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Citation: Selig, M.; Walz, K.; Lauer,
J.C.; Rolauffs, B.; Hart, M.L.
Therapeutic Modulation of Cell
Morphology and Phenotype of
Diseased Human Cells towards a
Healthier Cell State Using Lignin.
Polymers 2023,15, 3041. https://
doi.org/10.3390/polym15143041
Academic Editors: Erwann Guénin
and Vincent Terrasson
Received: 14 June 2023
Revised: 10 July 2023
Accepted: 12 July 2023
Published: 14 July 2023
Copyright: © 2023 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/).
polymers
Article
Therapeutic Modulation of Cell Morphology and Phenotype
of Diseased Human Cells towards a Healthier Cell State
Using Lignin
Mischa Selig 1,2, Kathrin Walz 1, Jasmin C. Lauer 1,2, Bernd Rolauffs 1and Melanie L. Hart 1, *
1G.E.R.N. Center for Tissue Replacement, Regeneration & Neogenesis, Department of Orthopedics
and Trauma Surgery, Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Engesserstraße 4,
79108 Freiburg, Germany; mischa.selig@web.de (M.S.); kathrin_walz@yahoo.de (K.W.);
jasmin.lauer@uniklinik-freiburg.de (J.C.L.); bernd.rolauffs@uniklinik-freiburg.de (B.R.)
2Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Germany
*Correspondence: melanie.lynn.hart@uniklinik-freiburg.de
Abstract:
Despite lignin’s global abundance and its use in biomedical studies, our understanding
of how lignin regulates disease through modulation of cell morphology and associated phenotype
of human cells is unknown. We combined an automated high-throughput image cell segmentation
technique for quantitatively measuring a panel of cell shape descriptors, droplet digital Polymerase
Chain Reaction for absolute quantification of gene expression and multivariate data analyses to
determine whether lignin could therapeutically modulate the cell morphology and phenotype of
inflamed, degenerating diseased human cells (osteoarthritic (OA) chondrocytes) towards a healthier
cell morphology and phenotype. Lignin dose-dependently modified all aspects of cell morphology
and ameliorated the diseased shape of OA chondrocytes by inducing a less fibroblastic healthier cell
shape, which correlated with the downregulation of collagen 1A2 (COL1A2, a major fibrosis-inducing
gene), upregulation of collagen 2A1 (COL2A1, a healthy extracellular matrix-inducing gene) and
downregulation of interleukin-6 (IL-6, a chronic inflammatory cytokine). This is the first study to
show that lignin can therapeutically target cell morphology and change a diseased cells’ function
towards a healthier cell shape and phenotype. This opens up novel opportunities for exploiting
lignin in modulation of disease, tissue degeneration, fibrosis, inflammation and regenerative medical
implants for therapeutically targeting cell function and outcome.
Keywords:
lignin; organosolv lignin; cell morphology; cell shape; fibrosis; health; disease; chondrocytes;
osteoarthritis; anti-inflammatory
1. Introduction
Lignin is the second most abundant biopolymer on the planet and is produced in
massive amounts as a by-product of the bioethanol, paper and pulp industries. Since
less than 2% is used for high value purposes [
1
,
2
], lignin’s natural abundance and global
availability has spurred new areas of lignin-based research in various biomedical fields
over the past decade [
3
,
4
]. Yet, how lignin modulates the cell morphology and function of
human cells and regulates disease through this modulation has never been assessed.
In nature, lignin is a polyphenolic polymer made up of random linkages that associate
with cellulose and hemicellulose [
5
] and functions as a protective barrier by making biomass
resistant to microorganisms and hence, disease. Lignin also greatly enhances the mechanical
strength of biomass [
6
,
7
]. In human health, lignins have been shown to have diverse
therapeutic properties such as antioxidant, anti-microbial and anti-viral effects and for
these reasons have been proposed to treat various diseases [
3
,
4
,
8
–
10
]. However, little is
known of how lignins can be used to therapeutically target the function of diseased cells
towards a healthier phenotype, e.g., through controlling cell morphology [11,12].
Polymers 2023,15, 3041. https://doi.org/10.3390/polym15143041 https://www.mdpi.com/journal/polymers
Polymers 2023,15, 3041 2 of 14
Organosolv lignins (OSL) are extracted via the organosolv method, which uses eco-
friendly solvents and enzymes and produces a high yield of lignin that is of high quality
and purity, making it ideal for use in biomedical and pharmaceutical applications [
2
].
Fractionation can further decrease lignin’s heterogeneity including the composition of
monomers and their linkages as well as functional groups and further standardize its prop-
erties [
13
–
16
]. Compared to higher molecular weight (MW) fractions, low MW fractionated
lignins are more capable of terminating oxidative chain reactions and oxidative stress due
to the increased availability of phenolic hydroxyl groups [
14
]. Other properties of low
MW lignins include anti-inflammatory, anti-elastase [
17
] and anti-microbial [
3
] effects. We
recently showed that, at a balanced concentration, low MW OSL is non-cytotoxic and
biocompatible with major cell types of cartilage (chondrocytes), bone (osteoblasts and
bone marrow-derived mesenchymal stromal cells (MSCs), skin (keratinocytes) and oral
(periodontal ligament and gingival fibroblasts) tissues [
16
]. We also showed that the low
MW fraction of OSL had increased interfacial adhesion/interactive forces compared to a
higher MW fraction of OSL [
13
,
16
]. Specifically, the lower MW fraction had more numer-
ous aliphatic hydroxyl functionalities, while having condensed phenolic structures and
a less branched conformation as well as an increased hydrogen bonding capacity, which
could hypothetically promote intermolecular interactions with cells and thereby modify the
phenotype and function of cells towards a healthier phenotype since these characteristics
would be expected to increase the availability of the lignin’s functional sites.
Osteoarthritis (OA) is one of the most common degenerative joint diseases and the
most frequent cause of physical disability worldwide [
18
]. To date, no early therapy for
this degenerative disease exists and early treatment strategies are needed. In the present
study, we used chondrocytes isolated from human diseased OA articular cartilage tissue as
a representative diseased cell type to determine if low MW OSL could modulate the cell
morphology and phenotype of diseased cells towards a healthier cell state. Chondrocytes
are the major resident cell of articular cartilage, the connective tissue that facilitates the
movement of joints such as the knee, hip and shoulder and the transmission of mechanical
loads applied to those joints during normal everyday activities [
19
]. Joint trauma (from
e.g., sports, accidents or work-related events), abnormal joint mechanics or increased joint
load due to increased body weight can damage cartilage and lead to early joint disease
characterized by long-term local and circulating low-grade inflammatory and degenera-
tive extracellular matrix (ECM) components produced from the breakdown of cartilage
tissue [
20
]. Importantly, due to cartilage’s avascularity, it has a limited healing and repair
capacity. When repair occurs, it generally results in the formation of a fibrocartilage tissue
that is biomechanically unstable and frequently undergoes degradation due to the high
amounts of collagen type 1 (COL1) produced by chondrocytes that form a weak ECM [
21
].
This inferior tissue can break down with time, leading to OA [
22
]. In addition to these
phenotypic effects, OA disease development is characterized by major cell morphological
changes with chondrocytes acquiring abnormal cell shape characteristics such as a loss of
their rounded or spherical morphology in favor of an elongated fibroblast-like cell shape,
which correlates with the expression of high levels of unhealthy fibrosis-inducing COL1
and inflammatory-inducing IL-6 and low levels of healthy collagen type 2 (COL2) [
23
].
Therefore, strategies to modulate the cell morphology and phenotype of diseased chon-
drocytes towards a healthier cell shape and phenotype would greatly benefit cartilage
tissue engineering strategies and also open up many new possibilities for using lignin in an
entirely new way.
