Comparison of repair characteristics of artificial dermis composite tissue with traditional prefabricated flap

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1 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. Comparison of repair characteristics of artificial dermis composite tissue with traditional prefabricated flap Yuchong Wang, M.D.* Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Mengyan Sun, M.D.* Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Haiying Dai, M.D. * Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Chuan Lv, M.D. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Jianguo Xu, M.D. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Ji Zhu, M.D. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Ran Tao, M.D. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Kai Wu, M.D.

2 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Xinwei Wang, M.D. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China Chunyu Xue, M.D. Chair and professor of Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China. *These three authors contribute equally to this work. Corresponding author: Chunyu Xue, M.D. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, , China xcyfun@sina.com tel fax Abstract Objective: We compared the application of artificial dermis composite tissue and the traditional prefabricated flap in a rat model of an exposed bone and tendon injury. Methods: Sprague Dawley rats were randomly divided into two groups (A and B, n = 40). Group A rats received artificial dermis composite tissue flaps while group B rats received traditional prefabricated flaps. Flap appearance, range of motion, degree of swelling, tissue histology, and imaging were compared between groups at 7, 14, 21, and 28 days. Results: There was no difference in flap appearance, range of motion, and degree of swelling between groups. However, blood perfusion of the artificial dermis composite tissue flap was better than in the traditional prefabricated flap; artificial dermis was also found to be thicker

3 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. than the traditional prefabricated flap. Conclusion: The artificial dermis composite tissue flap is an ideal method for repairing exposed bone and tendon, and displays a comparable repair effects and that it may be a better alternative compared with the traditional prefabricated flap. Key words: artificial dermis; composite tissue flap; prefabricated flap; wound; repair Introduction The repair of large skin and soft tissue injuries, especially those involving deep blood vessels, nerves, exposed tendons and bone, or abdominal wall defects due to war and natural disaster conditions is a difficult problem to solve. The traditional prefabricated flap has shortcomings, such as its thinness and lack of wear resistance. Therefore, we compared the use of artificial dermis and traditional prefabricated flaps in a rat model of exposed bone and tendon injury to assess their repair potential and characteristics. Methods Experimental Materials and Animal Models Eighty healthy SD rats (male, aged 6 to 8 weeks, g) were provided by the Experimental Animal Center of Second Military Medical University, Shanghai, China for this experiment. Artificial dermis provided by PELNAC LIMITED GUNZE, Kyoto, Japan was used. Rats were randomly divided into two groups (A and B, n = 40), with four subgroups of 10 rats each, to be assessed at four different time points (7, 14, 21, and 28 days). Operative Details Under isoflurane anesthesia, the perimeter of each rat s right hind ankle was measured. A 1 cm incision was made and the bone and tendon were exposed to simulate a complex wound. A 1 cm 3 cm skin graft was excised from the abdomen, placed in wet gauze, and set aside. In group A, the artificial dermis was fixed with sutures to the wound, followed by skin graft placement. After 4 weeks, the complete artificial prefabricated flap was taken off from the surrounding fascial flap and a fascial pedicle flap was formed using the superficial inferior epigastric artery and vein. A model of exposed tendon and bone was created. The fascial pedicle flap was tunneled subcutaneously and sutured. Then, the abdominal wound was sutured directly(fig1). In Group B, artificial dermis was not used; all other steps

4 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. remained the same. All wounds were dressed with sterile gauze and an adhesive bandage to fix the wound in a functional position. Outcome measures Flap appearance Postoperative flap appearance (congestion, swelling, fluid infiltration, secretion, ulceration, necrosis, crusting, scar formation, and exposure of tendon, bone, or artificial dermis) were recorded at different time points (7, 14, 21, and 28 days) in each group. Range of motion Ankle range of motion (flexion minus extension angles) was measured at 28 days. The average of three measurements was used for the analysis. Degree of swelling The average of three measurements of bilateral ankle joint circumference was performed at 7, 14, 21, and 28 days. Immunohistochemistry Paraffin sections of the wounds were made, capturing the different time points studied above (7, 14, 21, and 28 days) Anti-rabbit/mouse antibodies (REALTMEnVision+/HRP RABBIT/MOUSE, Dako, Glostrup, Denmark) were used for CD31+ immunohistochemical staining. Slices were observed under a high magnification optical microscope (10 slices, 200) for the presence of vascular structures. Masson staining At the 28 days postoperative time point, 40 paraffin sections of the wounds were made (two slices per wound). After conventional Masson staining, the sections were viewed under a high magnification optical microscope ( 40) to determine flap thickness. Imaging On postoperative day 28, rats were exsanguinated and infused with 10% barium sulfate (2 ml/min), via catheters placed into the inferior vena cava and abdominal aorta, respectively, until the death of rat. Rats were immediately placed in a refrigerator at 4 C for 24 h for radiographic imaging.

