[Link].

org Research Article

One-Step Gas Foaming Strategy for Constructing Strontium

Nanoparticle Decorated 3D Scaffolds: a New Platform for Repairing
Critical Bone Defects
Yujie Chen,*,# Yucai Li,# Xinyi Wang,# Xiumei Mo, Yicheng Chen, Zijun Deng, Xiaojian Ye,*
and Jiangming Yu*
Cite This: ACS Appl. Mater. Interfaces 2024, 16, 61664−61678 Read Online
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ABSTRACT: The management of critical-sized bone defects poses significant

clinical challenges, particularly in the battlefield and trauma-related injuries.
However, bone tissue engineering scaffolds that satisfy high porosity and good
angiogenic and osteogenic functions are scarce. In this study, 3D nanofiber
scaffolds decorated with strontium nanoparticles (3DS-Sr) were fabricated by
combining electrospinning and gas foaming. Sodium borohydride (NaBH4)
served a dual role as both a reducing and gas-foaming agent, enabling a one-
step process for expansion and modification. In vitro experimental results
demonstrated that 3DS-Sr possessed an integrated multilayered porous
structure. It promoted angiogenesis by upregulating the expression of
hypoxia-inducible factor-1α (HIF-1α) protein and phosphorylation of ERK
through the sustained release of Sr2+ and created a favorable microenvironment
for osteogenesis by activating the Wnt/β-catenin pathway. In vivo experiments
indicated that 3DS-Sr promoted cranial bone regeneration by synergistically promoting the effects of vascularization and
osteogenesis. In summary, this study proposed a bioactive bone scaffold in a “one stone, two birds” manner, providing a promising
strategy for bone defect repair.
KEYWORDS: bone tissue engineering, electrospinning, strontium, gas foaming, nanofibers

■ INTRODUCTION
Bone injuries and defects caused by trauma, tumors, and
environment through mechanical signals, and overly rigid
materials can negatively impact cell morphology and
infections represent prevalent clinical challenges.1 Current function.5,6 Furthermore, cells secrete ECM to support the
treatment methods, such as autologous or allogeneic bone tissue structure. However, dense fiber membranes can restrict
transplantation are limited by donor site scarcity, morbidity, the distribution and assembly of the ECM, thereby influencing
and immune rejection.2 Consequently, bone tissue engineering the formation of target tissues. Recently, various improved
has developed rapidly in recent years and emerges as a electrospinning techniques and postelectrospinning treatment
promising alternative.3 Ideally, bone scaffolds should mimic the methods have been developed for creating 3D nanofiber
extracellular matrix (ECM) structure of native bone to scaffolds.7−9 Yet, these technologies often achieve only
facilitate the adhesion, infiltration, and proliferation of adjacent marginal improvements in porosity, necessitate specialized
stem/progenitor cells, as well as a favorable environment for equipment, or entail lengthy production cycles, failing to
angiogenesis and osteogenesis.4 construct a highly porous, continuous, integrated 3D network.
Electrospinning enables the efficient fabrication of nano- Another postprocessing technique is the gas foaming method,
fibers with the resultant scaffolds capable of simulating the involving the in situ generation of bubbles through chemical
natural ECM structure. However, conventional electrospun reactions, such as sodium borohydride (NaBH4) decom-
membranes have dense layers of fibers with only surface pores, position.10 The simplicity of the experimental conditions and
lacking the internal three-dimensional (3D) networks neces-
sary for cellular infiltration and growth. Consequently, cells Received: August 4, 2024
face challenges in infiltrating these membranes, proliferating Revised: October 10, 2024
only on the surface and failing to form the desired 3D tissue Accepted: October 24, 2024
structures. Additionally, dense fiber membranes often exhibit a Published: October 30, 2024
higher mechanical stiffness, which may not match the
mechanical properties of natural tissues. Cells sense their

© 2024 American Chemical Society [Link]

