Development of a Composite Tissue Engineered Alveolar Bone−Mucosal Model Using Conventional and 3D Printed Scaffolding Techniques

Advances in tissue engineering have allowed the construction of various tissues of the oral and maxillofacial region for clinical and in vitro modelling proposes. Additive manufacturing, also known as three−dimensional printing (3DP) is an innovative technique that offers an entirely new method of fabricating geometrically precise 3D structures, allowing the opportunity to progress composite tissue engineering to the point where complex anatomical relationships can be accurately replicated. The aim of this study was to develop and characterise a novel 3D composite human alveolar bone−mucosal model (ABMM) based on conventional and 3D printed bone scaffolds. Two types of bone scaffold were used: firstly, a conventional hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold fabricated using an aqueous gel-casting method, and secondly, a 3D printed β−tricalcium phosphate (β−TCP) scaffold prepared using an extrusion−based Rapid Prototyping plotting system. In order to construct a composite bone−mucosal model, alveolar bone-derived osteoblasts were seeded into the respective scaffolds (both conventional and printed) and the resultant bone constructs were then attached to a tissue engineered, collagen−based oral mucosa. Histological, immunohistochemical, and ultrastructural features of the mucosal part as well as, the histology, genes expression, and proteins secretion of the composite models were examined to validate the ABMM as a representative analogue of combined oral hard and soft tissues. The mucosal component demonstrated a mature epithelium undergoing terminal differentiation similar to that of native oral mucosa, as confirmed using cytokeratin immunohistochemistry. Histological evaluation of ABMM confirmed an anatomically representative tri-layer consisting of distinct epithelial, connective tissue, and bone layers. Interrogation of osteogenic and epithelial−related gene expression within the models confirmed an osteogenic expression profile in the tri−layered model that was not observed in epithelial−stromal bilayers. Collectively, these data suggest that the developed composite model displayed characteristics similar to those of normal tissue counterparts. This novel tri−layered model, therefore, may offer great scope as a more advanced, and anatomically representative tool for a number of in vitro applications

Development of three-dimensional tissue engineered bone-oral mucosal composite models

Tissue engineering of bone and oral mucosa have been extensively studied independently. The aim of this study was to develop and investigate a novel combination of bone and oral mucosa in a single 3D in vitro composite tissue mimicking the natural structure of alveolar bone with an overlying oral mucosa. Rat osteosarcoma (ROS) cells were seeded into a hydroxyapatite/tri-calcium phosphate scaffold and bone constructs were cultured in a spinner bioreactor for 3 months. An engineered oral mucosa was fabricated by air/liquid interface culture of immortalized OKF6/TERET-2 oral keratinocytes on collagen gel-embedded fibroblasts. EOM was incorporated into the engineered bone using a tissue adhesive and further cultured prior to qualitative and quantitative assessments. Presto Blue assay revealed that ROS cells remained vital throughout the experiment. The histological and scanning electron microscope examinations showed that the cells proliferated and densely populated the scaffold construct. Micro computed tomography (micro-CT) scanning revealed an increase in closed porosity and a decrease in open and total porosity at the end of the culture period. Histological examination of bone-oral mucosa model showed a relatively differentiated parakeratinized epithelium, evenly distributed fibroblasts in the connective tissue layer and widely spread ROS cells within the bone scaffold. The feasibility of fabricating a novel bone-oral mucosa model using cell lines is demonstrated. Generating human ‘normal’ cell-based models with further characterization is required to optimize the model for in vitro and in vivo applications

Biomodification of a Class-V Restorative Material by Incorporation of Bioactive Agents

Restoring subgingival class-V cavities successfully, demand special biological properties from a restorative material. This study aimed to assess the effects of incorporating bioactive materials to glass ionomer cement (GIC) on its mechanical and biological properties. Hydroxyapatite, chitosan, chondroitin sulphate, bioglass, gelatine and processed bovine dentin were incorporated into a GIC restorative material. Compressive strength, biaxial flexural strength (BFS), hardness, setting and working time measurements were investigated. Biocompatibility of the new materials was assessed using both monolayer cell cultures of normal oral fibroblasts (NOF) and TR146 keratinocytes, and a 3D-tissue engineered human oral mucosa model (3D-OMM) using presto-blue tissue viability assay and histological examination. Significant reduction in the compressive strength and BFS of gelatine-modified discs was observed, while chondroitin sulphate-modified discs had reduced BFS only (p value > 0.05). For hardness, working and setting times, only bioglass caused significant increase in the working time. NOF viability was significantly increased when exposed to GIC-modified with bovine dentine, bioglass and chitosan. Histological examination showed curling and growth of the epithelial layer toward the disc space, except for the GIC modified with gelatine. This study has highlighted the potential for clinical application of the modified GICs with hydroxyapatite, chitosan, bioglass and bovine dentine in subgingival class-V restorations