TY - JOUR
AU1 - Kao, H-K
AU2 - Hsu, H-H
AU3 - Chuang, W-Y
AU4 - Chang, K-P
AU5 - Chen, B
AU6 - Guo, L
AB - Abstract Background The combination of fat grafting and negative pressure (VAC) therapy represents a synergistic interaction of all essential components for wound healing. The aim of this experimental study was to determine whether it could promote healing of wounds with exposed bone. Methods Full-thickness wounds with denuded bone in Sprague–Dawley rats were treated with either polyurethane foam dressing, fat grafting alone, polyurethane foam dressing with VAC, or polyurethane foam dressing with VAC combined with a single, or two administrations of fat graft. Wound healing kinetics, tissue growth, cell proliferation (Ki-67) and angiogenesis (platelet endothelial cell adhesion molecule 1 and α-smooth muscle actin) were investigated. Messenger RNA levels related to angiogenesis (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF)), profibrosis (platelet-derived growth factor A and transforming growth factor β), adipocyte expression (fatty acid-binding protein (FABP) 4 and peroxisome proliferator activated receptor γ), and extracellular matrix remodelling (collagen I) were measured in wound tissues. Results Wounds treated by VAC combined with fat grafting were characterized by cell proliferation, neoangiogenesis and maturation of functional blood vessels; they showed accelerated granulation tissue growth over the denuded bone compared with VAC- or foam dressing-treated wounds. Fat grafting alone over denuded bone resulted in complete necrosis. Expression of angiogenesis markers (VEGF and b-FGF) and adipocyte expression factors (FABP-4) was upregulated in wounds treated with VAC combined with fat grafting. Conclusion Fat grafting with VAC therapy may represent a simple but effective clinical solution for a number of complex tissue defects, and warrants testing in clinical models. Surgical relevance The combination of fat grafting and vacuum therapy represents a synergistic interaction of regenerative cells, hospitable wound matrix and stimulating micromechanical forces. It could accelerate complex wound healing through cell proliferation, neoangiogenesis and maturation of functional blood vessels. The efficacy of a multimodal wound healing approach is established in this experimental model; it could easily be translated into clinical trials of treatment for difficult wounds. Introduction Wounds with exposed bone stripped of periosteum present a challenging problem for reconstructive surgeons. Almost invariably, significant tissue transfer involves microsurgical techniques. Because of the cost and potential morbidity of free tissue transfer, there has been an increasing interest in identifying alterative treatment strategies. Cutaneous wound healing consists of two components, involving mesenchymal and epithelial tissues. Complete healing requires epithelial healing; however, if a well vascularized soft tissue (or mesenchyma) bed can be achieved over a full-thickness defect, the wound will heal eventually, sometimes aided by skin grafting. The vacuum-assisted closure (VAC) device applies negative pressure to a wound through an open-pore polyurethane foam. VAC has become a widely used method for treating a variety of complex wounds. It helps draw the wound together, removes excess fluid, keeps the wound environment stable, and provides stimulating micromechanical forces. It has been shown to induce a granulation tissue response and neoangiogenesis1. Soft tissue defects that used to require major flap coverage, or grafting, often respond to VAC treatment leading to healing with, or without a skin graft2–4. It is widely recognized that negative pressure alone does not work well when exposed bone is devoid of periosteum, or when other non-vascularized structures such as tendon or implanted prosthetics are exposed. Here tissue coverage is necessary. Autologous fat transplantation has become established as a method with both cosmetic and reconstructive indications. There is an easily accessible donor source, and fat grafting is a minimally invasive procedure with low morbidity. The most significant drawback to autologous fat grafting is its dependence on recipient bed neovascularization for initial survival. Adipose stem cells and stromal vascular fraction cells have been shown to aid cutaneous wound healing in different experimental models, as well as in humans5–8. Skin grafts will survive on well vascularized granulation tissue, but not over denuded bone. The present study used a novel bone-exposed murine model to evaluate whether the combination of fat grafting and