| BackgroundDue to the difficulty in self repair and regeneration of bone tissue, the treatment of bone defects especially treatment of large bone defects is a very difficult problem and a great challenge for clinical surgeons. Traditionally, the repairs of bone defects employ autologous bone graft, allogeneic bone grafts or artificial substitute implants. However, it tends to cause painness, dysfunction and other complications in donor site, also cause infection or spread of contagious disease. In the near 30 years, with the rapid development of cell biology, molecular biology, material science, tissue engineering and other subjects, stem cell therapy has developed quickly.The bone tissue engineering has become an ideal method to repair bone defect. The main study of bone tissue engineering includes the study of seed cells, scaffolds, cytokines and construction of tissue engineerd bone, with the focus of current research is seed cells. Ideal seed cells should have the following characteristics:1. wide range of sources, can be obtained in large numbers; 2. collection process is minimally invasive; 3. can differentiate into a variety of tissue by process control; 4. be safe and effective for implanting in autologous or allogeneic host. Mesenchymal stem cells have multipotent properties and good immune compatibility without ethical issues, has become an ideal choice of seed cells. Previous researches have focused on bone marrow mesenchymal stem cells with plenty of relevant studies. There are some limitations exist in bone marrow mesenchymal stem cells as seed cells. Firstly, bone marrow mesenchymal stem cell number is too small, with just one mesenchymal stem cell out of 1 million cells, far below the required number of cells for damaged tissue regeneration and repair. Secondly, the collection process is relatively difficult with donor site discomfort. Adipose derived stem cells (ADSCs) have become a hot spot in bone tissue engineering at present because of advantages as easy access, less trauma and large quantity. It is important to assess the feasibility of the transplantation of stem cells by monitoring the growth of the transplanted cells in vivo after the transplantation of adipose derived stem cells, and to understand the outcome of the transplanted cells. In recent years, with the rapid development of biological sciences and molecular imaging, with the advantages of good spatial and temporal resolution, high contrast, long effective imaging time and noninvasive operation, magnetic resonance imaging tracer technique become the ideal choice of evaluating effectiveness of stem cell transplantation and for treatment choice. Magnetic resonance imaging tracer technique can dynamiclly observe cell migration process, and gain molecular, physiological and anatomical information at the same time. Superparamagnetic magnetic iron oxide nanoparticles (SPIO) is a T2 weighted magnetic resonance imaging agent qualified by American FDA to use in clinical practice. SPIO can produce stronger local magnetic field under the action of an external magnetic field, strongly affect relaxation process of surrounding hydrogen proton particles in water molecules, thus significantly shorten the T2 time. T2 weighted images become dimmed and exhibit negative enhancement effect. A number of studies have indicated that mesenchymal stem cells labeled by SPIO will not change their morphological characteristics and will not inhibit their proliferation ability. At present, the magnetic resonance imaging use SPIO to track stem cells, including bone marrow mesenchymal stem cells and ADSCs, applied to many kinds of disease model such as myocardial infarction, cerebral ischemia, spinal cord injury, peripheral nerve injury and femoral head avascular necrosis. SPIO would be distinguished, phagocyted and degradated as exogenous foreign bodies by immune cell after entering the body. It had been found that SPIO significantly increased the proliferation and activity of Jurkat T cells. Similarly, when SPIO labels ADSCs, would it impact the proliferation and activity of ADSCs? When cocultured with osteogenic agents, would SPIO affect the osteogenic differentiation progress? And how is the possible mechanism? The present experiments is to study the interaction of SPIO with ADSCs from Sprague-Dawley rats in vitro, the effect of SPIO on ADSCs into osteogenic differentiation and the possible gene regulatory