JICDRO is a UGC approved journal (Journal no. 63927)

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Year : 2009  |  Volume : 1  |  Issue : 2  |  Page : 18-31

Use of autologous platelet - Rich plasma in the treatment of intrabony defects

1 Reader, Department of Periodontics & Oral Implantology, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune, India
2 Professor & H.O.D., Department of Periodontics & Oral Implantology, Government Dental College and Hospital, Bangalore, India
3 Professor & H.O.D., Department of Periodontics & Oral Implantology, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune, India
4 Lecturer, Department of Periodontics & Oral Implantology, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune, India

Date of Web Publication4-Mar-2011

Correspondence Address:
Sharath K Shetty
Reader, Department of Periodontics & Oral Implantology, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune
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Source of Support: None, Conflict of Interest: None

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Platelet Rich Plasma (PRP) has been combined with autologous bone or bone substitutes and used for periodontal regeneration. The clinical efficacy of this combination has been noted. However, it remains questionable whether this clinical efficacy is due to the PRP or the bone graft material alone with which PRP is used in combination. Objective was to assess the clinical effectiveness of autologous PRP alone in treating intrabony defects in humans. 6 patients were recruited having interproximal intrabony defects with pocket probing depth ≥ 5 mm at re-evaluation following initial therapy, and angular osseous defect depth ≥ 4mm as assessed by using Spiral Computed Tomography. Autologous PRP gel was obtained by mixing autologous PRP with autologous thrombin and placed in the intrabony defects after defect debridement. At 6 months postoperatively, mean probing pocket depth (PPD) noted at baseline (7± 1.27)mm reduced to3.67± 1.03 mm, clinical attachment level (CAL) gain was 3.33± 0.51mm, both of which were statistically significant. Mean distance from cemento-enameljunction (CEJ) to base of the defect (BOD) at baseline was 8.55 ± 0.89 mm. At 6 months it reduced to 6.58 ±1.13 mm showing a defectfillof 1.96±00.32 mm, which was statistically significant. Thepercentage of defect fill noted was 23.01 % and mean defect resolution was 2.05 ±0.30 mm, both of which were statistically significant.
Treatment of intrabony defects by autologous PRP gel alone caused significant soft tissue clinical improvement as well as hard tissue defect fill as evidenced by SSD view in spiral computed tomography.

How to cite this article:
Shetty SK, Pradeep A R, Deshmukh VL, Acharya A. Use of autologous platelet - Rich plasma in the treatment of intrabony defects. J Int Clin Dent Res Organ 2009;1:18-31

How to cite this URL:
Shetty SK, Pradeep A R, Deshmukh VL, Acharya A. Use of autologous platelet - Rich plasma in the treatment of intrabony defects. J Int Clin Dent Res Organ [serial online] 2009 [cited 2021 Oct 20];1:18-31. Available from: https://www.jicdro.org/text.asp?2009/1/2/18/77275

   Introduction Top

Regenerative treatment of periodontal defects with a procedure or an agent, requires that each functional stage of reconstruction be grounded in a biologically directed process. On the basis of current understanding of the molecular and cell biology of the periodontal development and regeneration, simplistic approach of introducing a filler material into a periodontal bony defect is no longer tenable. Rather, efforts must be made to recapitulate in wound healing, the crucial events that were associated with the original development and formation of the periodontium. Thus, serious efforts should be made to apply knowledge to the development of procedures based on sound biological principles rather than the current and widely held view of "See hole- must fill (with anything)". [1]

Also, today's understanding of bone science recognizes the pivotal role of growth factors in clinical bone grafting success. [2] The polypeptide growth factors (e.g. Platelet derived growth factor (PDGF), Transforming growth factor-beta (TGF- β), Insulin-like growth factor I, Fibroblast growth factor(FGF), and Bone morphogenetic proteins(BMP'S) alone or in combination, have been demonstrated to be effective in cell proliferation, chemotaxis, differentiation and extracellular matrix synthesis and consequently facilitate periodontal repair and/or regeneration in animal and human studies. [3],[4],[5] . Polypeptide growth factors (PDGF & TGF- β) are known to be abundant in the a granules of platelets. [6] For economical and biological reasons, platelet-rich plasma (PRP) preparation has been fractionated from the plasma of the patients and used as an autologous source of growth factors to regenerate periodontal tissue defects in the same patients.

