Rotator cuff injuries affect a large portion of the elderly population and disproportionally over-impact high performance athletes. Currently available surgical therapies often fall short of achieving long-term repair and healing, with re-tear rates estimated to occur in over half of all patients. One approach to tendon repair involves the application of mechanically sound biomaterials, capable of supporting cell attachment and proliferation, to address this clinical need. This work develops an innovative aligned porous scaffold produced entirely from natural polymers, suitable for applications in rotator cuff therapies. The developed material meets a number of design criteria: it is not only biocompatible and biodegradable, but it degrades slowly, at a rate that matches the formation of new tissue. Furthermore, it is key that the material enables cell attachment and proliferation, while promoting the tenogenic differentiation of cells seeded on its surface. Tendons are tissues with low vascularity and cellularity, characterized by their slow rate of self-healing and highly organized collagenous structure, where collagen assembles itself into aligned fibrils giving the tissue its characteristic high strength and stiffness. Matching the alignment of the tissue in the developed biomaterials is one of the design goals of this work. The scaffolds are produced through a unidirectional freezing technique, also known as ice segregation induced self-assembly, where a solute slurry is cast onto an insulated mould placed on top of a frozen surface, creating a temperature gradient between the bottom and top of the mould, facilitating the formation of aligned ice lamellae. The ice is rapidly removed through lyophilization, yielding a foam-like material, with a low density and aligned pore structure, mimicking the alignment present in the native tendon extracellular matrix. The polymeric composition of the scaffold is one of the focus of the first experimental chapter in this dissertation, as the development process was started from the basis of a gelatine-chitosan blend, which is a well-known, biocompatible composition for tissue engineering applications. This blend was found to yield large pores with low interconnectivity, which in turn results in a very fragile material. To further improve the material, we investigated the incorporation of microfibrillated cellulose (CMF) into the polymeric blend and found that even very low concentrations of CMF have a significant effect on the materials pore structure. Gradual increases in the CMF concentration yield reducing pore sizes and increasing mechanical properties, making the gelatine-chitosan-cellulose composition the better candidate for our intended application. A composition of 10% gelatine – 2% chitosan - 20% CMF was found to be optimal for both mechanical properties and cell attachment. Furthermore, we investigated the crosslinking conditions of the used biopolymers, as crosslinking is a vital step in the processing of natural polymer-based materials. For the design of our scaffold, obtaining a slow and gradual degradation was key to complement the slow regeneration of tendons. With this goal in mind, we evaluated two different chemical crosslinking alternatives: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and genipin (GP). While both compounds could effectively crosslink the scaffold, EDC yielded much lower degrees of crosslinking and resulted in the total degradation of the scaffold in under 21 days, whereas GP resulted in a material with about 20% of mass loss after 3 months. Moreover, adding water to the ethanol crosslinking solution was found to increase the degree of crosslinking and reduce the toxicity of the material, resulting in an optimal crosslinking with 0,25% w/v GP in 80% ethanol, for 24 hours at 37°C. Degradation studies showed that the functional groups present in the scaffold underwent no significant changes after 56 days of immersion in phosphate buffered saline (PBS), and the pore structure of the scaffold remained similarly unchanged. Mechanical testing resulted in an ultimate tensile strength of 3,9 MPa ± 1,4 MPa and 7,6% ± 0,9% strain at break when tested dry. The scaffold promoted cell attachment and proliferation for different cell types: human bone marrow mesenchymal stem cells (MSCs), porcine tendon progenitor cells (TDSCs), porcine tenocytes (TNCs) and human dermal normal fibroblasts, while exhibiting no signs of cytotoxicity. Both the TDSCs and the MSCs cultured on the surface of the scaffold retain their multi-lineage differentiation capacity, into bone, fat and cartilage lineages. Moreover, TNCs, TDSCs and MSCs produced tenomodulin, one of the most important extracellular matrix proteins in tendon tissue. When studied in a dynamic environment by inducing cyclic tensile strain on the scaffolds, increases in the gene expression of tenogenic gene markers, nuclear aspect ratio, cell alignment in the direction of strain and proliferation rate were found, indicating that the scaffold can effectively induce tenogenic differentiation of the cells. In summary, in this work we develop an aligned polymeric scaffold with appropriate mechanical properties and slow degradation rate, that enables cell attachment and tenogenic differentiation, suitable for tendon tissue engineering applications. Further developments and suggestions for required future evaluations of the scaffolds to achieve potential clinical translation are presented.
Florencia Diaz (Wed,) studied this question.