Objective To study the transfection and expression of pleiotrophin (Ptn) gene in mice adipose-derived stem cells (ADSCs) so as to provide a new approach for the treatment of ischemic injury. Methods ADSCs from clean inbred C57BL/6W mice (weighing, 15-20 g) were isolated and cultured in vitro. The cell surface markers (CD29 and CD44) of ADSCs were identified by flow cytometry. The ADSCs were transfected with plasmid pIRES2-LEGFPN1 (containing Ptn gene coding sequence) as experimental group (group A) and with plasmid pLEGFP-N1 (containing GFP gene coding sequence) as control group (group B). After ADSCs were transfected by different plasmids respectively, the cells containing Ptn gene were selected by G418 (the best selected concentration was 200 μg/mL), and the immunophenotype of the cells was identified by flow cytometry after transfection. Meanwhile, real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells. Results The mice ADSCs were isolated and cultured successfully in vitro. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%, respectively; the double positive rate of CD44 and CD29 was 93.6%. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%, respectively after transfection of Ptn gene; the double positive rate of CD44 and CD29 was 93.4%. The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B (P lt; 0.05). Conclusion The ADSCs can be stablely transfected by Ptn gene, the transfected ADSCs can express PTN protein highly, which is a new idea for tissue engineering of vascular reconstruction.
Objective To review the appl ication of collagen and biodegradable polymer composite scaffolds in vascular tissue engineering, and describe the multi-layering vascular scaffolds of collagen-based material in recent years. Methods The l iterature concerning collagen composite scaffold production for scaffold of vascular tissue engineering was extensively reviewed and summarized. Results As one of the structural proteins in natural blood vessel, collagen is widely used in vascular tissue engineering because of good biocompatibil ity, biodegradabil ity, and cell recognition signal. The vascular scaffolds with biological activity and good mechanical properties can be made by collagen-polymer composite materials. In addition, the structure and function of the natural blood vessel can be better simulated by multi-layering vascularscaffolds. Conclusion Collagen-polymer composite material is the hot spot in the research of vascular scaffolds, and multilayering vascular scaffolds have a brill iant future.
Objective To explore the method of preparing the electrospinning of synthesized triblock copolymers of ε-caprolactone and L-lactide (PCLA) for the biodegradable vascular tissue engineering scaffold and to investigateits biocompatibil ity in vitro. Methods The biodegradable vascular tissue engineering scaffold was made by the electrospinning process of PCLA. A series of biocompatibil ity tests were performed. Cytotoxicity test: the L929 cells were cultured in 96-wellflat-bottomed plates with extraction media of PCLA in the experimental group and with the complete DMEM in control group, and MTT method was used to detect absorbance (A) value (570 nm) every day after culture. Acute general toxicity test: the extraction media and sal ine were injected into the mice’s abdominal cavity of experimental and control groups, respectively, and the toxicity effects on the mice were observed within 72 hours. Hemolysis test: anticoagulated blood of rabbit was added into the extracting solution, sal ine, and distilled water in 3 groups, and MTT method was used to detect A value in 3 groups. Cell attachment test: the L929 cells were seeded on the PCLA material and scanning electron microscope (SEM) observation was performed 4 hours and 3 days after culture. Subcutaneous implantation test: the PCLA material was implanted subcutaneously in rats and the histology observation was performed at 1 and 8 weeks. Results Scaffolds had the characteristics of white color, uniform texture, good elasticity, and tenacity. The SEM showed that the PCLA ultrafine fibers had a smooth surface and proper porosity; the fiber diameter was 1-5 μm and the pore diameter was in the range of 10-30 μm. MTT detection suggested that there was no significant difference in A value among 3 groups every day after culturing (P gt; 0.05). The mice in 2 groups were in good physical condition and had no respiratory depression, paralysis, convulsion, and death. The hemolysis rate was 1.18% and was lower than the normal level (5%). The SEM showed a large number of attached L929 cells were visible on the surface of the PCLA material at 4 hours after implantation and the cells grew well after 3 days. The PCLA material was infiltrated by the inflammatory cells after 1 week. The inflammatory cells reduced significantly and the fiber began abruption after 8 weeks. Conclusion The biodegradable vascular tissue engineering scaffold material made by the electrospinning process of PCLA has good microstructure without cytotoxicity and has good biocompatibil ity. It can be used as an ideal scaffold for vascular tissue engineering.
