3D bioprinting for potential use in nasal cartilage reconstruction

3D printing is an additive manufacturing technique that is rapidly gaining traction in health and medical applications. This technique could potentially benefit plastic and reconstructive surgeries by fabricating patient-specific tissue replacements with tissue-like functions and mechanical properties. One specific example in the field of plastic and constructive surgery is nose reconstruction. Current gold standard for nasal reconstruction after rhinectomy or severe trauma involves a three stage surgery that requires a minimum of three and maximum of seven operations to achieve an acceptable result. The surgical procedure require transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvest of autologous rib cartilage requires a major additional procedure which creates donor site morbidity. Additionally, major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly-skill demanding and time-consuming. These drawbacks of the current approach for nasal reconstruction are some of the reasons why facial plastic and reconstructive surgeons are interested in the application of tissue engineering and 3D printing for reconstructive surgeries. To address these clinical challenges, the aim of the work presented in this thesis was to fabricate a personalised 3D bioprinted composite scaffold for nasal reconstruction mimicking the mechanical properties and architecture of nasal cartilage. The composite consists of biodegradable thermoplastic polycaprolactone (PCL) to provide structural support, and cell-laden thermoresponsive and UV crosslinkable gelatin methacrylate (GelMA) to act as a cell carrier. We first investigated the appropriate cell source to use for cartilage tissue engineering and 3D bioprinting. Primary sheep articular chondrocytes (ShCh) and sheep bone marrow derived Mesenchymal Stem Cells (ShMSCs) were isolated, expanded and differentiated; followed by an assessment of the effects of the 3D printing process on cell viability and functionality. From these studies it was observed that ShCh were easier to isolate and expand than ShMSCs because less steps are required and the doubling time is 50% shorter. Additionally, 80% of the ShCh survived the printing process compared to a 50% of the ShMSCs, suggesting that chondrocytes were able to tolerate higher stress caused by the 3D printing process. PCL and poly (lactic-co-glycolic acid) (PLGA) scaffolds were printed and seeded with chondrocytes post-printing. The printing process and the 3D printed structures of these polymers were characterised before and after printing by measuring their molecular weight, thermal and mechanical properties. It was found that the printing process reduced the molecular weight of PLGA by 50% percent due to thermal degradation. Consequently, its glass transition temperature and young’s modulus decreased post printing. On the contrary, PCL’s molecular weight remain unchanged after printing. Characterisation of the chondrocytes showed that whilst both scaffold materials supported cell attachment the ECM secreted deformed the PLGA whilst the PCL scaffolds were unaffected. Due to superior mechanical properties PCL was selected to 3D print the personalised nose scaffolds. Additional studies on the 3D printed scaffolds showed that controlling the surface pores of scaffolds was important for cell infiltration and proliferation Scaffolds with larger surface pores were 3D printed and these resulted in increased cell seeding and proliferation demonstrated by DNA quantification. Moreover, the printing process of the cell carrier GelMA was optimised by utilising its thermoresponsive properties. A rheological study of three different concentrations of GelMA was performed in order to identify the most suitable for bioprinting. GelMA 15% and 20% at 15 °C and 18 °C respectively were found the appropriate ones. Finally, multi-material 3D bioprinting of PCL and chondrocyte-laden GelMA was utilised for making cartilage constructs. The 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was evidenced by the presence of glycosaminoglycans and collagen type II after cultivation. The findings in this thesis therefore support the feasibility of using 3D bioprinted composite constructs for nasal reconstruction.