|Year : 2011 | Volume
| Issue : 4 | Page : 505-510
|Treatment of human periodontal infrabony defects with hydroxyapatite+ β tricalcium phosphate bone graft alone and in combination with platelet rich plasma: A randomized clinical trial
Bharadwaj T Kaushick, ND Jayakumar, O Padmalatha, Sheeja Varghese
Department of Periodontics, Saveetha Dental College and Hospital, Chennai, India
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|Date of Submission||25-Jul-2010|
|Date of Decision||11-Nov-2010|
|Date of Acceptance||05-Jul-2011|
|Date of Web Publication||26-Nov-2011|
| Abstract|| |
Background: The present study was aimed at comparing the clinical effectiveness of two regenerative techniques - platelet rich plasma (PRP) + bone graft (HA + β TCP) versus bone graft (HA + β TCP) + normal saline in the treatment of periodontal intrabony defects.
Materials and Methods: Ten patients diagnosed with chronic periodontitis were enrolled in a randomized split mouth clinical trial. Following phase I therapy the sites were randomly assigned to the test group - PRP + bone graft (HA + β TCP) and control group - saline + bone graft (HA + β TCP). Clinical parameters recorded at baseline and 6 months included plaque index, probing pocket depth, relative attachment levels, and relative gingival margin levels. Hard tissue evaluation was done using digital radiography to evaluate the image intensity and therefore the radioopacity of a desired region of interest in the intrabony defect. Pre- and postoperative comparisons were made between the treatment groups at 6 months.
Results: Test group sites showed a significantly higher reduction in pocket depth compared to control group sites. Test group sites showed a significantly higher amount of radioopacity in the regions of interest, indicative of better graft remodeling, compared to control group sites.
Conclusion: (HA + β TCP) bone graft appears to be a beneficial material in the treatment of human periodontal intrabony defects. When combined with platelet-rich plasma there is a significantly higher reduction in probing pocket depth, higher gain in attachment levels and higher amount of radio-density seen in the intrabony defects.
Keywords: Bone graft, infrabony defects, platelet rich plasma
|How to cite this article:|
Kaushick BT, Jayakumar N D, Padmalatha O, Varghese S. Treatment of human periodontal infrabony defects with hydroxyapatite+ β tricalcium phosphate bone graft alone and in combination with platelet rich plasma: A randomized clinical trial. Indian J Dent Res 2011;22:505-10
Periodontal surgery as a part of treatment of periodontal disease is mainly performed to gain access to diseased areas for adequate cleaning, to achieve pocket reduction or elimination and to restore periodontal tissues lost as a consequence of disease process.
|How to cite this URL:|
Kaushick BT, Jayakumar N D, Padmalatha O, Varghese S. Treatment of human periodontal infrabony defects with hydroxyapatite+ β tricalcium phosphate bone graft alone and in combination with platelet rich plasma: A randomized clinical trial. Indian J Dent Res [serial online] 2011 [cited 2019 Nov 12];22:505-10. Available from: http://www.ijdr.in/text.asp?2011/22/4/505/90278
The most favorable outcome for periodontal therapy is to regenerate the lost supporting tissues. For decades, a number of surgical procedures have been advocated, which include, open flap debridement; open flap debridement with bone grafts/bone substitutes, and guided tissue regeneration (GTR). Porous hydroxyapatite (HA) bone grafting material has been used to fill periodontal intrabony defects, which has resulted in clinically acceptable responses.  It has been shown that porous HA bone grafts have excellent bone conductive properties which permit outgrowth of osteogenic cells from existing bone surfaces into the adjacent bone material.  However true periodontal regeneration is not achieved because healing which occurs is a connective tissue encapsulation of the graft with a long junctional epithelium. 
Polypeptide growth factors (PGF) are biologic mediators that have the ability to regulate cell proliferation, chemotaxis, and differentiation.  Among these, platelet-derived growth factors (PDGF) and transforming growth factor β (TGF β) have been most extensively studied. These two growth factors are known to be abundant in the α granules of the platelets. A convenient and economical approach to obtain autologous PDGF and TGF β is the use of platelet rich plasma (PRP).
The use of porous HA + β TCP and PRP in a combination for periodontal regenerative therapy offers a potentially useful modality to the clinician in treating periodontal intrabony defects.
