Cartilage tissue engineering using funnel-like collagen sponges prepared with embossing ice particulate templates

Abstract

Three-dimensional porous scaffolds of collagen have been widely used for tissue engineering and regenerative medicine. In this study, we fabricated funnel-like collagen sponges with open surface pore structures by a freeze-drying method that used embossing ice particulates as a template. By controlling the size of the ice particulates and the temperature of freezing, collagen sponges with different pore structures were prepared. To investigate the effects of different pore structures on cartilage regeneration, the funnel-like collagen sponges were used to culture bovine articular chondrocytes. Scaffolds that were prepared with 400 μm ice particulate templates and a freezing temperature of −3 °C resulted in the best cell distribution, ECM production, and chondrogenesis. Although funnel-like collagen sponges prepared with 400 μm ice particulate templates and a freezing temperature of −1 °C and 720 μm ice particulates and a freezing temperature of −3 °C, showed even cell distribution, the cell seeding efficiencies and sGAG amount per cell were low. However, the scaffolds prepared with 400 μm ice particulate templates and a freezing temperature of −5 °C or −10 °C showed a limited effect on the improvement of cell distribution and chondrogenesis. Control collagen sponges without ice particulates failed to support the formation of homogenous cartilage-like tissue. These results indicate that funnel-like collagen sponges were superior to control collagen sponges and that scaffolds prepared with 400 μm ice particulate templates at −3 °C were optimal for cartilage tissue engineering.

Introduction

Adult articular cartilage has very limited intrinsic repair or regeneration capacity, due to the limited proliferation of highly differentiated chondrocytes in vivo, slow matrix turnover, low supply of progenitor cells, and a lack of vascular supply. Damaged or diseased cartilage tissue caused by developmental abnormalities, trauma, or aging-related degeneration results in disability and extensive pain. Autologous chondrocyte-based tissue engineering provides a promising therapy for cartilage damage [1], [2], [3]. In the most common approach, chondrocytes isolated from the patient are cultured in a three-dimensional (3D) porous scaffold, which provides the necessary support for cell adhesion and proliferation, guides new cartilage formation, and finally is replaced by the cells and their extracellular matrix (ECM).

Various types of scaffolds have been prepared for research and clinical applications of cartilage tissue engineering [4], [5]. The ideal scaffolds to repair damaged articular cartilage should meet many criteria. First, the ideal scaffolds should be prepared from a biocompatible and biodegradable substrate and have a suitable surface property for cell attachment, proliferation, and differentiation [6], [7]. Structurally, the ideal scaffolds should have an open and interconnected porous architecture, which helps cells easily penetrate into the inner part of the scaffolds and results in homogeneous cell distribution [8]. A homogeneous spatial distribution of cells within the scaffold is important because it lays the foundation for homogenous ECM deposition and hence uniform tissue growth [9]. However, a common problem encountered with porous scaffolds is the rapid formation of a thin layer of tissue on the outer edge caused by uneven cell seeding, which leads to the development of a necrotic core due to the limitations of cell penetration and nutrient exchange [10], [11], [12], [13].

We developed a method using embossing ice particulate templates to prepare funnel-like porous scaffolds that have open surface pores and interconnected pore structures. The funnel-like scaffolds facilitate cell penetration into the inner parts of the scaffolds and spatially homogeneous cell distribution [14], [15]. Although funnel-like scaffolds having different sizes of surface and inner bulk pores can be controlled by the sizes of the ice particulates and the temperature of freezing, their effect on cell function and chondrogenesis is unknown. However, pore size is one critical factor that influences cartilage tissue regeneration and chondrogenesis [16], [17], [18], [19].

In this study, we prepared 6 types of funnel-like collagen sponges having different pore structures and used them for the culture of bovine articular chondrocytes (BACs) to investigate the potential application of funnel-like sponges in cartilage tissue engineering and to compare the influence of different pore structures on cartilage regeneration. Type I collagen was used to prepare the scaffolds because it is one of the most widely used biomaterials in tissue engineering due to its abundant biorecognition, good biocompatibility, and hydrophilicity. Cell distribution was observed by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). The cells were cultured both in vitro and in vivo. Cell proliferation, sulfated glycosaminoglycans (sGAG) production, and chondrogenesis were investigated by biochemical, histological, immunohistological, and gene expression analyses.