Construction And Evaluation Of Tissue-engineered Composites Of Annulus Fibrosus And Nucleus Pulposus

Objective 1) To verify the feasibility of silk biphasic scaffold as a potential candidate for intervertebral disc(IVD) tissue engineering. 2) To fabricate oriented PCL microfiber scaffold by wet-spinning and construct tissue engineered annulus fibrosus compounding with AF cells of rabbit. 3) To explore the feasibility of construction of tissue engineered IVD based on oriented PCL microfiber/alginate hydrogel compounding with disc cells of rabbit.Methods 1) An integrated annulus ?brosus-nucleus pulposus(AF-NP) biphasic scaffold was fabricated with silk ?broin using two different uncomplicated methods. The scaffold was investigated by general observation, stereomicroscope, scanning electron microscopy(SEM) and compressive elastic modulus. The biocompatibility of the scaffold was evaluated by SEM, HE staining, live/dead staining and CCK-8 assay. 2) A new circumferentially oriented PCL microfiber scaffold was fabricated by innovative wet spinning strategy. The scaffold was evaluated by stereomicroscope, SEM and micro-CT. AF cells were isolated from rabbits and were seeded on the scaffold. The cell viability, attachment, proliferation and in?ltration were evaluated by SEM, live/dead, phalloid staining, Di I fluorescence labeling and MTT assay. After 1, 7 and 21 days of culture, AF cells secrete ECM was evaluated by quantitative biochemical analysis and the expression of AF-related ECM genes(aggrecan, Col Iα1 and Col IIα1) were examined. The mechanical properties of tissue-engineered AF were evaluated. 3) We construct a tissue-engineered IVD composed of circumferentially oriented PCL microfibers as AF scaffold and alginate hydrogels as NP scaffold and seeded with PKH26-labeled AF and PKH67-labeled NP cells, respectively, and then implanted subcutaneously in nude mice to monitor the metabolic activity of IVD cells by in vivo fluorescence imaging. The components and mechanical property of engineered AF-NP tissues was evaluated by histology, immunostaining and mechanical analysis.Results 1) Stereomicroscope and SEM showed that the AF and NP phase integrated perfectly. Both phases possessed highly interconnected porous structure(AF: 220.0±23.1 μm and NP: 90.0±17.8 μm) and highly porosity(AF:91% and NP: 93%).In addition, this scaffold had impressive mechanical properties(150.7±6.8) k Pa. SEM and HE staining revealed that disc cells attached to regions of pore walls, distributed uniformly and secreted ECM. Live/dead staining and CCK-8 analysis showed that the silk scaffold was non-cytotoxic to disc cells. 2) Stereomicroscope, SEM and micro-CT results showed that PCL micro?ber was smooth and regular shape. The micro?ber diameter was 16.13±2.77 μm and the porosity of scaffold was 69.33±6.67 %. Cell culture experiments demonstrated that this scaffold could support AF cell attachment, proliferation and in?ltration. Histological, immunohistochemical staining, biochemical quantitative analysis and RT-PCR showed that the AF cells inside scaffolds could spread along the microfiber direction and secrete AF-related extracellular matrix which also oriented along the microfiber direction. As a result, the compressive and tensile properties were enhanced with increasing culture time. 3) Gross morphology of tissue-engineered IVD was similar disc tissue. Strong red fluorescence in AF phase and weak green fluorescence in NP phase was observed by in vivo fluorescence imaging after 8 weeks culture. Fluorescence images showed that PKH26-labeled AF with red fluorescence were oriented along the direction of the microfibers and PKH67-labeled NP with green fluorescence were round or spindle shape after 4 and 8 weeks culture. No apparent cell migration and mixing in the junction transitional region.Complexes were positive in safranin-O staining. AF phase were positive in type I collagen immunohistochemistry and NP phase were positive in type II collagen immunohistochemistry staining, which were similar to native IVD. The compressive were enhanced with increasing culture time.Conclusion 1) Silk biphasic AF-NP scaffold is appropriate as an alternative for IVD tissue engineering. 2) Wet-spun microfibrous oriented scaffold holds great potential regeneration of AF, which mimicking native AF tissue. 3) Construction of tissue engineered IVD based on PCL microfiber/alginate hydrogel compounding with disc cells of rabbit is feasible.