Development and application of a 3D periodontal in vitro model for the evaluation of fibrillar biomaterials

Abstract

Background: Periodontitis is a chronic inflammation of the tooth supporting structures that finally can lead to tooth loss. As chronic periodontitis is associated with systemic diseases multiple approaches have been followed to support regeneration of the destructed tissue. But very few materials are actually used in the clinic. A new and promising group of biomaterials with advantageous biomechanical properties that have the ability to support periodontal regeneration are self-assembling peptides (SAP). However, there is still a lack of 3D periodontal models that can evaluate the migration potential of such novel materials.

Methods: All experiments were performed with primary human periodontal ligament fibroblasts (HPLF). Migration capacity was assessed in a three-dimensional model of the human periodontal ligament by measuring the migration distance of viable cells on coated (Enamel Matrix Protein (EMP), P11-4, collagen I) or uncoated human dentin. Cellular metabolic activity on P11-4 hydrogels was assessed by a metabolic activity assay. Deposition of ECM molecules in a P11-4 hydrogel was visualized by immunostaining of collagen I and III and fibrillin I.

Results: The 3D periodontal model was feasible to show the positive effect of EMP for periodontal regeneration. Subsequently, self-assembling peptide P11-4 was used to evaluate its capacity to support regenerative processes in the 3D periodontal model. HPLF coverage of the dentin surface coated with P11-4 increased significantly over time, even though delayed compared to EMP. Cell viability increased and inclusion of ECM proteins into the biomaterial was shown.

Conclusion: The presented results indicate that the 3D periodontal model is feasible to assess periodontal defect coverage and that P11-4 serves as an efficient supporter of regenerative processes in the periodontal ligament.

Clinical relevance: The establishment of building-block synthetic polymers offers new opportunities for clinical application in dentistry. Self-assembling peptides represent a new generation of biomaterials as they are able to respond dynamically to the changing environment of the biological surrounding. Especially in the context of peri-implant disease prevention and treatment they enable the implementation of new concepts.

Keywords: 3D model; Periodontal ligament; Regeneration; Self-assembling peptide.

Fig. 1

In vitro model set up. a) Illustration of the mould embedded in agarose gel (2 mg/ml) and the human dentin plate on the bottom with the collagen based cell donor compartment b) Cultivation for 4 and 8 days with exchange of the medium every other day c) Cell migration into the applied matrix, after 8 days incubation. Cells were visualized with a metabolic activity reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT)

Fig. 2

Proof of concept study. The interaction of human periodontal ligament fibroblasts (HPLF) with the different components used in the developed 3D in vitro model was first assessed by Scanning Electron Microscopy (SEM) and cell staining with MTT. a) SEM of HPLF grown on human dentin (scale bar 100 μm). Migration of HPLF was visualized by cell staining with MTT after 4 days of culture on cultured on b) collagen (2 mg/ml, scale bar 800 μm), c) dentin (scale bar 1000 μm) and d) Enamel Matrix Protein (EMP, 30 mg/ml, scale bar 1000 μm). e) Quantification of the maximal migration distance (μm) of HPLF on collagen, dentin and EMP (30 mg/ml) was determined by ImageJ analysis, n = 3. Data showed no statistical difference. f) Migration density calculated as cell number/cm²) with ImageJ analysis

Fig. 3

Time dependent penetration of P11–4 into dentin tubules. a) Confocal microscopy of human dentin plate (scale bar 50 μm). b) Penetration of labeled self-assembling peptide P11–4 (1:10 mix with a 20 mg/ml concentration) in dentin tubuli was tracked by confocal microscopy after 4 days incubation (scale bar 50 μm). A deep penetration of P11–4 into dentin tubuli can be observed

Fig. 4

Metabolic activity of HPLF. Cell viability was measured after 1, 3 and 6 days incubation on tissue culture polystyrene (TCPS), collagen (2 mg/ml), P11–4 (20 mg/ml) using a metabolic activity assay. Data were calculated to % TCPS control at day 1, n = 3, * p-value ≤0.05, *** p-value ≤0.001, **** p-value ≤0.001. HPLF cultured on P11–4 showed a significant increase in metabolic activity, compared to cells grown on collagen gels

Fig. 5

HPLF migration on P11–4 peptide hydrogel. (a) Cell migration was measured by MTT staining after 4 days, 7 days and 8 days culturing on P11–4 hydrogels (20 mg/ml). b) 3D Z-stack image of migrated HPLF on P11–4 hydrogel after 3 days, taken by confocal microscopy. Scale bar 60 μm. (c) Migration distance (μm) was measured with Axiovision software (Zeiss) from baseline after 4, 7 and 8 days incubation of HPLF on P11–4 (20 mg/ml), n = 3, *p-value ≤0.05, **p-value ≤0.01, *** p-value ≤0.001. (d) Cell migration density in number/cm² after 4 and 8 days incubation of HPDLF. e) Quantification of the maximal migration distance (μm) of HPLF on P11–4 (20 mg/ml) was determined by ImageJ analysis, n = 3, *p-value ≤0.05, **p-value ≤0.01, *** p-value ≤0.001. f) Migration density calculated as cell number/cm² with ImageJ analysis. Pictures proof a high migration capacity of HPLF into fibrillar P11–4 hydrogels

Fig. 6

Extracelluar matrix (ECM) protein expression. a) Collagen type I, b) Collagen type III and c) Fibrillin I was visualized with fluorescent immunostaining after 7 days incubation on P11–4 (20 mg/ml) hydrogels. Scale bar 100 μm. Pictures demonstrate the expression of relevant ECM proteins for periodontal regeneration, after the incubation of HPLF on P11–4 hydrogels

Fig. 7

HPLF collagen type Ι expression. HPLF were cultured on P11–4 hydrogels (20 mg/ml) up to 21 days. After 7, 14 and 21 days, supernatants were removed and analyzed for collagen type Ι expression by ELISA. Collagen amount were normalized to the cell proliferation rate. As a control, cells were cultured on tissue culture polystyrene (TCPS). Data represent the mean ± standard deviation, n = 3, *p-value ≤0.05, **p-value ≤0.01. Cells cultured on P11–4 hydrogels resulted in a significant increase in collagen type Ι production, compared to HPLF grown on TCPS