Clinical trial NCT04281901
Conditions: Otitis Media Chronic Temporal Bone
Keywords: Otitis Media Mastoidectomy Wound Healing Platelet-Rich Plasma Extracellular Vesicles Cholesteatoma Regenerative Medicine
Sponsor: University Medical Centre Ljubljana
Start date: 2019-03-20; Completion date: 2020-10-14
Enrollment: 25 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This clinical study evaluates the efficacy of the autologous blood-derived product called platelet- and extracellular vesicle-rich plasma (PVRP) for the treatment of chronically inflamed post-surgical temporal bone cavities. Half of the participants will be treated with platelet- and extracellular vesicle-rich plasma and another half with standard nonsurgical measures.

Detailed description:
The hypothesis of this study is that platelet- and extracellular vesicle-rich plasma (PVRP) may be efficient in the treatment of chronically inflamed post-surgical temporal bone cavities.

Platelet-rich plasma (PRP) is a well-known autologous blood-derived product with favourable immune, haemostatic and regenerative effects. It has been used in various medical fields including otorhinolaryngology.

In fact, PRP contains important concentrations of extracellular vesicles (EV) which are the main contributors to PRP effects. For that reason, PRP can be identified as platelet- and extracellular vesicle-rich plasma (PVRP). In this study, PVRP will be prepared by a unique non-commercial 2-step centrifugation protocol developed by this study researchers.

A radical cavity is a large post-surgical temporal bone cavity due to removal of the posterior external ear canal wall in open-technique cholesteatoma surgery. This technique is performed in approximately 40 per cent of cholesteatoma surgery.

Radical cavities become inflamed in 3-20 per cent, which leads to the formation of granulation tissue and multiple suppurative periods. The large spectrum of surgical and nonsurgical treatment options have been applied to reduce patient discomfort. This significantly worsens the patients' quality of life and puts a significant burden on health care.

Due to the knowledge of local immune response mechanisms in moist radical cavities and exhausted treatment options, new conservative treatment options have been researched. PVRP could present a promising treatment option for chronically inflamed radical cavities based on published preclinical and clinical studies.

PVRP will be administered to chronically inflamed radical cavities via PVRP-soaked ear wicks.

Arms: arm 1: Participants will receive PVRP in the chronically inflamed radical cavity at the baseline evaluation (day 0) and 1 month later (1. follow-up). There will be 2 additional follow-ups with a 1-month interval. arm 2: Participants will receive standard conservative measures for the chronically inflamed radical cavity at the baseline evaluation (day 0), 1 month later (1. follow-up), 2 months later (2. follow-up) and 3 months later (3. follow-up).
Interventions: intervention 1: Platelet- and extracellular vesicle-rich plasma intervention 2: Standard conservative treatment
Intervention descriptions: intervention 1: ear wick soaked in platelet- and extracellular vesicle-rich plasma intervention 2: standard conservative measures, including antimicrobials, antiseptics and aural toilette for treating a chronically inflamed radical cavity.

Protocol documents:
Prot_000.pdf: 
Study Protocol 
 
Efficacy of platelet- and extracellular vesicle-rich plasma in the treatment of chronic 
postoperative temporal bone cavity inflammations  
 
