3D nanofiber scaffolds from 2D electrospun membranes boost cell penetration and positive host response for regenerative medicine

Abstract

The ideal tissue engineering scaffold should facilitate rapid cell infiltration and provide an optimal immune microenvironment during interactions with the host. Electrospinning can produce two-dimensional (2D) membranes mimicking the extracellular matrix. However, their dense structure hinders cell penetration, and their thin form restricts scaffold utility. In this study, latticed hydrogels were three-dimensional (3D) printed onto electrospun membranes. This technique allowed for layer-by-layer assembly of the membranes into 3D scaffolds, which maintained their resilience impressively under both dry and wet conditions. We assessed the cellular and host responses of these 3D nanofiber scaffolds by comparing random membranes and mesh-like membranes with three different mesh sizes (250, 500, and 750 μm). It was found that scaffolds with a mesh size of 500 μm were superior for M2 macrophage phenotype polarization, vascularization, and matrix deposition. Furthermore, it was confirmed by subsequent experiments such as RNA sequencing that the mesh-like topology may promote polarization to the M2 phenotype by affecting the PI3K/AKT pathway. In conclusion, our work offers a novel method for transforming 2D nanofiber membranes into 3D scaffolds. This method boasts flexibility, allowing for the use of varied electrospun membranes and hydrogels in terms of structure and composition. It has vast potential in tissue repair and regeneration.

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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
https://doi.org/10.1186/s12951-024-02578-2 Journal of Nanobiotechnology
†Lingfei Xiao, Huifan Liu and Huayi Huang authors contribute equally
to this work.
*Correspondence:
Zhen Geng
nanboshan1987@163.com
Lin Cai
orthopedics@whu.edu.cn
Feifei Yan
yanfeifei0120@whu.edu.cn
1Department of Spine Surgery and Musculoskeletal Tumor, Zhongnan
Hospital of Wuhan University, Wuhan 430071, China
2Department of Anesthesiology, Research Centre of Anesthesiology and
Critical Care Medicine, Zhongnan Hospital of Wuhan University,
Wuhan 430071, China
3Department of Respiratory and Critical Care Medicine, Renmin Hospital
of Wuhan University, Wuhan 430071, China
4The Institute of Technological Science, School of Power and Mechanical
Engineering, Wuhan University, Wuhan 430072, China
5Institute of Translational Medicine, Shanghai University,
Shanghai 200444, China
6National Center for Translational Medicine (Shanghai) SHU Branch,
Shanghai University, Shanghai 200444, China
Abstract
The ideal tissue engineering scaold should facilitate rapid cell inltration and provide an optimal immune
microenvironment during interactions with the host. Electrospinning can produce two-dimensional (2D)
membranes mimicking the extracellular matrix. However, their dense structure hinders cell penetration, and
their thin form restricts scaold utility. In this study, latticed hydrogels were three-dimensional (3D) printed onto
electrospun membranes. This technique allowed for layer-by-layer assembly of the membranes into 3D scaolds,
which maintained their resilience impressively under both dry and wet conditions. We assessed the cellular and
host responses of these 3D nanober scaolds by comparing random membranes and mesh-like membranes
with three dierent mesh sizes (250, 500, and 750μm). It was found that scaolds with a mesh size of 500μm
were superior for M2 macrophage phenotype polarization, vascularization, and matrix deposition. Furthermore, it
was conrmed by subsequent experiments such as RNA sequencing that the mesh-like topology may promote
polarization to the M2 phenotype by aecting the PI3K/AKT pathway. In conclusion, our work oers a novel
method for transforming 2D nanober membranes into 3D scaolds. This method boasts exibility, allowing for the
use of varied electrospun membranes and hydrogels in terms of structure and composition. It has vast potential in
tissue repair and regeneration.
Keywords Electrospun nanober membranes, Three-dimensional scaolds, Tissue engineering, Macrophage
polarization, Mesh-like
3D nanober scaolds from 2D electrospun
membranes boost cell penetration
and positive host response for regenerative
medicine
LingfeiXiao1†, HuifanLiu2†, HuayiHuang1†, ShujuanWu3, LongjianXue4, ZhenGeng5,6*, LinCai1* and FeifeiYan1*
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 2 of 17
Xiao et al. Journal of Nanobiotechnology (2024) 22:322
Introduction
Tissue engineering offers a promising solution to tissue
repair and regeneration [1, 2]. e design and construc-
tion of tissue engineering scaffolds is essentially the art
of arranging specific spaces such as channels and pores
within the scaffold. Such porous scaffolds offer a 3D sup-
portive environment conducive to cell growth [3, 4]. e
scaffold’s porosity, pore dimensions, and surface attri-
butes optimize nutrient and oxygen delivery, guiding cel-
lular behavior and thereby directing tissue restoration
[5–7].
Electrospinning offers capabilities for fabricating mate-
rials at a much finer scale compared to other techniques
such as freeze-drying, sacrificial templating, 3D print-
ing, and melt electrowriting [8]. It is a versatile method
for generating ultrafine fibers ranging from nanometer
to micrometer scales. e resulting membranes support
cell adhesion, nutrient transfer, and new tissue forma-
tion [9, 10]. e topographical cues on the electrospin-
ning membrane can modulate individual cell morphology
and overall cell patterning, subsequently determining cell
fate [11]. For instance, electrospinning fibers with paral-
lel alignment have been found to promote the polariza-
tion of macrophages toward an M2 phenotype [12, 13].
Furthermore, a mesh-like electrospinning membrane
encouraged the secretion of anti-inflammatory and pro-
angiogenic cytokines by adipose-derived mesenchymal
stem cells [14]. In contrast, a latticed electrospinning
membrane could upregulate the HIF-1 signaling path-
way, which promotes vascularization and enhances bone
regeneration [15]. However, the density of the mem-
branes hinders cell infiltration, and their thinness, exist-
ing as a flexible two-dimensional surface, restricts their
use as scaffolds [16].
e conversion of two-dimensional nanofiber mem-
branes into 3D nanofiber scaffolds broadens the applica-
tions of electrospinning [17, 18]. Notably, such scaffolds
can be used to address defects in three-dimensional
spaces, specifically in bones and cartilage, which har-
ness the benefits of nanofibers creating a conducive envi-
ronment for cells mirroring their native in vivo [19]. A
variety of fabrication techniques exist for these 3D scaf-
folds, including multilayering electrospinning, sacrificial
agent electrospinning, wet electrospinning, ultrasound-
enhanced electrospinning, and several post-processing
methods like short fiber assembly, gas foaming, ultrason-
ication, and electrospraying [17–20]. Among these, the
short fiber assembly method stands out. In this method,
the electrospinning membrane is fragmented into short
fibers, which are then reconstructed into a sponge-like or
3D-printed scaffold. Such scaffolds have interconnected
pores fortified with these short fibers, optimizing them
for cell adhesion, nutrient transport, and immunomodu-
lation [21–23]. Gas foaming is another notable technique.
