Fibroblasts from HPV-negative oropharynx squamous cell carcinomas stimulate the release of osteopontin from cancer cells via the release of IL-6.

Introduction: HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) shows distinct biological and clinical behaviour when compared to HPV-negative OPSCC. The overall role of the tumour microenvironment (TME) in head and neck cancer progression and metastasis has been studied intensively, but differences in HPV-negative and HPV-positive OPSCCs are less understood. Objective: To investigate the role of cancer-associated fibroblasts (CAFs) and the functional interactions of normal tonsil fibroblasts (NTFs) and OP CAFs with HPV+ and HPV- OPSCC cells and explore novel candidates in tumour-fibroblast crosstalk. Materials and methods: A retrospective cohort of 143 primary OPSCCs was characterised using HPV16/18 RNAScope assay, p16 IHC and ɑ-SMA. Four OPSCC, three NTF and 2 new OPSCC CAF cultures were used to assess the cytokine-based interactions using cytokine arrays on conditioned media (CM), followed by co-culture approaches to identify the role of individual cell types and the role of OPN (SPP1) and IL-6 in SCC/fibroblast communication. Results: HPV status was associated with better overall survival. Although ɑ-SMA expression was observed in both OPSCC subtypes, it provided survival stratification only in the HPV-positive group (Log-Rank p = 0.02). Three normal tonsillar fibroblast cultures (NTFs) were characterised by induction of myofibroblastic and senescent phenotypes with similar reactivity to our published NOF phenotype. The OPSCC-derived CAF cultures were characterised and their baseline myofibroblastic and senescence phenotypes varied. Cytokine array analysis of CM to identify novel candidates in the crosstalk between OPSCC tumour cells and NTFs/CAFs identified differences in the cytokine profiles on comparison of HPV+ and HPV- OPSCC cells. Osteopontin (OPN/SPP1) was identified, particularly in HPV-negative OPSCC cell analyses. We have demonstrated that OPN was produced by the OPSCC cells and revealed an associated upregulation of IL-6 in fibroblasts. Treatment of NTFs with rOPN showed alteration in phenotype, including increased contraction and IL-6 production. Antibody-mediated inhibition of CD44v6 attenuated the production of IL-6 by OPN in NTFs. Conclusion: This investigation with OPSCC fibroblasts provides novel insights into the role of CAFs in OPSCC mediated by IL-6 stimulated release of OPN from HPV negative OPSCC cells. The details of HPV-positive SCC cell/fibroblast cytokine crosstalk remain elusive.

Personalised versus standard text message prompts for increasing trial participant response to telephone follow-up: an embedded randomised controlled retention trial.

BACKGROUND: Improving retention within randomised controlled trials is important. The effectiveness of different strategies can be assessed using a Study Within A Trial (SWAT). Previous research has shown personalised text message reminders improve clinic attendance rates; however, the results are mixed on improving postal questionnaire return. This SWAT aims to assess whether personalised text message reminders improve completion rates for scheduled telephone follow-ups. METHODS: This SWAT is a two-arm, multi-centre randomised controlled trial with equal allocation. The host trial was the Melatonin for Anxiety prior to General anaesthesia In Children trial (ISRCTN 18296119), where the child's caregiver was to answer a scheduled telephone follow-up 14 days post-surgery; participants for the SWAT were therefore the caregiver. Text messages were sent 24-48 h before the scheduled call and the personalised version contained the first name of the caregiver which was omitted in the non-personalised version. The primary outcome was questionnaire completion rate, defined as the proportion of caregivers successfully contacted, and completed any of the questionnaires, over the telephone within the follow-up window (day 14 + 7 days). RESULTS: The SWAT included 100 of the 110 (91%) participants randomised into the host trial. Randomisation within the SWAT was equal between non-personalised (n = 50) and personalised (n = 50) interventions. The overall questionnaire response rate was 73% with a difference between the two interventions of 68% in the non-personalised text message arm and 78% in the personalised text message arm. The adjusted absolute risk difference was 7.1% (95% confidence interval = -10.2%, 24.4%). There was no difference in either the time to response or the number of contact attempts between the two interventions. CONCLUSIONS: There is some evidence that personalised text messages could be effective at increasing response rates when data is collected via telephone and in a population of caregivers for paediatric trial participants. However, similar SWATs have shown mixed results. Given the low-cost and low risks associated with personalising text message reminders, this SWAT could be implemented easily in other RCTs scheduling telephone follow-up appointments. TRIAL REGISTRATION: ISRCTN 18296119 , SWAT 35 (MRC Northern Ireland Network for Trials Methodology Network).

