Biotechnology Advances 36 (2018) 68–91

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Silk fibroin/hydroxyapatite composites for bone tissue engineering T

a,⁎ b c a
Mehdi Farokhi , Fatemeh Mottaghitalab , Saeed Samani , Mohammad Ali Shokrgozar ,
Subhas C. Kundud, Rui L. Reisd, Yousef Fatahie, David L. Kaplanf
a
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
b
Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
c
Department of Tissue Engineering & Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
d
3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, AvePark, 4805-017
Barco, Guimaraes, Portugal
e
Department of pharmaceutical nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
f
Department of Biomedical Engineering, Tufts University, 4 Colby St, Medford, MA 02155, USA

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

Keywords: Silk fibroin (SF) is a natural fibrous polymer with strong potential for many biomedical applications. SF has
Silk fibroin attracted interest in the field of bone tissue engineering due to its extraordinary characteristics in terms of
Hydroxyapatite elasticity, flexibility, biocompatibility and biodegradability. However, low osteogenic capacity has limited ap-
Scaffold plications for SF in the orthopedic arena unless suitably functionalized. Hydroxyapatite (HAp) is a well-estab-
Bone tissue engineering
lished bioceramic with biocompatibility and appropriate for constructing orthopedic and dental substitutes.
However, HAp ceramics tend to be brittle which can restrict applications in the repair of load-bearing tissues
such as bones. Therefore, blending SF and HAp combines the useful properties of both materials as bone con-
structs for tissue engineering, the subject of this review.

1. Introduction biocompatibility, biodegradability, tunable structural properties and

Bone has an intrinsic capacity for regeneration; however, strategies
to improve the repair capacity of bone are still needed in many cir-
cumstances. Bone regeneration depends on the type of defect. For ex-
ample, about 10% of fractures are non-union that do not heal sponta-
neously and lead to inappropriate bone tissue regeneration and delayed
unions. The probability of normal bone healing in these fractures is
influenced by age, metabolic condition, and severity of the trauma.
Autologous bone or autografts are still considered the clinical “gold
standard” and the most effective method for bone regeneration. This
approach promotes bone formation by direct bone bonding (osteo-
conduction) and induces local stem cells to differentiate into bone cells
(osteoinduction) without any immune responses (Zhang et al., 2014).
About 2.2 million bone grafts are performed annually (Giannoudis
et al., 2005) and are met some limitations due to the limited supply,
morbidity of the donor site and are associated with > 50% failure in
specific regions (Bajaj et al., 2003; Clavero and Lundgren, 2003).
Therefore, many attempts have been made to develop suitable bone
constructs consisting of natural or synthetic biomaterials in order to
increase bone regeneration capacity and avoid the above limitations.
Some polymers are usually good candidates for this purpose due to their
ease of fabrication. Degradable polymers are important for these types
of repairs to avoid barriers to full regeneration of bone, and are cate-
gorized to natural polymers such as alginate, chitosan, collagen, silk
and synthetic polymers including polydioxanone (PDS), poly-
caprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), and
poly (lactic-co-glycolic acid) (PLGA) (Lee and Yuk, 2007; Rezwan et al.,
2006). Recently, more attention has been paid to natural polymers in
comparison to synthetic polymers for bone tissue engineering applica-
tions due to their unique biocompatibility, versatility, and biodegrad-
ability. These types of polymers provide well-patterned structures
consisting of ligands that can bind to cell surface receptors or provide
accessible enzymatic degradation sites (Swetha et al., 2010). Silk fi-
broin (SF) as a natural protein polymer has potent characteristics for
tissue engineering applications, as a biocompatible, biodegradable, and
low immunogenic polymer (Farokhi et al., 2014a; Farokhi et al., 2016a;
Mottaghitalab et al., 2013; Omenetto and Kaplan, 2010; Sugihara et al.,
2000). Moreover, SF possesses extraordinary properties for stimulating
bone repair; for example, the fibrous structure of SF is mostly similar to
collagen type I (Col I). The amorphous links between the β-sheets in the
structure of SF act as sites for deposition of HAp nanocrystals because it
can mimic the anionic structure of noncollagenous proteins (NCPs)

⁎
Corresponding author.
E-mail address: M_farokhi@pasteur.ac.ir (M. Farokhi).

http://dx.doi.org/10.1016/j.biotechadv.2017.10.001
Received 12 June 2017; Received in revised form 12 September 2017; Accepted 4 October 2017
Available online 07 October 2017
0734-9750/ © 2017 Elsevier Inc. All rights reserved.
M. Farokhi et al.
Fig. 1. Applications of (SF) as a biomaterial based on Scopus database. (A) SF-related articles (B) SF used for tissue engineering (C) SF used for bone tissue engineering (D) Silk/
hydroxyapatite (SF-HAp) composites. (E) to (H) remarkable studies related to parts (A) to (D).
(Marelli et al., 2012). However, there are also some reports believed
that β-sheets crystal can alone act as a nucleation site for deposition of
HAp nanaocrystals (Vetsch et al., 2015). Furthermore, this polymer is
introduced as a potential bone construct due to appropriate mechanical
behaviors and structural integrity (Mandal et al., 2012; Sofia et al.,
2001). The ultimate tensile strength of SF extracted from Bombyx mori
(B. mori) is 300–740 MPa (Cunniff et al., 1994; Shao and Vollrath,
2002) and it also has great breaking strain and high toughness more
than synthetic fibers such as Kevlar (Du et al., 2011; Gosline et al.,
1999). Upon searching the Scopus database for studies related to tissue
engineering and bone tissue engineering applications of SF from 2005
to Sep 2017, the number of peer-reviewed original articles per year and
the first ten scientific studies that attract the most attention and the
highest numbers of published articles were assembled (Fig. 1).
Various studies have also recently focused on the applications of
natural polymers as well as SF in bone repair. Although some of these
polymers have intrinsic capacities for the bone regeneration, this is
insufficient to repair large bone defects. For this reason, incorporating
particles into the polymeric matrix can be advantageous to improve the
mechanical behavior and tune the topographical features of the scaffold
in order to mimic the structure of natural bone (Abadi et al., 2010;
Aboudzadeh et al., 2010; Shokrgozar et al., 2010; Swetha et al., 2010).
HAp with similar properties to bone tissue and suitable biocompat-
ibility has been introduced as an appropriate bioceramic phase which
can be incorporated in different polymeric phases (Ginebra et al.,
2006a; Ginebra et al., 2006b). This is osteoconductive (Cai et al., 2009),
non-toxic (He et al., 2012a), low in immunogenicity with the ability to
directly bind to hard tissues (Azadi et al., 2016; Chen et al., 2015b; Li
et al., 2011). HAp is the most stable calcium phosphates (CaPs) under
neutral or basic pHs in the biological fluids and humid conditions (Aoki,
1991). Thus, this ceramic is usually formed in the fluids containing
phosphates in the bodies of vertebrates that has the pH between 7.2 and
7.4. The chemical structure of HAp is mostly similar to inorganic
compounds existed in the bone matrix. High-affinity of HAp to bone
tissue makes it a good bone replacement in comparison to allografts and
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Biotechnology Advances 36 (2018) 68–91
metallic implants (Bauer et al., 1991). There are two common struc-
tures of HAp including monoclinic and hexagonal constructs. In humans
and animal bones, HAp is mainly in the hexagonal structure and thus
synthetic HAp is the predominant form of CaP used in biomedical fields
(Figueiredo et al., 2010). However, HAp is weakly dispersed in aqueous
media which leads to aggregation. Thus, it is necessary to functionalize
the surface of HAp (Kim et al., 2014c). In addition, poor fracture
toughness of HAp (Azadi et al., 2016; Li et al., 2011; Wang et al.,
2004b), migration of nanoparticles from implanted sites (Azadi et al.,
2016; Wang et al., 2005) and challenging processability (Azadi et al.,
2016) restrict the clinical applications of this biomaterial. In order to
overcome these limitations, many efforts have been directed to develop
hybrid structures containing HAp and biopolymers or organic mole-
cules (Li et al., 2008; Li et al., 2011; Wang et al., 2004b; Wang et al.,
2005). Several natural polymers, including collagen, gelatin, chitosan,
and silk have been used in combination with HAp to enhance bone
regeneration (He et al., 2012c). Many structures based on SF/HAp
composites alone or in combination with bioactive molecules and stem
cells for bone tissue regeneration are schematically represented in
Fig. 2.
In this review, we focused on recent studies that used SF/HAp as
bone constituents. This review starts with a brief description about
bioceramics and CaPs, peruses structural properties of HAp and SF, and
continues with descriptions of the signaling responses. Finally, the
combination of SF/HAp for orthopedic applications will be discussed in
detail.
2. Bioceramics for bone tissue engineering applications
Ceramics are solid materials consisting of non-metallic, inorganic
substances in two crystalline and non-crystalline (amorphous) forms
(Hench and Jones, 2005). Ceramic and glasses have different applica-
tions in various fields of human health and in industry, such as diag-
nostic instruments, thermometers, eyeglasses, tissue culture flasks and
fiber optics for endoscopy (Dorozhkin, 2010). During past decades,
M. Farokhi et al. Biotechnology Advances 36 (2018) 68–91

Fig. 2. Silk/hydroxyapatite (SF-HAp) composites for bone tissue engineering.

Fig. 3. Different types and applications of bioceramics.

bioceramics have been used for repairing bone defects (Eshtiagh-
Hosseini et al., 2007) and hard tissues (Hench and Jones, 2005). Bio-
ceramics are included in the ceramic materials category that usually
have non-crystalline structures (Baino and Vitale-Brovarone, 2014).
The main classes of bioceramics and their subset groups and some ap-
plications are presented in Fig. 3.
Nontoxic and biologically inactive ceramics such as Al2O3 and ZrO2
as bioinert bioceramics (Park, 2009) are usually used for joint pros-
theses because of their mechanical properties. Bioactive glasses and
glass-ceramics directly bond to hard tissues and support new bone
formation (Baino and Vitale-Brovarone, 2014). In the biomaterial in-
dustry, CaPs are used predominantly as bone replacements due to their
biocompatibility, low density, chemical stability (Fathi et al., 2008),
chemical and crystallographic similarity to bone mineral (Ding et al.,
2016a, 2016b, 2016c), and integration into living tissue similar to the
healthy bone (Dorozhkin, 2010). These inorganic materials include
diverse types from bioresorbable tricalcium phosphate (TCP) to bioac-
tive and biostable HAp (Fathi et al., 2008). Most CaP bioceramics are
based on HAp, beta-tricalcium phosphate (β-TCP), alpha-tricalcium
phosphate (α-TCP) or biphasic calcium phosphate (BCP) containing a
mixture of α-TCP + HAp or β-TCP + HAp (Dorozhkin, 2010). The
major physical and structural properties of CaPs and inorganic com-
ponents of bone and tooth are listed in Table 1.
2.1. Hydroxyapatite
The term ‘apatite’, given by Wener in 1788, refers to inorganic
compounds with similar structure but different compositions (Kokubo,

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Table 1
Major properties of calcium phosphates and inorganic phases of adult human calcified tissues (Dorozhkin, 2012; Ducheyne et al., 2015; Park, 2009; Ratner et al., 2004; Wnek et al., 2008).

