Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

Securing IEEE 802.11 wireless LANs

As the acceptance and popularity of wireless networking technologies has proliferated, the security of the IEEE 802.11 wireless local area network (WLAN) has advanced in leaps and bounds. From tenuous beginnings, where the only safe way to deploy a WLAN was to assume it was hostile and employ higherlayer information security controls, to the current state of the art, all manner of improvements have been conceived and many implemented. This work investigates some of the remaining issues surrounding IEEE 802.11 WLAN operation. While the inherent issues in WLAN deployments and the problems of the original Wired Equivalent Privacy (WEP) provisions are well known and widely documented, there still exist a number of unresolved security issues. These include the security of management and control frames and the data link layer protocols themselves. This research introduces a novel proposal to enhance security at the link layer of IEEE 802.11 WLANs and then conducts detailed theoretical and empirical investigation and analysis of the eects of such proposals. This thesis �rst de�nes the state of the art in WLAN technology and deployment, including an overview of the current and emerging standards, the various threats, numerous vulnerabilities and current exploits. The IEEE 802.11i MAC security enhancements are discussed in detail, along with the likely outcomes of the IEEE 802.11 Task Group W1, looking into protected management frames. The problems of the remaining unprotected management frames, the unprotected control frames and the unprotected link layer headers are reviewed and a solution is hypothesised, to encrypt the entire MAC Protocol Data Unit (MPDU), including the MAC headers, not just the MAC Service Data Unit (MSDU) commonly performed by existing protocols. The proposal is not just to encrypt a copy of the headers while still using cleartext addresses to deliver the frame, as used by some existing protocols to support the integrity and authenticity of the headers, but to pass the entire MPDU only as ciphertext to also support the con�dentiality of the frame header information. This necessitates the decryption of every received frame using every available key before a station can determine if it is the intended recipient. As such, this raises serious concerns as to the viability of any such proposal due to the likely impact on throughput and scalability. The bulk of the research investigates the impacts of such proposals on the current WLAN protocols. Some possible variations to the proposal are also provided to enhance both utility and speed. The viability this proposal with respect to the eect on network throughput is then tested using a well known and respected network simulation tool, along with a number of analysis tools developed speci�cally for the data generated here. The simulator's operation is �rst validated against recognised test outputs, before a comprehensive set of control data is established, and then the proposal is tested and and compared against the controls. This detailed analysis of the various simulations should be of bene�t to other researchers who need to validate simulation results. The analysis of these tests indicate areas of immediate improvement and so the protocols are adjusted and a further series of experiments conducted. These �nal results are again analysed in detail and �nal appraisals provided.

Systematic variations in polymer gel dosimeter calibration due to container influence and deviations from water equivalence

There are a number of gel dosimeter calibration methods in contemporary usage. The present study is a detailed Monte Carlo investigation into the accuracy of several calibration techniques. Results show that for most arrangements the dose to gel accurately reflects the dose to water, with the most accurate method involving the use of a large diameter flask of gel into which multiple small fields of varying dose are directed. The least accurate method was found to be that of a long test tube in a water phantom, coaxial with the beam. The large flask method is also the most straightforward and least likely to introduce errors during setup, though, to its detriment, the volume of gel required is much more than other methods.

Colonization and osteogenic differentiation of different stem cell sources on electrospun nanofiber meshes

Numerous challenges remain in the successful clinical translation of cell-based therapies for musculoskeletal tissue repair, including the identification of an appropriate cell source and a viable cell delivery system. The aim of this study was to investigate the attachment, colonization, and osteogenic differentiation of two stem cell types, human mesenchymal stem cells (hMSCs) and human amniotic fluid stem (hAFS) cells, on electrospun nanofiber meshes. We demonstrate that nanofiber meshes are able to support these cell functions robustly, with both cell types demonstrating strong osteogenic potential. Differences in the kinetics of osteogenic differentiation were observed between hMSCs and hAFS cells, with the hAFS cells displaying a delayed alkaline phosphatase peak, but elevated mineral deposition, compared to hMSCs. We also compared the cell behavior on nanofiber meshes to that on tissue culture plastic, and observed that there is delayed initial attachment and proliferation on meshes, but enhanced mineralization at a later time point. Finally, cell-seeded nanofiber meshes were found to be effective in colonizing three-dimensional scaffolds in an in vitro system. This study provides support for the use of the nanofiber mesh as a model surface for cell culture in vitro, and a cell delivery vehicle for the repair of bone defects in vivo.

