Collagen Scaffolds Laden with Human Periodontal Ligament Fibroblasts Promote Periodontal Regeneration in SD Rat Model

Abstract

Periodontitis, a chronic inflammatory disease caused by microbial communities carrying pathogens, leads to the loss of tooth-supporting tissues and is a significant contributor to tooth loss. This study aims to develop a novel injectable cell-laden hydrogel consisted of collagen (COL), riboflavin, and a dental light-emitting diode (LED) photo-cross-linking process for periodontal regeneration. Utilizing α-SMA and ALP immunofluorescence markers, we confirmed the differentiation of human periodontal ligament fibroblasts (HPLFs) into myofibroblasts and preosteoblasts within collagen scaffolds in vitro. Twenty-four rats with three-wall artificial periodontal defects were divided into four groups, Blank, COL_LED, COL_HPLF, and COL_HPLF_LED, and histomorphometrically assessed after 6 weeks. Notably, the COL_HPLF_LED group showed less relative epithelial downgrowth (p < 0.01 for Blank, p < 0.05 for COL_LED and COL_HPLF), and the relative residual bone defect was significantly reduced in the COL_HPLF_LED group compared to the Blank and the COL_LED group (p < 0.05). The results indicated that LED photo-cross-linking collagen scaffolds possess sufficient strength to withstand the forces of surgical process and biting, providing support for HPLF cells embedded within them. The secretion of cells is suggested to promote the repair of adjacent tissues, including well-oriented periodontal ligament and alveolar bone regeneration. The approach developed in this study demonstrates clinical feasibility and holds promise for achieving both functional and structural regeneration of periodontal defects.

Keywords: collagen hydrogel; dental light-emitting diode; orientation of periodontal ligaments; periodontal regeneration; photo-cross-linking; riboflavin.

Figure 1

The three-wall defect of the first molar in SD rats. (a) The surgical field on the rat skull, indicating the selected sample area that was sectioned. (b) Three-wall defect procedures were performed rostral to the upper first molar using a 1.4 mm round tungsten carbide drill. The blue arrow and dotted circle on the left indicate intact bone, while the red arrow and dotted circle on the right indicate the postoperative bone defect, measuring 2 × 1.4 × 1.4 mm³. Note that the dash-dotted line in (b) indicates the histological section.

Figure 2

Anatomical reference and calculation method. The cementum–enamel junction and bone height were chosen as anatomical references for the assessment of epithelial downgrowth and residual bone defects, and tooth width and tooth height were also considered to correct for deviations in sample section angles. In addition, the angle of the epi-tooth axis was also evaluated.

Figure 3

HPLF-laden collagen scaffolds constructed from dental LEDs. (a) Strain test (%) of collagen structures cured with the dental LED at different times. The stiffness of the 3 s/9 s/21 s group was higher than that of the 0 s group (*** p < 0.001). (b) The cell viability under the LED light for 3 s was higher than that of the 9 s and 21 s groups (** p < 0.01). (c–e) Collagen cell scaffolds were illuminated with the dental LED for 3 s/9 s/21 s and cell viability was tested by Live-Dead (bar: 100 µm).

Figure 4

Immunofluorescence staining of α-SMA and ALP in the collagen scaffolds laden with HPLFs (n = 2 for each 1, 7, 14 day experiment). (a) Relative fluorescence intensity of DAPI, α-SMA/DAPI, and ALP/DAPI at 1, 7, 14 days (n = 6). (b) On the 7th day, both α-SMA and ALP expression were observed, indicating the differentiation of HPLF cells into myofibroblasts and pre-fibroblasts (bar: 100 µm). (* p < 0.05 and ** p < 0.01, compared with D1; # p < 0.05 significant between groups).

Figure 5

Histology of epithelium healing and bone growth are shown. Movement of the junctional epithelium is a key factor in the progression of periodontitis. The smaller residual bone defect and well-oriented periodontal ligaments indicated better postoperative periodontal regeneration capability. (a–c) Blank, (d–f) COL_LED, (g–i) COL_HPLF, (j–l) COL_HPLF_LED. (b,e,h,k) are enlarged pictures from the small box in (a,d,g,j), respectively. The 0° direction represents the orientation of the periodontal ligament fibers that are parallel to the cemento-enamel junction (CEJ) of the tooth. A higher peak at 0° indicates well-organized periodontal ligament fibers. In the COL_HPLF_LED group, osteoblasts aggregated near the newly formed bone, and the periodontal ligament was oriented in connection with the bone and root surfaces. This finding indicated that the periodontal ligament was effectively restored and functioned well in the COL_HPLF_LED group. (Arrows indicate periodontal ligaments, and arrowheads indicate osteoblasts accumulation.)

Figure 6

Combination of HPLF cells with LED-illuminated collagen scaffolds promotes periodontal tissue regeneration (Y-axis unit: %). Both (a) epithelial downgrowth and (b) relative epithelial downgrowth were significantly reduced in the COL_HPLF_LED group compared to the Blank group (** p < 0.01) and the COL_LED group (# p < 0.05, ## p < 0.01). Similarly, (c) residual bone defect and (d) relative residual bone defect were significantly reduced in the COL_HPLF_LED group compared to the Blank group (* p < 0.05) and the COL_LED group (# p < 0.05). (e) Compared with the COL_LED group, the angle between the epithelium and the tooth axis was significantly reduced in the COL_HPLF_LED group (# p < 0.05).