Compared to our understanding of positive prediction error signals occurring due to unexpected reward outcomes, less is known about the neural circuitry in humans that drives negative prediction errors during omission of expected rewards. While classical learning theories such as Rescorla–Wagner or temporal difference learning suggest that both types of prediction errors result from a simple subtraction, there has been recent evidence suggesting that different brain regions provide input to dopamine neurons which contributes to specific components of this prediction error computation. Here, we focus on the brain regions responding to negative prediction error signals, which has been well-established in animal studies to involve a distinct pathway through the lateral habenula. We examine the activity of this pathway in humans, using a conditioned inhibition paradigm with high-resolution functional MRI. First, participants learned to associate a sensory stimulus with reward delivery. Then, reward delivery was omitted whenever this stimulus was presented simultaneously with a different sensory stimulus, the conditioned inhibitor (CI). Both reward presentation and the reward-predictive cue activated midbrain dopamine regions, insula and orbitofrontal cortex. While we found significant activity at an uncorrected threshold for the CI in the habenula, consistent with our predictions, it did not survive correction for multiple comparisons and awaits further replication. Additionally, the pallidum and putamen regions of the basal ganglia showed modulations of activity for the inhibitor that did not survive the corrected threshold. While the field of reinforcement learning has generally focused on the role of reward prediction errors in training reward expectations, the mechanisms involved in learning about omission of expected reward delivery are less well understood. Classical models of learning such as Rescorla–Wagner and TD models suggest that prediction errors result from a simple subtractive computation, which also has been shown to match the firing of dopamine neurons. However, there is also recent evidence suggesting that brain areas projecting to dopamine neurons may provide input which contributes to specific parts of this computation, for example, some regions may encode the level of expected reward , while oIn appetitive Pavlovian conditioning, individuals learn expectations about stimuli that are reliably paired with rewards. This conditioning procedure causes the previously neutral cue to drive a conditioned response. In conditioned inhibition, a conditioned stimulus (CS) associated with reward is presented simultaneously with a conditioned inhibitor (CI), which causes the expected reward not to occur. Conditioned inhibition occurs because the unexpected omission of reward causes a negative reward prediction error. By learning theories like Rescorla–Wagner, if another sensory stimulus is reliably present during these unexpected omissions, the accumulation of negative prediction errors causes the CI to acquire negative value. This results in inhibitory conditioning, and a reduction of the conditioned response. For example, imagine that you enjoy drinking tea, but cannot make it when your kettle is broken. Over time, the broken kettle becomes a CI because it reliably predicts the omission of tea.Computationally, conditioned inhibition is an interesting problem, because it relies on the negative prediction errors that occur when the CS+ is unexpectedly followed by a reward omission in the presence of the inhibitor, which causes the CI to acquire negative value, even though the CI has never been paired with an aversive stimulus. Once inhibition is acquired, the inhibitor can pass the summation test, meaning there is a reduced conditioned response to a CS paired with the inhibitor compared to the CS alone . FurtherThe prediction error response in dopamine neurons includes both increases in firing for better than expected outcomes and decreases in firing, or dopamine dips, for worse than expected outcomes. However, few studies have focused on understanding the role of certain brain areas in the processes driving dopamine dip signals for worse than expected outcomes and how these areas are involved in learning about stimuli that predict reward omissions. In particular, an unanswered question remains about the extent to which brain areas involved in learning about reward omissions overlap with those involved in learning about aversive stimuli.Theories about how the positive and negative valence learning systems interact have proposed that something that stops a negative state leads to positive emotions, while the omission of a positive reward leads to negative emotions . HoweverWhile previous fMRI studies have looked at the neural mechanisms involved in monetary losses and the presentation of aversive stimuli, no human fMRI studies have focused on the learning about predictors of reward omissions in a conditioned inhibition experiment. Dopamine neurons respond to a CI with an inhibition, or pause in tonic firing, the same pattern of dopamine release in the substantia nigra seen to an aversive stimulus . IntriguTo examine the brain areas involved in each of these computations, we ran a novel fMRI study, adapting the conditioned inhibition paradigm from See Nineteen participants  ranging between 19 and 55 years old, from the University of Colorado, Boulder, and the local community volunteered for the study. All participants were right-handed and generally in good health. Participants were screened for MRI contraindications and provided informed written consent for protocols approved by the Institutional Review Board of the University of Colorado, Boulder. Participants were paid $48 for completing the study in addition to earnings from the task.The functional imaging was divided into six scanning runs, with an average length of 9 min, with brief 1–2 min breaks between blocks. The first 10 volumes of each run were discarded to account for equilibration of the scanner’s magnetic field. The experimental design is shown visually in In the first conditioning block of 48 trials, which lasted 8.4 min, participants were exposed to the initial CS – US contingencies, with equal number of CS+ and CS− trials. During Pavlovian conditioning, one fractal stimulus (CS+) was associated with reward (orange juice) 75% of the time, while another fractal (CS–) was deterministically associated with a neutral outcome  . This fiThis was followed by four conditioned inhibition blocks (blocks 2 -5), which consisted of 200 trials total, where the CS+ was paired with another stimulus, the CI, in 22% of these trials . Presentation of the CI was deterministically associated with the presentation of the neutral solution, negating the reward prediction elicited by the CS+. In another 22% of total trials, participants continued to experience the initial CS – US pairing, with reward presented in 75% of these trials in order to keep the reward association from being extinguished by the conditioned inhibition procedure. The remaining trials consistent of several different neutral control stimuli , and the CI viewed alone .This conditioned inhibition training was broken into several different blocks, based on the same sequence of trials as the original In blocks 3–5, which each lasted 10.4 min, and consisted of 168 total trials, participants saw five different stimuli, the CS+, CS–, CS+ paired with the Inhibitor, the CS– control consisting of two fractal images, the Inhibitor, and another CS– control consisting of a single fractal image. Each of these blocks consisted of 56 trials, 12 trials each of the CS+, CS–, CS+ paired with the Inhibitor, and the CS– control consisting of two images. Each block also included four trials where the Inhibitor was viewed alone and four trials of the CS– control consisting of a single fractal image.The inhibitor was shown less frequently alone  to minimize learning about the inhibitor in isolation, which would have reduced the strength of the inhibitory procedure, as done in a previous conditioned inhibition study in monkeys  which alIn the final block, we ran an inhibition test block, which lasted 8.4 min and consisted of 48 trials. In the test block, we followed the CI with an unexpected juice reward 75% of the time, in order to test for positive reward prediction errors. A control CS– was also paired with an unexpected juice reward 75% of the time in the conditioned inhibition test block, and we expected less of a prediction error signal for the CS– paired with reward than the inhibitor since it did not develop an association with reward omission.In each trial, there was a presentation of a fractal CS for 2 s. This was followed by 2 mL of orange juice (Tropicana brand) or the neutral-tasting solution, which was delivered by a taste pump connected to the stimulus computer. The onset of taste delivery was logged, and delivery of solution after receiving the trigger took about 3 s. It was a delay conditioning paradigm, where the CS remained onscreen until the US delivered was completed. After each trial of CS and US presentation, there was an ITI randomly sampled to be between 4 and 8 s. We selected participants who reported a preference for orange juice in the prescreening interview. Further, presentation of orange juice has been associated with higher pleasantness ratings than artificial saliva in prior studies . Visual There were several features of the design that were motivated by careful consideration of the learning problem. For example, we wanted to keep the duration between the CS and juice reward (US) consistent because there is evidence the striatum responds to temporal prediction errors . In addiMagnetic-resonance imaging (MRI) data were acquired at the Center for Innovation and Creativity at CU Boulder using a 3T Siemens Trio scanner and a 32-channel receive-only head coil. To guide the functional imaging, a structural volume of the entire brain was acquired first using a T1-weighted magnetization-prepared rapid gradient-echo (MPRAGE) sequence .