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Platelet-rich fibrin has a healing effect on chemotherapy-induced mucositis in hamsters
Vol. 117 No. 4 April 2014
Platelet-rich fibrin has a healing effect on chemotherapy-induced mucositis in hamsters Koichiro Horii, DDS,a Takeo Kanayama, DDS, PhD,b Hironori Miyamoto, DDS,a Tomoyuki Kohgo, DDS, PhD,c Tsukasa Tsuchimochi, DDS, PhD,b Toshio Shigetomi, DDS, PhD,d and Motoo Yokoi, DDS, PhDe Nagoya City University, Nagoya, Japan; Keiyukai Sapporo Hospital, Sapporo, Japan
Objective. The aim of this study was to evaluate the healing effect of topically applied platelet-rich fibrin (PRF) on experimental oral mucositis induced by chemotherapy in hamsters. Study Design. Oral mucositis was induced in 93 Syrian golden hamsters by an intraperitoneal injection of 5-fluorouracil, which was followed by light scratching of the cheek pouch. The hamsters were randomly divided into a PRF group, a fibrin group, and an untreated control group. The recovery stage of oral mucositis was evaluated through daily weighing, measurements of the ulcer area, histopathologic analysis, and a myeloperoxidase activity assay. Results. The PRF group exhibited significant improvements in the size and histologic features of the ulcer and in the myeloperoxidase activity compared with the control group (P < .05). Conclusions. The current findings suggest the consideration for future clinical trials in humans. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;117:445-453)
Oral mucositis is a painful and often debilitating side effect of chemotherapy and can substantially affect nutritional intake, daily functioning, and the quality of life. Approximately 20% to 40% of patients given cytotoxic agents for malignancies develop oral complications.1-4 These oral complications can result in increased infections, delays or interruptions in treatment, and dose reductions, and they can decrease disease remission and survival.1,2,5 The treatment costs can increase the economic burden on the patient, with costs increasing in proportion to the severity of the mucositis.1 The clinical interventions for oral mucositis are divided into 2 approaches: the prevention6-11 of oral mucositis or the treatment of established oral mucositis.12-16 According to the guidelines of the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/ International Society of Oral Oncology (MASCC/ISOO), several recommendations are provided for the management of established chemotherapy-related mucositis. The panel suggests that transdermal fentanyl may be effective in treating pain caused by oral mucositis in patients This work was supported by a Grant-in-Aid for Research, Nagoya City University, Nagoya, Japan. a Graduate Student, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. b Assistant Professor, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. c Division of Oral and Maxillofacial Surgery, Keiyukai Sapporo Hospital. d Associate Professor, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. e Professor, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. Received for publication Sep 2, 2013; returned for revision Nov 30, 2013; accepted for publication Dec 4, 2013. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2013.12.004
receiving conventional and high-dose chemotherapy with or without total body irradiation.12 Also, 0.5% doxepin mouthwash is suggested to be effective in treating pain from oral mucositis.13 Although not included in the guidelines, there are additional interventions for the management of established chemotherapy-related oral mucositis, such as polyvinylpyrrolidone-sodium hyaluronate gel (Gelclair),14 a supersaturated calcium phosphate mouthrinse,15 and antiseptic and antimicrobial mouthrinses.16 Many of these treatments are interventions to control pain, but there are few interventions to promote the healing of established oral mucositis directly. Inflammation is the first phase of wound healing and is marked by the formation of a blood clot containing red blood cells, macrophages, and a platelet plug.17 This blood clot provides a provisional extracellular matrix for cell migration. Fibrin glue has been developed to function as a provisional artificial extracellular matrix to stimulate healing, and it has been used to seal various types of wounds.18-20 In the oral cavity, fibrin sealant is applied to flap and graft procedures and promotes cellular migration and fibroblastic growth into the area of the fibrin seal application.20-23 The use of fibrin sealant remains controversial because of the complexity of the production protocols, the manufacturing
Statement of Clinical Relevance Oral mucositis is the most common adverse effect of chemotherapy and can hinder cancer care. Our studies suggest the healing effects of platelet-rich fibrin on oral mucositis in hamsters and support future clinical trials in patients. 445
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Fig. 1. The chemotherapy consisted of 2 intraperitoneal injections of 5-FU administered at 60 mg/kg on experimental days 0 and 2. The left cheek pouches were everted and irritated by superficial scratching on days 1 and 2. This light scratching was performed with 18-gauge needles within a 100-mm2 area. Treatment was performed from day 4 to the day when the samples were processed for hematoxylin-eosin staining or myeloperoxidase activity. (5-FU, 5-fluorouracil; ip, intraperitoneal; PRF, platelet-rich fibrin.)
costs, and the risks of cross-infections from the use of commercial products.18,24 As a natural fibrin, platelet-rich fibrin (PRF) is produced from biomaterials and contains several growth factors (GFs) and cytokines derived from autochthonous platelets and leukocytes. The natural concentrate is produced without anticoagulants or bovine thrombin.18,24-26 The PRF protocol, which was developed in France by Choukroun et al.,27 is simple and inexpensive. Recently, several reports have described applications of the PRF membrane for the improvement of soft tissue healing. In plastic surgery, PRF membranes applied to fresh postoperative hand wounds promote favorable tissue healing compared with the standard treatment in a randomized controlled clinical trial.25 The use of PRF membranes is highly efficient for root coverage28 and prevents peri-implant gingival recession.29 These reports were based on small samples and focused on normal gingival healing. To the best of our knowledge, there have not been any in vivo animal experiments evaluating the efficacy of PRF application in healing oral mucositis induced by a chemotherapy agent. This study aimed to evaluate whether PRF therapy can be extended as a curative approach in the treatment of oral chemotherapy-induced mucositis. To address this issue, the effect of locally applied PRF on chemotherapy-induced oral mucositis in a hamster model was studied by determining the body weight, ulcer area, histopathologic features, and myeloperoxidase (MPO) activity of the animals, which allowed for an assessment of their overall condition, healing, and inflammation.
MATERIALS AND METHODS Animals Male Syrian golden hamsters (Japan SLC, Nagoya, Japan), 7 weeks old and weighing 90 to 120 g, were
used in the experiments. All the animals were housed in a room maintained at 22 C 2 C under a 12-hour/ 12-hour light-dark cycle with the lights turned on at 8 AM. The hamsters were fed a standard rodent diet and given water ad libitum. This study was approved by the Institutional Animal Care and Use Committee of the Nagoya City University Graduate School of Medical Sciences, and the study was carried out in accordance with the guidelines for the center of experimental animal science. Oral mucositis model Hamsters were used in the present study based on the experimental mucositis model of Sonis et al.30 The chemotherapy involved 2 intraperitoneal injections of 5-fluorouracil (5-FU) (5-FU Injection 250 Kyowa; Kyowa Hakko Kirin Co Ltd, Tokyo, Japan) administered at 60 mg/kg on days 0 and 2 of the experiments (Figure 1). The 5-FU sterile solution was freshly prepared before each experiment. In combination with 5-FU to induce mucosal ulceration, the hamsters were anesthetized with pentobarbital sodium (Somnopentyl; Kyoritsu Seiyaku Co Ltd, Tokyo, Japan), and the left cheek pouches were everted and irritated by superficial scratching on days 1 and 2. This light scratching was performed with 18-gauge needles within a 100-mm2 area. Treatment groups Beginning on day 4 of the experiment, the hamsters with ulcerations induced by chemotherapy were randomly divided into 3 different treatment groups: the PRF, fibrin, and control groups. The hamsters were anesthetized with pentobarbital sodium before the treatments were applied. PRF group. PRF was prepared according to a previous protocol developed by Dohan et al.24 Blood collections were performed on 8 healthy volunteer men who were nonsmokers between the ages of 25 and 40.
