Background: The work of journal editors is essential to producing high-quality literature, and editing can be a very rewarding career; however, the profession may not be immune to gender pay gaps found in many professions and industries, including academia and clinical medicine. Our study aimed to quantify remuneration for journal editors from core clinical journals, determine if a gender pay gap exists, and assess if there are remuneration differences across publishing models and journal characteristics.Methods: We completed an online survey of journal editors with substantial editing roles including section editors and editors-in-chief, identified from the Abridged Index Medicus “Core Clinical” journals in MEDLINE. We analyzed information on demographics, editing income, and journal characteristics using a multivariable partial proportional odds model for ordinal logistic regression.Results: There were 166 survey respondents (response rate of 9%), which represented editors from 69 of 111 journals (62%). A total of 140 fully completed surveys were analyzed ; 50 (36%) editors did not receive remuneration for editorial work. No gender pay gap and no difference in remuneration between editors who worked in subscription-based publishing vs. open access journals were detected. Editors who were not primarily health care providers were more likely to have higher editing incomes . Editors who worked more than 10 hours per week editing earned more than those who worked 10 hours or less per week .Conclusions: We were unable to detect a gender pay gap and a difference in remuneration between editors who worked in subscription-based publishing and those in open access journals. More than one third of editors surveyed from core clinical journals did not get remunerated for their editing work. A rise in open-access journals is also evident; while in 2008 there were just 249 open-access journal titles in Web of Science, that number ballooned to 1431 in 20181. Growth in the journal industry comes with more opportunities to become a journal editor. Editors are often scientists, researchers, administrators, and clinicians who have expertise in a particular field and competencies in evaluating articles’ suitability for publication in scientific journals2. Editorial work can be a very satisfying full-time career or part-time job in addition to a clinical, research, or administrative careers.The number of academic journals continues to grow each year. In 2018, there were 5399 clinical journals tracked by Journal Citation Reports in comparison to only 3681 10 years prior5. For example, a 2012 US survey found that male physician researchers had higher average salaries  than females after adjusting for specialty, academic rank, and research productivity6. Other studies have shown salary deficits can be up to 12–28% for female physicians8 when compared to male counterparts. Similar statistics can be found in the UK, where the average pay gap for consultant physicians, including professors, was 13% in favor of men9. Several countries have mandated pay transparency laws in recent years. Denmark10 and Austria11 passed laws requiring companies to internally report gender-based wage statistics to it employees in 2006 and 2010 respectively. In 2018, the United Kingdom pushed for national pay transparency and equity by mandating public annual reporting of gender pay gap for any organization comprising more than 250 employees12. France followed suit with mandatory reporting, while Germany and Iceland enacted pay equity and transparency laws.Within medicine, substantial evidence of gender inequity in academia exists, including disparities in compensation13, there is currently very limited literature that evaluates how journal editors are compensated for their work. An international email survey of 88 editors of nursing journals found that their mean annual salary was $12,749 USD  for a mean of 13.4 hours worked per week14. A total of 8% of survey respondents (7 of the 88 editors) did not receive any monetary compensation and only 31% of participants felt that their compensation was adequate14. It is concerning that the critical job of editors to uphold the integrity of academic literature can be low-paid or voluntary. With mandatory data reporting, The Lancet reported gender pay gaps of 13–40% favouring men over women in major UK publishing companies in 201815. Specific data for journal editors were not available but some of these data do highlight the potential gaps.While there is an expanding body of literature on pay gaps in of academia and of medicine in general16. Though open access publishing comes at a large cost to authors, its popularity is supported by many benefits that may include faster publishing times and the ability to reach bigger audiences compared to subscription-based publishing. It is unknown whether a journal editor’s remuneration is affected by these journal variables.There is substantial heterogeneity among journals, such as publishing platform, scope, publication frequency, and Journal Impact FactorWith special interest in biomedical sciences, the objectives of this study were to quantify remuneration for journal editors from core clinical journals, determine if a gender pay gap exists, and assess if there are remuneration differences across publishing models  and other journal characteristics .18.We completed an international online survey of full-time and part-time journal editors identified from the Abridged Index Medicus “Core Clinical” journals in MEDLINE, which represented 111 peer-reviewed core medical journals with high clinical impact. Our target population of journal editors were those with substantial editing roles including editors-in-chief, deputy editors, executive editors, senior editors, associate editors, and editors of a specialty section. Editors were identified through each journal’s webpage in December 2018. We excluded roles classified as statistical editors, assistant editors, international editors and editorial board members. If the editors of a section or specialty section numbered greater than 200 for a single journal, these individuals were excluded based on the assumption that they may not have a substantial editorial role. Publicly available emails were found via the English-language search engine, Google. Major sources included journal web pages, academic institutional web pages, and corresponding author on recently published articles. Reporting of this online survey was guided by the CHERRIES reporting guideline19. We developed the survey to capture demographic data, editing remuneration in USD, editing experience, and journal characteristics. A blank copy of the survey is available asExtended data18. An online pilot test was sent out to our knowledge user team of three journal editors to identify poorly constructed questions, and to assess face validity before distribution. The survey included 10 questions on three pages, and adaptive questioning was used. Respondents were able to review their answers before submission. The following variables were collected in our survey and included in the final multivariable model: sex , gender identity, primary role , academic rank (any professorship vs. none), editing role , years in editing (>10 vs. ≤10 years), hours/week in editing role (>10 vs. ≤10 hours), and years worked for current journal (>5 vs. ≤5 years). Journal characteristics of publishing model  and frequency of publication (best fitted to monthly/bimonthly vs. weekly/biweekly) were extracted from the journal selected in the survey. A journal’s 2017 Journal Impact Factor was obtained from Journal Citation Reports1.The online survey was conducted using the subscription software, Qualtrics CoreXM Survey Tool20.Email invitations with the survey link were distributed via Qualtrics in February 2019. This was a voluntary survey, and no incentives were offered. We employed established methods to enhance survey completion rates with reminder emails at week 2 and week 4Only completed surveys were analyzed. Dichotomous baseline characteristics for male and female journal editors were presented as frequencies and percentages and compared with the chi-square test or Fisher’s exact test when expected sample sizes were 5 or less. Journal Impact Factor of the journal where male and female editors worked was a non-normally distributed continuous variable presented as a median with interquartile range. The Journal Impact Factor of each group was compared with the Wilcoxon rank sum test.Our outcome of interest was journal editor salary, which was modeled as an ordinal variable with three categories: ≤$10,000 per year, $10,001 to $50,000 per year, and ≥$50,000 per year. We derived adjusted odds ratios (OR) and 95% confidence intervals (CI) for our outcome of interest from a multivariable partial proportional odds model for ordinal logistic regression in SAS version 9.4 . The variables for editor sex and academic appointment did not satisfy the proportional odds assumption; therefore, these variables were assumed to have nonproportional odds in the final multivariable partial proportional odds model. Two-sided p-values were reported and p-values <0.05 were considered statistically significant.Institutional review board approval was obtained through the Unity Health Toronto Research Ethics Board in Toronto, Canada. The survey landing page included study information and consent to participate was implied by survey completion and submission.18. This was composed of 95 male and 45 female editors. All respondents identified as cis gender. A total of 50 (36%) editors did not receive remuneration for editorial work; 111 (79%) and 29 (21%) editors worked for a subscription-based journal versus an open/hybrid journal, respectively. A total of 90% of editors held an academic position. The median 2017 Journal Impact Factor was 4.9 . There was a larger proportion of female survey respondents who were fulltime journal editors compared to male counterparts . Details on baseline characteristics are provided inA total of 2165 editors were identified in December 2018, with 1948 having publicly available email addresses; of these, survey invitations were successfully sent to 1844 without bounce-back. A total of 193 surveys were started, and 166 surveys were submitted . We received survey responses from journal editors at 69 of 111 journals (62%). 140 completed surveys were included in our analyses . Data thIn univariate analyses , journalIn multivariable analysis , there wThe Lancet highlighting the presence of a gender pay gap of employees at major academic publishing companies15, numerous studies in the past have highlighted similar inequities across academia8. In our study of part-time and full-time journal editors from a focused subset of core clinical journals, we did not detect a gender pay gap. We found that annual remuneration for editing was higher for editors working more than 10 hours per week and if their primary occupation was not a health care provider. Full-time journal editing positions were disproportionately more likely to be held by a woman. While we hypothesized that the different revenue structure of subscription-based versus open access journals may translate to a difference in editor’s remuneration, we did not find it to be true after adjusting for predictor variables. A journal’s Impact Factor did not affect an editor’s remuneration. Although 36% of editors surveyed reported no direct earnings from their editorial work, there can be other benefits including subsidies for scientific meeting registration fees and travel costs.In addition to a recent study by13, our study only examined gaps specifically in biomedical sciences. Our study had several limitations. First, our results had limited generalizability due to a low response rate, which can magnify volunteer bias. However, our participants did represent 69% of the core clinical journals. Moreover, our sample size of 140 was comparable to an international survey of 148 scientific editors from biomedical journals to evaluate journal editing core competencies21. We were unfortunately not powered to compare subgroups of editing roles and journal characteristics. Second, given our approach to identifying email addresses for journal editors, our survey was biased towards editors who had either an English-language academic profile on an institutional website or if they were a corresponding author of a recently published