ArticlePDF Available

Equine Tenocyte Seeding on Gelatin Hydrogels Improves Elongated Morphology

Authors:

Abstract and Figures

(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 characteristics 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 (1H-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.
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
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
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 [1214]. 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 KruskalWallis 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).
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.
References
1.
Carpenter, J.E.; Hankenson, K.D. Animal models of tendon and ligament injuries for tissue engineering applications. Biomaterials
2004,25, 1715–1722. [CrossRef]
2.
Spaas, J.H.; Guest, D.J.; van de Walle, G.R. Tendon Regeneration in Human and Equine Athletes. Sport. Med.
2012
,42, 871–890.
[CrossRef] [PubMed]
3.
Burk, J. Mechanisms of Action of Multipotent Mesenchymal Stromal Cells in Tendon Disease. In Tendons; IntechOpen: London,
UK, 2019.
4.
Richardson, L.E.; Dudhia, J.; Clegg, P.D.; Smith, R. Stem cells in veterinary medicine—Attempts at regenerating equine tendon
after injury. Trends Biotechnol. 2007,25, 409–416. [CrossRef] [PubMed]
5.
Veronesi, F.; Salamanna, F.; Tschon, M.; Maglio, M.; Aldini, N.N.; Fini, M. Mesenchymal stem cells for tendon healing: What is on
the horizon? J. Tissue Eng. Regen. Med. 2017,11, 3202–3219. [CrossRef] [PubMed]
6.
Patel, D.; Sharma, S.; Bryant, S.J.; Screen, H.R.C. Recapitulating the Micromechanical Behavior of Tension and Shear in a
Biomimetic Hydrogel for Controlling Tenocyte Response. Adv. Healthc. Mater. 2017,6, 1–7. [CrossRef] [PubMed]
7.
Wang, T.; Chen, P.; Zheng, M.; Wang, A.; Lloyd, D.; Leys, T.; Zheng, Q.; Zheng, M.H.
In vitro
loading models for tendon
mechanobiology. J. Orthop. Res. 2017,36, 566–575. [CrossRef] [PubMed]
8.
Laternser, S.; Keller, H.; Leupin, O.; Rausch, M.; Graf-Hausner, U.; Rimann, M. A Novel Microplate 3D Bioprinting Platform for
the Engineering of Muscle and Tendon Tissues. SLAS Technol. 2018,23, 599–613. [CrossRef] [PubMed]
9.
Patterson-Kane, J.C.; Becker, D.L.; Rich, T. The Pathogenesis of Tendon Microdamage in Athletes: The Horse as a Natural Model
for Basic Cellular Research. J. Comp. Pathol. 2012,147, 227–247. [CrossRef] [PubMed]
10.
Dyment, N.A.; Barrett, J.G.; Awad, H.A.; Bautista, C.A.; Banes, A.J.; Butler, D.L. A brief history of tendon and ligament bioreactors:
Impact and future prospects. J. Orthop. Res. 2012. [CrossRef] [PubMed]
11.
Tan, S.; Selvaratnam, L.; Ahmad, T. A Mini Review on the Basic Knowledge on Tendon: Revisiting the Normal & Injured Tendon.
J. Health Transl. Med. 2015,18, 12–25.
12.
Kuo, C.K.; Marturano, J.E.; Tuan, R.S. Novel strategies in tendon and ligament tissue engineering: Advanced biomaterials and
regeneration motifs. BMC Sports Sci. Med. Rehabil. 2010,2, 20. [CrossRef] [PubMed]
13. Caliari, S.R.; Burdick, J.A. A practical guide to hydrogels for cell culture. Nat. Methods 2016,13, 405–414. [CrossRef] [PubMed]
14.
Wu, Y.; Han, Y.; Wong, Y.S.; Fuh, J.Y.H. Fibre-based scaffolding techniques for tendon tissue engineering. J. Tissue Eng. Regen.
Med. 2018,12, 1798–1821. [CrossRef] [PubMed]
15. Mũnoz, Z.; Shih, H.; Lin, C.C. Gelatin hydrogels formed by orthogonal thiol-norbornene photochemistry for cell encapsulation.
Biomater. Sci. 2014,2, 1063–1072. [CrossRef]
16. Ruoslahti, E. RGD and other recognition sequences for intergins. Annu. Rev. Cell Dev. Biol. 1996,12, 697–715. [CrossRef]
17.
