Carbohydrate Polymers 169 (2017) 366–375

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Biomacromolecular-based ionic-covalent hydrogels for cell

encapsulation: The atelocollagen − Oxidized polysaccharides couples
Andreea Luca a , Vasilica Maier b , Stelian S. Maier b,d,∗ , Maria Butnaru c , Maricel Danu a ,
Constanta Ibanescu a , Mariana Pinteala d , Marcel Popa a
a
Department of Natural and Synthetic Polymers, “Gheorghe Asachi” Technical University of Iasi, 700050, Romania
b
Department of Textiles and Leather Chemistry, “Gheorghe Asachi” Technical University of Iasi, 700050, Romania
c
Faculty of Medical Bioengineering, “Gr. T. Popa” University of Medicine and Pharmacy of Iasi, 700115, Romania
d
“Petru Poni” Institute of Macromolecular Chemistry, Centre of Advanced Research in Bionanoconjugates and Biopolymers, Aleea Grigore Ghica Voda 41A,
700487 Iasi, Romania

a r t i c l e i n f o a b s t r a c t

Article history: Mixed crosslinked networks of ionic-covalent entanglement type were prepared starting from ternary
Received 19 January 2017 mixtures of atelocollagen (aK; as fibrillary matrix generator), sodium hyaluronate (NaHyal; a microfib-
Received in revised form 29 March 2017 rillation assistant), and oxidized polysaccharides (OxPolys; as both cross-linkers and matrix fillers), and
Accepted 18 April 2017
were tested as hydrogels for eukaryotic cell encapsulation. Either oxidized gellan (GellOx) or pullulan
Available online 22 April 2017
(PullOx) were used. An original procedure and optimal hydrogel recipes were developed to encapsulate
fibroblasts and adipose-derived stem cells, while preserving their viability and proliferative ability dur-
Keywords:
ing ex vivo temporarily storage. Physical-chemical, rheological, and biocompatibility properties of the
Atelocollagen
Oxidized polysaccharides
prepared hydrogels were compared against the classic alginate hydrogel used for cell encapsulation. A
Hydrogels larger range of material characteristics (from lax to stiff) and better laboratory maneuverability were
Cell encapsulation demonstrated, which permit to design appropriate compositions for particular cell types. All hydrogels
Cell release undergo fast liquefaction at temperatures between 42 and 50 ◦ C, permitting the cell release after a short
Thermal liquefaction innocuous thermal shock.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Human cells reside, evolve, and potentially move inside complex
and dynamic morphological and biochemical environments. Except
in the case of some epithelial tissues, non-circulating cells are
hosted (either attached or entrapped) by the extracellular matrix
(ECM), which is intrinsically inhomogeneous, anisotropic and tridi-
mensionally structured. An essential role in cell accommodation
inside the ECM is played by the instructive biochemical and mor-
phological cues offered by the ECM macromolecular components
(Caliari et al., 2015). In the absence of such cues, cells limit or alter
their metabolism, cease their movement and/or proliferation, and
may deviate from their normal functions (Janson & Putnam, 2015).
Structured hydrogels are to some extent able to mimic the nat-
ural environment of cells. If at least some types of cues are present
in a minimal amount inside artificial or synthetic cytocompatible
hydrogels, cells “accept” to be hosted in, and remain biochemi-
cally, biomechanically, and proliferative active, as long as they can
∗ Corresponding author at: Department of Textiles and Leather Chemistry, “Ghe-
orghe Asachi” Technical University of Iasi, Romania.
E-mail address: smaier@tuiasi.ro (S.S. Maier).
make the necessary metabolic exchanges for in vitro survival. This
is why efforts are made to produce hydrogels bearing cell instruc-
tive cues, preferably starting from biomacromolecular precursors
isolated from tissues, or generated by cells cultured in controlled
environments. The envisaged applications are, among others, the
long term storage of cells (Chan, Zorlutuna, Jeong, Kong, & Bashir,
2010), cell therapy applications (Hashemi & Kalalinia, 2015), pro-
ducing bioartificial organs (Iacovacci, Ricotti, Menciassi, & Dario,
2016), and bioprinting (Murphy & Atala, 2014). Cell encapsulation
specifically benefits from this type of hydrogels.
One of the biomacromolecular edifices that abundantly include
cell instructive cues is represented by collagen supramolecular
aggregates, particularly of fibrillary type. Three kinds of cues
are common for them: (i) cell recognition domains (like the
GFOGER amino acid sequence (Malcor et al., 2016)), (ii) physical
and mechanical peculiarities of collagen assemblies (Moeinzadeh,
Shariati, & Jabbari, 2016), and (iii) microscopic-scale profile and
orientation of fibrillary collagen entities into the tri-dimensional
substrata (Chwalek et al., 2016). An easily available form of fibril-
lar aggregates-producing collagen is the atelocollagen (aK), which,
under appropriate conditions (of purity, concentration, tempera-
ture), generates physical hydrogels.