article. Third, we were unable to make direct comparisons between for-profit and non-profit journals, because several companies publish journals on behalf of non-profit organizations. This distinction may factor into a journal editor’s desire to edit for a journal at a given remuneration rate. In addition, we did not assess non-financial reasons that factor into the decision to be a journal editor. Lastly, there was substantial heterogeneity in editing roles and the lack of standardization in editing titles made it difficult to draw conclusions that were generalizable to the population. Future studies can include qualitative interviews to gauge the various roles and responsibilities of editors, and remuneration practices.While there is robust data on gender pay gaps in academiaWe conducted an international survey of journal editors from core clinical journals to understand how remuneration varied across editor’s demographics, professional experience, and journal characteristics. We did not detect a gender pay gap or a difference in remuneration between editors who worked in subscription-based publishing vs. open access journals. More than a third of editors surveyed were not remunerated for their work.https://doi.org/10.7910/DVN/AHMB8G18.Harvard Dataverse: Journal editors: How do their editing incomes compare?File ‘Journal editors deidentified data.tab’ contains a de-identified version of the dataset generated in this study.Due to the nature of this research, data provided is a limited de-identified dataset without potentially identifying information, i.e. clinical specialty (if applicable) and journal characteristics.Individual(s) wishing for access to the full de-identified dataset requires written support from the principal investigator  to be added to the study as study personnel(s), and subsequently needs approval from the Unity Health Toronto Research Ethics Board in Toronto, Canada.https://doi.org/10.7910/DVN/AHMB8G18.Harvard Dataverse: Journal editors: How do their editing incomes compare?File ‘Journal Editors Supplementary 2 - Survey.docx’ contains a copy of the survey used in this study.https://doi.org/10.7910/DVN/AHMB8G/XPMGJL18.Harvard Dataverse: CHERRIES checklist for ‘Journal editors: How do their editing incomes compare?’.Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the Amendments are minor and do not change my previous view of "Approving" the article.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Labour Economics; Applied Econometrics, including published works on gender in the workplace and the gender pay gap.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have addressed the vast majority of my previous comments in their current version.  Minor point: The authors state "2017 Journal Impact Factor, and frequency of publication (best fitted to monthly/bimonthly vs. weekly/biweekly), were extracted from the journal selected in the survey."  I would specify where journal impact factor was extracted from - many journals no longer list this information on their website. In these cases was this coded as missing? Or, was the data searched for elsewhere?Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Journalology; publication science; open scienceI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I am satisfied that the authors have taken on board my comments, as far as possible given their current limited access to the data. The authors are also fair and transparent with their methods, assumptions and the potential limitations of the study.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Labour Economics; Applied Econometrics, including published works on gender in the workplace and the gender pay gap.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This study aimed to quantify gender differences in pay for editing services within academia, specifically for “Core Clinical” journals. It also aimed to test for the presence of a gender pay gap, conditional on other observable (surveyed) characteristics of editors and their journals. Overall, the article is well-written, the methods are clearly explained, and some of the limitations are fairly outlined. Cobey (2020) has already provided a thoughtful review of this article, and I would echo those comments.In the introduction and the limitations, the authors may wish to dwell on the other reasons that editors choose to do what they do, besides direct financial gain . These motivations are all potentially endogenous factors (omitted variables) in the regression model. While in theory they may not be correlated with gender in the population of editors, the sample size here is small and the response to the survey probably non-random.1 in theBritish Journal of Industrial Relations. While that study focuses on the gender pay gap among economists, it also provides some prominent examples of the wider literature on the gender gaps in academia, including in Medicine and Science . The authors could look to reference more of this previous work, particularly since this journal is not field-specific.The literature cited for the gender pay gap is in academia is limited and very specific, focused on medical sciences. But there are good examples and literature reviews of gender pay gaps in academia more widely. One recent example I am aware of is Mumford & Sechel (2020)et al., 2020), there are some questions asked that are not being used in the analysis. Therefore, the article could be clearer on what information is being thrown away. For instance, for the unadjusted odds ratios, I am not sure why an editor preferring not to say what the frequency of their publication is should exclude them from the sample when estimating the raw gender pay gap. Overall, it is important that the authors explain why they don’t consider a larger sample for their statistics just because of some non-response to some survey questions.The authors state that “Only completed surveys were analyzed”. Elsewhere in the article it is mentioned that responses where editors “preferred not to say” were discarded. Having looked at the survey . Results following this alternative empirical strategy could be presented as a complement to those already shown in the article.Like the previous comment, I am also concerned about the amount of information lost in the chosen modelling strategy. The survey collects information on much narrower bands of pay (and hours). While I appreciate that in this field odds models are common and often preferred, in my own field, as an applied economist/econometrician, we would prefer not to lose all the information contained in the pay data collected. For example, I would suggest also estimating a censored linear least squares regression model , where the mid-point of each and every range in the survey is imputed as the pay value, which becomes the dependent variable . Similarly, combining the narrow pay bands and narrower hours bands in the survey could allow the authors to look at a measure of hourly pay, which is what matters most economically, being the compensation for a unit of labour input .The Introduction claims that the UK led the wag on gender pay gap reporting legislation. Denmark and Austria have legislation requiring firms to make their pay gaps available to their workers, which I believe substantially pre-date the UK policy  Thank you. Revised to address this limitation in both the introduction and limitations sections. Re: #2) Thank you. This was added to the limitations section. Mumford & Sechels 2020 and Connolly and Hodcroft 2009 were cited. Re: #3) Thank you. This was clarified in the manuscript. Of the 166 surveys, 140 were analyzed, excluding surveys that had incomplete responses. Responses of “prefer not to answer” were analyzed but often had small cell counts (n<5), and therefore the data were not reported to preserve privacy of participants. Re: #4) We did not perform the tobit regression, since we did not have exact salaries and cannot make an assumption regarding the mid-point values for the last category of salaries greater than $150,000 USD. In analyzing the zero/nonzero pay by male/female to explore censoring from the zeros, we found the following: chi-squared 1.35 (p=0.25) 2x2 table: Zero salary, male: 37 Zero salary, female: 13 Nonzero salary, male: 58 Nonzero salary, female: 32 This analysis has not been included in the article. Re: #5) We presented the data based on male/female as primary exposure and did not examine for possible interaction of gender with covariates. We are unable to return to the data at this time to update the analysis to explore this possibility. Re: Minor #1) Thank you. This has been revised to add Denmark and Austria’s reporting legislation. quantify remuneration for journal editors from core clinical journalsdetermine if a gender pay gap existsassess if there are remuneration differences across publishing models and journal characteristics. The authors addressed these aims through conducting an online survey of journal editors identified from the Abridged Index Medicus “Core Clinical” journals in MEDLINE.This study aimed to:The authors specify:2017 Journal Impact Factor, frequency of publication (monthly/ bimonthly vs. weekly/biweekly), editing role , years in editing (>10 vs. ≤10 years), hours/week in editing role (>10 vs. ≤10 hours), and years worked for current journal (>5 vs. ≤5 years) .”“The following variables were collected in our survey and included in the final multivariable model: sex , gender identity, publishing model , primary role , academic rank (any professorship vs. none), The paper is reported clearly and acknowledges relevant limitations. I describe a few suggestions for changes below:Further, the authors indicate the survey captured the frequency at which the included editors’ journals publish using the options: monthly/ bimonthly vs. weekly/biweekly. This is unlikely to apply uniformly across journals. For illustration, I don’t know how the F1000 journal editor would accurately respond to this item.Did the authors record bounce-backs among editors who they e-mailed their invitation to? In comparable work I have conducted this has been a fairly significant issue, especially when editor information is found via Google rather than through the journal website. If so, what was the N and could this be reflected in the study recruitment description and figure? The response rate may be somewhat higher if this is considered.  Minor comments:Given that this is not a sub-specialty journal, the authors could consider briefly addressing what the role and responsibilities of journal editors are in the introduction.The authors note: “The online survey was conducted using Qualtrics CoreXM Survey Tool software14, but an open access alternative such as SurveyMonkey15 could be used for replication of methods. ; I don’t think the free SurveyMonkey would be appropriate for replicating the survey described, I understand it allows for only 10 questions (each sub question ‘counts’) and 100 responses.The authors could provide rational for the journal sampling approach. When I review the survey provided by the authors, I don’t see any items asking about the 2017 journal impact factor or journal frequency of publication. Perhaps I missed something? If not, this leads me to think that perhaps the authors extracted this information online based on participants responses indicating which journal they are an editor at, or that the incorrect survey has been uploaded. This should be clarified in the paper.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Journalology; publication science; open scienceI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Re: Extracting journal information--Yes, revised to state that journal information was extracted online based on journal selection in the survey. Re: Journal frequency—Journal frequency was extracted from journals and rounded up/down to fit the categories. Re: Bounce-back emails—Yes, revised to clarify that there were 104 bounce-backs. The response rate was calculated based on the number of successful emails sent. Re: Minor comment on role of journal editors—Thank you, this has been added to the introduction. Re: Minor comment on free survey software—Thank you, this has been removed. To our knowledge, there are no easily accessible free survey software for survey replication. Re: Minor comment on the rational for journal sampling—Revised to state that we chose the core clinical journal for their high clinical impact on medicine.


