Biomimetic Micro-/nano-fibrous Scaffolds Inspired By Extracellular Matrix For Spinal Dura Mater Regeneration

Chapter I Hierarchical micro/nanofibrous bioscaffolds for structural tissue regenerationObjective Biomimetic scaffolds with hierarchical micro/nanostructures could provide nanoscale contact points for cells growth,and guide cell behaviors to generate hierarchical extracellular matrix for tissue regeneration and remodeling.In this study,a biomimetic designed micro/nanoscaled scaffold with integrated hierarchical dual fibrillar components is fabricated in order to repair dura mater and prevent the formation of epidural scars via collagen molecule self-assembly,electrospinning,and biological interface crosslinking strategies.Methods First,the electrospun fibrous membranes fabricated using poly L lactic acid(PLLA)and silk fibroin(SF)was prepared.After the polydopamine(PDA)coating was performed to modify the SF and PLLA electrospun membranes,acidic collagen solution self-assembled on PDA modified electrospun membranes to form collagen nano-fibers.The poly dopamine coating(PDA the interface reaction)technology can promote forming the stable covalent binding between natural or synthetic electrospun fibers and natural type I collagen self-assembled nanofibers,and maintain the stability of micro / nano structured scaffolds.The physicochemical property of different scaffolds was characterized via Scanning Electron Microscope(SEM)、 Atom Force Microscope(AFM)、 X-ray Photoelectron Spectroscopy(XPS)and water contact angle(WCA)measurement.As for in vitro assessment,viability and proliferation of fibroblast on the scaffold was assessed via Cell Counting Kit-8.Immunofluorescence staining was conducted to visualize cell spreading and distribution,along with expression of COL-Ⅰ,integrin-β1 and α-SMA.A rabbit lumbar duraplasty model was conducted to assess the in vivo performance of scaffold and SF based scaffold was employed to treat the dura defect considering its superior biocompability.Animals was devided into four groups,SF-PDA-COL group,SF-PDA group,SF group and control group,with 12 rabbits in each group.The regeneration of dura mater and formation of scar tissue was assessed through magnetic resonance image(MRI),macroscopic observing,histological evaluations and corresponding immunohistochemical staining.Results Micro/nano double-network hierarchical structure composed of SF or PLLA micro-scale fibrous network and self-assembled collagen nano-scale fibrous network could be observed via SEM and AFM,proving significant difference compared with single micro-scale net of SF and PLLA.Dopamine surface modification is not only responsible for covalent integration between collagen nanofiber and microfiber as shown by XPS data,but also found to significantly improve the hydrophilicity of scaffolds as indicated by measurement of water contact angle.Fibroblasts cultured on micro-nano hierarchical scaffold exhibit superior spreading and distribution of cytoskeleton,with proliferation rate and cell spreading area also significantly elevated when compared with bare microfibrous scaffold.Immunofluorescent staining indicated that the triple helix in collagen nanofibers contributing to activate integrin-β1 transmembrane receptors of fibroblasts,and promoting more deposition of collagen I.The soft and smooth surface of the hierarchical micro/nano fibrous scaffolds could inhibit the differentiation of myofibroblasts.In vivo duraplasty model further proved that the hierarchical micro/nano fibrous scaffolds could not only promote the regeneration of dura mater,but also suppress scar tissue formation.Conclusion We have developed a highly biomimetic hierarchical micro/nano-fibrous scaffold which is able to realize the interaction between cell and material matrix and further modulation on cell proliferation and differentiation activity.The scaffold could be employed for promoting the structural regeneration and remodeling of dura mater and mitigating epidural scar tissue formation.Chapter II Microstructural heterogeneity of fibrous scaffold promotes dura healing and suppresses scar adhesionObjective The main aim of this study is to investigate the influence of physicochemical property of electrospun fibers such as topological structure,chemical composition and fiber density on cell behaviors.Furtherly,we will fabricate a heterogeneous double-layered scaffold which inner layer composed of aligned or random SF/COL-Ⅰ nanofibers and outer layer consisting merely of random SF fibers.Through regulating topological structure and chemical composition of inner layer