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OSSEOUS REPAIR USING LEUKOCYTE – PLATELET RICH FIBRIN (L-PRF) ASSOCIATED OR NOT TO ANORGANIC XENOGRAFT (BIO-OSS®). HISTOMETRIC AND HISTOCHEMICAL STUDY IN RABBIT CALVARIA Vinicius Augusto Tramontina***, Anibal Soley Abbate Filho***, Luis Eduardo Almeida**, Sonia Mara Luczyszyn***, Vula Papalexiou***, Aline Cristina Batista Rodrigues Johann***, Sung Hyun Kim*** ** School of Dentistry, Marquette University Milwaukee, USA *** School of Life Sciences, Dentistry, Pontifical Catholic University of Parana , Curitiba (PUCPR-Brazil)
ABSTRACT
METHODOLOGY
Background:, Xenografts (bovine bone), obtained from chemical extraction of all organic material from the cortical bone or marrow is very similar to the mineral portion of human bone with similarities in internal surface area, porosity, particle size, and calcium/phosphate ratio.3 This bone substitute has demonstrated osteoconductive properties and has been shown to be highly biocompatible with oral hard tissues of animals and humans, causing no antigenic inflammatory or adverse responses.4.6 Bio-Oss can contribute to bone repair, as suggested by in vitro7 and histologic8 studies, which demonstrate a close relationship between the particle Bio-Oss and new bone formation. The platelet-rich fibrin (L-PRF) advocated by Choukroun et al.9 L-PRF belongs to a new generation of platelet concentrates, with simplified processing without biochemical blood handling. L-PRF components have synergistic effects on wound healing.10 Its use promotes a slow release of growth factors such as transforming growth factor β1 (TGF-β1), platelet-derived growth factor αβ (PDGF-αβ), vascular endothelium growth factor (VEGF), and glycoproteins (e.g., thrombospondin-1) for at least 7 days, reaching up to 14 days.12,13 The effects of L-PRF in bone regeneration have been widely studied both in vitro and in vivo: in alveolar ridge preservation,14 in maxillary sinus bone graft as sole material,15 in root coverage procedures,16 and in periodontal17 and furcation defects,18 with promising results. L-PRF has been shown to increase adhesion and proliferation of osteoblasts, and upregulate collagen-related protein, which could work together to promote bone regeneration. L-PRF can serve as a scaffold for migration of fibroblasts and angiogenesis and has been shown to be suitable for use as a matrix for in vitro replicating human periosteal cells, which can have applicability in bioengineering of bone tissue.20.
The present study aimed to evaluate bone healing using leukocytes and platelet rich fibrin (L-PRF) and xenograft (Bio-Oss) in surgically created defects
Methods: Four circular defects were made in the calvaria of 20 New Zealand rabbits. The defects were randomly filled with: L-PRF, L-PRF + Bio-Oss, Bio-Oss or clot (control). The animals were sacrificed after 60 days. Histological parameters evaluated: 1. Mean area of new bone; 2. Mean percentage of mature collagen; 3. Mean number of osteoclasts: 4. Mean percentage of contact bone/particle.
Results: Figures and Graphics
Conclusions:  The L-PRF + Bio-Oss biomaterials leads to increased formation of new bone;
The L-PRF + Bio-Oss provided a high percentage of mature collagen at 60 days of repair, showing a high degree of organization of new bone;
The L-PRF + Bio-Oss association showed a low average of osteoclasts, which indicates an advanced stage repair;
The addition of L-PRF to Bio-Oss did not interfere with the contact percentage of bone and particle in repaired defects at 60 days.
3. Mean number of Osteoclasts: TRAP staining was performed using the TRAP kit 387 (Sigma-Aldrich Co, St. Louis, MO) C D
Figure 1: (a) Filling of defects with L-PRF (A), L-PRF + Bio-Oss (B), Bio-Oss © and Clot (D). (b) Defects healed after 60 days.
Figure 4: Samples stained with Trap and with a magnification of 400x. The multinucleated osteoclast cells are stained in purple bluish tone (arrows, a and b).
Histomorphometrically Analysis
4. Mean percentage of contact bone/particle.
1. Mean area of new bone
Figure 2: (a) Sample of filled defect. (b) Sample with removal of all soft tissue keeping only the bone tissue, where total area was measured.
Figure 5: (a) Samples stained with Trap and with a magnification of 400X. Measurement of total perimeter of the particle. (b) Contact perimeter measuring bone/particle.
2. Mean percentage of mature collagen
RESULTS
Figure 3: (a) Samples stained with Sirius Red and taken under polarized light; areas with more reddish hues (arrows) are compatible with more mature collagen (lammelar bone); (b) Areas with greenish hues (arrows) demonstrate immature collagen (woven bone)