Indian Journal of Dental ResearchIndian Journal of Dental ResearchIndian Journal of Dental Research
Indian Journal of Dental Research   Login   |  Users online:

Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size         


Table of Contents   
Year : 2021  |  Volume : 32  |  Issue : 2  |  Page : 216-220
Assessment of pulp sensibility in the mature necrotic teeth using regenerative endodontic therapy with various scaffolds – Randomised clinical trial

Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Click here for correspondence address and email

Date of Submission16-Mar-2021
Date of Decision24-Jun-2021
Date of Acceptance13-Jul-2021
Date of Web Publication22-Nov-2021


Aim: This study aimed to evaluate and compare the possibility of regaining pulp sensibility and objectives of regeneration procedure in mature necrotic teeth using regenerative endodontics. Materials and Methods: A total of 36 mature necrotic teeth were selected and a regenerative endodontic procedure was performed. A total of four groups, each involving n = 9 teeth per group were randomly grouped as (1) periapical bleeding, (2) platelet-rich fibrin (PRF), (3) collagen, and (4) hydroxyapatite. Pulp testing and radiographic assessment of periapical healing (PH) were recorded at every 3-month interval for 1 year. Readings were compared as qualitative data using the Kruskal–Wallis test to see the inter-group significant difference. Results: Readings of pulp sensibility revealed non-significant results in all intervals. At 3 and 6 months, 22.3% in the PRF group and 11.15% in collagen group; and at 9 months, 44.4% in PRF group, 33.3% in the collagen group and 22.2% in hydroxyapatite group; and at 12-months, 66.6% in PRF, 44.4% in the collagen group, 33.3% in the hydroxyapatite group and 11.1% in the periapical bleeding group showed a positive response to the cold test. But all groups did not show a positive response to heat and electric pulp testing and showed good PH in all groups at the end of 12 months. Conclusions: The presence of sensibility to cold test is indicative of the formation of vital pulp-like tissues, which was the highest in the PRF group, followed by the collagen, hydroxyapatite and periapical bleeding groups.

Keywords: Collagen, hydroxyapatite, PRF, regaining pulp sensation, regeneration in mature teeth, scaffolds

How to cite this article:
Mittal N, Baranwal HC, Kumar P, Gupta S. Assessment of pulp sensibility in the mature necrotic teeth using regenerative endodontic therapy with various scaffolds – Randomised clinical trial. Indian J Dent Res 2021;32:216-20

How to cite this URL:
Mittal N, Baranwal HC, Kumar P, Gupta S. Assessment of pulp sensibility in the mature necrotic teeth using regenerative endodontic therapy with various scaffolds – Randomised clinical trial. Indian J Dent Res [serial online] 2021 [cited 2022 Aug 13];32:216-20. Available from:

   Introduction Top

The principal and predominant objective of every endodontic treatment is the conservation of natural structures of what is left and the healing of damaged tissues due to infection. Routine treatment considered for necrosed pulp varies from apexification for non-vital immature teeth and conventional root canal therapy for mature non-vital teeth.[1],[2] Thus, a traditional technique involves the removal of the necrosed pulp and filling the canal space with inert artificially produced filling materials. Despite saving millions of teeth every year, these conventional endodontic treatments have certain drawbacks such as removal of dentinal tissues, less fracture resistance, tooth discolouration and loss of pulp tissue that leads to proprioception mechanism loss, which will eventually lead to the absence of a natural defence system of the tooth. Tissue engineering applied in endodontics leads to regeneration of pulp–dentin complex, healing of periapical and periodontal tissues, regeneration of bone and regaining the vitality of the tooth. Regenerating nerve fibres of pulp have numerous benefits such as regaining proprioceptive mechanism, which acts as a primary alarm and prevents tooth fracture.[3],[4]

Regenerative endodontic procedure (REP) results in resolution of signs and symptoms and healing of periapical lesion, and in some patients the results are promising because of regaining of sensibility of the pulp. So, the only way to predict the formation of regeneration tissue inside the canal is to assess the sensibility periodically with follow-up every 3 months after REP. Fewer studies have been conducted to compare the outcome of regeneration procedures in mature teeth.[5],[6],[7] This study compares the possibility of treatment result and regaining of pulp sensation after regeneration procedure in teeth with necrosed pulp and apical periodontitis using various scaffolds.

