This double-blind, placebo-controlled comparative study was designed to investigate whether pranlukast dry syrup, a leukotriene receptor antagonist, has a protective effect against priming, controlled pollen exposure, and natural pollen exposure in children with Japanese cedar pollinosis.3) in an artificial exposure chamber (OHIO chamber).Thirty children aged 12–15 years with Japanese cedar pollinosis (positive skin test for Japanese cedar pollen), who had suffered from pollinosis for at least 2 years and developed severe nasal obstruction when exposed to Japanese cedar pollen, were enrolled in this study. They were randomly allocated to treatment with pranlukast or placebo orally after breakfast and dinner for 8 weeks during the Japanese cedar pollen season. Soon after the start of the pollen season, all subjects underwent a challenge by exposure for 3 h to Japanese cedar pollen .The effect of pranlukast was evaluated using self-rating of nasal symptoms by the subjects and measurement of eosinophil cationic protein in nasal discharge specimens.Scores for the symptoms of pollinosis were lower in the pranlukast group than in the placebo group during treatment in the priming state, as well as after controlled pollen exposure and natural pollen exposure. Pranlukast significantly improved the score for nasal obstruction, compared with placebo. A correlation was found between changes of the scores for symptoms of pollinosis and changes of the eosinophil cationic protein level.These results confirm a protective effect of pranlukast against both priming and challenge  with Japanese cedar pollen. The present findings suggested that pranlukast dry syrup may be useful for prophylaxis against pollinosis in children. It is caused by pollen dispersal in the early spring from Japanese cedar and cypress trees throughout Japan, which release a massive amount of pollen. In Japan, the prevalence of JC pollinosis increased by ∼10% over 10 years from 16.2% in 1998 to 26.5% in 20083. In 2008, the prevalence of JC pollinosis was 33.1–39.1% among adults in each decade from the 20s to the 50s, 31.4% in teenagers and 13.7% in children aged 5–10 years4. The prevalence of JC pollinosis in children appears to be much higher than that of allergic rhinitis, which was reported as 10% or less in children aged 6–7 and ∼15% in those aged 13–14 years6.Japanese cedar (JC) pollinosis is a form of seasonal allergic rhinitis peculiar to Japan that was first reported in 19647, and it is important to initiate treatment before or at the start of the pollen season to prevent the development of symptoms. Accordingly, the Japanese guideline for nasal allergy recommends initiation of prophylactic therapy before or early in the pollen season4.In Japan, the pollen season usually starts in early February and ends between mid-March and early April. JC pollinosis causes nasal and ocular symptoms that can significantly influence the quality-of-life (QoL)8, increase vascular permeability9, and promote the migration of inflammatory cells such as eosinophils and macrophages10–12. Thus, leukotrienes are important chemical mediators of allergic rhinitis, suggesting that usefulness of leukotriene receptor antagonists (LTRAs)13–15. Clinical studies have shown that LTRAs are as effective as antihistamines for pollinosis16–18. It has also been reported that initiating prophylactic LTRA therapy before or early in the pollen season can prevent symptoms of pollinosis19–22.Leukotrienes dilate vessels in the nasal mucosa23. It is also superior to placebo as prophylactic therapy for pollinosis22. Furthermore, pranlukast has an immediate effect against exposure to pollen in an artificial exposure chamber (OHIO chamber), improving the early and late phases of nasal obstruction24, and inhibits priming for pollinosis25. Moreover, pranlukast dry syrup (PLK-DS) is more effective than placebo for preventing nasal obstruction in children with pollinosis tested in an OHIO chamber27.The LTRA pranlukast is effective for perennial allergic rhinitis in adultsAccordingly, this study was performed to determine the efficacy of prophylactic therapy with PLK-DS vs placebo for JC pollinosis in children by investigating the inhibitory effect on priming for pollinosis and on the response to pollen exposure .This study enrolled 30 children with JC pollinosis who met the following inclusion criteria: aged from 10 years to younger than 15 years, class 2–6 when multiple antigen testing was performed to detect specific IgE antibodies for JC pollen in serum, a history of nasal symptoms with nasal obstruction following exposure to JC pollen in an OHIO chamber, development and exacerbation of nasal symptoms with nasal obstruction during the annual JC pollen season, and those determined to be eligible by an investigator or sub-investigator when clinical examination was done before the start of the study. The exclusion criteria were as follows: any lesion of the nasal or ocular mucosa, use of parenteral corticosteroids within 6 months before obtaining informed consent, nasal disease , systemic disease , a history of anaphylaxis, a history of hypersensitivity to any ingredient of the test drug, current immunotherapy , and any other reason that made patients ineligible for this study according to the investigator. We specified two persons who were responsible for patient enrollment (K.H. and S.M). It was also pre-determined that if they could not reach a consensus as to whether to include/exclude a child, an independent researcher  would be consulted to make the final decision.The subjects received a detailed explanation of this study and possible adverse reactions to the test drug, and their parents or guardians provided informed consent. The study was performed at Medical Corporation Shinanokai Samoncho Clinic according to the ethical guidelines in the protocol and the Declaration of Helsinki (2000) after receiving approval from the ethics committee of the clinic. This study was registered with the University Hospital Medical Information Network in Japan (registration No. UMIN000009840).This was a randomized, double-blind, placebo-controlled clinical trial. The investigational drugs included PLK-DS (10% pranlukast) and placebo, which were confirmed to be indistinguishable from each other. In late January 2013, 30 participants were selected based on the results of skin tests with JC pollen. For the skin test, a skin prick test was performed. When ‘the test site showed a wheal of at least 5 mm surrounded by at least 10 mm of erythema’, the judgment was positive. According to the schedule shown in 2 has been detected for at least 2 days by an automatic pollen measuring device (Durham pollen gathering device) at the Ministry of the Environment. In fact, a very low level of pollen dispersion occurs before the official start of the pollen season, and some patients develop symptoms before the starting date.‘The starting date of the pollen season’ is defined as the day when at least 1 pollen grain/cm3)28–30. Before the JC pollen season (January 24) and during the pollen season (March 3), eosinophil cationic protein (ECP) was measured in nasal discharge samples as an index of inflammation. Extraction of ECP from nasal discharge samples and measurement were done according to the manufacturer’s recommendations 27. Concomitant use of the following drugs was not permitted during treatment with the investigational drug: corticosteroids , antihistamines , anti-allergic drugs, leukotriene antagonists, anti-prostaglandin D2/thromboxane A2 drugs, vasoconstrictors, anticholinergics, and other drugs that could affect evaluation of efficacy. However, if a patient developed severe symptoms following the start of the pollen season, rescue medications could be used to treat the symptoms , provided that the total nasal symptoms score (Early in the JC pollen season (February 10), subjects were exposed to JC pollen for 3 h in an OHIO chamber , and changes from the mean score of Week 1 (including the first day of the pollen season and before controlled exposure in the OHIO chamber) were determined for each of the subsequent weeks. ECP levels were compared at two times, which were before the start of the JC pollen season and during the pollen season.In the nasal allergy diary, the severity of each symptom  was assigned a score from 0–4p ≤ 0.05 was accepted as the level of significance. Statistical analysis was performed using SAS version 8.02 .Data were analyzed with a two-sided U-test and the Wilcoxon test, and 3 in each week up to Week 8 (end of the study).The JC pollen season started on February 7 (7 days after commencing treatment) . In FebrOf the 30 patients enrolled, 25 completed this study and five were excluded due to insufficient data in the nasal allergy diary . Among tIn Week 2, subjects were exposed to JC pollen in the OHIO chamber. Subsequently, the sneezing score increased significantly in the placebo group, while there was no significant increase in the PLK-DS group. From Week 3 to Week 7, the sneezing score showed a significant increase in both groups .In Week 2 (OHIO chamber), the nasal discharge score increased significantly in the placebo group, while there was no significant increase in the PLK-DS group. In addition, the placebo group had a significantly higher nasal discharge score compared with the PLK-DS group. In Weeks 3 and 4 (before the peak of the pollen season), there was a significant increase of the score in the placebo group, but not in the PLK-DS group. Between Weeks 5 and 7 (peak pollen season), a significant increase of the score was observed in both groups .In the placebo group, a significant increase of the nasal obstruction score continued from Week 2 (OHIO chamber) to Week 7 of the JC pollen season. On the other hand, there was no significant increase of the score in the PLK-DS group from Week 2 to Week 8 (end of the study) .In the placebo group, the total nasal symptoms score was significantly increased between Week 2 (OHIO chamber) and Week 7 (peak pollen season). In the PLK-DS group, there was no significant increase between Week 2 and Week 4 (before the peak pollen season), although a significant increase of the score was observed during Weeks 5–7 (peak pollen season) .p = 0.042, Wilcoxon test). On the other hand, the PLK-DS group showed no significant increase of ECP in nasal discharge   to 12.97 ± 0.60 μg/L during the peak pollen season (March 3) (on test) .Rescue drugs were hardly used during the early pollen season (W1–4). The number of days when a dosage of 0.5 drops/day was exceeded was 0 days for Intal Nasal Solution 2% in both the placebo group and the pranlukast dry syrup group. For Intal Ophthalmic Solution 2%, a dosage of 0.5 drops/day was exceeded on 0 days in the placebo group and 1 day in the pranlukast dry syrup group. During the late pollen season (W5–8), there was a slight increase in use, with a dosage of 0.5 drops/day, for Intal Nasal Solution 2% being exceeded on 0 days in the placebo group and 1 day in the pranlukast dry syrup group. For Intal Ophthalmic Solution 2%, the dosage of 0.5 drops/day was exceeded on 2 days in the placebo group and 3 