| Cartilage play an important role in people’s daily life. Once thecartilage damaged it will be greatly affected people’s daily activities. Butcartilage is lack of sufficient blood and lymphatic circulation, the repairand regeneration ability is limited.Joint cartilage lesions caused by traumaand arthritis often leads to scar healing, eventually leads to the loss ofjoint function. It affected the quality of people’s lives seriously. Andfunction tissue-engineering has become one of the most prospectingapplication。In function tissue engineering, biological materials andmechanics cues have great effects on the structure and function of thefunction tissue-engineering cartilage. Silk fibroin (SF) is an attractivematerial for tissue-engineering because it is a fibrous protein that hashigh permeability to oxygen and water, low inflammatoryresponse,relatively low thrombogenicity, protease susceptibility, supportscell adhesion and growth,and, foremost, high tensile strength withflexibility. During normal joint movement, articular cartilage is inmechanical condition, including direct compression, hydrostatic pressure,shear force, etc. The researchers also found that mechanical cues couldimprove the mechanical properties of tissue engineering scaffolds. Manybioreactor was used in cartilage tissue engineering. Direct dynamiccompression and flow shear can increase the cell proliferation and matrixsynthesis of tissue-engineering scaffolds, and improve the mechanicalproperties.The load can mimic the pattern of stress in the joint greatly，sothe engineering cartilage could be in the condition of directcompression and shear forces by our bioreactor.Therefore, ourexperiment study the effect of the rolling load bioreactor and porous silk fibroin scaffolds on the tissue engineering cartilage.Objective： To explore the effect of the rolling load bioreactor onthe generation of tissue-engineered cartilage from rabbit chondrocytes-silk fibroin porous scaffold composite.Method： Chondrocytes were isolated from New zealand rabbits byenzymatic hydrolysis method, at approximately2.5months old. Passage3chondrocytes were used to make chondrocytes-silk fibroin constructs.Theexperimen sample were divided into mechanical load and static culturegroup. After the24hours of inoculation, samples were subjected touniaxial, cyclic Bionic mechanical stimulation at15%compressive strainamplitude and0.33Hz for2hours per day,5days per weeks; Controlsamples were cultured under conditions without rolling load compressionin incubator. At3,7,14,21days，chondrocytes viability，Real-time PCR,the amount of deoxyribonucleic acid (DNA) per sample,synthesis ofproteoglycan and collagen were detected to observe cells viable andproliferation，expression of chondrocytes gene and protein.Results： Silk fibroin porous scaffold has good biologicalcompatibility. The chondrocytes cell number and cell viable rate showedthat there is not difference between mechanical loading group andcontrol.After the24hours of inoculation, chondrocytes were evenlydistributed in the scaffolds, and the survival rate is greater than96%. At7,14,21days, the viability percentage is similar in the mechanical loadgroup and the control group，with90.26±2.3%of the mechanical loadgroup and89±1.2%of control group. In the control group，the aggrecangene expression was steady expressed; and the expression of collagentype I and collagen type II gene increased in the culture process anddecreased at7day and14day，respectively. And the expression ofcollagen protein, glycosaminoglycan increased steady.The expression of cartilage-specific gene expression in the rollingload group was significantly higher than the control group (P <0.05).Expression of aggrecan gene in the rolling load group were1.623 folds(P=0.04),3.298folds(P=0.002) of the control group at the7and14day，respectively.At3,21days, that were0.72folds (P=0.26),0.88folds(P=0.72)of the control group, with no significant difference. The collagentype II gene expression was also higher compared with the staticcultures(P <0.05).At3,7,14，21days, the expression of the collagen typeII in mechanical group were5.4folds (P <0.01) and13.7folds (P <0.01),2.05folds(P=0.107),4.51folds (P <0.01)compared to the control group.Collagen type I gene expression have a transient increase in the rollingload group at the3th days, relative to the control group by2.95folds (P<0.01). At7,14,21days,that were0.70folds(P=0.34),1.26folds (P=0.47),1.38folds(P=0.09)relative to control，but whitout statisticallydifferences.The synthesis of proteoglycans and total collagen in rolling loadgroup were higher than control(P <0.05). At3,7,14，21day，proteoglycansare12.17±1.835μg/(μg DAN)，17.367±2.48μg/(μg DAN)，25.83±1.56μg/(μg DAN)，30.25±3.41μg/(μg DAN)，respectively，which were1.71folds（P=0.01），1.66folds（P=0.06），1.62folds（P＜0.01），1.76folds（P＜0.01）of control. At3,7,14，21day，the total amount ofcollagen were1.23folds(P=0.08), and0.73folds(P=0.17),1.52folds(P=0.045)1.09folds(P=0.26)。The results revealed that a significantportion of the newly synthesized macromolecules was released into themedium， especially in mechanical group. Of the total amount(macromolecular in construct plus medium) of proteoglycans synthesizedat day21,16.5%of the newly synthesized proteoglycans were in theconstruct in free-swelling cultures，and10.5%in the rolling load group (P<0.05). And in the construct,the rolling group and static control groupwas no difference in the proteoglycan content.Tissue staining showed thatproteoglycan evenly distributed in the composite cultures, but rollinggroup and static control group was no significant difference in theproteoglycan，according with the results of the biochemical detection.Conlusion： Chondrocytes in the silk fibroin scaffolds have good viability. The cyclic Bionic mechanical stimulation at15%compressivestrain amplitude and0.33Hz for2hours per day,5days per weeks canmake the cell keep the chondrogenic phenotype and promote syntheticmetabolism, provide appropriate load mode for regeneration of thefunction tissue-engineering cartilage. |
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