Preparation Of Collagen-chitosan/fibrin Glue Asymmetric Scaffold And Its Application In Construction Of Tissue Engineered Skin

BackgroundIn clinical practice, skin defects caused by all kinds of factors such as burns, trauma,and chronic ulcers are common, and their traditional therapy method is skin transplantation. The main skin sources are autogenous skin, allogenous skin, or xenoskin. Among these, autogenous skin is the best choice because it has no rejection. However, autogenous skin transplantation has its deficiencies either. First there is not enough skin for the patients with extensive burns. Second, injury in the donor site is a problem, because of its possible scarring or pigmentation. In recent 20 years, tissue engineering rised quickly with the development of biomaterial and life science. Tissue engineering offers us a better method to repair and reconstruct tissue defects. Tissue engineered skin is a field that developed most quickly in tissue engineering. At present, many commercial products such as Integra, AlloDerm, Transcyte, Apligraf have been developed and obtained the permissions of FDA. These tissue engineered products have displayed a good therapeutic efficacy in wound repair and they can promote the regeneration of skin. However, the price of these products abroad is very high and beyond the endurance of common people of our country. It can't meet our people's demand. So, it is important to develop a tissue engineered-skin product by ourselves which not only meet the clinic requirement but also low in price. Collagen is a protein which occupies the highest content in mammal animals, with20—30%, and it has many characteristics: Good biocompatibility, degradation and low in antigenicity. So it has been widely used as an ideal scaffold in tissue engineering. Chitosan is extracted from chitin which distributes extensively in natural environment, and the cost price is low. The collagen-chitosan has good biocompatibility, proper degradation and mechanical intension, and it displays a porous structure with a pore size at 80-150μm which is suitable for the migration and proliferation of dermal fibroblasts. However, the complete tissue engineered-skin should contain epidermis and dermis. So if we want to develop composite skin substitutes, dermis and epidermis should be constructed simultaneously. But it seems that the homogeneous larger pore size of collagen-chitosan is not suitable for the construction of composite skin substitutes, so we must develop a scaffold which has a better microstructure.Fibrin glue is a polymer of fibrinogen in the presence of thrombin, and it has a good biocompatibility and degradation in vivo, and it was used widely in surgical operations as a hemostat and tissue adhesive. Researches have indicated that it could promote attachment, migration and proliferation of cells. At present, it is applied in the repair of the wound with tissue engineering methods. In addition, fibrin glue has a porous net-like microstructure which is related to the concentration of fibrinogen as well as thrombin.In this study, we successfully prepared a kind of asymmetric scaffold which possess a bilayer structures with different pore size in either side by combining fibrin glue to collagen-chitosan porous scaffold, and then we used it to construct tissue engineered composite skin, we found it can provide a good environment for cell growth, and has a potential prospect.ObjectiveThe aim of this study was to prepare an asymmetric scaffold which is fit for the construction of tissue engineered- skin with collagen-chitosan and fibrin glue, and then investigate the feasibility of using it to fabricate skin substitutes in vitro.Material and methods1. Isolation and culture of skin cells1.1 Cultures of human dermal fibroblastsPrimary human dermal fibroblasts were isolated by tissue culture technique from skin pieces that is obtained from donors after circumcision. Subculture was carried out. The 3-8 generation of cells were used as work cells.1.2 Culture of human epidermal cellsHunan keratinocyte cell line (HaCaT) was chosen in the study. The cells were cultured in DMEM (high glucose) medium supplemented with 100U/ml penicillin, 100U/ml streptomycin and 10% fetal bovine serum(FBS), at 37 ℃, 5%CO₂. The medium was changed every 3days.2. Preparation of collagen-chitosan/fibrin glue asymmetric scaffoldCollagen and chitosan were dissolved in 0.5 M acetic acid solution to form a 0.5% (w/v) solution respectively, and then these two kinds of solution were mixed in a mass ratio of 9:1 After deaeration under reduced pressure to evolve entrapped air bubbles, the collagen/chitosan composite was injected into a mold, frozen in a refrigerator at — 20℃ for 2h and then lyophilized for 24h to obtain a porous collagen/chitosan scaffold , and then this scaffold was crosslinked at 105℃ under reduced pressure for 24 h and then was further treated with EDAC/NHS solution for another 24h. After washed with double-distilled water (10min x 5times), the scaffold were freeze-dried again to obtain the EDAC/NHS treated collagen/chitosan scaffold. The scaffolds were immersed in 75% ethanol for over night for sterilization, followed with solvent exchange by PBS for 5 times at least. The fibrinogen solution was smoothly spread on the surface of collagen/chitosan scaffold (50μl/cm² ), and then the scaffold was put into incubator at 37℃ for 30min to make fibrinogen polymerize after thrombin was dropped onto it .When fibrin glue formed the asymmetric scaffold was obtained.3. Histology and microstructure observation of the asymmetric scaffoldFibrinogen solution was dilute to 40mg/ml and 8mg/ml with DMEM medium at the same time, thrombin solution was dilute to 300U/ml and 60U/ml with DMEM medium. Fibrinogen was spread onto the outside surface of collagen -chitosan porous scaffold which was sterilized , and then corresponding thrombin was drop onto it, and then the scaffold was put into incubator at 37℃ for 30min .The asymmetric scaffold was prepared after the fibrin glue was formed on the