Evaluation of Demineralized Dentin Matrix as a Scaffold for Revascularization in Immature Permanent Incisors: A Randomized Controlled Trial and an In -Vitro Study
Phase: NA
Conditions: Dental Trauma
Sponsor: Ain Shams University

1 
 
Evaluation of Demineralized Dentin Matrix as a 
scaffold for Revitalization in immature permanent 
incisors: A Randomized Controlled Trial and In Vitro 
Study 
A Project Submitted to the Department of Pediatric Dentistry and Dental Public 
Health, Faculty of Dentistry Ain Shams University 
 
 
In partial fulfillment of the requirements of Doctorate degree in 
Pediatric Dentistry 
 
By 
 
(Marwa Ahmed Abozed Mahmoud) 
B.D.S, Faculty of Dentistry, Benghazi University, 2009 
MSc (University of Ain Shams 2018), 
Assistant Lecturer in Benghazi University 
2019-2021 
 
 
Faculty of Dentistry 
Ain Shams University 
 
 
 
(2022) 

2 
 
Supervisors 
 
 
 
 
 
 
Prof. Dr. Nagwa Mohamed Ali Khattab 
Professor & Head of Pediatric Dentistry and Dental Public Health department 
Faculty of Dentistry 
Ain Shams University 
 
 
 
 
 
Dr. Mariem Osama Wassel 
Associate Professor of Pediatric Dentistry and Dental Public Health 
Faculty of Dentistry 
Ain Shams University 
 
 
 
 
Dr. Shaimaa Abu Elsadat, 
Associate Professor of oral and maxillofacial Radiology 
Faculty of Dentistry 
Ain Shams University 
 
 
Dr. Wael Elshater 
Lecturer at the department of Endodontics 
Faculty of Dentistry 
Ain Shams University 

3 
 
I. 
Introduction 
 
 
 
Necrosis of the immature pulp not only is one of the multiple factors affecting long-term tooth 
endurance and preservation, but also can act as a source of bacterial infection in periapical area 
and even the maxillofacial spaces (1). The use of regenerative endodontic techniques (RET) have 
gained popularity in the past decade with several endodontic and pediatric dentistry organization 
recognizing this technique as a viable technique for immature permanent teeth with necrotic 
pulps (2,3) 
Regenerative endodontics are defined as biologically based techniques planned 
to 
physiologically replace impaired tooth structure, including dentin and root constructions, as well 
as cells of the pulp–dentin complex (4). They offer the benefit of root lengthening, thickening, 
and subsequent apical closure which is not achievable with conventional nonsurgical endodontic 
treatment (NSET). The success rate of the blood clot (RET) in immature teeth has been reported 
to be 97.8% (5-7). 
 
The American Association of Endodontists, and European Society of Endodontology developed 
evidence based protocols. To help reducing some of the differences in the RET steps such as 
EDTA and sodium hypochlorite use, however, variations in terms of intracanal medicament, 
scaffold and assessment tools still exist due to the lack of high-quality evidence (8,9). 
A fundamental component of the regenerative endodontic process is the presence of a scaffold 
for stem cells from the apical papilla to adhere, multiply and differentiate. Based on the results of 
many studies, the use of blood clot Regenerative procedure is not considered a true scaffold 
system. But it is an essential part of any appropriately designed tissue engineering regenerative 
endodontic strategy (10-12). 
 
Different treatment options and scaffolds have been tested in regenerative endodontics. Only 
some of which exhibited clinical relevance such as blood clot, platelet rich plasma and platelet 
rich fibrin as RET scaffolds. Therefore, the use of suitable scaffold is an essential part of tissue 
engineering regenerative endodontic strategy (13-16). 
 
