Recent Advances in The Synthesis and Applications of Collagen Based

Mediterranean Journal of Basic and Applied Sciences (MJBAS)
(Peer Reviewed International Journal), Volume 3, Issue 2, Pages 54-98, April-June 2019
Recent Advances in the Synthesis and Applications of Collagen Based

Hydrogels: A Review
Jesús A. Claudio-Rizoa*, Laura Espíndola-Sernab, Juan J. Becerra-Rodriguezb, Lucia F. Cano-Salazara and Tirso E.
Flores Guíaa.
b Ingeniería en Biotecnología, Universidad Politécnica de Pénjamo, Carretera Irapuato-La Piedad Km 44, C.P. 36921, Pénjamo, GTO, México.
Article Received: 19 February 2018 Article Accepted: 07 March 2019 Article Published: 06 April 2019
ABSTRACT
Collagen is a triple helical protein present in the connective tissue and bones of mammals. The collagen has the ability to polymerize by adjusting the
pH and temperature, generating fibrillar matrices in the hydrogel state. Collagen hydrogels have shown great potential for applications related to
biomedicine, tissue engineering and regenerative medicine (BTERM), mainly due to their high biocompatibility and ease of processing. However,
collagen hydrogels have poor mechanical properties and high rate of proteolytic degradation, limiting their applicability for in vivo tests that demand
control in said properties. In order to combat the aforementioned disadvantages, systems of collagen-based hydrogels have been designed in
combination either with natural polymers, synthetic polymers, inorganic particles and/or complex biological molecules, demonstrating to regulate the
self-assembly processes of the collagen molecules, forming crosslinked and/or interpenetrated polymeric networks based hydrogels. In addition, the
physicochemical interactions among the collagen molecules and the functionalization agents regulate the structure and properties of the 3D hybrid
networks generated, enhancing their application in specific fields of BTERM. This work summarizes the most recent strategies for the synthesis and
applications of collagen-based hydrogels with diverse chemical components that have been designed for BTERM. Finally, this work also illustrates
the importance of the properties of collagen-based hydrogel systems and their potential use in other future biotechnological applications.
Keywords: Collagen hydrogels, chemical functionalization, biomedicine, Tissue engineering, Biotechnology
1. INTRODUCTION
Collagen hydrogels have demonstrated to have properties that make them candidates for application in
BTERM; however, their rapid degradation and weak mechanical properties limit their use. An accurate
control of the structural characteristics and properties of the collagen based hydrogels could provide the key
to regulate their behavior for in BTERM improved applications. Collagen has been widely used to prepare
biomaterials, since it represents the majority of the composition of the animal connective tissues [1]. From a
biochemical point, the main constituent of the extracellular matrix (EC M) is the collagen; this molecule is
formed in vivo by polymerization reactions in stages with enzymatic regulation among the conformer amino
acids [2]. To date, more than 20 types of collagen have been identified, varying in their amino acid sequence,
mainly [3]. The collagen polypeptide chains have a primary structure consisting of the sequence
Gly-X-Y-Gly-Pro-Hyp-Gly-X-Y where every third amino acid is glycine (Gly). Glycine comprises about
33% of the chain of amino acids while proline (Pro) and hydroxyproline (Hyp) comprise about 20%. The rest
of the percentage is composed of the polar amino acids, both acidic and basic, represented as X and Y [4].
The polypeptide chain of the collagen has amino functional groups ( -NH2), carboxylic acids (-COOH) and
hydroxyls (-OH) as substituents, which together with the amide bonds of the polymeric backbone represent
reactive centers. The secondary structure is defined by the local configuration of the chains resulting from
the satisfaction of stereochemical angles and the capacity of interaction via hydrogen bonding.
CITE THIS ARTICLE: Jesús A. Claudio-Rizo, Laura Espíndola-Serna, Juan J. Becerra-Rodriguez, Lucia F.
Cano-Salazar and Tirso E. Flores Guía., “Recent Advances in the Synthesis and Applications of Collagen Based
Hydrogels: A Review”, Mediterranean Journal of Basic and Applied Sciences, Volume 3, Issue 2, Pages 54-98,
April-June 2019
54 | Page
The tertiary structure gives rise to the collagen molecule consisting of three polypeptide chains arranged in
a triple helix configuration. The collagen molecules form a supramolecular structure comprising five
molecules packed in a specific arrangement, generating a collagen microfibril. The microfibrils are coupled
in specific series forming a collagen fiber; these fibers generate bundles of fibers to give rise to much larger
collagen fibers [5].
The collagen molecule, also called tropocollagen, has approximately a total of 240 ε-amino groups as well as
230 carboxylic groups and 300 nm in length [5]. Under physiological conditions these groups are charged. In
its natural state, collagen configurations are maintained by covalent chemical bonds, hydrogen bridge-type
interactions, ionic and hydrophobic interactions. The functional groups in the collagen interact with each
other to form intra or intermolecular bonds providing stability to the collagen fibers. Under these co nditions,
only a small number of charged groups are free but the electrostatic state of the collagen can be altered by the
change in pH of the medium, resulting in the weakening of intra- or intermolecular electrostatic interactions
[6]. The ends of the collagen triple helices are terminals with –NH2 and –COOH functional groups without
helical configuration called N- and C-terminal telopeptides, respectively, which produce crosslinking for the
stabilization of collagen and the formation of fibers. These non-helical ends are also related with the
formation of immune response reactions [4].
The versatility of collagen as a building biomaterial is mainly due to its complex hierarchical structure
originated from its molecular sequence. The tropocollagen molecules are assembled into complex
supramolecular aggregates by a polymerization process called fibrilogenesis; this is a process driven by
entropy, this process has also been presented for other protein self -assembly systems [7]. It is driven by the
loss of solvent molecules from the surface of the protein molecules and results in assemblies of longer
fibrillar structures with conical ends of circular cross section, which minimize the surface area to volume
ratio of the final assembly. The interactions that direct the association between tropocollagens are mostly
electrostatic and hydrophobic [8]. The stabilization of such interactions is ensured by the existence of
molecular crosslinking, which it occurs among the telopeptides of the collagen molecules with th e main
chain of the helix [9]. The process of fibrilogenesis can be carried out in situ by extracting collagen of
diverse ECMs to generate collagen-based hydrogels. The hydrogels are polymeric materials swollen in water
that maintain a specific three-dimensional structure. They are very suitable materials for BTERM
applications due to their good interaction with living tissues, since on the one hand they show good
biocompatibility properties, mainly due to their soft, elastic consistency and water content. Besides, their
characteristic of swelling brings them the property of absorbing, retaining and releasing under controlled
conditions quantities of water that regulates their structural conformation, desirable properties for BTERM
applications [10, 11].
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Specifically, the hydrogels formed from the partial digestion, solubilization and in situ polymerization of
decellularized tissues can retain the biological activity of the native ECM. Therefore, these hydrogels have
complex biochemical compositions, fibrous protein content (e.g., collagen, fibrin, elastin), proteoglycans
(PGs), and glycosaminoglycans (GAGs) that closely mimic the native 3D tissue environment, compared to
simple composition hydrogels [12]. The remaining ECM molecules after extracting the coll agen have a
direct relationship with the in situ polymerization process, structure and properties of the generated
hydrogels. A higher content of matrix proteins improves the mechanical properties and proteolytic
degradation rate, exhibiting enhanced biocompatibility properties [13]. The main ECMs used to extract
collagen and to generate hydrogels are: rat tail tendon [14], bovine Achilles tendon [15], bovine pericardium
[13], porcine intestinal submucosa [16], porcine bladder [17], fish skin [18], and porc ine skin [19].
These collagen based hydrogels have shown bioactivity and biocompatibility associated with their residual
composition. Recently, a review has been reported about diverse ECMs employed in the design of
biomedical hydrogels, describing the main role of collagen based hydrogel with characteristic composition
on the improved applicability [20]. Generally, the collagen hydrogels have been studied as substrates for
ophthalmology, sponges for burns/wounds, systems for controlled delivery of functio nal molecules or
nanoparticles, and matrices for 3D cell culture. They are also investigated for tissue engineering including
skin replacement, bone substitutes, and artificial blood vessels and heart valves [21].
Figure 1. Diverse strategies to generate collagen-based hydrogels for applications in BTERM
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Although collagen hydrogels show excellent biocompatibility and biological performance, there is the
challenge to tailor their physicochemical properties for an enhanced application in BTERM, as they ha ve
poor mechanical properties and fast degradation, limiting their use as cargo delivery systems or long -term
dressings, among others [22].
Therefore, it is still necessary to investigate methods to regulate the characteristics and to expand the use of
collagen based hydrogels in the medical biotechnology field. Recent approaches to combat this need include
functionalization with organic phases, such as biopolymers or biological molecules with specific activity,
synthetic polymers of defined chemical architecture and inorganic phases, such as nanoparticles, nanotubes,
bioglasses (Fig. 1).
The presence of these coupling phases on the collagen polymerization process has been shown to control the
kinetics and microstructure of the hybrid fibrillated matrices in the hydrogel state, where the concentration
of the coupling agent is determinant in the tailoring of the physicochemical and biological properties [23]. In
this sense, it is important to indicate that the alteration of the properties of the collagen base d hydrogels is
associated with the generation of interpenetrating network systems (IPNs), covalent crosslinking bonds and
inter- and/or intra-molecular interactions of the collagen fibers with the coupling molecules [24]. Few
reviews have been reported indicating strategies to generate functionalized collagen hydrogels with
diversity of chemical phases; therefore, the objective of this work is to show the relationship of the
composition of these hybrid matrices, detailing the strategies of the process of synthesis, crosslinking
methods, highlighting the improved properties of the systems and their application in some BTERM field.
Finally, we also mention the challenges, possibilities and perspectives of application of these hydrogel
systems in different areas of the biotechnological field.
2. COLLAGEN CROSSLINKING
The generation of collagen hydrogels with a variety of coupling agents requires strategies to reinforce their
basic properties for specific applications in the biotechnological field. Structurally, the fibrous collagen
network must be reinforced mechanically to support the load of the coupling materials. Generally,
crosslinking strategies have been developed for this purpose. Crosslinking, in a specific way, consists in the
generation of points of union among the triple helical chains of collagen. These int ra- or inter-molecular
interactions can be established physically; by means of programmed freezing-drying cycles to achieve a high
entanglement of the collagen fibers, considerably improving their mechanical properties and altering the
swelling capacity of the generated hydrogel [24]. In this strategy, the control of the degradation rate of the
hydrogel is not affected, so that the time of use of the materials under proteolytic conditions is limited [25].
The physical crosslinking can also carry out by the variation of the pH, temperature, electrical fields or other
physical stimuli [26]. This type of crosslinking offers advantages such as, relatively easy manufacture and
the reducing of the toxicity risks due to the absence of exogenous crosslinking molecul es [27]. The control of
57 | Page
the rate of collagen degradation and improvement in its mechanical properties are obtained by using
chemical crosslinking approaches (Fig. 2).
The chemical crosslinking approaches are based on the generation of covalent bonds amon g the functional
groups amine or carboxylic acid of the collagen with exogenous molecules. These methods increase the
resistance of the hydrogel toward both chemical and enzymatic degradation, reduce its immunogenicity,
sterilize and improve its mechanical properties. However, a direct evidence between the concentrations of
chemical crosslinkers with the biocompatibility has been determined in several works. In this sense, it is
advisable to use low concentrations of crosslinker in order not to reduce the c haracteristic biocompatibility
of collagen. The collagen crosslinkers most studied are the glutaraldehyde (GLU), carbodiimides
(specifically, 1-Ethyl-3-(3 -dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide) (EDC),
genipin (GEN), 1,3-phenylenediacetic acid (APH), polyethylene glycol diacrylate (PEGDA) and aqueous
polyurethane prepolymers (PPU). In the collagen hydrogels crosslinked with GLU, the ε -amine groups of
collagen yield an imine bond (so-called as Schiff base); the crosslinking reaction is relatively fast; reacting
with most ε-amine groups, improving both mechanics and degradation resistance, but it has been observed a
drastic reduction of the biocompatibility [28].
Figure 2. Approaches to chemically crosslink to the collagen to generate hydrogels with improved
properties.
When EDC is used as crosslinker, results as effective catalyst in the condensation of collagen carboxylic
acids with alcohols and amines, without presence of the chemical structure of carbodiimide in the
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crosslinking bonds, thus the degradation products of these hydrogels do not show cytotoxic character;
however, the crosslinking reaction is not taken out at physiological conditions (pH 7.4, 37 °C) [29].
Diverse strategies for the preparation of collagen hydrogels are based on the use of GEN as chemical
crosslinker, as a spontaneous crosslinking by formation of Schiff base is produced by a Michael reaction
involved in this process. The structure and properties of hydrogels show a direct relationship with the GEN
concentration; a notable disadvantage to consider is the generation of blue residues during the preparation of
hydrogels, limiting their transparence and use as 3D culture systems [30].
Hydrogels were successfully formed via functionalization of collagen with A PH as novel and bifunctional
segment crosslinker; the functionalization with the aromatic segment provided the formation of a triple
helical covalent network with tunable crosslinking density and swelling ratio, whereby enhanced mechanical
properties. Particularly based on their bioactivity and full biocompatibility at low APH concentrations, these
hydrogels have significant appeal for applications in mineralized tissue regeneration [31].
The use of synthetic polymers with reactive ends to generate covalent crosslinking bonds is an interesting
approach to tailor the properties of the collagen hydrogels and to functionalize with other components.
Strategies using PEGDA to induce photo crosslinking based on the formation of covalent linkages among the
functional groups acrylamide with the collagen-amines. Under this approach, the collagen hydrogels show
enhanced hydrolytic stability and susceptibility to collagen enzymatic degradation; showing also direct
dependency of the mechanical properties on time of UV irradiation. Limitations related with to use of UV
irradiation for applications to formation of hydrogel in situ or cell encapsulation must be considered [32].
Crosslinking agents with high solubility in water are required to potentiate the chemical crossli nking
reactions with hydrolyzed collagen precursors, thus avoiding the use of solvents or phases that can decrease
the biocompatibility. In this sense, recently the use of PPU based on poly(ethylene glycol) (PEG) and
aliphatic diisocyanates as crosslinker of the collagen chains has been reported. The process involves the
formation of urea linkages between end-blocked isocyanate of PPU and collagen amines. The crosslinking
process is taken out at physiological conditions. The structure and properties of coll agen hydrogels show a
direct relationship with the chemical structure of PPU. The use of soluble PPU around 15 % weight
accelerates the collagen polymerization forming consistent hydrogels in 20 minutes. Higher concentrations
of PPU inhibit the collagen polymerization, and decrease its biocompatibility [33].
The main component of the synthetic collagen crosslinkers discussed is PEG. Recently, a review of the state
of the art of physicochemical modifications made by PEG in collagen -based hydrogels has been established
[20]. This work highlights the use of PEG derivatives, widely used in formulations of pharmaceutical
products or in synthesis of biomedical polyurethanes, as a strategy to modulate both physical and biological
properties of natural ECM hydrogels, providing a comparison of the characteristics of hybrid hydrogels in
terms of the improvement of structure, mechanics, and degradation behavior for BTERM applications.
59 | Page
3. TYPES OF COLLAGEN-BASED HYDROGELS

