Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Breast cancer in its advanced stage has a high predilection to the skeleton. Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities. This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet. This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue. Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis. Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour. Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions. Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind. Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone. Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour. Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix. The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold. Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP scaffold, fabricated using a fused deposition modelling technique. After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique was chosen using the same polymer. Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice. Ectopic bone formation with sufficient vascularisation could be observed. After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed. Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment.

Preparation, characterization and in vitro angiogenic capacity of cobalt substituted β-tricalcium phosphate ceramics

Divalent cobalt ions (Co2+) have been shown to possess the capacity to induce angiogenesis by activating hypoxia inducible factor-1α (HIF-1α) and subsequently inducing the production of vascular endothelial growth factor (VEGF). However, there are few reports about Co-containing biomaterials for inducing in vitro angiogenesis. The aim of the present work was to prepare Co-containing β-tricalcium phosphate (Co-TCP) ceramics with different contents of calcium substituted by cobalt (0, 2, 5 mol%) and to investigate the effect of Co substitution on their physicochemical and biological properties. Co-TCP powders were synthesized by a chemistry precipitation method and Co-TCP ceramics were prepared by sintering the powder compacts. The effect of Co substitution on phase transition and the sintering property of the β-TCP ceramics was investigated. The proliferation and VEGF expression of human bone marrow mesenchymal stem cells (HBMSCs) cultured with both powder extracts and ceramic discs of Co-TCP was further evaluated. The in vitro angiogenesis was evaluated by the tube-like structure formation of human umbilical vein endothelial cells (HUVECs) cultured on ECMatrix™ in the presence of powder extracts. The results showed that Co substitution suppressed the phase transition from β- to α-TCP. Both the powder extracts and ceramic discs of Co-TCP had generally good cytocompatibility to support HBMSC growth. Importantly, the incorporation of Co into β-TCP greatly stimulated VEGF expression of HBMSCs and Co-TCP showed a significant enhancement of network structure formation of HUVECs compared with pure TCP. Our results suggested that the incorporation of Co into bioceramics is a potential viable way to enhance angiogenic properties of biomaterials. Co-TCP bioceramics may be used for bone tissue regeneration with improved angiogenic capacity.

Preparation, characterization, and in vitro bioactivity of nagelschmidtite bioceramics

The aim of this study is to prepare Ca, P and Si-containing ternary oxide nagelschmidtite (NAGEL, Ca7Si2P2O16) bioceramics and explore their in vitro bioactivity for potential bone tissue regeneration. We prepared dense NAGEL ceramics through high-temperature sintering of NAGEL ceramic powders. The apatite-mineralization ability, dissolution rate, and human osteoblast response (including cytotoxicity analysis, attachment, morphology, proliferation, and bone-related gene expression) to NAGEL ceramics have been systematically studied by comparing with conventional β-tricalcium phosphate (β-TCP) ceramics. The results showed that NAGEL ceramics possessed more obvious apatite mineralization and dissolution (degradation) and stimulated bone-related gene expression (OCN and OPN) of osteoblasts than β-TCP ceramics. NAGEL ceramics also showed no significant cytotoxicity. NAGEL ceramics supported osteoblast attachment, proliferation, and osteogenic gene expression, with a comparable cell proliferation activity with β-TCP ceramics. These results indicate that novel NAGEL bioceramics with the specific composition of Ca7Si2P2O16, are a promising biomaterial for bone tissue regeneration application.

Nagelschmidtite bioceramics with osteostimulation properties : material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells

Bioactive materials with osteostimulation properties are of great importance to promote osteogenic differentiation of human bone marrow stromal cells (hBMSCs) for potential bone regeneration. We have recently synthesized nagelschmidtite (NAGEL, Ca7Si2P2O16) ceramic powders which showed excellent apatite-mineralization ability. The aim of this study was to investigate the interaction of hBMSCs with NAGEL bioceramic bulks and their ionic extracts, and to explore the osteostimulation properties of NAGEL bioceramics and the possible molecular mechanism. The cell attachment, proliferation, bone-related gene expression (ALP, OPN and OCN) and WNT signalling pathways (WNT3a, FZD6, AXIN2 and CTNNB) of hBMSCs cultured on NAGEL bioceramic disks were systematically studied. We further investigated the biological effects of ionic products from NAGEL powders on cell proliferation and osteogenic differentiation of hBMSCs by culturing cells with NAGEL extracts. Furthermore, the effect of NAGEL bioceramics on the osteogenic differentiation in hBMSCs was also investigated with the addition of cardamonin, a WNT inhibitor. The results showed that NAGEL bioceramic disks supported the attachment and proliferation of hBMSCs, and significantly enhanced the bone-related gene expression and WNT signalling pathway of hBMSCs, compared to conventional beta-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic products from NAGEL powders also significantly promoted the proliferation, bone and WNT-related gene expression of hBMSCs. It was also identified that NAGEL bioceramics could bypass the action of the WNT inhibitor (10 μM) to stimulate the selected osteogenic genes in hBMSCs. Our results suggest that NAGEL bioceramics possess excellent in vitro osteostimulation properties. The possible mechanism for the osteostimulation may be directly related to the released Si, Ca and P-containing ionic products from NAGEL bioceramics which activate bone-related gene expression and WNT signalling pathway of hBMSCs. The present study suggests that NAGEL bioceramics are a potential bone regeneration material with significant osteostimulation capacity.

A reliable real-time transport protocol for control systems over wireless networks

Deploying networked control systems (NCSs) over wireless networks is becoming more and more popular. However, the widely-used transport layer protocols, Transmission Control Protocol (TCP) and User Datagram Protocol (UDP), are not designed for real-time applications. Therefore, they may not be suitable for many NCS application scenarios because of their limitations on reliability and/or delay performance, which real-control systems concern. Considering a typical type of NCSs with periodic and sporadic real-time traffic, this paper proposes a highly reliable transport layer protocol featuring a packet loss-sensitive retransmission mechanism and a prioritized transmission mechanism. The packet loss-sensitive retransmission mechanism is designed to improve the reliability of all traffic flows. And the prioritized transmission mechanism offers differentiated services for periodic and sporadic flows. Simulation results show that the proposed protocol has better reliability than UDP and improved delay performance than TCP over wireless networks, particularly when channel errors and congestions occur.

Effects of scaffold architecture on cranial bone healing

Scaffolds for bone tissue engineering should be designed to optimize cell migration, enhance new bone formation and give mechanical support. In the present study, we used polycaprolactone-tricalciumphosphate (PCL/TCP) scaffolds with two different fibre lay down patterns which were coated with hydroxyapatite and gelatine as an approach for optimizing bone regeneration in a critical sized calvarial defect. After 12 weeks bone regeneration was quantified using microCT analysis, biomechanical testing and histological evaluation. Notably, the experimental groups containing coated scaffolds showed lower bone formation and lower biomechanical properties within the defect compared to the uncoated scaffolds. Surprisingly, the different lay down pattern of the fibres resulted in different bone formation and biomechanical properties; namely 0/60/120° scaffolds revealed lower bone formation and biomechanical properties compared to the 0/90° scaffolds in all the experimental groups. The different architecture of the scaffold fibres may have an effect on nutrition supply as well as the attachment of the newly formed matrix to the scaffold. Therefore, future bone regeneration strategies utilising scaffolds should consider scaffold architecture as an important factor during the scaffold optimisation stages in order to move closer to a clinical application.

A reliable real-time transport protocol for networked control systems over wireless networks