   Materials and Methods Top

This clinical study was conducted in the Department of Conservative Dentistry and Endodontics after getting approval from the Ethical Committee of the Institute (No. Dean/2019/EC/1150). Based on the previous study, the sample size was calculated using software G* power.[8] The total sample size was 36 (nine per group), and the power of the study was 95%. A total of about 36 teeth grouped into four groups as Group 1 – periapical bleeding, Group 2 – platelet-rich fibrin (PRF), Group 3 – collagen, Group 4 – hydroxyapatite–collagen were included in the study conducted in 2019–2020. The treatment protocol and every procedure involved in this study were clearly explained to the patients, and their informed consent and signed consent were taken before commencing the treatment. The clinical trials are registered in the Clinical Trials Registry – India with the registration number REF/2019/03/024813 (H).

Inclusion criteria: Patients in the age group between 16 and 34 years were randomly selected with no sex predilection; those suffering from the necrotic pulp in the permanent tooth with a closed apex associated with or without periapical radiolucency less than 1.5 cm and no response to thermal test and electric pulp testing were included.

Exclusion criteria: Patients with any medical condition, those with a tooth that required post and core, those with severely curved canals or those who had any allergies to the materials used in this study were excluded.


Two-visit regeneration procedure

At the first appointment, under rubber dam isolation access, a cavity was prepared with number 4 round diamond bur. After entering inside the pulp chamber, straight-line access was made using Endo-Z bur. The canal was negotiated with stainless steel smaller number 15 K-file, which was confirmed with apex locator and radiograph. The canal was cleaned by standardised conventional technique with a range of 60 to 80 number K-file for upper anterior teeth and minimum 30 number K-file for posteriors and lower anterior teeth to remove microorganisms and necrotic tissues. During instrumentation, copious irrigation was done with 20 mL of 1.5% sodium hypochlorite (NaOCl) and 10 mL of saline. The intracanal medicament was done with double-antibiotic paste (DAP), which was prepared by taking metronidazole tablet 500 mg and ciprofloxacin tablet 500 mg crushed together and mixed with normal saline to form a uniform homogeneous paste. This creamy paste of DAP was injected into the prepared dried canal up to a level below the cement-enamel junction (CEJ). The cavity was closed after placing the cotton pellet at the level of CEJ with glass ionomer cement.

At the second appointment after 2 weeks, local anaesthesia was administered with 3% mepivacaine without epinephrine, and the tooth was re-accessed under rubber dam isolation. The canal was irrigated thoroughly with 1.5% NaOCl and normal saline to remove the antibiotic paste. If the tooth shows the absence of infection and seepage, then the final irrigation was done with 10 mL of 17% ethylenediamine tetra-acetic acid (EDTA), and the canal was dried completely with paper points. Revascularisation technique was carried by intentional overinstrumentation of 2–3 mm beyond the apex with 15 to 25 K-file to induce bleeding inside the canal from the periapical region. The bleeding was controlled till the CEJ level with the help of a cotton pellet soaked in saline. In the periapical bleeding group, after controlling the bleeding, the canal was sealed with biodentine 1 mm below the CEJ and then sealed with glass ionomer cement (GIC) and composite restoration. In the PRF group, scaffolds were prepared from the patient's blood and centrifuged in the PRF machine at 2,700 rpm for 14 minutes. This resulted in the formation of three distinct layers: bottom red blood cell layers, middle PRF layer and upper layer of plasma. The PRF was carefully taken out and inserted inside the canal carefully with the help of PRF instruments and a finger plugger. In the collagen group, a sterile granule of synthetic collagen marketed by Eucare Pharmaceuticals, Chennai, India, was used and in the hydroxyapatite group, the crystals marketed by Surgiwear Company (India) were used. These synthetic collagen and hydroxyapatite crystals were mixed with normal saline and inserted into the canal after inducing bleeding with the finger plugger till the CEJ. Then, the cavity was filled with biodentine over the clot and finally restored with a permanent restoration. After this, the cavity was sealed with GIC and finally with a composite restoration.

