Abstract
The potential use of extracellular matrix (ECM) as a source of wound dressing material has recently received much attention. The ECM is an intricate network of various combinations of elastin, collagens, laminin, fibronectin, and proteoglycans that play a key role in stimulating cell proliferation and differentiation. We evaluated the efficacy of an ECM sheet derived from human adipose tissue as a wound dressing material to enhance healing. We prepared a novel porous ECM sheet dressing scaffold from human adipose tissue. in vitro analysis of the ECM sheets showed efficient decellularisation; absence of immunostimulatory components; and the presence of a wide number of angiogenic and bioactive factors, including collagen, elastin, and proteoglycans. To evaluate in vivo efficacy, full‐thickness excisional wounds were created on the dorsal skin of a rat, and the ECM sheets; secondary healing foam wound dressing, Healoderm; or a conventional dressing were applied to each wound site. Photographs were taken every other day, and the degree of reepithelialisation of the wounds was determined. Application of an ECM sheet dressing enhanced the macroscopic wound‐healing rate on days 4, 7, and 10 compared with that in the control group. Microscopic analysis indicated that the reepithelialisation rate of the wound was higher in the ECM group compared with that in the control group; the reepithelialisation rate was better than that of the secondary healing foam wound dressing. Moreover, a denser and more organised granulation tissue was formed in the ECM sheet group compared with that in the secondary healing foam wound dressing and control groups. The ECM sheet also showed the highest microvessel density compared with the secondary healing foam wound dressing and control groups. Based on these data, we suggest that a bioactive ECM sheet dressing derived from human adipose can provide therapeutic proteins for wound healing.
Keywords: adipose tissue, extracellular matrix proteins, reepithelialisation, wound healing
1. INTRODUCTION
Impaired wound healing is a significant medical problem that can cause debilitating effects and tremendous patient suffering. Recent advances in tissue‐engineering approaches in the field of stem cell therapy have provided promising treatment options to meet the challenges of impaired wound healing, such as in diabetic foot ulcers.1 Adipose tissue has emerged as an attractive source of stem cells; the use of such stem cells has resulted in an improved outcome in wound‐healing studies.2, 3 In addition to adipocytes, adipose tissue also contains fibroblasts, smooth muscle cells, endothelial cells, immune cells, and adipose‐derived stem cells. Because of the ease of collection and potential to serve as a source of stem cells, stromal vascular fraction cells have been widely used in clinical applications.4, 5, 6 However, adipose tissue also contains various extracellular matrix (ECM) components, which might be attractive candidates in the manufacturing of wound‐healing materials.7, 8 The ECM is a natural ideal biological scaffold material, which helps retain cells together in tissues and protects and supports the cellular environment. The ECM represents an intricate network of various combinations of elastin, collagens, laminin, fibronectin, and proteoglycans9, 10; it plays key roles in stimulating cell proliferation and differentiation, regulating cell migration, and modulating cellular metabolism.11, 12, 13, 14, 15
Presently, intact ECM, derived from human skin, has been used for soft tissue repair and reconstruction under the trade names AlloDerm, Graft Jacket, and Axis dermis. The widespread use of intact ECM and ECM components is attributed to their excellent biocompatibility, biodegradability, and bioinductive properties.16 However, it should be noted that most intact ECMs and ECM components are isolated from animals or cadavers; consequently, concerns have been raised regarding immunogenicity and pathogen transmission. Recently, several studies have reported promising data describing the application of advanced dressing materials to treat chronic wounds.17, 18, 19, 20 Hyaluronic acid (HA) is a major component of the ECM. A pure HA dressing material, Healoderm, is a secondary healing foam wound dressing (Genewel, Seoul, Korea) that is commonly used and has already been shown to be effective in treating chronic wounds.21 The secondary healing foam wound dressing is composed mainly of HA (80 ± 5%) in combination with atelocollagen and poloxamer. This has led to the proposal that bioactive ECM proteins from human adipose tissue might also promote wound regeneration. Moreover, adipose tissue is the most prevalent tissue in the human body, and it can be easily collected by liposuction with minimal risk.22
In this study, we have successfully developed a novel human ECM sheet using adipose tissue. We proposed that the ECM sheet dressing might provide the structure for organised colonisation by dermal cells, along with the reconstruction of a well‐organised dermis. The efficacy of ECM sheets in inducing in vivo wound healing in a skin defect rat model was determined and compared with that of the commercial secondary healing foam wound‐dressing material, Healoderm. We hypothesised that the ECM sheet derived from human adipose tissue would be more effective in wound healing compared with a conventional dressing and would also be superior to the secondary healing foam wound dressing.
