CASE REPORT/CLINICAL TECHNIQUES

Claudia Brizuela Cordero, PhD,*

Allogeneic Cellular Therapy in a Gaston Meza Santander, DDS,*
Denisse Urrejola Gonza
lez,
Mature Tooth with Apical DDS,* Andrea Quezada, DDS,*
Carolina Inostroza Silva, PhD,*
Periodontitis and Accidental Camila Va
squez, DDS,*
Richard Jara, DDS,* Daniel Jara,
Root Perforation: A Case DSD,* and Maroun Khoury,
PhD†‡x
Report

ABSTRACT
SIGNIFICANCE
Introduction: Cell therapy in regenerative endodontics introduces an alternative option to
classic treatment strategies for complex endodontic cases. The aim of this case report was to A regenerative endodontic
describe cell-based therapy using allogeneic umbilical cord mesenchymal stem cells (UC- procedure using allogeneic
MSCs) encapsulated in a bioscaffold for a complex case of a mature permanent tooth with umbilical cord mesenchymal
apical periodontitis and accidental root perforation. Methods: A healthy 19-year-old man stem cells encapsulated in
undergoing orthodontic treatment was referred for endodontic treatment in tooth #7; he was platelet-poor plasma is an
diagnosed with apical periodontitis during a previously initiated treatment associated with innovative and successful
accidental perforation of the radicular cervical third. The root perforation was sealed with glass alternative treatment for a
ionomer and composite resin, and the root canal was instrumented, disinfected, and dressed complex clinical case such as a
with calcium hydroxide. After 3 weeks, allogeneic UC-MSCs were encapsulated in platelet- mature permanent tooth with
poor plasma and then implanted into the root canal, and Biodentine (Septodont, Saint-Maur- apical periodontitis and root
des-Fosses, France) was placed below the cementoenamel junction. Finally, the tooth was perforation.
restored with composite resin. Results: Follow-up examinations were performed 6 months
and 1 year later. The examinations included periapical radiography, cone-beam computed
tomographic imaging, and sensitivity and vitality tests. Radiographic and cone-beam
computed tomographic images indicated remission of the apical lesion. Clinical evaluations
revealed normal responses to percussion and palpation tests; the tooth was responsive to the
electric pulp test, and the vitality test indicated low blood perfusion units. Conclusions: This
case report reveals the potential use of allogeneic cellular therapy using encapsulated UC-
MSCS in a platelet-poor plasma scaffold for a complex case of a permanent tooth with apical
From the *Centro de Investigacio
n en
periodontitis and root perforation. (J Endod 2020;-:1–8.)
n Oral, Faculty of
Biología y Regeneracio
Dentistry, Universidad de los Andes,
KEY WORDS Santiago, Chile; †Laboratory of Nano-
Regenerative Medicine, Faculty of
Case report; cell engineering; mesenchymal stem cells; periapical periodontitis; regenerative Medicine, Universidad de Los Andes,
endodontics, tissue engineering Santiago, Chile; ‡Consorcio Regenero,
Chilean Consortium for Regenerative
Medicine, Santiago, Chile; and xCells for
Cells, Santiago, Chile
For a mature permanent tooth with infected necrotic pulp and a periapical lesion, the first treatment option
Address requests for reprints to
is nonsurgical root canal therapy, for which the outcome is considered predictable and reliable1,2.
Dr Claudia Brizuela Cordero, Centro de
However, an endodontically treated tooth loses its blood supply and innervation and hence has no
n en Biología y Regeneracio
Investigacio
n
sensitivity or immune system to alert and protect itself3. Therefore, the root canal may become reinfected Oral, Faculty of Dentistry, Universidad de
and/or susceptible to fracture, leading to either loss of the tooth or expensive and complex alternative

los Andes, Mons Alvaro del Portillo
treatments such as root canal retreatment, surgery, or implants. 12.455, Las Condes, Santiago, Chile.
E-mail address: clau@cibrizuela.com
Regenerative endodontic procedures (REPs) are an emerging branch of regenerative medicine that 0099-2399/$ - see front matter
aim to introduce new alternative options to classic root canal treatment4. REPs are defined as biologically
Copyright © 2020 American Association
based procedures designed to replace physiologically damaged tooth structures, restoring or of Endodontists.
regenerating pulpal tissue and allowing it to resume its sensory, immune, and dentin apposition roles1,5,6. https://doi.org/10.1016/
REPs apply the triad of tissue engineering, which comprises mesenchymal stem cells (MSCs), scaffold, j.joen.2020.04.007

