Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel.

Mellannie P Stuart,José M Granjeiro,Mayra S Azevedo,Karina R Silva,Isis Côrtes,Vladimir Mironov,Anderson Beatrici,Leandra S Baptista,Renata A M Matsui,Matheus F S Santos,Priscila Falagan-Lotsch,Paulo Emílio C Leite

doi:10.1155/2017/7053465

Abstract

The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 μm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-β1, TGF-β2, and TGF-β3 was significantly higher compared to monolayer (p < 0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p < 0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost).

Highlights

The classical tissue engineering relies on scaffold-based approaches in which the scaffold serves as a substitute for extracellular matrix
The micromolded nonadhesive hydrogel with 300 μm diameter formed cartilage progenitors cells (CPCs) spheroids with a reduced diameter, approximately twice smaller compared to 800 μm circular recess counterpart (Figure 2(b) and 2(d))
After 21 days of cell culture, CPC spheroids formed by the micromolded nonadhesive hydrogel with 300 μm diameter in each recesses have reduced their diameter

Summary

Introduction

The classical tissue engineering relies on scaffold-based approaches in which the scaffold serves as a substitute for extracellular matrix. 3D tissue constructs can be produced in the absence of scaffolds [2] This strategy is named scaffold-free tissue engineering, pointed out as having superior results since cells are responsible for synthesizing their own extracellular matrix optimizing cell-matrix and cell-cell interactions, recreating their native tissue microenvironment and recapitulating tissue morphogenesis [3]. The pellet culture, hanging-drop, and 96-well plate have been notably used for 3D cartilage constructs in scaffold-free tissue engineering [5]. The cellular self-organization is responsible for cell aggregate formation by applying an external force. In contrast to aggregates formed by external forces, spheroids are formed by self-assembling process using nonadherent hydrogel molds or platforms such as hanging-drop, 96-well plate, and recently micromolded nonadhesive hydrogels [7, 8]. The main advantage of recent platforms of micromolded nonadhesive hydrogel is seeding cell suspension with single pipetting, reducing substantially technical errors, and allowing automation [3]

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