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equipment required for gas foaming renders it an effective Sr(NO3)2 to strontium nanoparticles and expand to a 3D structure.
approach to fabricating continuously layered nanofiber After 10 min, the samples were rinsed and freeze-dried. The 3DS
matrices. Previous studies have shown that gas-foamed scaffolds were created using gas foaming without Sr(NO 3 ) 2
scaffolds enhanced cell infiltration, with successful construction immersion, following identical parameters as those used for 3DS-Sr.
Strontium salts increased the hydrolysis rate of NaBH4, which was
of 3D scaffold-cartilage composites.11−13 Nevertheless, the reduced to elemental strontium in the presence of NaBH4, as
osteogenic potential of these gas-foamed scaffolds is weak, illustrated in eq 1. The synthesized strontium, being unstable, reacted
necessitating improvements in bioactivity to leverage the with water to form a white precipitate of strontium hydroxide, as
advantages of their porous structure and provide the optimal shown in eq 2.
environment required for osteogenesis.
In addition to its role as a gas foaming agent, NaBH4 can Sr 2 + + BH 4 + H 2O intermediate + Sr + H 2 (1)
also serve as a reductant for the in situ reduction of inorganic Sr + 2H 2O Sr(OH)2 + H 2 (2)
and organometallic precursors.14 This capacity enables the
production of multilayered scaffolds decorated with inorganic 2.3. Characterization. The thickness of scaffolds was measured
nanoparticles in a single fabrication process. Various ions, such with a vernier caliper, and their volumes were determined by
as Ca, Mg and Sr, have been shown to have specific effects on multiplying the thickness by their lengths and width. To find the
volume percentage, the scaffold’s volume was divided by the initial
cell fate and physiological activities, including angiogenesis and membrane volume. Porosity was assessed by using the liquid
osteogenic differentiation.15 Strontium is a vital trace element displacement method.
that is pivotal in bone metabolism, enhancing osteoblast The morphology, functional groups, thermal stability, surface
activity and curtailing osteoclast resorption.16 Additionally, elements, and crystalline structure of scaffolds were examined using
strontium induces mesenchymal stem cells (MSCs) prolifer- scanning electron microscopy (SEM), Fourier transform infrared
ation and osteogenic differentiation, triggering the Wnt/β- spectroscopy (FTIR), Thermogravimetric analysis (TGA), X-ray
catenin pathway and boosting ECM deposition and mineral- photoelectron spectroscopy (XPS) and X-ray diffractometer (XRD),
ization.17,18 Therefore, the aim of this work is to develop a respectively. The specific brands and origins of the machines are
novel bioactive scaffold by in situ decoration of strontium shown in Table S1. The diameters of nanofibers were measured by
using ImageJ software based on SEM images. The lengths of pore
nanoparticles on the gas-foamed scaffold in a “one stone, two sides were measured and recorded using ImageJ and the pore area was
birds” manner, adopting dual benefits in terms of porosity and then calculated.
bioactivity in a single process. Mechanical properties were evaluated at room temperature by
Herein, we fabricated a two-dimensional nanofiber scaffold using a universal testing machine (HY-940FS, Hengyu Instrument,
(2DS) containing polycaprolactone and pluronic F127 (F-127) Shanghai, China). Tensile tests were conducted on rectangular
via electrospinning. Strontium nitrate salt saturated 2DS was samples of 2DS, 3DS, and 3DS-Sr in a wet state with a crosshead
treated with NaBH4, thus affording strontium-based nano- (speed: 5 mm/min) until rupture. Stress−strain curves were derived
particle-decorated 3D nanofiber scaffold (3DS-Sr). F-127 was from the recorded data to determine the ultimate tensile strength
(UTS), elongation at break (Eb), and Young’s modulus (E). A total of
an FDA-approved biocompatible amphiphilic polymer, con-
50 loading−unloading fatigue cycles were conducted at a constant
sisting of two hydrophilic poly(ethylene oxide) (PEO) blocks displacement rate of 5 mm/min. The compressive modulus of the
and a hydrophobic poly(propylene oxide) (PPO) block scaffold was analyzed and calculated by using the slope fitting method
positioned between the PEO segments.19 It enhanced the in the initial linear region of the stress−strain curve. To assess the
hydrophilicity of the nanofiber membrane and stabilized H2 release kinetics of Sr2+, supernatants collected at various intervals from
bubbles during gas foaming.20 We hypothesized that the 3DS- 3DS-Sr using inductively coupled plasma-atomic emission spectros-
Sr would possess an integrated multilayer structure to facilitate copy (ICP-AES, Leeman, USA). The scaffolds 2DS (length × width ×
cell infiltration. Introducing strontium was expected to thickness: 10 mm × 10 mm × 1 mm), 3DS (length × width ×
enhance the biological properties of the scaffolds, leading to thickness: 10 mm × 10 mm × 3 mm), and 3DS-Sr (length × width ×
thickness: 10 mm × 10 mm × 3 mm) were immersed in 10 mL of
improved vascularization and bone regeneration. Comprehen- PBS solution for 7 days, and the pH of the supernatant was
sive characterizations encompassing compositional, morpho- subsequently measured.
logical, mechanical, cytocompatibility analyses, and vasculari- 2.4. Cytocompatibility of Scaffolds In Vitro. Human umbilical
zation/osteogenesis experiments were conducted to validate vein endothelial cells (HUVECs) were cultured in DMEM high
the biomedical application of the scaffold for bone repair. glucose complete medium. SD rat-derived BMSCs (Servicebio,
Wuhan, China) were grown in specialized complete rat bone marrow
mesenchymal stem cell medium (Servicebio, Wuhan, China), with
2. MATERIALS AND METHODS media changes every 2 days. BMSCs from passages 3−5 were used for
2.1. Preparation of Nanofiber Mats (2DS). Poly(ε-caprolac- in vitro cellular experiments. Scaffold extracts were prepared by
tone) (PCL, Mw = 80 kDa, Sigma-Aldrich) at a concentration of 10 wt suspending 2DS, 3DS, or 3DS-Sr at 1 mg/mL in culture media for 1
% and Pluronic F-127 (Sigma-Aldrich) at 0.5 wt % were dissolved in a week; the supernatant was then collected for cell culture. Cells grown
solvent mixture of dichloromethane (DCM) and dimethylformamide in medium without scaffold extracts served as controls.
(DMF) in a 4:1 volume ratio. Load the solution was loaded into a The cytotoxicity of the scaffold was assessed using live/dead
syringe equipped with a blunt needle at the tip. Set the electro- staining (Beyotime, China). Briefly, BMSCs or HUVECs were seeded
spinning parameters were set to a rate of 1.2 mL/h, a voltage of 12 kV, into 48-well plates at a density of 2.0 × 104 cells per well. After 24 h of
and a receiving distance of 15 cm. The obtained 2DS on aluminum coculturing with the scaffold extract, staining was performed
foil was stored in a vacuum drying oven. according to the live/dead kit instructions. Cell proliferation was
2.2. Preparation of 3D Scaffolds. To fabricate 3D Sr NPs assessed after 1, 3, and 5 days of coculturing the cells with the scaffold
decorated nanofiber scaffolds (3DS-Sr), rectangular pieces of 2DS (1 extract, following the detailed steps provided from the cell counting
cm × 1 cm) were first submerged in 20 mL of strontium nitrate kit-8 (CCK-8) assay (Beyotime, China) instructions. Cell adhesion
(Sr(NO3)2) aqueous solution at concentrations of 2, 20, and 200 mM, experiments were conducted according to previous reports.12 Briefly,
respectively, for 10 min. Subsequently, the scaffold was transferred to after seeding cells on different scaffolds for 4 h, the culture medium
0.5 M sodium borohydride (NaBH4, Sinopharm Chemical Reagent, was collected, and the number of cells was recorded. The cell seeding
Shanghai, China) aqueous solution to facilitate the in situ reduction of efficiency (S) was calculated using eq 3:

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Figure 1. Schematic diagram illustrating the construction of 3DS-Sr for bone regeneration via electrospinning and gas foaming technology.

