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 Table of Contents  
Year : 2020  |  Volume : 15  |  Issue : 1  |  Page : 108-113

Autologous platelet-rich fibrin as a sole grafting material in regeneration of large periapical (palatal) defects: Report of four consecutive cases

Department of Periodontics, Sharad Pawar Dental College, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi(M), Wardha, Maharashtra, India

Date of Submission21-Nov-2019
Date of Decision30-Nov-2019
Date of Acceptance10-Dec-2019
Date of Web Publication13-Oct-2020

Correspondence Address:
Dr. Prasad Dhadse
Department of Periodontics, Sharad Pawar Dental College, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi (Meghe), Wardha, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jdmimsu.jdmimsu_186_19

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The maintenance of a tooth with periapical lesion is more often managed by periradicular surgery when the efforts using conventional endodontic (re) treatment prove insufficient to achieve desired outcome. The most critical elements of successful bone regeneration involve the production hermetic seal of the endodontic canal after the root end resection and promotion of early uneventful wound healing using desirable biomaterials, particularly in large-sized intraosseous periapical defects. Yet even after the use of different biomaterials available, the wide varieties of such defects have shown suboptimal outcomes and also took longer healing period. The simple procurement method, ready bioavailability, cost-effectiveness, ease of handling, and remarkable healing potential of platelet-rich fibrin (PRF) in filling the large-sized periapical intrabony defects is presented in this article, with the aim of providing optimal and most elusive environment for wound healing in regenerative procedure. The entire intrabony defects were condensed with autologous PRF as the only grafting material in all four different cases for regeneration. The same surgical technique successfully applied to all four selected cases resulted in an uneventful healing post surgically and almost complete defect fill over 6 months' follow-up. The use of autologous PRF can be recommended for further research in view of its therapeutic use in large-sized intraosseous defects even in different health disciplines; to support the successful outcome identified in this case series.

Keywords: Apicectomy, osseous regeneration, periapical cyst, periapical surgery, platelet-rich fibrin, radicular cysts

How to cite this article:
Dhadse P, Ragit G, Kale B, Sridhar S. Autologous platelet-rich fibrin as a sole grafting material in regeneration of large periapical (palatal) defects: Report of four consecutive cases. J Datta Meghe Inst Med Sci Univ 2020;15:108-13

How to cite this URL:
Dhadse P, Ragit G, Kale B, Sridhar S. Autologous platelet-rich fibrin as a sole grafting material in regeneration of large periapical (palatal) defects: Report of four consecutive cases. J Datta Meghe Inst Med Sci Univ [serial online] 2020 [cited 2020 Oct 23];15:108-13. Available from: http://www.journaldmims.com/text.asp?2020/15/1/108/297973

  Introduction Top

The most common lesions associated with inflammatory odontogenic cystic origin of the oral cavity are radicular cysts. Their most common locations are the root apices of endodontically affected teeth and may occur at any age, affecting any gender.[1] Frequently, they are symptomless and are direct sequel to chronic apical infections.[2] The availability of scant literature about natural history of radicular cysts presents an unclear view about what proportion of it regresses and grows. Initial small size cysts can even extend up to 5–6 cm, where larger lesions frequently require endodontic as well as surgical approach for their complete resolution. In the presence of long-standing large lesions that do not resolve after endodontic therapy, the teeth involved may undergo root resorption and displacement. Further progression of cysts may produce expansion of cortical plates either buccally or palatally,[1],[2] where palatal cortication often involves challenges in surgical management owing to poor accessibility.

To maximize the outcome of periapical surgery, prevention of leakage consisting predominantly of bacteria from the endodontic canal into the periradicular tissues involve placement of a compact root end filling material after the root resection.[3] This is usually achieved by removal of periapical tissue and by barring of any irritants inside the extended tangible confinement of the involved root followed by their resection and root end filling and simultaneously filling of the bony cavity with different biomaterials. Various biomaterials such as porous hydroxyapatite, demineralized freeze-dried cortical bone,[4] resorbable membrane,[5] and exogenous growth factors[6] have been demonstrated to produce successful outcomes when treating large lesions or situation where both cortical plates of the jaws are affected. However, certain disadvantages are inherently associated with each of these biomaterials such as suboptimal or delayed outcome resulting in recurrence and need for re-surgery.[3],[7] Outcomes of regenerative therapy in large-sized defects are affected by various factors such as patient's age and gender, group of teeth, quality of retrograde root filling, timing of root filling associated with surgery, the completeness of retrograde root fill, periodontal status of the involved teeth, extent of the periapical lesion, and experience of the operator.[8],[9]