http://dx.doi.org/10.1016/j.carbpol.2017.04.046
0144-8617/© 2017 Elsevier Ltd. All rights reserved.
 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375
To increase the versatility of cell-embedding/cell-interacting
substrata, aK-based mixed hydrogels are prepared. The second
generic component of these hydrogels is frequently a polysac-
charide or a functionalized polysaccharide, whose role is: (i) to
modulate the rheo-mechanical characteristics of the mixed-gels,
(ii) to diversify the morphologic entities of structured gels, (iii) to
add a supplementary (bio)chemical functionality, and, (iv) in the
case of crosslinking capable polysaccharides, to induce covalent
bridges between the hydrogel macromolecular entities.
The present work hypothesizes and investigates the importance of
recipe formulation on the physical-chemical and cell embedding char-
acteristics of ternary hydrogels of ionic-covalent entanglement type,
prepared starting from (i) aK, (ii) sodium hyaluronate (NaHyal)
as functional polysaccharide, and (iii) oxidized polysaccharides
(OxPolys) as macromolecular crosslinking agents. The rationale of
working with ternary mixtures of aK − NaHyal − OxPolys derives
from the constraints imposed by ECM mimicking, when anchoring-
dependent mammalian cells must be temporarily hosted: the need
to generate loose but rheo-mechanically stable hydrogels, able to self-
structure in the presence of cells, and to disintegrate in cell-protective
conditions. The role of aK is to produce the tri-dimensional fibrillar
structure of ECM, assisted by NaHyal in small but sufficient amounts
to consume the co-precipitation tendency of aK against larger
quantities of other polysaccharides having negative zeta poten-
tials. The crosslinking active OxPolys contributes to the expansion
of the hydrogel network, by inducing both physical and cova-
lent bridges between the fibrillary aK entities. As a consequence,
hydrogels able to be manipulated according to cell encapsula-
tion/culturing/release protocols are obtained.
In the attempt to select mixtures with optimal cell encapsulation
characteristics, the most important task is to perform reliable com-
parisons between hydrogels prepared according to distinct recipes.
Conferring of a “cyto-friendly” character of the embedding medium
is of paramount importance, because “cells feel and respond to the
stiffness of their substrate” (Discher, Janmey, & Wang, 2005), and
need to be assisted in their duro- and chemo-tactic migration into
the ECM substitutes, especially under the urge of imminent inter-
actions with other cells and with biochemical species (Herzmann,
Salamon, Friedler, & Peters, 2016).
If temporary cell encapsulation is required, the embedding
hydrogels need to be susceptible to disaggregation under the effect
of a cell-protective and easy to apply external “aggression”. In this
respect, we tested the ability of the investigated aK − NaHyal −
OxPolys hydrogels to be either enzymatically dissolved, or ther-
mally liquefied. The method of thermal liquefaction reported in
this paper is as effective as other techniques of releasing encapsu-
lated cells (Mak et al., 2015; Yildirimer & Seifalian, 2014), but not as
reproducible/predictable as those benefiting from the special and
sophisticated synthetic hydrogels (Deshayes & Kasko, 2013). It is,
however, safe for practical encapsulation applications because cells
are able to withstand thermal shocks, commonly in a ratio larger
than 90% after 5 min heating at about 50 ◦ C. Some of the most exi-
gently conducted experiments revealed none or minimal damages
when cells were heated up to 48 ◦ C for 2.5 min. (Reissis, García-
Gareta, Korda, Blunn, & Hua, 2013), up to 49 ◦ C for several minutes
in microfluidic systems (Ginet et al., 2011), and even near to the
protein denaturation threshold (60 ◦ C, in pulses of 2 s long) (Dams,
Liefde-van Beest, Nuijs, Oomens, & Baaijens, 2010).
2. Materials and methods
2.1. Materials
Type I atelocollagen was obtained according to a patented proce-
dure (Maier, Maier, Prunenanu, & Ignat, 2015), starting from bovine
367
tendons, and was purified and sterilized according the protocols
described in references (Maier, Maier, & Buciscanu, 2010) and (Xian,
2010), respectively. It was stored in lyophilized form, at −20 ◦ C, and
was prepared just before use, in sterile conditions, as a colloidal
solution with a concentration of 6 mg/mL in 0.01 M HCl, which was
maturated for 48 h, at 4 ◦ C.
Low acyl gellan was purchased from Kelco, and pullulan (Mw
of about 100 kDa) from Fluka. Prior to use, both polysaccharides
were purified and prepared as colloidal solutions with a concen-
tration of 10 mg/mL, by dissolving them in double distilled water
(gellan at 80 ◦ C). The final viscous solutions were degassed under
vacuum (100 millibar, 40 ◦ C, for 6 h), and maturated overnight, at
room temperature.
All the general and analytic reagents were purchased from
Sigma. All the aqueous solutions were prepared using fresh double
distilled water (ddH2 O).
2.2. Preparation of oxidized polysaccharides in stock solid form
Oxidation was conducted in an opaque reaction vessel, at room
temperature, according to a procedure derived from that described
in reference (Gong et al., 2009). A solution of 0.1 M sodium meta-
periodate (NaIO4 ) was added into each polysaccharides solutions,
to provide an amount of 160 mg NaIO4 for each 100 mL. After three
hours, an equimolar quantity of ethylene glycol was added (relative
to the NaIO4 amount) to stop the polysaccharides oxidation, and
the stirring was maintained for another half an hour. The resulted
solutions were dialyzed against ddH2 O, for three days, at room tem-
perature, in sterile conditions, using membranes with a MWCO of
12.000 Da. The oxidized polysaccharides were freeze-dried, and the
final products were stored under vacuum, at 4 ◦ C, until use.
2.3. Determination of oxidation degree of the oxidized
polysaccharides
A protocol derived from the one described in reference (Tang,
Sun, Fan, & Zhang, 2012) was used. Aqueous colloidal solutions of
raw and oxidized gellan and pullulan were analytically prepared
at a concentration of 10 ± 0.1 mg/mL, from the corresponding solid
stock forms, and then were titrimetrically investigated.
The number of moles of aldehyde groups per gram of sample
was calculated considering the initial mass of polysaccharide, w
(in grams), by using the formula:
1 1
CCH=O = · · TNaOH · (v − v0 ) , (1)
w ENaOH
where: ENaOH is the equivalent weight of NaOH (39.997), and
TNaOH is the titre of the analytical NaOH solution used for titra-
tion (in g/mL), v0 and v are the volume of 0.1 M NaOH consumed
for the titration of raw and oxidized polysaccharide, respectively.
Theoretically, by halving the calculated value of CCH = O , the num-
ber of cleaved bonds per gram of oxidized polysaccharide results.
Therefore, the nondimensional theoretical value of the percentage
degree of oxidation, Dox , is given by:
1  
DOX = · CCH=O · Mw · 100, (2)
2
were {Mw } is the molecular mass of the repeating unit of the treated
polysaccharide (645 g/mol for gellan, and 486 g/mol in the case of
pullulan). The degree of molecule fragmentation as a consequence
of oxidation process, FDOX , will be:
   
