Effect Of Nerve Growth Factor Isolated From Snake Venom On Rabbit Articular Chondrocytes And Their Application Into Preparation Of Tissue-engineered Cartilage In Vitro

Objective:we conducted this study to investigate the effect of nerve growth factor(NGF)isolated from snake venom on rabbit articular chondrocytes in 2 dimension culture and thereby was applied to preparation of cartilage-like tissue with rabbit articular chondrocytes in collagen hydrogel.Methods:(1)the articular chondrocytes were isolated from one-week-old New Zealand rabbits by re-modified enzyme digestion methods.We examined the biological characteristics and exclusive functions of primary and passaged chondrocytes for the purpose of acquiring enough chondrocytes that performed well in proliferation and maintenance of phenotype.(2)With these qualified chondrocytes,the cytotoxicity assay of NGF was conducted to identify the range of concentrations of no cytotoxicity,among which we chose the concentrations with relative obvious effect(35,70 and 140 ng/ml)and designed control group(0 ng/ml NGF)for the further research.DNA content,GAG/DNA,HE staining,immunohistochemical staining(collagen ? and ?)and PCR of cartilage-related genes were performed after 2,4 and 6 days of intervention respectively.(3)After determination of NGF positive effect on chondrocytes,we prepared 3D model of cartilage-like tissue with rabbit articular chondrocytes in collagen hydrogel.These boluses were randomized into NGF group(70 ng/ml NGF),positive group(10 ng/ml TGF-?)and control group(0 ng/ml NGF and TGF-?).After 7,14 and 21 days,the examinations in(2)and biomechanical analysis were performed.Results:(1)The primary articular chondrocytes from New Zealand could be obtained through the re-modified enzyme digestion method.With cells starting to be adherent after 24-36 h and regularly becoming triangular or fusiform,the toluidine blue O staining,PCR of collagen II gene showed high level,which would be opposite in the chondrocytes more than passage 3.collagen I which meant lose cartilage-related phenotype is low in cells not more than passage 3.Therefore,P3 chondrocytes is suitable for the further research with the virtues of high proliferation and cartilage-related phenotype.(2)The range of concentration with no cytotoxicity was 0-280 ng/ml,among which 17.5-240 ng/ml NGF increased the cell growth.DNA content showed NGF especially at the dose of 70 ng/ml enhanced proliferation and viability;GAG/DNA,IHC and PCR also the higher cartilage-related functions.But the HE staining showed no difference between all groups.(3)There was no difference with respect to Young's modulus for biomimetic material before and after culture(P>0.05).NGF group were similar to TGF-? group regarding promotion of proliferation and cartilage-related functions,while their effect were greater than control group.The HE staining showed no difference between all groups.Conclusion:The enough rabbit articular chondrocytes with the characteristics of high proliferation and cartilage-related phenotype could be obtained from New Zealand rabbit through re-modified enzyme digestion method and adherent culture.NGF from snake venom could enhance the proliferation and maintain the phenotype of the kind of chondrocytes in monolayer culture.Its effect also appeared in the preparation of tissue-engineered cartilage in vitro.