Biological Responses Of Osteoblast-like Cells In Three Dimensional Culture Under Cyclic Mechanical Stretch And Its Mechanism

1 Osteoblast-like cells in 3-demensional culture system and the morphological responses to mechanical stretch Objective: To investigate the 3-demensional culture system of colonal murine osteoblast-like cell line MC3T3-E1 and the morphological responses to the unique cyclic biomechanical stretch. The crisis of bone tissue engineering is to establish cell culture system in vitro in which the local environment is similar to the physiological environment in vivo. However, the cell culture system in vitro is single layer or two dimensional system before which is very different from the alive environment. At the present, the research of bone tissue engineering is to reconstruct three dimensional cell culture system. The vector of three dimensional cell culture system which is suitable for the specific tissue cell is different. In this experiment the ME3T3-E1 cells were bred in a novel three dimensional cell culture system which was loaded by dynamic force that is cyclic biomechanical stretch. The morphology of MC3T3-E1 cells was observed. Methods: Collagenous sponges which were the 3-dementional vector of MC3T3-E1 were cut to 2cmx2cmx0.25cm. 100μl of cell suspension was applied to the surface of every sponge and the cell number was 1.25x10⁵. The Bio-Stretch System was bought from ICCT Technologies Inc. in Canada. The stretch program was set to 5% elongation, 60cycles/min and 15 min/h on Bio-Stretch controller via a computer. At every time point 2d,4d,6d,8d,10d after performing stretch 3 samples were collected from stretch group and control group, respectively. Cell counting, HE staining were employed. The morphology of MC3T3-E1 were investigated by optical glass. Results: At the time point of 2d the cell number of stretch group was more than control group(P<0.05). Cell doubling time was reduced from 71hto 55h.In the early stage of cell culture the affinity between cells and the 3-dementional vector of MC3T3-E1 was weak. In the late stage, especially after stretch the extracellular matrix networks were produced and connected to the vector firmly. The cell shape was changed to shuttle by the influence of stretch. Conclusion: Collagen sponge can be the 3-dementional vector of MC3T3-E1. This cell culture system is very similar to the normal physiological environment of bone tissue. Cyclic stretch force can promote proliferation of MC3T3-E1 in 3-dimentional culture system, enhance the formation of matrix net between cells and influence the morphology of MC3T3-E1 which can change to shuttle. 2 Biological responses of osteoblast-like cells in 3-demensional culture system to mechanical stretch Objective: To investigate the effectiveness of unique cyclic biomechanical stretch on proliferation, alkaline phosphatase (ALPase) activity, OPN mRNA and matrilin-2 mRNA of colonal murine osteoblast-like cell line (MC3T3-E1) in 3-dementional culture system. According to Wolf Law bone lose and ossification occur when bone tissue responds to mechanical force, so the remodel of bone is achieved. Bone lengthening was performed in clinic by Ilizaov who first loaded mechanical stretch force to bone tissue. Meanwhile, the principle that bone ossification can occur under fair stretch force. However, it is not clear that what biological response occurs when bone tissue is loaded by mechanical force. This experiment is to study the biological response of ME3T3-E1 cells under cyclic biomechanical stretch. Methods: The experimental model is the same as the part . At every time point 2d,4d,6d,8d,10d after performing stretch 3 samples were collected from stretch group and control group, respectively. Cell counting, ALPase activity assay of cell and medium, OPN mRNA and matrilin-2 mRNA assay were employed. The probe of matrilin-2 cDNA is: 5 ËŠ-TCTGCCCGCTTATTCTCACA -3ËŠ, 3ËŠ-AG ACGGGCGAATAAGAGTGT-5ËŠ. Results: At the time point of 2d the cell number of stretch group was more than control group(P<0.05). Cell doubling time was reduced from 71hto 55h.In stretch group the ALPase activity of cell was in low level all the time. Besides 2d it was lower than control group(P<0.05). It was in low level in medium of both the stretch and control group. In stretch group OPN mRNA maintained high level all the time. At 2d the level of OPN mRNA was higher than in control group(P<0.05). In control group it reached peak at 4d.Then it decreased gradually and down to the lowest level at 8d. At 4d the level of matrilin-2 mRNA in stretch group was higher than in control group(P<0.01). Then matrilin-2 mRNA maintained high level in stretch group all the time. Conclusion: Cyclic stretch force can promote proliferation of MC3T3-E1 in 3-dimentional culture system, inhibit ALPase activity and promote expression of OPN mRNA andmatrilin-2 mRNA. So it also promotes differentiation of MC3T3-E1. 3 The directive differentiation of human bone marrow stromal cells transfected with ectogenic TGF-β₁ gene by liposome-mediated ex vivo Objective: To study the differentiation of human bone marrow mesenchymal stem cells(hBMSCs) transfected with TGF-β₁ gene through liposome-mediated ex vivo. Method: 1. The isolation and culture of hBMSCs. 