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.

Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with β-tricalcium phosphate microparticles (mPCL-TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL-TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.

Lithium release from ß-tricalcium phosphate inducing cementogenic and osteogenic differentiation for both hPDLCs and hBMSCs

It is accepted that the accelerated differentiation of tissue cells on bioactive materials is of great importance to regenerate the lost tissues. It was previously reported that lithium (Li) ions could enhance the in vitro proliferation and differentiation of retinoblastoma cells and endometrium epithelia by activating the Wnt canonical signalling pathway. It is interesting to incorporate Li ions into bioactive ceramics, such as β-tricalcium phosphate (Li-β-TCP), in order to stimulate both osteogenic and cementogenic differentiation of different stem cells for the regeneration of bone/periodontal tissues. Therefore, the aim of this study was to investigate the interactions of human periodontal ligament cells (hPDLCs) and human bone marrow stromal cells (hBMSCs) with Li-β-TCP bioceramic bulks and their ionic extracts, and further explore the osteogenic and cementogenic stimulation of Li-β-TCP bioceramics and the possible molecular mechanisms. The results showed that Li-β-TCP bioceramic disks supported the cell attachment and proliferation, and significantly enhanced bone/cementum-related gene expression, Wnt canonical signalling pathway activation for both hPDLCs and hBMSCs, compared to conventional β-TCP bioceramic disks without Li. The release of Li from Li-β-TCP powders could significantly promote the bone/cementum-related gene expression for both hPDLCs and hBMSCs compared to pure β-TCP extracts without Li release. Our results suggest that the combination of Li with β-TCP bioceramics may be a promising method to enhance bone/cementum regeneration as Li-β-TCP possesses excellent in vitro osteogenic and cementogenic stimulation properties by inducing bone/cementum-related gene expression in both hPDLCs and hBMSCs.

Osteoimmunomodulatory properties of magnesium scaffolds coated with β-tricalcium phosphate

The osteoimmunomodulatory property of bone biomaterials is a vital property determining the in vivo fate of the implants. Endowing bone biomaterials with favorable osteoimmunomodulatory properties is of great importance in triggering desired immune response and thus supports the bone healing process. Magnesium (Mg) has been recognized as a revolutionary metal for applications in orthopedics due to it being biodegradable, biocompatible, and having osteoconductive properties. However, Mg's high rate of degradation leads to an excessive inflammatory response and this has restricted its application in bone tissue engineering. In this study, β-tricalcium phosphate (β-TCP) was used to coat Mg scaffolds in an effort to modulate the detrimental osteoimmunomodulatory properties of Mg scaffolds, due to the reported favorable osteoimmunomodulatory properties of β-TCP. It was noted that macrophages switched to the M2 extreme phenotype in response to the Mg-β-TCP scaffolds, which could be due to the inhibition of the toll like receptor (TLR) signaling pathway. VEGF and BMP2 were significantly upregulated in the macrophages exposed to Mg-β-TCP scaffolds, indicating pro-osteogenic properties of macrophages in β-TCP modified Mg scaffolds. This was further demonstrated by the macrophage-mediated osteogenic differentiation of bone marrow stromal cells (BMSCs). When BMSCs were stimulated by conditioned medium from macrophages cultured on Mg-β-TCP scaffolds, osteogenic differentiation of BMSCs was significantly enhanced; whereas osteoclastogenesis was inhibited, as indicated by the downregualtion of MCSF, TRAP and inhibition of the RANKL/RANK system. These findings suggest that β-TCP coating of Mg scaffolds can modulate the scaffold's osteoimmunomodulatory properties, shift the immune microenvironment towards one that favors osteogenesis over osteoclastogenesis. Endowing bone biomaterials with favorable osteoimmunomodulatory properties can be a highly valuable strategy for the development or modification of advanced bone biomaterials.

Development of a cost-effective and flexible vibration DAQ system for long-term continuous structural health monitoring

In the structural health monitoring (SHM) field, long-term continuous vibration-based monitoring is becoming increasingly popular as this could keep track of the health status of structures during their service lives. However, implementing such a system is not always feasible due to on-going conflicts between budget constraints and the need of sophisticated systems to monitor real-world structures under their demanding in-service conditions. To address this problem, this paper presents a comprehensive development of a cost-effective and flexible vibration DAQ system for long-term continuous SHM of a newly constructed institutional complex with a special focus on the main building. First, selections of sensor type and sensor positions are scrutinized to overcome adversities such as low-frequency and low-level vibration measurements. In order to economically tackle the sparse measurement problem, a cost-optimized Ethernet-based peripheral DAQ model is first adopted to form the system skeleton. A combination of a high-resolution timing coordination method based on the TCP/IP command communication medium and a periodic system resynchronization strategy is then proposed to synchronize data from multiple distributed DAQ units. The results of both experimental evaluations and experimental–numerical verifications show that the proposed DAQ system in general and the data synchronization solution in particular work well and they can provide a promising cost-effective and flexible alternative for use in real-world SHM projects. Finally, the paper demonstrates simple but effective ways to make use of the developed monitoring system for long-term continuous structural health evaluation as well as to use the instrumented building herein as a multi-purpose benchmark structure for studying not only practical SHM problems but also synchronization related issues.

