Clinical Research
Assessment of Regaining Pulp Sensibility in
Mature Necrotic Teeth Using a Modified
Revascularization Technique with Platelet-rich
Fibrin: A Clinical Study
Mohamed Nageh, BDS, MSc, PhD,* Geraldine M. Ahmed, BDS, MSc, PhD,†
and Alaa A. El-Baz, BDS, MSc, PhD†
Abstract
Introduction: The aim of the present study was to
evaluate the possibility of regaining pulp sensibility in
mature necrotic teeth using modified regenerative
endodontic procedures by inducing bleeding in root
canals and using platelet-rich fibrin (PRF). Methods:
Fifteen patients with necrotic pulp with symptomatic
or asymptomatic apical periodontitis were included. At
the first visit, the tooth was anesthetized, and an access
cavity was performed. Mechanical preparation of root
canals was performed using the standardized technique
reaching apical canal preparation to K-file size #60–80.
Double antibiotic paste was injected into the canal, and
the cavity was temporarily sealed using glass ionomer
cement. Three weeks from the first visit, regenerative
endodontic procedures were performed by inducing
bleeding, and a freshly prepared PRF membrane was
placed in the canal. White mineral trioxide aggregate
was placed directly over the PRF matrix, and the tooth
was restored with a glass ionomer cement base and
resin composite restoration. The electric pulp test was
used to record if the teeth included in the study regained
sensibility or not every 3 to 12 months follow-up.
Readings at different times were compared as categoric
qualitative data using the chi-square test and compared
as means and standard deviations using the analysis of
variance test. Results: Readings of tooth sensibility
revealed a highly significant difference (P < .0001)
between baseline and the 12-month follow-up period.
Conclusions: The presence of sensibility is indicative
of the formation of vital pulplike tissue. Reestablishing
real pulp tissue after regenerative endodontic treatment
is debatable and still needs high level of evidence with
large-scale investigations. (J Endod 2018;-:1–8)
From the *Endodontic Department, Faculty of Oral and Dental Medicine, Fayoum University, Fayoum, Egypt; and †Endodontic Department, Faculty of Oral and Dental
Medicine, Cairo University, Cairo, Egypt.
Address requests for reprints to Dr Alaa A. El-Baz, Cairo University, Endodontic Department, Faculty of Oral and Dental Medicine, Cairo, Egypt. E-mail address: alaa.
elbaz@dentistry.cu.edu.eg
0099-2399/$ - see front matter
Copyright ª 2018 American Association of Endodontists.
https://doi.org/10.1016/j.joen.2018.06.014
JOE — Volume -, Number -, - 2018
Key Words
Modified revascularization technique, platelet-rich fibrin, pulp sensibility
P reservation of the natu-
ral dentition remains a
primary objective in end-
Significance
The gain of nerve function in regenerated pulp
tissues and maintaining possible proprioceptive
odontic practice. When
defense mechanisms of the pulp will provide an
the pulp is diseased or ne-
alarm system during tissue injury and protect the
cessitates removal for
pulp from further damage or the probability of tooth
restorative reasons, it is
fracture.
replaced with an artificial
filling material. Endodon-
tists are looking forward to using natural resources instead of artificial ones, especially
after the introduction of tissue engineering in many medical and dental fields (1).
Endodontics is a rich branch for applying tissue engineering science in many as-
pects, such as dentin-pulp complex regeneration, periodontal regeneration, and bone
healing and regeneration. The regenerative endodontic procedure is an example of tis-
sue engineering and was limited to immature teeth, but recently it extended to mature
teeth as an alternative to conventional endodontic treatment (2).
Conventional endodontic treatment has some drawbacks as highlighted by many
studies that concluded that endodontic treatment is a major etiologic factor for tooth
discoloration (3) and tooth fracture (4, 5). Generally, an endodontically treated
tooth undergoes coronal and radicular tissue loss because of prior pathology,
endodontic treatment and/or restorative procedures, and a loss of protective
mechanism of proprioception. There is evidence that these teeth have reduced levels
of proprioception (6, 7). Therefore, the gain of nerve function in regenerated pulp
tissues and maintaining possible proprioceptive defensive mechanisms of the pulp
will provide an alarm system during tissue injury and protect the pulp from further
damage or the probability of tooth fracture (7, 8).
According to numerous histologic studies (9–13) on extracted human teeth,
information regarding regenerated tissues is conflicting; some studies showed that
regenerated tissues are pulplike tissues, cementumlike tissues, bonelike tissues, and
periodontal-like tissues. Nerve regeneration was identified in some cases (14), so
reconstitution of the neurovascular system in root canals by regenerative endodontic
Regaining Pulp Sensibility in Mature Necrotic Teeth 1
Clinical Research
procedures will provide regenerated tissues with immune system cells,
which will function as the primary line of the defense mechanism against
microbial attacks (15–18). In addition to pulp regeneration, reparative
dentin may be deposited along the root canal walls that had been lost
during mechanical instrumentation after regenerative therapy (19)
and below mineral trioxide aggregate (MTA), which acts as a coronal
seal material above the regenerated tissues (20–22).
Regenerative endodontic therapy was limited to pulp
revascularization of immature necrotic teeth by disinfection of the
root canal system followed by establishing bleeding into the canals
via overinstrumentation (23–25). Shah and Logani (26) were the first
to attempt pulp revascularization in mature teeth followed by several
case reports (15–18). Their results were promising, relieving clinical
signs and symptoms and showing periapical healing. Some cases
showed regained tooth sensibility.
A second-generation platelet concentrate known as Choukroun’s
platelet-rich fibrin (PRF) is totally autologous in nature, a very simple
technique, and inexpensive. PRF contains platelets, growth factors,
and cytokines that might enhance the healing potential of both soft
and hard tissues (27). In the present study, modified regenerative
endodontic procedures were used to induce bleeding in the root canal
