Mesenchymal Stem Cells for Radiation-induced Xerostomia (MESRIX) in Previous HPV-positive Oropharyngeal Head and Neck Cancer Patients - A Safety and Feasibility Study
Phase: PHASE2
Conditions: Xerostomia
Sponsor: Rigshospitalet, Denmark

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Title: 
First-in-man mesenchymal stem cells for radiation-induced xerostomia 
(MESRIX): study protocol for a randomized controlled trial 
 
Christian Grønhøj Larsen (1), David H. Jensen (1), Peter V. Glovinski (2), Siri Beier Jensen (3), 
Allan Bardow (4), Roberto S. Oliveri (5), Lena Specht (6), Carsten Thomsen (7), Sune Darkner (8), 
Katalin Kiss (9), Anne Fischer-Nielsen (5), and Christian von Buchwald (1) 
 
Authors' information 
 
1: Department of Otorhinolaryngology - Head and Neck Surgery and Audiology, Rigshospitalet, University of 
Copenhagen, Section 2071, Blegdamsvej 9, 2100 Copenhagen, Denmark 
 
2: Department of Plastic Surgery, Breast Surgery and Burns, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 
2100 Copenhagen, Denmark 
 
3: Department of Dentistry and Oral Health, Vennelyst Boulevard 9, DK-8000 Aarhus C, Denmark 
 
4: Department of Oral Medicine, Department of Odontology, Faculty Of Health and Medical Sciences, University of 
Copenhagen, Nørre Allé 20, DK-2200 Copenhagen N 
Denmark 
 
 
 
5: Cell Therapy Facility, The Blood Bank, Dept. of Clinical Immunology, Rigshospitalet, University of Copenhagen, 
Blegdamsvej 9, 2100 Copenhagen, Denmark 
 
6: Department of Oncology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark 
 
7: Department of Radiology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark 
 
8: Department of Computer Science, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark 
 
9: Department of Pathology, Rigshospitalet, University of Copenhagen, Section 2071, Blegdamsvej 9, 2100 
Copenhagen, Denmark 
 
Email addresses: 
Christian Grønhøj Larsen: c.gronhoj@gmail.com 
David H. Jensen: david_hebbelstrup_jensen@hotmail.com 
Peter V. Glovinski: peter.viktor.vester-glowinski@regionh.dk 
Siri Beier Jensen: siri@dent.au.dk 
Allan Bardow: alba@sund.ku.dk 
Roberto S. Oliveri: mafioso_dk@yahoo.dk 
Lena Specht: lena.specht@regionh.dk 
Carsten Thomsen: carsten.thomsen@regionh.dk 
Sune Darkner: darkner@di.ku.dk 
Katalin Kiss: katalin.kiss@regionh.dk 
Anne Fischer-Nielsen: Anne.Fischer-Nielsen@regionh.dk 

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Christian von Buchwald: christian.buchwald@regionh.dk 
 
 
Corresponding author 
Christian Grønhøj Larsen 
Department of Otorhinolaryngology, Head and Neck Surgery and Audiology, Rigshospitalet, University of 
Copenhagen, Denmark 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Abstract 
Background 
Salivary gland hypofunction and xerostomia are major complications following radiotherapy for 
head and neck cancer and may lead to debilitating oral disorders and impaired quality of life. 
Currently, only symptomatic treatment is available. However, mesenchymal stem cell (MSC) 
therapy has shown promising results in preclinical studies. Objectives are to assess safety and 
efficacy in a first-in-man trial on adipose-derived MSC-therapy (ASC) for radiation-induced 
xerostomia.  
 
Methods 
This is a single-center, phase I and II, randomized, placebo-controlled, double-blinded clinical trial. 
A total of 30 patients are randomized in a 1:1 ratio to receive ultrasound-guided, administered ASC 
or placebo to the submandibular glands. The primary outcome is safety. The secondary outcomes 
are efficacy, change in quality of life, qualitative and quantitative measurements of saliva, as well as 
submandibular gland size, vascularization, fibrosis, and secretory tissue evaluation based on 
contrast-induced magnetic resonance imaging (MRI) and core-needle-samples. The assessments are 
performed at baseline (one month prior to treatment), and one and four months following 
investigational intervention. 
Discussion 
The trial is the first attempt to evaluate the safety and efficacy of ASCs in patients with radiation-
induced xerostomia. The results may provide evidence for the effectiveness of ASC therapy in 
patients with salivary gland hypofunction and xerostomia and deliver valuable information for the 
design of subsequent trials. 
 