The MAGIC trial: a pragmatic, multicentre, parallel, noninferiority, randomised trial of melatonin versus midazolam in the premedication of anxious children attending for elective surgery under general anaesthesia.

BACKGROUND: Child anxiety before general anaesthesia and surgery is common. Midazolam is a commonly used premedication to address this. Melatonin is an alternative anxiolytic, however trials evaluating its efficacy in children have delivered conflicting results. METHODS: This multicentre, double-blind randomised trial was performed in 20 UK NHS Trusts. A sample size of 624 was required to declare noninferiority of melatonin. Anxious children, awaiting day case elective surgery under general anaesthesia, were randomly assigned 1:1 to midazolam or melatonin premedication (0.5 mg kg-1, maximum 20 mg) 30 min before transfer to the operating room. The primary outcome was the modified Yale Preoperative Anxiety Scale-Short Form (mYPAS-SF). Secondary outcomes included safety. Results are presented as n (%) and adjusted mean differences with 95% confidence intervals. RESULTS: The trial was stopped prematurely (n=110; 55 per group) because of recruitment futility. Participants had a median age of 7 (6-10) yr, and 57 (52%) were female. Intention-to-treat and per-protocol modified Yale Preoperative Anxiety Scale-Short Form analyses showed adjusted mean differences of 13.1 (3.7-22.4) and 12.9 (3.1-22.6), respectively, in favour of midazolam. The upper 95% confidence interval limits exceeded the predefined margin of 4.3 in both cases, whereas the lower 95% confidence interval excluded zero, indicating that melatonin was inferior to midazolam, with a difference considered to be clinically relevant. No serious adverse events were seen in either arm. CONCLUSION: Melatonin was less effective than midazolam at reducing preoperative anxiety in children, although the early termination of the trial increases the likelihood of bias. CLINICAL TRIAL REGISTRATION: ISRCTN registry: ISRCTN18296119.

The K/HDEL receptor does not recycle but instead acts as a Golgi-gatekeeper.

Accurately measuring the ability of the K/HDEL receptor (ERD2) to retain the ER cargo Amy-HDEL has questioned earlier results on which the popular receptor recycling model is based upon. Here we demonstrate that ERD2 Golgi-retention, rather than fast ER export supports its function. Ligand-induced ERD2 redistribution is only observed when the C-terminus is masked or mutated, compromising the signal that prevents Golgi-to-ER transport of the receptor. Forcing COPI mediated retrograde transport destroys receptor function, but introducing ER-to-Golgi export or cis-Golgi retention signals re-activate ERD2 when its endogenous Golgi-retention signal is masked or deleted. We propose that ERD2 remains fixed as a Golgi gatekeeper, capturing K/HDEL proteins when they arrive and releasing them again into a subdomain for retrograde transport back to the ER. An in vivo ligand:receptor ratio far greater than 100 to 1 strongly supports this model, and the underlying mechanism appears to be extremely conserved across kingdoms.

In vitro Effect of Geranylgeraniol (GGOH) on Bisphosphonate-Induced Cytotoxicity of Oral Mucosa Cells.

