NR2F2 regulates chondrogenesis of human mesenchymal stem cells in bioprinted cartilage

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.

3D Bioprinting: from Benches to Translational Applications

“…They observed high cell viability, chondrogenic differentiation, as well as high robustness of the obtained constructs, indicating the capability of their tissue for the use as neocartilage. A similar approach was adopted by Gao et al, who cultured MSCs on a 3D‐bioprinted scaffold to investigate the importance of nuclear receptor subfamily 2 group F member 2 (NR2F2) . This factor plays a crucial role in the regulation of mesoderm‐derived tissues on chondrogenesis.…”

Chemical insights into bioinks for 3D printing

“…To induce cell differentiation, growth factors can also be added to the bioink, simply mixed [52][53][54][55][56] or chemically linked to the network. [57][58][59] Doping agents such as nanoparticles (e.g. hydroxyapatite [60][61][62][63][64] give robustness and can also improve cell differentiation into osteoblasts.…”

“…Upon MMPs release by cells from the culture, the peptide sequence is cleaved, specifically releasing the network and decreasing the degradation time. 57,58 It is also possible to add drugs, 60,65 peptide ligands improving cell adhesion like the integrin ligand RGD [57][58][59][60]105,106 and differentiation factors. 54 In these cases, the bioink itself acts as a reservoir for drug/bioactive compound delivery.…”

“…24 A) PEG-PCL-DA bioink, 170 and (B) acrylated-RGD. 57,58 Combination of PEG and gelatin, two of the most popular acrylated precursors, has been investigated for stereolithography 171 or extrusion. 172 The only other reported example of acrylated network precursors as bioink components, besides gelatin and PEG, is methacrylated silk fibroin (Fig.…”

“…PIs are mostly used at concentrations ranging from 1.7 to 0.01 mM depending on their respective toxicity. The most used is Irgacure 2959 (I2959), 10,55,[57][58][59]92,97,98,161,162,165,168 ([4-(2hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1propane-1-one, Fig. 25A), which absorbs at 257 nm.…”