Trial registration section 
EudraCT: 2014-004349-29. Registered: 1st of April 2015 
ClinicalTrials.gov Identifier: NCT02513238. First received: 2nd of July, 2015 
The trial is prospectively registered. 
 
Key words 
Mesenchymal stem cells, xerostomia 
 

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Background 
Xerostomia is the term used for a subjective feeling of dry mouth. Xerostomia can be coexisting 
with or without a reduced secretion of saliva, although xerostomia is generally perceived only when 
unstimulated whole saliva flow rate is reduced by more than 40-50% [1]. Thus, a decreased 
(salivary gland hypofunction) or pathologically reduced saliva secretion (hyposalivation) will 
severely impact quality of life and may lead to dental decay. The three main causes of severe 
xerostomia and hyposalivation are adverse effects of medication intake (polypharmacy and/or 
xerogenic medications), Sjögren's syndrome and radiation therapy for head and neck cancer. With 
regards to cancer chemotherapy, studies suggest that in some patients it may induce temporary 
salivary gland hypofunction and xerostomia during and following treatment, while other patients 
are not affected to a noticeable extent, however, no firm conclusions can be drawn from the 
literature [2]. Radiation therapy plays a major role in the curative treatment of most head and neck 
cancers, either as a single modality, or in combination with chemotherapy, surgery, or both. 
Radiation therapy significantly increases local tumour control and chance of survival, but despite 
more advanced methods (intensity-modulated radiation therapy, IMRT), a significant proportion of 
the radiation is deposited in the normal tissue surrounding the tumour. The long-term effect of 
radiation on salivary gland tissue is detoriation of gland function. [3,4]. Salivary gland dysfunction 
after radiation therapy predisposes to a variety of undesirable conditions directly or indirectly as a 
result of decreased salivary flow rate as well as changed composition and increased viscosity of 
saliva and include xerostomia, impairment of oral functions due to insufficient wetting (i.e. speech, 
chewing and swallowing) and reduced lubrication of mucosal surfaces and of the ingested food. 
Furthermore, the oral mucosa is prone to frictional trauma and ulceration. In addition, a reduced 
salivary flow rate results in a reduced clearance of the oral cavity, thus leading to microbial 
overgrowth, which in addition to other factors may result in rampant dental caries, dental erosion 
and oral candidiasis [2,5]. 
 
The average total dose range which represents the threshold for a significant reduction in salivary 
flow rate is 26-39 Gray (Gy) [6–10]. The dose causing toxicity in 50% of individuals (TD 50) is 
likely to be approximately 40 Gy [11], which is similar to the TD 50 [12], estimated for 
submandibular hypofunction [13]. The used methods to reduce the incidence of xerostomia after 
radiation is prevention (including more advanced radiation techniques [IMRT or proton therapy], 
radio-protective agents [amifostine and tempol [14]], and the more recently proposed stem cell 

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sparing radiation therapy [15]). 
 
A number of strategies to improve salivary gland function after radiation therapy have been 
developed, but all are symptomatic treatments only able to stimulate the function of the residual 
salivary gland tissue or provide short-term lubrication. These strategies include pharmacological 
agents, for example sialogogues (pilocarpine, cevimeline, etc), saliva stimulants (sugar-free, non-
erosive lozenges, chewing gum and custom-made, non-erosive weak-acidic candy) [16], and the use 
of oral lubricants and saliva substitutes [17]. 
 
