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Biomimetic porous scaffolds for bone tissue engineering

Shuilin Wu a, Xiangmei Liu a, Kelvin W.K. Yeung b,c,*, Changsheng Liu d, Xianjin Yang d,e
a
Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-of-Education Key Laboratory for the Green Preparation and
Application of Functional Materials, Hubei Province Key Laboratory of Industrial Biotechnology, Faculty of Materials Science & Engineering, Hubei University,
Wuhan, China
b
Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong, China
c
Shenzhen Key Laboratory for Innovative Technology in Orthopaedic Trauma, The University of Hong Kong Shenzhen Hospital, 1 Haiyuan 1st Road, Futian
District, Shenzhen, China
d
Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai, China
e
School of Materials Science and Engineering, Tianjin University, Tianjin, China

A R T I C L E I N F O A B S T R A C T

Article history: Increased use of reconstruction procedures in orthopedics, due to trauma, tumor, deformity,
Available online 5 May 2014 degeneration and an aging population, has caused a blossom, not only in surgical advancement, but
also in the development of bone implants. Traditional synthetic porous scaffolds are made of metals,
Keywords: polymers, ceramics or even composite biomaterials, in which the design does not consider the native
Biomimetic structure and properties of cells and natural tissues. Thus, these synthetic scaffolds often poorly
Scaffold integrate with the cells and surrounding host tissue, thereby resulting in unsatisfactory surgical
Bone implants outcomes due to poor corrosion and wear, mechanical mismatch, unamiable surface environment, and
Tissue engineering other unfavorable properties. Musculoskeletal tissue reconstruction is the ultimate objective in
Surface bio-functionalization
orthopedic surgery. This objective can be achieved by (i) prosthesis or fixation device implantation, and
(ii) tissue engineered bone scaffolds. These devices focus on the design of implants, regardless of the
choice of new biomaterials. Indeed, metallic materials, e.g. 316L stainless steel, titanium alloys and
cobalt chromium alloys, are predominantly used in bone surgeries, especially in the load-bearing zone of
prostheses. The engineered scaffolds take biodegradability, cell biology, biomolecules and material
mechanical properties into account, in which these features are ideally suited for bone tissue repair and
regeneration. Therefore, the design of the scaffold is extremely important to the success of clinical
outcomes in musculoskeletal surgeries.
The ideal scaffolds should mimic the natural extracellular matrix (ECM) as much as possible, since the
ECM found in natural tissues supports cell attachment, proliferation, and differentiation, indicating that
scaffolds should consist of appropriate biochemistry and nano/micro-scale surface topographies, in
order to formulate favorable binding sites to actively regulate and control cell and tissue behavior, while
interacting with host cells. In addition, scaffolds should also possess a similar macro structure to what is
found in natural bone. This feature may provide space for the growth of cells and new tissues, as well as
for the carriers of growth factors. Another important concern is the mechanical properties of scaffolds. It
has been reported that the mechanical features can significantly influence the osteointegration between
implants and surrounding tissues, as well as cell behaviors. Since natural bone exhibits super-elastic
biomechanical properties with a Young’s modulus value in the range of 1–27 GPa, the ideal scaffolds
should mimic strength, stiffness and mechanical behavior, so as to avoid possible post-operation stress
shielding effects, which induce bone resorption and consequent implant failure. In addition, the rate of
degradation and the by-products of biodegradable materials are also critical in the role of bone
regeneration. Indeed, the mechanical integrity of a scaffold will be significantly reduced if the
degradation rate is rapid, thereby resulting in a pre-matured collapse of the scaffold before the tissue is
regenerated. Another concern is that the by-products upon degradation may alter the tissue
microenvironment and then challenge the biocompatibility of the scaffold and the subsequent tissue
repair. Therefore, these two factors should be carefully considered when designing new biomaterials for
tissue regeneration. To address the aforementioned questions, an overview of the design of ideal

* Corresponding author at: Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong, China.
E-mail address: wkkyeung@hku.hk (Kelvin W.K. Yeung).

http://dx.doi.org/10.1016/j.mser.2014.04.001
0927-796X/ß 2014 Elsevier B.V. All rights reserved.
 Author´s Personal Copy
2 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36

biomimetic porous scaffolds is presented in this paper. Hence, a number of original engineering
processes and techniques, including the production of a hierarchical structure on both the macro- and
nano-scales, the adjustment of biomechanical properties through structural alignment and chemical
components, the control of the biodegradability of the scaffold and its by-products, the change of
biomimetic surface properties by altering interfacial chemistry, and micro- and nano-topographies will
be discussed. In general, the concepts and techniques mentioned above provide insights into designing
superior biomimetic scaffolds for bone tissue engineering.
ß 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1. The demand for musculoskeletal tissue engineering materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2. Characteristics of natural bone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1. Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.2. Natural bone architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.3. Biomechanics of bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3. The evolution of bone scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.1. Autograft bone scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.2. Allograft-based bone scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.3. Artificial and engineered bone scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2. Biomimetic biomechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1. Structural design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1.1. Porosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1.2. Pore shape, size and orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1.3. Hierarchical structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2. Choice of materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.1. Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.2. Bioglasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.3. Biopolymer and its derived composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3. Biomimetic surface design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.1. Surface functionalized chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.1.1. Coatings/film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.1.2. Biomineralization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.3. Surface polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2. Surface functionalized structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.1. Chemical treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.2. Physical methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.3. Synergistic regulation of surface structure and chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4. Biodegradability design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.1. Scaffold resorption/biodegradability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.2. Biodegradation rate control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5. Clinical applications of engineered tissue scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

