Introduction and review of literature
The therapeutic goals in the management of fracture mandible are to re-establish preinjury occlusion and restore anatomy, to provide fracture stabilization, and to restore function, fixation, and immobilization and to ensure the achievement of these therapeutic goals 1.
The objective of reduction is to restore the structures to a normal position in terms of function and contour. This is achieved by the placement of fracture segments of bone in the proper position. This can be achieved either by closed or by open reduction 2.
Platelets arise from cytoplasmic fragmentation of the megakaryocyte in the bone marrow. Like red blood cells, platelets enter the circulation as anuclear cells and they have a limited life span. The platelet lives for only about 7–10 days. The platelet synthesizes growth factors throughout its life span and actively secretes them in response to clotting. A platelet is about 2 µm at its largest diameter. It has numerous pseudopodial extensions, invaginations of its cell membrane, and internal vesicles (storage granules). The vesicles are composed of three types of granules: lysosomal, dense, and alpha. The lysosomal granules seem to function as storage for digestive enzymes. The dense granules mainly store and secrete ADP, which are potent recruiters and activators of other platelets 3.
Platelet-rich plasma (PRP) is a normal autogenous blood clot that contains a highly concentrated number of platelets. The minimum platelet count required for a blood clot to qualify as PRP may be arguable, but a concentration of about 1 million platelets/µl or about four to seven times the usual baseline platelet count has been shown to provide clinical benefits 3.
Anitua 4 reported improved epithelialization and bone density when PRP was placed into the extraction sockets. Similarly, Mancuso et al. 5 reported a low rate of alveolar osteitis, less pain, and more dense radiographic bone healing when PRP was placed into third molar extraction sockets.
However, Aghaloo et al. 6 showed no benefit for the use of PRP alone in rabbit cranial defects compared with untreated defects.
The use of PRP is one strategy available today that can modulate and enhance wound healing. The processing of PRP involves the sequestration and concentration of platelets and, therefore, the many growth factors they contain. This will amplify and accelerate the effects of growth factors present in platelets, which are universal initiators of almost all wound healing 7.
The source of the new preparation, known as PRP, consists of a limited volume of plasma enriched in platelets, which is obtained from the patient. Once the platelet concentrate is activated by thrombin generation with calcium, a three-dimensional and biocompatible fibrin scaffold is formed, and a myriad of growth factors and proteins are released, progressively, to the local environment, contributing toward accelerated postoperative wound healing and tissue repair 8.
Choukroun’s platelet-rich fibrin (PRF) is defined as an autologous leukocyte-rich and platelet-rich fibrin (L-PRF) biomaterial 9,10. This easy and open-access procedure was developed in France by Dohan et al. 10. Blood is collected in 9 ml tubes and gently centrifuged for 12 min to divide the blood sample into three layers: a base of red blood cells at the bottom, acellular plasma on the top, and a clot of PRF in the middle. The success of this technique entirely depends on the speed of blood collection and transfer to the centrifuge. If the duration required to collect blood and launch centrifugation is too long, fibrin will not be formed: the fibrin will polymerize in a diffuse manner in the tube and only a small blood clot without consistency will be obtained.
One of the main differences between the PRF concept and most PRPs systems is that the PRF production process is completely natural, with no use of an anticoagulant during blood harvest or bovine thrombin and calcium chloride for platelet activation and fibrin polymerization. PRF is often simply considered a natural optimized blood clot. PRF clots can easily be transformed in dense fibrin 12.
The aim of this study was to evaluate both clinically and radiographically the validity of a local application of PRF and PRP on mandibular fracture healing.
Patients and methods
The current study is a prospective comparative study carried on 16 patients ranging in age from 20 to 42 years. All surgical procedures were carried out at the Plastic Surgery Unit in the Department of Surgery at Suez Canal University Hospital over a period of 2 years (July 2011–July 2013).
The patients were divided into two equal groups:
- Group I (PRP group) included eight patients treated by open reduction and direct osteosynthesis using a 2.0 mm miniplate with a conventional screw where the PRP was locally added to the fracture line.
- Group II (PRF group) included eight patients treated by open reduction and direct osteosynthesis using a 2.0 mm miniplate with a conventional screw where the PRF was locally added to the fracture line.
Preoperative radiographic examination
Standardized preoperative panoramic or CT radiographs were performed for each patient to assess the number and location of line or lines of fracture, degree of displacement, and to localize the inferior dental canal or tooth in the fracture lines.
Preparation of platelet-rich plasma
A closed system of ordinary blood packs was used throughout the process. We used standard triple packs (Baxter KGR 7340; Milchstrasse, Hamburg, Germany) containing citrate phosphate dextrose as an anticoagulant for autologous (or homologous).
