Synthetic hydroxyapatite inhibits bisphosphonate toxicity to the oral mucosa in vitro

Medication-related osteonecrosis of the jaw (MRONJ) is a side effect of bisphosphonate therapy, characterised by exposed necrotic bone. The soft tissues of the oral mucosa no longer provide a protective barrier and MRONJ patients experience pain, infections and difficulties eating. We hypothesised that hydroxyapatite (Ca5(PO4)3(OH)) could reduce bisphosphonate concentrations and protect the oral mucosa by exploiting bisphosphonate’s calcium binding affinity. The effect of zoledronic acid (ZA) and pamidronic acid (PA) on the metabolism of oral fibroblasts, oral keratinocytes and three-dimensional oral mucosa models was investigated and then repeated in the presence of hydroxyapatite granules. Without hydroxyapatite, ZA and PA significantly reduced the metabolic activity of oral cells in a dose-dependent manner. Both drugs reduced epithelial thickness and 30 µM ZA resulted in loss of the epithelium. Hydroxyapatite granules had a protective effect on oral cells, with metabolic activity retained. Oral mucosa models retained their multi-layered epithelium when treated with ZA in the presence of hydroxyapatite granules and metabolic activity was comparable to controls. These results demonstrate hydroxyapatite granules protected oral soft tissues from damage caused by bisphosphonate exposure. Porous hydroxyapatite granules are currently used for socket preservation and this data suggests their potential to prevent MRONJ in at-risk patients

Isolation of stromal vascular fraction cell suspensions from mouse and human adipose tissues for downstream applications.

This protocol outlines a reliable and versatile approach to isolate stromal vascular fraction cells from different adipose tissues across human and mouse species. A number of downstream applications can then be performed to gain an appreciation of the functional activity of unique adipose tissue-resident cell populations. For complete details on the use and execution of this protocol, please refer to Macdougall et al. (2018)

Hybrid hydrogels based on polysaccharide gum karaya, poly(vinyl alcohol) and silk fibroin

This work focuses on preparation of a hybrid hydrogel consisting of both natural and synthetic polymers including the polysaccharide gum karaya which is both inexpensive and abundant, the protein silk fibroin which exhibits remarkable mechanical properties and poly(vinyl alcohol). These polymers were primarily selected due to their biocompatibility, but also through their ability to be combined together in an aqueous, non-toxic route, thus facilitating their potential future use as burn dressings. A range of structural, mechanical and practical techniques were employed to characterise the hydrogels including, FTIR, UV/VIS, phase contrast microscopy, XRD, DMA, swelling and hydrolytic stability. Finally, looking towards application as a dressing, these materials demonstrated low cell adhesion through a keratinocyte cell culture assay. The results support both the potential application of these hydrogels and provide insight into the role of each component polymer in the material. Therefore, we propose hybrid hydrogels such as these offer a unique combination of performance, ease of processing and low cost that can serve as inspiration for the next wave of bespoke medical products

A physiologically relevant, estradiol‐17β [E2]‐responsive in vitro tissue‐engineered model of the vaginal epithelium for vaginal tissue research

Aims There are many situations where preclinical models of the human vagina would be valuable for in vitro studies into the pathophysiology of vaginally transmitted diseases, microbicide efficacy, irritability testing, and particularly, for assessing materials to be inserted in the vagina for support of the pelvic floor. The aim of this study is to develop a physiologically relevant, low cost, and ethically suitable model of the vagina using sheep vaginal tissue (SVT) to reduce the need for animal testing in gynecological research. Methods Tissue-engineered (TE) vaginal models were developed by culturing primary vaginal epithelial cells and vaginal fibroblasts, isolated from the native SVTs on decellularized sheep vaginal matrices at an air–liquid interface. Morphological analyses of the models were conducted by performing hematoxylin and eosin staining and further characterization was done by immunohistofluorescence (IHF) of structural proteins and cytokeratins. Results Histological analysis of the models revealed a gradual formation of a stratified epithelium on our decellularized matrices and cell metabolic activity remained high for 21 days as measured by the resazurin assay. Our models showed a dose-dependent response to estradiol-17β [E2] with an increase in the vaginal epithelium thickness and cellular proliferation under higher E2 concentrations (100–400 pg/ml). The physiological relevance of these results was confirmed by the IHF analysis of Ki67, and cytokeratins 10 and 19 expression. Conclusion In this study, we have developed an estradiol-responsive TE vaginal model that closely mimics the structural and physiological properties of the native SVT

Inhibition and reversal of a TGF-β1 induced myofibroblast phenotype by adipose tissue-derived paracrine factors

Background Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation. Methods The transforming growth factor-β1 (TGF-β1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts. Results Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-β1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue. Conclusions Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism

Imaging of 3D tissue-engineered models of oral cancer using 890 and 1300 nm optical coherence tomography

