Three-Dimensional Porous Scaffolds Based On Gelatin/Polylactic Acid Nanofibers For Articular Cartilage Tissue Regeneration

Trauma,metabolic diseases and joint infection may lead to articular cartilage defect,which is one of common clinical disease in the whole world.The self-repair capacity of damaged cartilage is limited due to its avascularity.There are some traditional techniques for repairing cartilage defect,such as joint debridement,microfracture,cartilage transplantation and so on.However,each of these techniques has deficiency.For example,cartilage defect may not be completely repaired with hyaline cartilage by microfracture,the source of cartilage transplantation donor is limited.Therefore,seeking reliable and effective methods for repairing cartilage defect is of great significance.Recently,tissue engineering strategies are applied to replace or repair damaged cartilage tissue.Scaffold provides a microenvironment for chondrocyte growth and plays an important role in cartilage tissue engineering.Ideal cartilage tissue engineering scaffold should mimic the structure of cartilage matrix,be beneficial for cells proliferation and be able to function for a certain period of time under load-bearing conditions.It is supposed that nanofibrous scaffolds could mimic the structure of nature extracellular matrix(ECM).Currently,self-assembly,phase separation and electrospinning are three main methods to produce nanofibrous scaffolds,but the limitations of these approaches are also existed.For example,electrospinning has advantages in fabricating relatively 2D membranes,but the construction of scalable and controllable assembly of electrospun nanofibers for 3D structure is still a major challenge.In this study,3D porous scaffolds based on gelatin and polylactic acid(PLA)nanofibers were prepared by electrospinning and freeze drying.Generally,the robust preparation steps for creating 3D porous scaffold include:(1)gelatin and PLA composite nanofiber membranes were prepared by electrospinning technique;(2)electrospun nanofiber membranes were cut into small pieces,and then were uniformly dispersed in tert-butanol by high-speed homogenizer;(3)scaffold was formed after the dispersions were frozen and freeze dried;(4)crosslinking scaffold.Moreover,to improve the biocompatibility of 3D scaffolds,they were modified with hyaluronic acid(HA)or chondroitin sulfate(CS).Then,the physical properties and biocompatibility of 3D scaffolds were detected and characterized.Finally,3D scaffolds were subjected to an in vivo cartilage regeneration study on rabbits using an articular cartilage injury model.First of all,in order to verify the feasibility of this present preparation method,3D porous gelatin/PLA nanofibrous scaffolds were prepared by combining electrospinning and freeze drying,and were crosslinked with glutaraldehyde.By controlling the concentration of dispersion,scaffolds with different densities could be obtained.The shrinkage ratio of 3D scaffold after crosslinking decreased with increasing the density of scaffold.The fiber morphology and porous structure of 3D scaffolds which can mimic the structure of ECM were observed by scanning electron microscope(SEM).Water absorption study showed that scaffold present superabsorbent properties,the max water absorption decreased with increasing the density of scaffold.In wet sate,3D scaffold showed elastic property,which could bear a compressive strain as high as 80% and recover its original shape after absorbing water again.L-929 cells were seeded on 3D scaffold,which showed normal phenotypic morphology and excellent proliferation.The large pores could also be benefical for cells to infiltrate into the scaffold.Therefore,these results showed that the preparation method which combined electrospinning and freeze drying was feasible for fabricating tissue engineering scaffold.To avoid the toxicity issue of gelatin/PLA scaffold caused by glutaraldehyde,thermal crosslinking and water treatment(two-steps method)instead of crosslinking with glutaraldehyde,were used to crosslink scaffold.The influence of different heating temperature on the stability of mechanical property and morphology were discussed,the proper temperature for crosslinking scaffold was found.The results showed that the mechanical strength of the scaffold increased after crosslinked with high temperature.Moreover,after the heat treated scaffold absorbed water and freeze drying,the mechanical strength of scaffold would further improve.3D scaffold crosslinked by two-steps also present nanofibrous structure,excellent water absorption performance and elasticity in wet state.This scaffold could enhance the growth and proliferation of chondrocytes,indicating its good biocompatibility.Considering the chemical composition of cartilage matrix,gelatin/PLA scaffold was modified with HA,which is one of the components of cartilage matrix.HA was grafted on the nanofibers surface of scaffold by EDC/NHS.No significant changes on fibers morphology and water absorption of scaffold could be observed after the scaffold was modified with HA.However,crosslinking 3D scaffold with HA could enhance its compressive strength both in the dry and wet state.It indicated that HA plays an important role for increasing the strength of scaffold.In order to evaluate the repair capacity of 3D scaffold in vivo,an articular cartilage defect was created on rabbits and scaffolds were implanted into the defect.The in vivo study indicated that the cartilage repair capacity of scaffold without HA was limited,but scaffold modified with HA could enhance the repair of cartilage.At last,a novel porous PLA@gelatin scaffold based on groove gelatin fibers and PLA fibers was prepared.By coaxial electrospinning,PLA and gelatin were loaded in the core and shell of PLA@gelatin fibers,respectively.Under the action of mechanical agitation,inner PLA fiber would ?escape? from the PLA@Gelatin fiber and groove gelatin fibers formed.Scaffold prepared with groove gelatin fibers and PLA fibers also showed porous structure,excellent water absorption and elasticity in wet state.To further improve the biocompatibility of PLA@gelatin scaffold,CS was crosslinked on the surface of groove gelatin fibers.BMSC were cultured on the scaffolds with and without CS,the in vitro results showed that crosslinking the scaffold with CS could enhance BMSC promote collagen and proteoglycans synthesis,indicating scaffold modified CS could promote the differentiation of BMSC into chondrocytes.In addition,the most important thing was that scaffold modified with CS could enhance the repair of cartilage on rabbits.In brief,3D porous scaffolds were prepared by various techniques.Electrospinning could produce fibers which can mimic the collagen fibers in ECM;homogenate technology and freeze drying made fibers assemble to new porous structure;crosslinking made the morphology and mechanics of scaffold be more stable;functional modification of scaffold with HA or CS could improve the repair capacity of 3D scaffold in vivo.The present gelatin and PLA composite scaffolds would be promising for cartilage tissue regeneration application.