ASC/chondrocyte-laden alginate hydrogel/PCL hybrid scaffold fabricated using 3D printing for auricle regeneration

Highlights

• A 3D hybrid scaffold consisting of a PCL auricle framework and a cell-laden alginate hydrogel was fabricated.

• The ASCs/chondrocytes in the hybrid scaffold enhanced in vitro chondrogenesis.

• The in vivo results demonstrated promising results for cartilage regeneration.

Abstract

Tissue engineering using adipose derived stem cells (ASCs) has become one of the most promising treatments for defective articular cartilage owing to the stability and dynamic differentiation of ASCs. In this study, we fabricated a 3D hybrid scaffold using poly(ε-caprolactone) (PCL) to support the mechanical properties of the regenerating auricle cartilage, and injected a cell-laden alginate hydrogel, containing a mixture of ASCs and chondrocytes, into the PCL scaffold. Using the cell-laden 3D auricle structure, the in vitro chondrogenesis of the ASCs with and without the presence of chondrocytes was examined. Additionally, the feasibility of utilizing the 3D cell-laden auricle structure for cartilage tissue engineering was evaluated in a rat model. In our in vitro and in vivo experiments, we observed that as the ASCs were co-cultured with the chondrocytes, chondrogenic differentiation was encouraged, and the regeneration of cartilage was significantly increased.

Introduction

Auricular malformations and microtia, with or without preservation of the external auditory canal, traditionally require surgical reconstruction using rib cartilage autografts. The method has been a yardstick for the reconstruction of the total auricle for the last five decades (Reighard, Hollister, & Zopf, 2018). However, it is associated with some critical challenges. The surgery requires highly skilled and experienced surgeons as it is a complicated multi-step process consisting of (1) harvesting rib cartilage and (2) shaping the harvested autografts to the auricle shape. Especially, shaping the normal contralateral auricle with the autologous costal cartilage is a particularly exhausting procedure (Cho, Kim, & Byun, 2007; Ladani, Valand, & Sailer, 2019). Furthermore, side effects can occur on donor sites such as chest wall pain, clicking, and pneumothorax (Uppal, Sabbagh, Chana, & Gault, 2008), and the autologous rib cartilage can become calcified or susceptible to resorption over time (Fischer, Gubisch, & Sinha, 2010; Mori, Tanaka, Umeda, & Hata, 2002).

Recent developments in tissue engineering approaches for auricular reconstruction have demonstrated that it is possible to overcome the limitations associated with the use of rib autografts. The tissue engineering technique provides a tremendous advantage of simplified surgical steps that have been eliminated with the major troublesome caused by harvesting the rib cartilage and shaping it into an auricle shape. In details, the tissue engineering method has been supplemented with a 3D printer to artificially construct an auricle (Reighard et al., 2018). The 3D printing method allowed to attain more sophisticated auricle architecture by selecting proper biomaterials in terms of physical and biological properties, such as printability, sustainability, biocompatibility, and biodegradability (Bos et al., 2015; Haisch, Klaring, Groger, Gebert, & Sittinger, 2002; Markstedt et al., 2015).

The synthetic polymers, such as poly(glycolic acid), poly(lactic acid), and poly(ε-caprolactone) (PCL), have been broadly used for auricle tissue engineering owing to their reasonable mechanical properties and relatively good processability (Bichara et al., 2012). For instance, an auricle was engineered using polyethylene “Medpor” (Hempel, Braun, Patscheider, Berghaus, & Kisser, 2014). Although “Medpor” is commercially available and has been already used for auricular reconstruction, it is susceptible to minor trauma that leads to secondary infections, requiring a secondary operation using temporoparietal fascial flap for coverage and implant extrusion (Romo & Reitzen, 2008). Furthermore, synthetic polymers have a low level of bioactive components, which can restrain cell growth, neocartilage, inflammatory responses, or cell migration (Arevalo-Silva et al., 2000; Britt & Park, 1998; Cao et al., 1998; Santavirta et al., 1990; Shieh, Terada, & Vacanti, 2004).

