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Background or statement of the problem. Acrylic polymers, such as polymethyl methacrylate (PMMA), are widely used in the fabrication of removable dental prostheses due to their numerous advantages. These include adequate physical, mechanical, and aesthetic properties, low cost, easy fabrication, and biocompatibility. However, the lack of antimicrobial effects in denture base resins (DBRs) has garnered significant interest among dental researchers, particularly because PMMA lacks the ability to resist antimicrobial activity. Specifically, a recurring oral condition such as Candida-associated denture stomatitis (CADS) is difficult to eliminate because disease-causing yeast bacteria or fungi can penetrate the denture base materials. Furthermore, three-dimensional (3D) printing is rapidly emerging at the forefront of digital technology and permeating the dental profession, expanding treatment options for patients, such as fabricating dental prostheses. However, the surface properties of 3D printed materials have posed challenges in both general medicine and dentistry, primarily due to the potential for microbial adhesion to the material surface. To address this issue and mitigate the formation of biofilms on denture surfaces, previous studies have explored the incorporation of organic or inorganic antibacterial and antifungal agents into DBRs. Building upon this body of research, this study utilised phytochemical substances such as microencapsulated plant essential oils in 3D printing denture base materials. Objectives. The objectives of this project were: i. to incorporate microcapsules, containing biodegradable essential oil, into two 3D printed DBRs and investigate their effects on the physical and mechanical properties of the materials to ISO standards, ii. investigate the antimicrobial activity of the resulting materials, and to iii. investigate the cytotoxicity of the microcapsules. [...], Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Medicine & Dentistry, Griffith Health, Full Text

The development of bioactive materials that can mimic bone tissue has been an area of active research to try and improve the process of implant integration. Wool-derived keratin, a readily available natural polymer, has been shown in vivo to enhance bone healing and as such may be a novel material for use in the development of biomimetic devices. Coating titanium with keratin, therefore, may be a promising approach to achieve this, but effective bonding between keratin and titanium and the molecular response of osteoblasts to keratin has not been fully delineated. Using aminosilane (APTES) and glutaraldehyde (GA) to prepare the titanium surface, two coating strategies were employed in the present study i.e., solvent casting and molecular grafting, to try and improve the coating's chemical and mechanical properties and improve the interaction between keratin and adherent osteoblastic cells. This thesis had three main goals. First, to characterize the physiochemical and mechanical properties of the keratin coating using various analytical techniques. Second, to evaluate the cytocompatibility of the keratin coating with osteoblastic MG-63 cells in vitro. Finally, to investigate the bone healing response to keratin-coated titanium in vivo using a rat calvarial defect model. [...], Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Medicine & Dentistry, Griffith Health, Full Text

The present thesis aimed to study the interaction of neutrophils on titanium (Ti) and zirconia (Zr) surfaces having various surface roughness. Our specific aims were: 1) systematic review (SR) access the in-vitro studies that evaluated neutrophil interactions with different roughness levels in Ti and Zr implant surfaces; 2) Evaluate standardized polishing protocols for in vitro experimentation using a custom-made apparatus under controlled force to create consistent surface roughness on Ti and Zr discs; 3) Investigate the neutrophil expression of neutrophil elastase (NE), matrix metalloproteinase (MMP)-8 and (MMP)-9 after being exposed to rough, smooth, and very smooth Ti and Zr surfaces; and 4) To characterize neutrophils NETs release on Ti and Zr surfaces having different levels of surface roughness. [...], Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Medicine & Dentistry, Griffith Health, Full Text

