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 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 develop a functionalization method wherein the

scaffolds. PEDOT-POCO recovers bladder function and anatomical structure comparably to the cell-seeded POCO scaffolds and significantly better than non-cell-seeded POCO scaffolds. This

manuscript reports a functionalization method that confers electroactivity to a biodegradable elastic scaffold, facilitating the successful restoration of anatomical and physiological

function of an organ. SIMILAR CONTENT BEING VIEWED BY OTHERS EXTRACELLULAR MATRIX SHEET MODIFIED WITH VEGF-LOADED NANOPARTICLES FOR BLADDER REGENERATION Article Open access 02 December 2022

ADVANCING AUTOLOGOUS UROTHELIAL MICROGRAFTING AND COMPOSITE TUBULAR GRAFTS FOR FUTURE SINGLE-STAGED UROGENITAL RECONSTRUCTIONS Article Open access 20 September 2023 THE EFFECTS OF BONE

MARROW STEM AND PROGENITOR CELL SEEDING ON URINARY BLADDER TISSUE REGENERATION Article Open access 27 January 2021 INTRODUCTION Since the advent of tissue engineering in the late 1980s, the

promise of the field ignited substantial initial excitement, yet its clinical impact remains limited to date. Biomaterial innovation has explored the implementation of soluble and

immobilized biochemical cues, biomimetic scaffold architectures, and biomechanical and biophysical enhancements1,2,3,4,5. Despite these developments, many factors continue to hinder

translational success including high R&D costs, manufacturing challenges, clinical costs, and limited efficacy of most synthetic scaffolds6. The rigorous regulatory pathways accompanying

new materials also present a significant obstacle, and the inclusion of cells or active biologics, while helpful in regenerating tissue, complicates regulatory processes and user adoption.

The bladder is one of the few organs evaluated in humans that has been engineered to replace or augment function using synthetic scaffolds seeded with cells7,8,9,10,11,12,13,14. Bladder

tissue regeneration or augmentation is clinically required to address neurodegenerative diseases such as spina bifida, where bladder control and function become severely impaired, as well as

in cases of cancer or trauma15. Despite the clinical need, very few scaffolds for tissue regeneration have shown promise without culturing cells on the scaffold prior to implantation (cell

seeding)16. Shortcomings of currently used biomaterials are primarily attributed to (1) limited biomechanical suitability for dynamic tissue, (2) batch-to-batch reproducibility of natural

biomaterials, (3) pro-inflammatory 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 fields, 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 benefits of electroactive biomaterials remain vastly

underutilized. Increased implementation of electroactive biomaterials has shown promise to improve biomaterial bioactivity, even in the absence of external stimulation17,18,19. Conductive

polymers are a unique class of organic materials with mixed ionic/electronic conduction upon doping that are increasingly being incorporated into biomaterials17,20,21. Incorporation of

conductive polymers into a biological matrix tunes the ionic microenvironment, which mediates fundamental processes of life including cell adhesion, migration, and

proliferation21,22,23,24,25,26. Previous studies have documented that conductive polymer-mediated modulation of the ionic environment facilitates more favorable nerve, muscle, and epithelial

repair, all of which are required for bladder tissue regeneration27,28,29,30,31. 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 benefits of incorporating hydrophobic conductive

polymers into hydrophilic biomaterials have been well-documented, the lack of integration between the two materials poses significant complications, affecting structural stability and

hindering composite conductivity32. To address this issue in the context of bladder tissue engineering where mechanical properties are paramount, we developed a functionalization scheme,

wherein the hydrophobic conductive polymer is integrated into a similarly hydrophobic matrix. Although it has previously been demonstrated that modified 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 financially or logistically lend itself

toward commercialization33. Conversely, 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 Administration34. In particular, POLY(OCTAMETHYLENE-CITRATE-CO-OCTANOL) (POCO) exhibits mechanical properties under cyclic

tension that are suitable to tissues and organs with physically intensive requirements35. In previous studies, POCO scaffolds pre-seeded with both CD34+ hematopoietic stem/progenitor cells

and mesenchymal stromal cells (MSCs) demonstrated remarkable benefits for bladder tissue regeneration, but the need for pre-seeded cells complicates manufacturability and limits the

material’s overall translational potential36,37. Herein, we describe the successful synthesis of a biocompatible electroactive and bioactive elastomeric scaffold whereby the conductive

