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ORIGINAL ARTICLE
Year : 2022 | Volume
: 30
| Issue : 3 | Page : 69-75
Feasibility of microsurgery in burn injury and the effect of stem cell application
Ersin Gur1, Yigit Ozer Tiftikcioglu1, Gulinnaz Ercan2, Hafize Seda Vatansever3, Yakup Isik4, Hilal Kabadayi5, Ayfer Karlitepe6, Ecmel Songur1
1 Department of Plastic Reconstructive and Aesthetic Surgery, Ege University Faculty of Medicine, İzmir, Turkey
2 Department of Medical Biochemistry, Ege University Faculty of Medicine, İzmir, Turkey
3 Department of Histology and Embryology, Celal Bayar University Faculty of Medicine, Manisa; DESAM Research Institute, Near East University, Mersin, Turkey
4 Department of Plastic Reconstructive and Aesthetic Surgery, İstinye University, Bahçeşehir Liv Hospital, İstanbul, Turkey
5 Department of Histology and Embryology, Celal Bayar University Faculty of Medicine, Manisa, Turkey
6 Department of Medical Biology, Adnan Menderes University Faculty of Medicine, Aydın, Turkey
Correspondence Address:
Dr. Ersin Gur
Ege Universitesi Tip Fakultesi, Plastik Rekonstruktif ve Estetik Cerrahi AD, Kazim Dirik Mah. Universite Cad. No:9, Bornova/ Izmir
Turkey
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/tjps.tjps_25_22
Introduction: Burn is one of the most severe traumas and can affect skin, muscle, or bone. Although early debridement and use of skin grafts have proven their benefits; in some cases, free flaps are required for early reconstruction. It is undisputed that choosing recipient vessels from unburned or undamaged zones increase the success of anastomosis. However, it may not be possible, especially in large burns. Our study aims to investigate the success of anastomosis beneath burned tissues and the effects of stem cell application. Materials and Methods: Twenty-one Wistar albino male rats weighing 250 g on average were divided equally into three Groups. In Group I, femoral artery anastomoses were done as control group. Inguinal area of 7 rats was burned as deep 2nd degree depth, and femoral artery anastomoses were done immediately beneath the burned tissues. In Group III, after burning inguinal area and making anastomoses, adipogenic stem cells were applied on the anastomosis sites. All anastomoses were checked at postoperative 3rd day. Results: One rat of Group I died in the postoperative 3rd day and one rat of Group II had a wide wound dehiscence including many of foreign body on anastomosis zone so both were excluded from the study. One anastomosis in Groups I and 3 anastomoses in both Groups II and III failed and others were successful. Statistical analyzes showed that there was no difference between groups (P < 0.05). Stem cell application was not effective on anastomosis patency in burn injury but has benefits in wound healing according to our study. Conclusion: Recipient vessels can be chosen from adjacent area to burn or inflammation zone safely for microvascular anastomosis if there is no direct burn damage on vessels.
Keywords: Anastomosis, burn, free flap, microsurgery, stem cell
Burn is one of the most severe traumas and can affect skin, muscle, or bone through various etiologic factors. Due to benefits obtained in developed countries, especially in severe burns, early debridement of the burned tissues and use of the skin grafts reduce mortality and morbidity and so, have entered into the standard treatment algorithm.[1],[2],[3],[4],[5],[6] However, in a situation in which the skin grafting is unfeasible, such as a wound including exposed bone or tendon, free flaps are required as a powerful reconstructive option. There are many studies in the literature about the acute usage of free flaps for one-step coverage of complex wounds because of trauma and burns.[7],[8],[9],[10] Jabir et al.[11] reviewed studies about application of free flaps in burns, and emphasized reduced hospital stay and infection and providing limb salvage as advantages of acute-free flap application.
