The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue

In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no-matrix control. Poly-D,L-lactic-co-glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Dependence of second-harmonic signals generated in malignant human prostate tissue on excitation wavelength

The dependence of second harmonic generation (SHG) from hyperplastic parenchyma and stroma in maligant human prostate tissue on excitation wavelengths was measured. A femtosecond pulsed laser, a scanning microscope and a spectrograph were used to perform the measurements. The spectra were measured under excitation power of 10 mW at excitation wavelengths of 730 nm, 750 nm, 800 nm, 850 nm and 890 nm. Analysis suggested that the SHG in prostate tissue is highly structured and wavelength dependent signifying its ability to be used as an indicator for recognizing tissue components, ultrastructures, micro-environments and diseases.

Optimising the cell source and biomaterials for the generation of vascularized adipose tissue in vivo

We initially described a rat chamber model with an inserted arteriovenous pedicle which spontaneously generates 3-dimensional vascularized connective tissue (Tanaka Y et al., Br J Plast Surg 2000; 53: 51-7). More recently we have developed a murine chamber model containing reconstituted basement membrane (Matrigel®) and FGF-2 that generates vascularized adipose tissue in vivo (Cronin K et al., Plast Reconstr Surg 2004; in press). We have extended this work to assess the cellular and matrix requirements for the Matrigel®- induced neo-adipogenesis. We found that chambers sealed to host fat were unable to grow new adipose tissue. In these chambers the Matrigel® became vascularized with maximal outgrowth of vessels extending to the periphery at 6 weeks. A small amount of adipose tissue was found adjacent to the vessels, most likely arising from periadventitial adipose tissue. In contrast, chambers open to interaction with endogenous adipose tissue showed abundant new fat, and partial exposure to adjacent adipose tissue clearly showed neo-adipogenesis only in this area. Addition of small amounts of free fat to the closed chamber containing Matrigel® was able to induce neo-adipogenesis. Addition of small pieces of human fat also caused neo-adipogenesis in immunocompromised (SCID) mice. Also, we found Matrigel® to induce adipogenesis of Lac-Z-tagged (Rosa-26) murine bone marrow-derived mesenchymal stem cells, and cells similar to these have been isolated from human adipose tissue. Given that Matrigel® is a mouse product and cannot be used in humans, we have started investigating alternative matrix scaffolds for adipogenesis such as the PDA-approved PLGA, collagen and purified components derived from Matrigel®, such as laminin-1. The optimal conditions for adipogenesis with these matrices are still being elucidated. In conclusion, we have demonstrated that a precursor cell source inside the chamber is essential for the generation of vascularized adipose tissue in vivo. This technique offers unique potential for the reconstruction of soft tissue defects and may enable the generation of site-specific tissue using the correct microenvironment.

The role of vascularization in 3-D tissue engineering

The role of vascularization in 3-D tissue engineering was studied. Mouse fat, angiogenic growth factors, adult human stem cells and fat tissue have been inserted and subsequent tissue growth was monitored. Human fat grafts or human lipoaspirates in SCID mouse chambers induced mouse fat generation at 6 weeks. Tissue engineering models utilizing intrinsic vascularization have major advantages including rapid and appropriate vascularization of new tissues.

Adipose tissue induction in vivo

Engineering adipogenic tissue in vivo requires the concomitant induction of angiogenesis to generate a stable long-term three-dimensional construct. Histiocon-ductive tissue engineering strategies have been used. The disadvantage of using biodegradable scaffolds is a delayed angiogenic induction resulting in ischemic necrosis of the central cell population in the scaffold. We evaluated an histioinductive approach for adipose tissue engineering by combining essential key components for adipogenic induction: (1) a precursor cell source; (2) a vascular pedicle; (3) a supportive matrix, and; (4) a chamber to preserve space for the new tissue to develop. We observed concomitant adipogenic and angiogenic induction after 6 weeks in three-dimensional adipose tissue constructs.

Characterisation of the micro-architecture of direct writing melt electrospun tissue engineering scaffolds using diffusion tensor and computed tomography microimaging

This article describes the first steps toward comprehensive characterization of molecular transport within scaffolds for tissue engineering. The scaffolds were fabricated using a novel melt electrospinning technique capable of constructing 3D lattices of layered polymer fibers with well - defined internal microarchitectures. The general morphology and structure order was then determined using T 2 - weighted magnetic resonance imaging and X - ray microcomputed tomography. Diffusion tensor microimaging was used to measure the time - dependent diffusivity and diffusion anisotropy within the scaffolds. The measured diffusion tensors were anisotropic and consistent with the cross - hatched geometry of the scaffolds: diffusion was least restricted in the direction perpendicular to the fiber layers. The results demonstrate that the cross - hatched scaffold structure preferentially promotes molecular transport vertically through the layers ( z - axis), with more restricted diffusion in the directions of the fiber layers ( x – y plane). Diffusivity in the x – y plane was observed to be invariant to the fiber thickness. The characteristic pore size of the fiber scaffolds can be probed by sampling the diffusion tensor at multiple diffusion times. Prospective application of diffusion tensor imaging for the real - time monitoring of tissue maturation and nutrient transport pathways within tissue engineering scaffolds is discussed.

