Thermoresponsive, redox-polymerized cellulosic hydrogels undergo in situ gelation and restore intervertebral disc biomechanics post discectomy.

Back and neck pain are commonly associated with intervertebral disc (IVD) degeneration. Structural augmentation of diseased nucleus pulposus (NP) tissue with biomaterials could restore degeneration-related IVD height loss and degraded biomechanical behaviors; however, effective NP replacement biomaterials are not commercially available. This study developed a novel, crosslinked, dual-polymer network (DPN) hydrogel comprised of methacrylated carboxymethylcellulose (CMC) and methylcellulose (MC), and used in vitro, in situ and in vivo testing to assess its efficacy as an injectable, in situ gelling, biocompatible material that matches native NP properties and restores IVD biomechanical behaviors. Thermogelling MC was required to enable consistent and timely gelation of CMC in situ within whole IVDs. The CMC-MC hydrogel was tuned to match compressive and swelling NP tissue properties. When injected into whole IVDs after discectomy injury, CMC-MC restored IVD height and compressive biomechanical behaviors, including range of motion and neutral zone stiffness, to intact levels. Subcutaneous implantation of the hydrogels in rats further demonstrated good biocompatibility of CMC-MC with a relatively thin fibrous capsule, similar to comparable biomaterials. In conclusion, CMC-MC is an injectable, tunable and biocompatible hydrogel with strong potential to be used as an NP replacement biomaterial since it can gel in situ, match NP properties, and restore IVD height and biomechanical function. Future investigations will evaluate herniation risk under severe loading conditions and assess long-term in vivo performance.

Anaesthetic hypersensitivity reactions in France between 2011 and 2012: the 10th GERAP epidemiologic survey.

BACKGROUND: Immediate hypersensitivity reactions during anaesthesia are rare but potentially life-threatening. The epidemiology changes with time and evolving professional practice, and hence needs to be monitored. Our objective was to follow this epidemiology. METHODS: This was a retrospective, observational study in French hospital clinics, conducted by GERAP members (Groupe d'Étude des Réactions Anaphylactoïdes Périopératoires). Consecutive patients seen in allergo-anaesthesia outpatient clinics, who had experienced a hypersensitivity reaction during anaesthesia between 1 January 2011 and 31 December 2012, were included. Demographic data, allergy history, drugs received before the reaction, symptoms of the reaction, results of blood samples (histamine, tryptase, IgE-specific assays), and results of the allergy assessment were recorded. RESULTS: The most common causes of allergic reactions were (Neuromuscular Blocking Agents) NMBAs (N = 302; 60.6%), antibiotics (N = 91, 18.2%, Cephalosporin N = 49, 10%) and dyes (N = 27; 5.4%). Latex as an allergic agent was involved in 26 cases (5.2%), hypnotics in 11 cases (2.2%) and opioids in seven cases (1.4%). Of the NMBAs, Rocuronium had the highest proportion of reactions (13.8 reactions/100,000 vials sold) followed by Suxamethonium (13.3/100,000 vials sold). Cisatracurium had the lowest proportion of reactions (0.4/100,000 vials sold). Patients were sensitized to two or more NMBAs in 48.9% of cases and without testing, cross-sensitivity cannot be predicted. CONCLUSIONS: When compared with the previous GERAP studies, NMBAs are still the most frequently triggering allergens, with marked differences between individual NMBAs, but they are now followed by antibiotics (of which greater than 50% were cephalosporins) and dyes. Anaesthetists must be aware of the differences between drugs and of the pattern of emerging allergens. For the future of safe anaesthesia, allergy assessment is essential.

HPV immunisation and cervical screening--confirmation of changed performance of cytology as a screening test in immunised women: a retrospective population-based cohort study.

BACKGROUND: To document the effect of bivalent HPV immunisation on cervical cytology as a screening test and assess the implications of any change, using a retrospective analysis of routinely collected data from the Scottish Cervical Screening Programme (SCSP). METHODS: Data were extracted from the Scottish Cervical Call Recall System (SCCRS), the Scottish Population Register and the Scottish Index of Multiple Deprivation. A total of 95 876 cytology records with 2226 linked histology records from women born between 1 January 1988 and 30 September 1993 were assessed. Women born in or after 1990 were eligible for the national catch-up programme of HPV immunisation. The performance of cervical cytology as a screening test was evaluated using the key performance indicators used routinely in the English and Scottish Cervical Screening Programmes (NHSCSP and SCSP), and related to vaccination status. RESULTS: Significant reductions in positive predictive value (16%) and abnormal predictive value (63%) for CIN2+ and the mean colposcopy score (18%) were observed. A significant increase (38%) in the number of women who had to be referred to colposcopy to detect one case of CIN2+ was shown. The negative predictive value of negative- or low-grade cytology for CIN2+ increased significantly (12%). Sensitivity and specificity, as used by the UK cervical screening programmes, were maintained. CONCLUSIONS: The lower incidence of disease in vaccinated women alters the key performance indicators of cervical cytology used to monitor the quality of the screening programme. These findings have implications for screening, colposcopy referral criteria, colposcopy practice and histology reporting.

