Jessica Louise Roberts

Evaluation of the physical and biological properties of hyaluronan and hyaluronan fragments

Hyaluronan (HA) has been extensively used for various medical applications, including osteoarthritis, tissue augmentation and ocular surgery. More recently, it has been investigated for use in polymer therapeutics as a carrier for drugs and biologically active proteins, thanks to its biodegradability, biocompatibility and inherent biological properties. Such biological functions are strongly dependent on HAs chain length, yet the molecular weight of HAs used in polymer conjugates varies widely and is inconsistent with its intended application. Therefore, this study aimed to determine the ideal chain length of HA to be used in polymer conjugates for enhanced tissue repair. HA fragments (M(w) 45,000-900,000g/mol) were prepared by acid hydrolysis of rooster comb HA and their physicochemical and biological properties were characterized. Such HA fragments had a highly extended, almost rod-like solution conformation and demonstrated chain length- and concentration-dependent viscosity, while exposure to HAase caused a rapid reduction in HA viscosity, which was most significant for the native HA. Initial HA hydrolysis rate by HAase varied strongly with HA chain length and was dependent on the formation of a stable enzyme-substrate complex. When normal human dermal fibroblasts were exposed to the different HA fragments for 72h, only native (900,000g/mol) HA reduced proliferation at 1000μg/mL. Conversely, only the smallest HA fragment (70,000g/mol) reduced the proliferation of chronic wound fibroblasts, at 1000μg/mL. The 70,000g/mol HA fragment also promoted the greatest cell attachment. These observations demonstrate that low molecular weight (70,000-120,000g/mol) HA fragments would be best suited for the delivery of proteins and peptides with applications in chronic wound healing and paves the way for the rationalized development of novel HA conjugates.

An in vitro study of alginate oligomer therapies on oral biofilms.

OBJECTIVES The in vitro effect of a novel, oligosaccharide nanomedicine OligoG against oral pathogen-related biofilms, both alone and in the presence of the conventional anti-bacterial agent triclosan, was evaluated. METHODS The effect of OligoG±triclosan was assessed against established Streptococcus mutans and Porphyromonas gingivalis biofilms by bacterial counts and image analysis using LIVE/DEAD(®) staining and atomic force microscopy (AFM). The effect of triclosan and OligoG surface pre-treatments on bacterial attachment to titanium and polymethylmethacrylate was also studied. RESULTS OligoG potentiated the antimicrobial effect of triclosan, particularly when used in combination at 0.3% against S. mutans grown in artificial saliva. OligoG was less effective against established P. gingivalis biofilms. However, attachment of P. gingivalis, to titanium in particular, was significantly reduced after surface pre-treatment with OligoG and triclosan at 0.01% when compared to controls. Light microscopy and AFM showed that OligoG was biocidal to P. gingivalis, but not S. mutans. CONCLUSIONS OligoG and triclosan when used in combination produced an enhanced antimicrobial effect against two important oral pathogens and reduced bacterial attachment to dental materials such as titanium, even at reduced triclosan concentrations. Whilst the use of triclosan against oral bacteria has been widely documented, its synergistic use with OligoG described here, has not previously been reported. The use of lower concentrations of triclosan, if used in combination therapy with OligoG, could have environmental benefits. CLINICAL IMPORTANCE The potentiation of antimicrobial agents by naturally occurring oligomers such as OligoG may represent a novel, safe adjunct to conventional oral hygiene and periodontal therapy. The ability of OligoG to inhibit the growth and impair bacterial adherence highlights its potential in the management of peri-implantitis.

A Novel Ex vivo Culture Model for Inflammatory Bone Destruction

Pathological alterations in the balance of bone metabolism are central to the progression of inflammatory bone diseases such as periodontal disease. We have developed and characterized a novel ex vivo murine mandible model of inflammatory bone destruction. Slices of mandible were cultured for 14 days in the presence or absence of P. gingivalis lipopolysaccharide (LPS) or pro-inflammatory cytokines. Following culture, cell viability and tissue histomorphometry were assessed with quantification of matrix proteins, resident osteoclasts, ligament cells, monocytes, macrophages, and neutrophils. In the absence of inflammatory factors, culture viability, osteoclasts, and matrix components were maintained. LPS or TNFα stimulation demonstrated an increase in cellular proliferation, monocyte cells, osteoclast differentiation, and matrix degradation. Pathophysiological bone metabolism can be induced via exposure to LPS and direct influence of TNFα within the model despite the absence of systemic circulation, providing a model for inflammatory bone destruction and investigation of the effects of novel therapeutics.

