Comparison of Treatments With Local Mesenchymal Stem Cells and Mesenchymal Stem Cells With Increased Vascular Endothelial Growth Factor Expression on Irradiation Injury of Expanded Skin.

TRANSPLANTATION SURGERY AND RESEARCH

Comparison of Treatments With Local Mesenchymal Stem Cells and Mesenchymal Stem Cells With Increased Vascular Endothelial Growth Factor Expression on Irradiation Injury of Expanded Skin Sinan Öksüz, MD,* Murat Şahin Alagöz, MD, PhD,† Hüseyin Karagöz, MD, PhD,* Zafer Küçükodacı, MD,‡ Erdal Karaöz, PhD,§ Gökhan Duruksu, PhD,§ and Görkem Aksu, MDk Introduction: Radiation injury results in chronically ischemic tissue. Radionecrosis can be encountered in severe cases. Mesenchymal stem cells (MSCs) have a therapeutic effect on ischemia-related lesions. In here, effects of bone-marrow derived MSC and vascular endothelial growth factor (VEGF) gene-transfected MSC (VEGF-MSC) treatment on expanded skin with irradiation injury is investigated. Methods: Silicone tissue expander (50 cm3) was placed subcutaneously and expanded weekly up to 60 cm3 in 24 Sprague Dawley rats. Single fraction (30 Gy) radiotherapy was applied to the 2 2 cm area of the expanded skin. Dulbecco modified Eagle medium without cell component, MSCs, and VEGF-MSCs were injected subcutaneously at the irradiation-expansion sites. Skin samples were evaluated by histomorphometry and immunohistochemistry. Perfusion rate of the samples was assessed by scintigraphy. Results: Epidermal thickness of irradiated-expanded skin was increased after MSC and VEGF-MSC treatments, whereas dermal and capsule thicknesses did not change. The MSC and VEGF-MSC treatments were effective in preserving, respectively, CD31 and VEGF expressions at a similar level as expanded skin after irradiation injury. The VEGF-MSC treatment significantly elevated CD31 levels in the irradiated tissue. Skin perfusion results were consistent with the CD31 and VEGF expressions. The MSC and VEGF-MSC treatments were effective in increasing proliferating cell nuclear antigen (PCNA) expression in irradiation zone. The VEGF-MSC treatment was efficient in reducing both expansion- and irradiation-related apoptosis. Conclusion: Vascular impairment and dermal insufficiency due to tissue expansion and irradiation injury can easily result in a wound hard to repair. The MSCs and VEGF-MSCs can promote neovascularization, reverse the effect of irradiation, and provide more durable soft tissue for expansion/implant reconstruction. Key Words: gene transfection, irradiation injury, mesenchymal stem cell, radiotherapy, skin expansion, vascular endothelial growth factor (Ann Plast Surg 2015;75: 219–230)

M

esenchymal stem cells (MSCs) have a therapeutic effect on ischemia-related lesions. The MSCs potentially release angiogenic and antiapoptotic growth factors to facilitate the recruitment of endothelial progenitor cells.1–3

Radiodermatitis is the most frequent form of radiation injury. Subcutaneous fibrosis and radionecrosis can be encountered over time in severe cases.4 Radiation injury results in chronically ischemic tissue due to altered blood flow and reduced number of capillaries.5,6 Proliferation capacities of fibroblasts are also reduced by radiation.7 Severe irradiation injury potentially progresses to fibrosis and finally to ulceration without spontaneous recovery. Breaking the vicious circle of vascular problems, fibrosis, and increased ischemia should be the goal of treatment for irradiation injury.6 In irradiated breasts, autologous tissue reconstruction may be appealing. However, in immediate autologous tissue reconstruction, complications are reported at higher rates than delayed reconstructions performed after radiotherapy.8–11 For many patients who are not candidates for autologous tissue reconstruction, expander/implant is the single choice in the breast reconstruction. However, expanded skin is vulnerable to radiotherapy, and irradiation injury can be a limiting factor for expander/implant reconstruction.8,12 In this study, MSC treatment on expanded skin with irradiation injury is investigated. The effects of injections of MSCs and vascular endothelial growth factor (VEGF) gene-transfected MSCs (VEGF-MSCs) on irradiation injury are compared.

MATERIALS AND METHODS Institutional animal care and utilization committee approved the study protocol. Sprague Dawley rats (250–300 g) were divided into 3 groups (n = 8). In all groups, a 50-cm3 silicone tissue expander (Mentor) was placed subcutaneously. Radiotherapy was applied to the 2 2 cm area of the expanded skin. Ketamine (100 mg/kg) and chlorpromazine (5 mg/kg) were administered intraperitoneally for anesthesia. A single dose of cefazolin sodium (30 mg/kg) was administered for prophylaxis. All of the animals survived without any complication. The animals were euthanized with intravenous administration of thiopental sodium (100 mg/kg).