The purpose of this prospective, randomized, controlled clinical study is to compare a combination of PRP and HA + β TCP bone graft (OSSIFI) with a mixture of saline and HA + β TCP bone graft (OSSIFI) in the treatment of human periodontal infrabony defects.
| Materials and Methods|| |
Patients reporting to the Department of Periodontia, Saveetha Dental College and Hospitals Chennai were included in the study. Prior to initiating the study, the patients were informed of the purpose of this split mouth randomized clinical trial and were requested to sign an informed consent. Ethical clearance was taken for the study.
- Males and females aged between 20 and 50 years.
- Attachment loss > 3 mm as assessed by periodontal probe with a diagnosis of chronic periodontitis.
- The presence of infrabony defects (2/3 wall confirmed upon surgical exposure).
- Patients with a minimum of two intrabony defects in different quadrants.
- Vital teeth.
- Teeth with mobility less than grade I.
- Patients willing to comply with multiple recall schedules.
- Patients with systemic illness such as diabetes, hypertension, bleeding disorders, epilepsy, or abnormal blood picture.
- Pregnant/lactating women.
- Patients on medications known to cause gingival overgrowth or interfere with wound healing.
- Patients allergic to routine medications prescribed following surgery.
- Mucogingival problems.
- Aggressive periodontitis.
- Trauma from occlusion.
Clinical procedures and measurements
Ten healthy patients were recruited for the study.
Initial periodontal therapy was done and oral hygiene instructions were given. The patients were reviewed for adequate plaque control. Surgical periodontal therapy was done only when patients achieved a plaque score of zero. Patients were then randomized into the designated study groups.
Following measurements were noted at baseline and 6 months following surgery:
- Plaque index (Silness and Loe).
- Gingival index (Loe and Silness).
- Probing depth.
- Relative attachment levels (distance between the most apical portion of the stent and the base of the pocket).
- Relative gingival margin levels (distance between the apical most part of the stent and the coronal limit of the gingival margin).
- Radiographic measurements.
Acrylic stents were used to standardize the probing depth and relative attachment levels measurements.
Radiographic evaluation of the sites was done with the help of RVG (PLANMECA Dimaxis software version 2.4.8). The image intensity of the region of interest (ROI) was measured using the histogram application. The histogram, when applied to the ROI, provides values for image intensity which is a measure of the dark and white pixels present in the image. When all the variable parameters are standardized, an increase in the amount of white pixels in the desired region of interest indicates a greater amount of radioopacity in that particular region. This provides the operator a quantitative value of a qualitative measurement. 
Radiographs were made using a paralleling cone technique. All variable parameters associated with the digital radiographs were standardized. The overall intensity in the ROI was expressed in terms of percentage values.
Preparation of PRP
The preparation of platelet rich plasma was accomplished using a centrifuge capable of running at 1000-5000 rpm. On the day of surgery 12 ml of blood was drawn from each patient by venipuncture of the antecubital vein. Blood was collected in sterile glass tubes containing 3.8% sodium citrate as the anticoagulant (Vacutainer, Becton and Dickinson, USA) and centrifuged using a two-step procedure.  The initial centrifugation process was done at 5000 rpm for 10 minutes. This resulted in separation of the three basic fractions [Figure 1]:
- Platelet poor plasma (PPP) on the top of the preparation which contains few platelets as defined by American association of blood banks. 
- Middle layer comprising PRP. 
- The bottom most fraction comprising red blood corpuscles (RBC) and newly synthesized platelets at the very top.
|Figure 1: Following two rounds of centrifuge, three clear layers can be seen|
Click here to view
All of the plasma along with the top erythrocyte layer was collected using a 21 gauge spinal needle (Yale spinal needle, Becton and Dickinson, USA). This was then transferred to an empty vacutainer tube and centrifuged for 10 minutes at 2000 rpm. The PRP, thus obtained, was collected. One milliliter was set aside for platelet cell count of the prepared PRP.
All periodontal surgical procedures were performed by a single operator. Following the presurgical phase, the patients were anesthetized using lignocaine 2% with 1:100,000 epinephrine. Facial and palatal/lingual sulcular incisions were used to elevate mucoperiosteal flaps, extending to one tooth mesial and distal to the tooth included in the study. Defect debridement and root planing were carried out using hand curettes and ultrasonic devices. No osseous recontouring was done [Figure 2].