The anastomosis is one of the important phases of free flap surgery so that it is important to choose recipient vessels from unburned zones. However, especially in thermal burns, in a scenario of complete extremity burns, there may not be a completely undamaged area for anastomosis. Deep burns can affect directly skin, subcutaneous tissue, and recipient vessels which lies beneath the skin and, it is essential to avoid choosing directly damaged and affected vessels for anastomosis success in such cases. Although even if burn damage is superficial and limited in the skin, we know that there is a local and systemic response of burn damage.[2] Hence, there is a dilemma for a burned patient who is going to have benefits from acute-free flap coverage, while there are no undamaged or unburned areas to house recipient vessels. Should the surgeon wait until skin healing is completed? In presence of proper recipient vessels even in inflammation zone, is the anastomosis possible? How the local factors of burned tissue affect the anastomosis patency? These questions must be answered to decide if free flap surgery is the treatment of choice.
Adipose stem cells belong to the mesenchymal stem cell family, and today, we know that stem cells play a therapeutic role in wound healing.[12],[13],[14],[15],[16],[17] Many parameters can give the clues of healing process. For instance, vascular endothelial growth factor (VEGF) induces angiogenesis and accelerate wound healing.[18],[19] Superoxide dismutase (SOD)[20] and malondialdehyde (MDA)[21] values show oxidative stress. AT-III (Antithrombin III) inhibits thrombosis and has been used as an indicator of burn healing.[22] Finally, cutting a vessel and making an anastomosis also means creation of a new wound healing process at the anastomosis site.
Even there are too many studies about applicability of acute-free flaps in burned patients and the role of stem cells about wound or burn healing, as far as we can investigate and find in the literature, there is no detailed study about the possibility of microsurgical anastomosis beneath acute burns and also stem cell effect on anastomosis success under burned tissues. This study aims to investigate the influence of response of burned tissues and the effects of stem cells on anastomosis patency experimentally.
Materials and MethodsThis study was approved by Local Ethics Committee. Stem cells were obtained from Celal Bayar University Faculty of Medicine, Department of Histology and Embryology. This study was supported by Ege University Scientific Research Projects Coordination Unit with number: 2015-TIP-055.
Adipogenic stem cell preparation, culturing, and characterization
Five Wistar Albino male rats with 200–250 g were used for adipogenic stem cell preparation. After taking inguinal fat tissue, adipose tissues were washed with phosphate-buffered saline and 5% Penicillin–Streptomycin solution. Tissues were dissociated by lancet, incubated 30 min at 37°C, 5% CO2, alpha-MEM (Minimum Essential Medium), 0.075% collagenase type 1, and then were centrifugated and put into culture medium which included alpha-MEM, 20% fetal bovine serum, 1% L-glutamine for 2 weeks to proliferate. CD34 and CD44 markers were used for characterization. CD34 is a marker of hematopoietic stem cell and CD44 is positive for mesenchymal stem cells. Hence, immunoperoxidase analysis on cultured cells showed that cells were CD34(–) and CD44(+).
Burn model creation
For the prevention of direct damage to femoral vessels, 3rd degree burn creation was avoided. Deep 2nd degree burn was aimed for burn model creation. A device, which was working with electric and had a copper square tip, was used to create the burn model. A digital thermometer device was used to measure the temperature from the tip. The burn model was created by reaching to 100°C by the device [Figure 1]. To obtain a standardized deep 2nd degree burn model, 6 inguinal zone in 3 rats were burned for 1, 3, 5, 10, 15, and 20 s with the device, respectively [Figure 2] after applying general anesthesia. All six-burn models were examined histologically to provide the deep 2nd degree burn for standardization, and we found that 10 s was the proper duration for our purpose. All burn models were created by the same surgeon.
