To compare surgical results of endoscopic myringoplasty using double layer cartilage-perichondrium grafts versus single fascia grafts.Prospective, randomized, controlled.University-affiliated teaching hospital.In total,134 patients who underwent endoscopic myringoplasty were included in this study. Patients in group A received a double layer tragal cartilage-perichondrium graft and those in group B received a temporal muscle fascia graft. The groups were compared with respect to the pre- and postoperative air-bone gap (ABG) and the graft success rate.p = 0.362). However, the graft success rate was 97.0% (65/67) in the Group A and 85.1% (57/67) in the Group B at 12 months, the difference was statistically significant (p = 0.034).The graft success rate was 98.5% (66/67) in the Group A and 94.0% (63/67) in the Group B at 6 months, the difference wasn’t statistically significant  had small keratin pearls in the Group A, no patients developed cholesteatoma of middle ear in either group.The endoscopic double layer perichondrium-cartilage graft technique is feasible for repairing medium or larger perforations, it has a better long-term graft success rate and less operative time compared with the single layer fascia graft technique. However, long-term hearing outcomes were the same for the single and double layer closure techniques. Chronic tympanic membrane (TM) perforation is a common complication of chronic otitis media (COM) and trauma in adult; persistent perforation requires surgical closure by myringoplasty. Several graft materials and graft placement methods are used in myringoplasty. Fascia, perichondrium, cartilage and fat grafts are often used, but fascia is still the preferred graft material for most patients. However, cartilage grafts are favored in difficult cases, such as those with poor Eustachian tube function, retraction pockets, infection, or anterior perforations, as well as in revision surgery , 2. The Endoscopic inlay butterfly cartilage tympanoplasty, without the need for canalplasty or elevation of the tympanomeatal flap, has an excellent graft success rate, although it is technically more challenging in terms of achieving precise measurement of the perforation size and shape, and accurate sizing of the cartilage graft –12. In tThe study protocol was reviewed and approved by the Institutional Ethical Review Board of the affiliated yiwu hospital of wenzhou medical university , Yiwu city, Zhejiang provice, China. Informed consent was obtained from all participants.A prospective interventional randomized case series study was conducted from 2014 to 2018 in a tertiary care hospital. Inclusion criteria required patients to be older than 18 years, to have a diagnosis of unilateral TM perforation with COM, without cholesteatoma and with an intact ossicular chain, and to require TM repair. Patients with suspicious ossicular chain disruption, middle ear inflammation, myringitis and revision cases were excluded from the study.Pure-tone audiometry (PTA) was measured preoperatively and 6 months after surgery. Standard PTA was performed for the frequencies of 0.5, 1, 2 and 3 kHz. The air-bone gap (ABG) was calculated as the average difference between air conduction and bone conduction at 0.5, 1, 2, and 3 kHz. The patient with ossicular chain disruption was suspected if the preoperative ABG was more than 40 dB, who was then excluded from the study. Age, sex, presence of contralateral perforation or otitis media with effusion (OME), size, location of perforation, and preoperative and postoperative hearing levels were recorded for all patients. The location of perforation was described as marginal in cases in which the annulus was involved regardless of involvement of the umbo of the malleus; otherwise, it was described as central, then, the marginal perforations were classified as follows in relation to the handle of the malleus: antero-marginal, postero-marginal or inferomarginal. The TM perforation was classified according to size, as subtotal (> 75% of the eardrum), large (> 50%), or medium (25–50%). The small perforation wasn’t included in this study because fat graft or perichondrium graft myringoplasty was usually used for the small perforation in our department.Allocation of the technique and material of graft were performed by operating room nurse (ORN) using simple random sampling. Specifically, consecutive subjects who met the inclusion criteria and signed the consent form assigned random numbers generated by the SPSS for Windows software package  that allocated them to one of the two groups: double layer cartilage-perichondrium graft group (Group A) and temporal fascia graft group group (Group B). Surgery was performed by the same surgeon.A 0° angle, 4-mm-wide and 18-cm-long rigid endoscope  and high-definition monitor system (XiON GmbH) were used in all cases. All patients were operated on under general anesthesia. No postauricular approaches were performed. The perforation edges were visualized and deepithelialized with an angled pick.In the double layer cartilage-perichondrium group, tragal cartilage with a single layer perichondrium was used as the graft material and a 1-cm skin incision was made on the medial side of the ipsilateral tragus. An inferior cut was made as low as possible to gain the whole tragal cartilage-perichondrium; the graft was then shaped according to the perforation Figs. .Fig. 1DThe lateral perichondrium was peeled circumferentially and rolled up from the cartilage graft, forming a double layer graft with an interconnecting pedicle. The lateral graft is the perichondrium and the medial graft is the intact cartilage. The size of the pedicle of the perichondrium attached the center of the cartilage, which was kept intact, was equal to or less than that of the perforation.The cartilage graft was trimmed peripherally according to the size and type of perforation, but was not thinned and was at least 2 mm larger than the perforation. A notch was made in the cartilage graft to accommodate the exposed malleus handle, if applicable. The lateral free perichondrium was also trimmed peripherally, but was at least 1–2 mm larger than the medial cartilage graft  soaked in erythromycin ointment was used to support the graft, medially and laterally. The EAC was packed with gauze soaked in erythromycin ointment up to the tragus incision, which was not sutured Fig. .Fig. 3TIn the fascia graft group, a temporalis fascia graft of adequate size was harvested via a small incision just above the ear, and then pressed using a fascia clamp. The incision was sutured using 3–0 absorbable suture material. The fascia graft was placed medial to the TM remnant and the annulus in an underlay fashion without TM flap elevation in 31 patients with medium size central perforation. However, a tympanomeatal flap was elevated according to the localization and size of the perforation in 36 patients with marginal and large perforations. Then, the fascia graft was placed accordingly to close the perforation with the over-underlay technique or underlay fashion. The tympanomeatal flap was repositioned.Biodegradable Nasopore soaked in erythromycin ointment was used to support the graft medially and laterally in all the patients. The EAC was packed with gauze soaked in erythromycin ointment up to the tragus.Postoperatively, all the patients received oral amoxicillin/clavulanate potassium for 1 week to prevent infection. Postoperative follow-up visits took place at the hospital at weeks 2, 3 and 4 and at months 3, 6, 12, and 18. The packing gauze was removed from the EAC at 14 days post-surgery and biodegradable Nasopore fragments were aspirated from the EAC at 3–4 weeks post-surgery; this allowed the graft to be visualized endoscopically.In both groups, the operating time (from start of surgery after anesthesia induction to EAC packing) was recorded. Endoscopic examination was performed at each follow-up visit to view the graft change. The development of cholesteatoma and epithelial pearls were evaluated by each endoscopic examination and Computed tomography at 18 months postoperatively. An audiometric evaluation was carried out to measure the ABGs at the end of postoperative month 6 and 12. Any intraoperative or postoperative complications were also recorded. Graft success was defined as an intact graft without perforation, retraction, lateralization, significant blunting or medialization. The residual perforation was defined as that a perforation was seen following the removal of biodegradable Nasopore fragments. However, the re-perforation was defined as that the TM had healed completely following surgery but new perforation occured at postoperative 3 months.p-value < 0.05 were accepted as statistically significant.Statistical analyses were performed with SPSS software . The data are expressed as means (standard deviation [SD]) and percentages (%). The chi-squared test was used for comparison of categorical data. Wilcoxon and Mann-Whitney U tests were used for analyzing non-parametric variables; independent and paired-samples t-tests were used for parametric variables. Results with a p > 0.05). No ossicular chain disruption was found during surgery in all the patients in either group. Elevation of the tympanomeatal flap was performed in 36 (53.7%) patients in the B group. The average operating time was significantly shorter in the A group compared with the B group . In addition, the mean surgical time was also significantly shorter in the cases without TM flap elevation compared with the cases with TM flap elevation in the fascia group . All tragal incisions had healed by postoperative week 2 in the double layer graft group. All patients were followed up at least 18 months. The mean follow-up period was 20.2 ± 1.9 months in the Group A and 21.3 ± 2.6 months in the Group B  who met the above criteria were included in this study. The average age was 35.1 ± 1.29 years. The left ear was affected in 72 (53.7%) patients, and the right ear in 62 (46.3%) patients. Of the 134 ears, 67 were randomized to the double layer graft group (Group A) and 67 to the fascia graft group (Group B). Patient characteristics are shown in Table p = 0.001). In the Group B, the mean ABG was pre-24.15 ± 7.84 dB, post-6 months 10.12 ± 2.43 dB and post-12 months 9.43 ± 3.28 dB; again, the difference was statistically significant (p = 0.001).The pre- and post-operative ABG showed in the Table.p = 0.794) or post-6 months ABG values (p = 0.832) or post-12 months ABG values (p = 0.895). Also, no significant group difference was found in the mean post-6 months ABG gain (p = 0.836) or post-12 months ABG gain (p = 0.811).There was no statistically significant difference between the Group A and Group B in pre- (P = 0.742), however, the hearing success rate of ABG ≤ 10 dB in the Group B was significantly higher than that in the Group A on paired-samples test .At post-operative 6 months, the difference was not significant for the hearing success rate of ABG ≤ 20 dB between Group A and Group B  or ≤ 10 dB  . However, the graft success rate was 97.0% (65/67) in the Group A and 85.1% (57/67) in the Group B at 12 months, the difference was statistically significant (p = 0.034) (Table.The graft success rate was 98.5% (66/67) in the Group A and 94.0% 63/67) in the Group B at 6 months, the difference wasn’t statistically significant (/67 in thn = 1) and re-perforation (n = 5) occured in the other 6 patients  at 6 months between the double layer graft and fascia graft groups, the graft success rate in the Group A was significantly higer than that of the Group B  at 12 months. These results were similar to those of previous studies. Guler et al. [p = 0.048). Similarly, Kolethekkat et al. [p < 0.001) [In the present study, although the graft success rate wasn’t significantly different  . A carti< 0.001) . Althoug< 0.001) –20, we p< 0.001) .This technique is similar to the butterfly technique; however, rather than splitting the cartilage, the perichondrium graft is used to support the cartilage graft. On the other hand, the perichondrium graft covers the surface of remnant TM and serves as a scaffold to guide the migration of the outer squamous epithelium . The proSome studies reported a high graft success rate using butterfly cartilage myringoplasty –12, 22. Tympanomeatal flap elevation is one of the most fundamental steps during most of myringoplasty for repairing large or marginal perforations , 14, 26.P = 0.019) at 6 months. Nevertheless, the difference of the hearing success rate was not significant between Group A and Group B regardless of ABG ≤ 20 dB  or ≤ 10 dB  at post-operative 12 months. This fingings were also confirmed by other study [Almost all studies including fascia and cartilage groups reported no difference between them in terms of the functional results of the myringoplasty –20. Pre-er study . Wu PW eer study  suggesteA recent meta-analysis comparing cartilage and fascia grafts  showed tOne of the major concerns with lateral grafts is blunting. However, it is curious that this did not develop in this series. One possible reason is that Biodegradable Nasopore was packed for 3–4 weeks inn the EAC to prevent this from happening. Another possible reason is that the lateral perichondrium graft gradually formed the crust in all the patients in this study. We speculated that only the medial cartilage graft finally survived to close the perforation. Nevertheless, the exact mechanism is unclear, further study is expected.The development of cholesteatoma is another concern when using the double layer cartilage-perichondrium graft technique. A theoretical disadvantage of the double layer graft technique pertains to the graft coverage of the superficial layer of the TM, which can easily hide any pathology until significant damage occurs, after which complications may manifest. The superficial layer of the TM that initially abuts the graft margins might migrate below the graft instead of covering it, which can lead to cholesteatoma. However, the lateral perichondrium graft gradually formed the crust during the mean follow-up period of 20.2 ± 1.9 months, only one patient (1.49%) had small keratin pearls on the anterosuperior quadrant, that was removed under endoscopy. Olarieta et al.  underwenThe endoscopic double layer perichondrium-cartilage graft technique is feasible for repairing medium or larger perforations, it has a better long-term graft success rate and less operative time compared with the single layer fascia graft technique. However, long-term hearing outcomes were the same for the single and double layer closure techniques.Additional file 1


