Biomaterials are no longer considered innate structures and using functionalisation and biofabrication strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding of the host response will enable us to design appropriate strategies. The input from the host response needs to be weighed in depending on the host disease condition. Our current inputs have been through a thorough understanding of glyco-proteomics based tools which we are developing in our laboratory. In addition, biomaterials themselves provide immense therapeutic benefits which needs to be accounted in the design paradigm. Using functionalisation strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. The programmed assembly of biomolecules into higher-order self-organized systems is central to innumerable biological processes and development of the next generation of scaffolds. Recent design efforts have utilized a glycobiology and developmental biology approach toward both understanding and engineering supramolecular protein and sugar assemblies.

Discogenic low back pain affects 42% of patients suffering low back pain. Degenerative disc disease is described as failure in cellular response to external stresses leading to physiologic dysfunction. Glycosylation patterns of tissues give insights into the spatially and temporally regulated inflammatory and degenerative processes. These glycoconjugates participate in many key biological processes including molecular trafficking and clearance, receptor activation, signal transduction, and immunomodulation. We hypothesise that glycoprofile of the the intervertebral disc(IVD) is temporally and spatially distinct in health and degeneration. The glycoprofile of the IVD has been studied in murine, bovine and ovine models for injury and aging. In this study, healthy(n=2) and degenerated(n=2) human IVD samples received from Utrecht(UU, ND) with ethical approval(NUIG), were compared using lectin histochemistry. The N-glycan profile of human degenerated IVD samples was characterised by high resolution quantitative UPLC-MS. Healthy and degenerated human discs present distinct glycosylation trends intracellularly/extracellularly in annulus fibrosus(AF) and nucleus pulposus(NP) tissue. There are quantitative and spatial differences in glycosylation in healthy and degenerated tissue. These findings are consistent with previous studies of IVD in murine, bovine and ovine models. The human N-glycan profile of degenerated surgical tissues is distinct from other cited tissue profiles such as human plasma⁵. These studies offer validation of previous animal models of IVD injury and degeneration, demonstrating similar changes in the glycoprofile in both animals and humans. Glycoprofiling may offer insight into disease progression, offering new realms of disease classification in patient specific manner while also elucidating potentials therapeutic targets, inhibiting disease progression.

Electromechanical coupling (piezoelectricity) is present in all living beings and provides basis for sense, thoughts and mechanisms of tissue regeneration. Herein, we ventured to assess the influence of MMC in mesenchymal stem cell culture. In this study, we fabricated piezoelectric regenerative scaffolds to assess the role of electromechamical stimulation on tendon regeneration. Tendon cells were selectively stimulated in vitro by mechanical or electromechanical cues using non-piezoelectric or piezoelectric scaffolds and optimal mechanical loading (4% deformation at 0.5 Hz). This was followed up with an in vivo study to assess tendon regeneration in a rat Achilles tendon injury model. P(VDF-TrFE), scaffolds were observed to mimic the fibrous structure of tendon tissue (figure 1) and were capable of producing electrical charges up to 17 pC/N when mechanically loaded (figure 1. Genes associated with tendon specific markers (Col.I/Col III, Scx and Mkx) and mechanosensitive ion channels such as PIEZO1, TRAAK and TRPV1 were significantly upregulated (figure 2). The upregulated genes were validated with individual real time Q-PCR and bioinformatics revealed a possible regulated function. Those results were further validated in vivo. Protein expression of repaired tendons showed a correlation between increase in expression of tendon related proteins SCX, TNMD, Decorin and expression of ion channels KCNK2, TRAAK and TRPV1. Collectively, these data clearly illustrate that scaffolds made of PVDF-TrFE can produce electrical charges when mechanically loaded. Moreover, gene and protein analyses showed a positive regulation of tendon specific markers through activation mechanosensitive voltage-gated genes.

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The degeneration of the intervertebral disc (IVD) is the primary cause for low back pain, which is treated with surgical interventions such as spinal fusion. A strategy to develop a regenerative and non-invasive treatment requires an injectable cell carrier system. Our efforts have focussed on developing a hyaluronan (HA)-based hydrogel system that can be used as a carrier for therapeutic agents in annulus fibrosus (AF) repair. High molecular weight HA at 20mM is chemically crosslinked with varying concentrations of 4-arm PEG. Hydrogels were optimised for degree of crosslinking, stability and rheological properties. Subsequently, the morphology and viability of the human AF cells encapsulated in the hydrogels were studied. The highest crosslinking was seen with 4-arm PEG at 1:1 HA:PEG molar ratio. This was the most stable against enzymatic and hydrolytic degradation, and had greater swelling property, which is desired as the degeneration decreases the water retention capability of the IVD. The gelation time, important for in situ injectability, was under five minutes for all formulations. Storage modulus was between 0.4–1.1 kPa. Compared to 2D cultures, cells were rounder after encapsulation, mimicking the native microenvironment, and had the similar metabolic activity for seven days. AF cells encapsulated in HA/4-arm PEG hydrogel were stiffer compared to the nucleus pulposus (NP) cells encapsulated similarly as measured with Brillouin microscopy. The 4-arm PEG crosslinked HA-based hydrogel system promises to be a candidate for an injectable carrier for cells for AF repair and regeneration.