VAC therapy could achieve mesenchymal healing with granulation tissue, sufficient to provide coverage suitable for later skin grafting. Methods Sprague–Dawley rats weighing 250–300 g, aged 10–12 weeks, were obtained from the National Laboratory Animal Centre (Taipei, Taiwan). All animals were used in accordance with approved animal protocols (reference ID: 2009121109) and housed at the Chang Gung Memorial Hospital, Linkou Medical Centre. Wound healing model On the day before surgery, hair was removed from the scalp. On the day of surgery, under general anaesthesia, a 1·5 × 1·5-cm full-thickness wound was excised over the posterior scalp area and all the periosteum overlying the skull was removed. The wound was splinted with a 0·5-cm wide, 0·2-cm thick DuoDERM® CGF® frame (ConvaTec, Squibb & Sons, Princeton, New Jersey, USA) to stabilize the wound edge, and a digital photograph was taken. Wounds in all study groups were finally covered with an adhesive dressing (Tegaderm™; 3M, St Paul, Minnesota, USA). Wounds were redressed on days 4, 8 and 11 after wound creation. Each time, the wounds were cleaned and debrided of devitalized tissue, and all polyurethane foam dressings were changed under general anaesthesia. When the wounds in any study group reached 100 per cent of granulation deposition over the exposed bone, all animals from the group were killed. The entire wound was then harvested from each animal including 0·5 cm of surrounding skin and the underlying cranial bone. Tissue specimens were cut in half. One piece was snap-frozen in liquid nitrogen for further frozen sectioning and reverse transcriptase–PCR studies. The remaining piece was fixed in 10 per cent formalin solution overnight and then stored in 70 per cent alcohol at 4°C for paraffin-embedded histological analysis. Study groups To evaluate the optimal combination of autologous fat transplantation and VAC dressing, the following groups, each containing six to eight rats, were compared with regard to wound healing and wound healing-related growth factors (Fig. 1). In group 1 (foam control), the wounds were treated by polyurethane foam dressing alone. In group 2 (fat grafting), the wounds were treated by fat grafting alone. In group 3 (VAC), wounds were treated by polyurethane foam dressing with negative pressure of 125 mmHg. In group 4 (VAC with fat grafting once; V + F1), wounds were treated with a graft comprising minced fat from the right subcutaneous inguinal fat pad, and then covered with polyurethane foam dressing with negative suction at 125 mmHg starting on day 0; wounds were debrided on day 4, with resumption of the polyurethane foam dressing and negative pressure at 125 mmHg until the end of the study. The study design for group 5 (VAC with fat grafting twice; V + F2) was the same as that for V + F1, but on day 4 the wounds were debrided and covered with an additional minced fat graft from the left inguinal fat pad. Fig. 1 Open in new tabDownload slide Animal model and creation of wound with exposed bone. VAC, negative pressure Wound closure analysis Wound analysis was performed on days 1, 4, 8 and 11 after wound creation. The percentage reduction in wound size was quantified using planimetric methods (Image J; National Institutes of Health, Bethesda, Maryland, USA). Immunohistochemistry Paraffin-embedded sections were rehydrated and antigen retrieval for Ki-67 was performed by microwaving in 10-mmol/l sodium citrate (pH 6·0) for 10 min. Frozen sections were fixed with acetone and stained for platelet endothelial cell adhesion molecule (PECAM) 1 and α-smooth muscle actin (α-SMA). PECAM-1 (Pharmingen, San Jose, California, USA) and α-SMA (Sigma, St Louis, Missouri, USA) primary antibodies were incubated at 4°C overnight, whereas Ki-67 (Lab Vision, Freemont, California, USA) primary antibody was incubated for 1 h at room temperature. The PECAM-1 signal was intensified using the tyramide amplification system (PerkinElmer, Boston, Massachusetts, USA). Sections were counterstained with haematoxylin. Quantification of blood vessel density and diameter To quantify angiogenesis, three fields, including bilateral leading edges as well as the middle part of each PECAM-1-stained wound, were evaluated at ×100 magnification. The neovascular area (PECAM-1-positive cells) was measured using Image J, and expressed as a percentage of the total image area. The number and diameter of vessels with a definite lumen were determined in each wound using immunohistochemical staining for α-SMA. Each 1-mm2 area was counted and averaged for each slide in a blinded manner for statistical analysis with three randomly chosen