| Background and ObjectiveAs the largest and the most complex human organ,skin has a variety of physiological functions, such as barrier function, sensory function, absorption function, regulation of body temperature function, secretion and excretion function and so on, however, the skin is also the organ most vulnerable to trauma damage, a variety of reasons, such as burns, trauma, tumor resection, mole and refractory ulcers and others,can cause skin and soft tissue defects. Large area and deep layers of skin and soft tissue injury not only seriously affects patients function and appearance, but also cause psychological burden of patient and the decline in the quality of life for patients, brought in the family and society great economical burden and social burden. Therefore, the skin tissue defects repairing is always the primary issue for plastic surgery.With the development of tissue-engineered skin,it offered new strategy for skin tissue defects.Since1975,it make the development of tissue engineering skin substitute gradually widely as Rheinwald and Green cultured skin epidermic cells successfully in vitro.According to the anatomic structure, tissue engineered skin substitutes can be divided into three kind of the epidermal substitutes, dermal substitutes and epidermis/dermis composite skin substitute.Epidermal substitute lacks mechanical support and nutrition of the dermis, the epithelial scar formation after wound healing, poor resistance to infection, easy to rupture, poor flexibility, lack of flexibility, easy to drop out after transplantation, restoration effect is unsatisfactory.Dermal substitute can reduce wound contraction, has no high requirements for wound, but the lack of epidermal protection, will increase the risk of infection; the lack of epidermal protection, could easily lead to body water and electrolyte metabolism disorders, the appearance after repair with dermal substitute is poor, especially in exposed site. Epidermal/dermal composite skin substitutes in the chemical behavior and metabolism are similar to normal human skin, is more mature double-layer tissue-engineered skin substitutes, composite skin substitutes can achieve the purpose of the permanent repair of the wounds.The composite skin substitutes has good mechanical properties and graft acceptance rate, in the process of wound healing, because of the mesenchymal-epithelial interactions,the epidermis composite skin graft can short the machine time of dermis, inhibit scar formation and promote skin regeneration. The represent products of composite skin is Apligraf approved by U.S FDA in1998for treatment of venous stasis ulcers, which use a type Ⅰ bovine collagen matrix as scaffold, firstly plant neonatal foreskin fibroblasts in the bovine collagen, after a period of time to form a "dermal skin", and then planted neonatal foreskin epidermal cells on it, epidermal cells grow from a monolayer into a multilayer, and finally a layer of three-dimensional structure. Apligraf has been successfully used in the treatment of venous stasis ulcers and diabetic foot ulcers to promote wound healing. Apligraf does not contain Langerhans cells, endothelial cells, monocytes, white blood cells and other antigen-containing cells, clinical application prove that there is no rejection, immunological detection has not found a humoral or cellular immune response on the epidermal cells and fibroblasts.However, In clinical, skin graft similar to composite skin substitute is of wear resistance, poor elasticity, easy contracture, easy to blister formation and pigmentation. And compared with skin graft, the flap containing the subcutaneous fat layer can greatly improve the shortcomings. And for facial, functional site, bone, joint, neural stem, major vessels exposed wounds and chronic ulcer wound requires the use of skin flap transplantation in repairing. Comparison from the skin structure, flap with subcutaneous adipose tissue. Subcutaneous adipose tissue has a variety of physiological functions,such as the function of thermoregulatory, barrier, energy storage.and so on.In addition, as an endocrine organ,subcutaneous adipose tissue has a variety of pathophysiological role, such as those involved in inflammatory responses in vivo, vascular disease, obesity and other metabolic diseases. Subcutaneous adipose tissue and its stem cells can also secrete a variety of Adipocyte Factor and stem cell factor to regulate biological behavior of skin fibroblast and epidermal cell,thus have an impact on wound healing. Because the superiority of flap grafting and the importance of subcutaneous adipose tissue function, One