Regenerative Potential of Mature Teeth with Necrotic Pulp ... · • Preparation of the animal...

Regenerative Potential of Mature Teeth with Necrotic

Pulp and Apical Periodontitis

Thesis Submitted to Endodontic department Faculty of dentistry,

Ain Shams University In partial fulfillment of the requirements of Doctorate degree in Endodontics

BY

Hisham Mahmoud Hamdy Abada

B.D.S (2008) M.S.C (2015)

Faculty of Dentistry, Tanta University

2019

Supervised by

Dr. Ahmed Abdel Rahman Hashem Professor of Endodontics Faculty of Dentistry

Ain Shams University

Dr. Ashraf Mohamed Abdel Rahman Abu-Seida

Professor of Surgery, Anesthesiology and Radiology Faculty of Veterinary Medicine

Cairo University

Dr. Mohamed Mokhtar Nagy Associated Professor of Endodontics Faculty of Dentistry

Ain Shams University

Acknowledgements First of all, thanks to ALLAH for helping me to

complete this work.

I would like to express gratitude to Dr. Ahmed Abdel Rahman Hashem, professor of Endodontics ,Endodontic Department, Faculty of Dentistry, Ain shams University, for his valuable support, encouragement, supervision and useful suggestions through this research work. His moral support and continuous guidance enabled me to complete my work successfully.

My sincere thanks to Dr. Ashraf Mohamed Abdel Rahman Abu-Seida, Professor of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Cairo University, for his precious advices, wise opinions, cooperation, and continuous effort that paved the way for completing this work.

I am deeply grateful to Dr. Mohamed Mokhtar Nagy, Associated Professor of Endodontics, Faculty of Dentistry, Ain shams University, for her great support, sincere supervision efforts, giving me of her precious time and also for her brilliant comments and suggestions.

Last but not least, I would like to thank all my

professors, staff members and colleagues of the Endodontic Department for their spiritual encouragement and support.

Dedication

To my great Mother, whose shoulder is always there and without her encouragement,

this work could not be possible.

Full appreciation to my Father, who I always owe him for his usual support and

collaboration.

For my sister and brother, I express my gratefulness for their ongoing backup and

endorsement.

a

List of Contents

Subject Page I. Introduction. 1 II. Review. 3

II.1. Conventional endodontic therapy. 4 II.2. Regenerative endodontic therapy. 15

III. Aim of the work. 34 IV. Materials and methods. 35

• Animal models: 35 • Preparation of the animal model: 35 • Classification of samples 36 • Operative procedure 38 - Induction of periapical pathosis 38 - Root Canal preparation and

disinfection 39

- Treatment modalities: 39 o Root canal obturation. 40 o Regenerative endodontic

therapy. 41

• Tissue Preparation and Histologic Analysis 41

• Histopathologic evaluation: 42 - Inflammatory cell count: 42 - Bone resorption 42 - Presence or absence of vital

tissues inside the pulp space 42

- New hard tissue formation 43 • Statistical analysis 43

V. Results. 49 • Inflammatory cell counts 49

b

• Bone resorption. 53 • Nature and extent of tissue in-growth

in the pulp space. 58

• Formation of new hard tissue 62 VI. Discussion 67 VII. Summary and conclusion. 78 VIII. References. 81

Arabic summary

c

List of Tables

Table Table Title Page 1 showing mean inflammatory cell

count for all groups.

50

2 Score percentage of bone resorption among groups at two evaluation periods.

54

3 Score percentage of tissue in-growth among groups at two evaluation periods.

59

4 Score percentage of new hard tissue formation among groups at two evaluation periods

64

d

List of Figures

Figure Figure Title Page 1 Schematic diagram for samples

classification 37

2 Administration of general anesthesia 44 3 Pre-operative radiograph 44 4 Access opening 45 5 Working length determination 45 6 Root canal obturation. 46 7 Blood clot formation. 46 8 MTA coronal plug 47 9 Glass ionomer restoration 47

10 Radiograph representing RCT 48 11 Radiograph representing RET 48 12 Bar graph showing inflammatory cell

count among all groups at two time period

50

13 Photomicrograph showing sever inflammatory cell infiltration positive group after 1 month

51

14 Photomicrograph showing mild inflammatory cell infiltration negative group after 1 month (X400 magnification).

52

15 Photomicrograph showing moderate inflammatory cell infiltration RCT group after 3 month (X400 magnification).

52

16 Photomicrograph showing apical root resorption (score 1) positive group after 3 months evaluation period

56

17 Photomicrograph showing no apical root resorption (score 0) RCT group after 3 months evaluation period

57

e

18 Histogram representing percentage of bone resorption score for all groups at two evaluation period.

57

19 Photomicrograph showing tissue in-growth inside root canal at X40 magnification.

60

20 Histogram representing percentage of tissue in-growth score for all groups at two evaluation period

61

21 Photomicrograph showing nature of the tissue in-growth inside root canal.

61

22 Histogram representing percentage of new hard tissue formation score for all groups at two evaluation period.

65

23 Photomicrograph showing nature of the tissue in-growth inside root canal.

65

24 Photomicrograph intra-canal dystrophic calcification at 200X magnification

66

Introduction

Introduction

1

There are many challenge to perform a proper endodontic treatment include different root canal morphology, presence of extra canals, lateral and accessory canals. Also, root canal obturation is an exacting technique, demanding rigorous training and clinical skills. Each of the obturation technique and system (obturation equipments) require canals to be prepared accordingly. A large inventory of obturation systems and materials is required to obturate different canal systems; use of all these material also involves safety and biocompatibility with the tissues. All these factors put increased demand on operator's skill and time and finally increase the cost of treatment. All the above drawbacks are overcome by the new treatment approach proposed, i.e. achieving a biological seal rather than mechanical seal obtained by artificial obturating materials. A non-obturation method (regenerative endodontic treatment) of endodontic treatment of non-vital, infected teeth is highly desirable. It is highly cost saving and easier to perform and it will greatly simplify the endodontic treatment, the healing response after regeneration therapy in the apical third of the root canal had better resistance to infection and is advantageous over inert root filling materials, in addition to the disinfected canals are filled with vital biological tissue instead of nonvital, biocompatible obturating materials. In spite of these tissues are not true pulp tissue, they would those host’s identity or fundamental tissue, which will be inherited for immune defense mechanisms against a new starting foreign invaders.

The regenerative endodontic procedure was limited to immature teeth, but recently it extended to mature teeth as an alternative to conventional endodontic treatment, the

Introduction

2

ideal apical size diameter that permits regenerative endodontic procedure for mature teeth is still debates. There are few studies that compare apical size diameter for regenerative endodontic therapy, and also there is no study comparing histological assessment between regenerative endodontic therapy and conventional endodontic therapy.

Review

Review

3

If the pulpal vascular system becomes dysfunctional, pulpal infection typically progresses and causes pulpal disease (ranging from reversible to irreversible pulpitis), which will eventually lead to total necrosis, depending on how host defense mechanisms can cope with the increased numbers of (virulent) bacteria and their products. If left untreated, pulpal disease will spread beyond the apex of the tooth leading to periapical disease. Initially, only the periodontal ligament will be involved in the periapical reaction. However, resorption of cementum (and dentin) and breakdown of the alveolar bone ensues and all periodontal tissues end up affected.(1)

Apical periodontitis is an inflammatory lesion in the periodontal tissues that is caused mostly by bacterial elements derived from the infected root canal system of teeth (2). In non-treated teeth apical periodontitis represents a defensive response to a primary infection in a necrotic pulp.(3)

The primary objective of endodontic therapy should be to restore involved teeth to a state of normalcy nonsurgically. Different nonsurgical management techniques, namely, conservative root canal therapy, decompression technique, method using calcium hydroxide, aspiration-irrigation technique, lesion sterilization and tissue repair therapy, active nonsurgical decompression technique, and the apexum procedure have been advocated. New techniques which use drug-loaded injectable scaffolds, simvastatin, and epigallocatechin-3-gallate have been tried(4).

Review

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1- Conventional Endodontic Therapy:

Root canal treatment (RCT) may be defined as the combination of mechanical instrumentation of root canal system, its chemical debridement and filling with an inert material, designed to maintain or restore the health of the periradicular tissues. The manner of execution of treatment procedure(s) is so diverse even within prescribed protocols that it is difficult to define it any more precisely and it is accepted that this treatment intervention is not by its nature standardizable. In fact, the procedure is dominated by the tooth in question, in terms of its anatomical complexity and biological status, that is, its pre-operative status. The latter part of the definition alludes to the fact that essentially the same procedure is used to treat two distinct disease entities: (i) the vital but diseased pulp where the goal is to maintain existing periapical health and prevent periapical disease; or (ii) the nonvital or dying pulp associated with periapical disease where the goal is to restore the periradicular tissues back to health. (5)

Seltzer, et al (6) uncovered the relationships which might account for endodontic treatment failures. Teeth which had been treated by experienced endodontists and in which endodontic treatment was deemed to have failed were studied. Cases for which well-documented histories and roentgenograms were not available were excluded from the study. One hundred forty-six such failures were evaluated. Among those forty-one teeth were extracted. Complete root amputation and/or hemisections were performed on another fifteen teeth. Serial sections of the extracted teeth or roots were examined in an effort to find possible morphologic

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reasons for the failures. Periapical curettages were performed on eighty-seven teeth. Serial sections of the resected tissues were examined and classified as “radicular cyst,” “granuloma,” or “other lesions.” The findings and pathologic diagnoses were recorded on charts; after that, the clinical histories and roentgenograms were evaluated and the information was tabulated. Conclusions reached from analyses of these data must be tempered with the realization that any discernible patterns may not be characteristically representative of all endodontic treatment failures.

Katebzadeh, et al (7) analyzed histological periapical healing of infected roots obturated in one-step or with prior calcium hydroxide (Ca(OH)2) disinfection was compared. Seventy-two roots of vital dog teeth were instrumented to ISO size 45. Sixty roots were infected with dental plaque and closed. Six weeks later, apical periodontitis was radiographically confirmed in the infected roots. The teeth were divided into the following groups: group 1, one-step (n = 24) roots were irrigated with 10 ml of saline, obturated, and permanently restored; group 2, Ca(OH)2 (n = 24) roots were treated as in group 1, except that after saline irrigation, Ca(OH)2 dressing was placed in the canal for one weak before obturation; group 3, positive control (n = 12) the roots were irrigated with saline, but the canals were not obturated; and an additional group, group 4, served as a negative control (n = 12) these teeth that were not infected with plaque were aseptically obturated. The dogs were sacrificed after 6 months. The roots and surrounding apical tissues were prepared and histologically examined by two independent evaluators blinded to the treatment groups. A two-way ANOVA test demonstrated that the four treatment

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groups were significantly different from one another. The positive control showed the most inflammation, the negative control the least, and the Ca(OH)2 group had significantly less inflammation than the one-step group (p < 0.05). It is concluded that Ca(OH)2 disinfection before obturation of infected root canals results in significantly less periapical inflammation than obturation alone.

Sabeti, et al (8) compared the healing of instrumented and obturated versus instrumented and nonobturated root canal systems with apical periodontitis. Fifty-six root canals in 28 third and fourth bilateral lower premolar teeth with completely formed apices in seven mixed German Shepherd dogs were used. Apical lesions were created by accessing the canals, removing the pulp and leaving them open to the oral environment for 42 days. The teeth in the control group were instrumented, irrigated, and then obturated using a lateral condensation technique with gutta-percha cones and AH26 Plus as the endodontic sealer. The teeth in the experimental group were instrumented and irrigated without obturation. All teeth in both groups were sealed coronally. After 190 days, the animals were euthanized. The control group showed less cementum and dentin resorption in comparison with test group (p < 0.5). No statiscally significant difference was found in other parameters between the control and test groups (p<0.05). The noteworthy finding of this study was that there was no difference in healing of apical periodontitis between the instrumented and obturated and instrumented and nonobturated root canal system. The success of endodontic treatment ultimately depends on the elimination of the microorganism, host response and mechanical closure

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(coronal seal) of treated root canals that may provide a potential for future bacterial contamination

Ricucci, et al (9) compared the presence or absence of a radiopaque lamina of 60 human periapical lesions with the histological findings from a case study in dental practice. Paralleling radiographs were taken of 60 teeth with periapical radiolucencies and evaluated on a computer screen (13 inch) according to agreed criteria by 2 trained observers. They recorded the presence or absence of a radiopaque lamina on periapical lesions. The radiological examination was performed blind to the results of the histological evaluation. The specimens for histological examination were obtained from the extraction of 13 endodontically treated teeth and 27 nontreated teeth, as well as 20 post-treatment teeth with apical periodontitis removed during periapical surgical procedure. The specimens were classified, according to agreed criteria, as periapical abscess, granuloma, or cyst (true or pocket). The findings of the radiological and histological evaluations were compared. The results concluded that the diagnosis of periapical lesions cannot be made on the basis of the presence or absence of a radiopaque lamina, but requires histological examination of serial sections.

