Modular photoorigami-based 4D manufacturing of vascular junction elements

Four-dimensional (4D) printing, combining three-dimensional (3D) printing with time-dependent stimuli-responsive shape transformation, eliminates the limitations of the conventional 3D printing technique for the fabrication of complex hollow constructs. However, existing 4D printing techniques have limitations in terms of the shapes that can be created using a single shape-changing object. In this paper, we report an advanced 4D fabrication approach of a vascular junction, particularly the T-junction, using the 4D printing technique based on coordinated sequential folding of two or more specially designed shape-changing elements. In our approach, the T-junction is split into two components, and each component is 4D printed using different synthesized shape memory polyurethanes and their nanohybrids, which have been synthesized with varying hard segment content and by incorporating different weight percentages of photo-responsive copper sulfide-poly vinyl pyrrolidone nanoparticles. The formation of a T-junction is demonstrated by assigning different shape memory behaviors to each component of the T-junction. A cell culture study with human umbilical vein endothelial cells reveals that the cells are proliferating with time, and almost 90% of cells are viable on day 7. Finally, the formation of T-junction in the presence of near-infrared light has been demonstrated after seeding the endothelial cells on the programmed flat surface of the two components and the immunohistochemical analysis at day 3 and 7 reveals that the cells are adhered nicely and proliferating with time. Hence, the proposed alternative approach has huge potential and can be used to fabricate vascular junctions in the future.


Significance:
The role of fabrication techniques in driving the evolution of regenerative medicine cannot be overstated.In particular, 4D printing has emerged as a sophisticated technique that eliminates the limitations of conventional 3D printing.This article presents a novel solution for the fabrication of vascular junctions, particularly the 'T-Junctions' in the vascular network.Our approach utilizes the 4D printing technique and photo-responsive coordinated sequential folding of the printed elements to address challenges such as the need for supporting materials and cell viability of incorporated cells in high-resolution 3D printed structures.The successful formation of the T-junction with well-adhered cells, followed by cell proliferation on the inner surface of the tubular junction, serves as proof of concept of this proposed approach and its future applications.
Introduction.Tissue engineering and regenerative medicine are continuously evolving to fulfill the increasing demand for organ transplantation worldwide. 1The potential of different types of biomaterials, cells, and fabrication techniques, individually or collectively, is being studied to repair damaged tissues or to regenerate the structure or/and function of tissues. 2,3Among different factors, the development of fabrication methods plays an essential role in tissue engineering and regenerative medicine.Usually, additive, subtractive, and formative manufacturing techniques are used to fabricate structures with or without cells in tissue engineering 4,5 .However, all of them have certain limitations that restrict the field of their preferential application. 6,7Two strategies are commonly used to fabricate structures with cells: (i) fabrication of a structure incorporating the cells with the materials (cell-laden constructs) allows the construction of multicellular complex structures, but the issue with the viability of the incorporated cells restricts it to a few possible fabrication methods, while (ii) fabrication of a structure followed by cell seeding allows the use of almost all possible fabrication methods. 7,8ditive manufacturing (AM) or three-dimensional (3D) 9 printing has emerged as the most advanced and widely used technique for fabricating not only any complex structure in tissue engineering 10,11 as well as electronic devices and high-performance metamaterials. 12,13It allows the development of any 3D structure with a good resolution that helps to create new functionality or improves optimal performance.Recent advancement in 3D printing allows the printing of multimaterials that helps to develop heterogeneous structure or hybrids. 14,15Although these additional features of 3D printing resulted in new innovative applications, they are limited.In 3D bioprinting, the biological structures are fabricated layer-by-layer with accurate positioning of bioinks (biomaterials, cell, and bioactive molecules) through spatial control over the functional component placement. 11The most commonly used 3D printing techniques in tissue engineering or organ regeneration are inkjet printing, 16,17 extrusion-based 3D printing, 18,19 stereolithography, 20 and digital light processing (DLP), 21 etc.7][28] 4D printing allows shape transformation within the material or structural design of any 3D printed structure with time in the presence of a predetermined stimulus, including osmotic pressure, light, heat, current, magnetic field, etc. [29][30][31][32] 4D printing, being a new sophisticated technique, not only provides tremendous opportunities to design and fabricate smart or active structures efficiently and easily using shape-changing polymers (e.g.4][35][36] The main advantage of 4D printing over 3D printing is the possibility of fabrication of hollow structures without supporting materials.For example, the challenges with removing the rod-like support after 3D printing of tubular structures using a rotating rod or the issues with the attachment of the cells during seeding inside the fabricated tubes are eliminated using the 4D printing technique. 37In the 4D printing approach, cells are seeded on a flat surface, and cells have time to attach and proliferate.Finally, the shape transformation allows the formation of tubular or scroll structures with uniformly distributed cells in the inner wall of the tube. 37,389][40] Current 4D printing also has limitations regarding the shapes that can be created using a single shape-changing object.While a tube can easily be formed by rolling of rectangular film, achieving more complex shapes like, for example, T-and Y-junctions, which are essential elements of the vascular network, by shape-transformation of a single 2D object (film) is not possible.
