the surfaces of the two bones at the joint are covered in cartilage . articular cartilage is multi - layered . a thin superficial layer provides a smooth surface for the two bones to slide against each other . cartilage is composed of cells called condrocytes which are dispersed in a firm gel - like ground substance , called the matrix . the main purpose of cartilage is to provide a framework upon which bone deposition could begin . another important purpose of cartilage is to provide smooth surfaces for the movement of articulating bones . in order to illustrate the invention , the following examples are included . however , it is to be understood that these examples do not limit the invention and are only meant to suggest a method of practicing the invention . the following examples used hyaluronan prepared by the methods disclosed in japan patents 1284023 and 1353027 ( publication of examined application number : s60 - 009042 , title : separating method for acidic polysaccharides from connective tissues ; and patent number 1353027 , publication of examined application number : s61 - 021241 , title : separating method for acidic polysaccharides from connective tissues ), which concluding : hyaluronan of 6 , 000 , 000 dalton ( synvisc , biomatrix , usa ), of 600 , 000 - 1 , 200 , 000 dalton ( artzdispo , seikagaku , japan ), of 600 , 000 - 1 , 200 , 000 dalton ( hikamilon dispo , taisho pharmaceutical co ., ltd ., japan ), of 600 , 000 - 1 , 200 , 000 dalton ( lumisteron dispo , nissin , japan ), of 600 , 000 - 1 , 200 , 000 dalton ( unihylon dispo , uji , japan ), of 500 , 000 - 730 , 000 dalton ( hyalgan , fidia , italy ), and of 500 , 000 - 730 , 000 dalton ( suplasyn , bioniche , ireland ) hyaluronan reduced amounts of h 2 o 2 and inflammatory factors for demonstrating the effects of hyaluronan injected into patient &# 39 ; s articulation on the production of h 2 o 2 in synovial , we firstly drew the synovial from hyaluronan - treated patient and analyzed the content of free radical . the method for free radical analysis followed the method described in american journal of transplantation 2005 , 5 : 1194 - 1203 , which is incorporated herein by reference . briefly , the sampled synovial was colded on ice . 200 μl of the cold synovial was placed on iron plate , and then put into the detecting cell of chemiluminescence detector ( cla - fs1 , tohoku electronic ind . co ., sendai , japan ). after turning on the detector and measuring the backgroung for 50 sec , 500 μl of luminol ( from sigma corp ., usa , as 0 . 1 mm solution prepared by disolving the powder in phosphate buffer slution ( pbs ), stored at 4 ° c .) or lucigenin ( from sigma corp ., usa , as 0 . 1 mm solution prepared by disolving the powder in pbs , stored at 4 ° c . was added , and then the content of free radicals was measured in 300 sec , with one respective accumulative value obtained at the interval of 10 sec . the background integration in 300 sec was calculated by : ( average integration of time - count in 50 sec )× 30 . the average free radical count for each sample at the interval of 10 sec was calculated as follow : ( total area integration under time - count curve − background integration in 300 sec )÷ 25 . as showed in fig1 , hyaluronans with molecular weight of 500 , 000 to 8 , 000 , 000 dalton exhibited an effect on reducing the production of h 2 o 2 in synovial fluid . among the hyaluronans tested in this experiment , the effectiveness thereof in reducing the production of h2o 2 is 600 , 000 - 1 , 200 , 000 dalton & gt ; 500 , 000 - 730 , 000 dalton & gt ; 6 , 000 , 000 dalton . we also used the synovial fluid sampled f synovial fluid from untreated patient as a control . by the results showed in fig2 , the content of was significantly reduced in the synovial fluid from the hyaluronan - treated patients as compared to the control synovial fluid , which contained high level of free radicals such as o 2 −. , h 2 o 2 . the production of free radicals and mortality of chondrocytes were deminished by hyaluronan in vitro in order to realize the in vitro effects of hyaluronan on the reactivity of free radicals , such as h 2 o 2 , we took 200 μl of h 2 o 2 at various concentrations ( 0 , 100 , 200 , and 400 μm ) for analysing the presence of free radicals by chemiluminescence detector . additionally , 20 μl of hyaluronan was provided at the presence of 200 μm h 2 o 2 . the content of free radical was measured as described above . results were showed in fig3 a . the level of