Deploying wireless networks in networked control systems (NCSs) has become more and more popular during the last few years. As a typical type of real-time control systems, an NCS is sensitive to long and nondeterministic time delay and packet losses. However, the nature of the wireless channel has the potential to degrade the performance of NCS networks in many aspects, particularly in time delay and packet losses. Transport layer protocols could play an important role in providing both reliable and fast transmission service to fulfill NCS’s real-time transmission requirements. Unfortunately, none of the existing transport protocols, including the Transport Control Protocol (TCP) and the User Datagram Protocol (UDP), was designed for real-time control applications. Moreover, periodic data and sporadic data are two types of real-time data traffic with different priorities in an NCS. Due to the lack of support for prioritized transmission service, the real-time performance for periodic and sporadic data in an NCS network is often degraded significantly, particularly under congested network conditions. To address these problems, a new transport layer protocol called Reliable Real-Time Transport Protocol (RRTTP) is proposed in this thesis. As a UDP-based protocol, RRTTP inherits UDP’s simplicity and fast transmission features. To improve the reliability, a retransmission and an acknowledgement mechanism are designed in RRTTP to compensate for packet losses. They are able to avoid unnecessary retransmission of the out-of-date packets in NCSs, and collisions are unlikely to happen, and small transmission delay can be achieved. Moreover, a prioritized transmission mechanism is also designed in RRTTP to improve the real-time performance of NCS networks under congested traffic conditions. Furthermore, the proposed RRTTP is implemented in the Network Simulator 2 for comprehensive simulations. The simulation results demonstrate that RRTTP outperforms TCP and UDP in terms of real-time transmissions in an NCS over wireless networks.

The development of Monte Carlo techniques for the verification of radiotherapy treatments

Introduction: Recent advances in the planning and delivery of radiotherapy treatments have resulted in improvements in the accuracy and precision with which therapeutic radiation can be administered. As the complexity of the treatments increases it becomes more difficult to predict the dose distribution in the patient accurately. Monte Carlo (MC) methods have the potential to improve the accuracy of the dose calculations and are increasingly being recognised as the ‘gold standard’ for predicting dose deposition in the patient [1]. This project has three main aims: 1. To develop tools that enable the transfer of treatment plan information from the treatment planning system (TPS) to a MC dose calculation engine. 2. To develop tools for comparing the 3D dose distributions calculated by the TPS and the MC dose engine. 3. To investigate the radiobiological significance of any errors between the TPS patient dose distribution and the MC dose distribution in terms of Tumour Control Probability (TCP) and Normal Tissue Complication Probabilities (NTCP). The work presented here addresses the first two aims. Methods: (1a) Plan Importing: A database of commissioned accelerator models (Elekta Precise and Varian 2100CD) has been developed for treatment simulations in the MC system (EGSnrc/BEAMnrc). Beam descriptions can be exported from the TPS using the widespread DICOM framework, and the resultant files are parsed with the assistance of a software library (PixelMed Java DICOM Toolkit). The information in these files (such as the monitor units, the jaw positions and gantry orientation) is used to construct a plan-specific accelerator model which allows an accurate simulation of the patient treatment field. (1b) Dose Simulation: The calculation of a dose distribution requires patient CT images which are prepared for the MC simulation using a tool (CTCREATE) packaged with the system. Beam simulation results are converted to absolute dose per- MU using calibration factors recorded during the commissioning process and treatment simulation. These distributions are combined according to the MU meter settings stored in the exported plan to produce an accurate description of the prescribed dose to the patient. (2) Dose Comparison: TPS dose calculations can be obtained using either a DICOM export or by direct retrieval of binary dose files from the file system. Dose difference, gamma evaluation and normalised dose difference algorithms [2] were employed for the comparison of the TPS dose distribution and the MC dose distribution. These implementations are spatial resolution independent and able to interpolate for comparisons. Results and Discussion: The tools successfully produced Monte Carlo input files for a variety of plans exported from the Eclipse (Varian Medical Systems) and Pinnacle (Philips Medical Systems) planning systems: ranging in complexity from a single uniform square field to a five-field step and shoot IMRT treatment. The simulation of collimated beams has been verified geometrically, and validation of dose distributions in a simple body phantom (QUASAR) will follow. The developed dose comparison algorithms have also been tested with controlled dose distribution changes. Conclusion: The capability of the developed code to independently process treatment plans has been demonstrated. A number of limitations exist: only static fields are currently supported (dynamic wedges and dynamic IMRT will require further development), and the process has not been tested for planning systems other than Eclipse and Pinnacle. The tools will be used to independently assess the accuracy of the current treatment planning system dose calculation algorithms for complex treatment deliveries such as IMRT in treatment sites where patient inhomogeneities are expected to be significant. Acknowledgements: Computational resources and services used in this work were provided by the HPC and Research Support Group, Queensland University of Technology, Brisbane, Australia. Pinnacle dose parsing made possible with the help of Paul Reich, North Coast Cancer Institute, North Coast, New South Wales.

