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Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model
Acta Biomaterialia 9 (2013) 5243–5250
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Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model Yen-Chih Lin a, Tara Grahovac a, Sun Jung Oh a, Matthew Ieraci a, J. Peter Rubin a,b,c, Kacey G. Marra a,b,c,⇑ a
Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA c McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA b
a r t i c l e
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Article history: Received 14 April 2012 Received in revised form 13 September 2012 Accepted 21 September 2012 Available online 27 September 2012 Keywords: Adipose-derived stem cell Cell sheet Wound healing Full-thickness wound defect Vascularization
a b s t r a c t Cell sheet technology has been studied for applications such as bone, ligament and skin regeneration. There has been limited examination of adipose-derived stem cells (ASCs) for cell sheet applications. The specific aim of this study was to evaluate ASC sheet technology for wound healing. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue, and ASC cell sheets were created on the surface of fibrin-grafted culture dishes. In vitro examination consisted of the histochemical characterization of the ASC sheets. In vivo experiments consisted of implanting single-layer cell sheets, triplelayer cell sheets or non-treated control onto a full-thickness wound defect (including epidermis, dermis, and subcutaneous fat) in nude mice for 3 weeks. Cell sheets were easily peeled off from the culture dishes using forceps. The single- and triple-layer ASC sheets showed complete extracellular structure via hematoxylin & eosin staining. In vivo, the injury area was measured 7, 10, 14 and 21 days post-treatment to assess wound recovery. The ASC sheet-treated groups’ injury area was significantly smaller than that of the non-treated control group at all time points except day 21. The triple-layer ASC sheet treatment significantly enhanced wound healing compared to the single-layer ASC sheet at 7, 10 and 14 days. The density of blood vessels showed that ASC cell sheet treatment slightly enhanced total vessel proliferation compared to the empty wound injury treatment. Our studies indicate that ASC sheets present a potentially viable matrix for full-thickness defect wound healing in a mouse model. Consequently, our ASC sheet technology represents a substantial advance in developing various types of three-dimensional tissues. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Wound healing that will enhance skin regeneration is clinically needed. Skin damage caused by burns, cuts, abrasions, and ulcers varies in the degree of severity [1]. Creating a viable skin substitute in vitro by assembling individual components is still challenging [2]. The limitation of current skin substitutes are poor vascularization, microbial contamination, lack of drainage, lack of mechanical strength, blistering, poor healing times and inadequate cosmetic effects [3,4]. Stem cells may create a complex structure that overcomes the limitations of individual skin substitutes due to their ability to differentiate into various tissue types by asymmetric replication [2]. Every stem cell division, self-renewing capacity, whereas the other enters a differentiation pathway and joins mature nondividing populations are described as asymmetric re⇑ Corresponding author at: Department of Plastic Surgery, 1655E Biomedical Science Tower, 200 Lothrop Street, University of Pittsburgh, Pittsburgh, PA 15261, USA. Tel.: +1 412 383 8924; fax: +1 412 648 2821. E-mail address: [email protected] (K.G. Marra).
placed special property of stem cells [5]. Among the main sources of cells that might be used for repair and regeneration of injured skin are embryonic stem cells, induced pluripotent stem cells and adult stem cells [6]. Adipose-derived stem cells (ASCs) and mesenchymal stem cells (MSCs) can self-renew and differentiate into various cell lineages [7]. ASCs derived from discarded human adipose tissue are immunocompatible and multi-potent, rendering them ideal for regenerative medicine applications, such as cartilage, bone, soft tissue and nerve repair [8]. Adipose tissue may be harvested from patients in a minimally invasive manner to provide a large quantity of autologous cells [9]. ASCs possess the highest proliferation and differentiation potential, followed by MSCs derived from bone marrow and cartilage [10]. Kim et al. [11] reported that ASCs promoted human dermal fibroblast proliferation and secretion by direct cell-to-cell contact. Furthermore, ASCs can be used to treat photoaging and wound healing. ASCs accelerated the wound healing rate and decreased the numerical density of fibroblasts in diabetic rats. The mechanism by which ASCs enhance the diabetic wound healing rate is unknown [12].
1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.09.028
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Cell sheet technology has been used to enhance tissue-engineered organs in recent years [13]. The major advantage of using cell sheet technology in the tissue engineering field is customization and engineering of neo-tissue and an extracellular matrix (ECM) [13,14]. A temperature-responsive culture dish is used to make a viable cell sheet. The cell sheet can easily attach to other surfaces, such as culture dishes, other cell sheets and host tissues, due to an intrinsic ECM that is produced during in vitro culture [15]. Moreover, stem cell sheet technology has its own inherent potential for in vivo neovascularization [16]. Several studies have previously demonstrated the effects of cell sheets on wound healing [13,17,18]. In this study, we investigated the relationship of adipose stem cell sheets to wound regeneration. The use of a multi-layered cell sheet engineered in a critically sized athymic nude mice wound injury model was evaluated. Our results demonstrate that triple-layer ASC sheet treatment can significantly enhance wound healing. 2. Materials and methods 2.1. Cell isolation and cell sheet preparation Adipose tissue (100–200 g) was harvested from the superficial abdominal depots of Caucasian females (n = 3) undergoing elective abdominal reduction surgery. The patient age range was 40– 60 years old, and all were healthy according to clinical examination and laboratory tests. The University of Pittsburgh Institutional Review Board approved the adipose tissue sample collection procedure. Samples were not pooled, with each experiment using cells from three different patients. Abdominal adipose tissue was placed in a 50 ml centrifuge tube (10 g per tube) and immersed in a 1 mg ml1 solution of collagenase. The isolated tissue was chopped with scissors and incubated at 37 °C for 35 min. The digested specimen was filtered, then centrifuged at 1000 rpm for 10 min. The pellet was suspended in erythrocyte lysis buffer. The solution was centrifuged again at 178 gravities for 10 min. The pellet, which contained mesenchymal stem cells, was cultured in Dulbecco’s modified Eagle’s medium/F12 with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were passaged at confluency and characterized as previously described by our laboratory [19]. We followed the modified commercial product protocol provided by Thermo Scientific Nunc Upcell Surface to fabricate the cell sheets. To create the cell sheet, 1 106 human ASCs were seeded onto a temperature-responsive cell surface plate (Thermo Scientific Nunc Upcell Surface) to a form a confluent layer. All medium was aspirated and PBS was added to prevent the cells from drying out. A fibrin-coated membrane was gently placed on top of the cell layer. The plate was incubated at 25 °C for 10 min. The cell layer was detached with sterile forceps and the membrane was transferred to another cell sheet layer with the attached cell layer facing downwards. The two attached cell sheet layers were cultured at 37 °C for at least 30 min, then 1 ml of the culture medium was added on top of the membrane and the membrane was gently withdrawn from the cell layer. The previous steps were repeated to create a three-layer ASC cell sheet. 2.2. Gene expression RNA isolated during ASC cell sheet fabrication was used for quantitative polymerase chain reaction (qPCR). RNA was collected using a Qiagen RNeasy Mini Kit (Qiagen, USA). Approximately 200 ng of total RNA was reverse-transcribed into cDNA using a First Strand Transcription Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The PCR primers were designed using the Vector NTI (Invitrogen, Carlsbad, CA, USA) and
