| Background and ObjectionAs we all know, skin regeneration after full-thickness tissue injury is a challenge for the plastic surgeon. The traditional therapeutic method is autologous skin grafting or suitable flap transplantation. Although general clinical outcome is good, this technique has several limitations. The result of a skin graft is often lead to a hard scar tissue, which is not very beautiful and functional because of the pigmentation and contraction of the transplanted skin. Moreover, there are some circumstances that make this type of graft less operational, such as when wounds are located in areas of hands, joints, head, or neck. Another type of problem arises when wounds are so extensive that not enough donor sites can be obtained, and the wound site must be covered quickly to avoid fluid loss and potential infection. In order to avoid these difficulties, cultured epidermal sheets and bilayered cultured skin on scaffolds have recently been used clinically to treat various injuries.Tissue engineering is an emerging interdisciplinary field that aims to restore or improve impaired tissue function and the skin substitute engineering has been demonstrate a promising therapeutic method for the treatment of skin wound. Presently, skin substitute products have been divided into the following categories, depending on the skin layer which is replaced:epidermal, dermal and combined skin substitutes(or composite grafts). In the future, a fourth kind of skin substitute might need to be added:the combined skin substitute with a subcutaneous adipose layer.However, bilayered cultured skin is thin and unsuitable for the alleviation of soft tissue defects, such as those wounds that produced after deep burns, tumour resections or chronic non-healing ulcers. There is no effective therapy for the prevention of soft tissue defects except for suitable flap transplantation. Therefore, a bilayered dermal substitute containing an adipocyte-containing hypodermis is required.Before constructing a bilayered dermal substitute containing an adipocyte-containing hypodermis, the way to construct an adipocyte-containing hypodermis is pivotal for the whole construction, which is also the objective of our study.Porcine small intestinal submucosa (SIS) is a well-studied biomaterial that predominantly (>90%) consists of collagen types Ⅰ and Ⅲ; this composition is similar to that of the dermal component of skin. In randomized controlled trials, SIS has been shown to significantly increase the percentage of wounds healed and the healing rate compared to the current standard of care, especially for the treatment of chronic ulcers and difficult-to heal wounds. SIS functions by releasing growth factors to minimize the destructive activity of matrix metalloproteinases (MMPs) and to induce angiogenesis to support the growth of new blood vessels. As a naturally occurring extracellular matrix, SIS supports the adherence, proliferation, migration, and differentiation of numerous cell types. For example, bilayered cultured skin on an SIS scaffold has previously been shown to support the attachment, proliferation and differentiation of epidermal cells and fibroblasts in association with the deposition of basement membrane components.Recently, adipose tissue engineering using an artificial scaffold combined with adipose-derived stem cells (ASCs) has been reported. As the progenitors of adipose tissue, ASCs are a promising material for tissue engineering. One study reported that SIS accelerated the ASC-mediated secretion of factors such as vascular endothelial growth factor (VEGF) in vitro and that the SIS scaffold exhibited stronger synergistic proangiogenic effects on tissue regeneration than two other scaffolds (acellular dermal matrix [ADM] and collagen-chondroitin sulphate-hyaluronic acid [Co-CS-HA]). Although ASCs have been used extensively in soft-tissue reconstruction studies, especially in adipose tissue regeneration experiments, little attention has been devoted to combining ASCs with an SIS scaffold to serve as a sheet of adipocyte-containing hypodermal tissue.As fibrogenesis was seen only when ASCs cells were injected separately as a cell suspension, a nonphysiological microenvironment in which adherent adipose progenitor cells are suspended in saline solution may give rise to the unexpected differentiation and migration of the cells. Therefore, it was suggested that ASCs should be adhered to cells, tissue, ECM or biological scaffold before administration to avoid unexpected migration or differentiation.To induce the transplanted ASCs or host-invaded pluripotent cells to properly differentiate on the SIS scaffold, an optimized adipogenic microenvironment is required. An ASC-SIS scaffold was cultured in adipogenic media before transplantation because these culture conditions assign the fate of ASCs and change the scaffold, thereby rendering the artificial microenvironment more supportive of adipose tissue