fields, from both leading edges as well as the middle part of the specimen. Quantification of cell proliferation To quantify cell proliferation, high-power digital images of Ki-67-stained wound sections were obtained from both leading edges at ×200 magnification. The number of Ki-67-positive cells (proliferating nuclei) was expressed as a proportion of the total nuclei in the wound. Immunofluorescence imaging Paraffin-embedded formalin-fixed sections 3 µm thick were deparaffinized, rehydrated, then antigen retrieval was performed by microwaving twice with 10-mmol/l sodium citrate (pH 6·0) for 10 min. Bovine albumin solution (4 per cent) in phosphate-buffered saline (PBS) was applied to block non-specific endogenous antigens. The sections were incubated overnight at 4°C with unlabelled primary antibodies detecting perilipin (indicating viable adipocytes) (Cell Signaling, Danvers, Massachusetts, USA) and Ki-67 (indicating proliferating cells) (Lab Vision). After three 5-min PBS washes, fluorophore-labelled secondary antibodies were applied and the sections incubated overnight at 4°C. After three more 5-min PBS washes, the specimen was mounted and glass coverslips were attached for visualization under a fluorescence microscope (Axiovert 100; Zeiss, Thornwood, New York, USA). Real-time reverse transcriptase–PCR RNA was extracted from cultured cells or frozen tissue samples using a Rneasy® Mini Kit (Qiagen, Valencia, California, USA) according to the manufacturer's protocol. After DNase digestion, cDNA was synthesized using a SuperScript® III First-Strand Synthesis System (Invitrogen, Carlsbad, California, USA) for reverse transcriptase (RT)–PCR. RT–PCR was conducted with primers designed for this study (Table S1, supporting information) in an ABI Prism® 7300 system (Applied Biosystems, Foster City, California, USA) using a DyNAmo SYBR Green qPCR Kit (Finnzymes, Espoo, Finland). The amount of each RNA was normalized using glyceraldehyde-3′-phosphate dehydrogenase as an internal control. To compare across experimental groups, the results were normalized with respect to the value from the control group, which was designated as 1 unit. Statistical analysis The normality of data distribution was tested using the Shapiro–Wilk test and equal variance was tested by means of the F test. Student's t test was used for analysis of data with a normal distribution. When the null hypothesis of normality and/or equal variance was rejected, the non-parametric Mann–Whitney U test was used. Differences were deemed significant when P < 0·050. Statistical analyses were carried out with SPSS® version 13.0 (IBM, Armonk, New York, USA). Results Wound healing Macroscopic observation of the bone-exposed wounds revealed differences between the five groups. In the fat control group, there was complete necrosis of all the tissue, with wound infection (Fig. S1, supporting information). This group was therefore excluded from further analysis. There was a reduction in the area of exposed bone in wounds treated with fat grafting and VAC beyond day 4 (Fig. 2). Moreover, the overall time taken for complete granulation coverage of the exposed bone was shortened significantly (Fig. 3). The wounds treated with VAC and fat grafting had significantly thicker granulation tissue than those in the VAC and foam control groups. The wounds treated with fat grafting twice also had thicker granulation than wounds treated once with fat grafting (Fig. 4). Fig. 2 Open in new tabDownload slide Macroscopic images showing wound healing in the control, negative pressure (VAC), VAC with fat grafting once (V + F1) and VAC with fat grafting twice (V + F2) groups on days 0, 4, 8 and 11 Fig. 3 Open in new tabDownload slide Changes in area of exposed bone on days 4, 8 and 11 after wound creation in the control, negative pressure (VAC), VAC with fat grafting once (V + F1) and VAC with fat grafting twice (V + F2) groups. Values are mean(s.d.). *P < 0·050 (V + F1 and V + F2 versus control and VAC); †P < 0·050 (V + F2 versus V + F1, V + F2 versus VAC, and V + F2 versus control) (Student's t test) Fig. 4 Open in new tabDownload slide Thickness of granulation tissue relative to wound depth on day 11 in the control, negative pressure (VAC), VAC with fat grafting once (V + F1) and VAC with fat grafting twice (V + F2) groups. Values are mean(s.d.). *P < 0·050 (Student's t test) Cell proliferation The Ki-67 antigen is present only in actively dividing cells and is considered a robust marker of cell replication9,10. On day 11, the proliferation rate of cells populating the granulation tissue was assessed by nuclear staining for Ki-67 (upper