Cell-free biodegradable electroactive scaffold for urinary bladder regeneration

Abstract Tissue engineering heavily relies on cell-seeded scaffolds to support the complex biological and mechanical requirements of a target organ. However, in addition to safety and efficacy, translation of tissue engineering technology will depend on manufacturability, affordability, and ease of adoption. Therefore, there is a need to develop scalable biomaterial scaffolds with sufficient bioactivity to eliminate the need for exogenous cell seeding. Herein, we describe synthesis, characterization, and implementation of an electroactive biodegradable elastomer for urinary bladder tissue engineering. To create an electrically conductive and mechanically robust scaffold to support bladder tissue regeneration, we developed a phase-compatible functionalization method wherein the hydrophobic conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) was polymerized in situ within a similarly hydrophobic citrate-based elastomer poly(octamethylene-citrate-co-octanol) (POCO) film. We demonstrate the efficacy of this film as a scaffold for bladder augmentation in athymic rats, comparing PEDOT-POCO scaffolds to mesenchymal stromal cell-seeded POCO scaffolds. PEDOT-POCO recovered bladder function and anatomical structure comparably to the cell-seeded POCO scaffolds and significantly better than non-cell seeded POCO scaffolds. This manuscript reports: (1) a new phase-compatible functionalization method that confers electroactivity to a biodegradable elastic scaffold, and (2) the successful restoration of the anatomy and function of an organ using a cell-free electroactive scaffold.


Introduction
Since the advent of tissue engineering in the late 1980s, the promise of the eld ignited substantial initial excitement, yet its clinical impact remains limited to date.2][3][4][5] Despite these developments, many factors continue to hinder translational success including high R&D costs, manufacturing challenges, clinical costs, and limited e cacy of most synthetic scaffolds. 6The rigorous regulatory pathways accompanying new materials also present a signi cant obstacle, and the inclusion of cells or active biologics, while helpful in regenerating tissue, complicates regulatory processes and user adoption.
][9][10][11][12][13][14] Bladder tissue regeneration or augmentation is clinically required to address neurodegenerative diseases such as spina bi da, where bladder control and function become severely impaired, as well as in cases of cancer or trauma. 15spite the clinical need, very few scaffolds for tissue regeneration have shown promise without culturing cells on the scaffold prior to implantation (cell seeding). 16Shortcomings of currently used biomaterials are primarily attributed to (1) limited biomechanical suitability for dynamic tissue, (2) batchto-batch reproducibility of natural biomaterials, (3) pro-in ammatory responses, and (4) inadequate biological activity of synthetic biomaterials that do not support holistic tissue regeneration.Therefore, for bladder tissue engineering as well as the broader tissue engineering and regenerative engineering elds, there is a need for a mechanically durable, cell-free biomaterial with intrinsic bioactivity that can be feasibly and reproducibly manufactured.
Numerous approaches have been pursued to regenerate tissues and organs, yet the potential bene ts of electroactive biomaterials remain vastly underutilized.8][19] Conductive polymers are a unique class of organic materials with mixed ionic/electronic conduction that are increasingly being incorporated into biomaterials. 17,20,21 I8][29][30][31] We thus aim to evaluate whether incorporation of a conductive polymer into a biodegradable citrate-based elastomer will create an ionic electroactive environment that promotes safe and effective bladder regeneration.
Although the biological bene ts of incorporating hydrophobic conductive polymers into hydrophilic biomaterials have been well-documented, the lack of integration between the two materials poses signi cant complications, affecting structural stability and hinder composite conductivity. 32To address this issue in the context of bladder tissue engineering where mechanical properties are paramount, we developed a phase-compatible functionalization method, wherein the hydrophobic conductive polymer is integrated into a similarly hydrophobic matrix.Although it has previously been demonstrated that modi ed water-soluble conductive polymers can be reliably incorporated in hydrophilic water-based materials, the synthesis of these water-soluble units is complex, time consuming, and does not nancially or logistically lend itself toward commercialization. 33Conversely, hydrophobic monomer units, such as aniline, pyrrole, or 3,4-ethylenedioxythiophene (EDOT), are widely available yet require a hydrophobic substrate for functionalization.Citrate-based elastomers are a new class of hydrophobic biomaterial with versatile mechanical, chemical, and biological properties that have been recently used for biodegradable medical implants approved by the U.S. Food and Drug Administration. 34In particular, poly(octamethylene-citrate-co-octanol) (POCO) exhibits mechanical properties under cyclic tension that are suitable to tissues and organs with physically intensive requirements. 35In previous studies, POCO scaffolds pre-seeded with both CD34 + hematopoietic stem/progenitor cells and mesenchymal stromal cells (MSCs) demonstrated remarkable bene ts for bladder tissue regeneration, but the need for preseeded cells complicates manufacturability and limits the material's overall translational potential. 36rein, we describe the successful use of a phase-compatible functionalization method to create a biocompatible electroactive and bioactive elastomeric scaffold whereby the conductive polymer poly(3,4ethylenedioxythiophene) (PEDOT) is incorporated into POCO.We evaluated the safety and e cacy of the PEDOT-POCO scaffolds in a rodent bladder partial cystectomy model and demonstrate that we can restore bladder function to levels that are comparable to those achieved with a cell-seeded POCO scaffold.

