| Background and ObjectivesLarge area of skin tissue defect caused by severe burns and mechanical injury is difficult to manage, and has been a hot spot for Plastic and Reconstructive surgery. At present we usually use autologous flaps or split-skin to repair in clinical,but due to limited amount of tissue, autologous flaps or split-skin are unable to meet the clinical application. So we often use artificial skin or allogeneic skin to handle it. The allogeneic skin supply is limited, so we focused on the artificial skin. Recently due to advancement in tissue engineering, the use of tissue-engineering skin to repair large skin defect is spreading among many doctors. Although there are a lot of researches in tissue-engineering skin, but we have not yet found an ideal artificial skin. Therefore it become necessary to explore a tissue-engineering skin based on the structural and physiological characteristics of the skin.Nanotechnology provides a new idea for synthetizing and producing new bionic biological activity materials. Skin tissue engineering scaffold uses electrospun technology. Advantages compared with traditional scaffold include as follows:①special three-dimensional porous structure, interconnected between holes, nutritional and metabolic needs of growing cells on the scaffold; ②a specific surface than other forms of materials, and provide good conditions for the cell adhesion and the adhesion of the functional proteins; ③the epidermal thickness is 0.2～0.25mm, and the thickness of the dermal layer is 0.4～0.45mm. Electro spinning nano-fiber’s thickness may be in the range of 0.05～2mm, they are consistence with the microscopic structure of the extracellular matrix on the geometrical dimensions, and therefore the electro spinning nano-fibers can mimic the structure of the natural ECM and achieve a better simulation of the skin.This experiment use cultured adipose stem cells (adipose-derived stem cells, ADSCs) which is isolated from SD rats and electros pinning polylactic acid-polycaprolactone/Poloxamer (PLCL/P123)material to reconstruct tissue engineering skin, explore the possibility to enhance wound healing in rats, and then we tried to further explore the molecular mechanisms in electro spinning nano-scaffolds and its effects on stem cells differentiation and proliferation.Materials and Methods(A) Adipose-derived stem cells(ADSCs) isolation, identification, culture and induced differentiation1)The fat tissue is obtained from the groin of SD rats (6 weeks of age, weight about 180g), ADSCs are extracted by conventional enzyme digestion, then we observe the cell morphology and draw the growth curve.2) Use monoclonal antibody to label the ADSCs, then identify stem cells surface markers by flow cytometry:CD29, CD34, CD44, CD54, CD90.3) Use conditional induction medium to induce ADSCs to differentiate into fat, osteogenesis and epidermis, we stained oil red "O" to detect adipogenic; we use the alizarin red stain and Von kussa stain to detect osteogenesis; We use CK10 immunohistochemistry to detect epidermis.(B) Synthetize and properties determination of polylactic acid-polycaprolactone/poloxamer (PLCL/P123) electro spinning nano-scaffold1) Use electrostatic spinning machine to prepare four different proportions PLCL/P123 electro spinning nano-scaffolds (75:25,85:15, 90:10,95:5).2) Use scanning electron microscope to observe the electro spinning nano-fibers morphology and diameters of different proportions of PLCL/P123.3) Use SANS tensile to measure the mechanical properties of different proportions of PLCL/P123 electro spinning nano-fibers.4) The water contact angle determination of PLCL and different ratio PLCL/P123 electro spinning nano-fibers.(C) In vitro culture and biological activity studies of Adipose-derived stem cells with PLCL/P123 electro spinning nano-fibers1) This period includes six groups:ordinary cell culture dish plate group (control group), the PLCL group, and different ratio (75:25,85:15,90:10, 95:5) of PLCL/P123 electro spinning nano-fibers groups.2) In vitro co-culture of ADSCs (cell concentration of 5×104/ml) with six groups of materials, then use scanning electron microscope to observe of the growth of ADSCs on different scaffolds.3) In vitro co-culture of ASDCs (cell concentration of 2×105/ml) with six groups of materials, then detected Ki67 expression by RT-PCR after co-culture for 7 days.