Autologous Platelet- and Extracellular Vesicle-Rich Plasma Is an Effective Treatment Modality for Chronic Postoperative Temporal Bone Cavity Inflammation: Randomized Controlled Clinical Trial

Purpose To determine the efficacy of autologous platelet- and extracellular vesicle-rich plasma (PVRP) to treat chronic postoperative temporal bone cavity inflammation (CPTBCI) after exhausting surgical and standard conservative therapies. Materials and Methods Patients were randomly allocated to treatment with PVRP (PVRP group) or standard conservative methods (control group) in a setting of four once-monthly checkups and subsequent follow-up. The treatment outcome was measured with the Chronic Otitis Media Questionnaire-12 (COMQ-12), CPTBCI focus surface area, and CPTBCI symptom-free time after the fourth checkup. Results Eleven patients from each group completed the trial; 95% of patients suffered from chronically discharging mastoid cavity (the type of CPTBCI). Within four checkups, the COMQ-12 score decreased statistically significantly in the PVRP group (p < 0.001) but not in the control group (p = 0.339). The CPTBCI foci surface area decreased statistically significantly between the first and second checkups (p < 0.0005) but not between other checkups (p > 0.05) in the PVRP group. No statistically significant differences in CPTBCI foci surface area were detected between checkups in the control group (p = 0.152). Nine patients from the PVRP group and three patients from the control group were CPTBCI symptom-free at the fourth checkup. The median symptom-free time was 9.2 months (95% CI [7.4, 11.9]) in the PVRP group. Cumulatively, 49% of patients in the PVRP group remained CPTBCI symptom-free for 12.7 months after the fourth checkup. Conclusion Autologous PVRP represents a novel additional and successful treatment modality for a chronically discharging radical mastoid cavity when the surgical and standard conservative treatment methods have been exhausted. Trial Number https://clinicaltrials.gov (NCT04281901).


INTRODUCTION
The chronic postoperative temporal bone cavity inflammation (CPTBCI) typically implies a chronically discharging radical mastoid cavity, which results from canal wall down (i.e., radical) mastoidectomy (Gluth et al., 2013). This otological surgical procedure is performed in about 53% of cholesteatoma surgery (Wilkie et al., 2019). Although the CPTBCI affects only 1.5% of patients with the radical mastoid cavity (Pareschi et al., 2019), its burden on quality of life and healthcare is significant (Khalil and Windle-Taylor, 2003;Dornhoffer et al., 2008). The treatment is challenging, especially when the established treatment methods are exhausted. Considering that the CPTBCI is a subtype of chronic otitis media (COM), health-related quality of life can be assessed with patient-reported outcome measures, such as the Chronic Otitis Media Questionnaire-12 (COMQ-12) (Phillips et al., 2014;Demir et al., 2020).
The awareness of the burden of CPTBCI has induced research in novel treatment modalities, including the application of medical honey and the refinement of surgical techniques (Henatsch et al., 2015). On the other hand, the platelet-rich plasma (PRP) has been widely used to promote wound healing in many clinical settings, recently in skull-base reconstruction (Khafagy et al., 2018), nasal septal surgery, tonsillectomy (Gökçe Kütük and Özdaş, 2019), and laryngectomy (Eid et al., 2020) due to beneficial immune, hemostatic, and regenerative effects of platelets exerted by the release of growth factors from granules and several extra-granular molecules. Despite that, research, such as PRP in otorhinolaryngology, lacks randomized controlled clinical trials (Pachito et al., 2019;Vozel et al., 2020b). As plasma contains extracellular vesicles (EVs), which are considered to be important healing vectors, we refer to the preparation as plateletand extracellular vesicle-rich plasma (PVRP) in this study . EVs are a heterogeneous group of nanosized cellderived membrane vesicles with essential roles in intercellular communication and possess an exceptional potential to be used in diagnosis, treatment, or both (i.e., theranostics) (Yáñez-Mó et al., 2015). EVs are naturally present in the blood. In addition, they are produced from cells in blood during the sampling and processing Abbreviations: COM, chronic otitis media; COMQ-12, chronic otitis media questionnaire-12; CPTBCI, chronic postoperative temporal bone cavity inflammation; EVs, extracellular vesicles; PDGF, platelet-derived growth factor; PRP, platelet-rich plasma; PVRP, platelet-and extracellular vesicle-rich plasma.
of samples (Šuštar et al., 2011). Although the concentrations of EVs in PVRP are expected to be high, the literature describing this is scarce .
For the abovementioned reasons, the importance of research on the efficacy of PVRP in the treatment of CPTBCI has been recognized. As a result, this prospective randomized controlled clinical trial aims to compare the efficacy of autologous PVRP (administered via ear wick) and standard conservative methods in the CPTBCI treatment by measuring the outcome at 1, 2, and 3 months after the baseline evaluation with the CPTBCI surface area measurement and COMQ-12. The trial hypothesizes that the PVRP is efficient in the CPTBCI treatment when standard conservative and surgical methods have been exhausted. The exhaustion of methods to treat CPTBCI is the rationale behind this trial.

