Khademi Abbasali (Orcid ID: 0000-0002-0810-7908)

Nasr Esfahani Mohammad (Orcid ID: 0000-0003-1983-3435)

Improving pulp revascularization outcomes with buccal fat autotransplantation

Saber Khazaeia, Abbasali Khademia, Mahmoud Torabinejadb, Mohammad H. Nasr Esfahanic,

Mozafar Khazaeid, Sayed Mohammad Razavie

a
Department of Endodontics and Dental Research Center, Dental Research Institute, Isfahan
University of Medical Sciences, Isfahan, Iran
b
School of Dentistry, Loma Linda University, Loma Linda, CA, United States
c
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan
Institute for Biotechnology, ACECR, Isfahan, Iran
d
Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of
Medical Sciences, Kermanshah, Iran
e
Department of Oral and Maxillofacial Pathology and Dental Implant Research Center, Dental
Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran

Correspondence to: Dr. Abbasali Khademi

Address: Department of Endodontics, School of Dentistry, Isfahan University of Medical Sciences,
Hezar-Jerib Ave., Isfahan (81746-73461), Iran
Tel: +98-313-6692588
Email: abbaskhademi@hotmail.com

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/term.3094

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 ABSTRACT

Several techniques have been introduced to improve the pulp revascularization outcomes.
The use of the tissue graft can create more practical tissue regeneration, provides vascular
supply and enhances tissue healing. The aim of the present study was to investigate the
histologic and molecular outcomes of pulp revascularization with buccal fat
autotransplantation. Fifty six open apex roots from 4 dogs aged 4-6 months were randomly
allocated to 5 groups of endodontic regeneration models. Group 1 (negative control, n=4);
Group 2 (control and without intervention, n=4); Group 3 (blood clot, n=16); Group 4 (buccal
fat autotransplantation, n=16); Group 5 (blood clot plus buccal fat autotransplantation, n=16).
After three months, the extracted dog teeth were analyzed by histological and
immunohistochemical techniques. Furthermore, real-time quantitative polymerase chain
reactions were implemented to assess the gene expression profiles of dentin
sialophosphoprotein (DSPP), dentin matrix protein (DMP), collagen I (COL1) and alkaline
phosphatase (ALP) on regenerated tissue in the root canals. There were no significant
differences in the severity of inflammation and necrosis between intervention groups.
Immunohistochemical analysis showed significant differences among the study groups in
expression level of extracellular glycoproteins such as fibronectin, laminin and tenascin C.
Group 5 showed an increase in the expression of DMP1 and COL1 genes. The expression of
DSPP gene increased significantly in group 4. The expression of ALP gene increased
significantly in group 3. Using this procedure may open new fields of research for REP in
which tissue autotransplant particularly adipose tissue, may improve the outcomes of pulp
revascularization.
Key words: Regenerative endodontic procedure, revascularization, immature tooth

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 INTRODUCTION

Revascularization is defined as perfusion of blood for enhancement of tissue regeneration. In

regenerative endodontic procedure (REP), the current protocol is based on the formation of
clot in the pulp canal. It is assumed that the blood clot provides both scaffold and growth
factor that are necessary for pulp regeneration. On the other hand, the bleeding procedure
provides stem cells from periapical tissue, which is known to be a stem cell-rich tissue (M.
Khazaei, Bozorgi, Khazaei, & Khademi, 2016; Kim, Malek, Sigurdsson, Lin, & Kahler,
2018).

Accordingly, several techniques have been proposed to improve the pulp revascularization
outcomes. In this regard, research on revascularization with platelet-rich plasma (PRP) has
shown no difference between the use of blood clot and PRP as acellular scaffolds, both
causing the formation of bone-like or cement-like tissues along with intracanal connective
tissue (Torabinejad, Faras, Corr, Wright, & Shabahang, 2014). On the other hand, researchers
have used natural scaffolds like platelet-rich fibrin (Shivashankar, Johns, Vidyanath, &
Kumar, 2012) and injectable or other types of scaffolds impregnated with growth factor and
have shown a comparable achievement with a blood clot (Albuquerque, Valera, Nakashima,
Nor, & Bottino, 2014; Alexander et al., 2020).

The application of tissue grafts is believed to create more practical tissue regeneration (Chen
& Liu, 2016). The buccal fat grafts provide vascular supply, enhance tissue healing, and have
a soft-tissue hydrophobic layer which acts as a barrier owing to its physiologic composition
(Toshihiro et al., 2013). Besides, the buccal fat grafts have been used for the treatment of
periodontal defects, oral submucosal fibrosis, and intraoral malignant defects as well as
congenital cleft palate repair (Salehi-Nik et al., 2017).