Medication-related osteonecrosis of the jaw (MRONJ) is an often-severe complication found in patients receiving bisphosphonates in the management of Paget's, osteoporosis and metastatic bone cancer. Mucosal breakdown with bone exposure is a primary clinical presentation of MRONJ linked to the inhibitory effect of nitrogen-containing bisphosphonates (N-BP) on the mevalonate pathway. Geranylgeraniol (GGOH) has demonstrated a rescue effect on N-BP-treated osteoclasts but the biological effects on oral soft tissues and cells remain unclear. This study aimed to determine whether GGOH could prevent bisphosphonate induced toxicity to oral mucosa cells in vitro. Primary oral fibroblasts and keratinocytes were exposed to different GGOH concentrations or GGOH in combination with two nitrogen-containing bisphosphonates, zoledronic acid (ZA) or pamidronic acid (PA), for 72 h. The metabolic activity of each cell type was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GGOH without bisphosphonates significantly reduced the metabolic activity of oral mucosa cells. Fibroblasts treated with GGOH and ZA in combination showed a slight increase in metabolic status compared to fibroblasts treated with ZA alone, however this positive effect was not observed in keratinocytes. In the presence of PA, GGOH was unable to increase the metabolic activity of either cell type. These findings demonstrate that GGOH is toxic to oral mucosa cells and that GGOH was not able to prevent bisphosphonate induced toxicity. These data show that GGOH does not have therapeutic potential for bisphosphonate-induced soft tissue toxicity in MRONJ and the use of GGOH as an MRONJ treatment should be strongly reconsidered.

Continuous flow mechanochemistry: reactive extrusion as an enabling technology in organic synthesis.

Rapid and wide-ranging developments have established mechanochemistry as a powerful avenue in sustainable organic synthesis. This is primarily due to unique opportunities which have been offered in solvent-free - or highly solvent-minimised - reaction systems. Nevertheless, despite elegant advances in ball-milling technology, limitations in scale-up still remain. This tutorial review covers the first reports into the translation from "batch-mode" ball-milling to "flow-mode" reactive extrusion, using twin-screw extrusion.

Melatonin for pre-medication in children: a systematic review.

BACKGROUND: Melatonin's effectiveness as an anxiolytic medication has been confirmed in adults; however, its efficacy in a paediatric population is unclear. A number of small studies have assessed its use in children as a pre-operative anxiolytic, with conflicting results. METHODS: We undertook a systematic review of pre-operative melatonin use in children. Four databases (MEDLINE, Embase, the Cochrane Central Register of Controlled Trials and Web of Science), and ' ClinicalTrials.gov ' were searched for ongoing and completed clinical trials of relevance. Citation tracking reference lists and relevant articles were also accessed. The review was unrestricted by comparator or outcomes. Eleven studies were judged eligible for inclusion. There were high levels of heterogeneity in melatonin administration (in terms of dose and timing). Variable outcomes were reported and included: anxiety; anaesthetic success; analgesia; sedation; post-operative recovery; and safety. Outcomes were not always assessed with the same measures. RESULTS: Evidence to support melatonin's anxiolytic properties in this setting is conflicting. Melatonin was associated with reduced sedative effects, post-operative excitement and improved emergence behaviour, compared to comparator drugs. One study reported the benefit of melatonin use on sleep disturbance at two weeks post-surgery. No adverse safety events were identified to be significantly associated with melatonin, affirming its excellent safety profile. CONCLUSION: Despite potential advantages, including improved emergence behaviour, based on current evidence we cannot confirm whether melatonin is non-inferior to current "usual care" pre-medications. Further consideration of melatonin as an anxiolytic pre-medication in paediatric surgery is needed.

Ball-Milling-Enabled Reactivity of Manganese Metal*.

Efforts to generate organomanganese reagents under ball-milling conditions have led to the serendipitous discovery that manganese metal can mediate the reductive dimerization of arylidene malonates. The newly uncovered process has been optimized and its mechanism explored using CV measurements, radical trapping experiments, EPR spectroscopy, and solution control reactions. This unique reactivity can also be translated to solution whereupon pre-milling of the manganese is required.

Stakeholder perspectives on barriers and enablers to recruiting anxious children undergoing day surgery under general anaesthetic: a qualitative internal pilot study of the MAGIC randomised controlled trial.