Currently, along with titanium (Ti), zirconia is widely used as an abutment material for dental implants because it makes it possible to avoid gingival discoloration; however, the epithelial sealing capability of zirconia remains unknown. The purpose of the present study is to elucidate the localization of integrin β4 subunit (Inβ4), one of the main proteins in the attachment structure between gingival junctional epithelial (JE) cells and substrata. Maxillary first molars were extracted from rats, and implants were placed with Ti or zirconia transgingival parts; then, the localization of Inβ4 was observed. Morphological and functional changes in rat oral epithelial cells (OECs) cultured on a culture dish (Dish) and Ti and zirconia plates were also evaluated with Inβ4 immunofluorescence histochemistry and Western blotting. After four weeks of implant placement, the morphology of the peri-implant epithelium (PIE) and the localization of Inβ4 around the Ti and zirconia transgingival parts were similar. However, both exhibited markedly shorter Inβ4-positive bands in the PIE than in the JE around natural teeth. Decreased expression levels of Inβ4 were observed in OECs cultured on Ti and zirconia plates compared with those cultured on Dish. In conclusion, although inferior to natural teeth, zirconia implants are thought to have epithelial sealing properties comparable to those of titanium. Possessing excellent characteristics for osseointegration and soft tissue integration, titanium (Ti) is widely used as a dental implant material. However, its metallic color can occasionally be seen through the gingiva, and this gingival discoloration decreases soft tissue esthetics. In patient-defined success criteria for dental implant treatment, esthetics and occlusal function are emphasized. In this context, zirconia was introduced in implantology owing to its superb physical strength and white color, avoiding gingival discoloration ,2.In the case of natural teeth, a strong sealing structure termed “biologic width” is formed at the tooth–gingiva interface. Specifically, the junctional epithelium (JE) strongly attaches to the enamel surface with hemidesmosomes. Integrin (In) α6β4 is a transmembrane protein dimer of JE cells, and it is a main component of hemidesmosomes ,4. This The cervical portion of the implant component can be a source of inflammation around an implant because the implant penetrates the oral mucosa, and plaque control in this area is often insufficient. Although peri-Ti implant soft tissue was reported to possess biologic width and hemidesmosome–basal lamina connection, its sealing capability is inferior to that of periodontal biologic width ,7. By usWe therefore focused on the localization of integrin β4 subunit (Inβ4) at the interface between gingival soft tissue and zirconia. As mentioned above, Inβ4 is a hemidesmosome constituent, and can be an indicator for the secure attachment of PIE to zirconia. In the present study, the immunohistochemical localization of Inβ4 was investigated, and the distribution of Inβ4 with that around Ti and teeth was assessed using animal model and cell culture studies.All experiments were performed in accordance with the ARRIVE Guidelines for reporting animal research . All pro2O3-doped 3.0 mol% Y2O3-stabilized tetragonal zirconia polycrystal, Tosoh, Tokyo, Japan), were fabricated . The dimension of the transgingival column was 4 mm in height and 2 mm in diameter  after demineralization using a 5% ethylenediaminetetraacetate solution. For immunohistochemical staining, these sections were incubated with rabbit anti-Inβ4  and biotinylated anti-rabbit IgG , and the presence of Inβ4 was visualized using a diaminobenzidine (DAB) staining kit , as described in a previous paper . In the p < 0.05 were considered statistically significant.Data are indicated as means ± standard deviation (SD). One-way analysis of variance with post hoc Scheffe’s test was performed. Values of 2O3-doped 3.0 mol% Y2O3-stabilized tetragonal zirconia polycrystal) and pure Ti  plates  were used for this experiment. The surface roughness value (Ra) of each plate was identical to the implant surface for the animal study. Oral epithelial cells (OECs) were obtained from the oral mucosa of 4-day-old Wistar rats as reported previously [3 IU/mL) in Mg2+- and Ca2+-free Dulbecco’s phosphate-buffered saline for 12 h at 4 °C and removed from the connective tissue layer. Cells were cultured in Defined Keratinocyte Serum Free Medium  to eliminate contamination with cells other than epithelial on a culture dish  and Ti and zirconia plates  and fluorescein isothiocyanate-labeled secondary antibody . Actin filaments were stained with tetramethylrhodamine isothiocyanate-conjugated phalloidin . Imaging was performed using fluorescence microscopy .Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis, transferred to polyvinylidene difluoride membranes  and immunoblotted with the anti-Inβ4 antibody. Antibody-bound bands were visualized using an imaging device .A positive band of Inβ4 was observed at JE along the surface of the enamel B, left. The signal of Inβ4 was scattered in the cytoplasm of OECs seeded on Ti and zirconia plates A, left. Peri-implantitis and subsequent tissue breakdown are the main reasons for the loss of implants . WeaknesIn our animal study, there was no statistically significant difference in the length of the Inβ4-positive band between Ti and zirconia groups, both of which were significantly shorter than that for teeth. As previously reported, the length of the laminin-332-postive band on the substratum side of the attachment structure was significantly shorter around Ti than that around teeth ,11. The Our cell culture study indicated that the expression of Inβ4 in gingival epithelial cells was also similar between Ti and zirconia groups, and they were significantly inferior to those seeded on the culture dish. Specifically, in cells cultured on the culture dish, Inβ4 signals were observed around nuclei and in the cytoplasm. This means that Inβ4 was synthesized in cells cultured on the culture dish. In contrast, Inβ4 was not actively synthesized in cells seeded onto Ti or zirconia. Western blot analysis also showed a similar tendency. According to a previous report, the expression of Inβ4 on the Ti plate is weaker than that on the culture dish , and thiThe arrangement of actin fibers was also inferior in cells cultured on both materials. This also implies that the attachment of epithelial cells to both Ti and zirconia is weaker than that to the culture dish.The limitation of the present study is that rats were used as an in vivo oral implantation model. Although this model has been widely used ,11,15,20On the basis of our results, zirconia can also be used as a material for the transgingival part of implants from the point of view of epithelial attachment. Since this material can avoid gingival discoloration, further knowledge such as on mechanical properties and plaque retentivity should be accumulated, with the aim of using this material for the transgingival part of implants with excellent long-term prognosis.Within the limitation of the present study, we conclude that the expressions of Inβ4 in epithelial cells on Ti and zirconia were similar and inferior to those in periodontal epithelial cells. This implies that, from the viewpoint of epithelial attachment, although it is obvious that both materials are inferior to enamel, zirconia can be used as the transgingival part of an implant in an equivalent manner to that of Ti.


Analysis of our predicted epitopes for population coverage showed prominent population coverage (92.43%) around the world. Finally, molecular docking simulation of the foreseen T cell epitopes with respondent HLA alleles secured good HLA-epitope interaction. This study was directed towards the discovery of potential antigenic epitopes which can open up a new skyline to design novel vaccines for combating both of the diseases at the same time.Being a Positive sense RNA virus the recent reemergence of Chikungunya and Mayaro virus has taken the concern of the leading scientific communities of the world. Though the outbreak of Mayaro virus is limited to Neotropical region only, Chikungunya is already identified in over 60 countries around the world. Besides, the lack of a strong protective treatment, misdiagnosis issue and co-circulation of both the viruses calls for a new strategy which could potentially prevent these infections from spreading. In this study, we therefore, identified the peptide based vaccine candidates e.g. epitopes for B cell and T cell from Chikungunya virus which also showed to be homologous to the Mayaro virus through immuno-informatics and computational approaches. Final epitopes identified from the most antigenic structural polyprotein of both the viruses were 5 for CD8+ T cell Epitopes ( Chikungunya virus; Mayaro virus; Epitopes; Vaccine B as well . However as well . Previou as well . On the  as well . Further as well .CHIKV genome can be characterized as an (+)-sense ssRNA genome of 11.6 kb in length that encodes 5 structural proteins  and 4 non-structural polyproteins  . MAYV alAt present there are no approved definite vaccines at hand for CHIKV and MAYV, while scientist are trying hard to develop a specific treatment for these viruses. Only a live attenuated vaccine for CHIKV is now currently available in phase-2 trial but 85% of test showed resistance after one year . Therefo22.1The structural polyprotein of both of the viruses (Chikungunya and Mayaro) were targeted for epitope prediction/selection as the viral structural proteins are involved in cell entry and subsequent fusion of the virus inside host cells.https://www.uniprot.org/) and then saved in FASTA format for further antigenic properties analysis.Hence, the Swiss-Prot reviewed structural polyprotein sequences of both the viruses (Chikungunya and Mayaro) were regained from UniProt Knowledge Base (UniProtKB) database (2.2http://ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) which is able to calculate antigenic proteins more than 80% accurately [A toxin or other foreign components that stimulates the body's immune response, particularly in antibody production are known as antigens. To assess the best possible antigen, all the retrieved structural polyproteins of both of the viruses were subjected to computational antigenicity investigation in widely applied server known as VaxiJen2.0 (curately . The cut2.3http://expasy.org/cgi-bin/protpraram) named ProtParam was employed [To identify the functional physiochemical parameters of the selected antigenic polyprotein, a widely used