Engineering Shape-Memory Capable Electrospun Fibrous Scaffolds Of PLLA-PHBV For Bone Tissue Engineering

The native bone is a highly mechanosensitive tissue.While biomaterials-based bone tissue engineering（BTE）approach is applied for repairing bone defects/injuries,enabling the implanted biomaterial scaffolds per se capable of exerting in situ mechanical stimuli（i.e.,being mechanoactive）will improve the osteoinductive efficacy in bone regeneration.Shape memory polymers（SMPs）are a class of smart materials,whose shape recovery stress may be utilized to design mechanoactive BTE scaffolds.Electrospinning is a widely-recognized nanotechnology suitable for producing ultrafine fibers with fineness similar to the ones found in the native extracellular matrix（ECM）.Although a variety of shape memory polymers have been electrospun into fibers（SMPf）for BTE applications,there are few reports or cases in relation to making use of the shape recovery stress to regulate the stem cell fate in vitro and/or provide in situ mechanical forces for bone regeneration.In this dissertation research,using poly（3-hydroxybutyrate-co-3-hydroxyvalerate）（PHBV）-modified poly（L-lactic acid）（PLLA）（i.e.,PLLA-PHBV）as a demonstrating material,firstly the PLLA-PHBV hybrids were electrospun into fibrous mats to examine the shape memory capability and osteoinductive capacity.Then,influence of shape programming parameters on shape memory properties of the fibrous PLLA-PHBV mats was investigated.Finally,the stress stiffening effect of the shape-programmed fibrous mats under constrained recovery condition was employed to direct the osteogenic differentiation of BMSCs,and the shape recovery stress of implanted 3D scaffolds was utilized to promote bone regeneration in a rat cranial defect model.The main contents are detailed as follows:1)Firstly,the PHBV component was introduced into PLLA fibers by electrospinning to produce ultrafine composite fibers（i.e.,PLLA-PHBV）with the varied mass ratios of 10:0,9:1,8:2,7:3,6:4 and 0:10.Then,the shape memory capability and osteoinductive capacity were investigated based on the PLLA-PHBV（7:3）formulation.It was found that all the formulated PLLA-PHBV fibers could be electrospun into randomly oriented fibers with diameters of 2～3?m.When increasing the PHBV mass ratio to 30%,it gave rise to a T_g at 47.8?C,much lower than that of the pristine PLLA（56?C）.Tensile moduli of the produced fibers were increased from 41.5 MPa in PLLA to 73.5 MPa in PLLA-PHBV（7:3）.In view of the excellent mechanical performance,the electrospun PLLA-PHBV（7:3）mat was chosen to examine its shape memory properties and capacity in directing osteogenic differentiation of bone marrow mesenchymal stem cells（BMSCs）.It was revealed that the shape recovery ratio（R_r）of PLLA-PHBV（7:3）was superior to that of the PLLA fibers,and also favorably promoted the proliferation and osteogenic differentiation in BMSCs.2)In considering the strong connections between shape memory effect（SME）and shape programming process,differences in shape memory responsiveness compared between electrospun random and aligned fibrous mats were performed by varying shape programming temperature T_progand shape programming strain?_deform.The PLLA-PHBV（6:4）formulation was chosen for this purpose due to its lower T_g,close to the body temperature.The phase transition temperature range(（35）T_g,mix)was determined by thermal mechanical analysis（DMA）to be in the range of 36～54°C.Thereafter,two sets of shape-programming schemes,namely,varying T_prog at 37°C or 46°C and varying?_deform at 30%,50%or 100%,were used to examine their effects on shape memory properties.It was identified that the applied T_prog had less impact on fiber morphology,but increasing?_deformgave rise to attenuation in fiber diameters and bettering in fiber orientation,especially for the random ones.R_r and shape fixed ratio（R_f）were found to correlate with both the applied T_prog and?_deform,with the aligned fibers exhibiting relatively higher recovery ability than the random counterpart.Moreover,T_sw,corresponding to the temperature at which a maximal recovery speed can be observed,was found to be close to the T_prog,thereby revealing a temperature memory effect（TME）in the PLLA-PHBV fibers with the aligned showing a more proximity.The maximal shape recovery stress?