Amylose-lipid complex (ALC) was prepared with corn starch and stearic acid and used as a shortening replacement in white pan bread preparation. ALCs were prepared using various concentrations of stearic acid to corn starch  under different temperatures  and for different durations of time ; then, their complexing properties were assessed using iodine reagent and X-ray diffraction. The complexing reaction at 75 °C for 60 min showed the highest complexing index of the tested conditions; the in vitro digestibility of ALC was lower than that of corn starch. White pan bread was prepared with ALCs and their characteristics, including appearance, loaf volume, and starch retrogradation during storage at room temperature for four days, were compared with those of control bread. With increasing ALC replacement concentrations, loaf volume and shape were significantly affected; however, starch retrogradation was significantly retarded and energy value decreased by ALC replacement. Overall, 50% replacement of shortening by ALC appeared to be a reasonable level for retaining the basic characteristics of the bread while retarding the staling process. These results indicate that ALCs may be potentially useful in the bakery industry for preparing low calorie and low-fat products. Bread is the most important staple food worldwide and is recognized as a perishable food, best when consumed ‘fresh’. Bread is an unstable, elastic, solid foam. Its solid part contains a continuous phase comprising an elastic network of cross-linked gluten molecules and leached starch polymer molecules, primarily amylose, both uncomplexed and complexed with polar lipid molecules and a discontinuous phase of entrapped, gelatinized, swollen, and deformed (wheat) starch granules . ShortenRecently, with increasing consumer demand for healthy foods, such as low-calorie and low-fat products, a great deal of effort has been made to reduce the use of shortening owing to its high level of saturated fatty acids as well as the possible presence of trans fatty acids . High inAs fat has a higher caloric density than most nutrients in foods , reducinHowever, to our knowledge, no attempt to use amylose-lipid complexes (ALCs) as a replacement for fats, especially shortening, in bread preparation has been reported. The formation of starch-lipid complexes is a well-known interaction, and complexation mainly occurs between amylose and lipids . The chaRecently, ALC was recognized as a new type of resistant starch (resistant starch 5); a starch resistant to enzymatic digestion . ResistaIn this study, ALC was prepared from corn starch and stearic acid under different conditions including stearic acid-starch ratio , temperatures , and duration time  and its potential as a shortening replacement in bread making was investigated.Corn starch was purchased from Daesang Co., Inc., Seoul, Korea. Food-grade stearic acid was purchased from Sigma-Aldrich, St. Louis, MO, USA. All reagents were chemical-grade; iodine (99%) was obtained from Showa Chemical Industry Co., Ltd., Japan, and potassium iodine (99.5%) was purchased from Junsei Chemical Co., Ltd., Tokyo, Japan.w/w) starch slurry was prepared with corn starch and pre-warmed water (40 °C); then, a gelatinized starch-water mixture was prepared by stirring for 30 min at approximately 95 °C using an overhead stirrer . Gelatinized starch slurries were cooled to different temperatures , and a stearic acid solution (25 mL of 5%) was prepared with ethyl alcohol. The stearic acid solution was added to the gelatinized starch slurries, which were then continuously stirred for 30, 60, and 120 min at each temperatures . After the reaction, the starch-stearic acid mixture was stabilized by additional stirring for 30 min at 25 °C. Different concentration of stearic acid solutions in ethyl alcohol were also prepared , applied for ALC preparation at each temperatures , reacted for 60 min, and then used for the experiments.ALC was prepared as described in previous studies with some modification ,23. A 502 in 50 mL of distilled water and allowing them to dissolve overnight [g. The supernatant (250 μL) and iodine solution (1 mL) were added to distilled water (7.5 mL) in a 15 mL test tube. The tube was vortexed, and the absorbance was measured at 690 nm. CI was calculated using the following equation:The degree of ALC formation was determined by measuring the complexing index (CI) using an iodine solution, as described by Kaur and Singh . The iodvernight . Then, dg, 20 min) and successively washed with hot water and 50% ethanol (50 mL each) to remove free starch and free stearic acid. The final precipitates were dried at 40 °C overnight and milled using a cyclone mill.After sample preparation, each sample was washed to remove free stearic acid and corn starch using the methods described by Chang, et al.  and ReddThe crystalline structure of the complexes was assessed using an X-ray diffractometer  via the method described by Seo, Kim, and Lim . The scaIn vitro digestibility was determined using the Englyst procedure with modifications . The driBread samples were prepared according to the AACC standard straight dough method with slight modifications . The ingMoisture content was determined using an infrared moisture analyzer . The ash, crude protein, and crude fat content of the ALC bread samples were determined using the methods recommended by AOAC International . The calThe thermal properties of white pan bread were analyzed using a Diamond DSC . The gelatinization parameters, including the onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy change (ΔH), were calculated from the DSC thermograms. The ALC samples were measured after milling, and bread samples were ground into a powder after freeze-drying . The samples (7 mg) were weighed into the DSC sample pan , and each pan was sealed after adding 21 μL distilled water. All sample pans were equilibrated at room temperature for 1 h and then heated from 10 to 150 °C at a rate of 10 °C/min.p < 0.05). Each value shown is the mean ± standard deviation (SD), and different letters indicate significant differences (p < 0.05).All experiments were repeated at least three times. Statistical analysis was performed using SPSS® statistics program ver. 25 . Duncan’s multiple-range test in one-way analysis of variance (ANOVA) was carried out to identify significant differences  using an iodine solution as a function of temperature, time, and ratio of stearic acid/starch. The effect of temperature and time was investigated using a 5% stearic acid to corn starch ratio at different temperatures  and durations of time . The results are summarized in X-ray diffraction patterns of ALCs prepared under different conditions were compared to confirm the complexation of stearic acid into the starch structures , slowly digestible starch (SDS), and resistant starch (RS) after the in vitro digestion process . PolydexProximate analysis results and calorie content of ALC-containing bread are summarized in The overall appearance and volume of these loaves were also compared with those of the control bread . OverallQuality changes in the ALC-containing bread during storage were assessed in terms of starch retrogradation . DiffereOver the last several years, there has been an increase in the percentage of the population that is either overweight or obese. The growing concern about the relationships among health, feeding, and the maintenance of a healthy weight has boosted the market for foods with reduced energy values . AmyloseThe XRD spectra is a fingerprint for identifying the specific crystal structure of different types of starches. Corn starch, as an A-type starch, showed typical peaks at the Bragg angles (2θ) of 15.3, 17.2, 18.0, and 23.0° . ComplexDSC was used to compare the forming ability of ALC under each condition. The melting enthalpy (ΔH) of the ALC peak appeared over 90 °C; the melting enthalpy (ΔH) of the amylose-lipid complex in corn starch was 0.77 J/g. For the ALC samples used in this study, the melting enthalpy of the ALC peak was significantly affected by temperature and the concentration of stearic acid. After ALC complexation, the melting enthalpies were 2.18–4.13 times greater that of normal corn starch. Higher melting enthalpies were observed with increasing temperatures and stearate concentrations, with the highest ΔH at 75 °C with 5% stearate; however, no significant difference was observed with over 3% stearate, which we assumed was the saturating starch/lipid concentration ratio.ALC is recognized as resistant starch type 5 (RS5); Hasjim, et al.  summarizThe overall shape and appearance of the ALC bread was not affected by 50% shortening replacement; however, 100% of shortening-replaced bread exhibited significant changes in whole shape and appearance. The specific volume of the ALC 100% bread was 23% lower than that of the control, but only a 4% reduction was observed in the ALC 50% bread. This reduction was due to the role of shortening in the structure development of bread. The solid fraction  in shortening is known to play an important role in the formation of the structure of the dough as it influences the volume, grain, and texture of the finished product . Thus, tThe quality of bread is gradually reduced during storage and usually defined as “staling”, a term which indicates decreasing consumer acceptance of bakery products caused by changes in the crumb other than those resulting from the action of spoilage organisms . The mosALC addition at 50% replacement for shortening was expected to reduce fat content by 39% which can be clearly claimed as low-fat product, however, only a 4% reduction of calories was observed in ALC 50% bread. Further studies are required to optimize the formulation generating highly reduced calorie bakery product based on ALC replacement. Shortening replacement with other substitutes revealed that a certain level of each substitute can be applied in bakery products. Oatrim was used in cake preparation as a shortening replacement, and 20% of replacement was indicated as the maximum level without quality change  attempteThis study aimed to reduce the amount of shortening in white pan bread for industrial application by replacing it with amylose-lipid complexes without quality changes. The reaction time, temperature, and starch/lipid ratio of the corn starch and stearic acid complexes were examined to establish the optimum conditions. The corn starch and stearic acid complexing reaction at 75 °C for 60 min showed the highest complexing index of the tested conditions. The physical properties, proximate composition, and starch retrogradation during storage of ALC-containing breads were compared with those of the control bread. Crude fat and calories were decreased in the ALC-containing bread. However, the specific volume of the ALC 100% bread was significantly different from that of the control bread. The ALC 50% bread showed a reduced calorie content without volume changes, with a retardation in the starch retrogradation during storage. Collectively, the results indicate that 50% replacement of shortening with ALC has positive effects on white pan bread and could be used for the preparation of low calorie, low-fat bakery products without quality changes. Therefore, ALC was shown to be a promising shortening replacement in bread preparation.


