Establishment Of A Biological Delivery System In Bone Tissue Engineering

Background and Objective:Bone tissue engineering is a promising approach for treatment of defective and lost bone.ADSCs seems to be the ideal seed cell in bone tissue engineering because they can be obtained easily,have the ability of ossification and better amplification in vitro. RhBMP-2 and rhVEGF can induce ADSCs differentiate to bone tissue effectively. However,the half-life time of those bioactive factors are so short that couldn't produce sufficient effectiveness on the course of bone formation,when plant them in the bone tissue engineering simply.And all of the current release/delivery systems have their disadvantages when applied in bone engineering.Thus,it is important and necessary for us to explore a safe,stable and effective bioactive factor release/delivery system to meet the need of bone tissue engineering.RhBMP-2 and rhVEGF plasmids were transfected into the seed cells ADSCs.Then we observed on changes of ADSCs proliferation after transfection,the expression of rhBMP-2 & rhVEGF gene in ADSCs,and application the tissue-engineered bone with the transfected compound to repair the bone defect of GuiZhou minipigs' ulna.We managed to research a novel strategy of bone regeneration,in which rhBMP-2/rhVEGF genetically modified ADSCs were applied.Methods:1.The fractional cultivation and identification of ADSCsADSCs were obtained from the abdominal adipose tissues that come from the sixth-month-old Guizhou minipig by the improved enzyme digestion,cultivated consecutively,and observed the correlative growth characteristics under the convened microscope.ADSCs identification were performed by induction of ossification and lipoids formation,detection of marker vimentin. 2.The biological changes of transfected ADSCsThe ADSCs were transfected with rhBMP-2& rhVEGF by Lipofectamine^TM 2000.The cells were screened by G418 on 48 hours after transfection.The biological changes of ADSCs were estimated before and after transfection with rhBMP-2 and rhVEGF by cell counting and MTT methods,computed the colony doubling time,and draw the growth curve.3.The expression of rhBMP-2 & rhVEGF after transfectionImmunohistochemistry and RT-PCR were performed for detecting the expression of rhBMP-2 & rhVEGF gene in ADSCs in the 48^th h and 4^th week after transfection.4.Observation on the effectiveness of the tissue-engineered bone,which constructed with ADSCs modified by the rhBMP-2 and rhVEGF gene on repairing for the bone defectThe minipigs were divided into 3 groups to repair the bone defect that were constructed on the midpiece of ulna about 1.5cm.(group A) repaired by ACBM with the ADSCs that transfected by rhBMP-2 & rhVEGF,(group B) repaired by ACBM with the ADSCs only,(group C) no treatment for bone defect.And the effectiveness of repair were evaluated by X-ray on 1,2,4,12w and histological section on 4,12w.Results:ADSCs gained from adipose tissue could amplificated in vitro.Immunohistochemistry showed vimentin positive reaction.The results of Gomori stain,Von Kossa stain and oil red O stain were positive after induction.The proliferous speed of ADSCs became quickened after descendant culture in vitro, and tended to stabilize in the 4-6^th generation.Under microscope,ADSCs became fibroid and spindle shape from polygon,rotundity and abnormity.The colony doubling time increase 6 hours after transfection,but there was no difference in the growth proliferation(P＞0.05).The rhBMP-2 & rhVEGF gene could expression in 48h and 4th week after transfection.All of animals had no pantosomatous acute inflammatory reaction and focal sphacelism in two weeks after operation.The X-ray and histological section indicated: group A could be completely repaired,and their capacity of repairing were better than those of B group,but group C bone defect were filled by the self-fibrous connective tissue. Conclusions:ADSCs could be gained successfully from the adipose tissue by the improved enzyme digestion method.Those cells became stabilize when cultivated to the 4-6^th generation in vitro.Furthermore,there were no significant aberrance and apparent difference in their growth proliferation after transfection with rhBMP-2 & rhVEGF.As the seed cells,they can be used in the regeneration medicine carrying target genes.The rhBMP-2 & rhVEGF genes in ADSCs expressed stably within four weeks after transfection.The tissue-engineered bone that is constructed by ACBM and ADSCs with rhBMP-2 & VEGF genes seemed to be a satisfactory and effective osteogenetic repair substitute for bone defect.