A Facile and Cost-Effective Method to Prepare Biodegradable Poly(ester urethane)s with Ordered Aliphatic Hard-Segments for Promising Medical Application as Long-Term Implants

The article below describes a simple methodology to prepare cost-effective biodegradable poly(ester urethane)s (PEUs) with ordered hard segments (OHS) for medical application as long-term implants. A low-cost diurethane diol (1,4-butanediol-hexanediisocyanate-1,4-butanediol, BHB) was first designed and synthesized. Consequently, the BHB was employed as a chain extender to react with NCO-terminated poly(ε-caprolactone) to obtain PEUs. The molecular structural formats for BHB and PEUs were defined through NMR, FT-IR, and MS together with GPC, while the influence of OHS content on physical/chemical features for casted PEU films was investigated. The introduction of OHS could contribute to forming denser hydrogen-bonds, and consequently produce a compact network structure, resulting in great tensile capacity, low water absorption, and slow hydrolytic degradation rate by PEU films. PEU-2.0 films, which possessed the highest OHS content within PEUs, exhibited 40.6 MPa tensile strength together with 477% elongation at break, 4.3 wt % equilibrium water absorption and only 29.5% weight loss post-12 months’ degradation. In addition, cytotoxicity analysis of film extracts indicated that the cell viability of all PEUs containing OHS exceeded 75%, indicating good cytocompatibility. Due to outstanding tensile features, high biostability, nontoxic and absorbable degradation products and acceptable cytocompatibility, the cost-effective materials exhibited promising applications in the field of long-term implants.


Introduction
Polyurethane (PU), a type of polymer containing carbamate group, can be designed to have a large degree of freedom, and its physical/chemical features could become regulated by modifying the class and proportion of soft/hard regions [1,2]. Besides such adjustable tensile features, the micro-phase segregation stemming from incompatibility of soft/hard regions endows PU with adequate biocompatibility. These advantages make it widely employed within medicine, such as for artificial skin/tissues/organs, surgical sutures, vascular grafts, and drug carriers [3][4][5][6][7].
In an effort to diminish the influence of biological materials over human organs, medical materials employed in the body are required to have biological stability in the early stage of use and can be gradually degraded and absorbed in the later stage [8]. Therefore, both soft and hard segments of PU are expected to degrade into non-toxic and absorbable small molecules. Polyesters, especially poly(ε-caprolactone) (PCL) and polylactide, are susceptible to hydrolysis and are typically employed as soft segments to prepare biodegradable PUs [9][10][11][12]. However, the hard segments of PUs are based on diisocyanate, which can produce diamines as degradation products. One method for obtaining excellent tensile features, commercial medical-grade PUs for long-term medical implementations, including Biospan TM , Elasthane TM, and Biomer ® [13,14], is derived from aromatic diisocyanate. The π-π actions between adjacent aromatic rings could generate inter-chain actions and enhance PCL (M n = 1000 g/mol) and 1,4-butanediol (BDO, 99%) were procured through Aladdin Reagent Co.™ (Shanghai, China) and vacuum-dehydrated (five hours/100 • C) before utilization. Hexanediisocyanate (HDI, 99.6%) and stannous octoate (Sn(Oct) 2 , >95%) were procured through Sigma-Aldrich (Shanghai) Trading Co., Ltd., (Shanghai, China). N,N-Dimethylformamide (DMF), trichloromethane (CHCl 3 ), and acetone (Fuyu Fine Chemical Co., Ltd.™, Tianjin, China) were purified through standardized techniques.

Synthesis of BHB
The synthetic pathway of BHB is shown in Figure 1a. HDI was added dropwise into BDO (molar ratio of BDO and HDI was 8:1) at 75 • C under mechanical stirring. Subsequently, the reaction proceeded for another 4 h at the same temperature. The system was cooled to ambient temperature, with the resultant mixture filtrated under vacuum. The crude product was washed several times using anhydrous acetone to render BHB as a white powder, which was consequently vacuum-dried to constant weight. The yield was 89.0%.