Bristle stiffness was significantly reduced in soft and medium PBT toothbrushes after two months of use. We previously reported that polybutylene terephthalate (PBT) toothbrushes become less effective for plaque removal after two months of use. However, it remains unknown how the bristle stiffness of PBT toothbrushes changes after several months of use. We performed a single-center randomized controlled trial to evaluate the bristle stiffness and bristle splaying of soft and medium manual toothbrushes among dental and medical students of Hiroshima University. Subjects were 80 participants who met the criteria. Participants were randomly assigned to the soft toothbrush group (n = 40) or the medium toothbrush group (n = 40). We collected toothbrushes immediately after first use (T0), after one month of use (T1), after two months of use (T2), and after three months of use (T3). Bristle stiffness was measured according to the International Organization for Standardization (ISO) 22254. The mean bristle stiffness (N/cm Tooth brushing plays an essential role in the removal of dental plaque and the prevention of gingival inflammation . HoweverThere are several methods for measuring the bristle stiffness of a toothbrush . Rawls eSeveral laboratory studies have investigated the relationship between bristle stiffness and tooth abrasion ,10,11,12We performed a single-center randomized controlled trial (RCT) to evaluate the bristle stiffness and bristle splaying of soft and medium manual toothbrushes among dental and medical students of Hiroshima University . The stuTwo manual toothbrushes with different bristle stiffness (soft bristles and medium bristles) were used in this study. Participants in the soft toothbrush group used a soft toothbrush  and those in the medium toothbrush group used a medium toothbrush . We collected toothbrushes immediately after first use (T0: baseline), after 1 month of use (T1: month 1), after 2 months of use (T2: month 3), and after 3 months of use (T3: month 6), following the allocation of new toothbrushes. The toothbrushes had three rows of bristles with a total of 24 tufts and a height of 9.0 mm. The Tuft24 (soft) bristles contained approximately 37 monofilaments per tuft, and the diameter of the monofilaments was 0.15 mm. The Tuft24 (medium) bristles contained approximately 20 monofilaments per tuft and the diameter of the monofilaments was 0.2 mm.The resistance to deflection of the tufted portion was measured by ISO 22254. A toothbrush stiffness testing machine  was employed to measure the bending resistance of the tufted portion of the bristle. The testing machine conformed to the ISO 22254 standard and was composed of a brushing head, brushing table, gripping block, and load cell. The brushing table had five stainless steel bars with a diameter of 3.2 ± 0.1 mm. Before the bending resistance of the tufted portion was measured. The weight of the toothbrush head was adjusted to be approximately 5.0 g. Next, the toothbrush head was soaked in 37 ± 2 °C water for 90 seconds. The toothbrush head was shaken to remove excess water, and then fixed to the gripping block. We confirmed that the surface of the toothbrush head was parallel to the brushing table on the machine. This machine provided reciprocating movements of the toothbrush head at a speed of 10 mm per second. The toothbrush head was loaded with a force of 5 ± 0.05 Newtons (N) to make the bristles slip on the brushing table. The reaction force caused by the deflection of the tufted portion was recorded when the bristles moved back and forth five times. The average of the maximum value in the two directions  was calculated as the resistance to deflection force of the bristles. In accordance with previous study, the resistance to deflection force of the bristles (N) and the tufted area of the toothbrush  (cm²) was used to evaluate the toothbrush stiffness . The tooThe degree of toothbrush splaying was evaluated with the wear index developed by Rawls et al. and improved by Sforza et al. ,14. The p < 0.05 was considered to indicate statistical significance.The results were analyzed statistically using SPSS software . Tukey’s honestly significant difference (HSD) test or the Mann–Whitney U test with Bonferroni correction was used for multiple comparisons to compare toothbrush stiffness or wear index changes. Correlations between toothbrush stiffness and the wear index were analyzed using Spearman’s rank correlation coefficient. The bristle stiffness of toothbrushes was measured at T0, T1, T2, and T3. Medium toothbrushes exhibited higher bristle stiffness than soft toothbrushes at T0, T1, T2, and T3 . The stiWe examined the wear index of soft and medium toothbrushes at T0, T1, T2, and T3. Soft toothbrushes exhibited a higher mean wear index than medium toothbrushes at T0, T1, T2, and T3 . The meap < 0.001). Additionally, a significant negative weak correlation was found between bristle stiffness and the wear index in medium toothbrushes .A correlation between bristle stiffness and the wear index was investigated in soft and medium toothbrushes. There was a significant negative weak correlation between bristle stiffness and the wear index in soft toothbrushes  . AdditioNylon toothbrushes may be more effective in plaque removal than natural toothbrushes, because nylon bristles have higher stiffness than natural bristles . HoweverSeveral previous clinical studies have investigated the relationship between bristle splaying and plaque removal efficacy after toothbrush use ,23,24,25The American Dental Association recommends that toothbrushes are replaced approximately every 3–4 months . Long-teThere was a significant reduction in the bristle stiffness of medium and soft PBT toothbrushes after two months of use. Considering the reduced bristle stiffness and plaque removal efficacy, it is recommended that the PBT toothbrushes used in this study are replaced at least every two months.


Infectious bronchitis (IB) is a highly contagious respiratory disease in chickens and produces economic loss within the poultry industry. This disease is caused by a single stranded RNA virus belonging to Cronaviridae family. This study aimed to design a potential multi-epitopes vaccine against infectious bronchitis virus spike protein (S). Protein characterization was also performed for IBV spike protein.The present study used various tools in Immune Epitope Database (IEDB) to predict conserved B and T cell epitopes against IBV spike (S) protein that may perform a significant role in provoking the resistance response to IBV infection.1139KKSSYY1144,1140KSSYYT1145,1141SSYYT1145) were selected as surface, linear and antigenic epitopes.In B cell prediction methods, three epitopes (982YYITARDMY990 and 983YITARDMYM991 epitopes displayed high antigenicity, no allergenicity and no toxicity as well as great linkage with MHCI and MHCII alleles. Moreover, docking analysis of MHCI epitopes produced strong binding affinity with BF2 alleles.Many MHCI and MHCII epitopes were predicted for IBV S protein. Among them Five conserved epitopes were expected from spike glycoprotein of IBV as the best B and T cell epitopes due to high antigenicity, no allergenicity and no toxicity. In addition, MHC epitopes showed great linkage with MHC alleles as well as strong interaction with BF2 alleles. These epitopes should be designed and incorporated and then tested as multi-epitope vaccine against IBV. Gallus gallus). It’s a highly contagious respiratory disease in chickens that’s mainly severe for very young chicks. The signs of illness include tracheal rales, coughing, sneezing, nasal discharge and some strains may cause kidney damage [Infectious bronchitis virus (IBV) is a single Positive stranded RNA that belonging to coronavirus of the chicken , membrane (M), envelope (E), and nucleocapsid (N) protein . The spiInactivated and live-attenuated vaccines are employed to control the disease. However, inactivated vaccines often fail to induce strong cellular immunity, while live-attenuated vaccines can contribute to development of antigenic variant viruses . The incRNA viruses such as IBV have high mutational rates. Thus, the most important step in the design of cross-protective peptide vaccine against IBV is to target the conserved epitopes of different IBV serotypes .Presentation by MHC molecules is important for developing vaccinal immunity. MHC class I and class II molecules are typically highly polymorphic and polygenic . Avian MThe production of safer and more reliable vaccines for controlling IBV is important. Therefore, the aim of this study is to analyze strains of spike (S) glycoprotein of infectious bronchitis virus reported in NCBI database using immunoinformatics and computational approaches to select all possible epitopes that can be used as multi-epitopes vaccine. Protein characterization was also achieved for IBV spike protein.http://www.ncbi.nlm.nih.gov/protein/) database in March 2019. The sequences were saved in FASTA format (Table Spike (S) protein sequences of different infectious bronchitis virus (IBV) strains were retrieved from the GeneBank of National Central Biotechnology Information (NCBI) (Reference sequence of spike S protein (NP_040831.1) was analyzed to identify chemicals and physical properties including GRAVY (grand average of hydropathicity), half-life, molecular weight, stability index and amino acids atomic composition using an online tool Protparam https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl. TMHMM an online tool (http://www.cbs.dtu.dk/services/TMHMM/), used to examine the trans-membrane topology of S protein. Presence of disulphide-bonds were predicted through an online tool DIANNA v1.1. It makes prediction based on trained neural system [http://www.ncbi.nlm.nih.gov/BLAST/) [https://pfam.xfam.org/) [https://blast.ncbi.nlm.nih.gov/Blast.cgi) using reference sequence (refseq-protein) database was used to compare spike reference sequences of different coronaviruses in human and animals against IBV spike protein sequence. Phylogenetic tree was also constructed based on COBALT multiple alignment (https://www.ncbi.nlm.nih.gov/blast/treeview/treeView.cgi) [Secondary structure of spike S protein of IBV was analyzed through PSIPRED . The secl system . CDD-BLA/BLAST/) –21 and Pam.org/)  were useiew.cgi) , 20.http://www.jalview.org/about/jalview-scientific-advisory-committee) [http://tools.iedb.org/conservancy/) [The retrieved sequences of IBV S protein were aligned using Clustal program and consensus sequence was generated using the multiple sequence alignment (MSA) tool, Jalview version 2.10.5. (mmittee) . Epitopervancy/) . For calPhylogenetic tree of the retrieved sequences of spike (S) protein was performed using MEGA7.0.26 (7170509) software