Dynamic Mechanical and Nanofibrous Topological Combinatory Cues Designed for Periodontal Ligament Engineering

Abstract

Complete reconstruction of damaged periodontal pockets, particularly regeneration of periodontal ligament (PDL) has been a significant challenge in dentistry. Tissue engineering approach utilizing PDL stem cells and scaffolding matrices offers great opportunity to this, and applying physical and mechanical cues mimicking native tissue conditions are of special importance. Here we approach to regenerate periodontal tissues by engineering PDL cells supported on a nanofibrous scaffold under a mechanical-stressed condition. PDL stem cells isolated from rats were seeded on an electrospun polycaprolactone/gelatin directionally-oriented nanofiber membrane and dynamic mechanical stress was applied to the cell/nanofiber construct, providing nanotopological and mechanical combined cues. Cells recognized the nanofiber orientation, aligning in parallel, and the mechanical stress increased the cell alignment. Importantly, the cells cultured on the oriented nanofiber combined with the mechanical stress produced significantly stimulated PDL specific markers, including periostin and tenascin with simultaneous down-regulation of osteogenesis, demonstrating the roles of topological and mechanical cues in altering phenotypic change in PDL cells. Tissue compatibility of the tissue-engineered constructs was confirmed in rat subcutaneous sites. Furthermore, in vivo regeneration of PDL and alveolar bone tissues was examined under the rat premaxillary periodontal defect models. The cell/nanofiber constructs engineered under mechanical stress showed sound integration into tissue defects and the regenerated bone volume and area were significantly improved. This study provides an effective tissue engineering approach for periodontal regeneration-culturing PDL stem cells with combinatory cues of oriented nanotopology and dynamic mechanical stretch.

Fig 1. Schematic of experimental design.

Mechanical-stressed PDL cells supported on nanotopological-cued nanofiber membrane scaffolds. The Flexcell system equipped with the PDL cells supported on nanofiber matrix, where the dynamic mechanical tensional force was applied to the matrix/cell through equipment vacuum.

Fig 2. Surface morphology of the membranes.

Electrospun membranes made either random or oriented to provide different nanotopological alignment cues to PDL cells. SEM images of the (a) random and (b) aligned nanofibers. (c) Histograms of the orientation of the nanofibers.

Fig 3. Effects of the nanofiber membrane on the initial cellular adhesion behavior.

Fluorescent images of cell attachment on (a) random and (b) aligned nanofiber. PDL cells recognize the underlying nanofiber alignment, conforming the shape of cell spreading to the nanofiber orientation (Magnification x200, scale bar 100 μm). (c) Cell attachment level, and (d) subsequent proliferation (*p < 0.05, by student t-test).

Fig 4. Effect of the cyclic uniaxial stretch on the orientation of PDL cells.

Examination of cell shaping on the differently-aligned nanofibers with or without applying dynamic mechanical load. F-actins visualized with rhodamine-phalloidin (red) and nuclei stained with DAPI (cyan) for fluorescent images. Red arrows indicate stretch direction.

Fig 5. The orientation of and protrusion behaviors of the cells.

Analyses of the PDL cell orientation and cytoskeletal protrusion, after cell culture under the influence of nanofiber alignment and/or cyclic mechanical load. (a) Distribution of cell orientation angles. (b) Number of cytoskeletal protrusions, and the (c) distribution of protrusion length and (d) average protrusion length. Only bipolar cells were also analyzed in terms of distribution of (e) orientation angle and (f) protrusion length. (^ap < 0.05 compared to DA, by ANOVA).

Fig 6. Effects of the nanofiber alignment and cyclic loading on the osteogenic differentiation of PDL cells.

The effects were assessed by the ALP activity, an early osteogenic differentiation marker (^ap < 0.05 compared to DA, ^bp < 0.05 compared to DR, by ANOVA).

Fig 7. Expressions of proteins related with ligamentogenesis of the PDL cells, as analyzed by an ELISA.

(a) Periostin, (b) tenascin, and (c) TGF-β. Results presented when normalized to the static condition with random nanofiber. (^ap < 0.05 compared to DA, ^bp < 0.05 compared to SA, by ANOVA).

Fig 8. Tissue compatibility of the PDL cell/nanofiber constructs implanted in rat subcutaneous model for 4 weeks.

Histological images of HE and MT stains. Notable observation of PDL-like tissues with spindle-shaped oriented cells in the SA and DA groups, as revealed by MT stain. Direction of the PDL cells (two headed arrow) with randomly distributed collagen fibers were marked (Magnification x400, scale bar 100 μm).

Fig 9. Illustrative images showing the PDL defect models used in this study.

(a) Photograph of rat premaxillary operation field. Note the dimensions of the defect used to produce standardized 4 mm diameters round full-thickness defects on the lateral surface of premaxilla bone. Two defects were created on one animal and were covered with tissue-engineered construct. (b) Harvested specimens of rat premaxillary operation field after sacrifice. (c) Representative histology image of HE staining of new bone tissue formed within the defect at 4 weeks (black arrow: defect margins) (Magnification x40, scale bar 500 μm). (d) 2D and (e) 3D μCT images. The original outline of the 4 mm defect is clear (white arrow).

Fig 10. Micro-CT image analyses results of bone regeneration.

(a) % bone volume (b) bone surface, and (c) bone surface density. The graph represented statistically significant differences among the study groups on the quantification of new bone formation in premaxillary defects after 4 weeks of healing. (^ap < 0.05 compared to DA (remov); ^bp < 0.05 compared to SA (remov); ^cp < 0.05 compared to DA (sound), by ANOVA).

Fig 11. HE stained histological images, showing the tissue regeneration in the periodontal defect model.

(Magnification x400, scale bar 100 μm, NB: new bone, OB: old bone, TEM: tissue-e engineered matrix, RCT: reconstructed connective tissue).