Figure 1: The device working with electric energy, copper square tip, and thermometer (a), Device was turned on until the thermometer showed that the tip was 100°C (b)
Figure 2: Burn model. Use of copper tip to create burn model in inguinal area (a). The duration was 1, 3, 5, 10, 15, and 20 s, respectively (b-d)Study groups
Twenty-one Wistar albino male rats weighing 250 g on average, were equally divided into three groups. In Group I (control group), only femoral artery re-anastomosis was done. Seven inguinal zones of seven rats were examined as control group. After careful inguinal incision and dissection, the femoral artery was found and dissected gently from the surrounding tissues, and the approximator was positioned as usual. After cutting femoral artery with micro-scissors, it was re-anastomosed with 10/0 monofilament nylon sutures. Milking test was made to be sure that anastomosis was patent [Figure 3]. All stages were made under microscope. Skin incision was closed separately with 3/0 silk suture. In Group II (Anastomosis beneath burn tissue), seven burn model was created, as explained above. After burned skin was harvested as a flap (the incision did not cross the burned skin), the femoral arteries were identified like Group I [Figure 4]. Femoral artery was cut, anastomosed and incision was closed separately similar to Group I. Group III (burn + anastomosis and stem cell group) also included 7 rats. After creation of the inguinal burn model and finishing femoral artery anastomosis similar to Group II, 1 × 105 cell/ml stem cells, which were counted and examined by Thoma Counting Chamber and Trypan Blue, injected around the anastomosis site [Figure 4].
Figure 3: Group I. Preparing of inguinal area to surgery (a), Femoral artery identification after meticulous dissection (b), Inset of approximator and cutting femoral artery (c), Completion of anastomosis (d), Closure of the skin (e)
Figure 4: Preoperative marking for flap harvesting after completion of burn model for Group II, III (a), Harvesting of inguinal flap and femoral artery isolation and inset of approximator for Group II, III (b), Completion of femoral artery anastomosis for Group II, III (c), Microscopic image of anastomosis (d), Stem cells are ready for application for Group III (e), Seeding the stem cells on anastomosed vessel for Group III (f)All 21 anastomosis were made by the same surgeon. The surgery time was between 45 min to 1 h for each animal. No thrombolytic or vasodilative agent was given to rats. Rats were put into cages particularly. All anastomoses were re-examined on the postoperative 3rd day to determine anastomosis patency. The determination of the patency was made by milking test for each anastomosis. Before scarification, blood and femoral artery samples which include anastomosis sites were taken for further biochemical and histological examinations. “Zeiss Universal S3” model microscope was used for the study.
Statistical analysis
Chi-square test was used for statistical analyze of anastomoses' success between three groups, Kruskal–Wallis and Mann–Whitney U-tests were used for biochemical statistical analyzes. P < 0.05 was accepted as statistically significant.
ResultsOne rat of Group I died on the postoperative 3rd day and was excluded from the study. One rat of Group II which had a wide wound dehiscence including many of foreign body on anastomosis zone was also excluded. In all subjects, stitches were removed and anastomosis was examined by inspection and milking test under microscope on the postoperative 3rd day. Five anastomoses in Group I (n: 6), 3 in Group II (n: 6), and 4 in Group III (n: 7) were successful [Figure 5],[Figure 6],[Figure 7], others were failed. Anastomosis potencies on the 3rd day between three groups was not statistically significant (P = 0.448) [Table 1]. VEGF and AT-III serum values were not statically significant between the groups. However, MDA and SOD value differences were statistically significant [Table 2]. In histologic examination of anastomosed femoral artery samples with hematoxylin and eosin, vessel layers, tunica intima, media, and adventitia were distinguished easily. There were no differences among layers of vessels between three groups [Figure 8]. Immunohistochemical examinations showed that AT-III and VEGF stainings were moderate, especially in tunica adventitia and endothelial layers in Group I. For Group II, AT-III intensity was strongly positive in tunica adventitia and endothelial layers, and VEGF was weakly positive. In Group III, AT-III was strongly positive in tunica adventitia and moderate in endothelial layer. VEGF was strongly positive, and it was higher than Group I and II [Figure 8].
