The Use of Platelet Rich Fibrin and Nano Hydroxyapatite as Regenerative Treatment of Furcation Class II Defects (A Histomorphometric Study in Dogs)

Introduction

Periodontitis is one of the most common diseases affecting the periodontium. Its main cause is bacterial plaque, that can result in destruction of the gingival tissues and periodontal supporting apparatus (Wang et al., 2005). The difficulty of controlling plaque in furcations is responsible for the presence of extensive lesions in this area (Waerhaug, 1980). Different treatment modalities are used in management of furcation depending upon degree of furcation involvement including scaling and root planning, furca plasty, tunnel procedure, root resection, bicuspdization and regenerative approach as guided tissue regeneration and bone grafts (Kornman and Robertson, 2000).

Hydroxyapatites (HAs) is considered as a family of grafting materials that have a high level of biocompatibility. It has decreased osteoconductivity and poor degradation properties. To improve such shortcomings, nanocrystalline hydroxyapatite (nano-HA) paste containing 65% water and 35% of nanostructured apatite particles has been used for augmentation procedures in intrabony defects (Thorwarth et al., 2005).

Nano-HA has a stimulating effect on cultured PDL cells resulted in  increased proliferation rate of these cells which is linked to the activation of  epidermal growth factor receptor (Kasaj et al., 2008)

Heinz et al,. 2010 reported that nanocrystalline hydroxyapatite bone graft substitute in treatment of intrabony periodontal defects, showed more reduction in the probing pocket depth (PPD) in the study group compared with the control group.

Platelet-rich fibrin (PRF) was developed in France by Choukroun et al (2001) and considered as a second generation of platelet concentrates, it generally accelerates soft and hard tissue healing.  

The flaps were repositioned and secured by 4-0 bioresorbable sutures. Afer finishing the surgical procedure, all dogs were injected with antibiotics and pain killer as  a part of post-operative care. The animals were followed for 90 days healing period. During the first 28 days, all dogs fed on soft diet in order to protect the wound from any mechanical trauma. Topical application of 0.2% chlorhexidine solution were maintained to fight against plaque accumulation.

Figure (2): (a) showing defect treated with Nano hydroxyapatite alone (b) showing defect treated with PRF alone (c) defect treated with Nano hydroxyapatite and PRF (d) defect

treated with debridement only (controls)

Post-operative Evaluation and Sacrifice

Dogs were sacrificed by excess anesthesia and the mandibles were dissected and biopsies were collected for histopathological examination. The biopsy were preserved in jar containing formalin 10% and sent to the pathology lab. For histopathological examination.

I- Microscopic examination

The mandible was carefully dissected, immediately fixed in 10% formalin, after that decalcification was done using ethylenediaminetetraacetic acid (EDTA) for 4 weeks. Then, tissue blocks were processed and embedded in paraffin. Four μm sections were cut perpendicular to the long axis of the tooth and mounted on glass slides. The collagen and new bone formation were detected by Masson`s Trichrome stain (fig , 3) (fig , 4) (fig , 5) (fig , 6)  .

Figure (3): A photomicrograph of group A experimental specimen showing a mixture of woven bone and the more regular concentric lamellar bone (masson trichrome)

Figure (4): A photomicrograph of group B showing woven bone with wide osteocytes forming thickened irregular trabeculae. Note the widespread osteoblastic rimming and the dense fibrous background.(masson trichrome)

Figure (5) : A photomicrograph of group C experimental specimen showing mature lamellar bone (RED) surrounding marrow cavities and less organized woven bone (BLUE). (masson trichrome)

Figure (6):  A photomicrograph of group D control spaceman showing irregular woven bone spicules rimmed by osteoblasts as well as densely packed  Collagen fibers (masson trichrome).

II- Measuring the newly formed bone

The area of newly deposited bone was estimated using Leica Quin 500 analyzer computer system, (Leica Microsystems, Swizerland). The cursor was used to define the areas of newly deposited bone trabeculae or collagen, that were marked by a binary color which was measured by the computer. The image analyzer was calibrated automatically to transform pixels produced into actual micrometer units.

The percentage of newly-formed bone was evaluated in five different fields in each group

with magnification (x100). Both mean values and standard deviation (SD) were calculated for all groups.

Results

Histomorphometric analysis:

I- Area percent of collagen

The greatest area percent of collagen was recorded in Group D, whereas the lowest mean value was recorded in group A. Analysis of variance (ANOVA) test revealed that the difference was statistically significant (p<0.0001). Tukey’s post hoc test revealed a significant difference between each two groups, except between groups B, C (Table 1) (Table 2).

Table (1): Detailed results of Tukey’s post hoc test for difference between groups regarding the area percent of collagen

Table (2): Area percent of collagen in different groups and significance of the difference using ANOVA test

II- Area percent of bone

The greatest area percent of bone was recorded in Group A, whereas the lowest mean value was recorded in group D. Analysis of variance (ANOVA) test revealed that the difference was statistically significant (p=0.047). Tukey’s post hoc test revealed a significant difference between groups A and D only (Table 3) (Table 4).