NCT Number: NCT04281901 
 
Document date: February 1st, 2020 
 
 
1 
Introduction 
Platelet-rich plasma is a blood product, prepared from peripheral venous blood, which 
possesses beneficial immune, hemostatic, and regenerative effects. Since it is rich in 
extracellular vesicles (EV), it can be called platelet- and extracellular vesicle-rich plasma 
(PVRP) (1). In addition to platelets, EVs are thought to be the main mediators of the 
regenerative effects of PVRP (2). EVs are a heterogeneous group of nanometrically-sized 
membrane structures that play an important role in intercellular communication and can, 
therefore, be used in diagnosis and treatment (3–5). Although PVRP has been used in 
medicine for tissue regeneration for decades, there is a lack of trials regarding EVs in 
association with PVRP so far (6).  
Despite numerous clinical trials on the use of PVRP, research in otorhinolaryngology is 
currently scarce (7). A particular challenge is the treatment of chronic postoperative 
temporal bone cavity inflammation (CPTBCI). The postoperative temporal bone is most 
often the result of surgical treatment of cholesteatoma of the middle ear. Treatment of 
CPTBCI is often exhausted due to the failure of surgical procedures and standard 
conservative treatment methods. Up to 20 % of patients are expected to suffer from 
CPTBCI (8,9). Problems associated with CPTBCI not only impair quality of life, but also 
significantly impact the health care system (10–12). 
We aimed to test the efficacy of PVRP in the treatment of CPTBCI after exhausted surgical 
and standard conservative treatment in a prospective randomized controlled clinical trial. 
The outcome of treatment was assessed by comparing the quality of life in patients treated 
with PVRP and patients treated with standard conservative methods throughout four 
otorhinolaryngological examinations (i.e., check-ups). 
 
2 
Methods 
National Medical Ethics Committee, Republic of Slovenia approved the trial (No. 0120-
146 / 2019/5). 
The trial started by creating a list of patients or cases who met the inclusion criteria (i.e., 
recruitment) during visits at the Department of Otorhinolaryngology and Cervicofacial 
Surgery, University Medical Centre Ljubljana for six months (Table 1). CPTBCI on one 
ear (i.e., unilateral) was considered as one case of CPTBCI and CPTBCI on both ears (i.e., 
bilateral) as two cases of CPTBCI. Surgical treatment was considered as exhausted if an 
additional surgical treatment would not eliminate CPTBCI while preserving hearing. 
Standard conservative treatment was defined as exhausted if it was ineffective in treating 
CPTBCI for at least 8 weeks. 


 
Table 1: Inclusion and exclusion criteria of the trial.  
Inclusion criteria 
Exclusion criteria 
COM defined as a presence of ³ 1 of: venepuncture site inflammation  
• visible ear discharge 
pregnancy or breastfeeding  
• indirect ear discharge signs (e.g. on 
a pillow, clothes) 
chronic use of immunomodulatory and / or 
antimicrobial drugs 
• itching 
presence of systemic infectious disease 
• sensation of ear fullness 
presence of malignancy 
• clinical signs of inflammation 
exacerbation 
other experimental attempts to treat the 
consequences of radical COM surgery 
exhausted surgical and standard 
conservative treatment 
inability and / or refusal of the patient to 
participate in the trial 
age > 18 years 
presence of autoimmune disease 
COM – chronic otitis media. 
 
After recruitment, patients were randomly allocated one of two interventions: treatment 
with standard conservative methods (i.e., control group), and treatment with PVRP (i.e., 
PRVP group). 
After recruitment and allocation patients were examined at the Department of 
Otorhinolaryngology and Cervicofacial Surgery, University Medical Centre Ljubljana 
when they attended their regularly scheduled check-up. We informed each patient about 
the allocation and re-checked the same inclusion criteria (Table 1). If the patient met these 
criteria and wanted to participate in the trial voluntarily, he/she signed an informed 
consent, which meant the enrollment in the trial. If the patient refused to participate, he/she 
did not enter the survey, but this did not affect the regularly scheduled treatment. The latter 
included the continuation of standard conservative treatment methods, described below. 
Patients enrolled in the trial started the treatment according to the treatment protocol, either 
with standard conservative methods (i.e., control group) or with PVRP (i.e., PVRP group). 
Only patients who completed treatment according to the protocol were included in the final 
analysis. The researchers who treated patients and patients were informed about the 
allocation, therefore the trial was not blind. 
 