It involves stretching the dense electrospinning mat using
air bubbles. is action sporadically connects the lay-
ers to form a three-dimensional scaffold. Impressively,
this technique preserves the original aligned topologi-
cal characteristics, which play crucial roles in directing
cell migration and enhancing tissue growth and bone
regeneration [24, 25]. However, many of these methods
fall short in manifesting detailed topological features on
the scaffold’s surface, like precise porosity and pattern-
ing. e layer-by-layer stacking technique fills this gap.
It merges layers created via other methods with those of
electrospun ones. For instance, merging electrospinning
layers with 3D-printed ones could steer immune polar-
ization towards M2, which was conducive to vascular-
ization and bone regeneration [26]. Further, combining
the melt electrowriting mesh with electrospinning layers
yielded a lightweight scaffold, which minimally triggered
foreign body reactions while ensuring excellent biocom-
patibility [27]. However, in the layer-by-layer stacking
method, the electrospun layers in previous studies only
constituted a minor portion of the scaffold, failing to har-
ness the full potential of nanofibers in mimicking the 3D
extracellular matrix. Moreover, studies focusing on the
influence of membrane topography on the efficacy of tis-
sue engineering scaffolds remain sparse.
Here, we combined template-assisted electrospinning
with DLP (Digital Light Processing)-based 3D print-
ing technology. A small amount of latticed hydrogels
were printed on electrospun membranes, enabling the
membranes to be assembled layer by layer into a three-
dimensional scaffold, ensuring inter-layer spaces for cell
growth. In addition, the pattern of the monolayer mem-
brane can be tailored based on the received template
(Fig.1A). Considering the effects of membrane pores and
topological morphology on cell permeation and immune
response, we prepared three-dimensional scaffolds con-
sisting of random membranes and mesh-like nanofiber
membranes with three different mesh sizes (250, 500, and
750μm). rough in vitro cellular experiments and sub-
cutaneous embedding experiments, we have found the
most suitable 3D scaffolds for rapid tissue ingrowth and
good immune response, which will provide a reference
for future applications in tissue repair (Fig.1B).
Materials and methods
Materials
PCL (Mw ≈ 80,000) and silkworm cocoon were purchased
from Macklin Co., Ltd. (Shanghai, China). Sodium
bicarbonate, lithium bromide, hexafluoro isopropanol
(1,1,1,3,3,3-hexafluoro-2-propanol, HFIP), and lithium
phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were
obtained from Sigma-Aldrich (Shanghai, China). RPMI-
1640, α-MEM, Penicillin-streptomycin, Fetal bovine
serum (FBS), Phosphate buffer saline (PBS), and Trypsin
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Page 3 of 17
Xiao et al. Journal of Nanobiotechnology (2024) 22:322
were purchased from HyClone Laboratories Inc. (UT,
USA). Gelatin methacryloyl (GelMA) (Degree of sub-
stitution: 90%) was purchased from Cure Gel Co., Ltd.
(Wenzhou, China). For the cell culture experiment, fetal
bovine serum (FBS), Dulbecco’s modified Eagle’s medium
(DMEM), alpha-modified Eagle’s medium (α-MEM),
phosphate-buffered saline (PBS), trypsin-EDTA, and
penicillin/streptomycin (P/S) were purchased from
HyClone Laboratories Inc. (UT, USA). e Cell Count-
ing Kit-8 (CCK-8) was obtained from Dojindo Laborato-
ries, Kumamoto, Japan. e live/dead cell staining kit was
supplied by BestBio Biotechnologies (Shanghai, China).
Triton X-100 (Sigma–Aldrich), 4′,6-diamidino-2-phe-
nylindole (DAPI, Solarbio), and Cy3-labeled phalloidin
(Solarbio) were used for cell staining. All the antibodies
used in this study were purchased from Abclone Tech-
nologies Co., LTD. (Wuhan, China).
Extraction of silk broin
Silk cocoons were sectioned into approximately 25
mm2 segments and submerged in boiling 0.2M sodium
bicarbonate solution, stirred constantly to enhance the
Fig. 1 Schematic overview of the research. (A) Production of a mesh-like porous nanober membrane using a template-assisted electrospinning tech-
nique. This was followed by the fabrication of a latticed hydrogel layer on the nanober membrane using 3D printing technology, which was then
sequentially assembled to obtain a three-dimensional nanober scaold. (B) Three-dimensional scaolds composed of mesh membranes of appropriate
size proved advantageous for rapid cell inltration and macrophage polarization to the M2 phenotype when compared to those composed of unordered
membranes. Ultimately, this facilitates faster tissue integration and increased vascularization
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
interaction between the silk and the alkaline medium.
After degumming, the silk underwent rigorous rins-
ing with ultrapure water before drying for subsequent
applications. A concoction, comprising 20 g of lithium
bromide, 25g of water, and 6g of degummed silk, was
uniformly blended and then placed in a 55°C water bath
for 4h until it clarified. Dialysis was then performed on
this mixture using a bag with a molecular weight cut-off
of 8,000, against ultrapure water at 4°C. is four-day
procedure involved bi-daily water changes to expel the
lithium bromide. Following dialysis, the solution under-
went centrifugation at 4,500 rpm to discard insoluble
matter. e resulting solution was frozen, lyophilized,
and conserved at -20°C for subsequent applications.
Preparation of nanober membrane
A solvent was formulated by dissolving 0.8g of PCL and
0.2g of Silk Fibroin (SF) in 10ml of hexafluoroisopropa-
nol. en it was mixed up and down with a rotary mixer
for 12h to achieve a transparent solution. is solvent
was loaded into a 5ml plastic syringe for electrospinning.
