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Collagen-Based Hydrogels and Their
Applications for Tissue Engineering
and Regenerative Medicine
Sorina Dinescu, Madalina Albu Kaya, Leona Chitoiu, Simona Ignat,
Durmus Alpaslan Kaya, and Marieta Costache
Contents
1 Introduction ................................................................................... 2
2 Collagen-Based Hydrogels: Overview .. . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . 3
2.1 Methods of Obtaining Collagen Hydrogels ............................................ 4
2.2 Properties of Collagen Hydrogels ...................................................... 6
2.3 Collagen-Natural Polymer for Tissue Engineering .................................... 7
3 Applications of Collagen-Based Substituents in Tissue Engineering and Regenerative
Medicine ....................................... ............................................... 8
3.1 Bone and Cartilage Repair .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 8
3.2 Muscle Tissue Repair ................................................................... 11
3.3 Nerve Tissue Regeneration . . ........................................................... 12
3.4 Vascular Grafts .. . . . . . . . ................................................................ 13
3.5 Corneal Reconstruction ................................................................. 13
3.6 Other Biomedical Applications .. . ..................................................... 14
4 Original Collagen Hydrogels Designed and Tested for Adipose and Cartilage Tissue
Engineering ...................................... ............................................. 15
4.1 Collagen-Sericin Hydrogels .. . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 15
4.2 Collagen-Polysaccharides Hydrogels .. ................................................ 16
5 Conclusions ................................................................................... 17
6 Future Scope .................................................................................. 17
References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 18
S. Dinescu · L. Chitoiu · S. Ignat · M. Costache (*)
Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania
e-mail: marietacostache@gmail.com
M. Albu Kaya
Collagen Department, INCDTP –Leather and Footwear Research Institute, Bucharest, Romania
D. A. Kaya
Department of Medicinal and Aromatic Plants, Mustafa-Kemal University, Hatay, Turkey
#Springer International Publishing AG, part of Springer Nature 2018
Md. I. H. Mondal (ed.), Cellulose-Based Superabsorbent Hydrogels,
Polymers and Polymeric Composites: A Reference Series,
https://doi.org/10.1007/978-3-319-76573-0_54-1
1
Abstract
A promising solution for soft tissue regeneration is tissue engineering, a multi-
disciplinary field of research which involves the use of biomaterials, growth
factors, and stem cells in order to repair, replace, or regenerate tissues and organs
damaged by injury or disease. The success of tissue engineering (TE) depends on
the composition and microstructure of the used scaffolds. Ideally, scaffolds have to
be similar to natural tissues. Collagen is the major component of the extracellular
matrix of most soft tissues. The interactions between collagen and cells are vital
in the wound healing process and in adult tissue remodeling, collagen being able
to support differentiation and maintenance of cellular phenotype. As a natural
molecule, collagen possesses the major advantage of being biodegradable, bio-
compatible, easily available, and highly versatile and presents low antigenicity.
This chapter aims to present an overview on the structure, properties, and bio-
medical applications of collagen hydrogels. Moreover, it introduces the reader
to the latest research in the field of tissue engineering related to collagen. It also
displays the results we obtained as a joint bioengineering group on collagen
hydrogels designed for soft (ATE) or cartilage tissue engineering (CTE) applica-
tions: type I collagen hydrogels improved with either silk sericin (CollSS) or
with pro-chondrogenic factors –hyaluronic acid and chondroitin sulfate
(CollSSHACS). Results indicated in both cases the positive influence of sericin
on the interaction between cells and the surface of the hydrogels. In the absence
of HA and CS, specific chondrogenic inducers, CollSS hydrogel is adapted for
soft tissue reconstruction, whether the addition of HA and CS transforms
CollSSHACS into a suitable hydrogel formula for semihard tissue repair via
modern strategies in tissue engineering and regenerative medicine.
Keywords
Collagen hydrogels · Biomaterial · Regenerative medicine · Tissue engineering ·
Sericin · Hyaluronic acid · Chondroitin sulfate
1 Introduction
Hydrogels are three-dimensional (3D) polymer networks that are able to maintain
a large amount of water and biological fluids, whose physical and chemical cross-
links between polymer chains keep their structural integrity [1,2]. The polymer
chains can be natural, synthetic, or hybrid [3]. The natural polymers like collagen,
chitosan, elastin, alginates, hyaluronic acid, pullulan, and fibrin have been processed
from living organisms. Having a similar structure with the extracellular matrix,
natural polymers possess good biocompatibility properties with low cytotoxicity.
However, because of processing/extraction, they are not preserving mechanical
properties [4]. The synthetic polymers generally used in hydrogels preparation are
poly(ethylene glycol) (PEG), poly(acrylamide) (PAM), poly(vinyl alcohol) (PVA),
polyethylene oxide (PEO), poly(acrylic acid) (PAA), and poly(propylene fumarate-
co-ethylene glycol) P(PF-co-EG) [5,6], which possess controllable chemical and
2 S. Dinescu et al.
mechanical features but less biocompatibility properties. In the last 20 years, hybrid
hydrogels consisting in both natural and synthetic polymers have been developed in
order to provide a better quality of life, mechanical resistance, and water absorption
capacity [3].
The applications of hydrogels are multiple and in different research areas such
as pharmaceuticals, biomedical implants, drug delivery, tissue engineering, and
regenerative medicine [1]. The hydrogels were found to be ideal for clinical appli-
cations, in healing of wounds and burns and even necrotic tissue due to their ability
to transfer vapor and oxygen resulting from their high-water content applied to the
wound site [5].
Due to tissue-mimicking characteristics, the most recent research recommended
the use of hydrogels as scaffolds to provide a biomimetic 3D microenvironment for
the growth of cells [4].