Leonardo, et al (10) compared the periapical repair of teeth with periapical lesion following root canal treatment by using a calcium hydroxide-based intracanal dressing for several time periods or filling in a single visit. After induction of periapical lesions in 4 dogs, the root canals were prepared using 5.25% sodium hypochlorite for irrigation, and animals were separated into 4 experimental groups; in

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group I, root canals were filled in a single session; in groups II, III, and IV, a calcium hydroxide-based dressing was kept in place for 15, 30, or 180 days, respectively. Root canals from groups I, II, and III were filled with gutta-percha cones and AH Plus sealer. After 180 days, animals were killed and histological sections were stained with hematoxylin-eosin to evaluate periapical repair. Periapical repair was better in groups II, III, and IV (intracanal dressing) compared with group I (single session; P< .05).

Maria, et al (11) evaluated histopathologically different methods of experimental induction of periapical periodontitis. Fifty-seven root canals from dogs’ teeth were assigned to 4 groups. In GI (n=14) and GII (n=14), the root canals were exposed to oral environment for 180 days; in GIII (n=14) and GIV (n=15) the root canals were exposed for 7 days and then the access cavities were restored and remained sealed for 53 days. The root apices of GI and GIII were perforated, whilst those of GII and GIV remained intact. After induction of periapical periodontitis, the dogs were euthanized. Serial sections were obtained and stained with hematoxylin and eosin. Data of the histopathological evaluation were submitted to Kruskal-Wallis and Dunn’s tests at 5% significance level. The inflammatory periapical reaction and resorption of mineralized tissues were less intense in GII than in the other groups (p<0.05). There was no histopathological difference among the experimentally induced periapical lesions in the teeth with coronal sealing. On the other hand, when coronal sealing was not performed, greater intensity of induced periapical periodontitis was observed in the teeth with apical perforation.

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9

Ricucci, et al (12) evaluated the pulp healing process and the dentin-cementum complex in 51 endodontically treated human teeth after long observation periods and to correlate histologic observations with conventional periapical radiographic findings. The examined teeth were extracted either because the tooth was unrestorable due to extensive caries or fracture or because the root fillings had been exposed to oral environment for some time and the patient did not wish to undergo further treatment. The periapical conditions of the teeth were radiographically evaluated, immediately after extraction; the teeth were processed for histological evaluation. The results showed that in the majority of the cases, complete healing was observed, with no signs of acute or chronic inflammatory processes in the remaining apical tissue or periodontal tissue fragments. Some cases showed moderate inflammation in the root canal tissue. Debris intermixed with necrotic tissue and a sealer particle was a common finding in the pulp stump. Bacteria were present in the coronal portion of the root in almost all cases, but in only 1 case could bacteria be demonstrated in the coronal and apical portions of the root. This study concluded that apical tissue of properly treated teeth with no signs of periapical changes is only rarely significantly inflamed. When the tissue is inflamed, microbial causes can always be demonstrated. Despite the presence of microorganisms coronally in nearly all cases, apical tissue is seldom affected.

De Paula-Silva (13) evaluated the periapical repair after root canal treatment in the teeth of dogs using CT and conventional radiography and to compare these findings with the gold standard microscopic evaluation. The animals

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were divided into three groups according to endodontic treatment performed: Group 1, single-visit endodontic treatment in teeth without apical periodontitis; Group 2, single-visit endodontic treatment in teeth with apical periodontitis; and Group 3, endodontic treatment in teeth with apical periodontitis using calcium hydroxide as a root canal dressing. Group 4 consisted of teeth with apical periodontitis not submitted to root canal treatment and Group 5 consisted of healthy teeth without periapical disease. Radiographic, tomographic, and microscopic evaluations were performed by blind examiners. At 180 days experimental time, CT and radiographic measurements of periapical disease were compared with the gold standard microscopic measurement using intraclass correlation coefficient. Intergroup comparisons considering different methods of periapical lesions measurement or different clinical protocols of root canal treatment were performed by Kruskal Wallis test followed by Dunn. Integrity of lamina dura, presence of radiolucent areas, and presence of root resorption were analyzed by Fisher’s exact test. This study concluded that CT Scan evaluation of periapical repair following root canal treatment provided similar information than that obtained by microscopic analysis, whereas radiographic evaluation underestimated the size do periapical lesion.

Ricucci, et al (14) evaluated the prevalence of bacterial biofilms in untreated and treated root canals of teeth evincing apical periodontitis. Biopsy specimens from 106 (64 untreated and 42 treated) roots of teeth with apical periodontitis. Specimens were obtained by apical surgery or extraction and were processed for histopathologic and

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histobacteriologic techniques. The results of this study showed that intraradicular biofilm arrangements were observed in the apical segment of 77% of the root canals (untreated canals: 80%; treated canals: 74%). Biofilms were also seen covering the walls of ramifications and isthmuses. Bacterial biofilms were visualized in 62% and 82% of the root canals of teeth with small and large radiographic lesions, respectively.

Borlina, et al (15) evaluated the influence of apical foramen widening on the healing of chronic periapical lesions in dogs teeth after root canal filling with Sealer 26 or Endomethasone. Forty root canals of dogs teeth were used. After pulp extirpation, the canals were exposed to the oral cavity for 180 days for induction of periapical lesions, and then instrumented up to a size 55 K-file at the apical cemental barrier. In 20 roots, the cemental canal was penetrated and widened up to a size 25 K-file; in the other 20 roots, the cemental canal was preserved (no apical foramen widening). All canals received a calcium hydroxide intracanal dressing for 21 days and were filled with gutta-percha and 1 of the 2 sealers: group 1: Sealer 26/apical foramen widening; group 2: Sealer 26/no apical foramen widening; group 3: Endomethasone/apical foramen widening; group 4: Endomethasone/no apical foramen widening. The animals were killed after 180 days, histomorphologic analysis were performed for experimental teeth. The results showed that significantly better periapical healing was obtained when foramen widening was done and Sealer 26 was used.

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Silva, et al (16) evaluated the in vivo response of apical and periapical tissues of dogs teeth with apical periodontitis after one-session endodontic treatment with and without antimicrobial photodynamic therapy (aPDT). Sixty root canals with experimentally induced apical periodontitis were instrumented and assigned to 4 groups receiving aPDT and root canal filling (RCF) or not: group aPDT+/RCF+ (n = 20): aPDT (photosensitizer phenothiazine chloride at 10 mg/mL for 3 minutes and diode laser [λ = 660 nm, 60 mW/cm(2)] for 1 minute) and RCF in the same session; group aPDT+/RCF- (n = 10); group aPDT-/RCF+ (n = 20), and group aPDT-/RCF- (n = 10). Teeth were restored, and the animals were killed after 90 days. Results showed that In the aPDT-treated groups, the periapical region was moderately/severely enlarged with no inflammatory cells, moderate neoangiogenesis and fibrogenesis, and the smallest periapical lesions. Conclusions: Although apical closure by mineralized tissue deposition was not achieved, the absence of inflammatory cells, moderate neoangiogenesis, and fibrogenesis in the periapical region in the groups treated with aPDT indicate that this can be a promising adjunct therapy to cleaning and shaping procedures in teeth with apical periodontitis undergoing one-session endodontic treatment.

Cohenca et al (17) evaluated the efficacy of apical negative pressure irrigation (ANP), passive ultrasonic irrigation (PUI) and positive pressure irrigation (PP) in the reduction of intracanal bacteria of dogs teeth with pulp necrosis and apical periodontitis. Eighty root canals were randomly distributed into 3 experimental and 2 control groups according to the irrigation delivery system: group

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ANP (n=20), group PUI (n=20), group PP (n=20), group PC (positive control – sterile saline irrigation; n=10) and group NC (negative control - vital pulps not subjected to bacterial inoculation; n=10). The first sample (S1) was collected at baseline, and the second sample (S2) was collected after the disinfection protocols. All samples were seeded in culture media for anaerobic bacteria. CFU counts were statistically analyzed. Results of this study showed that experimental groups were effective in reducing Gram-positive bacteria compared with PC (p<0.05). Regarding the reduction of Gram-negative bacteria, group ANP was significantly better than PP (p<0.05). No statistically significant difference could be found between PP and PUI (p>0.05). In dog’s teeth with apical periodontitis, the use of ANP and PUI can be considered promising disinfection protocols as both delivery systems promoted a significant bacterial reduction.

López et al (18) investigated the correlation and the agreement between periapical radiography (PR) and cone-beam computed tomography (CBCT) correlating to histologic findings in the diagnosis of apical periodontitis (AP), One hundred thirty-four premolar root canals from 10 dogs were treated after AP induction. Four months later, the animals were killed, and standard digital PRs were obtained. The area of AP was measured by using ImageJ software. CBCT (i-CAT) images from each arch were obtained, and AP area and volume were measured by using Osiri-X software. The apical inflammatory infiltrate was evaluated under light microscopy. The correlation between imaging methods was evaluated by using the Pearson coefficient. The Bland-Altman method was used to assess the agreement between PR and CBCT data. The Spearman coefficient was

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used to correlate the imaging data and histologic findings. This study concluded that the diagnosis of AP based on PR data is clinically limited, and it should not be used for scientific investigations.

López et al (19) evaluated histologically the effect of irrigation with 400 ppm Sterilox, 2% and 5% sodium hypochlorite (NaOCl), with and without photo-activated therapy (PAD), in a single-visit root canal treatment of dog’s teeth with apical periodontitis (AP). Ten dogs were randomly divided into two groups (n=5): with and without PAD, and the root canals into four subgroups, according to the irrigating solution: SX (400 ppm Sterilox), SH2 (2% NaOCl), SH5 (5% NaOCl) and SS (saline solution) as positive control. A total of 134 root canals were opened and left exposed to the oral environment for 14 days and then sealed for 60 days for AP induction. Then, root canals were treated according to each proposed disinfecting protocol and filled in the same session. After 120 days, the dogs were euthanized and the periapical inflammatory events were evaluated under light microscopy. This study showed that PAD did not show any additional effect for the treatment of root canals with pulp necrosis and AP in a single visit and 400 ppm Sterilox may be considered an alternative to NaOCl in root canal treatment.

Hidalgo et al (20) evaluated one-session endodontic treatment with aPDT and two-session treatment with calcium hydroxide (CH)-based dressing in dog’s teeth with apical periodontitis. After experimental induction of apical periodontitis, 48 teeth were randomly assigned to the following groups: groups OS/ aPDT120d and OS/aPDT180d

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(one-session treatment with aPDT) and groups TS/CH120d and TS/CH180d (two-session treatment with CH-based dressing—control groups). The animals were euthanized after 120 and 180 days. After histotechnical processing, microscopic and radiographic analyses were performed. The results using aPDT were worse than those obtained with two-session endodontic treatment using a CH-based dressing in teeth with apical periodontitis.