mechanisms.ObjectiveFirstly, to investigate the effect of SPIO interacted with ADSCs on proliferation and viability.Secondly, SPIO labeled ADSCs, after the addition of osteogenic inducer co culture to induce ADSCs osteogenic differentiation, and the effect of SPIO on ADSCs induced osteogenic differentiation was studied.Thirdly, to study the effect of SPIO on the osteogenic gene expression of ADSCs.Methods1. Adipose tissues were isolated from SD rats and cultured in vitro. The morphological and structural changes of primary SD rats ADSCs and passage cultured ADSCs were observed under inverted microscope. The differences between different generations were compared.2. ADSCs from SD rat were labeled with CD34, CD44, CD45 and CD90 antibody respectively. Flow cytometry was used to detect and analyze their cellular immune phenotype.3.Take the SPIO labeled ADSCs as the experimental group and ADSCs without SPIO label as control group. The cells of the two groups were inoculated in 24 wells culture plate with the inoculum density is 5×104 cells per hole. Handheld cell counting instrument was used to count the cells number of the two groups in 1,2,3,4, 5,6,7 cultivation day respectively and the growth curve was depicted.4. ADSCs were induced osteogenic differentiation:both of ADSCs from the experimental group and the control group were cultured in 6-well plates, inoculation density is 3×104 cells/hole; adding enough conventional complete medium and placed at 37℃,5% CO2 incubator to culture for 24 hours. When the cells fused to 50%-70%, carefully removed the previous culture liquid and add 2ml osteogenic differentiation induced liquid culture medium (containing 0.1 mol/L dexamethasone, 10 mmol/L beta-glycerophosphate,50 umol/L ascorbic acid) into each hole. Replaced fresh osteogenic differentiation medium every 2-3 days and performed induction for two to four weeks.5. ALP activity determination:Took samples from two cell groups after every seven days of induction culture, discarded the culture liquid and rinsed quickly with PBS twice. Collected cells and used ultrasonic cell crusher instrument to perform cell lysis. Repeated the process of freezing and melting twice. Perform centrifugation with semi diameter of 10 cm,3000 R/min for 10 min. The supernatant was moved to a new auxiliary tube and used as sample. Add 100 μl substrate buffer and 20 μl sample to 96 microhole plate. Incubated for 15 minutes under 37℃. Added 80 μl reaction termination liquid and shake in a microplate shaker with fully oscillation for 1 minute. Measured absorbance OD value under wavelength at 520 nm with enzyme-labelling measuring instrument. Calculated ALP activity of each group (gold type unit/100 ml) according to the kit formula given with 3 samples in each group. The same method was used for blank (distilled water) control and standard test.6. To detect cell mineralization rate by Alizarin Red S staining method:Took slides from two groups after osteogenic differentiation culture for 14 d. Discarded the culture liquid, rinsed with l×PBS three times, each time 5 min. Fixed with 2 ml of 4% neutral formaldehyde for 30 minutes. Discarded the culture liquid 30 minutes later and rinsed with 1×PBS 3 times, each time 5 min. Added 1 ml 0.1% alizarin red staining liquid in each hole and mixed for 30 minutes under 37℃. Removed staining solution and rinsed with PBS three times, each time 5 min. Dried under natural environment and mounted with neutral gum. Observed extracellular calcium salt deposition under inverted phase contrast microscope and took photographs.7. To calculate osteoblast induced formation rate:After osteogenic differentiation culture for every seven days, fixed cells with formaldehyde and stained calcium nodules with Alizarin Red S. Selected four 100×vision samples from each group randomly and observed under microscope. Recorded with a digital camera. Enlarged the digital photos in computer to 11cm×14cm and printed on 70g copy paper. Cut carefully the bottom part of the hole and weighed accurately (s), then cut positive nodules by alizarin red staining and weighed them accurately too (SI). Finally calculate bone cells induced formation rate (%)=SI/s ×100%.8. Apoptosis rate detection by flow cytometry FCM:Made cells suspension containing 1×106 cells/ml from two groups with 1×binding buffer buffer. Added 100 μl cell suspension in 5ml culture tube (about 1×105 cells) and added 5-15 μg purified recombinant annexin