Results
In situ coacervation yields stable PEDOT-POCO composites For preparation of PEDOT-POCO, cured POCO lms were rst passively infused with a mixture of EDOT and uncured POCO pre-polymer (Fig. 1a).The addition of POCO serves as both a plasticizer and stabilizer in the functionalization of larger PEDOT-POCO lms (Fig. 1b).To optimize the addition of POCO in the EDOT solution, EDOT-POCO mixtures with various POCO dilutions were polymerized into PEDOT-POCO nanoparticles and subsequently characterized (Fig. 1c).Initial addition of POCO at a 1:1000 dilution increased PEDOT nanoparticle size, from 560.5 ± 60.8 nm initially to 955.6 ± 32.5 nm, indicative of complexation between these molecules.Further addition of POCO decreased the size of polymerized PEDOT-POCO particles to 736.6 ± 26.6 nm and 588.5 ± 18.8 nm with 1:100 and 1:10 dilution ratios, respectively.With more POCO added to the PEDOT nanoparticles, electrostatic interactions between the molecules are strengthened, resulting in smaller composite particles.The 1:100 POCO:EDOT ratio minimized PEDOT-POCO particle polydispersity, indicating that this dilution optimized molecular interactions and particle homogeneity (Supplementary Fig. 1).This dilution ratio was thus selected for further lm functionalization.
Polymerization of EDOT-POCO mixtures within the POCO lm was performed using an aqueous oxidative solution, which initiates polymerization of the EDOT complexed with POCO.This water-based polymerization approach initiates PEDOT-POCO coacervate within the POCO matrix, driving the formation of nanoparticle-like structures throughout the bulk of the lm.The presence of these PEDOT-POCO nanofeatures was visualized and con rmed through scanning electron microscopy (SEM) (Fig. 1d).Nanoparticles, similar to those that were generated ex situ, were apparent throughout the PEDOT-POCO composite.Visual inspection of the particulate dimensions revealed features that were comparable in size to those measured ex situ (~ 500-700nm).
POCO is rich in carboxyl groups, and we hypothesized that the negative charge of these functional groups contributed to stable, electrostatic hole interactions with the PEDOT backbone. 37Surface chemistry of PEDOT-POCO lms was evaluated and compared to POCO for further characterization of the effects of conductive polymer incorporation.PEDOT-POCO lms demonstrated signi cantly reduced hydrophobicity compared to POCO, measured through contact angle measurements (Fig. 1e).Contact angle for POCO scaffolds was 52.9 ± 8.9 and 36.5 ± 6.9 for PEDOT-POCO.It is feasible to expect that the addition of a hydrophobic ller (PEDOT) to an already hydrophobic matrix (POCO) would increase the composite hydrophobicity, yet in this case we observed the opposite effect in that PEDOT-POCO surfaces were more hydrophilic.This result supports the hypothesis that PEDOT-POCO complexes are driven by electrostatic interactions with carboxyl groups, since those functional groups are dominantly hydrophobic.Composite surface charge was also measured, as it plays a signi cant role in regulating adhesion and other cellular processes (Fig. 1f). 38,39 or PEDOT-POCO, surface charge was signi cantly more positive (-22.1 ± 0.9 mV) than that of the POCO lm alone (-29.6 ± 2.7 mV), which can again be attributed to PEDOT sequestering negative charges of the carboxyl groups within POCO (Fig. 1g).
PEDOT functionalization of POCO maintains desirable mechanical, degradation, and antioxidant properties POCO has been validated as a promising and effective biomaterial that facilitates tissue function restoration in mechanically intensive applications such as orthopaedic and cardiovascular engineering. 35e thus set out to maintain the mechanical and biological suitability of POCO when designing PEDOT-POCO lms, the end goal being to preserve favorable POCO mechanical, degradation, and antioxidant qualities while enhancing the cellular electronic/ionic microenvironments through PEDOT functionalization.Antioxidant properties of citric acid-based materials are among their most notable advantages for facilitating repair in vivo. 40These potent antioxidant characteristics are owed to the structure of citric acid, and we thus sought to determine whether PEDOT incorporation impaired the inherent antioxidant capabilities of POCO.A DPPH assay was performed to evaluate the free radical scavenging capabilities of PEDOT-POCO and POCO, with polytetra uoroethylene (PTFE) serving as a negative control (Fig. 2a).PEDOT-POCO exhibited higher free radical scavenging than POCO alone, with the materials exhibiting 23.1 ± 4.8% and 16.0 ± 5.6% scavenging respectively.[43] In addition to its antioxidant properties, POCO is an attractive biomaterial for its degradability.
Degradability continues to pose a major challenge in the landscape of conductive biomaterial design, as conductive polymers themselves are not typically degradable and may impair the degradability of host matrices. 18,44,45 A accelerated degradation assay was performed by incubating POCO and PEDOT-POCO in phosphate-buffered saline (PBS) at 70 C to evaluate whether the material was capable of degradation, despite the PEDOT functionalization.We found that PEDOT-POCO bulk, speci cally the POCO-based constituents, did dissolve while precipitated PEDOT nanoparticles remained, the sizes of which were comparable to those observed in the SEM images (Fig. 2b).The solution the material was degraded in was pipetted out from the solid PEDOT particles and analyzed through FTIR (Supplementary Fig. 2).The FTIR spectra of the leached solution showed no notable differences compared to the reference PBS spectra, indicating that the remaining PEDOT is the only signi cant by-product of lm degradation, which precipitates out of the solution.After verifying the maintenance of biodegradability, we screened PEDOT-POCO for cytotoxicity via alamarBlue cell activity assay.Human bone marrowderived mesenchymal stromal cells (MSCs) were seeded on bronectin-coated materials.No differences in cell viability were apparent with cells seeded on PEDOT-POCO compared to POCO alone (Fig. 2c).
Finally, we veri ed that phase-compatible PEDOT functionalization sustained favorable POCO mechanics and elastomeric behavior.In several biological applications, such as bladder or cardiac engineering, material robustness and low modulus are vital towards the success of the application.Few stretchable conductive materials demonstrate both elasticity and ~ kPa modulus.Tensile testing as well as cyclic biaxial testing was performed (Fig. 2d-f Supplementary Fig. 3).Modulus, elongation, and elastic behavior of PEDOT-POCO were all comparable to that of the pristine POCO, with Young's modulus of PEDOT-POCO measured as 630 ± 188 kPa, whereas the modulus of POCO is 445 ± 87 kPa.The increased stiffness was not unexpected, as conductive polymers have been well-documented to increase material modulus, yet it is not expected that this magnitude of difference in modulus will bear impact regarding the material's capability to facilitate regeneration.For bladder regeneration, the complex mechanical loads the tissue is subject to in conjunction with a diversity of tissue types require candidate materials to satisfy a stringent set of standards.We thus have demonstrated that PEDOT-POCO performs comparably to POCO as evaluated against key metrics including antioxidant activity, biocompatibility, and mechanical suitability.