1. Introduction materials for musculoskeletal tissue engineering is paramount in

1.1. The demand for musculoskeletal tissue engineering materials
The history of the restoration of injured tissues using biomater-
ials can be traced back to the prehistoric period, dating back 30,000
years [1]. There is evidence revealing that the Chinese and the
Romans used gold for false teeth as long as 2000 years ago [2].
Currently, orthopedic reconstruction procedures stemming from
trauma, tumor, deformity, degeneration and an aging population
have dramatically increased, triggering a high demand on the
advancement of bone implant technology. Modern clinical practice
in orthopedics has demonstrated that the autograft exhibits superior
therapeutic effect in bony fusion. However, donor site morbidity and
limited supply are major concerns. Allografts and xenografts may
raise other concerns in pathogen transmission and immunorejec-
tion, respectively [3,4]. Therefore, the development of synthetic
order to satisfy the booming demand of increased orthopedic
implantations.
Today, the need for bone implants is increasing, with more
than 400,000 bone-grafting operations performed annually in
Europe and over 600,000 in the United States [5,6]. According to
the Orthopedic Industry Annual Report and GlobalData’s report,
released in ORTHOWORLD, worldwide orthopedic product sales
exceeded $43.1 billion in 2012, increasing from $15 billion in 2002
[7,8]. The spending on bone graft substitutes in the U.S. market
had already reached $1.3 billion in 2010 and the forecasted
increase, with a compound annual rate (CAGR) of 7.4%, is about
$2.2 billion for the year 2017 [9]. Additionally, a recent report from
Medtech Insight revealed that bone graft substitute products for
spinal fusion totaled approximately $177.1 million in 2010 in
Europe and are predicted to increase at a CAGR of 17.3% [10].
According to the 2009 Asia Audit report released by the
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Table 1
Chemical composition of typical bone [16,17].
Element Ca2+ P Na+ Mg2+
a a a a
wt% 34.8 15.2 0.9 0.72
a
Obtained from an ashed sample.
b
Obtained from an unashed sample.
International Osteoporosis Foundation (IOF) [11], there are
almost 69.4 million people over the age of 50 suffering from
osteoporosis in the mainland of China, resulting in some 687,000
hip fractures and 1.8 million new vertebral fractures occurring per
year. Over the past four decades, the number of hip fractures
increased by 300% in Hong Kong and by about 500% in Singapore.
In Japan, the prevalence of osteoporosis is around 12 million and
the hip fracture incidence rate in the oldest population (over 75) is
increasing dramatically in both men and women [11]. TechNavio’s
analysts have forecasted that the global orthopedic contract
manufacturing market will grow by 11.05% of CAGR between the
years 2012 and 2016 [12].
1.2. Characteristics of natural bone
To design the ideal biomimetic artificial scaffold, a full
understanding of the compositions, structures and biomechanical
and biochemical properties of natural bone is required. These
qualitative and quantitative parameters may provide adequate
insights in the engineering of superior biomimetic scaffolds.
1.2.1. Composition
Generally, the living bone in the human musculoskeletal
system is composed of 10–20% collagen, 60–70% bone mineral,
and 9–20% water, by weight [13,14]. In addition, other organic
materials, such as proteins, polysaccharides and lipids, are also
included in small quantities [15].
The basic composition of the bone mineral component can be
approximately defined as hydroxyapatite (HA) with the chemical
formula Ca10(PO4)6(OH)2. However, when HA has a Ca:P ratio of
1.67 (5:3), the Ca:P ratio in bone minerals actually varies
between 1.37 and 1.87, indicating that these varied compositions
of bone minerals may contain other additional ions, such as
strontium, zinc and carbonate, etc. [14]. The typical chemical
composition of bone minerals has been summarized in Table 1
[16,17].
Fig. 1. Hierarchical structural organization of bone: (a) cortical and cancellous bone; (b) osteons with Haversian systems; (c) lamellae; (d) collagen fiber assemblies of collagen
fibrils; (e) bone mineral crystals, collagen molecules, and non-collagenous proteins [18].
3
K+ F Cl CO32 P2O74 H2O Sr2+, Zn2+, . . .
a a a b
0.03 0.03 0.13 7.4 0.07 10 trace
1.2.2. Natural bone architecture
As shown in Fig. 1(a) [17,18], a typical human long bone is
generally composed of cortical bone, cancellous bone, periosteum,
endosteum and articular cartilage in a macro scale. Cortical bone,
comprising 80% of the bone mass, forms a compact ectotheca
surrounding the central marrow cavity, whereas cancellous bone
has an open, honeycomb structure that accounts for roughly 20% of
the total mass of the skeleton. The cortical bone has a higher
Young’s modulus value in order to provide sufficient mechanical
strength to bear weight, while the cancellous bone possesses a
much lower Young’s modulus value than the cortical bone. The
special alignment of the cancellous bone structure, however, is
able to dampen the sudden stress. Cartilage is a type of collagen-
based connective tissue composed of very large protein–polysac-
charide molecules, providing a tough, flexible matrix made of
entangled collagen fibers, protein, and sugar. Mechanically,
articular cartilage has a very low coefficient of friction (<0.01),
indicating that it provides a smooth surface to allow for ease of
joint movement with the presence of synovial fluid [14].
Periosteum is a fibrous membrane of connective tissue found on
the surface of bone. It is rich in fibroblasts and progenitor cells,
which are critical to new bone formation. Endosteum is a thin layer
of lining cells found on the surface of the medullary cavity and is
important during the process of bone remodeling.
The cortical bone exhibits a hierarchical structural organization
composed by osteons on a micro scale, collagen fibers on a nano-
scale, and collagen molecules on a sub-nano-scale (Fig. 1). The
cross-section image of compact bone shown in Fig. 2(a) displays
the cylindrical osteons with blood vessels running along Haversian
canals (in the center of each osteon) [17,19]. The metabolic
substances can be transported by the inter-communicating
systems of canaliculi, lacunae, and Volkmann’s canals, which are
interconnected in the marrow cavity [13]. As shown in Fig. 2(b)
[20], the cancellous bone exhibits a cellular structure, which is
made up of an interconnected network of trabecula. A network of
trabecula rods provides a low density, open cell, while one
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4 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 2. Bone morphologies: (a) optical micrograph of transverse cross-section
showing the microstructure of compact lamellar bone–human femora [17,19]; (b)
SEM image of cancellous bone [20]; (c) high magnification histological section
image of trabecula in human cancellous bone by decalcified preparation; bm – bone
matrix; cc – calcified cartilage; end – endosteal osteoblasts [21].
trabecula gives high-density, virtually closed cells (shown in
Fig. 2(c)) [21], and Haversian canals are absent in trabecula,
implying that the cancellous structure lacks the Haversian system
[21]. Practically, the relative density of cancellous bone ranges
from 0.05 to 0.7 [22].
Fig. 3. Hierarchical organization of collagen fiber [5].
Collagen fibers are the main component forming the lamella on
the micro level. As shown in Fig. 3 [5], the diameter of these fibers
varies from 100 to 2000 nm, consisting of carefully arranged arrays
of tropocollagen molecules that are composed of three left-handed
helices of peptides, which are long, rigid molecules (300 nm long,
1.5 nm wide). Bone mostly contains type-I collagen and small
amounts of type-V collagen, whose molecules are organized into
collagen fibrils, which are formed by the assembly of tropocollagen
molecules in a 3/4 stagger with a parallel array [5] along the fibrils
[23]. The specific binding points serve as nucleation sites for the
bone mineral crystals. These hydroxyapatite (HA) crystals, which
appear in the form of plates or needles, are about 40–60 nm long,
20 nm wide, and 1.5–5 nm thick [13,19,22]. The mineral phase in
bone is made of a continuous phase of HA crystals, rather than a
discrete aggregation. Indeed, it has been proven that bones retain a
good mechanical strength after a complete removal of the organic
phase [13].
1.2.3. Biomechanics of bone
As shown in Fig. 4 [13], the stress–strain curves for compact
bones exhibit a linear elastic region, followed by a flat plastic
region at about a 0.8% strain at lower strain rates. It indicates that
bone exhibits a tough behavior at low strain rates but fractures
more like a brittle material at high strain rates [13,24]. The slope of
the stress–strain curve, i.e. the elastic modulus of bone, increases
with increasing mineral content [15,19]. At low strain rates, bone
exhibits excellent toughness, mostly due to its hierarchical, as well
as porous, structure, which is able to stop crack propagation after
small amounts of cracking [15]. The properties of compact human
bone are listed in Table 2 [17].
In comparison with cortical bone, cancellous bone exhibits a
different mechanical behavior. Fig. 5 demonstrates its compressive
stress–strain curve [25]. The curves exhibit three regimes of
behavior, namely linear elasticity, a plastic collapse-plateau and
densification area. The small strain, linear-elastic response of low-
density, near-isotropic cancellous bone derives from the elastic
bending of the cell walls [26–28]. The linear-elastic regime ends
when the units begin to collapse. Progressive compressive collapse
gives the long, horizontal plateau of the stress–strain curve, which
continues until opposite cell walls meet and touch, causing a steep
increase in stress [25]. The mechanical properties of cancellous
human bone also depend on porosities. The typical compressive
strength of cancellous bone ranges from 2 to 12 MPa and its
modulus is in the range of 0.1–5 GPa.
It is well known that the collagen phase, as the main component
of bone, plays an important role in mechanical behaviors of cortical
bone. Recent research by Leung et al. reveals that the Young’s
modulus value and ultimate strength of the demineralized bone
specimens decrease along with aging in both the longitudinal and
transverse orientations, indicating that the stiffness and strength
of the collagen phase are correlated to the age of the patient [29].
Table 2
Mechanical properties of compact human bones [17].
Test direction related to bone axis
Parallel Normal
Tensile strength (MPa) 124–174 49
Compressive strength (MPa) 170–193 133
Bending strength (MPa) 160
Shear strength (MPa) 54
Young’s modulus (GPa) 7.0–18.9 11.5
20–27 (random)
Ultimate tensile strain 0.014–0.031 0.007
Ultimate compressive strain 0.0185–0.026 0.028
Yield tensile strain 0.007 0.004
Yield compressive strain 0.010 0.011
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 4. Stress–strain curve for human compact bone [13].
Fig. 5. Compressive stress–strain curves for several relative densities of wet
cancellous bone [25].
The typical micro-tensile curve of collagen specimens from
demineralized femurs harvested from middle age male patients
is shown in Fig. 6. This study also found that the maximum
sustainable deformation of the collagen phase was independent of
age and orientation [29].
Apart from collagen, the bone mineral content also contributes
significantly to the mechanical properties of bone. As shown in
Fig. 7(a), the Young’s modulus value increases significantly with
increased Ca content: an increase from 200 mg calcium/g to
260 mg/g in dry bone induces an increase in Young’s modulus of
about 5–25 GPa. However, this correlation is not conclusive, as it
has not taken account of the variations in the porosity, the
relationship between bone ‘‘grain’’ and the direction of loading, as
well as the relative proportions of woven and lamellar bone
[30,31]. Additionally, the strain at yield decreases with increasing
mineral content. The yield strain is about 0.009 at 210 mg/g,
whereas it is about 0.005 at 270 mg/g. The higher value is about 1.8
times that of the lower value (shown in Fig. 7(b)) [30].
1.3. The evolution of bone scaffolds
1.3.1. Autograft bone scaffold
Clinically, the gold standard for bone defect repair is the use of
autograft (i.e. bone grafts taken from a donor site, such as the iliac
5
Fig. 6. The stress–strain curves of collagen specimens from demineralized middle
male femurs in both longitudinal and transverse axes [29].
crest or fibula, implanted to a recipient site during surgery) [32,33].
However, limited supply and donor site morbidity are a major
concern. Other factors, e.g. infection, chronic wound pain and
hypersensitivity, have also been reported [34].
1.3.2. Allograft-based bone scaffold
Bone allograft is an alternative, as the limitations in autograft
are no longer a concern. Its three-dimensional structure is similar
to that of host bone. However, the use of allograft presents
potential risks, including the transmission of viral diseases,
bacterial infection and immune rejection in the recipient body
[33]. In addition, superior integration with host bone is most likely
not due to its suboptimal osteoconductivity [35].
1.3.3. Artificial and engineered bone scaffold
Metallic implants like stainless steel have been developed and
applied for bone repair since the 1920s. Artificial bone implants
made of metals, ceramic, polymers and composites are synthesized
and widely used for bone reconstruction and regeneration [36–43].
These synthesized scaffolds can exhibit satisfactory mechanical
properties and biocompatibility, as well as other unique proper-
ties. However, as compared with bone autograft and allograft,
there are still some congenital deficiencies, such as ion release
from metallic scaffolds [44], poor strength of polymeric scaffolds
[45], friability of ceramic scaffolds [46], uncontrollable degrad-
ability of composites [47], and so on. Since the concept of tissue
engineering was formally proposed by Langer and Vacanti [3] in
1993, these synthesized scaffolds have been incorporated with
bone marrow stem cells [48,49], drugs and gene delivery, and
different growth factors, such as TGF-b [50], BMP [51], and VEGF
[51]. Recent research has revealed that the specific nano-
topography, chemistry and nanostructure of biomaterials could
not only enhance biocompatibility, but also regulate cell/tissue
behaviors without the use of growth factors [52,53]. This is
possible because these functionalized biomaterials mimic the
natural instincts of cells and tissues [54,55]. Moreover, Stupp et al.
[56] proposed that biomimetic systems could be achieved by
hydroxyapatite biominerization inspired by inorganic composition
of natural bone and enamel. In a contrast, Cui et al. discussed that
the advancement of bone grafts was initiated by the assembly of
mineralized collagen fibrils (i.e. the organic components of bone)
[57]. After all, the design of a synthetic scaffold is still the core
component in bone tissue engineering.
This paper, therefore, reviews the recent progress of bone
scaffolds from the perspective of bionics, including biomechanics
and biodegradability, as well as surface chemistry and topography.
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6 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 7. Diagrams showing the relationships between mineral content (as indicated
by mg calcium per g dry bone) and various tensile mechanical properties of compact
bone [30].
2. Biomimetic biomechanics
From a biomechanical and clinical point of view, the bone
scaffold should offer adequate mechanical integrity to provide
stable fixation, as mechanical loading is expected in patients
during bone regeneration. Secondly, the Young’s modulus value for
these scaffolds should be similar to that of host tissues, in order to
avoid potential post-operation stress shielding [58,59]. Most
importantly, recent studies reveal that mammalian cells are very
sensitive to the change in mechanical properties of the biomaterial
matrix, such as stiffness, density, and the surface chemical
composition of the matrix [60,61]. Zaman et al. demonstrated
that cell movement on a three-dimensional (3D) scaffold could be
altered by changing the matrix stiffness [62]. In comparison to the
stiff SiO2 substrate, the soft polydimethylsiloxane (PDMS) sub-
strates with the same topographical features might direct the
alignment of cell adhesion and enhance the elongation of
endothelial cells (ECs) [63]. Apart from the surface topography
and dimension of substrates, other groups reported that the
elasticity of scaffolds could also regulate the morphology,
proliferation and differentiation behavior of mesenchymal stem
cells (MSCs) [64–66]. For example, human MSCs (hMSCs) on a soft
scaffold (E  1 kPa) resulted in neuronal differentiation. The stiffer
scaffold (E  10 kPa) showed glial differentiation within 1 week
[65], while the substrate with a stiffness similar to bone (25–
40 kPa) enhanced osteogenic differentiation of hMSCs [64].
Therefore, taking account of the mechanical properties of natural
bone is both essential and critical when designing biomimetic
scaffolds.
2.1. Structural design
It is well accepted that a scaffold with a porous structure favors
tissue ingrowth, the mass-transportation of nutrients and
osteointegration with the host bone, as well as long-term stable
fixation of bone implants [67,68]. To facilitate desired bone tissue
regeneration, the structural design of the scaffold often considers
factors such as porosity, pore size and shape, the orientation of the
interconnected channel and a hierarchical control of structure.
2.1.1. Porosity
Some studies have suggested that the tangent elastic modulus
of natural bones generally decreases as their porosity increases
[69–71]. In other words, the tangent elastic modulus of porous
tantalum is reduced from 2.2 GPa to 373 MPa by adjusting the
porosity from 66 vol.% to 88 vol.% (shown in Fig. 8) and the yield
stress ranges from 4 to 12.7 MPa, which is similar to the
mechanical properties of bovine and human cancellous bone
[69]. In another example, a porous nickel–titanium (NiTi) scaffold
with 21–48 vol.% in porosity can be achieved by using a novel
metallurgical powder method and the space holder of ammonium
acid carbonate; the Young’s modulus value of these scaffolds is in
the range of 6–11 GPa [72], which is similar to that of wet compact
human bones [73]. The stress–strain curve (Fig. 9) indicates that
these porous scaffolds possess similar superelastic biomechanical
properties, similar to human hard tissues. This observation can be
attributed to the combined effects of the porous structure and the
intrinsic super-elasticity of NiTi [72]. Similarly, by varying the cold
compaction pressure and powder used, Guden et al. [74]
successfully worked out Ti6Al4V scaffolds in the porosity range
of 34–54%. The 40–42% yield strength of Ti6Al4V scaffolds is
comparable to that of human cortical bone [74]. Xu and his
colleagues [75] incorporated water-soluble mannitol crystals into
calcium phosphate cement (CPC) and subsequently removed the
mannitol by water dissolution to obtain macroporous CPC. By
changing the ratio of mannitol in the mixtures, the porosity of CPC
scaffolds can be realized in the range of 45–70.8 vol.%. Both the
flexural modulus and flexural strength were significantly de-
creased along with the increasing porosity [75]. For synthetic poly
Fig. 8. Tangent elastic modulus of porous tantalum scaffolds regressed against
scaffold porosity [69].
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 9. Compressive stress–strain curves of porous NiTi at room temperature [72].
(D,L-lactide-coglycolide) (PLG) scaffolds, the porosity can also be
adjusted by altering the content of dimethyl sulfoxide (DMSO) in
the mixture of PLG and DMSO. The scaffold with a porosity around
80 vol.% exhibits a higher compression modulus (0.26  0.08 MPa)
under 10% strain, whereas the scaffold with porosities of 85 vol.% and
92 vol.% are only 0.091  0.05 MPa and 0.0047  0.004 MPa, respec-
tively [76]. The scaffold with the higher modulus may provide more
sufficient mechanical support and, therefore, prevent the premature
collapse of the surrounding cartilage and subchondral bone near the
bone defect. However, it might also jeopardize the migration of bone
marrow cells, biomolecules, and enzymes, and then delay the
absorbance of the scaffold, resulting in a consequent occurrence of
cleft. Hence, if the mechanical modulus and porosity of the scaffold
are nicely defined, it will accelerate bone tissue regeneration.
Combining a similar modulus with a higher porosity encourages
the migration of bone marrow cells that erupted from the defect,
which is helpful for the rapid replacement of the scaffold with
subchondral bone and articular cartilage [76].
2.1.2. Pore shape, size and orientation
Weiner and Wagner reported that the mechanical properties of
natural bone were significantly influenced by the structure of bone
such as asymmetry and anisotropy [77]. For example, the Young’s
modulus of planar lamellar bone in the tibia of baboon was quite
different from that of osteonal bone. The highest modulus was
always seen, when the loading axis was perpendicular to the long
axis of the bone [77]. Although increased scaffold porosity may
increase permeability, the modulus will be sacrificed [68,69].
Alternatively, the scaffold mechanical properties can also be
adjusted by altering the structure of pores within the scaffold.
Fig. 10 shows the variation in modulus of two separate scaffolds
with different porous microstructures. The scaffold with a
spherical pore exhibits a higher elastic moduli that the one with
a cylindrical pore (Fig. 10(c)) [68]. Park et al. [78] fabricated
polycaprolactone (PCL) scaffolds with interconnecting pores, using
a 3D melt plotting system. By adjusting the ratio of nozzle size and
distance between strands, 3D PCL scaffolds with different pore
sizes were obtained (Fig. 11(a)). As the pore size increased from
125 mm to 300 mm, the corresponding modulus reduced from
10.3 MPa to about 3 MPa (Fig. 11(b)). These findings indicate that
the mechanical properties of these plotted scaffolds could be easily
manipulated by tuning the pore geometry, as compared with the
unordered structures in traditional salt-leaching scaffolds [78].
Furthermore, the orientation of pores in scaffolds could also
significantly contribute to mechanical behaviors [79,80]. For
7
Fig. 10. Modulus versus porosity for two scaffolds with designed spherical pore and
cylindrical pore microstructures. (a) The spherical pore, (b) cylindrical pore, (c) plot
of effective elastic moduli normalized by base moduli for spherical pore (dashed
line) and cylindrical pore (solid line). Results demonstrate that the modulus
increases as expected with volume fraction, and that, for a given volume fraction,
the spherical pore is stiffer. [68].
example, the porous poly(D,L-lactic acid) (PDLLA) foams that have
diversified structural morphologies were obtained by employing a
supercritical CO2 (scCO2) pressing technique. It was found that
their compression modulus significantly fluctuated; even the
porosity showed little change, indicating that the orientation of
porous structures could also play an important role in the
mechanical properties of scaffolds [79]. In addition to the
polymeric scaffolds, specific orientation of pores could also be
applied in metallic scaffolds [80–82]. For instance, highly porous
titanium with aligned pores up to 500 mm in diameter was
successfully fabricated using a modified reverse freeze casting
method (shown in Fig. 12(a)) [80]. The mechanism lies in the
spontaneous migrations of Ti powders along the pre-aligned
camphene boundaries, which then formed a titanium–camphene
mixture with an aligned structure after freeze drying and sintering.
As shown in Fig. 12(b), it is obvious that the compressive yield
strength mainly depends upon the orientation of pores and the
porosity of scaffolds. The sample, in which the porous geometry
was parallel in direction, exhibited much higher mechanical
strength than those structures cast in the usual direction [80].
2.1.3. Hierarchical structure
The mechanical properties and permeability are important in
engineered scaffolds. High porosity and large pore size benefit
permeability, but impair the mechanical properties of the scaffold.
Hierarchical scaffold design seems to be promising for desired
comprehensive properties. Indeed, the concept is to fabricate the
scaffold based on the basic structure of human tissues, such as
bone. Practically, there are seven hierarchical organization levels of
the bone family of materials including major components,
mineralized collagen fibril, fibril array, fibril array patterns,
cylindrical osteons, spongy versus compact bone, and whole bone,
and five levels have been thoroughly discussed by Weiner and
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8 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36

Fig. 11. Modulus versus pore size and distance between strands in the 3D PCL scaffolds: (a) SEM images of plotted scaffolds, (b) comparison of the compressive modulus for
various nozzle size/distance between strands values and pore sizes [78].