Table (3) Detailed results of Tukey’s post hoc test for difference between groups regarding the area percent of bone

Discussion

Animal studies are efficient complementary to in vitro studies prior to apply new clinical treatments because biopsy harvesting for histology is inapplicable in humans. Thus, animal experimentation is a necessary bridge prior to clinic application (Struillou et al, 2010; Seol et al, 2010).  Many animals have been used in evaluation of periodontal regeneration however; dogs and nonhuman primates are considered the most devilishly used in literature (Bogle et al,. 1997). A chronic class II furcation defect was used to hinder spontaneous regeneration that may reduce the sensitivity of testing any regenerative power in acute defects (Soares et al, 2005).

To create such chronic defect a rubber base impression material was used to prevent spontaneous repair and induce an inflammatory response as reported by (Burcak Simsek et al ,. 2012). The critical-sized defect is the minimal size of a defect that can not heal spontaneously (Liu et al, 2010).A surgically created furcation defect is better than natural plaque-induced chronic defect, as the surgical preparation saves more time and permits standardization of the defect as reported by Jung et al, 2011. In the present study, the defect size measured five mm in the apicoocclusal direction and two mm deep in horizontal direction and was performed on the buccal side of the mandibular second and third premolars as this defect size was reported in many studies to work as class II furcation defects (Plínio Regazzini et al ., 2004  ; Suaid  et al,. 2011 and Burcak Simsek et al ,. 2012 ). Histologic analysis was necessary in this study to evaluate the effectiveness of the regenerative techniques and to predict the presence and the extent of periodontal regeneration as done by Burcak Simsek et al., 2012.

The usage of PRF in regenerative  periodontal procedures is based on the presence of many growth factors within a homogenous, 3-dimensional organization of fibrin meshwork which contains circulating cytokines (Dohan Ehrenfest et al ,. 2009). Growth factors release from the PRF membrane could further augment the regenerative power of the bone cells, leading to considerable amount of bone formation (Sambhav Jain et al,. 2014). The combined use of both PRF and Nano-HA could accelerate healing processes getting use of the synergic effect of the powerful inherent regenerative potential of PRF and the osteoconductive property of Nano-HA. In the present study, Nano-HA was used based one the hypothesis that the Nano structured materials have a huge  number of molecules on their surface when compared to bulk materials. Although Nano-HA showed close contact with surrounding tissues and quick resorption characteristics, It was found that undisturbed osseous-integration and complete resorption occurred within 12 weeks (Schwarz et al., 2006).

The Nano-HA as bone graft substitute could result in rapid healing of critical sized defect in animal’s experiments and human trails. Schnettler et al., (2004) reported that Nano-HA stimulates bone healing by activation  of osteoblast. Nano-hydroxyapatite has been used successfully to fill in bone defects. It provides an ideal scaffold for new bone formation in animal studies, this bone substitute with small granules that allow more bone substitution. It has been widely used in bone regeneration, implant tooth replacement, and intra-bony defects as reported by Lekovic et al., 2012.

The present study showed that, the largest area percent of newly regenerated bone was observed in the (PRF+ NHA bone) group, followed by the PRF group, then the NHA group, with the least value recorded in the control group. The difference was not statistically significant except in comparing (PRF+ NHA group) with control group (p<0.05).  The maximum regenerative potential was noticed in combined PRF and NHA group than other groups. PRF has angiogenic properties that might lead to an increased probability of revascularization and enhances consolidation of the graft when used in combination with an osteoconductive medium. NHA application showed non-significant improvements compared with the control group. Non statistically significant improvement was observed in PRF group compared with bone group regarding % of newly formed bone in the defect, this was in accordance with Wang and Boyapati 2006 who reported that angiogenesis is a critical factor for success in furcation regeneration and as PRF play an important role in angiogenesis and contain growth factors such as PDGF and TGF β that cause better induction of osteoblastic differentiation as well, PRF clot provide a scaffold for recruitment of tissue cells to injured site (Dohan et al., 2006; Dohan et al., 2009 ). In the control group (group D) extensive fibrous and mesenchymal activity was found with densely packed granulation tissue that formed of fibroblasts, new collagen fibers, and blood vessels. The histomorphometric results within this group showed the least area percent of the bone and this was in agreement with many authors who had reported the difficulty of periodontal regeneration in furcation defects (Bowers et al ,. 2003 ; Verma et al ,. 2013). The present study revealed no inflammatory or foreign body reaction and no root resorption or ankylosis in all included four groups. Clinically, no allergic or toxic reaction recorded during the study period indicating biocompatibility of  Nano hydroxyapatite and the safety of platelet rich fibrin.

Conclusions

The use of platelet rich fibrin in treatment of furcation class II defects could result in a new bone formation in the furcation area .

Combining PRF with Nano hydroxyapatite bone graft in treatment of furcation class II defects could significantly accelerate healing processes.