Preparation of PEUs and PEU Films
A series of PEUs-with various chemical compositions-was prepared according to the formulations in Table 1, with a reaction overview illustrated in Figure 1b. Typical process: PCL and HDI were dissolved within anhydrous DMF (1.0 g/mL) at ambient temperature for obtaining a homogeneous solution. Post-purging with dry argon, Sn(Oct)2 (2 wt ‰ of PCL and HDI) was introduced and the reaction was conducted at 80 °C to prepare the prepolymer of NCO-terminated PCL (OCN-PCL-NCO). Once -NCO content (identified through di-n-butylamine-acetone titration [26]) reached the theoretical value range, DMF solution for BHB (0.2 g/mL) was introduced dropwise within the mixture, with such a reaction permitted to take place until absorption-peak for -NCO in FT-IR spectrum (2260~2275 cm −1 ) disappeared completely. Consequently, additional DMF was introduced to dilute the mixture. The diluted solution (0.05 g/mL) was de-aerated using diminished pressure and subsequently poured within a Teflon ® mold. The solvent was evaporated (40 °C) for obtaining PEU films (thickness: 0.3 ± 0.025mm), which were termed PEU-X (X: molar ratio for HDI and PCL).

Preparation of PEUs and PEU Films
A series of PEUs-with various chemical compositions-was prepared according to the formulations in Table 1, with a reaction overview illustrated in Figure 1b. Typical process: PCL and HDI were dissolved within anhydrous DMF (1.0 g/mL) at ambient temperature for obtaining a homogeneous solution. Post-purging with dry argon, Sn(Oct) 2 (2 wt ‰ of PCL and HDI) was introduced and the reaction was conducted at 80 • C to prepare the prepolymer of NCO-terminated PCL (OCN-PCL-NCO). Once -NCO content (identified through di-n-butylamine-acetone titration [26]) reached the theoretical value range, DMF solution for BHB (0.2 g/mL) was introduced dropwise within the mixture, with such a reaction permitted to take place until absorption-peak for -NCO in FT-IR spectrum (2260~2275 cm −1 ) disappeared completely. Consequently, additional DMF was introduced to dilute the mixture. The diluted solution (0.05 g/mL) was de-aerated using diminished pressure and subsequently poured within a Teflon ® mold. The solvent was evaporated (40 • C) for obtaining PEU films (thickness: 0.3 ± 0.025 mm), which were termed PEU-X (X: molar ratio for HDI and PCL).