New understanding of immune system biology has led a paradigm shift in the development of biomaterials away from classically ‘inert’, to ‘immuno-modulatory’ biomaterials that have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling responses as opposed to persistent inflammation and scar tissue formation. This project aims to examine macrophage behavior and in particular integrin expression at the cell–biomaterial interface in vitro, in order to delineate the underlying molecular events occurring during biomaterial-mediated osseous healing. After reviewing the current literature to identify research gaps that this thesis should focus on, the activation of macrophages with controlled timing, and modulation of their interactions with other cell types involved in wound healing, emerged as key strategies to improve biomaterial efficacy. Careful design of biomaterial structure and controlled release of immunomodulators can be employed to manipulate macrophage phenotype for the maximization of the wound healing response with enhanced tissue integration and repair, however, to elicit predictable immune responses there is a need for a thorough understanding of how the biomaterial properties can be tuned to harness a designed immunological outcome. Our systematic review of in vitro studies suggested that the initial immune response of macrophages to titanium may be modulated by its surface characteristics both topographically and physiochemically. We therefore assessed the potential effects of three novel titanium surfaces; Machined, Blasted and Fluoride-treated, on macrophage phenotype. We chose a monocytic cell-line as our macrophage source rather than using primary macrophages, as the inflammatory status of primary cells could not be readily defined nor reproduced between experiments. This was crucial to the overall study if we were to clearly assess any changes in macrophage phenotype as a result of their II interac, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Medicine & Dentistry, Griffith Health, Full Text

Full Text, Thesis (Masters), Master of Medical Research (MMedRes), School of Pharmacy & Med Sci, Griffith Health, Bisphosphonates are a group of drugs widely used in medical practice to prevent bone destruction, occurring in diseases like osteoporosis and bone cancer. An unfortunate sideeffect of these potent drugs is the subsequent death of areas of bone in the jaw following minor local trauma, commonly occurring during dental treatment. This unique, but welldocumented, adverse effect is termed medication-related osteonecrosis of the jaws (MRONJ). While the precise cause of MRONJ is still unclear, research suggests the inhibition of new blood vessel formation, limiting the capacity for bone healing, may be a significant factor. Unfortunately, treatment options for MRONJ are very limited, and despite successful development of bone mimetic materials, most available materials do not induce sufficient formation of blood vessels. Adequate vascularisation is crucial for timely and adequate transport of nutrients and waste removal, and the provision of progenitor cells for tissue remodeling and repair. This study provides a rationale for a new innovative approach using a human-adipose derived microvessel (HAMV)-osteoblast construct, which will potentially support local bone regeneration and healing. This project takes the important first steps in vitro, investigating the growth, migration, and viability of the HAMV within a 3D hydrogel construct and assessing the influence of HAMV development on osteogenesis, in order to provide the data necessary for subsequent testing in a pre-clinical animal model. In this study, HAMV were seeded into tissue culture plates and cultured over 28 days, to evaluate the proliferation potential of the HAMV in a 3-dimensional (3D) collagen hydrogel construct. Growth, proliferation and morphological changes, including cell death, of HAMV were also visualised and were examined via LIVE/DEAD® assay with the aid of confocal microscopy. The human osteoblast cell line, MG63, was cultured in conditioned media derived from the HAMV cultures to understand the inf

Periodontitis is a highly prevalent chronic inflammatory disease affecting more than 60% of the population, which leads to destruction of the tooth-supporting tissues. The periodontium is composed of both hard (bone and cementum) and soft tissues (periodontal ligament and gingiva), requiring a tissue-engineering approach to allow a precisely coordinated and compartmentalised healing response for subsequent structural and functional regeneration. The present study investigated the functionalisation of highly porous scaffolds with decellularised cell-laid extracellular matrix for periodontal regeneration. This novel technique allowed the combination of a three-dimensional scaffold providing mechanical support with a native ECM providing tissue specific biological activity. The decellularisation of such constructs allows the maintenance of an intact ECM structure and composition while removing the immunogenic cellular component, thus generating an acellular implant. By combining a bone-like ECM-decorated scaffold (bone compartment) with periodontal ligament cell-sheets (PDLcs), the aim was to achieve specific bone and periodontal ligament regeneration. [...], Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Medicine & Dentistry, Griffith Health, Full Text