Fracture in the Elderly Multidisciplinary Rehabilitation (FEMuR): study protocol for a phase II randomised feasibility study of a multidisciplinary rehabilitation package following hip fracture [ISRCTN22464643]

BackgroundProximal femoral fracture is a common, major health problem in old age resulting in loss of functional independence and a high-cost burden on society, with estimated health and social care costs of £2.3 billion per year in the UK. Rehabilitation has the potential to maximise functional recovery and maintain independent living, but evidence of effectiveness is lacking. Usual rehabilitation care is delivered by a multi-disciplinary team in the hospital and in the community. An ‘enhanced rehabilitation’ intervention has been developed consisting of a workbook, goal-setting diary and extra therapy sessions, designed to improve self-efficacy and increase the amount and quality of the practice of physical exercise and activities of daily living.Methods/designThis paper describes the design of a phase II study comprising an anonymous cohort of all proximal femoral fracture patients admitted to the three acute hospitals in Betsi Cadwaladr University Health Board over a 6-month period with a randomised feasibility study comparing the enhanced rehabilitation intervention with usual care. These will assess the feasibility of a future definitive randomised controlled trial and concurrent economic evaluation in terms of recruitment, retention, outcome measure completion, compliance with the intervention and fidelity of delivery, health service use data, willingness to be randomised and effect size for a future sample size calculation. Focus groups will provide qualitative data to contribute to the assessment of the acceptability of the intervention amongst patients, carers and rehabilitation professionals and the feasibility of delivering the planned intervention. The primary outcome measure is function assessed by the Barthel Index. Secondary outcomes measure the ability to perform activities of daily living, anxiety and depression, potential mediators of outcomes such as hip pain, self-efficacy and fear of falling, health utility, health service use, objectively assessed physical function and adverse events. Participants’ preference for rehabilitation services will be assessed in a discrete choice experiment.DiscussionPhase II studies are an opportunity to not only assess the feasibility of trial methods but also to compare different methods of outcome measurement and novel methods of obtaining health service use data from routinely collected patient information.Trial registrationCurrent Controlled Trials ISRCTN22464643, UKCRN16677.

Fracture in the Elderly Multidisciplinary Rehabilitation (FEMuR): A phase II randomised feasibility study of a multidisciplinary rehabilitation package following hip fracture

Objective To conduct a rigorous feasibility study for a future definitive parallel-group randomised controlled trial (RCT) and economic evaluation of an enhanced rehabilitation package for hip fracture. Setting Recruitment from 3 acute hospitals in North Wales. Intervention delivery in the community. Participants Older adults (aged ≥65) who received surgical treatment for hip fracture, lived independently prior to fracture, had mental capacity (assessed by clinical team) and received rehabilitation in the North Wales area. Intervention Remote randomisation to usual care (control) or usual care+enhanced rehabilitation package (intervention), including six additional home-based physiotherapy sessions delivered by a physiotherapist or technical instructor, novel information workbook and goal-setting diary. Primary and secondary outcome measures Primary: Barthel Activities of Daily Living (BADL). Secondary measures included Nottingham Extended Activities of Daily Living scale (NEADL), EQ-5D, ICECAP capability, a suite of self-efficacy, psychosocial and service-use measures and costs. Outcome measures were assessed at baseline and 3-month follow-up by blinded researchers. Results 62 participants were recruited, 61 randomised (control 32; intervention 29) and 49 (79%) completed 3-month follow-up. Minimal differences occurred between the 2 groups for most outcomes, including BADL (adjusted mean difference 0.5). The intervention group showed a medium-sized improvement in the NEADL relative to the control group, with an adjusted mean difference between groups of 3.0 (Cohens d 0.63), and a trend for greater improvement in self-efficacy and mental health, but with small effect sizes. The mean cost of delivering the intervention was £231 per patient. There was a small relative improvement in quality-adjusted life year in the intervention group. No serious adverse events relating to the intervention were reported. Conclusions The trial methods were feasible in terms of eligibility, recruitment and retention. The effectiveness and cost-effectiveness of the rehabilitation package should be tested in a phase III RCT. Trial registration number ISRCTN22464643; Results.

