This thesis reports on investigations carried out to identify the important antigens inducing a humoral immune response in periodontal disease. A number of different techniques were used. The first study examined the systemic immune response, specifically the effects of treatment on the immune response of patients with chronic periodontitis against a panel of periodontal pathogens and a large panel of antigen preparations from these bacteria. The results of the study indicated that there was no difference in antibody titre following treatment against any of the putative periodontal antigenic targets tested. These results conflict with many published reports, however, differences in factors such as length of treatment and antigen preparation often make the comparison of different studies futile. An investigation to examine the phenomenon of cross-reactivity between antibodies induced against putative periodontal pathogens was the second part of this overall project. The results suggested a high proportion of cross-reactivity between the 4 periodontal pathogens P. gingivalis, A. actinomycetemcomitans, P. intermedia and B. forsythus. Results indicating such a high cross-reactivity between these micro-organisms prove that the issue of cross-reactivity is an important one that should be considered when carrying out any immunological and microbiological investigations in this field. Following the above investigations of the systemic immune response the remainder of this thesis is a report of three studies examining the local immune response in periodontal disease.

Periodontal ligament inflammation or periodontitis is a common disease characterised by gradual destruction of connective tissue fibres that attach a tooth to the alveolar bone within which it sits. Diabetes and inflammation enhances periodontal bone loss through enhanced resorption and diminished bone formation. Periodontal ligament fibroblast attachment to collagen-I and function was impaired by methylglyoxal (MG) modification in vitro. The glyoxalase system is an anti-glycation defence in all cells that metabolises MG and thereby suppresses MG-mediated protein damage. Overexpression of Glo1 decreased the intracellular levels of MG The aim of this investigation was to improve the understanding of protein damage in PDL in diabetes, focusing on protein damage by MG in human periodontal ligament fibroblasts (hPDLFs) in hyperglycaemia and to evaluate the effects of high and low glucose concentrations on MG metabolism in hPDLFs with or without Glo1 inducers. The effect of high glucose concentration on the formation and metabolism of MG was studied in hPDLFs in vitro. The ability of two small molecule Glo1 inducers, individually and in synergistic combination, to counter dicarbonyl stress in hPDLFs in vitro was studied. Interactions between hPDLFs to the extracellular matrix protein, collagen-I, were investigated and impairments in hPDLFs adhesion to MG-modified collagen-I coated plates were assessed. Protein susceptible to MG modification and inactivation in the cytosol of hPDLFs were identified by high resolution mass spectrometry proteomics. The effect of clinical periodontitis on plasma protein glycation, oxidation and nitration was also investigated in a pilot clinical investigation. When hPDLFs were incubated with high glucose concentration in vitro there was a 45% decrease in Glo1 activity and 42% increase in D-lactate flux – surrogate indication of MG flux of formation, which contributed to increased cellular concentration of MG and increase in MG-H1 residue content of cell protein, compared to low glucose control. This indicated dicarbonyl stress was induced in hPDLFs by high glucose concentration in vitro, a model for hyperglycaemia in vivo. Decrease of Glo1 activity and increase in cellular MG concentration and MG-H1 residue content of cell protein was corrected with the addition of Glo1 inducers. The binding of hPDLFs to collagen-I was decreased by 30% in high glucose concentration and was corrected by addition of Glo1 inducers. Proteomics analysis of cytosolic extracts of hPDLFs indicated that high glucose incubations produced changes in MG-modified proteins and also up-regulated and down-regulated unmodified proteins in hPDLFs. The pilot investigation of clinical periodontitis suggested a systemic effect of this local inflammation which was associated with changes in plasma protein glycation, oxidation and nitration. This study reveals that dicarbonyl stress is a potential contributory pathogenic mechanism in hPDLFs in periodontitis and countering it may provide new treatment options to prevent and treat decline in periodontal health, particularly in diabetes. Small molecule inducers of Glo1 expression may in future contribute to improving periodontal health, particularly in diabetes.