Controller structure and design of firing angle controllers for (unit connected) HVDC systems

This paper investigates the control of a HVDC link, fed from an AC source through a controlled rectifier and feeding an AC line through a controlled inverter. The overall objective is to maintain maximum possible link voltage at the inverter while regulating the link current. In this paper the practical feedback design issues are investigated with a view of obtaining simple, robust designs that are easy to evaluate for safety and operability. The investigations are applicable to back-to-back links used for frequency decoupling and to long DC lines. The design issues discussed include: (i) a review of overall system dynamics to establish the time scale of different feedback loops and to highlight feedback design issues; (ii) the concept of using the inverter firing angle control to regulate link current when the rectifier firing angle controller saturates; and (iii) the design issues for the individual controllers including robust design for varying line conditions and the trade-off between controller complexity and the reduction of nonlinearity and disturbance effects

Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter×5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host–tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site. Keywords: Osteochondral tissue engineering; Scaffold; Bone marrow-derived precursor cells; Fibrin glue

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the pupillary light reflex and circadian rhythm

Recently discovered intrinsically photosensitive melanopsin retinal ganglion cells contribute to the maintenance of pupil diameter, recovery and post-illumination components of the pupillary light reflex and provide the primary environmental light input to the suprachiasmatic nucleus for photoentrainment of the circadian rhythm. This review summarises recent progress in understanding intrinsically photosensitive ganglion cell histology and physiological properties in the context of their contribution to the pupillary and circadian functions and introduces a clinical framework for using the pupillary light reflex to evaluate inner retinal (intrinsically photosensitive melanopsin ganglion cell) and outer retinal (rod and cone photoreceptor) function in the detection of retinal eye disease.

The provision of patient personal hygiene in the intensive care unit : a descriptive exploratory study of bed-bathing practice

Background/objectives The provision of the patient bed-bath is a fundamental nursing care activity yet few quantitative data and no qualitative data are available on registered nurses’ (RNs) clinical practice in this domain in the intensive care unit (ICU). The aim of this study was to describe ICU RNs current practice with respect to the timing, frequency and duration of the patient bed-bath and the cleansing and emollient agents used. Methods The study utilised a two-phase sequential explanatory mixed method design. Phase one used a questionnaire to survey RNs and phase two employed semi-structured focus group (FG) interviews with RNs. Data was collected over 28 days across four Australian metropolitan ICUs. Ethical approval was granted from the relevant hospital and university human research ethics committees. RNs were asked to complete a questionnaire following each episode of care (i.e. bed-bath) and then to attend one of three FG interviews: RNs with less than 2 years ICU experience; RNs with 2–5 years ICU experience; and RNs with greater than 5 years ICU experience. Results During the 28-day study period the four ICUs had 77.25 beds open. In phase one a total of 539 questionnaires were returned, representing 30.5% of episodes of patient bed-baths (based on 1767 bed occupancy and one bed-bath per patient per day). In 349 bed-bath episodes 54.7% patients were mechanically ventilated. The bed-bath was given between 02.00 and 06.00 h in 161 episodes (30%), took 15–30 min to complete (n = 195, 36.2%) and was completed within the last 8 h in 304 episodes (56.8%). Cleansing agents used were predominantly pH balanced soap or liquid soap and water (n = 379, 71%) in comparison to chlorhexidine impregnated sponges/cloths (n = 86, 16.1%) or other agents such as pre-packaged washcloths (n = 65, 12.2%). In 347 episodes (64.4%) emollients were not applied after the bed-bath. In phase two 12 FGs were conducted (three FGs at each ICU) with a total of 42 RN participants. Thematic analysis of FG transcripts across the three levels of RN ICU experience highlighted a transition of patient hygiene practice philosophy from shades of grey – falling in line for inexperienced clinicians to experienced clinicians concrete beliefs about patient bed-bath needs. Conclusions This study identified variation in process and products used in patient hygiene practices in four ICUs. Further study to improve patient outcomes is required to determine the appropriate timing of patient hygiene activities and cleansing agents used to improve skin integrity.

Nonprofit organizational and work unit identification and the occupational stress process

Research investigating the transactional approach to the work stressor-employee adjustment relationship has described many negative main effects between perceived stressors in the workplace and employee outcomes. A considerable amount of literature, theoretical and empirical, also describes potential moderators of this relationship. Organizational identification has been established as a significant predictor of employee job-related attitudes. To date, research has neglected investigation of the potential moderating effect of organizational identification in the work stressor-employee adjustment relationship. On the basis of identity, subjective fit and sense of belonging literature it was predicted that higher perceptions of identification at multiple levels of the organization would mitigate the negative effect of work stressors on employee adjustment. It was expected, further, that more proximal, lower order identifications would be more prevalent and potent as buffers of stressors on strain. Predictions were tested with an employee sample from five organizations (N = 267). Hierarchical moderated multiple regression analyses revealed some support for the stress-buffering effects of identification in the prediction of job satisfaction and organizational commitment, particularly for more proximal (i.e., work unit) identification. These positive stress-buffering effects, however, were present for low identifiers in some situations. The present study represents an extension of the application of organizational identity theory by identifying the effects of organizational and workgroup identification on employee outcomes in the nonprofit context. Our findings will contribute to a better understanding of the dynamics in nonprofit organizations and therefore contribute to the development of strategy and interventions to deal with identity-based issues in nonprofits.

