Highlights

A key challenge for the successful use of in vitro engineered tissues is long term stability. The ArtiVasc 3D project has broken new ground in manufacturing technologies for the generation and culture of perfusable bioartificial tissue.

In a multidisciplinary approach, experts in biomaterial development, simulation, freeform fabrication, automation and tissue engineering have developed methods to produce versatile perfusable scaffolds. We apply and combine biomolecules from the native extracellular matrix (ECM) and synthetic photocurable polymer materials with micro-scale laser-based polymerization, electro-spinning and dipcoating techniques to fabricate tubular structures which can be mounted in perfusion-bioreactors. The inner surface of such blood vessel equivalents can be coated with biomolecules to facilitate cell adhesion and an endothelial cell monolayer can be installed. For the generation of surrounding tissue, automated dispensing is used to deposit cell-matrix bioinks into the bioreactor. Crosslinking of chemically modified ECM bio-macromolecules, e.g. gelatin and hyaluronic acid, result in stable, non-shrinking tissue models.

In this study we tested electrospun poly(L-lactide-co-glycolide) (P(LLG)) and gelatine hydrogels together with adipose-derived stem cells (ASCs) in a perfusion bioreactor system under medium flow for a new approach in soft tissue engineering.
ASCs could be cultivated on P(LLG) scaffolds under static conditions and in gelatine hydrogels under flow conditions and showed good cell viability as well as the potential to differentiate. These results should be a next step to a physiological three-dimensional construct for soft tissue engineering and regeneration.

In the present study we synthesized two novel biodegradable polymers, poly(ε-caprolactone-co-urethane-co-urea) (PEUU) and poly[(L-lactide-co-ε-caprolactone)-co-(L-lysineethyl ester diisocyanate)-block-oligo(ethylene glycol)-urethane] (PEU), containing different types of hydrolytically cleavable bondings. Electrospun meshes made of these polymers were tested for cell compatibility with adipose-derived stem cells.
We could show that PEUU and PEU meshes show a promising potential as scaffold materials in adipose tissue engineering.

A first set of suitable biocompatible materials used in biofunctionali-sation was made available for the project. Cytotoxicity was assessed using Life/Dead and WST-1 assays. Both appropriate material prop-erties and biocompatibility make these materials promising candi-dates for further investigations in matrix-tissue interactions and the build-up of the ArtiVasc 3D vascularized composite tissue graft.

After the first 24 months of the project more than 22 polymers, copolymers and polymer mixtures suitable for electrospinning were developed.

14 out of 22 polymers were synthesized- the synthesized and purchased polymers were divided in two groups: non-biodegradable and biodegradable materials. In addition, 2 polymer mixtures containing collagen were created and 6 materials were purchased from commercial suppliers.

Laser based polymerisation technologies are responsible for the realisation of the small diameter vessels. In order to achieve this goal, laser based polymerisation processes have to be investigated to fit the needs of the vessels. The requirements can be met with different laser processes. Multiphoton polymerisation and UV induced polymerisation were investigated in order to find the best suited process for the project.

Large tissue constructs are needed as in vitro test systems and for tissue replacement, e. g. after tumor resection. For oxygen and nutrient supply of these tissues, vascular structures need to be integrated since oxygen is used by surrounding cells in a distance of 200 μm. Our strategy to achieve vascularisation of artificial tissues is the endothelial lining of small synthetic tubes with 2 mm inner diameter and subsequent culture in a fluid flow bioreactor.

Within the project, an in vitro fatty tissue will be generated using electro-spun scaffolds combined with hydrogel components, seeded with adipocytes and adipose derived stem cells (ASCs).

A CAD solid model (Figure 1) of a vascular system was generated using an automatic design tool developed by us. The solid CAD model was translated into an STL file for printing. Figure 2 shows an alternative design. This design uses a cubic wall to reduce the mechanical demands on thin walls during 3D manufacturing. The channels inside the cubic wall are the same as in figure 1. Samples from latter design are being used to identify the validity of blood vessel optimization with reduced outside wall accuracy.

Electrospinning is a process of forming fibers with diameters in the submicrometer range. Fiber fabrication occurs in a high-voltage electrical field using polymer solutions or melts.

A method to analyze diffusion properties of tracer molecules within a hydrogel scaffold was established. The results will help to develop optimized hydrogels.

The original “sandwich structure” concept for vessel systems includes such unnatural features as straight and long, completely parallel capillaries.