Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds.

Chih-Hao Chen,Jyh-Ping Chen,Chang-Yi Kuo

doi:10.3390/ijms19020370

Abstract

In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM) production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day) to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-β1 and IGF-1) genes by quantitative real-time polymerase chain reactions (qRT-PCR) and production of ECM (GAGs and Col II) by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-β1 and IGF-1) genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs) was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II) and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs) or co-culture (chondrocytes + ASCs) and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the chondrocytes replaced by ASCs for co-culture. This combined co-culture/dynamic culture strategy is expected to cut down the amount of donor chondrocytes needed for cartilage-tissue engineering.

Highlights

Cartilage is a unique connective tissue capable of enduring long-term tensile stress, compressive stress and heavy loads in the human body
We have previously reported the preparation of an elastic macroporous gelatin/chondoitin-6sulfate/hyaluronan/chitosan cryogel scaffold with high porosity, large pore size, and unique mechanical properties for cartilage tissue engineering [15]
Our study suggested that chondrocytes under dynamic compressive loading in the highly elastic cryogel scaffold can induce the chondrogenic differentiation of adipose-derived stem cells (ASCs)

Summary

Introduction

Cartilage is a unique connective tissue capable of enduring long-term tensile stress, compressive stress and heavy loads in the human body. The clinical practice for damaged articular cartilage repair includes microfracture, autologous chondrocyte implantation and mosaicplasty [2], which are autografting techniques that use cartilage from other parts of the body to repair damaged areas These practices cause additional damage; and the repair is subject to numerous restrictions, such as the required mechanical strength, and the amount, shape and size of the available cartilage tissue for replacement. To counter such shortcomings, tissue-engineering technique may offer a promising approach for further development. The use of dynamic culture in a bioreactor with scaffolds that mimic the nature of an ECM microenvironment of chondrocytes for cartilage tissue engineering development is a topic worthy of exploration

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Conclusion