BACKGROUND: The 'Melatonin for Anxiety prior to General anaesthesia In Children' (MAGIC) trial was designed to compare midazolam and melatonin as pre-medications for anxious children (aged five to fourteen), undergoing day-case surgical procedures under general anaesthesia. Low recruitment is a challenge for many trials, particularly paediatric trials and those in 'emergency' settings. A qualitative study as part of MAGIC aimed to gather stakeholder perspectives on barriers and enablers to recruitment. METHODS: Sixteen stakeholders from six sites participated in semi-structured interviews about their experiences of setting up the MAGIC trial and recruiting patients as part of the internal pilot. Data was analysed using framework analysis. RESULTS: Participants identified barriers and enablers to recruitment. Barriers and enablers related to the study, participants, the population of anxious children, practitioners, collaboration with other health professionals, ethics, specific settings and the context of surgical day units and the wider health system. Attempting to recruit anxious children from a surgical day unit is particularly challenging for several reasons. Issues include the practicalities of dealing with a child experiencing anxiety for parents/guardians; professional unwillingness to make things more difficult for families and clinicians and nurses valuing predictability within a busy and time-sensitive setting. CONCLUSIONS: Multi-site RCTs face recruitment barriers relating to study-wide and site-specific factors. There are multiple barriers to recruiting anxious children due to undergo day-case surgery. Barriers across domains can interrelate and reinforce each other, reflecting challenges relating to populations and settings. For example, in the case of anxious children, parents and other health professionals are concerned about exacerbating children's anxiety prior to surgery. They may look for ways to keep things predictable and avoid the uncertainty of an RCT. Pre-trial engagement work could help address concerns among collaborating health professionals. Using rapid ethnography during set-up or an internal pilot to focus on how the protocol will be or has been operationalised in practice may help identify issues. Allowing time to reflect on the findings of internal pilots and implement necessary changes could facilitate higher recruitment during the main phase of a trial. TRIAL REGISTRATION: NIHR Trial Registration Number: ISRCTN18296119 . Registered on October 01, 2019.

Increased Abundance of Tumour-Associated Neutrophils in HPV-Negative Compared to HPV-Positive Oropharyngeal Squamous Cell Carcinoma Is Mediated by IL-1R Signalling.

The incidence of human papillomavirus (HPV)-associated cancer is increasing and HPV is now implicated in the aetiology of more than 60% of all oropharyngeal squamous cell carcinomas (OPSCC). In OPSCC, innate immune cells such as neutrophils and macrophages generally correlate with poor prognosis, whilst adaptive immune cells, such as lymphocytes, tend to correlate with improved prognosis. This may, in part, be due to differences in the immune response within the tumour microenvironment leading to the recruitment of specific tumour-associated leukocyte sub-populations. In this study, we aimed to examine if differences exist in the levels of infiltrated leukocyte sub-populations, with particular emphasis on tumour-associated neutrophils (TAN), and to determine the mechanism of chemokine-induced leukocyte recruitment in HPV-positive compared to HPV-negative OPSCC. Immunohistochemical analysis showed that HPV-negative OPSCC contained significantly more neutrophils than HPV-positive tumours, whilst levels of CD68+ macrophages and CD3+ lymphocytes were similar. Using a 3D tissue culture model to represent tumour-stromal interactions, we demonstrated that HPV-negative tumour-stromal co-cultures expressed significantly higher levels of CXCL8, leading to increased neutrophil recruitment compared to their HPV-positive counterparts. HPV-negative OPSCC cells have previously been shown to express higher levels of IL-1 than their HPV-positive counterparts, indicating that this cytokine may be responsible for driving increased chemokine production in the HPV-negative 3D model. Inhibition of IL-1R in the tumour-stromal models using the receptor-specific antagonist, anakinra, dramatically reduced chemokine secretion and significantly impaired neutrophil and monocyte recruitment, suggesting that this tumour-stromal response is mediated by the IL-1/IL-1R axis. Here, we identify a mechanism by which HPV-negative OPSCC may recruit more TAN than HPV-positive OPSCC. Since TAN are associated with poor prognosis in OPSCC, our study identifies potential therapeutic targets aimed at redressing the chemokine imbalance to reduce innate immune cell infiltration with the aim of improving patient outcome.