panel, Fig. S2, supporting information). The Ki-67 signal within the granulation tissue was significantly enhanced in the groups with VAC and fat grafting (Fig. 5a). Most of the actively proliferating cells were fibroblasts by morphology and Masson's trichrome stain (Fig. S3, supporting information). Fig. 5 Open in new tabDownload slide Mean(s.d.) percentage positive staining for a Ki-67, a marker of cell proliferation, and b CD31, a marker of dermal/mesenchymal cell proliferation, and c vascular density (vessels larger than 50 µm) on day 11 in the control, negative pressure (VAC), VAC with fat grafting once (V + F1) and VAC with fat grafting twice (V + F2) groups. *P < 0·050 (Student's t test) Angiogenesis PECAM-1 (CD31) was used to stain for endothelial cells (middle panel, Fig. S2, supporting information). The wounds treated with VAC and fat grafting showed significant increases in blood vessel density compared with those in the VAC and control groups (Fig. 5b). Wounds treated with VAC and fat grafting also had a significant increase in vascular networks layered with smooth muscle cells. Specifically, wounds treated with VAC and fat grafting had approximately 16·5 blood vessels per mm2, compared with only 8 and 5 per mm2 in the VAC and control groups respectively (Fig. 5c). Expression levels of mRNA for basic fibroblast growth factor (b-FGF) and VEGF were significantly higher, and correlated with increased blood vessel density in wounds treated with VAC and fat grafting (Fig. S4, supporting information). Wounds treated with VAC and fat grafting also showed significantly upregulated mRNA expression of fatty acid binding protein 4, an adipocyte expression marker. Wounds treated twice with fat grafting had significant upregulation of peroxisome proliferator activated receptor γ, an adipocyte marker, compared with wounds in the VAC and control groups. Also significantly upregulated were the profibrotic factors platelet-derived growth factor A and transforming growth factor β. Wounds treated with VAC and fat grafting expressed significantly higher levels of collagen I than those in the control group. Adipose tissue fibrosis Dead adipocytes were not distinguished from living adipocytes by haematoxylin and eosin staining, whereas they could be distinguished easily on perilipin immunostaining. Round and intact adipocytes that stained strongly for perilipin were considered viable, whereas adipocytes weakly positive for perilipin or irregularly shaped were regarded as dead cells. Only adipocytes located close to the surrounding skin remained alive; the rest either died after grafting or were replaced by fibrosis (Fig. S5, supporting information). Discussion This experimental model of healing over denuded bone was devised to analyse a multimodal framework of mechanical forces and regenerative cells. The combination of fat grafting and VAC provided early wound coverage, avoiding desiccation of the exposed bone. The transplanted fat provided the scaffold and cellular support for a microenvironment that, under negative pressure, led to cell proliferation, neoangiogenesis and maturation of functional blood vessels. This suggests that the present method could be a simple alternative to significant soft tissue reconstruction for bone-exposed wounds. Granulation achieved in this way could allow easy skin graft coverage and lead to complete wound healing. Increased collagen content in the extracellular matrix is a change normally observed in the proliferative phase of wound healing. Once an adequate dermal bed is formed, the epidermis seems to be able to regenerate spontaneously by growing in from the edges. Therefore, granulation growth and deposition is critical because it may play an inducive and supportive role in keratinocyte migration, giving rise to re-epithelialization and eventual skin regeneration11. In this study, the wounds treated with fat grafting combined with a negative pressure dressing demonstrated increased granulation deposition over the exposed bone macroscopically and microscopically. Moreover, the granulation was thicker and more evenly distributed when fat grafting was done twice. Transplanted fat, which contains adipocytes, adipose stem cells and other cells, plays several key roles, including providing a biological scaffold to direct cells into the injury site as well as stimulating cell proliferation when combined with the application of VAC. Earlier studies2,12–13 on VAC therapy suggested that the mechanotransduction pathways that alter the biology of wound healing include angiogenesis, neurogenesis, granulation tissue formation, cellular