The buccal fat tissue contains mesenchymal stem cells (MSCs) with high proliferation rate
and differentiation potential which, unlike other MSCs, will not be affected by factors such as
age, gender, obesity and vascular diseases (DiMuzio & Tulenko, 2007). Furthermore, the
adipose tissue has extracellular matrix (ECM) molecules such as laminin, fibronectin,
collagen (Mori, Kiuchi, Ouchi, Hase, & Murase, 2014) and particularly tenascin, which is
linked to ECM remodeling and regulations (Catalan et al., 2012; Unamuno et al., 2018). On

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the other hand, the expression of these ECM molecules may have an effective role in
dentinogenesis (Fried et al., 2005; Martinez, Machado de Souza, Correa, & Cavalcanti de
Araujo, 2000). Considering the specific characteristics of adipose tissue, including a vast
number of MSCs and various ECM molecules, which can help tissue regeneration or maybe
dentin pulp complex, and suitability of this tissue (reduced risk of infection transmission,
simple oral access and low cost of procedure), the present study was conducted to investigate
the histologic and molecular outcomes of revascularization with buccal fat
autotransplantation.

MATERIALS AND METHODS

Ethical statement
The present study protocol was approved by the Regional Bioethics Committee affiliated to
Isfahan University of Medical Sciences (IUMS), Isfahan, Iran
(#Ir.mui.reacherch.REC.1397.188). The present study was reported based on the ARRIVE
guidelines for reporting animal research (Kilkenny, Browne, Cuthill, Emerson, & Altman,
2010).

Experimental animals and housing

This study was carried out on 4 Iranian dogs aged 4-6 months (Mean weight: 16.2 kg), all
were from the same mother and were kept in separate cages in the animal house in IUMS
under standard conditions such as air-conditioning (temperature: 25˚C), accessible food,
water and washing equipment. All dogs received a soft diet for three weeks after intervention.

Study design
The present study was performed on the first, second, third and fourth mandibular premolars
of four dogs (56 open apex roots were considered as an experimental unit). Roots were
randomly allocated to 5 groups. Group 1 (negative control, n=4): pulp exposure and
intervention were not done, so the normal tooth development was assessed; Group 2 (control
and without intervention, n=4): pulpectomy was carried out to cease the normal tooth
development; Group 3 (blood clot, n=16): pulpectomy was done and revascularization was
performed to induce blood clot formation in the root canal; Group 4 (buccal fat
autotransplantation, n=16): pulpectomy was done and canals were filled with fat tissue;
Group 5 (blood clot plus buccal fat autotransplantation, n=16): pulpectomy was done and

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revascularization was performed to induce blood clot formation and buccal fat tissue was put
on the established blood clot. In the experimental session, after each group-specific
intervention, a 3 mm-thick RetroMTA (BioMTA, Seoul, South Korea) was applied and the
teeth were filled with amalgam (Supplemental figure S1).

Experimental procedures
Experimental procedures were performed in the morning in a specific animal operation room.
For long‐term anesthesia (longer than 1 hour), ketamine–midazolam (Bremer Pharma GmbH,
Germany) (0.6 mg/kg/h) was administered intramuscularly 15–30 min before general
anesthesia. Subsequently, the animals were cannulated using a 22-G catheter and
administered 0.9% saline solution as a maintenance fluid at 10 mL/kg/h. Then the animals
were intubated using an endotracheal tube with cuff and maintained under anesthesia with
isoflurane (Piramal Healthcare, UK). After each procedure, the dogs were kept in the
recovery room for 24 hours. Postoperative medications consisted of Acepromazine
(AlfasanWoerden, Netherland) (0.01–0.05 mg/kg IV), Dexmedetomidine (0.5–1 μg/kg IV)
and Amoxiclav-VDM (500 mg, bid, oral) (V.M.D. HogeMauw, Arendonk, Belgium).

Obtaining and processing the buccal fat tissue

When animals were under general anesthesia, the surgical procedure for obtaining buccal fat
tissue was done after performing local anesthesia using 3% mepivacaine (Septodent, Novocol
Pharmaceutical, Ontario, Canada). Access to adipose tissue was achieved by flapping at the
surgical site. From each surgical site (each dog corresponds to one surgical site),
approximately 500 mg adipose tissue was isolated, which then irrigated with normal saline
(Darou Pakhsh, Tehran, Iran) to remove the extra blood. The flap was sutured subsequently.
Next, 10 to 20 mg adipose tissue was administrated to each root canal (group 4 and 5).