Trindade, et al (21) studied histopathological, microbiological, and radiographic analysis for the treatment of teeth with apical periodontitis in rats molars, root canals were assigned to four groups: one session photodynamic therapy PDT- [chemomechanical preparation (CMP)+root canal filling (RCF)]; two sessions/PDT- [CMP+calcium hydroxide (CH) for 14 days+RCF]; one session/PDT+ [CMP+PDT+RCF], and two sessions/ PDT+ [CMP+PDT+CH for 14 days+RCF]. The histological study revealed that lower scores for neutrophils/eosinophils in PDT-treated groups and macrophages/giant cells in calcium hydroxide groups. This study concluded that PDT may be indicated as an adjunct to conventional endodontic therapy for teeth with apical periodontitis, in association with an interappointment dressing with CH, in an attempt to produce better conditions to stimulate repair.

2- Regenerative Endodontic Therapy:

Non-vital infected mature teeth have long been treated with root canal therapy or doomed to extraction; this current treatment fails to reestablish healthy vital pulp tissue in these teeth. Regenerative endodontics is a field focus on replacing

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unhealthy infected pulp with new vital pulp tissue including pulp-dentin complex and it depend on tissue engineering and revitalization procedures. (22).

The Contrast between REPs over nonsurgical root canal therapy will be that the disinfected canals are filled with vital biological tissue instead of nonvital, biocompatible obturating materials. In spite of these tissues are not true pulp tissue, they would those host’s identity or fundamental tissue, which will be inherited for immune defense mechanisms against a new starting foreign invaders. (23).

Clinical REPs have been centered on immature necrotic teeth since they are expected to have a more prominent possibility of pulp tissue regeneration on the basis of the following biological reasons: Open apices of immature teeth permit more progenitor/stem cells to migrate into root canals space. Furthermore, immature root apices have stem cells of the apical papilla (SCAP) with a great regeneration endodontics potential (24,25). Clinically, regenerative endodontic therapy of immature roots cause thickening and lengthening of the roots that strengthen immature teeth.(26)

Regenerative endodontic procedures should not be restricted only to immature teeth but should be broadened to mature teeth as an alternative to conventional endodontic treatment to reconstitute the neurovascular system in root canals with an immune system that protect the pulp from further damage (27), but it will probably experience a greater number of difficulties than in immature teeth as there is less stem/progenitor cells and narrower apical diameter for these

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cell to migrate toward the root canals in mature teeth, in conjunction with disinfecting the root canals in mature teeth are more difficult (28), and tooth discoloration is reported as unfavorable results after REPs (29). Also, retreatment of mature necrotic teeth after attempted REP may be a problem because of the risk of root perforation while removing the MTA plugs (30).

Nygaard-Østby (31) tested the hypothesis that the presence of a blood clot within a root canal system promotes healing in experimental histologic study, mature teeth diagnosed with either vital or necrotic pulp received root canal therapy followed by foraminal enlargement, medicament dressing for the necrotic cases, evoked intracanal bleeding, and a kloroperka obturation placed coronal to the formed blood clot. Patients (n = 17) were followed for various time periods (17 days to 3.5 years), and then the treated tooth was extracted and the newly formed tissues were examined histologically. The outcomes were similar for all teeth: (i) resolution of symptoms of inflammation related to foraminal enlargement and over-instrumentation in as early as 17 days; (ii) resolution of signs and symptoms of pathosis for the necrotic cases; and, in certain cases, (iii) radiographic evidence of apical closure. From the histological analysis, it was observed that there was ingrowth of connective tissue into the canal space and varied levels of mineralized tissue along the canal walls as well as “islands” of mineralized tissue embedded within the newly formed tissue. This general finding was quite promising. However, the histological evidence of cementoblasts formation and absence of odontoblasts formation indicate that despite the inadequacies, this pioneer study established

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the framework for the subsequent studies in the regenerative endodontics field.

Hørsted and Nygaard-Östby (32), evaluated the tissue formation in the root canal after total pulpectomy and partial root filling. 20 human teeth were used in this study by removal of the pulp tissue and obturate the root canals using gutta- percha points and Kloroperka N-ö as a sealer 2 to 4 mm shorter from the foramen in single visit. After follow up periods 2, 6 and 10 months, the teeth were extracted and examined histologically. After 2 months, microscopic evaluation showed that connective tissue was occupy the space in the canals between the root filling and the foramen with a slight inflammation adjacent to the root filling. After 6 and 10 months, fully developed cell-rich fibrous tissue was fill the entire space and only a few lymphocytes were observed at the area close to the root filling material.

Annjka Skoglund (33) studied whether intentional fracturing of the root tip at the time of replantation or autotransplantation of mature teeth might improve the revascularization capacity of the pulp. The vascular system in apicoectomized and nonapicoectomized replanted and autotransplanted mature dog teeth was studied 10, 30 and 120 days postoperatively by means of a barium sulfate injection medium combined with microradiography. Pulpal revascularization occurred during a 120-day period in eight out of nine apicoectomized teeth. The pulp of the nonapicoectomized teeth did not revascularize.

Annjka Skoglund (34) evaluated the possibilities for rapid revascularization and original pulp tissue survival in replanted and auto transplanted mature dog teeth by

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increasing the contact area between the pulp and the vascularized periradicular tissues. In 12 teeth, the root tip was resected and 3 additional nutritional canals were prepared in the apical 1/3 of the root at the time of the operation. Another 12teeth were replanted or auto transplanted with intact roots. After an observation time of 120 days, the teeth were studied with microangiographic and histologic techniques. 11 of the teeth that were replanted or autotransplanted with intact roots showed total pulpal necrosis. Partial or total pulpal revascularization and repair was seen in all apicoectomized teeth with nutritional canals. The revascularized area of the pulp was somewhat larger in the replanted compared to the auto transplanted group of teeth. Signs of survival of the original pulp tissue were not seen. A comparison of the findings with those of previous studies on replanted and autotransplanted teeth with apicoectomized roots but without additional canals shows that the revascularization of the pulp did not proceed more rapidly in the apicoectomized teeth in the present study, although the contact area between the pulp and the periradicular tissues was enlarged through the preparation of the 3 artificial nutritional canals.

Kling et al. (35), investigate the frequency of pulp regeneration in reimplanted incisors and is its relationship with the apical foramen width, duration of extra-alveolar time, storage conditions and postoperative administration of antibiotics. The pulp was regenerated in 13 teeth from 72 immature teeth with apical foramen width 1.1–5.0 mm used in this study (18%), while no revascularization occurred in 88 mature teeth with apical foramen width 1.0 mm or less. Statistical analysis among parameters tested in immature

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teeth showed that there is a significantly increase in revascularization process in teeth reimplanted after 45 minutes extra-alveolar time. (p < 0.05) when compared with teeth reimplanted more than this time after avulsion. All teeth in which revascularization did not occur exhibited a periapical radiolucency and/or external inflammatory root resorption.

Shah and Logani (36) evaluated management of pulp and periapiacally involved teeth using non-surgical endodontic treatment protocol, based on "regenerative concept". 18 patients were selected in this study with signs and symptoms of pulp and periapical disease. After complete chemomechanical debridement and foramen widening of the teeth, bleeding were induced in the apical region to fill the canals space, and calcium-sulphate based cement was used to form orifice plug and the access cavities were sealed with a permanent restoration. The patients were followed-up clinically and radiographically after 6 months. 3 patients with 6 treated teeth were evaluated pre and post treatment using CBCT. The result of this study demonstrated that regenerative endodontic therapy was found to be effective clinically and radiographically in treatment of periapical infection.

Zhu et al. (37) used mature permanent dogs teeth to test the ability of dental pulp stem cells (DPSCs) and platelet-rich plasma (PRP) to regenerate dental pulp. After pulpectomy and apical foramen enlargement to #80 file, teeth were randomly divided into four experimental groups: (1) the blood clot group, (2) the autologous DPSCs group, (3) the PRP group, and (4) the DP + PRP group (a mixture

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of DPSCs and PRP). Four lower premolars with no further treatment after pulpectomy were utilized as the control group. MTA and composite was used to seal all teeth, and after 12 weeks from transplantation teeth were examined radiographically and histologically. The regenerated vital tissues was formed in 24 teeth from 32 experimental root canals, the histological examination demonstrated the formation of cementum and periodontal ligament–like tissues along the internal root canals walls in most cases. There is no significant difference between autologous DPSC transplantation group and other groups. Histologically, regenerative endodontics can be performed in permanent teeth after pulpectomy and apical foramen enlargement and this regeneration did not be enhanced by transplantation of DPSCs and/or PRP into root canals in compare to inducement of a blood clot formation alone.

Laureys et al. (38) evaluated the relation between apical foramen width and tissue regeneration in root canals after replantation. 3 adult beagle dogs with mature teeth were used. 15 single rooted teeth were extracted and underwent surgery for apicoectomy. The apical side was photographed to determine the foramen width (ranged from 0.24–1.09 mm.), then the teeth were replanted in infraocclusion. After evaluation period of 90 days, the teeth were histologically assessed. Smallest apical diameter teeth ranging between 0.24 and 0.53 mm demonstrated new tissue formation in at least one third of the root canals. However, teeth with apical diameter between 0.32 and 0.65 mm showed vital tissue regeneration in the entire root canals space. Teeth with a diameter between 0.31 and 1.09 mm showed vital tissue in at least one third of the pulp space. The apical foramen size

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of the root canals assumes not to be the definitive factor for successful regeneration after transplantation, and the minimum apical diameter has not been determined, but a size smaller than 1 mm does not prevent regeneration.

Gomes-Filho et al. (39) evaluated regenerative endodontic potential combining blood clot, platelet-rich plasma (PRP), and bone marrow aspirate (BMA) in closed-apex necrotic teeth in dogs. 20 upper and lower premolars of 2 dogs were used; after apical periodontitis inducement in all teeth, root canals were prepared and apical foramen enlarged to #60 file and teeth were disinfected with a triantibiotic paste for 28 days, roots were randomly divided into 4 treatment groups: blood clot (BC), BC + PRP gel, BC + BMA gel, and BC + BMA/ PRP gel and negative controls group. Teeth were histologically evaluated after 3-month follow-up period. Regenerated vital tissue was formed in 71.87% treated roots with no statistically significant difference between the treated groups; PRP and/or BMA did not improve tissue regeneration.

Paryani and kim (28) reported two cases of pulp revascularization using modified regenerative endodontic procedures in mature necrotic teeth. Firstly, the root canals were prepared and disinfected with antimicrobial irrigant, root canals were dried and intracanal medications (calcium hydroxide or ciprofloxacin) were filled the root canals. After that, the root canals were irrigated, and bleeding was induced into the root canals, collagen membranes matrix and MTA were placed at the canals orifice, and the access cavities were sealed with Glass ionomer. At recall visit, apical radiolucencies were reduced in size and clinical signs and

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symptoms were regressed. The mature necrotic permanent teeth can be treated with regenerative endodontic procedures; more clinical researches are needed to evaluate the effectiveness of regenerative endodontic therapy to treat mature necrotic teeth.

Saoud et al. (30) treated large cyst like inflammatory periapical lesion in mature necrotic permanent teeth using regenerative endodontic therapy. A female patient with 23 years old had her #8 with pulp necrosis and an acute apical abscess with slightly open apex and #7 with symptomatic apical periodontitis with completely formed apex. Teeth were chemomechanical debrided and treated with regenerative endodontic therapy, after follow up period the teeth showed evidence of periapical osseous healing radiographically and regression of signs and symptoms clinically which indicate regeneration of new vital tissue in the root canals.

Chrepa et al. (40) assessed the ability of evoked blood from the periapical tissues to deliver the MSCs into the root canal system of mature teeth. 20 patients with mature teeth with apical lesions were evaluated in this study. At the first appointment, teeth were chemomechanical prepared, bleeding from the periapical tissues was induced, and intracanal blood samples were collected. Also, positive blood aspirate was collected in the cartridges during local anesthesia. Total RNA was isolated and used as a template in quantitative RT-PCR using MSC-specific arrays. In addition, MSCs were isolated from blood sample evoked into root canals space and subjected to flow cytometry and quantitative osteogenesis assay. Results showed that the

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induced-bleeding technique delivers MSCs into the root canal system in mature teeth with apical lesions.

