OBJECTIVE: To observe the effects of silks on attachment, shape and function of chondrocytes cultured in vitro. METHODS: The silks from silk worm cocoons were digested by trypsin and coated with polylactic acid to from three dimensional scaffolds for rabbit rib chondrocyte culture. The growth and shape of chondrocytes were observed with phase contrast microscopy, scanning electron microscopy. RESULTS: The chondrocytes were adhered to silks slowly after chondrocytes were seeded into silk scaffolds and cells fixed on silks well 1 or 2 days later. Cells began to proliferate after 3 days and multiplicative growth was observed on the 6th day. Microholes of silk scaffolds were filled with chondrocytes 2 weeks later. Scanning electron microscopy showed that there was a lot of extracellular matrix surrounding cells. CONCLUSION: Silks are ideal for attachment, growth and function maintenance of chondrocytes, and silks can be used as scaffolds for chondrocytes in three dimensional culture.
Objective To develop three-dimensional (3D) porous nanofiber scaffold of PLGA-silk fibroincollagen and to investigate its cytocompatibil ity in vitro. Methods Method of electrostatic spinning was used to prepare 3D porous nanofiber scaffold of PLGA-silk fibroin-collagen (the experimental group) and 3D porous nanofiber scaffold of PLGA (the control group). The scaffold in each group was observed by scanning electron microscope (SEM). The parameters of scaffold fiber diameter, porosity, water absorption rate, and tensile strength were detected. SC harvested from the bilateral brachial plexus and sciatic nerve of 8 SD suckl ing rats of inbred strains were cultured. SC purity was detected by S-100 immunohistochemistry staining. The SCs at passage 4 (5 × 104 cells/mL) were treated with the scaffold extract of each group at a concentration of 25%, 50%, and 100%, respectively; the cells treated with DMEM served as blank control group. MTT method was used to detect absorbance (A) value 1, 3, 5, and 7 days after culture. The SC at passage 4 were seeded on the scaffold of the experimental and the control group, respectively. SEM observation was conducted 2, 4, and 6 days after co-culture, and laser scanning confocal microscope (LSCM) observation was performed 4 days after co-culture for the growth condition of SC on the scaffold. Results SEM observation: the scaffold in two groups had interconnected porous network structure; the fiber diameter in the experimental and the control group was (141 ± 9) nm and (205 ± 11) nm, respectively; the pores in the scaffold were interconnected; the porosity was 87.4% ± 1.1% and 85.3% ± 1.3%, respectively; the water absorption rate was 2 647% ± 172% and 2 593% ± 161%, respectively; the tensile strength was (0.32 ± 0.03) MPa and (0.28 ± 0.04) MPa, respectively. S-100 immunohistochemistry staining showed that the SC purity was 96.5% ± 1.3%. MTT detection: SC grew well in the different concentration groups and the control group, the absorbance (A) value increased over time, significant differences were noted among different time points in the same group (P lt; 0.05), and there was no significant difference between the different concentration groups and the blank control group at different time points (P gt; 0.05). SEM observation: in the experimental group, SC grew well on the scaffold, axon connection occurred 4 days after co-culture, the cells prol iferated massively and secreted matrix 6 days after co-culture, and the growth condition of the cells was better than the control group. The condition observed by LSCM 4 days after co-culture was the same as that of SEM. Conclusion The 3D porous nanofiber scaffoldof PLGA-silk fibroin-collagen prepared by the method of electrostatic spinning is safe, free of toxicity, and suitable for SC growth, and has good cytocompatibil ity and proper aperture and porosity. It is a potential scaffold carrier for tissue engineered nerve.
