Evaluation and Application of Silk Fibroin Based Biomaterials to Promote Cartilage Regeneration in Osteoarthritis Therapy

Abstract

Osteoarthritis (OA) is a common joint disease characterized by cartilage damage and degeneration. Traditional treatments such as NSAIDs and joint replacement surgery only relieve pain and do not achieve complete cartilage regeneration. Silk fibroin (SF) biomaterials are novel materials that have been widely studied and applied to cartilage regeneration. By mimicking the fibrous structure and biological activity of collagen, SF biomaterials can promote the proliferation and differentiation of chondrocytes and contribute to the formation of new cartilage tissue. In addition, SF biomaterials have good biocompatibility and biodegradability and can be gradually absorbed and metabolized by the human body. Studies in recent years have shown that SF biomaterials have great potential in treating OA and show good clinical efficacy. Therefore, SF biomaterials are expected to be an effective treatment option for promoting cartilage regeneration and repair in patients with OA. This article provides an overview of the biological characteristics of SF, its role in bone and cartilage injuries, and its prospects in clinical applications to provide new perspectives and references for the field of bone and cartilage repair.

Full text

Citation: Su, X.; Wei, L.; Xu, Z.; Qin,
L.; Yang, J.; Zou, Y.; Zhao, C.; Chen,
L.; Hu, N. Evaluation and
Application of Silk Fibroin Based
Biomaterials to Promote Cartilage
Regeneration in Osteoarthritis
Therapy. Biomedicines 2023,11, 2244.
https://doi.org/10.3390/
biomedicines11082244
Academic Editor: Elisa Belluzzi
Received: 6 June 2023
Revised: 27 July 2023
Accepted: 29 July 2023
Published: 10 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biomedicines
Review
Evaluation and Application of Silk Fibroin Based Biomaterials
to Promote Cartilage Regeneration in Osteoarthritis Therapy
Xudong Su 1, 2, †, Li Wei 1, 2, †, Zhenghao Xu 1,2, Leilei Qin 1,2, Jianye Yang 1,2, Yinshuang Zou 1,2, Chen Zhao 1,2,
Li Chen 1, 2, *,‡ and Ning Hu 1 ,2 ,*, ‡
1Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University,
Chongqing 400016, China
2Laboratory of Orthopedics, Chongqing Medical University, Chongqing 400016, China
*Correspondence: chenlicq@163.com (L.C.); huncqjoint@yeah.net (N.H.)
†These authors contributed equally to this work.
‡These authors also contributed equally to this work.
Abstract:
Osteoarthritis (OA) is a common joint disease characterized by cartilage damage and
degeneration. Traditional treatments such as NSAIDs and joint replacement surgery only relieve
pain and do not achieve complete cartilage regeneration. Silk fibroin (SF) biomaterials are novel
materials that have been widely studied and applied to cartilage regeneration. By mimicking the
fibrous structure and biological activity of collagen, SF biomaterials can promote the proliferation and
differentiation of chondrocytes and contribute to the formation of new cartilage tissue. In addition,
SF biomaterials have good biocompatibility and biodegradability and can be gradually absorbed and
metabolized by the human body. Studies in recent years have shown that SF biomaterials have great
potential in treating OA and show good clinical efficacy. Therefore, SF biomaterials are expected to
be an effective treatment option for promoting cartilage regeneration and repair in patients with OA.
This article provides an overview of the biological characteristics of SF, its role in bone and cartilage
injuries, and its prospects in clinical applications to provide new perspectives and references for the
field of bone and cartilage repair.
Keywords: silk fibroin; osteoarthritis; bone/cartilage repair; treatment
1. Introduction
Due to the gradual increase in the proportion of the aging population, obesity, and joint
injuries worldwide, the risks of bone and cartilage diseases have significantly increased,
seriously affecting people’s quality of life and physical health [
1
]. About 20~33% of the
world’s population suffers from bone and joint diseases, and their incidence in adults is
30~40% [
2
], among which the incidence of osteoporosis [
3
], bone fractures [
4
], cartilage
wear [
5
], and arthritis [
6
] gradually increases with age. Osteoarthritis (OA) is a progressive
degenerative joint disease and the main cause of disability in adults, characterized by
cartilage and subchondral bone degeneration [
7
]. Generally, cartilage and subchondral
bone degeneration are due to increased metalloproteases and inflammatory cytokines; the
excessive mechanical load and pathological factors cause bone and cartilage destruction,
resulting in an imbalance in the dynamic equilibrium of repair and leading to OA. Thus,
repairing damaged cartilage is a fundamental element in the treatment of OA [8,9].
Biomedicines 2023,11, 2244. https://doi.org/10.3390/biomedicines11082244 https://www.mdpi.com/journal/biomedicines

