Variations In The Ratios Of Co-cultured Adipose-derived Stem Cells And Chondrocytes Regulate The Expression Of Cartilaginous Phenotype In Cell Pellets

Objective: In the present study we established an in-direct Transwell co-culture system ofchondrocytes (ACs) and adipose derived stem cells (ADSCs) to promote chondrogenesis.And we used quantitative real-time PCR to evaluate gene expression level on both ofchondrogenic phenotype and hypertrophic phenotype in which way can we determine ifthe different proportion of ACs–ADSCs will change the inductive results. Depending onthat we tried to seek out the explicit mechanisms underlying the co-culturing in order toprovide cues for sustainable development of cartilaginous tissue engineering.Methods: In six independent experiments---group A(0.5：1)，group B（1：1），groupC（2：1），group D（3：1），group E（5：1），group F（7：1）)---co-cultures wereset up in a24-well Transwell system in which the two cell types were physicallyseparated and allowed to interact only by the means of diffusible factors, meanwhile weset up three control group, namely ADSCs negative group, growth factors group and ACsgroup. Except for the growth farctor control group,we used an incomplete chondrogenicmedium in our study and the inductive period is4weeks. After4-week induction,ADSCspellets were harvested and performed quantitative real-time RT-PCR to evaluate therelative mRNA expression level of Col2a1, ACAN, Sox-9, Col10a1and Runx2----throughwhich can we analysis the degrees of chondrogenic differentiation and hypertrophy.Results: Col2a1，ACAN and Sox-9genes expressed positively in every co-culture group.And that relative level of these three genes was tend to increase with the grow of ACsproportions. The relative mRNA level of Col2a1was significantly higher in the5:1group andthe7:1group compared with the other co-culture groups（P<0.01）.And therelative mRNA level of ACAN was significantly higher in5:1group compared with theother co-culture groups（P<0.05）.It is also notable that Sox-9mRNA had a mild increasein all of the co-culture groups. Despite the successful expression of chondrogenic makersin the co-culture groups, the hypertrophic marker----Col10a1and Runx2only had aslightly rise when compared with the ADSCs growth factors induction group. Inco-culture groups the Col10a1mRNA relative level were just beyond the baseline whilethat in growth factor inductive group was as high as85-fold of group C. Consistent with Col10a1, Runx-2gene expressed more strongly when growth factors were used, and thedifferences were significant when compared with group A,B,C and E（P<0.01）.Conclusion: We have demonstrated that the morphogenetic factors secreted by ACs caninduce chondrogenic differentiation of ASCSs pellet in the absence of external growthfactors.Additionally,we proved that the sufficient dose of soluble factors to differentiationsystem is needed, which is in a form of high ACs-ADSCs ratios.Sequentially we get thededuction that although soluble factors can and did direct undifferentiated ADSCs tochondrogenic lineage,while on the price of rather high ACs consumption,ACs itself or itsECM play an imperative role on co-culture system which soluble factors probably can notsubstitute.