|dc.description.abstract||Periodontitis is a highly prevalent chronic inflammatory disease affecting more than 60% of the population, which leads to destruction of the tooth-supporting tissues. The periodontium is composed of both hard (bone and cementum) and soft tissues (periodontal ligament and gingiva), requiring a tissue-engineering approach to allow a precisely coordinated and compartmentalised healing response for subsequent structural and functional regeneration.
The present study investigated the functionalisation of highly porous scaffolds with decellularised cell-laid extracellular matrix for periodontal regeneration. This novel technique allowed the combination of a three-dimensional scaffold providing mechanical support with a native ECM providing tissue specific biological activity. The decellularisation of such constructs allows the maintenance of an intact ECM structure and composition while removing the immunogenic cellular component, thus generating an acellular implant. By combining a bone-like ECM-decorated scaffold (bone compartment) with periodontal ligament cell-sheets (PDLcs), the aim was to achieve specific bone and periodontal ligament regeneration.
The first part of this study (Chapter 2) focused on the optimisation of cell seeding on highly porous scaffolds. Indeed, cell seeding on such structures is challenging, resulting in both poor and heterogeneous cellular attachment, impeding in vitro characterisation of the constructs and hence their clinical translation. Several parameters affecting the quality of cell seeding were investigated, and we successfully identified pre-incubation of the scaffolds in FBS as a reproducible and repeatable protocol, which significantly improved cell seeding efficiency and subsequent scaffold maturation.
The second part of the study (Chapter 3) investigated the effect of culture time on ECM deposition and its composition. To this end, human osteoblasts were seeded on 250 μm pore size polycaprolactone melt electrowritten scaffolds and cultured in osteogenic medium for 1, 2 or 4 weeks, allowing cell proliferation, differentiation and ECM deposition. The constructs were subsequently decellularised, using an in-house optimised protocol for PDLcs decellularisation. Cellularised and decellularised constructs were then extensively characterised in vitro to assess cellular and extracellular composition. The decellularised constructs were recellularised with osteoblasts to study their biological activity in vitro. In vivo performance of the different groups for bone regeneration was assessed in vivo in a rodent calvarial defect model. The various culture periods demonstrated a significant difference in ECM morphology and quantity between 1, 2 and 4 weeks. At the early time points, the fibres were decorated with collagen which mineralised over time and gradually obstructed the pores of the PCL scaffold. Although longer culture times resulted in higher osteogenic activity of reseeded cells, the more mature matrix impeded in vivo bone regeneration.
Scaffold porosity is crucial for host cell colonisation and vascularisation, which are indispensable for tissue regeneration. The decoration of the 250 μm pore size construct in the previous study altered its porosity and subsequent regeneration. In the third study (Chapter 4) scaffolds with different pore sizes (250, 500 and 750 μm) were cultured for 1, 2 and 4 weeks. The scaffolds with 750 μm pore sizes did not exhibit appropriate mechanical properties and were not further characterised. 250 and 500 μm scaffolds cultured for 1, 2 and 4 weeks were decellularised, characterised and recellularised with osteoblasts or macrophages. All decellularised constructs were implanted in a rodent calvarial defect and evaluated for bone regeneration. Although 500 μm pores enabled maintenance of the porosity even after 4 weeks of in vitro maturation, both pore sizes performed similarly in vivo. Again, shorter in vitro maturation was more beneficial for bone regeneration and more mature ECM impaired bone regeneration as observed 6 weeks post-implantation.
In the last part of this study (Chapter 5), the best performing bone compartment (250 μm pore scaffold maturated for 1 week) was combined with a PDLcs prior to decellularisation, in order to fabricate a biphasic scaffold for periodontal regeneration. Cell removal and ECM preservation were confirmed in vitro before implanting in a periodontal defect. Freshly decellularised constructs were compared before and after freeze drying and long-term storage. Freeze drying allows stabilisation of biological components, potentially increasing products stability, shelf life and therefore clinical translation. Although our biphasic construct did not induce bone regeneration in vivo, fresh and freeze dried constructs displayed a higher potential in periodontal regeneration. Both groups displayed enhanced cementum formation and periodontal attachment, and prevented the formation of ankylosis, as opposed to the control groups.
In conclusion, the ECM-decorated melt electrowritten scaffolds were shown to support bone and periodontal regeneration. Optimisation of the cell culture time was shown to be essential for efficient in vivo regeneration. Longer maturation time did not automatically increase scaffold performance, and indeed the more mature matrix appeared to inhibit in vivo bone regeneration. The combination of our optimised bone compartment with a mature periodontal ligament cell-sheet before decellularisation successfully generated a construct capable of promoting compartmentalised periodontal regeneration.||