Biomimetic Aligned Fibers With Shape Memory Capability For Tendon Tissue Engineering

Tendon tissue engineering?TTE?is a new treatment paradigm that can be used clinically to achieve regenerative repair of injured tendons.The basic principle of TTE is to biologically fabricate artificial tendon tissue in vitro for tendon transplantation by construction of biomaterial scaffolds,selection of appropriate seed cells,and application of physicochemical and/or mechanical signals to induce phenotype transitions in the seed cells.Amongst,constructing a biomimicking scaffold for cell survival and function realization is a key to the success of TTE.Electrospinning is a rapidly developing technique able to generate fibrous scaffolds with nano/micro-scale fineness and aligned fiber structure,resembling the natural extracellular matrix?ECM?of tendon.Such kind of fibrous scaffolds could provide ideal microenvironment for the cells.However,apart from the necessary matrix microenvironment,how to effectively induce seed cells to differentiate into tenogenic lineage is crucial.Biodegradable shape memory polymers?SMPs?are a new class of smart materials.Combining SMPs with electrospinning to produce oriented fiber scaffolds with shape memory capability can not only recapitulate the tendon matrix microenvironment,but also enable to apply appropriate mechanical stimulation with the shape memory effect of fibrous scaffolds to the cells,thereby promoting cells to differentiate into tendon.At present,research in this area is still very rare.This study selects poly?lactic acid??PLLA?with good biocompatibility,biodegradability and mechanical properties as the basic material to blend with the poly?3-hydroxybutyrate-co-3-hydroxyvalerate??PHBV?for producing aligned ultrafine PLLA/PHBV?6:4 w/w?fibers through a recently established stable jet electrospinning method.Morphology,composition/structure and thermal properties of the aligned PLLA/PHBV fibers are characterized with a focus on its shape memory performance.The results are presented as follows.PLLA/PHBV fibers show uniform morphology,high degree of orientation with a diameter ca.?1.38±0.15??m.Introduction of PHBV substantially improves the electrospinnability of PLLA and renders the glass transition temperature(T_g,as the shape memory transition temperature T_trans)reduced to⁴6?.Thus produced PLLA/PHBV fibers possess better shape memory effects with the shape fix and shape recovery rates higher than 96%and 61%,respectively.3-D structure constructed from the aligned PLLA/PHBV fibers demonstrates to return to its original shape in about 10 s at 50?.Secondly,based on the shape-programming principle of SMPs,above-prepared aligned PLLA/PHBV fibers are stretched at high temperature and cooled down to fix the shape,from which allows to obtain aligned fibers having different tensile strains for regulating the shape recovery force/stress.These stretched fibers are thoroughly characterized including morphology,tensile properties,molecular orientation,crystallinity and shape recovery.It is found that stretching followed by fixing the aligned PLLA/PHBV fibers at the tensile strains of 0%,40%,70%,100%gives rise to improved degree of fiber orientation and decreased fiber diameters from?1.38±0.15??m?0%?down to?0.91±0.08??m?100%?.Such a treatment also leads to increased tensile properties,that is,while varying the tensile strains in the predetermined range,Young's moduli of the aligned PLLA/PHBV fibers increase from?1486.27±86.31?MPa?0%?to?1758.01±94.99?MPa?40%?,?1971.83±73.57?MPa?70%?and?2011.99±97.45?MPa?100%?.However,since the fiber diameter after stretching becomes thinner,the fiber stiffness tends to decrease from?19.37±1.12?N/mm?0%?to?14.32±0.77?N/mm?40%?,?11.48±0.43?N/mm?70%?and?8.63±0.42?N/mm?100%?.By examining the molecular orientation and crystallinity of the PLLA/PHBV fibers stretched at different tensile strains,it is found that the greater the tensile strain,the better the degree of molecular orientation and crystallinity within the fibers,thus revealing the dependence of mechanical performance of the PLLA/PHBV fibers on the molecular orientation and crystallinity.Ultimate shape recovery stress of the aligned PLLA/PHBV fibers with different tensile strains are measured to vary from 0 to 5.32±0.32 MPa?40%?,7.63±0.26MPa?70%?and 9.24±0.13MPa?100%?,confirming the feasibility of tuning shape recovery stress via controlling tensile strain of the aligned fibers.Finally,effects of the fiber stiffness,from varying the tensile strain of the aligned PLLA/PHBV fibers,on the differentiation of murine bone marrow mesenchymal stem cells?rBMSCs?into tendon cells are studied.Fluorescence staining results reveal that rBMSCs grow well on the aligned fibers with different stiffness,showing a long fusiform morphology along the fiber direction which is typical to the tendon cell morphology.Functional assays indicate the expression of TNMD,a representative tendon marker protein,in rBMSCs,and the expression of TNMD protein increases with the decrease of fiber stiffness.At the gene level it shows that with decreasing the fiber stiffness expressions of the tendon cell differentiation markers,e.g.,TNMD,SCX,Collagen I,Collagen III,TNC and Dcn,are up-regulated as well.These results suggest that decreasing the stiffness of the shape memory capable PLLA/PHBV fibers facilitates the differentiation of stem cells into the tendon.In summary,based on the strategy of constructing a biomimetic cell-residing microenvironment for tendon scaffolding and regeneration,aligned PLLA/PHBV fibers with shape memory capability are prepared by a stable jet electrospinning method.It is proved that both the fiber stiffness and shape recovery force/stress can be tuned by varying the tensile strain in the process of shape-programming.The engineered PLLA/PHBV fiber scaffolds with different tensile strains are cytocompatibile and inducive in directing tendon differentiation,which thereby suggests a great potential in tendon tissue engineering application.This study paves the way for us to explore dynamic regulation of tendon cell functionality by making use of the shape memory effects of the newly developed biomimetic fiber scaffolds.