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Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study
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JORMAS-599; No. of Pages 7 J Stomatol Oral Maxillofac Surg xxx (2018) xxx–xxx
Available online at
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Original Article
Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study K.P. Nakkeeran a,*, K. Saravanan b, P. Babu c, R.R. John b a b c
Department of Oral and Maxillofacial Surgery, SRM Dental College and Hospital, Ramapuram, Chennai 600089, India Department Of Oral & Maxillofacial Surgery, Vinayaka Missions Sankaracharyar Dental College & Hospital, Salem 636308, India Department of Oral & Maxillofacial Surgery, Dr. Syamala Reddy Dental College Hospital and Research Centre, Marathahalli Bangalore, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 September 2018 Accepted 19 November 2018
Background: Osseous defects of the jaw occurs naturally by pathological conditions such as infections, tumors, cysts or iatrogenic causes induced during surgical treatment of preexisting periradicular bony pathologies. Management of these bone defects poses a great challenge for surgeons who are continually exploring ways to find new modalities to improvise bone substitutes. Purpose: To compare and evaluate bone regeneration with and without combining platelet rich plasma (PRP), calcium sulfate (CS) and autogenous bone graft in periapical defects of jaw. Methods: The study is a randomized prospective comparative study, in which 20 patients were assigned, with 10 patients each forming the study and control groups. In the first group (study group) the bone defect was filled with PRP, calcium sulfate and autologous bone graft. In the second group (control group) defect was allowed to heal without PRP, calcium sulfate and autogenous bone graft. Digital orthopantogram (OPG) were taken to analyze the bone density and bone regeneration. The parameters used to analyze the radiographs included grey scale analysis, residual bone defect calculation in pixels both performed in Corel Photopaint X31 software and radiopaque scoring scale. Statistical analysis was carried out using Kolmogorov Smirnov test, Independent t-test and Anova test. Results: The mean bone defect density (BDD) measured using grey scale was significant in the study group during 5th, 13th and 20th week than the control group (P < 0.0001). The percentage bone formation analyzed using residual bone defect calculation revealed significantly higher size reduction in the study group than with the outcome obtained in the control group (P < 0.0001). The mean radiopaque score given by blinded observers also showed significance in the study group. Conclusion: From the results of the study, it is clearly evident that radiographic bone density and rapid bone formation in the study group when compared to the control group. Thus the grouping of PRP, combined calcium sulfate with autologous bone graft proved to be a novel osteoinductive biomaterial.
C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Platelet rich plasma Calcium sulfate Bone defect density Orthopantogram
1. Introduction Osseous defects of the jaw occurs naturally by pathological conditions such as infections, tumors, cysts or iatrogenic causes, induced during surgical excision of preexisting intraosseous periradicular pathologies [1]. Management of these bone defects represents a great challenge for surgeons who are continually
* Corresponding author. E-mail addresses: [email protected] (K.P. Nakkeeran), [email protected] (K. Saravanan), [email protected] (P. Babu), [email protected] (R.R. John).
exploring ways to improve the success of bone grafting with bone substitutes [2]. Unfilled bone defects usually heal by initial resorption of the alveolar bone. Spontaneous bone regeneration in untreated defects is limited to small distance because of rapid proliferation of surrounding soft tissues. Further bone defects following cyst enucleation leads to loss of the concerned teeth. Filling these defects with bone or other substitute materials help to preserve the alveolar ridge and provides sufficient bone for immediate or subsequent implant placement [3]. Growth factors like platelet-derived growth factor (PDGF) play an important role in inducing proliferation of undifferentiated mesenchymal cells. It is an important mediator for bone healing
https://doi.org/10.1016/j.jormas.2018.11.008 C 2018 Elsevier Masson SAS. All rights reserved. 2468-7855/
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Abbreviations PRP Platelet rich plasma Calcium sulfate CS Platelet poor plasma PPP PDGF Platelet derived growth factor Bone defect density BDD Orthopantogram OPG
and remodeling. The therapeutic strategy of autologous PRP is based on acceleration of healing by the concentration of the growth factors, which are universal initiators of nearly all healing events [2]. It contains high concentration of thrombocytes, which in turn contains a number of growth factors namely PDGF, TGF-b, IGF and VEGF [5]. The use of autogenous bone graft is regarded as the gold standard for replacement of regenerated bone. Autologous bone provides osteoconduction combined with osteoinductive effects, but its availability is limited. Alloplastic (synthetic bone grafts) and xenogenic bone substitute materials have been used alone or in combination with autogenous bone graft [3,6]. Our study was designed to evaluate the efficacy of PRP when combined with alloplastic biomaterial like CS and autogenous bone graft in hastening bone regeneration in osseous defects of the jaw.