2. MATERIALS AND METHODS
2.1. Preparation of ECM sheets from human adipose tissue
Human adipose tissue was obtained from patients who had undergone liposuction or the excision of a lipoma. Informed consent was obtained from all patients, as approved by the Institutional Review Board of the Catholic University of Korea, College of Medicine, Seoul, Korea. The adipose tissue was washed several times with distilled water to remove blood components. Distilled water was added to the adipose tissue, and the tissue/water (2:1) mixture was homogenised for 3 minutes at room temperature using a commercial blender. The tissue suspension was centrifuged at 3000 rpm for 5 minutes, and the upper layer containing any oily components was discarded. The viscous suspension was then washed several times by gently pipetting in distilled water and centrifuging at 3000 rpm for 5 minutes. For sheet fabrication, the viscous suspension was cast in a shallow mould, frozen overnight at −20°C, and freeze‐dried for 48 hours. The freeze‐dried sheets were then treated with a buffered 0.5% sodium dodecyl sulphate solution (SDS; Sigma, St Louis, Missouri) for 1 hour at room temperature in a shaking water bath. The sheets were thoroughly rinsed with distilled water for 24 hours at 4°C with shaking. The medium was replaced every 2 hours with fresh distilled water. The acellular ECM sheets were lyophilised in a freeze dryer for at least 48 hours (Figure 1) and sterilised with ethylene oxide gas before use. Figure 1 was adapted from “Fabrication of Porous Extracellular Matrix Scaffolds from Human Adipose Tissue.”14
Figure 1.
Schematic representation of the fabrication process used to prepare extracellular matrix (ECM) sheets from human adipose tissue
2.2. Scanning electron microscopy
The inner structure of the ECM sheets prepared from human adipose tissue was observed using scanning electron microscopy (SEM) (Hitachi S‐4800 FE‐SEM, Tokyo, Japan). The ECM sheets were fixed to metal stubs and coated by sputtering them with platinum at an accelerating voltage of 15 kV.
2.3. Fluorescence microscopy
The ECM sheets were fixed in 4% paraformaldehyde at 4°C for 1 hour, embedded in paraffin (Merck, Darmstadt, Germany), and 10 μm‐thick sections were prepared. The sections were deparaffinised, dehydrated using a series of graded ethanol solutions, and stained with 4,6‐diamidino‐2‐phenylindole (DAPI; Thermo Scientific, Rockford, Illinois) to identify nuclear components, such as DNA and RNA. The stained specimens were examined using a fluorescence microscope (IX81; Olympus, Tokyo, Japan).
2.4. DNA quantification
DNA was isolated with a commercial extraction kit (G‐spin Kit; iNtRON Biotechnology, Seongnam, Korea). The total DNA content was assessed by measuring the absorption at 260 nm on a spectrophotometer (NanoDrop 1000; Thermo Fisher Scientific, Wilmington, Delaware). All measurements were normalised to the dry weight of the ECM sheet.
2.5. Growth factor antibody Array
Bioactive molecules in the ECM sheet were analysed using a growth factor antibody array kit (RayBiotech Inc., Norcross, Georgia) according to the manufacturer's protocol. The array glass chip containing 41 different human cytokine antibodies was blocked and incubated with the ECM sheet. The glass chip was washed and subsequently exposed to the biotin‐conjugated antibodies. After incubation with fluorescent dye‐conjugated streptavidin, cytokine signals were detected with a laser scanner (Axon Instruments, Union City, California) using the Cy3 channel. Signal intensities were quantified with the microarray analysis software (GenePix Pro; Molecular Devices, Sunnyvale, California).
