Dental cell sheet biomimetic tooth bud model

Abstract

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) - dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 10(6)/cm(2)) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation.

Keywords: Biomaterials; Dental stem cells; Regenerative medicine; Tissue engineering; Tooth development.

Figure 1. Experimental design and culture of 3D GelMA-CS tooth buds

A. DE and DM cells were seeded on thermo-responsive plates and cultured in normal DE and DM media, respectively, for 14 days. DE and DM CSs were detached by temperature reduction (20ºC) and layered over GelMA constructs to create experimental 3D tooth bud constructs (CSG = DE and DM CSs layered over dental cells encapsulated in GelMA; G = GelMA alone). For in vivo analyses, replicate constructs were cultured in osteogenic media for 4 days and implanted subcutaneously onto the backs of the rats. B. Bioengineered 3D CS - GelMA tooth bud model. The bottom layer mimics the pulp organ (5% GelMA encapsulating DM cells) and the top layer mimics the enamel organ (3% GelMA encapsulating DE cells). The DE and DM CS layers mimic polarized DE-DM cell layers normally observed in developing teeth. C. Steps used to prepare the constructs. DM cells (3×10⁷ cells/ml) were re-suspended in 100 μL of 5% GelMA and photo-crosslinked. DM and DE cell sheets were layered over the polymerized DM 5% GelMA. DE cells (3×10⁷ cells/ml) re-suspended in 100 μL 3% GelMA and 100 μL, layered over construct and photo-crosslinked.

Figure 2. DM and DE cell sheet formation at indicated cell-seeding densities and in vitro culture times, and formation of multilayered dental cell sheets constructs

A. Optimized cell-seeding densities for CS formation was 0.11 × 10⁶ cells/cm² for DM cells and 0.22 × 10⁶ cells/cm² for DE cells (scale bar 200μm). B. Two layered DM CSs. C. Three layered DE CSs.

Figure 3. 3D GelMA-CS constructs cultured in osteogenic media for 24 h, 4, 7 and 12 days

H&E images (A, B, C, D) revealed extracellular matrix formation and the morphology of the DE and DM cell sheets within the bilayer GelMA constructs. The arrows indicate the DE and the DM CSs. Pol images (E, F, G, H) show the organized collagen in the extracellular matrix. IF imaging (I, J, K, L) showed the expression of VM (green) by DM cells and CK18 (red) by the DE cells.

Figure 4. Dental CSs interaction in in vitro cultured GelMA constructs

High magnification H&E images and IHC analyses of multilayered DE DM CSs GelMA constructs cultured in osteogenic media for 24 h and 4 days, stained with FAK, TEN and SYN4. Arrows indicate expression. Cell sheets are identified as epithelial (DE) and mesenchymal (DM). A. H&E stained DE DM CSs GelMA constructs cultured in osteogenic media for 24 h. FAK, TEN and SYN4 staining (B, C and D) were detected in the DM CSs cultured in osteogenic media for 24 h. E. H&E image of DE DM CSs GelMA constructs cultured in osteogenic media for 4 days. F. FAK staining was detected in DE and DM CSs cultured in osteogenic media for 4 days. G. TEN was detected in the DM CSs cultured in osteogenic media for 4 days. H. Faint SYN4 staining was detected in DM CSs cultured in osteogenic media for 4 days. I. No staining was detected in the negative controls. Specific staining was detected on the natural tooth bud (J. FAK, K. TEN and L. SYN4).

