Introduction To in Situ Forming Hydrogels For Biomedical Applications
Introduction To in Situ Forming Hydrogels For Biomedical Applications
Introduction To in Situ Forming Hydrogels For Biomedical Applications
Bogyu Choi, Xian Jun Loh, Aloysius Tan, Chun Keat Loh, Enyi Ye,
Min Kyung Joo and Byeongmoon Jeong
B. Choi
Division of Advanced Prosthodontics, University of California, Los Angeles,
CA 90095, USA
X.J. Loh(*) A. Tan C.K. Loh E. Ye
Institute of Materials Research and Engineering (IMRE), A*STAR, 3 Research Link,
Singapore 117602, Singapore
e-mail: [email protected]
X.J. Loh
Department of Materials Science and Engineering, National University of Singapore,
9 Engineering Drive 1, Singapore 117576, Singapore
X.J. Loh
Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore
M.K. Joo
Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science
and Technology, Seoul, South Korea
B. Jeong(*)
Department of Chemistry and Nano Science, Ewha Womans University,
52 Ewhayeodae-Gil, Seodaemun-Gu, Seoul 120-750, South Korea
e-mail: [email protected]
1Introduction
2Chemical Hydrogels
visible light is less-thermogenic yet causes less cell damage. In addition, visible
light penetrating through human skin provided greater depth of cure than UV [19].
Riboflavin (vitamin B2) [20], eosin-Y [21, 22], or ruthenium (Ru (II))/sodium per-
sulphate (SPS) [23] have been used as a visible light initiator.
Crosslinking via reactions between functional groups present in the water-
soluble monomers or macromers produce hydrogels. Classical organic reactions
between functional groups such as the Michael addition, click reaction, Schiff
base formation, epoxide coupling, genipin coupling, and disulfide exchange
reaction have been used to prepare hydrogels. The Michael addition of nucleo-
philes (amine or thiol group) to ,-unsaturated carbonyl compounds or ,-
unsaturated sulfones in water forms hydrogels. Various functionalized polymers,
such as poly(ethylene glycol) (PEG) [2426], poly(vinyl alcohol) (PVA) [27],
N-isopropylacrylamide (NIPAAm) [28], and natural polymers [14, 29] have been
crosslinked via Michael addition and formed hydrogels. The copper [Cu(I)] cata-
lyzed azide-alkyne cycloaddition is one of the most popular click chemistry reac-
tions. Macromolecular derivatives of PVA [30], PEG [3133], NIPAAm [34], and
polysaccharides [35] with Cu(I) as a catalyst have been used to prepare in situ
forming hydrogels. However, cytotoxic problem of Cu(I) should be solved to use
these click chemistry induced hydrogels for biomedical applications. Thus, Cu(I)-
free click reactions have been developed to be used as a tissue engineering scaf-
folds [36, 37]. The Diels-Alder reaction, highly selective [4+2] cycloaddition
between a diene and a dienophile without a catalyst, is also known as a click type
reaction. Diels-Alder click crosslinked PEG [38], NIPAAm [39], or hyaluronic
acid (HA) [40, 41] based hydrogels have been investigated for tissue engineering
applications.1
There has been an increased interest in the enzymatically crosslinked hydro-
gels that shows few side reactions since their high specificity for substrates.
Horseradish peroxidase (HRP)/hydrogen peroxide (H2O2), transglutaminase (TG),
phosphatase (PP), tyrosinase, or thermolysin catalyzed crosslinking provides in
situ hydrogel formation of hydroxyphenyl propionic acid (HPA) functionalized
8-arm PEG [42], thiol functionalized poly(glycidol) [43], Tetronic-tyramine (Tet-
TA)/gelatin-HPA (GFPA) [44], dextran-tyramine (Dex-TA) [45], alginate-g-pyr-
role [46], or protein polymers containing either lysine or glutamine [47]. These
enzymatic crosslinks provide fast gelation.
