Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Contents lists available at ScienceDirect

Computational and Structural Biotechnology Journal

journal homepage: www.elsevier.com/locate/csbj

Review article

A comprehensive review of silk-fibroin hydrogels for cell and drug delivery

applications in tissue engineering and regenerative medicine
Alakananda Parassini Madappura a, Srinivas Madduri b, c, *
a
Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 300044 Hsinchu, Taiwan, Republic of China
b
Department of Biomedical Engineering, University of Basel, Basel, Switzerland
c
Department of Surgery, University of Geneva, Geneva, Switzerland

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

Keywords: Hydrogel scaffolds hold great promise for developing novel treatment strategies in the field of regenerative
Hydrogels medicine. Within this context, silk fibroin (SF) has proven to be a versatile material for a wide range of tissue
Biofabrication engineering applications owing to its structural and functional properties. In the present review, we report on the
Cell therapy
design and fabrication of different forms of SF-based scaffolds for tissue regeneration applications, particularly
Drug delivery
for skin, bone, and neural tissues. In particular, SF hydrogels have emerged as delivery systems for a wide range
Tissue engineering
Tissue regeneration of bio-actives. Given the growing interest in the field, this review has a primary focus on the fabrication,
characterization, and properties of SF hydrogels. We also discuss their potential for the delivery of drugs, stem
cells, genes, peptides, and growth factors, including future directions in the field of SF hydrogel scaffolds.

1. Introduction regeneration [6]. However, given that TE is deeply rooted in biomedi­

cine, it could also be argued that it is still in its infancy as it is neither
Tissue engineering (TE) stands at the crossroads of medicine, bio­ economical nor easy. To date, its clinical success is limited to small ar­
logical science, engineering and technology, thus implying an inter- and teries, skin grafts and the trachea among others, while the liver, lungs
multidisciplinary approach to the field. In the late 1980s, the entire and heart tissues are grown in laboratories. Although laboratory-grown
attention of medical science was focused on the genesis of the very tissues are far from clinical settings, they are of great help in studying
concept of TE at the National Science Foundation workshop held in basic mechanisms and drug screening, thereby significantly reducing
Washington, DC. TE demands a multidisciplinary attention to overcome animal experimentation.
the challenges and complexity of generating functional tissue substitutes Biomaterials play a key role in TE by providing structural support for
consisting of cells, scaffolding and biomaterials [1]. While TE has made the delivery of cells and therapeutic factors. The tissue environment is
substantial progress in the field, the functional regeneration of damaged mimicked by these materials by engineering a suitable architecture for
tissue and organs [2] remains challenging. TE and regenerative medi­ cell growth and proliferation [7]. Several classes of materials have been
cine reproduce the same idea, but with a different approach in which the identified and developed for this purpose, such as polyesters, poly­
former builds new tissues, while the latter restores functions using amides, polysaccharides, and their combinations. Among a multitude of
combinations of factors, both with the aid of foreign biological materials structural systems, hydrogels are particularly interesting due to their
[3,4]. A basic requirement for this is an engineered tissue construct versatility. They can be designed to support cell growth in a hydrated
combining scaffold, cells, and therapeutics or biomolecules to orches­ three-dimensional (3D) environment that permits oxygen and nutrient
trate and support the target tissue regeneration, either in vitro or in transport to replicate the microenvironment for regenerative tissue
humans [5]. This template provides the necessary microenvironment, growth [8]. Their popularity is also credited to their ability to sustain
such as biological, chemical, or biophysical signals, to support cell high water content and retain a porous structure, thus allowing them to
proliferation and neo-tissue formation. TE constructs are cultured in dispose of cellular waste and trade cell metabolites [9].
vitro [1] for transplantation into the damaged site in vivo for repair and Hydrogels provide important structural and functional support in the

* Correspondence to: Laboratory of Bioengineering and Neuroregeneration, Department of Surgery, University of Geneva, 1 Rue Michel-Servet, 1211 Geneva 4,
Switzerland.
E-mail address: [email protected] (S. Madduri).

https://doi.org/10.1016/j.csbj.2023.10.012
Received 15 May 2023; Received in revised form 7 October 2023; Accepted 8 October 2023
Available online 10 October 2023
2001-0370/© 2023 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

field of tissue engineering and regenerative medicine by controlling the exceptional thermal stability, with a transition temperature to 175 ◦ C.
sustained release of the desired factors and by regulating cell behaviour. Above this temperature, the material is stable up to 250 ◦ C due to stable
They can be used in different forms, such as injectable solutions, in situ β-sheet conformation [16]. Crystalline SF is insoluble in typical solvents.
gels, structured matrices, viscous gels and thin sheets. Hydrogels also However, electrolyte solutions, such as lithium bromide or calcium
facilitate an intricate cell-matrix interaction at the physiological length thiocyanate, can disrupt hydrogen bonds and dissolve SF [17]. Aqueous
scale. Therefore, hydrogels with their hierarchical structures have silk of a higher concentration tends to aggregate given its inter- and
gained much attention in the field. Furthermore, there is a growing in­ intramolecular interactions. SF hydrogels have poor swelling properties,
terest in developing hydrogels for regulating the target cell behaviour in which can be pointed to as a major drawback in the hydrogel system.
a spatiotemporal fashion. However, this issue can be tackled by blending with other polymers,
However, conventional hydrogels fall short when it comes to me­ including polyvinyl alcohol, gelatin, chitosan, and collagen.
chanical strength, structural stability, and electrical and magnetic SF is one of the strongest fibres and has a similar tensile strength
properties [10]. As a result, there has been a growing interest in compared to glass fibre or other synthetic organic fibres, allied with
multifunctional hydrogels to control cell functions. Different kinds of toughness, good resilience, and elasticity [17]. SF is more biocompatible
materials, both natural and synthetic, have been explored with and than materials like polylactic acid and polyglycolic acid and is less
without crosslinking using metallic, inorganic, organic or polymeric immunogenic and pro-inflammatory than collagen. SF degradation is
materials [11]. Among natural polymers, protein polymers such as silk predominantly due to the response to proteolytic enzymes [17]. It is
fibroin offer a bottom-up approach that helps to introduce novel features entirely unproblematic and products are non-toxic. The rate of degra­
rationally. dation is directly associated with the β-sheet content, degree of crys­
Silk is still considered as an excellent material in many industries due tallinity and molecular weight. Degradation of a lower β-sheet content
to its versatile biological properties. The silkworm has been domesti­ SF is much more pronounced. Enzymes, such as protease XIV,
cated for centuries to obtain silk fibres for commercialization purposes. αchymotrypsin, papain, and matrix metalloproteinases-1 and 11, spe­
From the Chinese tales of the unexpected unwinding of a silk cocoon by cifically act on cleavage sites like Tyr, Phe, Trp, His, Lys, Arg Val, Ile, and
the wife of the Yellow Emperor of China [12], silk has started rolling Leu to break down the chains to small polypeptides and amino acids in
ever since through human evolution, ranging from curtains in the textile due course [17]. Hence, with such an extensive understanding of the
industry to sutures in medicine. Further, silk-based scaffolds have degradation process, control over the performance of SF is feasible.
emerged for TE applications. Once identified, the particular properties SF is a versatile biomaterial due to its natural safety, biocompati­
of silk fibroin have offered the research world myriad opportunities to bility, and biodegradation properties. Recent developments of SF for
use silk in various industries (Figs. 1 and 2). molecular engineering, multifunctionalities, injectable formulations,
In the case of silk derived from the silkworm, the fibres consist of two conductive, stimuli responsive and bio-inks have widened the scope in
primary proteins: fibroin which forms the fibre, and sericin which acts as the field of regenerative medicine. In this review, we discuss the
a glue to bind the non-woven composite cocoon. A fibroin molecule importance and application of SF as a biomaterial and shed light on the
consists of a heavy chain of 391 kDa and a light chain of 26 kDa. [13] design of different forms of silk biomaterials for drug delivery and TE
that are connected by a single disulfide linkage, such as a covalent bond. applications. Among the various forms, hydrogels stand out as the better
The primary structure of fibroin has a crystalline domain of a few re­ candidate. Therefore, the focus of the review is on the production,
petitive sequences of hydrophobic (GAGAGS and less conserved repeats characterization and property of SF-based hydrogels, including their
of GAGAGX), aromatic residue, and polar amorphous regions (Figs. 3a, recent use for the delivery of drugs, stem cells, genes, peptides and
3b). Taken together, the crystalline region forms an antiparallel β-sheet growth factors.
secondary structure interspersed by an amorphous region [14] (Fig. 3c).
The amorphous region of silk is rich in glycine, less ordered with helical 2. Methods
structures [15]. The stability of silk fibroin (SF) is due to the crystallinity
of the material. Hence, altering the crystallinity would make silk a better Literature search was conducted using PubMed, EMBASE and Med­
and more approachable material for delivery systems. SF also possesses line databases according to the PRISMA guidelines for the articles

Fig. 1. Silk fibroin hydrogel, a versatile biomaterial for delivery application.

4869
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Fig. 2. Summary of the structure, properties and application of silk fibres.

published in English until February 2023. The following search terms are then used in various applications after aqueous or organic
were used: “silk fibroin biomaterials”, “silk fibroin hydrogel”, “silk solvent-processing methods. To achieve the dissolution of solid silk into
fibroin composite”, “tissue repair”, “tissue regeneration”, “drug de­ a fibroin solution, a process called ‘reconstitution of silk’ was developed.
livery”, “cell delivery”, “growth factors delivery”, and “gene delivery”. Here, the sericin coating of the fiber is removed to enhance its
Studies identified by the search outcomes were combined and duplicates biocompatibility as sericin can trigger immunological responses. Addi­
were removed. Screening of the title and abstracts were performed tionally, various methods, such as protein denaturation through chem­
before the extraction of full-text articles. Publications with inconclusive ical or thermal means and the modification of mechanical properties by
data and unrelated content were excluded. Selected articles were cate­ altering crystallinity, can be induced during the reconstitution phase.
gorized as those primarily based on hydrogel fabrication and, subse­ This can be induced after the process of immersing in alcohol or through
quently, the resulting outcome was highlighted as described in the water annealing. The technique can enable the fabrication of the new
Methods. Fabrication methods were evaluated for required controls and desired structure of the silk and helps to achieve the possibility of
(verification) and structural characterization (validation). The search other fabrication techniques. The property of the resulting structures can
yielded 1021 studies and a total of 193 articles were selected. Critical be finely controlled and customized to meet specific needs. Regenerated
review of the full text articles was conducted for those selected and SF exhibits exceptional versatility and biocompatibility, thus making it a
further categorized by considering the scaffold fabrication, hydrogel valuable resource for a wide range of biomedical applications [21].
properties and application of bioactive factors. Non-woven mats are of general interest given their increased surface
area and surface roughness for cell attachment. SF mats were prepared
from reprocessed native silk fibre by solubilizing silk fibres in formic
2.1. SF: a versatile material acid or calcium chloride prior to subcutaneous implant in the rat. The
materials showed biocompatibility with little or no immune response.
2.1.1. Design of SF for tissue engineering applications Vascularized, reticular and connective tissue formation was guided by
When designing a biomaterial, several elements should be consid­ the mat and growth was confirmed by histological and immunohisto­
ered: (i) the material should provide mechanical support parallel to the chemical characterization [22]. The homogenization technique made a
used tissue; (ii) no host immune response; (iii) the rate of degradation 10–30 µm mat with a diameter of 300 µm. Endothelial cells proliferated
should be proportional to the tissue formation; and (iv) the material when cultured for several weeks and micro-vessel-like structures were
must guide cells with the help of physical, chemical and biological cues formed. Due to the low infiltration of cells, no degradation was observed
[19]. Silk is a distinctive family of proteins with a primary set of amino during that time [23]. Electrospun fibres can range from a few nano­
acids having varied functional differences, with different structural meters to microns [24]. Human mesenchymal stem cells (hMSCs) were
sequencing [19]. Silk has been amenable to the human body in all cultured on uniform fibre < 0.8 µm diameter of aqueous SF sol­
possible ways and has been successfully investigated for countless ap­ ution/polyethylene oxide (PEO) and a lateral modulus of 8 GPa was
plications in the form of film, hydrogels, fibres and sponges, beginning observed. A 100 nm diameter SF mat from formic acid recorded a
with suture material that dates back several centuries [20]. Dry cocoons 515 MPa Young’s modulus and 7.25 MPa tensile strength [25].
produced through a sericulture process are used as raw materials. These

4870
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Fig. 3. Detailed illustration of the chemical structure for silk fibroin. (a) Basic structure and composition of amino acid sequences; (b) primary structure of silk
fibroin; (c) β-sheet secondary structure [18].

Electrospun non-woven mesh with random coils was prepared through sugar) determined the pore size when added with a solvent and cast to
methanol treatment, but adversely affected the structure by reducing molds [31]. A different porosity was achieved by controlling the varying
porosity due to dehydration [26]. stacking of salt/1,1 3,3 hexafluropropanol–silk solutions. RGD-modified
SF films can be prepared by many techniques, predominantly SF sponge was able to promote growth and differentiation of hMSCs,
through casting. Silk structure is reformed by treatment with 50% depositing hydroxyapatite and regulated bone markers. When a 900 µm
methanol, which in turn alters the water and oxygen permeability of the pore-sized sponge was used to culture hMSC in osteogenic media, the
film [24]. A nanoscale ultra-thin film made by layer-by-layer technique resulting structure had similarities to trabecular bone. In a study where
was shown to be stable due to the hydrophobic interactions and was able aqueous-based SF sponges were used for cartilage TE, chondrocytes
to support the adherence and proliferation of hMSCs. Silk films showed a proliferated much faster than collagen and a higher content of glycos­
better attachment to fibroblast and other mammalian cells than collagen aminoglycan was observed [32]. Tables 1 and 2 covers some of the SF
films [27]. For example, SF film healed full-thickness rat skin in 7 days scaffolds with their properties and applications. Although SF mats, film
with a low immune reaction compared to porcine-based wound dress­ and sponges have several advantages, a few disadvantages exist. For
ings [28]. Silk films modified with RGD or combined with bone example, electrospinning of SF mats or films involves the use of solvents,
morphogenic protein-2 [29] showed improved bone formation. In which can adversely affect biocompatibility. In addition, they usually
addition, SF films have been cast and blended with other polymers for have poor cell encapsulation and penetration ability. As for sponges,
improved or tailor-made properties. To increase the mechanical strength there is a need for the removal of added porogens, thus making the
and for better hepatocyte viability, silk was blended with cellulose and production process tedious. The uneven pore size and interconnectivity
recombinant human-like collagen, respectively [30]. can also influence cell growth. Therefore, a system with an ideal envi­
SF has been utilized for the preparation of porous sponges through ronment for cell survival that can mimic the microenvironment is
gas foaming and lyophilization or using porogen, which has great required.
importance for cell growth. In a solvent-based method, porogens (salt, Hydrogels are now considered structurally intelligent with a wide

Table 1
General production methods of SF scaffolds for biomedical applications.
Type of scaffold Fabrication method Application Characteristics Reference

Non-woven mat Electrospinning Wound management Anti-inflammatory, anti-adhesive and stretchable [33]
Coaxial bead- on-string fibre Electrospinning DOX release pH-sensitive drug release [34]
PEDOT− PSS functionalized SF Electrospinning Axonal regeneration Electroconducti-ve scaffolds [35]
Iontophoresis-stimulated SF films Casting Neurotensin delivery Sustained drug release [36]
Avidin-adsorbed SF film/RGD-SF Casting hMSCs culture Enhanced cell attachment and growth [37]
SF sponge Freeze-drying Tissue repair Tailored biomechanical properties [38]
Regenerated silk fibre Straining flow spinning Axonal guidance Enhancement of the guidance ability [39]

4871
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Table 2
SF hydrogels with various crosslinking strategies.
Crosslinking method Materials Crosslinking agent Properties Reference

Enzyme SF/gelatin Transglutaminase Enhanced mechanical properties [70]