HPV immunisation and increased uptake of cervical screening in Scottish women; observational study of routinely collected national data.

BACKGROUND: To measure the uptake of first invitation to cervical screening by vaccine status in a population-based cohort offered HPV immunisation in a national catch-up campaign. METHODS: A retrospective observational study of routinely collected data from the Scottish Cervical Screening Programme. Data were extracted and linked from the Scottish Cervical Call Recall System, the Scottish Population Register and the Scottish Index of Multiple Deprivation. Records from 201 023 women born between 1 January 1988 and 30 September 1993 were assessed. Women born in or after 1990 were eligible for the national catch-up programme of HPV immunisation. Attendance for screening was within 12 months of the first invitation at age 20 years. RESULTS: There was a significant decline in overall attendance from the 1988 cohort to the 1993 cohort with the adjusted attendance ratio of the 1988 cohort being 1.49 times (95% CI 1.46-1.52) that of the 1993 cohort. Immunisation compensated for this decrease in uptake with unvaccinated individuals having a reduced ratio of attendance compared with those fully vaccinated (RR=0.65, 95% CI 0.64-0.65). Not taking up the opportunity for HPV immunisation was associated with an attendance for screening below the trend line for all women before the availability of HPV immunisation. CONCLUSIONS: HPV immunisation is not associated with the reduced attendance for screening that had been feared. Immunised women in the catch-up cohorts appear to be more motivated to attend than unimmunised women, but this may be a result of a greater awareness of health issues. These results, while reassuring, may not be reproduced in routinely immunised women. Continued monitoring of attendance for the first smear and subsequent routine smears is needed.

Reduction of low- and high-grade cervical abnormalities associated with high uptake of the HPV bivalent vaccine in Scotland.

BACKGROUND: In Scotland, a national HPV immunisation programme began in 2008 for 12- to 13-year olds, with a catch-up campaign from 2008 to 2011 for those under the age of 18. To monitor the impact of HPV immunisation on cervical disease at the population level, a programme of national surveillance was established. METHODS: We analysed colposcopy data from a cohort of women born between 1988 and 1992 who entered the Scottish Cervical Screening Programme (SCSP) and were aged 20-21 in 2008-2012. RESULTS: By linking datasets from the SCSP and colposcopy services, we observed a significant reduction in diagnoses of cervical intraepithelial neoplasia 1 (CIN 1; RR 0.71, 95% CI 0.58 to 0.87; P=0.0008), CIN 2 (RR 0.5, 95% CI 0.4 to 0.63; P<0.0001) and CIN 3 (RR 0.45, 95% CI 0.35 to 0.58; P<0.0001) for women who received three doses of vaccine compared with unvaccinated women. CONCLUSIONS: To our knowledge, this is one of the first studies to show a reduction of low- and high-grade CIN associated with high uptake of the HPV bivalent vaccine at the population level. These data are very encouraging for countries that have achieved high HPV vaccine uptake.

Influence of HPV type on prognosis in patients diagnosed with invasive cervical cancer.

