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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 15  |  Issue : 3  |  Page : 148-163

Nanohydroxyapatite versus melatonin loaded on nanohydroxyapatite and nanohydroxyapatite with platelet rich fibrin on the treatment of intrabony defects


1 Department of Oral Medicine, Periodontology, Oral Diagnosis, and Oral Radiology; Faculty of Dentistry, Tanta University, Tanta, Egypt
2 Department of Oral Pathology; Faculty of Dentistry, Tanta University, Tanta, Egypt

Date of Submission28-Dec-2017
Date of Acceptance24-Jul-2018
Date of Web Publication10-Oct-2018

Correspondence Address:
Doaa A Yousef
Assistant Lecturer, Oral Medicine, Periodontology, Oral Diagnosis and Oral Radiology Department, Faculty of Dentistry, Tanta University, Tanta
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tdj.tdj_60_17

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  Abstract 

Aim
The aim of this study was to evaluate and compare, clinically, the effect of nanohydroxyapatite alone versus melatonin loaded nanohydroxyapatite and nanohydroxyapatite with platelet rich fibrin (PRF) in the treatment of intrabony defects in patients with severe chronic periodontitis.
Patients and methods
A total of 30 sites in patients with, severe chronic periodontitis, with clinical attachment loss (CAL) of at least five were included. Sites were allocated randomly to be treated with nanohydroxyapatite alone, melatonin loaded nanohydroxyapatite, nanohydroxyapatite with PRF. At baseline, 3, 6, and 12 months after surgery, the parameters are pocket depth, CAL, bleeding on probing. The experimental included six rabbits, where four identical bony defects, two in each mandible side, were created and each filled with one of the three materials. The fourth defect was left with no filler to serve as the control. Two rabbits were killed 2 weeks after the surgery, the second two rabbits, after 4 weeks and the third two rabbits, after 6 weeks. Samples were collected for histological and histomorphometric analysis.
Results
All groups showed significant improvement in all clinical parameters (pocket depth, bleeding on probing, and CAL) after 3, 6, and 12 months compared with baseline measures. Groups II and III showed statistically significant reduction when compared with group I. Histopathological findings were evidenced by histomorphometric analysis, as it revealed statistically significant difference in favor of group II when compared with the other groups at all evaluation periods except with group III at 6 weeks. Statistically significant difference in favor of group III when compared with group I and control group at all evaluation periods.
Conclusion
Either melatonin or PRF when combined with nanohydroxyapatite showed better comparable results, clinically, and histopathologically when compared with nanohydroxyapatite alone and can therefore be considered as a potentiating material for periodontal regeneration.

Keywords: intrabony defects, melatonin, nanohydroxyapatite, platelet rich fibrin, severe chronic periodontitis


How to cite this article:
Yousef DA, Al Hessy AA, Saeed AA, El Shamy ES. Nanohydroxyapatite versus melatonin loaded on nanohydroxyapatite and nanohydroxyapatite with platelet rich fibrin on the treatment of intrabony defects. Tanta Dent J 2018;15:148-63

How to cite this URL:
Yousef DA, Al Hessy AA, Saeed AA, El Shamy ES. Nanohydroxyapatite versus melatonin loaded on nanohydroxyapatite and nanohydroxyapatite with platelet rich fibrin on the treatment of intrabony defects. Tanta Dent J [serial online] 2018 [cited 2018 Dec 9];15:148-63. Available from: http://www.tmj.eg.net/text.asp?2018/15/3/148/243079


  Introduction Top


Periodontal regeneration is a complex multifactorial process involving biologic events like cell adhesion, migration, proliferation, and differentiation in an orchestrated sequence [1]. Periodontal regenerative procedures include soft-tissue grafts, bone grafts, root biomodifications, guided tissue regeneration, and combinations of these procedures [2]. Augmentation of the osseous defect with bone grafts has become one of the most common surgical techniques in recent years. The ideal synthetic bone graft should replicate the normal healing responses of autogenous bone by stimulating osteogenesis. An ideal osteogenic biomaterial should (i) support the attachment and migration of bone cells throughout the biomaterial (i.e. be osteoconductive), (ii) induce the differentiation of osteoprogenitor cells into bone-forming osteoblasts or promote osteoblast activity (i.e. be osteoinductive), and (iii) integrate with the newly formed bone while retaining similar mechanical properties as normal bone, that is, exhibit osteointegration [3],[4]. Many commercial products are limited in that they only provide for osteoconduction and osteointegration, exhibiting no other properties of natural bone. This can be overcome by adding growth factors and biomaterial, which act as mitogenic and chemotactic agents thus bring rapid regeneration [5],[6]. The introduction of biomimetic agents, such as enamel matrix derivatives, platelet-derived growth factor (PDGF), bone morphogenic proteins, platelet rich fibrin (PRF), and melatonin has given new promise for better outcomes in periodontal treatment [7],[8],[9].

Melatonin is an indolamine produced in various parts of the body, mainly in the pineal gland. This gland produces melatonin in a circadian manner, synchronizing a number of biologic processes in a 24-h, day–night rhythm [10]. Melatonin has several functions: it acts as antioxidant, a free-radical scavenger [8],[11],[12] and an immunomodulatory agent [13],[14]. Moreover, several researches showed beneficial effects of melatonin on fibroblast activity and bone regeneration [15],[16],[17].

