Vestibular rehabilitation therapy is an established treatment for patients with vestibular dysfunction. Virtual reality (VR) and augmented reality (AR) can be utilised in vestibular rehabilitation. Evidence of the efficacy of VR and AR delivered rehabilitation in patients with peripheral vestibular disorders is reviewed. MEDLINE, EMBASE, CENTRAL, CINAHL, PsychInfo, PsychBITE, OTSeeker, Ei Compendex, IEE, Clinical trials.gov and WebofScience databases were searched. Reduction in vestibular dysfunction symptoms 0–3 months post-intervention was the primary outcome. Secondary outcomes included long-term symptom improvement and side effects. Risk of bias assessment and meta analyses were planned. Five studies meeting eligibility criteria were included. Dizziness Handicap Inventory (DHI) scores 0–3 months post-intervention were reported by four studies. Meta-analysis identified a 1.13  standardized mean difference reduction in DHI in VR and AR treated patients compared to controls. Side effects reported by two studies were reduced by week four of VR intervention. Bias assessment identified DHI scores and side effects to be at high risk or of some concern. Adjunct VR interventions reduced patient DHI significantly more than vestibular rehabilitation alone 0–3 months post-intervention in adult patients diagnosed with unilateral vestibular disease. High quality studies are needed. In publicly funded healthcare systems 0.8–1.7% of general practitioner attendances are for symptoms of dizziness or vertigo3, 9–13% of whom are referred to other specialists such as neurologists, cardiologists and otolaryngologists4. Disorders of the vestibular system are identified in 50–65% of patients seen in specialist dizziness clinics6. The vestibular system consists of sensory organs, cortical and subcortical structures that contribute to balance alongside proprioception and vision. The vestibular apparatus of the inner ear is the primary input for the vestibular system and relays information on head position and motion to the midbrain. This sensory information leads to adjustments in body movements and posture to maintain balance7. The vestibular system can be affected by a variety of peripheral vestibular disorders (PVD), including benign paroxysmal positional vertigo (BPV), Menière’s disease (MD), vestibular neuritis (VN) and post-traumatic vestibular dysfunction. Chronic dizziness symptoms can lead to symptoms of anxiety and depression9. Dizziness and its sequelae can create a dizziness handicap for symptomatic patients and carries a $64,929 lifetime burden for affected older adults10.Dizziness is a common complaint affecting up to 23% of the population at any time in a first world setting11 Cochrane review concluded that there was moderate to strong evidence of its effectiveness in individuals with Unilateral Vestibular Hypofunction (UVH). Vestibular rehabilitation is an umbrella term that covers a range of exercise regimens from the generic such as Cooksey Cawthorne exercises to the customised11. The American Physical Therapy Association Clinical Practice Guidelines indicate that vestibular exercises are effective when compared to no or placebo exercises and that customized exercises are more effective than generic12. Vestibular rehabilitation can be done at home, in the clinic or in a combination of these settings. However, home exercises require the patient to be motivated to participate, which is an area where the playful activities utilized in virtual reality (VR) and augmented reality (AR) may add benefit. Seventy three percent of patients suffering from vestibular disease report more enjoyment and motivation with VR and AR interventions than vestibular rehabilitation13. Campbell et al.14 highlighted the importance of adherence to treatment in determining the benefit achieved through home based interventions aimed at preventing falls in the visually impaired. Interactive video gaming systems linked to an aerobic training regimen increase adherence and physical fitness attainment more than an aerobic training regimen alone15. Therefore, the use of VR/AR could improve vestibular rehabilitation compliance and overall outcomes17.Vestibular rehabilitation is the main therapeutic option for many patients with dizziness. McDonnell and Hillier's18. However, much of the current literature considers non-immersive commercially available video gaming systems (non-IGS) (ex. Wii Fit) to be VR. In an effort to focus on interventions that meet the true definition of virtual reality, non-IGS will be excluded from this systematic review. Immersive VR treats vestibular dysfunction by placing the subject in a simulated real world through two different strategies. One utilizes outpatient systems that use a head mounted display VR device and the second uses total body immersion inpatient systems such as the Immersive Rehabilitation Exercise System (Gesturetek Heath)19. In contrast to VR, AR augments the real-world environment instead of replacing it. Augmented reality adds to the subject’s real world sensory input through computer-generated sound, text and graphics that are projected onto the user’s natural visual and auditory fields20. Augmented reality platforms have been used to treat vestibular disorders using AR eyewear21.Virtual reality and AR are two ways of delivering potential reality type vestibular rehabilitative interventions. Virtual reality is defined as the immersion of the user in an interactive environment that mimics reality23. Virtual reality has been studied as a rehabilitation intervention and been found to elicit improvements in a variety of PVDs24. VR delivered vestibular rehabilitation has to the best of our knowledge not been assessed in current guidelines on the management of patients with vestibular disorders possibly due to its relative novelty. Furthermore, the previously published systematic review on the efficacy of VR in vestibular rehabilitation was inconclusive25. We aim to synthesize the evidence to test the hypothesis that patients suffering from a PVD who have received VR or AR vestibular rehabilitation interventions experience the same improvement in dizziness as patients who received vestibular rehabilitation alone or a comparable control treatment.Virtual reality’s utility in the treatment of vestibular dysfunction lies in the possibility of it achieving improved habituation, substitution, and adaptation through a more motivated vestibular rehabilitationThe primary objective was to assess the benefits of VR and AR interventions for vestibular dysfunction symptoms  in patients suffering from PVD within 3 months of treatment. Secondary objectives include the long term (> 3 months) benefits, post-intervention change in patient quality of life, determining the adverse events (harms) associated with use of VR or AR interventions and determining the number of patients who failed to complete treatment.26.This study is registered on the International prospective register of systematic reviews (PROSPERO) (CRD42020184674)Randomized controlled trials (RCT) focusing on VR or AR vestibular rehabilitation interventions were gathered from MEDLINE, EMBASE, CENTRAL, CINAHL, PsychInfo, PsychBITE, OTSeeker, Ei Compendex, IEE, Clinical trials.gov and WebofScience Fig. . The MEDTwo investigators (AH and MA) screened papers from these selected databases based on their title, abstract, and content. Shortlisted papers were identified based on exclusion and inclusion criteria. Studies were excluded if they were animal studies, review articles, non-RCTs, quasi-controlled trials, case–control studies, cohort studies, cross-sectional studies, case reports, case series, letters, abstracts alone, or books. Patients needed to be diagnosed with a PVD. No restrictions were placed on study subjects age or sex; publication date or language except that the abstract and title must be in English. Only studies of VR or AR experimental interventions alone, or in combination with vestibular rehabilitation or diet were included. Control interventions included no treatment, medications and diet, diet alone, medications alone, placebo and medications, placebo alone, surgery alone, vestibular rehabilitation alone, or vestibular rehabilitation in combination with placebo, medications, diet, medications and diet, or surgery. Disagreements between AH and MA on study inclusion were arbitrated by the senior investigator (DN). If multiple reports on the same study were identified they were synthesized into one study report to reduce bias.AH and MA independently extracted data using the Cochrane Data Collection Form. Trial characteristics extracted included objective(s), design details, study funding sources and possible conflicts of interest. Participant data extracted included study sample demographics, setting, method of recruitment, number of subjects invited to participate, number of subjects randomized, number of missing and moved patients, and subgroups measured and reported. The extracted intervention data included description of interventions and control groups, resource requirements and compliance. Outcome data and analysis included measurement tools, imputation of missing data, power, intervention and control results, unit of analysis, statistical methods used and primary and secondary outcome measures. If multiple symptomatology scales were used, data from the most standardized scale was used. Discrepancies in data extraction were settled by consensus, or by the senior author if a consensus could not be reached.27. It includes bias from the randomization process, intervention deviations, missing outcome data, outcome measurement and selection of reported results. Each of the study outcomes of interest  were independently RoB assessed according to Cochrane RoB2 tool recommendations. This was done independently by two investigators and disagreements were settled by the senior author. A study’s overall RoB was determined by the RoB in each domain. An overall low RoB requires that there is a low RoB in all domains. Some concerns of bias required there to not be a high RoB in any domain and for there to be at least one domain with some concerns of bias. Lastly, a study is judged to have an overall high RoB when the study is judged to have some concerns of bias in multiple domains or the study is judged to be at high RoB in at least one domain.Following data extraction each included RCT was assessed for their risk of bias (RoB). The RoB was assessed using version 2 of the Cochrane-RoB tool for RCTs28. Inter-trial statistical heterogeneity was assessed using the I2 test 29. When heterogeneity couldn’t be explained by characteristics of the RCTs, the data was incorporated into the meta-analysis using the random-effects model. When I2 < 50%, a fixed-effect model was used in the meta-analysis.Analysis of extracted data was completed using Cochrane RevMan 5.331. A summary intervention effect was then calculated as a weighted average with more weight given to values from studies with less variance and or more participants. Standard error of the summary intervention effect was used to derive a confidence interval (CI) and a p value. Significance cut off value (α) was set at a p value of 0.05. A random-effects model was planned and a forest plot display of results when studies did not use the same measure to estimate the same intervention effect.The summary statistics used in the meta-analysis were post-intervention mean values, and standard deviations. Standardized mean differences (SMD) were calculated for continuous data relevant to primary and secondary outcomes for change in Dizziness Handicap Index (DHI). The DHI score is a validated symptom index score created in 1990 that measures the functional, emotional and physical impacts of dizziness on the patient Fig. 30,31. A A sensitivity analysis was planned if a random effects model was adopted instead of a fixed effects model for the DHI-symptom index score–meta-analysis as recommendations on when to use each model is subject to much debate. The meta-analysis was undertaken twice, once using a fixed effects model and again using a random effects model to determine if and to what degree the result changed.33 and Viziano et al.23 reported initial and 12 month follow up reports on the same randomized controlled trial respectively. Their results were combined so they represented the same RCT, thus excluding the last of 9 articles. These two studies have been reported as Micerelli et al.33 in tables, figures and text. Therefore, a total of 5 RCT studies were eligible for this systematic review as listed in Fig. 136.The search yielded 1235 articles from the twelve research databases reviewed. WHO ICTRP was not used due to restricted access. These 1235 articles were analysed by title and abstract which resulted in 1221 articles being excluded due to duplicates and the inclusion and exclusion criteria. Next, a full text screen was conducted where 9 articles were excluded due to the intervention not meeting the definition of VR (n = 5), the VR device not being used as a PVD treatment (n = 1), article reporting irrelevant outcomes (n = 1) and the study design not meeting RCT criteria (n = 1)  and bilateral