Recently, platelet-rich fibrin (PRF) been shown to act as an appropriate scaffold for newly differentiating human periosteal cells which are proved to be worthy for bone tissue engineering applications.[10] The utilization of PRF in the disciplines of plastic surgery,[11] oral and maxillofacial surgery,[12] and implant surgery[13] for bone regeneration has demonstrated successful and rapid results.

The completely filled defect with autologous PRF is meant to stabilize the overlying flap and hasten the wound healing process. Due to limited information available in the scientific literature, concerning use of PRF in management of periapical intrabony defects, intent of the present case series was to assess clinical and radiographic effectiveness of the application of PRF as a sole grafting material in the regenerative management of large sized (palatal) intrabony defects.

Ethical clearance

Ethical clearance was obtained from the Institutional Ethical Committee of JNMC, Sawangi (Meghe), Wardha, on 5th Sept 2019. With ethical clearance no DMIMS(DU)/IEC/2019-20/355.

  Clinical Report Top

Four patients with good status of systemic health were included in this present study [Table 1]. Their chief complaint was persistent swelling and heaviness in the palatal region since 1–2 months. On examination, tenderness was present in teeth involved with radiographic evidence of large periapical lesion more than 10 mm in size. Initial presurgical therapy was performed to reduce the inflammation associated with periodontal tissues followed by endodontic treatment and restorations. The reevaluation of the patients was done 3 months after the completion of endodontic procedure both clinically as well as radiographically. The planning of surgical interventions was done at these reevaluation sessions due to the persistence of palatal swelling and associated large intrabony defect [Figure 1]a and b]. Clinical periodontal parameters recorded 3 months after completion of nonsurgical periodontal and endodontic therapy revealed no loss of periodontal support in the teeth involved. Final clinical and radiographic outcomes were recorded after 3 and 6 months' post periapical surgery.
Table 1: Patient's characteristics

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Figure 1: (a) Clinical view of periapical lesion in palatal region >10 mm (b) radiological view of periapical lesion in palatal region

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  Surgical Technique Top

Before surgery, patients face was painted with povidone iodine solution (Troydine, Troikaa Pharmaceuticals Ltd., Mumbai, Maharashtra, India). Presurgical mouth rinse with 0.2% chlorhexidine gluconate solution (Rexidine®, Indoco Remedies Ltd., India) was advocated for a minute. Bilateral greater palatine nerve and nasopalatine nerve block using 2% lignocaine with concentration of 1:80,000 adrenaline (Ligno-AD Local Anesthetic, Proxim remedies Mumbai, Maharashtra, India) was given to obtain local anesthesia. Using a surgical blade number 15 (Glassvan® Niraj Industries Pvt Ltd, Mumbai, Maharashtra, India), which has small, curved cutting edge facilitating short and precise incisions, crevicular incision was placed palatally preserving the entire papillary inclusions in the flap. No labial or buccal flap was contemplated owing to noninvolvement of the said side. The full thickness flap reflection was advanced till the defect could be seen with all its extent and with all the possible visual acuity [Figure 2]. Debridement was performed to eliminate the entire cystic lining and the granulomatous content. Utmost care was taken for not leaving any granulomatous tissue especially in the undermined areas at periphery of intraosseous defects. Sterile saline was used for irrigation of the surgical area. (University of North Carolina-15, Hu Friedy, Chicago, IL, USA) probe was used to record the intraoperative measurements. After complete debridement the diode laser of wavelength 980 nm (Doctor's smile Laser wiser, Doctor Smile, Italy. 16 watts 980 nm) was used in noncontact mode for decontamination of the infected intrabony wound [Figure 3]. This aided in resecting the apices of the offending teeth and more accurate placement of suitable root end filler material like Mineral Trioxide Aggregate (MTA), (Proroot® MTA, Dentsply Tulsa Dental Specialities Johnson City, TN USA). For the resection of the root apices small round 1 mm diamond bur (Strauss and Co. Haitech Medical Solutions, Mumbai, Maharashtra, India) was used along with the copious saline irrigation. Contra-angled handpiece was attached to the micro motor with medium speed for root cutting. Root end preparation was done obliquely which resected the apices of the involved teeth along with some lateral canals and also would allow the retention of root end filler material. The MTA was placed at root apices in isolation [Figure 4]. After the placement and setting of MTA, the bleeding was induced into the walls of intraosseous defect through micro-perforations using 1 mm round bur that would stimulate undifferentiated mesenchymal cells and help in regeneration through rapid acceleratory phenomenon (RAP) as bone tissue maintains a highly endogenous capacity for self-regeneration.[9] The preparation of PRF was done according to the guidelines given by Choukroun et al.[12] 10–20 ml of venous blood was drawn out from the patient's antecubital fossa. Thereafter, centrifugation was done at 3000 rpm for 12 min to obtain the buffy coat of PRF. The large-sized intrabony deficient defects were then filled only with PRF [Figure 5]a. The overfilling of the defect was avoided. Saline wetted gauze was applied with gentle pressure over the surgical area for 1 min to readapt the mucoperiosteal flap. Finally, the flap was approximated using 3-0 non resorbable silk suture material (Ethicon, Johnson and Johnson Ltd., Ethicon, US LLC) by simple interrupted suturing technique [Figure 5]b.
Figure 2: Elevation of the flap for access to facilitate debridement