Van Hoorick, J.; Tytgat, L.; Dobos, A.; Ottevaere, H.; Van Erps, J.; Thienpont, H.; Ovsianikov, A.; Dubruel, P.; Van Vlierberghe, S.
(Photo-)crosslinkable Gelatin Derivatives for Biofabrication Applications. 2019. Available online: http://www.tissue- regeneration.
at (accessed on 10 September 2020).
18.
Occhetta, P.; Visone, R.; Russo, L.; Cipolla, L.; Moretti, M.; Rasponi, M. VA-086 methacrylate gelatine photopolymerizable
hydrogels: A parametric study for highly biocompatible 3D cell embedding. J. Biomed. Mater. Res. Part A
2015
,103, 2109–2117.
[CrossRef]
19.
Rinoldi, C.; Costantini, M.; Kije ´nska-Gawro´nska, E.; Testa, S.; Fornetti, E.; Heljak, M.; ´
Cwikli ´nska, M.; Buda, R.; Baldi, J.; Cannata,
S.; et al. Tendon Tissue Engineering: Effects of Mechanical and Biochemical Stimulation on Stem Cell Alignment on Cell-Laden
Hydrogel Yarns. Adv. Healthc. Mater. 2019,8, 1801218. [CrossRef] [PubMed]
20.
Ramos, D.M.; Abdulmalik, S.; Arul, M.R.; Rudraiah, S.; Laurencin, C.T.; Mazzocca, A.D.; Kumbar, S.G. Insulin immobilized
PCL-cellulose acetate micro-nanostructured fibrous scaffolds for tendon tissue engineering. Polym. Adv. Technol.
2019
,30,
1205–1215. [CrossRef]
Polymers 2021,13, 747 15 of 16
21.
Tytgat, L.; Van Damme, L.; Van Hoorick, J.; Declercq, H.; Thienpont, H.; Ottevaere, H.; Blondeel, P.; Dubruel, P.; Van Vlierberghe,
S. Additive manufacturing of photo-crosslinked gelatin scaffolds for adipose tissue engineering. Acta Biomater.
2019
,94, 340–350.
[CrossRef]
22.
Van Hoorick, J.; Gruber, P.; Markovic, M.; Rollot, M.; Graulus, G.-J.; Vagenende, M.; Tromayer, M.; Van Erps, J.; Thienpont, H.;
Martins, J.C.; et al. Highly Reactive Thiol-Norbornene Photo-Click Hydrogels: Toward Improved Processability. Macromol. Rapid
Commun. 2018,39, 1800181. [CrossRef] [PubMed]
23.
Spicer, C.D. Hydrogel scaffolds for tissue engineering: The importance of polymer choice. Polym. Chem.
2020
,11, 184–219.
[CrossRef]
24.
Markovic, M.; van Hoorick, J.; Hölzl, K.; Tromayer, M.; Gruber, P. Hybrid tissue engineering scaffolds by combination of 3D
printing and cell photoencapsulation Hybrid Tissue Engineering Scaffolds by Combination of Three-Dimensional Printing and
Cell Photoencapsulation. J. Nanotechnol. Eng. Med. 2015,6, 0210011–0210017. [CrossRef] [PubMed]
25.
van den Bulcke, A.I.; Bogdanov, B.; de Rooze, N.; Schacht, E.H.; Cornelissen, M.; Berghmans, H. Structural and rheological
properties of methacrylamide modified gelatin hydrogels. Biomacromolecules 2000,1, 31–38. [CrossRef] [PubMed]
26.
Van Vlierberghe, S.; Schacht, E.; Dubruel, P. Reversible gelatin-based hydrogels: Finetuning of material properties. Eur. Polym. J.
2011,47, 1039–1047. [CrossRef]
27.
Lange-Consiglio, A.; Perrini, C.; Tasquier, R.; Deregibus, M.C.; Camussi, G.; Pascucci, L.; Marini, M.G.; Corradetti, B.; Bizzaro, D.;
De Vita, B.; et al. Equine Amniotic Microvesicles and Their Anti-Inflammatory Potential in a Tenocyte Model in Vitro. Stem Cells
Dev. 2016,25, 610–621. [CrossRef] [PubMed]
28.
De Schauwer, C.; Meyer, E.; Cornillie, P.; De Vliegher, S.; Van De Walle, G.R.; Hoogewijs, M.; Declercq, H.; Govaere, J.;
Demeyere, K.; Cornelissen, M.; et al. Optimization of the Isolation, Culture, and Characterization of Equine Umbilical Cord Blood
Mesenchymal Stromal Cells. Tissue Eng. Part C Methods 2011,17, 1061–1070. [CrossRef]
29.