JOE  Volume -, Number -, - 2020 Allogeneic Cellular Therapy 1

and growth factors to regenerate damaged perforation in the cervical root third (Fig. 1A) Second Appointment: Perforation
pulp tissue7,8. Cell therapy is an important part and a large temporary restoration on its palatal Treatment
of regenerative endodontics, and either surface. It was not sensitive to percussion or One week later, the periodontal dressing was
autologous or allogeneic MSCs may be used9. palpation testing. Pulp sensibility tests using removed, and the soft tissue migrated apically,
Although autologous MSCs represent a 1,1,1,2-tetrafluoroethane (Endo-Ice; Hygenic, leaving the perforation site totally exposed to
reliable source from the patient’s own tissues, Akron, OH), a hot gutta-percha bar (Dentsply the oral cavity (Fig. 2C). Local anesthesia was
allogeneic alternatives have more convenient Sirona, Konstanz, Germany), and vitalometer obtained with 2 mL 2% lidocaine hydrochloride
and practical features including cost efficiency (SybronEndo, Orange, CA) resulted in no with 1:100,000 epinephrine (Septodont).
and off-the-shelf immediate availability10. response. Periodontal probing affirmed normal Isolation of the operative field was performed
MSCs derived from human umbilical cord (UC- attachment with no probing depths .3 mm, with insertion of a gingival retraction cord (#000
MSCs) are obtained in a noninvasive way; they normal physiological mobility, and a soft tissue Ultrapack; Ultradent Products Inc, South
are stored in banking services worldwide and fenestration associated with the root Jordan, UT). Partial removal of the temporary
have odontogenic differentiation potential5. perforation area. The root canal presented an restorative material for the perforation was
Scaffolds from organic or synthetic origins are apical resorption, and an extensive apical performed. Acid etching with 37% phosphoric
used as support material for cell delivery but lesion was observed on radiographic acid for 30 seconds was performed, and a
can also promote cell proliferation depending examination (Fig. 1B) and cone-beam water rinse was followed by application of an
on their growth factor content. Some natural computed tomographic (CBCT) imaging, with adhesive system (Scotchbond Multi-purpose,
scaffolds are platelet derived or blood clots extension of 9.99 mm in the mesiodistal 3M ESPE). The adhesive was light cured, and
produced through the bleeding technique. direction, 16.53 mm in height, and 7.18 mm in the perforation was sealed externally with
Platelet-poor plasma (PPP) is the blood the buccopalatal direction (Fig. 1C). The initial composite resin (shade A2 body [Z350 XT, 3M
fraction with a reduced count of platelets. periapical index (PAI)14 score was 5, and the ESPE]) (Fig. 2D and Supplemental Fig. S1
Finally, growth factors stimulate cellular CBCT PAI15 was 5 1 D. The diagnosis for [available online at www.jendodon.com]).
proliferation and differentiation; they are placed tooth #7 was asymptomatic apical
in the dentin tubules and scaffold11. periodontitis on a previously initiated treatment
Third Appointment: Canal
REPs are more frequently used for associated with a perforation in the radicular
Disinfection
immature permanent teeth; however, some cervical third.
Local anesthesia was obtained with 2 mL 2%
reports have shown that these procedures
lidocaine hydrochloride with 1:100,000
have also been successful for the treatment of
epinephrine (Septodont). After rubber dam
mature teeth with apical periodontitis, First Appointment
isolation, temporary restorative material was
accomplishing resolution of the pathology and After an explanation of the different treatment
removed from the palatal surface. Serous
regression of symptoms and clinical signs12. options, the patient signed an informed
exudate was observed inside the canal, and
We recently presented the first clinical safety consent form specific for an experimental cell-
the canal was enlarged with a size #45 K-file
