Biomaterials and Nanomedicine for Mucosal Repair and Inflammation Control in Interstitial Cystitis/Bladder Pain Syndrome: An Extensive Review.

Interstitial cystitis (IC), also known as bladder pain syndrome (BPS), is a chronic and progressive common urinary system disease. Its etiology is still unknown, and symptoms often involve pelvic pain and lower urinary tract irritation. 1 IC/BPS often accompanies other chronic diseases both inside and outside the pelvic floor, such as endometriosis, rheumatoid arthritis, and asthma. 2 The estimated prevalence of IC/BPS worldwide is around 0.05%‒0.5%, with females being approximately 5‒9 times more likely to be affected by IC/BPS than males. About 90% of IC/BPS cases are female, which may be due to hormonal involvement and differences in pelvic anatomy. 3 Although the overall incidence rate of this disease is low, patients usually have very serious symptoms, such as severe pain, frequent urination and pregnancy, which greatly affect their quality of life. Cystoscopy is the main diagnosis method for this disease in clinical practice, and the detected inflammation may involve the entire or part of the bladder mucosa tissue, or may reach the bladder muscle layer. 4 At present, most of the speculated pathological mechanisms are the destruction and defects of the urinary tract urothelium and its barrier function, neurogenic inflammation, and possible immune system factors. 5 Therefore, patients with pelvic pain may also experience exacerbation of of bladder dilation. 6 The current clinical treatment methods for this disease are diverse, including behavioral therapy, oral or intravenous medication, and sometimes surgical treatment. The current theory on the pathogenesis of IC/BPS involves damage to the glycosaminoglycan (GAG) layer of bladder urethral epithelium, activation of bladder sensory nerves by leakage of toxins in urine, and inflammation and detrusor fibrosis. 7 Especially, bladder biopsies of affected patients show typical abnormalities in the homeostasis of urothelium, and the disruption of homeostasis in bladder mucosal layer is closely related to chronic inflammation. Usually, the bladder mucosa is covered with a dense protective layer of GAG, which can prevent urinary irritants from penetrating the bladder wall and activating bladder sensory nerves and inflammatory responses. In IC/BPS patients, the damage of GAG layer in bladder mucosa increases the permeability of the mucosal barrier. 8 The current clinical treatment for this disease mainly focuses on changing behavior and relieving pain through oral medication or bladder instillation medication. But these methods often have problems such as low efficacy, high recurrence rate, and limited drug retention time in the bladder. The development of biomaterials and nanomedicine provides the possibility of innovative therapies to overcome these limitations, which can be used as repair strategies to replace or regenerate defective bladder mucosal barriers, regulate inflammation, and directly support the regeneration of damaged tissues. 9 Therefore, using biomaterials and nanomedicine to repair or replace defective GAG layers, control and reverse inflammation, and regenerate effective physical and chemical barriers is a promising and practical strategy for IC/BPS therapy.

The bladder mucosa is a layer of transitional epithelial tissue on the inner wall of the bladder, which has protective, barrier, and sensory functions during urine storage. It also has functions such as mucus secretion, osmotic regulation, and immunity, and is closely related to many urinary tract diseases. The transitional epithelium consist of 3‒5 layers of cells: large and flat superficial cells, polygonal cells in the middle layer, and columnar or scaly cells in the basal layer. Superficial cells form a continuous barrier through tight connections and bridging arrangements to prevent the excretion of water and ions from urine into the interior of underlying tissues. A unique feature of this layered epithelium is its ability to perceive the adaptability of cell quantity and structure to bladder filling or emptying. When the bladder is stretched, the mucosa will stretch and become thinner because the cells will flatten with volume changes. On the contrary, when the bladder is emptied, it contracts and thickens, increasing its barrier function to resist harmful chemicals in urine. 10 The most common condition of classic IC/BPS patients is significant changes in the mucosa and submucosa, which were initially referred to as Hunner’s ulcers. This term was coined in the 19th century when Dr. Hunner first discovered ulcerative lesions during cystoscopy. 11 In fact, these so-called ulcers are not true ulcers, but discrete inflammatory lesions of the bladder mucosa and submucosa. Inflammatory disorders affect the mucosa and submucosa, manifested as moist patches on the bladder during cystoscopy. However, they are still referred to as Hunner’s ulcers, which can be seen on the bladder sidewall of approximately half of IC/BPS patients. 12 In addition to ulcers, IC/BPS patients often suffer from mucosal inflammation, including hyperemia, edema, and hemorrhage. In histopathology, inflammatory cells including lymphocytes, plasma cells, and histiocytes typically invade into the lamina propria of the bladder mucosa. Therefore, the mucosa is an important target for therapeutic intervention, alleviating and controlling mucosal inflammatory can help promote the repair of mucosal damage, as optimized mucosal permeability facilitates drug delivery. 13 With a deeper understanding of the role of mucosa in IC/BPS treatment, it is expected to overcome challenges such as drug loss caused by urination, short drug retention time, and insufficient drug concentrations in bladder tissue.