Stem Cell Isolation and Culture Received March 2, 2015, and accepted for publication, after revision May 8, 2015. From the *Department of Plastic, Reconstructive Surgery and Burn Unit, Haydarpasa Training Hospital, Gulhane Military Medical Academy, Istanbul; †Department of Plastic and Reconstructive Surgery, Kocaeli University Medical Faculty, Kocaeli; ‡Department of Pathology, Haydarpasa Training Hospital, Gulhane Military Medical Academy, Istanbul; §Kocaeli University Institute of Health Sciences Center for Stem Cell and Gene Therapies; and kDepartment of Radiation Oncology, Kocaeli University Medical Faculty, Kocaeli, Turkey. Conflicts of interest and sources of funding: The financial support of this study was granted by Gulhane Military Medical Academy, Haydarpasa Training Hospital scientific project support program. Reprints: Sinan Öksüz, MD, Gulhane Askeri Tip Akademisi Haydarpasa Egitim Hastanesi Plastik Rekonstruktif ve Estetik Cerrahi Servisi Tibbiye Cad. Uskudar, Istanbul, Turkey. E-mail: [email protected]. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.annalsplasticsurgery.com). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/15/7502–0219 DOI: 10.1097/SAP.0000000000000574

Annals of Plastic Surgery • Volume 75, Number 2, August 2015

Femur and tibia bones were cut, and the marrow was extruded by flushing with Minimum Essential Medium (MEM)-Earle medium (Biochrom, Germany) supplemented with 15% fetal bovine serum (Invitrogen/GIBCO), and 100 IU/mL penicillin-100, lg/mL streptomycin (Invitrogen/GIBCO). The same media was used as the culture medium. Derived marrow suspension was filtered and centrifuged. Resuspended cells were cultured in flasks and incubated in 5% CO2 atmosphere. Bone marrow-MSCs (BM-MSCs) were isolated on the basis of their ability to adhere to the culture plates. Adherent cells grown to 70% confluency were defined as passage 0 cells. The passage 0 MSCs were washed with phosphate-buffered saline (Invitrogen/GIBCO) and detached by incubating with 0.25% trypsin-EDTA (Invitrogen/GIBCO). The centrifuged and resuspended cells were then plated as passage 1 in flasks (BD Biosciences). Complete medium was replaced every 3 days over a 10- to 14-day period. The cells were plated similarly and grown to a confluency of 70% at each passage. www.annalsplasticsurgery.com

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FIGURE 1. Expander inflation with saline injection (A), expanded skin (B).

Characterization of BM-MSCs Flow cytometry was performed on a FACSCalibur flow cytometry system (BD Biosciences, San Diego, CA) for characterization. Immunophenotyping was performed with antibodies from Becton Dickinson against rat antigens CD29 (Integrin b1 chain; Ha2/5; fluorescein-isothiocyanate (FITC), CD45, MHC class I, MHC class II, and CD90 (Thy-1/Thy-1.1-FITC) and their isotype controls (IgG2aj; FITC).

FIGURE 3. Skin biopsy sites for immunohistochemistry and scintigraphy imaging. Normal skin (S), irradiated and expanded skin (R), nonirradiated expanded skin (E).

Passage 3 MSCs (3000 cells/cm2) were cultured in 6-well plates with osteogenic medium MEM (Invitrogen/GIBCO) and adipogenic medium MEM (Invitrogen/GIBCO) respectively for 4 weeks to induce osteogenic and adipogenic differentiation. Osteogenic differentiation was assessed with Alizarin Red staining (Sigma-Aldrich), and adipogenic differentiation was confirmed by Oil Red O (Sigma-Aldrich) staining (see Figure, Supplemental Digital Content 1, http://links.lww.com/SAP/A143, which illustrates culture and characterization of rBM-MSCs).

Green Fluorescent Protein Labeling The plasmid (Clontech, Palo Alto, CA) was amplified in Escherichia coli strain XL-1 and purified using Endofree Plasmid Maxi kit (Qiagen, Hilden, Germany). Neon Transfection System was used to transfect the plasmid (Invitrogen, Carlsbad, CA). Plasmid DNA was mixed with MSCs in transfer buffer. Real-time PCR was used to check the integration of green fluorescent protein (GFP) gene.

TABLE 1. Comparison of Scintigraphy Results Scintigraphy Results

Radioactivity uptake

FIGURE 2. Study protocol flow chart. 220

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D (n = 8)

M (n = 8)

V (n = 8)

Mean (SD)

Mean (SD)

Mean (SD)

S 100 (0.0) E 119.7 (3.14) R 74.45 (3.55) †P = 0.0001

100.0 (0.0) 119.7 (2.95) 88.30 (3.32) †P = 0.0001

100.0 (0.0) NA 120.8 (2.94) *P = 0.8270 98.34 (2.87) *P = 0.0001 †P = 0.0003

S indicates healthy skin; E, expanded but nonirradiated skin; R, expanded and irradiated skin; D, DMEM injection group; M, MSC injection group; V, VEGFMSC injection group. *Kruskal Wallis Test. †Freidman Test.