V. Gently mixed together. Added 5-15μg annexin V-FITC and 10 μg PI in each tube at room temperature for 15 min. Gentlely mixed at room temperature and placed in shady place. Added 400μl 1×binding buffer in the test tube after 15 minutes of incubation and. Placed in shady place for 5 minutes and processed to flow cytometry assay within 1 hours.9. Fluorescence quantitative RT-PCR detection of Runx2, Ocn, ALP and Opn gene transcription level:(1) Total RNA extraction (Trizol extraction)In the two groups, the cells were inoculated in 6 well plate with 1×105 cell counts. The cells were collected at DayO, Day7 and day14 respectively. Discarded the culture medium and rinsed with PBS once. Added 1ml Trizol reagent in each hole and placed in horizontal position for a moment till the pyrolysis liquid evenly distributed on the surface of cells. When cell lysis finished, used suction head several times to make cells fully dissolve. Ttransferred homogenate to 1.5ml centrifuge tube. Added 200μl chloroform, covered the lid and shaked the tube for 15s, incubated at 30℃ for 2-3min. Centrifugated at 2-8℃ by 12000×g force of 15 minutes to seperate RNA.Took a new 1.5ml centrifugal tube and added 500 μl isopropanol, then ransferred the supernatant in the tube and shaked to mix uniformly. After incubated for 10 min at 30℃, centrifuged with no more than 12000g force with high-speed for 15 minutes. A visible film sediment formed by RNA was seen to attach to the wall and bottom of the test tube. Removed the supernatant suspension and added 1 ml 75% alcohol. Flipped centrifuge tube 4-6 times and mixed samples by oscillating vortex. RNA was collected after centrifuging with no more than 12000g force with high-speed for 15 minutes. Removed the supernatant suspension and centrifugated at low speed for several seconds to make ethanol precipitation accumulate on the bottom of the tube. Removed the residual ethanol with 200 μl suction head and retained the white RNA sedimentation at the bottom of the tube. Placed RNA samples stilly under room temperature for 5-10min to air dry. Added 50-100 μl RNase-free water to dissolute RNA and stored at-70℃. Took 5μl RNA for computer RT-PCR experiments with transcription level of GAPDH mRNA as normal control.(2) RNA reverse transcription synthesis of the first strand cDNACyclophos phamide gDNA removal reaction:Defrosted RNA template on ice prepared gDNA removal reaction mixture according to instructions. Centrifugated after mixing thoroughly and incubated in 42℃ water, then placed on ice.Reverse transcription of the RNA:Prepared reverse transcription reaction mixture according to manual and added it to former reaction liquid, then mixed fully and incubated under 42℃. Placed on ice after incubation at 95℃ circumstance for 3 min., then stored the cDNA samples in-20℃ for subsequent experiments.Placed cDNA on ice and prepared fluorescence quantitative PCR reaction mixture according to manual. Added 2μl first strand cDNA template and the right amount of ddH2O in the fluorescent quantitative PCR reaction mixture to total volume as 50 μl. Gently and fully mixed and centrifuged. Set the PCR program and processed for quantitative PCR detection by amplified for 35 cycles under appropriate temperature parameters. This experiment was set as follows:Stage 1:predenaturation period (Reps:1) 95℃ lmin. Stage2:PCR reaction period (Reps:35 cycle) 95℃ 15s, 60℃ 35S,72℃ 30s. Stage 3:72℃ 7min.10. Statistical methods:The results are expressed as means standard deviation (SD). Comparisons between groups were analyzed using t-test or analysis of variance (ANOVA). SPSS 17.0 statistical software was used for statistically analysis. Differences were considered to be statistically significant at P<0.05.Results1.The primary ADSCs harvested with collagenase digestion method attached completely after 48 hours of culture with cells show long fusiform and more flat appearance like fibroblast cells. The primary cells began to proliferate after cultured for 48 hours and reach the peak of proliferation at third to fifth day, presenting colony like growth. Reached 80% confluence at sixth to eighth day. From the second generation, cell adherence time and proliferation time was significantly shortened than the primary generation. After subculture, the cells showed a long spindle fiber appearance, with morphology, arrangement and size tending to be consistent.2. Flow cytometry analysis showed that the vast