From the above summary and discussion, we are pleased to see that significant progress has been made in the basic and clinical research of biomaterials, nanomedicine and cell therapy for the treatment of IC/BPS ( Tables 1 and 2 ). However, there are still some urgent challenges that need to be addressed. The first obstacle faced by clinical doctors and materials scientists is the lack of an animal model that can fully recapitulate all disease symptoms of IC/BPS. This lack limits the in-depth understanding of the causes of bladder related symptoms (including pain and urinary dysfunction) that lead to the onset or worsening of IC/BPS. The second is to improve clinical research methods to more accurately investigate the morphological changes and functional recovery of bladder mucosa and submucosa. This is particularly important because symptoms of lower urinary tract disorders overlap with pain associated with interactions with pelvic organs such as the bladder, uterus, and colon. Third, standardization, stability, and reproducibility of the preparation and manufacturing process for biomaterials, nanomaterials, and stem cells are also crucial, especially for biomaterials used for drug delivery, stem cell therapy, stem cell-related nanomedicine, and PRP formulations. This mainly includes particle size control and batch consistency, sterile assurance, physical and chemical stability (such as aggregation, drug leakage, etc), and so on. As highly complex biological products, these products may exhibit inherent variability, and their unstable preparation can affect clinical efficacy. Fourthly, promoting innovative therapies based on biomaterials and stem cells in clinical applications still faces various challenges. Although these therapies have the advantages of personalized precision medicine, they may also incur high costs and manufacturing complexity, thereby hindering their clinical implementation. Finally, the fifth point is that some unsettled regulatory issues still exist: the classification of combination products involving biomaterials and stem cells, as well as the lack of clear regulatory guidance to keep up with these rapid technological innovations. Table 1 Summary of Biomaterials, Stem Cells, Nanomedicine, Namomaterials, Physical Therapies and Diagnostic Techniques for IC/BPS and Their Treatment Outcomes Biomaterials Administration Routes Forms/Combinations Species/Inducement Therapeutic Effects Ref. HA Intravesical instillation Solution Patients (clinical trial) Reduces urinary NGF levels [ 16 ] Intravesical instillation Solution Rats; CYP Suppresses secretion of cytokines IL-6 and IL-8; Increases sulfated GAG secretion [ 17 ] Intravesical instillation Nanoplatelets; α-CD; CS; Heparin Rats; LPS Strong anti-inflammatory effects; Reduces bladder inflammation; Promotes bladder mucosa regeneration [ 20 ] Intravesical instillation EGF (NewEpi) Rats; LPS Improves epithelial lining; Reduces inflammation; Diminishes oxidative stress; Extends bladder contraction intervals; Increases urine output [ 22 ] Intravesical instillation Thermosensitive hydrogel; Rabbit SIS ECM Rats; E. coli Prolongs HA residence; Superior antibacterial property; Reduces leukocyte migration and aggregation [ 23 ] Intravesical instillation Probiotic strain; Secreting HA; Ultrasound Rats; CYP Forms HA layer on bladder epithelium; Accelerates mucosal healing; Boosts tight junction protein expression; Lowers pro-inflammatory cytokines levels [ 25 ] CS Transurethral catheterization “SuperGAGs”; Diethylsulfone Mice; LPS Restores bladder impermeability [ 19 ] Stem cells Bladder submucosal injection; Tail vein injection; Transurethral