vestibular hypofunction (BVH) (n = 1)34 and one study did not indicate PVD laterality35. Four of the five included RCTs compared the use of VR vestibular rehabilitation interventions to drugs, diet, at home exercises, standard vestibular rehabilitation (in clinic and at home) or static VR interventions  scores which were both at a high RoB due to there being some concerns of bias in randomization, outcome measurement and reporting  scale and Simulator Sickness Questionnaire (SSQ) self-report measures. These DHI and ABC outcomes were both at high RoB due to some concerns of bias in measurement of the outcome and deviations from the intended interventions. The SSQ self-report data was also at a high RoB due to outcome measurement. The study published by Micarelli et al.32 also reported DHI, ABC and SSQ scores. The DHI and ABC outcomes had some concerns of bias due to outcome measurement, while SSQ was at a high RoB due to outcome measurement. Lastly, Pavlou et al.36 reported situational vertigo questionnaire (SVQ) scores, which had a high RoB due to some concerns of bias in outcome measurement and randomization  significantly favors VR and AR over control interventions (Z = 3.66 [p = 0.0003]). Next, the causes of heterogeneity were investigated through subgroup analyses.The DHI score was chosen as the main outcome measure for this meta-analysis as it was the most commonly reported patient symptomatology outcome among the included RCTs. Therefore, it meets the primary objective of a meta-analysis to amalgamate findings across several studies. Four studies reported DHI scores and these scores indicated that on average patients experienced moderate to severe functional impairment (DHI ≥ 31) prior to vestibular rehabilitation32. Augmented reality was also significantly favored over controls for reducing dizziness handicap for patients diagnosed with various PVDs . Furthermore, the confidence interval estimates of the effect size for all VR and AR reality interventions overlapped in keeping with there being no significant heterogeneity due to sub-group diagnosis. The second subgroup analysis, type of intervention demonstrated no subgroup differences with identical direction of effect and similar overlap of effect size estimates in AR, and in clinic virtual reality (ICVR) both being favoured over control interventions  studied by Micarelli, unilateral and bilateral MD studied by Garcia and a group of PVDs of unstated laterality  studied by Krueger. Virtual reality was favored over controls for reducing dizziness handicap in patients diagnosed with MD  and UVH   and heterogeneity could not be explained by the type of PVD or intervention. Therefore, a sensitivity analysis was completed to determine if the decision to use a random effects model instead of a fixed effects model for the meta-analysis changed the findings. When using a fixed effects model for the DHI score meta-analysis the I2 and Chi2p values changed from 67% and 0.03 to 79% and 0.002. Total SMD and 95% CI the values changed from − 1.13  to − 8.74 , hence the direction of the effect favouring VR and AR interventions over control interventions remained congruent though the effect size was accentuated.It is recommended that a random-effects model be used when the same outcome is assessed using different measures across included studies or if heterogeneity in the data cannot be explained40. One reported by Micarelli et al.33 and the second conducted by Marcarelli et al.32. Micarelli et al.33 indicated that 1 week post-intervention ABC scores for head mounted device (HMD) HBVR improved significantly (p = 0.0036) more than vestibular rehabilitation treatment. Micarelli et al.32 indicated that older adults completing HMD HBVR combined with vestibular rehabilitation had significant (p = 0.0082) improvements in ABC scores compared to vestibular rehabilitation alone 1 week after the intervention.Activities-specific balance confidence (ABC) scale is a validated measure of balance that was reported by two of the included RCTs35 and Pavlou et al.36 using validated MSQ and SVQ scores respectively (Table 42. Kreuger35 detected a significant (p < 0.05) improvement from baseline for AR and control interventions, however there were no significant between group differences (Table p ≤ 0.01) fewer rehabilitation sessions than vestibular rehabilitation alone. Pavlou et al.36 detected a significant (p = 0.001) between group difference in SVQ score change favouring dynamic (59.2% improvement) over static (1.6% improvement) VR interventions.Dizziness was measured by Kreuger23 reported a 12 month post-intervention ABC score for the HMD VR intervention that was significantly (p = 0.0022) more improved than with vestibular rehabilitation treatment alone.Viziano et al.33 and Micarelli et al.32 using the validated simulator sickness questionnaire  and Micarelli et al.32 (p < 0.05) studies  and one patient from the Pavlou et al.36 did not complete the trial due to non-compliance (94.1% adherence). Assuming each study is weighted equally, the VR and AR vestibular rehabilitation interventions had an overall adherence of 98.4% over a 4–6 week study period. Unfortunately the exact duration of the Krueger35 RCT was not specified, however the number of visits required to achieve improvement was 4 visits with AR and 6 visits without AR.Three of the five randomized controlled trials indicated a 100% adherence in both control and experimental intervention groups. However, one patient from the Garcia et al.2 assessed heterogeneity of 67%  with vestibular neuritis, who constituted up to 63% of Micarelli’s study’s participants, respond less to vestibular rehabilitation than middle aged patients46. Previous literature supports an improvement in dizziness handicap by HMD VR devices that lasted for 1 week before relapsing47. Viziano et al.23 refutes early relapse as they demonstrated that 12 months after 4 weeks of vestibular rehabilitation plus HMD HBVR intervention the DHI scores remained significantly (p < 0.05) lower than pre intervention scores23. Based on the results of our subgroup analyses it can also be hypothesized that immersive ICVR induces the most substantial reduction in the dizziness handicap experienced by patients diagnosed with PVD. This is supported by Whitney et al.37, who demonstrated a reduction in DHI after 6 vestibular rehabilitation sessions (6 weeks) using an immersive ICVR that was similar to the system used by Garcia et al.48.The favourability of VR and AR interventions was not shown on sub-group analysis for HMD HBVR. This is likely due to the small sample size of the Marcarelli et al. (2019) RCT, and the age seniority (avg age: 75.6 years) of the participants studied. The latter is supported by Yan et al.49. A second way of interpreting the VR and AR intervention effect size is to consider the minimally important difference (MID) in vestibular symptoms. The developers of the DHI scale determined that an 18-point score change represented a clinically important difference in vestibular symptoms31, hence this difference was adopted as the MID. The 1.13 SMD in DHI when converted into a DHI score using an estimate of the standard deviations of DHI scores across intervention and control groups in the studies in our meta-analysis as previously described49 yields a mean DHI difference of 14.07 . This suggests that the VR and AR additional intervention effect is clinically unimportant. The two effect size estimates described result in diametrically opposed conclusions. In our opinion the VR/AR effect size is more likely to lie between these two extremes of a very large and no discernable effect at all. More high quality RCTs are required to increase the number of patients studied because of the inherently large heterogeneity in aetiology, symptom severity, patient ages and comorbidity relevant to vestibular rehabilitation to better refine determination of the effect size of VR and AR.The 1.13 standardized mean difference in DHI score in favour of VR and AR interventions indicates a large intervention effect based on the rule of thumb that a 0.8 or greater standard deviation difference is consistent with a large treatment effect33. This improvement was significantly (p < 0.05) greater than vestibular rehabilitation alone at 1 week and 12 months post-intervention33. However, results should be interpreted with caution because their respective studies did not control for patient physical activity, which has been shown to promote central vestibular compensation and improve vestibular rehabilitation outcomes in patients diagnosed with VN51. This is relevant because VN makes up the largest fraction of patients participating in the included RCTs that reported ABC data. Therefore, physical activity differences between VR and control groups could have impacted study results and should be controlled for in future studies.The ABC scale shares some similarities to the DHI as it evaluates patient restrictions by measuring the subjective level of balance confidence experienced in 16 daily activities. The use of HMD HBVR was shown to improve balance confidence in elderly and middle aged patients diagnosed with neuritis, AN, Ramsay–Hunt syndrome or a history of petrous or cochlear surgery36 demonstrated that VR with a dynamic environment reduced vertigo symptoms  approximately 50 fold more than static VR. Similarly, AR has shown utility in reducing dizziness symptoms. Krueger35 determined that using AR during vestibular rehabilitation allowed patients with moderate to severe functional impairment to reach the same improvement level in motion provoked dizziness as vestibular rehabilitation alone. However, AR reached this level in four visits instead of six, which is one visit less than the optimal number of treatment sessions needed to treat chronic UVH52. This is clinically relevant as it could reduce financial and social barriers (e.g. time commitment) to rehabilitation adherence, thereby improving rehabilitation compliance and patient outcomes53.The use of VR or AR also improves vertigo symptoms experienced by patients suffering from PVD. However not all VR elicits the same response. Pavlou et al.54. These acute side effects are an intended part of vestibular rehabilitation interventions that aim to achieve habituation55. It is essential that patients are informed of these side effects and their purpose prior to implementing a VR vestibular rehabilitation intervention. Despite these side effects, 100% and 98.4% of participants were adherent to AR and VR interventions of 4–6 weeks duration. This could reflect the highly motivating nature of VR and AR interventions58.Side effects associated with VR interventions could be a barrier to the adoption of this technology. However, the two studies that reported VR side effects in this review noted that they occurred acutely but gradually reduced over time as patients habituate to the VR interventionDespite these positive results, this systematic review does have limitations. First, studies were also to be extracted from the WHO ICTRP database, but due to high COVID-19 search traffic the research team could not access the database. Moreover, the results of this systematic review and meta-analysis are more applicable to chronic unilateral vestibular disease rather than all PVD sufferers. This is because three of the five RCTs exclusively studied patients with UVH, and 98% of the patients in the fourth RCT studied were diagnosed with unilateral MD. Additionally, it is unsafe to extrapolate the review findings to patients with mild functional impairment secondary to UVH since the majority of patients in the included studies had moderate to severe vestibular functional impairment based on DHI. Similarly, only one AR RCT was included in this study, which means that there is lack of replicated RCT evidence to support benefit from AR in this patient group in the literature thus far. Lastly, 80% of the DHI, ABC, MSQ, SVQ and SSQ outcome measures were at a high RoB which weakens the review’s overall conclusions.59 meta-analysis supporting the use of adjunct VR in the vestibular rehabilitation of adult patients with unilateral vestibular disease. VR may also improve DHI and balance confidence (ABC) scores more than vestibular rehabilitation therapy at greater than 3 months post-intervention, however additional evidence is needed to support this statement. Side effects are also associated with VR sessions but these decreased significantly by the 4th week post-intervention. The validity of these conclusions are compromised by the high RoB in the data studied. Hence, while VR maybe a potential adjuvant to vestibular rehabilitation therapy alone for patients with unilateral vestibular disease additional high quality research is needed to support this assertion.In conclusion, this meta-analysis indicates that VR vestibular rehabilitation improves DHI scores significantly more than vestibular rehabilitation alone 0–3 months post-intervention in patients diagnosed with moderate (DHI 31–60) to severe (DHI ≥ 61) vestibular impairment secondary to unilateral vestibular disease. To our knowledge this is the first level 1a evidenced