Characterization and Instruments
Characterization: NMR spectra were registered through the VANCE II ® 400 MHz NMR spectrometer (Bruker™, Rheinstetten, Germany). FT-IR spectra were collected within the Bruker™ Alpha ® spectrometer carrying attenuated total reflectance (ATR ® , Bruker™) feature-at a frequency range of 4000-400 cm −1 , with 4 cm −1 resolution. Mass spectrum was obtained on a Bruker™ maXis ultra HR-TOF-MS system with a methanol solution of~10 µg/mL. Molecular weights (M n and M w )/molecular weight distribution (M n /M w ) were determined through GPC (Waters 2414 ® ) with THF as the continuous phase and monodisperse polystyrene serving as control.
Thermal features: Thermo-gravimetric analysis (TGA) was conducted for establishing the thermal stability of polymer materials. This was executed with a thermo-gravimetric analyzer (TGA 2950 ® , TA Instrument™, New Brunswick, NJ, USA). Thermal weight loss behaviors were supervised, with a temperature range of 50-550 • C (heating rate = 10 • C/min under nitrogen (50 mL/min)). Differential-scanning calorimeter (DSC) was adopted for analyzing thermal transitions of the polymers using a DSC2910 ® (TA Instrument™, USA) (−40 to 100 • C) under consistent nitrogen flow, while the heating rate was 10 • C/min. Individual samples were subjected to two back-to-back runs and registered, with such dataset outcomes obtained through a second heating iteration.
Tensile features: Tensile behavior for polymer films were obtained on a universal testing machine (UTM100 ® , Shandong Wanchen Instrument™, Jinan, China). Based upon ISO527-2 protocol, dumbbell-shaped films (L: 4 mm, H: 30 mm) were analyzed through a crosshead speed set at 50 mm/min at ambient temperature.
Water absorption: The water-absorbing properties of such films was determined through the gravimetric method. A film sample (weighted as M o ) was dipped into distilled water at 37 • C. At fixed time spans, the sample was removed, gently pressed between tissue papers, and weighed as M w . The measurement was performed until there was no increase in sample mass. The water absorption was obtained through the equation: Hydrolytic breakdown: Hydrolytic degradation for polymer films was determined gravimetrically within PBS solution at 37 • C. Film samples, which had reached the equilibrium for water absorption, were weighted (W o ) and kept in PBS solution (pH = 7.4). Post-incubation for a predetermined time, samples were collected and weighted (W d ). The percentage of remaining weight was ascertained according to the equation: All analyses were performed for 12 months or until the films became fragments. The typical breakdown sample was freeze-dried, and surface morphologies were observed through SEM (Hitachi™ SU8010 ® , Tokyo, Japan).
Cytotoxicity test: MTT methodology was employed for determining viability within L929 mouse fibroblast cultures and to evaluate PEU film cytotoxicity. Briefly, the film sample was rendered sterile through UV irradiation and consequently extracted in DMEM augmented through 10% FBS (37 • C/24 h). Cells (5 × 10 4 cells/mL) were seeded into the filtered extract and incubated at 37 • C for 24, 48, and 72 h. Post-continuous culturing in MTT solution for another 5 h, dimethyl sulfoxide was introduced for dissolving any developed formazan pigment. Absorbance at 570 nm was determined through a microplate reader, and cell viability was obtained according to the control (cells incubated only within a culture medium).

Synthesis and Characterization
BHB: The chain extender for aliphatic diurethane diol with ordered structure was generated from reacting HDI with a larger volume of BDO. The chemical structure of BHB was validated through 1 H NMR, 13 C NMR, and FT-IR. Within 1 H NMR spectrum for BHB (Figure 2a), the peaks observed at δ 4.42, 3.90, 3.39, and 2.91 belonged to the proton signals of -OH, -COOCH 2 -, -CH 2 OH, and -CH 2 NH-, respectively. In addition, the core regional ratio for peaks was approximately 1(0.46):2(0.98):2(1.00):2(0.98), which was reflective of the BHB molecular frame (Figure 1a), suggesting that there was nil extended segment present within the product. Furthermore, characteristic absorption peaks of -N-H, amide I/II appeared at 3312, 1680, and 1537 cm −1 in Figure 3a, which demonstrated the formation of the variation of urethane groups. Furthermore, broad absorption bands at~3565 cm −1 must be linked to terminated -OH for HBH. Such results in combination with 13 C NMR and MS analyses indicated the successful synthesis of diurethane diol (BHB).
Polymers 2022, 14, x FOR PEER REVIEW 5 of 1 the BHB molecular frame (Figure 1a), suggesting that there was nil extended segment pre sent within the product. Furthermore, characteristic absorption peaks of -N-H, amide I/I appeared at 3312, 1680, and 1537 cm −1 in Figure 3a, which demonstrated the formation o the variation of urethane groups. Furthermore, broad absorption bands at ~3565 cm −1 mus be linked to terminated -OH for HBH. Such results in combination with 13 C NMR and MS analyses indicated the successful synthesis of diurethane diol (BHB).  PEU: The PEUs, having varied OHS content, were generated through the classic two step method. PCL diol was first reacted with HDI to prepare a prepolymer of OCN-PCL NCO, which was consequently chain-extended with BHB to give PEUs. PEU films wer collected through the solvent-evaporation technique. The number-average molecula  Figure 3a, which demonstrated the formation the variation of urethane groups. Furthermore, broad absorption bands at ~3565 cm −1 mu be linked to terminated -OH for HBH. Such results in combination with 13 C NMR and M analyses indicated the successful synthesis of diurethane diol (BHB).  PEU: The PEUs, having varied OHS content, were generated through the classic two step method. PCL diol was first reacted with HDI to prepare a prepolymer of OCN-PCL NCO, which was consequently chain-extended with BHB to give PEUs. PEU films we collected through the solvent-evaporation technique. The number-average molecula PEU: The PEUs, having varied OHS content, were generated through the classic two-step method. PCL diol was first reacted with HDI to prepare a prepolymer of OCN-PCL-NCO, which was consequently chain-extended with BHB to give PEUs. PEU films were collected through the solvent-evaporation technique. The number-average molecular weight (M n ) and molecular weight distribution (M w /M n ) for PEU materials were 75~89 KDa and 1.49~1.62 (Table 1) Figures 2c and 3d), which was attributed to the increased OHS content.