Hence, developing a new dental material that retains the proteins or factors that can stimulate 
regenerative processes close to the natural process seems promising (17). Tissue-derived 
extracellular matrices have recently been known as naturally-derived scaffolds for tissue 
regeneration in various applications and have been revealed to serve as a cultural substrate on 
which cells tend to adhere to and proliferate well and can induce regeneration specific to tissues 
and sites (18,19) 

4 
 
A new extracellular matrix material, demineralized dentin matrix (DDM) derived from extracted 
teeth has been found to act as a biocompatible scaffold for the attachment, differentiation, and 
proliferation of human DPSCs (dental pulp stem cell) into odontoblast-like cells (20). Dentine 
matrix has proposed roles for directing mineralised tissue repair in dentine and bone; however, 
the range of bioactive components in dentine and specific biological effects on bone-derived 
mesenchymal stem cells (MSCs) in humans are less well understood (21). 
DDM is autogenous tooth dentin that has osteoconductive and osteoinductive potential since it 
contains extracellular collagen-1 and various growth factors. DDM as a solid blocky material has 
less physical stability and sealing ability as a material for regenerative endodontics. Therefore it 
has been further fabricated into their forms of DDM powder, gel, and as sheets in order to 
maintain bioactivity (22). 
DDM can be prepared with low risks of infection and rejection with noninvasive attainability; 
thus, it should be considered as a natural resource to be used to full advantage for other 
applications (23). 
 
In 2022 a meta-analysis performed by ( Grawish et al., 2022)(23) indicated that DDM as a 
regenerative biomaterial has potential roles in tissue regeneration. However, in order to confirm 
these trials, further well-designed analyses are needed, and evaluation of adverse events in 
observational studies is also needed. 
 
To our knowledge, DDM has not been investigated as scaffold in regenerative endodontics for 
permanent teeth. 
 
Several studies were evaluate the regenerative effect of DDM at dentin-pulp complex through 
direct pulp capping technique, and it was found to possess the ability to activate the odontogenic 
differentiation of stem cells resident in the pulp tissues and induce reparative dentin formation. 
DDM also considered for alveolar ridge, maxillary sinus floor augmentations, socket 
preservation, furcation perforation repair, guided bone, and bioroot regenerations as well as bone 
and cartilage healing (23-27). 
Hence, this study will be designed to evaluate regenerative effect of Demineralized Dentin 
Matrix as a scaffold in comparison to conventional blood clot in immature permanent teeth. 
 
II. The null hypothesis 
The null hypothesis is that there are no significance differences in the clinical and radiographic 
outcomes of regenerative endodontic of non-vital immature permanent teeth following the use of 
DDM in comparison to a blood clot scaffold. 

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III. Aim of the study 
 
 
This study aims to: 
Compare the clinical and radiographic outcomes of demineralized dentin matrix scaffold to 
blood clot scaffold in immature permanent incisors with non-vital pulps for one year. 
 
 
. 
 
IV. PICOTS 
 
 
 
 
P: Patients ranging age 7–13years 
 
I: Demineralized Dentin Matrix as a scaffold in Regenerative endodontic procedure 
 
C: Blood clot scaffold Regenerative endodontic procedure 
 
O: Clinical and radiographic success. 
 
T: One year 
 
S: outpatient’s clinic of the Pediatric Dentistry and Dental Public Health Department, Faculty of 
Dentistry, Ain Shams University. 
 
V. Methodology: 
i. Study design 
 
 
A Randomized, Controlled trial with 1:1 allocation ratio 
 
ii. Sample size estimation 
 
A power analysis was designed to have adequate power to apply a two-sided statistical 
test of the null hypothesis that there is no difference would be found between different groups. 
By adopting an alpha level of (0.05) a beta of (0.2) i.e. power=80% and an effect size (d) of 

6 
 
(1.51) calculated based on the results of a previous study (25). the predicted sample size (n) was a 
total of (24) cases (i.e. 12 cases per group). Sample size was increased by 20% to compensate for 
possible dropouts during follow-up intervals to be (30) cases (i.e. 10 cases per group). Sample 
size calculation was performed using G*Power version 3.1.9.7(28) 
iii. Assignment of intervention: 
a. Randomization and Allocation concealment. 
 
1- Type: block Randomization, with blocks numbers of 10 and1:1:1allocation ratio in to 3 
groups Group I: DDM (study) 
Group II: (control) blood clot 
Group III: (+  control) blood clot with collagen 
 
2- Sequence generation: the most senior supervisor will generate the sequence of random 
numbers using online randomization (www.random.org). 
 