While much is known about the influence of HPV type on the progression of pre-invasive cervical lesions, the impact of HPV type on cervical cancer prognosis is less evidenced. Thus, we assessed the impact of HPV type on the survival of women diagnosed with cervical cancer. A total of 370 cases of cervical cancer were assessed. Univariate analysis is presented using Kaplan-Meier survival curves and log-rank statistics and multivariable Cox proportional hazard models were generated using age group, socio-economic deprivation, FIGO stage, differentiation and HPV type. HPV grouping was considered in a number of ways with particular reference to the presence or absence of HPV 16 and/or 18. In the univariate analysis, FIGO, age at diagnosis and treatment were associated with poorer survival (p < 0.0001) as was absence of HPV 16 and/or 18 (p = 0.0460). The 25% mortality time in the non-HPV 16/18 vs. HPV16/18 positive group was 615 days and 1,307 days respectively. An unadjusted Cox PH model based HPV16/18 vs. no HPV 16/18 resulted in a hazard ratio of 0.669 (0.450, 0.995). Adjusting for deprivation, FIGO and age group resulted in a hazard ratio of 0.609 (0.395, 0.941) p = 0.025. These data indicate that cancers associated with HPV 16 and/or 18 do not confer worse survival compared to cancers associated with other types, and may indicate improved survival. Consequently, although HPV vaccine is likely to reduce the incidence of cervical cancer it may not indirectly improve cervical cancer survival by reducing the burden of those cancers caused by HPV16/18.

Prospective parallel randomized trial of the MultiCyte™ ThinPrep(®) imaging system: the Scottish experience.

BACKGROUND: Computer-assisted screening of cervical liquid-based cytology (LBC) preparations using the ThinPrep® Imaging System (TIS) has shown improved qualitative and quantitative gains. The use of Multicyte™ has not been described in a well-established national screening programme with a low incidence of high-grade dyskaryosis. OBJECTIVES: To assess the impact of computer-assisted screening within the Scottish Cervical Screening Programme (SCSP). METHODS: Two groups of three laboratories, each sharing a ThinPrep® Imager, screened 79 366 slides randomized to test and 90 551 to control arms by laboratory accession. Screeners were not blinded. Standard laboratory reporting profiles of the SCSP, sensitivity, specificity and false-negative rates of all grades of LBC abnormalities with respect to final cytology reports, predictive value for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) on histology; and screening rates were compared for both arms. RESULTS: Inadequate and negative reporting rates were significantly lower and low-grade reporting rates significantly higher in the imager arm. Imager-assisted screening showed significantly better specificity than manual screening with respect to the final cytology result. There was no evidence of a significant difference in the detection of CIN2+ or CIN3 +. Positive, abnormal and total predictive values (high-grade, low-grade and all abnormal cytology found to be CIN2+, respectively) were similar in both arms. Productivity was significantly higher in the imager arm. CONCLUSION: Computer-assisted screening in a well established screening programme showed significantly improved productivity without loss of quality. These findings should inform future policy for cervical screening programmes.

Distribution of HPV types associated with cervical cancers in Scotland and implications for the impact of HPV vaccines.

BACKGROUND/METHODS: This study evaluated human papillomavirus (HPV) type prevalence in 370 Scottish invasive cervical cancers (ICCs) using HPV genotyping and HPV mRNA detection. RESULTS: HPV 16 and/or 18 was detected in 72% of cancers overall and in 82% of HPV-positive cancers. HPV 45 and 16 were the most frequently transcribed types. CONCLUSION: A significant reduction in ICC in Scotland should be achieved through the HPV immunisation programme.

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells.

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.

Photo-crosslinked alginate hydrogels support enhanced matrix accumulation by nucleus pulposus cells in vivo.

OBJECTIVE: Intervertebral disc (IVD) degeneration is a major health concern in the United States. Replacement of the nucleus pulposus (NP) with injectable biomaterials represents a potential treatment strategy for IVD degeneration. The objective of this study was to characterize the extracellular matrix (ECM) assembly and functional properties of NP cell-encapsulated, photo-crosslinked alginate hydrogels in comparison to ionically crosslinked alginate constructs. METHODS: Methacrylated alginate was synthesized by esterification of hydroxyl groups with methacrylic anhydride. Bovine NP cells were encapsulated in alginate hydrogels by ionic crosslinking using CaCl(2) or through photo-crosslinking upon exposure to long-wave UV light in the presence of a photoinitiator. The hydrogels were evaluated in vitro by gross and histological analysis and in vivo using a murine subcutaneous pouch model. In vivo samples were analyzed for gene expression, ECM localization and accumulation, and equilibrium mechanical properties. RESULTS: Ionically crosslinked hydrogels exhibited inferior proteoglycan accumulation in vitro and were unable to maintain structural integrity in vivo. In further studies, photo-crosslinked alginate hydrogels were implanted for up to 8 weeks to examine NP tissue formation. Photo-crosslinked hydrogels displayed temporal increases in gene expression and assembly of type II collagen and proteoglycans. Additionally, hydrogels remained intact over the duration of the study and the equilibrium Young's modulus increased from 1.24+/-0.09 kPa to 4.31+/-1.39 kPa, indicating the formation of functional matrix with properties comparable to those of the native NP. CONCLUSIONS: These findings support the use of photo-crosslinked alginate hydrogels as biomaterial scaffolds for NP replacement.

