Airway management is a part of routine anesthetic procedures; however, serious complications, including hypoxia and death, are known to occur in cases of difficult airways. Therefore, alternative techniques such as fiberoptic bronchoscope-assisted intubation (FOB intubation) should be considered, although this method requires more time and offers a limited visual field than does intubation with a direct laryngoscope. Oxygen insufflation through the working channel during FOB intubation could minimize the risk of desaturation and improve the visual field. Therefore, the aim of this prospective randomized controlled study was to evaluate the utility and safety of oxygen insufflation through the working channel during FOB intubation in apneic patients.2) during FOB intubation  defined as the difference of PaO2 before and after intubation divided by the time to intubation. The secondary outcomes included the success rate for FOB intubation, time to intubation, visual field during FOB intubation, findings of arterial blood gas analysis, and occurrence of FOB intubation-related complications.Thirty-six patients were randomly allocated to an N group (no oxygen insufflation) or an O group (oxygen insufflation). After preoxygenation, FOB intubation was performed with (O group) or without (N group) oxygen insufflation in apneic patients. The primary outcome was the velocity of decrease in the partial pressure of oxygen , while the visual field was similar between groups. There were no significant intergroup differences in the secondary outcomes.We found that VThese findings suggest that oxygen insufflation through the working channel during FOB intubation aids in extending the apneic window during the procedure.ClinicalTrials.gov, NCT02625194, registered at December 9, 2015. In addition, the time to intubation, visual field, and occurrence of FOB intubation-related complications were investigated.Airway management is a part of essential anesthetic procedures. However, it becomes challenging in cases of difficult airways and may cause several complications, including airway trauma, hypoxia, and even death , 2. Therhttps://clinicaltrials.gov/ct2/show/NCT02625194?term=NCT02625194&rank=1). After the acquisition of written informed consent, 36 patients aged 20–60 years (American Society of Anesthesiolgists class I to III) who were scheduled for elective surgery under general endotracheal anesthesia with arterial cannulation at Yonsei University Gangnam Severance Hospital were included. The exclusion criteria included lung disease, anticipated difficult airway, inability to read or write, and pregnancy. The randomization table at www.random.org was used to allocate patients to an N group (no oxygen insufflation) or an O group (oxygen insufflation) at ratio of 1:1.This prospective randomized controlled study was approved by the Institutional Review Board of Yonsei University Gangnam Severance Hospital . The airway was opened by the jaw thrust maneuver, and endotracheal intubation was performed using a flexible FOB  with a preloaded endotracheal tube. In the O group, 100% oxygen was administered at 5 L/min through the working channel. The N group did not receive oxygen insufflation. Intubation success was confirmed by bronchoscopy, following which arterial blood was collected before the initiation of ventilation (T2). The first end-tidal carbon dioxide value after the resumption of ventilation following intubation was recorded.PaO2, defined as the difference of PaO2 between T1 and T2 divided by the time to intubation (mmHg/sec). Secondary endpoints included the FOB intubation success rate, time to intubation , visual field during FOB intubation , and occurrence of FOB intubation-related complications, including mucosal injury, abdominal distension, postoperative nausea and/or vomiting, and desaturation [peripheral capillary oxygen saturation (SpO2) < 90%]. In addition, vital signs and pH, partial pressure of carbon dioxide (PaCO2), and PaO2 values were recorded for all patients.The primary endpoint was VPaO2 of 0.9 mmHg/s with a standard deviation of 0.17, we found that 16 patients per group were required for detection of a 0.25 mmHg/s decrease in VPaO2 with a type 1 error and power of 0.05 and 80%, respectively [PaO2, Mann-Whitney U test was performed. Secondary endpoints including time to intubation, vital signs and arterial blood gas analysis data were also assessed with Mann-Whitney U test. Visual field and FOB intubation-related complications were analysed by Fisher’s exact test. In comparing the patients’ characteristics, continuous variables were assessed using Mann–Whitney U-test, while categorical variables were assessed using the chi-square test or Fisher’s exact test. The results from Mann-Whitney U test were expressed as mean with standard deviation. The categorical data was presented as number. The Bonferroni method was used to compensate the error of multiple comparisons. A P-value of < 0.05 was considered statistically significant.Considering the findings of Rosenstock, who reported a mean Vectively . Accordi2, PaO2 and SaO2 values also showed no significant intergroup differences throughout the procedure. The time to intubation was approximately 150 s in both groups. Although PaO2 showed no significant intergroup differences at T1 and T2, the decrease in PaO2 during FOB intubation was significantly greater in the N group than in the O group . In addition, VPaO2 was significantly greater in the N group than in the O group . An excellent visual field was obtained for 12 and 7 patients in the O and N groups, respectively, with no significant intergroup difference. None of the patients presented mucosal injury, abdominal distension, and desaturation. One patient in the O group exhibited postoperative nausea . If the hypoxia-free apnea time during FOB intubation is defined as the time from the discontinuation of mask ventilation at a PaO2 of 500 mmHg to the achievement of 90% SpO2 at a PaO2 of 60 mmHg, under the assumption that PaO2 exhibits a linear decrease during apnea, the hypoxia-free apnea duration could be approximately 10 min longer in the O group than in the N group .Several studies have attempted to overcome the limitations of FOB intubation. A direct laryngoscope or GlideScope can assist the placement of FOB near the glottis and facilitate easy intubation –18. In aThis calculation is based on a ventilatory mass flow, also known as apneic oxygenation. During regular breathing in an adult, oxygen and carbon dioxide are exchanged between the lungs and blood at a flow rate of 250 ml/min. During apnea, the carbon dioxide flow returning to the lungs is significantly reduced to 8–20 ml/min while the oxygen flow to the blood is maintained. Therefore, negative pressure is generated in the lungs according to the volume difference during oxygen and carbon dioxide exchange; this facilitates the movement of oxygen from the pharynx to the lungs .2 at 3 min during apnea [2 decreased to 92%. None of the patients in the apneic oxygenation group showed an SpO2 of < 97% until 10 min. On the other hand, the mean apnea time in the control group was 6.8 min and the lowest SpO2 value was 91% [Apneic oxygenation could be applied in various clinical situations involving different types of devices, including nasal prongs, nasopharyngeal catheters, and tracheal or bronchial catheters. The first two are commonly used because they are practical . Oxygenang apnea . In Tell was 91% . In pres was 91% .2O, and slow down the increase in the carbon dioxide level during apneic oxygenation [2 during the apneic period was similar in the O and N groups.One limitation of apneic oxygenation is that it cannot efficiently remove carbon dioxide from the blood, resulting in an increase in the carbon dioxide level at a rate of 1.1–3.4 mmHg/min and, eventually, hypercarbia and acidosis . Therefogenation , 26. In With regard to the visual field, the number of patients with an excellent visual field was not significantly different between groups, although the number was higher in the O group. This result was inconsistent with those of previous reports, and there are a few possible explanations for the discrepancy. Unlike Rosen’s study, which included pediatric patients with difficult airways and involved the use of smaller FOBs , the preThe two groups in our study showed a comparable intubation success rate and time to intubation; this could be attributed to the lack of differences in the visual field quality. The optimal intubation conditions may be another factor that repressed the influence of oxygen on the intubation-related parameters. We believe that different results may be derived if the measurements were recorded in emergency situations involving patients with unanticipated difficult airways. Further studies should take this aspect into consideration and assess the usefulness of oxygen insufflation during FOB intubation in different clinical scenarios.2 during FOB intubation. Oxygen insufflation through the working channel of FOB can cause rare but serious complications such as gastric rupture and pneumothorax [2O of pressure is required to open it [2O [From our results, it is evident that oxygen insufflation through the working channel of FOB can reduce VPaOmothorax , 11, 27.mothorax , 12. If mothorax , 28. How open it . Accordin it [2O , which in it [2O .2 measurement but not adequate to assess the occurrence of complications, which are anyways rare.This study has some limitations. First, FOB intubation was conducted under optimal conditions as described earlier. Therefore, the results cannot be applied to other clinical situations such as emergency difficult airway management. Second, our sample size was enough for VPaOIn conclusion, our findings suggest that oxygen insufflation through the working channel of FOB can help in extending the apneic window after the induction of general anesthesia during FOB intubation, with minimal complications.


