Establishment and characterization of an inflammatory cartilaginous organoids model for organoid transplantation study

Abstract

Background: Transplantation of cartilaginous organoids for repairing cartilage defects in osteoarthritis represents a novel treatment approach. However, A controversial argument remains about whether cartilaginous organoids derived from the differentiation of bone marrow mesenchymal stem cells (BMSCs) in the three-dimensional (3D) environment are strictly organoids and whether the inflammatory microenvironment would affect the success rate of organoid transplantation. This study characterized 3D BMSC-derived cartilaginous organoids and developed an inflammatory organoid model to better understand the transcriptomic changes in the organoids induced by the microenvironment when transplanted into the knee with osteoarthritis.

Methods: Spatial growth BMSCs were generated and cultured in the cartilage differentiation medium to establish cartilaginous organoids. The model was characterized in both morphology and biology aspects. Subsequently, IL-1β induced inflammatory cartilaginous organoids were established and the transcriptomic sequencing was performed to investigate gene expression changes.

Results: BMSC-derived cartilaginous organoids were characterized by histology and immunofluorescence. Both Alcian blue and Safranin O staining revealed abundant articular cartilage extracellular matrix (ECM) in the organoids. The expression of cartilage specific ACAN and Col2A1 was confirmed by immunofluorescence. The organoids had the biological ability to repair cartilage defects. IL-1β induced inflammatory cartilaginous organoids were established and mRNA sequencing revealed downregulation of pathways related to cell adhesion and extracellular matrix organization. Upregulation of IL-6, TNF-α, CCL2 and CXCL1 was confirmed.

Conclusion: We thoroughly validated and characterized BMSC-derived cartilaginous organoids and established the inflammatory cartilaginous organoid models. This study revealed that the attenuation in cell adhesion and ECM formation of organoids induced by inflammatory chemokines may decrease the success rate and effectiveness of organoids auto-transplantation for fixing cartilage defects in the inflammatory microenvironment of the OA joint.

The translational potential of this article: By establishing and validating an in vitro inflammatory cartilaginous organoid model, this study provides a robust platform to examine how inflammatory mediators influence cartilage-like constructs. These findings enable the identification of targeted interventions to enhance the organoids' resilience against the inflammatory environment commonly found in osteoarthritic joints. Ultimately, this strategy offers a novel avenue for improving transplant success and promoting cartilage defect repair in patients with OA, thereby contributing valuable insights and potential clinical applications in regenerative medicine.

Keywords: Cartilaginous organoid; inflammation; transplantation.

Graphical abstract

Fig. 1

Gross morphology and bright field microscopy of the cartilage organoids. (a–h) The gross morphology of cartilaginous organoid from day 0 to day 28. (i) The measurement of the size of the organoid. (j) The image of cartilaginous organoid was captured under bright field microscopy. Scale bar = 200 μm. N = 5.

Fig. 2

Characterization of cartilaginous and inflammatory organoids. (a) The Alcian blue staining was performed on cartilaginous organoids of day 28. Scale bar = 100 μm. (b) The Safranin O and Fast Green staining was performed on the cartilaginous organoids of day 28. The Immunofluorescence staining of the (c) DAPI, (d) ACAN and (e) merged image of cartilaginous organoids. The Immunofluorescence staining of the (f) DAPI, (g) COL2A1 and (h) merged image of cartilaginous organoids. Scale bar = 100 μm. N = 3.

Fig. 3

Transplantation of cartilaginous organoids in articular cartilage defect model. (a) The gross of rat femoral condyle of control group at day 0. (b–d) The H&E staining, Alcian blue staining and Safranin O - Fast Green (S–F) staining respectively of the rat femoral condyle on the sixth week. (e) The gross of rat femoral condyle of articular cartilage defect group at day 0. (f–h) The histological staining of the defect group on the sixth week. Area circled by dash line indicates defect area under microscope. (i) The gross of rat femoral condyle of cartilaginous organoid transplantation group at day 0. (j–l) The histological staining of the transplantation group on the sixth week. Area circled by dash line indicates repaired area after transplantation under microscope. Scale bar = 200 μm. N = 3.

Fig. 4

Transcriptomic analysis. (a) PCA of DEGs distributed in the cartilaginous organoids and inflammatory organoids. (b) Sample to sample cluster analysis between the cartilaginous organoids and inflammatory organoids. (c) Volcano plot representation of genes exhibiting significant expression changes exceeding a 4-fold difference between the two groups in transcriptome sequencing results. (d) Heatmap of gene expression clustering for DEGs. (e) GO analysis for down regulated genes in inflammatory organoids. (f) KEGG analysis for down regulated genes in inflammatory organoids. (g) WikiPathways enrichment analysis for down regulated genes related pathways in inflammatory organoids. (h) GSEA enrichment analysis for collagen fibril organization in organoids. (i) The Alcian blue staining of Il-1b induced inflammatory organoids of day 28. (j) The Safranin O and Fast Green staining of the inflammatory organoids of day 28. N = 3.

Fig. 5

Validation of inflammation level of IL-1β induced inflammatory organoids. (a) Relative gene expression of IL-6 between cartilaginous organoids (CO) and inflammatory cartilaginous organoids. (b) Relative gene expression of TNF-α between CO and inflammatory CO. Concentration of secreted (c) IL-6 and (d) TNF-α from CO and inflammatory CO was measured by ELISA. N = 5. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 6

PPI analysis and validation of key molecules (a) The potential interactions between encoded proteins were searched and protein interaction networks were constructed based on the DEG of organoids. The yellow circle indicates that the protein has fewer than five interactions, a green circle indicates five to ten interactions, and a red circle indicates more than ten interactions. Relative gene expression of (b) CCL2 and (c) CXCL1 between CO and inflammatory CO was measured by qPCR. Concentration of secreted (d) CCL2 and (e) CXCL1 from CO and inflammatory CO was measured by ELISA. N = 5. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001