The Preparation And Bioactivity Evaluation Of LGA/nHA/Gelatin Functionally Graded Bilayer Membrane For Bone Regeneration

Bone insufficiency or defects arising from tumor,trauma,or periodontitis frequently precludes the successful outcome of prosthodontics and dental implants.Guided bone regeneration(GBR)technology is commonly used in dentistry and bone field.The basic principle of GBR involves the placement of mechanical barriers to protect blood clots and to isolate the bone defect from the surrounding connective tissue,thus providing bone-forming cells with access to a secluded space intended for bone regeneration.According to this principle,GBR membranes would play a vital role in bone repair.Nowadays,there are still some problems in the clinical application of GBR membrane,for example,the stiffness of non-resorbable membranes would cause a higher number of exposures,whereas the low mechanical strength of resorbable membranes could significantly decrease the amount of bone formation.An ideal GBR membrane should address the requirements for biocompatibility,barrier action,space-making feature and clinical manageability.A proper membrane surface structure can facilitate the proliferation and migration of bone cells,and accelerate bone formation in defect area.In recent years,to meet the above requirements and promote osteogenesis,researchers have proposed the concept of functionally graded membrane(FGM),in which an asymmetrical membrane was prepared with different compositions and structures,toward bone tissue and epithelial tissue respectively.Therefore,the strategy of this study was to develop a novel PLGA/n HA/Gelatin functionally graded bilayer membrane(FGBM)with graded structure and composition gradients simultaneously by a combination of phase inversion and electrospining.Phase inversion layer served as a barrier for soft tissue and a supporting for nanofibers,while electrospun nanofibers layer facing bone tissues facilitated the activity of osteoblast.Meanwhile,different contents of n HA and gelatin were designed in each layer in order to improve mechanical strength and promote osteogenesis.The main contents are as followed:1.The dense layer with different ratios of PLGA/n HA was fabricated via phase inversion method.The surface morphology,hydrophilicity,mechanics and in vitro degradability were measured by SEM,contact angle test,tensile test and biodegradation test.Then,the in vitro barrier function of the phase inversion membrane to L929 cells was evaluated.It is found that the phase inversion membrane presented an asymmetric structure with one side being dense and smooth,and another porous.5wt.% n HA incorporation into PLGA would not compromise the mechanical properties and could perfectly prevent fibroblastic penetration,even in the process of membrane degradation.2.Base on phase inversion layer,a PLGA/n HA nanofiber layer was prepared via electrospinning method,resulting in a functionally graded bilayer membrane(FGBM).The surface morphology,hydrophilicity,mechanics and in vitro degradability of FGBM were investigated by SEM,EDX,TEM,contact angle test,tensile test and biodegradation test.It is found that the FGBM presented a graded porous structure,with the two layers integrating closely.However,the 40 wt.% n HA incorporation would lead to chaotic surface morphology.The mechanical properties and degradability of the FGBM can meet the requirements of GBR membrane.3.The formation of bone-like apatite on the surface of FGBM was examined by SBF immersion test.The in vitro osteogenic bioactivity of MC3T3-E1 cells on FGBM were investigated by evaluating cell adhesion,proliferation,differentiation,bone formation related gene and protein expression.The results shown that the porous layer with PLGA and 30 wt.% n HA had excellent capabilities of cell adhesion,proliferation and differentiation in vitro,and could promote the bone formation gene and protein expression,which might stimulate the cells to undergo an osteogenic process.4.In order to further facilitate osteogenesis and activities of osteoblast,different ratio of galatin was introduced into electrospun fiber layer.The corresponding properties were evaluated including surface morphology,hydrophilicity,mechanics,degradability,and in vitro osteogenic bioactivity.The results showed that the two layers of FGBM integrating closely.Gelatin could improve hydrophilicity,mechanics and degradability.FGBM PHG2 presented excellent osteoinductive activity.In conclusion,PLGA/n HA/Gelatin functionally graded bilayer membrane presented graded structure and composition gradients with excellent mechanical properties and fibroblastic barrier function.Meanwhile,the PLGA/n HA/Gelatin nanofiber layers of FGBM could mimic the dimensions and compositions of the extracellular matrix,exhibiting satisfactory cytocompatibility and favorable osteogenic effects.Therefore,this study showed that the designed bilayer membranes were promising for GBR therapy and other applications in bone tissue engineering.