Patent Application
20240352186
Provided herein are citrate-based elastomeric materials comprising conductive polymers, such as poly(3,4-ethylenedioythiophene) (PEDOT)-poly(1,8-octamethylene-citrate-co-octanol) (POCO), and methods of use therefor for medical applications, such as tissue engineering.
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.
Provided herein are citrate-based elastomeric materials comprising conductive polymers, such as poly(3,4-ethylenedioythiophene) (PEDOT)-poly(1,8-octamethylene-citrate-co-octanol) (POCO), and methods of use therefor for medical applications, such as tissue engineering.
In some embodiments, provided herein are compositions comprising a citrate-based elastomer functionalized with a conductive polymer. In some embodiments, the elastomer is poly(1,X-(CH2)X-citrate-co-(CH2)XOH), wherein X is 4-16. In some embodiments, the elastomer is poly(1,8-octamethylene-citrate-co-octanol) (POCO). In some embodiments, the conductive polymer is selected from polyaniline (PAni), polythiophene (PT), polypyrrole (PPy) and poly(3,4-ethylenedioythiophene) (PEDOT). In some embodiments, the conductive polymer is PEDOT. In some embodiments, the elastomer film is functionalized with the conductive polymer by in situ polymerization. In some embodiments, the composition comprises PEDOT-POCO.
In some embodiments, provided herein are compositions comprising a poly(1,8-octamethylene-citrate-co-octanol) elastomer film functionalized by in situ polymerization with a poly(3,4-ethylenedioythiophene) conductive polymer.
In some embodiments, provided herein are methods of tissue regeneration comprising contacting a subject, organ, or tissue in need of tissue regeneration with a citrate-based elastomer functionalized with a conductive polymer (as described herein). In some embodiments, provided herein are methods of bladder regeneration comprising contacting a bladder tissue in need of regeneration with a citrate-based elastomer functionalized with a conductive polymer (as described herein). In some embodiments, the citrate-based elastomer functionalized with a conductive polymer is PEDOT-POCO.
In some embodiments, provided herein are methods comprising in situ polymerizing of EDOT-POCO within a POCO matrix. In some embodiments, monomer units of the conductive polymer are polymerized in situ for the matrix.
In some embodiments provided herein are poly(1,8-octamethylene-citrate-co-octanol) elastomer films functionalized with a poly(3,4-ethylenedioythiophene) conductive polymer by the methods described herein.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.
As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.
As used herein, the term “about,” when referring to a value is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
Provided herein are citrate-based elastomeric materials comprising conductive polymers, such as poly(3,4-cthylenedioythiophenc)-poly(1,8-octamethylene-citrate-co-octanol) (PEDOT-POCO), and methods of use therefor for medical applications, such as tissue engineering.
A variety of materials have been explored for bladder engineering including acellular natural materials, synthetic polymers, and naturally derived polymers.54 Submucosal 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.55 Without the inclusion of cells, associated complications ensue such as fibrosis and stone formation, none of which were observed in our study.54, 56, 57 58 A 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 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 implantation.16 Offering surgeons a scaffold that has higher processability, manufacturability, and simplicity than the cell-seeded scaffold alternative is expected to simplify the translational pathway for tissue (e.g., bladder) engineering.
Provided herein is the synthesis, characterization, and implementation of electroactive biodegradable elastomers for tissue engineering (e.g., urinary bladder tissue engineering). To create an electrically conductive and mechanically robust scaffold to support bladder tissue regeneration, a phase-compatible functionalization method was developed wherein a hydrophobic conductive polymer (e.g., poly(3,4-ethylenedioxythiophene) (PEDOT)) was polymerized in situ within a similarly hydrophobic citrate-based elastomer (e.g., poly(octamethylene-citrate-co-octanol) (POCO) film). Experiments conducted during development of embodiments herein demonstrate the efficacy of an exemplary film (PEDOT-POCO) 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. Experiments conducted during development of embodiments herein demonstrate a phase-compatible functionalization method that confers electroactivity to a biodegradable clastic scaffold, and the successful restoration of the anatomy and function of an organ using a cell-free electroactive scaffold.
When evaluated in a bladder augmentation model, the PEDOT-POCO scaffold described herein, 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. 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 materials.32, 61-63 Previous research has complexed the popular PEDOT: polystyrene sulfonate (PSS) dispersion with rubber, polyurethane, or other co-polymers.61, 64, 65 While such composites show remarkable conductivity and stretchability, the modulus is typically higher than 1 MPa, and cytotoxicity often not reported. Experiments conducted during development of embodiments herein demonstrate a biocompatible electroactive elastomer with first-of-its-kind structural stability and bulk degradability.