∗-weighted gradient-echo echo-planar imaging (EPI) sequence . With these parameters, 24 contiguous slices were collected in interleaved-ascending order for each volume. Slices were aligned parallel to the base of the OFC. Due to the focus of our study on subcortical areas, we acquired limited coverage, which included the amygdala, insula, midbrain, thalamus, striatum, and ventral prefrontal cortex.High-resolution functional images were acquired with a blood-oxygen-level-dependent (BOLD) contrast using a T2The functional imaging was divided into six scanning runs, with an average length of 9 min. The first 10 volumes of each run were discarded to account for equilibration of the scanner’s magnetic field.In order to investigate whether there were behavioral effects of conditioned inhibition, we ran a follow-up study to look at how the conditioned inhibition procedure affected reward expectation. The study had an identical design, but used monetary rewards, and allowed us to look at the behavioral effects of conditioned inhibition by having participants rate their expectation of reward at the end of each training block. As our behavioral measure of conditioning, we assessed the reward expectation for each stimulus at the end of each block using a continuous rating scale for reward expectation, ranging from No Expectation to Strongest Expectation. Subject saw each CS in succession, followed by the rating scale depicted in 1, but with a distinct warping step. The first 10 images were discarded to account for the stabilization of the BOLD signal. Then, the functional images were motion-corrected using the realignment procedure in SPM 8, using a rigid-body, affine (six parameter) registration that helps correct for head movement during scanning. To identify outliers, we computed the mean and standard deviation across voxels for each image for all slices, and then calculated the Mahalanobis distance of each mean and standard deviation value, considering any volumes with a significant χ2 value as outliers, per the procedure described in The preprocessing pipeline followed a well-validated preprocessing pipeline that has been used in several other studies , and is Next, these motion-corrected functional images were co-registered to the structural images using FSL’s epi_reg script, an affine co-registration that improves registration by segmenting the structural and functional images . Each stThe functional images were corrected for slice timing to account for acquiring slices at slightly different timepoints and then motion corrected using the realignment procedure in SPM8. Each outlier image detected by the Mahalanobis distance method was modeled as a nuisance covariate, by inserting a dummy code variable of 1 where the spike occurred.In addition, we calculated several regressors of non-interest, which included an intercept for each run, dummy regressors for outlier images calculated by the spike detection method above, and motion-related covariates, which included 6 mean-centered motion parameter estimates, their squared values, successive differences and squared successive differences. Additional nuisance regressors were calculated by determining the first five principal components from the signal in the ventricles in the warped functional images with a 4 mm smoothing kernel.The fMRI analysis involved separate regressors for each of the different stimuli in the experiment, including separate regressors for each of the stimuli in the conditioning, conditioned inhibition and inhibition test phases, to allow us to assess the effects of Pavlovian conditioning and conditioned inhibition on brain activity. To this end, we generated separate first-level model task regressors for the CS+ and the CS– in the first conditioning block. In the three following conditioned inhibition blocks, we created first-level model task regressors for the CS+, CS+ paired with the Inhibitor, and Inhibitor viewed alone, as well as the two other CS– stimuli. In the conditioned inhibition test blocks, we generated separate regressors for the Inhibitor and the CS–.In the same first-level model, we also modeled each of the different outcomes following the CS with separate regressors, to allow us to examine the effects of expectation on outcome activity. Therefore, we generated separate regressors for the expected presentation of reward following the CS+, the unexpected reward omission resulting from presentation of the neutral solution following the CS+ (omission), and presentation of the neutral solution following each CS– (as a control stimulus for the effects of taste stimulation). In each case, the duration of each CS event was set to 2 s, while the duration of each US event was set to 3 s. The fixation cross, which was presented between each CS-US trial, was explicitly not modeled and considered the implicit baseline.Further, to assess the effectiveness of the conditioned inhibition procedure, we ran a conditioned inhibition test where the inhibitor and the CS– control were unexpectedly paired with reward in the last block of the experiment. Therefore, the first level model also included separate regressors for both cue and outcome activity in this inhibition test block. If conditioned inhibition was successful and caused the inhibitor to acquire negative value, positive prediction errors should result when it is unexpectedly followed by a reward.To specifically examine this, we looked separately at outcome activity when the Inhibitor was unexpectedly paired with reward and the trials where the Inhibitor was paired with no reward. Similarly, we also modeled the trials in the inhibition test block where the CS– was unexpectedly paired with reward separately from the trials where the CS– was followed by the expected neutral solution (no reward).q < 0.05, FDR .For the group level GLM analysis, we used a robust regression procedure, which has been shown to decrease sensitivity to outliers . Whole bWe defined ROIs according to probabilistic atlases, whenever possible. For each anatomical atlas, we used a threshold to include only voxels that had 75% or higher probability. For the habenula ROI, we used the habenula ROI from a high-resolution atlas of the thalamus based on histological data . Recent The basolateral and centromedial amygdala ROIs were derived from the CIT atlas, which is in the same standard space as the functional images . The anaa priori ROIs, results were corrected for multiple comparisons with q < 0.05, FDR  across a merged mask of all ROIs . To create this mask, we included voxels with 75% or higher probability from each probabilistic subcortical atlas and all voxels from the bilateral Insula and bilateral Orbital Frontal Cortex atlas.To visualize results in our a priori ROIs. When performing ROI analyses, we looked at mean activity in each ROI across subjects, calculated based on the individual subject-level beta images for each condition from the first-level analysis. For tests of ROI activity, p < 0.005 (Bonferroni corrected for comparisons across 10 ROIs) was considered significant, and for each test, we report whether it exceeded the Bonferroni correction.Additionally, we conducted comparisons of mean activity across different conditions in the p < 0.001 or p < 0.005. An additional result from the habenula ROI is shown at q < 0.05, FDR, small volume corrected with a binary mask of the ROI.Results that did not exceed the FDR threshold across the mask of all ROIs or survive Bonferroni correction are reported for information only. For visualization purposes for ROI results in the basal ganglia, substantia nigra and habenula, we plot the results at an uncorrected threshold of t(18) = 11.84, p < 0.001]. We also found that the ratings for the CS+ when presented concurrently with the Inhibitor were significantly lower than the ratings for the CS+ alone , indicating that the conditioned inhibition procedure had significantly reduced reward expectations, demonstrating conditioned inhibition. See In order to investigate the behavioral effects of conditioned inhibition, we ran a follow-up behavioral study using monetary rewards to look at how the conditioned inhibition procedure affected reward expectation. This study allowed us to examine the behavioral effects of conditioned inhibition by having participants rate their expectation of reward at the end of each training block. As outlined above, conditioned inhibition occurs if a CI presented concurrently with a CS+ is able to elicit a reduced conditioned response compared to the CS+ alone. To investigate whether there was a behavioral effect, we asked participants to rate how strongly they would expect reward on a continuous rating scale, ranging from No Expectation to Strongest Expectation after viewing the fractal stimulus. This behavioral test revealed that our conditioning procedure was successful, as mean ratings across blocks showed a significantly higher rating for the CS+ than the CS–  and insula ROIs  for juice compared to neutral solution survived FDR correction at q < 0.05 corrected across a mask of all ROIs, as shown in p < 0.005 and p < 0.001 uncorrected in these regions.We also conducted ROI analyses in a set of These regions are summarized in p < 0.001 in regions expected from prior studies of rewarding outcomes, including the insula, orbitofrontal cortex, basolateral amygdala and putamen for juice compared to the neutral solution, as shown in p-value are reported in the There was activity at a whole-brain uncorrected threshold of x = 40, y = 24, z = –10, k = 8, t = 5.83] and insula  surviving FDR correction at q < 0.05 correction across the whole brain.We further examined activity to an unexpected reward omission, examining the 25% of trials where the CS+ was unexpectedly followed by the neutral solution compared to neutral solution presentation following control trials (where it was expected). This revealed two peaks in the orbital frontal cortex , which did not survive the Bonferroni correction. The activity in the caudate ROI did not survive FDR correction across the mask of ROIs at q < 0.05, FDR. There were two peaks within the caudate at an uncorrected threshold of p < 0.005, one located within dorsal caudate , and the other in more ventral caudate , shown in An ROI analysis of mean activity averaged across the caudate ROI showed significant activity for juice compared to neutral solution , which survived Bonferroni correction across all ROIs. For visualization purposes, this is shown at an uncorrected threshold of p < 0.001 and p < 0.005 in Additionally, there was significant ROI activity in the central amygdala ROI for the juice reward compared to neutral solution , however, this did not survive the Bonferroni corrected threshold. The activity in the habenula ROI did not survive FDR correction across the mask of ROIs at q < 0.05, FDR. For visualization purposes, this is shown at an uncorrected threshold of p < 0.001 and p < 0.005 in An ROI analysis of mean activity in the habenula ROI showed a significant deactivation for juice presentation compared to the neutral solution , consistent with other studies that have found activity in the insula for food reward cues  and extending to the pallidum  for the CS+ compared to the CS–. However, this did not survive FDR correction across the mask of all ROIs, and is mentioned for information only, noting that it did not survive correction for multiple comparisons.Further peaks from the whole-brain threshold of t(18) = 3.17, Bonferroni corrected p = 0.053, p = 0.0053, mm center L = 8,–18,–14, R = –8,–20,–14] and VTA  for the CS+ than the CS–, as shown in p-value threshold in the SNc or VTA ROIs, it was just below the margin of significance in the SNc. For visualization purposes, the CS+ > CS– effect in substantia nigra is shown in p < 0.005. However, only a single voxel in this region  survived correction for multiple comparisons at q < 0.05, FDR across the mask of all ROIs. The CS+ > CS– effect