direction of the development of tissue-engineered skin substitutes are building multilayer skin composite tissue.with subcutaneous fat tissue layer.To build multi-layered tissue engineered skin substitutes containing the subcutaneous adipose tissue layer is still in the preliminary study stage. Maike etc placed Matriderm (?) stent above preadipocytes suspension, And in the other side of scaffold plant epidermal cells to construct the multi-layer skin substitute containing subcutaneous fat layer, and strive to improve functional effects and cosmetic results of wound healing of tissue defect left after the deep escharectomy,. But this method rely on the cells in preadipocytes suspension enter the scaffolds on their own, the amount and degree of enter cells can not control,The multi-layer skin substitutes built by this method has no "dermal layer", which is inconsistent with the normal skin structure Trottier etc use the adipogenic potential of adipose tissue-derived stem cells, adapt the way of "self assembly" to build the skin substitute containing three-tier structure:the epidermis, dermis and subcutaneous fat layer.However, the procedures of method are complicated and not conducive to the operation, only use adipose tissue-derived stem cells as seed cells,rely on stimulating the cells to secrete extracellular matrix as a scaffold, the structure is quite different to that of the normal skin. Domestic Zou yundong use rat adipose tissue slices as subcutaneous fat layer and with the composite skin to simulated a three-tier structure of skin and provide a model for constructing tissue-engineered skin substitute containing the layer of subcutaneous adipose tissue.The key link of constructing the multilayer skin complex tissue complex containing the subcutaneous fat layer is the construction of tissue-engineered fat. Using cells extracted from adipose tissue to construct tissue-engineered fat is more" economic". The SVF were a group of cells which contained ASCs, endotheliocytes, pericytes,and theCD31-/CD34+/CD45-/CD90+/CD105-/CD146-was ASCs subgroup. It has been proved that ASCs firstly isolated from liposuction fat tissue had the stem cell differentiation of adipogenic, osteogenic, chondrogenic by Zuk etl.The ASCs was a kind of mesenchymal stem cells,which were similar with BMCs in cell form and immunology phenotype.The cell surface characteristics were compared comprehensively and systemicly by Emanuele and the conclusion:CD29、CD44、 CD54、CD90、CD105、CD166、STRO-1were positive and CD31、CD34、CD45negtive both on ASCs and BMCs, CD49positive on ASCs and CD106on BMCs. The ASCs possessed many advantages:First of all, ASCs were easily harvested without causing secondary defects,and fat tissues were obtained from liposuction of abdomen, upper arm, thigh and buttocks.Second,a lot of fat could be obtained from liposuction and then lots of ASCs.Third, the ASCs could be obtained from patientself and avoiding of immunological rejection and ethics problems.Last, the ASCs could keep good hereditary capacity and lower aging as BMCs after passage. The adipose tissue had successfully constructed by ASCs as seed cells and placenta, PLGA,collagen sponge,fiber glue as scaffolds respectively.This study was to construct epidermal/dermal composite skin with epidermal cells,fibroblasts and type Ⅰ bovine collagen gel and tissue engineering fat with ASCs and type Ⅰ bovine collagen gel,and then the composite skin and tissue engineering fat are assembled to construct multilays composite skin which contained epiderm,derm and subcutaneous fat layer,and lay an experimental foundation for construction multilayer composite skin in tissue engineeringMethods1.Isolation, identification and labled of ASCs,fibroblasts, epidermis cells.Isolate of SVF from adipose tissue,and then gain ASCs from SVF。Induce the ASCs differentiation into fat, bone and cartilage to dentify the multidifferentiation potential.Isolate of fibroblasts and epidermis cells from foreskin. The fibroblasts were dentified by immunohistochemical staining of vimentin,and labled by Dil.2. To construct composite skin with fibroblasts and epidermis cellsTo construct derm substitute with fibroblasts and type I collagen gel,and then general observation,water content determination,microscopicobservation,fluorescence microscope,scanning electron microscopy,HEstaining,immunohistochemical staining and wound repair experiment were performed.3. To construct multilay composite skin with ASCs and composite skinTo construct tissue-engineered adipose with ASCs, and then assembled tissue-engineered adipose and composite skin according to normal