An electrospinning device, sourced from Yongkang Le
Industry Company, China, was utilized. Preset param-
eters included a steady ambient temperature and humid-
ity, a 22G needle, 1ml/h spinning rate, 16 kV voltage,
20cm spinning distance, and a collector of either tin foil
or stainless steel mesh with assorted pore diameters (250,
500, 750 microns). e random or mesh-like electros-
pun membrane underwent immersion in methanol for
15min, facilitating the transformation of the α-helix in
silk fibroin to a β-sheet configuration.
Preparation of three-dimensional nanober scaold
e random or mesh-like electrospun membrane was
placed in DLP (Digital Light Processing)-based 3D
printer (nanoArch S130, BMF Precision Tech Inc.).
e membrane was coated with a photocured hydro-
gel precursor solution (0.25% LAP in 5%GelMa aqueous
solution, a commonly used photocured hydrogel formu-
lation). e printer was then used to produce a lattice of
UV light projection areas (lattice strips: 0.2mm wide, lat-
tice sizes: 1.5 × 1.5mm squares). e UV wavelength in
the printer was 405nm, and it was irradiated for 5s at a
time, for a total of 10 times. en a latticed hydrogel layer
(approximately 250 microns thick) was crosslinked on
the electrospun nanofibers. e uncrosslinked precursor
solution was then thoroughly washed away. We carefully
manipulated two tweezers, stacking layers of electrospun
membrane with latticed hydrogel on top of each other
to create a 3D scaffold. e 3D scaffold was then further
crosslinked using a UV flashlight for the second time.
e latticed hydrogel dominated the thickness of scaffold,
so it taked about 20 layers to get a 5mm thick scaffold.
Characterization
e scaffolds were freeze-dried and then subjected to
various characterization tests. e chemical scaffolds
and crystalline structures of the materials were analyzed
using a Fourier transform infrared (FTIR) spectrometer
(Nicolet 5700, Perkin–Elmer) and an X-ray diffractom-
eter (Rigaku D/max 2400 diffractometer, Tokyo, Japan).
e surface of the gold-sputtered materials was observed
under a scanning electron microscope (MIRA3, LMH,
TESCAN), and the diameter of the fibers was measured
and documented using the FIJI software. e water con-
tact angle (WCA) was assessed by employing a video
contact angle tester (FM40MK2, Kruss). For mechani-
cal tests, a universal testing machine (Bose ElectroForce
3200, Bose CROP.) was employed where the dry samples
were cut into dumbbell shapes with a width of 5mm for
tensile testing and square shapes with 5 mm sides for
compression tests, with each set of samples being tested
five times.
In vitro biocompatibility
Before cell cultivation, the scaffolds were soaked in alco-
hol for 4h, and then were washed with PBS three times
for 15min each time. e extracts of the scaffolds were
obtained according to the international standard ISO
10993-12: 2012. e sterilized scaffold was immersed in
0.1g/mL (w/v) α-MEM culture medium and incubated at
37℃ for 3 days. Following this, FBS (10%, v/v) was added
to obtain scaffold extracts. e scaffold’s biocompatibil-
ity was subsequently assessed with Bone Mesenchymal
Stem Cells (BMSCs). Specifically, BMSCs were intro-
duced into 96-well plates at 5.0 × 103 cells/well. After the
cells adhered, the medium was replaced with the extract.
On days 2 and 4, the optical density at 450nm was deter-
mined after adding CCK-8 solution (Beyotime, China).
Meanwhile, some wells were stained with calcein-AM/
PI to mark live/dead cells, visualized under a fluorescence
microscope. e proportion of viable cells was calculated.
BMSCs were seeded onto sterile scaffolds in 24-well
plates at a density of 8.0 × 104 cells/scaffold. On days 2
and 4, live cells were stained with calcein-AM and scru-
tinized under a confocal microscope to observe adhesion
dynamics.
Single-layer electrospun membranes were placed and
fixed onto 24-well coverslips. BMSCs were seeded onto
these coverslips at a density of 8.0 × 104 cells/scaffold. On
days 2 and 4, after removing the membranes from the
coverslips, cells on the coverslips were fixed with a 10%
formaldehyde solution and stained with 0.1% crystal vio-
let, followed by imaging.
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
Quantitative reverse transcriptase polymerase chain
reaction (qRT-PCR)
RAW 264.7 cells were cultivated on scaffolds within
24-well plates at 8.0 × 104 cells/scaffold for a 3-day dura-
tion. RNA was subsequently isolated using TRIzol®
Reagent (Invitrogen, USA), and its concentration and
purity were ascertained via the ermo Scientific Nano-
DropTM 1000 UV-Vis Spectrophotometer. cDNA syn-
thesis ensued with the FastQuant RT kit. e qRT-PCR
was executed with the SYBR Green supermix (Bio-Rad,
USA) and recorded on the CFX96 real-time PCR detec-
tion system. Relative gene expression levels for IL-1β,
IL-6, IL-4, and IL-10 were standardized against GAPDH,
and computed employing the 2−ΔΔCT method. Primer
sequences can be found in Table S2.
Flow cytometric analysis
Post a 3-day culture period of RAW 264.7 cells on scaf-
folds in 24-well plates (8.0 × 104 cells/scaffold), the cells
were subjected to trypsinization and resuspension in
PBS. Cells underwent a 15–30 min primary antibody
incubation on ice, followed by two buffer washes. ey
were then incubated with a fluorescently labeled second-
ary antibody and subsequently washed twice more. e
cells were resuspended in 1X PBS for flow cytometric
analysis. FlowJo (Tree Star) software was employed for
data processing, setting a 1% gating for isotype controls
to negate non-specific staining and comparing percent-
ages of distinct cell populations displaying marker-posi-
tive staining.
Enzyme-linked immunosorbent assay (ELISA)
After culturing RAW 264.7 cells on scaffolds in 24-well
plates for 3 days at a density of 8.0 × 104 cells/scaffold, the
concentrations of VEGF, TNF-α, and TGF-β in the super-
natants were determined by ELISA kits (Elabscience Bio-
technology, Wuhan, China). e procedure involved the
initial addition of 40 µL of sample diluent to the ELISA
plate wells, followed by 10 µL of the sample. Subse-
quently, 100 µL of enzyme-labeled reagent was added
to each well, and the plate was sealed and incubated at
37℃ for 60min. Post-incubation, the wells were washed,
and 50 µL of color reagent A, followed by 50 µL of color
reagent B, were added and gently mixed. is was incu-
bated in the dark at 37℃ for 15min before the reaction
was halted with a stop solution. e absorbance was
measured at 450nm using an ELISA reader, and factor
concentrations were calculated from a standard curve.