PRF is a second-generation platelet concentrate which contains platelets and growth factors in the form of fibrin membranes prepared from the patient's own blood free of any anticoagulant or other artificial biochemical modifications. The PRF clot forms a strong natural fibrin matrix, which concentrates almost all the platelets and growth factors of the blood harvest [18],[19]. This unique structure may act as a vehicle for carrying cells, cell migration and proliferation, that are essential for tissue regeneration. Many growth factors, such as PDGF and transforming growth factor β (TGFβ), and insulin-like growth factors are released from PRF. Several, studies have demonstrated that the PRF membrane has a very significant slow sustained release of key growth factors for at least 1 week and up to 28 days, which means that the membrane stimulates its environment for a significant time during wound healing [20],[21]. It has been recently demonstrated to stimulate cell proliferation of the osteoblasts, gingival fibroblasts, and periodontal ligament cells but suppress oral epithelial cell growth. Lekovic et al. [22] demonstrated that PRF in combination with bovine porous bone mineral had ability to increase the regenerative effects in intrabony defects.

Accordingly, this study was conducted to clarify and evaluate clinically histologically and histomorphometrically the potential regenerative effects of melatonin loaded on nanohydroxyapatite or nanohydroxyapatite with PRF versus nanohydroxyapatite alone on the treatment of intrabony defects in patients with severe chronic periodontitis and in experimentally created mandibular bony defects in rabbits.


  Patients and Methods Top


The study population

All patients were selected from the Periodontology Clinic, Faculty of Dentistry, Tanta University. Their age range from 40 to 55 years old and diagnosed with severe chronic periodontitis according to the inclusion criteria [23]. Approval for this project was obtained from Tanta Faculty of Dentistry, Tanta University, Research Ethics Committee (REC).

Site selection

A total of 30 sites of two walls intrabony defects on the mesial or distal aspect of posterior teeth were selected from patients, which fulfill the inclusion criteria.

Inclusion criteria

  1. Presence of angular periodontal intrabony defects with a clinical attachment loss (CAL) of at least 5 mm measured from cementoenamel junction till the deepest probing depth (PD).
  2. Optimal compliance as evidenced by no missed treatment appointments and a positive attitude towards oral hygiene.


Exclusion criteria

  1. Patients with relevant medical conditions that may affect periodontal regeneration and periodontal surgery.
  2. Patients in whom periodontal surgery had previously been carried out on the selected site.
  3. Smokers.
  4. Pregnant and lactating women.
  5. Use of antibiotics agents 6 months before the study.


Materials

  1. Nanohydroxyapatite granules in hydrogel [NanoTech Egypt for Photo-Electronics (NTE), 6th October City, Giza Governorate, Egypt].
  2. Melatonin loaded nanohydroxyapatite granules in hydrogel (each 250 mg of nanohydroxyapatite loaded with 3 mg) [9].
  3. PRF (Laboratory Centrifuge, Model 800D; Pioway Medical Lab Equipment Co., Ltd. Jiangsu, China).
  4. Collagen membrane (Biotik Corporation).


The human study part

I: Pretreatment evaluation:

Clinical assessment:

  1. Probing PD [24].
  2. CAL [24]: Both clinical parameters were measured by a Williams periodontal probe from a fixed point to the deepest PD using occlusal stents.
  3. Bleeding on probing (BOP) [25]: The bleeding at each site was observed after probing. It was recorded positive if bleeding is observed within 30 s, if not it was recorded as negative. The above parameters were measured at baseline, 3, 6, and 12 months following surgery.


II: Treatment steps:

Phase I therapy:

  1. Full mouth scaling and root planning was carried out for all patients as well as, comprehensive oral hygiene instructions.
  2. Re-evaluation was conducted after 4 weeks to evaluate the patient response to phase I therapy and to confirm the need for periodontal surgery.


Patient grouping

One month following phase I therapy, patients or sites in the same patient were randomly classified into three groups using sealed envelope.

The patients or sites were treated with one of the following treatment modalities as follows:

  1. Group I: Nanohydroxyapatite granules in hydrogel.
  2. Group II: Melatonin loaded nanohydroxyapatite granules in hydrogel.
  3. Group III: Nanohydroxyapatite granules in hydrogel mixed with PRF particles.


Platelet rich fibrin preparation

Ten milliliters of blood was drawn from the patient, and then collected in a sterile glass test tube (10 ml) without any anticoagulant. Immediately, test tube was centrifuged using a refrigerated centrifugal machine at 3000 rpm for 12 min. Within few minutes, the absence of anticoagulant induced the activation of platelets contained in the sample, thus triggering a coagulation cascade. The result was a fibrin clot located in the middle of a mass of acellular plasma, with a maximum number of platelets and more than half of the leukocytes caught in the mesh of fibrin. The clot was removed from the tube with a forceps, and the attached red blood cells were shaved off and discarded. The fibrin clot could shaped freely.

Surgical procedure

All surgical procedures were performed by the same operator. Before surgery, patients were instructed to rinse with 0.1% chlorhexidine gluconate for 30 s. Anesthesia was obtained by administration of 2% lidocaine 1: 80 000 epinephrine. Buccal and lingual or palatal intrasulcular incisions were performed, and mucoperiosteal flaps on the facial and lingual or palatal aspects of each tooth, segment, or area involved were reflected. Incisions were designed to preserve as much of the interproximal tissue as possible. Soft-tissue debridement was performed as well as thorough scaling and root planing of the exposed root surfaces. The selected graft material was packed in the debrided intrabony defect, and collagen membrane was placed. Closure was accomplished and a periodontal dressing was placed.

