This trial was conducted in full accordance with the 2010 CONSORT guidelines of the World Medical Association Declaration of Helsinki. The study protocol was approved by the Ethic Committee of the the Affiliated Huai’an Hospital of Xuzhou Medical University (institutional approval number: HEYLL2020325). It was registered at Chinese Clinical Trial Registry (Registration umber: ChiCTR2000033420, http://www.chictr.org.cn/, Date of first registration: 31/5/2020). A written informed consent was provided to each subject prior to the treatments.
Patients were consecutively recruited referred to Department of Rheumatology, Stomatology and Ophthalmology, Affiliated Huai’an Hospital of Xuzhou Medical University. Eligible patients were diagnosed as having pSS according to the American College of Rheumatology (ACR) Diagnostic Criteria for Sjögren’s Syndrome. At least two syndromes with following three objective features will meet the criteria: (1)patients are newly diagnosed with pSS and have not received previous treatments; (2) Positive serum anti-SSA (Ro) and/or anti-SSB (La); (3) Labial salivary gland biopsy exhibiting focal lymphocytic infiltration with a focus score ≥ 1 focus/4 mm2; (4) Keratoconjunctivitis sicca with ocular staining score ≥ 3.
The exclusion criteria for all patients were: (1) not currently using daily eye drops for glaucoma, and has not had corneal surgery or cosmetic eyelid surgery in the last 5 years; (2) any of the following conditions: history of head and neck radiation treatment, hepatitis C infection, acquired immunodeficiency syndrome, sarcoidosis, amyloidosis, graft versus host disease, IgG4-related disease; (3) other systemic conditions that could affect the progression of pSS, such as Systemic lupus erythematosus and Rheumatoid arthritis; (4) a history disease of chronic obstructive sialadenitis disease; (5) status of pregnancy or lactation.
Patients were interviewed to provide of informations including their socio-demographic characteristics, life-style factors, compliance to drugs and self-care knowledge at study entry. Clinical assessments of disease activity were also performed at the first screening.
Randomization and masking
Patients were assigned to two groups via a computer-generated randomization schedule. The ADSCs Group was injected with ADSCs while Placebo Group was received 0.9% saline solution administration. Concealed allocated codes were kept in signed and sealed envelopes. An external investigator performed the randomization. Investigators, patients and investigators were masked to group assignment. Blinding was revealed after the treatments were performed and the data were analysed.
Sample size analysis
Sample size analysis using G Power version184.108.40.206 was determined with Walters’s method by considering two groups of subjects, an effect size of 0.30 and a standard deviation of 1.7 by a previous randomized controlled clinical study. With an assumption of normal sample distribution, we set power of 0.8 and an error of α = 0.05. Adding 10% with the compensation of drop-out during the trial period, we set an increase of 20%. The final sample including 26 subjects in each group were determined, so that a power of 0.8 was obtained.
The preparation of ADSCs
The drug in this trial was a solution of ADSCs. Adipose tissues were harvested with liposuction of abdomen from a healthy donor. They are washed in phosphate-buffered saline (PBS) and digested of fat aspirates with 0.075% collagenase. These stem cells were then packed in dimethyl sulfoxide and human platelet lysate and 1% antibiotics at a culture condition of 37 °C and 5% CO2. The surface markers of CD73, CD90 and CD105 were identified by flow cytometry analysis, as well as negative for expressing CD14, CD11b, CD45, CD19, CD79 and HLA-DR. Tumorigenicity study, sterility test and negative test for Mycoplasma were performed before use.
The final therapeutic drug solution was composed of 5 million cells per milliliter of solution. After disinfecting the surface projection of bilateral parotid glands with Iodophor and draping in a sterile fashion, the injection site was anesthetized with 1% lidocaine. A dose of 1 ml ADSCs solution was diluted in 5 ml 0.9% saline solution. The final stem cell solution was injected into bilateral glands with 0.05 ml ADSCs and saline mixed-solution (5 × 104 cells) per kilogram of the patient’s weight by a 5 ml-syringe and 0.45 mm- needle under ultrasonic guidance. The dosage and interval of ADSCs was injected according to our previous researches. Patients in Placebo Group were injected with 0.9% saline solution as forementioned proportion. Backflow should be performed against the blood to the syringe before injection. Patients were allowed to leave after resting for 15 min. The second injection was performed in bilateral glands in the second and fourth week under similar conditions. All subjects in this trial were reinforced with hygiene instructions and without any medical treatment. In this trial, the patients were clinical evaluated at four follow-up time points: before the intervention, 1-month, 3-month and 6-month after the third injection. During the follow-up, patients could withdraw in any time. Patients without a compliance rate more than two times were required to withdraw after approval from the principal investigator.