Objective To investigate the biological response and chemotaxis of endothel ial cells on template materials with different protein concentrations on the same surface, to provide the evidence for deep understanding of chemical induced cell motil ity. Methods Microcontact printing technique was employed to fabricate template materials with four different concentrations of collagen (50, 100, 200, 300 μg/mL) on the same substrate. Scanning electron microscopy was employed to characterize the qual ity of polydimethylsiloxane (PDMS) stamp. Confocal laser scanning microscopy (CLSM) was util ized to characterize the absorption of different concentrations of FITC conjugated collagen (50, 100, 200, 300 μg/mL) on the substrates surfaces. Software was used to analyze the fluorescence intensity of adsorbed protein on the substrates. Albumin was then used to block the substrates for cell culture of human umbil ical vein endothel ial cells (hUVEC). Substrates with no collagen adsorption were used as control samples. The influence of different concentrations of collagen on the prol iferation of hUVEC was investigated via MTT assay at 6, 24, 48 and 72 hours of culture. The cytoskeletal structures of cells were characterized by CLSM. The cell’ s migration speed and absolute displacement were measured by path measurement of single cell after 24 hours of culture. Results Fabricated PDMS stamps with complete pattern were flat. Template substrates were fully covered with evenly distributed collagen protein. The fluorescence intensities were 38.51 ± 1.63, 55.21 ± 3.88, 73.17 ± 3.59, and 80.95 ± 1.12 in adsorbed FTIC conjugated collagen with 50, 100, 200 and 300 μg/mL, respectively. Endothel ial cells spread better on various substrates coated with collagen than those of control samples. The prol iferation of endothel ial cells on collagen coated substrateswas significantly higher than that of control group (P lt; 0.05). With collagen concentration increasing from 50 µg/mL to 300µg/mL, the prol iferation abil ities and absolute displacements of endothel ial cells significantly increased (P lt; 0.05). Except for the group with 300 μg/mL, the migration speed of endothel ial cells on collagen coated substrates was significantly lower (P lt; 0.05) than that of control group. However, the migration speed of endothel ial cells on collagen coated substrates significantly increased (P lt; 0.05) along with collagen concentration increasing from 50 µg/mL to 300 µg/mL. Conclusion It is feasible to acquire domains with different protein concentrations on the same substrate using microcontact printing technique for investigating cell’s chemotaxis.
Objective To introduce the materials, preparative technique and endothel ial ization modification of scaffold. Methods The recent original articles about vascular tissue engineering were extensively reviewed and analyzed. Results The materials including natural materials, biodegradable polymers and composite materials were studied in the field of scaffold. The ways of casting, cell self-assembly, gel spinning and electrospinning were appl ied to prepare the scaffold of vascular tissue engineering. The modification of scaffold was one of the most important elements for vascular tissue engineering. Conclusion The recent researchs about scaffold of vascular tissue engineering focus on composite material and electrospinning, the modification of scaffold can improve the abil ity of adhesion to endothel ial cells.
Objective To investigate the current situation and developing trend of antithrombotic function study of endothelium in vasculartissue engineering. Methods The effect of several elements onthe antithrombotic ability of endothelium, including the source of endothelium,the characteristic of the matrix materials, the cell culture methods, and the endothelium’s gene modification were analyzed. Results The normal antithrombotic function of tissue engineered vascular relied on the source ofendothelium, gene modification of seeding cells, the cell culture methods in vitro, and the characteristic of the scaffolds. Conclusion The establishment of an ideal antithrombotic functional tissue engineering vascular still requires further studies in various aspects including seeding cells, matrix materials, and cell culture methods. Gene modification of vascular endothelium, which improves the antithrombotic ability, deserves more attention.