Robert E. Marx [7] used autologous platelet-rich plasma along with cancellous marrow graft and presented the evidence that addition of autologous PRP to bone grafts produced quantifiably enhanced results in comparison with grafts performed without its use. Juan et al. [8] used platelet gel biotechnology in combination with DFDBA and showed a significant reduction in the probing depth and new bone formation, which was evident and was confirmed by radiographs and surgical reentry.

Creeper et al [9] showed in an in vitro study that PRP alone appears to enhance proliferation of periodontal ligament and osteoblast cells and these cells are then induced to differentiate. However, few studies have shown PRP effects on periodontal regeneration as an adjunct to bone graft materials as non significant. [10],[11],[12],[13],[14] . PRP has been combined with autologous bone or bone substitutes such as inorganic bone mineral and organic bone matrix. [15] The data found in the literature regarding bone formation and maturation under the influence of PRP are contradictory.

Hence there exists a need to assess the clinical effects of autologous PRP alone used in periodontal and osseous regeneration.

Sarkar et al [16] evaluated bone formation in a long bone defect using a platelet-rich plasma-loaded collagen scaffold and suggested that PRP does not enhance new bone formation in a critical size defect with a low regenerative potential. Intrabony Periodontal defects on the other hand are known to have higher regenerative potential. In light of the above facts, this pilot study was carried out to compare the clinical effectiveness autologous PRP alone in treating intrabony defects in humans.

   Aim Top

To study the clinical effectiveness of autologous PRP in treating intrabony defects in humans in promoting wound healing, reduction in pocket probing depth, gain in clinical attachment level and bone fill in intrabony defects.

   Materials and Method Top

Patient Selection

Six Patients were selected for this study. It was made clear to the potential subjects that participation should be voluntary. Written informed consent was obtained from those who agreed for participation. Ethical clearance was taken for the study.

Inclusion criteria : Males and females between 21 and 62 years of age group with presence of a tooth with periodontal interproximal intrabony defects having pocket probing depth > 5 mm following initial therapy were selected for the study. Patients with angular osseous defects > 4mm in depth were taken for the study. Vital and asymptomatic teeth were included in the study. Systemically healthy patients and patients who agreed to give informed consent were included in the study. Patients who demonstrated acceptable oral hygiene prior to surgical therapy and able to return for the multiple follow up visits were selelcted for the study.

Exclusion criteria: Patients with history of aggressive periodontitis and lack of appropriate periodontal defect were excluded from the study. Presence of gingival recession at the surgical site were excluded from the study. Endodontic involvement and mobility of study teeth > Grade II were not selected. Patients currently suffering from any systemic disease or those who were on medication, which is known to interfere with wound healing were excluded. Patients having the habit of smoking and other tobacco products were not included in the study. Patients with unacceptable oral hygiene status were excluded. Patients unwilling to give informed consent and showing abnormal blood picture were excluded from the study.

Pre-Surgical Therapy

Prior to the surgery, each patient was given careful instructions on proper oral hygiene measures. A full mouth supra and subgingival scaling and root planing procedure were performed under local anesthesia. Occlusal adjustments were performed if trauma from occlusion was diagnosed. Trauma from occlusion was evaluated by examining the obvious presence of fremitus in centric occlusion or in working or balancing excursions.

6-8 weeks following phase I therapy, periodontal evaluation was performed to confirm the suitability of the sites for this study.

Pre-Surgical Measurements

Clinical parameters recorded before the surgical procedures included probing pocket depth (PPD), clinical attachment level (CAL), and gingival marginal level (GML), using customized acrylic stents with grooves to ensure a reproducible placement of the University of North Carolina no. 15 periodontal probe.[Figure 1] Site-specific plaque index (PI) [17] and sulcus bleeding index (SBI) were also measured. [18]
Figure 1: Pre operative SSD view-case 1

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Hard Tissue Measurement by using Spiral Computed Tomography