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Fig. 2. The animals in each group were weighed daily, and the data were expressed as the change in body weight compared with the initial body weight at the beginning of the experiment (day 0 of the trial). The data are expressed as the mean the standard error of the mean. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents significant differences (n ¼ 7 in the control group; n ¼ 8 in the fibrin group; n ¼ 10 in the PRF group). (PRF, platelet-rich fibrin.)
The protocol was conducted in accordance with the principles of the Declaration of Helsinki with good clinical practice standards. The previously fabricated PRF membrane was tailored to the size of the cheek pouch ulceration using sterile tweezers and scissors. To keep the PRF membrane on the cheek pouch ulceration, we used the fibrin sealant Bolheal (Chemo-Sero Therapeutic Research Institute, Kumamoto, Japan). This product is composed of solutions A and B: solution A contains human fibrinogen (80 mg/mL), human clotting factor XIII (75 U/mL), and bovine aprotinin (1000 kallikrein inactivator units); solution B contains human thrombin (250 U/mL) and calcium hydrochloride (5.9 mg/mL). Fifteen milliliters each of solutions A and B were applied to the PRF membranes, which were then applied to the cheek pouch ulcerations. Fibrin group. Fifteen milliliters each of solutions A and B of the fibrin sealant were applied directly to the cheek pouch ulcerations. Control group. The cheek pouch lesions of the control group hamsters without any treatments were observed. Assessments Body weight change. To assess the animals’ conditions, 10 animals in each group were weighed daily (from days 0 to 14), and the data were expressed as the change in body weight relative to the initial body weight on day 0. Macroscopic analysis of the ulcer area. From days 3 to 14, 10 animals in each group were anesthetized with pentobarbital sodium, and standard photographs were taken of the everted cheek pouch mucosa. The ulcer circumference was traced on the photograph using the
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method reported by Mitsuhashi et al.,31 and the area was determined from the product of the major and minor ulcer axes. The data of the calculated ulcer area were expressed as the relative data, and each experimental day was compared with day 3. Histopathologic analysis. Four animals with oral mucositis in each group had a cheek pouch sample removed on days 6, 10, and 14 for histopathologic analysis. The specimens were fixed in 10% (by volume) neutral-buffer formalin, dehydrated, and embedded in paraffin. Sections that were 5 mm in thickness were obtained for hematoxylin-eosin staining and examined by light microscopy (40). The histologic score was evaluated using the method reported by Lima et al.32 The parameters of inflammatory cell infiltration, vascular dilatation, and the presence of hemorrhagic areas, edema, ulcerations, and abscesses were determined in a single-blind manner and graded with scores of 0 to 3. MPO activity assay. MPO activity, which is a marker for neutrophils in inflamed tissue, was measured in the hamster cheek pouches using the MPO assay kit (Myeloperoxidase Coloring Activity Assay Kit; BioVision Inc, Milpitas, CA, USA). On day 6 of the experiment, 8 animals with oral mucositis in each group and 3 normal hamsters were humanely killed, and their cheek pouches were removed as samples for MPO activity analysis. The specimens were stored at 85 C before analysis. The mucosal tissue was weighed and titrated using a dispersing machine (Polytron Ultra-Turrax) in an ice-cold buffer solution, and the homogenate was centrifuged at 4 C for 10 minutes (12 000 rpm). The supernatant was stored at 0 C for the MPO assay using tetramethylbenzidine and H2O2, and the MPO concentration was determined by measuring the absorbance change at 412 nm. All the values are reported as the level of MPO activity per gram of cheek pouch mucosa. Statistical analysis The data are presented as the mean the standard error of the mean or as the median where appropriate. A univariate analysis of variance, followed by the Dunnett test, was used to compare the means. A value of P < .05 was considered to indicate a significant difference.
RESULTS Ratio of weight changes The body weight showed a tendency to decrease within the first 3 days of the experiment in all 3 groups. From days 4 to 14, the rate of weight gain was higher in the PRF and fibrin groups (8.78% and 6.21%, respectively) than in the control group (0.79%). These differences were not significant (P > .05) (Figure 2). Three animals in the control group and 2 animals in the fibrin group died on days 6 to 10. These deaths were caused by an
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Fig. 3. Macroscopic aspects of the cheek pouches of hamsters injected with 5-fluorouracil. The control group (A-C), the fibrin group (D-F), and the PRF group (G-I) are shown. (PRF, platelet-rich fibrin.)
infection of the ulcer area and excessive weight loss that exceeded 14% of the body mass. Macroscopic study Macroscopically, the cheek pouches of the hamsters injected with 5-FU followed by mechanical trauma showed ulcerated lesions on day 3. The control group showed ulcerations with a red coloration and mild cicatrization on day 5 (Figure 3, A). By days 9 and 14, the ulcerations were slightly reduced, but the cicatrization was severe (Figure 3, B and C). The fibrin group showed ulcerations with a red coloration, but the cicatrization was less severe than in the control group on day 5 (Figure 3, D). The ulceration area of the fibrin group was reduced by 6.25% and 40.87% compared with the control group on days 9 and 14, with only slight cicatrization seen in the fibrin group on day 14 (Figure 3, F). The PRF group showed ulcerations equivalent to those of the fibrin group on day 5 (Figure 3, G). On day 9, the ulceration areas of the PRF group were reduced by 69.54% and 71.55% compared with those of the fibrin and control groups, respectively (Figure 3, H). On day 14, the ulceration areas of the
PRF group were reduced by 98.4% and 99.05% compared with the fibrin and control groups, respectively, with minimal cicatrization (Figure 3, I). The relative ulcer area was expressed by the base of the ulcer area on day 3 (Figure 4). In the control group, the relative ulcer area reached its maximum on day 4 (1.32 0.09), remained essentially the same on days 5 and 6 (1.30 0.07 and 1.32 0.13, respectively), and decreased by day 7 (1.23 0.18). The fibrin group showed a maximum relative ulcer area on day 5 (1.23 0.07), with the area tending to diminish on and after day 6 (1.14 0.1). The relative ulcer area in this group was significantly diminished on days 8, 9, and 11 compared with that of the control group (0.77 0.08, 0.60 0.05, and 0.35 0.05, respectively) (P < .05). The PRF group showed a maximum relative ulcer area on day 4 (1.08 0.03), which decreased significantly compared with the control group on day 5 (0.88 0.04) and at all assessment time points thereafter (P < .05). Histologic analyses and microscopic scores In the control group, ulceration, vessel dilatation, and bleeding accompanied by a marked infiltration of
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Fig. 4. The circumference of an ulcer was traced, and the area was determined from the product of the major and minor axes. The data for the calculated ulcer area were expressed as relative data, and each experimental day was compared with day 3. The data are expressed as the mean the standard error of the mean of the relative ulcer area. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents significant differences (n ¼ 7 in the control group; n ¼ 8 in the fibrin group; n ¼ 10 in the PRF group). (PRF, platelet-rich fibrin.)
inflammatory cells were noted on day 6 of the experiment (Figure 5, A). On day 10, ulceration accompanied by a marked inflammatory cell infiltration persisted, and fibroblasts were present (Figure 5, B). On day 14, the ulceration was reduced, and infiltrating inflammatory cells and granulation tissue formation accompanied by fibroblasts were observed (Figure 5, C). In the fibrin group, ulceration, vessel dilatation, and bleeding accompanied by a marked infiltration of inflammatory cells were noted on day 6 (Figure 5, D). On day 10, inflammatory cell infiltration persisted, and granulation tissues accompanied by fibroblasts were observed (Figure 5, E). On day 14, changes were noted compared with the control group (Figure 5, F). In the PRF group, ulceration, vessel dilatation, and bleeding accompanied by inflammatory cell infiltration were noted on day 6, and the epithelium of the ulcer circumference indicated invasion into the granulation tissue (Figure 5, G). A slight infiltration of inflammatory cells, fibroblasts, vascularization, and collagenization were noted on day 10 (Figure 5, H). On day 14, more changes were noted, with nearly healthy tissues accompanied by slight scarring (Figure 5, I). These histologic findings were quantitatively evaluated according to the grade of recovery by employing the histologic score of Lima et al.32 The score differences on days 6 and 10 among the 3 groups were not significant (P > .05) (Figure 6). The score of the PRF group (0.5 0.29) was significantly reduced compared with that of the control group (1.75 0.25) on day 14 (P < .05).