Thermal Stability
TGA was widely employed for analyzing the thermal degradation by materials at differing temperatures. Figure 4 illustrates the TGA curves of PEU films with various OHS content. Such curves describe that thermal degradation for the entirety of samples took place across two weight-loss phases. An initial phase at a lower temperature was mainly ascribed to the scission of ester bonds, while the second phase took place at an elevated temperature that corresponded to carbamate bonding breakdown [27]. In addition, PEU film thermal stability was promoted upon increasing OHS presence (PEU-1.0~PEU-2.0), which might be ascribed to the physical-linking networked structure developed through Hbonding not just across hard segments but also across hard/soft segments. Starting-weight reduction temperature for PEU films was higher than 200 • C, which meant that they could be high-temperature sterilized, although additional studies should be carried out.   Figures 2c and 3d), which was attributed to the increased OHS content.

Thermal Stability
TGA was widely employed for analyzing the thermal degradation by materials at differing temperatures. Figure 4 illustrates the TGA curves of PEU films with various OHS content. Such curves describe that thermal degradation for the entirety of samples took place across two weight-loss phases. An initial phase at a lower temperature was mainly ascribed to the scission of ester bonds, while the second phase took place at an elevated temperature that corresponded to carbamate bonding breakdown [27]. In addition, PEU film thermal stability was promoted upon increasing OHS presence (PEU-1.0~PEU-2.0), which might be ascribed to the physical-linking networked structure developed through H-bonding not just across hard segments but also across hard/soft segments. Startingweight reduction temperature for PEU films was higher than 200 °C, which meant that they could be high-temperature sterilized, although additional studies should be carried out.

Thermal Transition
DSC thermograms for PEU films with various OHS content are shown in Figure 5. PEU films represented a sole glass-transition temperature (Tg), which indicated the homogenous nature of PEUs [28]. Furthermore, the Tg was altered gradually to an elevated temperature upon the increase of OHS content (PEU-1.0~PEU-2.0). The higher OHS con-

Thermal Transition
DSC thermograms for PEU films with various OHS content are shown in Figure 5. PEU films represented a sole glass-transition temperature (T g ), which indicated the homogenous nature of PEUs [28]. Furthermore, the T g was altered gradually to an elevated temperature upon the increase of OHS content (PEU-1.0~PEU-2.0). The higher OHS content not only meant an increase in molecular rigidity but also produced more hydrogen bonds which formed the network structure. These reasons induced the restriction of polymer chain mobility, therefore no obvious microphase separation was found within thermograms and T g was altered to elevated temperatures [29,30]. Thermograms also exposed a broad endothermic peak with melting temperatures (T m ) at~56 • C, which were distinctly due to the melting transition for crystallized PCL-soft/BHB-hard domains. Enthalpy for peaks decreased from 28.2 J/g to 15.9 J/g with an increase in OHS content (PEU-1.0~PEU-2.0). This should also be attributed to the denser H-bonds which hindered the rearrangement of soft and hard segments, leading to reduced crystallinity. A similar phenomenon had been found by Han in waterborne PU, based on PEG [31]. bonds which formed the network structure. These reasons induced the restriction of polymer chain mobility, therefore no obvious microphase separation was found within thermograms and Tg was altered to elevated temperatures [29,30]. Thermograms also exposed a broad endothermic peak with melting temperatures (Tm) at ~56 °C, which were distinctly due to the melting transition for crystallized PCL-soft/BHB-hard domains. Enthalpy for peaks decreased from 28.2 J/g to 15.9 J/g with an increase in OHS content (PEU-1.0~PEU-2.0). This should also be attributed to the denser H-bonds which hindered the rearrangement of soft and hard segments, leading to reduced crystallinity. A similar phenomenon had been found by Han in waterborne PU, based on PEG [31].

