Fabrication Of A Novel Electrospun Aligned Nanoyarn/ Nanofibrous Three-Dimensional Scaffold For Annulus Fibrosus Tissue Engineering

The high postoperative recurrence of intervertebral disc herniation usually results from defects of annulus fibrosus (AF) and limited repairing potential. It is proposed that tissue-engineering technology can effectively repair the ruptured AF. Ideal tissue-engineered functional replacements for damaged AF would support both neo-tissue formation and the restoration of biomechanical function. In combination with cells, biological materials such as scaffolds play a decisive role in AF tissue engineering. These scaffolds commonly provide a synthetic extracellular matrix (ECM) environment and a three-dimensional (3D) template for tissue regeneration. Electrospinning is an efficient and economical method to prepare scaffolds for tissue engineering applications. Nanofibers fabricated via electrospinning are similar in size to the elements of the native ECM. Microscale and nanoscale fibers improve cell proliferation and elicit superior metabolic and matrix forming activities. Electrospun nanofibrous scaffolds have been applied to engineer various musculoskeletal tissues. In a typical electrospinning process, electrospun polymer nanofibers are collected on a stationary grounded plate to form randomly oriented mats. The random nanofibrous scaffolds exhibit isotropic mechanical properties. However, annulus fibrosus that bears large mechanical loads in vivo and each lamella of the multi-lamellar AF consists of highly aligned collagen fibers which exhibit anisotropic mechanical properties. Methods have been devised to induce the alignment of electrospun fibers in nanofibrous scaffolds to mimic the ultrastructure of the aligned collagen fibers. The most common method for generating aligned fibers is to collect fibers on a rapidly rotating mandrel. The aligned nanofibrous scaffolds could recapitulate anisotropic mechanical properties and direct cell adhesion. Furthermore, the ability of cells to infiltrate nanofibrous scaffolds is limited due to the tightly packed nanofibers and small pore size. In the present study, we first characterized the organization and mechanical properties of the P(LLA-CL) aligned nanofirous scaffolds and evaluated cell proliferation and infiltration in them. Then, we introduce a novel electrospun aligned nanoyarn/nanofibrous scaffold composed of aligned nanoyarns and random nanofibers which were fabricated via electrospinning using a two-collector system. In addition, we characterized the organization and mechanical properties of the novel scaffolds and evaluated cell proliferation and infiltration in them. The results indicate that the aligned ANYNFS is a viable option for use in tendon tissue engineering.PARTI Fabrication of Electrospun AlignedPoly(L-Lactide-co-Caprolactone) Nanofibrous Scaffolds for Annulus Fibrosus Tissue EngineeringObjective:To fabricating electrospinning poly(L-Lactide-co-Caprolactone) aligned nanofibrous scaffolds for annulus fibrosus tissue engineering and investigate the structural morphologies, mechanical properties of the scaffolds and the cell behaviors on the scaffolds.Methods:Electrospinning aligned nanofibers were collected by a high-speed mandrel. The morphological feature of the scaffold was observed by a scanning electron microscopy (SEM). The tensile mechanical properties of the scaffolds were assayed by universal materials tester. Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured on the scaffolds, and the viability of the cells was determined using the Cell Counting Kit-8. Cell morphology was imaged by a SEM. Electrospinning random nanofibrous scaffold was served as a control.Results:The morphological images revealed that the nanofibers of the aligned nanofibrous scaffolds were highly oriented spread. The mechanical tests indicated that the tensile properties of the scaffold were reinforced in the parallel direction of nanofibers. Biocompatibility results showed that the BMSCs cultured on the aligned nanofibrous scaffolds were aligned distributed and elongated along the direction of nanofibers.Conclusion:With their superior physical properties and ability to replicate structure of the single lamella annulus fibrosus, the electrospun aligned P(LLA-CL) nanofibrous scaffolds warrant investigation as an option for annulus fibrosus tissue engineering.PART2 A Novel electrospun Aligned Nanoyarn/Nanofibrous Scaffold for Annulus Fibrosus Tissue EngineeringObjective:In the present study, we have fabricated a novel electrospun aligned nanoyarn/nanofibrous scaffold (ANYNFS) which mimics the structure of single lamellar AF. In addition, we characterized the organization and mechanical properties of the scaffolds and evaluated cell proliferation and infiltration in them.Methods:ANYNFSs were manufactured by electrospinning using the novel modified two-collector system. We used SF/P(LLA-CL) to fabricate the scaffolds and typical electrospinning methods to fabricate random and aligned nanofibrous scaffolds as controls. The surface morphologies of the scaffolds were examined using a scanning electron microscope and the cross-sectional microstructures of the specimens were examined using a transmission electron microscope. Tensile tests were performed using an optical microscope and digital image correlation testing device. Images of the cell morphologie on the scaffolds were acquired with a laser scanning confocal microscope and a scanning electron microscope. The viability of the BMSCs on the scaffolds was determined using the commercially available Quant-iTTM Pico Green(?) dsDNA assay. To evaluate the extent of cell migration into the scaffolds, cellular scaffolds were collected for histological analyses.Results:This novel 3D ANYNFSs composed of aligned nanoyarns and random nanofibers were fabricated via electrospinning using a two-collector system. The aligned yarns and random nanofibers of the ANYNFS provided a 3D structure with larger pores and greater porosity. The mechanical testing results indicated that the tensile properties of the ANYNFS were reinforced in the direction parallel to the nanoyarns. The structure of the aligned nanoyarns in the scaffold contributes to the improvement in the tensile properties. According to the Quant-iTTM Pico Green(?) dsDNA cell proliferation assay, the cell proliferation rate on the ANYNFS was significantly higher than those on the random and aligned nanofibrous scaffolds. The cells were randomly spread when cultured on the random nanofibrous scaffolds. However, the cells were highly evenly distributed and elongated along the direction of the fibers on the aligned nanofibrous scaffold. Interestingly, the BMSCs on the ANYNFSs exhibited an aligned and elongated growth pattern along the nanoyarns but diversely distributed among the yarns. Histological examination revealed that only one cell sheet formed on the surface of the aligned and random nanofibrous scaffolds, whereas the MSCs infiltrated into the ANYNFS.Conclusions:A novel 3D SF/P(LLA-CL) ANYNFS composed of aligned nanoyarns and random nanofibers was fabricated via electrospinning using a two-collector system. These scaffolds exhibited higher porosity than SF/P(LLA-CL) random and aligned nanofibrous scaffolds. Biocompatibility analysis demonstrated that the ANYNFS yielded improved cell proliferation and regulated cell morphology. In addition, the ANYNFS exhibited increased mechanical strength in the direction parallel to the nanoyarns. The ANYNFS mimics the structure of AF extracellular matrix and has good mechanical strength. More remarkably, the 3D structure of the ANYNFS enabled cell infiltration. This study indicates that the ANYNFS represents a balance between porosity and mechanical properties satisfying the requirements for annulus fibrosus tissue engineering applications.