was only visible an a lower, uncorrected threshold of p < 0.05 in the VTA ROI, which is shown in We expected activity for conditioned stimuli associated with reward in the midbrain, amygdala and striatum . We nextThere was not significant ROI activity in the amygdala, nucleus accumbens, caudate, pallidum or putamen for the CS+ compared to the CS–.q < 0.05, FDR.We expected that the CI would recruit activity in regions that have been shown to respond to predictors of reward omissions. However, we are unaware of other fMRI studies using a conditioned inhibition design with rewards, so it is unclear whether the same regions that have been shown to respond to predictors of monetary loss and aversive stimuli also respond to CIs, or predictors of reward omission. A CI has never explicitly been followed by a negative valence outcome, but acquires negative value by reliably signaling a reward omission. Based on computational theories of learning such as TD, Rescorla–Wagner and PVLV , this ocWhile there is little research on brain areas that encode CIs in humans . However, this activity did not survive correction at FDR q < 0.05 across the mask of ROIs or Bonferroni correction for multiple comparisons. In the whole-brain analysis for the inhibitor compared to controls, there was activity in the insula at p < 0.005 uncorrected, as shown in Consistent with this data, we also saw significantly more mean activity in the bilateral insula ROI for the inhibitor than the control stimuli . However, this activity for the CI did not survive the Bonferroni corrected threshold.Based on animal studies, we predicted that the habenula would show an increase in activity for the CI, as it showed an increase in activity for a CS that predicted omission of reward, accompanied by a reduction in SN/VTA activity for the Inhibitor . Consistt(18) = 1.119, p = .2779, Bonferroni corrected > 1, mm center L = 4,–24,2, R = –2,–24,2]. Habenula activity for the CI compared to the controls is shown at an uncorrected threshold of p < 0.005 and p < 0.001 in q < 0.05 , small-volume corrected with the habenula mask. However, this region did not appear when FDR correction was done across the mask of all ROIs, and therefore we strongly qualify this result, which awaits further replication before inference can be made.Further, this was not significant when the inhibitor was compared to only the neutral (Y–) control (consisting of a single cue shown at a similar rate to the inhibitor) that was always followed by the neutral solution . Further, limited evidence pointed towards the habenula encoding negative valence, as it significantly increased for an Inhibitor paired with a reward omission, but not the positively valenced CS+, though this comparison did not survive Bonferroni correction. However, there was not a significant difference between the Inhibitor > Control and the CS+ > CS– effect in the habenula .To compare the role of the habenula and substantia nigra in our learning task, we compared activity in both ROIs, as shown in x = 20, y = 4, z = –6, k = 53, t(18) = 11.29], which extended into the pallidum, for the inhibitor compared to control stimuli at p < 0.005, uncorrected, as shown in Along with the habenula, we also expected that regions of the basal ganglia would respond to the CI associated with reward omissions. The ventral striatum has been shown to be activated by predictors of aversive stimuli , and a cp < 0.005 uncorrected, but this activity did not survive correction for multiple comparisons. Only a single voxel in the SNc survived correction across the mask of all ROIs at q < 0.05, FDR . Further, an ROI analysis of mean ROI activity in the SNc showed more activity for the CS+ than the CI , but this did not survive Bonferroni correction for the number of ROIs.Additionally, shown in If the inhibitor acquired a negative association, there should be a prediction error when the inhibitor is paired with a reward, resulting in activity in dopamine regions. However, an analysis looking at the mean activity in the VTA, SNc or Accumbens ROIs for the inhibitor followed by a reward did not show significant activity in the trials where the Inhibitor was followed by an unexpected reward.q < 0.05, FDR . Further, an ROI analysis of mean activity in the putamen ROI showed an increase in the putamen ROI during taste presentation when the inhibitor was unexpectedly followed by reward, compared to when the inhibitor was followed by a neutral solution , but this did not exceed the threshold for Bonferroni correction.There was a significant increase in activity in the putamen ROI during taste presentation when the inhibitor was unexpectedly followed by reward which survived whole-brain FDR correction at p = 0.3305, Bonferroni corrected p > 1, t = 1.00]. The regions that survived whole brain correction for the Inhibitor compared to Controls and the Inhibition test are summarized in An additional, more sensitive test of conditioned inhibition may be the comparison of responses to the inhibitor followed by reward to the control stimulus followed by reward, as the Based on our discussion of the potential of conditioned inhibition to dissociate between representations of associations of a CS+ with reward and the representation of an inhibitor with reward omissions, we compared the activity for the CS+ and Inhibitor to that of the Inhibitor viewed alone. This should reveal regions that selectively reflected the associations of the CS+ with reward. Three clusters in visual cortex, including the lingual gyrus, showed more activity for the CS+ and Inhibitor compared to the Inhibitor alone, as described in p-value threshold, and thus should not be strongly interpreted. While other studies have found activity in habenula for predictors of aversive outcomes, such as shock . However, the activity in the habenula for the CI did not survive FDR correction across the mask of all ROIs, and the test of mean ROI activity for the inhibitor compared to controls in habenula did not exceed the Bonferroni-corrected as shock , or an aas shock , and oneas shock , none haas shock , which fas shock  were cruas shock , we cannThough increased activity in the habenula is associated with a dip, or pause in tonic dopamine firing , we did One potential reason that we did not see significant reductions in BOLD signals for the CI could be related to the physiology of the midbrain dopamine system. For example, inhibitory synaptic input has been shown to increase BOLD signals , and it We also replicated previous studies, which showed activation in the SN/VTA area for a rewarding outcome, and studies showing activity in the SN/VTA area for a CS+ paired with reward . While wWhile some studies have found activations in ventral striatum for pleasant taste presentation , other sWe also predicted that regions of the basal ganglia send signals to the lateral habenula encoding the level of reward expectation, allowing it to drive a dopamine dip if an expected reward is not received. While there was activity pallidum and putamen for the inhibitor compared to a control stimulus that was significant at a whole-brain uncorrected threshold, this activity did not survive correction for multiple comparisons and should not be strongly interpreted. As the behavioral ratings from the Monetary reward task demonstrated that the inhibitor in that study acquired negative value, and the imaging study found that the inhibitor led to activity in the pallidum, this is consistent with another study that observed pallidal activity increasing with the negative value of a shock cue . While aWhile we also saw a non-significant increase in BOLD activity for the CS+ in the habenula, such differences could potentially be explained by the complexities of mapping neuronal spiking in this region to BOLD signals, and reduced power due to the sample size. Several studies have shown that a CS+ associated with reward decreases neural firing in habenula neurons compared to cues associated with reward omissions . FurtherFurther, we observed activity in the putamen surviving correction for multiple comparisons when the CI was unexpectedly followed by a juice reward in the inhibition test at the end of the experiment. This may reflect a prediction error if the conditioned inhibition procedure caused the inhibitor to acquire negative value, consistent with other studies that have found putamen regions respond to prediction error signals . HoweverAlong with subcortical regions, we found that the orbital frontal cortex and anterior insula showed involvement in the reward learning task. We replicated prior studies showing activation of the anterior insula for taste stimuli . We alsoWe also observed activity for the CI in the anterior insula, but only at a whole-brain uncorrected threshold. This is interesting due to other papers which have suggested a role for the insula in safety signal processing in the aversive domain . ActivitConditioned inhibition provides an interesting way to examine the functioning of the dopamine system and can be used to look at how different brain regions are involved in learning about a CI that predicts not getting reward. It allows comparing the brain activity for stimuli associated with reward predictors to those associated with reward omissions and is interesting for examining how the subcortical areas projecting to the dopamine system, which have been primarily studied in animal learning studies, translate to humans in an fMRI task. Additionally, understanding the brain areas that drive this frustration signal for reward omissions can be translated to understand how disorders that involve persistent negative predictions, such as depression, may involve distortions in these systems. For example, recent research suggests that punishment prediction errors in the lateral habenula correlates with symptoms of depression , and futhttps://osf.io/njbmf/. Neuroimaging data used for the figures of second-level analysis results can be found on Neurovault: https://neurovault.org/collections/8676.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: all data used for the ROI analysis figures and behavioral rating data can be found in the Open Science Framework: The studies involving human participants were reviewed and approved by Institutional Review Board of the University of Colorado, Boulder. The participants provided their written informed consent to participate in this study.JM, TW, RO’R, and GF contributed to the conception and design of the study. JM performed the statistical analyses of the data. AK, LC, KK, TH, TW, GF, and RO’R contributed to interpretation and discussion of neuroimaging and behavioral results. JM wrote the first draft of the manuscript. All the authors contributed to manuscript revision, read, and approved the submitted version.RO’R is CSO and JM, TH, and KK are researchers at eCortex, Inc., Boulder, CO, United States, which may derive indirect benefit from the work presented here. The remaining 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.


