| Intervertebral disc herniation is one of the main causes of neck and back pain,as well as and radiating pain in the limbs.A small number of patients who fail conservative treatment need to be relieved by surgical treatment.Disc discectomy is one of the most commonly used surgical interventions to relieve the clinical symptoms of these patients.However,about 20% of patients still have poor outcome postoperatively because of the recurrence of disc herniation.The recurrence rate after surgery is related to the ineffective repair of the annulus fibrosus defect.In recent years,the tissue engineering technology for annulus fibrosus has attracted the attention of many researchers and is expected to become a new strategy for effectively repairing the annulus fibrosus.An ideal tissue engineering scaffold for annulus fibrosus repair not only needs to be able to fill the defect,replace the damaged tissue,but also can promote the formation of neo-tissue and form a functional entity.It keeps a difficult issue to construct a "biomimetic" scaffold based on the complex structure and mechanical prope-rties of the annulus fibrosus.The stress on the annulus fibrosus is complicated,and the internal collagen fibers are highly oriented,indicting its obviously anisotropic mechani-cal property.In order to simulate the mechanical feature,aligned nanofiber scaffolds with oriented,highly alignment nanofibers can be obtained through electrospin-ning.However,the infiltration rate of cells is very limited because of the dense packed fibers.In order to improve the ingrowth rate of BMSC in the aligned nanofibrous scaffold,we attempted to construct an aligned nanoyarn scaffold(AYS)by conjugated electrospin-ning.Mechanical properties and interactions with bone marrow mesenchymal stem cells(BMSC)in AYS were compared with conventional aligned nanofiber scaffold(AFS)and random nanofiber scaffold(RNS).In order to simulate the different mechanical and struct-ureal characteristics of the inner and outer layers of the annulus fibrosis and to further optimize the cell infiltration efficiency,we constructed a three-dimensional porous nano-fiber scaffold(3-DPS)through electrospinning and freeze-drying technique,and eventually a hybrid scaffold(HS)was prepared by combining AYS and 3-DPS together.Cell prolifer-ation,ingrowth,secretion of extracellular matrix,and differentiation towards annulus fibro-sus cells from BMSC in different scaffolds were evaluated.Besides,the association and underlying mechanism between different cell morphology and differentia-tion of BMSC in different scaffolds were explored.Finally,we evaluated the outcomes of the HS in repair-ing an annulus fibrosus defect in the beagle cervical annulus fibrosis defect model in vivo.Our results show that the AYS is consisted of highly oriented nanoyarn fibers and has anisotropic mechanical property.The maximum tensile strength in the direction of fiber alignment is slightly lower than that of AFS,and the infiltration rate of BMSC is higher in AYS than in AFS.A double-layered HS is constructed by combining 3-DPS with AYS,which has different pore sizes from nanometer to micrometer level,and can effectively promote the ingrowth of BMSC.The 3-DPS has a good elastic and axial compressive properties,and can promote the proliferation of BMSC and secrete extracellular matrix.BMSC show different differentiation characteristics in different scaffolds.It is confirmed that BMSC-scaffold could effectively repair the annulus fibrosus defects in beagle model in vivo,sustain the intervertebral height and reduce the loss of disc signal intensity,suggesting that it could be used as a promising tissue engineering scaffold for annulus fibular defect repair.Part I Fabrication and Evaluation of Electrospinning Aligned Nanofibrous Scaffolds for Annulus Fibrosus Tissue EngineeringObjective: Poly(L-Lactide-co-Caprolactone)and gelatin were used to construct an AYS by conjugated electrospinning and to analyze the morphology and mechanical properties of the scaffold.The proliferation and ingrowth of BMSC on the scaffold were observed,and the feasibility of its application to a monolayer annulus fibrosis tissue engineering scaffold was evaluated.Methods: The AYS was fabricated by conjugated electrospinning,and the structural characteristics of the scaffold were observed by scanning electron microscopy(SEM)and the tensile properties of the scaffolds were tested.BMSC were seeded on the scaffolds and cell proliferation was analyzed by CCK-8 assay while cell ingrowth was evaluated by histological staining.RNS and AFS were used as control groups.Results: The P(LLA-CL)/gelatin AYS had obvious anisotropic mechanical properties with highly oriented nanofibers.The AYS had a lower maximum tensile strength in the direction of fiber alignment than conventional oriented nanofibers(P<0.05).CCK-8 assay results showed that: the proliferation rate of BMSC in AYS was higher than other scaffolds at different time point(P<0.05).BMSC could be stretched along the direction of nanofibers.The infiltration depth of BMSC in the AYS was deeper than in other scaffolds(P <0.05).Conclusions: The AYS has highly oriented fiber structure and anisotropic mechanical property.Although its maximum tensile strength in the fiber alignment direction is slightly lower than that of the oriented nanofiber scaffold,the ingrowth rate of BMSC in the scaffold is improved.BMSC can be induced to stretch along the fiber direction,suggesting that it can be applied to construct a monolayer annulus fibrosus tissue engineering scaffold.Part II Fabrication of Nanoyarn/three-Dimensional Porous Nanofiber Scaffold for Annulus Fibrosus Regeneration and Its Influence on TheBehaviors of Bone Marrow Mesenchymal