3D Stem Cell Spheroids with 2D Hetero-Nanostructures for In Vivo Osteogenic and Immunologic Modulated Bone Repair

Abstract

3D stem cell spheroids have immense potential for various tissue engineering applications. However, current spheroid fabrication techniques encounter cell viability issues due to limited oxygen access for cells trapped within the core, as well as nonspecific differentiation issues due to the complicated environment following transplantation. In this study, functional 3D spheroids are developed using mesenchymal stem cells with 2D hetero-nanostructures (HNSs) composed of single-stranded DNA (ssDNA) binding carbon nanotubes (sdCNTs) and gelatin-bind black phosphorus nanosheets (gBPNSs). An osteogenic molecule, dexamethasone (DEX), is further loaded to fabricate an sdCNTgBP-DEX HNS. This approach aims to establish a multifunctional cell-inductive 3D spheroid with improved oxygen transportation through hollow nanotubes, stimulated stem cell growth by phosphate ions supplied from BP oxidation, in situ immunoregulation, and osteogenesis induction by DEX molecules after implantation. Initial transplantation of the 3D spheroids in rat calvarial bone defect shows in vivo macrophage shifts to an M2 phenotype, leading to a pro-healing microenvironment for regeneration. Prolonged implantation demonstrates outstanding in vivo neovascularization, osteointegration, and new bone regeneration. Therefore, these engineered 3D spheroids hold great promise for bone repair as they allow for stem cell delivery and provide immunoregulative and osteogenic signals within an all-in-one construct.

Keywords: 2D materials; bone repair; hetero‐nanostructures (HNSs); immunomodulation; stem cell spheroids.

Fig. 1

Schematic demonstration. a) The fabrication of sdCNTgBP 2D hetero-nanostructures by sdCNT and gBPNS followed by the loading of the DEX. b) The application of stem cell spheroids incorporated with sdCNTgBP 2D heterostructures loaded with DEX for bone regeneration through stem cell transplantation, phosphate ions supply, osteo-immunomodulation, neovascularization, and osteogenesis.

Fig. 2

SEM and TEM of 2D materials and in vitro cell viability. The TEM images of a) CNT, b) sdCNT, c) BPNS, d) gBPNS 2D materials, and e) sdCNTgBP hetero-nanostructures with schematic demonstrations. SEM images of f) CNT, g) sdCNT, h) BPNS, i) gBPNS 2D materials, and j) sdCNTgBP hetero-nanostructures. k) Photograph of a single stem cell spheroid formed within the microwell. l) Collected pure stem cell spheroids and the spheroids incorporated with sdCNTgBP 2D hetero-nanostructures. The SEM images of m) pure stem cell spheroids and n) spheroids incorporated with sdCNTgBP 2D hetero-nanostructures after critical-point drying (CPD) treatment. o) Flow cytometry analysis of the apoptosis of stem cells within the spheroids incorporated with varied concentrations of sdCNT materials. p) Live/dead staining of spheroids incorporated with varied concentrations of sdCNT and gBPNS 2D materials. q) Potential mechanism for the enhancement of stem cell viability within the spheroids after incorporating tiny amounts of sdCNT materials.

Fig. 3

In vivo bone formation of 2D hetero-nanomaterials incorporated 3D stem cell spheroids. a) Schematic demonstration of stem cell spheroids implantation to the bone injury sites with stem cell supply and osteogenesis induction. The photographs of initial implantation and rat defect sites after 4 weeks for b) stem cell spheroids and c) spheroids incorporated with sdCNTgBP 2D hetero-nanostructures. The analysis of d) BV/TV ratio, e) bone mineral density (BMD), and f) new bone area in rat calvarial defects with varied treatment. g) Micro-CT reconstruction images of the rat empty control calvarial defect and the defects implanted with spheroids and spheroids incorporated with sdCNTgBP 2D hetero-nanostructures at 4 weeks post-surgery. h) Immunohistology analysis of bone defects with H & E staining. i) Immunohistochemical staining of DAPI, ALP, and CD31 markers in the rat bone defect sites. Arrows indicate the sites with intensively expressed ALP and CD31. (*: p < 0.05).

Fig. 4

Osteo-immunomodulation. a) Fabrication of sdCNTgBP-DEX 2D hetero-nanostructures. b) DEX release kinetics from the 2D hetero-nanostructures. c) ALP activities in varied types of stem cell spheroids. d) Immunofluorescence staining of Runx2 osteogenic marker and F-actin in varied types of stem cell spheroids. Relative mRNA expression of e) OPN, f) BMP-2, g) Runx2, and h) OSX osteogenic marker genes. i) Schematic illustration of enhanced osteo-induction of stem cell spheroids by a synergistic effect of 2D hetero-nanostructures and DEX through elevating osteogenic gene expressions. j) Calcium minerals deposition in these spheroids incorporated with varied 2D materials. k) Attraction of calcium and phosphate ions on 2D carbon nanotube materials to form minerals. l) Macrophage phenotypic polarization by DEX stimulation. m) Flow cytometry analysis for the phenotypes of macrophages stained with CD68 and CD206 antibodies after co-culturing with the releasing medium from spheroids. n) Confocal microscopy images of macrophages stained with a pan-macrophage marker (CD68), M2 phenotypic marker antibodies (CD206), and DAPI with o) enlarged view of marker expression in single macrophages. (*: p < 0.05).

Fig. 5

In Vivo Osteo-immunomodulation and bone repair. a) Immunohistological staining of CD68 and CD206 markers expressions in rat empty calvarial defect and defects implanted with spheroids at two weeks post-surgery. b) Schematic demonstration of in vivo osteo-immunomodulation to help macrophage shift to pro-healing M2 phenotype. The c) micro-CT images, d) bone mineral density (BMD), and e) BV/TV ratio of the rat empty control calvarial defect and the defects implanted with spheroids incorporated with sdCNTgBP or sdCNTgBP-DEX 2D hetero-nanostructures at 8 weeks post-surgery. Immunohistological images of bone slices after being stained with f) H & E, g) Masson’s trichrome, and h) toluidine blue staining. (*: p < 0.05).

Fig. 6

In vivo neovascularization and osteogenesis. a) Schematic demonstration of in vivo neovascularization and osteogenesis in bone defects implanted with osteogenic spheroids incorporated with DEX-loaded 2D hetero-nanostructures. The in vivo expressions of b) OPN, c) BMP-2, d) Runx2, and e) OSX osteogenic markers. f) Immunohistochemical staining of osteogenic ALP and neovascular CD31 markers in the rat bone defect sites with g) enlarged view of the marker expression in the defect area. (*: p < 0.05).