We mainly proceed from the view of biological effect to study the acellular bovine bone matrix (ABBM) by the low concentration of hydrogen oxidation. After cleaning the bovine bone routinely, it was cleaned with different concentrations of NaOH and stained with hematoxylin-eosin (HE) to observe the effect of decellulization. The effect of bovine bone matrix treated with NaOH were observed by optical microscopy and scanning electron microscopy (SEM), and compared by DNA residue detection. Cell toxicity was also evaluated in MC3T3-E1 cells by CCK-8. For the in vitro osteogenesis detection, alkaline phosphatase (ALP) staining and alizarin red (AR) staining were performed in MC3T3-E1 cells. And the in vivo experiment, Micro CT, HE and Masson staining were used to observe whether the osteogenic effect of the materials treated with 1% NaOH solution was affected at 6 and 12 weeks. After the bovine bone was decellularized with different concentrations of NaOH solution, HE staining showed that ultrasonic cleaning with 1% NaOH solution for 30 min had the best effect of decellularization. The SEM showed that ABBM treated with 1% NaOH solution had few residual cells on the surface of the three-dimensional porous compared to ABBM treated with conventional chemical reagents. DNA residues and cytotoxicity of ABBM treated with 1% NaOH were both reduced. The results of ALP staining and AR staining showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis effect. The results of micro-CT, HE staining and Masson staining in animal experiments also showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis ability. The decellularization treatment of ABBM with the low concentration of NaOH can be more cost-effective, effectively remove the residual cellular components, without affecting the osteogenic ability. Our work may provide a novelty thought and a modified method to applicate the acellular bovine bone matrix clinically better. We developed a low-cost and improved method of acellular bovine bone matrix with NaOH and the biological evaluation in vitro and in vivo was performed.Our work may provide a novelty thought and a modified method to applicate the acellular bovine bone matrix clinically better. The global demand for bone replacements has been increasing , and manBased on the above, the scaffold materials used in surgery should be thoroughly acellular . At presFresh cow femur , Triton X-100 , hydrogen peroxide , pepsin , alpha galactose enzyme , DNase I .The soft tissue of fresh cow femur was removed and the bone cortex was excised by a high-speed cutting machine  to retain the cancellous bone, which was cut into cubes of 5 × 5 × 5 mm. Blood and bone marrow were cleaned by a water cannon. The samples were placed in a solution containing 1% triton X-100 and vibrated by ultrasound at 60 °C for 2 h, then residual triton solvent was rinsed thoroughly by warm water. The samples sequentially were cleaned with hydrogen peroxide and 75% alcohol for 2 h. The experimental group was treated with 0.5% NaOH, 1% NaOH and 3% NaOH solution in ultrasonic instrument for 30 min. Pepsinase, α galactoase and DNase I were used in turn. Before each treatment, it was fully cleaned with the distilled water. The materials were obtained after 24 h-freeze-drying in the vacuum freeze dryer.Natural bovine bone matrix (NBBM), ABBM, and ABBM treated by NaOH (ABBM-NH) were decalcemized with 10% EDTA solution. About 4 weeks later, the decalcemized samples were put into an automatic dehydrator for dehydration, then transparentized, wax dipped, and paraffin embedded. The samples were sliced with a thickness of 6 μm, placed on a slide and baked at 60 °C for 30 min. Then, hematoxylin-eosin (HE) staining was performed.Scanning electron microscope (SEM), the dried NBM, ABBM and ABBM-NH were fixed on the sample seat by the double-sided conductive adhesive and sputter-coated with gold. The microstructures of the scaffolds were observed using a scanning electron microscope.The tissue deoxyribonucleic acid content quantitative was detected by the kit  The tissue samples were weighed and freeze-dried, then were ground to powders and added in RNaseA solution, protease K and anhydrous ethanol, then centrifuged and the waste solution was discarded, then bleaching solution was added in. The cycle was repeated twice. At last, the scrubber was added to get the genomic DNA; Fluorescence was measured at 535 nm and DNA content was quantified by the standard curve.4/ml were divided into three groups and cultured in 5% CO2 incubator for 24 h. After the cells were attached, the extraction solution was added into the experimental group, and cultured for 1, 3, and 5 days respectively. The cytotoxicity was detected by CCK-8 kit . After 1 h reaction in 5% CO2 incubator, the absorbance was measured by a microplate analyzer at 450 nm wavelength. The calculated cytotoxicity formula was 100% × (ODtest − OD background)/(ODcontrol − ODbackground).After the preparation of the materials, the samples were weighed and packed, and then irradiated  for sterilization. The extracts were prepared with MEM medium containing 10% calf serum and samples at a volume ratio of 10:1. The suspension of mouse embryonic osteogenic precursor cells (MC3T3-E1) with the cell concentration of 2 × 104 cells/ml and inoculated in a 24-well plate with 1 ml/well. Each group was set up with three multiple wells and placed in the 37 °C and 5% CO2 incubator for 24 h. Osteogenic induction medium included MEM medium containing 10% serum, 10 mmol/l sodium β-glycerophosphine, 0.05 mmol/L vitamin C and 100 nmol/l dexamethasone; The experimental group was co-cultured with ABBM and ABBM-NH extracts, respectively. The complete medium containing osteogenic induction solution was replaced every 2–3 days, and the culture lasted for 14 days. The medium was gently washed with PBS for 3 times, fixed with 4% paraformaldehyde for 20 min, and gently washed with PBS for 3 times, 5 min each time. The staining was performed with BCIP/NBT kit and observed under a light microscope. ALP was quantified by ALP quantification kit .The cell suspension was diluted to a concentration of 2 × 104 cells/ml and inoculated in a 24-well plate with 1 ml/well. Each group was set up with three multiple wells and placed in the 37 °C and 5% CO2 incubator for 24 h. The experimental group was co-cultured with ABBM and ABBM-NH extracts, respectively. The above osteogenic induction solution was replaced every 2–3 days, and the culture lasted for 21 days. The medium was gently washed with PBS for 3 times, fixed with 4% paraformaldehyde for 20 min, and gently washed with PBS for 3 times, 5 min each time. The staining was performed with AR solution and observed under a light microscope. Add 200 μl 10% cetylpyridine chloride to each well and it was placed for 30 min at room temperature. After mixed thoroughly, 100 μl was absorbed and moved to a 96-well culture plate, the absorbance was measured at 590 nm by microplate analyzer, and the degree of mineralization was analyzed semi-quantitatively.The cell suspension was diluted to a concentration of 2 × 10Two-month-old male SD rats (180–220 g) were randomly divided into three groups with ten rats in each group. After feeding for 1 week, the operation was performed after the routine fasting and water prohibition. The rats were anesthetized with 3% sodium pentobarbital. After the effect of anesthesia, the rats were fixed on the operating table. The left mandible was peeled, and the surgical towel was spread. An oblique incision about 2–3 cm long was made along the lower edge of the mandible. Skin and subcutaneous tissue were cut layer by layer to separate muscles and expose the mandible body. A 5 mm diameter round defect was prepared with a mechanical drill, and normal saline was continuously added for cooling during the operation. ABBM and ABBM-NH scaffolds were placed in the mandibular defect, and no materials were placed in the blank control group. After the muscle and skin were sutured hierarchically, iodophor was used to disinfect the sutured area. Three days after the operation, 100,000 U penicillin sodium was intramuscularly injected continuously. The suture was removed 1 week after surgery, and the wound healing was observed. The rats were sacrificed at 6 weeks and 12 weeks, respectively.The mandibles of sacrificed rats at 6 weeks and 12 weeks were removed and fixed with 4% paraformaldehyde for 24 h. Inveon MM CT  was performed for the Micro-CT determination. Inveon Acquisition Workplace and Inveon Research Workplace  were separately selected as the scanning and analyzing softwares. The resolution of the images was 9.21 μm and the scan condition including an X-ray tube potential of 80 kV, an X-ray intensity of 500 μA, and an exposure time of 400 ms. COBRA_Exxim  was selected as the CT reconstruction software.After the mandibles of sacrificed rats were fixed with 4% paraformaldehyde for 24 h, then the decalcification was performed with 10% EDTA solution, which was changed once a week, and the decalcification of the samples was examined after 4 weeks. Paraffin sections of the decalcified specimens were made and HE and Masson stainings were performed to evaluate the defect repair.P < 0.05 was considered a significant difference.All data in this study were statistically analyzed using SPSS . One-way analysis of variance was used to analyze the data. HE staining results showed that some cells remained after treatment with 0.5% NaOH Fig. , and no HE staining results showed that there were a large number of cells in NBBM Fig. , some ceDNA residue detection of scaffold materials showed that the content of DNA in natural bovine bone matrix was significantly higher than that in other groups, while the content of DNA residue in ABBM-NH groups (1 and 3%) were significantly lower than that in ABBM Fig. .Fig. 3A P < 0.05), while there was no difference between the three groups on day 1 and day 3 , and there was no difference between ABBM-NH and ABBM . At 6 weeks and 12 weeks, compared with the control group, bone mineral density and new bone volume ratio were significantly increased, trabecular bone thickness and trabecular space in ABBM and ABBM-NH were better than the control group (Fig. The results showed that scaffolds of ABBM and ABBM-NH at 6 weeks and 12 weeks were dispersed in the bone defect area Fig. . With thoup Fig. .Fig. 5MiThe results showed that the defect was filled with a large amount of fibrous tissue at 6 weeks after surgery. Compared with the growth at 6 weeks, there was part of new bone ingrowth at the edge of the bone defect at 12 weeks, while a small amount of new bone formed at 6 weeks. At 12 weeks, the materials in ABBM and ABBM-NH were partially degraded, and few materials remained Fig. .Fig. 6ThIn this study, the low-concentration NaOH solution was used to improve the acellular effect of ABBM. The acellular effects of different concentrations of NaOH solution showed that some cells remained in ABBM after treatment with 0.5% NaOH solution, while the acellular effects of both 1 and 3% NaOH solutions were significant. However, the low concentration of NaOH can effectively treat the infectivity of the scrapie 263K , so 1% NThe results of HE staining and SEM showed that 1% NaOH solution effectively removed the remaining cells treated with the conventional reagent. Cells cultured with the material leaching solution, according to the results, on the first day and the third day, it had no obvious difference between groups Fig. , On the All the rats survived and were included for observation and analysis. There was no postoperative inflammation or infection at the surgical site. A three-dimensional image of the hard tissue of the rat mandible defect was shown in Fig. At present, it is necessary to reduce the immunogenic response and reduce the cost of ABBM in clinical application. Acellular method based on NaOH was similar to other reagents, in general, under study, 1% NaOH solution is likely to be better in terms of cost and safety among the acellular solutions. There are similar methods of decellularization, for example, using ammonium hydroxide with Tryton X as a high pH detergent has been successful for the decellularization of a human-sized liver , and higFrom the results of the present study, we can conclude that 1% NaOH solution effectively removed the cells remaining after conventional reagent treatment. In terms of removing extracellular matrix and eliminating DNA effectively, the acellular effect of NaOH solution is better than that of conventional acellular solution. The low concentration of NaOH acellular solution found by our group can be more cost-effective, effectively remove residual cells and reduce the occurrence of immunogenicity. Our work may provide a novelty thought to applicate the acellular bovine bone matrix clinically better.The acellular treatment of acellular bone matrix treated with the low concentration of NaOH can be more cost-effective, effectively remove the residual DNA components and reduce the immunogenicity without effect on the osteogenic ability of acellular bone matrix. Our work may provide a novelty thought and a modified method to applicate the acellular bovine bone matrix clinically better.