Wagner [77]. The Committee on Synthetic Hierarchical Structures
first proposed this in 1994, in order to develop novel processing
technologies to fabricate hierarchically structured materials with
proper control on an industrial scale [83]. In 2005, Hollister
proposed a clear definition stating hierarchical structure refers to
the features at scales from the nanometer to millimeter that are
able to determine how well the bioscaffold meets the conflicts
between mechanical function and mass-transport needs [68].
Almost all types of biomaterials, such as metals, polymers,
ceramics, hybrids and composites, can be machined into biomedi-
cal scaffolds for the purpose of tissue engineering [84–90]. For
example, by employing a liquid foaming method and subsequent
chemical treatments, three-dimensionally hierarchical porous
structures can be successfully achieved in titanium scaffolds with
pore size ranging from the nanometer to micrometer scale. This
scaffold also has a sufficient compressive strength to meet the
requirements of implantation [87]. Woodard et al. revealed that
the micro-pores in porous hierarchical hydroxyapatite (HA) bone
scaffolds are quite important to maintain the mechanical
satiability of the scaffolds, as the newly formed bone in the
scaffolds supported the load after a fracture. The mechanical
failure stress was significantly less than that of the scaffolds with
pure macro-pores (shown in Fig. 13). Furthermore, evidence also
suggested that bone could arrest crack propagation in hierarchical
HA scaffolds [89]. Practically, hierarchical bimodal or multi-modal
porous architectures with macro-, micro-, and nano-pores,
simultaneously, are also important in modulating the permeability
and compliance of polymeric scaffolds that favor various applica-
tions in tissue engineering [84,88,91]. Ferrer et al. developed
hierarchical macro- and micro-porous poly(L-lactic acid) (PLLA)
scaffolds by combining the fabrication processes, including the
dioxane solvent freeze extraction technique and leaching of
macroporogen spheres [88]. As shown in Fig. 14, an increase in
the PLLA/dioxane ratio decreased the size of micropores and also
influenced the macropore size to a certain extent. This phenome-
non can be attributed to the ability of dioxane to cause
macroporogen particles to swell, while also increasing the amount
of macroporogen. The porosity increases due to the expansion of

Fig. 12. Porous Ti sintered by a reverse freeze casting process. (a) SEM morphology along the longitudinal section, (b) compressive strength of the samples as a function of the
porosity, tested either parallel or normal to the direction of pore alignment [80].
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 9

Table 3
Metals used for orthopedic implant applications [93].

Metal Application

Stainless steel Osteosynthesis/(joint arthroplasty)

CoCrMo alloys Joint arthroplasty/(osteosynthesis)
Co–Ni alloys Osteosynthesis
CP–Ti Osteosynthesis
(a + b) Ti alloys Joint arthroplasty and osteosynthesis
(b/near-b) Ti alloys Osteosynthesis
NiTi Osteosynthesis
Ta Bone augmentation

Table 4 [92–96]. The general strategy for metallic materials is to

Fig. 13. Stress–strain curve of porous HA scaffolds, (solid curve) scaffolds with pure
macro-pores, and (dotted curve) hierarchical scaffolds with macro- and micro-
pores [89].
the pore dimension. Hence, the hierarchical structure can be
modulated by altering the PLLA/dioxane ratio and the amount of
macroporogen that directly contributes to the mechanical
properties and permeability [88].
2.2. Choice of materials
The matrix is a fundamental component of synthetic bone
scaffolds. It is essential to select appropriate materials to build up a
scaffold with the desirable mechanical properties to meet the
clinical need. For example, in addition to biocompatibility and
sufficient space for tissue ingrowth, the regeneration of bone
cartilage requires soft scaffolds with a lower elastic modulus [76],
while the hard tissue repair, like bone, requires scaffolds with a
higher elastic modulus [45].
2.2.1. Metals
Metallic biomaterials are widely used for load-bearing applica-
tion and have shown great success because of their excellent
mechanical properties and good machinability [92], which are
summarized in Table 3 [93]. However, dense metallic materials
have a much higher Young’s modulus value than natural bone,
which often induces the resorption of surrounding bone tissues,
which is ascribed to stress shielding during implantation. The
mechanical properties of typical metallic materials are listed in
reduce their stiffness to match that of natural bone by establishing
a porous structure and, thus, to eliminate stress shielding. For
example, by using powder metallurgical methods combined with
other techniques, porous metallic scaffolds like stainless steels
[97], titanium-based alloys [72,80–82], and magnesium-based
alloys [98], have been widely developed in the past decades. A
multi-stage rapid prototyping technique was developed to
produce porous titanium scaffolds with fully interconnected pore
networks and reproducible porosity and pore size, whose
characteristics were determined by a sacrificial wax template
that was fabricated using a commercial 3D-printer [82]. Powder
metallurgy processes were employed to generate the titanium
scaffolds by filling around the wax template with a titanium slurry.
Additionally, the pore sizes can be precisely modulated by altering
the wax design template from 200 to 400 mm, as shown in
Fig. 15(a) [82]. Consequently, the mechanical properties of Ti
scaffolds can satisfy the intended design, i.e. in comparison with
dense Ti. Both the Young’s modulus value (Fig. 15(b)) and yield
strength (Fig. 15(c)) are close to those of human cortical bone.
These low modulus, porous metallic scaffolds can also be prepared
by other routes. For example, by sintering the entangled titanium
wire with setting spatial architecture, porous titanium wire
scaffolds can be obtained [99]. The yield strength, ultimate tensile
strength, and elastic modulus of a 44.7% scaffold are 75 MPa,
108 MPa, and 1.05 GPa, respectively, while corresponding values
become 24 MPa, 47.5 MPa, and 0.33 GPa, respectively, when the
porosity increases to 57.9% [99]. This means that the mechanical
properties of this titanium scaffold can be precisely adjusted to
mimic those of human cancellous bone, which is ascribed to the
structural flexibility of the entangled titanium wire materials [99].
Besides the porous structure, the biomimetic biomechanics of a
metallic scaffold can be achieved by selecting alloys with

Table 4
Mechanical properties of typical metallic implants [92–96].

Materials E (GPa) syield(MPa) sult(MPa) % elong

Stainless steel: 316L type 193 172–690 485–860 12–40

Austenitic SS
F138 190–690 490–1350 <12–40
F1314 380–860 690–1035
F1586 430–1000 740–1100
F2229 590–1550 930–1730
Fe-based 200–205 170–690 540–1000 12–40
Co-based 220–230 450–1500 655–1900 5–30
Tantalum 188–190 138–345 205–517 1–30
CP–Ti 100–115 170–483 240–550 15–24
Ti6Al4V 110 860 930 10–15
Ti6Al7Nb 105 795 860 10
Ti5Al2.5Fe 110 820 900 6
Ti13Nb13Zr 79–84 863–908 973–1037 10–16
Ti35.5Nb7.3Zr5.7Ta 55–66 793 827 20
NiTi (martensitic/austenitic) 28–41/80 70–140 700–1100/1300–2000 9
Pure iron 200 150 210 40
Magnesium alloy 37.5–65 70–140 190–250 2–11
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Fig. 14. SEM images of hierarchical PLLA scaffolds at different concentrations. (a–c, g–i): low magnification showing the macro-porous structure for the two series (1:1 and
1:1.25-PLLA-solution/porogen weight ratio); (d–f, j–l): higher magnifications showing the micro-porosity and the trabecular structure of the scaffolds [88].

congenital superelasticity, such as nickel–titanium shape memory
alloy [72,90,100], and nickel-free Ti-based alloys [101–103]. As
shown in Figs. 4 and 5, generally, human hard tissues, like bone and
tendon, have high elasticity, low deformation forces and constant
force over wide ranges of strain, called superelasticity [104], which
is similar to the superelastic behavior of NiTi alloys caused by
stress-induced martensitic transformation depicted as follows: at a
temperature above the final austenitic transformation tempera-
ture (Af), the austenitic phase in the sample will start to transform
into martensite under a constant strain. After reaching critical

Fig. 15. Porous titanium scaffolds prepared by rapid prototyping and the 3D-template technique (a) SEM images of scaffolds with pore sizes of 200, 300, and 400 mm,
(b) Young’s modulus versus pore size, and (c) yield strength versus pore size [82].
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 11

Table 5
Features of typical bioglass scaffolds.

Materials Method Porosity (vol.%) Compressive strength (MPa) Young’s modulus (GPa) References

BioK PE burning-out 70–78 3.4–3.7 84.2 [113,114]

70S30C Sol–gel foaming 82–88 0.34–2.26 [106]
GC (SCK) PE burning-out 60–62 1.5–6.0 [115]
GC (Fa-GC) PE burning-out 23.5–50 20.1–56.3 [116]
Polymeric sponge replication 75 2 [117]
GC (CEL2) PE burning-out 48  3 7  0.6 [118]
Polymeric sponge replication 53.5–72.8 1.0–5.4 [119,120]
GC (ICEL2) Polymeric sponge replication 82  6.7 0.4  0.3 [120,121]
13–93 Polymer foam replication 85  2 11  1 3.0  0.5 [110]
Unidirectional freezing of suspensions 56–60 25  3 1.2 [122]
Unidirectional freezing of suspensions 20–60 16–180 4–25 [123]
Selective laser sintering 50 41–157 4.4  0.7 [124]
Freeze extrusion fabrication 50 140  70 5.5  0.5 [125]
45S5 Polymeric sponge replication 82–92 0.27–0.42 – [108]
Rice husk burning-out 43.5–47.2 5.4–7.2 – [112]
PE burning-out 83.4 [114]

stress, the stress at which the phase transformation begins, it glass scaffold exhibits almost the same biomechanical behavior as
remains roughly the same. Further straining will lead to elastic the cancellous bone, i.e. superior superelasticity and similar yield
loading of the martensite. Unloading before plastic deformation of strength, as shown in Fig. 17 [126]. Arcos and colleagues developed
the martensitic phase will induce a reverse transformation at a a highly ordered mesoporous bioactive glass (MBG) scaffold with
lower stress level and the strain can be fully recovered [92]. improved mechanical strength during the in vitro biomimetic
Combining this unique superelasticity with a porous structure or process in simulated body fluids (Fig. 18(a)), which is ascribed to
porous NiTi alloy scaffolds exhibits more impressive in vivo the unique internal mesoporous structure that facilitates ionic
biomechanical properties than porous titanium scaffolds after
implantation in the femur/tibia of rabbits for 15 weeks (Fig. 16),
thus shielding new bone tissues from large load stress, and
consequently favoring the deep penetration of new bone tissue
into the porous NiTi scaffolds compared to porous Ti scaffolds. This
reveals that the native superelastic biomechanical properties of
this porous metallic scaffold bode well for the fast formation and
ingrowth of new bones under load-bearing conditions [100].

2.2.2. Bioglasses
Bioactive glasses are becoming one of most promising scaffold
materials for bone regeneration since their discovery by Larry
Hench in 1969 [105]. They have a well recognized superior
osteoconductivity, controlled biodegradability, cell delivery capa-
bilities, the capacity for activation of osteogenic gene expression,
they favor the formation of bone mineral-like phases and have
drug delivery abilities, mainly stemming from their composition,
which is similar to bone mineral [106–111]. However, for clinical
use, the permeability and mechanical properties of 3D scaffolds
made from bioactive glass prepared by many conventional
methods is often conflicting and cannot satisfy the practical
application of repairing load-bearing bones. For example, most
traditional bioglass scaffolds are brittle and do not possess
superelastic performance [105,112–115]. In general, both the
compressive strength and the Young’s modulus value of these
scaffolds mismatch those of natural bones with the same porosity
and vary with the different fabrication process, structure and raw
materials, which can be found in Table 5 [108,110,112–125].
Consequently, the strategy is to control the structure and
composition by adjusting the process parameters and raw
materials during the bioglass scaffold fabrication process, so as
to achieve the satisfactory mechanical properties [126–129]. For
example, Xia and Chang [126] successfully fabricated a biomorphic
material with a similar structure and mechanical properties of
cancellous bone by using demineralized cancellous bone matrix
(DBM) as a template. DBM offers a template to allow entrapping of
nano-bioglass particles in the slurry, along the original channels
and gaps, thus ensuring the formation of the bone-like structure.
On the other hand, the addition of gelatin mimics the organic Fig. 16. Force–deflection curves obtained from the push-out tests: (a) Porous NiTi
composition of natural bone. Therefore, this synthetic bioactive and (b) porous Ti [100].
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Fig. 17. (a) Optical images of BG/gelatin scaffold from compression to restoration, (b) and (c) Force–extension curve for cancellous bone and bioactive glass (BG)/gelatin
scaffold [126].

transport through the bulk of the solid pieces. Consequently, the
newly crystallized apatite phase reinforces the grain boundary
interactions (Fig. 18(b)), resulting in a biomimetic nanocomposite
within the entire volume of the implant [128]. Apart from the
structural design, the components of inorganic phases are still
important for the improvement of bioactive glass scaffolds.
Recently, an MBG scaffold composed of CaO–MxOy–SiO2–P2O5
(M = Zr, Mg, Sr) was fabricated by combining polyurethane sponge
and block copolymer EO20PO70EO20 (P123) as co-templates and
an evaporation-induced self-assembly (EISA) process using Ca, P, Si
and M sources [129]. The evaluation of mechanical properties
indicates that the partial substitution of ZrO2, MgO or SrO for CaO
can adjust the comprehensive strength of the MBG scaffold.
Compared with the 15Ca MBG scaffold (52.8  6.4 kPa), 5Mg and
5Sr MBG scaffolds exhibit similar strengths of 57  6.4 and
62.7  17.8 kPa, respectively. However, with a similar porosity, the
compressive strength of the ZrO2-substituted MBG scaffold reached
138.8  15.2 kPa, which is much higher than the values of the CaO–
SiO2–P2O5 MBG scaffold. This is most likely due to the formation of a
tetrahedron network by Zn bonding with Si and O ions, which is more
stable than Ca, Mg or Sr ions bonding to the Si and O network [129].
2.2.3. Biopolymer and its derived composites
Owing to their excellent biocompatibility, tunable chemical
composition, good biological reorganization and adjustable
biodegradation, biopolymers, including both natural and synthetic
polymer composites, have been widely used as biomaterials for the
fabrication of medical devices and tissue-engineering scaffolds

Table 6
Mechanical properties of typical polymers for tissue engineering.