In our study, high resolution CT scanner (Siemen's Somotom Emotion, Spiral CT) equipped with 3 dimensional image reconstruction software package was used for CT scanning and image reconstruction.[Figure 1] and [Figure 2] The site of maximum bone loss was evaluated by using sagittal reconstructed images. Spiral CT images were reconstructed for 3-D images with built in software by accumulating sequential tranxial data. The measurements included the distances from the cemento-enameljunction (CEJ) to the base of the defect (BOD ) and to the alveolar crest(AC). The distance from the AC to the BOD was also measured.
Figure 2: Pre operative SSD view-case 2

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Autologous Platelet-Rich Plasma Preparation

10 ml blood was drawn from each patient by venipuncture of the antecubital vein. Blood was collected into sterile plastic test tubes that contained Citrate Phosphate Dextrose-Adenine as anticoagulant in the ratio of 1.4mlCPD-Ato 10 ml blood to achieve anticoagulation. The blood containing test tubes were shaken gently to enhance complete mixing of the blood with anticoagulant.

Later, blood containing test tubes were centrifuged by using refrigerated centrifugal machine (Remi's make) at 3000 rpm for 10 min which resulted in separation of 3 basic fractions: Red blood cells (RBC), Platelet-rich plasma (PRP) and Platelet-poor plasma (PPP). [Figure 3] Because of differential densities, the red blood cell layer forms the lowest layer, the PRP layer is the middle one, and the PPP layer is at the top. 2 -3 ml of the top layer corresponding to the PPP was aspirated with a Pasteur pipette and collected in a separate sterile plastic tube. The same aspirated PPP was used to get the autologous thrombin at the time of application. The PRP was collected in conjunction with the top 1 to 2 mm of RBC fraction since the latter is also rich in newly synthesized platelets. Thus PRP obtained is light red in color.
Figure 3

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Surgical Procedure

The surgical procedure was done after completion of presurgical phase following administration of local anaesthesia. Buccal and lingual sulcular incisions were used and mucoperiosteal flaps were elevated. Care was exercised to preserve as much interproximal soft tissue as possible. Complete debridement of the defects as well as scaling and root planing was achieved with the use of an ultrasonic device and hand curettes.

Preparation of Autologous PRP Gel

At the time of application, coagulation of autologous PRP was achieved. In previous studies, it was achieved by its combination with an equal volume of sterile saline solution containing 10% of Calcium chloride and 10 ul /ml of sterile bovine thrombin. Within a few minutes, it assumed a sticky gel consistency. In our study, we used autologous thrombin to get PRP gel. It was prepared in 2 steps of processing.

First Step of Processing

Autologous thrombin was recovered from a portion of platelet poor plasma. In brief, About 3 ml of PPP was taken in a sterile test tube. Freshly prepared Calcium chloride solution of 10% in the ratio of 33 ul /ml of PPP was added. It was ensured that PPP/CaC12 solution is well mixed. Then the mixture was poured into a bowl and left until the mixture coagulated. Once the coagulation was achieved [Figure 4] , clot was ringed out and skin was disposed. Remaining fluid is the autologous thrombin-rich plasma.
Figure 4

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Second Step of Processing

Autologous PRP gel was obtained by mixing autologous PRP with autologous thrombin. In brief, Autologous thrombin rich plasma was mixed with PRP in the ratio of 0.16ml/ml of PRP, on a sterile flat surfaced container. Allowed to coagulate for 3-5 min, the coagulated material was placed onto several sterile dry lint-free gauze pads, which absorbed the excess serum leaving well formed PRP gel. [Figure 5] . Following the defect debridement, once the autologous PRP gel is ready, the autologous PRP in the gel form of required size, was carried into the defect, and tightly packed into the defect by using amalgam condenser. Once again care was taken not to overfill the defects. After the defects were filled with the material, mucoperiosteal flaps were repositioned and secured in place using black braided (4-0) silk sutures. Sutures were placed in interrupted fashion and the surgical area was protected and covered with non-eugenol (Coe- pak) dressing. Later, suitable antibiotics and analgesics were prescribed.
Figure 5

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Post-Operative Care

Dressing and silk sutures were removed at 2 weeks post-operatively. Surgical wounds were gently cleansed with 0.2% of Chlorhexidine Gluconate on a cotton swab. Mechanical oral hygiene measures consisting of brushing and flossing or interproximal brushing were initiated by patients at the end of the second post­operative week. Patients were examined weekly up to 1 month after surgery and then at 3 and 6 months. Post-operative care included reinforcement of oral hygiene and mechanical plaque control whenever necessary.

