| The auricle represents only a minority of the total body surface area, but it is one of the most complex three-dimensional structures of the external body. Domestic literature reported that congenital external and middle ear malformation incidence of1/7000, male to female ratio of approximately2:1, bilateral10%, the deformity of right side is more. Total auricular reconstruction for congenital microbial or auricular traumatic amputation remains one of the greatest challenges for ENT surgeon. So far, the main body site from which cartilage can be obtained, in necessary quantity and suitable integrity, is the anterior region of the ribs. It was in1971that Tanzer advocated a plan for the pinna total reconstruction that required four stages for completion using costal cartilage as a skeletal scaffold. Autogenously reconstruction using cartilage frameworks has become a standard procedure of pinna reconstructions in most countries. But the surgery also has many unavoidable problems, such as surgical scars, trauma pain, and the risk of pneumothorax.Metals and organic materials stent experience from the initial iron and copper alloy tolatex and plastic and later to use silica gel. They are not accord with the body without adverse reaction, plasticity and biocompatibility principle,so have not been used widely. Nowadays, accompanying development of tissue engineering techniques, the most promising is biological tissue-engineered cartilage remodeling auricle. But, own cartilage derived small, it is hard to reach the required number of cells. Chondrocytes in vitro vulnerable to "dedifferentiation", cannot be achieved the need of cartilage tissue engineering. This is limiting development of auricular reconstruction with cartilage tissue engineering. This study will research factor--hepatocyte growth factor (HGF), using Ad-HGF transfection the New Zealand white rabbit’ s auricular cartilage cells in vitro. Observing survival and biological characteristics of auricular cartilage cells carrying with the human hepatocyte growth factor. Lay the foundation for auricular cartilage tissue engineering.Chapter one The isolation and culture of rabbit’s auricular chondrocytesThe purpose of this study is exploring method of isolation and culture of rabbit’s auricular chondrocytes and biological characteristics of rabbit’s auricular chondrocytes in vitro. Auricular chondrocytes were obtained from the cartilage of New Zealand rabbit aged2weeks adopting method of mechanical and sequential digestion by0.2%collagenase Ⅱ. We have observed chondrocyte morphology under an inverted microscope each day and drawn growth curve. Toluidine blue metachromatic staining and immunohistochemical staining were used to determine their biological characteristics. The results showed that:the time of rabbit’s auricular chondrocytes to a monolayer was about one week in primary culture. The time of subculture was about3to5days. Rounded and polygonal chondrocytes occupied most of cells, with the passage cell morphology changed to fibroblast-like. Toluidine blue staining confirmed that cells can synthesize proteoglycan, metachromatic reaction mainly concentrated in the colony growing areas. Before the third generations is obviously by dyed, and then gradually weakens. Combination of mechanical and enzymatic digestion in the isolated cell activity was more than85%. Collagen type Ⅱ immunohistochemical staining shows that type Ⅱ collagen content is almost completely lost. Conclusions:combination method of mechanical and enzymatic digestion in vitro rabbit’s auricular chondrocytes receive higher activity of chondrocytes. Type Ⅱ collagen of auricular cartilage have decreased and the cell morphology have changed, cartilage tissue have loss their biological properties.Chapter two Transfection and expression of Ad-HGF in rabbit’s auricular chondrocytes in vitro and its effect on cell proliferationThe purpose of this study is to explore the transfer efficiency of a recombinant adenovirus carry human hepatocyte growth factor (HGF) gene (Ad-HGF) in rabbit’s auricular chondrocytes and to observe the expression of HGF gene in cell and the effect of the transfection on the proliferation of rabbit’s auricular chondrocytes in vitro. Lay the foundation for auricular cartilage tissue engineering. Primary cultured chondrocytes were infected with Ad-GFP at different modified intensity (50,100,200). After infection72h, the transfer efficiency was evaluated by flow cytometry. After Ad-HGF infection at48h,72h when MOI=100, the HGF expression in supernatant was detected by ELISA. MTT was used to detect the proliferation of rabbit’s auricular chondrocytes and mmunohistochemical staining was used to determine their which carry HGF gene biological characteristics. Experiments show that transfer efficiency of Ad-GFP for rabbit’s auricular chondrocytes reached50%when the MOI=100. The level of HGF expression was about (513±20.77) pg/ml and (312.7±23.37) pg/ml at48h and72h respectively. HGF transfected in third generations of cartilage cells which.Type Ⅱ collagen immunohistochemical positive results in third generations of cells and transfected in third generations of cells were (0.652±0.0278),(0.682±0.039),p>0.05, no difference; in fifth generations of cells and transfected in fifth generations of cells were (0.23±0.0469),(0.35±0.0367),p<0.05. It is concluded that the recombinant adenovirus can infect efficiently rabbit’s auricular chondrocytes and the infected cells can express efficiently the aim protein. The HGF can promote the proliferation of rabbit ear cartilage cells and maintain their biological characteristics. |
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