Materials Compressive/tensile strength (MPa) Young’s modulus (GPa) Note References

PDLLA 35–150 Pellet [131]

29–35 1.9–2.4 Film or disk
PLLA 870–2300 10–16 Fiber
28–50 1.2–3.0 Film or disk
40–120 Pellet
PGA 340–920 7–14 Fiber
PLGA 41.4–55.2 1.4–2.8 –
PPF 2–30 – –
PHA and blends 0–43 – –
Poly(anhydrides) 25–27 0.14–1.4 –
Poly(ortho-esters) 4–16 2.5–4.4 –
Alginate 71  9 0.85  0.08 – [134]
Chitosan 0.0650 0.0001098 >80% porosity [135]
Collagen 0.00888 0.000332 – [136]
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Table 7
Typical ECM-like biopolymers for bone and cartilage scaffolds.

Materials Mechanical properties (MPa) Young’s modulus (MPa) Reference

Collagen – 0.31  0.04 1.27  0.12 [141]

poly(D,L-lactide-co-glycolide) (PLGA) 0.837  0.05 5.56  0.64
Aligned nano-fibrous PLGA 4.0–6.6 (dry) 54.2–187.7 (dry) [142]
0.3–3.4 (wet) 0.8–83.5 (wet)
Chitosan-polycaprolactone (CH-PCL) 0.59–0.69 (dry) 6.5–7.4 (dry) [143]
0.054–0.114 (wet) 0.39–0.74 (wet)
Hydrogel (Pluronic F127) Poly(D,L-lactic acid-co-glycolic acid) 1.2 15 [144]
(PLGA)/poly(ethylene glycol) (PEG) particles
Hydrogel (hyaluronic acid) 2.4 45
Hydrogel (Fibrin) 0.6 23
Collagen nano-fiber Poly-L-lactic acid (PLA) nano-fibers – 0.57 (in vivo) [145]
Collagen nano-fibers – – 0.33 (in vivo)
Collagen Glycosaminoglycan 0.005–0.019 (dry) 0.032–0.127 (dry) [146]
Hyaluronic acid Chitosan – 0.0209  0.013 (dry) [147]
0.00129  0.22 (wet)
Chitosan Alginate 0.46  0.022 MPa 8.16  1.57 [148]
Pure CH 0.125  0.015 2.56  0.41
Chitosan Poly(3-caprolactone) (PCL) – 0.00443–0.0236 [149]
RGD UV-cross-linked chitosan 6 – [150]
RGD Polycaprolactone (PCL) – – [151]
RGD Poly(ethylene oxide) – – [152]
Gelatin Chitosan [153]

[130–133]. However, the fatal drawback of pure polymers is the

lower and unstable mechanical strength, which makes them
inappropriate for a load-bearing bone scaffold. The mechanical
properties of typical biopolymers are summarized in Table 6
[131,134–136]. Therefore, the current strategy is to develop novel
synthetic biopolymer-based composites/hybrids for application
like bone scaffold materials, because these composites have the
required strength to match the properties of bone, which include a
low elastic modulus and high strength. The most important issue is
that the mechanical properties of these composites can be
modulated according to need by controlling the volume fraction
of the components, as well as the compositions [131–136]. In
addition to sufficient mechanical performance, biological recogni-
tion by host tissues and cells is quite important for engineered
scaffolds. In addition, the next generation of scaffolds is required to
have specific biological functions that provide cells with a variety
of physical, chemical, and biological cues to modulate their
behaviors [49,55,133,137]. As an extracellular part of a multicel-
lular structure, the extracellular matrix (ECM) typically provides
structural and biochemical support to the surrounding cells
without adverse immune responses, because it contains multiple
types of insoluble molecules, forming a meshwork of structural
proteins to which adhesive proteins, proteoglycans, and glycosa-
minoglycans are associated, despite the variation of ECM
composition between multicellular structures [138,139]. There-
fore, the ECM and ECM-like materials are often used as essential
components for synthesizing biopolymer-based composites or
hybrid scaffolds [138–140]. According to the components, these
synthetic polymer-based composites can be mainly grouped into
three categories, elaborated as follows.

2.2.3.1. ECM-like biopolymers. ECM-like natural biopolymers are

usually blended with some synthetic polymers, such as poly(lactic
acid) (PLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic
acid) (PLGA), and poly (e-caprolactone) (PCL) to form ECM-like
polymer composites/blends, which possess the needed mechanical
properties and biological functions. The typical ECM-like biopo-
lymers used for bone and cartilage scaffolds are listed in Table 7
[141–153]. In general, the mechanical properties of ECM polymer
Fig. 18. (a) Compressive strength values for the MBG-Si85 macroporous scaffold,
composites/blends are mainly regulated by changing the compo- before and after soaking in SBF. The inset shows the stress–strain plots before and
nent ratio and the structure of the scaffolds [142,144,148,153,154]. after soaking in SBF; (b) TEM images of grain boundaries of MBG-Si85 after soaking
For example, a pure collagen graft with a porosity of 70.69  6.52% in SBF for 7 days. [128].
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14 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 19. Evolution of compressive strength for PLGA/PEG-Pluronic F127, PLGA/PEG-
fibrin, PLGA/PEG-HyA and PLGA/PEG-saline (control) scaffolds, sintered for 2 h with
the variation of PLGA/PEG particle to hydrogel ratio [144].
exhibits a lower compressive strength and Young’s modulus value of
0.31 and 1.27 MPa, respectively [141]. The addition of PLGA
significantly enhances the corresponding values of the scaffold to
0.83 and 5.56 MPa, respectively, even at a much higher porosity of
94.79%. In addition, the average pore size of the latter is 121.84 mm,
which is much larger than that of the former, at 26.44 mm [141]. By
altering the ratio of the PLGA/PEG particles to hydrogel components,
such as Pluronic F127, hyaluronic acid and fibrin, the mechanical
properties of PLGA/PEG-hydrogel scaffold can be modulated flexibly
[144]. As shown in Fig. 19, it is obvious that, for all types of scaffolds,
the compressive strength increases with increasing hydrogel content,
up to a certain point. In addition, the mechanical properties are also
influenced by the hydrogel composition. The maximum compressive
strength is 1.2 MPa for PLGA/PEG-Pluronic F127 scaffolds, 2.4 MPa for
PLGA/PEG-HyA scaffolds, and 0.6 MPa for PLGA/PEG-fibrin scaffolds.
Recent research discloses that the structure and mechanical
properties of gelatin/carbohydrate chitosan (G/CC) scaffolds can be
adjusted by controlling the concentration of genipin, a cross-linker to
G/CC scaffolds. The 1% genipin-cross-linked G60/CC40 scaffolds,
prepared at room temperature for 24 h through the scaffold cross-
linking method, exhibit the best compressive strength and modulus
Fig. 20. (a) The elastic modulus and optical images of the collagen and EDC scaffolds; (b) histological analysis of new bone formation. EDC scaffolds significantly enhanced the
bone volume fraction (BVF). Moreover, the adipocyte numbers in the EDC group were significantly lower than in the CON group. [154].
value and could be a promising replacement in missing segments of
load bearing soft tissues [153].
The latest work by Sun et al. [154] shows that tailored
mechanical properties of a 3D gelatin scaffold can control stem cell
mediated bone regeneration by promoting endochondral ossifica-
tion. The mechanical strength of the scaffolds is significantly
increased after incubation in 1-ethyl-3-[3-dimethylaminopropyl]
carbodiimide hydrochloride (EDC), while the original microstruc-
ture of the scaffold is well preserved (shown in Fig. 20(a)) [154].
Further in vivo transplantation tests of mouse bone marrow
mesenchymal stem cell (BMSCs) scaffolds, implanted into pockets
in the dorsa, reveal that EDC scaffolds significantly enhance the
levels of new trabecular bone formation in vivo, but hinder the
formation of the adipocytes in the scaffolds, compared with pure
collagen scaffolds (shown in Fig. 20(b)), which suggests that the
mechanical properties of the matrix play a critical role in
osteogenic and chondrogenic differentiation of stem cells, in
addition to offering the essential mechanical support [154].
Micro and nano-fibrous structures have been proven to be
effective in enhancing the mechanical properties and osteogenesis
of ECM-like scaffolds [137,142,145,155–157]. Gentleman et al.
[155] shows that the mechanical properties of extruded bovine
Achilles tendon collagen fibers are significantly affected by fiber
diameter, with smaller fibers displaying greater tangent moduli
and peak stresses. Mechanical properties of 125 mm-diameter
extruded collagen fibers with a tangent modulus of
359.6  28.4 MPa and peak stress of 36.0  5.4 MPa are similar to
the reported values of human ligaments [155]. In comparison with
scaffolds made of other forms of gelatin, such as solid-walled gelatin
(SW-gelatin) and commercial gelatin foams (Gelfoam), nano-fibrous
gelatin (NF-gelatin) scaffolds exhibit much higher compressive
modulus values, despite pore sizes (shown in Fig. 21(a)) [157].
Moreover, this nano-fibrous structure can keep the long-term
mechanical and structural stability of the gelatin scaffolds in the
biological environment (Fig. 21(b)) [157].
2.2.3.2. ECM-HA/apatite materials. As a bone mineral component,
hydroxyapatite and its derived ceramics are often used to
strengthen ECM-like polymer scaffolds [158–162]. Generally, in
addition to porosity and structure, the mechanical properties of
these composite scaffolds are predominantly influenced by the
volume fraction, size, shape, and crystal form of HA/HA-derived
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 21. (a) The compressive modulus comparison of Gelfoam, NF-gelatin and SW-gelatin scaffolds; (b) The size variation of scaffolds with a macropore size of 250–420 mm
(NF-gelatin and Gelfoam) after culturing MC3T3-E1 osteoblasts for 2 and 4 weeks [157].
particles. Pek et al. [158] successfully prepared carbonated apatite
(CAP) by nanostructure processing and by employing a mixture of
CAP and HA nanocrystals as the inorganic component in scaffold
processing. The scaffolds, comprised of 32.5 wt% collagen and
67.5 wt% nanocrystalline apatite mixture, are best matched with
trabecular bone, in terms of molecular structure, crystalline phase
and crystallite size, achieved through tuning the weight ratio of
CAP and HA (shown in Fig. 22), and the optimal ratio is 4:1 of
CAP:HA (curve e in Fig. 22). The scaffold with optimized
compositions exhibits the highest compressive stiffness at
37.3  2.2 MPa and yield strength of 2.7  0.1 MPa, which is possibly
due to a similar crystal size as natural bone mineral, because the
synthetic HA and CAP sizes are about 40 nm and 20 nm, respectively,
while the natural apatite crystallite size is about 20 nm in trabecular
bone [158]. It is believed that the high volume fraction of grain
boundaries associated with nanocrystalline apatite will strengthen
the matrix, compared with conventional coarse-grained HA crystals
[158,163].
The ratio and shape of nano-HA particles in composites also
play an important role in the mechanical improvement of ECM-HA
composite scaffolds [159,160]. In the case of a freeze-dried
collagen scaffold, both HA whiskers and equiaxed HA powders
can be used to reinforce its mechanical properties [159]. As shown
in Fig. 23, in comparison with scaffolds with no reinforcement or
low reinforcement levels (1:1 of collagen:HA, by weight), the value
of the compressive modulus increases with increased HA
Fig. 22. XRD patterns of nanocomposite scaffold with 32.5 wt% of type j collagen and
67.5 wt% of nanocrystalline apatite, where the CAP:HAP weight ratio (a) 0:5, (b) 1:4,
(c) 2:3, (d) 3:2, (e) 4:1 and (f) 5:0, and (g) trabecular bone. [158].
15
reinforcement by weight, especially at a higher reinforcement
level (1:4 of collagen:HA, by weight), which induces a 10-fold
increase in the value of the compressive modulus, from 20 kPa to
200 kPa. Compared to equiaxed HA powders, HA whisker is more
effective for the improvement of the compressive modulus of the
scaffold, especially at 1:2 collagen:HA, by weight [159].
2.2.3.3. ECM-bioglass materials. As the aforementioned, bioglass is
a promising material for bone scaffolds because of its similar
make-up to bone mineral, as well as its apatite absorbance and
conversion abilities. In recent years, more and more focus has been
given to the development of ECM-bioglass composite scaffolds,
which not only mimic the natural ECM environment of human
bone, but also combine the individual advantages and minimize
the drawbacks intrinsic in each material choice [164–167]. Xu et al.
fabricated a novel biomimetic composite scaffold of bioglass-
collagen-phosphatidylserine (BG-COL-PS), using a freeze-drying
technique, the porosity and mechanical properties of which can be
modulated by changing the ratio between BG and COL-PS [164]. As
the bioglass content increases, the compressive strength also
increases, while the porosity decreases. The optimal porosity and
strength of the scaffold are 70.67% and 1.9724 MPa, respectively,
which favor the osteogenic differentiation of rat MSC, proven by
the radiographical images of femurs after surgery (shown in
Fig. 24) [164]. This phenomenon has also been confirmed by other
groups [165]. As shown in Fig. 25, the incorporation of nano-sized
Fig. 23. The apparent mean compressive modulus of freeze-dried collagen scaffolds
reinforced with HA whiskers or powder at 1:0, 1:1, 1:2, and 1:4 collagen:HA by
weight (n = 7/group) [159].
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16 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36

Fig. 24. X-ray photographs of rat femur defects treated with scaffolds or scaffold/MSC constructs at 3 days, 3 weeks, and 6 weeks post-surgery [164].

bioglass (nBG) into dense collagen (DC) gel improves the
compressive strength, especially at a larger strain, and this
difference becomes very distinct after incubation in SBF, i.e. a
13-fold increase in the modulus value after 7 days, which is
attributed to the mineralization triggered by nBG, because low
crystalline carbonated hydroxyapatite growth leads to a transition
from a soft to hard tissue-like scaffold response to compressive
stress [165].
Apart from the incorporation into ECM polymers to strengthen
the mechanical properties of composite scaffolds, the natural or
synthetic polymer coatings on bioglass can also affect the stiffness of
porous hybrid scaffolds [166]. The bioglass scaffolds are indepen-
dently coated with five types of biopolymers, such as collagen,
gelatin, PCL, alginate and PLLA by means of the solution-coating
technique. In comparison with an uncoated bioglass scaffold, both
PCL and collagen coatings can increase the scaffold stiffness by 58
and 38%, respectively, while no remarkable stiffness increase is
recorded for the other coatings. It is believed that the infiltration of
PCL and collagen into the cracks present in the struts is responsible
for the enhancement of the stiffness of the overall structure [166].
3. Biomimetic surface design
It is well accepted that surface characteristics are critical for the
successful design and medical application of biomaterials, because
the surface is the earliest contact with the biological environment.
The interactions between cells and the ECM are schematically
illustrated in Fig. 26 [168], which can be summarized into four
stages. The first stage is signal collection, exemplified by receptor
binding sites on the surface of ECM for the filopodia of cellular
transmembrane proteins. The second stage is signal transduction,
which delivers information, such as receptor conformation or
affinity for other cellular molecules. This initiates an intracellular
cascade of enzyme-mediated reactions, resulting in amplification
of the signal, which influences gene regulation and translocation of
the receptor [170]. The third stage is the attachment of integrins
and transmembrane heterodimers to specific binding sites, such as
the Arg–Gly–Asp (RGD) tripeptide found in cell adhesive proteins
like vitronectin, laminin, and fibronectin, as well as the mediation
of intracellular signals, mobility, cellular shape and cycles through
these connections [171]. Intercellular communication is the fourth
stage, which is controlled by cell recognition and adhesion to the
ECM [168,169].
Therefore, the design of scaffolds must take the interactions
between cells and the ECM into consideration; which is to say that
the surface affinity with receptors on the cytomembrane must also
be taken into consideration. Summarizing recent progress, the
surface design of scaffolds includes three types of categories, i.e.
tailoring the surface chemistry and regulating the surface
structure, as well as the synergistic effects of the two factors.

