3D Bioprinting for In Vitro Models of Oral Cancer: Toward Development and Validation.

The tumor microenvironment (TME) of oral carcinomas has highly complex contents and a dynamic nature which is difficult to study using oversimplified two-dimensional (2D) cell culture systems. By contrast, three dimensional (3D) in vitro models such as spheroids, organoids, and scaffold-based constructs have been able to replicate tumors three-dimensionality and have allowed a better understanding of the role of various microenvironmental cues in the initiation and progression of cancer. However, the heterogeneity of TME cannot be fully reproduced by these traditional tissue engineering strategies since they are unable to control the organization of multiple cell types in a complex architecture. 3D bioprinting is an emerging field that can be leveraged to produce biomimetic and complex tissue structures. Bioprinting allows for controllable and precise placement of multicomponent bioinks composed of multiple biomaterials, different types of cells, and soluble factors according to the natural compartments of the target tissue, aiming to reproduce the equivalent of the complex tissue. As such, 3D bioprinting provides a unique opportunity to fabricate in vitro tumor models with a complexity similar to that of the in vivo oral carcinoma. This will facilitate a thorough investigation of cellular physiology, cancer progression, and anti-cancer drug screening with unprecedented control and reproducibility. In this review, we discuss the role of 3D bioprinting in reconstituting oral cancer, the prospects of application to fill the literature gap, and the challenges that need to be addressed in order to exploit this emerging technology for future work in oral cancer research.

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.

Effects of electronic cigarette liquid on monolayer and 3D tissue-engineered models of human gingival mucosa.

Background: There is limited data available on potential biological effects of E-cigarettes on human oral tissues. The aim of this study was to evaluate the effects of E-cigarette liquid on the proliferation of normal and cancerous monolayer and 3D models of human oral mucosa and oral wound healing after short-term and medium-term exposure. Methods: Normal human oral fibroblasts (NOF), immortalized OKF6-TERET-2 human oral keratinocytes, and cancerous TR146 keratinocyte monolayer cultures and 3D tissue engineered oral mucosal models were exposed to different concentrations (0.1%, 1%, 5% and 10%) of E-cigarette liquid (12 mg/ml nicotine) for 1 hour daily for three days and for 7 days. Tissue viability was monitored using the PrestoBlue assay. Wounds were also produced in the middle surface of the monolayer systems vertically using a disposable cell scraper. The alterations in the cell morphology and wound healing were visualized using light microscopy and histological examination. Results: Statistical analysis showed medium-term exposure of TR146 keratinocytes to 5% and 10% E-liquid concentrations significantly increased the viability of the cancer cells compared to the negative control. Short-term exposure of NOFs to 10% E-liquid significantly reduced the cell viability, whereas medium-term exposure to all E-liquid concentrations significantly reduced the NOF cells' viability. OKF6 cells exhibited significantly lower viability following short-term and mediumterm exposure to all E-cigarette concentrations compared to the negative control. 3D oral mucosal model containing normal oral fibroblasts and keratinocytes showed significant reduction in tissue viability after exposure to 10% E-liquid, whereas medium-term exposure resulted in significantly lower viability in 5% and 10% concentration groups compared to the negative control. There was a statistically significant difference in wound healing times of both NOF and OKF6 cells after exposure to 1%, 5% and 10% E-cigarette liquid. Conclusion: Medium-term exposure to high concentrations of the E-cigarette liquid had cytotoxic effects on normal human oral fibroblasts and OKF6 keratinocytes, but a stimulatory cumulative effect on the growth of cancerous TR146 keratinocyte cells as assessed by the PrestoBlue assay and histological evaluation of 3D oral mucosal models. In addition, E-liquid exposure prolonged the wound healing of NOF and OKF6 oral mucosa cells.

3D printed tissue engineered model for bone invasion of oral cancer.

Recent advances in three-dimensional printing technology have led to a rapid expansion of its applications in tissue engineering. The present study was designed to develop and characterize an in vitro multi-layered human alveolar bone, based on a 3D printed scaffold, combined with tissue engineered oral mucosal model. The objective was to incorporate oral squamous cell carcinoma (OSCC) cell line spheroids to the 3D model at different anatomical levels to represent different stages of oral cancer. Histological evaluation of the 3D tissue model revealed a tri-layered structure consisting of distinct epithelial, connective tissue, and bone layers; replicating normal oral tissue architecture. The mucosal part showed a well-differentiated stratified oral squamous epithelium similar to that of the native tissue counterpart, as demonstrated by immunohistochemistry for cytokeratin 13 and 14. Histological assessment of the cancerous models demonstrated OSCC spheroids at three depths including supra-epithelial level, sub-epithelial level, and deep in the connective tissue-bone interface. The 3D tissue engineered composite model closely simulated the native oral hard and soft tissues and has the potential to be used as a valuable in vitro model for the investigation of bone invasion of oral cancer and for the evaluation of novel diagnostic or therapeutic approaches to manage OSCC in the future.

