Afgerond in 2019

Projectleider: Paul P.M. van Zuijlen – Rode Kruis Ziekenhuis

Beknopte samenvatting van de resultaten:

Tissue engineering demands a multidisciplinary approach. We need to understand all facets of structure, form and function. In chapter 2 we performed an extensive literature review of the challenges and limitations involved in the surgical reconstruction of burned ears. We further investigated the current state of cartilage tissue engineering strategies and the hurdles of tissue regeneration and 3D (bio)printing. As every burn victim poses an unique challenge there is not a single method. Skin coverage poses an important factor in ear regeneration and skin tissue engineering will need to evolve parallel to cartilage regeneration to provide a fundamental solution.

The extracellular matrix forms the scaffolding for the chondrocytes and is therefore essential to the form and function of cartilage. We therefore performed several mechanical studies on the cartilage extracellular matrix. In chapter 3.1 a novel approach to measure cartilage stiffness was investigated. A model was developed to serve as a surrogate for tissue development and used to validate a novel indentation device capable of measuring stiffness on the extracellular matrix level. Goat ears were subjected to different degradation processes removing the matrix components elastin and glycosaminoglycans. Good reproducibility was seen between consecutive measurements and a significant difference was seen between treatment groups.

As such, the method seems feasible for the monitoring of changes in cartilage stiffness on the extracellular matrix level. Based on these findings, in chapter 3.2 we extensively compared the biochemical composition, stiffness and 3D structure of the different facial cartilage types in order to get a better understanding of the nature of the tissue we aim to regenerate. Using nanoindentation, biochemistry and multiple-photon laser scanning microscopy, significant differences were seen not only between ear and nasal cartilage but also between the ala nasi and septal cartilage.

In chapter 4 we designed a novel approach to maintain scaffold form integrity during maturation. This chapter forms the culmination of our efforts, the integration of various scaffold materials with 3D printing and a combination of different cell types in vitro. 3D bioprinting was used to fabricate a framework of biodegradable Polycaprolactone to support cartilage development in a commercially available collagen I-III sponge scaffold (Optimaix). The constructs that were developed form the first step towards a clinically feasible reconstruction method. The materials and technologies involved have been either clinically proven or are in an advanced stage of validation for human use. Three different constructs were tested consisting of a fibrin/hyaluronic acid hydrogel, the hydrogel combined with a collagen I/III scaffold and a combination of the previous surrounded by a 3D-printed polycaprolactone scaffold. The scaffolds were seeded with different combinations of chondrogenic cell types and adipose derived mesenchymal stem cells. (Immuno)histological analysis, multiphoton laser scanning microscopy and biomechanical analysis showed increased extracellular matrix deposition stiffness and complete form integrity in the polycaprolactone scaffold group proving the feasibility of this approach.

After exploring the challenges of 3D printing and combining this technology with different materials we came to the conclusion that broader expertise was necessary to create a feasible solution for the engineering of appropriate ear cartilage. Design forms a field of expertise outside the scope of most researchers and we collaborated extensively with TU Delft to develop new approaches towards personalised ear scaffold shapes. This work is covered in chapter 5 demonstrating a parametric ear model combining personalized ear scaffold design with an adjustable architecture to anticipate potential surgical limitations. Computed tomography (CT) scans of 4 human cadaver ears were used to develop a fully adjustable parametric model based on the essential ear anatomy. The mean directed Haussdorff distance and the mean similarity coefficient (SC) of the model and scan surfaces showed good similarity between the parametric model and the ear cartilage scanning data. This supports the concept that a parametric standard model can be used to generate custom implants based on existing ear images.

The technical limitations of 3D printing which led to the development of the hybrid approach described in chapter 3 formed the basis for a novel technology to overcome certain hurdles regarding speed, dimensions and material choice in 3D (bio)printing. The concept evolved from the observation that it seems we try to print too much. In order to 3D (bio)print a certain material various concessions have to be made in regard of material traits and only select materials are eligible. In chapter 6 we propose a novel scaffold fabrication technology to overcome some of these limitations. The method comprises stacking multiple sheet elements of predefined dimensions on top of each other providing a fast and reproducible scaffold fabrication method. This method allows the integration of a broad range of other components such as hydrogels and cells offering a versatile platform for tissue engineering approaches. The suggested hybrid approach can potentially extend the possibilities of 3D printing technology and facilitate clinical implementation.

Resultaten: Uit de resultaten van dit fundamentele onderzoek komt een voorstel voor een hybride aanpak, een nieuwe technologie van het printen van de steiger in laagjes, welke daarna kunnen worden gecombineerd met biomaterialen zoals hydrogels en cellen. Deze hybride aanpak kan in potentie de toepassing van 3D print technologie uitbreiden en klinische implementatie faciliteren. Echter daarnaast is geconcludeerd dat een beter begrip van cel gedrag nodig is voordat 3D bioprinten volledig tot zijn recht kan komen.