3Physical Hydrogels
5Hydrogel Rheology
The basic principles and different aspects of hydrogels have been covered in sev-
eral reviews [4, 7577]. The mechanical properties of a hydrogel are important
considerations for specific biological applications [78]. The free-standing ability
10 B. Choi et al.
of the gel is an important consideration for cell growth scaffolds. The stiffness
of hydrogels has been reported to direct the differentiation of different cell types
[7981]. For drug delivery, hydrogels should preferentially reduce in viscosity
upon injection and undergo rapid recovery upon removal of the stress to form the
drug release gel depot. This design principle has been the basis of several in situ
thermogelling polymeric networks [5, 8284]. Finally, rheological measurements
allow for the understanding of the different gelation mechanisms which can be uti-
lized in the optimization of the properties of the hydrogels for tissue reconstruc-
tion and drug delivery applications.
The flow and strain properties of soft materials have been extensively investi-
gated since the 17th century. In the 1830s, scientists discovered that many materi-
als possess time-dependent mechanical properties under various conditions, which
cannot be explained by the classical theory of Newtonian fluid. For example, in
1835, Weber observed the phenomenon of elastic hysteresis when he studied the
uranium filament. In 1865, Lord Kelvin discovered the viscosity behavior of zinc,
and that its inner impedance was not proportional to the strain rate. Two years later,
Maxwell proposed a model for viscoelastic materials having properties both of vis-
cosity and elasticity. The Maxwell model can be simply represented by the series
connection of a purely elastic spring and a purely viscous damper. At the same time,
scientists also found many fluids, which were all called non-Newtonian fluid later
due to the nonlinear relationship between the shear stress and shear rate. Based on
the known constitutive equation, people proposed the concept of stress relaxation
time, suggesting the viscosity to be the product of the elastic modulus and the stress
relaxation time. In 1874, Boltzmann developed the linear viscoelasticity theory,
suggesting that the stress in a given time is not only related to the strain in the given
time, but also dependent on its previous deformation. In 1940s, Reiner pointed
out that in order to eliminate the Weissenberg effect (The Weissenberg effect is a
phenomenon that occurs when a spinning rod is inserted into a solution of liquid
polymer. Instead of being thrown outward, the solution is drawn towards the rod
and rises up around it), a stress proportional to the square of the spinning speed
needs to be applied [85]. Almost at the same time, Rivlins study on the torsion of
a rubber cylinder helped to solve the problem of Poynting effect [86]. The intrin-
sic significance of these two studies is to further apply the generalized approach
regarding the nonlinear constitutive equation, which brought in flourishing progress
in the field of rheology. With the advance in rational mechanics, from small defor-
mation theory to finite deformation theory, from linear theory to nonlinear constitu-
tive theory, from classical object model to microstructure theory, rheology rapidly
advanced after 1965, moving from phenomenological theory, which describes phe-
nomena only into the ontology, which considers the internal structure. The term
rheology was first coined by Bingham and Reiner in 1929 when the American
Society of Rheology was founded in Columbus, Ohio [87]. This term was inspired
by a Greek quotation, panta rei, everything flows. In the same year, Journal
of Rheology started its publication. In 1932, the Committee on Viscosity of the
Academy of Sciences at Amsterdam was founded, which was later renamed The
Dutch Rheological Society in 1951. The British Society of Rheology was founded
Introduction to In Situ Forming Hydrogels for Biomedical Applications 11
Fig.3Principle of a small
amplitude oscillatory shear
measurement
Strain
Stress
(t)
(t)
Time
Fig.4Typical graph
showing storage and loss G, Storage modulus
modulus
G, Loss modulus
Strain amplitude 0
material. If tan >1 (G>G), the sample behaves more like a viscous liquid
while, conversely, when tan <1 (G>G), the sample behaves more like an
elastic solid (Fig.4).
For gel samples, these parameters are often measured as a function of time,
strain and frequency. Observation of the gelation process can be achieved by mon-
itoring the temporal evolution of G and G. The linear viscoelastic region within
which G and G are independent of shear strain can be determined by monitoring
the moduli of the material as a function of the strain.
