Biotechnological
Communication
Biosci. Biotech. Res. Comm. 9(3): 463-470 (2016)
Effect of gel porosity and stiffness on culture of HepG2
cells encapsulated in gelatin methacrylate hydrogels
Mansi K. Aparnathi and Jagdish S. Patel*
P. D. Patel Institute of Applied Science, Charotar University of Science and Technology, Changa-388421,
Gujarat, India
ABSTRACT
Owing to in vivo applications, use of biodegradable three dimensional matrices to form implantable tissue constructs
has increased in recent times. Gelatin methacrylate gel (GelMA) is one such versatile matrix compatible for cell cul-
ture and has potential of in vivo implantation. Physical and mechanical properties of these hydrogels are extremely
crucial in modulating their rigidity, biodegradability and cellular compatibility. The present study involves testing
stiffness and porosity of GelMA at high and low methacrylation degrees and at different concentrations of pre-
polymer and itseffect on viability and proliferation of HepG2 cells cultured encapsulated in GelMA. Stiffness was
found to be directly proportional whereas porosity was found to be inversely related to degree of methacrylation and
concentration of GelMA pre-polymer. Softer gel with greater porosity was found to be favorable for proliferation of
cells encapsulated in the gel. In order to achieve stable implantable construct, it is important to ne tune its physical
properties such as stiffness and porosity since both these parameters are responsible for governing culture of cells
encapsulated within the gel matrix and the present study will be helpful in modulating these properties of GelMA as
per the intended application.
KEY WORDS: GELMA, PHYSICAL PROPERTIES, TMSPMA COATING
463
ARTICLE INFORMATION:
*Corresponding Author: jagdishpatel.biochem@charusat.ac.in
Received 27
th
June, 2016
Accepted after revision 14
th
Aug, 2016
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007
Thomson Reuters ISI ESC and Crossref Indexed Journal
NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006
© A Society of Science and Nature Publication, 2016. All rights
reserved.
Online Contents Available at: http//www.bbrc.in/
INTRODUCTION
Biomaterial tissue constructs that have found applica-
tions as in vivo implants are constructed by encapsu-
lating cells in three dimensional matrices. The cells in
constructs are either cultured prior to implantation or
directly implanted and allowed to grow in vivo post-
implantation (Bhatia & Chen, 1999).It has been reported
that the development of tissue within gel matrix is
modulated by surface chemistry of the scaffold, its pore
size, structure and mechanical properties, though gold
standard of parameters has not been de ned yet (Basu,
2004). Hollister et al. have explained the requirement
to modulate physical properties of gel matrix so as to
464 EFFECT OF GELMA POROSITY& STIFFNESS ON HEPG2 CULTURE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Aparnathi and Patel
suit the intended application (2002).A biodegradable,
architecturally exible, photocrosslinkable and biocom-
patible gel has been synthesized from methacrylated
gelatin gel GelMA (Van Den Bulcke et al., 2000) and
has been exploited for cell culture studies by various
groups (Chen et al., 2012; Nichol et al., 2010; Nikkah
et al., 2012; Samorezov et al., 2016).
Mechanical properties such as stiffness and rigidity of
cell matrix varies with the tissue type, being higher for
bones and lower for soft tissues (Tse and Engler, 2010).
It is important for tissue construct intended for implan-
tation to mimic the stiffness of the tissue found natu-
rally in the body. Tissue stiffness is important in deter-
mining cellular function, and changes in tissue stiffness
are commonly associated with brosis, cancer and car-
diovascular disease (Cretu et al., 2010).Mechanical prop-
erties of GelMA can be tuned to desired parameters by
altering the methacrylation levels of the gel (Hoch et al.,
2012) and percentage of GelMA in pre-polymer used for
crosslinking the gel (Chen et al., 2012).
Porosity of matrix is extremely important parameter
which determines the degree of solute diffusion, surface
properties, mechanical properties and surface mobility
(Peppas et al. 2006). Gel with higher porosity is reported
to be more suitable for cell proliferation(Hoch et al., 2012).
