3D Printed Wesselsite Nanosheets Functionalized Scaffold Facilitates NIR-II Photothermal Therapy and Vascularized Bone Regeneration

Abstract

Various bifunctional scaffolds have recently been developed to address the reconstruction of tumor-initiated bone defects. Such scaffolds are usually composed of a near-infrared (NIR) photothermal conversion agent and a conventional bone scaffold for photothermal therapy (PTT) and long-term bone regeneration. However, the reported photothermal conversion agents are mainly restricted to the first biological window (NIR-I) with intrinsic poor tissue penetration depth. Also, most of these agents are non-bioactive materials, which induced potential systemic side toxicity after implantation. Herein, a NIR-II photothermal conversion agent (Wesselsite [SrCuSi₄ O₁₀ ] nanosheets, SC NSs) with tremendous osteogenic and angiogenic bioactivity, is rationally integrated with polycaprolactone (PCL) via 3D printing. The as-designed 3D composite scaffolds not only trigger osteosarcoma ablation through NIR-II light generated extensive hyperthermia, but also promote in vitro cellular proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs), respectively, and the ultimate enhancement of vascularized bone regeneration in vivo owing to the controlled and sustained release of bioactive ions (Sr, Cu, and Si). The authors' study provides a new avenue to prepare multifunctional bone scaffolds based on therapeutic bioceramics for repairing tumor-induced bone defects.

Keywords: NIR-II; angiogenesis; osteogenesis; photothermal therapy; vascularized bone regeneration.

Figure 1

Schematic illustration of 3D printed SC/PCL composite scaffolds for bone tumor photothermal eradication and vascularized bone regeneration, respectively.

Figure 2

Characterization of SC NSs and 3D printed SC/PCL composite scaffolds. a,b) SEM images of SC bulk (a) and exfoliated SC NSs (b). c) AFM image and height profile of SC NSs. d) Optical images of 3D printed PCL and SC/PCL composite scaffolds. e–h) SEM characterization of macroporous structures of PCL (e), 2‐SC/PCL (f), 4‐SC/PCL (g), and 8‐SC/PCL (h) scaffolds. i) EDS elemental mapping analysis of SC NSs in 3D printed 4‐SC/PCL composite scaffold. j) SEM images of the cross‐section view of 3D printed 4‐SC/PCL composite scaffold. k) Compressive Young's moduli of different scaffolds. l–n) Ion release curves of Sr (l), Cu (m), and Si (n) ions from various composite scaffolds. Scale bar: 1µm (a), 200 nm (b,c), 2 mm (d), 500 µm (e–h), and 500 nm (j). Data are presented as mean ± s.d. (k) (n = 3), (l–n) (n = 4). ** For 0.001 < p < 0.01. One‐way ANOVA analysis.

Figure 3

In vitro osteogenesis and angiogenesis assay. a) rBMSCs proliferation on different scaffolds. b) HUVECs proliferation on different scaffolds. c,d) ALP staining (c) and the quantitative analysis (d) of rBMSCs on various scaffolds after cultivation for 7 days. e,f) HUVECs migration analysis using transwell method after co‐cultured with 3D printed PCL and 4‐SC/PCL composite scaffolds. g,h) In vitro tube formation assay of HUVECs after being co‐cultured with 3D printed PCL and 4‐SC/PCL composite scaffolds. i) Osteogenic gene (OCN, BMP2, RUN2) expression of rBMSCs cultured on 3D printed PCL and 4‐SC/PCL composite scaffolds for 7 days. k) Angiogenic gene (VEGF, HIF‐1α, BFGF) expression of HUVECs cultured on 3D printed PCL and 4‐SC/PCL composite scaffolds for 7 days. Scale bar: 1 mm (c), 100 µm (e), and 200 µm (g). Data are presented as mean ± s.d. (a,b,f,h,i) (n = 4), (d,k) (n = 3). * For 0.01 < p < 0.05, ** for 0.001 < p < 0.01, and *** for p < 0.001. One‐way ANOVA analysis.

Figure 4

In vivo vascularized bone regeneration. a–c) Typical 3D reconstruction of micro‐CT images (a) and analysis of new bone formation in defect areas after implantation of 3D printed PCL and 4‐SC/PCL composite scaffolds for 3 months: b) bone mineral density (BMD) and c) bone volume/total volume (BV/TV). d,e) Representative H&E staining (d) and Masson's trichrome staining (e) of the craniums with cranial defects after implantation of 3D printed PCL and 4‐SC/PCL composite scaffolds for 1 and 3 months. f) The immunohistochemistry staining targeting OPN, RUNX2, CD31, and HIF‐1α in new‐formed tissues after treating with 3D printed PCL and 4‐SC/PCL composite scaffolds for 1 and 3 months. Scale bar: 2 mm (a), 1 mm (low‐magnification images in d and e), 200 µm (high‐magnification images in d and e), 50 µm (f). Data are presented as mean ± s.d. (b,c) (n = 4). ** For 0.001 < p < 0.01 and *** for p < 0.001. One‐way ANOVA analysis.

Figure 5

In vitro photothermal antitumor killing activity. a) Photothermal heating (1064 nm, 0.6 W cm⁻²) curves of different 3D printed scaffolds. b) Photothermal heating curves of 3D printed 4‐SC/PCL scaffolds under 1064 nm laser irradiation at varying power densities (0.3, 0.6, 0.9, and 1.2 W cm⁻²). c) Photothermal stability of 3D printed 4‐SC/PCL scaffolds under sequential laser on/off cycles. d) Relative cell viability of Saos‐2 cells after different treatments as described. e) Laser power dependent relative cell viability of Saos‐2 cells treated with 3D printed 4‐SC/PCL scaffolds. f) The calcein AM/PI‐stained images of Saos‐2 cells on 3D printed PCL and 4‐SC/PCL composite scaffolds with/without NIR‐II laser irradiation. Scale bar: 100 µm. Data are presented as mean ± s.d. (d,e) (n = 5). *** For p < 0.001. One‐way ANOVA analysis.

Figure 6

In vivo antitumor efficacy. a,b) Thermal images (a) and the corresponding photothermal heating curves (b) of 3D printed PCL and 4‐SC/PCL composite scaffolds under 1064 nm laser irradiation (1 W cm⁻², 5 min). c) Relative tumor volume of the indicated different groups after treatments for 14 days. d) Histological analysis (H&E and TUNEL staining) of tumor sections collected from different groups. e) Body weight of the indicated different groups after treatments for 14 days. f) H&E staining of the major organs harvested from different groups. Scale bar: 50 µm. Data are presented as mean ± s.d. (c,e) (n = 5).