Allergic rhinitis (AR) is a highly prevalent chronic inflammatory disease of the respiratory tract. Previous studies have demonstrated that Bimin Kang Mixture (BMK) is effective in alleviating AR symptoms and reducing the secretion of inflammatory factors and mucin; however, the precise mechanisms underlying these effects remain unclear. κB signaling pathway through animal experiments.  We built target networks for each medication component using a network pharmacology technique and used RNA-seq transcriptome analysis to screen differentially expressed genes (DEGs) for AR patients and control groups. The overlapping targets in the two groups were assessed using PPI networks, GO, and KEGG enrichment analyses. The binding ability of essential components to dock with hub target genes was investigated using molecular docking. Finally, we demonstrate how BMK can treat AR by regulating the NF-κB signaling pathway. Molecular docking suggests that citric acid, deoxyandrographolide, quercetin, luteolin, and kaempferol are structurally stable and can spontaneously attach to IL-1β, CXCL2, CXCL8, CCL20, and PTGS2 receptors. Animal experiments have shown that BMK inhibits NF-κB transcription factor activation, reduces the expression of proinflammatory cytokines and chemokines IL-1β, CXCL2, IL-8, and COX-2, and exerts anti-inflammatory and anti-allergic effects.  Effective targets from network pharmacology were combined with DEGs from RNA-seq, with 20 intersections as key target genes. The construction of the PPI network finally identified 5 hub target genes, and all hub target genes were in the NF-κB signaling pathway improves inflammatory cell infiltration, regulates mucosal immune balance, and reduces airway hypersensitivity. These findings provide theoretical support for the clinical efficacy of BMK for AR treatment. BMK by regulating the NF- Allergic rhinitis (AR) is a noninfectious inflammatory disease of the nasal mucosa mediated by immunoglobulin E (IgE), developing after an atopic individual is exposed to an allergen. The typical clinical manifestations of AR include nasal congestion, nasal itching, mucosal drainage, and sneezing . EpidemiCurrently, common treatments for AR include reducing allergen exposure, drug therapy, allergen-specific immunotherapy (AIT), and biologics . Drug thChinese herbal medicine has been widely used in the clinical treatment of AR and plays an important role in alleviating symptoms and controlling inflammation. Bimin Kang Mixture (BMK) contains 18 herbal medicines, which is a formula based on two classic prescriptions, Yupingfeng San (YPFS) and Xiaoqinglong Decoction (XQLD), to which other Chinese herbs are added or subtracted .εRI) on the surface of mast cells, inducing a sensitized state. When exposed to the allergen again, mast cells release chemical mediators such as histamine, triggering a series of inflammatory responses [When the body is exposed to an allergen, immunoglobulin E binds to high-affinity receptors (Fcesponses . Previouesponses –13. In aesponses . Anotheresponses .α), thereby mitigating the inflammatory response. Research has also indicated that BMK induces relative increases in the CD3+ and CD4+ expression and a decrease in the CD8 + expression, further demonstrating its immunomodulatory function [BMK combines YPFS, XQLD, and several other Chinese herbs into one prescription, doubling treatment efficacy and addressing the fundamental cause of AR. Our previous studies have verified the clinical efficacy of BMK, highlighting its potential to significantly attenuate symptoms and improve quality of life in patients with AR , 17. Anifunction .The Chinese herbal formulae are characterized by diverse components and complex biological relationships, and similarly, allergic rhinitis involves the interaction of multiple target genes and functional proteins. Network pharmacology is a research tool that can construct complex synergistic relationships between drug components, disease-related targets, and molecular pathways to comprehensively assess the potential therapeutic effects of herbal formulations on the disease through multichannel modulation of signaling pathways , 21. In BMK contains numerous herbal components, and the specific anti-inflammatory and immunomodulatory mechanisms associated with its efficacy in the context of AR remain unclear. Therefore, we utilized network pharmacology and RNA-seq to provide evidence supporting the clinical application of BMK by performing a comprehensive analysis of its active components, potential targets, and relevant mechanisms of action in the context of AR .https://old.tcmsp-e.com/tcmsp.php) [https://www.uniprot.org/) [Pheretima and Cicadae Periostracum could not be retrieved from the TCMSP database, they were identified using SymMap (version 2.0) [Most of the chemical components of BMK were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform (msp.php) . Effectiot.org/) . Since tion 2.0) .http://db.idrblab.net/ttd/), DrugBank (https://go.drugbank.com/), GeneCards (https://www.genecards.org/), and Online Mendelian Inheritance in Man (OMIM) (https://www.omim.org/) [Target genes related to AR were identified by searching the following databases for the key term “allergic rhinitis”: Therapeutic Target Database (TTD) (im.org/) –29. Statim.org/) .The two target genes identified in the previous step were linked, and the duplicated region was considered an effective target for BMK in the treatment of AR. The overlap is represented as a Venny (version 2.1.0) diagram. Cytoscape software (version 3.7.2)  was usedn = 5) and patients with AR (n = 5). The research plan was approved by the Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine , and all participants provided written informed consent. Nasal epithelial cells were collected from each participant for RNA-seq using nasal swabs. First, total RNA was extracted from the collected samples using an RNAsimple Total RNA Kit 50 . An Agilent Bioanalyzer 2100  was used to verify RNA integrity. The purity and concentration of the total RNA were measured using a Qubit ® 3.0 Fluorometer  and Nanodrop One spectrometer . A library was constructed for the samples that met the detection criteria, following which mRNA was detected using an Illumina NovaSeq 6000 sequencer. Patients who satisfied the diagnostic criteria for AR were included in this study, according to the guidelines for the diagnosis and treatment of AR , 32. SpeR software package was used to analyze DEGs among the two groups (control vs. AR). Simultaneously, the differential expression multiple  was calculated according to the fragments per kilobase of exon per million reads mapped (FPKM) value, which is often represented as log2|FC|. Finally, due to the small number of DEGs obtained via screening, the FC threshold standard was appropriately adjusted to up 1.5 times or down 0.67 times, P < 0.05. DEGs were visualized using volcano plots and heat maps.The Edgein vivo or in vitro. The GO database defines genes and proteins based on cellular components (CC), biological processes (BP), and molecular functions (MF). GO enrichment analysis was performed using a P value <0.05 as the threshold. The KEGG database contains information related to genomic, chemical, and system functions and graphically illustrates the signaling pathways involved in the target genes. In this study, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (version 6.8) (https://david.ncifcrf.gov/) [The Gene Ontology (GO)  and Kyotrf.gov/)  was usedhttps://cn.string-db.org/) [PPI play a key role in disease, and PPI network analysis can aid in identifying the molecular mechanisms underlying disease process as well as new molecular targets .The STRIdb.org/)  was usedhttps://pubchem.ncbi.nlm.nih.gov/) and RCSB PDB (https://www.rcsb.org/) databases. Then, the ligand energy was minimized using Chem 3D (version 20.0), and the receptor was treated with dehydration and residue removal by PyMol software (version 1.4.1). After that, AutoDock Tools (version 1.5.7) was used to convert the files into (.pdbqt) format for molecular docking, the structure was evaluated by AutoDock Vina software (version 1.2.0), and the lower the affinity energies, the more stable the structure. Finally, visualization was performed using PyMol and Discovery Studio 2021 software.According to the degree value of the network construction, the five active ingredients with the highest scores were selected for molecular docking with the hub target genes. First, we obtained receptor and ligand structures from PubChem  were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.  20190003). The dose utilized in the present study was based on the results of our previous experiments, which verified the optimal efficacy of the medium dose [n = 6 in each): control, AR, BMK, and loratadine. The animals were raised at the Animal Experimental Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine . Drinking water and food were provided ad libitum. The mice were modeled after 1 week of quarantine and adaptive feeding. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Shandong University of Traditional Chinese Medicine .Female specific pathogen free (SPF) BALB/C mice 3; Sigma-Aldrich, USA, 239186, mixed suspension) on days 1, 8, and 15, respectively. The control group was intraperitoneally injected with the same volume of phosphate-buffered saline (PBS). On days 22 to 28, the AR, BMK, and loratadine groups received 1% inhaled OVA (10 mg/ml) for 20 min daily for 1 week. Simultaneously, the drug was administered intragastrically 1 h before OVA stimulation. The diluted drug concentration was determined using the body surface area method, and the BMK and loratadine groups were treated with a 200 μL BMK diluent (9.1 ml/kg)  and 200 μL loratadine tablets  dissolved in liquid (1.517 mg/kg), respectively. The blank and AR groups were administered 200 μL PBS via gavage  –43. At ta gavage .Twenty-four hours after the last OVA challenge, the mice were anesthetized via an intraperitoneal injection of 0.3% pentobarbital sodium (0.1 ml/10 g), following which the eyeball was removed for blood collection. The collected blood was centrifuged to obtain serum for enzyme-linked immunosorbent assays (ELISA), which was stored at -80°C. The nasal bone and maxilla were completely removed for hematoxylin and eosin (HE) staining. In other mice, the nasal mucosa was stripped for real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting.Thirty minutes after the last OVA challenge on day 28, the number of sneezing episodes and nasal scratches over a 10 min period was calculated by three blinded observers to assess improvements in nasal symptoms in AR mice treated with BMK.Total IgE levels in serum were determined using an ELISA kit , in accordance with the manufacturer's instructions. Four samples were taken from each group, and each assay was repeated three times.