MPO activity assay The MPO activity values of normal healthy mucosa, the control group, the fibrin sealant group, and the PRF group on day 6 were 0.39 0.15, 3.14 0.42, 3.26 0.39, and 1.79 0.39 104 g1 of the cheek pouch, respectively (Figure 7). The MPO activity value of the PRF group was significantly reduced compared with that of the control group (P < .05).
DISCUSSION The present experiment in hamsters with 5-FU-induced oral mucositis found that the PRF group (in which PRF sealed the mucositis site) showed a greater improvement of mucositis in terms of all of the assessed parameters compared with the fibrin group (in which only fibrin sealant was used) and the untreated control group. Because the percent change in body weight is closely related to the nourishment status, it serves as a proxy for both the animal’s overall physical condition and the pain associated with oral mucosal ulceration. The body weight tended to be reduced in all 3 groups within the first 3 days of the experiment. This reduction most likely occurred because the ulcer pain and associated traumatic intervention led to a decrease in food intake. From days 4 to 14, the rate of weight gain was 8.78% and 6.21% in the PRF and fibrin groups, respectively, and the weight gain rate was improved compared with the rate of weight gain of 0.79% in the control group. Accordingly, an increased food intake likely occurred as the pain from the oral mucositis subsided with healing.
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Fig. 5. Microscopic aspects of the cheek pouches of hamsters injected with 5-fluorouracil. The control group (A-C), the fibrin group (D-F), and the PRF group (G-I) are shown (hematoxylin-eosin, original magnification 40). (PRF, platelet-rich fibrin.)
These results suggest that the GFs and anti-inflammatory cytokines in the PRF might produce beneficial painrelieving effects in addition to the effects from the mechanical covering of the ulcers. Chignon-Sicard et al.25 reported that the patients treated with PRF for a hand wound had greater pain relief than those treated with a conventional petroleum-based wound dressing in a randomized controlled clinical trial. In a randomized clinical trial reported by Jankovic et al.,28 the subjective discomfort was reduced in severity among patients who had undergone PRF grafting compared with those who received connective tissue transplantation in the treatment of gingival recession. All of the findings reported in the aforementioned studies are consistent with the results of our study. In the control group, the relative ulcer area reached a maximum on day 4, remained essentially at a plateau on days 5 and 6, and decreased on and after day 7. This course was very similar to that of the oral mucositis usually observed as an adverse reaction to cancer chemotherapy in humans (i.e., in the clinical setting).1,3,30 The fibrin group showed a maximum relative ulcer area on day 5, with the area diminishing on and after day 6. The relative ulcer area in this group was significantly
diminished compared with the control group on days 8, 9, and 11, indicating the healing-promoting effects of fibrin (P < .05). Fibrin is believed to serve as a scaffolding for the healing of ulcers through the induction of fibroblasts and vascular endothelial cells by the adsorption of GFs on the wound, especially fibroblast growth factor basic and platelet-derived growth factor.26 Yücel et al.33 found that fibrin sealant was more useful for healing oral wounds than conventional suture treatment in their work with rats. In our study, the PRF group showed a maximum relative ulcer area on day 4, which was followed by a reduction in size that was significantly greater than that of the control group by day 5 and at all assessment times thereafter (P < .05). This indicates the marked healing-promoting effect of PRF. The reason for the more pronounced healing in the PRF group compared with the other groups is that PRF forms a characteristic 3-dimensional structure in the process of fibrin polymerization and contains high concentrations of platelets and leukocytes; PRF is likely to also contain GFs and cytokines in this structure.18,24,34 These GFs and cytokines promote ulcer healing, which most likely had a substantial effect on the experiments described here.35-37 It is also probable
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Fig. 6. The bar chart shows the mean of the microscopic score. The number of animals in each group was at least 4. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents significant differences. Score 0, normal epithelium and connective tissue without vasodilation; no or minimal cellular infiltration; no hemorrhagic areas, ulcerations, or abscesses. Score 1, discreet vascular dilatation with reepithelialization areas: discreet inflammatory infiltration with mononuclear prevalence; absence of hemorrhagic areas, edema, ulceration, or abscesses. Score 2, moderate vascular dilatation with areas of hydropic epithelial degeneration, inflammatory infiltration with neutrophil prevalence, and hemorrhagic areas with edema, eventual ulcerations, and the absence of abscesses. Score 3, severe vascular dilatation, inflammatory infiltration with neutrophil prevalence, hemorrhagic areas, edema and extensive ulcerations, and abscesses. (PRF, platelet-rich fibrin.)
Fig. 7. The MPO activity values on day 6 for the normal healthy mucosa group (n ¼ 3), the control group (n ¼ 8), the fibrin group (n ¼ 8), and the PRF group (n ¼ 8). The bars represent the mean the standard error of the mean of the concentration of MPO 104 g1 of the cheek pouch. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents a significant difference. (MPO, myeloperoxidase; PRF, platelet-rich fibrin.)
that the absorption of GFs from the wound surface occurred with both the fibrin sealant and natural fibrin. Given the known effects of GFs, one speculation is that the proliferation of fibroblasts and vascular endothelial cells was activated, triggering GF secretions such as keratinocyte growth factor and insulin-like growth factor. Such secretions could, in turn, stimulate epithelial cell growth with a consequent progression to epithelial closure (Figures 3 and 4). The 3-dimensional structure of fibrin undergoes a gradual degradation and
is ultimately absorbed into tissues. The consequence of this slow degradation is that GFs trapped into the reticular fibrin structure undergo a gradual and sustained release from the reticular fibrin structure over approximately 8 days.38 The GFs and cytokines taken up by the network inhibit the destruction of these factors by proteases, contributing to a sustained GF release.39-41 This sustained GF release may have the advantageous effect of promoting healing during 2 or more concurrent phases of mucositis. In another in vitro experiment on the cellular growth activity of PRF versus fibrin sealants, cultures of fibroblasts with medium containing fibrin showed a slight increase in cellular growth activity, although the difference from the control cultures did not reach statistical significance. The cultures with medium containing PRF showed significantly increased cellular growth activity compared with the control cultures.40 This finding is consistent with the results of the present study. The maximum relative ulcer area was 1.08 0.03 for the PRF group compared with 1.32 0.09 for the control group and 1.23 0.07 for the fibrin group. PRF was more effective than fibrin in halting ulcer exacerbation. The effects of anti-inflammatory cytokines, such as transforming growth factor b (TGF-b) and interleukin 4 (IL-4), that are taken into the 3-dimensional fibrin structure in PRF may account for this finding.42 We infer that these cytokines suppress the production of tumor necrosis factor a, IL-1b, and IL-6, which are factors that aggravate chemotherapy-induced mucositis,
