Harnessing the Regenerative Potential of Purified Bovine Dental Pulp and Dentin Extracellular Matrices in a Chitosan/Alginate Hydrogel.

When a tooth is diseased or damaged through caries, bioactive molecules are liberated from the pulp and dentin as part of the natural response to injury and these are key molecules for stimulating stem cell responses for tissue repair. Incorporation of these extracellular-matrix (ECM)-derived molecules into a hydrogel model can mimic in vivo conditions to enable dentin-pulp complex regeneration. Here, a chitosan/alginate (C/A) hydrogel is developed to sequester bovine ECM extracts. Human dental pulp cells (hDPCs) are cultured with these constructs and proliferation and cytotoxicity assays confirm that these C/A hydrogels are bioactive. Sequential z-axis fluorescent imaging visualizes hDPCs protruding into the hydrogel as it degraded. Alizarin red S staining shows that hDPCs cultured with the hydrogels display increased calcium-ion deposition, with dentin ECM stimulating the highest levels. Alkaline phosphatase activity is increased, as is expression of transforming growth factor-beta as demonstrated using immunocytochemistry. Directional analysis following phase contrast kinetic image capture demonstrates that both dentin and pulp ECM molecules act as chemoattractants for hDPCs. Data from this study demonstrate that purified ECM from dental pulp and dentin when delivered in a C/A hydrogel stimulates dental tissue repair processes in vitro.

A Porous Fluoride-Substituted Bovine-Derived Hydroxyapatite Scaffold Constructed for Applications in Bone Tissue Regeneration.

Hydroxyapatite is widely used in bone implantation because of its similar mineral composition to natural bone, allowing it to serve as a biocompatible osteoconductive support. A bovine-derived hydroxyapatite (BHA) scaffold was developed through an array of defatting and deproteinization procedures. The BHA scaffold was substituted with fluoride ions using a modified sol-gel method to produce a bovine-derived fluorapatite (BFA) scaffold. Fourier-transform infrared spectroscopy and X-ray diffraction analysis showed that fluoride ions were successfully substituted into the BHA lattice. According to energy dispersive X-ray analysis, the main inorganic phases contained calcium and phosphorus with a fluoride ratio of ~1-2 wt%. Scanning electron microscopy presented a natural microporous architecture for the BFA scaffold with pore sizes ranging from ~200-600 µm. The BHA scaffold was chemically stable and showed sustained degradation in simulated-body fluid. Young's modulus and yield strength were superior in the BFA scaffold to BHA. In vitro cell culture studies showed that the BFA was biocompatible, supporting the proliferative growth of Saos-2 osteoblast cells and exhibiting osteoinductive features. This unique technique of producing hydroxyapatite from bovine bone with the intent of producing high performance biomedically targeted materials could be used to improve bone repair.

Development and optimisation of hydroxyapatite-polyethylene glycol diacrylate hydrogel inks for 3D printing of bone tissue engineered scaffolds.

In the event of excessive damage to bone tissue, the self-healing process alone is not sufficient to restore bone integrity. Three-dimensional (3D) printing, as an advanced additive manufacturing technology, can create implantable bone scaffolds with accurate geometry and internal architecture, facilitating bone regeneration. This study aims to develop and optimise hydroxyapatite-polyethylene glycol diacrylate (HA-PEGDA) hydrogel inks for extrusion 3D printing of bone tissue scaffolds. Different concentrations of HA were mixed with PEGDA, and further incorporated with pluronic F127 (PF127) as a sacrificial carrier. PF127 provided good distribution of HA nanoparticle within the scaffolds and improved the rheological requirements of HA-PEGDA inks for extrusion 3D printing without significant reduction in the HA content after its removal. Higher printing pressures and printing rates were needed to generate the same strand diameter when using a higher HA content compared to a lower HA content. Scaffolds with excellent shape fidelity up to 75-layers and high resolution (∼200 µm) with uniform strands were fabricated. Increasing the HA content enhanced the compression strength and decreased the swelling degree and degradation rate of 3D printed HA-PEGDA scaffolds. In addition, the incorporation of HA improved the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) onto the scaffolds. 3D printed scaffolds with 2 wt% HA promoted osteogenic differentiation of hBMSCs as confirmed by the expression of alkaline phosphatase activity and calcium deposition. Altogether, the developed HA-PEGDA hydrogel ink has promising potential as a scaffold material for bone tissue regeneration, with excellent shape fidelity and the ability to promote osteogenic differentiation of hBMSCs.

Investigation of a Novel Injectable Chitosan Oligosaccharide-Bovine Hydroxyapatite Hybrid Dental Biocomposite for the Purposes of Conservative Pulp Therapy.