and efficacy phase I/II clinical trial for the based therapy approved by the Health Center
(Dentsply Sirona). The disinfection protocol
endodontic use of allogeneic UC-MSCs of Universidad de Los Andes Ethical Board.
was completed by irrigation with 2.5% sodium
encapsulated in a PPP matrix13. We The tooth was anesthetized using an infiltrative
hypochlorite (20 mL/canal for 5 minutes)
concluded that the innovative approach technique with 2 mL 2% lidocaine
followed by 17% EDTA (20 mL/canal for 5
presented a promising alternative for hydrochloride with 1:100,000 epinephrine
minutes) using a positive-pressure syringe
regenerative endodontic treatment of (Septodont, Saint-Maur-des-Fosses, France).
(Monoject 513; Kendall, Mansfield, MA). The
periapical pathology in mature teeth. However, The temporary restoration was removed, and a
canal was dried carefully with sterile paper
there are still no reports describing the use of size #40 hand K-file (Dentsply Sirona,
points and dressed with calcium hydroxide
our regenerative endodontic treatment Ballaigues, Switzerland) was placed into the
(Hertz Pharmaceutical, Santiago, Chile) mixed
approach in mature teeth with apical first two thirds of the root canal (Fig. 2A); the
with saline solution. It was placed in the root
periodontitis and an associated endodontic perforation was temporarily sealed externally
canal followed by a seal with a temporary
complication (eg, root perforation, breakage of with glass ionomer (Chemfill; 3M ESPE, St
restorative material (Chemfill, 3M ESPE).
instruments in the canal, etc). Paul, MN) with the aid of an operating
The aim of this case report was to microscope (OPMI PICO; Carl Zeiss AG,
describe a regenerative allogeneic cellular Go€ttingen, Germany). After the perforation was Expansion of Clinical Grade UC-
therapy using encapsulated UC-MSCs in a sealed, tooth #7 was isolated with a rubber MSC/PPP Storage and UC-MSC
PPP-based bioscaffold for a complex case of a dam (Hygenic). The working length was Encapsulation
mature permanent tooth with apical determined to be 24 mm using an apical UC-MSCs from batches that successfully
periodontitis and accidental root perforation. locator (Root ZX II; J Morita, Irvine, CA). The passed all quality control testing were
root canal was instrumented with a size #40 K- processed and encapsulated in PPP fractions
file (Dentsply Sirona) and irrigated with 20 mL as previously reported13 at the cell therapy
CASE REPORT 2.5% sodium hypochlorite and dried with facilities operated by Cells for Cells SA
A healthy 19-year-old male patient undergoing sterile paper points followed by a seal glass (Santiago, Chile), in an ISO 7 clean room. The
orthodontic treatment with a history of an ionomer temporary restorative material applied good manufacturing practice and
acute apical abscess in the permanent upper (Vitremer; 3M ESPE, St Paul, MN). Finally, a good tissue practice standards are compliant
right lateral incisor (tooth #7) was referred to noneugenol periodontal dressing (COE PAK; with 21 Code of Federal Regulations 1271
the Universidad de los Andes Dental Clinic, GC America Inc, Alsip, IL) was applied to guidelines (registration and listing, donor
Santiago, Chile, after a failed attempt at access protect the perforation site (Fig. 2B and eligibility, and good tissue practices) and
cavity preparation performed by a general Supplemental Fig. S1 [available online at www. additional requirements for manufacturers of
dentist. The tooth had an accidental jendodon.com]). human cells, tissues, and cell- and

2 Cordero et al. JOE  Volume -, Number -, - 2020

FIGURE 1 – (A ) Soft tissue fenestration associated with the root perforation area because of an accidental perforation in the cervical root third in tooth #7. (B ) The baseline periapical
radiograph showing an apical resorption and an extensive periapical lesion with a PAI of 5. (C ) The baseline sagittal image from CBCT imaging observing the magnitude of the periapical
bone lesion (PAI CBCT 5 5 1 D).