synthesized by Invitrogen. Efficiency was checked from tenfold serial dilutions of cDNA for each primer pair. A 2 SYBRÒ Green PCR Mastermix (Invitrogen, Carlsbad, CA, USA), 0.1 lM of each primer and the cDNA template were mixed in 25 ll volumes. qPCR was performed in triplicate in 96-well optical plates on Light Cycler 480 (Applied System, USA). b-Actin and mature adipocytes were used as inter-control and intra-control for qPCR analyses, respectively. The gene expression of mature adipocytes was normalized to 1. 2.3. In vivo studies Athymic nude mice (6 weeks old, n = 18) were housed individually and received standard rat chow (Rodent laboratory chow 5001, Purina Co., USA) and water ad libitum in the Division of Laboratory Animal Resources at the University of Pittsburgh. All in vivo experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. The mice were kept in a room with a constant temperature and a 12 h light–dark cycle. After a one-week acclimatization period, the animals were randomly divided into three groups: group 1: non-treated control (control) (n = 6); group 2: one-layer ASC cell sheet treatment (n = 6); and group 3: three-layer ASC cell sheet treatment (n = 6). 2.4. Surgical procedure Each animal was weighed and anesthetized with ketamine (80 mg kg1) and xylazine (12 mg kg1) intraperitoneally. The surgical area cleaned with 70% ethanol. While the mice were anesthetized, a sterile technique was used to create two 12 mm diameter full-thickness surgical skin wounds to include epidermis, dermis and subcutaneous fat to the level of the panniculus carnosus on the back, which were standardized by using a 12 mm biopsy punch. Each mouse received the same treatment in both of his wounds. A donut-shaped silicone splint with a 12 mm diameter was centered on the wound injury and fixed to the skin using 50 nylon sutures (Ethicon Inc., Somerville, NJ) until day 21. The ASC cell sheet was applied to the wounds once immediately after wounding. Non-occlusive dressings were not used on the wound. The wounds were digitally photographed on days 0, 7, 14 and 18, and until the wound was completely healed (21 days). These time points were selected based on our preliminary results. The photographs were analyzed for healing progress, as assessed by changes in the wound surface area. At each time point, animals from each group were sacrificed and a 3 cm 3 cm square was harvested from the center of each wound. The harvested wounds were fixed overnight in 4% paraformaldehyde at 4 °C for further analysis. An identical procedure was repeated for the triple-layer cell sheet wound treatment. 2.5. Histochemistry Five micron sections were prepared from all paraffin skin blocks. The sections were deparaffinized with three immersions in xylene, then hydrated with descending concentrations of ethanol (100%, 90% and 70% to distilled water). Slides then were stained using different methods. A modified protocol was used to stain sections with hematoxylin & eosin (H&E) [20], Masson’s trichrome [21] and Herovici’s stain [22]. 2.6. Human A/C protein To identify human ASCs, sections of tissue were blocked with 5% horse serum for 1 h at room temperature. Human lamin A/C protein is a type V intermediate filament protein. Mouse anti-human lamin A/C (Vector Laboratories) in 2.5% horse serum stain reagent
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was added to the slides and the slides were incubated overnight at 4 °C. The slides were then treated with biotinylated anti-mouse IgG (Vector Laboratories) for 90 min at room temperature. Streptavidin–fluorescein isothiocyanate (Invitrogen) was added and the slides were incubated for 20 min at room temperature. 2.7. Human and mouse CD31+ To determine the endothelial membrane protein of human or mouse blood vessels, samples were stained with anti-human or anti-mouse CD31+ protein, respectively. Sections were incubated in proteinase K (Millipore, USA) at room temperature for 20 min, then 5% bovine serum albumin (BSA) solution was added to sections for 10 min for blocking. Primary anti-human (GAH, Santa Cruz) and anti-mouse CD31+ (abcam) antibody in phosphate-buffered saline were added to the slides, which were then incubated for 2 h at room temperature. Secondary antibody, biotinylated rabbit anti-goat IgG antibody (RAG, Vector Labs) and polyclonal rabbit anti-rat immunoglobulins/horseradish peroxidase (Dako) were added and the slides further incubated for 1 h at room temperature. The samples were developed with DAB chromogen and substrate under microscope. Once desired signal to noise ratio is achieved, stop the reaction by washing slides in deionized water. The images were analyzed for vascular density (six images per biopsies, three biopsies from per group), then captured using the program MagnaFire 2.1B (Olympus). They were then analyzed with the software ImageJ to determine the extent of human and mouse CD31+ staining, showing the vascularization per square millimetre, in the injury site using the following formula:
vascular density ¼ the amount of total positive staining vascular=injury area 2.8. Keratinocyte staining Sections were blocked with 10% normal serum for 2 h at room temperature. Anti-cytokeratin 10 monoclonal antibody (abcam, ab9026) or Anti-Ki67 monoclonal antibody (abcam, ab16667) diluted in 1% BSA–Tris-buffered saline solution was added to the slides and the slides were incubated overnight at 4 °C. A secondary fluorescein IgG antibody Cy3 (Jackson Immuno Research Labs, USA) or IgG Chromeo™ 488 was added for the detection of the K10 or Ki67 protein, respectively. All stained samples were evaluated at 20 magnification using a microscope (Olympus Provis AX-70; Olympus America) equipped with a camera (Olympus U-MAD 2; Olympus Japan). The images
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were captured using the program MagnaFire 2.1B (Olympus) and analyzed with the software ImageJ. 2.9. Statistics All results are presented as mean ± standard deviation. All data was analyzed by a GLM programmer and the analysis of variance test contained in the SAS system (2000) was used to determine statistical significance between experimental groups. Statistical significance was set at a p 6 0.05. 3. Results Cell sheets were easily removed from the dishes using forceps (Fig. 1a). In vitro experiments investigated the gene expressed characterization of the ASC sheets. Fig. 2 shows qPCR results comparing gene expression ratios for FABP4 (a mature adipocyte marker), PPAR-c (a mature adipocyte marker) and CD34+ (MSC marker) gene expression detection from one-layer ASC cell sheets, three-layer ASC cell sheets and mature adipocytes. There were significant differences in FABP4 and PPAR-c gene expression between cell sheets and mature adipocytes. There were no significant differences in FABP4, PPAR-c and CD34+ gene expression between the cell sheets and two-dimensional (2-D) cultured cells. In vivo experiments consisted of implanting single-layer sheets, triple-layer sheets or non-treated control onto a full-thickness wound defect in nude mice for 3 weeks (Fig. 1b and c). The injury area was measured 7, 10, 14 and 21 days post-treatment to evaluate wound recovery (Fig. 3). The ASC sheet-treated group’s wound sizes were significantly smaller than those of the non-treated control group at all time points except day 21. The triple-layer ASC sheet treatment significantly enhanced wound healing compared to the single-layer ASC sheet at days 7, 10 and 14, demonstrating that the ASC cell sheet treatment significantly enhanced cell proliferation and migration compared to the non-treated control group. The three-layer ASC cell sheet increased healing by 1.10-, 1.06- and 1.07-fold compared to the one-layer ASC cell sheet at post-operation days 7, 10 and 14, respectively. Human lamin A/C protein immunostaining demonstrated that human adipose stem cells survived in athymic nude mice after post-operation day 21 (Fig. 4). ASC cell sheet structure was not observed in the wound injury site at day 21 post-wound. Histochemical staining (H&E (Fig. 5A), Masson’s trichrome (Fig. 5B) and Herovici’s (Fig. 5C)) showed the completed wound healing in all three groups after day 21. Moreover, more new collagen was identified in the ASC cell sheet treatment groups compared to non-treated control groups. The analysis of
Fig. 1. Representative macroscopic view of a cell sheet. ASC sheets were implanted on a 12 mm diameter wound injury in female athymic nude mice. After 21 days, wound grafts were explanted and fixed for histological analysis. (A) ASC cell sheet cultured in a Petri dish with a temperature-responsive surface. (B) Three-layer ASC cell sheet; white arrows indicate the edge of cell sheet. (C) Empty wound injury treatment (control).