using PRF as a scaffold and source of growth factors to treat necrotic
mature teeth with a closed apex and to evaluate the possibility of
regaining pulp sensibility.
Materials and Methods
Fifteen patients with necrotic upper central incisors were recruited
from the outpatient clinic of the Endodontic Department, Faculty of Oral
and Dental Medicine, Cairo University, Cairo, Egypt, between 2015 and
2017. The protocol of the trial was approved by the Ethics Committee of
Cairo University. The treatment procedures and the aim of the study
were thoroughly explained for all the patients. The patients were asked
about their dental and medical history; medically compromised patients
were excluded. Patients were asked to follow the general instructions,
sign a printed informed consent that explained the aim of the study, and
conform with the clinical and radiographic follow-up period every
3 months up to 1 year.
Inclusion criteria for the study were patients 18–40 years old with
no sex predilection, medically free, suffering from necrotic pulp in
maxillary permanent central incisor teeth with a closed apex associated
with or without periapical radiolucency, no response of vital pulp with
thermal (cold) and electric pulp testing, no tooth mobility, and a pocket
depth <3 mm. A full history of the chief complaint was taken including
intensity, quality, onset, duration, location, course, and initiating and
relieving factors of pain. Fifteen patients who fulfilled the inclusion
criteria were included in the study. Few of the included patients were
complaining of mild to moderate dull pain on percussion and palpation
tests with or without a fistula tract. Other patients were complaining of
moderate to severe pain with intraoral swelling and pain on biting and
palpation and percussion tests. Some patients were complaining of
tooth discoloration from previous trauma. The final diagnosis of the
included patients was necrotic pulp with symptomatic or asymptomatic
apical periodontitis.
At the first visit, the tooth was anesthetized with the buccal
infiltration technique using 1.8–3.6 mL 2% lidocaine with 1:100,000
epinephrine local anesthetic solution. An access cavity was performed,
and the tooth was isolated with a rubber dam. Negotiation of the canals
was performed using stainless steel hand K-files size #15. The working
length was determined using an electronic apex locator and then
confirmed with intraoral periapical radiography to be 0.5–1 mm
shorter than the radiographic apex. Mechanical preparation of the
2 Nageh et al.
root canals was performed using the standardized technique reaching
apical canal preparation to K-file size #60–80. The canals were
thoroughly irrigated between each successive file using 1.5% sodium
hypochlorite (NaOCl). The canal was dried with paper points. Double
antibiotic paste (DAP) was prepared by grinding 1 tablet of
metronidazole (500 mg) and 1 tablet of ciprofloxacin (500 mg), which
were then mixed with saline to form a homogenous paste of reasonable
creamy consistency. This mix was then injected into the canal to a level
just below the cementoenamel junction (CEJ). A cotton pellet was
placed, and the cavity was temporarily sealed with glass ionomer
cement.
At the second visit, the participants were recalled after 3 weeks
from the first visit for completion of the regenerative endodontic
procedures. A plain anesthesia 1.8-mL 3% mepivacaine buccal
infiltration was administered. The rubber dam was placed, temporary
filling was removed using a high-speed handpiece, and reirrigation of
root canals was performed with 20 mL 17% EDTA for 1 minute followed
by saline irrigation (22, 28). The canals were dried using paper points.
Intentional overinstrumentation 2–3 mm past the apex into the
periapical region was done with K-files #20–#40 to induce bleeding
near the apical foramen to a level below the CEJ. The file was gently given
2 to 3 clockwise turns and then withdrawn using counterclockwise
rotation. Excess blood reaching the pulp chamber was dried using a
small cotton pellet held with tweezers. A 5-mL sample of whole venous
blood was drawn from the patient’s forearm (right median cubital vein).
The blood sample was then transferred into a test tube
without anticoagulant and centrifuged immediately using a tabletop
centrifuge at 3000 rpm for 10 minutes (29). Three distinct layers
were formed in the tube: platelet-poor plasma at the top, a PRF clot
in the middle, and red blood cells at the bottom. Then, the freshly
prepared PRF membrane was fragmented, and the fragments were
placed incrementally in the canal using a hand plugger and a finger
spreader size 40 up to the level of the CEJ. A 3-mm-thick layer of
white MTA was placed directly over the PRF matrix. A moist cotton pellet
was placed over the MTA, and the tooth was temporized using a
temporary filling for complete setting of the MTA. The patient was
recalled after 2 days to remove the cotton pellet, and the tooth was
restored using a glass ionomer cement base and resin composite
restoration (Figs. 1–5).
Thermal (cold) and electric pulp tests were used to record if the
teeth included in the study regained sensibility or not. The preoperative
electric pulp test reading for the included tooth in the study was
recorded to ensure pulp necrosis of the involved tooth. The average
of 2 readings was recorded for each tooth with an interval of 5 minutes
according to the manufacturer’s instructions. Data were recorded every
3 months for a follow-up of 1 year.
Results
The mean age was 25 years, and there were more female patients
than male patients. However, the chi-square test revealed no significant
difference of sex distribution. Clinical evaluation results showed that
there was no pain, swelling, or fistula through the follow-up period
in all of the cases. Nine patients had sensitivity on the cold test. All
the cases with preoperative apical radiolucency showed radiographic
resolution of apical periodontitis, and no teeth without preoperative
radiolucency showed any bony changes after 12 months of follow-up.
According to the manufacturer’s electric pulp test instructions, if
the readings are between 0 and 39 and the patient feels ache, tingling, or
anesthesia, the result is a tooth with vital pulp. If the readings are
between 40 and 79 with ache or a tingling sensation, the result is
part of a tooth nerve is nonvital. Finally, if the readings are 80 with
JOE — Volume -, Number -, - 2018
 Clinical Research

