Efficiency of Triple Antibiotic Mixture and Propolis as Intracanal Medication in Revascularization process in immature apex: A clinical study

Introduction: The aim of this clinical study was to evaluate the effect of different antibacterial combinations on the outcome of revascularization process in permanent anterior immature teeth.

Methods: Immature necrotic permanent maxillary incisors (n = 40) of patients 8–18 years old were divided into 4 groups according to the intracanal medicament: group 1 was treated with Triple Antibiotic Paste (TAP), group 2 was treated with Ciprofloxacin + Propolis paste (CP), group 3 was treated with Ciprofloxacin + Metronidazole paste (CM), group 4 was treated with Propolis + Metronidazole paste (PM). Cases were followed at regular intervals up to 18 months clinically and radiographically.

Results: All cases showed radiographic evidence of periapical healing and continued root development. No statistically significant difference in root length, thickness, apical closure or periapical density between all groups through the whole follow up period.

Conclusion: Propolis is successful substitute for ciprofloxacin or metronidazole in DAP for effective disinfection in revascularization process.

Introduction

In the last 10 years, numerous published cases and case series described the revascularization or regenerative endodontics. Revascularization is a conservative method for inducing maturogenesis in necrotic immature teeth [1]. The favorable outcomes of regenerative endodontics are largely dependent on the adequate disinfection of the root canal [1]. These canals with compromised fragile underdeveloped dentinal walls represent a contraindication for mechanical instrumentation; thus, chemical debridement remains the main form of disinfection [1]. A mixture of ciprofloxacin, metronidazole, and minocycline, known as the triple antibiotic paste (TAP), has been shown to be very effective in eliminating endodontic pathogens in vitro and in situ [1,3-5]. However, TAP has an adverse effect on stem cell survival [6]. Discoloration of the tooth is a problem mostly related to the use of minocycline in TAP [7]. Moreover, TAP can demineralize dentin resulting in reduced microhardness and fracture resistance [8]. Propolis, a flavonoid-rich resinous product of honeybees, is ten times less cytotoxic than calcium hydroxide and has a well-known antibacterial effect [9]. Therefore, creating alternatives to the TAP for disinfecting the root canal of necrotic teeth during the process of pulp revascularization is thought to be of value. The null hypothesis in the current study stated that there is no difference between the tested intracanal medications on the outcome of the revascularization process in immature teeth.

Materials and Methods

Forty patients with immature, non-vital maxillary incisors were included in this study from the outpatient clinic of the Faculty of Dentistry, Ain Shams University, Cairo, Egypt. A detailed medical and dental history was obtained from each patient’s parents or guardians. Only medically free patients were included in this research. The clinical and radiographic exclusion criteria were teeth with vertical fractures, periodontally involved teeth, and nonrestorable teeth. All procedures were performed after obtaining proper institutional review board approval based on the regulations of the Ethical Committee of the Faculty of Dentistry, Ain Shams University (FDASU-RECID011508). Intraoral periapical radiographs revealed immature apices. The age of the patients ranged between 8 and 18 years. Informed consent was signed for each case by the patient’s parents or guardians including the proposed treatment and possible outcomes or complications.

Cases were divided randomly and equally into 4 groups according to the intracanal medicament (10 patients for each group):

TAP group: was treated with Triple Antibiotic Paste

CP group: was treated with Ciprofloxacin + Propolis paste

CM group: was treated with Ciprofloxacin + Metronidazole paste

PM group: was treated with Propolis + Metronidazole paste

1). Triple Antibiotic Paste (TAP): It consisted of Ciprofloxacin (Ciprocin 250 mg tablets; EPICO, Cairo, Egypt), Metronidazole (Flagyl 500 mg tablets; Sanofi Aventis Pharma, Cairo, Egypt), Doxycycline (Vibramycin 100 mg capsules; Pfizer, Cairo, Egypt). One Doxycycline capsule content was evacuated in a sterile mortar, one tablet of metronidazole and one tablet of ciprofloxacin were crushed and ground in the same mortar using a pestle into homogenous powder. Saline drops (Otrivin baby saline; Novartis, Cairo, Egypt) were added and mixed using the pestle until a creamy paste was achieved [4,10].