MATERIALS AND METHODS
The trial was registered, and results were reported on clinicaltrials.gov (No. NCT04281901) and approved by the National Medical Ethics Committee Slovenia (No. 0120-146/2019/5). Written informed consent was obtained from all individual participants included in the study.

Enrolment
The trial started by patient enrolment (Figure 1) during regularly scheduled checkups between March 20, 2019 and September 15, 2019 at the tertiary otorhinolaryngology referral center by leading researchers (SB and DV). This time is considerably longer than the period between two checkups in a patient with CPTBCI.
The inclusion criteria of the trial were: • Age > 18 years.
• COM defined as a presence of ≥ 1 of clinical signs of inflammation exacerbation, visible ear discharge, indirect ear discharge signs (e.g., on a pillow and clothes), itching, and the sensation of ear fullness. • Exhausted surgical treatment: additional surgical treatment of CPTBCI would not preserve serviceable hearing loss. • Exhausted standard conservative treatment: ineffective conservative treatment of CPTBCI for at least 8 weeks, which included administration of antimicrobial, antiinflammatory, antiseptic preparations (i.e., drops, FIGURE 1 | Flowchart of a prospective randomized controlled clinical trial for treating patients with chronic postoperative temporal bone cavity inflammation. Data are presented according to the CONSORT statement (Schulz et al., 2010). Control group represented intervention A and PVRP intervention B. CPTBCI: chronic postoperative temporal bone cavity inflammation; PVRP: platelet-and extracellular vesicle-rich plasma; COVID-19: coronavirus disease 2019.
ointments, pastes, and powders), and/or silver nitrate sticks (to perform chemocautery on the granulation tissue).
The exclusion criteria of the trial were: • Venipuncture site inflammation.
• Pregnancy or breastfeeding, chronic use of immunomodulatory, and/or antimicrobial drugs.
• Presence of systemic infectious disease, autoimmune disease, and malignancy. • Other experimental attempts to treat CPTBCI. • Inability and/or refusal of the patient to participate in the trial.
Unilateral CPTBCI was considered as one case and bilateral CPTBCI as two cases of CPTBCI.

Allocation and Treatment Initiation
After enrolment, patients were allocated treatment with standard conservative methods (i.e., control group) or treatment with PVRP (i.e., PRVP group) with simple random allocation (Machin and Fayers, 2010) using sealed envelopes by a senior researcher (VK-I) (Supplementary Material 1). Then, patients were informed by the leading researchers (DV and SB) about the treatment allocation at their next checkup scheduled regularly between September 18, 2019 and January 22, 2020. After re-evaluating the inclusion and exclusion criteria of the trial at this checkup, informed written consent was provided, and the patient started to participate in the trial. Later, the treatment regimen of the trial, which included four checkups, was initiated. After the first (i.e., baseline) checkup, three checkups with 4-week intervals were planned by the leading researchers (DV and SB). Subsequent checkups (i.e., after the fourth checkup) were considered as a followup.
The control group was treated with standard conservative methods (described in the inclusion criteria of the trial) at each checkup. The PVRP group was treated with the autologous PVRP at the first and second checkups. The aural toilet (i.e., cleaning by aspiration) was performed in all patients at each checkup. All patients were instructed to comply with the dry ear precautions.
Refusal of a patient to participate in the trial did not change the regular management of CPTBCI.