Figure 1. A mature carious tooth (#21) with a periapical radiolucency. (A) The preoperative periapical radiograph. (B) The 12-month postoperative periapical
radiograph after using the modified revascularization technique using PRF showing complete healing of the periapical lesion.

none of the previously mentioned reactions, the result is a tooth with
necrotic nonvital pulp.
The percentage of teeth regaining sensibility gradually increased
postoperatively to reach the highest level at 12 months. Readings at
different times were compared as categoric qualitative data using the
chi-square test and revealed an extremely significant different
(P < .0001) (Table 1 and Fig. 6).
Moreover, readings of tooth sensitivity were compared as means
and standard deviations using the analysis of variance test and also
revealed a highly significant difference (P < .0001). The Tukey post

Figure 2. A mature carious tooth (#21) with a periapical radiolucency. (A) The preoperative periapical radiograph. (B) The 12-month postoperative periapical
radiograph after using the modified revascularization technique using PRF showing complete healing of the periapical lesion.

JOE — Volume -, Number -, - 2018 Regaining Pulp Sensibility in Mature Necrotic Teeth 3
Clinical Research

Figure 3. A mature carious tooth (#21) with a periapical radiolucency. (A) The preoperative periapical radiograph. (B) The 3-month follow-up radiograph,
(C) the 9-month follow-up radiograph, and (D) the 12-month postoperative periapical radiograph after using the modified revascularization technique using
PRF showing complete healing of the periapical lesion. Teeth (#22, 11, and 12) with compromised restorability that needed root canal treatment, fiber posts,
and full-coverage crowns.

hoc test revealed no significant difference between 3 and 6 months. In
addition, there was no significance difference between 6 and 9 months
or between 9 and 12 months (Table 2 and Fig. 7).
Discussion
In the current study, a promising modified regenerative procedure
using induction of bleeding and PRF as a scaffold and source of growth
factors was performed. The treatment resulted in the elimination of all
clinical signs/symptoms and resolution of apical periodontitis in all the
teeth included in the study. Regaining tooth sensibility is important to
maintain possible proprioceptive defense mechanisms of the pulp,
which provides an alarm system against any tissue injury and protects
the pulp from further damage and tooth fracture (7, 8).
The rationale of cleaning and shaping should be based on the
ability to thoroughly remove the tissue, necrotic debris, and bacteria
through dentin removal. The larger preparation sizes have been shown
to provide adequate irrigation and debris removal as well as significantly
decreasing the number of microorganism. The apical foramina were
prepared to reach ISO K-file #60–80 for effective root canal disinfection
as mentioned previously (30). In order to allow the migration of
blood-borne cells and other cells including mesenchymal stem cells

Figure 4. A mature traumatized tooth (#11) with apical periodontitis. (A) The preoperative periapical radiograph and (B) the 12-month postoperative periapical
radiograph after using the modified revascularization technique using PRF.