© 2015 Wolters Kluwer Health, Inc. All rights reserved.



Irradiation Injury Treatment With MSCs

FIGURE 4. Scintigraphy images. Normal skin (S), irradiated and expanded skin (R), nonirradiated expanded skin (E).

TABLE 2. Comparison of Histomorphometry Analysis Results D (μm)

Histomorphometry Results

Epidermal thickness

S E R

Dermal thickness

S E R

Capsule thickness

E R

M (μm)

V (μm)

(n = 8)

(n = 8)

(n = 8)

Mean (SD)

Mean (SD)

Mean (SD)

19.9 (1.79) 21.96 (1.64) 26.6 (1.92) †P = 0.0001 458.1 (11.69) 407.03 (11.70) 367.3 (9.55) †P = 0.0001 234.2 (9.43) 226.8 (11.46) ‡P = 0.6406

19.18 (1.88) 23.22 (1.7) 30.13 (2.4) †P = 0.0001 453.6 (10.07) 391.5 (9.88) 364.2 (12.45) †P = 0.0001 230.7 (9.61) 232.7 (12.22) ‡P = 0.7422

19.83 (1.02) 21.78 (1.53) 29.74 (1.71) †P = 0.0001 464 (10.22) 398.1 (9.67) 372.1 (7.78) †P = 0.0001 233.8 (7.72) 237.1 (10.11) ‡P = 0.6406

*P = 0.5598 *P = 0.1210 *P = 0.0235 *P = 0.2815 *P = 0.0681 *P = 0.0905 *P = 0.9827 *P = 0.5726

*Kruskal Wallis Test. †Freidman Test. ‡Wilcoxon Test.




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FIGURE 5. Histomorphometry figures of irradiated skin samples (hematoxylin and eosin staining) and epidermal and dermal thickness graphs for all groups.

TABLE 3. Comparison of Immunohistochemistry Assessment Results

Immunohistochemistry Results

CD31

S E R

VEGF

S E R

PCNA

S E R

TUNEL

S E R

GFP

S E R

D (n = 8)

M (n = 8)

V (n = 8)

Mean (SD)

Mean (SD)

Mean (SD)

16.5 (0.96) 21.23 (1.25) 9.7 (0.97) †P = 0.0001 17 (2.69) 20.5 (2.58) 12.5 (1.92) †P = 0.0001 12 (1.12) 19 (1.11) 5.5 (1.37) †P = 0.0001 5.35 (0.44) 7.95 (0.48) 9.9 (0.57) †P = 0.0001 0 0 0 NA

17.5 (1.63) 23.9 (1.53) 25.9 (1.38) †P = 0.0009 18.5 (2.25) 22.5 (1.92) 25.6 (1.11) †P = 0.0001 13.15 (1.24) 28.25 (1.18) 27.85 (1.25) †P = 0.0004 5 (0.81) 7.75 (0.76) 7.3 (1.03) †P = 0.0005 1 (0.7) 8.5 (1.85) 24 (1.6) †P = 0.0001

17.75 (1.67) 27 (0.97) 31.8 (2.12) †P = 0.0001 19.75 (1.14) 31.5 (1.77) 34 (3.2) †P = 0.0001 13.15 (1.46) 36.25 (1.19) 37.10 (1.30) †P = 0.0003 5.25 (0.4) 6 (0.57) 5.5 (0.51) †P = 0.5444 1.5 (0.53) 8.5 (1.77) 22 (1.44) †P = 0.0001

*P = 0.1568 *P = 0.0001 *P = 0.0001 *P = 0.1922 *P = 0.0003 *P = 0.0001 *P = 0.1159 *P = 0.0001 *P = 0.0001 *P = 0.3368 *P = 0.0007 *P = 0.0001 *P = 0.0004 *P = 0.0003 *P = 0.0001

*Kruskal Wallis Test. †Freidman Test.

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VEGF Gene Transfection of MSCs The VEGF containing plasmid was generated after replacing the GFP gene into plasmid GFP vector with human VEGF gene (GenBank Acc. no. AF486837.1). The plasmid (pVEGF) was amplified in E. coli strain XL-1 and purified using the Endofree Plasmid Maxi kit (Qiagen). Neon Transfection System was used to transfect the plasmid (Invitrogen). The plasmid GFP and pVEGF plasmids were mixed with MSCs in transfer buffer. The VEGF secretion and cellular changes after gene transfection were demonstrated by immunostaining (see Figure, Supplemental Digital Content 2, http://links.lww.com/SAP/A144, which illustrates in vitro characterization of VEGF-MSCs by immunostaining) (see Figure, Supplemental Digital


Content 3, http://links.lww.com/SAP/A145, which illustrates in vitro immunofluorescence staining of VEGF-MSCs).

