Repair Of Goat Full-thickness Cartilage Defects Using The Co-culture System Of HUCSCs And PACs In Loading Area Of Knee

Objective: Ideal seed cell is the core element of cartilage tissue engineering, is the necessary prerequisite of regeneration of articular cartilage, and is also one of the current research interests.The purposes of this study are as followings: 1.Based on the need of clinic and existing research results, the 3D co-culture system was firstly constructed by using hUCSCs and pACs which were seed into the ACECM oriented scaffold; 2. To check and evaluate the biological behavior and histological characteristics of tissue engineering cartilage by co-culturing; 3. The big animal experiment was performed to explore the repair potential of co-culture system in the full-thickness cartilage defect of knee joint model to evaluate overall value of application in the future.Methods: 1. The isolation of hUCSCs and pACs were from fresh human umbilical cord and knee joint of adult goat respectively in sterile condition. The culture and identification of seed cells were performed to confirm the target cells and acquire the demand quantity. The hUCSCs and pACs were co-cultured in ACECM oriented scaffolds in different ratios(hUCSCs: pACs, 100:0?75:25?50:50?25:75 and 0:100); 2. After co-culturing for 1 week and 3 weeks, all the samples was tested the levels of genes and proteins, cellular biobehavior, and histological staining to analysis the biological characteristics of engineering tissue; 3. Adult goats were used in the experiment and the experimental model was created by punching a full-thickness cartilage defects in loading area of knees. This experiment was divided into 5 groups, including experimental group(co-culture group, CC), condition-control group(ACECM group, AC; microfracture group, MF;ACECM+hUCSCs group, ACUC; ACECM+ pACs, ACAC) and blank control group(BC), 3 goats per group. 2 and 4 weeks after first operation, the joint fluid were acquired to observe the inflammation reaction using H&E staining. 6 and 9 months after transformation operation, all goats were euthanasia and all samples were used to observe and score according to International Cartilage Repair Society Macroscopic Evaluation of Cartilage Repair(ICRS-MECR) and International Cartilage Repair Society Visual Histological Assessment Scale for Cartilage Repair(ICRS-VHASC ) , the quantity of special extracellular matrix of articular cartilage(glycosaminoglycan), imaging test(7.0 T nuclear magnetic resonance, 7.0T MRI), and biomechanical test(nano-indentation technology) to evaluate roundly the efficiency.Results: 1. The requiring cells derived from goat knees and human umbilical cores showed round-like and long spindle-like shape respectively. The immunofluorescence staining of collage land ? demonstrated pACs had perfect characteristics of articular chondrocytes. The flow cytometry and chondroblast induction found hUCSCs had the characteristic of stem cells and the ability to induce into chondrocytes. The co-culture systems were successfully constructed by pACs and hUCSCs in different ratios; 2. After co-culturing for 1 or 3 weeks, all the porous scaffolds were filled with cells and extracellular matrix in different extents. The dead-living staining showed good viability of cells and the fastest growth group was the ratio of 100:0 and the lowest group is 0:100 group. When two seed cells co-cultured in co-culture systems, the proliferation rate was changed significantly: pACs grew faster, while hUCSCs slew down the speed. The results of Rt-PCR revealed the chondrocytes-like genes were up-regulated, such as collagen ? and aggrecan. The histological staining(H&E, Safranine O and immunofluorescence)demonstrated amount of extracellular matrix and special cartilage matrix ingredients. The quantity of glycosaminoglycan showed the co-culture groups were significant higher than mono-culture groups. The results of western-blot showed collagen ? and aggrecan were expressed more intensive and collagen ? was weaker in co-culture groups than that of mono-culture groups. 3.There is no significant difference of inflammation between groups according to the H&E staining results of joint fluid and synovial membrane. Compared with control groups, both gross morphology and histological staining demonstrated the neo-cartilage tissue of CC group was closer to native tissue and better integrated with surrounding native tissue, while the subchondral bone was intact. The nano-mechanism test showed the mechanical strength of repair areas were significant higher in CC group than control group, especially in 9 months. In the results of MRI,the T2W1 signal of repair area were similar to native cartilage and subchondral bone and homogeneous, and the edema signal almost disappeared completely in 9months.Conclusions: the pACs and hUCSCs that were applied in co-culture systems were perfect cartilage-like phenotype and multi-differentiation ability respectively. In co-culture systems, the engineering tissue had better cartilage-like characteristic because hUCSCs were upregulated to express chondrocytes-like genes and the ability of pACs was enhanced by cell-cell interaction.The animal experiment showed the co-culture system consisted by hUCSCs and pACs achieved repair successfully in full-thickness articular cartilage defect model without subchondral bone damage. All in all, co-culture of hUCSCs and pACs is a promising strategy to overcome predicament of seed cells in the repair and regeneration of articular cartilage by complement the flaws of stem cells and chondrocytes in the future.