Development of Mineralised Gene-activated Matrices for Osteochondral Tissue Regeneration

Abstract

Full thickness osteochondral defects cannot naturally heal if critically sized due to the acellular, avascular, and aneural tissue structures of articular cartilage and subchondral bone. Despite a range of autograft and allograft implantations available to medical professionals to replace damaged tissue, many rely on the availability of donor tissue and – in the case of allografts - require deceullarisation prior to implantation. As an alternative to using donor tissue, gene-activated matrices (GAMs) are synthetic, acellular scaffolds that combine mimicry of the native extracellular matrix (ECM) of the osteochondral - via a multi-layered or gradient structure - with embedded genetic payloads to help spatially control stem cell differentiation and ECM deposition in-vivo.

Our group has recently improved upon an existing method to synthesise particles carrying genetic payloads within the nanoconfining matrix of agarose gels. This method, termed: ‘particle synthesis via agarose gel electrophoresis’ (PS-AGE), utilises modified agarose gel apparatus to synthesise calcium phosphate (CaP) particles in small layers < 2mm thick. In the literature, PS-AGE has previously been utilised to encapsulate proteins within CaP layers but has not yet been exploited for GAM fabrication due to the non-biomimetic geometry of CaP layers and overall gel that are inherently non-uniform in PS-AGE. Furthermore, agarose lacks any cell attachment motifs that are necessary for use as an osteochondral GAM material. The use of PS-AGE for osteochondral GAM fabrication is also hampered by a lack of exploration in the relationship between CaP particle size and biomolecular loadings (such as pDNA) or even basic experimental parameters (such as salt and gel concentration, or voltage). Despite these shortcomings in the literature, it is theorised that – with several modifications - PS-AGE could be used to fabricate GAMs suitable for a critically-sized pre-clinical osteochondral model.

The research in this work first investigates the functionalisation of agarose gels with polydopamine to establish cell attachment motifs across the gel surface. Particle characterisation of cryogel groups found that separate additions of pDNA loadings and a polydopamine coating reduced the primary modal diameter of CaP particles compared to control groups. Further, the combination of a pDNA loading and a polydopamine coating were symbiotic in their reduction of primary modal diameters, which was theorised to result from multimodal nucleation pathways of pDNA-CaP particles. However, no conclusive indication of enhanced cell attachment – or transfection – were provided by cytotoxicity and transfection assays respectively.

An ‘ideal’ subset of experimental conditions were obtained by systematically investigating the influence of individual experimental parameters (salt concentration and type, agarose concentration and type, temperature, and applied potential) on particle diameters. These ideal conditions were utilised in a modified PS-AGE method termed: particle synthesis via cylindrical agarose gel electrophoresis (PS-CAGE), with the aim of synthesising pDNA/CaP and pDNA-loaded magnesium phosphate (pDNA/MgP) particles with diameters ≤ 100 nm. This culminated in the fabrication of PDA-coated, cylindrical monolayer GAMs encapsulating cylindrically shaped and uniform pDNA/CaP or pDNA/MgP layers. In the case of pDNA/CaP GAMs, particle layers were greater than 2 mm in thickness and reflected the overall geometry required for an osteogenic layer in a critically-sized osteochondral defect rabbit model.

GAMs incubated with C2C12 cells showed evidence of transfection in-vitro via fluorescence microscopy due to green fluorescent protein (GFP) production. However, the high agarose concentration of GAM groups – required to reduce particle diameters – is thought to have significantly inhibited cellular infiltration and hence limit cell exposure to pDNA/CaP and pDNA/MgP particles. The use of dynamic cell seeding, as opposed to static, is expected to improve cellular infiltration into these cryogels and potentially enhance luciferase enzyme escape from the cryogel into the surrounding cell media, thus improving transfection assay sensitivity.

Overall, the PS-CAGE system was able to synthesise ~100 nm diameter pDNA/CaP particles as a macroscopically thick mineralised layer embedded within a PDA-coated cryogel. With further optimisation of the PS-CAGE system to produce bi-layers of genetic payloads, and with further development of in-vitro testing, there is great potential for the PS-CAGE to produce complete, multi-layered or gradient osteochondral GAMs. One could also pursue the synthesis of pDNA/MgP particles as osteogenic layers if layer thickness could be significantly increased.

Metadata

Supervisors:	Thomson, Neil Henderson and Fermor, Hazel Louise and Al-Jawad, Maisoon
Keywords:	tissue engineering; osteochondral; gene therapy; calcium phosphate nanoparticles; magnesium phosphate nanoparticles; non-viral vectors; agarose gel electrophoresis; pDNA; polydopamine; crystallisation in confinement; PS-CAGE; PS-AGE; template synthesis;
Awarding institution:	University of Leeds
Academic Units:	The University of Leeds > Faculty of Engineering (Leeds) > School of Mechanical Engineering (Leeds) > Institute of Medical and Biological Engineering (iMBE)(Leeds)
Date Deposited:	17 Jul 2025 09:32
Last Modified:	01 Dec 2025 01:06
Open Archives Initiative ID (OAI ID):	oai:etheses.whiterose.ac.uk:35598

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Development of Mineralised Gene-activated Matrices for Osteochondral Tissue Regeneration

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