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blood vessel density showed that the ASC cell sheet treatments slightly enhanced (no significant difference) total vessel amounts compared to the empty wound injury treatment (Figs. 6 and 8). One-layer ASC cell sheets and three-layer ASC cell sheets increased the total vessel amounts compared the non-treated control group, by 15% and 22.5%, respectively (no significant difference). Fig. 7 shows keratinocyte immunostaining (Ki67 and K10 antibody) of the ASC cell sheet treatment group and the non-treated control group at post-operative day 21. The results show the phenomena of keratinocyte proliferation and migration in both the cell sheet treatment and non-treated control groups.
4. Discussion
Fig. 2. qPCR results comparing gene expression ratios for FABP4 (a mature adipocyte marker), PPAR-c (a mature adipocyte marker) and CD34+ (an MSC marker) gene expression detection from one- and three-layer ASC cell sheets and mature adipocytes. The gene expressions of the mature adipocytes were normalized to 1 as the intra-control baseline. ⁄,#Significantly different between groups, p < 0.05.
Cell sheets created 3-D structures via the layering of individual sheets and were implanted directly to host tissues [23]. Traditional tissue engineering designs have focused on biodegradable scaffolds to support tissue formation [20,23]. However, biodegradable scaffolds still present many clinical challenges, such as poor vascularization, microbial contamination and lack of drainage [3] in critically sized wound injury areas. Cell sheets have been used
Fig. 3. The macroscopic pictures of the wounds at specific time points (7, 10, 14 and 21 days) and the area of full-thickness wound injury for the control and one- and threelayer ASC cell sheets implanted in athymic nude mice at 0, 7, 10, 14, 18 and 21 days. All data reported as mean ± SD, ⁄,#p < 0.05.
Fig. 4. Representative immunohistochemistry of a cross-section in the wound site at day 21 post-wound with human lamin A/C staining. Immunostaining (red) of one- and three-layer ASC cell sheets and control treatment (20 magnification) with human lamin A/C protein (red, human cell marker). (A) Control; (B) one-layer ASC cell sheet treatment; (C) three-layer ASC cell sheet treatment. Scale bar = 500 lm.
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Fig. 5. Representative histochemistry of cross-section in the wound site at day 21 post-wound with H&E staining (A), Masson’s trichrome staining (B) and Herovici’s staining (C) of one- and three-layer ASC cell sheets and control treatment. (A) Nuclei are stained blue; cytoplasm, pink to red; (B) collagen is stained blue; cytoplasm or keratin, red; and nuclei, black; (C) young collagen is stained blue; mature collagen, red; cytoplasm, yellow; and nuclei, black. Black arrows indicate the center of the wound injury. 20 magnification. Scale bar = 500 lm.
Fig. 6. Representative immunohistochemistry of a cross-section in the wound site at day 21 post-wound with human CD31+ staining. Immunostaining of one- and threelayer ASC cell sheets and control treatment (20 magnification) with human CD31+ marker (dark brown) and nuclei (black). White and black arrows indicate the human CD31- and mouse CD31-positive staining, respectively. E, epidermis; M, murine muscle. Scale bar = 500 lm.
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Fig. 7. Representative immunohistochemistry of a cross-section in the wound injury site at day 21 post-wound with keratinocyte staining. Immunostaining of one- and three-layer ASC cell sheets and control treatment (20 and 40 magnification) with Ki67 and K10 antibody (red). White triangles indicate the center of the wound injury. White arrows indicate the Ki67- and K10-positive staining, respectively. Scale bar = 500 lm. e, epidermal portion.
for wound healing due to their ability to self-assemble, allowing them to produce vascularization and skin equivalents [13,24,25]. Use of temperature-responsive surfaces for cultured cell sheets harvested without trypsin or dispase can result in cell damage and loss of differentiated phenotypes [26,27]. Yang et al. [27] further described that cell sheet technology seemingly provides several advantages over traditional regenerative therapies of cell injection and tissue reconstruction with biodegradable scaffolds. Stem cells have been used for bone, ligament and wound tissueengineering research in recent years [28]. ASCs derived from discarded human adipose tissue are immunocompatible and multipotent, rendering them ideal for regenerative medicine applications, such as cartilage, bone, soft tissue and nerve repair [8]. Zografou et al. [29] reported that autologous ASC transplantation increases full-thickness skin-graft survival and shows promise for use in skin-graft surgery due to in situ differentiation of ASCs into endothelial cells, and increased vascular endothelial growth factor and transforming growth factor b3 secretion by ASCs. Moreover, ASCs promoted dermal fibroblast proliferation by direct cell-to-cell contact and paracrine activation through secretory factors [11]. In
our study, we demonstrated that the ASCs in cell sheet types were significantly viable, supported the development of skin equivalents and enhanced wound healing compared to the non-treated control group (Fig. 5). Stem cells are defined by their ability to either self-renew or differentiate into multiple cell lineages [30]. Mesenchymal stem cells are often targeted for tissue engineering due to their vital role in native tissue formation and function [31]. Three-dimensional high-density primary adipose stem cell culture leads to completion of adipose tissue formation in vitro over a long-term period [28]. qPCR results compared gene expression ratios for FABP4 (a mature adipocyte marker), PPAR-c (a mature adipocyte marker) and CD34+(an MSC marker) gene expression detection from one-layer ASC cell sheets, three-layer ASC cell sheets and mature adipocytes. The results showed that the FABP4 and PPAR-c gene expression of cell sheet treatment are significantly lower than that of mature adipocytes. Our preliminary data showed that there is no significant difference between five-layer cell sheets and mature adipocytes in FABP4 and PPAR-c gene expression. Based on these results, one- and three-layer ASC cell sheets were investigated in
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are highly prolific and migratory at the wound boundaries, with the purpose of restoring the epidermis’s barrier function by closing the wound in the shortest time [13,39,40]. Langbein and Schweizer [41] reported that the epithelial keratins are expressed in various tissue constituents of the external sheaths and the companion layer of the follicle. Qi et al. [42] identified hair follicles on the epithelial surface in their nude mouse skin defect model. The keratinocytes and papilla cells may play important roles in the induction of hair follicle formation after full-thickness wound injury. Overall, we have demonstrated the utilization of multi-layer stem cell sheet technology to improve in vivo wound healing. 5. Conclusion
Fig. 8. The vascular density for control and one- and three-layer ASC cell sheets implanted in athymic nude mice at day 21 post-wound. All data reported as mean ± SD.
