Formation of Dentin-like Structure on 3D-Printed Scaffolds: Role of Microstructure

Objective: Tooth regeneration using stem/progenitor cells with biomaterial scaffolds has been attempted to overcome limitations of current dental prosthesis.  Microstructure of scaffolds, including diameter of strands, size of microchannels, interconnectivity, and micro-pattern, plays pivotal roles in the adhesion, proliferation and differentiation of stem/progenitor cells.  Extending from our recent work on the regeneration of anatomically shaped tooth-like structure with 3D printed scaffolds in vivo (Kim, et al., JDR 2010), the present study is designed to determine how scaffold design parameters, including microchannel size and surface tomography, affect odontogenic differentiation of human dental pulp stem/progenitor cells (DPSCs) in vitro.

Method: A composite of polycaprolactone (PCL) and hydroxylapatite (HA) (90:10wt%) was molten at 120°C and fabricated into 3D cylinders (10×3 mm²) via layer-by-layer fiber deposition using a 3D Bioplotter per our previous work.  The scaffolds comprised repeats of interlaid microstrands (~200 μm) and interconnected microchannels of specific sizes: 0 μm (no channels), 50 μm, and 100 μm.  Upon coating with collagen type I gel (2mg/mL), human DPSCs were seeded on surface of the scaffolds (100K cells/scaffold).  After 4wk culture in osteogenic/odontogenic differentiation medium, odontogenic differentiation of DPSCs was evaluated qualitatively and quantitatively using immuno-/histological analysis and confocal microscopy.

Result: Microscopy showed a unique surface tomography with parallel-aligned microgrooves and vertically oriented microchannels (50~100 μm) in the 3D printed scaffolds.  At 4wks, DPSCs seeded on scaffolds with 50-μm and 100-μm channels formed DSP⁺ mineralized matrix, in comparison with scaffolds without microchannels.  Interestingly, the mineralized structures formed on scaffolds with 100-μm channels were dense and polarized, reminiscent of native dentin.  Additional experiments showed that channels greater than 100 μm fails to yield the dentin-like, polarized mineralization.

Conclusion: Our findings demonstrate that scaffold’s microchannel size is one of the key valuables in promoting odontogenic differentiation of DPSCs in ways potentially useful for dentin regeneration.