Post-Surgical Measurements

6 months after initial surgery, plaque index, and gingival bleeding index were recorded. Soft and hard tissue evaluation was done. For soft tissue measurements, previously used acrylic stents were placed and all the measurements were repeated. A second CT scan of the study sites was carried out and all the hard tissue measurements and 3-D reconstruction were repeated for comparison. SSD views were shown. [Figure 6] and [Figure 7]
Figure 6: Post operative SSD view-Case 1

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Figure 7: Post operative SSD view-Case 2

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Statistical analysis

The clinical and radiographic parameters recorded pre and post treatment were compared by applying the Wilcoxon Signed rank test.

   Results Top
[Table 1]

Comparison Of Clinical And Radiographic Parameters(Baseline And 6 Month Values)
Table 1

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   Clinical Parameters Top

Plaque index (PI)

The mean plaque index score at baseline was 1.54 ± 0 .51. At 6 months, plaque index reduced to 0.49 ± 0.21 , showing a reduction of 1.06 ± 0.43 which was statistically significant (p<0.05). Comparison of mean plaque index between base line and at the end of 6 months was statistically significant.

Sulcus bleeding index (SBI)

The mean sulcus bleeding index score at baseline was 1.50 ±.0.46. At 6 months it reduced to 0.61 ± 0. 26 showing a reduction of 0.89 ±0.26 which was statistically significant (p<0.05). Comparison of mean sulcus bleeding index atthe baseline to 6 months was statistically significant.

Probing pocket depth (PPD)

The mean probing pocket depth (PPD) at baseline was 7± 1.26. At 6 months PPD reduced to 3.67 ±1.03, showing a reduction of 3.33 ± 0.51, which was statistically significant (p<0.05). Comparison of PPD between base line and at 6 months was statistically significant.

Clinical attachment level (C AL)

The mean CAL at baseline was 10 ±0.63 mm. At 6 months CAL reduced to 6.67 ± 0.52 mm showing a gain of 3.33± 0.51 which was statistically significant (p<0.05). Comparison of CAL between control and test group at 6 months was statistically significant.

Gingival Margin level (GML)

Control group: The mean distance from lower border of stent to the gingival margin at baseline was 2.5 ± 1.05 mm. At 6 months, this distance increased to 2.67 ± 1.21 mm, showing a recession of 0.17 ± 0.48mm, which was statistically not significant.

   Hard Tissue Parameters Top

Defect fill (DF): The mean distance from CEJ to BOD at baseline was 8.55 ± 0.89 mm. At 6 months it reduced to 6.58 ±1.13 mm showing a defect fill of 1.96 ± 0.32mm, which was statistically significant (p<0.05). The percentage of defect fill was 23.01%. Comparison of defect fill was statistically significant at the end of 6 months.

Defect resolution (DR): The mean distance from alveolar crest to BOD at baseline was 4.07 ± 0.23 mm. At 6 months, it reduced to 2.02 ± 0.20mm showing a defect resolution of 2.05 ± 0.30 mm, which was statistically significant. The percentage of defect resolution was 50.41 %. Comparison of defect resolution at 6 months was statistically significant.

Changes in Level of alveolar crest height (ACH): The mean distance from CEJ to the alveolar crest at baseline was 4.56 ± 0.10 mm. At 6 months, it remained same.

The difference was not statistically significant (p>0.05). Comparison of changes in relation to level of the alveolar crest height at base line to 6 months was not statistically significant.

   Discussion Top

Periodontal disease destroys the attachment apparatus leading to tooth loss. Until the recent past, the treatment of periodontal diseases included removal of etiological factors so as to arrest the progression of the disease process. Such therapies lead to healing by repair and do not restore the original structure and function of the periodontal structures. True periodontal regeneration however means healing after periodontal treatment that result in the regeneration of the lost supporting tissues including new cementum attachment to underlying dentin surface, a new periodontal ligament with functionally oriented collagen fibres inserting into new cementum as well as new alveolar bone attached to the periodontal ligament. [19] A different approach to periodontal regeneration is the use of polypeptide growth factors (PGFs). These biologic mediators have the ability to regulate cell proliferation, chemotaxis and differentiation. As preliminary evidence for their potential applications in periodontal wound healing, several PGFs have been identified in the human periodontal tissues by immunohistochemistry and in situ hybridization. Of all PGFs known, Platelet-derived growth factor (PDGF), Transforming growth factor- β (TGF- β) and Insulin-like Growth factor I (IGF I) were used in the field of Periodontics and with promising results. [20]

