polymers
Article
Equine Tenocyte Seeding on Gelatin Hydrogels Improves
Elongated Morphology
Marguerite Meeremans 1, * , Lana Van Damme 2, Ward De Spiegelaere 3, Sandra Van Vlierberghe 2, †
and Catharina De Schauwer 1, †
Citation: Meeremans, M.; Van
Damme, L.; De Spiegelaere, W.; Van
Vlierberghe, S.; De Schauwer, C.
Equine Tenocyte Seeding on Gelatin
Hydrogels Improves Elongated
Morphology. Polymers 2021,13, 747.
https://doi.org/10.3390/
polym13050747
Academic Editor: Vijay
Kumar Thakur
Received: 11 February 2021
Accepted: 24 February 2021
Published: 28 February 2021
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1Comparative Physiology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133,
B-9820 Merelbeke, Belgium; Catharina.DeSchauwer@ugent.be
2Polymer Chemistry and Biomaterials Group, Centre of Macromolecular Chemistry, Faculty of Sciences,
Ghent University, Krijgslaan 281 S4-Bis, B-9000 Ghent, Belgium; Lana.VanDamme@UGent.be (L.V.D.);
Sandra.VanVlierberghe@Ugent.be (S.V.V.)
3Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133,
B-9820 Merelbeke, Belgium; Ward.Despiegelaere@UGent.be
*Correspondence: marguerite.meeremans@ugent.be
† The authors contributed equally to this work.
Abstract:
(1) Background: Tendinopathy is a common injury in both human and equine athletes.
Representative
in vitro
models are mandatory to facilitate translation of fundamental research into
successful clinical treatments. Natural biomaterials like gelatin provide favorable cell binding charac-
teristics and are easily modifiable. In this study, methacrylated gelatin (gel-MA) and norbornene-
functionalized gelatin (gel-NB), crosslinked with 1,4-dithiotreitol (DTT) or thiolated gelatin (gel-SH)
were compared. (2) Methods: The physicochemical properties (
1
H-NMR spectroscopy, gel fraction,
swelling ratio, and storage modulus) and equine tenocyte characteristics (proliferation, viability,
and morphology) of four different hydrogels (gel-MA, gel-NB85/DTT, gel-NB55/DTT, and gel-
NB85/SH75) were evaluated. Cellular functionality was analyzed using fluorescence microscopy
(viability assay and focal adhesion staining). (3) Results: The thiol-ene based hydrogels showed a
significantly lower gel fraction/storage modulus and a higher swelling ratio compared to gel-MA.
Significantly less tenocytes were observed on gel-MA discs at 14 days compared to gel-NB85/DTT,
gel-NB55/DTT and gel-NB85/SH75. At 7 and 14 days, the characteristic elongated morphology of
tenocytes was significantly more pronounced on gel-NB85/DTT and gel-NB55/DTT in contrast to
TCP and gel-MA. (4) Conclusions: Thiol-ene crosslinked gelatins exploiting DTT as a crosslinker are
the preferred biomaterials to support the culture of tenocytes. Follow-up experiments will evaluate
these biomaterials in more complex models.
Keywords:
tenocytes; hydrogels; gelatin; cell proliferation; viability; morphology; gel fraction;
swelling ratio; storage modulus
1. Introduction
Overuse tendon injuries are a major cause of musculoskeletal morbidity in both human
and equine athletes [
1
]. Approximately 30–50% of all sport lesions in both professional
and recreational athletes are tendon injuries with increased age being an additional risk
factor [
2
,
3
]. Injuries to the tendons situated at the palmar/plantar side of the equine
distal limb are the most common orthopedic injuries in competition horses subjected to
high-intensity exercise [
4
]. The horse is also one of the most well-accepted, scientifically
supported animal models of human exercise-induced Achilles tendon injury [
5
]. The poor
success with conventional therapy supports the need to search for novel treatments to
restore the functionality and regenerate a tissue as close to the original tendon as possible,
instead of inferior scar tissue [2,4].
In vitro
models serve as important biological tools to study cell behavior under con-
trolled conditions, bypassing the confounding factors associated with
in vivo
clinical
Polymers 2021,13, 747. https://doi.org/10.3390/polym13050747 https://www.mdpi.com/journal/polymers
Polymers 2021,13, 747 2 of 16
trials [
6
,
7
]. A wide diversity of
in vitro
tendon models are used nowadays to study tendon
mechanobiology, tissue replacement processes, cell-based treatments, and drug screen-
ing applications [
8
,
9
]. Unfortunately, no generally accepted
in vitro
model or standard
tenogenic differentiation assay exists currently. Various biomaterials, bioreactors and
production technologies are randomly combined at this moment [
10
]. By establishing
a physiologically representative
in vitro
tendinopathy model, the use of experimental
animals and the number of in vivo experiments can be drastically reduced.
The tendon extracellular matrix (ECM) predominantly consists of collagen I [
2
,
11
] and
therefore, this natural biomaterial is mostly used for tendon applications [12–14]. Gelatin,
being denatured collagen, contains the same peptide sequences (e.g., arginine-glycine-
aspartic acid sequence) as collagen, critical for cell surface receptor recognition, and can be
used as an alternative to collagen [
15
,
16
]. When compared to collagen I, other advantages
of gelatin include lower immunogenicity, higher water solubility, lower cost, and wide
availability, which is important when considering large-scale
in vitro
studies [
8
,
17
]. To
provide more material strength, the gelatin backbone can be modified by introducing
different moieties at the level of different amino acid side-chain functionalities [
18
]. For
years, methacrylated gelatin (gel-MA) has been used as the gold standard [
8
,
19
,
20
]. Al-
though not yet reported in the context of tendon applications, norbornene-functionalized
gelatin (gel-NB) has already been proven suitable and even superior to gel-MA, in adi-
pose tissue engineering, for example [
21
,
22
]. Furthermore, the polymerization of gel-NB
combined with a thiolated crosslinker, occurs through a thiol-ene step growth polymer-
ization instead of a chain growth polymerization (as is the case for gel-MA). Step growth
polymerization is characterized by a higher reactivity, a more homogeneous end product
and a more cell-friendly approach towards cell encapsulation, because of reduced radical
formation [
15
,
17
,
23
]. It is therefore hypothesized that gel-NB combined with a thiolated
crosslinker (i.e., 1,4-dithiotreitol, DTT versus thiolated gelatin, gel-SH) could be a valuable
biomaterial to support the culture of tenocytes.
In order to identify a more physiologically representative hydrogel for tenocytes,
which enables the expression of the characteristic tenogenic morphology, four different
gelatin hydrogels were compared to tissue culture plastic (TCP) in this study
(Table 1)
. The
physicochemical characteristics, including the chemical structure via
1
H-NMR spectroscopy,
gel fraction, swelling ratio and mechanical properties of three gel-NB-based compositions
were evaluated and benchmarked against gel-MA. Furthermore, tenocyte morphology and
functionality were evaluated.
Table 1.