tool of Expasy server (employed . ProtPar2.4http://www.cbs.dtu.dk/services/NetCTL/) was used to identify CD8+ or cytotoxic T cell (CTL) epitopes from the selected structural polyprotein of Chikungunya virus [NetCTL 1.2 (ya virus  dependin2.5http://tools.iedb.org/mhcii/) which is well known prediction tool for MHC II Binding. A total of 7 reference set of human HLA alleles  from the same server were selected for CD4+ T cell epitope prediction [The 15 mer CD4+ or helper T cell epitopes (HTL) of the structural polyprotein of Chikungunya virus were identified using IEDB webserver (ediction . To asseediction  was util2.6http://ailab.ist.psu.edu/bcpred/) [Linear B cell lymphocyte (BCL) epitopes of the selected Chikungunya structural polyprotein were identified from the BCPRED webserver (bcpred/) . This sebcpred/)  and the 2.72.7.1The epitopes for vaccine candidate against Chikungunya virus that were preliminary identified by different computational studies only designed on the basis of CHIKV structural polyprotein for the selection of B cell and T cell epitopes. To show potency as vaccine candidate for MAYV, all of the predicted epitopes from CHIKV should be match as a conserved and homologous sequence in the structural polyprotein of MAYV as well. That's why all the predicted epitopes from CHIKV structural polyprotein were also screened through structural polyprotein of MAYV to recognize the conserved epitopes in both of the viruses.Furthermore after conservancy analysis, the desired conserved epitopes of both of the viruses were then again ran in the conservancy analysis tool against Dengue Virus  and Zika Virus's reference polyprotein to check if any of the epitope is conserved and similar in those polyprotein or not. If our predicted epitopes are matched in these polyproteins, there may be a cross reaction possibility can be occurred.2.7.2After conservancy analysis in the reference variants, we also screened the conserved and homologous epitopes of both of the viruses (CHIKV and MAYV) in the all representative sequence of all the variants. For this, first of all we have identified all the representative sequences of both of the viruses' structural polyprotein from NCBI database. A total of 100 and 64 sequences were found and retrieved as a representative sequence of CHIKV AND MAYV respectively.http://tools.iedb.org/conservancy/) for the mayaro virus structural polyprotein [However, to get epitope conservancy patterns of the shortlisted and promising epitopes, online based tool viz., conservancy analysis at the IEDB (Immune Epitope Database) was used , AllergenFP (https://ddg-pharmfac.net/AllergenFP/), and ToxinPred (http://crdd.osdd.net/raghava/toxinpred/) webservers were applied consecutively to find out the most appropriate antigenic epitopes, the allergenic and toxic activity of the short listed B cell and T-cell epitopes respectively [http://crdd.osdd.net/raghava/ifnepitope/) server was used to predict IFN-γ induction capability of the proposed epitopes [Thus, the VaxiJen /Human Leukocyte Antigens (HLA) interacting with selected individual CTL epitope from the structural polyprotein of CHIKV were selected by using B server . The IEDB server . The outB server .2.10The population coverage of a vaccine candidate indicates its wideness of fortification against the respective disease among various ethnics in the world for effective vaccination. But in reality it is quite difficult to make a broad spectrum vaccine that would cover all countries and races due to the high degree of polymorphism of major histocompatibility complex molecules (approximate 6000). Thus, the binding ability of epitopes with numerous HLA could raise population coverage as well as effective for further shortlisting of the epitopes.http://tools.iedb.org/population/) provided by the IEDB server [Therefore, the population coverage tool (B server  has used2.11https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD/) was exploited to build the precise three dimensional structure in PDB format of the selected CD8+ and CD4+ epitopes [PEP-FOLD3 (epitopes . PEP-FOL6AT5).The interaction of the obtained T cell epitopes with MHC-I allele of HLA-B∗07:02, HLA-B∗51:01, HLA-B∗53:01, HLA-B∗35:01, HLA-B∗08:01 and HLA-B∗44:02 were observed in all cases with minimum IC50 value. But, we chose one of the MHC-I allele (HLA-B∗07:02) from the above list for docking analysis as the availability of the crystal structure of the protein in Protein Data Bank (PDB ID: 1BX2). However, a refinement of the whole protein structure is required prior to docking for improving the docking analysis beyond the precision level. So, we followed a standardized Protocol for molecular docking used in previous studies e.g. elimination of previously attached peptide, protonation, energy reduction followed by elimination of H2O molecules from the retrieved crystal structure [https://servicesn.mbi.ucla.edu/PROCHECK/) for Structural validation of the refined HLA proteins [Further, the MHC- II alleles of HLA-DRB1∗15:01, HLA-DRB1∗07:01, HLA-DRB5∗01:01, HLA-DRB3∗02:02 were also observed to interact with the final CD4+ T cell epitopes. However, we chose the HLA-DRB1∗15:01 allele for docking analysis for the same reason describe above  on PyRx interface [6AT5) led us to choose these epitope as a control for MHC- I binding in our study. As well as, in case of MHC- II complex, the control epitope used was 15 amino acid long myelin basic protein (“ENPVVHFFKNIVTPR”) for HLA-DRB1∗15:01 recognition (PDB ID: 2Q6W).Though the interaction of the final CD8+ and CD4+ T cell epitopes with the corresponding HLA (MHC-I & MHC-II) can be predicted by IEDB webserver, the HLA-Epitope interaction should be examined by analyzing nterface , a colleFor docking, PDB files of both T cell epitopes  and corresponding alleles (MHC- I and MHC- II) were uploaded to the PyRx AutoDockvina wizard. After successful completion, the docked complexes were then visualized in discovery studio visualizer.33.1The current study was designed on the basis of the available proteomic and genomic data of CHIKV and Mayaro virus's polyproteins. A total of 4 Swiss-Prot reviewed CHIKV structural polyproteins were found in the Uniprot Database. Then the antigenicity values of all 4 reviewed proteins were computed by auto cross covariance (ACC) conversion at Vaxijen webserver. All subjected proteins exhibited different antigenic value ranging from 0.5163 in CHIKV structural polyproteins of strain S27-African prototype to 0.5319 in CHIKV Frameshifted structural polyprotein (strain S27-African prototype) . As Fram3.2in vitro  in mammalian reticulocytes. Further, ProtParam tools predicted other physicochemical characteristics of both of the polyproteins such as molecular weight, theoretical pI, aliphatic index and Grand average of hydropathicity (GRAVY) value which are shown in ProtParam tools of the Expassy server disclosed many crucial properties of the top ranked antigenic polyprotein of both of the viruses. For example, the instability indexes of both of the highest antigenic polyproteins were not found as satisfactory but both of the polyproteins showed long longevity 3.3For identification of T cell epitopes from both of the viruses, we used the CHIKV Frameshifted structural polyprotein as the referral protein. Then, in the conservancy analysis, we predicted the overlapped epitopes which found homologous to the Mayaro virus's structural polyprotein.Initially NetCTL 1.2 webserver predicted 245 potential CD8+ T cell epitopes or CTL (9 mer) contrary to 12 major histocompatibility complex super-classes (Supplementary Table 1) from the Frameshifted structural polyprotein of CHIKV. The threshold for the prediction was set at default (0.60).Further, IEDB webserver predicted 306 CD4+ T cell epitopes or HTL (15 mer) against 7 reference set of human HLA allele from CHIKV Frameshifted structural polyprotein (Supplementary Table 2).3.4At 0.8 cut-off value, BCPRED webserver predicted 22 B cell epitope from the Frameshifted structural polyprotein of CHIKV (Supplementary Table 3). However, the epitopes predicted by all of the webservers were filtered through several pipelines  to sort out the most potential epitopes in subsequent steps.3.53.5.1http://tools.iedb.org/conservancy/) was applied to observe the pattern of each of the B cell epitopes and T cell epitopes in the protein sequence of the Mayaro virus's structural polyprotein to filter out the conserved common homologous epitopes of both of the viruses (CHIKV and MAYV). Epitopes which showed 100% conservancy were selected for further analysis. Thus, after conservancy analysis, a total of 9 (nine) CTL, 10 (ten) HTL and 2 (two) B cell epitopes were found showing 100% conservancy in the structural polyprotein of both CHIKV and MAYV  to see if these epitopes were similar to this sequences or not. If any of the epitope was similar, then, there may be a cross reaction could occur. However, none of these epitopes showed conservancy against Dengue virus and Zika virus's referral proteome (Supplementary Table 4). So, it can suggest that there is no possibility of this epitope to be cross reactive with other arboviruses. The dataset for Dengue Virus and Zika virus's referral polyprotein can be found from Supplementary File 1.After conservancy analysis the predicted 9 (nine) CTL, 10 (ten) HTL and 2 (two) B cell epitopes were then again ran in the IEDB conservancy analysis tool against Dengue Virus and Zika virus's reference polyprotein  of CHIKV and MAYV in the all representative sequence of all variants. Maximum epitopes in our study were found as 100% conserved in all the 100 variants of CHIKV. However, for MAYV, out of 64 representative sequences, maximum 49 variants showed 100% conservancy for all the epitopes (Supplementary Table 5). As, none of our epitopes showed cross reaction against Dengue and Zika viruses referral proteome, we have omitted the analysis this for these viruses.3.6The predicted conserved epitopes from the previous steps needs to be analyzed for antigenicity, allergenicity, toxicity and immunogenicity properties prior to concede as potential vaccine candidates.105MCMKIENDCIFEVKH119 and 502DRTLLSQQSGNVKIT516) from the above list by eliminating the non-immunogenic epitope 504TLLSQQSGNVKITVN518  CTL (CD8+) epitopes, 3 (three) HTL (CD4+) epitopes and 1 (one) B cell epitopes . AdditioKITVN518 .Table 4A3.7The MHC-I and MHC-II binding prediction tool from the IEDB webserver predicted the corresponding MHC-I and MHC-II allele of the final 5 CD8+ (CTL) epitopes and final two HTL (CD4+) epitopes and shown in Tables 3.8IEDB provided population coverage analysis tool was applied to predict the