The impact of wrong-site surgery on dental undergraduate teaching: a survey of UK dental schools.

INTRODUCTION: Patient safety within dental education is paramount. Wrong-site surgery (WSS) tooth extraction is not uncommon and is a significant never event (NE) in dentistry. This study aimed to explore dental schools' undergraduate experience of NEs, safety interventions implemented and the impact on student experience. METHODS: All 16 UK dental schools were surveyed via email. RESULTS: The response rate was 100%. A modified World Health Organization (WHO) checklist was used within institutions (94%) including pre-operative briefings and recording teeth on whiteboards (81%, respectively). Students were directly supervised performing extractions (63%) utilising a 1:4 staff: student ratio. WSS by students was reported in 69% of schools, with student experience being impacted by an increased patient safety focus. DISCUSSION: This study demonstrated an increased utilisation of an adapted WHO checklist. Modification of practices to ensure patient safety was demonstrated at all schools, irrespective of student WSS occurrences. Institutions experiencing student NEs commonly implemented WHO checklists and recording teeth for extraction on whiteboards. Other strategies included direct staff supervision and pre-operative briefings. CONCLUSION: UK dental schools have increased the emphasis on patient safety by the implementation of national healthcare models, for example WHO checklists and pre-operative briefings. These strategies both aim to improve communication and teamwork. Increased levels of staff supervision foster greater quality of teaching; however, this has resulted in reduced student clinical experience. A proposed minimum standard for undergraduate surgery is suggested to ensure safe and competent dental practitioners of the future.

Human papillomavirus (HPV) can establish productive infection in dysplastic oral mucosa, but HPV status is poorly predicted by histological features and p16 expression.

AIMS: Previous studies have reported the presence of high-risk human papillomavirus (HR-HPV) in a subset of dysplastic oral epithelial lesions. Many cases show a histological spectrum of atypia similar to that seen in non-human papillomavirus (HPV) severe epithelial dysplasia, but some studies have suggested that HPV status can be inferred on the basis of histological features. We aimed to assess the utility of such histological features and p16 as surrogate markers of HPV infection in a retrospective cohort of 33 cases of severe epithelial dysplasia, with matched clinicopathological data and histological features. METHODS AND RESULTS: Tissue sections were assessed for the expression of p16, minichromosome maintenance 2, HPV E4 and HPV L1 by the use of immunohistochemistry. HPV16/18 E6 and E7 expression was assessed by the use of RNA in-situ hybridisation (RNAScope). In the cohort, 18.2% of cases (6/33) were HR-HPV-positive, with no age/gender differences between the HPV-positive and HPV-negative groups. HPV E4 and HPV L1 were expressed in surface keratinocytes in four of six (66%) HPV-positive cases, indicative of productive HPV infection. Lack of p16 expression was predictive of HPV-negative status, but sensitivity and specificity varied according to the cut-off. Histologically, the presence of karyorrhectic nuclei and abnormal mitotic figures was higher in HPV-positive lesions (P < 0.05), but the predictive specificity and sensitivity were suboptimal (sensitivity, 0.75; specificity, 0.52). CONCLUSIONS: This study demonstrates, for the first time, that a minority of severely dysplastic oral lesions harbour productive, biologically relevant HPV infection. Consideration should be given to the specific assessment of HPV status in severe epithelial dysplasia cases, as both p16 status and the presence of karyorrhectic cells are poor predictive markers of HPV status.

The IL-1/IL-1R axis induces greater fibroblast-derived chemokine release in human papillomavirus-negative compared to positive oropharyngeal cancer.