proliferation, differentiation and migration. The extracellular matrix could maintain cellular morphology and act as a conduit between extracellular stimuli and cells by regulating proliferation, differentiation, migration and survival14–16. Here, increased proliferation of wound matrix cells (mostly fibroblasts) and increased collagen and adipose tissue deposition were documented in wounds treated with fat grafting and VAC. Furthermore, the mechanical environment that the cells experience might influence biological processes that affect cell survival, migration or differentiation. The finding that fat grafting alone, without VAC, resulted in complete necrosis provides evidence of this. Angiogenesis in wounded tissue is controlled by a complex regulatory system and is critical for repair17. Among the variety of factors, VEGF and b-FGF are the major growth factors involved in angiogenesis18,19. The extracellular matrix is critical for normal vessel growth and maintenance. It is not only equipped with a scaffold support, through which endothelial cells may migrate, but also acts as a reservoir and modulator for growth factors to mediate intercellular signals20,21. In the present study, wounds treated with fat grafting and VAC showed significantly higher mRNA expression of VEGF and b-FGF than the VAC and control groups. This is consistent with the significantly greater vascularity of wound tissue demonstrated by CD31 staining. To identify vascular networks stabilized with smooth muscle cells, α-SMA staining was used; wounds treated with VAC and fat grafting had an increase in vascular networks layered with smooth muscle cells and a significantly larger blood vessel diameter, indicating sprouting of mature and functional blood vessels. The regulation of cell survival in the provisional wound matrix is critical to tissue development and remodelling. In the present study, angiogenesis and neovascularization were critical determinants of fat graft survival and wound healing. After non-vascularized fat grafting, parts of the connective tissue or extracellular matrix may be preserved as a scaffold, but all differentiated cells usually die and are replaced by those of the next generation derived from tissue-specific stem/progenitor cells, depending on the microenvironment22,23. Here, histology showed adipose tissue fibrosis with scar formation in the most central area of the wounds; however, live adipocytes could be identified in the periphery. The effects of repeat fat transplantation to enhance wound healing were also evaluated in the present study. Wounds treated with fat grafting twice (4 days apart) had increased wound matrix deposition, compared with wounds that received a single administration. This finding has the potential to extend the experimental research, to explore the optimal combination of fat grafting and VAC therapy. This approach could be translated relatively easily into clinical practice and could be a solution for treating difficult wounds with exposed bone. Acknowledgements This work was supported by grants from Chang Gung Memorial Hospital, Linkou Medical Centre (CMRPG 3B1631-3), and the Ministry of Science and Technology of Taiwan (NSC 99-2314-B-182A-018-MY3). Disclosure: The authors declare no conflict of interest. Supporting information Additional supporting information may be found in the online version of this article: Table S1 Primer sequences for real-time reverse transcriptase–PCR (Word document) Fig. S1 Wounding healing in the fat-only control group (Word document) Fig. S2 Wound healing tissues in each group stained for cell proliferation (Ki-67), platelet endothelial cell adhesion molecule 1 and α-smooth muscle actin (Word document) Fig. S3 Representative histology on day 11 after wounding in each group (Word document) Fig. S4In vivo reverse transcriptase–PCR quantification of RNA expression comparing wounds in each group on day 11 (Word document) Fig. S5 Perilipin and Ki-67 immunostaining of wounds treated with negative therapy and fat grafting twice (Word document) References 1 Scherer SS , Pietramaggiori G, Mathews JC, Prsa MJ, Huang S, Orgill DP. The mechanism of action of the vacuum-assisted closure device . Plast Reconstr Surg 2008 ; 122 : 786 – 797 . 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TI - Experimental study of fat grafting under negative pressure for wounds with exposed bone
JF - British Journal of Surgery
DO - 10.1002/bjs.9826
DA - 2015-06-10
UR - https://www.deepdyve.com/lp/oxford-university-press/experimental-study-of-fat-grafting-under-negative-pressure-for-wounds-aCckvtIfjB
SP - 998
EP - 1005
VL - 102
IS - 8
DP - DeepDyve
ER -