Stem cells have been identified as a potential treatment modality for a wide variety of cell 
degenerative disorders by virtue of their ability to differentiate into different specialized cell types. 
Most of the early work on stem cells has been performed in embryonic stem (ES) cells, which are 
derived from the inner cell mass of a blastocyst embryo. The clinical use of these cells as 
therapeutic agents is currently very limited, both due to histocompatibility problems and their 
potential ability to form malignant teratomas. Problems with histocompatibility have been improved 
with the development of induced pluripotent stem cells (iPS cells). In contrast to ES and iPS, which 
are both pluripotent stem cells i.e. ability to differentiate into cells deriving from all three primary 
germ layers, ecto-, meso-, and endoderm; adult stem cells are defined as multipotent, i.e. with a 
more narrow differential ability. In this respect, mesenchymal stem cells (MSCs) have gained 
considerable attention, since they are readily available from e.g. bone marrow and adipose tissue 
and have been characterized and investigated in a wide variety of pre-clinical studies and clinical 
trials. 
 
Originally described more than 40 years ago, MSCs reside in almost all connective tissues [18]. 
More recently, there has been an increasing awareness that MSCs have a number of interesting 
secretory paracrine bystander characteristics including anti-inflammatory, anti-apoptotic, 
immunomodulatory, angiogenic and trophic (tissue-regenerating) properties. Notably, MSCs have 
shown promising results in preclinical studies for the treatment of xerostomia, including radiation-
induced xerostomia [19]. 
 
The objectives of the current trial are to assess the safety and feasibility of autologous adipose 
tissue-derived MSCs administered for radiation-induced salivary gland hypofunction and 

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xerostomia in head and neck cancer patients. Head and neck cancer is the sixth most common 
malignancy worldwide, and the majority of these patients are due to advanced disease stage at 
presentation treated with chemo-, radiotherapy, or both [20,21]. The project can potentially help to 
develop a clinically relevant treatment option for the growing number of patients suffering from 
xerostomia after radiotherapy.  
 
Methods and materials 
Primary outcome 
Primary outcome is safety, including adverse events (AEs) and severe adverse events (SAEs). All 
measures of AEs will be graded according to Common Terminology Criteria for Adverse Events 
(CTCAE) [22]. Since this is a local treatment with ASCs the primary safety measures are: 
 Pain at injection site (Grade 1: Mild pain, grade 2: Moderate pain; limiting instrumental 
activities of daily living (ADL), grade 3: Severe pain; limiting self-care ADL) 
 Oral discomfort (Grade 1: Mild discomfort; not interfering with oral intake, grade 2: 
Moderate pain; interfering with oral intake, 3: Disabling pain; tube feeding or total parental 
nutrition indicated) 
 Infection (Grade 1: Localized; local intervention indicated, grade 2: Oral intervention 
indicated (antibiotic, antifungal, antiviral), grade 3: IV antibiotic, antifungal or antiviral 
indicated; or radiologic, endoscopic or operative intervention indicated, grade 4: life 
threatening consequences; urgent intervention needed) 
 
Secondary outcomes 
 Unstimulated and stimulated whole saliva and selective submandibular saliva flow rate and 
composition of saliva.  
 Complaints of xerostomia as evaluated by a physician and patient questionnaire. 
 Measurement of volume, fibrosis, and vascularisation change of submandibular glands 
based on magnetic resonance imaging (MRI).  
 
Morphological changes of the gland tissue, e.g. degree of atrophy, fibrosis, inflammation, 
and amount of specialized acinic cells including duct/acinic/myoepitelial cell ratio in 
histological sections from core-needle biopsies taken pre- (baseline) and post-interventional 
(4 months). An expert head and neck pathologist will blinded to treatment of participants 
evaluate the two sets of biopsies for changes.  

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Late complications – safety 
To detect late complications or late adverse events all study participants, who received ASC-
treatment are invited for a check-up one year and three years after treatment. Whether or not 
participants consent to a late check-up, the Principal Investigator (CGL) will contact participants by 
telephone to ensure that no subjective complaints have befallen. In case of subjective complaints, 
participants will be encouraged to meet for a physical examination. 
 