Inhibition of PI3K/AKT pathway
RAW 264.7 cells were seeded onto Mesh-500 scaf-
folds in 24-well plates at a density of 8.0 × 104 cells/
scaffold and cultured for 24h. en, cells were treated
with 10 µM of the PI3K inhibitor LY294002 (MedChem
Express, Princeton, USA) for 24h, followed by cultiva-
tion in a regular culture medium for an additional 24h.
e expression of CD206, ARG-1, IL-4, and IL-10 in cells
was detected using RT-PCR. Cells on the plate and those
on the Mesh-500 scaffold without inhibitor treatment
served as controls.
Western blotting
After 3 days of culturing RAW 264.7 cells on scaffolds in
24-well plates at a density of 8.0 × 104 cells/scaffold, total
proteins were collected using a Total Protein Extraction
Kit (Beyotime Biotechnology, China), and protein con-
centration was determined by the Bicinchoninic Acid
(BCA) method. Sample proteins underwent SDS-PAGE
and membrane transfer. e transferred membranes
were blocked and then incubated overnight at 4°C with
antibodies against Integrin β1, phosphorylated PI3K(p-
PI3K), PI3K, phosphorylated AKT (p-AKT), AKT, MCP-
1, and Glyceraldehyde 3-Phosphate Dehydrogenase
(GAPDH). After incubation with secondary antibodies
for 30min, the blots were visualized by a Tanon-5200
chemiluminescent imaging system (Tanon, Shanghai,
China). Results were reproduced in three independent
experiments using different samples.
Rat subcutaneous implantation
Animal experiments were conducted in accordance with
the guidelines for the care and use of experimental ani-
mals established by the National Institutes of Health
(NIH) and ethically approved by the Animal Care and
Use Committee of Zhongnan Hospital, Wuhan Univer-
sity (Approval No. ZN2021194). Nanofiber scaffolds
were cut into dimensions of 5mm x 5mm x 1mm and
sterilized using ethylene oxide gas, followed by immer-
sion in saline solution for reserve. Male SD rats weighing
between 250 and 300 g were utilized for subcutaneous
implantation. Briefly, the rats were anesthetized with a
constant delivery of 4% isoflurane. An area measuring
8cm ×4cm was shaved on the dorsal region of each rat,
and the povidone-iodine solution was applied thrice to
the exposed skin. A 1cm incision was made at the center,
followed by a slight separation of skin and muscles using
a toothless tweezer to create a subcutaneous pocket. e
scaffold was carefully placed into the pocket, ensuring a
distance of more than 3cm between adjacent scaffolds.
e incision was then sutured. Rats were euthanized with
CO2 at 1 and 2 weeks post-implantation, after which the
implanted materials were harvested for histological stain-
ing and transcriptome sequencing analyses.
Histology and immunohistochemical analysis
Specimens were fixed and underwent dehydration and
paraffin infiltration using an automated tissue proces-
sor, followed by manual embedding and sectioning in
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Page 6 of 17
Xiao et al. Journal of Nanobiotechnology (2024) 22:322
paraffin. Hematoxylin & Eosin (H&E) and Masson’s
Trichrome staining were performed on the samples.
For immunohistochemical staining, slides were depa-
raffinized, followed by endogenous peroxidase activity
elimination via incubation in 3% H2O2 at room tempera-
ture for 5–10min. is was followed by rinsing with dis-
tilled water and a 5-minute PBS soak. Antigen retrieval
was achieved by heating in citrate buffer (10 mM, pH 6.0,
95–100°C) for 10min. e slides were then blocked with
5–10% normal goat serum (diluted in PBS) for 10min
at room temperature. Appropriate primary antibod-
ies (e.g., CD68, CD206) were applied and incubated at
37°C for 1–2h. e biotin-labeled secondary antibody
was applied (diluted in 1% BSA-PBS) and incubated at
37°C for 10–30min, followed by horseradish peroxidase-
conjugated streptavidin (diluted in PBS) for an additional
10–30min. Visualization was done using a DAB chromo-
gen. After thorough rinsing with tap water, counterstain-
ing was performed, and the slides were sealed. Following
slide scanning, average optical density was analyzed
using the IHC TOOLBOX in FIJI software.
Transcriptome sequencing and data processing
Two weeks post-implantation, samples from both the
Random and Mesh-500 scaffolds were collected and
preserved in RNA Later™ stabilizing solution (ermo-
Fisher Scientific, USA). Bulk-RNA-Seq was conducted by
Biomarker Co., Ltd. (China). Key terms such as immune
response, inflammatory response, and angiogenesis were
subjected to KEGG and GO enrichment analyses. Heat-
maps of differentially expressed genes were constructed
to illustrate the levels of gene expression within distinct
scaffolds. Furthermore, key gene expressions (Acta2,
CCL2, CXCL1) were verified via RT-PCR.
Results and discussion
Preparation and characterization of 3D scaold
We proposed a strategy to fabricate a porous 3D nano-
fiber scaffold: creating a porous nanofiber membrane
through template-assisted electrospinning, followed by
the development of a latticed hydrogel layer using 3D
printing, and finally, layer-by-layer assembly to form a 3D
scaffold (Fig.1A). Tin foil and metal meshes of different
sizes (with pore sizes of 250μm, 500μm, and 750μm)
were used as receiving templates to produce random and
mesh-like membranes (Random, Mesh-250, Mesh-500,
Mesh-750). Given the significant impact of membrane
thickness on the scaffold’s post-implantation perfor-
mance, we carefully examined the reception time for a
single layer. When the time was too short, the electros-
pinning membrane became too thin and brittle, affecting
the 3D assembly; if too long, the mesh-like morphology
became blurred. Ultimately, the receiving duration was
set at 40 min. For the region of nanofiber deposition,
nanofibers tended to deposit both on the receiving metal
wires and the blank areas. e larger the mesh size, the
more nanofibers settled on the metal wires and fewer in
the blank areas. ose nanofibers deposited on the metal
wires aligned approximately parallel to the major axis of
the wire, while those in the blank areas exhibited an inter-
laced overlapping arrangement (Fig.2A, B). e nanofi-
bers of membranes have a diameter of 650 ± 200nm (Fig.