2. Materials and methods The study was performed at Department of Oral and Maxillofacial surgery, Vinayaka Missions Sankarachariyar Dental College and Hospital, Salem after obtaining institutional ethical committee clearance (REF:VMSDC/IEC/015). The prospective randomized control trial was performed on patients with periapical cystic lesions involving the maxillary and mandible anterior region. Twenty patients were randomly assigned into two groups with 10 patients each forming the study and control groups. The exclusion criteria were those patients of age less than 12 years and greater than 50 years, patients diagnosed with systemic diseases and given the history of smoking. Surgical consent was obtained for phlebotomy to collect blood for the development of platelet rich plasma, graft procurement and placement of CS. In the first group (study group) the bone defect was filled with PRP, combined CS and autologous bone graft. In the second group (control group) defect was allowed to heal normally without PRP and bone grafts. In the study group, PRP was prepared by table top centrifugation device using double centrifugation technique [7]. The autologous pre donated blood was collected in sufficient amounts of anticoagulation (EDTA) solution. The aspirated blood was gently agitated to thoroughly mix the anticoagulant with the blood. The first spin (called the hard spin or separation spin) separated the red blood cells from the plasma, which contains the platelets, white blood cells and clotting factors. The centrifugation speed was carried out at 5600 rpm for 3 to 4 minutes. The second spin (called the soft spin or concentration spin) finely separated the platelets and white blood cells together with a few red blood cells from the plasma. The centrifugation speed was carried out at 2400 rpm for 8 to 9 minutes. This soft spin produces the PRP and separates it from the platelet poor plasma (PPP) [7]. Ten milliliters of whole blood after centrifugation process roughly yielded 3 ml of PRP (see Fig. 1).
Fig. 1. Platelet rich plasma (PRP) obtained after double centrifugation technique.
Under local anesthesia, autologous cortical bone graft was procured from mandibular symphysis region and cystic defects were treated by creating a bony window with complete enucleation of the lesion. The final osseous defects varied from 1–1.5 cm to a maximum of 3–3.5 cm in diameter size. Endodontic treatment of the affected tooth was done prior, followed by apicoectomy. The derived platelet rich plasma was initially soaked with the procured autologous bone graft for some time and later mixed with CS (CapsetTM). Small amount of PRP was also applied on overlying soft tissue to enhance the healing process. It was then packed by careful stratification in a dry environment within the bone cavity (see Fig. 2). The control group defect was allowed to heal naturally without the application of PRP, CS and bone grafts. Radiographs (Digital OPG) were taken to assess the bone density and bone regeneration. The radiographs were evaluated preoperatively, followed by immediate postoperative period after 1 week and at a regular interval of 5th, 13th, 20th week. The radiological analysis was performed using Corel Photo-Paint X31 Software version-13.0. Bone density was measured on the radiographs through a gray scale of 256 tonalities as shown in the Figs. 3 and 4. The residual defect size of the cavity was measured in pixels using the same software at preoperative, immediate postoperative, 5th week, 13th week and 20th week respectively as depicted in Figs. 5 and 6. The measurement of bone formation was also performed subjectively but blindly, recording the score in a radiopacity increasing scale (see Table 1) by four independent blinded observers.
Fig. 2. Autologous bone graft procured from mandibular symphysis is combined with calcium sulfate (CS), and platelet rich plasma (PRP). The graft was packed by dry stratification technique.
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Fig. 3. Bone defect density (BDD) was measured within the grid corresponding to the radiolucent area of the lesion using the Corel- photo paint X3 software.
3. Results
Fig. 4. Normal bone density (NBD) was measured within the grid of the normal bone. The bone formation was observed by the decrease in the gray scale value between the NBD and BDD in subsequent follow up radiographs.
Fig. 5. Bone defect area marked using a freehand mask tool using the Corel photo-paint x3 software.
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Fig. 6. The delineated defect area is created as a separate object and its height and width is obtained in pixels. The residual defect cavity is measured by multiplying the height and width.
Table 1 Radiopacity scoring scale. Bone formation/healing
Score
Nil/failure Very minimal/doubtful Minimal Moderate Complete
0 1 2 3 4
Nil/ failure: complete radiolucency present. Very minimal/doubtful: very minimal radiopacity present at least in two consecutive follow-ups. Minimal: evidence of radiopacity at the periphery and noticeable decrease in defect size (radiopacity seen more than 1/4th up to 1/2 of the defect). Moderate: radiopacity covering at least 1/2 to 3/4th of the defect. Complete: radiopacity covering 3/4th to maximum size of the defect.
Among the total 20 patients who were included in the study, 12 comprised of males and 8 were females with a mean age of 24 years. By comparing the mean BDD of study group with control group the former showed significant increase in value (P < 0.0001). In the study group, the mean BDD measured in grey scale showed drastic increase from 60.6 in the 5th week to 73 in the 13th week and 91.6 in the 20th week postoperative. The mean BDD in the control group gradually increased from 40 in the 5th week to 52 in the 13th week and up to 62 in the 20th week postoperative. The above results have been summarized in Table 2.
