Preventing Graft Loss Caused by Hematoma: Experimental Study — Suleyman Tas PubMed

528 Graft surgery is the basis of reconstructive surgery to eliminate defects. Reasons for graft loss include hematoma (most common), infection, lack of immo- bilization, and unsuitable recipient site. Several studies have investigated the effect of hematomas on graft necrosis and associated preventative mea- sures. 1–3 Smahel 4 reported that hematomas could obstruct graft nutrition and revascularization and result in graft failure. Other reports highlight infec- tion as a reason for graft failure. 5,6 Creech et al7 used a porcine model to investigate this problem more closely, and previous studies showed that hematomas could even cause necrosis in flaps. 8,9 These investi- gators concluded that the major pathogenic agent involved is hemoglobin and/or iron8 because it cata- lyzes the chemical reaction leading to the production of highly destructive free radical species. 10 Mulliken and colleague 11,12 found that hematoma-induced strong vasospasms could assist in flap necrosis. Microporous polysaccharide hemospheres (MPH; Arista® AH; Medafor, Inc., Minneapolis, MN) is a novel absorbable agent that has been shown to be an effective hemostatic agent in cardiac, urological, and dermatological surgery. 13–16 Furthermore, it has been used to prevent adhesion formation in abdomi- nal surgery 17 and to aid in microvascular anastomosis with minimal sutures.18 To date, it has not been used in graft surgery. MPH particles are produced from purified potato starch and act as a molecular sieve to quickly extract fluids from blood. This osmotic action causes the microporous particles to swell and concentrate serum proteins, such as albumin, thrombin, fibrin- ogen, and platelets and other formed elements on their surfaces, to create a gelled matrix around the particles. It begins clotting the blood on contact by Copyright © 2014 by the American Burn Association 1559-047X/2014 DOI: 10.1097/BCR.0000000000000029 J Burn Care Res Hematoma is a common reason for graft loss. This study was intended to investigate the effects of microporous polysaccharide hemospheres (MPH; Arista® AH; Medafor, Inc.) on graft survival, the effect of MPH on graft loss caused by hematoma, and the correlation between neutrophil accumulation and graft survival. A total of 35 adult male Wistar rats were separated into five groups of seven as follows: control 1, saline, MPH, control 2 (hematoma group), and MPH + hematoma. All graft dressing was removed on the fifth postoperative day and graft survival percentage measured. Histopathological and semiquantitative analysis, including inflammatory cell infiltration and subcutaneous inflammation based on neutrophil count, was performed. Graft survival significantly improved in the MPH group (97.86 ± 1.676) compared with the control 1 (91.14 ± 3.671; P = .004) and saline groups (91.57 ± 4.791; P = .014). There was no significant increase in graft survival in the saline group compared with the control 1 group or in the MPH + hematoma group (19.57 ± 14.707) compared with the control 2 group (20.71 ± 16.869; P > .05). The neutrophil count was highest in the control 2 group (177.43 ± 22.464) and significantly decreased in the MPH group (33. 71 ± 8,674) compared with the control 1 group (66.14 ± 5.872; P = .001) and the saline group (65.57 ± 3.309; P = .001). There was no significant decrease in neutrophil count in the MPH + hematoma group (160.00 ± 27.952) compared with the control 2 group (P > .05). It seems that MPH can increase the graft survival, and there is an inverse relationship between graft survival and neutrophil accumulation. (J Burn Care Res 2014;35:528–533) From the Departments of *Plastic, Reconstructive and Aesthetic Surgery and †Pathology, Trakya University Medicine Faculty, Edirne, Turkey. Address correspondence to Süleyman Taş MD, Department of Plastic, Reconstructive and Aesthetic Surgery, Trakya University Medicine Faculty, 22030 Edirne, Turkey. Preventing Graft Loss Caused by Hematoma: Experimental Study Erol Benlier, MD,* Süleyman Taş, MD,* Ufuk Usta, MD†