Handala, L.; Fiore, T.; Rouillé, Y.; Helle, F. QuantIF: An ImageJ Macro to automatically determine the percentage of infected cells
after immunofluorescence. Viruses 2019,11, 165. [CrossRef]
30.
Skinner, B.M.; Johnson, E.E.P. Nuclear morphologies: Their diversity and functional relevance. Chromosoma
2017
,126, 195–212.
[CrossRef] [PubMed]
31.
Patterson-Kane, J.C.; Firth, E.C. Tendon, ligament, bone, and cartilage: Anatomy, physiology, and adaptations to exercise and
training. In The Athletic Horse: Principles and Practice of Equine Sports Medicine, 2nd ed.; Elsevier Inc.: Amsterdam, The Netherlands,
2014; pp. 202–242.
32.
Newman, P.; Galenano-Niño, J.L.; Graney, P.; Razal, J.M.; Minett, A.I.; Ribas, J.; Ovalle-Robles, R.; Biro, M.; Zreiqat, H. Relationship
between nanotopographical alignment and stem cell fate with live imaging and shape analysis. Sci. Rep.
2016
,6, 1–11. [CrossRef]
[PubMed]
33.
Roth, S.P.; Schubert, S.; Scheibe, P.; Groß, C.; Brehm, W.; Burk, J. Growth Factor-Mediated Tenogenic Induction of Multipotent
Mesenchymal Stromal Cells Is Altered by the Microenvironment of Tendon Matrix. Cell Transplant.
2018
,27, 1434–1450. [CrossRef]
[PubMed]
34. Ma, P.X. Scaffolds for tissue fabrication. Mater. Today 2004,7, 30–40. [CrossRef]
35.
Rodrigues, M.T.; Reis, R.L.; Gomes, M.E. Engineering tendon and ligament tissues: Present developments towards successful
clinical products. J. Tissue Eng. Regen. Med. 2013,7, 673–686. [CrossRef] [PubMed]
36.
Park, H.; Guo, X.; Temenoff, J.; Kasper, F.; Mikos, A. Effect of Swelling Ratio of Intectable Hydrogel Composites on Chondrongenix
Differentiation of Encapsculated Rabbit Marrow Mesenchymal Stem Cells In Vitro. Biomacromolecules
2010
,10, 541–546. [CrossRef]
[PubMed]
37.
Bertlein, S.; Brown, G.; Lim, K.S.; Jungst, T.; Boeck, T.; Blunk, T.; Tessmar, J.; Hooper, G.J.; Woodfield, T.B.F.; Groll, J. Thiol-Ene
Clickable Gelatin: A Platform Bioink for Multiple 3D Biofabrication Technologies. Adv. Mater.
2017
,29, 1703404. [CrossRef]
[PubMed]
38.
Shih, H.; Greene, T.; Korc, M.; Lin, C.C. Modular and Adaptable Tumor Niche Prepared from Visible Light Initiated Thiol-
Norbornene Photopolymerization. Biomacromolecules 2016,17, 3872–3882. [CrossRef] [PubMed]
39.
Van Hoorick, J.; Dobos, A.; Markovic, M.; Gheysens, T.; Van Damme, L.; Gruber, P.; Tytgat, L.; Van Erps, J.; Thienpont, H.; Dubruel,
P.; et al. Thiol-norbornene gelatin hydrogels: Influence of thiolated crosslinker on network properties and high definition 3D
printing. Biofabrication 2020,13, 015017. [CrossRef] [PubMed]
40.
Govoni, M.; Berardi, A.C.; Muscari, C.; Campardelli, R.; Bonafè, F.; Guarnieri, C.; Reverchon, E.; Giordano, E.; Maffulli, N.; Della
Porta, G. An Engineered Multiphase Three-Dimensional Microenvironment to Ensure the Controlled Delivery of Cyclic Strain
and Human Growth Differentiation Factor 5 for the Tenogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells.
Tissue Eng. Part A 2017,23, 811–822. [CrossRef] [PubMed]
41.
Egerbacher, M.; Gabner, S.; Battisti, S.; Handschuh, S. Tenocytes form a 3-D network and are connected via nanotubes. J. Anat.
