Impact of type-1 collagen hydrogel density on integrin-linked morphogenic response of SH-SY5Y neuronal cells.

Cellular metabolism and behaviour is closely linked to cytoskeletal tension and scaffold mechanics. In the developing nervous system functional connectivity is controlled by the interplay between chemical and mechanical cues that initiate programs of cell behaviour. Replication of functional connectivity in neuronal populations in vitro has proven a technical challenge due to the absence of many systems of biomechanical regulation that control directional outgrowth in vivo. Here, a 3D culture system is explored by dilution of a type I collagen hydrogel to produce variation in gel stiffness. Hydrogel scaffold remodelling was found to be linked to gel collagen concentration, with a greater degree of gel contraction occurring at lower concentrations. Gel mechanics were found to evolve over the culture period according to collagen concentration. Less concentrated gels reduced in stiffness, whilst a biphasic pattern of increasing and then decreasing stiffness was observed at higher concentrations. Analysis of these cultures by PCR revealed a program of shifting integrin expression and highly variable activity in key morphogenic signal pathways, such as mitogen-associated protein kinase, indicating genetic impact of biomaterial interactions via mechano-regulation. Gel contraction at lower concentrations was also found to be accompanied by an increase in average collagen fibre diameter. Minor changes in biomaterial mechanics result in significant changes in programmed cell behaviour, resulting in adoption of markedly different cell morphologies and ability to remodel the scaffold. Advanced understanding of cell-biomaterial interactions, over short and long-term culture, is of critical importance in the development of novel tissue engineering strategies for the fabrication of biomimetic 3D neuro-tissue constructs. Simple methods of tailoring the initial mechanical environment presented to SH-SY5Y populations in 3D can lead to significantly different programs of network development over time.

Bioengineered human skeletal muscle capable of functional regeneration.

BACKGROUND: Skeletal muscle (SkM) regenerates following injury, replacing damaged tissue with high fidelity. However, in serious injuries, non-regenerative defects leave patients with loss of function, increased re-injury risk and often chronic pain. Progress in treating these non-regenerative defects has been slow, with advances only occurring where a comprehensive understanding of regeneration has been gained. Tissue engineering has allowed the development of bioengineered models of SkM which regenerate following injury to support research in regenerative physiology. To date, however, no studies have utilised human myogenic precursor cells (hMPCs) to closely mimic functional human regenerative physiology. RESULTS: Here we address some of the difficulties associated with cell number and hMPC mitogenicity using magnetic association cell sorting (MACS), for the marker CD56, and media supplementation with fibroblast growth factor 2 (FGF-2) and B-27 supplement. Cell sorting allowed extended expansion of myogenic cells and supplementation was shown to improve myogenesis within engineered tissues and force generation at maturity. In addition, these engineered human SkM regenerated following barium chloride (BaCl2) injury. Following injury, reductions in function (87.5%) and myotube number (33.3%) were observed, followed by a proliferative phase with increased MyoD+ cells and a subsequent recovery of function and myotube number. An expansion of the Pax7+ cell population was observed across recovery suggesting an ability to generate Pax7+ cells within the tissue, similar to the self-renewal of satellite cells seen in vivo. CONCLUSIONS: This work outlines an engineered human SkM capable of functional regeneration following injury, built upon an open source system adding to the pre-clinical testing toolbox to improve the understanding of basic regenerative physiology.

Adjuvant statin therapy for oesophageal adenocarcinoma: the STAT-ROC feasibility study.