This study aimed to develop injectable chitosan oligosaccharide (COS) and bovine hydroxyapatite (BHA) hybrid biocomposites, and characterise their physiochemical properties for use as a dental pulp-capping material. The COS powder was prepared from chitosan through hydrolytic reactions and then dissolved in 0.2% acetic acid to create a solution. BHA was obtained from waste bovine bone and milled to form a powder. The BHA powder was incorporated with the COS solution at different proportions to create the COS-BHA hybrid biocomposite. Zirconium oxide (ZrO2) powder was included in the blend as a radiopacifier. The composite was characterised to evaluate its physiochemical properties, radiopacity, setting time, solubility, and pH. Fourier-transform infrared spectroscopic analysis of the COS-BHA biocomposite shows the characteristic peaks of COS and hydroxyapatite. Compositional analysis via ICP-MS and SEM-EDX shows the predominant elements present to be the constituents of COS, BHA, and ZrO2. The hybrid biocomposite demonstrated an average setting time of 1 h and 10 min and a pH value of 10. The biocomposite demonstrated solubility when placed in a physiological solution. Radiographically, the set hybrid biocomposite appears to be more radiopaque than the commercial mineral trioxide aggregate (MTA). The developed COS-BHA hybrid biocomposite demonstrated good potential as a pulp-capping agent exhibiting high pH, with a greater radiopacity and reduced setting time compared to MTA. Solubility of the biocomposite may be addressed in future studies with the incorporation of a cross-linking agent. However, further in vitro and in vivo studies are necessary to evaluate its clinical feasibility.

A review of current advancements for wound healing: Biomaterial applications and medical devices.

Wound healing is a complex process that is critical in restoring the skin's barrier function. This process can be interrupted by numerous diseases resulting in chronic wounds that represent a major medical burden. Such wounds fail to follow the stages of healing and are often complicated by a pro-inflammatory milieu attributed to increased proteinases, hypoxia, and bacterial accumulation. The comprehensive treatment of chronic wounds is still regarded as a significant unmet medical need due to the complex symptoms caused by the metabolic disorder of the wound microenvironment. As a result, several advanced medical devices, such as wound dressings, wearable wound monitors, negative pressure wound therapy devices, and surgical sutures, have been developed to correct the chronic wound environment and achieve skin tissue regeneration. Most medical devices encompass a wide range of products containing natural (e.g., chitosan, keratin, casein, collagen, hyaluronic acid, alginate, and silk fibroin) and synthetic (e.g., polyvinyl alcohol, polyethylene glycol, poly[lactic-co-glycolic acid], polycaprolactone, polylactic acid) polymers, as well as bioactive molecules (e.g., chemical drugs, silver, growth factors, stem cells, and plant compounds). This review addresses these medical devices with a focus on biomaterials and applications, aiming to deliver a critical theoretical reference for further research on chronic wound healing.

Fabrication and characterisation of melt-extruded chitosan/keratin/PCL/PEG drug-eluting sutures designed for wound healing.

Diclofenac potassium loaded sutures based upon PEG/PCL/chitosan-keratin blends were fabricated using the hot-melt extrusion technique. Polymer sutures were evaluated based on their physical, thermal and mechanical properties, while the drug-eluting sutures were evaluated for drug release properties. Lastly, the performance of the drug-loaded sutures in the contact with the human keratinocyte cell line HaCat were assessed. Results showed that the sutures extruded homogeneously at a temperature of 63 ± 1 °C providing a uniform thickness of fibres. Analysis by Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) showed that completely amorphous and miscible solid dispersions were created. Fourier transform infrared (FTIR) spectroscopy indicated that the presence of hydrogen bonds between the polymers improved material miscibility. Tensile properties of the sutures were clearly affected by the PEG, chitosan and keratin additions. The optimal formulation of tensile strength was obtained when PCL/PEG/chitosan-keratin were combined at a ratio of 80/19/1 w/w. Rapid and sustained drug release rates were achieved with the PEG/PCL/chitosan/keratin blends at various combinations. The composite of PCL/PEG/chitosan-keratin with 30 wt% of diclofenac potassium also exhibited high cell viability and wound healing rates in vitro cytotoxicity testing. The anti-inflammatory properties imparted by the PCL/PEG/chitosan/keratin/drug sutures may further the use of composite sutures for wound healing in clinical settings.

Untapped potentials of hazardous nanoarchitectural biopolymers.