using maximum likelihood parameter .http://tools.iedb.org/mhci/) was used to predict B and T cell epitopes of IBV reference sequence of S protein (NP_040831.1) [The Immune Epitope Database (IEDB) (40831.1) . Linear 40831.1) . Emini s40831.1) . Whereas40831.1) .Discontinuous epitopes were predicted using DiscoTope server . The parThe T cell epitopes were predicted in human among different alleles of major histocompatibility complex class I (MHCI) and class II (MHCII).http://tools.iedb.org/mhci. The binding affinity of peptides to MHC I molecules was measured using artificial neural networks (ANN) method [MHC-I binding epitopes were predicted by the IEDB MHC I prediction tool at ) method , 33. Prihttp://tools.iedb.org/mhcii/) [The IEDB MHCII prediction tool was used for MHC class II molecules at (/mhcii/) . Human M/mhcii/) .http://www.jenner.ac.uk/VaxiJen) [http://www.ddg-pharmfac.net/AllerTOP) [http://crdd.osdd.net/raghava/toxinpred/) [VaxiJen v2.0 server was used to predict the antigenicity of the conserved regions (VaxiJen) . The defllerTOP) . While Tinpred/) .http://raptorx.uchicago.edu/) to design their three D structures [http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) from amino acid sequences [IBV reference sequence and the protein sequences of BF alleles (BF2 *2101 and BF2*0401) were submitted to Raptor X server (ructures –40. PEPFequences –43. Chimequences .https://bioinfo3d.cs.tau.ac.il/PatchDock/) [To perform molecular docking, 3D structures of MHCI epitopes and BF alleles were submitted simultaneously to the PatchDock online autodock tools; an automatic server for molecular docking (chDock/) . The fivchDock/) . The reschDock/) .The physiochemical properties of the spike S protein, measured through Protparam, showed that it contained 1162 amino acids (aa) with a molecular weight of 128,046.70 kDa. The spike protein showed an antigenic nature when subjected to Vaxijen v2.0.Theoretical isoelectric point (PI) of spike protein was 7.71, indicating its positive in nature. An isoelectric point above 7 indicates the protein is charged positively. Near to 81 aa charges were found negative, whereas 84 aa found positive.Escherichia coli. The total numbers of Carbon (C), Oxygen (O), Nitrogen (N), Hydrogen (H) and Sulfur (S) were entitled by the formula: C5737H8847N1495O1718S56.Protparam computed instability-index (II) 35.53, this categorize the protein as stable. Aliphatic-index 86.05, which devotes a thought to the proportional volume holding by aliphatic side chain and GRAVY value of the protein sequence is 0.012. Half-life of S protein shown as the total time taken for its vanishing after it has been synthesized in cell, computed as 30 h for mammalian-reticulocytes, > 20 h for yeast, > 10 h for The secondary structure of IBV spike S protein was analyzed through PSIPRED and GOR IV server. The components of secondary structure prediction by GOR IV server are alpha helix (29.43%), extended strand (27.37%), beta turn (5.25%), and random coil (37.95%) Fig..Fig. 1 positions and assign them a score and makes prediction based on trained neural system. The trans-membrane protein topology was investigated via online tool TMHMM. Residues from 1 to 1093 were found to be exposed to the surface, residue from 1094 to 1116 were found inside trans-membrane-region and residues from 1117 to 1162 were buried within the core-region of the S protein  were identified. The conserved domains were sequenced by Conserved Domain (CDD) BLAST search. The results revealed that corona-S1 (pfam01600) is the only member of the superfamily cl03276 and corona-S2 domain (pfam01601) is the only member of the superfamily cl20218. The top associated sequences in both domains were Feline infectious peritonitis virus (strain 79–1146), Avian infectious bronchitis virus (strain Beaudette), and Human coronavirus 229E while Severe acute respiratory syndrome-related coronavirus sequences were associated only with corona-S2 domain. The closest homologue obtained from BLASTP (refseq-protein) results was the Turkey coronavirus S protein with E value 0.00 followed by Murine hepatitis virus strain JHM with E value 9e-109 when comparing various coronaviruses in human and animals with IBV spike protein sequence  software using maximum likelihood parameter see Fig. 1139KKSSYY1144,1140KSSYYT1145,1141SSYYTT1146,1141SSYYT1145,1142SYYTT1146,1142SYYT1145, and 1143YYTT1146. Based on the length and antigenicity score, three epitopes  were selected as top B cell epitopes.Several epitopes were predicted in B cell prediction methods using the Bepipred Linear Epitope Prediction tool. The conservancy percentages of these epitopes are presented in Table Discotope 2.0 server was used to predict the discontinuous epitopes from the 3D structure of S protein (PDB ID: 6CV0), 90% specificity, − 3.700 threshold and 22.000 Angstroms propensity score radius . Total 31115FFMTGCCGC1123,590FNLTVTDEY598,734GLLVLPPII742,1105IIFILILGW1113,1139KKSSYYTTF1147,1087KTYIKWPWY1095,166SVYLNGDLV174,985TARDMYMPR993,1145TTFDNDVVT1153,983YITARDMYM991,1144YTTFDNDVV1152,982YYITARDMY990,1143YYTTFDNDV1151.In this study, the Human MHC class-I HLA alleles were used to explore the interaction of epitopes with MHCI alleles as chicken MHC alleles don’t exists in IEDB database. MHC-1 binding prediction tool using IEDB database expected 13 conserved epitopes of spike protein (S) which were interacted with many cytotoxic T cell alleles. These epitopes were 694EDLLFTSVE702,1147FDNDVVTEQ1155,1115FFMTGCCGC1123,1116FMTGCCGCC1124,590FNLTVTDEY598,734GLLVLPPII742,1105IIFILILGW1113,902INECVKSQS910,984ITARDMYMP992,901KINECVKSQ909,1139KKSSYYTTF1147,1140KSSYYTTFD1148,1187KTYIKWPWY1195,735LLVLPPIIT743,592LTVTDEYIQ600,1014NKTVITTFV1022,893QQRELATQK901,894QRELATQKI902,895RELATQKIN903,589SFNLTVTDE597,1141SSYYTTFDN1149,166SVYLNGDLV174,1142SYYTTFDND1150,985TARDMYMPR993,1146TFDNDVVTE1154,593TVTDEYIQT601,1013VNKTVITTF1021,983YITARDMYM991,1144YTTFDNDVV1152,982YYITARDMY990 and 1143YYTTFDNDV1151.MHC-II binding prediction tool based on NN-align with half-maximal inhibitory concentration (IC50) ≤ 1000 was used. Thirty one conserved core sequences were predicted to interact with MHCII alleles. These cores were 985TARDMYMPR993, 983YITARDMYM991 and 982YYITARDMY990) showed a high linkage with MHCI alleles  using peptide-binding groove affinity. The chicken alleles were used as receptors, and the top MHCI epitopes Epitopes capable of inducing immunity in both types (B-cell and T-cell) are considered to be strong candidates for the vaccine . There aIBV vaccination studies have always focused on humoral immune responses regarding protection. Acquired immunity results in the activation of antigen-specific effector mechanisms including B-cells , T-cells (cellular) and macrophages, and memory cells production . ChickenIBV glycoprotein S1 is known to be responsible for virus neutralization (VN) and haemagglutination-inhibition HI antibodies and has been considered the most likely protective inducer . Multi-pIn the present study, IBV spike protein was analyzed using various prediction servers. Protein characterization of IBV spike S protein using Protparam confirmed its positive in nature and stable. The protein also exhibited good antigenic properties using Vaxijen 2.0v server.Corona-S1 and Corona S2 have been identified as major conserved domains in the IBV spike glycoprotein refseq. Conserved Domain (CDD) BLAST search revealed that corona-S1 (pfam01600) is the only member of the superfamily cl03276 and corona-S2 domain (pfam01601) is the only member of the superfamily cl20218. The main related sequences in both domains were Feline infectious peritonitis virus (strain 79–1146), Avian infectious bronchitis virus (strain Beaudette), and Human coronavirus 229E. However, Severe acute respiratory syndrome-related coronavirus sequences was only associated with corona-S2 domain .Prediction of B-cell epitopes is essential for the design vaccine components and immuno-diagnostic reagents. B-cell antigenic epitopes are either continuous or discontinuous in nature.Most epitope prediction methods are based on continuous epitopes . It has 1139KKSSYY1144,1140KSSYYT1145,1141SSYYTT1146,1141SSYYT1145,1142SYYTT1146,1142SYYT1145, and 1143YYTT1146) were predicted from B cell prediction methods as surface, linear and antigenic epitopes. The predicted epitopes were adjacent to each other from the position 1139–1146. In a similar study, only one epitope (YTSNETTDVTS175–185) was predicted within the S1 glycoprotein of M41 IBV strains using BepiPred epitope prediction server version 1, and three such epitopes  were predicted in CR88 IBV strains [In this study, seven shortened conserved epitopes  and only a minority of native antigens have linear B-cell epitopes . DiscotoEpitopes from the 3D structure of the spike IBV reference sequence. Around 30 discontinuous epitopes with a specificity of 90% were recognized at different exposed surface areas. These epitopes have a significant advantage in identifying the native well-structured protein Ag .Cytotoxic T lymphocytes (CTL) provide a critical arm of the immune system in eliminating autologous cells expressing foreign antigen. Unlike humoral immunity, the specificity of CTL activation depends on membrane receptors rather than secreted molecules, and antigen receptors of CTL interact with peptide determinants only in association with matched major histocompatibility complex (MHC) molecules. Virus-specific CTL have been shown to be important, if not critical, for resolution of infection and elimination of viral shedding .+ lymphocytes were mostly responsible for the observed protection [It is stated that, the major histocompatibility complex MHC restricted CTL response can be associated with decreases in viral load, and CD8otection , 56. Resotection .985TARDMYMPR993, 983YITARDMYM991 and 982YYITARDMY990 as they linked to 7 and 3 human MHCI alleles respectively and showed high antigenicity, no allergenicity and no toxicity. Recent studies showed that vigorous cytotoxic T lymphocyte (CTL) responses that correlate with initial decrease in infection and illness can be detected after IBV infection. It has been identified that the CD8+ T cells were exhausted without CD4+ helper T cells. CD4+ T cells do not seem important in the initial resolution of IBV infection in chickens [However, MHCI prediction methods showed three conserved CTL epitopes chickens .983YITARDMYM991 and 982YYITARDMY990 which were presented in MHCI prediction methods. They linked with 52 and 38 human alleles respectively. These epitopes showed high antigenicity, no allergincity and no toxicity.In MHCII prediction method, several core peptides were predicted to interact with MHCII alleles, but surprisingly the top core peptides were also 982YYITARDMY990,983YITARDMYM991 and 985TARDMYMPR993). The 3D structures of MHC I epitopes were designed using PEPFOLD and docked with BF alleles via Patchdock server. Docking the epitope 983YITARDMYM991 with both BF2 alleles produced strong binding affinity  followed by 982YYITARDMY990 . This indicates the strong interaction between the ligand and the receptor compared to other epitopes  and MHCI epitopes  and T cell epitopes (982YYITARDMY990 and 983YITARDMYM991). They showed high antigenicity, no allergenicity and no toxicity as well as great linkage of MHC epitopes with their alleles. The suggested epitopes should be designed, incorporated and tested as multi-epitopes vaccine against IBV. This vaccine may serve as a possible peptide vaccine to control IBV infection in chicken by inducing humoral and cellular responses.In this study, five epitopes were predicted from spike glycoprotein of IBV as the best B cell (Peptide vaccination against IBV spike protein (S) can strongly replace traditional vaccines as it is designed to cover all strains in different serotypes, which can reduce recurring outbreaks and their associated massive economic losses.


