This chapter will discuss the use of inkjet printing for the manufacture of barrier membranes. Barrier membranes can be used in dentistry for regenerative work such as improving alveolar bone augmentation. An ideal barrier membrane will have controllable resorption rates, be biocompatible, prevent surrounding tissues from collapsing into the defect space, as well as provide cell occlusivity. Current barrier membranes are produced from materials which are either non-resorbable, and require a secondary surgery for their extraction, or, made from resorbable materials which can have poor structural integrity or degrade into acidic by-products. Silk has had a long history of use as a biomaterial, it degrades into non-toxic components and has adaptable mechanical properties. Silk has several polymorphs; silk I and silk II. Silk I is non-crystalline and water soluble while silk II has a crystalline β-sheet structure that is non-water soluble. Silk I converts to silk II upon exposure to methanol. This process of conversion can be utilised in inkjet printing where multiple inks can be printed. Inkjet printing can produce complex samples with control over shape, surface topography and gradients and therefore may offer an alternative manufacturing method for the production of barrier membranes.

It has recently been shown that regenerated silk fibroin (RSF) aqueous solution can be printed using an inkjet printer. In this communication, we demonstrate an alternative reactive inkjet printing method that provides control over RSF crystallinity through β-sheet concentration. A biocompatible film has successfully been produced through the alternate printing of RSF aqueous solution and methanol using reactive inkjet printing. Control over the formation of the β-sheet structure was achieved by printing different ratios of RSF to methanol and was confirmed using Fourier Transform Infra Red spectroscopy. The biocompatibility of the printed silk scaffold was demonstrated by the growth of fibroblast cells upon its surface.

BACKGROUND AND OBJECTIVE:Studies of peri-implant soft tissue on in vivo models are commonly based on histological sections prepared using undecalcified or 'fracture' techniques. These techniques require the cutting or removal of implant during the specimen preparation process. The aim of this study is to explore a new impression technique that does not require any cutting or removal of implant for contour analysis of soft tissue around four types of titanium (Ti) surface roughness using an in vitro three-dimensional oral mucosal model (3D OMM). METHODS:The 3D OMM was constructed by co-culturing a keratinocyte cell line TR146 and human oral fibroblasts on to an acellular dermis scaffold. On the fourth day, a Ti disk was placed into the model. Four types of Ti surface topographies, i.e. polished, machined, sandblasted and anodized were tested. After 10 d of culture, the specimens were processed based on undecalcified (ground sectioning), electropolishing and impression techniques for contour analysis of the implant-soft tissue interface. RESULTS:Under light microscopic examination of the ground and electropolishing sections, it was found that the cell line-based oral mucosa formed a peri-implant-like epithelium attachment on to all four types of Ti surfaces. In contour analysis, the most common contour observed between the cell line-based oral mucosa and Ti surface was at an angle ranging between 45° and 90°. CONCLUSION:The in vitro cell line-based 3D OMM formed a peri-implant-like epithelium at the implant-soft tissue interface. The contour of the implant-soft tissue interface for the four types of Ti surface was not significantly different.

Aim:  The purpose of this study was to assess the effectiveness of combining freshly excised oral mucosa tissue with different biological and synthetic scaffolds for production of graft material for one-stage soft-tissue reconstruction. Material and Methods:  Small oral mucosa biopsies were finely minced and transplanted onto a larger circular subcutaneous wound bed in rats. Test groups included explants seeded onto (1) an electrospun scaffold, (2) acellular dermis, and (3) directly on the muscle fascia. The soft-tissue implants were examined using histology and immunohistochemical analysis for cytokeratins (CKs) 5, 10, 13, and 19. Results:  Mucosal explants grew into fully developed epithelium with keratinisation. Surprisingly, instead of spreading over the surface of the membranes, the epithelial explants curled up and formed cyst-like structures in all test groups. The epithelial layer of the cysts originated from mucosal explants grown on the electrospun scaffold showed weak positive staining for CK5, CK10, and CK19 similar to native rat oral mucosa. Other groups showed negative staining for the CKs. Conclusions:  Subcutaneous transplantation of freshly excised oral mucosa explants resulted in the growth of the explants and formation of inclusion cyst-like structures. The phenotype of the cyst epithelium was affected by the underlying connective tissue substrate.

Advances in tissue engineering have permitted the three-dimensional (3D) reconstruction of human oral mucosa for various in vivo and in vitro applications. Tissue-engineered oral mucosa have been further optimized in recent years for clinical applications as a suitable graft material for intra-oral and extra-oral repair and treatment of soft-tissue defects. Novel 3D in vitro models of oral diseases such as cancer, Candida, and bacterial invasion have been developed as alternatives to animal models for investigation of disease phenomena, their progression, and treatment, including evaluation of drug delivery systems. The introduction of 3D oral mucosal reconstructs has had a significant impact on the approaches to biocompatibility evaluation of dental materials and oral healthcare products as well as the study of implant-soft tissue interfaces. This review article discusses the recent advances in tissue engineering and applications of tissue-engineered human oral mucosa.