2). Ciprofloxacin + Propolis Paste: Ethanol extract of raw propolis (EEP; ElEzaby Co. Labs, Cairo, Egypt.) was prepared by adding 10 gm of propolis (Imtinan, Cairo, Egypt) to 40 gm of 70% ethanol (ElGomhorya Co., Cairo, Egypt) (for 20% tincture) in a dark container to prevent reduction of propolis. The container was sealed and placed at room temperature for a period of three weeks. The sealed container was manually shaken every 2 days to ensure proper mixing. After 3 weeks, the container was opened and ethanol extract of propolis was obtained. Ethanol-free EEP was made by evaporating the ethanol in a water bath [11,12]. EEP was then mixed with Ciprofloxacin powder in the ratio 1:1. Saline drops were added and mixed using the pestle until a creamy paste was achieved.

3) Ciprofloxacin + Metronidazole Paste: Ciprofloxacin powder was mixed with Metronidazole powder in the ratio 1:1. Saline drops were added and mixed using the pestle until a creamy paste was achieved.

4) Propolis + Metronidazole paste: EEP was mixed with Metronidazole powder in the ratio 1:1. Saline drops were added and mixed using the pestle until a creamy paste was achieved.

A preoperative radiograph was taken using the standardized paralleling technique by the Rinn XCP alignment system (Rinn Corporation Elgin, Illinois, USA) and Fona ScaNeo intra-oral digital imaging system (FONA Dental, Bratislava, Slovak Republic). Patients were treated as follows: Caries was excavated; access cavity was prepared. The rubber dam was then applied and working length was determined with a periapical radiograph taken with a file inserted into the canal. Canal space was debrided using K file size #80. Canal space was irrigated using 40 cc of NaOCl 2.6% solution and final flush of saline. Canal space was dried using paper points. The antibiotic paste was prepared as previously described. One cubic centimeter of the prepared paste was injected into the canals using a sterile plastic syringe with 20” gauge needle. Care was taken to avoid apical extrusion and to minimize placement in the coronal portion of the tooth. The access cavity was then sealed using temporary restoration (Coltosol F; Coltene Whaledent, Altstatten, Switzerland) over plain cotton. Coltosol F is a long-term temporary restoration. After the 3 weeks, under the same aseptic conditions, anesthesia without vasoconstrictor (Mepecaine, Alexandria Co., Alexandria, Egypt) was administrated. The tooth was re-entered, the antibiotic paste was removed and the canal was irrigated using a sterile saline and dried using paper points [4,13].

Sterile hand file size #25 was introduced into the root canal and placed at 2 mm beyond the working length to induce bleeding into the canal. The bleeding was allowed to reach a 3-mm level below the cemento-enamel junction, and teeth were left at rest for 5 minutes so that a blood clot could be formed [14]. Next, a 3-mm plug of MTA (Angelus; Londrina, Brazil) was inserted into the canals using a suitable-sized amalgam carrier to seal the root canal at the cervical level. The MTA plug was verified radiographically. The MTA plug was then covered by moist cotton and temporary filling. After one week, MTA setting was confirmed clinically, adhesive composite resin (Z250 Restorative; 3M ESPE, St Paul, Minnesota, USA) was used to seal the access cavity [15-17] Figure 1.

Figure 1: Photographs showing the clinical steps of pulp revascularization; A) after cleaning and irrigation with NaOCl solution, B) after intracanal application of TAP or CM, C) after intracanal application of CP or PM, D) dry clean canals after removal of intracanal medication, E) blood clot induced up to the level of canal orifice, F) MTA orifice plug.