Biomedicines 2023,11, 2244 2 of 21
Although clinical treatments for bone/cartilage-related diseases caused by OA include
pain management, physical therapy, joint injection, and surgical treatment, they only im-
prove the patients’ pain symptoms and do not achieve the goal of cartilage
repair [10,11]
.
Autologous chondrocyte transplantation and allogeneic/autologous cartilage transplanta-
tion have also been applied in clinical practice, but there are still limitations, such as donor
shortage, rejection reactions, and infections. Secondary surgery, limited collection sources,
and complications often make tissue transplantation ineffective as a clinical treatment,
especially for joint cartilage damage and key-sized bone defects [
12
–
15
]. Autologous and
allogeneic transplantation are common clinical techniques for replacing damaged tissues,
but they are limited by various factors, such as a lack of tissue that can be removed from
healthy areas, and a lack of suitable donors. Allogeneic transplant material from donor
tissue can cause immune responses, and, in cases of extensive injury and large surface
areas, it is difficult to obtain appropriate materials on time, leading to low success rates [
16
].
Tissue engineering (TE) relies on the use of a variety of biocompatible materials to restore,
maintain, and improve tissue function to regenerate injured tissues and organs. These
materials can be seeded with cells and contain various supportive components. In recent
years, TE has gained increasing attention as an alternative method for producing patient-
specific tissues for repair and replacement applications [
17
–
20
], but various biomaterials
have inherent limitations, so finding an excellent biomaterial has become the focus of
research in recent decades [21].
Silk fibroin (SF) is a common natural material with excellent mechanical properties,
low rejection reaction, tunable biodegradability, and good stability in the field of biomedi-
cal engineering, especially in tissue engineering [
22
–
24
]. SF is an important extracellular
matrix protein, widely present in bone and cartilage tissues, and has important biological
functions [25]
. SF plays an important role in tissue engineering. Due to its good biocompat-
ibility and biodegradability, SF can be used to build artificial tissues and organs. It can act
as a scaffold or matrix material to promote cell attachment, proliferation, and differentiation
in vivo
and support new tissues. Secondly, SF also promotes wound healing. It has good
biocompatibility and bioactivity to promote the regeneration and repair of traumatized
tissue. SF can promote angiogenesis, accelerate the wound-healing process, and reduce
inflammation. Therefore, SF has a wide range of applications in treating trauma, burns, and
ulcers. In addition, SF is used in the development of drug delivery systems. Due to its good
degradability and drug-modification properties, SF can be used as a carrier to control the
release of drugs. This property makes SF promising, improving traditional drug-delivery
systems, and developing novel drug delivery technologies. In conclusion, SF has a wide
range of roles in biomedical applications. It can be used in tissue engineering, wound repair,
and the development of drug delivery systems, providing valuable tools and materials for
medical research and clinical practice. [
26
–
28
]. Therefore, SF-based biomaterials have been
used as potential bio-polymer applications in bone/cartilage repair in tissue engineering
(Figure 1) [
29
–
31
]. This paper introduces the biological characteristics of SF, its role in
bone/cartilage injury, and its clinical applications.

Biomedicines 2023,11, 2244 3 of 21
Figure 1.
SF-based biomaterials in cartilage/osteochondral repair. (
A
) Sequential release of E7/KGN
from silk nanosphere matrix in osteochondral defect repair. Reprinted/adapted with permission
from Ref. [
32
], copyright 2020, Elsevier. (
B
) Mechanically strong silica-silk bioaerogel for bone regener-
ation. Reprinted/adapted with permission from Ref. [
33
], copyright 2019, American Chemical Society.
(
C
) Fabrication of silk/calcium silicate/sodium alginate composite scaffolds. Reprinted/adapted
with permission from Ref. [34], copyright 2018, Elsevier.
2. Biological Properties of SF
Silk is currently an SF raw material that can be mass-produced and has been applied
in clinical practice. As a natural fiber material, it mainly comprises two proteins: SF (SF)
and silk sericin (SS). SF accounts for about 75%, and silk sericin accounts for about 25%.
Silk has good biocompatibility and biodegradability [
35
,
36
]. To obtain pure SF, silk needs to
undergo processes such as degumming, washing, and drying [
37
]. SF is a large molecular
protein composed of 18 amino acids, of which glycine, alanine, and serine account for more
than 80% of the total amino acid content [
38
,
39
]. It is widely distributed in the extracellular
matrix and has various biological functions (Figure 2) [37,40].
Figure 2. SF structure and SF-based materials.