Histological and immunohistochemical analysis

After three months (Palma et al., 2017), histological and immunohistochemistry (IHC)
analysis was done on the entire of roots including regenerated tissue inside the root canals as
well, by an oral pathologist using a blind method in three different domains: 1) cellular
inflammatory response was defined as no inflammation, acute inflammation, chronic
inflammation and presence of both acute and chronic inflammation; 2) soft-tissue changes,
including hyperemia and necrosis; 3) hard tissue changes, including calcified block, its type,
consistency and thickness (Londero Cde et al., 2015; Palma et al., 2017). IHC was carried out

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to detect the expression of ECM glycoproteins such as fibronectin (Abcam, ab2413), laminin
(Abcam, ab11575) and tenascin C (GeneTex, GTX100558).

Real-time quantitative Polymerase Chain Reactions (PCR)

RAN from the formed root was extracted in cold condition, and about 500 ng of the extracted
RNA were reverse transcribed into cDNA by the cDNA synthesis kit (Prime Script TMcDNA
Synthesis Kit, Takara) according to the manufacturer’s instructions. The expression profiles
of dentin sialophosphoprotein (DSPP), dentin matrix protein (DMP), collagen I (COL1) and
alkaline phosphatase (ALP) genes were evaluated using the real-time PCR-based SYBR
GREEN I assay (SYBR Premix Ex Taq Master Mix, Takara). Table 1 shows the primer
sequences of the genes used in the present study.

Data analysis
The data were analyzed by EXCEL 2016, Graphpad PRISM (version 5; San Diego, CA) and
SPSS 23 (IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp) using
one-way ANOVA and chi-square statistical tests.

RESULTS
After three months follow up, root closure and increase of dentin wall thickness have been
confirmed radiographically (Supplemental figure S2). Out of 56 roots (unit of analysis), only
6 (three for group 4, and one each, in group 2, 3 and 5) roots have been missed during study
period owing to root fracture and loss of coronal seal (restoration loss).

Histological and immunohistochemical outcomes

There were significant differences among the study groups in the type of inflammation.
Group 3 showed no inflammation, in group 4, 50% of samples indicated both acute and
chronic inflammation and 25% of samples in group 5 showed chronic inflammation (Chi-
square, P-value <0.05). There were no significant differences in the severity of inflammation
among the study groups (Chi-square, P-value >0.05) (Table 2) (Figure 1).
There were significant differences among the study groups in terms of necrosis, and only all
root canals in group 2 showed tissue necrosis (Chi-square, P-value <0.05) (Figure 1). No
complete calcified bridge was detected in group 4, which was the same as control groups. In
addition, 75% and 50% of the samples in groups 3 and 5 had complete calcified bridge,

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respectively. The type of calcified bridge in these groups was almost osteodentin (Table 2)
(Figure 1).
IHC analysis showed significant differences among the study groups in expression level of
fibronectin, laminin and tenascin C (One-way ANOVA, P-value<0.05) (Figure 2 and 3).

Gene expression
The results of real time-PCR showed that the expression of DMP1 and COL1 genes increased
significantly in group 5 (One-way ANOVA, Tukey post hoc, P-value <0.05). Furthermore,
the expression of DSPP gene increased significantly in buccal fat autotransplantation group
(One-way ANOVA, Tukey post hoc, P-value <0.05). The expression of ALP gene increased
significantly in group 3 (One-way ANOVA, Tukey post hoc, P-value <0.05) (Figure 4).