S(%) = (S1 S2)/S1 × 100% (3) (Epizyme, Shanghai, China). Protein concentrations were determined
using a BCA protein assay kit (Thermo Scientific, USA). Proteins
where S1 is the total number of cells and S2 is the number of lost cells were separated via SDS-PAGE (Epizyme) and transferred to
(n = 3). polyvinylidene difluoride membranes (MilliporeSigma, Darmstadt,
The morphology and distribution of cells on different scaffolds Germany). Membranes were blocked with skim milk for 1 h at room
were stained with DAPI/phalloidin, which was observed and imaged temperature, followed by overnight incubation with primary antibod-
using confocal microscopy (Leica Microsystems, Germany). ies: pERK (1:1000, 28733-1-AP, Proteintech), ERK (1:2000, 11257-
2.5. Angiogenic Ability of Scaffolds In Vitro. HUVECs were 1-AP, Proteintech), HIF-1α (1:2000, 20960-1-AP, Proteintech),
maintained in extracts derived from various scaffolds over a period of CD31 (1:2000, 11265-1-AP, Proteintech), and VEGF (1:1000,
5 days. Subsequently, immunofluorescence staining was performed to 19003-1-AP, Proteintech). After incubation with secondary antibodies
detect platelet endothelial cell adhesion molecule-1 (CD31) and for 1 h, signal detection was performed by using enhanced
hypoxia-inducible factor-1α (HIF-1α). The cells were blocked and chemiluminescence (Epizyme) with a Tanon-5200 imaging system
incubated overnight with primary antibodies (anti-CD31, 1:200, (Tanon, Shanghai, China). Semiquantitative protein expression
ab222783, Abcam; anti-HIF-1α, 1:200, and 21960−1-AP, Protein- analysis was performed using ImageJ.
tech). The next day, cells were treated with fluorescence-labeled 2.6. Osteogenic Ability of Scaffolds In Vitro. BMSCs were
secondary antibodies and counterstained with DAPI. Imaging was maintained for a duration of 7 days using the Rat MSC Osteogenic
carried out by using a fluorescence microscope (Nikon, Tokyo, Differentiation Kit (Meilun, Dalian, China), which included extracts
Japan). from various scaffolds. To examine the expression of osteopontin
To evaluate the angiogenic potential further, a tube formation assay (OPN, 1:200, Thermo Fisher Scientific) and collagen type I (COL-I,
on Matrigel was conducted. HUVECs were seeded on Matrigel 1:200, Proteintech), immunofluorescence staining was conducted and
(Corning, USA) and exposed to different scaffold extracts for 6 h. The imaging was performed with a fluorescence microscope (Nikon,
formation of tubular networks was visualized by using light Tokyo, Japan).
microscopy. Quantification of the nodes, branches, and meshes was Quantitative PCR (qPCR) analysis was utilized to evaluate the
performed using ImageJ software (n = 3). mRNA levels of osteogenesis-associated genes, including runt-related
For gene expression analysis, HUVECs were cocultured with transcription factor 2 (RUNX2), COL-I, osteocalcin (OCN), and
scaffold extracts for 5 days. Quantitative real-time polymerase chain OPN. The specific primer sequences are provided in Table S3.
reaction (qPCR) was utilized to assess the expression of angiogenic Western blot analysis was employed to determine the expression of
genes. Total mRNA was isolated using the TRIzol reagent osteogenic proteins, using primary antibodies for frizzled 8 (FZD8,
(Invitrogen, Life Technologies, CA, USA). cDNA synthesis was 1:1000, A18393, ABclonal), β-catenin (1:5000, 66379-1-Ig, Protein-
performed with 500 ng of RNA using the PrimeScript RT reagent kit tech), RUNX2 (1:800, 29595, Signalway Antibody), COL-I (1:2000,
(TaKaRa, Japan), and qPCR was carried out with SYBR Premix Ex 66761-1-Ig, Proteintech), OPN (1:1000, 22952-1-AP, Proteintech),
Taq (TaKaRa, Japan) on an Applied Biosystems QuantStudio 5 Real- and OCN (1:800, A6205, ABclonal).
Time PCR System (Applied Biosystems, Waltham, MA, USA). The Alkaline phosphatase (ALP) activity was assessed by seeding
primer sequences are listed in Table S2. The expression levels of HIF- BMSCs in plates, followed by a 24 h incubation and subsequent
1α, CD31, and vascular endothelial growth factor (VEGF) were osteogenic induction with the Rat MSC Osteogenic Differentiation
measured with β-actin serving as the reference gene. Relative Kit along with scaffold extracts. After 14 days, the cells were subjected
expression levels were determined using the 2−ΔΔCT method. to staining using the BCIP/NBT Alkaline Phosphatase Color
Western blot analysis was conducted to assess the protein Development Kit (Beyotime, Shanghai, China). On the 14th day,
expression levels. HUVECs were cultured with the scaffold extracts cells were washed with PBS, lysed with RIPA buffer, and the total
for 5 days, and total proteins were extracted using RIPA lysis buffer protein concentration was measured using a BCA protein assay kit

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Figure 2. Morphology of 2DS, 3DS, and 3DS-Sr. (A) Gross and SEM images of 2DS, 3DS, and 3DS-Sr scaffolds after the reaction with 2 mM
(3DS-Sr2), 20 mM (3DS-Sr20), and 200 mM (3DS-Sr200) Sr(NO3)2. Quantification of the thickness (B), volume (C), density (D), and porosity
(E) of scaffolds (n = 3; #, $, %, and & indicate p < 0.05 vs 2DS, 3DS, 3DS-Sr2, and 3DS-Sr20).

(Thermo Scientific, USA). ALP activity was quantified according to staining. Additionally, immunofluorescent staining was conducted for
the instructions of the Alkaline Phosphatase Assay Kit (Beyotime, CD31, alpha-smooth muscle actin (α-SMA), OCN, OPN, COL-I,
Shanghai, China). interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α),
Following 21 days of culture in osteogenic medium supplemented with staining intensity quantitatively analyzed using ImageJ software.
with scaffold extracts, BMSCs were fixed and stained with Alizarin 2.8. Statistical Analysis. Statistical analyses were performed by
Red S (ARS). The stained cells were treated with acetic acid and utilizing OriginPro 8 software. The data were expressed as the mean
incubated overnight, after which the supernatant was collected and ± the standard deviation. To compare multiple groups, we employed
neutralized with ammonia. The absorbance of the neutralized solution a one-way analysis of variance (ANOVA) followed by Tukey’s post
was measured at 405 nm by using a microplate reader. hoc test. For comparisons involving only two groups, t tests were
2.7. In Vivo Bone Regeneration Evaluation. Animal experi- conducted. A p-value of less than 0.05 was deemed to indicate
ments were conducted with the approval of the Ethics Committee of statistical significance.
Shanghai Tongren Hospital (A2023-050-01). To evaluate the
osteogenic capabilities of various scaffolds, a cranial defect model 3. RESULT
using 8-week-old Sprague−Dawley (SD) rats was employed. The Two-dimensional PCL/F-127 nanofiber membranes (2DS)
animals were categorized into four groups: control (no scaffold), 2DS, were fabricated via electrospinning, which exhibited hydro-
3DS, and 3DS-Sr. A 5 mm cranial defect was generated using a
philic properties, allowing for the efficient adsorption of
trephine under pentobarbital anesthesia. The scaffolds were inserted
into the defect sites, followed by suturing of the skin. The rats were
Sr(NO3)2 into the membrane. NaBH4 served a dual role: on
sacrificed at 6 and 12 weeks postsurgery, and cranial samples were one hand, it reacted with water to produce hydrogen gas
harvested and fixed. Micro-CT scans were conducted to determine bubbles, facilitating the transformation of the scaffold into a
the structural parameters utilizing CT-Analyzer software for analysis. 3D multilayer structure; on the other hand, it reacted with
Postdecalcification, samples underwent dehydration, embedding, and Sr(NO3)2 to form uniform decorating of Sr NPs within the
sectioning. Bone regeneration across the groups was evaluated scaffold. The 3D porous nanofiber network provided an
through Hematoxylin and eosin (H&E) and Masson’s trichrome enhanced number of binding sites for cell attachment and
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Figure 3. Characterization of the scaffold composition, mechanical properties, and ion release behavior. ATR-FTIR (A), TG (B), XPS (C), and
XRD (D) spectra of 2DS, 3DS, and 3DS-Sr. Mechanical properties, including representative tensile stress−strain curves (E), Young’s modulus (F),
elongation at break (G), and ultimate tensile strength (H) of 2DS, 3DS, and 3DS-Sr (n = 3). (I) Release behavior of Sr2+ from 3DS-Sr in PBS at 37
°C (n = 3; # and $ indicate p < 0.05 vs 2DS and 3DS).