Priya et al. (41) evaluated the effectiveness of PRP to regenerate pulp tissue in mature avulsed incisor after delay replantation for a 11-years-old boy. The access cavity of the avulsed tooth was prepared and the root canal was disinfected and root apex was enlarged, then the tooth was placed 20 minutes in doxycycline solution. The tooth was replanted and splinted in its place, and PRP was injected up to the cementoenamel junction level and the access was sealed with glass ionomer cement. After 6-month follow-up, radiographic evaluation showed presence of internal and external root resorption with radiolucency at the root apex and winding of periodontal ligament space. Access was reopened; double antibiotic paste (minocycline and metronidazole) was inserted into the canal and sealed. After another evaluation period from 9 and 12 month, there was resolution of periapical radiolucency radiographically, and the tooth became positive to thermal and electric pulp tests. The outcomes of this case increase the demand for more research to assess the effectiveness of regenerative endodontic therapy.

Saoud et al. (42), used RET to manage 2 cases of mature teeth (#9 and #19) with apical periodontitis after root canal treatment in 2 patients. The gutta-percha fillings materials were removed from the root canals of the teeth using gutta-percha solvent and ProTaper retreatment rotary files. The canals were then prepared and irrigated with NaOCl and dressed with Metapaste, and the access cavity was sealed with intermediate restorative material. At the

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next treatment visit, RET was prepared by inducing periapical bleeding to fill the disinfected root canals with blood clot and covered it with MTA plugs and the access cavities were sealed with intermediate filling materials. After 13-month and 14-month follow-ups, teeth showed clinical and radiographical signs and/or symptoms of healing. Teeth with signs of apical periodontitis after root canal treatment can be retreated using RET.

Saoud et al. (43) used regenerative endodontic therapy for traumatized teeth after horizontal root fracture, avulsion, and perforating root resorption, a 15-year-old boy had a history of traumatic injury to mature tooth #8 resulting in horizontal root fracture and pulp necrosis of the coronal fragment. A 7-year- old girl suffered an avulsion injury to immature tooth #9, which developed inflammatory replacement resorption and subsequently root fractured 15 months later. Another 16-year-old boy also suffered a history of traumatic injury to mature tooth #8, resulting in perforating root resorption. All teeth were treated with regenerative endodontic procedures using chemomechanical debridement, calcium hydroxide/triple antibiotic paste dressing, EDTA rinse, induction of periapical bleeding into the canal space, and a coronal mineral trioxide aggregate plug. In the tooth presenting with horizontal root fracture, only the coronal fragment was treated to preserve pulp vitality in the apical fragment for possible pulp tissue regeneration. After regenerative endodontic procedures, clinical signs/symptoms subsided, and inflammatory osteolytic lesions resolved in all traumatized teeth. Two teeth were followed for 19 months and 1 tooth for 5 years. At the last review of the teeth with horizontal root fractures,

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the first case showed healing by calcified tissue and the second case showed healing by fibrous connective and hard tissue. Tooth with perforating root resorption demonstrated a decrease in size of the resorptive defect

Saoud et al. (23), presented a case series study consisted of 6 patients (4 females and 2 males) to evaluate the potential of using REPs to treat mature necrotic permanent teeth with apical periodontitis. Chemomechanical preparation of all canals was performed, and then dressed with Metapaste during treatment visits. Periapical bleeding was induced at all canals and MTA plug was placed over semicoagulated blood. The access cavities were sealed using composite or amalgam restorative materials. After Follow-up period ranged from 8 to 26 months, 2 teeth were reported healed and 5 teeth considered healing regarding the periapical lesions, and there were absent in clinical signs/symptoms in all teeth, and none of them responded to cold and electric pulp tests. This case series demonstrated the capability of using REPs to treat mature necrotic teeth with apical periodontitis.

Shah (44) provided scientific evidence on the outcome of a large number of cases treated by SealBio over the longer follow-up period. One hundred and thirty-four teeth in 116 patients presenting with pulp and periapical disease were randomly recruited between 2009 and 2014. SealBio was performed, and cases were followed up at regular intervals up to 6-year. Of the total 134 teeth treated, 16 teeth could not be followed up and 9 cases failed (7.62% of cases). In only 4 cases (approximately 3.38% of cases), the failure could be directly attributed to endodontic causes.

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In the remaining 5 cases, coronal leakage from under the crown margins or dislodged restoration was found after 3–5 years of treatment. SealBio was found to be a successful, nonobturation, regeneration-based endodontic treatment protocol. By cell homing of endogenous stem cells, a biological seal rather than an artificial seal with gutta-percha and sealer cement is possible to achieve. It is highly cost saving and easier to perform, in addition to other advantages, such as retreatment is much simpler.

Fahmy et al. (45) assessed the revascularization potential of necrotic mature teeth in a dog model following different protocols. Periapical infection was induced in 54 mature premolars. Teeth were distributed into seven groups: (1) Double-antibiotic-paste/Blood clot, (2) Ciprofloxacin/collagen, (3) Double- antibiotic-paste/Collagen, (4) Modified Tri-antibiotic-paste /collagen, (5) Ciprofloxacin/Gelfoam, (6) Double-antibiotic-paste/Gelfoam, and (7) Modified Triantibiotic- paste/Gelfoam. Positive and negative controls included infected and healthy teeth, respectively, (n = 12 roots/group). Canals were apically shaped to size 0.6 mm then disinfected for 1 month. Intra-canal bleeding was induced then scaffolds were applied for another month. Teeth and supporting bone were surgically sampled. Tissues were histologically scored and vimentin immuno-intensity was estimated. Ciprofloxacin and Double-antibiotic paste/ Collagen resulted in significantly better corono-apical tissue ingrowths, vascularity, cementum formation and significantly lower inflammatory extents (P < 0.05).These groups also showed significantly higher Vimentin intensities, (P < 0.05). The applied protocols revascularised

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necrotic mature canals and reduced inflammation particularly in the Ciprofloxacin/collagen and Double- antibiotic-paste/collagen groups.

Jacobs et al. (46) investigated the direct and residual antibacterial effects of intracanal antimicrobials against bacterial biofilms obtained from infected mature and immature teeth with necrotic pulps. Sterile dentin slabs (n = 100) were inoculated with bacterial biofilms obtained from root canals of an immature or a mature tooth with pulpal necrosis and incubated anaerobically for 3 weeks (n = 50 per biofilm). Dentin infected with each type of biofilm received 1 week of treatment with 1 or 5 mg/mL double antibiotic paste (DAP) in methylcellulose hydrogels, calcium hydroxide, or placebo paste or received no treatment (n = 10). The pastes were removed, and biofilm disruption assays were performed. Additional dentin slabs (n = 100) were pretreated with the same treatments (n = 20). The pastes were rinsed off, and the samples were immersed in phosphate-buffered saline for 1 week. Thereafter, samples from the treatment groups were infected with bacterial biofilm from both clinical sources mentioned earlier (n = 10 per biofilm) and incubated anaerobically for 3 weeks before conducting biofilm disruption assay. Uninfected dentin slabs were used for both antibacterial experiments as negative control groups (n = 20). Results: All antimicrobials showed significant direct antibacterial effects regardless of the biofilm source. Dentin pretreated with 5 mg/mL DAP provided significantly higher residual antibacterial effects in comparison with all other groups regardless of the source of biofilm. Dentin pretreated with calcium hydroxide did not show any residual antibacterial effects.

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Nageh et al. (47) evaluated 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). 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. 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.

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Nagas et al. (48) reported procedures and outcome of a regenerative endodontic treatment approach in 2 previously-traumatized incisors with closed apex with apical periodontitis. A 2-visit treatment procedure was employed. At initial visit, the root canals were copiously irrigated, followed by placement of a triple antibiotic paste containing ciprofloxacin, metronidazole, and clindamycin into the root canals. After 4 weeks, the antibiotic paste was removed, and apical bleeding was initiated with size 10 hand files beyond the apices. The root canals were coronally sealed with mineral trioxide aggregate, and the access cavities were restored with bonded resin composite. At post-operative 60 months, both teeth were remained asymptomatic, with the recall radiographs showing complete resolution of apical radiolucency and reestablishment of periradicular tissues. In both teeth, the dimensions of root space remained unchanged as verified by image analysis. The revitalization protocol utilizing root canal disinfection and induced apical bleeding in necrotic, closed-apex incisors may offer a clinically acceptable alternative to conventional root canal treatment.

Nagas (48) evaluated 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

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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.

Neelamurthy (49) evaluated the revascularization procedure both in immature and mature teeth with necrotic pulp and open apices, disinfected with triple antibiotic paste followed by inducing blood clot in the root canal. Fifteen patients were selected who presented with immature and mature permanent teeth with pulpal necrosis and open apices. In the first visit, the root canal was accessed with LA and rubber dam isolation; then the canal was disinfected using triple antibiotic paste containing ciprofloxacin, metronidazole, and clindamycin in the ratio of 1:1:1 and closed with IRM. In the second visit, after administering local anesthesia and isolating with a rubber dam, the triple antibiotic paste was washed out by saline irrigation, and apical papilla beyond the confines of the root canal was stimulated with sterile H file to produce a blood clot. Finally, the access was closed using a double seal with mineral

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trioxide aggregate (MTA) placed apical to cementoenamel junction and resin bonded cement over the MTA. Radiographic examination and pulp sensibility test was done during the follow-up period of 2,4,6,8 and 10 months. After 10 months follow-up, 10 out of 13 patients showed root development and apical closure. The eight patients out of 13 showed root development, apical closure and lateral thickening of radicular dentin and 2 out of 13 patients showed a positive response to electric sensibility test.

Urrejola (50) reported case to describe a regenerative autologous cellular therapy using mesenchymal stem cells from inflamed dental pulp and leukocyte platelet-rich fibrin (L-PRF) in a mature tooth. A healthy 50- year-old man consulting for spontaneous dental pain was referred for endodontic treatment in tooth #28, which was diagnosed with symptomatic irreversible pulpitis. Inflamed dental pulp was extracted and trans- ported to a good manufacturing practice laboratory for the isolation and culture of dental pulp stem cells (DPSCs). L-PRF was obtained from the patient’s blood and was introduced into the instrumented and disinfected root canal, and expanded DPSCs were inoculated into the clot. The cervical part of the root canal was sealed with Biodentine and a composite resin. Follow-up examinations were performed 6 months and 3 years later. The examinations included periapical radiographs (to measure the periapical index [PAI]), cone- beam computed tomographic (CBCT) imaging, sensitivity, and vitality tests. Clinical evaluations revealed normal responses to percussion and palpation tests. The tooth had a delayed response to cold, and the electric pulp test was responsive. The PAI and CBCT imaging revealed that the periapical area

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remained normal with a PAI score of 1 and a CBCT PAI score of 0. The vitality test performed indicated low blood perfusion units.

Aim of the work

Aim of the work

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The aim of the present investigation was to histopathological assessment of the regenerative potential of permeant mature teeth with necrotic pulp and apical periodontitis with different apical diameters in comparing to conventional endodontic treatment.

Materials and Methods

Materials and Method

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A- Materials: 1- Mineral Trioxide Aggregate. (MTA)1 2- AH plus sealer.2

B- Methods:

Animal models: A total of six healthy male dogs aged 2.5-3.5 years

with complete set of permanent dentition were recruited for this study. The selected dogs were of both sexes and clinically normal. Animals were quarantined in separate cages in the department of veterinary surgery, faculty of veterinary medicine, Cairo University. Animals were examined and kept under observation for two weeks before being used as experimental animals in the study. Diseased animals were excluded. The dogs were housed uniformly and were provided with healthy food (20g/Kg) and drinking bowls from the day of housing till the day of scarification. This study was conducted according to the ethical committee protocol at the Faculty of Dentistry, Ain Shams University, Egypt, and was performed observing the requirements for human attitude towards animals.

Preparation of the animal model: Before the beginning of the experimental procedures,

each dog was housed in a separate kennel and observed two weeks prior to the operative procedures to exclude any diseased dog. The kennels were sprayed by Neocidal agent (diazinon) with a concentration of 6/1000ml of water. Then the dogs were bathed by the Neocidal agent in concentration of 1/100ml of water. As a precaution, each dog was injected

1 - MTA Angelus, Brazil.