2020,236, 165–170. [CrossRef] [PubMed]
42.
Rowlands, A.S.; George, P.A.; Cooper-White, J.J. Directing osteogenic and myogenic differentiation of MSCs: Interplay of stiffness
and adhesive ligand presentation. Am. J. Physiol. Cell Physiol. 2008,295, 1037–1044. [CrossRef] [PubMed]
43.
Engler, A.J.; Sen, S.; Sweeney, H.L.; Discher, D.E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell
2006
,126, 677–689.
[CrossRef] [PubMed]
Polymers 2021,13, 747 16 of 16
44.
Liu, Y.; Ramanath, H.S.; Wang, D.A. Tendon tissue engineering using scaffold enhancing strategies. Trends Biotechnol.
2008
,26,
201–209. [CrossRef] [PubMed]
45.
Freshney, R.I. Basic Principles of Cell Culture. In Culture of Cells for Tissue Engineering; John Wiley & Sons, Inc.: Hoboken, NJ,
USA, 2006; pp. 1–22.
46.
Yao, L.; Bestwick, C.S.; Bestwick, L.A.; Maffulli, N.; Aspden, R.M. Phenotypic drift in human tenocyte culture. Tissue Eng.
2006
,
12, 1843–1849. [CrossRef] [PubMed]
47.
Shih, H.; Chien-Chi, L. Photo-click hydrogels prepared from functionalized cyclodextrin and poly(ethylene glycol) for drug
delivery and in situ cell encapsulation. Biomacromolecules 2015,16, 1915–1923. [CrossRef] [PubMed]
48.
Pavel, M.; Renna, M.; Park, S.J.; Menzies, F.M.; Ricketts, T.; Füllgrabe, J.; Ashkenazi, A.; Frake, R.A.; Lombarte, A.C.; Bento,
C.F.; et al. Contact inhibition controls cell survival and proliferation via YAP/TAZ-autophagy axis. Nat. Commun.
2018
,9, 1–18.
[CrossRef] [PubMed]
49.
McBeath, R.; Pirone, D.M.; Nelson, C.M.; Bhadriraju, K.; Chen, C.S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell
lineage commitment. Dev. Cell 2004,6, 483–495. [CrossRef]
50.
Waheed, A.; Mazumder, M.A.J.; Al-Ahmed, A.; Roy, P.; Ullah, N. Cell Encapsulation; Springer: Cham, Switzerland, 2019;
pp. 377–427.
... Both tenocytes and MSCs were harvested from a 15year-old Arabian horse. Equine tenocytes were isolated from the superficial digital flexor tendon using 0.1% collagenase type Ia digestion in high glucose DMEM [33]. MSCs were collected from abdominal adipose tissue using 0.1% liberase digestion in low glucose DMEM. ...
Article
Full-text available
Hand tendon injuries represent a major clinical problem and might dramatically diminish a patient’s life quality. In this study, a targeted solution for flexor tendon repair was developed by combining a mechanical and biological approach. To this end, a novel acrylate-endcapped urethane-based polymer (AUP) was synthesized and its physico-chemical properties were characterized. Next, tubular repair constructs were developed using electrospinning of the AUP material with incorporated naproxen and hyaluronic acid (i.e. anti-inflammatory and anti-adhesion compounds, respectively), and with a tubular braid as mechanical reinforcement. Tensile testing of the repair constructs using ex vivo sheep tendons showed that the developed repair constructs fulfilled the required mechanical properties for tendon repair (i.e. minimal ultimate stress of 4 MPa), with an ultimate stress of 6.4 ± 0.6 MPa. Moreover, in vitro biological assays showed that the developed repair tubes and the incorporated bioactive components were non-cytotoxic. In addition, when equine tenocytes and mesenchymal stem cells were co-cultured with the repair tubes, an increased production of collagen and non-collagenous proteins was observed. In conclusion, this novel construct in which a mechanical approach (fulfilling the required mechanical properties) was combined with a biological approach (incorporation of bioactive compounds), shows potential as flexor tendon repair application. Graphical abstract
... Other advantages are low cost and wide availability, especially when considering large-scale in vitro studies (Van Hoorick et al., 2019). Furthermore, we recently demonstrated the excellent biocompatibility of cross-linked gelatin (gelatin-methacrylamide and gelatin-norbornene) to support equine tenocyte cultures (Meeremans et al., 2021). Silk fibroin is a worthy alternative to collagen and gelatin for tendon and ligament tissue engineering and is collected from silkworms, mostly Bombyx mori. ...
