Three different methods, including autoclaving, autoclaving–debranching, and purification, were used to prepare Kudzu‐resistant starch (KRS) from Kudzu starch (KS). The physicochemical properties, such as thermodynamic properties, pasting properties, solubility, swelling, and coagulability, as well as the in vitro digestive characteristics of the three kinds of KRS were studied. The results showed that the morphology of starch granules of KRS prepared by autoclave, autoclave enzymatic hydrolysis, and purification methods was changed and the relative crystallinity was significantly decreased compared with the original starch. X‐ray diffraction (XRD) showed that KRS exists in the form of C and C+V crystalline form. There was a significant increase in the pasting temperature and a remarkable decrease in the peak viscosity and the expansion degree of the KRS prepared by all three methods. The solubility of the resistant starch (RS) obtained by autoclaving–debranching and that by purification were both increased compared to that of native KS, while the solubility of the RS obtained by autoclaving was decreased. Meanwhile, the retrogradation of the three RS was also improved to varying degrees. The contents of RS in the samples were: P‐KRS (71%) > DA‐KRS (43%) > A‐KRS (42%) > KS (9%). Simulated human in vitro digestion experiments showed that RS has stronger antidigestibility properties than native starch. Among them, the RS prepared by the purification method has stronger antidigestive properties, and it is predicted that it may have a better potential value in regulating blood glucose. These results indicated that the processing properties of KRS, especially the digestibility, are significantly improved and can be used as a new functional food ingredient, which deserves thorough study. Three different methods including autoclaving, autoclaving‐debranching, and purification were used to prepare Kudzu resistant starch from Kudzu powder. The physicochemical properties, such as thermodynamic properties, pasting properties, solubility, swelling and coagulability, as well as the in vitro digestive characteristics of the three kinds of kudzu resistant starch were studied. Kudzu has been reported to have various health‐maintaining pharmacological functions, including antidiabetic activity, antihyperlipidemic activity, and prevention of cardiovascular diseases, due to numerous active components such as isoflavones, saponins, polysaccharides, and starch , slowly digestible starch (SDS), and resistant starch (RS)  content but still very low (2.51% ~ 8.59%)  was purchased from Nanchang Hang Sheng Ecological Agriculture Co, Xinjian, Jiangxi Province, China. Pancreatic α‐amylase , pullulanase , high‐temperature‐resistant α‐amylase , glycosylase , D(+)‐Glucose . All the other chemicals were of analytical grade.2.2Autoclaving Kudzu‐resistant starch (A‐KRS): The KS suspension  was heated at 90°C in a water bath for 30 min. The gelatinized starch paste was heated at 121°C for 30 min, cooled at room temperature for 1 h, and refrigerated at 4°C for 12 h. Finally, the starch obtained was dried to a constant weight at 60°C and ground through a 100‐mesh sieve to obtain the resistant‐enhanced A‐KRS.g for 10 min. Later, the mixture was allowed to age for 24 h under refrigeration, dried at 60°C for 12 h, and then crushed and ground through a 100‐mesh sieve to obtain the DA‐KRS.Autoclaving–debranching Kudzu‐resistant starch (DA‐KRS): Following the autoclave method, take 100 g of KS and allow to cool at room temperature after the autoclave has been completed. Citrate buffer (0.135 mol/L) was used to adjust the pH to 4.5, followed by the reaction between prulanase (60 U/g) and suspension at 40°C for 4 h. After that, the starch suspension was inactivated at high temperature and cooled to room temperature, then the prulanase was removed by centrifugation at 892Purification Kudzu‐resistant starch (P‐KRS): After autoclaving–debranching, the treated starch was prepared with citric acid buffer . Then, 10 ml of pepsin (400 U/ml) was added to 15% starch milk, stirred in a water bath at 40°C, and adjusted the pH to 7, then 10 ml of high‐temperature‐resistant α‐amylase (400 U/ml) was added, stirred in a water bath at 95°C for 30 min, adjusted the pH to 4.5, and added 0.6 g of glycosylase , and stirred in a water bath at 60°C for 1 h. The enzyme was inactivated, and the supernatant was removed after repeated washing with 95% ethanol, stirred in the water bath at 60°C for 1 h. After repeated washing, the supernatant was removed, dried at 60°C for 12 h, crushed, and sieved, and the P‐KRS was obtained.2.3The morphology of the dried starch samples was examined using a Quanta 250 FEG scanning electron microscope (SEM)  with an accelerating voltage of 15 kV . For laser diffraction analysis, starch granules were suspended in distilled water and stirred. A dry method was used with a particle refractive index of 1.55. Duplicate measurements were carried out for each of the starch preparations. Particle size was obtained and expressed in terms of the mean particle size, D (0.1), D (0.5), D (0.9), D , and D .2.4−1 and at room temperature with 16 scans. Spectra were corrected and then deconvoluted in the range of 4000 ~ 400 cm−1  using a transmission mode. The starch sample (2 mg) was blended with potassium bromide (KBr) powder (150 mg) by grinding for 10 min under the infrared lamp and pressed into tablets before measurement. Each spectrum was recorded at a resolution of 4 cm2.5θ of 4° to 40° with step intervals of 0.02° at a scanning speed of 4°/min. The XRD data were analyzed by Origin, and the relative crystallinity was calculated by the following formula  operating at 40 KV and 40 mA at diffraction angle 22.6T0), peak temperature (Tp), conclusion temperature (Tc), and enthalpy values (∆H) were measured from the DSC thermogram . The dried starch sample was thoroughly mixed with distilled water in a ratio of 1:3. The pans were sealed, equilibrated for 24 h at room temperature, and then heated from 20 to 180°C at the rate of 10°C/min. An empty pan was used as a reference. Onset temperature  . Add 25 ml of distilled water into starch samples , then increase the temperature from 50°C to 95°C, and the whole procedure takes 13 min until the end . The supernatant and the sediment were separated, an aliquot of the supernatant was evaporated at 110°C, and the swollen starch sediment was weighed , W is dried starch weight (g), P is the swelling power, and B is the weight of sediment (g).Starch suspension (1%) was heated at 55°C, 65°C, 75°C, 85°C, and 95°C in a water bath for 30 min and then cooled to room temperature. The suspension was centrifuged at 30002.9Starch suspension (1%) was heated at 95°C for 30 min in a water bath, cooled to room temperature, and then poured it into a graduated cylinder and let it settle. Carefully observe the retrogradation of different starch samples by the ratio of the volume of starch paste supernatant to the total suspension at different times   method as mentioned above. Finally, the percentages of RDS, SDS, and RS were analyzed by the following equations:G20 and G120 indicate the glucose content (mg) released after 20 and 120 min, respectively, and TW is the total starch weight (mg).The in vitro starch digestibility was determined according to the literature, with minor modifications , k is the reaction kinetic constant (min−1), tf is reaction termination time (180 min), and t0 is initial reaction time (0 min).Goñi et al.  method wThe area of white bread under the hydrolysis curve (0 ~ 180 min) was used as the standard, and the HI (%) was calculated as the percentage of the integral area of the hydrolysis curve released from the sample compared to that of white bread. The GI (%) of the samples was estimated as follows:2.12p < 0.05).All analyses were carried out in triplicate, and the values were described as the means ± standard deviations. Data were subjected to the analysis of variance (ANOVA) by SPSS, and the significance level was determined at the 95% confidence level (CI)  of 8.54 ± 0.09 μm and d (0.9) of 17.95 ± 0.90 μm, as shown in Table 3.2The shape and surface characteristics of KS, A‐KRS, DA‐KRS, and P‐KRS starches are shown in Figure 3.3−1, the absorption curves of the three kinds of KRS were significantly different from that of KS. Due to the high‐pressure treatment during the preparation process, the amylose in the starch granules spilled, and the intermolecular hydrogen bonds weakened, so that the absorption strength of the A‐KRS was significantly reduced compared with the native KS. As to the DA‐KRS and P‐KRS, the process of enzymatic hydrolysis resulted in the precipitation of short‐chain molecules which reassembled then into a compact double helix structure with significantly enhanced amplitude. The three kinds of RS showed different vibration absorption peaks in the fingerprint area compared to KS, which may be related to the hydrogen bond association of RS in water  > DA‐KRS (1.046) > P‐KRS (1.044) > A‐KRS (1.016). Debranching and circulating temperature crystallization changed the short‐range structure, resulting in a higher degree of native starch order, as evidenced by a decrease in the 1047 cm−1/1022 cm−1 ratio of DA‐KRS and P‐KRS, and the results were agreement with those of Zeng et al. (−1/1022 cm−1 ratio is larger than those of DA‐KRS and P‐KRS.The ratio of the intensity of bands at 1047 cmg et al. . A‐KRS i−1/1022 cm−1 can be used to reflect the changes in the internal double helix structure of starch granules (Wang, Li, et al., −1 is correlated with the crystallization region (van Soest et al., −1/1022 cm−1 of KS, A‐KRS, DA‐KRS, and PA‐KRS were 1.031, 1.003, 0.998, and 0.974. DA‐KRS was enzymatically cleaved by prulanase, leading to the breakage of α‐1,6 glycosidic bonds and a decrease in amylopectin content, while P‐KRS was further cleaved by α‐amylase, breaking the α‐1,4 glycosidic bonds of both branched and straight‐chain amylose, leading to a further decrease in amylopectin content. Therefore, the 995 cm−1/1022 cm−1 P‐KRS was lower than that of DA‐KRS. The double helix structure of A‐KRS suffered some degree of damage at high temperatures, while KS was not damaged by high temperatures and enzymes and consequently had the most complete double helix structure.The ratio of absorption peak intensity at 995 cm3.4The XRD patterns and relative crystallinity of KS, A‐KRS, DA‐KRS, and P‐KRS are presented in Figure 3.5Starch gelatinization is a process of energy transfer, and the thermodynamic parameters of different starches have obvious differences. The thermodynamic parameters of different starch samples are listed in Table 3.6There were different changes in the starch granules of KRS prepared by different processing processes, such as heating and enzymatic hydrolysis. Whether the starch swells during heating or maintains its stable structure after enzymatic hydrolysis has a considerable influence on its viscosity after gelatinization Jay‐lin, . The pas3.7Swelling power and solubility behavior of KS, A‐KRS, DA‐KRS, and P‐KRS starches at different temperatures are shown in Figures 3.8The condensation of starch reflects the ability of starch molecules to hydrogen bond with water (Biduski et al., 3.9Digestion of starch granules is a complex process, which is influenced by the surface characteristics and internal structure of the starch granules and the starch crystal structure, as well as the interaction of many factors, such as starch source, straight‐chain starch content, and relative crystallinity (Ambigaipalan et al., 3.10The in vitro enzymatic hydrolysis rates of KS, A‐KRS, DA‐KRS, and P‐KRS are shown in Figure C∞, HI, and GI of native starch are the highest. A‐KRS and DA‐KRS had lower digestive kinetic parameters compared to KS, while purified P‐KRS had the lowest HI and GI. This