Biomedicines 2023,11, 2244 4 of 21
2.1. Structure of SF
The molecular structure of SF has certain characteristics, consisting of a heavy (H)
chain of 390 kDa and a light (L) chain of 26 kDa connected by disulfide bonds, as well as
a glycoprotein (P25/30 kDa) secreted into the posterior silk gland [
41
–
43
]. The H chain
accounts for most of the SF, with an amino acid composition of Gly (46%), Ala (30%),
Ser (12%), Tyr (5.3%), and Val (1.8%) [
44
]. Another 4 kDa peptide encoded by the P25
gene is mainly associated with the H-L complex via hydrophobic interactions [
45
]. The
genes encoding the three peptides are located on different chromosomes but appear to be
coordinately regulated in the posterior silk gland [
46
,
47
]. In addition, interactions between
the H chain and the L chain or P25 are crucial for the secretion of SF [
48
,
49
]. The main
body of the SF chain consists of alternating crystalline and non-crystalline regions [
50
,
51
].
The crystalline region is dominated by the GAGAG sequence (Gly-Ala-Gly-Ala-Ala-Gly-
Ser) with short side chains. The arrangement of amino acid residues in the amorphous
region is complex and contains many amino acid residues with long side chains, such as
tyrosine, lysine, and arginine. These residues are relatively hydrophilic, obstructing the
regular assembly and crystallization of the chain segment, resulting in an irregularly coiled
molecular conformation. The mechanical properties of SF can be regulated by changing
the size, number, orientation, and arrangement of the crystalline (silk) and amorphous
regions [
52
,
53
]. The primary crystal structures of SF are Silk I and Silk II, and the water-
soluble and unstable Silk I can be transformed into Silk II with a
β
-fold structure that is
insoluble in water under certain conditions [54,55].
2.2. Properties of SF
The stability and degradation of SF depend on factors such as temperature, pH, oxida-
tion, enzyme action, light, and humidity. Reasonable control of these factors can prolong
the stability of SF. The biological properties of SF are closely related to its structure [
53
,
56
].
Research has shown that SF can bind to receptors on cell membranes, thereby regulat-
ing biological processes such as cell proliferation, differentiation, and migration [
40
]. In
addition, SF can also regulate the synthesis and degradation of the extracellular matrix,
promoting the reconstruction and repair of the extracellular matrix [
57
,
58
]. In bone and
cartilage tissues, SF is an important component that can regulate biological processes such
as cell proliferation, differentiation, and migration [
59
]. The degradation properties of
biomaterials directly affect the speed and quality of cartilage/osteochondral repair. As a
kind of protein material, the degradation rate of SF-based biomaterials is mainly affected
by proteases. Most proteolytic enzymes tend to degrade non-crystalline SF. This suggests
that SF-based biomaterials with controlled degradability can be prepared by changing the
content of crystalline structures [
31
]. The stability and degradation of SF depend on factors
such as temperature, pH, oxidation, enzyme action, light, and humidity. Reasonable control
of these factors can prolong the stability of SF. Due to its biocompatibility, adjustable degra-
dation, unique biomedical and mechanical properties, ease of processing, and abundant
supply, SF can be processed into gels [
60
], films [
61
], nanofibers [
62
], nanoparticles [
63
],
and other materials that can be widely applied in drug delivery [
64
], tissue repair [
65
], and
other fields.
2.3. Preparation Method of SF-Based Biomaterials
At present, the preparation methods of SF-based biomaterials mainly include 3D bio-
printing, electrospinning, and freeze-drying. The main types of 3D bioprinting equipment
include inkjet printing, extrusion printing, and laser-assisted printing [
66
,
67
]. Bio-inks are
very important in 3D printing [
68
]. In general, when using 3D bioprinting to prepare SF-
based biomaterials, it is necessary to modify SF bio-inks to enhance the biological activity of
the SF, and the mechanical strength of SF-based biomaterials can be overcome by modifying
SF-based bio-inks to meet the needs of cartilage/osteochondral repair [
69
,
70
]. Electrospin-
ning can mix multiple matrices and combine the properties of the matrix to suit the needs
of cartilage tissue engineering [
71
]. In addition, electrospinning can maintain the elasticity

Biomedicines 2023,11, 2244 5 of 21
of SF, which is essential for cartilage/osteochondral cartilage tissue
engineering [72]
. In
the process of preparing biomaterials via freeze-drying, technology freezes the solvent and
then sublimates, with little effect on the solute, so freeze-drying also facilitates the carrying
of drugs and growth factors for SF-based biomaterials [73].
2.4. The Main Types of SF-Based Biomaterials
The main types of SF-based biomaterials are hydrogels, scaffolds, and microcarriers.
The native EMC-like microenvironment of hydrogels is suitable for loading cells to promote
cartilage/osteochondral repair [
74
]. At the same time, the mechanical properties, shape,
and swelling properties of hydrogels will change with the changes in temperature, pH
value, and ion concentration, which can achieve the intelligent release of chondrogenic
and osteogenic drugs and closely fit the interface of cartilage defects to improve the
integration effect [
75
]. Scaffolds promote cartilage/osteochondral repair and regeneration
by providing a specific microenvironment [
76
]. Compared with hydrogels, scaffolds have a
fixed shape and higher mechanical strength, which can be used as an adjunct to cell therapy
to promote cell attachment, growth, and differentiation [
77
]. Microcarriers generally refer
to small spherical scaffolds suitable for cell culture, growth, and transport, as they not
only promote cell growth and maintain the cell differentiation phenotype but also enable
tissue regeneration through direct injection into the target site, enabling microcarriers to
accelerate cartilage/osteochondral cartilage repair [
78
]. At the same time, microcarriers can
be loaded with growth factors to promote cartilage/osteoblast adhesion and growth [79].
3. Osteoarthritic Articular Cartilage Model
Articular cartilage plays an important role in joint movement and is very finely and
scientifically structured to suit different functional needs [
80
]. Articular cartilage mainly
comprises a hyaline cartilage layer and a calcified cartilage layer [
81
]. In addition, the
subchondral bone is located directly beneath the articular cartilage, and the hyaline car-
tilage, calcified cartilage, and subchondral bone together form the cartilage complex [
82
].
Hyaline cartilage is composed mainly of oval chondrocytes, an extracellular matrix (con-
taining large amounts of proteoglycan), and type III collagen [
83
]. Articular cartilage is
not innervated or vascularized, and its nutrients must be obtained from the joint fluid [
84
].
The core proteins that aggregate proteoglycans have covalently bound and strongly neg-
atively charged glycosaminoglycan side chains that interact with the surrounding fluid
environment through collagen and proteoglycan non-covalent linkages, giving articular
cartilage its unique biomechanical properties [
85
]. The cells in the calcified cartilage zone
are hypertrophic chondrocytes [
86
], and, because calcified cartilage is more dense and
mineralized, its modulus of elasticity is at the megapascal level, between the kilopascal
level of hyaline cartilage and the quarter-pascal level of bone tissue, which is equivalent
to being a mechanical transition zone that prevents cartilage tissue from being damaged
when subjected to excessive loading, and also effectively disperses the concentrated stress
on cartilage tissue under shear [
87
]. In addition, the presence of a physiological demarca-
tion line (tidal line) between the hyaline cartilage layer and the mineralized edge of the
calcified layer indicates that the articular cartilage has significantly developed and that the
growth-plate cartilage is fixed to the epiphysis, sometimes through a thin layer of calcified
cartilage and tidal markings, while the hypertrophic edge does not form tidal markings
and undergoes continuous vascular infiltration and endochondral ossification (EO) until
the bone matures, and the growth plate is completely resorbed and replaced by bone [88].
The pathological changes of OA initially occur in the hyaline cartilage layer, which in
turn causes changes in the calcified cartilage layer [
89
,
90
]. The onset of OA initially causes
alterations in the spatial structure of the proteoglycan and collagen fibers of the hyaline
cartilage layer, with swelling of the collagen fibers and an increase in free water [
91
,
92
]. The
calcified cartilage layer, an important structure for conducting stress with the tide line under
repeated compression, undergoes structural pathological changes, especially in the weight-
bearing area, where microfractures appear [
93
]. The persistent production of microfractures

