Ignite Creation Date:
2024-05-06 @ 6:36 PM
Last Modification Date:
2024-10-26 @ 2:51 PM
Study NCT ID:
NCT05720065
Status:
RECRUITING
Last Update Posted:
2023-10-05
First Post:
2023-01-07
Brief Title:
Peripheral TMD Pain Mechanisms and the Effect by Botulinum Toxin A
Sponsor:
Karolinska Institutet
Organization:
Karolinska Institutet
Study Overview
Official Title:
Peripheral TMD Pain Mechanisms and the Effect by Botulinum Toxin A A Randomized Controlled Double-blind Study
Status:
RECRUITING
Status Verified Date:
2024-10
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The goal of this clinical trial is to investigate the effect of botulinum toxin on neurons plasticity in the masseter muscle in humans with and without painful myogenous temporomandibular disorders TMDM The main questions it aims to answer are
does treatment with botulinum toxin alter gene expressions epigenetic signatures and cells plasticity in the masseter muscles of TMDM patients
do any such changes differ between patients with local and regional TMDM
does treatment with botulinum toxin influence pain characteristics intensity frequency and sensibility and other variables in patients with TMDM and are there correlations between significantly changed expression of biomarkers and other variables Participants will be examined with a questionnaire clinical examination and biopsy sampling from one of the masseter and are then randomized to treatment with botulinum toxin or control isotonic saline Follow-ups occur after one three and six months with questionnaire clinical examination and collection of post-treatment microbiopsies to see if botulinum toxin alter peripheral molecular events and clinical variables
does treatment with botulinum toxin alter gene expressions epigenetic signatures and cells plasticity in the masseter muscles of TMDM patients
do any such changes differ between patients with local and regional TMDM
does treatment with botulinum toxin influence pain characteristics intensity frequency and sensibility and other variables in patients with TMDM and are there correlations between significantly changed expression of biomarkers and other variables Participants will be examined with a questionnaire clinical examination and biopsy sampling from one of the masseter and are then randomized to treatment with botulinum toxin or control isotonic saline Follow-ups occur after one three and six months with questionnaire clinical examination and collection of post-treatment microbiopsies to see if botulinum toxin alter peripheral molecular events and clinical variables
Detailed Description:
Patient recruitment
The patients will be recruited among those referred to the Specialist Clinic for Orofacial Pain and Jaw Function at the University Dental Clinic Karolinska Institutet Huddinge Sweden or via advertisement
Randomization
Patients will be randomly assigned to treatment by a random generator wwwrandomizationcom For each participant the treatment will be written on a note and placed in a sealed envelope by a researcher not involved in any other part of data collection A second person not involved in patient examination will open the envelope prepare the syringe with the randomized solution and place it in the examination room before the examiner and patient enters it Botulinum toxin and saline have identical appearance so the participant and investigator will be masked to treatment assignment
Procedure
The participants complete an extended and slightly modified version of the Swedish Axis II questionnaire included in the Diagnostic Criteria for TMD DCTMD before the first visit The Axis II questionnaire contains sociodemographic question questions regarding TMD symptoms and headache presence duration and frequency used for diagnostic purposes Symptom Questionnaire and validated instruments to assess physical and emotional function A clinical examination is then performed according to the DCTMD Axis I which also includes recordings of pressure pain threshold temporal summation pain and conditioned pain modulation After having ensured that the participant fulfils the eligible criteria biopsies are obtained from a painful and a non-painful site for later analysis At least one week hereafter treatment are given
Biopsies
Microbiopsies will be obtained from the masseter and anterior tibialis muscle a non-treated pain-free internal control The microbiopsies are taken through the skin overlaying the most prominent part of the muscle under skin surface anesthesia A disposable biopsy instrument with a penetration depth of 11 mm and a diameter of 18 gauche G masseter or 16G tibialis will be used The biopsy instrument will be guided with a coaxial needle that will be inserted to a depth of 10 mm Three to four microbiopsies will be taken from each muscle to ensure that sufficient muscle tissue is obtained The coaxial needle will be inserted along the long axis of the muscles until the fascia is penetrated and the biopsy instrument is then inserted through the coaxial needle and a piece of the muscle with a size of approximately 012 cm 11 cm is collected The muscle section will be removed from the biopsy instrument using a sterile probe and immediately put in a cryotube which is snap frozen in liquid nitrogen After removal of the muscle section the biopsy instrument will be rinsed with isotonic saline This procedure will be repeated two to three times Each time the biopsy instrument will be rotated 45 so that the microbiopsy would be taken from a new portion of the muscle The biopsies will be stored -80ยบ in Karolinska Institutets dental biobank until analysis
Analyses of biopsies
The biopsies will be analyzed with bulk ribonucleic acid sequencing RNA-seq Multiome combined single-cell Assay for Transposase-Accessible Chromatin ATAC and RNA-seq immunohistochemistry IHC light sheet microscopy and flow cytometry FC at University of Texas Health Science Center in San Antonio UTHSCSA and at the Live Cell Imaging Facility at Karolinska Institutet Huddinge Sweden Data of the non-painful site anterior tibialis will be subtracted from data of the painful site masseter for every patient Then these data can be clustered