© 2015, Nizhny Novgorod State Medical Academy. All rights reserved. Optical coherence tomography (OCT) generates its primary form of contrast from elastic backscatter. It is now the gold standard technique for retinal screening and is emerging rapidly in cardiovascular research however it remains a research goal to establish it to the same degree in epithelial cancer detection and diagnosis. In this report we compare two different OCT systems: an 890 nm spectrometer-based OCT system with 2.5 µm axial resolution and a 1300 nm swept-source OCT system with 7.5 µm axial resolution to determine the effect of these different OCT parameters on the endogenous backscatter contrast of dysplastic/malignant oral mucosa models relative to normal mucosa models. Tissueengineered oral mucosa models constructed with a dysplastic cell line (DOK), a malignant cell line (Cal27) and normal cell were imaged with both of these OCT platforms and comparisons made with regard to apparent epithelial thickness and the visibility of the epithelium relative to the underlying stroma. For the Cal27’s, hematoxylin and eosin staining confirmed the formation of a keratinized layer superficial to a thickened layer of viable cells on top of the stroma. The keratinized layer presented as a hyperreflective thickened layer superficial to a darker region on both OCT platforms. The keratinized layer caused a steep fall in signal at 890 nm, making it difficult to visualise underlying structures, whereas 1300 nm OCT clearly visualized both the epithelial cells and the stroma lying beneath. For the DOK cells, hematoxylin and eosin staining confirmed the formation of an epithelial layer frequently presenting an abnormal morphology especially at the epidermal/stromal junction, with features such as infiltrating, bulbous rete pegs. These were more clearly visualized under 890 nm OCT. These observations show that 890 nm OCT retains some of its known advantages of higher contrast between anatomical tissue layers when used to observe dysplastic and malignant 3D oral mucosa constructs. However 1300 nm OCT is confirmed to possess a greater ability to image the full thickness of the model epithelia and in particular it is more suited to imaging through the keratinized layer

Estradiol-17β [E2] stimulates wound healing in a 3D in vitro tissue-engineered vaginal wound model

Childbirth contributes to common pelvic floor problems requiring reconstructive surgery in postmenopausal women. Our aim was to develop a tissue-engineered vaginal wound model to investigate wound healing and the contribution of estradiol to pelvic tissue repair. Partial thickness scalpel wounds were made in tissue models based on decellularized sheep vaginal matrices cultured with primary sheep vaginal epithelial cells and fibroblasts. Models were cultured at an airliquid interface (ALI) for 3 weeks with and without estradiol-17β [E2]. Results showed that E2 significantly increased wound healing and epithelial maturation. Also, E2 led to collagen reorganization after only 14 days with collagen fibers more regularly aligned and compactly arranged Additionally, E2 significantly downregulated α-SMA expression which is involved in fibrotic tissue formation. This model allows one to investigate multiple steps in vaginal wound healing and could be a useful tool in developing therapies for improved tissue healing after reconstructive pelvic floor surgery

Fabrication of hierarchical multilayer poly(glycerol sebacate urethane) scaffolds based on ice-templating

In this study, it was demonstrated that ice-templating via freeze drying with custom-made moulds, in combination with air brushing, allows for the fabrication of poly(glycerol sebacate urethane) (PGSU) scaffolds with hierarchical multilayer microstructures to replicate various native soft tissues. The PGSU scaffolds were either monolayered but exhibited an anisotropic microstructure, or bilayered and trilayered, with each layer showing different microstructures. By using freeze drying with custom-made moulds, the ice crystals of the solvent were grown unidirectionally, and after freeze-drying, the scaffolds had an anisotropic microstructure, mimicking tissues such as tendon and skeletal muscle. The anisotropic PGSU scaffolds were also examined for their tensile strength, and a range of mechanical properties were obtained by altering the reactants’ molar ratio and polymer concentration. This is of importance, since soft tissues exhibit different mechanical properties depending on their native location and functionality. By combining freeze drying with airbrushing, scaffolds were fabricated with a thin, non-porous layer on top of the porous layers to allow three-dimensional cell co-culture for tissues such as skin and oral mucosa. These results show that fabrication techniques can be combined to produce PGSU scaffolds with tailored hierarchical microstructures and mechanical properties for multiple tissue engineering applications

Banana Cultivar Field Screening for Resistance to Fusarium oxysporum f.sp. cubense Tropical Race 4 in the Northern Territory

Fusarium oxysporum f.sp. cubense, causal agent of Panama disease, is one of the biggest threats to global banana production, particularly the Cavendish competent tropical race 4 (Foc TR4). It continues to spread globally with detections occurring in regions of the Middle East and new continents such as Africa and South America in the last decade. As the search was on for new management strategies and resistant cultivars to combat the disease, a banana cultivar-screening trial took place in the Northern Territory of Australia, which examined the responses of 24 banana cultivars to the soil borne fungus. These cultivars included material from TBRI, FHIA and selections from Thailand, Indonesia and Australia and evaluated for their resistance to tropical race 4 for two cropping cycles. Several cultivars displayed considerable resistance to Foc TR4, including several FHIA parental lines and hybrids, the Cavendish (AAA) selections GCTCV 215 and GCTCV 247 from TBRI and an Indonesian selection CJ19 showed either very little to no plant death due to the disease