polymer POLY(3,4-ETHYLENEDIOXYTHIOPHENE) (PEDOT) is incorporated into POCO. This functionalization is performed by incubating cured POCO films in a complexation mixture consisting of EDOT

and uncured POCO (uPOCO). After the initial EDOT-uPOCO incubation, a downstream in situ polymerization is performed within the film matrix. We evaluated the safety and efficacy 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 COMPLEXATION YIELDS STABLE PEDOT-POCO COMPOSITES For preparation of PEDOT-POCO, cured POCO films (Fig. 1a) were first passively infused with a mixture of EDOT and

uncured POCO pre-polymer (uPOCO) (Fig. 1b). The addition of uPOCO to the EDOT solution during film incubation serves as both a plasticizer and stabilizer in the functionalization of larger

PEDOT-POCO films (Fig. 1c). To optimize the addition of uPOCO in the EDOT solution as well as to better understand the underlying mechanisms of EDOT-uPOCO interactions, EDOT-uPOCO mixtures

with various uPOCO dilutions were polymerized into PEDOT-uPOCO polyelectrolyte complexes and subsequently characterized (Fig. 1d). 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 uPOCO decreased the size of polymerized

PEDOT-uPOCO particles to 736.6 ± 26.6 nm and 588.5 ± 18.8 nm with 1:100 and 1:10 dilution ratios, respectively. With more uPOCO added to the PEDOT nanoparticles, electrostatic interactions

between the molecules are strengthened, resulting in smaller composite particles. The 1:100 uPOCO:EDOT ratio minimized PEDOT-uPOCO particle polydispersity, indicating that this dilution

optimized molecular interactions and particle homogeneity (Supplementary Fig. 1). This dilution ratio was thus selected for further film functionalization. Polymerization of EDOT-uPOCO

mixtures within the POCO film was performed using an aqueous oxidative solution, which initiates polymerization of the EDOT complexed with uPOCO. 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 film. The presence of these PEDOT-POCO nanofeatures

was visualized and confirmed through scanning electron microscopy (SEM) (Fig. 1e). Nanostructures, 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 (~500–700 nm). Energy dispersive x-ray spectroscopy (EDS)

analysis of a PEDOT-POCO cross-section demonstrated that sulfur, and thus PEDOT, was homogeneous throughout the material bulk (Supplementary Fig. 2). Surface chemistry of PEDOT-POCO films

was evaluated and compared to POCO for further characterization of the effects of conductive polymer incorporation. PEDOT-POCO films demonstrated significantly reduced hydrophobicity

compared to POCO, measured through contact angle measurements (Supplementary Fig. 3a). Contact angle for POCO scaffolds was 52.9 ± 8.9° and 36.5 ± 6.9° for PEDOT-POCO. The increase in

scaffold hydrophilicity is attributed to the water-based polymerization step. During this portion of functionalization, the films are exposed to an oxidative bath. Diffusion of the oxidative

species into the scaffold is hypothesized to be the primary driver of reduced hydrophobicity in the PEDOT-POCO scaffold compared to the pristine POCO material33. Composite surface charge

was also measured, as it plays a significant role in regulating adhesion and other cellular processes (Supplementary Fig. 3b)38,39. For PEDOT-POCO, surface charge was significantly more

positive (–22.1 ± 0.9 mV) than that of the POCO film alone (–29.6 ± 2.7 mV). 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

engineering35,37. We thus set out to maintain the mechanical and biological suitability of POCO when designing PEDOT-POCO films, 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 vivo40. These 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 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was performed to evaluate the free radical scavenging

capabilities of PEDOT-POCO and POCO, with polytetrafluoroethylene (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. These results are in line with previous studies documenting free radical scavenging capabilities of conductive

polymers including PEDOT41,42,43. In addition to its antioxidant properties, POCO is an attractive biomaterial for its degradability44,45. 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 matrices18,46,47. An 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, specifically the POCO-based constituents, did dissolve while precipitated PEDOT nanoparticles remained (Fig. 2b). The degraded material 

solution was pipetted out from the solid PEDOT particles and analyzed through Fourier Transform Infrared Spectroscopy (FTIR) (Supplementary Fig. 4a). 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 significant by-product of film degradation, which precipitates out of

the solution. In addition to the FTIR analysis, particle size of the degraded film particulates was analyzed (Supplementary Fig. 4b). The average particle size was 768.5 nm, which is within

the previously established range of the PEDOT-uPOCO complex measured outside the film. Based on these findings, the PEDOT-uPOCO complex likely remains intact as the bulk material degrades.

