Cells Tissues Organs : preface

This volume stems from the 1st International Conference on Epithelial-Mesenchymal Transitions (EMT), which was convened by the editors on October 5–8, 2003 in the beautiful setting of Port Douglas, Queensland, Australia. EMT, the name given to the transformation of cells arranged in a coherent layer – epithelial cells – to more individualistic and potential motile cells – mesenchymal cells – was recognized decades ago by Prof. Elisabeth (Betty) Hay (Harvard Medical School, Boston, Mass., USA) as a primary mechanism in embryogenesis for remodelling tissues. More recently EMT has been seen as crucial to the spread and invasion of carcinoma, and more recently still, EMT-like changes have been detected in various pathologies marked by fi brosis. Despite the basic and clinical importance of EMT, this extremely rapidly growing fi eld had never previously had a conference devoted to it, and indeed the disciplines of developmental biology, cancer and pathology rarely interact although they have much to share. The chapters assembled for this volume encompass these three major themes of the meeting, development, pathology and cancer, and further highlight the commonality in terms of mechanisms and outcomes among them...

Disparate companions : tissue engineering meets cancer research

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

Long-term persistence of tissue-engineered adipose flaps in a murine model to 1 year : an update

BACKGROUND Tissue engineering of patient-specific adipose tissue has the potential to revolutionize reconstructive surgery. Numerous models have been described for the production of adipose tissue with success in the short term, but little has been reported on the stability of this tissue-engineered fat beyond 4 months. METHODS A murine model of de novo adipogenesis producing a potentially transplantable adipose tissue flap within 4 to 6 weeks was developed in the authors' laboratory. In this study, the authors assess the ability of three-chamber (44-μl volume) configurations shown to be adipogenic in previous short-term studies (autograft, n = 8; open, n = 6; fat flap, n = 11) to maintain their tissue volume for up to 12 months in vivo, to determine the most adipogenic configuration in the long term. RESULTS Those chambers having the most contact with existing vascularized adipose tissue (open and fat flap groups) showed increased mean adipose tissue percentage (77 ± 5.6 percent and 81 ± 6.9 percent, respectively; p < 0.0007) and volume (12 ± 6.8 μl and 30 ± 14 μl, respectively; p < 0.025) when compared with short-term controls and greater adipose tissue volume than the autograft (sealed) chamber group (4.9 ± 5.8 μl; p = 0.0001) at 1 year. Inclusion of a vascularized fat flap within the chamber produced the best results, with new fat completely filling the chamber by 1 year. CONCLUSIONS These findings demonstrate that fat produced by tissue engineering is capable of maintaining its volume when the appropriate microenvironment is provided. This has important implications for the application of tissue-engineering techniques in humans.

Aberrant fibroblast growth factor receptor signaling in bladder and other cancers

Fibroblast growth factors (FGFs) are potent mitogens, morphogens, and inducers of angiogenesis, and FGF signaling governs the genesis of diverse tissues and organs from the earliest stages. With such fundamental embryonic and homeostatic roles, it follows that aberrant FGF signaling underlies a variety of diseases. Pathological modifications to FGF expression are known to cause salivary gland aplasia and autosomal dominant hypophosphatemic rickets, while mutations in FGF receptors (FGFRs) result in a range of skeletal dysplasias. Anomalous FGF signaling is also associated with cancer development and progression. Examples include the overexpression of FGF2 and FGF6 in prostate cancer, and FGF8 overexpression in breast and prostate cancers. Alterations in FGF signaling regulators also impact tumorigenesis, which is exemplified by the down-regulation of Sprouty 1, a negative regulator of FGF signaling, in prostate cancer. In addition, several FGFRs are mutated in human cancers (including FGFR2 in gastric cancer and FGFR3 in bladder cancer). We recently identified intriguing alterations in the FGF pathway in a novel model of bladder carcinoma that consists of a parental cell line (TSU-Pr1/T24) and two sublines with increasing metastatic potential (TSU-Pr1-B1 and TSU-Pr1-B2), which were derived successively through in vivo cycling. It was found that the increasingly metastatic sublines (TSU-Pr1-B1 and TSU-Pr1-B2) had undergone a mesenchymal to epithelial transition. FGFR2IIIc expression, which is normally expressed in mesenchymal cells, was increased in the epithelial-like TSU-Pr1-B1 and TSU-Pr1-B2 sublines and FGFR2 knock-down was associated with the reversion of cells from an epithelial to a mesenchymal phenotype. These observations suggest that modified FGF pathway signaling should be considered when studying other cancer types.