formation.Therefore, we hypothesized that the self-assembly combination of ASCs and the SIS scaffold would exert a stronger effect on adipogenesis by serve as an environment that promotes angiogenesis.In this experiment, we firstly compare the effect of three ways of acellular process on biocompatibility and immunogenicity of the small intestinal submucosa, preparing for the scaffold of tissue engineering skin. Secondly, we evaluated the adipogenic properties of the adipogenesis-induced ASC-SIS (AIAS) scaffold in vitro and in vivo. The results provide evidence of novel potential tools for adipose tissue engineering and the construction of an adipocyte-containing hypodermis, offer insight into the survival of the transplanted cell or the host cell source in the regenerated tissue and pave the way for the development of a more complete trilayered skin substitute.Methods and materials1、 Compare the effect of three ways of acellular process on biocompatibility and immunogenicity of the small intestinal submucosa,Fresh jejunum of pig was prepared by mechanical method, mechanical-chemical method and machanical-enzymic method into SIS, kept as groups A, B, C, respectively. Fibroblasts were seeded on the SIS scaffolds to construct the derm substitute. The comparative examinations were performed by histological observasion, MTT assay to observe the structure of the scaffolds and, proliferation and adhesion of the cell on the scaffolds. The inflammation cause by the scaffolds after subcutaneous implantation for 1,2,4 weeks were also analysed. Therefore, we can choose the better way to prepare SIS as a scaffold for cells in the tissue engineering skin.2、Evaluated the adipogenic properties of the adipogenesis-induced ASC-SIS (AIAS) scaffoldA series of acellular processes were performed to prepare an SIS scaffold and the histologic analysis is observed by hematoxylin and eosin (HE) staining and a scanning electron microscope(SEM). The inflammation cause by the SIS scaffold after subcutaneous implantation for 1,2,4 weeks were also analysed. The growing status of ASCs in the SIS scaffold was also investigated by the MTT assay and HE staining after culturing in the SIS for 1W. Assessment of the adipogenic properties of the pure SIS scaffold and adipogenesis-induced ASC-SIS in vitro and in vivo were also performed.3^ Statistical analysis.All results are reported as the mean ± standard deviation. Statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The data among three groups were statistically analyzed by using a One-way ANOVA and the camparions between three groups were performed by q test. Comparisons between two groups were performed using unpaired Student’s t-test. The confidence interval was set at 95%(p<0.05).Results:1、Histological observasion shows that there were no residual cells in groups B and C, but residual cells in group A. The apoptosis and cell-cycle test among three groups was similar, and the proliferation and adhesion test indicated that group A was better than the other two(P<0.05).The subcutaneous implantation analysis showed that group B caused a less serious inflammation,and the vascularization capacity of group C was greater than groups A and B.2、The HE and SEM show that SIS scaffold lacked cellular components and cellular debris. The SIS scaffold induced less severe inflammation at 1,2, and 4 weeks after subcutaneous implantation than the positive control, and also accompany with neovascularization in week 2 and additional new blood vessels appeared at 4 weeks. ASCs proliferated more rapidly when cultured on the SIS scaffold than when cultured without a scaffold. Furthermore, ASCs were strongly adherent to the scaffold and evenly distributed in the interior of the scaffold. Assessment of the adipogenic properties of the SIS scaffold showed that ASCs could differentiate into adipocytes in vitro and in vivo and that the number of adipocytes in the cell-matrix scaffold group was much greater than that in the pure scaffold group in vivo.DiscussionsCutaneous wounds after full-thickness tissue injury is still a challenging work for the plastic surgeon. The traditional treatment is autologous skin grafting or suitable flap transplantation. However, both of these two ways have their limitation. The treatment of cutaneous wounds has greatly benefited from the development of bioengineered skin substitutes. A combination of cells, scaffold materials, engineering methods, and biochemical and physiological factors is employed to generate the desired tissue substitute. As the structural building blocks of tissue-engineered constructs, extracellular matrix (ECM) scaffolds promote the proliferation, adhesion, and differentiation of various cells and provide frameworks for the regeneration of various tissues in vivo. The mechanism underlying these beneficial effects may be related to the preparation methods, degradation rate, surface structure, and growth factor release in the scaffold materialsAny