1) Sucked bone marrow 10 ml in greater trochanter of male adult femur, then used Percoll cells isolation liquid(1.073g/ml) dissociating hBMSCs and making primary culture. 2) Replaced DMEM medium contained 10%FBS every two or three days and observed cellular morphologic change and growth behaviors. 3) When hBMSCs were propagated to the fourth generation, taked parts of cells to be cultured with conditional medium (complete medium with dexaiafrma 10-8mol/L, ascorbic acid 50mg/L, beta-sodium glycerophosphate 100mmol/L), and haved alkaline phosphatase stain and economycin stain respectively in the given time. 2. The construction and amplification of pACCMV-hTGF-β₁ gene. 1) Recombinant plasmids were contructed by thesecond medical university of shanghai. 2) Amplification of recombinant plasmids. â‘ Pro-liferated the E.coli JM109 in the plate culture medium with the method of painting under 37 ℃to stay overnight. â‘¡Then, under 37℃, picked a mono-colony and put it into the liquid culture medium, shaking it to OD260=0.3-0.4 . â‘¢The germs were made into the sensitive state with the method of CaCL2. â‘£Added the plasmids contained the purpose gene into them. Thus, we could transfect the plasmids cDNA into the thallus by 42 ℃heat-shock . ⑤kept them staying overnight under 37 ℃in the liquid culture medium. â‘¥Spreaded the bacterio-liquid on the plate culture medium contained the Aminobenzylpenicillin,the X-gal and IPTG. ⑦Then picked out the needed germs by drug-resistance screening and the blue-white spots test. ⑧After it, we continued to culture the bacterio-liquid overnight. ⑨Purifyed the plasmids by Centrifugation, and measured the density and the purity by spectrophotography (the concentration and the purity of plasmid cDNA that drawed out is 0.067μg/μL and 1.782 respectively). â‘©Finally, we could evaluate plasmids cDNA extracted with mono-restriction endonuclease enzyme and the agar gel electrophoresis. â’ŠThe transfection of hBMSCs. 1) We divided hBMSCs into the transfected cells and the untransfected cells. One day before transfceting, we digested the fourth generation hBMSCs with 0.25%trypsin and 0.2% EDTA(the ratio of volum is 1:1), and inoculated it into petri dishes whose diameter is 35mm with the concentration of 5.0×104/ml, laid previously four pieces of cover glasses in petri dish. 2) Dispensed transfecting fluid referring to the introduction of cationic liposomes transfection reagent(TransFastTM Reagent), preservated at -20℃for use. 3) Transfected the plasmids contained GFP gene into hBMSCs to detect and optimize transfection efficiency. 4) Transfected pACCMV-hTGF-β₁ gene into hBMSCs at optimized transfection efficiency. 5) Finally, we could make immunocytochemical fluorescence stain about TGF-β₁, typeâ… and typeâ…¡collagen to observe the differentiation of transfected cells. Result: 1. 48 hours after inoculating hBMSCs, we could see cells and cellular colonies distributing rarefied by invert microscope. Fusiform cellsshowed typical fibroblast-like appearance. About two weeks later, cells fused on the whole, at the moment, the shape of cells colonies shows radiating or whirlpool-like. Detached primary cells showed alive 100% staining with 4% trypan blue. Digested cells appeared to be shrinked, however, the ratio of alive cells reached to 93%⁹8%. 2. When growing to about 50%, the fourth generation hBMSCs were cultured with conditional culture medium. After a week, there had brownish particles in celluar plasm with alkaline phosphatase stain. About two weeks later, hBMSCs formed Calcified nods, which were dyed into golden color with economycin when observed with confocal microscopy. 3. Immunocytochemical fluorescence staining indicated, after transfecting 48 hours, there were parts of cells emitting green fluorescence in the transfected cells; after four days or six days, fluorescence intensity of transfected cells began to weaken to certain extent, but the quantity of fluorescence cells did not decrease significantly. On the fourteenth day, fluorescence intensity of transfected cells became weaker and the quantity of fluorescence cells also decreased significantly, by chance, fluorescence intensity of a few cells showed highly yet. Both TGF-β₁ antibody and type â… collagen antibody stain showed that positive cells with TGF-β₁ antibody stain itself and peripheral cells indicated positive with â… t ype collagen immunocytochemical fluorescence stain. There showed that, after recombinant plasmid pACCMV-hTGF-β₁ genes had been transfected into hBMSCs, there were still some cells to secrete TGF-β₁ on the fourteenth day. 4. On the fourteenth day, we made use of both typeâ… and typeâ…¡collagen antibody to have immunocytochemical fluorescence stain in the transfected cells, which showed that the expression of the former is stronger than that of the latter, morever, there have not significantly expression of the both in the untransfected cells. Conclusion: 1. hBMSCs have potential ability to differentiate into osteoblast and form new bone. 2. the expression of TGF-β₁ in transfected hBMSCs approve to last for a longer time , for example, at least 2 weeks. 3. TGF-β₁ can make hBMSCs differentiate into osteoblast and chondroblast,...