may be due to the high RS content of P‐KRS of 70%, which makes the starch resistant to digestion. The k and C∞ of most starch samples showed the same change trend, the digestible starch content was high, and the value of C∞ and the speed of reaching the maximum equilibrium concentration increased correspondingly. The C∞ in KS is 0.94, which is much larger than that of KRS, but its k value is only 0.16, indicating that the hydrolysis of KS may be a process from fast to slow or uniform. Then according to the content of SDS in KS, the increase of SDS content has a direct impact, while the increase of RS content can significantly reduce C∞, HI, and GI (Jiang et al., The digestive kinetic parameter of different starch samples is listed in Table 4This study showed that the structural characteristics, physicochemical properties, and in vitro digestive characteristics of A‐KRS, DA‐KRS, and P‐KRS were different from those of KS. The KS, A‐KRS, DA‐KRS, and P‐KRS had similar FT‐IR spectra but have differences in absorption intensity indicating different crystallites and double helical structures. Furthermore, the KS, A‐KRS, and DA‐KRS all displayed typical C type crystallization; however, P‐KRS exhibited a complex C+V type crystallization, due to the formation of starch–lipid complexes after enzymatic debranching and recrystallization. The thermal properties and condensation of KS were lower than those of KRS. Among the three KRS, the thermal properties and condensation of P‐KRS were the highest, followed by DA‐KRS and then A‐KRS. On the contrary, the pasting properties and swelling power were in the order of KS > A‐KRS > DA‐KRS > P‐KRS. Compared to KS, KRS had higher RS content but lower RDS and SDS, and the ranking of RS content was P‐KRS > DA‐KRS > A‐KRS > KS. The preparation of KRS by the autoclaving method is simple and fast, and that by autoclaving–debranching is safer, while the starch prepared by the purification method has a high content of RS. The present study recommends that the purification of KS might be a better treatment for producing high RS, with improved pasting properties, better thermal stability, low swelling power, and low glycemic index. Also, the information obtained from this study can be used by manufacturers and researchers to produce Kudzu‐containing products.Conceptualization, Y.G, M.W., X.S., H.D., and W.Z.; methodology, Y.G., X.S., and H.D.; data curation, Y.G. and X.S.; writing–original draft preparation, Y.G., X.S., and M.W.; writing–review and editing, Y.G., H.D., and M.W.; visualization, S.Y. and C.J.; supervision, Y.G., H.D., and W.Z. All authors have read and agreed to the published version of the manuscript.The authors declare no conflict of interest.


Additionally, the results showed that the stiffness of RF (p < 0.01) and PPT (p = 0.03) were decreased immediately after SS intervention. These results suggested that SS intervention could be useful to increase knee flexion ROM and temporarily reduce the stiffness of specific regions of the knee extensor mechanism.Decreased muscle stiffness could reduce musculotendinous injury risk in sports and rehabilitation settings. Static stretching (SS) has been used to increase the flexibility of muscles and reduce muscle stiffness, but the effects of SS on the stiffness of specific regions of the knee extensor mechanism are unclear. The quadriceps femoris and patellar tendon are essential components of the knee extensor mechanism and play an important role in knee motion. Therefore, we explored the acute and prolonged effects of SS on the stiffness of the quadriceps femoris and patellar tendon and knee flexion range of motion (ROM). Thirty healthy male subjects participated in the study. Three 60-s SS with 30-s intervals were conducted in right knee flexion with 30° hip extension. We measured the ROM and stiffness of the vastus medialis (VM), vastus lateralis (VL), and rectus femoris (RF) and the proximal-(PPT), middle-(MPT), and distal-(DPT) region stiffness of the patellar tendon before and immediately after SS intervention, or 5 and 10 min after SS. The stiffness of the quadriceps muscle and patellar tendon were measured using MyotonPRO, and the knee flexion ROM was evaluated using a medical goniometer. Our outcomes showed that the ROM was increased after SS intervention in all-time conditions ( The knee extensor mechanism refers to the convergence of the four quadriceps muscles into the quadriceps tendon, which is attached to the proximal patella and extends distally to the tibial tuberosity through the patellar tendon . The musStatic stretching (SS) is commonly utilized to increase muscle flexibility, decrease muscle stiffness, and reduce musculotendinous injury risk in sports and rehabilitation settings . A systeIn addition, muscular function and integrity are close-knit and are related to variations in the mechanical properties of tendons . Some reA repeated measures experimental design was conducted to examine the acute and prolonged impacts of SS protocol (100% intensity and 3 min duration) on ROM, stiffness of the quadriceps , and patellar tendon  in the lead extremity . Our SS intensity and duration option refers to a previous study . The domThirty healthy male volunteers  were recruited for this experiment. Participants who regularly performed strength and flexibility training or had a history of neuromuscular diseases and lower limb musculoskeletal system injuries were excluded. All volunteers were requested not to conduct flexibility or resistance training of the lower extremities for the duration of the experiment. All volunteers signed written informed consent to participate in the present study. This research was approved by the Ethics Committee of the Luoyang Orthopedic Hospital of Henan Province (KY 2020-003-02), China, and conformed to the Declaration of Helsinki.The methodology for assessing ROM was the same as that of the previous study . SubjectThe MyotonPRO  is a non-invasive portable machine utilized to quantify tendon and muscle stiffness in this study. The device works by exerting a small mechanical impact on the tissue of interest area, perpendicular to the skin surfaces. The probe’s tip is then pushed to the measured site to reach the desired depth. After the red light turns green, the machine automatically implements five short pulses (0.8 s between taps) to cause mechanical oscillations of the tissue. According to the previous study, we found good intra- and inter-tester reliability for measuring the stiffness of the quadricep muscles and patellar tendon using the MyotonPRO . MyotonPWe measured the stiffness of the VM, VL, RF, PPT, MPT, and DPT using MyotonPRO. All subjects were tested in the same physical therapy room, and measurements were performed at the ambient temperature of 25°C in a relaxed position with knee and hip flexed at 90°. The stiffness of the RF at the reproducible location is about two-thirds of the distance between the anterior superior iliac spine and the patella apex . In ordeBefore the SS intervention maneuver, we randomly examined the test–retest reliability of stiffness measurement for the quadriceps femoris and patellar tendon using ten limbs in ten healthy male subjects.The SS maneuver was conducted similar to the ROM measurement. The 100% intensity of SS intervention was determined by the ROM in the PRE value just before the volunteers began to feel pain or discomfort . Three 6p values <0.05.All descriptive data are presented as means ± standard deviation. The statistical analyses were conducted using SPSS . The Shapiro–Wilk test was utilized to examine the normality of all data. Measurement test–retest reliability was analyzed using the intraclass correlation coefficient and coefficient variation. We executed a one-way repeated measure analysis of variance (ANOVA) to assess time effects (PRE vs POST vs POST 5 vs POST 10) for the ROM. For the stiffness, a two-way repeated measure ANOVA was utilized to evaluate [time (PRE vs POST vs POST 5 vs POST 10) and regions (RF vs VM vs VL vs PPT vs MPT vs DPT)] and analyze the main and interaction effect. Post-hoc analyses using Bonferroni multiple comparison tests were performed if a significant interaction effect was detected. Statistical significance was considered as Good test–retest reliability  was found for the stiffness measurements of the quadriceps and patellar tendon .p < 0.01). There was a significant increase in knee flexion ROM after the SS intervention (p < 0.01). In addition, the post-hoc test revealed that the knee flexion ROM was significantly higher in POST and POST 5 than in POST 10 (p < 0.01).The changes in the ROM before and after SS are shown in p = 0.02, F = 2.00, ηp2 = 0.05) and the significant time effect in the stiffness of RF and PPT . When comparing time points between PRE and POST in the SS intervention, the Bonferroni’s test revealed a significant decrease in the stiffness of RF and PPT , whereas no significant differences were found in POST 5 and POST 10 compared with PRE stiffness. In addition, there was no significant change in VM, VL, MPT, and DPT stiffness after the SS intervention compared with PRE stiffness.The stiffness changes of the patellar tendon and quadriceps muscles before and after SS are shown in The present research study explored the acute and prolonged effects of SS intervention on the ROM and VM, VL, and RF muscle stiffness and PPT, MPT, and DPT tendon stiffness. Accordingly, the major findings of our study were that 1) the SS intervention increased the knee flexion ROM, and this change was continued till 10 min after SS; and 2) the RF stiffness and PPT stiffness significantly decreased after the SS, whereas this reduction was not sustained till 5 min after SS. Although some studies have investigated the effects of SS intervention on the gastrocnemius , hamstriAccording to the study results, knee flexion ROM increased for 10 or more minutes after 100% intensity SS intervention. This acute effect was in alignment with the previous research , which rThe results of our study showed that RF muscle and PPT stiffness significantly decreased after SS, whereas there were no significant alterations in the VM, VL, MPT, and DPT stiffness. A previous study examined the effects of the 120% intensity with two different durations (1 and 3 min) and 110% intensity with 3-min duration SS intervention on RF muscle stiffness, and the authors reported a significant reduction in the 110% intensity SS group, but the stiffness of RF muscle was not changed in 120% intensity SS group . These rInterestingly, the SS intervention only significantly decreased the PPT stiffness, whereas there were no significant alterations in the MPT and DPT stiffness. Decreased tendon stiffness after SS training was also reported by There were several limitations in this study. First, the subjects were not regular exercisers. Thus, it is necessary to investigate the influences of SS intervention in athletes who regularly perform exercise training. Second, we did not consider female subjects in the study, although it is well known that there are differences in structure and function between the sexes. Third, the SS intensity was determined by reference to ROM . HoweverThe results showed that the SS intervention increased the knee flexion ROM, and this change was continued till 10 min after SS; the RF stiffness and PPT stiffness significantly decreased after the SS intervention, whereas this reduction was not sustained till 5 min after SS. It is suggested that SS intervention could be useful to increase ROM and temporarily reduce the stiffness of specific regions of the knee extensor mechanism. However, SS interventions with different stretching intensities and durations for further studies are necessary to investigate whether they effectively prevent injuries related to the quadriceps and patellar tendon.