3D-printed membrane for guided tissue regeneration.

Three-dimensional (3D) printing is currently being intensely studied for a diverse set of applications, including the development of bioengineered tissues, as well as the production of functional biomedical materials and devices for dental and orthopedic applications. The aim of this study was to develop and characterize a 3D-printed hybrid construct that can be potentially suitable for guided tissue regeneration (GTR). For this purpose, the rheology analyses have been performed on different bioinks and a specific solution comprising 8% gelatin, 2% elastin and 0.5% sodium hyaluronate has been selected as the most suitable composition for printing a structured membrane for GTR application. Each membrane is composed of 6 layers with strand angles from the first layer to the last layer of 45, 135, 0, 90, 0 and 90°. Confirmed by 3D Laser Measuring imaging, the membrane has small pores on one side and large pores on the other to be able to accommodate different cells like osteoblasts, fibroblasts and keratinocytes on different sides. The ultimate cross-linked product is a 150µm thick flexible and bendable membrane with easy surgical handling. Static and dynamic mechanical testing revealed static tensile modules of 1.95±0.55MPa and a dynamic tensile storage modulus of 314±50kPa. Through seeding the membranes with fibroblast and keratinocyte cells, the results of in vitro tests, including histological analysis, tissue viability examinations and DAPI staining, indicated that the membrane has desirable in vitro biocompatibility. The membrane has demonstrated the barrier function of a GTR membrane by thorough separation of the oral epithelial layer from the underlying tissues. In conclusion, we have characterized a biocompatible and bio-resorbable 3D-printed structured gelatin/elastin/sodium hyaluronate membrane with optimal biostability, mechanical strength and surgical handling characteristics in terms of suturability for potential application in GTR procedures.

Characterization of Multilayered Tissue-Engineered Human Alveolar Bone and Gingival Mucosa.

Advances in tissue engineering have permitted assembly of multilayered composite tissue constructs for potential applications in the treatment of combined hard and soft tissue defects and as an alternative in vitro test model to animal experimental systems. The aim of this study was to develop and characterize a novel three-dimensional combined human alveolar bone and gingival mucosal model based on primary cells isolated from the oral tissues. Bone component of the model was engineered by seeding primary human alveolar osteoblasts into a hydroxyapatite/tricalcium phosphate scaffold and culturing in a spinner bioreactor. The engineered bone was then laminated, using an adhesive tissue sealant, with tissue-engineered gingival mucosa consisting of air/liquid interface-cultured normal human gingival keratinocytes on oral fibroblast-populated collagen gel scaffold. Histological characterization revealed a structure consisting of established epithelial, connective tissue and bone layers closely comparable to normal oral tissue architecture. The mucosal component demonstrated a mature epithelium undergoing terminal differentiation similar to that characteristic of native buccal mucosa, as confirmed using cytokeratin 13 and cytokeratin 14 immunohistochemistry. Ultrastructural analysis confirmed the presence of desmosomes and hemidesmosomes in the epithelial layer, a continuous basement membrane, and newly synthesized collagen in the connective tissue layer. Quantitative polymerase chain reaction (qPCR) assessment of osteogenesis-related gene expression showed a higher expression of genes encoded collagen I (COL1) and osteonectin (ON) compared with osteocalcin (OC), osteopontin (OP), and alkaline phosphatase (ALP). Enzyme-linked immunosorbent assay quantification of COL1, ON, and OC confirmed a pattern of secretion, which paralleled the model's gene expression profile. We demonstrate in this study that, replicating the anatomical setting between oral mucosa and the underlying alveolar bone is feasible and the developed model showed characteristics similar to those of normal tissue counterparts. This trilayered model therefore offers great scope as an advanced and anatomically representative tissue-engineered alternative to animal models.

The significance of cell-related challenges in the clinical application of tissue engineering.

Tissue engineering is increasingly being recognized as a new approach that could alleviate the burden of tissue damage currently managed with transplants or synthetic devices. Making this novel approach available in the future for patients who would potentially benefit is largely dependent on understanding and addressing all those factors that impede the translation of this technology to the clinic. Cell-associated factors in particular raise many challenges, including those related to cell sources, up- and downstream techniques, preservation, and the creation of in vitro microenvironments that enable cells to grow and function as far as possible as they would in vivo. This article highlights the main confounding issues associated with cells in tissue engineering and how these issues may hinder the advancement of therapeutic tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3157-3163, 2016.

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.