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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5632_Библиотеки_им_академика_М_И_Перельмана.pdf
7 Graphene-Based Carbonaceous Materials: A Sustainable Biomaterial… 193
published in international peer-reviewed scientific journals
(recorded in Scopus and Web of Science database). In 2012, her
research work was nominated for ENI Award-2013 competition
on ‘Protection of the Environment’. She was associated with
e-COST, MP1301-NEW GEN project (2014–2017) as a working
group (WG) member, CA15216-ENBA project (2016–2020) as an
MC member, CA17107-CONTEXT project (2019–2022) as a WG
member. She is/was involved in many, internal/nationa
national projects
running at the Centre of Polymer Systems, TBU
l and inter-
in Zlin. She is a member of the Polymer Processing Society (PPS)
as well associated with the SPE-European Medical Polymer Division since 2009 as a Board member.
Prof. Petr Saha is the founder of Tomas Bata University in Zlín,
Czech Republic, where he served as Rector for four terms of
office. He currently holds the post of Director of the University
Institute. His expertise includes energy and composite materials.
His professional focus is on energy materials, polymer processes
and medical polymers. Completed his studies at the Faculty of
Technology of the Brno University of Technology, where he also
worked. He was employed at Chalmers University of Technology
in Sweden for more than 10 years. Moreover, he held the office of
Chairman of the Czech Rectors’ Conference. Prof. Sáha is also
active on the international scene at a number of universities (East
China University of Science and Technology, Clemson University
v USA, Ton Duc Thang University in Vietnam and the University
of Palermo in Italy). Between 2005 and 2007, he held the post of
President of the Polymer Processing Society located in the USA,
he focused on the growth of polymer science in the devel-
where
opment world. He was instrumental in the establishment of Bata
Centre in Vietnam, which supports cooperation between
institutions in Vietnam and Czech and European universities. At
present, Prof. Sáha is engaged in research into energy materials
from renewable resources; his team is mainly focusing on the
preparation of components for batteries and supercapacitors from
renewable resources, which will replace critical raw materials in
the future. Prof. Petr Sáha is the author and co-author of
435 publications (with more than 8000 citations on Web of Science and an H-index of 57 as of May 2024) and several dozen
patents, utility models, prototypes and industrial designs.
Green Approach for Synthesizing Silk Fibroin Biomaterial Scaffolds
8
Shristy Verma , Rishabha Malviya
, Lavanya Gupta ,
and Sathvik Belagodu Sridhar
Abstract
The protein called silk fibroin, which comes from the Bombyx mori silkworm,
has exceptional mechanical , biodegradable, and biocompatibility properties that
make it a great biomaterial for use in medical applications. Chemical-intensive
methods that endanger human health and the environment are used to create silk.
Green synthesis, a novel and environmentally friendly method, had to be developed for such a reason. Applications for silk fibroin include wound healing, tissue
engineering, bone engineering, and many more. The advantages of green synthesis over traditional methods for creating silk fibroin biomaterial scaffolds for
biomedical applications are explored in this chapter. Many techniques and
alternatives to conventional techniques are covered, including the use of DES,
enzymatic degumming, supercritical CO
the mentioned observations indicate that green synthesized silk fiber scaffolds,
which are also more environmentally friendly, can be used to produce enhanced
silk fiber (SF) for biomedical applications.
fluid, and many more. In conclusion,
2
S. Verma
Department of Pharmacy, School of Medical and Allied Sciences, Galgotias University, Greater
Noida, Uttar Pradesh, India
R. Malviya (
Department of Pharmacy, Galgotias University, Greater Noida, Uttar Pradesh, India
e-mail: rishabha.malviya@galgotiasuniversity.edu.in
L. Gupta
Faculty
e-mail: Lavanya.Gupta@medma.uni-heidelberg.de
S. B. Sridhar
RAK
Arab Emirates
e-mail: sathvik@rakmhsu.ac.ae
#
The Author(s), under exclusive license to Springer Nature Switzerland AG 2025
R. Malviya, S. Sundram (eds.), Sustainable Green Biomaterials As Drug Delivery
Systems, Biomaterials, Bioengineering and Sustainability 1,
https://doi.org/10.1007/978-3-031-79062-1_8
✉)
of Biosciences, Heidelberg University, Heidelberg, Germany
College of Pharmacy, RAK Medical & Health Sciences University, Ras Al Khaimah, United
195
196 S. Verma et al.
Keywords
Silk fibroin · Green · Degumming · Water annealing · Deep eutectic solvent
8.1 Introduction
Silk fibroin is a type of protein that comes from the silk of the Bombyx mori
silkworm. It is an excellent biomaterial for medical applications because of its
impressive mechanical properties, biocompatibility, and controlled biodegradability.