A preoperative intra-oral periapical radiograph and thermal and electric pulp testing (EPT) were done before commencing the treatment to ensure that the tooth was non-vital with a score of 0 in all cases, which was considered as the baseline record. The clinical, radiographic and pulp testing (thermal and EPT) evaluations were done according to the manufacturer's instruction at an interval of 3, 6, 9 and 12 months follow-up to assess the regaining of sensibility or not [Figure 1]. The values were noted as 0 – if the tooth showed absence of any response, 1 – if the tooth showed a partial response and 2 – if the tooth showed a normal response when compared with the normal contralateral tooth and an average of the two readings were taken at 5-minute intervals by two observers at every follow-up interval.
Figure 1: (A) Preoperative radiograph, (B) Working length radiograph, (C) 3 months radiograph, (D) 6 months radiograph, (E) 9 months radiograph and (F) 12 months radiograph showing resolution of periapical lesion

Click here to view

   Results Top

All 36 teeth were evaluated clinically, radiographically and by pulp testing at an interval of 3 months up to 12 months follow-up. Clinically, the patients were completely asymptomatic throughout the study period with no tenderness to palpation and percussion. The swelling and sinus tract had resolved completely and did not reappear. Radiographically, all 36 teeth showed improvement in terms of periapical healing (PH) and resolution of apical periodontitis.

For evaluation of pulp sensibility, qualitative analysis was done using a Kruskal–Wallis test to see the inter-group significant difference during all the follow-up periods. A score of 1 and 2 was considered as regaining of pulp sensibility. Results showed that there was no significant difference among the inter-groups in every 3 months follow-up period in terms of thermal and EPTs. At the end of 3 and 6 months, the PRF group showed 22.3% positive response and the collagen group showed 11.1% positive response to the cold test with a P value of 0.274, which was not significant for the cold test. None of the cases from the hydroxyapatite and the periapical bleeding groups showed any response to pulp sensibility till 6 months [Table 1]. At 9 months, the PRF group showed 44.4%, the collagen group showed 33.3% and the hydroxyapatite group showed 22.2% positive response to the cold test. There is no presence of any positive cases in the periapical bleeding group. The P value was 0.169, which was not significant. At 12 months, the PRF group showed 66.6%, the collagen group showed 44.4%, the hydroxyapatite group showed 33.3% and the periapical bleeding group showed 11.1% positive response to the cold test. The P value was 0.116, which was not significant. This study did not find any positive response to heat and EPT on all follow-up intervals [Table 2] and [Figure 2]. Considering all the four scaffolds and methods used in this study, the PRF group gave best results for regaining pulp sensibility followed by the collagen, hydroxyapatite and periapical bleeding groups, whereas on long-term follow-up of up to 12 months, all the four scaffolds showed better results without any statistically significant difference among the four groups.
Table 1: Comparative qualitative analysis of thermal testing and electric pulp testing at end of 3 months and 6 months among study groups

Click here to view
Table 2: Comparative qualitative analysis of thermal testing and electric pulp testing at end of 9 and 12 months among study groups

Click here to view
Figure 2: A chart showing the percentage of teeth with regaining of pulp sensibility to cold test in 1 year follow-up intervals

