Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo

Abstract

Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.

Fig 1. 3D-bioprinted grid.

Example of a cell-containing grid (5 × 5 × 1.2 mm) with a line spacing of 1.5 mm in three layers.

Fig 2. Chondrocyte proliferation.

Histological sections of 3D-bioprinted constructs with hNCs at day 30 (left) and day 60 (right) after implantation. Alcian blue and van Gieson staining allow visualization of the glycosaminoglycans in the chondrocyte extracellular matrix. Progressive proliferation and cluster formation were observed after 60 days as compared to that observed after 30 days. Encircled magnifications show the cluster formations. Bars = 1000 μm and 100 μm (magnification).

Fig 3. Chondrocyte proliferation.

Histological sections of 3D-bioprinted constructs with hNCs mixed with hBM-MSCs at day 30 (left) and day 60 (right) after implantation. The glycosaminoglycans in the chondrocyte extracellular matrix were visualized by Alcian blue and van Gieson staining. Progressive proliferation and cluster formation seen after 60 days as compared with that observed after 30 days. Encircled magnifications show the cluster formations. Bars = 1000 μm and 100 μm (magnification).

Fig 4. Number of chondrocytes per mm².

The proliferative increase in GAG-positive cells between days 30 and 60 was significant in both groups. The number of mature chondrocytes at the start of the experiment was 5-fold greater in the human nasal chondrocyte group as compared with the mixed group (10 M and 2 M, respectively), and the proliferative capacity was more pronounced in the mixed group.

Fig 5. Cluster formation.

Cluster area as a percentage of the entire area. Cluster formation represented a definitive sign of proliferation and resembled native cartilage.

Fig 6. Safranin-O staining.

Two consecutive sections from the mixed group (hNC/MSC) after 60 days. The GAG-positive extracellular matrix stains blue with Alcian blue and van Gieson (left). The proteoglycans in the extracellular matrix surrounding the chondrocytes are also visualized by Safranin-O staining (bright red) and nuclei are stained blue (right). The Safranin-O analysis confirm the results indicated by Alcian blue and van Gieson staining, i.e. similar areas are stained. Bars = 500 μm and 250 μm (magnification).

Fig 7. FISH analysis.

FISH analysis to determine human chromosome X (green) and Y (red) confirmed that the proliferating cells in the chondrocyte clusters were of human origin. This analysis also concluded that the cells in the mixed group originated mainly from the male-derived chondrocytes (XY) and not the female-derived stem cells (XX).

Fig 8. Immunohistochemical analysis.

Immunohistochemical analysis of the type 2 collagen distribution (bright red, left) in the hNC constructs correlated well with the glycosaminoglycan production visualized by Alcian blue and van Gieson staining (right). Bars = 250 μm.