days in the pranlukast dry syrup group.The main findings of the present study were as follows: First, prophylactic administration of PLK-DS to children with JC pollinosis demonstrated an inhibitory effect on priming in the early part of the pollen season. Second, prophylaxis with PLK-DS reduced the symptoms caused by exposure to JC pollen in an OHIO chamber during the early part of the pollen season. In particular, PLK-DS prophylaxis reduced nasal discharge and nasal obstruction after controlled exposure to JC pollen in the OHIO chamber. Third, prophylaxis with PLK-DS inhibited an increase of ECP in nasal discharge specimens during the peak pollen season. These findings indicated that PLK-DS prophylaxis was effective for reducing symptoms of pollinosis and nasal inflammation in children with JC pollinosis.26. In that study, we measured ECP levels in nasal discharge specimens at the end of pollen exposure, and found that PLK-DS inhibited an increase of ECP27. Although we demonstrated that PLK-DS suppressed inflammation and pollinosis symptoms, that study was not performed during the pollen season and did not confirm the effect of the drug on natural exposure to pollen. Also, prophylactic therapy is important in children as well as in adults, hence we considered it important to determine the efficacy of PLK-DS prophylaxis in children with pollinosis.We previously reported that PLK-DS was effective in children who were exposed to pollen using the OHIO chamber32. It is thought that repeated exposure to low levels of antigens causing no symptoms leads to chronic inflammation of the nasal mucosa. In fact, repeated out-of-season exposure of patients with seasonal allergic rhinitis to 1/100 of the antigen dose causing symptoms has been shown to increase histamine and ECP levels in nasal discharge33. It was also reported that symptom returned to baseline by 6 weeks after the pollen season, but the levels of interleukin-1, leukotriene, and ECP in nasal discharge remained high34. Assuming that patients have minimal chronic inflammation, we consider that initiation of prophylactic therapy with anti-inflammatory agents either before or early in the pollen season is a logical method to reduce the deterioration of symptoms and QoL during the pollen season.The pathogenesis of allergic rhinitis is considered to involve minimal chronic inflammation20–22, the nasal symptoms scores  of the PLK-DS group were significantly lower than those of the placebo group. Since use of PLK-DS as prophylactic therapy in children with pollinosis delays the onset of nasal symptoms as it does in adults22 and, thus, delays the deterioration of QoL and the need for symptomatic medication, this drug appears to be appropriate for prophylaxis because of its anti-inflammatory effect.The present placebo-controlled study demonstrated that PLK-DS prophylaxis is useful for children with pollinosis. As has been reported in adultsA limitation of this study was the small sample size, which was probably the reason for the few significant differences between the PLK-DS and placebo groups. Because the maximum number of patients who could fit in the pollen exposure chamber used in this study was 15 and it was important to test all subjects under similar conditions during the pollen season, we limited the number of subjects for this study. In the future, a larger multi-center, placebo-controlled, double-blind study should be performed to confirm the usefulness of PLK-DS as prophylactic therapy for JC pollinosis in children. Another limitation was that the dropout rate was rather high. The five dropouts were due to insufficient entries in the nasal allergy diary. Patients were instructed to make daily entries in this diary before bedtime about the most serious symptoms of that day. The method of evaluation was to calculate the weekly mean score and determine the change from the mean score of the first week. If entries were missed, it could have been possible to calculate the mean value from the remaining data. However, the severity of pollinosis is highly dependent on the amount of pollen dispersed in the atmosphere, and this varies from day to day. We, therefore, considered it inappropriate to calculate the mean score when data were missing.In children with JC pollinosis, we demonstrated an inhibitory effect of PLK-DS on priming in the early pollen season, as well as inhibition of symptoms caused by controlled exposure to pollen and symptoms during the pollen season. PLK-DS was also shown to suppress an increase of ECP in nasal discharge, indicating an anti-inflammatory effect. The present findings suggested that pranlukast dry syrup may be useful for prophylaxis against pollinosis in children.


Adolescence is associated with high impulsivity and risk taking, making adolescent individuals more inclined to use drugs. Early drug use is correlated to increased risk for substance use disorders later in life but the neurobiological basis is unclear. The brain undergoes extensive development during adolescence and disturbances at this time are hypothesized to contribute to increased vulnerability. The transition from controlled to compulsive drug use and addiction involve long-lasting changes in neural networks including a shift from the nucleus accumbens, mediating acute reinforcing effects, to recruitment of the dorsal striatum and habit formation. This study aimed to test the hypothesis of increased dopamine release after a pharmacological challenge in adolescent rats. Potassium-evoked dopamine release and uptake was investigated using chronoamperometric dopamine recordings in combination with a challenge by amphetamine in early and late adolescent rats and in adult rats. In addition, the consequences of voluntary alcohol intake during adolescence on these effects were investigated. The data show a gradual increase of evoked dopamine release with age, supporting previous studies suggesting that the pool of releasable dopamine increases with age. In contrast, a gradual decrease in evoked release with age was seen in response to amphetamine, supporting a proportionally larger storage pool of dopamine in younger animals. Dopamine measures after voluntary alcohol intake resulted in lower release amplitudes in response to potassium-chloride, indicating that alcohol affects the releasable pool of dopamine and this may have implications for vulnerability to addiction and other psychiatric diagnoses involving dopamine in the dorsal striatum. Adolescence is associated with high impulsivity and risk-taking behavior, making adolescent individuals more inclined to use drugs Drugs of abuse commonly act on the reward system and increase extracellular levels of dopamine in the nucleus accumbens acutely after intake Animal models are of great importance to our understanding of these mechanisms and the age window identified as adolescence in rodents is between postnatal day (PND) 28 and 50 The second objective of this study was to investigate the effect of environmental influence by voluntary alcohol intake during adolescence. The rationale behind this was that previous studies show that environmental factors during the adolescent period, such as intraperitoneally administered alcohol, increase basal extracellular levels of dopamine All animal experiments were performed under a protocol approved by the Uppsala Animal Ethical Committee and followed the guidelines of the Swedish Legislation on Animal Experimentation  and the European Communities Council Directive (86/609/EEC).ad libitum. The cages contained wood-chip bedding and paper sheets  and were changed once a week by animal care personnel. The animal room was kept at constant temperature (22±1°C) and humidity (50±10%) on a regular 12 h light/dark cycle with lights on at 06∶00 am. All rooms had a masking background noise to minimize unexpected sounds that could disturb the animals.Pregnant Wistar rats  arrived at the animal facility at gestation day 16. The animals arrived in batches over several weeks in order to accommodate the timing of the chronoamperometric recordings. The dams were single housed in macrolon cages (59 cm×38 cm×20 cm) with pellet food  and tap water An overview of the experimental outline can be found in Inactin, Nafion 5% solution, dopamine hydrochloride, L-ascorbic acid, potassium chloride, sodium chloride, sodium phosphate, calcium chloride and d-amphetamine sulfate were obtained from Sigma-Aldrich, LLC . Kerr sticky wax was obtained from DAB LAB AB . Carbon fibre microelectrodes  were purchased from Quanteon, LLC , the reference electrode silver wire  was purchased from A-M Systems Inc.  and glass capillaries (0.58 mm inner diameter) for the micropipettes were purchased from World Precision Instruments Ltd .Dopamine recordings were made at PND 28 (±1 day), PND 42 (±1 day) or PND 70 (±2 days). Surgery was performed immediately prior to the electrochemical recordings. A water-circulating heating pad  was used to maintain body temperature. The animals were anesthetized with Inactin 125 mg/kg intraperitoneally (i.p.) and placed in a stereotaxic frame . A hole in the skull was drilled overlying the recording site for the electrode, and another hole was drilled remote from the recording site for the placement of the Ag/AgCl reference electrode.in vitro in 0.05 M phosphate buffered saline to determine selectivity, limit of detection (LOD) and slope before use in vivoin vivo Ag/AgCl reference electrode High-speed chronoamperometric measurements  were performed using the FAST16-mkII recording system  according to a previously described procedure 2·2H2O) (pH 7.2–7.4) using a pipette-filling needle . The micropipette was affixed approximately 150–200 µm from the carbon fibre tip using sticky wax. The electrode was stereotactically placed in the dorsal striatum, AP: +1.0 mm, L: +3.0 mm from bregma, the incisor bar was adjusted according to age and weight A micropipette (10–15 µm inner diameter) was filled with isotonic potassium chloride solution  and after another 5 min release was again evoked every 10 min, producing peaks at 5, 15, 25, 35, 45, 55 and 65 min after the systemic injection, see The electrodes were cut off and left in place after the finished experiment and the brains were frozen. The placement was verified by sectioning of the frozen brains. From the 12 animals at PND28, 1 was excluded due to wrongful placement, and 2 because of recording errors. For the 12 animals at PND 42, 1 animal was excluded due to wrongful placement. For the 16 animals at PND 70, 3 were excluded due to recording errors. For the 16 ethanol-drinking animals at PND 70, 2 were excluded due to recording errors. Recording errors include pipette clogging and electrical disturbances such as power cuts and disturbances of the general power supply to the recording unit.The maximal amplitude of the evoked peaks and the time for the peak to decline to 80% of its amplitude (T80) were calculated using the FAST Analysis software version 4.4 , see Differences in reference amplitudes between the age groups are shown in No effects of age , time  or time and age  were found for the reference T80 values. The mean ± standard