These characteristics make it perfect for tissue engineering, especially for bone
regeneration and skin repair. However, the traditional methods for processing and
modifying silk fibroin often involve harsh chemicals and conditions that can be
harmful to the environment and health. Therefore, there is a growing interest in
developing more eco-friendly alternatives that are just as effective (Lu et al. 2010). It
is necessary to advance the field in alignment with ecological and health standards.
Three-dimensional porous matrices with robust mechanical characteristics support
cell adhesion and expansion in tissue engineering (Grabska-Zielińska et al. 2021).
The
new tissue. Porous scaffolds promote cell adhesion and expansion and often require
interconnected pores larger than 100 μm in diameter. Moreover, the scaffolds should
be structurally sound to hold and release growth factors at a regulated pace to
optimize cell functions and the development of particular tissues. Consequently,
developing porous scaffolds in an environmentally friendly manner by avoiding
harsh processing procedures and organic solvents would be a helpful strategy for
maximizing the functionalization of these scaffolds for cell interactions and possible
delivery of bioactive growth factors (Heseltine et al. 2022).
of silk fibroin’s remarkable mechanical, biodegradable, and biocompatibility
qualities, numerous other biomedical uses have been investigated in recent years.
As biomaterial scaffolds, silks offer support matrices for various cell types, such as
fibroblasts, osteoblasts, hepatocytes, stem cells, and ligaments. They also serve as
scaffolding for tissue engineering applications, including bone, cartilage, blood
vessels, ligaments, and cartilage. Additionally, silks can be used to create controlled
release systems for drugs and growth factors. Scaffolds made of silk have been
prepared using various techniques, including electrospinning, gas foaming, freezedrying, and salt leaching (Huang et al. 2017; Liu et al. 2017). Nevertheless, none of
the aforementioned techniques can eliminate the need for severe processing
conditions, high salt concentrations, or organic solvents like methanol or
hexafluoroisopropanol (HFIP). In some situations, scaffolds are needed to hold
labile medications or bioactive substances that would otherwise break down or
become denatured owing to any or all of the aforementioned factors. In these
situations, it would be helpful to prevent these problems (Tahamtan et al. 2015).
mechanical characteristics make silk fibroin a promising biomaterial. To employ
at which these scaffolds break down should also be similar to the growth of
rate
Biomed
ical s
utures made of silk fibroin have been in use for many years. Because
Moreover, biocompatibility, tunable degradability, minimal toxicity, and
8 Green Approach for Synthesizing Silk Fibroin Biomaterial Scaffolds 197
silk fibroin’s natural qualities, proteins and detritus from native silkworm cocoons
are removed to prevent inflammation. In purification and manufacturing, chemical
solvents, energy-intensive machinery, and lots of water are utilized to reverse
engineer silk fibroin into an aqueous solution and then the final substance. Greener
extraction and manufacturing processes replace toxic chemical additives and energyinefficient equipment while producing mechanically
et al.