Corrective osteotomy of a malunited distal radius conventionally relies on 2D imaging techniques for alignment planning and evaluation. However, this approach results in suboptimal bone repositioning, which is associated with poor patient outcomes. In this case series, we evaluate the use of novel patient-specific plates (PSPs), which feature navigation and fixation of bone segments as preoperatively planned in 3D.Ten participants with distal radius malunion underwent CT scans for preoperative alignment planning. Patient-specific guides and plates were designed, 3D-printed, and sterilized for use in corrective surgery of the distal radius. Pre- and postoperative results were compared in regard to clinical, functional, and radiographic outcomes.The application of a PSP was successful in 7 of the 10 cases. After treatment, the residual alignment error was reduced by approximately 50% compared with conventional treatment. The use of PSPs reduced pain significantly. Pre- and postoperative results were pooled and demonstrated significant correlations between: (1) pain and malpositioning, (2) the range of pro- and supination motion, the MHOQ score, the EQ-5D-5L score and dorsovolar angulation, and (3) MHOQ score and proximodistal translation.The correlation between malalignment and MHOQ score, EQ-5D-5L score, pain, and range of motion shows that alignment should be restored as well as possible. Compared to the conventional approach, which relies on 2D imaging techniques, corrective osteotomy based on 3D preoperative planning and intraoperative fixation with a PSP has been shown to improve bone alignment and reduce pain.IV. Fractures of the distal radius may result in symptomatic malunion of bone segments. Approximately 5% of patients with malunion of the distal radius  experienOver the last few decades, a number of techniques have emerged to plan the repositioning of bone segments preoperatively in 3D –21. The A novel approach to transfer the preoperative plan of a corrective distal radius osteotomy to the patient is based on a patient-specific plate (PSP). In this approach, a custom surgical guide is first used to predrill holes and to perform the osteotomy. Subsequently, the predrilled holes are used to fix the bone segments to a PSP using screws. The PSP is designed to fit the patient’s bone contour optimally; this has the advantage of aligning the bone segments according to the preoperative plan, which may limit tendon ruptures , and obvIn this case series, we evaluate the clinical, functional, and radiological outcomes of patient-specific plating in distal radius malunion. We hypothesize that the use of patient-specific plates improves the anatomical alignment of bone segments and therefore clinical outcomes.Patients who presented at our outpatient clinic with a malunion of the distal radius between June 2017 and June 2019 were CT scanned, and the deformity was evaluated using 3D modeling technology , 15, 16.Each patient underwent CT scanning of both the affected and healthy contralateral forearms. Scans were used for 3D preoperative planning and for postoperative evaluation at approximately 6 weeks and 6 months in order to evaluate initial and final positioning and consolidation, respectively. The scans were carried out using a Brilliance 64-channel CT scanner .Mdpre, and the proximal bone segment, Mppre. These matrices are used to quantify the preoperative malalignment, as will be discussed below.Preoperative planning began by carrying out a CT scan of both the affected and the contralateral bones for position planning. The contralateral bone in the mirrored image served as the reference bone, although a manual adaptation was sometimes needed to compensate for bilateral differences in length , 14. CusDuring preoperative planning, we further chose locations for osteotomy and screw placement. A patient-specific polyamide drilling/cutting guide and a plate were subsequently designed Fig.  10], an, an10], The drilling/cutting guide was fitted to the bone. Kirschner wires were used for temporary fixation of the guide, after which screw holes were drilled into the bone. The bone was cut using an oscillating surgical saw, first partially through the slit in the guide and then entirely after removal of the guide. After aligning the bone segments, the PSP is subsequently affixed, first to the distal bone segment using 2.4-mm locking screws  , and theImmobilization of the wrist was achieved using either a pressure bandage or a plaster cast, depending on patient preference. After surgery, the patients were instructed to keep the hand mobile by performing regular exercises but without exerting any additional external load, such as lifting. All patients started hand therapy on average 6 weeks after surgery, and received additional hand therapy if insufficient progress was observed.Hand function was assessed preoperatively and 6 months postoperatively by measuring range of motion parameters: flexion, extension, pronation, supination, radial deviation, ulnar deviation and grip strength. Grip strength was assessed using a Jamar™ dynamometer. Patients were asked to complete the Michigan Hand Outcomes Questionnaire (MHOQ) and the EuroQol EQ-5D-5L questionnaire (Dutch version) to rate their overall quality of life, expressed by an index in the range 0–1 (higher is better), and an overall Visual Analog Scale alternative (EQ-VAS) in the range 0–100 (higher is better).Mppre, we transformed the aforementioned distal and proximal bone segments to the target image containing the mirrored contralateral bone  was used for statistical analysis.Given the small sample size, we used nonparametric methods for statistical testing. A Wilcoxon signed-rank test for paired samples was used to evaluate the null hypothesis of the patient-specific method to improve positioning and clinical results. To evaluate if clinical parameters correlated with malalignment parameters, a Spearman’s product moment correlation coefficient (rho) was determined as well as its level of significance after pooling preoperative and postoperative results. A significance level of Patient characteristics are shown in Table In three of the first four cases, the 2.4-mm Synthes locking screws broke at the plate boundary, either during surgery when applying a torsional load while testing mobility (case #1) or during daily activities after approximately six weeks (case #3 and #4). In these cases, conventional treatment followed in which a standard plate was used for bone fixation. Given this issue, the plates for the remaining six patient cases (#5–#10) were designed for, and fixed with, 3.5-mm Synthes locking screws instead of their 2.4-mm counterparts. No screw breakage complications occurred after implementing this change to the surgical approach.In five other cases , secondary surgery was performed at an average of 13 months after the initial surgery. Four of these cases  were for hardware removal of plate and screws, and one patient  or the 6-week postoperative CT scan in cases where the screws broke . Figure N = 7 PSP cases. Table p = 0.016) and rotations about the radioulnar axis (p = 0.016). Pain and overall outcome of the hand (MHOQ) were significantly improved after using the PSP .Consolidation was confirmed by means of the 6-month postoperative CT scan. In patient #1, consolidation was confirmed using X-ray imaging. The preoperative and postoperative alignment errors were quantified by means of CT scan. To this end, the six-month postoperative CT scan was used . A significant positive correlation was found between pain and malalignment of the distal segment . Several parameters also showed a significant correlation with dorsovolar angulation of the distal segment . Finally, significant correlations were found between pronation and supination range, in percent of the healthy side and the TRE , and between the MHOQ score and proximodistal translation .To investigate the relationship between clinical and malalignment parameters, we pooled preoperative and postoperative results (U test with data from one of our previous studies [p < 0.001) than traditional treatment with a standard anatomical plate; at TTE = 6.8 mm and TRE = 17.5° for traditional treatment and TTE = 3.1 mm, TRE = 8.3° for the PSP approach. This observation is of the utmost importance, given the fact that the clinical outcome correlates with malalignment [In this study, we evaluated the use of a patient-specific osteosynthesis plate for transferring a preoperative 3D plan to the patient. Our proposed approach has the advantage of combining both navigated positioning and bone segment fixation by using a PSP. This could ultimately simplify bone repositioning and render the procedure more accurate and precise in comparison with alternative methods or conventional corrective osteotomies, which are based on 2D position planning using radiographic parameters . To be able to judge if positioning using a PSP performs better when compared with the conventional technique based on 2D imaging, we performed a groupwise Mann–Whitney  studies  in whichOur study further demonstrated a correlation between pain and malalignment parameters . Many clinical parameters also correlate with dorsovolar angulation of the distal segment . In a previous study, a correlation between clinical parameters and malalignment parameters was also established  . Volar aA number of different techniques have been proposed to transfer a preoperative 3D positioning plan for the distal radius to the patient. Athwal et al. proposed using tool-tracking equipment . Their sX, Y, and Z axes of a local coordinate system. The residual positioning errors that we found postoperatively  were in the same order of magnitude as in the aforementioned studies but seem slightly larger compared to the results of Oka et al. [Titanium PSPs have been 3D printed for a multitude of clinical applications, including reconstructive surgery of the skull , orbitala et al. . The difa et al.  or usinga et al. .Although the results of this case series seem promising, there are several limitations worth mentioning. The generalizability of our study may be limited due to a possible bias since a surgeon selected those cases that were eligible for treatment by PSP. We recommend carrying out a randomized controlled trial for further evaluation of the method and to quantify more parameters such as the ease of surgery and the overall costs including possible revision surgery and aftercare. This could confirm the finding of Bauer and coworkers  who repoWe have shown that preoperative pain, range of motion, MHOQ, and EQ-5D-5L scores correlate with malalignment of the distal bone segment. The PSP approach has been shown to improve bone alignment and reduce pain. It has further been shown to improve bone alignment in a way comparable to what can be achieved when using sophisticated 3D planning with a drilling/cutting guide, and the subsequent reduction guide. Overall, it performed better than conventional treatment solely based on planning using X-ray images and intraoperative fluoroscopic 2D evaluation.