For the first time, we recently showed that cell morphology could be used as a biologi-
cal fingerprint for describing healthy, inflamed, and degenerating/diseased chondrocyte
phenotype [
23
]. Since few studies have investigated the uses of OSL for biomedical appli-
cations and the effect of lignin on diseased cell morphology, in general, has never been
investigated, we combined an automated high-throughput method for quantitatively mea-
suring a panel of cell shape descriptors and absolute quantification of gene expression
using droplet digital PCR (ddPCR) to determine whether OSL could modulate the cell
Polymers 2023,15, 3041 3 of 14
morphology and phenotype (COL1, COL2, and IL-6) of inflamed, degenerating human
OA diseased cells towards a healthier cell morphology and phenotype. Using an image
cell segmentation technique on a large number of diseased chondrocytes, we quantified
single cell area, the major (length) and minor (width) axes of the cells, and their aspect
ratio, roundness, and the number of cytoplasmic processes depicted as a change in cell
circularity and solidity. Combining this data with multivariate data analyses (Figure 1), we
investigated the therapeutic potential of lignin in the modulation of cell morphology and
phenotype of diseased cells towards a healthier cell state. This is the first study to show
that lignin can be used to therapeutically target cell morphology which can change the
function of diseased cells towards a healthy cell shape and phenotype. This opens up new
possibilities for using lignin in the modulation of disease or tissue degeneration and in
tissue engineering strategies.
Polymers 2023, 15, x FOR PEER REVIEW 3 of 14
been investigated, we combined an automated high-throughput method for quantita-
tively measuring a panel of cell shape descriptors and absolute quantification of gene ex-
pression using droplet digital PCR (ddPCR) to determine whether OSL could modulate
the cell morphology and phenotype (COL1, COL2, and IL-6) of inflamed, degenerating
human OA diseased cells towards a healthier cell morphology and phenotype. Using an
image cell segmentation technique on a large number of diseased chondrocytes, we quan-
tified single cell area, the major (length) and minor (width) axes of the cells, and their
aspect ratio, roundness, and the number of cytoplasmic processes depicted as a change in
cell circularity and solidity. Combining this data with multivariate data analyses (Figure
1), we investigated the therapeutic potential of lignin in the modulation of cell morphol-
ogy and phenotype of diseased cells towards a healthier cell state. This is the first study
to show that lignin can be used to therapeutically target cell morphology which can
change the function of diseased cells towards a healthy cell shape and phenotype. This
opens up new possibilities for using lignin in the modulation of disease or tissue degen-
eration and in tissue engineering strategies.
Figure 1. Workflow for determining the effect of lignin on diseased cells. Diseased cells were iso-
lated from human cartilage tissue and treated with lignin. The quantification of gene expression was
measured using ddPCR, which allowed absolute quantification of genes in copies/µL by counting
the fluorescent positive (green) droplets above the threshold vs. negative (gray) droplets below the
threshold. For assessing cell morphology, single cells were detected using image segmentation, and
cell shape descriptors were quantified. Both sets of data were assembled for multivariate cell feature
analysis. The positive and negative relationship between features under lignin treatment was ana-
lyzed using a clustered image map and correlograms.
2. Materials and Methods
2.1. Preparation of OSL
Organosolv lignin (OSL, Batch No. KO22) derived from beechwood was kindly pro-
vided by the Fraunhofer Center for Chemical-Biotechnological Processes (CBP) (Leuna,
Germany). The selected method for the fractionating OSL at ambient temperature was
Figure 1.
Workflow for determining the effect of lignin on diseased cells. Diseased cells were isolated
from human cartilage tissue and treated with lignin. The quantification of gene expression was
measured using ddPCR, which allowed absolute quantification of genes in copies/
µ
L by counting
the fluorescent positive (green) droplets above the threshold vs. negative (gray) droplets below
the threshold. For assessing cell morphology, single cells were detected using image segmentation,
and cell shape descriptors were quantified. Both sets of data were assembled for multivariate cell
feature analysis. The positive and negative relationship between features under lignin treatment was
analyzed using a clustered image map and correlograms.
2. Materials and Methods
2.1. Preparation of OSL
Organosolv lignin (OSL, Batch No. KO22) derived from beechwood was kindly
provided by the Fraunhofer Center for Chemical-Biotechnological Processes (CBP) (Leuna,
Germany). The selected method for the fractionating OSL at ambient temperature was
based on solvent mixtures of acetic acid and water and fractionated into four different
fractions using a sequential precipitation method as previously reported [13,16,24].
The OSL fraction used in this study was previously characterized for structural and
physicochemical features and selected based on its high biocompatibility with chondrocytes
Polymers 2023,15, 3041 4 of 14
as well as other cell types commonly used in tissue engineering including human mesenchy-
mal stem cells (MSCs), osteoblasts, periodontal ligament fibroblasts, gingival fibroblasts
and keratinocytes [
16
]. In a step-wise approach, 30% (w/v) sodium hydroxide (NaOH),
which was chosen based on higher cell viability compared to the use of 40% ethanol [
16
],
was added to the low MW OSL fraction to create a stock solution of solubilized OSL. The
stock solution was heated to 85 ◦C while gently stirring and UV sterilized for 30 min.
2.2. Isolation of Human OA Chondrocytes from Articular Cartilage and Treatment with OSL
Human OA articular chondrocytes from n = 4–8 different donors were obtained
from the medial and lateral femoral condyles of articular cartilage during routine knee
replacement surgery with informed patient consent obtained by the Clinic for Department
of Orthopedics and Trauma Surgery, University Medical Center Freiburg, Germany, which
was conducted according to the guidelines of the Declaration of Helsinki and approved
by the Institutional Ethics Committee of the Albert-Ludwigs-University Freiburg (ethics
#418/19). These cells were previously characterized [
25
–
27
]. Under sterile conditions, the
cartilage was removed and covered with cartilage explant medium (DMEM low glucose,
GlutaMAX supplement, pyruvate, Thermo Fisher Scientific, Schwerte, Germany) containing
10 mM HEPES (Pan Biotech, Aidenbach, Germany), 10% FBS superior, 2% penicillin-
streptomycin, 1% amphotericin B, 0.1 mM nonessential amino acids, 0.4 mM L-proline
and 0.02 mg/mL L-ascorbic acid phosphate magnesium salt) and incubated for two days
at 37
◦
C and 5% CO
2
. Using 4 mL collagenase XI (1500 U/mL, Sigma Aldrich, St. Louis,
MO, USA), 2 mL dispase II (2.4 U/mL, Sigma Aldrich, St. Louis, MO, USA) in 6 mL
chondrocyte culture medium, cells were isolated for 6 h at 37
◦
C and stirred with a sterile
magnetic stirring bar at 250 rpm. The digest was filtered through a 100
µ
m cell strainer
(Thermo Fisher Scientific, Schwerte, Germany). The cell pellet was resuspended in media,
cultured in a 25 cm
2
tissue culture flask, and incubated at 37
◦
C and 5% CO
2
. When the
cells were approximately 70% confluent, they were split. After 24 h, passage 1 chondrocytes
(9375 cells/cm
2
) were treated with 20 or 80
µ
g/mL OSL, concentrations previously proven
to be non-cytotoxic and biocompatible with chondrocytes [
16
]. As a control, chondrocytes
were cultured in chondrogenic media without OSL. The cells were treated for 6 days
with a media change at day 3 with and without OSL. Two identical plates were prepared,
one for gene expression analysis and one for high-throughput quantitative single cell
morphological analysis.
2.3. Droplet Digital PCR for Absolute Quantification of Gene Expression
Ribonucleic acid (RNA) isolation and ddPCR for absolute quantification experiments
were performed as previously described in [
28
]. RNA was isolated using the RNeasy
Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The RNA
concentration was determined by measuring the optical density at 260 nm. Then cDNA
was synthesized from total RNA with oligo (dT) and random hexamer primers using the
Advantage RT-for-PCR Kit (Clontech, Mountain View, CA, USA) according to the manu-
facturer’s protocol. PCR duplex reactions are performed in 22
µ
L sample volumes with
11
µ
L ddPCR Supermix for Probes (no dUTP, Bio-Rad, Hercules, CA, USA), 1.1
µ
L of each
PrimePCR ddPCR Expression Probe Assay (Bio-Rad) labeled with HEX or FAM, 6.6
µ
L
cDNA with 1.5 ng RNA input and 2.2
µ
L DNase/RNase-free water. Primers and probes
specific for human COL1A2, COL2A1 and IL-6 genes were purchased from BioRad. PCR
was performed using the QX100 thermal cycler (Bio-Rad) with the following steps. The
polymerase activation at 95
◦
C for 10 min, followed by 40 cycles of denaturation at 94
◦
C
for 30 s and the annealing at 55
◦
C for 1 min. After cDNA extension, the polymerase was
denatured at 98
◦
C for 10 min and the PCR products were kept at 4
◦
C until droplet reading.