Glaucoma, a characteristic type of optic nerve degeneration in the posterior pole of the eye, is a common cause of irreversible vision loss and the second leading cause of blindness worldwide. As an optic neuropathy, glaucoma is identified by increasing degeneration of retinal ganglion cells (RGCs), with consequential vision loss. Current treatments only postpone the development of retinal degeneration, and there are as yet no treatments available for this disability. Recent findings in ocular regeneration have opened promising avenues to apply stem cell-based modalities to restore vision in progressive optic neuropathies. As such, the stem cell-based RGC replacement therapy, as a promising treatment of glaucoma, could be facilitated by using tissue-engineered scaffolds that provide a supportive structure. An appropriate selection of materials is a fundamental consideration in fabricating these scaffolds. My study investigates the structure, composition and properties of three most commonly used biopolymer materials blended with [polycaprolactone] (PCL) at 2:1 (wt.%) ratio, namely, poly(glycerol sebacate) (PGS)/PCL, polylactic-co-glycolic acid (PLGA)/PCL, poly-l-lactide (PLLA)/PCL and pure PCL as carrier vehicles for human embryonic stem cell derived retinal progenitor cell (hESC-RPC) attachment and proliferation. The physicochemical properties of PGS/PCL, PLLA/PCL, PLGA/PCL and pure PCL fibrous scaffolds, fabricated under the identical electrospinning conditions, were analysed employing scanning electron microscopy, contact angle analysis, Raman spectroscopy, electrical and ionic conductivity measurements, and supplemented by an in-vitro hESC-RPC adhesion and proliferation studies. My findings have shown that PGS/PCL scaffolds promote hESC-RPC attachment and growth more favourably compared to other polymeric blends and pure PCL, owing to a combination of advantageous surface and bulk properties, overall demonstrating a potential for PGS/PCL blend to become a suitab, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Eng & Built Env, Science, Environment, Engineering and Technology, Full Text

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry&Oral Hlth, Griffith Health, The search for an all-rounder “magic potion” to assist our body in healing and regeneration has driven alchemists and scientists for centuries. Our body has an amazing ability to heal, but in cases of critical size defects or wounds it will always need an exogenous source of help in the form of anti-microbials, enzymes or proteins that are either human, animals or even plant derived. By way of understanding how human and animal teeth develop, particularly in relation to the development of the tooth’s attachment apparatus, the group of Lindskog, Slavkin and Hammarstrom in the 1980s and 1990s were inspired by the role of enamel matrix proteins (EMPs) and amelogenin (AMEL) in particular, in cementogenesis and the formation of the periodontal attachment apparatus (Lindksog S 1981, Hammarstrom et al 1991, 1992, 1995). EMPs are proteins secreted by Hertwig’s epithelial root sheath (HERS) capable of promoting complete or partial periodontal regeneration (Gestrelius et al 1997, Heijl et al 1997). The commercial formulation of these enamel proteins, Emdogain® (EMD), is derived from the enamel layer of developing porcine teeth, with Propylene Glycol Alginate (PGA) as the carrier. The product is packaged in a syringe container to be injected directly into the defect or site where it is needed. Since the development of this product in the mid 1990s, hundreds of animal and human studies and clinical trials have been performed with varying results. The majority of the results are positive, showing that EMD that can assist in periodontal regeneration, recession coverage and possibly general wound healing and new bone formation. Among the hundreds of available studies already, why did I then choose to investigate this protein as the subject of my research? I believe that there is still a big gap in the knowledge of how EMD works and that we are only scratching the surface of the potential of this enamel-derived protein, which when added to an environment with inflammatory cells is