μm sections. The sections were then stained with HE , dehydrated, and sealed for further analysis. The structure of the nasal mucosa was observed under a light microscope, and three fields of view were randomly selected to assess the infiltration of eosinophils in each group.The complete nasal structure was first soaked in 4% paraformaldehyde  and fixed for 24 h, following which it was placed in ethylenediaminetetraacetic acid (EDTA) decalcification solution  for decalcification, embedded in paraffin, and cut into 5 https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used for primer design, which was performed by Qinke Biotechnology (Beijing) Co., Ltd. Finally, TaKaRa  was used for real-time PCR detection. Using β-actin as an internal reference, the relative levels of the mRNA expression for interleukin-1B (IL-1B/IL-1β), C-X-C motif chemokine ligand 8 (CXCL8/IL-8), C-X-C motif chemokine ligand 2 (CXCL2/MIP-2), and prostaglandin-endoperoxide synthase 2 (PTGS2/COX-2) genes were calculated using the 2-∆∆CT method. Primer sequences are shown in , and the quality (concentration and purity) of total RNA was determined using an Ultramicro spectrophotometer . Total RNA was then reverse-transcribed into cDNA using a TaKaRa reverse transcription kit . Reference to the NCBI database , Anti-COX-2 , Anti-p-NF-κB p65 , Anti-NF-κB p65 , and Anti-β-actin  . Finally, strip development is carried out. ImageJ was used to examine the band density.Protein levels were determined by western blotting. To extract total protein from mice nasal mucosal tissues, we employed RIPA lysate  and PMSF . Before denaturation, protein concentration was determined using a BCA protein concentration determination kit . To prepare a 10% gel for electrophoresis, an SDS-PAGE gel kit  was utilized. After blocking in 5% nonfat milk, antibodies were incubated: Anti-IL-1t-test was used for pairwise comparisons between groups. The results are presented as the mean ± standard error (SEM), and P values <0.05 were considered statistically significant . All statistical graphs were generated using GraphPad Prism (8.0).SPSS (version 26.0) software was used for statistical analysis. The degree of correlation between variables was expressed by Pearson correlation coefficient. The differences between groups conformed to a normal distribution and were analyzed using one-way analyses of variance (ANOVA). The least significant difference (LSD) Pheretima and Cicadae Periostracum were 19 and 29, respectively. In the process of searching for targets corresponding to each component, we noticed that some components had no relevant targets. Thus, these invalid components are not shown in the final chart. According to our statistical analysis, 244 effective components and 1,435 target genes in BMK were obtained after eliminating overlap.Candidate targets of 16 Chinese herbal medicines were obtained from the TCMSP database, and 257 components were screened according to their conditions (OB ≥ 30% and DL ≥ 0.18). According to the SymMap database, the numbers of effective components in A total of 761 disease-related target genes were collected from four databases: TTD (26), DrugBank (82), GeneCards (488), and OMIM (155) . After iA total of 168 overlapping target genes were obtained by integrating the data for the active components of BMK at AR targets . CytoscaκB, and Toll-like receptor signaling pathways were also involved , we identified 745, 329, and 416 DEGs of which were upregulated and downregulated, respectively. EdgeR software was used to analyze DEGs between the control and AR groups, which are displayed as a heat map . After treatment, BMK effectively reduced the number of sneezing and nasal scratches and effectively relieved the severity of AR symptoms. There was no significant difference in the treatment effect between the loratadine and BMK groups (P > 0.05)  .P < 0.001). Levels of the IgE expression were significantly lower in the BMK and loratadine groups compared to the AR group (P < 0.001)  . UltimatP < 0.001). When compared to the AR group, the number of eosinophils was considerably reduced in the BMK and loratadine groups (P < 0.001)  . These fβ, IL-8, CXCL2, and COX-2 in mice nasal mucosa were evaluated using RT-qPCR in each group to investigate the mechanism through which BMK relieves AR. The results demonstrate that the mRNA expression levels of these indicators were considerably greater in the AR group than in the control group (P < 0.001). The mRNA expression of the targets was considerably decreased in the BMK and loratadine groups when compared to the AR model group (P < 0.001) . BMK and loratadine, on the other hand, substantially reduced these effects. These findings show that BMK has anti-inflammatory effects via inhibiting the activation of the NF-κB signaling pathway, resulting in therapeutic effects.To further identify the importance of BMK in the therapy of AR via regulating the NF-B signaling pathway, we assessed the levels of NF- Figures . p-NF-κBAbnormal climate change and increased environmental pollution continue to increase the risk of AR. In addition to their short duration of efficacy and long treatment cycle, common treatments for AR are associated with high drug tolerance and several side effects. Identifying a reliable alternative therapy is key to overcoming these issues in AR treatment. YPFS and XQLD have been widely used in the treatment of the immune system and respiratory diseases for thousands of years, especially asthma and AR. These Chinese herbal formulations contain multiple components that act on various target proteins to affect multiple pathogenic pathways. BMK, which combines YPFS and XQLD, has been shown to inhibit the secretion of proinflammatory cytokines and chemokines, thereby exerting clinically significant anti-inflammatory effects. However, the specific mechanism by which these effects occur remains to be elucidated.κB, TNF, and IL-17 signaling pathways, promote CXCR chemokine or cytokine binding to receptors, and are involved in a variety of inflammatory or immune responses. Through PPI network construction, 5 hub target genes  were finally identified, which are closely related to BMK treatment of AR.Network pharmacology constructs ingredient-target-disease interaction networks by identifying multiple chemical components, targets, and signaling pathways, which can help reveal the pharmacodynamic mechanisms of TCM for the treatment of different diseases. Therefore, in this study, we used network pharmacology and RNA-seq to predict the active ingredients of BMK and the targets and signaling pathways for the treatment of AR. The results showed that validated targets from network pharmacology were combined with DEGs from RNA-seq, with a total of 20 intersections as key target genes. Enrichment analysis results revealed that key target genes have cytokine, chemokine, and signal receptor activator activities, activate cytokines and chemokines mainly through NF-β, a key proinflammatory cytokine produced primarily by monocytes and macrophages, activates the upregulation of adhesion molecules and promotes lymphocyte secretion and thus gets involved in the immune response [β expression in nasal secretions continues to rise, implying that IL-1β is involved in the entire inflammatory process [A variety of cytokines and chemokines mediate the autoimmune response. IL-1response . Bachert process . Inflamm process . CXCL2 a process .The resuIn order to preliminarily verify the effectiveness of BMK, 5 active ingredients were selected for molecular docking with 5 hub target genes. The results showed that these small molecules could freely bind to the target protein, indicating that BMK could play a certain therapeutic role. Jafarinia et al. and Mlcek et al. showed that quercetin exerts anti-inflammatory and immunomodulatory effects in allergic diseases by reducing IgE expression levels, regulating Th1/Th2 stability and inhibiting histamine production , 50. JanεRI on mast cells and other surfaces. Mast cells can release a variety of mediators when reexposed to allergens and simultaneously recruit and activate effector cells, thereby mediating inflammatory responses Continuous secretion of inflammatory factors leads to increased vascular permeability and exacerbates the inflammatory response of the upper and lower airways, further leading to nasal and bronchial mucosal edema, aggravating inflammatory cell infiltration, and ultimately leading to immune dysfunction and induced acute airway hyperreaction. Our experimental study found that the secretion levels of IgE in the serum were significantly higher in AR model mice than in control mice. Moreover, treatment with BMK significantly attenuated this increase in the IgE expression. The severity of allergic behaviors in AR mice, including sneezing and nasal scratching, was directly influenced by pathological changes. According to our analysis, BMK treatment significantly improved AR symptoms. In addition, HE staining verified that BMK reduced the release of inflammatory mediators, improved ciliated epithelium activity and integrity, reduced airway edema and mucus secretion, and relieved inflammatory infiltration. These findings indicate that BMK exerts antiallergic effects by inhibiting the secretion of inflammatory cells and reducing airway inflammation.In the pathogenesis of AR, IgE binds to the FcκB pathway. Therefore, we suggest that modulation of the NF-κB signaling pathway is the main mechanism of action of BMK in the treatment of AR. To better study the mechanism of BMK, we first looked at the mRNA expression levels of cytokines and chemokines. The results of the RT-qPCR detection divulged that BMK effectively reduced the mRNA expression levels of IL-1β, CXCL2, IL-8, and COX-2. What is more, the expression of various proinflammatory cytokines and chemokines is completely reliant on transcription factor activation [κB can be found in nearly all animal cell types. NF-κB p65 regulates DNA transcription and cytokine synthesis and is involved in inflammatory and immunological responses. Of course, NF-κB p65 phosphorylation regulates the transcriptional role of downstream components. Protein expression levels of NF-κB p65, p-NF-κB p65, IL-1β, and COX-2 were measured to better understand the mechanism of action. BMK suppressed the phosphorylation of NF-κB p65, lowered its transcriptional activity, and decreased the expression of inflammatory cytokines and chemokines.Network pharmacology analysis revealed that all five central target genes obtained from PPI network construction were enriched in the NF-tivation . NF-κB cκB signaling pathway and transcription factor activation, thereby reducing airway inflammation in AR mice. Results from animal experiments are consistent with our previous clinical studies. However, this study was only conducted in vivo, and further verification of the predicted results in vitro is required. Despite these limitations, our study provides a theoretical basis for the clinical efficacy of BMK in the context of AR, which may aid in the development of simpler, more effective treatment strategies.In this study, we explored the potential targets of BMK in the treatment of AR using network pharmacology, transcriptomics, and bioinformatics analyses. Our experiments verified that BMK reduces the expression of inflammatory cytokines and chemokines by inhibiting the NF-