Further, prosthetic height had no effect on maximum speed (p = 0.762). Faster maximum speeds were associated with reduced ground contact time, aerial time, and overall leg stiffness, as well as with greater stance-average vertical ground reaction force, contact length, and vertical stiffness . RSP shape, but not stiffness or height, influences the maximum speed of athletes with bilateral transtibial amputations.Running-specific prostheses (RSPs) have facilitated an athlete with bilateral transtibial amputations to compete in the Olympic Games. However, the performance effects of using RSPs compared to biological legs remains controversial. Further, the use of different prosthetic configurations such as shape, stiffness, and height likely influence performance. We determined the effects of using 15 different RSP configurations on the maximum speed of five male athletes with bilateral transtibial amputations. These athletes performed sets of running trials up to maximum speed using three different RSP models  each with five combinations of stiffness category and height. We measured ground reaction forces during each maximum speed trial to determine the biomechanical parameters associated with different RSP configurations and maximum sprinting speeds. Use of the J-shaped Cheetah Xtend and 1E90 Sprinter RSPs resulted in 8.3% and 8.0% (p<0.001) faster maximum speeds compared to the use of the C-shaped Catapult FX6 RSPs, respectively. Neither RSP stiffness expressed as a category (p = 0.836) nor as kN·m Athletes with bilateral transtibial amputations (TTAs) who use running-specific prostheses (RSPs) have achieved Olympic-qualifying 400 m performances  and havec) (the distance travelled by the center of mass during ground contact) and stance average vertical GRF relative to body weight. Thus, running speed also equals the product of Lc, stance average vertical GRF, and step frequency [c) [-1 during 40 m sprint trials over ground [-1 during brief treadmill sprints (20 steps) [There are many biomechanical variables that influence maximum speed and running performance . Runningrequency . Among trequency  and stepency [c) , 13. Elir ground , and up 0 steps) .-1 for a sprinter with bilateral TTAs [-1 for a sprinter with a unilateral TTA during brief treadmill sprints [Use of RSPs has enabled athletes with TTAs to achieve remarkable sprinting speeds , 14, 15. sprints . Similar sprints –5. Incre sprints –5 and ae sprints , 5 and h sprints  or simil sprints . It is n sprints , 4 in thvert) [leg) constant [The sprinting ability of athletes with TTAs may be limited by the stiffness of their RSPs. Unlike biological legs, the mechanical characteristics of RSPs, such as stiffness, cannot be actively modulated during running. Previous studies have found that to achieve faster running speeds, non-amputees increase vertical stiffness (kvert) , while econstant  or increconstant . RSPs arconstant . Manufacconstant –23. An aconstant –23. Howeconstant .vert), but decreased dimensionless leg stiffness (Kleg) in their affected legs at faster speeds. However, non-amputees increased Kvert and Kleg at faster running speeds. Across speeds ranging from 3 to 9 m·s-1, RSP stiffness affects the biomechanics of sprinters with bilateral TTAs [leg and decreases tc compared to use of less stiff RSPs, suggesting that more stiff RSP categories may permit increased Kvert and Kleg and thus elicit faster maximum sprinting speeds in athletes with bilateral TTAs. However, the effects of prosthetic stiffness on the biomechanical variables that determine running speed are mitigated at progressively faster speeds [-1), the differences in stance average vertical GRFs and tc between more stiff and less stiff RSPs are modest. Thus, it is unclear if prosthetic stiffness affects the maximum speeds of athletes with bilateral TTAs.McGowan et al.  found thral TTAs . Use of r speeds . Particu-1). Others [RSPs mimic the spring-like behavior of tendons and ligaments during running by storing elastic energy during the first half of the contact phase and returning most of this energy during the second half of the contact phase , 20, 24.. Others  claim thin-vivo Achilles tendon hysteresis in non-amputees report values between 6.8% [in-vitro measurements of mammalian tendons report hysteresis between 7% [Tendon and RSP hysteresis, defined as the percentage of elastic energy lost during recoil relative to the energy stored during loading , affectseen 6.8%  and 30% een 6.8% , while itween 7%  and 10% tween 7% . All of tween 7%  affect ttween 7%  and may -1. Slower step frequencies may be due to the greater rotational moment of inertia of the whole leg and possibly negate any effect of prosthetic height on maximum running speed. Yet, though we previously reported step frequency and step length for speeds of 3 to 9 m·s-1, it is not clear if this negative correlation between step length and step frequency due to height is retained at maximum sprinting speeds.Running performance may also be influenced by leg length. Hypothetically, longer legs could allow athletes to achieve longer steps and, for a given step frequency, faster speeds. Athletes with bilateral TTAs can alter their leg lengths by changing the height of their RSPs. However, these athletes must adhere to the International Paralympic Committee (IPC) guidelines , 31 to c-1) would allow faster maximum sprinting speeds in athletes with bilateral TTAs compared to use of more compliant RSPs (lower category and kN·m-1). Second, we hypothesized that use of taller RSPs would allow athletes to achieve faster maximum sprinting speeds compared to use of shorter RSPs. Third, we hypothesized that the RSP configuration that allows athletes to achieve faster maximum sprinting speeds would be associated with greater stance-average vertical GRF, longer contact length, and shorter ground contact time. Lastly, we hypothesized that the RSP configuration that allows the fastest maximum speed would be associated with the greatest mechanical energy return per step.We determined the maximum speeds elicited by athletes with bilateral TTAs using 15 different RSP configurations . First, we hypothesized that use of stiffer RSPs  participated and were the same subjects as in Beck et al. , 24. AllWe used a repeated-measures experimental design. On the first day, each participant completed an alignment and accommodation session. During this session, a certified prosthetist aligned participants to three different RSP models  to three per day and, based on subject’s feedback, scheduled additional rest days between testing sessions. The duration of the testing protocol  was typically 10–11 days.On subsequent days, we asked participants to run over a range of speeds up to their maximum speed using three different RSP stiffness categories per model: manufacturer-recommended, and one category more stiff and one category less stiff than recommended. We randomized the trial order . The stiffness category that elicited the fastest maximum speed for each RSP model was subsequently used with prosthetic heights that were increased or decreased by 0.02 m with respect to the maximum IPC height to determine the effects of prosthetic height on maximum speed. If the closest achievable height, based on an athlete’s residual limb lengths and the build height of the RSPs, was taller than the maximum IPC competition height, we increased prosthetic height by 0.02 m and 0.04 m. If the closest achievable height was shorter than the maximum IPC competition height, we decreased prosthetic height by 0.02 m and 0.04 m. In order to mitigate potential learning effects, we randomly inserted these trials into the trial order . To minimize the potential for fatigue, we limited the maximum number of series of trials  and aerial time (ta), then calculated step frequency (fStep) and step length (lstep). We measured peak and stance average vertical GRFs, and peak positive and negative horizontal GRFs and we calculated dimensionless stiffness, Kvert and Kleg [RSP, in W·kg-1) and energy return  during the second half of ground contact .We collected GRFs at 1000 Hz on a 3D force measuring treadmill . For each maximum speed trial, we used a custom MATLAB script  to filter the GRFs using a fourth order Butterworth filter with a 30 Hz cutoff. We measured ground contact time , and height on maximum speed. We used a third linear mixed model to determine the effects of contact and aerial times, peak and stance average vertical GRFs, peak positive and negative horizontal GRFs, and vertical and leg dimensionless stiffness on maximum speed. Step frequency and step length were excluded from this model given their intrinsic interaction with maximum speed  using the Össur Flex-Foot Cheetah Xtend RSPs, 9.31 ± 0.80 m·s-1 using the Ottobock 1E90 Sprinter RSPs and 8.68 ± 0.95 m·s-1 using the Freedom Innovations Catapult FX6 RSPs. While controlling for covariates, the use of the J-shaped Cheetah Xtend and 1E90 Sprinter RSPs facilitated 8.3% (p<0.001) and 8.0% (p<0.001) faster maximum speeds compared to use of the C-shaped Catapult FX6 RSPs, respectively. We found no significant differences in maximum speeds between the Cheetah Xtend and 1E90 Sprinter RSPs (p = 0.842). We found no effect of prosthetic stiffness when expressed as a category  on maximum speed. In addition, prosthetic height had no effect on maximum speed , aerial time (ta), stance average vertical ground reaction force , and contact length (Lc) together with leg and vertical dimensionless stiffness (Kleg and Kvert) were associated with maximum speed:We found that among all investigated biomechanical