Preliminary Study On The Biological Effects Of HA-Au NPs On Rat Osteoarthritic Chondrocytes

Objective:The hyaluronic acid-gold nanoparticle(HA-Au NPs)complex was prepared,and the mRNA expression of IL-1,TNF-? and MMP-13 and apoptosis on rat osteoarthritic chondrocytes were investigated in vitro.Method:1.Different kinds of Au NPs were prepared by three different methods.The color of the three Au NPs was observed.The UV-visible spectrophotometer was used to detect the UV absorption peak of Au NPs,the shape of Au NPs was observed by transmission electron microscope,and the cytotoxicity of Au NPs were detected by CCK-8;2.The HA-Au NPs complex was prepared,and the color of HA-Au NPs was observed.The UV-visible spectrophotometer was used to detect the UV absorption peak of HA-Au NPs,the shape of HA-Au NPs was observed by transmission electron microscope;3.Four 8-week-old SD rats were randomly divided into two groups.Two SD rats were modeled by the left knee anterior cruciate ligament + medial collateral ligament(group OA).The other two SD rats only cut the left knee joint and did not cut any ligaments(group Sham);Two groups of left knee osteoarthritis primary chondrocytes were obtained after 8 weeks;4.The primary chondrocytes were identified by type II collagen cytochemical staining,toluidine blue staining and alimin blue staining;5.The cultured second-generation chondrocytes were divided into 5 groups:group A(group sham control),group B(group OA control),group C(group OA +HA),group D(group OA + Au NPs)and Group E(group OA+HA-Au NPs).The expression of IL-1,TNF-? and MMP-13 mRNA in chondrocytes of 5 groups of osteoarthritis were detected by qRT-PCR;6.The apoptosis of chondrocytes was detected by flow cytometry.Results:1.The color,UV absorption peak,morphology and cytotoxicity of the three Au NPs prepared were different.The spherical Au NPs solution is burgundy,and the UV-visible absorption spectrum has an absorption peak at 525 nm.Its morphology is spherical,smooth and uniform in size;the rod-shaped Au NPs solution is brownish yellow,and the UV-visible absorption spectrum has two absorption peaks which is at500 nm and 820 nm,and its morphology is short and uniform.The snow-like Au NPs solution is grayish black,and the UV-visible absorption spectrum has an absorption peak at 560 nm,Its shape is snowflake and irregular in shape.The cytotoxicity of globular Au NPs is relatively small,the cytotoxicity of snow-shaped Au NPs is second,and the cytotoxicity of rod-shaped Au NPs is the strongest.2.The prepared HA-Au NPs solution is burgundy.The UV-visible absorption spectrum of HA-Au NPs has an absorption peak at 528 nm.The morphology of the HA-Au NPs is spherical,surrounded by a transparent thin layer.3.The knee joint fluid and articular surface of the two groups were different.The group that anterior cruciate ligament and medial collateral ligament were cut.The joint fluid was thick,yellow-brown,the joint surface was rough and partially worn,and a small amount of callus was visible on the outer edge of the joint.The other group had thin and light yellow joint fluid,smooth joint surface,and no obvious wear and osteophyte formation.4.After staining with toluidine blue,the chondrocytes werenucleus appeared dark blue.After staining with alimin blue,the cytoplasm was light blue.After immunohistochemical staining of type II collagen,the cytoplasm was yellowish brown.5.The results of qRT-PCR showed that the mRNA expression levels of IL-1,TNF-? and MMP-13 in group A(group sham control),group C(OA+HA),group D(OA+Au NPs)and group E(OA+HA-Au NPs)were significantly lower than those in group B(group OA control)(p<0.05).The mRNA expression of group E(group OA+HA-Au NPs)was lower than group C(group OA+HA)and group D(groupOA+Au NPs),the difference was statistically significant(p<0.05);The mRNA expression of group D(group OA+Au NPs)was lower than group C(group OA+HA),the difference was statistically significant(p<0.05).6.The apoptosis results showed that the apoptosis rate of chondrocytes in group A(group sham control),group C(group OA+HA),group D(group OA+Au NPs)and group E(group OA+HA-Au NPs)was lower than that in group B(group OA control),the difference between the groups was statistically significant(p<0.05).The apoptosis rate of chondrocytes in group E(group OA+HA-Au NPs)was lower than that in group C(group OA+HA)and group D(group OA+Au NPs),the difference was statistically significant(p<0.05).The apoptosis rate of chondrocytes in group D(group OA+Au NPs)was lower than that in group C(group OA+HA),the difference was statistically significant(p<0.05).Conclusion:1.The successfully prepared spherical Au NPs and HA-Au NPs have good stability,good biocompatibility and low cytotoxicity;2.HA-Au NPs,Au NPs and HA can inhibit the mRNA expression of IL-1,TNF-? and MMP-13 on osteoarthritic chondrocytes,and HA-Au NPs have the strongest inhibitory ability;3.HA-Au NPs,Au NPs and HA can inhibit osteoarthritis chondrocyte apoptosis,and HA-Au NPs have the strongest inhibition ability.