surface. Some collagen-chitosan/fibrin glue asymmetric scaffolds were fixed in 10% formaldehyde, embedded in paraffin, sections were stained with haematoxylin-eosin(HE). At the same time other scaffolds were fixed in glutaraldehyde and osmic acid, dried at critical vertex, and then observed under scanning electron microscope (SEM).4. Construction of tissue engineered skin with collagen -chitosan/fibrin glue asymmetric scaffoldThe HaCaT cells was digested from the flasks, and then were seeded onto the upper surface of the asymmetric scaffolds with a density of 1×10 6 /cm². After 2h , it was cultured in DMEM medium supplemented with 100U/ml penicillin, 100U/ml streptomycin and 10% FBS in a 5%CO₂ incubator, at 37℃ with medium change every 2 or 3 days , and then shifted to air-liquid interface after 3-7 days. At the same time the equivalent density of cells were seeded onto the surface of collagen-chitosan porous scaffold without fibrin glue in the same culture condition as the control.5. Construction of composite skin substitutes with the asymmetric scaffoldThe dermal fibroblast was digested from the flasks and then the density was adjusted to 5×10⁶ /ml. The cell suspension was inoculated into collagen-chitosan porous scaffold which was sterilized previously, and then incubated in DMEM medium supplemented with lOOU/ml penicillin, lOOU/ml streptomycin and 10% FBS under condition of 5%CO₂ 37℃. The medium was changed every 2-3 days. 1 week later , the collagen-chitosan porous scaffold containing Fbs was transferred to a new 6pore- plate and then was put into incubator at 37℃ for 30 minutes. When the surface of the scaffold is a somewhat dry, the fibinogen and thrombin were overlaid onto it. After the polymerization of the glue at 37℃ for about 0.5h, the epidermal cells were inoculated onto the upper surface of this collagen-chitosan porous scaffold containing Fbs with a density of 1×10⁶ /cm². After immersed culture for 3-7 days, the composite skin substitutes were shifted to a self-designed stainless steel mesh and cultured in the air-liquid interface conditions. Medium changes were performed every 2 or 3 days. The cultures were harvested 1, 2, 3 weeks later for paraffin embedded sections.6. Viability of cells in the asymmetric scaffoldThe asymmetric scaffold seeded dermal fibroblasts was cultured in DMEM medium (high glucose, 100U/ml penicillin, 100U/ml streptomycin and 10% FBS), and medium changes were performed every 2 or 3days. FDA-PI staining and SEM analysis were done after 1 and 2 week's culture respectively.7. Histological observation of the constructed skinThe cultures were fixed in 4% formaldehyde overnight, dehydrated in ethanol and embedded in paraffin; sections (5μm) were stained with haematoxylin-eosin(HE).8. Immunohistochemistrical examination2 weeks after transferred to air-liquid interface, the constructed skin were fixed in 4% formaldehyde and embedded in paraffin. After dewaxed, the sections were used to for immunohistochemistrical examination followed the instruction of Maxin's ultrasensitive SP kit.Results1. Isolation and culture of skin cells1. Dermal fibroblastsA great number of fibroblasts emigrated from the tissue pieces after cultivation for 1week, and they grew to 90% confluence after another week.1.2 HaCaT cellsThe HaCaT cells grew with a good state in DMEM medium and after seeded 4-5 days,the cell became confluence with a typical stone-like appearance.2. Histology and SEM observation of collagen-chitosan/fibrin glue asymmetric scaffoldIn HE staining images, the scaffold displayed an asymmetric bilayer structure, the top layer consisted of fibrin glue was a thin layer with a tight structure while the bottom layer consisted of collagen-chitosan scaffold was a thicker layer with a loose and porous structure. In some area, the top layer developed a rete ridge-like structure inserted into the bottom layer. With the SEM examination, a two-layer structure with different pore sizes and porosity was shown on the asymmetric scaffold clearly. The pore size of the bottom layer is at the rang of 80—150μm while the top layer had a smaller pore size, at the range of 5-20 μm.3. Viability of cells in the asymmetric scaffoldThe asymmetric scaffold seeded fibroblasts was stained with FDA and PI and then observed under fluorescence microscope. The image of fluorescence reveals a great number of cells (green fluorescence) grew in the dermal layer of the asymmetric scaffold with shuttle-like or irregular morphology, and the SEM image reveals that the fibroblasts were adhered on the walls of the scaffold with typical shuttle-like morphology, and much extracellular matrix was around.4. Histological observation of tissue engineered skinOn the surface of the asymmetric scaffold, 3-4 layers of cells were observed when cultured on air-liquid interface for 2 weeks, while no obvious multilayer were seen on the surface of collagen-chitosan porous scaffold without fibrin glue layer, even though they had been cultured for 3 weeks. The culturing of tissue engineered composite skin was terminated when cultured inair-liquid interface conditions for 1, 2and 3 weeks. The HE staining images reveal a great many fibroblasts grew in the bottom layer of the asymmetric scaffold while epidermal cells started to differentiate into a multilayer structure on its top layer. 3 weeks later, the epidermal cells developed into a epidermis-like structure with 7-10 layers of cells. The constructed composite skin substitutes possess a histological structure similar to the normal skin.5. Immunohistochemistry analysisAfter 2 weeks of cultivation, the multilayered epithelium of the tissue engineered-skin was obvious, The Pan- CK is positive in the whole layer of epithelium and the CK10 is positive only on the upper part of it.ConclusionsWe successfully developed a kind of asymmetric scaffold with different pore size on both sides by combining biopolymers: collagen-chitosan and fibrin glue, which is fit for the construction of tissue engineered skin in vitro and may have future application prospect.