A comparison of the efficacy of two ivermectin formulations against larval and adult Ascaris suum and Oesophagostomum dentatum in experimentally infected pigs.

A study was conducted to evaluate and compare the efficacy of two injectable formulations of ivermectin (IVM-1 and IVM-2) at a dose rate of 0.3 mg/kg bodyweight versus placebo in the treatment and control of larval and adult stages of Ascaris suum and Oesophagostomum spp. in experimentally infected pigs. Seventy helminth free pigs were allocated on a liveweight basis to 7 groups each comprising 10 pigs (A-G). Group A served as an untreated control group. Groups B and C were used to investigate the efficacy of both formulations against adult stages of A. suum and Oesophagostomum spp., Groups D and E for efficacy against larval stages of A. suum and Groups F and G for efficacy against larval stages of Oesophagostomum spp. Pigs of groups A, B, C, D and E were infected on Day-0 with 1000 infective A. suum eggs each. Infective larvae of Oesophagostomum spp. (10,000/pig) were given on Day-0 to pigs of Groups F and G and on Day-21 to pigs of Groups A, B and C. Treatment was given to pigs of Group A (saline as placebo) on Day-7 and -28, IVM-1 to pigs of Group F on Day-7, pigs of Group D on Day-14 and pigs of Group B on Day-49. IVM-2 was given to pigs of Group G on Day-7, Group E on Day-28 and Group C on Day-49. Pigs of Groups F and G were sacrificed on Day-28, pigs of Groups A, D and E on Day-49 and pigs of Groups B and C on Day-56. Post mortem worm counts showed the following efficacies: (IVM-1) against larval A. suum 100%, against adult A. suum 94.4%, against larval Oesophagostomum spp. 52.0% and against adult Oesophagostomum spp. 83.0%. (IVM-2) against larval A. suum 100%, against adult A. suum 90.3%, against larval Oesophagostomum spp. 94.0% and against adult Oesophagostomum spp. 94.7%.

The effect of nucleus pulposus crosslinking and glycosaminoglycan degradation on disc mechanical function.

Altered mechanical loading, secondary to biochemical changes in the nucleus pulposus, is a potential mechanism in disc degeneration. An understanding of the role of this altered mechanical loading is only possible by separating the mechanical and biological effects of early nucleus pulposus changes. The objective of this study was to quantify the mechanical effect of decreased glycosaminoglycans (GAG) and increased crosslinking in the nucleus pulposus using in vitro rat lumbar discs. Following initial mechanical testing the discs were injected according to the four treatment groups: PBS control, chondroitinase-ABC (ChABC) for GAG degradation, genipin (Gen) for crosslinking, or a combination of chondroitinase and genipin (ChABC+Gen). After treatment the discs were again mechanically tested, followed by histology or biochemistry. Neutral zone mechanical properties were changed by approximately 20% for PBS, ChABC, and ChABC+Gen treatments (significant only for PBS in a paired comparison). These trends were reversed with genipin crosslinking alone. With ChABC treatment the effective compressive modulus increased and the GAG content decreased; with the combination of ChABC+Gen the mechanics and GAG content were unchanged. Degradation of nucleus pulposus GAG alters disc axial mechanics, potentially contributing to the degenerative cascade. Crosslinking is unlikely to contribute to degeneration, but may be a potential avenue of treatment.

Modulation of proteoglycan and collagen profiles in human dermal fibroblasts by high density micromass culture and treatment with lactic acid suggests change to a chondrogenic phenotype.

Cartilage formation during embryonic development and in fracture healing in adult animals involves chondrogenic differentiation of mesenchymal precursors. Here we describe an in vitro model whereby human dermal fibroblasts, considered to be restricted to a fibroblast lineage, are apparently redirected toward a chondrogenic phenotype by high density micromass culture in the presence of lactic acid. Micromass cultures treated with 40 mM lactate exhibited increased levels of Alcian blue staining and sulfate incorporation, indicative of elevated sulfated glycosaminoglycan synthesis. Northern analysis revealed an up-regulation of chondroitin sulfate proteoglycan 1 (aggrecan) and transforming growth factor-beta 1 mRNA and a decrease in type I collagen expression. Type II collagen was detected by reverse transcription-PCR only in experimental cultures. Although the observed changes in biosynthesis and gene expression were consistent with differentiating chondrocytes, the cells displayed an elongated, fibroblast-like morphology. These findings suggest that dermal fibroblasts may be committed to differentiate along a chondrogenic pathway by in vitro culture under specific forcing conditions.