MHC I combined population coverage of the final five CD8+ T cell epitopes against a set of area around the world. However, the “HLA-DR” alleles in this server aren't updated for this analysis yet and that's why we skipped the MHC II population coverage analysis. The results showed the highest population coverage in the North America (94.82%) and lowest population coverage in the Central America (6.44%). Furthermore, Combination of the five epitopes can cover 92.43% of average world population predicted by the webserver .Figure 13.9To predict the 3D structure of the final CTL and HTL epitopes, PEP-FOLD3 Peptide Structure Prediction server was used . Further3.10209KPGDSGRPI217 has the highest epitope score, we selected these epitope and HLA for MHC-I binding Molecular docking analysis. The control epitope used here is 13 amino acid long NY-ESO-1 peptide (“APRGPHGGAASGL”) to compare with our proposed CTL epitope. After epitope selection, The PDB files of each epitopes and refined 3D structure of the HLA were uploaded in the PyRx AutoDock Vina interface. To give the maximal space to the ligand for their liberal movement inside the search space which were fixed to 58.52Å in the X, 44.56Å in the Y and 71.36Å in the Z direction, so we made the grid box large enough during docking simulation. After docking simulation, the binding energy for epitope 209KPGDSGRPI217 to the binding groove of the HLA-B∗07:02 was found -6.8 kcal/mol. This binding energy represents good HLA-Epitope interaction in contrary experimental epitope which was -4.6 kcal/mol. Further, to examine for MHC-II binding interaction we used the epitope 502DRTLLSQQSGNVKIT516 as it showed maximum allele binding affinity . However, we finally subjected the HLA-DRB1∗15:01 for docking analysis as the readily available crystal structure of it's in the PDB database. The control epitope used here is 15 amino acid long myelin basic protein (“ENPVVHFFKNIVTPR”). The actual size of the grid box was slightly varied from the previous simulation and was fixed at 43.92Å, 76.37 Å and 55.25 Å in X, Y, and X axes correspondingly. After docking completion, the free binding energy of the epitope 502DRTLLSQQSGNVKIT516 and HLA-DRB1∗15:01 was found - 4.5 kcal/mol whereas the control epitope showed -5.6 kcal/mol with HLA-DRB1∗15:01. The interacting residues of the docked complexes of HLA-Epitope are shown in A structure based molecular docking was performed for analyzing the free binding energy and docking pose of each of the epitope-HLA molecule by utilizing AutoDock Vina in PyRx 0.8. For MHC-I-epitope interaction, as most of the epitope showed good binding affinity with HLA-B∗07:02 and 4From the last decades, more than 55 countries around the world have reported about the outbreak of reemerging CHIKV which caused human fatal chikungunya fever with muscle pain, joint pain, joint swelling, and rash . Similarin silco peptide vaccine designing with reverse vaccinology approach is getting popular day by day because of its low threats of re-activation as like as live attenuated vaccine. The first epitope based vaccine was designed against N. meningitides using bioinformatics and after that many of the vaccines are designed for different infections including Dengue virus [However, several attempts based on designing of innovative subunit vaccine against CHIKV had failed, in the contrary, numerous epitope based vaccine candidates against particular microorganism are showing very promising in preclinical studies . For exaue virus , Nipah vue virus , Ebola vue virus , Herpes ue virus , human nue virus  and so o209KPGDSGRPI217, 219TGTMGHFIL227, 239ALSVVTWNK247, 98KPGRRERMC106 and 100GRRERMCMK108) out of 245, 2 HTL (105MCMKIENDCIFEVKH119 and 502DRTLLSQQSGNVKIT516) out of 306 and 1 BCL (504GGRFTIPTGAGKPGDSGRPI518) out of 22 as the most potential candidates. Furthermore, population coverage analysis of our 5 potential CTL epitopes showed outstanding average population coverage (92.43%) around the world where the standard world population coverage was at least 40% [Considering all the facts above, this study was conducted to identify the common epitopes between the structural polyproteins of CHIKV and MAYV which could assure a good protection against both of the viruses as vaccine candidates. The structural polyproteins were targeted in this study since both CHIKV/MAYV structural polyprotein plays a crucial role in viral attachment, penetration and subsequent fusion with host plasma membrane to form endosome within human cells. Theoretically, any vaccine substance must have two types of antigenic or immunogenic binding sites namely B cell and T cell epitopes to grow immunity in an individual . B cell east 40% . Lastly,One limitation of our approach is that the length of the epitope itself. Generally, the length of an epitope isn't sufficient enough to induce immunization in vivo. Besides, though epitope based vaccine is safer than conventional vaccine (e.g. live attenuated vaccine); there is a possibility of enzymatic degradation of the epitopes in vivo. So, the peptides found in our study needs to be structurally or physically modified prior to use as a vaccine candidate. This could be done by using different strategies like prodrug approach, peptidomimetic method, analogue developments, hydrophobic ion coupling, adjuvant linking, and joining with fatty acids. By applying these approaches predicted peptides physiochemical stability can be enhanced. Of note, researchers have been working to eliminate these viruses by understanding theirs pathophysiology and transmission. We hope that, our predicted epitopes may be evaluated for further in vivo research and clinical assay to develop a potential vaccine which can eliminate these neglected tropical diseases beyond any doubt.5This study discovered viable epitope candidates for designing an epitope-based peptide vaccine against Chikungunya and Mayaro virus concomitantly. Furthermore, this study found the predicted prominent epitopes won't have any cross reacting activity with other arboviruses e.g. Dengue and Zika virus. However, though epitope based vaccine has several limitations regarding the size of the epitopes, we anticipated that, after further wet lab based experiments, our predicted epitopes may develop secure, reproducible and genetically stable vaccine that could induce an effective immune response in future.Hammadul Hoque: Conceived and designed the experiments.Srijon Ghosh, Rahatul Islam: Performed the experiments; Wrote the paper.Mashiur Rahaman, Nurnabi Azad Jewel: Analyzed and interpreted the data.Abunasar Miah: Contributed reagents, materials, analysis tools or data.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


The purpose of this research was to investigate and improve the accuracy of the existing slab-track mat (STM) specifications in the evaluation of the vibration reduction effect. The static nonlinearity and dynamic mechanical characteristics of three types of STMs were tested, and then a modified fractional derivative Poynting–Thomson (FDPT) model was used to characterize the preload and frequency dependence. A modified vehicle–floating slab track (FST) coupled dynamic model was established to analyze the actual insertion loss. The insertion loss error evaluated by the frequency-dependent tangent stiffness increased with the increase in STM nonlinearity, and the error obtained by the third preload tangent stiffness was usually greater than that of the second preload. Compared with the secant stiffness, the second preload frequency-dependent tangent stiffness was more suitable for evaluating STMs with high-static–low-dynamics (HSLD) stiffness. In order to reflect the frequency dependence effect and facilitate engineering applications, it is recommended that second preload tangent stiffness corresponding to the natural frequency of the FST be used for evaluation. Furthermore, the insertion loss of the STMs with monotonically increased stiffness decreased as the axle load increased, and the opposite was true for the STMs with monotonically decreased stiffness. The vibration isolation efficiency of the STMs with HSLD stiffness was both stable and better than that of the STMs with monotonic stiffness. The convenience and energy-savings of urban rail transit (URT) have driven an increase in urban transportation-oriented development (TOD) construction. For instance, 46 cities in China have constructed 7142 km of URT lines, and the fastest operating speed has reached 160 km/h. However, the high density rail transit network and the fast operating speed inevitably cause more obvious environmental vibrations and noise problems , such asOptimizing the stiffness of the damping pad to reduce environmental vibration and structural noise has been extensively studied. Vogiatzis and Kouroussis ,12 estabSome theoretical models with a better performance have been widely used to accurately reflect the impact of the polymer’s mechanical properties on the track’s vibration characteristics. Wei et al. described the dynamic viscoelasticity and plasticity of a Vossloh 300 soft rail pad with a modified fractional derivative Zener (FDZ) model and a traditional Berg friction model, and analyzed the influence on the high-speed vehicle–slab-track coupled vibration . Zhu et In China, the provisional technical regulation TJ/GW 121-2014  indicateTo evaluate the VRE of the STMs conveniently and accurately, we improved the existing standard method by comparing the models that can reflect the actual dynamic characteristics. A modified FDPT model was proposed , which cTo describe pronounced nonlinear behavior depending on the preload and frequency, the modified KV  and PT  viscoelaP and the corresponding displacement is x. This model can be divided into two parts: the elastic spring part (K1) and the fractional derivative Maxwell (FDM) part consisting of the spring (K2) and Abel dashpot (K2γτ) in series. In this section, the classical FDPT model is first transformed into a temperature-dependent modified model, and then the preload-dependent nonlinear static stiffness and the frequency-dependent dynamic stiffness are respectively derived.The modified FDPT model is shown in τ is the relaxation time of the linear viscoelastic material and γ is the derivative order. It is worth mentioning that unlike the usual generalized fractional model, the spring stiffness in the FDPT model is considered separately. K1 and K2 reflect the static preload-dependent and dynamic frequency-dependent characteristics, respectively.The time-domain constitutive equation of the classic FDPT model and the complex stiffness obtained by Fourier transform (FT) are as follows:*K can be also written as the following equation. The specific calculation procedure can be found in [According to the time-temperature superposition (TTS)  of polymfound in .