To successfully execute 4D printing, the selection of materials is very crucial.2][43][44][45][46][47] Hydrogels generally exhibit shape-changing behavior utilizing their swelling behavior, while SMPs can recover their shapes from a temporarily programmed shape in the presence of stimuli.Despite having numerous advantages of hydrogels (like tunable swelling behavior, porous structure, and excellent biocompatibility), prolonged response rate (from a few minutes to a few days depending on the size), meager mechanical strength (ranging from ~ kPa to ~100 kPa) and fast degradation rate limiting its applications in 4D printing for biomedical applications. 48,49In that scenario, the fast response time (from a few sec to a few minutes), better mechanical properties (in the range of ~MPa to ~GPa), and slow degradation rate of SMPs make it a better choice as compared to hydrogels. 50,51Among different available environmental stimuli, light as a stimulus has distinct advantages, like i) it is possible to apply very accurately, ii) it can be applied remotely, and iii) it is possible to switch very quickly. 52Generally, one can introduce photo-responsiveness in shape memory polymers in two ways: i) using photo-responsive dyes or chromophores or ii) using photoresponsive fillers. 53Further, using photo-responsive fillers not only produces photoresponsiveness in SMP but will also help improve the viscoelastic properties during printing. 2,54ere, we introduce an advanced approach to fabricating vascular junctions, particularly Tjunction, using the sophisticated 4D printing technique based on coordinated sequential folding of two (and possibly more) shape-changing objects.While folding of single shape-changing objects can be compared with origami, coordinated folding of two or more shape-changing objects can be compared with the extension of traditional origami -modular origami.In this approach, the Tjunction of the vascular network is split into two hollow structures and is 4D printed using different shape memory polymers to introduce different responsive behaviors to the two hollow structures.This paper demonstrates the proof of concept of this approach, which can be extended to more than two objects.

Experimental Section
Materials.Polycaprolactone diol (PCL-diol, M n ~2,000 g mol -1 ), 1,6-hexamethylene diisocyanate (HDI), 1,4-butane diol (BD), dibutyltin laurate (DBTDL), N, N-dimethyl formamide (DMF) was purchased from Merck and was used as received.Copper sulfide-poly vinyl pyrrolidone(CuS-PVP) nanoparticles were synthesized as discussed in Ref . 55 Synthesis.Different shape memory polyurethanes (SMPUs) were synthesized through a two-step polymerization process in a three-neck round bottom flask.In the 1 st step, the prepolymer was atmosphere (N 2 atmosphere).While in the 2 nd step, the chain extender (BD) and catalyst (DBTDL: 0.1 ml of 1wt% toluene solution) were added to complete the polymerization (Figure S1a) The temperature was maintained at 70 o C throughout the polymerization.Different molar ratios of PCL-diol: HDI: BD were used to prepare polyurethanes with different hard segment content (HSC) (Table 1).The chain extension was performed for 24 hours to ensure complete polymerization and to obtain a high molecular weight polymer.The synthesized polymers were recovered through precipitation in double distilled water (non-solvent) and dried in a vacuum oven under reduced pressure at 60 o C for 72 hours.The molecular weight of different synthesized SMPUs was determined through Gel permeation chromatography (GPC), which is summarized in Table 1  Thermogravimetric analysis (TGA).The thermal stability of different synthesized SMPUs and their nanohybrids were investigated using Netzsch TG 209 thermogravimetric analyzer (TGA) in the temperature range of 25 to 600 o C at a heating rate of 20 o C min -1 under a nitrogen atmosphere.3D Printing of pure SMPUs and their nanohybrids.For fabricating the desired geometries with different pure SMPUs and their nanohybrids, a pneumatic pressure-assisted extrusion-based 3D printer of RegenHU (3D Discovery) was used.The samples were extruded above their melting temperature (T m ) and under sufficient pneumatic pressure to maintain continuous extrusion of the samples from the nozzle during printing.A nozzle of ~350 m diameters, a layer thickness of 0.1 mm, and a feed rate of 6-10 mm/s were used to print the desired geometries.The desired 3D geometries were created using Fusion 360 CAD software, and slicing was done using Bio-CAD software provided by the RegenHU 3D printer.