Allocation concealment and implementation: 
Allocation concealment will be ensured by preparing opaque envelopes containing one number 
from the generated random sequence for assigned group. The most senior supervisor will select 
an envelope and reveal the treatment group at the final regeneration visit. 
 
b.Blinding: 
double blind study, participants, outcome assessors, statistician will be blinded. 
iv. Study setting 
 
 
This study will be conducted at the Outpatients Clinic at the Department of Pediatric Dentistry 
and Dental Public Health, Faculty of Dentistry, Ain Shams University. 
ii. Study methodology 
 
 
ethical regulation: 
The study protocol will be submitted to the Research ethics committee, Faculty of Dentistry, Ain 
Shams University for approval. 

7 
 
v. Population 
All parent or legal guardian fulfill the eligible criteria will be signed an informed consent before 
beginning any procedure, explaining detailed treatment procedures, the possible outcomes, 
comprehensive discussions of the risks, complications, alternative treatment options, follow-up 
period needed, and sequel of no treatment (8-14). 
Inclusion criteria: 
(1) Age 7–13 years at the time of enrolment. 
(2) Provision of informed consent by one parent or legal guardian. 
(3) At least one immature permanent anterior tooth diagnosed with irreversible pulpitis or pulp 
necrosis with or without periapical lesions. 
(4) Restorable teeth. 
(5) Compliant patient/parent. 
(6) Patients not allergic to medicaments and antibiotics necessary to complete procedure (ASA 
1 or 2). 
(7)  Radiologic evidence of open apices (Teeth are considered immature when a minimum of 1 
mm apical foramen width is evident) 
 
Exclusion criteria 
(1) Teeth with root fractures or split roots. 
 
(2) Presence of root resorptions. 
 
(3) Presence of periodontal pockets. 
 
(4) Developmental anomalies 
 
(5) Presence of periapical radiolucency more than 10 mm 
 
(6) Tooth with class III mobility. 
 
vi. Clinical Procedures 
a. Screening Recruitment 
 
 
Patients attending the outpatient’s clinic of the Pediatric Dentistry and Dental Public Health 
Department, Ain Shams University will be screened for eligibility. Only patients who met the 
inclusion criteria and sign the informed consent will be conveniently enrolled in the study until 

8 
 
the predetermined sample size is reached. All examination procedures will be done by the same 
operator to ensure standardization. 
 
b. Informed Consent 
 
 
A signed informed consent from parent or legal guardian, and an oral assent to the child, as 
outlined by the Research Ethics Committee, Faculty of Dentistry, Ain Shams University will be 
obtained before the conduction of the study. 
. 
Grouping: 
 
Eligible immature anterior teeth will be randomly allocated in two groups: 
Group I: 10 teeth will be treated with demineralized dentin matrix as scaffold in regenerative 
endodontic procedure. 
Group II: 10 teeth will be treated with conventional regenerative endodontic procedure (blood 
clot scaffold). 
Group III: 10 teeth will be treated with regenerative endodontic procedure (blood clot and 
collagen scaffold). 
 
Clinical procedures: 
Pre-operative periapical radiograph and con beam computed tomography CBCT will be taken on 
the selected non-vital tooth in each child before the beginning of the regenerative endodontic 
treatment procedure (29, 30). 
RET will be carried out according to ASE(8) 
First Appointment 
 
• Local analgesia will be delivered (2% lidocaine with 1:100,000 adrenaline). 
• Following rubber dam isolation and access cavity preparation, pulp tissue 
extirpation/debridement 
• The root canal system will be minimally instrumented. 
• Copious irrigation will be performed, gently with 20ml NaOCl (1.5% NaOCl 
(20mL/canal, 5 min) using 0.3mm gauge single side vented needles, adjusted 3 mm short 
of the apex. 
• then canal will be irrigated with EDTA (20 mL/canal, 5 min), with irrigating needle that 
will be positioned about 1 mm shorter than the root end, to minimize cytotoxicity to stem 
cells in the apical tissues, followed by a final rinse 20 mL sterile saline . 
. 

9 
 
• Canals will be dried with paper points.

10 
 
double antibiotic paste, ciprofloxacin (Ciprocin 250mg tablets) and Metronidazole (Flagyl 500mg 
tablets) in a 1:1 ratio will be mixed with sterile distilled water in a 3:1 ratio, then will be introduced 
into the root canal systems using 18 G hypodermic needle and will be packed gently down the root 
canal using a hand plugger, ensuring that there will be no remains coronal to CEJ. 
 