In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild-type Ndc80 and three variants  domains mutated or Hec1 tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening; i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models that show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity. By time-tracing the stiffness of microtubule end-tracking Ndc80 complexes in optical trap experiments, we detect strain stiffening, and, thereby, provide new insights into the elastic properties of the Ndc80 complex. The strain stiffening is robust against mutations in the Ndc80 complex. We relate strain stiffening to the structure of the Ndc80 complex by means of a simple polymer model, to thermal fluctuations of the microtubule, and to the flexibility of force-generating flaring protofilaments at the tip of the microtubule. Since Ndc80 complexes play a major role in transmitting force from microtubule ends to the kinetochore, their elastic properties are of great interest for a deeper understanding of chromosome dynamics in the mitotic spindle.In the mitotic spindle, microtubules (MTs) attach to chromosomes via macromolecular structures called kinetochores . KinetocThe Ndc80 complex is a four-subunit rod-like coiled coil with a characteristic flexible kink at approximately one-third of its length ,4. MT biEarly theoretical models of spindle-kinetochore dynamics envisioned a static Hill sleeve with rigid linkers ,16 or moMeasurements of stiffness can confirm structural models if they are in agreement with elastic models derived from the available structural information on the Ndc80 complex. The elastic properties of the Ndc80 complex were studied experimentally by optical trapping methods introduced in . Here, wIn order to rationalize the experimental findings on strain stiffening of the Ndc80 links, we introduce a simple polymer model for the Ndc80 complex. Describing Ndc80 as an ideal chain with two bonds of different lengths, we show that the strain stiffening is a direct consequence of the characteristic Ndc80 structure with its two stiff rods that are connected flexibly. To match the measured stiffnesses, it is necessary to include the contributions from MT fluctuations and from protofilament (PF) bending to the total effective stiffness. Finally, to explain the shape of the experimental stiffness-force relations, we also introduce a positive correlation of the number of MT end-attached Ndc80 complexes with MT-generated force.1S3 modules  G (Roche). Non-specific binding of molecules to coverslips was prevented by an incubation with 1% Pluronic F-127. Then, GMPCPP-stabilized, digoxigenin-labeled MT seeds were attached to the coverslips. Finally, T−1. Position of the bead was continuously recorded at 10 kHz using a quadrant detector, and differential interference contrast (DIC) images were simultaneously recorded to monitor positions of the bead and the dynamics of MTs. The bead was held in a trap until the MT depolymerized, which resulted in two alternative outcomes: the bead-MT connection broke, or the bead rescued the MT shortening, and the MT started regrowing. In case of a rescue, recordings were stopped arbitrarily after 30 or more minutes by increasing the trap stiffness to 0.2–0.4 pN nm−1 and rupturing the bead-MT connection. Quadrant detector readings of the bead position were later turned so that one of the axes corresponded to bead displacement along MT to generate time traces Optical trapping experiments were performed as described previously . Before x by k of a certain length k. Then, the effective stiffness during the interval k is given by the inverse of the mean squared distanceAs long as the mean bead position is stationary, e.g., during MT stall, the effective stiffness can be easily calculated from the variance of the bead position Finally, we can assign a force c the stiffness. For our experiments, The time interval B. The stiffness of the optical trap will always be present in the measurements. Its value is determined during the calibration of the optical trap after the bead detached from the MT.Before modeling the outcomes of the stiffness measurements, we have to identify the elastic elements that contribute to the total stiffness as sketched in Further potentially elastic elements connect the bead and the MT seed that is fixed on the coverslip. On the one hand, this is the MT, which can exhibit both a mechanical (stretching or bending) and an entropic stiffness . On the other hand, the link between the MT and the bead has elastic properties. We attribute the elasticity of the link to its four constituents: first, the Ndc80 complex can act as an entropic spring with stiffness Each of these four stiffnesses may depend on the applied force and exhibit strain stiffening itself. Since the four elements are aligned in series, their inverse stiffnesses sum up to the inverse linker stiffness It is important to note that this reduces the total stiffness and that the softest elastic element dominates the total stiffness.F is shared equally between the parallel Ndc80-PF units with stiffness n parallelly attached Ndc80 complexes can be written asIt is possible that multiple Ndc80 complexes are attached parallelly to the MT. For the sake of simplicity, we will always assume that each PF can only attach one Ndc80 complex and that the parallel PFs and Ndc80 complexes have the same elongation, respectively. Then, the force Although parallel stiffnesses add up, the shared force can give rise to an overall reduction n parallel linkers A and B). This results in a total stiffnessThe ring see A and B.We note that the force dependence of the trap stiffness is negligible in the examined force ranges so that we can assume L  lay in a range between 3 and 10 nm. Given the values of 0.1–2 GPa for the Young’s modulus Y of an MT zL=T length : Lp=Lp∞/Δ80 and Ndc80CHmut compared with the wild-type Ndc80wt will allow us to address this issue in the “discussion” section. Regarding the linker stiffness The bond between individual Ndc80 complexes and the MT is established by the globular regions at the N terminus of the complex ,23,30 viC. In the cell, the long arm is bound to the kinetochore, in the experiment it is bound to the glass bead, while the short arm can attach to an MT. We describe the conformation of the two arms by two vectors C:In order to model the stiffness of the Ndc80 complex under force, we use a simple polymer model that is based on the known structure ,4: The NC). Experiments have shown that the two arms are connected flexibly within an angular range between z direction and favors small angles, we can neglect the constraint to angles below z direction,The impenetrable glass bead restricts these two vectors in the experiment. Since the diameter of the glass bead (1 μm) is much larger than the Ndc80 complex, we approximate the bead as a plane surface that confines the Ndc80 complex to the upper half-space, so that zb>0 see C. Exper kinked) . Since wRespecting the confinement to the upper half-space, we obtain the canonical partition function  as well as three variants, including Ndc80 complexes with a phosphorylated tail (Ndc80P), Ndc80 complexes, whose tail has been truncated (Ndc80Δ80), and Ndc80 complexes with mutated CH domains and a greatly reduced MT binding (Ndc80CHmut).The ability of different Ndc80 variants to track and stall depolymerizing MTs has been examined in two previous studies ,10. HereIn , the stiA–D for four representative examples, as well as in To settle this issue, we need to time trace the stiffness throughout a single experiment. We achieve this by binning the data of bead positions in intervals of 0.1 s and determining an effective stiffness and applied force for each interval separately using the procedure described in the “materials and methods” section. This allows us to determine the stiffness while the depolymerizing MT pulls the attached bead and thereby increases the force. In other words, we measure the stiffness for different forces in a single experiment and, thus, with a fixed Ndc80 density on the bead. The results of this analysis are shown in D. Based on previously observed absence of stiffening during lateral bead-MT attachment . Calibrations of QPD voltage to obtain bead displacement, as well as calibrations of trap stiffness as a function of bead’s displacement from the trap center, were linear within 200 nm from the trap center. Therefore, displacements of a bead from the trap center that exceeded 200 nm were excluded from the following analyses of the strain stiffening.For each of the Ndc80 variants, there are experiments in which we detect a smooth stiffening behavior when the tension increases, but there are also experiments that do not exhibit any stiffening at all, as in tachment , we intec80P see E. Calibwt and Ndc80P, but not for Ndc80Δ80 and Ndc80CHmut, where the bead often detaches before it can stall the MT (see B), and the stalls that occur are shorter  or MT growth is rescued (C). In the first case, the bead relaxes immediately to the equilibrium position of the optical trap and the measured stiffness is the trap stiffness A–D can be observed for each of the Ndc80 variants with frequencies as summarized in E.A stall can be interrupted in two possible ways: either the bead detaches from the MT A or MT  rescued C. In thwt were determined in . Finally, we evaluated the stiffness-force relations separately for different populations of beads. Since beads within the same population were prepared under the same conditions, they can be assumed to be coated with a similar number of Ndc80 complexes. Although the populations had very different mean Ndc80 densities, the separate evaluation does not reveal a correlation between Ndc80 density and the stiffening behavior, as shown by The stiffening exponents determined in ose to 4 G. FinalH by binning the time-traced stiffnesses of all experiments in bins of 1 pN width and averaging force and stiffness in each of these bins. We find that all variant Ndc80 complexes exhibit similar strain stiffening. Both truncating the tail or modifying the CH domain slightly decrease the stiffness compared with the wild type, especially for small forces. This indicates that, in all four Ndc80 variants, the remaining intact common parts play a central role in strain stiffening. Differences between variants are due to changes in the Ndc80-MT bond and will be addressed in the “discussion” section.For a better visibility and comparability of the strain stiffening of the different Ndc80 variants, we