2.6. ECM sheet protein content
Protein content in the ECM sheets was quantified using Sircol acid/pepsin‐soluble collagen and Fastin elastin assay kits according to the manufacturer's protocols (Koma Biotech, Inc., Seoul, Korea). For quantification of acid/pepsin‐soluble collagen, the ECM sheet was incubated with 1 mL of Sircol dye reagent for 30 minutes at room temperature. To determine the elastin content, the ECM sheet was mixed with 1 mL of Fastin dye. The absorbance was measured using a microplate spectrometer (BioTek Instrument, Winooski, Vermont). Laminin and fibronectin in the ECM sheet were quantified via enzyme‐linked immunosorbent assay according to the manufacturer's protocols (Koma Biotech, Inc.). Optical density was measured at 560 nm using a microplate spectrophotometer (BioTek Instrument).
2.7. In vivo wound‐healing study
The wound‐healing efficacy of the ECM sheets was evaluated using a full‐thickness cutaneous wound model. All experimental protocols were approved by the Institutional Review Board of the Catholic University of Korea. Male Sprague‐Dawley rats, 6 to 8 weeks old and weighing 200 to 250 g, were used in the study. Each rat was housed in a separate cage post‐operation with ad libitum access to food and tap water. The rats were subjected to a 12‐hour light/dark cycle. Prior to surgery, each rat was anaesthetised using isoflurane (2% isoflurane, 2 L/min oxygen). The dorsal surface of the rat was completely shaved with electric clippers, followed by the application of a depilatory agent (Nair; Church & Dwight Co, Princeton, New Jersey) for 2 to 3 minutes to remove any remaining hair. Povidone iodine was then applied on the exposed skin. A 2.0‐cm full‐thickness incisional wound was created on the upper back area of each rat. After this, a silicone ring (outer diameter 44 mm, inner diameter 30 mm, and thickness 2 mm) was fixed with eight interrupted sutures (4‐0 silk) to prevent contraction of the wound. The rats were then randomly divided into three groups (control, secondary healing foam wound dressing, and ECM sheet) with 10 rats assigned to each group. In all groups, a non‐adhesive foam dressing was applied over the primary dressing (ECM sheet and secondary healing foam wound dressing groups) or the wound lesion (control group) and fixed with tape (Hypafix; BSN Medical, Yorkshire, UK). Whenever the dressing was changed, photographs were taken with a digital camera for the macroscopic evaluation of wound healing. The unhealed wound areas were quantified using ImageJ software (version 2; National Institutes of Health, Bethesda, Maryland). From the macroscopic images, the margin of the advancing epithelium was traced, and the area within the margin of this epithelium was defined as the wound area. Subsequently, the animals were euthanised on postoperative day 14.
2.8. Histopathological examination
2.8.1. Reepithelialisation rate
For histological examinations, the tissue in the reepithelialisation area, scar tissue, and some area of normal skin were excised and fixed with 10% formalin on day 14. Paraffin sections from the centre of the wound, cut perpendicularly to the wound surface, were stained with haematoxylin and eosin (H&E) and Masson trichrome (MT) blue according to standard protocols. The reepithelialised wound area and collagen deposition in the regenerated skin tissue were digitally analysed using ImageJ software. The reepithelialisation rate was determined by measuring the distance of the regenerated keratinocyte cell layer from the original wound area as determined by the images of the stained histological sections.
2.8.2. Collagen deposition
Collagen deposition was measured by counting the pixels in the MT‐positive areas of granulation. The total area of granulation was counted as the sum of these regions. For quantitative analysis of collagen deposition, stained blue by MT, we assessed the staining intensity of the blue area using ImageJ software and evaluated the average intensity of three images.
2.9. Immunohistochemistry
2.9.1. CD31 expression
We assessed the expression of CD31 (platelet endothelial cell adhesion molecule‐1, PECAM‐1), which is a marker protein of mature vascular endothelium by immunohistochemistry. Sections (2.5 μm) were mounted on chromium‐coated slides, dewaxed, rehydrated, rinsed, and washed in phosphate‐buffered saline (PBS) solution for 30 minutes. Once endogenous peroxidase was inhibited, the specimens were treated with target retrieval solution (Dako, Atlanta, Georgia) equilibrated at 99°C. Tissue samples were then incubated for 40 minutes with a 1/50 dilution of anti‐CD31 antibody (Abcam, Cambridge, Massachusetts) in a background reduction solution (Dako). The immunohistochemical reactions were performed using the labelled streptavidin/biotin‐horseradish peroxidase conjugate method according to the manufacturer's instructions. Diaminobenzidine was used as the peroxidase substrate, after which the stained sections were counterstained with haematoxylin. Analysis was performed according to standard procedures.23 Sections were scanned at 200× magnification, and areas with the highest vascular density were identified. Vessels in three high‐power fields (400×) were counted by two independent observers, one of whom was blinded to the experimental conditions. The average vessel count was determined for each specimen. Six slides from each group were analysed, and the counts were averaged for statistical analysis.