Evaluation:

Patients were recalled for follow up at 3, 6, 9, 12, and 18 months. Follow-up included the clinical assessment of pain and/or swelling, and standardized radiographic assessment for:

1. An increase in root length


2. An increase in root thickness


3. A decrease in apical diameter


4. A change in periapical bone density

All follow-up radiographs were standardized using the Rinn XCP alignment system and Fona ScaNeo intra-oral digital imaging system. All radiographic measures were collected by the same investigator. All radiographic measurements were repeated after 1 week, and the mean of the 2 sets was considered as the final value.

Increase in root length: A measuring scale was set in the Image-J software (Image-J v1.44, US National Institutes of Health, Bethesda, MD) by measuring a known clinical dimension to its radiographic dimension. The scale was calculated as number of measured pixels per mm length. Root length was measured as a straight line from the cemento- enamel junction to the radiographic apex of the tooth [4,18] in millimeters. Pre and follow-up root lengths were measured using Image-J analysis software. Difference in length was calculated. Percentage of increase in length was calculated as follows: percentage of increase in length = [(postoperative length – preoperative length) / preoperative length] x 100.

Increase in root thickness: Using the preset measurement scale, the level of the apical third was determined and fixed from the cemento-enamel junction. The root thickness and the pulp width were measured mesiodistally at this level in millimeters. Mesiodistal dentin thickness was measured by subtraction of the pulp space from the whole root thickness. Pre and follow-up root thicknesses were measured. Measurements were done pre and post operatively at the same fixed level [18]. Difference in dentin thickness was calculated. Percentage of increase in dentin thickness was calculated as follows: percentage of increase in dentin thickness = [(postoperative thickness – preoperative thickness) / preoperative thickness] x 100.

Decrease in apical diameter: Using the preset measurement scale, the mesiodistal diameter of the apical foramen was measured in millimeters [4]. Measurements were done pre and postoperatively. Percentage of apical closure was calculated as follows: percentage of apical closure = [(preoperative apical diameter – postoperative apical diameter) / preoperative apical diameter] x 100.

Periapical bone density: Periapical bone density was estimated using Image-J software as follows: periapical area was located and analyzed for bone density. Average area density was measured in scale from 0 (black) to 255 (white) and recorded for each radiograph. The same area was then measured in subsequent radiographs and average densities were recorded for the follow up radiographs [4]. The difference between densities was calculated between subsequent radiographs [4,5]. Percentage of change in density was calculated from the original pre-operative radiograph density as follows: percentage of change in density = [(postoperative bone density - preoperative bone density)/preoperative bone density] x 100.

Data were collected, tabulated and statistically analyzed using statistical analysis software SPSS (Statistical Packages for the Social Sciences 19.0, IBM, Armonk, NY). Two-way analysis of variance was performed. The Tukey post hoc test was used in case of significance.

Results

A total of five cases were dropped out from the study, four of which was excluded due to poor patient compliance. Clinical and radiographic examination during the follow-up period showed signs and symptoms of failure in 1 case, which was re-evaluated, and the treatment plan was shifted to MTA apexification. Clinical outcomes are presented in Table 1.

Radiographic changes:

I. Increase in root length: Figure 2

Figure 2: Representative case in TAP; A: preoperative radiograph, B: 6 month follow up radiograph, C: 12 month follow up radiograph, D: 18 month follow up radiograph. Note the increase in root length from (a-b) in the preoperative radiograph to (a-c) in the postoperative radiograph after 18 months of follow up.

 Effect of type of intracanal medication:

Statistical analysis revealed significant effect of time on increase in root length. However, no statistically significant difference was found between 3 months and 6 months, nor between 6 months and 9 months, nor between 9 months and 12 months (P > 0.05) Table 2

 Effect of time:

Statistical analysis revealed significant effect of time on increase in root length. However, no statistically significant difference was found between 3 months and 6 months, nor between 6 months and 9 months, nor between 9 months and 12 months (P > 0.05) Table 2.

II. Increase in root thickness: Figure 3

Figure 3: Representative case in group CP; A: preoperative radiograph, B: 6 month follow up radiograph, C: 12 month follow up radiograph, D: 18 month follow up radiograph. Note the increase in root thickness indicated by narrowing of pulp space from (a) in the preoperative radiograph to (b) in the postoperative radiograph after 18 months of follow up.