our wound healing model. In order to characterize the ‘‘stemness’’ properties of the ASC sheet, CD34+ gene expression was investigated. CD34+ adipose-derived stem cells have been hypothesized to be resident pericytes that play a role in vascular stabilization by mutual structural and functional interaction with endothelial cells [32]. Li et al. [33] isolated four distinct stromal populations from human adult adipose tissue and characterized their adipogenic potential. Of the four populations, the CD31-/CD34+ group is the most prevalent and has the greatest potential for adipogenic differentiation. In our study, there is no significant difference in CD34+ gene expression between cell sheets and 2-D cultured cells. ASC sheets maintained the ability of differentiation. A donut-shaped silicone splint technology was used on the wound injury and fixed to the skin using 5-0 nylon sutures to prevent the potential wound contraction. Previously, Nie et al. [34] reported that the rat model with silicone splint fixed on the skin can prevent wound contraction and allows the wound to heal by epithelialization and granulation tissue formation. Boyce et al. [35] further demonstrated that keratinocyte proliferation can enhance the extracellular matrix secretion to reduce the skin contraction. One limitation of current skin substitutes is poor vascularization [3]. Branski et al. [2] point out stem cell therapy can create a suitable skin substitute to enhance vascularization in neo-tissue. Lee et al. [36] and Kim et al. [11] reported that the potential mechanisms of ASCs to promote wound healing are via angiogenic factor production and angiogenesis. In our study, ASC cell sheet treatment slightly enhanced vascular density compared to the nontreated control treatment (Fig. 8). However, there is no significant difference between all groups. This result also demonstrates that a possible mechanism of ASC cell sheet treatment in wound healing is increased vascularization of the injured site. Lai et al. [37] also reported that a PKH26-labeled human corneal endothelial cell sheet can significantly enhance injury healing and vascularization in a rabbit cornea model. Moreover, Herovici’s staining (Fig. 5C) showed that ASC cell sheet treatments significantly enhanced cell secretion at the epidermis/dermis interface compared to the nontreated control group. Maharlooei et al. [12] reported that adipose tissue-derived stromal cells improve the wound healing rate by collagen accumulation in the wound tissue. Kim et al. [38] further demonstrated that the wound healing and antioxidant effects of ASCs are mainly mediated by the activation of dermal fibroblasts and keratinocytes. In our study, keratinocytes had proliferated and migrated to the periphery of neo-dermis (Fig. 7). This phenomenon is in agreement with the understanding that keratinocytes
Our study demonstrates that multi-layer ASC sheets present a potentially viable matrix for full-thickness defect wound healing in a mouse model. Consequently, our ASC sheet technology provides an important advance in developing various types of tissue regeneration. While this study targets the use of autologous stem cells, future studies will determine if an allogeneic therapy would be possible, as some patients may not have sufficient adipose stem cells or be suitable for stem cell harvesting. Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1–8, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2012. 09.028. References [1] Walter MNM, Wright KT, Fuller HR, MacNeil S, Johnson WEB. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell Res 2010;316:1271–81. [2] Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns 2009;35:171–80. [3] Jones I, Currie L, Martin R. A guide to biological skin substitutes. Br J Plast Surg 2002;55:185–93. [4] Garcia Y, Wilkins B, Collighan RJ, Griffin M, Pandit A. Towards development of a dermal rudiment for enhanced wound healing response. Biomaterials 2008;29:857–68. [5] Cha J, Falanga V. Stem cells in cutaneous wound healing. Clin Dermatol 2007;25:73–8. [6] Chen M, Przyborowski M, Berthiaume F. Stem cells for skin tissue engineering and wound healing. Crit Rev Biomed Eng 2009;37:399–421. [7] Bajada S, Mazakova I, Richardson JB, Ashammakhi N. Updates on stem cells and their applications in regenerative medicine. J Tissue Eng Regen Med 2008;20:169–83. [8] Santiago LY, Nowak RW, Rubin JP, Marra KG. Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications. Biomaterials 2006;27:2962–9. [9] Wei Y, Wei YH, Han HY, Meng G, Zhang D, Wu Z, et al. A novel injectable scaffold for cartilage tissue engineering using adipose-derived adult stem cells. J Orthop Res 2008;26:27–33. [10] Peng L, Jia Z, Yin X, Zhang X, Liu Y, Chen P, et al. Comparative analysis of mesenchymal stem cells from bone marrow, cartilage and adipose tissue. Stem Cells Dev 2008;17:761–73. [11] Kim WS, Park BS, Sung JH, Yang JM, Park SB, Kwak SJ, et al. Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci 2007;48:15–24. [12] Maharlooei MK, Bagheri M, Solhjou Z, Jahromi BM, Akrami M, Rohani L, et al. Adipose tissue derived mesenchymal stem cell (AD-MSC) promotes skin wound healing in diabetic rats. Diabetes Res Clin Pract 2011;93:228–34. [13] Ng KW, Hutmacher DW. Reduced contraction of skin equivalent engineered using cell sheets cultured in 3D matrices. Biomaterials 2006;27:4591–8. [14] Laplante AF, Germain L, Auger FA, Moulin V. Mechanisms of wound reepithelialization: hints from a tissue-engineered reconstructed skin to long-standing questions. FASEB J 2001;15:2377–89. [15] Sasagawa T, Shimizu T, Sekiya S, Haraguchi Y, Yamato M, Sawa Y, et al. Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials 2010;31:1646–54.