Mw Mw
DOX 1
FDOX = = · 100 · · CCH=O = k ·
Mw,OxPolys 2 Mw,OxPolys
Mw,OxPolys · CCH=O , (3)
368 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375
where Mw,OxPolys represents the molecular mass of the oxidized
polysaccharide. The value of FDOX (in moles/g) also offers a mea-
sure of the amount of molecules of functionalized polysaccharide
bearing aldehyde groups.
2.4. Chromatographic characterization of oxidized
polysaccharides
The average molecular mass and the molar mass dispersity of
both raw and oxidized gellan and pullulan were determined by size
exclusion chromatography (SEC), on a PL-GPC 120 Varian system
equipped with refractive index detector and three PL-aquagel-OH
packed columns (8 ␮m particle size, and 20, 40 and 60 Å pore aver-
age diameters), connected in series. Polysaccharide stock solutions
were diluted with a saline neutral buffer (0.2 M NaNO3 , 0.01 M
NaH2 PO4 , pH 7) to a concentration of 0.1 mg/mL. The operating
parameters were as follows: the eluent was the same as the dilution
buffer, flow rate 1 mL/min, injected sample volume 100 ␮L, work-
ing temperature 25 ◦ C. Columns calibration was performed using a
pullulan-containing standard (P-82, Shodex Denko K.K., Japan, lot
01101) which includes the following standard molecular weights:
6.1, 9.6, 21.1, 47.1, 107, 194, 337 and 642 kDa. A five order polyno-
mial equation best fitted the calibration curve.
2.5. NMR characterization of oxidized gellan
Solid samples of raw and oxidized gellan were dried in a vac-
uum oven (100 mbar, 45 ◦ C, overnight) and dissolved in deuterated
water (D2 O). The solutions were degassed for 24 h, under vacuum,
and were analyzed using a Bruker Avance DRX400NMR spectrom-
eter. 1 H NMR spectra were recorded at a working frequency of
400.13 MHz.
2.6. Preparation of atelocollagen − oxidized polysaccharides
hydrogels
2.6.1. Recipes formulation
All the recipe variants consider the same final weight
of biomacromolecular components into the hydrogel sample
(1.92 mg), with a constant amount of NaHyal (0.06 mg) as the
unit part in the biomacromolecules mixtures. Table 1 presents the
tested recipe variants. Scheme 1 graphically describes the hydro-
gels preparation protocol.
2.6.2. Preparation procedure
Hydrogel samples were prepared by mixing the macromolecular
components solutions and some reagents solutions, in a pre-
cise order, in the central well of sterile organ culture dishes (BD
FalconTM 353037, 60 × 15 mm). Based on preliminary tests, the pre-
scribed volume of aK solution is diluted with a precise volume of
ddH2 O (for the mixed volumes, see Table SD-1, in Supplementary
Data file) (Lefter, Maier, Maier, Popa, & Desbrieres, 2013). After pH
adjustment of the aK solution at 7.0, using 1 M NaOH, this was sta-
bilized with 40 ␮L of 10 x PBS (pH 7.0). Then a constant amount
(20 ␮L) of NaHyal is added. In a separate sterile glass vessel, the
oxidized polysaccharide solution is adjusted to pH 8.0, and then
is pregelified with 0.1% v/v of a 1 mg/mL sterile solution of CaCl2 .
Immediately after the start of the agglutination process, the pre-
scribed volume is added to the mixture of aK and NaHyal, under
efficient stirring, then the prepared compositions are incubated at
37 ◦ C, for 30 min. During incubation, the mixture gelifies and turns
into a perfect translucent matrix, able to be manipulated according
to cell culturing protocols.
2.6.3. Direct encapsulation procedure
If the hydrogels must be prepared in the presence of the cells
to be embedded in, culture medium (Dulbecco’s Modified Eagle’s
medium, DMEM D6046, Sigma) is used for aK dilution. Cells are
added at a seeding density of 4·105 cells/mL relative to the final
hydrogel volume just before the solution of oxidized polysaccha-
ride. The compositions are incubated at 37 ◦ C, in an atmosphere
with 5% CO2 and 95% relative humidity.
Table SD-2 (in Supplementary Data) resumes the recipes of the
prepared hydrogels variants.
2.7. Evaluation of crosslinking between aK and the oxidized
polysaccharides
To put in evidence the covalent crosslinking mediated by the
condensation reaction between the carbonyl groups of oxidized
polysaccharides and the primary amino groups of lysine in ate-
locollagen, the amount of free ␧- and ␣-amino functions was
quantitatively determined, after gelation completion. The photo-
colorimetric method based on the reaction of the remnant primary
amino groups with 2,4,6-trinitrobenzen sulfonic acid (TNBS), was
used (Fields, 1972; Hermanson, 2008). The final solutions absorban-
cies were measured at 346 nm using a Anthos Zenyth 200rt
microplate reader (Biochrom Ltd., Cambridge, UK). Glycine was
used for method calibration and the crosslinking degree was
estimated as the percentage of free amino groups content of
crosslinked hydrogels (recipe variants R2–R7) against the content
in the hydrogel prepared according to recipe R1.
2.8. Rheological characterization of prepared hydrogels
Amplitude, frequency and temperature sweep, and also creep-
recovery tests were conducted using a Physica MCR501 rheometer
(Anton Paar) fitted with PP25 parallel plate system. Tests were per-
formed for the seven prepared hydrogels, and for a 0.5% alginate
hydrogel, considered as reference for the ability to be manipu-
lated in cell encapsulation protocols. The rheometric parameters
were as follows: (i) shear strain between 0 and 1% in the ampli-
tude sweep tests, (ii) angular velocity between 500 and 0.05 rad/s,
under a shear strain of 0.3%, in frequency sweep tests, (iii) tempera-
ture variation between 5 and 65 ◦ C, and 10 Hz oscillation frequency
in temperature sweep tests, and (iv) 5 Pa shear stress, 750 s in
loaded followed by 600 s in unloaded state, in creep-recovery tests.
Excepting the temperature sweep analyses, investigations were
conducted at 37 ◦ C.
Two distinct domains were considered for processing creep-
recovery data: the long-time creep and the early creep evolution. In
accordance, two types of parametric rheological models were fit-
ted: the classic Burgers model, and the Jeffreys inertial model (both
described in Supplementary Data file, Figure SD-1, and equations
Eq. SD-1 and Eq. SD-2).
2.9. Proteolytic degradation of the prepared hydrogels
The ability of the prepared hydrogels to be degraded and
destructured by proteolytic enzymes was tested using bacterial col-
lagenase. A ninhydrin-based assay to quantify the liberated amino
acids, adapted form reference (Quek et al., 2004), was applied
to the hydrogels prepared according to recipe variants R1 (not
crosslinked) and R4 (with best behavior in the preliminary tests
of cell encapsulation).
A collagenase solution (Gibco, Life Technologies, U.S.A., 220
units/mg) with a final proteolytic activity of 5 units/mL, and a 4%
ninhydrin solution in 2-methoxyethanol were used. The final solu-
 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375 369