Regenerative medicine is an emerging multidisciplinary field of research which
involves the use of biomaterials, growth factors, and stem cells in order to regener-
ate, repair, or replace tissues and organs damaged by injury or disease. Currently,
tissue engineering applications are focused toward the use of implantable biohybrids
consisting of scaffolds combined with stem or precursor cells and appropriate
inducers, as a regeneration strategy.
The objective of this chapter is to describe the structure, properties, and
main applications of collagen hydrogels for practical applications in Biology and
Medicine and to present our main findings in this field of research.
2 Collagen-Based Hydrogels: Overview
Collagen is the main natural protein of most soft, lax, semirigid, and rigid connective
tissues (skin, bones, tendon, basal membranes, etc.), providing mainly structural
integrity for tissues [7]. Collagen is found in proportion of 80% at skin level,
reported to all dermal dry substances [8,9].
Collagen is a polymer which is characterized by high hydrophilicity, variable
ionic character, and diverse functionality and can be involved in a large number of
interaction systems with other micro- and macromolecular components [10,11].
Collagen is an amphoteric macromolecule with rigid triple helix conformation,
containing both polar groups and hydrophobic portions derived from hydrophobic
amino acids. Consequently, at pH values outside the isoelectric range, between the
molecules, fibrils, and chain fragments of the non-denatured type I fibrillar collagen,
all types of intramolecular electrostatic, dipole-dipole, hydrogen and hydrophobic
bonds can be established in the aqueous medium. This may be the reason why
aqueous systems of this type of collagen become gels at low concentrations com-
pared to denatured collagen (gelatin or collagen hydrolysates), which have the
molecules in the form of statistical coils.
The most used collagen extracts which are basic resources for obtaining medical
biomaterials are type I collagen gels and solutions. A gel is defined as a system with
intermediate properties between a fluid and a solid. Thus, a gel can be a less viscous
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 3
fluid or a very viscous solid. At isoelectric pH, the fibers and fibrils are separated
from the collagen gel, making it dispersible. Collagen gels and solutions are poly-
dispersed colloidal systems, despite tropocollagen solutions which are mono-
dispersed, containing only molecules with triple helix conformation.
According to our experience, type I fibrillar collagen extracted from the calf
hide by acidic and basic treatments at temperatures between 18 C and 25 C, with
an average molecular weight of 3.1–3.3.10
5
g/mol, consists of native collagen
molecules, fibrils, and fragments of single chains.
The rheological study of non-denatured type I fibrillar collagen gels at pHs outside
the isoelectric range, acid (2.5–4), neutral (7.0), and alkaline (8.0), showed that they
have pseudoplastic behavior, more obvious at higher concentrations. Viscosity at zero
shear rate increases sharply with collagen concentration. The gels are easier to destroy
by the shear forces at lower concentrations but restore their structure if they are left at
rest long enough. This may suggest that intermolecular hydrogen bonds are carried
out, as with micromolecular amides, via water molecules.
The main forces that produce the coalescence of the type I fibrillar collagen gel
are electrostatic forces, the others contributing in a small extent to its consistency.
The collagen fibrils linked by hydrogen bonding and hydrophobic and electrostatic
interactions are not stable, being dissociated by variations in temperature, ionic
strength, pH, or collagenase [12]. In order to improve mechanical strength, thermal
stability, and biodegradation rate, covalent bonds are suggested for collagen gels.
Besides the gels, hydrogels are three-dimensionally hydrophilic polymeric net-
works obtained by cross-linking of gels. Cross-linking ensures the insolubility of
hydrogels in water because of the ionic interaction and hydrogen bonding, providing
mechanical strength as well as physical integrity to the polymeric hydrogels [1].
2.1 Methods of Obtaining Collagen Hydrogels
Collagen hydrogels are biomaterials obtained by cross-linking of corresponding
gels. Mechanical and biological properties are controllable and usually superior
compared to the gels from which they were obtained.
Natural in vivo cross-linking provides mechanical strength and proteolysis resis-
tance to collagen [13]. Used in vivo, collagen biomaterials are subject to enzymatic
degradation, which is why they are stabilized by in vitro cross-links, especially with
aldehydes [14–16]. Cross-linking methods lead to creation of additional chemical
bond between collagen molecules and/or fibrils, increasing mechanical and chemical
strength and, consequently, reducing biodegradability.
In vitro collagen cross-linking methods can be classified in two categories:
physical and chemical. The main disadvantage of chemical cross-linking is the
potentially toxic effect of residual cross-linking agent or of complexes formed in
the process of in vivo degradation. In order to eliminate this disadvantage, physical
treatment is applied, incl uding heat-drying and exposure to UV and γradiation. Both
dehydrothermal and UV exposure at 254 nm wave length increase the collagen
contraction temperature and also the enzymatic degradation strength. However,
these physical treatments cause partial denaturation in collagen [13].
4 S. Dinescu et al.
Chemical cross-linking consists of collagen reactions with aldehydes,
diisocyanates, acyl azides, polyepoxides, and polyphenolic compounds, which
lead to the formation of ionic or covalent bonds between molecules or fibrils.
The most used cross-linking agents are aldehydes, especially formic and glutaric.
From all cross-linking agents, glutaraldehyde (GA) is the most used due to its high
efficiency in stabilization of biomaterials made out of collagen. GA cross-linking
involves reactions of the free ε-amine groups of lysine or hydroxylysine from the
polypeptidic chains with the GA aldehyde groups [17]. Lysine and hydroxylysine
represent 3% from all collagen amino acids and, because of this, approximately 60%
of those amino acids residues react with GA under the conditions used to achieve
a certain degree of fixation [18].