Although the Asian population is growing globally, data in Asian subjects regarding differences between bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV) in aortic regurgitation (AR) remain unexplored.The aim of this study was to examine differences between Asian BAV-AR and TAV-AR in significant AR, including aorta complications.The study included 711 consecutive patients with chronic moderate to severe and severe AR from 2008 to 2020. Outcomes included all-cause death, aortic valve surgery (AVS), and incidence of aortic dissection (AD).P < 0.0001) patients; baseline indexed left ventricle and indexed aorta size were larger in TAV-AR. Total follow-up was 4.8 years (IQR: 2.0-8.4 years), 252 underwent AVS, and 185 died during follow-up; 18 cases (only 1 BAV) of AD occurred, with a mean maximal aorta size of 60 ± 9 mm. The 10-year AVS incidence was higher in TAV-AR (51% ± 4%) vs BAV-AR (40% ± 5%) even after adjustment for covariates (P < 0.0001). The 10-year survival was higher in BAV-AR (86% ± 4%) vs TAV-AR  and became insignificant after age adjustment (P = 0.33). Post-AVS 10-year survival was 93% ± 5% in BAV-AR and 78% ± 5% in TAV-AR, respectively (P = 0.08). The 10-year incidence of AD was higher in TAV-AR (4.8% ± 1.5%) than in BAV-AR (0.9% ± 0.9%) and was determined by aorta size ≥45 mm (P ≤ 0.015). Compared with an age- and sex-matched population in Taiwan, TAV-AR (HR: 3.1) had reduced survival (P < 0.0001).There were 149 BAV-AR (mean age: 48 ± 16 years) and 562 TAV-AR (mean age: 68 ± 15 years; AD rate as opposed to BAV-AR patients in Taiwan, emphasizing the importance of early referral for timely management. Surgery on the aorta with a lower threshold in TAV-AR should be considered.Our findings suggest that TAV-AR patients were at a later stage of AR course and had a high   Bicuspid aortic valve (BAV) is the most frequently seen congenital heart defectThe Asian population is growing and accounts for 60% of the global population. However, data that serve as the backbone of practice guidelines, which most Asian clinicians abide by, frequently come from Western populations. Understanding contemporary profiles of valvular heart disease in Asia helps to reduce global disease burden and promote health; hence, there is an urgent need to report Asian data. In Taiwan, health care is provided by National Health InsuranceThe current study compares consecutive patients with TAV-AR and BAV-AR identified from the TTE database in a tertiary university hospital in Taiwan regarding presentation, determinants for symptoms, surgical indication, surgical incidence, survival, and feared aorta complications.Between 2008 and 2020, all consecutive patients aged ≥18 years with chronic moderate to severe and severe AR according to TTE were retrospectively identified from an electronic echo database. All cases were manually reviewed to determine eligibility. Exclusion criteria included: mitral stenosis/regurgitation and aortic stenosis more than mild, prior mitral/aortic surgery, complex cyanotic congenital heart disease, and acute AR  . After e,In patients with multiple TTEs, the first eligible study was used for analysis. TTE was performed by trained sonographers using commercially available echo systems and de novo reviewed by cardiologists with level III echocardiography training (L.-T.Y.). Left ventricular (LV) volumes were derived from a biplane disk summation method or single plane if biplane not feasible.We compared BAV-AR vs TAV-AR regarding all-cause mortality during total follow-up (observation stopped at death or last follow-up), under medical surveillance  and post-AVS. Overall survival was also compared with expected survival from an age-and sex-matched general population in Taiwan. Mortality status, dates of death, and cause of death were retrieved from medical records and Taiwan’s National Health Insurance Research Database.2, and LV end-diastolic dimension[LVEDD] >65 mm) based on guidelinesWe compared BAV-AR vs TAV-AR regarding: 1) surgical indications  in the general population matched by age and sex.Continuous variables, expressed as mean ± SD or median (IQR) according to data distribution, were compared by using the Student’s P value of <0.05 was considered statistically significant.All statistical analyses were performed by using commercially available software . A 2-sided P < 0.0001), possibly reflecting its intrinsic stenosis.P ≥ 0.32) but larger aortic annulus (P = 0.017) compared with non–RL cusp fusion BAV-AR.Baseline characteristics of this cohort (n = 711), including 149 (21%) BAV-AR and 562 (79%) TAV-AR patients, are shown in P ≤ 0.0001) but similar chances of cusp restriction/retraction  and any dilatation of annulus or aortic root .Analysis was performed in 667 patients whose AR mechanisms were recognizable via TTE. Compared with patients with TAV-AR, BAV-AR patients had a higher prevalence of mixed mechanisms (60% vs 26%) and any cusp prolapse (57% vs 12%)  and pulse pressure, lower LVEF, and more cusp restriction with a trend toward larger LAVi and maximal tricuspid regurgitation pressure gradient. Multivariate determinants for symptomatic BAV-AR were: older age , higher systolic BP , and lower LVEF . In symptomatic TAV-AR, in addition to larger LAVi, higher E/e′, and tricuspid regurgitation pressure gradient, LV dimensions/volumes were proportionally related to symptoms. Multivariate predictors of symptomatic TAV-AR were older age, higher pulse pressure, larger LAVi, and one of the following: low LVEF, larger LVESDi, or larger LVEDDi .The relationships between baseline symptoms and chamber remodeling as well as AR mechanisms are shown in Supplemental Table 1(P = 0.09), respectively; TAV patients had 2.84-fold risk of having AVS after adjustment for age (P < 0.0001). Median time from baseline TTE to AVS was similar between BAV and TAV . As for BAV phenotypes, RL cusp fusion was associated with a trend toward higher AVS incidence . For surgical patients with concomitant aorta surgery (46%), BAV-AR patients had larger annulus and annulus/BSA, whereas TAV-AR patients had larger SOV and SOV/BSA  patients underwent AVS, including 51 BAV patients and 201 TAV patients; no patients had received transcatheter aortic valve replacement. The 10-year AVS incidence in TAV vs BAV was 51% ± 4% vs 40% ± 5%  for age  and afte SOV/BSA . Compari2), LVEDD >65 mm, and early surgery, compared with TAV-AR patients. The prevalence of LVESDi >25 mm/m2 was similar between BAV (16%) and TAV (17%) asymptomatic patients (P = 0.80). Concomitant aorta surgery was performed more in TAV-AR patients, reflected by their larger SOV. There were no differences regarding prosthetic valve size, although both were smaller than in patients from the United States (>40% used ≥27 mm prosthesis).(P = 0.08). All 30-day post-AVS mortality occurred in TAV-AR (n = 4).Regarding surgical indications , patientP < 0.0001). Regarding LVEF, TAV-AR patients had a significantly improved post-AVS LVEF , and BAV-TR patients exhibited a trend toward improvement   had a follow-up TTE between 6 and 18 months post-AVS (median time: 12 [IQR: 9.5-14.5] months). Post-AVS LVEDD, LVESD, and LVESDi decreased significantly compared with pre-AVS TTE in both groups  .During a median follow-up of 4.8 years (IQR: 2.0-8.4 years), 185 patients died (28 died post-AVS), including 170 (30%) TAV and 15 (10%) BAV patients. The mortality follow-up was 100% by December 2020.P < 0.0001) and follow-up under medical surveillance  as shown by Kaplan-Meier curves  and after additional adjustment for sex, CCI, LVEF, New York Heart Association functional class, and time-dependent AVS . Adjustment for year of baseline TTE did not change the results. Also, Kaplan-Meier curves showed that those with AVS and aortic surgery had better overall survival, whereas those without AVS had the worst survival , whereas BAV-AR patients had a similar risk of death  at 5 years ; however, after adjusting for age, survival was independent of valve anatomy . Also, those with AVS and aortic surgery had better post-AVS survival compared with those with AVS alone, mostly driven by TAV-AR (P = 0.07) . BAV patients (93% ± 5%) had better 10-year post-AVS survival compared with TAV patients (78% ± 5%) y TAV-AR ; after a = 0.07) .AD occurred in 18 patients , including 1 BAV (type A dissection) and 17 TAV patients. Type A AD occurred post-AVS in 3 TAV, non–Marfan syndrome patients: one received concomitant aorta plication at time of AVS (ascending aorta 45 mm) and the other 2 received AVS only (both had ascending aorta 47 mm). Twelve patients with type A AD were confirmed by emergent surgery, 3 confirmed by imaging studies, and 2 from death certificates.During follow-up, AD (12 patients had available data) were 53 ± 7 mm (range: 40-60 mm), 60 ± 10 mm (range: 45-80 mm), 60 ± 9 mm (range: 50-80 mm), and 35.5 ± 5.4 mm/m2, respectively. The annual growth rate of SOV and ascending aorta were 3.5 mm/year and 1.4 mm/year.The average dimensions of SOV, ascending aorta, maximal aorta size, and indexed maximal aorta size at the time of AD incidence was 3.7% ± 1.0% at 10 years ; the corresponding cumulative AD incidence in TAV-AR was 4.8% ± 1.5% (AD (n = 18) are presented in AD risk but not age, BP, presence of hypertension, and Marfan syndrome; increased body mass index showed a trend of association. When we classified SOV with the reference value <45 mm, SOV 45 to 55 mm and SOV ≥55mm exhibited 5.7-fold and 5.5-fold risk of AD, respectively. When maximal aorta size was classified with the reference value <45 mm, similar results showed that patients with an aorta >45 mm had higher risks of AD in AR.The overall cumulative % ± 1.5% . BaselinAD rate; and 4) maximal aorta size >45 mm was associated with higher AD risk.In this large contemporary Asian cohort, we compared differences between TAV and BAV patients with hemodynamically significant AR. Our major findings are: 1) compared with TAV-AR patients, BAV-AR patients were 2 decades younger, with similar absolute LV dimensions yet smaller BSA-normalized LV size, and were less symptomatic, resulting in lower AVS incidence; and 2) symptomatic TAV-AR patients had advanced cardiac remodeling, lower LVEF, higher pulse pressure, and tricuspid regurgitation pressure gradient. Conversely, except for higher systolic BP and lower LVEF, the associations between symptoms and cardiac remodeling were less prominent in BAV-AR. Other findings thus were: 1) although TAV-AR had a higher incidence of AVS, post-AVS LV recovery was good in both groups; 2) BAV-AR patients showed superior survival (mainly determined by age), and TAV-AR patients had excess risk of death compared with the general population, possibly caused by late referral; 3) TAV-AR patients had significantly larger BSA-normalized SOV and ascending aorta dimension, which led to a higher cumulative AD risk in BAV,AD rate in TAV-AR patients. Interestingly, in TAV-AR, there is a proportionate correlation between symptoms and chamber remodeling, different from the U.S. cohort,In our previous work from the United States, we found that, compared with TAV-AR patients, BAV-AR patients were less symptomatic and had good correlation between LV size and symptoms, a higher AVS incidence, more aortic valve repair, more concomitant aorta surgery, better overall survival caused by younger age, and survival similar to that expected.AD in patients with chronic moderate to severe and severe AR. Most prior studies on the incidence of AD focus on patients with genetic aortopathy, including Marfan syndrome, BAV, and nonsyndromic thoracic aneurysms,,AD than those with Marfan syndrome or other genetic-associated aortic aneurysms,,AD among BAV-AR, but the overall