A 3D ex vivo mandible slice system for longitudinal culturing of transplanted dental pulp progenitor cells

Abstract

Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell-lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the β-actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP-DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP-DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell-micro-community, phenotypically modified in the tooth (the "biology"); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high-content imaging (the biology in a "multiplex" format).

Keywords: high-content imaging; mesenchymal stem cell; organ culture.

Figure 1

“Organotypic model” to identify transplanted cells growing in their native tissue. Experimental methodology, showing the step‐by‐step process of cell injection (expressing constitutively‐active GFP) into rat mandible slices marked with an agarose bead (fiducial mark, to ensure tissue orientation), culture and subsequent 3D imaging at days 1 and 7 post‐injection. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

cGFP‐DPPC characterisation. A: The stability of GFP expression profiles were obtained for cell populations grown over 3 days on tissue culture plastic. Flow cytometry histogram to demonstrate the extent and levels of GFP‐expression per cell (Graph insert FSC/SSC gating of live cells to remove debris). Average GFP expression 80.66% ± 7.1 and geometric mean of GFP fluorescence 1463 ± 42.2 (arb units)). B: Time‐dependent population doubling levels for cGFP‐DPPCs (note cells are still expanding when used at passage 12). C: PCR analysis indicates positive expression of known mesenchymal markers; CD73, CD90, CD105, pericyte marker CD146 as well as osteopontin and CD34 and negative expression for CD45. D: Hematoxylin and eosin stained mandible slice cultured for 7 days shows different regions of a mandible slice (note the orientation is similar to images shown in Fig. 3). It also demonstrates that normal tissue architecture (pulp, dentine, odeontoblasts (OD), periodontal ligament (PDL) and bone) is maintained after injection and culture. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Representative confocal images from a mandible slice at days 1 and 7 post‐injection. A: Surface pulp region demonstrates cGFP‐DPPCs identification and location at day 1 post‐injection (tissue depth = 22 µm; GFP_TA = total area of GFP fluorescence in pixels). Magenta represents the reflected light channel and has been used to orientate the slice for image comparison, indicated by the white circles on each image. Approximate cell number is given and location of these cells has been plotted. The grey dashed outline is the approximate location of the dentine/pulp regions. A': Cell location in the surface pulp region at day 7 post‐injection. B: cGFP‐DPPCs in sub‐surface pulp region at day 1 post injection (tissue depth = 15 µm). B': cGFP‐DPPC expansion in sub‐surface pulp region at day 7 post‐injection. Scale bar = 100 µm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

(A) cGFP‐DPPCs migration/expansion in the dentine region of the slice at day 7 post‐injection (tissue depth = 37 µm). A': Reflected light. A": Merged image. (B) Higher magnification image to show changes in morphology to and patterning day 7 post injection. ** indicates location of cells in A". (C) Higher magnification image to demonstrate morphology of cGFP‐DPPCs at day 1 post‐injection. (D) A different mandible slice focusing on the dentine surface demonstrating cGFP‐DPPC columnar morphology. Scale bar = 100 µm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]