The need to regenerate bone remains a significant healthcare challenge both now and in the future to meet the needs of an ageing population. The research carried out in this thesis aimed to investigate the potential of using a combination of human dental pulp stromal cells (hDPSCs), and different architectures of 3D printed polylactic acid (PLA) scaffolds with/without self-assembling peptide P11-4 (SAP) for bone tissue engineering (BTE) in vitro and in vivo.
To evaluate hDPSCs multi-potential, considering donor variability, the cells were isolated from the dental pulp of upper/lower third molars from 3 different donors and characterised via trilineage differentiation (osteogenic, chondrogenic and adipogenic) approach. To investigate the effect of the architecture of 3D printed PLA scaffolds on cell attachment and bone matrix formation in vitro and in vivo, hDPSCs were statically seeded on 3D printed PLA scaffolds with fibres angled at either 45° or 90° and cultured in osteo-inductive medium for up to 5 weeks in vitro. At different time points, the constructs were examined using SEM, EDS, live/dead markers, histology and immunohistochemistry (IHC). For in vivo evaluation, the constructs were sealed within diffusion chambers (DCs), which were then implanted intraperitoneally in nude mice for a further eight weeks prior to retrieval and examination with SEM, histology and IHC. Based on the results from these experiments, 3D printed PLA scaffolds (45°) were infiltrated with SAP P11-4 to evaluate the potential of this novel combination for enhancing BTE in vitro and in vivo. HDPSCs were pre-mixed with SAP and seeded on PLA 45° scaffolds or scaffolds were directly seeded with hDPSCs alone. Constructs were then cultured in osteo-inductive medium for up to 5 weeks in vitro and examined using SEM, live/ dead markers, histology and IHC at different time points. For in vivo evaluation, 3D printed PLA 45° scaffolds were seeded with hDPSCs with/without SAP
iii
P11-4 and sealed within DCs, which were implanted in nude mice for up to 8 weeks prior to retrieval and examination with SEM, histology and IHC. HDPSCs isolated from all donors showed marked morphological changes and positively expressed different markers for osteogenic, chondrogenic and adipogenic differentiation after monolayer culture in different induction media in vitro. PLA 45° scaffolds showed greater hDPSCs attachment, macro-pores bridging/ closure and expression of IHC osteogenic markers in vitro with higher accumulation of mineral deposits in vivo compared to the PLA 90° scaffold group. PLA 45° /SAP P11-4 constructs showed greater hDPSC attachment, neo-tissue formation and enhanced expression of IHC osteogenic markers both in vitro and in vivo compared to the PLA 45° scaffold alone group.
The outcomes of this study verified the multilineage plasticity of hDPSCs. The data also demonstrated the significant effect of 3D printed PLA scaffold fibre geometry on hDPSCs osteogenic behaviour, suggesting that PLA 45° 3D printed scaffold layout is the design of choice for bone tissue engineering. In addition, incorporating SAP P11-4 into 3D printed PLA scaffolds further enhanced hDPSCs attachment and osteogenesis both in vitro and in vivo, illustrating the complementary benefits from both technologies into one scaffold entity and the promising use of this novel combination for bone tissue regeneration in the future.