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Figure 3: Complete debridement of the defect after application of diode laser

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Figure 4: Root end filling done using mineral trioxide aggregate

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Figure 5: (a) Placement of the autologous platelet rich fibrin into the intraosseous defect as a sole grafting material (b) primary wound closure of the surgical site using 3-0 silk suture

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  Postsurgical Care Top

After the surgery, patients were prescribed capsule amoxicillin 500 mg 8 hourly for 5 days to control bacterial contamination and tablet ibuprofen 400 mg also 8 hourlies for 5 days. The patients were asked to avoid mechanical oral hygiene measures post operatively until suture removal. Mouth rinse containing 0.12% chlorhexidine digluconate twice daily for 1 min was advised to the patient during this period. The sutures were taken out 10–14 days of the surgical period. The curetted specimen obtained was sent for histopathological examination which confirmed the diagnosis of infected dental cyst with 3 of the 4 cases and radicular cyst with the remainder [Figure 6].
Figure 6: Histopathological view suggesting dentigerous cyst (×40)

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  Clinical Outcomes Top

Primary wound healing was observed with good adaptation of flap margin, with interdental papillae showing integral continuity and almost 100% wound closure in all the cases during early healing period. All treated sites healed uneventfully with no evidence of pain, sensitivity, swelling, pus discharge, redness, or tenderness. A stringent plaque control protocol was instigated in these patients with weekly recall for the 1st month followed by bimonthly professional teeth cleaning for subsequent 6 months.

Baseline and 6 months' postoperative clinical parameters of all four cases treated are presented in [Table 2]. All the four cases showed uneventful healing and stable improvement in clinical periodontal parameters. Radiographs taken at 6 months' follow-up showed bone fill. Owing to the palatal defects occlusal radiographs taken with a standardized radiographic technique (XCP, Rinn, Dentsply Ltd., Surrey, UK) were independently and keenly assessed by the three examiners. Clinical signs and symptoms and also radiographic periapical healing were determined as per the criteria given by Rud et al.[14] and Molven et al.[15]
Table 2: Clinical parameters measured at baseline and at 6 months after surgery

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It was classified as successful when the radiograph demonstrated almost complete healing of the previous radiolucency or scar tissue formation, and no clinical signs or symptoms were present, doubtful when the radiographic healing was judged as uncertain (little reduction of the former radiolucency) and no clinical signs or symptoms were present and failure when the radiographic showed unsatisfactory healing (nil reduction or even an enlargement of the previous radiolucency), or clinical signs or symptoms were present.[16]

It was further classified as-complete healing, incomplete healing (scar tissue), uncertain healing and unsatisfactory healing (failures).[15] A specific healing was categorized when at least two examiners agreed on same healing criteria. The final healing classification was based on the radiographic assessment as well as on the presence or absence of clinical signs or symptoms [Table 3].
Table 3: Healing classification based on radiographic and clinical assessment