Higher porosity was achieved in calcium alginate/gelatin
hydrogels which were shown to possess enhanced compat-
ibility for cell culture (Cuadros et al., 2015). Even the oste-
ogenic differentiation of cells can be modulated by ne-
tuning porosity of hydroxyapatite (Tsurugaet al.,1997).
Porosity of GelMA as a function of its swelling ratio has
been extensively studied by Nichol et al. (2010). The degree
of swelling of gels relies on the porosity of the matrix and
the solvent-polymer interaction (Du et al., 2008).
In the present study, mechanical testing was to be
carried out in order to determine the compressive modu-
lus of GelMA at varying degrees of methacrylation and
pre-polymer concentration. Swelling analysis and SEM
imaging of gels for determination of porosity of GelMA
were undertaken. Compatibility of GelMA as a matrix
for culture of HepG2 cells was determined along with
the best degree of methacrylation via live-dead staining
and MTT proliferation assay. This appears to be the rst
study from India, where potential of GelMA as a tissue
engineering implant has been explored. The main aim
of the present study was to help in ne-tuning the gel
properties based on its intended application.
MATERIAL AND METHODS
SYNTHESIS OF GEIMA
High and low methacrylated GelMA were synthesized
by the protocol described by Nichol et al. (2010). Brie y,
Type-A porcine skin gelatin (Sigma)was dissolved in
DPBS (Sigma) yielding 100 ml 10% gelatin solutionat
60
0
C for 1 hr. Methacrylic anhydride (MA) (Sigma) was
added drop wise to the gelatin solution and was allowed
to react for 3 hrs at 60
0
C. 800 μl MA was added for the
synthesis of low methacrylated gelatin gel (GelMA
low
)
and 8 ml MA was added for the preparation high meth-
acrylated gelatin gel (GelMA
high
). Solution was diluted
5-X by adding warm DPBS and allowed to mix well at
50
0
C for 1 hr. GelMA solution was dialyzed using 12–14
kDa cut-off dialysis tubes (HiMedia) against deionized
water for 7 days at 50
0
C. The dialyzed GelMA solutions
were frozen at − 80
0
C for at least 5 days, freeze dried and
stored at room temperature until use.
GelMA pre-polymer
Freeze-dried GelMA (depending upon the nal % of
GelMA required) and 0.5% photoinitiator, 2-Hydroxy-
4’-(2-hydroxyethoxy)-2-methylpropiophenone (Sigma)
were dissolved in DPBS at 70
0
C to prepare pre-poly-
mer solution of GelMA. GelMA pre-polymer could be
lled in desired mold and photocrosslinked by expos-
ing it to 6.7 mW/cm
2
UV light for 1 min at room
temperature.
MECHANICAL TESTING
Fifty microliters of pre-polymer wasphotocrosslinked-
into cylindrical wells fabricated in PDMS mold. Dimen-
sions of the discs to be used for mechanical testinghave
approximately 0.75 mm diameter and 0.5 mm height.
Samples were detached from the mold and transferred
free oating at 37
0
C in DPBS. Immediately prior to test-
ing, the disc was blotted lightly with a wipe and test-
edon an Instron 5542 mechanical tester. The compres-
sive modulus was determined as the slope of the linear
region corresponding with Young’s Compressive stress
1 % - 10 %.
POROSITY ANALYSIS
Scanning Electron Microscopy(SEM)
GelMA sheets were made by crosslinking 30 μl of 10%
GelMA
high
and GelMA
low
pre-polymer between two glass
slides separated by a 1 mm spacer as demonstrated in
schematic Figure 1. Sheets were detached from slide,
frozen at -80
0
C overnight and lyophilized. These sheets
were further visualized under FEG-SEM (Nova Nano
SEM 450)tocompare porosity of both the gels.
Swelling analysis
Discs with 5 and 10% of GelMA
high
and GelMA
low
were
prepared and weighed. These discs were allowed to
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF GELMA POROSITY& STIFFNESS ON HEPG2 CULTURE 465
Aparnathi and Patel
hydrate in DPBS for 24 hrs in humidi ed CO
2
incubator
at 37
0
C. Discs were removed from PBS, blotted on wipes
and weighed. Percentage increase in weight was calcu-
lated. Experiment was repeated thrice and discs for each
set of experiment was made in triplicates.
