Periodontal ligament stem cell tissue engineering scaffolds can guide and promote canine periodontal tissue regeneration

Abstract

Introduction: Periodontal disease, including gingivitis and periodontitis, is caused by dental plaque invading the periodontal tissues and is the most common oral disease. The basic treatment methods applied in the clinic can destroy dental plaque, smooth the root surface, and reduce local inflammation, but it is difficult to completely regenerate and rebuild the complex three-dimensional periodontal tissues. The rapid development of periodontal tissue engineering has led to the development of new methods for the treatment of periodontal disease. Periodontal ligament stem cells (PDLSCs) are key seed cells in periodontal tissue engineering, which can provide strong support for tissue regeneration. Meanwhile, an engineering scaffold constructed from biomaterials provides a three-dimensional space for the growth and function of seed cells and can form a tissue engineering complex with the seed cells to repair periodontal tissue, which can guide consequently enable true three-dimensional periodontal structure regeneration and functional restoration.

Methods: This study established an effective way to isolate, culture, and identify canine PDLSCs. Using chitosan, β-glycerol phosphate, and biphasic calcium phosphate bone substitute as raw materials, a tissue engineering scaffold with good physical properties was prepared by freeze-drying method. Canine PDLSCs were co-cultured with the scaffolds to prepare canine PDLSC tissue engineering scaffolds with good biocompatibility in vivo and in vitro.

Results and discussion: The canine PDLSC tissue engineering scaffold was transplanted into the single wall bone defect of the first mandibular molar tooth of the dog without causing inflammatory reactions, and the tissue compatibility was satisfactory. The cell-scaffold complex can increase the content of related growth factors and immunomodulatory factors in the tissues, reduce the content of proinflammatory factors, and prevent the growth of binding epithelium in the defect area, thus forming new bone and new periodontal ligaments in the defect area, promoting the repair of periodontal defects, and improving the therapeutic effect of guided regeneration.

Keywords: canine PDLSC tissue engineering scaffold; guide periodontal regeneration; periodontal disease; periodontal ligament stem cells; periodontal repair.

Figure 1

Results of cell isolation and culture. (A) Cell growth state. The adherent cells covered the entire bottom of the dish. (B) The cell growth curve. The cells entered the exponential growth phase on the 3rd day. (C) After hematoxylin–eosin staining, the cells were spindle-shaped and adhered to the wall. After Giemsa staining, the colonies were observed. The boundaries between the central cells of the colonies were not clear, and the surrounding cells were tightly arranged radially. After alkaline phosphatase staining, brown and gray particles were observed among the closely packed cells, and more particles were found in the clonal colonies.

Figure 2

Results of Cell identification. (A) The isolated cells were subjected to immunofluorescence staining. The nucleus was blue, while no red fluorescence was observed to indicate keratin. The isolated cells were positive for vimentin expression. (B) The expression levels of STRO-1, CD73, CD90, CD105, CD44 and CD166 were 91.3, 87.3, 87.1, 86.7, 81.8 and 80.9%, respectively, according to flow cytometry. CD11a, CD31 and CD45 were not expressed. (C) Lipid-induced differentiation was performed on the isolated cells, lipid droplet particles were observed upon staining with oil red O. The isolated cells were differentiated by osteogenic induction, and alizarin red staining revealed a large area of tangerine-mineralized nodules. The isolated cells were subjected to chondroblast differentiation induction, and the cells were staining positive for Alcian blue.

Figure 3

Morphological characteristics of the scaffolds. (A) Using chitosan (CS), β-glycerol phosphate (β-GP) and biphase calcium phosphate bone substitute (HA/β-TCP) as raw materials, a tissue engineering scaffold was prepared by freeze-drying method. (B) Twelve groups of scaffolds were prepared after vacuum freeze-drying, all of which were cylindrical with regular external micromorphology. (C) The 12 groups of scaffolds had a three-dimensional spatial structure with interconnecting pores. With increasing β-GP content, the pores changed from a loose lamellar structure to a stable honeycomb structure, and the surface roughness increased after the addition of HA/β-TCP. (D) The porosity of the scaffolds was calculated using ImageJ, and a pore distribution map was constructed.