The crosslinking process of the collagen chains is important because it allows to generate functionalized
hydrogels with a variety of coupling agents, such as: natural polymers, synthetic polymers, inorganic
particles and complex biological molecules. This section covers the state of the art in the dev elopment and
study of the properties of these hybrid hydrogels in the field of BTERM, highlighting the innovation in the
formulation, the crosslinker used, the characterization techniques, and the main advantages offered for their
applications in some area of BTERM.
3.1 Natural polymers
The high both biocompatibility and bioavailability represented by natural polymers, such as chitosan, fibrin,
elastin, alginate, cellulose, cyclodextrin, poly(lactic acid) PLA, among others, represents significant
importance to develop systems of collagen-coupled hydrogels (Fig. 3).
Figure 3. Production of collagen hydrogels functionalized with natural polymers.
Natural polymers can be extracted from biomass, generating systems of materials with natural sustainability,
novel properties that can be exploited in the biotechnological field, ensuring low economic cost in their
production. The physicochemical combination of the hydrolysed collagen chains with biopolymer molecules
can generate hydrogels of IPNs. In this type of system, collagen chains are physically crosslinked, producing
entanglements that modify the properties of collagen and that of the coupling agent . If the coupling
biopolymer has reactive functional groups, it could generate chemical crosslinking, significantly improving
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the properties of the hybrid hydrogel. In table 1, the main systems of collagen hydrogels coupled with natural
polymers are summarized. Degradation rate control represents a major challenge in this hydrogels system,
since the components are naturally degraded. If the hydrogel is used for tissue repair or regeneration, it is
important that the hydrogel is degraded as the new tissue is formed. The by-products of degradation do not
show a cytotoxic character related to the biocompatibility of biopolymers. For long -term performance
applications, it is important to control their rate of degradation; which is achieved using exogenous
crosslinking molecules that ensure the formation of covalent bonds, inhibiting the proteolytic degradation of
the hydrogel. The reinforcement of the mechanical properties of these hydrogels should also be considered
when it is required to incorporate molecules with biological interest, such as drugs or cytokines that benefit
metabolic pathways of interest in the BTERM area; again the crosslinking process is important to ensure
mechanical performance of these hybrid hydrogels.
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Collagen Coupling agent Collagen Processing Novel features Characterization In vitro / In vivo Ref.
source crosslinker conditions methods results
Bovine Thiolated hyaluronic ------------- pH 7.4 at 37 The physical characteristics Mechanical The HA concentration [34]
Achilles acid °C, 5% CO2 of hydrogel can be analysis, confocal of the hydrogel
tendon for ∼1 h modulated through the reflectance induces cellular
variation of Hyaluronic Acid microscopy, SEM, morphological
(HA) concentration, which, cell spreading changes and variation
directly influences the assay and in spreading and
glioblastoma multiform cells migrations migration of GBM
(GBM) behavior. experiments. cells.
The collagen-gel offer a 3D
microenvironment to tumor
cell studies.
Soft coral Alginate EDC-NHS 48 h, RT The stiffness, strength of SEM, DIC and Hydrogel mechanical [35]
Sarcophyto alginate-collagen hydrogel mechanical properties was
n was more elevated than analysis dependent on the
alginate hydrogel. collagen concentration
Hyperelastic behavior similar and orientation and
to human tissues. arrangement of
collagen fibers.
Rat tail Alginate, CaSO4 and ------------- 4°C for 20 Increased mechanical SEM, DMA, TGA, Injectable [36]
tendon Na2HPO4 min, pH 7, properties and XDR and FTIR biocompatible
37°C for 1.5 osteoconductivity. hydrogel system with
h. The hydrogel contains gelation time variable,
collagen and apatite from 5 to 10 minutes
phosphate, components of for bone tissue
natural bone. regeneration.
Bovine Dialdehyde ------------- Centrifugate Spongy collagen cryogels of LLS spectroscopy, The swelling is [37]
Achilles carboxymethyl d at 2500 fast swelling rate. FTIR, SEM, DSC dependent of DCMC
tendon cellulose (DCMC) rpm, 4°C, 20 Improved thermostability. and swelling content and pH.
62 | Page
min, frozen Good blood compatibility analysis. Increased DCMC in