Table 1
The amounts of biomacromolecular components in the recipe variants for hydrogels preparation.

Recipe variant Components amounts (mg) Total amount (mg) Parts ratio aK:NaHyal:OxPolys (w/w/w) Total parts in the mixture

aK NaHyal OxPolys

R1 1.86 0.06 0.00 1.92 31:1:0 32

R2 1.77 0.06 0.09 29.5:1:1.5
R3 1.56 0.06 0.30 26:1:5
R4 1.38 0.06 0.48 23:1:8
R5 1.14 0.06 0.72 19:1:12
R6 0.93 0.06 0.93 15.5:1:15.5
R7 0.66 0.06 1.20 11:1:20

Scheme 1. The blueprint of the hydrogels preparing protocol.

With green color are marked the optional steps performed only when cells are embedded into the hydrogel during preparation.

tions absorbencies were determined at 570 nm, using a plate reader
(Tecan, Sunrise). Isoleucine was used for method calibration.
2.10. Cytotoxicity tests
The cytocompatibility of aK: OxPolys hydrogels prepared
according to R4 recipes was proved by MTT cell viability test,
performed in triplicate on adipose-derived stem cells, and on pri-
mary culture fibroblasts. Cells were seeded at a density of 5000
cells/well, in 24 well plates, in DMEM supplemented with 10% BFS
and 1% antibiotic. After 24 h of incubation (37 ◦ C, 5% CO2 ), cells
were directly exposed to 50 ␮L of hydrogel composition. As con-
trol, unexposed cells were further cultured. Tests were performed
after 24, 48 and 72 h of culture, in triplicate, using a 5% MTT solution.
2.11. Scanning electron microscopy
The microscopic morphology of the biomacromolecular matrix
and the accommodation of adipose stem cells to the hydrogel
embedding environment were studied by the SEM technique. The
samples of uncultured and cell embedding hydrogels were dried
at the critical point of carbon dioxide. Briefly, samples were first
fixed (2% glutaric dialdehyde, 12 h), abundantly washed with sterile
ddH2 O, dehydrated through a series of graded ethanol baths (70%,
80%, 90% and 100%) and finally dried using an EMS 850 Critical Point
Dryer. The dried samples were sputter-coated with gold, prior to
be visualized using a Tescan Vega II SBH electron microscope.
370 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375
2.12. Fluorescence microscopy
The viability and the morphology of the rabbit adipose-derived
stem cells cultured both in the presence of, and encapsulated in,
the R4 hydrogel were put in evidence by fluorescence microscopy.
After 7 days of culture, the medium was replaced with a solution of
2 ␮M Calcein AM in Hank’s Balanced Salt Solution (HBSS), and left
in contact for 30 min, at 37 ◦ C.
To put in evidence the cell morphology cells were stained
with rhodamine-phalloidin (Molecular Probes, Life Technologies,
U.S.A.), an indicator for actin filaments, and with 4 ,6-diamidino-2-
phenylindole dihydrochloride (DAPI), for nuclei observation. After
solution removal, the cells were visualized with a Leica DMI3000
microscope, using specific filters.
2.13. Release of cells from hydrogels by a short thermal shock
Fibroblasts of primary culture and adipose-derived stem cells
were separately encapsulated in hydrogels prepared according to
R4 recipe, at a seeding density of 1·105 cells/mL. After 72 h of
incubation, embedding hydrogels were transferred, in sterile con-
ditions, in a glass Petri dish (60 mm in diameter) preheated at 48 ◦ C
on a thermoblock (HLC Ditabis), and immediately covered with a
glass plate (20 × 20 × 3 mm) preheated at the same temperature.
After 4 min, the cell suspension was collected, re-suspended in cul-
ture media, and further cultured. After cell adherence, the hydrogel
debris were washed by changing the culture medium. After 48 h
of culture, the cells viability, proliferation, and morphology were
investigated using calcein AM, DAPI and rhodamine-phalloidin,
respectively.
2.14. Statistical analysis
Experimental data (three to five parallel measured values) were
statistically processed as follows: (i) possible outliers were iden-
tified and removed using Grubbs test; the removed outliers were
replaced by performing supplemental determinations; (ii) differ-
ences between the means of experimental data sets were evaluated
using one-way ANOVA, not assuming equal variances; (iii) where
applicable, post-hoc test was used to identify the specific data sets
which significantly differ; Games-Howell procedure for sets with
unequal variances and unequal sample sizes was considered. In
all cases, the evidence against the null hypothesis was declared
at p < 0.05.
3. Results and discussion
3.1. General remarks
When atelocollagen is mixed in appropriate conditions with
raw or functionalized polysaccharides, two processes evolve con-
currently: coacervation and gelation. The first process generates
tight associations between collagen and polysaccharides, which
may result in co-precipitation. The second leads to extended
supramolecular edifices, and concludes with a morphologically
defined network of local aggregates. In addition to coacervation and
gelation, the collagen microfibrillation continues to take place as a
parallel process, supplementary affecting the results of physical-
chemical interactions.
The inevitable interferences between the microfibrillation,
coacervation, and gelation, were considered when the recipes
and procedures of preparing aK-NaHyal-OxPolys hydrogels were
designed. While aK microfibrillation must be favored through
the procedure by slow temperature increase, the associative co-
precipitation of aK and the admixed polysaccharides (NaHyal and
OxPolys) must be prevented and/or controlled through both recipe
Fig. 1. The time evolution of the oxidation degree of gellan and pullulan. The isolated