Biomedicines 2023,11, 2244 6 of 21
causes the invasion of neovascular tissue and mineralization of the tideline, exacerbating
the degenerative process of the hyaline cartilage [
94
,
95
]. As OA progresses, the loss of
proteoglycans (PGs) from the cartilage tissue intensifies, and the collagen fibers continue to
swell, causing the calcified layer to thin, micro-fissures to increase, and new capillaries to
invade the calcified layer [
96
,
97
]. When cartilage tissue loses the protection of the calcified
layer, a vicious cycle is formed, and the OA process is accelerated (Figure 3) [98].
Figure 3.
Schematic diagram of articular cartilage. Reprinted/adapted with permission from Ref. [
31
],
copyright 2020, Ivyspring.
4. The Role of SF in Bone/Cartilage Damage
SF plays a key role in chondrocyte differentiation. SF is a protein found primarily
in collagen fibers that helps form and maintain the structure and function of cartilage
tissue. During chondrocyte differentiation, SF forms a complete extracellular matrix envi-
ronment by interacting with other extracellular matrix components such as collagen and
glycosaminoglycans. This environment provides the necessary support and structure for
chondrocytes to grow and differentiate. In addition, SF is also involved in key pathways
that regulate cell signaling and cell function. It can affect cell proliferation, differentiation,
and migration by binding to cell surface receptors (Figure 4). SF can also interact with
intracellular signaling pathways, such as TGF-
β
and BMP, to regulate the direction and
speed of cell differentiation [
99
,
100
]. Recent studies have shown that SF plays an important
role in the repair and regeneration of bone/chondrogenic tissue (Table 1) [101,102].
Figure 4. The role of SF in bone/cartilage damage.

Biomedicines 2023,11, 2244 7 of 21
Table 1. Application of SF in bone tissue regeneration.
Application Areas Mechanism of Action Application Results References
Bone regeneration
and repair
Promotes osteoblast proliferation and
differentiation, bone matrix production,
and epiphyseal migration
Promotes the speed of fracture healing,
enhances fracture stability, and
promotes bone defect repair
[31,69]
Cartilage regeneration
and repair
Promotes proliferation and differentiation
of chondroblasts and synthesis of collagen
and cartilage matrix
Promotes healing of cartilage defects
and improves cartilage tissue structure
and functional recovery
[99,100]
Bone implant repair
Provides an extracellular matrix scaffold to
improve the biocompatibility and adhesion
of bone implants
Enhances the bonding of the bone
implant to the surrounding tissue and
promotes stability and growth of the
bone implant
[101]
Oral periodontal
restoration
Promotes the growth of dental bone
attachment tissue and soft tissue repair
Improves the effectiveness of
periodontitis treatment and promotes
oral wound healing
[102]
Other applications Various tissue engineering repairs,
angiogenesis, immunomodulation, etc.
SF has potential for a wide range of
applications in tissue engineering and
regenerative medicine
[44]
4.1. The Role of SF in Bone Tissue
Bone tissue is one of the hardest tissues in the body, and damage and lesions often lead
to fractures, osteoporosis, and other diseases [
103
,
104
]. Recent studies have shown that SFs
play an important role in bone tissue [
105
–
108
]. As a natural polymer material with good
elasticity, tensile strength, biocompatibility, and biodegradability, SF can provide sufficient
space for the growth and differentiation of bone cells, thus promoting the generation of
new bone tissue [
109
–
113
]. SF has been extensively studied in bone TE because of its high
toughness, mechanical strength, and proven biocompatibility. Meinel et al. combined bone
tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs), and
SF biomaterials to investigate the effect of viral transfection on MSC osteogenic properties
in vitro
, and showed that RSF scaffolds promote osteogenic differentiation of human
mesenchymal stem cells (HMSCs)
in vitro
[
113
]. Meanwhile, Meinel et al. implanted porous
SF-based scaffolds into cranial defects in mice, demonstrating bone tissue regeneration
using silk-based implants with engineered bone, and expanding the selection of protein-
based bone implant materials through mechanical stability and durability [
114
]. RSF
scaffolds can be combined with other biomaterials, such as collagen or calcium phosphate-
based inorganic components, to enhance osteogenic properties [
115
]. To increase the success
of bone regeneration, accelerated angiogenesis is required [
116
]. Farokhi et al. used bio-
mixed SF/calcium phosphate/PLGA nanocomposite scaffolds as a vascular endothelial
growth factor (VEGF) to explore the efficacy of the delivery system, which also showed
good effects [117].
Bioactive molecules and active cells are a hot topic in tissue engineering research,
with various molecules and cells providing additional regulatory cues to guide cell dif-
ferentiation and functional bone regeneration [
118
]. Growth factors, drugs, and different
stem cells have been introduced into filament-based scaffolds to promote bone formation
(Figure 5) [119].
4.1.1. Bioactive Factor-Based Biomaterials
Bioactive factor-based biomaterials can release bioactive factors and promote bone
tissue repair by regulating cell proliferation and differentiation [
120
]. Drug-loaded beads
and colloidal crystals can be used to achieve a controlled release of bioactive factors through
microstructural alterations to improve bone repair [
121
,
122
]. Shen et al. developed an
SF/nano-hydroxyapatite (nHAp)-based scaffold with sequential and sustained release of
SDF-1 and BMP-2 in SF/nHAp scaffolds with synergistic effects on bone regeneration [
123
].