Although other cross-linking agents are preferred to reduce cytotoxicity, they
cannot equal GA in terms of stability [11]. The success of thousands bioprosthesis
implanted in last years has shown that GA cross-linking is clinically accepted,
despite reported cytotoxicity [16,19,20]. Biocompatibility of collagenic materials
cross-linked with GA can be increased by lowering its concentration or by combin-
ing it with others physical-chemical treatments [21]. As an alternative to aldehyde
treatment, resistant collagenic materials can be obtained using hexamethylene
diisocyanates (HDC) as cross-linking agents. HDC is solubilized in water with
surfactants or dissolved in 2-propanol. It forms bonds with two chemical ε-amine
groups passing through urea-like linkages, mainly used to obtain cross-linked
gelatin-based plasma substitutes.
Many studies have focused on the use of epoxide compounds (diglycidyl ethylene
glycol, polyglycidyl glycol, methylglycidyl ether) [22]. Epoxide compounds react
easily with amine groups of lysine, but they also pose the problem of
cytotoxicity [23].
Carbodiimide cross-linking, especially with 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), offers the advantage of forming amide linkages between the
carboxyl and amine groups of the collagen molecules without becoming part of the
effective linkage. Thus, dysfunctional cross-linking agents are avoided [13].
Collagen gel and collagen gel cross-linked with glutaraldehyde and with
carbodiimide obtained in our laboratory are presented in Fig. 1.
Intermediary forming of acyl azide is an alternative method of those with
carbodiimide, which has as result amidic bonds introduction [24]. Carboxyl groups
are transformed into hydrazide. Then, the reaction with sodium nitrite takes place to
form the acyl azide. Otherwise, the diphenylphosphoryl azide modification can be
made. Acyl azide treatment increases collagen-glycosaminoglycan matrix resistance
for up to 3 months and inhibits calcification in vivo, while collagen treated with
glutaraldehyde is completely calcified after 15 days due to calcium fixation at free
carboxyl groups [25].
Polyphenols have been used as vegetal tannins for tanning animal skins for a very
long time [26]. These are good cross-linking agents due to multiple interactions with
proteins, thus increasing the stability of collagen from hydrogels or spongious
matrices.
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 5
Genipin is a natural cross-linker which demonstrated lower cytotoxicity and
better biocompatibility compared with chemically synthetized cross-linking
agent [27].
Collagen properties can be controlled by using different type or amount of cross-
linkers named before and by varying cross-linking conditions. Cross-linking gener-
ally creates a covalently bonded gel that does not deform as a fluid during injection,
but it rather fractures and does not recover, leaving a mass of particles.
2.2 Properties of Collagen Hydrogels
Hydrogels are three-dimensional networks made of hydrophilic polymers obtained
from gels by covalent bonds cross-linking or by ionic forces, which in aqueous
medium swell to an equilibrium value.
Cross-linking degree is one of the most important factors which affects hydrogel
swelling. It is defined as a ratio between the number of moles of cross-linker and the
number of moles of units that are repeated from the polymer molecule. The higher
the degree of cross-linking, the more dense structure and the less intense swelling
due to reduction in polymeric chain mobility. The degree of swelling is also affected
by the chemical structure of the polymer: hydrogels containing large amount of
hydrophilic groups are swelling more intense than those containing hydrophobic
groups.
Hydrogel swelling may also be affected by the swelling environment temperature
or ionic strength and pH, as well as by other factors [28].
The kinetics of hydrogel swelling can be controlled by diffusion when applying
Fick’s law or by relaxation when the law is no longer respected. If the diffusion of
water in the hydrogel occurs faster than the relaxation of the polymeric chain, the
swelling kinetics is controlled by diffusion.
The mechanical properties of hydrogels are very important for pharmaceutical
applications and can be achieved by modifying their cross-linking degree. Increasing
cross-linking leads to more consistent but more fragile gels. There is an optimal
degree of cross-linking for which the hydrogel is elastic and resistant.
Fig. 1 Collagen gels: (a) un-cross-linked and cross-linked with (b) glutaraldehyde and
(c) EDC/NHS
6 S. Dinescu et al.
Collagen, alone or in combination with other molecules from the extracellular
matrix, plays an important role in the physiology and behavior of cells from
connective tissue. Keratinocytes and fibroblasts are important cells involved in
healing skin lesions. Therefore, the use of collagen hydrogels as a substrate for the
cell culture improves adherence, migration, growth, and cell differentiation under
normal and pathological conditions [4].
The interaction between collagen and blood platelets plays an important role in
the mechanism of hemostasis induction. Platelets adhere on the collagen surface;
their adhesion and aggregation activate clotting and initiate hemostasis. The reaction
depends on the cross-linking degree of collagen from hydrogels and on the positive
loads localized on the base groups from the side chain. If free collagen carboxylic
groups are blocked by chemical processes, more than 98% of hemostatic activity
disappears. Hemostasis is dependent also on the conformational structure of colla-
gen. Thus, denatured collagen (gelatin) does not activate platelet aggregation,
whereas non-denatured collagen in forms of fibrils can activate platelets, being
efficient in hemostasis induction.
Generally, the interactions between collagen and cells are important phenomena in
wound healing process and in adult tissue remodeling, collagen inducing differenti-
ation and maintaining cell phenotype [29,30]. Another important biological property
of collagen is biocompatibility, due to its low toxicity and poor immunogenicity.
Also, collagen is poorly antigenic. Low antigenicity can be due to the presence
of aromatic amino acids, especially tyrosine. In collagen, there is a low quantity of
aromatic amino acids, about three residues of tyrosine on a chain. To reduce or
eliminate collagen antigenicity, the N-terminal regions of the polypeptide chains
are removed during the collagen extraction process.
2.3 Collagen-Natural Polymer for Tissue Engineering
The most recent studies showed a continuing concern about obtaining biomaterials
which mimic extracellular matrix for further use in regenerative medicine. The
collagen, the main component of skin extracellular matrix, can be used as tissue
engineering scaffold in combination with other polymers or cross-linked [27].
