Fetalix in vivo validation and process scalability

Abstract

Low back pain (LBP) is a musculoskeletal condition that affects over 80% of the population. It is mainly caused by degeneration of the intervertebral disc (IVD), a core element for spinal mobility. As current therapies do not provide long-term solutions, there is an urgent need to develop novel treatments to restore a functional tissue. Our group has previously showed that fetal bovine nucleus pulposus (NP), the central part of the IVD, is enriched with collagen XII and XIV, two pro-regenerative proteins that disappear with age. Moreover, fetal decellularized NPs co-cultured with bovine NP cells, expressed higher levels of aggrecan and collagen II and reduced neovessel formation. From this work resulted Fetalix – the first fetal-inspired biomaterial to treat LBP. Thus, the goal of this thesis was to assess Fetalix potential in vivo through magnetic resonance imaging (MRI), histology and behavioural assessment, while optimizing its scalable production and injectability. Modified decellularization protocols enabled 6-8% time reduction and enabled an increase in processing capacity. However, further optimization is required to achieve a double strand DNA reduction of <50ng per mg of extracellular matrix dry weight. As expected, glycosaminoglycans content was reduced. Regarding biomaterial’s production and particle characterization, the 4-Place Mini Bead Mill homogeniser enabled the production of smaller and more porous particles, being selected for Fetalix production. 50 milling cycles were required to achieve an injectable solution. An injectability assay revealed peak and average forces around 200g, indicating easily injectable solutions. The in vivo study revealed that MRI (obtained through a developed algorithm) and histology results were consistent, expect hernias volume quantification. Fetalix administration induced decrease in IVD height, volume and brightness. Moreover, 2 out of 3 animals presented higher hernia volumes and degeneration scores, except one that did not have hernias. Von Frey test revealed that Fetalix group presented sensitivity similar to naïve and decreased compared to lesioned animals, suggesting that these animals were able to restore their sensitivity. Overall, these results showed that it was possible to produce Fetalix as an injectable solution. The in vivo analysis demonstrated that, despite not regenerating the IVD, biomaterial’s administration enabled sensitivity restoration to naïve levels.

Date of Award	17 Oct 2024
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Joana Caldeira Fernandes Frey Ramos (Supervisor), Raquel Gonçalves (Co-Supervisor) & Morena Fiordalisi (Co-Supervisor)