2.1 Treatment protocol 
The treatment included four check-ups by the leading researchers at the Department of 
Otorhinolaryngology and Cervicofacial Surgery, University Medical Centre Ljubljana. 
Four-week intervals between individual check-ups were planned, so the treatment was 
expected to last 12 weeks. The treatment protocol for both groups is shown in Table 2. 
 
Table 2: Chronic postoperative temporal bone cavity inflammation treatment protocol.  
 
Control group 
PVRP group 
1 
A 
review of inclusion and exclusion criteria 
B 
invitation to participate in the trial 
C 
enrollment in the trial 
D 
completion of the COMQ-12 questionnaire 


E 
toilet (i.e. cleaning) the middle ear 
  FA 
standard conservative methods 
F
treatment with PVRP 
2A (~ 4th week) 
1A, 1D, 1E, 1FA 
2
1A, 1D, 1E, 1FB 
3A (~ 8th week) 
1A, 1D, 1E, 1FA 
3
1A, 1D, 1E 
4A (~ 12th week) 
1A, 1D, 1E, 1FA 
4
1A, 1D, 1E 
Numbers (1-4) mark each check-up. Capital letters (A-F) mark interventions performed 
during the check-up, alphabetically. A refers to the control group check-up, B refers to the 
PVRP group check-up. Example: 1FB stands for treating the patient in a PVRP group with 
a PVRP. 3A contains measures 1A, 1D, 1E, 1FA. The second examination was scheduled 
for 4 weeks, the third 8 weeks and the fourth 12 weeks after the first examination. PVRP 
– Platelet- and extracellular vesicle-rich plasma; COMQ-12 – chronic otitis media 
questionnaire 12. 
 
Patients in the PVRP group received PVRP at the first and second check-up (Table 2). We 
performed a cleaning (i.e., toilet) of the ear at each check-up. The other standard 
conservative methods, described below, were not used in PVRP group. At the third and 
fourth check-up, we performed ear toilet only. The discontinuation of other standard 
conservative methods at the third and fourth check-up in the PVRP group did not pose any 
risk, as the patients' problems were chronic. 
Patients in the control group continued the treatment with standard conservative methods 
during each check-up. These methods included ear toilet, use of topical antimicrobial 
drugs, anti-inflammatory and antiseptic drugs (Table 2). 
 
2.2 Outcome measures 
The treatment outcome was measured with three outcome measures: 
• Inflammation surface area in postoperative temporal bone cavity at each check-up. The 
surface area in milimetres was transformed to the ratio of surface area according to the 
first check-up (i.e., baseline evaluation). Therefore surfaces at the first check-up were 
represented as 100 % different than 100 % in subsequent check-ups. 
• Slovenian version of COMQ-12 (Chronic Otitis Media Questionnaire-12) sum score 
(13) at each check-up. COMQ-12 is a chronic otitis media (COM)-related quality of 
life measure (12). It was completed by the patient himself. The questionnaire comprises 
12 questions scored 0-5. Seven questions relate to the severity of COM symptoms, two 
questions to the impact of COM on lifestyle and work, two questions to the burden on 
the health system due to COM, and one question is general. The maximum possible 
sum score is 60. Since the CPTBCI is a type of COM, the higher COMQ-12 sum score 
signifies the worse quality of life associated with COM or CPTBCI. 
• Microbiological analysis of temporal bone cavity ear swab performed at the first and 
third check-up. The cultivation of bacteria and fungi was performed. 
 


2.3 Platelet- and extracellular vesicle-rich plasma preparation protocol 
Blood was taken (i.e., venepuncture) at the Department of Otorhinolaryngology and 
Cervicofacial Surgery, University Medical Centre Ljubljana from the cubital vein by a 
vacuum method by a medical technician. 21 G wing needle (so-called butterfly) (Safety-
Lok Blood Collection Set, BD Vacutainer, Becton Dickinson, USA) was used to draw 
blood into four 4.5 mL citrate tubes. (9 NC sodium citrate 0.105 M, BD Vacutainer, Becton 
Dickinson, USA). The tubes were stored at room temperature before and after 
venepuncture. PVRP was prepared according to the protocol described in Table 3 on the 
day of venepuncture in the Laboratory of Clinical Biophysics, Faculty of Health, 
University of Ljubljana, for each patient in PVRP group at the first and second check-up 
(Table 2).  
 