Experiments conducted during development of embodiments herein 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 Yet, active stimulation regimes mask the passive influence of the electroactive material, which has been demonstrated herein to be independently effective. Electroactive materials have been predominantly examined for nerve regeneration.67-69 They have also shown promise for their ability to facilitate epidermal tissue and muscle regeneration.51, 67, 70, 71 Given that PEDOT-POCO scaffolds facilitated the simultaneous restoration of multiple tissue types, including urothelium, smooth muscle, nerve, and blood vessels, the experiments conducted during development of embodiments herein demonstrate the feasibility of electroactive polymer or scaffold systems to recapitulate the anatomical complexities of other organs and tissues.
One mechanism conductive polymers passively modulate biological activity is through rearranging the ionic microenvironment.72 Ions are responsible for regulating various cellular processes and can influence gene expression directly.73 Conductive polymer (e.g., PEDOT) incorporation within elastomeric (e.g., POCO) films creates scaffolds that modulate cellular electronic and ionic microenvironments, bolstering the scaffold's regenerative potential.21, 74 Conductive polymers can drive increased Ca2+ signaling as a key factor that regulates regenerative processes.22, 75, 76 PEDOT-POCO scaffolds facilitate higher passive Ca2+ concentrations than POCO scaffolds. There is evidence pointing to membrane depolarization as a key transducer in initiating differential cellular ion fluxes and influencing signaling.77-80
Preliminary examination of inflammatory cell populations revealed comparable M1:M2 ratios with both POCO and PEDOT-POCO bladders (
In some embodiments, provided herein are materials combining conductive polymers with elastomeric polymers. In some embodiments, a conductive polymer (e.g., PEDOT) is complexed to the surface of a film of an elastomer (e.g., POCO).
Exemplary conductive polymers that may find use in embodiments herein include polyaniline (PAni), polythiophene (PT), polypyrrole (PPy) and poly(3,4-ethylenedioythiophene) (PEDOT) (See
Elastomeric materials specifically are a promising platform to mitigate the mechanical limitations of typical CP-based materials considering their inherent stretchability. Citrate-based elastomers specifically have been validated towards a variety of biomaterials applications for their degradability, antioxidant benefits, and morphological versatility. Integrating CPs into citrate-based elastomers thus offers a promising approach to mitigate CP phase separation because elastomer hydrophobicity promotes CP integration into structurally stable composites.
In some embodiments, suitable elastomeric materials for use in embodiments herein are described in, for example, U.S. Pub. No. 2022/030,5176; incorporated by reference in its entirety. The elastomer described therein as poly (octamethylene-octanol citrates) (POOC) may be referred to synonymously as poly(1,8-octamethylene-citrate-co-octanol) (POCO).
In some embodiments, the elastomer is poly(1,X-(CH2)X-citrate-co-(CH2)XOH), wherein X is 4-16. Examples of such elastomers include, poly(1,4-butamethylene-citrate-co-butanol), poly(1,6-hexamethylene-citrate-co-hexanol), poly(1,8-octamethylene-citrate-co-octanol) (POCO), poly(1,10-decamethylene-citrate-co-decanol), poly(1,12-dodecamethylene-citrate-co-dodecanol), poly(1,14-quattuordecamethylene-citrate-co-quattuordecaanol), poly(1,16-sexdecamethylene-citrate-co-sexdecanol), etc.
In some embodiments, the materials described herein find use in tissue engineering, or more specifically, for use in urinary bladder tissue engineering; although, the compositions and methods herein are not so limited and may find use in regeneration of other tissues (e.g., soft tissues) or other applications. Bladder regeneration requires synergistic repair of multiple tissue types. In some embodiments, materials herein integrate conductive polymers into composites materials mechanically suited for bladder engineering.
The bladder is one of the few organs evaluated in humans that has been engineered in vitro to replace or augment function in vivo using synthetic scaffolds seeded with cells.7-14 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 trauma.15 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 (although the materials herein are not limited to cell-free applications).
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 stimulation.17-19 Conductive polymers are a unique class of organic materials with mixed ionic/electronic conduction that are increasingly being incorporated into biomaterials.17, 20, 21 Incorporation of conductive polymers into a biological matrix tunes the ionic microenvironment, which mediates fundamental processes of life including cell adhesion, migration, and proliferation.21-26 Previous studies have documented that conductive polymer-mediated modulation of the ionic environment facilitates more favorable regeneration for nerve, muscle, and epithelial repair, all of which are required for bladder tissue regeneration.27-31 Experiments were conducted during development of embodiments herein to evaluate whether incorporation of a conductive polymer into a biodegradable citrate-based elastomer creates an ionic electroactive environment that, for example, 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 hinder composite conductivity.32 To address this issue, for example, in the context of bladder tissue engineering where mechanical properties are paramount, experiments were conducted d to 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 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 commercialization.33 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 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.34 In particular, poly(octamethylene-citrate-co-octanol) (POCO) exhibits mechanical properties under cyclic tension that are suitable to tissues and organs with physically intensive requirements.35 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 potential.36 (although the materials herein may also be used with cells (e.g., pre-seeded).
