3D-printed CoSi/PCL composite scaffold with NIR-II photothermal ability and enhanced adipogenic activity for breast reconstruction after mastectomy

Abstract

Breast reconstruction after mastectomy aims to restore breast appearance and function. Current scaffolds often fail to meet the complex clinical demands of post-mastectomy breast reconstruction, which include personalized shaping, prevention of tumor recurrence and metastasis, and promotion of adipose tissue regeneration. This study aims to address these challenges by combining a readily processable biopolymer, polycaprolactone (PCL), with a multifunctional bioactive ceramic, cobalt orthosilicate (Co₂SiO₄, CoSi) to form a multi-functional CoSi/PCL composite scaffold through 3D printing technology. The scaffold exhibits a controllable shape with designed macroporous architecture. Its photothermal performances are well presented in both near-infrared (NIR) I and NIR-II regions, enabling effective tumor ablation with minimal side effects. Additionally, the release of bioactive silicate and cobalt ions promotes adipogenesis and angiogenesis, thereby enhancing the integration of the scaffold with surrounding tissues. This approach offers a promising strategy for combining tumor therapy with breast reconstruction, providing a clinically relevant solution for post-mastectomy patients.

Graphical abstract

Highlights

• •Developed a CoSi/PCL scaffold combining tumor therapy and breast reconstruction.
• •Scaffold features NIR-I and NIR-II photothermal capabilities for effective tumor ablation.
• •Bioactive ions from CoSi enhance adipogenesis and angiogenesis in tissue integration.
• •3D printing technology enables customizable, macroporous scaffold architecture.

1. Introduction

Breast cancer represents one of the most prevalent cancers diagnoses globally and is a leading cause of mortality among women [1,2]. Surgical intervention remains the primary treatment modality for breast cancer, leading patients facing the risk of partial or total mastectomy [3]. Such procedures often result in breast defects that profoundly affect both physical and psychological well-being. Additionally, incomplete surgical resection may lead to tumor recurrence or metastasis, necessitating adjuvant therapies such as chemotherapy or radiotherapy, which significantly diminish patients' quality of life [4]. The dual objectives of achieving complete tumor resection and effective breast reconstruction post-surgery continue to present significant challenges.

Implantable scaffolds with anti-tumor properties offer a promising avenue to address this challenge. The fundamental approach involves filling post-surgical defects with biomaterials that possess anti-tumor capabilities, thereby not only eradicating residual cancer cells and preventing recurrence but also replacing and potentially regenerating lost breast tissue [5]. Currently, several strategies aim to enhance the anti-tumor efficacy of these scaffolds, with near-infrared (NIR) photothermal therapy (PTT)-based scaffolds being the most frequently cited [6]. These scaffolds harness photothermal agents (e.g., gold nanoparticles, polydopamine, black phosphorus) to convert externally applied NIR light into heat, generating localized high temperatures to eliminate remaining cancer cells. However, the majority of photothermal conversion agents utilized in these anti-tumor scaffolds operate within the first NIR region (NIR-I, <1000 nm) [[6], [7], [8], [9], [10]]. Limited research exists on scaffolds functioning within the second NIR window (NIR-II, 1000–1700 nm), despite its potential for deeper tissue penetration and higher maximum permissible exposure (MPE) [11,12]. The development of NIR-II photothermal scaffold materials thus hold substantial promise for post-mastectomy breast reconstruction.

Beyond preventing tumor recurrence, an optimal breast reconstruction scaffold should exhibit appropriate shape, mechanical strength, and the capacity to promote adipose tissue regeneration [13]. Personalized customization technologies, such as 3D printing, are increasingly pivotal in breast reconstruction post-cancer surgery [14]. Typical 3D-printed scaffolds, like those composed of polycaprolactone (PCL), are extensively employed in adipose tissue engineering and breast reconstruction [15,16]. These PCL scaffolds offer excellent biocompatibility, adjustable mechanical properties, and degradation rates, supporting the adhesion and proliferation of various cell types, including adipocytes [17]. Preliminary large animal experiments and clinical trials have demonstrated the potential of PCL scaffolds in adipose tissue engineering and breast repair [18]. However, pure PCL scaffolds lack intrinsic anti-tumor properties and biological activity necessary for actively promoting adipose tissue regeneration.

Previous research has demonstrated that biodegradable silicate bioceramics can sustainably release silicate ions, which possess multiple biological functions, including promoting the differentiation of bone marrow mesenchymal stem cells or adipose precursor cells towards adipogenesis [13,19]. This makes them promising candidates for enhancing adipose tissue regeneration. Moreover, silicate bioceramics containing transmetallic elements often exhibit effective NIR photothermal conversion capabilities, making them suitable for incorporation into anti-tumor scaffolds^12,[20], [21], [22]. In this study, we have synthesized and characterized a novel biodegradable cobalt silicate (Co₂SiO₄, CoSi) bioceramic with NIR-II photothermal properties (see Scheme 1). To investigate its potential in both cancer therapy and adipogenesis, we combined it with polycaprolactone (PCL) to create a CoSi/PCL composite porous scaffold using 3D printing technology. We subsequently evaluated the photothermal and anti-tumor efficacy of these CoSi/PCL composite porous scaffolds through both in vitro and in vivo experiments. Additionally, we assessed the material's potential for tissue regeneration, focusing on its regulatory effects on mouse preadipocytes (3T3-L1), human umbilical vein endothelial cells (HUVECs), and its adipogenic activity in vivo. This research aims to provide both theoretical and empirical foundations for the design and development of innovative breast reconstruction scaffolds following breast cancer surgery.

Scheme 1.

Schematic illustration of the designed 3D-Printed CoSi/PCL composite scaffold with NIR-II photothermal ability and enhanced adipogenic activity for breast reconstruction after mastectomy.

2. Materials and methods

2.1. Materials

All chemical agents including tetraethoxysilane (TEOS), nitric acid, cobalt nitrate hexahydrate, ethanol, Oil Red O were purchased from Aladdin Co., Ltd. (Shanghai, China) except other mentioned. PCL was purchased from SUNP BIOTECH (Beijing, China). Cell counting kit-8 (CCK8) was purchased from Yeasen Biotechnology Co., Ltd (Shanghai, China). All primers were purchased from Sangon Biotech (Shanghai, China).