Contact with existing adipose tissue is inductive for adipogenesis in matrigel

The effect of adipose tissue on inductive adipogenesis within Matrigel (BD Biosciences) was assessed by using a murine chamber model containing a vascular pedicle. Three-chamber configurations that varied in the access to an adipose tissue source were used, including sealed- and open-chamber groups that had no access and limited access, respectively, to the surrounding adipose tissue, and a sealed-chamber group in which adipose tissue was placed as an autograft. All groups showed neovascularization, but varied in the amount of adipogenesis seen in direct relation to their access to preexisting adipose tissue: open chambers showed strong adipogenesis, whereas the sealed chambers had little or no adipose tissue; adipogenesis was restored in the autograft chamber group that contained 2- to 5-mg fat autografts. These showed significantly more adipogenesis than the sealed chambers with no autograft (p < 0.01). Autografts with 1 mg of fat were capable of producing adipogenesis but did so less consistently than the larger autografts. These findings have important implications for adipose tissue engineering strategies and for understanding de novo production of adipose tissue.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract (Matrigel™) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 μL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Second-harmonic generation from biological tissues : effect of excitation wavelength

We report on the measurement of second-harmonic signals from hyperplastic parenchyma and stroma in malignant human prostate tissue under femtosecond pulsed illumination in the wavelength range from 730 to 870 nm. In particular, the relationship of the second-harmonic generation to the excitation wavelength is measured. The result in these two regions behaves considerably differently and thus provides a possible indicator for identifying tissue components and malignancy.

Molecular aspects of tissue engineering in the dental field

Tissue engineering is a multidisciplinary field with the potential to replace tissues lost as a result of trauma, cancer surgery, or organ dysfunction. The successful production, integration, and maintenance of any tissue-engineered product are a result of numerous molecular interactions inside and outside the cell. We consider the essential elements for successful tissue engineering to be a matrix scaffold, space, cells, and vasculature, each of which has a significant and distinct molecular underpinning (Fig. 1). Our approach capitalizes on these elements. Originally developed in the rat, our chamber model (Fig. 2) involves the placement of an arteriovenous loop (the vascular supply) in a polycarbonate chamber (protected space) with the addition of cells and an extracellular matrix such as Matrigel or endogenous fibrin (34, 153, 246, 247). This model has also been extended to the rabbit and pig (J. Dolderer, M. Findlay, W. Morrison, manuscript in preparation), and has been modified for the mouse to grow adipose tissue and islet cells (33, 114, 122) (Fig. 3)...

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.

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.

Two-photon fluorescence spectroscopy for identification of healthy and malignant biological tissues

Two-photon fluorescence spectroscopy has been performed on rat skeletal muscles to investigate the effect of fixation processes on the micro-environments of the endogenous fluorophors in rat skeletal muscles. The two-photon fluorescence spectra measured for different fixation periods show a differential among those samples that were fixed in water, formalin and methanol, respectively. The results imply that two-photon fluorescence spectroscopy can be a potential technique for identification of healthy and malignant biological tissues.