(D) Adipose-derived stem cells and PLCL/P123 electro spinning nano-fibers reconstruct a tissue-engineering skin for wound repair in rats1) The group of SD rats are 8 weeks old, weighing about 250g, and then divided into three groups. Group A:PLCL/P123 with ADSCs; B group: PLCL/P123 without ADSCs; C groups:control group, use simple Vaseline gauze group.2) We prepare two round (diameter of 1.5cm) full-thickness skin wound on both sides of the back of 8-week-old SD rats, each resection of the 2cm interval. Then we compare them by A Group-B Group, A group-C group, B group-C group on the back of the rats, observe the rate of the dorsal wound healing.3) Remove the dorsal wound tissue after 21 days, and detect local wound healing by HE staining, CK10 immunohistochemical and immunofluorescence staining, CD31 immunohistochemical staining respectively.Results1. ADSCs were isolated from SD rats groin and obtained by conventional enzyme digestion, they have the morphology and characteristics of stem cells, and the expression of surface markers by flow cytometry for CD29, CD44, CD54, CD90 were 95%,97%,88%,94%, negative expression of CD34 expression (0%); After induction of ADSCs, they have the potential of differentiation into adipose, osteogenesis and epidermis.2 Scanning electron microscopy showed that the electrostatic spinning technology prepared PLCL/P123 electro spinning nano-scaffold has better fiber diameter, and the tensile modulus, tensile strength are more close to the normal skin; The water contact angle of PLCL is 131.5±8.9°, which of 75:25,85:15,90:10 three different group is 0, while the 95:5 group is 7.8±2.7°, showed that PLCL have poor hydrophilic capability, while P123 adding to blend of spun, can significantly improve the hydrophilic properties of the materials, is more conducive to the infiltration of the water on the material. The determination of PH value showed they are acidic, but 75:25,85:15,90:10 and 95:5 are weak acidity.3. After co-culture of ADSCs with ordinary cell culture dish (control group), pure PLCL group and 75:25,85:15,90:10,95:5 groups showed that the proliferation of ADSCs in PLCL/P123 is significantly better than the control group and the pure PLCL group, RT-PCR detection of Ki67 shows the same result.4. The wound healing rate of Group A (PLCL/P123 group with ADSCs) is faster than group B (PLCL/P123 group without ADSCs) and group C (simple Vaseline gauze group); Group B repaired quicklier than the control group, the difference was statistically significant, P<0.05. HE staining shows that group A wound tissue is more close to normal skin, and the CK10 staining positive suggesting ASDCs differentiate into epidermal like structure. The HE staining shows that the epidermal structure of group B is incomplete and CK10 staining is slightly positive, while C group shows no epidermal structure and CK10 negative expressed.5. HE staining of group A indicates the wound tissue is closer to normal skin than other groups, and CK10 staining positive. Group B HE staining of epidermal structure is incomplete, and CK10 staining found a small amount of positive expression. Group C showed no epidermal structure, and CK10 negative expression. Immunofluorescence staining of group A indicates the CM-Dil labeled ASDCs located in wound healing skin, which can be induced to differentiation into epidermal cells, a large number of micro-vessels can be detected subcutaneous, micro-vessel density was significantly greater than in group B and group C, the difference was statistically significant, P<0.05.Conclusions1. Adipose-derived stem cells (ADSCs) were simple to isolate and culture. They have potent stem cell characteristics, stable passaging and multi-differentiation potential.2. PLCL/P123 electro spinnig nano-fibers are more close to normal skin physiology, and suitable for ADSCs cell proliferation and differentiation.3. The ADSCs with PLCL/P123 of electro spun nano-scaffold for tissue engineering skin is closer to the skin structural and physiological characteristics, can promote wound healing, so it is worthy of clinical application. |
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