Enzyme SF/mHA Laccase Improved pore structure and swelling behavior [71]
Modulus= 204 kPa
Fracture energy= 56.6 kJ/m3
Enzyme SF/HA Horseradish peroxide High elasticity [72]
Storage moduli= 0.55 ± 0.03 kPa
Physical SF/MA UV Irradiation Structural stability [73]
G′ is larger than G″
Physical SF Gamma irradiation Improved gelation [74]
Physcial SF Sonication Controlled gelation [75]
Physical SF/gelatin Electrostatic blending Tailored biodegradation [76]
Chemical SF Glutaraldehyde Stiffness [77]
G′ and G″ increased by 10-fold
Chemical SF/gelatin Genipin Improved mechanical properties [78]
Chemical SF/PVA Dialdehyde starch Enhanced swelling ratio [79]
Chemical SF/collagen/ Glyoxal Improved porosity [80]
chitosan Young’s modulus 73.1 ± 3.4 kPa
Chemical SF/chitosan EDC/NHS Enhanced compressive strength and porous architecture [81]
G′ increased with respect to SF concentration.

range of applications in the field of. mechanical properties [48]. Self-assembly and gel formation are
TE and regenerative medicine. They can be described as water-rich, generally influenced by the concentration, processing temperature, and
3D polymer crosslinked networks that can swell to an extent in an pH of the solution [49]. Increasing temperature and concentration have
aqueous solution. Hydrogels derived from naturally occurring materials, an inverse relation to the time of SF gelation.
such as SF, chitosan, alginate, and collagen, are particularly appealing Temperature-induced gelation is a physical technique where
given their intrinsic biocompatibility and biodegradability. Silk hydro­ increasing the molecular collision with temperature decreases gelation
gels can be formed through physical and chemical crosslinking methods time [40]. Sonication induces pressure and temperature points leading
where their β-sheet structures play an important role [40]. During to aggregation and gelation [50]. The self-assembly of SF results in the
hydrogel preparation, the pH concentration of the SF solution has to be formation of different structures like microgels, micelles or vesicles due
kept in check as it can greatly impact gelation. While a 3% solution with to the hydrophopic-hydrophilic combination in SPF. Bono et al. used
pH 3–4 took only 2 days for gelation, a pH 5–12 solution took 8 days ultrasonification to fabricated 200 µm microgels in a water-oil emulsion
[41]. Gelation and pore size also depend on the concentration of the SF phase to encapsulate cells. [51] Applying shear force through vortexing
solution [40]. Supplementing SF with surfactants, such as poloxamer impels molecule-molecule interactions to form a gel [52]. To decrease
407, induces desirable gelation, but was found to reverse sol-gel tran­ the pH, an electric field is applied across the solution for silk to
sition [42]. Similarly, a semi-interpenetrating polymer network was agglomerate [53]. Photo-crosslinking falls under the better option of
formed to increase the mechanical property of the system [43]. physical crosslinking. In SF hydrogels, gelatin methacrylate that forms
SF/gelatin-blend hydrogel affected the rheological and mechanical hydrogels under UV light is commonly used. When SF (50 mg/Ml) was
properties as the helix coil transition of the gelatin was influenced by introduced into GelMA to form an interpenetrating composite hydrogel
temperature. In drug delivery applications, the drug release greatly combining photo-crosslinking and alcohol treatment, compressive
depends upon the concentration of the SF and the blended material, if modulus was 300kPa, consequently improving mechanical strength.
any, as in the case of benfotiamine in SF–glycerol hydrogels [44]. For TE [54] Methacrylated silk fibroin/laminin-acrylate hydrogel was formu­
application, osteoblast-like cells adhered to hydrogels and showed an lated in situ by Liu et al. to address spinal cord damage. The significant
enhanced proliferation rate with the addition of 30% glycerol. SF in aspect of the hydrogel is its adhesion with native spinal cord that fa­
bone TE is prominent as bone volume, thickness, minerals and the rate of cilitates molecular scale hydrogel-tissue suturing, thereby aiding axonal
bone formation were observed to be higher than in poly growth and guidance [55]. Although physical methods are attractive,
(lactide-co-glycolide) (PLGA) [45]. Compared to other structures, the resulting SF hydrogels are suboptimal due to a lack of controlled
hydrogels can be more effectively structurally modified for controlled properties. Consequently, chemical crosslinking-based hydrogels
drug release and for facilitating tissue regeneration. evolved.
Chemical crosslinking techniques are employed to form a more sta­
2.2. SF hydrogels and their fabrication ble hydrogel with better control over swelling and porosity, in addition
to being able to regulate hydrogel strength and biodegradability [56].
SF hydrogels emerged as an effective candidate for many biomedical Some fabrication techniques include salts like calcium (adding ions re­
applications given their excellent delivery properties for drug substances duces gelling) [57], PEO or polyethylene glycol (PEG) to promote
and cells. The popularity of silk has been ever-increasing for the past few protein-protein associations [40]. Organic solvents like alcohol helps to
decades and, recently, different functionalities have emerged to cement form β-sheet structures [58] and surfactants help to bind the proteins to
its properties and extend its application. Silk protein, together with form aggregates [59]. Chemical coupling can functionalize amino acids
natural/synthetic polymeric materials, usually forms hydrogel for on the SF chain to adjust the hydrophobic and hydrophilic properties
biomedical applications. A structural change from a coiled structure to a and thereby control hydrogel formation [60]. Due to the presence of
silk II β-sheet conformation has been observed during the sol-gel tran­ functional groups in silk, chemical crosslinkers can easily react with the
sition [46]. Silk hydrogels are generally formed through physical or –OH, –NH2, and –COOH moieties to form networks. Some of the com­
chemical crosslinking. In the former, silk molecules form a non-covalent mon chemical crosslinkers are genipin, glutaraldehyde and carbodii­
bond through hydrogen bonding, hydrophobic interaction or electro­ mide. Genipin react with the free amino groups in SF, whereas
static interaction, while in the latter, reactions are carried out with glutaraldehyde reacts with the amino group of lysine and phenolic group
chemical crosslinking agents forming a special network. [47]. Physically of tyrosine of the protein chain. Although the above-mentioned cross­
crosslinked hydrogels formed without chemical reactions have poor linkers are widely used, they appear to exert a cytotoxic effect [61,62].

4872
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Thus, there is a growing need for biocompatible reagents. Rehman et al. nanocellulose crosslinked with different mechanisms affects the prop­
synthesized citric acid crosslinked SF for biocompatibility. However, erties, thus varying the final outcome of the composite. Physical cross­
other crosslinking agents like gallic acid, ferulic acid and vanillin hold linking is mainly employed for fabricating SF/nanocellulose hydrogel,
promise for the production of non-toxic SF hydrogels [63]. The safest but has exhibited poor mechanical and water absorption properties
and most commonly used chemical crosslinking methods are enzymatic [91]. Mechanical properties can be improved by chemically crosslinking
crosslinking and radiation. The most used reagent is horseradish with different polymers like polyacrylamide to refine swelling behavior,
peroxidase [64], while carbonic anhydrase, tyrosinase and alcohol oxi­ rheological property and morphological changes obtained by varying
dase are some other reagents commonly used as crosslinking agents polymer ratios [92]. With a rapid gelation in approximately 3 min at
[65–67]. When SF was crosslinked to tyramine-substituted hyaluronic 37 ◦ C with 84% porosity and 25–66 µm pore size, semi-interpenetrating
acid (HA) with horseradish peroxidase, a tyramine–tyrosine bond was SF/polyacrylamide hydrogel was developed. These features enabled
formed with a relatable mechanical property to natural tissues [68]. The migration of keratocytes with no celluar toxicity for corneal tissue
abundant tyrosine in the SF can form di-tyrosine crosslinks through two regeneration [93]. Various other factors (stimuli sensitivity and
types of riboflavin (RF)-mediated oxidation mechanisms, direct oxida­ reversibility of sol-gel transition) can be manipulated to improvise
tion and singlet oxygen. In the former, tyrosyl radical is generated existing features. Collagen crosslinked with SF was studied for thermal,
through the direct reaction of photosensitized RF with tyrosine, while in viscoelastic, swelling, morphological properties and biocompatibility.
the latter, dissolved oxygen is used. Here, both reactions are between the The material proved to be stiff and thermally stable with the extended
adjacent tyrosyl radical forming the di-tyrosine link [69]. Similarly, mechanical property [94]. A variety of materials, such as collagen, hy­
laccase crosslinked the same materials to attain better structural sta­ droxyapatite, carbon nanotube and hyaluronic acid, have been added as
bility. Gelation kinetics, pore size and mechanical properties define the a reinforcement with SF for specialized applications and are summarized
success of the hydrogel as a system for a tissue engineering application. in Table 3.
Among these, a physical crosslinking technique such as temperature, Two-dimensional (2D) approaches by far could only poorly keep up
shear force and ultrasound leads to a simplicity of operation without with the complex tissue environment. With the possibilities brought by
toxic agent involvement. However, gelation kinetics and pore size are 3D models, the field has advanced for mimicking the physiological
not very easily controllable. Methods like using polar solvents and pH microenvironment by using the emerging 3D bioprinting technique.
promote the gelation process, but are mostly cytotoxic. Usage of sur­ Hydrogel bioinks are an emerging source for tissue biofabrication.
factants accelerated gelation, but their release leads to toxicity. Chem­ However, the precision, stability and reproducibility of the 3D-bio­
ical crosslinking helps to alter the gelation kinetics by the suitable printed structures are limited due to the lack of appropriate bio-inks.
selection of crosslinking agents. This method can yield elastic and Recently, multiple approaches through physical and chemical methods
porous gel structures, which are highly recommended for tissue growth. have been used to formulate robust bio-inks combing ionic, photo- and
New methods to improve functionalities are proposed very often by enzymatic-induced gelation processes for 3D bioprinting [95]. After
combining existing methods and this diversity can impart different consolidating according to previously mentioned information, it can be
characteristics according to needs. observed that SF alone cannot be 3D-printed, but should be blended with
other components to make a printable bio-ink [96]. The bio-ink should
2.3. SF composite hydrogels have the desired rheological property for it to be a successful 3D-print­
able compound. Since the bio-ink formulations including biological cues
Single and homopolymers may not be ideal for advanced TE appli­ are still under development, developing bio-ink with a natural material
cations as there may be a need for enhanced structural and functional like SF will boost its application in biofabrication. SF/gelatin after
properties in order to address the complex requirements of effective enzymatic crosslinking was developed for a cartilage TE application
tissue repair. Similarly, SF alone has its own limitation, which can be with a suitable mechanical strength and with the added benefit of cell
met by forming a composite, i.e., crosslinking a secondary material adhesion [97]. For materials like SF, it is also important to choose the
where the advantages of both materials increase the properties of the printing technique accordingly. Previously, inkjet printing, extrude
final product. Proteins/SF scaffolds are a type of SF composite in which printing and digital light printing were employed for the fabrication
collagen, keratin, or other proteins help to improve cell viability, pro­ using silk bio-ink [98–100]. The commonly used bioprinting technolo­
liferation, differentiation, and metabolism [82,83]. Polysaccharide/SF gies are based on digital light processing and laser-printing. Inkjet bio­
composites (chitosan/SF or HA/SF) alter surface properties, such as printing offers the advantages of cost-effectiveness and a moderate
roughness, to promote cell growth [84,85]. Synthetic polymers are printing speed similar to conventional 2D inkjet printing. However,
already well established in the biomedical industry and hence a syn­
thetic polymer/SF composite is used to fine-tune any desired property. Table 3
As an example, polymers such as polyethylene (PE), poly(methyl Properties of improved materials of SF composite hydrogel.
methacrylate) (PMMA), polylactic acid (PLA) and polyacrylamide are SF composite Application Properties Reference
already in use [86].
Graphene oxide BMSCs delivery Improved mechanical [33,103]
In a biocomposite, the important influences on mechanical proper­
nanosheet and structural properties
ties like stiffness and strength are the fibre volume fraction and the fibre Nanocellulose 3D-printing Micro-porous [91]
orientation. Aharonov et al. formulated a laminate of long silk fibres of biomimetic hydrogel
different fibre volume fraction and its orientation in alginate hydrogel. Polyacrylamide Nerve Controlled biomechanics [62]
Tailoring the matrix could mimic the tissue structures such as knee regeneration
Collagen Cell Improved biological [104]
meniscus. [87] Inorganic material like graphene can impart toughness to encaptulation functions
silk and integrate conductivity. Graphene oxide nanosheet was incor­ G’ > G’’
porated into SF hydrogel to improve the mechanical and structural Hydroxyapatite Bone tissue Highly porous scaffold [105]
integrity of hydrogel [88]. Graphene oxide/Fe3O4/carboxymethyl cel­ engineering
Carbon nanotube On-demand DOX Enhanced drug [106]
lulose/SF bionanocomposite was developed to address hyperthermia in
release circulation
cancer treatment. [89] Wu et al. produced a hydrogel by reducing Hyaluronic acid Vitreous humor Reduction of hydrogel [107]
HAuCl4 salt in situ to incorporate gold nanoparticles into chitosan and SF substitute stiffening
hydrogel with a much faster gelation time, thus indicating the strong G’ increased
intermolecular interaction of the polymers [90]. proportional to silk
concentration
SF/nanocellulose composites are being studied extensively as