TMSPMA COATING OF GLASS SLIDES
TMSPMA coating is essential for the adherence of GelMA
sheets to glass slide. 50 g of Sodium Hydroxide pellets
(NAOH, Sigma) were dissolved in 450 mL of distilled water.
Glass slides stacked in staggered manner were treated
with this NAOH solution overnight such that each surface
of every slide stays in contact with the solution. Slides
were then rinsed and rubbed under distilled water. Slides
were then sequentially plunged in ve tubes containing
reagent alcohol and allowed to air dry. Slides wrapped in
aluminium foil were baked at 80
0
C for 1 hr. Slides were
stacked vertically and 3-4 ml of TMSPMA (3-(Trimethox-
ysilyl)propyl methacrylate, Sigma) was dripped over the
stack with a syringe and allowed to ow over the slide
for 30 min. The stack was inverted and again coating was
allowed to go on for another 30 min. Beaker was cov-
ered with aluminum foil and baked overnight at 80
0
C.
Slides were again given 5 washes with reagent alcohol
and air dried. Slides were wrapped in aluminum foil and
baked for 1 hr at 80
0
C. The TMSPMA coated glass slides
wrapped in aluminum foil were stored at room tempera-
ture until use. These slides were cut into 1x1 cm pieces
with a glass cutter prior to use.
ENCAPSULATION AND CELL CULTURE
A con uent monolayer ofHepG2 cells was trypsi-
nized, spun down, trypsin was removed and pellet
was re-suspended in 3 ml Dulbecco’s Modi ed Eagle’s
Medium(DMEM, Sigma) with 10% Foetal Bovine Serum
(FBS, Gibco). Cells were counted under hemocytometer
and aliquots with 1 million cells were made. These ali-
quots were spun down, supernatant was removed and
pellet was re-suspended by gently pipetting pellet into
1 ml 10% GelMA pre-polymer solution so as to obtain 1
million cells per ml of GelMA.
Cell-GelMA suspension was crosslinked on a TMSPMA
coated glass slide as per the schematic diagram (Figure
1). Brie y, 20 μl cell suspension was placed between 150
μm spacers attached on a glass slide. 1x1 cm TMSPMA
coated glass slide was inverted on it. This system was
exposed to UV for crosslinking GelMA. Cells encapsu-
lated in GelMA layered on TMSPMA coated glass slide
was submerged in media and incubated in a humidi-
ed CO
2
incubator at 37
0
C. Media was changed every
alternate day. Cells were observed microscopically under
Leica DMIL inverted microscope.
VIABILITY AND PROLIFERATION ANALYSIS
Live dead staining
Viability of HepG2 cells cultured encapsulated in GelMA-
high
and GelMA
low
was determined by calcein-ethidium
homodimer Live-Dead assay (Invitrogen) of construct on
day 3 of culture using manufacturer’s protocol.
MTT assay
Proliferation of cultured cells was observed 1, 3 and 5 days
after cell seeding. Growth in 10% GelMA
high
was com-
pared with growth in 10% GelMA
low
. MTT Assay described
by Denizot and Lang method (1986) was employed.
RESULTS AND DISCUSSION
GelMA hydrogels are emerging biomaterials for bio-
medical applications, mainly owing to their biocom-
patibility, architectural exibility and biodegradability.
Crosslinked polymer gels can be fabricated with con-
trolled stiffness, pore size and other physical properties.
These controlled parameters can be achieved by varying
degree of methacrylate substitution or biopolymer con-
centration (Van Den Bulcke et al., 2000).
GELMA SYNTHESIS
High and low methacrylated gelatin gels were synthe-
sized (Figure 2a &2b, respectively), of which further 5%,
FIGURE 1. Schematic diagram of the steps involved in forming a layer of GelMA on TMSPMA coated glass slide.
466 EFFECT OF GELMA POROSITY& STIFFNESS ON HEPG2 CULTURE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Aparnathi and Patel
10% and 20% pre-polymers were used for each degree of
methacrylation for further study.