Journal of Burn Care & Research Volume 35, Number 6 Benlier, Taş, and Usta 529 accelerating the intrinsic blood clotting cascade and achieves complete hemostasis within minutes. The spherical particles with diameters ranging from 30 to 100 μm and their coating of compacted cells cre- ate a scaffold for the formation of a fibrin clot. By concentrating the larger particles within the site of activity through dehydration, it subsequently accel- erates fibrin mesh formation. 19 Importantly, MPH is fully resorbed and enzymatically cleared rapidly from the wound site within 24 to 48 hours. MPH has been approved by the U.S. FDA. In compari- son with other available hemostatic agents that are derived from human or bovine sources, MPH that is made from purified potato starch is hypoallergenic and carries no inherent risk of disease transmission. 13 MPH comes in a ready-to-use packet and does not require any time-consuming preparation, heating, or premixing. In previous studies, it has been shown to be safe and cost effective. 13–16 An indication for MPH use would be intraoperative bleeding.18 Prolonged tissue ischemia provides a favorable condition for the formation of oxygen-derived free radicals on reperfusion. 20 These highly reac- tive oxyradicals are released from various sources. Activated polymorphonuclear leukocytes seem to play a pivotal role in ischemia-induced reperfusion injury in various organs and species of laboratory animals. 20–22 The neutrophil has been implicated as a source of oxygen free radicals, provoking reper - fusion injury in various ischemic organs. The sig- nificant infiltration of neutrophils into skeletal muscle, 22 skin flaps, 20,21 bowel, 23 and liver 24 sub- jected to ischemia–reperfusion injury has also been demonstrated in animal models. A skin graft is vir - tually totally ischemic for several days after graft- ing until revascularization occurs, 10,25 and actually, even skin grafts undergo an ischemia–reperfusion period. To date, the accumulation of neutrophils in skin grafts has not been studied. This experiment was designed to evaluate the effects of MPH on graft survival, the effect of MPH on graft loss caused by hematomas, and the correlation between neutrophil accumulation and graft survival. METHODS The Institutional Animal Care and Ethics Commit- tee of Trakya University approved this study. A total of 35 male Wistar rats (weighing 300 to 350 g, 4 weeks of age) were used during the evaluation. All animals were provided rat chow and water through- out the experiment, and animal care was provided in accordance with ethics committee guidance. Animal Model Animals were sedated with ketamine hydrochloride 50 mg/kg and xylazine hydrochloride 15 mg/kg administered intramuscularly. After the dorsum of the animal was disinfected, the area was shaved and the skin lubricated with fat gauze to facilitate graft harvesting. A 2 × 2 cm split-thickness skin graft was harvested from the center of the back using a hand dermatome with the same thickness (0.2 mm) 26 (Hand Dermatome, Aesculap, Germany). After the skin graft harvest, skin graft donor areas were used as recipient beds. The grafts were positioned and fixed with sutures to the recipient beds, and sterile circum- ferential dressings were placed. Group Assignment Animals were divided randomly into five groups of seven rats, three experimental and two control groups. The control 1 group skin grafts were applied without any additional procedure. Saline group (Sham group) received skin grafts after washing the recipient site with 0.5 ml of solution. In the MPH group, skin was grafted after 1 g of MPH powder was sprinkled over the recipient site (Figure 1). The control 2 group (hematoma group) received skin grafts after a hematoma was produced over the recip- ient site using 0.5 ml of clotted blood obtained from the same donor rat by tail vein puncture (Figure 2). The MPH + hematoma group had skin grafted after a hematoma was produced as previously described, and 1 g of MPH was sprinkled over the recipient site. A single surgical team performed all procedures. Outcome Measurements All graft dressing was removed on the fifth postoper- ative day. Graft survival was determined by the inves- tigator in a blinded fashion. A map of each flap was drawn on acetate paper. The surviving and necrotic areas for each graft were determined and percent Figure 1. Microporous polysaccharide hemospheres (1 g) was sprinkled once over the donor site.

Journal of Burn Care & Research 530 Benlier, Taş, and Usta November/December 2014 survival documented.27 The necrotic area was evalu- ated by macroscopic observation and defined as areas stained black. Live area was defined as healed, viable, and adherent split-thickness skin grafts. The animals were killed by cervical dislocation, and the total wound sites with adjacent normal skin were excised and prepared for histological evaluation. Histological Diagnosis Skin specimens were stored in 4.5% buffered form- aldehyde solution for at least 24 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard procedures. The patholo- gist, who was blinded to study group distribution, performed the histological analysis and examined the specimens using a light microscope. Semiquantita- tive analysis including inflammatory cell infiltration and subcutaneous inflammation were evaluated by neutrophil count using high magnification (Hema- toxylin and eosin; ×50). Statistical Analyses All data are presented as mean ± SD in the text and figures. The results of percentage of graft survival and neutrophil count were statistically analyzed using a two-tailed Mann–Whitney test or Kruskal– Wallis one-way analysis of variance. Statistical signifi- cance was accepted at P < .05. RESUL TS Animals tolerated MPH treatment well without any apparent clinical adverse effects. No animals were lost during the experiment. Graft survival significantly improved in the MPH group compared with the control 1 (P = .004) and saline groups (P = .014); however, there was no statistically significant dif- ference between the control group 1 and the saline group. The difference between the control 2 group and the MPH + hematoma group was not significant (Figure 3A). Based on neutrophil count, there was no statis- tically significant difference between the control 1 group and the saline group, but the difference between the control 1 group and the MPH group was significant (P = .001). Again, there was no sta- tistically significant difference between the control 2 group and the MPH + hematoma group. How- ever, the difference between the control 1 group and the control 2 group was statistically significant (Figure 3B). Skin Graft Survival The split-thickness skin grafts in the experimental and control groups showed partial necrosis, total survival, or total necrosis. Skin graft survival was expressed as percent survival. The control 1 group had 91.14 ± 3.671% survival. Graft survival was 91.57 ± 4.791% (P > .05) in the saline group and 97.86 ± 1.676% (P = .004) in the MPH group. Graft survival was 20.71 ± 16.869% in the control 2 group and 19.57 ± 14.707% (P > .05) in the MPH + hema- toma group. Histopathological Analysis The number of neutrophils counted with the light microscope under high magnification (Hematoxy- lin and eosin; ×50) was 66.14 ± 5.872 in the con- trol 1 group. Neutrophil count was 65.57 ± 3.309 (P > .05) in the saline group and 33.71 ± 8.674 (P = .001) in the MPH group. The neutrophil count was 177.43 ± 22.464 in the control 2 group and 160.00 ± 27.952 (P > .05) in the MPH + hematoma group (Figure 4). DISCUSSION The skin graft model with associated hematoma has been described previously and involves incubating skin grafts by placing them in vitro for 48 hours. Some important recipient bed factors may be elimi- nated in the process, although this seems less likely because grafts placed over plasma had good survival and it would be more difficult to maintain aseptic conditions if the grafts were placed originally over hematomas in situ. 7,10 However, in the current study, we aimed to mimic conditions in the clinic, so the model of skin graft associated with hematoma used involved skin grafting after a hematoma was produced at the recipient site using 0.5 ml of clotted donor blood. Figure 2. Skin was grafted after a hematoma was pro- duced at the donor site using 0.5 ml of clotted donor blood obtained by tail vein puncture.

