| Cartilage reconstruction is still problematic for the otolaryngologist and head and neck surgeon. Although autologous or allogeneic cartilage, even artificial products are usually used as reparative materials to reconstruct laryngeal and tracheal function, there are many unsolved promblems including the damage to donor site, shortage of transplanted tissue, immune rejection from recipient and artificial materials hard to integrate into recipient. In resent years, the development of tissue engineering technology make it possible to avoid the above-mentioned demerits. Tissue engineering is a multi-disciplineary scientific field merging with biology, material science, engineeringand chemitry, and its main idea is to harvest minimal qualtity of tissue cells, and then expand them in vetro. Because bone marrowmesenchymal stem cells with stronger ability of proliferation are abundant and easy to get, they have become the most important cell seeds of tissue engineering. Many studies have demonstrated that bone morphoenetic protein, transforming growth factor-beta can induce BMSCs to myogenic differentiation, chondrogenesis, osteogenesis etc. Growth/ Differentiation Factor 5, as one of the inductive factors has more especial effects on chondrogenesis, skelatal growth, healing of muscles injuries, angeogenesis, and neuroprotection. Although exogenuous recombinant GDF5 could induce differentiation of mesenchymal stem cells, the growth factor's effects would last a very short time because of its easy to be digested in vivo. In this study, we designed to transfer Growth/Differentiation Factor 5 gene into BMSCs by liposome, and observed the expression of GDF5 by reverse transcription-polymerase chain reaction, westernbloting, immunohisto -chemistry, and compared the tranfected cells' biologic behavior with controls. Furthermore, we transplanted the compex of PGA and transfected cells into the thyroid cartilage defects of rabbits, and evaluated the rehabilitating effects.Materials and Methodes1. Isolation and culture of bone marrow mesenchymal stem cellsThe 5~6ml marrow was extracted from the New Zealand rabbits of 3 months, and the bone marrow mesenchymal stem cells were obtained by density gradient centrifugation with Percoll, and inoculated into low glucose DMEM medium containing 10% fetal bovine serus. The obtained bone marrow mesenchymal stem cells were planted into plastic culture utensil according to the cell density of 1×106/ml. Then the routine steps of adhere wall culture were adopted. The cells morphology were examined by invertmicroscope and Wright-Giemsa staining, and the primary and passage cells gowth curves were drawn respectively according to the invert microscope observing. In addition, the cells'ultrastructur was detected by transmissionelectron microscope.2. Transforming GDF5 Gene into mesenchymal stem cells with liposomeThe eukaryotic express vector plasmid (pcDNA3.1(+)/hGDF5)containinghuman GDF5 cDNA was presented by doctor Xu Peng, who are working in the Key Laboratory of Environment and Genes Related to Diseases of Education Ministry, Xi'an Jiaotong University. The plasmid was verified by digestion with restriction endonuclease, polymerase chain reaction. When the passage cells grew to 60% fusion, hGDF5 gene was transfected into mesenchymal stem cells through liposome. Subsequently the cells transfected with hGDF5 gene were screened out by G418, and the positive clones were collected and expanded. The expression of hGDF5 gene in mRNA and protein level was measured by reverse transcription-Polymerase Chain Reaction, immunobloting and indirect immunofluorescence methods.3. Effects of hGDF5 gene transfer on the proliferation and differentiation of rabbit bone marrow mesenchymal stem cells in vitroTo evaluate the effects of hGDF5 gene transfer on the differentiation and proliferation of rabbit BMSCs, activity of alkaline phosphates (ALP), expression of type II collagen (Col II), proteoglycan and growth of the cells were measured respectively with ALP measurement kit, MTT colorimetric assay, toluidine staining and anti-type II collagen immuostaining. Alkaline Phosphatase assay was also conducted by modified Gomori staining method.4. Repair of autologous thyroid cartilage defect using polyglycolic acid loaded BMSCs transfected with hGDF5 gene.10 New Zealand rabbits (3 months, 2.5-3.Okg) were made the mode of thyroid cartilage defect (0.5 cm length and 0.5 width). The autologous mesenchymal stem Cells were extrated from the rabbits'thighbone, and cultured in vitro. The animals were divided into 3 groups according to the implant used: group I , BMSCs transfected with hGDF5 gene/PGA, n=4; group II, BMSCs transfected with pcDNA3.1(+)/PGA , n=4; groupIII, PGA alone, n=2. Under aseptic condition, cells of the second generation after transfection and the control cells were regulated into 5><107/ml suspension. Then the prepared polyglycolic acid scaffolds(0.5cm><0.5cm) were soaked with the suspension lml for 24 hours in C02 incubator. To groupIII, the scaffolds were only wetted by saline without cells. A bit of scaffords with or without cells were fixed by 2.5% glutaraldehyde for electron microscope scanning. Other complexes were grafted into the defect areas, the morphologic observation was made by gross examination, HE staining, type II collagen immunohistochemistry, Toluidine blue staining after 4 and 8 weeks of operation.Results1. Cell cultureWhen the BMSCs were dissociated and seeded, the ingredients in cell suspension were not easily identified because of mixing blood cells. The media was changed first time 3 days later, the attached cells showed triangle, polygon or short fusiform shape. Tiny clones composed of several cells were formed in some region. 