2019; Gol
silk sources. They have excellent mechanical qualities, biostability, and biodegradability and can be processed greenly. Spider silk is mechanically superior to silkworm silk, although silkworm cocoons have been harvested for ages due to their
convenience, availability, and affordability (Smith and Doe
cocoons comprise mostly fibroin and sericin. Sericin is a globular gluelike protein
that keeps fibroin fibers together. Due to their inflammatory effects, the two proteins
are usual ly separated in biological applications (Rahimpour et al.
steriliz
ed silk fibroin is cytocompatible with poly(lactic acid) and collagen.
dberg and Kim 2023). Silkworms
robust biomaterials (Nguyen
and spiders are the most studied
2021). Silkworm
2023). Purified and
8.2 Properties of Silk Fibroin Scaffolds as a Biomedical
Application
Silk fibroin is immunologically inert and low-inflammatory after sericin silk protein
elimination (Diez-Echave et al.
with light and heavy chains, Mw = 26 and 390 kDa, respectively. The heavy chain
has 12 big repeating hydrophobic amino acid domains—glyci ne, alanine, serine,
valine, and tyrosine—separated by 11 short hydrophilic areas. Protein secondary
structure depends on hydrophobic domains (Asakura 2021). Silk fibroin has two
process-dependent secondary structures: silk I, an amorphous, water-soluble structure with random coils and α-helices, and silk II, characterized by at least 55%
crystalline antiparallel β-sheet structures. Processing processes cause protein rearrangement from α-helix to β-sheet structures. Hydrogen bond creation allows recurring hydrophobic domains in the heavy chain segment to produce antiparallel
β-sheet crystallites. Controlling the β-sheet composition can adjust the mechanical
characteristics of silk fibroin materials. Increased β-sheet content leads to stronger
material. The crystalline domains absorb impact pressure and distribute it along the
fibroin network, giving silk fibroin elasticity at significant deformations. At minor
deformations, the semi-amorphous matrix controls silk fibroin’s toughness and
extensibility due to its modulus, breaking strength, and elongation (Suzuki 2016;
H
A proteolytic enzyme breaks down silk fibroin into amino acids the body can absorb.
Adjusting β-sheet content and processing factors can influence the degradation rate,
similar to silk’s mechanical qualities. Material degradation slows with higher β-sheet
concentrations. Outside of material processing, the insertion site affects the degradation rate (Wang et al. 2021b)
over structural and morphological aspects is explained using a novel,
to et al. 2020). Silk biodegradability is another benefit as shown in Fig. 8.1.
ashimo
The production
of three-dimensional porous silk fibroin matrices with control
2021). Silk fibroin is a disulfide-bonded copolymer
.
198 S. Verma et al.
Fig. 8.1 Various properties of silk fibroin scaffold
environmentally friendly, all-aqueous method. Lyophilization is a method used to
create scaffolds made of silk. To modify the conformation of silk fibroin and the
interactions between silk fibroin and water, gelatin is added to the aqueous silk
fibroin solution (Liu et al. 2021). This is done by modifying the hydrophilic
interactions in silk fibroin-gelatin-water systems, which inhibits the formation of
distinct sheet-like structures in the material and produces a more homogenous
structure. By creating insolubility in the silk fibroin-gelatin scaffold system through
water annealing, the need for organic solvents like methanol to lock in the beta-sheet
structure is avoided (Wang et al.
2020; Rajput et al. 2020).
The morphological and
functional characteristics of the scaffolds can be controlled by varying the
concentrations of silk fibroin and gelatin. Depending on the amount of silk fibroin
utilized in the procedure, the scaffolds’ interconnecting pores ranged in size from
100 to 300 micrometers (Liu et al. 2020
scaffolds’ morp
hology altered at the same time from lamellar sheets to porous
). Due to the rise in gelatin content, the
structures. When compared to scaffolds obtained from aqueous salt leaching and
scaffolds derived from hexafluoroisopropanol (HFIP), these freeze-dried scaffolds
exhibited a greater degree of hydrophilicity due to their reduced beta-sheet concentration. Without the burst release in the phosphate buffer saline solution, the majority
of the gelat
in w
as trapped in the silk fi
broin
-gelatin scaffolds. These silk
fibroin-gelatin scaffolds were more cell-compatible during in vitro cell culture than
8 Green Approach for Synthesizing Silk Fibroin Biomaterial Scaffolds 199
the salt-leaching silk fibroin scaffolds. The following new method offers scaffold
structures based on silk fibroin that are beneficial for tissue engineering applications.
Moreover, the entire procedure is environmentally friendly, utilizing just water,
ambient temperature, pressure, and no hazardous chemicals or solvents. It opens
up new avenues for including growth hormones or bioactive medications in the
process (Farokhi et al.