The study group patients showed significantly higher bone defect size reduction than the control group patients (P < 0.0001). The mean percentage increase in bone formation in the study group stood at 52% at 5th week, 68% at 13th week and 87% at 20th week whereas for the control group percentage increase in bone formation was 16% at 5th week, 31.6% at 13th week and 49% at 20th week as valuated in Table 3. The mean radiographic score given by independent observers was significantly higher for the study group compared to the control group. Significant differences in the mean were found in the scores of four observers by using ANOVA test and between the study group and the control patients by using t-test. The mean radiographic score for study group was 2 at 5th week, 2.67 at 13th week & 3.57 at 20th week respectively where as for the control group it was 0.97 at 5th week, 1.47 at 13th week and 2.27 at 20th week respectively as summarized in Table 4.
4. Discussion The osteogenic potential of autogenous grafts with PRP is a well-established one. The bone substitute graft forms new bone via osteoconduction from adjacent osteoprogenitor cells. The autogenous bone graft transplants osteoprogenitor cells from distant site into the defect area and aid in new bone formation. In such
Table 2 Postoperative bone defect density (BDD) in grey scale. Patient No.
5th week
13th week
20th week
Study group
Control group
Study group
Control group
Study group
Control group
1 2 3 4 5 6 7 8 9 10 Mean bone defect density (BDD)
55 51 57 63 56 60 58 46 91 69 60.6
39 46 23 59 43 34 30 36 58 32 40
63 59 74 70 68 72 64 61 93 103 2.7
49 58 36 64 49 42 55 61 61 47 52.2
110 65 129 78 87 74 71 71 97 134 91.6
63 63 62 71 51 58 57 65 66 62 61.8
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Table 3 Residual bone defect size and percentage increase in bone formation. Patient No.
1 2 3 4 5 6 7 8 9 10 Mean percentage increase
Preoperative
5th week
13th week
20th week
Study group size
Control group size
Study group size (% increase)
Control group size (% increase)
Study group size (% increase)
Control group size (% increase)
Study group size (% increase)
Control group size (% increase)
27,566 18,126 27,238 45,666 9860 46,202 20,868 28,620 11,782 151,59
17,549 51,512 35,235 34,565 17,115 20,437 26,332 10,080 16,156 29,000
17,940 (35) 13,038 (28) 16,000 (41) 21,018 (54) 3354 (66) 19,933 (57) 4734 (77) 9313 (67) 7548 (36) 5846 (61) 52
14,157 (19) 47,880 (7) 29,190 (17) 30,250 (12) 14,994 (12) 20,776 (1.6) 19,474 (26) 8118 (19) 13,018 (19) 22,360 (23) 16
10,792 (61) 9156 (49) 5733 (79) 14,432 (68) 4837 (51) 6495 (86) 2788 (86.6) 7790 (73) 6487 (45) 2448 (84) 68
10,350 (41) 46,228 (10) 25,186 (28.5) 28,728 (17) 10,290 (40) 15,456 (24) 16,170 (38.5) 7533 (25) 6245 (62) 20,600 (29) 31.6
3303 1344 2980 1935 1914 3653 1518 4692 4090 1953 86.6
5694 (67.5) 27,360 (47) 24,948 (29) 25,320 (27) 9646 (43.6) 12,879 (37) 9576 (63.6) 5382 (46.6) 2787 (83) 14,295 (51) 49
(88) (92.5) (89) (96) (80.5) (92) (93) (83.6) (65) (87)
Percentage increase in bone formation is calculated by: postoperative defect size in pixels 100 preoperative defect size-100.
Table 4 Mean Radiopacity Score – study and control group. Observer
5th week
13th week
Study group
Control group
Study group
Control group
Study group
Control group
Observer 1 Observer 2 Observer 3 Observer 4 Mean score
2.6 2 2 1.4 2
1.5 0.7 1.1 0.6 0.97
3.1 2.5 2.9 2.2 2.67
2 1.2 1.7 1 1.47
3.9 3.4 3.8 3.2 3.57
2.9 2.3 2.4 1.5 2.27
conditions PRP upregulates osteoprogenitor cells into the grafts and helps in matrix formation [7]. The application of platelet rich plasma (PRP) in combination with bone and bone substitutes leads to the transformation of the surrounding cells in grafted bone cells and increases the integrity of the graft [8]. Recent studies of PRP have shown enhancement of nearly all bone substitute materials. Farrel et al. from their studies observed that no benefits when PRP alone has been placed into the bone defects without other grafting materials [2]. Marx [8] on his study on 88 patients with mandibular continuity defects using PRP concluded that the degree of maturation of the grafts was significantly greater with addition of PRP (1.6 to 2.2 times faster). PRP also acts as an anti-inflammatory agent by production of CCL5 (Chemokine ligand 5). CCL5 is a chemotactic for T cells, eosinophils, and basophils and plays an active role in recruiting leukocytes into inflammatory sites, blocking monocyte chemotactic protein-1 release from monocytes and its concentration of lipoxin A4, suggesting that PRP facilitates healing by controlling the local inflammatory response [9]. Calcium sulfate (CS) as a bone graft material is used in various skeletal sites including mandibular, craniofacial and long bone defects and benign bone lesions. It is highly biocompatible graft substitute material which is well tolerated by host tissues, not inducing any foreign body reactions. It has been used in treating periodontal disease, endodontic treatments, post extraction sites, maxillary sinus augmentation and as a barrier in guided tissue regeneration [10]. It is used as a barrier in guided tissue regeneration by physically halting soft connective tissue proliferation by means of the osteopromotion principle [1]. It is completely resorbable in a relatively short period and provides a local increase in Ca ion concentration which may stimulate osteoblastic activity and acts as a scaffold for bone growth [11–14]. The exothermic reaction generated by the mixture of CS with PRP activates the platelets contained within the PRP without the
