼ 10 kV 755� 294 Thermal, 110 8C [121]

Distance¼ 150 mm


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


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-

ADVANCED ENGINEERING MATERIALS 2010, 12, No. 9 � 2010 WILEY-VCH Verlag GmbH & Co. KG

ing materials with desired properties. For

example, Huang and co-workers[120] electro-

spun type I collagen and PEO to tailor fibre

morphology and mechanical properties of

scaffolds. The electrospinning of types I and

type III collagen blending in a 50:50 ratio was

investigated by Boland and co-workers[132]

because types I and III collagen are often found

together in many tissues, including blood vessel

ECM. Various other collagen blends such as

poly(lactide-co-glycolide) acid (PLGA)/col-

lagen[133] and poly-(L-lactide) (PLLA)/col-

lagen[121] have been produced by electrospin-

ning and utilized to culture cells. Their

advantage is obvious, because together with

the improvement of resistance to water and

collagenase, the biocompatibility of blends is the

same as pure collagen fibres.

5.3.3. Cross-Linking to Stabilize Electrospun

Fibres. As a principal structural element of the

native ECM in many native tissues, neat

collagen protein has emerged as an interesting

polymer to electrospin for diverse tissue engi-

neering applications. However, electrospun

collagen nanofibres still face many problems,

such as insufficient resistance in water and collagenase

environments, poor mechanical strength to bear loadings

and poor thermal stability.[134] Covalent cross-linking is a

good choice for increasing the dimensional, mechanical and

biological stability of collagen biomaterials.[135] Researchers

have developed a variety of cross-linking methods, including

chemical agents, physical heating and ultraviolet (UV)

irradiation to enhance the mechanical strength, thermal

stability and collagenase resistibility of collagen scaffolds,

thus increasing its overall biocompatibility.[121,136–138]

There are many physical cross-linking methods, such as

dehydrothermal (DHT) treatment, photo-oxidation, micro-

wave and UV irradiation.[130,139] Physical methods are

traditionally considered to be good cross-linking alternatives

because they do not require that materials come into contact

with solvents and, therefore, can be effective under solid-state

conditions. For instance, short exposures to UV light are

commonly known to affect the terminal telopeptide molecules

of collagen proteins with a high content of tyrosine, increasing

the shrinkage temperature, the resistance to collagenolytic

degradation and the durability in collagenase. However,

UV treatment may alter the polymer molecular weight and

chemistry and cannot ensure sufficient strength of the

matrices; this method has not been widely used in biomimetic

material treatment.[134]

Glutaraldehyde (GTA) is the most common cross-linking

agent in clinical use for fixing collagenous tissues. As a

widely used chemical cross-linker, GTA has been reported to

introduce a high degree of cross-linking and water-resistance

in the electrospun collagen-based fibres.[92,100,105,108]

aA, Weinheim http://www.aem-journal.com B459

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However, GTA can strongly increase the material cytotoxicity

by adverse reactions arising from residual and reversible

fixation.[134] Similar to GTA, 1,6-diisocyanatohexane (HMDI)

is another crosslinking agents that has been used in

electrospun protein fibres.[117] Carbodiimide is another

extensively used group of cross-linkers for several tissue

engineering applications, in use for over 30 years.[140–144]

N-[3-(dimethylamino)propyl]-N0-ethylcarbodiimide hydro-

chloride (EDC) is a kind of carbodiimide with relatively

low cytotoxicity; it facilitates the formation of amide bonds

between carboxylic and amino groups on the collagen

molecules with the advantage of not becoming part of the

resultant linkage. EDC has been currently used for enhancing

the biostability of collagen scaffolds in the presence of N-

hydroxysuccinimide (NHS), which can prevent the formation

of side products and also increase the reaction rate.[101,145–147]

Nevertheless, the aforementioned methods, based on

chemical or physical treatments, either can add potential

cytotoxic effects or can cause breakdown and proteolysis of

the collagen protein helical structures, respectively.[148] Thus,

cross-linking agents from natural organic tissues, such as

enzymes, are advantageous due to their low cytotoxicity.