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adipose-derived stem cells in rats. J Plast Reconstr Aesthet Surg 2011;64:1647–56. King JA, Miller WM. Bioreactor development for stem cell expansion and controlled differentiation. Curr Opin Chem Biol 2007;11:394–8. Liao J, Guo X, Grande-Allen KJ, Kasper FK, Mikos AG. Bioactive polymer/ extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for cartilage tissue engineering. Biomaterials 2010;31:8911–20. Traktuev DO, Merfeld-Clauss S, Li J, Kolonin M, Arap W, Pasqualini R, et al. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res 2008;102:77–85. Li H, Zimmerlin L, Marra KG, Donnenberg VS, Donnenberg AD, Rubin JP. Adipogenic potential of adipose stem cell subpopulations. Plast Reconstr Surg 2011;128:663–72. Nie C, Yang D, Xu J, Si Z, Jin X, Zhang J. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. Cell Transplant 2011;20:205–16. Boyce ST, Supp AP, Swope VB, Warden GD. Vitamin C regulates keratinocyte viability, epidermal barrier, and basement membrane in vitro, and reduces wound contraction after grafting of cultured skin substitutes. J Investigative Dermatology 2002;118:565–72. Lee SH, Lee JH, Cho KH. Effects of human adipose-derived stem cells on cutaneous wound healing in nude mice. Ann Dermatol 2011;23:150–5. Lai J-Y, Chen K-H, Hsiue G-H. Tissue-engineered human corneal endothelial cell sheet transplantation in a rabbit model using functional biomaterials. Transplantation 2007;84:1222–32. Kim WS, Park BS, Sung JH. The wound-healing and antioxidant effects of adipose-derived stem cells. Expert Opin Biol Ther 2009;9:879–87. Kirfel G, Herzog V. Migration of epidermal keratinocytes: mechanisms, regulation, and biological significance. Protoplasma 2004;223:67–78. Santoro MM, Gaudino G. Cellular and molecular facets of keratinocyte reepithelization during wound healing. Exp Cell Res 2005;304:274–86. Langbein L, Schweizer J. Keratins of the human hair follicle. International Review of Cytology 2005;243:1–78. Qi S-H, Liu P, Xie J-L, Shu B, Xu Y-B, Ke C-N, et al. Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells. Burns 2008;34:385–92.
Contents lists available at SciVerse ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model Yen-Chih Lin a, Tara Grahovac a, Sun Jung Oh a, Matthew Ieraci a, J. Peter Rubin a,b,c, Kacey G. Marra a,b,c,⇑ a
Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA c McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA b
a r t i c l e
i n f o
Article history: Received 14 April 2012 Received in revised form 13 September 2012 Accepted 21 September 2012 Available online 27 September 2012 Keywords: Adipose-derived stem cell Cell sheet Wound healing Full-thickness wound defect Vascularization
a b s t r a c t Cell sheet technology has been studied for applications such as bone, ligament and skin regeneration. There has been limited examination of adipose-derived stem cells (ASCs) for cell sheet applications. The specific aim of this study was to evaluate ASC sheet technology for wound healing. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue, and ASC cell sheets were created on the surface of fibrin-grafted culture dishes. In vitro examination consisted of the histochemical characterization of the ASC sheets. In vivo experiments consisted of implanting single-layer cell sheets, triplelayer cell sheets or non-treated control onto a full-thickness wound defect (including epidermis, dermis, and subcutaneous fat) in nude mice for 3 weeks. Cell sheets were easily peeled off from the culture dishes using forceps. The single- and triple-layer ASC sheets showed complete extracellular structure via hematoxylin & eosin staining. In vivo, the injury area was measured 7, 10, 14 and 21 days post-treatment to assess wound recovery. The ASC sheet-treated groups’ injury area was significantly smaller than that of the non-treated control group at all time points except day 21. The triple-layer ASC sheet treatment significantly enhanced wound healing compared to the single-layer ASC sheet at 7, 10 and 14 days. The density of blood vessels showed that ASC cell sheet treatment slightly enhanced total vessel proliferation compared to the empty wound injury treatment. Our studies indicate that ASC sheets present a potentially viable matrix for full-thickness defect wound healing in a mouse model. Consequently, our ASC sheet technology represents a substantial advance in developing various types of three-dimensional tissues. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Wound healing that will enhance skin regeneration is clinically needed. Skin damage caused by burns, cuts, abrasions, and ulcers varies in the degree of severity [1]. Creating a viable skin substitute in vitro by assembling individual components is still challenging [2]. The limitation of current skin substitutes are poor vascularization, microbial contamination, lack of drainage, lack of mechanical strength, blistering, poor healing times and inadequate cosmetic effects [3,4]. Stem cells may create a complex structure that overcomes the limitations of individual skin substitutes due to their ability to differentiate into various tissue types by asymmetric replication [2]. Every stem cell division, self-renewing capacity, whereas the other enters a differentiation pathway and joins mature nondividing populations are described as asymmetric re⇑ Corresponding author at: Department of Plastic Surgery, 1655E Biomedical Science Tower, 200 Lothrop Street, University of Pittsburgh, Pittsburgh, PA 15261, USA. Tel.: +1 412 383 8924; fax: +1 412 648 2821. E-mail address: [email protected] (K.G. Marra).
placed special property of stem cells [5]. Among the main sources of cells that might be used for repair and regeneration of injured skin are embryonic stem cells, induced pluripotent stem cells and adult stem cells [6]. Adipose-derived stem cells (ASCs) and mesenchymal stem cells (MSCs) can self-renew and differentiate into various cell lineages [7]. ASCs derived from discarded human adipose tissue are immunocompatible and multi-potent, rendering them ideal for regenerative medicine applications, such as cartilage, bone, soft tissue and nerve repair [8]. Adipose tissue may be harvested from patients in a minimally invasive manner to provide a large quantity of autologous cells [9]. ASCs possess the highest proliferation and differentiation potential, followed by MSCs derived from bone marrow and cartilage [10]. Kim et al. [11] reported that ASCs promoted human dermal fibroblast proliferation and secretion by direct cell-to-cell contact. Furthermore, ASCs can be used to treat photoaging and wound healing. ASCs accelerated the wound healing rate and decreased the numerical density of fibroblasts in diabetic rats. The mechanism by which ASCs enhance the diabetic wound healing rate is unknown [12].
