The present study will be performed to evaluate the efficacy of lyophilized platelet-rich fibrin alone and combined with nanocrystalline hydroxyapatite in treatment of periodontal intra-bony defects through:

1. Primary outcomes:

   • The clinical parameters including: Probing pocket depth (PPD) reduction and clinical attachment level (CAL) gain.
   • The radiographic parameters including: Marginal bone level (MBL), bone-defect fill.

2. Secondary outcome: The level of BMP-2 in Gingival crevicular fluid (GCF).

Periodontitis is a multi-factorial inflammatory process affecting the periodontal tissues. Among the characteristic signs of periodontal disease is loss of alveolar bone support. The risk of further alveolar bone loss and the probability of tooth loss increases with the presence of intra-bony defects.

Periodontal intra-bony defects (IBDs) are osseous defects with specific morphology, the bottom of these defects is located more apically than the alveolar crest and they are surrounded by bony walls on 1-, 2-, or 3- sides, with the tooth root forming the additional wall. The prevalence of these defects is significantly lower than supra-crestal periodontal defects.

Among the main goals of periodontal therapy is to regenerate the lost periodontal tissues after arresting the progress of the disease. Surgical management of periodontally affected sites includes conventional and regenerative procedures. The conventional modality of surgical debridement allows for reliable access to root surfaces leading only to healing by repair. While the regenerative treatment options allow for the regeneration of destroyed periodontal ligament and bone. Guided tissue regeneration (GTR), placement of bone grafting materials, the addition of biologic mediators and a combination of such techniques are the different forms of periodontal regenerative techniques.

Bone grafts (BGs) and substitutes are classified into autogenic, allogenic, xenogenic and synthetic bone grafts. Synthetic nanocrystalline hydroxyapatite (NCHA) has been extensively applied as bone graft replacement material and characterized by its osteoconductive properties and improved osseointegrative features. The size of the particle was found to be around 18 nm, allowing for quicker vital bone substitution. It has been proved that NCHA can enhance the adhesion of the bone formative cells (osteoblasts) more than micromaterials. Furthermore, the nano-sized particles provide special properties to NCHA such as ahydrated surface layer, a large surface/volume ratio, this layer is actively involved in the homeostasis process, ionic exchange and other techniques involved in the osteogenesis regulation. NCHA is osteoconductive but has inferior osteoinductive properties; therefore, one of the efforts to increase the ability of this material is to combine it with other bioactive materials such as platelet-rich fibrin (PRF).

Platelet-rich fibrin is a second-generation platelet concentrate that has been widely used in the treatment of bone deficiency and its main action is derived from its high content of platelets that release many growth factors (GFs) and cytokines. However, freshly prepared PRF must be used immediately in order to retain the bioactivity of growth factors. In addition, the bio-degradation rate of PRF is fast and irregular, along with the rapid release of growth factors, and then enzymatically hydrolyzed.

The lyophilization of PRF by vacuum freeze dryer was found to provide a dense three-dimensional fibrin network, better storage stability with a longer half-life and preservation of GFs. Lyophilized platelet-rich fibrin (Ly-PRF) showed adaptability as a viable biomaterial for application as a craniofacial bio-scaffold due to, the advantage of lowering the difficulty in application, providing a newly grown tissue with straightforward access to multiple GFs as well as, sustained release GFs presenting a better tissue regeneration process and bone tissue reconstruction.

It was proven that, Ly-PRF significantly improve the osteogenic differentiation of bone marrow mesenchymal stem cell (BMSC) in vitro through the upregulation of osteogenic markers: collagen type I, osteopontin (OPN), osteocalcin (OCN), and bone morphogenetic protein 2 (BMP-2). BMP-2 is described as being osteoinductive, which is capable of inducing and enhancing bone growth and formation, and it also promotes cell chemotaxis, proliferation and differentiation towards the osteogenic pathway.

The present clinical trial will be performed to clarify and shed some light on Ly-PRF in periodontal regeneration

Inclusion Criteria:

• All periodontitis patients to be included in this study should be diagnosed as having stage III periodontitis with probing pocket depth (PPD) ≥ 6 mm, and clinical attachment loss (CAL) ≥ 5mm.
• 2- or 3-wall intra-bony interproximal defect with depth ≥3 mm, a width of ≥3 mm at its most coronal part and 45-55-degree angulation will be selected in order to reduce defect variability.
• All patients will be free from any systemic diseases according to the American Dental Academy general guidelines for referring dental patients to specialists and other care settings

Exclusion Criteria:

• Any patients with any systemic condition that contraindicate any surgical intervention.
• Pregnant or lactating females, smokers, or alcoholic patients.
• Patients with intra-bony defects received drugs that affect bone turnover such as (chemotherapy and radiotherapy).
• History of periodontal therapy and antibiotic administration in the last 6 months.
• Patients taking any medicine which may affect the function of platelet (i.e., aspirin) in the previous 3 months.
• Miller grade II or greater mobility, furcation involvement as well as one-wall defects and interdental craters.
• Patients with unacceptable oral hygiene after the re-evaluation of phase I therapy.

The statistician and the evaluator were blinded, but the operator was not because of the nature of the study