Postoperative care

All patients received postoperative instructions, including rinsing with 0.1% chlorhexidine (twice daily for 2 weeks), a combination antibiotic therapy, amoxicillin/clavulanate 375 mg and metronidazole 250 mg both three times per day, and anti-inflammatory systemic medications for 1 week were administered. Periodontal dressing and suture removal were performed after 14 days. Supportive periodontal therapy were performed monthly which includes, periodontal evaluation (except for periodontal PD which will be measured beginning 3 months postoperative), reinforcement of plaque control, scaling, and root planing were carried out if needed and medical history was updated.

Statistical analysis

The collected data was organized, tabulated, and statistically analyzed, using the mean, SD, and χ2-test by SPSS, version 20 (SPSS Inc., Chicago, Illinois, USA). Comparison between the studied groups was performed using nonparametric tests.

Animal selection

Six rabbits were included in this experimental animal study, about 3–6 months of age and weight about 1.5–2 kg.

Experimental surgical protocol [26]

  1. The animals were anesthetized using an intramuscular injection of ketalar (30–40 mg/kg body weight).
  2. The area over the body of the mandible was shaved, and the skin was disinfected with an iodine solution (10% betadine).
  3. An incision was made through the skin 1 cm lower to the lower edge of the mandibular body with a lateral reflection, the underlying periostium was exposed. Additional incisions were made through the periostium in each side, and then elevated with a periosteal elevator to expose the bone.
  4. A round carbide bur in a slow hand piece with physiologic saline solution irrigation was be used to create four identical circular bony defects 6 × 6 mm (two in each side of mandible).
  5. The wound site was thoroughly irrigated with normal saline solution to remove any tissue debris or foreign matter.
  6. All defects were made in the same way and were filled each with one of the following materials:
  7. Defect with no filler to serve as the control (control group).
  8. Nanohydroxyapatite granules in hydrogel (group I).
  9. Melatonin loaded nanohydroxyapatite granules in hydrogel (group II).
  10. Nanohydroxyapatite granules in hydrogel mixed with PRF particles (group III).
  11. Tissues were carefully repositioned and sutured.
  12. After sutures stabilization the wound was again disinfected with betadine.
  13. To control postoperative pain and swelling, 0.1 ml of ketoprofen was administered daily for up to 3 consecutive days. In addition, 0.6 ml of norfloxacin was administered subcutaneously.
  14. Two rabbits were sacrificed 2 weeks after the surgery, another two rabbits were killed 4 weeks after the surgery and the remaining two rabbits were killed 6 weeks after the surgery. The animals were killed with an injection of 2 ml intravenous sodium thiopental into the marginal auricular venules.
  15. Samples were collected from the surgical areas of the experimental defects for histologic and histomorphometric examinations.


Histological evaluation

Sections were stained with hematoxylin and eosin stain following the routine technique. Histological findings were recorded.

Histomorphometric analysis

Bone histomorphometry was performed on IBM compatible computer using free software Image J (National Institutes of Health, Bethesda, Maryland, USA), a Java application which runs in most operative systems. The software calculated the area of newly formed bone in relation to the total defect area found in the digitalized histopathological photos [27].


  Results Top


The human tissue response to the different treatment modalities, Nanohydroxyapatite granules in hydrogel (group I) versus melatonin loaded nanohydroxyapatite granules in hydrogel (group II) or nanohydroxyapatite granules in hydrogel mixed with PRF particles (group III) in the treatment of intrabony defects in patients with severe chronic periodontitis, evaluated clinically. The experimental part illustrates the histological and histomorphometrical examination for evaluation and comparison the probable osteoinductive property of the above mentioned materials in experimentally created bony defects in the rabbit's mandible.

Baseline parameters

At baseline the three treatment groups showed no significant differences regarding the clinical parameters namely, PD, CAL, and BOP. This was evidenced by their baseline values as shown in the tables (P ≥ 0.05).

Clinical results

Pocket depth results

  1. Group I: It was found that the mean value of PD decreased with a statistically high significant difference between the 3, 6, and 12 months evaluation periods when compared with the baseline values (P1P2, and P3 ≤ 0.001) [Table 1].
  2. Group II: The mean value of PD decreased with a statistically high significant differences between the 3, 6, and 12 months evaluation periods when compared with the baseline values (P1, P2, and P3 ≤ 0.001) [Table 1].
  3. Group III: Results showed a high significant reduction in the mean value of PD along the study period (P1, P2, and P3 ≤ 0.001) [Table 1].
Table 1: Intergroup and intragroup comparison of mean pocket depth values at the different study period

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Intergroup comparison of the PD results between groups:

The differences between groups I, II, and III were not statistically significant at the baseline with P value of at least 0.05. At the 3, 6, and 12 months study intervals; however there was statistically significant difference in favor of groups II and III when compared with group I (Pa and Pb ≤ 0.05). Although at all study periods intervals (3, 6, and 12 months), there were no statistical significant difference between groups II and III (Pc > 0.05) [Table 1].

Clinical attachment loss results

  1. Group I: It was found that the mean value of CAL decreased with a high statistical significant difference at all study intervals when compared with baseline (P1, P2, and P3 ≤ 0.001) [Table 2].
  2. Group II: The mean value of CAL with a high statistical significant difference between the 3, 6, and 12 months evaluation periods measurements when compared with baseline values (P1, P2, and P3 ≤ 0.001) [Table 2].
  3. Group III: The mean values of CAL decreased. This decrease in measurements was statistically high significant with the 3, 6, and 12 months evaluation periods when compared with the baseline values (P1, P2, and P3 ≤ 0.001) [Table 2].
Table 2: Intergroup and intragroup comparison of mean clinical attachment loss values at the different study periods

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Intergroup comparison of the CAL results between groups:

The differences between groups I, II, and III were not statistically significant at the baseline with P value of at least 0.05. At the 3, 6, and 12 months study intervals, however there was statistically significant difference in favor of groups II and III when compared with group I (Pa and Pb ≤ 0.05). Although at all study periods intervals (3, 6, and 12 months), there were no statistical significant difference between groups II and III (Pc > 0.05) [Table 2].