Bamboo fibers are utilized for the production of various structures, building materials, etc. and is of great significance all over the world especially in southeast Asia. In this study, the extraction of microcrystalline cellulose (MCC) was performed using bamboo fibers through acid hydrolysis and subsequently different characterizations were carried out using various advanced techniques. Fourier transform infrared (FTIR) spectroscopy analysis has indicated the removal of lignin from MCC extracted from bamboo pulp. Scanning Electron Microscopy (SEM) revealed rough surface and minor agglomeration of the MCC. Pure MCC, albeit with small quantities of impurities and residues, was obtained, as revealed by Energy Dispersive X-ray (EDX) analysis. X-ray diffraction (XRD) indicates the increase in crystallinity from 62.5% to 82.6%. Furthermore, the isolated MCC has slightly higher crystallinity compared to commercial available MCC (74%). The results of thermal gravimetric analysis (TGA) demonstrate better thermal stability of isolated MCC compared to its starting material (Bamboo fibers). Thus, the isolated MCC might be used as a reinforcing element for the production of green composites and it can also be utilized as a starting material for the production of crystalline nanocellulose in future. The rapid industrial growth that we are witnessing today has led to an ever-increasing demand for different nano/micro functional and structural materials in various industries for applied research and developments. The modern industrial societies are increasingly extravagant in the use of nano/micro materials for various advance applications. Advanced materials generally have superior properties to conventional materials available, thus they could simply outperform conventional materials in terms of their properties and applications ,2. AdvanIn this context, cellulose is one important polymer, which could be tuned according to the requirements and could also be used as functional as well as structural material for the production of valuable composites. It is the most abundant organic compound found on earth that has long been a major reliable renewable source of materials ,7. FurthCellulose can be obtained from many natural resources, for example, wood, plant, bacteria, and algae. Through hydrolysis process, cellulose can be converted to microcrystalline form known as Microcrystalline Cellulose (MCC) or nanocrystalline form known as Nanocrystalline Cellulose (NCC).MCC, one of the cellulose derivates, is a naturally occurring particle. It is a fine, odourless white, crystalline powder and possesses important characteristics that include non-toxicity, biocompatibility, biodegradability, high mechanical strength, large surface area, and low density, etc. ,11. Due In this study, a very smooth, well known, highly tunable and straightforward approach was chosen for the isolation of MCC from bamboo fibers. Bamboo is by far the most important among the natural fiber plants because of its rapid growth rate and universality. Bamboo is a group of perennial evergreens in the true grass family Poaceae, subfamily Bambusoideae, tribe Bambuseae. More than 1,450 bamboo species from 70 genera are found in diverse climates-from cold mountains to hot tropical regions ,19. BambVarious sophisticated techniques have been used for the characterization of intermediate materials and final product, MCC.−1 for each sample within 650–4000 cm−1 range. The “find peak tool” functionality of Nicolet software  was used to determine the positions of significant transmittance peaks at a particular wave number.FTIR was carried out to understand chemical changes during the production of MCC. Perkin-Elmer 1600 Infrared spectrometer was used for the detection of various functional groups introduced during the isolation procedure. The spectra are collected by the spectrometer with 32 running scans at a resolution of 4 cmTo determine morphology of MCC samples, scanning electron microscope (SEM) (Hitachi Model S-3400N) was used. SEM model comes laced with energy dispersive X-ray (EDX) equipment, which has a 15kV accelerating voltage. To minimize the charging effect, the MCC samples were gold sputtered and subsequently the samples were observed. EDX diffraction was used for identification of elemental composition of the MCC samples. The analysis of the particle size of MCC samples was performed using Mastersizer 2000 particle size analyser.I002 represents the peak intensity corresponding to the crystalline domain (20° to 19.0°), and Iam represents the peak intensity corresponding to the amorphous domain (2° to 22.6°).X-ray diffraction (XRD) is a very powerful technique which is widely used for characterizing crystalline materials. In this study, XRD patterns were recorded by SIEMENS D5000 X-ray diffractometer using Ni-filtered Cu K-alpha radiation using an angular incidence of 5° to 40°. The percentage of crystallinity index (Crl) was calculated using Equation (1):Thermal gravimetric analysis (TGA) was performed to analyse the thermal properties of MCC using a Q500 Thermogravimetric analyser . The samples, weighing about 6 mg, were subjected to pyrolysis under nitrogen gas (N2) within 30 °C to 900 °C temperature with 20 °C/minute heating rate.In this study, a well-known procedure, acid hydrolysis, was followed for the isolation of MCC from bamboo. It is worth mentioning that the opted procedure is easily scalable and by changing the concentration of used solvents/chemicals, degree of polymerization, crystallinity of MCC, particle size distribution, and thermal properties etc. it could easily be controlled. The chemical composition of used bamboo fibers is mentioned in , change is color (brown to pure white) after every applied step indicates the separation of various hemicellulose, lignin, and other impurities from fibers. A pure white isolated MCC is shown in Figure 1Morphology of isolated MCC and intermediates (cooked and bleached pulp) have been reported in −1 to 500 cm−1 in order to understand the change in the chemistry of various samples during the isolation of MCC from bamboo fibers as shown in −1 in the case of cooked pulp and bleached pulp is attributed to the O−H stretching vibrations and the same O−H peak showed a red shift in the case of MCC at 3338 cm−1 while the peak frequencies at 2900 cm−1 and 2905 cm−1 correspond to C−H stretching vibrations. Furthermore, the MCC spectra shows almost the disappearance of characteristic C−O stretching vibration at 1722.16 cm−1 and 1533 cm−1, which corresponds to the acetyl and uronic ester groups of hemicelluloses as well as the ester linkage of the carboxyl groups of lignin and to the C=C vibration in lignin respectively. Additionally, significant peaks at 896 cm−1 (β-glycosidic bonds bending), 1050 cm−1 (C−OH stretching), and 1160 cm−1  represent unique fingerprint regions of all the processed samples.To study the conformational and physicochemical properties of polysaccharides, FTIR analysis is considered as one of the powerful tools. FTIR spectra was recorded for all the samples in order to observe the change in the peak frequencies of various functional groups. The chemical composition of all the samples does not show marked change as the samples go through the extraction process. The spectra were carried out from 4000 cmXRD pattern of CP, BP and MCC are presented in The EDX spectra for C-pulp, B-pulp and MCC are presented in The determination of thermal stability is crucial for applications where the processing temperature is higher especially in the production of biocomposites ,33. The Bamboo chips used in this study were provided by INTROP, UPM, Serdang, Malaysia. The initial moisture content of the bamboo chips was 8.3%. The other materials like sulphide, sodium hypochloride (NaClO), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Sigma Aldrich, Saint Louis, MO, USA. All chemicals used in this study were laboratory graded and were used as procured.The steps involved in the isolation of MCC from bamboo fibers are given Rotary digester having a capacity of 4 L was used for kraft pulping. The 20% of active alkali and 30% of sulphide, in the ratio of 1:7, was used in the process. Cooking was performed at 170 °C for 3 h. The utilized pressure was about 12–14 bar. The pulp was washed and screened using Somerville Shive content analyser and the black liquor was discarded. After that, the pulp was kept in an oven for 24 h at a temperature of 60 °C, resulting in dried pulp.2O to obtain white-yellowish fibre. Afterwards, it was dried in an oven at 60 °C for 24 h.Dried cooked pulp obtained from kraft pulping was subjected to bleaching process to separate lignin and hemicellulose using sodium hypochloride (NaClO). The process was carried out for 1 h at 70–80 °C. The pH of the solution was 12.0. Acetic acid was regularly added during the bleaching step and constantly stirred until its pH reached 4. Obtained bleached fiber was filtered and washed twice with distilled Hw/v) sodium hydroxide (NaOH) solution . Reaction procedure was carried out at room temperature for 30 min. Obtained pulp  was filtrated and washed followed by drying in an oven at 60 °C for 24 hrs.The bleached fibers were treated with 8% (−1). Hydrolysis was performed under agitation using a constant stirring speed. The obtained product was made to cool by keeping it at ambient temperature followed by filtration and washing to make pH neutral. The product obtained was dried in a vacuum at 80 °C until constant weight was achieved. Obtained white power was stored and denoted as MCC in the article.Acid hydrolysis is a well-known chemical process for removing the amorphous regions of cellulosic materials. In this process, strong acids like sulphuric acid, hydrochloric acid (HCl), etc. are used for dissolving the unlikely parts of cellulosic fibers. In this study, the fiber  was treated with 2.5 mol/L HCl. Hydrolysis process was carried out at 85 °C for 30 min. and the solid-to-liquor ratio being used was 1:30 (g/mLThis study revealed that bamboo fiber has the potential to be used as an alternative for the extraction of MCC by following processes that include bleaching, alkaline treatment and acid hydrolysis. FTIR spectra indicated that there was no change in the chemical structure of cellulosic components. Moreover, the chemical treatments have caused a significant amount of lignin removal from bamboo fiber. The SEM that provides the morphological analysis indicates that a rough rod-like and lighter structure of MCC were obtained after bleaching and acid hydrolysis of bamboo pulp. The prepared MCC also exhibited a high crystallinity index of 82.6%, which endorses its suitability for application in packaging materials. Based on the TGA analysis, MCC displayed greater thermal stability, confirming its suitability for application in polymeric composites of higher processing temperature. The MCC extracted in this research can find application in biocomposite packaging materials.