fostered cell infiltration and tissue ingrowth. The Sr NPs 3DS-Sr scaffold fabrication. At low Sr(NO3)2 concentrations
coating facilitated osteogenesis and angiogenesis by releasing (2 mM), a limited number of Sr NPs formed on the scaffold
strontium ions, thereby accelerating the bone regeneration fibers. Conversely, 20 and 200 mM Sr(NO3)2 treatment
process (Figure 1). resulted in extensive Sr NPs formation, occluding scaffold
Figure 2A presents both gross and SEM images of 2DS, pores and hindering both hydrogen bubble entry and nanofiber
3DS, and 3DS-Sr scaffolds. As revealed by SEM analysis, the expansion. Increasing salt concentrations yielded less signifi-
absence of Sr(NO3)2 precluded the formation of Sr NPs on cant changes in the thickness, volume, density, and porosity of
3DS. Conversely, the introduction of Sr(NO3)2 resulted in the 3DS-Sr scaffolds compared to their 2D counterparts (Figure
formation of Sr NPs, uniformly distributed on the nanofibers 2B−E). Therefore, a 2 mM Sr(NO3)2 solution was deemed
with an average size of 501 ± 163 nm (Figure S1). The optimal for maintaining multilayered morphology while
diameters of nanofibers in the 2DS, 3DS, and 3DS-Sr groups generating an appropriate density of Sr NPs.
exhibited no significant differences, measuring 532 ± 160 nm, FTIR spectroscopy revealed the chemical composition of
533 ± 178, and 527 ± 148 nm, respectively (Figure S2). In various scaffolds (Figure 3A). 2DS, 3DS, and 3DS-Sr all
addition, the pore area of 3DS and 3DS-Sr was 62 ± 20 μm2 exhibited characteristic PCL peaks. The peak at 1726 cm−1 was
and 58 ± 19 μm2, which was significantly higher than that of attributed to the carbonyl stretch vibration. Two peaks at 2945
2DS (13 ± 3 μm2; Figure S3). To investigate the impact of and 2866 cm−1 correspond to asymmetric and symmetric
Sr(NO3)2 concentration on 3D scaffold properties during gas- −CH2− stretching vibrations, respectively. Peaks at 1240 and
foaming, we examined a gradient of 2 mM, 20 mM, and 200 1170 cm−1 represented asymmetric C−O−C stretch vibrations
mM. This approach aimed to identify optimal parameters for and symmetric C−O−C stretch vibrations, respectively. The
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Figure 4. In vitro cytocompatibility assessment. (A) Live/dead assay of HUVECs and BMSCs cocultured with extracts from 2DS, 3DS, and 3DS-Sr
for 24 h. (B) DAPI-phalloidin staining of BMSCs cultured on different scaffolds for 3 days. (C) Cell seeding efficiency of HUVECs and BMSCs on
different scaffolds (n = 3). Proliferation of HUVECs (D) and BMSCs (E) cocultured with extracts from different scaffolds (n = 3; *, #, and $
indicate p < 0.05 vs control, 2DS, and 3DS, respectively).

FTIR data confirmed that no chemical reactions occurred at break (Eb) of 2DS were higher than those of the 3D
among components during the gas foaming process. At 800 °C, scaffolds. However, introducing Sr NPs to 3DS-Sr slightly
the residual weights of 2DS, 3DS, and 3DS-Sr were 0.66, 1.56, improved the mechanical properties of 3D scaffolds, albeit not
and 3.18 wt %, respectively, indicating the higher residue in significantly (Figure 3E−H). The loading−unloading cycle
3DS-Sr was likely from residual Sr NPs (Figure 3B). The XPS tests showed that after 50 compressions at 50% strain, both
analysis further illustrated the incorporation of strontium into 3DS and 3DS-Sr exhibited nonlinear, closed-loop compression
the scaffold. 2DS and 3DS were devoid of the Sr 3d spectrum, stress−strain curves, indicating low energy dissipation (Figure
whereas 3DS-Sr displayed the Sr 3d spectrum, indicating the S5A,B). Although the increase was not statistically significant,
decoration of Sr NPs on the nanofibers (Figure 3C). XRD the introduction of Sr particles led to a higher compressive
patterns of 2DS, 3DS, and 3DS-Sr exhibited characteristic modulus in the scaffolds. The compressive moduli of 3DS-Sr
peaks at 21.4 and 23.8°, attributed to the semicrystalline PCL and 3DS were 3.51 ± 0.17 and 3.26 ± 0.11 kPa, respectively
matrix.21 An additional peak at 25.3° in 3DS-Sr was attributed (Figure S5C). Figure 3I shows the release kinetics of strontium
to strontium hydroxide and strontium oxide (Figure 3D). ions from 3DS-Sr, with an initial release of 36.57% on day 1,
Figure S4 illustrates that the pH values of solutions containing increasing to 59.98% by day 3, and reaching a total of 78.81%
different scaffolds remained stable at approximately 7.4. by day 14. Figure S6 depicts the strontium ion release
Notably, 3DS-Sr did not induce significant pH changes, likely concentration of the 3DS-Sr, demonstrating its sustained ion
attributable to its low Sr(OH)2 content, which minimally release capacity. Figure S7 illustrates the 16-week degradation
affected the surrounding microenvironment. The Young’s trends of 2DS, 3DS, and 3DS-Sr scaffolds. By the eighth week,
modulus (E), ultimate tensile strength (UTS), and elongation the weight losses recorded for 2DS, 3DS, and 3DS-Sr were
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Figure 5. Angiogenic potential of different scaffolds on HUVECs. Immunofluorescence staining of CD31 (A) and HIF-1α (B) after 5 days of
coculture with scaffold extracts. Immunofluorescence quantification of CD31 (C) and HIF-1α (D) (n = 3). CD31 (E), VEGF (F) and HIF-1α (G)
mRNA expression levels in HUVECs after 5 days of culture with scaffold extracts (n = 3). (H) Formation of tubular networks in HUVECs after 8 h
of incubation. Quantitative assessment of angiogenesis parameters including node (I), branch (J), and mesh numbers (K). Representative Western
blot images (L) of protein expression in HUVECs cultured with scaffold extracts and quantitative analysis including CD31 (M), VEGF (N), HIF-
1α (O), and p-ERK/ERK ratio (P) (n = 3; *, #, and $ indicate p < 0.05 vs control, 2DS, and 3DS, respectively).