Chemical name Composition Lattice parameters Density Za Lattice structure Transformation Solubility pH
(g/cm3) temperature at 25 °C, stability
Formula Ca/P (°C)b g/L range in
molar (− logKa) aqueous
ratio solutions
at 25 °C

Monocalcium Ca(H2PO4)2·H2Oc 0.5 a = 5.6261(5), 2.23 2 Triclinic ~ 100 ~ 18 0.0–2.0

phosphate b = 11.889(2), transforms into (1.14)
monohydrate c = 6.4731(8) Å, MCPA
(MCPM) α = 98.633(6)°,
β = 118.262(6)°,
γ = 83.344(6)°
Monocalcium Ca(H2PO4)2 0.5 a = 7.5577(5), 2.58 2 Triclinic ~ 100 ~ 17 2
phosphate b = 8.2531(6), (1.14)
anhydrous c = 5.5504(3) Å,
(MCPA or α = 109.87(1)°,
MCP) β = 93.68(1)°,
γ = 109.15(1)°
Dicalcium CaHPO4.2H2O 1.0 a = 5.812(2), 2.32 4 Monoclinic ~ 80 ~ 0.088 2.0–6.0
phosphate b = 15.180(3), (6.59)
dihydrate c = 6.239(2) Å,
(DCPD), β = 116.42(3)°
mineral
brushite
Dicalcium CaHPO4 1.0 a = 6.910(1), 2.89 4 Triclinic ~ 100 ~ 0.048 1
phosphate b = 6.627(2), Sinter ~ 300 (6.90)
anhydrous c = 6.998(2) Å,
(DCPA or α = 96.34(2)°,
DCP), mineral β = 103.82(2)°,
monetite γ = 88.33(2)°
Octacalcium Ca8(HPO4)2(PO4)4.5H2O 1.33 a = 19.692(4), 2.61 1 Triclinic An unstable ~ 0.0081 5.5–7.0
phosphate b = 9.523(2), transient (96.6)
(OCP) c = 6.835(2) Å, intermediate
α = 90.15(2)°,
β = 92.54(2)°,
γ = 108.65(1)°
α-Tricalcium α-Ca3(PO4)2 1.5 a = 12.887(2), 2.86 24 Monoclinic – ~ 0.0025 3
phosphate (α- b = 27.280(4), (25.5)
TCP) c = 15.219(2) Å,
β = 126.20(1)°

β-Tricalcium β-Ca3(PO4)2 1.5 a = b = 10.4183(5), 3.08 21d Rhombohedral ~ 1125 ~ 0.0005 3

phosphate (β- c = 37.3464(23) Å, transforms into (28.9)
TCP) γ = 120° α-TCPe

Amorphous CaxHy(PO4)z.nH2O, n = 3–4.5, 1.2–2.2 – – – – – 5 ~5–12f

calcium 15–20% H2O
phosphates
(ACP)
Calcium-deficient Ca10 − x(HPO4)x(PO4)6 − x(OH)2 − x 1.5–1.67 – – – – ~ 700 ~ 0.0094 6.5–9.5
hydroxyapatite (0 < x < 1) transforms into (385)
(CDHA or Ca- β-TCP
def HA)g
Hydroxyapatite Ca10(PO4)6(OH)2 1.67 a = 9.84214(8), 3.16 4 Monoclinic ~ 250, ~ 0.0003 9.5–12
(HA, HAp or b = 2a, c = 6.8814(7) monoclinic to (116.8)
OHAp) Å, γ = 120° 2 hexagonal Sinter
(monoclinic) ~ 1000
a = b = 9.4302(5),
c = 6.8911(2) Å,
γ = 120° (hexagonal) Hexagonal

Fluorapatite (FA or Ca10(PO4)6F2 1.67 a = b = 9.367, 3.20 2 Hexagonal – ~ 0.0002 7–12

FAp) c = 6.884 Å, (120)
γ = 120°

(continued on next page)

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Table 1 (continued)

Chemical name Composition Lattice parameters Density Za Lattice structure Transformation Solubility pH
(g/cm3) temperature at 25 °C, stability
Formula Ca/P (°C)b g/L range in
molar (− logKa) aqueous
ratio solutions
at 25 °C

Oxyapatite (OA, Ca10(PO4)6O 1.67 a = b = 9.432, ~ 3.2 1 Hexagonal – ~ 0.087 3

OAp or OXA)h c = 6.881 Å, (369)
α = 90.3°,
β = 90.0°,γ = 119.9°

Tetracalcium Ca4(PO4)2O 2.0 a = 7.023(1), 3.05 4 Monoclinic – ~ 0.0007 3

phosphate b = 11.986(4), (38–44)
(TTCP or c = 9.473(2) Å,
TetCP), β = 90.90(1)°
mineral
hilgenstockite
Enamel – 1.63 a = 9.441, – – – – –
c = 6.880 Å
Dentine – 1.61 a = 9.421, – – – – –
c = 6.887 Å
Bone – 1.71 a = 9.41, c = 6.89 Å – – – – –

a
Number of formula units per unit cell.
b
Stable at temperatures above 100 °C.
c
Cannot be precipitated from aqueous solutions.
d
Per the hexagonal unit cell.
e
Cannot be measured precisely. However, the following values were found: 25.7 ± 0.1 (pH 7.40), 29.9 ± 0.1 (pH 6.00), 32.7 ± 0.1 (pH 5.28). The comparative extent of dis-
solution in acidic buffer is: ACP > > α-TCP > > β-TCP > CDHA > > HA > FA.
f
Always metastable.
g
Occasionally referred to as “precipitated HA” (PHA).
h
The existence of OA remains questionable.

2008). These minerals have lattice parameters a = 0.9432 nm and
c = 0.6881 nm and crystallize into hexagonal rhombic prisms (Park,
2009; Park and Lakes, 2007). Synthetic or pure HAp is introduced by
Ca10(PO4)6(OH)2 or Ca10 − z(HPO4)z(PO4)6 − z(OH)2 − z [0 < z < 1] as
a general formula. The chemical behavior of HAp depends on stoi-
chiometry status, which is basic for stoichiometric apatite (z = 1) and
acidic for the others (Faria et al., 2008).
Inorganic materials coexist with organic molecules in a well-ordered
manner in some tissues such as bone, cartilage and skin (Wang et al.,
2004a). Natural bone is a nanocomposite (Andiappan et al., 2013;
Bhattacharjee et al., 2016b) composed of organic components (~ 90%
type I collagen, ~5% NCPs,~2% lipids by weight), water and inorganic
CaP (Boskey, 2013; Šupová, 2015; Wu et al., 2014; Zhao et al., 2009).
Recently, NCPs have attracted attention due to their effect on bone
tissue behavior, including cell proliferation and attachment, HAp and
Ca2 + binding and degree of mineralization (Young et al., 1992). Os-
teocalcin, sialoprotein, alkaline phosphatase (ALP) and matrix Gla
protein are the most abundant NCPs that are a main focus of research;
however, it is not well-understood that which of these proteins may
have a crucial role in bone behavior (Fig. 4). It is reported that the
combination of these proteins may influence bone functions synergis-
tically more than each of them alone. It should be noted that some
features such as gender, age, health status and ethnicity affect the
proportion of these proteins in bone structure of an individual patient.
Therefore, the quantity and quality of NCPs can influence the char-
acteristics of bone tissue in terms of structure and function (Boskey,
1989, 2013).
The inorganic phase of bone consists of HAp nanocrystals (about
70 wt% (Andiappan et al., 2013; Wang et al., 2004a)) dispersed in
collagen type I (Col I) (Andiappan et al., 2013; Wang et al., 2004a). This
mineral phase is crystallized along the long axis of collagen fibrils (Hu
et al., 2015) and integrates together in a tight hierarchical organization
(Fig. 4) [28]. The X-ray diffraction (XRD) spectrum of sintered bone and
mineral apatite showed that HAp and fluorapatite are the main
minerals in bone and teeth. For many years, it was assumed that the
inorganic parts of calcified tissues were HAp. However, studies focused
on synthetic carbonated and biological apatites showed that the bone
mineral did not consist of pure apatite but contained carbonated apatite
(Kokubo, 2008) and some ions such as K+, Mg2 +, Na+, F− and CO32–
(Li et al., 2008; Webster et al., 2004). Moreover, bioactivity of bone
mineral is higher than synthetic HAp (Fathi et al., 2008). Therefore,
non-stoichiometric HAp substituted with suitable ions during synthesis
would be useful to mimic the function of bony apatite and os-
teoinductive activity when compared to pure HAp (Ren et al., 2009).
Different methodologies have been used to prepare HAp-based
products in micro- or nanoscale quantities. Co-precipitation (Cao et al.,
2013; Ming et al., 2014a), chemical precipitation (He et al., 2012a;
Kweon et al., 2011; Wang et al., 2002), solid-state reaction (Ming et al.,
2014a; Wang et al., 2002), hydrothermal methods (Cao et al., 2013;
Kweon et al., 2011; Ming et al., 2014a; Wang et al., 2002), sol-gel
synthesis (Cao et al., 2013; Kweon et al., 2011; Ming et al., 2014a), and
wet mechanochemical routes (Cao et al., 2013; Nemoto et al., 2001;
Wang et al., 2002) are common methods used for HAp synthesis. Mi-
croemulsions, spray drying and plasma melting are also applied for
synthesizing spherical HAp powders (Liu et al., 2013a). Structural
features of the synthesized powders are mainly related to the corre-
sponding preparation method (Kweon et al., 2011). Control of size
distribution and morphology of nano-sized HAp powders (nHAps) is
critical for tissue engineering and drug delivery systems, which are
often ignored in some methods (He et al., 2012a; Huang et al., 2014).
The different methods suitable for synthesis of nHAps are summarized
in Table 2.
2.2. Signaling responses of osteoblast cells to hydroxyapatite
Dynamic interactions between osteoblasts and their extracellular
matrix (ECM) regulate their behavior and differentiation potential.
Understanding the principal ECM cues promoting osteogenic

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Fig. 4. Ultrastructure of bone tissue. The extracellular matrix of bone is highly specialized. The organic part of bone is composed of collagen, noncollagenous proteins (NCPs) and lipids.
The inorganic phase of bone mainly consists of hydroxyapatite (HAp) nanocrystals. This mineral phase is crystallized along the long axis of collagen fibrils and integrates together in a
tight hierarchical organization. These crystals associate with the collagen fibers, making bone hard and strong. This matrix is organized into numerous thin layers, known as lamellae.

differentiation can be pivotal for both bone tissue engineering and re- interaction between HAp and osteoblast integrins such as BMP/Smad
generative medicine. HAp can trigger signaling cascades; however, the (Liu et al., 2013b), Wnt (Thorfve et al., 2014), TGF-β, MAPK, and Notch
mechanism of action is not well understood. The bioactivity of any signaling pathways (Lü et al., 2014).
material, here defined as the ability of osteoblasts to adhere and spread
upon it, depends on these signaling mechanisms (Kokubo, 1991;
3. Structure and characteristic of silk fibroin
Zambuzzi et al., 2011a). The signaling pathway involved in osteoblasts
adhering on HAp scaffolds was recently evaluated (Gemini-Piperni
Silk protein is found in the gland of silk producing arthropods such
et al., 2014). Adhesion of osteoblast cells on HAp substrates actives
as spiders, silkworms, mites, scorpions, and bees. During metamor-
such signaling pathway that leads to activation of protein kinase C
phosis, silk is spun to fibers. Cocoon silk, as a Food and Drug
(PKC), protein kinase A (PKA), Adducin 1 (ADD1) and vascular en-
Administration (FDA) approved biomaterial for some medical applica-
dothelial growth factor (VEGF).
tions, is produced by silkworm B. mori (Kaplan et al., 1994). B. mori silk
Moreover, kinase activation leads to increased phosphorylation of
is composed of two major types of proteins, silk fibroin (SF) and sericin.
Ser-421 in histone deacetylase 1 (HDAC1), a cyclin-dependent kinase 5
SF is located in the core of B. mori silk fibers and consists of light
(CDK5) substrate. The surface chemistry of HAp is crucial for promoting
(~ 26 kDa) and heavy (~ 390 kDa) chains; while, sericin coats the SF
the differentiation of osteoblasts via phosphorylation (Gemini-Piperni
chains. Silk fibers contain about 70–75% SF filaments and 25–30%
et al., 2014). In response to HAp, other signaling molecules such as
sericin. The primary sequence of sericin consists of some repeat motifs
extracellular regulated kinases (ERK) and SOX9 are also activated (Song
(Sutherland et al., 2010). The structure of the SF heavy chain is similar
et al., 2008b). The integrin receptors mediate the interaction between
to amphiphilic block copolymers because it contains several hydro-
osteoblast cells cultured on HAp substrates with some domains of ECM
phobic and hydrophilic domains or blocks. The hydrophobic blocks of
proteins that can activate the cytoskeletal and intracellular signaling
SF are comprised of highly conserved (GAGAGS) and less conserved
cascades like focal adhesion kinase (FAK). FAK is associated with Shc
(GAGAGX) repeats (where X is either valine or tyrosine) that are re-
protein activating Ras that stimulates the ERK signaling cascade
sponsible for the crystalline structure and β-sheet conformation of SF.
(Miyamoto et al., 1996). Activated ERK affects the expression level of
The hydrophilic part of SF core consists of short and non-repetitive
different osteoblast genes such as osteocalcin and Col I (Fig. 5) (Mimori
segments in comparison with hydrophobic parts (Bini et al., 2004;
et al., 2007; Xiao et al., 2002). Therefore, ERK signaling is essential for
Kaplan and McGrath, 2012). Silk fibers have useful mechanical prop-
cytoskeletal rearrangement and intracellular signaling (Zambuzzi et al.,
erties, including a tensile strength of 0.5 GPa, breaking elongation of
2011b). Various signaling cascades are also triggered upon the
15% and 62,104 J kg− 1 toughness (Li et al., 2012). The Young's

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 Table 2
Different methods for nHAps fabrication and main properties based on the materials used (El Briak-BenAbdeslam et al., 2008; Gedanken, 2004; Sadat-Shojai et al., 2013; Sasikumar and Vijayaraghavan, 2008, 2010; Tas, 2000).