Graphene-oxide-modified β-tricalcium phosphate bioceramics stimulate in vitro and in vivo osteogenesis

Graphene oxide (GO) has attracted much interest for applications in bone tissue engineering; however, until now the interaction between GO and stem cells, and the in vivo bone-forming ability of GO has not been explored. The aim of this study was to produce a GO-modified β-tricalcium phosphate (β-TCP-GRA) biceramics and then explore the material’s osteogenic capacity in vitro and in vivo, as well as unravel some of the molecular mechanisms behind this. β-TCP-GRA disks and scaffolds were successfully prepared by a simple GO/water suspension soaking method in combination with heat treatment. These scaffolds were found to significantly enhance the proliferation, alkaline phosphatase activity and osteogenic gene expression of human bone marrow stromal cells (hBMSCs), when compared to β-TCP without GO modification (controls). Activation of the Wnt/β-catenin signaling pathway in hBMSCs appears to be the mechanism behind this osteogenic induction by β-TCP-GRA. β-TCP-GRA scaffolds led to an increased rate of in vivo new bone formation compared to β-TCP controls, indicative of the stimulatory effect of GO on in vivo osteogenesis, making GO modification of β-TCP a very promising method for applications in bone tissue engineering, in particular for the regeneration of large bone defects.

A framework for generating realistic traffic for distributed denial-of-service attacks and flash events

An intrinsic challenge associated with evaluating proposed techniques for detecting Distributed Denial-of-Service (DDoS) attacks and distinguishing them from Flash Events (FEs) is the extreme scarcity of publicly available real-word traffic traces. Those available are either heavily anonymised or too old to accurately reflect the current trends in DDoS attacks and FEs. This paper proposes a traffic generation and testbed framework for synthetically generating different types of realistic DDoS attacks, FEs and other benign traffic traces, and monitoring their effects on the target. Using only modest hardware resources, the proposed framework, consisting of a customised software traffic generator, ‘Botloader’, is capable of generating a configurable mix of two-way traffic, for emulating either large-scale DDoS attacks, FEs or benign traffic traces that are experimentally reproducible. Botloader uses IP-aliasing, a well-known technique available on most computing platforms, to create thousands of interactive UDP/TCP endpoints on a single computer, each bound to a unique IP-address, to emulate large numbers of simultaneous attackers or benign clients.

Experiences With WM: A Centralised Scheduling Approach for Performance Management of IEEE 802.11 Wireless LANs

In our earlier work ([1]) we proposed WLAN Manager (or WM) a centralised controller for QoS management of infrastructure WLANs based on the IEEE 802.11 DCF standards. The WM approach is based on queueing and scheduling packets in a device that sits between all traffic flowing between the APs and the wireline LAN, requires no changes to the AP or the STAs, and can be viewed as implementing a "Split-MAC" architecture. The objectives of WM were to manage various TCP performance related issues (such as the throughput "anomaly" when STAs associate with an AP with mixed PHY rates, and upload-download unfairness induced by finite AP buffers), and also to serve as the controller for VoIP admission control and handovers, and for other QoS management measures. In this paper we report our experiences in implementing the proposals in [1]: the insights gained, new control techniques developed, and the effectiveness of the WM approach in managing TCP performance in an infrastructure WLAN. We report results from a hybrid experiment where a physical WM manages actual TCP controlled packet flows between a server and clients, with the WLAN being simulated, and also from a small physical testbed with an actual AP.

The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3-4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.

Porphyrins with multiple crown ether voids: novel systems for cation complexation studies

Porphyrins appended with crown ether, benzo-15-crown-5, at the methine positions have been synthesized and characterized. The fully and partially substituted porphyrins and their metallo (Co, Cu, and Zn) derivatives describe one or more ether cavities in the periphery that are capable of recognizing spherical cations. The ability of these macrocycles to complex cations (Na+, K+, Mg2+, Ca2+, Ba2', and NH4+) is investigated by use of visible, 'H NMR, ESR, and emission spectral studies. The tetrasubstituted crown porphyrin (TCP) exhibits very high selectivity for K+. The cations (K', Ba2+, and NH4+) that require two crown ether cavities for complexation promote dimerization of the porphyrins. The ESR study of the cation-induced porphyrin dimers reveals axial symmetry with the porphyrin planes separated by -4.2 A. This distance increases from the fully substituted to partially substituted porphyrins. The cations (K', Ba2+, and NH4') quench efficiently the fluorescence of the free base porphyrins and their metallo derivatives. The quenching process is attributed to the steric geometry of the dimers.