The fluorescence of the droplets was measured by the QX200 Droplet Reader (
Bio-Rad
)
and analyzed using QuantaSoft Software (Version 1.7.4) (Bio-Rad), which quantifies the
number of HEX- and FAM-positive and negative droplets and calculates the target concen-
tration for each HEX- and FAM-labeled target gene in copies/
µ
L. Data normalization was
Polymers 2023,15, 3041 5 of 14
achieved using a standardized amount of RNA for reverse transcription and, therefore, a
standardized amount of cDNA in the reaction volume.
2.4. Cell Staining and Microscopy
To accurately measure single cell morphology, we first stained the chondrocytes after
6 days of incubation with 1
µ
M calcein (Thermo Fisher Scientific, Schwerte, Germany)
and 1
µ
g/mL Hoechst (Thermo Fisher Scientific, Schwerte, Germany) to visualize the cell
body and nucleus. The cells were incubated in the staining solution for 30 min at 37
◦
C
and 5% CO
2
. Then, fresh chondrocyte culture medium was supplied and microscopical
images with a 20
×
magnification were taken with the Axio Observer Z1 microscope (Zeiss,
Oberkochen, Germany) in a tile format to image entire cell culture wells.
2.5. High-Throughput Quantitative Cell Morphometric Measurements Using a Panel of Cell
Shape Descriptors
The bioimage analysis tool QuPath [
29
] was used to convert large whole image samples
to a .tif file format and downsize the images by a factor of three. The images were split into
nine single image tiles, and three representative images used for analysis using an in-house
Fiji-based [
30
] single cell shape analysis algorithm and Trainable WEKA Segmentation
plugin [
31
] for segmentation of cells from the background. The WEKA classifier was
trained for pixel classification of three classes: nucleus, cytosol, and background. After
successfully segmenting the cells from the image background with the WEKA classifier,
neighboring cells were separated with a marker-based watershed algorithm. The resulting
single chondrocytes of a large number of cells were detected in the binary image maps and
single cells were assessed by calculating the following seven cell shape descriptors similar
to our previous studies [
32
–
35
]: area of the single cells (
µ
m
2
), major axis [
µ
m] representing
cell length; minor axis [
µ
m] representing cell width; circularity (4
×π
(area/perimeter
2
);
aspect ratio (ratio of major to minor axis), which is used an indicator of cell elongation
and different than cell length; roundness (4
×
area/(
π×
major axis length
2
); and solidity
(area/convex area (cell)), which measures the density of a cell with a solidity value of
1 representing a solid cell and a value less than 1 representing a cell with an irregular
boundary or a cell containing holes.
2.6. Correlation Analysis
Correlations were performed using the “R” [
36
] packages “Hmisc” [
37
] and “cor-
rplot” [
38
]. The Spearman Rank Order correlation method was used if one or more vari-
ables were categorical. Pearson product-moment was used when variables were numerical.
The classes were coded as 0 (control), 1 (20
µ
g/mL OSL), and 3 (80
µ
g/mL OSL treatment).
2.7. Clustered Image Map (CIM) Allowing Multi-Level Analyses
The CIM was generated using the “mixOmics” [
39
,
40
] package in “R” to determine
the standard deviation away from the mean on scaled and centered data. This allowed
assessment of the relationship between variables and donor variability across all donors in
response to treatment.
2.8. Statistical Analysis
The data was analyzed using Microsoft Excel (v. 2013) and SigmaPlot v.14.0 (Systat,
Chicago, IL, USA). ANOVA on ranks was performed using Dunn’s method as a post hoc
test. Statistical differences were considered significant for p< 0.05.
3. Results
3.1. Lignin-Mediated Modulation of Gene Expression in Human Diseased Chondrocytes
As a first step, we investigated whether OSL affected the health and inflammatory
profile of degenerating OA diseased chondrocytes. Cells were treated with or without
OSL for 6 days at biocompatible concentrations with a range of primary human cell types
Polymers 2023,15, 3041 6 of 14
isolated from various tissues [
16
]. OSL treatment of human OA chondrocytes resulted in
a significant dose-dependent decrease in COL1A2 expression, an unhealthy phenotypic
marker. It showed a trend in increasing COL2A1, a healthy phenotypic marker of cartilage
with 80
µ
g/mL OSL resulting in a 0.4-fold decrease in the expression of COLA12 and
a
0.6-fold
increase in the expression of COL2A1 (Figure 2A,B). OSL did not affect the
expression of IL-6 (Figure 2C).
Polymers 2023, 15, x FOR PEER REVIEW 6 of 14
2.8. Statistical Analysis
The data was analyzed using Microsoft Excel (v. 2013) and SigmaPlot v.14.0 (Systat,
Chicago, IL, USA). ANOVA on ranks was performed using Dunn’s method as a post hoc
test. Statistical differences were considered significant for p < 0.05.
3. Results
3.1. Lignin-Mediated Modulation of Gene Expression in Human Diseased Chondrocytes
As a first step, we investigated whether OSL affected the health and inflammatory
profile of degenerating OA diseased chondrocytes. Cells were treated with or without
OSL for 6 days at biocompatible concentrations with a range of primary human cell types
isolated from various tissues [16]. OSL treatment of human OA chondrocytes resulted in
a significant dose-dependent decrease in COL1A2 expression, an unhealthy phenotypic
marker. It showed a trend in increasing COL2A1, a healthy phenotypic marker of cartilage
with 80 µg/mL OSL resulting in a 0.4-fold decrease in the expression of COLA12 and a
0.6-fold increase in the expression of COL2A1 (Figure 2A,B). OSL did not affect the ex-
pression of IL-6 (Figure 2C).
Figure 2. Lignin effects on (A) COL1A2 (unhealthy phenotypic marker), (B) COL2A1 (healthy phe-
notypic marker), and (C) IL-6 (inflammatory marker ) in human diseased cells. OA chondrocytes
were treated for 6 days with or without lignin. Data is presented as mean fold change compared to
control of n = 4–8 donors per group +/− SEM. * p < 0.05.
3.2. Lignin-Mediated Modulation of a Diseased Cell Shape into a Healthier Cell Shape
Osteoarthritis disease development is characterized by major cell morphological
changes. Healthy chondrocytes are typically round or spherical, but as tissue degenera-
tion progresses, chondrocytes acquire an abnormal cell shape with the cells becoming less
round/less spherical and more elongated with cells acquiring a fibroblast-like cell shape,
increased cell volume and cell protrusions [12,23,41]. Next, we investigated how OSL in-
fluenced the cell morphology of already diseased OA chondrocytes using box plots, which
not only allows comparing treatment groups but also allows viewing the dispersion and
spread of data on a large number of cells (Figure 3A–G). OSL dose-dependently and sig-
nificantly decreased the area compared to control-treated cells by 3% and 11%, respec-
tively (Figure 3A). Similarly, OSL dose-dependently decreased the cell’s major axis (cell
length) by 3% and 13% (Figure 3B). Treatment with the lowest OSL concentration also
significantly decreased the cell’s minor axis (cell width) by 2% vs. control-treated cells
(Figure 3C). In line with this data, OSL dose-dependently decreased the aspect ratio (ratio
of major to minor axis) by 3% and 13%, respectively (Figure 3E). Together, this demon-
strates that OSL treatment caused the cells to become less elongated. This was compatible
with OSL effects on circularity and roundness, which showed that OSL significantly and
dose-dependently increased the cell’s circularity by 8% and 20% (Figure 3D) and round-
ness by 3% and 15% (Figure 3F) vs. control-treated cells. Similarly, OSL significantly and
dose-dependently increased the solidity by 1% and 2% vs. control (Figure 3G), indicating
few cell protrusions.