Bleeding on probing results

  1. Group I: At 3 and 6 months, the number of positive sites dropped which indicate high significant improvement when compared with baseline values as P value less than or equal to 0.001. Then at 12 months, the positive sites increased which indicate nonsignificant changes when compared with baseline values as P value of at least 0.05 [Table 3].
  2. Group II: At 3, 6, and 12 months the number of positive sites dropped which indicate significant improvement when comparing all study periods to baseline as P value less than or equal to 0.05 [Table 3].
  3. Group III: At 3, 6, and 12 months the number of positive sites dropped which indicate significant improvement when comparing all study periods to baseline as P value less than or equal to 0.001 [Table 3].
Table 3: Comparison of bleeding on probing percentage in groups I, II , and III at the different study periods

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Intergroup comparison of the BOP results between groups:

Comparing between the three studied groups at different study periods showed that, no statistically significant difference was observed at baseline, 3 months and at 6 months with P value of at least 0.05. At 12 months, there was statistically significant differences in favor of group II and group III when compared with groups I as P value less than or equal to 0.05 [Table 3].

Experimental results

Histopathological results

In the experimental part, 6 rabbits were included, about 3–6 months of age and weight was about 1.5–2 kg. Four identical circular bony defects, two in each side of mandible were created. All defects were made in the same way and each filled with one of the three materials which were examined and evaluated, namely: nanohydroxyapatite granules in hydrogel (group I), melatonin loaded nanohydroxyapatite granules in hydrogel (group II), nanohydroxyapatite granules in hydrogel mixed with PRF particles (group III). The fourth defect was left with no filler to serve as the control (control group).

Two rabbits were sacrificed 2 weeks after the surgery, another two rabbits were killed after 4 weeks and the last two rabbits were sacrificed after 6 weeks. Samples were collected from the surgical areas of the experimental defects for histological examination. Decalcified sections were stained with hematoxylin and eosin and Masson trichrome stains, followed by histomorphometric analysis.

Histopathological results revealed the following:

All defects, till the end of the study (after 6 weeks), were still opened. However, the quantity of new bone formation was different among those groups as follows:

The control group (after 2, 4, and 6 weeks)

After 2 weeks of the surgical procedure, there were moderate quantity of chronic inflammatory cell infiltration, fibroblastic activity with newly formed capillaries. No bone formation could be noticed [Figure 1].
Figure 1: Comparison of mean pocket depth (PD in mm) values in groups I, II, and III at the different study periods.

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Microscopic examination after 4 weeks, still showed moderate chronic inflammatory cell infiltration and fibroblastic activity. Besides, small focal areas of osteoid tissue started to develop [Figure 2].
Figure 2: Comparison of mean clinical attachment loss (CAL in mm) values in groups I, II, and III at the different study periods.

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After 6 weeks, thin newly formed bone trabeculae encircling the surgical defect margins surrounded by osteoid tissue, and fibrocellular tissue were detected. The thickness of the formed bone was found to be less than that formed in all other study groups [Figure 3].
Figure 3: Comparison of bleeding on probing percentage (BOP%) values in groups I, II, and III at the different study periods.

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Group I: nanohydroxyapatite granules in hydrogel (after 2, 4, and 6 weeks)

Microscopic examination after 2 weeks showed moderate chronic inflammatory cell infiltration, marked vascularity and fibrocellular condensation within and around the graft material as well as at the periphery of the surgical site forming a capsule. Osteoblasts and osteoclasts were observed at the periphery of some graft material [Figure 4].
Figure 4: A photomicrograph of the control group after 2 weeks shows moderate chronic inflammatory cell infiltration with a fibrocellular tissue proliferation. No bone formation can be observed (Masson trichrome, ×40).

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Microscopic findings after 4 weeks showed that, there was still moderate amount of chronic inflammatory cells and osteoblasts and osteoclasts at the periphery of the graft material with a reduction in the detected quantity of the graft material. A few osteoid tissues was detected in the connective tissue [Figure 5].
Figure 5: A photomicrograph of the control group after 4 weeks shows mild chronic inflammatory cell infiltration, fibrocellular tissue proliferation, and focal areas of osteoid tissue (arrows) (Masson trichrome, ×40).

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After 6 weeks, osteoid tissue was still present and thin trabecular newly formed bone with uneven thickness was detected around the graft material near to and far from the surgical bone margins. The condensation of fibrocellular tissue was still present with a minimal remnant of bone graft material. Moderate chronic inflammatory cell infiltration was still noticed [Figure 6].
Figure 6: A photomicrograph of the control group after 6 weeks exhibits newly formed bone trabeculae and osteoid tissue surrounding the surgical margins (Masson trichrome, ×40).

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Group II (Melatonin loaded nanohydroxyapatite granules) (after 2, 4, and 6 weeks)

Microscopic findings after 2 weeks showed less amount of chronic inflammatory cells in comparison to the other groups at the same evaluation period with marked vascularity and fibroblastic activity surrounding the graft material. Active osteoblasts encircled the graft material with osteoid tissue were observed. Thin area of newly formed bone were also detected at the surgical defect margins [Figure 7].
Figure 7: A photomicrograph of group I after 2 weeks shows chronic inflammatory cells and fibrocellular condensation around and within the graft material. Osteoblasts and few osteoclasts (arrows) are located at the periphery of some graft material (Masson trichrome, ×200).