1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.09.028
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Cell sheet technology has been used to enhance tissue-engineered organs in recent years [13]. The major advantage of using cell sheet technology in the tissue engineering field is customization and engineering of neo-tissue and an extracellular matrix (ECM) [13,14]. A temperature-responsive culture dish is used to make a viable cell sheet. The cell sheet can easily attach to other surfaces, such as culture dishes, other cell sheets and host tissues, due to an intrinsic ECM that is produced during in vitro culture [15]. Moreover, stem cell sheet technology has its own inherent potential for in vivo neovascularization [16]. Several studies have previously demonstrated the effects of cell sheets on wound healing [13,17,18]. In this study, we investigated the relationship of adipose stem cell sheets to wound regeneration. The use of a multi-layered cell sheet engineered in a critically sized athymic nude mice wound injury model was evaluated. Our results demonstrate that triple-layer ASC sheet treatment can significantly enhance wound healing. 2. Materials and methods 2.1. Cell isolation and cell sheet preparation Adipose tissue (100–200 g) was harvested from the superficial abdominal depots of Caucasian females (n = 3) undergoing elective abdominal reduction surgery. The patient age range was 40– 60 years old, and all were healthy according to clinical examination and laboratory tests. The University of Pittsburgh Institutional Review Board approved the adipose tissue sample collection procedure. Samples were not pooled, with each experiment using cells from three different patients. Abdominal adipose tissue was placed in a 50 ml centrifuge tube (10 g per tube) and immersed in a 1 mg ml1 solution of collagenase. The isolated tissue was chopped with scissors and incubated at 37 °C for 35 min. The digested specimen was filtered, then centrifuged at 1000 rpm for 10 min. The pellet was suspended in erythrocyte lysis buffer. The solution was centrifuged again at 178 gravities for 10 min. The pellet, which contained mesenchymal stem cells, was cultured in Dulbecco’s modified Eagle’s medium/F12 with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were passaged at confluency and characterized as previously described by our laboratory [19]. We followed the modified commercial product protocol provided by Thermo Scientific Nunc Upcell Surface to fabricate the cell sheets. To create the cell sheet, 1 106 human ASCs were seeded onto a temperature-responsive cell surface plate (Thermo Scientific Nunc Upcell Surface) to a form a confluent layer. All medium was aspirated and PBS was added to prevent the cells from drying out. A fibrin-coated membrane was gently placed on top of the cell layer. The plate was incubated at 25 °C for 10 min. The cell layer was detached with sterile forceps and the membrane was transferred to another cell sheet layer with the attached cell layer facing downwards. The two attached cell sheet layers were cultured at 37 °C for at least 30 min, then 1 ml of the culture medium was added on top of the membrane and the membrane was gently withdrawn from the cell layer. The previous steps were repeated to create a three-layer ASC cell sheet. 2.2. Gene expression RNA isolated during ASC cell sheet fabrication was used for quantitative polymerase chain reaction (qPCR). RNA was collected using a Qiagen RNeasy Mini Kit (Qiagen, USA). Approximately 200 ng of total RNA was reverse-transcribed into cDNA using a First Strand Transcription Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The PCR primers were designed using the Vector NTI (Invitrogen, Carlsbad, CA, USA) and
synthesized by Invitrogen. Efficiency was checked from tenfold serial dilutions of cDNA for each primer pair. A 2 SYBRÒ Green PCR Mastermix (Invitrogen, Carlsbad, CA, USA), 0.1 lM of each primer and the cDNA template were mixed in 25 ll volumes. qPCR was performed in triplicate in 96-well optical plates on Light Cycler 480 (Applied System, USA). b-Actin and mature adipocytes were used as inter-control and intra-control for qPCR analyses, respectively. The gene expression of mature adipocytes was normalized to 1. 2.3. In vivo studies Athymic nude mice (6 weeks old, n = 18) were housed individually and received standard rat chow (Rodent laboratory chow 5001, Purina Co., USA) and water ad libitum in the Division of Laboratory Animal Resources at the University of Pittsburgh. All in vivo experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. The mice were kept in a room with a constant temperature and a 12 h light–dark cycle. After a one-week acclimatization period, the animals were randomly divided into three groups: group 1: non-treated control (control) (n = 6); group 2: one-layer ASC cell sheet treatment (n = 6); and group 3: three-layer ASC cell sheet treatment (n = 6). 2.4. Surgical procedure Each animal was weighed and anesthetized with ketamine (80 mg kg1) and xylazine (12 mg kg1) intraperitoneally. The surgical area cleaned with 70% ethanol. While the mice were anesthetized, a sterile technique was used to create two 12 mm diameter full-thickness surgical skin wounds to include epidermis, dermis and subcutaneous fat to the level of the panniculus carnosus on the back, which were standardized by using a 12 mm biopsy punch. Each mouse received the same treatment in both of his wounds. A donut-shaped silicone splint with a 12 mm diameter was centered on the wound injury and fixed to the skin using 50 nylon sutures (Ethicon Inc., Somerville, NJ) until day 21. The ASC cell sheet was applied to the wounds once immediately after wounding. Non-occlusive dressings were not used on the wound. The wounds were digitally photographed on days 0, 7, 14 and 18, and until the wound was completely healed (21 days). These time points were selected based on our preliminary results. The photographs were analyzed for healing progress, as assessed by changes in the wound surface area. At each time point, animals from each group were sacrificed and a 3 cm 3 cm square was harvested from the center of each wound. The harvested wounds were fixed overnight in 4% paraformaldehyde at 4 °C for further analysis. An identical procedure was repeated for the triple-layer cell sheet wound treatment. 2.5. Histochemistry Five micron sections were prepared from all paraffin skin blocks. The sections were deparaffinized with three immersions in xylene, then hydrated with descending concentrations of ethanol (100%, 90% and 70% to distilled water). Slides then were stained using different methods. A modified protocol was used to stain sections with hematoxylin & eosin (H&E) [20], Masson’s trichrome [21] and Herovici’s stain [22]. 2.6. Human A/C protein To identify human ASCs, sections of tissue were blocked with 5% horse serum for 1 h at room temperature. Human lamin A/C protein is a type V intermediate filament protein. Mouse anti-human lamin A/C (Vector Laboratories) in 2.5% horse serum stain reagent
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was added to the slides and the slides were incubated overnight at 4 °C. The slides were then treated with biotinylated anti-mouse IgG (Vector Laboratories) for 90 min at room temperature. Streptavidin–fluorescein isothiocyanate (Invitrogen) was added and the slides were incubated for 20 min at room temperature. 2.7. Human and mouse CD31+ To determine the endothelial membrane protein of human or mouse blood vessels, samples were stained with anti-human or anti-mouse CD31+ protein, respectively. Sections were incubated in proteinase K (Millipore, USA) at room temperature for 20 min, then 5% bovine serum albumin (BSA) solution was added to sections for 10 min for blocking. Primary anti-human (GAH, Santa Cruz) and anti-mouse CD31+ (abcam) antibody in phosphate-buffered saline were added to the slides, which were then incubated for 2 h at room temperature. Secondary antibody, biotinylated rabbit anti-goat IgG antibody (RAG, Vector Labs) and polyclonal rabbit anti-rat immunoglobulins/horseradish peroxidase (Dako) were added and the slides further incubated for 1 h at room temperature. The samples were developed with DAB chromogen and substrate under microscope. Once desired signal to noise ratio is achieved, stop the reaction by washing slides in deionized water. The images were analyzed for vascular density (six images per biopsies, three biopsies from per group), then captured using the program MagnaFire 2.1B (Olympus). They were then analyzed with the software ImageJ to determine the extent of human and mouse CD31+ staining, showing the vascularization per square millimetre, in the injury site using the following formula:
vascular density ¼ the amount of total positive staining vascular=injury area 2.8. Keratinocyte staining Sections were blocked with 10% normal serum for 2 h at room temperature. Anti-cytokeratin 10 monoclonal antibody (abcam, ab9026) or Anti-Ki67 monoclonal antibody (abcam, ab16667) diluted in 1% BSA–Tris-buffered saline solution was added to the slides and the slides were incubated overnight at 4 °C. A secondary fluorescein IgG antibody Cy3 (Jackson Immuno Research Labs, USA) or IgG Chromeo™ 488 was added for the detection of the K10 or Ki67 protein, respectively. All stained samples were evaluated at 20 magnification using a microscope (Olympus Provis AX-70; Olympus America) equipped with a camera (Olympus U-MAD 2; Olympus Japan). The images
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were captured using the program MagnaFire 2.1B (Olympus) and analyzed with the software ImageJ. 2.9. Statistics All results are presented as mean ± standard deviation. All data was analyzed by a GLM programmer and the analysis of variance test contained in the SAS system (2000) was used to determine statistical significance between experimental groups. Statistical significance was set at a p 6 0.05. 3. Results Cell sheets were easily removed from the dishes using forceps (Fig. 1a). In vitro experiments investigated the gene expressed characterization of the ASC sheets. Fig. 2 shows qPCR results comparing gene expression ratios for FABP4 (a mature adipocyte marker), PPAR-c (a mature adipocyte marker) and CD34+ (MSC marker) gene expression detection from one-layer ASC cell sheets, three-layer ASC cell sheets and mature adipocytes. There were significant differences in FABP4 and PPAR-c gene expression between cell sheets and mature adipocytes. There were no significant differences in FABP4, PPAR-c and CD34+ gene expression between the cell sheets and two-dimensional (2-D) cultured cells. In vivo experiments consisted of implanting single-layer sheets, triple-layer sheets or non-treated control onto a full-thickness wound defect in nude mice for 3 weeks (Fig. 1b and c). The injury area was measured 7, 10, 14 and 21 days post-treatment to evaluate wound recovery (Fig. 3). The ASC sheet-treated group’s wound sizes were significantly smaller than those of the non-treated control group at all time points except day 21. The triple-layer ASC sheet treatment significantly enhanced wound healing compared to the single-layer ASC sheet at days 7, 10 and 14, demonstrating that the ASC cell sheet treatment significantly enhanced cell proliferation and migration compared to the non-treated control group. The three-layer ASC cell sheet increased healing by 1.10-, 1.06- and 1.07-fold compared to the one-layer ASC cell sheet at post-operation days 7, 10 and 14, respectively. Human lamin A/C protein immunostaining demonstrated that human adipose stem cells survived in athymic nude mice after post-operation day 21 (Fig. 4). ASC cell sheet structure was not observed in the wound injury site at day 21 post-wound. Histochemical staining (H&E (Fig. 5A), Masson’s trichrome (Fig. 5B) and Herovici’s (Fig. 5C)) showed the completed wound healing in all three groups after day 21. Moreover, more new collagen was identified in the ASC cell sheet treatment groups compared to non-treated control groups. The analysis of
Fig. 1. Representative macroscopic view of a cell sheet. ASC sheets were implanted on a 12 mm diameter wound injury in female athymic nude mice. After 21 days, wound grafts were explanted and fixed for histological analysis. (A) ASC cell sheet cultured in a Petri dish with a temperature-responsive surface. (B) Three-layer ASC cell sheet; white arrows indicate the edge of cell sheet. (C) Empty wound injury treatment (control).
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blood vessel density showed that the ASC cell sheet treatments slightly enhanced (no significant difference) total vessel amounts compared to the empty wound injury treatment (Figs. 6 and 8). One-layer ASC cell sheets and three-layer ASC cell sheets increased the total vessel amounts compared the non-treated control group, by 15% and 22.5%, respectively (no significant difference). Fig. 7 shows keratinocyte immunostaining (Ki67 and K10 antibody) of the ASC cell sheet treatment group and the non-treated control group at post-operative day 21. The results show the phenomena of keratinocyte proliferation and migration in both the cell sheet treatment and non-treated control groups.
4. Discussion
Fig. 2. qPCR results comparing gene expression ratios for FABP4 (a mature adipocyte marker), PPAR-c (a mature adipocyte marker) and CD34+ (an MSC marker) gene expression detection from one- and three-layer ASC cell sheets and mature adipocytes. The gene expressions of the mature adipocytes were normalized to 1 as the intra-control baseline. ⁄,#Significantly different between groups, p < 0.05.
Cell sheets created 3-D structures via the layering of individual sheets and were implanted directly to host tissues [23]. Traditional tissue engineering designs have focused on biodegradable scaffolds to support tissue formation [20,23]. However, biodegradable scaffolds still present many clinical challenges, such as poor vascularization, microbial contamination and lack of drainage [3] in critically sized wound injury areas. Cell sheets have been used
Fig. 3. The macroscopic pictures of the wounds at specific time points (7, 10, 14 and 21 days) and the area of full-thickness wound injury for the control and one- and threelayer ASC cell sheets implanted in athymic nude mice at 0, 7, 10, 14, 18 and 21 days. All data reported as mean ± SD, ⁄,#p < 0.05.
Fig. 4. Representative immunohistochemistry of a cross-section in the wound site at day 21 post-wound with human lamin A/C staining. Immunostaining (red) of one- and three-layer ASC cell sheets and control treatment (20 magnification) with human lamin A/C protein (red, human cell marker). (A) Control; (B) one-layer ASC cell sheet treatment; (C) three-layer ASC cell sheet treatment. Scale bar = 500 lm.
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Fig. 5. Representative histochemistry of cross-section in the wound site at day 21 post-wound with H&E staining (A), Masson’s trichrome staining (B) and Herovici’s staining (C) of one- and three-layer ASC cell sheets and control treatment. (A) Nuclei are stained blue; cytoplasm, pink to red; (B) collagen is stained blue; cytoplasm or keratin, red; and nuclei, black; (C) young collagen is stained blue; mature collagen, red; cytoplasm, yellow; and nuclei, black. Black arrows indicate the center of the wound injury. 20 magnification. Scale bar = 500 lm.
Fig. 6. Representative immunohistochemistry of a cross-section in the wound site at day 21 post-wound with human CD31+ staining. Immunostaining of one- and threelayer ASC cell sheets and control treatment (20 magnification) with human CD31+ marker (dark brown) and nuclei (black). White and black arrows indicate the human CD31- and mouse CD31-positive staining, respectively. E, epidermis; M, murine muscle. Scale bar = 500 lm.
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Fig. 7. Representative immunohistochemistry of a cross-section in the wound injury site at day 21 post-wound with keratinocyte staining. Immunostaining of one- and three-layer ASC cell sheets and control treatment (20 and 40 magnification) with Ki67 and K10 antibody (red). White triangles indicate the center of the wound injury. White arrows indicate the Ki67- and K10-positive staining, respectively. Scale bar = 500 lm. e, epidermal portion.