Coagulation of the obtained PRP was achieved using 10% calcium chloride mixed with equal volume of saline. Within a few minutes of the addition of calcium chloride the PRP assumed a cohesive nature indicating the onset of coagulation [Figure 3].
The test group sites received a mixture of bone graft (OSSIFI - porous hydroxypapatite in combination with tricalcium phosphate) and activated PRP [Figure 4]. The flaps were sutured using 3-0 black-braided silk suture in an interrupted fashion to obtain maximum closure. A periodontal pack (COE-PACK) was placed. Postoperative care included oral administration of amoxicillin 500 mg, TID for 5 days, diclofenac sodium (50 mg) TID for 3 days and 0.12% chlorhexidine gluconate mouth rinse for a period of 2 weeks commencing from the day of surgery.
Control group sites were treated with a mixture of bone graft (OSSIFI) and normal saline using the same surgical procedure and postoperative care as described for the test group sites.
Sutures were removed 1 week postoperatively. Patients were instructed to avoid flossing of the surgical sites for a period of 4 weeks following surgery.
Patients were examined twice in the first month following surgery and thereafter once a month for the remainder of the study period. Reinforcement of oral hygiene measure was done if and when required.
All measurements noted at baseline were recorded at 6 months following surgery [Figure 5] and [Figure 6].
Calculation of percentage of platelet concentration
The average platelet count of the patients was 200,000 cells/cu mm. The average concentration obtained was 12.5 million cells/cu mm. The average increase in platelet counts above baseline was sixfold [Figure 7]. A concentration of four to five times has been deemed enough for cell proliferation. 
The enrichment percentage calculated from obtained average values in the study is approximately 500% which is an acceptable level of concentration. 
The Kolmogorov Smirnov Goodness-of-Fit test was used to test the normality of the data in each study group. The Mann - Whitney U-test was used to compare the mean values between the two study groups and the Wilcoxson matched-pair signed-rank test was used to test the significance of change between the pre- and postoperative measurements for each study group.
In the present study P < 0.05 was considered as the acceptable level of significance.
| Results|| |
All enrolled patients successfully completed the study with uneventful healing.
Soft tissue parameters:
The mean plaque index score at baseline, for the control group, (0.3 ± 0.45) was not significantly different from the test group (0.1 ± 0.3) (P = 0.17).
The mean plaque index score at 6 months following therapy, for the control group(1 ± 0.0) was not significantly different from the test group (0.8 ± 0.4) (P = 0.18).
The mean gingival index, at baseline, for the two groups was 0.0 ± 0.0.
The mean gingival index, at 6 months following therapy, for the control group (1.0 ± 0.0) was not significantly different from the test group (0.8 ±0.4). (P = 0.18).
Probing pocket depth
The mean PPD, at baseline, was similar in both control group (7.3 ± 0.5) and test group (7.3 ± 0.7) (P = 0.9).
The mean PPD 6 months post-therapy in the control group (4.0 ± 0.7) was significantly higher than the test group (3.0 ± 0.7) (P = 0.006) [Table 1].
|Table 1: Probing pocket depth (mm) - table showing the mean pre- and postoperative probing pocket depth recorded for both groups|
Click here to view
Relative attachment levels
The mean relative attachment levels (RAL), at baseline, for the control group (10.0 ± 0.0) was similar to the test group (9.9 ± 0.3) (P = 0.32).
The mean RAL, at 6 months following therapy, for the control group (7.1 ± 0.7) was significantly higher than the test group (5.5 ± 0.8) (P = 0.002) [Table 2].
|Table 2: Relative attachment level (mm) - table showing the mean pre- and postoperative relative attachment level recorded for both groups, with the apical most portion of the acrylic stent being the point of reference|
Click here to view
Relative gingival marginal levels
The mean relative gingival marginal levels (RGML), at baseline, for the control group (2.7 ± 0.46) was similar to the test group (2.6 ± 0.66) (P = 0.68).
The mean RGML, at 6 months following therapy, for the control group (3.1 ± 0.54) was similar to the test group (2.8 ± 0.87) (P = 0.27) [Table 3].
|Table 3: Relative gingival marginal levels (mm) - table showing the mean pre- and postoperative relative gingival marginal levels recorded for both groups, with the apical most portion of the acrylic stent being the point of reference|
Click here to view
Intensity values (indicative of radio-density)
The mean intensity values, at baseline, for the control group (5.7 ± 1.4) were similar to the test group (5.6 ± 1.3) (P = 1.0).