2 - Dentsply Maillefer ,Ballaigues ,Switzerland


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with Ivermectin3 0.1 ml/Kg body weight to protect against internal and external parasites.

Classification of samples: 12 premolars in each dog were included to sum 72

teeth and 144 root canals, in which each root was used as a unit of measure for statistical analysis. These teeth were divided into four equal experimental groups and two equal control groups (24 teeth each), these groups were randomly represented in each animal, and all groups were further subdivided into two subgroup according to post treatment evaluation period (Figure 1).

• Group I (RCT): Root canal obturation for root canals prepared up to #F3 ProTaper file.

• Group II (RET-F3): Regenerative endodontic therapy for root canals prepared up to #F3 ProTaper file.

• Group III (RET-F4): Regenerative endodontic therapy for root canals prepared up to a #F4 ProTaper file.

• Group IV (RET-F5):: Regenerative endodontic therapy for root canals prepared up to #F5 ProTaper file.

• Group V (P): Positive control represent infected teeth. • Group VI (N): Negative control represent untouched

teeth.

subgroups classification:

• subgroup A: Post- treatment evaluation period one month.

• subgroup B: Post- treatment evaluation period three months.

3 - Ivomec, MSdMerk&Co. Inc, USA


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Figure 1: Schematic diagram for samples classification.

6 Dogs 60 Teeth (120 roots)

Gp I: RCT with # F3

apical size. 12 teeth (24 roots)

Sub Gp (A): one-month evaluation period.

period Sub Gp (B): three-month evaluation period.

Gp II: RET with # F3 apical size.

12 teeth (24 roots)


Sub Gp (B): three-month evaluation period.

Gp III: RET with # F4 apical size.

12 teeth (24 roots)


Sub Gp (B): three-month evaluation period

Gp IV: RET with # F5 apical size.

12 teeth (24 roots)

Gp V: Positive control. 12 teeth (24 roots)

)



Sub Gp (A): one-month

evaluation period.


Gp VI: Negative control. 12 teeth (24 roots)


Sub Gp (A): one-month evaluation period


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Operative procedure:

A- Induction of periapical pathosis General anesthesia was administrated after fasting the

dogs for 12 hours as the following protocol, the dogs were premedicated by using 0.05 mg/kg by weight atropine sulphate4 injected subcutaneous and 1mg/kg by weight Xylazine HCl5 injected intramuscular. The anesthesia was induced by using Ketamine HCl6 injected intravenous after using intravenous cannula in the cephalic vein at a dose of 5 mg/kg by weight. The anesthesia was maintained by using Thiopental sodium7 at a dose of 25 mg/kg by weight 2-5 % injected intravenous (dose to effect). (Figure 2).

After general anesthesia, all teeth were examined radiographically to confirm complete root formation (Figure 3). Endodontic access cavity was done in all experimental and positive control teeth. Exposing the pulp chamber was done using size #2 diamond stone with conventional speed hand piece8 mounted on electric micro motor9 (Figure 4). A sterile file size #15 was used to disrupt the pulp tissue in the canals. Supra-ginigval plaque was applied in pulp chambers (51), a piece of cotton was inserted into the entrance of each canal and the access was left open for one month.

4 - Atropine Sulphate: ADWIA Co., Egypt. 5 - Xylaject: ADWIA Co., Egypt. 6 - Keiran: EIMC pharmaceuticals Co., Egypt. 7 - Thiopental sodium: EIPICO, Egypt. 8 - NSK handpiece, Japan. 9 - NSK micromotor, Japan.


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B- Root Canal preparation and disinfection All surfaces were coated with tincture iodine, and all

experimental teeth were re-entered under completely aseptic conditions and cotton roll isolation. The soaked cotton was removed, and the working length of each root was determined using apex locator10 (Figure 5), the root canals will be instrumented up to #F3 ProTaper rotary files11 for group I and group II, and up to #F4 and #F5 for group III and IV respectively. All canals were irrigated with 1.5% sodium hypochlorite12 (NaOCl) (20 mL/canal, 5 min) and 17% EDTA13 (1 mL/canal, 1 min) with a 27-gauge side vented needle after every instrument during all steps of preparation. Canals will be finally rinsed with saline (20 mL/canal, 5 min), canals dried with paper points and dressed with Ca(OH)214. Then, the access cavity will be sealed using glass ionomer restoration15 for 2 weeks to allow disinfection of the root canals.

C- Treatment modalities: After 2 weeks of using the Ca(OH)2 paste, under the

same general anesthesia and aseptic techniques, the experimental teeth will be re-entered; the glass ionomer restoration will be removed using diamond stone and Ca(OH)2 will be removed with copious saline irrigation and 17% EDTA (1 mL/canal, 1 min). The root canals will be

10 Ipex II, NSK, Japan. 11 Dentsply Maillefer, Ballaigues, Switzerland. 12 Clorox Co., Egypt. 13 Prevest Denpro, Digiana, Jammu – India. 14 Metapaste, Metabiomed, Republic of Korea. 15 Riva Self Cure, SDI Limited, Australia.


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dried with sterile paper points and treated according to different treatment protocols as follows:

Root canal obturation (RCT): (Group I) Root canals were obturated using continuous wave

condensation technique with gutta-percha/ AH Plus (Figure 6), using Dia-Gun and Dia-Pen obturation system16. Continuous wave obturation was carried out in two steps. First, the master gutta- percha cone was placed in the apical third of the root canal. The gutta-percha was adjusted with a scalpel 1 mm short of working length until tug-back was achieved. A medium- sized plugger was marked at its binding point with a rubber stop within 4 mm of working length. After coating the root canal walls with sealer, the apical part of master cone was coated with a thin film of sealer and placed into the canal. The heat source was adjusted to 200 oC at full power and touch mode was activated. The heated plugger was driven through the gutta-percha to approximately 3–4 mm before the rubber stop approached the reference point. During the downpack the plugger was moved apically slowly. The plugger was then deactivated, and firm apical pressure held for 10 s. the backfilling step was completed as follow, the Dia-Gun’s activated tip contacted with the upper part of filled master cone in the canal to transfer heat for 3 to 5 seconds to softened the material. The melted gutta-percha Obturator extruded to the root canal while withdrawing the tip from the canal, after that hand plugger was used to compact obturation material and Glass ionomer filling was used to seal the access cavity.

16 DiaDent Group International, Republic of Korea.


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Regenerative endodontic therapy (RET): (Group II, III and IV)

A sterile hand #20 K-file was inserted 2 mm beyond the canal terminus until bleeding was induced to fill the canal space (Figure 7), after blood clot had formed at the cervical portion of the canal, MTA was mixed according to the manufacturer instructions and inserted into the canal using a MTA carrier and then packed over blood clot using a suitable size plugger to form 3mm coronal plug (Figure 8), and the access cavity was sealed using Glass ionomer filling (Figure 9). During all steps of operative procedures radiograph were taken to check the accuracy of procedures (Figure10-11).

Tissue Preparation and Histologic Analysis: The animals were sacrificed according to the post

treatment evaluation period; one month for subgroup I and three months for subgroup II using anesthetic overdose. Jaws were separated and bone segments including the experimental and control teeth were resected. Blocks were fixed using 10% buffered Formalin solution with ratio 1:50 (fixative: tooth mass). After two weeks of fixation, blocks were decalcified using 17% EDTA. The decalcifying solution was renewed on daily basis for about 120 days.

After decalcification, samples were dehydrated in 70% ethanol and then embedded in paraffin blocks. Blocks were sectioned in bucco-lingual sections of 6µm thickness. Sections were stained using hematoxylin and eosin dye and prepared for histopathologic evaluation.

All sacrificed animals were safely discarded by burning them through the disposal unit of dead animals in faculty of Veterinary Medicine, Cairo University.


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Histopathologic evaluation: Parameters were evaluated according to the following

scoring systems:

1- Inflammatory cell count: For each slide, three representative fields were

analyzed at X 400 magnification (52,53). Fields were selected conforming to the following criteria;

i) well preserved tissue with good architecture and no artifacts,

ii) intense inflammatory cells infiltration. Total inflammatory cell number was counted using image analysis software17 as follows: Color coding threshold was adjusted to select the perimeter of the whole range of inflammatory cells in order to exclude other non-desired structures. Then binary threshold of the selected color-coded inflammatory cells was done prior to calculation. Data were collected, tabulated for statistical analysis.

2- Bone resorption:(54) It will be evaluated as follows:

• Score 0: absence of signs of resorption. • Score 1: presence of signs of active resorption including

osteoclasts, Howship’s lacunae and areas of resorption.

3- Presence or absence of vital tissues inside the pulp space (51,54):

• Score 0: No tissue in-growth into the canal space. • Score 1: evidence of tissue in-growth to the apical third

of the canal space.

17 Image J, 1.41, NIH, USA


43

• Score 2: evidence of tissue in-growth extending to the middle third of the canal space.

• Score 3: evidence of tissue in-growth extending to the cervical third of the canal space. 4- New hard tissue formation: a- Qualitative analysis: Criteria for histologic

identification of hard structure (55) included: • Dentin: presence of dentinal tubules. • Cementum: presence of cementocyte-like cells adherent

to dentin. • Bone: presence of Haversian canals with uniformly

distributed osteocyte-like cells. • Periodontal ligament: presence of Sharpey’s fibers

bridging cementum and bone.

b- Quantitative analysis (54) :This will be evaluated as follows:

• Score 0: absence of new hard tissue formation. • Score 1: partial formation of new hard tissues. • Score 2: complete formation of new hard tissues.

Statistical analysis:

Data were collected, tabulated and statistically analyzed using statistical analysis software SPSS18 version 23 for mac. ANOVA test was used for parametric variables, Tukey post hoc tests was used in case of significance. Kruskal Wallis test was used for non-parametric results, Chi square test was used for non-numeric variables.

18 SPSS: Statistical Packages for the Social Sciences, IBM, USA


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Figure 2: Administration of general anesthesia

Figure 3: Pre-operative radiograph. A) Upper teeth, B) Lower teeth.


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Figure 4: Access opening

Figure 5: Working length determination.


46

Figure 6: Root canal obturation. A)-Vertical condensation with dia-pen, B)-Backfill obturation with dia-gun.

Figure 7: Blood clot formation.


47

Figure 8: MTA coronal plug

Figure 9: Glass ionomer restoration


48

Figure 10: Radiograph representing RCT (group I), A)Preoperative, B) Ca(OH)2 medication, C) Root canal obturation.

Figure 11: Radiograph representing RET (group II), A)Preoperative, B) Ca(OH)2 medication, C) MTA Plug.

Results

Results

49

Histopathological changes were observed in terms of:

1- Inflammatory cell counts 2- Bone resorption. 3- Nature and extent of tissue in-growth in the pulp

space. 4- Formation of new hard tissue.

Histopathological changes were observed among all groups at the two periods of observation.

1- Inflammatory cell counts ( Table 1, Figure 12-15):

After the first month evaluation period, the highest inflammatory cell counts was recorded for positive control group (208±13.5), while the lowest count was recorded for negative control group (45±11.2), among the experimental groups the highest inflammatory cell counts was recorded for RCT (190.3±16.4) group followed by RET-F3 (124.3±10.7) and RET-F4 (123±8.5), and the lowest count was recorded for RET-F5 (112±11.4). Statistical analysis showed that there was statistical significant difference between all groups (p≤0.05) except between regenerative endodontic groups (RET-F3, RET-F4 and RET-F5) .

After the three month evaluation period the highest inflammatory cell counts was recorded for positive control group (281.6±23.4) followed by RCT (99.3±16.6) group and RET-F3 group (87.5±5.8), the RET-F4 (82.1±14.7) showed higher inflammatory cell count than RET-F5 group (66.7±16.7), the lowest count was recorded for negative control group (47.1±8.4). Statistical analysis showed that there was statistical significant difference between all groups

Results

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Table 1: showing mean inflammatory cell count for all groups.