The behavior of the hydrogel at short and long timescales can be studied by
measurement of the moduli of the material as a function of frequency. The fre-
quency dependence of the moduli is a critical hydrogel parameter since a single
material can look quite solid-like (GG) at a high frequency (short timescale)
but behave much more liquid-like (G>G) at low frequency (long timescale).
Gelation kinetics and final gel stiffness are critical material properties that directly
impact the application of the material.
Besides small perturbation measurements, creep and creep recovery tests are
also employed to investigate the time-dependent evolution of compliance. This
aids in the critical understanding of the long-term viscoelastic behavior of hydro-
gels. Different mammalian cells exert different stress levels on the hydrogel scaf-
folds and they behave differently in response to the compliance of the gel material.
In typical experimental setups, creep and creep recovery tests are performed con-
secutively. For this experiment, there is an instantaneous increase in the stress
from 0 to 1. This is kept constant from t0 to t1 in the creep phase to subject the
material to a prolonged period of stress. Then the stress is completely removed
in the subsequent recovery phase. The resulting strain is recorded as a function
of time (t0<t<t2) in both tests. The creep compliance is defined as J(t)=(t)/
0 which has a unit of reciprocal modulus (Pa1). Within the linear viscoelastic
region, the creep compliance is independent of applied stress and all J(t) curves
obtained under various stresses should overlap with each other. Sometimes creep
Introduction to In Situ Forming Hydrogels for Biomedical Applications 15
Fig.6Graph illustrating
shear thickening and shear
thinning
16 B. Choi et al.
Fig.7Graph of an G, G (Pa)
oscillatory stress sweep
LVR
G, Storage modulus
G, Loss modulus
Suggested
measurement
range here
general guide, this section should cover the general rheological characterization
of the different unknown materials, however, it also requires certain amount of
creativity on the part of the rheologist to design the most appropriate protocol. A
point to note is that these recommended conditions could be independently used
to further evaluate a materials rheological response. In all the experiments, it is
important that the sample is well conditioned to a particular temperature before
proceeding with the measurements.
Most of the time, the rheological properties of a viscoelastic material are strain-
independent up to a critical strain level. When the strain exceeds the critical level, the
storage modulus of the material declines and a non-linear behavior is observed. The
measurement of the strain amplitude dependence of the storage and loss moduli (G,
G) is a usually the first step taken to characterize a materials viscoelastic behav-
ior and to determine the pseudo-linear viscoelastic region (LVR) of the material. An
oscillatory stress sweep (OSS) will give a general range of where the LVR is located.
The range of the stress sweep should be tested over the allowable shear stress
(torque~110,000Nm) range of the instrument. In future experiments, the shear
stress range can be adjusted appropriately to collect only reliable data. As the allow-
able shear stress range is dependent on the geometry used, torque will be used as the
controlled variable. The frequency should be set to a value of about 1Hz (Fig.7).
After the materials LVR has been defined by a strain sweep, its structure can
be further characterized using a frequency sweep at a strain below the critical
strain. This experiment provides more information about the effect of colloidal
forces and the interactions among particles.
In a frequency sweep, measurements are made over a range of oscillation fre-
quencies at a constant oscillation amplitude and temperature. Below the critical
strain, the elastic modulus G is often nearly independent of frequency, which is a
characteristic of a structured or solid-like material. On the other hand, frequency-
dependent elastic modulus is a characteristic of a more fluid-like material.
Introduction to In Situ Forming Hydrogels for Biomedical Applications 17
Fig.8Frequency sweep
test in the sol (10C) and
gel (37C) phases of the
CS-g-(PAF-PEG) polymer
aqueous solution (6.0wt%).