Biomedicines 2023,11, 2244 8 of 21
Exosome-encapsulated silk frames have also been shown to promote the recovery of bone
defects in vivo [124].
Figure 5. The role of silk fibroin in cartilage tissue engineering.
4.1.2. Biodegradable Polymer-Based Biomaterials
Biodegradable polymeric biomaterials are biomaterials with good biocompatibility and
tunability, which can be modified in terms of composition, structure, and physicochemical
properties to achieve a variety of different functions, such as cell adhesion, biodegrada-
tion, and drug retardation [
125
–
127
]. Currently, common biodegradable polymer-based
biomaterials include polylactic acid and polycaprolactone [
128
,
129
]. Diaz-Gomez et al.
prepared composite scaffolds using various combinations of PCL, SF, and nanohydrox-
yapatite (nHA), confirming the synergistic effect of silk and nHA on the extent of bone
repair [
130
]. As a biodegradable polymer film, the silk in protein/chitosan composite film
can be used not only as a metal implant coating for bone injury repair but also as a tissue
engineering scaffold for skin, cornea, adipose, and other soft tissue injury repair, while the
silk in protein/chitosan film not only provided a comparable environment for the growth
and proliferation of rat bone marrow-derived mesenchymal stem cells but also promoted
their osteogenic and lipogenic differentiation [
131
]. Biodegradable polymeric composites
of SF are widely used in tracheal tissue engineering [
132
], wound dressings [
133
], and
materials for biomedical applications [134].
4.1.3. Calcium- and Phosphorus-Based Biomaterials
Calcium and phosphorus biomaterials are biomaterials that can form,
in vivo
, similar
to bone tissue, and can promote bone tissue repair, for example, by promoting the prolif-
eration and differentiation of bone cells [
135
]. For example, hydroxyapatite and calcium
tri-calcium phosphate can be used as carriers of bone growth factors to promote bone tissue
repair through the slow release of bone growth factors [
136
,
137
]. At the same time, the
significant anti-stress properties of SF in concert with
β
-tricalcium phosphate provided a
good bionic environment for bone marrow MSCs [
138
]. The SF/calcium phosphate material
has good biocompatibility and mechanical properties as a bone repair scaffold, providing a
good growth space for osteoblast differentiation. In addition, the injectability exhibited by
the hydrogel and its use as a drug carrier allows the hydrogel to fit closely to the interface
of the cartilage defect, thus improving the integration effect [139].
4.2. The Role of SF in Cartilage Tissue
Cartilage tissue is an elastic connective tissue whose main role is to cushion and
support bone [
140
,
141
]. Injuries and lesions to cartilage tissue often lead to diseases such
as OA and cartilage lesions [
142
]. SF is associated with the extracellular matrix widely
distributed in cartilage tissue, and its main role is to maintain the structure and function