4873
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

highly viscous materials cannot be used due to nozzle clogging. The genetically modified to express sequences of collagen and fibronectin
extrusion bioprinter is a modification of inkjet technology that uses an where the cell adhesion and calcium-binding activity were higher than
air pump or screw plunger to dispense bio-inks, this allowing the natural silk [116], but further studies to investigate its hydrogel
adaption of a range of viscous materials. The disadvantage is the me­ formulation are necessary.
chanical stresses on encapsulated cells when dealing with thicker
hydrogels. However, these issues can be overcome by digital light pro­ 2.5. Characterization of SF hydrogel
cessing (DLP) bioprinters. The ultraviolet (UV) light-induced photo­
polymerization technique allows a layer-by-layer model formation with The determination and characterization of hydrogel network pa­
approximately single micron resolution and a printing speed of rameters are crucial in the design of hydrogels in order to obtain the
30 mm/s, irrespective of size and complexity. DLP printing significantly desired structure and performance. Characterization methods aim to
enhances cell viability (85–90%) due to the short printing duration and quantify swelling, mesh size, bound and free water content, pore
nozzle-free technique [73]. Tao et al. formulated a regenerated SF so­ structure, chemical composition, the strength of chemical bonds, and
lution functionalized with gold nanoparticle and inkjet printed to be mechanical strength. Hydrogel characterization can be carried out by
used in applications ranging from photonics to bioimaging and therapy physiochemical methods (solubility, swelling measurements, rheology,
[95]. SF was photo-crosslinked with methacryloyl using lithium phenyl ultraviolet absorption spectroscopy, infrared spectroscopy, mass spec­
(2,4,6-trimethylbenzoyl) phosphinate (LAP) photo initiator at 365 nm troscopy, nuclear magnetic resonance [NMR], small-angle X-ray scat­
with an UV DLP printer. Kim et al. were able to successfully print several tering [SAXS], X-ray diffraction [XRD]) as well as by morphological/
structures that could mimic heart, lungs, trachea and blood vessels. The structural methods (scanning electron microscope [SEM], transmission
researchers were also able to compose human chondrocytes and human electron microscopy [TEM], differential scanning calorimetry [DSC],
turbinate-derived MSCs with tissue engineered trachea made of silk- atomic force microscope [AFM], and dynamic mechanical analysis
methacryloyl hydrogel for use in a rabbit trachea damage model [101]. [DMA]).
Xu et al., 3D-printed a hydrogel with gelatin, SF and methacrylate. A The most explored characterization technique in hydrogels is used to
prepolymer, photo-crosslinkabe SF-gelatin was first formed and formed understand their structural and functional properties. SEM is the
a hybrid hydrogel with the reactive methacrylate group. The matrix was exemplar of structural analysis in microscopy to analyze surface
used as a skin patch, which had adjustable mechanical behavior that morphology, roughness fracture [117], porous/non-porous architecture,
improved the fibroblast growth [102]. pore size [105,118], interconnecting network structure, [74] cross­
linking status [119] and effects of loading compounds [119,120].
2.4. Recombinant SF-based hydrogels Scanning probe microscopy is also used to observe the mechanical
properties. Hydrogel microstructure is specifically assessed using the
Traditionally, harvested silk has many limitations to be used in such SAXS technique [121]. Similarly, laser scanning confocal microscopy
broad and commercial industries as biomedicine. The structural (func­ (LSCM), together with fluorescent dye, generates Z-stacks to evaluate
tional) variation of each batch, impurities and the threat of other disease similar features [122,123]. Structural conformation in SF hydrogels can
transmissions always persist [108]. An alternative to tackle these risks is be determined using Fourier transfer infrared spectroscopy (FTIR),
to use biotechnological advances and genetic engineering to develop a while functional groups can be analyzed by FTIR and NMR. Absorbance
safe and consistent quality protein. In addition, incorporating the most at different infrared structural regions confirms the secondary and
modern computational methods to model and analyze crystalline structures of silk I and silk II. DSC can confirm structural
structure-function relations enable predictions. Production of recombi­ properties (silk I, silk II) through thermal degradation, which can also
nant protein is now well established, although it is a massive process to provide insight into thermal stability. Moreover, DSC allows a percep­
follow from DNA sequencing, designing recombinant DNA, cloning, host tion of the glass transition behavior of gel [124]. DSC and DMA
transformation (a commonly-used host is Escherichia coli) and induction contribute to characterizing the molecular chain dynamics. DMA was
to purification of the protein [109]. The entire framework can be also used to analyze the effect of hydration on mechanical properties
considered to be a hybrid system. With recombinant technology, silk­ [125] (Table 4).
–collagen, –laminin and –reflectin constructs have been explored to Rheological properties are equally important as mechanical and
understand the various options of combinational and structural designs biological properties for injectable hydrogels. To comprehend fluid
[110,111]. This synergistic approach enables SF to become a tailorable behavior, rheological measurements are performed to determine the
versatile material that can be turned into films, capsules, particles, crosslinking density and viscoelastic properties [124,126]. Water ab­
foams, hydrogels, microfibres and other therapeutic agents [112]. sorption influences the mechanical property and chain dynamics. The
Silk-elastin-like protein (SELP) is a genetically engineered polymer stability, hydration capability and tunability of the mechanical property
with amino acid motifs from silk (Gly-Ala-Gly-Ala-GlySer) and elastin of the formed hydrogel have been studied with respect to water intake. A
(Gly-Val-Gly-Val-Pro) that could control solubility, gelation, stimuli- water uptake study of hydrogels was performed in a controlled chamber
sensitivity, material strength and biodegradation [113]. Variants of a at specific conditions including temperature and percent relative hu­
SELP hydrogel chemoembolization agent results in blocking blood ves­ midity (% RH) [125]. The swelling ratio (Q) and water uptake (h) were
sels, thereby shrinking the tumor and delivering chemotherapy drugs. It determined by the following equation:
was observed that the sol-gel transition occurred at the point of interest
when tested in vivo in the rabbit. Hatefi et al. studied the prolonged Q=
Ws
release of the bioactive adenoviral vector by carrier SELP hydrogel Wd
where the controlled release profile was an impressive 28 days.
Ws − Wd
Hydrogels could also keep the infectivity of the cargo intact. Of interest, h=
Wd
SELP can be designed to be temperature sensitive [114]. Minimally
invasive injection of the liquid polymer in situ showed that the firm and Ws - weight of swollen SF gel at varying time intervals and
irreversible hydrogel was formed when stimulated by body temperature, conditions.
later releasing the encapsulated therapeutics upon gradual degradation Wd - dry weight of crosslinked SF gel.
of the matrix. When the SELP hydrogel carrying DNA in vitro for 28 days Functional analysis is a significant characterization segment where
was studied, no significant loss in molecular weight or bioactivity was the effect of solutes or external stimuli, such as absorption-related pa­
observed, thus confirming the potential of this material for controlled rameters, solute/monomer levels, and photostability, are examined. In a
gene delivery [115]. In other studies, transgenic silkworm was hydrogel delivery system, drug loading, diffusion and retention are

4874
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Table 4 characterization techniques, SF can be crafted into an ideal delivery

Silk fibroin hydrogel characterization technique. vehicle to carry drugs, cells and other molecules as discussed below
Characterization Parameters Characteristics and Reference (Table 5).
technique measured properties

Fourier Transform Molecular Secondary [127] 3. SF hydrogel as delivery vehicle

Infrared vibration and structures of silk
Spectroscopy (FTIR) rotation proteins 3.1. Drug
information
Raman Spectroscopy Vibration and Secondary structure [128]
rotation of content in silk
The basic necessity of a drug delivery system is the controlled release
molecules of the therapeutics at the targeted region in a predetermined course of
Circular Dichroism Conformational Secondary structure [129] time. Hydrogels in general are considered as an efficient model for drug
(CD) transition kinetics and conformational release, enabling controlled release approach for which SF is found to be
transitions of SF
more suitable. SF hydrogel has been considered as pH-responsive drug
Wide-Angle X-Ray Crystallite Crystal size, density [130]
Diffraction orientation and and ordering delivery material as it controls the passage of molecules with SF acting
(WAXD)/ Small- intensity of parameters as an amphoteric ion-exchanger. The chemotherapy drug, doxorubicin,
Angle Angle X-Ray diffraction was delivered with injectable silk hydrogels for a pH-dependent release,
Scattering (SAXS) patterns which lasted 8 weeks and provided a good antitumor effect [134]. The
AFM Force Degree of Mechanical [131]
Spectroscopy deflection of the information and the
release profile can be controlled by the molecular weight of SF or of the
cantilever versus internal blended material, if any. Pore size essentially influences the release
piezo movement nanostructure property by slowing down the release with smaller pores and can be
Imaging techniques Detect reflected or Morphological, [132] measurably formed by adding materials like glycerol to SF. They can
1. Scanning electron knocked off topological and
also reduce gelation time by increasing interchain interaction [135]. An
microscopy (SEM) electron compositional
2. Transmission Transmitted information analysis showed that a silk composite offers a more sustained release
electron microscopy electrons than SF alone and this was confirmed by a study of the physiochemical
(TEM) property of SF/polyacrylamide hydrogels [136]. SF has also shown a
Mechanical technique Enthalpy Mechanical [133] potential control release in cancer treatment by reducing the dosage
1. Differential Deformation characterization
while releasing the chemotherapeutic agent (bevacizumab) [137]. For
scanning (storage modulus
calorimetry (DSC) (E’) and tan (δ)) and pulp regeneration, a complex injectable hydrogel was formulated in
2. Dynamic hydration which initially Tideglusib was released followed by melatonin release.
mechanical analysis The hydrogel has electrospun fibers of melatonin (Mel)-PMMA/Tide­
(DMA)
glusib (Td)-SF carried by GelMA-thiolated pectin hydrogel [138]. The
sequential release of Mel and Td was studied in vitro and the biological
equally significant. Drug concentration in respective media is dynami­ performance of the released factors was confirmed by the proliferation
cally probed to measure the parameters (plotting concentration) against and differentiation of dental pulp stem cells into odontogenic differen­
time/percentage. The degradation property of the materials also tiation. These results encourage further studies in animals to promote
shadows the delivery system, thus aiding in controlled release. The dental pulp regeneration and hold great promise for the future. Song
material itself, or the added molecules, can bring about degradation. et al. addressed articular cartilage repair by forming SF/pullulan
Cell differentiation and migration are related to the elastic modulus of hydrogel embedding etanercept. The drug was dissolved in
the material and the degree of crosslinkage of the hydrogel. tyramine-SF/carboxymethylated pullulan and enzyme crosslinked. The
Given the large number of processing, crosslinking and system could regulate catabolic and anabolic dynamics in the

Table 5
SF hydrogel as a delivery vehicle for therapeutic factors.
Delivery system Active substance Application Fabrication method Experimental model Remarks Reference

pH responsive SF hydrogel DOX Breast cancer Self-assembling Nude mice tranplanted with Long-term antitumor [33,134]
MDA-MB-231 cells efficacy
SF hydrogel releasing anti- Bevacizu mab Ocular drug Sonication Intravitreal injection into Release over 91 days [137]
VEGF delivery the eyes of Dutch-belted
rabbits
SF-SWCNT-FA/DOX DOX- loaded folic Cancer therapy Blending KB cell line pH and temperature- [106]
acid dependentDOX release
SF hydrogel for photo- Biliverdin Glioma tumor Ultrasonication Balb/c nude mice with Photo-acoustic and [141]
thermal therapy subcutaneous glioma photo-thermal
properties
SELPs 815 K Thymidine kinase Oral cancer Direct loading CD-1 mice with squamous Prolonged release [148]
and luciferase cell carcinoma
SF-PEGDMA hydrogel TGF-β1 and bFGF Articular cartilage Entrapment Dental pulp Site specific release [154]
regeneration
SF hydrogel VEGF and BMP-2 Sinus floor Entrapment Rabbit sinus cavity Slow release of dual [176]
augmentation factors
Injectable SF hydrogel Insulin Diabetes Direct loading Diabetic T1DM Wistar rats Controlled release from [160]
porous scaffold
SF and phenylboronic acid Insulin Diabetes Polymerization Mouse skin Transdermal insulin [161]
/acrylamide hydrogel delivery
microneedle
SF hydrogel BMSCs Bone regeneration Blended and sonicated Calvarial defects in rats Prolonged survival of [167]
BMSCs
SF/pullan composite MSCs - HRP and HP enzyme- - Improved mechanical [170]
hydrogel mediated polymerization properties,

4875
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

neighboring chondrocytes and were able enhance bone marrow-derived the development of an intrinsic multi-stimuli-responsive hydrophilic SF
SC chondrogenesis in a rabbit osteochondral defect model [139]. hydrogel. The hydrogels were able to perform reversible thixotropic
In a very recent work, SF was blended with single-walled carbon gel− sol transition with a cycled shearing stress and resting procedure. In
nanotubes-folic acid/doxorubicin (SWCNT-FA/DOX) to form a nano­ addition, the drug release was guided by the acidity and reactive oxygen
composite injectable hydrogel. Here, silk was the carrier and the species (ROS) of the tumor environment and hyperthermia from the
hydrogel was made into a near-infrared (NIR), light stimuli-responsive external NIR irradiation. DOX/Cy7 demonstrated a long-term retention
system by SWCNT-FA/DOX. The NIR-stimulated low pH tumor area characteristic on intra-tumoral injection. This synergistic treatment
helped to release SWCNT-FA/DOX into the environment. Consequently, showed a better outcome and has great potential as a multi-responsive
the folate receptor-positive cancer cells will take up the content and drug delivery system (Fig. 4c) [142]. In a recent study, silk sericin is
undergo programmed cell death (Fig. 4a) [106]. In a similar study, drug also being explored and has showcased a wound healing capacity with
release was controlled through the application of an electric field and no inflammatory responses and an excellent cell internalization profile.
NIR laser. Additional heat was also produced in parallel with the The chitosan/alginate-silk sericin hydrogel was orally administered to
exposure to the radiation. The localized accumulation of the chemo­ locally accumulate in the colitis tissues.
therapeutic drug, apoptosis induced by external stimuli, and the suffi­
cient heat generated by the system combined to form a synergistic
strategy to eliminate the tumor (Fig. 4d) and pave the way for clinical 3.2. Genes
application [140].
SF/chitosan-based hydrogel formed by 1-ethyl-3-(3-dimethylamino­ For the last several decades, gene therapy has emerged as a potential
propyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) chemical treatment strategy for treating genetic disorders. Furthermore, gene
crosslinking carrying chlorhexidine digluconate (CHD) was developed therapy also holds potential to address the issues related to the clinical
against bacteria and fungi for wound healing applications. The high rate tissue reconstruction of the traumatic form of injuries. The major issue
of drug loading ensured long-term drug release. The combination of with this delivery system that still needs to be effectively addressed is
materials promoted antibacterial activity and reduced cytotoxicity the targeted delivery and controlled gene expression. SF-based systems
[143]. In most solid tumors where surgical resection and multidrug carrying biological cues, drugs and genes can be effectively manipulated
chemotherapy is essential, the local and perioperative delivery of che­ to form a more stabilized vehicle required to achieve the desired ther­
motherapeutics opens up a favorable route. A bioinspired biliverdin/SF apeutic effects and targeted release [145]. However, without a
hydrogel was developed for anti-glioma photo-thermal treatment (PTT) carrier-cargo stable interaction, it is almost impossible to achieve the
and subsequent tissue regeneration using photo-thermal effects. Under stability of active ingredients. In addition, apart from a few growth
NIR irradiation, the temperature of hydrogel rises above 45 ◦ C due to the factors, the stability is entirely dependent on the entrapment of the
presence of biliverdin, killing cancer cells in vitro and subsiding further cargo into the matrix [146]. SF-based biomaterials can withstand tem­
growth in vivo. Alongside, wound repair and tissue regeneration is su­ perature, humidity and physiological variations. Hence, antibiotics like
pervised by the system Fig. 4b [141]. To induce magnetic hypothermia erythromycin with low stability in water are stabilized in SF sponge
and for cancer elimination, a nanobiocomposite was prepared consisting [147].
of GO and Fe3O4 MNPs along with chitosan/SF crosslinked hydrogel SELPs with a controllable sequence and composition are soluble in an
[144]. A recent in situ synergistic robust cancer treatment study reported aqueous solution at room temperature. Viral gene carriers are mixed
with the material to form an insoluble hydrogel on intra-tumoral

Fig. 4. (a) Schematic representation of injectable hydrogel consisting of a carbon nanotube/silk fibroin for the localized release of an anticancer drug [106]. (b)
Representation of biliverdin/silk fibroin as a bifunctional hydrogel with photothermal and pro-regenerative properties [141]. (c) Injectable silk fibroin hydrogel with
a multi-responsive capacity for effective tumor therapy [142]. (d) Silk fibroin hydrogel consisting of a photo-electro nanocomposite for the chemotherapeutic de­
livery of breast cancer treatment [140].