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and this suppression inhibits the progression of tissue destruction.43,44 Consistent with this inference, Clarke et al.45 found that TGF-b in a whey growth factor extract prevents chemotherapy-associated oral mucositis in a similar hamster mucositis model. In the fibrin group, it was possible that factors (e.g., TGF-b) released from macrophages in the wound were adsorbed. The same event may have occurred when fibrin was contained in the PRF, such that a greater suppression of ulcer aggravation was obtained in the PRF group. The results were histologically evaluated on days 6, 10, and 14. Intact healthy cheek pouch tissues presented as a thin layer of smooth keratinized epithelium with a muscle bundle layer. The submucosa was primarily fibrous, with scant cells and few blood vessels.46 There was evidence of an earlier invasion of epithelial cells around the ulcer into the granulation tissue in the PRF group compared with the other 2 groups. Excessive inflammatory cell infiltration was inhibited at an earlier stage in the PRF group (Figure 5). A possible explanation for this is that the various GFs contained in the PRF may have affected the growth of the epithelial cells, and anti-inflammatory cytokines, such as TGF-b and IL-4, were taken up by these cells. These histologic findings were evaluated using the histopathologic scoring criteria,32 and no significant intergroup difference was observed on day 6 of the experiment. On day 14 of the experiment, the PRF group exhibited a significant score decrease compared with the control group (P < .05) (Figure 6). These histologic findings indicated accelerated ulcer healing and the suppression of excessive inflammation in the PRF group. The PRF treatment had a noticeable influence on the degree of epithelialization and inflammatory cell infiltration, indicating that PRF had a beneficial effect. An assessment of the degree of inflammatory cell infiltration based on the MPO activity assay found an approximately 43% reduction in MPO activity on day 6 in the PRF group compared with the control and fibrin groups. The inhibition of inflammatory cell infiltration, especially neutrophil infiltration, was evident in the PRF group. The degree of inflammatory cell infiltration seemed to be related to the degree of wound pain and tended to coincide with the weight change data. This study presented data suggesting that the healing of 5-FU-induced oral mucositis can be promoted by the use of topically applied PRF. In contrast to many existing management strategies of established oral mucositis that are interventions to control pain, the use of topically applied PRF may be able to help promote the healing of established oral mucositis. Our current findings support future clinical trials in patients. Certain aspects of the mechanism by which PRF promotes the healing of oral mucositis have not yet been clarified. Further investigation will be required to resolve these
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issues. Additional studies aimed at improving the method of PRF application onto lesions and further developing the practical clinical utilization of this system are necessary. REFERENCES 1. Sonis ST. Oral mucositis in cancer therapy. J Support Oncol. 2004;2:3-8. 2. Loury DJ, Embree JR, Steinberg DA, Sonis ST, Fiddes JC. Effect of local application of the antimicrobial peptide IB-367 on the incidence and severity of oral mucositis in hamsters. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1999;87:544-551. 3. Sonis ST. Mucositis as a biological process: a new hypothesis for the development of chemotherapy-induced stomatotoxicity. Oral Oncol. 1998;34:39-43. 4. Sonis ST, Elting LS, Keefe D, et al. Perspectives on cancer therapy-induced mucosal injury. Cancer. 2004;100(9 suppl): 1995-2025. 5. Sonis ST. Oral mucositis. Anti-Cancer Drugs. 2011;22:607-612. 6. Peterson DE, Öhrrn K, Bowen J, et al. Systematic review of oral cryotherapy for management of oral mucositis caused by cancer therapy. Support Care Cancer. 2013;21:327-332. 7. Raber-Durlacher JE, Logan RM, Bowen J, et al. Systematic review of cytokines and growth factors for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:343-355. 8. Migliorati C, Hewson I, Lalla RV, et al. Systematic review of laser and other light therapy for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:333-341. 9. Nicolatou-Galitis O, Sarri T, Bowen J, et al. Systematic review of anti-inflammatory agents for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:3179-3189. 10. Qutob AF, Allen G, Gue S, Revesz T, Logan RM, Keefe D. Implementation of a hospital oral care protocol and recording of oral mucositis in children receiving cancer treatment: a retrospective and a prospective study. Support Care Cancer. 2013;21: 1113-1120. 11. Cheng KKF, Molassiotis A, Chang AM, Wai WC, Cheung SS. Evaluation of an oral care protocol intervention in the prevention of chemotherapy-induced oral mucositis in paediatric cancer patients. Eur J Cancer. 2001;37:2056-2063. 12. Saunders DP, Epstein JB, Elad S, et al. Systematic review of antimicrobials, mucosal coating agents, anesthetics, and analgesics for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:3191-3207. 13. Epstein JB, Epstein JD, Epstein MS, Olien H, Truelove EL. Doxepin rinse for management of mucositis pain in patients with cancer: one week follow-up of topical therapy. Spec Care Dentist. 2008;28:73-77. 14. Vokurka S, Skardova J, Hruskova R, et al. The effect of polyvinylpyrrolidone-sodium hyaluronate gel (Gelclair) on oral microbial colonization and pain control compared with other rinsing solutions in patients with oral mucositis after allogeneic stem cells transplantation. Med Sci Monit. 2011;17:572-576. 15. Markiewicz M, Dzierzak-Mietla M, Frankiewicz A, et al. Treating oral mucositis with a supersaturated calcium phosphate rinse: comparison with control in patients undergoing allogeneic hematopoietic stem cell transplantation. Support Care Cancer. 2012;20:2223-2229. 16. McGuire DB, Fulton JS, Park J, et al. Systematic review of basic oral care for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:3165-3177. 17. Singer AJ, Clark RAF. Cutaneous wound healing. N Engl J Med. 1999;341:738-746.
OOOO Volume 117, Number 4 18. Dohan D, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends in Biotechnology. 2009;27:158-166. 19. Laurens N, Koolwijk P, Maat MPM. Fibrin structure and wound healing. J Thromb Haemost. 2006;4:932-939. 20. Kin H, Nakajima T, Okabayashi H. Experimental study on effective application of fibrin glue. Gen Thorac Cardiovasc Surg. 2012;60:140-144. 21. Greenhalgh DG, Sprugel KH, Murray MJ, Ross R. PDGF and FGF stimulate wound healing in the genetically diabetic mouse. Am J Pathol. 1990;136:1235-1246. 22. Matras H. Fibrin seal: the state of the art. J Oral Maxillofac Surg. 1985;43:605-611. 23. Gibble JW, Ness PM. Fibrin glue: the perfect operative sealant? Transfusion. 1990;30:741-747. 24. Dohan D, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: Technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:37-44. 25. Chignon-Sicard B, Georgiou CA, Fontas E, et al. Efficacy of leukocyte- and platelet-rich fibrin in wound healing: a randomized controlled clinical trial. Plast Reconstr Surg. 2012;130: 819-829. 26. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: Clinical effect on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:56-60. 27. Choukroun J, Adda F, Schoeffler C, Vervelle A. Une opportunité en paro-implantologie le PRF: (Platelet Rich Fibrin). Implantodontie. 2001;42:55-62. 28. Jankovic S, Aleksic Z, Klokkevold P, et al. Use of platelet-rich fibrin membrane following treatment of gingival recession: a randomized clinical trial. Int J Periodont Restor Dent. 2012;32: 41-50. 29. Saadoun AP, Touati B. Soft tissue recession around implants: is it still unavoidable? Part II. Pract Proced Aesthet Dent. 2007;19: 81-87. 30. Sonis ST, Tracey C, Shklar G, Jenson J, Florine D. An animal model for mucositis induced by cancer chemotherapy. Oral Surg Oral Med Oral Pathol. 1990;69:437-443. 31. Mitsuhashi H, Suemaru K, Li B, Cui R, Akira H. Evaluation of topical external medicine for 5-fluorouracil-induced oral mucositis in hamsters. Eur J Pharmacol. 2006;551:152-155. 32. Lima V, Brito GAC, Cunhan FQ, et al. Effect of the tumour necrosis factor-a inhibitor pentoxifylline and thalidomide in short-term experimental oral mucositis in hamsters. Eur J Oral Sci. 2005;113:210-217. 33. Yücel EA, Oral O, Olgaç V, Oral CK. Effects of fibrin glue on wound healing in oral cavity. J Dent. 2003;31:569-575. 34. Dohan D, Corso MD, Diss A, Mouhyi J, Charrier JB. Threedimensional architecture and cell composition of a Choukroun’s platelet-rich fibrin clot and membrane. J Periodontol. 2010;81: 546-555. 35. Sommeling CE, Heyneman A, Hoeksema H, Verbelen J, Stillaert FB, Monstrey S. The use of platelet-rich plasma in plastic