The first biosimilar of recombinant follicle stimulating hormone (rFSH) launched in Europe was Bemfola® in 2014 following a clinical development programme demonstrating efficacy and safety to the satisfaction of the European Medicines Agency. Since then the increasing use of biosimilar rFSH has provided the opportunity to study both effectiveness across the whole population and the variation of rFSH use during routine clinical care in a real-world setting in Spain.This is a real-world study of 1222 women treated in 26 assisted reproduction treatment centres throughout Spain providing experience of the use of a biosimilar recombinant follicle stimulating hormone in four distinct populations. The four populations studied were poor responders, suboptimal responders, normal responders and oocyte donors. The primary endpoint was the total number of oocytes retrieved. Secondary endpoints included number of days of rFSH stimulation, total dose of rFSH administered, number of MII oocytes, number of fertilized oocytes, quality of embryos, number of embryos transferred, implantation rates, clinical pregnancy rates following embryo transfer, number of multiple pregnancies and number of serious adverse reactions, including moderate-to-severe OHSS.Differences were seen across the populations both in the characteristics of the women and ART outcomes suggestive of a continuum of fertility prognosis. In the poor responders, suboptimal responders, normal responders and oocyte donor populations the mean age in years was 39.9 (±SD 3.4), 38.4 (±SD 2.9), 34.4 (±SD 3.3) and 26 (±SD 4.6) respectively and number of oocytes retrieved was 4.1 (±SD 2.7), 8.6 (±SD 6.0), 12.2 (±SD 7.2) and 19.5 (±SD 9.5) respectively. The proportion of embryos graded as best quality was 18.5%, 33.0% and 43.8%, and graded as worst quality was 20.4%, 5.8% and 5.8% for poor responders, suboptimal responders and normal responders respectively. In a similar pattern, for poor responders, suboptimal responders and normal responders the implantation rates were 16.0%, (8/50), 22.4% (49/219), 30.6% (97/317) respectively and clinical pregnancy rates were 23.2% (10/43), 30.4% (59/194) and 37.0% (114/308) respectively. Adverse events were reported in only 7 of 1222 women (0.6%).Overall the results were consistent with the national ART results reported for Spain, hence this study provides reassurance of the clinical effectiveness of a biosimilar rFSH used in a real world setting.NCT02941341.ClinicalTrials.gov identifier -  The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE) estimated that in 2014 one in every 50 children born in Europe were the result of Assisted Reproductive Technology (ART) treatments , which hThe basis of the demonstration of equivalence between a biosimilar and the reference product primarily relies on exhaustive, highly sensitive physicochemical and biological activity comparability assessments later supported by clinical studies leading to a total development time of typically 6 to 12 years . For bioBeyond the demonstration of efficacy in carefully controlled phase 3 clinical trials on selected populations, the value of assessing the effectiveness of new treatments across the whole population undergoing routine clinical care in a real-world setting is becoming increasingly recognised . The EU The BIRTH study was a post EMA approval, non-interventional study of women who had undergone oocyte retrieval after ovarian stimulation with Bemfola® with GnRH antagonist pituitary-suppression, either as part of an autologous in vitro fertilisation (IVF) / intracytoplasmic sperm injection (ICSI) treatment cycle or for oocyte donation. Other than the latter requirements treatment was according to the centres’ standard of care. In 28% of autologous embryo transfers all embryos had been cryopreserved and embryo transfer delayed. The study was carried out in 26 Spanish Assisted Reproduction centres, both public and private, belonging to 10 regions throughout Spain.The study was designed by the sponsor  supported by a group of Spanish ART experts. The study was approved by the Ethics Committee of Clinical Research of Euskadi (CEIC-E). Enrolment was conducted from September 2016 until April 2018 with the data collected and analysed by Dynamic, Azcona, 3,128,028 Madrid, Spain. All authors were involved in the preparation of the paper and validate the accuracy of the data.Women aged ≥18 years at recruitment, either patients undergoing an IVF/ICSI procedure or egg donors who completed a controlled ovarian stimulation receiving at least 5 doses of Bemfola® were included in the study. Pituitary suppression was achieved by administering GnRH antagonists. All participating women signed an Informed Consent Form, prior to any data collection. Women reporting hypersensitivity to follitropin alfa or to any of the excipients of Bemfola® were excluded. The presence of a pituitary or hypothalamic tumour was also an exclusion criteria.Patients were divided into four populations  defined as follows. Poor responders were defined according to the Bologna definition of having at least two of the following criteria: advanced reproductive age (≥40 years) or any other risk for poor ovarian response; previous poor ovarian response  < 0.5–1.1 ng/ml . SuboptiThe primary endpoint was the total number of oocytes retrieved. Secondary endpoints included number of days of rFSH stimulation, total dose of rFSH administered, number of MII oocytes, number of fertilized oocytes, quality of embryos, number of embryos transferred, implantation rates, clinical pregnancy rates following embryo transfer and number of multiple pregnancies. To support routine post marketing pharmacovigilance monitoring moderate-severe ovarian hyperstimulation syndrome (OHSS) and serious adverse reactions were recorded.Since the purpose of this study was purely descriptive, no formal sample size calculations based on comparative hypothesis testing were conducted but rather the sample size was based on ensuring a suitable size to describe the effectiveness of Bemfola® in routine use. As this was a real world study doctors were free to treat patients as they felt appropriate and there was no randomisation nor stratification into study groups. It would thus be anticipated that there would be considerable heterogeneity within groups, which may be subject to a number of biases. Accordingly, the data is only provided descriptively using appropriate methodology to avoid misleading the reader with any attempt at comparative analyses, which would be inherently flawed.n = 96), suboptimal responders (n = 301), normal responders (n = 386) and oocyte donors (n = 439). Data were not collected from the oocyte recipient treatment cycles and patient flow for each population throughout treatment is summarised in Table A total of 1222 women were available for data analysis divided into four populations: poor responders , in whom adverse events were reported. Ovarian hyperstimulation syndrome (OHSS) of at least moderate severity occurred in 1 case in the suboptimal responder population and in 4 cases of the normal responder population, of which 1 case was reported as severe OHSS. In addition, two serious adverse events were reported. One patient (out of 386), in the normal responder population, reported severe pain after egg retrieval. One oocyte donor (out of 439 women) reported a Bell’s palsy, which was considered unrelated to medication. All adverse events reported during the study were collated with adverse events reported directly to the Gedeon Richter pharmacovigilance department and the consolidated pharmacovigilance data is presented in this manuscript.By studying four populations of women  the BIRTH study shows a clear and related continuum of ovarian response and fertility among women. The majority of patients were not classified consistently across all centres with a clear sole cause of infertility illustrating the complexity of understanding the cause of infertility and agreeing to a common diagnostic terminology . NeverthAlthough this study adopted the Bologna criteria  to definn = 6473) and 44.5%, 36.3% and 21.2% for ICSI  according to ages < 35 years, 35–39 years and ≥ 40 years respectively [No lack of efficacy nor pharmacovigilance signals were revealed in this real-world study of the use of this new product and in fact the differences between patient groups was far more relevant to whether a patient was successful with ART or suffered an adverse event than the choice of gonadotrophin. Furthermore, the pregnancy rates in the BIRTH study were consistent to the overall pregnancy rates per embryo transfer reported in 2017 to the national Spanish ART registry of 42%, 36.5% and 25.6% for IVF  , 25. TheAlthough a real world study has the advantage of reporting on the whole highly heterogeneous population receiving a very diverse range of clinical ART practices, inevitably this leads to study limitations of low internal validity, lack of quality control and susceptibility to multiple sources of bias. Further the BIRTH study did not present spontaneous abortion rates, live birth rates and rates of congenital anomalies. Hence, real world studies such as the BIRTH study should be viewed as part of a wider body of evidence to guide clinical practice.Fertility is a continuum from poor responders, suboptimal responders, normal responders through to fertile oocyte donors. Bemfola® produced the anticipated number of oocytes according to patients’ populations with adequate embryo quality and pregnancy rates in a real world setting which were consistent with Spanish national ART reports. In addition a low rate of adverse reactions were reported. The results seen regarding the use of combination protocols reflect the medical literature and recent guidance from ESHRE questioning the value of adding LH activity to gonadotrophin stimulation protocols .