Tensile Features
Tensile property proves essential for the application of medical PU materials [32]. The representative stress-strain curves for PEU films having various OHS content are presented in Figure 6, with signature datasets of tensile strength and elongation at the break depicted in Table 2. No defined yielding point was denoted from curves, indicating an elastic deformation [33]. Upon increasing OHS content (PEU-1.0~PEU-2.0), elongation-atbreak diminished, while tensile strength was enhanced. Similar tendencies had been found in the other PU materials. Dataset outcomes demonstrated tensile features can be regulated by modifying BHB dosage. In addition, all the PEU films showed good tensile features, especially for PEU-2.0 film, which contained the highest OHS content (BHB content: 20.7 wt %) and exhibited outstanding tensile features-having a tensile strength of 40.6 MPa and elongation-at-break of 477%. Such values were comparable to those of commercial medical PU, based on polycarbonate diol and 4,4′-diphenylmethane diisocyanate (Chronoflex AR TM , tensile strength: 41 MPa, elongation at break: 448%) [34]. The well-defined chain extender of BHB contained two carbamate groups, thus they would generate six carbamate groups in each hard segment containing BHB, which could form increaseddensity inter/intramolecular H-bonding and supply additional energy dissipation throughout tensile tests, resulting in outstanding tensile features.

Tensile Features
Tensile property proves essential for the application of medical PU materials [32]. The representative stress-strain curves for PEU films having various OHS content are presented in Figure 6, with signature datasets of tensile strength and elongation at the break depicted in Table 2. No defined yielding point was denoted from curves, indicating an elastic deformation [33]. Upon increasing OHS content (PEU-1.0~PEU-2.0), elongation-at-break diminished, while tensile strength was enhanced. Similar tendencies had been found in the other PU materials. Dataset outcomes demonstrated tensile features can be regulated by modifying BHB dosage. In addition, all the PEU films showed good tensile features, especially for PEU-2.0 film, which contained the highest OHS content (BHB content: 20.7 wt %) and exhibited outstanding tensile features-having a tensile strength of 40.6 MPa and elongation-at-break of 477%. Such values were comparable to those of commercial medical PU, based on polycarbonate diol and 4,4 -diphenylmethane diisocyanate (Chronoflex AR TM , tensile strength: 41 MPa, elongation at break: 448%) [34]. The well-defined chain extender of BHB contained two carbamate groups, thus they would generate six carbamate groups in each hard segment containing BHB, which could form increased-density

Water Absorption
Regarding the breakdown of polyester-based polyurethane, this was predomi occurring through ester bond hydrolysis, where water absorbency has an importan on the degradation rate [35]. The water absorption of PEU films was tested as a fu of immersion duration (Figure 7). All PEU films presented Fickian diffusion be [36,37] and reached the equilibrium level post-36 h. Although the equilibrium wa sorption (EWA) was marginally raised upon OHS content reduction (PEU-2.0~PE all the films displayed a low EWA of less than 5%. This could be attributed to two f one is the hydrophobic chain structure; while another reason could be the comp bond-crosslinked network structure, which restricted water molecules from enteri matrix.