Journal of Burn Care & Research Volume 35, Number 6 Benlier, Taş, and Usta 531 The most common cause of graft loss is hema- toma, and many studies have indicated the impor - tance and influence of hematomas on graft necrosis. The contact between the skin graft and the recipient site is particularly important during the revascular - ization phase. To achieve this contact, the applica- tion of pressure to the graft using tie-over dressing is a well-known technique. However, it is not an appli- cable method for difficult and delicate anatomical sites such as the face, eyelids, fingers, and toes and for covering over muscle flaps. 2 Fibrin sealant is used for graft fixation and for reducing hematoma, but it is expensive and not available in all countries. 1 In graft surgery, while providing hemostasis in recipient bed, graft contact with the recipient bed should not be blocked. Thus, a rapidly absorbable agent is required. MPH is fully resorbed from the wound site within 24 to 48 hours, which is why we chose to use it in this experiment. We were able to observe the efficiency of MPH on graft loss com- pared with the control 1 group and the saline group. These results demonstrate that MPH can decrease the graft loss. However, based on the results from the control 2 group and the MPH + hematoma group, MPH is not effective after hematoma has already occurred. Therefore, clinically if a hematoma is detected, it should first be removed, and then MPH could be applied. We have considered that the viable graft percentage in the control group may be smaller because of the fact that no bleeding control process such as pressing was applied. A skin graft is virtually totally ischemic for several days after grafting. 10 It is believed that free radicals are important mediators of destruction during isch- emic injury, which can participate in chain reactions and cause peroxidation of cellular and intracellular membranes and intracellular proteins, resulting in irreversible cell injury. 28 There are multiple potential sources of free radicals in the skin, such as xanthine oxidase, iron, other intracellular enzyme proteins, and neutrophils. 29 The only oxygen that can reach the graft would be through diffusion from the graft Figure 3. A. Percentage area of graft survival at the fifth postoperative day. There is a significant increase in graft survival in MPH group compared with the control 1 group (*P = .004). B. Neutrophil count in the MPH group was significantly lower than that in the other groups (*P = .001). MPH, microporous polysaccharide hemospheres.

Journal of Burn Care & Research 532 Benlier, Taş, and Usta November/December 2014 bed. Angel et al 10 observed that a small amount of oxygen from the bed could result in a low amount of free radicals. Reactive oxyradicals are released from various sources, among which activated neutrophils seem to play a pivotal role in ischemia–reperfusion injury. 27 The role of neutrophils in ischemia–reperfusion injury has been widely investigated, and the impor - tance of this role is emphasized. 20,27,29 Angel et al performed a study examining the effect of free radi- cals on graft survival with biochemical analysis of lipoperoxidation; however, this study did not have a histopathological component demonstrating neu- trophil accumulation. We investigated the role of neutrophil accumu- lation in graft survival as in ischemia–reperfusion injury by correlating neutrophil accumulation with graft loss. We observed that there is an inverse rela- tionship between graft survival and neutrophil accu- mulation. We did not investigate any biochemical parameter such as malondialdehyde or myeloperoxi- dase in this experiment. However, the results showed the importance of neutrophils on graft survival and beg the question “Can drugs such as deferoxamine, cyclosporine A, FK506, and allopurinol that are used in ischemia–reperfusion injury be used in graft sur - geries?” The risk of infection could be increased, and ultimately further detailed studies are required to investigate this issue. CONCLUSION It seems that neutrophils play an important role in skin graft survival as seen in ischemia–reperfusion injury, and MPH can increase the graft survival. We believe that it is because of the hemostatic effect of MPH. However, further investigations should be performed before clinical use. 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