cumulative incidence of AD, driven by TAV-AR patients, was high in this study . The incidence of AD in Taiwan was reported as 5.6 per 100,000 persons,AD risk, suggesting that surgical intervention could be considered at this point in patients with significant AR in both BAV and TAV, although prospective studies are required. Current guideline recommendations on a lower threshold  for surgery on ascending aortic aneurysms included patients with Marfan syndrome plus risk factors,For the first time, we report the cumulative incidence of AD risks during observation in the absence of other risk factors , similar life expectancy to general population, good post-AVS survival, and good LV recovery, which again emphasizes that age and not valve anatomy determined AR survival. Second, for patients with AR, regular TTE surveillance and clear reporting for AR progression and most importantly, aorta progression both before AVS and post-AVS is paramount because fatal AD may be prevented through early intervention once the aorta size was ≥45 mm. Third, attention should be paid to indexed LV size, not absolute LV size, in Asian patients with AR for timely surgical referral. In the face of culture-related surgery hesitancy, good post-AVS survival in both BAV-AR and TAV-AR patients should reassure the patient for prompt intervention.From this large Asian cohort, important clinical implications are: first, clinicians can reassure BAV patients with AR about low AD cumulative incidence although not every deceased patient underwent autopsy.In this retrospective, tertiary referral center study, selection bias was possible. However, because of good accessibility to TTE and health care in Taiwan, our cohort reflected real-world conditions in patients with AR in Taiwan. The year of “baseline TTE” and related factors could play a role in outcomes; however, after adjusting it as a covariate , resultsPerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: Differences between Asian TAV-AR and BAV-AR patients were not entirely the same as in the Western population. This cohort exhibited a larger baseline indexed left ventricle and aorta size for TAV-AR, suggesting that TAV-AR patients were at later stage of the AR natural course. Superior survival was noted in BAV-AR vs TAV-AR patients, who had a higher surgical incidence. Cumulative 10-year incidence of AD was higher in TAV-AR and was associated with baseline aorta size >45 mm.TRANSLATIONAL OUTLOOK: Future research should explore whether a lower cutoff prompting concomitant aorta surgery in Asian patients with TAV-AR could improve outcomes by lowering the rate of AD.Findings from this large contemporary Asian cohort comparing TAV-AR and BAV-AR are both reassuring and concerning. We showed that post-AVS survival in both groups and overall survival in BAV-AR were comparable to those of the U.S. cohort . HoweverThis study was supported by the Ministry of Science and Technology of Taiwan (MOST 110-2314-B-002-026) and the National Taiwan University Hospital (111-N0028). The authors have reported that they have no relationships relevant to the contents of this paper to disclose.


Successful prosthetic rehabilitation is essential to improve the physical and mental outcomes of people with lower-limb amputation. Evaluation of prosthetic services from a prosthesis user perspective have been published and commissioned by the national bodies, however, the perspectives of clinicians working with service users during rehabilitation have not to date been sought. We sought to determine factors impacting lower-limb prosthetic rehabilitation from a clinician's perspective to inform studies focusing on prosthetic and socket design and fitting. Six clinician  interviewees were self-selected from a survey exploring issues and frustrations during lower-limb prosthetic rehabilitation. Semi-structured interviews explored the impactors on and frustrations with rehabilitation and the prosthetic socket. A thematic analysis was subsequently conducted to identify themes in the responses. Five themes were identified: Service Disparity, Body Impactors, Consequences of Ill-Fit, Prosthesis Irritants, and Limitations of Practice. Each theme, though distinct, relates to the others either as a cause or consequence and should be viewed as such. Addressing the themes will have benefits beyond the issues addressed but also expand into the other themes. This study provides an insight into the clinician perspectives on lower-limb prosthetic rehabilitation, which has not been formally documented to date. Successful prosthetic rehabilitation of people with lower-limb amputations is vital to their ability to live and function independently. Some evaluation of prosthetic services from a prosthesis user perspective have been published in literature –3, and aThroughout rehabilitation, patients interact with various clinicians, including occupational therapists and surgeons, amongst others. However, most of their interactions will be with prosthetists, who prescribe and fit the prosthesis, and physiotherapists, who guide strength training and movement re-education.In most settings, after initial recovery from the amputation surgery the patient is discharged from inpatient treatment. Prosthetic rehabilitation is done primarily in an outpatient setting, with regular visits to the prosthetists and physiotherapists. Depending on the setting, prosthetists and physiotherapists may work in the same location and meet regularly to determine the best course of action for their patients. In other settings, they may be geographically separated and discuss care paths only as required e.g., if an issue with the prosthesis arises in a physiotherapy session.Of the literature on the evaluation of prosthetic care, most is from the perspectives of the service users and based on questionnaire data –3. WhileOne study evaluated prosthetic service provision in the UK from the perspective of clinicians . HoweverThis study aims to start to highlight the experiences of rehabilitation clinicians, and how they impact lower-limb prosthetic rehabilitation. The understanding of clinician experiences is important to gain a broader picture of the potential impactors on rehabilitation as they are involved in aspects that service users are not. Due to the clinician roles, they are part of many people's rehabilitation and therefore see a broader picture than an individual's personal experience and are able to draw trends from their practice.To explore the factors impacting rehabilitation first-hand, in particular socket-fit, a questionnaire with an optional follow-up telephone interview, approved by Joint Research and Compliance Office at Imperial College London (ICREC Reference: 18IC4485), was deployed for prosthesis users and rehabilitation clinicians. Both prosthesis users and rehabilitation clinicians were recruited for the surveys and interviews, however, in this paper only the interviews of rehabilitation clinicians are discussed. The analysis of the survey results and prosthesis user interviews have been published previously , 7.The survey was created with input from rehabilitation physiotherapists as standard outcome measurement tools did not meet the needs of the study. The survey started with a free text question about rehabilitation frustrations to collect uninfluenced views, before questions being introduced specifically about sockets. The interview questions, however, were based on the responses given in the survey, related to any of the topics mentioned as having the most impact or being a particular frustration through rehabilitation.At the end of the survey, participants could provide their contact details to participate in the telephone interviews . The goal of the semi-structured interviews was to understand the reasons and impacts surrounding the answers given in the survey. Telephone interviews were chosen to minimize expenses and travel difficulties, facilitating higher participation.The survey was created and shared online using Qualtrics, a web-based survey tool. The Participant Information Sheet was provided, followed by a checkbox to confirm participant consent, before the survey was displayed. The survey link was distributed via social media and emailed to personal contacts in relevant clinical teams, charities, and professional organizations relevant to the professions sought, to recruit relevant rehabilitation clinicians. Participants had to be 18 years or older; currently not taking medication that affected their cognitive function; have a good understanding of written and spoken English; and be a current member of a lower-limb prosthetic rehabilitation team.All those who provided their details were contacted to complete the follow up interview, a total of eight clinicians completed the telephone interview. Recruitment was limited by the number of willing participants in the study period. Telephone interviews lasted no more than one h and were dictated by the length of answers given by participants. Relationships between participants and professional body membership were not monitored.To analyse the issues experienced during rehabilitation, a thematic analysis was conducted on the interviewee responses. All transcriptions were anonymised before analysis. Steps for analysis were taken, based on Braun & Clarke's methodology: familiarization, coding, generating themes, reviewing themes, defining and naming themes, and reporting . ProfessOf the 44 clinicians that completed the survey, eight UK based clinicians participated in the telephone interviews, however, two of the recordings were corrupted before the start of the thematic analysis. Therefore, the responses of six clinicians have been included in the presented results and discussion. Participants were varied in terms of years of experience and role, with two of the participants being prosthetists and the other four physiotherapists, and the vast majority being female .Service Disparity, Body Impactors, Consequences of Ill-Fit, Prosthesis Irritants, and Limitations of Practice. Quotations are accompanied by a role, participant number and sex to allow the range of views to be seen. The data was sex disaggregated for transparency, as females tend to be underrepresented in research though this is not the case in this study. Themes were not identified for or compared between the different professional roles due to the small sample size.Five interrelated themes were identified : ServiceService Disparity between private and military settings, compared to the NHS regarding longer waiting times for appointments and socket refitting.There are a variety of settings for prosthetic provision, including military rehabilitation centres with inpatient and outpatient care, NHS inpatient and outpatient care, and private clinics providing mainly outpatient care. Clinicians noted the “I'm a bit different because I work privately; our sockets are generally a bit better” “In the NHS, I guess it's worse as well-because you've got a bigger time (to wait for an appointment)…patients may be on that ill-fitting socket for six weeks, eight weeks whilst they get the appointment to get a new one started” .Clinicians also noted a lack of continuity in care; the change in management and number of temporary clinicians could make it difficult for continuity of patient care. Time to update new clinicians on patient history means that there is less time to for clinicians to spend with patients or work on self-development to improve their practice.