Experimental set-up: four different gelatin biomaterials were evaluated as hydrogels for
tenocyte culture. Tissue culture plastic was included as control group. DS: degree of substitution;
DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin;
gel-SH: thiolated gelatin; LAP: lithium(2,4,6-trimethylbenzoyl) phenylphosphinate.
Hydrogel Concentration DS Photo-Initiator Cross-Linker Abbreviation
Gel-MA 10% w/v97% 2 mol% LAP NA Gel-MA
Gel-NB 10% w/v85% 2 mol% LAP DTT Gel-NB85/DTT
Gel-NB 10% w/v55% 2 mol% LAP DTT Gel-NB55/DTT
Gel-NB 10% w/v85% 2 mol% LAP Gel-SH 75% Gel-NB85/SH75
2. Materials and Methods
2.1. Materials
Gelatin type B, isolated from bovine skin was kindly supplied by Rousselot (Ghent, Bel-
gium). N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide (EDC), N-acetyl-homocysteine
thiolactone, ethylene diamine tetra-acetic acid (EDTA), acetone, methacrylic anhydride, 5-
norbornene-2-carboxylic acid, collagenase type Ia, antibiotic-antimycotic solution, trypsin-
EDTA, calcein-acetoxymethyl (Ca-AM), propidium iodide (PI), the focal adhesion staining
kit, paraformaldehyde, and secondary goat anti-mouse antibody were purchased from
Sigma-Aldrich (Diegem, Belgium). High glucose Dulbecco’s Modified Eagle Medium
Polymers 2021,13, 747 3 of 16
(DMEM), L-glutamine, 4-well plates, Tween-20, Triton-X, and bovine serum albumin (BSA)
were bought from Fisher Scientific (Merelbeke, Belgium). Dimethyl sulfoxide (DMSO) and
N-hydroxysuccinimide (NHS) were obtained from Acros (Geel, Belgium). The Spectrapor
dialysis membranes MWCO 12–14 kDa were purchased from Polylab (Antwerp, Belgium).
Lithium (2,4,6-trimethylbenzoyl) phenylphosphinate (LAP) was synthesized according to
a protocol described earlier [24].
2.2. Disc Development
2.2.1. Development of Gel-MA
Gel-MA was obtained following a previously reported protocol by Van den Bulcke
et al. [
25
]. Briefly, 100 g gelatin type B was dissolved in 1 L of a 0.1 M phosphate buffer
(pH 7.8) at 40
◦
C (Figure 1). Afterwards, 2.5 equivalents of methacrylic anhydride were
added dropwise and the mixture was allowed to react for 1 h under continuous mechanical
stirring. Next, the reaction mixture was diluted with 1 L double distilled water (ddH
2
O),
followed by dialysis (Spectrapor 12–14 kDa cutoff) against distilled water (dH
2
0) for 24 h
at 40
◦
C, changing the water at least five times. The pH of the solution was adjusted to
~7.4. The obtained gel-MA was then frozen and lyophilized (Christ freeze-dryer alpha
I-5;
−
80
◦
C; 0.37 mbar). The degree of substitution (DS) was determined using
1
H-NMR
spectroscopy (Bruker Avance WH 500 MHz).
Polymers 2021, 13, x FOR PEER REVIEW 3 of 16
dride, 5-norbornene-2-carboxylic acid, collagenase type Ia, antibiotic-antimycotic solu-
tion, trypsin-EDTA, calcein-acetoxymethyl (Ca-AM), propidium iodide (PI), the focal ad-
hesion staining kit, paraformaldehyde, and secondary goat anti-mouse antibody were
purchased from Sigma-Aldrich (Diegem, Belgium). High glucose Dulbecco’s Modified
Eagle Medium (DMEM), L-glutamine, 4-well plates, Tween-20, Triton-X, and bovine se-
rum albumin (BSA) were bought from Fisher Scientific (Merelbeke, Belgium). Dimethyl
sulfoxide (DMSO) and N-hydroxysuccinimide (NHS) were obtained from Acros (Geel,
Belgium). The Spectrapor dialysis membranes MWCO 12 -14 kDa were purchased from
Polylab (Antwerp, Belgium). Lithium (2,4,6-trimethylbenzoyl) phenylphosphinate (LAP)
was synthesized according to a protocol described earlier [24].
2.2. Disc Development
2.2.1. Development of Gel-MA
Gel-MA was obtained following a previously reported protocol by Van den Bulcke
et al. [25]. Briefly, 100 g gelatin type B was dissolved in 1 L of a 0.1 M phosphate buffer
(pH 7.8) at 40 °C (Figure 1). Afterwards, 2.5 equivalents of methacrylic anhydride were
added dropwise and the mixture was allowed to react for 1 h under continuous mechan-
ical stirring. Next, the reaction mixture was diluted with 1 L double distilled water
(ddH2O), followed by dialysis (Spectrapor 12–14 kDa cutoff) against distilled water (dH20)
for 24 h at 40 °C, changing the water at least five times. The pH of the solution was ad-
justed to ~7.4. The obtained gel-MA was then frozen and lyophilized (Christ freeze-dryer
alpha I-5; −80 °C; 0.37 mbar). The degree of substitution (DS) was determined using 1H-
NMR spectroscopy (Bruker Avance WH 500 MHz).
Figure 1. Reaction scheme depicting the development of gel-MA, gel-NB and gel-SH precursors. The crosslinked hydrogel
films are obtained through chain growth polymerization for gel-MA and via step growth polymerization when gel-NB is
combined with either DTT or gel-SH. DTT: 1,4-dithiotreitol; EDC: N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hy-
drochloride; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; NHS:
N-hydroxysuccinimide.
Figure 1.
Reaction scheme depicting the development of gel-MA, gel-NB and gel-SH precursors. The crosslinked hydrogel
films are obtained through chain growth polymerization for gel-MA and via step growth polymerization when gel-NB
is combined with either DTT or gel-SH. DTT: 1,4-dithiotreitol; EDC: N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; NHS:
N-hydroxysuccinimide.
2.2.2. Development of Gel-NB
A protocol described earlier by Van Hoorick et al. was used to obtain gel-NB [
22
].
Briefly, 2.5 equivalents of 5-norbornene-2-carboxylic acid (relative to the primary amines of
gelatin) were converted into an activated succinimidyl ester using carbodiimide coupling
chemistry. To this end, 2 equivalents of EDC followed by 3 equivalents of NHS were
added to dry DMSO under inert atmosphere at room temperature. Following 25 h of
Polymers 2021,13, 747 4 of 16
reaction, the mixture was heated to 50
◦
C. In parallel, gelatin type B was dissolved in
dry DMSO at 50
◦
C (Figure 1). The reaction mixture was added to the dissolved gelatin
and left to react overnight at 50
◦
C under Argon atmosphere. Next, the mixture was
precipitated in a tenfold excess of acetone. The precipitate was isolated and washed with
acetone, before dissolving the residue in ddH
2
O. Afterwards, the mixture was dialyzed
(Spectrapor MWCO 12–14 kDa cutoff) for 24 h at 40
◦
C. The pH of the mixture was adjusted
to ~7.4, before freezing and lyophilization of the product. The DS was assessed using
1
H-NMR spectroscopy. In order to obtain gel-NB with a lower DS, the same protocol was
applied using 1.2 equivalents of 5-norbornene-2-carboxylic acid, 0.75 equivalents EDC and
1.5 equivalents NHS.