4876
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

injection, which is apt for localized delivery. SELP 815 K was designed platform can also serve as a vessel for in vitro cell culture [153].
to carry and release adenoviral vectors in a head and neck tumor model For more efficient work progress, combination therapy with different
based on studies by Price et al. Gene-directed enzyme prodrug therapy growth factors can contribute to SC maturation following differentia­
was the treatment method adapted by delivering genes for a therapeutic tion. For example, studies have been conducted with a focus on different
enzyme to targeted cells. In this case, the administered non-toxic pro­ growth factors acting simultaneously, mainly in 3D hydrogels
drug was converted into a cytotoxic metabolite to kill transfected cells, mimicking natural tissue regeneration. To improve articular cartilage
which resulted in a remarkable reduction in tumor size, the longest time tissue regeneration, a semi-degradable SF-PEGDMA hydrogel system
to tumor rebound, and prolonged survival in a mouse tumor model. through a physically and photo-crosslinking mechanism for chondro­
Compared to the virus administered in poloxamer 407, SELP 815 K in­ genesis was developed [154]. The system carried transforming growth
jection showed an increased duration of expression, thus avoiding factor (TGF)-β1 loaded PLGA nanoparticles, dental pulp stem cells, and
possible immunological responses and lowered local toxicity caused by bFGF. This dual delivery system helped cell viability, proliferation and
adenoviral administration, although it did result in an encapsulated chondrogenic differentiation at the defect site to form a cartilage-like
hydrogel mass in the tissues [148]. structure. This synergy was brought into effect with the mechanical
Similarly, investigations on the spatial and temporal control over superiority of PEGDMA and the biological activity of fibroin, conse­
gene expression influenced by the concentration and structural confor­ quently controlling cellular responses. In parallel, the simultaneous
mations of SELP were conducted. In the series of studies conducted on a delivery of growth factors (bFGF and TGF-β1) increased glycosamino­
murine model of a head and neck tumor xenograft, the structure- glycan (GAG) and collagen type II production, thus boosting chondro­
dependent changes in degradation of SELP hydrogels were analyzed genic differentiation. The system was attested as a cost-effective,
and 815 K at 4 wt% polymer concentration showed the highest trans­ target-specific, tuneable delivery vehicle for cartilage tissue regenera­
fection efficiency and prolonged gene expression for 21 days. Viruses tion [154] and in situ-forming SF-gelatin hybrid hydrogel was developed
disseminating to the liver were also minimized. Among the samples, for the delivery of bFGF. However, at physiological temperature, the
SELP-815 K had the lowest degradation rate at equivalent concentra­ system was less stable and showed a significant release of growth factor.
tions of polymers and elastase. Further research is currently being car­ The sonicated SF hydrogel leached 50% of bFGF in 24 h, which was
ried out to evaluate the extent of the applications based on the polymer reduced to 42% after 72 h of incubation [153].
to deliver therapeutic adenoviruses [149]. Combination therapies using inorganic materials have also been used
to tune all properties, including the release of biological molecules.
3.3. Growth factor Magaz et al. designed a biohybrid composite in which SF was the
continuous phase. The electro-responsive element of the composite was
The cells attune themselves and their function by communicating reduced graphene oxide (rGO), which was employed as the dispersed
with each other through a series of molecular interactions, hormonal phase. With the conductive rGO, a long-term sustained release of nerve
activity, and locally and systematically acting mediators. Growth factors growth factor-β is made possible with the application of a pulsatile
are one such element regarded as a class of proteins that promotes cell electrochemical stimulus. The application level of this system was very
proliferation and differentiation in response to the requirements [150, impressive due to its ability to support cell survival in vitro and tissue
151]. These factors have to be protected and carefully delivered as they regeneration in vivo. At present, tailorable biohybrids (Fig. 5a) are a step
are sensitive to proteolytic degradation. Due to their instability under ahead of other approaches currently in use in the field of TE [155]. The
high temperatures and altered physiological conditions, novel ap­ study by Cai et al. of GelMA/silk graphene-based double-sided tapes
proaches are employed for the controlled release of growth factors for represents a novel approach for promoting diabetic peripheral nerve
enhancing their therapeutic efficacy. repair, where the tape was loaded with gradients of netrin-1 growth
SC-based TE holds great promise in regenerative medicine. A typical factor for directional alignment. A diabetic mouse model was used for
cell proliferates, differentiates and survives in its niche, which is the repair of a 7.5 mm sciatic nerve defect. Interestingly, animals treated
orchestrated by extracellular matrix (ECM). SF is a unique natural pro­ with a gradient of netrin-1 resulted in enhanced directional axonal
tein polymer that perfectly fits most criteria for such an approach. regeneration and concomitant myelin recovery and functional restora­
Kaplan and Zhang et al. assessed the in vitro and in vivo behavior of rat tion as evidenced by the sciatic functional index. Thus, these results
BMSCs by the lenti-green fluorescent protein tracking of porous silk demonstrate the benefits of the gradient formation of netrin compared to
scaffolds formed after gelation in cranial bone defects. An increase in the all other animal groups for nerve repair applications. Notably, this
number of seeded cells from 2 days to 2 weeks after implantation was system showcases many aspects of a potentially good design with good
observed and a consequent moderate decrease at 8 weeks. Emphasizing mechanical support, biocompatibility, quick curing, and extended
the duration of cell survival, some cells also differentiated into endo­ growth factor delivery. The design also protected the animals from
thelial cells and osteoblasts. Moreover, angiogenesis and osteogenesis muscle atrophy [156].
were promoted by the addition of vascular endothelial growth factor
(VEGF) and bone morphogenic protein-2 in the scaffolds, thus proving 3.4. Peptide
the efficiency of the system [152].
Hybrid hydrogels are a typically heterogeneous system that can Peptides are profusely found in enzymes, hormones, structural ele­
ensure adequate cell organization, cell-cell interactions and adhesion. ments and antibodies. They are unable to easily move across cells due to
An in situ crosslinked hydrogel hybrid system modelled by Bragg et al. their poor penetration capability and thus need assisted transport for
demonstrated controlled growth factor sequestering and release over therapeutic strategies. Given their structural network, porosity, sus­
time. The striking difference from other platforms was that sonicated SF tainability and stimulated responses, hydrogels play a significant role as
was used to entrap bioactive gelatin or heparin-conjugated gelatin, thus the carrier of peptides [157,158]. For the continuous supply of insulin, a
employing a secondary crosslinking mechanism. Genipin was used to self-regulated injectable hydrogel was developed with optimum
reinforce the design and gelation kinetics were systematically moni­ mechano-physical properties. Such systems help in minimally invasive
tored. Notably, genipin additionally contributed to a ~40% increase in delivery with no surgical implications and accompanying risk [159].
the sequestration of basic fibroblast growth factor (bFGF) from SF/ Maity et al. prepared an injectable hydrogel for insulin delivery
heparin-conjugated gelatin (SSF-GH) to SF/genipin crosslinked (Fig. 5b) and also investigated the effect of adding ethylene glycol (EG)
heparin-conjugated gelatin (SSF-GH-GN) after 72 h. The crosslinks also and triethylene EG for the gelation process. By introducing the additives,
slowed the release of bFGF by 15% from SSF-GH to SSF-GH-GN. In the transition from the random coil to the β-sheet structure was facili­
addition to its ability for the sustained delivery of growth factors, the tated. At varying concentrations, a quick gelation with retention of the

4877
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Fig. 5. (a) Electro-responsive materials consisting of β-NGF loaded silk fibroin and graphene oxide [155]. (b) Formation of injectable silk fibroin hydrogel as a
sustained delivery system for insulin [160].

injectable property with a mesoporous nature was developed for sub­ for a wide range of diseases and injuries (Table 6). However, advance­
cutaneous injection. Human recombinant insulin was encapsulated in SF ments are impeded by the low rate of cell survival and the uncontrolled
hydrogel and injected without affecting its structural and functional differentiation of the SCs. Hydrogels can be regarded as a scaffold sys­
integrity in streptozotocin-induced T1DM Wistar diabetic rats. The in­ tem for cell encapsulation, which can even recapitulate the ECM with
sulin was slowly deployed from under the skin for 4 days to maintain additional fine-tuning of the elastic modulus, chemical functional
glucose levels [160]. Chen et al. developed hydrogel-forming micro­ groups and other properties of the material. Apparently, non-modified
needles by a two-layer fabrication strategy to deliver insulin in a silk hydrogels can easily be one such material, given their adsorption
non-invasive manner transdermally. The base layer of microneedles was of proteins such as fibronectin and their ability to mimic endogenous
the stable and mechanically stiff SF and glucose-responsive SF combined ECM. Although silk does not possess integrin binding domains, the
with a semi-IPN network hydrogel formed the needle region. The system meticulously functionalized silk molecules can open up new possibilities
showed microporosity with interconnected structures. The hydrogels for the use of silk with novel functions [162].
retained their structural integrity in an aqueous environment for 7 days Davis et al. entrapped islets and mesenchymal stromal cells in silk
and released insulin on demand accordingly, thus maintaining the hydrogels to study how the combination improves islet transplantation
glucose level. Their biodegradation addressed all safety concerns for type 1 diabetes. In the in vitro studies performed, an improved insulin
regarding the residues and could replace subcutaneous self-injection for response was noted, due to the increase in gene expression for insulin I,
diabetes management [161]. insulin II, glucagon and pancreatic and duodenal homeobox 1 (PDX-1)
[163]. In a related work, an in vivo evaluation on a streptozotocin-in­
duced diabetic mouse model was conducted where marginal pancreatic
3.5. Cells
islets and pelleted pancreatic islets were co-encapsulated with MSCs in a
SF matrix. Both were able to control the blood glucose level, the former
Researchers have made massive efforts to find cell-based solutions
in 4 days and the latter within 15 days. The combination of the two types
of islets with MSCs required 9 days to control blood glucose. Addition­
Table 6 ally, the system was able to induce the expression of VEGF, which helped
Silk fibroin hydrogel for the encapsulation of therapeutic cells.
the hydrogels to appropriately function and survive. MSCs aided in the
Hydrogel Cell type Application Outcome Reference expression of trophic and angiogenic factors, in turn supporting the
delivery
function of the graft. Without the added SCs, the grafts failed to control
system
the blood glucose [164].
SF hydrogel BMSCs Bone Prolonged [167] MSCs can self-renew and differentiate to specialized cells and hence
regeneration survival of
BMSCs
are used frequently for regenerative purposes. MSCs have a unique
SF/pullan MSCs Cell delivery Improved cell [170] cross-talk with hematopoietic cells, which empowers them with immu­
composite survival nosuppressive characteristics. Therefore, MSCs blend in perfectly for
hydrogel autologous and allogeneic transplantation, which is also due to their low
SF hydrogel Muscle- Angiogenesis Enhanced tissue [174]
immunogenicity. Martín-Martín et al. proved that 3D SF hydrogel matrix
derived Muscle regeneration
MSCs regeneration releases neuroprotective and neuroregenerative factors, although the
SF-Alginate MSCs Tissue Enhanced cell [194] release rate was higher for non-encapsulated MSCs. Encapsulated MSCs
Neural engineering survival survived for several days in the matrix, but an over-secretion of TFG- β1
progenitor (anti-inflammatory factor) was observed. Thus, further investigation is
cells
necessary to understand the MSCs secretome in functionalized SF, as
SF-DMPG L929 Tissue Biocompatible [175]
NIH/323 engineering Cytoprotective well as in their composites with organic and inorganic materials and
SaOS-2 polymers (Fig. 6c). When taking a step forward to pre-clinical and
CaSki clinical analysis, it is imperative to contemplate the therapeutic effect of
SF-DMPG SaOS-2 Bone tissue Osteogenic [173]
the desired therapeutic molecule to know to what extent these molecular
engineering differentiation
SF-chitosan Hepatocytes Tissue Enhanced [172] groups are responsible for secretions and to identify their release profiles
engineering functional in order to open doors for their sustainable application [165]. To cater
phenotype for two distinct functions: sustained release of the drug and adipogenic
SF microgel L929 Tissue VEGF release [51] inducer, an injectable and bioresorbable SF was developed. Inter­
Myoblast engineering
estingley, DOX had a sustained release to 21 days, which contributed for
cells

4878
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

Fig. 6. (a) Preparation of porous silk fibroin hydrogel for the delivery of bone marrow SCs for bone regeneration applications [167]. (b) Microscopic data showing
the cardiac mesenchymal SCs cultured on PEGylated silk fibroin hydrogel substrate over 14 and 21 days. Scale bar = 10 µm [168]. (c) Secretome profile resulting
from silk fibroin hydrogels carrying hMSCs [165]. (d) Differentiation of hMSCs into smooth muscle cells using silk fibroin hydrogel features with different mechanical
properties [169].

tumour supression and overcame recurrence, while dexamethasone was monitored. Furthermore, after the gelation process, the inner seeded
released for 30 days, which helped in the adipose tissue-derived SC cells with the scaffold were implanted in calvarial defects in rats after
differentiation for tissue reconstruction. The molecules retained their osteogenesis (Fig. 6a) [167]. Chena et al. developed a pullulan-SF
stability and bioactivity during the slow and sustained release [166]. copolymer hydrogel for MSC delivery to provide cell-specific epitopes
Floren et al. studied the effect of SF stiffness and TGF-β1 on the that are lacking in SF alone. The advantages of both materials highlight
vascular commitment of hMSCs. Providing the required stiffness and the importance of hydrogel as a biodegradable in situ forming an
TGF-β1 and the upregulation of myosin heavy chain expression was injectable carrier system. This hydrogel formed through
demonstrated in 72 h. Additionally, by modulating stiffness, a direc­ enzyme-mediated polymerization was studied for its gelation time,
tional hMSC differentiation was made possible. Hence, a well-defined swelling behavior, rheological properties and the mechanical property
and specialized biomaterial can be made out of SF that fits the TE of different pullulan concentrations used. The composite increased
portfolio [169]. Ciocci et al. developed a PEGylated injectable SF fracture strength, breaking elongation and compressive reproducibility
hydrogel with PEG diacrylated (PEGDa) through a sol-gel reaction. when individual materials were considered. The potential of this cell
Molecular characterizations of the SF and PEGylated SF (molecular delivery system in musculoskeletal TE was assessed in vitro with rabbit
weight of 20 and/or 6 kDa) were carried out by reverse-phase high-­ BMSCs [170]. Ni et al., 3D-printed hydrogel with SF and hydrox­
performance liquid chromatography circular dichroism, and FTIR). ypropylmethylcellulose of methacrylation (HPMC-MA) embedded with
During the mild photochemical sol-gel reaction, a pliable encapsulation BMSC. The material had a modulus of 0.1–10 MPa, similar to that of
of resident human cardiac MSCs (cMSCs) was possible, demonstrating natural bones. As for any 3D-printed hydrogel, SF and HPMC-MA ensure
the cytocompatibility of the system. The albumin-perfluorohexane mi­ a sufficient nutrient supply, great biochemical supportabilit, and
crospheres were embedded inside the matrix of SF and this bio­ excellent mechanical properties. The survival and proliferation of
functionalization encouraged biological cues to promote cell BMSCs in the printed scaffolds have been studied to understand the
attachment, growth and function. This natural micro-environment hel­ chondrogenesis-related gene expression for their application in cartilage
ped in the expression of myosin and actinin (Fig. 6b). Microspheres TE [171]. Tan et al. studied the effect of SF hydrogel stiffness and
increased the viability of the stem cells and helped in their proliferation TGF-β1 in upregulating myosin heavy chain expression (Fig. 6d). hMSC
in the microsphere modified PEGylated SF 3D hydrogel matrix [168]. differentiation for TE applications was also investigated [169].
BMSCs were delivered using an SF hydrogel, which was earlier sonicated Gholami et al. developed a 3D delivery vehicle for hepatocytes using
for β-sheet structure into a specifically shaped 3D system for their SF and chitosan. This injectable system is also thermoresponsive due to
growth. The transportation of molecules and proteins into the gels was the addition of glycerophosphate that can further undergo sol-gel