tissue-based products. The PPP fractions incubated at 37 C for 5 minutes. After #10 K-file (Dentsply Sirona). Once the apical
were obtained from AB Rh–positive universal checking the jellification, the end product was portion of the root canal was filled with blood,
irradiated plasma from healthy donors stored and transported at 4 C until its PPP UC-MSCs were introduced into the root
collected from the Blood Bank Unit of the implantation. canal using a sterile tweezer (Fig. 3A) followed
Clínica Universidad de los Andes. All the by the application of gentle compaction
donors were screened for mandatory Fourth Appointment: Regenerative movements using hand pluggers (Fig. 3B). A
reportable diseases, and samples were stored Procedure with PPP-UC-MSC membrane of collagen (CollaPlug; Zimmer,
at 280 C. The UC-MSCs were harvested Implantation Warsaw, IN) was placed on the cervical third of
using TrypLE Express 1! (Thermo Fisher The patient returned asymptomatic after 2 the tooth (Fig. 3C), and Biodentine (Septodont)
Scientific, Waltham, MA), washed twice with weeks. Local anesthesia was obtained with 2 was prepared and placed below the
phosphate-buffered saline 1!, and mL 3% mepivacaine. After rubber dam cementoenamel junction, protecting the PPP
resuspended in 175 mL saline solution. The isolation, intracanal medication was removed UC-MSCs and reinforcing the root perforation
resuspended cells were then mixed with 760 by irrigating with 17% EDTA (20 mL/canal for 3 site internally (Fig. 3D). The cavity was sealed
mL freshly thawed PPP, 15 mL tranexamic acid, minutes). The root canal was then carefully with glass ionomer (Vitrebond, 3M ESPE) and
and 50 mL 2% solution of CaCl2 in a 1.5-mL dried with sterile paper points. Controlled restored with a composite resin (Supplemental
microfuge tube. All the components were microbleeding in the canal was induced by Fig. S1 is available online at www.jendodon.
mixed by pipetting, and the suspension was careful laceration of the apical tissues with a com).

JOE  Volume -, Number -, - 2020 Allogeneic Cellular Therapy 3

FIGURE 2 – (A ) A size #40 hand K-file was placed into the first two thirds of the root canal, and the perforation was temporarily sealed externally with glass ionomer. (B ) After the
perforation was sealed, a noneugenol periodontal dressing was applied to protect the perforation site. (C ) One week later, the periodontal dressing was removed, and the soft tissue
migrated apically, leaving the perforation site totally exposed to the oral cavity. (D ) Perforation was sealed externally with composite resin.

Follow-up showed dentin bridge formation in the middle rate and can prolong tooth survival, the loss of
Follow-up examinations were at scheduled at third of the root canal. Vitality testing using the vital pulp and hard tooth structure,
1, 6, and 12 months after treatment. The REP laser Doppler flowmetry (Moor LAB/FloLAB; because of the nature of the therapy, may still
outcome was evaluated radiographically and Moor Instruments, Axminister, UK) showed accelerate tooth loss16. The use of tissue
clinically. The patient remained asymptomatic that the tooth presented a lower mean value of engineering to regenerate the pulp-dentin
during the 12 months of follow-up. Clinically, perfusion units with respect to the control complex overcomes the disadvantages of
no tenderness to percussion or palpation was tooth (#10). Despite this, it had a pulse nonsurgical root canal therapy because it
noted; the periodontal examination revealed characteristic, indicating blood perfusion in the recovers the sensory, immune, and dentinal
no pocket depths .3 mm and normal tooth. No adverse events were recorded apposition roles lost in the tooth17.
physiological mobility (Fig. 4A). At 12 months, during the development of this therapy. A preliminary clinical trial in young
the tooth was nonresponsive to the cold and patients using REPs with the bleeding
hot tests. The electric current test was technique demonstrated the potential use of
responsive. Radiographic examination
DISCUSSION this treatment as an option for mature teeth18.
showed a minimal apical lesion (PAI 2) This is the first case report to evaluate the use REPs using only the apical bleeding technique
(Fig. 4B). CBCT imaging showed a periapical of allogeneic encapsulated UC-MSCs in a PPP in mature permanent teeth have limitations
radiolucency of a diameter of 1.02 mm in the matrix for a complex case of a mature compared with those in immature teeth
mesiodistal direction, 2.38 mm in height, and permanent tooth with apical periodontitis and because of the low and variable number of
1.26 mm in the buccopalatal direction, with no root perforation. stem/progenitor cells and a narrower apical
cortical bone involvement (CBCT PAI 5 3) Nonsurgical root canal therapy aims to foramen for cell migration and because
(Fig. 4C and Supplemental Fig. S1 [available prepare, disinfect, and fill the root canal space. disinfection is more challenging because of the
online at www.jendodon.com]). In addition, it Although this treatment has a high success complex root canal anatomy19. A study