After verifying the maintenance of biodegradability, we screened PEDOT-POCO for cytotoxicity via alamarBlue cell activity assay. Human bone marrow-derived MSCs were seeded on

fibronectin-coated materials. No differences in cell viability were apparent with cells seeded on PEDOT-POCO compared to POCO alone (Fig. 2c). Both scaffold types demonstrated inherent

antimicrobial properties, which was evaluated through visual examination of S_taphylococcus_ _aureus_ growth (Supplementary Fig. 5)48,49. Finally, we verified that 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. 6). 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 doped conducting polymers, including PEDOT, is widely cited as a quality that functionally

differentiates these materials from others21. For biological applications, ionic conduction is particularly attractive considering the vital influence of ions in life processes. Regardless

of this recognized benefit, the specific changes in ionic microenvironment following conductive polymer incorporation remains largely uncharacterized. During material preparation, films are

subjected to a leaching protocol to ensure that unreacted reagents, such as oxidant or polymer, are removed. Prior to functionalization with PEDOT, POCO films are leached in solutions with

gradually increasing osmolarity, with the final step being cell media. After PEDOT functionalization, PEDOT-POCO is again leached in cell media. This leaching protocol establishes a specific

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 films was

altered by PEDOT incorporation, EDS was performed on film 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 films 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

films after PEDOT incorporation, the electrochemical properties of the films 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 capabilities50. As 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 film 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 films, 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 films 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, film resistance increased outside the measurable range prior to 500 deformation cycles. In contrast, resistance of

PEDOT-POCO films 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 significant damage to the film 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 films and verifying their preliminary suitability for bladder repair applications, we evaluated efficacy for promoting

regeneration in a rat bladder augmentation model (Fig. 4a). Since acellular scaffolds have historically demonstrated limited success for facilitating bladder regeneration as efficiently 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 quality16. With cell-seeded scaffolds,

human MSCs were seeded in conjunction with CD34+ hematopoietic progenitor cells as previously described51. This approach has demonstrated significant potential to facilitate functional

bladder regeneration, including when used in conjunction with citrate-based biomaterials such as POCO36,51. Bladder regeneration was assessed at 4 weeks post augmentation. At this time

point, the scaffold is not yet fully degraded, but utilizing this endpoint ensures that advantages of early-stage regeneration are captured. 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 (Fig. 4d–f). Increased void frequency is associated with overactive

bladder conditions and has been associated with dysregulated bladder sensation52,53. Assessment 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 significantly 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 significantly 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 significant benefits throughout the regenerative engineering field, 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 studies17,25,54,55. However, 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 influenced regeneration of specific tissues subtypes (Supplementary Figs. 7, 8). 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 quantified by immunofluorescence staining of β-III tubulin (Supplementary Fig. 9). The urothelium

is a critical anatomical feature that enables the bladder to safely interface with the nitrogenous waste it contains56. An inability for scaffolds to withstand this harsh environment has

been one longstanding limitation in the design of biomaterials for bladder regeneration16. Furthermore, 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 is associated with inflammation 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 filling as well as subsequent voiding,

or bladder emptying. Furthermore, the muscle:collagen ratio is quantified by analyzing the levels of red (muscle) to the levels of blue (collagen) throughout the trichrome images (Fig. 5c).

Image quantification confirms this qualitative observation, with the PEDOT-POCO tissue having a significantly 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 ~0.58, demonstrating that PEDOT-POCO recapitulates tissue organization that is closer to the initial physiological

state. Vasculature is another component that is vital for bladder regeneration. Conductive polymers have remained largely unstudied in the context of vasculature regeneration, making this

study among the first to examine their potential to promote new blood vessel infiltration and development (Fig. 5d)57. Area 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 significantly