2.2. Synthesis of CoSi

A typical sol-gel method was employed to produce Co₂SiO₄. Initially, TEOS ( 2.23 mL) was added to a mixture of ethanol (10 mL) and nitric acid (1M, 1 mL). This solution was stirred for 1 h. Subsequently, cobalt nitrate hexahydrate (5.8206 g) was dissolved in 10 mL of deionized water and then introduced dropwise into the TEOS solution. The resulting mixture was sealed and maintained at 60 °C for 24 h until a gel formed. Following the gelation process, the lid was removed, and the gel was aged for an additional 2 h. The gel was then dried at 120 °C for 48 h before undergoing calcination. The calcination was performed with a heating rate of 2 °C/min, reaching a final temperature of 750 °C, which was maintained for 3 h. The resulting particles were subsequently milled and sieved for further use.

2.3. 3D printing of CoSi/PCL composite scaffold

The CoSi/PCL composite scaffold was fabricated using an extrusion-based 3D printing technique. Initially, CoSi (at weight ratios of 0.1, 0.25, and 0.5 wt%) and PCL were thoroughly mixed in chloroform and cast onto the inner wall of a beaker to form a thin film. After the chloroform had evaporated, the CoSi/PCL composite film was melted at 80 °C and transferred to a syringe for 3D printing using the BioMakerV2 (SUNP BIOTECH, Beijing, China). The dispensing pressure was maintained between 600 and 700 kPa at a temperature of 90 °C. The printing speed was set at 6 mm/s with an extrusion speed of 0.6 mm³/s. Scaffolds were printed in dimensions of 20 mm × 20 mm × 1 mm with a line spacing of 1 mm, a layer height of 0.15 mm, and a line routing design of 0°–45°–90° staggered straight lines. These printed samples were then cut into cylinders with an 8 mm diameter for subsequent use. For control purposes, pure PCL scaffolds were also 3D printed following the same protocol. The obtained CoSi/PCL composite scaffolds with weight ratios of 0.1, 0.25, and 0.5 wt% were designated as 0.1CoSi, 0.25CoSi, 0.5CoSi, respectively.

2.4. Characterization of CoSi and CoSi/PCL composite scaffold

Scanning electron microscopy (SEM, HITACHI SU8010, Japan) was employed to examine the morphology and elemental distribution of the particles and composite scaffolds. The phase composition and crystal structure of the synthesized CoSi powder were analyzed using X-ray diffraction (D8 ADVANCE, Bruker, Germany). The compressive mechanical properties of the composite scaffolds were assessed with an Instron machine (Instron 5944, USA) at a displacement velocity of 3 mm/min. The absorption spectra of CoSi were tested using a UV–vis–NIR spectrometer equipped with an integrating sphere (CARY5000, Agilent, USA). To evaluate the photothermal properties, the real-time surface temperature of the samples was recorded under irradiation from both NIR I (808 nm) and NIR II lasers (1064 and 1550 nm) at various power densities using an infrared thermal imager.

For the accelerated degradation experiment, all scaffolds were immersed in 5 M NaOH solution at 37 °C in a shaken bath. At specified time points (1, 3, and 5 days), the remaining samples were dried and weighed. To investigate the ion release properties of the composite scaffolds, each sample was soaked in Tris-HCL solution (pH 7.4) at a weight-to-volume ratio of 1 g/20 mL at 37 °C for 7 days at 120 rpm in an incubator shaker. At predetermined intervals (1, 3, and 7 days), Tris-HCL solutions were collected, and the release of silicate ions was quantified using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7850, USA).

2.5. Cell culture

3T3-L1 preadipocytes, human umbilical vein endothelial cells (HUVECs), and 4T1 mouse breast cancer cells were obtained from the Chinese National Immortalized Cell Bank (Shanghai, China), EK-Bioscience (Shanghai, China). American Type Culture Collection (ATCC, USA), respectively. All these cells were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin (P/S) in a humidified atmosphere of 5 % CO₂ at 37 °C.

2.6. Cell proliferation

The proliferation of 3T3-L1 preadipocytes and HUVECs on various scaffolds was assessed using a Cell Counting Kit-8 (CCK-8) assay at 1, 3, and 5 days. Briefly, cells were seeded at a density of 2 × 10³ cells per well in 96-well plates. Following incubation, the culture medium was replaced with CCK-8 solution and incubated for 1 h. Absorbance at 450 nm was measured using a microplate reader (Epoch2, Bio-Tec Instruments, USA) to quantify cell proliferation.

2.7. Cell morphology

The morphology of 3T3-L1 preadipocytes and HUVECs on the scaffold was examined using SEM. Briefly, cells were fixed on the scaffold with 2.5 % glutaraldehyde for 3 h, followed by dehydration through a graded ethanol series (30 %, 50 %, 70 %, 80 %, 90 %, and 100 %). The dehydrated samples were then sputter-coated with gold and imaged using an SEM (Hitachi SU8010, Japan).

2.8. Cell migration

The migration performance of 3T3-L1 preadipocytes and HUVECs co-cultured with scaffolds was evaluated using a scratch assay. Briefly, cells were seeded at a density of 5 × 10⁵ cells per well in a 6-well plate and cultured for 24 h to form a monolayer. A straight line was then scratched across the cell monolayer using a 200-μL pipette tip. The scaffold was subsequently placed into the medium using a transwell chamber. After 24 h of co-culture with different scaffolds, the cells were fixed with 4 % paraformaldehyde and stained with crystal violet for 10 min. Images were captured using a microscope (OLYMPUS CKX53, Japan), and cell migration was quantified using ImageJ software.