Carbon nanostructures for hard tissue engineering

The primary goal in hard tissue engineering is to combine high-performance scaffold materials with living cells to develop biologically active substitutes that can restore tissue functions. This requires relevant knowledge in multidisciplinary fields encompassing chemical engineering, material science, chemistry, biology and nanotechnology. Here we present an overview on the recent progress of how two representative carbon nanostructures, namely, carbon nanotubes and graphene, aid and advance the research in hard tissue engineering. The article focuses on the advantages and challenges of integrating these carbon nanostructures into functional scaffolds for repairing and regenerative purposes. It includes, but is not limited to, the critical physico-chemical properties of carbon nanomaterials for enhanced cell interactions such as adhesion, morphogenesis, proliferation and differentiation; the novel designs of two- and three-dimensional nanostructured scaffolds; multifunctional hybrid materials; and the biocompatible aspects of carbon nanotubes and graphene. Perspectives on the future research directions are also given, in an attempt to shed light on the innovative and rational design of more effective biomedical devices in hard tissue engineering.

Diabetic peripheral neuropathy and retinal tissue thickness

Using retinal imaging, the nature and extent of compromise of retinal structural integrity has been characterized in individuals suffering from diabetic peripheral neuropathy. These findings extend our understanding of the pathological processes involved in diabetic neuropathy and offer novel ophthalmic approaches to the diagnosis and monitoring of this debilitating condition.

Mechanical model and superelastic properties of carbon microcoils with circular cross-section

Here we report on an unconventional Ni-P alloy-catalyzed, high-throughput, highly reproducible chemical vapor deposition of ultralong carbon microcoils using acetylene precursor in the temperature range 700-750 °C. Scanning electron microscopy analysis reveals that the carbon microcoils have a unique double-helix structure and a uniform circular cross-section. It is shown that double-helix carbon microcoils have outstanding superelastic properties. The microcoils can be extended up to 10-20 times of their original coil length, and quickly recover the original state after releasing the force. A mechanical model of the carbon coils with a large spring index is developed to describe their extension and contraction. Given the initial coil parameters, this mechanical model can successfully account for the geometric nonlinearity of the spring constants for carbon micro- and nanocoils, and is found in a good agreement with the experimental data in the whole stretching process.

Web crippling behaviour of C-section beams with web ribs

SupaCee section is one of the cold-formed steel members which is increasingly used in the construction sector. It is characterized by unique ribbed web and curved lip elements, and is claimed to be more economical with extra strength than the traditional channel sections. SupaCee sections are widely used in Australia as floor joists, bearers, purlins and girts. Many experimental and numerical studies have been carried out to evaluate the behaviour and design of conventional channel beams subject to web crippling. To date, however, no investigation has been conducted into the web crippling behaviour and strength of SupaCee sections. Current cold-formed steel design equations do not include any design procedures for SupaCee sections. Hence experimental studies were conducted to assess the web crippling behaviour and strengths of SupaCee sections under ETF and ITF load cases. Thirty six web crippling tests were conducted and the capacity results were compared with the predictions from the AS/NZS 4600 and AISI design rules developed for conventional channel sections. Comparison of ultimate web crippling capacities from tests showed that AS/NZS 4600 and AISI design equations are unconservative for SupaCee sections under ETF load case, but are overly conservative for ITF load case. Hence new equations were proposed to determine the web crippling capacities of SupaCee sections based on the experimental results from this study. Suitable design rules were also developed within the direct strength method format. This paper presents the details of this experimental study of SupaCee sections subject to web crippling and the results.

A novel approach for numerical simulation of plant tissue shrinkage during drying

Drying is a key processing techniques used in food engineering which demands continual developments on advanced analysis techniques in order to optimize the product and the process. In this regard, plant based materials are a frequent subject of interest where microstructural studies can provide a clearer understanding on the fundamental physical mechanisms involved. In this context, considering numerous challenges of using conventional numerical grid-based modelling techniques, a meshfree particle based model was developed to simulate extreme deformations of plant microstructure during drying. The proposed technique is based on a particle based meshfree method: Smoothed Particle Hydrodynamics (SPH) and a Discrete Element Method (DEM). A tissue model was developed by aggrading individual cells modelled with SPH-DEM coupled approach by initializing the cells as hexagons and aggregating them to form a tissue. The model also involves a middle lamella resembling real tissues. Using the model, different dried tissue states were simulated with different moisture content, the turgor pressure, and cell wall contraction effects. Compared to the state of the art grid-based microscale plant tissue drying models, the proposed model is capable of simulating plant tissues at lower moisture contents which results in excessive shrinkage and cell wall wrinkling. Model predictions were compared with experimental findings and a fairly good agreement was observed both qualitatively and quantitatively.

Delivery of therapeutic molecules using electrosprayed polymeric particles for applications in tissue engineering

This thesis has developed an innovative technology, electrospraying, that allows biodegradable microparticles to deliver pharmaceuticals that aid bone regeneration. The establishment, characterisation and optimisation of the technique are a step forward in developing an affordable and safe alternative to the products used currently in the clinical setting for the treatment of musculoskeletal disorders. The researcher has also investigated electrospraying as a coating technique on biodegradable structures that are used to replace damaged tissues, in order to provide localised and efficient drug delivery in the site of the defect to help tissue reconstruction.