Fig. 25. (a) Mechanical characterization of DC and DC-nBG at different conditioning times in SBF; (b) representative unconfined compressive stress/strain curves of DC and
DCnBG rolls, as made, and at days 1, 3, and 7 in SBF, respectively. Inset in (a) A schematic of the compression test, and in (b) an image of a hybrid specimen at day 7 in SBF prior
to testing [165].
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 17

Fig. 26. Schematic diagram of the interaction between the cell and ECM; Adherens junction (AJ) and focal adhesion (FA) as mechanosensors. Calcium-dependent homophilic
interactions between cadherins results in binding of the actin cytoskeleton via catenin (b), R-catenin (a), and vinculin (Vin) complexes. Heterodimeric integrin receptors bind
ECM proteins via their extracellular domains, while their cytoplasmic domains are associated with a supramolecular plaque containing talin (Tal), vinculin (Vin), paxillin
(Pax), focal adhesion kinase (FAK), etc. The plaque, in turn, is connected to the termini of actin filament bundles [168].

3.1. Surface functionalized chemistry
3.1.1. Coatings/film
Numerous surface techniques are employed to produce
different types of coatings to functionalize the surface of the
scaffolds and implants. According to their surface chemistry, these
coatings can be mainly categorized into three types: inorganic,
with HA-derived coatings as preventatives [172–185], ECM-
derived organic coatings [186–194], and other derived hybrid
coatings.
3.1.1.1. HA-derived inorganic coatings. Bone mineral-like ceramics,
such as apatite and its derived ceramics, are usually deposited on
the surface of bone scaffolds to achieve specific biological functions
[172–176]. There are numerous fabrication techniques for bioac-
tive calcium phosphate coatings, which have been summarized in
detail in recent review papers [172,174]. Herein, we mainly focus
on the effects of these coatings on the improvement of biological
functions for bone implants or scaffolds.
The most well-known biofunction is the earlier osteoinduction
and osteointegration of HA and its derived coatings. Employing
a molecular precursor method, a thin hydroxyapatite (HA) film
can be successfully coated onto the titanium fiber web
without sacrificing the complex interior structure [175]. In vivo
implantation in rat cranial bone defects displays that the amount
of newly formed bone is significantly higher in the HA coated
titanium fiber mesh than in the uncoated scaffold 2 weeks after
implantation, and the corresponding new bone formation ratio is
35.8% and 7.1%, respectively, while the values rise to 45.1% and
56.2% two weeks after implantation, indicating the earlier
osteoinduction of thin HA coatings, which is relevant to the
ability of HA to enhance the formation of apatite crystals produced
by human osteoblasts during the culturing process (shown in
Fig. 27). Osteoblasts are osteogenic cells associated with bone
formation and cause osteoid and mineralization of the osteoid
matrix for calcification [175]. Recent research shows that the form
of HA coating significantly influences its biological function [176].
A mesoporous hydroxyapatite (M-HA) coating can be prepared on
the surface of a porous b-tricalcium phosphate (b-TCP) scaffold by
using a sol–gel dip-coating process, using the block copolymer
Pluronic F127 (EO106PO70EO106) as the template (shown in
Fig. 28) [176]. The osteoblasts exhibit a relatively round shape with
sphere-like surface evaginations and the microvilli on the non-
mesoporous HA (N-HA) coating, while more cells are strongly
attached to M-HA and spread in an elongated shape, indicating that
the latter is more suitable for osteoblast adhesion and spreading,
which is in accordance with the much higher alkaline phosphatase
(ALP) and bone sialoprotein (BSP) expression on M-HA. This is

Fig. 27. (a) Stereoscope finding of apatite crystal in HA-coated titanium fiber web after 14 days of human osteoblast culture. Apatite crystals are deposited on titanium fiber
(arrows). Scale bar 200 mm. (b) Scanning electron microscope findings of human osteoblast cultured in HA-coated titanium fiber web for 7 days. Human osteoblast extends
projections to adhere to titanium fibers, secreting calcification matrix (arrows). Scale bar 20 mm [175].
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18 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 28. Schematic illustration of the formation of mesoporous HA particles. Reaction (1) shows the formation of F127-phosphate micelles by hydrogen bonding. Reaction (2)
reveals the formation of mesoporous HA precursor micelles by electrostatic force. The spherical precursor micelles induced the formation of spherical mesoporous HA
particles after calcination. [176].
possibly due to the large surface area of M-HA, providing higher
availability of binding sites for receptors in osteoblast filopodia,
and thus stimulating cell spread and proliferation [170,176]. In
addition to the shape of the HA coating, the Ca substitution in HA
by other metallic elements like Sr and Si can also be helpful for
osteointegration and osteoconduction, which can be fabricated by
an aqueous precipitation reaction process or the sol–gel dip-
coating method [177–180]. For example, the in vivo tests of
commercial HA-coated Ti implants and 10% SrHA-coated implants
(10 mol% Ca2+ replaced by Sr2+) in the proximal tibiae of rats show
that the latter is more favorable for the osseointegration than the
former, with the bone area ratio and bone-to-implant contact
increased by 70.9% and 49.9% in histomorphometry after a 2-week
healing period, as shown in Fig. 29 [179]. In vitro tests disclose that,
in comparison with pure HA, Sr substitution increases the levels of
proliferation, alkaline phosphatase, and collagen type I, and
decreases the level of interleukin-6 in both osteopenic osteoblasts
(O-OB) and normal osteoblasts (N-OB), demonstrating that the
partial substitution of Sr for Ca in HA promotes osteoblast
differentiation and benefits the therapy of bone lesions and
defects in the presence of osteoporotic bone [180].
Another typical ceramic coating is the use of inorganic
antibacterial agents like Ag, Zn, Sr, or dual/multi-metal co-doped
HA coatings for improving the antibacterial performance and
biocompatibility of scaffolds, which can be prepared by combined
biomimetic deposition [181], plasma spraying [182], the sol–gel
method [183], pulsed electrochemical deposition (PED) [184], or
the spin coating technique [185]. As is already known, both
excellent osteoinduction and self-antibacterial capabilities are
equally important for bone scaffolds or implants, because the
former accelerates the osteoblast differentiation and bone tissue
regeneration, while the latter protects implants from microbial
infection during the operation and post-surgery by resisting
bacterial invasion [182,195]. Due to the drug-resistance of some
bacteria to antibiotics, broad-spectrum antimicrobial agents like
nano-Ag, -Zn and related metallic ions are attracting much
attention [181–185,195–197]. One of the most promising strate-
gies is to combine these inorganic antibacterial agents with bone
mineral-like ceramics, thus ensuring both good biocompatibility
and self-bacterial performance of bone scaffolds. For example, the
HA coating doped with 2.0 wt% Ag2O exhibits much better
effectiveness against Pseudomonas aeruginosa colonization than
a pure HA coating, but the addition of Ag2O impairs the original
osteogenic capability of HA, due to its cytotoxicity. Simultaneously,
the co-doping of Ag and Sr can not only improve antibacterial
performance, but also enhance the osteoinduction of coatings,
which can be clearly seen in Fig. 30 [182]. This is most likely due to
the mediation function of Sr that directly interacts with the
calcium sensing receptor in osteoblast cells to trigger mitogenic
signals in the protein kinase c/d signaling pathways, finally
resulting in increased cell division [182,198].
3.1.1.2. ECM-derived organic coatings. It is well known that organic
components of the ECM are mainly composed of proteins, such as
elastin, collagen, fibronectin, laminin, etc., which play an impor-
tant role in controlling specific cell behaviors, including adhesion,
migration, proliferation, differentiation, and apoptosis, depending
upon the specific tissue type [140]. Therefore, a variety of ECM-
derived organic coatings, such as collagen [186,187], peptides
[188,189], gelatin [190], fibrin/alginate scaffold coating [191],
diamines and heparin [192], have been developed to achieve
specific biological functions by modulating the surface bio-
molecule structures.
In general, building these ECM organic coatings is usually
carried out in two stages. The first is to develop a suitable surface
environment on the implant/scaffold to enable the connection
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 19

Fig. 29. Histological images of the proximal tibiae with implants approximately 4 mm below the epiphyseal plate, 12 weeks after implantation ((a) HA; (b) 10% SrHA;
toluidine blue stain, original magnification: 100); and results of the BA (c) and BC (d) in histomorphometry. Data are expressed as mean  SD, n = 10 specimens per group.
*
p < 0.05 versus HA group (by t-test) [179].

Fig. 30. (a) Live/dead confocal images of bacteria after 24 h exposure. Dead bacteria appear red, live bacteria appear green; (b) Confocal micrographs showing ALP expression
in hFOB cells at 5 days. Green fluorescence indicates active ALP; blue fluorescence indicates PI bound to the nuclei of cells [182]. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
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20 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
between these natural molecules and substrates by some
anchoring processes like cross-linking [187,190], benzyl-protected
phosphonic anchors [188], thiol and phosphonate anchors [189],
dopamine anchoring [191,199], photopolymerization [200], and
layer by layer assembly [201]. The second is to immobilize the
targeting compounds onto the surface, in order to form coatings/
films; the typical techniques are illustrated in Fig. 31 [202].
Practically, the different ECM organic molecules exhibit obvious
distinction in bio-functions, due to their unique molecular
structure. The in vivo comparison of three typical ECM molecule
coatings on titanium implants confirms this variation, despite their
common tendency to accelerate bone healing and new bone
formation [186]. For example, in comparison with a collagen and
chondroitin sulfate (CS) coating, an RGD coating stimulates the
most obvious increase and decrease of ED 1-positive macrophages
around the implants at an earlier and in the later period, while
collagen and chondroitin sulfate co-coatings exhibit the greatest
ability to promote the formation of new bone [186]. More recent
research also reveals that human mesenchymal stem cells (hMSC)
exhibit the best osteogenic differentiation on the surface of a
collagen/sulfated hyaluronan (sHya) coating, compared with pure
collagen and collagen/CS coatings, by the highest alkaline
phosphatase (ALP) activity and the greatest mineral deposition
[187]. Some biocompatible scaffold materials like polyurethane
(PU) and titanium are bioinert and lack cell recognition sites,
delaying cell adhesion and differentiation, as well as bone
remodeling, and some ECM coatings, such as diamines, heparin
or peptide coatings, can increase the anchoring sites for the
receptor protein by tailoring surface hydrophilicity and stiffness
[191–193]. Further studies reveal that the binding behaviors of
integrin can be modulated by functionalizing the peptides or
proteins using selective ligands or proteins, and thus regulating
cell adhesion behaviors [188,194]. For instance, the immobilized
a5b1-selective RGD peptidomimetic coating exhibits a 9-fold
increased affinity for a5b1 compared to the uncoated titanium
[188], and the co-modification of titanium binding and RGD
peptide for a silk fibroin protein coating enhances the chimeric
Fig. 31. Overview of the most common techniques to functionalize implants with biomolecules [202].
protein deposition by more than 35% on Ti implants, with a
consequent 60% increase in the adhering cell number [194].
Another major bio-function of ECM organic coatings is to block
the fouling from pathogens, in order to eliminate the inflammation
and infection of tissues around implants after surgery, which is
usually achieved by the controlled release of antimicrobial
peptides from coatings, which are found as part of the innate
immune system in eukaryotes against the invasion of pathogens
[201,203]. For example, employing a layer-by-layer technique, the
antimicrobial peptide ponericin G1 can be incorporated into a
hydrolytically degradable polyelectrolyte multilayer film, ensuring
rapid release within the initial 4 days and a subsequent stable
linear release within 10 days, which significantly inhibits bacterial
attachment [201]. The most interesting part is that these
antibacterial peptides are not only biocompatible with the relevant
wound healing cells, NIH 3T3 fibroblasts, and human umbilical
vein endothelial cells [201], but can also regulate angiogenesis and
the recruitment of stem cells to promote bone regeneration [203].
Therefore, in terms of the cytocompatibility processes, these
antibacterial peptide coatings exhibit much greater advantages
than Ag/Zn-containing inorganic coatings.
3.1.1.3. Hybrid coatings. Coatings comprised of biomolecules and
calcium phosphate are the most common hybrid coatings, which
can give implants/scaffold either multiple biological functions or
much more efficiency, such as improved bone inductivity
[204,205], good antibacterial performance [205], desirable surface
stiffness [166,192,206,207], and controlled drug release, as well as
enhanced protein adsorption [207–209], which stems from the
combined biological abilities of each component in the coatings.
For example, although carbonated hydroxyapatite (CHA) alone can
promote the differentiation of porcine bone marrow stromal cells
(BMSCs) on poly(e-caprolactone)/tri-calcium phosphate (PCL/TCP)
scaffolds, compared to uncoated, the hybrid coating composed of
CHA and gelatin significantly enhances the proliferation rate of
BMSCs, at a 1.7-fold higher rate than the CHA coating (Fig. 32(a))
[204]. The expression of osteonectin (ON), osteocalcin (OCN) and
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 21