CellsObjective: 1)To construct a double-layered composite scaffold to simulate the different characteristics of the microstructure and mechanical properties of the annulus fibrosus;2)To optimize the efficiency of cell infiltration in the scaffold furtherly;3)To evaluate the proliferation rate and secretion of extracellular matrix of BMSC in different scaffolds and to explore the impact and underlying mechanism of different scaffolds on the differentiation of BMSC toward annulus fibrosus cells.Methods: Three-dimensional porous nanofiber scaffold(3-DPS)was prepared by electrospinning,homogenization,and freeze-drying,while hybrid scaffolds(HS)was constructed by combine 3-DPS and AYS together.AFS and AYS were compared as control groups.SEM was used to observe the surface morphology of these scaffolds.Mechanical test was performed to evaluate the compression performance of 3-DPS.Rat BMSC were seeded on the scaffolds,and cell proliferation was evaluated by CCK-8 kit and Quant-iT? PicoGreen? dsDNA assay.Histological staining was used to analyze the growth of cells in the scaffolds.SEM and confocal microscopy were used to observe the morphology of the cells in the scaffolds.Glycosaminoglycan and hydroxyproline assay kits were used to detect the extracellular matrix secreted by BMSC.Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK,chondrocyte-like phenotypes,and downstream signaling molecules ERK1/2 and p38 after ROCK inhibitors intervention.Results: AFS was composed of oriented nanofibers,while AYS was composed of highly oriented nanofibers and nanoyarns.The 3-DPS was consisted of many short nanofibers and contained different sizes of pores.Mechanical tests show that 3-DPS had a good elasticity and axial compressive properties.BMSC were seeded into different scaffolds for 14 days,the number of BMSC in HS was greater than that of the other three scaffolds(P<0.05).The depth of ingress of BMSC in HS was significantly greater than that of AFS and AYS(P<0.05).BMSC were appeared significant difference in the cytoskeletal system in different morphological scaffolds observed by scanning electron microscopy and confocal microscopy.After three weeks of chondrogenic induction in vitro,BMSC exhibited different differentiation and produced diverse levels of extracellular matrix in these scaffolds.HS facilitated the induction differentiation of BMSC into the inner annulus fibrosus cells with higher expression level of COL II,Aggrecan and produced more glycosaminoglycans(P<0.05),while AYS was more favorable for inducing differentiation of BMSC into the outer annulus fibrosus cells with higher expression of COL I,and synthesis of more hydroxyproline(P<0.05).The ROCK signaling pathway may participate in this process by regulating the expression of ERK1/2.Conclusions: The three-dimensional porous structure layer in the HS has pore structures with different sizes from nanometer to micron level,and the cell growth rate of BMSC was effectively promoted.The 3-DPS has good elasticity and axial compressive property,and promote the proliferation and secretion of extracellular matrix of BMSC.BMSC show diverse differentiation characteristics in different scaffolds.HS can induce differentiation of BMSC into inner annulus fibrosus cells,while AYS can induce differentiation of BMSC into the outer annulus fibrosus cells.ROCK signaling pathway may participates in this process by regulating downstream ERK signaling.The fabrication of the scaffolds and regulation of the ROCK signaling pathway may lay the foundation for the further construction of multi-layer annulus fibrosus tissue engineering scaffolds.Part III Evaluation of Bone Marrow Mesenchymal Stem Cells Combined with Hybrid Scaffold for Repairing Annulus Fibrosus Defects in Beagle Dog’s Model in VivoObjective: To conducted an annulus fibrosis defect model in the cervical intervertebral disc of Beagle dog and to evaluate the performance of BMSC combined with HS for repairing annulus fibrosus defects in vivo.Methods: BMSC were harvested,cultured and seeded into HS to construct a cell-scaffold complex.A Beagle dog model of annulus fibrosis defect in cervical disc was constructed.The cell-scaffold complex was implanted in the defect.Cervical MRI and histological staining of the cervical disc were performed at 4 and 12 weeks after operation.Intervertebral disc height index and signal intensity ratio of intervertebral discs on T2-weighted MRI were evaluated.The HS bare scaffold and the defect group were used as controls.Results: BMSC-HS complex was successfully constructed.After implantation for 12 weeks,the signal intensity ratio and the relative disc height index of the intervertebral disc in the control group decreased compared with the BMSC+HS group and the HS group(P<0.05),The signal intensity ratio of intervertebral disc in HS group was slightly lower than that of BMSC+HS group,but the difference was not statistically significant(P>0.05),while the relative disc height index of intervertebral disc was lower than that of BMSC+HS group,and the difference was statistically significant(P<0.05).Histological observation revealed that the scaffold can effectively fulfill the annulus fibrosus defects,and cells in the outer layer of the scaffold(AYS layer)grow along the direction of the fibers or in the large pore in the inner layer of the scaffold(3-DPS layer).Conclusions: Both the BMSC+HS complex and HS can block the annulus fibrosus defect,which can sustain the disc height and the signal intensity of intervertebral disc.The repair effect of BMSC+HS complex is better than that of HS.HS may become a potential tissue engineering scaffold for repair of annulus fibrosus defect. |
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