2.2.3. Development of Gel-SH
Gel-SH was developed by dissolving 20 g of gelatin type B in 200 mL carbonate
buffer (pH 10) at 40
◦
C under inert argon atmosphere, as described earlier (Figure 1) [
26
].
When the gelatin was completely dissolved, 1.5 mM of EDTA was added in order to
chelate any metals which could catalyze the oxidation of the sulfhydryls into disulfide
bonds. Afterwards, 5 equivalents (relative to the primary amines of gelatin) of N-acetyl
homocysteine thiolactone were added and the mixture was left to react for 3 h under inert
atmosphere at 40
◦
C. Next, the obtained gel-SH was purified using dialysis (SpectraPor
12–14 kDa cutoff) for 24 h under inert atmosphere at 40
◦
C. The water was changed
5 times. Following dialysis, the gel-SH was immediately frozen in liquid nitrogen followed
by lyophilization. The DS was determined using an ortho-phthalic dialdehyde amine
determination assay.
2.2.4. Hydrogel Film Casting
Prior to determining the physicochemical properties of the hydrogel discs and seeding
cells, the starting materials were processed into films. To this end, a 10 w/v% aqueous
solution of the gelatin derivatives, containing 2 mol% LAP as photoinitiator, was made
at 40
◦
C. The thiolated crosslinkers, either DTT or gel-SH, were added in an equimolar
quantity (1:1 thiol-ene ratio). When complete dissolution was obtained, the mixture was
poured between two glass plates covered with release foil and separated by a 1 mm silicone
spacer. Crosslinked hydrogels were obtained by putting the plates at +4
◦
C for 30 min,
followed by exposure to UV-A light (365 nm, 9 mW/cm2) for 30 min.
2.3. Physico-Chemical Characterization
2.3.1. Gel Fraction Assessment
To determine the gel fraction, the crosslinked hydrogel discs (d = 8 mm) were freeze-
dried determining the dry mass (m
d1
). Next, the dried hydrogel discs were incubated in
ddH
2
O at 37
◦
C for 24 h, followed by freeze-drying to determine the second dry mass
(md2). The gel fraction was determined using the following Equation:
Gel fraction (%)=md2
md1
∗100 (1)
2.3.2. Mass Swelling Ratio Determination
To determine the swelling ratio, the hydrogel discs (d = 8 mm) were incubated imme-
diately after crosslinking for 24 h in ddH
2
O at 37
◦
C to obtain equilibrium swelling. Next,
the hydrated mass of the samples was measured (m
h
) and the samples were lyophilized
to determine their dry mass (m
d
). The mass swelling ratio was determined using the
following formula:
Mass swelling ratio =mh
md
(2)
Polymers 2021,13, 747 5 of 16
2.3.3. Mechanical Properties’ Assessment
To obtain insight into the hydrogels’ mechanical properties, a rheometer (Physica
MCR-301; Anton Paar, Sint-Martens-Latem, Belgium) was used. Punched out discs (14 mm
in diameter) of equilibrium swollen hydrogel films (1 mm in height, incubated for 24 h
in ddH
2
O at 37
◦
C) were placed between the two parallel plates. Differences in storage
moduli were assessed using a strain of 0.1% with an oscillation frequency ranging from
0.01 Hz to 10 Hz at 37
◦
C, whilst applying a normal force of 1 N. All measurements were
performed in triplicate.
2.4. Cell Isolation, Culture and Seeding
Briefly, equine superficial digital flexor tendons were aseptically collected in the
slaughterhouse and transported within 2 h to the lab. Representative tendon samples of
2
−
3 g were obtained and washed in PBS containing 0.5% gentamycin. After three washing
steps with PBS, the tendon samples were minced into small pieces, and subsequently
digested overnight in high glucose DMEM supplemented with 0.075% collagenase type
Ia at 38
◦
C in a humidified atmosphere containing 5% CO
2
. After blocking collagenase
activity with fetal bovine serum (FBS) containing medium, the suspension was filtered on
a 70
µ
m cell strainer to remove undigested tissue. After centrifugation (400 g, 10 min, RT),
two more washing steps with PBS were performed. Isolated cells were incubated at 38
◦
C
in a humidified atmosphere containing 5% CO
2
in tenocyte medium consisting of high
glucose DMEM, 10% FBS, 1% antibiotic-antimycotic solution, and 2mM L-glutamine [
27
].
After 72h, the medium was removed and the cells were washed with PBS. Thereafter, the
medium was replaced twice weekly. When they reached 80
−
90% confluency, the tenocytes
were passaged using 0.25% Trypsin-EDTA. Cell viability was determined by trypan blue
exclusion using the improved Neubauer hemocytometer [
28
]. The tenocytes of passage
three (P3) were plated at a density of 40,000 cells/cm
2
in tenocyte medium in either 4-well
plates of TCP or gelatin discs. Gelatin discs of 14 mm in diameter were punched out and
sterilized by submerging them in ethanol 70% for 24 h (refreshed after 12 h) followed by
UV
−
C irradiation (30 min) before cell seeding. Tenocyte medium was added onto the discs
following 1 h of incubation and was carefully refreshed twice weekly. All experiments
were performed in triplicate.
2.5. Viability Assay
The dye solution, consisting of 2
µ
L Ca-AM and 2
µ
L PI per 1 mL PBS, was used
to assess cell viability at days 7 and 14 after seeding, using an inverted fluorescence
microscope (Axio Observer 7, ZEISS, Jena, Germany). Four random images per hydrogel
per replicate at 488/509 nm (Ca-AM, live cells) and 584/608 nm (PI, dead cells) were
evaluated. The area occupied by the cells was measured per image with an ImageJ macro
(adapted from [29]) and cell viability was determined with the following formula:
Area live cells (%)=Area live cells
Area live cells +Area dead cells ×100 (3)
2.6. Cell Morphology
A focal adhesion staining kit was used at days 7 and 14 to (1) confirm cellular align-
ment by visualizing the actin filaments and the focal contacts between cells, (2) identify
the nuclei for counting and (3) determine their shape. Briefly, cell-seeded hydrogel discs
were fixed with 4% paraformaldehyde (in PBS) for 20 min, washed twice with washing
buffer (PBS + 0.05% Tween-20) and permeabilized using 0.1% w/vTriton-X (in PBS) for
4 min. After double washing, tenocytes were blocked with 1% BSA (in PBS) for 30 min,
subsequently incubated with the primary anti-vinculin antibody (1:400, in blocking solu-
tion) for 1 h. After three washes of each 5 min, the secondary goat anti-mouse antibody
(FITC-conjugated) (1:200, in PBS) and the TRITC-conjugated phalloidin (1:200, in PBS) were
incubated simultaneously for 1 h. Counterstaining of the nuclei with DAPI (1:1000, in
Polymers 2021,13, 747 6 of 16
PBS) for 4 min was performed after three washes (5 min each). After three final washing
steps (5 min each), the cells were covered with PBS for visualization. All incubations were
performed at room temperature.