BACKGROUND: Statins inhibit proliferative signalling in oesophageal adenocarcinoma (OAC) and their use is associated with better survival in observational studies. The present study was undertaken to examine the feasibility of assessing adjuvant statin therapy in patients with operable OAC in a phase III RCT. METHODS: For this multicentre, double-blind, parallel-group, randomized, placebo-controlled feasibility trial, adults with OAC (including Siewert I-II lesions) who had undergone oesophagectomy were centrally allocated (1 : 1) to simvastatin 40 mg or matching placebo by block randomization, stratified by centre. Participants, clinicians and investigators were blinded to treatment allocation. Patients received treatment for up to 1 year. Feasibility outcomes were recruitment, retention, drug absorption, adherence, safety, quality of life, generalizability and survival. RESULTS: A total of 120 patients were assessed for eligibility at four centres, of whom 32 (26·7 per cent) were randomized, 16 in each group. Seven patients withdrew. Participants allocated to simvastatin had lower low-density lipoprotein cholesterol levels by 3 months (adjusted mean difference -0·83 (95 per cent c.i. -1·4 to -0·22) mmol/l; P = 0·009). Median adherence to medication was greater than 90 per cent between 3 and 12 months' follow-up. Adverse events were similar between the groups. Quality-of-life data were complete for 98·3 per cent of questionnaire items. Cardiovascular disease, diabetes and aspirin use were more prevalent in the non-randomized group, whereas tumour site, stage and grade were similar between groups. Survival estimates were imprecise. CONCLUSION: This RCT supports the conduct and informs the design considerations for a future phase III trial of adjuvant statin therapy in patients with OAC. Registration number: ISRCTN98060456 (www.isrctn/com).

ANTECEDENTES: Las estatinas inhiben las señalizaciones proliferativas en el adenocarcinoma de esófago (oesophageal adenocarcinoma, OAC) y su uso se asocia con mejor supervivencia en estudios observacionales. El presente estudio se llevó a cabo para examinar la viabilidad de evaluar el tratamiento adyuvante con estatinas en pacientes con OAC operable en un ensayo aleatorizado y controlado de fase III. MÉTODOS: En este ensayo de viabilidad controlado por placebo, aleatorizado, de grupos paralelos, doble ciego y multicéntrico, los pacientes adultos con OAC (incluyendo lesiones Siewert I/II) que fueron sometidos a esofaguectomía se asignaron de forma centralizada (1:1) a tratamiento con simvastatina 40 mg o placebo equivalente mediante aleatorización en bloques, estratificados por centro. Los participantes, los clínicos y los investigadores desconocían la asignación del tratamiento. Los pacientes recibieron el tratamiento hasta un año. Los resultados de viabilidad fueron reclutamiento, retención, absorción del fármaco, adherencia, seguridad, calidad de vida, generalización, y supervivencia. RESULTADOS: Un total de 120 pacientes fueron evaluados para elegibilidad en 4 centros, de los cuales 32 (26,7%) fueron aleatorizados, 16 en cada grupo. Siete pacientes abandonaron el ensayo. Los pacientes asignados a tratamiento con simvastatina tenían niveles de colesterol LDL más bajos a los 3 meses (diferencia media ajustada, −0,83 mmol/L, i.c. del 95% −1,4 a −0,22, P = 0,009). La mediana de la adherencia a la medicación fue mayor del 90% entre los 3-12 meses de seguimiento. Los eventos adversos fueron similares entre los grupos. Los datos de calidad de vida estaban completos en el 98,3% de las preguntas del cuestionario. Enfermedad cardiovascular, diabetes y uso de aspirina eran más prevalentes en el grupo no aleatorizado, mientras que la localización del tumor, el estadio y el grado fueron similares entre los grupos. Las estimaciones de supervivencia fueron imprecisas. CONCLUSIÓN: Este RCT apoya la realización e informa de las consideraciones de diseño para un futuro ensayo de fase III de tratamiento adyuvante con estatinas en pacientes con OAC.

The academic-practice gap: Strategies for an enduring problem.

The academic-practice gap in nursing is well documented. Academe is criticized for producing nurses insufficiently prepared to fully participate in patient care. Practice settings are criticized for having unrealistic expectations of new graduates. This article is based on a review of the literature and an exploration of contemporary practices used to bridge academic and practice partnerships. Differences in outcome expectations for new graduates between academe and practice are outlined and consequences of the gap for new graduates, patients, and employers are discussed. Five specific strategies to address the gap are discussed, with the primary realm and responsibility for two falling to education, one to practice, and two to both. Strategies discussed include increased use of simulated learning in nursing education; disruptive innovations in education that promote learner-centered active learning; extended orientation/Transition to Practice Programs for new graduates; dedicated education units; and academic service partnerships. Current literature suggests the viewpoints of academic and practice leaders continue to appear divergent. Closing the gap will require a dedicated and coordinated response from both academe and clinical practice.