Trial design 
Randomized, placebo-controlled, phase I/II trial with double blinding (Figure 1, Figure 2, SPIRIT 
Checklist (Additional file 1)). All participants will undergo a mini lipo-suction from which ASCs 
will be ex vivo expanded in a good manufacturing practice (GMP)-approved clean room facility. 
The Good Clinical Practice committee performs data monitoring, and is independent from the 
sponsor and has no competing interests. 
 
One month prior to the liposuction, participants will undergo a MRI scan of the submandibular 
glands, fill-out questionnaires on quality of life and undergo saliva measurements. Also, all patients 
will have blood samples analyzed for anti-HIV I/II, HBsAg, anti-HBc, anti-HCV and syphilis to 
fulfil the requirements in the Danish Tissue Act, as well as relevant kidney parameters to secure 
adequate renal function when using MRI contrast, see below. A core-needle-sample of one of the 
submandibular glands and a liposuction of the abdomen (approx. 60 ml of adipose tissue) are 
performed for all participants. Approximately 14 days following liposuction, participants will have 
ASCs or placebo injected in the submandibular glands on an outpatient basis. Subjects will 
subsequently undergo a saliva measurement, contrast-induced MRI and fill out questionnaires at 
baseline, 4 weeks post-intervention and 4 months post-intervention. Finally, at the 4-month visit, a 
small biopsy is taken from one of the two submandibular glands. The procedure takes place under 
local anaesthesia. Histology is determined (samples are blinded to the pathologist) (Figure 1). 
 
Randomization 
At inclusion to the study protocol, each participant will be given consecutive numbers starting with 
number 1. To randomize participants to either placebo or MSCs we will use a table of random 
numbers, generated by a computer program (http://www.randomization.com). This table with 

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randomization numbers will only be available to specified personnel at the Tissue Center, 
Department of Clinical Immunology (Rigshospitalet). 
 
Amount of ASCs required for injection 
Based on published animal studies the amount of cells given for xerostomia to mice varies from 2 x 
105 to 2 x 106 [23]. To convert these mice data to a realistic dose in humans we have extrapolated 
numbers based on the following data: The volume density of submandibular glands very closely 
approximates 1 mg/mm3 (1.06-1.07 mg/mm3) [24]. In the mice studies on radiotherapy the mean 
gland weight was approximately 350 mg, which corresponds to an approximate volume of 350 
mm3. Accordingly, the mouse dose pr. gland volume is approximately 2.86 x 105 to 2.86 x 106 
ASC/cm3 gland (when calculated from either 1 x 105 to 1 x 106). The volume of the submandibular 
gland in human subjects after radiotherapy is 6.6-7.9 cm3 [25]. This corresponds to an approximate 
dose pr. patient of between 1.9 x 106 to 2.3 x 106, or 1.9 x 107 to 2.3 x 107 cells pr. submandibular 
gland. From the above assumptions, and in order not to administer an inadequate number of cells, 
we have chosen to proceed with the maximum dose corresponding to 2.8 x 106 ASC/cm3 gland, i.e. 
a maximum total dose per patient of approximately 4.48 x 107 ASCs (see below). However, to 
standardize the dose administered to patients, the amount administered will be standardized to the 
size of their submandibular glands, as described below. 
 
Injection of the ASCs or placebo-suspensions into submandibular glands will be performed by the 
principal investigator (CGL) under local anesthesia using ultrasonic guidance and sterile technique. 
The ASCs will be suspended in isotonic NaCl (0.9 mg/ml) and human albumin (HA) 1% to a final 
volume of 2 ml. Placebo will be 2 ml of isotonic NaCl (0.9 mg/ml) and HA 1%. After receiving the 
ASC-suspension or placebo-suspension, the surgeon (the principal investigator) will identify the 
submandibular glands and inject the ASC suspension. Calculation of injected number of ASCs pr. 
participant rests on the following calculation: 𝑆𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛𝑚𝑙= 2.8 × 106 
𝐴𝑆𝐶
𝑐𝑚3 × 𝑣𝑜𝑙𝑢𝑚𝑒𝑐𝑚3 , 
where volume is the volume of the submandibular gland, and a gland-volume of approx. 7-8 cm3 is 
the norm. Therefore, the amount of ASCs given to each participant will be approx. 2.8 ×
 106 𝐴𝑆𝐶
𝑐𝑚3  ×  8𝑐𝑚3 ×  2 = 4.48 106 𝐴𝑆𝐶𝑠 in total. If the expanded number of ASCs does not fulfil 
the above criteria, it is accepted to diminish the number to a minimum of 50% of the calculated 
dose.  
 