Biomedicines 2023,11, 2244 9 of 21
of the cartilage tissue [
143
]. SFs can promote the proliferation and differentiation of
chondrocytes and the generation of new cartilage tissue [
144
]. In addition, SFs can regulate
the apoptosis and survival of chondrocytes, and promote the repair and regeneration of
cartilage tissue [145].
4.2.1. Application of SFs in Cartilage Tissue Engineering
Cartilage tissue engineering is a method of repairing and reconstructing cartilage
tissue using biomaterials such as cells, matrix materials, and growth factors [
146
]. SF is a
natural protein that has many similarities with the protein structure of human skin and
has good biocompatibility and biodegradability [
28
]. In cartilage tissue engineering, SF
can be used to construct biocompatible, biodegradable scaffold materials, and can likewise
be used as surface modifiers to enhance scaffold surface cell adhesion and proliferation
properties (Figure 6) [
147
]. Studies have shown that SFs can promote the proliferation
and differentiation of chondrocytes, thereby promoting the growth and repair of cartilage
tissue [
101
]. In addition, SFs can improve the strength and stability of cartilage tissue, thus
increasing the success rate of cartilage tissue engineering [
148
]. Additionally, silk in proteins
has good water retention and can provide a good environment under growth conditions
to aid chondrocytes in nascent tissue growth and repair [
149
]. Saha et al. evaluated the
role of mulberry and non-mulberry laminar filament biomaterials in cartilage or bone
induction using human bone marrow stromal cells (hBMSC)
in vivo
and
in vitro
, showing
good bone induction [
150
]. In cartilage tissue engineering, combining SF with other
biomaterials, such as gelatin and alginate, supports the fabrication of scaffold materials
with good biocompatibility and biodegradable properties that can provide skeletal support
for the generation of new tissue, and can degrade into harmless metabolites with reduced
adverse effects on the human body [
151
,
152
]. Li et al. developed a silk-composite hydrogel
of SF and carboxymethyl chitosan (CMCS), which supported the adhesion, proliferation,
glycosaminoglycan synthesis, and chondrogenic phenotype of rabbit articular chondrocytes,
and the subcutaneous implantation of the hydrogel in mice showed no infection or local
inflammatory response, indicating good biocompatibility
in vivo
[
153
]. Liu et al. used
electrostatic spinning to prepare a fibrillar SF/poly L-lactic acid (PLLA) scaffold, showing
good adhesion, biocompatibility, and cytocompatibility [
154
]. Overall, SF, as an important
matrix material, has good prospects for application in cartilage tissue engineering. With a
better understanding of silk, we will be better able to apply this natural protein to promote
cartilage tissue repair and regeneration.
4.2.2. SF in the Treatment of Patients with OA
OA is a serious disease derived from the degeneration of cartilage tissue and, if nec-
essary, requires surgical intervention. Tissue engineering using stem cell graft scaffolds
is an attractive approach and a challenge for orthopedic surgery [
137
,
138
]. SFs can also
reduce pain and inflammatory responses in patients with arthritis, thereby relieving the
symptoms of arthritis. Wang et al. prepared silk/BDDE hydrogel balls using oil/water
(o/w) emulsification and evaluated their biocompatibility and biodegradability
in vivo
.
The silk/BDDE hydrogel ball was demonstrated to be biocompatible and can be used as
a lubricant for the treatment of OA, as well as for pain relief and the sustained release
of drugs for future OA treatment [
155
]. In clinical trials, Sharafat-Vaziri et al. evaluated
an autologous chondrocyte and collagen/silk heart protein scaffold consisting of newly
engineered tissues to repair osteochondral defects, showing great coverage and integra-
tion of the grafts in patients without effusion, edema, and reduced cartilage formation
signals [
156
]. Jaipaew et al. prepared SF/hyaluronic acid (HA) scaffolds, with different
SF/HA (w/w) ratios via freeze-drying, which were suitable for OA surgery [
157
]. Thus,
SF-based biomaterials have great promise in OA surgery.

Biomedicines 2023,11, 2244 10 of 21
Figure 6. In vivo
implantation of SF-based biomaterials. (
A
) Schematic diagram illustrating the
3D fabrication of a scaffold made via bioprinting for
in vivo
implantation. Reprinted/adapted
with permission from Ref. [
151
], copyright 2017, John Wiley and Sons. (
B
) Silk-GMA hydrogel
transplantation loaded with chondrocytes. Reprinted/adapted with permission from Ref. [
140
],
copyright 2020, Elsevier.
4.2.3. Application of SFs in Drug Delivery
SF is a biomaterial that has been extensively studied in tissue engineering and drug
delivery (Figure 7). As a carrier material, SF has high permeability, and its microporous
structure can promote the penetration of drugs and improve the delivery efficiency of
drugs. At the same time, SF can be degraded and absorbed by the human body, avoiding
the risk of secondary surgery. Additionally, SF can protect the drug from degradation
and inactivation during delivery and improve the stability of the drug. Moreover, SF
has low immunogenicity and histocompatibility, reducing rejection of the drug delivery
system [
158
]. At present, SF can be used as a carrier for drugs in OA drug delivery,
enveloping drugs in nanoparticles or microspheres to increase the efficiency and stability of
drug delivery. Additionally, it can be combined with growth factors or cytokines through
controlled release, promote the proliferation and differentiation of chondrocytes, and
promote the repair and regeneration of joint cartilage. At the same time, it can combine
with stem cells or osteoblasts to form a tissue-engineered scaffold for the repair and
regeneration of joint cartilage [
159
–
161
]. In addition, SFs can be used in combination
with other biomaterials to further enhance their drug delivery [
162
]. Ratanavaraporn
loaded previously developed gelatin/SF microspheres with curcumin, and the gelatin/SF
microspheres encapsulated with curcumin delayed cell destruction in joint and synovial
tissue, which showed prolonged anti-inflammatory effects compared to rapidly degrading
gelatin microspheres [
163
]. Red-modified silk nanoparticles were fabricated by Sharma
et al. and loaded with gentamicin as a deposition material on titanium surfaces, which
showed better killing of S. aureus on the titanium surfaces [
164
]. Hassani Besheli et al.
constructed a sustained drug delivery system by loading vancomycin (VANCO) in silk
nanoparticles to treat severe osteomyelitis [
165
]. Thus, in OA drug delivery, SF can play an
important role (Table 2).