2020).
The two main crystalline structures of SF are called silk I and silk II. Silk I is a
metastable crystalline structure that contains bound water molecules, while silk II is
the most stable state because of strong hydrogen bonds between neighboring peptide
blocks, which increase mechanical properties like tensile strength and stiffness
(Qi et al. 2017). The crystalline and amorphous structures found in the secondary
structure derived from regenerated silk fibroin (RSF) solutions will be covered in the
sections that follow. Silk comprises β-turns (silk I) and insoluble structures made of
folded β-sheets (silk II) when it has a crystalline structure, and α-helices, turns, and
random coil structures when it is in an amorphous state. Silk I can be easily
converted to silk II using methanol or potassium chloride; this process is widely
used in biomaterial engineering applications. At the air-water interface of RSF
solutions, Silk III is the unstable crystal structure of SF (Cho et al.
2015; Cebe
et al. 2017; Jaramillo-Quiceno et al. 2017). To guarantee that RSF has good
mechanical properties, numerous approaches have been tested. For instance,
the breaking stress of RSF fibers produced by a dry-spinning method contrasts
with the breaking stress of composite silk fibers made of graphene oxide and RSF.
The mechanical qualities of silk scaffolds made from RSF can be enhanced, among
other things, by crosslinking, porogens, and 3D bioprinting technologies. As a result,
the resulting SF-based scaffolds are strong enough to be handled during the surgical
procedures required for implantation (Valluzzi et al. 1999)
.
Furthermore, the ability of cells to adhere to scaffold surfaces and migrate into the
scaffold while undergoing proliferation and differentiation within the scaffold is a
critical component of successful scaffold implementation. This is made possible by
biocompatibility. Furthermore, the scaffold must induce either no immune response
at all or a very mild one following implantation (Zhang et al. 2016; Floren et al.
2016; Nazarov et al. 2004
).
Since SF is a naturally occurring polymer, it is known to
be biocompatible and biologically inert. In in vivo experiments, SF has
demonstrated blood compatibility since 1989. Successful scaffold materials should
also have the ability to degrade and resorb biologically since during recovery, the
patient’s cells and extracellular matrix should gradually replace the scaffolds. It is
crucial that biodegradation byproducts are non-toxic and do not affect other tissues,
organs, or bodily functions during bodily metabolism (Gregory et al.
2016;
Cao and
Wang 2009). It has been demonstrated that SF, an enzymatically degradable polymer, does not elicit an immunogenic response. Enzymes are adsorbed onto the
surface of the SF scaffold through surface-bonding domains, initiating the degradation process (Arai et al. 2004).
200 S. Verma et al.
8.3 Various Approaches for Green Synthesis of Silk Fibroin
Biomaterial Scaffolds
The silk fibroin synthesis process, traditionally reliant on chemical-intensive
methods, has been increasingly adapted towards greener approaches due to environmental concerns and sustainability goals. Silk fibroin, a natural polymer obtained
from the Bombyx mori silkworm, is highly valued in biomedical applications for its
remarkable biocompatibility, strength, and biodegradability (DeBari et al.
Traditional methods, however, often utilize harsh chemicals and processes not
aligned with green chemistry principles. There are various green techniques for
silk fibroin synthesis that not only reduce environmental impact but also enhance
the material’s applicability in medical fields. Degumming is the initial critical step in
the processing of silk fibroin, where sericin, the sticky protein surrounding fibroin
fibers, is removed. Traditional methods like sodium carbonate or soap solutions are
effective but environmentally demanding, involving high water and energy consumption (Lujerdean et al. 2022). Using a freeze-drying approach, researchers
created scaffolds with good porosity architectures by observing that collagen
interacted with silk fibroin in an aqueous solution in some research, although the
exact process remains unknown. Drugs might be included in these scaffolds and
released at predet ermined times at the same time. But to get the scaffolds ready to
produce insolubility in water, methanol was needed. Researchers have demonstrated
that a water-an nealing technique can convert soluble silk films into water-stable silk
films with a modified β-sheet composition. This method was not applied to the
manufacture of porous scaffolds (Ansari and Sheikh
2023). The advantages and
disadvantages of green synth esized silk fibroin biomaterial scaffold are summarised
in Table 8.1.