Collagen-elastin cross-linked squaric acid created a 3D transparent hydrogel
highly resistant to enzymatic degradation to be used in medicine and tissue engi-
neering [31]. Other hydrogels based on collagen, hyaluronic acid (HA), and sericin
in different ratios showed that HA played the role of cross-linking agent which were
proved to be nontoxic and very consistent against enzymatic treatment [32]. Collagen
and chitosan in the presence of α,β-glycerophosphate formed hydrogels which
proved their ability to be biocompatible substrates with L929 cells, being promising
scaffolds for tissue engineering [33]. Drug delivery systems and hybrid hydrogels
based on collagen such as collagen-hydroxyapatite-alendronate hydrogel for
MC3T3-E1 osteoblastic cells [33], collagen-polyvinylpyrrolidone superabsorbent
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 7
hydrogels cross-linked by γ-irradiation [34], chitosan-collagen coated with
poly(L-lactic acid) by electrospinning [35] for tendon regeneration, and collagen-
poly(N-isopropyl acrylamide) with montmorillonite nanoparticles incorporated
hydrogel [36] were also developed.
Our experience in natural collagen hydrogels allowed the development of func-
tional hydrogel scaffolds biocompatible with different types of cells and designed
for different tissue engineering applications. These hydrogels are further presented
in Sect. 4.
3 Applications of Collagen-Based Substituents in Tissue
Engineering and Regenerative Medicine
Collagen is the key component of the extracellular matrix in most tissues and is
involved in the maintenance of tissue three-dimensional architecture, as well as in
the development of new tissues and organs. Consequently, one of the targets in tissue
engineering is to develop substitute biomaterials that can closely resemble the
extracellular matrix and, thus, to be integrated both structurally and functionally in
the newly formed tissue. Due to the high prevalence of collagen in all tissues and
organs, the engineered collagen-based substituents have wide applications, as further
described.
3.1 Bone and Cartilage Repair
Collagen is the main structural protein in the extracellular matrix of hard and
semihard tissues [37]. Consequently, orientation toward collagen-based biomaterials
for cartilage and bone tissue engineering came as a natural choice; however, because
collagen alone is not a suitable substituent for bone tissue engineering (BTE)
applications in terms of mechanical properties, the usage of a new combination of
materials in the pursuit of a more biomimetic scaffold was reported [38]. The team
created a nanosized hydroxyapatite (nHA) and collagen-based hydrogel as a support
for the proliferation and differentiation of human mesenchymal stem cells (hMSCs)
isolated from either adipose tissue (AT-MSCs) or bone marrow (BM-MSCs).
Although their hypothesis stated that osteogenic differentiation should be dependent
on nHA concentration, the results of the study showed that AT-MSCs display a
higher osteogenic potential compared to BM-MSCs, irrespective of nHA amount in
the constructs.
Similarly, in 2016, Gurumurthy et al. [39] reported the attempt to find a more
suitable scaffold for guided bone regeneration. Since collagen alone is characterized
by mechanical weaknesses, composites with elastin-like polypeptide (ELP) were
analyzed as a support for human adipose-derived stem cells (hASCs) in osteogenic
differentiation processes. After 3 weeks of evolution, the ELP-collagen constructs
proved to be superior to pure collagen scaffolds in terms of tensile strength, elastic
modulus, osteogenic activity, and mineralization.
8 S. Dinescu et al.
A new technique in BTE [40] offers a deeper perspective in long-term repair
of bone defects. Using an iterative layering freeze-drying method of assembling
gradual amounts of collagen (Col) and hyaluronic acid (HA), the group has created
a biomimetic bone substitute which presents a top-bottom gradient of both
interconnected porosity and HA content. The similarities with the physiological
structure of the bone were easily observed, and the chemical composition resembled
the natural model, with 1
=
3organic and 2
=
3inorganic elements. The biocompatibility of
this Col/HA scaffold was assessed via the evolution of a seeded BM-MSCs culture.
Compared to a collagen scaffold, the Col/HA composite indicated better rates of
success in bone tissue regeneration, and thus, it could be considered an advanced
strategy in tissue engineering.
One of the main implementation problems in BTE is to ensure the blood supply to
the construct. According to a recent study [41], this issue may have found its solution
in cocultivation of cells. As well as bone grafting, bone tissue engineering is a feasible
approach toward minimizing extended bone defects in the fields of orthopedics and
oncology. In order to regenerate bone fragments, the team studied the advantages of
cocultivating endothelial cells together with an osteogenic cell lineage in the context
of a mouse calvarial defect model. Using a negative control represented by a pure
collagen scaffold, a positive control of a collagen scaffold enhanced with bone
morphogenetic protein 7 (BMP-7), and three collagen scaffolds seeded with human
osteoblasts (hOBs) only, CD34+ cells only, and a coculture of hOBs and CD34+ cells,
respectively, Hertweck et al. concluded through a series of radiographic, histological,
immunohistological, and statistical methods that the cocultivation of hOBs and CD34
+ cells or even the monoculture of CD34+ cells offers a better perspective in bone
regeneration than monocultures of osteoblasts.
More recently, Nguyen et al. [42] reported the analysis of a suitable biomaterial
for prevascularized bone fabrication prior to implantation in a patient. The group
determined the importance of specific environmental parameters in human umbilical
vein endothelial cells (HUVECs) and hMSCs coculture. Different scaffold types,
from alginate hydrogels for hMSCs differentiation into osteoblasts to collagen type I
hydrogels for HUVECs angiogenesis, were assessed. In addition, shear stress was
applied to this coculture system in a tubular perfusion system bioreactor in order to
advance the progression toward osteogenesis and prevascularization. Moreover, it
was observed that cells attached, proliferated, and differentiated with better rates due
to specific peptide sequences as native content of cell-binding sites in collagen
fibrils, leading to the conclusion that collagen would be better for future bone tissue
engineering applications.