20th week
need for an agonist like bovine thrombin. Due to the activation process, the multiple biological factors released by activated platelets are present in high concentrations within the matrix [16].The CS-PRP is initially moldable and later solidifies thus preventing the loss of the material. It acts as a scaffold needed by the bone regenerative process. It was also proven that CS can slowly release recombinant human platelet- derived growth factor (rhPDGF) over time; therefore supporting the hypothesis that CSPlatelet behaves as time-controlled releasing matrix for all platelet-derived factors [17]. Dario De Leonardis et al. [12] confirmed that careful stratification and dry compaction of calcium sulfate was effective in reducing the resorption rate and the extent of mass contraction
Fig. 7. Graphical representation of mean bone density comparing the study and control group in grey scale.
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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The interpretation of the observers coincided with the analysis obtained using computed imaging software and showed rapid bone regeneration by 5th week in study group when compared to the control group. Even though significance in bone regeneration was witnessed in normal healing of bony defects, the study group demonstrated faster and maximum bone formation by 20th week. 5. Conclusion
Fig. 8. Graphical representation of the mean percentage increase in bone formation comparing the study and control group.
during healing. The studies conducted by Pecora et al. [18], Kelly et al. [21,22], Park et al. [23], Kim et al. [24], Liu et al. [25] confirmed the osteo conductive potential of CS. In our study, to avoid large donor site defect we procured minimal autogenous bone graft just to cover 1/4th of the defect leaving the maximum remaining defect to be filled with PRP-CS complex. The PRP–CS complex acted as a reservoir of calcium ions for bone mineralization. When packed gently in cases of periapical lesions extending up to 2/3 of the root having clinical grade I tooth mobility, there showed a drastic improvement with complete mobility receding by the end of 5th week. In the study group the affected teeth were treated with apicoectomy, retrograde filling and grafting of the defect with CS. The treatment was same for the control group without placement of PRP and graft materials. Bone healing is radiographically expressed as an increase in radiopacity, resulting in a higher optical density of the bone image. Several studies have incorporated the use of grey scale values for measuring the bone density in radiographs using computed imaging software. The size of the residual bone cavities can also be measured in pixels using the same imaging software. In our study we had adopted the same methodology for radiographic evaluation of bone regeneration [4,19,20]. Radiographic evaluation at 6th and 12th month showed better and complete bone healing in the study group. In our cases, the mean grey scale value of the study group patients had increased drastically from 60.6 from 5th week to 91.6 in the 20th week post op, whereas in the control group bone density has gradually increased from 40 at 5th week to 62 in the 20th week post op as illustrated in Fig. 7. The mean percentage increase in bone formation in the study group was significantly higher with 52% in the first 5 weeks postoperative and 87% by 20th week (P < 0.0001) as visualized in Fig. 8. The control group has achieved only 16% increase in bone formation in the 5th week and 49% by 20th week. The mean increase in bone formation what the control group gained in the 20th week (49.48%) was attained by the study group in the 5th week itself (52%) as seen in Table 3. Apart from the software analysis of digital radiographs, we had included four independent blinded observers to evaluate the OPG and calibrate scores based on a radiopacity increasing scale (0-nil to 4-maximum). The mean radiopacity scores of the study group given by 4 blinded independent observers increased from 2 in the 5th week to 3.57 in the 20th week indicating maximum to complete radiopaque formation. The mean radiopacity scores in the control group had just increased from 0.97 in the 5th week to 2.27 in the 20th week indicating minimal bone formation as noted in Table 4. In the study group by 5th week itself there was noticeable decrease in defect size where radiopacity covered more than 1/4th to 1/2 of the defect size and with reference to the control group there was only very minimal radiopacity present.
This comparative study revealed increased radiographic bone density and rapid bone formation in the study group than the control group. Though our sample size is smaller, the combination of PRP–CS complex with autologous bone graft proved to be a novel osteoinductive biomaterial and can be used for mild to moderate osseous defects to promote bone regeneration. Funding No external funding was obtained for this study. Disclosure of interest The authors declare that they have no competing interest.
