A novel inherent fluorescence biodegradable polyurethane
Juan Liu, Shun Li, Zhengwei Li, Jinfeng Wang, Zhigang Chen, Pinpin Wang, Haobo Pan, Yanfeng Luo, Changshun Ruan
TL;DR
This paper introduces a new type of biodegradable polyurethane with built-in fluorescence to track and adjust its degradation for tissue regeneration.
Contribution
The novelty lies in integrating a fluorescent molecule into the polyurethane structure for real-time degradation tracking and tunability.
Findings
IFPU materials showed excellent fluorescence and adjustable degradation in vitro and in vivo.
3D-printed IFPU scaffolds had high porosity, mechanical strength, and supported cell growth and biocompatibility.
Hard segments were found to regulate degradation rates through hydrophilic properties.
Abstract
In situ tissue regeneration using biodegradable biomaterials provides a viable strategy to harness endogenous regenerative potential for tissue repair. Ideally, biomaterials should have appropriate biodegradability to accommodate new tissue formation. However, the limited adjustability and trackability of their degradation behaviors pose a considerable challenge in achieving suitable biodegradability that matches the tissue regeneration process. Herein, we developed a series of novel inherent fluorescence biodegradable polyurethane (IFPU) materials by incorporating a fluorescent small molecule (thiazolpyridinic acid) as both the hard segment composition and the fluorescent probe to adjust and track their degradation behaviors. These IFPUs, both in solid films and porous scaffolds, exhibited outstanding fluorescence performance, enabling the rapid visualization of their adjustable…
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Figure 10- —National Key Research and Development Program of China10.13039/501100012166
- —National Natural Science Foundation of China10.13039/501100001809
- —Fundamental Research Funds for the Central Universities10.13039/501100012226
- —Natural Science Foundation of Guangdong Province in China
- —Shenzhen Fundamental Research Foundation
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Taxonomy
TopicsWound Healing and Treatments · Nanoplatforms for cancer theranostics · Polymer composites and self-healing
Introduction
Biodegradable biomaterials provide a promising approach for in situ tissue regeneration [1–3]. An ideal tissue substitute should have appropriate biodegradability to accommodate new tissue formation [4–6]. Tissue regeneration rates vary with age and pathology, imposing different requirements for the degradation rates of biomaterials [7, 8]. Accordingly, establishing a precise correlation between the degradation behavior of biomaterials and tissue regeneration is highly necessary, which, however, remains a major challenge. This challenge is primarily attributed to the lack of biomaterials with adjustable degradation behaviors and effective real-time monitoring methods, especially in vivo. Therefore, it is crucial to develop a biomaterial capable of adjusting and tracking degradation behavior to promote tissue regeneration.
Biodegradable polyurethanes (BPUs) are multi-block polymers typically composed of flexible polyester soft segments and rigid urethane hard segments [9, 10]. By adjusting the proportion and structure of these segments, BPUs can be tailored to have desired mechanical properties [11], biocompatibility [12] and biodegradability [13]. In recent years, they have been widely used in 3D-printed scaffolds [14, 15]. Particularly, since urethane and ester bonds have different hydrolysis rates [16], the degradation rate of BPUs can be controlled by modifying the ratio of the soft and hard segments. Moreover, the hard segments of BPUs have a dual role in regulating degradation. Under low content (<4 wt%), the hard segments are difficult to assemble into hard domains, leaving more free urethane groups in the hard segments. The free urethanes form hydrogen bonds with water, enhancing hydrophilicity and water absorption and thus promoting BPU hydrolysis [17]. Contrarily, when their content is high, the strong hydrogen bonding between the urethane groups facilitates the formation of tight hard segment domains, decreasing water absorption and inhibiting BPU degradability [18]. Despite numerous investigations demonstrating the adjustable degradation behaviors of BPUs in vitro, they require sacrificing a large number of samples and taking a long time to obtain dry samples for studying degradation. The diversity in chemical structures of BPUs and shapes of substitutes further increases the time cost for studying degradation behavior. Moreover, the actual degradation behavior in vivo is poorly understood as well. The in vivo degradation behaviors could differ from those observed in vitro since various biological and physical factors involving enzymes, cells, mechanical stress, etc., are involved in vivo. Therefore, effectively tracking the in vivo degradation of BPUs in real time is of great importance.
Fluorescence visualization is a fast, efficient, non-destructive and real-time tracking technique. It has been widely used in various fields, including life science [19], drug development [20] and polymer research [21]. Han [22] and Wang et al. [21] designed aggregation-induced emission fluorogens to visualize the phase separation and gelation process by incorporating fluorescent probes into polymers. Artzi [23] and Chen et al. [24] covalently grafted fluorescent probes at the ends of polymer chains to fluorescently track hydrogel degradation. Mixing an exogenous fluorescence probe into BPUs may track the in vivo degradation behaviors of BPUs in real time. However, the additional fluorescence probe may inevitably alter the biodegradability and biocompatibility of BPUs [25].
In this study, as illustrated in Figure 1, a series of new inherent fluorescent biodegradable polyurethanes (IFPUs) were designed and developed by incorporating a biocompatible fluorescent molecule (5-oxo-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-3,7-dicarboxylic acid, TPA) [26, 27] as the chain extender. The TPA fragment serves a dual role as both a fluorescence probe and a hard segment component, with the expectation of granting IFPUs with trackable and adjustable degradation behaviors. The fluorescent visualization of IFPUs enabled tracking the degradation behavior of both IFPU films and 3D-printed IFPU scaffolds. The degradation mechanism of BPU and the regulation of degradation rate by hard segments were comprehensively elucidated as well.
