Glukozamin (Ga) Bağlı Hyaluronik Asit (Ha) Nanopartiküllerin (Ga-Nha) Geliştirilerek Yapısal Özelliklerinin Belirlenmesi Ve In Vıtro Hücre Kültüründeki Etkilerinin Araştırılması

Abstract

Osteoarthritis (OA) is a articular joint disease which causes pain, limitation of movement, deformity and can lead to disability and mortality. Glucosamine (GA) and chondroitin sulfate (CS) are alter the course of the disease but it is lesser known that how they are reach to the synovial fluid and show their effects. Research questions of this study whether (a) we could produce and use as a drug delivery system hyaluronic acid nanoparticles (nHA). (b) GA would transport to joint cavity using with nHAs. (c) GA-nHA would increase cell proliferation, (d) GAnHA would stimulate chondrogenesis of bone marrow mesenchymal stem cells (BM-MSC), and (e) GA-nHA would improve extracellular matrix (ECM) production and reduce OA markers of normal and osteoarthritic chondrocytes. After production of nHA’s its structural property determined with SEM, DLS and ¹H NMR. After conjugating CS and GA to nHA’s, release dynamics of these molecules determined with HPLC. CS didn’t release from nHA’s so studies contiuned with GA. Effects of 1, 10, 100 µg/ml GA-nHA’s on chondrosarcoma cell line (SW-1353) measured by WST-1 Cell Proliferation Assay. Efficacy of early chondrogenic markers including cartilage oligomeric matrix protein (COMP) and SRY-Box-9 (SOX-9) expression on BM-MSCs of GA-nHAs measured by RT-PCR. Efficacy of extracellular matrix components including type II collagen (COL2A1) and COMP expression and OA marker matix metalloproteinase-13 (MMP-13) iv

expression on healthy and osteoarthritic human chondrocytes of GA-nHAs mesaured by RT-PCR, again. Peaks between 0.6 and 5.0 ppm of ¹H NMR results showed that nHAs succesfully synthesised. SEM and DLS results showed particule size dispersed between 200 and 600 nm homogeneously. HPLC results showed us GA released in the ratio of 20% in 21 days. Three doses of GA-nHA’s didin’t increase proliferation of chondrosarcoma cells according to WST-1 Proliferation Assay outcomes. However, application of GA alone increased the proliferation significantly compare to control group. GA-nHAs did not significantly change the SOX-9 and COMP expressions. On OA chondrocytes, 1 µg/ml GA-nHA administration significantly decreased MMP-13 expression, 10 µg/ml GA-nHA administration decreased MMP-13 expression and incresed COMP and COL2A1 expressions, yet there is no significant difference was found. On healthy chondrocytes, three doses of GAnHA administration repressed the MMP-13 expression compared with the control group, but no significant changes were observed. When GA applied alone, MMP13 levels significantly decreased and chondrogenic marker COMP levels significantly increased compared with the control group. Also, 10 µg/ml HA group increased COMP and COL2A1 levels of healthy chondrocytes but there is no significant difference with control group. In conclusion, GA-nHAs did not effect cell proliferation and chondrogenic differentiation but decreased MMP-13 expression, increased COMP and COL2A1 expression of OA chondrocytes. Although these results are not statistically significant, they may be clinically significant. When we apply GA and HA alone, some of the positive results may be due to the fact that GA-nHA is in a form that cells can not use with nanoparticle form. In order to determine whether GA-nHAs is an effective method in the treatment of OA, the dynamics of cell culture medium, cellular uptake, cell processing mechanisms of GA-nHA should be monitored and long term effects of cell culture should be investigated.