PEDOT incorporation into POCO differentially affects ionic and electronic microenvironments
The mixed ionic/electronic conductivity of conducting polymers, including PEDOT, is widely cited as a quality that functionally differentiates these materials from others. 21For biological applications, ionic conduction is particularly attractive considering the vital in uence of ions in life processes.Regardless of this recognized bene t, the speci c changes in ionic microenvironment following conductive polymer incorporation remains largely uncharacterized.During material preparation, lms are subjected to a leaching protocol to ensure that unreacted reagents, such as oxidant or polymer, are removed.Prior to functionalization with PEDOT, POCO lms are leached in solutions with gradually increasing osmolarity, with the nal step being cell media.After PEDOT functionalization, PEDOT-POCO is again leached in cell media.This leaching protocol establishes a speci c ionic microenvironment that varies between POCO and PEDOT-POCO, despite the primary source of ions (cell media) being consistent.To better understand how ionic content of the lms was altered by PEDOT incorporation, energy dispersive x-ray spectroscopy (EDS) was performed on lm cross-sections.Levels of magnesium, sodium, chlorine, and calcium were examined in POCO and PEDOT-POCO (Fig. 3a-b).We found that compared to POCO, PEDOT-POCO lms contained lower levels of magnesium and sodium but higher levels of chlorine and calcium.We hypothesize that POCO retains higher levels of small, monovalent ions due to trapping effects of the negatively charged carboxyl groups, which are unencumbered in the pristine POCO case (Fig. 3c).Chlorine levels were also dramatically elevated in PEDOT-POCO as compared to POCO likely due to interactions of negative chlorine ions and the positively charged PEDOT backbone.After characterizing differences in the ionic makeup of the lms after PEDOT incorporation, the electrochemical properties of the lms were characterized using a two electrode-setup with both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) (Fig. 3d-f).POCO CV demonstrated oxidative and reduction peaks that are characteristic of citric acid and its scavenging capabilities. 46As expected, PEDOT incorporation elevated conductivity, evidenced by both CV and EIS results.PEDOT-POCO conductivity is estimated to be ~ 0.3 S/m, based on 2-point resistance probing via CV.After validating that PEDOT-POCO bolstered passive lm conductivity, we sought to evaluate the electronic performance of the material under mechanical stress, which it would experience in vivo (Fig. 3g).To assess electromechanical durability of the lms, chronoamperometry was performed during tensile and biaxial testing (Fig. 3h,i).During tensile testing to 30% strain, PEDOT-POCO demonstrated ~ 5-times higher sensitivity to deformation than the POCO lms alone, the resistance of which did not change remarkably after a single deformation up to 30% strain.Changes in resistance were also measured throughout cyclic testing.During POCO biaxial testing, lm resistance increased outside the measurable range prior to 500 deformation cycles.In contrast, resistance of PEDOT-POCO lms was lower than that of POCO throughout the duration of testing.We have thus demonstrated PEDOT-POCO is mechanically and electronically robust for sustaining repeated cyclic deformation without undergoing signi cant damage to the lm integrity or electrochemical performance.