In summary, OSL treatment led to (i) dose-dependent effects on cell morphology and
(ii) ameliorated the diseased shape of OA chondrocytes and reverted the cells to a healthy
cell shape.
Figure 2.
Lignin effects on (
A
) COL1A2 (unhealthy phenotypic marker), (
B
) COL2A1 (healthy
phenotypic marker), and (
C
) IL-6 (inflammatory marker) in human diseased cells. OA chondrocytes
were treated for 6 days with or without lignin. Data is presented as mean fold change compared to
control of n = 4–8 donors per group +/−SEM. * p< 0.05.
3.2. Lignin-Mediated Modulation of a Diseased Cell Shape into a Healthier Cell Shape
Osteoarthritis disease development is characterized by major cell morphological
changes. Healthy chondrocytes are typically round or spherical, but as tissue degeneration
progresses, chondrocytes acquire an abnormal cell shape with the cells becoming less
round/less spherical and more elongated with cells acquiring a fibroblast-like cell shape,
increased cell volume and cell protrusions [
12
,
23
,
41
]. Next, we investigated how OSL
influenced the cell morphology of already diseased OA chondrocytes using box plots,
which not only allows comparing treatment groups but also allows viewing the dispersion
and spread of data on a large number of cells (Figure 3A–G). OSL dose-dependently
and significantly decreased the area compared to control-treated cells by 3% and 11%,
respectively (Figure 3A). Similarly, OSL dose-dependently decreased the cell’s major axis
(cell length) by 3% and 13% (Figure 3B). Treatment with the lowest OSL concentration also
significantly decreased the cell’s minor axis (cell width) by 2% vs. control-treated cells
(Figure 3C). In line with this data, OSL dose-dependently decreased the aspect ratio (ratio
of major to minor axis) by 3% and 13%, respectively (Figure 3E). Together, this demonstrates
that OSL treatment caused the cells to become less elongated. This was compatible with
OSL effects on circularity and roundness, which showed that OSL significantly and dose-
dependently increased the cell’s circularity by 8% and 20% (Figure 3D) and roundness by
3% and 15% (Figure 3F) vs. control-treated cells. Similarly, OSL significantly and dose-
dependently increased the solidity by 1% and 2% vs. control (Figure 3G), indicating few
cell protrusions.
In summary, OSL treatment led to (i) dose-dependent effects on cell morphology and
(ii) ameliorated the diseased shape of OA chondrocytes and reverted the cells to a healthy
cell shape.
3.3. Positive and Negative Correlations between Measured Features under Treatment
As the next step, correlation analysis was performed to understand how lignin induced
changes in human diseased OA morphology and phenotype and how these features related
to one another under treatment (Figure 4). Importantly, all features significantly correlated
suggesting a strong relationship between cell morphology and phenotype with lignin
treatment. As expected, correlations among gene expression markers showed that COL1A2
expression negatively correlated with COL2A1 and positively with IL-6 expression. These
findings agree with the known phenotype of OA chondrocytes (1). There was a strong
(indicated by the correlation coefficient and the larger circle size) negative correlation
between lignin treatment and the expression of COL1A2 in line with Figure 2showing
that lignin can significantly decrease its expression. There was also a negative correlation
Polymers 2023,15, 3041 7 of 14
between lignin treatment and the expression of IL-6, supporting the idea that lignin can
reduce IL-6. Surprisingly, there was a small negative correlation between lignin treatment
and the expression of COL2A1. This suggested that some donors differed in their response
to lignin as explained in more detail in the next section.
There was a strong significant positive correlation between lignin treatment and
circularity, roundness and solidity, indicated by the correlation coefficient and the circle
size in Figure 4, and a significant negative correlation with cell length showing that, similar
to Figure 2, lignin treatment induced more circular, rounded and less fibroblastic-like cells
and thereby induced a healthier cell morphology.
Importantly, the expression of the unhealthy COL1A2 marker significantly and strongly
negatively correlated with all of the cell shape descriptors. In most cases, IL-6 followed a
similar trend. The expression of COL2A1 significantly and strongly negatively correlated
with area, length and width and positively with circularity and solidity. Collectively, this
shows co-occurring morphological and phenotypical changes indicative of lignin-mediated
modification of cell morphology and gene expression towards a less dedifferentiated and,
hence, healthier morphology and phenotype.
Polymers 2023, 15, x FOR PEER REVIEW 7 of 14
Figure 3. Lignin effects on the cell morphology of human diseased chondrocytes. The following cell
morphology descriptors were measured: (A) area, (B) major axis (length), (C) minor axis (width),
(D) circularity, (E) aspect ratio, (F) roundness, and (G) solidity in OA chondrocytes treated for 6
days with or without lignin. Data is expressed as raw data of n = 4–8 donors per group with 11,726,
6088, and 4495 cells analyzed in control, 20, and 80 µg/mL lignin-treated groups, respectively. The
boxplots demonstrate the median (central line) and the data’s 25th and 75th percentile values. The
whiskers below and above the box plots represent the 10th and 90th percentile values and the black
points illustrate the 5th and 95th percentiles. Significant differences (p < 0.05) are indicated as fol-
lows:
a
between the control vs. 20 µg/mL lignin-treated groups,
b
between the control vs. 80 µg/mL
lignin-treated groups, and
c
between the 20 and 80 µg/mL groups.
3.3. Positive and Negative Correlations between Measured Features under Treatment
As the next step, correlation analysis was performed to understand how lignin in-
duced changes in human diseased OA morphology and phenotype and how these fea-
tures related to one another under treatment (Figure 4). Importantly, all features signifi-
cantly correlated suggesting a strong relationship between cell morphology and pheno-
type with lignin treatment. As expected, correlations among gene expression markers
showed that COL1A2 expression negatively correlated with COL2A1 and positively with
IL-6 expression. These findings agree with the known phenotype of OA chondrocytes (1).
There was a strong (indicated by the correlation coefficient and the larger circle size) neg-
ative correlation between lignin treatment and the expression of COL1A2 in line with Fig-
ure 2 showing that lignin can significantly decrease its expression. There was also a nega-
tive correlation between lignin treatment and the expression of IL-6, supporting the idea
that lignin can reduce IL-6. Surprisingly, there was a small negative correlation between
lignin treatment and the expression of COL2A1. This suggested that some donors differed
in their response to lignin as explained in more detail in the next section.
Figure 3.
Lignin effects on the cell morphology of human diseased chondrocytes. The following cell
morphology descriptors were measured: (
A
) area, (
B
) major axis (length), (
C
) minor axis (width),
(
D
) circularity, (
E
) aspect ratio, (
F
) roundness, and (
G
) solidity in OA chondrocytes treated for 6 days
with or without lignin. Data is expressed as raw data of n = 4–8 donors per group with 11,726,
6088, and 4495 cells analyzed in control, 20, and 80
µ
g/mL lignin-treated groups, respectively. The
boxplots demonstrate the median (central line) and the data’s 25th and 75th percentile values. The
whiskers below and above the box plots represent the 10th and 90th percentile values and the black
points illustrate the 5th and 95th percentiles. Significant differences (p< 0.05) are indicated as follows:
a
between the control vs. 20
µ
g/mL lignin-treated groups,
b
between the control vs. 80
µ
g/mL
lignin-treated groups, and cbetween the 20 and 80 µg/mL groups.
Polymers 2023,15, 3041 8 of 14
Polymers 2023, 15, x FOR PEER REVIEW 8 of 14
Figure 4. Correlograms depicting correlations in diseased cells under all conditions. Significant (p <
0.05) positive (white circles) or negative (black circles) correlations between features and the
strength of the correlation is indicated by the size of the circle (larger circles indicate a stronger
correlation having higher correlation coefficients). A blue empty box indicates a lack of correlation.
Data is representative of the average gene expression and cell morphology values of 22,309 cells
measured in the control, 20, and 80 µg/mL lignin-treated groups, respectively, for each of the cell
morphology descriptors of n = 4 individual experiments.
