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Cellular Senescence Program is Sensitive to Physical Differences in

Polymeric Tissue Scaffolds
Parul Yadav, Rahul Shah, Anindo Roy, Sibani Jani, Kaushik Chatterjee,* and Deepak Kumar Saini*
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ABSTRACT: A typical cellular senescence program involves exposing

Downloaded via Pascal Richard Niño Rodriguez on December 6, 2023 at 00:53:44 (UTC).

cells to DNA-damaging agents such as ionization radiation or chemo-

therapeutic drugs, which cause multipronged changes, including increased
cell size and volume, the onset of enhanced oxidative stress, and
inflammation. In the present study, we examined if the senescence onset
decision is sensitive to the design, porosity, and architecture of the
substrate. To address this, we generated a library of polymeric scaffolds
widely used in tissue engineering of varied stiffness, architecture, and
porosity. Using irradiated A549 lung cancer cells, we examined the
differences between cellular responses in these 3D scaffold systems and
observed that senescence onset is equally diminished. When compared to
the two-dimensional (2D) culture formats, there were profound changes
in cell size and senescence induction in three-dimensional (3D) scaffolds.
We further establish that these observed differences in the senescence state can be attributed to the altered cell spreading and cellular
interactions on these substrates. This study elucidates the role of scaffold architecture in the cellular senescence program.
KEYWORDS: Aging, Scaffolds, Biomaterials, Cellular Senescence, Tissue Engineering