Critically ill patients often require mechanical ventilation (MV) to facilitate treatment for respiratory failure or airway protection when consciousness is impaired. Whilst the endotracheal tube (ETT) is an essential interface between the patient and ventilator, it may promote VAP by impeding host defence mechanisms and by translocating microorganisms from dental plaque to the lower airways. Ventilator-associated pneumonia (VAP), which may be challenging to diagnose, is the most frequent hospital-acquired infection in critical care. It has been reported that when patients receive MV the composition of dental plaque changes to include respiratory pathogens such as Meticillin-Resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. The primary aim of this research was to confirm that dental plaque communities altered during MV and to identify the potential causes of these changes. A combination of culture-based microbiology, community profiling molecular techniques and proteomic analysis of saliva was performed to analyse the microbiological content of the oral cavity, and to also quantify changes in dental plaque composition and saliva. For the first time, this study comprehensively analysed the dental plaque of mechanically ventilated patients and documented considerable species richness and diversity. Numerous potential respiratory pathogens were detected including Staphylococcus aureus, P. aeruginosa and Streptococcus pneumoniae in approximately one-third of mechanically ventilated patients. In addition, salivary flow rate was decreased and both the salivary pH and concentration of pro-inflammatory cytokines were significantly elevated during intubation. Dental plaque is a reservoir for VAP, and preventing or reducing respiratory colonisation may play a role in the management of ventilated patients. Interventions to prevent colonisation could include the modulation of salivary parameters such as pH and volume and further work may lead to the identification of specific proteins that are significant. Reducing the incidence of VAP will not only reduce mortality in the ICU, but will also have a great impact on hospital economics by reducing inpatient stay.

The novel epoxy-tiglianes, EBC-46 and ‘lesser activity’ EBC-211, are sourced from seeds of the Fountain’s Blushwood Tree (Fontainea picrosperma), indigenous to Queensland Tropical Rainforest. Australian biotechnology company, QBiotics Ltd.,has demonstrated that EBC-46 stimulates exceptional dermal wound healing responses in vivo, following cancer treatment and tumour destruction in domesticated animals. Consequently, QBiotics is developing EBC-46 as a veterinary anti-cancer pharmaceutical and performing human clinical trials. However, little is known on how EBC-46 induces its exceptional healing effects, manifested as accelerated wound re-epithelialisation, closure and reduced scarring. This study aimed to elucidate how EBC-46 and EBC-211 mediates these exceptional wound healing effects in vitro, through analysis of HaCaT keratinocyte and dermal fibroblast/myofibroblast genotypic and phenotypic responses, following epoxytigliane treatment (0.001-100μg/ml). A number of key wound healing responses were assessed, including proliferation, cell cycle progression, scratch wound repopulation; and transforming growth factor-β1 (TGF-β1)-driven, fibroblastmyofibroblast differentiation. Studies demonstrated that both EBC-46 and EBC-211 induced fibroblast and HaCaT cytotoxicity at 100μg/ml. EBC-46 and EBC-211 (0.001-10μg/ml) significantly retarded fibroblast proliferation and delayed S/G2 cell cycle transition, but exerted no significant effects on fibroblast migratory responses. Although EBC-46 had no effects on α-smooth muscle actin (α-SMA) expression, stress fibre organization and myofibroblast formation (0.001-0.01μg/ml and 1-10μg/ml), EBC-46 significantly inhibited α-SMA expression and stress fibre formation at 0.1μg/ml, with cells retaining normal fibroblast morphologies. EBC-211 induced similar effects at 10μg/ml Both EBC-46 and EBC-211 (0.001-10μg/ml) stimulated significant HaCaT proliferation, G1/S and S/G2 cell cycle transitions; and accelerated scratch wound repopulation, even with mitomycin C. Microarray analysis and protein level validation, identified numerous differentially expressed genes in epoxy-tiglianetreated, HaCaTs. Up-regulated genes included certain keratins and others associated with promoting cell cycle progression, proliferation and migration. Down-regulated genes included other keratins and genes associated with inhibiting cell cycle progression and proliferation, including certain cytokines and chemokines. This study has provided evidence to explain the enhanced re-epithelialisation and reduced scarring responses observed in epoxy-tigliane-treated skin. Furthermore, it highlights the potential of epoxy-tiglianes as novel therapeutics for impaired dermal wound healing and excessive scarring situations.