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After 4 weeks, microscopic examination revealed that, marked vascularity and little amount of chronic inflammatory cell infiltration still present. There is a large quantity of well-organized newly formed trabecular bone with basophilic reversal lines. Osteoid tissues encircled with active osteoblasts were also detected. Remnants of graft material with crenate borders encircled with osteoclasts were detected [Figure 8].
Figure 8: A photomicrograph of group I after 4 weeks displays osteoblastic activity and new bone formation as well osteoblasts and osteoclasts bordering the remnants of the graft material. Spicules of osteoid tissue are easily detected in the connective tissue (Masson trichrome, ×200).

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After 6 weeks, microscopic examination of the surgical defect showed more quantities of well-organized newly formed bone trabecules with reversal lines. These bone trabecules appeared thicker and more organized lamellated bone than that the previous groups at this stage. Osteoid tissues were still formed in the surgical defect. Tiny spicules of the graft material were still seen [Figure 9].
Figure 9: A photomicrograph of group I at 6 weeks shows osteoid tissue, newly formed bone and condensation of fibrocellular tissue around them. Remnants of the graft material are seen (arrows) (hematoxylin and eosin, ×40).

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Group III (nanohydroxyapatite granules in hydrogel mixed with PRF particles) (after 2, 4, and 6 weeks)

Microscopic examination after 2 weeks showed marked fibrous tissue proliferation near surgical margins with mild to moderate chronic inflammatory cell infiltration and marked vascularity. Osteoid tissue and a few spicules of new bone formation could be detected away from the surgical margins. The graft material was seen in the bone defect [Figure 10].
Figure 10: A photomicrograph of group II at 2 weeks shows thin trabeculae of newly formed bone and osteoid tissues at the surgical margin (hematoxylin and eosin, ×100).

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After 4 weeks, connective tissue proliferation in the form of collagen fibers, osteoid tissue, osteoclasts and osteoblasts were observed in and around the periphery of the graft material. New bone formation near the surgical margins was progressed toward the center of the surgical defect that has the smallest thickness of new bone [Figure 11].
Figure 11: A higher magnification of the previous field exhibits fibroblastic activity, well-organized newly formed trabecular bone with basophilic reversal lines and osteoid tissues. Active osteoblasts on the peripheries of bone trabeculae are seen (hematoxylin and eosin, ×100).

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Microscopic findings after 6 weeks, exhibited marked vascularity and well-developed lamellar bone trabeculae with basophilic reversal lines. Osteoid tissue was still formed. The center of the defect had the smallest thickness of new bone. Remnants of graft material could be detected with many osteoclasts at their peripheries [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16].
Figure 12: A photomicrograph of group II after 6 weeks shows increased trabecular bone density, some bony spicules and osteoid tissues (hematoxylin and eosin, ×40).

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Figure 13: A photomicrograph of group III after 2 weeks shows fibrous tissue proliferation near surgical margins with marked vascularity. Osteoid tissue and a few spicules of new bone formation could be detected away from the surgical margins (Masson trichrome, ×40).

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Figure 14: A photomicrograph of group III after 4 weeks shows connective tissue proliferation, marked vascularity, Osteoid tissue and newly formed bone as well as remnants of the graft material (Masson trichrome, ×100).

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Figure 15: A photomicrograph of group III after 6 weeks shows newly formed well-organized lamellar bone trabeculae with marked vascularity. The center of the defect has the smallest thickness of new bone (Masson trichrome, ×40).

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Figure 16: Comparison of the mean percentages of new bone formation in groups I, II, III, and control at the different study periods.

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Histomorphometrical results

  1. Control group: It was demonstrated that there was not new bone formation at 2 weeks. Although at 4 weeks and at 6 weeks when compared with 2 weeks, there was highly statistically significant (Pa and Pb ≤ 0.001) [Table 4].
  2. Group I: It was demonstrated that, there was not new bone formation at 2 weeks after using nanohydroxyapatite bone graft alone. Although, at 4 weeks, and at 6 weeks the mean percentage of new bone formation increased with high statistically significant difference when compared with 2 weeks (Pa and Pb ≤ 0.001) [Table 4].
  3. Group II: After treatment with melatonin loaded on nanohydroxyapatite on hydrogel, the amount of new bone formation showed higher percentage histomorphometrically at 2 weeks. This percentage continued to increase at 4 weeks and at 6 weeks. These data showed statistically significant difference between both evaluation periods (2 and 4 weeks) (Pa ≤ 0.5), and high statistically significant difference between both evaluation periods (4 and 6 weeks) as (Pb ≤ 0.001) [Table 4].
  4. Group III: After treatment with melatonin PRF mixed with nanohydroxyapatite on hydrogel, the amount of new bone formation showed high percentage histomorphometrically at 2 weeks. This percentage continued to increase to reach at 4 weeks and at 6 weeks. These data showed high statistically significant difference between both evaluation periods (4 and 6 weeks) when compared with 2 weeks (Pa and Pb ≤ 0.001) [Table 4].
Table 4: Intragroup and intergroup comparison of the mean percentages of new bone formation in at the different study periods

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Intergroup comparison regarding the percentage of new bone formation between groups:

It was observed that, at all evaluation periods, there was a high significant increase in the percentage of new bone formation in (group II) when compared with control, groups I and III (P2, P4, P6 ≤ 0.001), except, at 4 weeks the percentage of new bone formation decreased but still significant when compared with group III and at 6 weeks, there was no statistically significant differences between groups II and III. Also at all evaluation periods group III showed high statistically significant difference when compared with group I and control group P3 and P5 value less than or equal to 0.001. Finally group I showed no significant differences when compared with control group with P1value more than 0.05 at 2 and 4 weeks but become significant at 6 weeks [Table 4].