Slc38a4 at d18.5. Neither placental and fetal weights, nor placental DNA methylation levels were affected. Thus, total caloric restriction beginning in the periconceptional period does have a lasting impact on placental development in the mouse, but without changing placental efficiency.Maternal undernutrition has detrimental effects on fetal development and adult health. Total caloric restriction during early pregnancy followed by adequate nutrition for the remainder of gestation, is particularly linked to cardiovascular and metabolic disease risks during adulthood. The placenta is responsible for transport of nutrients from the maternal to fetal circulation, and the efficiency with which it does so can be adjusted to the maternal nutrient supply. There is evidence that placental adaptations to nutrient restriction in early pregnancy may be retained even when adequate nutrition is restored later in pregnancy, leading to a potential mismatch between placental efficiency and maternal nutrient supplies. However, in the mouse, 50% caloric restriction from days 1.5–11.5 of gestation, while temporarily altering placental structure and gene expression, had no significant effect on day 18.5. The periconceptional period, during which oocyte maturation, fertilization, and preimplantation development occur may be especially critical in creating lasting impact on the placenta. Here, mice were subjected to 50% caloric restriction from 3 weeks prior to pregnancy through d11.5, and then placental structure, the expression of key nutrient transporters, and global DNA methylation levels were examined at gestation d18.5. Prior exposure to caloric restriction increased maternal blood space area, but decreased expression of the key System A amino acid transporter  Chronic diseases are leading causes of morbidity and mortality. For example, in the United States more than 29.1 million people 9.3%) are diabetic, and at least one-third of these cases in adults and 17% in children are associated with obesity % are dia and obesIn the classic Dutch Hunger Winter Study, higher rates of obesity and cardiovascular disease were found in adult sons of mothers that received only about 50% of normal calories during the first trimester, followed by an adequate diet later in gestation , 9. In cThe mechanisms by which the particular combination of early gestation undernutrition and adequate nutrition in mid-late gestation programs poor adult outcomes remain poorly understood. The placenta is the organ responsible for the transfer of nutrients from the mother to the fetus. It is also in direct contact with both the maternal and fetal circulations. The placenta therefore can respond to maternal environmental stimuli to in turn affect the growing fetus . There iHere, we test the hypothesis that caloric restriction will permanently impact the mouse placenta if it occurs over the periconceptional period. Periconception is defined as the period from oocyte maturation and selection through early embryo development . Particuad libitum for one week to acclimatize them to their surroundings. Specialized feeding containers were used so that daily food consumption could be recorded [Fig 1. Beginning three weeks prior to mating, female mice were randomly separated into two groups: a control group fed ad libitum and a restricted group fed 50% of previous average daily food consumption for each individual. Mice were fed at 2 pm each day. During the mating period, males were placed in the female cages each night at 8 pm and returned to their own cages each morning, with free access to food until a copulatory plug was detected for up to five nights. Females on the nutrient restricted diet consumed their entire food allotment each day before males were added to the cage. The day a copulatory plug was observed was termed d0.5 of gestation. Dietary treatments continued until d11.5, and then all mice were fed ad libitum from d11.5 to d18.5. All animals were allowed free access to water. At d18.5, mice were sacrificed, and placentae collected to examine placental morphology and gene expression. In total, tissues were collected from five mice in the control group and six in the restricted group.All animal procedures were approved by University of Missouri Institutional Animal Care and Use Committee. Swiss Webster mice 6–8 weeks old and ≥ 25g were obtained from Envigo . They were individually housed in a 12hr dark, 12hr light cycle and fed an AIN-93G  growing rodent diet recorded . The exp2 asphyxiation and cervical dislocation, and fetuses were euthanized by decapitation. Placentae were collected, and placental and fetal weights were recorded. One half of each placenta was fixed in 4% paraformaldehyde in PBS overnight for histological analysis. The second half of each placenta was divided into two pieces, one for gene expression analysis and the second for DNA methylation analysis. Both were snap frozen in liquid nitrogen and stored at -80°C until needed.At d18.5, pregnant females were euthanized by COFixed placentae were dehydrated and embedded in paraffin. Placentae were cut into 6 serial mid- sagittal sections, 5um thick, 50um apart, mounted on glass slides and stained with hematoxylin and eosin (H&E) . The largest cross-section on each slide was chosen for morphological analysis. For determination of total cross-sectional area and that of each of the major zones, overlapping images were photographed with a 4x objective lens on a Zeiss Axiovert 200M microscope, and arranged into a single, high-resolution image of the entire placental cross-section. Junctional zone and labyrinth zone areas were measured by manual outlining using the freehand selection tool in ImageJ software (NIH) by an operator blinded to treatment groups. Three placentas were chosen at random from each litter for the morphometric analysis by drawing three tubes from a bag.To identify fetal blood vessels in the labyrinth zone, cross sections were immunohistochemically stained for PECAM1 (CD31) by using VECTASTAIN Elite ABC HRP Kit . Briefly, sections were rehydrated in a graded alcohol series, and antigen retrieval was performed by boiling in sodium citrate buffer  for 3 minutes in a pressure cooker, and then incubated in normal goat serum diluted according to manufacturer’s instructions to block non-specific binding. Placental sections were then incubated with anti-PECAM1 (Abcam ab28364) overnight at 4C. Secondary antibody and color development were carried out according to Vectastain instructions, and nuclei were counterstained with hematoxylin. Five representative images were photographed per placenta with a 40x objective on an Olympus IX81 microscope. The total cross-sectional areas of maternal blood sinuses and fetal blood vessels were determined by using the freehand selection tool within ImageJ software.Three placentas were chosen at random from each litter for analysis of gene expression. Placental tissue was homogenized in Trizol Reagent (Sigma Aldrich) with an Omni GLH Homogenizer. After phase separation, RNA was further purified using Qiagen RNeasy kit  according to manufacturer’s protocol. RNA concentration was determined by absorbance using the Take3 Plate (Bio-Tek Instruments). Two μg of total RNA was reversed transcribed with SuperScript III First-Strand Synthesis System , using random hexamer primers, according to manufacturer’s protocol.Hand1) was selected as a marker for trophoblast giant cells [Tpbpa) for spongiotrophoblast and glycogen cells [Esx1) for synctiotrophoblast cells [Pecam1, CD31) for endothelial cells [Prl3b1) marks spongiotrophoblast, parietal and sinusoidal trophoblast giant cells, and has been found to be regulated by fasting at the protein, but not mRNA level [Ctsq) is a marker of sinusoidal trophoblast giant cells [Real-time RT-PCR was used to measure relative transcript levels of genes specific to the major trophoblast lineages: heart and neural crest derivative 1 , a key fatty acid transporter, and Srebp1c¸ (Sterol regulatory element binding transcription factor 1), a regulator of fatty acid synthesis [Slc2a3 (GLUT3) is the major placental glucose transporter and has previously been shown to be regulated by maternal nutrition [Slc7a5 (LAT1) and Slc38a4 (SNAT4) [Additionally, messenger RNAs encoding six key placental nutrient transporters were examined. The placenta expresses ynthesis , 43. Slcutrition –46, as h (SNAT4) , 47, 48.Table 1) were synthesized by Integrated DNA Technologies. Primers for Slc7a5 were predesigned by IDT (Mm.PT.58.10563809). The specificity and efficiency of each reaction was validated by using serial dilutions of placental cDNA. PCR was conducted in triplicate using RT2 SYBR Green ROX qPCR Master Mix (Qiagen) in a final volume of 12μl on a CFX Connect Thermocycler (Bio-Rad). Cycling conditions were as follows: 95°C for 10 minutes, 40 cycles of 95°C for 15 secs, 60°C for 1 minute. No-template and no reverse transcriptase controls were performed. Data from each gene of interest were normalized to the average values of housekeeping genes hypoxanthine phosphoribosyltransferase 1 (Hprt) and Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh), which were determined not to change with nutrient restriction. Fold change differences in gene expression and error bars were calculated using the ΔΔCt method [Primers (t method .Genomic DNA was isolated as previously described  from thrE.Coli (used for the standard curve) genomic DNA, according to manufacturer’s instructions.The level of global DNA methylation was measured using the 5-mC DNA ELISA kit (Zymo Research) according to the manufacturer's protocol, with the following exceptions: (1) Antibody incubation was performed overnight at 4⁰C; (2) 200ng of DNA was used per standard and sample well. To construct a standard curve, methylated DNA (Zymo Research) and unmethylated Lambda DNA (New England BioLabs) were mixed in various proportions . Intra-assay coefficient of variation was 5.41%. Samples were run in duplicate. To calculate the percentage of CpG that are methylated, the %5-methylcytosine (mC) calculated from the standard curve was corrected for the differences in average CpG density between mouse and Rbm31x and the corresponding 353 bp region of Rbm31y by PCR (Table 1) [RNA was isolated and reverse transcribed or DNA was isolated, as described above for each placenta used for the morphometry, RT-PCR, or methylation analyses. Sex was then determined by amplification of a 269 bp region of Table 1) .Fetal and placental weights were averaged for each litter, and these averages were compared between control and restricted groups by using Student’s t-test in GraphPad Prism software. Morphological features were analyzed by using the Mixed Procedure in SAS, with treatment group and placental sex as independent variables and litter nested within treatment group as a covariate, to account for the interdependence of placentas from the same litter. The same method was used to compare delta Ct values for analysis of real-time RT-PCR data. Data were termed statistically significant if p< 0.05.Fig 2A–2D). No structural abnormalities, hemorrhaging, or necrosis were observed . Similarly, there were no significant differences in the appearance or cross-sectional areas of the junctional zone . While the total cross-sectional area of the labyrinth zone also was not affected , the structure of blood spaces within it was altered . Specifically, there was an approximately 20% increase in the cross-sectional area of maternal blood sinuses per unit area following periconceptional exposure to food restriction . Maternal blood sinus area also differed by sex, with male placentas having greater area than female placentas, but both sexes responded similarly to food restriction.The overall size of the placenta near term, measured either by weight or cross-sectional area, was not different in dams fed