Reproduced with permission
from [91]
These measurements have been used to determine the sol-gel properties of ther-
mogelling polymers. Jeong etal. studied the sol gel behavior of thermogelling
polymers with this approach [91]. The frequency sweep test showed that the sol
and gel phases of the PEG-PAF grafted chitosan (CS-g-(PAF-PEG)) aqueous solu-
tion were characterized by fluid-like behavior and solid-like behavior (Fig.8). At
10C, the elastic modulus and loss modulus of the aqueous polymer solution were
proportional to 2.1 and 1.1, respectively, indicating a typical viscoelastic fluid-
like phase of the sol [9294]. In addition, the loss modulus was greater than elastic
modulus at 10C. At 37C, the elastic modulus was greater than the loss modu-
lus by an order of magnitude at 37C. The elastic modulus was nearly independ-
ent of frequency, whereas the loss modulus slightly decreased as the frequency
increased in the investigated frequency range of 0.110rads1. In the solution
state, the thermogelling system showed viscous fluid-like behavior with G>G
and a frequency-dependent modulus, whereas in the gel state, G>G and G was
independent of the frequency.
Next, it is important to determine if the material requires pre-treatment (such
as pre-shearing) before measurements. This can be determined from the pseudo-
viscosity profile of the material. Pre-shearing will determine a zero-time of shear,
thereby eliminating any structure history prior to loading. This is done by perform-
ing a continuous flow test under the broad torque range. The data can be viewed as
viscosity versus torque/stress and converted to viscosity versus shear rate.
Most food formulations, cosmetics, pharmaceuticals and paints are structured
fluids, containing droplets of an immiscible fluid or particle suspended in a liq-
uid matrix. The viscosity of the liquid matrix in the dispersions plays an impor-
tant role on the flow properties of the material. When there are repulsive forces
between particles they do not settle rapidly, forming a network structure, which
stabilizes the suspension. The delicate network structure can be destroyed by
shearing, resulting in decreased fluid viscosity.
Most structured fluids do not obey a simple linear relationship between applied
stress and flow (Newtonian fluid behavior). Most of these materials have viscosi-
ties, which decrease with increasing stress. Such an observation is known as shear
thinning which becomes progressively significant as the volume concentration of
solid particles increases.
18 B. Choi et al.
Another aspect that has to be ascertained is the stability of the material prop-
erties over the time of testing. For this experiment, an oscillatory time sweep of
about 15min, with oscillation shear stress/torque within the LVR and a frequency
of 1Hz can be carried out. The material can be pre-sheared at a shear rate beyond
the 1st Newtonian plateau determined in the previous step. The experiment is
allowed to run and a plot of modulus against time is obtained. The point where the
modulus plateaus off is judged to be the minimum time required for the recovery
of the material structure. This was applied by Moura etal. to understand the gela-
tion kinetics and gel properties upon crosslinking the hydrogel. Both the compo-
nents G and G moduli, was monitored. Figure9 shows the time sweep profiles
of elastic (G) and viscous (G) moduli near the gel point for pure chitosan solu-
tion (A) and for 0.10% (B) and 0.15% (C) genipin concentration networks. At
the beginning, G was larger than G, which was expected because the samples
were still in a liquid state and, thus, viscous properties dominated. As the solutions
began to turn into a gel-like state due to the formation of the cross-linked network,
both moduli increased. However, the rate of increase of G (G/t) was higher
than that of G because the elastic properties started to dominate. This difference
in the rates leads to a G and G crossover. The time required to achieve this cross-
over is, as mentioned above, the gelation time. From the figure, it is can be seen
that higher genipin concentrations lead to lower gelation times. It should also be
stressed that the gelation time decreases from about 8min to about 2min when the
genipin concentration is increased from 0 to 0.15%.
The creep test probes the time-dependent nature of a sample. Creep and recov-
ery tests allow the differentiation between viscous and elastic responses when the
viscoelastic material is subjected to a step constant stress (creep) and then the
applied stress is removed (recovery). A standard creep experiment provides criti-
cal parameters such as zero shear viscosity (o) and equilibrium compliance (Jeo),
which measures the elastic recoil of a material.
After a sample is allowed to creep under load, the materials elastic behavior
can be obtained by abruptly relieving the imposed stress and measuring the extent
the sample recovers. A creep/recovery test can be carried out as follows.
First, standard temperature conditioning and pre-shearing beyond the 1st
Newtonian plateau is performed.
The sample is then equilibrated for a set time necessary to obtain a stable struc-
ture as determined earlier in the judgment of the material stability.