The effect of experimental diabetes and glycaemic control on guided bone regeneration: histology and gene expression analyses.

OBJECTIVES: To investigate the effect of experimental diabetes and metabolic control on intramembranous bone healing following guided bone regeneration (GBR). MATERIAL AND METHODS: Ninety-three Wistar rats were allocated to three experimental groups, healthy (H), uncontrolled diabetes (D) and controlled diabetes (CD). Twenty one days following diabetes induction, a standardised 5-mm defect was created at the mid-portion of each parietal bone. In 75 animals (25H, 25D, 25CD), one defect was treated with an intracranial and extracranial membrane according to the GBR principle, and one defect was left empty (control); five animals per group were then randomly sacrificed at 3, 7, 15, 30 and 60 days and processed for decalcified histology. In 18 animals (6H, 6D, 6CD), both defects were treated according to the GBR principle; three animals from each group were then randomly sacrificed at 7 and 15 days of healing and employed for gene expression analysis. RESULTS: Application of the GBR therapeutic principle led to significant bone regeneration even in the D group. However, at 15 and 30 days, the osteogenesis process was impaired by uncontrolled diabetes, as shown by the significant reduction in terms of defect closure (38-42%) and newly formed bone (54-61%) compared to the healthy group. The comparison of the D vs. H group at 15 days of healing yielded the largest number of genes with significantly differential expression, among which various genes associated with the ossification process (bmp4, ltbp4, thra and cd276) were identified. CONCLUSIONS: Uncontrolled diabetes seems to affect early phases of the bone regeneration following GBR. A misregulation of genes and pathways related to cell division, energy production, inflammation and osteogenesis may account for the impaired regeneration process in D rats. Further studies are warranted to optimise the GBR process in this medically compromised patient population.

Annexin-enriched osteoblast-derived vesicles act as an extracellular site of mineral nucleation within developing stem cell cultures.

The application of extracellular vesicles (EVs) as natural delivery vehicles capable of enhancing tissue regeneration could represent an exciting new phase in medicine. We sought to define the capacity of EVs derived from mineralising osteoblasts (MO-EVs) to induce mineralisation in mesenchymal stem cell (MSC) cultures and delineate the underlying biochemical mechanisms involved. Strikingly, we show that the addition of MO-EVs to MSC cultures significantly (P < 0.05) enhanced the expression of alkaline phosphatase, as well as the rate and volume of mineralisation beyond the current gold-standard, BMP-2. Intriguingly, these effects were only observed in the presence of an exogenous phosphate source. EVs derived from non-mineralising osteoblasts (NMO-EVs) were not found to enhance mineralisation beyond the control. Comparative label-free LC-MS/MS profiling of EVs indicated that enhanced mineralisation could be attributed to the delivery of bridging collagens, primarily associated with osteoblast communication, and other non-collagenous proteins to the developing extracellular matrix. In particular, EV-associated annexin calcium channelling proteins, which form a nucleational core with the phospholipid-rich membrane and support the formation of a pre-apatitic mineral phase, which was identified using infrared spectroscopy. These findings support the role of EVs as early sites of mineral nucleation and demonstrate their value for promoting hard tissue regeneration.

Does the site of anastomosis for esophagectomy affect long-term quality of life?