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry&Oral Hlth, Griffith Health, Biomaterial scaffolds engineered to facilitate osteogenesis which can subsequently remodel in a similar fashion to that of natural tissue, is an ideal clinical strategy for treating bone defects in the maxillofacial region. This study has developed, using a novel melt electrowriting (MEW) technique, various graded porous polycaprolactone (PCL) scaffolds able to imitate the bimodal structure of cortical and cancellous bone tissue in terms of their morphological pore structure. These scaffolds were subsequently shown to facilitate the proliferation of osteoblasts with significant alkaline phosphatase (ALP) activity. Scaffolds with a staggered architecture i.e. those where the corresponding fibers in different layers are offset horizontally during the printing process, increases the number of contact points, enables larger pores, facilitates cell attachment and creates a highly porous structure with interconnected networks favourable for improved cell migration and vascularization. MEW is a relatively new technology that fills a gap between conventional fused deposition modelling (FDM) 3D printing and solution electrospinning by affording significantly better control over the fabrication of the porosity in scaffolds. This is due to the high-resolution during printing of the fibers which enables small pore sizes in the desired porosity. PCL polymer with its advantages of less immunoreactivity following implantation and mechanically suitability for the support of bone cells, has flexibility in design and is widely used in bone applications. However poor bioactivity and cellular affinity as well as long-term degradation issues with PCL have been reported. A promising solution to overcome these drawbacks is fiber surface modification such as coating with bioactive inorganic components. hydroxyapatite (HAP) is the most stable form of calcium phosphate (CaP) used to improve the hydrophobic characteristics of PCL and enhance the binding affinity to the host tissue and promote

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry&Oral Hlth, Griffith Health, Successful osseous healing is achieved by highly organised and controlled biological processes. Inflammation is an initial and integral part of these biological processes, influencing the fate of overall healing outcomes. Indeed, it has been well established that the skeletal and immune systems closely interact and influence each other in order to regulate bone homeostasis, both in healthy and diseased conditions. Compromised wound healing is one of the major clinical complications of diabetes, often leading to high morbidity and mortality. Pathophysiological changes, such as chronic hyperglycaemia and a resultant pro-inflammatory microenvironment, are considered to be the main aetiological factors in diabetes, contributing to a compromised immune system and poor wound healing potential. Macrophages play a pivotal role in both the initiation and resolution of inflammation by expressing an array of functional phenotypes (e.g. M1 and M2) in response to diverse local microenvironmental chemical cues. A timely transition of inflammatory status from ‘proinflammatory (M1)’ to ‘anti-inflammatory/pro-healing (M2)’, is believed to be a crucial biological step in order to achieve successful wound healing, and there is increasing evidence supporting the notion that macrophages are responsible for this ‘switch’ of microenvironment. This important cellular phenomenon of M1 and M2 phenotypic transition is disrupted in diabetes due to various systemic and local factors, however the underlying cellular mechanisms are yet to be fully elucidated. Recently, numerous in vitro and in vivo studies have investigated the influence of different biomaterial surface characteristics on the resultant immune responses, demonstrating that the expression of macrophage phenotypes can be modulated by surface topography, chemistry and hydrophilicity, thus resulting in enhanced osseous healing. However, there is insufficient evidence in the current literature to support whether such biomaterial enhanc

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry&Oral Hlth, Griffith Health, Maxillary sinus pneumatization is a frequently encountered problem in the dental implant rehabilitation of the posterior maxilla. Maxillary sinus augmentation (MSA) using a lateral wall approach is a well-established and commonly utilized surgical technique for overcoming this bone deficiency and allowing implant placement. MSA is still in the refinement process with a large degree of variation in all aspects of the technique including; the surgical protocol, the anatomical site, the choice of material, the site of autogenous bone harvesting, the timing of implant surgery and the use of barrier membranes. Generally, however, prospective controlled clinical trials critically assessing these domains remain rare, especially studies applying well defined success criteria for implant supported restorations placed in sites of MSA. The maxillary sinus grafting procedure is invasive and surgically demanding, but appears to have limited interference with maxillary sinus physiology when performed well. In addition, reported surgical complications are generally well tolerated and followed by normal recovery in the vast majority of cases. Most of this data however is derived from medium to low level evidence (clinical case series, retrospective analyses). Patients undergoing this procedure also expect to be counselled about their expectations regarding pain and the impact of this procedure on their daily life in the post-operative period. Such information is not available. There is also limited long term outcome data on implants placed in MSA. Historically, autogenous bone grafts have been considered the gold standard due to their inherent osteoinductivity. Their significant limitations in MSA however has driven intense research into various bone substitutes. Anorganic bovine bone mineral (ABBM) is a very well documented xenograft in MSA when used alone or as a composite graft with autogenous bone (AB) (ABBM + AB). More recently, collagen stabilized ABBM using 10% porcine type

