Hyperelastic, shape‐memorable, and ultra‐cell‐adhesive degradable polycaprolactone‐polyurethane copolymer for tissue regeneration

Abstract Novel polycaprolactone‐based polyurethane (PCL‐PU) copolymers with hyperelasticity, shape‐memory, and ultra‐cell‐adhesion properties are reported as clinically applicable tissue‐regenerative biomaterials. New isosorbide derivatives (propoxylated or ethoxylated ones) were developed to improve mechanical properties by enhanced reactivity in copolymer synthesis compared to the original isosorbide. Optimized PCL‐PU with propoxylated isosorbide exhibited notable mechanical performance (50 MPa tensile strength and 1150% elongation with hyperelasticity under cyclic load). The shape‐memory effect was also revealed in different forms (film, thread, and 3D scaffold) with 40%–80% recovery in tension or compression mode after plastic deformation. The ultra‐cell‐adhesive property was proven in various cell types which were reasoned to involve the heat shock protein‐mediated integrin (α5 and αV) activation, as analyzed by RNA sequencing and inhibition tests. After the tissue regenerative potential (muscle and bone) was confirmed by the myogenic and osteogenic responses in vitro, biodegradability, compatible in vivo tissue response, and healing capacity were investigated with in vivo shape‐memorable behavior. The currently exploited PCL‐PU, with its multifunctional (hyperelastic, shape‐memorable, ultra‐cell‐adhesive, and degradable) nature and biocompatibility, is considered a potential tissue‐regenerative biomaterial, especially for minimally invasive surgery that requires small incisions to approach large defects with excellent regeneration capacity.