Water Absorption
Regarding the breakdown of polyester-based polyurethane, this was predominantly occurring through ester bond hydrolysis, where water absorbency has an important effect on the degradation rate [35]. The water absorption of PEU films was tested as a function of immersion duration (Figure 7). All PEU films presented Fickian diffusion behavior [36,37] and reached the equilibrium level post-36 h. Although the equilibrium water absorption (EWA) was marginally raised upon OHS content reduction (PEU-2.0~PEU-1.0), all the films displayed a low EWA of less than 5%. This could be attributed to two factors: one is the hydrophobic chain structure; while another reason could be the compact H-bondcrosslinked network structure, which restricted water molecules from entering the matrix.

Hydrolytic Degradability
The high biostability represents a paramount requirement to ensure long-term implant biomaterial quality. Hydrolytic degradability for PEU films was measured by the weightloss method within PBS (pH 7.4) at body temperature, while weight-loss curves as a function of time are displayed in Figure 8. The weight-loss rate for films decreased drastically upon increased OHS level (PEU-1.0~2.0). The PEU-1.0 film, which did not contain OHS, had a weight loss of 41.2% post-7 months of degradation and became fragmented. While only 29.5% weight loss was detected for PEU-2.0 film (the highest OHS content across all samples) on assay termination, the film still maintained its primary shape and seemed to retain tensile features up to a certain degree. Undoubtedly, such a breakdown was predominantly induced through ester-bond hydrolysis within soft segments. When BHB was introduced into the main chain, six urethane groups contained in each OHS. The additional urethane groups could form denser H-bonds and produced a compact networked Polymers 2022, 14, 1674 9 of 12 structure, which restricted water molecules from approaching ester groups, leading to a gradual hydrolysis rate. Such results were consistent with the water-absorbing capacity. The materials expressing slow biodegradability (or high biostability) can meet the requirement of long-term implants, although additional measurements need to be performed.
Regarding the breakdown of polyester-based polyurethane, this was predominan occurring through ester bond hydrolysis, where water absorbency has an important eff on the degradation rate [35]. The water absorption of PEU films was tested as a functi of immersion duration (Figure 7). All PEU films presented Fickian diffusion behav [36,37] and reached the equilibrium level post-36 h. Although the equilibrium water a sorption (EWA) was marginally raised upon OHS content reduction (PEU-2.0~PEU-1 all the films displayed a low EWA of less than 5%. This could be attributed to two facto one is the hydrophobic chain structure; while another reason could be the compact bond-crosslinked network structure, which restricted water molecules from entering t matrix.

Hydrolytic Degradability
The high biostability represents a paramount requirement to ensure long-term i plant biomaterial quality. Hydrolytic degradability for PEU films was measured by t weight-loss method within PBS (pH 7.4) at body temperature, while weight-loss curv as a function of time are displayed in Figure 8. The weight-loss rate for films decreas drastically upon increased OHS level (PEU-1.0~2.0). The PEU-1.0 film, which did not co tain OHS, had a weight loss of 41.2% post-7 months of degradation and became fra mented. While only 29.5% weight loss was detected for PEU-2.0 film (the highest O content across all samples) on assay termination, the film still maintained its prima shape and seemed to retain tensile features up to a certain degree. Undoubtedly, such breakdown was predominantly induced through ester-bond hydrolysis within soft s ments. When BHB was introduced into the main chain, six urethane grou contained in each OHS. The additional urethane groups could form denser H-bonds a produced a compact networked structure, which restricted water molecules from a proaching ester groups, leading to a gradual hydrolysis rate. Such results were consiste with the water-absorbing capacity. The materials expressing slow biodegradability high biostability) can meet the requirement of long-term implants, although addition measurements need to be performed. Weight loss for films during degradation-processing meant that several small mo cules were formed and dissolved in water, thus the degradation process could be embo ied by changes in surface morphology. The SEM images for PEU-2.0 films at differi degradation periods are displayed in Figure 9. A smooth and level surface was observ within the pristine film (Figure 9a). However, the relatively rough surface appeared po degradation for 4- (Figure 9b) and 8-months (Figure 9c), the film also kept its starti shape and had no apparent structural change. Upon termination of measurements months, Figure 9d), the surface exhibited distinct sunken morphology with considera weight loss, though the films still maintained their integrity, demonstrating that the PE 2.0 film possessed higher hydrolytic stability. Weight loss for films during degradation-processing meant that several small molecules were formed and dissolved in water, thus the degradation process could be embodied by changes in surface morphology. The SEM images for PEU-2.0 films at differing degradation periods are displayed in Figure 9. A smooth and level surface was observed within the pristine film (Figure 9a). However, the relatively rough surface appeared post-degradation for 4- (Figure 9b) and 8-months (Figure 9c), the film also kept its starting shape and had no apparent structural change. Upon termination of measurements (12 months, Figure 9d), the surface exhibited distinct sunken morphology with considerable weight loss, though the films still maintained their integrity, demonstrating that the PEU-2.0 film possessed higher hydrolytic stability. Polymers 2022, 14, x FOR PEER REVIEW 10 of 12