DISCUSSION
The main findings of the present study were that revascularization with buccal fat
autotransplantation enhanced the outcome of REP. To our knowledge, this is the first study
that evaluated both histological and molecular outcomes of pulp revascularization. Using this
procedure may open new fields of research for REP in which tissue autotransplant,
particularly adipose tissue, may improve and enhance the outcomes of revascularization
(Sultan et al., 2012).
The results of histological analysis showed that all root canals in groups 3 and 5 showed
apical closure after their specific intervention. This finding was similar to previous reports
(Khademi, Dianat, Mahjour, Razavi, & Younessian, 2014; Thibodeau, Teixeira, Yamauchi,
Caplan, & Trope, 2007). Moreover, use of buccal fat autotransplantation alone and
revascularization did not increase the tissue inflammatory responses.
The results of the present study showed no difference between the intervention groups in the
term of soft tissue changes. This fact supports the concept of tissue autotransplantation into
the root canal. Based on current evidence, the application of tissue autotransplantation is
vastly used in reconstructive oral surgeries with promising results (Hammond, Samuels, &
Thaller, 2019; Karmali, Hanson, Nguyen, Skoracki, & Hanasono, 2018).
Hard tissue variables such as continuity and tissue type of the coronal calcified bridge were
evaluated. It has been demonstrated that the development of coronal calcified bridge may
suggest a process of repair (Hasheminia, Feizi, Razavi, & Feizianfard, 2007). Furthermore,
the most newly formed hard tissues inside the root canal in group 3 and 5 were connective
tissue and bone-like tissue, similar to the findings of Thibodeau et al. (Thibodeau et al., 2007)

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and Wang et al. (Wang, Thibodeau, Trope, Lin, & Huang, 2010). In these groups, the blood
clot was established contrary to the assigned protocol of group 4. It has been shown that the
provocation of bleeding into the canal results in an influx of MSCs. The formation of bone-
like tissue in the necrotic canals after the regeneration procedure may be due to the presence
of MSCs that go through environmental signaling and differentiate these cells into
odontoblast-, osteoblast- and cementoblast-like cells.
The results of IHC indicted that expression level of ECM molecules such as fibronectin,
laminin and tenascin C were higher in revascularization with buccal fat autotransplantation
than revascularization alone. Fibronectin is one of the extracellular non-collagenous
glycoproteins. Numerous roles such as cell adhesion, migration, growth, and differentiation
have been suggested for fibronectin. It also has been reported that fibronectin may have a
critical role in the differentiation, polarization and migration of odontoblasts (Martinez et al.,
2000). The expression level of fibronectin is dependent on the dose of calcium ions (Mizuno
& Banzai, 2008). Hence, the expression of this glycoprotein may be due to the use of the
calcium silicate-based cements in REP.
Tenascin has a pivotal role in odontoblast differentiation, and it has been suggested that this
large oligomeric glycoprotein may play a role in the formation of mineralized tissues
(Moradi, Saghravanian, Moushekhian, Fatemi, & Forghani, 2015). Laminin is another
glycoprotein that is commonly found in the basement membranes. It has been reported that
laminin takes part in the neuronal outgrowth, maturation and regeneration (Fried et al., 2005).
The increased expression of these glycoproteins in comparison to other intervention groups
may suggest that using buccal fat tissue can contribute to differentiation and mineralization.
The findings of this study also showed that all these glycoproteins were expressed in the
healthy pulp tissue and may play several roles.
Several theories have been proposed for the tissue formation in the root canal after
revascularization (Kim et al., 2018). Different studies have shown that connective- and
periodontal ligament-like tissues and hard tissues, including bone-, cementum-like tissue, are
formed in the root canal, even in the teeth without necrosis or periapical disease (Nosrat et
al., 2015; Torabinejad et al., 2014). In the present study, real-time PCR was used to evaluate
the expression profiles of DSPP, DMP, COL1 and ALP genes in the tissue formed in the root
canal. ALP gene expression increased significantly in the group with induced blood clot
formation (group 3), which might show the increase of calcium deposition or ectopic
calcification (Karwowski, Naumnik, Szczepanski, & Mysliwiec, 2012; Savinov et al., 2015).
However, a question that still remains unanswered is, which signals correspond to this