PEDOT-POCO enables functional bladder recovery that is comparable to cell-seeded scaffolds
After performing in vitro characterization on the PEDOT-POCO lms and verifying their preliminary suitability for bladder repair applications, we evaluated e cacy for promoting regeneration in a rat bladder augmentation model (Fig. 4a).Since acellular scaffolds have historically demonstrated limited success for facilitating bladder regeneration as e ciently as their cell-seeded counterparts, we investigated 3 experimental groups: POCO, cell-seed POCO, and PEDOT-POCO.By comparing the PEDOT-POCO scaffold to a cell-seeded, non-conductive scaffold, we benchmark the regenerative potential of PEDOT-POCO scaffolds to a condition that is considered the optimal approach to maximize regenerated tissue quality. 16With cell-seeded scaffolds, human MSCs were seeded in conjunction with CD34 + hematopoietic progenitor cells as previously described. 47This approach has demonstrated signi cant potential to facilitate functional bladder regeneration, including when used in conjunction with citratebased biomaterials such as POCO. 36,47 dder regeneration was assessed at 4 weeks post-augmentation.Urodynamics measurements are the key clinical indicator for bladder performance and were thus the assessment weighed most heavily when evaluating the performance of regenerated bladder tissue (Fig. 4b-c).Compliance and void frequency were derived from these measurements while capacity, another vital indicator of bladder performance, was measured directly (Figure d-f).Increased void frequency is associated with overactive bladder conditions and has been associated with dysregulated bladder sensation. 48,49 ssessment of void frequency in POCO, cell-seeded POCO, and PEDOT-POCO groups showed that both cell-seeded POCO and PEDOT-POCO reduced void frequency to comparable degrees, which was signi cantly lower than the POCO only group.In addition to void frequency, compliance and capacity were evaluated, with the former typically serving as the clinical indicator of limited bladder performance requiring intervention.Similarly to void frequency, both cell-seeded POCO and PEDOT-POCO comparably improved bladder compliance and capacity while signi cantly exceeding the tissue quality achieved by animals augmented using POCO only.
Taking these results together, we have demonstrated the potential for a purely synthetic biodegradable biomaterial, in this case PEDOT-POCO, to perform comparably to a cell-seeded scaffold in facilitating functional bladder regeneration.The use of cell-seeded materials has demonstrated signi cant bene ts throughout the regenerative engineering eld, yet we show here that conductive polymer-elastomers have the potential to match the quality of regenerated tissue while reducing cost and complexity of manufacturing such materials.