Experimental Protocol After a 15-mm skin incision on midsacral region, a subcutaneous pouch (8 5 cm) was prepared underneath the panniculus carnosus muscle along the right dorsolateral side of spine. A 50-cm3 silicone tissue expander was placed into pouch using sterile surgical techniques. The port was placed subcutaneously and sutured to the sacrum. The expander was initially inflated with 15 mL saline after placement. Dulbecco Modified Eagle Medium (DMEM) without cell component, BM-MSCs, and VEGF gene-transfected BM MSCs (VEGF-MSCs)

FIGURE 6. Endothelial marker (CD31) expression in the tissue. The tissue sections were stained against CD31 (PECAM-1). Although the number of CD31-positive cells was higher in MSC-treated tissues than in control tissues (DMEM), the staining intensity of VEGF-MSC groups against CD31 was significantly much higher. Scale bar: 100 μm. © 2015 Wolters Kluwer Health, Inc. All rights reserved.



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were injected subcutaneously at the expansion sites of the animals in the DMEM (Control), MSC, and VEGF-MSC groups, respectively. The expansion sites were inflated weekly with a volume of 15 mL saline for 3 times. Final volume was 60 mL at postoperative day 21 (Fig. 1A, B).

anesthesia with a 6 MV Electron linear accelerator unit at a sourceskin distance of 100 cm. Dose prescription was 30 Gy at a single fraction (see Figure, Supplemental Digital Content 4, http://links.lww.com/ SAP/A146, which illustrates irradiation zone marking at the dome of expander (A), CT plan for irradiation (B), irradiation under the electron linear accelerator unit (C).).

Radiation Injury A 2 2 cm dome of expanded dorsolateral skin was irradiated at postoperative day 7. Computed tomography (CT) scans of the animals were taken under anesthesia in the irradiation position to delineate the irradiation protocol. The CT data were used to determine the irradiation planning. Animals were irradiated in the supine position under

Treatment Protocol The control group received 1 mL DMEM injections without cells. The MSC and VEGF-MSC groups received 1 106 MSCs and VEGFMSCs, respectively, in 1 mL of DMEM at each injection. In all groups,

FIGURE 7. VEGF-expressing cells in the tissue. As part of the regeneration process, VEGF secretion and formation of new vessels are important. The involvement of tissue-specific MSCs in the regeneration process in the control group (DMEM) might be the source of VEGF. Transplantation increased the VEGF+ cells in the tissue, which improved the small vessel formation in the tissue. The increased VEGF secretion by ectopic expression of VEGF gene in MSCs further improved the number of vessels. Scale bar: 100 μm. 224





the media was subcutaneously injected at the 2 2 cm dome of the expansion site. The first injection was done after the expander placement and initial inflation at day 0. At day 7, the expander was inflated with 15 mL saline, and 2 2 cm dome of the expansion site was irradiated. The second injection was administered at the same expansion zone after the irradiation. The animals received the third injection at the irradiated 2 2 cm zone after the inflation of the expander at day 14 (see Figure, Supplemental Digital Content 5, http://links.lww.com/SAP/A147, which illustrates subcutaneous injection of MSCs.). In total, 3 mL of DMEM, 3 106 MSCs, and VEGF-MSCs were administered to the irradiated and expanded 2 2 cm zones of all groups by day 14 (Fig. 2) (see Table, Supplemental Digital Content 6, http://links.lww.com/SAP/A148, which illustrates timetable of interventions for study groups).


Scintigraphic Examination At postoperative day 30, all groups were injected with 3 mCi of technetium-99m methoxyisobutylisonitrle (99Tcm MIBI) via tail vein. Thirty minutes after the injection, the rats were euthanized. Three different skin samples with underlying panniculus carnosus were collected from the expanded and irradiated skin sites, expanded but nonirradiated skin sites, and a distant nonirradiated and nonexpanded healthy skin site (Fig. 3). The specimens were laid in a container including 10% formalin solution without distortion. Scintigraphic images of the formalin fixed samples were taken with a 256 256 matrix under the Gamma camera pinhole collimator (Philips Forte Dual-Head Gamma Camera) for 30 minutes as described by Zor.13 The average radioactivity uptake

FIGURE 8. Proliferating cells after cell injection. The cells were stained for proliferating cells in the tissue against PCNA. The cells were observed to divide in the treatment area. The number of stained cells was higher in VEGF-MSCs compared to the cells of both the control (DMEM) and MSC groups. Scale bar: 50 μm. © 2015 Wolters Kluwer Health, Inc. All rights reserved.