Primary Outcome Measures
At each of the four checkups, the CPTBCI focus surface area was measured for each CPTBCI case, and every patient completed the COMQ-12 (Phillips et al., 2014). The latter was validated and cross-culturally adapted in the target language (Vozel et al., 2020c).
The CPTBCI foci (e.g., granulation tissue, redness, and edema) were detected and photographed by the leading researchers (DV and SB) with a diagnostic otomicroscope (OPMI pico/S100, Carl Zeiss Surgical GmbH, Oberkochen, Germany) at 5.1× and 8.5× magnification, for which calibration was performed. The focus of inflammation was instantly anatomically classified (Supplementary Material 2). Later, the independent blinded outcome assessor (NS), unaware of treatment allocation, outlined the focus and measured the CPTBCI focus surface using the ZEN 3.0 blue edition software (© Carl Zeiss Microscopy GmbH, 2019). Areas in mm 2 were converted into a percentage with respect to the baseline CPTBCI surface area (i.e., 100% at the first checkup) as depicted in Figure 2.

Secondary Outcome Measure
The secondary outcome measure was CPTBCI symptom-free time measured in the follow-up period. It was analyzed in patients who did not suffer from CPTBCI-related symptoms between the fourth checkup and subsequent checkup(s) in the followup period. These symptoms were ear discharge, indirect ear discharge signs (e.g., on a pillow and clothes), itching, pain, and the sensation of ear fullness.

Platelet-and Extracellular Vesicle-Rich Plasma-Related Adverse Events
The PVRP-related adverse events were considered as aggravation of CPTBCI when intensification of ear discharge, an itch, and a sensation of ear fullness occurred within 3 days after PVRP administration. To monitor the adverse events, patients were instructed to contact the leading researchers (DV and SB) or tertiary otorhinolaryngology referral center.

Platelet-and Extracellular Vesicle-Rich Plasma Preparation Protocol
Blood was drawn into four 4.5-ml evacuated blood tubes with sodium citrate anticoagulant (9 NC sodium citrate 0.105 M, BD Vacutainer, Becton Dickinson, San Jose, CA, United States, stored at room temperature) using a 21-G wing needle (Safety-Lok Blood Collection Set, BD Vacutainer, Becton Dickinson, San Jose, CA, United States) (Figure 3, panel 1). Two filled blood tubes were used to prepare one unit of PVRP, and two units were prepared in parallel. Blood tubes were immediately transported at ambient temperature (22-24 • C) to the laboratory, where the PVRP preparation started. First, the soft spin centrifugation (i.e., 5 min, 300 × g, 18 • C) of blood tubes was performed, and two fractions were distinguished (Figure 3, panels 2, 3A): hematocrit (red layer in the bottom) and plasma with platelets (yellow upper layer). Occasionally, a buffy coat (thin opaque whitish layer of leukocytes) on top of hematocrit could be observed. Plasma directly above the buffy coat was transferred by a sterile pipette (Figure 3, panel 3A) into a sterile polypropylene tube (Figure 3, panel 3B). Caution was required to prevent mixing layers, i.e., disruption of hematocrit and buffy coat and consequent leukocyte and erythrocyte harvesting. Collected plasma was equally distributed among two sterile polypropylene tubes and then centrifuged at hard spin (i.e., 17 min, 700 × g, 18 • C) to sediment the platelets plasma (Figure 3, white arrows in panels 4, 5A, and 5B). The upper half of the centrifuged plasma (bordered by the white dashed line Figure 3, panel 4), called platelet-poor plasma, was carefully removed and discarded without disruption of layers (Figure 3, panel 5A). The remaining pellet was resuspended in the remaining plasma volume (Figure 3, panel 5B) in the same tube (Figure 3, panel 5C) to produce PVRP. Two units of PVRP were pooled and administered to the same patient (i.e., autologously) via ear wick (i.e., gauze tamponade 1 cm × 10 m cut on a 7-cm long wick, Tosama d.o.o., Domzale, Slovenia) into the area of CPTBCI (Figure 3, panel 6). The patient was instructed to remove the ear wick after 2 days. Autologous PVRP administration was performed at the first and second checkups in the PVRP group.