In Vitro Evaluation of the Interaction of Dextrin–Colistin Conjugates with Bacterial Lipopolysaccharide

Dextrin-colistin conjugates have been developed with the aim of achieving reduced clinical toxicity associated with colistin, also known as polymyxin E, and improved targeting to sites of bacterial infection. This study investigated the in vitro ability of such dextrin-colistin conjugates to bind and modulate bacterial lipopolysaccharide (LPS), and how this binding affects its biological activity. These results showed that colistin and amylase-activated dextrin-colistin conjugate to a lesser extent induced aggregation of LPS to form a stacked bilayer structure with characteristic dimensions, although this did not cause any substantial change in its secondary structure. In biological studies, both colistin and dextrin-colistin conjugate effectively inhibited LPS-induced hemolysis and tumor necrosis factor α (TNFα) secretion in a concentration-dependent manner, but only dextrin-colistin conjugate showed no additive toxicity at higher concentrations. This study provides the first direct structural experimental evidence for the binding of dextrin-colistin conjugates and LPS and gives insight into the mode of action of dextrin-colistin conjugates.

Development of an evidence-based complex intervention for community rehabilitation of patients with hip fracture using realist review, survey and focus groups

Objectives To develop an evidence and theory-based complex intervention for improving outcomes in elderly patients following hip fracture. Design Complex-intervention development (Medical Research Council (MRC) framework phase I) using realist literature review, surveys and focus groups of patients and rehabilitation teams. Setting North Wales. Participants Surveys of therapy managers (n=13), community and hospital-based physiotherapists (n=129) and occupational therapists (n=68) throughout the UK. Focus groups with patients (n=13), their carers (n=4) and members of the multidisciplinary rehabilitation teams in North Wales (n=13). Results The realist review provided understanding of how rehabilitation interventions work in the real-world context and three programme theories were developed: improving patient engagement by tailoring the intervention to individual needs; reducing fear of falling and improving self-efficacy to exercise and perform activities of daily living; and coordination of rehabilitation delivery. The survey provided context about usual rehabilitation practice; focus groups provided data on the experience, acceptability and feasibility of rehabilitation interventions. An intervention to enhance usual rehabilitation was developed to target these theory areas comprising: a physical component consisting of six additional therapy sessions; and a psychological component consisting of a workbook to enhance self-efficacy and a patient-held goal-setting diary for self-monitoring. Conclusions A realist approach may have advantages in the development of evidence-based interventions and can be used in conjunction with other established methods to contribute to the development of potentially more effective interventions. A rehabilitation intervention was developed which can be tested in a future randomised controlled trial (MRC framework phases II and III). Trial registration number ISRCTN22464643, Pre-results.

Hip fracture in the elderly multidisciplinary rehabilitation (FEMuR) feasibility study: testing the use of routinely collected data for future health economic evaluations

BackgroundHealth economic evaluations rely on the accurate measurement of health service resource use in order to calculate costs. These are usually measured with patient completed questionnaires using instruments such as the Client Service Receipt Inventory (CSRI). These rely on participants’ recall and can be burdensome to complete. Health service activity data are routinely captured by electronic databases.The aim was to test methods for obtaining these data and compare with those data collected using the CSRI, within a feasibility study of an enhanced rehabilitation intervention following hip fracture (Fracture in the Elderly Multidisciplinary Rehabilitation: FEMuR).MethodsPrimary care activity including prescribing data was obtained from the Secure Anonymised Information Linkage (SAIL) Databank and secondary care activity (Emergency Department attendances, out-patient visits and in-patient days) directly from Betsi Cadwaladr University Health Board (BCUHB), North Wales, UK. These data were compared with patient responses from the CSRI using descriptive statistics and the intraclass correlation coefficient (ICC).ResultsIt was possible to compare health service resource use data for 49 out of 61 participants in the FEMuR study. For emergency department (ED) attendances, records matched in 23 (47%) cases, 21 (43%) over-reported on electronic records compared with CSRI and five participants (10%) under-reported, with an overall ICC of 0.42. For out-patient episodes, records matched in only six cases, 28 participants over-reported on electronic records compared with CSRI and 15 (12%) under-reported, with an overall ICC of only 0.27. For in-patient days, records matched exactly in only five cases (10%), but if an error margin of 7 days was allowed, then agreement rose to 39 (66%) cases, and the overall ICC for all data was 0.88.It was only possible to compare prescribing data for 12 participants. For prescribing data, the SAIL data reported 117 out of 118 items (99%) and the CSRI only 89 (79%) items.ConclusionsThe use of routinely collected data has the potential to improve the efficiency of trials and other studies. Although the methodology to make the data available has been demonstrated, the data obtained was incomplete and the validity of using this method remains to be demonstrated.Trial registrationTrial registration: ISRCTN22464643 Registered 21 July 2014.

