Encapsulated Mesenchymal Stem Cells for Endodontic Treatment of Permanent Teeth With Apical Lesion: a Controlled Clinical Trial.
Phase: NA
Conditions: Periapical Periodontitis
Sponsor: Universidad de los Andes, Chile

 
 
 
 
 
 
 
 
Encapsulated Mesenchymal Stem Cells for 
Dental Pulp Regeneration. (RanoKure) 
 
 
 
 
 
 
 
 
 
 
 
 
 
Clinical trial register: NCT03102879. 
Date: 01/05/2019. 
 
 
 
 
 
 
 
 
 
 
 

PROTOCOL STUDY 
 
Manufacturing and quality control of clinical grade UC-MSC  
 
Umbilical cords were obtained from full-term human placentas delivered by cesarean 
section after informed consent, from healthy donors, and processed within 4 hours 
after birth. To elaborate the end product, UC-MSCs were cultured from the Master 
Cell Bank (MCB) of Cells for Cells S.A. and cryopreserved in aliquots of 1x106 cells 
in FBS + 10% DMSO.  For quality control of the MCB and end cellular product, UC-
MSCs were evaluated for identity and purity for the following surface markers: 
CD105, CD90, CD73, CD34, CD45, CD19 and HLA-DR by Flow Cytometry in a 
FACS Canto II Flow Cytometer (BD Biosciences) and analyzed with FlowJo analysis 
software. Furthermore, sterility of the product was evaluated by Hemoculture, Fast 
Gram Test, Endotoxin (Endosafe PTS, Charles River, Wilmington, MA, USA) and 
Mycoplasma (MycoAlert, detection kit, Lonza, Basel, Switzerland). Finally, cell count 
and viability were determined by Trypan Blue exclusion assay. In addition, MCB was 
evaluated for tri-differentiation potency and tumorigenicity. 
 
 
Platelet-Poor Plasma (PPP) storage and UC-MSCs encapsulation 
PPP fractions were obtained from AB Rh-positive universal irradiated plasma from 
healthy donors collected from the Blood Bank Unit of the Clínica Universidad de los 
Andes, Chile. All the donors were screened for mandatory reportable diseases and 
stored at –80°C.  At 48 hours before treatment, cells were thawed and seeded in a 
T150 flask with aMEM supplemented with 5% Plasma AB+.  MSCs were then 
harvested using TrypLE Express 1X (Walthan, MA, USA) and washed twice with 
PBS 1X and resuspended in 175uL de saline solution. The resuspended cell was 
then mixed with 760uL of freshly thawed PPP, 15uL of Tranexamic acid and 50uL of 
a 2% solution CaCl2 in a 1,5ml microfuge tube. All the components were mixed by 
pipetting and the suspension was incubated at 37°C for 5 minutes. After checking 
the gelification, the end-product was stored and transported at 4°C for 
transplantation.  
 
 
Clinical Procedure 
The operators were two endodontic specialists who had been previously calibrated 
with a standard operating procedure. The first session was the same for every 
patient. Tooth sensitivity (cold, heat and electric) was assessed. Tooth vitality was 
tested using a Laser Doppler flowmetry (LDF) (Moor VMS-LDF; Moor Instruments 
Limited, Axminster, UK). Then intervention was started by the administration of local 
anesthesia (2% lidocaine hydrochloride with epinephrine 1:100,000; Septodont, 
Saint-Maur-des-Fosses, France). The access cavity was prepared using sterile high-

speed round burs, size 018 (Dentsply Sirona, Ballaigues, Switzerland). In all cases, 
rubber dam isolation was maintained (Hygienic; Coltene/Whaledent AG, Altstätten, 
Switzerland). A glide file was established with a size 10 hand K-file (Dentsply, 
Ballaigues, Switzerland). The working length to the apical constriction was confirmed 
using an electronic apex locator (Root ZX; J Morita, Tokyo, Japan) and verified 
radiographically. All root canals were enlarged using Reciproc® (VDW, GmbH, 
Munich, Germany) selected according to manufacturer’s recommendation. All teeth 
were irrigated with 20 ml 2.5% sodium hypochlorite and Endoactivator system 
(Dentsply Tulsa Dental Specialties, Tulsa, OK) was used for final sonic activation. 
Finally, calcium hydroxide (Hertz Pharmaceutical, Santiago, Chile) was used as an 
intracanal medication and closed with glass ionomer (Vitrebond; 3M ESPE, St Paul, 
MN, USA). After three weeks in the second session, the medication was removed 
by irrigation with 20 ml 17% EDTA and canal dried with sterile paper points. 
Subsequently, each tooth was randomized and assigned to each group following a 
specific protocol. ENDO Group: canals were filled using gutta-percha cones 
(Reciproc® VDW, GmbH, Munich, Germany) and Topseal® sealer (Dentsply Sirona, 
Switzerland) through a continuous wave condensation technique with an ElementsTM 
Obturation Unit (Sybronendo, Orange County, CA, USA). REP Group: apical 
bleeding was induced by using a size 8 stainless steel hand K-file (Dentsply Sirona) 
3 mm beyond the apex. Once the apical portion of the root canal was filled with 
blood, PPP-UC-MSCs was implanted and absorbable gelatin sponge hemostats 
(Gelita-Spon® GmbH, Eberbach, Germany) was used to contain the Biodentine™ 
(Septodont, France) filling material. Final restorations in both groups were done with 
resin (Filtek™ Z350 XT Universal Restorative; 3M ESPE, St Paul, MN, USA). 
Patients were blinded to treatment group. All clinical procedures were performed 
under an operating microscope (OPMI® Pico; Carl Zeiss AG, Göttingen, Germany). 
 
Statistical Analysis  
The categorical variables were described by absolute frequencies and percentages 
by group of treatment and were evaluated by the Chi-square test of independence 
or Fisher’s Exact test at each time point. The quantitative variables were described 
by the median and interquartile range and they were compared between treatment 
groups using the Mann Whitney test at each time point. Lesion size differences 6 
months versus basal time point and 12 months versus 6 months were compared 
between treatment group through Mann Whitney test. The evolution over time in 
each treatment arm of perfusion, dimensions of lesion, and cortical compromise was 
evaluated through a generalized estimating equation (GEE) model assuming 
repeated measures over time. It was reported 95% confidence interval and p-values. 
For variables with zeros as a result, as is the pain to percussion and sensitivity test, 
differences between basal point and 12 months was used McNemar test and p-value 
was reported. It was considered significant a p-value<0.05. Effect size by 

bootstrapping estimation was reported if it was >0.4. The analysis was performed 
using the software Stata® (StataCorp 2015-Stata Statistical software 14.2, College 
Station. TX: StataCorp LP).