In 2015, Lee et al. [43] studied the fabrication process of biomimetic three-
dimensional structures with preosteoblasts and hASCs. Their team engineered a
bioink that apart from being nontoxic, biocompatible, and printable, also has cell-
activating properties. By pre-cultivating cells in collagen, they enriched the tradi-
tional collagen/alginate composite with extracellular matrix. After being printed, the
resulted cell-embedded bioink undergone a cell viability and differentiation study:
preosteoblasts successfully started displaying osteogenic activity; whereas hASCs
followed a hepatogenic differentiation pathway, expressing liver-specific genes.
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 9
Cartilage is an avascular, aneural, and alymphatic tissue, with a very low number
of progenitor cells and a very slow turnover of the extracellular matrix. Therefore,
this tissue has a very limited auto-regeneration potential and represents one of the
most addressed tissues by tissue engineering and reconstruction procedures. In this
context, collagen-based biomaterials have been widely used for applications, such as
repair of articular cartilage lesions, replacement of intervertebral discs, knee menisci,
anatomical disorders of the temporomandibular joint, deformities of the midface and
ears, etc.
To address one of the challenges of cartilage engineering, several collagen-
mimetic hydrogels with chondrogenesis-inducing properties have been proposed.
For example, Parmar et al. suggested a novel biodegradable collagen hydrogel
with incorporated heparin-binding, integrin-binding, and hyaluronic acid-binding
sequences at the backbone of a streptococcal collagen-like 2 protein. Meta-
lloproteinase 7 (MMP7) and aggrecanase (ADAMTS4)- cleavable peptides were
later used in the process as cross-linkers. Their conclusion was that this new
collagen-based hydrogel can be used for cartilage regeneration therapies, since it
promoted cell viability, cell adhesion, and chondrogenesis [44].
One of the treatment courses adopted for focal cartilage lesions involves matrix-
induced chondrocytes implantation (MACI). As this method requires autologous
chondrocytes prone to dedifferentiation during in vitro expansion, Fensky et al.
proposed the implantation of predifferentiated MSCs seeded in a type I collagen
hydrogel. The MSCs were cultured in collagen hydrogels, and predifferentiation was
initiated by adding TGF-β1 in the culture media. After 10 days, the hydrogels were
switched to a TGF-β1-free media for another 11 days. In both histochemical assay and
chondrogenic markers, gene expression confirmed the evolution of chondrogenesis.
Therefore, the collagen hydrogel could represent a reliable method in delivering
predifferentiated cells to the needed implantation site [45]. Additionally, Chen
et al. [46] concluded that a type I collagen hydrogel seeded with mesenchymal
stem cells isolated from the Wharton’s jelly of human umbilical cord (UC-MSC)
could be a suitable 3D model for cartilage healing.
The suitability of collagen hydrogels in CTE was also confirmed in a study
focused on material’s immunomodulatory properties [47]. The group investigated
the immunogenicity of neonatal rabbit chondrocytes seeded in a collagen type I
hydrogel. Even though the immunogenicity markers showed a tendency to increase
over the first 14 days of in vitro culture, after 28 days, the immunogenic effects were
lower. The collagen hydrogel promoted the synthesis of extracellular matrix over the
28 days in culture. This led to the accumulation of extracellular matrix, and it was
suggested that it isolated the seeded cells from the host immune cells and from the
hydrogel itself, providing some immunogenicity-reducing effects. In addition,
in 2016 Yang et al. [48] published a study that compared the evolution of
chondrogenesis and the secretion of immunoregulatory factors of MSCs in 2D and
3D culture. The cells expanded in the collagen hydrogels (3D) showed higher
chondrogenic markers at gene and protein levels of expression than the ones
on plastic (2D), simultaneously with an enhanced secretion of immunoregulatory
factors. This could suggest that the collagen hydrogel promoted the chondrogenic
differentiation of MSCs.
10 S. Dinescu et al.
3.2 Muscle Tissue Repair
Collagen constitutes 1–2% of the muscular tissue, where it serves as a major
component of the endomysium. Several studies have been performed in order to
reconstruct muscle tissue assisted by collagen substituents, as further described.
Recently a study was conducted regarding the usage of vascularized tissue scaffolds
in large volumetric muscle defects [49]. Working on a rat biceps femoral model, the
team developed a series of vascularized collagen hydrogels with adipose-derived
microvessels. After seeding these scaffolds with myoblasts and implanting them in
the existent limb injury, Li et al. observed the revascularization and muscle regen-
eration processes, as well as the differentiation of myoblasts in myotubes. However,
as compared to a vascularized scaffold only, which showed a similar regeneration
rate after a 2-week evolution, the team concluded that while the vascular network of
the construct may support the regeneration of volumetric muscle defects, other
factors have an important role as well.
With respect to the cardiac muscle, studies have been developed to test
the efficiency of hybrid hydrogels based on collagen in myocardial regeneration.
Injectable bioactive hydrogels were developed from thiolated collagen (Col-SH) by
adding multiple acrylate containing oligo(acryloyl carbonate)-b-poly(ethylene gly-
col)-b-oligo(acryloyl carbonate) (OAC-PEG-OAC) copolymers for improving the
material’s mechanical properties [50]. The cellular lineage selected for this study
consisted of BM-MSCs, due to their rapid spreading rate and extensive network-
forming capacity. After the implantation of BM-MSC-encapsulating hybrid hydro-
gel in a rat infarction model, Xu et al. observed a significant improvement in cardiac
function in the case of the hydrogels as compared to the PBS control. Moreover, the
echocardiography analysis showed an increase in the ejection fraction at 28 days,
while the histological assessments demonstrated a decrease in the infarct size and an
increase in ventricular wall thickness.