1. INTRODUCTION be taken into consideration. Most of the preclinical and even

Aging is an inevitable process leading to a progressive decline clinical testing of the currently available biomaterials are largely
in the organism’s physiology and functionality. It is driven by performed in young individuals where the immune system,
the accumulation of DNA damage in cells, which leads to cell repair, and healing capacity are efficient.20−22,22−24 However,
cycle arrest and initiates a process called cellular senescence the regenerative capacities tend to be compromised in the
that leads to systemic aging if left unresolved.1 Cellular geriatric population leading to delayed recovery. This differ-
senescence has been implicated in many age-associated ence may be attributed to the altered host response to
ailments and is a major driver of several degenerative diseases, biomaterials in aged individuals25 which differs from that in
including cardiac and neuronal disorders, fibrosis, etc.,2 where younger individuals.
Aging is typically associated with chronic health problems,
changes in the mechanical properties of tissues are invariably
and geriatric patients are principal recipients of several
recorded. Besides, the removal of senescent cells has been
biomaterial-based assistive therapies. Despite the increasing
shown to improve organ function in mice and human
usage of biomaterials in aged patients, few studies examine the
tissues.3−6 Furthermore, several of these pathologies require
effect of aging on the host response to the material.26 Given
interventions that utilize some form of biomaterial−cell
that senescent cells accumulate with increased age, they add
interaction. This includes but is not limited to invasive devices,
another dimension to a large number of other factors which
catheters,7 pacemakers,8 long-term biosensors,9 and implants10
affect the biocompatibility of the introduced material. It is,
ranging from soft polymers to hard metals. The interactions
therefore, imperative to investigate the interaction of senescent
between cells and biomaterials play a critical role in modulating
cells with biomaterials to engineer implants and tissue scaffolds
tissue homeostasis in the biological response to materials. It
to promote tissue regeneration in older patients.
has been shown that such interactions influence cell
proliferation,11 differentiation,12 migration,13 immune re-
sponse,14 and survival to influence the repair or regeneration Received: July 3, 2023
at the implanted site. For any biomaterial to perform its Revised: September 19, 2023
intended function and have good biocompatibility in vivo, Accepted: September 22, 2023
several parameters, including its composition, surface top-
ography,15 mechanical properties,16,17 and infection resist-
ance,18 among others,19 are essential requirements that need to
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Numerous in vitro studies have underscored the roles of 2. MATERIALS AND METHODS
chemical, biomolecular, and physical properties of biomaterials
on cell function and fate. Fibroblasts exhibit broader and flatter 2.1. Scaffold Synthesis
morphology and generate more traction force on stiffer 2.1.1. Salt Leaching. Porous scaffolds of poly(ε-caprolactone)
substrates than softer ones.27 The growth of transformed (PCL, average molecular weight = 80,000, Sigma-Aldrich) were
cells has been demonstrated to be supported by gelatin-coated fabricated by salt leaching. PCL pellets were dissolved in 2,2,2-
substrates softer than 100 Pa; however, the same could not be trifluoroethanol (TFE, Spectrochem, India) at 10% (w/v) concen-
tration. The solution was stirred continuously for 12 h for uniform
observed in untransformed cells.28 Surface topography such as
mixing. Salt size ranging from 235 to 425 μm was selected as the
roughness and patterning−anisotropic or isotropic, in the form porogen, and the solution was poured into cylindrical molds of 48
of grooves, fiber, pits, pillars, etc., have been reported to well plates. 135 μL of the solution was poured into each well
markedly influence cellular fate, including for stem,29,30 containing 0.39 g of salt. The plate was dried for 24 h, and the salt was
cardiac,31 muscle,32,33 epithelial,34,35 and neuronal cells.36,37 leached out in a water bath, followed by air drying. Further, the
We and others have elucidated the effect of architecture on the scaffolds were sterilized by exposing them to ultraviolet (UV- 254
growth, migration, and metastasis of cancer cells.38 Other nm) light for 1 h, followed by 70% (v/v) ethanol washes. Hereafter,
reports revealed the effect of surface patterning and stiffness on these scaffolds will be referred to as SL.
cardiac cell function39 and osteogenic differentiation of human 2.1.2. Electrospinning. Nanofibers of PCL were fabricated by
electrospinning using the ESPIN NANO V1VC instrument. 12% (w/
mesenchymal stem cells,40 among others. The architecture of v) feed solution was prepared by dissolving PCL pellets in TFE and
the 3D scaffold can serve as a biophysical cue to influence cell stirring for 12 h. The parameters used to draw neat PCL fibers were as
response through a combination of altered geometry and follows: voltage of 12 kV, a distance of 15 cm between the electrode
mechanical properties (stiffness).41 tip and collector plate, and a flow rate of 0.5 mL/h. The fibers were
For most of these studies, the effect was recorded for collected on aluminum sheets. The nanofibrous mats were then cut
