Fabrication And Research Of Poly(L-lactide-co-Caprolactone)/Silk Fibroin Nanoyarn Scaffold For Tissue Engineered Annulus Fibrosus

Background:Disc degeneration disease (DDD), a main cause of lower back pain, is an endemicproblem and a big economic burden for the health care system. DDD not only bringsenormous social and economic burden, but also seriously affects people’s normal productionand life. The current main treatments of DDD include physics therapy, medicine and surgery.Physics and medicine treatments are symptom relieving, and surgery is mainly to removedegeneration of intervertebral disc tissue, but which do not address underlyingproblems—biological and structural deterioration of the disc. However, tissue engineeringcan repair the natural tissue for the treatment of intervertebral disc degeneration andprovides a new thought of exploration. Annulus fibrosus (AF) tissue engineering is one ofthe keys to prepare the intervertebral disc tissue engineering. Scaffolds for tissueengineering are the basis for the repair of the annulus fibrosus. Thus, the scaffold materialselection and preparing methods have important biological significance in the annulus tissueengineering research.Objective:To mimic the shape and structure of the extracellular matrix of the annulus fibrosus,Poly(L-lactide-co-caprolactone)(P(LLA-CL))/silk fibron nanoyarn scaffold withthree-dimensional orientation and large porosity was prepared by a dynamic liquidelectrospinning method. Physical and chemical properties of the scaffold were evaluated. Invitro studies were performed to evaluate the biological properties of the tissue-engineeredannulus fibrosus.Methods:1. P(LLA-CL)/silk fibron random nanofiber scaffolds and aligned nanofiber scaffoldsas control groups were fabricated by traditional electrospinning method; a novel P(LLA-CL)/silk fibron nanoyarn scaffold with three-dimensional orientation and largeporosity was prepared by a dynamic liquid electrospinning method.These scaffolds were characterized in terms of fiber morphology, fiber diameter, poresize, porosity, fourier transforms infrared spectroscopy and mechanical properties.2. Rabbit annulus fibrosus cells (AFCs) isolated by two-step enzyme digestion methodwere cultured and stained with DAPI and phalloidine. They were observed under invertedmicroscope. AFCs were identified by phenotype identification after Ⅰ, Ⅱ collagenimmunohistochemical staining.3. AFCs were seeded on the P(LLA-CL)/silk random nanofibers,and orientednanofibers,and nanoyarn scaffold for tissue-engineered annulus fibrosus. After hours ordays of cell culture, the following items were test:After4hour,1,4, and7days of culture, the viabilities of AFCs on the samples and24-well tissue-culture polystyrene plates (TCP) control were determined using thecommercially available Cell Counting Kit-8(CCK-8; Beyotime). The morphology of AFCson the samples was observed by SEM.After4,7and14days of culture, attachment, proliferation and infiltration of AFC onthe scaffolds were examined by Hematoxylin and eosin stain (HE staining) and laserscanning confocal microscopy (LSCM).After7and14days of culture, the specific gene (type I collagen, type Ⅱ collagen,and Aggrecan) expressions of AFCs were assessed using Real-time polymerase chainreaction (PCR). The Hydroxyproline content in regenerative AF was determined with aHydroxyproline quantification kit. The mechanical properties of tissue-engineered AF wereexamined as well.Results:1. Physical and chemical properties of three different electrospinning P (LLA-CL)/silkfibrous scaffolds.(1) The surface of random nanofibrous scaffold presented compact reticular structureand random fibers arrangement. Surface of aligned nanofibrous scaffold presented compactreticular orientation arrangement. Surface of nanoyarn scaffold presented loose porousstructure and certain orientation arrangement.(2) The results indicated the average fiber diameter of the random and aligned nanofibrous scaffold was (460±75) nm and (471±77) nm, respectively. The average fiberand yarn diameter of the nanoyarns scaffold was (456±64) nm and (13.20±4.16) μm,respectively. And the pore size and porosity of the nanoyarn scaffold was (31.93±15.48)μmand (87.16±4.27)%,respectively. They were significantly better than those of the randomnanofibrous scaffold [(4.41±1.87)μm，(71.47±6.42)%] and the aligned nanofibrous scaffold[(4.41±1.87)μm，(70.7±6.45)%].