Abstract
Poly-lactic acid (PLA) scaffolds are widely used in bone tissue engineering. The introduction of 3D printing has greatly increased the ability for tailoring different geometrical designs of these scaffolds for improved cellular attachment, growth and differentiation. This study aimed to investigate the effect of PLA fibre angle in 3D printed PLA scaffolds on hDPSC attachment and growth in vitro.
Two types of PLA scaffolds were prepared via 3D printing containing fibres angled at either 45° or 90°. hDPSCs (P4, 2*105 cells per scaffold) were statically seeded for 4 hours on to the scaffolds (7×3.5×3 mm3, n=3). Cellular attachment was checked using fluorescence microscopy and the number of unattached cells was counted using a haemocytometer (HCM). The cell-scaffold constructs were then cultured in osteogenic medium for up to 5 weeks. ALP staining and SEM were performed for one construct from each group at week 3. Cellular viability was determined using CMFDA/EHD1 live/dead labelling at week 4. After 5 weeks, constructs were processed for histology.
Fluorescence micrographs showed high numbers of hDPSCs attached to scaffold surfaces in both groups after seeding irrespective of fibre angle. However, HCM cell count revealed that the 45° angled PLA scaffolds had significantly greater cell attachment compared to the 90° angled PLA group (p<0.0001). After 3 weeks in osteogenic culture, both types of construct showed strong ALP staining. SEM showed that in the 45° angled PLA group, almost all macro-pores were fully closed with newly formed cell sheets. In comparison, in the 90° angled group, most of the macro-pores remained open although a limited amount of cellular bridging was present. SEM also detected crystal deposits in different areas within the cell sheets for both construct groups. Most hDPSCs were alive in both groups at week 4 of culture with few dead cells present. After 5 weeks, histology showed marked cellular growth and new matrix formation, with detectable Van Kossa +ve crystal deposits in different areas within all constructs irrespective of PLA fibre angle.
This study showed that 45° angled PLA 3D printed scaffolds enhanced hDPSC attachment and cellular bridging, which may help to rapidly close the macro-pores within the scaffold compared to the 90° angled group. This illustrates the potential of 45° angled 3D printed PLA scaffolds as good candidates for bone tissue engineering.