8 days later, the cells got larger and uniform, and exhibited typical fibroblastlike morphology . After 10-12 days, the attached cells increased, contacted to each other to form cell sheets. The cells arrayedlike fishes group. Giemsa's staining showed plenty of cytoplasm, deep-stainednucleus and 1-3 nucleolus.2. Analyse of growth curve of primary and passage cellsAfter being seeded for 12 hours, a few cells got attached and the attached cells increased significantly at 24 hours later. Within the first 7 days, the cells grew slowly relatively and this time is called lag period; After 7 days, the cells grew quickly and entered into the increased logarithmic phase. When the cells formed monolayer on 10-12th day, the cells entered into plateau phase and grew slowly again. Compared to primary cells, the passaged cells increased quickly. Cells got attached at 6-8 hours later and entered into the increased logarithmic phase after 3 days and overgrew after 7 days. Before 5th passage, the cells grew quickly and passaged every 4~5 days. And then, the cells were passaged every 7-9 days. At 10th passage, the cells overgrew in 12 days and began aging. 2.Observation under transmission electronic microscopeCytoplasm of BMSCs was abundant with quite a few ribsome, endoplasts and Goli complexes.Many microvilli existed on cells' surface. 3.Observation from cell growth curveprimary cells enter logarithm growth period after 7 days incubation period and during 10-12 days completed conergence and sticked to the bottom of culture utensil. Passage cells took shorter time(about 3 days) to enter logarithm growth period, and overspreaded the culture utensil's bottom. After 5 passages, cells'growth slowered markedly. 4. Cells ultrastructureUnder transmission electron microscope(TEM), we found microvilli on the surface of 4th passage cells. There were numerous ribosome and moremitochondria in cytoplasm. Rough endoplasmic reticulum(RER) were swollen lightly.5. Verification of recombinant eukaryotic expression vector pcDNA3.1(+) /hGDF5The reliability of 6.8 kb expression vector was confirmed by digestion with restriction endonuclease Hind III/ Xho I or Hind III, the sizes of fragments were fully compatible to that of the expected fragments determined by the restriction endonuclease sites in the map of expression vector pcDNA3.1(+)/hGDF5. PCR amplification by using pcDNA3.1(+)/hGDF5 as template, an anticipative electrophoresis band (443bp) can also be obtained .5. Growth and morphology of transfected BMSCs12h after transfected , a small part of BMSCs showed denaturalized, but 48h after transfected, cells growth became normal.6. Expression of hGDF5 mRNA and protein after transfectionFor the cells transfected with hGDF5 gene, hGDF5 positive expression could be detected in the cytoplasm with anti-GDF5 antibody by immunofluorescence staining, while no positive staining found in cells with sham transfection or without transfection. The results of RT-PCR showed that cells with hGDF5 gene presented more hGDF5 mRNA expression, but the control group had no expression. By Westernblot, the transfected group showed 55.6KD protein band, while no obvious positive result detected in the control group.7. Proliferation of BMSCs after transfectionMTT method was used to measure cell proliferation in all the groups. The BMSCs transfected with recombinant hGDF5 gene proliferated more quickly than the control cells did, but the difference was not significant in statistics.Population doubling time is 51.39h day in hGDF5gene transfected group, 49. 48h in sham transfected group, and 49. 48h in non-transfected group. 8. Detection of alkaline phosphates (ALP) activity after transfection.ALP activities of different groups were measured with ALP kit in different time-points. The results were presented by optical absorbance (A). Statistical analysis showed that there were no significant difference among different groups in all the time-points. 9.Expression of ECM in BMSCsCompuer image analysis showed that the expression of ECM (COL II and proteoglycan) in BMSCs transfected with hGDF5 gene increased significantly than control cells. 10. Observation of the complex of BMSCs and PGAElectromicroscope scanning show that Cells combined tightly with materials. 11 .Observation of reparative effect with BMSCs transfected with hGDF5gene-PGA complex4 weeks after operation, gross inspection showed that cartilage defect areas were initially repaired. In the group I, neoregenerated tissue had significantly better histological appearance than group II and III. 8 weeks after operation,in the defect areas of group I , many cartilage-like cells formed, and type II collagen immunostaining and toluidine blue staining were both positive , While in the control goups, there was no evidence of cartilage formation. No PGA fibers and inflammatory cells were found.Conclusion 1. Combination of density gradient centrifugation and adhere wall culture is asimple and pragmatic method for cell dissociation and culture. By this...
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