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 tissue engineering including acellular natural materials, synthetic polymers, and naturally derived polymers37,58. Small intestinal submucosa and bladder acellular matrix are among

the most widely explored scaffolds for bladder regeneration, yet these scaffolds present poor mechanical properties for this application and have demonstrated suboptimal outcomes59. A

variety of cell types have previously been investigated for bladder regeneration including urothelial cells, smooth muscle cells, adipose-derived stem cells, and MSCs51,60,61. Results 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 field’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 efficacy. Cell manufacturing is expensive and

difficult 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 implantation16. Offering surgeons a scaffold that has better processability, manufacturability, and simplicity than

the cell-seeded alternative is expected to simplify the translational pathway for bladder tissue engineering. However, potential problems often attributed to cell-free scaffolds for bladder

tissue regeneration include fibrosis and stone formation. We did not observe either problem at the 4 week timepoint of our study58,62,63,64. Citrate-based materials are a highly advantageous

platform for bladder regeneration as citrate limits stone formation through its intrinsic capability to chelate oxalate, a significant driver of stone formation and subsequent

encrustation65,66. These benefits of POCO were demonstrated in the study from our research group that showed POCO regenerated bladder tissue and preserved bladder function, without stone

formation or encrustation, for two years in non-human primates37. As infection is another significant driver of encrustation, preliminary demonstration of PEDOT-POCO’s antimicrobial

properties is a key result pointing to PEDOT-POCO sustaining POCO’s resistance to encrustation67. Additional studies are needed to verify PEDOT-POCO’s long-term safety and efficacy for

bladder tissue regeneration. 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

first implementation of an electroactive scaffold in a bladder augmentation model. These results are especially notable considering the low loading levels of PEDOT within the films. EDS

shows that the total loading of sulfur in the films is less than 5% total weight (Supplementary Fig. 2). 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. Other studies have sought different approaches for improving the mechanical

properties of PEDOT-based materials32,68,69,70. Previous research has complexed the popular PEDOT:POLYSTYRENE SULFONATE (PSS) dispersion with rubber, polyurethane, or other

co-polymers68,71,72. While 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 biocompatible electroactive elastomer with first-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 processes17,54,73. Yet, active stimulation regimes mask the passive influence of the electroactive material, which we

have demonstrated here to be independently effective. Electroactive materials have been predominantly examined for nerve regeneration74,75,76. They have also shown promise for their ability

to facilitate epidermal tissue and muscle regeneration55,74,77,78. Given 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. Results from

in vivo experiments can be very variable and our histomorphometry data reflect that variability. The deviations may be a function of the mechanisms behind PEDOT-POCO benefits and will be

studied in more detail through future work. It should be highlighted, however, that the true benefits of PEDOT-POCO are observed with regards to functionality of the regenerated bladder

tissue, in which case the magnitude of error was comparable to the other groups analyzed. One mechanism by which conductive polymers passively modulate biological activity is through

rearranging the ionic microenvironment79. Ions are responsible for regulating various cellular processes and can influence gene expression directly80. We incorporated PEDOT within POCO films

to create scaffolds that modulate cellular electronic and ionic microenvironments, bolstering the scaffold’s regenerative potential21,81. Conductive polymers can drive increased _Ca__2+_

signaling as a key factor that regulates regenerative processes22,82,83. PEDOT-POCO scaffolds facilitate higher passive _Ca__2+_ concentrations than POCO scaffolds. The specific biophysical

relationship between conductive polymers and upregulated _Ca__2+_ induction has yet to be elucidated, although there is evidence pointing to membrane depolarization as a key transducer in

initiating differential cellular ion fluxes and influencing signaling84,85,86,87. 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 inflammatory cell populations revealed comparable M1:M2 ratios with

both POCO and PEDOT-POCO bladders (Supplementary Fig. 10). Gross examination and histopathology of the kidneys was performed to screen for signs of damage due to the PEDOT-POCO scaffold

(Supplementary Fig. 11). The kidney anatomy of animals treated with both POCO and PEDOT-POCO were comparable. These results are promising as it suggests that the incorporation of PEDOT into

the POCO matrix does not cause severe acute inflammatory 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 efficacy 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 ETHICAL STANDARDS Animal studies were conducted according to the guidelines established and

approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University. The study was executed under IACUC protocol IS00011571. Animals had 24/7 access to food and

water and were housed in Northwestern University’s animal facility. POCO POLYMER & SCAFFOLD/FILM SYNTHESIS POCO was synthesized through stirring and mixing citric acid, 1,8-octanediol,

and octanol at 160 °C with liquid nitrogen flow until the mixture turned transparent, ~15 min, then the temperature was lowered to 145 °C35. Stir speed started at 500 rpm, and was

subsequently reduced until the stir bar could not spin smoothly in the solution. After synthesis, the polymer was purified three times by dissolving in ethanol and precipitating out in