Human papillomavirus (HPV) is now recognised as a major aetiological agent in the pathogenesis of oropharyngeal carcinoma (OPC). HPV-positive tumours are associated with better outcomes compared to HPV-negative tumours, possibly due to differences in their aetiology and/or the tumour microenvironment. Increased numbers of tumour-associated leukocytes have been observed in many cancers including OPC, with variable influence on prognosis depending on the leukocyte subpopulation investigated. Whether HPV status influences leukocyte recruitment to OPC remains unknown. This in-vitro study examined differences in the chemoattractant capacity of HPV-positive and HPV-negative OPC cell lines. Gene and protein expression analysis demonstrated that whilst both monocultures of HPV-positive and HPV-negative cell lines, along with normal tonsillar fibroblasts (NTF), expressed low chemokine levels, NTF cultured with conditioned medium from HPV-negative OPC cells expressed significantly higher levels of all chemokines tested compared to NTF incubated with the medium from HPV-positive OPC cell lines. HPV-negative OPC lines expressed IL-1ß mRNA whereas HPV-positive cells did not, and NTF constitutively expressed IL-1R1. Pre-treatment with the IL-R antagonist, anakinra or siRNA to IL-1R1 significantly reduced chemokine secretion from NTF stimulated with conditioned medium from HPV-negative tumour cells or recombinant IL-1ß (p < 0.05). These data suggest that secretion of chemokines is driven by the interaction between HPV-negative OPC cells and stromal fibroblasts through an IL-1/IL-1R-mediated mechanism that is less prominent within the HPV-positive tumour microenvironment. These observations may explain differences in leukocyte sub-populations recruited to HPV-positive versus negative OPC and indicate that HPV status is a key determinant in controlling the inflammatory tumour microenvironment.

3D printed tissue engineered model for bone invasion of oral cancer.

Recent advances in three-dimensional printing technology have led to a rapid expansion of its applications in tissue engineering. The present study was designed to develop and characterize an in vitro multi-layered human alveolar bone, based on a 3D printed scaffold, combined with tissue engineered oral mucosal model. The objective was to incorporate oral squamous cell carcinoma (OSCC) cell line spheroids to the 3D model at different anatomical levels to represent different stages of oral cancer. Histological evaluation of the 3D tissue model revealed a tri-layered structure consisting of distinct epithelial, connective tissue, and bone layers; replicating normal oral tissue architecture. The mucosal part showed a well-differentiated stratified oral squamous epithelium similar to that of the native tissue counterpart, as demonstrated by immunohistochemistry for cytokeratin 13 and 14. Histological assessment of the cancerous models demonstrated OSCC spheroids at three depths including supra-epithelial level, sub-epithelial level, and deep in the connective tissue-bone interface. The 3D tissue engineered composite model closely simulated the native oral hard and soft tissues and has the potential to be used as a valuable in vitro model for the investigation of bone invasion of oral cancer and for the evaluation of novel diagnostic or therapeutic approaches to manage OSCC in the future.

Altered Toll-like receptor expression and function in HPV-associated oropharyngeal carcinoma.

Toll-like receptors (TLRs) have been widely investigated due to their importance in the inflammatory response and possible links to tumor promotion/regression and prognosis. In cancers with an infective etiology, such as human papillomavirus (HPV)-associated Oropharyngeal Squamous Cell Carcinoma (OPSCC), TLR responses may be activated and play a role in tumorigenesis. Our aim was to assess the expression of all TLRs in OPSCC cell lines (both HPV+ and HPV-) by qPCR, Western Blot and flow cytometry and assess their response to TLR ligands lipopolysaccharide (LPS), LPS ultra-pure (LPS-UP) and peptidoglycan (PGN) by analyzing IL-8 and IL-6 production. We also immunostained 61 OPSCC tissue samples with anti-TLR4. Results showed lower TLR1 and TLR6 mRNA expression and higher TLR9 protein expression in HPV+ when compared to HPV-OPSCC cells. TLR4 expression did not vary by HPV status in OPSCC cells, but TLR4 expression was significantly lower in HPV+OPSCC tissues. After stimulation with PGN, only one cell line (HPV+) did not secrete IL-6 or IL-8. Furthermore, HPV+OPSCC lines showed no IL-6 or IL-8 production on treatment with LPS/LPS-UP. The data suggest changes in TLR4 signaling in HPV+OPSCC, since we have shown lower tissue expression of TLR4 and no pro-inflammatory response after stimulation with LPS and LPS-UP. Also, it suggests that OPSCC may respond to HPV infection by increased expression of TLR9. This study demonstrates differences in expression and function of TLRs in OPSCC, which are dependent on HPV status, and may indicate subversion of the innate immune response by HPV infection.