at -20°C for 5 cryogel decreases
days and hemolysis ratio and
thawed at increases
RT. antithrombotic
activity.
Porcine Microcrystalline ------------- 100°C under Structure macroporous 3D SEM, FTIR, The absorption [38]
dermis cellulose and 1-butyl, N2 for 2 h with actives amino groups to mechanical capacity is related to
3-methylimidazolium increased binding Cu(II). analysis and collagen/cellulose
chloride ([C4mim]Cl) Reusable until 4 absorption/desor mass ratio. The pH
absorption/desorption cycles ption assays for optimum to
before their broken. Useful Cu(II) absorption is 6 in all
for wastewater quantification. study cases. The
bioremediation hydrogels were
desorbed at 95%
efficiency.
Rat tail Bacterial cellulose EDC-NHS RT for 24 h, The hydrogel induced cell SEM, FTIR, 31P The hybrid hydrogel [39]
tendon Gluconoacetobacter dried to 37°C growth for bone NMR, TGA, containing osteogenic
xylinus regeneration purposes. mechanical growth peptide
Its uses do not show analysis and stimulated the early
cytotoxic, genotoxic and cytotoxicity, development of
mutagenic effect. genotoxicity and osteoblastic
mutagenic assay. phenotype and
enhanced cell growth.
Collagen Chitosan ------------- 37°C for 30 Mechanical properties SEM, rheological Immobilizing of [40]
(BD min in a suitable for survival, analysis, Angiopoietin-1
Biosciences humidified maturation and performance live/dead, peptide, allowing to
) 5% CO2 of cardiomyocytes. enzymatic the cardiomyocytes
incubator Stable for 7 days in vivo degradation and can survive within the
LDH cytotoxicity hydrogel both in vitro
assay, and and in vivo conditions.
histological and The hydrogel injected
immunofluoresce in Lewis rat model was
63 | Page
nt staining. localized in the

injection site one
week after.
Fish skin Chitosan and gelatin GLU -20°C for 24h The component influence to SEM, Oscillatory The gels with salmon [41]
and gel properties, so the largest rheology, components have the
lyophilize swelling is for gel with higher mechanical higher solubility and
until dried. content of chitosan, collagen analysis and lower absorbency
confer less compressed and biodegradation while African catfish
more flexibility while gelatin assay. shows more hardness,
more compressible and less less flexibility and
flexibility. better action
antioxidant.
Collagen Chitosan and 1 sugar Triethyl pH 10, -20°C 2-deoxy-D-ribose sustainable FTIR, SEM, Superficial wounds of [42]
(Mian (ribose, fucose or orthoformat for 24 h, and prolonged delivery of microbiological rats was completely
Scientific rhamnose) e then washed chitosan/collagen hydrogel and biochemistry closed and the skin
suppliers in absolute promotes angiogenesis to analysis, and looked healthy after
Lahore) ethanol and wound healing. chick chorionic 17 days of treatment
dried RT allantoic with 2-deoxy-D-ribose.
membrane (CAM)
assay
Rat tail Chitosan and silica of GEN pH 7.4, mix Hybrid hydrogels have SEM, cell viability Collagen-chitosan-silic [43]
tendon 210 nm and 438 nm all pro-osteogenic properties assay, a gels promotes
components without additional biochemical and osteogenic
by vortexed, osteogenic inductor. molecular biology differentiation of
and incubate analysis human bone
at 37°C for marrow-derived
24 h mesenchymal stromal
cells (hBMSCs) in vitro
cultures. The
differentiation of
hBMSCs is influenced
for particle silica size.
1
Bovine β -cyclodextrins β-CD and 4°C for 48 h, Improved mechanical H NMR, XRD, The composition is an [44]
64 | Page
Achilles (β-CD) polyrotaxane stirred at 37 properties. The biogels FTIR, SEM, effective
tendon multialdehydes °C for 15 exhibit good swelling, mechanical and biocrosslinker than
min, frozen suitable for cell adhesion biochemical conserve the
at -20 °C for and proliferation. The analysis, properties of collagen.
48 h and biodegradation rate has enzymatic L929 fibroblasts cell
thawed at been improved, and show degradation and culturing in hydrogel
25°C lower cytotoxicity similar to cytotoxicity assay had good adhesion,
gels with carbodiimide growing and
proliferation after 5
days.
Porcine Chitosan EDC-NHS pH 7, 4°C for The hydrogel permits SEM, XRD, TGA Wounds of treated in [45]
dermis Alginate 2h sustained released of mechanical vivo with curcumin
Curcumin curcumin for diabetic wound analysis and cell loaded hydrogel
healing. viability healed and closed
Improved properties of /morphology faster than control
hydrogel. assays. and placebo scaffold
groups. Also the
hydrogel aids to
reducing the
persistent
inflammation in
wounds.
Rat tail Fibrin PEG ether pH 7, 37°C Fibrin-collagen hydrogel FTIR, SEM, cell Astrocytes had good [46]
tendon tetrasuccini and 5% CO2 maintains good cell viability viability/motility viability in hydrogels.
midyl and permits the astrocytes assays and The height and weight
glutarate migration. molecular of gels did not change
(4S-StarPEG) biology. significantly after 9
days of cell culture.
Fibrin regulates the
astrocyte migration.
Porcine Fibrin ------------- pH 7, 37°C Injectable fibrin hydrogel SEM, In vitro and in vivo [47]
articular for 30 min functionalized with ECM biochemical, ECM-hydrogel was
joint microparticles and histological and able to supported
65 | Page
transforming growth factor immunohistoche chondrogenesis by