S1). Subsequently, Gelatin methacryloyl (GelMA) hydro-
gel was selectively solidified onto the membrane using a
3D printer. is design ensures the separation of adja-
cent electrospun membranes, offering space for tissue
growth. erefore, the dimensions of the latticed hydro-
gel were not designed too densely, preserving the effec-
tiveness of the electrospinning membrane. e latticed
hydrogel was uniformly distributed on the membrane
(Fig. 2C). In the non-photo-cured areas of GelMA, no
residual GelMA was found; they were completely washed
away with water without damaging the appearance of the
membrane. Sequentially, composite layers of the electro-
spinning membrane and hydrogel were stacked to form
a 3D scaffold. It can be seen that the layers are well iso-
lated and loose and porous (Fig.2D). en the 3D scaf-
folds were freeze-dried to facilitate long-term storage at
room temperature. e weight of the scaffolds increases
to about four times its original weight after water absorp-
tion, which may be related to its high porosity (70–85%)
(Table S1). In addition, the scaffolds were super hydro-
philic and the droplets were absorbed the moment they
were dropped on the scaffolds (Fig. S2), which was attrib-
uted to the addition of silk fibroin in the electrospinning
(Fig. S3). e addition of silk fibroin to the electrospin-
ning polymer has been reported to improve the biocom-
patibility of the material, modulate the immune response,
and accelerate tissue repair [28, 29].
Mechanical properties of the 3D scaold
e scaffolds were trimmed into cubes of 5 × 5 × 5 mm,
showcasing good resilience. Regardless of their dry or
wet state, they returned to their original form after being
pressed with a 50g weight (Fig.3A, Video 1). Mechani-
cal performances of the scaffolds were assessed via cyclic
compression and tensile testing. Cyclic compression
tests revealed that after 20 compression cycles under a
maximum strain of 60%, the stress-strain curves of the
scaffolds almost matched the initial cycle (Fig. 3B, C),
with recovery rates being Random (96 ± 4%), Mesh-250
(97 ± 3%), Mesh-500 (98 ± 2%), and Mesh-750 (97 ± 2%).
is resilience might be attributed to the latticed hydro-
gel undergoing deformation upon compression, while
the electrospun membrane provided support, prevent-
ing easy rupture. Tensile tests indicated that the Random
scaffold exhibited the best tensile properties, followed by
Mesh-250, Mesh-500, and Mesh-750 (Fig.3D). is could
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
be because as the mesh size increases, the nanofibers are
more orderly aligned, but there are imperfections at the
metal mesh nodes, resulting in less tight binding of the
nanofibers, causing them to break at these defect sites.
For tissue engineering scaffolds, good elasticity ensures
the scaffold conforms better to tissue defects, achieving
better integration with the tissue, and a certain degree of
tensile resistance ensures the scaffold won’t shatter in the
human body as soft tissues deform.
Various strategies for nanofiber three-dimensionaliza-
tion have been explored over the years. e methods of
short fibers and gas foaming have garnered significant
attention [19]. Compared to the short fibers method, our
method retains the original appearance of the nanofiber
membrane, allows for customized surface structures,
Fig. 2 Fabrication and characterization of the three-dimensional nanober scaold. (A) Schematic of the fabrication process (with corresponding physi-
cal images on the right). (B) SEM images of the unordered and three dierent mesh specications of the electrospun membranes (Random, Mesh-250,
Mesh-500, Mesh-750). The top row displays low magnication, while the bottom row showcases high magnication images. (C) Photographs of the
electrospinning membrane integrated with a latticed hydrogel layer. (D) Lateral and top-down views of the three-dimensional nanober scaold
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
and does not require cross-linking, while the mechanical
properties of the short fibers method depend on the final
cross-linking method. For three-dimensional scaffolds
made by the gas expansion method. Its structure was not
easy to maintain according to our previous attempts. e
scaffold relies on the intermittent contact between the
membranes after expansion thus supporting the whole
three-dimensional form.
Eects of materials on cells in vitro
e morphology and structure of scaffolds play pivotal
roles in directing cellular responses such as adhesion,
migration, and proliferation. We first assessed the cyto-
toxicity of individual scaffold components. Compared to
the control scaffold with standard culture medium, the
live/dead cell ratios in extracts of the electrospinning
membrane and the 3D scaffold incorporated with hydro-
gel were both over 95% (Fig. 4A), with no significant
difference on day 2 and 4 (Fig.4B). Similarly, the OD
values reflecting cell numbers were consistent on days
2 and 4 (Fig.4C). is suggests the extracts were non-
toxic. Subsequently, upon examining cell-seeded mem-
branes, we found that cell morphology aligned closely
with the nanofiber orientation of the substrate. In regions
where fibers were orderly aligned, cells adopted an elon-
gated form. And the cells’ distribution often mirrored
the scaffold’s mesh pattern (Fig. 4D). CCK-8 experi-
ments revealed no significant difference in cell counts
on the various membranes on day 2. By day 4, the Ran-
dom scaffold had fewer cells compared to Mesh-250 and
Mesh-500, while Mesh-750 had fewer than Mesh-250
(Fig.4E). is suggests that the mesh structure promotes
cell proliferation, but overly large pores may reduce cell
growth area, leading to a decrease in cell count. Addi-
tionally, we placed the membranes on round cover-
slips, exploring the number of cells that penetrated the
Fig. 3 Mechanical properties of the three-dimensional nanober scaold. (A) Images illustrating the scaold’s ability to return to its original form after
compression by a 50g weight, in both dry and wet conditions. (B) Cyclic compression tests of scaolds under a 60% compressive strain (1st cycle). (C)
Cyclic compression tests of scaolds under a 60% compressive strain (20th cycle). (D) Tensile testing of the scaolds
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
electrospun membrane and attached to the coverslips.
On day 2, numerous cells had penetrated the Mesh-750
membrane, attaching to the coverslip. Some cells had
penetrated Mesh-500 and Mesh-250, but almost none
from the Random scaffold. By day 4, a large number of
cells were observed on coverslips from the Mesh scaf-
folds (Fig. 4F), with a sequence of Mesh-750 > Mesh-
500 > Mesh-250, while the Random scaffold still exhibited
very few cells (Fig.4G). is indicates that larger pores
are more conducive for cell penetration through electros-
pun membranes, but this difference diminishes over time
in mesh-like membranes.