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Available online at
ScienceDirect www.sciencedirect.com
Original Article
Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study K.P. Nakkeeran a,*, K. Saravanan b, P. Babu c, R.R. John b a b c
Department of Oral and Maxillofacial Surgery, SRM Dental College and Hospital, Ramapuram, Chennai 600089, India Department Of Oral & Maxillofacial Surgery, Vinayaka Missions Sankaracharyar Dental College & Hospital, Salem 636308, India Department of Oral & Maxillofacial Surgery, Dr. Syamala Reddy Dental College Hospital and Research Centre, Marathahalli Bangalore, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 September 2018 Accepted 19 November 2018
Background: Osseous defects of the jaw occurs naturally by pathological conditions such as infections, tumors, cysts or iatrogenic causes induced during surgical treatment of preexisting periradicular bony pathologies. Management of these bone defects poses a great challenge for surgeons who are continually exploring ways to find new modalities to improvise bone substitutes. Purpose: To compare and evaluate bone regeneration with and without combining platelet rich plasma (PRP), calcium sulfate (CS) and autogenous bone graft in periapical defects of jaw. Methods: The study is a randomized prospective comparative study, in which 20 patients were assigned, with 10 patients each forming the study and control groups. In the first group (study group) the bone defect was filled with PRP, calcium sulfate and autologous bone graft. In the second group (control group) defect was allowed to heal without PRP, calcium sulfate and autogenous bone graft. Digital orthopantogram (OPG) were taken to analyze the bone density and bone regeneration. The parameters used to analyze the radiographs included grey scale analysis, residual bone defect calculation in pixels both performed in Corel Photopaint X31 software and radiopaque scoring scale. Statistical analysis was carried out using Kolmogorov Smirnov test, Independent t-test and Anova test. Results: The mean bone defect density (BDD) measured using grey scale was significant in the study group during 5th, 13th and 20th week than the control group (P < 0.0001). The percentage bone formation analyzed using residual bone defect calculation revealed significantly higher size reduction in the study group than with the outcome obtained in the control group (P < 0.0001). The mean radiopaque score given by blinded observers also showed significance in the study group. Conclusion: From the results of the study, it is clearly evident that radiographic bone density and rapid bone formation in the study group when compared to the control group. Thus the grouping of PRP, combined calcium sulfate with autologous bone graft proved to be a novel osteoinductive biomaterial.
C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Platelet rich plasma Calcium sulfate Bone defect density Orthopantogram
1. Introduction Osseous defects of the jaw occurs naturally by pathological conditions such as infections, tumors, cysts or iatrogenic causes, induced during surgical excision of preexisting intraosseous periradicular pathologies [1]. Management of these bone defects represents a great challenge for surgeons who are continually
* Corresponding author. E-mail addresses: [email protected] (K.P. Nakkeeran), [email protected] (K. Saravanan), [email protected] (P. Babu), [email protected] (R.R. John).
exploring ways to improve the success of bone grafting with bone substitutes [2]. Unfilled bone defects usually heal by initial resorption of the alveolar bone. Spontaneous bone regeneration in untreated defects is limited to small distance because of rapid proliferation of surrounding soft tissues. Further bone defects following cyst enucleation leads to loss of the concerned teeth. Filling these defects with bone or other substitute materials help to preserve the alveolar ridge and provides sufficient bone for immediate or subsequent implant placement [3]. Growth factors like platelet-derived growth factor (PDGF) play an important role in inducing proliferation of undifferentiated mesenchymal cells. It is an important mediator for bone healing
https://doi.org/10.1016/j.jormas.2018.11.008 C 2018 Elsevier Masson SAS. All rights reserved. 2468-7855/
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Abbreviations PRP Platelet rich plasma Calcium sulfate CS Platelet poor plasma PPP PDGF Platelet derived growth factor Bone defect density BDD Orthopantogram OPG
and remodeling. The therapeutic strategy of autologous PRP is based on acceleration of healing by the concentration of the growth factors, which are universal initiators of nearly all healing events [2]. It contains high concentration of thrombocytes, which in turn contains a number of growth factors namely PDGF, TGF-b, IGF and VEGF [5]. The use of autogenous bone graft is regarded as the gold standard for replacement of regenerated bone. Autologous bone provides osteoconduction combined with osteoinductive effects, but its availability is limited. Alloplastic (synthetic bone grafts) and xenogenic bone substitute materials have been used alone or in combination with autogenous bone graft [3,6]. Our study was designed to evaluate the efficacy of PRP when combined with alloplastic biomaterial like CS and autogenous bone graft in hastening bone regeneration in osseous defects of the jaw.