osteopontin (OPN) is 1.21, 1.07 and 1.73 times higher in the
hybrids than in the pure CHA coating (Fig. 32(b)). ON, OCN and OPN
are important proteins for bone formation, closely related to
mineralization at the early stage, calcification of the bone matrix
and crystal growth, respectively. The enhanced proliferation rate of
BMSCs and protein expression confirm that hybrids can achieve
much higher rates of osteoinduction, favoring rapid bone healing
and reconstruction.
One of the most important objectives in the development of
hybrid coatings is to achieve dual or multiple biological functions,
which is difficult to accomplish by coatings with a single
component. It has recently been shown that a graphene-containing
hybrid coating exhibits the greatest potential for bone scaffolds
with a variety of functions [206,207]. The addition of graphene into
a PEG/SiO2 hybrid coating can favor the osteoblast adhesion and
growth around the pores of porous 45S5 Bioglass1-based scaffolds
(Fig. 33(a)) and, furthermore, in comparison with a PEG/SiO2
hybrid coating, the graphene/PEG/SiO2 hybrid coating exhibits
enhanced surface mechanical properties, as shown in Fig. 33(b)
[206]. Research confirms that graphene-containing hybrid coat-
ings like graphene/HA markedly enhance the attachment and
proliferation of osteoblast cells, which is most likely attributable to
the fast adsorption of key serum proteins like fibronectin, with an
elongated stretching conformation on graphene (shown in
Fig. 33(c)) [207]. These kinds of hybrid coatings can also have
good surface electrical conductivity, imparted by incorporated
graphene, which might be used in the electrically stimulated
biological environment. Some other hybrid coatings like HA/PCL
[208], PLGA/PVA [209], and gelatin/chitosan [210] can also be used
as effective carriers of some drugs like bone morphogenetic protein
(BMP) and antibiotics to obtain some biological functions for
scaffolds.
3.1.2. Biomineralization
Since the invention of simulated body fluid (SBF) by Kokubo
et al. [211] in 1990, the apatite formation ability on the surface of
biomaterials in SBF has become a ‘‘gold standard’’ to ascertain
whether materials are biocompatible or not. Meanwhile, biomin-
eralization of biomaterials in SBF has become one of most
important surface techniques to adjust surface components, so
as to mimic bone minerals and consequently enhance the surface
biocompatibility. In the case of bone tissue engineering, biomin-
eralization is a process by which bone-mineral like crystals, such as
hydroxyapatite, apatite and CaP compounds, are deposited or grow
on the scaffolds or in the matrix of living organisms, inducing the
formation of bone mineral-like skeletal structures during devel-
opment, which includes two categories, depicted as follows.
3.1.2.1. SBF-determined biomineralization. Numerous biomimetic
approaches have been developed to achieve surface biominerali-
zation in SBF, which is the typical SBF-determined biomineraliza-
tion [212–219]. The typical method is a soaking process in SBF,
including Kokubo SBF [212,213], revised SBF (R-SBF) [214], and
supersaturated SBF [215]. In general, the biomineralization in
Kokubo SBF is usually assisted by a pretreatment process, such as
plasma pretreatment [212], carboxymethyl cellulose pre-attach-
ment [213], or deposition of precursor phases like dicalcium
phosphate dehydrate (DCPD) and amorphous calcium phosphate
(ACP) [219]. For example, apatite can form easily on the surface of
three-dimensional (3D) fibrous PCL scaffolds after a vacuum

Fig. 32. (a) Confocal laser microscopy with depth projection images reconstructed from multiple horizontal images shows the 3D distribution of cells within the scaffolds of
PCL/TCP, PCL/TCP-CHA and PCL/TCP-CHA-gelatin after 3 days; (b) osteonectin (ON), osteopontin (OPN) and osteocalcin (OCN) protein extracts from cell-scaffold constructs
after culture with BMSCs for 31 days on PCL/TCP, PCL/TCP-CHA and PCL/TCP-CHA-gelatin scaffolds [204].
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22 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36

Fig. 33. Enhanced surface functions of graphene-containing hybrid coatings: (a) CLSM-images of osteoblast-like MG-63 cells on the surface after 3 weeks of incubation. The
cells were stained red and the bioactive glass surface can be seen in green; (b) typical nanoindentation traces on PEG/SiO2 hybrid coating (solid line) and PEG/SiO2 hybrid
coating added with 1.5 wt% graphene (dotted line) (coating thickness ca. 20 mm) [206]; (c) TEM image of the negative stained fibronectin on GN. Typical individual fibronectin
molecule is surrounded by a red line loop, while the areas enveloped by the red rectangular boxes show multiple fibronectin molecules tangled together. The inset image is of
selected individual fibronectin molecules indicating the strands are in the coiled stretching state, with a length of 100–135 nm; the images were Gaussian low-pass filtered
for clearer presentation of the molecule. The white arrows point to the molecule [207]. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)

nitrogen plasma pretreatment, ascribing to the formation of –CH3,
–CO–, and –NH2 groups, which is induced by nitrogen plasma. This
biomineralization favors cell proliferation and enhancement of
surface hydrophilicity [212]. R-SBF involves the difference in pH,
chemical components and ion concentration of Kokubo SBF, which
can lead to the formation of low crystallized apatite with various
shapes on b-TCP scaffolds, due to the high supersaturation of the
solution with a P/Ca molar ratio of about 6.0 [214], while pure b-
TCP bioceramics have no apatite-forming ability on their surfaces
in Kokubo SBF [216]. Supersaturated SBF favors the formation of
plate-like apatite on alginate/chitosan microparticles that can be
used as effective bone morphogenetic proteins carriers to the local
microenvironment [215]. Besides tailoring surface chemistry,
other techniques can also be used to accelerate the biomineraliza-
tion process, such as the hydrothermal route at 150 8C in SBF [217],
and continuous CO2 bubbling into SBF [218].
3.1.2.2. SBF-free induced biomineralization. This type of biominer-
alization is carried out in the absence of SBF and can be performed
by electric filed assisted synthesis and deposition on scaffolds
[220], and by the calcification of osteoblasts, stem cells and tissues
on the scaffolds by tailoring the surface chemistry or topography
[221,222]. For example, in the case of multi-carbon nanotubes/
chitosan scaffolds, the initially deposited dicalcium phosphate
dehydrate can transform into HA, via octacalcium phosphate as an
intermediate, by changing the voltage during the electrodeposition
process. Meanwhile, it promotes faster and significant osteoblast
terminal differentiation on the scaffolds [220]. The electrolyte with
a pH of 4.2, adjusted by HCl, is composed of 20 mM Ca(NO3)2,
12 mM NH4H2PO4 and 0.1 M NaNO3, which is completely different
from Kokubo SBF [220]. Habibovic et al. published their research
showing that the sustained localized release of orthophosphate
(Pi) can supersaturate tissue surrounding the PCL implants and
induce the mineralization of collagen (shown in Fig. 34) [221]. This
recent study displays that the appropriate surface treatment with
oxygen plasma combined with collagen grafting can enhance the
biomineralization of unrestricted somatic stem cells (USSC) on
polyethersulfone (PES) nano-fibrous scaffolds [222].
3.1.3. Surface polymerization
Owing to the diversity of biomolecules, almost all types of
biomaterials, including biometals, biopolymers, bioceramics and
hybrids, can be modified to achieve the desired surface chemistry
by a surface polymerization process, which can be accomplished
by various methods like aminolysis [223], hydrolysis [224],
electron beam irradiation [225], UV-induced grafting [226], laser
ablation [227], alternative polymerization [228], covalent immo-
bilization [229,230], plasma polymerization [231–238], or surface-
initiated atom transfer radical polymerization (ATRP) [239–242].
In view of the wide applications, herein, we only briefly introduce
their biomimetic applications through tailoring the surface
chemistry of bone scaffold materials.
3.1.3.1. Plasma polymerization. Plasma polymerization is a process
in which the polymerization reaction is initiated by plasma, which
is usually used to modulate the surface chemistry of biomaterials
by selective deposition of biomolecules or functional radicals. This
process is composed of two stages of plasma-assisted chemical
vapor deposition of a gaseous species (monomer) and polymeri-
zation during deposition, which involves the activation of the
starting materials (monomer) by the interaction between the gas
phase molecules and plasma, and the subsequent deposition on
the substrate. The fundamental components of a reactor for
plasma polymerization consist of a vacuum system, plasma
generator, controllable gas inlet, and a system for in situ analysis
[231,232].
A nanotube is often used as the biological interface on Ti-based
implants or components of tissue scaffolds [41,220,233] whose
surface can be functionalized by plasma polymerization of an n-
allylamine polymer (AAPP) combined with other processes, due to
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 23

Fig. 34. TEM images showing regions of mineralized collagen (arrows) in soft tissue, found in the proximity of phosphate-loaded implants. SAED confirmed the presence of an
apatitic phase [221].

the formation of NH2 facilitating the subsequent grafting of titanium can favor fibronectin adsorption and consequent osteo-
poly(sodium styrenesulfonate) (PSS) and poly(ethylene glycol)(- blast adhesion in the early stage, ascribing to the formation of Si–H,
PEG) (shown in Fig. 35) [233]. Hayakawa’s research shows that a Si–C, C–H, C55O, and Si–O–Si bonds on the plasma-polymerized
plasma polymerized hexamethyldisiloxane (HMDSO) thin film on HMDSO film [234]. Our recent work shows that a stable and

Fig. 35. Scheme for the tailoring of the surface functionalities on TiO2 nanotube films: (a) bare TiO2 nanotube arrays and the inset corresponding SEM image, (b) plasma
polymerization of n-allylamine polymer (AAPP) and the inset corresponding SEM image (1), (c) electrostatic adsorption of poly(sodium styrenesulfonate (PSS) on AAPP (3), (d)
covalent attachment of polyethylene glycol (PEG) on APPP (3), and (e) contact angles on TiO2 nanotube film (bare), TiO2 nanotube film modified by AAPP (120 s deposition),
PSS-AAPP and PEG-AAPP and control Si modified by AAPP, PSS-AAPP and PEG-AAPP [233].
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24 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36

Fig. 36. (a) High-resolution XPS-analysis of C1s; (b) Vinculin organization in GFP vinculin-transfected MG-63 osteoblasts in the time frame 60 min on functionalized Ti
surfaces [237].

uniform allylamine (PPAAm) film with a thickness of around
300 nm can be prepared on NiTi by low temperature plasma
polymerization [235]. In vitro tests show that tailored interfacial
chemistry of PPAAm films by plasma polymerization significantly
enhances osteoblast adhesion, compared with untreated titanium,
with an adhesion rate increasing from 18  2% to 54  5% [236].
Meanwhile, plasma-polymerized PPAAm favors the immobilization
of type I collagen, thus accelerating the formation of adaptor proteins
like vinculin that play a key role in cell adhesion and signaling, due to
the formation of hydrophilic radicals, such as –C–NH2, –C5 5N, 55C55N,
–C–N–C, O5 5C–NH2, and O5 5C–NHR (shown in Fig. 36) [237]. In
addition to metallic implants, the surface chemistry of bioceramics
like HA can also be modulated by plasma polymerization, which
significantly influences the differentiation behavior of human adipose
derived stem cells (hASCs) [238].
3.1.3.2. Surface imitated ATRP. As one of the living radical
polymerization methods, atom transfer radical polymerization
(ATRP) is attracting extensive attention from biomaterial scientists
and engineers because of its simplicity and broad applicability,
especially for preparing well-defined nanostructured polymer-
based materials [239–242]. Most of all, this process can be readily
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 37. The reaction scheme for ATRP, including the activator regeneration methods
ICAR (initiators for continuous activator regeneration) – where the activator is
regenerated using a radical initiator and ARGET (activators regenerated by electron
transfer) – where reducing agents are used. Pn represents the polymer (with degree
of polymerization n), Mtz is the metal species in oxidation state z, L is a ligand and X
is a halogen atom. Changes in oxidation state are represented by changes in color.
The kinetic parameters kact, kdeact, kp and kt represent the rate constants of
activation, deactivation, propagation and termination, respectively. [239].
initiated from various types of substrates, regardless of their
chemical compositions and topographies, such as planar surfaces,
three-dimensional topographies, inorganic particles/colloids,
polymer chains and networks to produce a variety of polymer
brushes, so as to modulate the surface chemistry for the desired
biological functions [239,240]. The fundamentals of ATRP are
schematically illustrated in Fig. 37 [239]. Briefly, alkyl halide
initiators or dormant species (RX or PnX) react with activators
consisting of low-oxidation-state metal complexes MtzLm, (Mtz
represents the metal species in oxidation state z, and L refers to a
ligand) to achieve a reversible equilibrium between growing
radicals (active species) and dormant species and, during ATRP
equilibrium, the dormant species can have polymer chains that are
able to grow in one or many directions, or polymers attached to
functional colloidal particles, surfaces, and biomolecules [239]. It is
obvious that the attachment of initiators on the substrate plays a
critical role in the ATRP process. Xua et al. [240] reviewed several
processes for immobilizing ATRP imitators on various substrates in
their recent paper.
Using this method, HA-poly(L-lactide) (PLLA) nanohybrids can
be successfully prepared and exhibit excellent dispersibility in
composites, which makes the PLLA/HA-PLLA nanocomposites
induce apatite formation much faster than PLLA/HA composites,
compared to the aggregated and unmodified HA [241]. Biopoly-
mers can also be easily functionalized by ATRP. For instance,
functional polymer brushes of glycidyl methacrylate (GMA) can be
grafted on the surface of polycaprolactone (PCL) film and their
dense and reactive epoxide groups can effectively couple with cell-
adhesive collagen and Arg–Gly–Asp–Ser (RGDS) peptides, which
significantly improves the cell-adhesion properties of the PCL film
[242].
3.2. Surface functionalized structure
It is well accepted that surface structures, such as topographies,
roughness, and nano-architectures, influence the cell and tissue
behaviors of biomaterials. Many surface modification techniques
have been developed to achieve desired structures on biomaterials
to cater to particular biological applications, some of which are
complex and can be generally categorized into chemical or
physical treatments.
25
Fig. 38. Illustration for the MNTs initiating b-catenin signaling. The topographical
cue initiates b-catenin expression resulting in more b-catenin product and
promotes the amount of p-GSK3b, thereby inhibiting b-catenin degradation,
through some temporarily unknown mechanism. [243].
3.2.1. Chemical treatment
The surface structure of scaffolds or implants can be function-
alized by chemical treatment, which includes two kinds of routes
according to the reaction between substrate and environment. The
typical route is through a direct reaction between biomaterials and
surrounding media, such as anodization [243,244], chemical etching
[245], hydrothermal treatment [90], hydrolysis [224], oxidation
techniques [246,247], plasma immersion ion implantation (PIII)
(except for inert gases like argon as PIII sources) [248–250],
electrochemical etching [251], lithographic techniques [252–254],
and nano-imprinting [255]. Another pathway is to establish coatings
with controllable micro/nano-architectures on biomaterials depos-
ited by products from chemical reactions that have occurred in the
surrounding media, including chemical vapor deposition (CVD)
[256], biomineralization [257], electrodeposition [220], sol–gel, and
so on [258–260]. Generally, the surface structure prepared by the
first route exhibits better bonding stability, due to its integration
with the substrate, while the surface coating from the second
pathway has an interfacial bonding with substrates.
Using traditional anodization in a electrolyte composed of
0.5 wt% hydrofluoric acid and 1 M phosphoric acid [243], two kinds
of micro/nano-textured topographies (MNTs) combined with
nanotubes and a micropitted topography can be built on the surface
of titanium implants, which exhibit the ability to trigger a higher
expression of integrin b1 and b3 and integrin-linked kinase (ILK),
thus activating the b-catenin signaling pathway by initiating b-
catenin expression and inhibiting its degradation by phosphorylat-
ing GSK3b, consequently favoring human osteoblasts differentia-
tion. The related initiation mechanism is illustrated by Fig. 38 [243].
Novel anodization combined with block copolymer templates can
create highly ordered and tunable oxide nanopatterns on planar Ti
platen (Fig. 39), which benefits the higher expression of osteocalcin
in MSCs cultured on nanopatterned beads (Fig. 39(b)). In addition to
the size of the surface topography, the arrangement of nano/micro
patterns, such as ordering/disordering and density of nanopatterns
significantly influences the differentiation behaviors of human MSCs
[55,255]. For instance, a defined lithography approach can be
employed to define five different levels of nanodisorder and random
surfaces: (1) square array (SQ), (2) hexagonal array (HEX), (3)
disordered square array with dots displaced randomly by up to
50 nm on both axes from their position in a true square (DSQ50), (4)
disordered square array with dots displaced randomly by up to
20 nm on both axes from their position in a true square (DSQ20), and
(5) pits placed randomly over a 150 mm  150 mm field, repeated to
fill a 1 cm2 area (RAND) [55]. The in vitro culturing tests of human
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26 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36