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activity? In fact, the future research should open new lines to evaluate the activity of ALP.
On the other hand, using calcium silicate-based cements in REP may provide an environment
for differentiation of MSCs to osteoblast and cementoblast due to release of calcium and
phosphorus ions (Viti et al., 2016).
Using buccal fat autotransplantation in the root canal with induced blood clot improved the
activity of DMP and COL1. It has been shown that type I collagen is the key protein of dentin
ECM and regulates the expression level of DMP1 as well. On the other hand, Studies have
established that active odontoblasts express both type I collagen and DMP1 (Mizuno,
Miyamoto, Wada, Watatani, & Zhang, 2003). The results of the present study indicated that
using buccal fat enhanced the expression of both DMP and COL1. This finding supports this
fact that if adequate signals and ECM are provided, the fate of each stem cell may alter.
Our results showed that the expression of DSPP increased in all intervention groups in
comparison to the control groups. This shows that some mineralization may occur. DSPP is
produced by odontoblasts and either osteoblasts can produce this protein as well. It also has
been verified that DSPP plays an essential role in mineral deposition (Yamakoshi, 2009).
The benefits of adipose tissue autotransplant are no immunologic reactions and no risk of
microbiological contamination. Autotransplantation of adipose tissue can be considered as
source of mesenchymal stem cell that is able to grow and proliferate in great numbers. In
addition, the effectiveness of ASCs, unlike other MSCs, does not change with age, and is not
affected by gender, obesity and different metabolic diseases (DiMuzio & Tulenko, 2007).
Furthermore, in our previous study, it was also demonstrated that adipose tissue-derived stem
cells can easily and efficiently be isolated from the buccal fat pad and differentiated into
odontoblast-like cells (S. Khazaei et al., 2021).
One of the limitations of the present study was that apical periodontitis was not induced and
the site of revascularization and transplant was not necrotic. Because this study was a
preliminary step to evaluate the fate of fat tissue autotransplant through the root canal, the
current protocol was considered. Moreover, assessment of tissue interaction without necrosis
was another goal. For future research, we suggest using the adipose tissue autotransplant with
REP in root canal by means of apical periodontitis. Furthermore, randomized subsequent
randomized control trials are highly recommended.

CONCLUSION
Pulp revascularization by buccal fat autotransplantation enhanced the outcome of REP,
including the increased expression of dentin matrix protein and collagen I genes. In addition,

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the expression level of ECM molecules such as fibronectin, laminin and tenascin C increased.
Furthermore, histological assessment showed no tissue necrosis and no increase in the tissue
inflammation severity. Moreover, 75% of the formed coronal calcified bridge was
osteodentin.

ACKNOWLEDGEMENT
The authors declare no potential conflicts of interest.
Author contributions:
Saber Khazaei: contributed to conception, design, data acquisition, analysis, interpretation, drafted,
critically revised the manuscript and gave final approval.
Abbasali Khademi: contributed to data acquisition, analysis, interpretation, critically revised the
manuscript and gave final approval.
Mahmoud Torabinejad: contributed to data acquisition, analysis, interpretation, critically revised the
manuscript and gave final approval.
Mohammad H. Nasr Esfahani: contributed to conception, design, data acquisition, analysis,
interpretation, drafted, critically revised the manuscript and gave final approval.
Mozafar Khazaei: contributed to conception, design, data acquisition, analysis, interpretation, drafted,
critically revised the manuscript and gave final approval.
Sayed Mohammad Razavi: contributed to data acquisition, analysis, interpretation, critically revised
the manuscript and gave final approval.

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 Gene Accession Annealing Size Primer sequence
Number temperature (°C) (bp)
F: 5ˊ-TGTCCCCACCCCCAATGTATC-3ˊ
GAPDH NM_001003142 60 100
R: 5ˊ-CTCCGATGCCTGCTTCACTACCTT-3ˊ
F: 5ˊ-AGATGTGGAGTATGAGATGGA-3ˊ
ALPL NM_001197137 60 110
R: 5ˊ-CGTAGTGAGAGTGCTTGTG-3ˊ
F: 5ˊ-GGCTCTGGTGATGATGAAGGT-3ˊ
DSPP XM_003434003 60 187
R: 5ˊ-TGTTGTCTCCACCGATGTCA-3ˊ
F: 5ˊ-GAATCCAACGAAAGCCTCAG-3ˊ
DMP1 XM_005639164 60 158
R: 5ˊ-GCATCATCTTCCTCAGACTG-3ˊ
F: 5ˊ-CACCTCAGGAGAAGGCTCAC-3ˊ
COL1A1 NM_001003090 60 124
R: 5ˊ-ATGTTCTCGATCTGCTGGCT-3ˊ

Table 1: Primer sequence of genes

The primers were designed and used for expression of ALPL, DSPP, DMP1 and COL1A1 genes by AllelelD 7.5
software and https://www.ncbi.nlm.nih.gov/tools/primer-blast.

