Recombinant human bone morphogenetic protein -2 in combination with β-Tricalcium Phosphate and Platelet Rich Fibrin for Socket Preservation before Implant Placement

–Periosteum is a rich source of osteoprogenitor cells, and apless extraction technique allows for preservation of periosteum. Recombinant Bone Morphogenetic Protein-2 has osteoinductive capacity and has been exploited for bone augmentation. The aim of this study is to determine the effectiveness of rhBMP-2 for socket preservation along with β-TCP and PRF to enhance bone quality, clinically and radiographically. Results- Minimal crestal bone changes were seen at 3 months compared to baseline values; bone loss of 0.6 ± 0.13 mm at mesio-distal aspect, 0.5 ± 0.13 mm at bucco-lingual aspect. At 3 months reentry, no residual bone particulate graft were seen in any of site. Clinical hardness of bone evaluated through tactile perception while drilling, prior to implant placement, was analogous to drilling into spruce or white pine wood. Histologic analysis conrmed the absence of residual bone graft indicating new bone formation .Complete radiographic ll was seen at 3 months. The patients in current study showed adequate bone formation in grafted sockets and required no additional augmentation during implant placement. Therefore, based on the results and within the limitations of study it can be suggested that, rhBMP-2 with PRF and β-TCP can be recommended for socket preservation prior to implant placement


Introduction
Currently endosseous implants have become the choice of therapy for the management of edentulous site .(1)(2) (3).Following extraction, however the alveolar process undergoes resorption as a result of osteoclastic activity leading to diminished alveolar ridge dimensions in both the vertical and horizontal planes.(4)Alveolar ridge alterations ranging from 40-60% have been reported to occur as early as three months after extraction.(5) This altered height and width present functional as well as aesthetic problems resulting in compromised rehabilitation.Non-grafted extraction sites may lose up to fty percent of ridge width in rst 12 months following removal of tooth.(6) An average reduction reported in width and height of the grafted extraction sites is minimal in contrast to non-grafted extraction sites, with the majority cases requiring no additional augmentation to have the implants placed.(7)Therefore, surgical approaches aiming at maintaining ridge volume by ridge preservation before implant placement have been proposed to be effective.
Platelet -rich brin (PRF) comprises of brin matrix enriched with platelets and growth factors.Hence is a powerful bioscaffold for tissue regeneration.(8)Alloplastic materials like tri-calcium phosphate (TCP) is regarded as a viable substitute to the autogenous bone or other bone substitutes.(9)An ability of PRF for regeneration can be intensi ed in a blend with bone substitutes.The Recombinant human bone morphogenetic protein-2 (rhBMP-2) reported to be promising in various bone augmentation procedures.(10) An animal study has reported noteworthy bone formation escorted with rhBMP-2 impregnated β-TCP scaffold in cranial bone defect model.(11) The utility of rhBMP-2 and Acellular Collagen Sponge (ACS) in extraction defects reported to be bene cial in reconstruction of buccal plate.It aid to maintain ridge con gurations favoring implant placement 5 months later.(12) The alveolar ridge dimensions are crucial for implant placement.Similarly, the inherent amount of vital bone formation in healing socket is also important.Therefore, the study was undertaken to evaluate clinically, radiographically, and histologically the effectiveness with ridge preservation performed using rh-BMP-2 in combination with β-TCP and PRF.

Materials And Method
Following informed consent, two patients who required extraction of teeth for reasons other than periodontitis were recruited.After proper examination and diagnosis, initial therapy consisting of oral hygiene instructions, supragingival and subgingival scaling were performed.(Table 1) Four sites were treated in two patients.The selected sites were examined, both clinically and radiographically (Table 2).Allen Way, Carlsbad, California) to enhance wound healing by lling the socket up to the level of the alveolar crest.The remaining clot was compressed to a membrane of high tensile strength.The membrane was then carefully placed covering the graft material.Approximation of the surgical site was done using a 3-0 silk suture.(Centisilk Non-Asorbable Surgical Suture U.S.P).

Implant placement
The patients were recalled after 3 months for implant placement.Following administration of local anaesthesia (Xicaine®, ICPA Health Products Limited), an incision was given from line angle of adjacent teeth to elevate mucoperiosteal ap and expose the underlying bone.Alveolar ridge measurements were taken intrasurgically, and were compared with the preoperative alveolar dimensions (Table 2).A 2 mm trephine drill was initially used to harvest bone core for histologic examination.The bone tissue was moved to a xture containing 10% neutral buffered formalin.Further it was sent to the laboratory for histologic evaluation.Osteotomy preparation was completed using sequential drills based on the size of the implant selected.An implant (Equinox, Myriad Plus TM implant system) of the largest possible dimension was placed according to manufacturer's recommendations and further follow-up was carried out .Four implants were placed in two patients.The biopsied tissue from the four sites, obtained prior to implant placement were evaluated, histologically (Fig. 1)

