Comparative Study On Rabbit Bone Marrow Mesenchymal Stem Cell Co-culture With Articular Chondrocytes Versus Non Co-culture In Fabrication Of Scaffold-free Tissue Engineering Cartilage

ObjectiveIn order to deepen the thought of developing scaffold-free tissue engineering cartilage for the repair of articular cartilage defects in clinical pactice,we observe the size and biological characteristics of cartilage tissue formation produced by co-culture and non co-culture systems in four groups of high-volume cell clusters in centrifuge tube without scaffold materials,and then make an evaluation on the methodology.MethodAfter the bone marrow fluid was obtained from the bone marrow cavity of rabbit,the bone marrow mesenchymal stem cells(BMSCs)were isolated by density gradient centrifugation,and purified BMSCs were obtain on the third generation.The rabbit articular cartilage was isolated and digested with trypsin and collagenase type?in order to obtain relatively pure articular chontrocytes(ACs).ACs were identified through alcian blue staining,safranin“O”-light green staining and type II collagen immunohistochemically.The molecular surface markers ofCD34,CD44,CD45 and CD105 on BMSCs were identified by immunofluorescence.We developed four groups for comparative study as following.BMSCs and ACs were mixed into co-culture(CO)groups at the ratio of 1:3 and 2:2 respectively,1500r/min centrifugation for 5 minutes,then gathered in 15ml centrifuge tube,the number of cell in each centrifuge tube is 4*10~7.The AC group was composed of equivalent numbers of chondrocytes only,and the BMSCs induction group was composed of the equivalent numbers of BMSCsinduced into chondrocytes by adding TGF-beta 1 and so on in the culture medium.The cells of the four groups were cultured for 1 week,2 weeks,3 weeks and 4 weeks respectively,and collected for paraffin tissue sections.HE staining was used to observe the morphological structure of cartilage tissue in each group.After dewaxing,the sections were processed for alcian blue staining,saffron"O"-light green staining and type II collagen immunohistochemical staining.The amount of Glycosaminoglycan(GAG)in the supernatant was quantitatively determined by Alcian blue colorimetric assay.The amount of type?collagen in supernatant was quantitated by Enzyme-Linked Immunosorbent Assay(ELISA).Results ACs'alcian blue,safranin“O”-light green and type II collagen immunohistochemical staining were positive.BMSCs'immunofluorescence staining outcome was CD34 negative,CD44 positive,CD45 negative,CD105 positive.In the AC group,2:2 co-culture group and 1:3 co-culture group,the results of alcian blue staining,safranin O-light green staining and type II collagen immunohistochemical staining were positive,while the staining was weakly positive for BMSCs induced group.The cells in AC group and 1:3 co-culture group were fused gradually in the first three weeks,and could be observed to fused into initial cartilage tissue on HE staining in four weeks,while the cells in 2:2 co-culture group and BMSCs induction group were observed to be integrated gradually in the first three weeks,but no embryonic form of cartilage tissue was formed in the fourth week.In the first week,the difference in GAG content among AC group(61.68+32.62ug/ml),2:2 co-culture group(34.82+17.06ug/ml)and 1:3 co-culture group(42.86+23.69ug/ml)was not statistically significant(F=1.170,p>0.05);in the second week,the difference in GAG content among AC group(96.70±44.56ug/ml),2:2co-culture group(107.05±44.56ug/ml)and 1:3 co-culture group(92.83±26.71ug/ml)was not statistically significant(F=1.170,p>0.05);in the third week,the difference in GAG content among ACgroup(157.19±48.07ug/ml),2:2co-culture group(132.75±19.56ug/ml)and 1:3 co-culture group(121.01±29.92ug/ml)was not statistically significant(F=0.352,p>0.05);after four weeks,the difference in GAG contentamongACgroup(179.43±59.19ug/ml),2:2co-culture group(158.06±23.77ug/ml)and 1:3 co-culture group(160.06±30.58ug/ml)was not statistically significant(F=0.210,p>0.05).In the first week,the GAG content difference between AC group(61.68±32.62 ug/ml)and BMSCs induced group(10.71±6.12ug/ml)was significant statistically(t=3.697,p<0.05);in the second week,the GAG content difference between AC group(96.70±44.56 ug/ml)and BMSCs induced group(25.29±14.41ug/ml)was significant statistically(t=3.765,p<0.05);in the third week,the GAG content difference between AC group(157.19±48.07 ug/ml)and BMSCs induced group(56.14±43.67ug/ml)was significant statistically(t=4.209,p<0.05);after four weeks,the difference in GAG content between AC group(179.43±59.19 ug/ml)and BMSCs induced group(70.74±40.17ug/ml)was significant statistically(t=3.980,p<0.05).In the first week,the difference in type II collagen content among AC group(130.68±27.19ng/ml),2:2 co-culture group(101.05±19.91ng/ml)and 1:3 co-culture group(77.16±29.01ng/ml)was significant difference(F=22.106,p<0.05);in the second week,the difference in type II collagen content among AC group(145.51±40.37ng/ml),2:2co-culture group(127.80±31.72ng/ml)and 1:3 co-culture group(101.13±24.43ng/ml)was of no significant difference(F=1.443,p>0.05);in the third week,the difference in type II collagen content among AC group(162.19±38.07ng/ml),2:2 co-culture group(152.25±28.23ng/ml)and 1:3 co-culture group(133.91±32.29ng/ml)was of no significant difference(F=2.241,p>0.05);in the last week,the difference in type II collagen content among AC group(185.13±35.96ng/ml),2:2 co-culture group(169.72±32.56ng/ml)and 1:3co-culture group(148.91±33.16ng/ml)was of no significant difference(F=0.680,p>0.05).In the first week,type II collagen content between AC group(145.51±40.37ng/ml)and BMSCs induced group(52.36±5.37ng/ml)was of statistical difference(t=7.859,p<0.05);inthesecondweek,typeIIcollagencontentbetweenAC group(130.68±27.19ng/ml)and BMSCs induced group(69.25±13.98ng/ml)was of statistical difference(t=4.610,p<0.05);in the third week,type II collagen content between AC group(162.19±38.07ng/ml)and BMSCs induced group(90.49±39.90ng/ml)was of statistical difference(t=3.503,p<0.05);in the last week,type II collagen content between AC group(185.13±35.96ng/ml)and BMSCs induced group(116.83±40.17ng/ml)was of statistical difference(t=3.434,p<0.05).Conclusion BMSCs could be induced into ACs through aggregation in centrifuge tube by co-culture,manifesting with the biological characteristics of the AC.In the AC group and 1:3 co-culture group,the initial cartilage tissue was partly observed on HE staining after four weeks,however not seen in the BMSCs induced group and 2:2co-culture group.