The Study Of Promoting Adipose-derived Mesenchymal Stem Cells Adhere To Freeze-dried Bone Tissue Scaffold By Engineering Dynamic Culture

ObjectiveThe adhesion was observed of adipose-derived mesenchymal stem cells onfreeze-dried bone scaffold by two different static and dynamic culture methods. Itcan provide experimental data and theoretical basis for repairing bone defect.MethodsAD-MSCs of rat was extracted and purified by the enzyme digestion method.The expressions of surface antigen CD31/CD44/CD49d/CD106were detected.Proliferation activity was detected by MTT method. Fresh bovine cancellous bonewas prepared and cut into small pieces of45mm3, and the sterile FDB wasobtained by degreased, dehydrated, freezed and dried. The FDB was cultured inculture medium, and PH value of the medium was detected at0,1,2,3days. Threeexperimental groups were divided into:(1) cells and bone (CB group);(2) cellgroup;(3) freeze-dried bone group. AD-MSCs was cultured together with FDB bytwo different methods: static methods of cultivation (two-dimensional-2D) anddynamic culture method (three-dimensional-3D). After co-cultured24h, thecultured tissue was fixed with formalin, decalcified in HNO3, embedded inparaffin and stained with HE. The cells adhered to the FDB was detected withsemi-quantitative analysis software.9visual field was randomly selected under amicroscope of200times. After the dynamic co-cultured24h, FDB was incubatedin12-well plate for6days. CB group was dynamic cultured for24h, and incubatedin12-well plate for3days and6days. AD-MSCs and FDB were digested and isolated with0.05%trypsin. And the live cells were counted by trypan bluestaining. Cellular activity of CB group under static cultured for0day,3days and6days was detected by the same method.ResultsPrimary cells adhere to the culture flask and present spindle-shaped inmorphology. CD44was high expression, CD49d was low, CD31and CD106wasnegative. MTT results showed that proliferation capacity didn’t decreasedobviously of AD-MSCs after repeated subculture. Compared with the simplemedium, pH of medium with FDB over time didn’t change significantly (P>0.05).Co-cultured AD-MSCs and FDB with dynamic or static for24h, cells was3.01±0.16of static culture, while the cells was4.15±0.11of dynamic culture, there wasa significant difference between the two groups(P <0.05). And there was multi-celllayer in dynamic cultured FDB surface. While there wasn’t this phenomenon instatic culture. Trypan blue staining result showed that living cells decreased whendynamic FDB changed into static culture3days and6days. Living cells appearedreduced after static culture6days. There was a significant difference between thetwo groups.ConclusionsCompared with static culture method, dynamic culture can increaseadipose-derived mesenchymal stem cells number adhere to freeze-dried bonesurface, and improve its adhesion activity.