_max generated was?_deform-dependent and 2.1-3.4 folds stronger for the aligned in comparison with the random.Overall,the aligned fibers generally demonstrated better shape memory properties,which can be attributed to the macroscopic structural orderliness and increased molecular orientation and crystallinity imparted during the shape-programming process.3)Based on the findings shown in 2),impact of applying stress stiffening effect on osteogenic differentiation of BMSCs was examined by having the shape-programmed fibrous mat of PLLA-PHBV recovered under constrained condition.To this end,gelatin（Gt）component was introduced into the PLLA-PHBV（6:4）matrix to generate randomly oriented fibrous mat of PLLA-PHBV-Gt formulated at the mass ratio of 3:2:1.Next,fibrous mats of Aligned 37 and Aligned 46 were prepared by stretching the PLLA-PHBV-Gt mats to?_deform=100%at T_prog 37?C and 46?C,respectively.Using the as-electrospun PLLA-PHBV-Gt mat（named Random）as the control,it was found that the stiffness measured in warm water（37?C）followed the order of Aligned 37（29）Aligned 46（29）Random.This is mainly due to the stress stiffening effect in Aligned 37 under constrained recovery condition.Improvements in fiber alignment and stiffness promoted the adhesion and proliferation of BMSCs as evidenced by the upregulated expression in Integrin a5?1,C-Myc,and Vinculin genes.The SME-resultant substrate stiffening promoted the ALP expression,mineral deposition,and expression of osteogenic genes（e.g.,Runx2,Alp,and Ocn）and proteins（e.g.,Runx2,Col I,and OPN）.Moreover,inhibition of Rho A expression significantly suppressed the expressions of Rock1,Rock2 and Vinculin,thus suggesting a correlation between the observed osteogenic differentiation and Rho A-Rock signaling pathway in BMSCs.The shape recovery enabled stress stiffening effect was proved to be effective in improving the efficiency of BMSC osteodifferentiation,which thereby provides a new paradigm in using SMPf for regulating the fate of stem cells.4)Based on the findings shown in 2),proof of concept animal test in a rat cranial defect model was performed to evaluate the efficacy of applying engineered 3D fibrous scaffolds capable of providing shape recovery stress(?_rec)in situ upon being implanted.To this end,3D cylindrical fibrous scaffold of PLLA-PHBV（6:4）was prepared by a stacking-winding-hot pressing process,and then fibrous mat of PLLA-PHBV-Gt（3:2:1）was adhered to the top surface of the cylindrical scaffold to form a combination（or an integration）similar to the bone-periosteum structure.Effects of?_rec,modulated by varying the?_deform with a compressive strain of 30%,50%,and 70%in the 3D cylindrical fibrous scaffold of PLLA-PHBV（6:4）,on cytocompatibility,macrophage polarization（RAW264.7）,functional expression in human endothelial cells（huv ECs）,and BMSC osteodifferentiation were investigated.Finally,the integrated bone-periosteum biomimicking structure with an optimized?_deform（50%）in the cylindrical part was implanted into the rat cranial defect model（diameter:5 mm）,and the in vivo repair efficacy was evaluated by Micro-CT,histological staining and immunofluorescence staining.The results showed that the integrated scaffolding structure with a proper?_rec at 50%of?_deform did not affect the cytocompatibility,promoted the M2 phenotype in RAW264.7,functional expression in huv ECs and osteogenic differentiation in BMSCs in vitro,and also significantly augmented bone regeneration in vivo.These results proved our hypothesis that the shape recovery force of SMPs can be used as a novel modality to exert in situ mechanical stimulation for augamenting the functionality of BTE scaffolds,thus providing a new paradigm for the application of SMPs in bone regeneration.In conclusion,by using the electrospun fiber system of PLLA-PHBV as a demonstrating SMPf for engineering mechanoactive scaffolds,this study could provide a unique platform for future research relating to cellular mechanobiology.The results obtained may also benefit for designing new mechanoactive scaffolds to stimulate in vivo bone regeneration,from which real clinical applications can be expected in future.