Scheme illustration. IFPU was developed based on TPA functioning as both a hard segment component and a fluorescent probe. Further, the IFPU scaffolds, constructed by 3D printing, can not only track their degradation behaviors by fluorescence visualization but also regulate their degradation rates by their hard segment contents. Moreover, IFPUs presented outstanding printability, and their 3D printed scaffolds not only showed high mechanical properties but also promoted cell recruitment, adhesion and proliferation, making them a promising alternative as a tissue-regenerative biomaterial.
Materials and methods
Preparation of IFPUs
First, a diol was synthesized by the ring-opening polymerization of D, L-lactide at 140°C, in which stannous caprylate (1/5000) was the catalyst and PEG400 (1/50) was the initiator. Then, IFPUs were prepared similarly to our previously reported procedure [9]. To be more specific, the diol was capped with hexamethylene diisocyanate (HDI) at 75°C for 3 h in toluene, and chain extended by thiazolpyridinic acid (TPA) at 50°C for another 12 h. Subsequently, the products were precipitated three times using the dichloride-alcohol system. The purified samples were vacuum-dried at 25°C for 72 h to obtain IFPU materials. The molar ratio of diol/HDI/TPA was regulated to control the content of hard segments. The IFPUs for molar ratios of diol/HDI/TPA = 1/1.1/0.1, 1/1.2/0.2 and 1/1.3/0.3 were designated as IFPU1, IFPU2 and IFPU3, respectively (Table S1).
Chemical structure and physical property characterization of IFPUs
The ^1^H NMR spectra were collected by an AVANCE III 400 spectrometer with dimethyl sulfoxide-d6 as solvent. Fourier transform infrared (FTIR) spectra were recorded on a Spectrum GX FTIR spectrometer with a scanning range from 4000 to 400 cm^−1^ at 25°C. The molecular weights were measured by a VISCOTEK 270MAX gel penetration chromatography (GPC) with N, N-dimethylformamide (DMF) as a mobile phase. Elemental analyses were detected by X-ray photoelectron spectroscopy (XPS). Water absorptions were measured in water at 37°C. Hydrophilicities were indicated by the water contact angle. Thermal properties were measured by thermogravimetric analysis (TGA) from 30 to 600°C at 10°C·min^−1^ heating rate and differential scanning calorimetry with two cycles at a constant heating/cooling rate of 10°C·min^−1^.
Fluorescence characterization
The ultraviolet-visible (UV-vis) absorption spectra were collected using a Lambda25 UV-vis spectrophotometer with DMF as solvent. All fluorescence spectra were obtained by an FLS920 fluorescence spectrometer. Both the excitation and emission slit widths were set at 1 nm for all samples unless otherwise stated. The reference method was used to measure the fluorescent quantum yield with quinine sulfate aqueous solution as a control. The photostability of solutions was evaluated by recording the changes in the fluorescence intensity under continuous white light. Fluorescence imaging of IFPU films and scaffolds was observed by a fluorescence microscope.
Fabrication of IFPU films and scaffolds
IFPUs were dissolved in chloroform to form a polymer solution of 5 wt%. The solution was poured into a Teflon mold (80 mm×80 mm×8 mm) to obtain IFPU films by the solvent evaporation method. IFPUs were dissolved in 1,4-dioxane to prepare IFPU inks (Table S2). The IFPU scaffolds were fabricated by 3D printing in a Bioscaffolder 3.1 and freeze-drying. The strand width and distance were set to 500 μm, and the strand height was set to 300 μm.
Physical properties characterization of IFPU scaffolds
Surface topological morphologies were observed by the ZEISS SUPRA^®^ 55 scanning electron microscope (SEM) at 5 kV. Mechanical properties of scaffolds (5 × 5×5 mm^3^) were tested by uniaxial compression test at 25°C, 50% humidity with a constant compression strain rate of 5 mm·min^−1^. The compressive strength and elastic modulus were determined by stress–strain curves. Porosities were detected by the liquid displacement method.
Fluorescence visualization of degradation behavior in vitro and in vivo
Firstly, the model of fluorescence visualization tracking degradation behavior in vitro was built using 0.2 M NaOH alkali solution and PBS solution to accelerate degradation and simulate the in vivo environment. The initial mass (m0) and initial fluorescence intensity (F0) of films (diameter 10 mm, thickness 0.1 mm) and scaffolds (9 × 9×1.5 mm^3^) were recorded. The samples were put into 2 ml alkali solution or 4 ml PBS solution and incubated at 37°C. The alkali solutions were replaced daily, and the PBS solutions were replaced weekly. Secondly, the model of fluorescence visualization tracking degradation behavior in vivo was constructed by subcutaneous implantation in mice. Initial mass (m0) and initial fluorescence intensity (F0) of the scaffolds (diameter 8 mm, height = 1.5 mm) were recorded. The scaffolds were implanted subcutaneously into 4-week-old male Balb/c mice (the specific surgical operations presented in the animal model section). Finally, residual samples and degradation media were collected at a predetermined time in each model.