MECHANICAL PROPERTIES
GelMA discs of 5% and 10% of GelMA
high
and GelMA
low
were compressed on instron for determining the effect
of gel concentration and degree of methacrylation on
the mechanical properties of the GelMA (Figure 3). At a
given degree of methacrylation, both 5% and 10% gels
demonstrated a dramatic difference in their compressive
modulus. At 10% concentration, signi cant difference
was observed in compressive modulus of both, high and
low methacrylated GelMA. On the contrary, there was
no drastic difference in compressive modulus of both the
gels at 5% concentration. No sample failure was reported
before the maximum 50 N load was reached, providing
testimony to the elastomeric properties of the GelMA.
Chen et al. have demonstrated increase in the compres-
sive modulus of the GelMA with its methacrylation
degree (2012). Nichol et al. have reported that increas-
ing the degree of methacrylation increased the stiffness
at all the gel concentrations and at a constant degree
of methacrylation, increase in the GelMA concentration
signi cantly increased the compressive modulus (2010).
In accordance with their ndings, the present study
also has shown direct proportionality between degree
of methacrylation of GelMA and its compressive modu-
lus, which is further proportional to the stiffness of the
hydrogel. Moreover, similar proportionality has also
been observed between concentration of GelMA and
compressive modulus. But, at a given concentration
of GelMA, stiffness of GelMA
high
is always found to be
higher than GelMA
low
. In contrast with the study reported
by Nichol et al., in present study, the instron analysis
of 5% low methacrylated GelMA was successful, prob-
ably on account of different dimension of GelMA disc
being used for the test and PDMS mold based fabrication
of the disc instead of punching sheets, making the gel
easier to handle.
POROSITY ANALYSIS
SEM
SEM imaging of the surface of freeze dried 10%
GelMA
high
(Figure 4a) and GelMA
low
(Figure 4b) sheets
demonstrate distinct difference in porosity of both the
gels. GelMA
low
possesses visibly much larger pores as
compared to GelMA
high
.
Swelling analysis
The degree of swelling of hydrogelsrelies on the porosity
of the polymer. Change in mass swelling ratio of GelMA
with respect toits concentrationand its methacrylation
level was studied. 5%, 10%and20% (w/v) GelMA
high
and
GelMA
low
were compared in terms of their swelling prop-
erties (Figure 4c). At any given concentration of GelMA,
the mass swelling ratioof GelMA
low
was signi cantly
greater than that of GelMA
high
. On the other hand, at a
given degree of methacrylation, mass swelling ratio was
inversely dependent on the concentration of GelMA.
Hoch et al. have demonstrated that the average size
of pores in the scaffold matrix decreases with higher
degrees of methacrylation and the degree of methacryla-
tion was found to affect the physical and mechanical
properties of the synthesized GelMA hydrogels, with
higher methacrylation resulting in stiffer and more dura-
ble hydrogels, with smaller pore sizes (2012). In accord-
ance with these studies, we observed that GelMA
low
dem-
onstrated signi cantly greater swelling as compared to
GelMA
high
at any given concentration of GelMA. Swell-
ing was also found to increase with decrease in concen-
tration of GelMA. The results of swelling analysis were
also in accordance with the porosity observed by SEM
imaging, with GelMA
low
demonstrating larger pores in
comparison with GelMA
high
.
HEPG2 CULTURE IN GELMA
Highest porosity and least stiffness for both degrees
of methacrylation was observed in 5% GelMA. There-
FIGURE 2. Photographs of freeze dried (a)
GelMA
high
and (b) GelMA
low
.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF GELMA POROSITY& STIFFNESS ON HEPG2 CULTURE 467
Aparnathi and Patel
fore, taking into consideration intermittent stiffness and
porosity, 10% GelMA
low
andGelMA
high
were used for fur-
ther encapsulation and cell culture study. HepG2 cells
encapsulated in 10% GelMA
high
and GelMA
low
were cul-
tured for a span of 5 days in DMEM (Figure 5) so as
to form 3d sheet of 150 μm thickness and was inter-
mittently observed microscopically. Cells depicted mor-
phology similar to what they demonstrate on 2d cul-
ture. Therefore, encapsulation process, UV exposure and
presence of photoinitiator didn’t alter the morphology of
HepG2 cells in GelMA.