More recently, von Marion et al. [51] published a study regarding a different
method of addressing cardiac stem cell therapy by culturing cells directly in the
biomaterial for better survival, proliferation, and differentiation rates. Cardiomyocyte
progenitor cells (CMPCs) were seeded in a unidirectional constrained and a stress-
free collagen hydrogel. The three-dimensional environment highly supported cellular
growth and maturation, and an enhancement was observed in specific markers
(Nkx2.5, myocardin). The proof that the CMPCs were actively involved in the matrix
secretion and remodeling processes was given by levels of collagen type I, collagen
type III, elastin, fibronectin, and matrix remodeling enzymes. Furthermore, when
constrained, CMPCs became mechanosensitive, adopted a rod-shaped morphology
and responded to mechanical stimuli in addition to developing sarcomeres and
contractility.
In 2017, Ketabat et al. [52] identified collagen and alginate injectable conductive
hydrogel systems as promising candidates for cardiac muscle regeneration in terms
of cardiomyocyte viability and syringeability. Other tissue engineering applications
of such hydrogels rely on their specific property that allow delivery to the requested
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 11
site through a catheter. Collagen is therefore used for its self-assembly characteristic,
resulting fibrous forms upon warming up to 37 C, while alginate is recommended
for its non-thrombogenic nature.
3.3 Nerve Tissue Regeneration
Currently, nerve injuries or diseases that affect the nervous system are found more and
more often and seriously affect patients’lives. These nerve injuries could be effi-
ciently addressed by modern strategies in tissue engineering, namely, by designed
scaffolds that would represent the necessary support for nerves to regenerate. A few
of these strategies are further discussed.
In 2015, a study regarding a novel combination of PuraMatrix 3D nanofibrous
hydrogel and a honeycomb collagen sponge in creating a scaffold suitable for
neuronal regeneration after complete transection of the spinal cord in adult rats
was published. After the implantation of the construct in the 5 mm spinal gap, its
evolution was recorded during 24 days to 4 months, as compared to the PBS-injected
control group. The histological and western blot analyses have indicated the migra-
tion of both neurons and astrocytes toward the implant site, as well as their
differentiation and maturation stages, therefore suggesting functional recovery,
spinal repair, and neuronal regeneration [53].
Using carbon nanotubes and collagen in generating three-dimensional micro-
environmental conditions for MSCs in neural tissue regeneration, Lee et al. [54]
highlighted once again the impact of collagen scaffolds on bioengineering. Studying
the differentiation process of MSCs in neural lineages, the team has selected collagen
as the ideal support for MSCs growth and expression of neural phenotypes, while the
addition of small amounts of carbon nanotubes (0.1–1 wt%) has improved cellular
proliferation rate. Moreover, CNTs were associated with improvements in both
expression of neuronal markers and secretion of neurotrophic factors, especially
nerve growth factor and brain-derived neurotrophic factor.
Similarly, Park et al. [55] introduced the concept of 3D collagen scaffolds
for human umbilical cord blood (UCB) cells in order to promote the secretion
of therapeutic factors, such as neurotrophic factors. Collagen proved to be an
advantageous biomaterial, not only being a suitable 3D microenvironment but also
stimulating the secretion of neurotrophins, nerve growth factor, brain-derived
neurotrophic factor, and ciliary neurotrophic factor. Moreover, the 3D scaffold has
also been described as an efficient reservoir of neurotrophic factors by storing and
slowly delivering them toward the target cells. To confirm their hypothesis, Park
et al. have facilitated indirect interaction of UCB environment with human neural
precursor cells, observing afterward neurite outgrowth. Future implications of such
scaffolds include therefore neural repair and regeneration processes.
12 S. Dinescu et al.
3.4 Vascular Grafts
Aiming to study vascular biology in health and disease, Roberts et al. published
in 2016 a study concerning the process of generating microvessels by seeding
endothelial cells in a microfluidic channel embedded with a native type I collagen
hydrogel. Their three-dimensional model serves as a solution for the inadequate
planar cell culture and uses injection molding methods to generate engineered blood
vessels. The study of seeded HUVECs has already offered an insight in pericyte
migration, platelets adhesion to activated endothelium, fluid shear stress modulation
of endothelial activity, and von Willebrand factor (VWF) secretion and assembly.
Moreover, by adding parenchymal cells in the collagen matrix, the researchers have
created a tissue microenvironment which allows them to further study processes
related to the intestinal lining, lung alveoli, kidney tubule, and many others [56].
The developing process of an injectable allogenic collagen-phenolic hydroxyl
(collagen-Ph) hydrogels in mice that supports de novo generation of a three-
dimensional vascular network was recently documented [57]. Specifically, they
injected in subcutaneous murine models a prepolymer solution of collagen-Ph,
human blood-derived endothelial colony-forming cells (ECFCs), bone marrow-
derived mesenchymal stem cells (MSCs), horseradish peroxidase, and hydrogen
peroxide and analyzed the course of tissue forming. After 7 days, the ECFCs have
generated extensive vascular networks and functional anastomoses with the existing
vasculature of the host tissue; the collagen has improved MSCs differentiation, while
phenolic hydroxyl has favored an increase in the number of adipocytes present in the
targeted tissue [57].
3.5 Corneal Reconstruction
The cornea may suffer from diseases, injury, infections, or inflammation, and these
can lead to the need of a corneal replacement. To avoid total replacement by surgery,
modern approaches in tissue engineering identified the opportunity to use adapted
hydrogels to assist corneal regeneration. Latest research in this field is further
presented.