(TGF)-β3, promote mical analysis. controlated release of
chondrogenesis and is (TGF)-β3, generating
chondroinductive, for more cartilage-like
articular cartilage tissue.
regeneration
Bovine skin Bovine fibrinogen ------------- Mix at 4°C, Hydrogels with low HAP Mechanical, In vitro, the [48]
and hidroxiapatite add HAP, 37 concentration promote ultrasound vasculogenesis was
(HAP) °C for 45 min perfusion, growth cellular imaging, 3-D LDPI, increased at low
and add cell and vasculogenesis. histologycal and concentration of HAP
The HAP improves the immunohistoche of 5 mg ml-1, showing
mechanical properties of mistry analysis. perfusion in the
hydrogels but its increased implant site, cellular
inhibits the endothelial growth, capillary
network formation. formation and
inflammatory
reaction.
Porcine Silk fibroin and HAP GLU Gently The hydroxyapatite particles SEM, TEM, XRD, Assays in vitro shows [49]
dermis stirred to gel with size ranging from 30 to FTIR, mechanical good biocompatibility
formation, 100 nm were uniformly analysis, cell between MG63
RT for 24 h, distributed along the viability assays. osteoblast-like cells
pH 7.0 polymer matrix without and hydrogel.
regular crystallographic
orientation.
Elastic modulus was
improved with silk fibroin
adding.
Recombina 2-methacryloyloxyet EDC-NHS Mixed to Soft hydrogel that contains SEM, The gels were cut with [50]
t human hyl 0°C, curated 85% water showing robust fluorescence laser, fitting precisely
collagen phosphorylcholine 100% mechanical properties, microscopy, and welding together
type III humidity suitable to cutting with optical and with excised porcine
(RHCIII) under femtosecond laser and mechanical corneas.
produced nitrogen at posterior microcontact analysis, In vitro, fibronectin
66 | Page
in yeast RT. printing. refractive indices promoted cell

(Pishia and cytotoxicity adhesion and highest
pastoris) assays. mitosis rate of
GFP-HCEC cell line.
Fish skin Pullulan Sodium pH 9.0, RT Translucent, clear and soft SEM, FTIR, TGA, The swelling ratio [51]
trimetaphos for 90 min super absorbent hydrogel. DSC, reached was 320%.
phate and 50°C for Improved mechanical compatibility The gels show good
30 min, pH properties and assay, swelling biocompatibility,
7.0 biocompatible for wound and biochemical enhanced adhesion
healing analysis and proliferation of
fibroblast cell and
promote angiogenesis
in the chick
choriallantoic
membrane.
Rats wound was
closed to 96% after
11±2 days.
Bovine Sodium hyaluronate Oxidized Mix to pH 7, The hydrogels allowed SEM, NMR, Fibroblasts and [52]
Achilles gellan change to pH encapsulation, maintained fluorescence adipose-derived stem
tendon (GellOx) or 8 to viability and proliferation of microscopy, cells were
oxidized pregelifield, eukaryotic cell during ex vivo crosslinking encapsulated in
pullulan 37°C for 30 temporarily storage. analysis for oxidized
(PullOx) min. photocolorimetry polysaccharides-hydro
, rheological gels. The best cell
analysis, stability viability was for
and cytotoxicity PullOx-hydrogel. The
assay hydrogel with GellOx
limit the cell metabolic
activity.
Genetically Genetically ------------- Mixed for 4h, Stability for long time, FTIR, rheological Rat bone MSC [53]
engineered engineered gelation for self-healing behavior and analysis, oxidized cultured in direct
silk–collage silk–collagen-like overnight at tunable mechanical degree, cell contact with the
67 | Page
n-like copolymer produced RT, pH 7.4 properties. viability/proliferat hydrogels are viable
copolymer by Pichia pastoris ion assay and for 21 days of assay,
produced molecular biology however proliferation
by Pichia and mineralization
pastoris were lower than
controls.
Bovine Aldehyde-functionali ------------- 4°C, pH 7.0 Aldehyde-functionalized NMR, SEM, Fibroblast culture [54]
Achilles zed dextran and gelation Dextran/Collagen hydrogel rheological and shows cytoplasmic
tendon to 37°C, 24h are stronger, more mechanical extensions. Cell
thermostable and with a analysis oxidized cultures are alive on
better regular structure than degree and the surface of
pristine collagen hydrogels. cytotoxicity assay hydrogels, without
Biocompatible with differences between
fibroblast and Hela cell. ones.
Grass carp Polydopamine Dopamine pH 7.4, 4°C Improved thermal stability FTIR, XDR, AFM, L929 fibroblasts [55]
(Ctenophar hydrochlorid and and swelling, improved physicochemical cultures were able to
yngodon e centrifuged resistant to enzymatic and adhered and
idella) skin for10 min. degradation For use in biocompatibility proliferate for 5 days.
37°C for 4 h biomedical applications analysis and
to initiate enzymatic
gelation stability
Collagen I Poly(γ-glutamic acid) Fibronectin-li 4°C to mix Injectable scaffold, whose CD and UV-VIS The composition does [56]
(Biolad) (γ-PGA) ke and storage. gelation occurs at body spectroscopy, not affect the growth
engineered temperature in optical and of mMSCs in the
protein approximately 2 min. fluorescence hydrogel at 4 days of
Suitable for regenerative microscopy, SEM culture.
medicine, by delivery of and rheological In vivo mMSCs were
MSCs and anti-oxidant drugs analysis able to remain in the
into injured sites. injection site and have
protective effects
against renal
dysfunction.
Human-like Carboxyl pullulan 1,4- pH 8.0, RT Improved mechanical FTIR, SEM, In vitro, BHK-21cell [57]
68 | Page
collagen butanediol for 30 min, properties, high swelling mechanical were attachment and
(HLC) diglycidyl sol-gel capacity, increased cell analysis and transfected, the cells
expressed ether transition at adherence and cell viability swelling behavior viability increased,
in 50°C for 3h, with anti-inflammatory without cytotoxic
Escherichia washed in effects. This soft hydrogel is effect. In vivo were
coli hot bath biocompatible, proved
containing cytocompatible and anti-inflammatory
pyrogen-free non-biodegradable, suitable effects,
water and to tissue engineering. biocompatibility and
stirred for 72 anti-biodegradability
h, refreshing in mice for 24 weeks.
water every
2 h.
Table 1. Collagen hydrogels coupled with biopolymers
69 | Page
3.2 Synthetic polymers
Diverse formulations of collagen-based hydrogels functionalized with synthetic polymers have been
developed (Fig. 4). Synthetic polymers, such as PEG, polycaprolactone (PCL), PPU, polyacrylamide
(PAM), polymethylmethacrylate (PMMA), polyvinyl alcohol (PVA) , polyvinylpyrrolidone (PVP), among
others, are characterized by giving control in the degradation, mechanics and swelling of hybrid hydrogels;
however, these polymers significantly decrease the biocompatibility associated with collagen.
Figure 4. Synthesis of collagen based hydrogels coupled with synthetic polymers.
Chains of synthetic polymers can alter the entanglement of collagen chains, producing IPNs hydrogels, and
chemical crosslinking systems if they have reactive groups. The mass ratio of the synthetic polymer to the collagen
is an important variable for generating hybrid hydrogels with improved properties for applications in BTERM. In
concentrations of around 10 to 15% of weight of synthetic polymer, the biocompatibility of collagen is not
significantly affected, ensuring control in the mechanics and degradation of the hybrid material. Table 2 shows the
collagen hydrogels formulations using synthetic polymers as coupling agents. The systems of hydrogels with
properties of charge-controlled release of molecules with biological interest are based on collagen matrices
reinforced with synthetic polymers; this mainly because the mechanical properties and swelling can be adapted,
promoting a controlled release, thus improving their applicability in BTERM. Within this system, PEG derivatives
have been shown to be excellent regulators of the properties of collagen hydrogels, indicating a direct relationship
of the concentration of these derivatives with cellular metabolism and phenotype, therefore generating novel
70 | Page
strategies for the tailoring of signaling and cellular fates required in BTERM strategies, as reported in a recent
review [20]. Various synthetic chemical routes can be designed to polymerize and / or copolymerize the monomers
of synthetic polymers with hydrolyzed collagen molecules, generating complex architectures of hydrogels with
adjusted properties; however, in these approaches it is important to control that the polymerization processes are
carried out at temperatures below 40 ºC, thus avoiding the denaturation of the collagen and the loss of its properties.
71 | Page
Collagen Coupling Collagen Processing Novel features Characterization In vitro / In vivo results Ref.
source agent crosslinker conditions methods
Rat tail Hydroxylam Methacrylic Mix vigorously The hydroxylamine CD and UV-vis Hydrogels induced the [58]
tendon ine anhydride stirred for 10 hydrochloride -methacrylated spectroscopy, reduction of MMP-9 and
hydrochlori (MA), min and 24 h collagen conjugate with mechanical and MMP-3 up to ~13 and
de hydroxylamine to reaction, integrated matrix biochemical ~32 RFU% respectively
–methacryla hydrochloride precipitation metalloproteinase analysis for 4 days of incubation
ted and EHC-NHS with ethanol (MMP)-modulating capability in vitro.
for 8 h. hydrogel are suitable for G292 osteosarcoma cell
regulation of culture on hydrogel did
inflammation-response, stable not show toxic effects
in structure and chemical during 4 days of
composition, without cytotoxic incubation.
effect
1
Porcine Methacrylat Glycidyl pH 7.0 mixed The material entirely derived H NMR, These materials were [59]
knees ed methacrylate centrifuged at from native cartilage ECM with biomechanics, able to induced
solubilized 3000 rpm, and similar properties to native bioactivity, chondrogenesis and
decellulariz stored at 4 °C cartilage. Suitable to related biochemistry, matrix synthesis. The
ed cartilage overnight issues of cartilage tissue. histological and MeSDCC concentration
(MeSDCC) molecular biology affects the cellular
analysis. behavior, promoting the
ECM synthesis.
Bovine skin Gelatin-met MA 50°C to The GelMA-hydrogel contains a Oscillatory BAEC cells and [60]
hacrylate completely IPN of gelatin-methacrylate rheology, MDA-MB-231 cells were
(GelMA) mix until 24 h, GelMA that permits wide confocal adhered to collagen
pH 7.0, at 37°C variation of shear modulus microscopy, fibers of GelMA
for 30 min to while retaining the fibrillary mechanical, hydrogel.
polymerize, structure of collagen. biochemical and BAEC cell exhibited a
UV exposure Appropriate for studies of cell image analysis spread and fibroblastic
at 365 nm, behavior. and cell morphology in both the
37°C for 5 min viability/morphol absence and presence of
ogy assay, collagen fibers, although
increased of stiffness
72 | Page
decreased sprouting
Equine knee GelMA, MA 50°C for 1 h, The hydrogel stimulates the Confocal-vis-micr Chondrocytes and MSCs [61]
cartilage stirring and formation of cartilage template oscopy, XRD, µCT, cells showed high
derived add MA, via endochondral, in vivo is mechanical and viability and
matrix dialyzed for 7 biodegradable and reusable into image analysis, chondrogenesis in
(CDM) days at 40°C, mineralized bone new-tissues. and cell viability GelMA-MSC-hydrogel
pH 7.4, assay regardless of the present
freeze-dried. or absence of CDM, after
6 weeks of culture.
1
Porcine Type II MA Dissolution of Photo-crosslinkable type II H NMR, FTIR, CD, After 2 weeks of [62]
articular collagen Col-II-Ma for collagen methacrylamide DMA, CLSM and subcutaneous
cartilage methacryla 12h, add (Col-II-MA) hydrogel presented cell viability/ implantation of hydrogel
mide photoinitiator, gelatinous form with improved morphology/proli in athymic mice,
(Col-II-MA) pH 7.0, 4°C, mechanical strength. feration assay. histological samples
and UV light Gel-II-MA hydrogel promote the showed more formation
(8W ) growth, proliferation and of cartilage lacuna and
chondrogenic differentiation of cartilaginous matrix.
BMSCs cells.
Collagen Nano-hydro PEG and poly PECE solution Injectable thermo-sensible, SEM, rheological Histological observation [63]
from xyapatite ε-caprolactone at 60°C for 2 biocompatible and analysis, optical of composite implanting
Mingrang (n-HAP) and (PLC) min, ice-water biodegradable for bone microscopy, XRD, into dorsal muscle
Biological PEG-PCL-PE bath for 5 min, regeneration. µ-CT, and pouches of rats showed
Science-Tec G (PECE) add collagen, histological assay. degradation of implant
hnology and HAP, mix and complete
Company and disappearance of
ultrasonicated inflammation after 14
at least for days of treatment.
30min New bone tissue were
formed at 20 weeks of
the surgery in rat skulls.
Bovine PAM, PAM pH 7.4, 37°C The matrix of hydrogel can NMR, U2OS human [64]
73 | Page
Achilles 2-Pyridineca for 12-16 h anchor full-length proteins of High-resolution osteosarcoma cells were
tendon rboxaldehyd ECM electrospray able to assembled and
e and ionization mass remodeled large
fibronectin spectrometry collagen fiber.
(HR-ESI-MS),
fluorescence
microscopy, SEM,
biochemical and
rheological
analysis.
Collagen MA PEG-thiol pH 8.0 - 8.2, Versatile hydrogel for uses in NMR, UV-Vis/CD The cell HCECs were able [65]
type I the time with tissue engineering either as spectroscopy, to attachment and
(without gelation time implantable scaffolds or SEM, structural proliferate for 5 days of
specified variable. injectable hydrogel. and mechanical culture.
origin) analysis. After 3 days of culture
the cells CPCs are viable
and spreading, showing
elongated morphology.
Porcine PPU PPU subunits pH 7.4, 37 ºC The polymerization rate and the FTIR, SDS-PAGE, The hybrid hydrogels [22]
intestinal for 30 min gel network parameters such as SEM, UV-Vis formulated with 15 wt%
submucosa fiber diameter, porosity, turbidimetry, of oligourethane exhibit
crosslinking degree, mechanics, crosslinking assay enhanced storage
swelling, in vitro degradation and oscillatory modulus and
and cell proliferation, keep a rheology. degradation resistance,
direct relationship with the while maintaining the
oligourethane concentration. cell viability and
impeding the
fibroblast-induced
contraction.
Type I PVA and --------------- RT for 1h. Increased mechanical XRD, SEM, Embryonic [66]
collagen HAP properties. The composites are mechanical keratocyto-like cells
solution micro-porous sponge like analysis and cell were able to attached,
structure, suitable to corneal attachment- spread and proliferated
74 | Page
cell adhesion and proliferation. proliferation on PVA-COL-HAP