At present, the prime candidate for augmentation of bone defects are autologous bone grafts (ABGs), owing to their ability to emulate the physiological signalling environment to induce bone marrow-derived, mesenchymal stem cells (BMMSCs) to differentiate into functional osteoblasts. However, the uses of ABGs are negated due to patient complications. Dentine matrix has recently become increasingly recognised as possessing bioactive nature and has demonstrated the ability to augment bone repair in vivo. However, the constituents of dentine that confer efficacy for bone formation are poorly understood. The aim of this study was to determine the potential for demineralised dentine matrix (DDM) to induce biological responses of BMMSCs and elucidate the key mediators required for bioactivity. Isolation of a sub-population of BMMSCs yielded a population of cells with enhanced expansive capacity in vitro, with maintained mesenchymal stem cell marker expression. Application of DDM to BMMSCs significantly reduced cell expansion (0.1-10μg/mL), reduced apoptotic activity (10μg/mL) and enhanced migration (0.1μg/mL). Importantly, DDM at 10μg/mL directed osteogenesis of BMMSC by enhancing RunX2 gene expression after 5 days and deposition of a mineralised matrix after 28 days. Western blot and enzyme-linked immunosorbent assay (ELISA) analyses of DDM identified a plethora of growth factors associated with directing osteogenesis of BMMSCs, including; transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-2 (BMP-2) and vascular epidermal growth factor (VEGF). Fractionation of DDM by heparin-affinity, to concentrate growth factors, resulted in diminished potential of DDM to enhance RunX2 expression and mineral deposition of BMMSCs. Depletion of decorin from DDM had no effect on osteogenic potential, however, depletion of biglycan attenuated RunX2 expression and mineral deposition. Substrates composed of silk-fibroin/gelatin (SF/G) at ratios of 75:25 supported attachment and expansion of BMMSCs with no discernible changes in morphology, compared to cells cultured on plastic. Addition of DDM to SF/G substrates resulted in enhanced BMMSC expansion (20μg/mL) and evidence of mineralised matrix deposition (5-20μg/mL), compared to un-loaded SF/G substrates. In conclusion, DDM possesses bioactive potential towards BMMSCs, which is attributable to a range of synergistically acting growth factors along with additional matrix constituents. The ability of DDM to stimulate osteogenesis demonstrates potential for future developments in the field of tissue engineering of bone.

Background: Craniofacial morphology has been reported to be highly heritable, but prior to this research, little was known as to which genetic variants influence normal lip phenotypes. Much of the previous genetic research involved assigning rare genetic mutations to craniofacial abnormalities, giving little insight as to what causes normal variation. More recent studies have attempted to assign genetic variants to facial genotypes using landmarking methods, however, these markers are sparse in the lip region. Considerable variation of lip morphology exists, which is not amenable to landmarking methods. The aim of this study was to investigate the biological basis of lip phenotypes. Objectives: • Develop a robust, reproducible classification system of lip phenotypes; • Measure the prevalence of lip phenotypes within a population sample of 4,747; • Assess for associations of lip phenotypes with other lip phenotypes; • Assess if there were any sex variations of lip phenotypes; •Assess the reproducibility of the classification system with other acquisition methods; •Perform a nome-wide association study (GWAS) of lip phenotypes; • Assess for associations of non-syndromic cleft lip/palate (NSCL/P) SNPs to lip phenotypes; • Perform a case-control study to assess the prevalence of lip phenotypes amongst unaffected biological parents of cleft and non-cleft children; • Assess the predictive capability of lip phenotypes as a precursor of cleft risk. Methods: Three-dimensional laser scanned facial images were obtained of 4,747 subjects recruited from the Avon Longitudinal Study of Parents and Children (ALSPAC). A total of 102 individuals were recruited from the University Dental Hospital, Cardiff to assess the reproducibility of the classification system with other acquisition methods. Genetic data was available for 3,687 ALSPAC subjects for the discovery phase of GWAS, and 3,215 digital photographs of Australian twins for the replication phase. Images of 597 3dMD case-parent trios and controls were obtained