5 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. Statistical Analysis The student's t-test was used to compare mean values for range of motion, degree of swelling, and flap thickness. SPSS version 18.0 (International Business Machines Corp, Armonk, New York) was used for statistical analysis. A p-value of less than 0.05 was considered statistically significant. Results Flap appearance All flaps survived well in the postoperative period at all four time points, with a rich epidermal blood supply. There were no obvious signs of ulceration, infection, or artificial dermal exposure at each time point. Hair growth was seen after 21 days in all rats(fig2). Range of motion There was no significant difference in range of motion between right and left hind ankles in the same rats, or between groups, at 28 postoperative days (p>0.05). Degree of swelling Degree of swelling, as measured by ankle perimeter, was statistically different between groups at different time points. In both groups, this difference was shown in the following comparisons: preoperative vs. 7 d; 7 d vs. 14 d; 14 d vs. 21 d; and 21 d vs. 28 d (p<0.05). There was no significant difference in swelling when comparing preoperative and 28 d time points (p<0.05). However, no significant difference was observed between groups when comparing degree of swelling the four time points. Immunohistochemistry In Group A, the artificial dermis was visible between the skin and fascia with an obvious boundary and neovascularization at postoperative day 7. Over time, cell morphology and arrangement became less disordered and neovascularization gradually increased. In group B at the same time point, fascial flaps also displayed increasing neovascularization.(fig3) CD31+ immunohistochemical positive cumulative integrated optical density (IOD) values were significantly different between groups at the 7 and 14 d time points (p<0.05); this difference was no longer significant when comparing flaps at 21 and 28 d (p>0.05).(fig6,7) Masson staining Masson staining for flap thickness at 28 d were significantly different between groups

6 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. (1.94±0.25 mm vs. 1.19±0.05 mm, p<0.05), with a 63% increase in group A(Fig4,8). Imaging Radiographic assessment of vascular branches showed no significant differences between groups at 28 postoperative days. Abdominal blood vessels and branches were visible bilaterally without significant differences between limbs. However, it was noted on radiography that the right sided flaps in group A were thicker than in group B, with visible neovascularization in the artificial dermis(fig5). Discussion Prefabricated flaps were first successful in animal experiments in the 1970s. With continued improvements in research models, these flaps have been increasingly applied in the clinical setting. There is great utility in constructing a new flap at a site where direct flap transfer is not possible. The original random flap can be constructed for use as a pivot flap, to ensure a more adequate and stable blood supply. In spite of the many advantages of prefabricated flaps, thickness is a limiting factor in its practical application. Prefabricated flaps cannot meet repair requirements such as greater range of motion and the ability to withstand friction, due to lack of wear-resistance and tendency to form contractures. Thus, increasing thickness of prefabricated flaps has become a research direction. Other researchers increased the flap thickness by adding the dermis, fascia, muscle and other tissues, or by injecting stem cells into the flap to promote the thickness in the past. Presently, artificial dermis, which is easy to obtain, is widely used in clinical practice due to its curative effect. Advantages of artificial dermis include increased tissue thickness, as well as wear and contracture resistance. Herein, we combined the use of a prefabricated flap and artificial dermis in a rat model of an open bone and tendon injury, and compared the repair characteristics of artificial dermis composite tissue and traditional prefabricated flaps. We found no obvious differences in flap appearance, range of motion, or degree of swelling between groups. However, blood perfusion was superior in the artificial dermis composite tissue compared with the traditional prefabricated flap. The reason for this may be the layer of artificial dermis present in the former type of flap, in which neovascularization and cell organization can occur, which together increase vascular density and flap thickness, which was confirmed by statistically significant comparisons. Conclusion