Human transglutaminase 2 (TGase2) has various functions, including roles in various cellular processes such as apoptosis, development, differentiation, wound healing, and angiogenesis, and is linked to many diseases such as cancer. Although TGase2 has been considered an optimized drug target for the treatment of cancer, fibrosis, and neurodegenerative disorders, it has been difficult to generate TGase2-targeted drugs for clinical use because of the relatively flat and broad active site on TGase2. To design more specific and powerful inhibitors, detailed structural information about TGase2 complexed with various effector and inhibitor molecules is required. In this review, we summarized the current structural studies on TGase2, which will aid in designing drugs that can overcome the aforementioned limitations. Transglutaminases (TGases) are enzymes that catalyze the cross-linking reaction between the γ-carboxamide of a glutamine residue and the ε-amino group of a lysine residue; they also catalyze the deamidation reaction of glutamine residues to generate glutamic acid in the presence of water ,2. TheseHuman TGase2  is a multi-functional, ubiquitous enzyme that is important for various cellular processes, including apoptosis ,7,8, devOwing to its biologically and pathogenically important functions, TGase2 has been considered a major therapeutic target for the treatment of many human diseases caused by dysregulated TGase2 ,54,55,56TGase2 is a multi-functional protein. Cross-linking, deamination, and amine incorporation activities are the main functions of TGase2, GTPase, disulfide isomerase, and kinase. Scaffolding activities are also reported as minor functions of TGase2 ,57. The 2+ (activator of transamidase activity) nor guanosine triphosphate  affect the PDI activity of TGase2 . A. A67]. AAmino acid sequence analysis of TGase2 indicates that the nucleotide-binding pocket generated by the residues R476, R478, V479, S482, M483, R580, and Y583 is only conserved in mammals C. This sWhile nucleotides maintain the closed form and inhibit TGase2 activities, substrate derived-peptide mimetics, which work as irreversible inhibitors, promote the open, active conformation of TGase2 A 79]. T. T79]. TWith important roles in many pathological conditions and various activities, TGase2 has been considered an optimized target for therapeutic intervention ,54,81. IThe screening and discovery of TGase2-targeting drugs by examining small molecules was not initially successful. Owing to its relatively flat and broad active site, finding small molecules that target the active site of TGase2 has always been challenging. To overcome this issue, many researchers have used peptide mimetics to cover the broad active site. Because of this effort, several potential peptide mimetic inhibitors are currently in clinical trials . The nucBesides finding small molecules and peptide mimetics that target the active site or the nucleotide-binding site of TGase2 for pharmaceutical purposes, various approaches to discover drugs for TGase2 inhibition are undergoing. One of the representative cases is the search for an antibody against TGase2. Celiac disease is caused by an autoimmune response to dietary gluten antigens. This antigen produces autoantibodies against TGase2 . The eff


To compare the mechanical characteristics of a percutaneous superior pubic intramedullary screw, percutaneous bridging plate and percutaneous screw-rod system of the anterior ring for the treatment of unilateral vertical pubic fractures to provide a reference for clinical application.A finite element model of pelvic anterior ring injury  was produced. The fractures were fixed with a percutaneous superior pubic intramedullary screw, percutaneous bridging plate and percutaneous screw-rod system of the anterior ring and their combinations in 5 types of models. The fracture stabilities under vertical, bilateral and anterior-posterior load were quantified and compared based on the displacement of the hip joints’ midpoint as quantificational index of fracture stability.In the condition of bilateral and anterior-posterior load, the vertical, bilateral and anterior-posterior displacements of the hip joints’ midpoint of different models were significantly different respectively. In general, the displacements of the 5 pelvic anterior ring fixations were ranked from maximum to minimum as follows: bridging plate, pelvic anterior screw-rod system, combination of bridging plate and pelvic anterior screw-rod system, superior pubic intramedullary screw and combination of superior pubic intramedullary screw and pelvic anterior screw-rod system.For the fixation in unilateral pubic fractures of pelvic ring injury, the percutaneous superior pubic intramedullary screw is optimal, the percutaneous pelvic anterior screw-rod system is the second choice, and percutaneous bridging plate ranks the third. The percutaneous pelvic anterior screw-rod system can significantly increase fixation stability of the percutaneous superior pubic intramedullary screw and the percutaneous bridging plate. In order to preserve life, it is impossible to perform time-consuming final reduction and internal fixation with more surgical trauma and much blood loss, at the early stage of treatment of pelvic fracture with combined injuries of important organs, blood vessels, and nerves. Furthermore, due to the distinctive anatomical and physiological characteristics of the pelvis, the pelvic injury has become timeworn when the patient’s general conditions turn to be stable. In this case, the difficulty of reduction and fixation has increased significantly. The deformity is difficult to be corrected, and the risk of intraoperative vascular and neurologic injury significantly increases. This situation is common in Tile C pelvic fracture with poor stability caused by severe injury. Therefore, in order to minimize the initial mortality rate and late disability rate of pelvic injury, it is particularly essential to perform early, rapid and minimally invasive replacement and internal fixation after an injury and on the precondition that the body can tolerate it.The importance of reconstruction of posterior ring stability is self-evident in the treatment of pelvic fractures. However, in terms of some Tile B and all Tile C pelvic injuries, it has been proved that only posterior ring fixation cannot satisfy the requirements of maintaining bone position. Previous biomechanical researches have demonstrated that the stability of any posterior ring-internal fixator complex does not match the normal intact pelvis , 2. TherSo far, primary minimally invasive treatments of the final internal fixation for the pelvic anterior ring includes percutaneous superior pubic intramedullary screw, percutaneous bridging plate and percutaneous screw-rod system of the anterior ring . How to The ethics committee of Yantai Shan Hospital approved the study. Informed consents were obtained from the individual participant included in the study.In this study, the three-dimensional finite element model of pelvis established by our series of researches was used, and the related parameters were in line with the relevant literature , 2, 4. TAccording to the size of screw, plate and screw rod, parametric design models was established to simulate the operation modes of the percutaneous bridging plate, anterior ring screw-rod system and superior pubic intramedullary screw to fix the pubic superioris. The superior pubic intramedullary screw was a cannulated screw with a diameter of 7.3 mm and a length of 100 mm. The screw was placed retrograde and located in the center of the safe area of the superior ramus of pubis, and the screw thread passed through the fracture line.The percutaneous bridging plate was a reconstructed plate on the surface of the pelvic anterior ring. Its proximal part was located nearby right anterior superior iliac spine, and the distal portion was located at superior ramus of pubis beside the pubic symphysis. Three cortical screws with 3.5 mm diameter and 15-32 mm length were placed at the plate proximal and distal ends respectively.The screw of the anterior ring screw-rod system was 6.5 mm in diameter and 80 mm in length. It was inserted from the anterior inferior iliac spine and pointed to the posterior inferior iliac spine. Boolean operations were performed on the pelvic bone and internal fixators to form fixation models.Simulated fixation modes were as follows: right percutaneous anterior ring bridging plate (P), right percutaneous superior pubic intramedullary screw (S), percutaneous anterior ring screw-rod system (R), the combination of right percutaneous anterior ring bridging plate and percutaneous anterior ring screw-rod system (P + R), the combination of right percutaneous superior pubic intramedullary screw and percutaneous anterior ring screw-rod system (S + R). Table  Figs. 1, 4 and 5Tie constraints were applied between the bone-implant interfaces except for the cannulated screw stem regions where the frictionless sliding contact was applied. Penalty contact with a friction coefficient of 0.3 was applied between the interaction surfaces of fractures. The longitudinal downward load, anteroposterior load and transverse load were simulated to imposed to pelvis respectively, and all of them were 600 N.Under three different loads, the displacements of the virtual right hip midpoint in vertical, transverse and anteroposterior directions were taken as the evaluation index of stability. The smaller the displacement, the better the stability.In the simulated bipedal standing situation, there was no significant difference among the vertical, transverse and anteroposterior displacements of the virtual hip midpoint in the models of bridging plate, superior pubic intramedullary screw, anterior ring screw-rod system and their combined fixations. Similarly, the results of the simulation of standing on 1 foot were almost the same as those of the simulation of standing on 2 feet mentioned above.Under the simulated anteroposterior loading condition, there were obvious differences among the virtual hip midpoint’s displacements in anteroposterior, transverse and vertical directions in three models of pelvic anterior ring single fixation, in which bridging plate was the largest, anterior ring screw-rod system was the second, and the intramedullary screw was the smallest. Comparing the models of single with combined fixations, it demonstrated that the assistant of the anterior ring screw-rod system significantly reduced the displacement of three directions in single bridging plate fixation and sole superior pubic intramedullary screw fixation, and the displacement of the model of superior pubic intramedullary screw combined with anterior ring screw-rod system was the smallest.Under the simulated left-right loading condition, there were obvious differences among the virtual hip midpoint’s displacements in anteroposterior, transverse and vertical directions in three models of pelvic anterior ring single fixation, in which bridging plate was the largest, and anterior ring screw-rod system was the second, and intramedullary screw was the smallest. Comparing the models of single and combined fixations, it illustrated that the assistant of the anterior ring screw-rod system significantly reduced the displacements of three directions in single bridging plate fixation and the displacements of left-right and anteroposterior directions in single intramedullary screw fixation, however there is no effect on the displacement of vertical direction in single intramedullary screw fixation. The displacement in the model of the combination of superior pubic intramedullary screw and anterior ring screw-rod system was the smallest.Generally speaking, under simulated anteroposterior and transverse loading conditions, the multidirectional displacements in all models were ranked from small to large as follows: the combination of superior pubic intramedullary screw and anterior ring screw-rod system, superior pubic intramedullary screw, the combination of bridging plate and anterior ring screw-rod system, anterior ring screw-rod system and pelvic anterior bridging plate. Tables , 3 and 4Antegrade and retrograde percutaneous pubic intramedullary screw technique avoids extensive exposure during operation via ilioinguinal approach etc., and achieves ideal clinical results. However, because of the risk of bladder injury, iliac artery and vein injury, and hip joint involvement, the cannulated screw is a relatively safe choice –7. If thCompared with conventional plate internal fixation, anterior ring bridging plate has the advantage of minimally invasive percutaneous implantation, which effectively avoids the pain of surgical site and incision complications,  and signAs an ‘inbuilt external fixator’, the pelvic anterior screw-rod system creates the principle of fixation above the acetabulum in order to stabilize the anterior pelvic ring by minimal screw and more minimally invasive means and to help fracture reduction by lateral compression and stretching. Although a biomechanical study concluded that the anterior subcutaneous pelvic ring fixator had no biomechanical advantage over the traditional external pelvic fixation,  the fixaThus, three kinds of pelvic anterior ring minimally invasive internal fixation methods have their own pros and cons. Further studies are needed about how to choose an effective and safe fixation method according to the specific traumatic condition, available hardware and technical level, and maximize the biomechanical advantages of various anterior ring internal fixations, so as to achieve the best effect of minimally invasive internal fixation, which helps to define the respective indications of the anterior ring minimally invasive internal fixation methods, pre-plan the operation pertinently and effectively shorten the preoperative preparation and operation time.In this study, it was found that under the simulated bipedal standing state, there were no significant differences in the vertical, bilateral and anteroposterior stabilities of the fractures between the models of the percutaneous superior pubic intramedullary screw, percutaneous bridging plate and percutaneous anterior ring screw-rod system and their combined fixations. Then, the state of standing on a single foot was simulated, and the results were similar to those of the state of standing on two feet. This suggested that the anterior pelvic ring contributed little to the overall vertical stability of the pelvis. Therefore, backward loads and right-and-left loads were applied to the pelvis to simulate the mechanism of open-book and lateral injuries. By comparing the displacements of the virtual acetabular midpoint in vertical, transverse and anteroposterior directions, the translation and rotation stabilities of three kinds of anterior ring fixations and their combined fixations on coronal, sagittal and horizontal planes were determined. The results show a significant consistency. Comparing the multidirectional stabilities of three kinds of anterior ring internal fixators’ independent fixation, the percutaneous superior pubic intramedullary screw is the best, the percutaneous anterior ring screw-rod system is the second, and the percutaneous bridging plate is the worst, which shows the significant mechanical advantage of intramedullary centraxonial fixation. Although the fixation effect of anterior ring screw rod system is affected by indirect fixation across pubic symphysis, it still shows better mechanical characteristics than bridging plate with eccentric fixation. Considering the material consistency of the three internal fixators, it can not be excluded that this result may be related to the bridging plate’s ‘congenital deficiency’ of thin and flat plate and short screws.In order to maximize the effect of internal fixation, it was planned to combine three kinds of anterior ring minimally invasive internal fixation devices in pairs. However, the superior pubic intramedullary screw and bridging plate could not be fixed simultaneously because of the limitation of anterior ring anatomy, so only the anterior ring screw-rod system was combined with the intramedullary screw and bridging plate respectively. The experiment shows that, compared with the independent fixation modes, the combined anterior ring internal fixation modes show obvious mechanical advantages, among which the combination of the superior pubic intramedullary screw and anterior ring screw-rod system achieves the most stable fixation effect. Although the combination of bridging plate and anterior ring screw-rod system is superior to their independent fixation, it is still inferior to the single superior pubic intramedullary screw. This suggests that in the clinical practice of fixing unilateral pubic fracture, superior pubic intramedullary fixation should be preferred as far as possible. In order to minimize the failure risk of internal fixation, it is suggested to combine superior pubic intramedullary fixation with anterior ring screw-rod system fixation.However, it is undeniable that in some cases, smaller pubic diameter, a variation of pubic curvature or non anatomical reduction of pubic fractures result in the unavailability of 7.3 mm and 6.5 mm screws, so 4.5 mm or 3.5 mm diameter screws are also used by some orthopedic surgeons. As mentioned above, it was reported that the risk of cannulated screw breakage increased when the screw’s diameter was less than 6.5 m m. In ordeFor unilateral anterior ring injury of type pelvic fracture, in general, there are obvious consistent differences in transverse, vertical and anteroposterior stabilities of the three internal fixations and their combinations; In terms of stability, the combination of percutaneous superior pubic intramedullary screw and percutaneous anterior ring screw-rod system is the best, percutaneous superior pubic intramedullary screw ranks second, percutaneous bridging plate with percutaneous anterior ring screw-rod system is the third, percutaneous anterior ring screw-rod system is the fourth, and percutaneous bridging plate is the worst; Percutaneous anterior ring screw-rod system can significantly increase the stability of percutaneous bridging plate fixation and superior pubic intramedullary screw fixation.