(3)K*, and the corresponding densities, respectively. α(T) is the conversion coefficient of the temperatures, in accordance with the following equation:In Equation (3), Tg) is regarded as the reference temperature, C1 and C2 are 17.44 and 51.6, respectively. If another temperature is selected as the reference temperature, C1′ and C2′ at the corresponding reference temperature can be calculated by using Equations (5)–(7).When the glass transformation temperature  measured by the harmonic excitation and the static nonlinear K1 obtained by fitting, the stiffness K2 associated with frequency dependence under different preload conditions can be shown by the following equations:rP and K2′ are a reference preload and the corresponding stiffness value, respectively. rβ is an exponential function related to the preload.After obtaining the complex stiffness (K′), loss stiffness (K″), and loss factor (tan δ) can be obtained as follows:The temperature-, frequency-, preload-dependent storage stiffness , three kinds of damping pads to be used by Guangzhou URT were adopted as the test objects—see The instrument is shown in During the test, the minimum load applied was the FST weight supported within the sample area, and the maximum load was the sum of the minimum load and axle load supported within the sample area when the vehicle ran above the track. It is worth noting that when the speed reaches 160 km/h, the axle load needs to consider a certain dynamic coefficient of 1.3. Three adjacent floating slabs were established by Ansys software , and the3, and the corresponding maximum load increases from 4.0 to 4.2 kN. To facilitate testing, the minimum load and maximum load were determined to be 0.8 and 4.1 kN, respectively.The maximum loads obtained by the different STMs’ stiffness were slightly different. The stiffness of the STMs increases from 0.01 to 0.03 N/mmA series of experiments were carried out to scientifically test and characterize the dynamic properties of the STMs see . Case 1 According to the test procedures, the static and dynamic characteristics of the test samples of the STMs were tested.Static loading with a nearly zero velocity was carried out on the STMs within the load range of 0 kN to 5 kN at an interval of 0.5 kN. The measured data and fitting results of the nonlinear force–displacement and stiffness–force curve are shown in a1, a2, a3, and a4 of STM-I, STM-II, and STM-III are shown in Although the force–displacement curve of STM-I in In order to avoid repetition, only part of the dynamic hysteresis loops within the service load range are shown, because its load and displacement are larger and the hysteresis loop area is more obvious see .It can be observed that the frequency-dependent stiffness and loss factor of STM-I and STM-II are basically the same, while STM-III not only has obvious frequency-dependent stiffness but also a higher loss factor . According to these measured hysteresis loops, the stiffness at different preloads and frequencies can be obtained. The test data and fitting results of secant stiffness and tangent stiffness of the STMs are shown in −5 and 6.83 × 10−5, respectively) are relatively small. The frequency-dependent effect of STM-III with strong nonlinearity in the service load range is obvious, and the stiffness growth rate is 1.25 × 10−4. For STM-III, which has a relatively small static stiffness in the load range of 2–3 kN, a moderately large frequency change effect can also effectively suppress the dynamic displacement of the floating slab. It is worth noting that the secant stiffness of STM-I and STM-II is between the preloaded tangent stiffness of 2 and 4 kN, while the secant stiffness of STM-III is greater than the preloaded tangent stiffness of 1 and 5 kN.Under different preloads, the stiffness of the STMs at a frequency approaching 0 is basically consistent with the static stiffness in K2′ and rP in the fitting. The fitting parameters K2′, r, τ, and γ are shown in The fitting process under a single preload can be directly calculated using Equation (2), and the stiffness and preload force values with a reference preload of 0.5 kN are selected as Using the modified FDPT model in In order to accurately evaluate the influence of the preload- and frequency-dependent characteristics of the STMs on the VRE of the rail transit system, a vertical vehicle–DPFST coupled dynamic model combined with the FDPT model for the STMs was used to investigate a URT under construction in Guangzhou see . In the The railway vehicle was generally simulated as a multi-rigid-body model with two suspensions. The degrees of freedom mainly included the up and down and nodding movement of a vehicle body, two bogies, and four wheel sets. The differential equations of the vehicle system can be found in . The parThe DPFST dynamic model consisted of a rail, fasteners, slabs, STMs, concrete bases, and subgrade. The floating slab was simulated as a free beam with discrete supports according to the sample sizes of the STMs. The concrete base was laid on the subgrade, which was simulated as a continuously supported free beam. The fasteners were simply modeled with the Kelvin model, and the STMs were modeled by the FDPT model proposed in this paper.mr, Er and Ir are the mass, modulus, and rotational inertia of the rail, respectively; Zr is the vertical displacement of the rail; xi and xwj are the positions of the i-th fastener and the j-th wheelset, respectively; Nf is the total number of fastenings; δ(x) is the Dirac delta function; Frsi is the vertical force applied to the rail by the i-th fastener, as shown in Equation (14); and pj is the vertical force applied to the rail by the j-th wheel.The vertical vibration equation for the rail can be written askq (t) is the kth generalized coordinate of the rail vertical deflection, lr is the simulation length of the rail, and kY(x) is the kth orthogonal function, defined as followsBy applying Ritz’s method and normalized shape functions of Euler beams, the partial derivative equation (Equation (14)) can be transformed into the time-dependent differential equations, as follows:Ms, Es, Ls and Is are the mass, modulus, length, and rotational inertia of the floating slab, respectively; Np and Ns are the total number of fasteners and STMs on each slab, respectively; and Fsg is the vertical supporting force applied to the floating slab by the gth STM, as shown in Equation (18). The detailed derivation process of the supporting force of the fractional-order model is detailed in Reference [sgN is the total number of the past integral steps , which makes the calculated support force take into account the loading history process. The fractional parameters G1–G3 are functions of K1(P) and K2(P), which makes the FDPT model not only reflect the frequency dependence of the STMs but also consider the influence of the imposed preload. Δt is the integral step and mA is the mth Grünwald–Letnikov coefficient, defined as follows:The differential equations of the floating slab supported by the STMs can be written as follows:eference .(18)FsgnT(t) is the nth generalized mode coordinate of the floating slab, and nX is the nth modal function, defined as follows:nC and nβ are constants of the free beam function.Similarly, the dynamic equation of the floating slab can be transformed into a second-order differential equationMc, Ec, Lc, and Ic are the mass, modulus, length, and rotational inertia of the concrete base, respectively.The differential equation of continuously supported concrete base is similar to Equation (21), and the second-order differential equation is directly givenFor investigation into the influence of the different evaluation methods on the train-induced vibrations and the VRE of the FST, some calculation cases were designed, as shown in Case 1 in La between the DPFST and the monolithic track in the 1/3 frequency band from 1 to 200 Hz, defined as follows:The VRE is usually evaluated by the insertion loss ΔVLq(i) and VLh(i) are the frequency division vibration level (dB) of the vertical vibration acceleration of the monolithic track and the DPFST at the ith center frequency in the 1/3 octave band. The frequency weightings in the 1/3 octaves refer to ISO 2361-1:1997 [1-1:1997 .To avoid repetition, the calculation results under the full load of the vehicle are illustrated. The vertical displacements of the rail and floating slab obtained by different evaluation methods in Cases 1–5 are shown in the 2, respectively. It can be found that the vibration acceleration of the slab without considering the frequency dependence of STM-I is the smallest among all the results, and the acceleration of the concrete base is the largest . The time-domain acceleration results obtained by considering the frequency dependence of STM-I in Cases 2–5 show only minor differences.The time-domain vertical acceleration and vibration acceleration levels (VALs) of the slab with STM-I and concrete base in Cases 1–5 are shown in It is evident from In Case 1, due to the fact that the frequency- and preload-dependent characteristics of STM-I are not considered, the natural frequency of the floating slab is relatively low, which is mainly reflected in the value of 20 Hz. Although Cases 3 and 4 consider the influence of frequency characteristics, the stiffness under different preloads also has a slight difference in the vibration in the range of 20–31.5 Hz at the 1/3 octave frequency. The greater the stiffness of the STMs, the greater the vibration energy of the higher frequency within the resonant frequency range of the system. Since the stiffness of STM-I increases monotonically, compared with the second preload parameter (Case 3), the VALs of the slab obtained by the third preload parameter are slightly higher at the 1/3 octave frequency of 31.5 Hz and slightly lower at the 1/3 octave frequency of 20 Hz. When the preload dependence is further considered in the Case 5, the lower stiffness nonlinear segment between the first preload and second preload of STM-I is introduced into the calculation. Therefore, the VAL of the slab decreases at the 1/3 octave frequency of 31.5 Hz and increases at the 1/3 octave frequency of 20 Hz compared with Cases 3 and 4. The above influence is also reflected in the VALs of the concrete base, which is also the reason for the difference in the insertion loss calculated by different stiffness evaluation methods. The insertion losses obtained by different evaluation methods for STM-I under the conditions of empty load and full load are shown in In contrast, the evaluation error in Case 4 is relatively large, regardless of whether the vehicle is empty or fully loaded, because the force that STM-I bears is less than, or has just reached, the third preload see . TherefoThe subsequent sections focus on the differences in the VRE of the STMs obtained by different evaluation methods; thus, the figures of the dynamic displacement that meet the requirements of safe operation are omitted. The maximum dynamic displacement of the slab in Cases 6–10 under the full load of vehicle was 2.7 mm. The time-domain vertical acceleration and vibration acceleration levels (VALs) of the slab and concrete base in Cases 6–10 are shown in 2, respectively. Similarly, when using STM-II, the impact of different evaluation methods on the DPFST is mainly reflected in the frequency band near the natural frequency, especially at the 1/3 octave frequency of 31.5 Hz. Since the stiffness of the third preload of STM-II is less than that of the second preload, the maximum frequency division vibration levels (VLmax) of Cases 8 and 9 are mainly concentrated at 31.5 and 20 Hz, respectively. Compared with Case 9, the nonlinear segment with higher stiffness before the third preload is considered in Case 10; thus, the VAL at 31.5 Hz is higher than Case 9 but lower than that in Case 8 when only considering high stiffness. The insertion losses obtained by the different evaluation methods are shown in The maximum vibration accelerations of the concrete base in Cases 6–10 are 3.69, 2.27, 2.53, 1.95, and 2.18 m/sThe effect of the frequency-dependent stiffness on the insertion loss is almost uniform for all of the STMs; thus, the insertion loss of Case 6 is the largest. The secant stiffness weakens the difference between different preloads of STM-II, meaning that the result of Case 7 is between that of Case 8 and Case 9. However, considering the nonlinear segment with the higher stiffness before the third preload, the actual VRE of Case 10 is less than that of Case 9. Moreover, unlike STM-I, the insertion loss of STM-II increases by 0.8 dB when the vehicle is fully loaded. This is because the increased load makes the lower stiffness segments of STM-II perform better. It can be concluded that the insertion loss error obtained by the third preload evaluation method is not negligible, but different from the evaluation for STM-I, and, thus, the VRE of STM-II will be overestimated.The maximum dynamic displacements of the rail and slab in Cases 11–15 under the full load of vehicle are 2.6 and 1.8 mm respectively, which is basically the same as the results of STM-I and STM-II. 2, respectively. Despite the fact that the frequency dependence is considered in Cases 12–15, the true strong nonlinearity of STM-III results in a significant difference between the maximum vibration acceleration in Case 15 and the results of other evaluation methods (Case 12–14). The VALs of the slab and concrete base in Cases 11–15 are shown in The maximum vibration accelerations of the concrete base in Cases 11–15 are 2.82, 3.52, 2.32, 3.12, and 2.45 m/sVLmax of Cases 12 and 14 are about 7 dB larger than Cases 13 and 15, which is very important for the insertion loss. As described in Compared with other STMs, the most significant difference between different evaluation methods for STM-III is that the frequency-dependent secant stiffness can no longer approximately reflect its true VRE. In addition, compared with STM-II, the third preloading did not overestimate its VRE of STM-III, but rather underestimated it. Another interesting finding is that the VRE of STM-III has little change in the no-load or full-load conditions, which is also what we hope to achieve under different axle loads. Compared with most STMs whose force–displacement curve is approximately linear , this type of STM with high-static–low-dynamic (HSLD) stiffness in the service load range is a good choice for improving the efficiency of vibration isolation.3, respectively. As the vibration level of an empty vehicle is low and the vehicle is almost always in a loaded state, we evaluated the VRE of the vehicle under full load according to the method proposed in this article. The vertical VALs of the concrete base and insertion loss calculated by the new method for the three types of STMs are shown in From the results of the above three STMs, it can be concluded that the insertion loss calculated by using the second preload frequency-dependent tangent stiffness is the closest to the real result. In order to facilitate its application, it is practical to adopt a single frequency parameter that can accurately reflect the vibration characteristics of the floating slab track and the VRE. Therefore, we proposed an improved calculation method for the second preload evaluation method, which uses the corresponding stiffness at the natural frequency to replace the frequency-dependent stiffness. It is worth mentioning that this natural frequency is not the tuning frequency under no load, but the natural frequency considering the P2 resonance during vehicle operation ,41. The VLmax that has a greater impact on the insertion loss).It is precisely because the parameters at the natural frequency can more accurately reflect the natural vibration characteristics of the DPFST that the VALs of the concrete base calculated by using the new method are relatively close to the actual results (especially the It is obvious that the maximum error between the result of the new method and the actual insertion loss calculated by considering the preload and frequency dependence is only 0.5 dB, which meets the error requirements of the engineering design. On the whole, this single parameter evaluation method at the natural frequency under the second preload is the most practical and accurate method at present.In specification TJ/GW 121-2014 , the metThe disadvantage of DIN 45673-7:2010  is that Among the above different evaluation methods, the evaluation method using the second preload frequency-dependent stiffness of the DIN 45673-7:2010  is the mIn terms of the VRE of the different STMs, the insertion loss of STM-I with a monotonically increased stiffness decreases slightly with the increase in axel load, while the insertion loss of STM-II with a monotonically decreased stiffness improves with the increase in axel load. The VRE of STM-III with HSLD stiffness characteristics is both better and stable. This advantage of STM-III comes from the lower stiffness under the second preload. In addition, this also coincides with the evaluation and design idea of using the second preload tangent stiffness to evaluate the VRE.A series of performance tests were conducted to study the preload dependence and frequency dependence of different STMs. The FDPT model was used instead of the KV model to accurately calculate the actual VRE obtained from the vehicle–DPFST coupled dynamic model. Next, the differences in the existing STM specifications in the evaluation of the VRE were analyzed. Finally, based on the above analysis, an accurate and practical single parameter evaluation method for guiding engineering design was proposed. The four main conclusions are as follows:


Stimulus-responsive therapy permits precise control of therapeutic effect only at lesion of interest, which determines it a promising method for diagnosis and imaging-guided precision therapy. The acid environment and overexpressed matrix metalloproteinases-13 (MMP-13) are typical markers in osteoarthritis (OA), which enables the development of stimulus-responsive drug delivery system with high specificity for OA. We herein demonstrate a nano-micelle based stimuli-responsive theranostic strategy with reporting and drug release controlled by acidic pH and MMP-13 for OA therapy. Such nanoplatform is incorporated with a motif specifically targeting on cartilage, a motif responsive to matrix metalloproteinases-13 to specifically report OA condition and biodynamics of nano-micelles, an anti-inflammatory drug ) from traditional Chinese medicine, and a biocompatible polymeric skeleton for sustainable drug release in response to the acidic OA condition. The high effectiveness of this targeted precision therapy is demonstrated comprehensively by both in vitro and vivo evidences. As the most common chronic joint disease, osteoarthritis (OA) is regarded as the primary cause of disability among older adults , 2. SystDuring the process of OA, the increase of active protease can degrade the cartilage ECM, which is considered as the main cause of cartilage destruction, such as matrix metalloproteinases (MMPs), adamalysin-like metalloproteinases with thrombospondin motifs (ADAMTSs), and cathepsins. Among them, matrix metallopeptidase 13 (MMP-13) that can cleave cartilage type II collagen is considered as one of the major therapeutic targets. MMP-13 is highly expressed in the hyaline cartilage of OA patients in comparison to healthy cartilage . With th2N–GPLGVRGC–SH) [Poly (2-ethyl-2-oxazoline)-poly (ε-caprolactone) (PEOz-PCL or PPL) is a biocompatible and biodegradable polymer . It has VRGC–SH) , 21 labeVRGC–SH) , 23.As schematically illustrated in Fig. PPL copolymers self-assembled to nano-micelles (120.3 ± 22.81 nm), as revealed by transmission electron microscopy  in vitro. As shown in Fig. μM PSO didn’t exert any cytotoxicity. Therefore, this dosage was chosen for the following experiments.The cytotoxicity of micelles to chondrocytes isolated from C57BL6/J mice was investigated in vitro using CCK8 assay. Even at a high concentration (200 μg/mL) of MRC-PPL, the cell viability is  > 85% after 48 h of incubation, indicating its good biocompatibility Fig. a. Then, TNF-α, MMP-3 and MMP-13 with promoting cartilage destruction effect in each treatment group, as well as the chondroid-specific marker, Col2a1. As shown in Fig. TNF-α, MMP-3 and MMP-13 are elevated by 115.72, 3.33 and 2.59 times respectively after IL-1β stimulation whereas the expression of Col2a1 was decreased to 23.78%. The upregulation of inflammatory factors and down-regulation of Col2a1 were significantly reversed by MRC-PPL@PSO treatment.High level of interleukin 1 beta (IL-1β) is a marker for OA and is commonly used to induce inflammation of chondrocytes –27. As sAs shown in hematoxylin and eosin (HE) staining Fig. e, chondrTaking the observations in Fig. During the progression of osteoarthritis, IL-1β levels are elevated, which activates the nuclear factor NF-κB pathways to induce the expression of matrix metalloproteinases (MMPs) in cultured chondrocytes, leading to ECM degradation, abnormal bone metabolism and inflammatory disease , 29. In