2. Materials and methods The study was performed at Department of Oral and Maxillofacial surgery, Vinayaka Missions Sankarachariyar Dental College and Hospital, Salem after obtaining institutional ethical committee clearance (REF:VMSDC/IEC/015). The prospective randomized control trial was performed on patients with periapical cystic lesions involving the maxillary and mandible anterior region. Twenty patients were randomly assigned into two groups with 10 patients each forming the study and control groups. The exclusion criteria were those patients of age less than 12 years and greater than 50 years, patients diagnosed with systemic diseases and given the history of smoking. Surgical consent was obtained for phlebotomy to collect blood for the development of platelet rich plasma, graft procurement and placement of CS. In the first group (study group) the bone defect was filled with PRP, combined CS and autologous bone graft. In the second group (control group) defect was allowed to heal normally without PRP and bone grafts. In the study group, PRP was prepared by table top centrifugation device using double centrifugation technique [7]. The autologous pre donated blood was collected in sufficient amounts of anticoagulation (EDTA) solution. The aspirated blood was gently agitated to thoroughly mix the anticoagulant with the blood. The first spin (called the hard spin or separation spin) separated the red blood cells from the plasma, which contains the platelets, white blood cells and clotting factors. The centrifugation speed was carried out at 5600 rpm for 3 to 4 minutes. The second spin (called the soft spin or concentration spin) finely separated the platelets and white blood cells together with a few red blood cells from the plasma. The centrifugation speed was carried out at 2400 rpm for 8 to 9 minutes. This soft spin produces the PRP and separates it from the platelet poor plasma (PPP) [7]. Ten milliliters of whole blood after centrifugation process roughly yielded 3 ml of PRP (see Fig. 1).
Fig. 1. Platelet rich plasma (PRP) obtained after double centrifugation technique.
Under local anesthesia, autologous cortical bone graft was procured from mandibular symphysis region and cystic defects were treated by creating a bony window with complete enucleation of the lesion. The final osseous defects varied from 1–1.5 cm to a maximum of 3–3.5 cm in diameter size. Endodontic treatment of the affected tooth was done prior, followed by apicoectomy. The derived platelet rich plasma was initially soaked with the procured autologous bone graft for some time and later mixed with CS (CapsetTM). Small amount of PRP was also applied on overlying soft tissue to enhance the healing process. It was then packed by careful stratification in a dry environment within the bone cavity (see Fig. 2). The control group defect was allowed to heal naturally without the application of PRP, CS and bone grafts. Radiographs (Digital OPG) were taken to assess the bone density and bone regeneration. The radiographs were evaluated preoperatively, followed by immediate postoperative period after 1 week and at a regular interval of 5th, 13th, 20th week. The radiological analysis was performed using Corel Photo-Paint X31 Software version-13.0. Bone density was measured on the radiographs through a gray scale of 256 tonalities as shown in the Figs. 3 and 4. The residual defect size of the cavity was measured in pixels using the same software at preoperative, immediate postoperative, 5th week, 13th week and 20th week respectively as depicted in Figs. 5 and 6. The measurement of bone formation was also performed subjectively but blindly, recording the score in a radiopacity increasing scale (see Table 1) by four independent blinded observers.
Fig. 2. Autologous bone graft procured from mandibular symphysis is combined with calcium sulfate (CS), and platelet rich plasma (PRP). The graft was packed by dry stratification technique.
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Fig. 3. Bone defect density (BDD) was measured within the grid corresponding to the radiolucent area of the lesion using the Corel- photo paint X3 software.
3. Results
Fig. 4. Normal bone density (NBD) was measured within the grid of the normal bone. The bone formation was observed by the decrease in the gray scale value between the NBD and BDD in subsequent follow up radiographs.
Fig. 5. Bone defect area marked using a freehand mask tool using the Corel photo-paint x3 software.
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Fig. 6. The delineated defect area is created as a separate object and its height and width is obtained in pixels. The residual defect cavity is measured by multiplying the height and width.
Table 1 Radiopacity scoring scale. Bone formation/healing
Score
Nil/failure Very minimal/doubtful Minimal Moderate Complete
0 1 2 3 4
Nil/ failure: complete radiolucency present. Very minimal/doubtful: very minimal radiopacity present at least in two consecutive follow-ups. Minimal: evidence of radiopacity at the periphery and noticeable decrease in defect size (radiopacity seen more than 1/4th up to 1/2 of the defect). Moderate: radiopacity covering at least 1/2 to 3/4th of the defect. Complete: radiopacity covering 3/4th to maximum size of the defect.
Among the total 20 patients who were included in the study, 12 comprised of males and 8 were females with a mean age of 24 years. By comparing the mean BDD of study group with control group the former showed significant increase in value (P < 0.0001). In the study group, the mean BDD measured in grey scale showed drastic increase from 60.6 in the 5th week to 73 in the 13th week and 91.6 in the 20th week postoperative. The mean BDD in the control group gradually increased from 40 in the 5th week to 52 in the 13th week and up to 62 in the 20th week postoperative. The above results have been summarized in Table 2.