Characterization of Multilayered Tissue-Engineered Human Alveolar Bone and Gingival Mucosa.

Advances in tissue engineering have permitted assembly of multilayered composite tissue constructs for potential applications in the treatment of combined hard and soft tissue defects and as an alternative in vitro test model to animal experimental systems. The aim of this study was to develop and characterize a novel three-dimensional combined human alveolar bone and gingival mucosal model based on primary cells isolated from the oral tissues. Bone component of the model was engineered by seeding primary human alveolar osteoblasts into a hydroxyapatite/tricalcium phosphate scaffold and culturing in a spinner bioreactor. The engineered bone was then laminated, using an adhesive tissue sealant, with tissue-engineered gingival mucosa consisting of air/liquid interface-cultured normal human gingival keratinocytes on oral fibroblast-populated collagen gel scaffold. Histological characterization revealed a structure consisting of established epithelial, connective tissue and bone layers closely comparable to normal oral tissue architecture. The mucosal component demonstrated a mature epithelium undergoing terminal differentiation similar to that characteristic of native buccal mucosa, as confirmed using cytokeratin 13 and cytokeratin 14 immunohistochemistry. Ultrastructural analysis confirmed the presence of desmosomes and hemidesmosomes in the epithelial layer, a continuous basement membrane, and newly synthesized collagen in the connective tissue layer. Quantitative polymerase chain reaction (qPCR) assessment of osteogenesis-related gene expression showed a higher expression of genes encoded collagen I (COL1) and osteonectin (ON) compared with osteocalcin (OC), osteopontin (OP), and alkaline phosphatase (ALP). Enzyme-linked immunosorbent assay quantification of COL1, ON, and OC confirmed a pattern of secretion, which paralleled the model's gene expression profile. We demonstrate in this study that, replicating the anatomical setting between oral mucosa and the underlying alveolar bone is feasible and the developed model showed characteristics similar to those of normal tissue counterparts. This trilayered model therefore offers great scope as an advanced and anatomically representative tissue-engineered alternative to animal models.

Use of a Rho kinase inhibitor to increase human tonsil keratinocyte longevity for three-dimensional, tissue engineered tonsil epithelium equivalents.

The generation of tissue-engineered epithelial models is often hampered by the limited proliferative capacity of primary epithelial cells. This study aimed to isolate normal tonsillar keratinocytes (NTK) from human tonsils, increase the lifespan of these cells using the Rho kinase inhibitor Y-27632 and to develop tissue-engineered equivalents of healthy and infected tonsil epithelium. The proliferation rate of isolated NTK and expression of c-MYC and p16INK4A were measured in the absence or presence of the inhibitor. Y-27632-treated NTK were used to generate tissue-engineered tonsil epithelium equivalents using de-epidermised dermis that were then incubated with Streptococcus pyogenes to model bacterial tonsillitis, and the expression of pro-inflammatory cytokines was measured by cytokine array and ELISA. NTK cultured in the absence of Y-27632 rapidly senesced whereas cells cultured in the presence of this inhibitor proliferated for over 30 population doublings without changing their phenotype. Y-27632-treated NTK produced a multi-layered differentiated epithelium that histologically resembled normal tonsillar surface epithelium and responded to S. pyogenes infection by increased expression of pro-inflammatory cytokines including CXCL5 and IL-6. NTK can be isolated and successfully cultured in vitro with Y-27632 leading to a markedly prolonged lifespan without any deleterious consequences to cell morphology. This functional tissue-engineered equivalent of tonsil epithelium will provide a valuable tool for studying tonsil biology and host-pathogen interactions in a more physiologically relevant manner.

