Prefabrication of 3D Cartilage Contructs: Towards a Tissue Engineered Auricle – A Model Tested in Rabbits

Abstract

The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15×8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.

Long-Term Morphological and Microarchitectural Stability of Tissue-Engineered, Patient-Specific AuriclesIn Vivo

“…Color images available online at www.liebertpub.com/tea perichondrial layer were consistent with previous research, 3,6,10,12,23 and a continuous elastin network was found similar to only one other tissue-engineered ear study at a comparable time point. 23 In contrast to other long-term studies, auricular constructs at 6 months displayed no signs of osteophyte development, 6,10 necrosis, 10 fibrocartilage formation, 10 or degradation at the periphery, 26 despite the production of thicker cartilage tissue. 23 Beyond the microstructure, biochemical and mechanical properties of native cartilage were also either reached or maintained through 6 months and were comparable to previously reported values.…”

“…25 Although our previous study demonstrated the potential of utilizing photogrammetric and CAD/CAM methods to produce patient-specific molds and constructs, the long-term stability (greater than 3 months) of the developing tissue in vivo has not been verified. In general, few studies utilizing complete human ear geometries have exceeded this time point, 3,6,10,12,23,26 and none of these featured a collagen scaffold. Additionally, the biochemical composition of these constructs must be characterized.…”

Bone Graft Prefabrication Following the In Vivo Bioreactor Principle

“…Interestingly, this term seems to have “off-label” uses in the literature. For instance, by implantation of an avascular construct, such as a three-dimensional (3D) auricular cartilage construct (von Bomhard et al, 2013) or a tracheal allograft (Vranckx et al, 2014), into a vascular territory for a period, the avascular construct would be revascularized and could be transferred as a composite flap for tissue defect reconstruction (Fig. 3B).…”

“…This method was first described by Erol and Spira, who successfully constructed a prefabricated skin graft in rats using an AVL between femoral vessels (Erol and Spira, 1980). Employing the AVL strategy, formation of a significant neovascular network and new tissue, such as cartilage tissue (von Bomhard et al, 2013), cardiac tissue (Morritt et al, 2007), and muscle tissue (Bach et al, 2006), originating from the arteriovenous fistula have been described.…”

Engineering of scaffold‐free tri‐layered auricular tissues for external ear reconstruction

“…The auricular perichondrium contains cartilage progenitor cells necessary for neocartilage formation and promotes the growth of fibrous cells over damaged tissue in cartilage wound healing . It is important for the implantation of autologous or engineered cartilage because the rapid vascular invasion of the perichondrium is essential for supplying oxygen and nutrients to the avascular cartilage layer . It is also necessary for the integration of implants to the native tissue and acts to protect implants from immune response and resorption .…”