an adequate diet throughout pregnancy and dams restricted during the periconceptional period-mid gestation . There were no significant differences in relative mRNA levels of placental lineage markers Esx1 (synctiotrophoblast), Tpbpa (spongiotrophoblast and glycogen cells), Hand1(trophoblast giant cells), Prl3b1 [Ctsq  [Pecam1 [Prl3b1 expression did differ with placental sex, with males expressing more than females (p = 0.01). Tpbpa expression also was significantly higher in males than in females, but only in the restricted group (p = 0.003) .In order to further characterize the permanent impacts of periconceptional food restriction on placental development, semi-quantitative real-time RT-PCR was used to compare steady-state mRNA levels of transcription factors that are specifically expressed in the major trophoblast sublineages within the placenta (t cells) , Ctsq (st cells)  or Pecaml cells) . HoweverFig 4B). In the placenta from previously food restricted dams, there was a three-fold decrease in mRNA encoding Slc38a4, which transports neutral amino acids such as glutamine, glycine, serine and alanine [Slc38a4 mRNA levels were also higher in males than females, although food restriction had the same effect regardless of sex. In contrast, there were no differences in mRNA levels for the L-type, branched-chain amino acid transporters Slc7a5 and Slc7a8 . Levels of mRNA for fatty acid transporter Cd36 and Srebp1c, a transcription factor that regulates genes involved in sterol biosynthesis, were not changed, nor were there differences in expression of glucose transporter Slc2a3 between control and restricted placentae.Real-time PCR was also used to compare expression of key placental nutrient transporters at d18.5 following periconceptional food restriction . There was no significant difference in the overall percentage of methylated CpGs between placentas from control dams and those exposed to periconceptional undernutrition (p = 0.8), or between male and female placentas (p = 0.4).An ELISA was used to quantify the effects of periconceptional food restriction on the total level of placental DNA methylation (Slc38a4 (SNAT4), even in late pregnancy.The goal of this study was to determine whether the periconceptional period is a critical time in which maternal total caloric restriction can permanently alter placental development and function. Whereas studies in humans and sheep found that total caloric restriction during early pregnancy leads to placental overgrowth near term , 22, 23,These results are consistent with the hypothesis that adverse exposures during the periconceptional period, including balanced, total, caloric restriction, have irreversible effects on the placenta. Exposure to a low protein diet from conception to implantation results in a smaller placenta, with greater glucose transport and reduced amino acid transport near term , 31. AlcThe current study, combined with our previous work, shows that in the mouse a period of nutrient restriction that includes periconception (-3 weeks to d11.5) but not the period immediately following (d1.5–11.5), is sufficient for lasting effects on the placenta, strongly suggesting the importance of periconception. However, one limitation to this study is that nutrient restriction was not limited to the periconception period. We cannot rule out the possibility that nutrient restriction both before and after d1.5 are needed. Furthermore, the overall length of the nutrient restriction was longer than in than in the previous study, which could contribute to the more long-lasting effects. Future experiments targeted to just -3 weeks through d1.5, or an even narrower window, would help to more precisely pinpoint the period of susceptibility.Nonetheless, the window of susceptibility identified in the present study provides some insight into possible mechanisms of action. From -3 weeks to d1.5, the oocyte, and then the zygote, undergo major rewriting of DNA methylation. Maternal imprints are established in the oocyte, and methylation of most non-imprinted genes is erased shortly following fertilization. Evidence from monozygotic twins further shows that many DNA methylation marks are established very early in embryonic development, prior to twinning . A diet ad libitum feeding in the calorie restricted mice. However, it is known that caloric restriction can induce later hyperphagia in non-pregnant mice, and that estrogen plays a role in acute refeeding after fasting [ad libitum feeding at day 11.5, that they consumed more calories than the control mice. Additional experiments, including pair-feeding, would be needed to determine whether post-restriction eating plays a role in the placental phenotype on day 18.5.Another unknown variable is the effect of refeeding on the growth and function of the placenta. Very little is known about the dynamics of refeeding after nutrient restriction in pregnant mice, and here food consumption was not determined after the resumption of  fasting , 74. ThuSlc2a3 is reduced in the late gestation mouse placenta following assisted reproduction procedures in the periconceptional period [Slc2a3 expression in rodents. However, here no significant differences in late gestation gene expression of Slc2a3 were detected in placentae from mothers that were nutrient restricted during early pregnancy. Along with the previous observation that placental Slc2a3 levels are acutely regulated by maternal glucose concentrations [Placental adaptations in response to maternal undernutrition that outlast the period of undernutrition are potentially harmful because they produce a mismatch between the placenta and the environment. The placenta is “nutrient sensing,” adjusting structure and nutrient transport to preserve fetal growth in the face of reduced maternal nutrient supplies . For exal period , 76. Call period  and stiml period  placentatrations , these rSlc38a4 mRNA levels increase with periconceptional exposure to assisted reproduction procedures [Slc38a4 in placentas from previously nutrient restricted dams was only one-third that of controls. This reduction would be predicted to decrease amino acid transport and thereby limit fetal growth. Conversely, placentas from dams that had been nutrient restricted also had increased maternal blood space area within the sampled regions of the labyrinth zone, providing a greater surface area for nutrient exchange between the maternal blood and surrounding syncytiotrophoblast; this would support greater growth. Thus, the overall consequences of the combined placental adaptations for fetal growth and development are difficult to predict, but the observed lack of change in fetal weights could be a result of these placental alterations canceling one another. While both alterations were present at d18.5, it is possible that one occurred first, triggering a compensatory response to maintain consistent amino acid transport.Amino acid transport is also regulated by maternal nutritional status during pregnancy . The Sysocedures . In the Offspring exposed to undernutrition during early pregnancy have greater chronic disease risk as adults, even without changes in birth weight . Our dat


Biofilms formed on skin wound lead to inflammation and a delay of healing. In the present work, a novel textile pile debridement material was prepared and treated by plasma. Samples before and after plasma treatment were characterized by a series of methods, including scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and water uptake capacity. Besides, mechanical, coagulation, and in vitro biofilm removal performances of the textile pile debridement material were evaluated, with a medical gauze as a control. The results demonstrate that the plasma treatment produced corrosions and oxygen-containing polar groups on the fiber surface, offering an enhanced water uptake capacity of the textile pile debridement material. In addition, compressive tests certify the mechanical performances of the textile pile debridement material in both dry and wet conditions. The results from a kinetic clotting time test suggest a favorable ability to promote blood coagulation. Furthermore, the results of an MTT cell viability assay, SEM, and confocal laser scanning microscopy (CLSM) illustrate that the textile pile debridement material demonstrates a more superior in vitro biofilm removal performance than medical gauze. All of these characterizations suggest that the textile pile debridement material can offer a feasible application for clinical wound debridement. Skin wound care has attracted worldwide attention because of its importance for public health and social development . The firVarious kinds of wound debridement techniques are currently available in clinical practice, such as autolytic , enzymatMedical cotton, gauze, and bandages are the conventional textile-based materials applied for wound cleaning and debridement, due to their convenience in operation, as well as having an acceptable price for the public. However, patients frequently complain of pain when they are removed from or replaced on a skin wound ,12. A noBiofilms, adhered to the surface of inert or active objects, are bacterial communities with constructive and coordinative functions . CompareIn this work, a novel textile pile debridement material, consisting of polyester fibers, was designed and prepared. Afterwards, plasma treatment was implemented to further promote the liquid absorption ability of the textile pile debridement material. The compressive strength and elastic recovery of the textile pile debridement material in both dry and wet conditions were studied. Furthermore, the coagulation and in vitro biofilm removal performances of the textile pile material were assessed, with a medical gauze as a control, to explore its potential application for debridement.Staphylococcus aureus (ATCC 25923) was provided by JK Microorganism Co., Ltd., China. Tryptic soy broth (TSB) was prepared with tryptone Lp0042, yeast extract Lp0041 , and sodium chloride . Methylthiazolyldiphenyl-tetrazolium bromide, 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Shanghai Bio-Gene Technology Co., Ltd., Shanghai, China. Fluorescein isothiocyanate (FITC) and propidium iodide (PI) were obtained from Sigma, USA. CaCl2, NaOH, glutaraldehyde, ethanol, and isopropanol were all purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. All chemicals were analytical grade and were prepared with deionized water (DIW).Polyester staple fibers and draw textured yarn (DTY) were acquired from Jiangsu Chemical Fiber Co., Ltd., Wuxi, China. Biocompatible polyacrylate coating latex was supplied by Shanghai Huasen Chemical Co., Ltd.,Shanghai, China. Commercial medical gauzes from Winner Industries Co., Ltd., Shenzhen, China, were employed as control samples. A series of sliver knitted pile fabrics with a double faced structure were manufactured on a circular knitting machine, followed by back-coating, thermosetting, and shearing, as reported in our previous work ,17. In oScanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to inspect changes in the surface morphology of samples before and after plasma treatment. The micrographs were obtained from TM-3000 SEM  with an accelerating voltage of 15 kV and a FastScan AFM  with a Scanasyst-Air probe in light tapping, scanning, and imaging mode. The contact force was 0.4 cN/m and the resonance frequency was 70 kHz. Finally, the surface topography of samples with an area of 4 μm × 4 μm were obtained. The root mean square roughness (Rq), average roughness (Ra), and the maximum peak valley roughness (Rmax) were used as indexes to analyze the microstructure of the sample surface.An X-ray photoelectron spectrometer  was used to investigate the surface chemical composition and chemical groups of the textile pile debridement material. An Al-Kα (hv = 1486.6 eV) X-ray