Phase-separated Micro/Nanostructures And Recovery Strain Of Shape Memory Polyurethane And Their Regulation To Cell Morphology

Shape memory polyurethane(SMPU)has attracted increasing attentions in biomedical fields due to its sound biocompatibility,strong mechanical properties and shape memory properties.The phase-separated micro/nanostructures,which are formed due to the thermodynamic incompatibility between the soft and hard segments,are the structural basis for SMPU to possess the potential of shape memory effect(SME),while programming,fixation,and recovering are the indispensable procedures for SMPU to demonstrate SME.Programming and recovering can alter the micro/nanostructures and recovering process can further produce recovery strain.To deeply understand and control the biocompatibility of SMPU and further guide the structural design and processing of SMPU,it is essentially important to explore the micro/nanostructures and the recovery strain during the shape memory process and their regulation to cell behavior.Unfortunately,it has received few attentions.In the present study,the effects of shape memory process on the micro/nanostructures of SMPU were investigated by using wide angle X-ray diffraction(WAXD),small angle X-ray scattering(SAXS),attenuated total internal reflectance Fourier transform infrared spectroscopy(ATR-FTIR),Differential Scanning calorimeter(DSC),atom force microscope(AFM),and scanning electron microscope(SEM).The bioglical effects of the resulting micro/nanostructures and recovery strain were further evaluated in terms of osteoblasts morphology.The main research contents and conclusions were listed as follows:(1)The effects of programming on the micro/nanostructures of SMPU films.Block copolymer equipped with PDLLA-PEG-PDLLA(poly(DL-lactic acid)-polyethylene glycol-poly(DL-lactic acid)triblock macrodiol)as the soft segments and HDI/PPZ(hexamethylene isocyanate/piperazine)as the hard segments was adopted as a model SMPU,and PDLLA was selected as the control.SMPU and PDLLA films were prepared through solvent casting and then programmed by stretching at different stretching ratios(0%,50%,100% and 200%).The effect of stretching on the phase separation and the resulting micro/nanostructures were thoroughly investigated,which may help for clarifying the regulation of programming to cell morphology.(1)On the as-cast SMPU films,hard segments sequentially assembled into nano-scale hard domains,isolated islands,and nano fibers.The obivous phase separation drove SMPU into two-phase morphology in which nano fibers represented unordered chemcial patterns.In contrast,PDLLA films were uniform and smooth.A low degree of phase separation was seen to result from weak crystallization in PDLLA film.(2)Stretching produced nano apophyses on PDLLA films,which arrayed perpendicularly to the stretching direction.On SMPU films,however,both the nano-scale hard domains and islands all tended to orientate perpendicularly to yet pack along the stretching direction.As a result,the nano fibers on the stretched SMPU films became parallel to the stretching direction.These results verify that streching could increase the ordering of nano fibers on SMPU films,suggesting the potential role of programming in the fabrication of ordered chemical patterns.(2)The effects of stretching-induced micro/nanostructures on osteoblast morphology and protein adsorption.To investigate the biological effects of the stretching-induced micro/nanostructures,the morphology of osteoblasts and the protein adsorption of fibronectin(Fn)were visualized by using immunofluorescence staining.It was found that both the long axis of ostoblasts and assembled Fn fibrils were parallel on SMPU films yet perpendicular on PDLLA films to the stretching direction,indicating the decisive role of streching-induced micro/nanostructures in regulating cell orientation and protein assembly.These results confirmed the vital role of programming in controlling material-cell interactions.Besides,it is suggested that programming might be a potential new method to fabricate ordered chemical patterns on SMPU.