skin to construct multilay composite skin with subcutaneus adipose tissue.4. StatisticsThe data were analysised by statistical software SPSS13, and the data were mean±standard deviation, cell growth curve by regression analysis method, the accepted level of significance was P<0.05.Results1. Isolation, identification and labled of ASCs,fibroblasts and epidermis cells.The SVF could be harvested by digestion and centrifugation of adipose tissues,and then ASCs could be harvested by culture SVF.The ASCs could be induced into fat, bone and cartilage cells.The fibroblasts were long spindle,and cytoplasm appeared brown and nuclei blue by immunohistochemical staining of vimentin. The fibroblasts appeared strong red fluorescent,but the nuclei without fluorescent after marked by DiI.The DiI were nontoxic to fibroblasts,and the most survival rate were97.12%.The epidermis cells appeared round and bright when just isolation,and then cobblestone when confluence.2. To construct composite skin with fibroblasts and epidermis cellsThe live derm subsititute performed pink translucence just preparation and then the diameter,color,thickness and toughness changed with the period of culture elongated,The diameter were18.2%of original after14days.The water content of live derm subsititute decreased straightly in culture period, and performed69.3%,67.1%,65.5%,64.3%at1st d、4th d、7th d、11th d respectively The water content sustained above60%in the period of culture.The fibroblasts interlaced and connected network form without direction in gel.The number of fibroblasts increased at2w than1w.The DiI-fibroblasts appeared red fluorescent in gel,and with clear shape,the intensity of fluorescent did not decrease obviously with culture.The contracted live derm subsititute were with loose surface and compact inside.The cells were layed in interspace of collagenous fiber.The collagenous fiber became more regulation and tight,the number of cells increased and were parallel arrangement with fiber.The fibroblasts grew well in gel,and kept secretion activity,type Ⅰ collagen, fibronectin,VEGF werw all positive.The wound were contraction slightly and with mild scar after4w transplanted with live derm subsititute.The live derm subsititute were covered by more thickness stratified epiderm,compared with mormal skin, the collagenous fiber arranged regulation, homogeneous and without layering, with smaller inflammatory cells and vascular, lack of follicle and sebaceous.The fibroblasts in gel grew well and kept activity after4w transplanted on wound.The fibroblasts in gel kept secreting activity, reformed the structure of gel by secreting extracellular matrix, the "derm" were cultured under air-liquid condition after the epiderm cells were planted on it, and then epiderm cells stratified and differentiated and formed a suprabasal layer.3. To construct multilay composite skin with ASCs and composite skin The ASCs were layed in different dimension in gel.The cell were full of round lipid droplets,the lipid droplets appeared bright under inverted microscope and red spheroidal stained by oil red O or transparent bubble stained by HE.The composite skin and fat tissue were assembled according normal skin to construct multilay composite skin tissue which contained epiderm,derm and subcutaneous fat.The epiderm were continuous and multilay,the cells in derm performed homogeneous, dedifferentiated fat cells which contained lipid droplet in gel performed homogeneous.The derm adhered to subcutaneous fat strongly,and the cells gathered between two laysConclusions1. The SVF could be harvested from adipose tissues,and then ASCs from SVF. The ASCs possessed the proficiency of multiplex differentiation which could adipogenic, osteogenic and chondrogenic differentiation.2. The fibroblasts cultured in type Ⅰ collagen gel could performe good cell activity. The live derm substitute constructed with fibroblasts and type Ⅰ collagen gel could reduce wound contraction and scar formed,but lack of cutaneous appendages compared with normal skin.The composite skin were constructed by dermal substitutes planted epidermis cell on its surface.3. The ASCs mixed with type Ⅰ collagen gel and then adipogenic induction could form fat tissues in vitro and in vivo.4. The composite skin which constructed with fibroblasts,epiderm cells and type Ⅰ collagen gel and tissue-engineered adipose which constructed with dedifferentiated fat cells and type Ⅰ collagen gel were assembled according to normal skin to construct multilays skin composite tissue which contained epiderm,derm and adipose layer. |
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