Click here to view

   Discussion Top

In this present study, which was conducted by REP in the mature teeth using induction of periapical bleeding, a standard technique along with PRF as a natural scaffold and collagen and hydroxyapatite as an artificial scaffold was performed. Regaining sensation in the regular follow-up period to the cold test in some of the teeth explains the possible proprioceptive defence mechanism of the pulp, which acts as an alarm system and prevents tissue injury/tooth fracture against pulp, thus promising the tertiary objective of the treatment.[9] To further promote the longevity of natural teeth and to overcome the limitations of the conventional endodontic treatment, the regeneration concept was implemented in the mature teeth and obtained excellent results. If the dental pulp or nearly pulp-like tissue is regenerated, then the canal will be filled with natural innate immune system defence cells which will act efficiently against the remaining bacteria and restore the natural function compared with the root canal treatment.[10] The protocols used in the REP of mature necrotic teeth are almost similar to that used in immature teeth. There have been many studies that attempted revascularisation in mature teeth with slight modification in the mechanical instrumentation of the root canal. In the immature teeth, slight instrumentation along the canal walls is needed, but in the mature teeth, complete instrumentation and disinfection of the canal are required before inducing bleeding. There are many challenges and difficulties in REP of mature teeth compared with immature teeth: First, there is less population of SCAP (stem cells from the apical papilla) or other stem cells such as periodontal ligament stem cells, bone marrow mesenchymal cells or some residual dental pulp stem cell in the mature teeth. The stimulation and migration of any of these stem cells inside the canal for regeneration is the most important procedure. The 1 to 2 mm over instrumentation beyond the apex is required with the help of a smaller number of K-files. Lovelace et al. concluded that there was 600-fold increase in stem cell markers after inducing periapical bleeding in the canal of immature teeth than in the systemic level.[11] Second, the apical foramen is very narrow compared with the immature teeth. Another major concern is the apical foramen diameter, because it was suggested that there should be at least 1.1 mm diameter for regeneration procedure in the earlier studies. Therefore, REP was not considered for mature teeth previously. However, animal studies suggested that the size of 0.32 mm diameter does not prevent ingrowth of new tissue and revascularisation mechanism.[12] The average size of the stem cells is 10 to 100 microns, which is smaller than the average apical foramen size. Human studies by Shah and associates enlarged till 30 K-file, Saoud and associates enlarged to 35 K-file resulted in successful regeneration in mature teeth. The third challenging step is disinfection of the canal before REP, which includes the concentration of NaOCl, EDTA and DAP used in this study.[13] This study necessitates a rationale on a scientific basis, protocols and difficulties in the procedure. There is a limited potential of the pulp to regenerate in the mature teeth due to various reasons. The current protocols by the American Association of Endodontists (AAE) suggested the use of 1.5% concentration, thus having a balance between disinfection and also the stem cell survival property.[14] EDTA was used as a canal irrigant because it partially reverses the effect of NaOCl on stem cells and the release of entrapped growth factors from the dentin matrix.[15],[16] In this study, the use of DAP excludes the disadvantages of triple-antibiotic paste and calcium hydroxide. The current AAE recommends the use of 0.1 mg/mL of the antibiotic mixture in regenerative procedures.[17],[18] Another important key factor in REP is achieving a tight coronal seal, which provides favourable environment for the regeneration of new tissues. The gain of sensibility to cold test indicates the formation of pulp-like tissue with neurovascular bundles which was the highest in the PRF group. Regaining of pulp sensibility to cold test and negative result in case of heat and EPT is achieved in all groups might be due to various reasons: (1) Cold test has high sensitivity when compared with other tests and detects the vitality of the pulp. (2) In the regeneration mechanism, there is an improper formation of neurovascular bundles mostly embedded in the connective tissue. There is no formation of proper pulp–dentin complex along the dentinal walls. (3) Cold test is more reliable in case of stimulating nerve proper, whereas EPT and heat test will stimulate nerves along dentinal wall, which might be a reason. (4) For EPT, the placement of electrode is also important; it should be placed along the thinnest portion of the enamel walls, but in this study, the use of thick coronal seal material might have prevented the conduction of electric current. (5) The final reason is the most important. Because of a short follow-up period of 1 year, at 3 months, only the PRF and collagen groups showed pulp sensibility, but at the end of 9 and 12 months, almost every group started showing pulp sensibility, which indicates the maturation and proper formation of neurovascular bundles in a long time.[19],[20] These are the various hypothesised reasons attributed to explain the regaining of pulp sensation. For better outcomes and results, a long-term follow-up and large sample size are always required.

   Conclusion Top

Tissue engineering can be applied effectively in mature necrotic teeth compared with conventional root canal therapy. Thus, REP in this study proved to be effective in the resolution of clinical signs and symptoms and achieved PH. Despite histologic evidence of regenerated tissue, the regaining of sensitivity suggested that the canal space filled with pulp-like tissue and natural defence mechanism, which fought against the remaining bacteria, and the neural response protected the tooth from further injury. Further large clinical trials and histologic investigations are needed to assess the outcomes of regeneration evidence in mature necrotic teeth.

   Acknowledgements Top

The authors are grateful to ICMR, New Delhi, India, who supported this research financially.

Financial support and sponsorship

This research was funded by the Indian Council of Medical Research, Grant Number: No. 3/2/March-2019/PG-Thesis-HRD (8D).

Conflicts of interest

There are no conflicts of interest.