through FaceBase, to assess the prevalence of lip phenotypes amongst unaffected biological parents of cleft and non-cleft children. Results: Twenty-five reproducible lip phenotypes have been described. Most phenotypes occur in combinations, except mentolabial fold and chin dimple, which are distinct. Prevalence of features and sex dimorphism are also explained. A modification of the lip scale is required for facial shells with reduced surface resolution taken with 3dMD or digital photographs. A discovery GWAS of 3,687 ALSPAC subjects revealed two new GWAS significant hits: chin dimple and DOCK1 (P=2x10⁻⁸) and mentolabial fold and CDH4 (P=1x10⁻⁸). Both genes have been reported to play a role in tumour pathways (Du, C. et al, 2011, Laurin, M. et al, 2013), and CDH4 may have a role to play in muscle development (Nogueira, J.M. et al, 2015). In addition, 29 near-hit associations were found with 18 lip phenotypes (P < 10⁻⁷). Replication was attempted for 2 lip phenotypes (chin dimple and mentolabial fold). However, it was not possible to replicate these findings. Genotype analysis discovered associations with 14 out of 17 candidate NSCL/P SNPs with 21 out of 25 lip phenotypes. The main findings were the association of NOG and skeletal II pattern (P=1.58x10⁻⁶) and V-shaped Cupid's bow (P=2.48x10⁻³) with 8q24. A generated NSCL/P genetic allele score demonstrated association with V-shaped Cupid's bow (P=2x10⁻⁴), narrow philtrum (P=3x10⁻⁴), upturned commissures (P=0.03) and deep philtrum (P=0.045). The prevalence of 12 out of 23 lip phenotypes was found to be higher amongst case-parent trios compared with control parents. Case mothers had increased prevalence of convex upper lip contours, upper lip groove, absent lower lip groove, flat lower lip contour and angular lower lip tone with bumping. Case fathers had absent lower lip vermilion borders and double borders. Conclusions: A robust and reliable scale has been presented, which allows categorisation of lip phenotypes. Considerable variation exists within the ALSPAC population of 15 year olds, including some rare phenotypes and evidence of sex dimorphism. Lip phenotypes tend to appear in combinations with other lip phenotypes, whilst chin dimple and mentolabial fold are generally distinct. Discovery GWAS indicated genotype/phenotype associations with chin dimple and DOCK1* (P=2x10⁻⁸) and mentolabial fold and CDH4 (P=1x10⁻⁸). However, this was not replicated in an independent sample. NSCL/P SNPs and combined high-MNSCL/P genetic alleles affect lip phenotypes, and appear to induce a narrow philtrum, V-shaped Cupid's bow and a skeletal II pattern. Parents of cleft children had higher prevalence of some lip phenotypes compared with control parents. As such, this study proposes that certain lip phenotypes may be utilised as subphenotypic markers of cleft risk.

IL-33 and IL-17 family cytokines (IL-17A – IL-17F) have been shown to play roles in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. However knowledge of their role in periodontal disease pathogenesis is limited. The aim of this study was therefore to determine clinical associations between IL-33 and IL-17 family cytokines and chronic periodontitis. In addition, to begin to investigate the biological significance of these associations using in vitro model systems. 97 patients with chronic periodontitis and 77 healthy volunteers were recruited in Glasgow and Newcastle. Serum, gingival crevicular fluid (GCF) and saliva were analysed for levels of IL-33 and IL-17 family cytokines by ELISA. Periodontal tissues from 17 chronic periodontitis patients and 10 healthy subjects from Glasgow were also investigated for IL-33 and IL-17 family cytokines mRNA expression by real time PCR. Immunohistochemical analysis was also performed on tissue to investigate expression of IL-33 and IL-17E at the protein level. In vitro experiments were performed using the OKF6/TERT-2 oral keratinocyte cell line and primary human gingival epithelial (PHGE) cells. The cells were stimulated with either a live Porphyromonas gingivalis monospecies biofilm or recombinant cytokines and changes in expression of cytokines, chemokines and their receptors evaluated by real-time PCR, immunocytochemical analysis or ELISA. In addition, transcriptional activity was monitored by analysis of changes in the phosphorylation (activation) of the NF-κB p65 subunit transcription factor using serum, GCF and saliva. IL-17A and IL-17A/F levels were higher in chronic periodontitis patients, but serum IL-17E was lower. IL-17A, IL-17A/F and the serum IL-17A:IL-17E ratio correlated positively with clinical