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surgery: a systematic review. J Plast Reconstr Aesthet Surg. 2012;66:301-311. Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg. 2006;118:147-159. Anitua E, Aguirre JJ, Algorta J, et al. Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers. J Biomed Mater Res B Appl Biomater. 2008;84B:415-421. Dohan D, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: Platelet-related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:45-50. Lundquist R, Holmstrøm K, Clausen C, Jørgensen B, Karlsmark T. Characteristics of an autologous leukocyte and platelet-rich fibrin patch intended for the treatment of recalcitrant wounds. Wound Repair Regen. 2013;21:66-76. Lundquist R, Dziegiel MH, Ågren MS. Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin. Wound Repair Regen. 2008;16:356-363. Pradeep AR, Rao NS, Agarwal E, Bajaj P, Kumari M, Naik SB. Comparative evaluation of autologous platelet-rich fibrin and platelet-rich plasma in the treatment of three-wall intrabony defects in chronic periodontitis: a randomized controlled clinical trial. J Periodontol. 2012;83:1499-1507. Dohan D, Choukroun J, Dohan SL, Dohan AJJ, Mouhyi J, Gogly B. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part III: Leucocyte activation: a new feature for platelet concentrates? Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:51-55. Suemaru K, Cui R, Watanabe S, Okihara K, Hashimoto K, Yamada H, Araki H. Topical application of royal jelly has a healing effect on 5-fluorouracil-induced experimental oral mucositis in hamster. Methods Find Exp Clin Pharmacol. 2008;30:103-106. von Bültzingslöwen I, Brennan MT, Spijkervet FKL, Logan R, Stringer A, Keefe D. Growth factors and cytokines in the prevention and treatment of oral and gastrointestinal mucositis. Support Care Cancer. 2006;14:519-527. Clarke J, Butler R, Howarth G, Read L, Regester G. Exposure of oral mucosa to bioactive milk factors reduces severity of chemotherapy-induced mucositis in the hamster. Oral Oncol. 2002;38:478-485. Morvan FO, Baroukh B, Ledoux D, Caruelle J-P, Barritault D, Saffar J-L. An engineered biopolymer prevents mucositis induced by 5-fluorouracil in hamsters. Am J Pathol. 2004;164:739-746.
Reprint requests: Koichiro Horii Department of Oral and Maxillofacial Surgery Nagoya City University 1-Kawasumi Mizuho-ku Nagoya 467-8601 Japan [email protected]
Platelet-rich fibrin has a healing effect on chemotherapy-induced mucositis in hamsters Koichiro Horii, DDS,a Takeo Kanayama, DDS, PhD,b Hironori Miyamoto, DDS,a Tomoyuki Kohgo, DDS, PhD,c Tsukasa Tsuchimochi, DDS, PhD,b Toshio Shigetomi, DDS, PhD,d and Motoo Yokoi, DDS, PhDe Nagoya City University, Nagoya, Japan; Keiyukai Sapporo Hospital, Sapporo, Japan
Objective. The aim of this study was to evaluate the healing effect of topically applied platelet-rich fibrin (PRF) on experimental oral mucositis induced by chemotherapy in hamsters. Study Design. Oral mucositis was induced in 93 Syrian golden hamsters by an intraperitoneal injection of 5-fluorouracil, which was followed by light scratching of the cheek pouch. The hamsters were randomly divided into a PRF group, a fibrin group, and an untreated control group. The recovery stage of oral mucositis was evaluated through daily weighing, measurements of the ulcer area, histopathologic analysis, and a myeloperoxidase activity assay. Results. The PRF group exhibited significant improvements in the size and histologic features of the ulcer and in the myeloperoxidase activity compared with the control group (P < .05). Conclusions. The current findings suggest the consideration for future clinical trials in humans. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;117:445-453)
Oral mucositis is a painful and often debilitating side effect of chemotherapy and can substantially affect nutritional intake, daily functioning, and the quality of life. Approximately 20% to 40% of patients given cytotoxic agents for malignancies develop oral complications.1-4 These oral complications can result in increased infections, delays or interruptions in treatment, and dose reductions, and they can decrease disease remission and survival.1,2,5 The treatment costs can increase the economic burden on the patient, with costs increasing in proportion to the severity of the mucositis.1 The clinical interventions for oral mucositis are divided into 2 approaches: the prevention6-11 of oral mucositis or the treatment of established oral mucositis.12-16 According to the guidelines of the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/ International Society of Oral Oncology (MASCC/ISOO), several recommendations are provided for the management of established chemotherapy-related mucositis. The panel suggests that transdermal fentanyl may be effective in treating pain caused by oral mucositis in patients This work was supported by a Grant-in-Aid for Research, Nagoya City University, Nagoya, Japan. a Graduate Student, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. b Assistant Professor, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. c Division of Oral and Maxillofacial Surgery, Keiyukai Sapporo Hospital. d Associate Professor, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. e Professor, Department of Oral and Maxillofacial Surgery, Nagoya City University Graduate School of Medical Sciences. Received for publication Sep 2, 2013; returned for revision Nov 30, 2013; accepted for publication Dec 4, 2013. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2013.12.004
receiving conventional and high-dose chemotherapy with or without total body irradiation.12 Also, 0.5% doxepin mouthwash is suggested to be effective in treating pain from oral mucositis.13 Although not included in the guidelines, there are additional interventions for the management of established chemotherapy-related oral mucositis, such as polyvinylpyrrolidone-sodium hyaluronate gel (Gelclair),14 a supersaturated calcium phosphate mouthrinse,15 and antiseptic and antimicrobial mouthrinses.16 Many of these treatments are interventions to control pain, but there are few interventions to promote the healing of established oral mucositis directly. Inflammation is the first phase of wound healing and is marked by the formation of a blood clot containing red blood cells, macrophages, and a platelet plug.17 This blood clot provides a provisional extracellular matrix for cell migration. Fibrin glue has been developed to function as a provisional artificial extracellular matrix to stimulate healing, and it has been used to seal various types of wounds.18-20 In the oral cavity, fibrin sealant is applied to flap and graft procedures and promotes cellular migration and fibroblastic growth into the area of the fibrin seal application.20-23 The use of fibrin sealant remains controversial because of the complexity of the production protocols, the manufacturing
Statement of Clinical Relevance Oral mucositis is the most common adverse effect of chemotherapy and can hinder cancer care. Our studies suggest the healing effects of platelet-rich fibrin on oral mucositis in hamsters and support future clinical trials in patients. 445
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Fig. 1. The chemotherapy consisted of 2 intraperitoneal injections of 5-FU administered at 60 mg/kg on experimental days 0 and 2. The left cheek pouches were everted and irritated by superficial scratching on days 1 and 2. This light scratching was performed with 18-gauge needles within a 100-mm2 area. Treatment was performed from day 4 to the day when the samples were processed for hematoxylin-eosin staining or myeloperoxidase activity. (5-FU, 5-fluorouracil; ip, intraperitoneal; PRF, platelet-rich fibrin.)
costs, and the risks of cross-infections from the use of commercial products.18,24 As a natural fibrin, platelet-rich fibrin (PRF) is produced from biomaterials and contains several growth factors (GFs) and cytokines derived from autochthonous platelets and leukocytes. The natural concentrate is produced without anticoagulants or bovine thrombin.18,24-26 The PRF protocol, which was developed in France by Choukroun et al.,27 is simple and inexpensive. Recently, several reports have described applications of the PRF membrane for the improvement of soft tissue healing. In plastic surgery, PRF membranes applied to fresh postoperative hand wounds promote favorable tissue healing compared with the standard treatment in a randomized controlled clinical trial.25 The use of PRF membranes is highly efficient for root coverage28 and prevents peri-implant gingival recession.29 These reports were based on small samples and focused on normal gingival healing. To the best of our knowledge, there have not been any in vivo animal experiments evaluating the efficacy of PRF application in healing oral mucositis induced by a chemotherapy agent. This study aimed to evaluate whether PRF therapy can be extended as a curative approach in the treatment of oral chemotherapy-induced mucositis. To address this issue, the effect of locally applied PRF on chemotherapy-induced oral mucositis in a hamster model was studied by determining the body weight, ulcer area, histopathologic features, and myeloperoxidase (MPO) activity of the animals, which allowed for an assessment of their overall condition, healing, and inflammation.