error of the mean (SEM) reference T80 values were 17.3±1.3 for the PND 28, 19.5±0.9 for the PND 42 and 20.5±1.0 for the PND70.Differences between the age groups in amplitude response to amphetamine are shown in The T80 response to amphetamine is shown in 2 = 9.80; p = 0.08] towards differences driven by the intake during the second week (PND 35–37), which was slightly higher than the following weeks. A Friedman ANOVA of the preference showed an increase over time , mainly as a result from increases over the first three weeks, see Ethanol intake data for the 14 rats that were used in the chronoamperometric recordings are shown in Differences in reference amplitudes between the ethanol- and water-drinking groups are shown in No effects of drinking group , time  or time and drinking group  were found for the reference T80 values. The mean ± SEM reference T80 values were 20.5±1.0 for the water-drinking rats, and 19.1±1.3 for the ethanol-drinking rats.The response to amphetamine in the ethanol- and water-drinking groups is shown in For the T80 values, Age-dependent effects on dopamine release and uptake was investigated under basal conditions and in response to amphetamine in early and late adolescent, as well as adult, rats. The impact of alcohol drinking during adolescence was also examined and is, to our knowledge, the first study investigating release and uptake in voluntary drinking adolescent rats with a chronoamperometric technique.2 density The age-dependent differences in reference amplitudes are in agreement with a previous study using voltammetry in combination with electrical stimulation, which showed that adult rats released more dopamine upon stimulation than young rats max should therefore not be reached. Using the linear part of the peak curve to calculate uptake rate under these conditions will only produce uptake rates dependent on amplitudes The uptake measure, T80, did not reveal any differences between the ages in the current study, whereas Stamford (1989) found that the rate of uptake was higher in adult rats. This can be due to methodological differences in the measure of uptake; T80 includes both the linear and the curvilinear part of the curve, whereas Stamford used the linear part of the curve The greater potassium-evoked release in adulthood could be due to a larger releasable pool of dopamine No age-dependent effects on T80 after amphetamine was found, which indicates that amphetamine exerts similar effects on dopamine uptake in all ages. This is again supported by the results from Stamford (1989), showing no differences in the degree of uptake blockade after nomifensine between age groups. There are also studies suggesting that age-related differences in dopamine transporter structure and function are related to the cocaine-binding site on the transporter, but not the amphetamine-binding site Voluntary adolescent alcohol intake for six weeks resulted in lower reference amplitudes compared to water-drinking controls. The amplitudes were similar to those seen in early adolescent rats. Since the effects were seen in amplitudes and not uptake time, it is conceivable that alcohol affects factors controlling the releasable pool of dopamine rather than the dopamine transporter and there are data supporting unaffected uptake after adolescent alcohol When treated with amphetamine, there were no significant differences between the alcohol- and water-drinking groups in amplitudes or T80. However, there was a trend towards an effect on amplitudes, due to the increase displayed by the alcohol-drinking group. There is also more variation in the response to amphetamine in the alcohol-drinking group, which could be due to a variation in alcohol intake, although this variation is not correlated to the response (data not shown). This also points to a limitation with this study, namely that blood alcohol levels were not measured. The study was based on undisturbed access for 24 h and for blood alcohol levels to be measured the access would have had to be limited and the stress involved in blood sampling would have risked to disturb the intake patterns of the animals. Thus, correlations between response and individual blood alcohol levels cannot be ruled out. However, the intake data presented in this study is similar to other studies, showing neurobiological effects of alcohol, using Wistar rats in similar ages or intake paradigmsNo differences in uptake time after amphetamine suggests that adolescent alcohol has no effect on dopamine transporter function in response to amphetamine, but would also benefit from investigation by application of exogenous dopamine Furthermore, two interesting observations were made. Firstly, the reference amplitudes after alcohol intake are similar to those seen in animals at the beginning of the alcohol intake period, i.e. PND 28. Secondly, the size of the increase in amplitudes after amphetamine in the alcohol-drinking animals is similar to the late adolescent rats, i.e. PND 42. Whether these findings relate to altered development of the releasable pool and the storage pool of dopamine in the neurons remains to be elucidated. The current study did not include a group of adult alcohol-drinking rats so conclusions about the possibility of age-specific effects cannot be drawn. However, indications of age-specific effects can be found in the discrepancies between studies of adolescent alcohol-exposed rats that show unaffected dopamine uptake The data show a gradual increase of evoked dopamine overflow with age, supporting previous studies suggesting that the pool of releasable dopamine increases with age. In contrast, a gradual decrease in evoked overflow with age was seen in response to amphetamine, supporting a proportionally larger storage pool of dopamine in younger animals, making them potentially more sensitive to dopamine-releasing drugs. Adolescent alcohol intake resulted in overflow lower than in water-drinking controls., indicating that alcohol affects the releasable pool of dopamine and this may have implications for vulnerability to addiction and other psychiatric diagnoses involving the dopamine system in the dorsal striatum.


Following extensive surgical debridement in the treatment of infection, a “dead space” can result following surgical closure that can fill with hematoma, an environment conducive to bacterial growth. The eradication of dead space is essential in order to prevent recurrent infection. This study describes a novel small animal model to investigate dead-space management in muscle tissue. Two absorbable test materials were implanted in each animal; beads of calcium sulfate alone, and beads loaded with vancomycin and tobramycin. In-life blood samples and radiographs were taken from each animal following implantation. Animals were sacrificed at 1, 7, 21, 42, and 63 days post-operatively (n = 4), and implant sites were analysed by micro-computed tomography, histology and immunohistochemistry. Complete resorption was confirmed radiographically at 3 weeks post-implantation. Histologically, the host tissue response to both materials was identical, and subsequent healing at the implant sites was observed with no dead space remaining. Vancomycin was not detected in blood serum. However, peak tobramycin levels were detected in all animals at 6 hours post-implantation with no detectable levels in any animals at 72 hours post implantation. Serological inflammatory cytokine expression for IL-6, TNF-α and IL-1β indicated no unusual inflammatory response to the implanted materials or surgical procedure. The model was found to be convenient and effective for the assessment of implant materials for management of dead space in muscle tissue. The two materials tested were effective in resolving the surgically created dead space, and did not elicit any unexpected adverse host response. When extensive debridement is required, patients are left with a dead space that, when closed, can fill with a hematoma or seroma, an environment conducive to the growth of bacteria. Clinical results suggest that the major factor for successful eradication of infection is surgical debridement, dead space management  and prevLocal tissue flaps or free flaps may be used to fill dead space , 4. HowePMMA may have some disadvantages. As a non-resorbing material, in many circumstances it must be removed in an additional surgical procedure when its function has been completed, due to the risk of becoming a nidus for future infection , 13. AlsThe use of alternative materials has been reported. Mackey et al reported results with antibiotic with calcium sulfate beads, adding them to reduce the dead space remaining following surgery, therefore reducing post-operative hematoma formation. The authors believed that this reduction in hematoma formation, a potential culture medium for any remaining bacteria, possibly explained the positive outcomes when using calcium sulfate in the treatment of osteomyelitis cavities. They also reported that when used in the presence of a draining wound, calcium sulfate is either resorbed or drains in the pus, and does not remain as a sequestrum .The technique of transmetatarsal amputation has also described the placement of antibiotic-impregnated calcium sulfate beads with careful attention to soft-tissue technique, placing them into the dead space between the skin flaps. This was reported to prevent recontamination of the deep space should wound necrosis occur .A logical decision regarding whatever filler is used, is the need for some kind of protection against colonisation with pathogenic bacteria . Device-Animal models have been used previously to demonstrate the efficacy of materials for dead-space management and tissue regeneration in bone tissue –26. HoweImplant materials  were placed into intramuscular sites following retraction of the longissimus muscles at 4 non adjacent implant sites in 24 adult New Zealand white rabbits to mimic implantation for dead-space management in muscle tissue following approval from the Animal Care and Ethics Committee of the University of New South Wales (ACEC #: 13/67A). Two test materials were implanted; beads of calcium sulfate alone (CS), and beads of calcium sulfate combined with vancomycin and tobramycin (CSVT).Under sterile conditions, 10 mL kits containing 20g of pharmaceutical grade calcium sulfate alpha-hemihydrate powder  were used to prepare the beads for implantation; 20g of calcium sulfate alpha-hemihydrate was mixed with 6 mL of the provided mixing solution for unloaded beads. For the antibiotic containing beads, 20g of calcium sulfate alpha-hemihydrate was mixed with 1000 mg vancomycin hydrochloride powder (Hospira UK Limited) and thoroughly blended prior to the addition of 6 mL of tobramycin sulfate solution .In each case, all components were mixed thoroughly for 30 to 60 seconds to form a smooth paste which was pressed into 6.0mm diameter hemispherical cavities in the flexible mould . The beahe mould .The beads were hemispherical with a diameter (D) of 6mm and a length (L) of 4.8mm .A homogenous mixture of antibiotic and calcium sulfate alpha-hemihydrate was achieved as a result of