Abstract
The maintenance and expansion of the cells required for formation of tissue-engineered cartilage has, to date, proven difficult. This is, in part, due to the initial solid phase extracellular matrix demanded by the cells inhabiting this avascular tissue. Herein, we engineer an innovative alginate-fibronectin microfluidic-based carrier construct (termed a chondrobag) equipped with solid phase presentation of growth factors that support skeletal stem cell chondrogenic differentiation while preserving human articular chondrocyte phenotype. Results demonstrate biocompatibility, cell viability, proliferation and tissue-specific differentiation for chondrogenic markers SOX9, COL2A1 and ACAN. Modulation of chondrogenic cell hypertrophy, following culture within chondrobags loaded with TGF-β1, was confirmed by down-regulation of hypertrophic genes COL10A1 and MMP13. MicroRNAs involved in the chondrogenesis process, including miR-140, miR-146b and miR-138 were observed. Results demonstrate the generation of a novel high-throughput, microfluidic-based, scalable carrier that supports human chondrogenesis with significant implications therein for cartilage repair-based therapies.
Highlights
Cartilage lesions represent an important technical challenge for tissue engineering given the avascular nature of this tissue, which often leads to defective intrinsic healing [1]
Microfluidic fabrication of mono-dispersed alginate-fibronectin chondrobags and encapsulation of skeletal stem cells (SSCs) and human articular chondrocytes (HACs) SSCs and HACs were encapsulated in pearl-lace like chondrobags using co-flow focusing glass capillary devices (figures 1(A)–(C) and supplementary video 1 and 2)
The cell counts at each time point are the result of eight measurements sequentially acquired at 30-min intervals at room temperature for 2 h. (B) Volume distribution of produced chondrobags of two miscible fluid streams under laminar flow conditions using flow rates of 125–250 μl h−1 for the two inner phases and 3000 μl h−1 for the outer phase
Summary
Cartilage lesions represent an important technical challenge for tissue engineering given the avascular nature of this tissue, which often leads to defective intrinsic healing [1]. Chondrocytes, derived from the native tissue, have been examined as an apparent cell source of the formation of neocartilage. Chondrocytes or chondroprogenitors, derived from stromal progenitors, need to be guided in the formation of native hyaline cartilage, to avert dedifferentiation and, critically, to ensure the generation of pre-eminent tissue reconstruction in vivo that resembles the complex cellular and extracellular matrix (ECM) make-up in the native tissue. More advanced cartilage-like tissues exhibiting improved mechanical properties have been made exploiting 3D models for cell proliferation and differentiation [6]. These systems facilitate interactions between the cell and the requisite cellular environment/matrix [7]. These 3D systems still display an overall lack of homogeneity in cell responses related to the necrotic cores [8], that could be assigned to a list of contributing factors including: (i) the non-physiological closely compacted cells that are formed during initial stages of culture, (ii) the existence of necrotic cores enclosed by in vitro/ex vivo cell clusters as a consequence of high cell number, (iii) sub-standard physiological culture conditions and, (iv) diffusion limitations by uncontrolled establishment of chemical gradients throughout samples [9]
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