proliferating cells wherein the effect is dominated by changes into circular shapes (depending on the size of the well plate being
in the rate of cell proliferation as an outcome of the effect of used), washed with 70% (v/v) ethanol and sterilized by placing them
the substrate changes. However, during senescence, the cells under UV (254 nm) light for 1 h on each side, top, and bottom.
are in a state of irreversible growth arrest driven by presence of Hereafter, the nanofibrous mats will be referred to as NF.
2.1.3. Compression Molding. Flat PCL discs were fabricated by
persistent DNA damage,42 high concentration of reactive compression molding. PCL pellets were placed in cylindrical molds of
oxygen species (ROS),43 mitochondrial dysfunction,44 and an 2.5 cm diameter or 1 cm with 0.24 cm height. The PCL pellets were
overall inflammatory milieu.45,46 Effects of senescent cells and preheated in the mold for 30 min, followed by compression under a
associated inflammation include the recruitment of immune pressure of 2 MPa for 2 h. The fabricated discs were sterilized under
cells for their clearance,47 which disrupts normal tissue UV (254 nm) light for 1 h, followed by 70% (v/v) ethanol washes.
structure and function if left unresolved. Furthermore, Hereafter, the fabricated PCL discs will be referred to as CM.
implantation of a biomedical device/product in a geriatric 2.1.4. 3D Printing. 3D printed scaffolds were fabricated with a
patient can induce additional stress over and above the existing CELLINK BIO X 3D printer, using PCL pellets as the feed material.
A cylindrical 3D model of 12 mm diameter and 2.5 mm height was
senescent microenvironment due to aging.48−51 There is a modeled with Microsoft 3D Builder, and the structure was sliced with
limited understanding of how biomaterial properties influence CELLINK Heartwave and SLIC3R software. The infill pattern and
the senescence status of the cells. Previously, inherent density were set to rectilinear and 45%, respectively. Printhead and
differences were reported in senescent cell behavior when print bed temperatures were set to 130 and 12 °C, respectively. The
cultured on soft, porous scaffolds compared to conventional polymer was extruded with a pressure of 200 kPa, and the printhead
two-dimensional (2D) tissue culture plates.52 It was demon- speed was set to 3 mm/s. The printed scaffolds were sterilized under
strated that there is a fundamentally aberrant impact of UV (254 nm) light for 1 h followed by 70% (v/v) ethanol washes.
culturing cells on 2D platforms, which results in the Hereafter, the 3D-printed PCL constructs will be referred to as 3DP.
exacerbation of overall senescence signatures unlike what is 2.2. Scaffold Characterization
recorded in 3D culturing platforms. Scanning electron microscopy (SEM) imaging was carried out using a
The goal of this work was to investigate the effect, if any, of Carl Zeiss Ultra 55 FE-SEM instrument to study the surface
changes in 3D scaffold architectures on the senescence state of topography of the fabricated scaffolds. The scaffolds were gold-
the cells. We selected one of the most widely utilized sputtered before imaging. The images were analyzed using ImageJ
biomaterials, polycaprolactone (PCL), which has been used software to obtain scaffold surface parameters such as pore size, fiber
in several products and devices approved for clinical use. PCL diameter, interstrut distance, and diameter of struts. A universal
is cytocompatible, biodegradable, inexpensive, and easily testing machine (Instron 5967) was used for mechanical analysis of
3DP samples. Compression test was performed on cylindrical sample
processable, with excellent mechanical and degradation
with 5 kN load cell and crossover speed of 1 mm/min. The
properties affording the fabrication of a wide range of three- compression modulus was evaluated by calculating the slope of the
dimensional (3D) substrates for hard and soft tissues. In this stress−strain curve in the linear region. The storage modulus of the
study, scaffolds with different architectures and stiffness nanofibers (NF) was determined by carrying out dynamic mechanical
(ranging from ≈8 kPa to ≈320 MPa) were fabricated from analysis (DMA) using a TA Instruments Q 800 in film tension-
PCL such that their effect on the cellular response can be controlled force mode with a constant force of 3 N/min. Analysis was
analyzed. The fabricated scaffolds were characterized for their carried out with a rectangular-shaped sample of dimensions 15 mm ×
morphology using microscopy and various physical techniques. 10 mm × 0.2 mm, and the modulus was determined from the linear
Further, the cellular response of senescent cells was evaluated part of the stress−strain graph. Rectangular strips (15 mm × 5 mm ×
0.5 mm) of PCL were fabricated by compression molding and used to
using cell morphology and gene expression profile analysis. calculate the storage modulus of PCL. A frequency sweep from 0.1 to
This study can aid in designing biomaterials that reduce the 100 Hz was performed with the measurement at 1 Hz and a preload
adverse effects of senescent cells on an already compromised of 0.01 N. The same DMA instrument was used to determine the
environment and provide a template framework for screening elastic modulus of the SL scaffolds in compression mode. An
for appropriate material properties for geriatric applications. amplitude of 10 μm with a preload force of 0.01 N was used. All the