Shape memory behavior of pure SMPUs and their nanohybrids.The shape memory behavior of different pure polymers and their nanohybrids was examined using 4D printed geometries.First, the 3D printed geometries (permanent shape) of different samples were deformed into temporary geometries (temporary shape) above their transition temperature (T r ~ melting temperature of soft segments of SMPUs, ~ 40 o C), and the temporary shapes were fixed, lowering the temperature (T f ~ 10 o C) below the transition temperature (T r ).This process is called "Programming"; the temporary shape is called the programmed shape.Further, by increasing the temperature above the T r temperature or using near-infrared (NIR) light (Philips R95 IR 100W near-infrared light), recovery of the permanent shape from the temporary shape of different pure SMPUs and their nanohybrids was investigated.The shape fixity and shape recovery efficacy of different SMPUs and their nanohybrids were calculated using Equations 1 and 2.
Small-angle x-ray scattering (SAXS)measurement of SMPUs and their nanohybrids.For SAXS analysis, the SMPUs and their nanohybrids were used as obtained, whereby the 3D printed samples had a permanent and the programmed ones a temporary shape.The measurements were performed at ambient conditions using a Double Ganesha AIR system (SAXSLAB/Xenocs), providing monochromatic radiation with a wavelength of λ = 1.54 Å (produced by a rotating Cu anode, MicroMax 007HF, Rigaku corporation) on the lateral area of the samples.The position-sensitive detector (Pilatus 300K, Dectris) was placed at a different sample to detector distances to cover a wide range of scattering vectors q ( ; θ scattering angle).The 1-dimensional intensity I(q) was obtained by radial averaging and normalized to the intensity of the incident beam, the sample thickness (1.5 mm), and the accumulation time.
Spectroscopic Measurement.The synthesized polymers ' proton nuclear magnetic resonance (1H-NMR) spectra were recorded using Bruker Avance 500 spectrometer (500 MHz).The samples were completely dissolved in DMSO-d6 solvent and were equilibrated in the magnetic field for 10 minutes before recording the spectrum.The chemical shifts were reported in ppm units relative to tetramethylsilane (TMS).Infrared spectra of solid films of different SMPUs and their nanohybrids were recorded using Fourier transform infrared (FT-IR) spectrometer of Bruker Tensor 27, USA.
The spectra were taken in the spectral range of 800 to 4000 cm -1 with a spectral resolution of 4 cm -1 at room temperature.
Morphological Investigation.The morphologies of the 3D printed structures of different SMPUs and their nanohybrids were investigated using a scanning electron microscope (SEM) of Thermo Fisher Scientific, USA.The samples were coated with platinum before measurement.
Cell culture.Human Umbilical Vein Endothelial cells (HUVECs) were seeded on the flat surfaces of different pure SMPUs and nanohybrids.The samples were 3D printed and prepared under aseptic conditions.Before culturing the cells, the surface of the samples was coated with a laminin solution to support the adhesion and growth of the endothelial cells.The excess laminin solution was removed, letting the sample dry under the hood for 15 minutes.A Live-Dead assay was performed to investigate the viability of cells with time on the surface of 7 days) with a mixed solution of calcein AM and ethidium homodimer-1 after removing the culture media from the 48 well plates.Then, the well plate was incubated in dark conditions for 30 minutes at room temperature.Finally, the fluorescence images were taken using a Nikon Ti2 microscope.
The mixed solution was prepared by adding 3.6 l of calcein AM and 4 l of ethidium homodimer-

Result and Discussion
The approach of fabricating the vascular junction utilizing the sequential folding of the 4D printed structure is illustrated in Figure 1.The fabrication consists of three steps.The first step is the 3D printing of the components of vascular junctions individually using an extrusion-based 3D printer.
In the second step, the 3D structures are programmed into the flat structure.In the third step, the sequential folding of the arranged flat structures with or without seeded cells produces a vascular junction (T-junction).Both flat objects must fold at different times because simultaneous folding results in their collapse.To achieve separation of actuation of the objects in time, the materials used for their fabrication must possess either (i) different sensitivities that will allow the use of different signals to trigger their shape transformation or (ii) one sensitivity but different sensitivity thresholds that will allow triggering with one signal but at different time moments.We have explored both possibilities using different synthesized thermoresponsive shape memory polyurethanes (SMPUs), which can be mixed with different amounts of light-sensitive nanoparticles.
The SMPUs are synthesized through two-step condensation polymerizations mixing hexamethylene diisocyanate (HDI), polycaprolactone diol (PCL-diol), and 1,4-butane-diol (BD), and varying hard segment contents (Figure S1a).The synthesized SMPUs are designated as HPB15, HPB20, and HPB30; as shown in Table 1; the number means the mass percentage of the hard segment.The molecular weight of the synthesized SMPUs is measured using gel permeable chromatography (GPC) with respect to polystyrene reference that shows an unimodal distribution of the molecular weight for all synthesized SMPUs (Figure S1b).The weight average molecular weight (M w ) of HPB15, HPB20, and HPB30 are 68, 94, and 53 kDa, respectively (Table 1).The chemical structure of the synthesized polyurethanes is characterized using 1 H NMR (Figure S1c).