free radical was increased as the increasing concentrations of h 2 o 2 . however , the production of free radical was inhibited at the presence of hyaluronan ( with 200 μm h 2 o 2 ), which was almost at the same level in the h 2 o 2 - untreated group ( i . e ., the group at the presence of 0 μm h 2 o 2 ). for knowing that the increased contents of free radical will cause cell death , we also investigated effects of hyaluronan on the mortality caused by free radicals . the cartilage tissue was collected from a patient older than 60 years old . the primary chondrocytes were isolated and cultured in appropriate medium and conditions described as follow . the collected cartilage tissue was weighed in a centrifuge tube , and then washed with 10 ml pbs twice . the washed cartilage tissue was transferred onto a 10 cm cell culture dish by a blunt forceps and washed with 10 ml pbs once again . the soft tissue and bone tissue attaching to the cartilage were excised with a scalpel . the separated white cartilage tissue was transferred onto a 10 cm cell culture dish , and then divided into pieces having volume less than 1 mm 3 by using a scalpel . to the cell culture dish containing cartilage pieces 60 μl of typr ii cellulose ( 100mg / ml ) was added , and then shaken on a shaker ( 50 rpm ) at 37 ° c ., 5 % co 2 for 4 hr . the digested cartilage tissue was washed with 5 ml of culture medium . the separated cells were transferred into a sterile 15 ml centrifuge tube , and washed with 10 ml pbs twice . the cells were resuspended in 10 ml of culture medium . 50 μl of the cell culture was sampled for counting viable chondrocytes . 40 μl pbs and 10 μl of 10 × trypan blue were added to the sampled cell culture , mixed thoroughly and 10 μl of the mixture was dropped into hemocytometer . the cell density was calculated as follow : total counted cell no in 9 lattices x 2 × 10 4 / 9 = cell no ./ ml . chondrocytes were inoculated into a flask at the density of 1 × 10 4 / ml , and cultured at 37 ° c ., 5 % co 2 , with changing fresh medium every three days . for the displacement of fresh medium , the culture medium was drawn off by a disposable pipette , and 10 ml pbs was carefully added along the wall of flask . after shaking the culture flask gently , pbs was drawn off and 12 ml of fresh medium was added , and then the culture flask was incubated at 37 ° c ., 5 % co 2 . for the in vivo test , various concentrations ( 0 , 100 , 200 , and 400 μm ) of h 2 o 2 were added to the cell culture respectively , with the two additional groups added 0 and 200 μm h 2 o 2 each plus 20 μl of hyaluronan . the chondrocytes were cultured for one day under the h 2 o 2 treatment , and then collected for detecting their viability . as shown in fig3 b , the viability of chondrocyte was decreased with the increasing concentrations of h 2 o 2 . on the contrary , however , the viability of chondrocyte was increased at the presence of hyaluronan . accordingly , it is suggested that hyaluronan possesses the ability to inhibit the production of free radicals , decrease the mortality of chondrocytes , and further improve the proliferation of chondrocytes . this experiment is provided to evaluate the effects of hyaluronan on the growth of chondrocytes in old patient &# 39 ; s joint . the chondrocytes from old patient were isolated and cultured as described in example 2 . chondrocytes were cultured to 80 % confluence and plated in 60 mm dish ( each contained about 8 × 10 4 cells ), then further cultured over night . the culture medium was replaced with fresh medium containing 1 mg / ml hyaluronan ( ha group ) or fresh medium only ( control group ), and the cultivation was continued till 12 days , with a replacement of culture medium ( with or without hyaluronan ) at day 5 . the initial cell number was counted at day 0 , and taken a count every 2 days . the growth curves of cultured cells were shown in fig4 . in day 2 to day 4 , the cell number of chondrocytes grew at the presence of hyaluronan ( ha group ) was significantly greater than control group of about 1 . 