points marked with unfilled symbols represent the values of aldehyde content after
two months of storage of the oxidized products.
(by using a limited amount of the acidic polysaccharide) and proce-
dure (by a distinctive stage of NaHyal admixing), in order to obtain
macroscopically homogenous hydrogels.
3.2. Physical-chemical characteristics of oxidized gellan and
pullulan
The oxidation process of polysaccharide with metaperiodate
concluded with a noticeable decrease of the viscosity of gellan solu-
tion (due to the decrease of average molar mass from 1486.3 kDa
and 1.79 mass dispersity, Di , to 848.1 kDa and 1.64 Di ), but with
only a slight decrease in the case of pullulan (associated with a
molar mass decrease from 85.9 kDa and 1.76 Di , to 50.8 kDa and
2.07 Di ). Also, the intrinsic gelation ability of GellOx significantly
decreased.
The proof of the presence of carbonyl groups on the chain of
GellOx was done by analyzing its 1 H NMR spectrum (Figure SD-3,
in Supplementary Data file) in comparison with the spectrum of
native low acyl gellan (Figure SD-2). The peak at about 9.5 ppm is
characteristic for aldehyde proton. The oxidation process resulted
in some degradation of the polysaccharide chain, which is empha-
sized by the abundance of the peaks in the range 3.0–0.5 ppm.
In order to estimate the crosslinking ability of the oxidized
polysaccharides, the content of sterically available and reactive
aldehyde groups of the samples was analytically determined
through a titrimetric technique (Zhao & Heindel, 1991). Based on
the evolutions in Fig. 1, the optimal duration of oxidation is con-
sidered to be of 45 min for gellan functionalization, and of 75 min
for pullulan processing, when the relative yield of the oxidation
processes (reported as percentage degree of oxidation) is of about
48.96% for GellOx, and of 13.52% for PullOx. The percentage degree
of oxidation determined after two months of storage (hermeti-
cally sealed, at 4 ◦ C) was of 45.97± 0.11%, in the case of GellOx,
and of 11.81± 0.26% in the case of PullOx. Accordingly, when used
as crosslinker, the prepared GellOx contains three times as much
free aldehyde groups (1.42 ± 0.025 mM/g), as compared with Pul-
lOx (0.47 ± 0.0124 mM/g).
3.3. Quantitation of crosslinking degree of the prepared hydrogels
The characteristics of the prepared hydrogels are directly influ-
enced by the degree of crosslinking between the atelocollagen and
 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375
Fig. 2. The evolution with temperature of the internal organization of investigated
hydrogels, as reflected by their complex viscosity. Hydrogels prepared according to
R1 and R4 recipes are compared.
the functionalized polysaccharides. The number of covalent bridges
developed during the preparation is pointed out by the consump-
tion of the free amino groups of protein partner, and is correlated
with the mass ratio of oxidized polysaccharide in the mixture recipe
(see Figure SD-4). Figure SD-5 depicts the values of the relative
covalent crosslinking degree determined for the hydrogels pre-
pared according to the recipe variants presented in Table SD-1,
considering the physical crosslinked hydrogel (which only contains
aK and NaHyal) as reference for the absence of covalent bridges.
Because of the low volume of PullOx molecules, the mixing with aK
molecules is more intimate, and crosslinking evolve more rapidly,
at smaller amount in recipes. To facilitate the comparison between
the crosslinking efficacy of GellOx and PullOx, the dependence of
crosslinking degrees on the amount of functionalized polysaccha-
ride was fitted by using a power growth law (y = a·xp ; see Figure
SD-5). Then, for the amount related with the recipe of the most
versatile hydrogels (which is 1.039 mg/mL OxPolys), the param-
Fig. 3. Dependence of the values of fitted Burgers models, according the preparing recipes of the investigated hydrogels. Lateral isolated segments correspond to the values
calculated for 5 mg/mL alginate hydrogel.
371
eter a was calculated as being 19.55 for GellOx, and 42.19 for
PullOx. Accordingly, the difference is of 22.64% in the benefit of
PullOx, which means that, in practical terms, the same degree of
crosslinking can be obtained using about 20% less PullOx, than Gel-
lOx, provided the two polysaccharides were functionalized using
the same recipe, and were further post-processed identically.
In correlation with the final remark of Section 3.2, even if, in
abstracto and per se, PullOx should be about four times more active
as crosslinker when compared with GellOx, in the real conditions of
hydrogels preparing it is only 1.2 fold more efficient. Paradoxically,
the measured amounts of aldehyde groups are in opposite rela-
tion (1.42 ± 0.025 mM/g for GellOx, and only 0.47 ± 0.0124 mM/g
in the case of PullOx). The reason of the discordance between
the abundance of reactive groups and the efficacy as crosslinkers
resides in the difference of fragmentation degrees of the function-
alized polysaccharides. The smaller PullOx molecules are prone
to self-disentanglement, and to better entangle with the fibrillary
structured aK, thus maximizing the reciprocal interaction.
3.4. Rheological behavior of the prepared hydrogels
Organoleptic observation made during the maneuverability
tests (like those presented in Figure SD-19) revealed that the R4
hydrogels from the GellOx-crosslinked series, and the R3 hydro-
gels from the PullOx-crosslinked series, were the most versatile
in responding to various physical-mechanical stresses. In order to