4 Cordero et al. JOE  Volume -, Number -, - 2020

FIGURE 3 – (A ) Once the apical portion of the root canal was filled with blood by controlled microbleeding induced by careful laceration of the apical tissues, PPP UC-MSCs were
introduced into the root canal using a sterile tweezer (B ) followed by the application of gentle compaction movements using hand pluggers. (C ) A membrane of collagen was placed on
the cervical third of the tooth, and (D ) Biodentine was prepared and placed below the cementoenamel junction, protecting the PPP UC-MSCs and reinforcing the root perforation site
internally.

published in 2015 by Chrepa et al20 showed of donors. Chrepa et al20 reported a high cells and leukocyte platelet-rich fibrin provides
that the evoked bleeding technique delivers variability among patients in the concentrations a new alternative therapy for pulpitis in mature
undifferentiated MSCs into the root canal of MSC markers in samples collected from permanent teeth6. In a clinical trial, Xuan et al27
systems of adult patients with mature teeth; blood clots of mature teeth. This suggests that demonstrated that implantation of autologous
nonetheless, the exact source of these cells factors such as age, sex, tooth type, and human deciduous pulp stem cells may
was not directly addressed. In adult patients, others modulate the relative abundance of regenerate the lost 3-dimensional pulp tissue,
the apical papilla does not exist, and the these cells. These uncontrollable variables lead restore pulp function, and promote root
potential stem cells made available for REPs to inconclusive or contrasting results. development in immature permanent teeth that
using this technique could come from bone Therapeutic transplantation of MSCs is had undergone trauma. However, the
marrow stem cells and periodontal ligament considered safe and has been experimentally translation of the autologous cellular therapy
stem cells (PLSCs)6,19. Bone marrow stem evaluated in clinical trials for cardiovascular, transplantation to daily endodontic clinical
cells have a lower capacity to regenerate a neurologic, and immunogenic conditions25. practice is hampered by the costs and limited
highly vascularized and innervated pulpal With advances in tissue engineering through cell sources. Given that there is a need for off-
volume21, and their ability to differentiate is scaffolds, growth factors, and MSCs, the-shelf immediate availability, there is
variable for each donor22. PLSCs have shown particular attention has been paid to pulp substantial interest in the application of
their ability to differentiate into osteogenic and regeneration mediated by stem cell allogeneic MSCs28, and UC-MSCs have
cementoblastic lineages23,24, but under natural transplantation26. gained significant attention. First, UC-MSCs
conditions, converting PLSCs into an In a previous report from our group, we are multipotent, with high capabilities for
odontoblast phenotype may be difficult. described a regenerative autologous cellular differentiation and proliferation. Second, they
The major critical limitation for REPs therapy using MSCs from inflamed pulp and are obtained in a noninvasive way and can be
using the bleeding technique in mature teeth, leukocyte platelet-rich fibrin. It was concluded used immediately in emergency treatment
as for the autologous therapies, is the variability that the use of autologous dental pulp stem because they can be cryopreserved and