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percentage compared to healthy normal skin uptake represented the average tissue perfusion rate.2 Specimens were processed for histomorphometry and immunohistochemistry assays after imaging.

Paraffin sections of skin samples were stained with hematoxylin and eosin for histomorphometry. A blinded pathologist calculated the average epidermal, dermal, and capsule thicknesses after measuring 5 randomly selected fields of each slide (magnification, 10).

used to detect cellular proliferation. ApopTag Apoptosis Detection kit (Chemicon, Temecula, CA) was used for terminal deoxynucletiyl transferase-mediated dUTP nick end labeling (TUNEL) to detect apoptotic cells. Fluorescence microscope (Leica DMI 4000B, Wetzlar, Germany) and a conventional light microscope (Olympus CX21, Hamburg, Germany) were used for immunofluorescence and immunohistochemistry stainings, respectively. The positive-stained cell count was blindly determined by calculating the average count of 5 randomly selected fields under 40 magnification.

Immunohistochemistry-Immunofluorescence Assay

Statistical Analysis

The GFP (sc-9996), CD31 (sc-1506), and VEGF (sc-507) antibodies were supplied by Santa Cruz Biotechnology (Heidelberg, Germany) to detect the angiogenesis, endothelial proliferation, and GFP activities. PCNA (ab 29; Abcam, Cambridge, MA) antibody was

The results were compared with a statistical program between 3 different groups and within the same group. The perfusion rate, histomorphometry, and immunohistochemistry results between all 3 groups were compared using the Kruskal-Wallis and Mann-Whitney

Histomorphometry

FIGURE 9. Apoptotic cells in the tissue shown by TUNEL. The number of apoptotic cells was higher in the non-stem cell treated group. MSC treatment decreased the apoptosis level, but the VEGF-MSC group tissue had the lowest number of apoptotic cells compared to the other groups. Scale bar: 50 μm. 226






U test. The perfusion rate, histomorphometry, and immunohistochemistry results of irradiated, nonirradiated, and healthy skin zones within the same group were compared using the Freidman test. Wilcoxon test was used to compare paired groups. The significance level was set as P less than 0.05.

different between all groups; the highest perfusion rate was observed in the VEGF-MSC group (P < 0.05) (Table 1) (Fig. 4).

RESULTS Scintigraphic Examination The mean tissue perfusion rates and standard deviations for the expanded and irradiated sites of the DMEM, MSC, and VEGF-MSC groups were 74.45 (3.55), 88.30 (3.32), and 98.34 (2.87), respectively. Tissue perfusion rate was increased in the expanded but nonirradiated sites of all groups without any statistical difference between groups. The perfusion rates of expanded and irradiated sites were significantly

Histomorphometric Analysis The epidermal thickness of the healthy skin and expanded but nonirradiated skin samples did not differ between groups. The epidermal thickness of the expanded and irradiated skin of the MSC and VEGF-MSC groups did not statistically differ, whereas both groups statistically differed compared with the DMEM group. Comparison of epidermal thickness within the same group revealed significant differences for all groups. The highest epidermal thicknesses were observed in the expanded and irradiated samples of all groups. The dermal thicknesses did not have difference between groups. However, there was a significant difference between the dermal thicknesses within each group. The lowest dermal thicknesses were measured in the expanded and irradiated samples of all groups. The capsule

FIGURE 10. The GFP-positive cells infiltrated into the tissue. The transplanted cells were located by staining against GFP. The numbers of GFP+ cells in both the MSC and VEGF-MSC groups were significantly higher. The low number of stained cells in the surrounding tissue might be explained by migration of MSCs and VEGF-MSCs. Scale bar: 200 μm. © 2015 Wolters Kluwer Health, Inc. All rights reserved.



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thickness comparison did not reveal any statistical difference between the groups (Table 2) (Fig. 5).