substantiate this finding, the variation of gels strength was first
determined, by comparing the ratio between G’ and G” shear mod-
uli with the value accepted as threshold between the weak and
the strong covalently crosslinked gels (which is G’/G” = 10 (Clark
& Ross-Murphy, 1987), or, equivalently, G”/G’ = 0.1 (Borzacchiello
& Ambrosio, 2009). Figure SD-6 depicts the linear variation of the
conventional strength of the prepared hydrogels in relation with
the recipes details. With a G’/G” ratio of 7.1998, the purely phys-
ically crosslinked aK − NaHyal hydrogel represents the reference
in evaluating the increase of hydrogels strength. The increase of
the strength of PullOx-based hydrogels is about three times faster
in comparison with those of the GellOx-based ones. GellOx- and
PullOx-crosslinked hydrogels from the R4 series produce gels with
relatively similar rheological behavior. At PullOx amounts higher
372 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375
than 1 mg/mL, resulting hydrogels become increasingly stronger
due to the increasing number of covalent linkages. Compara-
tively, all the GellOx-based hydrogels seem to be predominantly
crosslinked by means of physical forces, to the detriment of cova-
lent bridging (even if chemical crosslinking is present, as confirmed
by the relative crosslinking degree; see Figure SD-5). For compari-
son purpose, the strength of an alginate hydrogel of physical type,
produced in the presence of Ca2+ ions, is also represented in Figure
SD-6.
Classical frequency sweep tests were performed to put in evidence
the ranges of oscillatory stresses on which elastic responses still
occur under low shear strain (see the amplitude sweep data, Fig-
ure SD-7). The first column of Figures SD-8 and SD-9 includes the
results. All the investigated samples reveal specific behaviors of
stable gels, dominantly elastic (G’ > G” up to 80–100 rad/sec, with
a poor dependence on the frequency), and mostly crosslinked by
physical interactions. At higher oscillation frequency, the trends of
the two moduli reverse, indicating that the macromolecular matrix
of the hydrogels cease to respond elastically (as a solid), but dump
the propagation of the shears train, acting as highly viscous fluids.
The rheological characteristics of the covalent crosslinked hydro-
gels with best maneuverability, the alginate-based hydrogel mostly
used for mammalian cell encapsulation, and the purely physically
crosslinked aK hydrogel are comparatively presented in Figure SD-
10.
Temperature sweep tests, included in the second columns of
Figures SD-8 to SD-10, allow the evaluation of the variation of
hydrogels complex viscosity in the range of temperature in which
cell encapsulation is usually performed (30–40 ◦ C). Complex viscos-
ity is proportionally related with the degree of regular organization
of the macromolecular networks, especially of those physically
crosslinked (Soares et al., 2014).
Fig. 2 graphically resumes the results of temperature sweep
tests performed on the most representative hydrogels discussed
in the present paper. Two peculiarities are evident, both due to the
presence of atelocollagen in the gelling mixtures: (i) an increase
of structural order in the temperature range of collagen fibrillation
(30–42 ◦ C), and (ii) a liquefaction threshold at about 50 ◦ C.
The supramolecular association of triple-helical aK macro-
molecules is promoted by the slow heating, with a slope of
0.5 ◦ C/min. The presence of GellOx and PullOx has negligible influ-
ence on the process as far as the physical crosslinking between
polysaccharides and protein remain predominant (up to an aK:
OxPolys ratio of about 3:1), but increasingly interfere when cova-
lent crosslinking becomes significant (see the series of SD-8 to
SD-10 Figures). In the latter case, a noticeable difference between
GellOx and PullOx crosslinking action is revealed. Even if both tend
to diminish the supramolecular aggregation of aK, the smaller and
more active PullOx reagent delay the aK microfibrillation and the
protein network generation, mainly due to earlier and more intense
crosslinking. Larger GellOx macromolecules exhibit a lower chem-
ical reactivity, but entangle in a more efficient manner with aK,
maximizing the physical crosslinking.
The hydrogels liquefaction is induced by the thermal denatu-
ration of atelocollagen, concluded with the complete unfolding of
helical domains, by helix-coil transition. The liquefaction starting
temperature is practically the same for both pure aK and mixed
hydrogels, suggesting that, after gelation, OxPolys are unable to
protect the supramolecular aggregation of protein. The slope of liq-
uefaction process seems to be related with the ratio between the
physical and of covalent crosslinkages. For comparison purposes,
Figure SD-11 also includes the temperature sweep behavior of the
alginate hydrogel. In this case, thermal liquefaction does not occur,
but contrary, the complex viscosity increases at temperature val-
ues larger than 32 ◦ C, which supplementary constrict the embedded
cells.
At 37 ◦ C, the complex viscosity of aK and of 3: 1 aK: Gel-
lOx hydrogels are quasi-equal, suggesting that the regularity of
their internal organization is the same, being dominated by the
resulted aK microfibrils. Advantageously, up to about 45 ◦ C, the 3:1
aK:GellOx hydrogel maintains its ordered organization in the par-
ticular state which was achieved at physiologic temperature. Such
an effect could be exploited in cell encapsulation techniques, when
cell release is to be done by a short thermal shock (as it will be