Some researchers provided a novel approach for the production of 3D silk fibroin
scaffolds that would be stable in aqueous systems and not require the use of harsh
2021).
Table 8.1 Various advantages and disadvantages of green synthesized silk fibroin biomaterial
scaffold
S.no. Green approach Advantages Disadvantages
1 Green degumming Non-immunogenic Expensive
2 Water annealing Environment friendly Low viscosity
3 Green lyophilisation Biocompatible Low production
4 Use of supercritical CO
5 Inducing secondary
structures
6 Enzymatic degumming Good transparency Rheology needs to be
7 Use of alternative dissolution
medium
8 Use of DES Higher c
fluid Biodegradable No data regarding clinical
2
Excellent mechanical
properties
Well characterised Allergic response to silk
iability
ellular v
efficacy
Cannot incorporate cells
within
optimized
8 Green Approach for Synthesizing Silk Fibroin Biomaterial Scaffolds 201
processing conditions or organic solvents for cell and tissue culture requirements.
Therefore, by combining lyophilization and water annealing, a revolutionary green
technology was devised. During the procedure, silk-based scaffolds were made
without using salt or organic solvents at room temperature utilizing an all-aqueous
processing technique. Notably, as compared to earlier silk fibroin scaffold systems,
these new scaffolds based on silk fibroin have a lowe
them advantageous
characteristics for tissue engineering (Guo et al.
r β-sheet content, which gives
2021).
8.3.1 Green Alternatives for Degumming
Supercritical CO2 Fluid is the alternative which utilizes high-pressure carbon dioxide to remove sericin without water. This method is praised for its minimal environmental impact and ability to recycle the CO2 used in the process. It reduces water
usage and energy compared to traditional methods. It also enables the extraction of
clean sericin, which can be used in other industries, thus promoting zero-waste
principles (Lo 2021). Enzymatic Degumming is another alternative as shown in
Fig. 8.2 that employs natural enzymes from sources such as papaya skin, pineapple
skin, and microbial cultures to selectively break down sericin without damaging
fibroin fibers. It is highly efficient and specific, this method lowers the environmental
burden by avoiding harsh chemicals and reducing energy usage. It is particularly
effective in preserving the mechanical integrity of fibroin fibers (Zhu et al. 2022).
Citric Acid is another one that uses a mild, biodegradable organic acid that is less
harsh on fibers and the environment than synthetic chemicals. It is comparable to
traditional methods in efficacy, citric acid is less resource-intensive and more
Fig. 8.2 Schematic representation of various alternative approaches to synthesizing silk fibroin
scaffold
202 S. Verma et al.
environmentally friendly, enhancing the sustainability of the silk processing (Wöltje
et al.
2021). Sodium Hydroxide and Urea is another one that offers an alternative
alkali treatment that is more energy-efficient than sodium carbonate, though still
demanding in terms of chemical use. While not the greenest, modifications in
process design, such as reduced temperature and optimized reagent concentrations,
can enhance its eco-friendliness (DeBari et al. 2021
degummed, silk fi
solvents like lithium bromide are effective but environmentally harmful.
broin must be dissolved into a processable solution. Traditional
; Medronho et al. 2019). Once
8.3.2 Green Alternative to Dissolution Techniques
Deep Eutectic Solvents (DES) involve a mixture of biodegradable compounds that
dissolve silk under less harsh conditions than typical chemical solvents. DESs can be
tailored for specific applications and are reusable, significantly reducing solvent
waste and energy consumption (Cao and Wang
as both a degumming agent and a dissolution medium, simplifying the silk
processing pipeline. It uses less water and energy than other methods, though it
requires careful management to avoid excessive fiber damage (Hu et al. 2020).
Inducing secondary structures is another technique where its secondary structure
largely determines the final material properties of silk fibroin, typically the β-sheet
configuration, which provides the material with its needed stabili ty and strength for
medical applications (Wang et al. 2021a).
