Development of a porous, biphasic, resorbable osteochondral scaffold for the functional regeneration of osteochondral lesions

Pitrolino, Katherine (2020) Development of a porous, biphasic, resorbable osteochondral scaffold for the functional regeneration of osteochondral lesions. PhD thesis, University of Nottingham.

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Tissue engineering and regenerative medicine research investigate the ability to repair and restore function in the body using biological cells, signals or biomimetic scaffolds. In particular, bone and cartilage tissue engineering has led to the development of many products reaching clinical trials and some obtaining regulatory approval as a class III medical device. Unfortunately, despite a large effort by researchers and industry, there is a lack of cost effective and scalable treatments to regenerate and restore function in an osteochondral defect.

This PhD utilises regenerative medicine tools and techniques to develop a biphasic scaffold that can be placed in the body to locally regenerate cartilage and bone tissue which has previously been damaged by trauma or disease. The scaffold is manufactured from a biomaterial, chitosan, which is derived from chitin, a long chain polysaccharide, found in crustacean shells and the inner membrane of the cell wall in fungi. Chitosan provides a more cost effective and abundant choice of biomaterial than collagen, which is more traditionally used for this type of device.

Key scaffold properties have been assessed and characterised in order to comply with the requirements of a class 3 medical device suitable for tissue repair. The manufacturing process has been optimised to produce a bioactive, resorbable chitosan scaffold that supports the body’s natural repair processes. The removal of excess solvent waste during a freeze gelation stage has resulted in a freeze drying process that is more sustainable and environmentally friendly. A bilayer structure provides a cue for regeneration by mimicking the architecture of articular cartilage and cancellous bone and unlike other osteochondral bilayer scaffolds, the layers are integral to the scaffold’s structure resulting in a strong interface between the two regions. A novel porogen made of polycaprolactone microspheres has allowed a well defined pore size and porosity to be achieved in each layer.

The development process includes an evaluation of the type and concentration of crosslinker required to ensure the scaffold is biocompatible. Genipin was used as a crosslinker as it enhances cell attachment and proliferation when compared to glutaraldehyde; which was shown to be cytotoxic to mesenchymal stem cells. The structure and strength of the scaffold was enhanced by nano-hydroxyapatite rods which also increased the biocompatibility of the scaffold.

The degradation properties of the scaffold have been assessed to ensure the product is not only safe within the body but it has a programmed time for breakdown. Identification and quantification of the degradation products has allowed a unique insight into the causes surrounding adequate cell integration and proliferation. Glucosamine is known to be the major product of chitosan degradation, however, this work has uniquely determined that the cytotoxicity of a chitosan scaffold can be influenced by the rate of degradation.

It was found that high concentrations of glucosamine exert a cytotoxic effect on the surrounding cells that could be problematic when the scaffold is tested in vivo. The conditions in the body during degradation have been assessed using a novel combination of enzymes such as lysozyme and N-acetyl-glucosaminidase (NAG) which have shown that although lysozyme is the major cause of structural breakdown, NAG has a role in the later stages of degradation.

The scaffold has been tested in vitro using mesenchymal stem cells and the specific differentiation of these cells towards the osteogenic and chondrogenic lineages has been tested on each phase of the scaffold. A perfusion bioreactor system has been used with differentiation protocols to mimic the flow conditions seen in vivo.

This work has demonstrated that chitosan has the ability to provide a cost effective and more abundant source of biomaterial for an osteochondral scaffold than collagen.

The strength and structure of the scaffold can be improved using a porogen of PCL microspheres and adding nHA rods into the bone phase, while the type of cross-linker can be selected to improve biocompatibility. The work also shows how the scaffold degradation rate determines long term cytocompatibility, thus providing a promising solution for a medical device to repair and regenerate osteochondral tissue.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Sottile, Virginie
Scotchford, Colin
Grant, David
Keywords: Regenerative medicine; Biphasic scaffold; Bone regeneration; Cartilage regeneration; Chitosan; Biocompatibility
Subjects: W Medicine and related subjects (NLM Classification) > WE Muscoskeletal system
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 63302
Depositing User: Pitrolino, Katherine
Date Deposited: 03 Nov 2023 08:46
Last Modified: 03 Nov 2023 08:46

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