Analyses of Blood and Platelet-and Extracellular Vesicle-Rich Plasma
Concentration of leukocytes, erythrocytes, hemoglobin, C-reactive protein (CRP), differential blood cell count, and erythrocyte sedimentation rate (ESR) was determined in blood samples by standard automatic cytometry methods.  Additionally, blood and PVRP samples were analyzed by flow cytometry (flow cytometer MACS QUANT, Miltenyi, Bergisch-Gladbach, Germany, and the related MACSQuantify software) to classify PVRP as given in DeLong et al. (2012) according to the platelet concentrations in PVRP (DeLong et al., 2012). Particles were characterized based on forwarding and side scattering signals of the flow cytometer. The gates were set in a preliminary study, in which the identity of cells in the presented regions was confirmed by immuno-labeling and by scanning electron microscopy analysis. The latter also provided evidence on the presence of vesicles. By flow cytometry approaches used in this study, a fraction of vesicles (bigger than the threshold 400 nm) is detected and evaluated as a part of the population P2 (Supplementary Material 3). Later, the yield of platelets and the detectable fraction of EVs in PVRP was calculated by the following equation: concentration of platelets and EVs in PVRP concentration of platelets and EVs in blood (1) The PVRP was prepared for sterility analysis using the preparation protocol described above from volunteers aged over 18 years and without CPTBCI and associated diseases and signs of infection. Chemicals, additional materials, and methods used in the PVRP analysis are described in Supplementary Material 4.

RESULTS
Results of the interim analyses of the trial have been partially presented elsewhere (Vozel et al., 2020a).

Patients
The trial flowchart is depicted in Figure 1 and reported according to the CONSORT statement in Supplementary Material 6 (Schulz et al., 2010). Twenty-five patients or 27 cases of CPTBCI (two patients with bilateral involvement) were enrolled and allocated. One patient allocated to the PVRP group refused to participate in the trial, and two (i.e., one from each group) were lost to follow-up. The final analysis included 22 patients or 24 cases of CPTBCI (i.e., two patients with bilateral CPTBCI) ( Table 1 and Supplementary Material 7).

Alcohol consumption
Yes ( the control group), radical mastoidectomy was performed due to middle ear cholesteatoma (Supplementary Material 7). The intervals between checkups did not show statistically significant difference between groups (Mdn = 28 and p > 0.05 for each interval between checkups) (Supplementary Material 9).
Blood laboratory results determined by standard automatic cytometry methods were normal in all patients. The average platelet and EV concentration measured by flow cytometry in the blood samples of the PVRP group was 199 × 10 9 /L (SD = 62 × 10 9 /L) (Supplementary Material 8).

Treatment Outcome
Chronic Otitis Media Questionnaire-12 Intervention allocation had a statistically significant effect on the COMQ-12 sum scores throughout four checkups according to the two-way mixed ANOVA [F(3, 60) = 8.755, p < 0.0005, partial

Follow-up
At the fourth checkup, 82% (i.e., nine patients) from the PVRP group and 27% (i.e., three patients) from the control group were CPTBCI symptom-free; therefore, the CPTBCI symptom-free time was measured for these patients (Supplementary Material  9). Data were cut off on February 22, 2021 for the patients who were symptom-free at that date. The median symptom-free time was 9.2 months (95% CI [7.4,11.9]) in the PVRP group. According to the Kaplan-Meier analysis, cumulatively, 49% of patients in the PVRP group remained symptom-free for 12.7  graphs (A,B). Graph (C) is based on the data of 9 of 11 (82%) patients treated with PVRP who were symptom-free after the fourth checkup of the trial. Cumulatively, 49% of patients from the PVRP group remained symptom-free for 12.7 months after the fourth checkup. COMQ-12: chronic otitis media questionnaire 12; CPTBCI: chronic postoperative temporal bone cavity inflammation; PVRP: platelet-and extracellular vesicle-rich plasma; *statistically significant difference between groups within checkup; #statistically significant difference between the first and second checkups in the PVRP group.

Platelet-and Extracellular Vesicle-Rich Plasma-Related Adverse Events
No PVRP-related adverse events were detected.
Scanning electron microscopy of PVRP samples showed an abundance of platelets, as demonstrated in Figure 5A. We detected no resting disk-like platelets in PVRP. Instead, observed platelets came in different sizes and shapes and exhibited tubular protrusions. Fragmented or deformed platelets that have lost a considerable part of their membrane are considered EVs (Figures 5C,D)   since there is no sharp boundary between platelets and EVs.
The PVRP preparation protocol sterility analyses of the PVRP prepared from eight volunteers showed the absence of bacterial or fungal growth in all eight samples.