The features of the spectra of three SMPUs are in good agreement with each component of polyurethane chains, and the peaks are assigned according to the literature. 56Generally, the peaks that appear between 1 to 2 ppm belong to the methylene groups present in the HDI, PCL, and BD.
The peak appears at 7 ppm, corresponds to >N-H protons of the hard segment, while the peak appears at 2.92 ppm, belongs to >N-CH 2 protons of the hard segment.The intensities of these peaks increase from HPB15 to HPB30, supporting the increase of hard segment content in SMPUs.
Different nanohybrids of SMPUs are prepared by dispersing 0.2, 0.5, 0.75, and 1 wt% of the CuS-PVP nanoparticles in the pure SMPU (HPB20) matrix, and the prepared nanohybrids are designated as HPB20_0.2NH,HPB20_0.5NH,HPB_0.75NH, and HPB20_1NH, respectively.The presence of nanoparticles (NPs) in the SMPU matrix and the interaction of the NPs with the polymer matrix is confirmed through the investigation of shifting of the vibrational frequencies of different functional groups in nanohybrids using FT-IR spectroscopy (Figure S1d).The absorption band appearing at 1731 and 1688 cm -1 are responsible for the stretching frequencies of free carbonyl groups (>C=O) of ester and urethane moieties present in SMPU, while the absorption bands appearing at 1717 and 1668 cm -1 are responsible for the stretching frequencies of hydrogenbonded carbonyl groups of ester and urethane moieties present in SMPU. 51In the nanohybrids (HPB20_0.2NHand HPB20_0.5NH), a single band appears at 1726 cm -1 instead of two individual bands at 1731 and 1717 cm -1 for the ester carbonyl group (>C=O) and the bands for free and Hbonded urethane carbonyl groups (>C=O) shifts to lower stretching frequencies to 1684 and 1665 cm -1 from 1688 and 1665 cm -1 , respectively.Further, the deformation frequency band of >N-H group of urethane linkage shifts to 1539 from 1546 cm -1 in nanohybrids.This suggests a strong interaction between the nanoparticles and polymer chains occurs, which helps lower the stretching and deformation frequencies of >C=O and >N-H groups in nanohybrids. 57Thus, the results confirmed the successful synthesis of the polycaprolactone-polyurethane copolymers and their hybrids with the nanoparticles.40Jg -1 ) for HPB20 and then decreases slightly to 41 o C (35 Jg -1 ) for HPB30.Hence, the melting temperature increases with increasing the hard segment content (HSC) of polyurethane.A similar trend is also observed in the case of crystallization temperature and the corresponding heat of fusion (H) of both soft and hard segments.The H-bonding between urethane linkages of the hard segments as well as between the ester ˃C=O group of the soft segment and amide ˃N-H group of the hard segment of the polyurethane chains increases with increasing HSC that helps to improve the melting temperature SMPUs with increasing HSC. 56However, incorporating the NPs in the polymer matrix decreases both melting and crystallization temperatures for the soft and hard segments of SMPU nanohybrids.The interaction of the polymer chains with dispersed CuS-PVP NPs (c.f.evident from FT-IR measurement) is responsible for the depression of the melting temperature (T m ) in nanohybrids. 51,57Hence, synthesized SMPUs and nanohybrids have two melting and crystallization temperatures, and the value of melting and crystallization temperature increases with increasing hard segment content and decreases with increasing concentration of NPs.The melting of soft segments in the 30-40 o C temperature range will allow the SMPUs to show shape memory behaviors in the range of human body temperatures.In contrast, melting the hard segment at high temperatures will help to 3D print the SMPUs and nanohybrids.