Biomaterials are no longer considered innate structures and using functionalisation strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding the host response will enable us to design proper functionalisation strategies. The input from the host response need to be weighed in depending on the host disease condition. In addition, biomaterials themselves provide immense therapeutic benefits which needs to be accounted for when using functionalisation strategies. Using strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. Our recent design efforts have harnessed the therapeutic effects of biomaterials and mapped the molecular fingerprint of this specific host response in a disease target. This approach allows us to rethink functionalisation strategies currently employed in the field. This talk will elucidate some of these ongoing strategies that have applications in the development of the next generation of orthopaedics devices.

Cell-based therapies require removal of cells from their optimal in vivotissue context and propagation in vitroto attain suitable number. However, bereft of their optimal tissue niche, cells lose their phenotype and with it their function and therapeutic potential. Biophysical signals, such as surface topography and substrate stiffness, and biochemical signals, such as collagen I, have been shown to maintain permanently differentiated cell phenotype and to precisely regulate stem cell lineage commitment (1, 2). Herein, we developed and characterised substrates of variable rigidity and constant nanotopographical features to offer control over cellular functions during ex vivoexpansion.

PDMS substrates with varying ratios of monomer to curing agent (0:1, 1:1, 5:1) were fabricated based on established protocols. Grooved substrates were created using a silinated wafer with groove dimensions of 2µm × 2µm × 2µm; planar control groups were created using flat glass. The aforementioned PDMS solutions were poured onto the wafer/glass, cured at 200 ºC and treated with oxygen plasma. Substrates were then investigated with/without collagen I coating. (0.1, 0.5, and 1 mg/ml). Atomic force microscopy (AFM) and optical profilometry were used to assess the topographical features of the substrates. Dynamic mechanical analysis (DMA) was used to determine the mechanical properties of the substrates. The simultaneous effect of surface topography / substrate rigidity on cell phenotype and function was assessed using human permanently differentiated cells (dermal fibroblasts, tenocytes) and stem cells (human bone marrow stem cells) and various morphometric and gene / protein assays.

PDMS substrates of varying stiffness (1000 kPa, 130 kPa, 50 kPa) can be made by varying the Sylgard ratio, while maintaining topographical features. Human adult dermal fibroblasts, tenocytes, and tenocytes attach, align, elongate and deposit aligned extracellular matrix on the grooved PDMS substrate surface of all 3 stiffnesses.

Preliminary in vitrodata indicate that surface topography and substrate stiffness play crucial role in maintaining cell phenotype and the prevention of phenotypic drift in vitro.

Cell-based tissue engineering strategies for tendon repair have limited clinical applicability due to delayed extracellular matrix (ECM) deposition and subsequent prolonged culture periods, which lead to tenogenic phenotypic drift. Deposition of ECM in vitrocan be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the intra- and extra-cellular milieu of multicellular organisms², which has been described to accelerate ECM deposition in human tenocytes¹. A variety of cell sources have been studied for tendon repair including tenocytes, dermal fibroblasts and mesenchymal stem cells (MSCs)³and various biophysical, biochemical and biological tools have been used to mimic tendon microenvironment and induce phenotype maintenance in long term cultures or differentiation⁴. Therefore, we propose to assess the combined effect of macromolecular crowding and mechanical loading on different cell sources to determine their suitability for the in vitro fabrication of tendon-like tissue.

Human dermal fibroblasts, tenocytes and bone marrow mesenchymal stem cells were cultured for 3 days with 100 µg/ml of carrageenan (MMC) under static and dynamic culture conditions. Cyclic uniaxial strain was applied using a MechanoCulture FX (CellScale) at 1 Hz and 10% strain for 12 hours a day. Cell morphology and alignment were evaluated by fluorescein isothiocyanate (FITC) labelled phalloidin and 4’,6-diamidino-2-phenylindole (DAPI) staining. Extracellular matrix composition was evaluated by immunocytochemistry. Cell phenotype maintenance/differentiation (tenogenic, chondrogenic and osteogenic lineages) were assessed by gene and protein analysis.

After 12 hours of exposure to the uniaxial load, permanently differentiated cells are strictly aligned in the direction perpendicular to the load while the MSCs do not show preferential alignment. ECM deposition (e.g. collagens type I, III, V, VI) is increased in the presence of MMC and this effect is maintained under mechanical loading. ECM deposited under mechanical loading is also aligned in the direction perpendicular to the load. Tenogenic, osteogenic and chondrogenic markers are being tested to assess cell phenotype.

Mechanical loading and macromolecular crowding can induce cell and ECM alignment and increased ECM deposition without affecting cell metabolic activity or viability. Cell and ECM alignment alongside ECM composition and tenogenic marker expression suggest this approach might be suitable to maintain or differentiate towards tenogenic lineage.