7.16%, 10.51%, and 11.97%, respectively. By the 16th week, scaffolds, while growing on the surface of the 2D scaffolds
these values increased to 15.53%, 17.76%, and 19.31%. (Figure 4B). Cells cultured on 2D and 3D nanofiber scaffolds
Live/dead assays indicated that the cytotoxicity of all exhibited more stereoscopic morphologies compared to those
scaffolds was negligible (Figures 4A and S8). DAPI-phalloidin cultured on control plates. The increased space of 3D scaffolds
staining images demonstrated that the cells cultured on both facilitated cellular proliferation and a wider distribution of cells,
2D and 3D scaffolds exhibited a more stereoscopic consistent with previous findings.22 Quantitative analysis of
morphology compared to the cells cultured on the culture confocal 3D reconstruction images revealed an average
plate. Cells displayed a 3D distribution within the gas-foamed infiltration depth of 13 ± 3 μm for the 2DS. In contrast, the
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Figure 6. Osteogenic effects of the scaffolds on BMSCs. Expression of OPN (A) and COL-I (B) after coculture of BMSCs with scaffold extracts for
7 days. Quantification of immunofluorescent staining for OPN (C) and COL-I (D) (n = 3). Relative expression levels of RUNX2 (E), COL-I (F),
OCN (G), and OPN (H) mRNA in HUVECs cultured with scaffold extract for 7 days (n = 3). Representative digital images and micrographs of
ALP staining (I) and ARS staining (J) for BMSCs. Quantitative analysis of ALP activity (K) and ARS staining (L) (n = 3). Western blot images
(M) and quantitative analyses of COL-I (N), OPN (O), RUNX2 (P), OCN (Q), FZD8 (R), β-catenin (S) (n = 3; *, #, and $ indicate p < 0.05 vs
control, 2DS, and 3DS, respectively).

3DS and 3DS-Sr demonstrated significantly higher cell ± 9 μm, respectively (Figure S9). The 3D scaffolds showed a
infiltration depths, reaching average depths of 75 ± 7 and 76 higher cell seeding efficiency compared to 2DS (Figure 4C).
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Figure 7. Bone regeneration of different scaffolds in vivo. (A) Schematic illustration of scaffolds implantation and repair of calvarial defect. Micro-
CT images (B) and BV/TV values (C) of regenerated cranial bone after 6 and 12 weeks of scaffold implantation (n = 4). (D) H&E staining and
Masson’s trichrome staining of regenerated bone tissues. The blue and red arrows point toward the new bone (*, #, and $ indicate p < 0.05 vs
control, 2DS, and 3DS, respectively).

HUVECs proliferated faster in scaffold groups compared with formation compared to other groups, with increased numbers
the control groups, and BMSCs exhibited the highest of nodes and mesh structures and elongated branching (Figure
proliferation rate in 3DS-Sr at day 5 (Figure 4D,E). These 5H−K). As shown in Figure 5L-P, the p-ERK, HIF-1α, CD31,
results suggested that the Sr NPs-modified 3D scaffolds with and VEGF protein levels were significantly higher in the 3DS-
multilayered structures featured good cytocompatibility and Sr group compared with the others. The results indicated that
can support cell adhesion and proliferation. 3DS-Sr elicited increased phosphorylation of ERK, enhancing
Immunofluorescence staining was performed after 5 days of the expression of CD31, HIF-1α, and VEGF in HUVECs,
culturing HUVECs with different scaffold extracts to explore resulting in a favorable effect on angiogenesis.
their pro-angiogenic effects. The HUVECs cocultured with After coculturing BMSCs with different scaffold extracts for
3DS-Sr extracts showed the strongest fluorescence signal of 7 days, immunofluorescent staining was employed to
CD31 and HIF-1α compared to other groups (Figure 5A−D). investigate the osteogenic effects. The 3DS-Sr scaffold notably
The mRNA levels of angiogenesis-related genes, including increased the expression of OPN and COL-I in BMSCs
CD31, VEGF, and HIF-1α were significantly elevated in the compared to other groups (Figure 6A−D). 3DS-Sr signifi-
3DS-Sr group compared to other groups, as detected by qPCR cantly upregulated RUNX2, COL-I, OCN, and OPN
(Figure 5E−G). In vitro tube formation assays on Matrigel expression in BMSCs (Figure 6E−H). The osteogenic
demonstrated that 3DS-Sr induced more tube-like network potential of the scaffolds was further validated through ALP
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Figure 8. Immunofluorescence staining of regenerated bone tissue in vivo. (A) Immunofluorescence staining of repair bone sections including
CD31 (green) and α-SMA (yellow) after 6 weeks of scaffold implantation, OCN (red), OPN (red), and COL-I (red) after 12 weeks of scaffold
implantation. (B−F) Quantitative analysis of the fluorescent-positive staining areas using ImageJ software (n = 4; *, #, and $ indicate p < 0.05 vs
control, 2DS, and 3DS, respectively).