For bulk RNA seq differentially expressed genes DEGs will be selected and then paired analysis will be run within empirical analysis of DEGs in R edgeR software to select significantly different p005 DEGs This is an established and well standardized approach Selection criteria for further analysis are fold charge FC15 and Reads Per Kilobase of transcript per Million mapped reads RPKM10 Finally biological processes will be defined by wwwpantherdborg on base of DEGs Substation of internal controls from experimental data will also be done followed by clustering of patients using DEGs Both approaches produce similar results Power analysis based on preliminary results from skin biopsies showed that 19 paired samples will obtain 836 power with a standard deviation SD of 08 However RNA from muscle tissue is usually of higher quality than skin samples Hence the investigators anticipate that bulk RNA-seq will have lesser SD and require 14 paired samples per group this includes samples after treatments
Combined gene and epigenetic signature plasticity on cellular levels will be evaluated by Multiome using Genomic x10 platform Multiome analysis is standardized by Genomic x10 For visual presentation Seurat will be used Power analysis for Multiome showed that 3-4 paired samples per group is needed Currently accepted analysis is that paired samples belonging to the same participant group combined and multiple t-test-like analysis for each cell cluster run against another group
The IHC analysis will focus on sensory neurite plasticity using markers for subsets of sensory neurons and use Light Sheet microscopy to measure neurite length and branching for whole biopsies The investigators also plan to use the Glyclick method which generates stable and homogenous antibody conjugates for immunoglobin G from several species and subclasses If possible also proteomics will be done FC will be used to profile immune and vasculature cells Power analysis showed that 8 samples per group will be needed for IHC and FC
Analyses of clinical data
P005 will be used as significance level The Shapiro-Wilks test will test for normality of data Mean SD or median interquartile range IQR depending on normality will be used to describe the data For normally distributed and continuous data repeated measures Analysis of Variance ANOVA will be used to analyze differences in treatment effect with Group and Time as factors If significant differences are found these will be further analyzed with Dunnets posthoc test Data that are skewed or ordinal will be analyzed with Friedman ANOVA for each group separately with Dunns test as posthoc test for time differences The Mann-Whitney U-test will be used to analyze differences between groups at different time points using Bonferroni correction for multiple testing
Multivariate statistics will be used to identify correlations between significantly changed biomarkers and other variables Principal component analysis PCA will be used to identify moderate or strong outliers Orthogonal partial least squares discriminant analysis OPLS-DA will then be used to regress group membership using the biomarkers as regressors For correlations between biomarkers and clinical variables OPLS modelling will be performed The Variable Influence on Projection VIP value indicates the relevance of each X-variable pooled over all dimensions and the Y-variables indicate the group of variables that best explain Y VIP 10 is considered significant R2 describes the goodness of fit ie the fraction of sum of squares of all the variables explained by a principal component whereas Q2 describes the goodness of prediction ie the fraction of the total variation of the variables that can be predicted by a principal component using cross validation methods R2 should not be considerably higher than Q2 if substantially higher 03 the robustness of the model is poor To validate the model an obtained cross-validated analysis of variance CV-ANOVA p-value will be used The OPLS-DA model is considered of significant importance if the CV-ANOVA has a p-value 005
The patients will be recruited among those referred to the Specialist Clinic for Orofacial Pain and Jaw Function at the University Dental Clinic Karolinska Institutet Huddinge Sweden or via advertisement
Randomization
Patients will be randomly assigned to treatment by a random generator wwwrandomizationcom For each participant the treatment will be written on a note and placed in a sealed envelope by a researcher not involved in any other part of data collection A second person not involved in patient examination will open the envelope prepare the syringe with the randomized solution and place it in the examination room before the examiner and patient enters it Botulinum toxin and saline have identical appearance so the participant and investigator will be masked to treatment assignment
Procedure
The participants complete an extended and slightly modified version of the Swedish Axis II questionnaire included in the Diagnostic Criteria for TMD DCTMD before the first visit The Axis II questionnaire contains sociodemographic question questions regarding TMD symptoms and headache presence duration and frequency used for diagnostic purposes Symptom Questionnaire and validated instruments to assess physical and emotional function A clinical examination is then performed according to the DCTMD Axis I which also includes recordings of pressure pain threshold temporal summation pain and conditioned pain modulation After having ensured that the participant fulfils the eligible criteria biopsies are obtained from a painful and a non-painful site for later analysis At least one week hereafter treatment are given
Biopsies