Article
Overuse tendon injuries are a major cause of musculoskeletal morbidity in both human and equine athletes, due to the cumulative degenerative damage. These injuries present significant challenges as the healing process often results in the formation of inferior scar tissue. The poor success with conventional therapy supports the need to search for novel treatments to restore functionality and regenerate tissue as close to native tendon as possible. Mesenchymal stem cell (MSC)-based strategies represent promising therapeutic tools for tendon repair in both human and veterinary medicine. The translation of tissue engineering strategies from basic research findings, however, into clinical use has been hampered by the limited understanding of the multifaceted MSC mechanisms of action. In vitro models serve as important biological tools to study cell behavior, bypassing the confounding factors associated with in vivo experiments. Controllable and reproducible in vitro conditions should be provided to study the MSC healing mechanisms in tendon injuries. Unfortunately, no physiologically representative tendinopathy models exist to date. A major shortcoming of most currently available in vitro tendon models is the lack of extracellular tendon matrix and vascular supply. These models often make use of synthetic biomaterials, which do not reflect the natural tendon composition. Alternatively, decellularized tendon has been applied, but it is challenging to obtain reproducible results due to its variable composition, less efficient cell seeding approaches and lack of cell encapsulation and vascularization. The current review will overview pros and cons associated with the use of different biomaterials and technologies enabling scaffold production. In addition, the characteristics of the ideal, state-of-the-art tendinopathy model will be discussed. Briefly, a representative in vitro tendinopathy model should be vascularized and mimic the hierarchical structure of the tendon matrix with elongated cells being organized in a parallel fashion and subjected to uniaxial stretching. Incorporation of mechanical stimulation, preferably uniaxial stretching may be a key Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 May 2021 | Volume 9 | Article 651164 Meeremans et al. In vitro Tendon Models element in order to obtain appropriate matrix alignment and create a pathophysiological model. Together, a thorough discussion on the current status and future directions for tendon models will enhance fundamental MSC research, accelerating translation of MSC therapies for tendon injuries from bench to bedside.
Article
Full-text available
Photocrosslinkable gelatin hydrogels are excellent bioinks or biomaterial ink components to serve biofabrication applications. Especially the widely investigated gelatin-methacroyl hydrogels hold an impressive track record. However, over the past decade, increasing attention is being paid to thiol-ene photo-click chemistry to obtain hydrogel networks benefitting from a faster reactivity (i.e. seconds vs minutes) along with superior biocompatibility and processability. In order to exploit this photo-click chemistry, often an ene-functionality (e.g.. Norbornene) is introduced onto gelatin followed by crosslinking in the presence of a multifunctional thiol (e.g.. DTT). To date, very limited research has been performed on the influence of the applied thiolated crosslinker on the final hydrogel properties. Therefore, the present work assesses the influence of different thiolated crosslinkers on the crosslinking kinetics, mechanical properties and biological performance of the hydrogels upon encapsulation of primary adipose tissue-derived stem cells in which indicated a cell viability exceeding 70%. Furthermore, the different formulations were processed using two-photon polymerisation which indicated, in addition to differences in processing window and swelling ratio, a previously unreported phenomenon. At high intensities (i.e. ≥ 150 mW), the laser results in cleavage of the gelatin backbone even in the absence of distinct photo-cleavable functionalities. This can have potential to introduce channels or softer regions in gels to result in zones characterized by different degradation speeds or the formation of blood vessels. Consequently, the present study can be used to provide guidance towards tailoring the thiol-ene system towards the desired applications.