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Figure 1. SEM micrographs of the various scaffolds. (A) CM disc, (B) NF mat, (C) SL scaffolds, and (D) 3DP scaffolds. Scale bar represents 200
μm in A, C, and D and 2 μm in B.

DMA measurements were performed at 37 °C. We further performed (Amresco, USA) in PBS for 15 min at room temperature. Three PBS
atomic force microscopy (AFM) on only salt leached scaffolds, the washes were given, followed by the addition of a freshly prepared
details of which are mentioned in the Supporting Information (SI). staining solution. The staining solution comprised of 1 mg mL−1 X-gal
Differential scanning calorimetry (DSC) was also performed on all the (GoldBio Technology, USA) in 40 mM citric acid/sodium phosphate
samples (details in SI). (pH 6.0) with 150 mM NaCl, 2 mM MgCl2, 5 mM potassium
ferrocyanide and 5 mM potassium ferricyanide (all chemicals from
2.3. Cellular Studies
SRL, India). The cells were incubated in the staining solution
2.3.1. Cell Culture. The human lung epithelial carcinoma cell line, overnight, washed with 1X phosphate-buffered saline (PBS) twice,
A549, was procured from ATCC. Cells were cultured in Dulbecco’s and imaged using an inverted IX81 microscope equipped with a DP72
minimal essential media (DMEM, Sigma) supplemented with 10% color camera (Olympus, Japan). The presence of a blue-colored stain
(v/v) fetal bovine serum (FBS, Gibco, Invitrogen) and 1% antibiotics- in the cells confirmed senescence induction.
penicillin and streptomycin. Cells were cultured at 37 °C in a 5% CO2 2.3.5. Senescence-Associated Markers. NS and Sen A549 cells
humidified incubator. All the scaffolds used in the study were soaked were cultured either on 2D TCPS or scaffolds for 4 days. RNA was
in serum-containing medium (10% FBS+DMEM) for 24 h prior to isolated using the RNeasy Mini Kit (Qiagen) as per the protocol
cell seeding. described by the manufacturer. Several scaffolds were pooled for
2.3.2. Cellular Senescence Induction. A549 cells were exposed extracting RNA to obtain sufficient amounts from each scaffold
to 8 grays of gamma radiations (60Co) using a blood irradiator, BI system. Lysis buffer was added directly on NF, CM, and 3DP
2000 machine. Cells were trypsinized, the cell suspension (in scaffolds. For SL scaffolds, each scaffold was cut into 4 smaller pieces
DMEM) was irradiated in a 15 mL sterile polypropylene tube, and using a scalpel, which was followed by the addition of lysis buffer. 500
cells were immediately used for experimental seeding. Irradiated ng of RNA was used to synthesize cDNA using the Verso cDNA
(senescent, Sen) cells and nonirradiated (nonsenescent, NS) cells synthesis kit (Thermofisher Scientific) according to the manufac-
were counted and seeded on tissue culture polystyrene (TCPS) turer’s instructions. A quantitative real-time polymerase chain reaction
dishes. Similarly, cells were seeded on different scaffolds. (qRT-PCR) was performed using QIAquant 96 real-time PCR
2.3.3. Morphology Analysis. A549 cells, Sen and NS, were (Qiagen) using PowerUP SYBR Green Master Mix (Applied
stained for F-actin and nucleus 4 days after culture in various matrices. Biosystems). Gene expression was determined by calculating fold
Briefly, cells on scaffolds and 2D TCPS were fixed using 4% change using 2−ΔΔct, and GAPDH was used as the housekeeping gene.
paraformaldehyde for 15 min, followed by permeabilization using The list of primers used, and their sequence is mentioned in Table S1.
0.2% TritonX-100. The cells were then stained using phalloidin 2.3.6. Western Blot. NS and Sen A549 cells were cultured on 2D
conjugated with Alexa Fluor 488 (Invitrogen) for 1 h at 25 °C, and TCPS (60 mm Petri dish) for 4 days. We varied the cell density for
DAPI (Sigma-Aldrich) was used at 1 μg/mL for 5 min at 25 °C to Sen A549 cells from 1.5, 3, 6, and 12 x105 on the 60 mm Petri dish to
stain the nuclei. The samples were imaged using a laser scanning restrict cell spreading. Since NS A549 will exhibit proliferation, these
confocal microscope (Zeiss LSM 880), and the maximum intensity cells were seeded at 1.5 x105 as control. Cell lysate was prepared using
projections were used to calculate the cellular area using ImageJ Mammalian cell lysis buffer (GoldBio, USA) as per manufacturer’s
software. protocol. The total amount of protein in the lysate was estimated
2.3.4. Senescence-Associated β-Galactosidase Activity using Bradford’s protein estimation reagent. For Western blot, 50 μg
(SAβGal). To confirm senescence in the irradiated cells, 4 days of total protein was resolved on 12% SDS-PAGE gel. This was then
postcell seeding, both the NS and Sen cells cultured on the 2D TCPS transferred to PVDF membrane (Merck, India) using a semidry
were stained for SaβGal., as per the protocol described by Dimri et transfer unit (Power Blotter System, Invitrogen, USA) at 25 V for 12
al.53 Briefly, the cells were fixed using 0.2% (v/v) glutaraldehyde min. The membrane with transferred protein was blocked using Tris

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buffered saline with Tween 20 (TBST) buffer containing 5% BSA

protein (MP Biomedicals, USA) for 1 h. The membrane was further
incubated in primary antibody diluted in TBST at 4 °C overnight.
Membrane was washed thrice for 10 min using TBST. This was
followed by incubation with appropriate HRP tagged secondary
antibody (Jackson Laboratories Inc., USA) diluted as per
manufacturer’s instructions and incubated for 1 h at 27 °C.
Membrane was again washed thrice using TBST and subjected to
chemiluminescence detection using ECL substrate (Clarity Western
ECL substrate, Bio-Rad, USA). The blots were imaged in a
ChemiDoc MP imaging system (Bio-Rad Inc., USA) at multiple
exposure settings. The details of antibodies used have been mentioned
in Table S2.
2.4. Statistical Analysis
The data were statistically analyzed by performing either one-way
ANOVA or unpaired t test to evaluate the significant differences
between the samples. Data are presented as mean ± standard error of
the mean for n = 3.