and ARS staining. On day 14, the 3DS-Sr scaffold significantly degree of bone accumulation compared to the 2DS group, and
boosted ALP activity in BMSCs and promoted the formation the 3DS-Sr group presented the highest amount of new bone
of more mineralized nodules by day 21 compared to the formation in the defect area (Figure 7B).
control and other scaffold groups (Figure 6I,J). Quantitative Quantitative analysis of the bone volume fraction (new bone
analyses of ALP and ARS were consistent with these volume/total volume, BV/TV) demonstrated a gradual
observations (Figure 6K,L). Furthermore, the expression levels increase in the amount of new bone tissue over time (Figure
of COL-I, OPN, RUNX2, OCN, FZD8, and β-catenin were 7C). After 12 weeks of scaffold implantation, the control group
markedly higher in the 3DS-Sr group (Figure 6M−S). These had the lowest BV/TV of 15.75 ± 2.21%, the 2DS group
findings suggested that the 3DS-Sr scaffold can activate the exhibited a slightly higher BV/TV of 23 ± 2.44% compared to
Wnt/β-catenin signaling pathway, thereby enhancing the the control group, the 3DS group had a BV/TV of 33.25 ±
expression of osteogenic genes and proteins in BMSCs 2.63%, and the 3DS-Sr group displayed the highest BV/TV of
2DS, 3DS, and 3DS-Sr scaffolds were implanted into 44.75 ± 2.5%. Furthermore, quantitative results of bone
calvarial defects of SD rats. After 6 weeks and 12 weeks mineral density (BMD), bone volume (BV), trabecular
postimplantation, the rats were sacrificed to observe the cranial separation (Tb.S), trabecular number (Tb.N) and trabecular
regeneration in different groups (Figure 7A). After 6 weeks of thickness ([Link]) of regenerated bone tissue demonstrated
scaffold implantation, the control group exhibited relatively that 3DS-Sr exhibited the best repair effect compared to the
low bone regeneration in the defect area. The 2DS and 3DS control groups and other scaffold groups (Figure S10).
groups displayed a limited amount of regenerated bone tissue, Histological evaluation using H&E and Masson’s staining
while the 3DS-Sr group displayed partial fusion of new bone. was conducted to assess the formation of the regenerated bone
After 12 weeks, the control group exhibited defects with largest tissue (Figure 7D). The control group accumulated a
areas in the center. The 2DS group showed a small amount of substantial amount of fibrous tissue in the bone defect cavity.
newly formed bone tissue, the 3DS group displayed a higher The 2DS group exhibited only a small amount of newly formed
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bone tissue, while the 3DS group displayed new bone the osteogenic microenvironment for bone tissue remodeling,
regeneration in the central area. The 3DS-Sr group elucidating the potential applications and mechanisms of 3D
demonstrated predominantly new bone tissue covered in the nanofiber scaffolds modified with bioactive ions in bone tissue
defect area, performing significantly better than the other regeneration.
groups. Several techniques, including 3D printing, thermally induced
Immunofluorescence staining was employed to further assess phase separation (TIPS), and electrospinning, are currently
the neovascularization, osteogenesis, and inflammation of the employed to develop scaffolds for bone tissue engineering.23,24
regenerated bone tissue (Figures 8A and S11A). CD31 and α- However, these methods present limitations. While 3D
SMA staining were performed on the 6-week regenerated bone printing enables precise scaffold fabrication, it often fails to
tissue, revealing that the 3DS-Sr group exhibited a greater replicate the nanofibrous morphology of the natural ECM,
extent of neovascularization compared to other groups. thereby limiting cell adhesion and requiring expensive
Immunofluorescence staining of the 12-week regenerated equipment. TIPS, on the other hand, relies on potentially
bone tissue was performed to assess the expression of toxic organic solvents and offers limited control over the
osteogenic markers (OCN, OPN, COL-I) and pro-inflamma- scaffold pore size. Electrospinning generates ultrafine nano-
tory cytokines (IL-1β, TNF-α). The 3DS-Sr group exhibited fibers that mimic the natural ECM, making them ideal for
the highest fluorescence intensity for osteogenic markers and tissue engineering scaffolds. Conventional electrospun 2D
the lowest for pro-inflammatory cytokines compared to other nanofiber membranes, while providing good surface character-
groups. Quantitative analysis of fluorescence intensity con- istics, are mainly limited by their thickness and lack of a 3D
firmed this trend (Figures 8B−F and S11B,C). These results structure. This limitation may result in cells growing only on
suggested that the 3DS-Sr group promoted vascularization and the surface of the membrane rather than within the 3D space
bone regeneration, while mitigating inflammatory responses. of the material, thereby restricting the cultivation of more
Figure 9 describes the overall clues of 3DS-Sr in promoting complex and thicker tissues in tissue engineering.25 Gas
bone regeneration. 3DS-Sr displayed a multilayered structure foaming technology creates porous structures with uniformly
distributed pores in nanofiber scaffolds. These porous scaffolds
provide a 3D environment conducive to cell growth. Although
gas foaming technology improves the applicability of nanofiber
scaffolds, their bioactivity in bone tissue engineering remains
limited, particularly in promoting angiogenesis and osteo-
genesis, which are crucial for providing nutrients and
enhancing bone tissue regeneration. We utilize NaBH4 as a
gas foaming and reducing agent, enabling the simultaneous
formation of 3D scaffolds and the in situ synthesis of anchored
Sr NPs on their surface. The innovation lies in leveraging the
gas-foaming and -reducing capabilities of NaBH4 to simulta-
neously enhance the dimensionality and bioactivity of the
scaffold in a single step. This approach for producing bioactive
3D scaffolds addresses the limitations associated with nano-
fiber membranes. This facile and efficient preparation method
avoids the use of toxic organic solvents and specialized
equipment. The uniform dispersion of Sr NPs within the
polymer matrix endows the scaffolds with the capability to
promote angiogenesis and bone regeneration, demonstrating a
promising therapeutic effect in bone tissue repair.
An increase in the Sr(NO3)2 concentration was inversely
correlated with 3D scaffold expansion. This phenomenon likely
Figure 9. Schematic diagram illustrating the mechanism of 3DS-Sr arose from the preferential reaction of NaBH4 with water in the
scaffold induced angiogenesis and osteogenesis. absence of Sr(NO3)2, leading to gas formation and maximizing
the 2D mat porosity. Conversely, in the presence of high
that provided more binding sites for cell adhesion and Sr(NO3)2 concentrations (20 and 200 mM), a rapid reaction
proliferation. By releasing strontium ions, it elicited increased between NaBH4 and Sr(NO3)2 occurred prior to the NaBH4−
phosphorylation of ERK, promoting angiogenesis in HUVECs, water reaction. This generated large Sr NPs on the nanofibers,
and activated the Wnt/β-catenin signaling pathway to promote effectively coating the scaffold, sealing surface pores, and
osteogenesis in BMSCs. preventing hydrogen bubble penetration, thereby inhibiting 3D
structure formation. Consequently, increasing the Sr(NO3)2
4. DISCUSSION concentration resulted in a shift from 3D to 2D sample
In this study, we presented an approach for fabricating bone morphology. This suggested that the relatively loose 3D
tissue engineering scaffolds that enabled the 3D structure structure formed at lower Sr(NO3)2 concentrations allowed for
construction and in situ synthesis of Sr NPs. The 3D scaffolds uniform Sr NPs distribution throughout the nanofiber
provided more space for cell adhesion and proliferation due to framework. Therefore, an optimal Sr(NO3)2 concentration (2
their multilayered structure. 3DS-Sr released Sr2+, creating a mM) was crucial, as excessive salt concentrations hindered the
microenvironment conducive to HUVEC vascularization and 3D structure development. The Young’s modulus of the 3D
BMSCs osteogenic differentiation. Our research underscored scaffold was significantly lower than that of the pristine mats.
the importance of constructing 3D structures and enhancing The elongation at break decreased rapidly due to the reduction
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in the dense fiber arrangement caused by gas foaming, where strontium onto the surface of titanium implants enhanced
H2 gas separated the fibers, reducing mechanical performance. ERK1/2 phosphorylation. This enhancement further boosted
The Young’s modulus of 3DS-Sr was marginally higher than HIF-1α expression, ultimately facilitating angiogenesis and
that of 3DS, suggesting that the incorporation of Sr NPs osteogenesis for accelerated vascularized bone repair.
enhanced the mechanical strength of the scaffolds. In vitro BMSCs have the potential to differentiate into various cell
cytotoxicity assays indicated that the presence of Sr NPs did types, including adipocytes and chondrocytes, but directing
not adversely affect cell viability or growth, showing negligible them toward osteogenic differentiation remains challenging.34
cytotoxicity to HUVECs and BMSCs. FDA-approved F-127 Research by Li et al. revealed that strontium enhances cell
has been documented to facilitate cell adhesion at low cycle progression, particularly in the S and G2/M phases,
concentrations, possibly accounting for the increased pro- which may trigger osteogenic differentiation.35 This could
liferation of HUVECs on the scaffold compared to the explain the higher cell proliferation observed in strontium-
control.26 We speculated that this phenomenon was not containing scaffolds. Moreover, strontium can activate the
observed in the BMSCs proliferation experiment due to the Wnt/β-catenin signaling pathway, which is essential for the
slower proliferation rate of BMSCs compared with HUVECs, osteogenic behavior including proliferation, differentiation, and
thus diminishing the cell adhesion-promoting effect of F-127. mineralization of BMSCs.36 It has been reported that
The porous multilayer structure of the gas-foamed scaffold strontium ions can interact with calcium-sensing receptors
likely contributed to improved cell adhesion and growth, which (CaSR), activating the ERK, Wnt, and p38 pathways, thereby
could expedite bone tissue remodeling. The degradation of the promoting the proliferation and osteogenic differentiation of
scaffolds was assessed in vitro by immersion in PBS for 16 osteoblasts and accelerating bone matrix mineralization.37
weeks. As detailed in the Supporting Information, the Previous studies have shown that the incorporation of
degradation rates for 2DS, 3DS, and 3DS-Sr were 15.53%, strontium into bioactive borate glass bone cement can activate
17.76%, and 19.31%, respectively. PCL is a biodegradable the Wnt/β-catenin signaling pathway to enhance osteogenic
polymer widely employed in biomedical applications, partic- capacity in vivo.36 Similarly, calcium phosphate bioceramics
ularly for scaffold fabrication in tissue engineering. Its incorporating strontium have been found to activate the Wnt/
degradation, primarily via hydrolysis, is influenced by factors β-catenin and BMP-2/Smad pathways, creating a favorable
such as molecular weight, scaffold morphology (e.g., porosity, microenvironment for promoting osteogenesis.38 Consistent
thickness), and the biological environment (e.g., pH, enzyme with previous studies, our study demonstrated that 3DS-Sr
activity).27 Due to its long aliphatic chains, PCL degrades upregulated key proteins in the Wnt/β-catenin pathway,
relatively slowly, typically requiring 1−2 years for complete
specifically β-catenin and FZD8, thereby promoting osteogenic
degradation in vivo. While multilayered scaffold structures may
differentiation and facilitating bone remodeling.
accelerate localized degradation and enhance interaction with
Optimal bone regeneration was observed in rat cranial
bodily fluids, even highly porous gas-foamed scaffolds are
defects treated with 3DS-Sr, in alignment with the in vitro
unlikely to degrade completely within a short time frame.
findings. The 3D bioactive nanofiber scaffold enhanced tissue
Importantly, PCL degradation products are nontoxic and
repair via its multilayer structure and combined effects of Sr2+
generally do not elicit significant immune responses in vivo.
Furthermore, the extended degradation period does not release, which induced vascularization and bone regeneration.
preclude the scaffold’s function as a temporary matrix during We found that 3D nanofiber scaffolds enhanced the integration
bone remodeling. Given the high porosity and nanofiber with natural matrix. Following implantation, the sustained
morphology of the gas-foamed 3DS-Sr scaffold, we speculate release of strontium ions favored cell for osteogenic differ-
that it can integrate with nascent tissue and become embedded entiation. This synergistic interaction between the 3D structure
within newly formed bone. and the osteogenic microenvironment enhanced bone
Strontium, a trace element found predominantly in bone regeneration. However, the current study has some limitations.
tissue, plays a vital role in bone regeneration, despite its exact The precise roles and mechanisms of the scaffold in in vivo
mechanisms being not fully understood.28 Recent research has bone regeneration require further investigation; the roles of
shown that Sr2+ enhances the viability of endothelial cells, strontium in osteogenesis, osteoclastogenesis, and immune
fibroblasts, and smooth muscle cells, which are key regulation necessitate more detailed future investigations.
components of blood vessels.29 Strontium has been extensively Although strontium shows promise for bone regeneration, it
reported to promote angiogenesis. Chen et al.30 developed is associated with potential off-target effects and long-term
strontium-functionalized gelatin hydrogels that accelerated stability issues. Excessive presence of strontium ions can
endothelial progenitor cell-driven angiogenesis and enhanced adversely affect the cardiovascular system, potentially increas-
wound tissue regeneration. Strontium also promotes angio- ing the risk of cardiovascular diseases with prolonged high
genesis by stimulating the secretion of pro-angiogenic factors. intake.39 Since strontium is primarily excreted through the
This mechanism likely involves the ERK1/2 pathway, a critical kidneys, elevated strontium levels could burden renal function,
upstream regulator of HIF-1α. Activation of the ERK1/2 especially in individuals with compromised kidneys.29,40 For
pathway enhances HIF-1α expression, a crucial step in effective bone regeneration, strontium ion release must be
hypoxia-induced angiogenesis.31 Our findings indicated that stable over the long-term; a rapid release may result in
3DS-Sr elicited faster vascularized bone repair through excessively high concentrations, causing off-target effects.
activation of the ERK1/2 signaling pathway in HUVECs. Future research should focus on developing biomaterials with
Previous research aligned with this study, demonstrating that controlled release mechanisms that adjust strontium release in
mesoporous bioactive glass composite scaffolds stimulated the response to external stimuli such as pH changes. Additionally,
ERK1/2 signaling pathway. This activation promoted both local implants or coating technologies could confine strontium
HIF-1α protein expression and the formation of H-type blood release to specific areas, thereby minimizing systemic off-target
vessels.32 Similarly, Zhang et al.33 found that incorporating effects.
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Strontium is known to promote osteoblast proliferation and