2.9. Oil red O staining

3T3-L1 preadipocytes were seeded on various scaffolds and initially cultured in a standard medium for 2 days. The cells were subsequently cultured in alternating adipogenic media types A and B. Adipogenic medium type A comprised 0.5 × 10⁻³ M IBMX, 10 mg/mL insulin, 100 × 10⁻⁶ ml indomethacin, and 1 × 10⁻⁶ M dexamethasone, while adipogenic medium type B contained 10 mg/mL insulin. The cells were treated with adipogenic medium type A for 3 days followed by medium type B for 1 day, for a total of 2 cycles, before switching to medium type B for an additional 4 days, resulting in a total adipogenesis induction period of 14 days.

Post-induction, 3T3-L1 cells on the scaffolds were stained with oil red O solution for 20 min and rinsed three times with phosphate-buffered saline (PBS). The stained cells were observed using microscope (OLYMPUS CKX53, Japan). For quantification of lipid accumulation, the cells were lysed in 100 % isopropanol for 15 min, and absorbance was measured at 492 nm using a microplate reader (Epoch2, Bio-Tec Instruments, USA).

2.10. Tube formation assay

To evaluate the tube formation ability of HUVECs treated with different scaffolds, Matrigel was thawed at 4 °C overnight. At the following day, 120 μL of Matrigel was placed into each well of a pre-chilled 48-well plate and incubated at 37 °C for 1 h. HUVECs (4 × 10⁴ cells/well) were then seeded onto the Matrigel substrate and incubated with various scaffolds for 6 h. The formation of tubular structures was subsequently observed using an OLYMPUS CKX53 microscope (Japan) and quantified with ImageJ software.

2.11. In vitro anticancer effect

To investigate the in vitro antitumor effects of the scaffolds, 4T1 breast cancer cells were seeded on PCL and CoSi/PCL composite scaffolds in 48-well plates, with a cell density of 5 × 10⁴ cells per well. The cells were cultured in a medium within an incubator set at 37 °C with 5 % CO₂ for 24 h. Subsequently, the scaffolds were subjected to photothermal ablation using NIR-II lasers (1064 nm) at various power densities (0, 0.2, 0.4, 0.6, 0.8 W/cm²). After 5 min of laser irradiation, cell viability was assessed using a CCK-8 assay kit.

For live/dead cell staining, 4T1 cells seeded on the different scaffolds were incubated in a medium containing calcein AM (5 μM) and propidium iodide (PI, 5 μM) for 20 min. The cell/scaffold constructs were then washed twice with PBS and observed using a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems, Germany).

2.12. Real-time quantitative PCR (qRT-PCR)

Total RNA was extracted from 3T3-L1 cells cultured on scaffolds for 14 days or from HUVECs cultured on scaffolds for 3 days using a total RNA assay kit. The extracted RNA was then reverse transcribed into cDNA using the SYBR Premix Ex Taq (Takara, China). The quantitative real-time PCR (qRT-PCR) was employed to quantify the mRNA expression levels of fatty acid binding protein 4 (Fabp4), leptin, peroxisome proliferator-activated receptor gamma (Pparγ), and perilipin A in 3T3-L1 cells. While in HUVECs, the mRNA expression levels of vascular endothelial growth factor (VEGF), platelet endothelial cell adhesion molecule (CD31), hypoxia-inducible factor 1α (HIF-1α), and endothelial nitric oxide synthase (eNOS) were measured. Gene expression levels were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

2.13. In vivo antitumor effect

Eight-week-old female nude mice were obtained from the Zhejiang Provincial Laboratory Animal Center. The animal experimental protocol for this study was approved by the Animal Research and Ethics Committee of Wenzhou Research Institute, University of Chinese Academy of Sciences (Grant No.: WIUCAS23082403). To establish a tumor-bearing mouse model, 1 × 10⁷ 4T1 cells were injected subcutaneously into the dorsal region of each mouse. When the tumor volume reached approximately 100 mm³, the mice were randomly assigned to four groups: PCL scaffolds without laser irradiation (PCL), PCL scaffolds with NIR-II laser irradiation (PCL + NIR II), 0.25CoSi/PCL scaffolds without laser irradiation (0.25CoSi), 0.25CoSi/PCL scaffolds with NIR-II laser irradiation (0.25CoSi + NIR II). The temperature of the tumors was continuously monitored in real-time using a near-infrared thermal imaging system.

Tumor volume (V) was measured every 2 days and calculated using the formula: V = (length × width²)/2. Tumors from each group were subsequently stained for hematoxylin and eosin (H&E) and TUNEL to assess tissue morphology and apoptosis, respectively. After 14 days of treatment, the major organs (heart, liver, spleen, lungs, and kidneys) from each group were also stained with H&E to evaluate the systemic effects of the anti-tumor treatment.

2.14. In vivo adipose tissue restoration

Six-week-old female nude mice were procured from the Experimental Animal Center of Zhejiang Province. The animal experimental protocol was approved by the Animal Research and Ethics Committee of the Wenzhou Institute, University of Chinese Academy of Sciences (Approval No. WIUCAS23082403). The implantation experimental groups consisted of four sets: a PCL scaffold group (PCL), a 0.25CoSi/PCL composite scaffold group (0.25CoSi), a PCL scaffold with 3T3-L1 cells pre-cultured in lipid differentiation medium for 14 days (PCL + Cell), and a 0.25CoSi/PCL composite scaffold with 3T3-L1 cells pre-cultured in lipid differentiation medium for 14 days (0.25CoSi + Cell). The scaffolds were implanted between the skin and myofascia on both sides of the mice's backs. After 3 weeks, the mice were sacrificed, and the implanted scaffolds were removed for analysis of adipose tissue and blood vessel formation. The volume of the adipose tissue/scaffold constructs were evaluated by ImageJ software.