Transglutaminases are a group of enzymes that can catalyse

several types of post-translational modifications of proteins

and result in the cross-linking of peptides or proteins to form

multimers via an e-(g-glutamyl)lysine linkage using the side

chains of lysine and glutamine residues. Transglutaminases

are also able to covalently attach primary amine containing

compounds to peptide-bound glutamine, facilitating mod-

ification of the physical, chemical and biological properties of

proteins.[149] So, transglutaminase has been used to crosslink

various biomaterials to increase their resistance to load and

degradation.[136,137,150–152] The enzymatically cross-linked

collagen matrices, which are considered to be a more

biological treatment, can promote adequate cell adhesion

and proliferation.[134]

Besides, there are other natural cross-link reagents,

D,L-glyceraldehyde is a natural product of a metabolic

process.[153,154] Studies suggest that gelatin crosslinked with

glyceraldehyde is well tolerated in vivo.[154] Genipin is

derived from geniposide, which is extracted from the fruit

of Gardenia jasminoides Ellis. It was reported that genipin

cross-linked gelatin is about 10 000 times less cytotoxic than

glutaraldehyde cross-linked gelatin.[155]

5.3.4. Functionalization of Electrospun Collagen Matrix. After

fabrication of nanofibres by electrospinning, we may use

coating or immobilization technologies to modify the fibre

surface with some functional molecules, which may improve

biocompatibility and some tissue-specific inductivity of the

matrix. Many electrospun collagen fibres have been coated

before cell culture. For example, collagen-coated PLLA/

collagen nanofibres have demonstrated higher cell attach-

ment, spreading and viability than the unmodified nanofi-

bres.[156] PLGA/collagen blended nanofibre scaffolds func-

tionalized with E-selectin achieved a rapid, rich and specific


capture of bone marrow-derived hematopoietic stem cells

(BM-HSCs).[131] In many reports, some functional molecules

blended into the collagen solution before electrospinning.

Fortunately, the mild aqueous process required for electro-

spinning offers an important option for delivery of labile

biomolecules into the system. The mineralized collagen/PLA

three-dimensional scaffold with bone morphogenetic pro-

tein-2 (BMP-2) had already shown some exciting healing

effects in vivo.[127] In principle, the nanofibre materials can

easily absorb more growth factors on their surface, and

possibly result in an optimal healing effect. Silk fibroin

nanofibre scaffolds containing BMP-2 and/or nanoparticles of

HA prepared via electrospinning were selected as a matrix for

in vitro bone regeneration.[157] Scaffolds with the co-spun

BMP-2 supported higher calcium deposition, higher crystal-

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

5.5. Responses of Bone Cells to

Electrospun Collagen Fibres

Electrospun collagen fibres, due to

their inherently native biocompatibility,

are proposed as an ideal environment for

living cells. Various bone cells, including

MSCs,[101,121] human osteosarcoma cells (MG

63),[130] and human adipose stem cells[159]

have been utilized to evaluate the effects of

fibre size,[101,130] components[121] and surface

characteristics[133] on cell adhesion, viability,

migration and osteogenic differentiation.

Shih and co-workers[101] reconstituted

type I collagen nanofibres prepared by

electrospinning technology and examined

the morphology, growth, adhesion, cell

motility, and osteogenic differentiation of

MSCs on fibrous matrix of different sizes.

SEM showed that cells on the nanofibres

had a more polygonal and flattened cell

morphology (Fig. 5). Also, they concluded

that type I collagen nanofibres support


the growth of MSCs without compromising their osteogenic

differentiation capability and can be used as a scaffold for

bone tissue engineering to facilitate bone formation. Similarly,

a three-dimensional electrospun nanofibre matrix of type I

collagen significantly enhanced the proliferation and osteo-

genic differentiation and mineralization behaviours of human

adipose stem cells.[159] Compared with pure collagen

nanofibres, the blended fibres show excellent stability in the

medium environment; for example, Schofer and

co-workers[121] compared electrospun type I collagen and

PLLA nanofibres with regard to their stability and ability to

promote growth and osteogenic differentiation of human

MSCs in vitro. During 28 d of incubation in the medium, the

PLLA nanofibres remained stable, while the presence of cells

resulted in an attenuation of the collagen nanofibre mesh. Do

the nanofibres mineralized with HA show better biocompat-

ibility? Ngiam and co- workers[133] answered this question.