described in a further section).
Creep-recovery tests were performed to confirm the covalent
crosslinking between the aK and OxPolys, and to evaluate the
parameters describing the viscoelastic behavior of the investigated
hydrogels. Because all samples exhibited specific “tremor” immedi-
ately after the constant shear stress was applied, the creep-ringing
formalism was also considered (Ewoldt & McKinley, 2007). Two
classes of idealized rheomechanical models were fitted: Burgers
model for long term creep, and Jeffreys model for the early time
period after shear stress application. Figure SD-12 comparatively
presents the creep-recovery curves determined for the hydrogels
prepared according to the seven recipes. In Figure SD-13, the early
stage of creep is zoomed, to put in evidence the creep-ringing.
Tables SD-3 to SD-6 systematize the values of the fitted models
coefficients, together with the time and rheological constants cal-
culated based on these coefficients.
Fig. 3 depicts the dependence of Burgers coefficients values on
the amount of OxPolys crosslinker used to prepare hydrogels. For
comparison, the corresponding values fitted for the alginate hydro-
gel (5 mg/mL) are also represented.
Based on the evolution of Burgers parameters with the aK:
OxPolys ratio (Fig. 3), optimal recipes can be formulated according
to different types of constraints imposed to the prepared hydro-
gels. Ratio values of 3: 1, in the case of using GellOx as crosslinker,
and of 1.6: 1 in the case of working with PullOx, seem to repre-
sent particular compositions of aK − NaHyal − OxPolys hydrogels.
Even if covalent crosslinking bridges were induced, these hydro-
gels tend to resemble with the pure physical aK hydrogel, from
the point of view of their rheological behavior. For both lower and
larger ratios, hydrogels are stiffer (larger G1 values), tend to pro-
long the duration of reversible flow in parallel with a slow evolution
of deformation during this period (larger ␩2 values), also tend to
slowly reach the irreversibly flow domain (larger G2 ), and slowly
deform irreversibly afterwards (larger ␩1 values). The retardation
time (␭ret ) is the shortest in the case of the above mentioned ratios
(see Table SD-3), but still more than three times longer as compared
with the same parameter measured for alginate hydrogel (see Table
SD-4).
Fitting of Jeffreys mathematical model to the early-creep data
allows a finer discrimination between the real rheological char-
acteristics of hydrogels. Therefore, because of the complicated
dependency between the recipe formulations and the results of
classical creep measurements, we decided to check whether the
general allure and the peculiarities of the curves in Fig. 3 are also
found for early-creep stages. Of special interest was the signifi-
cant decline of elastic moduli for R4/R5 recipes (aK:OxPolys ratio of
about 3:1). The results are resumed in Figures SD-14 and SD-15 and
Tables SD-5 and SD-6, confirming the general evolution of Burgers
parameters over the recipes series.
A graphical comparison between the creep-recovery curves of
the most representative hydrogels is presented in Figure SD-16. The
investigated covalent aK − NaHyal − OxPolys hydrogels have inter-
mediate behavior between two extrema of purely physical ones: (i)
alginate-based, which are stiffer, and flow in limited but essentially
irreversible amounts, and (ii) protein-based, which are approxi-
mately six fold more elastic, and significantly recover more than
half of the induced deformation, after stress unloading.
 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375
Fig. 4. Results of cytotoxicity evaluation, performed on the investigated hydrogels
by using the MTT assay.
3.5. Cytotoxicity assays
The in vitro cytotoxicity tests were performed according to the
ISO 10993-5:2009 standard, with the results depicted in Fig. 4.
Because the viability of adipose stem cells cultured in the presence
of aK − GellOx hydrogel slightly decreased after 72 h of exposure
(from 98.75% to 95.72% viability compared to control), the same
hydrogel was further evaluated against fibroblasts of primary cul-
ture. All investigated hydrogels proved to be non-cytotoxic. As a
general remark, it seems that, in time, hydrogels crosslinked with
GellOx tend to limit the cells metabolic activity, in comparison with
those containing PullOx. This effect could be also induced by the
increased ability of functionalized gellan to retain cations, both by
complexation or as counter-ions.
To confirm that the hydrogels do not release cytotoxic com-
pounds in the culture medium, cells were incubated for seven more
days in the presence of the samples. The incubated cells preserved
their normal morphology and viability, and proliferated to the cul-
ture plate surface.
3.6. Microscopic investigation
The morphological details of the hydrogels fibrillary matrix are
revealed by the SEM images in Figs. 5 a and 6 a. Fibers with mean
diameters of 200 nm compose a dense but little entangled network.
The apparent density of the microscopically observed fibrillary net-
work usually increases during the drying protocol we used (at
critical point of CO2 ) (Ulrich, Jain, tanner, MacKay, & Kumar, 2010),
but the gross morphology is preserved (Branco da Cunha et al.,
2014). Salts crystals are abundantly present on the samples pre-
pared using cell culture media. Adipose stem cells in active state
were visualized at the surface of hydrogels samples and in rupture
areas, as can be observed in Figs. 5b and 6b.
3.7. Tests on cells release from the embedding hydrogels