PEDOT-POCO supported the formation of various tissue types integral to bladder function
The capability for conductive polymers to induce differentiation and regeneration of tissue types including nerve, muscle, and epithelium has been well-documented in several previous studies. 17,25,50,51 Hover, their use for the regeneration of multiple tissue types within an organ, such as the bladder where regeneration of distinct cell lineages is required for restoration of holistic function has not been reported.Furthermore, after observing that PEDOT-POCO improves bladder function comparable to the cell-seeded POCO, we examined the regenerated organ more closely to determine how the conductive polymer in uenced regeneration of speci c tissues subtypes.A variety of histological analyses were performed to determine the quality of regenerated tissue subtypes.Trichrome staining was used to quantify urothelium, or epithelial bladder lining, thickness, muscle to collagen ratio, and blood vessel size (Fig. 5a).Nerve regeneration was quanti ed by immuno uorescence staining of β-III tubulin (Supplementary Fig. 4).
The urothelium is a critical anatomical feature that enables the bladder to safely interface with the nitrogenous waste it contains. 52An inability for scaffolds to withstand this harsh environment has been one longstanding limitation in the design of biomaterials for bladder regeneration. 16Furthermore, the success of a material depends heftily on its capability to facilitate urothelium regrowth (Fig. 5b).PEDOT-POCO promoted the growth of thicker urothelium (44.5 ± 24.6 µm), on average, than POCO alone (19.7 ± 12.2 µm), and was statistically indeterminate from cell-seeded POCO (48.4 ± 6.5 µm).Despite the statistically comparable performance, cell-seeded POCO regenerated thicker urothelium on average when compared to the PEDOT-POCO scaffold.
In addition to urothelium regeneration, muscle to collagen ratio of regenerated bladder tissue was analyzed.Excessive collagen production can be associated with in ammation and overall improper tissue regeneration.Smooth muscle tissue, however, is a critical constituent of the bladder wall and is vital for facilitating passive low-pressure bladder lling as well as subsequent voiding, or bladder emptying.Furthermore, the muscle:collagen ratio is quanti ed by analyzing the levels of red (muscle) to the levels of blue (collagen) throughout the trichrome images (Fig. 5c).Image quanti cation con rms this qualitative observation, with the PEDOT-POCO tissue having a signi cantly higher muscle:collagen ratio than the POCO alone, with values of 0.48 ± 0.18 and 0.22 ± 0.09 respectively.Native muscle:collagen ratio is typically approximately 0.58, demonstrating that PEDOT-POCO recapitulates tissue organization that is closer to the initial physiological state.
Vasculature is another vital component that is vital for bladder regeneration.Conductive polymers have remained largely unstudied in the context of vasculature regeneration, making this study among the rst to examine their potential to promote new blood vessel in ltration and development (Fig. 5d). 53Area of the average regenerated vessel in PEDOT-POCO bladders was ~ 30% larger than those in POCO only bladders yet ~ 25% smaller than those in cell-seeded POCO bladders.PEDOT-POCO's capability to increase regenerated vessel size compared to the POCO-only group is promising and points toward the capabilities for conductive polymers to facilitate vasculature regeneration in future applications.
Finally, we examined the average length of the peripheral nerve elements regenerated throughout the bladder tissue (Fig. 5e).Nervous system regeneration is one of the most popular applications that electronic materials are currently implemented towards, in part due to the widely-recognized electrogenic nature of this tissue type.As was observed with urothelium, smooth muscle, and vasculature, PEDOT-POCO signi cantly improved the average length of the nerves in regenerated bladder tissue compared to POCO alone.The cell-seeded POCO again performed the best among all of these conditions promoting growth of nerves that were on average 32.7 ± 5.2 µm long compared to 26.5 ± 5.1 µm with PEDOT-POCO and 20.2 ± 2.6 µm with POCO alone .

