Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing

Access routes, devices and guidance methods for intrapericardial delivery in cardiac conditions

Drug deposition into the intrapericardial space is favourable for achieving localised effects and targeted cardiac delivery owing to its proximity to the myocardium as well as facilitating optimised pharmacokinetic profiles and a reduction in systemic side effects. Access to the pericardium requires invasive procedures but the risks associated with this have been reduced with technological advances, such as combining transatrial and subxiphoid access with different guidance methods. A variety of introducer devices, ranging from needles to loop-catheters, have also been developed and validated in pre-clinical studies investigating intrapericardial delivery of therapeutic agents. Access techniques are generally well-tolerated, self-limiting and safe, although some rare complications associated with certain approaches have been reported. This review covers these access techniques and how they have been applied to the delivery of drugs, cells, and biologicals, demonstrating the potential of intrapericardial delivery for treatments in cardiac arrhythmia, vascular damage, and myocardial infarction

Superparamagnetic iron oxide nanoparticles regulate smooth muscle cell phenotype

Superparamagnetic iron oxide nanoparticles are used for an increasing range of biomedical applications, from imaging to mechanical actuation of cells and tissue. The aim of this study was to investigate the loading of smooth muscle cells with superparamagnetic iron oxide nanoparticles and to explore what effect this has on the phenotype of the cells. Adherent human smooth muscle cells were loaded with ∼17 pg of unconjugated, negatively charged, 50 nm superparamagnetic iron oxide nanoparticles (SPION). Clusters of the internalized SPION particles were held in discrete cytoplasmic vesicles. Internalized SPION did not cause any change in cell morphology, proliferation, metabolic activity, or staining pattern of actin and calponin, two of the muscle contractile proteins involved in force generation. However, internalized SPION inhibited the increased gene expression of actin and calponin normally observed when cells are incubated under differentiation conditions. The observed change in the control of gene expression of muscle contractile apparatus by SPION has not previously been described. This finding could offer novel approaches for regulating the phenotype of smooth muscle cells and warrants further investigation. This article is protected by copyright. All rights reserved

The regulation of bone turnover in ameloblastoma using an organotypic in vitro co-culture model

Ameloblastoma is a rare, odontogenic neoplasm with benign histopathology, but extensive, local infiltrative capacity through the bone tissue it originates in. While the mechanisms of ameloblastoma invasion through the bone and bone absorption are largely unknown, recent investigations have indicated a role of the osteoprotegerin/receptor activator of nuclear factor kappa-B ligand regulatory mechanisms. Here, we present results obtained using a novel in vitro organotypic tumour model, which we have developed using tissue engineering techniques. Using this model, we analysed the expression of genes involved in bone turnover and detected a 700-fold increase in receptor activator of nuclear factor kappa-B ligand levels in the co-culture models with ameloblastoma cells cultured with bone cells. The model described here can be used for gene expression studies, as a basis for drug testing or for a more tailored platform for testing of the behaviour of different ameloblastoma tumours in vitro

Prospective regenerative medicine therapies for obstetric trauma-induced fecal incontinence.

Fecal incontinence is a major public health issue that has yet to be adequately addressed. Obstetric trauma and injury to the anal sphincter muscles are the most common cause of fecal incontinence. New therapies are emerging aimed at repair or regeneration of sphincter muscle and restoration of continence. While regenerative medicine offers an attractive option for fecal incontinence there are currently no validated techniques using this approach. Although many challenges are yet to be resolved, the advent of regenerative medicine is likely to offer disruptive technologies to treat and possibly prevent the onset of this devastating condition. This article provides a review on regenerative medicine approaches for treating fecal incontinence and a critique of the current landscape in this area

TIPS to manipulate myogenesis: retention of myoblast differentiation capacity using microsphere culture

Cell therapy is an emerging option for regenerating skeletal muscle. Improved delivery methods for anchorage-dependent myoblasts are likely to improve integration and function of transplanted muscle cells. Highly porous microspheres, produced using thermally induced phase separation (TIPS), have features ideally suited for minimally invasive cell delivery. The purpose of this study was to investigate, for the first time, the use of TIPS microspheres as highly porous microcarriers for manipulation of human skeletal muscle myoblasts (HSMM) under defined culture conditions. HSMM cells readily attached to the surface of poly (DL-lactide-co-glycolide) (PLGA) TIPS microcarriers, where they were induced to continue proliferating or to be driven towards differentiation whilst under static-dynamic culture conditions for 7 days. Switching from proliferation medium to differentiation medium for 7 days, resulted in increased protein expression of skeletal muscle cell contractile apparatus components, MyoD and skeletal muscle myosin heavy chain, compared with cells cultured on conventional culture plasticware for the same duration (p < 0.001). Growth of myoblasts on the surface of the microcarriers and their migration following simulated delivery, caused no change to the proliferative capacity of cells over 7 days. Results from this study demonstrate that TIPS microspheres provide an ideal vehicle for the expansion and delivery of myoblasts for therapeutic applications. Transplantation of myoblasts anchored to a substrate, rather than in suspension, will reduce the amount of ex vivo manipulation required during preparation of the product and allows cells to be delivered in a more natural state. This will improve the ability to control cell dosage and increase the likelihood of efficacy

