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Objective To investigate the influence of dynamic mechanical stimulation on the annulus fibrosus (AF) cells seeded on silk scaffolds.Methods AF cells were isolated from rabbits and were seeded on the scaffold,then cultured for 3,7,14 days with different range of dynamic compression.Stereomicroscope and scanning electron microscope (SEM) was used to observe the surface morphology of tissue engineering annulus fibrosus cells (TE-AFs).After fixation,samples were harvested for histological staining.AF cells related extracellular matrix (ECM) was evaluated by the quantitative analysis of total DNA,proteoglycan and collagen I.The mechanical properties were compared within different groups.Results Stereomicroscope and SEM results showed that the colors of TE-AFs in all groups were deepening with time going.SEM showed cell adhesion on the scaffold and the secretion of extracellular matrix.Histological,immunohistochemical staining,biochemical quantitative analysis and total DNA content showed that the AF cells inside scaffolds could support AF cell attachment,proliferation and secretion.As a result,the compressive properties were enhanced with increasing culture time.Stereomicroscope showed that the colors of TE-AFs in all groups were deepening with time going after dynamic compression.HE staining,Safranin O staining and Type Ⅰ collagen staining showed that cell proliferation and secretion,GAG secretion and collagen secretion were increased with time going within different groups.Quantitation of GAG achieved maximum in 15％ strain group,and quantitation of collagen achieved maximum in 10％ strain group.The total DNA content achieved maximum in 5％ strain group,and compression elastic modulus achieved maximum in 15％strain goup.The height of TE-AFs did not change after mechanical stimulation for 14 days.Conclusion Suitable mechanical stimulation is a positive factor for new AF tissue engineering that will tend to the nature tissue.Excessive compression can accelerate the progress of cell apoptosis.
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Objective To establish an animal model of annulus fibrosus (AF) partial defect for the repairing of interver?tebral disc (IVD) defect. Methods Image J 1.46r software was used to measure the T12/L1-L6/S1 intervertebral height in ovine lumbar spine X-ray films. AF thickness was measured by axial split disc. A 11 blade was used to make a trapezoid de?fect of upper bottom 3 mm, lower bottom 5 mm, height 5 mm and thickness 3 mm, whose lower bottom toward the nucleus pulposus (NP) in the left front of ovine lumbar IVD in vitro. The minimally invasive lateral approach was used to make the same type of trapezoid defect in the left front of the ovine lumbar IVD in vivo. The trapezoidal defect length of the axial divid?ing disc was measured, AF and a small amount of NP from trapezoidal defect in IVD were weighed, and the production of trapezoidal defect in IVD was evaluated. Results The lumbar intervertebral space height of ovine was (4.45 ± 0.28) mm. There were significant differences in the thickness of AF (4.08±0.50) mm , thickness (3 mm) and height (5 mm) of trapezoidal defect (P0. 05). The weights of the AF and NP taken out from ovine lumbar IVD in vitro and in vivo were (0.162 ± 0.011) g and (0.166 ± 0.014) g, and there was no significant difference between them (P > 0.05). Conclusion Through the operation of minimally invasive lateral approach, the method of making a trapezoidal defect in the experiments can establish animal model of AF partial defect, which meets the requirements for the repairing of IVD defect, and is simple, safe and reliable.
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Objective To assess the prospect of integrated biphasic silk fibroin scaffold made by annulus fibrosus-nu?cleus pulposus tissue engineering in application as integrated intervertebral disc(IVD). Methods An integrated annulus fi brosus-nucleus pulposus(AF-NP)biphasic scaffold was made by silk fi broin using two different uncomplicated methods which were paraffin spheres-leaching method(outer AF phase)and phase separation method(inner NP phase). The scaf?fold was investigated by general observation, stereomicroscope and scanning electron microscopy(SEM). Its pore size, poros?ity, and compressive elastic modulus were determined. AF and NP cells were isolated from rabbit IVD and seeded into the corresponding phase of the scaffold respectively. The cell-scaffold complex was cultured for 48 hours. The biocompatibility of the scaffold was evaluated by SEM, live/dead staining while CCK-8 assay was used to assess cell proliferation. Results Stereomicroscope and SEM showed that AF phase and NP phase integrated perfectly without cross-linking. Both phases pos?sessed highly interconnected porous structure [pore size of AF and NP phase were(220.0±23.1)μm and(90.0±17.8)μm, re?spectively] and highly porosity(AF and NP phase were respectively 91%and 93%). In addition, this silk biphasic scaffold had impressive mechanical properties(150.7 ± 6.8)kPa. SEM revealed that disc cells attached to regions of pore walls, dis?tributed uniformly and secreted extracellular matrix. Live/Dead staining and cell count kit-8(CCK-8)analysis showed that the silk composite scaffold was non-cytotoxic to disc cells. Conclusion This silk biphasic AF-NP scaffold has satisfied pore size, porosity, biomechanical properties and biocompatibility, so it is ideal candidate for IVD tissue engineering.
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Objective To investigate the application of PKH26 and molecular light imaging system in cartilage en?gineering. Methods Canine chondrocyte was labeled by fluorescent dye PKH26 and seeded into the porous cartilage acel?lular matrix scaffold. The cells/scaffold constructs were cultured in vitro for 1 week. Then the constructs were implanted into the dorsal pocket of nude mice. We utilized a molecular light imaging system to macroscopically observe cells/scaffold con?structs in vivo with fluorescence at the 4th weeks, and compared with X-rays taken at the same position. The fluorescence im?ages were compared with the immunohistochemical and immunofluorescent results of cartilage-like tissue in vivo. Results Luminescent images were acquired at the 4th weeks, a red color enhanced overlay of the luminescent image over X-ray photo?graphic image demonstrated the location of the implants and the cell viability and cell growth on porous CACM scaffold in vivo were very well. Histological results show that the safranin O, anti-collagenⅡimmunohistochemistry and toluidine blue stain of cartilage-like tissue is positive. Immunofluorescence examination demonstrated chondrocytes in the constructs whitch is showen red fluorescence, and anti-collagenⅡimmunofluorescent staining was showen in green while the overlap?ping image is showen in yellow. Conclusion This study outlines an applicable non-destructive method to evaluate cell growth in tissue engineering constructs in vivo using PKH26 and molecular light imaging system.