assays. composites after 7 day
culture.
Bovine skin PVP and ------------- Shaker 30 min, Improved physical and SEM, Oscillatory Human corneal epithelial [67]
ZnO incubated mechanicals properties in rheological, TEM, cell were cultivated on
nanoparticl overnight for comparison with collagen FTIR, TGA, hydrogel and showed
es (NPs) gelation. hydrogels. thermal stability low decrease of cell
analysis and, viability compared to
cytotoxicity assay. collagen control after 24
h of exposure, due to
minimal Zn2+ ion release.
Table 2. Use of synthetic polymers to design hybrid hydrogels with collagen.
75 | Page
3.3 Inorganic phases
Composite hydrogels have been generated by coupling inorganic phases to the 3D collagen matrix (Fig. 5). The
mechanical properties of the collagen matrix must be optimized using crosslinking strategies, in order that the
inorganic phase be adequately supported, avoiding deformation of the network in the hydrogel state. The main
inorganic phases that have been coupled in collagen-based hydrogels are: hydroxyapatite, Zn, Ag, Si, Fe, Ti
nanoparticles, bioglasses, carbon nanotubes, graphene and clays such as kaolinite.
Figure 5. Design of collagen composite hydrogels using different inorganic phases.
The table 3 lists the collagen-based hydrogel systems coupled with inorganic phases. The presence of these inorganic
phases in the 3D fibrous matrix has generated biomaterials with high osteoinductive capacity, systems with potential
to be disease detection devices, biosensors and materials with antimicrobial capacity. The design of these
biocomposites in the hydrogel state requires that the charge of the inorganic phase inside the collagen matrix be well
dispersed, without significant modification of the typical fibrous structure. High concentrations of inorganic phase
generate brittle granular matrices, decreasing the biocompatibility of collagen. The magnetic properties of Fe
nanoparticles have been used to generate materials with biomapping capacity, thus monitoring the processes of bone
76 | Page
tissue generation. The different crystalline phases that the inorganic particles present also show a relationship in the
regulation of cell fate and cytotoxicity.
Specifically, Ti nanoparticles, in anatase phase, have shown excellent bone biocompatibility, while rutile phase
increased cytotoxicity has been observed. Si particles with mesoporous structure have shown excellent
biocompatibility for applications in tissue repair, modulating cell fate and phenotype. In addition, the intracellular
adsorption of this type of particles is necessary to direct specific metabolic pathways. It has been reported that the
heater the size of the inorganic Si particle, the intracellular degradation products show increment cytotoxicity,
limiting their use in BTERM applications. The introduction of carbon nanotubes in collagen hydrogels allows the
generation of materials with controlled electrical potential that can be used as detection systems for cellular
biochemical processes related to diseases. The antimicrobial capacity of the Ag nanoparticles has been exploited to
generate collagen hydrogels with bacterial infection control, generating biomaterials for applications related with the
reparation of dermal tissue. The encapsulation of molecules with biological interest within inorganic particles that are
coupled to collagen hydrogels represents another research approach. The swelling of the hydrogel would lead to a
controlled release of the loaded particles, and the subsequent hydrolysis would release the encapsulated molecules to
a specific site, thus improving their performance in biotechnological applications.
77 | Page
Collagen Coupling agent Collagen Processing Novel features Characterization In vitro / In vivo Ref.
source crosslinker conditions methods results
Bovine Magnetic --------- pH 7.0, Control gel orientation Oscillatory Neurons within the 3D [68]
Achilles nanoparticles based RT, under dynamically and remotely in rheology, TEM magnetically induced
tendon on ferrite 162G to situ. SEM, gels exhibited normal
2110G electrical activity and
magnetic viability.
fields.
Rat tail Silica nanoparticles ----------- pH 7.0, RT Significantly improved the Oscillatory Favor the metabolic [69]
tendon and sodium silicate mechanical and thermal rheology, activity of immobilized
stability. ICP-AES, SEM, human dermal
DSC. fibroblasts while
decreasing the
hydrogel contraction.
Colonizing and
vascularizing without
inducing strong
inflammatory
response in murine
model.
Bovine Bioglass® ------------ pH 7.0, 37 Bioactive and osteoinductive FTIR, XRD, SHG, Collagen remodeling [70]
dermis (45)SiO2-(24.5)Na2O-(2 °C, 30 min. properties, as indicated by SEM, µ-CT associated with
4.5)CaO-(6)P2O5 the formation of CHA in SBF mineralization in
and bone-like material in an subcutaneously
ectopic environment, in vivo. injected scaffolds into
rats.
Rat tail HAP ------------- pH 7.0, Preserves the structural TEM, SEM, XRD, Supports the [71]
tendon 25 °C for 1 h integrity and great tensile TGA, DMA and attachment and
and 40 °C for strength of collagen. Oscillatory spreading of
22.5 h. Unidirectional aligned rheology MC3T3-E1 osteoblasts.
78 | Page
macro-pores with improved