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry and Oral Health, Griffith Health, Single tooth implant placement in the anterior maxilla has been reported to achieve predictable survival rates. Implant supported single tooth restorations in the anterior maxilla are expected to mimic their natural counterparts in order to achieve aesthetic harmony. The overall success of an implant reconstruction in the anterior maxilla must not only consider implant survival and measures of implant success, such as peri-implant tissue health, but also achieve favourable aesthetic outcomes. This has led to the development of aesthetic indices for the evaluation of the peri-implant mucosa (Pink Esthetic Score – PES) and the implant supported crown (White Esthetic Score – WES). To meet the growing needs and expectations of patients for the completion of treatment in a shorter timeframe, implant placement has been modified from a conventional delayed placement approach to abbreviated placement protocols. Although these protocols reduce the total treatment time, their outcomes are nevertheless influenced by the dynamic bone remodelling changes that occur after tooth extraction. These ridge dimensional alterations could lead to compromised outcomes, especially in the anterior maxilla, where aesthetics is of prime importance. The effect of timing of implant placement on these tissue dimensional changes has not been fully explored in the existing literature.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry and Oral health, Griffith Health, Bisphosphonates (BPs) are a group of anti-resorptive agents that possess high binding affinity to bone mineral. They accumulate in bone and are released during bone turnover to inhibit resorption after their internalisation by osteoclasts. This has been described as the principal mechanism of action of BPs, warranting their use in various bone depleting conditions such as osteoporosis, Paget’s disease, metastatic bone diseases (breast cancer, prostate cancer and multiple myeloma primary sites) and paediatric osteogenesis imperfecta. However, BPs are associated with a significant adverse effect that selectively occurs in jaw bones, termed Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ). While the pathogenesis of BRONJ is not yet clearly understood, recent focus of research has been on their anti-angiogenic properties that could significantly contribute to the aetiopathogenic process by which the avascular necrosis of the jaw bone ensues. The focus of this project was to explore the mechanism by which the anti-angiogenic properties of BPs can lead to the occurrence of BRONJ lesions and subsequently formulate a localised approach for prevention of this painful and debilitating condition.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry and Oral Health, Griffith Health, The periodontium is a complex structure that is comprised of soft tissue components the gingiva and periodontal ligament, as well as hard tissue components in the form of alveolar bone and cementum. Human periodontal ligament cells (HPDLCs) and their extracellular matrix are regarded as essential components to achieve successful periodontal regeneration when treating periodontal lesions. In the present study, cell sheet technology was utilized to fabricate periodontal ligament cell sheets. These cell sheets were subsequently decellularized to isolate and preserve the extracellular component structural and functional characteristics, and were evaluated in vitro and in vivo. In all experiments that were undertaken in this study, melt electro-spun polycaprolactone (PCL) scaffolds were used as a carrier for the cell sheets, in order to support their fragile nature during the processes of decellularization. The combined cell sheet-PCL scaffold structure is referred to as a decellularized cell sheet construct.

OBJECTIVES: Published information regarding the use of rat jawbones for dental implant osseointegration research is limited and often inconsistent. This study assessed the suitability and feasibility of placing dental implants into the rat maxilla and to establish parameters to be used for dental implant research using this model. MATERIALS AND METHODS: Forty-two customized titanium implants (2 × 3 mm) were placed bilaterally in the maxillary first molar area of 21 Sprague-Dawley rats. Every animal received two implants. The animals were subsequently sacrificed at days 3, 7, 14, 28 and 56 post-surgery. Resin-embedded sections of the implant and surrounding maxilla were prepared for histological and histomorphometric analyses. RESULTS: The mesial root of the first molar in the rat maxilla was the optimal site to place the implant. Although the most apical 2-3 threads of the implant penetrated into the sinus cavity, 2 mm of the remaining implant was embedded in the bone. New bone formation at day 7 around the implant increased further at day 14, as measured by the percentage of bone-to-implant contact (%BIC) and new bone area (%BA) in the implant thread chambers (55.1 ± 8.9% and 63.7 ± 7.7%, respectively). There was a further significant increase between day 14 and 28 (P < 0.05), however, no significant differences were found between day 28 and 56 in either %BIC or %BA. CONCLUSIONS: The mesial root socket of the first molar in the rat maxilla is a useful model for dental implant research. Osseointegration following implant placement as measured by BIC plateaued after 28 days. The recommended implant dimensions are 1.5 mm in diameter and 2 mm in length.