2.15. Histological analysis and ions detection in vivo

The samples were first fixed in 4 % paraformaldehyde, embedded at the optimal cutting temperature and sectioned into 10 μm slices, stained with Oil Red O solution (counterstained with hematoxylin). The red-stained fat areas were circled, and their ratio to the total field was quantified using ImageJ. Immunofluorescence staining was performed to analyze adipose tissue using PPARγ as the primary antibody and to analyze blood vessels using CD31 as the primary antibody. Briefly, the sliced samples were rinsed with PBS three times for 3 min each. The sections were then treated with methanol containing 3 % H₂O₂ for 15 min to block endogenous peroxidase activity and subjected to antigen retrieval by incubation in an oven at 37 °C for 2 h. After rinsing with PBS, the sections were blocked with 5 % bovine serum albumin (BSA) for 30 min. The sections were then incubated at 4 °C overnight with anti-PPARγ (1:200) or anti-CD31 (1:400) primary antibodies. At next day, the sections were incubated with a fluorescent secondary antibody diluted 1:200 for 1 h at room temperature. Images were captured using a ZEISS Axio Vert.A1 microscope and the quantitative analysis of PPARγ (area ratio) and CD31 (vessel number) was conducted using ImageJ software.

To detect the ions released from the 0.25CoSi scaffold in vivo, tissues surrounding the scaffolds were collected, weighed, and digested with aqua regia for ICP-MS measurement (Agilent 7850, USA). Additionally, the concentrations of Si and Co in normal tissues before scaffold implantation were measured and set as the baseline.

2.16. Statistical analysis

All data are presented as mean ± standard error of mean (SEM) using one-way analysis of variance (ANOVA) between groups. Statistical significance was defined as ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. All statistical analyses were conducted using GraphPad Prism software.

3. Results

3.1. Characterization of CoSi and CoSi/PCL composite scaffold

As shown in Fig. S1, the synthesized ceramics via the sol-gel method, exhibited a black hue. Scanning electron microscopy (SEM) images (Fig. 1A) further revealed a particle size of approximately 5–20 μm. The energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD) analyses (Fig. 1B) further confirm that the synthesized particles were Co₂SiO₄ (CoSi) ceramics. The absorption spectra of CoSi, shown in Fig. S2, indicated that CoSi exhibits broad and strong absorption in both the NIR-I and NIR-II regions, highlighting its potential as a photothermal agent for NIR PTT. Then, we employed 3D printing technique to fabricate CoSi/PCL composite scaffolds with varying CoSi contents. The mass fractions of CoSi within the scaffolds were 0.1, 0.25, and 0.5 wt%, respectively, resulting in the designations of 0.1 % CoSi, 0.25 % CoSi, and 0.5 % CoSi. Fig. 1C presents the overall morphology of the different composite scaffolds. Pure PCL scaffolds appeared semi-transparent milky white, while the inclusion and increased concentration of CoSi progressively darkened the color of the CoSi/PCL composite scaffolds from grey to black. The macroporous structure and surface morphology of the scaffolds were further characterized by SEM images (Fig. 1D), which demonstrated that the addition of CoSi didn't alter the overall pore structure, with macropores measuring approximately 600 μm and printed fiber diameters around 400 μm, suggesting scaffold porosity exceeding 60 %. Enlarged SEM images (top right) indicate that CoSi was predominantly encapsulated within the scaffold, with no significant impact on surface morphology. Moreover, the cross-section SEM images of the scaffolds (Fig. 1E and F) revealed the encapsulation of CoSi particles, with EDS mapping confirming their relatively uniform distribution.

Fig. 1.

Characterization of CoSi particle and 3D printed CoSi/PCL composite scaffolds. (A) SEM and EDS mapping of synthesized CoSi particle. (B) XRD pattern of CoSi powders. (C) Overview of 3D printed CoSi/PCL composite scaffolds with various concentrations of CoSi powders. (D) SEM images of macropore structures of the 3D printed scaffolds. Top right corner presents the high magnification SEM images. (E) SEM images and (F) EDS mapping of the cross-view of CoSi/PCL composite scaffold. (G) Accelerated degradation of different scaffolds. n = 6. (H) Co and (I) Si ion release profile from different scaffolds. n = 3.

Subsequent characterization of the scaffolds' compressive properties (Fig. S3) indicated a decrease in compressive strength and Young's modulus with increasing CoSi content. Given the slow degradation rate of PCL, NaOH solution was used to expedite degradation process. Over a period of 7 days, significant degradation was observed across all scaffold compositions (Fig. 1G). The degradation rates of PCL, 0.1%CoSi, 0.25%CoSi, and 0.5%CoSi were 28.34 ± 4 wt%, 29.95 ± 2 wt%, 30.55 ± 6 wt%, and 31.13 ± 5 wt%, respectively, with no significant differences among the groups. Ion release profiles demonstrated that Co and Si ion release increased with higher CoSi content (Figure H and I). After 7 days, the cumulative Co ion concentrations released from 0.1%CoSi, 0.25%CoSi, and 0.5%CoSi were 0.211 ± 0.017, 0.665 ± 0.064, and 1.169 ± 0.054 ppm, respectively, while the Si ion concentrations were 1.264 ± 0.136, 2.29 ± 0.058, and 4.448 ± 0.303 ppm, respectively.

3.2. NIR photothermal performance and in vitro antitumor killing activity of CoSi/PCL composite scaffold

Subsequently, the NIR photothermal properties of the CoSi/PCL composite scaffolds in both NIR I windows and NIR II windows were characterized. First, the temperature elevation of various scaffolds under NIR I (808 nm, 0.4 W/cm²) laser irradiation was analyzed. As depicted in Fig. S4A, the temperature rise curve increased with higher CoSi content. After 180 s of irradiation, the temperature of the 0.5%CoSi scaffold reached 83.1 °C. Then, the effect of different laser intensities on the photothermal conversion of the scaffold (taking the 0.25%CoSi scaffold as an example) was examined (Fig. S4B). As expected, the rate of temperature increased correlated with the laser power. At a laser power of 0.9 W/cm², the temperature of the 0.25%CoSi scaffold reached 135.9 °C after 180 s of irradiation. Fig. S4C further demonstrates the excellent photothermal stability of the scaffold, as six consecutive photothermal cycles did not significantly affect its photothermal performance. Similarly, the temperature elevation curves of different scaffolds under NIR II (1064 nm, 0.4 W/cm², Fig. 2A; 1550 nm, 0.4 W/cm², Fig. S5A) laser irradiation exhibited a similar trend to that observed under NIR I irradiation. The temperature of the 0.5CoSi scaffold reached 74.8 °C (1064 nm) and 131.3 °C (1550 nm) after 180 s of irradiation, respectively. Furthermore, the rate of temperature increase under NIR II laser irradiation was positively correlated with laser power (Fig. 2B and Fig. S5B), and the scaffolds also demonstrated good photothermal stability under NIR II laser irradiation (Fig. 2C and Fig. S5C). Although the 0.5CoSi scaffold exhibits stronger absorption and heating effects at 1550 nm compared to 1064 nm, we selected 1064 nm for subsequent experiments. This decision was based on the strong absorption of water in the near-infrared region, where the absorption coefficient of water at 1550 nm is nearly 80 times higher than at 1064 nm [23]. To minimize the risk of direct tissue damage from light exposure, 1064 nm was deemed more appropriate.