MATERIALS AND METHODS Animals Male Syrian golden hamsters (Japan SLC, Nagoya, Japan), 7 weeks old and weighing 90 to 120 g, were
used in the experiments. All the animals were housed in a room maintained at 22 C 2 C under a 12-hour/ 12-hour light-dark cycle with the lights turned on at 8 AM. The hamsters were fed a standard rodent diet and given water ad libitum. This study was approved by the Institutional Animal Care and Use Committee of the Nagoya City University Graduate School of Medical Sciences, and the study was carried out in accordance with the guidelines for the center of experimental animal science. Oral mucositis model Hamsters were used in the present study based on the experimental mucositis model of Sonis et al.30 The chemotherapy involved 2 intraperitoneal injections of 5-fluorouracil (5-FU) (5-FU Injection 250 Kyowa; Kyowa Hakko Kirin Co Ltd, Tokyo, Japan) administered at 60 mg/kg on days 0 and 2 of the experiments (Figure 1). The 5-FU sterile solution was freshly prepared before each experiment. In combination with 5-FU to induce mucosal ulceration, the hamsters were anesthetized with pentobarbital sodium (Somnopentyl; Kyoritsu Seiyaku Co Ltd, Tokyo, Japan), and the left cheek pouches were everted and irritated by superficial scratching on days 1 and 2. This light scratching was performed with 18-gauge needles within a 100-mm2 area. Treatment groups Beginning on day 4 of the experiment, the hamsters with ulcerations induced by chemotherapy were randomly divided into 3 different treatment groups: the PRF, fibrin, and control groups. The hamsters were anesthetized with pentobarbital sodium before the treatments were applied. PRF group. PRF was prepared according to a previous protocol developed by Dohan et al.24 Blood collections were performed on 8 healthy volunteer men who were nonsmokers between the ages of 25 and 40.
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Fig. 2. The animals in each group were weighed daily, and the data were expressed as the change in body weight compared with the initial body weight at the beginning of the experiment (day 0 of the trial). The data are expressed as the mean the standard error of the mean. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents significant differences (n ¼ 7 in the control group; n ¼ 8 in the fibrin group; n ¼ 10 in the PRF group). (PRF, platelet-rich fibrin.)
The protocol was conducted in accordance with the principles of the Declaration of Helsinki with good clinical practice standards. The previously fabricated PRF membrane was tailored to the size of the cheek pouch ulceration using sterile tweezers and scissors. To keep the PRF membrane on the cheek pouch ulceration, we used the fibrin sealant Bolheal (Chemo-Sero Therapeutic Research Institute, Kumamoto, Japan). This product is composed of solutions A and B: solution A contains human fibrinogen (80 mg/mL), human clotting factor XIII (75 U/mL), and bovine aprotinin (1000 kallikrein inactivator units); solution B contains human thrombin (250 U/mL) and calcium hydrochloride (5.9 mg/mL). Fifteen milliliters each of solutions A and B were applied to the PRF membranes, which were then applied to the cheek pouch ulcerations. Fibrin group. Fifteen milliliters each of solutions A and B of the fibrin sealant were applied directly to the cheek pouch ulcerations. Control group. The cheek pouch lesions of the control group hamsters without any treatments were observed. Assessments Body weight change. To assess the animals’ conditions, 10 animals in each group were weighed daily (from days 0 to 14), and the data were expressed as the change in body weight relative to the initial body weight on day 0. Macroscopic analysis of the ulcer area. From days 3 to 14, 10 animals in each group were anesthetized with pentobarbital sodium, and standard photographs were taken of the everted cheek pouch mucosa. The ulcer circumference was traced on the photograph using the
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method reported by Mitsuhashi et al.,31 and the area was determined from the product of the major and minor ulcer axes. The data of the calculated ulcer area were expressed as the relative data, and each experimental day was compared with day 3. Histopathologic analysis. Four animals with oral mucositis in each group had a cheek pouch sample removed on days 6, 10, and 14 for histopathologic analysis. The specimens were fixed in 10% (by volume) neutral-buffer formalin, dehydrated, and embedded in paraffin. Sections that were 5 mm in thickness were obtained for hematoxylin-eosin staining and examined by light microscopy (40). The histologic score was evaluated using the method reported by Lima et al.32 The parameters of inflammatory cell infiltration, vascular dilatation, and the presence of hemorrhagic areas, edema, ulcerations, and abscesses were determined in a single-blind manner and graded with scores of 0 to 3. MPO activity assay. MPO activity, which is a marker for neutrophils in inflamed tissue, was measured in the hamster cheek pouches using the MPO assay kit (Myeloperoxidase Coloring Activity Assay Kit; BioVision Inc, Milpitas, CA, USA). On day 6 of the experiment, 8 animals with oral mucositis in each group and 3 normal hamsters were humanely killed, and their cheek pouches were removed as samples for MPO activity analysis. The specimens were stored at 85 C before analysis. The mucosal tissue was weighed and titrated using a dispersing machine (Polytron Ultra-Turrax) in an ice-cold buffer solution, and the homogenate was centrifuged at 4 C for 10 minutes (12 000 rpm). The supernatant was stored at 0 C for the MPO assay using tetramethylbenzidine and H2O2, and the MPO concentration was determined by measuring the absorbance change at 412 nm. All the values are reported as the level of MPO activity per gram of cheek pouch mucosa. Statistical analysis The data are presented as the mean the standard error of the mean or as the median where appropriate. A univariate analysis of variance, followed by the Dunnett test, was used to compare the means. A value of P < .05 was considered to indicate a significant difference.
RESULTS Ratio of weight changes The body weight showed a tendency to decrease within the first 3 days of the experiment in all 3 groups. From days 4 to 14, the rate of weight gain was higher in the PRF and fibrin groups (8.78% and 6.21%, respectively) than in the control group (0.79%). These differences were not significant (P > .05) (Figure 2). Three animals in the control group and 2 animals in the fibrin group died on days 6 to 10. These deaths were caused by an
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Fig. 3. Macroscopic aspects of the cheek pouches of hamsters injected with 5-fluorouracil. The control group (A-C), the fibrin group (D-F), and the PRF group (G-I) are shown. (PRF, platelet-rich fibrin.)