MilliQ water. After purification, the pre-polymer was then diluted to 40 wt% in ethanol for film synthesis. Glass slides were prepped for POCO films 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

 h until slides were dry. 1.5 mL of POCO prepolymer was pipetted onto the PVA slides and left out at room temperature overnight to allow for excess solvent evaporation. After the overnight

evaporation, films were cured through polycondensation post-polymerization at 65 °C for 4 days. In citric acid-based films, this curing occurs via covalent crosslinking88,89. After complete

curing, POCO films were incubated in DI water overnight to dissolve the PVA and lift the POCO from the glass slide. Subsequent leaching of the film was performed to remove unreacted carboxyl

groups from the film. Films were leached in 20% ethanol in PBS with 1% penicillin/streptomycin for 24 h at 37 °C, followed by PBS with 1% penicillin/streptomycin for 24 h at 37 °C. Films

were then leached in low glucose DMEM with 1% penicillin/streptomycin for 2 h, followed by a brief rinse in DI water, and low glucose DMEM was readded to the film and leached overnight.

Finally, the film was leached in high glucose DMEM with 10% FBS and 1% penicillin/streptomycin. At this step, films were ready for PEDOT functionalization. To sterilize the POCO and

PEDOT-POCO films for downstream use, they were cut into desired shape, incubated in 70% ethanol for 20 min, and leached overnight at 37 °C in high glucose DMEM with 10% FBS and 1%

penicillin/streptomycin to remove excess ethanol remaining on the film. SYNTHESIS OF PEDOT:UPOCO POLYELECTROLYTE COMPLEXES To better evaluate the PEDOT-POCO complexation dynamics, PEDOT and

uPOCO interactions were evaluated by analyzing the colloidal nanoparticles outside cured films. uPOCO was added in various concentrations to an EDOT polymerization mixture. Polymerization

solution consisted of 14.2% 1.24 M ammonium persulfate, and 57.3% phytic acid added directly to pure EDOT. uPOCO was added in 1:1000, 1:100, and 1:10 volumetric ratios to the EDOT

polymerization mixture. The resulting solution was then mixed vigorously at 4 ⁰C overnight. Subsequent analysis was performed after diluting the PEDOT:uPOCO in ethanol at a 1:100 ratio. IN

SITU PEDOT-POCO OXIDATIVE POLYMERIZATION After complete leaching, POCO films were functionalized with PEDOT first by incubating the films in EDOT with a 1:100 dilution of uncured POCO

pre-polymer (uPOCO) for 72 h 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. The

incubation was performed in a Falcon tube, which was placed on a shaker where the films were vigorously mixed at 4 °C overnight. After polymerization, films were incubated in 70% ethanol for

15 min 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 h, and the next day, leaching solution was replaced and films were transferred to 37 °C

and leaching was continued for 24 h. FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY Fourier transform infrared (FTIR) spectroscopy was performed on a Nicolet iS50 spectrometer using

attenuated total reflection spectroscopy to analyze the composition of degraded PEDOT-POCO leach solution. Spectra were collected with OMNIC Software. ZETA POTENTIAL MEASUREMENTS Zeta

potential and particle size measurements were evaluated on a Malvern Zeta Sizer. For measurements of PEDOT particle size with uPOCO dilutions, PEDOT polymerization solution was prepared as

described in the PEDOT-POCO in situ polymerization section and uPOCO 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 film surface charge, PEDOT-POCO and POCO films were frozen in liquid nitrogen

then samples were pulverized, suspended in PBS, and filtered through a 70 µm mesh. Surface charge was quantified in triplicates and three different samples of solution were evaluated.

SCANNING ELECTRON MICROSCOPY (SEM) & ENERGY DISPERSIVE X-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 film. Data was collected and processed using Aztec

software. FREE RADICAL SCAVENGING Free radical scavenging was measured to assess PEDOT-POCO antioxidant activity. Films were cut into circles using a 6-mm biopsy punch. Samples were

incubated for 24 h 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.