Synthesis method Raw materials Advantages Disadvantages Synthetic conditions Shape Size-Size distribution Product features
M. Farokhi et al.

categories1

Solid-state reaction
Mechanochemical
(Mechanosynthesis)
Chemical precipitation
Hydrolysis
Few Ca and PO4 containing Relatively simple Weak precise control over 900–1300 °C, usually with water 1, 3 Usually micron-Wide Diverse morphology
chemicals Mass production product features vapor flowing Very high crystallinity
Presynthesized CaP salts Using organic templates to Heterogeneous composition Usually low purity
control morphology of powder Variable Ca/P
Low cost Irregular shape
Cannot be exploited for
biomimetic synthesis
Few Ca and PO4 containing Simplicity – Wet or dry medium 1, 2, 3 Nano-Usually wide Diverse morphology
chemicals Reproducibility Reaction activated Very high crystallinity
e.g.: CaHPO4.2H2O + CaO Production of by mechanical milling Low purity
CaCO3 + NH4H2PO4 nanocrystalline alloys and Effective factors: reagents, Non-stoichiometric Ca/P
Ca3(PO4)2 + Ca(OH)2 ceramics milling medium, atmosphere,
Ca(OH)2 + P2O5 Well-defined structure of rotational speed, diameter of
CaO + Ca(OH)2 + P2O5 powder milling ball
Mass production
Low cost
Frequently few Ca and PO4 The simplest route Precise control over Usually at room temperature 1, 2, 3, 4, 5, 7, Usually nano- Variable Diverse morphology
containing reagents Using organic templates conditions necessary to Atmospheric pressure 8, 11 Frequently low
and molecules to control minimize defects Aqueous medium crystallinity
morphology Phase impurities (DCPAa, pH 7–12 Variable purity
Low cost OCPb) Usually non-
stoichiometric Ca/P
Few chemical substances Distinct method to convert Usually high cost pH 6–7 for acidic phases (DCPD, 1, 3, 5, 7 Variable- Variable Diverse morphology
Other CaP phase (DCPDc, DCPA a CaP into HAp DCPA) Variable crystallinity

74
TCP) Method to modify a Conversion depends on pH, T and Usually high purity
prepared HAp other ions Stoichiometric Ca/P
Sol-gel Variable chemical reagents Molecular level mixing of High cost of some raw Aqueous or organic media 1, 2, 3 Nano-Narrow Diverse morphology
Ca(OEt)2, Ca(NO3)2, P(OEt)3, P2O5, reactants materials Aging at room temperature Variable crystallinity
(NH4)2HPO4 Improving chemical Possible impurities (CaO, Drying at ~ 100–150 °C Variable purity
homogeneity Ca2P2O7, Ca3(PO4)2, CaCO3) Heat treatment at ~ 300–900 °C Stoichiometric Ca/P
Low temperature for Long aging to complete Higher bioresorbability
various steps reaction than chemical
Stoichiometric structure Thermal treatment to get precipitation
pure HAp
Hydrothermal Variable reagents Using organic modifiers to Expensive equipment for Aqueous solution 1, 2, 3, 4, 5, 6, Nano and micron- Frequently needle-like
Wet chemically prepared HAp, control process high temperature and High temperature and pressure 8, 9 Wide Usually irregular
other calcium phosphates, seeding Regulating more pressure 100–200 °C (1–2 MPa), Very high crystallinity
large crystals predictable reactions as Poor control over 300–600 °C (1–2 kbar) Usually high purity
crystal nucleation, morphology and size Stoichiometric Ca/P
growth, and aging distribution
Emulsion Many raw materials Simplicity High cost Low temperature 1, 2, 3, 5 Nano-Narrow Frequently needle-like
More effective control over Different surfactants Frequently low
morphology and Mild conditions crystallinity
microstructure Variable purity
Agglomerate-free Non-stoichiometric Ca/P
Sonochemical Few Ca ad PO4 containing Increasing rate of crystal – Aqueous environment 1, 2, 3 Nano-Usually narrow Diverse morphology
chemicals growth Chemical reactions activated by (usually needle-like)
e.g.: Ca(OH)2 + Ca(H2PO4)2 More uniform, smaller and powerful ultrasound radiation Variable crystallinity
purer crystals Heterogeneous Usually high purity
Minimal agglomeration reactions between liquid and Variable Ca/P
Using organic modifiers to solid reactants
control or change process
Usually low cost
(continued on next page)
Biotechnology Advances 36 (2018) 68–91
M. Farokhi et al. Biotechnology Advances 36 (2018) 68–91

1-Irregular sphere, 2-(micro and nano)Sphere or ball-shaped, 3-rod- or needle-shaped or strip-like, 4-plate or sheet, 5-self-assembled or bundled nanorod, 6-dendelion or rosette, 7-bundled sheets or leaves, 8-Flower, 9-porous microsphere or
modulus of silk obtained from forced silking of silkworm in the la-

Usually stoichiometric Ca/

boratory is about 12.4–17.9 GPa, with an ultimate tensile strength of

Variable crystallinity
Diverse morphology

Diverse morphology
360–530 MPa and 18–21% breaking elongation (Pérez-Rigueiro et al.,

Usually high purity

(usually irregular)

High crystallinity
2001). Silk fibers have been used for many years as sutures and for
Product features

Variable purity
Variable Ca/P
textile engineering due to their high toughness (Nova et al., 2010).
Some studies have reported comparable mechanical behavior for silk
filaments with nylons (Lucas, 1964). These unique mechanical char-

P
acteristics make SF a useful polymer candidate for load-bearing appli-
cations, such as for bone tissue engineering in composite systems with
Size-Size distribution

embedded in micron
aggregates- Variable
HAp.
Usually nano-Wide

Nano particles

3.1. Osteogenic signaling responses to silk fibroin

While silk scaffolds do not inherently contain any cell-specific sig-

naling epitopes in the primary sequence, silk-based scaffolds provide
suitable substrates for supporting the proliferation and adhesion of
mesenchymal stem cells (MSCs) (Mandal and Kundu, 2009; Mauney
categories1

1, 2, 3, 5

1, 2, 3, 5

et al., 2007). The ability of hydrolyzed silk protein to improve the ALP
Shape

activity of osteoblast cells and their osteogenic differentiation has also

been reported (Seo et al., 2011). Moreover, B. mori silk induces mineral
deposition from rat bone marrow stromal cells (BMSCs) during 12 days
Particles formed from aqueous

Heat sources: flame or furnace

of culture in vitro (Nikbakht Dastjerdi, 2006). This study demonstrated

combustion of a fuel and
solution via spontaneous

that by using silk, no dexamethasone additives are required for pre-

osteogenic factor, although the compound can accelerate the outcomes.
Synthetic conditions

oxidiser at elevated

Dexamethasone triggers Wnt/β-catenin signaling dependent Runx2

expression (Langenbach and Handschel, 2013). Furthermore, other
temperatures

signaling factors like Wnt, Notch, fibroblast growth factor (FGF), bone
morphogenetic protein/transforming growth factor beta (BMP/TGFβ),
insulin-like growth factor (IGF), and platelet derived growth factor
(PDGF) regulate the osteogenic differentiation of stem cells on silk-
TCP at high temperature of
HAp decomposition into α-

based substrates (Midha et al., 2016). Based on pharmacologic and

Secondary aggregations

molecular studies, Notch signaling was responsible for differentiation of

leading to decrease in
temperature reaction
Mixed phases due to

Particle morphology

specific surface area

uncontrollable high

mesenchymal progenitor cells into osteoblast cells. Silk protein pro-

dependent on fuel

voked the upregulation of some osteoblast differentiation markers (e.g.

Disadvantages

ALP, osteorix and Runx2) by inhibiting Notch signaling (Jung et al.,

2013). Additionally, the mass of trabecular bone in the skeletogenic
flame
front

used

mesenchyme of mice limbs was significantly increased by inhibiting the

Notch signaling pathway in adolescent mice. Severe osteopenia was
Well chemical homogeneity

observed in mice during aging period when they were without me-
Inexpensive raw materials

senchymal progenitors in their bone marrow. The inhibition of osteo-

Fairly low temperature

continuous production
Single step operation
Quick production of

genic differentiation by Notch signaling is triggered by Hes and Hey

Easily scaled-up for
Relatively simple

Usually low cost

Usually low cost

proteins, which can suppress Runx2 activity through physical interac-

tion (Fig. 6a) (Hilton et al., 2008). Thus, for optimal osteogenic in-
Advantages

of powder

duction of silk scaffolds, suppression of the Notch signaling pathway is

powder

advantageous (Fig. 6b).

3.2. Silk fibroin-based scaffolds for bone tissue engineering

sucrose, citric acid, succinic acid)
Oxidants (Ca(NO3)2, (NH4)2HPO4

The outstanding properties of SF such as biocompatibility, biode-

Organic fuels (glycine, urea,

e.g.: Ca(NO3)2, (NH4)2HPO4

Variable chemical materials

gradability, low immunogenicity, and suitable processability make it a

useful scaffold system for various tissue engineering applications, in-
Few raw materials

cluding bone constructs. Another advantage of SF as a bone substitute is

Raw materials

its high mechanical strength (Kundu et al., 2013; Mottaghitalab et al.,

mesoporous sphere, 10-Bowknot, 11-Dumbbell.
and HNO3)

2015b; Nourmohammadi et al., 2017); it can bear the force produced in

vivo (Nazarov et al., 2004). These properties of SF have attracted many
researchers to use this biomaterial in bone tissue engineering applica-
Anhydrous dicalcium phosphate.

Dicalcium phosphate dihydrate.

tions. Recent investigations on the SF-based scaffold as a bone construct

are summarized in Table 3.
Self-propagating combustion

Octacalcium phosphate.

4. Silk fibroin/hydroxyapatite for bone tissue engineering

Synthesis method
Table 2 (continued)

4.1. Silk fibroin/hydroxyapatite composites for bone tissue engineering

synthesis

Pyrolysis

Bone tissue engineering offers suitable constructs for restoring and

repairing bone defects (Ma et al., 2005). Mimicking the structure and
1

b
a

composition of natural tissues by engineering biomaterials, such as

75
M. Farokhi et al.
Fig. 5. Signaling pathway of osteoblast cells seeded on hydroxyapatite (HAp) scaffold. Adhesion of osteoblast cells on HAp substrates triggers some signaling pathways like extracellular
regulated kinases (ERK). Due to interaction of integrin receptors with such extracellular matrix of bone e.g. fibronectin, focal adhesion kinase (FAK) as an intracellular signaling cascade
can be activated. This causes activation of ERK pathway and then expression of some osteoblast specific genes e.g. osteocalcin and collagen type I.
developing inorganic-organic composites, has grown in interest and
focus over the past decades. Some typical ceramics such as bioactive
glass, HAp, β-TCP, and calcium citrate are suitable candidates for bone
tissue engineering applications (Chouzouri and Xanthos, 2007; Maquet
et al., 2004). The most common organic material used as bone con-
structs is collagen. This protein polymer can be obtained from some
animals (e.g. skin of pig and cow) or generated through transgenic
techniques. However, collagen is not cost-effective, provoke immune
responses, and retains a risk for contamination (Du et al., 2009). For
these limitations, some researchers have tried to use other natural
biomaterials such as silk for bone tissue engineering.
Blending silk with HAp enhanced crystal formation of HAp along
the c-axis and the coordinative effect on the structure and properties
between SF and HAp was found during the composite film fabrication.
The nucleation of HAp could enhance the molecular orientation and
crystallinity of SF (Du et al., 2009). Recently, Jo et al. evaluated algi-
nate/HAp/SF composites as a bone replacement (Jo et al., 2017). Four
weeks after implantation, the rate of bone formation in the defected site
was significantly higher using alginate/HAp/SF scaffold than alginate
and control (unfilled defects) groups. The suitable biocompatibility of
alginate/HAp/SF scaffold led to less immunogenic response or forma-
tion of giant cells around the degraded grafts. Significantly lower ex-
pression level of tumor necrosis factor-alpha (TNF-α) was also observed
in alginate/HAp/SF group compared with alginate and alginate/HAp
groups; while, the osteogenic markers such as Runx2, osteoprotegerin,
and FGF-23 showed higher expression rate. The alginate/HAp/SF
scaffold was introduced as a potential bone construct for repairing
76
Biotechnology Advances 36 (2018) 68–91
calvarial defects in rat model that was stably biodegraded without
provoking the immunogenic responses (Jo et al., 2017).
The homogenous dispersion and interfacial bonding between poly-
meric matrix and HAp is still challenging in fabricating composites. The
interfacial bonding between HAp and the organic phase is dependent to
the density of contact numbers and the chemical interactions. In this
context, pretreatment of SF fibrils with an alkali increased the exposed
active sites on the surface that further improved the interactions be-
tween the organic and mineral phases. The alkali pretreatment effected
the crystallographic characteristics of HAp in comparison to pretreat-
ment with proteinase K, a protease that digests the SF. Applying both
pretreatments enhanced the exposed active sites to interact with the
mineral phases. It was suggested that SF surface fibrils were mostly
disentangled by alkali, while the enzymatic pretreatment was effective
in disentanglement of the SF blocks into smaller fragments (Wang et al.,
2007). An increase in number of contact sites enhanced the interactions
between organic and inorganic phases to improve microhardness
(> 50%) (Wang et al., 2007; Wang et al., 2004b; Wang et al., 2005).
However, the remaining sodium ions from the alkali reagent may have
unexpected side effects on crystallographic properties of HAp due to
replacement of calcium with sodium ions.
During the past decade, many efforts have been attempted to de-
velop hybrid biomaterials based on organic and inorganic materials to
mimic the structure and function of natural biomaterials. The existence
of organic materials like polymers and proteins in the structure of hy-
brid materials regulate the nucleation of inorganic crystals, and en-
hance the physicochemical properties of the microstructure. As
M. Farokhi et al. Biotechnology Advances 36 (2018) 68–91