The fluorescence intensities of samples in wet (F1) and dry (F2) states were recorded in vitro, in vivo and ex vivo by an IVIS^®^ Spectrum imaging system (excitation at 430 nm, emission at 500 nm, exposure time of 1 s). The fluorescence residual ratios of degraded samples in wet and dry states were calculated according to Equations (1) and (2). The wet weight (m1) and dry weight (m2) of the degraded sample were recorded. Similar to the traditional methods [28], the mass residual ratio and water absorption of the degraded sample were calculated according to Equations (3) and (4). The molecular weights of residual samples were measured by GPC in DMF. In addition, the degradation media were collected to characterize the UV-visible absorption spectrum, fluorescence spectrum and pH value by a pH meter.
Characterization of cytocompatibility in vitro
Sterilized samples were placed in 24-well plates. Human bone mesenchymal stem cells (hBMSC) were seeded on samples at a density of 3 × 10^4^ cells per scaffold and cultured with α-MEM medium containing 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C, 5% CO_2_ to assess the cytocompatibility in vitro. FITC-phalloidin and DAPI were employed to stain the cytoskeleton and nuclei of cells in the samples. Fluorescein diacetate (FDA) and propidium iodide (PI) were used to stain live and dead cells, respectively. Cell counting kit-8 (CCK-8) was used to characterize cell activity.
hBMSCs were seeded in 24-well plates and cultured for 3 days. Then sterilized scaffolds were lightly placed on hBMSCs and cultured with α-MEM medium at 37°C and 5% CO_2_. The scaffold recruitment cells were detected by FDA/PI staining and CCK-8.
Animal models
All experimental protocols in this animal study were approved by the Ethics Committee for Animal Research, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. And the accreditation number of the laboratory is SIAT-IACUC-200820-YYS-RZS-A0765-01. Four-week-old male Balb/c mice were randomly divided into three groups, which were recorded as control, PLA and IFPU groups, respectively. Mice were anesthetized with isoflurane gas, and a 10 mm incision was made on the back. Sterilized scaffolds were placed bilaterally, and the wound was sutured and sterilized with iodine. The control group only suffered incised and sutured, without any scaffolds.
Characterization of biocompatibility in vivo
The whole blood of experimental mice was collected after implantation for 1 week to detect parameters of white blood cells (number of white blood cells, number of lymphocytes (Lymph), number of monocytes, number of neutrophils), red blood cells (number of red blood cells, Hematocrit, Hemoglobin, red blood cell distribution width coefficient of variation) and platelets (platelet number, mean platelet volume, platelet distribution width, platelet count). The serums of experimental mice were collected after implantation for 16 weeks to detect parameters of liver function (aspartate aminotransferase, alanine aminotransferase, albumin, γ-glutamyltransferase, total bilirubin, direct bilirubin) and kidney function (blood urea nitrogen, creatinine).
The heart, liver, spleen, lung and kidney of experimental mice were harvested after implantation for 12 and 16 weeks, and the tissues around scaffolds were harvested after implantation for 12 weeks. Paraffin embedding and H&E staining were employed to detect the biocompatibility in vivo.
Statistical analysis
All the statistical analyses were performed by one-way analysis of variance with t-test and expressed as mean ± SD. A P-value <0.05 was considered to be statistically significant.
Results
Novel IFPUs with various hard segment contents
To obtain biodegradable IFPUs with various hard segment content (Figure 2A), we first prepared a biodegradable macromolecule diol (PDLLA-PEG-PDLLA, designated as diol) that we previously reported as a soft segment [18]. Concretely, D, L-lactide with strong molecular rigidity was polymerized by the PEG400 block as the open-ring initiator to improve the hydrophilicity of BPU. Peak d’ at 1.25 ppm and peak c’ at 4.22 ppm, respectively, corresponded to the –CH_3_ protons and –CH protons of the lactic acid fragment connected with the end –OH, and peak e at 5.45 ppm corresponded to end –OH protons in ^1^H NMR spectra of diol (Figure S1A). The relative ratio of the peak c’ and peak c indicated that the molecular weight of the diol was 7816 Da. The wide and strong peak at 3700–3500 cm^−1^ belonged to –OH stretching vibration, and the peak at 1755 cm^−1^ belonged to C = O stretching vibration in the FTIR spectra of diol (Figure S1B). These results indicate that diol has been synthesized successfully.
*The structure and hydrophilia of IFPUs. (A) The synthetic routes of IFPUs and the hard segment structure for adjustability and trackability of their degradation behaviors. (B) The 1H NMR spectra of IFPUs for calculating the hard segment contents. (C) The FTIR spectra of IFPUs with various hard segment contents. (D) N–H stretching vibration and amide II bands of the hard segment in IFPUs in FTIR spectra. (E) The percentages of N–H stretching vibration bond (3550–3200 cm−1) and amide II bond (1560–1490 cm−1) intensities compared to the corresponding C–H intensity (3050–2750 cm−1) in various IFPUs. (F) Water contact angle of IFPU materials, and PLA as a control. (G) Water absorption of the IFPU materials in water at 1 and 3 days. (H) Gel permeation chromatography (GPC) curves of IFPUs in DMF and the obtained weight-average molecular weight (P < 0.05 vs PLA, #P < 0.05 vs IFPU1, $P < 0.05 vs IFPU2; n = 4).