Several regions of interest per condition per replicate were evaluated, images were
taken at 495/519 nm, 548/561 nm and 353/465 nm, for FITC, TRITC and DAPI using an
inverted fluorescence microscope (Axio Observer 7, ZEISS, Germany), respectively. An
ImageJ macro (adapted from [
29
]) was used to quantify cell numbers per image using the
DAPI channel. Additionally, cell shape was qualitatively evaluated on the actin channel,
combined with quantitative determination of nucleus’ circularity and aspect ratio (AR),
being major indications for an elongated cell shape (Figure 2) [
30
–
32
]. ImageJ analysis was
performed four times and the mean of each parameter was used for statistical analysis.
Circularity =4π×[Area]
[Perimeter]2Aspect Ratio =[Major Axis]
[Minor Axis](4)
Polymers 2021, 13, x FOR PEER REVIEW 6 of 16
were fixed with 4% paraformaldehyde (in PBS) for 20 min, washed twice with washing
buffer (PBS + 0.05% Tween-20) and permeabilized using 0.1% w/v Triton-X (in PBS) for 4
min. After double washing, tenocytes were blocked with 1% BSA (in PBS) for 30 min,
subsequently incubated with the primary anti-vinculin antibody (1:400, in blocking solu-
tion) for 1 h. After three washes of each 5 min, the secondary goat anti-mouse antibody
(FITC-conjugated) (1:200, in PBS) and the TRITC-conjugated phalloidin (1:200, in PBS)
were incubated simultaneously for 1 h. Counterstaining of the nuclei with DAPI (1:1000,
in PBS) for 4 min was performed after three washes (5 min each). After three final washing
steps (5 min each), the cells were covered with PBS for visualization. All incubations were
performed at room temperature.
Several regions of interest per condition per replicate were evaluated, images were
taken at 495/519 nm, 548/561 nm and 353/465 nm, for FITC, TRITC and DAPI using an
inverted fluorescence microscope (Axio Observer 7, ZEISS, Germany), respectively. An
ImageJ macro (adapted from [29]) was used to quantify cell numbers per image using the
DAPI channel. Additionally, cell shape was qualitatively evaluated on the actin channel,
combined with quantitative determination of nucleus’ circularity and aspect ratio (AR),
being major indications for an elongated cell shape (Figure 2) [30–32]. ImageJ analysis was
performed four times and the mean of each parameter was used for statistical analysis.
Circularit
y
= 4π ×
Aspect Ratio =
(4)
Figure 2. Visualization of nuclear shape on DAPI stained nuclei. If the ratio of the major nuclear
axis to the minor nuclear axis, Table 1, a more elongated shape is seen. Circularity (Circ.) is meas-
ured between 0 and 1, the value being close to 1 represents a rounder cell shape.
2.7. Statistical Analysis
Data are shown as mean (M) ± standard deviation (SD). Statistical analysis was per-
formed with GraphPad Prism, (GraphPad Software; San Diego, CA, USA) and in R Studio
(Version 1.3.1093, RStudio, PBC, Boston, MA, USA). For physicochemical properties one-
way ANOVA tests were executed. Post hoc Tukey’s test displayed pairwise differences
between the conditions. Cell characteristics, i.e., cell numbers, viability and cell morphol-
ogy, were assessed with non-parametric Kruskal−Wallis tests, combined with Dunn’s
multiple comparison test. Reported p-values were corrected for the multiple comparisons
by the Bonferroni correction method.
3. Results
3.1. Materials
3.1.1. Gelatin Modification
Gelatin type B was used as starting material onto which photo-crosslinkable func-
tionalities were introduced along the backbone (Figure 1). The frequently reported gel-
MA was used as a benchmark throughout the present work.
1
H-NMR spectroscopy was
performed to determine the DS of the developed gel-MA and gel-NB (Figure 3). To this
end, either the characteristic peaks of the methacrylamide moieties (5.6 and 5.8 ppm) or
of the norbornene functionalities (6.33 and 6.00 ppm (endo derivative); 6.28 and 6.28 ppm
Figure 2.
Visualization of nuclear shape on DAPI stained nuclei. If the ratio of the major nuclear
axis to the minor nuclear axis, the aspect ratio (AR), is bigger than 1, a more elongated shape is seen.
Circularity (Circ.) is measured between 0 and 1, the value being close to 1 represents a rounder
cell shape.
2.7. Statistical Analysis
Data are shown as mean (M)
±
standard deviation (SD). Statistical analysis was
performed with GraphPad Prism, (GraphPad Software; San Diego, CA, USA) and in R
Studio (Version 1.3.1093, RStudio, PBC, Boston, MA, USA). For physicochemical properties
one-way ANOVA tests were executed. Post hoc Tukey’s test displayed pairwise differences
between the conditions. Cell characteristics, i.e., cell numbers, viability and cell morphology,
were assessed with non-parametric Kruskal
−
Wallis tests, combined with Dunn’s multiple
comparison test. Reported p-values were corrected for the multiple comparisons by the
Bonferroni correction method.
3. Results
3.1. Materials
3.1.1. Gelatin Modification
Gelatin type B was used as starting material onto which photo-crosslinkable function-
alities were introduced along the backbone (Figure 1). The frequently reported gel-MA was
used as a benchmark throughout the present work.
1
H-NMR spectroscopy was performed
to determine the DS of the developed gel-MA and gel-NB (Figure 3). To this end, either the
characteristic peaks of the methacrylamide moieties (5.6 and 5.8 ppm) or of the norbornene
functionalities (6.33 and 6.00 ppm (endo derivative); 6.28 and 6.28 ppm (exo derivative))
were compared to the methyl signals of the side chains of the inert Val, Leu, Ile peak at
1.01 ppm. Gel-MA was successfully developed with a DS of 97%. Furthermore, gel-NB
with both a high DS of 85% and a low DS of 55% were obtained, which was in agreement
with previously reported literature [
21
,
22
]. The DS of gel-SH was determined using an
ortho-phthalic dialdehyde assay, yielding a DS of 75%. These results are in line with earlier
reports, obtaining gel-SH with a DS of 72% [21].
Polymers 2021,13, 747 7 of 16
Polymers 2021, 13, x FOR PEER REVIEW 7 of 16
(exo derivative)) were compared to the methyl signals of the side chains of the inert Val,
Leu, Ile peak at 1.01 ppm. Gel-MA was successfully developed with a DS of 97%. Further-
more, gel-NB with both a high DS of 85% and a low DS of 55% were obtained, which was
in agreement with previously reported literature [21,22]. The DS of gel-SH was deter-
mined using an ortho-phthalic dialdehyde assay, yielding a DS of 75%. These results are
in line with earlier reports, obtaining gel-SH with a DS of 72% [21].