An ex-vivo model to determine dental pulp responses to heat and light-curing of dental restorative materials

AIM Based on histological studies from the 1960s, it is recommended that dental pulp temperature increases should not exceed 5.5 °C. However, no contemporary reliable models exist to explore the effects of heat on living dental pulp. The aim of this project was to develop a clinically valid model for studying temperature increases caused by three commonly-used light curing units (LCUs). METHODS Temperature increases caused by LCUs at varying exposure times and via various thicknesses of dentine were recorded using traditional approaches (i.e. thermocouple device on a laboratory bench) and an ex-vivo tooth slice model. Histomorphometric and immunohistochemical (IL-1β, HSP70, caspase-3) analysis was performed of the tooth slice model following varying exposure and culture times. RESULTS Reduced dentine thickness and increased exposure time led to increases in temperature. Whilst the majority of temperature increases recorded using the traditional approach (53 of 60) were greater than the recommended 5.5 °C, 52 of the 60 reference points recorded using the ex-vivo tooth slice model resulted in temperature increases of less than 5.5 °C. Temperature increases of 5.5 °C or more that are prolonged for 40 s caused an immediate decrease in cell number. IL-1β was not detected in any samples, while HSP70 was detectable immediately after exposure to a temperature increase of 6 °C or more. Higher levels of HSP70 were detected after 24 h culture in tooth slices that experienced a temperature increase of 7.5 °C or more. Low levels of caspase-3 were detected in tooth slices exposed to temperature increase of 7.5 °C or more. CONCLUSION Experimental arrangements for assessing LCU performance that measure temperature increases using a thermocouple device on a laboratory bench should no longer be used. Future studies in this area should include replication of the clinical environment using greater sophistication, such as the use of an ex-vivo tooth slice model as described here. Temperature increases of 5.5 °C or more for 40 s caused an immediate decrease in cell number, which supports previous findings. However, complex interactions at an immunohistochemical level suggest that while temperature increases of 5 °C or less are ideal, there may be some cell damage between 5-7 °C which might not result in pulpal death. Further investigations are indicated.

Enterococcus faecalis Demonstrates Pathogenicity through Increased Attachment in an Ex Vivo Polymicrobial Pulpal Infection

ABSTRACT This study investigated the host response to a polymicrobial pulpal infection consisting of Streptococcus anginosus and Enterococcus faecalis, bacteria commonly implicated in dental abscesses and endodontic failure, using a validated ex vivo rat tooth model. Tooth slices were inoculated with planktonic cultures of S. anginosus or E. faecalis alone or in coculture at S. anginosus/E. faecalis ratios of 50:50 and 90:10. Attachment was semiquantified by measuring the area covered by fluorescently labeled bacteria. Host response was established by viable histological cell counts, and inflammatory response was measured using reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. A significant reduction in cell viability was observed for single and polymicrobial infections, with no significant differences between infection types (∼2,000 cells/mm2 for infected pulps compared to ∼4,000 cells/mm2 for uninfected pulps). E. faecalis demonstrated significantly higher levels of attachment (6.5%) than S. anginosus alone (2.3%) and mixed-species infections (3.4% for 50:50 and 2.3% for 90:10), with a remarkable affinity for the pulpal vasculature. Infections with E. faecalis demonstrated the greatest increase in tumor necrosis factor alpha (TNF-α) (47.1-fold for E. faecalis, 14.6-fold for S. anginosus, 60.1-fold for 50:50, and 25.0-fold for 90:10) and interleukin 1β (IL-1β) expression (54.8-fold for E. faecalis, 8.8-fold for S. anginosus, 54.5-fold for 50:50, and 39.9-fold for 90:10) compared to uninfected samples. Immunohistochemistry confirmed this, with the majority of inflammation localized to the pulpal vasculature and odontoblast regions. Interestingly, E. faecalis supernatant and heat-killed E. faecalis treatments were unable to induce the same inflammatory response, suggesting E. faecalis pathogenicity in pulpitis is linked to its greater ability to attach to the pulpal vasculature.