Fig. 2.

NIR photothermal performance and in vitro antitumor killing activity of CoSi/PCL composite scaffold. (A) Photothermal heating (1064 nm, 0.4 W/cm²) curves of different 3D printed scaffolds. (B) Photothermal heating curves of 3D printed 0.25CoSi scaffolds under 1064 nm laser irradiation at varying power densities (0.4, 0.6, 0.8, and 1 W/cm²). (C) Photothermal stability of 3D printed 0.25CoSi scaffolds under sequential laser on/off cycles. (D) Schematic diagram for the measurement of the penetration effect of NIR light passing through tissues. (E, F) The output intensity of 808 nm (E) and 1064 nm (F) laser passing through tissues with different thickness (0–10 mm). (G) Schematic diagram for measuring photothermal heating by NIR light passing through different tissues. (H, I) Photothermal heating images (H) and curves (I) under NIR I (808 nm) and NIR II (1064 nm) light irradiation for 5 min. (J) Relative cell viability of 4T1 cells after different treatments as described. n = 5. (K) The live-dead stained images of 4T1 cells co-cultured with 3D printed PCL and 0.25%CoSi composite scaffolds with/without NIR-II (1064 nm) laser irradiation. Scale bar: 100 μm. (L) Laser power dependent relative cell viability of 4T1 cells treated with 3D printed 0.25%CoSi scaffolds. n = 5.

Considering the influence of tissue on laser energy of different wavelengths, we compared the residual laser intensity after passing through varying thicknesses of chicken breast tissue for NIR I (808 nm) and NIR II (1064 nm) laser irradiation. As shown in Fig. 2D–F, the residual laser intensity decreased with increasing tissue thickness, where 1064 nm laser exhibiting better tissue penetration than 808 nm laser. Building on this, the photothermal effects on the scaffold when irradiated with NIR I (808 nm) and NIR II (1064 nm) lasers through the same tissue (chicken breast or mouse skin) were examined. Fig. 2G–I indicate that, regardless of the tissue type, the temperature rise rate of the scaffold under 1064 nm laser irradiation was significantly higher than under 808 nm. Based on these results, we chose the 1064 nm laser for the following studies.

To further substantiate the potential of CoSi/PCL composite scaffolds in combination with NIR II laser for therapeutic applications, we analyzed the in vitro anti-tumor capabilities of the scaffolds. First, the killing ability of different scaffolds on 4T1 cancer cells were conducted. As shown in Fig. 2J, neither NIR II (1064 nm) irradiation alone, pure PCL, PCL combined with NIR II, nor the CoSi/PCL scaffold alone could achieve significant tumor cell eradication. Only the CoSi/PCL scaffold in conjunction with NIR II irradiation demonstrated a marked tumor cell elimination effect. Live/dead cell staining results further confirmed the in vitro anti-tumor efficacy of the CoSi/PCL + NIR II group (Fig. 2K). Additionally, varying laser intensities yielded a correlation between laser power and cell viability inhibition, underscoring the controllability of the photothermal anti-tumor strategy. These findings collectively highlight the enhanced anti-tumor potential of CoSi/PCL composite scaffolds when used in combination with NIR II laser irradiation.

3.3. Effects of CoSi/PCL composite scaffold on 3T3-L1 preadipocytes

3T3-L1 preadipocytes are commonly used to assess the adipogenic potential of materials in vitro. Here, the proliferative capacity of cells on various scaffolds was first validated using the CCK-8 assay. As illustrated in Fig. 3A, cell proliferation was observed on all scaffolds from day 1 to day 5. Notably, the 0.25%CoSi composite scaffold demonstrated the most significant improvement, with a marked increase in cell proliferation compared to PCL at day 3 and day 5. Consequently, the 0.25 % CoSi composite scaffold was selected for subsequent cellular experiments. Fig. 3B presents SEM images of cells adhered to PCL and 0.25%CoSi composite scaffolds after 24 h, showing good cell attachment and spreading. Next, a scratch assay was employed to evaluate the effect of different scaffolds on cell migration. Results indicated that the 0.25%CoSi composite scaffold significantly enhanced the migration rate of 3T3-L1 cells compared to the pure PCL scaffold (Fig. 3C).

Fig. 3.

CoSi/PCL composite scaffold promotes proliferation, migration and adipogenesis of 3T3-L1 cells. (A) The proliferation of 3T3-L1 cells on different scaffolds for 1, 3, and 5 days, respectively. n = 6. (B) Representative SEM images of 3T3-L1 cells cultured on different scaffolds for 24 h. (C) Representative images and quantitative analysis of cell migration with the treatment of 3D printed PCL and 0.25%CoSi composite scaffolds for 24 h using the scratch assay. n = 6. (D) Representative Oil red O staining micrographs and quantitative analysis of 3T3-L1 cells after adipogenic induction for 14 days. n = 3. (E) Adipogenic gene (Fabp4, Leptin, Pparγ and PerilipinA) expression in 3T3-L1 cells cultured on 3D printed PCL and 0.25%CoSi composite scaffolds after adipogenic induction for 14 days. n = 3.