Long-term survival after esophagectomy is improving, and hence, quality of life (QOL) of these patients has become a priority. There has been extensive debate regarding the optimal site of surgical anastomosis (cervical or intrathoracic). We aimed to evaluate the impact of anastomotic site on long-term QOL postesophagectomy. Quality of life questionnaires (European Organisation for Research and Treatment of Cancer [EORTC] C-30 and OG-25) were sent to patients surviving over 3 years following esophagectomy. The data were analyzed by site of esophagogastric anastomosis: intrathoracic or cervical. EORTC C-30 data were compared against the reference population data. Of the patients, 62 responded (82%) with a median time postsurgery of 6.1 years (range 3-12 years). Patient demographics were comparable. There was no significant difference between cervical or intrathoracic anastomosis groups for functional or symptom scores, focusing on dysphagia (cervical = 8.8 vs. intrathoracic = 17.6, P = 0.24), odynophagia (cervical = 13.4 vs. intrathoracic = 16.1, P = 0.68) and swallowing problems (cervical = 8.1 vs. intrathoracic = 13.4, P = 0.32). There was no difference in overall health score between groups (cervical = 70.5 vs. intrathoracic = 71.6, P = 0.46). Overall general health score was comparable with the reference population (esophagectomy group P = 70.9 ± 22.1 vs. reference population = 71.2 ± 22.4, P = 0.93). There is no difference in long-term QOL after esophagectomy between patients with a cervical or intrathoracic anastomosis. Scores compare favorably with EORTC reference data. Survival after esophagectomy is associated with recovery of QOL in the long term, regardless of site of anastomosis and despite worse gastrointestinal-related symptoms.

Identifying the Cellular Mechanisms Leading to Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating condition defined by the de novo development of bone within non-osseous soft tissues, and can be either hereditary or acquired. The hereditary condition, fibrodysplasia ossificans progressiva is rare but life threatening. Acquired HO is more common and results from a severe trauma that produces an environment conducive for the formation of ectopic endochondral bone. Despite continued efforts to identify the cellular and molecular events that lead to HO, the mechanisms of pathogenesis remain elusive. It has been proposed that the formation of ectopic bone requires an osteochondrogenic cell type, the presence of inductive agent(s) and a permissive local environment. To date several lineage-tracing studies have identified potential contributory populations. However, difficulties identifying cells in vivo based on the limitations of phenotypic markers, along with the absence of established in vitro HO models have made the results difficult to interpret. The purpose of this review is to critically evaluate current literature within the field in an attempt identify the cellular mechanisms required for ectopic bone formation. The major aim is to collate all current data on cell populations that have been shown to possess an osteochondrogenic potential and identify environmental conditions that may contribute to a permissive local environment. This review outlines the pathology of endochondral ossification, which is important for the development of potential HO therapies and to further our understanding of the mechanisms governing bone formation.

A dielectrophoretic method of discrimination between normal oral epithelium, and oral and oropharyngeal cancer in a clinical setting.

Despite the accessibility of the oral cavity to clinical examination, delays in diagnosis of oral and oropharyngeal carcinoma (OOPC) are observed in a large majority of patients, with negative impact on prognosis. Diagnostic aids might help detection and improve early diagnosis, but there remains little robust evidence supporting the use of any particular diagnostic technology at the moment. The aim of the present feasibility first-in-human study was to evaluate the preliminary diagnostic validity of a novel technology platform based on dielectrophoresis (DEP). DEP does not require labeling with antibodies or stains and it is an ideal tool for rapid analysis of cell properties. Cells from OOPC/dysplasia tissue and healthy oral mucosa were collected from 57 study participants via minimally-invasive brush biopsies and tested with a prototype DEP platform using median membrane midpoint frequency as main analysis parameter. Results indicate that the current DEP platform can discriminate between brush biopsy samples from cancerous and healthy oral tissue with a diagnostic sensitivity of 81.6% and a specificity of 81.0%. The present ex vivo results support the potential application of DEP testing for identification of OOPC. This result indicates that DEP has the potential to be developed into a low-cost, rapid platform as an assistive tool for the early identification of oral cancer in primary care; given the rapid, minimally-invasive and non-expensive nature of the test, dielectric characterization represents a promising platform for cost-effective early cancer detection.

Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds.

Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment.

Aspirin may prevent cholangiocarcinoma: a case-control study from the United kingdom.