2.9.2. Statistical analysis
The results are expressed as means ± SD. Analysis was performed using the Statistical Program for Social Science (SPSS 11.0 software; SPSS Inc. IBM, Armonk, New York). Statistically significant differences (P < 0.05) among the different groups were evaluated by one‐way or two‐way anova, followed by a Bonferroni test.
3. RESULTS
3.1. Fabrication of the ECM sheet from human adipose tissue
The ECM sheets were prepared without any chemical treatment. The volume of the ECM components extracted from adipose tissue was approximately 5% of the original adipose tissue volume. Macroscopic and microscopic images of the manufactured bioactive protein ECM sheet dressings derived from human adipose tissue are shown (Figure 2). The ECM sheets possessed a highly porous structure within complex three‐dimensional (3D) shapes. Such a porous structure allows the mass transport of cell nutrients and provides channels for cell migration and surfaces for cell attachment.
Figure 2.
Extracellular matrix (ECM) sheets derived from human adipose tissue. A, Macroscopic view of ECM sheets showing a round 3D shape with a porous structure. B, Microscopic images of an ECM sheet obtained by scanning electron microscopy
3.2. Composition analysis of the ECM sheet
DAPI (4,6‐diamino‐2‐phenylindole) staining confirmed the removal of immunogenic cells and nuclei (Figure 3A). The extracted DNA concentration was less than 0.5 μg/μL, which indicates that there were almost no cellular components remaining (Figure 3B). Endogenous growth factors in the ECM sheet were detected using different human growth factor antibody arrays. Among the 41 growth factors, 25 growth factors were detected in the prepared ECM sheet (Table 1). Notably, hepatocyte growth factor, platelet‐derived growth factor‐BB, endothelial growth factor, insulin‐like growth factor, vascular endothelial growth factor, and transforming growth factor‐β1, which are all involved in the regulation of wound healing and angiogenesis, were detected in the ECM sheet. The major proteins in the ECM sheet were soluble elastin (16.463 ± 1.440 mg/mL), acid/pepsin‐soluble collagen (4.572 ± 0.136 mg/mL), laminin (1.038 ± 0.064 mg/mL), and small amounts of fibronectin (0.144 ± 0.054 mg/mL) (Figure 3C).
Figure 3.
A, DAPI staining of extracellular matrix (ECM) sheets before and after decellularisation. The blue colour indicates residual nucleic acids. B, DNA content before and after the decellularisation process. Samples were normalised to the dry weight of the ECM sheet. Data are shown as means ± SD. *P < 0.05. C, The major protein components in the ECM sheet. The ECM sheet contained large amounts of soluble elastin (16.463 ± 1.440 mg/mL), acid/pepsin‐soluble collagen (4.572 ± 0.136 mg/mL), laminin (1.038 ± 0.064 mg/mL), and small amounts of fibronectin (0.144 ± 0.054 mg/mL). Data are shown as means ± SD
Table 1.
Profile of growth factors in the ECM sheet derived from human adipose tissue using growth factor antibody assays. Adapted with permission from “Injectable and thermosensitive soluble extracellular matrix and methylcellulose hydrogels for stem cell delivery in skin wounds”.24 Copyright 2016 American Chemical Society. [Correction added on 05 April 2019, after first online publication: The table caption has been revised in this version.]