Fig. 39. (a) Schematic showing the processes during formation of the nanopatterns. Dark gray = Ti, red = P4VP, green = PS, and light gray = titania. (1) After spin coating the
BCP onto the Ti substrate, a thin film of arranged micellar structures is formed. (2) When the sample is immersed in the electrolyte, the P4VP segments of the BCP are
protonated and thus swell with electrolyte. (3-1) During anodization anodic oxide grows within the P4VP segments at the same time that Ti ions interact directly with the
P4VP. (3-2) Enlarged view of the surface illustrating how Ti4+ ions react with the P4VP (4) Oxygen plasma treatment degrades the polymer template and oxidizes the surface
further; (b) FE-SEM images of the nanopatterned Ti microbeads; (c) MSCs immunostained for the bone marker osteocalcin (green) and actin (red) after 21 days of culture.
Scale bar = 100 mm [244]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

MSCs on these surfaces disclose that nano-scale disorder structures
exhibit a marked ability to stimulate MSCs to produce bone mineral,
despite the absence of osteogenic supplements; DSQ is the best
biomimetic structure (shown in Fig. 40(a)) [55]. It has recently been
shown that the size and distribution density of nano-pattern can also
determine human MSC behavior [255]. As shown in Fig. 40(b), two
kinds of nanostructures labeled D150S350 and D80S110 can be
fabricated by nano-imprint lithography with features (diame-
ter  interdot gap width) of 150 nm  350 nm, and
80 nm  110 nm for the former and the latter, respectively.
Immunofluorescent staining shows the enhanced vinculin expres-
sion of MSCs, compared to the untreated surface [255]. The surface
structure of bone scaffolds or implants can also be tailored by
changing the topography of the coating [257,260]. HA coatings with
three kinds of topographies can be prepared on PCL discs by a
biomineralization method in supersaturated SBF and revised SBF;
the corresponding roughness (Ra) is 1.0, 1.3 and 2.0 mm (by
profilometry), respectively [257]. The in vitro cell culture tests reveal
that osteoblast attachment and differentiation are greater on more
complex, micro-rough HA surfaces (Ra  2 mm) than on smoother
topographies (Ra  1 mm), while the activity of osteoclast marker
tartrate-resistant acid phosphatase is greater on smoother than on
micro-rough surfaces [257]. In the case of porous scaffolds, solution
treatments like hydrothermal and hydrolysis processes can
effectively modify the whole exposed area to mimic the nature
structure of bone tissues, due to the line of sight nature [90,224]. In
our previous work, one-dimensional titanate nanowires/nanobelts
were successfully fabricated on three-dimensional porous Ti and
nickel–titanium scaffolds to mimic the hierarchical nanostructure of
natural bone by a low temperature hydrothermal route [90].
3.2.2. Physical methods
The surface structure of scaffolds and implants, like topogra-
phies and roughness, can also be modified by physical methods
without any change of chemical compositions, which can be
performed through a direct mechanical process to the substrate or
depositing coatings, without chemical reactions. The typical
methods include surface mechanical attrition treatment (SMAT)
[261,262], plasma spraying [263], magnetron sputtering [264],
physical vapor deposition [265], inert-ion texturization [266], and
porogen-induced modification [267]. It has been reported that
surface nanocrystallized metallic biomaterials like Ti-based alloys
can exhibit many novel properties, such as enhanced cell adhesion
and differentiation behaviors compared to their coarse-grained
counterparts, which is ascribed to the numerous nanometer-sized
grains with a large number of grain boundaries [261,262], which
can be achieved by surface mechanical attrition treatment (SMAT)
that is often performed using ultrasonic shot peening or sand
blasting [268–270]. For example, two kinds of crystalline layers, i.e.
nano- and ultrafine-grained b-Ti (labeled as NG and UFG,
respectively) can be formed on the surface of Ti–25Nb–3Mo–
3Zr–2Sn alloy, which exhibits a similar surface roughness and
topography as the coarse-grained surface [261]. However, in
comparison with the conventional coarse-grained (CG) surface, the
former significantly accelerates human fetal osteoblast adhesion,
proliferation, maturation and mineralization while little improve-
ment is found in the latter, which is mostly attributed to the
improved surface stiffness and hydrophilicity by nano-grain size
(shown in Fig. 41) [261]. Similarly, SMAT treated commercial
titanium is also beneficial for the growth of MSCs, including
adhesion, filament orientation, proliferation and gene expression
[262].
3.3. Synergistic regulation of surface structure and chemistry
Since both surface chemistry and structure can play important
roles in regulating tissue and cell behaviors, the ideal surface
strategy is to encompass the two aspects as far as possible to obtain
a particular chemistry and structure, so as to achieve the desired
biomimetic scaffold. It is well known that HA-derived coatings
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 27

Fig. 40. Biological functions determined by the arrangement of nanopatterns: (a) effects of different disorder level of nanostructure on OCN and OPN expression of human
MSCs [55]; (b) effects of size and distribution density of nanopattern on vinculin expression of human MSCs [255].

with different topographies can be prepared on implants using
thermal spray, biominerization, and sol–gel by changing the
parameters [176,257,263,271], and the chemistry of these coatings
can be regulated by the addition of metallic elements like Ag [181],
Sr [182,263], Zn [183,184], carbon-like materials like graphene and
carbon nanotubes [207,272], and ECM-like biomolecules [40,208]
at the same time. The details of coatings have been discussed in
section 3.1.1, and herein, we only focus on the synergistic function
of surface structure and chemistry. Using a high temperature spray
system with ground, sieved and sintered powders as starting
materials, micron-rough strontium-substituted hardystonite (Sr-
HT) ceramic coating can be prepared successfully, which exhibits
hierarchical topographies with superimposed nano-sized grains in
the coating (Fig. 42(a)) [263]. In vivo tests of canine bone marrow
mesenchymal stem cells (BMMSCs) disclose that Sr-HT-coated Ti-
alloy implants have the highest osseointegration, while the
uncoated implants have the lowest; meanwhile, the classic
hydroxyapatite (HA) coating shows worse osseointegration ability
than HT and Sr-HT coatings. This was determined by a push-out-
test and new bone formation area (Fig. 42(b)), which indicates the
biological regulation of released Sr for enhanced osteointegration.
In vitro cell cultures and related protein expression of integrin b,
BMP-2, ALP and OCN show that BMMSCs exhibit similar adhesion
behavior on the surface of both Sr-HT and HT coatings, while
different differentiation behaviors are observed (Fig. 42(c)), which
makes the deduction reasonable that the topography, instead of
the ion release, is the contributing factor in the promotion of
cellular responses at early stages [263].
Alongside hybrid coatings, synergistic factors can be encom-
passed together during the fabrication of scaffolds. For example,
surface-modified nano-fibrous poly(L-lactic acid) (NF-PLLA) scaf-
folds with a porous topography can be fabricated by a one-step
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28 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 41. (a) Surface images of three kinds of surface layers; (b) triple staining of actin (red), vinculin (green), and nucleus (blue) of the osteoblasts after 24 h culture on the CG,
UFG and NG–Ti surfaces [261]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
process with gelatin spheres as porogen that displays dual
functions [264]. One aspect is the production of a scaffold with
a porous structure by gelatin spheres, and another is that gelatin
molecules are entrapped onto the scaffold surface during the
fabrication process [264]. Consequently, a porous topography on
the surface benefits osteointegration, while the gelatin molecule
modified surface favors initial cell adhesion and proliferation
[264].
4. Biodegradability design
4.1. Scaffold resorption/biodegradability
Biodegradation/bioresorption is the chemical dissolution or
decomposition of biomaterials under physiological environments,
which is an essential factor in ideal tissue engineering scaffolds
that are required to be absorbed by the surrounding tissues
without the need for surgical removal. As was aforementioned in
Section 1.2, natural bone is a porous composite, completely
constructed of biodegradable collagen and apatite; consequently,
the ECM-like biomolecule- and bone mineral-derived materials are
ideal biodegradable materials for bone scaffolds. However, the low
mechanical strength of these natural materials cannot match the
demand for bone reconstruction, especially under load-bearing
conditions. Therefore, synthetic biodegradable polymers, ceramics,
hybrids and some biodegradable metals like Mg-based alloys
exhibit great potential for bone scaffolds.
Generally, the degradation depends upon the chemical
composition of the scaffold. In the case of hydrolytically
degradable polymeric scaffold materials that have hydrolytically
unstable chemical bonds in their backbone, such as esters,
orthoesters, anhydrides, carbonates, amides, urethanes, urea,
etc., their degradation course comprises chemical hydrolysis and
enzymatic decomposition [273,274]. Representative materials
include poly(a-esters), polyurethanes, poly(ester amide), poly(-
orthoesters), polyanhydrides, poly(anhydride-co-imide), cross-
linked polyanhydrides, poly(propylene fumarate), pseudo poly(-
amino acid), poly(alkyl cyanoacrylates), polyphosphazenes, and
polyphosphoester. Briefly, water penetrates the scaffold bulk,
preferentially attacking the chemical bonds in the amorphous
phase and converting long polymer chains into shorter, ultimately
water-soluble fragments. Enzymatic attack of these fragments
occurs almost simultaneously, resulting in a rapid loss of polymer
mass, which can be illustrated by Fig. 43 [273,275]. Several factors
are responsible for the degradation rate, including the hydrolysis
rate constant of the chemical bonds, the diffusion coefficient of
water in the matrix, the diffusion coefficient of the chain
fragments, the solubility of the degradation product, the presence
of catalysts, structure of scaffolds, and the location of implantation
[275,276]. For porous scaffolds, the degradation rate is not
consistent, perhaps because hydrolysis on the surface is intensive
at the initial stage due to the greater availability of water and lower
concentration of fragments, while degradation products diffuse
slowly in the interior of the scaffold, inducing a local acidic
environment that promotes further degradation [275], a theory
which is currently disputed. For example, it has been reported that
low-porosity implants from 50/50 DLPLG degrade faster than high-
porosity implants, due to the quick diffusion of low pH degradants
from the interior of the high-porosity samples [277]. However, a
72-week degradation study of PCL scaffolds with various porosities
disclosed that a minimal acid autocatalysis happens in the
scaffolds with low porosities (less than 85%) and a higher porosity
leads a more severe loss of weight, molecular weight and a
decreased compressive modulus [278]. Apart from hydrolytically
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 29

Fig. 42. (a) SEM photographs of the surface morphology of sprayed coatings; (b) the expression measured by real time PCR after incubating on different coatings for 14 days;
(c) new bone area by histomorphometric measurements, and biomechanics measured by push-out test (right). [263].