mechanical and thermal
stability.
Porcine Bioglass GEN pH 3.0, RT 24 Improve angiogenesis FTIR, DSC-TGA, EnSCs could be [72]
dermis 45% SiO2, 24.5% CaO, h, -20 °C for through differentiating XRD and SEM programmed into
24.5% 16 h and -80 endometrial stem cells cardiomyocyte linage
Na2O and 6% P2O5 °C for 5 h. (EnSCs) toward endothelial and considered a
mol% lineage and increasing level suitable cell source for
of vascular endothelial myocardial
growth factor secretion. regeneration
Rat tail Superparamagnetic ------------- pH 7.0, 37 Great potential to visualize MR imaging, Did neither influence [73]
tendon iron oxide particles °C, 20 min. hMSCs and track their histological the viability nor the
migration after analysis and proliferation potential
transplantation for articular molecular of hMSCs.
cartilage repair. biology. Furthermore, iron
incorporation did not
affect hMSCs in
undergoing
adipogenic,
osteogenic or
chondrogenic
differentiation.
Chicken Iron oxide -------------- pH 5.0, 37 °C Tailored swelling behavior SEM, VSM, Enhanced osteogenic [74]
sternum nanoparticles using and flexibility. ICP-MS, CLSM differentiation of the
cartilage electrodepos Osteogenic differentiation and mechanical stimulated MC3T3-E1
ition process. activity being promoted with analysis. cells originated from
alteration on mechanical magnetically actuated
properties. mechanotransduction
signaling pathway,
embodying the
upregulated
79 | Page
expression of
osteogenic-related
and
mechanotransduction-
related genes
Rat tail Fullerol derivatives -------------- pH 7.4, RT Enhanced bioactivity of is SEM, AFM, Greatly suppress the [75]
tendon nanoparticles for 1 h. highly related with its TR-PCR, malignant progression
Gd@C82(OH)22 surface-structure relation. oscillatory of cancer cells in vitro,
rheology and and the
turbidity assays. metallofullerol can
efficiently reduce the
mechanical property
of collagen matrix.
Rat tail TiO2 nanoparticles GEN pH 7.4, 37 °C Significant influence on the Oscillatory The materials are [76]
tendon for 1 h. swelling properties and their rheology and biocompatible as they
impact is strongly dependent SEM. can support
on the concentration of mitochondrial activity
nanoparticles. of MEFs as well as
Successfully induced MG-63 cells.
formation of apatite-like
structures.
1
Fish HAP / alendronate GEN pH 7.4, 37 °C The gelation time of H NMR, FTIR, Supported the [77]
dermis for 2 h. hydrogels ranged from 5 to DRX, TGA. adhesion and growth
37 min. Notably, exhibited of murine MC3T3-E1
markedly improved osteoblastic cells.
mechanical property.
Tunable degradation
behaviors against
collagenase
Rat tail Surface-aminated ------------- pH 7.0, RT, Improved physicochemical SEM, TEM, Increasing chemical [78]
80 | Page
tendon mesoporous until properties and mechanical ATR-FTIR, TGA, N2 stabilization.

nanoactive glass gelation. stability adsorption–desor Hydrolytic and
ption, ICP-AES, enzymatic degradation
XPS, Laser reducing.
Doppler Mesenchymal stem
electrophoresis cell highly viable with
and DMA unclear cell
differentiation.
Hydrolyz Kaolin Acrylic acid Mechanical The maximal swelling FTIR, SEM, APS concentration is [79]
ed (AA), stirred 200 capacity of superabsorbent swelling capacity the factor that most
collagen ammonium rpm, 80°C, polymer (SAP) hydrogel is and absorbency effect over swelling
(from persulfate for 20 min, 674 g/g (g water/ g dried under load (AUL). capacity.
Parvar (APS) at 55°C. gel). The time for the
Novin-E N,N´-methyl highest AUL is reached
Tehran ene a 150 min.
Co.) bisacrylamid
e (MBA),
Rat tail Chitosan, Iron (II) Dialdehyde RT for 1h, 3D hydrogels have highly SEM, TEM, The composite [80]
tendon sulfate heptahydrate, starch (DAS) adding swelling capacity and are ATR-FIR hydrogels in PBS
iron (III) chloride magnetic hydrophilic with magnetic mechanical, decreased the swelling
hexahydrate particles and properties. Its rigidity physical, swelling capacity, Young’s
stirred for 20 increases according to an and moisture Modulus and moisture
min and increase in the concentration content analysis. content decrease with
frozen for 2 of magnetic particles. increasing of magnetic
days. particles.
Porcine ZnO, EDC-NHS TR for 16 h, Improved mechanical FTIR, The expression of [81]
dermis 2-Acrylamido-2-methy then 37°C for strength and stability. fluorescence reporter genes in cells
l-1-propanesulfonic 5 h. Reduced cytotoxicity of ZnO spectroscopy, of rabbit corneal
acid sodium salt due to add of PNaAMPs, enzymatic anterior stromal
(PNaAMPS), PEGDA useful in ophthalmic degradation and fibroblasts (RCFBF)
and Irgacure (2959). research as a possible novel molecular contained in
81 | Page
corneal substitute. biology. composite hydrogels is

efficient after 5 days
of culture, offering
resistance to
degradation of
hydrogel by inhibition
of collagenase.
Rat tail Mesoporous silica ------------- 37°C for 30 The system is useful to load TEM, N2 A NGF was linked to [82]
tendon nanoparticles (MSNs) min and CO2 and deliver large protein adsorption–desor MSNs and embedded
incubated. molecules such as a nerve ption, FTIR, LDE, in collagen hydrogel.
growth factor (NGF). Brunauer–Emmet The system
t–Teller (BET) NGF/MSN/Collagen
method, confocal was able to release
microscopy, cell sustainably the NGF
viability assay and for over a week
molecular following a similar
biology. pattern of NGF-MSN,
improving the
outgrowth of neurites
and stimulating the
neuritogenesis.
Bovine Silica with either ------------- Orbital Morphology rougher. SEM, confocal Monocytes was [83]
Achilles mineral phaseHAP, stirring at The osteoblast resorption microscopy, differentiated to
tendon calcium carbonate 37°C, 95% was carried out in fluorescence osteoclasts in the
(CaCO3), strontium relative Silica/Collagen and spectroscopy, surface of all xerogels
phosphate (Sr3(PO4)2 humidity for Ca+2/Silica/Collagen xerogels. complexometry and in the presence of
or strontium 3 days assays, osteoclast
carbonate (SrCO3) biochemical and differentiation factors
histological (M-CSF and RANKI).
analysis. The xerogel with
82 | Page
strontium components
stopped the activity of
osteoclast, while
calcium favored.
Rat tail 12 nm Silica ------------- pH 7.0, RT, The hydrogels are suitable SEM, TEM, DSC, Fibroblasts entrapped [84]
tendon nanoparticles or 80 adding cell for fibroblast growth and cell Oscillatory in gel were able to
nm Silica suspension viability. rheology, perform vital functions
nanoparticles and medium inmunodetection such as metabolism
with PBS. techniques, and cell division.
biochemical and
histological
analysis
Rat tail 300 nm bare silica ------------- Acid pH, RT The drug-loaded hydrogel SEM, FTIR and Streptomycin and [85]
tendon nanoparticles exposed to has antimicrobial effect due microbiological rifampicin was
ammonium to the prolonged release of analysis homogeneously
vapors antibiotics. Did not provoke loaded in
overnight irritation or inflammation in silica-collagen gel,
then pH 7.4, test realized, for uses in allowing sustained
RT development of cutaneous antibacterial effect
wound healing systems. against Staphylococcus
aureus for 10 days and
in vivo decreasing the
bacterial population in
a model of infected
wound.
Rat tail Silica nanoparticles of ------------- pH 7, 37°C Increased mechanical SEM, TEM, The increase in silica [86]
tendon several sizes properties, major proteolytic Oscillatory size, decreased the
stability in fibroblast for rheology, cytotoxicity of
silica of 500 nm. enzymatic fibroblast.
degradation, Gentamycin-loaded in
histological the gel was
83 | Page
assays, sustainable releases