3. RESULTS
To investigate the role of scaffold architecture on senescent
cell behavior, PCL scaffolds with different architectures were
fabricated using three techniques: salt leaching (variable
pores), electrospinning (unaligned nanofibers), and 3D Figure 2. Quantification of scaffold parameters. (A) SL pore diameter,
printing (fixed pores). TCPS, which is the conventional (B) interstrut distance in 3DP, (C) strand diameter of 3DP, and (D)
substrate of choice, was used as the 2D control (flat surface). NF fiber diameter. Error bars indicate the standard error of the mean.
Compression-molded (flat) discs of PCL sheets served as an
additional 2D control to assess the differences in material very well established that 2D culture systems do not
composition between TCPS and PCL. The fabricated scaffolds recapitulate several of the in vivo properties of human tissues,
exhibited a wide variety of topographies with varying structural including architecture, stiffness, cellular interactions, matrix
parameters such as porosity and fiber diameter. modeling, etc. Therefore, the scaffold systems provide us with
a better mimic of the microenvironment and thus would be an
3.1. Scaffold Characterization
appropriate/suitable alternative to conventional culture
The topography of the fabricated scaffolds was characterized techniques.
using SEM. SEM images of the compression-molded discs 3.2. Ionization Radiation-Based Cellular Senescence
revealed a smooth surface (Figure 1A). SEM micrographs of Induction
the nanofibers showed the random (unaligned) arrangement of
To optimize senescence induction by irradiation in A549 cells,
fibers deposited by electrospinning (Figure 1B), wherein the
a preliminary analysis was done by comparing the cell size of
mean fiber diameter of the fibers was 585 ± 58 nm (Figure
NS A549 cells with those of Sen A549 cells on 2D TCPS using
2D). The fibers were of uniform sizes throughout the
ImageJ. Bright-field microscopic images of NS and Sen A549
nanofibrous sheet. SEM images of the salt-leached scaffolds
cells are presented in Figure 3A and B, respectively. On
revealed the roughness and high porosity of the scaffold
average, the NS samples had an area of 1513 ± 82 μm2 and a
(Figure 1C). The average pore diameter was 320 ± 77 μm,
perimeter of 164 ± 14 μm, whereas the Sen cells had an area of
with pore sizes ranging from 130 to 480 μm (Figure 2A). SEM
7737 ± 70 μm2 and a perimeter of 358 ± 14 μm (Figure S1),
images of the 3D printed scaffolds showed their smooth
implying a significant increase in cellular area and spreading.
surface and uniform strut diameters and pore sizes (Figure
These trends corroborate the observation in the literature
1D). The average strand diameter was 340 ± 23 μm (Figure
where the senescent cells are characterized by their larger size
2C), and the interstrut distance was 240 ± 17 μm (Figure 2D).
in 2D culture. To further verify senescence induction, samples
All scaffolds depicted porous structures with good intercon-
were stained for SAβGal activity, which is known to be higher
nectivity which is essential for cell proliferation and survival.
in the Sen cells compared to the NS cells53 (Figure 3D and E).
DSC result showed all samples to be semicrystalline (Figure S2
NS showed, on average, 21% staining, whereas Sen exhibited
and Table S4).
78% staining (Figure 3F). Moreover, the Sen cells exhibited
The mechanical properties of the scaffolds were determined
increased expression of known markers, cell-cycle inhibitor
by UTM and DMA. The compressive modulus of the 3DP
p21, and senescence-associated inflammatory markers IL-6 and
scaffolds evaluated from the stress−strain curve obtained from
IL-8, as compared to NS, confirming the senescent state of the
universal testing machine (UTM) was 54 ± 5 MPa. The
A549 cells postirradiation (Figure 3G−I).
storage modulus of the NF assessed from the data obtained
from DMA was 1.61 ± 0.2 MPa, whereas that of SL porous 3.3. Effect of Substrate Architecture on Cell Morphology
scaffolds was 7.82 ± 1.5 kPa, and that of CM sheets was 315 ± The effect of the scaffold architecture was studied by staining
2 MPa. The 3DP ones displayed the highest stiffness of all the the cells for F-actin and nucleus. Figure 4 shows the cellular
scaffolds, followed by NF and SL, owing to their architectural distribution and morphology of each type of scaffold. We
differences. Solid PCL CM discs further had high stiffness observed that the Sen A549 cells on all the culture platforms
compared to other samples; however, they were still had a significantly higher area than their NS counterpart
significantly lower than 2D TCPS (≈2.79 GPa). It is now corroborating well-established trends on planar 2D substrates.
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Figure 3. Assessment of senescence induction in A549 cells. Bright-field images of (A) NS and (B) Sen A549 cells (scale bar represents 80 μm).
Quantification of area (C) of the cells. SaβGal staining of (D) NS and (E) Sen cells and quantification of the percentage of positively stained cells
(F) (scale bar represents 100 μm). Gene expression analysis of (G) p21, (H) IL6, and (I) IL8. Error bars indicate the standard error of the mean.
An unpaired t test was performed. **** indicates p < 0.0001, and * indicates p < 0.05.