Described herein is the use of a phase-compatible functionalization method to create a biocompatible electroactive and bioactive elastomeric scaffold whereby the conductive polymer (e.g., poly(3,4-ethylenedioxythiophene) (PEDOT)) is incorporated into a citrate-based elastomer (e.g., POCO). The safety and efficacy of the scaffold (e.g., PEDOT-POCO) was evaluated in a rodent bladder partial cystectomy model and it was demonstrated that the scaffold (without cell seeding) restores bladder function to levels that are comparable to those achieved with a cell-seeded POCO scaffold.
In some embodiments, the materials herein find use as a scaffold for tissue regeneration. In some embodiments, the material (e.g., comprising PEDOT-POCO or another material herein) is placed on the site of damage tissue (e.g., bladder tissue) and provides a matrix or scaffold for regrowth of tissue. In some embodiments, the material (e.g., comprising PEDOT-POCO or another material herein) is placed on the site of damage tissue (e.g., bladder tissue) without exogenous cells and/or without being pre-seeded with cells. In some embodiments, the material (e.g., comprising PEDOT-POCO or another material herein) is placed on the site of damage tissue (e.g., bladder tissue) with cells and provides a substrate upon which to transplant a desired cells or mixture of cells. In some embodiments, a scaffold (e.g., comprising PEDOT-POCO or another material herein) provides a growth surface and/or material for a desired cell mixture upon transplantation. In some embodiments, a scaffold (e.g., comprising PEDOT-POCO or another material herein) is configured to remain as part of new tissue (e.g. urinary bladder tissue) following transplant. In some embodiments, a scaffold (e.g., comprising PEDOT-POCO or another material herein) is configured to remain associated with transplanted cells and/or regenerated tissue (e.g. urinary bladder tissue). In some embodiments, a scaffold (e.g. POOC scaffold) is configured to degrade following transplantation (e.g. hours after transplantation, days after transplantation, weeks after transplantation, months after transplantation, years after transplantation, etc.). In some embodiments, a scaffold (e.g., comprising PEDOT-POCO or another material herein) is configured to degrade following tissue regeneration (e.g. hours after transplantation, days after transplantation, weeks after transplantation, months after transplantation, years after transplantation, etc.).
In some aspects, provided herein are systems comprising a scaffold as described herein. In some embodiments, provided herein are cell transplantation systems. The cell transplantation system comprises a scaffold as described herein (e.g., comprising PEDOT-POCO or another material herein) and a population of cells. Suitable populations of cells are described in U.S. Patent Publication No. 20150231303, U.S. Patent Publication No. 20100316614, U.S. Patent Publication No. 20150265749, and U.S. Patent Publication No. 20180154045, the entire contents of each of which are incorporated herein by reference. In some embodiments, the population of cells are autologous (i.e., derived from the subject for which administration of the scaffold is intended). In some embodiments, at least a portion of the population of cells are derived from a different donor (i.e., not derived from the subject for which administration of the scaffold is intended). In some embodiments, the population of cells comprises a population of mesenchymal stem cells (MSCs). In some embodiments, the population of cells comprises a population of hematopoietic stem/progenitor cells (HSPCs). In some embodiments, the population of cells comprises bone marrow derived HSPCs. In some embodiments, the population of cells comprises a mixture of MSCs and HSPCS.
In some embodiments, the scaffold is seeded with the population of cells. Seeding may be performed by any suitable method, including the methods described herein. The proper number of cells to be added to the scaffold, selection of culture media, duration of cell seeding, etc. may be adjusted as necessary depending on the specific cell type used.
The scaffold (e.g., without cell seeding) and/or cell transplantation systems described herein may find use in a method of regenerating tissue in a subject in need thereof. Although the methods provided herein are frequently discussed in relation to regeneration of urinary bladder tissue, the scaffolds and systems described herein may be provided to a subject to regenerate other soft tissues. Other soft tissues that may be regenerated using a scaffold as described herein include, for example, muscles, tendons, ligaments, fascia, nervous, fibrous tissues, blood vessels, and synovial membranes. For regeneration of such tissue, an appropriate cell population may be selected to be used in combination with a scaffold disclosed herein to promote tissue regeneration. Selection of the appropriate cell types may depend on the exact soft tissue to be regenerated.