Understanding the ice recrystallisation inhibition (IRI) activity of antifreeze biomimetics is crucial to the development of the next generation of cryoprotectants. In this work, we bring together molecular dynamics simulations and quantitative experimental measurements to unravel the microscopic origins of the IRI activity of poly(vinyl)alcohol (PVA)—the most potent of biomimetic IRI agents. Contrary to the emerging consensus, we find that PVA does not require a “lattice matching” to ice in order to display IRI activity: instead, it is the effective volume of PVA and its contact area with the ice surface which dictates its IRI strength. We also find that entropic contributions may play a role in the ice-PVA interaction and we demonstrate that small block co-polymers (up to now thought to be IRI-inactive) might display significant IRI potential. This work clarifies the atomistic details of the IRI activity of PVA and provides novel guidelines for the rational design of cryoprotectants. Understanding ice re-crystallization is key to improve the current cryopreservation technologies. Here, the authors bring together experiments and simulations to unravel the atomistic details of the ice re-crystallization inhibition (IRI) activity of poly(vinyl)alcohol—the most potent biomimetic IRI agent. IR is a form of Ostwald ripening3, where larger ice crystals are formed at the expense of smaller ones and typically occurs during the thawing process of a frozen substance. The damage arises because the formation of the larger ice crystals causes osmotic stress and can rupture and pierce cells, resulting in cell death4. IR damage therefore poses a threat to the viable recovery of cryobiologically stored material, leading to the waste of precious resources (e.g. cells and tissue samples) used in regenerative medicine7. IR damage can be mitigated by the use of cryoprotectants, but their usage suffers from extensive work protocols and potential cytotoxicity10. Consequently, a substantial body of work17 has been devoted to finding alternative cryoprotectants that are biocompatible, relatively straightforward to produce and which display exceptional ice recrystallisation inhibition (IRI) activity at comparatively low concentrations.Being able to understand and control ice recrystallisation (IR) is of fundamental importance for a wide range of applications21. PVA has been found to be capable of protecting proteins during freezing, by preventing irreversible protein aggregation on growing ice crystals, as well as enabling solvent-free cryopreservation of bacteria12. Similarly, PVA has been shown to mitigate IR during cryopreservation and enables some increase in post-thaw recovery for mammalian cells7. Thus, understanding the underlying mechanism by which PVA confers IRI activity is key for the development, refining and optimisation of the next-generation cryoprotectants that can be fine-tuned for a diverse range of applications .The main obstacle to the rational design of new IRI-active compounds is our patchy understanding of their structure-function relation: in fact, even in the case of prototypical IRI agents such as the synthetic polymer poly(vinyl)alcohol (PVA), there is much speculation about the molecular-level interactions between PVA and ice22 have suggested that the IRI activity of PVA might be due to the “distance matching” between the hydroxyl groups of the polymer and water molecules at the ice surface, in which PVA would require to adopt a linear/extended conformation to achieve such a distance match. In a similar vein, computational work by Weng et al.23 indicated that PVA did not have to bind to ice in a linear conformation via a geometric lattice matching per se: rather, the study showed a stereoscopic match of hydroxyls to water molecules in the ice lattice which then facilitated binding and IRI activity. The IRI properties of PVA seem to be closely linked to its degree of polymerisation (DP), i.e., the number of monomeric units in a polymer chain, with shorter chain PVA (DP = 10) showing a significant reduction in activity compared to PVA with DP = 1920. In fact, PVA is thought to be significantly IRI active only for DP values greater or equal than this critical threshold of DP = 19. Recent simulations24 have rationalised this in terms of a size dependence of the ice-binding efficiency of PVA, showing that short-chain polymers are less likely to bind to ice and that the polymer-ice hydrogen bond life-times for small polymers (DP < 10) is rather short compared to the time taken for ice to grow. Thus, while the microscopic motivations at the heart of the IRI activity of PVA are still hotly debated, it would seem there is a growing consensus that a minimum number of hydroxyl groups is required for PVA to bind ice long enough to stop the growth of an advancing ice front.Recent molecular dynamics simulations25, we show that PVA can bind ice in any conformation and that lattice matching is not a prerequisite for ice inhibition. We also show that short-chain PVA10 has reduced IRI activity not because it cannot bind ice sufficiently well, but rather because it becomes overgrown by the ice front and that sustained IRI activity is dependent on the volume occupied by a polymer at the ice interface. We also show that both enthalpic and entropic contributions may play a part in PVA-ice interactions. Lastly, we explore the role small chain block copolymers such as PVA-b-PVAm  could have in designing IRI active polymers with DP as small as 10, which has been previously thought of as unattainable.In this work, we use molecular dynamic (MD) simulations to investigate the IRI activity of PVA by systematically probing the impact of conformation, number of functional groups and degree of polymerisation  to extended (Rg = 0.95 nm). The free energy barriers separating the minima are of the same order of thermal fluctuations at room temperature (kBT ~ 25 meV). In addition, the most favourable conformation (Rg = 0.74 nm) corresponds to a random coil, which is consistent with what we observe in our unbiased simulations of PVA20 at 265 K.In order to understand the impact of the PVA conformation on its ice-binding activity, we performed well-tempered metadynamics simulations (see Methods section) using the radius of gyration see Fig. ; we find26 and theoretical works25 have suggested that a linear/extended conformation is crucial for the polymer to adopt in order to bind ice and display IRI activity. The rational for this argument is based on a “lattice matching” of the hydrogen bonding between the hydroxyl functional groups of PVA to the water molecules of the ice crystal. Recent computational work22 on PVA has suggested a specific 2:1 binding pattern: two adjacent hydroxyls groups bind to ice, followed by an unbound group; the distance between the two binding hydroxyls is comparable to the nearest oxygen–oxygen distance within the primary prismatic ice surface. Other recent simulations23 have suggested that the binding between PVA and ice is based on the stereoscopic matching of the hydroxyl groups to the underlying ice lattice. Our metadynamics simulations suggest however that given the very flexible nature of PVA, the probability that this polymer binds to an advancing ice front by adopting a linear/extended conformation is vanishingly small. Furthermore, the lattice matching argument assumes the growing ice front to be a smooth surface: while relatively smooth ice surfaces can develop via a spiral growth regime based on screw dislocations, this scenario is characteristic of much stronger supercooling than those considered here – and indeed is not thought to be relevant in cryopreservation conditions, see e.g. refs. 29. In contrast, when dealing with ice-water interfaces characterised by some degree of roughness (as considered in this work), the probability of PVA binding thanks to specific structural patterns consistent with the ice lattice is even further reduced.The role of PVA conformation has been a central argument surrounding the mechanistic details of its IRI activity. In particular, previous experimental20 where we have either let the polymer free to adopt any conformation or we have restrained it by applying a bias to its radius of gyration so as to enforce a specific orientation of the polymer with respect to the ice surface. We discuss in the Methods section the computational setup as well as the details of the order parameters we have used to identify ice-like molecules. These simulations start with PVA being in solution which is in contact with a seeded ice crystal  as well, as illustrated in Supplementary Fig. 22 put forward a 2:1 binding pattern for PVA on ice which we do not observe in our simulations. This contradiction is likely due to the fact that the investigation reported in Naullage et al.22: (i) assumed that PVA behaves like a linear molecule when biding to ice, which appears not to be the case at the supercooling investigated here; (ii) utilised the mW30 and a united-atom force field31 to model water molecules and PVA, respectively. The coarse-grained nature of these force fields is unlikely to capture the complexity of hydrogen bonding at heterogeneous interfaces such as the PVA-ice one. We use a fully atomistic force field instead, as discussed in the Methods section; (iii) the distance between the two binding hydroxyls characterising this 2:1 pattern was thought to be comparable to the nearest oxygen–oxygen distance characterising the pristine crystalline structure of the primary prismatic ice surface. As no simulations of the actual growth of the ice front were included (i.e. PVA bound on a static ice surface only) the roughness of the growing ice front was not taken into account. In contrast, in this work we have considered the PVA interactions with a dynamically growing ice front.These results hold for MD simulations of unrestrained PVA10 along with two identical PVA10 polymers within the same volume of water  have shown that a 1-mg/ml solution of PVA10 lowers the mean largest grain size (MLGS) of ice crystals by ~20% if compared to a phosphate-buffered saline (PBS) control. In other words, PVA10 displays some IRI activity: however, the same concentration of PVA19 reduces the MLGS by ~80% – a much stronger impact on ice growth. The much weaker IRI activity of PVA10 has often been attributed to the polymer not being able to bind ice well enough (or for a prolonged period of time) due to relatively low number of hydroxyl groups available to bind ice. To be specific, it has been suggested in the past that at least 6–8 units of PVA are needed for it to bind to ice26 and recent computational work24 has shown that there is a size dependence on the ice-binding efficiency of PVA – or, along the same lines, that the number of functional