Figure 3. To confirm the degree of substitution,
1
H-NMR spectra were assessed of (A) Gel-MA DS 97%, (B) Gel-NB DS
85%, and (C) Gel-NB DS 55%. Gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin.
3.1.2. Physicochemical properties
An overview of the assessed physicochemical properties of the obtained films is
shown in Figure 4. To gain insight into the amount of potentially harmful precursors
leaching out, which can be harmful to the cells, the gel fraction of the hydrogel films was
determined. As anticipated, films with a higher DS of gel-NB resulted in a higher gel frac-
tion due to its denser crosslinked network. In addition, it was observed that the applied
thiolated crosslinker did not influence the gel fraction, as both the gel-NB85/DTT and gel-
NB85/SH75 resulted in similar gel fractions (87.5 ± 2.4% vs. 89.6 ± 1.8%). However, the gel
fraction of the thiol-ene based films was significantly lower compared to gel-MA (98.8 ±
1.3%), p < 0.0001.
The mass swelling ratio gives an indication of the ability of the hydrogel to mimic the
aqueous cellular environment of the ECM. A significantly lower mass swelling ratio was
observed for gel-MA (13.7 ± 0.5) compared to the thiol-ene based films, p < 0.0001. The
thiol-ene films were characterized by at least a 1.6 times higher swelling ratio, varying
between 22.3 and 25.2. Furthermore, a higher swelling ratio was obtained for gel-NB with
a lower DS compared to a higher DS, p = 0.0246.
Considering the mechanical properties of the hydrogel films, a significantly higher
storage modulus (up to 1.9 times higher) was observed for gel-MA (31.3 ± 3.6 kPa) com-
pared to the thiol-ene films (varying between 16.3–17.7 kPa), p = 0.0007, p = 0002, and p =
Figure 3.
To confirm the degree of substitution,
1
H-NMR spectra were assessed of (
A
) Gel-MA DS 97%, (
B
) Gel-NB DS 85%,
and (C) Gel-NB DS 55%. Gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin.
3.1.2. Physicochemical Properties
An overview of the assessed physicochemical properties of the obtained films is
shown in Figure 4. To gain insight into the amount of potentially harmful precursors
leaching out, which can be harmful to the cells, the gel fraction of the hydrogel films was
determined. As anticipated, films with a higher DS of gel-NB resulted in a higher gel
fraction due to its denser crosslinked network. In addition, it was observed that the applied
thiolated crosslinker did not influence the gel fraction, as both the gel-NB85/DTT and
gel-NB85/SH75 resulted in similar gel fractions (87.5
±
2.4% vs. 89.6
±
1.8%). However,
the gel fraction of the thiol-ene based films was significantly lower compared to gel-MA
(98.8 ±1.3%), p< 0.0001.
The mass swelling ratio gives an indication of the ability of the hydrogel to mimic
the aqueous cellular environment of the ECM. A significantly lower mass swelling ratio
was observed for gel-MA (13.7
±
0.5) compared to the thiol-ene based films, p< 0.0001.
The thiol-ene films were characterized by at least a 1.6 times higher swelling ratio, varying
between 22.3 and 25.2. Furthermore, a higher swelling ratio was obtained for gel-NB with
a lower DS compared to a higher DS, p= 0.0246.
Considering the mechanical properties of the hydrogel films, a significantly higher
storage modulus (up to 1.9 times higher) was observed for gel-MA (31.3
±
3.6 kPa) com-
pared to the thiol-ene films (varying between 16.3–17.7 kPa), p= 0.0007, p= 0002, and
p= 0.0014, respectively. No significant differences were observed between the thiolated
crosslinkers used. Furthermore, as anticipated, there was an inverse correlation between
the obtained swelling ratio of the films and their mechanical properties.
Polymers 2021,13, 747 8 of 16
Polymers 2021, 13, x FOR PEER REVIEW 8 of 16
0.0014, respectively. No significant differences were observed between the thiolated cross-
linkers used. Furthermore, as anticipated, there was an inverse correlation between the
obtained swelling ratio of the films and their mechanical properties.
Figure 4. Physicochemical properties of the obtained hydrogel films: (A) gel fraction, (B) mass swelling ratio, and (C)
mechanical properties of 10 w/v% films containing 2 mol% LAP photoinitiator. Significant differences are shown with
adjusted p-values and error bars representing standard deviation. DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin;
gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; LAP: lithium(2,4,6-trimethylbenzoyl) phe-
nylphosphinate.
3.2. Cell Characteristics
3.2.1. Cell Proliferation
Mechanical properties, structure and composition of the hydrogel are all features
which impact cellular function [13]. To assess cell proliferation on the different hydrogel
compositions, the number of DAPI positive cells per image was determined (Figure 5A).
At day 7, significantly more cells were observed on gel-NB55/DTT (415.54 ± 213.1) when
compared to gel-NB85/DTT (153.44 ± 215.13), p = 0.0052. After 14 days in culture, however,
the number of cells on gel-NB85/DTT (340.42 ± 84.19) was comparable with those on gel-
NB55/DTT (383.08 ± 27.71). Cell numbers measured on gel-MA (187.92 ± 91.55) were sig-
nificantly lower compared to thiol-ene based hydrogels (gel-NB85/SH75: 326.92 ± 80.63),
p < 0.0001, p = 0.0048, and p = 0.0203, respectively. It can therefore be concluded that gel-
Figure 4.
Physicochemical properties of the obtained hydrogel films: (
A
) gel fraction, (
B
) mass swelling ratio, and (
C
) me-
chanical properties of 10 w/v% films containing 2 mol% LAP photoinitiator. Significant differences are shown with adjusted
p-values and error bars representing standard deviation. DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB:
norbornene-functionalized gelatin; gel-SH: thiolated gelatin; LAP: lithium(2,4,6-trimethylbenzoyl) phenylphosphinate.
3.2. Cell Characteristics
3.2.1. Cell Proliferation
Mechanical properties, structure and composition of the hydrogel are all features
which impact cellular function [
13
]. To assess cell proliferation on the different hydrogel
compositions, the number of DAPI positive cells per image was determined (
Figure 5A)
.
At day 7, significantly more cells were observed on gel-NB55/DTT (415.54
±
213.1) when
compared to gel-NB85/DTT (153.44
±
215.13), p= 0.0052. After 14 days in culture, however,
the number of cells on gel-NB85/DTT (340.42
±
84.19) was comparable with those on gel-
NB55/DTT (383.08
±
27.71). Cell numbers measured on gel-MA (187.92
±
91.55) were sig-
nificantly lower compared to thiol-ene based hydrogels (gel-NB85/SH75:
326.92 ±80.63
),
p< 0.0001, p= 0.0048, and p= 0.0203, respectively. It can therefore be concluded that gel-MA
is certainly not optimal to support tenocyte cultures. Gel-NB55/DTT also significantly
differed from TCP (241.57
±
45.87), p= 0.0024, which indicates a superior biocompatibility
of gel-NB55/DTT, especially when considering TCP is specifically designed for cell culture.