responses in vitro, biodegradability, compatible in vivo tissue response, and healing capacity were investigated with in vivo shape-memorable behavior. The currently exploited PCL-PU, with its multifunctional (hyperelastic, shape-memorable, ultra-celladhesive, and degradable) nature and biocompatibility, is considered a potential tissue-regenerative biomaterial, especially for minimally invasive surgery that requires small incisions to approach large defects with excellent regeneration capacity. and polyurethanes (PU) and toward the design of more tailorable polymeric materials with enhanced biological and mechanical properties. [1][2][3][4][5] PU-based polymers have recently attracted substantial interest for use in various tissue-regenerative processes, including muscle, cartilage, blood vessel and bone regeneration, because of their tunable properties, such as biodegradability, elasticity and resistance to flex fatigue. [6][7][8][9][10] However, poor cell adhesiveness and the need for further bio-friendly surface modification due to the innate absence of functional groups from their original structures remain obstacles to the use of PU-based polymers as substrates for clinical tissue regeneration along with low biodegradability, [11][12][13][14][15] while cell attachment is considered the essential first step to trigger proliferation, migration and differentiation, which are prerequisites for tissue regeneration. 16,17 Myriad strategies have been attempted to increase the cell adhesiveness of PU-based polymers for use in clinical settings. [18][19][20] The aromatic diisocyanates, which has an isocyanate (N C O) group directly attached to the aromatic ring, could improve the cell-adhesive functionalities when used in synthesizing PU polymers and enhance mechanical properties, [21][22][23][24][25][26] but the utilization of these materials in clinical settings is limited due to the low degradation rate and to tumorigenesis induced by degradation or unreacted monomers. 27,28 Instead, aliphatic diisocyanates has been introduced as a component of PU polymers due to its superior biocompatibility without toxic degradation byproducts, but they had compromised mechanical properties with moderate cell adhesiveness. 29,30 Although there have been a lot of efforts to enhance biological and mechanical properties of aliphatic isocyanate-based PU polymers by structural or component modifications, they are still suboptimal. 31,32 Shape-memory biomaterials have also received attention as morphologically responsive materials with a variety of potential applications, particularly in biomedical devices for minimally invasive surgery and the delivery of therapeutics and cells for tissue engineering. [33][34][35][36][37] Shape-memory features have been associated mainly with metals and detected with synthetic polymer materials and some biological substrates. [38][39][40][41] To combine the merits of both elasticity and shape-memory properties, shape-memorable PUs has been developed, but the mechanical strength, shape-memory behavior, and cell-adhesive properties are unsatisfactory for clinical utilization. 37,42 To solve the above issues, PCL diol-1,6-hexamethylene diisocyanate (HDI)-based PU polymers, as an aliphatic isocyanate-based PUs, were developed for biocompatible shape-memory PUs and isosorbide was added as reinforcement for enhanced mechanical properties, but the cell-adhesive property was still subprime. 22,25,43 In particular, even though PCL and isosorbide-based PUs with shape-memorable, degradable, and elastic properties have been reported, those were still lack of mechanical and biological evaluations from in vitro and in vivo studies for being utilized in tissue regeneration, leading to the investigation of new PCL-PU copolymer for biomedical application.
The isosorbide is known as chiral and rigid molecules, and the innocuous nature ensures its use as an biocompatible alternative to petroleum-based polymer, opening up the possibility of utilization for medical devices. 44 But bare isosorbide had the low reactivity of the secondary hydroxyl groups, limiting participation in the copolymer synthesis. 45,46 To tackle above issues, various isosorbide derivatives with enhanced reactivity have been developed, but those were still suboptimal in the aspect of mechanical properties as well as cellularfunctionality for tissue regeneration. 47,48 Here, two newly synthesized isosorbide derivatives (propoxylated or ethoxylated isosorbide) with the enhanced reactivity were introduced for fabricating polyurethanes polymer over bare isosorbide. By increasing the reactivity and participation in the polymer structure, they had enhanced elastic properties as well as a stable shape transition temperature (T m ) around body temperature. Interestingly, specific isosorbide-derivative (propoxylated isosorbide)-incorporated PCL-PU (renamed as ISB-P) was shown to have significantly enhanced cell-adhesive properties for various cell types, including mesenchymal stem cell, compared with that of other shape-memory PUs and even that of FDA-approved PCL. A novel stem cell-adhesive mechanism was elucidated with RNA sequence analysis, siRNA study and functional inhibitor study, revealing adhesion by heat shock protein-mediated integrin α5 and αV. Comparison of the in vitro tissue (muscle and bone) regenerative potential with that of PCL after matching initial cell-adhesive properties presented similar muscle maturation and bone differentiation. In vivo studies revealed biocompatibility, absence of major organ toxicity, tissue (bone)-regenerative efficacy as well as shape-memory properties in sinus and femur of live rabbit. Thus, the current isosorbide-derivative PCL-PU (ISB-P) is considered a promising platform for tissue regeneration, especially for tissue defects with small openings connected to large defects, and is envisaged to extend to other biomedical applications that require repair and regeneration with volumetric selfadjustment.
2 | RESULTS AND DISCUSSION 2.1 | Synthesis of versatile, hyperelastic, shapememorable PCL-PU biomaterials PCL diol-HDI-based PUs were synthesized by one-shot catalyst-free bulk polymerization and named ISB-2 0 (from original isosorbide), ISB-P (from propoxylated isosorbide) and ISB-E (from ethoxylated isosorbide), depending on their isosorbide derivatives (Table S1 and Figure S1). Isosorbide-free PCL-PU was also fabricated as synthesis due to the higher primary alcohol reactivity of isosorbide derivatives for polymerization (Table S2). In addition, this difference in molecular weight among groups has the potential to affect the physicochemical properties, especially the mechanical performances. [53][54][55] Polydispersity index was~2 in ISB-2 0 because secondary alcohol in isosorbide is less reactive for PCL-PU polymerization than primary alcohol and 2propanol in ISB-E and ISB-P, respectively. 56 Next, the mechanical properties of PCL-PU were investigated in tensile mode with a rectangular film (50 Â 5 Â 0.2 mm), and a stressstrain curve was plotted to visualize the hyperelasticity, as approximately determined by (maximum stress) Â (strain at break) values ( Figure 1a,b In addition, the maximum stress and strain could be controlled by the ratio between the PCL diol and HDI of ISB-P with an inverse correlation ( Figure 1b). 25  . ISB-free was excluded for further experiment due to poor elasticity. (c) Bulk elastic recovery test using tension and compression was performed on film and 3D scaffold, respectively, revealing enhanced elasticity in PCL-PU (ISB-P) than PCL (n = 3, ***p < 0.001, ****p < 0.0001 by t test). (d) Dynamic mechanical behavior under cyclic tensile loading conditions (10 cycles, 100%-1100% strain, 10 mm/min), displaying maintenance of hyperelasticity of ISB-P (80%-95% of initial strength) during 10 times cyclic tension against up to maximum tensile strain (~1100%). In contrast, PCL was torn at 2nd cycle tensile with 800% strain. Referring to Figure S4 for dynamic mechanical behavior of other PCL-PUs (ISB-E and ISB-2).