Table 3: PVRP preparation protocol: 
Step 
Description 
1 
Venepuncture of cubital vein to draw blood into four 4.5 mL sodium citrate tubes  
2 
Weighing of four 4.5 mL citrate tubes. If the weights differed > 0.02 g (incomplete 
blood collection), they were balanced by the addition of counterweight tubes. 
3 
First centrifugation step: 300 g, 5 min and 18 °C. 
4 
Transfer of the supernatant (i.e., plasma or yellow top layer without buffy coat) 
with a sterile pipette from four centrifuged citrate tubes into two sterile 
polypropylene tubes. 
5 
Second centrifugation step: 700 g, 17 min and 18 °C. 
6 
Removal of approximately half of the supernatant (i.e., platelet-poor plasma) from 
two sterile polypropylene tubes with a sterile pipette. 
7 
Resuspension or homogenization of the remaining halves in sterile tubes with a 
sterile pipette to obtain half of PVRP. 
8 
Fusion of two halves of PVRP. 
9 
PVRP resuspension 
Step 9 was followed by administration of PVRP in the ear via PVRP-soaked ear wick. 
Weighing was performed with Sartorius, T2145-OCE, Goettingen, Germany and 
centrifugation with Tehtnica, Centric 400R, Domel, d.o.o., Železniki, Slovenia. 
Termofisher scientific, USA (ref.: 225-1S, lot: 19100819) sterile pipettes and Simport 
scientific, Canada (ref.: T405-1A lot: 904175849) sterile tubes were used to prepare 
PVRP. PVRP – platelet- and extracellular vesicle-rich plasma. 
 
PVRP was brought to a patient, waiting at the Department of Otorhinolaryngology and 
Cervicofacial Surgery, University Medical Centre Ljubljana. Immediately before 
administration, PVRP was resuspended, as it sedimented during the transport. Then the 
ear wick (i.e. gauze tamponade 1 cm x 10 m cut on a 7 cm long strip, Tosama d.o.o., 
Slovenia) was soaked with PVRP and inserted into the ear. The patient was instructed to 
pull the wick out of his ear after two days. 
 
2.4 Statistical analysis plan 
We used the computer program Microsoft Excel for Mac (versions 16.9.0-16.36) to record 
and edit the trial data, and the computer program SPSS (statistical package 23, IBM Corp., 


Armonk, New York, USA) for statistical analysis. The difference between the groups was 
defined as statistically significant if the probability of rejecting the null hypothesis was 
greater than 95 % (p < 0.05). Statistical analysis plan is in a separate document. 
 
References 
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SAP_001.pdf: 
Statistical Analysis Plan 
 
Official title: Efficacy of platelet- and extracellular vesicle-rich plasma in the treatment of 
chronic postoperative temporal bone cavity inflammations  
 
NCT Number: NCT04281901 
 
Document date: February 1st, 2020 
 
 
Statistical analysis was performed by the principal investigator. We used the computer program 
Microsoft Excel for Mac (versions 16.9.0-16.36) to record and edit the survey data, and the 
computer program SPSS (angl. Statistical Package for the Social Sciences, version 23, IBM 
Corp., Armonk, New York, USA) for statistical analysis. The difference between the groups 
was defined as statistically significant if the probability of rejecting the null hypothesis was 
greater than 95% (p <0.05). 
 