Discussion
A variety of materials have been explored for bladder engineering including acellular natural materials, synthetic polymers, and naturally derived polymers. 54Submucosal intestinal submucosa (SIS) and bladder acellular matrix (BAM) are among the most widely explored scaffolds for bladder regeneration, yet these scaffolds present poor mechanical properties for this application. 55Without the inclusion of cells, associated complications ensue such as brosis and stone formation, none of which were observed in our study. 54, 56, 57 58A variety of cell types have been investigated for bladder regeneration including urothelial cells, smooth muscle cells, adipose-derived stem cells, and MSCs. 47,59,60 Rsults from these studies support the need for exogenous cells seeded on scaffolds for the recapitulation of bladder function and have thus served as a beacon guiding the eld's standards.Despite the clinical potential for this approach, cell-seeded scaffolds do present with regulatory, manufacturing, and adoption barriers to widespread commercialization.From a regulatory point of view, the inclusion of cells on a scaffold is likely considered a combination product, requiring extensive clinical trials to demonstrate safety and e cacy.Cell manufacturing is expensive and di cult to reliably implement on a large scale.Regarding clinical adoption, cell-seeded scaffolds require special controls for transport and storage and in the case of an autologous cell source, require operations for both cell/tissue harvesting and scaffold implantation. 16Offering surgeons a scaffold that has higher processability, manufacturability, and simplicity than the cell-seeded scaffold alternative is expected to simplify the translational pathway for bladder tissue engineering.
When evaluated in a bladder augmentation model, the PEDOT-POCO scaffold, in the absence of seeded cells, demonstrated the capability to regenerate bladder tissue and restore organ function.The PEDOT-POCO scaffold properties achieved, including low modulus, elasticity, stretchability, electrical conductivity, and bulk degradability, enabled the rst implementation of an electroactive scaffold in a bladder augmentation model.Use of conductive polymers in tissue engineering has been limited due to biocompatibility and mechanical property mismatch challenges for the tissue or organ regeneration application.][63] Previous research has complexed the popular PEDOT:polystyrene sulfonate (PSS) dispersion with rubber, polyurethane, or other co-polymers. 61,64,65 Wile such composites show remarkable conductivity and stretchability, the modulus is typically higher than 1 MPa, and cytotoxicity often not reported.In our study, we introduce a novel biocompatible electroactive elastomer with rst-of-its-kind structural stability and bulk degradability.
In this study, we also demonstrate the capability for conductive materials to promote regeneration intrinsically, without external stimulation.Many regenerative engineering studies implement electroactive materials in conjunction with external electronic stimulation such as applied currents or potentials to modulate cellular processes. 17,50,66 Yt, active stimulation regimes mask the passive in uence of the electroactive material, which we have demonstrated here to be independently effective.8][69] They have also shown promise for their ability to facilitate epidermal tissue and muscle regeneration. 51,67,70,71 Gien that PEDOT-POCO scaffolds facilitated the simultaneous restoration of multiple tissue types, including urothelium, smooth muscle, nerve, and blood vessels, we demonstrate the feasibility of electroactive polymer or scaffold systems to recapitulate the anatomical complexities of other organs.
One mechanism conductive polymers passively modulate biological activity is through rearranging the ionic microenvironment. 72Ions are responsible for regulating various cellular processes and can in uence gene expression directly. 73We incorporated PEDOT within POCO lms to create scaffolds that modulate cellular electronic and ionic microenvironments, bolstering the scaffold's regenerative potential. 21,74 onductive polymers can drive increased Ca 2+ signaling as a key factor that regulates regenerative processes. 22,75,76 PDOT-POCO scaffolds facilitate higher passive Ca 2+ concentrations than POCO scaffolds.8][79][80] The exact mechanism and source of differences in ionic microenvironments are thus under continued investigation.Enhanced understanding of material-regulated ionic dynamics would be instrumental in advancing the design of more effective biological scaffolds.
Future work should aim to investigate longer-term outcomes and mechanisms associated with PEDOT-POCO degradation in vivo.Preliminary examination of in ammatory cell populations revealed comparable M1:M2 ratios with both POCO and PEDOT-POCO bladders (Supplementary Fig. 5).This result is highly promising as it suggests that the incorporation of PEDOT into the POCO matrix does not cause severe acute in ammatory responses that could be detrimental.While the short-term examination of immune response is promising, future investigation is warranted to better understand the physiological processing and longer-term risks of PEDOT-POCO as it degrades.One limitation of the present study is the use of an immunocompromised small animal model due to the need to include scaffolds seeded with human cells for comparison.To further screen the safety and e cacy of the PEDOT-POCO scaffold, its regenerative potential should be evaluated in a large animal model, which will be the focus of future studies.