There was a strong significant positive correlation between lignin treatment and cir-
cularity, roundness and solidity, indicated by the correlation coefficient and the circle size
in Figure 4, and a significant negative correlation with cell length showing that, similar to
Figure 2, lignin treatment induced more circular, rounded and less fibroblastic-like cells
and thereby induced a healthier cell morphology.
Importantly, the expression of the unhealthy COL1A2 marker significantly and
strongly negatively correlated with all of the cell shape descriptors. In most cases, IL-6
followed a similar trend. The expression of COL2A1 significantly and strongly negatively
correlated with area, length and width and positively with circularity and solidity. Col-
lectively, this shows co-occurring morphological and phenotypical changes indicative of
lignin-mediated modification of cell morphology and gene expression towards a less de-
differentiated and, hence, healthier morphology and phenotype.
3.4. Lignin-Mediated Modulation of the Gene Expression and Cell Morphology at the Sample
Level
As a final step, we used CIM, which allows multivariate data comparisons at the
sample level (Figure 5). Hierarchical clustering was used because it allows viewing mul-
tivariate data over a variety of scales by creating a cluster tree or dendrogram with the
height of the branch points indicating how similar or different the relationship between
the entities is from one another: the greater the height, the greater the difference. This
enabled us to explore relationships between linkages of clusters. As shown on the left side,
Figure 4.
Correlograms depicting correlations in diseased cells under all conditions. Significant
(
p< 0.05)
positive (white circles) or negative (black circles) correlations between features and the
strength of the correlation is indicated by the size of the circle (larger circles indicate a stronger
correlation having higher correlation coefficients). A blue empty box indicates a lack of correlation.
Data is representative of the average gene expression and cell morphology values of 22,309 cells
measured in the control, 20, and 80
µ
g/mL lignin-treated groups, respectively, for each of the cell
morphology descriptors of n = 4 individual experiments.
3.4. Lignin-Mediated Modulation of the Gene Expression and Cell Morphology at the Sample Level
As a final step, we used CIM, which allows multivariate data comparisons at the sam-
ple level (Figure 5). Hierarchical clustering was used because it allows viewing multivariate
data over a variety of scales by creating a cluster tree or dendrogram with the height of
the branch points indicating how similar or different the relationship between the entities
is from one another: the greater the height, the greater the difference. This enabled us to
explore relationships between linkages of clusters. As shown on the left side, with the ex-
ception of one of the controls (sample 3), the control samples clustered together. Treatment
with low (20
µ
g/mL) lignin and high (80
µ
g/mL) lignin samples clustered into two and
three groups, respectively, discussed in more detail below. As shown in the lower part of
Figure 5, cell roundness and its inverse shape descriptor aspect ratio clustered, which we
previously identified as key shape descriptors of fully diseased human OA chondrocytes
and as early de-differentiating chondrocytes, induced by IL-1
β
[
23
]. COL1A2 and IL-6 gene
expression also clustered demonstrating a relationship between these unhealthy fibrotic-
and inflammatory-inducing markers. Whereas COL2A1 expression clustered with cell
circularity and solidity, COL1A2 and IL-6 gene expression clustered with roundness and
aspect ratio. This further demonstrates a close relationship between gene expression and
cell morphology.
Polymers 2023,15, 3041 9 of 14
Polymers 2023, 15, x FOR PEER REVIEW 9 of 14
with the exception of one of the controls (sample 3), the control samples clustered to-
gether. Treatment with low (20 µg/mL) lignin and high (80 µg/mL) lignin samples clus-
tered into two and three groups, respectively, discussed in more detail below. As shown
in the lower part of Figure 5, cell roundness and its inverse shape descriptor aspect ratio
clustered, which we previously identified as key shape descriptors of fully diseased hu-
man OA chondrocytes and as early de-differentiating chondrocytes, induced by IL-1β
[23]. COL1A2 and IL-6 gene expression also clustered demonstrating a relationship be-
tween these unhealthy fibrotic- and inflammatory-inducing markers. Whereas COL2A1
expression clustered with cell circularity and solidity, COL1A2 and IL-6 gene expression
clustered with roundness and aspect ratio. This further demonstrates a close relationship
between gene expression and cell morphology.
Figure 5. CIM plot demonstrating the effects of lignin on diseased OA chondrocytes at the sample
level. The gene expression data from Figure 2 and the cell morphology data from Figure 3 was scaled
and centered, allowing comparisons at the sample level using a clustered image map (CIM). The
dendrograms cluster the biological samples based on parameter similarities. The scale on the upper
left side of the figure describes the standard deviation below (blue) or above (red) the overall mean
across all samples with the intensity representing increases and decreases of the measured feature
from the overall mean. The samples were coded as follows: 0 (control-treated cells), 1 (low lignin =
20 µg/mL lignin-treated cells), and 2 (high lignin = 80 µg/mL lignin-treated cells).
Using the CIM (the scale is shown in the upper left corner) allowed us to see how
individual donors responded to lignin on the cell shape and gene expression level. Except
sample 3, the control samples clustered together and generally showed the same trend
under control treatment. As mentioned above lignin-treated samples clustered into sev-
eral groups showing that, in some cases, individual samples differed in response to lignin.
Importantly, with the exception of sample 9, all lignin-treated cells responded by decreas-
ing COL1A2, the most important unhealthy marker of diseased OA chondrocytes and an
important fibrotic-inducing gene. COL2A1, one of the most important healthy markers of
chondrocytes and cartilage ECM, increased in two of four samples (samples 10 and 11)
treated with the higher dose (80 µg/mL) of lignin. Lignin decreased IL-6 in four of eight
samples (samples 6 and 8 treated with 20 µg/mL lignin and samples 10 and 12 treated
with 80 µg/mL lignin). Overall, heat maps in samples 6, 8, 10 and 12 were relatively similar
showing that regardless of dose, lignin modified the gene expression towards being
healthier and less inflammatory while simultaneously regulating the cell shape into a
Figure 5.
CIM plot demonstrating the effects of lignin on diseased OA chondrocytes at the sample
level. The gene expression data from Figure 2and the cell morphology data from Figure 3was
scaled and centered, allowing comparisons at the sample level using a clustered image map (CIM).
The dendrograms cluster the biological samples based on parameter similarities. The scale on
the upper left side of the figure describes the standard deviation below (blue) or above (red) the
overall mean across all samples with the intensity representing increases and decreases of the
measured feature from the overall mean. The samples were coded as follows: 0 (control-treated cells),
1 (low lignin = 20 µg/mL lignin-treated cells), and 2 (high lignin = 80 µg/mL lignin-treated cells).
Using the CIM (the scale is shown in the upper left corner) allowed us to see how
individual donors responded to lignin on the cell shape and gene expression level. Except
sample 3, the control samples clustered together and generally showed the same trend
under control treatment. As mentioned above lignin-treated samples clustered into several
groups showing that, in some cases, individual samples differed in response to lignin.
Importantly, with the exception of sample 9, all lignin-treated cells responded by decreasing
COL1A2, the most important unhealthy marker of diseased OA chondrocytes and an
important fibrotic-inducing gene. COL2A1, one of the most important healthy markers
of chondrocytes and cartilage ECM, increased in two of four samples (samples 10 and 11)
treated with the higher dose (80
µ
g/mL) of lignin. Lignin decreased IL-6 in four of eight
samples (samples 6 and 8 treated with 20
µ
g/mL lignin and samples 10 and 12 treated
with 80
µ
g/mL lignin). Overall, heat maps in samples 6, 8, 10 and 12 were relatively
similar showing that regardless of dose, lignin modified the gene expression towards
being healthier and less inflammatory while simultaneously regulating the cell shape into
a healthier cell shape, which was more apparent using the higher dose of lignin. This
demonstrates that low or high doses of lignin decreased COL1A2 expression in all donors
but in half of the donors, low or high doses of lignin increased COL2A1 and decreased IL-6,
which corresponds with a healthier cell shape.
4. Discussion
Despite the fact that lignin is the second most abundant global biopolymer on Earth [
1
],
our understanding of how lignin modulates the cell morphology and function of diseased
cells remains fragmentary. This is the first study to show that lignin modulated the cell
morphology of human cells and concurrently decreased a major marker of disease, COL1A2.