Subgroup A Subgroup B p-Value Mean±SD Mean±SD

RCT a16.4±190.3 a 16.6±99.3 <0.0001 RET-F3 b10.7±124.3 a 5.8±87.5 <0.0001 RET-F4 b8.5±123 ba 14.7±82.1 <0.0001 RET-F5 b11.4±112 b 16.7±66.7 <0.0001

P c13.5±208 c 23.4±281.6 <0.0001 N d11.2±45 d 8.4±47.1 0.61

p-Value 0.001 0.001 Significant at p ≤0.05. Different small letters indicate a significant difference between groups within the same follow-up period

Figure 12: Bar graph showing inflammatory cell count among all groups at two time period.

0

50

100

150

200

250

300

RCT RET-F3 RET-F4 RET-F5 P N

Subgroup A Mean±SD Subgroup B Mean±SD

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Figure 13 : Photomicrograph showing sever inflammatory cell infiltration positive group after 1 month (a) at X200 magnification, (B) at X400 magnification.

A

B

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52

Figure 14: Photomicrograph showing mild inflammatory cell infiltration negative group after 1 month (X400 magnification).

Figure 15: Photomicrograph showing moderate inflammatory cell infiltration RCT group after 3 month (X400 magnification).

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(p≤0.05) except between RCT group with RET-F3 and RET-F4 groups (p=0.423, 0.081 respectively), and between RET-F3 and RET-F4 groups (p=0.954), and between RET-F4 and RET-F5 groups (p=0.155).

Comparing the inflammatory cell counts for each groups between the 1st and 2nd evaluation period showed that the 2nd evaluation period had lower mean inflammatory cell count than that recorded for the same group in the 1st evaluation period except for the positive group. Statistical analysis showed that there was statistical significant difference between inflammatory cell count between 1st and 2nd evaluation period (p≤0.05) except between negative group (p=0.61).

2- Bone Resorption.

Histological sections were observed for signs of resorption including presence of osteoclasts or odontoclasts, Howship’s lacunae and areas of active bone resorption. Data were recorded according to the previously described scoring system. Data were tabulated in table 2 (Figure 16-18).

Regarding the 1st evaluation period, 100% of positive control group showed bone resorption (score 1), while the negative control group did not showed any sign of bone resorption (0% score 1), the percentage of bone resorption for experimental groups were ranked ascendingly as 25%, 33.4%, 58.4% and 75% for groups RCT, RET-F5, RET-F4, and RET-F3 respectively. Statistical analysis showed that there was statistical significant difference between all groups (p≤0.05). Mann-Whitney test comparisons resulted in statistical significance differences between positive control

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Table 2: Score percentage of bone resorption among groups at two evaluation periods.

Subgroup A Subgroup B

p-Value 0 1 0 1

RCT

N 9 3 11 1 0.284

% 75% 25% 91.6% 8.4% Mean rank

ae 28 ac 27.5

RET-F3

N 3 9 7 5 0.105

% 25% 75% 58.4% 41.6% Mean rank 46 bc a 39.5

RET-F4

N 5 7 8 4 0.229

% 41.6% 58.4% 66.6% 33.4%

Mean rank

ba 40 a 36.5

RET-F5

N 8 4 10 2 0.356

% 66.6% 33.4% 83.3% 16.7%

Mean rank

a 31 ca 30.5

P

N 0 12 0 12 1

% 0% 100% 0% 100%

Mean rank

c 55 b 60.5

N

N 12 0 12 0 1

% 100% 0% 100% 0%

Mean rank

de 19 c 24.5 Kruskal-Wallis

Test 31.196 34.76

p-Value 0.0001 0.0001 Significant at p ≤0.05. Different small letters indicate a significant difference between groups within the same follow-up period

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55

group and RCT, RET-F4, RET-F5, and negative control (p≤0.05), there were also significant differences between negative control group and RET-F3, RET-F4 and RET-F5 groups (p≤0.05), there were no significant differences between all experimental groups (p³0.05) except between RCT and RET-F3, and between RET-F3 and RET-F5 groups.

Regarding the 2nd evaluation period, the percentage of bone resorption for the control groups showed the same results of the 1st evaluation period (100% and 0% for positive and negative groups respectively), for experimental groups the highest percentage of bone resorption was recorded for RET-F3 (41.6%) while the lowest percentage was recorded for RCT group (8.4%), and the percentage of bone resorption for RET-F4 group (33.4%) was higher than that for RET-F5 group (16.7%). Statistical analysis revealed that there was statistical significant difference between all groups (p≤0.05). Mann-Whitney test comparisons showed that there were no significant differences between all experimental groups and between negative control with both RCT and RET-F5 (p³0.05).

The percentage of bone resorption recorded for all experimental groups at the 2nd evaluation period showed lower percentage value compared to that recorded at the 1st evaluation period and remained the same value for control groups with no statistical significant difference between the two periods.

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Figure 16: Photomicrograph showing apical root resorption (score 1) positive group after 3 months evaluation period (a) at X40 magnification, (B1 and B2) at X400 magnification showing active odontoclast.

A

B1 B2

B1

B2

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Figure 17: Photomicrograph showing no apical root resorption (score 0) RCT group after 3 months evaluation period (A) at X40 magnification, (B) at X400 magnification.

Figure 18: Histogram representing percentage of bone resorption score for all groups at two evaluation period.

75%

25.00%41.60%

67%

0%

100% 92%

58% 67%83%

0%

100%

25%

75.00%58.40%

33%

100%

0% 8%

42% 33%17%

100%

0%

0%10%20%30%40%50%60%70%80%90%

100%

RCT

RET-

F3

RET-

F4

RET-

F5 P N

RCT

RET-

F3

RET-

F4

RET-

F5 P N

Sub group (A) Sub group (B)

0 1

A B

Results

58

3- Nature and extent of tissue in-growth in the pulp space.

The histopathological analysis showed connective tissue in-growth in some samples. The nature of this tissue resembles periodontal connective tissue with variable amounts of inflammatory cell infiltration and noticeable angiogenic activity in some samples. This parameter did not analyzed among RCT and negative groups as root canal space were filled by root canal obturations materials and normal pulp tissue respectively. The extent of tissue in-growth was recorded according to the previously described scoring system. Data were tabulated in table 3. (Figure 19-21).

When the 1st evaluation period was observed, the highest mean rank for tissue in-growth was recorded for RET-F5 group (36.75) and the lowest rank was recorded for positive group (8), RET-F4 group showed higher mean rank value compared to RET-F3 group (33.25 and 20 respectively), Statistical analysis showed that there was statistical significant difference between all groups (p≤0.05). Mann-Whitney test comparisons resulted in statistical significance differences between all groups except between RET-F4 and RET-F5 groups (p³0.05).

After the 2nd evaluation period (3 months interval), the mean rank for tissue in-growth was ranked ascendingly as 6.75, 20.75, 34 and 36 for group positive, RET-F3, RET-F4 and RET-F5 respectively. Statistical analysis revealed that there was statistical significant difference between all groups (p≤0.05), when Mann-Whitney test was performed between pairs of groups it showed that there were statistical

Results

59

significance differences between all groups except between RET-F4 and RET-F5 groups (p³0.05).

The mean rank for tissue in-growth after the 2nd evaluation period showed higher value than that recorded at the 1st evaluation period with no statistical significant difference between the two periods.

Table 3: Score percentage of tissue in-growth among groups at two evaluation periods.

Subgroup A Subgroup B P-

Value 0 1 2 3 0 1 2 3

RET-F3

N 0 8 4 0 0 6 6 0 1 % 0% 66.6% 33.4% 0% 0% 50% 50% 0%

Mean rank a 20 a 20.75

RET-F4

N 0 1 7 4 0 0 6 6 0.324 % 0% 8.3% 58.3% 33.4% 0% 0% 50% 50%

Mean rank be 33.25 be 34

RET-F5

N 0 0 6 6 0 0 4 8 0.418 % 0% 0% 50% 50% 0% 0% 33.3% 66.7%

Mean rank be 36.75 be 36

P

N 8 4 0 0 11 1 0 0 0.140 % 66.6% 33.4% 0% 0% 91.7% 8.3% 0% 0%

Mean rank c 8 c 6.75

Kruskal-Wallis Test 34.456 37.353

P-Value 0.0001 0.0001 Significant at p ≤0.05. Different small letters indicate a significant difference between groups within the same follow-up period

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Figure 19: Photomicrograph showing tissue in-growth inside root canal at X40 magnification.

A) Score 0: positive control group subgroup A. B) Score 1: RET-F3 group subgroup A. C) Score 2: RET-F4 group subgroup A. D) Score 3:RET-F5 group subgroup B.

A B

C D

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Figure 20: Histogram representing percentage of tissue in-growth score for all groups at two evaluation period.

Figure 21: Photomicrograph showing nature of the tissue in-growth inside root canal.

A)-Tissue in-growth inside root canal, RET-F5 subgroup-B (X40 magnification).

B)- Nature of the tissue in-growth at X400 magnification showing: a) cementoid structure, b) dilated blood vessels, c) connective tissue.

0% 0.00% 0.00%

67%

0% 0% 0%

92%67%

8.30% 0.00%

33%

50%

0% 0%

8%33%

58.30%50.00%

0%

50%

50%33.30%

0.00%0%

33.40%50.00%

0% 0.00%

50.00%67%

0%

0%

20%

40%

60%

80%

100%

120%

RET-F3 RET-F4 RET-F5 Positive RET-F3 RET-F4 RET-F5 Positive

Sub group (A) Sub group (B)0 1 2 3

A B

a

b

c

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62

Newly formed tissues (Figure 21) characterized as loosely to moderately dense connective tissue with a low to moderate number of blood vessels at central portion that were engorged by red blood cells (RBCs), inflammatory cell and islands of dystrophic calcification that sometimes it may showed feature of cementoid or dentinoid structure.

4- Formation of new hard tissue.

The histopathological analysis showed variety of hard tissue formation inside root canal space, like the tissue in-growth inside root canal space this parameter did not analyzed among RCT and negative control groups as root canal space were filled by root canal obturations materials and normal pulp tissue respectively. Data were collected and tabulated according to the previously described scoring system (Table 4, Figure 23-25).

Regarding subgroup A; the highest mean rank of new hard tissue formation was equally recorded for RET-F4 and RET-F5 (31.5) in which 100% of sample for both group formed partially new hard tissue (score 2), while the lowest mean rank was recorded for positive control group (7.5), the mean rank for RET-F3 group was 27.5. statistical analysis for this data revealed that there was statistically significance difference among all groups, mann-whitniy test showed that there were significant difference between positive group with all regenerative groups (p≤0.05), and no significant difference between pairs of experimental groups (p³0.05).

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Regarding subgroup B; the mean rank for RET-F4 and RET-F5 were equal and it was the highest value (31), followed by RET-F3 group (29), and finally positive group (7), there were statistically significance difference between all groups (p≤0.05), comparing pairs of groups revealed that there were significance difference between positive group with other experimental groups (p≤0.05), and no significance difference between pairs of experimental groups (p≤0.05).

Statistical analysis between subgroup A and subgroup B for the same group resulted in no significance difference between the 1st and 2nd evaluation period.

The new hard tissue that have been formed in most of sample were in the form of dystrophic calcification, in which islands or patches of calcified material were present, these islands could not be classified as cementum, dentin or bone like tissue, some sample showed presence of cementum like and/or dentin like tissue inside root canal space. (Figure 23-24). This cementum-like tissue or ectopic cementum was deposited on the inner aspect of the radicular dentin, covered by a thin layer of cementoid tissue. Cementoblasts-like cells and cementocytes-like cells were also observed.

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Table 4: Score percentage of new hard tissue formation among groups at two evaluation periods.