variables, contact time , a 0.173 s decrease in ta , a 1.09 BW increase in GRFavg,z , a 0.13 m increase in Lc , a 50 unit decrease in Kleg , and a 333 unit increase in Kvert .For example, while controlling for covariates, a 0.01 second decrease in tp<0.001, , a 0.01 p<0.001, , a 0.01 p<0.001, , a one up<0.001, , and a op<0.001, . And, whc compared to use of the 1E90 Sprinter and Catapult FX6 RSPs, respectively (p<0.001 for both comparisons). Use of the Catapult FX6 RSPs resulted in 6.9% shorter ta compared to use of the Cheetah Xtend RSPs (p = 0.002), while there were no differences in ta when using the Cheetah Xtend versus 1E90 Sprinter RSPs (p = 0.100) and the Catapult FX6 versus 1E90 Sprinter RSPs (p = 0.146). Use of the Cheetah Xtend RSPs resulted in 4.3% and 5.4% greater GRFavg,z compared to use of the 1E90 Sprinter (p = 0.026) and Catapult FX6 (p = 0.027) RSPs, respectively. Use of the 1E90 Sprinter RSPs resulted in 4.0% longer Lc compared to use of the Cheetah Xtend RSPs (p<0.001), which in turn resulted in 3.1% longer Lc compared to use of the Catapult FX6 RSPs (p<0.001). Use of the 1E90 Sprinter RSPs resulted in 9.4% lower Kleg compared to use of the Cheetah Xtend RSPs (p = 0.02), which in turn resulted in 17.2% lower Kleg compared to use of the Catapult FX6 RSPs (p<0.001). Use of the 1E90 Sprinter RSPs resulted in 12% lower Kvert compared to use of the Catapult FX6 RSPs (p = 0.001), but Kvert was not different compared to use of the Cheetah Xtend RSPs (p = 0.118).Using a post-hoc analysis, we found that at their respective maximum speeds, use of the Cheetah Xtend RSPs resulted in 5.0% and 5.1% shorter tstep, step, We found that across all RSP configurations, both step length lstep,  and stepstep compared to use of the Catapult FX6 RSPs (p<0.001 for both comparisons). We found no differences in fstep between RSP models .We found that use of the Cheetah Xtend and 1E90 Sprinter RSPs resulted in 9.0% longer lRSP, RSP, We found that maximum speed was positively correlated with both prosthetic mechanical power return PRSP,  and elasRSP ranged from 3.92 W·kg-1 (corresponding to an 8 m·s-1 maximum speed) to 8.22 W·kg-1 (corresponding to a 9.5 m·s-1 maximum speed), while ERSP ranged from 2.11 J·kg-1·m-1 (corresponding to an 8 m·s-1 maximum speed) to 4.05 J·kg-1·m-1 (corresponding to a 9.5 m·s-1 maximum speed). Use of the 1E90 Sprinter RSPs resulted in 15.9% and 20.3% greater mechanical power return compared to use of the Catapult FX6 and Cheetah Xtend RSPs, respectively (p = 0.001 for both comparisons) and resulted in 11.8% and 16.3% higher elastic energy return compared to use of the Catapult FX6 (p = 0.016) and Cheetah Xtend (p<0.001) RSPs, respectively.Among all maximum speed trials, PHysteresis (H) was negatively correlated with maximum speed across all RSP configurations :MaximumWe found that based on the equations of Beck et al. , the CheContrary to our first hypothesis, alterations in prosthetic stiffness alone did not affect maximum sprinting speed for athletes with bilateral transtibial amputations. Our results agree with and extend the findings of Beck et al.  who repostep = 4.39−0.026×Δh; p = 0.078). This trend is supported by the findings of Beck et al. [-1) speeds. Our findings challenge the IPC regulations that limit prosthetic height for athletes with bilateral TTAs [Contrary to our second hypothesis, altering prosthetic height alone by ±0.02 m did not affect maximum sprinting speed for athletes with bilateral TTAs. A ±0.02 m change in prosthetic height is approximately a ±2% change in leg length, which could potentially change running speed by ±2% for athletes if contact time and aerial time remain unchanged , 35. We ral TTAs . A recenc), higher stance-average vertical ground reaction forces , and longer contact lengths (Lc) were associated with faster maximum sprinting speeds, indicating that these biomechanical variables likely have similar associations with sprinting performance for both non-amputees and sprinters with bilateral TTAs. Contact times for sprinters with bilateral TTAs across RSP configurations (0.116 ± 0.012 s) were similar to those reported for non-amputees [-1; non-amputees [-1). Stance average vertical ground reaction forces for sprinters with bilateral TTAs across RSP configurations (1.90 ± 0.15 BW) were lower than those reported for non-amputees [Our third hypothesis was supported. Similar to previous studies of non-amputee sprinters , 13 and We found that faster maximum speeds are associated with increased vertical stiffness for all of the prosthetic configurations tested in sprinters with bilateral TTAs. This is similar to what previous studies report for non-amputees  and athlleg) compared to the affected kleg, and the values were constant for running speeds between 2.5 and 3.5 m/s. McGowan et al. [leg) and relative sprinting speed in non-amputees, but a negative correlation for the affected legs of sprinters with unilateral and bilateral TTAs. Beck et al. [leg) and absolute running speeds between 3 and 7 m·s-1 for athletes with bilateral TTAs. Both studies [-1). Our linear mixed model did not evidence a direct association between the angle of the leg at touch down and maximum speed. However, there is a clear relationship between θ, leg length (L0) and contact length (Lc) (Lc = L0sin(θ)) [-1). These findings indicate that the association of faster maximum speeds and decreased leg stiffness in athletes with bilateral TTAs is associated with an increased angle of the leg at touch down.We found that decreased leg stiffness is associated with faster maximum speeds for all of the prosthetic configurations in sprinters with bilateral TTAs. Previous studies of non-amputees report contrasting findings regarding leg stiffness and running speed. While some studies found no relationship , others n et al.  reported studies , 5 found0sin(θ)) , 41, 42.-1) to progressively faster (up to 9 m·s-1) speeds for any given RSP configuration.We found that increases in step length and step frequency were associated with faster maximum speeds for all of the prosthetic configurations in sprinters with bilateral TTAs. Previous research on non-amputee sprinters  and spriUse of the two J-shaped RSP models that resulted in the fastest maximum speeds, the Cheetah Xtend and 1E90 Sprinter RSPs, was associated with all of the biomechanical parameters associated with maximum speed except for aerial time and step frequency. At maximum speed, the use of both RSP models resulted in longer contact lengths, lower leg stiffness, and longer step lengths compared to use of the Catapult FX6 RSPs. In addition, maximum speeds were associated with different biomechanical variables for the J-shaped RSPs compared to the C-shaped Catapult FX6 RSPs. In particular, at maximum speed use of the Cheetah Xtend RSPs resulted in shorter contact times and higher stance average vertical GRFs, while use of the 1E90 Sprinter RSPs resulted in lower vertical stiffness, compared to the C-shaped Catapult FX6 RSPs. Other factors such as different sagittal plane alignment, the width, thickness and geometry of each RSP model, and the RSP moment of inertia may elicit different biomechanics that are associated with faster maximum speeds.RSP) and mechanical power return (PRSP). Our results indicate that faster maximum speeds are associated with greater ERSP and PRSP when athletes with TTAs use J-shaped RSPs. Further, use of RSP models with low compared to high hysteresis values, i.e. more energy return, may elicit faster maximum speed in athletes with TTAs.We accept our fourth hypothesis that the use of the prosthetic configuration that results in the fastest maximum speed maximizes the energy return per step. Lower hysteresis values in J-shaped RSPs compared to C-shaped RSPs, as shown by Beck et al. , allow gWe encountered some limitations in our study. We did not find statistically significant interactions among the biomechanical variables included in Prosthetic model (shape), but not stiffness or height, is associated with maximum sprinting speed in athletes with bilateral transtibial amputations. Use of prosthetic models with a J-shaped design resulted in 8.0–8.3% faster maximum sprinting speeds compared to a prosthetic model with a C-shaped design. Among different model, stiffness and height prosthetic configurations, shorter contact and aerial times, higher stance average vertical ground reaction forces, longer contact lengths, lower leg stiffness, higher vertical stiffness, longer step lengths and higher step frequencies were associated with faster maximum sprinting speeds in athletes with bilateral transtibial amputations. Our findings that RSP height changes of ±0.02 m do not affect maximum speed, one of the key aspects of performance, are in contrast with current International Paralympic Committee (IPC) regulations that limit the competition height of athletes with bilateral transtibial amputation. Based on our findings, we encourage the governing bodies of athletics to adopt regulations based on scientific evidence.c) and aerial time (ta) using a vertical GRF threshold of 20 N and calculated step frequency (fStep) as:step) as:-1.We identified ground contact time  divided by the peak vertical displacement of the center of mass :leg as the peak vertical GRF  divided by the peak leg compression :We also calculated peak and stance average vertical GRFs, and peak positive and negative horizontal GRFs . We calculated k0) for each trial as the distances from the greater trochanters to the most distal locations of the unloaded RSPs. We then calculated ΔL as:We calculated Δz by integrating the vertical acceleration of the center of mass twice . We measGonzales , for a d−cosθ)Eq where θ c2×L0)Eq  of each subject:We calculated dimensionless stiffness, KRSP) and energy return (eRSP) during the second half of the ground contact phase as a function of prosthetic stiffness (kRSP), peak prosthetic displacement (Δd), prosthetic hysteresis (HRSP) [step):Body weight included the weight of each athlete and his RSPs and was measured by averaging the vertical GRF data over 10 strides for each trial. For each RSP configuration, we calculated mechanical power (ps (HRSP) , and stemax,z for each trial, multiplied by 1000 and divided by the calculated displacement to obtain prosthetic stiffness , which is utilized in We used the equations reported in Beck et al.  to calcuRSP by running speed (s), equals the energy return per meter travelled:Dividing pRSP is measured in W·kg-1 and ERSP in J·kg-1·m-1.To compare different subjects, we divided both quantities by each subject’s body mass including his RSPs (m) :PRSP=pRS1 FileMaximum sprinting speed and biomechanical parameters for each subject in each tested condition.(XLSX)Click here for additional data file.