Furthermore, oil red O staining was used to characterize adipogenic differentiation of 3T3-L1 cells on different scaffolds. As depicted in Fig. 3D, after 14 days of adipogenic induction, the 0.25%CoSi composite scaffold facilitated a 33 % increase in lipid droplet formation compared to the pure PCL scaffold. Subsequently, the impact of the scaffolds on adipogenic gene expression was assessed using qRT-PCR, with results shown in Fig. 3E. Compared to the pure PCL scaffold, the 0.25 % CoSi composite scaffold upregulated the expression of multiple adipogenic relative genes, including Fabp4, Leptin, Pparγ, and Perilipin A. These findings collectively suggest that CoSi/PCL composite scaffolds possess superior potential for promoting adipogenesis.

3.4. Effects of CoSi/PCL composite scaffold on HUVECs

Given the importance of vascularization for adipose tissue regeneration, we also evaluated the effects of the scaffolds on HUVECs. Cell proliferation results (Fig. 4A) indicated that the 0.25%CoSi composite scaffold exhibited the highest pro-proliferative effect, and thus, the 0.25%CoSi scaffold was selected for subsequent cell experiments. Similar to 3T3-L1 cells, HUVECs were able to adhere and spread on both PCL and 0.25 % CoSi composite scaffolds (Fig. 4B). The scratch assay (Fig. 4C) demonstrated that HUVECs treated with the 0.25%CoSi composite scaffold exhibited significantly enhanced cell migration capability, with a migration rate of 39.37 ± 4.09 % compared to 8.30 ± 4.24 % for the pure PCL scaffold. An in vitro tube formation assay further revealed that after 6 h of co-culture with the 0.25 % CoSi composite scaffold, a significantly greater number and length of capillary-like structures formed in the Matrigel compared to the pure PCL scaffold (Fig. 4D and Fig. S6). Additionally, qRT-PCR analysis was conducted to assess the expression of angiogenic genes in HUVECs treated with different scaffolds. As shown in Fig. 4E, the 0.25%CoSi composite scaffold significantly upregulated the expression of VEGF, CD31, HIF-1α, and eNOS in HUVECs after 3 days of stimulation compared to the pure PCL scaffold. Collectively, these results indicate that the CoSi/PCL composite scaffold positively influences angiogenesis.

Fig. 4.

CoSi/PCL composite scaffold promotes proliferation, migration and angiogenesis of HUVECs. (A) The proliferation of HUVECs on different scaffolds for 1, 3, and 5 days, respectively. n = 6. (B) Representative SEM images of HUVECs cultured on different scaffolds for 24 h. (C) Representative images and quantitative analysis of cell migration with the treatment of 3D printed PCL and 0.25%CoSi composite scaffolds for 24 h using the scratch assay. n = 6. (D) Representative formed tube in Matrigel and quantitative analysis of HUVEC after being treated with PCL and 0.25%CoSi composite scaffolds for 6 h n = 5. (E) Angiogenic genes (VEGF, CD31, HIF-1α, and eNOS) expression in HUVECs after co-cultured with PCL and 0.25%CoSi composite scaffolds for 3 days. n = 3.

3.5. In vivo anti-tumor effects of CoSi/PCL composite scaffold assisted with NIR II light

To further validate the in vivo anti-tumor efficacy of the scaffolds, we established a subcutaneous breast cancer model in nude mice and implanted PCL scaffolds and 0.25%CoSi composite scaffolds. Under NIR II (1064 nm, 0.4 W/cm²) irradiation, the 0.25%CoSi + NIR II group exhibited a more pronounced local tumor temperature increase (Fig. 5A), stabilizing at 56 °C after 2 min, whereas the temperature for the PCL + NIR II group was approximately 41 °C (Fig. 5B). With the photothermal ablation effect, tumors in the 0.25%CoSi + NIR II group gradually shrank and became completely undetectable after 14 days (Fig. 5C). Further histological analysis using H&E and TUNEL staining confirmed the anti-tumor efficacy of the 0.25 % CoSi + NIR II group. This group showed more extensive cell death and apoptosis compared to the other groups. The relative tumor volume was measured every 2 days over a 14-day period, which indicated that while PCL, 0.25 % CoSi, and PCL + NIR II groups did not exhibit significant tumor inhibition, the tumor volume in the 0.25 % CoSi + NIR II group progressively decreased over time (Fig. 5D). Importantly, apart from a slight increase in body weight in the 0.25%CoSi + NIR II group after one week, no other significant changes were recorded during the treatment (Fig. 5E). H&E staining of major organs (Fig. 5F) revealed no apparent abnormalities, confirming the good biocompatibility of the scaffolds, thus underscoring their potential as a safe and effective anti-tumor platform.

Fig. 5.

In vivo anti-tumor effects of CoSi/PCL composite scaffold assisted with NIR II light. (A, B) Representative thermal images (A) and the corresponding photothermal heating curves (B) of 3D printed PCL and 0.25CoSi composite scaffolds under 1064 nm laser irradiation (0.4 W/cm², 5 min). (C) Optical images of the mice with different treatments treated and the histological analysis (H&E and TUNEL staining) of tumor sections collected from different groups. (D) Relative tumor volume of the indicated different groups after treatments for 14 days. n = 5. (E) Body weight of the indicated different groups after treatments for 14 days. n = 5. (F) H&E staining of the major organs harvested from different groups.

3.6. In vivo adipogenesis of CoSi/PCL composite scaffold

To evaluate the in vivo adipogenic potential of the materials, a subcutaneous adipose tissue formation experiment was conducted. The experimental groups were divided into four: PCL scaffold alone (PCL), 0.25%CoSi composite scaffold alone (0.25%CoSi), PCL scaffold loaded with 3T3-L1 cells (PCL + Cell), and 0.25%CoSi scaffold loaded with 3T3-L1 cells (0.25%CoSi + Cell). After subcutaneous implantation of the scaffolds for 3 weeks, the samples were harvested and photographed. Fig. 6A shows the engineered adipose tissue formed by the scaffolds and the newly formed adipose-like tissue. It is evident that, compared to the acellular scaffolds (PCL and 0.25%CoSi), the scaffolds containing 3T3-L1 cells (PCL + Cell and 0.25%CoSi + Cell) exhibited more tissue formation and visible vascular networks. Notably, the 0.25 % CoSi + Cell group demonstrated the most significant adipose-like tissue regeneration. Quantitative analysis of the engineered adipose tissue, including height and area (Fig. 6B and C), further confirmed that the cellular groups exhibited superior adipose formation compared to the acellular groups, with the 0.25%CoSi composite scaffold showing a stronger adipogenic capacity than the PCL scaffold.