groups present on the chain dictates the degree of IRI activity 23. Most recently, it has been proposed that PVA10 might bind reversibly to ice, but lacks any IRI activity because the time bound to ice is shorter than the time it takes for ice to grow: thus the longer the PVA chain, the greater the chance of it to staying bound to ice and hence displaying IRI activity.24.In order to explore the relationship between the chain length of the polymer and its IRI activity we have also simulated PVA10 has reduced IRI activity, here we show that this is due to the polymer becoming overgrown once bound to ice, rather than not being able to bind for a sufficiently long enough time. Similar results have been obtained for PVA10 by Weng et al.23. In fact, out of the twenty MD simulations we have performed, PVA10 bound to ice in nineteen of them: a representative example is illustrated in the left panel of Fig. 20, PVA10 is quickly overgrown, as shown in the middle panel of Fig. 10 gets entirely embedded in the growing ice front within ~100 ns . Importantly, these molecular-level details translate into a rather stark difference observed, experimentally, between the IRI activity of PVA10 and PVA20 – as shown in the right panels of Fig. 20 or PVA10: note the significant reduction of the MGS caused by PVA20 in comparison to the much larger ice crystals observed for PVA10.Further insight into the weaker IRI activity of PVA10 displays comparable IRI activity to that of PVA20 (i.e. those lines not highlighted blue). This is consistent with experimental evidence20 showing that PVA10 does display limited IRI activity: for instance, PVA10 in concentrations between 2 and 5 mg/ml yields a MGS reduced by ~20–40% compared to PBS control. Thus, we argue that the IRI activity of PVA10 is largely affected by the kinetics of ice growth, i.e., the time period during which PVA10 manages to hold back the ice front before becoming engulfed in the ice phase might result in some IRI activity observed experimentally – but due to its eventual engulfment in ice its activity will be much weaker compared to PVA20.We do observe, however, situations in which PVA26. As PVA tends to be found in the form of a random coil, the number of the monomers interacting with the ice surface does not increase linearly with the polymer length. Indeed, the binding affinities of PVA10 and PVA20, albeit difficult to quantify when dealing with dynamically growing ice surfaces, appear to be very similar.In light of these results, it is important to clarify the role of the “binding affinity” of PVA with respect to ice, which is often thought of as a linear function of the number of monomers10 and the minimum distance (rMI hereafter) between the periodic images of the polymer. Specifically, PVA10 polymers which became overgrown by the ice front are characterised, on average, by rMI = 3.7 ± 0.30 nm, while PVA10 polymers which showed some extent of IRI activity are characterised by an average value of 3.5 ± 0.30 nm. This trend holds for different surface areas as well; in particular, we have obtained, in the case of smaller simulation box . We have found that the growth of the ice front is largely unhindered by the two short-chain polymers, similarly to what we observe when considering a single chain of PVA10  shows the very same IRI activity as that of a single PVA10 interacting with an ice surface of ~13 nm2 , followed by PVA20 polymers free to adopt any conformation – and thus on average found to be as random coils; note the substantial spread of volumes, indicative of the variety of conformation that “unrestrained” PVA can assume when binding to ice. At lower volumes we find the “linearly restrained” polymers, i.e. extended, rod-like conformations that we force to be either perpendicular or parallel to the growing ice front  compared to any PVA20 conformations. The random coil, compact and linear perp. conformations display the stronger IRI activity. On the other hand, the lower volume occupied by PVA10 at the ice-water interface translates in a much weaker IRI activity. In addition, the linearly restrained PVA20 conformations show another interesting trend: while linear perp. and para. are characterised by very similar occupied volumes, linear para. is much more likely to become overgrown by the ice phase, as illustrated in Fig. Instead, we argue that it is the ice-PVA contact area, as well as the volume occupied by a bound PVA on ice, that rules the strength of its IRI activity. This proposition is illustrated in Fig. 10 or, equivalently, specific orientations of the polymer with respect to the ice surface (e.g. linear para. conformation), create “defects” in the ice phase which in some cases are clearly not extended enough to prevent the ice from growing further and eventually engulfing the polymer. , 26.1 nm2 , 53.4 nm2 (size M/L) and 124.1 nm2 (size L). The results are summarised in Supplementary Fig. 20 is IRI active for surface coverages of 0.04 polymers/nm2 or higher. In addition, the IRI activity of both PVA20 and PVA10 increases in the case of size S (compared to size M), albeit PVA20 remain much more effective than PVA10 even in this regime.Finally, we also note that the activity of any given IRI agent strongly depends on its coverage of the ice surface: in this case, lower numbers of PVA polymers per (ice) unit area would lead to a lower IRI activity. This is the reason why, experimentally, a certain IRI agent displays activity above a certain concentration but it is not straightforward to translate (bulk) IRI agent concentration to the effective ice surface coverage. For completeness, we have investigated the IRI activity of both PVA20. The system without PVA is used as a control: the advancement of the ice front ; note the sharp decrease in the growth rate due to the binding of PVA20 to ice (shown in blue).It is clear from our simulations that the IRI activity of PVA relies on its ability of binding to ice. Here, we investigate the driving forces behind the PVA-ice interactions. To begin with, Fig. 22 has suggested that the PVA-ice-binding arises from a linear, extended arrangement of the polymer which should be lying flat on top of the ice: if we take into account the 2:1 binding pattern also discussed in Naullage et al.22, we should observe, on average, ~70% of hydroxyl groups forming hydrogen bonds to ice. However, our results . Thus, we have investigated the desolvation of the methylene (-CH2) hydrophobic groups of PVA, which might lead to an entropic contribution in addition to the enthalpic gain originating from the PVA-ice hydrogen bonding. In solution, the first solvation shell of the methylene groups contains on average five water molecules , via MD simulations as well as splat-assay measurements, summarised in Fig. To probe this possibility in greater detail, we have investigated the IRI activity of polypropylene (PP), an entirely hydrophobic polymer. While PP is in reality insoluble in water, this computational experiment allows us to probe the potential effect of the desolvation of methylene groups in absence of hydrogen bonding. We find that PP does indeed interact with the growing ice front via purely hydrophobic interactions originating from the desolvation of methylene groups  and one with DP = 10 (PVA5-b-PVAm5). As illustrated in Fig. It is interesting to observe what happens when only a fraction of the PVA’s -OH groups are substituted with -NH38 on PVA-b-PVP block copolymers has shown that the addition of PVP (polyvinylpyrrolidone) had little influence on the IRI activity, with the block polymers presenting roughly the same activity levels as the PVA homopolymers. In particular, a range of PVA10-b-PVPn block copolymers were examined where the MLGS of PVA10 homopolymer was roughly the same as that of the block copolymers. Outside the realms of synthetic polymers, it has also been shown39 that a particular 7 kDa anti-freeze protein (AFP) has identical IRI activity to a 7 kDa AFP with a further 42 kDA protein appended to it. Hence, both studies suggest no increase in activity by appending further monomeric units or protein segments to the initial starting structure.Previous experimental work40, who showed that even a low dispersity mixture (with a dispersity index of e.g. 1.2), obtained via RAFT polymerisation, can contain small amounts of high molecular weight polymers which dominate the IRI activity, thus yielding false positives in terms of the IRI activity of low molecular weight PVA. Ultra-low dispersity indexes (as low as 1.005) could be obtained via column chromatography: the resulting polymers indicate that a DP = 12 represents the minimum value required to observe a significant IRI activity – albeit at much higher concentrations than PVA20, i.e., to observe similar MLGS for 5 mg/ml of PVA20, 25 mg/ml of PVA12 would be needed.At this stage we would like to point out that, as opposed to the previous experimental works discussed above, here we are not adding extra units to the polymer: rather, we wish to assess whether the IRI activity of block copolymers (with DP of either 10 or 20) can be affected by substituting some of their hydrogen bonding functional groups with -NH5-b-PVAm5  is capable of slowing down the growth of the ice front as shown in Supplementary Fig. 10. The non-negligible IRI activity of PVA5-b-PVAm5 compared to PVA10 originates from the fact that the primary protonated amines such as the NH5-b-PVAm5 form fewer hydrogen bonds compared to hydroxyl groups (as discussed above in the case of PVAm). As such, it is much more difficult for the growing ice front to form hydrogen bonds with the PVAm section of the block copolymer, see Fig. In contrast, our results indicate that even the short block copolymer PVAAm5 DP = , <12 is 13-b-PVAm7 copolymer (which has the same DP as PVA20) displays similar IRI activity compared to the unrestrained PVA20 polymer  lowers the probability of observing potential side effects associated with supercooling beyond the thermal hysteresis gap, such as dynamic ice shaping (see refs. 41). We do however, note that 3/10 simulations for PVA5-b-PVAm15 and 2/10 simulations for PVA5-b-PVAm5 showed no binding activity, whereas the copolymers with 13 units of PVA (PVA13-b-PVAm7) bound to ice in all simulations constrained MD simulations. This is a very important result, as it removes a long-standing design constraint, that of lattice matching, from the rational design of IRI active polymers. It is important to stress that lattice matching might be playing an important role for different classes of polymers and/or different types of IRI agents. In particular, the recent body of work on ice-binding proteins10 compared to PVA20) is due to the shorter polymer becoming overgrown by the advancing ice front, rather than – as previously