The flow behavior and moduli of different SMPUs and nanohybrids, which are relevant for processing via 3D printing, are investigated at different temperatures using a Rheometer varying strain rate ( ), the amplitude of strain ( ), and angular frequency ().(Figure 2c and Figure S3) The investigation of the effect of shear rate on the viscosity of the pure HPB20 and its nanohybrid HPB20_0.5NH at 150 o C shows a continuous decrease of the viscosity with increasing shear rate for HPB20, suggesting typical shear thinning behavior due to break of hydrogen bonds between polymer chains and contacts between particles. 58,59Hence, the nanohybrid needs a critical level of stress (yield stress, which is around 50 Pa) to start the flow, and below this critical stress ( ) it   behaves like a solid (inset of Figure 2b) because of the interaction between nanoparticles.This type of flow behavior (high viscosity at low stress) helps the extruded materials retain their shape during printing and increases the resolution of the printed structure. 60The investigation of the effect of amplitude sweeps on the moduli of both pure polymer and its nanohybrids at 150 o C shows that the storage and loss moduli of pure HPB20 and HPB20_1NH remained linear with increasing the amplitude of the shear strain and started to decrease after 13% of the shear strain (Figure S3b).Further, in frequency sweep measurement at different temperatures, the Storage (G′) and Loss (G′′) modulus increases for all pure SMPUs and nanohybrids with increasing angular frequency () as well as with decreasing temperature (Figure S3c).The HPB15 and HPB30 polymers show terminal flow behavior above 130°C.HPB20 shows more complex rheological behavior: either G´ or G´´ dominate depending on the frequency due to different kinds of relaxation processes.Interestingly, the increase of G´ was observed upon a decrease in frequency.Any model of viscoelastic behavior cannot explain this behavior.We believe that the origin of this behavior is the re-establishing of hydrogen bonds between polymer chains with the time-points at low frequencies that are measured at the end of the measurement and take longer.However, the most important observation is that the rheological properties of HPB20 are different from those of HPB15 and HPB30: the last two polymers flow (nearly pure viscous behavior), while HPB20 is viscoelastic.The reason for this difference is the high molecular weight of HPB20 that results in a large relaxation time of polymer chains and observation of viscoelastic behavior on the time scale studied by rheology.However, in nanohybrids, especially with a higher concentration of nanoparticles (0.75 and 1wt %), G′′ remains higher than G′ at 160 and 150 o C within the whole frequency range (0.1 to 100 rad/s).Crossing between G′ and G′′ happens at lower temperatures (140 o C and 130 o C) because of the lowering of the melting temperature (T m ) in the presence of NPs (c.f.evident from DSC thermogram).This is to mention that all the frequency sweep measurements have been done within the linear region of the modulus.
The thermal stability of polymers is a very important factor for melt extrusion-based 3D printing.
The thermal stability of pure shape memory polyurethanes and nanohybrids is investigated using thermogravimetric analysis (TGA) (Figure S4 c) The decrease of viscosity with increasing shear rate is associated with the shear thinning effect for both HPB20 and HPB20_0.5NH.Higher slop for HPB20_0.5NHcompared to HPN20 refers to the greater shear thinning for nanohybrids.Similar behavior of viscosity is also observed with increasing shear stress (inset).
The dynamical mechanical analysis (DMA) has been performed with increasing temperature and at a constant frequency of 1 Hz to understand the phase/relaxation/structural transitions of the programmed shapes of the 3D printed SMPUs and nanohybrid.Figure 3a and Figure S5a show changes in storage (E′) and loss (E′′) modulus for all the SMPUs and nanohybrid with increasing the temperature from 20 o C to 50 o C. A significant lowering of the moduli has been observed within 30 to 40 o C for HPB15, HPB20, and HPB20_0.5NH with a plateau before and after that temperature range, while no significant changes in moduli have been observed for HPB30 within that temperature range (Figure S5b).Hence, a clear transition taking place for HPB15, HPB20, and HPB20_0.5NHwithin the temperature range of 30 to 40 o C that will allow them to demonstrate the shape memory behavior within the physiological temperature range of most mammals, including humans. 51,61Further, it is also observed that the dropping of moduli in the case of HPB15 and HPB20_0.5NHtakes place at a relatively lower temperature as compared to HPB20, supports the early recovery of the programmed shape of HPB15 and HPB20_0.5NH as compared to HPB20 during the formation of T junction through the sequential folding of the two components.
In general, in SMPUs three types of blocks (diisocyanates, diols, and chain extenders) are linked together.These blocks are, however, able to undergo phase segregation.Small-angle X-ray scattering (SAXS) is suited to address the individual small units inside the SMPUs and their hybrids, is important for understanding phase separation.For the SAXS study (Figure . 3b-c), samples that show the intended shape-changing behavior in a temperature range suited for biomedical applications were chosen, in detail HTP15, HTP20, and HTP20_0.5NH.All samples are investigated in the permanent shape (after 3D printing) and in the temporary shape (after 3D printing and programming).An anisotropic scattering pattern is observed in the 2D SAXS diffraction pattern for the permanent shapes of pure SMPUs and nanohybrid, indicating alignment of the lamellae in a particular direction during 3D printing. 62,63Except for HTP20 in temporary shape, all samples show a relatively small spatial/angular distribution of lamellae exhibit a high degree of order compared to the mainly randomly dispersed ones of programmed HPB20.