This pioneering work shows the therapeutic potential of lignin-mediated modulation of
Polymers 2023,15, 3041 10 of 14
cell morphology and phenotype of diseased cells towards a healthier phenotype. Thus,
lignin treatment led to (i) dose-dependent effects on cell morphology and (ii) ameliorated
the diseased shape of OA chondrocytes by reverting the cells to a healthier cell shape,
which correlated with positive changes in disease-, ECM- and inflammatory-regulating
gene expression. This shows that lignin can change degenerative and inflamed diseased
cells towards a healthier cell state.
While it is not known how lignin directly modulates the cell morphology of diseased
human cells, we can extract from lignin’s role in nature and postulate how lignin could be
capable of cell phenotype modulation. Lignin affects plant development by strengthening
a plant’s robustness via adding a significant reinforcement to cell walls [
6
,
7
] as it does to
tissue engineering cell scaffolds as we [
16
] and others [
42
–
45
] have shown. The mechanical
stability that lignin provides to plants has recently been shown to correlate with lignin
content and the cell morphology of plant cells [
46
]. We previously showed that other types
of biomaterials, as well as their nanoscale surface stiffness and surface topography lead to
significant changes in cell morphology and, importantly, large phenotypic
effects [32–35].
The present study extends these findings and shows, for the first time, that lignin is a
biomaterial that can modulate the cell morphology and phenotype of human cells. We
previously showed that the low MW fraction of OSL used in this study had more numerous
aliphatic hydroxyl functionalities, while having condensed phenolic structures and a
less branched conformation as well as an increased hydrogen bonding capacity. This
could hypothetically promote intermolecular interactions with the cells themselves or
even the pericellular matrix of e.g., chondrocytes and potentially other cell types [
16
] and
thereby modify the phenotype and function of cells towards a healthier phenotype as
these characteristics would be expected to increase the availability of lignin’s functional
sites and provide reinforcement. By reinforcing the cartilage pericellular matrix, OSL
could also potentially enhance the mechanical properties of the artificial regenerative
cartilage environment. This is important because the mechanical properties of cartilage
play a critical role in its function and are often compromised in degenerative joint diseases.
We have shown how the stiffness of the cartilage tissue degenerates in the progression
of OA [
47
] and that tissue and biomaterial stiffness are key regulators of chondrocyte
phenotype [
48
]. OSL could help to stabilize an artificial cartilage environment and help
stabilize a chondrogenic phenotype.
Importantly, lignin significantly modified multi-factorial aspects of cell morphology in-
cluding the area (decreased), length (decreased), width (decreased), roundness (increased),
circularity (increased), solidity (increased), and the number of cytoplasmic processes (de-
creased). Thus, lignin induced a less fibroblastic cell shape, causing the cells to become
more circular and less elongated. We previously showed that IL-1
β
caused early diseased
chondrocytes to become morphologically and phenotypically more de-differentiated [
23
].
In the present study, we showed that lignin produced the opposite effect, causing dis-
eased chondrocytes to become morphologically and phenotypically less de-differentiated.
Therefore, lignin induced a less fibroblastic cell morphology and, therefore, a healthier
chondrocyte shape, which correlated with a shift in phenotype from an unhealthy to a
healthier cell state. Hence, lignin transformed the diseased cell morphology and shifted
the diseased phenotype by significantly downregulating the expression of the unfavorable
COL1A2, which leads to fibrotic ECM (fibrosis) and, in cartilage, a biomechanically instable
weak ECM and joint instability [
22
]. Besides cartilage, the effect of lignin on the downregu-
lation of COL1A2 may also be important in the regulation of other fibrotic diseases where
the expression of type I collagen is increased such as in pulmonary, liver, and bone marrow
fibrosis and scleroderma [
49
] or in tumor invasion and progression [
50
–
52
]. Moreover, in
half of the donors, in addition to lignin-induced changes in cell morphology and inhibition
of COL1A2 expression, treatment with the higher dose of lignin significantly upregulated
the expression of COL2A1, which promotes a biomechanically-stable ECM and tissue
homeostasis in cartilage [
21
]. It simultaneously downregulated the expression of IL-6,
a cytokine deeply involved in uncontrolled inflammation in chronic inflammatory and
Polymers 2023,15, 3041 11 of 14
autoimmune diseases, cancer, and the cytokine storm induced by viral diseases such as
coronavirus disease 2019 (COVID-19) [
20
,
53
,
54
]. Together, this suggests novel therapeutic
use of lignin to convert the cell morphology and phenotype of human diseased cells into
healthy cells, which could benefit tissue engineering strategies but also control fibrosis
and inflammation.
Lignin could be used alone (e.g., pharmaceutically) as a therapeutic modulator of
diseased cells to therapeutically target cell function by altering diseased and inflamed
cells towards a healthy status by modification of cell morphology and function. Lignin
could also be used in a cell scaffold. We previously showed that the incorporation of
organosolv lignin used in the present study significantly and dose-dependently increased
scaffold stiffness and viscosity as well as chondrocyte cell attachment. Hansen solubility
physiochemical parameters also showed high compatibility and interactive forces between
lignin and agarose, demonstrating biocompatibility in a tissue engineering scaffold [
16
].
Moreover, the lignin and concentrations used in the present study were non-cytotoxic
and biocompatible with other cell types including fibroblasts, MSCs, osteoblasts, and
keratinocytes [16].
Therefore, incorporating lignin could improve tissue regeneration, e.g., in degenerative
diseases related to cartilage such as cartilage defect repair, osteoarthritis, or intervertebral
disc disease and, potentially, in other diseases and tissues as well via the effects shown in
this study.
5. Conclusions
This is the first study to show that lignin can modulate many aspects of cell morphol-
ogy and induce a healthier cell shape, which correlates with positive changes in ECM- and
inflammatory-regulating genes and a concurrent decrease in a major marker of disease,
COL1A2, in human cells. This study demonstrates that lignin can be exploited in an en-
tirely new way, allowing the development of pharmaceutical and biomedical products that
could give rise to versatile and innovative technologies by using lignin to therapeutically
modulate cell morphology and phenotype of diseased cells towards a healthier cell state.
Author Contributions:
Conceptualization, M.L.H. and B.R.; methodology, M.L.H., B.R., M.S. and
J.C.L.; formal analysis, K.W., B.R., M.L.H. and M.S.; investigation, K.W.; resources, B.R.; data curation,
K.W. and M.S.; writing—original draft preparation, M.L.H.; writing—review and editing, M.L.H.,
B.R. and M.S.; supervision, M.L.H., B.R. and J.C.L. All authors have read and agreed to the published
version of the manuscript.
Funding:
The article processing charge was funded by the Baden-Württemberg Ministry of Science,
Research and Art and the University of Freiburg in the funding program Open Access Publishing.
Institutional Review Board Statement:
The study was conducted according to the guidelines of
the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the Albert-
Ludwigs-University Freiburg (418/19).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The datasets used and/or analyzed during the current study are
available from one of the corresponding authors upon reasonable request.
Acknowledgments:
We would like to thank the Leuna Biorefinery: Fraunhofer Chemical-Biotechnological
Processes (CBP) for kindly providing the original organosolv lignin as well as Marie-Pierre Laborie for
our collaboration in discussing/planning for this lignin preparation/utilization of lignin as well as her
team including Robert Gleuwitz, Mehmet Yapa, Jian Chen, and Lisa Ebers from the Department of Forest
Biomaterials at the University of Freiburg for their support in the preparation of fractionated lignin and
insightful tips.
Conflicts of Interest: The authors declare no conflict of interest.
Polymers 2023,15, 3041 12 of 14
References
1.
Bajwaa, D.S.; Pourhashem, G.; Ullah, A.H.; Bajwac, S.G. A concise review of current lignin production, applications, products and
their environmental impact. Indus. Crops Prod. 2019,139, 111526. [CrossRef]
2.
Melro, E.; Filipe, A.; Sousa, D.; Medronho, B.; Romano, A. Revisiting lignin: A tour through its structural features, characterization
methods and applications. New J. Chem. 2021,45, 6986–7013. [CrossRef]
3.