(e) Versatility of PCL-PU (ISB-P) in surgical procedures. The material could be stretched, sutured, bent, twisted, and cut. Scale bar is 10 mm. After suturing to muscle tissue, a stable suture interface between ISB-P and the tissue was observed in Video S1. dynamic force conditions. An example of the surgical compatibility of PCL-PU (ISB-P) is illustrated in Figure 1e, revealing elastically stretchable, bendable, twistable without visible deformation and surgical compatibility in terms of cutting and suturing (Video S1).
Next, shape-memory characteristics of PCL-PU were determined by differential scanning calorimetry (DSC). T m values, where memorybased shape-changes theoretically occur in polymer, was initially determined near body temperature (37.1-38.3 C) for the newly synthesized isosorbides (ISB-P and ISB-E) slightly higher than that of bare isosorbide (36.9 C, ISB-2 0 ) ( Figure S5A). When bulk shape memory property under tension was manually performed on film to confirm shape-memory characteristics briefly, the most recovery property (~82%) was detected in ISB-P than others (30%-70%), which was selected for further shape-memory analyses ( Figure S5B). Thermodynamic mechanical analysis similarly showed~78% recovery of the original shape from the temporarily deformed (10%) PCL-PU specimen (ISB-P as representative) at 40 C in four independent temperature cycles (40 C $ 0 C) ( Figure S6), a typical characteristic of shape memory polymer near body temperature, which is comparable to values obtained from other PU-based shape memory polymers working at 40 or 60 C (32%-95% for shape-recovery and 40%-95% for shape-fixity). 22 After the shape consisting of English letters was memorized at 37.5 ± 0.5 C, the English letters were randomly deformed at room temperature (RT, 20 ± 0.4 C) for 24 h. When the temperature was increased again to 37.5 ± 0.5 C, the original shape of the English letters was recovered from plastically deformed status ( Figure 2b). Similarly, when thin thread at RT was stretched up to 200% for 24 h at 37.5 ± 0.5 C, the thread recovered almost to its original length (118%,~82% recovery) (Figure 2c). When the prestretched (~200%) thread at RT was inserted into pig skin to mimic skin wound suturing and the temperature was increased, the thread tightened to close the wound gap (Video S2). Finally, when the cylindrical 3D scaffold was compressed from the original length down to 39% (À61%) at RT for 24 h to allow plastic deformation and the temperature was increased, the length partially recovered to 81% (~70% of compressed deformation, Figure 2d). However, PCL did not show any shape-memory property in the all conditions above. Unfortunately, the processability of ISB-P by 3D printing was limited due to its thermoset property, similar to other PU polymers, and 3D printable, PU based shape-memory polymer remain elusive. Combined together, regardless of the shape (film, thread, 3D scaffold) and stress direction (random, tension, and compression), shape-memory characteristics were successfully detected in PCL-PU (ISB-P). F I G U R E 2 Shape-memory properties of PCL-PU with various forms and force directions. (a) Stress-strain response of PCL-PU (ISB-P) across a range of temperatures. A total of 4 cycles (40 C $ 0 C, 10%) were performed and measured by thermodynamic mechanical analysis, showing~78% recovery of the original strain from the temporarily deformed (10%) PCL-PU specimen (ISB-P) at 40 C, a typical characteristic of shape memory near body temperature. Blue arrow indicating recovery of strain as shape-memory property.
(b) Shape memory of the letters cut from the membrane form: English letters (ITREN) were randomly deformed at room temperature (RT) for 24 h to allow plastic deformation, and shape recovery was detected at 37.5 C. (c) Shape memory of the thread form; thin thread (2 Â 100 mm) was stretched at RT up to 200% for 24 h and recovered to its original length (118%,~82% recovery) at 37.5 ± 0.5 C. Loose suturing using prestretched (~200%) thin thread at RT became tight at 37.5 C (n = 5, Video S2). (d) Three-dimensional scaffolds (40 mm h Â 5 mm d) recovered by~70% at 37.5 C after compression to 39% of the original length (À61%) at RT for 24 h. PCL was used as a negative control without shape-memory behavior (n = 5). ***p < 0.001 and ****p < 0.0001 by t test. (a)
bone, which are the most abundant target tissues for regeneration in the biomedical field. First, to investigate the possibility as tissue-engineering platform, C2C12 myoblast cells were cultured on ISB-P.
C2C12 myoblast cells matured better on ISB-P than PCL@OP after 7 days based on the levels of myosin heavy chain (MHC)-stained myotubes and their widths as muscle cell differentiation markers ( Figure S16A). Next, to utilize the elasticity of ISB-P, stretching culture could be applied to achieve biomimetically aligned skeletal muscle tissues, as skeletal muscle fiber construction for tissue-engineered implants requires the assembly of unidirectionally aligned juxtaposed myotubes. 92,93 C2C12 cells were seeded on unidirectionally electrospun ISB-P nanofibers (700-1300 nm), indicating 90% F I G U R E 4 Legend on next page. alignment along the fiber direction ( Figure S16B), and stretching culture conditions (4 s stretching (10%) and 6 s rest per cycle, 1 h per day) were then applied. 94 The number and width of MHC-stained cells increased at the early differentiation time point (day 4), revealing possibility of ISB-P for accelerated muscle tissue engineering under dynamic tensional culture conditions due to elasticity ( Figure S16C).
To investigate the eligibility of PCL-PU (ISB-P) as implanted biomaterials for tissue regeneration, an osteogenic capacity was revealed as representative. In vitro osteogenesis study using MSCs cultured on 2D film structure revealed that ISB-P had higher expression of early and late osteogenic genes (RUNX2, ALP, BSP, and OCN) than PCL@OP with enhanced biomineralization, as revealed by qPCR and Alizarin red S (ARS) staining (Figure 6a). To assess the 3D-bone forming ability, 3D ISB-P scaffold with interconnected porous was fabricated by the salt-leaching method with pore sizes (50-500 μm) similar to that of PCL ( Figure S17). When the cell adhesion and mineralization of MSCs were tested, cell adhesion was well recognized even inside pores, and penetration of cells to inner scaffold structures through interconnected pores over 5 days of culture was similarly visualized ( Figure 6b). Biomineralization at 14 days of differentiation was comparably detected between groups unlike the data from 2D film. The discrepancy between 2D and 3D culture data might come from bulk elasticity and molecules diffusion. [95][96][97] In summary, myoblast cells and osteoprogenitors differentiation on ISB-P was confirmed with 2D film, pseudo3D electrospun or 3Dporous structures even under tensile stress (for muscle differentiation), showing the versatility of ISB-P for tissue regeneration due to its elasticity, cell-adhesive and differentiation-conductive properties with appropriate cytocompatibility for long-term culture (up to 14 days). Although the mechanism study might be needed to understand the significant increase of osteogenic differentiation in ISB-P (even though it was observed only in 2D films), the engagement of heat shock proteins upregulated in ISB-P during cell adhesion is suggested as one of the possible biological machinery. Because there was no significant difference in surface characteristics, including roughness, hydrophilicity, and chemical structure, between ISB-P and PCL@OP, while, HSP gene levels were higher in ISB-P.  100 and ultrasound (for HSP70 and 90). 101 Overall, the osteogenic differentiation might be promoted via upregulation of osteogenic signaling, which was enhanced by continuously higher heat shock proteins levels on ISB-P from initial cell attachment.