Then, a series of IFPUs (Figure 2A and Table S1) with various hard segment contents (designated as IFPU1, IFPU2 and IFPU3) were successfully obtained using diol as the soft segments, along with HDI and TPA as the hard segments. The ^1^H NMR spectra of IFPUs (Figure 2B and Figure S1C and D) revealed peaks i, j, l and k at 7.45, 7.20, 4.90 and 4.30 ppm, corresponding to the –CH and –CH_2_ protons of the TPA fragment. Their relative ratios indicated that the hard segment contents of IFPU1, IFPU2 and IFPU3 were 2.9 wt%, 3.6 wt% and 4.0 wt%, respectively (Table S1), which were expected to regulate the degradation behaviors of IFPUs. The FTIR spectra of IFPUs (Figure S1E) revealed peaks of N–H stretching vibration at 3550–3250 cm^−1^ and amide II band at 1525 cm^−1^ for the newly formed urethane and amide groups. The peak of –NCO stretching vibration at 2275 cm^−1^ disappeared completely (Figure S1E), avoiding the biotoxicity of the isocyanate. Relative ratios of N–H in IFPUs increased from 10.88% to 15.84% with the increase of hard segment content (Figure 2C–E). XPS spectra further indicated the gradual increase of the N content (IFPU1: 0.38%, IFPU2: 0.67%, IFPU3: 0.92%) in IFPUs (Figure S1F and G). Moreover, the hydrophilia of the surface (Figure 2F) and water absorption (Figure 2G) gradually increased with the increase of hard segment content in IFPUs, despite similar surface topological morphologies (Figure S1H). This indicated the N–H in the hard segment could enhance the hydrophilicity of IFPUs, which was expected to regulate the degradability of IFPUs by the hard segment contents. The high molecular weights of IFPUs ranging from 190 to 316 kDa were expected to give them excellent mechanical properties (Figure 2H). In addition, IFPUs possessed strong thermal stability (Figure S2A and B) and a glass transition temperature of ∼43°C (Figure S2C and D), making them suitable for implantations in vivo.
Inherent fluorescence properties of IFPUs
Fluorescence property is a prerequisite for fluorescence visualization. All TPA and IFPU solutions in DMF emitted strong fluorescence. The strong absorption bands and emission bands of IFPUs appeared at 368 and 432 nm, resembling those of TPA (366 and 430 nm) (Figure 3A and B and Figure S3A–H). The maximum absorption wavelength and emission wavelength of TPA aqueous solutions were 346 nm (Figure 3A) and 426 nm (Figure 3B), respectively. Moreover, all IFPUs inherited excellent photostability (Figure 3C) and high fluorescence quantum yield (>0.2) (Figure 3D) from TPA, which were higher than traditional organic fluorescent dyes [29].
The inherent fluorescence property of IFPUs. (A) The UV-Vis absorption spectra of IFPUs solutions and TPA and diol as controls. (B) Fluorescence emission and excitation spectra of IFPU solutions. (C) Photostability of IFPU solutions under continuous excitation in white light and rhodamine B (RB) aqueous solution as a control. (D) Fluorescence intensity-absorbance intensity curves and corresponding quantum yield, along with a quinine aqueous solution as a control (n = 4).
Printability of IFPUs and their fluorescence imaging performance
The IFPU films were fabricated by a solution casting method (Figure 4A). Interestingly, IFPU films also emitted bright blue, green and red light (Figure 4B). Further, various IFPU scaffolds were fabricated via low-temperature deposition 3D printing (Figure 4C) to explore the traceability by fluorescence visualization and adjustability of degradation of IFPU scaffolds. The rheological results showed that IFPU inks (Table S2) exhibited shear thinning (Figure 4D), stable viscosity at a constant shear rate (Figure 4E) and rapid recovery within 1 s after shear cessation (Figure 4F). These rheological characteristics benefited the precise fabrication of IFPU scaffolds. The 3D-printed IFPU scaffolds inherited the outstanding fluorescence properties from IFPU materials. All scaffolds emitted strong fluorescence, varying from blue to red, especially bright blue fluorescence (Figure 4G), providing the potential for tracking the inherent degradation of IFPUs via fluorescence visualization.
The printability of IFPUs and their fluorescence imaging performance. (A) Schematic illustration of IFPU films fabricated by solution casting. (B) Fluorescence imaging performance of IFPU films, and PLA film as a control. (C) Schematic illustration of IFPU scaffolds fabricated by low-temperature deposition 3D printing. (D–F) Rheological properties of IFPU inks. (D) Viscosity of ink as a function of shear rate (0.01–100 1/s: solid; 100–0.01 1/s: hollow). (E) Ink viscosity at a constant shear rate (10 1/s). (F) Viscosity of ink during simulating printing, i.e. suffering from shear force for 10 s and then keeping static. (G) Fluorescence imaging of 3D-printed IFPU scaffolds.