The PRP was separated from the blood by centrifugation for 15 min at 327g and at ambient temperature (soft spine). Recentrifugation at 3000g for 15 min (hard spine) was performed to concentrate platelets. The platelet concentration target was preset at more than 6×1010/unit and a plasma volume of 40–60 ml, and then kept for 48 h at +20°C with continuous shaking on a horizontal shaker (Forma Scientific, Marietta, Ohio, USA) 13,14.
Platelet-poor plasma was mixed with calcium gluconate (1/0.2 rate) in a sterile Falcon tube and then incubated at +37°C for 15–30 min; the final supernatant, full of thrombin precursors, was recovered and stored in a syringe at −40°C. It was important to remove the thrombin solution from the clot as soon as possible as thrombin adsorbs to it 15.
Platelet gel activation
From the platelet concentrate pack, amount of blood was withdrawn into a syringe. The thrombin syringe was thawed at +37°C and added to a sterile Falcon tube with calcium gluconate in the following proportions: three parts of platelet concentrate, one part of thrombin, and 0.5 part of calcium gluconate. The suspension was exposed to slow shaking and it was ensured that a 360° tube revolution was completed 10–12 times, and this was allowed to stand for about 15 min 16. Immediately before preparing the PG, the total volume was adjusted to a level suitable for use in the fracture line. The mixing of platelet gel (GL) with 10 ml of thrombin solution and 5 ml calcium gluconate was acceptable.
Preparation of platelet-rich fibrin
PRF was prepared according to the technique described by Dohan et al. 10. Twenty minutes before starting surgery, 10 ml of venous blood was collected into a sterilized dry, neutral glass tube without an anticoagulant. After immediate centrifugation at 300g for 10 min, the platelet-poor plasma, which accumulated at the top, was discarded. PRF was dissected ∼2 mm below its connection to the red corpuscle beneath to include the remaining platelets, which have been proposed to localize below the junction between PRF and the red corpuscle.
Patients were kept fasting for 8 h before surgery. The surgical area was shaved and cleaned preoperatively. The patient was anesthetized using nasotracheal intubation. The oral cavity was first scrubbed with povidone iodine, then all around the extraoral surgical site, followed by draping with sterile towels, exposing only the area of surgery.
Group I (platelet-rich plasma group)
Patients in this group were treated by open reduction and direct osteosynthesis using miniplates with the application of PRP in the fracture line.
Group II (platelet-rich fibrin group)
Patients in this group were treated by open reduction and direct osteosynthesis using miniplates with the application of PRF in the fracture line.
Clinical and radiographic follow-up was performed for all patients for 6 months postoperatively; the patients were recalled immediately the day after the operation, and 1, 3, and 6 months postoperatively.
Postoperative care and follow-up
Clinical follow-up was carried out every week during the first month, then at 2, 3, and 6 months postoperatively. Clinical assessment was performed on the basis of the following parameters:
- Postoperative pain: it was measured using a visual analogue scale to assess pain with the end point marked score 0 (no pain) and score 10 (worst pain) for 1 week postoperatively.
- Postoperative trismus mandibular movements: Using calipers to measure the maximum interincisal mouth opening, these measurements were performed preoperatively and after 1, 3, and 6 months.
- Soft tissue infection: the wound was evaluated for signs and symptoms of infection including swelling, redness, hotness, discharge, and pain in addition to observation for any signs of wound-healing disturbance.
- Occlusion: it was checked in the maximal intercuspal position to ensure a proper occlusal relationship including molar relation and midline centralization. Any occlusal disturbance including open bite or improper tooth contact was noted.
- Stability of the fractured segments.
- Teeth related to the fractured line (tooth damage).
- Assessment of the sensory and motor nerve function: assessment of the sensory function of the inferior alveolar nerve was performed subjectively by asking the patient about any alteration in sensation; in addition, an objective examination was performed using a dental probe to assess the sensory changes along the distribution of the mental nerve through examination of lip sensation in comparison with the contralateral side.
- Nonunion, malunion, and malocclusion.
Radiographic follow-up was carried out through a digital panoramic radiograph at the following time points: immediately postoperatively, and 1, 3, and 6 months postoperatively. Radiographic assessment was performed on the basis of the following parameters:
- Width of the fracture line.
- Bone surrounding plate.
- Teeth related to the fracture line.
All radiographs for every patient were imaged using the same electronically controlled panoramic machine; the exposure parameters were considered fixed for all patients at 70 kV and 10 A for 15 s.
