Fig 1.
Dentin acellular scaffold preparation.
Extracted specimens were washed with chlorhexidine solution and periodontal bacterial biofilm and dental calculus were removed. Cross-sectioning to the cementoenamel junction was performed and the apex was removed (panel A). Pulp tissue and predentin were completely removed (panel B). Sections of teeth 1.5-mm thick were obtained and cut into two to three fragments (panel C). Three different surfaces were considered: surface A (SA: dentin tubules perpendicular or oblique to the surface of the scaffold, i.e., accessible tubules), surface B (SB: dentin tubules parallel to the surface of the scaffold, i.e., non-accessible tubules), and surface C (SC: cells grown far from the scaffold, i.e., cells grown on the culture plate surface). Human dental pulp stem cells (hDPSCs) were cultured on the scaffolds for up to 6 weeks and processed for light microscopy. Samples were embedded in paraffin, cut into sections, and subjected to Masson’s trichrome staining (panel D). Representative results of 15 different specimens are shown. Scale bar equals to 100 μm.
Fig 2.
Cell culture kinetics on acellular dentin scaffolds.
hDPSCs were cultured on the scaffolds for up to 6 weeks and processed for light microscopy. Confluence was estimated by phase contrast microscopy. Representative images obtained at 1 week (panel A) and 6 weeks (panel B) are shown. Samples were embedded in paraffin, cut into sections, and subjected to Masson’s trichrome staining. Representative images obtained at 2 weeks (panels C and D) and 6 weeks (panels E and F) of cells grown on SA (panels C and E) and SB (panels D and F) are shown. All experiments were performed in six replicates using hDPSCs isolated from two different donors. Scale bar equals to 20 μm.
Fig 3.
Extracellular matrix secreted by hDPSCs cultured on SA.
hDPSCs were cultured on scaffolds for 6 weeks and processed for light microscopy. Samples were embedded in paraffin, cut into sections, and subjected to Masson’s trichrome staining (panel A) or immunohistochemistry for dentin sialophosphoprotein (DSPP) (panel B). The area between SA (center) and SB (left and right) is shown. Representative results of 12 separate experiments are shown. Scale bar equals to 100 μm.
Fig 4.
Cell morphology of hDPSCs cultured on SA.
hDPSCs were cultured on scaffolds for 6 weeks and processed for light microscopy. Samples were embedded in paraffin, cut into sections, and subjected to Masson’s trichrome (panel A), hematoxylin and eosin staining (panel B), or immunohistochemistry for DSPP (panel C). Black arrows indicate cellular processes inside dentin tubules. Representative results of 12 separate experiments are shown. Scale bar equals to 20 μm.
Fig 5.
Ultrastructure of hDPSCs cultured on SA.
hDPSCs were cultured on scaffolds for up to 6 weeks and processed for transmission electron microscopy (TEM). dt, dentin tubule; id, intertubular dentin; pd, peritubular dentin; cp, cellular process; a, actin filaments; g, glycogen; n, nucleus; sv, secretory vesicles. Representative results of 12 separate experiments are shown in panel A and B. Scale bar equals to 5 μm.
Fig 6.
Ultrastructure of hDPSCs cultured on SA.
hDPSCs were cultured on scaffolds for up to 6 weeks and processed for TEM. r, rough endoplasmic reticulum; a, actin filaments; g, glycogen; id, intertubular dentin; pd, peritubular dentin; dt, dentin tubule; sv, secretory vesicles; cp, cellular process. Representative results of 12 separate experiments are shown in panel A, B and C. Scale bar equals to 5 μm.
Fig 7.
Ultrastructure of hDPSCs cultured on SA.
hDPSCs were cultured on scaffolds for 6 weeks and processed for TEM. r, rough endoplasmic reticulum a, actin filaments; g, glycogen; id, intertubular dentin; pd, peritubular dentin; dt, dentin tubule; sv, secretory vesicles; cp, cellular process; nem, neo-secreted extracellular matrix. Representative results of 12 separate experiments are shown in panel A, B and C. Scale bar equals to 5 μm.
Fig 8.
Cell morphology of hDPSCs cultured on SA.
hDPSCs were cultured on scaffolds for 6 weeks and processed for light microscopy. Samples were embedded in paraffin, cut into sections, and subjected to hematoxylin and eosin staining (panel A) or immunohistochemistry for DSPP (panel B). Black arrows indicate stellate cells negative for DSPP. Representative results of 12 separate experiments are shown. Scale bar equals to 20 μm.
Fig 9.
Cell morphology of hDPSCs cultured on SB.
hDPSCs were cultured on scaffolds for 6 weeks and processed for light microscopy. Samples were embedded in paraffin, cut into sections, and subjected to Masson’s trichrome staining (panel A) or immunohistochemistry for DSPP (panel B). Representative results of 12 separate experiments are shown. Scale bar equals to 20 μm.
Fig 10.
Ultrastructure of hDPSCs cultured on SB.
hDPSCs were cultured on scaffolds for 6 weeks and processed for TEM. r, rough endoplasmic reticulum; a, actin filaments; g, glycogen; sv, secretory vesicles; n, nuclei. Representative results of 12 separate experiments are shown in panel A, B and C. Scale bar equals to 5 μm.
Fig 11.
Cellular morphology and extracellular matrix characteristics of hDPSCs grown on SC.
hDPSCs were cultured on the surface of the culture plate far from the dentin acellular scaffold for 6 weeks and processed for light microscopy and TEM. Samples were embedded in paraffin, cut into sections, and subjected to Masson’s trichrome staining (panel A) or immunohistochemistry for DSPP (panel B). Samples were also analyzed by TEM (panel C). Representative results of 12 separate experiments are shown. Scale bar equals to 20 μm.