Articular cartilage is a highly specialized, hierarchically organized tissue covering the articular surfaces of diarthrodial joints that absorbs and distributes forces upon mechanical loading and enables low-friction movement between opposing bone ends. Despite a strong resilience towards mechanical stress, once damaged cartilage is generally not regenerated due to a limited repair potential of the residing cells (chondrocytes) and the local absence of vascularized blood vessels and nerves. Eventually, this may lead to osteoarthritis, a chronic degenerative disorder of the synovial joints which has a strongly growing prevalence worldwide. Modern regenerative therapies that aim to rebuild cartilage tissue in vivo and in vitro using chondrocyte- and stem cell-based methods are still not able to produce tissue constructs with desired biomechanical properties and organization for long-term repair. Therefore, cartilage tissue engineering seeks for new ways to solve these problems. In this regard, the application of hydrogel-based scaffolding materials as artificial matrix environments to support the chondrogenesis of embedded cells and the implementation of appropriate biofabrication techniques that help to reconstitute the zonal structure of articular cartilage are considered as promising strategies for sophisticated cartilage regeneration approaches.
In this thesis, a modular starPEG-heparin hydrogel platform as cell-instructive hydrogel scaffold was used in combination with a custom-designed 3D inkjet bioprinting method with the intention to develop a printable 3D in vitro culture system that promotes the chondrogenic differentiation of human mesenchymal stromal cells (hMSC) in printed cell-laden hydrogels with layered architectures in order to fabricate cartilage-like tissue constructs with hierarchical organization. Firstly, the successful bioprinting of horizontally and vertically structured, cell-free and -laden hydrogel scaffolds that exhibit layer thicknesses in the range of the superficial zone, the thinnest
articular cartilage layer is demonstrated. The long-term integrity of the printed constructs and the cellular functionality of the plotted cells that generally had a high viability after the printing process are shown by a successful PDGF-BB-mediated hMSC migration assay in a printed multilayered hydrogel construct over a culture period of 4 weeks. Secondly, when the established printing procedures were applied for the chondrogenic differentiation of hMSCs, it was found that the printed cell-laden constructs showed a limited potential for in vitro chondrogenesis as indicated by a weaker immunostaining for cartilage-specific markers compared to casted hydrogel controls. In order to increase the post-printing cell density to tackle the limited printable cell concentration which was regarded as the primary reason for the impaired performance of the printed scaffolds, different conditions with varying culture medium and hydrogel compositions were tested to stimulate 3D cell proliferation. However, a significant 3D cell number increase could not be achieved which ultimately resulted in shifting the further
focus to casted hMSC-laden starPEG-heparin hydrogels. Thirdly, the chondrogenic differentiation of hMSCs in casted hydrogels proved to be successful which was indicated by a uniform deposition of cartilage-specific ECM molecules comparable with the outcomes of scaffold-free MSC micromass cultures used as reference system. However, the quantitative analysis of biochemical and physical properties of the engineered hydrogel constructs yielded still significant lower values in relation to native articular cartilage tissue.
Fourthly, in order to improve these properties and to enhance the chondrogenesis in starPEGheparin hydrogels, a dualistic strategy was followed. In the first part, specific externally supplied stimulatory cues including a triple growth factor supply strategy and macromolecular crowding were applied. As second part, intrinsic properties of the modular hydrogel system such as the crosslinking degree, the enzymatic degradability and the heparin content were systematically and independently altered. It was found that while the external cues showed no supportive benefits for the chondrogenic differentiation, the reduction of the heparin content in the hydrogel proved to be a key trigger that resulted in a significantly increased cartilage-like ECM deposition and gel stiffness of engineered constructs with low and no heparin content. In conclusion, this work yielded important experiences with regards to the application of inkjet bioprinting for hMSC-based cartilage tissue engineering approaches. Furthermore, the obtained data provided valuable insights into the interaction of MSCs and a surrounding hydrogel-based microenvironment that can be used for the further development of chondrosupportive scaffolding materials which may facilitate the fabrication of cartilage-like tissue constructs.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:36519 |
| Date | 12 December 2019 |
| Creators | Schrön, Felix |
| Contributors | Werner, Carsten, Zenobi-Wong, Marcy, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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