Protocol for engineering bone organoids from mesenchymal stem cells

Abstract

Bone organoids are emerging as powerful tools for studying bone development and related diseases. However, the simplified design of current methods somewhat limits their application potential, as these methods produce single-tissue organoids that fail to replicate the bone microarchitecture or achieve effective mineralization. To address this issue, we propose a three-dimensional (3D) construction strategy for generating mineralized bone structures using bone marrow-derived mesenchymal stem cells (BMSCs). By mixing BMSCs with hydrogel to create a bone matrix-mimicking bioink and employing projection-based light-curing 3D printing technology, we constructed 3D-printed structures, which were then implanted subcutaneously into nude mice, away from the native bone microenvironment. Even without external stimulation, these implants spontaneously formed mineralized bone domains. With long-term culture, these structures gradually matured into fully differentiated bone tissue, completing both mineralization and vascularization. This in vivo bone organoid model offers a novel platform for studying bone development, exploring congenital diseases, testing drugs, and developing therapeutic applications.

Keywords: 3D bioprinting; Bioink; Bone organoids; Mineralization; Vascularization.

Graphical abstract

Fig. 1

Schematic overview of this protocol. (A) Preparation of bioink: hybrid hydroxyapatite hydrogels were synthesized, and rat BMSCs were isolated, extracted, and expanded in vitro. (B) 3D bioprinting of bone organoids. (C) In vivo cultivation and assessment of bone organoids.

Fig. 2

Characterization of bone matrix-inspired hybrid hydroxyapatite bioink. (A) NMR spectroscopy of hybrid hydroxyapatite bioink. (B) XRD analysis of hybrid hydroxyapatite bioink. (C) SEM analysis of hybrid hydroxyapatite bioink. (D) Solution state of bioink before and after photopolymerization. (E) Elemental composition evaluation of bioink using EDS. (F) Elemental mapping of bioink.

Fig. 3

3D bioprinting and evaluation of bone organoids: structural integrity, cell viability, and morphological analysis. (A) Schematic of 3D bioprinting process. (B) 3D Max constructed model for 3D bioprinting. (C) Panoramic view of 3D printed bone organoids. (D) Cell viability and biocompatibility assessment of bone organoids. (E) High-resolution fluorescence imaging of the cytoskeleton in bone organoids.

Fig. 4

In vivo mineralization evaluation of bone organoids. (A) Schematic of micro-CT scanning for bone organoids. (B) Surgical retrieval of bone organoids. (C) Stereomicroscopic analysis of vascularization in bone organoids. (D) In vivo mineralization progress of bone organoids assessed by micro-CT scanning.

Fig. 5

Comprehensive morphological and structural analysis of bone organoids: SEM and 3D immunofluorescence imaging. (A) Experimental design for in vivo SEM evaluation of bone organoids. (B) Microarchitecture analysis of bone organoid via SEM scanning. (C) Cell morphological diversity of bone organoids via SEM scanning. (D) Identification of cell types in bone organoids via SEM scanning and morphological analysis. (E) 3D immunofluorescence analysis of specific markers in bone organoids. (F) Immunofluorescence analysis of CD31, collagen II, and periostin in bone organoids.

Fig. 6

Ultrastructural characterization and validation of mineralization crystals in bone organoids. (A) Experimental design for TEM examination of bone organoids. (B) Ultrastructural analysis of bone organoid mineralization using TEM scanning. (C) Electron diffraction analysis of crystalline mineral components in bone organoids. (D) Fibrous reticular structure of mineralized region in bone organoids. (E) Elemental composition analysis of bone organoids via EDS spectrum. (F) Elemental enrichment and distribution in bone organoids revealed by EDS mapping.

Fig. 7

Histological staining of bone organoids. (A) HE staining of bone organoids. (B) Masson's Trichrome staining of bone organoids. (C) ALP staining of bone organoids. (D) ARS staining of bone organoids. (E) TRAP staining of bone organoids. (F) Oil Red O staining of bone organoids. (G) Toluidine blue staining of bone organoids. (H) Immunofluorescence staining of OPN in bone organoids. (I) Immunofluorescence staining of OCN in bone organoids.