Tissue integration status of the scaold in rat
subcutaneous implantation
3D scaffolds were implanted subcutaneously in rat
dorsa to observe tissue integration speed and potential
immune reactions. In sections stained with Hematoxy-
lin and Eosin (HE), the cytoplasm appeared red while
nuclei were stained purple. Non-specific staining was
observed on undegraded material: the electrospun mem-
brane was pink and the hydrogel presented a light purple
hue (Fig.5A). On Day 7, a greater number of cells were
found surrounding the scaffold than within it, and the
infiltration areas expanded with the increment in mesh
Fig. 4 Biocompatibility of the scaold and its capability to permit cellular migration through the electrospinning membrane. (A) Live/dead cell staining
of cells cultivated in the scaold component’s extraction uid. (B) Statistical representation of the live cell percentage from live/dead staining. (C) CCK-8
assay of cells cultivated in the scaold component’s extraction uid. ns, no signicance. (D) Confocal microscopy images of live/dead stained BMSC cells
post-cultivation on the scaold. (E) CCK-8 assay post BMSC cell cultivation on the scaold. ns, no signicance. *P < 0.05. (F) Crystal violet staining images
of cells after migration through a single layer of the electrospinning membrane onto a cell round coverslip. (G) Percentage representation of the cellular
region on the round coverslips. (* and ** represent P < 0.05 and P < 0.01 when compared with Random. ## represent P < 0.01 when compared with Mesh-
250. $$ represent P < 0.01 when compared with Mesh-500.)
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
size. On Day 14, both Mesh-500 and Mesh-750 scaffolds
were densely populated by cells, save for some remaining
undegraded hydrogel. Conversely, the Random scaffold
and Mesh-250 exhibited regions without any cells. e
microvascular area was measured, there was no statisti-
cal significance on day 7 across samples. However, by the
second week, the Mesh-500 scaffold displayed the high-
est vascularization, registering significant differences
Fig. 5 Tissue integration status of the scaold in rat subcutaneous implantation. (A) HE staining of tissue sections. (red arrow: new vessels) (B) Masson’s
staining of tissue sections. (C) Statistical analysis of vascular area within the scaold. (D) Quantication of collagen deposition within the scaold. (Data
are represented as mean ± standard deviation, n = 5, ns, no signicance, *P < 0.05, **P < 0.01, ***P < 0.001)
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
when compared to the Random and Mesh-250 scaf-
folds (Fig.5C). Using Masson’s trichrome stain, collagen
deposits were detected within the material. Collagen
deposition was faint on day 7, evidenced by the predom-
inance of light blue regions. On day 14, this deposition
surged notably, both Mesh-500 and Mesh-750 showed
intense blue collagen areas. In contrast, the Random scaf-
fold demonstrated minimal collagen presence, closely
followed by Mesh-250 (Fig.5B). e Integral optical den-
sity (IOD) of blue collagen was measured. On day 7, the
Random scaffold had the least IOD. On day 14, Mesh-
750 showed the largest IOD, followed by Mesh-500 and
Mesh-250 (Fig. 5D). is data indicates that scaffolds
with larger mesh pores are more conducive to rapid cell
infiltration and collagen deposition, yet Mesh-500 offers
the most substantial microvascular area.
Immune response of the scaold in rat subcutaneous
implantation
To assess the immunogenic responses triggered by scaf-
folds of diverse structures when placed subcutaneously,
immunohistochemical analyses were performed for
macrophage polarization markers and their correlating
cytokines. CD68 is a pan-macrophage marker (Fig.6A).
iNOS and TNF-α typify the M1 macrophage phenotype
(Fig.6B, D), whereas CD206 and IL-10 are representa-
tive of the M2 macrophage phenotype (Fig.6C, E). On
Day 7, Mesh-500 and Mesh-750 displayed elevated IOD
values for CD68 compared to Random and Mesh-250,
presumably due to enhanced cell infiltration through
the expansive pores of these scaffolds (Fig.6F). A diver-
gence in CD206 and iNOS levels indicated that by Day 7,
Mesh-500 presented a higher concentration of M2 cells
and fewer M1 cells than Mesh-750 (Fig.6G, I). On Day
14, there was an absence of significant variation in the
CD68 values across the scaffolds, suggesting substantial
Fig. 6 Immune response of the scaold in rat subcutaneous implantation. (A-E) Immunohistochemistry staining evidencing the presence of CD68,
iNOS, CD206, TNF-α, and IL-10. (F-J) Integrated optical density (IOD) measurements for CD68, CD206, IL-10, iNOS, and TNF-α. (Data are represented as
mean ± standard deviation, n = 5, ns, no signicance, *P < 0.05, **P < 0.01, ***P < 0.001.)
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Fig. 7 RNA-seq, GO/KEGG analysis, and verication of the PI3K/AKT pathway. (A) The volcano plot of dierential expression of genes between Random
and Mesh-500 group: the up-regulated, unchanged genes, and down-regulated were doted in red, black, and green, respectively. (B) GO enrichment
analysis for dierential expressed genes (biological process). (C) A cluster heatmap showed dierentially expressed genes involved in PI3K/AKT signal-
ing pathway (|Log2 FC|>1). (D) The top twenty enriched signaling pathways following KEGG enrichment analysis for dierential expressed genes. (E)
Relative mRNA levels of genes, including Acta2, CCL2, and CXCL1, as determined by qRT-PCR. (F) Representative Western blot images of RAW264.7 cells
cultured on the Random and Mesh-500 group. (n = 3). (Data are represented as mean ± standard deviation, n = 3, ns, no signicance, *P < 0.05, **P <0.01,
***P < 0.001.)
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
macrophage migration (Fig. 6A). CD206 demonstrated
elevated expression in the Random, Mesh-250, and
Mesh-500 scaffolds (Fig.6C, G), with IL-10 peaking in
Mesh-500 (Fig.6E, H). Both the Random and Mesh-750
scaffolds revealed heightened iNOS expression, con-
trasted by Mesh-250 and Mesh-500 (Fig.6B, I). TNF-α
exhibited a pronounced expression solely in the Random
scaffold (Fig. 6D, J). In summary, while Mesh-500 and
Mesh-750 promoted swift immunocyte migration into
scaffolds, Mesh-500 appeared to favor the M2 macro-
phage polarization. e Random scaffold, however, sup-
ported slower cell infiltration paired with a significant
expression of M1 macrophage indicators.