Polymers 2021,13, 747 9 of 16
Polymers 2021, 13, x FOR PEER REVIEW 9 of 16
MA is certainly not optimal to support tenocyte cultures. Gel-NB55/DTT also significantly
differed from TCP (241.57 ± 45.87), p = 0.0024, which indicates a superior biocompatibility
of gel-NB55/DTT, especially when considering TCP is specifically designed for cell cul-
ture.
Figure 5. Tenocyte characteristics as determined on thiol-ene based hydrogels and compared to
both TCP and gel-MA: (A) cell proliferation: cell numbers per image are shown, (B) viability assay:
the percentage of the area occupied by living cells is shown in relation to the total cell area cul-
tured per image. Significant differences are shown with adjusted p-values. After 14 days in culture
the number of cells on all thiol-ene based hydrogels were comparable and significantly higher
than on gel-MA. It can therefore be concluded that gel-MA is not optimal to support tenocyte cul-
tures. Gel-NB55/DTT also significantly differed from TCP which indicates superior biocompatibil-
ity of gel-NB55/DTT. Viability on all hydrogels exceeded 95%, indicating excellent biocompatibil-
ity (>70%, ISO 10993-5 (2009)). The decreased viability on gel-NB55/DTT can be explained by con-
tact inhibition. DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-func-
tionalized gelatin; gel-SH: thiolated gelatin; TCP: tissue culture plastic.
3.2.2. Viability Assay
As both living and dead cells are stained with DAPI, an additional fluorescent stain-
ing with Ca-AM/ PI was performed to assess the viability of the equine tenocytes. Viability
Figure 5.
Tenocyte characteristics as determined on thiol-ene based hydrogels and compared to both
TCP and gel-MA: (
A
) cell proliferation: cell numbers per image are shown, (
B
) viability assay: the
percentage of the area occupied by living cells is shown in relation to the total cell area cultured per
image. Significant differences are shown with adjusted p-values. After 14 days in culture the number
of cells on all thiol-ene based hydrogels were comparable and significantly higher than on gel-MA. It
can therefore be concluded that gel-MA is not optimal to support tenocyte cultures. Gel-NB55/DTT
also significantly differed from TCP which indicates superior biocompatibility of gel-NB55/DTT.
Viability on all hydrogels exceeded 95%, indicating excellent biocompatibility (>70%, ISO 10993-5
(2009)). The decreased viability on gel-NB55/DTT can be explained by contact inhibition. DTT:
1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH:
thiolated gelatin; TCP: tissue culture plastic.
3.2.2. Viability Assay
As both living and dead cells are stained with DAPI, an additional fluorescent staining
with Ca-AM/ PI was performed to assess the viability of the equine tenocytes. Viability
on all hydrogels exceeded 95%, indicating excellent biocompatibility (ISO 10993-5 (2009)).
It must be mentioned however that the viability for gel-NB55/DTT (97.65%
±
2.24) and
gel-NB85/SH75 (97.16%
±
1.83) was significantly lower at 14 days when compared to
gel-MA (99.47% ±0.52), p= 0.0156, and p= 0.0011, respectively (Figure 5B).
Polymers 2021,13, 747 10 of 16
3.2.3. Cell Morphology
To confirm the tenogenic phenotype, morphological characterization is usually per-
formed as no unique tendon marker has been identified yet [
11
]. Tenocytes should be
spindle-shaped, elongated and organized in a parallel fashion [
33
]. Qualitative evaluation
of the fluorescent images however showed a rounder cell morphology on gel-MA discs
and an intermediate morphology, i.e., between round and spindle-shaped, when cultured
on TCP and gel-NB85/SH75 (Figure 6).
Polymers 2021, 13, x FOR PEER REVIEW 10 of 16
on all hydrogels exceeded 95%, indicating excellent biocompatibility (ISO 10993-5 (2009)).
It must be mentioned however that the viability for gel-NB55/DTT (97.65% ± 2.24) and gel-
NB85/SH75 (97.16% ± 1.83) was significantly lower at 14 days when compared to gel-MA
(99.47% ± 0.52), p = 0.0156, and p = 0.0011, respectively (Figure 5B).
3.2.3. Cell Morphology
To confirm the tenogenic phenotype, morphological characterization is usually per-
formed as no unique tendon marker has been identified yet [11]. Tenocytes should be
spindle-shaped, elongated and organized in a parallel fashion [33]. Qualitative evaluation
of the fluorescent images however showed a rounder cell morphology on gel-MA discs
and an intermediate morphology, i.e., between round and spindle-shaped, when cultured
on TCP and gel-NB85/SH75 (Figure 6).
Figure 6. Representative images of the qualitative evaluation of tenocyte morphology and cell alignment based on fluo-
rescent staining of the actin filaments (orange) with DAPI nuclear counterstaining (blue). Experiment was performed in
triplicate. Tenocytes should be spindle-shaped, elongated and organized in a parallel fashion (as seen on gel-NB55/DTT
days 7 and 14, and gel-NB85/DTT day 14). A rounder cell morphology on gel-MA discs is seen (both timepoints) and an
intermediate morphology (between round and spindle-shaped) on TCP and gel-NB85/SH75 (both timepoints). DTT: 1,4-
dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; TCP:
tissue culture plastic.
To confirm these findings, the nuclear shape was analyzed, as it is representative for
cell shape [30,31]. If the ratio of the major nuclear axis to the minor nuclear axis, the so-
called AR, is bigger than 1, this is indicative for a more elongated nuclear shape (Figure
2). A significantly higher AR was measured for gel-NB55/DTT (1.71 ± 0.11) at day 7 com-
pared to the other materials evaluated (TCP: 1.41 ± 0.10; gel-MA: 1.44 ± 0.07; gel-
NB85/DTT: 1.40 ± 0.20), p = 0.0004, p = 0.0052, and p = 0.0009, respectively (Figure 7A). At
day 14, a significantly lower AR was measured for gel-MA (1.40 ± 0.07) when compared
to other hydrogels (gel-NB85/DTT: 1.90 ± 0.12; gel-NB55/DTT: 1.87 ± 0.16; gel-NB85/SH75:
1.77 ± 0.17), p < 0.0001, p < 0.0001, and p = 0.0031, respectively. Circularity, indicated by a
Figure 6.
Representative images of the qualitative evaluation of tenocyte morphology and cell alignment based on
fluorescent staining of the actin filaments (orange) with DAPI nuclear counterstaining (blue). Experiment was performed in
triplicate. Tenocytes should be spindle-shaped, elongated and organized in a parallel fashion (as seen on gel-NB55/DTT
days 7 and 14, and gel-NB85/DTT day 14). A rounder cell morphology on gel-MA discs is seen (both timepoints) and
an intermediate morphology (between round and spindle-shaped) on TCP and gel-NB85/SH75 (both timepoints). DTT:
1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; TCP:
tissue culture plastic.