differentiation in bone tissue engineering. However, continu-
■ AUTHOR INFORMATION
Corresponding Authors
ous, excessive strontium ion release poses potential risks, Yujie Chen − Laboratory of Key Technology and Materials in
including skeletal overstimulation and abnormal growth.40 This Minimally Invasive Spine Surgery, Tongren Hospital,
duality arises from strontium’s concentration-dependent effects Shanghai Jiao Tong University School of Medicine, Shanghai
on osteoblasts. Appropriate concentrations promote differ- 200336, China; Center for Spinal Minimally Invasive
entiation and accelerate bone formation. However, excessive or Research, Shanghai Jiao Tong University, Shanghai 200336,
prolonged exposure to high strontium levels can lead to China; Department of Orthopaedics, Tongren Hospital,
osteoblast overactivation, increasing bone tissue deposition.41 Shanghai Jiao Tong University School of Medicine, Shanghai
This uncontrolled bone formation may manifest as bone 200336, China; [Link]/0000-0002-6285-4074;
thickening, spurs, or calcification, potentially impairing skeletal Email: yjchen@[Link]
structure and function and even triggering pathological Xiaojian Ye − Laboratory of Key Technology and Materials in
conditions. 3DS-Sr developed in this study aimed to mitigate Minimally Invasive Spine Surgery, Tongren Hospital,
these risks through controlled, sustained strontium ion release, Shanghai Jiao Tong University School of Medicine, Shanghai
avoiding burst release. Cellular experiments confirmed that the 200336, China; Center for Spinal Minimally Invasive
scaffold’s strontium ion release effectively promoted BMSCs Research, Shanghai Jiao Tong University, Shanghai 200336,
proliferation and differentiation without cytotoxicity. In vivo, China; Department of Orthopaedics, Tongren Hospital,
3DS-Sr effectively promoted bone regeneration in animal Shanghai Jiao Tong University School of Medicine, Shanghai
models without observable bone overgrowth or abnormalities. 200336, China; Email: 732004380@[Link]
Compared with strontium-free scaffolds and the blank control Jiangming Yu − Laboratory of Key Technology and Materials
group, 3DS-Sr demonstrated the most favorable bone in Minimally Invasive Spine Surgery, Tongren Hospital,
regeneration capacity. While this study primarily focused on Shanghai Jiao Tong University School of Medicine, Shanghai
short-term bone regeneration outcomes, long-term monitoring 200336, China; Center for Spinal Minimally Invasive
of strontium ion effects was crucial. Future research will Research, Shanghai Jiao Tong University, Shanghai 200336,
evaluate bone quality, density, and mechanical properties over China; Department of Orthopaedics, Tongren Hospital,
extended periods following scaffold implantation. Shanghai Jiao Tong University School of Medicine, Shanghai
200336, China; Email: yjm_st@[Link]
5. CONCLUSION
Authors
In conclusion, by exploiting the dual roles of NaBH4 in gas
foaming and reduction, we developed an advanced porous Yucai Li − Laboratory of Key Technology and Materials in
bioactive scaffold, designated as 3DS-Sr. This scaffold was Minimally Invasive Spine Surgery, Tongren Hospital,
characterized by its multilayered architecture, which enabled Shanghai Jiao Tong University School of Medicine, Shanghai
the prolonged release of bioactive Sr2+ ions. The 3D structure 200336, China; Center for Spinal Minimally Invasive
offered extensive space for cell and tissue proliferation. In vitro Research, Shanghai Jiao Tong University, Shanghai 200336,
analyses revealed that Sr2+ ions facilitated angiogenesis and China; Department of Orthopaedics, Tongren Hospital,
osteogenesis through activation of the ERK and Wnt/β-catenin Shanghai Jiao Tong University School of Medicine, Shanghai
signaling pathways, thereby promoting bone regeneration. 200336, China
Additionally, in vivo experiments verified that the 3DS-Sr Xinyi Wang − Shanghai Engineering Research Center of Nano-
scaffold effectively supported bone regeneration. Collectively, Biomaterials and Regenerative Medicine, College of Biological
these findings suggested that the 3DS-Sr scaffold was a Science and Medical Engineering, Donghua University,
promising candidate for the treatment of bone defects. Songjiang, Shanghai 201620, China
Xiumei Mo − Shanghai Engineering Research Center of Nano-