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Interestingly, on radiographic assessments all the comparative occlusal views taken at 3 and 6 months revealed marked radio opacity seen with intrabony region suggestive of almost complete bone fill. There was no evidence of recurrence of the lesion at 6 months' follow-up [Figure 7]a and [Figure 7]b.
Figure 7: (a) Postoperative clinical view 6 months after surgery (b) postoperative radiographic view 6 months after surgery

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  Discussion Top

Cysts of odontogenic origin are the most common form of cystic lesions that affect the maxillofacial region which are divided conventionally into two groups. Developmental group is usually asymptomatic. However, they can become extremely large thus causing expansion of the cortical plates and erosion, for example, keratocyst and dentigerous cyst.[1] The second group includes inflammatory group like radicular cyst. Radicular cysts are traditionally believed to be the most common cystic lesions[1],[2],[3] and prevalence ranges from 52% to 68% of all the cysts affecting the human jaws.[2] Male patients show higher prevalence as compared to females, especially in the third decade of their life. The anterior region of the maxilla appears to be more prone to the development of cysts whereas in the mandible the radicular cysts tend to occur more frequently in the premolar region.[1] Abramowitz et al.[9] discussed guidelines of case selection for apical surgery where they emphasized that the management of 24.5% of the cases was not possible without surgical therapy. Success rate up to 90% or above have been documented in several clinical studies.[8]

Thorough evaluation is substantially significant to reduce surgical trauma that helps to develop optimal conditions for debridement and subsequent root-end filling. Clinical parameters such as esthetic demands of the patient, periodontal status, biotype of the gingiva and width of keratinized gingivae, level of margin/s of the restoration, extent and condition of lesion needs to be evaluated prior to surgery. Radiographic evaluation of the parameters consists of location and size of the periapical lesion and health of the marginal periodontium.[8] The incision and flap design should be selected according to clinical and radiographic parameters. In situ ation, where the cystic lesion encompasses toward the alveolar crest, an intrasulcular incision is used for reflection of the flap to gain access to periapical lesion.

In the present case series, palatal intrabony defect could be seen immediately after flap reflection as a result of loss of cortical plate. It is known that the putrefying and proteolytic end products of the dying cells in the cystic lumen release a huge number of molecules resulting in increase in the osmotic pressure within the cyst fluid. With rise in the osmotic pressure comparable to that of the tissue fluid, the latter diffuses into the cyst cavity thereby raising the intraluminal hydrostatic pressure significantly above the capillary pressure. This leads to rise in the pressure within the cyst may leading to resorption of the bone and expansion of the cyst. Furthermore, the presence of T-lymphocytes and macrophages within the cyst wall may provide a constant source metabolite responsible for bone resorption, cytokines, and matrix metalloproteinase-1 and-2. This explain the possible mechanism that could have resulted in resorption of palatal bone.[1] Following flap reflection, thorough debridement of bony cavity was performed to remove all the granulation tissue. This also aided in better visualization of defect margins and tooth apices. After debridement intra-surgical measurements were made to correlate defect size with that of radiographic measurements. It was found that the measurements in both cases were almost similar. This aided in appreciating postsurgical bone fill. The debrided tissue was sent for histopathologic examination to determine type of cyst. This is considered as gold standard for diagnosis as it not only aids in providing final diagnosis but also helps to determine prognosis and recurrence.[4],[8] Diode laser of 810 nm was used in noncontact mode in the bone cavity to achieve hemostasis, minimize the risk of contamination, and also for its indirect benefits in reduction of postoperative pain.[4]

Oblique root amputation was carried out to facilitate expulsion of apical delta (root canal ramification) surgically, widening of entry to apex, creation of a suitable operational surface for retrograde preparation, allow debridement of periapical tissue and provide clear view of resected root end for existence of vertical fractures.[8] MTA was used as root end filler, whereas a multiplicity of materials has been promulgated in the past for the same. However, MTA is considered as the benchmark for a root-end filling material. Clinical (comparative) studies have stated excellent success rates for MTA ranging from 90% to 92% (with a follow-up periods from 1 to 5 years)[8] as it has excellent biocompatibility,[15] fantastic adherence to the cavity walls,[8] and reduced solubility,[17] and promote cementogenesis at the rendered root face, where newly formed cementum is observed to be deposited over the exposed dentin and MTA surfaces.[17],[18] Micro-perforations were made into the bone cavity to induce bleeding that could aid as source of stem cells during healing. The RAP thus induced makes bone tissue maintain a highly endogenous capacity for self-regeneration.[9]