Figure 4

Physical performance test results of the scaffolds. (A) Compressive properties and porosity of the scaffolds. The scaffold prepared with 12% β-GP and 2% HA/β-TCP was most in line with practical needs. (B) Results of the swelling performance test. When the HA/β-TCP content was constant, the β-GP content had no significant effect on the swelling rate of the scaffold (p > 0.05). When the content of β-GP was constant, the swelling rate of the scaffold decreased significantly with increasing HA/β-TCP content (p < 0.05). (C) Degradation performance of the scaffolds. Changes in the β-GP content had no significant effect on the degradation performance of the scaffolds. When the β-GP content was constant, the degradation rate in the 2% HA/β-TCP and 4% HA/β-TCP groups was lower than that in the no-HA/β-TCP group and gradually stabilized after the 14th day.

Figure 5

Cytotoxicity of scaffolds. (A) Cell proliferation on the scaffold. On the 1st day after the inoculation of canine PDLSCs, the number of cells attached to the bottom of the petri dish was significantly greater than that attached to the scaffold (p < 0.05). On Day 3, there were more cells attached to the scaffold than to the bottom of the dish, but the difference was not significant. On the 5th day, the number of cells attached to the scaffold and their proliferation were significantly greater than on the bottom of the Petri dish (p < 0.05). (B,C) Effects of the scaffold extract liquid on cell migration. At 48 h, the migrating cells were observed to cover almost the entire scratched area. According to the quantitative analysis of the scratch area, there was no significant difference in the cell migration area between the experimental group and the control group at 24 h or 48 h. (D) Adhesion and growth of cells on scaffolds. On the 3rd day, the number of cells attached to the scaffold had gradually increased, and the scaffold was moved to a new well to continue to observe the cell growth. On the 5th day, a large number of cells with red fluorescence could be observed, and the red fluorescence was aggregated into clumps.

Figure 6

In vivo biocompatibility test results. (A,B) After the two groups of scaffolds were implanted into the subcutaneous tissue, the healing process and the speed of the wound were not affected, and blood circulation at the implantation site was not affected. (C) There was no obvious inflammatory cell infiltration around the scaffolds, new capillaries could be observed, and there was no pathological damage, such as tissue necrosis or hyperplasia, around the scaffolds. Both groups of scaffolds had good histocompatibility in vivo. Red arrows indicate remaining scaffolds, yellow arrows indicate blood vessels.

Figure 7

Evaluation of the effectiveness of periodontal defect repair and regeneration. (A) The gingival epithelium covered the whole wound in both the tissue engineering scaffold group and the scaffold group, while the periodontal soft tissue in the control group was still concave at the defect site. (B) The levels of TSG-6, PGE2, IDO, IGF-1, TGF-β, VEGF, SDF-1 and bFGF in the tissue engineering scaffold group were significantly greater than those in the scaffold group and control group (p < 0.01). The levels of IGF-1, TGF-β, VEGF, SDF-1 and bFGF in the scaffold group were significantly greater than those in the control group (p < 0.01, p < 0.05). (C) IL-2, IL-6, TNF-α, TNF-β and INF-γ, were significantly decreased after implantation of the tissue-engineered scaffold containing PDLSCs (p < 0.01). TNF-β expression in the scaffold group was significantly lower than that in the control group (p < 0.05). (D) The results for the junctional epithelium, new bone height, new bone area and periodontal ligament regeneration.

Figure 8

Evaluation of the effectiveness of periodontal defect repair and regeneration. At the 8th week after the operation, the tissue from the dog transplantation site was collected, and hard tissue sections 8 ~ 10 μm in thickness were obtained by grinding the slices. The results from top to bottom were HE staining, Masson staining and toluidine blue staining. D represents teeth, NB represents new alveolar bone, the black arrow points to the enamel bone boundary, the red box represents periodontal tissue, and the green box represents osteoblasts. A black round frame indicates a residual support.