The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration

Abstract

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Transient supplementation of growth factor TGF-β1 effectively initiates chondrogenic redifferentiation of human chondrocytes

“…A wide range of treatment strategies were developed involving replacement of injured tissue by osteochondral graft (autologous, allogenous or synthetic), marrow stimulation and transplantation of autologous chondrocytes into the defect [1,2]. For the latter cell-based regeneration technique autologous chondrocytes were harvested from articular cartilage obtained from non-weight bearing region of the joint and cultured in vitro to increase cell number.…”

Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

“…Radial heterogeneity in both cell phenotype and matrix composition is common [421,422] and a necrotic core is sometimes observed [423]. We have previously manufactured thousands of smaller diameter cartilage microtissues using the AggreWell (StemCell Technologies) [337] and our own in--house fabricated microwell platform [395]. The reduced diameter enhanced metabolite and signal molecule diffusion in the resulting microtissues, enabled the formation of more homogenous tissue, and significantly enhanced chondrogenic differentiation [337].…”

“…The silicone glue anchoring the microwell disc inserts was cured for 60 minutes on an 80°C hot plate. PDMS was selected for the manufacture of the base microwell platform as PDMS rapid fabrication is inexpensive, versatile, and has been successfully used to manufacture microwells in similar studies [395,409]. Nylon (6/6) was selected for use in the mesh as nylon is a non--toxic biocompatible material commonly used in clinical applications including formation of gastrointestinal segments, vascular grafts, and sutures [413].…”

“…Microtissues are appealing building blocks in tissue engineering applications [334,341,351,395,409], including the manufacture of cartilage microtissues from either mesenchymal stem/stromal cells (MSC) [337] or articular chondrocytes [395]. The fundamental motivation for using a microwell platform to manufacture cartilage microtissues is its capacity to reproducibly generate hundreds of uniform tissue building blocks.…”

“…This is achieved either through forced aggregation (centrifugation) or gravity settling of a cell suspension into an array of microwells placed at the bottom of a larger tissue culture well [410]. Once the cells have settled into discrete microwells they generally amalgamate within a few hours, and then mature into uniform cartilage microtissues over the subsequent culture period [337,395]. Complete medium exchange is often required to feed microtissues, to sequentially modify the exogenous signal microenvironment, and/or to guide the appropriate development of artificial cartilage tissue.…”