Cytotoxicity Assay
Aimed at biomedical applications, good biocompatibility is the most fundamental requirement, and consequently, cytotoxicity analysis is essential for assessing such a property. As shown in Figure 10, cell viability-as assessed through MTT assays-was slightly elevated upon increased OHS presence, indicating that the introduction of OHS contributed to cytocompatibility. This could be due to the compact networking structural formation through hydration bonds, which inhibited toxic substances from entering the culture medium. All PEU films containing OHS (PEU-1.25~2.0) exhibited cell viability > 75%, meaning that cytotoxicity was class 1 (ISO 10993. ) and that the acceptable cytocompatibility could meet the requirement of implant biomaterials [38,39].

Cytotoxicity Assay
Aimed at biomedical applications, good biocompatibility is the most fundamental requirement, and consequently, cytotoxicity analysis is essential for assessing such a property. As shown in Figure 10, cell viability-as assessed through MTT assays-was slightly elevated upon increased OHS presence, indicating that the introduction of OHS contributed to cytocompatibility. This could be due to the compact networking structural formation through hydration bonds, which inhibited toxic substances from entering the culture medium. All PEU films containing OHS (PEU-1.25~2.0) exhibited cell viability > 75%, meaning that cytotoxicity was class 1 (ISO 10993. ) and that the acceptable cytocompatibility could meet the requirement of implant biomaterials [38,39].

Cytotoxicity Assay
Aimed at biomedical applications, good biocompatibility is the most fundamen requirement, and consequently, cytotoxicity analysis is essential for assessing such a pr erty. As shown in Figure 10, cell viability-as assessed through MTT assays-was sligh elevated upon increased OHS presence, indicating that the introduction of OHS contr uted to cytocompatibility. This could be due to the compact networking structural f mation through hydration bonds, which inhibited toxic substances from entering the c ture medium. All PEU films containing OHS (PEU-1.25~2.0) exhibited cell viability > 75 meaning that cytotoxicity was class 1 (ISO 10993. ) and that the acceptable cytoco patibility could meet the requirement of implant biomaterials [38,39].

Conclusions
Within this study, ordered structure was introduced to the hard segments of PEUs by a facile and cost-effective method. The BHB, which contained an ordered structure, was synthesized and employed as a chain extender to react with NCO-terminated PCL to prepare the PEUs. The influences of OHS content on physicochemical features of the casted PEU films were investigated. Regarding each OHS containing six urethane groups, it was beneficial to form higher-density H-bonds and consequently produce a compact network structure, resulting in great tensile features, low water absorption, and a slow hydrolytic degradation rate for such PEU films. PEU-2.0 films, which possessed the highest OHS content, exhibited tensile features with a tensile strength of 40.6 MPa and elongationat-break of 477%, equilibrium water absorption of 4.3 wt %, and weight-loss of 29.5% after post-12 months' degradation. Furthermore, the cytotoxicity test for film extracts showed that cell viability for all PEUs containing OHS exceeded 75%, indicating a good cytocompatibility. Regarding the outstanding tensile features, high hydrolytic stability, non-toxic and absorbable degradation products, and acceptable cytocompatibility, these cost-effective materials (especially PEU-2.0) exhibited promising applications as long-term implants, although additional biological assessments are required.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.