DISCUSSION
This randomized controlled clinical trial compares the efficacy of autologous sterile PVRP and standard conservative methods in the CPTBCI treatment. Autologous PVRP possesses beneficial effects when administered two times in a monthly interval via ear wick according to the results of the CPTBCI-related quality of life assessment ( Figure 4A) and CPTBCI foci surface area measurements ( Figure 4B).
The treatment with PVRP resulted in a more significant improvement of the CPTBCI-related quality of life than the treatment with standard conservative methods ( Figure 4A). Additionally, the CPTBCI foci surface areas decreased statistically significantly within 4 months in the PVRP group but not in the control group ( Figure 4B). This trial may indicate long-term effects of PVRP-based treatment of CPTBCI according to the absence of symptoms in most patients for many months after the last (i.e., fourth) checkup of the treatment regime ( Figure 4C). This corresponds to the long-term effects of platelet-rich preparations described by Altamura et al. (2020). No PVRP-related adverse events were detected in this trial, which is consistent with the findings of low adverse events rate in the meta-analysis of Wang et al. (2019). Therefore, PVRP is a safe method to treat CPTBCI.
The tolerable withdrawn blood volume (i.e., 18 ml) and the time required to prepare PVRP (i.e., M = 80 min, SD = 26 min) favor the treatment of other otorhinolaryngological diseases with PVRP on an outpatient or inpatient basis. To optimize platelet recovery and reduce withdrawn blood volume, theoretical models based on whole blood volume, hematocrit, and centrifugal conditions could be considered (Piao et al., 2017). Since there are heterogeneity and 10% replicability of PVRP preparation protocols, there is an urge to describe the PVRP and its preparation steps (Chahla et al., 2017). The thoroughly described sterile PVRP preparation protocol used in this trial yielded moderate platelet concentrations, which were identified as advantageous in the regeneration of PVRP as studied by DeLong et al. (2012). Based on the CPTBCI treatment outcomes and identification of EVs in PVRP (Figure 5), it is believed that EVs, along with platelets, positively impact the regenerative properties of PVRP . It has been reported that plateletderived EVs play significant roles in tissue regeneration, hemostasis, and immune response (Vozel et al., 2020b). A twofold mechanism could mediate these roles. EVs exhibit a larger surface-to-volume ratio than platelets and provide an extended catalytic area for biologically important reactions. By mediating interactions between cells, EVs carry signaling molecules for phenotypic transformation and serve as a cell mechanism to get rid of unwanted constituents (Ogorevc et al., 2014). In addition, platelet-derived EVs encapsulate principal growth factors from platelets  and are especially rich in TFG-β (transforming growth factor beta), bFGF (basic fibroblast growth factor), PDGF (platelet-derived growth factor), and VEGF (vascular endothelial growth factor) (Vozel et al., 2020b). Regenerative roles of platelet-derived EVs from PVRP were confirmed in a recent in vitro study by Otahal et al. (2020), where the induction of chondrogenic gene expression in chondrocytes and prevention of the release of proinflammatory cytokines were found. This study substantiates the regulatory roles of EVs in inflammation and metabolism of the cartilage extracellular matrix (Otahal et al., 2020). The above-listed properties of EVs could explain the regenerative, hemostatic, and immunological roles of PVRP in the treatment of CPTBCI. These roles could be regulated by choosing an appropriate PVRP activation procedure (i.e., exogenous or endogenous) and optimizing PVRP preparation protocol since the formation of EVs depends on in vivo or ex vivo platelet shedding (Vozel et al., 2020b).
There were no differences in baseline demographic, clinical characteristics, and COMQ-12 scores between groups, which confirm randomization (Table 1). To the best of our knowledge, there is no literature reporting the COMQ-12 scoring to assess CPTBCI-related quality of life. Moreover, the median COMQ-12 score of patients with CPTBCI (Mdn = 34) resembles the score of discharging COM without cholesteatoma (M = 33) as reported by Demir et al. (2020). For that reason, this trial confirms the significant socioeconomic and healthcare burden of CPTBCI (mainly chronically discharging radical mastoid cavity), comparable to COM (Khalil and Windle-Taylor, 2003). This burden, along with a long-term commitment to the patient and exhaustion of standard conservative treatment methods, is confirmed by the median time to treatment (i.e., 8.1 years) in our trial and was already reported by Dornhoffer et al. (2008). Analysis of performed procedures confirmed the exhaustion of surgical treatment methods. More radical surgical procedures (i.e., subtotal petrosectomy) would prevent serviceable hearing loss and not necessarily treat the CPTBCI (Yung, 2016). Additionally, the percentage of radical mastoidectomy performed to treat cholesteatoma (92%) and the predominance of male patients (82%) are consistent with the study of Khalil and Windle-Taylor (2003).
The results of risk factors of the patient (i.e., comorbidities, regular medicine intake, and harmful habits) are unique since there are no such data so far, to the best of our knowledge. However, there is no apparent predisposition to suffer from CPTBCI, according to the risk factor analysis. In contrast, two patients with coagulopathies (i.e., regular acetylsalicylic acid intake) were efficiently treated with PVRP. This also complements the study results by Smith et al. (2007), which reported that acetylsalicylic acid does not decrease the release of PDGF, which possesses essential regenerative roles of PVRP (Smith et al., 2007).
However, it is essential to balance the effects and limitations of PVRP-based therapies due to the high implicit hype of PVRP (Rachul et al., 2017). Since only 12% of patients were excluded from the trial after enrolment, the internal validity due to loss to follow-up is not at risk (Dettori et al., 2011). Issues related to the selection bias were avoided by the allocation concealment, and issues related to the performance bias were avoided by simple random allocation. Additionally, the blinded independent treatment outcome assessor measured the CPTBCI foci surface areas to minimize the detection bias. It is challenging to photograph with an otomicroscope under the same exposure at every checkup; therefore, CPTBCI foci surface area measurements could be biased.
Nonetheless, this objective measurement tool has reportedly not been utilized in inflammation area measurement so far. For that reason, it could provide the basis for further research in otology. Due to non-blind treatment allocation, the treatment outcome measured with COMQ-12 could present the detection bias. However, this disease-specific patient-reported outcome measure is possibly the most appropriate tool to assess CPTBCIrelated quality of life, as already discussed. To the best of our knowledge, COMQ-12 has not been utilized to measure CPTBCI treatment outcome yet. In addition, to the best of our knowledge, the application of PVRP in the treatment of CPTBCI has been reported for the first time. Lastly, our trial is based on a relatively small sample collected in a single institution. CPTBCI is a rare disease that affects about 1.5% of patients with a radical mastoid cavity (Pareschi et al., 2019) or 0.8% after cholesteatoma surgery (Pareschi et al., 2019;Wilkie et al., 2019). Moreover, the risk of developing therapeutically resistant CPTBCI is even lower. Multicentric research could be conducted to overcome the limitation of a small sample size.