4879
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

transition. Hydrogels with varying amounts and compositions of poly­ and TE scaffold. The primary factors are: 1) load-bearing capacity; 2)
mer and gelling agents were formed and evaluated for mechanical porosity; 3) cell-matrix interactions; and 4) the possibility to adjust the
properties and cytocompatibility. The entire structure of gel composi­ release profile to achieve the therapeutic concentration of the drug
tion of 1.3 and 1 wt/volume percentage chitosan and SF, respectively, [178]..SF is such a protein fibre and can be considered as an efficient
with 0.05 M sodium hydrogen carbonate and 30% glycerophosphate, controlled delivery material in view of its controllable crystallinity and
were layered and featured with 100 µm pores, which makes it ideal for a molecular conformation.
variety of tissue engineering applications. Modulus was increased by 6 SF hydrogels have been highlighted for a variety of applications due
kPa for this composition, while the addition of genipin also further to their excellent physical and chemical properties [75,179–182] and
increased the modulus. The sample did not compromise the viability of much effort is being made to comprehend the drug release mechanism of
cells and had a modest haemolytic effect. More importantly, the liver- different concentrations and formulations. To perceive an exact point of
specific activity was studied to understand the functioning of the he­ release is almost impossible, leaving only near-zero order guidance to
patocytes used. A significantly enhanced synthesis of urea in hydrogel elucidate the kinetics. However, given the number of studies carried out
encapsulating HepG2 was confirmed using quantitative analysis by 3 on SF delivery systems, the factors affecting release can be narrowed
days after cell encapsulation [172]. down to diffusion, swelling and degradation. Fick’s laws of diffusion
To promote angiogenesis and muscle regeneration, a SF scaffold was describe drug mass transport in diffusion-controlled release through the
prepared with 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol following equation:
(DMPG) sodium salt with comparable mechanical properties to natu­
dCA
ral soft tissue. The encapsulated muscle stem cells were extracted from JA = − D
dx
sheep. The resulting scaffold was tested in an adult sheep model. The
seeded cells formed new muscle cells with a faster growth rate until day dCA d 2 CA
9 and then at a slower rate. They also promoted angiogenesis when a dt
=D 2
dx
10% DMPG SF solution scaffold was used and confirmed by immuno­
histochemical staining of CD34. However, the study was conducted only JA - Diffusive flux of the drug.
for a limited time of two weeks. In brief, the scaffold holds the potential D - Diffusion coefficient.
to be a viable substitute for ECM [174]. Similarly, DMPG and SF were CA - Concentration of drug in the release medium.
used to form a hydrogel with controlled gelation varying the lipid con­ x - Position.
centration (5,10, and,15 mM DMPG/3% SF). As the concentration of t - Time.
DMPG increased, the degradation profile decreased. Four different cell Here, the drug diffusion coefficient is assumed to be constant and the
types were encapsulated in the matrix: L929, NIH/3T3 (fibroblastic cell initial drug concentration is observed to be lower than drug solubility.
lines), SaOS-2 and CaSki (cancer cell lines). The fibroblast had a normal Among other parameters, an ideal sink condition should be established
proliferation rate, but cell adhesion was of concern. SaOS-2 maintained for this model. The conditions to be satisfied can vary with different
their shape and proliferated for 7 days and remained viable for 21 days. geometrical shapes and edges. Swelling behavior, polymer dissolution/
CaSki cell growth decreased with a higher concentration of DMPG degradation and chemically-controlled delivery have been studied in
[175]. Subsequently, DMPG-induced SF hydrogel was prepared and complex mechanistic models [183], but these models have yet to be fully
loaded with dexamethasone and osteoblastic SaOS-2 cells. The drug implemented for silk-based delivery systems.
loaded underwent a burst release in the first 6 h. The remaining drug The Peppas model (or power law model) has been adapted to
was released gradually over 28 days, indicating the capability of compare the resulting release kinetics of different formulation parame­
hydrogels for the prolonged release of entrapped molecules. Cell growth ters, although the model is based on empirical or semi-empirical
increased steadily during the first week and then plateaued at 28 days mathematical models and not on true release mechanisms [184]. The
with cells mostly in S phase. The APL (alkaline phosphatase) activity test Peppas equation is as follows:
confirmed the osteogenic differentiation of the cells. The study also Mt
confirmed calcium production through alizarin red staining. Impor­ = ktn
M∞
tantly, this study reveals that hydrogels can carry drugs and cells effi­
Mt - Cumulative amount of drug released at time ‘t’.
ciently with their functionalities intact [173].
M∞ - Cumulative amount of drug released at infinite time.
L929 was also encapsulated in SF microgel by Bono et al. using the
k - Constant for the drug delivery system.
batch emulsion method. Their proliferation and VEGF secretion were
n - Release exponent.
evaluated. Survival of myoblasts was confirmed by the expression of
As a matrix for drug delivery, SF hydrogels were prepared by de
Ncam1 and Pax3 proliferation markers by qRT-PCR. VEGF levels were
Moraes et al. by accelerating the gelation kinetics of fibroin by adding a
found to increase up to 22 pg mL− 1 by day 7. Similar to a previous work,
varying content of ethanol. Ethanol acts on SF by dehydrating the fibroin
the shape and size were well maintained, thus meaning that SF micro­
molecules, thus preserving the chemical and conformational properties
gels are stable and can exchange metabolites [51]. It is evident from the
of fibroin hydrogel, despite the amount of ethanol. When evaluating the
above-mentioned studies that SF is an excellent candidate for cell
release profile of diclofenac sodium dissolved in (a) ethanol and (b)
encapsulation for cell structural and functional preservation. With
water, the former presented a more sustained drug release from SF
different approaches, SF can be fabricated into matrices for cell growth
hydrogel [117].
and delivery.
A double-release study investigated SF hydrogels containing fluo­
rescent dye-incorporated SF nanoparticles, but the release of nano­
3.6. SF hydrogel and its drug release profile
particles from hydrogels showed a prolonged release, while the dye was
released quickly and sustained the release for 5 days [182]. However,
The mastery of polymers that artistically deliver films, gels, capsules,
although the dye was incorporated, its influence over the material
tablets, creams and other therapeutic agents as carrier systems is key to
interaction was overlooked in the study. Tomado et al. also studied the
the most important aspects of controlled drug release, such as long-
properties of dye-incorporated SF microparticles containing SF hydro­
duration sustained drug release, low toxicity, manipulation of the car­
gel. A study of the evaluation of matrix interaction and release aimed at
rier and easy administration of cargo. Polymer stands as an experienced
using different dyes and properties. Dyes such as methylene blue (MB),
and well-qualified delivery system and has been abundantly studied
rose bengal (RB), rhodamine B (RhB), and neutral red (NR) with a
since its well-deserved recognition [177]. Each of these materials should
different charge, hydrophilicity, molar mass, physicochemical
be developed and reinforced to cater to the needs for controlled delivery

4880
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

properties and interaction were studied to expand knowledge on their body parts. Silk hydrogel 3D-printing is carried out primarily through
application as a delivery vehicle (Fig. 7) [185]. extrusion and inkjet printing among other methods that are recently
In a further study, the influence of hydrophilic and electrostatic in­ being investigated [188,189]. The shape fidelity of the extrusion 3D-
teractions over the common hydrophobic interactions was evident in the printed silk cannot be guaranteed due to silk viscosity. Silk nanofibers in
loading of dyes, thereby performing a more effective fusion with mi­ combination with several other materials can improve the mechanical
croparticles. Indeed, a 10-fold longer release time was observed in a outcome of printed silk and modify rheological properties to make silk
hydrogel containing SF microparticles with dye (~900 min) compared desirable for printing [190–192]. Devising new formulations, charac­
to dye release from microparticles (~90 min). In contrast to the Fickian terizing and standardizing them can give access to different aspects
diffusion observed in the release of dyes from SF microparticles, the including shape fidelity, avoiding cytotoxicity and other consequences
anomalous mechanism and transport of Case II mechanism were pre­ from the additives used if any, to allow their use in clinical application
dominant, thus suggesting that the matrix degradation directly influ­ [193].
enced dye release. The study confirmed that the scheme of double Among other forms, SF hydrogel is the “pick of the litter” for delivery
release prolonged the release and a better understanding of the influence applications for drugs, cells and growth factors to name but a few, due to
of other factors, such as load, hydrophilicity and the size of the mole­ its biofriendly nature, multifunctionality and mechanical superiority.
cules in dye release from the matrix [185]. Certain aspects that add to their extensive use include injectability, self-
In another release study, Fang et al. characterized the drug release healing property, adhesion, environmental responsiveness, anti-
pattern and the physicochemical properties of low (18 kDa) and high bacterial activity and 3D printability. SF hydrogels have already been
molecular weight (76 kDa) silk protein (SP) hydrogels. Silk protein used in established areas (adhesives, sutures, wound healing, myocar­
hydrogels are composed of hydrophilic amino acids with both beta-sheet dial patches, wearable biosensor) and unmet needs with existing tech­
and random coil conformation; formed hydrogels had interconnecting nologies and thus adding novel approaches. From delivering and
pores. Hydrogen bonding is important for the linkage of hydrogels and releasing single components to a complex multi-component system
the study showed a structural conversion from crosslinks to sheet shape controlled spatially and temporally, SF hydrogel systems have evolved
when SPL was incorporated into SPH hydrogel [179]. to respond to internal triggers and external stimuli, with localized
controlled release corresponding to enzymes and cellular events. SF
4. Challenges and future perspectives could nest and nurture therapeutic drugs, SCs and other cargo
mimicking extracellular matrix and allow the release of optimal dosage
The fundamentals of silk in itself stand out with its overall appeal, by modulating the concentration over time. Moving forward at this rate
thus making state-of-the-art research of silk still relevant. Notably, the of precision, SF hydrogel can optimize the implant structure, maintain
curiosity revolving around the performance of silk in cross-disciplinary material property, and control release behavior conveniently and safely,
research has never ebbed throughout several decades. These abundantly which otherwise cannot be achieved through traditional approaches.
available fibres, once used as garments, have evolved impressively to The near future of the use of SF hydrogels will certainly be the surge
heal wounds and treat cancer and more. Regardless, thus hinting that no in clinical applications. Although recognized as an exceptional material,
material is perfect, silk comes with disadvantages. The necessity for SF hydrogels have yet to be moved towards clinical trials and US Food
purification to remove type 1 allergic sericin residues is a requisite for it and Drug Administration approval. Although the materials lack certain
to be used for clinical applications. For instance, SF sutures perform aspects, such as poor attachment of certain cell types and mechanical/
better than sutures made by synthetic materials [186]. Silk-based physical properties, crosslinking, blending or the conjugation of other
products have the tremendous potential to be an ideal healthcare ma­ moieties help to eliminate these issues and enhance functionality. The
terial, mainly due to their ability to mimic extracellular matrix [187]. challenges posed by the significant toxicity of chemical crosslinking can
However, despite numerous clinical translations and research products, be overcome by various physical crosslinking techniques. As high­
its real world application has yet to be endorsed by regulatory lighted, silk possesses the great property to be a perfect biomedical
authorities. material. However, to reach clinical studies, many hurdles remain. Cost-
Processed silk fibers molded to diverse forms and designs, including effectiveness has still to be achieved and bulk-produced SF with a
fibers, films, scaffolds and hydrogels, have emerged as an inevitable consistent property has yet to be accomplished and therefore new so­
representative in biomedical science. 3D printing being a revolution per lutions should be explored. To satisfy the demands of commercializa­
se, bioprinting is the immediate future in creating human tissue and tion, SF should meet criteria such as prolonged shelf life, and ease of

Fig. 7. Inference of the investigation conducted by Tomado et al. on assessing the interaction within silk fibroin matrix and its subsequent release properties.

4881
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

application according to need (in situ gelation for wound healing, constructs. Mater Today Bio 2019;1:100008. https://doi.org/10.1016/j.
mtbio.2019.100008.
injectability for tissue regeneration/drug delivery).
[7] Nicolas J, Magli S, Rabbachin L, Sampaolesi S, Nicotra F, Russo L. 3D
extracellular matrix mimics: fundamental concepts and role of materials
5. Conclusion chemistry to influence stem cell fate. Biomacromolecules 2020;21:1968–94.
https://doi.org/10.1021/acs.biomac.0c00045.
[8] Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell
This review provides an overview of the emergence of SF hydrogels culture. Biotechnol Bioeng 2009;103:655–63. https://doi.org/10.1002/
as a drug carrier system in biomedicine, including their evolution, bit.22361.
[9] El-Sherbiny IM, Yacoub MH. Hydrogel scaffolds for tissue engineering: progress
fabrication, characterization and application in tissue engineering.
and challenges. Glob Cardiol Sci Pr 2013:2013. https://doi.org/10.5339/
Compared to other materials, SF has a strong potential regarding gcsp.2013.38.
biodegradability, cytocompatibility and ease of availability. As a natural [10] Sun TL, Kurokawa T, Kuroda S, Ihsan ABin, Akasaki T, Sato K, et al. Physical
material, silk has some drawbacks, but a focus on the improvement of its hydrogels composed of polyampholytes demonstrate high toughness and
viscoelasticity. Nat Mater 2013;12:932–7. https://doi.org/10.1038/nmat3713.
properties has significantly widened its range of applications. SF can be [11] Appel EA, Tibbitt MW, Webber MJ, Mattix BA, Veiseh O, Langer R. Self-
considered as a safe biomaterial from an excellent natural source that assembled hydrogels utilizing polymer–nanoparticle interactions. Nat Commun
can be gathered, processed, manipulated and developed to meet a wide 2015;6:6295. https://doi.org/10.1038/ncomms7295.
[12] Das S, Bora U, Borthakur BB. 2 - Applications of silk biomaterials in tissue
range of medical needs. Future in vivo studies should be conducted to engineering and regenerative medicine. Kundu SCBT-SB for TE and RM, editor.
further explore the potential of SF in drug delivery and tissue engi­ Woodhead Publishing,; 2014. p. 41–77. https://doi.org/10.1533/
neering applications in order to connect the bridge between laboratory 9780857097064.1.41.
[13] Kon’kov AS, Pustovalova OL, Agapov II. Biocompatible materials from
success and the clinical application. regenerated silk for tissue engineering and medicinal therapy. Appl Biochem
Microbiol 2010;46:739–44. https://doi.org/10.1134/S0003683810080028.
Funding [14] Gobin AS, Froude VE, Mathur AB. Structural and mechanical characteristics of
silk fibroin and chitosan blend scaffolds for tissue regeneration. J Biomed Mater
Res Part A 2005;74A:465–73. https://doi.org/10.1002/jbm.a.30382.
The study was financially supported by a EUROSTARS (E!10668) [15] Lefèvre T, Rousseau M-E, Pézolet M. Protein secondary structure and orientation
research grant to SM. Further funding to SM, was provided by the in silk as revealed by raman spectromicroscopy. Biophys J 2007;92:2885–95.
https://doi.org/10.1529/biophysj.106.100339.
Department of Surgery at Geneva University Hospitals and the Univer­ [16] Tomeh MA, Hadianamrei R, Zhao X. Silk fibroin as a functional biomaterial for
sity of Geneva, and the Geneva University Hospitals (HUG) Private drug and gene delivery. Pharmaceutics 2019:11. https://doi.org/10.3390/
Foundation (RC08–21) for Research, Geneva, Switzerland. pharmaceutics11100494.
[17] Phillips DM, Drummy LF, Conrady DG, Fox DM, Naik RR, Stone MO, et al.
Dissolution and regeneration of bombyx mori silk fibroin using ionic liquids.
Declaration of Competing Interest J Am Chem Soc 2004;126:14350–1. https://doi.org/10.1021/ja046079f.
[18] Reizabal A, Costa CM, Pérez-Álvarez L, Vilas-Vilela JL, Lanceros-Méndez S,
Reizabal A, et al. Silk fibroin as sustainable advanced material: material
The authors declare that the research was conducted in the absence properties and characteristics, processing, and applications. Adv Funct Mater
of any commercial or financial relationships that could be construed as a 2023;33:2210764. https://doi.org/10.1002/ADFM.202210764.
potential conflict of interest. [19] Mohammad E. Khosroshahi. Applications of Biophotonics and Nanobiomaterials
in Biomedical Engineering. 2017. https://doi.org/https://doi.org/10.1201/9781
315152202.
Acknowledgements [20] Vollrath F, Knight DP. Liquid crystalline spinning of spider silk. Nature 2001;410:
541–8. https://doi.org/10.1038/35069000.
[21] Rockwood DN, Preda RC, Yücel T, Wang X, Lovett ML, Kaplan DL. Materials
The authors would like to thank the team of the Department of fabrication from Bombyx mori silk fibroin. Nat Protoc 2011 610 2011;6:1612–31.
Plastic, Reconstructive and Aesthetic Surgery, Geneva University Hos­ https://doi.org/10.1038/nprot.2011.379.
pitals, for their scientific and technical support. Special thanks to Karin [22] Dal Pra I, Freddi G, Minic J, Chiarini A, Armato U. De novo engineering of
reticular connective tissue in vivo by silk fibroin nonwoven materials.
Uelfeti, Patricia Berset-Neuhaus and Rosemary Sudan for their support. Biomaterials 2005;26:1987–99. https://doi.org/10.1016/j.
The authors would like to thank the copyright owners for enabling the biomaterials.2004.06.036.
use of their images in this manuscript. [23] Unger RE, Wolf M, Peters K, Motta A, Migliaresi C, James Kirkpatrick C. Growth
of human cells on a non-woven silk fibroin net: a potential for use in tissue
engineering. Biomaterials 2004;25:1069–75. https://doi.org/10.1016/S0142-
Authors’ contribution 9612(03)00619-7.
[24] Reneker DH, Chun I. Nanometre diameter fibres of polymer, produced by
electrospinning. Nanotechnology 1996;7:216–23. https://doi.org/10.1088/0957-
SM conceived the idea and designed the study. APM performed a 4484/7/3/009.
review of the literature, and wrote the manuscript. SM critically [25] Ayutsede J, Gandhi M, Sukigara S, Micklus M, Chen H-E, Ko F. Regeneration of
Bombyx mori silk by electrospinning. Part 3: characterization of electrospun
reviewed the article, revised the article and provided the feedback and nonwoven mat. Polym (Guildf) 2005;46:1625–34. https://doi.org/10.1016/j.
edits on all aspects of the manuscript. SM and APM read and approved polymer.2004.11.029.
the submitted version. SM acquired the funding and supervised the [26] Chen C, Chuanbao C, Xilan M, Yin T, Hesun Z. Preparation of non-woven mats
from all-aqueous silk fibroin solution with electrospinning method. Polym
project. (Guildf) 2006;47:6322–7. https://doi.org/10.1016/j.polymer.2006.07.009.
[27] Minoura N, Aiba SI, Higuchi M, Gotoh Y, Tsukada M, Imai Y. Attachment and
References growth of fibroblast cells on silk fibroin. Biochem Biophys Res Commun 1995;
208:511–6. https://doi.org/10.1006/bbrc.1995.1368.
[28] Dong-Oh Han. Healing effects of astragali radix extracts on experimental open
[1] O’Brien FJ. Biomaterials & scaffolds for tissue engineering. Mater Today 2011;14:
wounds in rats. J Physiol Pathol Korean Med 2005;19:92–7.
88–95. https://doi.org/10.1016/S1369-7021(11)70058-X.
[29] Karageorgiou V, Meinel L, Hofmann S, Malhotra A, Volloch V, Kaplan D. Bone
[2] Dzobo K, Thomford NE, Senthebane DA, Shipanga H, Rowe A, Dandara C, et al.
morphogenetic protein-2 decorated silk fibroin films induce osteogenic
Advances in regenerative medicine and tissue engineering: innovation and
differentiation of human bone marrow stromal cells. J Biomed Mater Res Part A
transformation of medicine. Stem Cells Int 2018;2018:2495848. https://doi.org/
2004;71A:528–37. https://doi.org/10.1002/jbm.a.30186.
10.1155/2018/2495848.
[30] Freddi G, Romanò M, Massafra MR, Tsukada M. Silk fibroin/cellulose blend films:
[3] Katari R, Peloso A, Orlando G. Tissue engineering and regenerative medicine:
preparation, structure, and physical properties. J Appl Polym Sci 1995;56:
semantic considerations for an evolving paradigm. Front Bioeng Biotechnol 2015;
1537–45. https://doi.org/10.1002/app.1995.070561203.
2:1–6. https://doi.org/10.3389/fbioe.2014.00057.
[31] Nazarov R, Jin H-J, Kaplan DL. Porous 3-D Scaffolds from regenerated silk
[4] Williams D. Benefit and risk in tissue engineering. Mater Today 2004;7:24–9.
fibroin. Biomacromolecules 2004;5:718–26. https://doi.org/10.1021/
https://doi.org/10.1016/S1369-7021(04)00232-9.
bm034327e.
[5] Howard D, Buttery LD, Shakesheff KM, Roberts SJ. Tissue engineering: strategies,
[32] Chen C-H, Liu JM, Chua C-K, Chou S-M, Shyu VB, Chen J-P. Cartilage tissue
stem cells and scaffolds. J Anat 2008;213:66–72. https://doi.org/10.1111/
engineering with silk fibroin Scaffolds fabricated by indirect additive
j.1469-7580.2008.00878.x.
manufacturing technology. Mater 2014:7. https://doi.org/10.3390/ma7032104.
[6] Ashammakhi N, Ahadian S, Xu C, Montazerian H, Ko H, Nasiri R, et al. Bioinks
and bioprinting technologies to make heterogeneous and biomimetic tissue

