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Objective:To explore the possibility of constructing tissue-engineered cartilage three-dimensional nanoscaffolds with collagen Ⅱ (COLⅡ), hyaluronic acid (HA) and chondroitin sulfate (CS).Methods:The tissue-engineered cartilage scaffolds were prepared by electrospinning techniques with the mixture COLⅡ-HA-CS solvent, which dissolved by 3-trifluoroethanol-water. The surface topography was observed under electron microscope (SEM). And the diameter of nanofibers, the water absorption rate, contact angle and degradation rate were also detected. Generation 2 rabbit chondrocytes were seeded into the scaffold. The cell survival rate and proliferation were evaluated by Cell Counting Kit-8.Results:When the concentration range of electrospinning was 80-120 mg/ml and the mixing ratio of Col, HA and CS was 6-8∶1∶1-2, the tissue engineered cartilage nanoscaffolds could be successfully prepared. Their diameters were mainly distributed between 126.5±23.3 nm and 374.7±14.1 nm. The scaffolds had satisfactory hydrophilicity and degradability. The chondrocytes could well adhere and proliferate on the scaffold.Conclusions:The COLⅡ-HA-CS tissue-engineered cartilage nanoscaffolds have good physical and biological properties, which suggests its promising application in tissue-engineered cartilage.
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BACKGROUND: Previous studies have shown that a variety of materials can be used for the construction of tissue engineering scaffolds. The topological structure of the scaffold surface has a regulatory effect on the biological behaviors such as stem cell proliferation and differentiation, but the specific mechanism is still unclear. OBJECTIVE: To investigate the role of P38 and Akt pathways in the oriented differentiation of bone marrow mesenchymal stem cells in nanofiber scaffolds. METHODS: Three kinds of nanofiber scaffolds (AFS, AYS, 3-DPS) with different structures were constructed. Rat bone marrow mesenchymal stem cells were inoculated on the surface of three kinds of nanofiber scaffolds. After osteogenic induction, cell morphology, adhesion and proliferation were detected. mRNA expression levels of key phenotype molecules (COLIα1, COLIIα1, Aggrecan, Sox-9) were measured using qRT-PCR. Intracellular P38, AKT, ERK1/2 and JNK expression was detected by western blot assay. RESULTS AND CONCLUSION: After 4 and 8 hours of culture, cell adhesion rate of the 13-DPS scaffold group was higher than that of the AFS and AYS scaffold groups (P<0.05). After 7 days of culture, cells of the 13-DPS scaffold group proliferated faster than those of AFS and AYS scaffold groups (P<0.05). Bone marrow mesenchymal stem cells adhered firmly and grew well on three kinds of scaffolds. Fibroblast-like growth was observed on the AFS and AYS scaffolds and chondrocyte-like growth was observed on the 3-DPS scaffold. After 3 weeks of cartilage induction, mRNA expression of COLIIα1, Aggrecan and Sox-9 was higher, and the mRNA expression of COLIα1 was lower, in the 3-DPS scaffold group compared with the other two groups (both P<0.05). After 3 weeks of cartilage induction, relative expression level of p-AKT and p-P38 in the 3-DPS scaffold group was significantly higher than that in the other two groups (both P<0.05). There were no significant differences in AKT total protein and ERK1/2, JNK, P38, p-ERK1/2, p-JNK and p-P38 protein expression levels among three groups. These findings suggest that nanofiber annulus fibrosus scaffolds with different spatial structures can induce the oriented differentiation of bone marrow mesenchymal stem cells through the P38 and AKT pathway, which were the downstream of the Integrin-FAK signaling pathway.
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BACKGROUND: Statins plays a significant role in regulating blood lipids, treating and preventing cardiovascular and cerebrovascular diseases. Studies have shown that statins has certain potential in promoting bone formation and treating osteoporosis. OBJECTIVE: To prepare the drug release scaffolds for the sustained release of atorvastatin calcium, which consist of bovine serum albumin microspheres and polycaprolactone electrostatic spinning fibers, and to investigate the effects of the drug sustained release scaffolds on osteoblast adhesion and proliferation. METHODS: Bovine serum albumin microspheres containing atorvastatin calcium were prepared by desolvation. A layer of chitosan was coated on the surface of the bovine serum albumin microspheres by electrostatic adsorption, which can increase the stability of the microspheres. Bovine serum albumin microspheres were purified and lyophilized for later use. The lyophilized powder of microspheres was dissolved in organic solvent. An appropriate amount of hydroxyapatite was added in the solvent. The nanofiber scaffolds for sustained release of atorvastatin calcium were prepared via electrospinning. The micromorphology, degradation performance, and sustained-release performance of the nanofiber scaffolds were characterized. The prepared nanofiber scaffolds for sustained-release of atorvastatin calcium were co-cultured with MC3T3-E1 cells to observe cell adhesion and proliferation. RESULTS AND CONCLUSION: (1) Transmission electron microscopy revealed that the shape of the bovine serum albumin nanospheres was regular and circular. Bovine serum albumin nanospheres were discarded in the electrostatic spinning fibers. The basic morphology of the microspheres was retained. (2) Scanning electron microscopy revealed that the nanofibers used for preparation of nanofiber scaffolds for sustained-release of atorvastatin calcium were composed of filaments with uniform diameters and continuous smooth surface. Filaments were intertwined to form a network structure. (3) The nanofiber scaffolds exhibited the fastest degradation in the first month. The material was incomplete when degraded for 3 months. (4) The nanofiber scaffolds had the ability to slow down the release of drugs. The effect could last for more than 1 month. The overall process of drug release was similar to the zero-order kinetic process. (5) The nanofiber scaffolds for sustained-release of atorvastatin calcium can promote MC3T3-E1 cell adhesion and proliferation. (6) These results suggest that the nanofiber scaffolds for sustained-release of atorvastatin calcium have good biocompatibility and can promote the adhesion and proliferation of osteoblasts.