Background Osteocytes, the most abundant cells in bone, havemultiple functions, including acting as mechanosensors and regulating mineralization. It is clear that osteocytes influence bone remodeling by controlling the differentiation and activity of osteoblasts and osteoclasts. Determining the relationship between titanium implants and osteocytes may therefore benefit our understanding of the process of osseointegration. Purpose The aim of this study was to visualize the ultrastructural relationship between osteocytes and the titanium implant surface following osseointegration in vivo. Materials and Methods Titanium implants were placed in the maxillary molar regions of eight female Sprague Dawley rats, 3 months old. The animals were sacrificed 8 weeks after implantation, and undecalcified tissue sections were prepared. Resin-cast samples were subsequently acid-etched with 37% phosphoric acid prior to examination using scanning electron microscopy. Results Compared with mature bone, where the osteocytes were arranged in an ordered fashion, the osteocytes appeared less organized in the newly formed bone around the titanium implant. Further, a layer of mineralization with few organic components was observed on the implant surface. This study shows for the first time that osteocytes and their dendrites are directly connected with the implant surface. Conclusions: This study shows the direct anchorage of osteocytes via dendritic processes to a titanium implant surface in vivo. This suggests an important regulatory role for osteocytes and their lacunar-canalicular network in maintaining long-term osseointegration.

The periodontal ligament is the key tissue facilitating periodontal regeneration. This study aimed to fabricate decellularized human periodontal ligament cell sheets for subsequent periodontal tissue engineering applications. The decellularization protocol involved the transfer of intact human periodontal ligament cell sheets onto melt electrospun polycaprolactone membranes and subsequent bi-directional perfusion with NH4OH/Triton X-100 and DNase solutions. The protocol was shown to remove 92% of DNA content. The structural integrity of the decellularized cell sheets was confirmed by a collagen quantification assay, immunostaining of human collagen type I and fibronectin, and scanning electron microscopy. ELISA was used to demonstrate the presence of residual basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in the decellularized cell sheet constructs. The decellularized cell sheets were shown to have the ability to support recellularization by allogenic human periodontal ligament cells. This study describes the fabrication of decellularized periodontal ligament cell sheets that retain an intact extracellular matrix and resident growth factors and can support repopulation by allogenic cells. The decellularized hPDL cell sheet concept has the potential to be utilized in future "off-the-shelf" periodontal tissue engineering strategies.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Dentistry and Oral Health, Griffith Health, Titanium dental implants are currently one of the best treatment alternatives for replacing missing teeth. The direct bond that forms between the jawbone and titanium in a process defined as osseointegration has made titanium the material of choice for such devices. Whilst the underlying biological mechanisms responsible for the osseointegration process have not been fully elucidated, high demand by both patients and clinicians has driven the clinical application of titanium implants. Modification of the surface properties of titanium has been proven to be effective in promoting osseointegration, and thus understanding the fundamental cellular and molecular mechanisms responsible for the clinically observed outcomes as a result of titanium surface modification is the focus of considerable research. Previous research in this field has, as expected, focused on osteoblasts as they are the bone forming cells. This however, ignores the possible fundamental role of early mediators of the healing process, such as platelets and macrophages, which may interact with the implant surface well ahead of osteoblasts. As platelets and macrophages have well documented roles in modulating the function of other cells including osteoblasts, it is reasonable to postulate that the events that occur during the very early stages of the healing process may modulate repair in later stages, and ultimately influence the final osseointegration outcome.