thought – the limited number of hydroxyl groups binding to ice. In fact, PVA10 does bind to ice and we have established that the IRI activity of PVA10 is lower than that of PVA20 because the former occupies a much smaller effective volume/surface than the latter at the ice-water interface. These results are particularly interesting as they provide an explanation for the lower – and yet non-negligible – IRI activity of PVA10 compared to longer polymers.Furthermore, we have showed that the weaker IRI activity of short polymers  have the potential to exhibit significant IRI activity. This finding is of great relevance in the context of the rational design of novel cryoprotective agents, as it shows that it is possible to synthesise very short (DP < 20) IRI active polymers. From a practical standpoint, this is especially intriguing, as smaller cryoprotectants are less likely to cause dynamic ice shaping – a potentially lethal occurrence which can exacerbate the problem of cryopreservation.Lastly, we have probed the IRI activity of PVA-We believe these findings are not only of great relevance to cryobiology and medical applications, but they are bound to make an impact on many other areas where ice recrystallisation plays a role, such as the development of freeze resistant crops and the food industry – as well as furthering our fundamental knowledge within the realms of atmospheric sciences and climate change. We are also confident that our results will foster future experimental and computational works aimed at probing the IRI activity of more complex copolymers, thus laying the foundations toward the rational design of the next generation of cryoprotectants.43 along with the TIP4P/Ice water model44, using the MD package GROMACS 5.1.345. There is a large body of evidence indicating that the CHARMM36 - TIP4P/Ice combination of force fields is especially well suited to perform MD simulations of biomolecules in supercooled water (and in contact with ice)48. In our simulations, we have investigated the atactic poly(vinyl)alcohol (PVA) polymer with various degree of polymerisation  and conformational flexibility. We have found that the tacticity of PVA does not have an impact on our results. We have also simulated a range of polyvinylalcohol/polyvinylamine block copolymers (PVA-b-PVAm) characterised by different ratios of hydroxyl to amine functionalisation.All the molecular dynamics simulations reported in this work have been preformed using the all-atomistic CHARMM36 forcefield20, PVA10 and 2xPVA10 polymers  and equilibrated for 30 ns at room temperature and ambient pressure within the NPT ensemble: to this end, we have employed the Bussi-Donadio-Parrinello thermostat50 and the Berendsen barostat51, with coupling constants of 0.5 and 4 ps, respectively. Periodic boundary conditions (PBCs) were applied in three dimensions and the integration time-step for the leap-frog algorithm was set to 2 fs. In parallel, a separate ice/water box was created, starting by preparing an ice crystal (of dimensions 4.7 × 5.9 × 3.9 nm) cleaved so as to have its primary prismatic face lying in the xy-plane of the simulation box: this crystal acted as the seed for the ice phase to grow along the ±z direction  demonstrates that PVA chiefly interacts with the primary prismatic face of hexagonal ice. Thus, in this work we have chosen to focus on this particular surface, in line with previous computational work23.To begin with, the geometry of a given polymer was optimised using a steepest descent algorithm52 positioned at the top and bottom of the box. This strategy enabled us to use the Yeh and Berkowitz correction term53 to the Ewald summation: in turn, this approach mitigates any potential artefacts due to the treatment of electrostatic interactions when dealing with a slab geometry54. The cutoff for the van der Waals and electrostatic interactions was set to 12 Å and 10 Å, respectively: a switching function was used to bring the van der Waals interaction to zero at 12 Å. The geometry of the water molecules was constrained using the SETTLE algorithm55 while the P-LINCS algorithm56 was used to constrain the hydrogen bonds for the polymers at their equilibrium value. This setup has been extensively validated in some of our previous works, see e.g. Sosso et al.48. The ice/water system was subsequently equilibrated  at 300 K for 50 ns. Once the polymer and the ice/water system were independently equilibrated, two polymers were then placed into the ice/water box within the two water slabs, one above and one below, as per Fig. 57 with a coupling constant of 4 ps. The starting configurations of PVA in the water slab depended on the last frame in the equilibration run and as such all trajectories had independent starting configurations. To prevent the polymers interacting with the water/vacuum boundary, a constraint for the centre of mass of the polymer was set to be at least 1 nm below the water-vacuum boundary layer. Configurations were collected every 2000 MD steps.The oxygen atoms within the ice slab were subjected to position restraints via a harmonic potential characterised by a spring constant of 10,000 kJ/mol; the system was then solvated, adding two layers (each one ~11 nm thick) of water below and above – a total of 90,818 atoms. The simulation box was elongated along the z direction to include ~22.5 nm of vacuum: at this point we switched to 2D (xy) PBCs in conjunction with 9-3 Lennard-Jones “walls”58: as such, we have conducted a systematic investigation, discussed in detail in the Supplementary Information  as:rij is the distance vector between the ith and jth atom, Ym6, is a spherical harmonic of order {6, m} and σ is a switching function which determines the extent of the of coordination shell. Then, we combine these Steinhardt vectors to obtain the following order parameter:59, we identify the largest connected cluster of water molecules which oxygen atoms display a value of s6(i) greater than a certain threshold . Thus, the result of this procedure provides the number of water molecules found within the largest ice cluster per trajectory frame, which invariably will be the seeded ice crystal that grows over time. Concerning the analysis of hydrogen bonds, we have used a geometric criterion based on an acceptor-donor bond distance of <0.3 nm and a donor-hydrogen-acceptor angle between 160∘ and 200∘. The analysis on the solvation shell around the methylene groups was based on the first minimum of the radial distribution function (g(r) see Supplementary Fig. s6 parameter described above we were able to distinguish which water molecules surrounding the methylene groups can be classified as either ice-like or liquid-like.To determine whether a certain water molecule belongs to the ice crystal or the supercooled liquid phase, we have employed a strategy based on the Steinhardt order parameters61, an enhanced sampling technique which forces the system to explore the whole free energy surface relative to a particular set of collective variables (CVs). This is achieved by depositing Gaussian potentials V at a defined time interval, t:s is a given CV, τ is the Gaussian deposition stride and σ the width of the Gaussian for the ith CV and W(kτ) the height of said Gaussian potential. We have used PLUMED63 (version 2.4.2) to perform metadynamics simulations. We have chosen the radius of gyration (Rg) as our CV to bias: the width, height and deposition stride of the Gaussian potentials has been set  to σ = 0.02 nm, W = 0.6 kJ/mol, and 500 steps, respectively. The bias factor was set to γ = 100. The uncertainty associated with our estimate of the free energy profile reported in, e.g., Fig. 64. We have also used PLUMED to perform the MD simulations where the polymers conformation was restrained  was enforced to fluctuate around values of 4.9 and 1.2 nm in the case of a compact and elongated conformation, respectively.To probe the conformational space of the PVA polymer we use well-tempered metadynamicsentclass1pt{minima65: by treating the atoms within the polymer as a set of points in the Euclidean space, the alphaShape function implemented in MATLAB66 identifies the smallest shape enclosing said atoms – which in turn gives us access to the volume of the molecule as a whole as well as the surface area of the polymer occupied on the ice slab (obtained by projecting the atomic positions of the polymer bound to the ice onto the xy plane). The key parameter in this procedure is the so-called alpha radius, which basically determines how loose or tight the fit of the alpha shape is around the set of points under investigations: we have set this value to 0.17 nm, but we note that different choices do not affect the qualitative trends we report in e.g. Fig. To measure the average volume occupied as well as the surface area for a given polymer we employed the concept of alpha shape67, and the area of the field of view divided by this number of crystals gave the average crystal size per wafer , and reported as a percentage of area compared to the PBS control. We note that along with MGS another quantity MLGS (mean largest grain size) can be computed which is the largest grain in the field of view and therefore slightly biased towards lower activity, however this quantity can be measured without image analysis software. For further details about the physical, analytical and synthesis methods, please refer to the Experimental Section in the SI.A 10 μL sample of polymer was dissolved in phosphate-buffered saline  and then dropped from 1.40 m onto a glass microscope cover slip, which was sat on top of an aluminium plate cooled to −78° using dry ice. The droplet froze instantly upon impact with the plate, spreading out and forming a thin wafer of ice. This wafer was then placed on a liquid nitrogen cooled cryostage held at −8°. The wafer was then left to anneal for 30 min at −8°. Next, the number of crystals in the image were counted using ImageJSupplementary InformationPeer Review File