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Justification for patient population 
In order to standardize the participant population, we choose to only include participants treated for 
a human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinoma, and exclude 
patients with severe salivary gland hypofunction and already manifest or near-manifest xerostomia, 
as this population most likely will not benefit from this treatment. This will be assessed by a 
preliminary questionnaire and a subsequent saliva flow rate measurement before inclusion.  
 
Eligibility criteria 
Evaluation of eligibility criterias and consent forms are collected by the Primary Investigator. 
 
Inclusion criteria: 
 Previous radiotherapy for a T1-T2 and N0, N1 or N2a, HPV-positive oropharyngeal 
squamous cell carcinoma with bilateral irradiation of the neck 
 2 years follow-up without recurrence 
 Clinically reduced salivation and hyposalivation, evaluated by a screening 
o Unstimulated whole saliva flow rate in the range of 0.05-0.20 ml/min 
 Informed consent 
 Grade 1-3 xerostomia as evaluated by the “Udvalg for Kliniske Undersøgelser” (UKU) side 
effect rating scale[26]. 
 
Exclusion criteria: 
 Any cancer in the previous 2 years 
 Ongoing xerogenic medications 
 Any other diseases of the salivary glands, e.g. Sjögrens syndrome, sialolithiasis, etc. 
 Pregnancy or planned pregnancy within the next 2 years 
 Breastfeeding 
 Any other disease/condition judged by the investigator to be grounds for exclusion 
 Treatment with anticoagulant that cannot be stopped during the intervention period 
 Failure of expanding up to 50% of the calculated dose of ASC 
 Withdrawal of informed consent 
 

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Criteria for withdrawal of subjects under study 
 Pregnancy 
 Infection of the transplanted site 
 Allergy to local anesthetic 
 Withdrawal of consent from participants 
 In case of withdrawal or dropouts before assessment of efficacy patients will be replaced to 
ensure that a total of 30 patients complete the study. 
 
Surgical method for mini-liposuction  
The procedure is done in an outpatient setting, and performed under sterile conditions according to 
local guidelines. 6 ml of local anesthesia (lidocaine 1 %) is injected subcutaneously at two injection 
sites of the lateral abdomen. Two 5 mm incisions are then made and Klein's solution is injected with 
a blunt infiltrator through the incisions. The fatty tissue is harvested from the abdomen with a 3 mm 
blunt cannula 23 cm in length, coupled to the MonoJect 60 ml syringe with a blunt and Toomey tip 
(Teico Healthcare). The harvested lipoaspirate is sedimented in the syringe. Any oil layer at the top 
or aqueous layer at the bottom is removed. By this procedure the middle layer of fat tissue is 
obtained and will be transported in a sterile plastic bag for the isolation of ASCs.  
 
Surgical method for submandibular gland biopsy 
This procedure takes place in an outpatient settings. A biopsy will be taken 14 days before injecting 
the ASC and four months after injection. The biopsy procedure is carried out under sterile 
conditions; in local anesthesia with lidocaine with epinephrine 0.5% an ultra-sound-guided, core 
biopsy of one of the glands is taken. Participants are randomized for biopsy of either the right or left 
gland. Each biopsy will be fixed in 4% formalin and sent for histology.  
 
Surgical method for injection of ASC or placebo in submandibular gland 
The procedure will be performed without local anesthesia in an outpatient setting. The surgeon will 
receive two syringes of the sterile ASC-suspension or placebo of each 1 ml (total of 2 ml). The 
suspensions are injected into each submandibular gland guided by ultrasound. The ASC-suspension 
will be deposited in two areas (superficial and deep lobe) with equal volume per site (0.5ml) to 
ensure equal distribution of the suspension. The subject will afterwards receive a small band aid, 

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which can be removed the same day. The participant will be administered over the counter pain 
relief. 
 