4 Nageh et al. JOE — Volume -, Number -, - 2018


Figure 5. A mature carious tooth (# 21). (A) The preoperative periapical radiograph, (B) the 3-month follow-up radiograph, (C) the 9-month follow-up
radiograph, and (D) the 12-month postoperative periapical radiograph after using the modified revascularization technique using PRF.
and thin blood vessels into the canal from the periapical area through
the apical foramen, an ISO K-file #20–40 was used to violate the apex by
overinstrumentation (16, 31).
In the present study, copious irrigation with 20 mL 1.5% NaOCl for
5 minutes was used with a closed-end and side-vented needle to
minimize the possibility of extrusion of irrigant into the periapical
space. A concentration of 5.25% NaOCl is a tissue solvent and
antimicrobial agent, but it has a proteolytic action on the dentin collagen
matrix, decreasing the elastic modulus and flexural strength of human
dentin (32). Moreover, 5.25% NaOCl has an insignificant antimicrobial
effect compared with 0.5% (33). Also, a 5.25% concentration was
found to be cytotoxic to stem cells in the apical tissues and decreased
odontoblastic differentiation (34). NaOCl was then flushed away from
the root canal with saline in an attempt to reduce any lingering toxicity
that can reduce the regeneration responses as recommended by the
American Association of Endodontists (28). Based on the current
available evidence, there is no significance for using hypochlorite
solutions at concentrations over 1.5%.
DAP that consists of only ciprofloxacin and metronidazole
(eliminating minocycline) was used in the present study. Case reports
and studies have shown that minocycline causes visible crown
discoloration (35–37) and a significant reduction in tooth fracture
resistance (38, 39) because it causes demineralization of
intertubular dentin by binding to calcium ions via chelation to form
an insoluble complex and become incorporated into the tooth matrix
causing the discoloration (40). The acidity of triple antibiotic paste
(pH = 2.9) is more than that of DAP (pH = 3.4), having a more
demineralizing effect on the intertubular dentin and causing a
significant reduction in the phosphate/amide ratio and a decrease in
the dentin microhardness and fracture resistance (41, 42).
TABLE 1. The Number and Percentage of Teeth Regaining Sensitivity
Pre-operative, 3 months,
Reading n (%) n (%)
Between 0 and 39 0 2 (13.3)
Between 40 and 79 1 (6.7) 12 (80)
80 14 (93.3) 1 (6.7)
JOE — Volume -, Number -, - 2018
Clinical Research
Plain anesthesia 3% mepivacaine was used in the second visit to
facilitate bleeding. A solution of 17% EDTA was used because its
chelating effect promotes the release of dentin-derived growth factors
that were previously embedded into dentin during dentinogenesis
(43–45).
PRF, a second-generation platelet concentrate, has been
developed to overcome the limitations associated with the use of
platelet-rich plasma. PRF preparation eliminates the process of adding
anticoagulants or bovine thrombin, making it similar to centrifuged
natural blood. Furthermore, a highly resistant and elastic membrane
of fibrin is obtained, which does not dissolve quickly after application
(46), allowing cellular migration, cytokine enmeshment, and slow
continuous release of cytokines such as platelet-derived growth factor,
transforming growth factor beta 1, fibroblast growth factor, and
vascular endothelial growth factor from 7 to 28 days, achieving the
peak level on day 14 and coinciding with cell ingrowth. Therefore, it
directs stem cell migration, proliferation, and differentiation and
supplements angiogenesis (46–48). The presence of leukocytes and
cytokines along with small amounts of lymphocytes in PRF can play a
significant role in the self-regulation of inflammatory and infectious
phenomenon (49).
MTA was placed directly over the PRF to obtain a coronal seal by
the formation of calcium hydroxide and hydrated silicate gel. Calcium
hydroxide gives rise to numerous biological and physicochemical