Direct digital panoramic radiographs for groups I and II were carried out to assess the radiodensitometric bone changes in the fracture site immediately postoperatively, and 1, 3, and 6 months postoperatively.
The patients were positioned (for panoramic exposure) according to the standard procedure as follows:
The patient’s head was aligned so that the dental arches were located in the middle of the focal trough.
Data management and analysis
The data were collected and the significance of differences between groups was assessed by analysis of variance, followed by an independent t-test.
The collected data were coded and entered into the statistical package of social sciences (SPSS-17; Chicago, Illinois, USA) program for statistical analysis.
Each panoramic image was evaluated for width of the fracture line after reduction and alignment of the fractured segments.
Quantitative analysis (radiodensitometric analysis)
Densitometric (radiometric) measurement: digitized images were manipulated using the specially designed software of the Digora (Soredex).
On each digital image, the mean gray value of the marked region of interest was calculated using the following steps:
- Point A was selected at the fracture line and the pixel density of this point was measured on a scale from 0 to 255 according to its radiopacity, where the maximum radiopacity is 255. A zero scale was assigned to the totally black regions (totally radiolucent) and 255 for totally white regions; values in between were represented by different shades of gray.
- A second point (point B) was selected at the same level and just next to the first area, but at sound bone, and its pixel density was also measured as before.
- The difference between these two areas was calculated, representing the difference between bone density (pixel density) at the fracture line (point A) and at sound bone (point B).
To standardize the position of the point of interest under investigation, the exact coordinates (X and Y coordinates) for each of points A and B were calculated for each case and repeated during the follow-up radiography.
One month postoperatively
- Wound dehiscence or plate exposures were not observed in any case. The mucosa overlying the miniplates appeared healthy and of normal color and texture. Extraoral wound scars became hidden in the shadow of the inferior border of the mandible and became fainter with time.
- This occur normally after one month of IMF.
- Bimanual examination of the fractured segments showed absolute stability. A smooth uniform inferior border of the mandible could be palpated in all cases.
Three and 6 months postoperatively
- All cases showed stability of the bony segments, with no detected mobility of the bony segments.
- During clinical follow-up, there was no abnormal swelling or discoloration, except for case no. 1 in group II; there was abnormal swelling after 3 months, which was treated by antibiotics and anti-inflammatory medications.
- Interincisal distant is increase compared with last months.
- At the end of the follow-up period, all cases presented with normal occlusion, healthy soft tissue, and proper alignment of the mandibular inferior border. Also, extraoral wound scars became unnoticeable in all cases by the end of follow-up. Neither infection nor mobility was observed in any of the teeth related to the fracture line of all patients. The teeth remained vital without the need for any therapeutic interference, except in case no. 1 in group I; we extracted the tooth after 6 months postoperatively.
- No sensory or motor nerve dysfunction was reported or observed in any of the patients, except case no. 5 (group I) and case no. 1 (group II), who still had a complaint of numbness of the lower lip and chin.
Qualitative radiographic results
- Immediate postoperative radiograph: immediate radiographic examination of all cases showed properly reduced fractured segments with narrowing of the interfragmentary gap. In all cases, the inferior border of the mandible was properly aligned.
- One-month postoperative radiograph: the interfragmentary gap was also seen with no significant difference appearing.
- Three-month postoperative radiograph: radiographic examination showed greater healing of fracture lines, with proper alignment of the inferior border of the mandible and signs of disappearance of interfragmentary gap.
- Six-month postoperative radiograph: the fracture line had become unidentifiable in all cases; no abnormal radiographic changes were observed in relation to both the plate and the teeth within the fracture line in all cases.
Quantitative radiographic results
Figures 1–8 show a comparison of pixel density values among the three groups in terms of the bone density immediately postoperatively, and 1, 3, and 6 months postoperatively.
- Radiographic analysis results for the fracture line:
- From Table 1, it can be seen that the mean and standard deviations of bone mineral densities (BMD) for three groups were as follows:
- In group I (PRP), the mean BMD was 59.7±8.02 immediately postoperatively, 47.5±6.3 at 1 month, 19.3±3.6 at 3 months, and 10.38±2.4 at 6 months, whereas in group II (PRF), the mean BMD was 59.5±6.7 immediately postoperatively, 43.1±3.4 at 1 month, 13.3±3.4 at 3 months, and 5.1±2.1 at 6 months.
- Changes in density over time:
- Group I (PRP group): from Table 2, it can be seen that a highly significant differences was detected during study period between immediately postoperatively and at 3 months, immediately postoperatively and 6 months, at 1 and 3 months, and at 1 and 6 months.
- A significant difference was detected in the same group between 3 and 6 months, whereas no significant difference was detected in the same group between immediately postoperatively and at 1 month.