previously irradiated skin or when skin is irradiated during expansion. Evans et al21 conclude that irradiation has a negative impact on the outcome of implant reconstruction independent of its timing. Autologous tissue reconstruction does not have a protective effect for the implant in irradiated tissue. In single-stage immediate breast reconstruction, postmastectomy radiotherapy may adversely affect reconstruction; also, reconstructed breast may impair the delivery of radiotherapy. Usually, tissue expander placement during mastectomy and replacing it with a permanent implant before radiotherapy is also not feasible due to ongoing chemotherapy. Thus, radiotherapy is usually applied after completing the expansion and chemotherapy. To avoid complications, the expander is replaced with a permanent implant after radiotherapy.8,12,22–24 Still the complication rate of implant-based reconstruction in irradiated breast is 40.7%; and 18.5% of irradiated cases necessitate the removal or replacement of the implant. The implant extrusion rate is almost 15% in irradiated cases.8,18 Deflating the expander completely for effective radiotherapy results in extrusion and loss of expander during reinflation after radiotherapy.25 In skin-sparing mastectomies, placing large volume filled expanders or implants for the first stage of breast reconstruction can impair circulation and induce skin necrosis.26,27 Thus, 2 major concerns arise about delayed/immediate breast reconstruction. Radiated and reexpanded mastectomy skin may have elasticity problems and cause contracture over the long term and mastectomy skin can have an increased risk of necrosis due to irradiation injury and pressure from an inflated expander.23 Thereof, irradiation injury is usually considered an indication for surgical removal. However, MSCs have a potential therapeutic and protective effect on these injuries by rendering tissue preservation.6 In postmastectomy expander/implant reconstruction, fat grafting is beneficial in increasing the quality of skin for patients who receive radiotherapy.19 Transplant of autologous lipoaspirates containing adiposederived stem cells have a therapeutic effect on radiotherapy-induced lesions. Microvascular alterations observed in the irradiation injury, similar to chronic ischemic diseases or systemic sclerosis, could be reversed by the neovascularization effect of MSCs in the lipoaspirates.6 Tissue expansion causes thickening in the epidermis; however, the dermis and subcutaneous tissue become thinner. Similarly, the thickness of the dermis and subcutaneous tissue decreases and the epidermis becomes thicker in irradiated and expanded skin. However, irradiation has a prominent impact when both tissue expansion and irradiation are combined.28–30 In tissue expansion, blood flow in the capsule of an expander increases with no effect on cutaneous blood flow. Cutaneous or subdermal blood vessels and flow do not increase, and neovascularization is not observed. The deflation of expanded skin temporarily increases blood flow, with a negative impact on circulation after the initial increase.31,32 We aimed to promote angiogenesis in the dermis via MSC treatment. Thus, by increasing neoangiogenesis and proliferation capacity of the skin, the reconstruction process can be accomplished rapidly without complications. The epidermal thickness of irradiated and expanded skin was increased after MSC and VEGF-MSC treatment compared to the control group. However, MSC and VEGF-MSC treatment did not significantly change the dermal and capsule thickness of the irradiated and expanded skin. The increase in epidermal thickness in the MSC-treated groups can be attributed to the regenerative capacity of MSCs. Irradiation causes endothelial injury in a dose-dependent manner and higher doses damage the endothelial cells. After higher doses of radiation, microvessel density and CD31 expression in the skin are constantly decreased except for the transient peaks between 0 to 2 and 8 to 10 weeks.33,34

Immunohistochemistry Assays CD31, VEGF, PCNA, and TUNEL expressions of healthy skin samples did not statistically differ between all three groups. Comparison of CD31 expression within the same group revealed a difference for the DMEM and VEGF-MSC groups. There was no statistical difference between the expanded and irradiated samples and expanded but nonirradiated samples of the MSC group. Comparison of expanded and irradiated samples and expanded but nonirradiated samples between all 3 groups showed a significant difference. The VEGFMSC group expressed the highest CD31 levels against the other groups in expanded and irradiated samples (Table 3) (Fig. 6). Comparison of VEGF expression within the same group revealed a significant difference for both the DMEM and MSC groups. No statistical difference was noted between the expanded and irradiated samples and the expanded but nonirradiated samples of VEGF-MSC group. The expanded but nonirradiated samples of the DMEM and MSC groups did not differ. The expanded and irradiated samples of all 3 groups did statistically differ. The VEGF-MSC group expressed the highest VEGF levels among all groups in expanded and irradiated samples (Table 3) (Fig. 7). The PCNA levels statistically differed within the DMEM group. No statistical differences were found between the expanded and irradiated samples and expanded but nonirradiated samples of both the MSC and VEGF-MSC groups. Comparison of both expanded and irradiated samples and expanded but nonirradiated samples between all 3 groups demonstrated a significant difference. The VEGF-MSC group expressed the highest PCNA level among all groups in expanded and irradiated samples (Table 3) (Fig. 8). Comparison of apoptosis results revealed a statistical difference within the DMEM group, whereas there was no statistical difference within the VEGF-MSC group. Apoptosis results in expanded and irradiated samples and expanded but nonirradiated samples did not differ in the MSC group. There was a significant difference between the TUNEL results of expanded and irradiated samples of all 3 groups. The highest apoptosis count was determined in the expanded and irradiated sites of the DMEM group. There was no statistical difference between the expanded but nonirradiated samples of the DMEM and MSC groups (Table 3) (Fig. 9). The samples of the DMEM group did not express GFP. No statistical difference was found between GFP expression levels of the MSC and VEGF-MSC groups in all samples. Comparison of GFP expression levels between expanded and irradiated samples and expanded but nonirradiated samples of both the MSC and VEGF-MSC groups revealed a significant difference. The highest GFP expression was found in the irradiated and treated samples of the MSC and VEGF-MSC groups (Table 3) (Fig. 10).