properties; it creates an antibacterial environment by its alkaline pH
and promotes reparative dentinogenesis or mineralized tissue
formation by uncoupling and activating growth factors nested in the
proximal dentin that control the growth and differentiation of cells
(50). Signaling molecules, including transforming growth factor beta,
macrophage colony-stimulating factor, and interleukins 1a and b,
6 months, 9 months, 12 months, p
n (%) n (%) n (%) c2 Value
4 (26.7) 6 (40) 9 (60) 72.38 .0001
11 (73.3) 9 (60) 6 (40)
0 0 0
Regaining Pulp Sensibility in Mature Necrotic Teeth 5
Clinical Research

100%
90%
80%
70%
60%
50% 80
40% Between 40-79
30% Between 0-39
20%
10%
0%

Figure 6. A column chart showing the percentage of teeth regaining sensitivity.

are stimulated via the continued release of calcium ions during the
setting process of MTA (51).
The objective of the electric pulp test is to stimulate intact A delta
nerves in the pulp-dentin complex by applying an electric current on the
tooth surface. A positive response results from an ionic shift in the
dentinal fluid within the tubules, causing local depolarization and
subsequent generation of an action potential from intact A delta nerves
(52). In normal healthy teeth, an area of high neural density in the pulp
horns should have a relatively fast and strong response and requires the
least electric current. Therefore, the most desirable area of assessment
in incisor teeth is at the incisal edge, where the enamel is thinnest or
absent.
In this study, 3 mm MTA was placed directly over the PRF. The
tooth was then double sealed with 2 mm glass ionomer and resin
composite restoration. A total of 5 mm was used for coronal
sealing. Because the distance between vital tissue and the coronal
region is 5 mm, the electric pulp test was applied in the middle third
of the labial surface of the tooth; because it is principally the fluid in
the tubules that conducts electrical impulses from the pulp tester
electrode to the vital tissues, the shorter the distance between the
electrode and the vital tissues, the lower the resistance to the flow of
current (53).
Nine of the 15 cases (60%) have regained tooth sensibility at the
end of the 12-month follow-up period, indicating the presence of
vital tissues within the root canal. Such results are in agreement with
that of Shivashankar et al (54) and Keswani and Pandey (29) who
reported that immature teeth with necrotic pulp treated with PRF
revascularization responded positively to sensibility testing after a
12-month follow-up. Paryani and Kim (15) reported that 50% of
necrotic teeth with a closed apex responded positively to pulp sensibility
test after a 22-month follow-up. The current results were not in
agreement with Bakhitar et al (55), who reported that all of the
4 immature teeth with necrotic pulp responded negatively to the
electric pulp test after 12 months of PRF revascularization
treatment. Several studies using blood clot revascularization
showed a negative response to pulp sensibility after follow-up periods
from 8 to 26 months in cases of necrotic teeth with a closed apex
(16–18).
Nerve regeneration may be attributed to several mechanisms. The
first possible mechanism could be caused by the PRF technique, which
acts as a rich source of various growth factors that are enrolled in
neurogenesis. Transforming growth factor beta regulates mitogenic
effects of other growth factors (nerve growth factor and
brain-derived neurotrophic factor), stimulates neurite outgrowth,
and regulates differentiation of Schwann cells (SCs) (56). Platelet-
derived growth factor (PDGF) induces SC proliferation, differentiation,
and myelin formation (57). Vascular endothelial growth factor
promotes blood vessel growth, neurogenesis, and neuroprotection
(58). Insulin growth factor 1 supports the forward extension of the
nerve fibers and suppresses apoptosis in motor, sensory, and
sympathetic neurons (59). Basic fibroblast growth factor facilitates
neuroprotection and SC regeneration (60).