JOE  Volume -, Number -, - 2020 Allogeneic Cellular Therapy 5

FIGURE 4 – (A ) After 12 months of follow-up, the periodontal examination revealed no pocket depths .3 mm and healthy gingiva. (B ) Radiographic examination showed a minimal
apical lesion (PAI 5 2). (C ) The CBCT image shows a periapical radiolucency with no cortical bone involvement (CBCT PAI 5 3) and dentin bridge formation in the middle third of the
root canal.

stored in banking services worldwide29. Chen health of periapical tissue in mature teeth with migration, and differentiation of dental pulp
et al10 reported that UC-MSCs can be induced apical lesions. stem cells31.
to form odontoblastlike cells in vitro and in vivo; Martínez et al30 compared the growth The defined amount of allogeneic UC-
induced UC-MSCs expressed dentin-related factor composition of platelet-rich plasma and MSCs (106 cells) from a single donor
proteins including dentin sialoprotein and PPP from the same donors and assayed the encapsulated in a PPP-based matrix used in
dentin matrix protein 1, and their gene effects of these formulations on the this report was complemented with a
expression levels were similar to those in native differentiation potential of periodontal ligament controlled microbleeding only in the apical
pulp tissue cells, indicating their potential stem cells to osteoblastic phenotype. The third, providing the adequate vasculature for
odontogenic differentiation. In a previous trial, authors concluded that platelet-rich plasma ensuring oxygen and nutrition to the construct.
our team demonstrated for the first time the and PPP contain a similar profile of growth Although the beneficial effects should be
safety of PPP UC-MSCs implants and factors and that both formulations exerted a attributed to the cellular product, a synergy
evaluated their effectiveness13. This novel similar stimulus on bone differentiation. PPP is effect probably occurs between microbleeding
strategy represents an innovative alternative a potential scaffold for pulp regeneration and PPP UC-MSC product13.
treatment based on biological principles that because of the growth factors present and its A possible limitation of this therapy is the
promotes dentin-pulp regeneration and the previously reported effect on the proliferation, need for a good manufacturing practice

6 Cordero et al. JOE  Volume -, Number -, - 2020

laboratory within close reach for the production of outcome shows that this clinical protocol is a Maroun Khoury is the chief scientific
the encapsulated UC-MSCs. There is need for a feasible alternative therapy for complex and officer of Cells for Cells and REGENERO.
focus on the development of a direct thaw-and- accidental clinical cases, such as teeth with apical
use approach that might help to overcome the periodontitis and root perforation.
need for a specialized laboratory in close
SUPPLEMENTARY MATERIAL
proximity. This limitation may also be
counterbalanced by the expanding use of
ACKNOWLEDGMENTS Supplementary material associated with this
allogeneic cellular sources in different applications Supported by Corporacio
n de Fomento de la article can be found in the online version at
and the recent market authorization of many
n, Santiago, Chile (project number
Produccio www.jendodon.com (https://doi.org/10.1016/
autologous cell therapies. The successful L2 14IDL2-30051). j.joen.2020.04.007).

REFERENCES
1. Saoud TM, Ricucci D, Lin LM, Gaengler P. Regeneration and repair in endodontics-a special issue
of the regenerative endodontics-a new era in clinical endodontics. Dent J (Basel) 2016;4:E3.

2. Jung C, Kim S, Sun T, et al. Pulp-dentin regeneration: current approaches and challenges. J
Tissue Eng 2019;10:2041731418819263.

3. Morotomi T, Washio A, Kitamura C. Current and future options for dental pulp therapy. Jpn Dent
Sci Rev 2019;55:5–11.
4. Bakhtiar H, Mazidi SA, Mohammadi Asl S, et al. The role of stem cell therapy in regeneration of
dentine-pulp complex: a systematic review. Prog Biomater 2018;7:249–68.

5. Diogenes A, Ruparel NB, Shiloah Y, Hargreaves KM. Regenerative endodontics. J Am Dent

Assoc 2016;147:372–80.

