Influence Of Mechanical Stimulation On The Annulus Fibrosus Cells Seeded On Circumferentially Oriented Silk Fibrous Scaffolds

Objective:1 To investigate the feasibility of fabricating oriented silk fibroin(SF) scaffold by wet-spinning for annulus fibrosus(AF) tissue engineering. 2 To explore the feasibility of constructing tissue engineering annulus fibrosus(TE-AFs) by oriented SF scaffold with rabbit annulus fibrosus cells(AFs) in vitro. 3 To investigate the influence of dynamic mechanical stimulation on the annulus fibrosus cells(AFC) seeded on silk scaffolds.Methods:1 A new oriented SF scaffold was fabricated by innovative wet spinning strategy using silk fibroin solution. Stereomicroscope and SEM was used to observe the surface morphology and fiber diameter of scaffold. A micro-material mechanical testing system was used to analyze the compressive elastic modulus(Young’s modulus) of the scaffold. 2 AF cells were isolated from rabbits and were seeded on the scaffold, then culture for a period of time. The cell viability, attachment, proliferation and infiltration were evaluated by SEM, live/dead and DNA contents. After 1, 7 and 21 days of culture, AF cells secrete ECM was evaluated by quantitative biochemical analysis. The mechanical properties of tissue-engineered AF were evaluated. 3 The TE-AFs were loaded into a biodynamic bioreactor with a dynamic cyclic compression stimulation, which was controlled by computer-control system. The TE-AFs received a sinusoidal, dynamic compression of 5, 10, 15 and 20% strain at 1 Hz for 2 hours per day for 3, 7 and 14 days. After fixed, samples were cuted for histological staining, and AFCs related ECM was evaluated by the quantitative analysis of total DNA, proteoglycan, collagen I and II. The compression elastic modulus and height of TE-AFs were compared in different groups.Results:1 Stereomicroscope and SEM results showed that the scaffold was milky white, hollow circular, diameter 4 mm, diameter 8 mm, 2 mm high. SF fibers were banded and ring parallel orientation, and has smooth surface, good uniformity. The fiber diameter was(51.06±9.65) μm, the porosity of scaffold was(60.37 + 0.08)%.2 Stereomicroscope showed that the color of TE-AFs were deepen with time growing. SEM showed cell adhesion on the scaffold, and the secretion of extracellular matrix. Cell culture experiments demonstrated that this scaffold could support AF cellattachment, proliferation and infiltration. Histological, immunohistochemical staining, biochemical quantitative analysis and total DNA content showed that the AF cells inside scaffolds could spread along the microfiber direction and secrete AF-related extracellular matrix which also oriented along the fiber direction. As a result, the compressive properties were enhanced with increasing culture time. 3 Stereomicroscope showed that the colors of TE-AFs in all groups were deepen with time growing. After 14 days, 5%, 10% and 15% strain showed the deepest color. HE staining showed of cell proliferation and secretion were increased with time in different groups, and after stimulation for 14 days, cell proliferation and secretion seemed like more in 5%, 10%, 15% strains. Toluidine blue and safranin O staining showed GAG secretion increased with the time increased, and after stimulation for 14 days, gag accumulation was seen in 5%, 10%, 15% strains groups. Type I and type II collagen staining of collagen showed cell secretion increased with time, and after stimulation for 14 days, obvious collagen accumulation were oberseved in 5%, 10% strain groups. Quantitation of collagen proved the maximum collagen secretion was in 10% strain group after stimulation for 14 days. Quantitation of GAG proved TE-Afs in 15% strain group secreted maximum GAG, but 10% strain group was no significant difference with 15% strain group. The maximal total DNA content was seen in 5% strain group. Compression elastic modulus achieved maximum in 20% strain group, but there was no statistical difference between the 15% strain groups and 20% strain group. The height of TE-AFs did not change after mechanical stimulation for 14 days.Conclusion :1 Wet-spun oriented SF fibrous scaffold has suitable porosity and cell compatibility and mechanical properties, is appropriate as an alternative for IVD tissue engineering. 2 The oriented SF fibrous scaffold holds great potential regeneration of AF, which mimicking native AF tissue. 3 AFCs proliferation and secretion is related to mechanical environment closely. Suitable mechanical stimulation is a positive factor for AFCs, excessive compression can accelerate cell apoptosis. Additionally, the maximum collagen concentrations were produced under 10% strain, the maximum GAG content and elastic modulus were produced under 15% strain, and the maximum DNA content was produced under 5% strain.