for wound healing due to their ability to self-assemble, allowing them to produce vascularization and skin equivalents [13,24,25]. Use of temperature-responsive surfaces for cultured cell sheets harvested without trypsin or dispase can result in cell damage and loss of differentiated phenotypes [26,27]. Yang et al. [27] further described that cell sheet technology seemingly provides several advantages over traditional regenerative therapies of cell injection and tissue reconstruction with biodegradable scaffolds. Stem cells have been used for bone, ligament and wound tissueengineering research in recent years [28]. ASCs derived from discarded human adipose tissue are immunocompatible and multipotent, rendering them ideal for regenerative medicine applications, such as cartilage, bone, soft tissue and nerve repair [8]. Zografou et al. [29] reported that autologous ASC transplantation increases full-thickness skin-graft survival and shows promise for use in skin-graft surgery due to in situ differentiation of ASCs into endothelial cells, and increased vascular endothelial growth factor and transforming growth factor b3 secretion by ASCs. Moreover, ASCs promoted dermal fibroblast proliferation by direct cell-to-cell contact and paracrine activation through secretory factors [11]. In
our study, we demonstrated that the ASCs in cell sheet types were significantly viable, supported the development of skin equivalents and enhanced wound healing compared to the non-treated control group (Fig. 5). Stem cells are defined by their ability to either self-renew or differentiate into multiple cell lineages [30]. Mesenchymal stem cells are often targeted for tissue engineering due to their vital role in native tissue formation and function [31]. Three-dimensional high-density primary adipose stem cell culture leads to completion of adipose tissue formation in vitro over a long-term period [28]. qPCR results compared gene expression ratios for FABP4 (a mature adipocyte marker), PPAR-c (a mature adipocyte marker) and CD34+(an MSC marker) gene expression detection from one-layer ASC cell sheets, three-layer ASC cell sheets and mature adipocytes. The results showed that the FABP4 and PPAR-c gene expression of cell sheet treatment are significantly lower than that of mature adipocytes. Our preliminary data showed that there is no significant difference between five-layer cell sheets and mature adipocytes in FABP4 and PPAR-c gene expression. Based on these results, one- and three-layer ASC cell sheets were investigated in
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are highly prolific and migratory at the wound boundaries, with the purpose of restoring the epidermis’s barrier function by closing the wound in the shortest time [13,39,40]. Langbein and Schweizer [41] reported that the epithelial keratins are expressed in various tissue constituents of the external sheaths and the companion layer of the follicle. Qi et al. [42] identified hair follicles on the epithelial surface in their nude mouse skin defect model. The keratinocytes and papilla cells may play important roles in the induction of hair follicle formation after full-thickness wound injury. Overall, we have demonstrated the utilization of multi-layer stem cell sheet technology to improve in vivo wound healing. 5. Conclusion
Fig. 8. The vascular density for control and one- and three-layer ASC cell sheets implanted in athymic nude mice at day 21 post-wound. All data reported as mean ± SD.
our wound healing model. In order to characterize the ‘‘stemness’’ properties of the ASC sheet, CD34+ gene expression was investigated. CD34+ adipose-derived stem cells have been hypothesized to be resident pericytes that play a role in vascular stabilization by mutual structural and functional interaction with endothelial cells [32]. Li et al. [33] isolated four distinct stromal populations from human adult adipose tissue and characterized their adipogenic potential. Of the four populations, the CD31-/CD34+ group is the most prevalent and has the greatest potential for adipogenic differentiation. In our study, there is no significant difference in CD34+ gene expression between cell sheets and 2-D cultured cells. ASC sheets maintained the ability of differentiation. A donut-shaped silicone splint technology was used on the wound injury and fixed to the skin using 5-0 nylon sutures to prevent the potential wound contraction. Previously, Nie et al. [34] reported that the rat model with silicone splint fixed on the skin can prevent wound contraction and allows the wound to heal by epithelialization and granulation tissue formation. Boyce et al. [35] further demonstrated that keratinocyte proliferation can enhance the extracellular matrix secretion to reduce the skin contraction. One limitation of current skin substitutes is poor vascularization [3]. Branski et al. [2] point out stem cell therapy can create a suitable skin substitute to enhance vascularization in neo-tissue. Lee et al. [36] and Kim et al. [11] reported that the potential mechanisms of ASCs to promote wound healing are via angiogenic factor production and angiogenesis. In our study, ASC cell sheet treatment slightly enhanced vascular density compared to the nontreated control treatment (Fig. 8). However, there is no significant difference between all groups. This result also demonstrates that a possible mechanism of ASC cell sheet treatment in wound healing is increased vascularization of the injured site. Lai et al. [37] also reported that a PKH26-labeled human corneal endothelial cell sheet can significantly enhance injury healing and vascularization in a rabbit cornea model. Moreover, Herovici’s staining (Fig. 5C) showed that ASC cell sheet treatments significantly enhanced cell secretion at the epidermis/dermis interface compared to the nontreated control group. Maharlooei et al. [12] reported that adipose tissue-derived stromal cells improve the wound healing rate by collagen accumulation in the wound tissue. Kim et al. [38] further demonstrated that the wound healing and antioxidant effects of ASCs are mainly mediated by the activation of dermal fibroblasts and keratinocytes. In our study, keratinocytes had proliferated and migrated to the periphery of neo-dermis (Fig. 7). This phenomenon is in agreement with the understanding that keratinocytes
Our study demonstrates that multi-layer ASC sheets present a potentially viable matrix for full-thickness defect wound healing in a mouse model. Consequently, our ASC sheet technology provides an important advance in developing various types of tissue regeneration. While this study targets the use of autologous stem cells, future studies will determine if an allogeneic therapy would be possible, as some patients may not have sufficient adipose stem cells or be suitable for stem cell harvesting. Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1–8, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2012. 09.028. References [1] Walter MNM, Wright KT, Fuller HR, MacNeil S, Johnson WEB. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell Res 2010;316:1271–81. [2] Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns 2009;35:171–80. [3] Jones I, Currie L, Martin R. A guide to biological skin substitutes. Br J Plast Surg 2002;55:185–93. [4] Garcia Y, Wilkins B, Collighan RJ, Griffin M, Pandit A. Towards development of a dermal rudiment for enhanced wound healing response. Biomaterials 2008;29:857–68. [5] Cha J, Falanga V. Stem cells in cutaneous wound healing. Clin Dermatol 2007;25:73–8. [6] Chen M, Przyborowski M, Berthiaume F. Stem cells for skin tissue engineering and wound healing. Crit Rev Biomed Eng 2009;37:399–421. [7] Bajada S, Mazakova I, Richardson JB, Ashammakhi N. Updates on stem cells and their applications in regenerative medicine. J Tissue Eng Regen Med 2008;20:169–83. [8] Santiago LY, Nowak RW, Rubin JP, Marra KG. Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications. Biomaterials 2006;27:2962–9. [9] Wei Y, Wei YH, Han HY, Meng G, Zhang D, Wu Z, et al. A novel injectable scaffold for cartilage tissue engineering using adipose-derived adult stem cells. J Orthop Res 2008;26:27–33. [10] Peng L, Jia Z, Yin X, Zhang X, Liu Y, Chen P, et al. Comparative analysis of mesenchymal stem cells from bone marrow, cartilage and adipose tissue. Stem Cells Dev 2008;17:761–73. [11] Kim WS, Park BS, Sung JH, Yang JM, Park SB, Kwak SJ, et al. Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci 2007;48:15–24. [12] Maharlooei MK, Bagheri M, Solhjou Z, Jahromi BM, Akrami M, Rohani L, et al. Adipose tissue derived mesenchymal stem cell (AD-MSC) promotes skin wound healing in diabetic rats. Diabetes Res Clin Pract 2011;93:228–34. [13] Ng KW, Hutmacher DW. Reduced contraction of skin equivalent engineered using cell sheets cultured in 3D matrices. Biomaterials 2006;27:4591–8. [14] Laplante AF, Germain L, Auger FA, Moulin V. Mechanisms of wound reepithelialization: hints from a tissue-engineered reconstructed skin to long-standing questions. FASEB J 2001;15:2377–89. [15] Sasagawa T, Shimizu T, Sekiya S, Haraguchi Y, Yamato M, Sawa Y, et al. Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials 2010;31:1646–54.
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