Fig 1.
(A) The DLP process starts with a light curable resin and a powder, which are combined to make a photocurable slurry. We utilized a premixed hydroxyapatite photocurable slurry. (B) Slurry is poured into the vat where the (C) light is projected from below onto the build plate, curing one layer at a time of the part (e.g., coupons). (D) The printed part (coupon) is removed from the build plate and cleaned, then placed in a furnace where the light curable resin is burned out and the ceramic densifies, (E) creating the final part (coupon).
Fig 2.
Scanning Electron Microscopy (SEM) of DLP print builds.
(A) Macro view of coupons built using DLP process, which were rinsed with a propylene carbonate/tripropylene glycol blend and then fired. Micro view and analysis of coupons using SEM imaging was conducted on coupons (B) transversely sectioned mounted and polished, (C) revealing innate porosity. (D) SEM image of coupon before firing (green state, not sintered) reveals particle size of LithaBone HA400 ~2–6 microns and (E) after firing (heat treatment at 1300°C),densification of particles is seen as a result of sintering.
Fig 3.
MSCs morphology on DLP printed coupons.
MSCs seeded onto tissue culture polystyrene (12-well plate) and DLP printed HA coupon were fixed and stained for S6 ribosomal protein (red, conjugated primary antibody), actin (green, phalloidin dye), and nuclei (blue, DAPI) after 24 hours of culture. Image taken at 20x objective magnification using IN CELL analyzer, demonstrates a typical spindle morphology on coupons. Scale bar 50μm.
Fig 4.
U2OS proliferation on DLP printed HA coupons.
Relative luminescence unit (RLUs) were measured from U2OS cells seeded at ~25% (1.75x103 cells/cm2) density on polystyrene plates or DLP printed HA coupons using RealTime-GloTM MT Cell Viability Assay. Cell counts were enumerated based on RLU measurements for the 4 and 48 hour time points using a standard curve correlating cell count and RLU (S7 Fig). Direct cell count was done at 66 hours of incubation by image analysis (counted cell nuclei). U2OS cells proliferation rates were similar on coupons and tissue culture polystyrene plates (P>0.5; using T-test, differences were not statistically significant).
Fig 5.
MSC proliferation on DLP printed coupons.
(A) MSCs were seeded at an approximate density of 40% (1x104 cells/cm2) onto tissue culture polystyrene (12-well plates) and coupons. Cells were cultured for 3 days in the presence of RealTime-GloTM MT Cell Viability Assay (Promega). Relative luminescence unit (RLUs) was measured at various time points and plotted using a logarithmic y-axis. (B) Cell doubling times were comparable to cells attached on tissue culture polystyrene plates.
Fig 6.
Calcium deposition from osteogenic differentiated MSCs on DLP printed coupons.
(A) Calcium deposits stain red when cells are stained with Alizarin Red S and (B) gray to black with von Kossa. (B) The von Kossa counter stain, nuclear fast red, stains cells a fuchsia color. The observation of punctate staining in MSCs cultured in osteogenic differentiation medium on DLP printed coupons and polystyrene plates demonstrates calcium deposition on both surfaces using both Alizarin Red S and von Kossa staining. In contrast, MSCs cultured in growth media lack similar punctate calcium staining with either Alizarin Red S or von Kossa. Images were taken using Lecia dissection scope at 4x magnification, scale bar 1.25mm.
Fig 7.
Expression of alkaline phosphatase and RT-PCR analysis for MSCs differentiated on DLP printed HA coupons.