In the present study, it has been hypothesized that PRF may provide a suitable scaffold material for periodontal tissue regeneration. The aptness of PRF as a biologically active scaffold has been illustrated in a number of studies enlightening proliferation and differentiation of osteoblasts and gingival fibroblasts.[8],[9] Clinical studies have confirmed that PRF promotes soft tissue and bone regeneration[10],[11] and also periodontal tissue regeneration.[12],[13] Shilpa et al.[16] in their case series evaluated the effectiveness of PRF in ridge preservation and concluded that use of PRF was successful in preserving the alveolar ridge by limiting the amount of bone resorption after tooth extraction resulting in radiographic bone fill in 3 months.

PRF is produced from centrifuged blood and is strictly autologous. PRF derived after centrifugation consequences from a natural and progressive polymerization occurring during the process. The predominant contains a fibrin matrix opulent in platelet and leukocyte cytokines such as interleukin-1β,-4, and-6, and growth factors such as transforming growth factor beta 1, platelet-derived growth factor, and vascular endothelial growth factor.[5] The last stage of the coagulation torrent in which fibrinogen molecules form indigenous dimensional fiber network is exploited by the fibrin gel.[6] Thus, PRF is configured of a fibrin matrix polymerized in a tetra molecular assembly, with incorporation of leukocytes, cytokines, platelets, and circulating stem cells.[6],[12] The fabulous intrinsic inclusion of cytokines inside the fibrin mesh allows for their progressive and consequential release over time (7–11 days), as the network of fibrin disintegrates.[9] This desired configuration provides an increased lifespan for these cytokines, because they will be released and utilized only at the time of initial cicatricial matrix remodeling (prolonged effect). Therefore, the effortlessly applied PRF membrane serves much like a bandage of fibrin, acting as a milieu to hasten the healing of wound edges and hence was used as sole grafting material. Patient in the present case series reported less discomfort. This could be attributed to prescription of antibiotics and analgesics postsurgery.

The accountability of the healing outcome closely correlates with the classification of healing that is built on definite clinical and radiographic healing criteria.[8] In the literature, healing associated with soft tissue following apical surgery has rarely been mentioned, where the emphasis has always been on the periapical healing. In the present study, soft-tissue healing was satisfactory with no evidence of any pain, infection, swelling, or postsurgical recession. The bone fill as adequate which was determined by radiographic evaluation at 3- and 6-month recall.[19],[20],[21]

  Conclusion Top

The utility of PRF as a sole grafting material in regeneration of large intraosseous defects within a short span, almost leads to improved patient-based outcomes. The application of good root end sealing material, sound surgical principles, use of laser decontamination, micro-perforations to induce RAP, and provision of simultaneous hydration of PRF promises an uneventful healing phase. However, there is a need for long-term comparative studies with other biomaterials either alone or in combination with PRF to better characterize this therapy.