Figure 5. Activation of signaling pathways in dental CSs in vitro

H&E images and IF analyses of multilayered DE DM CSs GelMA constructs cultured in osteogenic media for 24 h and 4 days, stained with SHH, RUNX2 and BMP2 in green, and VM positive DM cells in red. The red staining identifies the DM CSs, while, the absence of red staining identifies the DE cells. Arrows indicate expression. H&E stained DE DM CSs GelMA constructs cultured in osteogenic media for 24 h (A) and 4 days (E). SHH staining was detected in DE and DM CSs after 24 h (B) and 4 days (F). RUNX2 staining was faintly detected at the interface of DE and DM CSs (C), but strongly detected at the second layer of DE CSs after 24 h (inset in the image C), and detected in DE and DM CSs after 4 days (G). BMP2 staining was detected in DE and DM CSs after 24 h (D) and 4 days (H). No staining was detected in the negative controls (I, J and K).

Figure 6. In vivo implanted bioengineered 3D CSG tooth bud constructs

A. In vivo implanted 3 week constructs at harvest (G is acellular GelMA, CSG is biomimetic 3D CSs GelMA construct). B. Bright field images of an in vivo CSG construct. C. Bright field image of an in vivo acellular GelMA constructs.

Figure 7. MicroCT analyses of in vivo bioengineered CSG constructs

A. No mineralized tissue formation was observed in the acellular GelMA constructs (G). B. Mineralized tissue formation was observed in the CSG constructs. C. 3D model of the mineralized tissue. D. Quantification of mineral density (g/cm³) of the CSG constructs. E. Comparison of mineral densities from engineered and natural mineralized tissues (pig spine, trabecular bone, cortical bone and human enamel) [1, 2]. F. Percent volume of mineralized tissue within ranges of mineral density (ROI – region of interest corresponds to the whole mineralized tissue). G. Representation of areas of mineralized tissue within the ranges of mineral densities (white color represents areas within the range). Abbreviations: MD, mineral density.

Figure 8. In vivo implanted bioengineered 3D CSG tooth bud constructs exhibited elaborate extracellular matrix formation after 3 weeks

No tissue formation was observed in the acellular GelMA constructs, H&E (A) and Pol (B) images. H&E stained embedded paraffin and sectioned constructs exhibited high cellularity (C, D), extensive extracellular matrix and dentin/bone-like tissue formation at the DM GelMA layer. The dashed line separates the biomimetic pulp organ (DM in the bottom layer) from the biomimetic enamel organ (DE in the top layer). Pol images (E, F) revealed organized collagen formation within the CSG constructs. IF images (G, H) show the expression of VM (green) by DM cells in the biomimetic pulp organ layer, and ECAD (red) by the DE cells in the biomimetic enamel organ of the CSG constructs.

Figure 9. Dental cell differentiation within in vivo grown CSG tooth bud constructs after 3 weeks

IHC analyses of dentin, enamel and bone specific markers. The odontoblast differentiation marker DSPP was highly expressed throughout the biomimetic pulp organ layer (A, B). Odontoblast/Osteoblast differentiation marker OC was expressed in the centers of the CSG constructs (C, D). Ameloblast differentiation marker AM was expressed throughout the CSG constructs in both biomimetic pulp and enamel organ layer (G, H), while AMBN was not detected in the CSG constructs (E, F). TEN was detected in the both biomimetic pulp and enamel organ layers of the CSG constructs (I, J).

Figure 10. Activation of signaling pathways and host blood vessel detection within in vivo grown CSG tooth bud constructs after 3 weeks

IF analyses of SHH, RUNX2, BMP2 and CD31 in green and VM positive DM cells in red. The red staining identifies the biomimetic pulp organ layer where the DM cells are localized, while, the absence of red staining identifies the biomimetic enamel organ layer where the DE cells are localized. SHH was expressed throughout the CSG constructs in both biomimetic enamel and pulp organ layer (A, B). BMP2 was also expressed throughout the CSG constructs, but stronger BMP2 expression was observed in biomimetic pulp organ layers (C, D). RUNX2 was expressed in both biomimetic enamel and pulp organ layer (E, F), however, stronger RUNX2 expression was observed in the biomimetic epithelial organ layer (E, arrows). No CD31 positive staining was detected in the constructs (G, H). No positive staining was detected in the negative controls (I).