Interestingly, NS cells on NF, SL, and 3DP scaffolds had a hundred folds in humans, ranging from red blood cells54 (≈8
lesser area than 550 μm2; however, cells on CM discs had an μm) to neurons (≈100 μm). However, within each cell type,
area of 1267 μm2 (≈2.3 times higher), suggesting that the there is a tight regulation of standard size with little deviation.
substrate architecture does play a role in determining the Cell spread area has been shown to be correlated with several
cellular shape and spread. When irradiated cells were allowed important cell functionalities, and deviation from this has been
to become senescent on these platforms, we observed that Sen shown to slow down cell processes. The work of Neurohr et al.
cells on CM displayed the highest cell spreading (≈5000 μm2) showed that, with increasing cell size, the cell could not meet
among the fabricated substrates. All the other scaffolds had a the demand of synthesizing enough proteins to maintain
cellular area lesser than 1500 μm2. On the TCPS control normal cell functions.55 This results in dilution of the
(Figure 4I), we found that the NS cells measured ≈1500 μm2, cytoplasm and disruption of cell division. With cellular DNA
whereas the Sen cell population averaged around ≈7700 μm2. already damaged during senescence, it leads to disruption of
These trends suggest that the Sen cells cultured on 3D the surface area to volume ratio, resulting in overall functional
scaffolds measure (<1500 μm2) nearly equal to those of NS decline. The 3D scaffold systems used in this study partially
cells (≈1500 μm2) on the 2D (TCPS and CM). This restrict this phenomenon, thereby indirectly reducing the
important observation indicates that the enlargement or detrimental effects or the extent of damage to Sen cells.
spreading of senescent cells (widely recognized as a marker Notably, Sen cells were well spread on both 2D formats
of senescence induction in vitro) is indeed a culture-induced (TCPS and CM), indicating that the architecture is dominant
(2D format) artifact and that this could possibly be a reason over differences in material composition and/or hydro-
for elevated senescence signatures on TCPS that have been phobicity. The putative differences in composition, wettability,
reported to date in the literature. We have reported a similar and possibly minor differences in surface roughness seem to
observation wherein irradiated HeLa cells cultured on soft, have induced only modest differences in cell spreading. We
porous PCL scaffolds had lower cell area and subdued propose that the design of a biomedical device must account
senescence signatures than TCPS.52 Cell size can vary a for the differences in the cellular response to biomaterials by
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(Figure 5A). Inflammation was present in all the scaffold