Patients were asked to attend at 8:00–10:00 am in the same room with constant temperature and humidity to minimize diurnal variation. Eating/chewing, drinking and brushing teeth were refrained 90 min prior to each appointment. After resting for 10 min, unstimulated whole saliva (UWS) was collected by a sterile and pre-weighed plastic container every 30 s for a 5 min period. Stimulated whole saliva (SWS) was collected by asking patients to chew a piece of sterilized silicone rubber tubing (4*8 mm) and to spit all the saliva into another container as the same frequency and period of UWS. UWS and SWS were determined by the result of reweighting each container after collection subtracting the weight of empty container14.
Schirmer I test
We introduced the Schirmer I test to measure lacrimal flow (LF) without previous topical anesthetic to measure the tear secretion. A Whatman special filter paper strip of 20 mm*5 mm (Cytiva®, Shanghai, China) were placed in the lateral 1/3 of the lower bilateral eyelid. The length of the moistened portion of the strip was measured at the fifth minute15.
ESSDAI and ESSPRI16
Disease activity was assessed using and European League Against Rheumatism Sjögren’s Syndrome Disease Activity Index (ESSDAI). These questionnaires were filled out by the patients at baseline and the follow-ups. The ESSDAI includes 12 domains, including cutaneous, respiratory, kidney, articular, muscular, peripheral nervous system, central nervous system, hematological, glandular, constitutional, lymphadenopathic, and immunological, with a total score ranging from 0–3 points.
European Alliance of Associations for Rheumatology SS Patient Reported Index (ESSPRI) is a patient-administered questionnaire to assess disease symptoms on a 10-point scale for pain, fatigue and dryness. The patients were required to show the maximal scale they had experienced during the last 3 days on the VAS ruler. The scores on the ruler were at least 10 cm with 10 visual analogue scales, indicating dryness, pain, and fatigue.
Patients’ venous blood samples were drawn at 6:00–8:00 am prior to administration of ADSCs injection and at each follow-up. Serum samples were transported at − 20 °C and kept at − 80 °C in a freezer until analysis. Immunological indexes, including IgA, IgG, IgM, complement 3 (C3), complement 4 (C4) and erythrocyte sedimentation rate (ESR), were analyzed using a flow cytometer (Beckman Coulter®, Fullerton, CA).
To evaluate safety and toxic effects, clinical laboratory values, vital sign, physical examination and 12-lead electrocardiography were recorded at baseline and follow-ups. A treatment-emergent adverse events (AE) was defined as any untoward symptom, occurred from the first dose of medication until the end of the study. Those were possibly, probably or definitely related to the study medication in the judgment of the investigator.
In this study, data were statistically analyzed using SPSS v20.0 software (IBM Corp, Armonk, NY, USA) and visualized using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). The demographic and health status information at baseline was summarized using Means (Standard Deviation, SD) for continuous variables and Frequencies (Percentages) for categorical variables. Data were first examined for normality by the Kolmogorov–Smirnov test and, the data that achieved normality were analyzed using parametric methods while the others used non-parametric methods. Differences in continuous variables between groups were tested by t-test or MannWhitney U test, while differences in categorical variables were tested by Chi-squared tests or Fisher’s exact tests. In our trial, Unpaired t-test was applied to test UWS, SWS, LF, ESSDAI and ESSPRI, immunological indexes to determine significant difference between baseline and follow-up. Paired t-test was used to analyze significant differences of intergroup at each data point. All reported p-values were from two-side tests and compared with a significance level of 0.05 (α = 0.05).