Interestingly, the presence of the NPs improves the ordering of the lamellae after programming compared to others. 64,65This is to mention that Bragg reflections are predominantly visible in 90° to print/programming direction.The corresponding 1-dimensional data (1D SAXS, Fig. 3c) show a correlation peak in the range of q ≈ 0.25-1.0nm -1 (maximum around q max ≈ 0.4 nm-1) characteristic of the polyurethanes' phase-segregated morphology.According to Bragg's law, the average size of the interdomains is ca.l ≈ 15 ± 3 nm ( ) for all samples.Note that the 2   ≈  correlation length (l) value is slightly smaller for the programmed samples compared to the corresponding 3D printed ones, indicating alignment of the lamellae of the actuating domain (soft segment) during programming.Comparing HPB20 in its permanent and temporary shape, the alignment of the lamellae is more regular in the permanent shape.Additionally, after adding 0.5 wt% CuS-PVP NPs as filler to programmed HPB20, the regular alignment of interdomains is significantly enhanced compared to the data of programmed HPB20 (more pronounced Bragg peaks), allowing the formation of smaller but more easily orientable crystalline domains.Two-dimensional small angle X-ray scattering (2D SAXS) pattern of different pure SMPUs and nanohybrid showing the alignment of the lamellae after 3D printing (permanent shape) and after 3D printing followed by programming (temporary shape).The anisotropy indicates the orientation of the lamellae of SMPUs in the direction of drawing; c) One-dimensional (1D) SAXS patterns of different pure SMPU and nanohybrid exhibits that the characteristic length decreases after programming that supports the alignment of the polymer chains during programming.Here 'P' refers to the permanent shape, and 'T' refers to the temporary shape.
To fabricate the T-junction, the individual components are 3D printed using a commercially available pneumatic pressure-assisted melt extrusion-based 3D printer (Figure S6a and Video SV1).The digital model of individual components is prepared using commercial CAD software (Autodesk Fusion360) and modified using the native printer software.The surrounding temperature of the extruder that is required to melt the polymer as well as to maintain the continuous flow of the polymers through the nozzle of melt extrusion-based 3D printing increases from 140 o C for HPB15 to 180 o C for HPB30 (Figure S6b).Hence, the printing temperature increases with the increasing HSC of SMPUs because of the increase of the melting temperature from 105 to 150 o C with increasing the HSC of the SMPUs (c.f.evident from DSC thermogram).
printing temperature as well as with increasing the pneumatic pressure (Figure S6c and Figure S6d) For HPB20, the wall thickness increases from 0.3 mm to 1.1 mm with increasing temperature from 140 o C to 165 o C when pressure is constant.While the wall thickness increases from 0.25 mm to 1.5 mm with increasing pressure from 0.3 mm to 0.7 mm at a constant temperature.It is also observed that the concentration of the nanoparticles (up to 1wt%) does not affect the printing conditions.The smallest achievable diameter of the components using a nozzle of 350 m diameter is 2 mm.(Figure S7a).The morphological investigation of the printed structure using a scanning electron microscope shows a microgroove pattern on the surface of the printed structure.
Interestingly, the diameter of this individual microgroove is higher (50 m) in HPB20_0.5NHnanohybrid (Figure S7b) as compared to pure HPB20 (40 m).The high yield stress in HPB20_0.5NHrestricts the spreading of the nanohybrid compared to pure HPB20 after extrusion and helps to retain the diameter of the microgrooves. 66 After printing the components (C1 and C2) of T-junction using different shape memory polyurethanes (SMPUs) and their nanohybrids, their shape-changing efficacy was investigated in terms of shape-changing temperature, shape fixity, shape recovery, and recovery kinetics for the successful construction of the T-junction through the sequential folding of the two components.
To check the shape-changing behavior of different SMPUs and their nanohybrids, the 3D printed cylindrical hollow components were cut manually and made flat at the above transition temperature of polymers, and the flat shapes were frozen by decreasing the temperature below the transition temperature.This whole process is called programming. 67The programmed or temporary shapes started to recover their permanent shape with time in the presence of an appropriate stimulus.Here, the temperature is used as a stimulus for pure SMPUs, while nearinfrared light is used as a stimulus in nanohybrids.It is observed that the shape recovery temperature (T r ) for pure SMPUs increases from 33 o C for HPB15 to 70 o C for HPB30.(Figure S8a) Hence, the shape recovery temperature increases with increasing the hard segment content (HSC) of SMPU.Further, the shape recovery efficacy is also increased initially from 81% for HPB15 to 90% for HPB20 and then decreases to 87% for HPB30 (Figure S8b).Similarly, the shape fixity increases initially from 84% for HPB15 to 97% for HPB20 and then decreases to 90% for HPB30.Although the shape fixing of HPB20 is also possible at 20 o C, the shape fixing of all the SMPUs has been done at 10 o C to achieve maximum shape fixity.Hence, HPB20 is showing better shape memory behavior in terms of better shape fixity and higher recovery ratio as compared to HPB15 and HPB30.The better temporary shape retention efficacy of HPB20 as compared to HPB15 and HPB30 is because of its higher molecular weight, which ensures elastic behavior in a broader time scale range.Further, only HPB15 and HPB20 are showing shape recovery behavior within the physiological temperature range.In the case of nanohybrids, the recovery of the permanent shape occurs under illumination with light because of the photothermal effect of the CuS-PVP nanoparticles.The CuS-PVP nanoparticles produce heat, absorbing near-infrared light (~800 nm) that helps the melting of the crystalline soft segments (actuating segment) of the SMPUs.Figure 4a shows that the shape recovery percentage increases from 90% for pure HPB20_0.2NH to 93% for HPB20_0.5NH, and then it decreases to 86% for HPB20_1NH, while the shape fixity continuously decreases from 99% for HPB20_0.2NH to 86% for HPB20_1NH.