The First Industrial Revolution began when manual labour transitioned to machines. Fossil fuels and steam eventually replaced wood and water as an energy source used predominantly for the mechanized production of textiles and iron. The emergence of the required numerous enormous factories gave rise to smoke pollution due to the immense growth in coal consumption. The manufactured gas industry produced highly toxic effluent that was released into sewers and rivers polluting the water. Many pieces of legislation were introduced to overcome this issue, but with varying degrees of effectiveness. Alongside our growth in world population, the problems that we had with waste remained, but together with our increase in number the waste produced has also increased additionally. The immense volume of waste materials generated from human activity and the potentially detrimental effects on the environment and on public health have awakened in ourselves a critical need to embrace current scientific methods for the safe disposal of wastes. We are informed daily that our food waste must be better utilized to ensure enough food is available to feed the world's growing population in a sustainable way (Thyberg and Tonjes, 2016). Some things are easy, like waste food and cellulose products can be turned into compost, but how do we recycle sheep's wool? Or shrimp shells? Despite the fact that both these substances are hazardous, and have caused environmental and economic impact from being incinerated; but we anticipate that those substances may have the potential to convert into added value applications.We have been working in this area for over 15 years, working towards managing them and seeking their added value applications. We take the biological products, process (reconstitute) and engineer them into added value products such as functional and nanostructure materials including edible films, foams and composites including medical devices useful in the human body. Anything that we can ingest, should not cause an immune response in the human system. Natural biomacromolecules display the inherent ability to perform very specific chemical, mechanical or structural roles. Specifically, protein- and polysaccharide-based biomaterials have come to light as the most promising candidates for many biomedical applications due their biomimetic and nanostructured arrangements, their multi-functional features, and their capability to function as matrices that are capable of facilitating cell-cell and cell-matrix interactions.

Biodegradable methacrylated casein for cardiac tissue engineering applications.

Casein is a naturally derived amino group (-NH2) rich protein, that enables surface functionalization leading to hydrophilicity, which in turn facilitates better cell adhesion. Casein obtained from either commercial ß-casein rich skim milk (A2 milk) or dissolved air flotation (DAF) technology was tested for its potential for tissue engineering applications in a comparative study. A novel biodegradable biomaterial was synthesized from casein by chemically modifying with methacrylic anhydride (MA) and combined with polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) blend. The resulting methacrylated casein (CasMA) with the two polymers was processed into porous scaffolds with low and high MA concentrations to demonstrate CasMA's ease of modification and reproducibility. Fourier Transform Infrared Microscopy (FTIR) and Proton Nuclear Magnetic Resonance (1H NMR) revealed the presence of all the components and the successful modification of casein. The rheological and morphological analysis presented viscous behaviour and columnar hollow tube-like microstructures in agreement with the biomaterials' swelling and biodegradation behaviour. The live/dead in vitro assay showed high cell viability that agreed with the cell proliferation (MTT) assay in vitro, which indicated increased proliferation upon casein modification at appropriate biomaterial concentrations and volumes. This study not only showed a possible mechanism of casein methacrylation but also presented the potential use of waste materials like DAF-casein as a value-added product for tissue engineering applications.

Critical Role for Cold Shock Protein YB-1 in Cytokinesis.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, ß-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

Development of an Organic-Inorganic Nanostructured Hybrid Dental Biocomposite.

Dental pathologies such as caries is one of the most prevalent diseases worldwide. Dental pulp contains stem cells capable of regenerating the dentine in the tooth, consequently, healthy dental pulp is essential for long term tooth survival. The aim of this study was to incorporate a variety of polymers that provide strength, an antibacterial substance and a protein-based polymer to provide cell support. These components were combined into a triphasic hybrid dental biocomposite (3HB), that together could provide regenerative properties for the pulp tissue. The 3HB biocomposite was incorporated into Organic-inorganic nanostructured materials such as Mineral Trioxide Aggregate (MTA) as a base to assemble a hybrid dental biocomposite. The effects of the 3HB on cytotoxicity was examined in mouse dental pulp cells, MDPC-23. In vitro studies showed that 3HB supported the proliferative growth of the cells significantly more than the no treatment control. 3HB also caused little stress to the cells and supported cell viability. Fourier transform infrared (FTIR) spectra confirmed the presence of polymer functional groups within the 3HB biocomposite. Therefore, 3HB compound has the potential to be applied as a pulp wound dressing providing superior cytocompatibility than the present options but also may be indispensable for the regeneration of dental pulp.

Dephosphorylation of YB-1 is Required for Nuclear Localisation During G2 Phase of the Cell Cycle.

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB1 determines its cellular location.

Spatial and temporal changes in myogenic protein expression by the microenvironment after freeze injury.