4882
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

[33] Wang Y-C, Bai M-Y, Hsu W-Y, Yu M-H. Evaluation of a series of silk fibroin [59] Wang W, Nema S, Teagarden D. Protein aggregation—pathways and influencing
protein-based nonwoven mats for use as an anti-adhesion patch for wound factors. Int J Pharm 2010;390:89–99. https://doi.org/10.1016/j.
management in robotic surgery. J Biomed Mater Res Part A 2018;106:221–30. ijpharm.2010.02.025.
https://doi.org/10.1002/jbm.a.36225. [60] Murphy AR, John PSt, Kaplan DL. Modification of silk fibroin using diazonium
[34] Xi H, Zhao H. Silk fibroin coaxial bead-on-string fiber materials and their drug coupling chemistry and the effects on hMSC proliferation and differentiation.
release behaviors in different pH. J Mater Sci 2019;54:4246–58. https://doi.org/ Biomaterials 2008;29:2829–38. https://doi.org/10.1016/j.
10.1007/s10853-018-3137-z. biomaterials.2008.03.039.
[35] Magaz A, Spencer BF, Hardy JG, Li X, Gough JE, Blaker JJ. Modulation of [61] Zhou H, Wang Z, Cao H, Hu H, Luo Z, Yang X, et al. Genipin-crosslinked polyvinyl
neuronal cell affinity on PEDOT–PSS nonwoven silk scaffolds for neural tissue alcohol/silk fibroin/nano-hydroxyapatite hydrogel for fabrication of artificial
engineering. ACS Biomater Sci Eng 2020;6:6906–16. https://doi.org/10.1021/ cornea scaffolds—a novel approach to corneal tissue engineering. J Biomater Sci
acsbiomaterials.0c01239. Polym Ed 2019;30:1604–19. https://doi.org/10.1080/09205063.2019.1652418.
[36] Lemos CN, Cubayachi C, Dias K, Mendonça JN, Lopes NP, Furtado NAJC, et al. [62] Ferrero F, Periolatto M, Burelli S, Carletto RA. Silk grafting with chitosan and
Iontophoresis-stimulated silk fibroin films as a peptide delivery system for wound crosslinking agents. Fibers Polym 2010;11:185–92. https://doi.org/10.1007/
healing. Eur J Pharm Biopharm 2018;128:147–55. https://doi.org/10.1016/j. s12221-010-0185-7.
ejpb.2018.04.019. [63] Rehman MS ur, Raza ZA, Rehan ZA, Bakhtiyar MJ, Sharif F, Yousaf M. Citric acid
[37] Abbott A, Oxburgh L, Kaplan DL, Coburn JM. Avidin adsorption to silk fibroin crosslinked biocompatible silk fibroin-mediated porous chitosan films for
films as a facile method for functionalization. Biomacromolecules 2018;19: sustained drug release application. Mater Today Commun 2023;34:105373.
3705–13. https://doi.org/10.1021/acs.biomac.8b00824. https://doi.org/10.1016/J.MTCOMM.2023.105373.
[38] Liu J, Chen H, Wang Y, Li G, Zheng Z, Kaplan DL, et al. Flexible water-absorbing [64] Hasturk O, Jordan KE, Choi J, Kaplan DL. Enzymatically crosslinked silk and silk-
silk-fibroin biomaterial sponges with unique pore structure for tissue engineering. gelatin hydrogels with tunable gelation kinetics, mechanical properties and
ACS Biomater Sci Eng 2020;6:1641–9. https://doi.org/10.1021/ bioactivity for cell culture and encapsulation. Biomaterials 2020;232:119720.
acsbiomaterials.9b01721. https://doi.org/10.1016/j.biomaterials.2019.119720.
[39] Castro-Domínguez C, Lozano-Picazo P, Álvarez-López A, Garrote-Junco J, [65] Han Y, Yu S, Liu L, Zhao S, Yang T, Yang Y, et al. Silk fibroin-based hydrogels as a
Panetsos F, Guinea GV, et al. Axonal guidance using biofunctionalized straining protective matrix for stabilization of enzymes against pH denaturation. Mol Catal
flow spinning regenerated silk fibroin fibers as Scaffold. Biomimetics 2023:8. 2018;457:24–32. https://doi.org/10.1016/j.mcat.2018.07.009.
https://doi.org/10.3390/biomimetics8010065. [66] Burrs SL, Vanegas DC, Bhargava M, Mechulan N, Hendershot P, Yamaguchi H,
[40] Kim U-J, Park J, Li C, Jin H-J, Valluzzi R, Kaplan DL. Structure and properties of et al. A comparative study of graphene–hydrogel hybrid bionanocomposites for
silk hydrogels. Biomacromolecules 2004;5:786–92. https://doi.org/10.1021/ biosensing. Analyst 2015;140:1466–76. https://doi.org/10.1039/C4AN01788A.
bm0345460. [67] Kang GD, Lee KH, Ki CS, Nahm JH, Park YH. Silk fibroin/chitosan conjugate
[41] Ayub ZH, Arai M, Hirabayashi K. Mechanism of the gelation of fibroin solution. crosslinked by tyrosinase. Macromol Res 2004;12:534–9. https://doi.org/
Biosci Biotechnol Biochem 1993;57:1910–2. https://doi.org/10.1271/ 10.1007/BF03218439.
bbb.57.1910. [68] Raia NR, Partlow BP, McGill M, Kimmerling EP, Ghezzi CE, Kaplan DL.
[42] Kang G-D, Nahm J-H, Park J-S, Moon J-Y, Cho C-S, Yeo J-H. Effects of poloxamer Enzymatically crosslinked silk-hyaluronic acid hydrogels. Biomaterials 2017;131:
on the gelation of silk fibroin. Macromol Rapid Commun 2000;21:788–91. 58–67. https://doi.org/10.1016/j.biomaterials.2017.03.046.
https://doi.org/10.1002/1521-3927(20000701)21:11<788::AID- [69] Indrakumar S, Joshi A, Dash TK, Mishra V, Tandon B, Chatterjee K.
MARC788>3.0.CO;2-X. Photopolymerized silk fibroin gel for advanced burn wound care. Int J Biol
[43] Yoo M-K, Kweon HY, Lee K-G, Lee H-C, Cho C-S. Preparation of semi- Macromol 2023:233. https://doi.org/10.1016/J.IJBIOMAC.2023.123569.
interpenetrating polymer networks composed of silk fibroin and poloxamer [70] Boudreau R. Injectable Gelatin-Silk Fibroin Composite Hydrogels for In Situ Cell
macromer. Int J Biol Macromol 2004;34:263–70. https://doi.org/10.1016/j. Encapsulation. Honor Theses Capstones 2021.
ijbiomac.2004.06.002. [71] Wang L, Wang F, Xu B, Zhou M, Yu Y, Wang P, et al. Efficient regulation of the
[44] Miyazaki S, Takahashi A, Itoh K, Ishitani M, Dairaku M, Togashi M, et al. behaviors of silk fibroin hydrogel via enzyme-catalyzed coupling of hyaluronic
Preparation and evaluation of gel formulations for oral sustained delivery to acid. Langmuir 2021;37:478–89. https://doi.org/10.1021/ACS.
dysphagic patients. Drug Dev Ind Pharm 2009;35:780–7. https://doi.org/ LANGMUIR.0C03136/SUPPL_FILE/LA0C03136_SI_003.MP4.
10.1080/03639040802592443. [72] Raia NR, Partlow BP, McGill M, Kimmerling EP, Ghezzi CE, Kaplan DL.
[45] Motta A, Migliaresi C, Faccioni F, Torricelli P, Fini M, Giardino R. Fibroin Enzymatically crosslinked silk-hyaluronic acid hydrogels. Biomaterials 2017;131:
hydrogels for biomedical applications: preparation, characterization and in vitro 58–67. https://doi.org/10.1016/J.BIOMATERIALS.2017.03.046.
cell culture studies. J Biomater Sci Polym Ed 2004;15:851–64. https://doi.org/ [73] Kim SH, Yeon YK, Lee JM, Chao JR, Lee YJ, Seo YB, et al. Precisely printable and
10.1163/1568562041271075. biocompatible silk fibroin bioink for digital light processing 3D printing. Nat
[46] Yin Z, Wu F, Xing T, Yadavalli VK, Kundu SC, Lu S. A silk fibroin hydrogel with Commun 2018 91 2018;9:1–14. https://doi.org/10.1038/s41467-018-03759-y.
reversible sol–gel transition. RSC Adv 2017;7:24085–96. https://doi.org/ [74] Kim MH, Park WH. Chemically cross-linked silk fibroin hydrogel with enhanced
10.1039/C7RA02682J. elastic properties, biodegradability, and biocompatibility. Int J Nanomed 2016;
[47] Zheng H, Zuo B. Functional silk fibroin hydrogels: preparation{,} properties and 11:2967–78. https://doi.org/10.2147/IJN.S106467.
applications. J Mater Chem B 2021;9:1238–58. https://doi.org/10.1039/ [75] Wang X, Kluge JA, Leisk GG, Kaplan DL. Sonication-induced gelation of silk
D0TB02099K. fibroin for cell encapsulation. Biomaterials 2008;29:1054–64. https://doi.org/
[48] Wu N, Yu H, Sun M, Li Z, Zhao F, Ao Y, et al. Investigation on the structure and 10.1016/j.biomaterials.2007.11.003.
mechanical properties of highly tunable elastomeric silk fibroin hydrogels cross- [76] Jetbumpenkul P, Amornsudthiwat P, Kanokpanont S, Damrongsakkul S. Balanced
linked by γ-ray radiation. ACS Appl Bio Mater 2020;3:721–34. https://doi.org/ electrostatic blending approach – an alternative to chemical crosslinking of Thai
10.1021/acsabm.9b01062. silk fibroin/gelatin scaffold. Int J Biol Macromol 2012;50:7–13. https://doi.org/
[49] Chung C, Burdick JA. Engineering cartilage tissue. Adv Drug Deliv Rev 2008;60: 10.1016/J.IJBIOMAC.2011.08.028.
243–62. https://doi.org/10.1016/j.addr.2007.08.027. [77] Lin Y, Xia X, Shang K, Elia R, Huang W, Cebe P, et al. Tuning chemical and
[50] Paulusse JMJ, Sijbesma RP. Ultrasound in polymer chemistry: revival of an physical cross-links in silk electrogels for morphological analysis and mechanical
established technique. J Polym Sci Part A Polym Chem 2006;44:5445–53. reinforcement. Biomacromolecules 2013;14:2629–35. https://doi.org/10.1021/
https://doi.org/10.1002/pola.21646. BM4004892/SUPPL_FILE/BM4004892_SI_001.PDF.
[51] Bono N, Saroglia G, Marcuzzo S, Giagnorio E, Lauria G, Rosini E, et al. Silk fibroin [78] Xiao W., Liu W., Sun J., Dan X., Wei D., Fan H. Ultrasonication and Genipin Cross-
microgels as a platform for cell microencapsulation. J Mater Sci Mater Med 2023; Linking to Prepare Novel Silk Fibroin–Gelatin Composite Hydrogel. Http://
34:1–12. https://doi.org/10.1007/S10856-022-06706-Y/FIGURES/5. DxDoiOrg/101177/0883911512448692 2012;27:327–341. https://doi.org/1
[52] Yucel T, Cebe P, Kaplan DL. Vortex-induced injectable silk fibroin hydrogels. 0.1177/0883911512448692.
Biophys J 2009;97:2044–50. https://doi.org/10.1016/j.bpj.2009.07.028. [79] Kuchaiyaphum P, Chotichayapong C, Butwong N, Bua-ngern W. Silk fibroin/poly
[53] Lu Q, Huang Y, Li M, Zuo B, Lu S, Wang J, et al. Silk fibroin electrogelation (vinyl alcohol) hydrogel cross-linked with dialdehyde starch for wound dressing
mechanisms. Acta Biomater 2011;7:2394–400. https://doi.org/10.1016/j. applications. Macromol Res 2020 289 2020;28:844–50. https://doi.org/10.1007/
actbio.2011.02.032. S13233-020-8110-4.
[54] Zheng K, Zheng X, Yu M, He Y, Wu D. BMSCs-seeded interpenetrating network [80] Grabska-Zielińska S, Sionkowska A, Coelho CC, Monteiro FJ. Silk fibroin/
GelMA/SF composite hydrogel for articular cartilage repair. J Funct Biomater collagen/chitosan scaffolds cross-linked by a glyoxal solution as biomaterials
2023, Vol 14 2023;14:39. https://doi.org/10.3390/JFB14010039. toward bone tissue regeneration. Mater 2020 2020;Vol 13(13):3433. https://doi.
[55] Liu Y, Zhang Z, Zhang Y, Luo B, Liu X, Cao Y, et al. Construction of adhesive and org/10.3390/MA13153433.
bioactive silk fibroin hydrogel for treatment of spinal cord injury. Acta Biomater [81] Xu Z, Tang E, Zhao H. An environmentally sensitive silk fibroin/chitosan
2023;158:178–89. https://doi.org/10.1016/J.ACTBIO.2022.12.048. hydrogel and its drug release behaviors. Polym (Basel) 2019;11:1980. https://
[56] Kapoor S, Kundu SC. Silk protein-based hydrogels: promising advanced materials doi.org/10.3390/POLYM11121980.
for biomedical applications. Acta Biomater 2016;31:17–32. https://doi.org/ [82] Hu K, Lv Q, Cui FZ, Feng QL, Kong XD, Wang HL, et al. Biocompatible fibroin
10.1016/j.actbio.2015.11.034. blended films with recombinant human-like collagen for hepatic tissue
[57] McPherson A. Introduction to protein crystallization. Methods 2004;34:254–65. engineering. J Bioact Compat Polym 2006;21:23–37. https://doi.org/10.1177/
https://doi.org/10.1016/j.ymeth.2004.03.019. 0883911506060455.
[58] Stevenson LC. Characterization of protein and peptide stability and solubility in [83] Vasconcelos A, Freddi G, Cavaco-Paulo A. Biodegradable materials based on silk
non-aqueous solvents. Curr Pharm Biotechnol 2000;1:165–82. https://doi.org/ fibroin and keratin. Biomacromolecules 2008;9:1299–305. https://doi.org/
10.2174/1389201003378942. 10.1021/bm7012789.