BACKGROUND: The proliferation of cholangiocarcinoma cells is suppressed in cell culture by nonsteroidal antiinflammatory drugs (NSAIDs) through the inhibition of cyclo-oxygenase-2 enzyme and also by statins which decrease the production of mediators of the cell cycle. AIMS: To investigate whether there is an inverse association between NSAIDs, including aspirin, and the development of cholangiocarcinoma and, for the first time in a Western population, between statin use and the development of cholangiocarcinoma. METHODS: This epidemiological study had a case-control design in which cases of cholangiocarcinoma diagnosed in Norwich between 2004 and 2010 and in Leicester in 2007 were identified from clinical databases. Controls were patients with basal cell carcinomas treated in the respective dermatology departments. The case notes of all subjects were reviewed to confirm diagnoses and obtain information on medication use. The data were analyzed using unconditional logistic regression to calculate odds ratios (OR) with 95 % confidence intervals (CI). RESULTS: In total, 81 cases of cholangiocarcinoma and 275 controls were identified. For all cases there was radiological evidence of cancer and 86 % of the cases involved the extrahepatic biliary system. Aspirin use was inversely associated with the development of cholangiocarcinoma (OR 0.45, 95 % CI 0.22-0.92), but there were no significant associations between the development of cholangiocarcinoma and NSAIDs (OR 0.39; 95 % CI 0.11-1.42) or statins (OR 0.58; 95 % CI 0.28-1.19). CONCLUSIONS: The epidemiological data from this study support the biological evidence for aspirin having a protective effect against the development of cholangiocarcinoma. Aspirin use should be measured in future etiological studies and assessed as a chemoprevention agent in those at high risk of developing this type of cancer.

Acute mechanical overload increases IGF-I and MMP-9 mRNA in 3D tissue-engineered skeletal muscle.

Skeletal muscle (SkM) is a tissue that responds to mechanical load following both physiological (exercise) or pathophysiological (bed rest) conditions. The heterogeneity of human samples and the experimental and ethical limitations of animal studies provide a rationale for the study of SkM plasticity in vitro. Many current in vitro approaches of mechanical loading of SkM disregard the three-dimensional (3D) structure in vivo. Tissue engineered 3D SkM, that displays highly aligned and differentiated myotubes, was used to investigate mechano-regulated gene transcription of genes implicated in hypertrophy/atrophy. Static loading (STL) and ramp loading (RPL) at 10 % strain for 60 min were used as mechano-stimulation with constructs sampled immediately for RNA extraction. STL increased IGF-I mRNA compared to both RPL and CON (control, p = 0.003 and 0.011 respectively) whilst MMP-9 mRNA increased in STL and RPL compared to CON (both p < 0.05). IGFBP-2 mRNA was differentially regulated in RPL and STL compared to CON (p = 0.057), whilst a reduction in IGFBP-5 mRNA was found for STL and RPL compared to CON (both p < 0.05). There was no effect in the expression of putative atrophic genes, myostatin, MuRF-1 and MAFBx (all p > 0.05). These data demonstrate a transcriptional signature associated with SkM hypertrophy within a tissue-engineered model that more greatly recapitulates the in vivo SkM structure compared previously published studies.

Randomized clinical trial of fluid and salt restriction compared with a controlled liberal regimen in elective gastrointestinal surgery.

BACKGROUND: Excessive intravenous fluid prescription may play a causal role in postoperative complications following major gastrointestinal resectional surgery. The aim of this study was to investigate whether fluid and salt restriction would decrease postoperative complications compared with a more modern controlled liberal regimen. METHODS: In this observer-blinded single-site randomized clinical trial consecutive patients undergoing major gastrointestinal resectional surgery were randomized to receive either a liberal control fluid regimen or a restricted fluid and salt regimen. The primary outcome was postoperative complications of grade II and above (moderate to severe). RESULTS: Some 240 patients (194 colorectal resections and 46 oesophagogastric resections) were enrolled in the study; 121 patients were randomized to the restricted regimen and 119 to the control (liberal) regimen. During surgery the control group received a median (interquartile range) fluid volume of 2033 (1576-2500) ml and sodium input of 282 (213-339) mmol, compared with 1000 (690-1500) ml and 142 (93-218) mmol respectively in the restricted group. There was no significant difference in major complication rate between groups (38·0 and 39·0 per cent respectively). Median (range) hospital stay was 8 (3-101) days in the controls and 8 (range 3-76) days among those who received restricted fluids. There were four in-hospital deaths in the control group and two in the restricted group. Substantial differences in weight change, serum sodium, osmolality and urine : serum osmolality ratio were observed between the groups. CONCLUSION: There were no significant differences in major complication rates, length of stay and in-hospital deaths when fluid restriction was used compared with a more liberal regimen. REGISTRATION NUMBER: ISRCTN39295230 (http://www.controlled-trials.com).