Fig. 6.

Characterization of the engineered adipose tissues in a nude mice subcutaneous transplantation model after treatment with PCL, PCL + Cell, 0.25%CoSi, and 0.25%CoSi + Cell for 3 weeks, respectively. (A) Representative photos of taken-out engineered adipose tissues after different treatments. (B, C) Quantitative analysis of the height (B) and area (C) of the engineered adipose tissues. n = 6.

Subsequent histological analysis of the scaffolds was conducted. Oil red O staining demonstrated that the cellular groups (PCL + Cell and 0.25%CoSi + Cell) formed significantly more lipid droplets compared to the acellular groups, with the 0.25 % CoSi + Cell group forming the most (Fig. 7A and Fig. S7). Additionally, to assess the quality of the newly formed adipose-like tissue, immunofluorescence staining for adipogenic marker PPARγ and angiogenic marker CD31 was performed. As shown in Fig. 7B, C and Figs. S8 and S9, the PCL + Cell group exhibited stronger PPARγ expression, and a higher number of newly formed blood vessels compared to the PCL and 0.25%CoSi groups. No surprisingly, the 0.25%CoSi + Cell group showed the most substantial vascularized adipose formation in groups. Considering that the continuous release of Si and Co ions from the scaffold is likely the primary source of its bioactivity, we also evaluated the in vivo ion release performance of the 0.25CoSi scaffold (Fig. S10). As expected, within three weeks of scaffold implantation, the Si and Co ion concentrations in the surrounding tissue were consistently much higher than those in normal tissue, demonstrating the scaffold's ability to continuously release active ions in vivo. All these results suggest that the 3D-printed 0.25%CoSi composite scaffold holds great potential as a graft for vascularized adipose tissue regeneration.

Fig. 7.

Histological analysis of the new formed adipose tissues. (A) Representative Oil Red O staining images of the engineered adipose tissues after treatment with PCL, PCL + Cell, 0.25%CoSi, and 0.25%CoSi + Cell respectively. (B) Representative immunofluorescence staining targeting PPARγ of the new-formed tissues after the treatment with different scaffolds. (C) CD31 immunohistochemistry staining of represents the new formed vessels in the obtained tissues after different treatments.

4. Discussion

For patients undergoing post-mastectomy breast reconstruction, achieving the best possible restoration of the breast's appearance is of paramount concern. Commercial breast implants, such as silicone scaffolds, offer some degree of shaping but fall short in meeting the highly personalized clinical needs of breast reconstruction [24]. Ideal breast reconstruction scaffolds must meet two critical criteria: the macroscopic structure (shape, size, and symmetry) must be customizable to the patient's specific requirements, and the internal structure must promote adipose tissue regeneration for better integration with surrounding tissues [14,25]. In this regard, 3D printing technology is indispensable as it allows for the customization of implants that perfectly match the defect size and enables the creation of porous structures that enhance nutrient transfer and cell/tissue ingrowth^[[26], [27], [28]^]. Clinically, 3D-printed scaffolds have been reported for use in post-mastectomy breast reconstruction. For instance, Wayne A. Morrison and colleagues customized non-degradable acrylic porous scaffolds for five patients requiring breast reconstruction, providing a protective cavity for autologous tissue implants [29]. Six months post-implantation, these scaffolds facilitated the formation of new vascular, fibrous, and adipose tissues [29]. Similarly, Ju-Liang Zhang's group used biocompatible, easily moldable, and degradable polycaprolactone (PCL) to create a personalized 3D-printed PCL porous scaffold for a patient with left invasive breast cancer, enabling successful single-stage breast reconstruction [30]. Nine months post-surgery, the scaffold repaired the breast with satisfactory shape, size, and symmetry, showing new granulation tissue and good integration with autologous tissue without significant side effects [30]. These studies demonstrate the feasibility of 3D printing technology in post-mastectomy breast reconstruction. Our study demonstrated that the incorporation of small amounts of CoSi into 3D-printed PCL scaffolds does not significantly alter their printability, making 3D-printed CoSi/PCL composite scaffolds a potential biodegradable option for breast reconstruction.

Apart from considering the adaptability of the scaffold, its antitumor activity is also important. Surgical treatment for breast cancer carries the risk of incomplete resection, leading to tumor recurrence or metastasis. Consequently, adjuvant local radiotherapy or systemic chemotherapy is often required post-mastectomy. However, clinical reports indicate significant conflicts between local radiotherapy and immediate breast reconstruction. Breast implants may alter chest wall anatomy, distorting the geometric design of the radiation field and causing under- or overdosing of the target and underlying tissues, thereby compromising radiotherapy efficacy [31]. Additionally, local radiotherapy can exacerbate side effects such as increased inflammation, fibrosis, and delayed wound healing [32]. Conversely, chemotherapy does not hinder immediate breast reconstruction but can cause systemic side effects like bone marrow suppression and gastrointestinal stress, which may impair wound healing and lead to reconstruction failure [33]. To mitigate systemic side effects, localized chemotherapy delivery via scaffolds has been explored. For instance, Phong A. Tran etc. used salt-leaching to load doxorubicin (DOX) into PCL porous scaffolds, achieving prolonged drug release over 28 days [34]. In murine models, these scaffolds, with only 1/20th the DOX dose of systemic administration, effectively prevented tumor recurrence and demonstrated lower cytotoxicity and systemic toxicity [34].