Sugiarto, S.; Leow, Y.; Tan, C.L.; Wang, G.; Kai, D. How far is Lignin from being a biomedical material? Bioact. Mater.
2022
,8,
71–94. [CrossRef]
4.
Witzler, M.; Alzagameem, A.; Bergs, M.; Khaldi-Hansen, B.E.; Klein, S.E.; Hielscher, D.; Kamm, B.; Kreyenschmidt, J.; Tobiasch, E.;
Schulze, M. Lignin-Derived Biomaterials for Drug Release and Tissue Engineering. Molecules
2018
,23, 1885. [CrossRef] [PubMed]
5.
Zhou, S.; Jin, K.; Buehler, M.J. Understanding Plant Biomass via Computational Modeling. Adv. Mater.
2021
,33, e2003206.
[CrossRef]
6.
Ozparpucu, M.; Ruggeberg, M.; Gierlinger, N.; Cesarino, I.; Vanholme, R.; Boerjan, W.; Burgert, I. Unravelling the impact of lignin
on cell wall mechanics: A comprehensive study on young poplar trees downregulated for Cinnamyl Alcohol Dehydrogenase
(CAD). Plant J. 2017,91, 480–490. [CrossRef]
7.
Ozparpucu, M.; Gierlinger, N.; Cesarino, I.; Burgert, I.; Boerjan, W.; Ruggeberg, M. Significant influence of lignin on axial elastic
modulus of poplar wood at low microfibril angles under wet conditions. J. Exp. Bot. 2019,70, 4039–4047. [CrossRef]
8.
Domínguez-Roblesa, J.; Cárcamo-Martínez, A.; Stewart, S.A.; Donnelly, R.F.; Larrañeta, E.; Borrega, M. Lignin for pharmaceutical
and biomedical applications—Could this become a reality? Sustain. Chem. Pharm. 2020,18, 100320. [CrossRef]
9.
Vinardell, M.P.; Mitjans, M. Lignins and Their Derivatives with Beneficial Effects on Human Health. Int. J. Mol. Sci.
2017
,18, 1219.
[CrossRef]
10.
Liu, R.; Dai, L.; Xu, C.; Wang, K.; Zheng, C.; Si, C. Lignin-Based Micro-and Nanomaterials and their Composites in Biomedical
Applications. ChemSusChem 2020,13, 4266–4283. [CrossRef]
11.
Hart, M.L.; Lauer, J.C.; Selig, M.; Hanak, M.; Walters, B.; Rolauffs, B. Shaping the Cell and the Future: Recent Advancements in
Biophysical Aspects Relevant to Regenerative Medicine. J. Funct. Morphol. Kinesiol. 2018,3, 2. [CrossRef]
12.
Lauer, J.C.; Selig, M.; Hart, M.L.; Kurz, B.; Rolauffs, B. Articular chondrocyte phenotype regulation through the cytoskeleton and
the signaling processes that originate from or converge on the cytoskeleton: Towards a novel understanding of the intersection
between actin dynamics and chondrogenic function. Int. J. Mol. Sci. 2021,22, 3279. [CrossRef]
13.
Gleuwitz, F.R.; Sivasankarapillai, G.; Chen, Y.; Friedrich, C.; Laborie, M.G. Lignin-Assisted Stabilization of an Oriented Liquid
Crystalline Cellulosic Mesophase, Part B: Toward the Molecular Origin and Mechanism. Biomacromolecules
2020
,21, 2276–2284.
[CrossRef] [PubMed]
14.
Pang, T.; Wang, G.; Sun, H.; Su, W.; Si, C. Lignin fractionation: Effective strategy to reduce molecule weight dependent
heterogeneity. Indus. Crops Prod. 2021,165, 113442. [CrossRef]
15.
Izaguirre, N.; Robles, E.; Llano-Ponte, R.; Labidi, J.; Erdociac, X. Fine-tune of lignin properties by its fractionation with a sequential
organic solvent extraction. Indus. Crops Prod. 2022,175, 114251. [CrossRef]
16.
Menima-Medzogo, J.A.; Walz, K.; Lauer, J.C.; Sivasankarapillai, G.; Gleuwitz, F.R.; Rolauffs, B.; Laborie, M.P.; Hart, M.L.
Characterization and In Vitro Cytotoxicity Safety Screening of Fractionated Organosolv Lignin on Diverse Primary Human Cell
Types Commonly Used in Tissue Engineering. Biology 2022,11, 696. [CrossRef]
17.
Saluja, B.; Thakkar, J.N.; Li, H.; Desai, U.R.; Sakagami, M. Novel low molecular weight lignins as potential anti-emphysema
agents:
In vitro
triple inhibitory activity against elastase, oxidation and inflammation. Pulm. Pharmacol. Ther.
2013
,26, 296–304.
[CrossRef]
18.
Brown, T.D.; Johnston, R.C.; Saltzman, C.L.; Marsh, J.L.; Buckwalter, J.A. Posttraumatic osteoarthritis: A first estimate of incidence,
prevalence, and burden of disease. J. Orthop. Trauma 2006,20, 739–744. [CrossRef]
19.
Herzog, W.; Federico, S. Considerations on joint and articular cartilage mechanics. Biomech. Model. Mechanobiol.
2006
,5, 64–81.
[CrossRef]
20.
Khella, C.M.; Asgarian, R.; Horvath, J.M.; Rolauffs, B.; Hart, M.L. An evidence-based systematic review of human knee post-
traumatic osteoarthritis (PTOA): Timeline of clinical presentation and disease markers, comparison of knee joint PTOA models
and early disease implications. Int. J. Mol. Sci. 2021,22, 1996. [CrossRef]
21.
Armiento, A.R.; Alini, M.; Stoddart, M.J. Articular fibrocartilage—Why does hyaline cartilage fail to repair? Adv. Drug Deliv. Rev.
2019,146, 289–305. [CrossRef]
22.
Hunziker, E.B. Articular cartilage repair: Are the intrinsic biological constraints undermining this process insuperable? Osteoarthr.
Cartil. 1999,7, 15–28. [CrossRef]
23.
Selig, M.; Azizi, S.; Walz, K.; Lauer, J.C.; Rolauffs, B.; Hart, M.L. Cell morphology as a biological fingerprint of chondrocyte
phenotype in control and inflammatory conditions. Front. Immunol. 2023,14, 1102912. [CrossRef] [PubMed]
24.
Kubo, S.; Uraki, Y.; Sano, Y. Preparation of carbon fibers from softwood lignin by atmospheric acetic acid pulping. Carbon
1998
,
36, 1119–1124. [CrossRef]
25.
Felka, T.; Rothdiener, M.; Bast, S.; Uynuk-Ool, T.; Zouhair, S.; Ochs, B.G.; De Zwart, P.; Stoeckle, U.; Aicher, W.K.; Hart, M.L.;
et al. Loss of spatial organization and destruction of the pericellular matrix in early osteoarthritis
in vivo
and in a novel
in vitro
methodology. Osteoarthr. Cartil. 2016,24, 1200–1209. [CrossRef] [PubMed]
Polymers 2023,15, 3041 13 of 14
26.
Rolauffs, B.; Williams, J.M.; Aurich, M.; Grodzinsky, A.J.; Kuettner, K.E.; Cole, A.A. Proliferative remodeling of the spatial
organization of human superficial chondrocytes distant from focal early osteoarthritis. Arthritis Rheum.
2010
,62, 489–498.
[CrossRef]
27.
Aurich, M.; Hofmann, G.O.; Best, N.; Rolauffs, B. Induced Redifferentiation of Human Chondrocytes from Articular Cartilage
Lesion in Alginate Bead Culture After Monolayer Dedifferentiation: An Alternative Cell Source for Cell-Based Therapies? Tissue
Eng. Part A 2018,24, 275–286. [CrossRef] [PubMed]
28.