Influence of osteocytes in the in vitro and in vivo β-tricalcium phosphate-stimulated osteogenesis

Osteocytes, known to act as the main regulators of bone homeostasis, have become a major focus in the field of bone research. Bioactive ceramics have been widely used for bone regeneration. However, there are few studies about the interaction of osteocytes with bioceramics. The effects of osteocytes on the in vitro and in vivo osteogenesis of bioceramics are also unclear. The aim of this study was to investigate the role of osteocytes on the b-tricalcium phosphate (b-TCP) stimulated osteogenesis. It was found that osteocytes responded to the b-TCP stimulation, leading to the release of Wnt (wingless-related MMTV integration site), which enhanced osteogenic differentiation of bone marrow stromal cells via Wnt signaling pathway. Receptor activator of nuclear factor kappa B ligand, an osteoclast inducer, was also upregulated, indicating that osteocytes would also participated in activation of osteoclasts, which played a major role in the degradation process of b-TCP and new bone remodeling. In vivo studies further demonstrated that when the material was completely embedded by newly formed bone, the only cell contacting with the material was osteocyte. However, the material would eventually be degraded and replaced by the new bone, requiring the participation of osteoclasts and osteoblasts, which were demonstrated by using immunostaining in this study. As the only cell contacting with the material, osteocytes probably acted in a regulatory role to regulate the surrounding osteoclasts and osteoblasts. Osteocytes were also found to participate in the maturation of osteoblasts and the mineralization process of biomaterials, by upregulating E11 (podoplanin) and dentin matrix protein 1 expression. These findings indicated that osteocytes involved in bone biomaterial-mediated osteogenesis and biomaterial degradation, providing valuable insights into the mechanism of material-stimulated osteogenesis, and a novel strategy to optimize the evaluating system for the biological properties of biomaterials.

Controlling whole blood activation and resultant clot properties on various material surfaces : a possible therapeutic approach for enhancing bone healing