Assessment of xerostomia and subjective treatment outcome 
The subjective effect of the treatment is assessed by a 100 mm visual analogue scale of xerostomia 
filled out by the patient [27] and a physician-rated questionnaire [28], both carried out at baseline as 
well as one and four month after treatment (Figure 1).  
 
Assessment of salivary flow rate and objective treatment outcome 
Changes in the secretion rate of the unstimulated whole saliva in the oral cavity is probably the 
most important parameter for the biological development of xerostomia and accompanying 
pathological oral conditions. Whole saliva, e.g., the secretions from the major and minor salivary 
glands are mixed in the oral cavity. A precise determination of this value is crucial for the 
assessment of efficacy in this trial. For determination of the saliva flow rate, whole saliva will be 
collected between 2 p.m. and 3 p.m. for all collections to take the salivary diurnal variation into 
account. Participants will refrain from eating, drinking, smoking and oral hygiene for 2 hrs. prior to 
collection. After being seated upright in a chair, subjects relax for 5 min and are then instructed to 
make as few movements as possible, including swallowing, during the collection. 
At baseline, one and four months after treatment, unstimulated whole saliva will be collected using 
the spitting method [29] where participants spit their saliva into a collection container over a period 
of 15 minutes. The salivary flow rate (SFR) (ml/min) is determined as the increase in weight of the 
container divided by the collection time in minutes. After the collection of unstimulated saliva, the 
subjects are instructed to chew on 1 g of sterile paraffin wax. Participants will chew for 60 s, and 
clear the oral cavity for saliva. Subsequently, as the glands are now in a stimulated state, 
participants will continue chewing the paraffin wax and expectorate into a saliva collector for the 
duration of five minutes. Subsequently, testing of the submandibular glands will be performed. 
To assess ASC efficacy on the submandibular glands, saliva is also collected directly from the floor 
of the mouth in an unstimulated and stimulated state. The flow rate of the submandibular glands 
will be assessed by the swab method with cotton rolls placed buccally in each maxillary molar 
region to block the orifices of the parotid ducts and cotton rolls under the tongue in the floor of the 
mouth to collect submandibular/sublingual saliva. Immediately prior to the start of collection 
participants will be asked to swallow. Unstimulated saliva testing will be performed with neutral 

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cotton rolls(Salivette ®, Sarstedt, Nümbrecht, Germany) and stimulated saliva testing with cotton 
rolls with 20 mg citric acid (Salivette with citric acid ®, Sarstedt, Nümbrecht, Germany). For both 
measurements, collections takes place during a period of 3 min. Saliva flow rates are determined by 
weight (1 g equals 1 ml of saliva) with cotton rolls weighed before collection and reweighed after. 
The flow rates are calculated as the increase in weight during collection and expressed as millilitres 
per minute. Saliva from the cotton rolls will be extracted by centrifugation (1500 g) and analyzed 
for its composition. From each of these collections, saliva will be aliquoted and stored at -80°C. 
 
Chemical analysis of saliva 
Whole saliva contains a large number of bacteria and epithelial cells, as well as gingival crevicular 
fluid. Therefore, whole saliva is normally not suited for the analysis of sensitive chemical 
parameters. For this purpose, non-contaminated selectively collected saliva from individual glands: 
i.e. parotid and submandibular/sublingual saliva is more suitable. The following analyzes will be 
performed on the collected saliva: pH and bicarbonate by ionic balance estimation [30], sodium, 
potassium, calcium, phosphate, chloride and fluoride [16], total protein, selected proteins [31] and 
amylase [32]. Chemical analysis will provide an estimate of dental and mucosal protective capacity 
of the saliva before and after treatment. 
 