Methods
POCO polymer and scaffold/ lm synthesis POCO was synthesized according to previously published methods. 35Brie y, citric acid, 1,8-octanediol, and octanol were stirred and melted at 160 C with liquid nitrogen ow until the mixture turned transparent, approximately 15 minutes, then the temperature was lowered to 145 C. Stir speed started at 500 rpm, and stir speed was subsequently reduced until the stir bar could not spin smoothly in the solution.After synthesis, the polymer was puri ed three times by dissolving in ethanol and precipitating out in MilliQ water.After puri cation, the pre-polymer was then diluted to 40 wt% in ethanol for lm synthesis.
Glass slides were prepped for POCO lms by rinsing in DI water and drying with nitrogen.Polyvinyl alcohol (PVA) solution was prepared at a concentration of 50 mg mL − 1 in MilliQ water, and 2 mL of PVA was pipetted onto each glass slide and then cured at 65 C for 1.5-2 hours until slides were dry. 2 mL of POCO was pipetted onto the PVA slides and left out at room temperature overnight to allow for excess solvent evaporation.After the overnight evaporation, lms were cured at 65 C for 4 days.POCO lms were then incubated in DI water overnight to dissolve the PVA and lift the POCO from the glass slide.Subsequent leaching of the lm was performed to remove unreacted carboxyl groups from the lm.Films were leached in 20% ethanol in PBS with 1% penicillin/streptomycin for 24 hours at 37 C, followed by PBS with 1% penicillin/streptomycin for 24 hours at 37 C. Films were then leached in low glucose DMEM with 1% penicillin/streptomycin for 2 hours, followed by a brief rinse in DI water, and low glucose DMEM was readded to the lm and leached overnight.Finally, the lm was leached in high glucose DMEM with 10% FBS and 1% penicillin/streptomycin.At this step, lms were ready for PEDOT functionalization.To sterilize the POCO lms for downstream use, they were cut into desired shape, incubated in 70% ethanol for 20 minutes, and leached overnight at 37 C in high glucose DMEM with 10% FBS and 1% penicillin/streptomycin to remove excess ethanol remaining on the lm.

In situ PEDOT-POCO oxidative polymerization
After complete leaching, POCO lms were functionalized with PEDOT rst by incubating the lms in EDOT with a 1:100 dilution of POCO pre-polymer for 72 hr at room temperature.Films were then moved to a polymerization solution containing 28.5% EDOT, 14.2% 1.24 M ammonium persulfate, and 57.3% phytic acid, and vigorously mixed at 4 C overnight.After polymerization, lms were incubated in 70% ethanol for 15 minutes and rinsed in high glucose DMEM with 10% FBS and 1% penicillin/streptomycin until the solution stopped changing color, to remove excess polymerization solution.Films were then incubated at room temperature in high glucose DMEM with 10% FBS and 1% penicillin/streptomycin for 24 hours, and the next day, leaching solution was replaced and lms were transferred to 37 C and leaching was continued for 24 hours.
Fourier Transform Infrared (FT-IR) Spectroscopy Fourier transform infrared (FT-IR) spectroscopy was performed on a Nicolet iS50 spectrometer using attenuated total re ection (ATR) spectroscopy to analyze the composition of degraded PEDOT-POCO leach solution.Spectra were collected with OMNIC Software.

Zeta Measurements
Zeta potential and particle size measurements were evaluated on a Malvern Zeta Sizer.For measurements of PEDOT particle size with POCO dilutions, PEDOT polymerization solution was prepared as described in the PEDOT-POCO in situ polymerization section and POCO was added at either 1:1000, 1:100, or 1:10 dilution levels.The resulting solution was vigorously mixed overnight at 4 C.The polymerized solution was then diluted at 1:100 in ethanol for particle size measurements.To analyze lm surface charge, PEDOT-POCO and POCO were frozen in liquid nitrogen then samples were pulverized, suspended in PBS, and ltered through a 70 µm mesh.Surface charge was quanti ed in triplicates and three different samples of solution were evaluated.

Scanning Electron Microscopy (SEM) & energy dispersive ray spectroscopy (EDS)
Samples were dried overnight at room temperature and then coated in carbon with a Denton III Desk Sputter Coater.EDS was performed on a Hitachi SU8030 to visualize the distribution of sulfur throughout the cross-section of the PEDOT-POCO lm.Data was collected and processed using Aztec software.

Free Radical Scavenging
Free radical scavenging was measured to assess PEDOT-POCO antioxidant activity.Samples were incubated for 24 hours at 37 C in 200 µM DPPH dissolved in ethanol.PTFE was used as a negative control, and ascorbic acid was used as a positive control.The positive control measurement was subtracted from all samples and normalized to the blank.Absorbance was measured at 320 nm to minimize precipitate background effects.All samples were measured in triplicate.

Contact Angle Measurements
Sample contact angles were measured with an Ossila Contact Angle Goniometer and measurements were collected with the corresponding Ossila software.Reports of contact angle were logged and measured in triplicate.

Electrochemical Measurements
EIS and CV were conducted using a two-probe set up with reference and working electrodes.The counter electrode was shorted to the reference.Pogo pins 4 mm apart were used to conduct measurements.EIS was executed from 0.1 to 10 6 Hz.CVs were performed by cycling from − 0.6 to 0.6 V 5 times.Measurements were collected with a Palmsens 4 and analyzed in the PS Trace software.