  Discussion Top


The rationale for using melatonin in this study is on the basis of its positive aspects: it is endogenously produced, nontoxic, plays a regulatory role in many physiological processes. It acts as an antioxidant and free-radical scavenger, as an immunomodulatory agent and as a promoter of bone formation [16],[17],[28],[29].

In this study, PRF was used. It is found to be superior to other platelet concentrates like PRP because of its ease and inexpensive method of preparation as it obtained from an anticoagulant free-blood harvest without any artificial biochemical modification. Several researches also suggested that, it is more advantageous than autogenous graft, because an autograft requires a second surgical site and procedure [2]. The PRF clot forms a strong natural fibrin matrix, which concentrates almost all the platelets and growth factors of the blood harvest and shows a complex architecture as a healing matrix with unique mechanical properties which make it distinct from other platelet concentrates. It does not dissolve quickly at the application site, and its structure is preserved. So, growth factors contained within PRF are released slowly from 7 days up to 28 days [22],[30],[31],[32],[33]. PRF has been recently demonstrated to stimulate cell proliferation of the osteoblasts, gingival fibroblasts and periodontal ligament cells but suppress oral epithelial cell growth [34],[35],[36].

Evaluation of clinical results

The material used in this study were found to be well tolerated by both human and animals periodontal tissues with no signs of inflammation. No adverse reactions such as allergies or abscesses were observed in any patients during study period. These findings are in agreement with the results of other clinical and histologic studies which have shown that neither melatonin nor PRF elicited allergic reactions throughout their study periods.

At baseline, statistical analysis of data revealed no significant differences between the three treatment modalities in terms of PD, CAL, and BOP measurements. Accordingly, any difference during the study period between the groups at the assigned intervals, would be due to the type of graft material used.

There was a highly significant reduction in PD and CAL in all studied groups at all evaluation periods as compared with baseline. Furthermore intergroup comparison for PD and CAL showed that at the 3, 6, and 12 months study intervals, there was statistically significant difference in favor of groups II and III when compared with group I. Although at all study periods intervals (3, 6, and 12 months), there were no statistical significant differences between groups II and III.

The results of all test groups were favorable in terms of BOP, it diminished throughout the study period except in (group I) that return to increase at 12 months evaluation period and showed nonsignificant changes when compared with baseline values. Comparing between the three studied groups at different study periods, there was no statistically significant difference at baseline, 3 months, and at 6 months. At 12 months, there was statistically significant differences in favor of groups II and III when compared with groups I.

Several mechanisms may explain the results of group II, Some of these are, the influence of melatonin on fibroblastic activity and its stimulatory effect on the synthesis of type I collagen fibers which was documented by Nakade et al. [37]. Also its potent antioxidant properties that would be beneficial in reducing the degree of tissue damage [38]. Moreover, melatonin could contribute to protect and recover the integrity of gingival tissues, as it was not toxic for human gingival fibroblasts, it downregulated the expression of profibrotic markers during wound healing, suggesting a more-regenerative/less-scarring response [39]. In addition, melatonin has a significant role in preventing diseases induced by oral bacteria [40].

The factors very likely contributing to these more favorable clinical results with group III would be the significant increase in the number of matured blood vessels as well as angiogenic growth factors such as PDGF and vascular endothelial growth factor which would provide greater regeneration potential of the graft [41].

Also, PRF demonstrates a slow sustained release of growth factors ranging from 7 to 28 days ensuring a continuous stimulation for a significant time during tissue remodeling [42],[43]. Moreover, PRF increases cell attachment, proliferation and collagen related protein expression of human osteoblasts, gingival fibroblasts and periodontal ligament cells [44]. The presence of leukocytes and cytokines in the fibrin network can play a significant role in the self-regulation of inflammatory and infectious phenomena within the grafted material [19].

These results were supported by Cutando et al. [45], which examined the possible links between salivary melatonin levels and the severity of periodontal disease using the community periodontal index (CPI) and elucidated that the amount of serum melatonin secreted by salivary glands varied according to the severity of the periodontal disease: the higher the CPI score (worsening of periodontal disease), the lower the salivary melatonin level and salivary/plasma melatonin ratio, a negative association between the salivary melatonin level and periodontal disease severity. Consequently, the reduction in saliva production with higher age and the decreased melatonin production in older adults reflected in low salivary melatonin levels predispose these individuals to an increased risk of developing oral and periodontal disease.

In addition, our results were consistent with Srinath et al. [46], that evaluated the presence of melatonin in gingival crevicular fluid (GCF) and assessed the levels of salivary and GCF melatonin in periodontal health and disease. Their study showed the presence of melatonin in GCF in a concentration; 60% less than that of serum melatonin (14–60 pg/ml). When the levels of melatonin in GCF were analyzed, there was a reduction in melatonin levels from healthy participants to patients with periodontitis. These values ran inversely proportional to the values of clinical indices (i.e. the more severe the inflammation, the higher the index score and the lower the melatonin level) supporting the antioxidant and anti-inflammatory effects of melatonin.