Despite their potential usefulness, animal-derived or genetically engineered PGFs are still not available for routine use in practice because their safety and effectiveness have not been completely confirmed. Also, short shelf life, inefficient delivery to target cells and increased expense are major concerns associated with local administration of recombinant human growth factors.

The polypeptide growth factors like PDGF and TGF-β are known to be abundant in the alpha-granules of platelets. A convenient approach to obtain autologous PDGF and TGF-β is the use of PRP. It basically involves the sequestration and concentration of platelets in plasma with subsequent application of that preparation to wound healing sites. It has been shown that the application of PRP to the wound-healing site increases the concentration of platelets (and theoretically of PDGF and TGF-β) by up to 338%.7 The aim and objectives of the present clinical study was to compare the clinical effectiveness of PRP in treating infrabony defects in humans. Investigators such as Zechner et al. [21] (2003) suggest a time-dependent action on the part of PRP, with a favouring of bone regeneration in the early stages of healing in animal studies.

For the study, six sites in 6 patients were selected. All the patients who fulfilled the selection criteria were considered for the study. Six months time frame seems to be standard for evaluating the success of regenerative periodontal therapy [22] Furthermore, since the success of periodontal therapy is dependent on maintenance of optimum oral hygiene, thorough oral prophylaxis was performed at subsequent follow-up visits.

Pre and post-surgical clinical measurements included Plaque index, Sulcus bleeding index, PPD, CAL, and GML. For standardization of soft tissue parameters, custom-made acrylic stent and UNO 15 probe were used. To date, most of the studies used IOPA to evaluate the hard tissue changes, both pre- and post­operatively, but in our study, we used Spiral CT (3-D) images to evaluate the hard tissue changes.

In the present study, there was a statistically significant reduction in plaque index and sulcus bleeding index scores at 6 months when compared with pre-operative measurements. Comparison of mean plaque index and sulcus bleeding index scores between two groups were not statistically significant. These results are in accordance with the study by Lekovic et al. (2002). [23] Low levels of these indices indicate the stringent plaque control regime followed in the present study and also improvement in gingival condition of the sites treated respectively.

Periodontal pocket is considered to be a pathognomonic sign of periodontal disease and reduction in PPD is one of the parameters used to evaluate the success of periodontal therapy. In the control group, the mean probing pocket depth reduced by 3.67±0 1.03 mm after 6 months showing statistically significant reduction (p<0.05). On comparing the probing pocket depth, statistically significant difference was found at the end of 6 months.

The aim of periodontal therapy is to achieve a gain in clinical attachment level and return the periodontal tissues to the state of health. At the end gain in clinical attachment level was found to be 3.33 ±0.51 mm showing a statistically significant improvement (p<0.05). On comparing, the gain in CAL revealed a significant difference at the end of 6 months.

Quritero et al. [24] speculated that increase in clinical soft tissue attachment represented a new attachment composed of new bone, cementum, and periodontal ligament fibres. But without histological data it is impossible to verify the gain and type of attachment that has actually occurred.

After periodontal therapy, reduction in probing pocket depth is often due to a combination of gain in attachment as well as gingival recession. Hence, it is important to assess the amount of gingival recession. Analysis of data indicated that there was no statistically significant change in the gingival margin position after an interval of 6 months.

   Hard Tissue Evaluation Top

Limitations of conventional two-dimensional radiographs have been emphasized in many studies. Because the image is a two-dimensional map of a three-dimensional tooth and periodontal tissues, complex anatomical structures such as cortical plates or teeth may be superimposed on the region of interest. In addition, the exact form of many periodontal defects including infrabony defects, furcation involvements and hemiseptum cannot be determined from radiographs. 3-D image analysis by computed tomography (CT) was rarely used in the field of periodontics to evaluate the outcome of periodontal regeneration. Few studies have used radiographic evaluations in experimental animal models and most studies make use of visual measures based on different tones of gray. They indicated that while radiography is able to differentiate mineralization patterns, the results should not be overestimated.

