The Development And Application Of β-TCP Porous Bioceramic For The Scaffold Of Cartilaginous Tissue Engineering

【Objective】:β-TCP is an ideal scaffold for bone tissue engineering of inorganicmaterials with attractive applications. However, the traditionalmaking-method ofβ-TCP bioceramics is hard to control the microstructureof the scaffold, soβ-TCP bioceramics were seldom applied to thecartilage tissue engineering.Recently,French Mediterranean Universityhas successfully developed a controllable porous structure ofβ-tricalcium phosphate porous ceramic materials.This material not onlyhas good biocompatibility and high mechanical strength,but aslo can caterto the cartilage tissue engineering reconstruction needs. With thecollaboration of Shanghai Bio-lu biomaterialcompany,by the means of theobservation of the results of chondrocytes cultured in differentmicrostructure bioceramic scaffolds in vitro,A suitable micro-structureof bioceramic scaffolds can be found. Through the measurement ofanatomical structure of rabbits,the rabbit model of articular cartilagedefects can be built up. Via animal experiments, the cartilagereconstruction effect ofβ-TCP material was observated and assessed,sothat suitable microstructure bioceramic scaffold can be found in vivo.The aim of this study is to provide an experimental basis for furtherclinical application ofβ-TCP bioceramic scaffold in articular cartilagedefect reconstruction.【Methods】:(1) Select the healthy New Zealand white rabbits age 2 months,ears fateof intravenous air embolism death,take bilateral femur,humerus and tibia,remove soft tissue respectively,and use of vernier caliper and 64-rowCT to to obtain the anatomical data.(2) With the patented technology andon the basis of preliminary studies, the sintering of suitabletissue-engineered cartilage constructed with appropriate micro-structure ofβ-TCP bioceramic scaffolds.(3) Isolated adult rabbitcostal cartilage and articular cartilage cells and cultured in vitro toobserve the two types of cartilage cells to obtain the rate,survival rate,the rate of cell adhesion,morphological changes,MTT determined cellproliferation rate,and to carry out a toluidine blue staining,saffron"0" stain, methylene blue dimethyl sulfate glycosaminoglycandetermination.Optimize the cartilage suitable algebra cartilage cellsand their mass in order to fit the study of tissue-engineering. (4)Composite of costal cartilage cell biology of different micro-structureof ceramic materials in vitro, the calculation of cartilage cellsproliferation of different growth trends within stent,optimize thesuitable microstructure ofβ-TCP bioceramic scaffold for the bettergrowth of cartilage cells.(5) Put composite of the scaffolds with optimalmicrostructure for growth of cartilage cells and costal cartilage cellsinto rabbit articular cartilage defect model in vitro,to verify thefeasibility of the micro-structure of the animals to repair articularcartilage defect.【Results】:(1) Anatomic measurement results obtained in rabbit femur,humerus andtibia of the data and conducted a three-dimensional reconstruction of CT,the measurement results show that humeral head cartilage from theepiphyseal line to 4.5±0.2mm.Medial tibial platform width of 6.1±0.4mm,width of lateral platform 6.4±0.3m m,but the surface coverageof the tibial plateau have meniscus tissue,surgery can not be revealed,the two parts of the body can not meet the required tests.Medial femoralcondyle width of 5.0±0.2mm,lateral condylar width of 4.0±0.1mm,the width of both sides of the condyle is smaller and less structuredsurface,the same test does not meet the needs of the body.Patellarsurface width of 6.5±0.5m m,the surface of condylar cartilage to marrowdistance of 9.1±0.6 mm,and more regular surface morphology,theseresults can fit the needs of the body.(2) The use of patented technology is suitable to the study oftissue-engineered cartilage. (3) The results of two types of chondrocytes cultured in vitro showed thatthe articular cartilage:digest time 6.49±1.87h,cells were 5.79±1.7×105/100 mg.Costal cartilage:digest time 4.14±1.45h,cells were4.76±1.2×105/100 mg,costal cartilage cells and articular cartilagecells in the cell to obtain the rate of survival rate on the wall did nothave statistical significance,and digestion time of costal cartilagecell was less than that of articular cartilage cells,P value of 0.0032.Dimethyl methylene blue determination of cell culture medium in passageof the GAG content showed that first generation of cartilage cells andsecond generation cartilage cells in thea mount of GAG was similar withoutsignificant difference,but there is statistical difference from the 3rdgeneration of cartilage cells.(4) The cartilage cells were cultured in the scaffolds with differentmicro-structure,and the results showed that ,in the first week of culture,rib chondrocytes cultured in different pore and interconnection had nosignificant difference in growth rate,but from the beginning of thesecond week,,the growth rate of the scaffold with the 120μm diameterinterconnection was quicker than that of other interconnections withinthe scaffold material,and this situation lasted until the fourth week.(5) The results of animal experiment verification in vivo show that costalChondrocytes compounded withβ-TCP ceramic scaffold can repair rabbitarticular cartilage defects.4 months later,the histology score of 20.76±2.13,toluidine blue staining,saffron "0" stain,and staining of typeⅡcollagen stain all showed a good complete repair of cartilage tissue.【Conclusion】:Cartilage defect in rabbit femoral trocheae groove is a suitable animalmodel for cartilage tissue engineering research.Resultes of optimization accounted that costal chondrocytes culturedin vitro had more rapid growth rate, secreted more amount ofglycosaminoglycans. Second generation costal chondrocytes are moresuitable for cartilage tissue engineering.The costal chondrocytes cultured in bioceramic scaffolds with different microstructure had different results.The bioceramic scaffolds withmicrostructures of pore 500-630μm and interconnection 120μm were moresuitable for the growth of chondrocytes in vitro.The results of animal experiments in vivo accounted that composite indifferent ways produced different results of the cartilage reconstruction.Second generation rib chondrocytes cultured in scaffolds were better thanthe first generation without culture.Hard materials of bioceramics can be used as the scaffold for cartilageengineering.The chondrocytes of the upper level of bioceramic scaffolddifferentiated to cartilage tissue.The chondrocytes of the lower levelof bioceramic scaffold did not differentiate to cartilage tissue and hadthe possibility to differentiate to bone tissue.