The success of dental implant treatment depends on the healing of both hard and soft tissues. While osseointegration provides initial success, the biological seal of the peri-implant soft tissue is crucial for maintaining the long term success of implants. Most studies of the biological seal of peri-implant tissues are based on animal or monolayer cell culture models. To understand the mechanisms of soft tissue attachment and the factors affecting the integrity of the soft tissue around the implants, it is essential to obtain good quality histological sections for microscopic examination. The nature of the specimens, however, which consist of both metal implant and soft peri-implant tissues, poses difficulties in preparing the specimens for histomorphometric analysis of the implant-soft tissue interface. We review various methods that have been used for the implant-tissue interface investigation with particular focus on the soft tissue. The different methods are classified and the advantages and limitations of the different techniques are highlighted.

Modified fluorcanasite glasses were fabricated by either altering the molar ratios of Na(2)O and CaO or by adding P(2)O(5) to the parent stoichiometric glass compositions. Glasses were converted to glass-ceramics by a controlled two-stage heat treatment process. Rods (2 mm x 4 mm) were produced using the conventional lost-wax casting technique. Osteoconductive 45S5 bioglass was used as a reference material. Biocompatibility and osteoconductivity were investigated by implantation into healing defects (2 mm) in the midshaft of rabbit femora. Tissue response was investigated using conventional histology and scanning electron microscopy. Histological and histomorphometric evaluation of specimens after 12 weeks implantation showed significantly more bone contact with the surface of 45S5 bioglass implants when compared with other test materials. When the bone contact for each material was compared between experimental time points, the Glass-Ceramic 2 (CaO rich) group showed significant difference (p = 0.027) at 4 weeks, but no direct contact at 12 weeks. Histology and backscattered electron photomicrographs showed that modified fluorcanasite glass-ceramic implants had greater osteoconductivity than the parent stoichiometric composition. Of the new materials, fluorcanasite glass-ceramic implants modified by the addition of P(2)O(5) showed the greatest stimulation of new mineralized bone tissue formation adjacent to the implants after 4 and 12 weeks implantation.

Bone graft procedures typically require surgeons to harvest bone from a second site on a given patient (Autograft) before repairing a bone defect. However, this results in increased surgical time, excessive blood loss and a significant increase in pain. In this context a synthetic bone graft with excellent histocompatibility, built in antibacterial efficacy and the ability to regenerate healthy tissue in place of diseased tissue would be a significant step forward relative to current state of the art philosophies. We developed a range of calcium–strontium–zinc–silicate glass based bone grafts and characterised their structure and physical properties, then evaluated their in vitro cytotoxicity and in vivo biocompatibility using standardised models from the literature. A graft (designated BT109) of composition 0.28SrO/0.32ZnO/0.40 SiO2 (mol fraction) was the best performing formulation in vitro shown to induce extremely mild cytopathic effects (cell viability up to 95%) in comparison with the commercially available bone graft Novabone® (cell viability of up to 72%). Supplementary to this, the grafts were examined using the standard rat femur healing model on healthy Wister rats. All grafts were shown to be equally well tolerated in bone tissue and new bone was seen in close apposition to implanted particles with no evidence of an inflammatory response within bone. Complimentary to this BT109 was implanted into the femurs of ovariectomized rats to monitor the response of osteoporotic tissue to the bone grafts. The results from this experiment indicate that the novel grafts perform equally well in osteoporotic tissue as in healthy tissue, which is encouraging given that bone response to implants is usually diminished in ovariectomized rats. In conclusion these materials exhibit significant potential as synthetic bone grafts to warrant further investigation and optimisation.

Glass-ionomer Cements (GICs) have been employed extensively in the repair of tooth tissue since the 1970s and this long history suggests that they are among the most biocompatible dental materials available. Their apparent safety and history of good biocompatibility led scientists and clinicians to consider them for wider surgical uses in the 1980s and 1990s. Conventional GICs are formed from the combination of high molecular weight polymeric acids, a basic floroaluminsilicate glass powder and water. GICs have some advantages for orthopedic applications, including their truly adhesive nature and non-exothermic setting reaction, which does not result in shrinkage and might improve the release of incorporated therapeutic compounds. Their mechanical properties are, however, inferior to those of acrylic bone cements, limiting their load-bearing capacity. It is their interaction with the biological environment that is both encouraging and problematic in equal measure. Biological evaluation has provided some encouraging results, for example, the so-called bioactive nature of GICs, by which a composition, site and tissue-dependent ion exchange has encouraged an appropriate host response.