4883
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

[84] Kweon H, Ha HC, Um IC, Park YH. Physical properties of silk fibroin/chitosan [108] Aamodt JM, Grainger DW. Extracellular matrix-based biomaterial scaffolds and
blend films. J Appl Polym Sci 2001;80:928–34. https://doi.org/10.1002/ the host response. Biomaterials 2016;86:68–82. https://doi.org/10.1016/j.
app.1172. biomaterials.2016.02.003.
[85] Foss C, Merzari E, Migliaresi C, Motta A. Silk fibroin/hyaluronic acid 3D matrices [109] Rosano GL, Ceccarelli EA. Recombinant protein expression in microbial systems.
for cartilage tissue engineering. Biomacromolecules 2013;14:38–47. https://doi. Front Microbiol 2014;5:341. https://doi.org/10.3389/fmicb.2014.00341.
org/10.1021/bm301174x. [110] An B, DesRochers TM, Qin G, Xia X, Thiagarajan G, Brodsky B, et al. The influence
[86] Whitesides GM, Wong AP. The intersection of biology and materials science. MRS of specific binding of collagen–silk chimeras to silk biomaterials on hMSC
Bull 2006;31:19–27. https://doi.org/10.1557/mrs2006.2. behavior. Biomaterials 2013;34:402–12. https://doi.org/10.1016/j.
[87] Aharonov A, Mordechai HS, Sharon SE, Sharabi M. The mechanical behavior of biomaterials.2012.09.085.
silk-fibroin reinforced alginate hydrogel biocomposites - toward functional tissue [111] Bracalello A, Santopietro V, Vassalli M, Marletta G, Del Gaudio R, Bochicchio B,
biomimetics. J Mech Behav Biomed Mater 2023;138:105598. https://doi.org/ et al. Design and production of a chimeric resilin-, elastin-, and collagen-like
10.1016/J.JMBBM.2022.105598. engineered polypeptide. Biomacromolecules 2011;12:2957–65. https://doi.org/
[88] Nilogal P, Uppine GB, Rayaraddi R, Sanjeevappa HK, Martis LJ, Narayana B, et al. 10.1021/bm2005388.
Conductive in situ reduced graphene oxide–silk fibroin bionanocomposites. ACS [112] Aigner TB, DeSimone E, Scheibel T. Biomedical applications of recombinant silk-
Omega 2021;6:12995–3007. https://doi.org/10.1021/acsomega.1c00013. based materials. Adv Mater 2018;30:1704636. https://doi.org/10.1002/
[89] Ghafori Gorab M, Aliabadi HAM, Kashtiaray A, Mahdavi M, Bani MS, Etminan A, adma.201704636.
et al. Decoration of graphene oxide nanosheets with carboxymethylcellulose [113] Megeed Z, Cappello J, Ghandehari H. Genetically engineered silk-elastinlike
hydrogel, silk fibroin and magnetic nanoparticles for biomedical and protein polymers for controlled drug delivery. Adv Drug Deliv Rev 2002;54:
hyperthermia applications. Nanoscale Adv 2022;5:153–9. https://doi.org/ 1075–91. https://doi.org/10.1016/S0169-409X(02)00063-7.
10.1039/D2NA00394E. [114] Hatefi A, Cappello J, Ghandehari H. Adenoviral gene delivery to solid tumors by
[90] Wu Z, Li W, Cheng S, Liu J, Wang S. Novel fabrication of bioengineered injectable recombinant silk–elastinlike protein polymers. Pharm Res 2007;24:773–9.
chitosan hydrogel loaded with conductive nanoparticles to improve therapeutic https://doi.org/10.1007/s11095-006-9200-5.
potential of mesenchymal stem cells in functional recovery after ischemic [115] Megeed Z, Haider M, Li D, O’Malley BWJ, Cappello J, Ghandehari H. In vitro and
myocardial infarction. Nanomed Nanotechnol, Biol Med 2023;47:102616. in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene
https://doi.org/10.1016/J.NANO.2022.102616. therapy. J Control Release 2004;94:433–45. https://doi.org/10.1016/j.
[91] Dorishetty P, Balu R, Athukoralalage SS, Greaves TL, Mata J, de Campo L, et al. jconrel.2003.10.027.
Tunable biomimetic hydrogels from silk fibroin and nanocellulose. ACS Sustain [116] Xu H. The advances and perspectives of recombinant protein production in the
Chem Eng 2020;8:2375–89. https://doi.org/10.1021/acssuschemeng.9b05317. silk gland of silkworm Bombyx mori. Transgenic Res 2014;23:697–706. https://
[92] Li G, Kong Y, Zhao Y, Zhao Y, Zhang L, Yang Y. Fabrication and characterization doi.org/10.1007/s11248-014-9826-8.
of polyacrylamide/silk fibroin hydrogels for peripheral nerve regeneration. [117] de Moraes MA, Albrecht Mahl CR, Ferreira Silva M, Beppu MM. Formation of silk
J Biomater Sci Polym Ed 2015;26:899–916. https://doi.org/10.1080/ fibroin hydrogel and evaluation of its drug release profile. J Appl Polym Sci 2015:
09205063.2015.1066109. 132. https://doi.org/10.1002/app.41802.
[93] Bhattacharjee P, Ahearne M. Silk fibroin based interpenetrating network hydrogel [118] Burger D, Beaumont M, Rosenau T, Tamada Y. Porous silk fibroin/cellulose
for corneal stromal regeneration. Int J Biol Macromol 2022;223:583–94. https:// hydrogels for bone tissue engineering via a novel combined process based on
doi.org/10.1016/J.IJBIOMAC.2022.11.021. sequential regeneration and porogen leaching. Molecules 2020:25. https://doi.
[94] Lv Q, Hu K, Feng Q, Cui F. Fibroin/collagen hybrid hydrogels with crosslinking org/10.3390/molecules25215097.
method: Preparation, properties, and cytocompatibility. J Biomed Mater Res Part [119] Elliott WH, Bonani W, Maniglio D, Motta A, Tan W, Migliaresi C. Silk hydrogels of
A 2008;84A:198–207. https://doi.org/10.1002/jbm.a.31366. tunable structure and viscoelastic properties using different chronological orders
[95] Tao H, Marelli B, Yang M, An B, Onses MS, Rogers JA, et al. Inkjet printing of of genipin and physical cross-linking. ACS Appl Mater Interfaces 2015;7:
regenerated silk fibroin: from printable forms to printable functions. Adv Mater 12099–108. https://doi.org/10.1021/acsami.5b02308.
2015;27:4273–9. https://doi.org/10.1002/ADMA.201501425. [120] Akrami-Hasan-Kohal M, Eskandari M, Solouk A. Silk fibroin hydrogel/
[96] Wang Q, Han G, Yan S, Zhang Q. 3D printing of silk fibroin for biomedical dexamethasone sodium phosphate loaded chitosan nanoparticles as a potential
applications. Mater 2019, Vol 12 2019;12:504. https://doi.org/10.3390/ drug delivery system. Colloids Surf B Biointerfaces 2021;205:111892. https://doi.
MA12030504. org/10.1016/j.colsurfb.2021.111892.
[97] Das S, Pati F, Choi YJ, Rijal G, Shim JH, Kim SW, et al. Bioprintable, cell-laden [121] Kim HH, Kim JW, Choi J, Park YH, Ki CS. Characterization of silk hydrogel
silk fibroin-gelatin hydrogel supporting multilineage differentiation of stem cells formed with hydrolyzed silk fibroin-methacrylate via photopolymerization.
for fabrication of three-dimensional tissue constructs. Acta Biomater 2015;11: Polym (Guildf) 2018;153:232–40. https://doi.org/10.1016/j.
233–46. https://doi.org/10.1016/J.ACTBIO.2014.09.023. polymer.2018.08.019.
[98] Xiong S, Zhang X, Lu P, Wu Y, Wang Q, Sun H, et al. A gelatin-sulfonated silk [122] Ribeiro VP, Silva-Correia J, Gonçalves C, Pina S, Radhouani H, Montonen T, et al.
composite scaffold based on 3D printing technology enhances skin regeneration Rapidly responsive silk fibroin hydrogels as an artificial matrix for the
by stimulating epidermal growth and dermal neovascularization. Sci Rep 2017 71 programmed tumor cells death. PLoS One 2018;13:1–21. https://doi.org/
2017;7:1–12. https://doi.org/10.1038/s41598-017-04149-y. 10.1371/journal.pone.0194441.
[99] Compaan AM, Christensen K, Huang Y. Inkjet bioprinting of 3D silk fibroin [123] Ziadlou R, Rotman S, Teuschl A, Salzer E, Barbero A, Martin I, et al. Optimization
cellular constructs using sacrificial alginate. ACS Biomater Sci Eng 2017;3: of hyaluronic acid-tyramine/silk-fibroin composite hydrogels for cartilage tissue
1519–26. https://doi.org/10.1021/ACSBIOMATERIALS.6B00432/ASSET/ engineering and delivery of anti-inflammatory and anabolic drugs. Mater Sci Eng
IMAGES/MEDIUM/AB-2016-00432C_0008.GIF. C 2021;120:111701. https://doi.org/10.1016/j.msec.2020.111701.
[100] Kim SH, Yeon YK, Lee JM, Chao JR, Lee YJ, Seo YB, et al. Precisely printable and [124] He W, Li P, Zhu Y, Liu M, Huang X, Qi H. An injectable silk fibroin nanofiber
biocompatible silk fibroin bioink for digital light processing 3D printing. Nat hydrogel hybrid system for tumor upconversion luminescence imaging and
Commun 2018 91 2018;9:1–14. https://doi.org/10.1038/s41467-018-03759-y. photothermal therapy. N J Chem 2019;43:2213–9. https://doi.org/10.1039/
[101] Kim S.H., Hong H., Ajiteru O., Sultan M.T., Lee Y.J., Lee J.S., et al. 3D bioprinted C8NJ05766D.
silk fibroin hydrogels for tissue engineering n.d. https://doi.org/10.1038/s [125] Whittaker JL, Choudhury NR, Dutta NK, Zannettino A. Facile and rapid
41596–021-00622–1. ruthenium mediated photo-crosslinking of Bombyx mori silk fibroin. J Mater
[102] Xu L, Zhang Z, Jorgensen AM, Yang Y, Jin Q, Zhang G, et al. Bioprinting a skin Chem B 2014;2:6259–70. https://doi.org/10.1039/C4TB00698D.
patch with dual-crosslinked gelatin (GelMA) and silk fibroin (SilMA): An [126] Wu J, Liu J, Shi Y, Wan Y. Rheological, mechanical and degradable properties of
approach to accelerating cutaneous wound healing. Mater Today Bio 2023;18: injectable chitosan/silk fibroin/hydroxyapatite/glycerophosphate hydrogels.
100550. https://doi.org/10.1016/J.MTBIO.2023.100550. J Mech Behav Biomed Mater 2016;64:161–72. https://doi.org/10.1016/j.
[103] Wang L, Lu R, Hou J, Nan X, Xia Y, Guo Y, et al. Application of injectable silk jmbbm.2016.07.007.
fibroin/graphene oxide hydrogel combined with bone marrow mesenchymal stem [127] Ma L, Liu Q, Wu R, Meng Z, Patil A, Yu R, et al. From molecular reconstruction of
cells in bone tissue engineering. Colloids Surf A Physicochem Eng Asp 2020;604: mesoscopic functional conductive silk fibrous materials to remote respiration
125318. https://doi.org/10.1016/j.colsurfa.2020.125318. monitoring. Small 2020;16:2000203. https://doi.org/10.1002/SMLL.202000203.
[104] Buitrago JO, Patel KD, El-Fiqi A, Lee J-H, Kundu B, Lee H-H, et al. Silk fibroin/ [128] Qiu W, Patil A, Hu F, Liu XY. Hierarchical structure of silk materials versus
collagen protein hybrid cell-encapsulating hydrogels with tunable gelation and mechanical performance and mesoscopic engineering principles. Small 2019;15:
improved physical and biological properties. Acta Biomater 2018;69:218–33. 1903948. https://doi.org/10.1002/SMLL.201903948.
https://doi.org/10.1016/j.actbio.2017.12.026. [129] Yang Y, Shao Z, Chen X, Zhou P. Optical spectroscopy to investigate the structure
[105] Kim MH, Kim BS, Lee J, Cho D, Kwon OH, Park WH. Silk fibroin/hydroxyapatite of regenerated bombyx mori silk fibroin in solution. Biomacromolecules 2004;5:
composite hydrogel induced by gamma-ray irradiation for bone tissue 773–9. https://doi.org/10.1021/BM0343848.
engineering. Biomater Res 2017;21:12. https://doi.org/10.1186/s40824-017- [130] Du N, Xiang YL, Narayanan J, Li L, Lim MLM, Li D. Design of superior spider silk:
0098-2. from nanostructure to mechanical properties. Biophys J 2006;91:4528–35.
[106] Gangrade A, Mandal BB. Injectable carbon nanotube impregnated silk based https://doi.org/10.1529/BIOPHYSJ.106.089144.
multifunctional hydrogel for localized targeted and on-demand anticancer drug [131] Wu R, Ma L, Liu XY. From mesoscopic functionalization of silk fibroin to smart
delivery. ACS Biomater Sci Eng 2019;5:2365–81. https://doi.org/10.1021/ fiber devices for textile electronics and photonics. Adv Sci 2022;9:2103981.
acsbiomaterials.9b00416. https://doi.org/10.1002/ADVS.202103981.
[107] Raia NR, Jia D, Ghezzi CE, Muthukumar M, Kaplan DL. Characterization of silk- [132] Gong Z, Huang L, Yang Y, Chen X, Shao Z. Two distinct β-sheet fibrils from silk
hyaluronic acid composite hydrogels towards vitreous humor substitutes. protein. Chem Commun 2009:7506–8. https://doi.org/10.1039/B914218E.
Biomaterials 2020;233:119729. https://doi.org/10.1016/j. [133] Mu X, Sahoo JK, Cebe P, Kaplan DL. Photo-crosslinked silk fibroin for 3D printing.
biomaterials.2019.119729. Polym 2020, Vol 12 2020;12:2936. https://doi.org/10.3390/POLYM12122936.