7 - fold . the cell number in ha group was greater than control group of above twofold at day 6 . hyaluronan promoted the growth of chondrocytes by controlling their cell cycle the chondrocytes from old patient were isolated and cultured as described in example 2 . chondrocytes were cultured to 80 % confluence and plated in 100 mm dish ( each contained about 3 × 10 5 cells ). the culture medium was replaced with medium containing 1 mg / ml hyaluronan ( ha group ) or fresh medium ( control group ) on next day , and the cultivation was continued till 8 days . cell sampling was begun at day 0 and kept at intervals of 2 days . the harvested cells were treated as described below for the analysis of cell cycle by a flow cytometery . the cultured chondrocytes were washed with pbs , trypsinized and suspended in 5 ml of culture medium containing 5 % fetal calf serum ( fcs ). the cells were washed with 5 ml cold pbs , and then fixed in 1 ml of 70 % alcohol at − 20 ° c . for more than 1 hr . the fixed cells were washed with 5 ml pbs and spun at low speed ( 1200 rpm / min ), then stained with 1 ml of propidium iodide ( pi )/ triton x - 100 solution ( which containg 0 . 1 % triton x - 100 , 0 . 2 mg / ml rnase a , and 20 μg / ml pi at final concentration , respectively ) at room temperature in the dark for 30 min . the sample was mixed throughly and filtered through 35 - μm nylon membrane before run on the flow cytometery ( fl2 - a ) for detecting the fluorescence expressed on cells . the results were shown in fig5 . referring to the detection of g0 / g1 phase in cell cycle ( fig5 a ), the % of cells in g0 / g1 phase in the control group was higher than those in ha group at day 4 , otherwise , there is no significant difference between those two groups . by the results in s phase detection ( fig5 b ), there is no statistical difference between ha and control groups from day 0 to day 8 . on the other hand , it was found that the % of cells in g2 / m phase in the ha group was much higher than those in control group at day 4 , but showed no difference after day 6 ( see , fig5 c ). hyaluronan promoted the growth of chondrocyte by up - regulating the expression of cyclin b1 ( g2 / m ) for further understanding the mechanism of hyaluronan action in modulating the cell cycle of chondrocyte , we investigated the expression of various cell cycle regulating factors at the prescence of hyaluronan ( 1 mg / ml ). the chondrocytes cultured and treated as described in example 2 were harvested at day 2 , 4 , 6 , and 8 and prepared for western blotting as follow . the harvested cells were washed with 1 × pbs three times , and ripa buffer ( containing 50 mm tris ( ph7 . 5 ), 150 mm nacl , 1 % np40 , 0 . 1 % sds , 0 . 5 % sodium deoxycholic acid , and proteinase inhibitor ) was added to lyse cell and release proteins . the cell lysate was collected in an eppendorf tube and spun at 12 , 000 rpm , at 4 ° c . for 30 min . the quantified supernatant containing 20 μg protein was mixed with sampling buffer ( with glycerin ) at the rate of 1 : 4 , and boiled in 100 ° c . water bath for 5 min . the prepared protein sample was loaded on sds - polyacrylamide gel for electrophoresis , and then transferred onto a nylon paper . the nylon paper was blocked with 5 % skimed milk in tris buffer at room temperature for 1 hr . the nylon paper was washed with tris buffer three times , each for 5 min . the primary antibody was added and the mixture was shaked at room temperature for 1 hr or at 4 ° c . over night . the nylon paper was washed with tris buffer five times , each for 5 min . the secondary antibody was added and the mixture was shaked at room temperature for 40 min . the nylon paper was washed with tris buffer as described above . the chemiluminescence detecting kit ( ecl kit ) was added and reacted at room temperature for 1 min , and then developed in dark room . as shown in fig6 , the expression of g0 / g1 phase regulating proteins , such as cyclin d1 , cdk4 , and cdk6 , exhibited no difference between the control and ha groups . however , the g2 / m phase regulating protein , cyclin b1 , exhibited higher expression level in ha - treated cells than those in control group . furthermore , the expression of cyclin b13 - associated protein cdc2 was unchanged . the present invention has been illustrated by the embodiments and examples described above . the skilled in the art will appreciate that any modification or change can be made as if not depart from the spirit and scope of the invention . the present invention was encompassed in the appended claims .
the present invention relates to the use of hyaluronan in protecting chondrocytes against oxidative damages and further promoting their proliferation . particularly , the present invention relates to the use of hyaluronan with a molecular weight of 600 , 000 - 1 , 200 , 000 dalton in protecting chondrocytes against reactive oxygen damages and further promoting their proliferation .