Mechanical & Electromechancial Testing
Tensile testing was performed using an Instron and data was collected via the Bluehill Universal software.All experiments were conducted in triplicate.Young's modulus was calculated by measuring the slope in the linear region of the tensile curve.Strips were cut into 1 mm rectangles, and a strain rate of 15 mm min − 1 was utilized for testing.For biaxial testing, a maximum strain of 30% was applied and material was elongated for 1000 cycles.To collect electromechanical measurements, copper tape was attached to the Instron grips which served as contact points for Palmsens 4 alligator clips.Chronoamperometry was then performed to measure changes in potential with the application of a constant current.Electromechanical data was collected using the PS Trace software.

Animal Surgeries & Urodynamics Studies (UDS)
Charles River 10-week old athymic rats weighing approximately 200 g underwent bladder augmentation procedures as previously described (n = 6 per group). 47,81 or POCO and PEDOT-POCO augmentations, all animals were female.For cell-seeded POCO, 4 females and 2 males were used.Brie y, rats were anesthetized and the bladder dome was excised through a 1-cm midline abdominal incision.Rats underwent a 50-60% partial cystectomy and the appropriate scaffold was sutured around the bladder defect using 7 − 0 polyglactin suture.An omental wrap was then sutured around the scaffold-augmented bladder.The rat abdomen was then closed with 5 − 0 ethibound and nally with 9-mm autoclips at the skin surface.
UDS were performed prior to augmentation procedures and 4-weeks post-augmentation.Animals were anesthetized and catheterized with the abdomen closed to measure capacity.The bladder was manually massaged to facilitate emptying.A 1 mL syringe was then used to ll the bladder until a voiding event was observed.The total volume infused into the bladder at the time of voiding was measured to determine bladder capacity.After measuring capacity, the rat abdomen was opened with a 1 cm incision and a 20 gauge cannula (Becton Dickinson) was inserted into the bladder wall to assess urodynamics.
The needle was secured to a transducer and syringe pump for consistent uid injection.The syringe pump was set to a ow rate of 150 µL min − 1 .Recordings of pressure over time were collected in a custom LabView program that included at least 5 voiding events.From this data, void frequency and compliance were analyzed.Compliance is calculated as the fraction of time during voiding in which the bladder pressure is less than 20 cmH 2 O. 82

Histological Assessments
Animals were euthanized 4 weeks post-augmentation and bladder tissue was collected and xed in 10% buffered formalin (Fisher Scienti c) overnight.After xation, samples were gradually dehydrated and embedded in para n.Sectioning was performed in 5 µm slices.Samples were then depara nized accordingly through well-established protocols for either trichrome or immuno uorescence staining.
Portions of the regenerated tissue were then imaged using a 40x objective on an Eclipse Ti2 Nikon microscope.For each histological assessment, 3 images were evaluated per tissue section.Urothelium thickness was measured in at least 3 unique locale per image from the trichrome-stained slides in ImageJ.For each animal, all measurements were averaged and taken to represent the average urothelium thickness.Muscle:collagen ratio and vasculature quanti cation was determined as previously described. 81muno uorescence was performed to evaluate peripheral nerve regeneration as well as macrophage (M1:M2) ratios.To evaluate bladder tissue peripheral nerve regeneration, an anti-β-III tubulin antibody (Biolegend) was utilized to stain bladder tissue at a 1:250 dilution for regenerating peripheral nerves.Peripheral nerve length was measured in ImageJ.For a signal to be measured as peripheral nerve, at least two distinct nuclei were required to be considered an element.This ensured that only elements within the appropriate plane were quanti ed.For M1:M2 quanti cation, an anti-CD86 antibody (Thermo, 1:100 dilution) was used to stain for M1 macrophages and an anti-CD163 antibody (Thermo, 1:200 dilution) was used to stain for M2 macrophages.Blood vessels were removed from the images due to their auto uorescence potentially confounding quanti cation, and cell area was measured using the Analyze Particles function in ImageJ.

Statistical Analysis
Results reported in the text are shown as mean ± standard deviation.Graphs show data set quartiles, with the medians shown as "X" and the mean being indicated as a solid line within the box.Statistical signi cance was determined using a two-tailed t-test where p < 0.05 was considered statistically signi cant.Signi cance was indicated with the following indications: *p < 0.05, **p < 0.01, ***p < 0.001.Declarations ECCS2025633), the International Institute for Nanotechnology, and Northwestern's MRSEC program (Grant No. NSF DMR-1720139).

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