| Growth factor | Functions |
|---|---|
| HGF | Regulation of cell growth, cell mortality, and morphogenesis |
| TGF‐β1 | Control of cell proliferation, differentiation, adhesion, and migration |
| TGF‐β3 | Control of cell differentiation and embryogenesis |
| EGF | Stimulation of cell growth, proliferation, and differentiation |
| EGFR | Receptor for EGF |
| HB‐EGF | Mediation of cell adhesion, migration, and cell cycle progression; predominant growth factor for epithelialisation of skin wound |
| PDGF‐AA, AB, BB | Regulation of cell growth, division, and angiogenesis |
| PDGF Rβ | Receptor for PDGF |
| VEGF | Regulation of angiogenesis and lymphangiogenesis |
| VEGF R2, R3 | Receptor for VEGF |
| IGF1 | Promotion of cell proliferation, the inhibition of cell apoptosis, and regulation of neural development |
| IGF1R | Receptor for IGF1 |
| IGFBP2 | A carrier protein for IGF‐1 |
| IGF2 | Organ development and function in foetal stage |
| CSF1 | Stimulation of neutrophil survival, proliferation, differentiation, and function |
| CSF1R | Receptor for CSF1 |
| CSF3 | Stimulation of neutrophil survival, proliferation, differentiation, and function |
| SCFR | Receptor for SCF |
| PLGF | A key molecule in angiogenesis and vasculogenesis during embryogenesis |
| AREG | Regulation of mitogen of astrocytes, Schwann cells, fibroblasts, and T‐cells |
| NT‐3 | Encouragement of neuron growth and differentiation |
Abbreviations: AREG, amphiregulin; CSF1, colony‐stimulating factor 1; CSF1R, CSF 1 receptor; CSF3, colony‐stimulating factor 3; EGF, epidermal growth factor; EGF‐R, EGF receptor; HB‐EGF, heparin‐binding EGF‐like growth factor; HGF, hepatocyte growth factor; IGF 1, insulin‐like growth factor 1; IGF1R, IGF‐1 receptor; IGF2, insulin‐like growth factor 2; IGFBP2, IGF‐binding proteins‐2; NT‐3, neutrophin‐3; PDGF‐AA, AB, BB, platelet‐derived growth factor subunit AA, AB, BB; PDGF‐Rβ, platelet‐derived growth factor‐receptor β‐polypeptide; PLGF, placenta growth factor; SCF, stem cell factor; SCFR, stem cell factor receptor; TGF‐β, transforming growth factor‐β; VEGF, vascular endothelial growth factor; VEGF R2, R3, VEGF receptor 2, 3.
3.3. Effect of ECM sheets on wound healing
3.3.1. In vivo wound‐healing study
There was no sign of inflammation or infection in any of the groups (Figure 4A). Wound sizes were reduced over a period of time in all groups (Figure 4A). The ECM sheets, which were applied on the site of the wound, significantly improved wound healing on 4, 7, and 10 days compared with that in the control group (P < 0.05) (Figure 4B). On day 4, the ECM sheet group showed an enhanced wound‐healing rate, which was significantly higher (35.7 ± 6.6%), compared with the rate of the control group, which was 22.2 ± 5.3%. Moreover, the healing rates of the ECM sheet group were higher compared with those of the control group on day 7 (57.6 ± 5.6% vs 39.7 ± 4.8%) and also on day 10 (74.3 ± 6.2% vs 59.1 ± 4.6%). In addition, on day 14, the ECM sheet group showed the highest healing rate, as determined by macroscopic evaluation, compared with other groups; however, the differences in the healing rates were not statistically significant. Thus, the wound‐healing ability of the ECM sheet tended to be greater than that of the secondary healing foam wound dressing, although there was no statistical significance.
Figure 4.
In vivo wound‐healing study: A, Gross observation of wound healing on days 0, 2, 4, 7, 10, and 14 in the extracellular matrix (ECM) sheet, Healoderm (secondary healing foam wound dressing), and control groups. B, Wound areas were quantified using ImageJ software and are expressed as the percentage of the respective initial wound area. Each value represents the mean ± SD of 10 experiments. *P < 0.05
3.3.2. Histopathological examination
On day 14, a histological examination of the healed tissue by H&E staining showed that the ratio of the area of totally epithelialised tissue to that of wounded tissue was the highest in the ECM sheet group (92.4%) compared with that in the control group (31.8%) (P < 0.05); however, there was no statistically significant difference observed between the ECM sheet and secondary healing foam wound dressing (88.4%) groups (Figure 5A,B). The wounds treated with the ECM sheet groups were covered with a continuous epidermal layer, and the regenerated dermis was much thicker than that of the control group. In contrast, on day 14, the control group wounds were partially covered with epidermis.