To confirm these findings, the nuclear shape was analyzed, as it is representative
for cell shape [
30
,
31
]. If the ratio of the major nuclear axis to the minor nuclear axis,
the so-called AR, is bigger than 1, this is indicative for a more elongated nuclear shape
(Figure 2)
. A significantly higher AR was measured for gel-NB55/DTT (1.71
±
0.11) at day
7 compared to the other materials evaluated (TCP: 1.41
±
0.10; gel-MA: 1.44
±
0.07; gel-
NB85/DTT: 1.40
±
0.20), p= 0.0004, p= 0.0052, and p= 0.0009, respectively
(Figure 7A)
. At
day 14, a significantly lower AR was measured for gel-MA (1.40
±
0.07) when compared to
other hydrogels (gel-NB85/DTT: 1.90
±
0.12; gel-NB55/DTT: 1.87
±
0.16; gel-NB85/SH75:
1.77 ±0.17)
,p< 0.0001, p< 0.0001, and p= 0.0031, respectively. Circularity, indicated by
a value close to 1, represents an aberrant cell shape when discussing tenocytes
(Figure 2)
.
After 7 days, cells cultured on TCP (0.77
±
0.02) displayed a significantly rounder nuclear
shape than cells on gel-MA (0.70
±
0.05), gel-NB85/DTT (0.65
±
0.11) and gel-NB55/DTT
(0.70
±
0.04), p= 0.0154, p= 0.0018, and p= 0.0008, respectively (Figure 7B). On the other
Polymers 2021,13, 747 11 of 16
hand, circularity measured at day 14 was significantly lower for all thiol-ene based films
(gel-NB85/DTT: 0.65
±
0.02; gel-NB55/DTT: 0.64
±
0.02; gel-NB85/SH75: 0.69
±
0.04)
compared to gel-MA (0.76
±
0.03), p< 0.0001, p< 0.0001, and p= 0.0165, respectively. In
addition, cells cultured on gel-NB85/DTT and gel-NB55/DTT displayed a significantly
more elongated cell shape when compared to TCP (0.73
±
0.03), p= 0.0125 and p= 0.0047. In
conclusion, qualitative assessment, AR and circularity data showed a favorable morphology
of tenocytes when cultured on thiol-ene based films, especially for gel-NB85 and gel-NB55
combined with the bifunctional thiolated crosslinker DTT, while tenocytes cultured on TCP
and gel-MA showed a less desirable morphology.
Polymers 2021, 13, x FOR PEER REVIEW 11 of 16
value close to 1, represents an aberrant cell shape when discussing tenocytes (Figure 2).
After 7 days, cells cultured on TCP (0.77 ± 0.02) displayed a significantly rounder nuclear
shape than cells on gel-MA (0.70 ± 0.05), gel-NB85/DTT (0.65 ± 0.11) and gel-NB55/DTT
(0.70 ± 0.04), p = 0.0154, p = 0.0018, and p = 0.0008, respectively (Figure 7B). On the other
hand, circularity measured at day 14 was significantly lower for all thiol-ene based films
(gel-NB85/DTT: 0.65 ± 0.02; gel-NB55/DTT: 0.64 ± 0.02; gel-NB85/SH75: 0.69 ± 0.04) com-
pared to gel-MA (0.76 ± 0.03), p < 0.0001, p < 0.0001, and p = 0.0165, respectively. In addi-
tion, cells cultured on gel-NB85/DTT and gel-NB55/DTT displayed a significantly more
elongated cell shape when compared to TCP (0.73 ± 0.03), p = 0.0125 and p = 0.0047. In
conclusion, qualitative assessment, AR and circularity data showed a favorable morphol-
ogy of tenocytes when cultured on thiol-ene based films, especially for gel-NB85 and gel-
NB55 combined with the bifunctional thiolated crosslinker DTT, while tenocytes cultured
on TCP and gel-MA showed a less desirable morphology.
Figure 7. Tenocyte characteristics as determined on thiol-ene based hydrogels and compared to
both TCP and gel-MA: (A) nuclear aspect ratio (AR): ratio of the major nuclear axis to the minor
nuclear axis. An elongated cell shape is represented by a higher aspect ratio, and (B) circularity: a
perfect circle has a value of 1. Significant differences are shown with adjusted p-values. Favorable
Figure 7.
Tenocyte characteristics as determined on thiol-ene based hydrogels and compared to
both TCP and gel-MA: (
A
) nuclear aspect ratio (AR): ratio of the major nuclear axis to the minor
nuclear axis. An elongated cell shape is represented by a higher aspect ratio, and (
B
) circularity: a
perfect circle has a value of 1. Significant differences are shown with adjusted p-values. Favorable
tenocyte characteristics are high AR and low circularity, as expressed on all thiol-ene based hydrogels,
most clearly pronounced on day 14. DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB:
norbornene-functionalized gelatin; gel-SH: thiolated gelatin; TCP: tissue culture plastic.
Polymers 2021,13, 747 12 of 16
4. Discussion
As it is generally accepted that standard cell culture materials lack biomimetic capac-
ities, current research is focusing on the evaluation of different biomaterials, especially
when considering specific applications [
13
]. Parameters which are evaluated include cor-
rect biochemical composition and structure, biocompatibility towards appropriate cell
populations, and appropriate mechanical strength and elasticity [12,34,35].
Indeed, physicochemical characteristics, mechanical properties, and cell characteristics
are intrinsically linked. A significantly lower mass swelling ratio was observed for gel-MA
discs, which resulted in inferior cell characteristics, as could be observed in Figures 5A and 7
(i.e., low cell proliferation, lower AR and higher circularity). The effect of the swelling ratio
has been demonstrated before on chondrogenic differentiation. Mesenchymal stem cells
were encapsulated in different hydrogel compositions showing a superior chondrogenic
differentiation for the hydrogel compositions with a higher swelling ratio [
36
]. The higher
mass swelling ratio of the thiol-ene discs indeed showed superior cell proliferation (day 14)
and morphology (higher AR and lower circularity), indicating that the aqueous ECM
environment was well mimicked using both biomaterials. The lower swelling properties of
Gel-MA can be attributed to the hydrophobic oligo-methacrylamide chains present in the
crosslinked gel-MA network [
37
]. The higher swelling ratio obtained for gel-NB55/DTT can
be explained by the fact that a lower DS results in a less densely crosslinked network [
38
].
Moreover, since norbornene is hydrophobic, a higher DS results in less swelling. The results
are in agreement with previous literature reports, describing a lower mass swelling ratio of
gel-MA hydrogels compared to thiol-ene films [17,21,39].