(3)The effect of recovery medium on shape recovery and micro/nanostructures of SMPU films.The shape recovery process,as an indispensable procedure for SMPU to show SME,generally happens in body fluids for biomedical applications.Investigating the effects of body fluids on the micro/nanostructures and recovery strain may help for a more comprehensive and in-depth understanding of the biocompatibility of SMPU.Accordingly,the stretched SMPU films were strain-held by clamping the two ends of films and incubated at 32°C for 24 h in air(denoted as “Air-clamped”)or in DMEM(denoted as “DMEM-clamped”);thereafter,the films were un-fixed and allowed to recover in air(denoted as “Air-recovered”)or in DMEM(denoted as “DMEM-recovered”).The “clamping” treatment of the films was designed to simulate the cell culture condition of osteoblasts before shape recovery process of SMPU-cell composites(see(4)),so that the obtained micro/nano structures and recovery strain could be effectively employed to clarify the role of shape recovery in regulating cell behavior.(1)The shape recovery stress and the recovery ratio were detected.It was found that all films demonstrated a quick recovery within the initial 20 min and completed the recovery process within 2 h.“Clamping” treatment had no obvious effect on the micro/nanostructures yet decreased the shape recovery stress and recovery ratio.DMEM led to lower shape recovery stress and recovery ratio than air.The mechanism is,according to the DSC result,that water in DMEM acts as a plasticizer for SMPU to enhance the chain mobility and thus the stress relaxation.These results indicate that the recovery media could affect the shape recovery of SMPU films.(2)The recovery process was found to change the micro/nanostructures of SMPU films with a dependence on the recovery medium.In air,the assembly of hard domains did not recover to its originally unordered state.Instead,most of the nanofibers took an angle of > 40° with the strethcing direction,which should be caused by the stress relaxation and the resulting low recovery ratio.In DMEM,on the other hand,the aggregation of hard domains was enhanced during the recovery process to produce more fibrous and punctiform hard domains,and the angles between the fibers and the stretching direction was > 40° too.The plasticization of water and the hydrogen bonds between water and the soft segments of SMPU might be the responsible factors.These results demostrate that the recovery media could significantly regulate the micro/nanostructures of recovered SMPU films.(4)The regulation of recovery strain and micro/nanostructures to osteoblasts morphology.Recovery strain of SMPU may exert a lasting mechanical loading to the cells adhered on films.Accordingly,exploring the regulation of recovery strain to cell behavior may provide a new perspective to evaluate the biocompatibility of SMPU.In order to evaluate the regulation of recovery strain and micro/nanostructurs on osteoblasts morphology,the stretched films were strain-held by clamping the two ends of the films and osteoblasts were seeded thereon at 32°C for 24 h.Then,the films were un-fixed and the films-cells composites were placed at 37°C for shape recovery.The morphological evolution of osteoblasts during shape recovery process was traced using “Live Cell Imaging System” and immunofluorescence staining.In addition,cell proliferation was also examined using CCK-8.(1)It was found hat the total recovery strain(%)of SMPU films increased with the increase of stretching ratio: 17.55±3.34(50%),27.87±2.93(100%)and 34.80±2.60(200%).(2)The “Live Cell Imaging System” and immunofluorescence staining revealed that,during the intial period of shape recovery process(? 4h),osteoblasts tended to orientate perpendicularly to the stretching direction with a dependence on the stretching ratio,indicating that shape recovery strain may act as a mechanical stimulus to regulate the cell orientation.(3)After 24 h of recovery process,osteoblasts tended to orientate along the nanofibers on the films,suggesting that the micro/nanostructures might become the determinant factor for cell orientation with the extension of incubation time.(4)CCK-8 detection displayed that,after 1-7 d of recovery process,the cell population on the recovered films was higher than those on un-recovered films,suggesting that the shape recovery strain could act as a mechanical stimulus to promote cell proliferation and SMPU could serve as a new dynamic cell culture system.In summary,the effect of programming,recocvery and recovery media on the micro/nanostructures of SMPU films were thoroughly explored,and the regulation mechanism of micro/nanostructures and recovery stain to cell morphology was uncovered.The obtained results provide a theoretical basis for understanding the biocompatibility of SMPU and help to guide the design and processing of SMPU.In addition,the potential applications of SMPU in fabricating ordered chemical patterns and dynamic cell culture system were also preliminarily verified.