MRI analysis of the submandibular glands 
A 3.0-Tesla 4-channel head coil and a dual-channel neck coil are employed to obtain hsMRI images 
of subjects (Siemens Magnetom Verio; Erlangen, Germany).  
Volumetric and tissue-specific analysis will be performed based on axial and coronal sequences 
with 72 slices and 4-mm slice thickness including diffusion weighted and dynamic contrast-induced 
sequences. The contrast agent used is Gadolinium (“Gadovist”). 
Statistical analyses 
Data will be analyzed with SPSS v. 24 (IBM) or R statistics. We will use Excel or Access databases 
for collecting and entering data. All data will be double-entered. End of trial is defined as: last 
patient’s last visit (LPLV). 
The results on salivary flow rate will be calculated as a percentage change in salivary flow rate 
(from baseline) in the group of participants given ASCs compared to the change-score in the control 
group.  
Histologically, the composition of glandular tissue in the submandibular gland will be calculated 

13 
 
from before (baseline) and four months after the intervention (Figure 1). The differences between 
the intervention and the placebo group will be calculated by a non-paired t-test, alternatively a non-
parametric test, if the conditions for parametric tests are not present. Differences are considered 
statistically significant if the two-sided p-value is less than 0.05. 
 
Sample size calculation  
The following sample size determination is based on a non-paired t-test. The minimal change in 
salivary flow rate that we want to be able to determine is 50%. 
 
 
Standard deviation (SD) of a salivary flow rate measurement can, using the spitting method in 
healthy participants and under completely standardized conditions, be minimized to about 20%. The 
SD can vary greatly and we have, therefore, in this test used a value from the published literature 
[33] for persons with reduced unstimulated whole salivary flow rate (from 0.00 to 0.20 ml/min) 
and/or hyposalivation (< 0.10 mL/min) indicating a relative SD of 58% of these groups. However, 
since we are interested in a comparison of change scores (change from baseline to after treatment) 
between the intervention and placebo group we apply the rule of thumb that the SD of the change 
score equals SD/20.5, which equals an SD of approximately 41%. The power is set at (1-β): 90%. 
Significance level (α) is set at 0.05. This is a trial with a continuous response variable from 
independent control and experimental research unit with one control per experimental research unit. 
If the true difference in the change score between experimental and control means is 50%, we will 
need to study 15 participants for the experimental intervention and 15 patients as control population 
in order to to reject the null hypothesis that the population means of the experimental and control 
groups are equal with with a study power of 90%. The Type I error probability associated with this 
test of this null hypothesis is 0.05. 
All investigators will have access to the final trial dataset. 
 
Discussion 
This first-in-man trial aims to establish the safety, efficacy and feasibility of autologous, adipose-
derived stem cell therapy for patients with radiation-induced xerostomia. In addition, it provides an 
opportunity to advance trial methodology for the assessment of the putative role of stem cell 
therapy and provide suggestions for the design of subsequent trials to verify potential efficacy. To 
establish safety profile of the intervention, selection and timing of appropriate outcomes have been 
guided by similar intervention studies [34], and our trial design facilitates conditions for assessment 