The study group patients showed significantly higher bone defect size reduction than the control group patients (P < 0.0001). The mean percentage increase in bone formation in the study group stood at 52% at 5th week, 68% at 13th week and 87% at 20th week whereas for the control group percentage increase in bone formation was 16% at 5th week, 31.6% at 13th week and 49% at 20th week as valuated in Table 3. The mean radiographic score given by independent observers was significantly higher for the study group compared to the control group. Significant differences in the mean were found in the scores of four observers by using ANOVA test and between the study group and the control patients by using t-test. The mean radiographic score for study group was 2 at 5th week, 2.67 at 13th week & 3.57 at 20th week respectively where as for the control group it was 0.97 at 5th week, 1.47 at 13th week and 2.27 at 20th week respectively as summarized in Table 4.
4. Discussion The osteogenic potential of autogenous grafts with PRP is a well-established one. The bone substitute graft forms new bone via osteoconduction from adjacent osteoprogenitor cells. The autogenous bone graft transplants osteoprogenitor cells from distant site into the defect area and aid in new bone formation. In such
Table 2 Postoperative bone defect density (BDD) in grey scale. Patient No.
5th week
13th week
20th week
Study group
Control group
Study group
Control group
Study group
Control group
1 2 3 4 5 6 7 8 9 10 Mean bone defect density (BDD)
55 51 57 63 56 60 58 46 91 69 60.6
39 46 23 59 43 34 30 36 58 32 40
63 59 74 70 68 72 64 61 93 103 2.7
49 58 36 64 49 42 55 61 61 47 52.2
110 65 129 78 87 74 71 71 97 134 91.6
63 63 62 71 51 58 57 65 66 62 61.8
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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Table 3 Residual bone defect size and percentage increase in bone formation. Patient No.
1 2 3 4 5 6 7 8 9 10 Mean percentage increase
Preoperative
5th week
13th week
20th week
Study group size
Control group size
Study group size (% increase)
Control group size (% increase)
Study group size (% increase)
Control group size (% increase)
Study group size (% increase)
Control group size (% increase)
27,566 18,126 27,238 45,666 9860 46,202 20,868 28,620 11,782 151,59
17,549 51,512 35,235 34,565 17,115 20,437 26,332 10,080 16,156 29,000
17,940 (35) 13,038 (28) 16,000 (41) 21,018 (54) 3354 (66) 19,933 (57) 4734 (77) 9313 (67) 7548 (36) 5846 (61) 52
14,157 (19) 47,880 (7) 29,190 (17) 30,250 (12) 14,994 (12) 20,776 (1.6) 19,474 (26) 8118 (19) 13,018 (19) 22,360 (23) 16
10,792 (61) 9156 (49) 5733 (79) 14,432 (68) 4837 (51) 6495 (86) 2788 (86.6) 7790 (73) 6487 (45) 2448 (84) 68
10,350 (41) 46,228 (10) 25,186 (28.5) 28,728 (17) 10,290 (40) 15,456 (24) 16,170 (38.5) 7533 (25) 6245 (62) 20,600 (29) 31.6
3303 1344 2980 1935 1914 3653 1518 4692 4090 1953 86.6
5694 (67.5) 27,360 (47) 24,948 (29) 25,320 (27) 9646 (43.6) 12,879 (37) 9576 (63.6) 5382 (46.6) 2787 (83) 14,295 (51) 49
(88) (92.5) (89) (96) (80.5) (92) (93) (83.6) (65) (87)
Percentage increase in bone formation is calculated by: postoperative defect size in pixels 100 preoperative defect size-100.
Table 4 Mean Radiopacity Score – study and control group. Observer
5th week
13th week
Study group
Control group
Study group
Control group
Study group
Control group
Observer 1 Observer 2 Observer 3 Observer 4 Mean score
2.6 2 2 1.4 2
1.5 0.7 1.1 0.6 0.97
3.1 2.5 2.9 2.2 2.67
2 1.2 1.7 1 1.47
3.9 3.4 3.8 3.2 3.57
2.9 2.3 2.4 1.5 2.27
conditions PRP upregulates osteoprogenitor cells into the grafts and helps in matrix formation [7]. The application of platelet rich plasma (PRP) in combination with bone and bone substitutes leads to the transformation of the surrounding cells in grafted bone cells and increases the integrity of the graft [8]. Recent studies of PRP have shown enhancement of nearly all bone substitute materials. Farrel et al. from their studies observed that no benefits when PRP alone has been placed into the bone defects without other grafting materials [2]. Marx [8] on his study on 88 patients with mandibular continuity defects using PRP concluded that the degree of maturation of the grafts was significantly greater with addition of PRP (1.6 to 2.2 times faster). PRP also acts as an anti-inflammatory agent by production of CCL5 (Chemokine ligand 5). CCL5 is a chemotactic for T cells, eosinophils, and basophils and plays an active role in recruiting leukocytes into inflammatory sites, blocking monocyte chemotactic protein-1 release from monocytes and its concentration of lipoxin A4, suggesting that PRP facilitates healing by controlling the local inflammatory response [9]. Calcium sulfate (CS) as a bone graft material is used in various skeletal sites including mandibular, craniofacial and long bone defects and benign bone lesions. It is highly biocompatible graft substitute material which is well tolerated by host tissues, not inducing any foreign body reactions. It has been used in treating periodontal disease, endodontic treatments, post extraction sites, maxillary sinus augmentation and as a barrier in guided tissue regeneration [10]. It is used as a barrier in guided tissue regeneration by physically halting soft connective tissue proliferation by means of the osteopromotion principle [1]. It is completely resorbable in a relatively short period and provides a local increase in Ca ion concentration which may stimulate osteoblastic activity and acts as a scaffold for bone growth [11–14]. The exothermic reaction generated by the mixture of CS with PRP activates the platelets contained within the PRP without the
20th week
need for an agonist like bovine thrombin. Due to the activation process, the multiple biological factors released by activated platelets are present in high concentrations within the matrix [16].The CS-PRP is initially moldable and later solidifies thus preventing the loss of the material. It acts as a scaffold needed by the bone regenerative process. It was also proven that CS can slowly release recombinant human platelet- derived growth factor (rhPDGF) over time; therefore supporting the hypothesis that CSPlatelet behaves as time-controlled releasing matrix for all platelet-derived factors [17]. Dario De Leonardis et al. [12] confirmed that careful stratification and dry compaction of calcium sulfate was effective in reducing the resorption rate and the extent of mass contraction
Fig. 7. Graphical representation of mean bone density comparing the study and control group in grey scale.
