| Medical polyurethane(MPU)is a kind of copolymers composed of flexible soft segments and rigid hard segments.The soft segments are generally polyether or polyester and the hard segments are rich in urethane groups.MPUs may demonstrate good shape memory properties,biodegradability,biocompatibility and mechanical properties so that they have attracted wide attention in biomedical fields.Shape memory properties are important for MPU to act as a smart material and the microphase separation between soft and hard segments is the structural basis.Therefore,visually tracing the microphase separation in a real-time manner may help to understand the underlying mechanism and regulate the microphase separation.Biodegradability is a precondition for MPUs to act as a tissue regeneration material.Visually tracing the biodegradability in a real-time manner may help to clarify the underlying mechanism and regulate the tissue regeneration.However,most of the existing methods cannot directly visualize the microphase separation and biodegradability and they are generally time-and labor-consuming and need to sacrifice samples.This work aims to develop a new method capable of fast,efficiently,low-cost,nondestructively tracing the microphase separation and biodegradability and further to form a novel MPU material integrating fluorescence tracing and good bone formation.It is known that the microphase separation in MPUs is induced by the thermodynamic incompatibility between soft and hard segments while the urethane groups in hard segments are the structural basis for biodegradability of MPUs.Both are related to hard segments.Therefore,we hypothesize that incorporating a fluorescent molecule into the hard segments of MPUs should promise a fluorescent visualization of the microphase separation and biodegradability.To test this hypothesis,a reactive fluorescent molecule5-oxo-2,3-dihydro-5H-[1,3] thiazolo [3,2-a] pyridine-3,7 dicarboxylic acid(TPA)was selected as the chain extender to design and synthesize a series of novel MPUs possessing inherent fluorescence in the hard segments(IFPUs).Further,the fluorescent visualization of the microphase separation and biodegradability by using the covalently incorporated TPA was investigated and the matching of biodegradability to bone regeneration was evaluated.The main contents and conclusions are summarized as follows:Ⅰ Design,preparation and characterization of IFPUsBy using TPA as the chain extender,1,6-hexamethylene diisocyanate(HDI)as the coupling agent,and a flexible polytetramethylene glycol(PTMG)or a biodegradable poly(D,L-lactic acid)(PDLLA)as the soft segment,two types of novel IFPUs,i.e.PTMG-IFPUs and PDLLA-IFPUs,were designed and prepared.The incorporated TPA acts as both a component of hard segments and a fluorescence probe.(i)The chemical structures and molecular weights were characterized by using Nuclear Magnetic Resonance Spectrometer(NMR),Fourier Transform Infrared Spectrometer(FT-IR),and Gel Permeation Chromatography(GPC).The results indicated that,by controlling the molar ratios of soft segment/HDI/TPA,PTMG-IFPUs with a variable hard segment content of 10-20 wt% and a molecular weight of no less than 130 k Da and PDLLA-IFPUs with a variable hard segment content of 2.5-4.0 wt%and a molecular weight of no less than 190 k Da could be successfully prepared.(ⅱ)The fluorescence properties of various IFPUs were detected by using a UV-visible spectrophotometer and a fluorescence spectrometer.The results demonstrated that both PTMG-IFPUs and PDLLA-IFPUs have similar fluorescence properties to TPA molecules,with the maximum excitation and emission wavelength at368 nm and 432 nm respectively.Moreover,they both have good light resistance and high quantum yield,which lays a foundation for the following fluorescent visualization study on the microphase separation and degradation behavior.Ⅱ Fluorescent tracing of the microphase separation of IFPUs(i)Since the higher hard segments contents and more flexible soft segments of PTMG-IFPUs are good for microphase separation,PDLLA-IFPU films were employed as a model IFPU to fluorescently trace the microphase separation at static states and during programming(stretching)and shape recovering.It was found that,by means of the fluorescent effects of TPA in hard segments,the soft segments matrix,the hard segments domains and the mixing phase of soft and hard segments could be directly visualized.Moreover,semi-quantitative statistical analysis of the fluorescence images could obtain some parameters to indicate the degree of microphase separation,including the size,aspect ratio and orientation of hard segment domains.This verifies that covalent incorporation of TPA into the hard segments of MPUs is a fast,efficient,low-cost,nondestructive,semi-quantitative method to trace the microphase separation of MPUs in a real-time manner.(ⅱ)According to the results from the fluorescence images and semi-quantitative statistical analysis,the hard segments content,the stretching process,and the shape recovering process could all regulate the microphase separation of IFPUs.The underlying mechanism is related to the altered urethane-urethane physical distance and chain mobility and the inherent directionality of hydrogen bonding,suggesting that this developed fluorescence visualization method may help to clarify the mechanism for microphase separation of MPUs as well in a more comprehensive and detailed way.