Abstract
Objective The aim of this study was to examine the potential of periodontal ligament (PDL) cells sheet and arginine-glycyl-aspartic acid (RGD)-modified chitosan scaffold for periodontal tissue regeneration in horizontal periodontal defect model. Materials and Methods PDL cell cytotoxicity was tested with 3–[4,5- dimethylthiazol-2yl]–2,5-diphenyl-2H-tetrazolium bromide assay. Cell migration toward the chitosan-based materials was analyzed with trans-well migration assay. Horizontal periodontal defect model was created in four maxillary and mandibular lateral incisors of Macaque nemestrina . Following periodontal therapy, the sites were transplanted with various regenerative materials: (1) chitosan, (2) RGD-modified chitosan, (3) PDL cell sheet with chitosan, (4) PDL cell sheet with RGD-modified chitosan. The periodontal tissue regeneration was evaluated clinically and radiographically. Gingival crevicular fluids were collected each week to evaluate cementum protein-1 (CEMP-1) expression with enzyme-linked immunosorbent assay, while the biopsies were retrieved after 4 weeks for histological and microcomputed tomography evaluation. Statistical Analysis Data was statistically analyzed using GraphPad Prism 6 for MacOS X. Normality was tested using the Shapiro–Wilk normality test. The Kruskal–Wallis test was used to compare the groups. Significance was accepted when p < 0.05. Results Clinical examination revealed more epithelial attachment was formed in the group with PDL cell sheet with RGD-modified chitosan. Similarly, digital subtraction radiography analysis showed higher gray scale, an indication of higher alveolar bone density surrounded the transplanted area, as well as higher CEMP-1 protein expression in this group. The incorporation of RGD peptide to chitosan scaffold in the group with or without PDL cells sheet reduced the distance of cement–enamel junction to the alveolar bone crest; hence, more periodontal tissue formed. Conclusions Horizontal periodontal defect model could be successfully created in M. nemestrina model. Combination of PDL cell sheet and RGD-modified chitosan resulted in the higher potential for periodontal tissue regeneration. The results of this study highlight the PDL cell sheet and RGD-modified chitosan as a promising approach for future clinical use in periodontal regeneration.
Highlights
Periodontal defects are frequently found in severe p eriodontitis patients and various attempts have already been performed to regenerate the new periodontal tissues.[1,2,3] The current treatments provide predictable clinical outcome of the improvement in clinical parameters; these approaches are restricted in appropriate case selection of small-to-medium-size defect
Combination of periodontal ligament (PDL) cell sheet and arginine-glycyl-aspartic acid (RGD)-modified chitosan resulted in the higher potential for periodontal tissue regeneration
More than 50% of the seeded cells were migrated toward lower compartments in the Boyden chamber in both chitosan and RGD-modified chitosan group (p < 0.01) (►Fig. 3)
Summary
Periodontal defects are frequently found in severe p eriodontitis patients and various attempts have already been performed to regenerate the new periodontal tissues.[1,2,3] The current treatments provide predictable clinical outcome of the improvement in clinical parameters; these approaches are restricted in appropriate case selection of small-to-medium-size defect. In critical size defect such as in one-wall or horizontal periodontal defect, periodontal tissue regeneration is still a challenge and it often results in unpredictable clinical outcome. As such focus has shifted to the potential use of tissue engineering technique for periodontal regeneration in large bone defect cases.[4,5,6,7,8,9]. Cells from various sources such as periodontal ligament (PDL), bone marrow, and adipose tissues have been tested for the synthesis of cell sheet materials.[6,7,8,9] the initial concept of cell sheet technology is to eliminate the need for scaffold materials, due to delicate nature of the cell sheet, the use of biodegradable support matrices is still necessary to support the cell sheet to be transplanted to the defect areas such as periodontal tissue defects
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