ObjectiveTo investigate the effect of silk fibroin-poly-L-lactic acid (SF-PLLA) microcarriers on the expansion and differentiation of adipose-derived stem cells (ADSCs).MethodsADSCs were extracted from adipose tissue donated voluntarily by patients undergoing liposuction by enzymatic digestion. The 3rd generation ADSCs were inoculated on CultiSpher G and SF-PLLA microcarriers (set up as groups A and B, respectively), and cultured in the rotary cell culture system. ADSCs cultured in normal two-dimensional plane were used as the control group (group C). Scanning electron microscope was used to observe the microcarriers structure and cell growth. Live/Dead staining and confocal fluorescence microscope was used to observe the distribution and survival condition of cells on two microcarriers. DNA quantification was used to assess cell proliferation on two microcarriers. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect chondrogenesis, osteogenesis, and adipogenesis related gene expression of ADSCs in 3 groups cultured for 18 days. Flow cytometry was used to identify the MSCs surface markers of ADSCs in 3 groups cultured for 18 days, and differential experiments were made to identify differentiation ability of the harvested cells.ResultsADSCs could be adhered to and efficiently amplified on the two microcarriers. After 18 days of cultivation, the total increment of ADSCs of the two microcarriers were similar (P>0.05). qRT-PCR results showed that chondrogenesis related genes (aggrecan, cartilage oligomeric matrix protein, SOX9) were significantly up-regulated for ADSCs on SF-PLLA microcarriers and adipogenesis related genes (peroxisome proliferator-activated receptor γ, lipoprotein lipase, ADIPOQ) were significantly up-regulated for ADSCs on CultiSpher G microcarriers, all showing significant differences (P<0.05). Flow cytometry and differentiation identification proved that the harvested cells of the two groups were still ADSCs.ConclusionThe ADSCs can be amplified by SF-PLLA microcarriers, and the chondrogenic differential ability of harvested cells was up-regulated while the adipogenic differential was down-regulated.
Test of tissue tolerance to domastic prosthetic materials (carbon fiber mesh, siliconized velvet, silk cloth and dacron cloth) as a subcutaneos transplant was performed in the adcominal wall of rabbit. These implants and their surroundding tissues were excied for studies at second , fourth, eighth and the twelfth weeks after operation. Ratio of fibroblast count to inflammatory cells count which is a common parameter of tissue tolerance was calculated in these four groups. The result shows that fibroblastic cell reaction elicited by carbon fiber mesh is the greates among the four prosthetic materials, the second one is dasron cloth. The inflammatory cell reaction elicited by silk is the greatest among the four materials, the second is carbon fiber mesh, and the dacron cloth the least. Tissure tolerance of dacron cloth is the best in the four prosthetic materials for implantation while sick is the worst.
Objective To investigate the diagnostic efficacy of silkworm larvae plasma (SLP) colorimetry in the accurate diagnosis of periprosthetic joint infection (PJI). Methods Ninety healthy male New Zealand white rabbits were used for knee arthroplasty with Swanson prosthesis. Then they were randomly divided into 3 groups according to different pathogenic bacteria: group A (Staphylococcus aureus group), group B (Staphylococcus epidermidis group) and group C (Escherichia coli group), with 30 rats in each group. The PJI model was prepared by knee injection with 1 mL of pathogenic bacteria of different concentrations. Samples were taken before inoculation and at 7, 14, and 21 days after inoculation, and based on the 2018 PJI Philadelphia International Consensus diagnostic criteria, the success rate of modeling among 3 groups of experimental animals was determined. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficiency of SLP colorimetry were calculated. Results At 21 days after inoculation, 26, 18, and 23 rabbits in groups A, B, and C were diagnosed as infection, respectively. The success rates of modeling were 86.7%, 60.0%, and 76.7%, respectively, showing no significant difference among the 3 groups (χ2=5.724, P=0.073). The results of PJI colorimetry showed that 1 false-positive animal (specificity 75.0%) appeared in group A at 7 days, and the specificity of SLP increased to 100.0% over time (on 14 and 21 days); on 14 and 21 days, another animal appeared false-negative results (sensitivity decreased from 100.0% to 96.2%). One false-positive animal appeared in group B at 7 days (specificity 91.7%), the specificity returned to 100.0% over time; 1 and 4 false-negative animals appeared at 14 and 21 days, respectively (sensitivity 94.4% and 83.3%, respectively). In group C, two false-positive animals (specificity 71.4%) were found at 7 days, and then returned to 100.0%. The diagnostic efficiency of groups A and C was very high at 21 days (96.7% and 100.0%), even for the low virulence Staphylococcus epidermidis in group B, the diagnostic efficiency could be maintained at 90.0% (21 days), and the overall diagnostic efficiency was very good (95.6%). Conclusion SLP colorimetry has high sensitivity, specificity, and diagnostic efficiency in the diagnosis of PJI, which is a potential diagnostic method.