Fig. 6. The role of Notch signaling pathway in osteoblast differentiation of stem cells. a). Notch signaling blocks RUNX2 expression and consequently inhibition of osteocalcin and
osteopontin. b). Blocking of Notch signaling cascade after seeding of stem cells on silk fibroin based scaffold.
EGF: epidermal growth factor, LNR: Lin-12/Notch repeats, HD domain: heterodimerization domain, TMD: transmembrane domain, RAM: RBPjκ association module, ANK: ankyrin, NLS:
nuclear localization sequences, TAD: transactivation domain, PEST: proline (P), glutamic acid (E), serine (S), threonine (T) rich motif, NICD: Notch intracellular domain, ADAM: a
disintegrin and metalloproteinase, MAMAL: Mastermind-like, HES: hairy and enhancer of split, HEY: HES-related with YRPW motif.

mentioned above, adding HAp to SF improve the biological and me-
chanical properties of SF. HAp/SF composites are so similar to the
natural bone structure that mimic the inorganic and organic phases of
native bone tissue. Thus, combination of inorganic and organic mate-
rials in a unique hybrid composite can improve the flexibility, me-
chanical strength, and the toughness of the bone tissue scaffolds. Some
applications of SF/HAp composites for bone tissue engineering are
listed in Table 4.
4.2. Silk fibroin/nano-hydroxyapatite composites for bone tissue
engineering
Recently, nanoparticles have attracted attention in biomedical fields
due to their exceptional properties such as high surface to volume ratio,
tunable structural properties in terms of surface chemistry, size and
shape (Brannon-Peppas and Blanchette, 2012). Today, different appli-
cations are considered for nanoparticles including theranostics therapy,
molecular imaging, drug delivery and cell labeling (Hassani Besheli
et al., 2017; Kim et al., 2014a; Mottaghitalab et al., 2015a;
Mottaghitalab et al., 2017). It is also observed that the alterations in
nanoscale features can affect cellular responses. For example, nano-
patterned structures were potent in directing stem cell differentiation
without the using of exogenous biochemical agents (Kim et al., 2012a,
2012b). Controlling the cellular behavior by using nano-patterned
structures is a key application for nanotechnology in biomedical fields.
In the orthopedic field, HAp nanocrystals were sintered and improved
densification because of the high surface area. The high surface area
can also increase fracture toughness and mechanical strength of HAp
nanocrystals. However, agglomeration is the most challenging issue in
using nano-powders (LeGeros, 1993). nHAp showed better bioactivity
in comparison to coarser crystals that broaden its applications in tissue-
engineered implants when compared to other constructs (Stupp and
Ciegler, 1992). Moreover, nHAp have high bioresorption properties and
similar crystallographic and chemical characteristics to the natural
apatite in bone (Murugan and Ramakrishna, 2005). Composites con-
taining nHAp induced both osteogenesis and angiogenesis by stimu-
lating the proliferation, adhesion, and differentiation of osteoblasts;
therefore, these composites are potent for bone tissue engineering ap-
plications (Kilian et al., 2008; Nageeb et al., 2012; Wei and Ma, 2004).
Besides, in many studies SF protein extracted from B.mori alone or
in blended with CaP has been used for bone tissue engineering.
However, there is no RGD motifs or sufficient arginine amino acids in
the structure of SF derived B.mori to support cellular adhesion (Sen and
Babu, 2004). Some reports suggesting the presence of RGD epitopes and
high arginine content in the structure of non-mulberry silk fibroins
(NSF) that reduced the cytotoxicity and inflammatory responses (Datta
et al., 2001; Mandal and Kundu, 2008). Moreover, presence of hydro-
philic amino acids in the structure of NSF promotes cellular attachment
to the surface of the materials (Meinel et al., 2005b; Sen and Babu,
2004; Zhang et al., 2005b). The in situ reinforcement of NSF-nHAp
scaffold and the NSF scaffold with external nHAp deposition showed
higher mechanical properties and biocompatibility in comparison to
NSF-nHAp reinforced fibroin scaffolds. Both composite scaffolds also
showed less immunogenicity by using osteoblast-macrophage co-cul-
ture model (Behera et al., 2017). However, no significant anti-in-
flammatory differences were observed through the modified scaffold
indicating the native anti-inflammatory properties of fibroin structures.
It was also observed that by culturing the macrophages on the modified
scaffolds, the level of interleukin 1 beta (IL1-β) increased that might be
related to the role of HAp (Loi et al., 2016). Similarly, nHAp/SF pow-
ders were synthesized using a co-precipitation method, added to SF
solution in order to fabricate nHAp/SF composite material (Liu et al.,
2011). These composites showed improved compressive properties. The
good compressive properties were due to the uniform dispersion of
nHAp/SF in SF solution, the high surface area of needle-like nHAp/SF
powders, and good molecular interaction between SF molecules in the

77
 M. Farokhi et al.

Table 3
SF based scaffolds for bone tissue engineering applications.

Material Processing method Scaffold structure Cell Key findings Ref.

TSFa/PLAb Electrospinning Nanofibers mMSCsc Inducing the proliferation and attachment of MSC, increasing the rate of ALPd production, (Shao et al., 2016a)
(90:10) osteogenesis, and bone mineralization
NSFe/PCLf Electrospinning Nanofibers – Stimulating the early bone formation, infiltration of implants and formation of strong bonds at the (Bhattacharjee et al., 2016a)
interface of bone-implant, activating the initial immune reaction which reaching to normal after
4 weeks post-implantation
SFg/CMCh Electrospinning Nanofibers hMSCsi Increased proliferation, adhesion and differentiation of hMSCs confirmed by ALP production, (Singh et al., 2016)
transcription of RUNX2, and expressions of osteocalcin and type1 collagen
SF/Decellularized pulp/Collagen/ Freeze-drying 3D scaffold MG-63 Capability of the modified scaffold for alveolar bone repair due to outstanding biofunctionality (Sangkert et al., 2016)
Fibronectin
SF/Diopside Freeze-drying 3D scaffold MC3T3-E1 Increased wettability, suitable porosity, high mechanical strength, and good biocompatibility by (Ghorbanian et al., 2013)
incorporating diopside nanopowders into the SF matrix
j k l
SF/CS /Nano ZrO2 Freeze-drying 3D scaffold HGF Formation of a porous scaffold with appropriate pore size for cell infiltration, improved compressive (Teimouri et al., 2015)
strength and water uptake, and decreased porosity after adding zirconia
SF/CaPm/PLGAn Electrospinning/Freeze- 3D scaffold hOBso Providing an optimal microenvironment for the biological behavior of osteoblast cells due to (Farokhi et al., 2014b)
drying acceptable porosity, chemical and physical properties with ability to sustain the release rate of VEGFp
within 28 days, stimulating the new bone formation at the site of injury 10 weeks after implantation
SF Casting Film BMSCq More efficient delivery of BMP-2r after immobilizing this biomolecule on the surface of biomaterial in (Karageorgiou et al., 2004)
comparison to soluble delivery, retained in vitro biological activity of BMP-2 based on its potential to
induce the osteogenic markers of BMSCs
SF – Mesh OECs/hOBs Extracellular matrix deposition by hOBs during 4 weeks, enhancing expression of osteogenic markers, (Fuchs et al., 2009)
retained functionality of both cell types in provoking the formation of capillary like structures and

78
differentiation of hOBs
SF Salt leaching 3D scaffold hMSC Increased bone formation in calvarial critical size defects in mice during 5 weeks by using tissue- (Meinel et al., 2005a)
engineered bone implants, detecting less bone formation by using stem cell loaded SF scaffolds and
scaffolds alone

a
Tussah silk fibroin.
b
Polylactic acid.
c
Mouse mesenchymal stem cells.
d
Alkaline phosphatase.
e
Non-mulberry silk fibroin.
f
Silk fibroin.
g
Poly(ε-caprolactone).
h
Carboxymethyl cellulose.
i
Human mesenchymal stem cells.
j
Chitosan.
k
Zirconia.
l
Human gingival fibroblast.
m
Calcium phosphate.
n
Poly (lactic-co-glycolic acid).
o
Human osteoblast cell.
p
Vascular endothelial growth factor.
q
Bone marrow mesenchymal stem cells.
r
Bone morphogenetic protein-2.
s
Outgrowth endothelial cells.
Biotechnology Advances 36 (2018) 68–91
 Table 4
Applications of SF/HApcomposites for bone tissue engineering.

Materials Processing method Cell Structural properties In vitro/in vivo key findings Ref.
M. Farokhi et al.

a b c −1 −1
SF /HAp MAPLE SaOs2 Exhibition of 1540 cm amide II, 1654 cm Improved proliferation of cells with normal (Miroiu et al., 2010)
amide I, 1243 cm− 1 amide III peaks in the FTIR morphology after 72 h culture on SF and HAp/SF
spectrum of SF, and observing of 1027 cm− 1 only films
for HAp and HAp/SF composite groups
SF/HAp Layer solvent casting/Freeze BMSCsd Decreasing pore average size with increasing SF No variation in the proliferation rate of BMSCs on (Gholipourmalekabadi et al., 2015)
drying content from 5% (350 ± 67 μm) to 10% HAp/SF and no toxicity, good in vivo
(112 ± 89 μm), more uniform pores in the biocompatibility of SF containing 5% HAp, no
scaffolds containing 5% HAp significant increase in the average number of
lymphocytes in long and short periods
SF/HAp Casting – Exhibition of silk II conformation in the secondary – (Ming et al., 2014b)
structure of SF films, no impact of HAp content in
the solution on the structure of SF films, Significant
effect of HAp content on rheological behavior of SF
solution
NSFe/HAp Electrospinning and soaking Osteoblast Higher crystallinity and thermal stability of Higher cell viability on electrospun NSF/HAp (Sekar et al., 2016)
electrospun NSF/HAp scaffold compared with pure scaffold than pure NSF and NSF/HAp formed by
NSF and NSF/HAp formed by soaking method soaking method
CSf/SF/nHAp Salt fractionation MG-63 Scaffolds exhibited 90.5–96.1% porosity with Suitable for increasing the proliferation and (Qi et al., 2014)
100–300 μm pore size, better mechanical behavior adhesion of cells, higher efficacy of CS/SF/nHA
of tri-component scaffold compared with bi- composite porous scaffold than other groups
component scaffolds
CS/SF/HAp Co-precipitation – Characteristics of primary HAp crystals: needle- – (Wang and Li, 2007)
like shape, 20–50 nm long, and ~ 10 nm wide,
Chemical interactions between amino group of
chitosan or amid bands of SF with Ca2 + ions