14 
 
of short and long-term changes and complications. 
We have strived to standardize saliva testing by performing tests at the same time of the day, and 
instructed and supervised by the same person (CGL). All participants are fasting two hours before 
the testing. This period is deemed useful to exclude any stimulants (e.g. lunch and drinking liquid) 
before testing, but might especially for participants with hyposalivation (e.g. participants with 
unstimulated whole saliva flow rate ≤0.10 ml/min) induce almost no secretion. Further, we perform 
in total four saliva measurements per visit (two tests for whole saliva (unstimulated/stimulated), and 
two tests for the submandibular glands (unstimulated/stimulated)) totaling to an effective period of 
26 minutes of saliva testing. This is possibly too extensive for some participants meaning that the 
glands will when testing the submandibular glands not be capable of further secretion. Testing of 
the submandibular glands is performed following the stimulated testing of all glands (whole saliva). 
It might be an advantage to test the submandibular glands in a post-stimulated state, but it should be 
noted that the glands are stimulated. Testing of the submandibular glands is performed by 
placement of cotton rolls at the orifices under the tongue, and consequently the rolls will also 
collect saliva from the sublingual glands. 
We have adjusted numbers of cell according to gland size based on experience from preclinical 
studies, but whether this is transferable to humans, and the dose is adequate remains unknown.  
Following the liposuction 12-14 days prior to treatment, a core-needle biopsy of one of the 
submandibular glands (participants are randomized for biopsy of either the right or left gland) is 
performed. It cannot be excluded out that this procedure might inflict salivation and influence the 
effect of the ASCs. It is well-documented that ASCs secret bioactive molecules that provide a 
regenerative microenvironment for a variety of injured tissues to limit damage and facilitate a 
regenerative response[35]. In this context, it is unknown whether injection to the submandibular 
glands regenerates only damaged tissue from the biopsy or also generates new or improved tissue. 
In connection with the injection of the MSCs into the salivary glands, there is a minimal risk of 
adverse events, such as pain, infection and bleeding. The risk of these adverse events is less than 
0.5% using similar procedures. A theoretical risk of a possible carcinogenic effect in the treatment 
of MSC has been discussed due to their production of growth factors such as epidermal growth 
factor (EGF). These considerations are particularly relevant, since MSCs are used in participants 
previously diagnosed with cancer. However in model systems of cancer, the effect of MSCs on 

15 
 
cancer growth is controversial as they have both been shown to be inhibitory and stimulatory 
[36,37]. Vascular endothelial growth factor (VEGF) is a known facilitator of angiogenesis, and 
might be a mediator of the potential effect of ASCs[38]. 
Based on numerous trials including studies with local injection of MSCs with a total 
of more than 1,000 participants with MSCs in and no increase in the incidence of cancer is detected 
[39,40]. Data on ex vivo expanded MSCs have not, despite extensive research, indicated malignant 
transformation [41], including a study that demonstrated that the injection of a very high dose of 
ASC (240 x 106 MSC/kg) revealed no signs of cancer, organ toxicity or change in body-weight after 
12 months in mice [42].  
It is estimated that the risks of adverse events in the experiment is outweighed by the benefits of 
participation. If the treatment has a clinically significant effect and is safe, patients suffering from 
radiation-induced xerostomia could be offered a simple, minimally invasive procedure with few 
adverse events and risks, which is in contrast to the current sub-optimal treatments.  
 
Trial status  
This is an investigator-initiated phase 1/2 trial funded by the Candy’s Foundation. The ethics committees have given 
full approval. Date of first enrollment was 22th May 2015. The trial is in the recruitment phase.  
Abbreviations 
Mesenchymal stem cell (MSC)  
Intensity-modulated radiation therapy (IMRT) 
adipose-derived MSC-therapy (ASC) 
magnetic resonance imaging (MRI) 
epidermal growth factor (EGF) 
Vascular endothelial growth factor (VEGF) 
Standard deviation (SD) 
milliliter (mL) 
Kilogram (kg) 
last patient’s last visit (LPLV). 
 
 
Declarations 
Ethical Approval and Consent to participate 
Ethics approval (The Danish National Board): 1406653  
We have obtained informed consent from all participants in the study. 

16 
 
 
Consent for publication 
Not applicable 
 
Availability of supporting data 
Not applicable 
 
Competing interests 
No competing interests 
 
Funding 
CGL is funded by Candy’s Foundation (non-profit; no reference number) 
 
Authors' contributions 
CGL, DHJ, DBJ, AB, AFN, RO, LS, PG and CvB conceived of the study, and participated in its design and 
coordination. All authors helped to draft the manuscript. CGL is principal investigator and carries out all interventions. 
All authors read and approved the final manuscript. All authors provided conceptual input and contributed in signicant 
ways to the article.  
 
Acknowledgements 
None  
 
 
Sponsor  
Christian von Buchwald  
Department of Otorhinolaryngology, Head and Neck Surgery and Audiology, Rigshospitalet, University of 
Copenhagen, Denmark 
 
 
 
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Additional files: 
Additional file 1.docx 
Spirit Checklist 
 
 
 
Figures 
Figure 1 Overview of the study process 
 
 
Figure 2 Schedule of enrolment, interventions, and assessments.