Tissue Engineering Approach To Articular Cartilage Repair: Autologous Growth Factors Or Differentiated Embryonic Stem Cells With Bilayer Collagen Scaffold For Cartilage Repair In Vivo

Background:Articular cartilage injuries are common clinical diseases in orthopedics,but articular cartilage has very limited self-repair capability.Once injuried,if unreated,osteoarthritis will occur in longer time,which causes great pain for patients.Conventional methods for cartilage repair include debridement,microfracture,osteochondral graft and so on.While these methods can not maintain long-term therapeutic effect and can not treat larger cartilage defects.Tissue engineering method can treat larger cartilage defects.But current scaffolds and cells of cartilage tissue engineering are accompanied with disadvantages.To overcome those disadvantages,this study designed a novel bilayer collagen scaffold and explored autologous PRP as well as human embryonic stem cell derived mesenchymal stem cells(hESC-MSCs)to improve cartilage repair.This study included three parts.PartⅠ:To design a bilayer collagen scaffold and investigate their efficiency on cartilage repair;PartⅡ:To determine whether the collagen matrix with autologous platelet rich plasma(PRP)have synergistic effect to repair cartilage defects;PartⅢ:To determine the efficiency of the hESC-MSCs with bilayer collagen scaffold on cartilage repair.PartⅠ:Use of bilayer collagen scaffold for articular cartilage defects repair in a rabbit modelAIM:Bilayer scaffolds have been widely used for researches on cartilage repair.When compared to other kinds of bilayer scaffolds,collagen scaffold will have much perspective of clinical application because of its good biocompatibility of collagen.In this study we investigated the efficiency of this bilayer collagen scaffold and determine which layer should be adjacent to the surface of the subehondral boneMETHODS:Full-thickness cartilage defects(diameter=4 mm,thickness=3 mm)were made in the patellar grooves of New Zealand White rabbits(n=11).Bilayer collagen scaffold was implanted into the defect.Group A:dense layer was adjacent to the surface of the subchondral bone(n=11);Group B:loose layer was adjacent to the surface of the subchondral bone(n=11). Rabbits were sacrificed at 6 and 12 weeks after operation.The repaired tissues were processed for histology(n=3)at each time point and for mechanical test(n=5)at 12 weeks.RESULTHS:The results showed that at both 6 and 12 weeks,group B had more amounts of hyaline cartilage and had more glycosaminoglycans(GAGs)content.At 6 weeks,the histological scores of group A and B were 5.7+/-2.1,4.3+/-1.8 respectively,at 12 weeks,the histological scores of group A and B reached 10+/-1.4,8.7+/-2.1.The modulus of the repaired tissue in group B(0.133+/-0.023MPa)was significantly higher than that in groupA(0.169+/-0.057MPa)at 12 weeks post-surgery(P＜0.05).CONCLUSIONS:Bilayer collagen scaffold can repair cartilage defects.The dense layer to the bone marrow cavity is superior on cartilage regeneration compared to that of the loose layer.PartⅡ:Autologous platelet rich plasma(PRP)with bilayer collagen scaffold repair cartilage defects in a rabbit modelAIM:Microfracture is a routine therapy to release bone marrow from subchondral bone for in-situ cartilage repair.But it can only treat the small size of cartilage defects(＜2 cm~2).The study aims to investigate whether autologous platelet rich plasma(PRP)transplantation in collagen matrix can synergically improve the in-situ bone marrow initiated cartilage repair.METHODS:Full-thickness cartilage defects(diameter=4 mm,thickness=3 mm)were made in the patellar grooves of New Zealand White rabbits and treated with bilayer collagen scaffold (group B)and PRP with bilayer collagen scaffold(group C)and untreated(group A), respectively(n=11).The rabbits were sacrificed at 6 and 12 weeks after operation.The repaired tissues were processed for histology(n=3)at each time point and for mechanical test(n=5)at 12 weeks.RESULTS:The results showed that at both 6 and 12 weeks,group C had the most amounts of hyaline cartilage,which recovered larger cartilage area of surface at the cartilage defects.Also group C had higher histological scores and more glycosaminoglycans(GAGs)content than those in other two groups(p＜0.05).At 6 weeks,the histological scores of group A,B and C were 3.0+/-1.41,5.3+/-2.12,8+/-2.12 respectively,at 12 weeks,the histological scores of group A,B and C reached 5.0+/-1.41,10.3+/-2.12,12.7+/-3.51.The modulus of the repaired tissue in group B(0.124+/-0.024MPa)and group C(0.140+/-0.037 MPa)were significantly higher than that in groupA(0.087+/-0.012 MPa)(p＜0.05).CONCLUSIONS:Autologous PRP and bilayer collagen matrix synergistically stimulated the formation of cartilage tissues.The findings implicated that the combination of PRP with collagen matrix may repair certain type of cartilage defects larger than 2 cm~2 which currently require the complex autologous chondrocyte implantation(ACI)or osteochondral grafting.PartⅢ:Human embryonic stem ceil derived mesenehymal stem ceils(hESC-MSCs)with bilayer collagen scaffold repair cartilage defects in a rat modelAim:Chondrocytes and MSCs are often used as seed cells for cartilage tissue engineering.But they are accompanied with some disadvantages such as limited donor sources and lifespan.This study explored the use of hESC-MSCs for cartilage repair.METHODS:Full-thickness cartilage defects(diameter=2 mm,thickness=2 mm)were made in the patellar grooves of SD rats and treated with bilayer collagen scaffold(group A)and bilayer collagen scaffold with hESC-MSCs(group B),respectively(n=16).The rats were injected with cyclophosphamide(150mg/kg,ip)for immunosuppression and sacrificed at 4 and 8 weeks after operation.The repaired tissues were processed for histology(n=3)and for mechanical test(n=5)at each time point.RESULTS:The results showed that at both 4 and 8 weeks,group B had more amounts of cartilage and more glycosaminoglycans(GAGs)content than group A.The modulus of the repaired tissue in group B was higher than that in group A at 4 weeks(group A,0.031+/-0.0082MPa;group B, 0.0322+/-0.014MPa)and 8 weeks(group A,0.035+/-0.012 MPa;group B,0.040+/-0.014 MPa). CONCLUSIONS:hESC-MSCs with bilayer collagen scaffold can improve the cartilage formation of the defects and the hESC-MSCs can be alive,hESC-MSCs can be considered as candidate seed cells for cartilage tissue engineering.