 Effect of type of intracanal medication:

Statistical analysis showed that there was no statistically significant difference between all groups through the whole follow up period (P > 0.05) Table 3.Effect of time:

No statistically significant difference was found between 6 months and 9 months (P > 0.05). However, statistically significant difference was found among other follow up periods Table 3.

III. Decrease in apical diameter: Figure 4

Figure 4: Representative case in group CM; A: preoperative radiograph, B: 6 month follow up radiograph, C: 12 month follow up radiograph, D: 18 month follow up radiograph. Note the decrease in apical diameter from (a) in the preoperative radiograph to (b) in the postoperative radiograph after 18 months of follow up.

 Effect of type of intracanal medication:

Statistical analysis showed that there was no statistically significant difference between all groups through the whole follow up period (P > 0.05) Table 4. Effect of time:

Statistically significant difference was found among all follow up periods Table 4.

IV. Periapical bone density: Figure 5

Figure 5: Representative case in group PM; A: preoperative radiograph, B: 6 month follow up radiograph, C: 12 month follow up radiograph, D: 18 month follow up radiograph. Note the increase in periapical bone density from (a) in the preoperative radiograph to (b) in the postoperative radiograph after 18 months of follow up.

 Effect of type of intracanal medication:

Statistical analysis showed that there was no statistically significant difference between all groups through the whole follow up period (P > 0.05) Table 5.

 Effect of time:

Statistically significant difference was found among all follow up periods. Significant improvement in bone density was found after 9 months of follow up in each group Table 5.

Discussion

Triple antibiotic paste (TAP) has been found to have antimicrobial properties and to be biocompatible [19]. Ciprofloxacin inhibits DNA gyrase synthesis, metronidazole inhibits DNA synthesis, and minocycline inhibits protein synthesis of microbes [15]. Because case reports have shown that minocycline causes visible crown discoloration [20,21]. In our study, Saline drops were added and mixed with the antibiotic combination until a creamy paste was achieved. This is consistent with most case reports, in which there was no attempt to deliberately deliver a specific concentration of the drugs. Instead, drugs were mixed until a certain physical consistency was achieved that was deemed suitable by clinicians [4].

Propolis was used as an intra-canal irrigant and has been found to be effective in reducing endotoxins and inhibiting E. faecalis, Streptococcus aureus, Candida albicans and E. coli [22]. Also, the co-adjuvant activity of propolis in antimicrobial therapy was confirmed [23]. In vitro experiments have shown that propolis presents a high level of artepillin-C, inhibiting the cyclo-oxygenase pathway and eicosanoid synthesis [24]. In addition, studies have observed that propolis promotes regeneration of dental pulp [25].

Immature permanent teeth with necrotic pulp and an apical opening that measures 1 mm or larger were selected because they are considered suitable candidates for treatment [26,27]. Mechanical instrumentation is contraindicated, not only to avoid further weakening the thin root canal walls, but also to protect the vitality of stem cells of apical tissues [2]. However, Lin et al. 2014 published a failed clinical case of regenerative endodontic therapy due to the bacterial biofilms that remained firmly attached to the apical canal walls because of the lack of mechanical debridement [28]. Therefore, there is a trend for even slightly mechanical instrumentation in recent publications [4,29], which was made in our study.

Our clinical observations are in agreement with Paryani and Kim [30]. Our clinical results are also in full agreement with a retrospective study, which demonstrated 100% survival rate of teeth after regenerative treatment [31]. In a retrospective study of 30 cases, followed for up to 1 year, only two cases had complications, which were restricted to discomfort or discoloration [17], which is consistent with our study.

The bone density results in our study showed an increase in the radio-opacity of previously rarefacted areas. This indicates a favorable healing response to the treatment. In the majority of our cases, improvement or resolution of the apical lesion was evident in approximately 6 months. Root elongation and apical closure, with thickening of the root canal walls, was evident after 12-16 months postoperatively.