79
SF/COLg/HAp Particulate leaching MG-63 Displaying porous structure with adjustable Significantly higher biocompatibility at days 7 and (Mou et al., 2013)
macropores with high interconnectivity, uniform 14 compared with day 3, suitable features of
dispersion of SF and collagen within the scaffold scaffold in inducing the cellular proliferation, and
potential of the scaffold to stimulate the cellular
infiltration and migration
PVAg/SF/HAph Freezing and thawing – No agglomeration and uniform distribution of silk – (Zhang et al., 2012)
in the composite hydrogel, high impact of silk on
elastic modulus of PVA-HAp-Silk composite
hydrogel

a
Silk fibroin.
b
Hydroxyapatite.
c
Matrix Assisted Pulsed Laser Evaporation.
d
Bone marrow stromal cells.
e
Non-mulberry silk fibroin.
f
Chitosan.
g
Collagen.
h
Polyvinyl alcohol.
Biotechnology Advances 36 (2018) 68–91
M. Farokhi et al.
structure of nHAp/SF powder and SF matrix. The good interfacial in-
teractions between the nHAp/SF powders and the SF matrix promoted
cellular proliferation, attachment, and differentiation (Liu et al., 2011).
The superior biological behavior of nHAp powders when compared to
micro-scaled particles provides options for many biomedical applica-
tions. Recently, nHAp particles 30–100 nm were homogeneously dis-
persed in a collagen/silk fibroin (Col-SF) matrix and the electrostatic
interaction between Ca2 + of the inorganic phase and carboxyl or amino
groups of organic phase generated nanocomposites (Col-SF/HAp) with
suitable structural properties and biocompatibility. Additionally, the
existence of SF in the structure of nanocomposites enhanced elastic
modulus in comparison to Col/nHAp (Chen et al., 2014b). In another
study, it was observed that SF affected the mineralization of HAp na-
nocrystals. SF was able to significantly increase the growth of HAp
nanocrystals at pH 8 in room temperature. The strong chemical inter-
action between HAp and the SF protein shifted the amide II peak from
1517 to 1539 cm− 1 in FTIR spectra, which indicated that HAp crystals
were replaced by carbonated HAp (Kong et al., 2004). SF not only in-
fluenced the morphological aspects of HAp crystals but also affected the
water dispersibility of HAp. Moreover, SF surrounded HAp nanocrystals
to form a layer with a negative charge to prevent aggregation of nHAp
particles in aqueous solution (Huang et al., 2014). Previous studies also
confirmed the regulatory activity of SF on biomineralization of calcium
salts (Takeuchi et al., 2005; Wang et al., 2012). However, the distance
between SF and HAp nanocrystals played an important role in the in-
tensity of their interactions. At longer distances, the interactions be-
came weaker (Zhang et al., 2013). Thus, SF and nHAp particle ratios are
important factors in controlling the growth of nanocrystals and crystal
morphology. Accordingly, regular SF nanostructures were implemented
in order to evaluate their effect on the formation and morphology of
HAp nanocrystals. SF nanospheres induced the formation of rice-like
nHAp, while SF nanofibers stimulated the formation of HAp nanofibers
(He et al., 2012a). Therefore, it was suggested that different nanos-
tructures of SF can be considered as templates for the formation of
different morphologies of HAp. Silk fibroin/sodium alginate (SF/SA)
hydrogels induced the formation of HAp nanorods with rectangular
column morphologies at room temperature. The strong interaction be-
tween SF/SA nanofibers and Ca2 + ions in HAp had significant effect on
the morphology of nanocrystals (Ming et al., 2014a). Generally, the
phosphorylated motifs on the surface of SF play an important role in
triggering the nucleation and formation of apatite crystals. Each of
apatite crystals grew in size to form a thick mineralized layer during
mineralization. Adding foreign molecules may trigger the initial for-
mation of HAp crystals; however, mineral proliferation may be delayed
due to inhibition of the growth sites (Boskey, 1998; LeGeros, 1990).
Furthermore, one of the main challenges of using biomaterials to induce
the process of bone regeneration is the bacterial resistance before
complete bone repair. Thus, the biomaterials should have the ability to
stimulate the regeneration before the adhesion of bacterial species. For
this purpose, SF/nHAp hydrogen was modified with gold nanoparticles
(AuNPs) and AgNPs in order to induce the antimicrobial effect (Ribeiro
et al., 2017). The obtained results revealed that the hydrogel containing
high concentration of AgNPs had strong capability to reduce the
number of different gram positive and gram negative bacterial strains
such as S. aureus (MSSA), S. aureus (MRSA), S. epidermidis, Escherichia
coli (E. coli), and P. aeruginosa. However, the hydrogen comprising
AuNPs did not show a linear antimicrobial effect against S. epidermidis.
The hydrogels containing AuNPs ≥ 0.5% had suitable antibacterial
activity toward MRSA and P. aeruginosa; while, those containing
AuNPs ≥ 0.1% reduced the number of MSSA and E. coli. It seems that
the higher surface activity of AgNPs in comparison to AuNPs might be
responsible for this observation (Ribeiro et al., 2017).
Taken together, the biological HAp existed in the natural bone has
nanometric thickness and length with nanoscopic plate-like or rod-like
shape. Thus, applying nHAp would be a good bone substitute with high
regeneration capacity that highly mimic the mineral phase of natural
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Biotechnology Advances 36 (2018) 68–91
bone tissue (Vallet-Regi and González-Calbet, 2004). nHAp possess
great characteristics compared with HAp such as high sinterability and
densification as a result of high surface area that lead to improved
mechanical strength and toughness (LeGeros, 1993). Additionally, the
appropriate biocompatibility, osteoconductivity, the ability to promote
the angiogenesis, and inducing the attachment, migration, and differ-
entiation of osteoblast make it useful for bone regeneration (Hassan
et al., 2016). However, nHAp is fragile, less flexible with intrinsic
hardness that make it difficult to shape into the desired form which
limits its applications in repairing load-bearing bone defects. In order to
hamper the aforementioned drawbacks, nHAp in blended with other
polymers such as SF to form nanocomposites with high osteo-
conductivity suitable for orthopedic surgery (Sun et al., 2011).
4.3. Silk nanofibers containing hydroxyapatite for bone tissue engineering
application
The high surface area of nanofiber scaffolds makes them useful for
protein absorption and binding to cell membrane receptors. The ab-
sorbed proteins can change the structural conformation of the na-
noscale scaffolds along with generating more binding sites that make
them more suitable for tissue engineering. Electrospinning is one of the
most useful techniques for fabricating nanofibrous scaffolds.
Electrospun mats are 3D structures with appropriate porosity and high
surface area to volume ratio that mimic to some extent the structure of
the ECM. Therefore, electrospun nanofibers are considered as potential
scaffolds for tissue engineering applications due to their ability to
support cellular proliferation, adhesion, and differentiation (Agarwal
et al., 2008; Liang et al., 2007). However, using electrospun SF nano-
fibers is still limited for bone tissue applications because of their low
mechanical properties. In order to overcome this limitation, inorganic
ceramics can be incorporated into the polymeric matrix. However, the
aggregation of a ceramic particle in the SF matrix can interrupt the
electrospinning process. Therefore, further investigations are needed to
fabricate appropriate SF/HAp nanofibers for bone tissue engineering
purposes. In several studies, the aggregation of HAp particles in SF/HAp
nanocomposites was reported (Kim et al., 2008; Li et al., 2006; Zhang
et al., 2010). The aggregation can reduce the mechanical strength of the
SF/HAp composites (Kim, 2007; Song et al., 2008a) and can result in
non-uniform physical and chemical structures in these scaffolds.
A useful strategy for enhancing the uniform dispersion of HAp
particles in the SF matrix is the surface modification of the SF polymer
by γ-glycidoxypropyltrimethoxysilane (GPTMS). The epoxide func-
tional groups of GPTMS are able to attach to amino groups in the side
chains of SF. Concomitantly, another side group of GPTMS can interact
with the hydroxyl groups on the HAp surface (Arafat et al., 2011; Kim
et al., 2014b). Electrospun SF nanofibers containing < 20% of well-
dispersed nHAp modified by GPTMS showed high mechanical strength
for load bearing applications. However, higher concentrations of HAp
(> 20%) disrupted the polymeric chains that affect the mechanical
behavior of the scaffolds (Kim et al., 2014b). Similarly, using > 20%
HAp content showed decreased bending modulus due to local ag-
gregation of nHAp (Yang et al., 2016).
Electrospinning of SF/HAp blends faces another limitation such as
immediate precipitation of nHAp before ejection. A three-way stopcock
connector was developed for rapid mixing of various polymeric solu-
tions and then rapidly injecting under the electric field to form blended
nanofibers (Sheikh et al., 2013). Based on FTIR analysis, the chemical
interaction between SF and nHAp changed the conformation of SF from
random coil to β-sheet, which improved the mechanical strength of SF
nanofibers (Sheikh et al., 2013). It was also showed that combining
electrospun SF nanofibers with high pure HAp and silver nanoparticles
(AgNPs) produced a suitable scaffold with antimicrobial properties for
tissue engineering (Sheikh et al., 2014). AgNPs enhanced the anti-
microbial activity of nanofibrous structures. SF nanofibers containing
0.5% silver nitrate showed no toxicity toward NIH 3 T3 fibroblast cells;
M. Farokhi et al.
however, using higher concentrations (1.0 or 1.5%) of silver nitrate
revealed typical toxicity (Sheikh et al., 2014). New biocomposites based
on electrospun SF nanofibers containing mesoporous bioactive glass/
hydroxyapatite nanocomposite (MGHA) were reported (Liu et al.,
2014). Recently, mesoporous bioactive glass (MBGs) has been in-
troduced as potential candidates for drug delivery and bone tissue en-
gineering applications. The ternary SiO2–CaO–P2O5 structure of MBG
possess useful properties such as high surface area, degradability, and
large pore volume in comparison to non-mesoporous bioactive glasses
that make them suitable for inducing bone formation (Wu et al., 2013;
Xia and Chang, 2006). Moreover, adding MGHA particles into SF so-
lution increased fiber diameter related to conductivity affecting fiber
diameter and particle dispersion. The decrease in the conductivity of
the blend solution was controlled by using higher MGHA content and
less SF solution in the matrix. Using MGHA as a reinforcing component
in the structure of SF-based nanocomposites improved swelling, surface
hydrophilicity, and pore size, while tensile strength decreased. SF/
MGHA scaffolds induced the formation of larger bones than pure SF
after 4 and 8 weeks post-implantation. Additionally, in both implants,
new bones containing osteocytes were directly formed near the scaf-
folds. The capability of SF/MGHA composites in bone regeneration may
be related to the chemical interactions between the implant and host
tissue, along with their surface bioactivity (Liu et al., 2014).
In a recent study, coaxial electrospinning was applied in order to
prepare a core-shell structure consisting of HAp and tussah silk fibroin
(TSF) (Shao et al., 2016b). The crystalline region of TSF has a higher
amount of alanine (Ala) in comparison to B. mori silk; therefore, Ala
repeats are the main sequences in the structure of TSF, while domes-
ticated silk is mostly comprised of Gly-Ala-Gly-Ser repeats. In addition,
the existence of an Arg-Gly-Asp motif in TSF structure induced specific
cellular attachment, useful for biomedical purposes (He et al., 2013;
Zhang et al., 2009). An increased content of nanoparticles in the core of
prepared fibers increased the tensile strength. In other words, the
content of core nanoparticles can be used to gradually change the
mechanical properties of the composite from soft to rigid. A similar
trend was also seen in the Young's modulus of the composites. This
scaffold showed promising properties for bone tissue engineering ap-
plications due to potential in inducing cellular proliferation, attach-
ment, ALP production, and biomineralization (Shao et al., 2016b). It
was also found that the robust interaction between nHAp and SF na-
nofibrills induced the formation of flower-like structure that potentially
facilitated the mineralization of HAp in situ. Moreover, the good me-
chanical properties and thixotropy with storage modulus (G’) make SF/
HAp hydrogel useful to recover to over 85% in 50 s after applying a
large shearing strain (5000%) (Mi et al., 2016).
Generally, it is necessary to use nanofibrous structure for bone
tissue engineering applications due to the nanometric nature of ECM
components existed in the natural bone. The bone is a natural nano-
composite comprising nanoscale HAp with about 20–80 nm in length
that are dispersed in an organic collagenous matrix (Rho et al., 1998).
The individual collagen chains have the length about 10 nm which
reach to 500 nm after collagen fiber formation. Thus, using nanoscale
bone constructs not only mimic the structure of natural bone but also
can provoke the cellular activities in terms of adhesion, growth, dif-
ferentiation, and expression of various proteins (Jiang et al., 2015).
Despite the potential of electrospun mats in resembling the structure of
collagenous fibers, the weak mechanical behavior compared with
porous materials might be challenging for bone tissue engineering ap-
plications. Therefore, it is crucial to find a way for increasing the me-
chanical strength of fibrous SF nanofibers to reach to adequate ten-
sional or compressional stresses similar to skeletal bone (Fu et al., 2011;
Wei and Ma, 2004). It seems that addition of ceramic phases in SF fibers
are good strategies to improve the mechanical properties of fibrous
scaffolds (Ito et al., 2005).
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Biotechnology Advances 36 (2018) 68–91
4.4. Silk fibroin/hydroxyapatite for drug delivery in bone tissue engineering
To date, many attempts have been made to develop an appropriate
carrier based on novel materials for drug delivery purposes. These
systems control the release rate of drugs in a more sustained manner
that can be helpful for decreasing side effects. Different structures such
as membranes, micro- and nanoparticles, fibers and three-dimensional
scaffolds have been introduced as carriers for different biomolecules
(Balmayor, 2015; Zarrabi et al., 2014). Most of these structures are
mainly comprised of polymers; however, using inorganic materials can
be also useful.
Bioceramics are attractive inorganic materials to incorporate
bioactive molecules or even cells, without denaturation or loss of
bioactivity during preparation or implantation (Ginebra et al., 2012).
Large surface area, narrow pore size distribution, and well-ordered pore
systems make CaPs useful candidates for drug delivery (Bose and
Tarafder, 2012; Vallet-Regí et al., 2007). Moreover, CaP is useful for
controlling the local release rate of antibiotics (Arcos and Vallet-Regí,
2013; Verron et al., 2010), anticancer, anti-inflammatory, and analgesic
drugs (Ginebra et al., 2006a) and growth factors (Bose and Tarafder,
2012; Ginebra et al., 2006b). Oral administration of CaPs is impossible
because of rapid degradation at low pH of the gastric system. Therefore,
drug delivery systems for the intestinal system (pH 8–9) with a con-
trolled and targeted release profile are useful. However, current in-
vestigations have been focused more on drug delivery to locally or di-
rectly to bone tissue or as coatings (Kolmas et al., 2016).
CaPs also absorb many chemical molecules on their surface, which
is useful for applications such as chromatography, and different bio-
molecules isolation and purification (Ginebra et al., 2006a; Tiselius
et al., 1956). For instance, this criterion has been used for purification
of bone growth factor through HAp chromatography (Urist et al.,
1984). The high affinity of HAp to different biomolecules can provide
options for developing novel matrixes for drug delivery systems.
However, applying HAp-based carriers faces challenges due to their low