On the other hand, naturally-derived hydrogels, such as alginate (Bichara et al., 2010; Kuo & Ma, 2001; Mooney, 2003), fibrin (Ahmed, Dare, & Hincke, 2008; Silverman, Passaretti, Huang, Randolph, & Yaremchuk, 1999; Ting et al., 1998; Xu et al., 2005, 2004), collagen (Zhou et al., 2011), hyaluronic acid (HA) (Chung et al., 2006; Inbal, Lemelman, Millet, & Greensmith, 2017), chitosan (Jeon et al., 2007), and gelatin (Xue et al., 2013), have been widely studied for auricle construction because they have higher level of bioactive components and cell-carrying capability than the synthetic polymers even though they exhibit limitations in their biomechanical properties and shape retention ability (Bichara et al., 2012; Yamaoka et al., 2006). Consequently, hybrid structures using synthetic polymers and naturally derived hydrogels have been studied in tissue engineering especially for auricle reconstruction (Jiang et al., 2019; Schuurman et al., 2011; Shen et al., 2019; Thomas, Biggs, O’Brien, & Pandit, 2018). Table 1 shows some studies about the hybrid scaffolds for cartilage regeneration with their methods, results, and limitations. Although our method to fabricate the hybrid structure in our study has not been much different from the fabricating methods, this study has some advantages of (1) simpler fabrication method for obtaining a hybrid (auricular) structure consisting of cell-laden alginate bioink, (2) co-culture of chondrocytes and stem cells, (3) various in vitro cellular results demonstrating the synergistic effect of the stem cells on the auricle regeneration, and (4) in vivo results of the cell-laden hybrid structure.

In addition, the cell sources have been also important for auricle regeneration. Although the ideal cell source is autologous auricular chondrocytes from affected ear or contralateral side via biopsy, the use of patient auricular cartilage is limited by their availability (Mandl, Van Der Veen, Verhaar, & Van Osch, 2004). Therefore, co-culture of stem cells with chondrocytes have been spotlighted to enlarge the number of cells for cartilage regeneration by inducing the chondrogenesis of the stem cells (Hwang et al., 2011; Shi et al., 2017; Zhao et al., 2017). Co-culturing of ASCs and chondrocytes also enhanced cartilage proliferation and chondrogenic differentiation owing to the stimulation of TGF-β1 pathway (Shi et al., 2017).

In this study, PCL and alginate have been selected to fabricate a hybrid auricle scaffold, which are the most frequently used materials for the auricle reconstruction (Bichara et al., 2012). PCL has been reported to show the most stable shape preservation compared with other polymers like poly-l-lactic acid and poly-4-hydroxybutyrate because of its slower degradation rate (Shieh et al., 2004). The natural polysaccharide, alginate, has been extensively applied in tissue engineering scaffolds owing to non-antigenicity, reasonable biocompatibility, and controllable biodegradability. Furthermore, with divalent cations, the alginate can be rapidly crosslinked; thus, it has been used as a drug-releasing system and a cell carrier for various cells including chondrocytes (Beekman, Verzijl, Bank, von der Mark, & TeKoppele, 1997; Bichara et al., 2010; Chang et al., 2001; Chia et al., 2005; Dobratz, Kim, Voglewede, & Park, 2009; Hauselmann et al., 1992, 1994; Mooney, 2003). Although the hydrogel has been applied as an important supplementary material for various tissue engineering applications, further studies are required to improve the mechanical properties of alginate-based engineered tissues including cartilages.

Here, we hypothesized that a hybrid structure can be one of innovative platforms to facilitate high degree of chondrogenesis by adapting a synthetic polymer to support the low mechanical properties of alginate. The hypothesized structure can be consisted of PCL mesh structure and the alginate loaded with various cells. Using a 3D printing process, we fabricated a 3D auricle-shaped PCL framework for mechanical and structural maintenance and injected a cell-laden alginate hydrogel in the PCL structure. In the alginate hydrogel, human ASCs and chondrocytes derived from rabbit were laden in a ratio of 1:1. The cell-laden alginate/PCL structure was used to regenerate the auricle cartilage and evaluate the in vitro and in vivo feasibility of the structure.