2009). Also, sodium hydroxide acts
8.3.3 Green Alternative to Fabrication Techniques
Water Annealing involves exposing silk films or fibers to moisture, which induces
the formation of β-sheets. It eliminates the need for harmful chemicals like methanol,
using a simple and environmentally benign process that can be easily scaled (Egan
et al. 2022). Mechanical Stimulation uses physical forces, such as stretching or
ultrasonication, to induce alignment and crystallization of fibroin molecules. Water
annealing reduces reliance on chemical treatments and emphasizes physical processes that can be energy-efficient and minimize waste. Moreover, adopting green
synthesis methods for silk fibroin not only aligns with global sustainability goals but
also potentially improves the biocompatibility and utility of silk in various biomedical applications. These green approaches, ranging from enzymatic degumming to
water annealing for structural induction, represent a significant advancement in the
field of biomaterials. By continuing to develop and refine these methods, the silk
industry can significantly reduce its environmental footprint while enhancing the
functionality and application scope of silk fibroin products (Hu et al. 2011).
The synth
application of principles that reduce or eliminate the use or generation of hazardous
substances. Key principles include the use of safer solvents and renewable materials,
energy efficiency, and designing for degradation. These principles aim to minimize
esis of silk fibroin scaffolds through green chemistry involves the
8 Green Approach for Synthesizing Silk Fibroin Biomaterial Scaffolds 203
the ecological footprint of biomaterial production while ensuring the functionality
and safety of the scaffolds for biomedical use. Traditionally, silk fibroin is extracted
from silk using sodium carbonate or other harsh chemicals that can harm the
environment (Li et al.
processes that reduce toxic waste and energy consumption. For instance, the
degumming of silk, necessary to remove sericin (another protein in silk), can be
achieved using enzymatic treatments that are less polluting and preserve the integrity
of the fibroin fibers better than chemical methods (Wang et al.
synthesis, cross-linking
mechanical properties—is achieved using non-toxic agents such as genipin, a natural
crosslinker derived from the gardenia fruit. This contrasts with commonly used
chemical cross-linkers like glutaraldehyde, which are effective but potentially harmful. Additionally, enzyme-base d methods for cross-linking, such as using
transglutaminase, offer specificity and can be c
reduci
ng
energy
use
2024). A greener approach is a milder, aqueous-based
2023). In green
of silk fibroin—necessary to improve its stability and
onducte
and avoiding toxic by-products (Dorishetty et al. 2020).
d under mild conditions,
8.4 Comparing the Traditional Approach to the Green
Approach
Silk fibroin, a biomaterial used in many medical applications, is synthesized using
chemical-intensive techniques that pose environmental and health hazards. As
sustainability becomes a top priority in industries, green methods are being
embraced. These green methods reduce ecological footprints while maintaining or
improving silk fibroin functioning for tissue engineering, wound healing, and
medication delivery (Kostag et al.
Silk fibroin is traditionally produced by degumming with sodium carbonate,
dissolving with lithium bromide, and chemically forming secondary structures.
Each stage has serious environmental impacts. Conventional degummin g uses a
lot of water and strong chemicals that pollute the environment and kill aquatic life.
Due to the use of hazardous, volatile solvents that emit significant VOCs, the
dissolution process poses serious occupational and environmental health concerns
(Khan et al. 2013). While, green silk fibroin synthesis uses safer chemicals, less
waste, and energy-efficient techniques to reduce environmental consequences.
Greener degumming methods like enzymatic degumming have replaced sodium
carbonate. Natural enzymes target sericin proteins, conserving fibroin integrity and
decreasing chemical inputs and energy use. Studies show that papaya enzymes can
degum similarly, if not better while being biodegradable and safer for workers and
the environment (Zhou et al. 2019b)
solvents (DESs), a blend of biodegradable chemicals that break down silk fibroin
at lower temperatures without the toxicity of standard solvents. Reusing and
recycling these DESs reduces waste and exposure to dangerous compounds (Zhou
et al. 2001).
The tradi
conditions that are not environmentally friendly. For example, the use of organic
tional synthesis of silk fibroin scaffolds often involves solvents and
2021).
. G
reen synthesis also uses deep eutectic