Abstract
Congenital birth defects, trauma, inflammation, and cancer surgery are the main causes of craniofacial deformities. Cleft lip and palate (CLP) contribute to the most common craniofacial birth defects. Platelet-rich fibrin (PRF) is recognised in craniofacial surgery for providing essential autologous growth factors (GF) required for bone regeneration. However, its low elastic modulus, poor storage potential, and limitations in emergency therapy cause its short-term clinical application, thus restricting the use of PRF for broader clinical applications.
Lyophilised PRF (LyPRF) has demonstrated superior storage and transport capabilities, greater tissue compatibility, and osteogenic regenerative potential. This thesis aimed to develop and characterise a novel three-dimensional (3D) bone substitute using collagen, bioglass and LyPRF (C-BG-LyPRF) scaffold and assess its biocompatibility for potential applications in craniofacial tissue engineering and regeneration.
Ethical approval for this study was obtained from the University of Otago Human Ethics Committee (Health) and consultation with Māori has been undertaken with the Ngāi Tahu Research Consultation Committee. PRF was prepared from venous blood obtained from healthy volunteers according to Choukroun’s protocol. To prepare LyPRF, a fresh PRF was frozen and stored at -80°C for 30 minutes before being freeze-dried overnight. After being ground into granules with a mortar and pestle, LyPRF granules were combined with collagen
and bioglass to construct the C-BG-LyPRF scaffold. Following that, the C-BG-LyPRF scaffold was characterised based on physicochemical and biological evaluations. In addition, the biomimetic apatite formation on the C-BG-LyPRF scaffold after immersion in simulated body fluid (SBF) was examined to ascertain the scaffold's bioactivity qualities. This is accomplished by determining and characterising the formation of a hydroxyapatite layer on the C-BG-LyPRF scaffold following immersion in the SBF. In vitro studies using MC3T3-E1 cells were conducted to assess the biocompatibility and differentiation potential of the C-BG-LyPRF scaffold. This cell line was chosen because it has been extensively used to assess the in vitro regeneration of craniofacial bone.
The findings of this study showed that LyPRF demonstrated versatility as a potential biomaterial for a growth factor reservoir, as well as a potential craniofacial bioscaffold. The CBG-LyPRF scaffold revealed an interconnected pore system with an average pore diameter of 146 µm and fell within the acceptable bone scaffold porosity of 87.26%. Fourier-transform-infrared-spectroscopy (FTIR) spectra confirmed the presence of amide I, amide II, and amide III functional groups in the C-BG-LyPRF scaffold. Energy-dispersive X-ray analysis (EDX) showed that the inorganic phase of the C-BG-LyPRF scaffold was composed of sodium, silicon, calcium, phosphorous, chlorine, and sulphur. The X-Ray Diffraction (XRD) pattern confirmed the crystalline appearance of the C-BG-LyPRF scaffold. The C-BG-LyPRF scaffold
demonstrated a swelling rate of 157% at 50 minutes and a sustained release of PDGF-AB delivery for approximately 28 days. Furthermore, the apatite formation on the C-BG-LyPRF scaffold’s surface after immersion in SBF demonstrated superior physical and chemical properties compared to the control groups based on Scanning Electron Microscopy (SEM), XRD, FTIR, and EDX analyses. The apatite phase formed on the C-BG-LyPRF scaffold’s surface was also comparable to the hydroxyapatite phase observed in natural bone.
Furthermore, C-BG-LyPRF scaffold extract medium was found to be biocompatible with MC3T3-E1 cells in both the Live/Dead and proliferation assays. The differentiation of MC3T3-E1 cells was supported by the observation of mineralised nodules and bone matrix protein by Alkaline Phosphatase (ALP), Alizarin Red S Staining (ARS), and Osteocalcin (OCN) ELISA Assays. It is critical to highlight that, when compared to other control groups, the C-BG-LyPRF scaffold extract medium exhibited the strongest mineralisation potential as determined by ARS staining, ALP, and OCN assays.
In summary, the aforementioned results demonstrate the C-BG-LyPRF scaffold's exceptional biocompatibility and regenerative potential, establishing it as a promising biomaterial for craniofacial tissue engineering.