4884
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

[134] Wu H, Liu S, Xiao L, Dong X, Lu Q, Kaplan DL. Injectable and pH-responsive silk [158] Zhao F, Wu D, Yao D, Guo R, Wang W, Dong A, et al. An injectable particle-
nanofiber hydrogels for sustained anticancer drug delivery. ACS Appl Mater hydrogel hybrid system for glucose-regulatory insulin delivery. Acta Biomater
Interfaces 2016;8:17118–26. https://doi.org/10.1021/acsami.6b04424. 2017;64:334–45. https://doi.org/10.1016/j.actbio.2017.09.044.
[135] Floren M, Migliaresi C, Motta A. Processing techniques and applications of silk [159] Huynh DP, Nguyen MK, Pi BS, Kim MS, Chae SY, Lee KC, et al. Functionalized
hydrogels in bioengineering. J Funct Biomater 2016:7. https://doi.org/10.3390/ injectable hydrogels for controlled insulin delivery. Biomaterials 2008;29:
jfb7030026. 2527–34. https://doi.org/10.1016/j.biomaterials.2008.02.016.
[136] Mandal BB, Kapoor S, Kundu SC. Silk fibroin/polyacrylamide semi- [160] Maity B, Samanta S, Sarkar S, Alam S, Govindaraju T. Injectable silk fibroin-based
interpenetrating network hydrogels for controlled drug release. Biomaterials hydrogel for sustained insulin delivery in diabetic rats. ACS Appl Bio Mater 2020;
2009;30:2826–36. https://doi.org/10.1016/j.biomaterials.2009.01.040. 3:3544–52. https://doi.org/10.1021/acsabm.0c00152.
[137] Lovett ML, Wang X, Yucel T, York L, Keirstead M, Haggerty L, et al. Silk hydrogels [161] Chen S, Matsumoto H, Moro-oka Y, Tanaka M, Miyahara Y, Suganami T, et al.
for sustained ocular delivery of anti-vascular endothelial growth factor (anti- Smart microneedle fabricated with silk fibroin combined semi-interpenetrating
VEGF) therapeutics. Eur J Pharm Biopharm 2015;95:271–8. https://doi.org/ network hydrogel for glucose-responsive insulin delivery. ACS Biomater Sci Eng
10.1016/j.ejpb.2014.12.029. 2019;5:5781–9. https://doi.org/10.1021/acsbiomaterials.9b00532.
[138] Atila D, Keskin D, Lee YL, Lin FH, Hasirci V, Tezcaner A. Injectable methacrylated [162] Seib FP. Reverse-engineered silk hydrogels for cell and drug delivery. Ther Deliv
gelatin/thiolated pectin hydrogels carrying melatonin/tideglusib-loaded core/ 2018;9:469–87. https://doi.org/10.4155/tde-2018-0016.
shell PMMA/silk fibroin electrospun fibers for vital pulp regeneration. Colloids [163] Davis NE, Beenken-Rothkopf LN, Mirsoian A, Kojic N, Kaplan DL, Barron AE, et al.
Surf B Biointerfaces 2023;222:113078. https://doi.org/10.1016/J. Enhanced function of pancreatic islets co-encapsulated with ECM proteins and
COLSURFB.2022.113078. mesenchymal stromal cells in a silk hydrogel. Biomaterials 2012;33:6691–7.
[139] Song X, Wang X, Guo L, Li T, Huang Y, Yang J, et al. Etanercept embedded silk https://doi.org/10.1016/j.biomaterials.2012.06.015.
fibroin/pullulan hydrogel enhance cartilage repair in bone marrow stimulation. [164] Hamilton DC, Shih HH, Schubert RA, Michie SA, Staats PN, Kaplan DL, et al.
Front Bioeng Biotechnol 2022;10:2312. https://doi.org/10.3389/ A silk-based encapsulation platform for pancreatic islet transplantation improves
FBIOE.2022.982894/BIBTEX. islet function in vivo. J Tissue Eng Regen Med 2017;11:887–95. https://doi.org/
[140] Gangrade A, Gawali B, Jadi PK, Naidu VGM, Mandal BB. Photo-electro active 10.1002/term.1990.
nanocomposite silk hydrogel for spatiotemporal controlled release of [165] Martín-Martín Y, Fernández-García L, Sanchez-Rebato MH, Marí-Buyé N, Rojo FJ,
chemotherapeutics: an in vivo approach toward suppressing solid tumor growth. Pérez-Rigueiro J, et al. Evaluation of Neurosecretome from Mesenchymal Stem
ACS Appl Mater Interfaces 2020;12:27905–16. https://doi.org/10.1021/ Cells Encapsulated in Silk Fibroin Hydrogels. Sci Rep 2019;9:8801. https://doi.
acsami.0c02470. org/10.1038/s41598-019-45238-4.
[141] Yao Q, Lan Q-H, Jiang X, Du C-C, Zhai Y-Y, Shen X, et al. Bioinspired biliverdin/ [166] Jaiswal C, Gupta T, Jadi PK, Moses JC, Mandal BB. Injectable anti-cancer drug
silk fibroin hydrogel for antiglioma photothermal therapy and wound healing. loaded silk-based hydrogel for the prevention of cancer recurrence and post-
Theranostics 2020;10:11719–36. https://doi.org/10.7150/thno.47682. lumpectomy tissue regeneration aiding triple-negative breast cancer therapy.
[142] Gou S, Xie D, Ma Y, Huang Y, Dai F, Wang C, et al. Injectable, thixotropic, and Biomater Adv 2023:145. https://doi.org/10.1016/J.BIOADV.2022.213224.
multiresponsive silk fibroin hydrogel for localized and synergistic tumor therapy. [167] Ding X, Yang G, Zhang W, Li G, Lin S, Kaplan DL, et al. Increased stem cells
ACS Biomater Sci Eng 2020;6:1052–63. https://doi.org/10.1021/ delivered using a silk gel/scaffold complex for enhanced bone regeneration. Sci
acsbiomaterials.9b01676. Rep 2017;7:2175. https://doi.org/10.1038/s41598-017-02053-z.
[143] Xu Z, Chen T, Zhang K-Q, Meng K, Zhao H. Silk fibroin/chitosan hydrogel with [168] Ciocci M, Cacciotti I, Seliktar D, Melino S. Injectable silk fibroin hydrogels
antibacterial, hemostatic and sustained drug-release activities. Polym Int 2021; functionalized with microspheres as adult stem cells-carrier systems. Int J Biol
70:1741–51. https://doi.org/10.1002/pi.6275. Macromol 2018;108:960–71. https://doi.org/10.1016/j.ijbiomac.2017.11.013.
[144] Eivazzadeh-Keihan R, Pajoum Z, Aghamirza Moghim Aliabadi H, Ganjali F, [169] Floren M, Bonani W, Dharmarajan A, Motta A, Migliaresi C, Tan W. Human
Kashtiaray A, Salimi Bani M, et al. Magnetic chitosan-silk fibroin hydrogel/ mesenchymal stem cells cultured on silk hydrogels with variable stiffness and
graphene oxide nanobiocomposite for biological and hyperthermia applications. growth factor differentiate into mature smooth muscle cell phenotype. Acta
Carbohydr Polym 2023;300:120246. https://doi.org/10.1016/J. Biomater 2016;31:156–66. https://doi.org/10.1016/j.actbio.2015.11.051.
CARBPOL.2022.120246. [170] Li T, Song X, Weng C, Wang X, Wu J, Sun L, et al. Enzymatically crosslinked and
[145] Dong Y, Dong P, Huang D, Mei L, Xia Y, Wang Z, et al. Fabrication and mechanically tunable silk fibroin/pullulan hydrogels for mesenchymal stem cells
characterization of silk fibroin-coated liposomes for ocular drug delivery. Eur J delivery. Int J Biol Macromol 2018;115:300–7. https://doi.org/10.1016/j.
Pharm Biopharm 2015;91:82–90. https://doi.org/10.1016/j.ejpb.2015.01.018. ijbiomac.2018.04.046.
[146] Yucel T, Lovett ML, Kaplan DL. Silk-based biomaterials for sustained drug [171] Ni T, Liu M, Zhang Y, Cao Y, Pei R. 3D bioprinting of bone marrow mesenchymal
delivery. J Control Release 2014;190:381–97. https://doi.org/10.1016/j. stem cell-laden silk fibroin double network Scaffolds for cartilage tissue repair.
jconrel.2014.05.059. Bioconjug Chem 2020;31:1938–47. https://doi.org/10.1021/acs.
[147] Pritchard EM, Valentin T, Panilaitis B, Omenetto F, Kaplan DL. Antibiotic- bioconjchem.0c00298.
Releasing Silk Biomaterials for Infection Prevention and Treatment. Adv Funct [172] Gholami M, Tajabadi M, Khavandi A, Azarpira N. Synthesis, optimization, and
Mater 2013;23:854–61. https://doi.org/10.1002/adfm.201201636. cell response investigations of natural-based, thermoresponsive, injectable
[148] Price R, Gustafson J, Greish K, Cappello J, McGill L, Ghandehari H. Comparison of hydrogel: An attitude for 3D hepatocyte encapsulation and cell therapy. Front
silk-elastinlike protein polymer hydrogel and poloxamer in matrix-mediated gene Bioeng Biotechnol 2023:10. https://doi.org/10.3389/FBIOE.2022.1075166.
delivery. Int J Pharm 2012;427:97–104. https://doi.org/10.1016/j. [173] Laomeephol C, Ferreira H, Kanokpanont S, Luckanagul JA, Neves NM,
ijpharm.2011.09.037. Damrongsakkul S. Osteogenic differentiation of encapsulated cells in
[149] Gustafson J, Greish K, Frandsen J, Cappello J, Ghandehari H. Silk-elastinlike dexamethasone-loaded phospholipid-induced silk fibroin hydrogels. Biomater
recombinant polymers for gene therapy of head and neck cancer: from molecular Transl 2022;3:213–20. https://doi.org/10.12336/
definition to controlled gene expression. J Control Release 2009;140:256–61. BIOMATERTRANSL.2022.03.005.
https://doi.org/10.1016/j.jconrel.2009.05.022. [174] Li AY, Shi XY, You W, Yue W. Muscle-derived stem cells in silk fibroin hydrogels
[150] Nimni ME. Polypeptide growth factors: targeted delivery systems. Biomaterials promotes muscle regeneration and angiogenesis in sheep models: An
1997;18:1201–25. https://doi.org/10.1016/S0142-9612(97)00050-1. experimental study. Eur Rev Med Pharm Sci 2022;26:787–98. https://doi.org/
[151] Babensee JE, McIntire LV, Mikos AG. Growth factor delivery for tissue 10.26355/EURREV_202202_27987.
engineering. Pharm Res 2000;17:497–504. https://doi.org/10.1023/A: [175] Laomeephol C, Guedes M, Ferreira H, Reis RL, Kanokpanont S, Damrongsakkul S,
1007502828372. et al. Phospholipid-induced silk fibroin hydrogels and their potential as cell
[152] Zhang W, Zhu C, Ye D, Xu L, Zhang X, Wu Q, et al. Porous silk Scaffolds for carriers for tissue regeneration. J Tissue Eng Regen Med 2020;14:160–72. https://
delivery of growth factors and stem cells to enhance bone regeneration. PLoS One doi.org/10.1002/TERM.2982.
2014;9:1–9. https://doi.org/10.1371/journal.pone.0102371. [176] Zhang W, Wang X, Wang S, Zhao J, Xu L, Zhu C, et al. The use of injectable
[153] Bragg JC, Kweon H, Jo Y, Lee KG, Lin C-C. In situ formation of silk-gelatin hybrid sonication-induced silk hydrogel for VEGF165 and BMP-2 delivery for elevation
hydrogels for affinity-based growth factor sequestration and release. RSC Adv of the maxillary sinus floor. Biomaterials 2011;32:9415–24. https://doi.org/
2016;6:114353–60. https://doi.org/10.1039/C6RA22908E. 10.1016/j.biomaterials.2011.08.047.
[154] Fathi-Achachelouei M, Keskin D, Bat E, Vrana NE, Tezcaner A. Dual growth factor [177] Mandal BB, Mann JK, Kundu SC. Silk fibroin/gelatin multilayered films as a
delivery using PLGA nanoparticles in silk fibroin/PEGDMA hydrogels for articular model system for controlled drug release. Eur J Pharm Sci 2009;37:160–71.
cartilage tissue engineering. J Biomed Mater Res Part B Appl Biomater 2020;108: https://doi.org/10.1016/j.ejps.2009.02.005.
2041–62. https://doi.org/10.1002/jbm.b.34544. [178] Wenk E, Merkle HP, Meinel L. Silk fibroin as a vehicle for drug delivery
[155] Magaz A, Ashton MD, Hathout RM, Li X, Hardy JG, Blaker JJ. Electroresponsive applications. J Control Release 2011;150:128–41. https://doi.org/10.1016/j.
silk-based biohybrid composites for electrochemically controlled growth factor jconrel.2010.11.007.
delivery. Pharmaceutics 2020:12. https://doi.org/10.3390/ [179] Fang J-Y, Chen J-P, Leu Y-L, Wang H-Y. Characterization and evaluation of silk
pharmaceutics12080742. protein hydrogels for drug delivery. Chem Pharm Bull 2006;54:156–62. https://
[156] Cai Y, Huang Q, Wang P, Ye K, Zhao Z, Chen H, et al. Conductive hydrogel doi.org/10.1248/cpb.54.156.
conduits with growth factor gradients for peripheral nerve repair in diabetics with [180] Chao P-HG, Yodmuang S, Wang X, Sun L, Kaplan DL, Vunjak-Novakovic G. Silk
non-suture tape. Adv Health Mater 2022;11:2200755. https://doi.org/10.1002/ hydrogel for cartilage tissue engineering. J Biomed Mater Res B Appl Biomater
ADHM.202200755. 2010;95:84–90. https://doi.org/10.1002/jbm.b.31686.
[157] Matsumoto A, Tanaka M, Matsumoto H, Ochi K, Moro-oka Y, Kuwata H, et al. [181] Guziewicz N, Best A, Perez-Ramirez B, Kaplan DL. Lyophilized silk fibroin
Synthetic \&\#x201c;smart gel\&\#x201d; provides glucose-responsive insulin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies.
delivery in diabetic mice. Sci Adv 2017;3:eaaq0723. https://doi.org/10.1126/ Biomaterials 2011;32:2642–50. https://doi.org/10.1016/j.
sciadv.aaq0723. biomaterials.2010.12.023.

4885
A.P. Madappura and S. Madduri Computational and Structural Biotechnology Journal 21 (2023) 4868–4886

[182] Numata K, Yamazaki S, Naga N. Biocompatible and biodegradable dual-drug 0300820720181553959 2018;61:163–173. https://doi.org/10.1080/03
release system based on silk hydrogel containing silk nanoparticles. 008207.2018.1553959.
Biomacromolecules 2012;13:1383–9. https://doi.org/10.1021/bm300089a. [190] Lee H, Yang GH, Kim M, Lee JY, Huh JT, Kim GH. Fabrication of micro/
[183] Siepmann J, Siepmann F. Mathematical modeling of drug delivery. Int J Pharm nanoporous collagen/dECM/silk-fibroin biocomposite scaffolds using a low
2008;364:328–43. https://doi.org/10.1016/j.ijpharm.2008.09.004. temperature 3D printing process for bone tissue regeneration. Mater Sci Eng C
[184] Peppas NA. Analysis of Fickian and non-Fickian drug release from polymers. 2018;84:140–7. https://doi.org/10.1016/J.MSEC.2017.11.013.
Pharm Acta Helv 1985;60:110–1. [191] Huang L, Yuan W, Hong Y, Fan S, Yao X, Ren T, et al. 3D printed hydrogels with
[185] Tomoda BT, Pacheco MS, Abranches YB, Viganó J, Perrechil F, De Moraes MA. oxidized cellulose nanofibers and silk fibroin for the proliferation of lung
Assessing the influence of dyes physico-chemical properties on incorporation and epithelial stem cells. Cellulose 2021;28:241–57. https://doi.org/10.1007/
release kinetics in silk fibroin matrices. Polym (Basel) 2021:13. https://doi.org/ S10570-020-03526-7/FIGURES/6.
10.3390/polym13050798. [192] Zhang J, Allardyce BJ, Rajkhowa R, Zhao Y, Dilley RJ, Redmond SL, et al. 3D
[186] Jamali H, Abuali M, Khalili M. Clinical outcomes of silk versus nylon sutures for printing of silk particle-reinforced chitosan hydrogel structures and their
suturing of conjunctival autograft in pterygium surgery. Middle East Afr J properties. ACS Biomater Sci Eng 2018;4:3036–46. https://doi.org/10.1021/
Ophthalmol 2020;27:110. https://doi.org/10.4103/MEAJO.MEAJO_166_19. ACSBIOMATERIALS.8B00804/ASSET/IMAGES/MEDIUM/AB-2018-00804T_
[187] Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci 2007;32:991–1007. 0013.GIF.
https://doi.org/10.1016/J.PROGPOLYMSCI.2007.05.013. [193] Agostinacchio F, Mu X, Dirè S, Motta A, Kaplan DL. In situ 3D printing:
[188] Dorishetty P, Balu R, Gelmi A, Mata JP, Dutta NK, Choudhury NR. 3D printable opportunities with silk inks. Trends Biotechnol 2021;39:719–30. https://doi.org/
soy/silk hybrid hydrogels for tissue engineering applications. Biomacromolecules 10.1016/J.TIBTECH.2020.11.003.
2021;22:3668–78. https://doi.org/10.1021/ACS.BIOMAC.1C00250/SUPPL_ [194] Hasturk O, Smiley J, Arnett M, Sahoo J, Staii C, Kaplan D. Cytoprotection of
FILE/BM1C00250_SI_001.PDF. human progenitor and stem cells through encapsulation in alginate templated,
[189] DeBari M.K., Keyser M.N., Bai M.A., Abbott R.D. 3D printing with silk: dual crosslinked silk and silk-gelatin composite hydrogel microbeads. Adv Health
considerations and applications. Https://DoiOrg/101080/ Mater 2022;11(17):e2200292. https://doi.org/10.1002/adhm.202200293.

4886