- Group II (PRF group): from Table 2, it can be seen that a highly significant differences was detected during the study period between immediately postoperatively and at 3 months, immediately postoperatively and 6 months, at 1 and 3 months, at 1 and 6 months, and at 3 and 6 months.
- No significant difference was detected in the same group between immediately postoperatively and at 1 month.
- Comparison between the three study groups:
- Table 3 shows the similarity between the changes in density in the three study groups as there was a highly significant difference (P<0.01) between group I compared with group II at 6 months.
- There was a significant difference (P<0.05) between group II and group III at 3 months, whereas there was no significant difference (P>0.05) in group I and group II immediately postoperatively and at 1 month (Fig. 9).
The present study showed that PRF application in the fracture line mandible led to greater bone formation than PRP application. This may have been because of higher levels of growth factors in PRF than PRP 13. The PRF growth factor contents were logically expected to be much higher than the PRP contents because most platelets were activated in the PRF clots (this is the definition of PRF) 17. Another reason for this may be related to the intrinsic nature, content, and architecture of the PRF biomaterial. Moreover, the fibrin seems to be a relevant matrix to support osteoblastic growth and differentiation, and it is used frequently during bone tissue-engineering experimentations 18. This finding was in agreement with that of Shi Zhu et al. 19 in their experimental study on rats. The aim of that study was to compare the effects of PRP and platelet-enriched fibrin glue on bone formation in bone tissue engineering. Histomorphometric analysis showed that the nodules contained 14.9±4.1% newly formed bone when using PRP and 19.8±3.6% newly formed bone when using platelet-enriched fibrin glue.
The results indicated that the osteogenic characteristics of platelet-enriched fibrin glue are superior to those of PRP in bone tissue engineering. Moreover, this finding was not in agreement with an in-vitro human cell culture study. The aim of that investigation was to examine the growth factor release from PRP and PRF. They found that in osteoblast cultures, cytokine concentrations were significantly higher for PRP than for PRF; they showed that PRP application in cell cultures leads to higher levels of growth factors than PRF application 20. The difference from our results may be because of differences in the study model.
The results of the current study are in agreement with those of Diss et al. 21; the study was carried out to document, radiographically, changes in the apical bone levels on microthreaded implants placed in subsinus residual bone height according to a bone-added osteotome sinus floor elevation technique with PRF as a grafting material. Measurements of the changes in the endosinus level on the mesial and distal sides showed that all implants gained endosinus bone. The mean endosinus gain was 3.2±1.5 mm, with 3.5±1.4 mm on the mesial side and 2.9±1.6 mm on the distal side. The lowest bone gain was 0.9 and 0.1 mm on the mesial and distal sides, respectively. The highest gain was 5.8 and 5.2 mm on the mesial and the distal sides, respectively. They concluded that the use of PRF as a grafting material can lead to endosinus bone gain.
The results of the present study were also in agreement with those of Pripatnanont et al. 22; their study aimed to investigate the effect of PRF on bone regeneration of various grafting materials in rabbit calvarial defects. Two bicortical skull defects were prepared in 20 New Zealand white rabbits; 10 rabbits were treated with PRF and the other 10 were not treated. The mean optical density and histomorphometric analysis of the percentage of new bone showed that the PRF groups had significantly greater autogenous bone graft than the non-PRF groups. They concluded that PRF had a positive effect on bone formation when used alone or combined with autogenous bone.
The scientific rationale behind the use of these preparations lies in the fact that the platelet α-granules are a reservoir of many growth factors that are known to play a crucial role in hard and soft tissue repair mechanisms 23,24. This study found that PRF application in fracture line mandibles showed good bone formation. This finding was in agreement with one previous in-vitro study carried out to analyze the effects of Choukroun’s PRF, a leukocyte and platelet concentrate clinically usable as fibrin membrane or clot, on human primary cultures of gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts. They found that PRF induced significant and continuous stimulation of proliferation in all cell types. Moreover, PRF induced a strong differentiation in the osteoblasts under all culture conditions 25.
The results of the present study indicated that PRF application in fracture line mandibles led to higher bone formation than PRP application. This observation was in agreement with a previous in-vitro study carried out by He et al. 26. The aim of that study was to evaluate the effect of biologic characteristics of PRP and PRF on the proliferation and differentiation of rat osteoblasts. They found that PRF is superior to PRP in terms of expression of alkaline phosphatase and induction of mineralization. They concluded that PRF released autologous growth factors gradually and exerted a stronger and more durable effect on the proliferation and differentiation of rat osteoblasts than PRP in vitro.
Conflicts of interest
There are no conflicts of interest.
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