| In vivo biocompatibility and bone induction of the PCL-PU biomaterial
Degradation study using base chemical (NaOH, 5 M), hydrolyzing esters by OH radical into a salt and alcohol, was performed to reveal degradation ability. It showed a degradation rate of~14.3 wt%/day from ISB-P, much faster than that of PCL (~4.5 wt%/day) along with other PCL-PUs (7-12.5 wt%/day, Figures 7a and S18A) due to asgiven hydrophilic characteristics, increasing the water penetration into the matrix and give increased rates of hydrolysis of the ester and urethane bonds. 25 In challenge with enzymatic degradation using esterase (2 mg/ml), enzymatically splitting esters into an acid, and alcohol, 102  The in vivo results together showed that ISB-P was highly effective, even comparable to PCL, in accelerating early neobone regeneration, suggesting potential applications as an implantable biomaterial for bone repair and regeneration.
To investigate the possible clinical applicability of ISB-P based on its shape memory characteristics, two in vivo clinical models were suggested: maxillary sinus bone lift and a bone defect with a small outer exposure but a large inner space to fill in, possibly caused by a bullet or sharp weapon. After memorization of the original shape at 37 C, the ISB-P scaffold, coated with radiopacifier (iodoform) for visualization by x-ray, was compressed at RT (~30% from the original height) and inserted into the rabbit maxillary sinus and tibia bone.
Comparison of x-ray images immediately after implantation and 60 min later in the rabbit body (37 C) showed that the ISB-P scaffold was enlarged compared to the condensed structure, which was a unique characteristic of the shape-memory polymer, unlike the PCL derivatives ( Figure 7g). Although the bone regenerative potential of the recovered ISB-P was not directly investigated, it was assumed to have similar bone-forming ability to that in a rat calvarial defect model, due to identical chemical structure before and after the shape change. The in vitro and in vivo results together showed that ISB-P accelerated regeneration of tissue, including bone, which might result from the collective effects of ultra-cell adhesiveness, biocompatibility without distinct immune response, target-tissue conductivity, and shape-memory properties in various forms, indicating the potential applications of ISB-P in tissue-regenerative implantable biomaterials.