Cellular uptake and MMP-13 responsive behaviors of MRC-PPL micelles were investigated after 24 h incubation with normal chondrocytes and IL-1β stimulated chondrocytes. As shown in Fig. Two weeks after IA injection of papain solution, OA developed in the knee joint of mice, showing mild swelling and deformity. As demonstrated by in vivo fluorescence imaging Fig. a and quaAfter the mice receiving different treatments and being sacrificed at week 2 or 6, the femoral condyle and tibial plateau were collected and evaluated according to the criteria described by Lydia Wachsmuth et al. for the depth of erosion in articular cartilage . CompareThe cartilage tissues were then evaluated by hematoxylin–eosin (H&E) staining and Safranin O-fast green staining. As shown in Fig. TNF-α, MMP-3 and MMP-13, and significantly alleviated the cartilage lesions with the lowest OARSI score, as confirmed by histological staining and MMP-13 expression after 2 and 6 weeks post-treatment, indicating the protective and the targeting therapeutic effect on cartilage. Furthermore, MRC-PPL@PSO exerts antiarthritic effects by regulation of PI3K/AKT, MAPK and NF-κB signaling pathway.OA affected joints are presented by overexpressed MMP-13 and acidic microenvironment, which enables the development of ‘smart’ dual-stimuli-responsive probes for highly-efficient and controlled delivery and release of theranostic agents. Herein we demonstrated a theranostic nanoplatform for osteoarthritis, activated by microenvironment of acidic and MMP-13 enzyme overexpression in OA joints. The incorporated collagen type Il binding peptide facilitates the targeting and retention in the joint. Such disease specific stimulus-responsive strategy improves the efficiency and minimizes the side-effects. Upon activation, the theranostic nano-micelles (MRC-PPL@PSO) produce fluorescence signal and sustainably release the anti-inflammatory drug molecules. In IL-1β-treated chondrocytes, MRC-PPL@PSO promoted cell proliferation and inhibited inflammatory responses by down-regulating In principle, multiple drug compounds can be loaded to achieve synergistic effects. In addition, such biocompatible PEOz-PCL based polymeric system can be readily modified for diagnosis and treatment of other diseases  by varying the targeting motif, enzyme substrates, and drugs.2, Mw 5000 Da) was purchased from Ruixibiotech (China). Psoralidin (PSO) and CCK8 were obtained from Sigma-Aldrich (US). Cy5.5-NHS and BHQ-3000S-5 was purchased from Seebio Biotech (China). N-Hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC•HCl) and N-succinimidyl-4-maleimide butyrate (GMBS) were supplied by J&K Scientific (China).Poly(ε-caprolactone)-Poly(2-ethyl-2-oxazoline)-amine  followed by the addition of N-succinimidyl-4-Maleimidobutyrate (30 mmol in 100 μL DMSO) to form MR-Cy5.5. The reaction solution was incubated at room temperature for 4 h in a 1.5 mL eppendorf tube with gentle shaking. Then, to conjugate with the MMP-13 responsive peptide, EDC and NHS were added in MR-Cy5.5 solution, followed by the addition of 50 mg PPL. Subsequently, BHQ-3 was added in dark to form MR-Cy5.5-BHQ-3-PPL (MR-PPL), followed by the purification using HPLC. In parallel, WYRGRL peptide (10 mg in 100 μL DMSO) which specifically binds with collagen type II was coupled with PPL (50 mg in 100 μL DMSO) to obtain C-PPL. With addition of N, N-diisopropyl ethylamine  and overnight reaction, MRC-PPL micelles self-assembled from C-PPL and MR-PPL were finally obtained after ultrasonication and filtration through a 0.8-μm membrane.MMP-13/pH responsive and cartilage targeting MRC-PPL micelles was obtained by self-assembly of C-PPL and MR-PPL . Specifi2O was added and vortexed. The morphology of MRC-PPL was uncovered by a transmission electron microscope . We used Malvern Zetasizer Nano ZS90 to measure the size distribution and zeta potential of the micelles in aqueous suspension. Absorption spectra were obtained from a microplate reader (Thermo Scientific Multiskan GO Microplate Spectrophotometer). And the fluorescence from Cy5.5 was detected using a Fluorescence spectrophotometer (RF-5301PC).Polymeric micelles can encapsulate drug compounds using the film hydration method , 35. TheThe loading capability of PSO in MRC-PPL micelles was determined using the HPLC. Briefly, Lyophilized MRC-PPL@PSO powder was dissolved in HCl (0.1 mol/L) and moderate methanol was added to release PSO. After the supernatant was collected by centrifugation , the PSO concentration in the supernatant was detected by ACQ-BSM HPLC system .The system parameters were set to wavelength 347 nm, and the C18 analytical column  was used at 37 °C. The mobile phase was consisted of methanol/water/0.05 M acetic acid  and the flow rate was 1.0 mL/min. The loading content (LC) of PSO were calculated according to the formula: The pH-dependent release behavior of PSO from the MRC-PPL@PSO micelles was monitored at 37 °C. Briefly, 2 mL of the PSO-loaded micelles were placed in a dialysis bag (MWCO 3500), which was then immersed in PBS  containing 1% Tween 80. The release device was continuously shaken at 37 °C. Remove 1 mL of release media at predetermined intervals and immediately replace it with 1 mL of fresh media. The accumulative release amount of PSO was finally determined by HPLC.The chondrocytes was extracted from the knee joints of new born (3-5 days) C57BL6/J mice  after the articular cartilage were minced, digested with trypsin for 30 min, and digested with type II collagenase . Chondrocytes were cultured in Eagle medium (Gibco) containing 1% (v/v) penicillin/streptomycin (Solarbio) and 10% (v/v) fetal bovine serum (Gibco). Cell experiments were constructed according to our previous work.4 cells/wells were cultured in 24-well plates, which were treated with 10 ng/mL IL-1β for 2 h to establish an in vitro model of osteoarthritis. Incubated with the PSO (15 µM), MR-PPL@PSO or MRC-PPL@PSO for 24 h. The latter two treatments equivalently contained 15 µM PSO. The remaining steps are based on the CCK8 method.The cytotoxicity of different formulations to chondrocytes cells were using Cell Counting Kit-8 . Briefly, the cell at a density of 5000 per well were seeded in 96-well plate overnight. Thereafter, MRC-PPL@PSO with the various amount of PSO was added and further incubated for 48 h. MRC-PPL micelles without PSO at different concentrations (0 ~ 200 μL/mL) were also tested for comparison. After treatment, 10 μL of CCK-8 were added to each well and cultivated for 4 h. Finally, the absorbance at 450 nm was measured using a microplate reader (Thermo Scientific Multiskan GO Microplate Spectrophotometer). The viability of MRC-PPL@PSO on the proliferation of IL-1β-induced chondrocytes was measured by CCK8 assay. Chondrocytes with density of 1.5 × 10Cellular uptake of MRC-PPL was examined in chondrocytes. IL-1β (10 ng/mL) treated cells were added MRC-PPL or MR-PPL (1 mg/mL). Meanwhile, the cells were stained with immunofluorescence against COL2A1 . After the nuclei were stained with DAPI, the images were captured using a laser scanning confocal microscope . The excitation wavelengths for DAPI and Cy5.5 were 405 nm and 640 nm, respectively.4 cells/cm2) or 24-well plates (density of 2 × 104 cells/cm2). The cells pretreated with IL-1β (10 ng/mL) for 2 h were incubated with the PSO (15 µM), MR-PPL@PSO or MRC-PPL@PSO for 24 h. The latter two treatments equivalently contained 15 µM PSO. The cells were then washed with saline and fixed with 95% ethyl alcohol for 30 min, followed by staining with hematoxylin and eosin .The immunofluorescence was used to detect the protein expression level of MMP-13 and TNF-α. Samples were incubated with primary antibodies against MMP-13  and TNF-α  at 37 °C for 4 h. The second antibody FITC-anti-rabbit IgG  was incubated in dark for 1 h. Finally, the nuclei were stained with DAPI, and the cells were observed with fluorescence microscope .The chondrocytes were seeded in 6-well  were used to extract total RNA. The reverse transcription is done by a reverse transcription kit . All qRT-PCR reactions were performed by a light Cycle 96 system  for 10 min at 95 °C, followed by 40 cycles with 10 s duration at 95 °C and then 60 s duration at 60 °C. The primers were used as follows in the Table Proteins were extracted from chondrocytes with RIPA lysis buffer. Lysates were sonicated on ice and centrifuged at 12,000 rpm for 30 min at 4 °C. The protein concentration of the supernatant was determined using a BCA protein assay kit. Equal amounts of protein (40 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with blocking buffer 5% non-fat milk in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature and then probed with the primary antibodies against p65, Akt and p-Akt, P38, p-P38 (dilution 1:1000) overnight at 4 °C. After washing three times with TBS containing 0.1% Tween-20 for 5 min, the membranes were then incubated with the secondary antibody  and visualized using the Odyssey Infrared Imaging System (LI-COR USA) according to the manufacturer’s instructions. Data were normalized by housekeeping protein (GAPDH).l-cysteine  three times a week. Mice were randomly divided into different experimental groups (n = 5): healthy control group, OA group and treatment groups. For OA group, mice were IA injected with 20 μL of PBS once per week. The treatments included IA injection of 20 μL PSO (15 µM), 20 μL MR-PPL@PSO solution (containing 15 µM PSO), or 20 μL MRC-PPL@PSO (containing 15 µM PSO), once per week for 2 or 6 weeks.A total of 100 C57BL/6 J mice (aged 6-10 weeks) were used for the in vivo experiments, which were performed in accordance with the ethical approval of the Institutional Ethics Committee of Guangxi Medical University. Arthritis was induced by articular injection of a mixture of papain solution  with IVIS 200 system with a Cy5.5 filter set . The mice were anesthetized and imaged at 30 min, 0.5, 1, 3, 7, 14, 21 d after injection.After anesthetized with 2% isoflurane , mice were injected with treatment solution through the articular cavity (n = 5 for each group). In vivo fluorescence imaging was performed on an At research endpoints, mice were euthanized and their hind knee joints were collected for fluorescence imaging by an IVIS-200 system. The treated articular cartilages were harvested. Grading of articular cartilage surface was performed by specifically evaluating a nine-area grid of each medial and lateral tibia plateau  . MoreoveAll data were reported as mean ± standard deviation of at least three experiments. The two-sided student t test was used for statistical analysis. P value less than or equal to 0.05 was considered statistically significant.