Next, for the retardation step, a shear stress is again selected from within the 1st
Newtonian plateau and performed for about 15min or enough time for slope to
be constant.
Then the recovery of the sample is affected by setting the shear stress to zero
and duration for the sample to recover is examined.
During the creep test, the stress causes a transient response, including the elastic
and the viscous contributions. By following the recovery phase after the release
of the applied stress, one can separate the total strain into the instantaneous elastic
part, the recovered elastic part, and the permanently viscous part.
Introduction to In Situ Forming Hydrogels for Biomedical Applications 19
Fig.9Dynamics of
elastic, G, and viscous, G,
moduli near the gel point,
at 1Hz (a, pure chitosan;
b and c, 0.10% (w) and
0.15% (w) genipin chitosan
concentration network,
respectively). The gelation
time is determined as the time
at which G and G intersect
each other. Reproduced with
permission from [95]
20 B. Choi et al.
Fig.10Creep (open
symbols) and recovery (full
symbols) curves for the
poly(isopropyl lactate diol)-
based polyurethane hydrogels
at 37C when a stress of
5Pa was applied for 60s.
Reproduced with permission
from [96]
Viscoelastic creep data can be presented by plotting the creep modulus (con-
stant applied stress divided by total strain at a particular time) or the strain, as
a function of time. Gradinaru etal. studied the creep response of thermogelling
poly(isopropyl lactate diol)-based polyurethane hydrogels [96].
Figure 10 shows the curves that represent the viscoelastic response at an
applied stress of 5Pa for the three hydrogels obtained at 37C, in a creep test fol-
lowed by recovery. The creep curves comprise three parts: the instantaneous strain,
the retardation strain, and the viscous strain. When the applied stress is removed,
the recovery process starts, and first the instantaneous strain is recovered, then the
retardation one, and finally remains the viscous part. The high elasticity of the
hydrogels can be observed, where the reached strain after the stress of 5Pa was
applied for 60s is very high, and the recovered strain represents 52% from the
maximum value reached by the strain in the creep test.
Changes in modulus of thermogelling polymer aqueous solutions can be deter-
mined by dynamic rheometry (Fig.11).
First, standard temperature conditioning at the lower solution temperature and
pre-shearing beyond the 1st Newtonian plateau is performed.
The sample is then equilibrated for a set time necessary to obtain a stable struc-
ture at the lower temperature as determined earlier in the judgment of the mate-
rial stability.
Next, the material is subjected to a temperature ramp at a fixed stress and a
fixed frequency rate.
The point at which the elastic and loss modulus intersects is defined as the gel
transition temperature.
Jeong etal. reported poly(alanine-co-leucine)-poloxamer-poly(alanine-co-leucine)
(PAL-PLX-PAL) aqueous solution [62]. As the temperature increased, the polymer
aqueous solution underwent sol-to-gel transition at 2040C in a polymer con-
centration range of 3.010.0wt%. The sol-gel transition of the polymer aqueous
Introduction to In Situ Forming Hydrogels for Biomedical Applications 21
solution was investigated by the test tube inverting method. The aqueous polymer
solution (1.0mL) was put in the test tube with an inner diameter of 11mm. The
transition temperatures were determined by a flow (sol)-no flow (gel) criterion
with a temperature increment of 1C per step. Each data point is an average of
three measurements. Changes in modulus of the polymer aqueous solutions were
investigated by dynamic rheometry. The aqueous polymer solution was placed
between parallel plates of 25mm diameter and a gap of 0.5mm. To minimize the
water evaporation during the experiment, the plates were enclosed in a water-satu-
rated chamber. The data were collected under a controlled stress (4.0 dyn/cm2) and
a frequency of 1.0rads1. The heating rate was 0.5C/min.
