Regenerative Medicine Utrecht

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Vivian Mouser

Thesis:  Bio-inks for 3D Printing of Cartilage Implants

 

PhD Supervisor: Jos Malda, PhD, UMC Utrecht.
Defense date: 15 June 2017.

Articular cartilage defects can cause major pain for a patient and often result in arthritic changes of the whole joint if no interventions are taken. Cartilage is the tissue covering the bone extremities in a joint. Its main functions are to reduce the surface frication and to absorb peak loads. The tissue consists of predominantly collagen type II, proteoglycans, water and chondrocytes (cartilage cells). Furthermore, the matrix composition and orientation is depth-dependent and can be divided into three zones: the superficial, the middle, and the deep zone.

Cover of Vivian’s thesis book.

A promising approach to treat articular cartilage defects is the implantation of stratified cell-laden hydrogel implants that mimic the native tissue. To fabricate such constructs, three-dimensional (3D) bioprinting techniques are promising, as they allow accurate deposition of (cell-laden) biomaterials, the so-called bio-inks, as well as biological cues and reinforcement structures. Several hydrogels have been suggested as bio-inks, including hydrogels based on the natural polymer gelatin-methacryloyl (gelMA) with gellan gum or hydrogels based on synthetic triblock copolymers of polyethylene glycol (PEG) and partially methacrylated poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate (polyHPMA-lac). However, to bioprint successful constructs with a high resolution, the bio-ink properties are crucial. Therefore, the research of Vivian aimed to investigate the application of gelMA and polyHPMA-lac-PEG based hydrogels, as bio-ink platforms for the 3D bioprinting of cell-laden organized cartilage implants. Here, the optimal cartilage bio-ink properties are based on the ability to print the material with high shape-fidelity and the ability of the material to support chondrogenesis.

During her research Vivian discovered that the feasibility of bioprinting and cell encapsulation with gelMA/gellan hydrogels was governed by the yield stress of the polymer mixture. Moreover, the yield stress could be increased with the addition of gellan gum and/or methacrylated hyaluronic acid (HAMA) in gelMA hydrogels and with HAMA in polyHPMA-lac-PEG hydrogels. Especially the incorporation of HAMA forms an interesting strategy to improve the bio-inks as specific concentrations of HAMA improved the chondrogenic potential of embedded chondrocytes. To increase the stiffness of the final constructs towards the range of native cartilage, co-printing of a hydrogel with medical grade poly-ε-caprolactone was found to be sufficient.

For the generation of stratified implants, Vivian investigate the potential of three different cell types for the production of zone-specific cartilage (chondrocytes, articular cartilage progenitor cells, and multipotent mesenchymal stromal cells) and the effect of different cell delivery approaches on tissue repair. The latter was studied in an ex vivo osteochondral plug model. The most suitable cell type to produce superficial zone-like matrix are the articular cartilage progenitor cells. Additionally, the highest amount of cartilage-like tissue was produced by multipotent mesenchymal stromal cells, which are therefore interesting for the fabrication of middle/deep zone cartilage. Moreover, a homogeneous spatial cell distribution within a hydrogel construct was beneficial for defect filling with hyaline-like cartilage, while a dense cell layer at the bottom of a cartilage defect improved construct integration in full thickness cartilage defects. Therefore a combination of both cell delivery strategies may be beneficial for cartilage repair.

Altogether, the work in this thesis resulted in two optimized cartilage bio-inks: gelMA/gellan/HAMA and polyHPMA-lac-PEG/HAMA hydrogels. Although several steps towards the bioprinting of organized cartilage implants have been made, some challenges still need to be overcome, such as finding the optimal combination of factors to stimulate zone-specific cartilage production by embedded cells. However, the results of this thesis encourage further development of organized cartilage implants using gelMA/gellan/HAMA and polyHPMA-lac-PEG/HAMA bio-inks.