Skeletal muscle has the remarkable capability to regenerate itself following injury. Adult myogenic stem cells (MSCs) are responsible for the repair and regeneration, and their activity is controlled by intrinsic and extrinsic factors. The aim of this study was to examine and compare the expression levels of Pax3, Pax7, MRF and p38 proteins during the course of regeneration and in different areas of the focal freeze-lesion damaged adult rat TA muscle. Using the focal freeze injury model, immunohistochemistry, laser-capture micro-dissection and Western blot analysis were performed. The results show that (1) in the severely damaged area, the focal freeze-lesion injury significantly activated Pax7 and myogenin expression within 7 days and down-regulated Pax3, MyoD and Myf-5 within 1 or 3 days, and (2) the level of the p38 protein was strongly and transiently up-regulated in the whole muscle on day 7 following injury, whereas the level of the pp38 protein was down-regulated within 3 days in the severely damaged and non-damaged areas. These findings indicate that the temporal (e.g. the time course of regeneration) and spatial (e.g. three zones created by the focal freeze-lesion) cues in a regenerating muscle have a significant impact on the activity of the adult MSCs.

Dynamic patterns of mononucleated myogenic cell populations in the developing rat hindlimb.

Formation of an organ is governed by both the genetic programming of individual cells and dynamic interactions amongst different cell communities or the 'community effect'. Using the developing vertebrate limb muscle, we identified myogenic stem cell communities derived from migratory somitic cells. These cells express Pax3, a gene from the paired box (PAX) family of transcription factors and Pax7, a paralog of Pax3. Both Pax genes act upstream of myogenic regulatory factor (MRF) whose activation marks a specified myogenic lineage and subsequent differentiation. Quantitative analyses on the size of the individual cell populations revealed that Pax3 and MRF compartments remained constant. Further analysis showed that the size of the Pax7 cell population increased significantly. The pool of foetal MRF populations contained decreasing Pax3 and increasing Pax7 proportions. This increase is dynamic at the developmental stage. Upon abrupt disruption of the p38 regulatory pathway for myogenic differentiation, established kinetic patterns were significantly altered. Changes in the proportions of these myogenic subpopulations imply that a community effect involving dynamic interactions among differentiating cell communities may play a crucial role in correct maintenance and propagation of myogenic stem cells.

Segmental Distribution of Myosin Heavy Chain Isoforms Within Single Muscle Fibers.

Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY, and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the three EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I⇔IIA⇔IIX⇔IIB, however, in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be because of the myonuclear domain. Anat Rec, 300:1636-1642, 2017. © 2017 Wiley Periodicals, Inc.

Development and characterization of a xenograft material from New Zealand sourced bovine cancellous bone.

A xenograft (bovine hydroxyapatite [BHA]) was developed from New Zealand sourced bovine cancellous bone by a successful defatting and deproteinizing procedure. The BHA was chemically, compositionally and structurally characterized. Fourier transform infrared spectroscopy confirmed the removal of organic matter from the bone matrix and the presence of carbonate ( CO32-), hydroxyl (OH- ), and phosphate ( PO43-) functional groups. X-ray diffraction analysis suggested that the processed bone corresponds characteristically to hydroxyapatite (HA). SEM analysis showed that the BHA has an interconnected porous architecture with a pore diameter ranging from 100 to 700 µm while µCT analysis calculated the total porosity as 73.46% ± 1.08. Furthermore, the BHA was stable up to 1000°C and lost only 1.8% of its weight. The Ca/P molar ratio of the BHA was 1.58, which is comparable with commercially available natural HA-Endobon® . After 28 days of incubation in simulated body fluid (SBF), the pH value only fluctuated between 7.1 and 7.5 and the BHA scaffold did not degrade significantly by weight indicating the scaffold had excellent chemical and structural stability. In vitro studies showed the BHA was cytocompatible and supported the proliferative growth of Saos-2 osteoblast cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1054-1062, 2017.

Development and characterization of hydroxyapatite/ß-TCP/chitosan composites for tissue engineering applications.

Calcium phosphate ceramics that mimic bone composition provide interesting possibilities for the advancement in bone tissue engineering. The present study reports on a chitosan composite reinforced by hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) obtained from waste mussel shells and cross-linked using tripolyphosphate (TPP). The ratios of the ceramic components in composites were 20/10/70, 30/20/50 and 40/30/30 (HA/ß-TCP/CH, w/w %). Biodegradation rate, structural properties and in-vitro degradation of the bone-like composite scaffolds were investigated. The optimum amount of TPP required for composite was 2.5% and glycerol was used as plasticizer at an optimized concentration of 1%. Tripolyphosphate cross-linked chitosan composites were developed by freezing and lyophilisation. The Young's modulus of the scaffolds was increased from 4kPa to 17kPa and the porosity of composites dropped from 85 to 68% by increasing the HA/ß-TCP ratio. After 28days in physiological solution, bone-like composite scaffolds with a higher ratio of HA/ß-TCP (e.g. 40/30/30) showed about 2% lower biodegradation in comparison to scaffolds with a lower ratio of HA/ß-TCP (i.e. 20/10/70). The obtained data suggest that the chitosan based bone-like composites could be potential candidates for biomedical applications.