Discussion
Healthy osseous contour provides a skeleton for peri-implant soft tissues.The purpose of atraumatic extraction is to minimize bone resorption and to maintain alveolar ridge dimensions during healing.In present study, socket preservation was done by using a combination of PRF, β-TCP and rh-BMP-2.After 3 months of ridge preservation clinical examination of the ridge revealed minimal dimensional changes ( mesio-distal bone loss 0.6 ± 0.13 mm and bucco-lingual bone loss 0.5 ± 1.13 mm).The clinical hardness of bone evaluated during drilling, prior to implant placement, provided a tactile sensation analogous to drilling into spruce or white pine wood at all the four sites.(13) Although the density of bone could not be evaluated using Houns eld units, dense to thick porous cortical bone on the crest and coarse trabecular bone underneath was seen on radiographic examination, suggesting D2 bone quality (14).Histologically, presence of vital, mineralized osteoid trabeculae along with soft tissue containing spindle nuclear cells indicating new bone formation was noted.All four implants achieved good primary stability.
As soft tissue grows six times faster than the bone tissue (15), in the present study the sockets were covered with a saddle of PRF membrane which prevented the gingival connective tissue down-growth.It prevented epithelial migration into the defect.It also held osseous graft in place.( 16) Additionally, the membrane also aided in clot stabilization.PRF principally contain brin matrix rich in platelets, leukocytes.Cytokines such as IL-1, -4, and − 6, and growth factors such as PDGF, TGF-beta, IGF, EGF and VEGF are other important components.These are the key elements in bone regeneration.(17) Fibrin gels in coagulation cascade aids in brinogen molecules collaboration.This leads to a highly biocompatible three-dimensional ber network.(18)Therefore, PRF resulted in improved wound healing and enhanced bone graft density and maturation.(19) β -TCP is (puri ed, microcrystalline porous) form of calcium phosphate.A remarkable resorption of β -TCP particulate is estimated around 3 to 6 months after placement.Besides, particles become well-incorporated into new bone formation.This creates a dense cancellous matrix.Biodegradation of graft takes place by osteoclastic activity as well as chemical dissolution by tissue uids.During implant placement, a solid new bone formation was evident which favored the placement of an ideal implant size.
The use of rhBMP-2 necessitates the use of a carrier for its oseoinductivity (20) Misch emphasized advantages of rhBMP-2 in new bone growth in patients requiring extraction.In present case series rhBMP-2 was used as an agent to promote socket repair as well to aid in ridge augmentation when combined with a part of β -TCP.Fiorellini et al (21) determined the e cacy of varying concentrations of rhBMP-2 in socket defects.The results showed profound bone growth when rhBMP-2 concentration was increased.Author also stated an ideal concentration for rhBMP-2 in grafting could be 1.5 mg/mL.Therefore, in the present study 1.5 mg/mL concentration of rhBMP-2 was used.
Various studies (22) ( 23)have supported the use of a mixture of β-TCP and rh-BMP-2 to facilitate increased bone formation in the defects.Therefore, in this study rhBMP-2 was used in combination with β-TCP to promote bone growth.

Limitations
The present study describes results obtained in a small sample.Although there is evidence of good quality bone formation in a short time period following the use of a combination of rhBMP-2, β-TCP and PRF, larger population studies may further improve the signi cance of the results obtained.PRF was used to cover and hold the graft in place.The increase in keratinized tissue thickness following the PRF membrane placement was not evaluated.Additionally, only six months follow-up results have been reported, further long term evaluation of the patients would be bene cial.Even though pre-operative CBCT was taken but due to cost concerns and unwillingness of the patient the post-operative CBCT could not be taken.

Table no 1
Full Mouth Papillary Bleeding Index (FMPBI) and Full Mouth Plaque Index (FMPI) After administration of local anaesthesia (Xicaine®, ICPA Health Products Limited) a minimal ap re ection was done.The ap re ection was not extended beyond 2 mm of alveolar crest.Minimally invasive periotomy was carried out with a Bard Parker Surgical blade number #15 (Glassvan®, Niraj Industries Pvt Ltd) to facilitate atraumatic extraction without causing damage to the buccal plate.Consecutively, walls of the socket were verified for integrity.Twenty millilitres of venous blood was withdrawn in two test tubes from the antecubital vein.The blood was centrifuged (3000 rpm for 12 minutes) to obtain PRF.One part of PRF was mixed with β-TCP (OSTEON TM II Genoss Co.,Ltd, Korea) and rh-BMP2 (GibcoR, Recombinant Human Bone Morphogenetic protein-2, Life Technologies,Van Table no 2-Measurements at ridge prior tooth/root piece extraction (at baseline) and at 3 months (Post-operative)