Hence, the temporary shape retention efficiency of SMPU decreases with the increasing concentration of CuS-PVP NPs.In contrast, the shape recovery efficiency is high at lower concentrations of CuS-PVP NPs, but at high concentrations of the NPs, it decreases even more than pure HPB20.The reason for this trend is the formation of elements of particle network with yield behavior -structures formed by particles slow down (high viscosity) and even oppose recovery of polymer (particle network has elastic properties below yield stress).
The shape recovery kinetics of different nanohybrids in the presence of light is investigated by plotting the recovery percentage of shapes at different time intervals during the recovery process.
The shape recovery profile of different nanohybrids with time (Figure 4b and For future biomedical applications, it is very important to investigate the cell-material interactions in terms of cell adhesion, cell proliferation, and cell viability on top of the development materials. As endothelial cells are responsible for the interior of blood vessels. 68Hence, we performed all the in-vitro cell culture studies using endothelial cells (HUVECs).Two sets of samples are prepared for cell culture studies: 3D printed flat structures of HPB20 and HPB20_0.5NH are prepared for investigating the cell proliferation and viability, while 3D printed T-junction was used for investigating the adhesion and monolayer formation of cells inside the T-junction after folding.
The proliferation of the endothelial cells with time on the 3D printed flat surface has been investigated using an alamarBlue reduction assay.The intercellular metabolic reduction of resazurin produces a highly fluorescent form of resazurin, and the fluorescence intensity is directly proportional to the number of living cells respiring. 69Figure 5a shows that the fluorescence intensities on day 1 are 43 and 51% for HPB20 and HPB_0.5NH,respectively, increasing to 62 and 67% on day 7. Hence, the metabolic rate of the endothelial cells increases with time for both pure SMPU and nanohybrid, indicating cells are proliferating on the surface of both materials.
Further, the viability of the endothelial cells on the surface of the pure SMPU and nanohybrid has been investigated using a live-dead assay in which calcein AM is used to stain the live cells.In contrast, ethidium bromide-1 is used to stain the dead cells only.The cell viability (%) has been determined using the number density of live cells with respect to total live and dead cells.
Typically, the calcein AM is hydrolyzed by the esterase of living cells only and produces green fluorescence remaining within the cytoplasm of the live cells, while ethidium homodimer-1binds with the nucleic acid of dead cells and produces red fluorescence.Figures 5b & 5c show high viability of the endothelial cells on day 7 with slightly higher viability of cells on HPB20_0:5NH (90%) compared to pure HPB20 (85%).
To prove the folding behavior of T-junction with cells (Figure 5d) followed by the proliferation of the cell on the inner side of the tube, 3D printed T-junction was programmed into a flat structure and placed in a petri dish.This is to mention that for this study, T-junctions are 3D printed using

Conclusions
In this paper, we reported a novel approach to fabricating vascular junctions, particularly the Tjunction of the vascular network, using the advanced extension 4D printing technique that resembles modular origami.Our approach permits the construction of a T-junction through coordinated sequential folding of two shape-changing elements with different shape-changing behavior.To successfully execute our objective, the T-junction is split into two tubular hollow components and 4D printed using synthesized shape-memory polyurethanes (SMPUs) and their blends with nanoparticles.We, in detail, characterized the properties of the polymers and blends that allowed the finding of optimal conditions to achieve the best printability and proper stimuliresponsive properties, which allow coordinated shape transformation, as well as shape fixation and recovery.Here, we demonstrated three approaches to achieve the T-Junction through coordinated sequential folding of two components by combining differential shape memory behaviors of different synthesized SMPUs and nanohybrids.Among these approaches, the combination of two different nanohybrids with different shape recovery responses is able to construct the T-junction more efficiently and accurately as compared to other combinations.The used polymers, blends as well as printed structures were found to be biocompatible, which is expressed in high cell viability.
Finally, the T-junction with attached cells has been fabricated through coordinated sequential folding in the presence of NIR light.The histochemical analysis reveals that the cells adhere nicely on the surface of the T-junction and proliferate with time.Hence, the proposed modular-origami-inspired 4D printing approach will open new possibilities for the fabrication of complex structures, which are important not only for tissue engineering in general but also for other technical fields.
. The nanohybrids of different polyurethanes were prepared by dispersing the CuS-PVP nanoparticles homogeneously into the DMF solution of SMPUs and drying the casted solution at 80 o C under reduced pressure in a vacuum oven.Different nanohybrids were prepared with varying wt% of the nanoparticles in SMPU.