The mean intensity values, 6 months following therapy, for the control group (6.2 ± 1.3) was significantly lower than the test group (8.6 ± 2.5) (P = 0.03) [Table 4].
|Table 4: Intensity percentage (radio-density) - table showing the pre- and postoperative intensity percentage recorded for both groups. The values are those obtained from the application of the histogram analysis|
Click here to view
| Discussion|| |
For the study, 20 sites in 10 patients were selected according to a split mouth design to null the differences arising due to variation among individuals. No complications were observed at any of the treated sites. Six-month time frame seems to be the standard for evaluating the success of periodontal regeneration.  Conventional radiographs and reentry methods depict bone fill in a purely quantitative aspect. They do not describe the quality of the newly formed bone. Defect resolution following bone grafting can be a result of connective tissue encapsulation of the bone graft and long junctional epithelium formation or because of remodeling of the graft and replacement by host bone.  The use of gray-scale pixel values has been used and advocated as a reliable method to measure the density of the bone. 
In the present study plaque index and gingival index scores recorded at baseline and at 6 months following therapy did not show any statistically significant difference between group I and group II before and after therapy. Thus the change in probing depths and attachment levels could not be attributed to any significant differences in the levels of oral hygiene of the control and test sites.
Periodontal pocket is considered as the pathognomonic sign of periodontal disease and reduction in probing pocket depth is one of the requisites for successful periodontal therapy. The results of this study compare well with other trials using porous HA as a grafting material, alone or in conjunction with PRP done by Okuda et al.  who found a mean reduction in PPD of 4.7±1.6 mm in test sites (PRP + porous HA) and 3.4 ± 2.0 in control sites (porous HA + saline). 
Clinical attachment levels represent a new attachment composed of new bone, cementum and periodontal ligament fibers.  However without histologic data it is impossible to verify this observation. The mean gain in attachment levels, following therapy are also consistent with the results obtained by Okuda et al. 
Following periodontal therapy the reduction in PPD is often due to a combination of gingival recession and gain in attachment levels. Hence the relative gingival marginal levels were assessed (statistically not significant) (P = 0.75).
Hard tissue evaluation (analysis of image intensity in % values)
In the control group there was a mean gain of 0.6 ± 0.2%. This change was statistically significant (P = 0.008). This gain is indicative of an increase in radio-opacity of the defect region following therapy.
In the test group there was a mean gain of 3.0 ± 1.4%. This change was statistically significant (P = 0.005). There is an increase in intensity values for the test group vis-à-vis control group. This can be interpreted as an increased remodeling of the graft material due to the addition of PRP. PRP delivers a highly concentrated source of autologous platelets containing a variety of biological mediators that can be directly applied to the periodontal wound. Studies have shown that more dense and more mature bone with better organized trabeculae and greater bone regeneration takes place when PRP is added to allografts.  Most of these reports also suggest that PRP improves the handling properties of the graft material with which it is combined, facilitating graft placement, and stability. 
PRP utilizes the patients own blood in a significantly small quantity and is therefore not harmful to the patient. Preparation of PRP takes about 30 minutes and is best performed by a surgical assistant under the supervision of a trained dental surgeon. This can be done simultaneously, while performing the surgery, and therefore does not significantly increase the chair time of the operator and the patient. Activation of PRP was done using calcium chloride. No adverse effects liking the use of calcium chloride have been reported in the literature.
PRP has several advantages including being safe as it is an autologous preparation, provides clot stability by promoting production of fibronectin which enhances adhesion of fibroblasts to the root surface thereby favoring regeneration of the osseous defect especially in the early stages, is biologically acceptable, contains myriad growth factors, contains dense fibrin network, promotes angiogenesis, has hemostatic properties, improves wound healing, and is economically viable. 
For all the reasons mentioned above and from the results obtained from our study, in sites treated with only bone graft (HA + β TCP) and bone graft with PRP our clinical impression is that platelet rich plasma significantly enhances periodontal wound healing. In conclusion, within the limits of the present study, the two-tested therapies seem to be effective in the treatment of periodontal intrabony defects. Also further studies need to be carried out to confirm the effects of PRP in periodontal regeneration and need to be corroborated by investigations on a larger population size and include patients with compromised wound healing.
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Bharadwaj T Kaushick
Department of Periodontics, Saveetha Dental College and Hospital, Chennai
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4]
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