In 2016, Rafat et al. [58] assessed the suitability of collagen-based hydrogels
as biomaterials in corneal regeneration. Their team engineered a construct with
a transparent core and an adjustable periphery characterized by faster degradability
rates. The material was used in in vitro cultivation of human epithelial cell popul-
ations and transplanted in an in vivo mice model. After a 3 months’time evolution,
the collagen construct was declared suitable for corneal transparency restauration,
maintaining the overall corneal shape and integrity.
Previously, other studies [59] presented an alternative to the cross-linkage
method of generating transparent collagen hydrogels. By substituting the
usage of N-(3-dimethylaminopropil)-N0-ethylcarbodiimide (EDC) with a sterically
bulky carbodiimide, N-cyclohexyl-N0-(2-morpholinethyl)carbodiimide metho-p-
toluensulfonate (CMC), the group obtained corneal replacements with longer, easier
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 13
to control gelation time, as well as superior resistance to collagenase. Liu et al. [60]
started using collagen in developing corneal substitutes since 2008. Their hydrogels
were synthesized as interpenetrating polymeric networks (IPNs) and were constituted
of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide, N-hydroxysuccinimide cross-
linked porcine atelocollagen, and poly(ethylene glycol) diacrylate cross-linked
2-methacryloyloxyethyl phosphorylcholine. Compared to each individual com-
pound, the hydrogel presented greater mechanical strength, as well as enzymatic
stability and low UV degradation rates. Due to collagen, the corneal substitute
appeared to be cell-friendly, promoting nerve ingrowth and regeneration. Moreover,
its optical properties resemble the human model. When implanted in pigs, the
allografts indicated in vivo compatibility, the experiment being declared a success.
After previously postulating porcine collagen hydrogels as corneal substitutes,
another study [61] assessed the potential use of recombinant human type I and type
III collagen in such applications in order to minimize the possible immune reactions
toward animal origin implants. By cross-linking the collagen fibrils with 1-ethyl-3-
(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS),
the team created a corneal substitute with light transmission properties comparable to
superior to that of a human model. Not only that these substitutes had adequate
tensile strength and elasticity for surgical procedures, but type III collagen proved
to be superior in terms of mechanical characteristics. During a 12-month post-
implantation assessment in pigs, the hydrogels maintained their optical clarity,
promoted regeneration of corneal cells, nerves, and tear film and proved to be
eligible for further clinical applications.
McLaughlin et al. [62] addressed the problem of tissue regeneration by extracel-
lular mimics in the context of cornea, a three cellular layered structure in a hydrated
extracellular matrix, delimited by a nonkeratinizing epithelium and an inner endo-
thelium. Proposing a corneal substitute made out of carbodiimide cross-linked
porcine type I collagen, they have demonstrated that simplicity in fabrication
might be the key to future biomaterial applications. During a 12-month integration
and development of the collagen implant in the host tissue, it was observed that the
substitute supported the regeneration of corneal cells, nerves, and the tear film.
Moreover, it had been described as maintaining its optical clarity.
3.6 Other Biomedical Applications
Collagen scaffolds can also be used as apoptotic sites when conjugated with
cyclopamine, according to a study of Jain et al. published in 2014. The team
analyzed the migration of the cells constituting a glioblastoma, an aggressive brain
tumor which invades adjacent cerebral regions along white matter tracts and blood
vessels. After engineering aligned polycaprolactone-based nanofibers as an invasion
site, they mediated the transfer of the tumor toward the extracortical cyclopamine
drug-conjugated, collagen-based hydrogel. Cyclopamine was preferred because it
only affects cells which are dependent on the sonic hedgehog pathway of survival,
i.e., cancer stem cells. Tumor loads were significantly higher in the case of nanofiber
14 S. Dinescu et al.
implants compared to smooth film or empty conduit implants. Moreover, tumor
loads outside of the collagen-based hydrogel were significantly lower in the case of
nanofiber implants [63].
In 2013, Rao et al. developed an unprecedented study of a series of three-
dimensional collagen –hyaluronan composite hydrogel in order to achieve an
accurate model of glioblastoma tumor cell migration. Their work focused on using
biomaterials to understand tumor cell biology rather than promote tissue engineer-
ing. Collagen scaffolds were selected due to the material’s association with cell
migration, collagen types I, III, and IV being found in glial lamina externa and
vascular basement membranes, while collagen types I and III as well as hyaluronan
are the main constituents of the native brain extracellular matrix. The team observed
that glioblastoma migration occurs in inverse function of hyaluronan concentration;
therefore hyaluronan has a delaying effect on cell movement [64].
4 Original Collagen Hydrogels Designed and Tested
for Adipose and Cartilage Tissue Engineering
Based on the positive results reported by other groups and on the versatility
of collagen-based hydrogels for different tissue engineering applications, our
group has studied two different composites designed for cartilage and soft tissue
reconstruction.
4.1 Collagen-Sericin Hydrogels
Hydrogels based on type I fibrillar collagen (Coll) with different ratio of silk sericin
(SS) presented a superporous and absorbent structure (Fig. 2). The pore size varied
between 2 and 90 μm, the structure being denser with increasing amount of sericin.
The absorbent capacity showed values between 3000% and over 4000% at acidic,
alkaline, and neutral pH and classified hydrogel as being very absorbent. Even the
high amount of sericin showed more compact structures. The mechanical properties
and biodegradation rates demonstrated that the best scaffold was Coll:SS =5:2
ratios [65]. The physicochemical results were confirmed by biological ones which
showed that preadipocytes colonizing the scaffold containing 40% sericin, being a
good candidate for future applications in soft tissue engineering [66].