Biomedicines 2023,11, 2244 11 of 21
Figure 7. The role of SF in drug delivery.
Table 2. Application of SF in drug delivery.
Application Areas SF in Drug Delivery References
Oncology treatment To deliver drugs to tumor tissue, SFs are used as carriers to improve
the stability and bioavailability of drugs [166]
Wound healing
SFs promote cell migration, proliferation, and repair during wound
healing and can be used to prepare drug-delivery systems to promote
wound healing
[167]
Treatment of blood disorders
SFs provide reliable carriers for the transport and release of drugs for
the treatment of blood disorders, such as anticoagulants and
anti-platelet agents
[168]
Treatment of neurological disorders
SFs can be used to deliver drugs for the treatment of neurological
disorders, such as neuroprotective agents and anti-epileptic drugs, to
promote the protection and repair of nerve cells
[169]
Skin beauty and treatment
SFs are widely used in cosmetic skin products and delivery systems
for therapeutic drugs to improve skin texture, promote wound
healing, reduce scar formation, etc.
[170]
Infectious disease control
SFs are used as carriers for drug delivery systems to deliver antiviral,
antibacterial, and antifungal drugs, improving their efficacy
and bioaccessibility
[171]
Treatment of orthopedic diseases
SFs are used to deliver drugs for treating orthopedic diseases, such as
bone growth factors and anti-inflammatory drugs, and to promote
the growth and repair of bone cells
[106,107]
Cardiovascular disease treatment
SFs are used as carriers in drug delivery systems for the delivery of
drugs treating cardiovascular disease, such as anti-hypertensives and
anti-heart failure drugs, to alleviate the symptoms of
cardiovascular disease
[172]
Immune disease treatment
SFs can be used in drug delivery systems to deliver drugs for treating
immune diseases, such as anti-inflammatory drugs and
immunomodulators, to regulate the function of the immune system
and treat diseases
[102]
Dental treatment
SFs are widely used in dental therapeutic drug delivery systems for
the delivery of antibacterial drugs, natural anti-inflammatory agents,
and bone growth factors to promote dental restoration and healing
[173]
4.3. Prospects for SF in Clinical Applications
As a natural polymer with good biocompatibility, SF has been widely used in biomed-
ical applications, including ophthalmic surgical sutures, artificial corneas, artificial tendons,

Biomedicines 2023,11, 2244 12 of 21
orthopedic ligaments, cartilage engineering, artificial skin for wound surfaces in trauma-
tology, and anticoagulation stents in cardiology [
174
–
176
]. Although there is extensive
research into the application of SFs in different base materials, most of it is still at the
laboratory research stage, and few products have been successfully commercialized and are
actually used in clinical treatment (Table 3). SERI surgical scaffolds, Silk Voice injections,
and silk-substituted isoserine trauma dressings are the products in routine clinical use
today (Table 4).
Table 3. Selected SF material development companies.
Company Name Region SF Products
Sofregen Inc. US SERI surgical stents
Injectable fillers
Vaxess Technologies Inc. US Drug and vaccine delivery
Evolved by nature US
Skincare products, textile coatings, topical ophthalmic treatments, etc.
Cocoon Biotech Inc. US Drug delivery systems (hydrogels, osteoarticular microspheres, etc.)
Kraig Biocraft Laboratories Inc. US Special textiles
Oxford Biomaterials Ltd. UK Artificial blood vessels
Orthox Ltd. UK Meniscal repair stents, tissue stents
Suzhou Semtex Biotechnology Co. China Injectable gels, stents, and dressings
Suzhou Suhao Biomaterials Technology Co.
China Trauma dressings
Zhejiang Xingyue Biotechnology Co. China Raw materials such as SF gels, microspheres, solutions, and sponges
SF skin rejuvenation mask
Table 4. Some approved SF products.
Product Type Trademarks Uses Time to Market
SERI Surgical Stent Allergan
Full body contouring, brachioplasty,
abdominoplasty, breast fixation, breast
reconstruction, etc.
2013
Silk Voice Injection Sofregen Vocal cord dielectricity and vocal cord
insufficiency United States, 2018
Silk-substituted isoserine
wound dressing Soho Biotechnology Co. Wound healing China, 2012
With increasing in-depth research on SF, its application in the treatment and repair
of bone and cartilage tissues is becoming more promising. In regenerative medicine, the
binding of SF to cells or growth factors can promote cell attachment, proliferation, and differ-
entiation, while providing structural support to help rebuild tissues. The biocompatibility
and biodegradability of SF fibers make them ideal materials for manufacturing artificial
blood vessels, and SF can be used in the preparation of biomedical dressings to promote the
healing process of wounds. SF dressings provide a protective physical barrier to promote
cell regrowth and tissue repair, and SF can be used as a vehicle for drug delivery systems.
SF has good drug adsorption and sustained release performance, which can control the
release rate and dose of drugs and improve the efficacy and bioavailability of drugs; SF
can also be used to prepare artificial bone substitutes for bone-tissue engineering and bone
repair. Moreover, an SF scaffold can promote the attachment and growth of bone cells and
accelerate the process of bone regeneration and repair. SF has wide application prospects
in regenerative medicine, which can promote tissue regeneration and repair and provide
new treatments and means for disease treatment and rehabilitation [
155
,
177
,
178
]. In terms
of applications, most are concentrated in the fields of wound repair dressings [
179
,
180
]
and orthopedic repair materials [
31
]; in terms of morphology, SF membranes [
181
] and SF
scaffolds [
182
] are the materials with more applications. SF can be combined with other
biomaterials to form composite scaffolds, mimic the natural
in vivo
environment, increase
the
in vivo
fusion potential of scaffolds, implant destination cells to accelerate the healing
and regeneration of trauma sites, etc. It can be modified into different scaffolds such as