To overcome the inherent difficulties of conventional radiography in the present study, we made an attempt to use spiral CT (3-D) images [25] to evaluate and measure all the hard tissue changes at baseline and after 6 months. The CT provided the excellent quality images for evaluating the morphology of the periodontal defects. Till date, only one case report was documented in literature by Koichi et al. [26] where they used new compact computed tomography system for evaluating the outcome of regenerative therapy in the furcation defect of lower left first molar.

The amount of defect fill was measured from CEJ to base of the defect where the CE J served as the fixed reference point.

The mean defect fill was 1.96±0.32 mm after 6 months showing a statistically significant defect fill. The original defect fill was found to be 33.33% after 6 months. These values are lower when compared with Lekovic et al [23] , who showed a mean defect fill of 4.82± 1.34 mm after 6 months by using PRP/BPBM.

Depth of the defect was measured as the distance between the crest of the alveolar bone and the base of the defect. This defect resolution may occur by defect fill at the base of the defect as well as crestal resorption.

In sites treated with PRP mean defect resolution was 2.05. ±0.30 mm (50.40%) showing statistically significant improvement (p<0.05). Comparison revealed a statistically significant (p<0.05) difference after 6 months.

Following periodontal surgery, there is remodeling of the alveolar bone leading to slight resorption and loss of alveolar crest height. After a few days, however, the rate of resorption is exceeded by the rate of bone formation resulting regeneration of alveolar bone. However, crestal resorption occurs to a greater extent when no regenerative therapy is performed i.e., when only open flap debridement is performed.

Comparison of mean crestal resorption or change in alveolar crestal height was not statistically significant (p>0.05).

The practical aspect of PRP use in clinical practice needs to be examined carefully. Because the autologous PRP preparation utilizes patient's own blood, the risk of human to human disease is virtually eliminated, making it a safe treatment modality. Preparation of PRP, however, adds another step to the periodontal surgical procedure and increases the time for procedure by 20 to 30 mins. PRP preparation can be performed by a properly trained surgical assistant under the supervision of a Periodontist.

Activation of PRP in the present study, in contrast to previous studies, was achieved by using autologous thrombin. Bovine thrombin and calcium chloride has been used to activate coagulation and precipitate gel formation. The use of topical bovine thrombin has been associated however, with development of antibodies to clotting factors V, XI, and thrombin, resulting in the risk of life threatening coagulopathies. Topical bovine preparation contains factor V, which results in reaction of the immune system when challenged with a foreign protein. The factor V deficiency after thrombin exposure is thought to be caused by the cross-reactivity of anti-bovine factor V antibodies with human factor V. [27]

In our study we used autologous thrombin to achieve PRP gel, thereby; avoiding the chances of topical bovine thrombin-associated coagulopathies. In the present study, autologous PRP was used to achieve periodontal regeneration. A favorable response was observed in all the clinical parameters recorded. Furthermore, Spiral CT images revealed significant bone-fill.

Where PRP alone was used, it exhibited the following properties and advantages. 1) It is safe because of an autologous preparation; 2) It provides adhesiveness and tensile strength for clot stabilization; 3) It is biologically acceptable to the root surface; 4) It contains important GFs such as PDGF and TGF-β released by platelets; 5) Promotes angiogenesis; 6) Contains a dense fibrin network that is highly osteoconductive; 7) It has haemostatic properties; 8) It contains high concentrations of leukocytes which reduces the risk of infections; 9) It improves wound healing; and is also an affordable treatment modality.

For all of the reasons mentioned above and from the results we obtained in our study treated with PRP alone, our clinical impression is that it significantly enhances periodontal wound healing. However, it has been noted that different concentrations of platelet concentrates may result in varying clinical outcomes and Weibrich et al. (2004) [28] suggests that while concentrations below l,000,000ul exert suboptimum effects, higher concentrations paradoxically could exert inhibitory effects. Appropriately designed clinical studies comparing different concentrations of PRP, as well as PPP, are indicated to determine the effect of various PRP concentrations on clinical outcomes.[29]

   References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

  [Table 1]


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