CONCLUSION
Autologous PVRP represents a novel additional and successful treatment modality for a chronically discharging radical mastoid cavity when the surgical and standard conservative treatment methods have been exhausted. This disease is a type of CPTBCI and resembles COM without cholesteatoma in its impact on health-related quality of life, according to  This trial contributes to further research on PVRP-based treatment for hearing loss (Stolle et al., 2018), facial nerve palsy (Sánchez et al., 2018), dysphonia (Cobden et al., 2016;Özgürsoy et al., 2018), dysphagia (Vidovic and Delling, 2017), anosmia (Yasak et al., 2018), soft-tissue defects (Eryılmaz et al., 2016;Tambella et al., 2018), or laryngotracheal reconstruction (Sahin Onder et al., 2020). In addition, sterile PVRP could be administered to the sterile human body areas. Since it is believed that platelets and EVs carry main benefits of PVRP, EV isolates from PVRP or various body fluids could be used to treat various diseases (Fais et al., 2016), including otological in the future (Kalinec et al., 2019).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by National Medical Ethics Committee, Slovenia. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
DV contributed to the conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, validation, visualization, and writing -original draft preparation, review, and editing. DB was involved in the data curation, funding acquisition, investigation, methodology, and writing -review and editing. MJ, ZJ, MP, and LP participated in the funding acquisition, investigation, and writing -review and editing. NS was involved in investigation and writingreview and editing. VK-I contributed to the conceptualization, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, and writing -original draft preparation, review, and editing. SB contributed to the conceptualization, investigation, methodology, project administration, resources, software, supervision, visualization, and writing -original draft preparation, review, and editing. All authors contributed to the article and approved the submitted version.