capacity for loading drugs and the inability to control the release ki-
netics of such biomolecules. In contrast, silk is a useful biomaterial with
high drug loading capacity and capability in controlling the release rate
of drugs. Different drugs and biological macromolecules can be also
loaded on silk-based structures without changing their bioactivity
(Kundu et al., 2010; Shi et al., 2013a). For this reason, considerable
efforts have been devoted to developing combinations of the intrinsic
bone regeneration potential of HAp blended with SF, with the ability to
incorporate drugs or other active molecules relevant for different
therapeutic needs.
Electrospun SF fibers containing nHAp/bone morphogenetic protein
2 (BMP-2) were used to induce bone formation in vitro using human
bone marrow-derived mesenchymal stem cells (hMSCs) (Li et al., 2006).
The authors found that it was impossible to electrspin the SF solution
with the concentration > 18% because the insufficient viscosity and
surface tension of this solution. Higher concentrations of SF solution
induced the gel formation during the electrospinning process. Fur-
thermore, nHAp had a tendency to form aggregations after suspending
in aqueous media and to overcome to this issue the SF and polyethylene
glycol (PEG) solutions were prepared in 0.001 M phosphate buffer
(pH 6.8) that reduced the aggregation. It was observed that the nHAp
were effectively embedded in the SF/PEO fiber scaffold. The nano-
particles showed different fashions in dispersing in the polymeric ma-
trix in which they were well-oriented along the fiber axis in some re-
gions while agglomerated in the other parts. It seems that high viscosity
of SF/PEO blend solution led to non-homogenous dispersion of in-
dividual particles. Moreover, this study revealed that co-processed
BMP-2 supported more calcium deposition and more upregulation of
bone-specific markers (BMP-2, Col I and bone sialoprotein-II) in com-
parison to control groups. This observation suggested that silk nanofi-
bers were useful for BMP-2 delivery, which broadened the application
of this biomaterial in bone tissue engineering applications. Based on
M. Farokhi et al.
XRD spectra, the crystallinity of apatite on SF/BMP-2 scaffolds was
higher than bulk SF scaffolds. Additionally, the incorporation of nHAp
with SF/BMP-2 during electrospinning induced bone formation (Li
et al., 2006). In another study, silk was used as a surface stabilizer and
template in order to enhance surface properties of nHAp (Ding et al.,
2016a, 2016b, 2016c). The core-shell structure of these composite
materials facilitated the interaction between silk on the outer surface of
nanoparticles with BMP-2 resulting increase the loading capacity of
nHAp. This study showed that removing silk from nHAp had no effect
on the morphological aspects of nHAp; however, aggregation particles
were observed because of the elimination of repulsive forces from SF.
Several studies confirmed that the high loading capacity of silk was
related to electrostatic interactions with BMP-2 (Shi et al., 2013a; Shi
et al., 2013b). Moreover, HAp particles alone showed 90% release of
BMP-2 during 2 h, it was about 50% and 70% for silk alone and silk-
nHAp composite groups after 21 days, respectively. Despite the faster
release kinetics of BMP-2 from silk-nHAp compared to silk particles, the
trend was linear. Moreover, BMP-2 loaded silk-nHAp showed more
potential for inducing osteogenesis from bone mesenchymal stem cells
(BMSCs) in comparison to BMP-2 loaded HAp particles or silk scaffolds
(Ding et al., 2016a, 2016b, 2016c).
In a study, a BCP consisting of HAp/β-TCP (60/40% ratio) and
rhBMP-2/SF microsphere was developed which were then integrated
into a BCP/rhBMP-2/SF composite (Chen et al., 2014a, 2014b). Based
on SEM, HAp particles showed a needle-like structure, while β-TCP had
hexahedral-like morphology. The rhBMP-2/SF had the mean diameter
of 398.7 ± 99.86 nm with loading capacity of 4.53 ± 0.08%. For
pore formation, different concentrations of sodium chloride (NaCl; 50,
100, 150, and 200 w/v) were applied. It was observed that using NaCl
with 50, 100 and 150% (w/v) concentrations had no effect on the
structure materials during the desalination process; while, 200% NaCl
easily damaged and degraded into particles or powder. Moreover,
rhBMP-2 showed a dual-phase release profile, with both fast and slow
rates, from the blended composites during 28 days. In the first three
days, a burst and the fast release of protein (about 47.3 ± 3.1%) were
observed. Afterwards, the cumulative release was about 72.2 ± 4.6%
detected between days 3 to 14, and about 90.4 ± 5.3 between days 14
and 28 (Chen et al., 2014a, 2014b). The addition of organic materials
into inorganic phases exhibits many advantages, such as increasing
degradability, promoting migration and recruitment of osteoblast cells
on the surface of the materials, and increasing rates of mineralization.
The efficacy of BCP/rhBMP-2/SF in a sheep lumbar fusion model
was also evaluated (Chen et al., 2015a, 2015b). For this purpose, BCP
and rhBMP-2/SF microspheres were blended to form BCP/rhBMP-2/SF
composites that were implanted into the disc spaces of 30 sheep at the
levels of L1/2, L3/4 and L5/6, randomly. The growth factor exhibited
an initial burst release about 39.1 ± 2.8% during the first 4 days, and
then had sustained release over 28 days (cumulative release
rate ~ 81.3 ± 4.9%). After implantation, the BCP/rhBMP-2/SF
showed significantly better histological aspects such as in semi-quan-
titative CT scores, fusion rates, histologic scores and fusion stiffness in
bending in all directions than the BCP or BCP/rhBMP-2 study groups.
The results recommended that using low doses of rhBMP-2 in the
structure of SF microspheres improved bone fusion in sheep via BCP
constructs (Chen et al., 2015a, 2015b).
Despite the beneficial characteristics of controlled release systems in
biomedical fields, the important parameters for mimicking the natural
process of bone repair remain challenging to emulate. In many ex-
periments, a single growth factor with controlled release kinetics is
used; however, developing a system with the ability to control the re-
lease of multiple growth factors is more useful for bone regeneration
(Kolambkar et al., 2011). Therefore, attempts have been pursued to
develop delivery systems containing multiple growth factors for the
synergistic effect on bone healing (Chen et al., 2010; Farokhi et al.,
2016b; Shah et al., 2011).
The electrospinning method was used to produce nanofibrous NSF/
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Biotechnology Advances 36 (2018) 68–91
PCL for dual delivery of rhBMP-2 and TGF-β (Bhattacharjee et al.,
2016b). The 4-methacryloyloxyethyl trimellitate anhydride (4-META)
was used to modify the NSF-PCL matrix and the nHAp were deposited
on them by electrodeposition at two separate potentials (3 V and 5 V).
The strong ionic interaction between nHAp and 4-META promoted the
deposition of nHAp on the NSF-PCL matrix. The rhBMP-2 and TGF-β
with a 1:1 ratio were loaded on the surfaces of the scaffold. After
4 weeks, rhBMP-2 and TGF-β exhibited about 20.02% and 20.93% re-
lease kinetics from NSF-PCL scaffolds, respectively. It seemed that the
covalent interaction between the growth factors and HAp nuclei re-
duced the release kinetics. This structure had many favorable properties
for bone tissue engineering because it contained SF with RGD motifs
with the ability to induce cellular attachment and proliferation, calcium
phosphate deposition with high potential in promoting the proliferation
of osteoblasts, and the ability for dual growth factor delivery. The re-
sults also revealed that the prepared structures increased ALP produc-
tion, intensified mineralization and improved the expression of bone-
related genes. Random actin-stress fibers with dense cell colony de-
posits were observed across the nanofibers loaded with dual growth
factors via confocal laser microscopy. However, these outcomes were
lower in the single growth factor-loaded scaffolds, which confirmed the
potential utility of dual growth factor delivery in bone tissue en-
gineering applications (Bhattacharjee et al., 2016b).
Similarly, chemical deposition method was used for preparing nHAp
particles. Electrospun PCL nanofibers were prepared with different
contents of nHAp (0%, 25% and 50%) (Bhattacharjee et al., 2016c).
NSF was then grafted on the nHAp-PCL composites. It was observed
that initial addition of nHAp had a positive effect in increasing the
mechanical strength of NSF grafted PCL nanofibers. However, the
higher tensile strength, toughness, and ductility was detected in those
matrices containing 25% nHAp. Although the samples containing 50%
nHAp showed higher mechanical properties and toughness than NSF-
PCL nanaofibers without nHAp, but it was weaker than samples con-
taining 25% nHAp. In addition, rhBMP-2 and TGF-β were coupled to
the scaffolds by using carbodiimide coupling. The release rate of
rhBMP-2 and TGF-β was about 21.34% and 22.6% after 4 weeks, re-
spectively. These low release rates may be related to the strong inter-
actions between the functional groups of growth factors with active
binding sites of SF. In the first 3 days, a burst release profile of both
growth factors was observed, which may be due to the presence of non-
conjugated or physically conjugated growth factors (about 22%). After
3 days, the remaining growth factors that were covalently bonded to
the scaffolds were slowly released during 4 weeks. The fabricated
scaffolds containing both growth factors provided optimal micro-
environments for cellular migration, proliferation, differentiation, and
enhanced expression of bone specific markers (Bhattacharjee et al.,
2016c). The cross-talk between signaling of these growth factors and
different signaling cascades such as Wnt, Hedgehog, Notch, MAP, and
FGF play important roles in triggering the differentiation of osteoblasts
and consequently, bone formation (Chen et al., 2012). As mentioned
earlier, multiple biomolecules can help to improve the bone healing
process. In addition to growth factor delivery, other biomolecules such
as some drugs would be useful in bone tissue engineering applications.
Recently, silk–HAp films were prepared using casting method for in-
corporation of anti-osteoporotic drugs like clodronate (non-nitrogenous
bisphosphonate) and alendronate (nitrogenous bisphosphonate)
(Hayden et al., 2014). Biological aspects of THP-1 human acute
monocytic leukemia cell line-derived osteoclasts and human mesench-
ymal stem cell-derived osteoblasts such as calcium deposition, meta-
bolic activity and ALP production on the prepared films were evaluated
during 12 weeks. Clodronate-complexed scaffolds showed higher me-
tabolic activity and roughness after culturing osteoblasts and co-cul-
tures of osteoblasts with osteoclasts. Although the low dose of alen-
dronate enhanced the metabolic activity and calcium deposition of
osteoblasts (Fig. 7), some toxicity was observed using the high doses
against osteoclasts, osteoblasts and co-cultures. These data may result
M. Farokhi et al.
Fig. 7. Effects of bisphosphonates on calcium deposition. Calcium content of films was measured after 4, 8 and 12 weeks of culture and expressed as mean ± SD. Top left: osteoblasts
cultured on clodronate-loaded films. Top right: osteoblasts cultured on alendronate-loaded films. Bottom left: co-cultures cultured on clodronate-loaded films. Bottom right: co-cultures
cultured on alendronate-loaded films. ∗P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Copyright © 2014 Elsevier B.V. Reprinted with permission from (Hayden et al., 2014).
from different mechanisms of action of nitrogenous and non‑ni-
trogenous bisphosphonates. The toxicity may be related to drug class.
Nitrogenous bisphosphonates cause apoptosis in various cell types at
concentrations similar to those that cause osteoclast apoptosis, and by
the same mechanisms (Idris et al., 2008). Moreover, the inhibitory ef-
fect of nitrogenous bisphosphonates on mineralization is independent
of the mechanism, which leads to cell apoptosis (Idris et al., 2008).
Additionally, the ALP production was not changed using clodronate;
however, alendronate decreased the ALP activity in a dose-dependent
manner (Hayden et al., 2014).
Generally, drugs can be loaded in CaPs such as HAp by tailoring the
porosity with large surface area during synthesis (Arcos and Vallet-
Regí, 2013; Vallet-Regí et al., 2007). Although high porosity may de-
crease mechanical strength, pores with an appropriate size can act as a
host for drug molecules (Arcos and Vallet-Regí, 2013). The release of
drug can be controlled by altering the amount of porosity of CaP
structures (Kim and Tabata, 2015). Furthermore, a homogeneous dis-
tribution of drug can be achieved in well-ordered pores (Vallet-Regí
et al., 2008). In addition to the beneficial features of CaPs as drug
carriers, nHAp retains an advantage as a major constituent of natural
bones. The degradation of CaP produces Ca2 + and PO43 − ions that are
found in high concentrations in the bloodstream. Moreover, CaPs,
particularly HAp, are relatively insoluble at pH 7.4, thus there is no
immunogenic response, and no cytotoxic degradation products (Bose
and Tarafder, 2012).
4.5. Silk fibroin/hydroxyapatite for stem cell differentiation
Several tissues are important source of therapeutically relevant
differentiated cells but the inevitable difficulties are in harvesting suf-
ficient cells for implantation. Lineages such as neurons and cardiac
cells, being terminally differentiated and non-regenerative, impose the
biggest challenge. Among different stem cell lineages, pluripotent cells
have been focus in biomedical applications due to high self-renewal
capacity, prolonged undifferentiated options, and the ability to differ-
entiate into multiple cell types under suitable stimuli (Zhao et al.,
2013). Adult stem cells are other potential cells useful for tissue en-
gineering because they can fully regenerate damaged tissue and im-
prove the process of tissue repair. Recently, autologous adult stem cells,
such as cardiac stem cells (CSCs), were found favorable when compared
to induced pluripotent stem cells (iPSCs) and embryonic stem cells
(ESCs) for treating myocardial infarction (MI) and chronic heart failure
(CHF) (Shafiq et al., 2016). Moreover, adult stem cells do not face the
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Biotechnology Advances 36 (2018) 68–91
challenges of using ESCs such as ethical, religious and immune rejection
or the high costs of isolating and culturing iPSCs. However, iPSCs and
ESCs are still good candidates for biomedical and tissue engineering
applications due to their beneficial characteristics (Nori et al., 2011;
Song et al., 2010; Takahashi et al., 2007). In recent years, using dif-
ferent stem cell sources for different kinds of tissue engineering pur-
poses has been introduced. Besides, it is more cost-effective to promote
the differentiation of stem cells using appropriate scaffolds without
growth factors for optimal tissue repair. For example, silk/HAp com-
posies have been introduced as a potential scaffold for promoting the
stem cell differentiation. Accordingly, the NSF/nHAp scaffold showed
better biological and physiochemical properties for stimulating the
differentiation of bone marrow-derived stem cells (CBhMSC) into an
osteogenic lineage in comparison to NSF electrospun scaffolds because
the existence of well-dispersed nHAp over NSF matrix. After 24 h, the
cells were attached and aligned along the axis of fibers that form or-
iented morphology. The ability of the scaffold to promote the osteo-
genic differentiation of CBhMSC cells were confirmed by the slight
decrease in the proliferation rate and the increase in the expression
level of ALP and osteogenic genes (Panda et al., 2014). Recently, NSF
composites containing silk fibers and HAp was fabricated as bone
substitutes (Gupta et al., 2016). The HAp was uniformly dispersed in
the structure of highly porous and interconnected composite scaffold
that formed an appropriate substrate for inducing the proliferation and
migration of cells by preparing the suitable environment for transfer-
ring oxygen and nutrients. Moreover, the great mechanical behavior of
the composite provided a supportive matrix for growth and differ-
entiation of MG63 and hBMSCs cells due to high surface area/rough-