This validated composition of CollSS hydrogel was developed for adipose tissue
engineering (ATE) and revealed good biocompatibility and supported differentiation
of human adipose-derived stem cells (hASCs) [67]. Silk sericin (SS) is a natural
sticky protein isolated from B. mori silk fibers, which proved to be responsible for
proliferation and attachment of several mammalian cell lines [68] as well as for the
activation of collagen production, both in vitro and in vivo [69].
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 15
4.2 Collagen-Polysaccharides Hydrogels
Furthermore, in order to direct this biocompatible composite (CollSS) toward
cartilage tissue engineering (CTE), two pro-chondrogenic molecules, components
of cartilage extracellular matrix –hyaluronic acid (HA) and chondroitin sulfate (CS)
–were added in the composition, resulting in a pro-chondrogenic collagen hydrogel
(CollSSHACS). Several ratios between HA and CS were tested to identify the
most efficient one for CTE: CollSS-10%HA, CollSS-5%HA, CollSS-10%CS, and
CollSS-5%CS. HA increased the thermal stability of scaffolds; the absorbent prop-
erties showed a higher capacity to uptake water (4200–4300%) than the control
samples (CollSS) and the pore size between 20 and 150 μm, being larger for CollSS
samples which contain HA and smaller for samples which contain CS [70].
Cytocompatibility studies with hASCs and their differentiation toward the carti-
lage lineage revealed the collagen hydrogel improved with 10% HA and 5% CS
to be the most equilibrated formula to support cell proliferation [70] and hASCs
chondrogenesis during in vitro studies. Briefly, 3D cell-scaffold culture was
achieved by seeding hASCs in CollSSHACS and then exposed to chondrogenic
induction cocktail for 28 days. The in vitro differentiation process was monitored at
7, 14, and 28 days post-induction. Scanning electron microscopy (SEM) revealed
that cells distributed in the volume of the hydrogel through the network of pores and
adhered to the CollSSHACS better than to control Coll, probably due to the sticky
properties of sericin. Gene expression studies revealed upregulated chondrogenic
markers such as SRY (sex determining region Y)-box 9 (Sox9) and cartilage
oligomeric matrix protein (COMP). These results were also confirmed at protein
level by confocal microscopy.
Fig. 2 SEM image of
collagen: sericin (5:2)
hydrogel, x 200
16 S. Dinescu et al.
To conclude our experience with collagen hydrogels, we have evaluated
hASCs/CollSS bioconstruct for ATE [67] and hASCs/CollSSHACS for CTE.
Results indicated in both cases the positive influence of sericin on the interaction
between hASCs and the surface of the hydrogels and on the differentiation pro-
cesses. Evaluation by immunohistochemistry and microscopy showed the formation
of cellular aggregates in CollSSHACS, which is specific for the first steps of
chondrogenesis. Nevertheless, in the absence of HA and CS specific chondrogenic
inducers, the conditions offered by CollSS hydrogel favor hASCs differentiation
toward the adipogenic lineage, thus confirming CollSS to be appropriate for soft
tissue engineering applications.
5 Conclusions
Collagen-based hydrogels incorporating other natural polymers (derived from extra-
cellular matrix tissue) showed very good biocompatibility with different types of
cells, but request controllable biodegradability and improved mechanical properties
using suitable cross-linker agents (preferably natural). The hydrogels consisting in
collagen and synthetic polymer have controllable physical-chemical properties but
poor biocompatibility and request surface functionalization with natural polymers in
order to improve their biocompatibility. Biopolymers such as hyaluronic acid, chon-
droitin sulfate, and sericin in combination with collagen formed 3D hydrogels with
high biocompatibility and potential to support cells differentiation. Furthermore,
CollSS could prove useful for soft tissue engineering, while CollSSHACS could
be a good candidate for cartilage reconstruction.
6 Future Scope
For future studies, more attention should be focused on smart biomaterials such as
drug delivery systems with antimicrobial properties based on collagen hydrogels.
The drug delivery systems request complex structure so that the drug can be released
from hydrogels in a controlled way to avoid the risk of not being effective from the
pharmaceutical point of view. A high amount of drug could be toxic for cells –the
hydrogel will not be biocompatible, while a small amount of drug would not
influence the biocompatibility nor be effective for the proposed aim.
Other challenge in tissue engineering is the risk of infection at the tissular level.
For this reason, we focused to establish a balance between antimicrobial and
biocompatibility properties, using natural ingredients such as essential oils and
zeolite [71]. Previous results showed that it is possible to open such a “barrier”but
extensive research needs to be performed in the future.
Another opportunity to further improve the collagen hydrogel would be to bring
together the benefits of collagen with the benefits of autologous platelet-rich plasma
(PRP). Rich in growth factors that positively influence cell growth, proliferation,
adhesion, and other cellular processes, PRP would ensure better biocompatibility,
lower inflammatory potential, and better acceptance rate at the implant site of
Collagen-Based Hydrogels and Their Applications for Tissue Engineering and... 17
collagen. One recent study [72] approached this idea and used it to create a collagen-
based construct dedicated for skin regeneration. Researchers developed a type I
collagen, fractionated platelet-rich plasma (PRP)-supplemented scaffold, able to
recruit dermal stem cells from adjacent tissue, fully disclaiming the need of cell
seeding. As compared to a collagen –fetal bovine serum control, the new biomaterial
proved to be better when implanted into rat wound models in terms of epitheliali-
zation and neovascularization. Not only the number of recruited stem cells was
higher when PRP was used, but also the dermis-like tissue formation proceeded at
a faster rate, leading to hair growth and sweat gland production [72].
Acknowledgments The authors would like to acknowledge the funding sources that supported
this work: grant 65PCCDI/2018 (REGMED), Project 3- dedicated to regeneration of soft tissues and
to national project Bridge Grant PNIII-P2-2.1-BG-2016660 0458 (123BG/2016), as well as the
National Executive Agency for Research Funding.
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