thorough mixing in the preparation of the beads, enabling the calculation of concentration of antibiotic, as weight/weight of beads.Peripheral blood was taken pre-operatively, 1, 6, 12, 24 hours and 3, 7, 14, 21, 42 and 63 days throughout the study for standard blood panel haematology/biochemistry , serum cytokine levels  and to determine systemic Vancomycin and Tobramycin levels.Blood was collected into a Vacutainer Plain Clot 4mL tube and allowed to clot at room temperature for 2 hours before centrifugation for 15 minutes at 2000xg. The serum was carefully removed and stored at -80°C until needed.Serum IL-1β (E04I0010), IL-6 (CSB-E06903Rb) and TNF-α (CSB-E06998Rb) levels were analysed by enzyme-linked immunosorbent assay (ELISA) using commercially available kits  according to the manufacturer’s instructions. Each sample was measured in duplicate and standards were also run on each 96-well plate. The detection limits within serum for each of these tested cytokines are 1 pg/ml, 15.6 pg/ml and 78 pg/ml respectively.For all 24 animals in the study, four implant sites were used per animal, two sites each side of the spine, in non-adjacent intramuscular sites (longissimus muscles) above the levels in L1-L2, L2-L3, L3-L4 and L4-L5 . The intFor each sacrifice time point in the study the allocation of material to implant sites is as outlined in in-vivo absorption of the test materials was quantified by analysis of standard radiographs and micro-computed tomography (μCT).Posteroanterior radiographs were used to visualise the appearance of the beads in the soft tissue at the time of surgery. Radiographs were taken with a mobile xray machine  and digital plates . These images were used as a baseline for examination at later time points. During the study, in life blood samples and in life postero-anterior and lateral radiographs were taken from each animal following implantation. The levels of vancomycin and tobramycin in the blood serum samples versus time are also reported. The After the scheduled duration was complete, the animals were euthanized, radiographs and μCT was performed, and the implant sites were inspected and harvested for evaluations to assess healing, local tissue reactions and local vancomycin and tobramycin levels.in-vivo release kinetics of vancomycin and tobramycin in local tissues as well as serum levels were evaluated. The general integrity of the skin incision was noted along with the macroscopic reaction of the underlining subcutaneous tissues as normal or abnormal. If abnormal this was further assessed for evidence of infection or macroscopic signs of inflammation/foreign body reaction.Animals were sacrificed according to the study design: each time point had 4 animals with 4 implantation sites per animal. The At the time of harvest the heart, liver, kidney, spleen, and lungs were examined and any abnormalities noted, for each animal. A portion of the distant organs was processed for routine paraffin histology and evaluated in a blinded fashion for any abnormalities. Microscopic changes were graded on a severity grade based upon the following: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe, N = normal.The animals were radiographed in the same manner as performed at the completion of surgery. The images were used to determine resorption based on radiographic appearance. Radiographic status of the implant sites was compared to radiographs obtained immediately after implantation to determine absorption of the implant material. The radiographs were graded in a blinded fashion for implant absorption on a scale of 0–4 . Data wain-vivo scanner  in order to obtain high resolution images of the implant absorption. The surgical sites were scanned and the raw images reconstructed resulting in effective pixel size of 53.12 μm. 3D models were created using Siemens image analysis software  to assess in-vivo absorption as well as any adverse tissue reactions. Images were examined in the axial, sagittal and coronal planes. The resultant images were graded in a blinded fashion for implant absorption on a scale of 0–4 (Micro-computed tomography (μCT) was performed for all animals using an e of 0–4 . Data waThe surgical sites were carefully dissected and examined for the presence of any residual beads. A portion of any residual beads present at the surgical sites was harvested. This material was allowed to air dry and placed in a desiccator for 24 hours and then morselized using a mortar and pestle. 0.1g of the powder was immersed in 1ml of serum for 24 hours. Vancomycin and tobramycin levels in the samples per gram of material were determined using a standard antibody assay.A muscle sample (1x1cm) from the implantation site was harvested, air dried for 72 hours and then morselised using a mortar and pestle. 0.1g of the morselised tissue was immersed in 1ml of serum for 24 hours. The local concentration of vancomycin and tobramycin in the muscle sample was measured using a standard antibody assay.Rabbit serum was harvested from rabbits from other studies at the time of sacrifice to perform a dose response curve for rabbit serum with a known concentration of vancomycin and tobramycin. Standard vancomycin and tobramycin levels in saline were also run for comparison. Concentration levels of 0.1, 1, 5, 10, 20, 50, 100, 200, 500 and 1000 μg/ml were determined to provide a standard curve. This portion of the study was completed with a standardised assay . The assay method was based on a homogeneous enzyme immunoassay technique used for the quantitative analysis of antibiotic in human serum or plasma. .The serum samples collected post implantation were then analysed using this assay.In-vivo response and biocompatibility to the materials was assessed at the implant site/ host tissue boundary.Harvested implant sites allocated for histology were immediately fixed in phosphate buffered formalin for a minimum of 48 hours. They were then decalcified in 10% formic acid–phosphate buffered formalin at room temperature. The decalcified samples were placed into embedding blocks for paraffin processing. Paraffin blocks were sectioned to 5 microns using a microtome  and placed onto super-frosted slides for routine haematoxylin and eosin (H&E) staining. Stained sections were photographed using an Olympus Microscope  and Olympus DP72 Camera. Histology was qualitatively assessed versus time at each time point. Immunohistochemistry was performed on paraffin sections from all animals at all sites for markers of inflammatory response including IL-1β, CD3, CD79a and IL-6 using standard techniques –32. ParaSurgery was completed without any adverse events. All animals recovered well following surgery. Macroscopic reactions at the level of the surgical incisions were monitored for each animal. There was no evidence of wound breakdown at any time point. Bead absorption was observed at day 7 and day 21. A slight reaction was noted at day 21 in one site in one animal where there was elevation of the subcutaneous tissues but the overall reaction appeared normal. All subcutaneous sites were normal at day 42 and day 63 and the surgical implantation sites appeared well healed. The distant organs appeared normal in all animals.Peripheral blood samples were collected prior to surgery (pre-op) and 1, 6, 12, and 24 hours following surgery as well as 3, 7, 14, 21, 42, and 63 days post implantation to determine the systemic serum vancomycin and tobramycin levels following implant material placement. Although the vancomycin assay was confirmed in preliminary validation of methodology using rabbit serum dilution experiments, no vancomycin was detected in blood serum in any animal at any time point in this study. Tobramycin levels were detected in 6 animals at 1 hour post implantation, following with detectable levels in all animals at 6 hours post implantation. The highest serum level detected at this time point was 0.88mg/L, with average values across all animals of 0.69mg/L. Serum levels of tobramycin then dropped in all subsequent time points for all animals, with detectable serum levels in only 4 animals at 24 hours post-implantation, and no detectable serum levels in any animals 72 hours post implantation .By comparison with standard curves for each of the factors measured , the levels of systemic cytokines were quantified preoperatively (baseline) and postoperatively at each of the allocated time points: 1hr, 6hrs, 12hrs, 24hrs, 72hrs, 7 days, 14 days, 21 days, 42 days and 63 days.IL-1β levels were detected at all the allocated time points with a significant difference between pre-op and day 63 expression (p<0.05), however IL-6 levels were only detected from 12hrs to 42 days. These are shown in Figs During harvest the surgical sites were carefully dissected and examined for the presence of any residual calcium sulfate beads. A portion (0.1g) of any residual calcium sulfate bead present was harvested. Residual beads were only found in animals allocated for 24 hours and day 7 time points. Vancomycin and tobramycin levels in the samples per gram of material (CSVT) were determined. Results are represented in Muscle samples (1x1cm) from the implantation sites were harvested and then minced and air dried. The local concentration of vancomycin and tobramycin in the muscle sample was measured using a standard antibody assay. Antibiotic was below the level of detection in any samples analysed for 24 hours and day 7 harvested samples, thus later time points were not examined.Posterior anterior radiographs were taken as per schedule at the time of harvest and post operatively. Radiographs were graded from 0 (81–100% implant remaining) to a grade of 4 (0–20% implant remaining).CS and CSVT beads were apparent in day 0, 24 hours, and day 7 radiographs. Some softening of the beads as well as some areas of lucency was observed on day 7. Complete absorption of all beads was confirmed by day 21, and only scarce areas of bead consolidation were noticeable. Beads were not evident at the 42 and 63 day radiographs . RadiogrA faint halo of material was noted in the muscle tissue around both CS and CSVT beads on the radiographs at 24 hours and 7 days. The halo remained at 21 days despite complete absorption of the beads. However, this halo of material was no longer present on the 42 and 63 day radiographs.