Detection of herpes viruses in clinical samples using real-time PCR.

Herpes viruses including cytomegalovirus, varicella zoster and herpes simplex are an important cause of morbidity and mortality, especially in immunocompromised patients. Real-time PCR assays were developed with the aim of introducing a rapid and sensitive test to replace culture, and as a surveillance system for high-risk patients. The assays were optimised using cell culture derived material, and the sensitivity ascertained using cloned product before applying to extracted and non-extracted clinical samples. The sensitivity was between 1--100 virus copies with increased sensitivity to detect less than 10 copies possible when an initial round of amplification was carried out using external primers. Results were available within four hours of receipt compared with a median of 4.4 days for culture and immunofluorescence. Real-time PCR was found to be a sensitive and rapid method of detecting these viruses and will be a valuable tool for the surveillance of immunosuppressed patients.

Detection of T. gondii in tissues of sheep and cattle following oral infection.

It has been reported in the literature that cattle are more resistant to toxoplasmosis than sheep. Congenital disease due to T. gondii infection is rarely reported in cattle whereas the parasite is a major cause of abortion and neonatal mortality in sheep. It is believed that sheep remain chronically infected for life. Undercooked meat from infected sheep is an important source of infection for man. In contrast cattle are thought to harbour fewer parasite tissue cysts which may not persist for the lifetime of the host. Therefore, cattle are believed to pose less of a risk for human infection. In this study we examined the presence of T. gondii within a range of tissues in sheep and cattle at 6 weeks and 6 months following oral infection with 10(3) or 10(5) sporulated oocysts of T. gondii. The presence of parasite was determined by bioassay in mice and using polymerase chain reaction (PCR). The results from this study show that T. gondii was more frequently and consistently detected in sheep, in particular within brain and heart tissues, whereas parasites were not detected in the samples of tissues taken from cattle. T. gondii was more frequently detected in sheep given the higher dose of T. gondii. Examination of tissues at either 6 weeks or 6 months after infection did not appear to affect the distribution of T. gondii. The polymerase chain reaction has more specificity and sensitivity when detecting the presence of T. gondii in large animals than histological detection.

Validation of computer-assisted manufacture of titanium plates for cranioplasty.

We have constructed 300 titanium cranioplasty plates, over 150 cases using a computerised technique, the remainder by external impression. The clinical follow-up of these cases over 8 years has shown consistently good results that justify our simple low-cost method of manufacturing these plates. Both techniques require the provision of a model on which to construct the plate. In the traditional technique, an approximate model is derived from the resected bone or a direct impression of the defect over the patient's scalp. Using the computerised technique, a more accurate model of the defect and the surrounding bone is milled in polyurethane foam from cross-sectional computerised tomographic (CT) scans. Sheet titanium is pressed to shape from a design outlined on a counterdie. The subsequent stages of the plate construction are then the same for both methods. This study describes the stages of the model manufacture, the validation of its accuracy and the plate construction that follows. Use of the computerised method has resulted in a reduction of errors, enabling the manufacture of a smaller plate than was possible previously. It has also enabled design changes through the achievement of greater accuracy in fit.

Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures.

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.

A prospective study of computer-aided design and manufacture of titanium plate for cranioplasty and its clinical outcome.

The use of computerized three dimensional imaging and automated milling of models to produce accurate titanium plates for the reconstruction of craniofacial defects is described. A total of 148 patients have had extensive calvarial defects repaired using this (computer aided design and manufacture) technique developed in our unit. Of these, 141 were repaired secondarily (delayed cranioplasty), whilst seven were repaired immediately following craniectomy (single stage cranioplasty). All cases were assessed for accuracy of fit, restoration of natural skull contour and aesthetics. Seventy-two patients were reviewed after 1 year to determine the effect on adverse preoperative symptoms. Of the plates 97% had an excellent or good intraoperative fit. The modal insertion time was only 15 minutes. Postoperatively 98% resulted in the restoration of natural skull shape and symmetry. After 1 year, 82% of patients had complete resolution or diminution in severity of the adverse symptoms. A staphylococcus infection necessitated the temporary removal of one plate.