Figure 5: Postoperative 3rd day of Group I. Incision of operation area (a), isolated femoral artery (b). Milking test shows the successful anastomosis (c and d)
Figure 6: Postoperative 3rd day of Group II. Image of burned inguinal area (a), Flep re-harvesting and femoral artery isolation (b), Microscopic image of a failed anastomosis of Group II (c), A successful anastomosis of femoral artery (d), Milking test shows the success of anastomosis (e and f)
Figure 7: Postoperative 3rd day of Group III. Image of burned inguinal area (a), Anastomosed femoral artery (b), Milking test shows patent femoral artery anastomosis microscopely (c and d)
Figure 8: Vessel specimens with hematoxylin and eosin staining, postoperative 3rd day. Tunica intima, media and adventitia are distinguished easily for Group I (a and b), Group II (a and b) and Group III (a and b). There are no differences for all layers of vessels between 3 groups. Immunohistochemical examinations of vessels specimens, postoperative 3rd day. Group 1; Middle intensity AT-III (c) and VEGF (d) involvement specially in tunica adventitia and endothelial layers. Group II, strongly positive ATIII in tunica adventitia and endothelial layers (c) and weakly positive VEGF (d). Group III, strongly positive ATIII in tunica adventitia and middle intensity in endothelial layer (c) and strongly positive VEGF (d). VEGF: Vascular endothelial growth factor
Table 2: Comparison of Groups through P values of malondialdehyde, superoxide dismutase, vascular endothelial growth factor, and antithrombin III Discussion Burn is a serious health problem which can result in limb loss, mortality, psychologic complications, and a huge financial burden in Turkey, as all around the world. Especially facing burn injury more in the productive population, during the working years of life,[23] amplify the physical and mental devastation.
Today, we know that early treatment of burn injury decreases complications and hospital stay. Early debridement reduces the stress factors and free radicals created by burned tissue, and accelerate healing process.[2],[3] Reconstructive choice depends on the remaining wound bed, and local and free flaps are needed when tendons, nerves, vessels, or bones are exposed, especially in extremities. Considering that extremities have already poorly donor site for local flaps, for example, distal lower extremity, the addition of burn scenario makes local flaps' applicability more difficult, so free flaps remain as a powerful tool for early reconstruction of complex wounds. There are many studies in the literature about the benefits and usage of free flaps for early coverage of burn injury or traumatic wounds of extremities.[7],[8] In these studies, we found that undamaged zones' vessels were first used as recipient, as expected. However, in some cases, the extremity could have been burned totally and there may not be an undamaged or clean area for recipient vessels of a free flap anastomosis.
Total extremity burns can be uniform degree or mixed, for example, 2nd and 3rd degree burns together. 3rd degree burns can result in a devastation, injury of the whole skin and subcutaneous tissues including nerves, bone, or vessels. It is unfeasible to apply anastomosis to burned vessels. However, 2nd degree burns are classified as superficial and deep. Superficial burns are seen red, erythematous and can heal spontaneously, but deep 2nd degree burns are seen pale pink to brown and can require surgical treatment as 3rd degree burns. Whether deep or superficial, 2nd degree burn is limited to skin, so vessels can be protected from direct burn damage, but at the same time, they can be affected from its local response. Although the literature has some studies about use of recipient vessels beneath damaged tissue, our study is a detailed experimental study focusing this topic with use of stem cells.
Stefanacci et al.[24] published their retrospective clinical cases and mentioned that, for some of their cases, recipient vessels had been chosen at the injured zone. However, their study is about a general approach for the usage of free flap reconstruction, so there was no detailed discussion about our topic, normally. Chick et al.[25] presented 5 patients which included three electrical, one heat-press, and one both electrical and avulsion injury. They mentioned that the anastomosis was possible near the wound if the vessels were patent through visualization. Ibrahim et al.[26] emphasized the controversy in the literature about choosing recipient vessels from injured zones. Georgette et al.[27] also published a review article about burn reconstruction and mentioned that the vessels from burn damage could be used for anastomosis if the vessels had patency and quality, referring both Chick's and Stefanacci's study.
Shen et al. published their clinical experience with 70 flaps in 1988[28] and reconstructed burn wounds between different time intervals as urgent to late period. They defended that, vessels without burn damage could be used as recipient vessels and referred another study for the details, which was published in Chinese Journal of Plastic Surgery and Burns in 1987, so we could not reach to original text in PubMed. Kuo[29] published an experimental study in 1990 which was about electrical burns on a rabbit model and mentioned that 3 cm beyond the margin of the wound is feasible for anastomosis in abstract. This article was also in Chinese language so the original study could not be examined.