parameters. IL-33, and IL-17 family cytokine (except IL-17B) gene transcripts were higher in tissue of chronic periodontitis patients. In addition, IL-33, ST2, IL-17E and IL-17RB proteins are expressed in periodontal tissues. Furthermore, IL-33 protein expression is upregulated in tissue of chronic periodontitis patients. In vitro models showed that IL-33 and its receptors (ST2 and ST2L) are expressed by oral keratinocytes (OKF6/TERT-2 cells and PHGE cells) and IL-33 expression up-regulated in response to P. gingivalis. However, IL-33 failed to induce expression of a range of inflammatory mediators and receptors in OKF6/TERT-2 cells. In vitro, IL-17E inhibited P. gingivalis monospecies biofilm and IL-17A induced expression of chemokines (IL-8 and/or CXCL5) by OKF6/TERT-2 cells at the transcriptional level by blocking the phosphorylation (activation) of the NF-κB p65 subunit. This study demonstrates clinical associations between IL-33 and IL-17 family cytokines and chronic periodontitis. The expression of IL-33 by oral keratinocytes and its up regulation upon exposure to P. gingivalis suggest it plays a role in the innate immune response to pathogens within the periodontium. However, the role of IL-33 in the periodontal inflammatory response remains to be elucidated. The negative correlations between serum levels of IL-17A and IL-17E and correlations with disease parameters, combined with their differing effects on the induction of expression of key neutrophil chemoattractants (CXCL5 and CXCL8), suggest opposing roles in periodontal immunity. Indeed, it can be hypothesised that the differential regulation of chemokine expression is due to IL-17A having pro- and IL-17E having anti-inflammatory properties. Indeed, as neutrophils play a key role in the early events associated with periodontal disease progression, the data suggests IL-17E is a rational target for therapeutic intervention.

The overall aim of this thesis was to examine the kinetics of proton-promoted dissolution of the dental hard tissue enamel as a non-bacterial process and the evaluation of inhibitors with the intent of minimising the dissolution process and effectively protecting the surface. A novel approach was taken, utilising scanning electrochemical microscopy (SECM) to galvanostatically generate controllable and well defined proton fluxes in defined areas of the surface. The resulting etch pits formed on the surface were characterised by optical microscopy and white light interferometry (WLI), which quantitatively determined etch pit dimensions. A theoretical finite element model (FEM) was used to elucidate the kinetics of dissolution based upon the analysis of the shape and dimensions of etch pits produced. A heterogeneous rate constant of dissolution of 0.08 ± 0.04 cm s-1 was attributed to untreated enamel, whereas 2 min treatment with 1000 ppm sodium fluoride (NaF) decreased this rate constant slightly to 0.05 ± 0.03 cm s-1. The impact of fluoride on the rate of proton attack was evident from the formation of shallower broader etch pits. In relation to both acid erosion and caries, the two most relevant acids pertinent to enamel dissolution are citric acid and lactic acid. These acids were investigated by protonating their respective sodium salts in-situ to produce localised weak acid directly under the probe tip. This permitted the surrounding enamel sample to remain largely unaltered giving a true surface for comparison, whilst allowing evaluation of the kinetics in the presence of each weak acid. Etching in the presence of lactic acid, showed a surface controlled process with a rate constant of 0.1 ± 0.03 cm s-1. Etching in the presence of the triprotic citric acid, also yielded a surface controlled process with a rate constant of 0.35 ± 2.6 cm s-1. Calcite was also investigated using SECM, WLI and FEM to validate the use of these techniques. The kinetic data extrapolated was comparable to rate constants found in literature, confirming the validity of these methods. In this case, a novel approach was the use of experimental data to parameterise the finite element model directly. Confocal laser scanning microscopy (CLSM) coupled with SECM was used to visualise proton fluxes from the tip of the UME. This allowed, not only, correlation of the current applied to the probe tip with the pH, but also quantitative data on the spread of protons across a particular surface. Rate constants found for untreated and fluoride-treated enamel were comparable to those found in SECM etching, however, zinc ion treatment proved to result in much greater inhibition of dissolution than fluoride.

