Biomimetic Tubular Matrix Induces Periodontal Ligament Principal Fiber Formation and Inhibits Osteogenic Differentiation of Periodontal Ligament Stem Cells

Abstract

Periodontal ligament (PDL) is assembled from highly organized collagen fiber bundles (PDL principal fibers) that are crucial in supporting teeth and buffering mechanical force. Therefore, regeneration of PDL needs to reconstruct these well-ordered fiber bundles to restore PDL functions. However, the formation of PDL principal fibers has long been a challenge due to the absence of an effective three-dimensional (3D) matrix to guide the growth of periodontal ligament stem cells (PDLSCs) and to inhibit the osteogenic differentiation of PDLSCs during the PDL principal fibers deposition. In this work, we designed and fabricated a bio-inspired tubular 3D matrix to guide the migration and growth of human PDLSCs and form well-aligned PDL principal fibers. As a biomimetic 3D template, the tubular matrix controlled PDLSCs migration inside the tubules and aligned the cells to the designated direction. Inside the tubular matrix, the PDLSCs expressed PDL markers and formed oriented fiber bundles with the same size and density as those of natural PDL principal fibers. Furthermore, the tubular matrix downregulated the osteogenic differentiation of PDLSCs. A mechanism study revealed that the Yap1/Twist1 signaling pathway was involved in the inhibition of PDLSCs osteogenesis within the tubular matrix. This work provides an effective approach to induce PDLSCs to form principal fibers and gives insight into the underlying mechanism of inhibiting the osteogenic differentiation of PDLSCs in biomimetic tubular matrices.

Keywords: osteogenic differentiation; periodontal ligament; periodontal regeneration; principal fibers; tubular matrix.

Figure 1.

Characterizations of 3D tubular matrices. (A) An SEM image of a nanofibrous tubular matrix. (B) High magnification of the tubular matrix showing that the matrix was composed of collagen-like nanofibers. (C) Top view of a tubular matrix under a confocal microscope. (D) Cross-section views of the tubular matrix showing that the tubules were open at both ends.

Figure 2.

Migration of PDLSCs in the tubular matrices (A, C) and on the flat matrices (B, D). (A) and (B) are top view images, (C) and (D) are cross-section view images. Green: gelatin matrices. Red: actin. Blue: DAPI.

Figure 3.

(A−D) Dynamic migration process of PDLSCs in the tubular matrix from 2 to 24 h. Green: gelatin matrix. Red: actin. Blue: DAPI. (E) Migration distances of PDLSCs from 2 to 24 h. (F) Migration ratios of PDLSCs from 2 to 24 h. (∗) Compared to the 2 h group; (∗∗) compared to the 6 h group, and (∗∗∗) compared to the 12 h group.

Figure 4.

PDL-like tissue formation in tubular matrices. (A) The expressions of PDL markers (Collagen I, Collagen III, and Periostin) in the tubular matrices. Green: gelatin matrices. Red: actin. Blue: DAPI. Yellow: PDL markers. (B) Quantitative analyses of the expressions from Collagen I, Collagen III and Periostin. (C) Sirius red staining of the tubular construct after 21 days. Collagen fiber bundles formed inside of the tubules (yellow arrow). Images were taken under bright light (left) and polarized light (right) conditions, separately.

Figure 5.

Expressions of osteogenic genes on the tubular and flat matrices on day 3 and day 7. The three osteogenic genes are (A) Ocn, (B) Runx2, and (C) Sp7.

Figure 6.

Yap1 expressions on the tubular and flat matrices on day 3 and day 7. (A) Immunofluorescence of PDLSCs on the tubular and flat matrices. Green: gelatin matrices. Red: actin. Blue: DAPI. Yellow: Yap1. (B) Relative Yap1 gene expressions on the tubular and flat matrices on day 3 and day 7. (C) Ratios of Yap1expression in nuclei/total Yap1 expression on the tubular and flat matrices, separately.

Figure 7.

(A−D) Yap1 and osteogenic gene expressions on the tubular and flat matrices after the treatment of ROCK inhibitor for 7 days. (E−H) Twist1 and osteogenic gene expressions on the tubular and flat matrices after the treatment of Twist1 esiRNA for 7 days. P < 0.05 was considered statistically significant. (∗) Compared to the control group.

Figure 8.

Gene ontology enrichment analysis of the gene expressions of PDLSCs in the tubular matrix using PDLSCs on the flat matrix as a control.

Scheme 1.

Illustration of the Fabrication of the 3D Nanofibrous Tubular Matrix and Use of the 3D Tubular Matrix for PDL Principal Fiber Regeneration