majority of rat ADSCs expressed with the phenotype of CD44+/CD90+/CD34-/CD44, indicating that ADSCs had mesenchymal stem cell phenotype different from hematopoietic stem cell lines and angiogenesis stem cells.3. The growth curve of two groups was S-like at Dayl, Day2, day3, day4, Day5, Day6, and Day7. The cells proliferation of first two days were in the incubation period. From the third day on, the cell began to enter the logarithmic growth period. Statistical analysis showed that cell number differences in two groups at Day2 was without statistical significance and cells number of Day3, Day4, Day5, Day6 and Day7 had differences with statistical significance. The number of cells in the experimental group was significantly higher than that of the control group. So the proliferation ability of ADSCs was not inhibited but obviously enhanced after labeled with SPIO.4. ALP in seventh days had a significant vitality and reached the peak at 21st day, then maintained a high level to twenty-eighth days. At each test point (7,14,21,28 day), the ALP activity of both groups had a statistically significant difference (P< 0.05), with ALP activity increased after labeled with SPIO.5. After seven days of osteogenic differentiation, ADSCs cells of experimental group marked with SPIO began to emerge small mineralized nodules and increased with the induction time passing on. At 14 days of induction, both of the experimental group and the control group formed mineralized nodules significantly and reached the peak at 28th day with the cell showing a lot of calcium nodules. But the mineralized nodules in the ADSCs of the experimental group increased significantly with large amounts of calcium salt depositing in the cell center.6. From Day 7 onwards, the differences of osteoblast induced formation rate in the two groups were statistically significant with the experimental group significantly higher than that of the control group, indicating that SPIO could promote the osteogenic differentiation process of ADSCs.7. Flow cytometry analysis showed that ADSCs apoptosis rate was less than 1% regardless of whether there is no marker SPIO. The experimental group was slightly higher than control group, suggesting that the ADSCs had good self-renewal ability, and low subculture cell apoptosis rate. The suitable concentration of SPIO was basically harmless to ADSCs, which would not cause a large number of apoptotic cells.8. Whether or not labelled with SPIO, after seven days of osteogenic differentiation, ADSCs expressed bone related gene like Runx-2, OPN, OCN and ALP gene. The expression level was stable and persistent, but the bone related gene expression level was significantly increased in the experimental group labeled with SPIO, significantly higher than that of the control group which not labeled with SPIO, with statistical significance (P< 0.05).Conclusions1. The original ADSCs harvested by collagenase digestion method had a mesenchymal stem cell phenotype with good proliferation ability. The biological characteristics of passage cells were stable.2. The proliferation of ADSCs could be promoted after labeled with SPIO without affecting the cell apoptosis rate. The ALP activity, the osteoblast formation rate and mineralization nodule numbers all increased at same time.3. Whether or not labeled with SPIO, the third generation of ADSCs can be induced into osteoblasts when applying osteogenic differentiation inducing medium.4. SPIO could enhance the expression of Runx-2, Ocn, ALP and Opn on ADSCs and promote the osteogenic differentiation.5. The inadequacies of this research include that Prussian blue staining method and electron microscope detection perspective should be further applied to confirm that SPIO particles located within the cytoplasm of ADSCs and understand intracellular distribution of SPIO, so as to show the reliability of the coculture system. Immunohistochemistry and Western blotting methods should be further used to understand the protein expression level of Runx-2, Ocn, ALP and Opn gene after ADSCs labeled with SPIO were osteogenically induced. Further researches on the signal pathway of proliferation, differentiation and aging of ADSCs labeled with SPIO should be carried on. This in vitro research was made only in cell level and gene level, bone defect model should be designed and animal experiment should be further carried on. |
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