A hybrid pedicle screw system for minimally invasive spinal fixation was developed based on the uniplanar pedicle screw construct and a new intermediate screw. Its biomechanical performance was evaluated using finite element (FE) analysis.  A T12-L2 FE model was established to simulate the L1 vertebral compression fracture with Magerl classification A1.2. Six fixation models were developed to simulate the posterior pedicle screw fracture fixation, which were divided into two subgroups with different construct configurations: (1) six-monoaxial/uniplanar/polyaxial pedicle screw constructs and (2) four-monoaxial/uniplanar/polyaxial pedicle screw constructs with the new intermediate screw. After model validation, flexion, extension, lateral bending, and axial rotation with 7.5 Nm moments and preloading of 500 N vertical compression were applied to the FE models to compare the biomechanical performances of the six fixation models with maximum von Mises stress, range of motion, and maximum displacement of the vertebra.  Under four loading scenarios, the maximum von Mises stresses were found to be at the roots of the upper or lower pedicle screws. In the cases of flexion, lateral bending, and axial rotation, the maximum von Mises stress of the uniplanar screw construct lay in between the monoaxial and polyaxial screw constructs in each subgroup. Considering lateral bending, the uniplanar screw construct enabled to lower the maximum von Mises stress than monoaxial and polyaxial pedicle screw constructs in each subgroup. Two subgroups showed comparable results of the maximum von Mises stress on the endplates, range of motion of T12-L1, and maximum displacement of T12 between the corresponding constructs with the new intermediate screw or not.  The observations shown in this study verified that the hybrid uniplanar pedicle screw system exhibited comparable biomechanical performance as compared with other posterior short-segment constructs. The potential advantage of this new fixation system may provide researchers and clinical practitioners an alternative for minimally invasive spinal fixation with vertebral augmentation. Spinal fixation with the posterior pedicle screw-rod system has been considered the mainstream treatment method for degenerative and traumatic spinal diseases. A number of in vitro, finite element (FE) and clinical studies have been performed to figure out the influencing factors for the fixation stability and modification or alternative methods to improve the biomechanical or functional outcomes, which included bone mechanical properties , disc arRegarding thoracic and lumbar vertebral fractures, minimally invasive short-segment pedicle screw instrumentation is an easy procedure and gaining momentum as a routine option. However, limited exposure increases the difficulty of coupling pedicle screws with rods, which will lead to a superficial position of longitudinal rods and decreased strength of the entire construct. Benefited by a decreased force arm, the polyaxial pedicle screw (PAPS) allows easier assembling pedicle screws with rods and provides theoretically better biomechanical strength. Nevertheless, prior studies demonstrated that PAPS tended to slippage through the universal joint, and its mechanical strength was always less than the monoaxial pedicle screw (MAPS) in the sagittal plane , 8. ThesAs the head of UPPS that can only swivel freely in one specific plane while fixed in other directions, the freedom of movement of the screw's head in the axial plane of body can facilitate the pedicle screw-rod assembly, without sacrificing the stiffness of the construct in the sagittal plane, whereas only two studies performed the biomechanical investigation of UPPS for spinal fixation to date, which verified its biomechanical advantage over PAPS in the sagittal plane , 8. MeanIn addition to these modifications to the instrument design, for most thoracolumbar bursts or compression fractures, vertebral augmentation has been used as a means to mechanically support the fractured vertebra anteriorly. Nonetheless, most clinical trials utilized one-above and one-below 4-MAPS short-segment construct, of which some reported excellent reduction and lordosis maintenance , 12, whiIn the present study, a hybrid UPPS fixation system was introduced. With this short-segment construct, a new intermediate screw (NIS) was developed to facilitate vertebroplasty and reduction maneuver. Different from the traditional parallel configuration of pedicle screws, this NIS is inserted in a more outside-in direction. This configuration allows bilateral intermediate screws to achieve the center of the injured vertebra, elevate the depressed endplate, and then maintain its reduction position more efficiently. To demonstrate the biomechanical performance of this hybrid UPPS system, an FEA was performed to compare the monoaxial, uniplanar, and polyaxial screw short-segment constructs with or without the NIS.The geometric 3D model of UPPS was drawn using the Solidworks software . The UPPS is barrel designed, with the outer and inner diameter of 6 mm and 4.1 mm, respectively. The screw-tulip joint of our UPPS allows about ±30° angular motion of the screw in the axial plane of the body (perpendicular to the plane in which the slot of the gasket is located), which is greater than Ye et al.'s UPPS of ±25° . During The geometry model of T12-L2 spine was reconstructed using Mimics software  based on the CT-scan data in Digital Imaging and Communications in Medicine format  of a 32-year-old healthy male without spine pathologies. The established model in Stereo-Lithography format was inputted into Geomagic Studio software  for reverse engineering reconstruction. After a series of image processing, a 3D model of T12-L2 in Initial Graphics Exchange Specification format was established. The measurement method and results of dimensions of the vertebra and intervertebral disc (IVD) were shown in The material properties of the T12-L2 FE model were assigned based on previous studies , 15 Tab. The corThe bone and intervertebral disc were meshed with Solid 187 . Due to Before FEA was performed on the spinal fixation model, the FE model of the intact spine was validated with range of motion (ROM) of T12-L1 and L1-L2, respectively. The inferior surface of the inferior vertebra was fixed in all directions. A reference point was created on the superior surface of the T12 and L1 vertebrae for force and moment application, respectively. Flexion, extension, right lateral bending, and right axial rotation with pure 7.5 Nm moment were applied to T12-L1 and L1-L2, respectively. The ROM of two segmental units was calculated at the endpoint of loading.Following the previously established methods , 17, 18,To analyze the biomechanical properties of these six fixation models, the moments of 7.5 Nm in each direction with preloading of vertical 500 N compression were applied to the reference point of the T12 vertebra . Under dThe ROMs of the intact T12-L1 model were shown in As shown in Disregarding the sporadic stress concentration point caused by the screw-tulip mobile joint of UPPS, which was out of the scope of this study , the maxUnder flexion loading, the maximum von Mises stresses were seen on the upper screws of the monoaxial and uniplanar screw constructs and the lower screws of the polyaxial screw constructs. Moreover, the maximum stress was the lowest in the 6-PAPS construct, followed by the 6-UPPS and 6-MAPS construct in sequence, and the NIS subgroup displayed the same varying rule.In the case of extension, the maximum von Mises stresses were shown on the lower screws in all six construct groups. The maximum stress was the lowest in the 6-MAPS construct, followed by the 6-UPPS and 6-PAPS construct in sequence, and the NIS subgroup displayed similar varying rule.For the condition of lateral bending, the maximum von Mises stresses were found at the lower screws in uniplanar and polyaxial screw constructs but the upper screws in monoaxial screw constructs. Besides, the varying rule was opposite to the flexion condition.The right axial rotation movement resulted in similar changing trends like the flexion loading, except for the pair of 4-PAPS/2-NIS and 6-PAPS. The possible reason might be that the fixed NIS altered the force-bearing condition of the polyaxial screw constructs and caused stress concentration.As known to all, lack of adequate anterior column support is an important reason for rekyphosis and early implant failure after the traditional posterior pedicle screw fixation . To incrThe intermediate screw has been verified to be an efficient reinforcer to the biomechanical stability of the whole construct. Baaj et al.  and MahaLi et al.'s FEA study compared the 6-MAPS versus 4-MAPS/2-NIS construct on the T11-L1 FE model and concluded that the maximum stress was located at the root of the upper pedicle screw under all loading conditions with 500 N compression and 6 Nm moments . Liao etUntil now, there were limited FEA studies on the UPPS system and hardly any intermediate screw used in the UPPS short-segment construct either. To facilitate vertebral augmentation, we designed a new intermediate screw with a new screw configuration. Our FEA results showed that the biomechanical performance of the UPPS short-segment construct with the NIS was acceptable by comparing with 6-UPPS or 4-MAPS/2-NIS. Our results were not entirely the same as the previous FEA studies because of inconsistent conditions and parameters. We did not see the maximum stress on the rod like Wang et al. . The posOne potential advantage of our new construct is that the smooth middle one-third shank of the NIS allowed increased ROM of the screw within the injured vertebra, which could conveniently elevate the endplate of the injured vertebra and strengthen the fracture fixation simultaneously. Another advantage is the combination of vertebroplasty and the intermediate screw. The conventional short-segment construct does not allow additional vertebroplasty without interfering its standard procedure. The current solutions include a short-segment construct with vertebroplasty but without the intermediate screw  or partiOur study had several limitations. First, although there existed several potential advantages, including vertebroplasty and the elevation and augmentation of the depressed endplate with the help of the NIS, we did not prove it in the present study due to the study design and space limitation. The application in minimally invasive spinal fixation with vertebral augmentation should be conducted in further simulated and clinical studies. Second, the FE model was based on a healthy male. How our system performs in the vertebral osteoporotic compression fracture is still needed to be determined. Third, our FE model was simplified without considering all the influencing factors. For example, the endplates were considered a part of the cortical bone rather than an independent structure. In vitro experiments on cadaveric specimen should be performed to verify the results achieved in the present study.The observations shown in this study verified that the developed hybrid uniplanar pedicle screw system exhibited comparable biomechanical performance than the current posterior short-segment constructs. Considering the biomechanical performance and superiority of vertebral augmentation and operation maneuvers, this new fixation system may provide researchers and clinical practitioners an important alternative for minimally invasive spinal fixation in patients with thoracolumbar vertebral compression fracture.