| CONCLUSION
Here, the currently exploited PCL-PU (especially ISB-P) were synthesized for the fabrication of a biocompatible, and multifunctional (hyperelastic, shape-memorable, ultra-cell-adhesive, regenerative, and degradable) tissue-regenerative biomaterial (Figure 8). The unique combination of notable mechanical performance, shape-memory effects, and ultra-cell-adhesive properties make appropriate forms (e. g., patch, thread, electrospun, 3D scaffold) of the engineered material suitable as a regenerative substrate for various tissues (e.g., bone, muscle, and skin) in the biomedical field. Cell adhesion mechanism of ISB-P was distinctly revealed as heat shock protein-mediated integrin α5 and αV activation, leading to further development of newly synthesized, tailored polymer for enhancing specific biological behavior. The

| Fabrication of films, nanofibers, and 3D scaffolds
The

| Chemical characteristics
The 1 H NMR spectra for the synthesized PU as a 5% (w/v) polymer solution in CDCl 3 were recorded by a Bruker Avance 500 × F I G U R E 8 Summary of optimal PCL-PU (ISB-P) characteristics for tissue regeneration. Biodegradable PCL-PUs were newly synthesized with PCL, HDI and isosorbide or its derivatives. From four candidates, ISB-P was selected as optimal polymer in tissue regeneration or engineering. Currently optimized PCL-PU (ISB-P) is hyperelastic (~1100% strain Â~50 MPa), shape-memorable (room temperature (RT) $~37 C), ultracell-adhesive (better than bare PCL) via HSP mediated integrin activation, and tissue regenerative with great biocompatibility against tissue and major organs. HDI, 1,6-hexamethylene diisocyanate; HSP, heat shock protein; PCL, polycaprolactone; RT, room temperature; U, urethane group.

| Physical properties
The surface roughness was measured by a surface roughness meter

| Shape-memory test
ISB-P was dissolved in N,N-dimethyl-formamide at 20 wt%, stirred at room temperature for 24 h, and dried in the oven with appropriate polytetrafluoroethylene mold (0.2 mm Â 5.0 mm Â 10 mm) at 60 C.
The shape-memory test was initially performed using a dynamic  Bioneer. The Ct value was used to determine the efficiency of different genes with respect to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the endogenous reference amplified from the samples.
The internal control ΔCt was calculated by (ΔCt = Ct a target gene ÀCt a GAPDH), and then, the relative transcript quantities were calculated using the ΔΔCt (ΔΔCt = ΔCt a target sample ÀΔCt a control sample) method. The fold change for each gene was subsequently determined from 2 ÀΔΔCt . Each measurement was performed in triplicate (n = 3). The primer sequences of the genes are shown in

| In vitro biocompatibility test
Human monocytes (THP1, ATCC) were differentiated into adherent macrophages by PMA (50 ng/ml, Peprotech) treatment for 24 h and seeded on PCL@OP, ISB-P, and TCP. THP-1 cells were lysed immediately after a 48 h polarization step, and qPCR was performed to measure inflammatory gene (TNF-a, IL-1b, and IL-6) expression. Primer sequences are listed in Table S3. After 2 and 4 weeks of implantation, the surrounding tissue was collected for histological analysis after immediate fixation in 10% neutral buffered formalin for 24 h at RT. And after 8 weeks of implantation, the major organs (kidney, liver, spleen, lung, and heart) were obtained to access the organ toxicity based on histological analysis. Next, a long-term bone regeneration study was performed in a rat calvarium regular-shape defect model established as described previously. 109,110 The calvarial skin and periosteum were exposed by a sagittal incision

| Histological and immunohistochemical analysis
All the samples were dehydrated in an ethanol series, embedded in paraffin, and sectioned at 5-μm thickness. 111 Especially the tissue samples obtained from calvarial defects were completely decalcified in RapidCal™ solution (BBC Chemical Co.) before dehydration. For histology analysis, the tissue slides were stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT) and visualized by light microscopy after hydration. Inflammation scores were further determined by pathologist using high-magnified H&E stained images. 112 For IF examination, the sections were incubated with primary antibodies targeting iNOS (ab15323, Abcam), TNF-α (52B83, Santa Cruz), CD206 (6A598, Santa Cruz), and Arg-1 (ab203490, Abcam) to detect proinflammatory and anti-inflammatory markers. Secondary antibodies conjugated with rhodamine (Alexa Fluor 594) and FITC (Alexa Fluor 488) and DAPI were applied for counterstaining. The IF-stained sections were observed using a fluorescence microscope (IX71, Olympus, Japan), and quantitative analysis was performed using ImageJ software.