Injured bone initiates the healing process by forming a blood clot at the damaged site. However, in severe damage, synthetic bone implants are used to provide structural integrity and restore the healing process. The implant unavoidably comes into direct contact with whole blood, leading to a blood clot formation on its surface. Despite this, most research in bone tissue engineering virtually ignores the important role of a blood clot in supporting healing. Surface chemistry of a biomaterial is a crucial property in mediating blood-biomaterials interactions, and hence the formation of the resultant blood clot. Surfaces presenting mixtures of functional groups carboxyl (–COOH) and methyl (–CH3) have been shown to enhance platelet response and coagulation activation, leading to the formation of fibrin fibres. In addition, it has been shown that varying the compositions of these functional groups and the length of alkyl groups further modulate the immune complement response. In this study, we hypothesised that a biomaterial surface with mixture of –COOH/–CH3(methyl), –CH2CH3 (ethyl) or –(CH2)3CH3 (butyl) groups at different ratios would modulate blood coagulation and complement activation, and eventually tailor the structural and functional properties of the blood clot formed on the surface, which subsequently impacts new bone formation. Firstly, we synthesised a series of materials composed of acrylic acid (AA), and methyl (MMA), ethyl (EMA) or butyl methacrylates (BMA) at different ratios and coated on the inner surfaces of incubation vials. Our surface analysis showed that the amount of –COOH groups on the surface coatings was lower than the ratios of AA prepared in the materials even though the surface content of –COOH groups increased with increasing in AA ratios. It was indicated that the surface hydrophobicity increased with increasing alkyl chain length: –CH 3 > –CH2CH3 > –(CH2)3CH3, and decreased with increasing –COOH groups. No significant differences in surface hydrophobicity was found on surfaces with –CH3 and –CH2CH3 groups in the presence of –COOH groups. The material coating was as smooth as uncoated glass and without any major flaws. The average roughness of material-coated surface (3.99 ± 0.54 nm) was slightly higher than that of uncoated glass surface (2.22 ± 0.29 nm). However, no significant differences in surface average roughness was found among surfaces with the same functionalities at different –COOH ratios nor among surfaces with different alkyl groups but the same –COOH ratios. These suggested that the surface functional groups and their compositions had a combined effect on modulating surface hydrophobicity but not surface roughness. The second part of our study was to investigate the effect of surface functional groups and their compositions on blood cascade activation and structural properties of the formed clots. It was found that surfaces with –COOH/–(CH2)3CH3 induced a faster coagulation activation than those with –COOH/–CH3 and –CH2CH3, regardless of the –COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces decreased the rate of activation. Moreover, all material-coated surfaces markedly reduced the complement activation compared to uncoated glass surfaces, and the pattern of complement activation was entirely similar to that of surface-induced coagulation, suggesting there is an interaction between two cascades. The clots formed on material-coated surfaces had thicker fibrin with a tighter network at the exterior when compared to uncoated glass surfaces. Compared to the clot exteriors, thicker fibrins with a loose network were found in clot interiors. Coated surfaces resulted in more rigid clots with a significantly slower fibrinolysis after 1 h of lysis when compared to uncoated glass surfaces. Significant differences in fibrinolysis after 1 h of lysis among clots on material-coated surfaces correlated well with the differences in fibrin thickness and density at clot exterior. In addition, more growth factors were released during clot formation than during clot lysis. From an intact clot, there was a correlation between the amount of PDGF-AB release and fibrin density. Highest amount of PDGF-AB was released from clots formed on surfaces with 40% –COOH/60% –CH 3 (i.e. 65MMA). During clot lysis, the release of PDGF-AB also correlated with the fibrinolytic rate while the release of TGF-â1 was influenced by the fibrin thickness. This suggested that different clot structures led to different release profiles of growth factors in clot intact and degrading stages. We further validated whether the clots formed on material-coatings provide the microenvironment for improved bone healing by using a rabbit femoral defect model. In this pilot study, the implantation of clots formed on 65MMA coatings significantly increased new bone formation with enhanced chondrogenesis, osteoblasts activity and vascularisation, but decreased inflammatory macrophage number at the defects after 4 weeks when compared to commercial bone grafts ChronOSTM â-TCP granules. Empty defects were observed when blood clot formation was inhibited. In summary, our study demonstrated that surface functional groups and their relative ratios on material coatings synergistically modulate activation of blood cascades, resultant fibrin architecture, rigidity, susceptibility to fibrinolysis as well as growth factor release of the formed clots, which ultimately alter the healing microenvironment of injured bones.

Osteogenic differentiation of bone marrow MSCs by β-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway

Immune reactions play important roles in determining the in vivo fate of bone substitute materials, either in new bone formation or inflammatory fibrous tissue encapsulation. The paradigm for the development of bone substitute materials has been shifted from inert to immunomodulatory materials, emphasizing the importance of immune cells in the material evaluation. Macrophages, the major effector cells in the immune reaction to implants, are indispensable for osteogenesis and their heterogeneity and plasticity render macrophages a primer target for immune system modulation. However, there are very few reports about the effects of macrophages on biomaterial-regulated osteogenesis. In this study, we used b-tricalcium phosphate (b-TCP) as a model biomaterial to investigate the role of macrophages on the material stimulated osteogenesis. The macrophage phenotype switched to M2 extreme in response to b-TCP extracts, which was related to the activation of calcium-sensing receptor (CaSR) pathway. Bone morphogenetic protein 2 (BMP2) was also significantly upregulated by the b-TCP stimulation, indicating that macrophage may participate in the b-TCP stimulated osteogenesis. Interestingly, when macrophageconditioned b-TCP extracts were applied to bone marrow mesenchymal stem cells (BMSCs), the osteogenic differentiation of BMSCs was significantly enhanced, indicating the important role of macrophages in biomaterial-induced osteogenesis. These findings provided valuable insights into the mechanism of material-stimulated osteogenesis, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of bone substitute materials.

Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer : preparation, formation mechanism, improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells

The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel’s adhesive versatility, which is thought to be due to the plaque–substrate interface being rich in 3,4-dihydroxy-L-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of b-tricalcium phosphate (b-TCP) bioceramics by soaking b-TCP bioceramics in Tris–dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris–HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of b-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the b-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of b-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by significantly improving their surface physicochemical properties and cellular bioactivity for bone regeneration application.