Fluorescent visualization for tracking the degradation behaviors of IFPUs
To investigate whether the inherent fluorescence of IFPUs could be used to visualize and track the degradation behaviors of IFPUs, accelerated degradation tests were first conducted for both solid IFPU films and porous IFPU scaffolds. The fluorescence regions and intensities of the wet and dry IFPU films gradually decreased during the accelerated degradation process (Figure 5A); the curves for the fluorescence residual ratios were consistent with the corresponding curves for the mass residual ratios (Figure 5B), indicating that the inherent fluorescence of IFPUs can be used to visually and quantitatively track the degradation behaviors of IFPU films. Additionally, the degradation media exhibited similar fluorescence properties to those of aqueous TPA solution, including a maximum absorption wavelength at 346 nm (Figure S4A) and maximum excitation and emission wavelengths at 346 and 426 nm (Figure S4B), which should be attributed to the soluble degradation products from IFPUs. The cumulative fluorescence intensities of the media gradually increased with the extension of degradation time (Figure 5B); the curves for the fluorescence release rate of the media were consistent with the curves for the mass degradation rate of the films (Figure 5C). These results indicate that the TPA fragments released from IFPU chains do not dissociate in the degradation media, so that the fluorescence behaviors of the media can reflect the degradation behaviors of IFPU films as well. Compared with IFPU films, IFPU scaffolds also demonstrated a decrease in fluorescence regions and intensity (Figure 5D). Moreover, the curves for the fluorescence residual ratios corresponded well with the curves for the mass residual ratios as well (Figure 5E), confirming the feasibility of fluorescence visualization for tracking the degradation behaviors of IFPU scaffolds. The degradation rate of IFPU scaffolds was faster than that of corresponding IFPU films, which should be related to the high porosity and specific surface area of scaffolds.
Fluorescent visualization for tracking the degradation behaviors of IFPUs. (A) Fluorescence visualization for tracking accelerated degradation behaviors of IFPU films. (B) Comparison of fluorescence residual ratio in wet and dry states and the mass residual ratio of IFPU films, along with cumulative fluorescence intensity in the media. (C) Comparison of fluorescence release rate in media and mass loss rate of films. (D) Fluorescence visualization for tracking accelerated degradation behaviors of IFPU scaffolds. (E) Comparison of fluorescence residual ratio in wet and dry states and the mass residual ratio of IFPU scaffolds (n = 4).
Trackable and adjustable degradation behavior of IFPU scaffolds
After confirming the feasibility of fluorescence visualization for tracking the degradation behaviors of IFPU films and scaffolds, the in vitro degradation behaviors of IFPU scaffolds in a PBS solution were further studied via fluorescence visualization. Meanwhile, the adjustable degradation behavior was explored. The fluorescence regions and intensities decreased with degradation time (Figure 6A). Similar to the accelerated degradation tests, both the fluorescence residual ratios and the fluorescence degradation rates of the wet and dry IFPU scaffolds in PBS were consistent with the corresponding mass residual ratios (Figure 6B) and mass degradation rates (Figure 6C). Despite these, their specific degradation behaviors were significantly different. In PBS, the scaffolds presented three degradation periods, including a plateau, a rapid decline and a slow decline within 16 weeks. Besides the scaffolds themselves, the degradation media demonstrated excellent fluorescence properties (Figure S5A and B) and consistent fluorescence release rates with the corresponding mass degradation rates as well (Figure 6C). An increase in the hard segment contents promoted the degradation of IFPU scaffolds, mainly reflected in the earlier time of starting degradation (IFPU1: 8th week, IFPU2: 7th week, IFPU3: 6th week) and shorter half-life (IFPU1: 11.7 weeks, IFPU2: 10.5 weeks, IFPU3: 9.6 weeks) (Figure 6D). These results indicated that the fluorescence residual ratios and the fluorescence degradation rates of the wet and dry IFPU2 scaffolds, as well as the fluorescence release in the degradation media, can be used to quantitatively and visually track their degradation behaviors in vitro.
Trackable and adjustable degradation behavior of IFPU scaffolds. (A) Fluorescence visualization for tracking degradation behaviors of IFPU scaffolds in vitro. (B) Comparison of fluorescence residual ratio in wet and dry states and the mass residual ratio of IFPU scaffolds, along with cumulative fluorescence intensity in degradation media. (C) Comparison of fluorescence degradation rate in wet and dry states and mass degradation rate of IFPU scaffolds, along with fluorescence release rate in degradation media. (D) Regulation of the degradation behavior of the scaffolds by hard segment contents. (E) Illustration of the IFPU degradation mechanism suggested by the fluorescence visualization (n = 4).
During the rapid degradation stage of IFPU scaffolds, the pH value of the media first decreased and then increased (Figure S5C). As the hard segment content increased, the pH valley value gradually decreased. The water absorption ratio of IFPU scaffolds increased slowly within the first 5 weeks (about 20%), and then increased rapidly (Figure S5D), indicating that many hydrophilic groups, such as –COOH, –OH and –NH_2,_ were produced by the dissociation of IFPU polymer chains. The water absorption of IFPU scaffolds gradually increased along with the hard segment content. Although the mass of IFPU scaffolds changed negligibly within 4 weeks, the molecular weight of IFPUs decreased significantly, from >300 kDa (Mw) to 20–30 kDa, showing an exponential decline pattern, and then maintained at 20 kDa (Figure S5E and F). The degradation rates of relative molecular weight in IFPU2 and IFPU3 were higher than those in IFPU1 (Figure S5K and L) due to high water absorption. Moreover, the fluorescence loss of scaffolds and fluorescence intensity in the media were very low. Therefore, the TPA fragments were still retained in the solid degradation product of IFPU scaffolds, and the decay of molecular weight was caused by the ester dissociation in the soft segment (Figure 6E), further implying that fluorescence behaviors can be used to indicate the degradation of IFPU scaffolds.
