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The Experimental Study of the Repairing of Cartilage Defects with Chondrocyte and Osteoblast Composites Using Bioreactor and Micromass Culture in Vitro

Author: WuShuai
Tutor: SunShui
School: Shandong University
Course: Surgery
Keywords: cartilage defect bMSCs micromass culcure bioreactor β-TCP scaffold osteochondral composites cartilage repairing
CLC: R684
Type: Master's thesis
Year: 2009
Downloads: 68
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Abstract

ObjectiveArticular cartilage is a non-vascular tissue which is composed of chondrocyte and cartilage extracellular matrix.Articular cartilage injury is a common disease in the field of orthopedics,articular wear,degeneration,trauma and sports injury is increasing.It has been a very important factor leading to disability and reduced quality of life.The previous methods to repair the articular cartilage defects do not always attain satisfying results.Reconstruction of cartilage defects still remains one of the most difficult problems for joint surgeons.Significant achievements in co-culturing chondrocytes and scaffolds to repair the cartilage defects by using techniques of cartilage tissue engineering have been obtained.In the meantime,the work to seek after suitable seed cells and scaffold materials for cartilage tissue engineering is ongoing strenuously.The goal of our study is to evaluate the effect of treating the cartilage defects with cell-β-TCP composites implanted into osteochondral defects on canine models by using techniques of mosaicplasty, chondrocyte-β-TCP scaffold composites in the top of the defect and osteoblast-β-TCP scaffold composites in the bottom of the defect,osteochondral composites were constructed in vivo,looking forward to acquire desirable seed cells and scaffold materials. Materials and Methods1.Three healthy Beagles,approximately 4-10 months of age were used in this study. A 6-7ml of bone marrow was isolated from the iliacs of each carrie.The bMSCs suspension was aspired after bone marrow was centrifuged by percoll separating medium,cultured primarily,and then generated.The bMSCs was identitied by flow cytometry.The third generation bMSCs were induced using micromass culture method.Three groups were divided:GroupⅠreceiving 200ng/ml BMP-2,IGF-I 10ng/ml,dexamethasone 10^-7M/ml,transferrin 6.25ug/ml,Vit C 50ug/ml; GroupⅡreceiving dexamethason 100nmol/L,β—Glycerin phosphoric acid sodium 10mmol/L,Vitamin C 0.05mmol/L;GroupⅢas the control group receiving no cell growth factors,and three groups were cultured conventionally in H-DMEM medium containing 10%fetal bovine serum.The induced bMSCs were viewed at different stages with phase contrast microscope.After 14-day culture,chondrocyte cell viability was estimated by the MTT assay,and cell function was assessed by measureing sulfated glycosaminoglycan（GAG） secreted by chondrocytes.Immunocytochemistry was applied to detect the secretion of collagen typeⅡ.Osteoblast cell viability was estimated by alkaline phosphatase dye.2.Adjust the cell density of 14-day induced cultured bMSCs to 5×10⁶/ml,co-clture chondrocyte osteoblast andβ-TCP scaffolds for 1 day.Then culture the biorcomposites in bioreactor for 21 days.The biocomposites were ready for animal model experiment.Theβ-TCP scaffolds and chondrocyte -osteoblast -β-TCP scaffolds constructs were harvested for scanning electron microscopic examination（SEM）.3.Nine Beagles,approximately 10-12months of age,were used in this animal experiment.A full- thickness cylindrical osteochondral defect of 4.5mm diameter and 4.5mm depth was created in both femoral humeris in every canie.These canies were randomized and divided into 3 groups:Group A（n=9）,as experimental group, implanted with osteoblast-β-TCP scaffold composites in the base and induced chondrocyte-β-TCP scaffold composites in the top into the defects of the left knees; Group B（n=9）,as negative group,implanted with chondrocyte-β-TCP scaffold composites into the defects of the left knees;Group C,as control group,implantedβ-TCP scaffolds into the defects of the right knees.The canies（5 of each group） were sacrificed at 12,16 weeks.Each detect area was evaluated both with naked eyes and histology.Results1.Changes of cell morphology The growth of the bMSCs was observed with the phase contrast microscope.During the beginning of 1-3 days,sparse primary cells fastened wall,the shape of the primary bMSCs was short spear-like.After changing medium of 2-3 times,most of the floating cells were cleared.Three days later,the clone cluster of the cells showed up.After 7-8 days,the cells reached confluence. After 12-14 days,monolayer cells were established.The subcultured cells grew faster. After 7-8 days,monolayer cells,with high nuclears and many grains in cytoplasm, were observed.After adding cell growth factors,cells proliferated significantly.2.MTT OD value,GAG content in culture mediums and positive secretion of collagen typeⅡwith Immunohistochemistry in 200ng/ml BMP-2 experimental group was obviously higher than the control group.With Immunohistochemistry of collagen typeⅡ,cytoplasm of positive bMSCs was stained yellow or brown-yellow. Electron microscope observation.β-TCP scaffold had multi-pore shape,with a good pore of 200-300um.The induced bMSCs had fine adhesion progression and proliferation in theβ-TCP scaffold.3.Animal experiment resultsObservation with naked eyes at 16 weeks after transplantation the defects of Group A were covered with semi-transparent smooth white tissue and the margins between the repair tissue and the surrounding cartilage were not recognized.The defects of Group B were covered with repair tissues,partial of them were conformed with original cartilage.Only in limited area some little pores were noticeable.Compared to Experimental Group,the luster of repair tissues of Negative Group was not good. However,the defects of Control Group were repaired with soft fibrous tissues without luster,and an obvious boundary between reparative and original cartilage was seen.4.Histological observations At 16 weeks after operation:the repair tissues of Experimental Group maintained their thickness to the full depth of the original defects.Chondrocytes arranged regularly.Arrangement of chondrocytes on the surface tended to be parallel to the joint line,while that of the deeper layer liable to be vertical.The defects were repaired with hyaline-like cartilage.The subchondral bone and tidemark were well remodeled. There was no clear gap between reparative and surrounding cartilage.In the Negative Group chondrocytes arranged irregularly,partial fibrous repair tissue was viewed. However,the defects of Control Group were only repaired with fibrous tissue.Conclusion1 bMSCs can be easily obtained,percoll separation method can get huge bMSCs, their characteristics were stable in vitro,easy to generate.Under some conditions, bMSCs can be induced to cartilage cells in vitro.Therefore bMSCs are the suitable seed cells for constructing tissue engineered cartilage.2 It was demonstrated that combination of growth factors played an important role in chondrogenic differentiation.Proliferation and expression of chondrogenic phenotype of bMSCs induced by BMP-2 were obviously higher than the control group.3 Micromass culture is the best method of inducing bMSCs to cartilage cells in vitro.4β-TCP scaffold,with a good pore rate and biocompatibility,can be used as satisfying scaffold materials for cartilage tissue engineering,bMSCs in this scaffold have a fine adhesion,progression and proliferation.5 Chondrocyte -osteoblast -β-TCP scaffold composite was the ideal construct for repairing cartilage defects.Animal experiments demonstrated that the effect of osteoblast in the bottom and chondrocyte on the top composited withβ-TCP scaffolds repairing cartilage defects was obviously better than with chondrocyte -β-TCP compostites.The repair tissue was well-differentiated into nearly normal morphology of cartilage.6 Perfusion culture permitted the persistent nutrition supply and gas exchange into the centre of large scaffold.This perfusion bioreactor makes chondrocyte and osteoblast survive and proliferate through a three-dimensional scaffold.

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