A prospective, randomized, single-blind trial of 5-mm versus 3-mm ports for laparoscopic cholecystectomy: is smaller better?

BACKGROUND: Single-incision laparoscopic cholecystectomy (SILC) is said to provide improved cosmesis with a reduction in postoperative pain, but SILC involves a change in operative technique. A single-blind, randomized controlled trial compared cosmetic outcomes and postoperative pain between 3- and 5-mm ports used for laparoscopic cholecystectomy (LC). METHODS: For this study, 80 patients with symptomatic gallstones were recruited from a single center and randomized to a LC using either a 5-mm port and three 3-mm ports (group A) or a 10-mm port and three 5-mm ports (group B). Operative details; pain scores at 1 h, 6 h, and 1 week; and analgesia required during the first week were collected. Cosmetic outcome was assessed at 6 months using a validated questionnaire. RESULTS: For each group, 40 patients were recruited. The two groups were well matched except for sex. Group A had 11 males, and Group B had 4 males. The mean operative time was 49 ± 12 min (range, 24-120 min) in the 3-mm group versus 46 ± 19 min (range, 21-124 min) in the control group (p = 0.40). The two groups did not differ statistically in the day case rate. The pain scores in Group A were 2.5 ± 2.1 at 1 h, 3.2 ± 2.2 at 6 h, and 0.8 ± 2.2 at 1 week versus 4.2 ± 2.9 at 1 h, 3.3 ± 2.4 at 6 h, and 2.1 ± 2.4 at 1 week in Group B (p = 0.003, 0.63, and 0.002, respectively). No difference in the analgesia consumption was observed during the first postoperative week. The patients in Group A had significantly better cosmetic outcome scores at 6 months. CONCLUSION: The use of 3-mm ports is technically feasible in patients undergoing LC for gallstones. The operating times are comparable with those for conventional LC, whereas the pain scores are reduced, and the cosmetic outcome is better.

Systematic review: potential preventive effects of statins against oesophageal adenocarcinoma.

BACKGROUND: The incidence of oesophageal adenocarcinoma (OAC) has risen dramatically in recent decades, and its prognosis remains extremely poor. There is emerging evidence that statins may prevent OAC. AIM: To systematically review both the experimental and epidemiological evidence to determine whether statins reduce the risk of developing OAC. METHODS: Relevant laboratory and epidemiological studies were identified by systematically searching the PUBMED and EMBASE electronic databases for data on statins and oesophageal cancer (OC). The evidence was assessed according to the nine Bradford Hill criteria (BHC) of causality. Pooled effect sizes (ES) were calculated for the risk of OC with prior statin use. RESULTS: Many of the BHC were supported including: 'plausible biological mechanisms', 'coherence', 'strong associations', 'consistency', 'biological gradient', 'analogy' and 'temporality'. Three experimental studies reported that statins inhibited proliferation, induced apoptosis and may limit metastatic potential in OAC cell lines. Fixed effects meta-analysis of two prospective studies in Barrett's oesophagus cohorts, involving 1382 participants, showed an ES of 0.53 (95% CI = 0.36-0.78, P = 0.001, I(2)  = 0%) for risk of OAC with prior statin use. Meta-analysis of three prospective studies in general population cohorts, involving 35 214 participants, showed an ES of 0.86 (95% CI = 0.78-0.94, P = 0.001, I(2)  = 0%) for risk of OC with prior statin use. The most important criterion, 'experiment', is as yet unfulfilled as to date there are no clinical trials which investigate this hypothesis. CONCLUSION: There is some evidence that statins may protect against the development of OAC, although to be conclusive, data from randomised clinical trials are required.

Characterization and optimization of a simple, repeatable system for the long term in vitro culture of aligned myotubes in 3D.

