Redifferentiation of High-Throughput Generated Fibrochondrocyte Micro-Aggregates: Impact of Low Oxygen Tension

Abstract

In meniscus tissue engineering strategies, enhancing the matrix quality of the neomeniscal tissue is important. When the differentiated phenotype of fibrochondrocytes is lost, the quality of the matrix becomes compromised. The objective of this study was to produce uniform fibrochondrocyte micro-aggregates with desirable phenotype and tissue homogeneity in large quantities using a simple and reproducible method. Furthermore, we investigated if hypoxia could enhance the matrix quality. Porcine fibrochondrocytes were expanded at 21% oxygen until passage 3 (P3) and a gene expression profile was determined. P3 fibrochondrocytes were cultivated in chondrogenic medium at 5 and 21% oxygen in high-throughput agarose chips containing 2,865 microwells 200 µm in diameter. Evaluation included live/dead staining, histological examination, immunohistochemistry, dimethylmethylene blue assay and real-time reverse transcriptase quantitative polymerase chain reaction of the micro-aggregates. Gene expression analysis showed a drastic decline in collagen II and high expression of collagen I during monolayer culture. After 4 days, uniform and stable micro-aggregates could be produced. The redifferentiation and matrix quality of the hypoxic cultured micro-aggregates were enhanced relative to the normoxic cultures. Sulfated glycosaminoglycan synthesis was significantly higher, and collagen II expression and the collagen II/collagen I ratio were significantly upregulated in the hypoxic cultures. High-throughput production of uniform microtissues holds promise for the generation of larger-scale tissue engineering constructs or optimization of redifferentiation mechanisms for clinical applications.

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids

“…Spheroids are generated using a high-throughput nonadhesive agarose microwell system, as previously described (Gevaert et al, 2014;Berneel et al, 2016;Roosens et al, 2017;De Moor et al, 2018. In brief, polydimethylsiloxane (PDMS) molds (NaMiFab, Ghent University) with a diameter of 18 mm and a height of 3 mm, containing 1585 micropores of 400 µm diameter each, are used as a negative replica to create microwells ( Figure 1A).…”

“…Next to culturing MSC in 3D, differentiation into the chondrogenic lineage can be induced by applying a chondrogenic culture medium containing growth factors such as transforming growth factor-β (TGF-β) and/or bone morphogenetic protein (BMP) (Ude et al, 2017). Further mimicking of the natural environment can be done by culturing in a low oxygen environment as hypoxia results in an increase of cartilage ECM molecules such as collagen II and aggrecan (Tan et al, 2011;Berneel et al, 2016).…”

Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide‐Modified Gelatin Hydrogel Characteristics

“…Generation of Spheroids by Micromolding: Spheroids were generated using a non-adhesive microwell system, as previously described. [5,10,65,66] In brief, customized patterned negative polydimethylsiloxane (PDMS) molds (ø = 18 mm, NaMiFab, Ghent University) were placed in the wells of a 6-well plate. A sterile 4.5 w/v% Ultrapure agarose (Life technologies) solution was poured on top of the negative PDMS molds and after solidification at room temperature, agarose microwells were separated from the molds with sterile gloves.…”

“…[ 5–7 ] In order to regain phenotype, the native cartilage microenvironment can be mimicked in several ways, for example, by adding growth factors to the culture medium such as TGF‐β [ 8,9 ] or by incubating the cells in a low oxygen environment, as hypoxia results in an increase of cartilage ECM components such as collagen II and aggrecan. [ 10,11 ] The creation of a 3D culture environment, by either encapsulation of cells in a hydrogel or cellular self‐aggregation in 3D cell pellets or spheroids, can also induce chondrogenic (re)differentiation due to the high cell density and the formation of cell–cell and cell–ECM contacts. [ 5,12 ] The latter gives promising prospects to use self‐assembled spheroids as building blocks to generate tissue‐engineered cartilage instead of the conventional single cell suspension.…”

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

“…ASC/HUVEC co‐culture spheroids were generated by using a non‐adherent microwell culture system, as previously described (Berneel, Philips, Declercq, & Cornelissen, 2016; De Moor et al, 2018; Gevaert et al, 2014). In brief, a 3 w/v% ultrapure agarose solution (Life Technologies) dissolved in sterile PBS was heated and poured on top of a negative polydimethylsioloxane (PDMS) customized mould with a diameter of 18 mm and a height of 3 mm.…”