infection of the ulcer area and excessive weight loss that exceeded 14% of the body mass. Macroscopic study Macroscopically, the cheek pouches of the hamsters injected with 5-FU followed by mechanical trauma showed ulcerated lesions on day 3. The control group showed ulcerations with a red coloration and mild cicatrization on day 5 (Figure 3, A). By days 9 and 14, the ulcerations were slightly reduced, but the cicatrization was severe (Figure 3, B and C). The fibrin group showed ulcerations with a red coloration, but the cicatrization was less severe than in the control group on day 5 (Figure 3, D). The ulceration area of the fibrin group was reduced by 6.25% and 40.87% compared with the control group on days 9 and 14, with only slight cicatrization seen in the fibrin group on day 14 (Figure 3, F). The PRF group showed ulcerations equivalent to those of the fibrin group on day 5 (Figure 3, G). On day 9, the ulceration areas of the PRF group were reduced by 69.54% and 71.55% compared with those of the fibrin and control groups, respectively (Figure 3, H). On day 14, the ulceration areas of the
PRF group were reduced by 98.4% and 99.05% compared with the fibrin and control groups, respectively, with minimal cicatrization (Figure 3, I). The relative ulcer area was expressed by the base of the ulcer area on day 3 (Figure 4). In the control group, the relative ulcer area reached its maximum on day 4 (1.32 0.09), remained essentially the same on days 5 and 6 (1.30 0.07 and 1.32 0.13, respectively), and decreased by day 7 (1.23 0.18). The fibrin group showed a maximum relative ulcer area on day 5 (1.23 0.07), with the area tending to diminish on and after day 6 (1.14 0.1). The relative ulcer area in this group was significantly diminished on days 8, 9, and 11 compared with that of the control group (0.77 0.08, 0.60 0.05, and 0.35 0.05, respectively) (P < .05). The PRF group showed a maximum relative ulcer area on day 4 (1.08 0.03), which decreased significantly compared with the control group on day 5 (0.88 0.04) and at all assessment time points thereafter (P < .05). Histologic analyses and microscopic scores In the control group, ulceration, vessel dilatation, and bleeding accompanied by a marked infiltration of
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Fig. 4. The circumference of an ulcer was traced, and the area was determined from the product of the major and minor axes. The data for the calculated ulcer area were expressed as relative data, and each experimental day was compared with day 3. The data are expressed as the mean the standard error of the mean of the relative ulcer area. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents significant differences (n ¼ 7 in the control group; n ¼ 8 in the fibrin group; n ¼ 10 in the PRF group). (PRF, platelet-rich fibrin.)
inflammatory cells were noted on day 6 of the experiment (Figure 5, A). On day 10, ulceration accompanied by a marked inflammatory cell infiltration persisted, and fibroblasts were present (Figure 5, B). On day 14, the ulceration was reduced, and infiltrating inflammatory cells and granulation tissue formation accompanied by fibroblasts were observed (Figure 5, C). In the fibrin group, ulceration, vessel dilatation, and bleeding accompanied by a marked infiltration of inflammatory cells were noted on day 6 (Figure 5, D). On day 10, inflammatory cell infiltration persisted, and granulation tissues accompanied by fibroblasts were observed (Figure 5, E). On day 14, changes were noted compared with the control group (Figure 5, F). In the PRF group, ulceration, vessel dilatation, and bleeding accompanied by inflammatory cell infiltration were noted on day 6, and the epithelium of the ulcer circumference indicated invasion into the granulation tissue (Figure 5, G). A slight infiltration of inflammatory cells, fibroblasts, vascularization, and collagenization were noted on day 10 (Figure 5, H). On day 14, more changes were noted, with nearly healthy tissues accompanied by slight scarring (Figure 5, I). These histologic findings were quantitatively evaluated according to the grade of recovery by employing the histologic score of Lima et al.32 The score differences on days 6 and 10 among the 3 groups were not significant (P > .05) (Figure 6). The score of the PRF group (0.5 0.29) was significantly reduced compared with that of the control group (1.75 0.25) on day 14 (P < .05).
MPO activity assay The MPO activity values of normal healthy mucosa, the control group, the fibrin sealant group, and the PRF group on day 6 were 0.39 0.15, 3.14 0.42, 3.26 0.39, and 1.79 0.39 104 g1 of the cheek pouch, respectively (Figure 7). The MPO activity value of the PRF group was significantly reduced compared with that of the control group (P < .05).
DISCUSSION The present experiment in hamsters with 5-FU-induced oral mucositis found that the PRF group (in which PRF sealed the mucositis site) showed a greater improvement of mucositis in terms of all of the assessed parameters compared with the fibrin group (in which only fibrin sealant was used) and the untreated control group. Because the percent change in body weight is closely related to the nourishment status, it serves as a proxy for both the animal’s overall physical condition and the pain associated with oral mucosal ulceration. The body weight tended to be reduced in all 3 groups within the first 3 days of the experiment. This reduction most likely occurred because the ulcer pain and associated traumatic intervention led to a decrease in food intake. From days 4 to 14, the rate of weight gain was 8.78% and 6.21% in the PRF and fibrin groups, respectively, and the weight gain rate was improved compared with the rate of weight gain of 0.79% in the control group. Accordingly, an increased food intake likely occurred as the pain from the oral mucositis subsided with healing.
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Fig. 5. Microscopic aspects of the cheek pouches of hamsters injected with 5-fluorouracil. The control group (A-C), the fibrin group (D-F), and the PRF group (G-I) are shown (hematoxylin-eosin, original magnification 40). (PRF, platelet-rich fibrin.)
These results suggest that the GFs and anti-inflammatory cytokines in the PRF might produce beneficial painrelieving effects in addition to the effects from the mechanical covering of the ulcers. Chignon-Sicard et al.25 reported that the patients treated with PRF for a hand wound had greater pain relief than those treated with a conventional petroleum-based wound dressing in a randomized controlled clinical trial. In a randomized clinical trial reported by Jankovic et al.,28 the subjective discomfort was reduced in severity among patients who had undergone PRF grafting compared with those who received connective tissue transplantation in the treatment of gingival recession. All of the findings reported in the aforementioned studies are consistent with the results of our study. In the control group, the relative ulcer area reached a maximum on day 4, remained essentially at a plateau on days 5 and 6, and decreased on and after day 7. This course was very similar to that of the oral mucositis usually observed as an adverse reaction to cancer chemotherapy in humans (i.e., in the clinical setting).1,3,30 The fibrin group showed a maximum relative ulcer area on day 5, with the area diminishing on and after day 6. The relative ulcer area in this group was significantly
diminished compared with the control group on days 8, 9, and 11, indicating the healing-promoting effects of fibrin (P < .05). Fibrin is believed to serve as a scaffolding for the healing of ulcers through the induction of fibroblasts and vascular endothelial cells by the adsorption of GFs on the wound, especially fibroblast growth factor basic and platelet-derived growth factor.26 Yücel et al.33 found that fibrin sealant was more useful for healing oral wounds than conventional suture treatment in their work with rats. In our study, the PRF group showed a maximum relative ulcer area on day 4, which was followed by a reduction in size that was significantly greater than that of the control group by day 5 and at all assessment times thereafter (P < .05). This indicates the marked healing-promoting effect of PRF. The reason for the more pronounced healing in the PRF group compared with the other groups is that PRF forms a characteristic 3-dimensional structure in the process of fibrin polymerization and contains high concentrations of platelets and leukocytes; PRF is likely to also contain GFs and cytokines in this structure.18,24,34 These GFs and cytokines promote ulcer healing, which most likely had a substantial effect on the experiments described here.35-37 It is also probable
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Fig. 6. The bar chart shows the mean of the microscopic score. The number of animals in each group was at least 4. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents significant differences. Score 0, normal epithelium and connective tissue without vasodilation; no or minimal cellular infiltration; no hemorrhagic areas, ulcerations, or abscesses. Score 1, discreet vascular dilatation with reepithelialization areas: discreet inflammatory infiltration with mononuclear prevalence; absence of hemorrhagic areas, edema, ulceration, or abscesses. Score 2, moderate vascular dilatation with areas of hydropic epithelial degeneration, inflammatory infiltration with neutrophil prevalence, and hemorrhagic areas with edema, eventual ulcerations, and the absence of abscesses. Score 3, severe vascular dilatation, inflammatory infiltration with neutrophil prevalence, hemorrhagic areas, edema and extensive ulcerations, and abscesses. (PRF, platelet-rich fibrin.)