Burns have a systemic and local response in human body. Both of them are very complex and important for treatment. Acute burn includes coagulation, stasis, and hyperemia zones locally. The coagulation zone occurs where the heat and damage are maximum and ends up with coagulation necrosis. Coagulation zone is encircled with stasis and hyperemia zones, respectively, and edema and inflammation processes begin with many mediators. Our study shows that especially local response has no important role on anastomosis success. Even Group II and III have more failure rate, differences are not statistically significant (P > 0.05). Major problems after microvascular anastomosis, particularly of artery, usually occur in hours after operation. Thrombosis regarding overlooked microsurgical mistakes is seen generally after 12–24 h, and after this period, possibility of thrombosis is a rare situation for artery as in our clinical experience, so we decided to check our anastomosis patency at the 3rd day. We chose inguinal region for that femoral artery, as it has proper calibration for microsurgical examination and lies beneath to skin so it was suitable for our purpose. No anticoagulant or anti thrombotic agent has been used for anastomoses success.
Stem cells have a big potential of differentiation to other cells and there are many publications about benefits on wound healing, even burn wounds. These cells can be produced from adipose tissue, bone marrow, umbilical cord, etc., We used adipose stem cells because it is easy to obtain, compared by the others. These cells play a key role to accelerate re-epithelization by secretion of some mediators such as PDGF, fibroblast growth factor (FGF), transforming growth factor-beta, and also stimulate neo-angiogenesis, wound healing and fibroblast proliferation by secretion of VEGF, FGF-2, etc., Furthermore, it brings benefit on ischemia-reperfusion injury, so it enhances flap survival. However, in spite of the presence of many positive effects of stem cells on wound healing, there was no statistical difference on anastomosis success between groups in our study.
VEGF is secreted from many cells, especially from endothelial cells, and induce vascular permeability, neutrophil migration, and growth of granulation tissue. Another effect of VEGF is about endothelial cells' regeneration and mitotic activity. Infanger et al.[30] investigated the effects of intraluminal VEGF usage and found a positive effect on the healing of microvascular anastomosis in a rat model. In a normal coagulation system, ATIII inactivates thrombin and inhibits thrombosis. Decrease of ATIII is one of the responsible factors for thrombosis in burn patients. Kritikos et al.[31] have emphasized the anticoagulant and anti-inflammatory benefits of ATIII in their study and worked ATIII as one of the parameters on partial-thickness burn healing. In inflammation zone, free radicals also play roles. MDA, which shows lipid peroxidation, and SOD, which transforms superoxide-free radical to hydrogen peroxide, were also examined in our study. Blood samples and anastomosis site biopsies were examined immunohistochemically and biochemically. Our findings showed that in Group II, ATIII was strongly positive comparing with Group I and Group III, and it could be a result of the defense mechanism, or stem cells may have decreased the secretion of ATIII. VEGF reactivity was strongly positive in Group III, which can be explained the effect of stem cells. Blood samples showed that there is no statistically significant difference for ATIII and VEGF between three groups. However, for SOD and MDA, the differences were statistically significant between Group II and III. This result shows that stem cells decreased oxidative stress and had a positive effect on wound healing in our study, similar to the literature.
The limitation of our study is that it includes small experimental animals in groups. We believe that future experimental studies with large sample numbers will be beneficial. However, we think that our study is important in terms of guiding and giving ideas about the topic.
ConclusionThere are many benefits of acute one-step free flap coverage. Choosing recipient vessels from undamaged zones must be the priority to minimize risks. However, our study shows that in absence of suitable donor sites, recipient vessels can be chosen from an adjacent area to burned tissues, or inflammation zone safely for microvascular anastomosis, if there is no direct burn damage on vessels. Even if stem cells have many of benefits, there is no significant difference on anastomosis success in our study. We believe that advanced clinical studies are required for this controversial subject because of the prevalence and seriousness of burns.
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Conflicts of interest
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