Subgroup A Subgroup B P-value

0 1 2 0 1 2

RET-F3

N 2 10 0 1 11 0

0.546 % 16.7% 83.3% 0% 8.3% 91.6% 0% Mean rank a 27.5 a 29

RET-F4

N 0 12 0 0 12 0

1 % 0% 100% 0% 0% 100% 0% Mean rank a 31.5 31 a

RET-F5

N 0 12 0 0 12 0

1 % 0% 100% 0% 0% 100% 0% Mean rank a 31.5 a 31

Positive

N 12 0 0 12 0 0

0.317 % 100% 0% 0% 100% 0% 0% Mean rank b 7.5 b 7

Kruskal-Wallis Test 39.101 42.455

P-Value 0.0001 0.0001 Significant at p ≤0.05. Different small letters indicate a significant difference between groups within the same follow-up period

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65

Figure 22: Histogram representing percentage of new hard tissue formation score for all groups at two evaluation period.

Figure 23: Photomicrograph showing nature of the tissue in-growth inside root canal.

A)-Tissue in-growth inside root canal, RET-F3 subgroup-B (X40 magnification). B)- Nature of the tissue in-growth at X400 magnification showing dentinoid like structure with dentinal tubule formation.

17%0.00% 0.00%

100%

8% 0% 0%

100%83%

100.00% 100.00%

0%

92% 100% 100%

0%0% 0.00% 0.00% 0% 0% 0% 0.00% 0.00%

0%

20%

40%

60%

80%

100%

RET-F3 RET-F4 RET-F5 Positive RET-F3 RET-F4 RET-F5 Positive

Sub group (A) Sub group (B)

0 1 2

A B

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Figure 24: Photomicrograph intra-canal dystrophic calcification at 200X magnification. A)- RET-F3 subgroup B, B)- RET-F4 subgroup A.

A B

Discussion

Discussion

67

The primary goals of endodontic treatment of mature and immature permanent teeth are prevention and/or elimination of apical periodontitis and resolution of patient symptoms (56).

The aim of this animal study was to histopathological evaluation of the regenerative potential of permeant mature teeth with necrotic pulp with different apical diameters in comparing to conventional endodontic treatment. The null-hypothesis is that the regenerative endodontic therapy has healing ability over the conventional endodontic therapy, and increase the apical size diameter influence the healing of apical periodontitis after regenerative endodontic therapy.

The choice of dog as an animal model for biological experiments in endodontics is based on the fact that dogs have similar apical repair compared with humans in shorter duration (average one sixth of human) due to higher growth rate (57). Double rooted premolars were selected summing up a total of 12 teeth in each dog comprising 24 roots increasing the whole number of samples for a reliable statistical analysis. Premolars are accessible for endodontic procedures, also having average-sized canals for endodontic manipulation, Age range selected was 2.5-3.5 years which was suitable for the study of mature teeth.

Sample were divided into four groups; group I resample RCT, and group II, III and IV that respmple RET with different apical size diameter. Positive control group (group V) represent infected teeth while and negative control group (group VI) represent infected teeth. These groups were farther subdivided into two subgroup based up on observation periods; one month and three months which are

Discussion

68

equivalent to six and eighteen months respectively in relation to their human analogue.

For many years, RCT used traditional protocols involves chemo-mechanical preparation of the root canal system to eliminate organic, inorganic and bacterial products and sealing of the radicular space with a biocompatible material (obturation). Root canal sealers are used in conjunction with the core filling material to establish an adequate three-dimensional seal and induce hard tissue formation in healing outcomes(58). The optimal apical enlargement during conventional root canal preparation remains controversial. The anatomical diameter of root canals has been investigated extensively in an effort to identify the optimal size of apical canal instrumentation. Previous studies suggest that the apical diameter is larger than often assumed, which may hamper proper cleaning of the root canal system (59,60). An average apical preparation of 300-350 µm, equivalent to file sizes 30-35, has been usually recommended for endodontic therapy (61).

The RET is defined as a biologically based procedure designed to replace damaged structures such as dentin/ root structure and cells of pulp dentin complex, regenerative endodontics emerged as a non- traditional method for management of immature teeth and more recently extended for management of mature teeth. In regenerative endodontic therapy, the migration of dental pulp stem cells into the pulp chamber might be influenced by the width of the apical foramen. The foramen allows the ingrowth of blood vessels and cell migration of the surrounding periapical tissues into the root canal space after regenerative endodontic therapy. This new clinical procedure relies on creating a blood clot in

Discussion

69

the pulp chamber, delivering and activating growth factors and stem cells passing through the apical foramen (38,62,63).

Laureys et al.(38) concluded that the minimum diameter of the apical foramen to allow ingrowth is not determined, but a diameter much smaller than 1 mm (0.31mm) does not prevent revascularization and ingrowth of new vital tissue, Estefan et al (64) showed that regeneration procedures were successful with apical diameters as small as 0.5 mm, Fang et al (65) concluded that the teeth with apical diameters <1.0 mm achieved clinical success after regenerative endodontic treatment. Meanwhile, the teeth with apical diameters of 0.5– 1.0 mm attained the highest clinical success rate, which may be related to other potential factors, including patient age, pulp necrosis etiology, preoperative apical radiolucency, procedure details, follow-up period, and sample size. So that in this study three different apical diameter were used to evaluate the regenerative potential of mature teeth with necrotic pulp and apical periodontitis.

Induction of periapical infection was done in order to simulate clinical conditions. Access cavies were left open for one month in order to leave sufficient duration for establishment of periapical pathosis (66,67). Radiographic monitoring for periapical pathosis was not essential because the infection time was insufficient to establish definite detectable periapical radiolucency.

Failure to use rubber dam for isolation was attributed to the morphology of premolar teeth in which it was very difficult to stabilize the rubber dam clamp to the tooth.

Discussion

70

Isolation was done using cotton rolls and atropine injection to decrease salivary flow during manipulation (51). Working length was measured using electronic apex locator as it define the working length by measuring the impedance with different frequencies between the file tip and the canal fluid, so this method are reliable, accurate and highly reproducible in locating the apical constriction regardless of electrolyte concentration (68). Biomechanical preparation is done with rotary instrumentation because it is easier to perform and provides a more appropriate standardization of root canals space (69,70), A volume of 20 mL/canal of 1.5% NaOCl for 5 min was used for root canal irrigation using irrigating needle with a closed end and side-vents positioned about 1 mm coronally from the root end to minimize cytotoxicity to the stem cells in the apical tissues (71), 17% EDTA (1 mL/canal, 1 min) irrigant was used at it stimulates the release of growth factors contained in the dentine matrix (72,73).

calcium hydroxide Ca(OH)2 paste was used as disinfectant material for two weeks, The earliest case reports describing the disinfection protocol in the treatment with REPs involved medicating the canals with a combination of antibiotics (74,75). However, other earlier reports advocated the use of calcium hydroxide exclusively (76,77). The vast majority of published cases seem to have employed a combination of antibiotics as the preferred intracanal medicament (78). However, these antibiotic pastes have been shown to be cytotoxic to the survival of SCAP in concentrations equal to or higher than 1 mg/mL in in vitro studies. In contrast, the disinfection of root canal space with calcium hydroxide promoted the proliferation of SCAP (79,80). Calcium hydroxide also increased the release of

Discussion

71

growth factors from dentine, whereas antibiotic pastes negatively influenced growth factor release after the use of EDTA (72,81). The AAE clinical considerations for a regenerative endodontic procedure advocate the use of either a combination of antibiotic paste or calcium hydroxide paste (71). A retrospective radiographic study employing a quantitative analysis of teeth treated with REPs showed that teeth medicated with calcium hydroxide had a significantly greater increase in root length than the teeth medicated with the combination antibiotic paste . The placement of calcium hydroxide within the canal appeared to favorably affect the outcome of the treatment (82).

GIC were used for interim restoration as it has long term sealing ability and it should be used when placed for variable periods of time, the sealing ability is due to electrochemical bonding ability to tooth structure (83,84).

AH Plus is an epoxy-resin based sealer that was developed as a substitute of its former AH26 which is toxic as it gives off some formaldehyde during the setting reaction. It showed better long-term sealing ability compared to conventional sealers due to its reported expansion over time. It is biocompatible and more radiopaque, has a shorter setting time (approximately 8 hours), lower solubility, and a better flow characteristics compared with AH26 (85,86). Continuous wave of condensation technique were used for root canal obturation as it provide better adaptation of gutta-percha to canal walls with fewer voids and greater density, this technique has faster obturation time compared to other techniques (87,88), also it has been found superior to some other canal filling techniques in terms of apical leakage and procedural time (89,90).

Discussion

72

Histological evaluation included assessment of periapical reaction and tissue in-growth inside pulp space for regenerative groups. Periapical assessment included inflammatory tissue reaction and bone resorption for all groups. Pulp space evaluation included new tissue in- growth inside the canal in terms of extension and characteristics. New hard tissue deposited on inner dentinal walls was also evaluated. Summing up these histologic assessment is needed for obtaining comprehensive conclusions regarding management of mature necrotic teeth with apical periodontitis.

Results of the animal study showed that the RCT group showed the highest inflammatory cell count among experimental groups, it may be attributed to root canal obturation technique used as the studies demonstrated that continuous wave obturation associated with more apical sealer extrusion than other methods of root canal obturation methods (89,90). Other factor may be related to biocompatibility of AH Plus root canal sealer, Silveira et al. (91) revealed that AH Plus showed mild inflammatory reaction after 15 and 30 days, Scarparo et al. (92) found that AH Plus promotes a mild inflammatory reaction, mainly after longer periods (60 days). The regenerative endodontic groups showed less inflammatory cell counts compared to RCT group, the presence of inflammatory cells are attributed to the immunological reaction against the previously induced infection and to the immediate inflammatory reaction of the periradicular tissues to traumatic insult via over instrumentation to induce bleeding.

The reduction of number of inflammatory cells for all experimental groups after the 2nd time evaluation period for

Discussion

73

RCT group could be attributed to resolving of inflammatory response of sealer over time being composed mainly by plasma cells and macrophages (91,93), and for regenerative endodontic groups could be attributed to immune-competent cells of regenerated vital tissue can function effectively overtime, the regenerated vital tissue in the canals after RET is also endowed with immune defense mechanisms. Therefore, healing could occur in the infected canals after RET depending on the numbers of bacteria and their virulence and the host’s immune defense (36,42,45,47).

In both evaluation period results have revealed that increase size of apical diameter were associated with decrease the number of inflammatory cell for regenerative endodontic groups and this is in argument with Borlina et al (94) who documented that in cases of apical periodontitis, intra-canal bacteria can penetrate dentin to a depth of 150-250 μ, where they remain protected from the action of medicament and irrigants. Therefore, apical canal widening to 300-500 μ is required to thoroughly cleanse the apical portion of the canal. Positive control group showed significantly higher inflammatory reaction due to progression of the infection induced previously without any mean of eradication. These findings concerning the presence of inflammatory cell were in argument with da Silva et al (54), and Wang et al (95), who stated that mild inflammatory reaction exists regardless of new tissue in-growth following revascularization protocol.

Regarding the bone resorption; root resorptions have two basic mechanisms of occurrence, inflammation or replacement. Any cause that is active in the site where cementoblasts are found and that removes them rom the

Discussion

74

surface will expose the mineralized root surface; the bone cells, which are very close, will promote root resorption, though maybe only temporary. Root resorptions have local causes that eliminate cementoblasts from the root surface, and no systemic cause is known to produce the same effect. (96).

Bone/root resorption was highly evident in the majority of samples within all groups at the 1st evaluation period, this can be attributed to the inflammatory reaction associated with release of inflammatory mediator as prostaglandins and interleukins that result in bone resorption (97). Due to slightly subsided inflammation in all groups after three month evaluation period, the percentage of root resorption also declined without significant difference between both subgroups. These findings concerning the presence of bone resorption were in argument with da Silva et al (54), who stated that presence of sign of resorption in some revascularization samples could occur up to three months postoperatively.

Concerning to the tissue in-growth inside root canal space; the tissue in-growth mechanism is still mysterious. Many mechanisms (98) have been postulated explaining the mechanism of tissue regeneration inside the canal space.

One possible mechanism postulates that few vital pulp cells remain at the apical canal end (74). These cells might have the ability to proliferate and differentiate into odontoblasts guided by intact epithelial root sheath of Hertwig. Epithelial root sheath of Hertwig are thought to be resistant to destruction even in presence of inflammation.