Please cite this article in press as: Nakkeeran KP, et al. Evaluation of bone regeneration in periapical osseous defects with and without platelet rich plasma, combined calcium sulfate and autologous bone graft – A comparative study. J Stomatol Oral Maxillofac Surg (2018), https://doi.org/10.1016/j.jormas.2018.11.008
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The interpretation of the observers coincided with the analysis obtained using computed imaging software and showed rapid bone regeneration by 5th week in study group when compared to the control group. Even though significance in bone regeneration was witnessed in normal healing of bony defects, the study group demonstrated faster and maximum bone formation by 20th week. 5. Conclusion
Fig. 8. Graphical representation of the mean percentage increase in bone formation comparing the study and control group.
during healing. The studies conducted by Pecora et al. [18], Kelly et al. [21,22], Park et al. [23], Kim et al. [24], Liu et al. [25] confirmed the osteo conductive potential of CS. In our study, to avoid large donor site defect we procured minimal autogenous bone graft just to cover 1/4th of the defect leaving the maximum remaining defect to be filled with PRP-CS complex. The PRP–CS complex acted as a reservoir of calcium ions for bone mineralization. When packed gently in cases of periapical lesions extending up to 2/3 of the root having clinical grade I tooth mobility, there showed a drastic improvement with complete mobility receding by the end of 5th week. In the study group the affected teeth were treated with apicoectomy, retrograde filling and grafting of the defect with CS. The treatment was same for the control group without placement of PRP and graft materials. Bone healing is radiographically expressed as an increase in radiopacity, resulting in a higher optical density of the bone image. Several studies have incorporated the use of grey scale values for measuring the bone density in radiographs using computed imaging software. The size of the residual bone cavities can also be measured in pixels using the same imaging software. In our study we had adopted the same methodology for radiographic evaluation of bone regeneration [4,19,20]. Radiographic evaluation at 6th and 12th month showed better and complete bone healing in the study group. In our cases, the mean grey scale value of the study group patients had increased drastically from 60.6 from 5th week to 91.6 in the 20th week post op, whereas in the control group bone density has gradually increased from 40 at 5th week to 62 in the 20th week post op as illustrated in Fig. 7. The mean percentage increase in bone formation in the study group was significantly higher with 52% in the first 5 weeks postoperative and 87% by 20th week (P < 0.0001) as visualized in Fig. 8. The control group has achieved only 16% increase in bone formation in the 5th week and 49% by 20th week. The mean increase in bone formation what the control group gained in the 20th week (49.48%) was attained by the study group in the 5th week itself (52%) as seen in Table 3. Apart from the software analysis of digital radiographs, we had included four independent blinded observers to evaluate the OPG and calibrate scores based on a radiopacity increasing scale (0-nil to 4-maximum). The mean radiopacity scores of the study group given by 4 blinded independent observers increased from 2 in the 5th week to 3.57 in the 20th week indicating maximum to complete radiopaque formation. The mean radiopacity scores in the control group had just increased from 0.97 in the 5th week to 2.27 in the 20th week indicating minimal bone formation as noted in Table 4. In the study group by 5th week itself there was noticeable decrease in defect size where radiopacity covered more than 1/4th to 1/2 of the defect size and with reference to the control group there was only very minimal radiopacity present.
This comparative study revealed increased radiographic bone density and rapid bone formation in the study group than the control group. Though our sample size is smaller, the combination of PRP–CS complex with autologous bone graft proved to be a novel osteoinductive biomaterial and can be used for mild to moderate osseous defects to promote bone regeneration. Funding No external funding was obtained for this study. Disclosure of interest The authors declare that they have no competing interest.
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