ObjectiveTo prepare composite scaffold of different quality ratio of silk fibroin to collagen,analyze the scaffold performance,and optimize the quality ratio for chondrocyte tissue engineering. MethodsThe silk fibroin/collagen composite scaffold was made using a freeze-drying technique in different quality ratios of silk fibroin to collagen:4:2(group A),4:4(group B),and 4:8(group C).The porosity,water absorption expansion rate,mechanical properties,and pore size of composite scaffold were detected.The bone marrow mesenchymal stem cells (BMSCs) were isolated from 4-week-old male Wistar rats by density gradient centrifugation,and the third generation BMSCs were seeded onto the scaffolds at 2×107 cells/mL density,and were cultured for 14 days.The cell proliferation was detected using MTT assay at 1,3,5,7,9,11,and 13 days,the cell morphology and distribution were observed by HE staining and scanning electron microscopy (SEM). ResultsThe porosity of groups A,B,and C was 94.6%±1.6%,80.6%±1.1%,and 60.6%±1.0% respectively;and significant differences were found between group A and groups B and C,and between group B and group C (P<0.05).The water absorption expansion rates of groups A,B,and C were 1 523.7%±186.6%,1 091.0%±151.6%,and 659.6%±161.4% respectively,showing no significant difference among 3 groups (F=6.67,P=0.08).The elasticity modulus of groups A,B,and C were (23.1±2.5),(25.1±2.3),and (29.8±2.6) kPa respectively,showing no significant difference among 3 groups (F=2.00,P=0.28).The pore size of groups A,B,and C was (103±12),(80±15),and (60±16)μm respectively,showing no significant difference among 3 groups (F=2.22,P=0.26).MTT results showed that the cell proliferation in the group A at 7,9,11,and 13 days were better than those in groups B and C (P<0.05);at 14 days after cultivation,even pore size,good intercommunicating of holes,and good cells growth on the scaffolds with full extension and more extracellular matrix were seen under SEM in group A,but small pore size,poor intercommunicating of holes and poor cell growth on the scaffolds in groups B and C.HE staining and SEM results showed that the cells on the scaffold in group A was obviously more than those in groups B and C. ConclusionThe scaffold prepared in a quality ratio 4:2 of silk fibroin to collagen has better porosity,water absorption expansion rate,elasticity modulus,and pore size,on which the cells can grow well,so it is more suitable for cartilage tissue engineering.
ObjectiveTo investigate the differentiation of rat adipose-derived stem cells (ADSCs) into neuronlike cells by indirect co-culture with Schwann cells (SCs) in vitro so as to look for the ideal seed cells for tissue engineering. MethodsSCs were isolated from sciatic nerves of 1-2 days old Sprague-Dawley rats with enzymatic digestion method. Immunofluorescence staining was used to identify SCs with the marker S-100. ADSCs were isolated from the epididymal fat pads of adult male Sprague-Dawley rats by means of differential attachment. And the cell phenotypes (CD29, CD34, CD45, CD73, CD90, and CD105) of ADSCs at passage 3 were determined by flow cytometry analysis. Primary SCs and ADSCs at passage 3 were co-cultured at a ratio of 2:1 in Transwell culture dishes (experimental group), and ADSCs cultured alone served as control group. Immunofluorescence and flow cytometry were adopted to investigate the neural differentiation of ADSCs at 14 days. The expression differences for neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), neuronal nuclei protein (NeuN), and glial fibrillary acidic protein (GFAP) were detected, and the percentage of positive cells was calculated. ResultsADSCs were successfully extracted and can passage in a considerable large amount. Flow cytometry analysis showed that ADSCs at passage 3 were positive for CD29, CD90, CD73, and CD105 expression, but negative for CD34 and CD45 expression. The ADSCs of the experimental group showed contraction of nucleus, increasing of soma refraction, and several long and thick protrusions of cell body. The cell shape had no obvious change in the control group. Both immunofluorescence and flow cytometry analysis results showed the expressions of MAP2, NSE, NeuN, and GFAP at 14 days after co-cultured with SCs, and the positive cell ratios were significantly higher than those in the control group (P<0.01). ConclusionCo-culture with SCs not only can promote the survival regeneration of ADSCs, but also can induce the differentiation of ADSCs into neuron-like cells.
ObjectiveTo study the preparation and cytocompatibility of bone tissue engineering scaffolds by combining low temperature three dimensional (3D) printing and vacuum freeze-drying techniques. MethodsCollagen (COL)and silk fibroin (SF) were manufactured from fresh bovine tendon and silkworm silk. SolidWorks2014 was adopted to design bone tissue engineering scaffold models with the size of 9 mm×9 mm×3 mm and pore diameter of 500μm. According to the behavior of composite materials that low temperature 3D printing equipment required, COL, SF, and nano-hydroxyapatite (nHA)at a ratio of 9:3:2 and low temperature 3D printing in combination with vacuum freeze-drying techniques were accepted to build COL/SF/nHA composite scaffolds. Gross observation and scanning electron microscope (SEM) were applied to observe the morphology and surface structures of composite scaffolds. Meanwhile, compression displacement, compression stress, and elasticity modulus were measured by mechanics machine to analyze mechanical properties of composite scaffolds. The growth and proliferation of MC3T3-E1 cells were evaluated using SEM, inverted microscope, and MTT assay after cultured for 1, 7, 14, and 21 days on the composite scaffolds. The RT-PCR and Western blot techniques were adopted to detect the gene and protein expressions of COL I, alkaline phosphatase (ALP), and osteocalcin (OCN) in MC3T3-E1 cells after 21 days. ResultsCOL/SF/nHA composite scaffolds were successfully prepared by low temperature 3D printing technology and vacuum freeze-drying techniques; the SEM results showed that the bionic bone scaffolds were 3D polyporous structures with macropores and micropores. The mechanical performance showed that the elasticity modulus was (344.783 07±40.728 55) kPa; compression displacement was (0.958 41±0.000 84) mm; and compression stress was (0.062 15±0.007 15) MPa. The results of inverted microscope, SEM, and MTT method showed that a large number of cells adhered to the surface with full extension and good cells growth inside the macropores, which demonstrated a satisfactory proliferation rate of the MC3T3-E1 cells on the composite scaffolds. The RT-PCR and Western blot electrophoresis revealed gene expressions and protein synthesis of COL I, ALP, and OCN in MC3T3-E1 cells. ConclusionLow temperature 3D printing in combination with vacuum freeze-drying techniques could realize multi-aperture coexisted bionic bone tissue engineering scaffolds and control the microstructures of composite scaffolds precisely that possess good cytocompatibility. It was expected to be a bone defect repair material, which lays a foundation for further research of bone defect.