Ⅲ Fluorescent tracing of the in vitro and in vivo biodegradability of IFPUsBoth the soft and hard segments of PDLLA-IFPUs are degradable,so that PDLLA-IFPUs were employed to fluorescently trace the in vitro and in vivo degradation behaviors.(i)It is known that the content of hard segments and addition of β-tricalcium phosphate(β-TCP)can both regulate the biodegradability of MPUs.Moreover,β-TCP can promote bone formation.Therefore,PDLLA-IFPUs with various contents of hard segments were first fabricated into films and porous scaffolds by using a solution casting method and a low temperature deposition 3D printing/freeze-drying technique respectively.The composite scaffolds of PDLLA-IFPU(hard segments content of 3.6%)and β-TCP with β-TCP content as 10% and 30%(PU/T10,PT/T30)were further fabricated by using the same method for PDLLA-IFPU scaffolds.All scaffolds had good pore structures with a wire diameter of ～450 μm,a pore size of ～450 μm and a large quantity of micron-sized pores.More importantly,all films and scaffolds were observed to demonstrate good fluorescent properties,which lays a foundation for the following fluorescent visualization study on the biodegradability and the regulation of biodegradability.(ⅱ)The fluorescent tracing of the in vitro and in vivo biodegradation behavior was investigated by visualizing the fluorescence intensity of PDLLA-IFPU films and scaffolds.It was observed that the fluorescence intensities of both wet and dried samples decreased with prolonging degradation time.The ratios of residual fluorescence intensities of wet samples indicated similar degradation behavior to that by the ratios of residual fluorescence intensities of dried samples and that by the ratios of residual weights,suggesting that the ratios of residual fluorescence intensities of wet samples can well indicate the degradation behavior of PDLLA-IFPUs and IFPU-based scaffolds.This not only saves the drying time,but also greatly reduces the required sample quantities.Therefore,it can be concluded that the introduction of fluorescence probe TPA into the hard segment is a fast,efficient,low-cost,nondestructive,visual method to trace the degradation behavior of MPU.(ⅲ)The degradation mechanism of PDLLA-IFPU in various scaffolds was clarified by combining the fluorescence residual ratios of scaffolds,the fluorescence intensity of degradation medium,the p H values of degradation medium,the water absorption of residual scaffolds,and the molecular weight of PDLLA-IFPU.Specifically,both the hard segments content and the β-TCP content can regulate the degradation rate of PDLLA-IFPU in the scaffolds,yet they will not change the degradation mode of the polymer chains.The hard segments content regulates the degradation behavior via affecting the water absorption whereas addition of β-TCP regulates the degradation behavior via inhibiting the fast increase of local acidity.Ⅳ Cytocompatibility and in vivo osteogenesis of PDLLA-IFPU and scaffoldsThe controllable biodegradability and good pore structures and mechanical properties of PDLLA-IFPU scaffolds and PU/T scaffolds suggest their potential application as bone regenerative scaffolds.To verify this possibility biologically,the in vitro cytocompatibility,in vivo osteogenesis,and biosafety were investigated and the matching of degradation behavior and osteogenesis was analyzed.(i)First,the in vitro cytocompatibility was evaluated by using human bone marrow mesenchymal stem cells(h BMSCs)as model cells and PDLLA(marked as PLA)as control.It was observed that h BMSCs on PDLLA-IFPU films and PDLLA-IFPU-based scaffolds all demonstrated better cell adhesion,spreading,proliferation and osteogenic differentiation than on PLA.Besides,both PDLLA-IFPU scaffolds and PU/T scaffolds could recruit h BMSCs to grow into the scaffolds.These results reveal that PDLLA-IFPUs and their scaffolds have good cytocompatibility with h BMSCs.(ⅱ)The in vivo osteogenesis of PDLLA-IFPU scaffolds and PU/T scaffolds was examined by using a rat femur defect model.The scaffolds were observed to promote the regeneration of defected bone and the osteogenesis was dependent on the content ofβ-TCP,taking an order of PDLLA-IFPU scaffolds ≥ PU/T30 scaffolds > PU/T10 scaffolds.Further analysis of the visualized degradation behavior and the osteogenesis indicated that the regulation of β-TCP to biodegradability is responsible for this dependence.Specifically,PDLLA-IFPU scaffolds had a matching biodegradability with the osteogenesis rate,which,together with the good osteogenesis of PDLLA-IFPU material,produced the highest bone amount.On the other hand,the low biodegradation of PU/T10 could not spare enough room for new bone formation,leading to slow bone regeneration.Regarding PU/T30 scaffolds,despite their low biodegradation,the strong osteoconduction endowed by high β-TCP content promoted the formation of higher quality of new bone.(ⅲ)Finally,the in vivo biosafety was evaluated by subcutaneously implanting the scaffolds.The results on the inflammatory reactions,blood compatibility,organ toxicity,and the reactions of surrounding tissues revealed that both PDLLA-IFPU scaffolds and PU/T scaffolds have good in vivo biosafety.In summary,covalent incorporation of fluorescence molecule TPA into the hard segments of MPU is a new visualization method capable of fast,efficiently,low-cost,nondestructively and quantitatively tracing the microphase separation and biodegradation of MPU,and the 3D printed PDLLA-IFPU scaffolds and PU/T30 scaffolds are promising bone regeneration materials with both fluorescence tracing and good osteogenesis. |
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