They electrospun PLGA and PLGA/collagen nanofibrous

composite scaffolds and coated these scaffolds with nano-HA

and then investigated the effects of HA-coating on osteoblastic

behaviour for bone tissue engineering. The mineralized

PLGA/collagen fibres had a greater surface area than

non-mineralized controls. Cells captured on mineralized

PLGA/collagen fibres were comparable to non-mineralized


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controls. Although nano-HA impeded proliferation during

the culture period, cellular functionality, such as ALP, was

ameliorated on mineralized nanofibres.

Hsu and co-workers[130] found that the sizes of collagen

nanofibres significantly influence the growth and migration of

MG 63 cells. The growth of MG 63 cells on different sized

fibres showed higher growth than those cultured on poly-

styrene. Interestingly, the migration speed of MG 63 cells

decreased as the diameter of nanofibres increased. In addition,

variation in the size of collagen nanofibres apparently has

more impact on cell migration distance and cell morphology

than on cell growth. Besides collagen, gelatin has also

been co-electrospun with poly[(L-lactide)-co-(e-caprolactone)]

(PLCL) and its ability to promote cell differentiation tested[160]

– the incorporation of gelatin in the nanofibres stimulated the

adhesion and osteogenic differentiation of MSCs.

5.6. Comparison between Electrospun Nanofibres Based

on Collagen and Synthetic Polymers

As mentioned above, electrospun collagen nanofibres show

excellent osteoconductivity[101,121,130] and osteoinductivity[121]

in bone tissue engineering. However, as naturally derived

material, collagen scaffolds may exhibit immunogenicity and

contains pathogenic impurities. There is also less control over

their mechanical properties, biodegradability and batch-

to-batch consistency.[161] Many of them are also limited in

supply and can therefore be costly. In contrast, biodegradable

synthetic polymers, such as PLLA or PLGA, could be

electrospun in large scale with controlled properties, includ-

ing strength, degradation rate and microstructure. Further-

more, polymers have great design flexibility because the

composition and structure can be tailored to meet the specific

needs.[161] The disadvantages of synthetic polymers is their

poor biocompatibility, release of acidic degradation products,

poor processability and loss of mechanical properties during

degradation.[162] Therefore, single-component natural (col-

lagen) or synthetic polymers have drawbacks that simply

cannot be overcome. The best strategy is to combine their

advantages. The co-spinning of collagen and synthetic

polymer,[120,121,131–133] and coating collagen on polymer

fibres[91,156] are successful examples with optimized functions

in bone tissue engineering.

6. Collagen Nanofibres Fabricated by PhaseSeparation

Phase separation has been used for several years as a

technique to create porous polymer membranes.[163] This

technique was recently utilized to fabricate biodegradable

three-dimensional polymer scaffolds.[164] In this approach, the

polymer is first dissolved in a solvent at a high temperature,

liquid–liquid or solid–liquid phase separation is induced by

lowering the solution temperature. Subsequent removal of the

solidified solvent-rich phase by sublimation leaves a porous

polymer scaffold.[164,165] Polymer scaffolds obtained by the


phase separation method usually have a sponge-like porous

morphology with microscale spherical pores. However, if the

conditions, including the solvent type, polymer concentration,

gelation temperature and time, are precisely controlled,

micro- or nanoscale polymer fibres can be obtained.[166]

To mimic the nanofibrous architecture, Zhang and

co-workers[164] developed a liquid–liquid phase separation

technique to create three-dimensional interconnected fibrous

networks of PLLA. The fibres have a diameter ranging from 50

to 500 nm, which is the same as that of the collagen matrix.[2]

The nanofibrous scaffolds preferentially absorb cell adhesion

proteins, such as fibronectin, and promote osteoblast attach-

ment.[164,167] Liu and co-workers[168] prepared highly porous

collagen–HA scaffolds by using a solid–liquid phase separa-

tion method. The collagen–HA scaffolds were porous with a

three-dimensional interconnected fibre microstructure, the

pore sizes 50–150mm, and HA particles were dispersed evenly

among the collagen fibres. Their results showed that the

porous collagen–HA composite has good biocompatibility

and is suitable as a scaffold for bone tissue engineering.