Our findings concerning the increase in length and thickness were in agreement with many authors [5,19,26,32]. Our findings were also in agreement with other authors [33,34], who reported an increase in length and thickness without quantitative measurements. The results of our study are in disagreement with Lin et al., [28], where the failure was attributed to inadequate root canal disinfection without mechanical removal of biofilm and bacteria in dentinal tubules.

All of groups, except group 3 (DAP-2, Ciprofloxacin+Metronidazole), have shown cervical discoloration, which can be due to using minocycline or propolis in the intracanal medicament [35,36]. There are reports that white MTA may also discolor the tooth structure [7,37,38].

Conclusions

All tested intracanal medicaments paved the way for successful pulp revascularization process in immature permanent anterior teeth, which was evident by continued root growth and resolution of periapical radiolucency. Thereofore, the null hypothesis was accepted.

Recommendations

Follow up visits should be scheduled every 6 months, rather than 3 months, to observe root changes after pulp revascularization procedure. Longer follow up period (>18 months) is needed for monitoring of complete root maturogenesis.

The authors deny any conflict of interest related to this study.

1. Law A (2013) Considerations for Regeneration Procedures. J Endod 39:44–56. Link: https://bit.ly/2YALJjQ  
2. Wigler R, Kaufman AY, Lin S, Steinbock N, Hazan-Molina H, et al. (2013) Revascularization: A Treatment for Permanent Teeth with Necrotic Pulp and Incomplete Root Development. J Endod 39: 319–326. Link: https://bit.ly/2yuyp1x  
3. Hoshino E, Kurihara-Ando N, Sato I, Uematsu H, Sato M, et al. (1996) In Vitro Antibacterial Susceptibility of Bacteria Taken from Infected Root Dentine to a Mixture of Ciprofloxacin, Metronidazole and Minocycline. Int Endod J 29:125-130. Link: https://bit.ly/2LT9Zrj
4. Nagy M, Tawfik H, Hashem A, Abu-Seida A (2014) Regenerative Potential of Immature Permanent Teeth with Necrotic Pulps after Different Regenerative Protocols. J Endod 40: 192–198. Link: https://bit.ly/2T0LWHD
5. Tawfik H, Abu-Seida A, Hashem A, Nagy M (2013) Regenerative Potential following Revascularization of Immature Permanent Teeth with Necrotic Pulps. Int Endod J 46: 910–922. Link: https://bit.ly/2ovU59e  
6. Ruparel N, Teixeira F, Ferraz C, Diogenes A (2012) Direct Effect of Intracanal Medicaments on Survival of Stem Cells of the Apical Papilla. J Endod 38: 1372–1375. Link: https://bit.ly/338epA7  
7. Nosrat A, Homayounfar N, Oloomi K (2012) Drawbacks and Unfavorable Outcomes of Regenerative Endodontic Treatments of Necrotic Immature Teeth: A Literature Review and Report of a Case. J Endod 38: 1428–1434. Link: https://bit.ly/2OBGdtt  
8. Berkhoff J, Chen P, Teixeira F, Diogenes A (2014) Evaluation of Triple Antibiotic Paste Removal by Different Irrigation Procedures. J Endod 40: 1172–1177. Link: https://bit.ly/2SYeBx9  
9. Al-Shaher A, Wallace J, Agarwal S, Bretz W, Baugh D, et al. (2004) Effect of Propolis on Human Fibroblasts from the Pulp and Periodontal Ligament. J Endod 30: 359–61. Link: https://bit.ly/2SZAaNG  