ness and osteoconductivity. The osteogenic potential of the scaffolds
were further confirmed by assessment the expression level of osteogenic
genes like Col I, osteopontin, osteocalcin, and sialoprotein, ALP pro-
duction, and ECM deposition (Gupta et al., 2016).
Another study showed that adding NSF (10 wt%) into PLGA/gra-
phene oxide (GO) nanofibers and then mineralization in simulated body
fluid (SBF) can significantly enhance the hemocompatibility, protein
absorption, surface hydrophilicity, and the mechanical properties.
Besides, the conductivity of the electrospinning solution was increased
from 0.39 to 0.78 mS/cm after adding 1 wt% GO into PLGA/NSF na-
nofibers that significantly decreased the fiber diameter from 321 nm to
89 nm. Moreover, biological tests confirmed that scaffolds not only
support hMSC adhesion and proliferation, but also promote osteogen-
esis and ALP production (Shao et al., 2016c).
Similarly, GO-HAp/SF composite was prepared by biomineralizing
M. Farokhi et al.
carboxylated GO sheets, blending with SF, and freeze-drying for eval-
uating the differentiation of MSCs. The GO-HAp (1:4)/SF composites
showed significantly higher mechanical strength in comparison to
HAp/SF scaffolds that had better compressive strength (85 MPa) and
compressive modulus (938 MPa). The protein absorption of GO-HAp
(1:4)/SF were also 1.8-fold higher than HAp/SF scaffolds. Furthermore,
the GO-HAp/SF composite was able to potentially improve the adhesion
and proliferation of MSCs, increase the expression level of osteocalcin,
and differentiation of osteoblast cells (Wang et al., 2017).
Aqueous-derived porous SF scaffolds were fabricated and con-
sidered as templates to deposit apatite on the pore surfaces via mixing
with polyaspartic acid (PA) during processing, followed by miner-
alization with CaCl2 and Na2HPO4 (Kim et al., 2008). The salt-leaching
method using granular NaCl with particle size of 850 ∼ 1000 μm was
used to prepare the scaffolds. Moreover, the ratios of SF-PA (w/w)
blends were as 100/0 (group I), 95/5 (group II), 90/10 (group III) and
80/20 (group IV). The scaffolds were then used for seeding hMSCs
under osteogenic condition in the presence or absence of BMP-2 for
6 weeks. After 24 h, the aqueous solution of SF/PA converted to hy-
drogel and formed a porous and water-stable structure. The apatite
crystals were deposited using salt deposition because PA produced
nucleation sites at the interfaces of the hydrophobic silk structures. It
was found that the increased polyaspartic content increased HAp de-
position on the scaffolds. Accordingly, it is well-established that pro-
teins containing aspartic acid-rich motifs are able to interact with cal-
cium salts in a specific fashion (Addadi and Weiner, 1985; Fujisawa
et al., 1996). It was also showed that the calcium deposition and ALPase
activity were almost the same in mineralized silk scaffolds in the ab-
sence of BMP-2. Moreover, the osteogenic media, whether using BMP-2
or not, also stimulated similar ALPase activity of MSCs after 6 weeks in
groups I, II, and III. The group IV showed higher ALPase activity in the
presence of BMP-2. All the mineralized scaffolds revealed more calcium
deposition with BMP-2, while no calcium deposition was detected in
those scaffolds without mineralization (control groups) after 6 weeks.
Based on van Kossa staining, more calcium deposition led to higher
osteoconductivity of the scaffolds. Immunocytochemistry demonstrated
Col I was expressed in all groups. Additionally, the expression of Col I
was increased in groups II and III in the presence of BMP-2 (Fig. 8). It
was concluded that higher amounts of apatite in the structure of porous
silk constructs along with the existence of BMP-2 increased the osteo-
conductivity of the scaffolds (Kim et al., 2008).
Despite the effectiveness of using silk/HAp as bone constructs, the
injectable form of bone materials is less investigated. The main draw-
back of bone substitutes is the lack of suitable structure for filling the
irregular bone defects. To provide an injectable biomaterials with
Fig. 8. Immunohistochemical staining of collagen type I. Extracellular matrices (*) stained positive for Col I within the scaffold pores. The Col I matrix covered the entire mineralized
aqueous-based silk lattice (#) after 6 weeks of culture with BMP-2 (100 ng/mL; b, d, f, h) or without BMP-2 (a, c, e, g). Copyright © 2008 Elsevier B.V. Reprinted with permission from
(Kim et al., 2008).
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Biotechnology Advances 36 (2018) 68–91
osteogenic properties, the silk-HAp composite with silk nanofibers in
hydrogels was prepared. For this, an injectable system was fabricated
by blending thixotropic silk nanofiber hydrogels with water dispersible
silk-HAp nanoparticles to form injectable nanoscale systems. The
composites had about 60% w/w HAp content for optimally mimic the
bone environment. The physical strength of the system was also im-
proved to 21 kPa by adding of HAp that was suitable for stimulating the
osteo-differentiation. The osteogenesis was more enhanced by using
composite hydrogels in comparison to silk nanofiber hydrogel without
HAp. The silk-HAp composite hydrogel was able to treat irregular bone
defects after injection (Ding et al., 2017).
The main purpose of tissue engineering is to develop functional
materials with suitable biocompatibility that highly mimics the char-
acteristics of the target tissue. For this, using engineered biomaterials is
of interest due to rational design, adequate biocompatibility and bio-
degradability, tunable structural properties, and functionality.
Recombinant silk fusion proteins are an example of engineered mate-
rials that have these features. Mineral-binding sequences are rich in
acidic residues (aspartate, glutamate, phosphoserines) that can increase
the net negative charge of engineered materials. These sequences can
interact with positively charged HAp nanocrystals (Addison et al.,
2010). Recently, an organic-inorganic hybrid system was developed
based on genetically engineered spider silks. The organic phase of
spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYG-
GLGSQGT)15 maintained the stability of the scaffold and facilitated
different modes of processing; while, the inorganic domain of SF
VTKHLNQISQSY (VTK) for HAp binding was involved in regulating the
osteogenesis and biomineralization. The VTK peptide localized on re-
combinant fusion protein was able to provoke the calcification but it
did not any effect in improving the physical strength of the recombinant
structure. It was also observed that adding VTK peptide on both c-
terminal and N-terminal sides of recombinant silk could significantly
enhance the formation of crystalline HAp. All the prepared constructs
was able to provoke the attachment, proliferation, and differentiation
of hMSCs (Dinjaski et al., 2017). Salt-leached SF scaffolds were also
prepared followed by functionalization and mineralization in CaCl2
ethanol solution/K2HPO4 aqueous solution. The scaffolds were then
mineralized by immersing in the simulated body fluid (SBF) for 4 days.
The authors claimed that the duration of soaking in SBF would be de-
creased by using this method that consequently protect the SF-nHAp
scaffold from degradation during the mineralization process. The mi-
crospores of nHAp with a size range of 100 to 250 nm were formed on
the surface wall of SF. Incorporating of nHAp particles with the SF
scaffolds significantly improved the compressive strength and stiffness
of SF (Liu et al., 2015a, 2015b). Furthermore, the cDNA microarray
M. Farokhi et al.
demonstrated changes in 23 calcium ion binding genes and 17 cell
adhesion genes of BMSCs seeded on nHAp-SF scaffolds compared with
bulk SF constructs. Among these genes, about 15 genes corresponded to
cellular adhesion and 20 genes were attributed to calcium ion binding
were decreased in expression level. Based on these observations, the
cultured BMSCs on nHAp-SF scaffolds had round shapes due to less
surface adhesion. The nHAp-SF scaffolds showed higher os-
teoinductivity in vitro, significantly higher ALP activity on day 14, and
more osteogenesis related to signaling pathway genes on day 7 com-
pared with the SF scaffolds. Moreover, the nHAp-SF scaffolds demon-
strated higher capability to induce bone regeneration in cranial bone
defects than bulk SF scaffolds. It was also shown that soluble IL-1a
factor and biomolecules on the surface of BMSCs were played an im-
portant role in provoking osteogenesis. Upon interaction between
BMSCs and nHAp, BMSCs regulated the activity of IL-1a by IL IR2. This
study helped to understand better the possible mechanism of os-
teoinductivity by nHAp (Liu et al., 2015a, 2015b).
Electrospun SF nanofibers containing nHAp and BMP-2 were pre-
pared as bone construct with the ability to induce the differentiation of
BMSCs (Niu et al., 2017). The nHAp particles were successfully dis-
persed in SF fibers. The increase in the content of HAp resulted in more
aggregations of this particles in the SF matrix. The mechanical prop-
erties of the composite containing 20% HAp such as tensile strength,
yield strength and Young's modulus were almost two-fold more than
pure SF scaffolds. BMSCs showed significantly more ALP activity on SF/
HAp/BMP-2 scaffolds compared with SF scaffolds. Thus, SF/HAp/BMP-
2 scaffolds were considered as a potential structure in inducing osteo-
genesis (Niu et al., 2017). It is assumed that synergistic effect of HAp
and BMP-2 improves the osteogenic capacity of the composite scaffold.
This issue was also confirmed by Lu et al. (2015) or Liu et al. (2015a,
2015b)) claimed that degradation of SF can increase the exposure of
HAp to the cells during time.
Similarly, the bioactivity and the sustained release rate of BMP-2
was preserved in the nHAp-embedded SF scaffold by adjusting the ratio
of loaded BMP-2 on SF and nHAp. The suitable BMP-2 release behavior
inside the structure of SF-nHAp scaffold increase the expression level of
Col I, ALP and Runx2 protein that resulted in higher osteo-conductivity.
Based on the results from in vitro and in vivo, the release of BMP-2 had
positive effect in inducing osteogenesis of the scaffold (Ding et al.,
2016a, 2016b, 2016c).
Shen et al. fabricated porous SF/nHAp composites containing BMP-
2 and SDF-1 loaded SF microspheres to synthesis the cell-free scaffold.
Laminar jet break-up technology was used for SF microsphere pre-
paration which provides optimal condition for high encapsulation ef-
ficiency (Shen et al., 2016). It was reported by Wenk et al. that pre-
paring SF microspheres using this method can preserved the bioactivity
of IGF-I and controlling the release rate of growth factor in a sustained
manner (Wenk et al., 2008). SDF-1 showed a rapid release rate during
the first few days, while it presented a sustained profile in the following
three weeks. The scaffold significantly provoked the osteogenic differ-
entiation and recruitment of BMSCs (Shen et al., 2016).
In another study, electrospun SF nanofibers containing HAp were
fabricated for periosteum regeneration (Ding et al., 2016c). Periosteum
is the main source of osteo-progenitor cells around bone, therefore, this
tissue plays a crucial role in the initial phase of bone formation and
regeneration (Tiyapatanaputi et al., 2004; Zhang et al., 2005a). The
HAp particles were seen on the surfaces of SF nanofibers containing
10%, 20%, and 30% HAp. Moreover, more HAp was observed on the
scaffolds with higher content of HAp. Some aggregations of HAp par-
ticles were observed on the surface of interconnected pores of SF/HAp
nanofibers which may be responsible for increasing the diameter of
10%, 20%, and 30% SF/HAp nanofibers. The Young's modulus of the
scaffolds was also increased with the increase in the content of HAp.
Moreover, the results demonstrated that SF scaffolds containing 30%
HAp induced the metabolic activity of rBMSCs more than those scaf-
folds containing 0% HAp after 4 days. SF scaffolds containing 30% HAp
85
Biotechnology Advances 36 (2018) 68–91
promoted the proliferation of rBMSCs and upregulated mRNA levels of
ALP and Runx2 more than the other groups. Moreover, rBMSCs showed
higher levels of ALP expression when cells were cultured on SF scaffolds
containing 30% HAp in comparison to 0% HAp-SF scaffolds. Higher
calcium deposition was found by Alizarian red assay when 30% HAp-SF
scaffolds were used versus 0% HAp-SF scaffolds (Ding et al., 2016c).
Therefore, it was suggested that SF scaffolds containing 30% HAp were
potent structures to promote bone formation due to their ability to
induce mineral deposition and consequently, cell differentiation.
Co-cultures of MSCs between ligament and bone cells on suitable
sections of hybrid silk scaffolds induced cellular differentiation into
fibrocartilage lineage (He et al., 2012b). The section of the scaffold for
seeding osteoblast cells was pre-coated with HAp. Real-Time PCR re-
vealed that both osteoblast and fibroblast cells maintained their normal
phenotype during co-culture. However, co-cultured BMSCs differ-
entiated toward the fibrocartilage lineage. The co-cultured BMSCs on
the hybrid SF scaffolds exhibited up-regulation of Col II, Sox9, aggrecan
and fibrocartilage gene markers in comparison to mono-cultured
BMSCs. The data were confirmed by histological analysis and im-
munohistochemistry. The immunohistochemistry staining revealed that
aggrecan was the most prominent marker in the enthesis (the con-
nective tissue between tendon or ligament and bone). In addition, the
trilineage co-culture was supplemented with 10 ng/mL TGF-β3 and the
fibrocartilaginous differentiation of BMSCs by the TGF-β3-treated
group was similar to those groups without TGF-β3. No calcium de-
position was observed in the TGF-β3-treated group and it was suggested
that TGF-β3 could postpone the process of mineralization in chon-
drocytes (He et al., 2012b).
In another study, leaching paraffin microsphere with the modified
temperature gradient-guided thermal-induced phase separation (TIPS)
methods were applied to fabricate SF/HAp scaffolds with the potential
to mimic the integrated trilayered osteochondral substrates (Ding et al.,
2014). Based on the Micro-CT analysis, the consecutive tri-layer
structure consisting a chondral (top) layer, intermediate layer, and
bony (bottom) layer was observed in the integrated scaffold. Using TIPS
method resulted in the formation of oriented microtubularlike porous
structure in the chondral layer and the paraffin-sphere leaching led to
formation of an interconnected macropore structure in the bony layer.
The chondral and bony layers had porosities of 85.30% ± 1.80% and
90.25% ± 2.05%, respectively. Adipose-derived stem cells (ADSCs)
were used under chondrogenic (with TGF-β1 and IGF) and osteogenic
(with dexamethasone) conditions in chondral and bony layers of the
scaffold in order to evaluate the potential of these cells for differ-
entiation to cartilage and bone lineages. In the chondral layer which
was previously seeded with ADSCs, chondrocyte-like cells were ob-
served, which were enclosed by ECM components, especially collagen
type II and glycosaminoglycan (GAG). The amounts of ECM compo-
nents were also increased during 21 days in the chondral layer. More-
over, chondrogenic-related genes were also up-regulated during cul-
ture. Generally, it was proposed that ADSCs seeded chondral layer
provided suitable environments for inducing chondrogenesis under
chondrogenic conditions. The ADSCs also showed higher expression of
osteogenic related genes and bone ECM components such as Col I and
calcium. Therefore, osteogenic differentiation also could be promoted
under osteogenic conditions. Consequently, the pores were filled with
more cartilage-related and bone-related matrices as the culture time
increased, resulting in an increase in the compressive elastic modulus
(Ding et al., 2014). The results showed promise for the use of novel 3D
integrated SF scaffolds in osteochondral tissue engineering in vitro.
However, whether ADSCs can differentiate into cartilage and bone in
the corresponding chondral and bony layers of integrated scaffolds
needs to be further investigated through in vivo implantation. The
isolating role of the intermediate layer needs to be confirmed in vivo.
Other important studies using SF/HAp for stem cell differentiation are
listed in Table 5.
Totally, primary osteoblast cells are the mostly used cells for bone
 Table 5
M. Farokhi et al.