Article
Full-text available
Cells use different cell adhesion and communication structures to promote tissue development, maintenance of tissue integrity as well as repair and regenerative processes. Another recently discovered way of information exchange is long‐distance thin cellular processes called nanotubes (NTs), mainly studied in vitro. Information on the existence and relevance of NTs in vivo is sparse. Building on two references which hint at the potential existence of longitudinally directed cell processes resembling NTs, we investigated tendons from young (3 weeks) and adult (9 weeks, 4 and 8 months) Fisher rats. Whole mounts of rat tail tendon fascicles (RTTfs) and sections of Achilles, flexor, extensor and patellar tendons were stained with Deep Red plasma membrane and DAPI nuclear stain and immunolabelled with Connexin43 (Cx43). In addition, 3‐D reconstruction of serial semithin sections and TEM was used to verify the presence of NTs. We were able to demonstrate NTs as straight thin longitudinal processes (Ø 100–500 nm) reaching up to several 100 μm in length, mainly originating from lateral sheet‐like cell processes or cell bodies in all tendon types investigated. NTs were observed to distend between tenocyte rows at the same level but also connect cells of different rows, thus leading to a complex 3‐D cellular scaffold. Shorter NTs connected lateral cell sheets of tenocytes in the same row, omitting one or two cells. In addition, we detected links or potential branching of NTs. Cx43 immunostaining for the detection of gap junctions revealed Cx43‐positive foci at the end‐to‐end contacts of tenocyte cell bodies as well as along their contacting sheet‐like processes. Only rarely, we found clear Cx43 signals at their potential contact points between NTs and tendon cells as well as along the course of NTs, and most NTs appeared completely devoid of Cx43 signals. Therefore, we conclude that NTs in tendons could have a twofold function: long‐distance communication as well as stabilization of a mechanically challenged tissue. From in vitro studies it is known that NTs allow intercellular transmission of various cell components, offering potential protective effects for the respective tissue. Further studies on functional properties of NTs in tendons under changing mechanical loading regimens are required in the future. The fact that NTs are present in tendons may necessitate the reconsideration of our traditional understanding of cell‐to‐cell communication. Nanotubes (NTs), a recently discovered way of information exchange comprising long‐distance thin cellular processes, are present in tendons. Together with the well‐known flat sheet‐like lateral processes, NTs form a complex three‐dimensional cellular network. NTs in tendons could have a twofold function: long‐distance communication as well as stabilization of a mechanically challenged tissue, and may require the reconsideration of our traditional understanding of cell‐to‐cell communication.
Article
Full-text available
Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells.
Article
Full-text available
Use of growth factors as biochemical molecules to elicit cellular differentiation is a common strategy in tissue engineering. However, limitations associated with growth factors, such as short half‐life, high effective physiological doses, and high costs, have prompted the search for growth factor alternatives, such as growth factor mimics and other proteins. This work explores the use of insulin protein as a biochemical factor to aid in tendon healing and differentiation of cells on a biomimetic electrospun micro‐nanostructured scaffold. Dose response studies were conducted using human mesenchymal stem cells (MSCs) in basal media supplemented with varied insulin concentrations. A dose of 100‐ng/mL insulin showed increased expression of tendon markers. Synthetic‐natural blends of various ratios of polycaprolactone (PCL) and cellulose acetate (CA) were used to fabricate micro‐nanofibers to balance physicochemical properties of the scaffolds in terms of mechanical strength, hydrophilicity, and insulin delivery. A 75:25 ratio of PCL:CA was found to be optimal in promoting cellular attachment and insulin immobilization. Insulin immobilized fiber matrices also showed increased expression of tendon phenotypic markers by MSCs similar to findings with insulin supplemented media, indicating preservation of insulin bioactivity. Insulin functionalized scaffolds may have potential applications in tendon healing and regeneration.
Article
Bioreactors are powerful tools with the potential to model tissue development and disease in vitro . For nearly four decades, bioreactors have been used to create tendon and ligament tissue‐engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of 2‐ and 3‐dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell‐cell and cell‐matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades. This article is protected by copyright. All rights reserved.
Article
Hydrogel scaffolds that can repair or regrow damaged biological tissue have great potential for the treatment of injury and disease. These biomaterials are widely used in the tissue engineering field due to their ability to support cell proliferation, migration and differentiation, to permit oxygen and nutrient transport, and mimic native soft tissue. Careful design of the underlying polymer scaffold is therefore critical, dictating both the physical and biological properties of a hydrogel. In this review, we will provide a critical overview of hydrogel design from the perspective of the polymer chemistry, highlighting both the advantages and limitations of particular polymer structures, properties, and architectures. By doing so, we hope to equip researchers with the tools to design new polymer systems and hydrogel scaffolds that address current limitations in the field and limit clinical translation.