Biomedicines 2023,11, 2244 13 of 21
injectable and printable gels, porous sponges, and electrostatically spun two- and three-
dimensional structures [
130
,
134
]. The advantages of SF therapy are its obvious effects, low
side effects, and high safety, and more possibilities for SF-based scaffolds can be expected
through new modification techniques. In the future, the application of SF in the treatment
and repair of bone and cartilage tissues will be even more promising, while more research
is needed to explore its mechanism of action and dose determination.
5. Conclusions and Outlook
Repairing cartilage and osteochondral damage due to OA has always been a challenge
for clinicians. Currently, tissue engineering techniques have been the subject of much
research and have great potential in bone/cartilage repair. SF, as a natural material, is
widely used in tissue engineering due to its inexpensive availability, excellent biocom-
patibility, unique mechanical properties, and desirable processing properties. This paper
summarizes the progress of research on SF biomaterials in the field of bone/cartilage repair.
Compared with other types of biomaterials, SF has great research value and broad appli-
cation prospects. SF-based biomaterials have more robust mechanical properties through
loading peptides, gene-editing means, exosomes, nanoparticles, and growth factors, which
are more conducive to cell adhesion and growth and enhance bone/cartilage repair. For
example, Ding et al. significantly improved the cell recruitment ability of SF/HA scaffolds
by loading bone morphogenetic protein-2 (BMP-2) onto SF/HA scaffolds to accelerate
osteochondral repair [
183
], proliferation, and differentiation, promoting bone repair and
cartilage repair [184].
SF has good biocompatibility, biodegradability, and bio-absorbability, and has been
widely used in the medical field in recent years. Currently, clinical applications regarding
SF biomaterials are mainly focused on wound healing and wound dressings [
114
]. In
addition, there have also been clinical studies on SF scaffolds for meniscal cartilage repair,
with some success in recent years [
185
]. However, for OA and cartilage regeneration, SF
has some limitations. First of all, OA is a joint disease whose main features are cartilage
degeneration and joint inflammation. While SF can help maintain cartilage structure,
it does not prevent or reverse cartilage degeneration. This means that SF has a limited
therapeutic effect on OA. Secondly, SF also has certain limitations on cartilage regeneration.
Cartilage regeneration is a complex biological process that includes steps such as stem
cell differentiation, cell proliferation, and secretion of a matrix. Although SF has a certain
effect on cartilage structure, it does not directly promote cartilage regeneration. When
treating cartilage damage or joint degenerative diseases, stem cell therapy, biomaterial
implantation, or surgery are often required to promote cartilage regeneration and repair.
Overall, while SF plays an important role in maintaining cartilage structure and function, it
has certain limitations in OA and cartilage regeneration. The treatment of these conditions
often requires a combination of factors and treatments to achieve the best results [186].
In conclusion, SF-based biomaterials have broad application potential in the treatment
of OA, and future research can combine SF biomaterials with other bioactive molecules,
such as growth factors and gene therapy, to improve the effect of cartilage regeneration.
At the same time, according to the specific situation of patients, the personalized design
of SF biomaterials can help achieve better treatment results. More importantly, as the
preparation process of SF biomaterials is further improved, the mechanical properties and
stability of the materials are improved, and their degradation rate
in vivo
is prolonged.
Although SF biomaterials have achieved some success in the treatment of OA, they still face
some challenges and limitations. More clinical research and scientific exploration will help
further develop and refine this treatment, providing better treatment options for patients
with OA.

Biomedicines 2023,11, 2244 14 of 21
Author Contributions:
X.S., L.W. and L.Q.: data curation, formal analysis, writing—original draft,
software. J.Y.: resources, formal analysis. C.Z.: writing—review and editing. Z.X.: resources, formal
analysis. Y.Z.: resources, formal analysis. L.C.: conceptualization, formal analysis,
writing—review
and editing. N.H.: conceptualization, formal analysis, funding acquisition,
writing—review
and
editing, project administration. All authors have read and agreed to the published version of
the manuscript.
Funding:
General Project of National Natural Science Foundation of China (Project number: 82072443),
Excellent Project of Chongqing Overseas Returnee Entrepreneurship and Innovation Support Program
(Project number: CX2022032), General Project of Chongqing Technology Innovation and Applica-
tion Development Special Project (Project number: Cstc2020jscx-msxmx0094), the key project of
Chongqing Science and Health Joint Medical Research Project (Project number: 2019ZDXM014).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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