DISCUSSION Radiotherapy improves the survival rate of breast cancer patients. Despite controversies, radiotherapy is used widely, even for lower stage tumors. Thus, an increasing number of patients are likely to face irradiation injury in the process of cancer management.8,12,14 Reports about the optimal timing and methods of postmastectomy reconstruction, and complications arising from radiotherapy along with expander/implant reconstruction particularly for cases requiring radiotherapy, are conflicting.12,15–18 However, several studies indicate an increased incidence of surgical complications and unfavorable outcomes after irradiation. Reconstruction with expander/implant after radiotherapy is posed to increase the risk of capsular contracture and extrusion.19,20 Goodman et al16 implicated adverse effects of radiotherapy when expanders are placed in 228






The VEGF, a proangiogenic growth factor, can be expected to decrease after radiation injury; however, in some studies VEFG is increased after radiation exposure.35–37 In this study, VEGF and CD31 were increased in the expanded skin sites of all groups, and both were decreased in the irradiated skin site of the control group. The MSC treatment was effective in preserving CD31 expression at a similar level as expanded skin after irradiation injury. However, VEGF-MSC treatment significantly elevated CD31 levels in the irradiated tissue. The VEGF-MSC treatment was effective in preserving VEGF expression at a similar level as expanded skin after irradiation injury. The perfusion results were consistent with the CD31 and VEGF expressions. Radiation injury increases apoptosis while decreasing the expression of PCNA in the skin. Higher apoptosis rate and reduced PCNA expression is associated with arrested wound healing in irradiated tissue.38,39 The PCNA as a marker of proliferation also reflects DNA repair activity in the tissue.40 The PCNA levels were significantly decreased in the irradiated zone of the control group. The MSC and VEGF-MSC treatments were effective in increasing PCNA expression to the same level as the expanded skin in the irradiation injury zone. The MSC treatment was successful in preventing irradiationrelated apoptosis. The VEGF-MSC treatment was efficient in reducing both expansion- and irradiation-related apoptosis to the healthy skin sample level.