7 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. In summary, the artificial dermis composite tissue flap is an ideal method for repairing exposed bone and tendon injuries, with a comparable repair effects and that it may be a better alternative due to the ease of acquisition of artificial dermis than harvested dermis. Its longterm repair effect requires further study. References [1] Orgill DP, Ogawa R. Current methods of burn reconstruction. Plast Reconstr Surg 2013; 131(5): [2] Agaoglu G, Erol OO. A prefabricated, tissue-engineered integra free flap. Plast Reconstr Surg 2008;122(2): [3] Lukasz K, Adam M, Cezary S, et al. The use of prefabrication technique in microvascular reconstructive surgery. Wspolczesna Onkol 2012;16: [4] Hajime M, Masahide G, Ryutaro I, et al. Chronological histological findings of cultured epidermal autograft over bilayer artificial dermis. Burns 2013; 39: [5] Rami AK, Joon FO, Elof E, et al. Tissue engineering of skin. J Am Coll Surg 2013;217(3): [6] Alyssa RG, Karina AH, Jason AS. Tissue engineering for plastic surgeons: a primer. Aesthetic Plast Surg 2014;38(1): [7] Thomas B, Sophie BH, Ernst R. Tissue engineering of skin for wound coverage. Eur J Pediatr Surg 2013;23(5): [8] Dennis PO, Rei O. Current Methods of Burn Reconstruction. Plast Reconstr Surg 2013;131: [9] Matthew LI, John S, Christopher EA. Wound healing in the upper and lower extremities: a systematic review on the use of acellular dermal matrices. Plast Reconstr Surg 2012;130(5) Fig1 Operation process of group A a: artificial dermis, which is made of the same size of the wound, is covered in the wound of the abdominal wall. b: skin grafts were implanted in the artificial dermis surface. c:suture fixation the skin. d: after 28 days, cut the artificial dermis composite tissue flap to be a pedicled fascia flap with the superficial artery and vein. e:

8 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. making bone and tendon exposed wound of right leg. f: the inner side after operation. g: the lateral side after operation. Fig2 Flap of group A in different time. a: 7 days after operation. b: 14 days after operation. c: 21 days after operation. d: 28 days after operation. Flap of group B in different time. e: 7 days after operation. f: 14 days after operation. g: 21 days after operation. h: 28 days after operation. Fig3 Observation of CD31+ immunohistochemical staining flap sections of group A and group B at different time(200 ), in which the red arrow indicates the vascular structure. Group A a: 7 days after operation. b: 14 days after operation. c: 21 days after operation. d: 28 days after operation. Group B e: 7 days after operation. f: 14 days after operation. g: 21 days after operation. h: 28 days after operation. Fig4 Observation of masson staining flap sections of group A and group B at 28 days after operation(40 ). a: flap section of group A. b: flap section of group B. Fig5 X ray radiography results after perfusion of 10% barium sulfate of group A and group B at 28 days after operation. a: right side of group A. b: left side of group A. c: right side of group B. d: left side of group B.

9 JAPMA Article In Press This article has been reviewed and accepted for publication. Please note that it has not been copyedited, proofread, or typeset and is not a final version. Fig6 CD31+ immunohistochemical staining IOD value of group A at different time. * indicate comparison of 7 days and 14 days after operation in group A, P < # indicate comparison of 14 days and 21 days after operation in group A, P < Fig7 The curve of CD31+ IOD values of group A and group B at different time. Fig8 The flap thickness of group A and group B at 28 days after operation. * indicate thickness comparison of group A and group B, P < 0.05.

Management of Complex Avulsion Injuries of the Dorsum of the Foot and Ankle in Pediatric Patients by Using Local Delayed Flaps and Skin Grafts

Management of Complex Avulsion Injuries of the Dorsum of the Foot and Ankle in Pediatric Patients by Using Local Delayed Flaps and Skin Grafts Ahmed Elshahat, MD Plastic Surgery Department, Ain Shams University,