HPV-negative, but not HPV-positive, oropharyngeal carcinomas induce fibroblasts to support tumour invasion through micro-environmental release of HGF and IL-6.

Human papillomavirus (HPV) infection is causally related to a subset of oropharyngeal carcinomas (OPC) and is linked to a more favourable prognosis compared to HPV-negative OPC. The mechanisms underlying this effect on prognosis are not fully understood, but interactions with the tumour microenvironment may be pivotal. Here, we investigated the role of the tumour microenvironment in HPV-positive compared to HPV-negative cancer using 2D and 3D modelling of OPC interactions with stromal fibroblasts. HPV-negative, but not HPV-positive, OPC-derived cell lines induced a rapid fibroblast secretory response that supported 2D cancer cell migration and invasion in vitro. Array profiling of this HPV-negative induced fibroblast secretome identified hepatocyte growth factor (HGF) as the principal secreted factor that promoted cancer cell migration. The interaction between HPV-negative cell lines and fibroblasts in 2D was prevented using c-Met (HGF receptor) inhibitors, which further restricted both HPV-negative and positive cell invasion in 3D co-culture models. Furthermore, we discovered a synergistic relationship between HGF and IL-6 in the support of migration that relates JAK activation to HGF responsiveness in HPV-negative lines. In summary, our data show significant differences in the interactions between HPV-positive and HPV-negative OPC cells and stromal fibroblasts. In addition, we, provide in vitro evidence to support the clinical application of c-MET inhibitors in the control of early HPV-negative OPC.

Long-term results of arthroscopically assisted anatomical single-bundle anterior cruciate ligament reconstruction using patellar tendon autograft: are there any predictors for the development of osteoarthritis?

PURPOSE: The primary purpose of our study was to analyse the long-term outcome of patients treated for anterior cruciate ligament (ACL) tears by anatomical single-bundle ACL reconstruction with patellar tendon autograft. The secondary purpose was to identify predictive factors for good outcome and occurrence of osteoarthritis. METHODS: Sixty-three patients (m:f = 54:9; mean age at surgery, 27 ± 7 years) treated by ACL reconstruction were evaluated with a mean follow-up of 16 ± 1 years using IKDC2000, the SF36, Lysholm and Tegner score, Knee Society score, visual analogue scale for pain and satisfaction and KOOS. The femoral tunnel position was evaluated according to Sommer. It was also assessed in percentage of the Blumensaat line and the tibial tunnel position in percentage of the total anterior-posterior plateau length. The extent of osteoarthritis was graded according to the Kellgren-Lawrence score. RESULTS: The total IKDC2000 was normal in 20 (32 %), nearly normal in 29 (46 %), abnormal in 12 (19 %) and severely abnormal in 3 (5 %) of patients. The mean total SF-36 was 89 ± 13, the Lysholm score 95 ± 12, the Knee Society score 191 ± 16 and the total KOOS 84 ± 19. The Tegner score decreased from pre-injury 7(4-10) to 6 (2-10) at follow-up. The Kellgren-Lawrence score was normal in 17 (27 %), suspected osteoarthritis in 25 (40 %), minimal osteoarthritis in 5 (8 %), moderate osteoarthritis in 9 (14 %) and severe osteoarthritis in 3 patients (5 %). The femoral tunnel was in zone A in 43 patients (68 %), in zone B in 16 (25 %) and in zone C in 4 patients (7 %). The femoral tunnel position in percentage of the Blumensaat line was 49 ± 3 (range, 44-57), and the tibial tunnel position in percentage of the total anterior-posterior plateau length was 32 ± 6 (range, 21-46). Patients with meniscal lesion at the time of ACL tear showed significantly less favourable outcomes than those without. CONCLUSIONS: Patients treated by the proposed ACL reconstruction technique showed on average good to excellent long-term results. A meniscal lesion at the time of ACL tear was highly predictive for less favourable outcome.