8. Kronowitz SJ, Robb GL. Radiation therapy and breast reconstruction: a critical review of the literature. Plast Reconstr Surg. 2009;124:395–408. 9. Tran NV, Chang DW, Gupta A, et al. Comparison of immediate and delayed free TRAM flap breast reconstruction in patients receiving postmastectomy radiation therapy. Plast Reconstr Surg. 2001;108:78–82. 10. Rogers NE, Allen RJ. Radiation effects on breast reconstruction with the deep inferior epigastric perforator flap. Plast Reconstr Surg. 2002;109:1919–1924. 11. Spear SL, Ducic I, Low M, et al. The effect of radiation on pedicled TRAM flap breast reconstruction: outcomes and implications. Plast Reconstr Surg. 2005; 115:84–95. 12. Cordeiro PG, Pusic AL, Disa JJ, et al. Irradiation after immediate tissue expander/ implant breast reconstruction: outcomes, complications, aesthetic results, and satisfaction among 156 patients. Plast Reconstr Surg. 2004;113:877–881. 13. Zor F, Ozturk S, Deveci M, et al. Saving the zone of stasis: Is glutathione effective? Burns. 2005;31:972–976. 14. Woodward WA, Strom EA, Tucker SL, et al. Locoregional recurrence after doxorubicin-based chemotherapy and postmastectomy: implications for breast cancer patients with early-stage disease and predictors for recurrence after postmastectomy radiation. Int J Radiat Oncol Biol Phys. 2003;57:336–344. 15. McCarthy CM, Pusic AL, Disa JJ, et al. Unilateral postoperative chest wall radiotherapy in bilateral tissue expander/implant reconstruction patients: a prospective outcomes analysis. Plast Reconstr Surg. 2005;116:1642–1647. 16. Goodman CM, Miller R, Patrick CW Jr, et al. Radiotherapy: effects on expanded skin. Plast Reconstr Surg. 2002;110:1080–1083. 17. Collier P, Williams J, Edhayan G, et al. The effect of timing of postmastectomy radiation on implant-based breast reconstruction: a retrospective comparison of complication outcomes. Am J Surg. 2014;207:408–411. 18. Ascherman JA, Hanasono MM, Newman MI, et al. Implant reconstruction in breast cancer patients treated with radiation therapy. Plast Reconstr Surg. 2006; 117:359–365. 19. Serra-Renom JM, Muñoz-Olmo JL, Serra-Mestre JM. Fat grafting in postmastectomy breast reconstruction with expanders and prostheses in patients who have received radiotherapy: formation of new subcutaneous tissue. Plast Reconstr Surg. 2010;125:12–18. 20. Forman DL, Chiu J, Restifo RJ, et al. Breast reconstruction in previously irradiated patients using tissue expanders and implants: a potentially unfavorable result. Ann Plast Surg. 1998;40:360–363. 21. Evans GR, Schusterman MA, Kroll SS, et al. Reconstruction and the radiated breast: is there a role for implants? Plast Reconstr Surg. 1995;96:1111–1115. 22. Pierce LJ. Postmastectomy radiotherapy: future directions. Semin Radiat Oncol. 1999;9:300–304. 23. Kronowitz SJ, Hunt KK, Kuerer HM, et al. Delayed-immediate breast reconstruction. Plast Reconstr Surg. 2004;113:1617–1628. 24. Kronowitz SJ. Delayed-immediate breast reconstruction: technical and timing considerations. Plast Reconstr Surg. 2010;125:463–474. 25. Kronowitz SJ, Lam C, Terefe W, et al. A multidisciplinary protocol for planned skin-preserving delayed breast reconstruction for patients with locally advanced breast cancer requiring postmastectomy radiation therapy: 3-year follow-up. Plast Reconstr Surg. 2011;127:2154–2166. 26. Crosby MA, Dong W, Feng L, et al. Effect of intraoperative saline fill volume on perioperative outcomes in tissue expander breast reconstruction. Plast Reconstr Surg. 2011;127:1065–1072. 27. Celet Ozden B, Guven E, Aslay I, et al. Does partial expander deflation exacerbate the adverse effects of radiotherapy in two-stage breast reconstruction? World J Surg Oncol. 2012;10:44. 28. Leighton WD, Russell RC, Feller AM, et al. Experimental pretransfer expansion of free-flap donor sites: II. Physiology, histology, and clinical correlation. Plast Reconstr Surg. 1988;82:76–87. 29. Austad ED, Pasyk KA, McClatchey KD, et al. Histomorphologic evaluation of guinea pig skin and soft tissue after controlled tissue expansion. Plast Reconstr Surg. 1982;70:704–710. 30. Kao JT, Dagum AB, Mahoney JL, et al. The histopathological changes in irradiated vs. nonradiated tissue-expanded skin in the porcine model. Ann Plast Surg. 1997;39:287–291. 31. Goding GS, Cummings CW, Trachy RE. Tissue expansion and cutaneous blood flow. Laryngoscope. 1988;98:919–922. 32. Manders EK, Schenden MJ, Furrey JA, et al. Soft-tissue expansion: concepts and complications. Plast Reconstr Surg. 1984;74:493–507. 33. Zeng L, Yan Z, Wang L, et al. Irradiation is an early determinant of endothelial injury during hematopoietic stem cell transplantation. Transplant Proc. 2008;40: 2661–2664. 34. Kim JW, Lee DW, Choi WH, et al. Development of a porcine skin injury model and characterization of the dose-dependent response to high-dose radiation. J Radiat Res. 2013;54:823–831.

CONCLUSIONS Histomorphometry analysis showed that MSC and VEGF-MSC treatments did not change the dermal thickness, whereas the epidermal thickness was increased with the treatments in the irradiated and expanded skin. Scintigraphy, CD31, and VEGF results showed that MSC and VEGF-MSC treatments were effective to promote angiogenesis and increase vascularity in the irradiated and expanded skin. Based on PCNA and TUNEL results, MSC and VEGF-MSC treatments also increased the proliferation and decreased the apoptosis in the skin. Vascular impairment and dermal insufficiency due to tissue expansion and irradiation injury can easily result in a wound hard to repair. The MSCs and VEGF-MSCs can promote neovascularization, reverse the effect of irradiation, and provide more durable soft tissue for expansion/implant reconstruction. ACKNOWLEDGMENT The authors thank Dr Muammer Urhan for his contribution in Scintigraphic evaluation. REFERENCES 1. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003;9:702–712. 2. Öksüz S, Ülkür E, Öncül O, et al. The effect of subcutaneous mesenchymal stem cell injection on statis zone and apoptosis in an experimental burn model. Plast Reconstr Surg. 2013;131:463–471. 3. Rehman J, Traktuev D, Li J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 2004;109:1292–1298. 4. Bentzen SM, Thames HD, Overgaard M. Latent-time estimation for late cutaneous and subcutaneous radiation reactions in a single-follow up clinical study. Radiother Oncol. 1989;15:267–274. 5. Perbeck LG, Celebioglu F, Danielsson R, et al. Circulation in the breast after radiotherapy and breast conservation. Eur J Surg. 2001;167:497. 6. Rigotti G, Marchi A, Galiè M, et al. Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived adult stem cells. Plast Reconstr Surg. 2007;119:1409–1422. 7. Rudolph R, Vande Berg J, Schneider JA, et al. Slowed growth of cultured fibroblasts from human radiation wounds. Plast Reconstr Surg. 1988;82:669–677.




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