Microbiopsies will be obtained from the masseter and anterior tibialis muscle a non-treated pain-free internal control The microbiopsies are taken through the skin overlaying the most prominent part of the muscle under skin surface anesthesia A disposable biopsy instrument with a penetration depth of 11 mm and a diameter of 18 gauche G masseter or 16G tibialis will be used The biopsy instrument will be guided with a coaxial needle that will be inserted to a depth of 10 mm Three to four microbiopsies will be taken from each muscle to ensure that sufficient muscle tissue is obtained The coaxial needle will be inserted along the long axis of the muscles until the fascia is penetrated and the biopsy instrument is then inserted through the coaxial needle and a piece of the muscle with a size of approximately 012 cm 11 cm is collected The muscle section will be removed from the biopsy instrument using a sterile probe and immediately put in a cryotube which is snap frozen in liquid nitrogen After removal of the muscle section the biopsy instrument will be rinsed with isotonic saline This procedure will be repeated two to three times Each time the biopsy instrument will be rotated 45 so that the microbiopsy would be taken from a new portion of the muscle The biopsies will be stored -80ยบ in Karolinska Institutets dental biobank until analysis
Analyses of biopsies
The biopsies will be analyzed with bulk ribonucleic acid sequencing RNA-seq Multiome combined single-cell Assay for Transposase-Accessible Chromatin ATAC and RNA-seq immunohistochemistry IHC light sheet microscopy and flow cytometry FC at University of Texas Health Science Center in San Antonio UTHSCSA and at the Live Cell Imaging Facility at Karolinska Institutet Huddinge Sweden Data of the non-painful site anterior tibialis will be subtracted from data of the painful site masseter for every patient Then these data can be clustered
For bulk RNA seq differentially expressed genes DEGs will be selected and then paired analysis will be run within empirical analysis of DEGs in R edgeR software to select significantly different p005 DEGs This is an established and well standardized approach Selection criteria for further analysis are fold charge FC15 and Reads Per Kilobase of transcript per Million mapped reads RPKM10 Finally biological processes will be defined by wwwpantherdborg on base of DEGs Substation of internal controls from experimental data will also be done followed by clustering of patients using DEGs Both approaches produce similar results Power analysis based on preliminary results from skin biopsies showed that 19 paired samples will obtain 836 power with a standard deviation SD of 08 However RNA from muscle tissue is usually of higher quality than skin samples Hence the investigators anticipate that bulk RNA-seq will have lesser SD and require 14 paired samples per group this includes samples after treatments
Combined gene and epigenetic signature plasticity on cellular levels will be evaluated by Multiome using Genomic x10 platform Multiome analysis is standardized by Genomic x10 For visual presentation Seurat will be used Power analysis for Multiome showed that 3-4 paired samples per group is needed Currently accepted analysis is that paired samples belonging to the same participant group combined and multiple t-test-like analysis for each cell cluster run against another group
The IHC analysis will focus on sensory neurite plasticity using markers for subsets of sensory neurons and use Light Sheet microscopy to measure neurite length and branching for whole biopsies The investigators also plan to use the Glyclick method which generates stable and homogenous antibody conjugates for immunoglobin G from several species and subclasses If possible also proteomics will be done FC will be used to profile immune and vasculature cells Power analysis showed that 8 samples per group will be needed for IHC and FC
Analyses of clinical data
P005 will be used as significance level The Shapiro-Wilks test will test for normality of data Mean SD or median interquartile range IQR depending on normality will be used to describe the data For normally distributed and continuous data repeated measures Analysis of Variance ANOVA will be used to analyze differences in treatment effect with Group and Time as factors If significant differences are found these will be further analyzed with Dunnets posthoc test Data that are skewed or ordinal will be analyzed with Friedman ANOVA for each group separately with Dunns test as posthoc test for time differences The Mann-Whitney U-test will be used to analyze differences between groups at different time points using Bonferroni correction for multiple testing
Multivariate statistics will be used to identify correlations between significantly changed biomarkers and other variables Principal component analysis PCA will be used to identify moderate or strong outliers Orthogonal partial least squares discriminant analysis OPLS-DA will then be used to regress group membership using the biomarkers as regressors For correlations between biomarkers and clinical variables OPLS modelling will be performed The Variable Influence on Projection VIP value indicates the relevance of each X-variable pooled over all dimensions and the Y-variables indicate the group of variables that best explain Y VIP 10 is considered significant R2 describes the goodness of fit ie the fraction of sum of squares of all the variables explained by a principal component whereas Q2 describes the goodness of prediction ie the fraction of the total variation of the variables that can be predicted by a principal component using cross validation methods R2 should not be considerably higher than Q2 if substantially higher 03 the robustness of the model is poor To validate the model an obtained cross-validated analysis of variance CV-ANOVA p-value will be used The OPLS-DA model is considered of significant importance if the CV-ANOVA has a p-value 005
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
False
Is an FDA AA801 Violation?:
None