The biodegradability of IFPU scaffolds could be more truly reflected by the testing in vivo. The subcutaneously implanted IFPU2 scaffold presented excellent fluorescence properties as well (Figure 7A and B). The declines of in situ and ex vivo fluorescence signals were also positively correlated to the degradation behaviors of IFPU2 scaffold (Figure 7C and D), confirming that tracking the in vivo degradation behaviors of IFPU scaffolds via fluorescence visualization is feasible and effective. The mass of the IFPU2 scaffold began to decline after implantation for 8 weeks, and the degradation rates increased gradually (half-life: 11.2 weeks). This should be caused by the dissociation of IFPU to generate hydrophilic groups, thus increasing water absorption after 6 weeks (Figure S6A) and decreasing molecular weights (Figure S6B). The molecular weight curves of IFPU2 in vivo were similar to those in vitro, suggesting that the same degradation pattern of IFPUs in vitro applies to the in vivo case.
Fluorescence visualization of the degradation behavior of IFPU scaffolds in vivo. (A, B) Fluorescence visualization for tracking degradation behaviors of the IFPU2 scaffold in situ (A) and ex vivo (B). (C) Comparison of fluorescence residual ratio in situ, in the wet and dry states ex vivo, and mass residual ratio of the IFPU2 scaffold. (D) Comparison of fluorescence degradation rate in situ, in the wet and dry states ex vivo, and mass degradation rate of the IFPU2 scaffold (n = 4).
Physical properties of IFPUs scaffolds
Various IFPU scaffolds showed interconnected macropores with a pore size and strand width of both ∼450 μm, along with a strand height of ∼400 μm (Figure 8A). The strands were observed with abundant micropores (<20 μm, marked by blue arrows) and wrinkles (marked by green arrows), which would provide abundant cell adhesion sites. The macropore and micropore structures endowed the scaffolds with a porosity of 71.0 ± 2.6–74.7 ± 2.0%, along with micro-porosity accounting for 30–40% (Figure 8B). High porosity (>70%) facilitates cell growth, angiogenesis, nutrient transport and tissue regeneration [30, 31].
*Physical properties of IFPUs scaffolds. (A) SEM images (blue arrows meant micropores and green arrows marked wrinkles on the strands of scaffolds). (B) Porosity. Mechanical properties, including (C) stress–strain curves of uniaxial compression, (D) compressive modulus and (E) compressive strength of scaffolds (P < 0.05 vs PLA, #P < 0.05 vs IFPU1, $P < 0.01 vs IFPU2, n = 4).
All scaffolds exhibited elastic deformation at a low strain of less than 2% (Figure 8C). The compression moduli were 108.7 ± 3.9, 117.9 ± 8.5 and 127.7 ± 17.5 MPa for IFPU1, IFPU2 and IFPU3 scaffolds, respectively; the IFPU scaffolds presented greater compression moduli than the control PLA scaffold (87.8 ± 10.2 MPa) (Figure 8D) and gradually enhanced with the increase in hard segment content due to the gradual formation of the hard segment network. However, when the strain exceeded 10%, the real-time modulus of each scaffold decreased (Figure 8C), indicating that the pore structures were damaged. The corresponding compressive strengths were 7.0 ± 0.5, 14.0 ± 0.7 and 10.1 ± 0.9 MPa for IFPU1, IFPU2 and IFPU3, respectively; similarly, the IFPU scaffolds presented greater compression strength than the PLA scaffold (5.5 ± 0.2 MPa) (Figure 8E). Accordingly, the IFPU scaffolds had favorable mechanical properties in a low-strain environment, suitable as bone and cartilage substitutes.
Cytocompatibility of IFPU scaffolds in vitro
To investigate the feasibility of IFPU scaffolds as tissue scaffolds, their bioactivities were first investigated in vitro (Figure 9A). The IFPU scaffolds supported hBMSC adhesion and led to a negligible number of dead cells after culture for 3 and 7 days (Figure 9B). The hBMSCs continuously proliferated within 7 days (Figure 9C) and formed a dense cell layer on the strands (Figure 9D). hBMSCs on IFPU scaffolds showed better proliferation than those on PLA scaffolds, which was mainly because IFPU materials could promote cell adhesion, activity and proliferation (Figure S7A–C). Recruitment of surrounding cells through scaffolds would provide a cell source for tissue regeneration [32]. When the IFPU scaffolds with a height of 1.5 mm were placed on the TCPS plates containing hBMSCs, the hBMSCs were recruited and further migrated upward through the pores of the IFPU scaffolds, forming abundant cell networks on the top surfaces of the scaffolds after 14 days (Figure 9E). The cell number continuously increased on the scaffolds yet decreased on the plates (Figure 9F), indicating that the cells on the scaffolds are recruited from the plates. IFPU scaffolds demonstrated better cell recruitment than PLA scaffolds (Figure 9F).
*Cytocompatibility of IFPU scaffolds in vitro. (A) Schematic illustration of in vitro cytocompatibility evaluation of IFPU scaffolds. (B) Live/dead staining of hBMSCs seeded on the IFPU scaffolds for 3 and 7 days, along with the PLA scaffold as a control. (C) Cell proliferation activity of hBMSCs on the IFPU scaffolds measured by CCK-8. (D) F-actin and nucleus staining for 7 days. (E) Live/dead staining of hBMSCs recruited by scaffolds from the bottom to up. (F) Cell viability was measured by CCK-8 (I: Recruited by scaffolds, II: on the well plate) (P < 0.05 vs PLA, # P < 0.05 vs IFPU1, n = 4).