Prosthetist subjects were also considerably less consistent than amputee subjects in their preferences . Mutual preference seemed to be dictated by the specific patient-prosthetist dynamic, and no clear trends emerged.When fitting prosthetic feet, prosthetists fuse information from their visual assessment of patient gait with the patient’s communicated perceptions and preferences. In this study, we sought to simultaneously and independently assess patient and prosthetist preference for prosthetic foot stiffness using a custom variable-stiffness prosthesis. In the first part of the experiment, seven subjects with below-knee amputation walked on the variable-stiffness prosthetic foot set to a randomized stiffness, while several prosthetist subjects simultaneously observed their gait. After each trial, the amputee subjects and prosthetist subjects indicated the change to stiffness that they would prefer (increase or decrease). This paradigm allowed us to simultaneously measure amputee subject and prosthetist subject preferences, and provided a reliability index indicating the consistency of their preferences. In the second part of the experiment, amputee subjects were instructed to communicate verbally with one prosthetist subject to arrive at a mutually preferred stiffness. On average, prosthetist subjects preferred a 26% higher stiffness than amputee subjects ( While there is relative agreement about the important input variables , answers about what to focus on or optimize are less clear. Researchers tend to base their recommendations on biomechanical analyses that are difficult to perform in a clinical setting, whereas prosthetists tend to rely on qualitative feedback from patients and visual assessment of gait to guide their decisions, which can be difficult to quantify. High quality perceptive analyses in the field of prosthetics are generally lacking. That is, it is difficult to design experiments free from subject biases, such as acquiescence bias and the placebo effect, and subjective rating scales are often coarse, unvalidated, and not easily analyzed using conventional statistical metrics5.The clinical process of matching and fitting lower-limb amputation patients with prosthetic componentry is as much art as it is scienceIn this investigation, we focus on developing a more rigorous understanding of how people with below-knee amputation (BKA) and certified prosthetists (CP) develop and communicate preferences for prosthetic foot stiffness. Stiffness is a key determinant of overall prosthetic foot behavior; it defines how energy is absorbed and returned during collision with the ground, and how the foot provides support and ‘push-off’ in the terminal stance phase of gait. Stiffness varies by prosthetic foot model and category, and manufacturers often provide tables of recommended categories, based on patient weight and activity level. Based on discussions with clinical and industrial collaborators, we have found that these recommendations are often informally derived from internal testing with highly experienced users.7. Conversely, when paired with an overly compliant foot, people with BKA may incur foot-slap in early stance and loss of anterior support in terminal stance, leading to increased quadriceps activity to maintain stability8. The exacerbation of gait abnormalities and asymmetries can lead to long-term side effects, including chronic back pain and osteopenia9. Researchers have sought specific biomechanical markers that indicate an appropriate stiffness, but these experiments have tended to be inconclusive, ultimately yielding tentatively issued and often contradictory guidelines12. More holistic functional outcomes, such as self-selected walking speed, metabolic cost, or balance, have also been proposed as optimizable metrics, but don’t appear particularly sensitive to foot mechanics15. Thus, while we know prescribing the right stiffness is important to overall patient quality of life, researchers have not converged on clinically viable methods for doing so.Pairing a patient with the appropriate stiffness is critical to their overall mobility, and possibly their long-term health. When paired with an inappropriately stiff foot, people with BKA may incur increased shock loading and prolonged heel contact during early stance, knee hyperextension during terminal stance, and reduced energy storage and returne.g. socket comfort, smoothness of motion, trust in balance, or local muscle fatigue). It is unlikely that these outcomes will be perceptibly improved using more measurable variables, such as gait symmetry or metabolic cost of locomotion, as proxy. However, patients are able to sense these factors, and can often provide verbal feedback to prosthetists. This feedback may not be in the form of the intended variable, but instead in the form of the sensation, and it is the prosthetist’s responsibility to map that sensation into an appropriate change .Patient experience is dominated by outcomes that are difficult to quantify  differ in both their values and their reliability. We accomplish this goal by implementing a two-alternative forced choice task, in which a BKA subject walks on a variable-stiffness prosthetic ankle set to an unknown stiffness level, and both the observing prosthetist subjects and the BKA subject silently report the directional change to stiffness they would prefer. Following these trials, we conducted a subsequent set of trials where communication between the BKA subject and a prosthetist subject was permitted, to see if mutual preference is more strongly biased towards either individual preference of the patient or prosthetist.In this set of experiments, we sought to understand how stiffness preference differs between patients and prosthetists; specifically, how the independent sources of information available to a prosthetist (Seven certified prosthetist (CP) subjects were recruited from the Shirley Ryan AbilityLab via word-of-mouth, and seven high-activity (K3-K4) subjects with below-knee amputation (BKA) were recruited through the facility’s prosthetics and orthotics clinic. Further details for the BKA subjects are presented in the Appendix. The study consisted of a complete 4 × 4 block and an incomplete 3 × 3 block, in which multiple prosthetist subjects were present for each BKA subject visit, and each prosthetist subject observed at least two BKA subjects. The split experimental blocks were chosen out of necessity, due to the difficulty in scheduling many prosthetists to be available simultaneously. Prior to the study, prosthetist subjects completed a questionnaire designed to elucidate the importance of prosthesis stiffness in clinical decision-making. This study was approved by the Northwestern University Institutional Review Board, and the study was carried out in accordance with the regulations and guidelines of the Northwestern University Institutional Review Board. Written informed consent was obtained by all BKA subjects and prosthetist subjects prior to participating in the experiment, as was written informed consent to publish their images.18. This experimental ankle–foot consists of a rigid footplate (23.5 cm or 25.3 cm based on subject height) with attached crepe shoe material, and a rotating ankle joint. To change ankle stiffness, a small motor in the keel repositions the fulcrum of a propped cantilever spring embedded in the structure20. Notably, the torque–angle relationship is linear, and plantarflexion stiffness is 33% of dorsiflexion stiffness19. The experimenter commanded stiffness changes to the prosthesis wirelessly. To mask auditory clues of the magnitude and direction of stiffness changes, the motor controller separated the movement of the moving fulcrum into multiple jogs. A small degree of accuracy may be lost in simulating the mechanics of prosthetic feet with a rigid foot and dynamic ankle, but this approach reduces the complicated mechanical behavior of prosthetic feet to a single controllable, reproducible, and reportable variable: the angular stiffness of the ankle joint.A quasi-passive ankle prosthesis capable of continuous stiffness variation was used to modify ankle stiffness between trials19, the subject practiced walking on the prosthesis set to stiffness levels covering a range of 66–150% of the starting stiffness. During practice, the experimenter indicated the directionality of the changes to stiffness, to educate subjects on the relationship between changes to stiffness and the associated sensations. Subjects typically walked on each stiffness level for a couple minutes, until they were comfortable and ready to try a new stiffness. After the subject practiced walking at a range of stiffnesses, the subject provided verbal feedback regarding their preferred stiffness, which then served as the reference stiffness for the experiment. This process gave the subject approximately 30 min of time walking on the prosthesis.A prosthetist on the research team (not included as a subject in the study) attached and aligned the variable-stiffness ankle–foot to the BKA subject’s customary socket. After familiarization with the prosthesis set to a stiffness based on a previously found weight-normalized mean preferred stiffnessBefore the trials began, prosthetist subjects observed the BKA subject walking at several stiffness levels, spanning the range of levels to be tested. The experimenter described the direction of the stiffness changes, and indicated the highest and lowest stiffness. Prosthetist subjects were instructed not to communicate with one another. In two of the familiarization sessions, the range of stiffness levels to be tested was shifted upwards based on the prosthetists’ request. Prosthetist subjects were informed that alignment could not be changed either before the experiment began or between trials.For all trials, prosthetist subjects sat with a sagittal view of a 10 m walkway  to write comments after each trial, describing what visual cues prompted them to select their answer. The BKA subjects indicated their preference by pointing to either an up-facing or down-facing arrow on a sheet placed on the experimenter’s desk, shielded from the view of the prosthetists. Since plantarflexion and dorsiflexion stiffness could not be independently varied, prosthetists were instructed to base their responses primarily on mid- and late-stance (dorsiflexion stiffness) in the event of competing preferences. Each trial took approximately 15 s, with an additional ~ 15 s between trials for the stiffness to be changed and for prosthetists to write comments.To obtain a better estimate of the BKA subject’s consistency of preference, the subject completed an additional 14–23 trials after the 39 trials were completed. These trials were drawn from seven logarithmically-spaced levels, with half the spacing of the previous trials. The stimuli were centered around an estimate of the subject’s preferred stiffness data, based on the first 39 trials. During this portion, the prosthetist subjects were not in the experiment room; all but one of the prosthetists had completed the day’s experiment, with the remaining prosthetist (randomly chosen prior to the experiment) briefly waiting in a separate room prior to participation in Part 2.In this portion of the experiment, the prosthetist subject and BKA subject were instructed to communicate to find their mutually preferred stiffness. They were encouraged to work collaboratively, as if they were fitting the BKA subject with a new prosthesis