In some embodiments, diseased tissue (e.g., diseased urinary bladder tissue) is removed from the subject and replaced with a cell transplantation system as described herein (e.g., comprising a scaffold of PEDOT-POCO or another material herein). In some embodiments, the cell transplantation system is administered to the subject by surgical techniques, such as anastomosis. For example, administering the cell transplantation system to the subject may comprise anastomosing the cell-seeded scaffold to the urinary bladder of the subject.
POCO was synthesized according to previously published methods.35 Briefly, citric acid, 1,8-octanediol, and octanol were stirred and melted at 160° C. with liquid nitrogen flow 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 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 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, films were cured at 65 C for 4 days.
POCO films were then 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 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 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 films 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 film.
After complete leaching, POCO films were functionalized with PEDOT first by incubating the films 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, films 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 films were transferred to 37 C and leaching was continued for 24 hours.
Fourier transform infrared (FT-IR) spectroscopy was performed on a Nicolet iS50 spectrometer using attenuated total reflection (ATR) spectroscopy to analyze the composition of degraded PEDOT-POCO leach solution. Spectra were collected with OMNIC Software.
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 film surface charge, PEDOT-POCO and POCO 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.
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 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.
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.
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 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.
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 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.
Charles River 10-week old athymic rats weighing approximately 200 g underwent bladder augmentation procedures as previously described (n=6 per group).47, 81 For POCO and PEDOT-POCO augmentations, all animals were female. For cell-seeded POCO, 4 females and 2 males were used. Briefly, 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 finally 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 fill 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 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 Lab View 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 cmH2O.82
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 accordingly through well-established protocols for either trichrome or immunofluorescence staining. Portions of the regenerated tissue 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 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 quantification was determined as previously described.81 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 (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 autofluorescence potentially confounding quantification, and cell area was measured using the Analyze Particles function in ImageJ.
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 indicated with the following indications: *p<0.05, ** p<0.01, *** p<0.001.
For preparation of PEDOT-POCO, cured POCO films were first passively infused with a mixture of EDOT and uncured POCO pre-polymer (
Polymerization of EDOT-POCO mixtures within the POCO film 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 film. The presence of these PEDOT-POCO nanofeatures was visualized and confirmed through scanning electron microscopy (SEM) (
POCO is rich in carboxyl groups, experiments conducted during development of embodiments herein indicate that the negative charge of these functional groups contributed to stable, electrostatic hole interactions with the PEDOT backbone.37 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 (
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.35 Experiments were conducted during development of embodiments herein 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 vivo.40 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 DPPH assay was performed to evaluate the free radical scavenging capabilities of PEDOT-POCO and POCO, with polytetrafluoroethylene (PTFE) serving as a negative control (
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 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. Experiments conducted during development of embodiments herein demonstrated that PEDOT-POCO bulk, specifically the POCO-based constituents, did dissolve while precipitated PEDOT nanoparticles remained, the sizes of which were comparable to those observed in the SEM images (
Experiments were conducted during development of embodiments herein to verify 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 (
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.21 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, energy dispersive x-ray spectroscopy (EDS) was performed on film cross-sections. Levels of magnesium, sodium, chlorine, and calcium were examined in POCO and PEDOT-POCO (
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) (
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, experiments were conducted to evaluate efficacy for promoting regeneration in a rat bladder augmentation model (
Bladder 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 (
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 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, the regenerated organ was examined more closely to determine how the conductive polymer influenced regeneration of specific 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 (
The urothelium is a critical anatomical feature that enables the bladder to safely interface with the nitrogenous waste it contains.52 An inability for scaffolds to withstand this harsh environment has been one longstanding limitation in the design of biomaterials for bladder regeneration.16 Furthermore, the success of a material depends heftily on its capability to facilitate urothelium regrowth (
In addition to urothelium regeneration, muscle to collagen ratio of regenerated bladder tissue was analyzed. Excessive collagen production can be 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 (
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 first to examine their potential to promote new blood vessel infiltration and development (
Experiments were conducted during development of embodiments herein to examine the average length of the peripheral nerve elements regenerated throughout the bladder tissue (
The following references, some of which are cited above by number, are herein incorporated by reference in their entireties.
The present invention claims priority to U.S. Provisional Patent Application Ser. No. 63/497,093, filed Apr. 19, 2023, which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant number EB026572 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63497093 | Apr 2023 | US |