1. Statistical analysis of continouous variables 
 
To compare the differences between the two groups of independent continouous numerical 
variables (e.g. the comparison between PVRP and the control group), we first determined the 
outliers in groups. Outliers were defined as values greater than 1.5 times the length of the 
boxplot (i.e., the length of the box without the whiskers). We then determined the distribution 
in the groups using the Shapiro-Wilk test. At p-value (p) <0.05, the distribution was considered 
as non-normal. If there were no outliers in both groups and the distribution was non-normal, 
we used an independent samples t-test. The homogeneity of the variances between the groups 
was checked by the Levene test. If the variances were different (i.e., heterogeneous) between 
groups (p <0.05), we used an independent samples t-test, adapted for the case of violation of 
the homogeneity of variances. If the of variances were homogeneous, we used a normal 
independent samples t-test. Descriptive t-test statistics for independent samples were described 
with mean (M), standard deviation (SD), and 95% confidence interval (95% CI), and the 
statistical signicifance was defined by the value of test statistics (t) at degrees of freedom and 
with p-value (p). 
 
If outliers and / or a non-normal distribution were present in at least one of the two independent 
groups with continuous numerical variables, the groups were analysed with the Mann-Whitney 
U-test. Data transformation or elimination of outliers was not used due to significant impact on 
results. We first compared the shape of distributions in both groups; if the shapes were similar, 
we compared the medians with the Mann-Whitney U-test. Descriptive statistics of the Mann-
Whitney U-test were presented with median (Mdn) and statistical characteristics with the value 
of Mann-Whitney test statistics (U), standardized test statistics (z) and p-value (p). 
 
To compare the differences between four groups of dependent and two groups of independent 
continuous numerical variables (eg comparison of COMQ-12 questionnaire scores between the 
four check-ups and at the same time between PVRP and the control group) we checked the 
assumptions for using two-way mixed analysis of variance, i.e., two-way mixed ANOVA). We 
first determined the outliers, the shape of the distribution, and the homogeneity of the variances 
in all groups. In the absence of outliers, normal distribution, and homogeneity of variances, the 
homogeneity of covariances was analyzed by the Box M-test. At p> 0.05, the covariances of 
the groups were homogeneous, so the sphericity was determined by the Mauchly test. Since 


sphericity was taken into account at a value of p> 0.05, a two-way mixed analysis of variance 
with post-hoc testing was performed in the final analysis. Descriptive statistics of two-way 
mixed analysis of variance were presented with mean (M) and standard error (SE), and the 
statistical characteristic with the value of test statistics F at degrees of freedom (in parentheses), 
with p-value and with effect size measure (partial η2 at p > 0.05). 
 
To compare the differences between at least four groups of dependent continuous numerical 
variables (eg comparison of the surface area between four check-ups), we first tested the 
assumptions for using one-way repeated measures ANOVA. Outliers, shape of distributions, 
and sphericity were determined in all groups. If the assumption of sphericity was met, we 
performed a standard one-way analysis of the variance with repeated measures, and when the 
assumption of sphericity was violated, we performed a Greenhouse-Geisser correction of the 
one-way analysis of the variance of repeated measures by determining the epsilon (ε) value. 
We then identified the differences between the pairs of groups by post-hoc analysis using the 
Bonferroni correction. Descriptive statistics of one-way analysis of the variance of repeated 
measurements were described with mean (M), standard deviation (SD) and 95% confidence 
interval (95% CI), and statistical significance with the value of test statistics F at degrees of 
freedom (in parentheses), with p -value and with a measure of the effect size (partial ω2 at p> 
0.05). 
 
2. Statistical analysis of dichotomous variables 
 
To compare the differences between the two groups of independent dichotomous variables (e.g. 
gender differences between PVRP and the control group), we used the Fisher exact test for 
samples of size < 5 and the χ2 test of homogeneity for samples of size > 5. The McNemar test 
was used to compare the differences between the two groups of dependent dichotomous 
variables (e.g., the presence of bacteria in the postoperative temporal bone cavity at the first 
and at the third check-up). Descriptive statistics of the analysis of dichotomous variables were 
presented with a percentage and statistical significance with p-value.