The phase diagram of PAL-PLX-PAL aqueous solutions determined by the test
tube inverting method is shown in Fig.11. Aqueous solutions of PAL-PLX-PAL
undergo sol-to-gel transition as the temperature increases in a concentration range
of 3.010.0wt%. The sol-to-gel transition temperature decreased from 38 to 23C
as the concentration increased from 3.0 to 10.0wt%. At concentrations lower than
3.0wt%, the viscosity of the polymer aqueous solution increased as the tempera-
ture increased; however, it was not large enough to resist the flow when the test
tube was inverted, and thus they were regarded as a sol state. At polymer concen-
trations higher than 10.0wt%, the polymer aqueous system formed a gel in a tem-
perature range of 060C.
22 B. Choi et al.
Fig.12Variation of
viscosity of chitosan-
ammonium hydrogen
phosphate solution with
time as measured using an
oscillatory rheometer at a
fixed frequency of 1Hz and
fixed temperature of 37C.
Reproduced with permission
from [97]
Sharp increases in both the storage modulus (G) and loss modulus (G) of
PAL-PLX-PAL aqueous solutions were observed as the temperature increased
(Fig. 11). G and G are an elastic component and a viscous component of the
complex modulus of a system, respectively. When G is greater than G, the sys-
tem is considered to be a gel, and the crossover point was defined as the sol-to-gel
transition temperature. The sol-to-gel transition temperatures defined by the test
tube inverting method coincided with those defined by dynamic mechanical analy-
sis of G and G within 2 to 3C. By varying the polymer concentration, not only
sol-to-gel transition temperature but also modulus of the gel could be controlled.
The control of gel modulus (G) has a significant effect on 3D cell culture as well
as the differentiation of the stem cell. In the case of chondrocytes, the modulus of
3002,500Pa showed a cytocompatible microenvironment for proliferation of the
cells. The gel prepared from 10.0wt% aqueous solution of PAL-PLX-PAL formed
a gel with a G of 380Pa at 37C, thus being recommendable as a 3D culture
matrix for chondrocytes.
By raising the temperature above the gelation temperature, the time required
for the gelation can be determined. Nair etal. demonstrates the thermogelation
of chitosan-ammonium hydrogen phosphate solution determined as a function of
time using oscillatory rheometers [97]. The viscosity of the chitosanammonium
hydrogen phosphate solution was found to increase after 8min and showed a sig-
nificant increase within 15min of incubation at 37C, demonstrating the sol-gel
transition (Fig.12).
6Biomedical Applications
In situ forming hydrogels have been increasingly studied for various biomedical
applications such as drug delivery, gene delivery, wound healing, tissue engineer-
ing, and microfluidics [83, 98105]. To use hydrogel systems as drug or gene
delivery systems or tissue regeneration matrices, (1) drugs, genes, and/or cells
Introduction to In Situ Forming Hydrogels for Biomedical Applications 23
+ Drugs
(a) or genes
Polymer solution
(c)
In-situ formed physical
and/or chemical hydrogels
(b) + Cells
& bioactive molecules
Fig.13a As injectable drug or gene delivery systems, drugs or genes were dispersed in the pol-
ymer solution and then injected to form in situ hydrogel depots. b As injectable tissue regenera-
tion matrices, cells and bioactive molecules were mixed with polymer solutions and then injected
to the defect area. c Injected solutions to the target sites form in situ hydrogels via physical and/
or chemical crosslinking
(a)
120 20 nm
Loading and photopolymerization
PEG-PE
PC/NH 2-PEG-DSPE/
Cholesterol 3:1:1 Rehydration
Extrusion/freeze drying UV washing
PLA PEG PLA
CD-Sb505 complex
IL-2
(b) (c)
IL-2 released (ng mg-1 mLG)
IL-2
Drug No treatment
Soluble SB
Tumor area (mm 2 )
Sol SB+IL-2
nLG-SB
nLG-IL-2
nLG-SB+IL-2
Fig.14a nLGs were formulated from lyophilized liposomes loaded with biodegradable PLA-
PEG-PLA diacrylate, acrylated-CD-TGF- inhibitor complex, and IL-2 cytokine. After loading,
photopolymerization of the polymer and acrylated-CD induced gel formation. b Simultaneous
release of IL-2 and TGF- inhibitor released from co-loaded nLGs. c Tumor area growth versus
time. Reproduced from [107]
(a)
(b) 70
PI PII PIII Matrigel
60
g sGAG/(10 cells)
50
6
40
30
20
10
0
14d 28d
Type II Collagen
Type I Collagen
GAPDH
terminal groups modified PNIPAAm brushes were fabricated for improving cell
adhesion and cell sheet harvest [126]. Specifically, carboxyl-terminated PNIPAAm
brush surface most enhanced cell adhesion and cell sheet harvesting. (L/DL)-
PA-Pluronic-(L/DL)-PA thermogel showed a higher population of nanofibrous
structures as the L-Ala content increased and chondrocytes cultured in these ther-
mogels expressed higher chondrogenic markers compared to commercially avail-
able MatrigelTM (Fig.15) [3].