   References Top

Gher ME, Dunlap RM, Anderson MH, Kuhl LV. Clinical survey of fractured teeth. J Am Dent Assoc 1987;114:174–7.  Back to cited text no. 1
Ahmed HM, Abbott PV. Discolouration potential of endodontic procedures and materials: A review. Int Endod J 2012;45:883–97.  Back to cited text no. 2
Wang X, Thibodeau B, Trope M, Lin LM, Huang GT. Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 2010;36:56–63.  Back to cited text no. 3
Iohara K, Imabayashi K, Ishizaka R, Watanabe A, Nabekura J, Ito M, et al. Complete pulp regeneration after pulpectomy by transplantation of CD105+stem cells with stromal cell-derived factor-1. Tissue Eng Part A 2011;17:1911–20.  Back to cited text no. 4
Paryani K, Kim SG. Regenerative endodontic treatment of permanent teeth after completion of root development: A report of 2 cases. J Endod 2013;39:929–34.  Back to cited text no. 5
Saoud TM, Huang GT, Gibbs JL, Sigurdsson A, Lin LM. Management of teeth with persistent apical periodontitis after root canal treatment using regenerative endodontic therapy. J Endod 2015;41:1743–8.  Back to cited text no. 6
Saoud TM, Martin G, Chen Y-H, Chen KL, Chen CA, Songtrakul K, et al. Treatment of mature permanent teeth with necrotic pulps and apical periodontitis using regenerative endodontic procedures: A case series. J Endod 2016;42:57–65.  Back to cited text no. 7
Nageh M, Ahmed GM, El-Baz AA. Assessment of regaining pulp sensibility in mature necrotic teeth using a modified revascularization technique with platelet-rich fibrin: A clinical study. J Endod 2018;44:1526-33.  Back to cited text no. 8
Ou KL, Chang CC, Chang WJ, Lin CT, Chang KJ, Huang HM. Effect of damping properties on fracture resistance of root filled premolar teeth: A dynamic finite element analysis. Int Endod J 2009;42:694–704.  Back to cited text no. 9
Xavier AC, Martinho FC, Chung A, Oliveira LD, Jorge AO, Valera MC, et al. One-visit versus two-visit root canal treatment: Effectiveness in the removal of endotoxins and cultivable bacteria. J Endod 2013;39:959–64.  Back to cited text no. 10
Saoud TM, Sigurdsson A, Rosenberg PA, Lin LM, Ricucci D. Treatment of a large cystlike inflammatory periapical lesion associated with mature necrotic teeth using regenerative endodontic therapy. J Endod 2014;40:2081–6.  Back to cited text no. 11
Kling M, Cvek M, Mejare I. Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol 1986;2:83–9.  Back to cited text no. 12
Laureys WG, Cuvelier CA, Dermaut LR, De Pauw GA. The critical apical diameter to obtain regeneration of the pulp tissue after tooth transplantation, replantation, or regenerative endodontic treatment. J Endod 2013;39:759–63.  Back to cited text no. 13
Harrison JW, Wagner GW, Henry CA. Comparison of the antimicrobial effectiveness of regular and fresh scent Clorox. J Endod 1990;16:328–30.  Back to cited text no. 14
Harrison JW, Hand RE. The effect of dilution and organic matter on the anti-bacterial property of 5.25% sodium hypochlorite. J Endod 1981;7:128–32.  Back to cited text no. 15
Vianna ME, Horz HP, Gomes BP, Conrads G. In vivo evaluation of microbial reduction after chemo-mechanical preparation of human root canals containing necrotic pulp tissue. Int Endod J 2006;39:484–92.  Back to cited text no. 16
Stojicic S, Shen Y, Haapasalo M. Effect of the source of biofilm bacteria, level of biofilm maturation, and type of disinfecting agent on the susceptibility of biofilm bacteria to antibacterial agents. J Endod 2013;39:473–7.  Back to cited text no. 17
Yassen GH, Chu TM, Eckert G, Platt JA. Effect of medicaments used in endodontic regeneration technique on the chemical structure of human immature radicular dentin: An in vitro study. J Endod 2013;39:269–73.  Back to cited text no. 18
Lin J, Chandler NP, Purton D, Monteith B. Appropriate electrode placement site for electric pulp testing first molar teeth. J Endod 2007;33:1296–8.  Back to cited text no. 19
Mainkar A, Kim SG. Diagnostic accuracy of 5 dental pulp tests: A systematic review and meta-analysis. J Endod 2018;44:694-702.  Back to cited text no. 20

Correspondence Address:
Dr. Praveen Kumar
3rd floor, Department of Conservative Dentistry and Endodontics, FODS, Trauma Centre Complex, BHU, Varanasi - 221 005, Uttar Pradesh
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdr.ijdr_253_21

Rights and Permissions


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

    Materials and Me...
    Article Figures
    Article Tables

 Article Access Statistics
    PDF Downloaded70    
    Comments [Add]    

Recommend this journal