Subcutaneous implantation of materials has been lev-
eraged to investigate tissue ingrowth into materials, the
ensuing immune responses, material degradation, and
neo-tissue formation [30, 31]. Post-implantation, mate-
rials undergo sequential phases including injury, hemo-
stasis-material interaction, provisional matrix formation,
acute inflammation, chronic inflammation, granulation
tissue development, and fibrous capsule development
[32, 33]. In brief, during protein adsorption and acute
inflammation, as a biomaterial is integrated into the host,
proteins from the bloodstream swiftly adsorb onto the
material’s surface, recruiting leukocytes like neutrophils
and macrophages, and instigating an acute inflammatory
reaction. If macrophages fail to phagocytize and degrade
the biomaterial, they might fuse, giving rise to multinu-
cleated foreign body giant cells. ese cells persistently
attempt to disintegrate the material, releasing various
cytokines and enzymes. Fibrous encapsulation emerges
during prolonged inflammation and foreign body reac-
tions, where fibroblasts and smooth muscle cells are
recruited, synthesizing and secreting collagen, thereby
forming a dense fibrous capsule, and isolating the bio-
material from adjacent healthy tissue [34, 35]. Notably,
no prominent fibrous capsules were discerned around
these four scaffold scaffolds. is could be attributed
to the nanofibrous scaffolds minimizing inflammatory
responses, coupled with the porous structure fostering
cellular infiltration [36]. Moreover, the thin electrospun
membrane enables macrophages and foreign body giant
cells to effectively encapsulate the spun layer, reducing
the release of indigestible foreign signals.
Macrophages, intrinsic immune cells, play pivotal roles
in foreign body reactions and wound healing following
biomaterial implantation. In foreign body reactions, mac-
rophages can polarize into distinct phenotypes, primar-
ily M1 (pro-inflammatory) and M2 (anti-inflammatory)
[37]. ese two macrophage phenotypes distinctly influ-
ence tissue repair. M1 macrophages are predominantly
active during initial injury and inflammatory responses,
releasing pro-inflammatory cytokines like Tumor Necro-
sis Factor-alpha (TNF-α) and Interleukin-1β (IL-1β),
along with antimicrobial and destructive oxidants and
enzymes. While M1 macrophages mitigate pathogens
and dead cells in the wounded area through inflamma-
tion, an excessive M1 response might lead to exacer-
bated inflammation and tissue damage. Conversely, M2
macrophages, primarily active during the latter stages of
wound healing, emit anti-inflammatory cytokines such as
Interleukin-10 (IL-10) and Transforming Growth Factor-
beta (TGF-β), and factors that boost tissue repair and
remodeling. By dampening excessive inflammation, M2
macrophages promote cellular proliferation, matrix syn-
thesis, and subsequently, tissue regeneration and repair.
However, an overdriven M2 response might culminate
in excessive matrix deposition and fibrosis [38, 39]. Prior
research intimates that mesh-like membranes, in com-
parison to random and aligned topography membranes,
were advantageous to immunocyte recruitment and
angiogenesis. Moreover, in the bone microenvironment,
they favored the upregulation of M2 macrophage marker
gene expression [15]. is aligns with our empirical find-
ings, wherein Mesh-500 notably fostered macrophage
polarization towards the M2 phenotype. is phenom-
enon may be linked to the orderly alignment of nanofi-
bers on the Mesh-500 membrane and the membrane’s
porosity. Furthermore, overlaying multiple membranes
intensifies the topographical differences instigated by
the membrane. Previous studies have reported that per-
forated CO2 expanded nanofibrous scaffolds favored an
increased M2/M1 ratio four weeks post-implantation,
highlighting the importance of the porosity of nanofi-
brous scaffolds [40]. In contrast with Mesh-500, while the
sparser threads in the Mesh-750 scaffold facilitated faster
cellular penetration to the scaffold’s center, M2 macro-
phage marker expression was not as elevated as in Mesh-
500. is suggests that rapid cellular infiltration isn’t
necessarily optimal. However, this might also be related
to the thicker edges of the Mesh-750 grid, compelling
infiltrating macrophages to expend more time degrading
these edges. Earlier studies indicated that angiogenesis
and scaffold vascularization necessitate the coordinated
efforts of both M1 and M2 macrophages [41, 42]. e
augmented vascular area in the Mesh-500 scaffold might
result from its elicited harmonized immune responses.
Mesh-500 scaold modulates macrophage polarization by
activation of PI3K/AKT signaling pathway in vivo
We conducted RNA-seq analysis on both the Random
scaffold and Mesh-500 scaffold two weeks post-subcuta-
neous removal to decipher the underlying mechanisms
accounting for their divergent outcomes. Differential
gene expression analysis revealed that in the Mesh-500
scaffold, 1031 genes were upregulated and 1261 genes
were downregulated with a significance level of p < 0.05
and an absolute log2 fold change greater than 1 (Fig.7A).
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
Fig. 8 The phenotype of the macrophages cultured on the Random and Mesh-500 group. (A, B) Relative mRNA levels of genes, including IL-1β, IL-6, IL-4,
and IL-10, as determined by qRT-PCR. The data were normalized to that of the control cells seeded on tissue cell plates. (C) Quantitative results of FCA. (D)
Expression of CD206 and CD86 in RAW264.7 cells by FCA. (E-G) The concentrations of TGF-β, TNF-α, and VEGF in the cell supernatant were determined by
ELISA. (H, I) The fold change of mRNA levels of genes, including CD206, Arg-1, IL-4, and IL-10. (Data are represented as mean ± standard deviation, n = 3,
ns, no signicance, *P < 0.05, **P < 0.01, ***P < 0.001.)