Fig. 7. The MPO activity values on day 6 for the normal healthy mucosa group (n ¼ 3), the control group (n ¼ 8), the fibrin group (n ¼ 8), and the PRF group (n ¼ 8). The bars represent the mean the standard error of the mean of the concentration of MPO 104 g1 of the cheek pouch. The data were analyzed using an analysis of variance and the Dunnett test. *P < .05 represents a significant difference. (MPO, myeloperoxidase; PRF, platelet-rich fibrin.)
that the absorption of GFs from the wound surface occurred with both the fibrin sealant and natural fibrin. Given the known effects of GFs, one speculation is that the proliferation of fibroblasts and vascular endothelial cells was activated, triggering GF secretions such as keratinocyte growth factor and insulin-like growth factor. Such secretions could, in turn, stimulate epithelial cell growth with a consequent progression to epithelial closure (Figures 3 and 4). The 3-dimensional structure of fibrin undergoes a gradual degradation and
is ultimately absorbed into tissues. The consequence of this slow degradation is that GFs trapped into the reticular fibrin structure undergo a gradual and sustained release from the reticular fibrin structure over approximately 8 days.38 The GFs and cytokines taken up by the network inhibit the destruction of these factors by proteases, contributing to a sustained GF release.39-41 This sustained GF release may have the advantageous effect of promoting healing during 2 or more concurrent phases of mucositis. In another in vitro experiment on the cellular growth activity of PRF versus fibrin sealants, cultures of fibroblasts with medium containing fibrin showed a slight increase in cellular growth activity, although the difference from the control cultures did not reach statistical significance. The cultures with medium containing PRF showed significantly increased cellular growth activity compared with the control cultures.40 This finding is consistent with the results of the present study. The maximum relative ulcer area was 1.08 0.03 for the PRF group compared with 1.32 0.09 for the control group and 1.23 0.07 for the fibrin group. PRF was more effective than fibrin in halting ulcer exacerbation. The effects of anti-inflammatory cytokines, such as transforming growth factor b (TGF-b) and interleukin 4 (IL-4), that are taken into the 3-dimensional fibrin structure in PRF may account for this finding.42 We infer that these cytokines suppress the production of tumor necrosis factor a, IL-1b, and IL-6, which are factors that aggravate chemotherapy-induced mucositis,
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and this suppression inhibits the progression of tissue destruction.43,44 Consistent with this inference, Clarke et al.45 found that TGF-b in a whey growth factor extract prevents chemotherapy-associated oral mucositis in a similar hamster mucositis model. In the fibrin group, it was possible that factors (e.g., TGF-b) released from macrophages in the wound were adsorbed. The same event may have occurred when fibrin was contained in the PRF, such that a greater suppression of ulcer aggravation was obtained in the PRF group. The results were histologically evaluated on days 6, 10, and 14. Intact healthy cheek pouch tissues presented as a thin layer of smooth keratinized epithelium with a muscle bundle layer. The submucosa was primarily fibrous, with scant cells and few blood vessels.46 There was evidence of an earlier invasion of epithelial cells around the ulcer into the granulation tissue in the PRF group compared with the other 2 groups. Excessive inflammatory cell infiltration was inhibited at an earlier stage in the PRF group (Figure 5). A possible explanation for this is that the various GFs contained in the PRF may have affected the growth of the epithelial cells, and anti-inflammatory cytokines, such as TGF-b and IL-4, were taken up by these cells. These histologic findings were evaluated using the histopathologic scoring criteria,32 and no significant intergroup difference was observed on day 6 of the experiment. On day 14 of the experiment, the PRF group exhibited a significant score decrease compared with the control group (P < .05) (Figure 6). These histologic findings indicated accelerated ulcer healing and the suppression of excessive inflammation in the PRF group. The PRF treatment had a noticeable influence on the degree of epithelialization and inflammatory cell infiltration, indicating that PRF had a beneficial effect. An assessment of the degree of inflammatory cell infiltration based on the MPO activity assay found an approximately 43% reduction in MPO activity on day 6 in the PRF group compared with the control and fibrin groups. The inhibition of inflammatory cell infiltration, especially neutrophil infiltration, was evident in the PRF group. The degree of inflammatory cell infiltration seemed to be related to the degree of wound pain and tended to coincide with the weight change data. This study presented data suggesting that the healing of 5-FU-induced oral mucositis can be promoted by the use of topically applied PRF. In contrast to many existing management strategies of established oral mucositis that are interventions to control pain, the use of topically applied PRF may be able to help promote the healing of established oral mucositis. Our current findings support future clinical trials in patients. Certain aspects of the mechanism by which PRF promotes the healing of oral mucositis have not yet been clarified. Further investigation will be required to resolve these
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issues. Additional studies aimed at improving the method of PRF application onto lesions and further developing the practical clinical utilization of this system are necessary. REFERENCES 1. Sonis ST. Oral mucositis in cancer therapy. J Support Oncol. 2004;2:3-8. 2. Loury DJ, Embree JR, Steinberg DA, Sonis ST, Fiddes JC. Effect of local application of the antimicrobial peptide IB-367 on the incidence and severity of oral mucositis in hamsters. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1999;87:544-551. 3. Sonis ST. Mucositis as a biological process: a new hypothesis for the development of chemotherapy-induced stomatotoxicity. Oral Oncol. 1998;34:39-43. 4. Sonis ST, Elting LS, Keefe D, et al. Perspectives on cancer therapy-induced mucosal injury. Cancer. 2004;100(9 suppl): 1995-2025. 5. Sonis ST. Oral mucositis. Anti-Cancer Drugs. 2011;22:607-612. 6. Peterson DE, Öhrrn K, Bowen J, et al. Systematic review of oral cryotherapy for management of oral mucositis caused by cancer therapy. Support Care Cancer. 2013;21:327-332. 7. Raber-Durlacher JE, Logan RM, Bowen J, et al. Systematic review of cytokines and growth factors for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:343-355. 8. Migliorati C, Hewson I, Lalla RV, et al. Systematic review of laser and other light therapy for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:333-341. 9. Nicolatou-Galitis O, Sarri T, Bowen J, et al. Systematic review of anti-inflammatory agents for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:3179-3189. 10. Qutob AF, Allen G, Gue S, Revesz T, Logan RM, Keefe D. Implementation of a hospital oral care protocol and recording of oral mucositis in children receiving cancer treatment: a retrospective and a prospective study. Support Care Cancer. 2013;21: 1113-1120. 11. Cheng KKF, Molassiotis A, Chang AM, Wai WC, Cheung SS. Evaluation of an oral care protocol intervention in the prevention of chemotherapy-induced oral mucositis in paediatric cancer patients. Eur J Cancer. 2001;37:2056-2063. 12. Saunders DP, Epstein JB, Elad S, et al. Systematic review of antimicrobials, mucosal coating agents, anesthetics, and analgesics for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:3191-3207. 13. Epstein JB, Epstein JD, Epstein MS, Olien H, Truelove EL. Doxepin rinse for management of mucositis pain in patients with cancer: one week follow-up of topical therapy. Spec Care Dentist. 2008;28:73-77. 14. Vokurka S, Skardova J, Hruskova R, et al. The effect of polyvinylpyrrolidone-sodium hyaluronate gel (Gelclair) on oral microbial colonization and pain control compared with other rinsing solutions in patients with oral mucositis after allogeneic stem cells transplantation. Med Sci Monit. 2011;17:572-576. 15. Markiewicz M, Dzierzak-Mietla M, Frankiewicz A, et al. Treating oral mucositis with a supersaturated calcium phosphate rinse: comparison with control in patients undergoing allogeneic hematopoietic stem cell transplantation. Support Care Cancer. 2012;20:2223-2229. 16. McGuire DB, Fulton JS, Park J, et al. Systematic review of basic oral care for the management of oral mucositis in cancer patients. Support Care Cancer. 2013;21:3165-3177. 17. Singer AJ, Clark RAF. Cutaneous wound healing. N Engl J Med. 1999;341:738-746.
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Reprint requests: Koichiro Horii Department of Oral and Maxillofacial Surgery Nagoya City University 1-Kawasumi Mizuho-ku Nagoya 467-8601 Japan [email protected]