Gene-activated Biomimetic Matrices Regulated The Osteoblastic Differentiation Of Bone Marrow Stromal Cells In Vitro

PartⅠIsolation and Identification of Rabbit-derived Bone Marrow Stromal CellsObjective To obtain rabbit autogenous bone marrow stromal cells (BMSC) and provide seed cells for our researches on bone defect restoration in bone tissue engineering. Methods The bone marrow tissues were fetched from rabbit and BMSCs were isolated by the method of density centrifuge .Inverted phase contrast microscope and BrdU label were employed to observe the cell morphologic features and growth statuses. Flow cytometry was also used to analyze the CD markers on cell surfaces. Results The cultured BMSC grew well and displayed strong capabilities of proliferation until 10th to 12th generations. Above 70 percentage of the cells were labelled by Brdu. FCM analyses showed the positive rates of CD44, CD90, CD34, CD45, CD11b and Lamine on the cell membranes were 93.85%, 75.28%, 2.98%, 1.70%, 0.45% and 0.24% respectively.Conclusion The cultured cells were mostly composed of undifferentiated BMSCs, being of the property of stem cells. The cells were suitable for tissue engineering.PartⅡDesign and synthesis of a bifunctional oligopeptide and specific adhesion by the synthetic peptide Objective To design and sythesize a bifunctional oligopeptide and explore the specifility of cell adhesion mediated by the synthetic peptide.Methods Design a RGD-containing peptide consisting of 23 amino residues. The peptide was synthesized by solid-phase synthesis method and characterized by mass spectrometry and high pressure liquid chromatography. The feasibility and speicifility of cell adhesion mediated by the synthetic peptide was examined by cell attachment inhibition assay.Results MS showed the average molecular weight of the synthetic peptide was 2741.54 m/z, which was accorded with the theoretic molecular weight. HPLC showed the purity of the synthetic peptide was 99.2796%, which was pure enough for our experiments. RGD-containing peptides of GRGDSPC and (K)16GRGDSPC and BSA, (K)16 all promoted the attachments of BMSCs, especially (K)16GRGDSPC. Only the RGD-containing peptides of GRGDSPC and (K)16GRGDSPC could significantly inhibit the cell attachments to pretreated flusk by (K)16GRGDSPC. Non-RGD-containing peptide of GRGESPC displayed no effects and (K)16 and BSA showed not inhibitory effects but further promoting the attachments.Conclusion Our designed peptide (K)16GRGDSPC was easy to synthesize and could mediate BMSC cell adhesion specifically via RGD tripeptide motif.PartⅢGene delivery by the synthetic peptide and analysis of influence factorsobjective To evaluate the feasibilty and the specifility of gene delivery mediated by the synthetic peptide and to analyze the related influence factors on gene delivery. Methods The synthetic peptide was used as vector and two kinds of reporter genes of luciferase and EGFP were individually transfected into BMSCs. The gene expressions were observed by Liquid Scintillation Counting detector and fluorescent microscope. The transfection efficiency was compared with another commercial liposome Lipofectamine 2000. The targeted specifility of gene delivery by the peptide was examined by the gene delivery inhibition tests. On the conditions of keeping other factors constant, one factors was changed and its influences on the gene delivery was also evaluated and optimized.Results Both the two reporter genes of luciferase and EGFP were expressed in BMSCs. The transfection efficiency of synthetic peptide by FCM was 19.47%, which had no sigficant difference with that of Lipofectamine 2000(19.98%). RGD- containing peptides GRGDSPC and (K)16GRGDSPC decreased the transfection efficiency significantly, but non-RGD contaiing peptide GRGESPC failed. Chloroquine and PEI could facilitate the gene delivery by the synthetic peptide, especially Chloroquine. Endosomal buffer chloroquine was essential for the peptide-mediated gene delivery and optimal exposure time to chloroquine is more than 8 hours and its optimal final concentration was 100μM. When the concerntration of DNA is 2～4μg and the weight to weight ratio of vector to DNA was 3 to 1 the optimal transfection effects were received. The time exposure to vector/DNA complex shorter than 4 hours or longer than 24 hours was adverse to the targeted gene expression. Serum significantly inhibited the combination of vector/DNA complexes with cells. Fusion peptide based on hemagglutinin N terminum 20 residues and cationic liposome Lipofectamine 2000 facilitated the gene delievery, especially the fusion peptide had significant synergistic effects with the synthetic RGD-containing peptide.Transferrin increased TGFβ1 expression greatly as well and the optimal effects were obtained when 25μg/ml.Conclusion The synthetic peptide (K)16GRGDSPC not only mediated specific cell adhesion but also mediated gene delivery targetedly and specifically obtaining ideal transfection efficiency. That optimizing kinds of influence fators could provide tactics for improving peptide-mediated gene delivery efficiency and provide foundations for the wide application of peptide vector in gene delivery fields and next experiments on the gene modification of bone matrix materials.PartⅣFibrication of gene-loaded biomimetic matrices and gene delivery mediated by the novel gene activated matricesObjective To fibricate transforming growth factor beta 1 gene-loaded biomimetic matrices and evaluate whether the modified matrices could mediate gene delivery into BMSCs.Methods The synthetic peptide vector (K)16GRGDSPC was conjugated with PLGA-[ASP-PEG] matrices by cross linker Sulfo-LC-SPDP and examined by XPS. Then BMSCs obtained from rabbit were mixed cultured onto the scaffolds and observed by scanning electonic microscope. Cell adhension behaviors of the cells were analyzed by conventional precipitation method and micropipette aspiration technique. The feagures of cytoskeleton F-actin were observed under laser scanning confocal microscope. Then the modified mtrices were mixtured with transforming growth factor beta 1 gene and BMSCs were cultured on them. The expressions of transduced gene were examined by RT-PCR, western blot, ELISA and immunohistochemistry.Results XPS patterns showed that the synthetic peptide were successfully conjugated into PLGA-[ASP-PEG] mitrices and significantly improved their adhesion capabilities. RT-PCR, western blot, ELISA and immunohistochemistry all showed TGF-β1 gene expressed in BMSCs.Conclusion TGF-β1 gene-loaded matrices was in fact a gene-activated matrix and could mediate exogenous genes into BMSCs as a novel nonviral vector.PartⅤgene-loaded matrices regulated the osteoblastic differentiation of bone marrow stromal cellsObjective To evaluate the osteoblastic differentiation of bone marrow stromal cells regulated by TGF-β1 gene-loaded matrices.Methods TGF-β1 gene-loaded matrices were fabricated and BMSCs were mixed cultured onto them. The prolification behaviors were examined by MTT assay, Coomassie Brilliant Blue dyes and flow cytometry analysis. With osteoinductive medium culture, the osteoblast-associated markers ALP, OCN, OPN, CollagenⅠand Cbfa1 mRNAs and mineralization were assayed. The safety of the gene-loaded matrices were evaluated by DNA contents and cytoskeleton staining.Results The proliferation activities of BMSCs cultured on the gene-loaded matrices were significantly higher than the control groups. The osteoblastic phenotype expressions of ALP, OCN, OPN, CollagenⅠand Cbfa1 and minerization of experimental group were also significantly higher than the control groups.The cells mixed cultured with gene-loaded matrices were found no heteroploids and normal cytoskeletons.Conclusion TGF-β1 gene-loaded matrices were capable of regulating the osteoblastic differentiation of bone marrow stromal cells and TGF-β1 could be an ideal inducer for BMSCs.