Increased recent research activity in exercise physiology has dramatically improved our understanding of skeletal muscle development and physiology in both health and disease. Advances in bioengineering have enabled the development of biomimetic 3D in vitro models of skeletal muscle which have the potential to further advance our understanding of the fundamental processes that underpin muscle physiology. As the principle structural protein of the extracellular matrix, collagen-based matrices are popular tools for the creation of such 3D models but the custom nature of many reported systems has precluded their more widespread adoption. Here we present a simple, reproducible iteration of an established 3D in vitro model of skeletal muscle, demonstrating both the high levels of reproducibility possible in this system and the improved cellular architecture of such constructs over standard 2D cell culture techniques. We have used primary rat muscle cells to validate this simple model and generate comparable data to conventional established cell culture techniques. We have optimized culture parameters for these cells which should provide a template in this 3D system for using muscle cells derived from other donor species and cell lines.

Cancer, pre-cancer and normal oral cells distinguished by dielectrophoresis.

Most oral cancers are oral squamous cell carcinomas (OSCC) that arise from the epithelial lining of the oral mucosa. Given that the oral cavity is easily accessible, the disease lends itself to early detection; however, most oral cancers are diagnosed at a late stage, and approximately half of oral cancer sufferers do not survive beyond five years, post-diagnosis. The low survival rate has been attributed to late detection, but there is no accepted, reliable and convenient method for the detection of oral cancer and oral pre-cancer. Dielectrophoresis (DEP) is a label-free technique which can be used to obtain multi-parametric measurements of cell electrical properties. Parameters such as cytoplasmic conductivity and effective membrane capacitance (C(Eff)) can be non-invasively determined by the technique. In this study, a novel lab-on-a-chip device was used to determine the cytoplasmic conductivity and C(Eff) of primary normal oral keratinocytes, and pre-cancerous and cancerous oral keratinocyte cell lines. Our results show that the electrical properties of normal, pre-cancerous and cancerous oral keratinocytes are distinct. Furthermore, increasing C (Eff) and decreasing cytoplasmic conductivity correlate with disease progression which could prove significant for diagnostic and prognostic applications. DEP has the potential to be used as a non-invasive technique to detect oral cancer and oral pre-cancer. Clinical investigation is needed to establish the reliability and temporal relationship of the correlation between oncologic disease progression and the electrical parameters identified in this study. To use this technique as an OSCC detection tool in a clinical setting, further characterisation and refinement is warranted.

Stretching skeletal muscle in vitro: does it replicate in vivo physiology?

Skeletal muscle is highly adaptable and responds to changes in loading through exercise or resistance training through a number of mechanisms resulting in increased muscle mass and changes in contractile phenotype. To further understand and study the molecular mechanisms underlying the adaptive response of muscle, a number of in vitro culture systems have been developed that utilise mechanical loading or stretching of the cultured muscle to recapitulate the adaptations observed in vivo. Here we review the use of such stretching regimes for engineered muscle constructs and assess how well these in vitro systems mimic in vivo muscle physiology and adaptation.

The effect of cell density on the maturation and contractile ability of muscle derived cells in a 3D tissue-engineered skeletal muscle model and determination of the cellular and mechanical stimuli required for the synthesis of a postural phenotype.

The successful engineering of a truly biomimetic model of skeletal muscle could have a significant impact on a number of biomedical disciplines. Although a variety of techniques are currently being developed, there is, as of yet, no widely available and easily reproducible culture system for the synthesis of 3D artificial muscle tissues. In attempting to generate such a model it is essential to optimise any protocol in order to generate a tissue that best represents the in vivo environment. Since the maturation of muscle derived cells in culture is critically dependent on density, a major factor to be addressed in the development of these models is the ideal concentration at which to seed cells in order to generate an optimal response. In studying the effect of cell density on the performance of cells in an established 3D collagen based model of skeletal muscle, we demonstrate that an optimum density does exist in terms of peak force generation and myogenic gene expression data. Greater densities however, lead to the formation of a more physiologically relevant tissue with a phenotype characteristic of slow, postural muscle.