A particle based model to simulate microscale morphological changes of plant tissues during drying

Fundamental understanding on microscopic physical changes of plant materials is vital to optimize product quality and processing techniques, particularly in food engineering. Although grid-based numerical modelling can assist in this regard, it becomes quite challenging to overcome the inherited complexities of these biological materials especially when such materials undergo critical processing conditions such as drying, where the cellular structure undergoes extreme deformations. In this context, a meshfree particle based model was developed which is fundamentally capable of handling extreme deformations of plant tissues during drying. The model is built by coupling a particle based meshfree technique: Smoothed Particle Hydrodynamics (SPH) and a Discrete Element Method (DEM). Plant cells were initiated as hexagons and aggregated to form a tissue which also accounts for the characteristics of the middle lamella. In each cell, SPH was used to model cell protoplasm and DEM was used to model the cell wall. Drying was incorporated by varying the 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 can be used to simulate tissues under excessive moisture content reductions incorporating cell wall wrinkling. Also, compared to the state of the art SPH-DEM tissue models, the proposed model better replicates real tissues and the cell-cell interactions used ensure efficient computations. Model predictions showed good agreement both qualitatively and quantitatively with experimental findings on dried plant tissues. The proposed modelling approach is fundamentally flexible to study different cellular structures for their microscale morphological changes at dehydration.

Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation

An essential step for therapeutic and research applications of stem cells is their ability to differentiate into specific cell types. Neuronal cells are of great interest for medical treatment of neurodegenerative diseases and traumatic injuries of central nervous system (CNS), but efforts to produce these cells have been met with only modest success. In an attempt of finding new approaches, atmospheric-pressure room-temperature microplasma jets (MPJs) are shown to effectively direct in vitro differentiation of neural stem cells (NSCs) predominantly into neuronal lineage. Murine neural stem cells (C17.2-NSCs) treated with MPJs exhibit rapid proliferation and differentiation with longer neurites and cell bodies eventually forming neuronal networks. MPJs regulate ~. 75% of NSCs to differentiate into neurons, which is a higher efficiency compared to common protein- and growth factors-based differentiation. NSCs exposure to quantized and transient (~. 150. ns) micro-plasma bullets up-regulates expression of different cell lineage markers as β-Tubulin III (for neurons) and O4 (for oligodendrocytes), while the expression of GFAP (for astrocytes) remains unchanged, as evidenced by quantitative PCR, immunofluorescence microscopy and Western Blot assay. It is shown that the plasma-increased nitric oxide (NO) production is a factor in the fate choice and differentiation of NSCs followed by axonal growth. The differentiated NSC cells matured and produced mostly cholinergic and motor neuronal progeny. It is also demonstrated that exposure of primary rat NSCs to the microplasma leads to quite similar differentiation effects. This suggests that the observed effect may potentially be generic and applicable to other types of neural progenitor cells. The application of this new in vitro strategy to selectively differentiate NSCs into neurons represents a step towards reproducible and efficient production of the desired NSC derivatives. © 2013.

Nutrient element-based bioceramic coatings on titanium alloy stimulating osteogenesis by inducing beneficial osteoimmmunomodulation

A paradigm shift has taken place in which bone implant materials has gone from being relatively inert to having immunomodulatory properties, indicating the importance of immune response when these materials interact with the host tissues. It has therefore become important to endow the implant materials with immunomodulatory properties favouring osteogenesis and osseointegration. Strontium, zinc and silicon are bioactive elements that have important roles in bone metabolism and that also elicit significant immune responses. In this study, Sr-, Zn- and Si-containing bioactive Sr2ZnSi2O7 (SZS) ceramic coatings on Ti–6Al–4V were successfully prepared by a plasma-spray coating method. The SZS coatings exhibited slow release of the bioactive ions with significantly higher bonding strength than hydroxyapatite (HA) coatings. SZS-coated Ti–6Al–4V elicited significant effects on the immune cells, inhibiting the release of pro-inflammatory cytokines and fibrosis-enhancing factors, while upregulating the expression of osteogenic factors of macrophages; moreover, it could also inhibit the osteoclastic activities. The RANKL/RANK pathway, which enhances osteoclastogenesis, was inhibited by the SZS coatings, whereas the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) was significantly enhanced by the SZS coatings/macrophages conditioned medium, probably via the activation of BMP2 pathway. SZS coatings are, therefore, a promising material for orthopaedic applications, and the strategy of manipulating the immune response by a combination of bioactive elements with controlled release has the potential to endow biomaterials with beneficial immunomodulatory properties.