Table 1 :
Molar ratio, hard segment content (HSC, %), and molecular weight of different synthesized SMPUs (Unit of molecular weight is Da or gmol -1 ).HMDI is hexamethylene diisocyanate, PCL is polycaprolactone, and BD is butane diol.(M n refers to the number average molecular weight, M w refers to the weight average molecular weight and PDI refers to poly dispersity index.)Rheological Properties.The rheological behavior of pure SMPUs and their nanohybrids was investigated using a multidrive rheometer of Anton Paar (MCR 702 MultiDrive).The samples were placed between two parallel plain plate geometry of 25 mm diameter.The samples' complex viscosity, storage modulus, and loss modulus were recorded by sweeping the angular frequency from 0.1 to 100 rad/s and varying the temperature from 200 o C to 100 o C. Dynamic Mechanical Analysis (DMA) of pure SMPUs and nanohybrids.The mechanical properties of 3D printed samples (dimension: 0.30 × 4 × 7 mm 3 ) of different shape memory polyurethanes and their nanohybrids were investigated using Anton Paar MCR 702 MultiDrive.The change in elongational storage and loss modulus was recorded with increasing temperature from 20 to 50 o C at a constant frequency of 1 Hz and varying the frequency from 0.1 Hz to 10 Hz at a constant temperature of 40 o C.During the measurement, samples were stretched by 5% and stress was kept at 0.5 MPa.Differential Scanning Calorimetry (DSC).The melting temperature, crystallization temperature, and heat of fusion value (H) of 3D printed samples of different SMPUs and their nanohybrids were investigated using a Mettler differential scanning calorimeter.The samples were scanned in the temperature range of -40 to 200 o C keeping the heating and cooling rate 10 o min -1 and 5 o min - 1 , respectively.
2×10 5 cells per square centimeter were seeded on the 3D printed samples to check the cell adhesion, viability, and metabolic activity.Cell Proliferation and Cell Viability.The proliferation of the cells on the samples' surface was investigated by measuring the metabolic activity of the seeded endothelial cells at predetermined time intervals using an alamarBlue assay.10 % (v/v) of the alamarBlue reagent in HUVEC cell culture media was prepared and added to the 48 well plates to cover the cells on the 3D-printed samples.After 90 minutes of incubation in a CO 2 incubator and gentle shaking in all directions to ensure a homogeneous distribution, the media containing reduced almarBlue were taken out and kept under dark and icy conditions.After that, 100 l of aliquots were pipetted into 96 well plates, and the fluorescence intensity was measured at 600 nm after excitation at 535 nm using the plate reader (TriStar² S LB 942 Multimode Microplate Reader, Berthold Technologies GmbH & Co.KG, Germany).The negative and positive controls were non-reduced and 100% reduced alamarBlue without cells, respectively.

1 in 2
ml of DPBS.Staining of Actin Filaments (F-actin) and Nucleus.The adhesion and monolayer formation of endothelial cells over the surface of the samples were investigated by staining the fibronectin and nucleus of endothelial cells with Phalloidin daylight 488 conjugated 300 units (prod # 21833 ThermoFisher) and 4′, 6-diamidino-2-phenylindole (DAPI) at day 7.The staining solution is prepared by adding 500 l of DAPI (concentration:0.1 mg/ml) and 250 ml of Phalloidin (200 units/ml) stock solution in 10 ml of DPBS.Before staining the cells, media was aspirated from the well plate, and cells were washed with DPBS twice.Then, cells were treated with freshly prepared 3.7% formaldehyde for 5 minutes to fix the cells.After fixing the cells, the cell membrane was permeabilized by treating the cells with 0.1% Triton 100X solution for 5 min at ambient conditions.Finally, cells were washed twice with DPBS and stained with DAPI/phalloidin solution.Statistical Analysis.Obtained data were shown as the mean ± standard deviation (SD) (3-7 replicates were used).Student t-test and one-way analysis of variance (ANOVA) were performed to analyze the differences between the experimental groups.A value of P≤ 0.05 was considered statistically significant.

Figure 1 .
Figure 1.Illustration of fabrication of T-junction through the sequential folding of 4D printed

Figure 2 .
Figure 2. Thermal and flow properties of SMPUs and nanohybrids: a) and b) DSC thermograms

Figure 3 :
Figure 3: Thermomechanical properties and structural characterization: a) Dynamic mechanical

Figure
S6e shows two different 3D-printed components of the T junction, which are hollow tubular self-standing polymeric structures.The bigger component is Component 1 (C1) and the smaller component is Component 2 (C2).Proper arrangements of C 1 and C 2 can construct a T junction.Further, it is also possible to construct the T junction of different diameters using two individual 3D printed components of different diameters.

Figure 5 :
Figure 5: Cell culture studies with human umbilical vein endothelial cell line (HUVEC): a) Almar