Discussion

75

These odontoblasts are thought to be responsible of apexogenesis in cases of Revascularization

Another hypothesized mechanism: Abundance of dental pulp stem cells in immature teeth found at the apical end (99) might be seeded onto the dentinal walls. These cells are thought to be the precursors of new odontoblast responsible for continuity of root maturation.

The third possible mechanism depends on periodontal ligament stem cells (100,101). This hypothesis is valid in case of destruction of epithelial root sheath of Hertwig and apical papilla tissues. These cells have the ability to proliferate and grow within the apical end of the canal lumen through the wide open apex. In this case, it is not un common to see periodontal structure inside the canal lumen e.g. ectopic bone or cementum.

The fourth possible mechanism rely on stem cells of apical papilla (SCAP), in which instrumentation beyond apical limit of the canal leads to transplantation of SCAP cells into the canal lumen. SCAP cells may survive the infection and retain the capacity for proliferation and differentiation into bone or dentin forming cell (102,103).

The fifth possible mechanism involves the blood clot itself. The established blood clot is considered a reservoir of growth factors; Platelet derived growth factor, vascular endothelial growth factor, and tissue growth factors. This rich supply of growth factors stimulates differentiation, growth and maturation of fibroblasts, odontoblasts and cementoblasts from their undifferentiated precursors. Expression of vascular endothelial growth factor in immature teeth has been documented (104).

Discussion

76

In our study, Histologic evaluation of the newly formed tissue showed that the new regenerated tissue resembles periodontal tissue in structure. The newly deposited hard tissue resembled cementum, characterized by inclusion of cementocyte-like cells, direct attachment to dentin and fibrous attachment to the neighboring connective tissue. These finding are in agreement with the third and fourth hypothesis in which the source of the implanted cells is periodontal. The role of blood clot is considered beneficial as it supply the newly growing micro-organ with the necessary supply of growth factors.

Our histological evaluation revealed that increasing apical diameter was associated with increase the tissue in-growth inside root canal, this was in agreement with Kling et al (35) who showed that teeth with large apices involve increased revascularization rates, mainly because of increased possible ingrowth of newly formed blood vessels. The results showed that there were significance difference between tissue in-growth for RET-F3 and both RET-F4 and RET-F5 groups, while there were no significance difference between RET-F4 and RET-F5 group indicated that the size of the apical foramen enlargement has direct effect of tissue in-growth after regenerative endodontic procedure. These results is in argument with Laureys et al. (38) who showed that smallest apical diameter teeth ranging between 0.24 and 0.53 mm demonstrated new tissue formation in at least one third of the root canals. However, teeth with apical diameter between 0.32 and 0.65 mm showed vital tissue regeneration in the entire root canals space.

Positive group recorded significantly lower scores for tissue in-growth. This may be due to absence of blood clot

Discussion

77

(scaffold) rendering the conditions unfavorable for tissue growth within the canals. Also higher level of inflammation reported may prevent regenerative ability of tissues for growth and proliferation. Few samples showed limited growth inside the canal lumen. This may be due to a higher self-growing capacity of the periapical granulation tissues in some samples.

Some samples show signs of hard tissue deposition at the MTA – tissue interface. Few samples showed formation of complete calcific barrier. This tissue response is believed to be a result of good sealing ability (105), bio-mineralizing ability (106) and alkaline pH (107). Calcium and phosphate ions had the capacity to attract blastic cells and promote a favorable environment for cementum deposition (108,109).MTA has osseous and cemento conductive effect via induction of release of mediators as osteocalcin, some interleukins and alkaline phosphatase (110).

Our finding showed that the majority of experimental groups form partial new hard tissue (score 2) with no statistically significance different between them, this may be attributed to the similarity in the repair mechanism in all groups. The positive control group showed no hard tissue deposition, this may be due to absence of regenerated tissue responsible for hard tissue deposition. This results were in agreement with da Silva et al (54) and Wang et al (95) they reported deposition of cementum like at the apical third regarding revascularization samples.

Summary & Conclusion


78

The aim of the present investigation was to histopathological assessment of the regenerative potential of permeant mature teeth with necrotic pulp and apical periodontitis with different apical diameters in comparing to conventional endodontic treatment.

72 premolars in six health dogs (12 premolars in each dog) to sum 72 teeth and 144 root canals were collected for this study. Teeth were distributed into sex groups: root canal obturation (RCT) with continuous wave compaction for root canals prepared up to #F3 ProTaper file, Regenerative endodontic therapy for root canals prepared up to #F3 ProTaper file (RET-F3), RET for root canals prepared up to a #F4 ProTaper file (RET-F4), RET for root canals prepared up to a #F4 ProTaper file (RET-F5), positive (P) and negative (N) controls included infected and healthy teeth respectively. Each group was further subdivided into two subgroups according to evaluation periods (one and three months).

Induction of infection was done followed by canal disinfection using the Ca(OH)2 paste. Treatment protocols were performed according to the previous classification and samples were left for follow up according to the period for each sup-group.

Histological evaluation included inflammatory reaction, bone/resorption, tissue in-growth, hard tissue formation, quantitatively results were evaluated using appropriate scoring system. Data were collected, tabulated and statistically analyzed.

Histological evaluation of inflammatory cell count showed that the RCT group had the highest count comparing


79

to RET groups, and increasing the apical size diameter among RET groups resulted in decrease in inflammatory cell count in both evaluation period. After three month evaluation period there was no statistical significance difference between RET-F4 and RET-F5 groups.

Regarding percentage of bone resorption, RCT group showed less bone resorption percentage than the rest of experimental groups. After three month evaluation period there were no statistical significance difference among all experimental groups.

Concerning tissue in-growth inside root canal space; increasing the apical diameter was associated with more tissue in-growth inside root canal space with no statistical significance difference between RET-F4 and RET-F5 groups. The nature of this tissue resembles periodontal connective tissue with variable amounts of inflammatory cell infiltration and low to moderate number of blood vessels at central portion that were engorged by red blood cells (RBCs).

When hard tissue formation was investigated, results showed variety of hard tissue formation inside root canal space, there were no statistical significance difference among all experimental groups regarding scores of new hard tissue formation.

There were no statistical significance difference between 1st and 2nd evaluation period for our tested parameters except inflammatory cell counts that showed significate reduction after the 2nd evaluation period.


80

Conclusion :

Under the circumstances of this study, it can be concluded that:

1- Regenerative endodontic therapy was successfully decrease the inflammatory cell count for mature teeth with necrotic pulp and apical periodontitis,

2- Revascularization procedure induced tissue resembling pulp-like tissue and not true pulp tissue inside the root canal space.

3- The minimum apical diameter that could be used for RET was 0.4 mm.

Recommendations:

1- Longer follow up period is needed for monitoring of the repair process.

2- Further studies are recommended for using different irrigates to disinfect the canal space and release growth factors found in dentin.

3- Further studies are recommended for using tissue engineering protocol (cell based approach).

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يبرعلا صخلملا


ھمدقم اًرخؤم دتما ھنكلو ، ةجضانلا ریغ نانسألا ىلع يدیدجتلا يبللا ءارجإلا رصتقا .يدیلقتلا يلخادلا جالعلل لیدبك ةجضانلا نانسألا لمشیل تایدحتلا نم ریثكلا ھجاوی ومنلا ةلماكلا نانسألا يف يدیدجتلا يبللا جالعلا نانسألا يف ةیعذجلا ایالخلا ةلقل كلذو ,ومنلا ةلمتكم ریغلا نانسألاب ةنراقم يف ةیعذجلا ایالخلا ةرجھل ةمزاللا ةیرذجلا تارمملا قیضلو ومنلا ةلمتكملا هذھ يف ةیرذجلا تاونقلا ریھطت يف ربكأ ةبوعص يلإ ةفاضإلاب ،يمقلا هاجتالا نانسألا ثحبلا اذھ نم فدھلا ةجضان نانسأل يبللا ددجتلا ةیلامتحال يجیسنلا مییقت وھ ثحبلا اذھ نم فدھلا .ةیدیلقتلا ةیبللا جالعلا عم اھتنراقمو ةفلتخم ھیمق راطقأ عم يمقلا ةثللا باھتلا عم

ثحبلا ھقیرط عمجل )بلك لك يف كحاوضلا ١٢( ھمیلس بالك ةتس يف كحاض ٧٢ عمج مت

ىلع نانسألا عیزوت مت .ةساردلا هذھل رذجلا تاونق ١٤٤ و نانسألا 72 :يتالاك تاعومجم

مم ٠.٣ يمق رطقب يدیلقتلا يلخادلا جالعلا-١ مم ٠.٣ يمق رطقب يدیدجتلا يبللا جالعلا -٢ مم ٠.٤ يمق رطقب يدیدجتلا يبللا جالعلا -٣ مم ٠.٥ يمق رطقب يدیدجتلا يبللا جالعلا -٤ .ةباصم ھنراقم ھعومجم-٥ .ةیحص ھنراقم ھعومجم -٦

دحاو رھش( مییقتلا تارتف بسح ةیعرف تاعومجم ىلإ ةعومجم لك میسقت مت .)رھشأ ةثالثو جئاتنلا يلخادلا جالعلا ةعومجملا نأ ةیباھتلالا ایالخلا ددعل يجیسنلا مییقتلا رھظأ ةدایز نأو ، يدیدجتلا يبللا جالعلا تاعومجمب ةنراقم ددع ىلعأ اھیدل يدیلقتلا يف ضافخنا ىلإ ىدأ يدیدجتلا يبللا جالعلا تاعومجم نیب يمقلا مجحلا رطق

.مییقتلا ةرتف يف ةیباھتلالا ایالخلا ددع يدیدجتلا يبللا جالعلا ةعومجم ترھظأ ، مظعلا لكآتل ةیوئملا ةبسنلا صوصخب ةثالث ةدمل مییقتلا ةرتف دعب .ةیبیرجتلا تاعومجملا يقاب نم لقأ ماظع لكآت ةبسن .ةیبیرجتلا تاعومجملا عیمج نیب ةیئاصحإلا ةلالدلا قرف كانھ نكی مل رھشأ


يمقلا رطقلا ةدایز طبترا ؛ رذجلا ةانق ةحاسم لخاد ةجسنألا ومنب قلعتی امیف ةیئاصحإ ةلالد قرف دوجو مدع عم رذجلا ةانق ةحاسم لخاد ةجسنألا نم دیزمب .ةعبارلا و ةثلاثلا نیتعومجملا نیب نكی ملو ، رذجلا ةانق ةحاسم لخاد ةبلصلا ةجسنألا نیوكت عونت جئاتنلا ترھظأ .ةیبیرجتلا تاعومجملا عیمج نیب ةیئاصحإلا ةلالدلا يف فالتخا كانھ

بل تاذ ومنلا ةلمتكم نانسأل ددجتلا ةیناكمإ

يمق ھثل باھتلابو تیم

بط ةیلكب روذجلا جالع مسق يلا مدقم ھلاسر عورشم ةجرد ىلع لوصحللً ادیھمت سمش نیع ةعماج نانسألا

روذجلا جالع يف هاروتكدلا

بیبطلا نم ةمدقم

ةدابع يدمح دومحم ماشھ

)٢٠٠٨( نانسألاو مفلا ةحارجو بط سویرولاكب )٢٠١٥( روذجلا جالع ریتسجام

٢٠١٩

فارشإ تحت

مشاھ نمحرلا دبع دمحأ /د.أ

روذجلا جالع ذاتسأ نانسألاو مفلا بط ةیلك

سمش نیع ةعماج

هدعس وبأ نمحرلا دبع دمحم فرشأ /د.أ

ةعشألا و ریدختلاو ھحارجلا ذاتسأ يرطیبلا بطلا ةیلك

ةرھاقلا ةعماج

يجان راتخم دمحم /د.أ روذجلا جالع دعاسم ذاتسأ

نانسألاو مفلا بط ةیلك سمش نیع ةعماج

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