TABLE 2. The Mean Value Measurements of Teeth Sensitivity

Minimum Maximum Mean SD F value p value
a
Preoperative 78.00 80.00 79.87 0.51 19.697 .000
3 months 34.00 80.00 61.73b 14.66
6 months 26.00 76.00 51.40b,c 16.11
9 months 21.00 70.00 45.13c,d 16.10
12 months 14.00 70.00 37.53c,d 17.12
Means sharing the same superscript letter are not significantly different.

6 Nageh et al. JOE — Volume -, Number -, - 2018


79.87
80
70 61.73
60
50
Mean
40
30
20
10
0
pre_operaƟve 3 months
Figure 7. A column chart showing the mean value of teeth regaining sensitivity.
The second hypothesized mechanism depends on the survival of
dental pulp stem cells from residual vital apical pulp tissue; in mature
teeth, some vital pulp tissue might remain despite the presence of a
periradicular lesion. Dental pulp stem cells have the potential to
differentiate into neuronal cells and can induce axon guidance (61).
The third hypothesized mechanism postulates that stem cells might
differentiate from the periodontal ligament. Human periodontal
ligament stem cell subpopulations have been reported to express the
markers of undifferentiated neural crest cells and to exhibit the
potential to differentiate into neurogenic lineages (62). The fourth
possible mechanism relies on bone marrow mesenchymal stem cells,
which can differentiate into neurons and astrocytes under appropriate
conditions (63).
The fifth possible mechanism is that irrigation with 17% EDTA in
regenerative endodontic treatment is known to promote neurogenesis
by locally released growth factors from the dentin matrix such as the
brain-derived neurotrophic factor, the glial cell line–derived
neurotrophic factor, and growth/differentiation factor 15. These factors
promote neuronal growth and axonal regeneration and play an
important role in neuronal maintenance (64).
The sixth possible mechanism is that the pulp canal likely received
a collateral reinnervation by the sprouting or ingrowth of neighboring
ipsilateral nerves, similar to the reinnervation of replanted and
autotransplanted immature teeth after surgery. In addition, nerve fibers
are known to regenerate after the transection of the inferior alveolar
nerve and to grow into the tooth pulp by the sprouting and ingrowth
of intact nerves in adjacent tissues. However, the underlying mechanism
of this action needs further investigation (14).
Although histologic examination was not performed, the presence
of sensitivity and a positive response to electric testing are indicative of
the formation of vital pulplike tissue inherited with innate and adaptive
immune defense mechanisms and innervated by sensory nerve fibers to
detect and protect themselves from foreign invaders. Reestablishing real
pulp tissue after regenerative endodontic treatment is debatable and still
needs a high level of evidence with large-scale investigations.
Acknowledgments
The authors deny any conflicts of interest related to this study.
JOE — Volume -, Number -, - 2018
Clinical Research
51.4
45.13
37.53
6 months 9 months 12 months
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JOE — Volume -, Number -, - 2018