Both
in vitro
and
in vivo
, tenocytes respond to mechanical stimuli via cell
−
cell and
cell
−
matrix interactions, and mechano-transduction pathways [
7
,
40
]. When tenocytes are
exposed to appropriate mechanical stimulation, they arrange in a parallel fashion [
41
]. In
the study of Rowlands et al., the effect of mechanical stiffness was evaluated by expos-
ing mesenchymal stem cells to different acrylamide substrates, with increasing stiffness,
of which the surface was modified using various proteins, i.e., collagen I, collagen IV,
fibronectin, and laminin. Osteogenic differentiation was significantly more pronounced
on stiffer materials (80 kPa), regardless of the protein surface used, whereas myogenic
differentiation was affected by the protein coating and was observed on both stiff (80 kPa)
and soft materials (25 kPa) [
42
]. As tenocytes are specialized fibroblasts, just as myoblasts,
a similar response of tenocytes on gelatin might be expected. Another research group
reported the highest expression of myogenic markers by mesenchymal stem cells when
cells were cultured on 11kPa hydrogels, while 34 kPa gels are preferred for osteogenic
differentiation [
43
]. Thiol-ene based hydrogels with lower storage moduli (16.3–17.7 kPa)
also resulted in favorable cell characteristics in this study. Furthermore, a higher stiffness
for chain-growth hydrogels (such as gel-MA) has been described in literature due to the
presence of nondegradable kinetic chains of an uncontrolled length [21,37,39].
Although gel-MA hydrogels were characterized by a higher storage modulus (31.3 kPa),
usually desirable for tendon tissue engineering [
44
], this resulted in both an inferior biocom-
patibility and cell morphology. It could therefore be hypothesized that the heterogeneous
nature of the chain growth polymerization resulted in different mechanical cues throughout
the construct which were less preferred by the tenocytes. This can be substantiated by the
cells’ morphology and alignment seen in Figure 6as the tenocytes seemed more clustered
compared to the other compositions. It can be concluded that the more homogeneous step
growth polymerization could offer additional beneficial results irrespective of the lower
storage moduli.
In general, cell proliferation curves are sigmoidal. Immediately after seeding cells, a
lag phase is observed for approximately 48 h, depending on the cell type, during which
cells adapt to the new culture conditions [
45
]. Subsequently, cells enter an exponential
growth phase, a so-called log phase. For human tenocytes, a lag phase of approximately
4 days was reported on TCP [
46
]. The lower cell numbers observed for gel-NB85/DTT at
day 7 might be explained by an extended lag phase. However, an initial cytotoxic effect of
Polymers 2021,13, 747 13 of 16
DTT could also explain the results observed. The latter is in agreement with the literature,
reporting on a potential cytotoxic effect of DTT especially for encapsulated cells [
39
,
47
].
These results can be substantiated by a gel fraction <90% and the lower cell density present
at day 7, as shown in Figure 6. Nevertheless, due to the favorable material properties of
gel-NB85/DTT (e.g., high swelling ratio), cells are able to proliferate and obtain a similar
cell density during the log phase of the other constructs, indicating that any undesirable
effects only occurred in the initial phase.
The results of the viability assay support the characteristic biocompatibility of natural
biomaterials, e.g., gelatin [
16
]. The decreased viability observed (Figure 5B) comparing
day 7 to day 14 when using gel-NB55/DTT can be explained by contact inhibition, a
well-known process in which cells stop proliferating when confluency is reached [
48
]. As
already mentioned, tenocytes cultured on gel-NB55/DTT proliferated faster, and, as such,
reached confluency faster when compared to the other biomaterials evaluated in this study.
Furthermore, the high proliferation rate of tenocytes cultured on gel-NB55/DTT, creating a
higher cell density, also resulted in a favorable cell morphology [49].
Tenocyte dedifferentiation, indicating that tenocytes lose their typical morphology and
eventually their functionality [
46
], is frequently reported for TCP cell cultures. The current
experiment strengthened this statement by the detrimental nuclear shape on TCP (low AR
and high circularity). This is possibly explained by the unphysiologically stiff characteristics
of TCP [
13
]. In contrast, the elongated nuclear shape (high AR and low circularity) on
thiol-ene based hydrogels can be explained not only by its favorable physicochemical
properties, but furthermore by the tripeptide arginine-glycine-aspartic acid sequence
present in gelatin, which was facilitating cell adherence and therefore cell spreading by
connecting with integrins in the cell membrane [16,49].
In this study, there were two limitations which would be interesting to evaluate in
future research. First, an advanced technique such as qPCR or immunostaining might
be used to further evaluate cell functionality. Second, the effect of cell encapsulation
should be verified. Cell encapsulation is more physiologically relevant than seeding the
cells on the biomaterial and as such, more appropriate for assessing both the advantages
and the disadvantages of the individual biomaterials in relation to tenocyte culture [
50
].
The current study focused on the evaluation of different chemically crosslinked gelatin
derivatives (i.e., realized via step growth versus chain growth polymerization) and their
potential to support tenocyte adhesion, viability and proliferation. Assessing functionality
and encapsulating tenocytes in future experiments will improve our knowledge in order
to identify the preferred gelatin derivative which can be combined with tenocytes in a
3D-environment. From this study, it is clear that gel-MA is not eligible as an appropriate
biomaterial to support the culture of equine tenocytes.
5. Conclusions
In the present study, the physicochemical characteristics, mechanical properties, and
cell characteristics of four gelatin hydrogels were evaluated. The thiol-ene based hydro-
gels showed a significantly lower gel fraction/storage modulus and a higher swelling
ratio compared to gel-MA. Although gel-MA is frequently used as the gold standard for
tissue engineering purposes, detrimental tenocyte characteristics support the search for
alternative biomaterials. Thiol-ene crosslinked gelatins exploiting DTT as a crosslinker
(gel-NB85/DTT and gel-NB55/DTT) emerged as the preferred biomaterials to culture
tenocytes when considering both their physicochemical and the cell characteristics. The
current research improves our knowledge on the interaction between natural biomaterials
and tenocytes, which is essential to establish a representative tendon model.
Polymers 2021,13, 747 14 of 16
Author Contributions:
Conceptualization, M.M., L.V.D., S.V.V. and C.D.S.; data curation, M.M. and
L.V.D.; formal analysis, M.M., L.V.D. and W.D.S.; funding acquisition, S.V.V. and C.D.S.; investigation,
M.M. and L.V.D.; methodology, M.M., L.V.D. and W.D.S.; project administration, S.V.V. and C.D.S.;
supervision, S.V.V. and C.D.S.; visualization, M.M. and L.V.D.; writing—original draft, M.M. and
L.V.D.; writing—review and editing, W.D.S., S.V.V. and C.D.S. All authors have read and agreed to
the published version of the manuscript.
Funding:
Marguerite Meeremans was funded by a BOF starting grant provided by Ghent University
to Prof. Catharina De Schauwer. The research was partly funded by the Research Foundation—
Flanders (FWO, application nr: I003318N)
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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