A range of calcium—strontium—zinc—silicate glass grafts are developed. Following characterization, their ability to form an apatite layer in simulated body fluid (SBF) is evaluated. Concurrently, their in vivo biocompatibility is determined. These glasses are incapable of forming an apatite layer in SBF. However, in vivo, each glass is well tolerated with new bone formation apparent in close apposition to implanted particles and no evidence of an inflammatory response. Such results are contrary to much of the literature and indicate that forecasting a materials ability to bond to bone based on SBF experiments may provide a false negative result.

INTRODUCTION: An alternative approach to bone tissue engineering may be to find a suitable cell source to engineer a cartilage-based construct (e.g. hypertrophic cartilage) that can mineralise in vitro or in vivo. Cartilage has the advantage that it can survive in a relatively hypoxic environment, which may allow more time for vascularisation of the engineered graft to develop post-implantation. The aim of this research was to investigate the conditions that promote generation of a cartilage construct with characteristics of hypertrophic tissue

Restorative dental materials and oral health care products come into direct contact with oral mucosa and can cause adverse reactions. In order to obtain an accurate risk assessment, the in vitro test model must reflect the clinical situation as closely as possible. The aim of this study was to develop and optimize a three-dimensional full-thickness engineered human oral mucosal model, which can be used for biological assessment of dental materials. In this study human oral fibroblasts and keratinocytes were isolated from patients and seeded onto a number of collagen-based and synthetic scaffolds using a variety of cell seeding techniques and grown at the air/liquid interface to construct human oral mucosa equivalents. Suitability of 10 different scaffolds for engineering human oral mucosa was evaluated in terms of biocompatibility, biostability, porosity, and the ability to mimic normal human oral mucosa morphology. Finally an optimized full-thickness engineered human oral mucosa was developed and characterized using transmission electron microscopy and immunostaining. The oral mucosa reconstruct resembled native human oral mucosa and it has the potential to be used as an accurate and reproducible test model in mucotoxicity and biocompatibility evaluation of dental materials.

Tissue-engineered oral mucosal equivalents have been developed for clinical applications and also for in vitro studies of biocompatibility, mucosal irritation, disease, and other basic oral biology phenomena. This paper reviews different tissue-engineering strategies used for the production of human oral mucosal equivalents, their relative advantages and drawbacks, and their applications. Techniques used for skin tissue engineering that may possibly be used for in vitro reconstruction of human oral mucosa are also discussed.

Components released from dental composite resins are essential factors in the assessment of biocompatibility of these materials. The effect of different extraction media on monomer release from composite resins based on different monomer types was evaluated. Three types of visible light cured composite resins were formulated based on the following monomers: triethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerolate dimethacrylate (BisGMA), and urethane dimethacrylate (UDMA). Seventy-five composite resin discs were fabricated and light cured for 1 min in the absence of oxygen. Extraction media used were: distilled water, saline solution, artificial saliva, serum-free culture medium, and culture medium with 10% fetal calf serum. The analysis of extracts from the composite resins was carried out by High Performance Liquid Chromatography (HPLC). Quantifiable amounts of TEGDMA were released into the aqueous media. However, BisGMA and UDMA were not detectable in any of the extracts from the composite resins. Statistical analysis by one-way ANOVA followed by Tukey’s test showed that there was a significant difference in TEGDMA release between culture media and other media (p < 0.05). From the results of this experiment it can be concluded that TEGDMA-based composite resins can release a high quantity of monomer into aqueous environments. The type of extraction medium may have a significant effect on monomer release from composite resins.

Fluorcanasite glass-ceramics were produced by controlled two stage heat-treatment of as-cast glasses. These glasses were modified from stoichiometric fluorcanasite composition by either adding P(2)O(5) or altering the molar ratios of Na(2)O and CaO. Commercial bioactive 45S5 Bioglass(R) was also prepared in-house to evaluate the relative in vitro biocompatibility of fluorcanasite glass-ceramics. The scanning electron microscopy (SEM) images showed that cells had colonized the surfaces of fluorcanasite glass-ceramics to form a confluent sheet. Quantitative MTT assay results were in good agreement with the qualitative SEM observations. It was concluded that incorporation of excess calcium oxide or P(2)O(5) in stoichiometric glass composition improved in vitro biocompatibility. Controlled heat-treatment further improved the biological response of cultured bone cells to modified fluorcanasite glass-ceramics when compared with their parent glasses. Ion release and pH data suggested a strong correlation between solubility (in particular, Na ion release) and biocompatibility. Reduced solubility, Na ion release, and related pH effects appeared to be the principal mechanisms responsible for improvement in in vitro biocompatibility.