10. Sabrah A, Yassen G, Gregory R. (2013) Effectiveness of Antibiotic Medicaments against Biofilm Formation of Enterococcus Faecalis and Porphyromonas Gingivalis. J Endod 39: 1385-1389. Link: https://bit.ly/2MAfD1g  
11. Sosnowski, Z. (1983) Method for Extracting Propolis and Water Soluble Dry Propolis Powder. United States Patent Link: https://bit.ly/2LWo4V8  
12. Savickas A, Ramanauskiene K, Pukalskas A, Giedrė Kasparavičienė, et al. (2010) The Influence of Solvent on The Quantity and Antioxidant Activity of Ethanolic Extracts of Lithuanian Propolis. Chemija 21: 118-122. Link: https://bit.ly/2MAphkq
13. Lenzi R, Trope M (2012) Revitalization Procedures in Two Traumatized Incisors with Different Biological Outcomes. J Endod 38: 411–414. Link: https://bit.ly/2SYgU3h
14. Nagata J, Soares A, Souza-Filho F, Zaia AA1, Ferraz CC, et al. (2014) Microbial Evaluation of Traumatized Teeth Treated with Triple Antibiotic Paste or Calcium Hydroxide with 2% Chlorhexidine Gel in Pulp Revascularization. J Endod 40: 778–783. Link: https://bit.ly/2SZCy78  
15. Rodriguez-Benitez S, Stambolsky C, Gutierrez-Perez J, Torres-Lagares D, Segura-Egea J (2015) Pulp Revascularization of Immature Dog Teeth with Apical Periodontitis Using Triantibiotic Paste and Platelet-rich Plasma: A Radiographic Study. J Endod 41: 1299–1304. Link: https://bit.ly/2KnEnHj  
16. Lovelace T, Henry M, Hargreaves K, Diogenes A (2011) Evaluation of the Delivery of Mesenchymal Stem Cells into the Root Canal Space of Necrotic Immature Teeth after Clinical Regenerative Endodontic Procedure. J Endod 37: 133–138. Link: https://bit.ly/2Yzjb61  
17. Garcia-Godoy F, Murray PE. (2012) Recommendations for Using Regenerative Endodontic Procedures in Permanent Immature Traumatized Teeth. Dent Traumatol 28: 33–41. Link: https://bit.ly/2KjxDKn  
18. Bose R, Nummikoski P, Hargreaves K (2009) A Retrospective Evaluation of Radiographic Outcomes in Immature Teeth with Necrotic Root Canal Systems Treated with Regenerative Endodontic Procedures. J Endod 35: 1343–1349. Link: https://bit.ly/2K9McBs  
19. Gomes-Filho J, Duarte P, de Oliveira C, Watanabe S, Lodi CS, et al. (2012) Tissue Reaction to a Triantibiotic Paste Used for Endodontic Tissue Self-Regeneration of Nonvital Immature Permanent Teeth. J Endod 38: 91–94. Link: https://bit.ly/2ZqwDKQ  
20. Jeeruphan T, Jantarat J, Yanpiset K, Suwannapan L, Khewsawai P, et al. (2012) Mahidol Study 1: Comparison of Radiographic and Survival Outcomes of Immature Teeth Treated with either Regenerative Endodontic or Apexification Methods - A  Retrospective Study. J Endod 38: 1330-1336. Link: https://bit.ly/2KkqcT9  
21. Kim J, Kim Y, Shin S, Park JW, Jung IY et al. (2010) Tooth Discoloration of Immature Permanent Incisor Associated with Triple Antibiotic Therapy: A Case Report. J Endod 36: 1086–1091. Link: https://bit.ly/2T2cYhU  
22. Arslan S, Ozbilge H, Kaya E, Ozgur E. (2011) In Vitro Antimicrobial Activity of Propolis, Biopure MTAD, Sodium Hypochlorite, and Chlorhexidine on Enterococcus Faecalis and Candida Albicans. Saudi Med J 32: 479-483. Link: https://bit.ly/2YwFI7J  
23. Victorino F, Franco S, Svidzinski T, Mário Julio Avila-Campos, Roberto Kenji Nakamura Cuman, et al. (2007) Pharmacological Evaluation of Propolis Solutions for Endodontic Use. Pharmaceutical Biology 45: 721–727. Link: https://bit.ly/2ZpxtaA  