ELECTROCHEMICAL MEASUREMENTS EIS and cyclic voltammetry (CV) were conducted using a two-probe set up with reference and working electrodes. Circular films were cut with a 6-mm biopsy punch

and subsequently measured. 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 106 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 5 × 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 the 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 while the material was subject to mechanical testing90,91.

Electromechanical data was collected using the PS Trace software. CELL VIABILITY & ANTIMICROBIAL PROPERTIES Cytocompatibility analysis was performed by culturing MSCs on POCO and

PEDOT-POCO scaffolds and utilizing alamarBlue to assess cell health. Scaffolds were coated with 100 µg mL–1 fibronectin for one hour at room temperature prior to cell-seeding. Cell health

was analyzed at 24-h and 48-h post seeding and normalized to the POCO-only condition. Antimicrobial properties of the scaffolds were analyzed against _S. aureus_. Bacteria were cultured with

the scaffolds in DMEM + 10% LB Broth overnight at 106 CFU mL–1. After 24 h, the scaffolds were visually examined for evidence of biofilm growth. ANIMAL SURGERIES & URODYNAMICS STUDIES

(UDS) Athymic rats (Charles River), each being ~10 weeks old as well as weighing 200 g, were subject to bladder augmentation (_n_ = 6 per group)51,92. For POCO and PEDOT-POCO augmentations,

all animals were female. For cell-seeded POCO, 4 females and 2 males were used. After being anesthetized, an excision was made to excise the bladder from the abdomen. A 50–60% partial

cystectomy was performed by removing the top portion of the bladder dome, and 7–0 polyglactin suture was used to secure the appropriate scaffold to repair the bladder. After completion of

the cystectomy, omental wrap was secured around the scaffold-augmented bladder. The rat abdomen was then closed with 5–0 ethibound suture, 9-mm autoclips were applied at the skin surface,

and the wound was covered with antibacterial ointment. UDS measurements were performed to measure bladder function at the completion of the study, which was concluded at 4 weeks post

augmentation. Animals were anesthetized and catheterized with the abdomen closed to measure the bladder volume capacity. A 1 mL syringe was used to fill the bladder until a voiding event was

observed. The total volume infused into the bladder at the time of voiding was recorded to determine bladder capacity. After measuring capacity, the rat abdomen was opened to access the

bladder, and a 20-gauge needle (Becton Dickinson) was inserted into the bladder wall to further assess urodynamics and thus bladder performance. The needle was secured to a transducer for

collecting measurements, and a syringe pump was used to ensure consistent fluid injection. The syringe pump was set to a flow rate of 150 µL min–1. Recordings of pressure over time were

collected in a custom LabView program, and recordings were collected until at least 5 voiding events were noted. From this data, void frequency and compliance were analyzed. Compliance was

calculated by determining the fraction of time during voiding where the bladder pressure remains below 20 cmH2O93. HISTOLOGICAL ASSESSMENTS Animals were euthanized 4 weeks post augmentation

and bladder tissue was collected and fixed in 10% buffered formalin (Fisher Scientific) overnight. After fixation, samples were gradually dehydrated and embedded in paraffin. Sectioning was

performed in 5 µm slices. Samples were then deparaffinized for either trichrome, hematoxylin and eosin (H&E), or immunofluorescence staining. The regenerated tissue regions were then

imaged using a 40× objective on an Eclipse Ti2 Nikon microscope. For each histological assessment, 3 images were evaluated per tissue section. Urothelium thickness was measured in ImageJ by

randomly selecting at least 3 unique locations per image from the trichrome-stained slides. For each animal, all measurements were averaged and taken to represent the average urothelium

thickness. Muscle:collagen ratio and vasculature quantification was determined by measuring the ratio of blue (representing collagen) and red (representing muscle) levels of three images per

animal in Adobe Photoshop92. The vasculature sizes were also measured through Adobe Photoshop. All identifiable vessel areas were measured from the trichrome images with at least 3 images

analyzed per animal. Immunofluorescence 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 quantified. For M1:M2 quantification, an anti-CD86 antibody (Abcam ab220188, 1:100 dilution) was used to stain for M1 macrophages and an anti-CD163 antibody (Abcam ab182422, 1:200

dilution) was used to stain for M2 macrophages. Blood vessels were removed from the images due to their autofluorescence potentially confounding quantification, 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 significance was determined using a two-tailed _t_-test where _p_ < 0.05 was considered statistically

significant. Significance was illustrated with the following indications: *_p_ < 0.05, **_p_ < 0.01, ***_p_ < 0.001. REPORTING SUMMARY Further information on research design is

available in the Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY The raw and analyzed datasets generated during the study are too large to be publicly shared,

yet they are available from the corresponding authors upon reasonable request. Any of the data documented in the figures or the Supplementary Information may be provided to the requester.