microbiological, for 7 days showed
biochemical and efficiently
cytotoxicity antibacterial activity
analysis. to 500 nm silica
particles.
Rat tail Carboxyl-functionalize ------------- pH 7.0, and Improved mechanical TEM, SEM, AFM, Improved mechanical [87]
tendon d multiwalled carbon mix, then strength electrical electrical and electrical
nanotubes 37°C for 15 conductivity and native properties properties of
min for submicron fibrous structure analysis and cell hydrogel. Cultures of
gelation of hydrogels. viability assay rat cardiomyocytes
showed increased
rhythmic contraction
after 5 days of culture.
Type I Multiwalled carbon ------------- Frozen by 3D matrix highly porous (> XRD, SEM, EDS, MG-63 cell culture [88]
collagen nanotube, chitosan cooling to 95%), improvement in its FTIR, TGA, BET, into hydrogel did not
(Sigma and HAP. -40 °C at 0.9 mechanical properties and optical show a significant
Aldrich, °C/min, biomineralization. High microscopy, difference of cell
CAS: lyophilization bioactivity in vitro, mechanical viability compared
9007-34- at 0 °C, 200 appropriate for use in bone analysis, and cell with control group.
5) mtorr for tissue. viability assay.
48h
Bovine Silver doped bioactive Bovine Mix all Induction of proliferation Reflectance DPSCs encapsulated in [89]
Achilles glass fibrinogen solution and differentiation of dental confocal microbeads were
tendon components pulp stem cells (DPSC) microscope, and viable, spread,
and encapsulated. It showed antimicrobial proliferated and
centrifuge strong bacteria inhibition. analysis changed to fibroblastic
4°C, 600 rpm For applications in tissue morphology both in
for 5min and regeneration. vitro as in vivo assays.
gelation to Microbeads showed
37°C. antibacterial activity,
84 | Page
but the presence of Ag