Furthermore, our results were confirmed by multiple studies investigated the clinical and radiological (bone fill) effectiveness of autologous PRF along with use of bone graft [xenogenic bone mineral, β-tricalcium phosphate (βTCP), hydroxyapatite]. Six and 12 months evaluation resulted in statistically significant (P < 0.01) favorable changes in PD, clinical attachment level [35],[47],[48]. In addition, in 2015, a total of 40 sites with intrabony defects were selected and were assigned to the test group (open flap debridement and PRF, n = 20) and the control group (open flap debridement + demineralized freeze-dried bone allograft, n = 20). Clinical parameters were measured just before surgery (baseline) and at 6 months postsurgery. PRF has shown significant results after 6 months, which is comparable to demineralized freeze-dried bone allograft for periodontal regeneration in terms of clinical parameters [49].

Evaluation of histological and histomorphometric results

Our histopathologic findings suggested that group II had superior results in terms of the amount of bone formed when compared with group I, III and control groups at all evaluation periods. By the end of 6 weeks of surgery bone maturation with thicker and well-organized (lamellated) trabecules was obvious. This was evidenced by histomorphometric analysis, as it revealed statistically significant difference in favor of group II when compared with the other groups at all evaluation periods except with group III at 6 weeks. Osteoid tissues were still formed till the end of evaluation period, which mean that, bone formation capacity is still.

The best results of melatonin regarding the amount of bone formed may be attributed to the stimulatory effects of melatonin on the proliferation and differentiation of osteoblasts [15],[50]. It also could be explained by the direct action of melatonin on the osteoblasts cells as it induces a higher rate of maturity of preosteoblasts to osteoblasts, both in quantity and velocity [reduces the period of differentiation of preosteoblast line cells into matured osteoblasts from 21 days (which is normal) to 12 days], with a higher rate of production of the osseous matrix and its corresponding calcification [15],[50],[51]. Furthermore, it may also be explained by the action of melatonin on osteoprotegerin (OPG) and RANKL molecules. It was demonstrated that, melatonin application in-vitro upregulated the expression of OPG and downregulated the expression of RANKL in mouse osteoblast line MC3T3-E1 cells [52]. Consequently, inhibition of osteoclasts maturation, differentiation, activation and increased apoptosis occurred [53],[54],[55].

Another interesting explanation is that bone marrow cells of mice and humans are capable of synthesizing melatonin and that high concentrations of melatonin are found in bone marrow (100 times higher than that in plasma) which suggested that melatonin has local regulatory actions in bone [56].

In addition, melatonin acted on the bone as a local growth factor, it has been found to be a significant modulator of the metabolism of calcium, and prevents osteoporosis and hypocalcemia in certain cases, probably because of its interaction with other bone regulatory factors, such as parathormone, calcitonin, or prostaglandins [57],[58].

Of interest, it was observed that, in group II, less amount of chronic inflammatory cells in comparison to the other graft materials with marked vascularity and fibroblastic activity surrounding the graft material in all evaluation periods. This may be explained by the influence of melatonin on fibroblasts activity and the stimulatory effect of melatonin on the synthesis of type I collagen fibers which was documented by Nakade et al. [37]. Also, it may be attributed to the well-known potent antioxidant and anti-inflammatory properties of melatonin [38].

Several studies in vivo on this indolamine concluded that, the antioxidant properties of melatonin might justify its therapeutic use in diseases in which free-radical damage is a major aspect of the condition like infectious and inflammatory disorders [59],[60],[61],[62],[63].

These results are in agreement with many recent studies that have dealt with the effect of local application of melatonin on osteointegration around dental implants, using histomorphometric examination to assess the amount of newly formed bone as Cutando et al. [51], who applied 1.2 mg of lyophilized powdered melatonin to the bone hole at each side of the mandible of 12 Beagle dogs before implanting. None was applied to the control sites. After 2-week treatment period, histomorphometric analysis revealed that melatonin significantly increased the perimeter of bone that in direct contact with the treated implant, bone density, new bone formation and interthread bone. Their results were in accordance with our results which revealed significant increase of newly formed bone (P < 0.05) with melatonin treatment.

Similarly, the same study was done by Guardia et al. [64], however, instead of 2 weeks evaluation period in the study of Cutando et al. [51], they evaluated their results after 5- and 8-week treatment periods. Their findings demonstrated that melatonin significantly increased interthread bone and new bone formation. They also noticed an increase bone implant contact and peri-implant bone, however, it was insignificant. The difference of their results with those of Cutando et al. [51], was attributed to the time of evaluation where at 2 weeks evaluation period, the melatonin has maximum activity over bone parameters. These observations supported our results regarding the marked significant amount of bone formation reported at 2 weeks evaluation period. It worthy-noted that although the differences in the methodology as, site of melatonin application, the form of melatonin used, periods of examination and the method of calculation of the percentage of new formed bone between our study and the previous studies [51] and we can postulated that the actions of melatonin on bone tissue are of interest as it is superior as a biomimetic agent enhancing bone formation.

In accordance with these results, Tresguerres et al. [65] demonstrated that local application of melatonin (3 mg) around dental implants inserted in the rabbit tibia significantly increased the trabecular bone density. In addition, they detected newly osteoid tissues using sections stained with Masson trichrome after 4 weeks from implant insertion.

Also, Ramírez-Fernández et al. [66] histomorphometrically studied melatonin that promotes angiogenesis during repair of bone defects, in rabbit tibiae. The authors observed complete repair of the bone defects. Statistically significant differences were found in the melatonin group in relation to the length of cortical formation and in relation to the number of vessels observed when compared with the control group.