A stimulatory effect of Ca3ZrSi2O9 bioceramics on cementogenic/osteogenic differentiation of periodontal ligament cells

The regeneration of periodontal tissues to cure periodontitis remains a medical challenge. Therefore, it is of great importance to develop a novel biomaterial that could induce cementogenesis and osteogenesis in periodontal tissue engineering. Calcium silicate (Ca–Si) based ceramics have been found to be potential bioactive materials due to their osteostimulatory effect. Recently, it is reported that zirconium modified calcium-silicate-based (Ca3ZrSi2O9) ceramics stimulate cell proliferation and osteogenic differentiation of osteoblasts. However, it is unknown whether Ca3ZrSi2O9 ceramics possess specific cementogenic stimulation for human periodontal ligament cells (hPDLCs) in periodontal tissue regeneration in vitro. The purpose of this study was to investigate whether Ca3ZrSi2O9 ceramic disks and their ionic extracts could stimulate cell growth and cementogenic/osteogenic differentiation of hPDLCs; the possible molecular mechanism involved in this process was also explored by investigating the Wnt/β-catenin signalling pathway of hPDLCs. Our results showed that Ca3ZrSi2O9 ceramic disks supported cell adhesion, proliferation and significantly up-regulated relative alkaline phosphatase (ALP) activity, cementogenic/osteogenic gene expression (CEMP1, CAP, ALP and OPN) and Wnt/β-catenin signalling pathway-related genes (AXIN2 and CTNNB) for hPDLCs, compared to that of β-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic extracts from Ca3ZrSi2O9 powders also significantly enhanced relative ALP activity, cementogenic/osteogenic and Wnt/β-catenin-related gene expression of hPDLCs. The present results demonstrate that Ca3ZrSi2O9 ceramics are capable of stimulating cementogenic/osteogenic differentiation of hPDLCs possibly via activation of the Wnt/β-catenin signalling pathway, suggesting that Ca3ZrSi2O9 ceramics have the potential to be used for periodontal tissue regeneration.

Strong and bioactive tri-calcium phosphate scaffolds with tube-like macropores

Calcium phosphate ceramic scaffolds have been widely investigated for bone tissue engineering due to their excellent biocompatibility and biodegradation. Unfortunately, they have the shortcoming of low mechanical properties. In order to provide strong, bioactive, and biodegradable scaffolds, a new approach of infiltrating the macro-tube ABS (acrylontrile butadiene styrene) templates with a hydroxyapatite/bioactive glass mixed slurry was developed to fabricate porous Si-doped TCP (tri-calcium phosphate) scaffolds. The porous Si-doped TCP ceramics with a high porosity (~65%) and with interconnected macrotubes (~0.8mm in diameter) and micropores (5-100 m) had a high compressive strength (up to 14.68+0.2MPa), which was comparable to that of a trabecular bone and was much higher than those of pure TCP scaffolds. Additional cell attachment study and MTT cytotoxicity assay proved the bioactivity and biocompatibility of the new scaffolds. Thus a potential bioceramic material and a new approach to make the potential scaffolds were developed for bone tissue engineering.

Analysis of temporary traffic control cost items in transportation construction bidding process

Temporary Traffic Control Plans (TCP’s), which provide construction phasing to maintain traffic during construction operations, are integral component of highway construction project design. Using the initial design, designers develop estimated quantities for the required TCP devices that become the basis for bids submitted by highway contractors. However, actual as-built quantities are often significantly different from the engineer’s original estimate. The total cost of TCP phasing on highway construction projects amounts to 6–10% of the total construction cost. Variations between engineer estimated quantities and final quantities contribute to reduced cost control, increased chances of cost related litigations, and bid rankings and selection. Statistical analyses of over 2000 highway construction projects were performed to determine the sources of variation, which later were used as the basis of development for an automated-hybrid prediction model that uses multiple regressions and heuristic rules to provide accurate TCP quantities and costs. The predictive accuracy of the model developed was demonstrated through several case studies.