Background: It is reported that hospitalised adults require daily oral care to prevent respiratory infections and maintain oral health but patient oral health declines in hospital. Enhancing knowledge and attitudes has not proven effective for changing behaviours or improving oral health. Reports suggest that some nurses find providing oral care unpleasant, therefore, emotions may influence care provision. Aim: To understand how nurses’ and student nurses’ emotional experiences and reactions influence the provision of oral care for hospitalised adult patients. Methods: The initial study explored emotional experiences, reactions and oral care practices. Eight focus groups and ten one-to-one semi-structured interviews with 48 subjects were used to collect data. These were analysed with Grounded Theory. A second study developed and tested methods to measure student nurses’ emotions towards oral care. This used a self-report questionnaire, interviews and Stroop tests. In the final study, 248 student nurses completed a revised self-report questionnaire, a disgust sensitivity questionnaire and two oral care attitude measures; 41 participants additionally completed emotional Stroop tests, implicit association tests and interviews. Qualitative data were analysed with thematic analysis. Χ2 tests, correlations, and Principal Component Analysis were used to analyse quantitative data. Results: Nurses and student nurses experience emotions towards the social, moral and physical aspects of providing oral care; emotions vary with different situations. Unclean mouths are associated with unpleasantness. Failure to provide oral care evokes moral disgust and anxiety. Providing oral care can evoke anxiety and disgust in unpleasant situations, this leads to student nurses reporting altering oral care procedures. Conclusions: Nurses’ and student nurses’ emotions of disgust and anxiety influence oral care. Although these emotions can motivate nurses to provide care, anxiety and disgust can lead to the selection of procedures that avoid aspects of oral care thus reducing the quality of care provided. Nurses’ oral care training programmes need to address these emotions to improve the quality of oral care for patients in hospitals.

The reconstruction of critical-size bone defects following tumour resection or bone loss due to trauma is topical today and relates to the complexity of the treatment involved and poor healing outcomes. In bone bioengineering, the current trends are to explore novel methods of repairing these defects by using various bone substitutes. Various graft materials have been used for the restoration of these defects. A graft ideally needs to promote osteogenesis, osteoinduction and osteoconduction. The aim of this investigation was to assess the histological, radiographic and mechanical properties of the tissue regenerate following the application of tricalcium phosphate (TCP) scaffolding and recombinant human bone morphogenetic protein 7 (rhBMP-7) for the reconstruction of a critical-size osteoperiosteal mandibular continuity defect in the rabbit model. Highly purified and freeze dried recombinant human BMP-7 was used. It was produced by Chinese hamster ovary cells in culture and purified from the culture media. All the TCP samples had a porosity of 80% and average pore size of 100 – 500µm. For the rhBMP-7 loaded scaffolds; rhBMP-7 was reconstituted according to a recommended specification and 400ng were loaded by adsorption into the TCP scaffolds. Nine adult New Zealand white rabbits (3.0-4.0kg) were used for the planned study. In each case a unilateral osteoperiosteal mandibular body critical-size defect was created. In six cases the critical-size defect was filled with the rhBMP-7 on the TCP scaffolding, and in three cases the TCP was used alone. Assessments were made with plain radiographs at 0, 4, 8, and 12 weeks follow-up. Three months post-operatively the animals were sacrificed, the mandibles removed and the surgical sites were assessed with cone beam CT radiography, tested mechanically and analysed histologically. More bone regeneration was seen radiographically and histologically within the mandibles that received rhBMP-7 in the TCP, with evidence of both woven and lamellar bone formation. Union was obtained at the surgical site with no cartilage formation. The regenerated bone was confined to the area that had received the scaffold, with no calcification of the surrounding soft tissues. The TCP was also resorbed more completely in this experimental group. Very little bone was formed in the cases where the defect was filled with TCP alone. The mechanical properties of the regenerate in the group that received the rhBMP-7 and TCP were also significantly superior to those of the cases that received TCP alone. Histologically the overall mean of the percentage regenerated bone volume in the rhBMP-7 and TCP cases was 29.41% ± 6.25, while that for the TCP alone cases was 6.35% ± 3.08. The difference between the groups was statistically significant (p = 0.014). Mechanically the failure moments for the TCP alone cases were found to be very low (0-48mNm) while those for the rhBMP-7 and TCP cases were higher but there was considerable variation between the cases (55-2115mNm). Some of the cases in this group achieved failure moments comparable to normal untreated bone. In conclusion TCP scaffolding and rhBMP-7 can be used successfully for the reconstruction of critical-size mandibular defects in the rabbit model and TCP loaded with rhBMP-7 was significantly superior in its capacity for bone regeneration histologically when compared to TCP alone. The resultant bony regenerate could also at times have mechanical properties similar to those of natural bone. But due to the variability of the mechanical properties further investigations are required before clinical application.