Simultaneous use of SF/HAp composites and stem cell for bone tissue engineering.

Material Processing method Structure and properties of the scaffold Cell Key findings Ref.

SFa/HApb Direct-write assembly Formation of 3D scaffold with an interpenetrating gradient network of hMSCsc/ Formation of complex network of hMSCs matrix within the 3D scaffold ((Sun et al., 2012)
pore channels, suitable mechanical integrity hMMECsd by hMSCs and hMMECs based on histological analysis, observation of
vascular-like structures only in hMSCs and hMMECs co-culture groups
SF/HAp Freeze-drying/co- Formation of dense flake-like HAp crystals on the surface and the pore BMSCse Increasing the surface roughness and proliferation of BMSCs by (Jiang et al., 2013)
precipitation walls of the 3D silk structure incorporating HAp in SF matrix, improved in vitro differentiation of
BMSCs toward osteoblast cells
SF/nHAp Graft polymerization High and uniform coupling of nHAp on the surface of nano-HAp/SF MMCsf Good cellular proliferation and attachment, higher ALPg and bone- (Tanaka et al., 2007)
sheet specific osteocalcin production under osteogenic condition after
14 days of cell cultured on nano-HAp/SF sheets compared with controls
SF/HAp Salt-leaching Altered surface chemistry, increased surface roughness and stiffness hMSCs Higher surface osteoconductivity of SF scaffold by using HAp and (Bhumiratana et al.,
after incorporating HAp in the structure of 3D scaffold suggesting HAp microparticles as nucleation sites for directing 2011)
mineralization, enhancing the formation of trabecular structure,
increasing the mechanical behavior and conductivity of tissue grafts
SF/Gelatin/ Salt-leaching/ Formation of 3D scaffold with BMSCs/ Higher proliferation rate of mouse MC3T3-E1 cell line seeded on the (Sinlapabodin et al.,
HAp co-precipitation approximately 52 ± 5.5% porosity and pore size of 52.3 ± 5.5 MC3T3E1 scaffolds with perfusion flow rate of 1 mL/min than the static culture, 2016)
increased differentiation of BMSCs into osteoblast using perfusion flow
rate of 3 mL/min compared to 1 and 5 mL/min flow rates, statistically
higher Ca/P ratio, ALP activity, and calcium contents in flow rate of
3 mL/min compared to other flow rates
Ti/SF/nHAp Co-precipitation/ Induction of HAp nanocrystals aggregation in the presence of SF, intact MG-63 Better cellular attachment and osteogenic differentiation based on ALP (Lin et al., 2015)
electrostatic spray crystalline structure of HAp in the structure of SF/nHAp composites activity, BGPh contents, and Col Ii of cells in SF/nHAp compared with
deposition nHAp groups

86
SF/HAp Freeze-drying Formation of 3D structure with interconnected pores, varied pore size hDPCsj/ Remaining mostly SF scaffold after 4 weeks at the defected site, (Park et al., 2015)
between 20 and 80 μm with the diameter of 65 μm hPDLCsk supporting cellular attachment on SF scaffold and producing
extracellular matrix, inducing new bone formation in the edges of
defects by using SF fibers
SF/PHBVl/HAp Electrospinning Reduction in the tensile Young's modulus of the composite fibers with MC3T3-E1 Increased cellular proliferation and attachment on composites (Paşcu et al., 2016)
the increase in the content of the nHAp and SF phase from 2% to 5% compared with PHBV control, higher expansion and elongation of cells
on the surface of fiber composites after 1 day of culturing, incomplete
elongation of cells seeded on PHBV scaffolds
SF/HAp Salt-leaching – PMSCsm Secretion of calcium crystals by exposing PMSCs into osteogenic (Jin et al., 2014)
medium, secreting extracellular matrix when cell cultured on SF/HAp
scaffolds, increasing tissue regeneration by using PMSCs seeded SF/
HAp scaffolds in radius segmental bone of rabbit

a
Silk fibroin
b
Hydroxyapatite
c
Human bone marrow derived mesenchymal stem cells
d
Human mammary microvascular endothelial cells
e
Bone marrow derived mesenchymal stromal cells
f
Marrow mesenchymal cells
g
Alkaline phosphatase
h
Bone gla protein
i
Collagen type I
j
Human dental pulp cells
k
Human periodontal ligament cells
l
Polyhydroxybutyrate–polyhydroxyvalerate
m
Placenta-derived mesenchymal stem cells.
Biotechnology Advances 36 (2018) 68–91
M. Farokhi et al.
regeneration; however, their limited number and dedifferentiation
properties might limit applications. Using potential stem cells can
overcome these drawbacks (Wang et al., 2006). Stem cells have been
largely investigated for creating functional tissues and organs but it is
still at infant stage. Bone marrow is the favored source of MSCs for both
experimental and clinical application. The biological features of stem
cell such as self-renewal and differentiation capacity are regulated by
ECM components. ECM also serves as a supporting structure for various
growth factors and biomolecules which can act as structural support
with the ability to control signal transduction (Kundu and Kundu,
2010). The suitable morphological aspects of SF/HAp composites make
it an appropriate structure for stimulating the differentiation of stem
cell into desired lineage and thus promoting the regeneration of de-
fected tissue.
5. Conclusions and future perspectives
Silk scaffolds are biocompatible structures with tunable properties
in terms of morphology, degradability, and conformation that make
them useful candidates for tissue engineering applications. Recently,
investigations have been performed to better understand the structure
and processing of silk-based structures that broadened the applications
of different types of silk in regenerative medicine. The unique char-
acteristics of silk such as exceptional mechanical and physical behavior,
and versatile processability to form different structures make it poten-
tially useful for ligament, bone and musculoskeletal tissue engineering.
Silk has many suitable properties for various applications; however, it
has some limitations. For example, the properties of silk protein, as a
natural polymer, are different based on species source. Moreover, in-
consistence degumming processes and different fabrication methods
may produce silk structures with various properties if the process is not
tightly controlled. It is well established that genetically modified silk
proteins may overcome the mentioned limitations pending scale up and
cost efficiencies. In order to enhance the potential of silk-based scaf-
folds for bone tissue engineering applications, this polymer can be
blended with other materials such as HAp to enhance osteogenic po-
tential of the scaffolds. HAp has a similar chemical structure to mi-
neralized bone of human tissue. This ceramic material demonstrates
affinity to hard tissues. More investigations are to be carried out in
order to understand better the application of nanoscale constructs that
can be degraded over time and replaced by endogenous hard tissues. It
is also necessary to focus on modifying the surfaces of biomaterials with
different biomolecules with the aim of improving their biological be-
havior and interfacial functions. There are many studies that describe
the benefits of using nHAp-SF scaffolds for bone tissue engineering
applications in vitro and in vivo. However, there is no comprehensive
study that shows the adverse effects of using these structures. It is es-
sential to develop effective constructs for clinical use despite advanta-
geous properties of SF/HAp in this area. It is noteworthy that many
investigations have confirmed the potential of SF scaffolds to induce
osteogenesis in vivo. Most of these studies are performed on small an-
imal models, thus the results may not fully be extended for human use.
More investigations are needed to be performed in the future to identify
the requirements of using silk-based scaffolds in clinical trials and
constructing suitable commercialized structures for bone tissue re-
generation.
Acknowledgments
This work is supported by Pasteur Institute of Iran (grant number
984).
Disclosure statement
No potential conflict of interest was reported by the authors.
87
Biotechnology Advances 36 (2018) 68–91
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