A novel squid pen chitosan/hydroxyapatite/ß-tricalcium phosphate composite for bone tissue engineering.

Squid pen chitosan was used in the fabrication of biocomposite scaffolds for bone tissue engineering. Hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP) obtained from waste mussel shells were used as the calcium phosphate source. The composite was prepared using 2.5% tripolyphosphate (TPP) and 1% glycerol as a cross-linker and plasticizer, respectively. The weight percent (wt.%) ratios of the ceramic components in the composite were 20/10/70, 30/20/50 and 40/30/30 (HA/ß-TCP/Chi). The biodegradation rate and structural properties of the scaffolds were investigated. Scanning electron microscopy (SEM) and microCT(µCT) results indicated that the composites have a well defined lamellar structure with an average pore size of 200 µm. The porosity of the composites decreased from 88 to 56% by increasing the ratio of HA/ß-TCP from 30 to 70%. After 28 days of incubation in a physiological solution, the scaffolds were degraded by approximately 30%. In vitro investigations showed that the composites were cytocompatible and supported the growth of L929 and Saos-2 cells. The obtained data suggests that the squid pen chitosan composites are potential candidates for bone regeneration.

Altered transcription of murine genes induced in the small bowel by administration of probiotic strain Lactobacillus rhamnosus HN001.

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli (Lactobacillus-free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus-free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1, angptl4, and hspa1b, associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus-free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Seasonal changes in morphology and steroid receptor expression in the prostate of the brushtail possum (Trichosurus vulpecula): an animal model for the study of prostate growth?

The prostate of the brushtail possum undergoes growth and regression during the year. The present study investigated the morphological changes and expression of androgen and oestrogen receptors during the breeding and non-breeding seasons. Prostate tissue was collected from adult possums at 2-monthly intervals. The periurethral and outer glandular areas were separated and the volume of stromal, epithelial and luminal tissues measured in each area. Immunohistochemistry was used to investigate cell proliferation with proliferating cell nuclear antigen (PCNA) and to localise androgen receptor (AR) and oestrogen receptors α and ß (ERα, ERß). Seasonal changes in expression of the three receptors were investigated using quantitative PCR and western blot analysis. During the breeding season the volume of stromal tissue in the periurethral area and the luminal volume in the glandular area significantly increased. The change in periurethral volume was associated with increased PCNA-immunopositive cells. While the localisation of AR to the stromal and epithelial cells did not change, there was a significant increase in receptor expression before the main breeding season. ERα and ERß expression and localisation did not alter during the year. Similarities in receptor expression and localisation suggest that the possum may be a suitable animal model for the study of human prostate growth.

Interstitial cells of Cajal are present in human extrahepatic bile ducts.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) are distributed with smooth muscle throughout the gastrointestinal tract and are involved in regulating motility. ICC were recently discovered in the wall of the human gallbladder. This study sought to determine whether ICC are present in human bile ducts. METHODS: Biliary tract samples were obtained from several sources: surgical specimens (n = 16, 11 women, mean age 61 years); archival post-mortem specimen (n = 1, 86 years, man); and cadavers (n = 2, 68 and 80 years, men). Paraffin-embedded sections (3 microm) from the gallbladder (fundus, body and neck) and both extrahepatic and intrahepatic bile ducts were investigated. A double immunofluorescence protocol using polyclonal and monoclonal c-kit antibodies and mast cell tryptase was used to distinguish c-kit-positive cells with typical ICC morphology from c-kit-positive mast cells. Small bowel samples were used as positive controls. ICC in the gallbladder were confirmed by ultrastructural study. RESULTS: c-kit-positive cells with characteristic ICC morphology were identified in the subepithelial and muscular layers of the gallbladder and extrahepatic bile ducts. They were most prominent within the muscle layer of the extrahepatic bile ducts where they were organized into loosely arranged laminae running parallel to circular smooth muscle fibers. ICC were not found in intrahepatic bile ducts. CONCLUSION: This study demonstrates for the first time that ICC are present in human extrahepatic bile ducts where they are more densely aggregated than in the gallbladder. This cellular network is likely to be involved in biliary tract motility and its related disorders.