systems as senescent cells are known to maintain slow chronic
inflammatory status.51
Among the scaffold systems, SL and 3DP exhibited the
lowest upregulation in senescence pathway induction (p21).
Cells on CM had the highest levels of p21 expression since the
stiffness of the substrate was high with no structural variations.
In comparison, 3DP scaffolds with relatively lower stiffness but
porous architecture depicted lower induction of p21, implying
that architecture contributes to the cellular response. Softer
scaffolds, SL and NF, each displayed lower inflammatory status
too. Several studies have highlighted the importance of stiffness
in regulating senescence via cytoskeletal changes (mechano-
transduction), and that could be a plausible reason for the
effects seen in these various scaffolds. Further, pore size
(architecture) is also reported to alter cellular function. Since
several factors can modulate cellular response, we envision that
the effect we see in the NS and Sen cells on different structural
modalities is a multifactorial outcome. Since the cellular area
on scaffolds had decreased markedly, we hypothesized if cell
spreading (and thereby cell area) plays a role in senescence
induction. We performed a simple experiment of restricting
cellular spread by seeding Sen A549 on a 60 mm Petri dish and
varying cell seeding density by seeding at 1.5, 3, 6, or 12 (×
105) cells/dish. This allowed for abundant spread area in the
lowest cell density to crowding and thus restricted spreading
for the highest cell density. We then probed p21 protein levels
by Western blotting and found that the available cell spread
area has a direct effect on senescence induction. The lower cell
densities (1.5 and 3 × 105) demonstrated upregulation of p21
whereas the higher cell densities (6 and 12 × 105) showed a
decrease (Figure 5B). This again reiterates the fact that cell
size is a crucial determinant of cellular functionality. The
aberrant increase in cell size during senescence seen in vitro is a
culture artifact and thereby the effects seen are misrepre-
sentation of the in vivo mechanisms. 3D scaffolds help
minimize these effects and offer important design parameters
for engineering tissue scaffolds for regeneration in vivo and
better biomimetic in vitro model systems. This study
demonstrates that culture substrate plays an important role
in senescence induction program.
Figure 4. Cell distribution and spread on the scaffolds. (A−H) NS
and Sen A549 cells were stained for F-actin (green) and nucleus 4. DISCUSSION
(blue) with the corresponding quantification of the cellular area on
Aging is a significant contributor to several pathologies and is
each scaffold. An unpaired t test was performed. **** indicates p <
0.0001. Error bars indicate the standard error of the mean. (I) the greatest risk factor for most chronic diseases,56 and cellular
Comparative analysis of the cellular area of the culture platforms. senescence is a major contributing factor to this.57−61
Error bars indicate the standard error of the mean. Detailed statistical Senescence is a stress response to numerous distinct stimuli
analysis of each group is shown in Table S3 of the SI. leading to the progressive deterioration of cellular functions at
multiple levels. It is essentially an irreversible growth-arrested
state when cells are exposed to a high but sublethal dose of
careful design to prevent further damage and distress to an DNA damage.1 Senescent cells are characterized by stable cell
existing senescent milieu/microenvironment. cycle arrest,62,63 altered gene expression, resistance to
3.4. Senescence and Inflammation Status of Senescent apoptosis, chromatin reorganization64−66 as well as morpho-
Cells on Scaffolds logical and metabolic changes.67 In vitro, these cells have a
Cells actively respond to their surrounding environment, which larger and flatter body as compared to healthy cells with
includes both the chemical and physical environment they enhanced actin stress fibers.55,68 They are also characterized by
reside in and modulate their behavior accordingly. It is now large nuclei, multinuclei, and chromatin reorganization and are
established that substrate stiffness plays a vital role in this relatively more granulated than their nonsenescent counter-
process and can lead to different outcomes of cellular part.69−71 Several of the age -associated diseases require
physiology. When senescent cells were cultured on the various treatments in the form of replacement, support, or intervention
scaffolds, we found that despite upregulation of p21, in using some form of biomaterials. It could range from the
response to the DNA damage the cells were subjected to, the insertion of pacemakers,72 stents, plates, screws,73 etc., to
levels were consistently lower than the Sen cells on 2D TCPS breast implants,74 catheters,75 dental implants,10 and even
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Figure 5. Senescence and inflammation-associated gene expression analysis. (A) Gene expression analysis of p21, IL6, and IL8 in NS and Sen A549
cells cultured on various scaffold systems. Dotted line represents NS of individual scaffold (which has been normalized to 1). Error bars indicate the
standard error of the mean. (B) Protein levels of p21 analyzed through Western blotting and quantification.