6. Meza G, Urrejola D, Saint Jean N, et al. Personalized cell therapy for pulpitis using autologous
dental pulp stem cells and leukocyte platelet-rich fibrin: a case report. J Endod 2019;45:144–9.
7. Pinto N, Harnish A, Cabrera C, et al. An innovative regenerative endodontic procedure using
leukocyte and platelet-rich fibrin associated with apical surgery: a case report. J Endod
2017;43:1828–34.
8. Kim SG, Malek M, Sigurdsson A, et al. Regenerative endodontics: a review. Int Endod J
2018;51:1367–88.

9. Al-Daccak R, Charron D. Allogenic benefit in stem cell therapy: cardiac repair and regeneration:
allogenic benefit in stem cell therapy. Tissue Antigens 2015;86:155–62.

10. Chen Y, Yu Y, Chen L, et al. Human umbilical cord mesenchymal stem cells: a new therapeutic
option for tooth regeneration. Stem Cells Int 2015;2015:549432.
11. Diogenes A, Henry MA, Teixeira FB, Hargreaves KM. An update on clinical regenerative
endodontics. Endod Topics 2013;28:2–23.

12. Saoud TM, Sigurdsson A, Rosenberg PA, et al. Treatment of a large cystlike inflammatory
periapical lesion associated with mature necrotic teeth using regenerative endodontic therapy. J
Endod 2014;40:2081–6.

13. Brizuela C, Meza G, Urrejola D, et al. Cell-based regenerative endodontics: a randomized

controlled clinical trial. J Dent Res 2020;99:523–9.
14. Ørstavik D, Kerekes K, Eriksen HM. The periapical index: a scoring system for radiographic
assessment of apical periodontitis. Dent Traumatol 1986;2:20–34.
15. Estrela C, Bueno MR, Azevedo BC, et al. A new periapical index based on cone beam computed
tomography. J Endod 2008;34:1325–31.

16. Caplan DJ, Cai J, Yin G, White BA. Root canal filled versus non-root canal filled teeth: a
retrospective comparison of survival times. J Public Health Dent 2005;65:90–6.

17. Kumar H, Al-Ali M, Parashos P, Manton DJ. Management of 2 teeth diagnosed with dens
invaginatus with regenerative endodontics and apexification in the same patient: a case report
and review. J Endod 2014;40:725–31.
18. Arslan H, Ahmed HM, S x ahin Y, et al. Regenerative endodontic procedures in necrotic mature
teeth with periapical radiolucencies: a preliminary randomized clinical study. J Endod
2019;45:863–72.

JOE  Volume -, Number -, - 2020 Allogeneic Cellular Therapy 7

19. Paryani K, Kim SG. Regenerative endodontic treatment of permanent teeth after completion of
root development: a report of two cases. J Endod 2013;39:929–34.

20. Chrepa V, Henry MA, Daniel BJ, Diogenes A. Delivery of apical mesenchymal stem cells into root
canals of mature teeth. J Dent Res 2015;94:1653–9.
21. Murakami M, Hayashi Y, Iohara K, et al. Trophic effects and regenerative potential of mobilized
mesenchymal stem cells from bone marrow and adipose tissue as alternative cell sources for
pulp/dentin regeneration. Cell Transplant 2015;24:1753–65.
22. Peng L, Ye L, Zhou X. Mesenchymal stem cells and tooth engineering. Int J Oral Sci 2009;1:6–12.

23. Yang Z-H, Zhang X-J, Dang N-N, et al. Apical tooth germ cell-conditioned medium enhances the
differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like
tissues. J Periodontal Res 2009;44:199–210.

24. Huang GT-J, Garcia-Godoy F. Missing concepts in de novo pulp regeneration. J Dent Res
2014;93:717–24.
25. Albuquerque MT, Valera MC, Nakashima M, et al. Tissue-engineering-based strategies for
regenerative endodontics. J Dent Res 2014;93:1222–31.

26. Iohara K, Murakami M, Takeuchi N, et al. A novel combinatorial therapy with pulp stem cells and
granulocyte colony-stimulating factor for total pulp regeneration. Stem Cells Transl Med
2013;2:521–33.