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
Notably, the Mesh-500 scaffold augmented the expression
of pivotal immune-related genes, including Acta2, CCL2,
and CXCL1 (Fig.7C, E). Gene Ontology (GO) analysis
highlighted a pronounced enrichment of immune-asso-
ciated genes, primarily clustered into categories such as
“immune response,” “inflammatory response,” “extra-
cellular matrix organization,” and “positive regulation
of monocyte chemotaxis.” (Fig. 7B) Subsequent KEGG
pathway analysis identified twenty significant associated
targets and pathways. Notably, the PI3K/AKT signal-
ing pathway, a known regulator of M2 polarization, saw
a marked increase (Fig.7D). It’s established that a mate-
rial’s topological morphology influences the adherence of
macrophages on its surface. is adherence subsequently
fosters macrophage differentiation, primarily driven by
integrin expression. Phosphatidylinositol 3 kinase (PI3K),
a precursor regulator of protein kinase B (AKT), modu-
lates the M2 macrophage phenotype [43]. Activation of
PI3K catalyzes the conversion of phosphatidylinositol
4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-tri-
sphosphate (PIP3). is latter molecule is pivotal for Akt
activation as it binds to the PH domain, phosphorylating
and activating Akt, thereby amplifying M2-associated
gene expression [44]. Further assessment via the Western
Blot assay of macrophage integrin/PI3K/AKT pathway-
related proteins on the scaffolds revealed a progressive
increase in the relative expression of integrin, p-PI3K/
PI3K, and p-AKT/AKT across the Control, Random, and
Mesh-500 scaffolds (Fig. 7F, Fig. S4). is pattern sug-
gests that the Mesh-500 scaffold more effectively acti-
vates the PI3K/AKT pathway within macrophages.
Mesh-500 scaold modulates macrophage polarization by
activation of PI3K/AKT signaling pathway in vitro
We subsequently sought to discern the influence of the
scaffold on macrophages, following a seven-day in vitro
intervention. PCR analyses revealed that the expression
of M1 macrophage-specific markers, IL-1β and IL-6
mRNA, exhibited a decline across the Control, Random,
and Mesh-500 cohorts (Fig.8A). Conversely, M2 macro-
phage indicators, namely IL-4 and IL-10, demonstrated
an ascending trajectory in the aforementioned groups
(Fig.8B). Flow cytometric analysis further corroborated
that the Mesh-500 cohort was more conducive to the
augmentation of CD206 markers, while simultaneously
witnessing a decline in CD86 markers on the macrophage
surface, in comparison to the Control and Random group
(Fig.8C, D). Sequentially, we evaluated the secretion lev-
els of TGFβ, TNFα, and VEGF in the supernatant via the
ELISA methodology (Fig.8E, F, G). It was discerned that
while TGFβ and VEGF were substantially augmented in
the Mesh-500 group, TNFα was markedly diminished.
Introducing the PI3K inhibitor, LY294002, to the Mesh-
500 cohort for macrophage treatment led to intriguing
observations. Seven days post-treatment, the mRNA
expression levels of CD206 and Arg-1 in macrophages
mirrored those of the Control group (Fig.8H). Concur-
rently, the excretion of IL-4 and IL-10 in the supernatant
realigned to concentrations analogous to the Control
group (Fig.8I). Cumulatively, these observations provide
compelling evidence that the Mesh-500 scaffolds modu-
late macrophage polarization towards an M2 phenotype
via the PI3K/AKT signaling pathway in vitro.
is study acknowledges certain limitations. Specifi-
cally, the hydrogel’s geometrical attributes, which func-
tion as a foundational pillar in the electrospun membrane
layer, significantly influence the scaffold’s structure. Fac-
tors such as pattern shape, pattern density, and gel layer
height were not rigorously examined in this research.
Furthermore, extending the observation period for the
subcutaneous implantation model might have offered
insights into tissue attributes post-complete material
degradation.
Conclusions
In conclusion, we have successfully demonstrated the
feasibility of 3D printing hydrogels as pillars on electros-
pun membranes to enable the assembly of membrane lay-
ers to obtain three-dimensional scaffolds. We fabricated
four kinds of porous 3D nanofiber scaffolds with random
membranes and three kinds of mesh-like membranes
with different mesh sizes. e nanofibers are composed
of PCL and silk fibroin, the addition of silk can improve
the hydrophilicity and biocompatibility of PCL, and
modulate the negative immune response. ese scaffolds
have good mechanical properties to meet the needs of
tissue engineering. In the in vitro cell inoculation experi-
ments, the mesh-like membrane was more favorable to
cell proliferation than the random electrospinning mem-
brane, and the larger the mesh size was more favorable to
cell penetration through the electrospinning membrane.
During subcutaneous trials, the larger the mesh pores
were more favorable for rapid inward cell penetration.
Nevertheless, our findings suggest that an optimal mesh
pore dimension, specifically Mesh-500, optimally directs
macrophage polarization within the scaffold toward the
M2 phenotype. is phenomenon potentially fosters
a conducive microenvironment for tissue restoration,
expediting tissue growth, matrix deposition, and scaffold
vascularization. Consequent RNA sequencing tests and
wet lab experiments inferred that the Mesh-500’s dis-
tinct topology might modulate macrophage polarization
towards the M2 phenotype via the PI3K/AKT pathway’s
interaction. Recognizing the unique microenvironments
of various implantation sites (e.g., bone, skin, cartilage,
muscle), future research will delve into the 3D scaffold’s
applicability as a tissue-engineering platform.
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Xiao et al. Journal of Nanobiotechnology (2024) 22:322
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12951-024-02578-2.
Supplementary Material 1
Supplementary Material 2
Acknowledgements
This work was supported by the Fundamental Research Funds for the
Central Universities (2042022kf1121) and the Program of Excellent Doctoral
(Postdoctoral) of Zhongnan Hospital of Wuhan University (Grant No.
ZNYB2021005).
Author contributions
L.X.: Conceptualization, Methodology, Formal analysis, Investigation, Data
curation, Writing – original draft, Writing – review & editing. H.L.: In vivo and
in vitro experiments and data analysis. S.W.: Investigation, Visualization, Data
curation. L.X.: Investigation, Visualization. Z.G.: Methodology, Resources,
Writing – review & editing. F.Y.: Conceptualization, Methodology, analysis,
Formal analysis, Resources, Writing – review & editing, Funding acquisition.
L.C.: Conceptualization, Resources, Writing – review & editing, Supervision,
Funding acquisition. All authors reviewed the manuscript.
Data availability
The raw data related to this work are available from the corresponding author
upon reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Received: 10 November 2023 / Accepted: 23 May 2024
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