Effects of the architecture of tissue engineering scaffolds on cell seeding and culturing

The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work we assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer‐designed gyroid architecture fabricated by stereolithography to a random‐pore architecture resulting from salt‐leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than tenfold higher permeability due to the absence of size‐limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds, and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture, gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt‐leached scaffolds were covered with a cell‐sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolongs the time of static culture before overgrowth of cells at the scaffold periphery occurs. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds, and to create tissue engineering grafts with designed, pre‐fabricated vasculature.

Organ printing

Organ printing techniques offer the potential to produce living 3D tissue constructs to repair or replace damaged or diseased human tissues and organs. Using these techniques, spatial variations along multiple axes with high geometric complexity can be obtained.. The level of control offered by these technologies to develop printed tissues will allow tissue engineers to better study factors that modulate tissue formation and function, and provide a valuable tool to study the effect of anatomy on graft performance. In this chapter we discuss the history behind substrate patterning and cell and organ printing, and the rationale for developing organ printing techniques with respect to limitations of current clinical tissue engineering strategies to effectively repair damaged tissues. We discuss current 2-dimensional and 3-dimesional strategies for assembling cells as well as the necessary support materials such as hydrogels, bioinks and natural and synthetic polymers adopted for organ printing research. Furthermore, given the current state-of-the-art in organ printing technologies, we discuss some of their limitations and provide recommendations for future developments in this rapidly growing field.

Three-dimensional hydrogen-bonded structures in the guanidinium salts of the monocyclic dicarboxylic acids rac-trans-cyclohexane-1,2-dicarboxylic acid (2:1, anhydrous), isophthalic acid (1:1, monohydrate) and terephthalic acid (2:1, trihydrate).

The structures of bis(guanidinium)rac-trans-cyclohexane-1,2-dicarboxylate, 2(CH6N3+) C8H10O4- (I), guanidinium 3-carboxybenzoate monohydrate CH6N3+ C8H5O4- . H2O (II) and bis(guanidinium) benzene-1,4-dicarboxylate trihydrate, 2(CH6N3+) C8H4O4^2- . 3H2O (III) have been determined and the hydrogen bonding in each examined. All three compounds form three-dimensional hydrogen-bonded framework structures. In anhydrous (I), both guanidinium cations give classic cyclic R2/2(8) N--H...O,O'(carboxyl) and asymmetric cyclic R1/2(6) hydrogen-bonding interactions while one cation gives an unusual enlarged cyclic interaction with O acceptors of separate ortho-related carboxyl groups [graph set R2/2(11)]. Cations and anions also associate across inversion centres giving cyclic R2/4(8) motifs. In the 1:1 guanidinium salt (II), the cation gives two separate cyclic R1/2(6) interactions, one with a carboxyl O-acceptor, the other with the water molecule of solvation. The structure is unusual in that both carboxyl groups give short inter-anion O...H...O contacts, one across a crystallographic inversion centre [2.483(2)\%A], the other about a two-fold axis of rotation [2.462(2)\%A] with a half-occupancy hydrogen delocalized on the symmetry element in each. The water molecule links the cation--anion ribbon structures into a three-dimensional framework. In (III), the repeating molecular unit comprises a benzene-1,4-dicarboxylate dianion which lies across a crystallographic inversion centre, two guanidinium cations and two water molecules of solvation (each set related by two-fold rotational symmetry), and a single water molecule which lies on a two-fold axis. Each guanidinium cation gives three types of cyclic interactions with the dianions: one R^1^~2~(6), the others R2/3(8) and R3/3(10) (both of these involving the water molecules), giving a three-dimensional structure through bridges down the b cell direction. The water molecule at the general site also forms an unusual cyclic R2/2(4) homodimeric association across an inversion centre [O--H...O, 2.875(2)\%A]. The work described here provides further examples of the common cyclic guanidinium cation...carboxylate anion hydrogen-bonding associations as well as featuring other less common cyclic motifs.

Cell invasion with proliferation mechanisms motivated by time-lapse data

Cell invasion involves a population of cells which are motile and proliferative. Traditional discrete models of proliferation involve agents depositing daughter agents on nearest- neighbor lattice sites. Motivated by time-lapse images of cell invasion, we propose and analyze two new discrete proliferation models in the context of an exclusion process with an undirected motility mechanism. These discrete models are related to a family of reaction- diffusion equations and can be used to make predictions over a range of scales appropriate for interpreting experimental data. The new proliferation mechanisms are biologically relevant and mathematically convenient as the continuum-discrete relationship is more robust for the new proliferation mechanisms relative to traditional approaches.