27. Xuan K, Li B, Guo H, et al. Deciduous autologous tooth stem cells regenerate dental pulp after
implantation into injured teeth. Sci Transl Med 2018;10:eaaf3227.

28. Fawzy El-Sayed KM, Ahmed GM, Abouauf EA, Schwendicke F. Stem/progenitor cell mediated
pulpal tissue regeneration: a systematic review and meta-analysis. Int Endod J 2019;52:1573–
85.

29. Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma: in
situ and in vitro surveys. Stem Cells 2007;25:319–31.
30. Martínez CE, Gonza
lez SA, Palma V, Smith PC. Platelet-poor and platelet-rich plasma stimulate
bone lineage differentiation in periodontal ligament stem cells. J Periodontol 2016;87:e18–26.

31. Altaii M, Kaidonis X, Koblar S, et al. Platelet rich plasma and dentine effect on sheep dental pulp
cells regeneration/revitalization ability ( in vitro ). Aust Dent J 2017;62:39–46.

8 Cordero et al. JOE  Volume -, Number -, - 2020

 Paent male 19 years old, ASA 1, undergoing orthodonc treatment. Referred by maxillary right lateral
TIMELINE incisor with an accidental perforaon of radicular cervical third.

REGENERATIVE PROCEDURE WITH PPP-UC-

MSC IMPLANTATION:
PERFORATION TREATMENT:
1. Paent asymptomac.
1. So ssue migrated apically, 2. Irrigaon with 17% EDTA (20 mL/canal for 3
perforaon site totally minutes).
exposed to the oral cavity. 3. Inducon of controlled micro-bleeding.
2. Perforaon was sealed 4. Introducon of PPP-UC-MSCs into the root canal.
externally with composite 5. Biodenne TM was placed on cervical third.
resin. 6. Seal of cavity with composite resin.

11/04/2018 10/05/2018

06/04/2018 26/04/2018 1, 6 and 12 months

FIRST APPOINTMENT: CANAL DESINFECTION: FOLLOW UP:

1. Temporarily seal of 1. Canal was enlarged with - Asymptomac during the 12 months of follow-up.
perforaon with glass size #45 k-file. - No response to the cold and hot tests.
ionomer. 2. Dressed with calcium - The electric current test was responsive.
2. Root canal was hydroxide mixed with - Radiographic examinaon showed a reducon of
instrumented with a size saline soluon. the apical lesion.
#40 k-file. - CBCT PAI= 3
3. Non eugenol periodontal - Denn bridge formaon in the middle third of the
dressing was applied. root canal.
- Laser Doppler flowmetry showed that the tooth
presented a lower mean value of perfusion units
with respect to the control tooth (#10).
- Despite this, it had a pulse characterisc, indicang
blood perfusion in the tooth.

SUPPLEMENTAL FIGURE S1 – Timeline. First appointment: Temporarily seal of perforation with glass ionomer. Root canal was instrumented with a size #40 k-file. Non eugenol
periodontal dressing was applied. Perforation treatment: Soft tissue migrated apically, perforation site totally exposed to the oral cavity. Perforation was sealed externally with composite resin.
Canal desinfection: Canal was enlarged with size #45 k-file. Dressed with calcium hydroxide mixed with saline solution. Regenerative procedure with PPP-UC-MSC implantation: Patient
asymptomatic. Irrigation with 17% EDTA (20 mL/canal for 3 minutes). Induction of controlled micro-bleeding. Introduction of PPP-UC-MSCs into the root canal. Biodentine was placed on
cervical third. Seal of cavity with composite resin. Follow up: Asymptomatic during the 12 months of follow-up. No response to the cold and hot tests. The electric current test was responsive.
Radiographic examination showed a reduction of the apical lesion. CBCT PAI5 3. Dentin bridge formation in the middle third of the root canal. Laser Doppler flowmetry showed that the tooth
presented a lower mean value of perfusion units with respect to the control tooth (#10). Despite this, it had a pulse characteristic, indicating blood perfusion in the tooth.

JOE  Volume -, Number -, - 2020 Allogeneic Cellular Therapy 8.e1