BACKGROUND:There are some disadvantages in harvesting and transferring cells in the traditional tissue engineering technique,and it is difficult to form dense tissues,which significantly limits the development of tissue engineering.OBJECTIVE:To explore the culture and fabrication of dog bone marrow mesenchymal stem cell(BMSC)sheet in vitro.METHODS:Bone marrow was extracted from dogs following anesthesia.BMSCs were isolated with the method of density gradient centrifugation in vitro.BMSCs at passage 4 at a density of 1×10~9/L were incubated in the temperature-responsive culture dishes with a diameter of 3.5 cm,and cultured in an incubator at 37 ℃,5% CO_2 and saturated humidity.The temperature of the incubator was changed from to 37 V to 20 ℃ to prepare BMSCs cell sheet for 20 minutes.Cell morphological changes and cell sheet formation were observed under an inverted microscope.RESULTS AND CONCLUSION:Dog BMSCs following 24 hours of primary culture presented ellipse or polygonal shape.Most cells adhered at hour 72,and cell colonies were visible at day 7.Cells showed long spindle and completely confluence at day 12,with unclear boundary.BMSCs in the temperature-responsive culture dishes presented short spindle shape,and gradually separated from the dish bottom,forming entire cell sheet containing extracellular matrix at 20 V.These verified that dog BMSCs can be effectively obtained with method of density gradient centrifugation.Complete cell sheet layer can be fabricated with temperature-responsive culture dishes.

BACKGROUND:Conventional methods,including trypsin digestion and cells transfer using single call suspension,have many drawbacks,which limit the development of bone tissue engineering.OBJECTIVE:To culture bone marrow stromal stem calls,induce osteoblastic differentiation,and prepare cell sheets.METHODS:Canine bone marrow stromal calls were isolated by density gradient centrifugation technique,inoculated into DMEM medium,and induced to differentiate into osteoblasts.Complete call sheets were harvested by call sheet engineering based on the temperature change of temperature-responsive medium.RESULTS AND CONCLUSION:Immediately after inoculation,primary calls were scattered on the bottom of culture flask,presenting a transparent spherical body with a good refractive capacity.At 12 hours,calls exhibited a long shuttle shape,reached complete confluency,and grew in a whirlpool-like fashion.After osteoblastic induction,the majority of bone marrow stromal stem calls appeared tetragonal,polygonal,and squamose.At 21-28 days,round or oval-shaped calcified nodules formed.When the bone marrow stromal stem calls in the temperature-responsive culture dishes were cooled below the critical temperature 32℃,cells were gradually detached from the bottom of culture flask and formed complete bone marrow stromal stem call sheets.These findings indicate that density gradient cantrifugation technique can be used to successfully isolate and culture canine bone marrow stromal stem cells to differentiate into osteoblasts and call sheet engineering enables to harvest complete bone marrow stromal stem call sheets.

BACKGROUND: How to reconstruct tissue-engineered bone with structure similar to natural bone iS a problem in the development of tissue engineering. Cell sheet engineering technology enables novel approaches to construction of tissue-engineered bone. OBJECTIVE: To observe the biocompatibility of call sheets to decalcified bone matrix (DBM) and their growth on DBM. DESIGN, TIME AND SETTING: An in vitro observation was performed at the Central Laboratory, Affiliated Hospital, Qingdao University Medical College between June and September 2009.MATERIALS: Dog bone marrow stromal cell sheets were prepared using temperatura-responsive medium. Dog DBM was prepared by defatting, decalcification, and noncotlagen protein removal procedures. METHODS: DBM surface was covered by call sheets prepared by temperature-responsive technology and cultured with DMEM containing 10% fetal bovine serum and osteoinductive agent.MAIN OUTCOME MEASURES: Under scanning electron microscope, DBM structure, as well as the attachment and growth of cell sheets on DBM surface, was observed. Porosity and aperture size of DBM were calculated. RESULTS: DBM exhibited a three-dimensional latticed structure, with a porosity of approximately 75%. The mean aperture size was (250.11±98.89) μm, exhibiting a normal distribution. Cell sheets well attached to and grew on DBM surface, and rapidly proliferated.CONCLUSION: Cell sheets show good biocompatibility to DBM. DBM/cell sheets complex can be applied in tissue-engineered bones, which promotes the construction of tissue-engineered bone with structure similar to natural bone.

Objective:A new recombinant baculovirus transfer vector was constructed, in whic h a recombinant gene fragment encoding HIV-1 gag-gp120 chimeric gene was inse rted.Methods:After HIV-1 gag gene and gp120 gene were linked,the recombinant baculov irus vector was constructed,and the DNA recombinant technique and the E coli /baculovirus system were used.Results:Gel electrophoresis of DNA analysis showed that the genes were recombine d correctly.Electron microscope showed that the recombinant baculovirus reproduc ed in a great quantity.Conclusion:Recombinant baculovirus vector which HIV-1 gag-gp120 chimeric gene fragment was inserted in was constructed successfully.This vector is useful in study of the expression and the biological function of the HIV-1 Gag-gp120 ch imeric protein.