Beyond localized chemotherapy, scaffolds can also facilitate novel tumor therapies. Zhuang Liu etc. developed an immunotherapy scaffold based on fibrin hydrogel encapsulating cyclophosphamide (CTX) and an immune checkpoint inhibitor (aPDL1 antibody) [35]. This combination selectively eliminated regulatory T cells and enhanced immune checkpoint blockade efficacy, showing significant anti-recurrence effects in an incomplete resection breast cancer model [35]. However, both chemotherapy and immunotherapy have drawbacks, including high costs, low clinical response rates (20–30 % for PD-1/PD-L1 inhibitors), and difficulty in dose control [36]. In contrast, hyperthermia offers a more universally applicable approach. Tumor cells are more heat-sensitive than normal cells, undergoing autolysis and necrosis at temperatures of 39–42 °C, while normal cells only experience functional decline without death [37]. This provides the basis for hyperthermia as a cancer treatment. NIR PTT has emerged as a novel method, utilizing NIR irradiation of materials with high photothermal conversion efficiency to convert light into heat, selectively killing tumor cells. As a non-invasive treatment, NIR PTT allows precise spatiotemporal control of tumor irradiation, enhancing therapeutic efficiency and reducing side effects. Filling post-mastectomy breast defects with scaffolds possessing NIR photothermal properties and using NIR PTT to eliminate residual tumors around the scaffold is a current research focus. Studies have shown that nanocomposites like AuNP can achieve significant tumor ablation under NIR-I (808 nm) irradiation, but they suffer from limited tissue penetration and low safety power [38]. In contrast, NIR-II (1000–1700 nm) light offers deeper tissue penetration and higher safety, making materials with high photothermal conversion efficiency in NIR-II more clinically promising [39]. In our study, the synthesized CoSi endowed the CoSi/PCL composite scaffolds with excellent photothermal conversion efficiency in NIR II regions, making CoSi/PCL composite scaffolds superior in vivo and in vitro anti-tumor functions under the irradiation of NIR II light.

In addition to timely removal of residual tumors, it is crucial to repair defective breast tissue promptly. Adipose tissue, being the most abundant component of the breast, plays a crucial role in maintaining breast shape and size and protecting other breast tissues [40]. Adipose tissue regeneration is therefore vital in breast reconstruction. Clinically, autologous fat grafting is a common method but carries risks such as infection, fat nodules, and embolism [41]. The proposal of adipose tissue engineering provides a new approach to solving this problem, specifically including two methods: in situ tissue engineering and ex vivo tissue engineering. The former involves directly implanting acellular tissue engineering scaffolds at the defect site, stimulating the proliferation and differentiation of surrounding adipose stem cells or preadipocytes to form new adipose tissue. The latter first forms a cell-scaffold composite resembling adipose tissue through ex vivo culture, and then implants it into the defect site to fuse and grow with the surrounding adipose tissue [42]. Regardless of the type of adipose tissue engineering, the choice of scaffold materials is crucial. Acellular dermal matrix (ADM) is currently the only type of active scaffold material approved for breast reconstruction. For example, AlloDerm®, developed by Life Cell in the United States, is an acellular dermal scaffold derived from human skin. The active components in its extracellular matrix and its three-dimensional structure are conducive to the regeneration of tissues such as fat and blood vessels, and it is widely used in various types of breast reconstruction surgeries, playing a positive role in enhancing fat regeneration, accelerating implant integration, and promoting wound healing [43]. However, ADM is expensive, has low mechanical strength, and degrades quickly, so it is usually only used as an auxiliary tool in conjunction with other implants for breast reconstruction, and cannot be applied as an independent scaffold material in breast reconstruction surgeries. Compared to the widely used ADM scaffolds, synthetic scaffold materials offer advantages in cost, biosafety, mechanical strength, and degradability. For example, PCL scaffolds provide good biocompatibility, mechanical strength, and controlled degradation rates, with studies showing their potential in adipose tissue engineering for breast repair [44]. However, pure PCL scaffolds lack bioactivity and cannot actively promote adipose regeneration. Previous studies found that calcium silicate (CaSiO₃) ceramics release bioactive Si ions, which promote adipogenic differentiation and enhance adipose regeneration through angiogenesis [13,19]. Here, CoSi ceramics can release both Si and Co ions, with Co ions known to promote angiogenesis by stabilizing HIF-1α [45]. The CoSi ceramic combined with PCL to form CoSi/PCL composite scaffolds demonstrated significant effects on vascularized adipose formation, whether or not the cells are preincubated, highlighting the potential of 3D-printed 0.25 % CoSi composite scaffolds as grafts for vascularized adipose tissue regeneration.

5. Conclusion

This study designed 3D-printed CoSi/PCL composite scaffolds capable of simultaneous NIR-II PTT for breast tumors and promotion of vascularized adipose tissue regeneration. The CoSi component exhibits high photothermal conversion efficiency in the NIR-II region, enabling the scaffold to achieve superior photothermal therapeutic effects on deep-seated breast tumor cells without significant adverse reactions. Additionally, the released bioactive Si and Co ions not only effectively promote the differentiation of adipocytes and endothelial cells in vitro but also enhance vascularized adipose tissue regeneration in vivo. This study provides scientific insights for the further application of PTT dual-functional scaffolds in tumor therapy.

CRediT authorship contribution statement

Jupei Zhang: Writing – original draft, Project administration, Methodology, Data curation, Conceptualization. Hangbin Xia: Writing – original draft, Methodology, Formal analysis, Data curation. Xuerui Zhou: Writing – original draft, Methodology, Formal analysis, Data curation. Zhaoxu Meng: Data curation. Qishu Jin: Data curation. Dongmin Chen: Data curation. Xiaojuan Xia: Data curation. Yiren Jiao: Writing – review & editing. Jiang Chang: Supervision, Project administration. Zhihong Dong: Writing – review & editing, Supervision. Zhen Zeng: Writing – review & editing, Project administration, Methodology, Conceptualization. Hongshi Ma: Writing – review & editing, Supervision, Funding acquisition. Chen Yang: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

The data that supports this study is available from the authors upon reasonable request.