Introduction to In Situ Forming Hydrogels for Biomedical Applications 27
or cells. These side reactions might induce immunogenicity and damage cells or
the efficacy of drugs. (5) Residual enzyme after enzymatic crosslinking also can
provide unexpected reactions with incorporated protein drugs. (6) Development
of various hydrogel-based drug delivery systems with non-modified original drug
is one of the best ways to produce the improved versions of biologics. Biobetters
can advance the efficacy, pharmacokinetic parameters, and safety profile of drugs
than original biologics or Biosimilars (subsequent versions of off-patent biolog-
ics with demonstrated physicochemical similarity). In addition, Biobetters can
also improve patient compliance due to a reduced rate of side effects and enhanced
convenience. (7) Sustained-release systems without initial burst release should be
considered. The charge interaction or the inclusion complex formation between
polymer and drug can improve these problems. (8) Incorporated drugs or cells
should be stable during the implantation period. Acidic degradation-products
released from polyester-based hydrogels raise the local acidity inside and around
the hydrogels, which lead an inflammatory response and a decrease of cell via-
bility or drug stability. (9) For tissue regeneration application, ECM mimicking
design of the hydrogel system is a key factor. Polypeptides have unique second-
ary structures of -helix, triple helix, -sheet, or random coil, etc. Different
combinations of polypeptide-based hydrogel systems allow various nanostruc-
tures in the hydrogels that affect proliferation and/or differentiation of encapsu-
lated cells. (10) Duration of in situ formed hydrogels should be adjusted to match
with drug release profile or tissue regeneration rate. (11) Macromers should be
selected based on application route. NIPAAm copolymer has been used for cell
sheet (a tissue-like cellular monolayer) development that already showed suc-
cessful applications to human clinical studies. However, in vivo application of
PNIPAAm hydrogel still has limitation on the toxicity of the residual monomer.
(12) Gel modulus, degradability, functional groups can affect stem cell fate. Soft
gel improves neurogenesis or adipogenesis, while stiff gel enhances osteogenesis.
Degradable or non-degradable hydrogels induces osteogenesis or adipogenesis,
respectively. Phosphate groups or alkyl groups can stimulate osteogenesis or adi-
pogenesis, respectively.
Challenging design of hydrogels with these understands and considerations
about various hydrogel systems will advance the development of smart bioactive
in situ gelling hydrogels for specific biomedical applications.
Also, study of flow properties of liquids is important for pharmacists working in
the manufacture of several dosage forms, such as simple liquids, ointments, creams
and pastes. The flow behavior of liquids under applied stress is of great relevance in
the field of pharmacy. Flow properties are used as important quality control tools to
maintain the superiority of the product and reduce batch-to-batch variations.
The clinically approved systems by using Pluronics/alginate (Guardix-SG)
and chitosan/glycerol phosphate (BST-CarGel) based thermal gels are interest-
ing examples. Alginate and Pluronics form interpenetrating network (IPN) by
adding calcium salt, where it forms a temperature sensitive gelling system. The
system was successfully applied as an antiadhesive agent after the surgery. BST-
CarGel was applied for articular cartilage repair on the microfractured treatment.
Introduction to In Situ Forming Hydrogels for Biomedical Applications 29
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