was selected as the excitation source. Peaks of the C 1s spectrum were fitted using XPS peak 4.1 software to analyze the percentage content of the chemical functional groups on the surface of each sample.wC) was quantified by the following equation, where w0 and w1 are the dry and wet weight of the samples, respectively.A water absorption study was conducted to estimate the exudate holding capacity of the textile pile debridement material before and after plasma treatment, according to ISO 9073-6. Three dry samples with a size of 1 cm × 1 cm were immersed in distilled water at room temperature for 10 s, then were taken out and drained for 10 s to remove excess water. The water uptake capacity  was designated as 1 cm, and the velocity of the presser (v) was set as 1 cm/min. Compression strength was obtained when the presser dropped to its lowest position, as illustrated in The compression strength and elastic recovery rate were employed to evaluate the compression properties of the textile pile debridement material in both dry and wet states. The typical test process of a LLY-06D compression tester  is exhibited in 2 solution of 0.2 mol/L to initiate the blood coagulation cascade. After incubation at 37 °C for a predetermined period of time, specimens were inundated with 2.5 mL distilled water at the end of 5, 10, 30, and 60 min, and placed back into the chamber for another 5 min. Then 200 μL resultant blood solution were transferred into a 96-well plate, and the optical density at 540 nm was obtained on an ELISA plate reader . To prepare samples for SEM observation, specimens were fixed in 2% glutaraldehyde solution at 4 °C for 30 min, dehydrated with a series of gradient alcohol solution of 55%, 70%, 80%, 90%, 95%, and 100%, and then dried at 4 °C.The coagulation activity of the textile pile debridement material was evaluated using a kinetic clotting time method. Commercial gauze and glass cover slips were taken as commercial and positive controls, respectively. Specimens measuring 10 mm × 10 mm were placed in 24-well tissue culture plates in triplicate. Afterwards, 10 μL mixed solution of whole human blood and trisodium citrate with a volume ratio of 9:1 were dropped onto the surface of each specimen, followed by a 5 μL CaCl3 per well and cultured for 4 h. Then the culture medium was replaced by fluid extracts and cultured for 1, 3, 5, and 7 days. Twenty microliters of MTT solution were added to each well and incubated for 4 h. After discarding the supernatant, 100 μL of DMSO were added to each well before measuring the optical density (OD) at 570 nm with a microplate reader .The in vitro cytotoxicity of the textile pile debridement material was evaluated by an MTT assay . Fluid eS. aureus was diluted in TSB to 106 cfu/mL. Then 600 μL diluted bacterial liquid were added into each well of 24-well plates with cell growing glass slides . Cells were cultured at 37 °C for 24 h. At the end of incubation, the supernatant was removed and the formed biofilms were washed with phosphate buffered solution  to remove planktonic and loosely attached bacteria.An isolated single bacterial colony picked from an agar plate was transferred to 4 mL of TSB medium and then incubated at 37 °C for 14 h at a speed of 180 rpm. An overnight culture of 2), medium (12.25 cN/cm2), or low (8.71 cN/cm2). PBS solution was used to fully soak the debridement material or cotton gauze in the wet state test.An in vitro biofilm removal test was conducted on a self-built experimental device under different velocities and pressures in both dry and wet states, as illustrated in An MTT cell viability assay, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) were employed to compare the viability and morphology before and after the biofilm removal test.MTT cell viability assayAn MTT cell assay was used to assess the viability of bacteria in the biofilm. Only live bacteria in the biofilm were counted in the MTT assay, by measuring the metabolic activity of each individual bacterial cell . Cell grS. aureus biofilmSEM of S. aureus biofilm architecture, SEM images were taken. After fixation by glutaraldehyde for 2 h at 4 °C, samples, before and after the biofilm removal tests, were dehydrated by a series of ethanol with increasing concentrations  for 10 min each time. Dehydrated specimens were coated with gold–palladium and observed.For the visualization of the S. aureus biofilmCLSM of Biofilms were prepared and fixed by glutaraldehyde for 2 h at 4 °C. After washing three times with PBS, cells within the biofilm were fluorescently stained in the dark at 4 °C by 50 μg/mL FITC for 30 min and 10 μg/mL PI for 15 min, which identified polysaccharide and DNA in the biofilm structure, respectively . BiofilmXPS overall scans of the untreated and plasma-treated textile pile materials are illustrated in To further explore the types of polar groups introduced on the fiber surface by plasma treatment, XPS peak 4.1 software was employed to analyze the C 1s photoelectron scanning pattern. The C 1s spectra and functional group percentage of the untreated and plasma treated textile pile materials are demonstrated in The influence of the plasma treatment parameters on the water uptake capacity of the textile pile debridement material is shown in To achieve effective removal of necrotic tissues, it is always necessary to wipe the wound with textile pile debridement material several times. Therefore, a repeated compression test was performed ten consecutive times to study the impact of repetition on the compression strength and elastic recovery rate of the textile pile debridement material. The compression strength and elastic recovery rate of the textile pile debridement material in dry and wet states at each compression time are displayed in To further quantificationally study the influence of the wet condition and repetition on the compression strength and elastic recovery rate of the textile pile debridement material, the percentage of wet/dry compression strength and elastic recovery rate under different compression repetitions are listed in The absorbance OD value at 540 nm of aqueous blood solutions at different time points is displayed in SEM images of the textile pile debridement material and gauze at 30 min in the blood coagulation test are shown in The relative cell viability was employed to evaluate the in vitro cytotoxicity results of the textile pile debridement material. Cell viability was expressed as the percentage of the OD value of the experimental group to the control group. The MTT results for the in vitro biofilm removal test are shown in It can be seen from The morphologies of biofilms before and after debridement are shown in 2 (The SEM morphology results of biofilms under different in vitro debridement conditions  are cons2 e1. Moreo2 b is noti2 a. Thus, 2, as well as the control before debridement, are illustrated in CLSM images of biofilms debrided with the textile pile material and gauze under a pressure of 15.68 cN/cmS. aureus stained in green, suggesting the typical characteristic of glycocalyx formation in the biofilm. In contrast, green bacteria are barely observed in FITC is a green nucleic acid probe with the ability to make polysaccharide in an extracellular matrix green. PI is a red fluorescent dye which can only stain DNA in bacteria. It can be clearly observed from In addition, it was reported that a silver-containing dressing is the first choice to treat chronic wound biofilms, which can remove 90% of the bacteria within 24 h and eradicate biofilms within 48 h . BesidesHerein, we prepared a novel textile pile debridement material treated by plasma. The experimental results indicated that the hydrophilicity and water uptake capacity of the textile pile debridement material were improved by plasma treatment. Meanwhile, the textile pile debridement material displayed satisfactory mechanical properties in wet conditions. Moreover, the textile pile debridement material also presented an ideal blood clotting performance and excellent biofilm removal capacity. The novel textile pile debridement material will find its potential application in wound debridement.


Ascophyllum nodosum (AN), a brown macroalgae, on performance and gut health as compared to either a non-supplemented diet  or a diet supplemented with 2500 ppm ZnO (PC) in piglets weaned at 28 days of age. At any of the amounts supplemented, FRM sustained growth performance similar to the PC group during the first 10 days before weaning (18–27 days of age) and improved performance better than the PC from 28–41 days of age when fed at 8%. Inclusion of AN (0.6% or 1.0%) on top of 10% FRM did not affect growth performance. The percent of piglets that completed the experiment was increased at all levels of FRM (maximum of 91% at 8% FRM) or a combination of 10% FRM with AN (maximum of 90% at 10% FRM + 0.6% AN). Maximum intestinal development  was observed at lower levels (8–10%) of FRM supplementation, but this was abolished by inclusion of AN. Feeding of FRM with or without AN increased some hematological parameters at all doses and enhanced immunoglobulin and interleukin-6 titers at lower doses (8% or 10%). In conclusion, FRM sustained piglet growth performance and intestinal development similar to ZnO with an optimal dietary inclusion level at 8–10% of dietary DM. This study investigated the effects of dietary addition of increasing amounts  of pre-fermented rapeseed meal (FRM) or 10% FRM with the inclusion of 0.6% or 1.0% Ascophyllum nodosum (AN) on weaner piglets’ performance and gut development. Ten days pre-weaning, standardized litters were randomly assigned to one of nine isoenergetic and isoproteic diets comprising (on DM basis): no supplement , 2500 ppm ZnO , 8, 10, 12, 15 or 25% FRM, and 10% FRM plus 0.6 or 1.0% AN. Fifty piglets receiving the same pre-weaning diets were weaned at 28 days of age and transferred to one pen, where they continued on the pre-weaning diet until day 92. At 41 days, six piglets per treatment were sacrificed for blood and intestinal samplings. The average daily gain was at least sustained at any dose of FRM  from 18–41 days similar to PC but unaffected by inclusion of AN. The percentage of piglets that completed the experiment was increased by FRM compared to NC, despite detection of diarrhea symptoms. FRM showed quadratic dose-response effects on colon and mid-jejunum crypts depth, and enterocyte and mid-jejunum villus heights with optimum development at 8% or 10% FRM, respectively, but this was abolished when AN was also added. In conclusion, FRM sustained piglet growth performance and intestinal development similar to ZnO with an optimum inclusion level of 8–10% of dietary DM.This study evaluated the effects of increasing doses of pre-fermented rapeseed meal (FRM) without or with inclusion of the brown macroalgae  Weaning is the most challenging event in modern pig production, which often results in reduced feed intake, decreased growth, increased risks of post-weaning diarrhea (PWD) and mortality in newly weaned piglets ,2,3. ThuEschdurierichia coli and overall coliforms throughout the gastrointestinal tract  × Duroc & Pietrain hybrid [DP] crossbreed) were recruited for the study from 45 sows  in their second or third parity. The piglets came from litters that were standardized to 14 piglets per sow. Standardization of litters to 14 piglets per sow was made by transferring piglets among sows until 14 days of age. Thereafter, no movement of piglets among sows was allowed. At 18 days of age (10 days before weaning), piglets were introduced to one of nine experimental diets comprising a basal diet  with: no supplement (NC), 2500 ppm (PC), increasing levels of FRM , and a combination of 10% FRM with either 0.6 or 1.0% AN of dietary DM while still with the sows, with piglets from five sows receiving the same experimental diet. At 