summarize the stiffness measurements in C, the time-traced stiffness follows the same stiffness-force relation in the shrinking and growing states before and after rescue, respectively. The same observation can be made in experiments with several consecutive rescues and catastrophes. This indicates that the stiffness is independent of the MT state; i.e., on whether the Ndc80 complexes are attached to a shrinking, stalled, or growing MT. To address this question systematically, we identify the phases of shrinking, stalled, and growing MTs in the single experiments, and bin the stiffnesses analogously to H but separately for each of the three MT states. The results in I do not show a significant dependence on the MT state for any of the four Ndc80 variants. The independence of Ndc80-MT linker stiffness from MT state indicates that PFs either do not contribute to the total stiffness  step by step to discuss their relevance for the observed strain stiffening. We start with the Ndc80 complex, which is the most promising and interesting candidate because of its flexible structure and its force transmitting role in the mitotic spindle.C), resulting in a stiffness n Ndc80 complexes attached in parallel. For this situation, we assume that each Ndc80 complex has the same elongation so that the force F is shared equally by the attached complexes and the total stiffness is given by A, we compare the total stiffness of n parallel Ndc80 complexes with the measured stiffnesses of Ndc80wt. For any n, the model results are about a factor of 2–4 larger than the measured values. Moreover, the two-segment Ndc80 model results in a strain stiffening exponent E. We conclude that the stiffness of the Ndc80 complex alone can explain strain stiffening but is not sufficient to explain the measured stiffnesses quantitatively.We model the Ndc80 complex as an FJC with two flexibly connected stiff arms see C, resulF/n) see . In Fig.−1.We note that the experimental values in The fact that the Ndc80 model gives larger stiffnesses than measured motivates the consideration of the additional elastic elements from the MT, the PFs, and the Ndc80-MT bonds in the total stiffness in Next, we consider the contribution of the MT, which can be attached to several Ndc80 complexes. While a purely mechanical MT stiffness that follows from stretching or bending the MT can be ruled out to contribute significantly see , the MT n parallel Ndc80 complexes alone is significantly reduced when the MT stiffness is added in series (see A). Interestingly, the combination of a single MT with stiffening exponent 3/2 and several  in φ of the PF, its persistence length Since the PFs are bent out of their preferred curvature, a restoring force on the Ndc80 complexes builds up, governed by an effective stiffness odel see D in Eq.in vitro , a fixedB, we plotted the stiffnesses of n parallel Ndc80 complexes with In in vitro .fixed number n of parallel Ndc80 complexes over the whole range of applied forces. We now analyze how our model behaves when the number n of attached Ndc80 complexes depends on the applied force, So far, we have assumed a  tension . In our n in A and B. The envelope of the F. The resulting C and increases linearly, which also implies a catch-bond behavior. For long PFs . Our theory, on the other hand, predicts exponents of 3/2, 2, and 5/2 for the MT, the Ndc80 complexes and the PFs, respectively. The relation Apart from the absolute stiffness values, a model should reproduce the observed roughly linear strain stiffening behavior with the exponent nity see E. Our tFor a slip bond, on the other hand, where E, 79 % of the stiffening exponents lie below 2.We conclude that any kind of slip bond will increase the stiffening exponent for large forces. Stiffening exponents below the values The novel analysis of time traces of bead positions in optical trapping experiments allows us to time trace the stiffness during single experiments and go beyond an analysis limited to the MT stall state . This enWe found that all mutant Ndc80 complexes exhibit strain stiffening hinting at a central role of the Ndc80-Nuf2 coiled coil, rather than the MT binding sites in the CH domain and the N-terminal tail. The measured stiffnesses of the entire Ndc80-MT link are lower than theoretical predictions from our Ndc80 model. Therefore, additional elastic elements in the Ndc80-MT link or a force-dependent number of attached Ndc80 complexes should be relevant. We could exclude the PLL-PEG connection between Ndc80 complexes and the bead, which is too stiff to contribute. Effects from the MT and from protofilament flexibility are necessary for the model to conform to experimentally measured stiffnesses, and the observed linear stiffening may be explained by an increasing number of attached Ndc80 complexes under force (catch bond). Moreover, stiffness differences between variants allow us to draw some conclusions on the Ndc80-MT bond.C and H), leading to the conclusion that the stiffness of the bent PFs is either too large to contribute significantly to the total stiffness or that it does not depend on the MT’s polymerization state.Time tracing of the stiffness allowed us to study strain stiffening selectively in the shrinking, stalled and growing state of MTs. We found no significant stiffness differences between MT states see C and H,E, which show that Ndc80Δ80 with truncated tail enters a state of stall, which requires binding to the MT, less frequently. There are, however, differences. Our stiffness measures derive from experiments where Ndc80 multimers were used that were able to tip-track, in contrast to the single Ndc80 complexes in . This is consistent with the conclusion that PFs contribute to the total stiffness but have identical elastic properties during MT shrinkage, stall, and growth, and is also in agreement with the recent observation that the curvature of PFs is the same during polymerization and depolymerization (Attachment of Ndc80 to long flaring PFs with attachment lengths around ffnesses B. This rization .n of simultaneously attached Ndc80 complexes could not be directly inferred from the experiments in (The number ments in  and 10)n of simuments in .wt complex, timescales of From the equal stiffness-force relations before and after a rescue, we also conclude that the number of attached Ndc80 complexes is the same during polymerization and depolymerization. Dynamically, it is possible that the number of attached Ndc80 complexes changes in a force-dependent manner between MT catastrophes and rescues. For force-free detachment and attachment of a single Ndc80en found . With a  smaller . Since tE) are best reproduced by a force-dependent number B) implying a catch-bond mechanism. Among the stiffening exponents for the time traces from single experiments in F, 79% of the strain stiffening exponents lie below 2, which is indicative of a catch-bond-like behavior. As already noted above, the linear stiffening may also be an apparent phenomenon that results in the examined force range from the combination of the different stiffening behaviors of a single MT and a sufficiently large constant number of Ndc80 complexes and PFs.Our modeling results showed that the absolute stiffness values as well as the roughly linear strain stiffening relation see E are beorce see B implyiSaccharomyces cerevisiae, however, were shown to build catch bonds with MTs in vitro even without Aurora B activity , an effect we ascribed either to a softened Ndc80-MT bond or to a reduced number of attached Ndc80 complexes. In the elastic toy model in A, both the CH domain and the tail of Ndc80wt bind to the PF, so that they can be represented by two parallel springs that add up to the total bond stiffness Δ80 or Ndc80CHmut reduces the bond stiffness, predicting overall stiffnesses as depicted in G. If both the CH domain and the N-terminal tail bind directly to the MT lattice, the two Ndc80 mutants probably have a reduced MT binding affinity. Therefore, in the direct binding model, the reduction of the overall stiffness could be a consequence of a reduced number of attached Ndc80 complexes as well as of a reduction of the individual bond stiffnesses.Our comparative study of Ndc80ild type G, an efwt complexes, see B. Deletion of the tail or mutation of the CH domain may impair the cooperative behavior, with the result that fewer linkers connect the bead (or the kinetochore) with the MT and the overall stiffness decreases  also reproduces the observed roughly linear strain stiffening relation. Ndc80, by itself or in series with PFs, gives absolute stiffness values that are still above the measured stiffnesses. Including a force-dependent number of bound Ndc80 complexes gives values that are close but still slightly higher than measured. As already mentioned, the missing part might be the Ndc80-MT bond, which is additionally in series with the Ndc80 complex and the PF, as illustrated in Regarding the Ndc80, we have also investigated possible effects from finite flexibility of the two Ndc80 arms by modeling both arms as semiflexible worm-like chains. We performed Monte Carlo simulations, in which each Ndc80 arm had the same persistence length pomyosin , our simpomyosin .Finally, we need to discuss the accuracy of our experimental method. While the trap stiffness was calibrated by fitting the power spectrum based on Brownian motion in a harmonic potential , 39, 40,T/Var(x) , 57, 58.In conclusion, we accumulated extensive stiffness data of the Ndc80-MT link by optical trapping methods in combination with a novel time-tracing analysis. We were able to study wild-type Ndc80 complexes and three variants. Our theoretical model for the Ndc80-MT link, which includes structure-based models of the Ndc80 complex, the MT, and flaring PFs, is able to explain the strain stiffening observed when Ndc80 is bound to shortening MT plus ends, and reproduces the correct order of magnitude of the stiffness. Thus, our results on the elastic properties further support these structural models from the mechanical point of view. Our model also reproduces the roughly linear strain stiffening behavior when taking a force-dependent binding affinity into account.A.M., M.D., V.A.V., and P.J.H. conceived and designed the experiments. P.J.H. contributed reagents. V.A.V. performed and analyzed the experiments. J.K. and F.S. developed the theoretical model. F.S. analyzed the data and performed calculations. F.S. and J.K. wrote the original draft. A.M., V.A.V., and P.J.H. reviewed and edited the manuscript.