24. Pileggi R, Antony K, Johnson K, Zuo J, Holliday L (2009) Propolis Inhibits Osteoclast Maturation. Dental Traumatology 25: 584–588. Link: https://bit.ly/2KdqPPM  
25. Neiva K, Catalfamo D, Holliday S, Wallet S, Pileggi R. (2014) Propolis Decreases Lipopolysaccharide-Induced Inflammatory Mediators in Pulp Cells and Osteoclasts. Dental Traumatology 30: 362–367. Link: https://bit.ly/2KjF9oz  
26. Estefan B, El-Batouty K, Nagy M, Diogenes A. (2016) Influence of Age and Apical Diameter on the Success of Endodontic Regeneration Procedures. J Endod 42: 1620–1625. Link: https://bit.ly/332CUPb
27. Kling M, Cvek M, Mejare I (1986) Rate and Predictability of Pulp Revascularization in Therapeutically Reimplanted Permanent Incisors. Endod Dent Traumatol 2: 83–89. Link: https://bit.ly/2ywvIwt
28. Lin L, Shimizu E, Gibbs J, Loghin S, Ricucci D, et al (2014). Histologic and Histobacteriologic Observations of
Failed Revascularization/Revitalization Therapy: A Case Report. J Endod 40: 291–295. Link: https://bit.ly/2YBrkuS
29. Alobaid A, Cortes L, Jeffery Lo, Nguyen TT, Albert J, et al. (2014) Radiographic and Clinical Outcomes of the Treatment of Immature Permanent Teeth by Revascularization or Apexification:
A Pilot Retrospective Cohort Study. J Endod 40: 1063–1070. Link: https://bit.ly/2yxYLQs
30. Paryani K, Kim S (2013) Regenerative Endodontic Treatment of Permanent Teeth after Completion of Root Development: A Report of 2 Cases. J Endod 39: 929–934. Link: https://bit.ly/2YB44Jr
31. Cehreli ZC, Ishitiren B, Sara S, Erbas G (2011) Regenerative Endodontic Treatment (Revascularization) of Immature Necrotic Molars Medicated with Calcium Hydroxide: A Case Series. J Endod 37: 1327-1330. Link: https://bit.ly/332Fc0t
32. Nosrat A, Seifi A, Asgary S (2011) Regenerative Endodontic Treatment (Revascularization) for Necrotic Immature Permanent Molars: A Review and Report of Two Cases with New Biomaterial. J Endod 37: 562-567. Link: https://bit.ly/31eeMaC
33. Banchs F, Trope M (2004) Revascularization of Immature Permanent Teeth with Apical Periodontitis: New Treatment Protocol. J Endod 30: 196-200. Link: https://bit.ly/2LY87gQ
34. Iwaya S, Ikawa M, Kubota M (2011) Revascularization of an Immature Permanent Tooth with Periradicular Abscess after Luxation. Dent Traumatol 37: 55-58. Link: https://bit.ly/31d26k9
35. Nagata J, Gomes B, Lima T, Murakami LS, de Faria DE (2014) et al. Traumatized Immature Teeth Treated with 2 Protocols of Pulp Revascularization. J Endod 40: 606–612. Link: https://bit.ly/2MxfwDq
36. Akcay M, Arslan H, Yasa B, Kavrık F, Yasa E, et al. (2014) Spectrophotometric Analysis of Crown Discoloration Induced by Various Antibiotic Pastes Used in Revascularization. J Endod 40: 845–848. Link: https://bit.ly/338crzo
37. Miller E, Lee J, Tawil P, Teixeira FB, Vann WF Jr, et al. (2012) Emerging Therapies for the Management of Traumatized Immature Permanent Incisors. Pediatr Dent 34: 66–69. Link: https://bit.ly/2GHaQr4
38. Lenherr P, Allgayer N, Weiger R, Filippi A, Attin T, et al. (2012) Tooth Discoloration Induced by Endodontic Materials: A Laboratory Study. Int Endod J 45: 942–949. Link: https://bit.ly/2LWe6Ts