Objective To construct a recombinant adenovirus vector pAdxsi-GFP-NELL1 that co-expressing green fluorescent protein (GFP) and homo sapiens NEL-l ike 1 (NELL1) protein (a protein bly expressed in neural tissue encoding epidermal growth factor l ike domain), to observe its expression by transfecting the recombinant adenovirus into rat bone marrow mesenchymal stem cells (BMSCs) so as to lay a foundation for further study on osteogenesis of NELL1 protein. Methods From pcDNA3.1-NELL1, NELL1 gene sequence was obtained, then NELL1 gene was subcloned into pShuttle-GFP-CMV (-)TEMP vector which was subsequently digested with enzyme and insterted into pAdxsi vector to package the recombinant adenovirus vector (pAdxsi-GFP-NELL1). After verified by enzyme cutting and gel electrophoresis, pAdxsi-GFPNELL1 was ampl ified in HEK293 cells and purified by CsCl2 gradient purification, titrated using 50% tissue culture infective dose (TCID50) assay. The rat BMSCs were cultured and identified by flow cytometry and directional induction, then were infected with adenoviruses (pAdxsi-GFP-NELL1 and pAdxsi-GFP). NELL1 expression was verified by RT-PCR and immunofluorescence; GFP gene expression was verified by the intensity of green fluorescence under fluorescence microscope. Cell counting kit-8 (CCK-8) was used for investigate the influence of vectors on the prol iferation of rat BMSCs. Results Recombinant adenoviral vector pAdxsi-GFP-NELL1, which encodes a fusion protein of human NELL1, was successfully constructed and ampl ified with titer of 1 × 1011 pfu/mL. The primary BMSCs were cultured and identified by flow cytometric analysis, osteogenic and adipogenic induction, then were used for adenoviral transfection efficiency and cell toxicity tests. An multipl icity of infection of 200 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Three days after transfection with 200 pfu/cell pAdxsi-GFP-NELL1 or pAdxsi-GFP, over 60% BMSCs showed green fluorescent by fluorescence microscopy. Imunofluorescence with NELL1 antibody also revealed high level expression of human NELL1 protein in red fluorescent in these GFP expressing cells. RT-PCR analysis confirmed that the exogenous expression of NELL1 upon transfection with pAdxsi-GFPNELL1 at 200 pfu/cell, whereas NELL1 remained undetectable in Ad-GFP-transfected rat BMSCs. The prol iferative property of primary rat BMSCs after adenoviral NELL1 transfection was assayed by CCK-8 in growth medium. Growth curve demonstratedno significant difference among BMSCs transfected with pAdxsi-GFP-NELL1, pAdxsi-GFP, and no treatment control at 7 days (P gt; 0.05). Conclusion Recombinant adenovirus vector pAdxsi-GFP-NELL1 can steady expressing both GFP and NELL1 protein after being transfected into rat BMSCs. It provides a useful tool to trace the expression of NELL1 and investigate its function in vitro and in vivo.
ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
Objective Surface modification of nitinol (NiTi) shape memory alloy is an available method to prevent nickel ion release and coating with titanium-niobium (TiNb) alloy will not affect the superelasticity and shape memory of NiTi. To evaluate the bone histocompatibil ity of NiTi shape memory alloy implants coated by TiNb in vivo. Methods NiTi memory alloy columns which were 4 mm in diameter and 12 mm in length were coated with Ti (Ti-coating group) and TiNb alloy (TiNb-coating group) respectively by magnetron sputtering technique. And NiTi group were not coated on the surface. Fifteen mongrel dogs were divided into 3 groups randomly with 5 dogs in each group. NiTi, Ti-coating and TiNb-coating columns were implanted into the lateral femoral cortex of each group, respectively. There were 10 columns embedded in eachdog’s femur whose distance was 1.0 cm to 1.5 cm from each other. The materials were obtained 12 months after operation. After X-ray photography, only those columns which were perpendicular to the cortex of the femur shaft were selected for subsequent analysis. Push-out tests were performed to attain the maximum shear strength (the number of specimens of TiNi group, Ticoating group, and TiNb-coating group were 12, 10, and 14, respectively). Undecalcified sections were used for histological observation and the calculation of osseointegration rate (the number of specimens of TiNi group, Ti-coating group, and TiNb-coating group were 8, 5, and 10, respectively). Results The maximum shear strength of Ti-coating group (95.10 ± 10.03) MPa, and TiNb-coating group (91.20 ± 15.42) MPa were significantly higher than that of NiTi group (71.60 ± 14.24) MPa (P lt; 0.01). Gimesa staining showed that no obvious macrophage and inflammation cell was observed in 3 groups. The osseointegration rates of NiTi group, Ti-coating group, and TiNb-coating group were (21.30% ± 0.23%), (32.50% ± 0.31%), and (38.60% ± 0.58%), respectively; there were significant differences among 3 groups (P lt; 0.01). Conclusion The implants of 3 groups all have good bone histocompatabil ity. But the osseointegration rate and the shear strength in the Ti-coating group and the TiNb-coating group were better than those in the NiTi group, the TiNb-coating group is the best among them.