Philip Alexander, MD, is a native Texan, retired physician, and accomplished musician and artist. After 41 years as an internal medicine physician, Dr. Phil retired from his practice in College Station in 2016. A lifelong musician and former music professor, he often performs as an oboe soloist for the Brazos Valley Symphony Orchestra. He began exploring visual art in 1980, evolving from pencil sketches—including an official White House portrait of President Ronald Reagan—to the computer-generated drawings featured in this journal. His images, which first appeared in this journal in the spring of 2012, are his own original creations.Methodist DeBakey Cardiovascular Journal, submit your creation online at journal.houstonmethodist.org as a “Humanities” entry.If you would like to see your art published in the


In moyamoya disease (MMD) with direct or combined revascularization, the initially hemodynamic recipient features are likely one of the main causes of acute hemodynamic disruption. Previous studies have explored the relationship between recipient diameter or flow velocity and postoperative complications, but there are still no optimal selection criteria with multiple potential recipient vessels. Cerebral edema is one of the most common radiological manifestations in the acute postoperative period. This study assessed the hemodynamic characteristics of cortex vessels related to postoperative cerebral edema.All patients who had undergone direct or combined revascularization with preoperative digital subtraction angiography (DSA) between 2019 and 2021 were eligible for inclusion in this study. The application of DSA was performed and regular radiological examinations were employed after surgery. DSA was analyzed with the hemodynamic features within chosen recipient vessels. Cerebral edema was identified as a low-density image on CT or high signaling in the MRI T2 phase. The recipient hemodynamic characteristics and demographic presentation, as well as clinical data, were retrospectively analyzed in this study.p = 0.078), recipients observed in angiography (p = 0.002), and surgery on the left (p = 0.097) may be associated with cerebral edema. The following regression analysis confirmed low occurrence of cerebral edema was accompanied by recipients observed in angiography (p = 0.003). After subdividing by flow direction and hemodynamic sources, the incidence rate of anterograde direction, anterior sources, and posterior sources were significantly lower than undetected recipients.A total of 103 patients underwent direct or combined revascularization with preoperative DSA. The mean age of this enrolled cohort was 44.31 ± 10.386 years, in which bilaterally involved MMD accounted for the main part. The preliminary correlation analysis found preoperative disease period (Cerebral edema is a common radiological manifestation in MMDs after surgery. In this study, the observation in angiography reliably identifies a variety of physiological or pathological recipient detection, flow direction, and hemodynamic sources in patients with MMD after revascularization, which indicates the selection strategy of potential recipients and highlights the importance of recipient observability in DSA. Meanwhile, vascular conditions determined by recipient hemodynamics meditate the occurrence of postoperative cerebral edema. Moyamoya disease (MMD) is a chronic cerebrovascular disease that is characterized by progressive steno-occlusive in bilateral internal carotid arteries and their proximal branches . SurgicaThe radiologic incidence of stroke was higher than symptomatic stroke, in which 33.3% of MMDs were reported with postoperative diffusion-weighted imaging (DWI)-detected lesions . CerebraThis study included 103 MMDs with surgical revascularization at our hospital between November 2019 and February 2021 after 15 patients were excluded . The dia(1) Age ≥ 18 years;(2) Definite diagnosis of MMD confirmed by DSA;(3) The application of direct or combined revascularization;(4) Radiological examinations, including computed tomography (CT) or magnetic resonance imaging (MRI), were performed within 4 days after surgery.The exclusion criteria were as follows:(1) The employment of dual direct revascularization;(2) Lack of preoperative angiography in our hospital.Direct revascularization indicates superficial temporal artery (STA)—middle cerebral artery (MCA) bypass . CombineThe hemodynamic characteristics of the recipient's were assessed through preoperative DSA by two experienced neurosurgeons. Of note, the flow direction of the recipient's vessels was categorized as follows:(1) Anterograde direction: blood flow from M3 segment to M4 segment;(2) Retrograde direction: blood flow from M4 segment to M3 segment;(3) Undetected direction: no or little flow direction was detected in angiography.The hemodynamic sources of the recipient's vessels were categorized as follows:(1) Anterior sources: blood supply was from an anterior cerebral artery (ACA) or MCA;(2) Posterior sources: blood supply was from a posterior cerebral artery (PCA) or vertebrobasilar artery;(3) Undetected sources: no or few hemodynamic sources were detected in angiography.The evaluation of cerebral edema was relatively fussy. The first step is to identify signal changes around the anastomotic area of postoperative radiological examinations, which presented as either slightly low-density on CT or high-signaling in the MRI T2 phase. Subsequently, the comparison among perioperative imaging was applied to confirm whether the changes were recent, which means occurring or not. The occurrence of cerebral edema was confirmed by two experienced neurosurgeons independently and the results were reached after consensus, as shown in t-test, Pearson's chi-squared, or Fisher's exact test correlation analysis was performed. Afterward, the logistic regression analysis was conducted for significant factors achieving P < 0.10 in the univariate analysis. All tests were two-tailed. P < 0.05 was considered to be statistically significant.The SPSS 24.0  was used for statistical analysis. A descriptive summary is presented as mean ± standard deviation (SD), median (interquartile range), frequency, or percentage, as appropriate. For comparison between the non-postoperative cerebral edema group and postoperative cerebral edema group, an independent-sample Of the 118 consecutive patients with MMD, who underwent surgical revascularization from November 2019 to February 2021, we identified 103 MMDs who met the inclusion and exclusion criteria of this research . The meap = 0.078). Remarkably, the proportion of recipient's vessels observed in preoperative angiography was less in the postop edema group significantly, compared with the non-postop edema group (p = 0.002), which presented as 54.3 vs. 82.4%. Meanwhile, the duration of the postop edema group was prone to be longer than the non-postop edema group statistically . Also, the time of postoperative imaging was irrelevant to cerebral edema (p = 0.181). After possible factors were included in logistic regression, we found recipient vessels observed in preoperative angiography were an independent factor associated with postoperative cerebral edema .Initially, we analyzed basic features between the non-postoperative (non-postop) edema group and the postoperative (postop) edema group. As shown in p = 0.012; p = 0.002, K = 0.906; K = 0.903; K = 0.916, p = 0.004, p = 0.008; p = 0.005; Afterward, we further investigated the flow direction and hemodynamic sources of recipient vessels on the occurrence of cerebral edema. The two different features had obvious statistical differences in the two groups  and Zhejiang Provincial Natural Science Foundation of China (LQ19H160039).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Growing evidence shows that arterial stiffness measurement provides important prognostic information and improves clinical stratification of cardiovascular risk. Thyroid and parathyroid diseases are endocrine diseases with a relevant cardiovascular burden. The objective of this review was to consider the relationship between arterial stiffness and thyroid and parathyroid diseases in human clinical studies. We performed a systematic literature review of articles published in PubMed/MEDLINE from inception to December 2021, restricted to English languages and to human adults. We selected relevant articles about the relationship between arterial stiffness and thyroid and parathyroid diseases. For each selected article, data on arterial stiffness were extracted and factors that may have an impact on arterial stiffness were identified. We considered 24 papers concerning hypothyroidism, 9 hyperthyroidism and 16 primary hyperparathyroidism and hypoparathyroidism. Most studies evidenced an increase in arterial stiffness biomarkers in hypothyroidism, hyperthyroidism and primary hyperparathyroidism, even in subclinical and mild forms, although heterogeneity of measurement methods and of study designs prevented a definitive conclusion, suggesting that the assessment of arterial stiffness may be considered in the clinical evaluation of cardiovascular risk in these diseases. Growing evidence shows that arterial stiffness measurement provides important prognostic information and improves clinical stratification of cardiovascular disease ,2. The sThyroid and parathyroid diseases should be considered as endocrine diseases with relevant implications in the cardiovascular system. Patients with overt hyperthyroidism or hypothyroidism show several alterations, caused either by effects of thyroid hormones in the heart and in the vasculature or by cardiovascular risk factors , which may increase cardiovascular risk and lead to cardiovascular morbidity and mortality ,8. SimilIn recent years, numerous clinical studies have investigated the relationship between thyroid and parathyroid diseases and arterial stiffness markers. New evidence explains the molecular and physiological pathways leading to vascular disease in conditions of hormonal excess or deficiency. The use of arterial stiffness measures has been proposed to improve clinical management of these conditions in several clinical studies.In this review, we aimed to perform a literature search for the effects of thyroid and parathyroid hormone excess or deficiency on the structural and functional alterations of the arterial wall which leads to arterial stiffening, focusing on clinical studies conducted in humans.A PubMed/MEDLINE search was performed to select peer-reviewed articles published from inception to 31 December 2021. The complete search string included inclusive keywords regarding arterial stiffness  and inclusive keywords regarding the evaluated hormones and their imbalance (“hypothyroidism” or “thyroid hormones” or “thyrotoxicosis” or “levothyroxine” or “Hashimoto” or “Graves” or “Basedow” or “dysthyroidism” “hyperparathyroidism” or “PTH”). Papers written in languages other than English, not pertinent with the present review, or whose full text was not available were excluded. The pertinent references were evaluated and eventually included in the final manuscript. We considered all papers in open-access and non-open access journals. The included papers were summarized and their results discussed in the text, offering an overview of current literature. Clinical studies in adult humans were organized in Tables . From an overall evaluation, variable results about the impact of thyroid hormones on arterial stiffness are reported. In addition, arterial stiffness was assessed using different measurement methods, varying from ultrasounds to tonometry. Considered measures of arterial stiffness included pulse wave velocity (PWV), augmentation index and other surrogate measures. PWV is the velocity at which the pressure pulsation propagates through the circulatory system, and is defined as the distance travelled by the pulse wave divided by the time , and theThe prevalence of subclinical hypothyroidism, defined as a serum thyroid stimulating hormone (TSH) level above the upper normal limit despite normal levels of serum free thyroxine (fT4), in the general population is 5–15% . Of thesRegarding subclinical hypothyroidism, evidence observing an increase in arterial stiffness is conflicting and heterogeneous, both in methods used to evaluate arterial stiffness and in results. A number of small studies were conducted to evaluate arterial stiffness in subclinical hypothyroidism, in small cohorts of patients and with different methodologies. Earlier studies mainly considered the evaluation of brachial-ankle PWV, which was found to be increased  but not More recent studies explored the relationship between the PWV in aorta and subclinical hypothyroidism. Three studies found an increase in aortic PWV in this condition ,34,35, aSimilarly to subclinical, most studies conducted in overt hypothyroidism found an increase in arterial stiffness, which has been evaluated across studies with a variety of biomarkers. Earlier studies evaluated markers related to reflected waves, as augmentation index ,41. TimiOne study conducted in thyroidectomized patients on long-term replacement therapy, did not find an increase in arterial stiffness, suggesting that targeting TSH in the reference range does not seem to cause adverse cardiovascular effects .Studies conducted in patients with overt hyperthyroidism suggest an increase in structural arterial stiffness biomarkers. An increase in β index in carotid arteries was found, which was reduced by antithyroid drug or radioiodine treatment ,48. AddiTwo studies evaluated the algorithm-derived PWV calculated from Mobil-O-Graph device in patients with hyperthyroidism, finding an increase in PWV in the office setting , which iThe evaluation of markers of reflected waves magnitude and the analysis of central pressure waves gave variable results among studies. In thyrotoxicosis, Obuobie et al. found a decrease in AIx despite an increase in central pulse pressure , thus suPrimary hyperparathyroidism (pHPT) is another very common endocrine disorder, affecting between 0.4% and 11% of the population . The higBased on this background, several works have evaluated the effect of mild pHPT and parathyroidectomy on arterial stiffness. PTH has been found associated with arterial stiffness in the general population ,64, and Interestingly, hypoparathyroidism has also been associated with an increase of arterial stiffness ,82, consOver recent years, the role of thyroid hormones in cardiovascular health has been widely studied. Several mechanisms of action of fT3, fT4 and TSH on arterial stiffness have been hypothesized. Thyroid hormones may affect the cardiovascular system by direct effects on arterial vessels , on the heart  or indirectly by influencing cardiovascular risk factors .Focusing on effects on arterial vessels, thyroid hormones have both genomic and non-genomic mechanisms affecting vascular tone, by ion channel activation and regulation of specific signal transduction pathways.Firstly, they have a cellular action on endothelium, causing production of nitric oxide via the phosphatidylinositol 3-kinase and serine/threonine protein kinase pathways . VasodilSecondly, due to its positive inotropic and chronotropic effect, fT3 is directly responsible for acute hemodynamic changes , which aThirdly, by acting on cardiovascular risk factors, thyroid hormones may indirectly lead to an increase in arterial stiffness. Notably the main determinant of aortic stiffness are blood pressure levels, which may be influenced by thyroid hormones. In hyperthyroidism, caused by inotropic effect on the heart and reduction in systemic vascular resistance, an increase in systolic and pulse pressure is often seen. Hypothyroidism is conversely associated with diastolic hypertension, induced by increased peripheral vascular resistance and by changes in circulating volume . VariatiAn additional cause that may affect the promotion of cardiovascular disease in the presence of thyroid disease is the autoimmune process of disease per se ,93. RegaRegarding the effect of treatment of thyroid and parathyroid diseases in relation to thyroid disease, most studies observed an improvement in vascular markers after correction of hormone levels. In hypothyroidism, an improvement in AIx  followed correction with hormone replacement therapy. In hyperthyroidism, treatment with antithyroid drugs may restore both AIx and PWV levels, although the evidence is scarce.We should consider an important limitation in our work, which is not having considered articles not in the English language. Considering the important heterogeneity of studies considering effects of thyroid hormones on stiffness and the complex interactions of factors determining vascular dysfunction, a further contribution is given by a recent meta-analysis, which found that both hypothyroidism and thyrotoxicosis are associated with an increase of aortic stiffness . The schIn pHPT, a prolonged exposure to high levels of PTH may affect the vascular system in different ways, leading to vascular functional and structural changes and to arterial stiffness. Arterial remodeling is induced by PTH with a few mechanisms. A direct effect of PTH on endothelial and vascular smooth muscle cells was hypothesized  and confAn elevation of blood pressure levels, which is a hallmark of pHPT, is associated with functional changes in arterial viscoelastic properties of the large arteries and an increase in the blood pressure-dependent component of arterial stiffness . In expeRegarding possible therapeutic approaches, surgical treatment of pHPT may improve arterial stiffness , and thuThe factors and the mechanisms influencing arterial stiffness in pPHT are schematically represented in Our review of clinical studies prompts that thyroid and parathyroid diseases are able to affect arterial stiffness biomarkers, thus leading to possible adverse cardiovascular outcomes. In hypothyroidism, most studies agree that an increase in arterial stiffness is present in both subclinical and overt hypothyroidism, although there is no complete agreement among methodologies evaluating the different aspects of vascular stiffening in the arterial vasculature. Levothyroxine replacement therapy in most studies has shown to lead to an improvement in arterial stiffness markers. Regarding hyperthyroidism, few studies observed an increase in biomarkers of structural arterial stiffening, although a relevant methodological heterogeneity prevents a definite conclusion. The increased heart rate typical of hyperthyroidism significantly alters the blood pressure profile, affecting biomarkers of functional arterial stiffness. In primary hyperparathyroidism, an increase in arterial stiffness is evident also in the mild forms. Arterial stiffening is reversed by parathyroidectomy, suggesting a role of surgery in reducing cardiovascular risk. Regarding methods used to quantify arterial stiffness, most studies focused on PWV, which represents the gold standard method to evaluate this parameter and may thus represent the preferred method in the vascular evaluation of thyroid and parathyroid diseases.Thyroid and parathyroid diseases are systemic diseases, characterized by an increase of cardiovascular risk, which present an increase in stiffness of the large arteries. Arterial stiffness measurement can be effectively used in the clinical evaluation of these conditions in order to quantify and possibly reduce the risk of cardiovascular events.