28 days of age, the piglets were weaned and 50 of the piglets already receiving the same dietary treatment, excluding the runts, were then transferred from five sows to one slatted floor pen in the weaner unit per treatment and continued on the same dietary treatment until they exited the weaner unit at 92 days of age. All diets were based on the same basal diet, where wheat and soybean products were replaced with increasing amounts of FRM and AN. All sows received the same standard lactation diet commonly used at the farm. Piglets were fed pre-starter diets from 18 (10 days pre-weaning) to 64 days of age, and a starter diet from 65 to 91 days of age, i.e., until the end of the experiment, ad libitum from open troughs along with ad libitum access to water from drinking nipples during the entire trial.Pediococcus acidilactici (DSM 16243), Pediococcus pentosaceus (DSM 12834) and Lactobacillus plantarum (DSM 12837) where ground rapeseed meal (80% on DM basis) was mixed with the inoculant broth in a one-step fermentation process with constant stirring, followed by decanting and incubation at 38 °C for four days. FRM  was obtained from European Protein . It was prepared with solid-state fermentation using inocula from three lactic-acid fermentative bacteria, namely, Lactobacillus spp. containing FRM product. Formulation of the diets was performed by AgroSoft®  according to the Danish nutritional recommendations for piglets [The mixing of the final experimental diets was conducted by a certified feed company where no heat treatment was involved after the addition of  piglets . AnalysePiglets were ear-tagged at birth, and body weights of individual piglets were recorded 10 days before weaning (18 days of age), at weaning . Thereafter, body weight was measured in each treatment on pen-basis until the piglets exited the experiment at 92 days of age. Feed consumption in each experiment was recorded daily on pen-basis throughout the experiment.Piglets were examined daily by farm staff for signs of diarrhea or signs of any other ailments including mortality and if possible, its causes, and any changes observed were recorded. Piglets with poor health conditions due to, among others, diarrhea were removed from the pen and experiment to be treated with antibiotics elsewhere. No antibiotics treatment was allowed for any piglet remaining in the experiment. Fecal samples were collected from each pen and analyzed for lactic acid content at the veterinary diagnostic laboratory, ALAB Weterynaria . A completion rate was calculated in percentage for each treatment based on the number of piglets remaining in a pen at the end of the experiment and the number of piglets assigned to pens at weaning. Fourteen days after weaning (41 days of age), six piglets were randomly selected from each treatment or pen, and transported to a commercial slaughter facility. Piglets were sacrificed by exsanguination following captive bolt stunning. Whole blood samples were collected during exsanguination from the jugular vein into ethylene diamine tetra acetic acid (EDTA) tubes for hematological analysis and plain vacutainer tubes for analysis of serum immunoglobulins and interleukin-6. Whole blood samples from EDTA tube were stored at +4 °C and the blood samples in plain tubes were allowed to clot to separate the serum and the serum samples were then stored at –20 °C until analyses the following day. The abdominal wall was opened, and the cardiac sphincter, pyloroduodenal, ileocecal and anorectal junctions were ligated, and the gut segments were cut and taken out. Tissue sections of approximately 2 cm length were sampled from the middle of the small intestine and the apex of the ascending colon. Sampled tissue sections were gently rinsed for intestinal contents with saline and fixed in 10% buffered formalin. Histological analyses of mid-jejunum and colonic tissue were conducted at the commercial laboratory ALAB Weterynaria  using a standard light microscope Olympus BX41 and CellSens software . All microscopic evaluations were performed in a blinded fashion. Initially, tissue samples fixed in 10% buffered formalin were dehydrated using graded ethanol and xylene baths and embedded in paraffin wax. Tissue sections of 3–4 µm were mounted on the microscopic slide and stained with hematoxylin and eosin (HE). General histopathological examinations were evaluated at a magnification of 10×, 40× and 100× (objective lens) and 10× (eyepiece), and photographic documentation was made. For the mid-jejunum the following were examined: the mucosa and submucosa, intestinal crypts, mucosal and submucosal blood vessels, enterocyte height and brush border integrity as well as the presence of goblet cells. The morphometric analysis delivered the following quantitative results: mid-jejunum villus height (JVH) at 10× magnification, mid-jejunum crypt depth (JCD) and colonic crypt depth (CCD) both at 10× magnification, and mid-jejunum enterocyte height (JEH) at 40× magnification. Two types of scoring analyses were performed: for intraepithelial lymphocytes infiltration (IEL) and stromal lymphocytes infiltration (SL). The following scale was used in both scoring evaluations: 0—normal; 1—slight; 2—moderate; 3—severe. Hematological analysis of whole blood samples and serum immunoglobulin analysis were conducted at the ALAB Weterynaria . Analysis of red blood cell counts (RBC), hematocrit value (HCT), hemoglobin content (Hb), white blood cell counts (WBC) and differential leucocytes count were carried out using a Sysmex XT 2000i analyzer . Commercially available swine-specific enzyme-linked immunosorbent assay (ELISA) kits  were employed to quantify the concentrations of serum immunoglobulins such as immunoglobulin G , immunoglobulin A  and immunoglobulin M  as well as the pro-inflammatory cytokine interleukin-6  according to the manufacturer protocols.j = male or female), k = 1,2,3,…, 50), Data for growth performance, hematology and serum immunoglobulins, as well as a dose response effect of FRM on gut morphometry were analyzed using Linear Mixed Models of R version 3.6.0 . Dietaryp < 0.05.Linear and quadratic relations for dietary inclusion level of FRM (FRM dose as % of dietary DM) and effects on gut morphometric parameters were analyzed using the following model: p < 0.05) during the 10 days before weaning (18 to 27 days of age) and at 27 days of age, respectively, compared to the NC piglets. FRM sustained similar ADG during 18–27 and 18–41 days of age, and BW at 27 and 41 days of age as the PC at all doses, and significantly increased (p < 0.05) ADG during 28–41 days of age when supplemented at 8%. ADG was increased during 18–27 days of age, and demonstrated no discernable pattern (despite significant increment at 8% FRM) during 28–41 days of age, and showed no difference during 18–41 days of age with increasing doses of FRM. Inclusion of AN on top of 10% FRM at either dose did not affect ADG compared to the 10% FRM. Results for BW and ADG after 41 days of age (42–92 days of age) were pen-based single observation per treatment, and hence, could only provide an inconclusive rough estimation of growth  resulted in a numerically lower incidence and higher completion rates than NC. At any of the doses supplemented, FRM without or with AN resulted in a higher completion rate than the NC but achieved similar completion rates to that of PC . DietaryThe effects of dietary supplementation on intestinal morphologies are shown in Supplementation of FRM at any dose or its 10% combination with any dose of AN resulted in numerically lower gut-associated lymphoid tissue (GALT) counts, and reduced immune cell infiltrations of the jejunal and colonic mucosa compared to the NC and PC piglets, which had similar levels of infiltration .Assessment of histopathological changes revealed weak to strong stromal and intraepithelial immune cell infiltrations of the small intestine in both NC and PC piglets. A few piglets demonstrated disruption of the epithelial barrier and moderately visible brush borders in areas of significant intraepithelial lymphocyte infiltration in both controls. There were negligible immune cell infiltrations of the small intestine and colon at any doses of FRM compared to NC and PC, and infiltrations were limited only to the stroma in both intestinal segments. Inclusion of AN did not have significant impacts except for a slight increment in colonic stromal immune cell infiltration.p < 0.05), but reduced granulocyte (GRA) counts (except for 25% FRM) (p < 0.05) at any dose administered compared to NC and PC piglets. Inclusion of AN had no additional impact on hematological parameters except for a significant increment of RBC counts when 1.0% AN was supplemented. There were no differences in hematological values between PC and NC piglets except for higher WBC in NC (p < 0.05). As shown in As shown in In the current study, FRM demonstrated the same desirable effects on ADG, and intestinal development and barrier function as we have previously observed and optimum effects were observed when 8% or 10% FRM was included in the weaner diets . SimilarIn this study, AN did not have any impacts on ADG at any of the doses provided on top of the FRM. This is in contrast to Turner et al. , who repInclusion of FRM at 8% or 10% of dietary DM in weaner diets in the current study resulted in improved intestinal morphometric indices , intestinal mucosal integrity and barrier functions consistent with observed improvements in ADG. Long villus height and intact mucosal integrity are essential for nutrient digestion and absorption as well as prevention of invasion of intestinal tissues by pathogenic microbes. Similar findings were reported when fermented rapeseed was fed to broilers where intestinal villus height and villus-to-crypt ratio showed significant improvements ,37,38. FLaminaria digitata [The desirable effects of FRM on gut histomorphometry and GALT were counteracted by the addition of AN on top of 10% FRM. A similar result was reported where intestinal villus height was reduced in response to feeding of weaned piglets with a seaweed extract from digitata . In contdigitata ,47.Salmonella typhimurium and E. coli, which are known etiologic agents for enteric infections and diarrhea [E. coli which are the causative agent of infectious diarrhea during weaning [Although a high percentage of piglets completed the experiment in response to feeding of FRM (highest at 8% FRM) or a combination with AN (highest at 0.6% AN), piglets were not protected from diarrhea in the current study. We speculate that this diarrhea could be linked to osmotic pressure triggered by possibly high intestinal lactic acid concentrations as confirmed by detection of high fecal lactic acid concentration amidst intact intestinal mucosal integrity and barrier functions. In contrast, fermented feed such as soybean has been reported to prevent diarrhea in piglets weaned at 28 days of age . Studiesdiarrhea ,50. Regadiarrhea , while ndiarrhea . However weaning ,51. RBC indices are essential markers for efficient oxygen transport to tissues and nutrient utilization for energy release. Although all of the RBC indices investigated were in the normal ranges for all of the treatments in the current study , they weIn the current study, we found out that WBC indices were generally within normal ranges compared to reference values , and feeAscophyllum nodosum (AN) did not show any additional benefits. Thus, FRM could be a potential alternative for medicinal ZnO when supplemented at ≤ 10% dietary DM. However, a comprehensive dose-response study is deemed necessary to assure optimum inclusion rate for desirable effects on performance and gut health. FRM improved piglet performance (ADG) and intestinal histomorphometric indices demonstrating optimum effects at 8% or 10% inclusion, but further inclusion of the brown macroalgae