Combining Innovative Bioink and Low Cell Density for the Production of 3D-Bioprinted Cartilage Substitutes: A Pilot Study

Abstract

3D bioprinting offers interesting opportunities for 3D tissue printing by providing living cells with appropriate scaffolds with a dedicated structure. Biological advances in bioinks are currently promising for cell encapsulation, particularly that of mesenchymal stem cells (MSCs). We present herein the development of cartilage implants by 3D bioprinting that deliver MSCs encapsulated in an original bioink at low concentration. 3D-bioprinted constructs (10×10×4 mm) were printed using alginate/gelatin/fibrinogen bioink mixed with human bone marrow MSCs. The influence of the bioprinting process and chondrogenic differentiation on MSC metabolism, gene profiles, and extracellular matrix (ECM) production at two different MSC concentrations (1 million or 2 million cells/mL) was assessed on day 28 (D28) by using MTT tests, real-time RT-PCR, and histology and immunohistochemistry, respectively. Then, the effect of the environment (growth factors such as TGF-β1/3 and/or BMP2 and oxygen tension) on chondrogenicity was evaluated at a 1 M cell/mL concentration on D28 and D56 by measuring mitochondrial activity, chondrogenic gene expression, and the quality of cartilaginous matrix synthesis. We confirmed the safety of bioextrusion and gelation at concentrations of 1 million and 2 million MSC/mL in terms of cellular metabolism. The chondrogenic effect of TGF-β1 was verified within the substitute on D28 by measuring chondrogenic gene expression and ECM synthesis (glycosaminoglycans and type II collagen) on D28. The 1 M concentration represented the best compromise. We then evaluated the influence of various environmental factors on the substitutes on D28 (differentiation) and D56 (synthesis). Chondrogenic gene expression was maximal on D28 under the influence of TGF-β1 or TGF-β3 either alone or in combination with BMP-2. Hypoxia suppressed the expression of hypertrophic and osteogenic genes. ECM synthesis was maximal on D56 for both glycosaminoglycans and type II collagen, particularly in the presence of a combination of TGF-β1 and BMP-2. Continuous hypoxia did not influence matrix synthesis but significantly reduced the appearance of microcalcifications within the extracellular matrix. The described strategy is very promising for 3D bioprinting by the bioextrusion of an original bioink containing a low concentration of MSCs followed by the culture of the substitutes in hypoxic conditions under the combined influence of TGF-β1 and BMP-2.

Hydrogel‐Based 3D Bioprinting for Bone and Cartilage Tissue Engineering

“…A low MSC concentration was identified in healthy hyaline cartilage, indicating viable alternatives and the induction of chondrogenic genes by TGF-1. [80,81] Considering the importance of biomaterials' type and viscosity for 3D bioprinting of cell-laden bioinks, using a combination of gelatin, alginate, and fibrinogen was successful for cartilage 3D printing in the presence of a cocktail of BMP-2 and TGF-1 during a 56 day period. Gelatin and alginate have been previously applied as 3D systems to enhance the regeneration of hyaline-like cartilage tissue following the chondrogenic differentiation of MSCs within an ECM enriched in proteoglycans and type II collagen.…”

“…To address this issue, researchers have engineered 3D-printed agarose hydrogels with tailored properties by means of nano silicate additives. [80,107,108] The mechanical test and the bright field images of NIH/3T3 cells indicated that not only the structural integrity and shape fidelity of the extrusion-based bioprinted nanocomposite, but also the metabolic activity and morphology of the cells could be tailored using different concentrations of nano silicate (Figure 3A,B). [109] Collagen, as a triple-helix protein and respect to its natural and biological origin, has found wide attraction for gelbased skeletal tissue regeneration.…”

Effects of Processing Parameters of 3D Bioprinting on the Cellular Activity of Bioinks

“…A low cell density of MSC (1 million) is beneficial for the chondrogenic differentiation potential. [ 116 ] Tröndle et al. revealed that high cell density (25 × 10 6 ± 3 × 10 6 cells mL −1 ) is beneficial while bioprinting primary endothelial cells in the DoD technique.…”

Stem Cells and Extrusion 3D Printing for Hyaline Cartilage Engineering

“…The last important factor is the environment. Chondrogenic matrix synthesis is driven by growth factors [ 57 , 58 , 59 , 60 , 61 ], oxygen levels [ 62 , 63 , 64 , 65 , 66 ], maturation time [ 67 ], and mechanical stimuli, including dynamic compression and shear stress, to mimic the natural diarthrodial environment [ 68 , 69 , 70 , 71 , 72 , 73 ]. Classical TE usually produces homogenous constructs.…”

“…To further reproduce the calcified layer, calcium can be added to the bioink to increase the expression of hypertrophic cartilage markers [ 166 ]. To fine-tune the chondrogenesis of the embedded MSCs in 3D-printed constructs, the use of growth factors, especially TGF-β family members, is essential [ 60 ].…”