degradable polymers, numerous polymeric materials are enzy- product of the necks connecting these powders after crystallization
matically sensitive, usually inducing another kind of degradation, [283]. Consequently, the dissolution of necks induces the
termed surface erosion. ECM-derived natural and synthetic breakdown of bulk materials into the original particles.
biopolymers, such as collagen, poly(amino acids), elastin, elas- In view of the requirement of tissue engineering scaffolds for
tin-like peptides, fibrin and polysaccharides often degrade in this mechanical properties, biocompatibility and biodegradability,
form, and the degradation rate varies significantly with the site of magnesium-based alloys are the most promising metallic material
implantation, which is closely affinitive with the availability and for load-bearing bone scaffolds, due to their low Young’s modulus
concentration of enzymes. Generally, surface erosion can lead to value, low density, high toughness and rapid degradation, as well
thinning of a scaffold wall while maintaining its bulk integrity,
which is schematically illustrated in Fig. 43 [273,275,279].
In the case of apatite-derived bioceramics/bioglasses, their
degradation can be summarized by the following factors. The first
is physiochemical dissolution, depending upon the solubility of
degraded products and the local pH of the surrounding environ-
ment. During this reaction, some newly formed phases like
amorphous calcium phosphate, dicalcium phosphate dihydrate,
octacalcium phosphate, and anionic-substituted HA may be
deposited onto the surface of the scaffolds. The second is the
physical disintegration of bulk materials into small particles,
inducing the erosion or break of grain boundaries [280]. The third
is determined by biological factors, such as phagocytosis and
resorption from osteoclasts and macrophages, which induce a
decreased local pH concentration, accelerating the degradation of
small particles [281,282]. Therefore, the corresponding degrada-
tion rates are determined by chemical compositions, crystallinity,
and physical forms like porosity, pore size and distribution, and
grain size [280,281]. For example, in the case of scaffolds made
from powders, degradation is predominantly governed by the Fig. 43. Ester bond hydrolysis reaction mechanism and schematic of nonporous and
disintegration of particles, which depends upon the solubility porous polymeric device degradation [275].
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30 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 44. Degradation mechanisms of Mg alloys in physiological solution: (a) Bare Mg–Zn–Ca bulk metallic immersed in SBF [286]; (b) coated Mg-based materials [288].
as the necessity of trace Mg for human bone tissue and metabolism
[284–292]. The in vitro degradation of pure magnesium in a
physiological environment stems from the direct corrosion
reactions, depicted as follows [285,292]:
MgðsÞ þ 2H2 O ! MgðOHÞ2 ðsÞ þ H2 ðgÞ
 RpðMgÞ þ RpðCÞ þ Rs þ RMgC
MgðsÞ þ 2Cl ðaqÞ ! MgCl2 ;
MgðOHÞ2 ðsÞ þ 2Cl ! MgCl2 þ 2OH :
This degradation rate induces the rapid collapse of scaffolds.
The large hydrogen bubbles have an adverse effect on osteoblast
adhesion and osteointegration. Therefore, the current strategy of
magnesium scaffold materials is to control the corrosion process
by alloying and surface modification [293–301], which are related
to different degradation mechanisms. Recently, MgZn-derived
alloys are exhibiting the greatest potential for the development of a
new generation of biodegradable bone scaffold, due to their
controllable degradation rate and highly reduced hydrogen
reduction [284,286,287,291,292,300]. The corresponding degra-
dation process in SBF is schematically illustrated in Fig. 44(a),
proposed by Gu et al. [286]. The first stage involves the anodic
dissolution of magnesium and subsequent formation of a
magnesium hydroxide layer that is easily subject to the attack
of Cl at a weak site, inducing the transformation of magnesium
hydroxide into soluble magnesium chloride and the consequent
release of Mg2+ and Zn2+ from the exposed fresh substrate, eroded
directly by the medium. The second stage involves a self-repairing
process from Zn(OH)2 precipitates, produced by locally supersatu-
rated Zn2+ during the first stage. These precipitates will repair the
defects in the surface layer because of the higher content of OH,
thus forming a continuous layer. As the immersion time increases,
the thickened and spread Zn(OH)2 and Mg(OH)2 precipitates
provide suitable sites for apatite nucleation, inducing the
continuous and spontaneous growth of an apatite layer on the
corrosion product layer by consuming the calcium and phosphate
from the surrounding medium [286,302]. In the case of a coated
Mg-based alloy modified by various surface techniques [294–298],
galvanic corrosion is usually the prevailing degradation mecha-
nism and is schematically shown in Fig. 44(b) [288]. Firstly, the
discontinuous coating often leaves some small pits exposed to
physiological solution, inducing the formation of a galvanic cell
between the coating and substrate. The galvanic current can be
calculated by the following equation [303]:
(1) EC  EMg
I¼ (4)
(2)
where EC and EMg represent the corrosion potentials of the cathode
(3)
and anode, respectively, Rp(Mg) and Rp(C) are the polarization
resistance of the anode and the polarization resistance of the
cathode, respectively, Rs is the electrical resistance of the
electrolyte, and RMg–C corresponds to the electrical resistance
between the anode and cathode. In a stable system, Rp(Mg) and Rs
are constant and the corrosion current between the anode and
cathode is determined by EC  EMg, Rp(C) and RMg–C. If the value of
RMg–C is large enough, the coating and substrate will corrode
independently. It is evident that a higher potential difference
between coatings and Mg alloy substrates induces rapid degrada-
tion [288,303].
4.2. Biodegradation rate control
As with the aforementioned degradation/resorption mecha-
nism of three kinds of scaffold materials, in order to obtain a
desired and controllable biodegradation rate for practical
application, the scaffold design should consider the chemical
compositions and structure of bulk materials, as well as the
properties of coatings and the specific biological environment.
This is in order to obtain the desired biodegradation rate, which
offers cells within the scaffold enough time to produce new tissue
while providing stable mechanical strength to support the system
until its entire degradation after complete bone regeneration. It
has recently been demonstrated that surface modification is quite
effective in regulating the degradation rate of polymeric scaffolds.
For example, electron beam irradiation can be utilized to regulate
the degradation rate of some polymeric materials, such as PLLA,
poly(L-lactide-hydroxyapatite) (PLLA-HA), poly(L-lactide-glyco-
lide) co-polymer(PLG) and poly(L-lactide-DL-lactide) co-polymer
(PLDL) by changing the irradiation dose, because e-beam
 Author´s Personal Copy
S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36
Fig. 45. (a) In vitro degradation studies in lysozymes show that a composite scaffold
degrades more slowly than CG scaffolds. *p < 0.05 [306]; (b) In vitro biodegradation
in SBF: BG-COL (&); BGCOL-HA-PS (*); and cross-linked BG-COL-HA-PS (~) [308].
irradiation leads to chain scission in all polymers due to the high
radiation energy breaking the attractive forces between the
atoms. Active species, such as alkyl free radicals and peroxyl free
radicals are formed during irradiation, and the propagation of
these radicals greatly enhances chain scission, finally accelerating
the surface erosion of the materials during hydrolytic degradation
[304]. Hybrids/composites comprising different kinds of poly-
mers or polymer/ceramic/bioglass can not only enhance mechan-
ical properties and biological functions, but can also modulate
Fig. 46. (a) Perspective drawings indicating different sections/locations of the implant; (b) Resorption ratios of CPC implanted in rabbit femurs at different sections for
different implantation times [312].
31
their biodegradation behavior [305–309]. The covalent interac-
tions between silica and gelatin can well modulate the degrada-
tion rate of the hybrid scaffold [305]. ECM-derived biomolecules
are accessible to surface erosion due to their high enzymatic
sensitivity, which induces the rapid degradation of scaffolds made
from these materials, like chitosan and collagen [306,308]. As
shown in Fig. 45(a), in comparison with chitosan-gelatin (CG)
scaffolds with pore sizes ranging from 150 to 300 mm, the hybrid
scaffolds composed of GC and nano-bioglass ceramic (nBGC)
exhibit a much lower degradation rate when immersed in
lysozyme-containing PBS for 1 week, which is possibly caused
by the neutralization reaction between the acidic degradation
products of chitosan and the alkali groups leaching from nBGC,
since the released products of bioactive glass systems are known
to be alkaline in nature, thus reducing the degradation rate of the
scaffold. In addition, the higher concentration of gelatin leads to a
faster degradation because the macromolecular chains of gelatin
polymer hydrolyze quickly under the attack of water, due to its
higher hydrophilicity [306]. In addition to the incorporation of
inorganic nanoparticles, the addition of dual organic components
can also favor control of the degradation rate. As shown in
Fig. 45(b), bioglass-collagen (BG-COL) scaffolds show a rapid
degradation rate, with 48% weight loss within the initial 24 h and a
subsequent equilibrium of mineralization and degradation after
immersion for 48 h, while the co-addition of hyaluronic acid
(HYA) and phosphatidylserine (PS) inhibits the degradation
obviously. This reveals that the interaction of several macro-
molecules in scaffolds causes more resistance to degradation in
SBF and accelerates mineralization [308]. Cross-linking can
further enhance this resistance to degradation, due to the amide
linkages through cross-linkage and the protective hydrolysis
barrier of inorganic glass particles [308]. Some other factors, such
as incorporated inorganic particle size [307], content [309], and
scaffold porosity [277,278] also play important roles for the
degradation of polymer-based scaffolds. In the case of ceramic/
bioglass scaffolds, incorporation of nanoparticles [310], composi-
tions [311], surface modifications like elemental doping
[280,292], and the physiological environment [311,312] can
change the degradation rate. For example, calcium phosphate
cement (CPC) has a strong degradation capability, but its
resorption rate is quite sensitive to the implant site/location.
When implanted into rabbit femurs at different locations (shown
in Fig. 46(a)), the resorption ratios of CPC samples are entirely
different and decrease from the exterior to the interior portion of
the implants, although the resorption ratio values increase with
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32 S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36

Table 8
Typical engineering products for bone repair [316].

Product Regulatory Description Materials Use Form

status

Xelma, Molnlycke 2005 EU ECM protein (amelogenins) in Resorbable; Animal derived; Plant or Leg ulcers Gel
propylene glycol alginate carrier bacteria derived
INFUSE Bone Graft, 2002 Bovine type I collagen sponges soaked Synthetic; Resorbable; Animal derived; Spinal fusion Solid
Medtronic in rhBMP-2 in LT-CAGE Lumbar Growth factor
Tapered Fusion Device
OP-1, Stryker 2001 Bovine type I collagen with rhBMP-7 Resorbable; Animal derived; Growth Bone injury Paste
factor
PuraMatrix, 3DM Preclinic Synthetic 16-amino-acid peptide Resorbable; animal derived Dental bone Gel
forming nanofibres defects
Vitoss Scaffold FOAM, 2004 Porous foam comprising b-TCP and Synthetic; Resorbable; Animal derived Bone injury Foam
Orthovita bovine type I collagen
Bioset IC, Pioneer surgical 2008 Human demineralized bone matrix Resorbable; Animal derived; Human Bone injury Paste
with bovine bone chips in type I derived
collagen carrier
FortrOss, Pioneer Surgical 2008 Nanocrystalline hydroxyapatite and Synthetic; Resorbable; Animal derived; Bone injury Paste
E-matrix (porcine collagen Plant or bacteria derived
co-polymerized with dextran)
Regenafil, Regeneration 2005 Human mineralized bone matrix in Resorbable; Animal derived; Human Bone injury Paste
Technologies/Exatech porcine gelatin carrier derived
GEM 21S, BioMimetic 2005 b-TCP particles and recombinant Synthetic; Resorbable; Growth factor Dental bone/ Paste
Therapeutics human platelet-derived growth gum defects
factor-BB (PDGF-BB)
BCT001, Bioceramic Preclinic Strontium releasing bioactive glasses Synthetic; Resorbable Bone defects Granules,
Therapeutics paste

implantation time at all implant locations (shown in Fig. 46(b)),
which is possibly attributed to different stress distribution under
loading and the physiological environment [312]. The biodegra-
dation of Mg-based scaffolds can mainly be controlled by the
alloying of multiple metallic elements like Ca, Zn, Mn, Sr and Yb
[37,96,284,286,287,301], plasma surface modification using gas
and metal ion sources [249,290,296,297,300], and inorganic or
organic coatings [288,293,295,298,313–315], which have been
reviewed in detail by Li and Zheng [96], and Wu et al. [288].
5. Clinical applications of engineered tissue scaffolds
In the past decade, bone tissue engineering has been seeking to
regenerate osseous tissue by designing biomimetic scaffolds with
the additions of multipotent adult stem cells (human bone marrow
stromal/stem cells and adipose stem cells) or growth factor.
However, only a few of biomimetic products have been approved
for clinical trail or even in regular clinical applications for bone
regeneration/repair (shown in Table 8) [316]. The results of such
clinical trials disclosed that the constructs containing multipotent
stem cell exhibited good curative effect for the cases of large bone
defect and nonunion. The typical cases were summarized in Table 9
[317]. It evidenced that the biomimetic scaffolds demonstrated
great advantages over the traditional implants. One example was
the recent clinical treatment in mandibular ameloblastoma
resection and reconstruction of a 55-year-old gentleman. The
tissue-engineered constructs consisting of synthetic b-TCP gran-
ules, BMP2, and adipose stem cells were successfully applied to

Table 9
Representative clinical trials of engineered bone products [317].

Bone defects and nonunions Number Results

Nonunions of the tibia 20 Case series reporting on injection of unprocessed bone marrow. Healing occurred in 18 of 20 cases
after a mean of 6–8 months (1.25)
Mandibular defect 1 Heterotopic bone induction to form a mandible replacement inside the latissimus dorsi muscle after
application of 20-ml bone marrow suspension delivery together with 7 mg BMP-7 in a human being
Nonunions of the tibia 60 Positive correlation between the volume of mineralized callus at 4 months and the number
(p = 0.04) and concentration (p = 0.01) of fibroblast colony-forming units in the graft
Patients undergoing 33 Significantly increased osteoid areas in bones treated with addition of a platelet gel or a platelet gel
high-tibial osteotomy combined with bone marrow stromal cells to lyophilized bone chips
Bone defects of lower limb, primary, 125 Bone grafting with composite transplants composed of autogenous marrow cells and industrial
and revision THA and fractures processed bone allografts was a good alternative to conventional bone grafting
Acetabular defects in revision THA 78 No difference in functional outcome after impaction bone grafting using a frozen nonirradiated
bone bank allograft or a freeze-dried irradiated bone allograft, vitalized with autologous marrow
Bone defects: 101 Safety study and surveillance, no control group, use of BMAC as suspension or via a matrix was
37 avascular necrosis hip shown to be safe at 2–24 month follow-up
32 avascular necrosis others
12 pseudarthrosis
20 other defects
Bone defects 39 Comparative efficacy study comparing BMAC delivered together with a matrix of collagen (n = 12) or
hydroxyapatite (n = 27), with superior results for the HA group
Nonunions of long bones 3 After ex vivo expansion BMSCs on macroporous hydroxyapatite scaffolds showed abundant callus
formation 2 months after surgery
Distraction osteogenesis of long bones 20 Bones treated with culture-expanded BMCs and PRP showed a significantly higher healing index
than a control group treated with no cell-based therapy
Distraction osteogenesis of long bones 46 Bones treated with culture expanded BMCs and PRP showed accelerated bone healing and less
complication than bones treated without a cell-based approach
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S. Wu et al. / Materials Science and Engineering R 80 (2014) 1–36 33

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