processing step intended to remove cells will alter the native three-dimensional architecture of the ECM. The most commonly utilized methods for decellularization of tissues involve a combination of physical and chemical treatments. The physical treatments can include agitation or sonication, mechanical massage or pressure, or freezing and thawing. These methods disrupt the cell membrane, release cell contents, and facilitate subsequent rinsing and removal of the cell contents from the ECM. These physical treatments are generally insufficient to achieve complete decellularization and must be combined with a chemical treatment. The goal of a decellularization protocol is to efficiently remove all cellular and nuclear material while minimizing any adverse effect on the composition, biological activity, and mechanical integrity of the remaining ECM.In this study, SIS was selected to prepare scaffolds by three methods, and its histomorphology and biocompatibility were evaluated. The results showed that SIS scaffold prepared by the pure mechanical method is more beneficial for the cells to adhension and proliferation, and the SIS scaffold prepared by the mechanical-chemical method caused a less serious inflammation, and the SIS scaffold prepared by the machanical-enzymic method has a better vascularization capacity. Therefore, we considered that SIS scaffold prepared by the mechanical-chemical method and machanical-enzymic method have a better histomorphology and biocompatibility, which can be choosed to use as the scaffold of skin substituted engineering.After acquiring the optimization project of acellular processes, the adipogenic properties of the adipogenesis-induced ASC-SIS (AIAS) scaffold was evaluated. In this study, SIS was selected to prepare scaffolds, and its histomorphology and biocompatibility were evaluated. The results showed that no residual cells were observed and that the main components of the scaffolds were collagen fibres. The ASCs were strongly adherent and were evenly distributed in the interior of the scaffold, and the growth curve of ASCs showed that the ASC proliferation rate was higher in the SIS scaffold than in the absence of a scaffold. Furthermore, the angiogenesis and inflammation induced by the SIS scaffold in 1,2, and 4 weeks was evaluated after transplantation in vivo, and the results indicated that SIS caused less severe inflammation than NSIS and that new capillaries formed at 2 weeks and additional new blood vessels appeared at 4 weeks in both scaffolds. Because vascularization is a key factor in adipogenesis, we chose 5 weeks as the time point to evaluate adipose tissue formation after transplantation.We first demonstrated that ASCs could differentiate into adipocytes on the SIS scaffold based on oil red O staining in vitro. These results suggested that the SIS scaffold was suitable to promote ASC adipogenesis. Because ASCs develop multiple lipid droplets approximately 1 week after exposure to the induction medium24, to assign the fate of ASCs on the scaffold, which serves as an artificial microenvironment that supports adipose tissue formation, the ASC-SIS scaffold was cultured in adipogenesis-inducing media for 1 week before transplantation. The results of the in vivo experiments were consistent with those of the in vitro experiments. Adipocytes contained many lipid droplets in the AIAS scaffold based on HE and perilipin staining. Furthermore, the staining of adipogenesis-induced ASCs with CM-Dil remained apparent, and the ASCs differentiated into adipocytes, although this population accounted for only a small percentage of the total adipocytes. the expression levels of LPL and FABP4 in the AIAS group were higher than those in the pure scaffold group. These results are consistent with the HE and perilipin staining results, further confirming that the microenvironment provided by the AIAS scaffold was superior to that provided by the pure SIS scaffold in terms of adipose tissue formation.Conclusion1、SIS scaffold prepared by the mechanical-chemical method and machanical-enzymic method have a better histomorphology and biocompatibility, which can be choosed to use as the scaffold of skin substituted engineering.2、ASCs can differentiate into adipocytes in the SIS scaffold following adipogenic induction in vitro and in vivo. These results suggest that the artificial microenvironment provided by the AIAS scaffold is similar to the physiological adipogenic microenvironment, thereby promoting the differentiation of invading host stem cells and ASCs into adipocytes. Our results demonstrate the potential utility of ASCs combined with SIS as a sheet of an adipocyte-containing hypodermis beneath a bilayered skin substitute. This method will pave the way to construct a more complete skin substitute with an adipocyte-containing hypodermis for the treatment of full-thickness injuries with soft tissue defects. |
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