Generally, cell encapsulation aims to physically isolate cells for
a certain period of time, without altering their viability and spe-
cific functionality, and frequently, in conditions that they can be
“quantitatively” released in “active” state, by simple techniques.
Therefore, some applications require that cells encapsulation be
reversible. Two feasible methods to extract cells from their host-
ing matrices were tested: (i) a conventional one, by slow enzyme
erosion/hydrolysis of the embedding hydrogels, and (ii) an exper-
373
imental one, of shorter duration, by applying a thermal shock on
the cell populated macromolecular matrices.
3.7.1. Degradation of hydrogels under collagenolytic attack
The action of collagenase was investigated for three types of
non-populated hydrogels: one prepared according R1 recipe, and
two according R4 recipe, using, respectively, GellOx and PullOx as
crosslinker agents. The amount of solubilized polypeptides of colla-
gen origin during the proteolytic treatment is represented in Figure
SD-17. All three hydrogels are completely destructured after 24 h
of enzyme attack, but during the first eight hours, their behavior
is quite different. The investigated hydrogels partially lose their
encapsulating action after about three hours, and are completely
destructured after more than five hours. Such long durations could
be detrimental for the encapsulated cells, this is why we decided
to capitalize the liquefaction ability of the aK − NaHyal − OxPolys
hydrogels, under short thermal shocks.
3.7.2. Cell release by the liquefaction of aK − OxPolys hydrogels
under thermal shock
Temperature sweep rheological tests revealed that a complete
loss of physical integrity of aK − NaHyal − OxPolys hydrogels occur
in a relatively narrow thermal range (between 44 and 50 ◦ C), and
in short intervals of time (2–8 min.). The irreversible liquefaction
ability was exploited to rapidly release the encapsulated cells, and
the effect of the necessary thermal shock on the viability of released
cells was assessed.
The viability of released cells was assessed by means of their
biochemical activity and morphologic and ultrastructural charac-
teristics, in comparison with those not thermally exposed. The
tested fibroblasts and adipose-derived stem cells preserved their
viability, and kept their proliferative ability unaltered.
Figure SD-18 summarizes the morphological and structural
characteristics of heat exposed adipose-derived stem cells, after
re-culturing. The microscopic aspect of cells does not differ from
that of not-exposed ones (see Figure SD-18.a; hematoxylin − eosin
staining). The cellular metabolic activity and the integrity of cells
membrane were put in evidence by calcein staining of cytoplasm
(Figure SD-18.b). DAPI (4 ,6-diamidino-2-phenylindole) staining
indicates that cells nuclei are intact (see Figure SD-18.c), without
nuclear blebbing that usually suggests the start of apoptotic pro-
cesses. Rhodamine −phalloidin staining (Figure SD-18.c) revealed
that the heat shock did not affected cells integrity, fact confirmed
by the presence and abundance of the intracellular actin filaments.
Figure SD-20 graphically summarizes the liquefaction tests.
4. Conclusions
Atelocollagen − oxidized polysaccharides hydrogels are of ionic-
covalent entanglement type. Their biomacromolecular network is
predominantly based on physical interactions, but covalent bridges
consolidate the tri-dimensional edifice, and provide the rheo-
mechanical properties imposed by cell encapsulation protocols.
The present work investigated the dependence of the char-
acteristics of aK − NaHyal − OxPolys hydrogels on the recipe
formulation, in order to identify the optimal substrata for cell
embedding. Considering a constant amount of NaHyal in the recipes
series, the hydrogels prepared at an aK: OxPolys ratio of 3: 1
offered the best correlations between the physical-chemical and
rheo-mechanical characteristics.
In the case of optimal recipes, oxidized gellan with relatively
low amount of free aldehyde groups (1.42 ± 0.025 mM/g) is able to
consolidate the atelocollagen-based hydrogels by ensuring a cova-
lent crosslinking yield of about 20%, and the rheological behavior
of a weak hydrogel (expressed by the G’/G” ratio of about 8). When
compared with the hydrogels crosslinked with oxidized pullulan,
374 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375

Fig. 5. GellOx crosslinked hydrogel (Recipe 4). The fibrillary structure and the accommodation of the encapsulated cells.

Fig. 6. PullOx crosslinked hydrogel (Recipe 5). The fibrillary structure and the accommodation of the encapsulated cells.

or with the 5 mg/mL alginate hydrogel, the GellOx crosslinked ones
have the most similar rheological characteristics to those prepared
with aK only, but an increased maneuverability during cell encap-
sulation and culturing.
The cells encapsulated in aK − NaHyal − OxPolys hydrogels can
be released by applying a short thermal shock (48 ◦ C, 4 min), with-
out compromising their viability and their proliferation ability. This
effect is based on the liquefaction capacity of the investigated gels,
induced by the irreversible denaturation of aK component.
 A. Luca et al. / Carbohydrate Polymers 169 (2017) 366–375
Acknowledgements
The research works have been supported by a grant of the Roma-
nian National Authority for Scientific Research, CNCS-UEFISCDI,
project number PNII-ID-PCCE-2011-2-0028.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.04.
046.
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