The effect of cryotherapy on the vascular regeneration following closed soft tissue trauma

INTRODUCTION Icing (cryotherapy) is being widely used for the treatment of closed soft tissue trauma (CSTT), such as those resulting from sport injuries. It is believed that cryotherapy induces vasoconstriction and through this mechanism reduces inflammation [1]. However, the impact of this technique on the healing of impaired vasculature and muscle injuries following trauma remains controversial. Recent evidence suggests that the muscle regeneration is delayed after cryotherapy [2]. Consequently, we aimed to investigate the effect of cryotherapy on the vascular morphology following CSTT using an experimental model in rats by contrast-enhanced micro-CT imaging. METHODS Fifty four rats were divided into three main groups: control (no injury, n=6), sham (CSTT but no icing treatment, n=24) and icing (CSTT, treated with one session of ice block massaged directly on the injured muscle for 20 minutes, n=24). The CSTT was induced to the left thigh (Biceps Femoris) of anaesthetised rats (Male, Wistar) to create a standardized and reproducible vascular and muscle injury using an impact device [3]. Following trauma, animals were euthanized after 1, 3, 7, and 28 days healing time (n=6 for each time point). For a three-dimensional vascular morphological assessment, the blood vessels of euthanised rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil, MV 122, Flowtech, USA) using an infusion pump. Both hind-limbs were dissected, and then the injured and non-injured limbs were imaged using a micro-CT scanner (µCT 40, Scanco Medical, Switzerland) and total volume of the perfused blood vessels (TVV) was calculated. More detailed morphological parameters such as vessel volume (VV), diameter (VD), spacing (VSp), number (VN) and connectivity (VConn) were quantified through high resolution (6 µm), micro-CT-scanned biopsy samples (diameter: 8mm) taken directly from the region of the injured muscles. The biopsies were then analysed histologically to confirm the results derived from contrast-enhanced micro-CT imaging. RESULTS AND DISCUSSION The TVV was significantly higher in the injured legs compared to the non-injured legs at day 1 and 7 in the sham group and at day 28 in both sham and icing groups. The biopsies from the injured legs of the icing group showed a significant reduction in VV, VN, VD, VConn and an increase in VSp compared to those in the sham and control groups at days 1, 3 and 7, post injury. While the injured legs of the sham group exhibited a decrease in VN and VConn 28 days post trauma, indicating a return to the original values prior to trauma, these parameters had increased in the icing group (Figure 1). Also, at day 1 post injury, VV and VD of the injured legs were significantly higher in the sham group compared to the icing group, which may be attributed to the effect of vasoconstriction induced by icing. Further histomorphological evaluation of day 1 post injury, indicated that although cryotherapy significantly reduced the injury size and influx of inflammatory cells, including macrophages and neutrophils, a delay in vascular and muscle fiber regeneration was found at later time points confirming other reports from the literature [2]. CONCLUSIONS We have demonstrated using micro-CT imaging that the vascular morphology changes after CSTT, and that its recovery is affected by therapeutic modalities such as icing. This may be useful for the development of future clinical monitoring, diagnosis and treatment of CSTT. While icing reduces the swelling after trauma, our results suggest that it may delay the recovery of the vasculature in the injured tissue.

Assessment of cone beam CT registration for prostate radiation therapy : fiducial marker and soft tissue methods

Introduction This investigation aimed to assess the consistency and accuracy of radiation therapists (RTs) performing cone beam computed tomography (CBCT) alignment to fiducial markers (FMs) (CBCTFM) and the soft tissue prostate (CBCTST). Methods Six patients receiving prostate radiation therapy underwent daily CBCTs. Manual alignment of CBCTFM and CBCTST was performed by three RTs. Inter-observer agreement was assessed using a modified Bland–Altman analysis for each alignment method. Clinically acceptable 95% limits of agreement with the mean (LoAmean) were defined as ±2.0 mm for CBCTFM and ±3.0 mm for CBCTST. Differences between CBCTST alignment and the observer-averaged CBCTFM (AvCBCTFM) alignment were analysed. Clinically acceptable 95% LoA were defined as ±3.0 mm for the comparison of CBCTST and AvCBCTFM. Results CBCTFM and CBCTST alignments were performed for 185 images. The CBCTFM 95% LoAmean were within ±2.0 mm in all planes. CBCTST 95% LoAmean were within ±3.0 mm in all planes. Comparison of CBCTST with AvCBCTFM resulted in 95% LoA of −4.9 to 2.6, −1.6 to 2.5 and −4.7 to 1.9 mm in the superior–inferior, left–right and anterior–posterior planes, respectively. Conclusions Significant differences were found between soft tissue alignment and the predicted FM position. FMs are useful in reducing inter-observer variability compared with soft tissue alignment. Consideration needs to be given to margin design when using soft tissue matching due to increased inter-observer variability. This study highlights some of the complexities of soft tissue guidance for prostate radiation therapy.