In addition, Calvo-Guirado et al. [67] radiographically and histomorphometrically examined new bone formation in bone defects after melatonin and porcine bone grafts. Histomorphometric values indicated that, melatonin has proven to regenerate the width and length of cortical bone in tibiae rabbits more quickly than collagenized porcine bone. Melatonin acts as a bone stimulator compared with porcine bone and control sites.

Furthermore, Shino et al. [68] evaluated the effects of topical application of melatonin on vertical bone augmentation in rat calvaria secluded spaces. They demonstrated that, new bone regeneration within the plastic cap was increased significantly in the melatonin versus the control group. Also, the number of blood vessels increased significantly in the melatonin group versus the control group. Which mean that, melatonin enhanced angiogenesis during the repair of bone defects in rabbit.

Other several studies showed that melatonin administration had positive effects on both angiogenesis and wound healing, angiogenesis preceded bone regeneration in bone defects and significantly induced angiogenesis during the first 4 weeks [68],[69],[70].

In this study, the second best results were observed with group III. Bone formation represented by osteoid tissue and a few spicules of new bone could be detected at 2 weeks. Although at 4 weeks, osteoid tissue and calcified bone formation were seen around and within the graft material. By the end of 6 weeks well-developed lamellar bone trabeculae with basophilic reversal line were observed. Osteoid tissue was still formed, whereas graft material remnants were still present. Histomorphometric analysis in this study, revealed statistically significant difference in favor of group III when compared with group I and control group at all evaluation periods.

These results may be explained by the release of growth factors such as PDGF, fibroblast growth factor (FGF), TGFβ1, and vascular endothelial growth factor from the PRF. Growth factors such as FGF and TGFβ are multifunctional cytokines and key regulators of bone development. TGFβs are member of the TGFβ superfamily includes bone morphogenic proteins. TGFβ are existed at the highest rate in bone matrix of all tissues. TGFβ1 promoted chemotactic attraction of osteoblasts, enhancement of osteoblast proliferation whereby increase new bone formation. Also FGF positively enhance osteoblast differentiation and bone formation. However, the mechanisms of TGFβ and FGF on bone formation are still poorly understood [71].

Also these results may be attributed to the fibrin matrix structure of PRF that acts as a scaffold for growth factors thus aiding in cytokine retention for extended periods. We think that also stem cell in blood circulating trapped within fibrin matrix may also enhance new bone formation [71].

PRF has been shown to significantly increase the proliferation of human osteoblasts in vitro, depending on the dose. In addition, it increases alkaline phosphatase expression in a time-dependent manner and it is believed that both these properties are because of the presence of growth factors and the fibrin matrix structure.

In this study, marked vascularity and fibroblastic activity was observed in group III at all evaluation periods. This may be related to PRF that may consider an abundant source of vascular endothelial growth factor, which is a key player in angiogenesis. Also, polypeptide growth factors may enhance soft-tissue healing by increasing the angiogenesis and matrix biosynthesis during the wound healing process. PRF increase cell attachment, proliferation and collagen related protein expression of human osteoblasts. It also enhances phosphorylated extracellular signal regulated kinases, OPG, and alkaline phosphatase expression which benefits periodontal regeneration by influencing human periodontal ligament fibroblasts [72].

In accordance with these results, Kim et al. [73] suggested that PRF in combination with βTCP increased new bone formation when compared with βTCP alone. Also, Acar et al. [32] showed that by histomorphometric and microcomputed tomographic analysis, the Straumann Bone Ceramic + PRF group exhibited more new bone formation at the end of weeks 4 and 8 compared with the Straumann Bone Ceramic group.

Furthermore, these current results were confirmed by the study of Oliveira et al. [36], who investigated the effects of PRF associated or not with Bio-Oss on bone defects in the calvaria of rats. Bone regeneration was evaluated by histomorphometric analysis. The highest mean percentages of new bone formation at 30 days were observed in the Bio-Oss associated with PRF group with significant differences than that of all of the other groups. However, in disagreement with our results, at 60 days, the Bio-Oss associated with PRF and Bio-Oss groups had similar results. This may be attributed to the difference in evaluation periods, the type of bone graft, and the size of its partials.

In an in-vitro study by He et al. [21], on rat osteoblasts, it was found that cells treated with exudates of PRF collected at day 14 reached peak mineralization significantly more than both negative control and positive (PRP) control groups. They concluded that PRF is superior to PRP, from the aspects of expression of alkaline phosphatase and induction of mineralization, because of markedly released TGFβ1 and PDGF-AB which supported our results.

In this study, residual parts of all the graft materials were still present up till the end of study period. This was in accordance with Gab Allah et al. [74] who reported that, nanohydroxyapatite needed 12 weeks to be completely degraded. Thorwarth et al. [75] and Spies et al. [76], also reported, undisturbed osseous integration and complete resorption of nanohydroxyapatite graft occurred within 12 weeks. So, this apparently was an inadequate period for such an assessment.


  Conclusion Top


On the basis of the results of this study, there is a strong correlation between clinical, histological and histomorphometrically analysis. Melatonin loaded on nanohydroxyapatite in hydrogel and nanohydroxyapatite mixed with PRF treatment were found to improve clinical and histological outcomes. On the basis of these findings, it can be claimed that, the two tested graft materials of melatonin loaded on nanohydroxyapatite in hydrogel and nanohydroxyapatite mixed with PRF are considered a promising alternative grafts for treatment of intrabony defects in patients with severe chronic periodontitis by adding osteoinductive properties to osteoconductive scaffolds.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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