The Effect Of Hydrogen And Preservation Solution’s Update Frequency On Preservation Of Articular Cartilage In Vitro

ObjectiveAs an ideal antioxidant and anti-apoptotic substance,hydrogen can selectively neutralize the most toxic hydroxyl radicals（-OH）and peroxynitrite anions（ONOO-）.Moreover,hydrogen has a good biofilm permeability and enter cells and organelles quickly.Now hydrogen has protective effects on many isolated organs,such as the heart,lung and kidney.In this study,we explore whether H₂ improves the preservation effect of osteochondral allograft by adding it to storage solution during the prolonged tissue culture stage.And explored the effect of preservation solution’s update frequency on preservation of articular cartilage in vitro.And it can provide effective experimental basis and guidance for the further exploration of the optimal preservation method of osteochondral allograft.MethodThis study have two parts.Part Ⅰ,Storage solution containing hydrogen improves the preservation effect of osteochondral allograftThe storage solution containing different hydrogen concentration was prepared by hydrogen generator.The local healthy pig was selected as the research subject and the ARTHREX osteochondral graft apparatus was used to obtain 120 pieces of cylindrical bone cartilage（l=10.0mm,d=6.0mm）in the lateral loading area of the femoral condyle from the pig knee joint in the aseptic condition.Randomly the grafts were divided into 4groups:Control group;H-1 group（hydrogen concentration:0.2mmol/L）;H-2 group（hydrogen concentration:0.2mmol/L）;H-3 group（hydrogen concentration:0.8mmol/L）.The survival rate of chondrocyte,histological changes（H&E staining,safranine O,fast green staining,toluidine blue staining）,biomechanical properties and protein expression（SOD-2,Caspase-3 and 8-OHdG）were investigated after 28 days’storage.Part Ⅱ,The effect of preservation solution’s update frequency on preservation of articular cartilage in vitro.Choosing the local healthy pig as the research subject and using the ARTHREX osteochondral graft apparatus to obtain 60 pieces of cylindrical bone cartilage（l=10mm,d=6mm）in the lateral loading area of the femoral condyle from the pig knee joint in the aseptic condition.Randomly the grafts were divided into 3 groups:Group A（Don’t change liquid during preservation）20;Group B（exchange of liquid per 7 days）20;Group C（exchange of liquid per 2 days）20.The survival of chondrocytes,histological（Safranine O-fast green staining）and biomechanical properties were examined after 28 days preservation.ResultPart Ⅰ,（1）Chondrocyte viability:after 28 days,the chondrocytes viability in each group was determined by EB-FDA fluorescence staining.The results showed that the survival rate of each group decreased significantly.In the H-3 group,the survival rate was the highest,and the H-3 group was superior to the H-2 group（P<0.05）.The cell survival rates of H-3 and H-2 groups were significantly better than the control and H-1 group（P<0.05）.There was no significant difference between the control group and the H-1 group（P>0.05）.（2）Histology:H&E staining showed that the cartilaginous surfaces of each group were smooth,and the superficial chondrocytes were small and mostly flat.The deep chondrocytes are larger in size,and are clustered in clusters.Chondrocalcification layer cells are less,and bone is closely connected with cartilage.The safranine O-fast green staining and toluidine blue staining showed that the cartilage surface of the h-3 group was smooth,the cell structure was clear,the color was the deepest,and the protein polysaccharide content was the highest.Collagen II immunohistochemical staining showed that the collagen content of group H-3 was higher than other groups（P<0.05）.（3）Biomechanics:There was no significant difference in each group（P>0.05）about Young’s modulus.（4）Key proteins:Western Blot results showed that SOD-2 expression levels:H-3group was superior to H-2 group,H-2 group was superior to Control group and H-1 group（P<0.05）,and there was no significant difference between the Control group and H-1group（P>0.05）.Apoptotic factor Caspase-3 and the main marker of the DNA damage8-OHdG:H-3 group was significantly lower than the Control group,H-1 group and H-2group（P<0.05）.There was no significant difference between the Control group and H-1group（P>0.05）.Part Ⅱ:（1）Chondrocyte viability:the viability of chondrocytes in each group was decreased.Group A was higher than group B and group C（P<0.05）.Group B and group C was similar（P>0.05）.（2）Histology:group C was higher than group A and group B on proteoglycan content（P<0.05）.There was no obvious difference between group A and group B（P>0.05）.（3）Biomechanics:Young’s modulus,group C was higher than group A and group B（P<0.05）.Conclusion1.Hydrogen as an additive of storage solution improves the preservation effect of osteochondral allograft by inhibiting the oxidative stress and the apoptosis.The protective effect of H₂ was concentration dependent.2.It is better to replace the preservation solution every two days during the period of preservation.The application of hydrogen and the choice of preservation solution’s update frequency provide a new way for the exploration of the preservation of articular cartilage in vitro,which provides a new plan and guidance for the establishment of cartilage tissue bank.