An Expeimental Study Of The Optimal Induction Ratio About Indirectly Pellet Co-culture Adipose-derived Stem Cells Of Rabbits And Chondrocytes

Cartilage defects show lack of proper regeneration whereas tissue engineeringmethods shine a new light on that. Adipose-derived stem cells (ADSCs) are perfectseed cells for they can be found in abundant quantities and can be harvested by aminimally invasive procedure. While the problem for now is that an efficientchondrogenic inductive methods has not been established.Growth factors such asTGF-βs、BMPs are used in traditional protocols but the hypertrophy following theinduction as well as the short half-life period of growths factors troubled people for along time. Previous studies demonstrated that chondrocytes (ACs)can lead ADSCs tochondrogenic lineage and there are growing numbers of studies which are aboutACs-ADSCs co-culturing. Besides of that it is still unclear what is the best ratios ofACs-ADSCs in this kind of co-culturing.Objective: This study is undertaken to demonstrate:（1）in which proportion ofACs/ADSCs can we harvest the chondrogenic tissue without significant expression ofhypertrophic markers such as collagen type Ⅹ;（2）comparing the differencesbetween the growth factors and co-culturing on chondrogenesis effect.Methods:1、Isolation,culture and identification of of Adipose derived stem cells (ADSCs) andchondrocytes:Aseptically removed perirenal adipose tissue, digested withcollagenaseⅠ of2-4weeks old healthy New Zealand rabbits, combined withdifferential adhesion in vitro isolated and purified rabbit Adipose derived stemcells(rADSCs). Taken the3rd passage of monolayer culture of ADSCs to beosteogenic, adipogenic and finally measured by Alizarin red staining and oil red Ostaining. Take the articular cartilage, which was digested with typeⅡ collagenasedigestion overnight, collected by centrifugation hyaline cartilage cells were cultured in vitro, Toluidine blue staining detect the results.2、Preparation of Pellet:5xl05cells were respectively placed in an Eppendorf tube wascentrifuged unit in an incubator of common medium cultured for2-3days.3、Co-cultured chondrocytes and Adipose derived stem cells (ADSCs) and groups:Take prepared adipose derived stem cells and chondrocytes Pellets in the upper andlower layers of tranwell system, co-cultures were set up in a system which the two celltypes were physically separated and allowed to interact only by the means of diffusiblefactors. Experiments were divided into nine groups:#1, Pellet of chondrocytes werecultured in the lower layer in a common medium (H-DMEM,1%ITS,40μg/mlL-proline,50μg/ml ascorbic acid,1%Pen/Strep,3μg/ml amphotericin B), as apositive control group;#2，Pellet of ADSCs were cultured in the upper layer in amedium identical to#1,as a negative control group;#3, Pellet of Adipose derived stemcells (ADSCs) were cultured in the upper layer in a medium in chondrogenic inductionmedia(added of10ng/ml TGF-β3+10ng/ml BMP-6+0.1mM dexamethasone), asthe positive control group2; and#4-#9served as the experimental groups. The ADSCsand chondrocytes Pellets in different proportions (0.5:1;1:1;1:2;1:3;1:5;1:7) wereco-cultured in a common medium(as#1and#2). Detection measures: After28days ofculture were performed histology (morphology), protein level (COL-Ⅱ; GAG;COL-Ⅹ) qualitative and quantitative detection.Results:1、Extracted adipose derived stem cells proliferated rapidly, The primary ADSCsexpanded in colonies, and were mostly as fusiform shape after passages withorderliness permutation as whirlpool. Multilineage differentiation potentiality werecomfired in vitro, which was a performance that lipid droplets showed red though OilRed O staining, calcium nodules showed dark pink; Hyaline cartilage culturedadhered rapidly and grew well with a clear outline, toluidine blue staining showedproteoglycan aggregate were blue, Cartilage cells showed typical morphology, bluecytoplasm；2、Indirectly Pellet co-culture adipose-derived stem cells and chondrocytesfor28days, toluidine blue staining and immunofluorescence staining of collagen typeⅡ display in addition to the negative control group, all results were positive.However, compared to the strong positive performance of positive control group1,the ratio of0.5:1and1:1groups showed a weak positive. Ⅹcollagen in each group were shown to be positive, but the positive control group showed the growth factorsmost strongly. Protein quantitation detection: the tested result of total GAG contenabout induced ADSCs by dimethyl methylene blue spectrophotometry (DMMB)showed that the total GAG content in co-culture groups have increased compared withthe control group, the differences had statistically significant (P<0.05), and increasethe amount and proportion of chondrocytes positive correlation, but instead declinedafter ADSC-AC ratio to1:5. OHP measured by changes in the total collagen similar toGAG change. The total collagen content in Transwell co-culture groups5:1accumulated collagen more than any other co-culture groups.which increased7.3-foldcompared with ADSCs mono-culture.Conclusion:1、Adipose derived stem cells (ADSCs) have the ability to adipogenesis(oil red Ostaining), osteogenesis (Alizarin red staining) and chondrogenesis (toluidineblue staining)；2、The different ratios about indirectly co-culture of chondrocytes and Adiposederived stem cells (ADSCs) impacted the capacity of adipose derived stem cells todifferentiate into chondrocytes and the characters neochondrocytes greatly; Theoptimal ratio that made adipose derived stem cells express the maximum cartilagespecific extracellular matrix is1:5；3、Co-culture chondrocytes and Adipose derived stem cells (ADSCs) can inhibits thehypertrophic compared with growth factor induction (COL-Ⅹexpression wasdecreased).