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 Histologic criteria Group 1 Group 2 Group 3 Group 4 Group 5 P-value
(N=4) (N=3) (N=8) (N=8) (N=8)
Type of inflammation 0.027*
None 100% 0% 100% 50% 75%
Acute 0% 0% 0% 0% 0%
Chronic 0% 35% 0% 0% 0%
Acute and Chronic 0% 65% 0% 50% 25%
Severity of inflammation 0.075
0 -30 ICs 100% 0% 100% 50% 75%
30-60 ICs 0% 65% 0% 0% 25%
>60 ICs 0% 35% 0% 50% 0%
Necrosis 0.002*
Yes 0% 100% 0% 25% 0%
No 100% 0% 100% 75% 100%

Table 2: Histologic criteria using Hematoxylin and Eosin (H & E) stain

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Coronal calcified bridge 0.02*
Complete 0% 0% 75% 0% 0%
Incomplete 0% 0% 25% 0% 50%
No calcified bridge 100% 100% 0% 100% 50%
Type of calcified bridge 0.011*
No calcified bridge 100% 100% 0% 100% 0%
Osteodentin 0% 0% 50% 0% 75%
Resemble dentin 0% 0% 50% 0% 25%

*Statistically significant (Chi-square, P<0.05); Group 1 (negative control): without pulp exposure and
intervention, Group 2 (control and without intervention): pulpectomy without any intervention, Group 3 (blood
clot): revascularization to induce blood clot formation, Group 4 (buccal fat autotransplantation): canals filled
with buccal fat tissue, and Group 5 (blood clot plus buccal fat autotransplantation); ICs: inflammatory cells; N:
sample size correspond to histologic examination.

Figures legend

Figure 1: Histologic examination (hematoxylin-eosin [h&e] stain) of study groups. D: dentin, OD: odontoblast
layer, AT: adipose tissue, CT: connective tissue. (a) Group 1 (negative control): without pulp exposure and
intervention; arrows show blood vessel (original magnification ×10). (b) Group 2 (control, without
intervention): pulpectomy alone; lymphocytes infiltration (arrows) (original magnification ×10). (c) Group 3
(blood clot): revascularization to induce blood clot formation; calcify deposition (bone-like tissue) inside root

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canal (arrows) (original magnification ×10). (d) Group 4 (buccal fat autotransplantation) (original magnification
×10). (e) Group 5 (blood clot plus buccal fat autotransplantation): blood clot formation and buccal fat
autotransplantation; arrows show blood vessel (original magnification ×10) (f) Group 5 and arrows blood vessel
(original magnification ×40).

Figure 2: Bar chart presents the results of immunohistochemical (IHC) examination. Groups (Columns) with
different superscript letters are statistically significant (One-way ANOVA, Tukey’s Post Hoc: P<0.05). Group 1
(negative control): without pulp exposure and intervention; Group 2 (control, without intervention): pulpectomy
alone; Group 3 (blood clot): revascularization to induce blood clot formation; Group 4 (buccal fat
autotransplantation): canals filled with buccal fat tissue; Group 5 (blood clot plus buccal fat
autotransplantation): blood clot formation and buccal fat autotransplantation. (A) Expression of fibronectin, (B)
Expression of laminin, (C) Expression of tenascin C.

Figure 3: Immunohistochemical (IHC) examinations correspond to group 5 (blood clot plus buccal fat
autotransplantation): blood clot formation and buccal fat autotransplantation. D: dentin, CT: connective tissue.
(a) Expression of fibronectin (original magnification ×10). (b) Expression of fibronectin (arrows) (original
magnification ×40). (c) Expression of tenascin C (original magnification ×10). (d) Expression of tenascin C
(arrows) (original magnification ×40). (e) Expression of laminin (original magnification ×10). (f) Expression of
laminin (arrows) (original magnification ×40).

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Figure 4: The expression profile of dentin sialophosphoprotein (DSPP), dentin matrix protein (DMP), collagen
I (COL1) and alkaline phosphatase (ALP) genes evaluated using Real-Time PCR. Stars show the statistically
significant differences (One-way ANOVA, Tukey’s Post Hoc: *P<0.05, **P<0.01, ***P<0.0001, ****
P<0.00001). Numbers correspond to the study groups, as follows: Group 1 (negative control): without pulp
exposure and intervention; Group 2 (control, without intervention): pulpectomy alone; Group 3 (blood clot):
revascularization to induce blood clot formation; Group 4 (buccal fat autotransplantation); Group 5 (blood clot
plus buccal fat autotransplantation): blood clot formation and buccal fat autotransplantation.

Supplemental Figures legend

Supplemental Figure S1: Schematic representation of blood clot formation and buccal fat autotransplantation
in the present study. (A) Immature permanent tooth. (B) Procedure used in the present study.

Supplemental Figure S2: (A) Preoperative radiograph of dog teeth. (B) Postoperative radiograph. (C) Three-
month follow-up.

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