contact lenses.76 With the decline in overall functionality and modulating cellular properties. When variation in pores and
regenerative capabilities of geriatric populations, the success geometry was introduced, there was little difference between
rates of biomaterials also fall.77 Thus, investigating the the fabricated scaffolds. We observed that all PCL substrates
interactions between aged tissue/cells and biomaterials (2D and 3D) were semicrystalline. Though there were some
remains essential. Understanding the response of aged cells differences in the degree of degree of crystallinity, there were
to different topological cues and structures needs to be no clear trends correlating it with the senescence markers,
evaluated when developing materials that directly interact with which further indicates that differences in architecture
aged cells for applications, as both topical and interventional primarily affected the cell response.
forms of biomaterials lead to foreign body reactions which act Studies have shown that pore diameter and geometry affect
as an added stress on an already existing inflammatory the alignment of cells and, ultimately, the structure of the
microenvironment.78,79 desired tissue.80 Different studies have shown that higher cell
This study examined the effect, if any, of the 3D scaffold viability and proliferation are associated with porous
architecture on the senescent cells. We recorded that substrate biomaterials with bigger pores, which can be related to better
properties affected the cell morphology of the A549 cells and diffusion of nutrients and gases in the interior region of these
scaffold stiffness varied from kPa to MPa compared to 2D structures.81 Nanoscale topographies are also known to elicit
TCPS (GPa). For a given material, i.e, PCL, we varied the diverse cell behavior, ranging from cell adhesion, orientation,
structural properties of the scaffolds resulting in various pore motility, and cytoskeletal changes, and modulate intracellular
sizes, stiffness, and architecture. These led to significant signaling pathways.82 The type of topography, such as fibers,
changes in cellular morphology and arrangement, thereby ridges, pillars, grooves, and pits, and their symmetry, e.g.,
modulating senescence signatures. The cellular area between aligned, random, or orthogonal, have also been reported to
scaffolds did not vary much in between 3D, but there was a modulate cell behavior.15 In a study by Kumar et al., it was
significant difference between NS and Sen cell populations. On demonstrated that human bone marrow stromal cells expressed
CM discs, NS cell areas are similar to those of Sen cells in the unique gene expression signatures depending on the type of
scaffolds. Compared to the 2D TCPS, there was no difference scaffold structure.83 Scaffolds with nanofibers could drive these
between the NS cell area of CM; however, a lower stiffness of cells to osteogenic lineage without additional supplements. In
CM did render a decreased area in the Sen cell population. our study, all the scaffolds depicted an increase in the cell cycle
This suggests that stiffness is an essential parameter in inhibition gene, p21; however, the levels were lower than those
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ACS Mater. Au XXXX, XXX, XXX−XXX
ACS Materials Au pubs.acs.org/materialsau Article

for 2D TCPS. The presence of an inflammatory milieu was Rahul Shah − Department of Materials Engineering, Indian
evident in all the cultural systems. This implies that senescent Institute of Science, Bangalore, India 560012
cells respond to the inherent material property rather than Anindo Roy − Department of Materials Engineering, Indian
architecture in macro-sized scaffold systems. This study further Institute of Science, Bangalore, India 560012; orcid.org/
strengthens the fact that biological processes vary significantly 0000-0001-7307-3436
between substrates, and there is a need to investigate aging/ Sibani Jani − Department of Bioengineering, Indian Institute of
senescence on more platforms. Since the cellular response Science, Bangalore, India 560012
depends on multiple components such as substrate stiffness, Complete contact information is available at:
architecture, pore size, presence of fibers, alignment, etc., we https://pubs.acs.org/10.1021/acsmaterialsau.3c00057
believe that several/combinations of these are involved in
invoking a specific outcome. We also established that cell Author Contributions
morphology plays an important role in determining biological
response.84 However, additional studies will be necessary to CRediT: Parul Yadav conceptualization, data curation, formal
design effective implants, materials, and tissue engineering analysis, writing-original draft; Rahul Shah conceptualization,
scaffolds to meet the need of an increasingly aged population. data curation, formal analysis, writing-original draft; Anindo
Roy data curation, formal analysis, writing-review & editing;
5. CONCLUSION Sibani Jani data curation, formal analysis; Kaushik Chatterjee
conceptualization, funding acquisition, project administration,
We prepared a library of culture platforms using PCL to test supervision, writing-review & editing; Deepak Kumar Saini
differences in the cellular senescence induction program. This conceptualization, project administration, supervision, writing-
was achieved by varying the 3D scaffold stiffness and porosity review & editing.
and comparing it with the conventional culture method of 2D
culture dishes and 2D substrates of PCL. We observed that Notes
while there were subtle differences between the different 3D The authors declare no competing financial interest.
scaffold systems, marked alterations were found in comparison
with 2D substrates. We attributed these differences to changes
in cell size which leads to changes in cellular functionality. This
study highlights the role of cellular size, substrate architecture,
■ ACKNOWLEDGMENTS
The authors acknowledge support from the Science and
and stiffness in regulating cellular senescence. Further, these Engineering Research Board (SERB), Government of India
3D scaffolds can be utilized for testing of drugs to eliminate (IPA/2020/000025). The authors acknowledge the central
senescent cells such as senolytics and senostatics, as they imaging facility of the Division of Biological Sciences and the
mimic the in vivo tissue architecture more closely than 2D mechanical testing facility at CeNSE. Authors also thank Dr.
culture plates. Sagar Nilawar for his help with DMA.

■
*
ASSOCIATED CONTENT
sı Supporting Information
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■ AUTHOR INFORMATION
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of Science, Bangalore, India 560012; orcid.org/0000- Aged-senescent Cells Contribute to Impaired Heart Regeneration.
0002-6257-7783 Aging Cell 2019, 18 (3), No. e12931.

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