• A sterile dry cotton pellet will be placed over the paste after which the tooth will be 
sealed with 3-4mm of a temporary restorative material. 
• Patient will be dismissed for 2week 
Second Appointment (two week after 1st visit) 
• Response to initial treatment will be assessed. If there are signs/symptoms of persistent 
infection, additional treatment time with antimicrobial. 
• Local Anesthesia without vasoconstrictor will be administered; dental dam isolation will be 
achieved. 
• Copious gentle irrigation will be performed with 20ml of 17% EDTA, followed by a final rinse 
20 mL sterile saline. 
• Canals will be dried with paper points. 
 
In group I: teeth will be treated with demineralized dentin matrix as scaffold in regenerative 
endodontic procedure 
Teeth, in this group, will be anaesthetised using a local anaesthetic without vasoconstrictor. 
Demineralized dented matrix will be introduced inside the root canal and bleeding will be 
created into root canal by over-instrumentation by rotating K-file at 2 mm past the apical 
foramen with the goal of having the entire canal filled with blood to the level of the cemento– 
enamel junction. 
A lightly moistened sterile cotton pellet will be placed into the canal, 3-4 mm apical to the CEJ, 
for 7-10 minutes to allow blood clot formation. 
Placement of the bioactive and restorative materials: A bioactive material such as tricalcium 
silicate will be placed over the blood clot as a capping material. A 3–4 mm layer of glass 
ionomer will be gently placed over the capping material. 
Followed by resin composite restoration (16). 
In group II: teeth will be treated with conventional regenerative endodontic procedure (blood clot 
scaffold). 

11 
 
Teeth, in this group, will be anaesthetised using a local anaesthetic without vasoconstrictor. 
Bleeding will be created into root canal by over-instrumentation 
A lightly moistened sterile cotton pellet will be placed into the canal, 3-4 mm apical to the CEJ, 
for 7-10 minutes to allow blood clot formation. 
Placement of the bioactive and restorative materials as mentioned above. 
In group III: teeth will be treated with conventional regenerative endodontic procedure (blood clot 
and collagen scaffold). 

12 
 
Teeth, in this group, will be anaesthetised using a local anaesthetic without vasoconstrictor. 
Bleeding will be created into root canal by over-instrumentation 
A collagen will be placed into the canal, apical to the CEJ. 
Placement of the bioactive and restorative materials as mentioned above 
e. Follow up 
Clinical and Radiographic examination will be carried out during follow up period (15, 16). 
1- Clinical evaluation: Treatment success will be assessed using following criteria of 
success. 
Criteria of success: Absence of 
 
• Pain 
• Soft tissue swelling 
• Soft tissue inflammation 
• Sinus tract 
• tenderness to percussion 
• tooth mobility 
2- Radiographic evaluation: 
A-Conventional radiographic evaluation: 
Criteria of success: 
• 
Apical foramen width reduction 
• Increased dentinal wall thickness 
• Increased root length 
• Conventional radiograph showing progress bone healing and presence of a normal 
periodontal ligament space. 
• Reduction in size of the lesion. 
B- CBCT evaluation: 
• CBC with limited field of view will be carried out at 12 month to asses dentin 
width and length(23, 28) 

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f. Outcome measurement 
 
 
 
Outcome 
assessment 
Assessment 
method 
Primary outcome 
1- pain 
2- pain percussion 
3- pathological mobility 
4-gingival becket 
1-visual assessment 
2-percussion test 
3-blunt ends of two 
examination mirrors 
4-graduated metal 
periodontal probe 
Secondary out com 
1- Root length 
2- Root dentinal thickness 
3- Apical foramen width 
4- Total radiographic root 
area (absence of internal 
and external root 
resorption, widening of 
periodontal ligament 
space, new preiabical 
radiolucencies or 
enlargement of pre- 
existing lesion, pulp 
canal calcification) 
Digital periapical radiograph 
examination with using 
paralleling device 
Secondary outcome 
Increased root wall thickness 
and increased root length. 
CBCT with limited field of 
view 