Objective To prepare the silk fibroin (SF)-chitosan (CS) scaffolds by adjusting the mass ratio between CS and SF, and test and compare the properties of the scaffolds at different mass ratios. Methods According to the mass ratios of 6 ∶ 4 (group A), 6 ∶ 8 (group B), and 6 ∶ 16 (group C) between SF and CS, CS-SF scaffolds were prepared by freeze-drying method, respectively. The material properties, porosity, the dissolubility in hot water, the modulus elasticity, and the water absorption expansion rate were measured; the aperture size and shape of scaffolds were observed by scanning electron microscope (SEM). Density gradient centrifugation method was used to isolate the bone marrow mesenchymal stell cells (BMSCs) of 4-week-old male Sprague Dawley rats. The BMSCs at passage 3 were seeded onto 3 scaffolds respectively, and then the proliferation of cells on the scaffolds was detected by MTS method. Results The results of fourier transform infrared spectroscopy proved that with the increased content of CS, the absorption peak of random coil/α helix structure (1 654 cm-1 and 1 540 cm-1) constantly decreased, but the absorption peak of corresponding to β-fold structure (1 628 cm-1 and 1 516 cm- 1) increased. The porosity was 87.36% ± 2.15% in group A, 77.82% ± 1.37% in group B, and 72.22% ± 1.37% in group C; the porosity of group A was significantly higher than that of groups B and C (P lt; 0.05), and the porosity of group B was significantly higher than that of group C (P lt; 0.05). The dissolubility in hot water was 0 in groups A and B, and was 3.12% ± 1.26% in group C. The scaffolds had good viscoelasticity in 3 groups; the modulus elasticity of 3 groups were consistent with the range of normal articular cartilage (4-15 kPa); no significant difference was found among 3 groups (F=5.523, P=0.054). The water absorption expansion rate was 1 528.52% ± 194.63% in group A, 1 078.22% ± 100.52% in group B, and 1 320.05% ± 179.97% in group C; the rate of group A was significantly higher than that of group B (P=0.05), but there was no significant difference between groups A and C and between groups B and C (P gt; 0.05). SEM results showed the aperture size of group A was between 50-250 μm, with good connectivity of pores; however, groups B and C had structure disturbance, with non-uniform aperture size and poor connectivity of pores. The growth curve results showed the number of living cells of group A was significantly higher than that of groups B and C at 1, 3, 5, and 7 days (P lt; 0.05); and there were significant differences between groups B and C at 3, 5, and 7 days (P lt; 0.05). Conclusion The CS-SF scaffold at a mass ratio of 6 ∶ 4 is applicable for cartilage tissue engineering.
ObjectiveThe properties and characteristics of different types of silk fibroin (SF) drug-loaded sustained-release carriers and their effects on the drug release behavior were reviewed, and the existing problems and development prospects of SF drug-loaded sustained-release carriers in tissue engineering drug delivery system were discussed.MethodsThe literatures about drug-loaded SF sustained-release carriers in recent years were extensively consulted, and the types of sustained-release carriers, characteristics of drug release, range of applications, advantages and disadvantages, and solutions were summarized and analyzed.ResultsAt present, the SF drug-loaded sustained-release carriers are mainly divided into SF microparticles, SF scaffolds, SF membranes, SF hydrogels, SF nanofibers, SF sponges, and so on. These types of SF drug-loaded sustained-release carriers have their own advantages and problems, of which the most prominent problem is the burst release of drugs at the initial stage. While, the initial burst release of drugs can be effectively solved by improving the preparation process and adjusting the material ratio. Different types of drug-loaded sustained-release carriers can be prepared by combining different materials to achieve different application scopes and drug release behaviors under different conditions.ConclusionSF is a good drug-loaded carrier for tissue engineering, the burst release of drugs at the initial stage can be solved by improving the preparation process and changing the material structure; through the combination of the advantages of various types of SF drug-loaded sustained-release carriers, it is expected to prepare SF drug-loaded sustained-release carriers that meet different clinical needs.