Barisic, D.; Erb, M.; Follo, M.; Al-Mudaris, D.; Rolauffs, B.; Hart, M.L. Lack of a skeletal muscle phenotype in adult human bone
marrow stromal cells following xenogeneic-free expansion. Stem Cell Res. Ther. 2020,11, 79. [CrossRef]
29.
Bankhead, P.; Loughrey, M.B.; Fernández, J.A.; Dombrowski, Y.; McArt, D.G.; Dunne, P.D.; McQuaid, S.; Gray, R.T.; Murray,
L.J.; Coleman, H.G.J. QuPath: Open source software for digital pathology image analysis. Sci. Rep.
2017
,7, 16878. [CrossRef]
[PubMed]
30.
Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid,
B.J. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012,9, 676–682. [CrossRef]
31.
Arganda-Carreras, I.; Kaynig, V.; Rueden, C.; Eliceiri, K.W.; Schindelin, J.; Cardona, A.; Sebastian Seung, H.J.B. Trainable Weka
Segmentation: A machine learning tool for microscopy pixel classification. Bioinformatics 2017,33, 2424–2426. [CrossRef]
32.
Walters, B.; Turner, P.A.; Rolauffs, B.; Hart, M.L.; Stegemann, J.P. Controlled Growth Factor Delivery and Cyclic Stretch Induces a
Smooth Muscle Cell-like Phenotype in Adipose-Derived Stem Cells. Cells 2021,10, 3123. [CrossRef]
33.
Uynuk-Ool, T.; Rothdiener, M.; Walters, B.; Hegemann, M.; Palm, J.; Nguyen, P.; Seeger, T.; Stockle, U.; Stegemann, J.P.; Aicher,
W.K.; et al. The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the
stiffness of the biomaterial and by cyclic tensile forces. J. Tissue Eng. Regen. Med. 2017,11, 3508–3522. [CrossRef]
34.
Rabel, K.; Kohal, R.J.; Steinberg, T.; Tomakidi, P.; Rolauffs, B.; Adolfsson, E.; Palmero, P.; Furderer, T.; Altmann, B. Controlling
osteoblast morphology and proliferation via surface micro-topographies of implant biomaterials. Sci. Rep.
2020
,10, 12810.
[CrossRef]
35.
Walters, B.; Uynuk-Ool, T.; Rothdiener, M.; Palm, J.; Hart, M.L.; Stegemann, J.P.; Rolauffs, B. Engineering the geometrical shape of
mesenchymal stromal cells through defined cyclic stretch regimens. Sci. Rep. 2017,7, 6640. [CrossRef]
36. R Core Team. R: A Language and Environment for Statistical Computing; R Core Team: Vienna, Austria, 2013; pp. 275–286.
37. Harrell, F.E., Jr.; Harrell, M.F.E.J.C., Jr. Package ‘hmisc’. R Package Version 4.2-0. CRAN2018 2019,2019, 235–236.
38.
Wei, T.; Simko, V.; Levy, M.; Xie, Y.; Jin, Y.; Zemla, J.J.S. R Package “Corrplot”: Visualization of a Correlation Matrix (Version 0.84).
Statistician 2017,56, e24.
39.
Rohart, F.; Gautier, B.; Singh, A.; Le Cao, K.A. mixOmics: An R package for ’omics feature selection and multiple data integration.
PLoS Comput. Biol. 2017,13, e1005752. [CrossRef] [PubMed]
40.
Welham, Z.; Dejean, S.; Le Cao, K.A. Multivariate Analysis with the R Package mixOmics. Methods Mol. Biol.
2023
,2426, 333–359.
[CrossRef]
41.
Hall, A.C. The Role of Chondrocyte Morphology and Volume in Controlling Phenotype-Implications for Osteoarthritis, Cartilage
Repair, and Cartilage Engineering. Curr. Rheumatol. Rep. 2019,21, 38. [CrossRef]
42.
Zhang, Y.; Jiang, M.; Zhang, Y.; Cao, Q.; Wang, X.; Han, Y.; Sun, G.; Li, Y.; Zhou, J. Novel lignin-chitosan-PVA composite hydrogel
for wound dressing. Mater. Sci. Eng. C Mater. Biol. Appl. 2019,104, 110002. [CrossRef]
43.
Belgodere, J.A.; Zamin, S.A.; Kalinoski, R.M.; Astete, C.E.; Penrod, J.C.; Hamel, K.M.; Lynn, B.C.; Rudra, J.S.; Shi, J.; Jung,
J.P. Modulating Mechanical Properties of Collagen-Lignin Composites. ACS Appl. Bio. Mater.
2019
,2, 3562–3572. [CrossRef]
[PubMed]
44.
Eivazzadeh-Keihan, R.; Moghim Aliabadi, H.A.; Radinekiyan, F.; Sobhani, M.; Farzane, K.; Maleki, A.; Madanchi, H.; Mahdavi,
M.; Shalan, A.E. Investigation of the biological activity, mechanical properties and wound healing application of a novel scaffold
based on lignin-agarose hydrogel and silk fibroin embedded zinc chromite nanoparticles. RSC Adv.
2021
,11, 17914–17923.
[CrossRef]
45.
Silva-Barroso, A.S.; Cabral, C.S.D.; Ferreira, P.; Moreira, A.F.; Correia, I.J. Lignin-enriched tricalcium phosphate/sodium alginate
3D scaffolds for application in bone tissue regeneration. Int. J. Biol. Macromol. 2023,239, 124258. [CrossRef] [PubMed]
46.
Polo, C.C.; Pereira, L.; Mazzafera, P.; Flores-Borges, D.N.A.; Mayer, J.L.S.; Guizar-Sicairos, M.; Holler, M.; Barsi-Andreeta,
M.; Westfahl, H., Jr.; Meneau, F. Correlations between lignin content and structural robustness in plants revealed by X-ray
ptychography. Sci. Rep. 2020,10, 6023. [CrossRef] [PubMed]
47.
Tschaikowsky, M.; Selig, M.; Brander, S.; Balzer, B.N.; Hugel, T.; Rolauffs, B.J.O. Proof-of-concept for the detection of early
osteoarthritis pathology by clinically applicable endomicroscopy and quantitative AI-supported optical biopsy. Osteoarthr. Cartil.
2021,29, 269–279. [CrossRef]
48.
Selig, M.; Lauer, J.C.; Hart, M.L.; Rolauffs, B.J. Mechanotransduction and stiffness-sensing: Mechanisms and opportunities to
control multiple molecular aspects of cell phenotype as a design cornerstone of cell-instructive biomaterials for articular cartilage
repair. Int. J. Mol. Sci. 2020,21, 5399. [CrossRef]
49.
Ramirez, F.; Tanaka, S.; Bou-Gharios, G. Transcriptional regulation of the human alpha2(I) collagen gene (COL1A2), an informative
model system to study fibrotic diseases. Matrix Biol. J. Int. Soc. Matrix Biol. 2006,25, 365–372. [CrossRef]
Polymers 2023,15, 3041 14 of 14
50.
Van Kempen, L.C.; Rijntjes, J.; Mamor-Cornelissen, I.; Vincent-Naulleau, S.; Gerritsen, M.J.; Ruiter, D.J.; van Dijk, M.C.; Geffrotin,
C.; van Muijen, G.N. Type I collagen expression contributes to angiogenesis and the development of deeply invasive cutaneous
melanoma. Int. J. Cancer 2008,122, 1019–1029. [CrossRef]
51.
Liang, Y.; Diehn, M.; Bollen, A.W.; Israel, M.A.; Gupta, N. Type I collagen is overexpressed in medulloblastoma as a component of
tumor microenvironment. J. Neurooncol. 2008,86, 133–141. [CrossRef]
52.
Li, J.; Ding, Y.; Li, A. Identification of COL1A1 and COL1A2 as candidate prognostic factors in gastric cancer. World J. Surg. Oncol.
2016,14, 297. [CrossRef] [PubMed]
53. Gabay, C. Interleukin-6 and chronic inflammation. Arthritis Res. Ther. 2006,8, S3. [CrossRef] [PubMed]
54. Hirano, T. IL-6 in inflammation, autoimmunity and cancer. Int. Immunol. 2021,33, 127–148. [CrossRef] [PubMed]
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