in the clinic.To simulate the prosthetist as the final decision-maker regarding prosthesis purchasing and fitting, the prosthetist subject communicated directly to the experimenter the changes that the pair wished to be made. The prosthetist subject could indicate both the direction and magnitude of change to be made, with “large,” “moderate,” “small,” and “tiny” changes translating to changes of 28%, 18%, 7%, and 3.5% respectively. The specific magnitudes of the changes were unknown to the subjects. Trials ended when the BKA subject and prosthetist subject arrived at the ankle stiffness they considered optimal. Four trials were completed for all subjects except BKA #1 and BKA #2, who only completed three trials due to time constraints. The starting stiffness levels were block randomized to start with either the lowest or highest tested stiffness levels, with the third and fourth starting levels equal to 33% or 66% of the range.22. We assume both the percept of walking at a specific stiffness level and the internal reference of preferred stiffness are subject to Gaussian noise, and that participants follow a consistent decision rule: participants will report preferring stiffness to be increased if they perceive the present stimulus to be of lower stiffness than their preferred stiffness, and vice versa.Our statistical analysis was directed by several assumptions about preference variability and decision-making22 (lapse rate set to 1%). The Point of Subjective Equality (PSE) signifies the preference . The steepness of the cumulative normal is inversely proportional to the standard deviation of the underlying probability density function, which describes the difference between stimulus levels and their internal notion of an ideal stimulus. Normalizing this standard deviation by the preference, we describe the consistency of each participant by the coefficient of variation (CV). For an intuitive interpretation of the CV: a CV of 0.10 indicates that if the stiffness is 10% higher than their preference, the subject will respond they would prefer stiffness be decreased 84% of the time.To obtain each participant’s preferred stiffness, their raw preference data were fit with a psychometric function  using maximum likelihood estimationThe additional 14–23 trials the BKA subjects completed around their preference allowed adequate estimation of their CV. To improve the estimation of the CP subjects’ consistency, each CP’s raw data was normalized by their PSE from each session, and pooled across sessions. A new cumulative normal was fit to this pooled data. While the spacing of the stimuli was logarithmic, the fitted curves are non-logarithmic to aid in interpretability.one with a two-sided, one-sample t-test. BKA-specific effects on the difference between BKA and prosthetist preferences were investigated with an imbalanced one-way ANOVA, in which there were 2–4 observations for each of the seven BKA subjects (random factor). Differences in consistency (CV) between BKA subjects and prosthetist subjects were tested with a Welch’s unequal variances t-test. Finally, to see if either the BKA subject or prosthetist subject had a stronger impact on the mutual preference, the mean logarithmic distances between mutual preference and the individual preferences . The few comments that did not fit in these categories were excluded. Comments were then organized by their distance from the prosthetist’s preferred stiffness for the corresponding patient. For example, if CP #2 preferred a stiffness of 500 Nm/rad for BKA #3 and they wrote “prolonged heel contact” during a trial of stiffness 600 Nm/rad, then that comment would be logged at 1.2 (600/500).In all but two cases, the participant’s response data could be fit with a cumulative normal function, providing a preferred stiffness and reliability score. One prosthetist subject’s responses for one BKA subject had a reversed trend and could not be fit with a cumulative normal function; this data was excluded. The responses of BKA #7 were highly consistent; the test was too short  and the tested levels too coarse to accurately estimate reliability, so this subject’s CV was excluded from analysis , with them sometimes in large disagreement , and sometimes in relative agreement (BKA #3 and BKA #4).The prosthetist subjects and BKA subjects typically did not have the same preference Fig. a. Prosthest Fig. b. The dip = 0.014, Welch’s t-test). Notably, there was high inter-prosthetist variability in consistency (SD: 14%).Normalized preference data with fitted psychometric functions are shown in Fig. p = 0.67). In one instance, BKA #2 was highly consistent individually but was markedly pulled towards the stiffness the prosthetist preferred, later stating that they trusted the prosthetist’s assessment, and that the prosthetist possibly knew what would be best long-term. In another session, the prosthetist deferred largely to the patient to suggest the changes, acknowledging the changes in stiffness were too small to see.The nature of the interaction between BKA subject and prosthetist subject in Part 2 varied Fig. . In fiveIn their written comments, prosthetist subjects were more likely to indicate effects associated with too low of a stiffness, though this may be due in part to where their preferences fell in the tested range Fig. . Of partTo understand the complementary and competing effects of prosthetist and BKA preferences, we sought to determine their preferences for ankle–foot stiffness during level ground walking, both independently , and mutually  Our paradigm allowed us to simultaneously assess both absolute preferences and consistency of preferences.One of the primary results of this study is that the prosthetist subjects preferred a higher stiffness (+ 26%) than the BKA subjects. The higher prevalence of written comments by the prosthetist subjects corresponding to a low stiffness, specifically “foot slap” and “drop-off effect,” may imply that a low stiffness may be more visually perceptible or obvious. Prosthetists may also deem a stiffness that is too low to be more detrimental than a stiffness that is too high, and thus erred on the side of an overly stiff foot. Additionally, limitations of the ankle prosthesis used in the study may have altered the experience in ways that were more easily felt by the BKA subjects than seen by the prosthetists. For example, BKA subjects may have preferred a more compliant ankle to reduce impact loading during heel-strike, or for increased energy return in late stance to help propel the higher weight of the prosthesis. Of course, in the absence of a ground truth, we cannot comment on whose preference was truly “optimal,” and would caution against over-interpretation of our result that the preferences were different.Variflex is approximately 11%23. The consistency of BKA subjects was higher in this experiment than in a previous study, in which subjects tuned the stiffness via a handheld dial while walking on a treadmill19; this finding suggests that perception of stiffness may be improved walking over-ground at a self-selected pace, or may be the result of fundamental differences between the method of adjustment and a two-alternative forced choice paradigm.The BKA subjects were highly consistent in selecting their preference (CV of 5.6%), with prosthetist subjects less consistent overall (CV of 23%). For a reference regarding how these sensitivities compare to foot availability, the difference between categories for the common Össur The higher consistency of the BKA subjects may have been due to several factors. The BKA subjects may have modified their kinetics, which are difficult to visually assess, to maintain more consistent kinematics across stiffness levels. Several prosthetist subjects commented anecdotally that certain BKA subjects in particular tended to mask changes by altering their knee kinetics . This may be particularly true in high-level ambulators, such as in this study, and could potentially limit the extrapolation of our results to lower-level ambulators. Natural stride-to-stride variation in gait mechanics may also mask the influence of foot stiffness to external observers, particularly given the short trial length.25. Thus, while highly consistent within the same experimental session, their preference may change across longer time scales. These topics will be the subject of future investigations.Though the BKA subjects were more consistent than the prosthetist subjects, this study does not seek to quantify the quality or level of satisfaction with non-preferred stiffness levels. The wide distribution (standard deviation of 18%) of stiffness levels that prosthetist subjects commented “looked good” indicates that a large range of stiffness levels appeared acceptable. We do not have an analogous description from BKA subjects, meaning that although they have a highly specific and reliable preference, deviating from this preference may not cause a steep drop-off in satisfaction. It’s also important to note that preferences may deviate with larger accommodation times; individuals with BKA alter their gait over the course of several weeks as they adapt to a new foot16. Based on our questionnaire, the prosthetists in this study fit patients with one of their three most-prescribed feet 81% of the time. Similarly, and perhaps due to the impracticality of ordering multiple feet for testing purposes, the prosthetists responded that 84% of the time a patient is eligible for a new foot, they only trial a single foot.The results of this study indicate that allowing patients to test and learn the effects of stiffness enable them to provide reliable feedback of preference; however, without clinical tools or processes that allow patients to efficiently explore prosthesis mechanical behavior, patient feedback may be more limited. In the United States, clinics often do not carry a large assortment of test feet; instead, prosthetists order individual prosthetic feet that they believe will be most appropriate for a given patient. Accordingly, prosthetists familiarize themselves well with a small number of modelse.g., foot-slap and knee extension moment), but one prosthetist subject mentioned competing compensations during mid- and late-stance in Part 2 of the experiment, which may have indicated that changes to alignment, foot length, or nonlinear response were needed. Another limitation is the short accommodation times, both within individual trials, and with the foot and tested range in general. The BKA subjects also received more training with the variable stiffness ankle prosthesis than the prosthetist subjects, which was done to ensure they were comfortable with the experiment and understood the tested variable, but may have given them extra time to refine their preference. Finally, the participants were not representative of their full populations: the BKA subjects participating in this study were highly active, K3–K4 ambulators, and may be more sensitive to prosthesis mechanics than lower K-level ambulators, and the prosthetist subjects all worked at the same rehabilitation hospital.One limitation in our study is the use of the Variable Stiffness Prosthetic Ankle–Foot to simulate changing stiffness of a conventional energy-storage-and-return prosthetic foot. In particular, the linear shape of the torque–angle response and the ratio between dorsiflexion and plantarflexion stiffness were fixed. In none of the written comments were competing effects mentioned (Supplementary file1