“(Contracted clinicians) might change every…8–12 weeks…when I'm talking to the prosthetist about an issue for a patient, then you're obviously effectively starting again in terms of supplying them with information about the patient's…history… you're not necessarily getting the clinician knowing as much about the client as ideally, they would do” .Changes in the team had an impact on how different clinical teams worked together, and team setups varied in settings and locations. The settings where prosthetists and physiotherapists were in the same location was an advantage. They allowed for small issues with the prosthesis discovered during physiotherapy to be solved quickly. There was a view that the clinical teams that worked together when seeing patients, whether in the civilian or military environment, meant that patients could be treated and their issues solved with multiple perspectives considered.“I think the biggest frustration in the NHS which I don't have so much (in the private setting) was time constraints; so, not being able to work jointly with the Prosthetists as well…the patient adapts and gets stronger with the physio there's a delay between the Prosthetist seeing them again to adapt the alignments of their new body position“ .Changes to the shape and volume of the residual limb are common and are varied with changes to weather and activity levels; even during one physiotherapy session a residual limb can change enough for the socket to become uncomfortable or prevent it from being worn. A once well-fitting socket may become loose, allowing for limb movement in the socket causing residual limb damage. The socket may become so tight, the prosthesis cannot be worn due to pain or skin conditions.”(Patients) can't get into their socket if they're particularly swollen…I've had a few patients that can't get their limb on for, say, a week at a time” .Another issue is that the residual limb gets hot in the prosthesis, due to the fact sockets are typically made of non-breathable materials, with the addition of socks or liners to aid comfort and fit. Temperature build up in the socket is often accompanied by moisture build up due to sweat which can cause further issues for residual limb health , but also for prosthesis use.“As soon as people get sweaty, things change…it causes skin problems and then they can't be on their leg” .Even when the limb shape and volume have stabilized, the tissue in the residual limb may not be suited to prosthesis use. Adherent scars and skin grafts are not uncommon, particularly for those with traumatic etiology. The internal tissue, that is fused to skeletal structures, can become stiff and immobile, leading to significant pain and skin breakdown, which must be addressed before continued prosthesis use.“An adherent scar, it sort of sticks when you put a prosthesis on…if the prosthesis is moving over the stump and over the scar itself, and the scar is bound down to the underlying bone; then you can get friction and tissue breakdown” .The body's healing process is not easily controlled and is different for each person. Healing can be facilitated by clinicians but is not guaranteed. Open wounds generally prevent the prosthesis being worn, impeding prosthetic rehabilitation and much sought-after progress.“The soft tissue will mature post-surgery or post-injury, or just after commencing use of a prosthesis; it will mature in its own time…so we have to go at the rate of the body” .“You can do exercises until you're blue in the face, but you can't actually beat walking” .Alongside the physical impactors of the body, the psychology of prosthetic rehabilitation is a crucial factor in success. Individuals are facing a big change in their lives, and many do not want to be impeded by their amputations. It is not uncommon for people to become demotivated when they are not making the progress they hoped.“There's the psychological aspect of being held back, just when they think they're going to be making progress” .consequences of ill-fitting sockets are focused on the physical health of the residual limb and ability to wear the prosthesis. Due to sockets' rigidity they do not shape to the individual's changes, which can lead to severe pain and discomfort.The socket is a crucial component of the prosthesis and a complex one due to the interface with the body. The direct “(The socket is) the one factor which is likely to prevent them wearing it entirely, and therefore the comfort is critical to that point” .Ill-fitting sockets compound issues of residual limb health, facilitating skin breakdown and pressure sore development, which can have lasting impacts on residual limb health, and that of the rest of the body.“If the prosthesis rubs in any way, shape, or form, then, again, you've got to stop using the prosthesis” .Continued prosthesis use with an ill-fitting socket, despite pain or skin conditions, can have adverse effects across the body. Individuals tend to compensate for pain and discomfort by changing their weight distribution or movement. The change can impact the body's biomechanics, potentially leading to musculoskeletal overuse injuries, impacting rehabilitation, function, and long-term health.Irritations with the prosthesis are not easily solvable due to their make-up and design. The socket is one factor that causes frustration, in particular, its static nature. The rigid nature of the socket means that it does not change with the continual changes to the user's residual limb, which can lead to an ill-fitting socket and associated ramifications.“(The socket is) only one shape and size at any point. I mean, we can make adjustable sockets now as well, but they're limited. You know, we can't make them change in any way they want to at any point” .“It's the rigid factor, if you like; that doesn't change as the patient's body changes.” .The liners used in some prostheses are also reported to cause issues, particularly around the build-up of heat and sweat in the socket. When liners are paired with socks inside the socket, to increase comfort or minimize movement in the socket, the issues are exacerbated and can lead to residual limb health issues if not dealt with.“We use a lot of the silicon liners and sealant liners just because you're…enclosed…and they find that even if it's not an hour (exercise) session…they quite often have to stop and take the leg off, dry it down, and then put it back on again” .While fixing known issues may not need significant time, building a rapport between clinician and patient does. The time required for an appointment is often restricted, particularly in the NHS, and so there is not always time to build the clinician-patient relationship, which may mean patients do not feel comfortable talking about personal issues affecting them which may impact their rehabilitation.“I think it's building your relationship with (patients) as well. But I think that patients in any capacity just seem to want time” .It was also felt that gaps in support provision contribute to clinicians' time need with patients. Individuals with amputations have undergone a significant life change, requiring physical and emotional support. If the only opportunity for support is during prosthetist or physiotherapist consultations, this adds to the required time.“Patients don't always get…the mental health input and things like that that they need so sometimes like they will come in and talk to… there's a lot going on for them especially when they're new amputees.” .When it comes to solving issues, there is no standard practice; e.g., some prefer to remove the prosthesis for wound healing, whereas, others believe it helps to wear it and encourage activity to increase blood-flow and healing for minor wounds. Sometimes, it is unknown what the correct way is to proceed, resulting in a trial and error process.“It's difficult to know…just by wearing the socket, whether you're going to improve the blood flow to that area, or more typically…worsen the healing rate” .Similarly, for ill-fitting sockets, increasing the number or thickness of socks is suggested, but specifics cannot be predicted, so mitigation is reactive rather than proactive.“It's the rigid factor if you like; that doesn't change as the patient's body changes. So, you're constantly playing catch-up” .Although patients can be referred back to the physiotherapist by their prosthetist in the UK, this typically happens only when new components have been prescribed. In the NHS, the goal is for individuals to walk and perform daily tasks. However, once initial goals are achieved, there is no standard scheduled check-up. If goals or lifestyles change, individuals will not see a physiotherapist to help them adapt or check good practice.“(Patients) get up and walking and then that seems to be it really and then it's up to us to then refer them back because if we think they need a bit more but I just don't feel like they get quite enough” .This study aimed to understand the frustrations and impacts of clinicians through lower-limb prosthetic rehabilitation. Generally in published literature, the views of service users and their carers are sought, and rehabilitation itself is not the specific interest, rather life using a prosthesis is the focus. As a result, this study highlights some of the experiences and impacting factors associated specially with early care of people with amputations.Analyzing transcripts of the interviewee responses is advantageous to understand the reasoning behind their survey answers; using their own words enhances the impact of their meaning rather than being paraphrased or solely grouped into a statistic.Service Disparity, Body Impactors, Consequences of Ill-Fit, Prosthesis Irritants, and Limitations of Practice. The individual themes are related closely, with some being the consequences of or exacerbated by others. The identified themes are similar to those identified from the analysis of the prosthesis users' perspectives , can have a significant inhibiting effect on the rehabilitation progress as individuals cannot wear their prosthesis. Whilst other aspects of physiotherapy can continue, such as strength training, gait re-education is often halted if the prosthesis is too painful  or if there is an infection or skin breakdown. The volume change of the residual limb, which can be between 17 and 35% in the 6 months can take around 100 days to stabilize .The datasets presented in this article are not readily available because of ethics restrictions. Requests to access the datasets should be directed to Dr Shruti Turner . The patients/participants provided their written informed consent to participate in this study.ST: study design, data collection, data analysis, and article writing and editing. AB: data analysis, article review, and article editing. AM: supervisor of study, principle investigator, article writing, and article editing. All authors contributed to the article and approved the submitted version.This work was conducted under the auspices of the Royal British Legion Centre for Blast Injury Studies at Imperial College London.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.