Harnessing the Native Extracellular Matrix for Periodontal Regeneration Using a Melt Electrowritten Biphasic Scaffold

Abstract

Scaffolds have been used to promote periodontal regeneration by providing control over the spacio-temporal healing of the periodontium (cementum, periodontal ligament (PDL) and alveolar bone). This study proposes to enhance the biofunctionality of a biphasic scaffold for periodontal regeneration by means of cell-laid extracellular matrix (ECM) decoration. To this end, a melt electrowritten scaffold was cultured with human osteoblasts for the deposition of bone-specific ECM. In parallel, periodontal ligament cells were used to form a cell sheet, which was later combined with the bone ECM scaffold to form a biphasic PDL-bone construct. The resulting biphasic construct was decellularised to remove all cellular components while preserving the deposited matrix. Decellularisation efficacy was confirmed in vitro, before the regenerative performance of freshly decellularised constructs was compared to that of 3-months stored freeze-dried scaffolds in a rodent periodontal defect model. Four weeks post-surgery, microCT revealed similar bone formation in all groups. Histology showed higher amounts of newly formed cementum and periodontal attachment in the fresh and freeze-dried ECM functionalised scaffolds, although it did not reach statistical significance. This study demonstrated that the positive effect of ECM decoration was preserved after freeze-drying and storing the construct for 3 months, which has important implications for clinical translation.

Keywords: ECM decoration; additive manufacturing; extracellular matrix; melt electrowriting; polycaprolactone.

Figure 1

Scanning electron microscopy images of the cellularised (cell) and decellularised (decell) biphasic constructs. Images were taken at 50, 100 and 250 magnifications to characterise the surface of the bone compartment, the PDL cell sheet and the cross-section of the construct. The PDL and bone compartment of the cross-sectional view have been delimited by the dotted line and, respectively, labelled with “PDL” and “Bone”.

Figure 2

Confocal image with ×10 and ×20 magnification of the biphasic scaffold, displaying views from the bone side and the periodontal ligament side. Samples were stained for cellular DNA in blue (DAPI), collagen VI in red and the main ECM component, collagen I, in green.

Figure 3

(A) Schematic of the periodontal defect (B) MicroCT images of the different groups along with (C) the quantification of the newly formed bone in the defect area (as indicated by the white dash line). Values are presented as individual data points, superimposed by the mean (horizontal line) and the whiskers representing one standard deviation (SD).

Figure 4

(A) Masson’s trichrome staining of the different groups at magnification ×2 (left panel), where the areas designated by the box have been magnified ×10 (right panel). Newly formed cementum is indicated by “C”, bone by “B”, PDL by “PDL” and the PCL fibres by “*”. (B) The percentage of newly formed cementum on the surgically denuded roots was quantified, along with (C) the percentage of regenerated periodontal attachment.

Figure 5

Masson’s trichrome staining of the different groups showing periodontal regeneration in the defect area at magnification ×4, where the areas designated by the box have been magnified ×20. Newly formed cementum is indicated by “C”, new bone by “NB”, periodontal ligament by “PDL” and the PCL fibres by “*”. The white dashed lines represent the periodontal gap.