■
*
ASSOCIATED CONTENT
sı Supporting Information
Biomaterials and Regenerative Medicine, College of Biological
Science and Medical Engineering, Donghua University,
Songjiang, Shanghai 201620, China; [Link]/0000-
The Supporting Information is available free of charge at 0001-9238-6171
[Link] Yicheng Chen − Laboratory of Key Technology and Materials
SEM images of 3DS-Sr and size distribution of Sr NPs in Minimally Invasive Spine Surgery, Tongren Hospital,
(Figure S1); Nanofiber diameter distribution of scaffolds Shanghai Jiao Tong University School of Medicine, Shanghai
(Figure S2); Pore size distribution of scaffolds (Figure 200336, China; Center for Spinal Minimally Invasive
S3); pH analysis of scaffolds immersed in PBS (Figure Research, Shanghai Jiao Tong University, Shanghai 200336,
S4); Comprehensive mechanical testing of 3D scaffolds China; Department of Orthopaedics, Tongren Hospital,
(Figure S5); Sr2+ release behavior of 3DS-Sr (Figure Shanghai Jiao Tong University School of Medicine, Shanghai
S6); In vitro degradation studies (Figure S7); Live/Dead 200336, China
assay of BMSCs on the scaffold (Figure S8); Cell Zijun Deng − Laboratory of Key Technology and Materials in
infiltration depth on scaffolds (Figure S9); Evaluation of Minimally Invasive Spine Surgery, Tongren Hospital,
the microstructure of regenerative bone tissue (Figure Shanghai Jiao Tong University School of Medicine, Shanghai
S10); Evaluation of the inflammation response of 200336, China; Center for Spinal Minimally Invasive
regenerative bone tissue (Figure S11); Equipment, Research, Shanghai Jiao Tong University, Shanghai 200336,
brand, and origin for scaffold characterization (Table China; Department of Orthopaedics, Tongren Hospital,
S1); Primer sequences for qPCR on HUVECs (Table Shanghai Jiao Tong University School of Medicine, Shanghai
S2); Primer sequences for qPCR on BMSCs (Table S3) 200336, China
(PDF) Complete contact information is available at:
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[Link] Electrospun Scaffolds for Cartilage Regeneration. Biomater. Adv.

2022, 134, 112643.
Author Contributions (13) Chen, Y.; Xu, W.; Shafiq, M.; Tang, J.; Hao, J.; Xie, X.; Yuan,
# Z.; Xiao, X.; Liu, Y.; Mo, X. Three-Dimensional Porous Gas-Foamed
These authors contributed equally to this work (Yujie C., Y.L.,
Electrospun Nanofiber Scaffold for Cartilage Regeneration. J. Colloid
and X.W.). Interface Sci. 2021, 603 (4), 94−109.
Notes (14) Amarjargal, A.; Goudarzi, Z. M.; Cegielska, O.; Gradys, A.;
The authors declare no competing financial interest. Kolbuk, D.; Kalaska, B.; Ruszczynska, A.; Sajkiewicz, P. A Facile One-
Stone-Two-Birds Strategy for Fabricating Multifunctional 3D Nano-

■ ACKNOWLEDGMENTS
This research was supported by the Science and Technology
fibrous Scaffold. Biomater. Sci. 2023, 11, 5502−5516.
(15) Wan, Z.; Yuan, Z.; Li, Y.; Zhang, Y.; Wang, Y.; Yu, Y.; Mao, J.;
Cai, Q.; Yang, X. Hierarchical Therapeutic Ion-Based Microspheres
Commission of Shanghai Municipality (22Y11912000), with Precise Ratio-Controlled Delivery as Microscaffolds for In Situ
Medical and Industrial Cross Research Foundation of “Star Vascularized Bone Regeneration. Adv. Funct. Mater. 2022, 32 (23),
of Jiaotong University” Program of Shanghai Jiao Tong 2113280.
University, China (Grant No. YG2022ZD030), Laboratory (16) Cai, Z.; Li, Y.; Song, W.; He, Y.; Li, H.; Liu, X. Anti-
Open Fund of Key Technology and Materials in Minimally Inflammatory and Prochondrogenic in Situ-Formed Injectable
Hydrogel Crosslinked by Strontium-Doped Bioglass for Cartilage
Invasive Spine Surgery (2024JZWC-YBA01), Tongren Hospi- Regeneration. ACS Appl. Mater. Interfaces 2021, 13 (50), 59772−
tal Introduces the Talented Person Scientific Research Start 59786.
Funds Subsidization Project (TR2023rc08). (17) Yang, F.; Yang, D.; Tu, J.; Zheng, Q.; Cai, L.; Wang, L.

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