VIABILITY AND PROLIFERATION ANALYSIS
Live-dead staining analysis
Viability of HepG2 cells encapsulated in GelMA
high
(Fig-
ure 6a) and GelMA
low
(Figure 6b) was observed micro-
scopically on day 3 of culture by staining the sheets with
calcein-ethidium homodimer. Live cells were stained
green with calcein and dead cells appeared red stained
with ethidium homodimer. Number of dead cells in Gel-
MA
high
were visually much higher as compared to Gel-
MA
low
.Viability of cells in GelMA
low
were comparatively
higher.
MTT assay
Proliferation of HepG2 cells cultured in 10% GelMA-
high
and GelMA
low
for day 1, 3 and 5 was assessed using
MTT assay (Figure 6c). In GelMA
low
, cells proliferated to
achieve three times the number of cells from day 1 to
day 5. On the contrary, some cells initially grew in Gel-
MA
high
, but failed to proliferate and demonstrated decline
in cell number from day 1 to day 5. Therefore, GelMA
high
proved to be hostile for culture of HepG2 cells as com-
pared to GelMA
low
. On the other hand, GelMA
low
proved
to be much more favorable for HepG2 cell culture.
Cell viability has been shown to decrease with
increasing polymer concentration (Hoch et al., 2012).
Better proliferation was observed in GelMA
low
as com-
pared to GelMA
high
as observed via live-dead staining
and MTT cell proliferation assay. The results were in line
with the results previously reported by Occhettaet al.
(2012). Nichol et al. also observed that in 5% (w/v) gels,
cell elongated, migrated and formed interconnected net-
works with neighboring cells, but not at higher concen-
trations of GelMA (2010). This explains that cells grow
more effortlessly in softer gel matrices with large pores,
where as in stiffer gel with smaller pore size, cells nd it
dif cult proliferate and eventually die.
FIGURE 3. Mechanical properties of GelMA with varying gel percentage and
degree of methacrylation. Compressive modulus for 5% and 10% (w/v) GelMA
at low and high degree of methacrylation. Error bars represent the SD of meas-
urements performed on 4 samples.
468 EFFECT OF GELMA POROSITY& STIFFNESS ON HEPG2 CULTURE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Aparnathi and Patel
FIGURE 4. SEM images of freeze dried GelMA discs (a) low methacrylated and (b) high methacrylated. (Photo-
graphs of the discs used for imaging are on top left corner of images)(c) Swelling analysis of 5% and 10% of
high and low methacrylated GelMA. Error bars represent the SD of measurements performed on 3 samples.
FIGURE 5. Representative image of the culture of HepG2
cells encapsulated in GelMA.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF GELMA POROSITY& STIFFNESS ON HEPG2 CULTURE 469
Aparnathi and Patel
FIGURE 6. Representative images for Live-Dead staining of HepG2 cells on day 3 of culture encapsulated in (a) GelMA
low
(b) GelMA
high
. (c) MTT assay for the proliferation of HepG2 cells encapsulated in GelMA
low
and GelMA
high
measured on day
1, 3 and 5. Error bars represent the SD of measurements performed on 3 samples.
Therefore, present study puts forth the correlation
between degree of methacrylation and concentration of
GelMA with its porosity and stiffness. These very impor-
tant physical matrix parameters can be ne-tuned and
modulated as per the requirement and application for
which this versatile biodegradable biocompatible hydro-
gel, GelMA, is intended to be used.
ACKNOWLEDGEMENTS
Authors are highly obliged to Dr. Ali Khademhosseini,
Professor, Harvard University, Cambridge, USA for giving
Mansi K. Aparnathi an opportunity to learn working with
GelMA as a Pre-Doctoral Research Fellow in his esteemed
laboratory at Harvard-MIT Health Science & Technology
division. Authors are grateful to Anand Agricultural Uni-
versity, Anand, Gujarat, India for providing infrastruc-
ture. Authors are thankful to Sophisticated Analytical
Instruments Facility Supported by Department of Science
& Technology (Govt.of India), Vallabh Vidyanagar, Guja-
rat, India for facilitating SEM imaging.
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