Article
Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for biofabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their crosslinking mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies applying inkjet, deposition and light-based additive manufacturing techniques, and the applications of the obtained 3D constructs. STATEMENT OF SIGNIFICANCE: Gelatin and more specifically gelatin-methacryloyl has emerged to become one of the gold standard materials as an extracellular matrix mimic in the field of biofabrication. However, also other modification strategies have been elaborated to take advantage of a plethora of crosslinking chemistries. Therefore, a review paper elaborating on the different modification strategies and processing of gelatin is presented. Particular attention is paid to the underlying chemistry along with the benefits and drawbacks of each type of crosslinking chemistry. The different strategies were classified based on their basic crosslinking mechanism including chain- or step-growth polymerization. Within the step-growth classification, a further distinction is made between click chemistries as well as other strategies. The influence of these modifications on the physical gelation and processing conditions including mechanical properties is presented. Additionally, substantial attention is put to the applied photoinitiators and the different biofabrication technologies including inkjet, extrusion or light-based technologies.
Article
There exists a clear clinical need for adipose tissue reconstruction strategies to repair soft tissue defects which outperform the currently available approaches. In this respect, additive manufacturing has shown to be a promising alternative for the development of larger constructs able to support adipose tissue engineering. In the present work, a thiol-ene photo-click crosslinkable gelatin hydrogel was developed which allowed extrusion-based additive manufacturing into porous scaffolds. To this end, norbornene-functionalized gelatin (Gel-NB) was combined with thiolated gelatin (Gel-SH). The application of a macromolecular gelatin-based thiolated crosslinker holds several advantages over conventional crosslinkers including cell-interactivity, less chance at phase separation between scaffold material and crosslinker and the formation of a more homogeneous network. Throughout the paper, these photo-click scaffolds were benchmarked to the conventional methacrylamide-modified gelatin (Gel-MA). The results indicated that stable scaffolds could be realized which were further characterized physico-chemically by performing swelling, mechanical and in vitro biodegradability assays. Furthermore, the seeded adipose tissue-derived stem cells (ASCs) remained viable (>90%) up to 14 days and were able to proliferate. In addition, the cells could be differentiated into the adipogenic lineage on the photo-click crosslinked scaffolds, thereby performing better than the cells supported by the frequently reported Gel-MA scaffolds. As a result, the developed photo-click crosslinked scaffolds can be considered a promising candidate towards adipose tissue engineering and a valuable alternative for the omnipresent Gel-MA. Statement of Significance The field of adipose tissue engineering has emerged as a promising strategy to repair soft tissue defects. Herein, Gel-NB/Gel-SH gelatin-based hydrogel scaffolds were produced using extrusion-based additive manufacturing. Using a cell-interactive, thiolated gelatin crosslinker, a homogeneous network was formed and the risk of phase separation between norbornene-modified gelatin and macromolecular crosslinkers was reduced. UV-induced crosslinking of these materials is based on step growth polymerization which requires less free radicals to enable polymerization. Our results demonstrated the potential of the developed scaffolds, due to their favourable physico-chemical characteristics as well as their adipogenic differentiation potential when benchmarked to Gel-MA scaffolds. Hence, the hydrogels could be of great interest towards future development of adipose tissue constructs and tissue engineering in general.
Article
Fiber‐based approaches hold great promise for tendon tissue engineering enabling the possibility of manufacturing aligned hydrogel filaments that can guide collagen fiber orientation, thereby providing a biomimetic micro‐environment for cell attachment, orientation, migration, and proliferation. In this study, a 3D system composed of cell‐laden, highly aligned hydrogel yarns is designed and obtained via wet spinning in order to reproduce the morphology and structure of tendon fascicles. A bioink composed of alginate and gelatin methacryloyl (GelMA) is optimized for spinning and loaded with human bone morrow mesenchymal stem cells (hBM‐MSCs). The produced scaffolds are subjected to mechanical stretching to recapitulate the strains occurring in native tendon tissue. Stem cell differentiation is promoted by addition of bone morphogenetic protein 12 (BMP‐12) in the culture medium. The aligned orientation of the fibers combined with mechanical stimulation results in highly preferential longitudinal cell orientation and demonstrates enhanced collagen type I and III expression. Additionally, the combination of biochemical and mechanical stimulations promotes the expression of specific tenogenic markers, signatures of efficient cell differentiation towards tendon. The obtained results suggest that the proposed 3D cell‐laden aligned system can be used for engineering of scaffolds for tendon regeneration. Highly aligned hydrogel yarns are fabricated using a novel system employing a wet‐spinning technique to recapitulate the structure and morphology of tendon tissue. Scaffolds are loaded with human bone‐marrow‐derived stem cells and subjected to mechanical stretching and biochemical stimulation. The combined effect of these stimuli results in cell preferential orientation, tenogenic differentiation, and collagen expression.