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ACKNOWLEDGEMENTS This work was primarily supported by a National Institutes of Health awards R01EB026572 (AKS and GAA) and R01DK109539 (AKS). AKS also acknowledges the Michelon Family for

their support. RK was supported in part by the National Institutes of Health Training Grant (Grant No. T32GM008449) through Northwestern University’s Biotechnology Training Program. RK

gratefully acknowledges support from the Ryan Fellowship and the International Institute for Nanotechnology at Northwestern University. This work made use of the EPIC facility of

Northwestern University’s NUANCE Center, which has received support from the SHyNE Resource (Grant No. NSF ECCS2025633), the International Institute for Nanotechnology, and the Northwestern

MRSEC program (Grant No. NSF DMR−1720139). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA Rebecca L. Keate, 

Maria Mendez-Santos, Andres Gerena, Madeleine Goedegebuure, Jonathan Rivnay, Arun K. Sharma & Guillermo A. Ameer * Center for Advanced Regenerative Engineering, Northwestern University,

Evanston, IL, USA Rebecca L. Keate, Maria Mendez-Santos, Andres Gerena, Madeleine Goedegebuure, Jonathan Rivnay, Arun K. Sharma & Guillermo A. Ameer * Division of Pediatric Urology,

Department of Surgery, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA Matthew I. Bury & Arun K. Sharma * Stanley Manne Children’s Research Institute, Louis A.

Simpson and Kimberly K. Querrey Biomedical Research Center, Chicago, IL, USA Matthew I. Bury & Arun K. Sharma * Center for Regenerative Nanomedicine, Northwestern University, Chicago,

IL, USA Jonathan Rivnay & Arun K. Sharma * Department of Materials Science, Northwestern University, Evanston, IL, USA Jonathan Rivnay * Querrey Simpson Institute for Bioelectronics,

Northwestern University, Evanston, IL, USA Jonathan Rivnay & Guillermo A. Ameer * Chemistry Life Processes Institute, Northwestern University, Evanston, IL, USA Jonathan Rivnay & 

Guillermo A. Ameer * International Institute for Nanotechnology, Evanston, IL, USA Jonathan Rivnay & Guillermo A. Ameer * Department of Urology, Feinberg School of Medicine, Northwestern

University, Chicago, IL, USA Arun K. Sharma * Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA Guillermo A. Ameer Authors * Rebecca L. Keate

View author publications You can also search for this author inPubMed Google Scholar * Matthew I. Bury View author publications You can also search for this author inPubMed Google Scholar *

Maria Mendez-Santos View author publications You can also search for this author inPubMed Google Scholar * Andres Gerena View author publications You can also search for this author inPubMed

 Google Scholar * Madeleine Goedegebuure View author publications You can also search for this author inPubMed Google Scholar * Jonathan Rivnay View author publications You can also search

for this author inPubMed Google Scholar * Arun K. Sharma View author publications You can also search for this author inPubMed Google Scholar * Guillermo A. Ameer View author publications

You can also search for this author inPubMed Google Scholar CONTRIBUTIONS R.L.K., J.R., G.A.A., A.K.S. designed the experiments conducted here. R.L.K. led material design, synthesis,

characterization, and histological analysis. M.I.B. performed animal surgeries and assisted with the histological experiments. A.G. helped synthesize the material and generate POCO films.

M.M.S. and M.G. assisted with histological staining. CORRESPONDING AUTHOR Correspondence to Guillermo A. Ameer. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Communications_ thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

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http://creativecommons.org/licenses/by-nc-nd/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Keate, R.L., Bury, M.I., Mendez-Santos, M. _et al._ Cell-free biodegradable

electroactive scaffold for urinary bladder tissue regeneration. _Nat Commun_ 16, 11 (2025). https://doi.org/10.1038/s41467-024-55401-9 Download citation * Received: 23 January 2024 *

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