μCT provided an assessment of bead absorption in the axial, sagittal, and coronal anatomical planes and confirmed the radiographic findings. All beads were absorbed by the 21 day time point . No adveFor implantation sites at day 0, no local reaction to the implant material was noted at any of the implantation sites. For implantation sites at 24 hours, the reaction to CS and CSVT was identical in nature and magnitude, with lymphocytes and red blood cells observed at the implant /host tissue interface. At day 7, once again, the reaction to both materials was identical in nature and magnitude. Lymphocytes and red blood cells were observed at the implant /host tissue interface. In addition some multinucleated cells were present and some infiltration of fibrous tissue was observed. At days 21, 42 and 63, the response to both materials was identical in nature and magnitude, all with lymphocytes and red blood cells at the implant /host tissue interface, and multinucleated cells present. The materials had completely resorbed and were replaced by fibrous tissue.in-vivo response to the materials was the same . Some expression for these markers were found in the CS as well as the CSVT groups at days 7, 21, 42 and 63 and was similar between CS and CSVT at each time point. The overall the same .IL-6 was expressed at day 1 in adjacent blood vessels. Expression was noted at day 7 in endothelial cells as well as multi-nucleated cells (macrophage lineage) .Dead space can be defined as a space remaining in a wound after surgical closure. It is frequently a result of the removal of implants and the extensive debridement of necrotic tissue following joint revision surgery. If the dead space is not eradicated, it quickly fills with hematoma and wound exudate immediately following surgery, which provides ideal conditions for bacterial growth for residual pathogens present in the surgical site. Once bacteria are established in this space, infection is difficult to eradicate due to the limited ability of parenterally administered antibiotics to penetrate the space as it is not vascularised. Therefore it is paramount that dead space is minimised. This can be achieved with the transfer of muscle flaps –37 or fiA number of animal models have been published which enable the evaluation of implant materials to fill voids in bone following osteomyelitis , 43–45, The model offers a number of advantages. Firstly, it allows multiple surgical sites per animal which makes the comparison of host response to device / material variations possible in the same animal, and allows fewer animals to be studied considering the internal controls. Also, after scaling up to human clinical subjects, the size of the intramuscular site is comparable to potential dead space observed in clinical scenarios following extensive debridement. Finally, the model is large enough to easily allow a comprehensive range of analysis techniques, including radiographic analysis (standard radiographs and μCT), multiple blood serum analysis, tissue analysis, and complete histological analysis to characterise and quantify the host response to the implant material under test.The absorbable calcium sulfate material chosen to evaluate the model has been reported previously in literature as a means of dead space management in joint revision surgery –51 and iVancomycin is a glycopeptide antibiotic used in the prophylaxis and treatment of infections caused by Gram-positive bacteria. Tobramycin is an aminoglycoside antibiotic used to treat various types of bacterial infections. The combination of vancomycin with tobramycin is favourable due to the synergistic activity of the two antibiotics with respect to Gram-negative and Gram-positive bacterial coverage , 54. VanElution characteristics of tobramycin impregnated calcium sulfate beads have previously been evaluated –61. SystAnalysis of residual CSVT beads harvested at 7 and 21 days contained some residual vancomycin and tobramycin, with a higher quantity of vancomycin present. As a result of the large standard deviation in the measured levels for both antibiotics, a comparison of relative levels of antibiotic present in residual beads with the original ratio in the beads as prepared is of limited relevance. When the local concentration of vancomycin and tobramycin in the harvested muscle samples was measured, levels were below the level of detection in all samples analysed for 24 hours and day 7 post implantation. This contradiction may be a result of a lack of antibiotic binding to the harvested muscle tissue and warrants further investigation.in-vivo response to the implantation of a calcium sulfate which is a well-tolerated implant material, with the host tissue response graded as mild to moderate. This is an interesting observation when considering the nature of the implant materials and their constituents. Tobramycin sulfate is safe for intramuscular administration at the correct dosage and is subsequently rapidly absorbed. In contrast, vancomycin hydrochloride solutions have a low pH (2.8–4.5), and are irritating to tissue, caused drug fever, pain and possible necrosis. As such it is not administered intramuscularly, with intravenous administration the recommended route. Stimulan is produced synthetically from pharmaceutical purity reagents. It has a neutral to mildly alkaline physiological pH. This is different to other CS which are produced from gypsum rock and tends to have an acidic pH. Clinical studies using Stimulan with vancomycin and tobramycin show very low levels IL-1β plays a role in tissue repair and the elevated levels at day 63 are consistent with the histological response of fibrous tissue at the sites were the material had completely resorbed [The histological response to the implanted materials was in keeping with the expected resorbed .The host tissue response to both CS and CSVT was the same, indicating no additional adverse local tissue response to the presence of both vancomycin and tobramycin in the CSVT beads. The potential additional tissue irritation that could be caused by the presence of vancomycin was not evident. This may be due to the low levels of vancomycin hydrochloride present in the CSVT beads, or as a result of some change to a characteristics of the antibiotic that elicit an adverse reaction. A possible cause is a buffering of the acidic pH of vancomycin hydrochloride as a result of its combination with calcium sulfate.The study had a number of limitations. Although a dead space is typically created by resection of a volume of tissue in a clinical situation, in our model we retracted the muscle and created the dead space. Cutting out a piece of muscle to elicit a blood filled muscular/soft tissue defect would have elicited a different type of tissue response. No sham procedure was employed in the model to determine the effect of the creation of an unfilled intramuscular site. It was felt that this would have been of limited use, as the intramuscular site would have closed with the formation of dead space if no implant material was placed. The model as described is not an infection model. Therefore it was not possible to determine the in-vivo efficacy of the antibiotic in the CSVT implant on protecting the material from bacterial colonisation, although this has recently been shown in-vitro . The intThe model was found to be a convenient and effective model for the assessment of implant materials for management of dead space in muscle tissue. The two materials tested were effective in resolving the surgically created dead space, and did not elicit any unexpected adverse host response.


Scaffolds made of aligned nanofibers are favorable for nerve regeneration due to their superior nerve cell attachment and proliferation. However, it is challenging not only to produce a neat mat or a conduit form with aligned nanofibers but also to use these for surgical applications as a nerve guide conduit due to their insufficient mechanical strength. Furthermore, no studies have been reported on the fabrication of aligned nanofibers and randomly-oriented nanofibers on the same mat. In this study, we have successfully produced a mat with both aligned and randomly-oriented nanofibers by using a novel electrospinning set up. A new conduit with a highly-aligned electrospun mat is produced with this modified electrospinning method, and this proposed conduit with favorable features, such as selective permeability, hydrophilicity and nerve growth directional steering, were fabricated as nerve guide conduits (NGCs). The inner surface of the nerve conduit is covered with highly aligned electrospun nanofibers and is able to enhance the proliferation of neural cells. The central part of the tube is double-coated with randomly-oriented nanofibers over the aligned nanofibers, strengthening the weak mechanical strength of the aligned nanofibers. The anatomical recovery of damaged peripheral nerves with their proper neurological functions after injury remains a significant clinical challenge23568101213151718In this study, we have developed a method to fabricate highly-aligned electrospun NGCs with selective permeability using various materials including poly lactic-co-glycolic acid (PLGA) and polyurethane (PU). The external side of the central part of the nerve conduit is coated with randomly-aligned nanofibers to increase its mechanical strength, and the internal side of the conduit is coated with growth factor-bearing aligned nanofibers for improved nerve regeneration186Aligned and randomly oriented nanofibrous mats were prepared in one step using a modified electrospinning method as shown in Fast Fourier Transfer (FFT) approach is applied to measure the alignment rates of the nanofibers, and ImageJ, image analysis software was used to analyze the FFT results21Multiple angled U shaped patterns were made on a single mat using the modified electrospinning method. Each single angled U-shaped pattern was used as the final layout for the NGC; the schematic is shown in The transparencies of the scaffolds were carried out using spectrum analysis in the wavelength range from 380 nm to 780 nm, and 23A universal testing machine  was used to carry out the tensile properties of the matrices with woven, aligned PU and PLGA nanofibers at room condition. The testing samples were dog-bone shaped with a gauge length of 40 mm and a width of 10 mm, and tested at a speed of 5 mm/min. The sample seen in the inset of Cell adhesion to the biocompatible material, which is an essential prerequisite for healthy cell growth, cell migration and proper cell differentiation, is greatly affected by the physical surface properties and the chemical composition of the scaffolds. Since the cell adhesion to the scaffolds is mandatory for cell regeneration, the contact angle measurements is an important assessment to evaluate the cell behavior on the scaffolds. The wettability was influenced by various factors, such as the mat homogeneity, the type of fluid, and the surface tension measured according to the contact angle. As shown in 25The presence of the electric charges makes the electric force and the electric field to become the area where an electric force exists. This is a significant point to account for the interaction between two sites of a different electric charge, and if there are more sites that affect the electric field in the system, there is potential to change the electric charge27in vitro biocompatibility evaluations were done by using CCK-8 assay, SEM and confocal laser scanning microscopy. It has been ascertained from the CCK-8 assay result, as shown in 303132The surface matrix of the electrospun scaffolds can provide a supportive condition for adhesion and proliferation of the cells due to the similarities in the physical properties and neural tissue structureConsequently, scaffolds with aligned-oriented nanofibers can facilitate and accelerate nerve regeneration due to the nature of the nerve cells that tend to flourish (grow well) along with