Figure 5.
Histological evaluation of wound sections in the extracellular matrix (ECM) sheet, Healoderm (secondary healing foam wound dressing), and control groups after dermal excision on day 14. A, The blue arrows indicate the wound edges. The insets are magnified images of the indicated rectangles and represent the outer layer of the skin. The long black arrow indicates the regenerated dermis. B, Average reepithelisation ratio, expressed as a percentage of baseline. Each value represents the mean ± SD of 10 experiments. *P < 0.05
Consistent with the above data, MT staining also showed a fully formed epidermis covering the granulation tissue in the ECM sheet group (Figure 6A). In particular, the ECM sheet group showed excellent collagen alignment on the regenerated skin tissues compared with the other groups. Wounds in the control group showed 29 ± 3.0% collagen deposition. In contrast, the wounds treated with the ECM sheet and secondary healing foam wound dressing showed 47 ± 3.0% and 39 ± 3.0% collagen deposition, respectively (Figure 6B). This analysis confirmed that the ECM sheet induced more collagen deposition in the wound sites than the other treatments.
Figure 6.
Masson trichrome histology analysis of epithelial regeneration and dermal repair. A, Masson trichrome‐stained sections shown at 20× magnification on day 14 after wound treatment. The blue areas indicate the newly formed collagen tissue. The insets are magnified images of the indicated rectangles. B, The images were analysed using ImageJ software to quantify collagen deposition in the wound area. Each value represents the mean ± SD of 10 experiments. *P < 0.05
3.3.3. Immunohistochemistry
To examine the wound vasculature, we performed immunohistochemistry using an anti CD31 antibody (PE‐CAM‐1), an endothelial cell marker. CD31 immunostaining was rarely detected in the control group on day 14 (Figure 7A), which is consistent with the lack of blood vessels observed by H&E or MT staining. In contrast, numerous CD31‐positive vessels were present in the ECM sheet and secondary healing foam wound dressing groups. A microvessel density analysis was performed on CD31‐stained tissue sections to quantitate the vascularity in the different groups. The average microvessel counts, in the ECM sheet and secondary healing foam wound dressing groups, per ×400 power field, on day 14 were 28 ± 5 and 23 ± 3, respectively (Figure 7B). In contrast, the average microvessel count in the control group was 5 ± 2 (Figure 7B). This 5.6‐fold increase in vascularisation in the ECM sheet group was statistically significant (P < 0.001).
Figure 7.
Neovascularisation is improved by extracellular matrix (ECM) sheet dressing. A, Immunohistochemical analysis of endothelial cells on day 14 after wound treatment. The arrows indicate capillary vasculature. The scale bar represents 100 μm. B, Microvessel density analysis on day 14. Three high‐power fields (400×) of the highest vascular density were examined for each group, and all counts were averaged for statistical analysis. Each value represents the mean ± SD of 10 experiments. *P < 0.001
4. DISCUSSION
Adipose tissue, which is a major endocrine and secretory organ, can be relatively easily collected through the liposuction procedure.1, 2, 3, 4, 5, 6 It is rich in ECM components such as collagen, elastin, and fibronectin. Recently, there has been considerable interest in the tissue engineering of scaffolds fabricated from various living tissues.25 These natural scaffolds consist mostly of ECM components such as collagen and contain various cytokines, but they are derived from animal tissues or are isolated from cadavers, and their usage may be limited by serious concerns regarding adverse immune responses and viral infection.
However, our novel ECM sheet derived from human adipose tissue was fabricated using only physical stimuli without the addition of chemical or enzymatic factors. Before the decellularisation process, abundant levels of nucleic acids were apparent, shown by the DAPI‐positive staining. However, after the decellularisation process, the nucleic acids were effectively removed from the ECM sheets, thus decreasing the possibility of an immunogenic reaction.
The major components of the ECM sheet are collagen and elastin. Collagen dressing has been extensively used recently as it is known to promote cell adhesion and growth.18, 21 Healoderm, a secondary healing foam wound dressing (HA: 80 ± 5%, in combination with atelocollagen and poloxamer), which is currently the most common collagen dressing21 used in the clinic, was selected to compare the wound‐healing efficacy of the ECM sheet.