Biocompatibility of IFPU scaffolds in vivo
In addition, the in vivo biocompatibility of the IFPU scaffolds was investigated using a mouse subcutaneous implantation model. After 1 week of implantation, leukocyte (Figure 10A), erythrocyte (Figure 10B) and platelet (Figure 10C) related parameters fell into the normal range, indicating that the IFPU scaffolds were safe without leading to long-term inflammatory responses. After 16 weeks of implantation, the liver (Figure 10D) and kidney (Figure 10E) function parameters were normal as well, indicating that the degradation products of the IFPU scaffolds did not adversely affect liver and kidney metabolic functions. Similarly, the degradation products were safe for the major organs according to the H&E staining of the heart, liver, spleen, lung and kidney tissue sections after 12 and 16 weeks of implantation (Figure 10F) and for the surrounding tissues according to the H&E staining of the subcutaneous and surrounding tissue structures (Figure 10G). In detail, the heart muscle cells were arranged in order; the liver cells were normal and closely connected; the white pulp, red pulp and trabecular structure of the spleen were intact; the alveolar structures were clear and complete and the nephron structures were normal. The subcutaneous and surrounding tissue structures were intact and negligibly oedematous and inflammatory after 12 weeks of implantation.
Biocompatibility of IFPU scaffolds in vivo. (A) Inflammatory response, (B) red blood cell parameters and (C) platelet parameters about hemocompatibility of the IFPU2 scaffold implanted subcutaneously in Balb/c mice for 1 week, and the PLA scaffold as a control. (D) Parameters of liver function and (E) kidney function when the IFPU2 scaffold was implanted subcutaneously in Balb/c mice for 16 weeks. (F) H&E staining of heart, liver, spleen, lung and kidney tissue sections when the IFPU2 scaffold was implanted subcutaneously in Balb/c mice for 12 and 16 weeks. (G) H&E staining of surrounding tissue sections of the IFPU2 scaffold when scaffolds were implanted subcutaneously in Balb/c mice for 12 weeks. (n = 4).
Discussion
Novel IFPU scaffolds suitable for potential regenerative biomaterials
The degradation behavior of biomaterial-based scaffolds plays a crucial role in regulating tissue regeneration in situ. However, the laws and mechanisms of regulation are not yet fully understood due to the challenges in tracing and regulating the degradation behaviors of biomaterials. In this study, a series of novel BPUs possessing inherent fluorescence (IFPUs) have been successfully synthesized, in which the TPA fragments serve as both a component of the hard segment and a fluorescence probe to regulate and track the inherent degradation behaviors of biomaterials (Figure 2A). Subsequently, 3D IFPU scaffolds were fabricated to track the adjustable degradation behavior by fluorescence visualization and explore the potential as a regenerative biomaterial.
The degradation behavior of BPUs is closely related to the structure and ratio of the hard and soft segments, especially for the hard segment with abundant urethane groups [9, 33]. When the hard segment content is low (<4 wt%), the free urethane easily binds to water through hydrogen bonds, promoting BPU degradation with the increase of hard segment content [34]. However, when the hard segment content is high, urethane forms hydrogen bonds by itself, leading to the formation of a hard segment domain and microphase separation, inhibiting BPU degradation with the increase of hard segment content [18].
Therefore, a series of novel IFPUs (Figure 2A) were used to track the degradation behaviors through fluorescence visualization and to explore the potential as a regenerative biomaterial, taking into account the structure, molecular weight and hard segment content of BPU. First, PDLLA diol containing PEG and with a molecular weight of 7816 Da (Figure S1A) was selected as the soft segment to meet the biodegradability and sufficient mechanical properties required for tissue regeneration scaffolds. Second, the hard segment contents of IFPUs with a molecular weight >190 kDa (Figure 2H) were 2.9 wt%, 3.6 wt% and 4.0 wt% (Figure 2B), respectively, to regulate the degradation behaviors, as it promotes degradation when the hard segment content is below 4.0 wt%. Third, TPA, a fluorescent small molecule with biocompatibility [27, 35], was selected as a chain extender, serving as both a hard segment component and a fluorescent probe, endowing IFPU with outstanding fluorescence properties (Figure 3). Finally, IFPUs presented outstanding printability (Figure 4C–F), and their 3D printed scaffolds showed excellent fluorescence imaging properties to track the adjustable degradation behavior of IFPU scaffolds through fluorescent visualization ([Figures 5–7](#rbag006-F5 rbag006-F6 rbag006-F7)). Moreover, outstanding physical and biological properties supported IFPU scaffolds as promising regenerative tissue scaffolds, especially for bone and cartilage, including their macropores/micropores structure and >70% porosity favorable for cell adhesion and tissue ingrowth (Figure 8A and B) [28, 36], adequate mechanical properties supporting tissue regeneration (Figure 8C–F) [37, 38], and excellent cytocompatibility (Figure 9) and biocompatibility (Figure 10).
Trackability and adjustability of the IFPU scaffold degradation behaviors and their mechanisms
The traditional methods used to track the degradation behaviors of biomaterials not only require the sacrifice of many experimental samples but also are expensive and time-consuming. There is still a lack of scaffolds with adjustable and traceable degradation behaviors. The IFPU scaffolds showed adjustable and trackable degradation behaviors, which not only provided a new way to track the degradation of scaffolds using fluorescence visualization but also developed a new scaffold with adjustable degradation behavior for tissue regeneration.