Bernhardt and co-workers[169] fabricated porous three-

dimensional structures from mineralized collagen by apply-

ing a procedure in which collagen fibril reassembly and

precipitation of HA occur simultaneously. The in vitro

experiments indicated that the collagen/HA scaffolds pro-

moted the proliferation and osteogenic differentiation of

MSCs. Their data suggest that porous collagen/HA scaffolds

are promising candidates for application as bone grafts to

improve the property of the collagen matrix. Wang and

co-workers[170] crosslinked chitosan, a positive charged

polysaccharide, into the scaffolds using solid–liquid phase

separation. The ability of the porous collagen/chitosan

scaffold to repair bone was investigated by orthotope bone

defect reparation in vivo. Their results indicated that the

repaired bone was obviously remodeled and revascularized

post-operatively and the artificial bone matrix could be used

as a bone substitute with excellent properties.

7. Comparison of Different CollagenNanofibre Fabrication Methods

The three techniques mentioned above – self-assembly,

electrospinning and phase separation – are all successful

methods for the fabrication of collagen nanofibres. Compared

with electrospinning and phase separation, self-assembly can

produce much thinner nanofibres, only a few nanometres in

diameter, and mineralized collagen assemblies closely

resembling the D-periodicity of collagen fibrils.[61] The

in vitro self-assembly structure of nano-HA/collagen com-

posite have hierarchical structures similar to those found in

nature.[63] Aligned ECM structures, fabricated by self-

assembly of collagen, can maintain and allow multilineage

differentiation of stem cells and lead to the deposition of

ordered matrix mineralization.[80,81] However, the realization

of precisely fabricated structures using self-assembly requires

much more complicated procedures and elaborate techniques.


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The low productivity of the self-assembly method is another

limitation.[166] Comparatively, electrospinning can produce

nanofibres with various parameters, such as the components,

diameter, thickness of scaffold and alignment, by tailoring the

proportion of collagen and synthesized polymer, the con-

centration of collagen solution, the volume of solution and the

collection manner, respectively.[171] Nanofibres electrospun

with pure collagen or blended collagen provide ideal niches

for cell living, growth, migration and differentiation. How-

ever, the denaturation of collagen during the electrospinning

process and the relatively poor biomechanical properties

of the fibres are major drawbacks of the electrospinning

method; these problems should be fully addressed in future

research.[102] Phase separation is a convenient method for

fabricating three-dimensional porous polymer scaffolds. The

mechanical properties and architecture of the scaffold can be

easily modified by varying the polymer constituents and

concentration, solvent exchange, thermal treatment and order

of procedures.[172] In addition, phase separation is a simple

technique that does not require much specialized equipment.

It is also easy to achieve batch-to-batch consistency. However,

this method is limited to being effective with only a select

number of polymers and is strictly a laboratory-scale

technique.[163]

8. Conclusions

Collagen, one of the most abundant proteins in the body

and the structural and functional basis of hierarchical bone

organization, has been regarded as an increasing important

biomaterial for bone tissue engineering. The hierarchical

organization of collagen composite in the body provides us

with cues for fabricating biomimetic collagen matrices

mimicking its in vivo counterpart. On the other hand, the

development of new techniques, such as electrospinning,

provides us with more opportunities to easily create ECM

analogous nanofibres, promoting our methods from the

simply concept of placing cells in a degradable scaffold to

building native tissue either in vivo or in vitro. It is anticipated

that, with the promotion of new concept and technology,

current research in bone tissue engineering is approaching a

major breakthrough in the treatment of injury and disease.

Received: December 31, 2009

Final Version: March 26, 2010

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