To clarify the effect of ECM sheet dressing on the wound‐healing process, we used a skin wound model in rats. On macroscopic examination, the ECM sheets, which were applied on the site of the wound on the first day, showed a significantly higher wound‐healing rate on days 4, 7, and 10 compared with that in the control group. The beneficial effects of the ECM sheet were also confirmed by microscopic examination. The reepithelialisation rate was higher in both the ECM sheet and secondary healing foam wound dressing groups, indicating that the ECM sheet dressing could be a good candidate as a dressing material for wound reepithelialisation. Using MT stain, we also observed a more organised accumulation of granulation tissue in the ECM sheet group. Wounds in the control group showed 28.8% collagen deposition, whereas the wounds treated with the ECM sheets and secondary healing foam wound dressing showed 46.8% and 38.9% collagen deposition, respectively. The ECM sheets induced more collagen fibre deposition in the wound site than the other groups. The alignment and synthesis of collagen are essential during the maturation process of wound healing because the severity of scar formation can be determined by this process.26 Finally, in the wounds treated with ECM sheets or secondary healing foam wound dressing, the microvessel density was much higher than in the control group.
The study showed that ECM sheets from human adipose tissue accelerate wound healing rapidly through reepithelialisation and neovascularisation of wounds and also efficiently enhance collagen alignment and deposition in the maturation process of wound healing.
We propose that ECM fragments are released via degradation of the ECM scaffold sheet as a result of the action of numerous proteolytic enzymes in the tissue, and this could play an important role by releasing the raw materials necessary for the production of a new matrix in wounds. Generally, ECM‐based scaffolds show relatively fast degradation in vivo and are compensated by the ECM proteins secreted by the ingrowing cells.24, 27 The ability to promote wound healing is probably because of the high growth factor content of the ECM sheet. These ECM components are fundamental to each phase of wound healing, including haemostasis, inflammation, proliferation, and the remodelling process. The ECM components recruit macrophages, fibroblasts, and endothelial cells into the wound sites, modulating epithelialisation, collagen accumulation, and angiogenesis.10, 11, 12
The wound‐healing effects are highly influenced by the ECM component interactions with cells and growth factors in a dynamic, reciprocal process. In chronic wounds, the normal healing process is delayed because of underlying systemic dysfunction. These wounds exhibit deficiencies or dysfunction in the ECM, and such wounds are characterised by a distorted ECM that cannot support wound healing.28, 29, 30 Because of the consequences of a distorted ECM, wound‐healing approaches that compensate for the dysfunctional ECM may be valuable.
As mentioned above, our novel ECM sheets degrade within the wound, and these degraded ECM fragments can be used to reconstruct granulation tissue. However, a further detailed investigation of the underlying mechanism is necessary to support our data on the efficacy of the ECM sheet in wound healing.
In this study, we successfully manufactured a decellularised non‐immunogenic ECM sheet dressing. Above all, the ECM sheet dressing significantly improved wound‐healing ability, probably by accelerating reepithelisation and new vessel formation. A combination of histology and immunohistochemistry assessments in the in vivo rat study confirmed the safety and efficacy of the ECM sheet in the treatment of a simple wound model. The ECM sheet dressing should be further refined in more complex wound models in order to provide further evidence for establishing a clinically beneficial dressing material.
CONFLICTS OF INTEREST
The authors have no conflicts of interest to report.
ACKNOWLEDGEMENTS
This work was financially supported by the Ministry of Trade Industry and Energy of Korea (10062127 and 10063334) and by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017M3A9E2060428).
Lee YJ, Baek SE, Lee S, et al. Wound‐healing effect of adipose stem cell‐derived extracellular matrix sheet on full‐thickness skin defect rat model: Histological and immunohistochemical study. Int Wound J. 2019;16:286–296. 10.1111/iwj.13030
[Correction added on 05 April 2019, after first online publication: Author Yong W. Cho and the affiliation have been removed in the this version.]
Funding information Ministry of Trade Industry and Energy of Korea, Grant/Award Number: 1006212710063334; National Research Foundation of Korea, Grant/Award Number: 2017M3A9E2060428
Contributor Information
Young J. Jun, Email: joony@catholic.ac.kr.
Jong W. Rhie, Email: rhie@catholic.ac.kr.
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