Patient-reported POSAS showed better values for colour for all three measurements, i.e., 3, 6, and 12 months, and the observers rated both vascularity and pigmentation better on these occasions (p < 0.01). At the recipient site, (n = 21) the mesh patterns were avoided as the DG covered the donor site due to its elastic properties and rendered the meshing procedure unnecessary. Scar formation was seen at the dermal donor and recipient sites after 6 months as in the standard scar healing process. The dermis graft technique, besides potentially rendering a larger graft yield, reduced donor site morbidity, as it healed faster than the standard STSG. Due to its elastic properties, the DG procedure eliminated the meshing requirement (when compared to a 1:1.5 meshed STSG). This promising outcome presented for the DG technique needs to be further explored, especially regarding the elasticity of the dermal graft and its ability to reduce mesh patterns.To investigate if donor and recipient site morbidity  could be reduced by a novel, modified split-thickness skin grafting (STSG) technique using a dermal component in the STSG procedure (DG). The STSG technique has been used for 150 years in surgery with limited improvements. Its drawbacks are well known and relate to donor site morbidity and recipient site cosmetic shortcomings . The Dermal graft technique (DG) has emerged as an interesting alternative, which reduces donor site morbidity, increases graft yield, and has the potential to avoid the mesh procedure in the STSG procedure due to its elastic properties. A prospective, dual-centre, intra-individual controlled comparison study. Twenty-one patients received both an unmeshed dermis graft and a regular 1:1.5 meshed STSG. Aesthetic and scar assessments were done using The Patient and Observer Scar Assessment Scale (POSAS) and a Cutometer Dual MPA 580 on both donor and recipient sites. These were also examined histologically for remodelling and scar formation. Dermal graft donor sites and the STSG donor sites healed in 8 and 14 days, respectively  12/01/2022. The STSG is defined as a cutaneous free tissue transfer that is intentionally separated from a donor site and transplanted to a recipient site, in which the graft depends on capillary ingrowth for its survival. Although skin grafting has been known for 2500 years, the technique that represents the current STSG technique was first described in 1872 by Ollier de Lyon2. This technique has remained essentially the same for 150 years. The technique is far from perfect, and shortcomings relate to donor site morbidity, which has been in focus recently3, as well as recipient site scarring. This is a problem well known in the field of burns, for example, where the problem presents not only as a contracture of the scar, but the mesh pattern most often seen post-transplantation contributes especially to non-optimal healing, i.e., scarring. The meshing procedure is regularly undertaken to obtain a graft that covers larger areas than, e.g., the donor site, as the STSG is inelastic and impossible to expand. The meshing procedure is almost always used, except for in the face, where due to cosmetic reasons the STSG graft is unmeshed to avoid the unfavourable mesh pattern and the concomitant risk of worse scarring. A non-meshed STSG graft is called a sheet graft and is well known to improve scar quality at the cost of donor site size.Skin grafting is an important and useful reconstruction option for covering skin defects, irrespective of their cause or site. Amongst skin grafts used for reconstructive surgery, such as split-thickness skin grafts (STSGs), full-thickness skin grafts (FTSGs), free cartilage grafts, and composite grafts; the STSG is the most commonly used and has the broadest application in everyday surgical care7. Especially promising is the fact that the dermal graft is very elastic, and so it may lead to a graft that is expandable. In this case, the need for performing a mesh procedure is reduced or at best avoided.The use of only the dermis portion as a graft for the recipient site has also been reported in the literature. The skin is then harvested in two layers from the same site, a traditional split-thickness skin graft of the upper layer including the corneal layer of the epidermis, and a dermal graft of the lower layer. The upper part is returned to the donor site, whereas the dermal portion is added to the recipient site. This procedure has gained significant interest lately, as it has favourable characteristics that address the donor site as well as issues at the recipient area in the traditional STSG procedureThe aim of this prospective, , exploratory, dual-centre study was to compare a DG technique with a conventional STSG. The patient group selected for the trial had burns of a limited size. The primary endpoint was donor site morbidity . The secondary exploratory endpoints were: first to examine the dermal graft elasticity and therefore the potential for it to be expanded without a meshing procedure; and secondly to examine the transplant effect of this un-meshed DG at the recipient site .From February 2017 to December 2020, 23 patients with thermal and chemical burns that fulfilled the eligibility criteria (see below) were consecutively enrolled from the Helsinki Burn Center, Helsinki, Finland and the Linköping Burn Center, Linköping, Sweden.Thermal & chemical burns < 30% TBSA.Males and females between 18 and 80 years.At least one coherent full-thickness burn measuring 15 × 7.5 cm.A minimum of 15 × 7.5 cm coherent undamaged skin located at the front or lateral side of one of the thighs for graft harvest.Inclusion criteria were:Full-thickness burns of the head, genitalia, axilla, or upper third of the medial upper arm.Patients with severe cutaneous trauma at the same site as the burn injury.Previous burns at the treatment site.Severe cognitive dysfunction or psychiatric disorder.Chronic skin disorder.Use of systemic or local corticosteroid.Use of anticoagulants or platelets.Autoimmune disease.Active hepatitis.Exclusion criteria were:10.Two patients died due to severe burn injuries and sepsis and were excluded, leaving a total of 21 included patients. Patients were treated according to a standard burn care protocol in both centresAll patients were treated with both methods for skin grafting (STSG and dermis graft). The locations of the skin graft of each method at the donor sites as well as the recipient sites were selected to avoid local heterogeneity in the donor and the recipient site. The latter was the burn injured area, and it was selected if the size was larger than 15 × 7.5 cm and the burn depth was considered not to vary significantly within the area. The areas within the burn wound allocated to the DG or STSG were randomly assigned using closed envelopes just before surgery. The envelopes contained information about where on the wound the dermis graft should be placed and from which part of the donor site  the graft would be harvested. Intact skin at the thigh was used as a donor site in all patients. The study was approved by the Swedish Ethical Review Authority 2016-401-31.8.Initially, an 8/1000 in. (0.203 mm) thick STSG was left attached to one end and folded up after the harvest, and a subsequent 12/1000 in. (0.305 mm) thick dermal split-thickness graft was harvested at the same site using Zimmer Air Dermatome . A surgical assistant aided in the harvest process to maintain tissue tension as well as provide more pressure, which is crucial to get a satisfying DG. To properly harvest a useful DG is a challenge, as it requires training and the extra hands of an assistant. This challenge imposed limitations on the patient recruitment process and was the initial reason why two centres were asked to participate. The Finish centre had already published their experience in the DG procedureAdjacent to the DG donor site a 12/1000 in. (0.305 mm) thick STSG graft was harvested according to the standard method. After the harvest, this graft was meshed 1:1.5 using a Zimmer II Mesher.Directly after surgical debridement, before reinstating the other half of the STSG at the donor site, the donor site was treated with adrenaline-soaked dressings for a minimum of 15 min for haemostatic purposes. The DG was then expanded to cover the excised area and fixed with sutures or staples. Figures After haemostasis, the STSG that had been left attached along one edge at the dermal donor site, was reinstated and sutured with a non-absorbable running technique to the wound edges using Prolene® 4-0 (Ethicon Inc.) leaving a covered dermal donor site, and an adjacent standard STSG donor site for comparison Fig. . Figure Small drainage holes were made to minimise the risk of haematoma underneath the DG. Both donor sites and recipient sites were dressed using conventional paraffin gauze, and finally, pressure was applied using fixed pressure pads and elastic fixation bandage.A total of four 3 mm punch biopsies were collected, perioperatively and at 6 months’ follow-up, from the dermal and split-thickness graft donor sites as well as the dermal and split-thickness graft recipient sites. The biopsies were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm. The sections were stained with haematoxylin and eosin as well as Masson’s Trichrome Stain  and blindly evaluated for histomorphology.Follow-up healing assessment was done by two independent, experienced burn surgeons. Epithelialisation was considered when > 90% of the wound area had epithelial cover on recipient sites and on the STSG donor site. The healing of the DG donor site was assessed by adherence of the returned skin-flap to the wound bed. Healing of both donor and recipient sites were assessed according to the standard of care within a week after surgical intervention. Healing assessment was done at least twice weekly until completed. Dressings were removed whenever an infection was suspected.11. The recipient and donor sites were assessed separately.Assessment of scar quality and the aesthetic outcome was done by an experienced occupational therapist and a burn surgeon at the trial site using two different reliable and validated tools; The Patient and Observer Scar Assessment Scale (POSAS) and the Cutometer Dual MPA 580The observer scale (objective) of the POSAS consists of six items . The patient scale (subjective) of the POSAS also consists of six items . All items are scored on a scale ranging from 1  to 10 (“worst scar imaginable”) .12. Firmness: The R0 variable (Uf) shows the highest point (maximum amplitude) of the first curve implies the firmness of the skin and represents the passive behaviour of the skin to force. Elasticity: The R2 is calculated by dividing the maximum drop of the curve (Ua) when removing the vacuum (the relaxation phase) with the R0 value (Ua/Uf).A cutometer was used in this study to assess the viscoelasticity properties of the different grafts and the different donor sites. This was done at the Linköping burn centre only. A 6 mm probe was used, said to best reflect the viscoelasticity of the dermis. A controlled vacuum of 450 mbar was applied in the central area of the scar of interest for two seconds, called “on-time” followed by a relaxation time  of two seconds called the “off-time”. This was automatically repeated three times, and the results for the corresponding variables R0 and R2 were displayedData are presented as median (25th to 75th percentile) or number (in percent) unless otherwise stated. The significance of the difference between variables was assessed with the aid of the Wilcoxon Matched Pair Test. Probabilities of less than 0.05 were accepted as significant.All methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Swedish Ethical Review Authority 2016-401-31. Informed consent was obtained from all participants.Median age was 53 (44–67) (n = 21) years at admission, 17 (81%) were males. Median (range) percentage total body surface area (TBSA %) burned was 10 (2–16) %. The median (range) duration of hospital stay was 13 (5–30) days, four patients (19%) were treated in the outpatient clinic (minor injuries). The aetiology of the burns were 13 (62%) flame burns, six (29%) contact with hot surfaces, one (5%) chemical burn, and one (5%) scalding. Excision of devitalised tissue was performed a median (range) five (1–11) days after injury. Vast majority of the recipient site was upper and lower extremities, 16 (76%), followed by four (19%) on the abdomen and one (5%) on the back. The excision depth after surgical revision of the wound was recorded on 13 patients and it varied from fascia/muscle (n = 4), subcutis (n = 7) and the deepest layer of dermis (n = 2). Most of the patients were categorised as ASA I or II   eight (7–14) days as compared to the STSG donor site; median (range) 14, (12–17) days,  was successful in 18 of 21 cases. Two STSG grafts failed due to infection and one because of patient’s poor compliance: In two of the patients a wound infection with p < 0.001) of the corresponding STSG graft  healing time of the DG (recipient site) was longer, 22, (14–31) days compared with eight (7–14) days, (The scar assessment (POSAS) was done at 3, 6, and 12 months, separately, for the donor and recipient sites. Fifteen (71%), 18 (86%), and 14 (67%) participants had POSAS recordings at the 3, 6, and 12 months follow up, respectively. Figures p = 0.03) and 12 months 2.9 (DG) and 3.8 (STSG))  for colour at all three measurements, the only significant difference was at 6 months (3 months 3.8 (DG) and 4.8 (STSG); 6 months 2.7 (DG) and 3.8 (STSG) (p < 0.01) and pigmentation (2.6 (DG) and 3.0 (STSG) 3 months, 2.2 (DG) and 2.9 (STSG), 1.9 (DG) and 1.9 (STSG)) lower at 3 and 6 months, although not significantly so (Table The observers rated both vascularity (2.0 (DG) and 2.9 (STSG) at 3 months; 1.8 (DG) and 2.9 (STSG) at 6 months; 1.6 (DG) and 2.4 (STSG) at 12 months, all There were no differences noted in the POSAS evaluation when comparing the DG and STSG grafts at the recipient site.   and 4.3 (STSG), 6 months 3.8 (DG) and 4.1 (STSG), 12 months 2.8 (DG) and 3.6 (STSG). Not statistically significantly different but a tendency at 3 and 12 months (p = 0.008). Other than that, we found no differences in the viscoelastic properties between the different grafts and the different donor sites. The analysis of differences in viscoelasticity over time in grafts and donor sites showed that the STSG became less elastic between 3 and 6 months (mean (SD) R2 decreased from 0.88 (0.07) to 0.74 (0.11), p = 0.04), and that the dermal grafts became less firm between 3 and 12 months (mean (SD) R0 increased from 0.59 (0.26) to 0.82 (0.25), p = 0.04), and less elastic between six and 12 months (mean (SD) R2 decreased from 0.85 (0.08) to 0.79 (0.13), p = 0.01).  