fibers,it will promote the natural healing of dura mater.While the density of SF electrospun fibers in the outer layer of double-layered scaffold plays an important role in inhibiting adhesion and ingrowth of fibroblasts and reducing the formation of epidural scar.Methods Various ratios of SF/COL-Ⅰelectrospun membranes with random(RSCF)or aligned(ASCF)topological feature was fabricated,meanwhile silk fibroin electrospun scaffolds with different fiber density were also prepared.Scanning electron microscope(SEM)was employed to observe the morphological and structural characters of scaffolds with different parameters.Flow cytometry was applied to evaluate the expression of activated integrinβ1 receptor.SEM was also adopted together with cytoskeleton staining to characterize the adhesion,spreading,morphology and cytoskeleton remodeling of fibroblasts cultivated in different scaffolds.The proliferation rate of fibroblasts on scaffold was determined by cell counting kit-8(CCK8).Immunofluorescence staining was conducted to visualize vinculin,α-SMA,collagen-I and YAP on different scaffolds in order to observe the formation of focal adhesive,fibroblast differentiation,deposition of extracellular matrix collagen typeⅠand activity of YAP(Yes-associated Protein).We fabricated heterogeneous double-layered scaffolds which inner layer composed of aligned or random SF/collagen nanofibers and outer layers with random silk fibroin fibers.A rabbit laminectomy and duraplasty model was used to evaluate the effectiveness of the double-layered hierarchical scaffolds with anisotropic properties to promote dura mater regeneration and suppress scar formation.Animals were divided into following groups:(1)Normal group,(2)Control group(without scaffold implanted),(3)D-RSCF/RSF group(Dura defect implanted using double-layered scaffolds with random inner layer)(4)D-ASCF/RSF group(Dura defect implanted using double-layered scaffolds with aligned inner layer).Magnetic resonance image(MRI)and Immunohistochemistry staining were employed at 8 weeks post-surgically to assess the regeneration of dura mater and the formation of epidural scar tissue.Results Smooth surface and homogeneous fiber distribution could be observed via SEM on all groups of electrospin scaffolds.ASCF exhibited aligned fiber distribution while RSCF shown disorderly distributed fibers and interwoven into network.Fibroblasts cultured on ASCF shown elongated and spindle morphology,cytoskeleton arrangement,and growth with its long axis in line with the orientation of aligned fiber.While fibroblasts seeded on RSCF displayed larger spreading area with the formation of lamellipodia.The cell spreading area increase in both ASCF and RSCF with increase of collagen ratio,however,at the same ratio of collagen,ASCF could significantly reduce the cell spreading area when compared with RSCF.As for another factor,with the increase of fiber density on RSF scaffold,morphology of fibroblast adhered on the scaffold gradually turned from flat,well-spread into round or oval morphology,together with significant reduction on adhesive cell numbers and spreading area.Quantified data from CCK8 test implied that nanofibrous collagen played an important role in promoting proliferation of fibroblast.On the other hand,both anisotropic topology and higher fiber density was found to compromise proliferation activity of fibroblast.Immunofluorescence staining verified that oriented topology and chemical composition had a significant effect on cytoskeleton arrangement,cell adhesion and distribution of focal adhesion.Topological feature was thought to be responsible for promoting aligned deposition of collagen I of extracellular matrix and tend to preserve the static phenotype of fibroblast,which could lead to the suppressed differentiation of fibroblast into myofibroblast.The formation of the active α-SMA stress fibers in myofibroblasts is closely related to the nuclear localization of YAP.In vivo model further verified heterogeneous double-layered scaffold(D-ASCF/RSF group)not only promote dura mater regeneration,but also prevent epidural scar formation at the same time.Conclusion The SF/COL-Ⅰcomposite fiber matrix established in this study could not only promote cell proliferation via its aligned topology and chemical composition but also tend to preserve the static phenotype of fibroblast.Proliferation of fibroblast could be further modulated via increasing density of silk fibroin fibers.Taken together,the heterogeneous double-layered scaffold could not only guide the regeneration of dura mater,but also reduce the epidural scar formation.