A Hydrogel-Integrated Culture Device to Interrogate T Cell Activation with Physicochemical Cues

The recent rise of adoptive T cell therapy (ATCT) as a promising cancer immunotherapy has triggered increased interest in therapeutic T cell bioprocessing. T cell activation is a critical processing step and is known to be modulated by physical parameters, such as substrate stiffness. Nevertheless, relatively little is known about how biophysical factors regulate immune cells, such as T cells. Understanding how T cell activation is modulated by physical and biochemical cues may offer novel methods to control cell behavior for therapeutic cell processing. Inspired by T cell mechanosensitivity, we developed a multiwell, reusable, customizable, two-dimensional (2D) polyacrylamide (PA) hydrogel-integrated culture device to study the physicochemical stimulation of Jurkat T cells. Substrate stiffness and ligand density were tuned by concentrations of the hydrogel cross-linker and antibody in the coating solution, respectively. We cultured Jurkat T cells on 2D hydrogels of different stiffnesses that presented surface-immobilized stimulatory antibodies against CD3 and CD28 and demonstrated that Jurkat T cells stimulated by stiff hydrogels (50.6 ± 15.1 kPa) exhibited significantly higher interleukin-2 (IL-2) secretion, but lower proliferation, than those stimulated by softer hydrogels (7.1 ± 0.4 kPa). In addition, we found that increasing anti-CD3 concentration from 10 to 30 μg/mL led to a significant increase in IL-2 secretion from cells stimulated on 7.1 ± 0.4 and 9.3 ± 2.4 kPa gels. Simultaneous tuning of substrate stiffness and stimulatory ligand density showed that the two parameters synergize (two-way ANOVA interaction effect: p < 0.001) to enhance IL-2 secretion. Our results demonstrate the importance of physical parameters in immune cell stimulation and highlight the potential of designing future immunostimulatory biomaterials that are mechanically tailored to balance stimulatory strength and downstream proliferative capacity of therapeutic T cells

Biomaterials for hollow organ tissue engineering

Tissue engineering is a rapidly advancing field that is likely to transform how medicine is practised in the near future. For hollow organs such as those found in the cardiovascular and respiratory systems or gastrointestinal tract, tissue engineering can provide replacement of the entire organ or provide restoration of function to specific regions. Larger tissue-engineered constructs often require biomaterial-based scaffold structures to provide support and structure for new tissue growth. Consideration must be given to the choice of material and manufacturing process to ensure the de novo tissue closely matches the mechanical and physiological properties of the native tissue. This review will discuss some of the approaches taken to date for fabricating hollow organ scaffolds and the selection of appropriate biomaterials

A Comparison of Human Neutrophils Acquired from Four Experimental Models of Inflammation

Defects in neutrophil function have been implicated in a wide spectrum of clinical conditions. Several models are employed to study activated human neutrophils akin to those found at a site of inflammation. These include whole blood (WB) ex vivo stimulation with lipopolysaccharide (LPS) and in vivo techniques: cantharidin blister, skin windows and intra-dermal injection of UV-killed E.coli (UVKEc). Neutrophils obtained from these have never been compared. We compared the activation status of neutrophils from each technique in order to inform the optimal model for use in human studies. Healthy male volunteers were randomised to undergo one of the four techniques (n = 5/group). LPS: WB stimulated with 1ng/ml of LPS for 4 hours. Cantharidin: 12.5μl of 0.1% cantharidin elicited a single blister, aspirated at 24 hours. Skin windows: four 6mm mechanical-suction blisters created, de-roofed and an exudate-collection chamber placed over the windows for 4 hours before aspiration. UVKEc: 1.5 x 107 UVKEc injected intra-dermally. A single 10mm mechanical-suction blister formed and aspirated at 4 hours. Unstimulated WB used as the control. Flow cytometry was used to determine activation status using CD16, CD11b, CD54, CD62L and CD88. Functional status was assessed with a phagocytosis assay. The pattern of neutrophil activation was similar in all models. Neutrophil CD11b was elevated in all models, most markedly in UVKEc (p<0.0001), and CD54 was also elevated but only significant in the LPS model (p = 0.001). CD62L was significantly reduced in all 4 models (p<0.0001) and CD88 was also suppressed in all. There were no changes in CD16 in any model, neither was there any significant difference in the phagocytic capacity of the neutrophils. In summary, there are no significant differences in activation marker expression or phagocytic capacity in the neutrophils obtained from each technique. Therefore we believe whole blood stimulation is the best model in experimentally challenging inpatient populations

Genomics as a practical tool in sport - have we reached the starting line?

The genetic component of athletic performance approximates 50%, depending on which specific element of performance is considered. Limited genetic testing is already available commercially and genetic tests are likely to become powerful tools to improve sport performance in the future. Currently, however, selection of athletes for training squads or competition based on genomic data is premature. Larger volumes of longitudinal data within individual sports are needed to determine the efficacy of using genomic data in the management of elite athletes via manipulation of training load and diet based on personal genomic information