the aligned nanofibers. The advanced hydrophilicity and the adhesive property of the aligned nanofibrous matrix might play other key roles for better cell proliferation. These results indicate a superiority of the aligned nanofibrous scaffold to randomly-oriented nanofibrous scaffolds as a substrate for tissue engineering, especially regarding cell proliferation and guidance. Therefore, aligned-oriented nanofibrous scaffolds can be a candidate for extensive use in nerve tissue engineering.In summary, we have successfully invented a modification of the electrospinning process in order to fabricate highly-aligned nanofibers and randomly-oriented nanofibers on a single mat via a single step process. In addition, the electrospun aligned and random oriented nanofibers can be easily rolled into an angled U-shape to form an NGC that can be used as a practical way to assist nerve regeneration. In this work, the fabricated NGCs show high transparency and could allow the incised nerve fibers in the conduits to be seen directly by the naked eyes during implantation. This improvement in transparency of both the edges of the tube with aligned nanofibrous mat could potentially facilitate the ease of nerve reconstructive surgery. The central exterior portion of the tube where the random nanofiber is located could prevent scar tissue invasion while the inner coating aligned nanofibers can help vital active nerve regeneration. The fabricated scaffold with aligned nanofibers has superb hydrophilic properties that can facilitate in cell proliferation and cell growth guidance. The aligned nanofibers acted as a guide for neural cells and were able to achieve a higher cell proliferation and migration compared to randomly oriented nanofibers. This processing method can be a promising candidate for fabricating scaffolds for nerve tissue engineering.Co-polymers of poly (lactic- co -glycolic acid) (PLGA) and polyurethane (PU) were obtained from Sigma Aldrich, Korea to produce the aligned oriented nanofibrous mat. Tetrahydrofuran (THF) and N, N dimethylformamide (DMF) were obtained from Showa (Japan) and were prepared for use as a solvent. Mouse Schwann cells (S42) and Rat pheochromocytoma cells (PC12) were obtained from the Cell Line Bank. Culture reagents including Dulbecco’s modified Eagle’s medium (DMEM), antibiotic solutions and fetal bovine serum (FBS) were purchased from Sigma. Mouse nerve growth factor (NGF) was obtained from Sunlong Biotech (Korea).In this experiment, the 10 wt% concentration of PLGA and PU solutions were dissolved with mixed solvent containing 1:1(by weight percent) of THF and DMF with continuous magnetic stirring. Random, aligned electrospun nanofibrous scaffolds made of co-polymer of poly (lactic- co -glycolic acid) (PLGA) and polyurethane (PU) were fabricated using the electrospinning set-up shown in R, USA) in a wavelength range from 380 nm to 780 nm. The FFT method was used to evaluate the fiber alignment of the fabricated mats. The alignment of the fibers in the images was analyzed using the ImageJ software. During the FFT analysis, the alignment degree of the data image was reported by the FFT peak shapes and the height. The FFT utilization has the point to systematically evaluate the correlation and causality between the alignment degree and the other structural and material properties of the scaffold by setting up the relative numerical values of the alignment of the electrospun fibers34The fiber morphology and the cell adhesion of the electrospun PU and PLGA nanofibers were observed using field-emission scanning electron microscopy  and scanning electron microscopy . Samples collected on from the rotating collector with a polyethylene sheet were prepared for each experiment. The mechanical properties of the mats in the longitudinal direction were measured by using a universal testing machine  at room temperature. To determine the wettability of the electrospun nanofibrous mats, the water contact angle was measured with deionized water using a contact angle meter . All experiments were conducted in room conditions and were performed at different time intervals of 3, 6 and 9 s for more than 5 times per sample. The permeability and the mechanical properties of the nanofibrous scaffolds were determined according to their porosity, and the porosity of the PU fibrous mats was measured using MicroActive AutoPore V 9600 . The measurements of the film scaffold transparency was carried out using a spectrum analysis method by SYNERGY Mx spectrophotometer  were cultured in DMEM medium including 10% FBS(Sigma), 1% penicillin/streptomycin. The electrospun nanofibrous scaffolds were cut to their proper size (1.2 cm × 1.2 cm) to cover the cell culture plate . After sterilization under UV radiation for 12 hours, the scaffolds were washed 3 times with phosphate buffered saline solution (PBS), and cells with density of 2 × 104 cells per well were seeded on the electrospun mats and were cultured in DMEM medium in a humidified atmosphere with 5% CO2 at 37 °C. The samples were then transferred to individual 48-well tissue culture plates. The adhesion and proliferation of the cells on the nanofibrous scaffolds was measured using the cell counting kit-8 after 1, 3 and 5 days of cell culture. At desired points in time, the CCK-8 solution was added to each well and was incubated for 2 hours in 37 °C incubator with 5% CO2. The measurement of the absorbance was checked at 450 nm using a microplate reader . A morphological study was carried out for the in vitro cultured PC12 cells and S42 cells on aligned and randomly-oriented electrospun nanofibrous scaffolds for 1 day, 3 days and 5 days. First, the cells were grown on scaffolds for 1, 3 and 5 days and were washed three times with PBS to remove the non-adherent cells. The attached cells were fixed with 2.5% glutaraldehyde solution, and were then dehydrated in ethanol solution through a graded series of alcohol  and were dried for 12 hours in a clean bench. The morphology was then examined by using SEM and FE-SEM.PC-12 cells  were cultured in DMEM (Gibco) medium supplemented with 1% penicillin/streptomycin, 5% horse serum(Gibco) and 5% calf serum(Gibco) and 1% NGF  at 37 °C with 5% CO × 104 cells per well and were incubated for 1, 3 and 5 days. The media with the cultured cells was aspirated from wells, and the attached cells were rinsed with PBS solution. The cells were then fixed with 4% paraformaldehyde (PFA) solution for 10 min at room temperature and were permeabilized in 0.2% triton X-100 in PBS for 2 min. After blocking with 1% HSA/PBS dilution for 30 min, the nanofibrous scaffolds were stained with Actin-green488 solution and DAPI in each of the 24 wells and were incubated in the dark for cell nuclei and actin filament staining. Cells fluorescence was then observed using a fluorescence microscope.The PC12 cells and S42 cells were seeded on aligned and randomly-oriented electrospun nanofibrous scaffolds  with a density of 2How to cite this article: Kim, J. I. et al. A Controlled Design of Aligned and Random Nanofibers for 3D Bi-functionalized Nerve Conduits Fabricated via a Novel Electrospinning Set-up. Sci. Rep.6, 23761; doi: 10.1038/srep23761 (2016).


We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using  The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR - associated protein (Cas) system, an adaptive immune system found in many archaea and bacteria, has recently emerged as an efficient and precise tool for genome engineering1246Streptococcus pyogenes is the most commonly used based on its relative simplicity12To date, three CRISPR-Cas subtypes have been classified in prokaryotesTo direct the Cas9 complex to a desired locus for genetic manipulation, a 20-nucleotide guide sequence found within the sgRNA must be complementary to the target DNAGiven the relatively short length of the guide sequence in the sgRNA (20 nt), targeting specificity has become one major concern in using CRISPR-Cas9, and the off-target effects of the system have been extensively investigatedet al. evaluated the efficiencies of a total of 1,841 sgRNAs in inducing complete loss of a protein and demonstrated that the nucleotide composition at specific positions, especially the one adjacent to the protospacer-adjacent motif (PAM), contributes to the activity of the sgRNAet al. analyzed the effects of sequence context on sgRNA efficiency and generated models that achieved reasonable predicative power in which the Area Under Curve (AUC) scores were greater than 0.7 in Receiver Operating Characteristic (ROC)Despite extensive research on off-target effects, only a limited number of studies have focused on analyzing the on-target cleavage efficiency of the sgRNA/Cas9 complex. It has been observed that the mutagenesis rate of the CRISPR/Cas9 system varies greatly20212223in vitro systems available to evaluate sgRNA performance, the Surveyor nuclease assay is the most commonly used and reliable method. This assay utilizes an enzyme mismatch cleavage system in which heteroduplex DNA with mismatches and indels are cleaved. However, despite its high reliability, the procedure is tedious and time-consuming: it usually takes 10 days from the design of sgRNA to obtain the final assay results. Additionally, it is difficult to multiplex the procedure since cell culture, transfection, and genomic PCR are required for each individual sgRNA assay. Currently, systematic evaluations of sgRNA on-target efficiency based on Surveyor assay are still limited, especially using mammalian cell lines. In this study, we reported the evaluation of the on-target activity for 218 sgRNAs based on the mouse Neuro2A cell line.Among the host of We designed and successfully cloned 218 sgRNAs into expression vectors. The experimental design and procedure are briefly outlined in We next set out to explore whether the nucleotide composition of the sgRNAs affected the cleavage results. First, we separated the sgRNA sequences into two groups: Surveyor positive and Surveyor negative. For each group, the occurrences and frequencies of nucleotides  at each position were calculated . We thenP values of 0.031 and 0.022, respectively .We next asked whether these frequency changes are statistically meaningful or merely represent chance observations. Chi-square analysis was performed for each position of the guide sequence to test if the overall nucleotide composition is different between Surveyor positive and negative sequences. Statistically significant changes were observed at positions 3 and 20 with ectively . PositioSince the GC percentage of sgRNAs, particularly that of the 6 PAM-proximal bases, has been previously reported to be positively correlated with on-target efficiency2025As a follow-up to the Chi-square test, we tried to evaluate the effect of each nucleotide on cleavage efficiency through logistic regression analysis. The nucleotides in each position were coded as dummy variables, and the nucleotide that showed the lowest frequency change at each given position was set as the baseline level. Similarly, the genomic contexts of the target sequence were also included in the regression analysis where