(3) Fourier transform infrared spectrum analysis:FTIR analysis revealed the compositions of the random nanofiber, aligned nanofiber,and nanoyarn scaffolds. There were two main absorption peaks at1,653cm-1and1,552cm-1in pure collagen, which was corresponding to amide I and amide II, respectively. Therewere two representative absorption peaks at1,756cm-1and1,735cm-1, which wascorresponding to C-O stretching in poly(L-lactide) and poly(e-caprolactone), respectively.(4) Mechanical test results:The tensile strength and elastic modulus of the nanoyarn scaffold was (7.31±0.96) MPaand (70.58±10.66) MPa, respectively. They were significantly higher than those of therandom nanofibrous scaffold (P<0.05). But the other values were lower than those of thecontrol groups.2. Separation of the annulus digestive cells in monolayer culture.AFCs showed polygonal, irregular shape or spindle under laser scanning confocalmicroscopy (LSCM). Their nucleuses were dyed in blue color by DAPI and the celluarplasms were stained in red by phalloidine. Immunohistochemical staining results showedthe cells with positive stain of collagen type Ⅰ and collagen type Ⅱ.3. The observation of tissue-engineered AF morphology, cell compatibility tests,histologic assessment, the analysis of related gene expressions and the test ofbiomechanical:(1) Gross observation: morphologically similar to natural disc AF structure.(2) By day4, AFCs spread well on the random nanofiber scaffold and exhibitedpolygonal shape and random orientation. However, the AFCs seeded on the alignednanofiber and nanoyarn scaffold exhibited a spindle-shaped morphology and spread alongwith substrate fibers/nanoyarns. In addition, some cells were immgrated into the pores ofnanoyarn scaffold. By day7, all cell-seeded scaffolds were apparently with cell sheets and ECM secreted by AFCs.(3) CCK-8assay results showed that cell proliferation on the nanoyarn scaffold wasobviously higher than that on the random nanofibrous and aligned nanofibrous scaffolds (P<0.05).(4) H&E-staining and LSCM results showed that cellular activities on the random andaligned nanofibrous scaffolds were mainly confined to the dense upper layer. In contrast, thecells almost had infiltrated into the entire nanoyarn scaffold and the cells attached to theporous skeleton of the nanoyarn scaffold.(5) Real-time PCR detection results showed that the levels of the five tendon-specificECM genes expressed by the cells on/in the nanoyarn scaffold were significantlyupregulated when compared with those on the random and aligned nanofibrous scaffolds (P<0.05).(6) Mechanical test: After14days of cell culture, both the Young’s modulus and tensilestrength of the cell-seeded nanoyarn scaffold were significantly improved when comparedwith the unseeded nanoyarn scaffold. The Young’s modulus and tensile strength ofcell-seeded random and aligned nanofibrous scaffolds also increased when compared withthe unseeded random and aligned nanofibrous scaffolds, respectively (P <0.05).Conclusion1.Basing on traditional electrospinning method, a novel P(LLA-CL)/silk fibroin nanoyarn scaffold was prepared by a dynamic liquid electrospinning. This three dimensional porous scaffold had higher porosity and orientation. Elastic modulus and break strength in nanoyarn horizontal orientation was significantly enhanced when compared with random nanofibrous scaffold. It has a great potential application in AF tissue engineering.2. The yarn diameter of the nanoyarn scaffold is similar to native collagen fibers in AFtissue. The nanoyarn scaffold with higher orientation could mimic the native extracellularmatrix of AF tissue.3. The nanoyarn scaffold promoted the proliferation and immigration of AFCs whencompared with that of control groups. AFCs showed an oriented growth pattern andenhanced cell infiltration in the nanoyarn scaffold. The results of biocompatibility test,histological evaluation, ECM-related gene expression test and biomechanical test show thatit is feasible to construct tissue-engineered AF by AFCs and P(LLA-CL)/silk fibroin nanoyarn scaffold. This study lays an experimental foundation for the further constructionof tissue-engineered intervertebral disc.