Author would like to thank Dr. Pavan Bajaj, Dr. Tulika Soni, Dr. Bindu Singh Priya for their assistance in the study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Raval RD, Nyklesh V, Patel HM, Naik PS, Patel PP. Management of infected radicular cyst in maxillary anterior region: Case report. Int J of Adv Health Sci 2015;1:8-11.  Back to cited text no. 2
von Arx T, Jensen SS, Hänni S. Clinical and radiographic assessment of various predictors for healing outcome 1 year after periapical surgery. J Endodont 2007;33:123-8.  Back to cited text no. 3
Bowen EM, Penarrocha M. An update in periapical surgery. Med Oral Patol Oral Cir Buccal 2006;11:503-9.  Back to cited text no. 4
Escoda CG, Mendez UM, Sanchez MA, Berini L. Applicacion de los ultrasonidos en cirugia periapical. Rev Eur Odontoestomatol 1996;8:207-14.  Back to cited text no. 5
Regan JD, Gutmann JL, Lacopino AM, Diekwisch T. Response of peri-radicular tissues to growth factors introduced into surgical site in the root end filling material. Int Endodon J 1999;32:171-82.  Back to cited text no. 6
Pompa DG. Guided tissue repair of complete buccal dehiscence associated with periapical defects: A clinical retrospective study. J Am Dent Assoc 1997;128:989-97.  Back to cited text no. 7
von Arx T. Periapical surgery: A review of current techniques and outcome. Saudi Dent J 2011;23:9-15.  Back to cited text no. 8
Abramowitz PN, Rankow H, Trope M. Multidisciplinary approach to apical surgery in conjunction with the loss of buccal cortical plate. Oral Surg Oral Med Oral Pathol 1994;77:502-6.  Back to cited text no. 9
Gassling V, Douglas T, Warnke PH, Acxil Y, Willfang J, Becker ST. Platelet rich fibrin membranes as scaffold for periosteal tissue engineering. Clin Oral Implants Res 2010;21:543-9.  Back to cited text no. 10
Sclafani AP. Application of platelet rich fibrin matrix in facial plastic surgery. Facial Plast Surg 2009;25:270-6.  Back to cited text no. 11
Choukroun J, Diss A, Simopieri A, Girard MO, Schoeffler C, Dohan SL, et al. Platelet rich fibrin (PRF): A second generation platelet concentrate. Part V: Histologic evaluations of PRF effects on bone allograft maturation in sinus lift. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:299-303.  Back to cited text no. 12
Mazor Z, Horowitz RA, Del Corso M, Prasad HS, Rohrer MD, Ehrenfest DM. Sinus floor augmentation with simultaneous implant placement using Choukroun's platelet rich fibrin as the sole grafting material: A radiologic and histologic study at 6 months. J Periodontol 2009;80:2056-64.  Back to cited text no. 13
Rud J, Andreasen JO, Jensen JM. Radiographic criteria for the assessment of healing after endodontic surgery. Int J Oral Maxillofac Surg 1972;1:195-214.  Back to cited text no. 14
Molven O, Halse A, Grung B. Observer strategy and the radiographic classification of healing after endodontic surgery. Int J Oral Maxillofac Surg 1987;16:432-9.  Back to cited text no. 15
Shilpa BS, Dhadse PV, Bhongade ML, Puri S, Nandanwar J. Evaluation of effectiveness of platelet-rich fibrin for ridge preservation after atraumatic extraction: A case series. J Datta Meghe Inst Med Sci Univ 2017;12:294-300.  Back to cited text no. 16
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Poggio C, Lombardini M, Conti A, Rindi S. Solubility of root end filling materials: A comparative study. J Endodont 2007;33:1094-7.  Back to cited text no. 17
Camilleri J, Fort TR. Mineral trioxide aggregate: A review of the constituents and biologic properties of the material. Int Endodon J 2006;39:747-54.  Back to cited text no. 18
Dambhare A, Bhongade ML, Dhadse PV, Sehdev B, Ganji KK, Thakare K, et al. A Randomized Controlled Clinical Study of Autologous Platelet Rich Fibrin (PRF) in Combination with HA and Beta-TCP or HA and Beta-TCP Alone for Treatment of Furcation Defects. J Hard Tissue Biol 2019;28:185-90. Available from: https://doi.org/10.2485/jhtb.28.185. [Last accessed on 2019 Oct 12].  Back to cited text no. 19
Jagati A, Chaudhary R, Rathod S, Madke B, Baxi K, Kasundra D. Preparation of Platelet-Rich Fibrin Membrane over Scaffold of Collagen Sheet, Its Advantages over Compression Method: A Novel and Simple Technique. J Cutan Aesthet Surg 2019;12:174-8. Available from: https://doi.org/10.4103/2543-1854.267617. [Last accessed on 2019 Oct 12].  Back to cited text no. 20
Dhote VS, Thosar NR, Baliga SM, Dharnadhikari P, Bhatiya P, Fulzele P. Surgical Management of Large Radicular Cyst Associated with Mandibular Deciduous Molar Using Platelet-Rich Fibrin Augmentation: A Rare Case Report. Contemp Clin Dent 2017;8:647-9. Available from: https://doi.org/10.4103/ccd.ccd_370_17. [Last accessed on 2019 Oct 12].  Back to cited text no. 21


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

  [Table 1], [Table 2], [Table 3]


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