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VII. Statistical analysis 
Categorical data will be represented as frequency (n) and percentage (%) and will be analyzed 
using chi square test. Numerical data will be explored for normality by checking the data 
distribution, calculating the mean and median values and using Kolmogorov-Smirnov and 
Shapiro-Wilk tests. If the data was found to be normally distributed, it will be presented as mean 
and standard deviation values and paired t-test will be used for the analysis. If the assumption of 
normality was found to be violated; the data will be presented as median and range values and 
will be analyzed using Signed rank test. The significance level will be set at p ≤0.05 for all tests. 
Statistical analysis will be performed with IBM SPSS Statistics Version 26 for Windows. 
VIII. Study timeline 
 
This study will be conducted from October 2023 to October 2024. 
X. Patient withdrawal 
 
 
- 
Patients who aren’t willing to continue in the study will have the right to quit at any time 
without loss of any benefits. 
- 
Patients who don't comply with the oral hygiene measures given will be excluded from the 
study and will be offered to continue dental treatment at the departments’ outpatient clinic. 
- 
Any adverse event will be recorded and managed promptly. In addition, in failure occurs, 
the use of the experimental material will be stopped and patients will be treated with 
another modality. 
xi. Data management 
 
 
Patient information will be guarded as confidential information that should never be revealed at 
all times. 
All information will be kept as a hard and soft copy. 
xii. Adverse event reporting 
 
 
Regenerative treatment is not expected to have any adverse side effects on health but if failure 
occurs in the regenerative treatment, conventional regenerative endodontic treatment will be 
fabricated. 

15 
 
Random allocation of immature 
teeth in two groups 
Clinical assessments at 
3, 6, 9 and 12 months 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Schematic presentation for the study design 
Signing of informed consent by caregivers. 
Selection of the study sample, following the inclusion 
criteria 
Group I: 10 teeth will 
be treated with (DDM 
scaffold). 
Group II: 10 teeth 
will be treated with 
(blood 
clot 
scaffold). 

16 
 
xiii. Ethics and dissemination 
 
 
a. Research ethics approval: 
 
 
This protocol and the template informed consent form contained in Appendix are written 
following the guidelines outlined by the Research Ethics Committee and Institutional Review 
Board, Faculty of Dentistry, Ain Shams University. 
b. Protocol amendments: 
 
 
In case of protocol modifications, the Research Ethics Committee and Institutional Review 
Board, Faculty of Dentistry, Ain Shams University will review and approve the changes of 
protocol before proceeding and implementation of changes. 
c. Informed consent: 
 
 
A verbal as well as a written consent will be obtained from the caregivers of the participants after 
explaining the objectives of the study and assuring complete confidentiality of data. All 
caregivers and participants will be informed that they have the right to withdraw from the study 
at any time. Moreover, participants 6 years and older will be requested to sign an assent form 
after an age appropriate explanation of the trial procedures and their objectives. All consent and 
assent forms are translated into Arabic (APPENDIX I). 
 
 
d. Confidentiality 
 
 
All study-related information will be stored securely at the study site. Personal information, 
consent forms of enrolled participants will be collected by the primary investigator (candidate) 
and stored in hard copy examination sheets in locked file cabinets with limited access. All local 
databases will be secured on password-protected computers. Patients’ identity will not be shared 
at any stage of the study. Participants receiving treatment will be given codes (identification 
numbers) for statistical analysis. 

17 
 
e. Access to data: 
 
 
Primary investigator, supervisors and biostatistician will have access to the final trial dataset. 
 
 
f. Post-trial care 
 
 
All patients with premature loss of teeth in the patients’ mouth will be treated with the proper 
treatment protocol. According to our knowledge, no side effects have been reported in literature 
from using both materials under study in patients with non-vital immature permanent teeth. 
 
 
g. Dissemination policy 
 
 
After completion of the study, trial results will be communicated to participants – if interested- 
via phone calls. Results, as well as some radiographs and clinical pictures of treated teeth, will be 
available for healthcare professionals and the public via publications. 
Guidelines for authorship will follow the four criteria stated by the International Committee of 
Medical  Journal  Editors  ―ICMJE‖.  Contributors  meeting  less  than  the  four  criteria  will  be 
acknowledged. 
h. Declaration of interest 
 
 
The investigators declare no conflict of interest. 

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