The IFPU, in the form of solutions (Figure 3), solid films (Figure 4B) and porous scaffolds (Figure 4G), showed excellent fluorescence properties, noninvasively tracking their degradation behaviors (Figures 5A, 5D, 6A, 7A and 7B). The fluorescence residual ratio and fluorescence degradation rate of the wet and in situ scaffold can directly indicate the mass residual ratio and mass degradation rate during the degradation, because they were consistent with the corresponding fluorescence residual ratio and fluorescence degradation rate of the dry scaffold, and the corresponding mass residual ratio and mass degradation rate (Figures 5B, 5C, 5E, 6B, 6C, 7C and 7D), achieving real-time tracking of the degradation behavior. The degradation behaviors of scaffolds are influenced by various factors, making it difficult to accurately evaluate the degradation process. However, the method of fluorescence visualization tracking BPU degradation behavior based on IFPU was found to be feasible and effective in broad circumstances, i.e. chemical and physical cues (IFPU1/2/3, and formed as solid films or porous scaffolds) and surrounding environments (alkaline medium, in vitro, and in vivo) ([Figures 5–7](#rbag006-F5 rbag006-F6 rbag006-F7)). Therefore, the fluorescence visualization method based on IFPU can directly track the degradation behaviors of the scaffolds in real time, fast, non-destructive, visualized, convenient, time-saving, efficient and inexpensive manner. In addition, the media were endowed with fluorescent properties by soluble degradation products containing TPA fragments (Figures S4A, B and S5A, B). The fluorescence of the media can also indicate the degradation behaviors of the scaffolds, as the fluorescence release rate in the media was consistent with the corresponding fluorescence degradation rate and the mass degradation rate of the scaffolds (Figures 5B, 5C, 6B and 6C).
The molecular weight of IFPU decreased exponentially to 20–30 kDa (nearly 20% of the initial molecular weight) within 4 weeks (Figures S5E–H and S6B). However, the water solubility of the degradation products remained low, resulting in minimal mass loss and fluorescence loss (Figure 6B), as well as weak fluorescence intensity in the media (Figure 6C). Therefore, the TPA fragments are still retained in the solid degradation product of IFPUs, and the decay of molecular weight is caused by the ester dissociation in the soft segment rather than urethane in the hard segment (Figure 6E), further proving that fluorescence behaviors of IFPU scaffolds can be used to indicate their degradation behaviors. The hydrophilic groups (–COOH and –OH) formed by ester dissociation enhance water absorption (Figures S5D and S6A) and local acidity (Figure S5C), promoting the acidic autocatalytic degradation of the soft segment. As a result, a lot of soluble degradation products dissolve into the media, leading to rapid degradation with decreases in pH value, mass residual ratio and fluorescence residual ratio, as well as increases in the mass degradation rate, fluorescence degradation rate and fluorescence intensity of the media (Figure 6 and Figure S5). Finally, the TPA fragments are completely released into the media, leaving only a few soft fragments. Importantly, this degradation model of IFPU is independent of hard segment content, and in vitro and in vivo environments ([Figures 5–7](#rbag006-F5 rbag006-F6 rbag006-F7) and Figures S5 and S6).
To promote tissue regeneration, the degradation rate of the scaffold must align with the rate of tissue regeneration [39]. In this study, the degradation behaviors of IFPU scaffolds are enhanced by increasing the hard segment content, resulting in decreased half-life (IFPU1: 11.7 weeks, IFPU2: 10.5 weeks, IFPU3: 9.6 weeks) (Figure 6D). The hard segment content of <4 wt% (IFPU1: 2.9 wt%, IFPU2: 3.6 wt%, IFPU3: 4.0 wt%) (Figure 2B) makes it difficult to form hard segment aggregation by the hydrogen bond association of urethane [33, 34], so that free urethane easily binds with water by hydrogen bonds to increase hydrophilicity (Figure 2F and G) and promote polymer dissociation.
Although TPA, as a fluorescent probe, can be used to visually track the in vitro and in vivo degradation behavior of IFPU scaffolds in this study, it is impossible to visually track the degradation behavior of IFPU scaffolds in deep tissue in vivo due to the limited penetration of fluorescence. Therefore, some probes for in situ in vivo tracking in deep tissues also need to be further considered. Fluorescent probes at near-infrared wavelengths can penetrate up to 10 mm into the tissue [40]. Alternative imaging modalities possess better penetration, such as micro-CT imaging, nuclear magnetic resonance imaging [41], ultrasound [42] and so on. In future studies, these visualized imaging methods are expected to be used to track the degradation of BPU scaffolds in situ.
Conclusion
In this study, a series of biodegradable IFPUs was successfully prepared by TPA functioning as both a fluorescence probe and the hard segment component. The IFPUs showed excellent printability, and their 3D printed scaffolds possess an interconnected pore network, suitable mechanical properties, excellent cytocompatibility and biocompatibility, and adjustable and trackable degradation behaviors. Not only do the fluorescence residual ratios and the fluorescence degradation rates of the IFPU scaffolds well correspond to the corresponding mass residual ratios and the mass degradation rates, but also the fluorescence release rates in the media are consistent with the mass degradation rates, confirming that a fast, effective, non-destructive, time-saving and real-time method for tracking the degradation behavior of scaffolds is established based on IFPU fluorescence visualization. As novel regenerative biomaterials, the IFPU scaffolds could also promote cell recruitment, adhesion, spreading, proliferation and show excellent biocompatibility.
Supplementary Material
rbag006_Supplementary_Data
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