the intergenic region was set as the baseline level. In addition to the sequential features of the sgRNAs, we also evaluated the impacts of several structural features on cleavage efficiency. We assessed the overall secondary structure of each sgRNA measured as single minimum free energy (MFE). Additionally, we analyzed the local secondary structure of the seed region and the effects of the guide sequence on tracrRNA structure. We further speculated that the relationship between GC percentage and cleavage efficiency is likely to be a non-linear one, where sgRNAs with GC percentages that are too high or too low are unfavorable. Thus, we labeled sgRNAs with GC percentages within the range of 40%–60% as “GC normal” and those with GC percentages below 40% or above 60% as “GC abnormal.” These variables were incorporated into the logistic regression model.P values are illustrated in After logistic regression analysis, a total of 14 variables were found to be significantly correlated with the cleavage results, including the nucleotide present at 10 distinct positions, the condition of being targeted to a promoter-transcription start site (TSS), having a normal range GC, as well as several features of the secondary structures of sgRNA. The position-dependent nucleotide et al.’s studyP value is marginal at 0.07. We then assessed the ROC curve of the model fitted based on this score and calculate the area under the ROC curve (AUC) to be 0.57. As a comparison, the AUC score based on our current logistic model is 0.91 and 0.67 when the fitted model is applied to the total training data and from a 20-fold cross-validation, respectively.To evaluate the performance of the current model, we first tried to examine how well the cleavage activities of sgRNAs used in this study can be predicted by previous methods. We calculated “on-target scores” for our sgRNAs using the standalone Python software proposed in Doench In this study, we performed Surveyor assays to evaluate the on-target efficiency of 218 sgRNA sequences. We found that 41% of the sgRNAs showed no cleavage effects based on our assays. To understand what sequence features influence the cleavage outcome, we performed comprehensive statistical analyses that revealed the position-dependent nucleotide preferences associated with positive cleavage results. We further revealed that the genomic contexts of target DNA as well as the GC percentage and secondary structure of sgRNAs also contribute to sgRNA performance. As such, these factors should be considered when designing guide sequences.Based on Chi-square analysis, we found that position 3 and position 20, a base adjunct to the PAM, are associated with cleavage efficiency. The significant association at position 20 is in line with previous reports20In addition to the PAM-proximal region, we also observed significant correlations between positions in the PAM-distal region and cleavage efficiency. Unlike the proximal region, the PAM-distal region has been considered less important in determining the sgRNA specificity. However, in our study, we show that this region may actually contribute to the on-target efficiencies of sgRNAs. At position 2 and 3, The T and G were found to have a negative effect on cleavage efficiency. Additionally, A at position 6 was identified as significant nucleotide correlated with the cleavage outcome. It has been shown that the backbones of position 2 and 4–6 interact with the REC1 domain of the sgRNA, which is critical for sgRNA:DNA recognitionDrosophila, respectively. However, in our study, which is based on a mammalian cell line, we found that sgRNAs with very high or low GC contents are less effective rather than a simple model in which a greater GC percentage always lead to higher activity. In our logistic regression analysis, we demonstrate that sgRNAs with a GC percentage within the range of 40%–60% are favored for efficient on-target cleavage.Previous studies have shown that the GC percentage of overall sgRNAsIntriguingly, we found that if a target DNA sequence is located at the promoter-TSS region, the sequence has a greater chance to be successfully cleaved compared with a sequence located in the intergenic region. This result is likely related to local chromatin accessibilities in different types of genomic loci. A recent genome-wide Cas9 binding analysis based on Chip-Sequencing demonstrated that chromatin inaccessibility decrease the dCas9 binding and genomic loci commonly accessible in large number of cell types have a significantly higher probability binding to the sgRNA/Cas9 complex28Furthermore, our analysis also suggested that the secondary structure of the guide sequence is also an important parameter that should be considered for designing sgRNAs. In particular, the probability of the seed region to form an unfolded structure was identified as the most significant feature. Counterintuitively, our analysis revealed that if the seed sequence is more likely to form secondary structure, the sgRNA has a higher chance of cleaving the target sequence. Loading of the guide RNA into Cas9 has been demonstrated to be a crucial step in converting Cas9 into an active conformation capable of executing its nuclease functionet al. evaluated 1,841 sgRNAs’ efficiency in inducing complete loss of the proteinSp together with 82 and 69 sgRNAs for Cas9St1 were determined and were used to build the predictive modelP = 2.7 × 10−11). This observation confirms that the promoter/TSS regions have a higher level of chromatin accessibility. This link was further supported by a genome-wide survey of chromatin accessibility of human genome using 125 diverse cell and tissue types, in which it was found that promoters typically exhibit high accessibility across various cell typesRecently, two large-scale studies have been reported with the aim to improve the sgRNA design21in vivo mutagenesis study of CRISPR/Cas9 in Drosophila, the protein level of Cas9 is unlikely to be a critical factor for mutagenesis efficiency, while the amount sgRNA has a more profound impactAlthough our study offers key insights into sgRNA design, attention should be paid for interpreting the results. First, we used cleavage outcome data, which is binary in nature, for our statistical analysis. Although binary responses are easy to understand and interpret, and by this criterion we can clearly separate the sgRNAs into 2 distinct groups, the efficiencies of individual guide sequences might differ within the same group of sgRNAs that showed positive cleavage results. Thus, quantitative outcomes such as cleavage percentage and number of mutations induced by each sgRNA are needed to provide further insight into sgRNA optimization. Secondly, we used 800 ng plasmids for each transfection, which is commonly used for 24-well platein vitro Surveyor assay. We found that 41% of sgRNAs in our study showed negative results for cleavage, further emphasizing the need to improve the design of the sgRNA. Through statistical analysis, we found that nucleotide preferences at positions both adjunct and distal to the PAM sequence are significantly correlated with on-target performance. Furthermore, we showed that the genomic contexts of the target region, the optimal GC percentage, and secondary structure of sgRNA are important factors contributing to the cleavage efficiency. Taken together, our study reveals crucial parameters for the design of sgRNAs to achieve high on-target efficiency, particularly in the context of high throughput applications. Future studies are warranted to further replicate our study and improve the state-of-the-art CRISPR/Cas9 technology.Here we report a systematic evaluation of on-target performance of 218 sgRNAs based on http://crispr.mit.edu). Then, candidate sgRNAs with the highest scores  were selected and synthesized. Two complementary oligonucleotides of sgRNAs were annealed, phosphorylated, and cloned into the BbsI sites of pX330 CRISPR/Cas9 vector (Addgene plasmid ID 42230).The sgRNAs were designed to target the flanking sites of various loci that harbor copy number variations (CNVs) associated with autism spectrum disorder (ASD). The top 100 most frequently occurred ASD CNVs were retrieved from the SFARI CNV database2. The cells were seeded into 24-well plates (FALCON) to reach 1 × 105 cells per well. Plasmids (800 ng) were transfected using Lipo3000 reagents. N2A cells were harvested 48 hours post-transfection.Neuro2A (N2A) cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (Life Technology), 100 units penicillin, and 100 μg Streptomycin  and incubated at 37 °C with 5% CON2A cells transfected with both empty and sgRNA-containing PX330 vectors were treated with buffer containing proteinase K, and genomics DNA was then extracted by ethanol precipitation. Genomic PCR was conducted to amplify a 400–700 bp region containing the sgRNA target. PCR products were gel purified with Wizard SV Gel and the PCR CleanUp kit (Promega). 800 ng of each purified PCR product was mixed and re-annealed to form heteroduplexes, which were subsequently treated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics) following the manufacturer’s recommended protocol. The final product was separated on a 3% TAE Agarose gel and stained with ethidium bromide.P value < 0.05 was regarded as the level of statistical significance. Categorical variables were analyzed using Chi-square test. Independent two-sample t-tests and Kolmogorov-Smirnov test were used in the comparison of means between groups. Logistic regression was used to determine factors independently correlated with cleavage efficiency. To adjust for multiple testing, we further calculate permutation P values based on 10,000 times randomization. In each cycle of the permutation test, 129 and 89 sgRNAs were randomly assigned as positive and negative sequences, standard Chi-square test was followed and the smallest P value among all 21 positions was recorded to construct an empirical frequency distribution of the smallest P values. After 10,000 repeats of this procedure, the permutated P value is determined by comparing the original P value from the real data with the empirical P value distribution. We used annotatePeaks.pl program from the Homer Chip-Seq software to annotate the genomic context of each sgRNA targetThe R environment (version 3.1.3) was used for statistical analyses20), and U = 12 which is the length of the seed sequence. Finally, we also estimated the effect of the guide sequence on the tracrRNA structure using the dot plot of the base-pairing matrix predicted by RNAfold. In brief, for each nucleotide on the tracrRNA, we calculated its maximum and average base pairing probability with nucleotides on the guide sequence from the base-pairing matrix. We then averaged each individual probability over all nucleotides on tracrRNA and calculated the overall probability that the tracrRNA structure interacts with the guide sequences.The MFE of each sgRNA was predicted using RNAfold with the default parametersHow to cite this article: Liu, X. et al. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system. Sci. Rep.6, 19675; doi: 10.1038/srep19675 (2016).


Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder which can affect multiple organs of the body, requiring ongoing disease management and healthcare resource utilization. The economic impact of SLE has not been evaluated in a Medicare population to date. This study was conducted to assess the prevalence of SLE and its burden in terms of healthcare resource utilization and costs in a US Medicare population.This was a retrospective observational study using Medicare medical claims data (5% random sample) for the period spanning 2003 to 2007. SLE patients were identified by having ≥2 medical claims with a primary or secondary diagnosis of ICD-9 code 710.0X. The earliest quarter of SLE diagnosis was defined as the index quarter. Prevalence of SLE, the proportion of SLE cases on disability benefits, and the contribution of SLE to new disability cases were evaluated. Healthcare resource utilization and direct medical costs (2008 US dollars) over 12 months were compared between a cohort of patients with SLE and a cohort without SLE matched on key demographics. Differences in outcomes between cohorts were assessed using McNemar’s test for dichotomous variables and paired t-tests for continuous variables.A total of 13,348 patients with SLE were identified. The prevalence of SLE was approximately 3 per 1000 Medicare beneficiaries. After matching, the sample consisted of 6,707 SLE and 13,414 non-SLE patients. On average, the SLE cohort compared with the non-SLE cohort had 2.4 times more physician visits, 2.7 times more hospitalizations, 2.2 times more outpatient visits, and 2.1 times more emergency room visits. A medical cost surplus of approximately $10,229 per patient per year in the SLE cohort relative to the non-SLE cohort was driven largely by inpatient hospitalization costs (p < 0.001).SLE prevalence was 3 per 1,000 Medicare patients. Patients with SLE consumed significantly more health care resources with significantly greater costs compared with those without SLE. Added costs were largely attributable to inpatient hospitalizations. The Medicare population is an important target for efforts to improve SLE disease management and reduce costs. Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disorder that causes significant morbidity and mortality . SLE hasThe economic impact of SLE has been evaluated in managed care or Medicaid populations -25, but This retrospective analysis (GHO-09-1623) assessed the prevalence of SLE, the proportion of SLE cases on disability benefits, the contribution of SLE to new disability cases each year, and healthcare resource utilization and costs in a Medicare population from 2003–2007.This analysis was conducted using a 5% random sample of Medicare’s medical claims database obtained from the Centers for Medicare and Medicaid Services (CMS). This database is a nationally representative sample of patients seeking medical care through Medicare providers and can be extrapolated to the entire Medicare population. It contains all inpatient fee-for-service claims (Part A) and outpatient fee-for-service claims (Part B) for beneficiaries. Prescription data (Part D) was not available for this study. Actual dates of service are not available in the database, therefore the quarter in which the service was rendered is utilized.th edition (ICD-9) code 710.0x) during the identification period (first quarter of 2003 through the fourth quarter of 2006). The index quarter was defined as the earliest quarter of SLE diagnosis for the SLE cohort. For the non-SLE control group, inclusion criteria required that patients never had SLE during the study period. The index quarter for non-SLE patients was defined as the first quarter of Medicare enrollment for patients 65 years and older or the first quarter with a qualifying disability for patients under the age of 65 years. Patients were excluded from the non-SLE control group if they were <65 years of age with end stage renal disease (ESRD). Patient-level data were de-identified and thus exempt from institutional board review and ethics committee approval. The study was conducted in accordance with the principles of the Health Insurance Portability and Accountability Act (HIPAA).Inclusion criteria for the target SLE cohort were patients having 2 or more medical claims with a primary or secondary diagnosis of SLE  were deemed to receive disability based on the entitlement code  listed on claims. The proportion of new disability cases attributed to SLE was calculated as the number of new SLE cases with qualifying disability in a given year from 2003–2007 divided by the number of new disability cases in that year. The date of the first SLE claim was used in the disability analyses.Healthcare resource utilization and healthcare costs were compared between cohorts with and without SLE matched 1:2 on the basis of age (±2 years), gender, race, region, and index quarter (±2). To be eligible for the matched sample, patients had to have a first SLE diagnosis that occurred from the first quarter of 2003 through the fourth quarter of 2006 (index quarter) and had to be continuously eligible for medical services during the 2 quarters before and the 1 year after the index quarter.Outcomes were evaluated for 1 year after the index quarter and included all-cause healthcare utilization and costs (inflated to 2008 US dollars) for inpatient hospitalizations, emergency room visits, physician visits, and outpatient visits. Outpatient drug costs were not determined because these records are not included in the Medicare Part A or Part B datasets. Healthcare utilization and costs were summarized both for the matched sample as a whole and for the subset of the matched sample on disability benefits. Healthcare costs were also summarized in the matched sample as a whole as a function of race and gender. Re-hospitalization rates and average length of stay were also calculated.Inferential statistics was used to describe and quantify inter-cohort differences in baseline characteristics before the cohorts were matched. After matching, baseline characteristics were compared between cohorts using paired t-tests or Wilcoxon signed rank tests (continuous variables) and McNemar’s test . SLE prevalence, the proportion of SLE cases on disability benefits, and the contribution of SLE to new disability cases were summarized with descriptive statistics. Outcomes were compared statistically between cohorts using paired t-tests for continuous variables and McNemar’s test for categorical variables. Outcome comparisons were conducted for the sample as a whole and stratified by race and gender. Differences between cohorts were considered statistically significant at p < 0.001. SAS software was used for statistical analysis .The number of Medicare beneficiaries for the study period (5% random sample) was 2,719,310 of whom 13,348 had 2 or more claims for SLE. The prevalence of SLE in the overall Medicare population from 2003–2007  from 2003–2007. The proportion of new disability cases attributed to SLE ranged between 1.4-2.0%. Of the patients in the overall SLE cohort, the proportion receiving disability benefits increased from 45.0% in 2003 to 49.7% in 2007.A total of 13,348 patients met criteria for inclusion in the SLE cohort and 2,705,962 patients met criteria for the non-SLE cohort. Patient characteristics prior to matching are shown in Table In the matched sample overall, the percentage of patients in the SLE cohort who utilized all categories of healthcare, including physician visits, in-patient visits, outpatient visits, and emergency room visits, was significantly larger than the percentage utilized by the non-SLE cohort Figure . The bigIn post-hoc analyses of patients with at least one hospitalization, the average length of hospital stay was 6.4 days for both the SLE and non-SLE cohorts. Re-hospitalization rates were numerically higher (not tested for significance) in the SLE cohort (53%) compared with the non-SLE cohort (43%).Patients with SLE incurred significantly greater average annual healthcare costs than matched controls without SLE Figure . The cosAlthough several cost of illness studies have been conducted in patients with SLE in managed care or Medicaid populations, this is the first study to evaluate the prevalence, healthcare resource utilization and cost of SLE in a sample of Medicare beneficiaries. The prevalence of SLE in this large US Medicare population was found to be approximately 3 per 1000 across the 5-year study period spanning 2003 to 2007. This prevalence rate exceeds the approximate 1 per 1000 persons estimate obtained from several studies conducted in various settings during the past 3 decades [The findings that approximately half of SLE patients in this study were on disability benefits and that approximately 2% of new disability cases were attributed to SLE are consistent with previous reports linking SLE to functional impairment, poor quality of life, reduced productivity, and unemployment -20. In aIn the current study, patients with SLE in the Medicare population were significantly more likely to use healthcare resources than the matched control group without SLE, and the higher rate of healthcare resource utilization translated into significantly higher healthcare costs. On average, patients in the SLE cohort had at least two times more physician visits, hospitalizations, outpatient visits, and emergency room visits when compared to the non-SLE cohort —excess utilization that translated into $10,229 greater annual healthcare costs per patient. The annual per-patient healthcare cost of SLE in this study  was similar to or lower than the annual costs found in other studies of a managed care or Medicaid population  ,22-25. WThe difference in healthcare costs between the SLE cohort and the non-SLE cohort was similar between the matched sample as a whole and the subset of the matched sample on disability benefits. The difference in costs between the SLE cohort and the non-SLE cohort was also similar between males and females in the matched sample as a whole but was magnified in blacks compared with whites. Factors such as (a) greater disease severity attributable to genetic and non-genetic  factors in blacks and other minority groups ,30 and  was not available for inclusion in the study, which would increase the total economic burden of SLE. Third, clinical and disease-specific parameters that could impact study outcomes are not readily available in claims data. Despite the lack of clinical parameters, claims data are very useful in evaluating real-world resource utilization and cost patterns.This study quantifies the prevalence and economic burden of SLE in a large Medicare population. The prevalence of SLE in this Medicare study sample , was higher than the national prevalence estimate of 1 per 1000 people. More than 40% of all SLE cases in the Medicare sample (before matching) were patients <65 years of age receiving disability benefits. SLE is associated with high healthcare resource utilization and costs, on average 2 times higher utilization and $10,000 greater annual costs than non-SLE patients, with inpatient hospitalization as the largest cost driver. Thus, the Medicare population is an important patient segment to direct efforts to improve disease management strategies and reduce costs of SLE.