R2 was 0.85 (0.08) and 0.74 (0.11), respectively, p = 0.07) although not significantly so. The dermal donor site thickness was 99 µm with IQR 73–117, p = 0.87). The split thickness donor site was 100 µm and IQR 87–114, p = 0.87).The histological analysis was blinded to the pathologist. The microscopic architecture was similar between the donor and recipient sites. The histological evaluation of the biopsies confirmed skin healing, irrespective of the biopsy site. The epidermal thickness was slightly larger in the dermal recipient site (median 124 µm and IQR 102–247) in comparison to the split-thickness recipient site  this could be of real benefit.The new and important findings in this prospective, dual-centre, intra-individual, controlled (within patient donor/recipient site) study are that healing time and cosmesis were improved at the donor site, findings that have been suggested previously in uncontrolled case series16. Other indications of improved viscoelastic effects at the dermal graft recipient site in this study were the tendency to a better elasticity as noted by cutometer measurements. This improved elasticity measure was also supported by the more pronounced collagen deposition seen in the histopathological examinations. However, these findings, especially at the recipient site, need to be further examined as the cutometer assessments were only made at one site (Sweden) and patients were lost at follow up. The explorative finding at the recipient site further suggests, in line with the finding at the donor site, that pigmentation also appeared better, as judged by POSAS (observer), but the findings did not reach statistical significance. The healing time was significantly longer for the DG at the recipient site, similar to our previous study8. This may be due to the thickness chosen for the DG and can probably be shortened using a thinner DG. Another explanation would be that the dermal graft needs time to vascularise prior to epithelialisation, which then results in a prolonged healing time. Other attempts, such as negative pressure treatment, may be tried to reduce the healing time at the recipient site and are needed to further optimise this new approach to skin grafting. Other options would be covering the dermal graft with an epidermal skin substitute such as Suprathel ® 17, cell suspension therapy19 allograft skin, or another novel skin grafting technique20.Further, during the study, it was appreciated early that the elasticity of the non-meshed DG was considerable, reducing the need for a mesh procedure. The result of which may be important for the future, as the mesh pattern after “meshed” regular STSG procedure is well-known to negatively affect post-surgical cosmesis21. After this attempt, two publications described a modified technique by which the dermal graft was applied upside down at the recipient site23. The graft was thereafter left to vascularise and as it did not epithelialise at the surface, a second STSG was applied on top after 2 weeks. It needs to be stressed that the STSG taken at the donor site at the time of the dermal graft was returned to its original position in our study. In the publication by Querings et al. in 2002, it was emphasised that both the functional and cosmetic results at the recipient site were favourable23.The previous experience of using a strict and isolated dermal component in the STSG technique was first introduced by Hynes already in 1954, as an alternative to the direct or tubed flap7 used the dermal grafting technique to reconstruct palmar skin defects of the digits and hand using plantar dermal grafts. The dermal graft healed in 12–16 days when there was a good wound bed. In this case series of 17 patients, there was excellent graft take (99.1%), with favourable cosmetic results and no marginal scarring. The STSG to the donor sites healed in 7–8 days with no marginal scarring and good cosmetic results. Histological observation 1 year after dermal grafting, the stratum lucidum had been remodelled and sweat gland tissues were observed7.Tanabe et al.6 in 2002, in a pig model, they used two layers of dermal grafts and concluded that the epithelialisation was slower at the dermal recipient site than the donor site as there is a need for vascularisation of the graft prior to epithelialisation. Despite this, all grafts were healed in 2 weeks (90% area covered) and the cosmetic results were good.An experimental study by Rubis et al.13. It was concluded that in all eight patients included in the study, there were good dermal grafts “take” with rapid or slow epithelialisation.Another attempt to increase the availability of autologous skin, by use of dermal grafts in the treatment of burns was the study by Kogan et al.4. They compared dermal grafting to STSG in a plastic surgery setting. The wounds of the dermis (n = 53) and skin grafts (n = 33) had re-epithelialised after 15 and 12 days, respectively. Scarring on the recipient site of the dermis graft was found to be less severe than that on the regular skin graft in terms of pigmentation, height, and vascularity . It was concluded that the dermal grafting technique was superior both aesthetically and functionally to the regular skin grafting technique in both the recipient and donor sites4. These findings are similar to ours.The largest study yet, using dermal graft for wound coverage, is the study by Han et al.8.In the dermal graft study in the burn setting performed by the senior author, 16 dermal grafts performed in nine patients were compared to regular STSG. As a control donor site (STSG) an adjacent area on the back close to the dermal graft donor was selected. The focus of this study was primarily to generate more autologous skin for burn-injured patients. Time to dermis graft epithelialisation ranged from 12 to 35 days (median 21 days). All grafts recorded had > 90% epithelialisation by 4 weeks. There was no significant difference in donor site healing times when comparing the dermal graft “deeper” donor site  to the standard control SSG donor site 24. Han et al. further demonstrated, in a retrospective study, the satisfying cosmetic as a functional outcome of the dermis graft after coverage of small skin defects postoperatively after tumour resection involving the face25. Most of the patients had satisfactory results in both functional and cosmetic matters with high-quality skin characteristics. Patients’ satisfaction with the dermis graft was also excellent.In a small (n = 4) patient case series by Kang et al., it was shown that dermal grafts may be successfully applied on top of exposed bone and tendonsThe strength of this investigation is that it is, to the best of our knowledge, the first dual-centre, prospective, intra-individual, controlled,  study of dermal grafting to examine both donor and recipient site morbidity and cosmesis as assessed by both observer and patient (POSAS). The evaluation tools used, the POSAS instrument, and the cutometer are well-known and properly validated instruments. Although the difference was small, as can be seen in the figures, it was measurable by the POSAS. Histological analysis was also performed. The small study population in the present study is a significant short coming and to detect aesthetic differences, especially at the recipient site, further studies with larger samples sizes will be required. The viscoelasticity measurements and biopsy follow ups were not investigated on all patients due to missed appointments and follow up. Another limitation is that the meshing procedure of the STSG was different between the centres (13 meshed 8 unmeshed), which can have affected our results. However, considering that the aesthetic result likely would be better with an unmeshed STSG the advantage of the DG may have been more pronounced if all STSG had been meshed.The technique is new and there is no established instrument yet for the dermal graft harvest. Therefore, significant training is required to get satisfying grafts yield with a regular dermatome and this contributed to the small sample size. The technique of taking a DG is more demanding technically compared to a standard STSG harvest. The first layer, the STSG, is usually managed rather easily by a trained plastic surgeon. When taking the second layer, the DG, it needs to be appreciated that the tissue is much less firm and as such poses a significant challenge. The challenges can be summarised by: the lack of firmness which makes it difficult to ascertain the thickness of the graft; and secondly, to harvest a proper DG the procedure needs to be adjusted to the four borders of the first STSG. The research group has, for the future development of this technique, developed a new dermatome that simultaneously collects two layers of skin in a pre-set fashion. We hope that this will be advantageous for further studies as it will make the technical learning curve of the DG procedure less steep, and as the method becomes less challenging it becomes more established and it will be easier to recruit patient for future studies. This technical issue significantly contributed to the challenges of including patients. The inter-rater reliability was not evaluated, which may affect the results in a multicentre study with several investigators. The POSAS reviewers were not blinded towards which site received which graft type, which could have introduced bias. However, despite this, the POSAS instrument has been claimed to be adequate.Additionally, regarding the elasticity of the DG, it was appreciated early during the study that the DG had a prominent elasticity, not necessitating a meshing procedure to cover the larger recipient areas. It is a significant limitation of the present protocol that we cannot present data on the expansion rate of the DG. This is an important topic to address for the next study. However, we think that the fact that the unmeshed DG could be expanded so that it covered the recipient area of a similar size as the meshed STSG supports the idea of the potential of a favourable cosmetic outcome for the recipient site of the DG as compared to a regular STSG.The dermis graft reduced donor site morbidity, because returning the STSG component to the donor area heals faster than the standard STSG technique. The long-term outcomes of the recipient sites were also favourable because the elasticity of the dermal graft facilitated the graft expansion without necessitating the mesh procedure. An inherent advantage of the dermal graft technique (not examined in this study) is the double or triple graft yield that can be accomplished to cover large burn areas. However, the strength of the conclusions in this study is hampered by the limited number of patients, the loss of observations between sites, and patient drop-out at follow up. Larger studies should therefore be initiated to explore the advantages and disadvantages of the technique, particularly at the recipient site.Supplementary Information.