increase the microbial
inhibition
Porcine Silver nanoparticles BDDGE pH 11, 100% Collagen-coated AgNPs ICP-MS, DSC, Keratinocytes had [90]
dermis (AgNPs) humidity, RT hydrogel with, mechanical SEM, mechanical good proliferating in
for 24 h and properties similar to skin analysis, AgNPs composites
37°C for 1 tissue and anti-bacterial enzymatic After 24 or 72 h no
day effect. The hydrogels are degradation visual inflammation
biocompatible and biosafe assay, were registered in
for use in regeneration of biocompatibility, samples of hydrogels
chronically cytotoxicity, implanted
infected/inflamed tissues. antimicrobial and subcutaneously in
biochemical mice. AgNP
analysis composites showed
bactericide effect over
Gram (+) and Gram (-)
bacteria.
Table 3. Inorganic phases coupled inside collagen based hydrogels.
85 | Page
3.4 Biological molecules with complex functionality
The modulation of the biological response required for improved applications in BTERM using collagen hydrogels
can be achieved when a biomolecule with specific biochemical function is coupled in the 3D matrix. In these
approaches, complex and selective hydrogels have been designed by coupling peptides of specific functionality,
growth factors and another proteins. Table 4 exemplifies these strategies. In general, each complex biological
molecule can control cell signaling processes by inhibiting or segregating of important cytokines from biochemical
pathways involved in BTERM strategies. The modulation of cell signaling processes represents an effective key for
successful application in the biotechnology area. However, the extraction and purification of the biological
molecules with functionality of interest represents a determining challenge for the reproducibility of these
strategies. The cell-hydrogel interaction is also benefited if there are biochemical components that allow to the cells
to regulate their metabolism within the matrix allowing them to grow, reproduce and proliferate. Peptides of
different proteins of interest can be coupled in collagen-based hydrogels to mimic cell- extracellular matrix
interactions, favoring the cell growth and proliferation. To guarantee the desired performance in these systems with
controlled biological functionality, the hydrogel should be stable at periods of cellular recognition, should have
antimicrobial capacity and tailored degradation and swelling. The strategies reviewed in the previous sections
provide tools to design hydrogels with these required properties.
86 | Page
Collagen Coupling agent Collage Processing Novel features Characterization In vitro / In vivo results Ref.
source n conditions methods
crosslin
ker
Streptococc Matrix Acrylate Mix all The HIHA-Scl2 hydrogel, FTIR, SEM, hMSCs embedded in [91]
al metalloproteinase component at is biodegradable, fluorescent HIHA-Scl2 hydrogel showed
collagen-lik 7 (MMP7) and RT until gelling. contains binding sites to spectroscopy, high metabolic activity and
e 2 (Scl2) aggrecanase HA, heparin (H) and mechanical, higher gene expression of
protein, (ADAMTS4) integrase (I) that serves swelling, chondrogenetic markers
expressed cleavable peptides as attractors to carry out biochemical, from 2 to 6 weeks
in chondrogenesis from histology and Accumulation of GAGs
Escherichia embedded hMSCs and immunohistochemi were reached and more
coli cleavable peptides that stry analysis, and elevated synthesis of
BL21-DE3 act for degradation and molecular biology. collagen type II than
(HIHA-Scl2) remodeling of collagen. collagen type I or type X,
while mechanical integrity
were maintained.
Recombina HA or chondroitin PEG RT, pH 8.5 and Biocompatible, bioactive FTIR, CD, (CP-MAS) Hydrogels contained [92]
nt Scl2 sulfate or RGDS acrylate mix in several and biodegradable solid-state 13C MMP7-cleavage peptide
protein peptides -NHS points. hydrogels with possible NMR, fluorescence were degraded for
expressed and application for articular confocal recombinant human
in MMP7- cartilage regeneration. microscopy, MMP7, the weight lost
Escherichia cleavag mechanical, were ~75% at 7 days.
coli e biochemical, hMSCs embedded into
BL21-DE3 peptide histology and hydrogel had good viability
immunohistochemi and underwent enhanced
stry and gene chondrogenic
expression analysis. differentiation. The
hydrogel promoted the
endogenous MMP7
activity.
Types I and Globular domains ----------- 37°C for 2h, and The CLG3/CLP hydrogel CD, phase-contrast A laminin-derived cell [93]
III of C-terminal 2h for binding promote la survival of optical microscopy, adhesive heterodimer
87 | Page
atelocollage region of laminin CLG3/CLP NSCs SDS-PAGE and cell (CLG3/CLP) was binding to
n (Nippon alfa1 chain viability/adhesion/ collagen hydrogel and
Meat (LG3)-9-redisue proliferation assay. promote adhesion, survival
Packers, peptide of the and proliferation of Neural
Inc., Osaka, C-terminal region stem cells (NSCs) for 7 days
Japan) of the laminin γ1 of assay
chain (LP). with
collagen binding
peptide
(CLG3/CLP)
Bovine Fusion protein ---------- 37°C for 1 h R136K-CBD collagen Biochemical R136K-CBD collagen [94]
Achilles consist of R136K hydrogel were stable for analysis, hydrogel stimulated the
Tendon (relatively 14 day of assay, slowly proliferation assay, proliferation of smooth
thrombin-resistant released the growth fluorescent and muscle cell (SMC) since the
mutant derivative factor and increased cell reflection confocal day 3.
of FGF-1) and proliferation of SMCs, microscopy
collagen-CBD suitable for grow factor
delivery for vascular
tissue engineering.
Table 5. Biological molecules of specific functionality coupled in collagen hydrogels
88 | Page
It is worth mentioning that the cell-hydrogel interaction is a determining factor in the modulation of the biological
response, favoring the applicability in BTERM areas. The chemical composition of the hydrogel can also direct cell
signaling processes, thus it is important to select the ideal components that will form the 3D matrix, in order to
potentiate the effects on the modulation of the cellular response required for an efficient application in BTERM.
4. PERSPECTIVES AND CONCLUSIONS
The diversity of collagen-based hydrogel systems varying the coupling agent has been widely exploited in BTERM
strategies. The biocompatibility of collagen, inherent to its properties and functions of structural protein and
signaling of the extracellular matrix, is the main reason to include it in these hydrogels systems. The coupling with
natural polymers, synthetic polymers, inorganic phases and / or complex biological molecules has allowed adapting
the properties of these hydrogels to improve their performance in BTERM applications. Mainly, the regularization
of the degradation rate and the control of the mechanical properties of the 3D fibrillar network of collagen are
achieved producing hybrid systems. These systems can be structurally as interpenetrated networks, presenting
physical and / or chemical crosslinking that modify the entanglement of collagen fibers and their physicochemical
properties.
These novel systems of hybrid hydrogels could be applied in diverse biotechnological strategies, not only in
BTERM, due to the adaptation of their properties, which it has been studied in detail using diverse physicochemical
techniques. This work visualizes successful performance of collagen-based hydrogels in the biotechnological field,
being applied as: i) substrates for vegetable tissue, ii) hydrogels for encapsulation of microorganisms for
environmental bioremediation, iii) matrices for heavy metal ion adsorption for remediation of water, iv) 3D
matrices to regulate and study the growth of microorganisms with biotechnological interest, v) base substrates for
the formulation of products in the food and/or cosmetological areas and vi) matrices for solid state and semisolid
fermentation.
Collagen-based hydrogels for application as substrates for vegetal tissues could be developed using cellulose,
alginate and / or dextran derivatives as coupling polymers. The degradation and swelling of these materials can be
regulated controlling the concentration of the natural polymer. The diffusion of nutrients and moisture would be
regulated modifying the composition of the hybrid hydrogel. The growth of vegetal tissue could be studied varying
the structure of the hydrogels, and new substrates could be generated with potential application in the agroindustrial
sector. Hydrogels with genes or encapsulated plasmids mimic the behavior of a virus, controlling the cellular
metabolism.
Collagen as a protein, represents a source of C and N, for microorganisms that have potential application in the area
of bioremediation of soils and waste water contaminated by heavy metals and/or environmentally dangerous
chemical substances. These microorganisms could be encapsulated in hybrid matrices with controlled properties
that ensure the bioavailability of the organism, potentizing its bioremediating effect. In this approach, synthetic
89 | Page
polymers are effective for the tailoring of the degradation of collagen, swelling and adapted mechanical properties,
regulating the diffusion, absorption and release processes important for the optimization and effectiveness of
environmental bioremediation processes.
The porosity and surface chemistry of hybrid collagen hydrogels could be used to promote the adsorption of heavy
metal ions present in contaminated aqueous systems. These type of materials would represent a potential alternative
for environmental strategies focused on the remediation of aqueous systems. The novel production of composite
hydrogels that include inorganic phases to potentiate the effect of chemisorption, it could represent a promising
area of research. The composite hydrogel with the ion of interest adsorbed could be treated with microorganisms to
generate chemical species of these contaminants that can be reincorporated into the earth crust. In this way, the
study of the application of this type of hydrogels for biotechnological purposes represents a current
multidisciplinary challenge. The collagen source tissue and the residual composition of the ECM molecules present
in the hydrolyzed collagen solution would directly affect the effectiveness of the biotechnological application, so it
is essential to study the influence of the composition of the collagen matrix in the hydrogel state on the application
of interest. For this, it would be convenient to continue developing collagen extraction and purification techniques
that allow formulating hydrogels with high functionality. Various industrial processes involving tissues rich in
collagen represent abundant sources of easily accessible of raw material for the production of these hydrogel
systems.
The ease of manufacture of these hybrid systems, and the control in the sol-gel transition of the formulations, could
be take advantage to encapsulate microorganisms, with which it would be possible to study the alteration of their
phenotypes, induction of specific biochemical processes, related with the microorganism-matrix interactions and
the stimulation of specific metabolic pathways, when a biological agent of complex functionality is coupled in the
hydrogel. Therefore, the generation of metabolites of biotechnological interest could be implemented if the effects
of the hybrid matrix on the growth of microorganisms are known. As it has been reviewed in this work, eukaryotic
cells that constitute different tissues have regulated their metabolic processes interacting with the matrix in the
hydrogel state, potentiating its applicability in the BTERM field. This approach would be interesting if the type of
microorganism is modified, and the relationship of its metabolic activity is studied by the interaction with the
matrix. Thus, the production of hydrogel matrices with adapted biotechnological functionality would be possible
opening new research panoramas.
In another approach, several formulations of these hydrogels based on collagen that have shown promising results,
they could be used as bases for the formulation of products of biotechnological interest, specifically in the food and
cosmetological fields. Hydrogels can be dosed with additives to generate creams, gels, sprays, everyday products
that improve the quality of life of people with dermatological problems, skin wounds, and burns, among others. In
the food industry, hydrogels could be used as thickeners or fillers for food for daily consumption. In this sense,
90 | Page
researching and developing formulations based on collagen hydrogels with daily application represents another
current challenge.
Collagen in combination with agro-industrial wastes is a good option for production of inert support and / or
nutritional supplier in solid state fermentation (SSF) or semisolid fermentation, because it has been proved that
matrices of collagen are biocompatible and biosafe with several types of cells. Also, the mechanical properties of
collagen hydrogels can be adjusted for reached the optimal kinetics parameters of microorganisms or cells during
SSF or semisolid fermentation, for production of several products of interest biotechnological such as enzymes,
organic acids, flavors, colorants, and aromas. Besides, matrices based collagen can be utilized to immobilization of
cells that not supporting high cutting effort for production of interleukins, interferons, hormones and other
biopharmaceuticals whose cost of production are elevated. Some advantages of SSF and semisolid fermentation
over submergible fermentation (SmF) are their higher products yield, environmental friendly (lower energy
consumption, less wastewater generation) and the easer recovery product, so collagen and other type of hydrogels
can be very useful in this field.
Finally, it is essential to inform the advances and the different approaches in the synthesis and application of
collagen-based hydrogels, where the physicochemical aspect and the application in BTERM areas have been
covered mainly; however, the panorama for the application of these novel materials in the biotechnological field is
a promising area of research and development, which it should be started to study to know the potential of these
innovative matrices in another fields.
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Recent Advances in The Synthesis and Applications of Collagen Based

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Mediterranean Journal of Basic and Applied Sciences (MJBAS)

Recent Advances in the Synthesis and Applications of Collagen Based

Figure 1. Diverse strategies to generate collagen-based hydrogels for applications in BTERM

3. TYPES OF COLLAGEN-BASED HYDROGELS

3.1 Natural polymers

Figure 3. Production of collagen hydrogels functionalized with natural polymers.

min, frozen Good blood compatibility analysis. Increased DCMC in

nt staining. localized in the

transforming growth factor immunohistoche chondrogenesis by

in yeast RT. printing. refractive indices promoted cell

Table 1. Collagen hydrogels coupled with biopolymers

3.2 Synthetic polymers

Figure 4. Synthesis of collagen based hydrogels coupled with synthetic polymers.

cell adhesion and proliferation. proliferation on PVA-COL-HAP

Table 2. Use of synthetic polymers to design hybrid hydrogels with collagen.

3.3 Inorganic phases

Figure 5. Design of collagen composite hydrogels using different inorganic phases.

macro-pores with improved

tendon mesoporous until properties and mechanical ATR-FTIR, TGA, N2 stabilization.

corneal substitute. biology. composite hydrogels is

assays, sustainable releases

but the presence of Ag

Table 3. Inorganic phases coupled inside collagen based hydrogels.

3.4 Biological molecules with complex functionality

4. PERSPECTIVES AND CONCLUSIONS

[8] Whitesides G M, Boncheva M. Beyond molecules: Self-assembly of mesoscopic and macroscopic

[23] Claudio-Rizo J A, Rangel-Argote M, Muñoz-González P U, Castellano L E, Delgado J, Gonzalez-García G,

[39] Saska S, Teixeira L N, de Castro Raucci L M S, Scarel-Caminaga R M, Franchi L P, Dos Santos R A,

[52] Luca A, Maier V, Maier S S, Butnaru M, Danu M, Ibanescu C, Pinteala M, Popa M.

[91] Parmar P A, Chow L W, St-Pierre J P, Horejs C M, Peng Y Y, Werkmeister J A, Ramshaw J A, Stevens M M.

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