ObjectiveTo prepare the small intestinal submucosa (SIS)-silk composite scaffold for anterior cruciate ligament (ACL) reconstruction, and to evaluate its properties of biomechanics, biocompatibility, and the influence on synovial fluid leaking into tibia tunnel so as to provide a better choice in the clinical application of ACL reconstruction. MethodsThe silk was used to remove sericin and then weaved as silk scaffold, which was surrounded cylindrically by SIS to prepare a composite scaffold. The property of biomechanics was evaluated by biomechanical testing system. The cell biocompatibility of scaffolds was evaluated by live/dead staining and the cell counting kit 8 (CCK- 8). Thirty 6-week-old Sprague Dawley rats were randomly assigned to 2 groups (n=15). The silk scaffold (S group) and composite scaffold (SS group) were subcutaneously implanted. At 2, 4, and 8 weeks after implanted, the specimen were harvested for HE staining to observe the biocompatibility. Another 20 28-week-old New Zealand white rabbits were randomly assigned to the S group and SS group (n=20), and the silk scaffold and composite scaffold were used for ACL reconstruction respectively in 2 groups. Furthermore, a bone window was made on the tibia tunnel. At last, the electric resistance of tendon graft in the bone window was measured and recorded at different time points after 5 mL of 10% NaCl or 5 mL of ink solution was irrigated into the joint cavity recspectively. ResultsThe gross observation showed that the composite scaffold consisted of the helical silk bundle inside which was surrounded by SIS. The maximal load of silk scaffold and composite scaffold was respectively (138.62±11.41) N and (137.05±16.95) N, showing no significant difference (P>0.05); the stiffness was respectively (24.65±2.62) N/mm and (24.21±2.39) N/mm, showing no significant difference (P>0.05). The live/dead staining showed that the cells had good activity on both scaffolds. However, the cells on the composite scaffold had better extensibility. In addition, the cell proliferation curve indicated that no significant difference in the absorbance (A) values was founded between groups at various time points (P>0.05). HE staining showed less inflammatory cells and much more angiogenesis in SS group than in S group at 2, 4, and 8 weeks after subcutaneously implanted (P<0.05), indicating good biocompatibility. Additionally, the starting time points of electric resistance decrease and the ink leakage were both significantly later in SS group than in S group (P<0.05). The duration of ink leakage was significantly longer in SS group than in S group (P<0.05). ConclusionThe SIS-silk composite scaffold has excellent biomechanical properties and biocompatibility and early vacularization after in vivo implantation. Moreover, it can reducing the leakage of synovial fluid into tibia tunnel at the early stage of ACL reconstruction. So it is promising to be an ideal ACL scaffold.
ObjectiveTo review the application of silk fibroin scaffold in bone tissue engineering. MethodsThe related literature about the application of silk fibroin scaffold in bone tissue engineering was reviewed, analyzed, and summarized. ResultsSilk fibroin can be manufactured into many types, such as hydrogel, film, nano-fiber, and three-dimensional scaffold, which have superior biocompatibility, slow biodegradability, nontoxic degradation products, and excellent mechanical strength. Meanwhile these silk fibroin biomaterials can be chemically modified and can be used to carry stem cells, growth factors, and compound inorganic matter. ConclusionSilk fibroin scaffolds can be widely used in bone tissue engineering. But it still needs further study to prepare the scaffold in accordance with the requirement of tissue engineering.
Objective To investigate the biocompatibil ity of silk fibroin nanofibers scaffold with olfactory ensheathing cells (OECs) and to provide an ideal tissue engineered scaffold for the repair of spinal cord injury (SCI). Methods Silk fibroin nanofibers were prepared using electrospinning techniques and were observed by scanning electron microscope (SEM). Freshly isolated OECs from SD rats purified by the modified differential adherent velocity method were cultured. The cells at passage 1 (1 × 104 cells/cm2) were seeded on the poly-l-lysine (control group) and the silk fibroin nanofibers (experimental group) coated coversl ips in Petri dish. At desired time points, the morphological features, growth,and adhesion of the cells were observed using phase contrast inverted microscopy. The OECs were identified by the nerve growth factor receptor p75 (NGFR p75) immunofluorescence staining. The viabil ity of OECs was examined by l ive/dead assay. The prol iferation of OECs was examined by MTT assay. The cytotoxicity of the nanofibers was evaluated. Results The SEM micrographs showed that the nanofibers had a smooth surface with sol id voids among the fibers, interconnecting a porous network, constituted a fibriform three dimensional structure and the average diameter of the fibers was about (260 ± 84) nm. The morphology of OECs on the experimental group was similar to the cell morphology on the control group, the cells distributed along the fibers, and the directions of the cell protrusions were in the same as that of the fibers. Fluorescence microscopy showed that the purity of OECs was 74.21% ± 2.48% in the experimental group and 79.05% ± 2.52% in the control group 5 days after culture. There was no significant difference on cell purity between two groups (P gt; 0.05). The OECs in the experimental group stained positive for NGFR p75 compared to the control group, indicating that the cells in the experimental group still maintained the OECs characteristic phenotype. Live/dead staining showed that high viabil ity was observed in both groups 3 days after culture. There was no significant difference on cell viabil ity between two groups. The prol iferation activity at 1, 3, 5, 7, and 10 days was examined by MTT assay. The absorbency values of the control group and the experimental group had significant differences 3 and 5 days after culture (P lt; 0.05). The relative growth rates were 95.11%, 90.35%, 92.63%, 94.12%, and 94.81%. The cytotoxicity of the material was grade 1 and nonvenomous according to GB/T 16886 standard. Conclusion Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI.