Differentiation Behaviour of Adipose-Derived Stromal Cells (ASCs) Seeded on Polyurethane-Fibrin Scaffolds In Vitro and In Vivo.

Katrin Radeloff,Dorothee Weiss,Norbert Kleinsasser,Andreas Radeloff,Rudolf Hagen

doi:10.3390/biomedicines9080982

Abstract

Adipose-derived stromal cells (ASCs) are a promising cell source for tissue engineering and regenerative medicine approaches for cartilage replacement. For chondrogenic differentiation, human (h)ASCs were seeded on three-dimensional polyurethane (PU) fibrin composites and induced with a chondrogenic differentiation medium containing TGF-ß3, BMP-6, and IGF-1 in various combinations. In addition, in vitro predifferentiated cell-seeded constructs were implanted into auricular cartilage defects of New Zealand White Rabbits for 4 and 12 weeks. Histological, immunohistochemical, and RT-PCR analyses were performed on the constructs maintained in vitro to determine extracellular matrix (ECM) deposition and expression of specific cartilage markers. Chondrogenic differentiated constructs showed a uniform distribution of cells and ECM proteins. RT-PCR showed increased gene expression of collagen II, collagen X, and aggrecan and nearly stable expression of SOX-9 and collagen I. Rabbit (r)ASC-seeded PU-fibrin composites implanted in ear cartilage defects of New Zealand White Rabbits showed deposition of ECM with structures resembling cartilage lacunae by Alcian blue staining. However, extracellular calcium deposition became detectable over the course of 12 weeks. RT-PCR showed evidence of endochondral ossification during the time course with the expression of specific marker genes (collagen X and RUNX-2). In conclusion, hASCs show chondrogenic differentiation capacity in vitro with the expression of specific marker genes and deposition of cartilage-specific ECM proteins. After implantation of predifferentiated rASC-seeded PU-fibrin scaffolds into a cartilage defect, the constructs undergo the route of endochondral ossification.

Highlights

Cartilage defects of the head and neck region are due to trauma, infections, congenital malformations, or surgical procedures, e.g., for malignancies
The use of chondrocytes for cartilage tissue engineering has been extensively studied and transferred to clinical applications [6,10], many groups have focused on mesenchymal stem cells (MSCs) from bone marrow (BMSCs) [11] or adipose tissue (ASCs)
PU-fibrin composites without Human Adipose-Derived Stem Cells (hASCs) (Figure 2B) and cell-seeded constructs maintained in EM-DMEM showed no blue staining (Figure 2C), whereas cell-seeded PUfibrin constructs maintained in chondrogenic differentiation medium with the growth factor combinations BBT (Figure 2D), BT (Figure 2E), and TI (Figure 2F) were typically blue-stained

Summary

Introduction

Cartilage defects of the head and neck region are due to trauma, infections, congenital malformations, or surgical procedures, e.g., for malignancies. Common therapeutic methods include the use of autologous cartilage from other locations, such as rib cartilage for the reconstruction of the auricle [1] or auricular cartilage for closure of nasal septal defects [2]. Complex reconstruction procedures of laryngotracheal defects, for instance, include the transplantation of autologous cartilage tissue combined with local or microvascular flaps [3]. Tissue engineering and regenerative medicine approaches may be promising methods for replacing cartilage tissue of the larynx and trachea, auricle, or nose [4,5,6,7,8,9]. The use of chondrocytes for cartilage tissue engineering has been extensively studied and transferred to clinical applications [6,10], many groups have focused on mesenchymal stem cells (MSCs) from bone marrow (BMSCs) [11] or adipose tissue (ASCs) 4.0/).

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