instillation Human USCs; Human ADSCs; Human BMSCs; Human AFSCs Rats; Uroplakin II Reduces inflammatory cell accumulation; Enhances bladder function recovery [ 27 ] Bladder submucosal layer injection Human UCB-MSCs Rats; HCl Reduces irregular and shortened urinary intervals; Controls bladder epithelial injury; Alleviates inflammation and mast cell infiltration [ 28 ] Bladder wall injection Rat ADSCs Rats; HCl Decreases inflammation and fibrosis; Inhibits mast cell infiltration; Prevents collagen fiber accumulation [ 30 ] Anterior and posterior bladder wall injection Human Muse cells Rats; HCl Enhances epithelial repair; Controls inflammation; Functional recovery [ 31 ] Intravenous injection Rat BMSCs Rats; Loxoribine Reduces mucosal hemorrhage, leukocyte infiltration and mucosal edema; Exerts mucosal repair [ 32 ] Intravenous injection ADSCs; Rats; Irradiation Decreases vascular lesions; Restores urinary tract epithelial barrier function [ 33 ] Intravenous injection Human UC-MSCs Rats; CYP Reduces inflammation; Inhibits mast cell degranulation inflammatory cytokines production [ 35 ] Indwelling catheter infusion Human DPSCs Rats; HCl Improves inflammation; Heals damaged epithelium [ 36 ] Intravesical instillation BMSCs; μgelbot Mice; CYP Inhibits mast cell infiltration, collagen deposition, and bladder cell apoptosis; Reconstructs mucus layer [ 37 ] Bladder anterior wall injection Human ESCs; NAC Rats; LPS Restores urinary function; Repairs damaged urethral epithelium; Alleviates inflammation [ 38 ] Bladder anterior wall and dome injection Human ESCs; Two-photon in vivo imaging; Single-cell transcriptome analysis Rats; HCl Precise in vivo behavior of the engrafted cells; Tracks gradual integration of ESCs into perivascular structures; Identifies key genes regulating ESC homing, migration, and anti-inflammatory functions [ 40 ] Bladder submucosal injection Human ESCs Patients (clinical trial, Phase 1/2a) Improves symptoms and life quality; Reduction or complete disappearance of Hunner lesions [ 41 ] Intravenous injection (ADSCs); Intraperitoneal injection (Melatonin) Rat ADSCs; Melatonin Rats; CYP Improves collagen deposition in submucosal and muscular layers; Alleviates inflammatory responses and oxidative stress; Reduces proteinuria and urine amount [ 50 ] Oral (PPS); Bladder submucosal injection (ADSCs) Human ADSCs; PPS Rats; Uroplakin3A Enhances urothelial regeneration; Reduces fibrosis; Decreases mast cell infiltration; Downregulates inflammatory markers [ 51 ] EVs Intrathecal injection Derived from Human UC-MSCs Rats; CYP Attenuates pain and urinary frequency; Suppresses glial cell activation; Decreases neuroinflammation [ 45 ] Intrathecal injection Derived from Human UC-MSCs Mice; Protamine Alleviates pain and urinary frequency; Reduces glial cell activation; Lowers systemic and central inflammation; Mitigates neuroinflammation [ 46 ] Bladder submucosal injection Derived from human Wharton’s jelly MSCs Mice; LPS Improves anti-inflammatory responses; Restores bladder function; Repairs urethral epithelium [ 47 ] MSC-CM Intravesical infusion Derived from human UC-MSCs; PLGA/CM-TMC NPs Rats; CYP Supports urinary epithelium regeneration; Reduces infiltration of inflammatory and mast cells; Restores bladder’s protective barrier [ 49 ] SVF Intravenous injection (half) and pelvic floor injection (half) Human adipose tissues Patients (clinical trial) Feeling improvement; Reduces pain [ 54 ] PRP Intravesical infusion Derived from rat blood Rats; CYP Promotes urethral epithelial repair; Decreases urinary frequency [ 59 ] Intravesical injection Derived from human blood Patients (clinical trial) Improves symptoms; Changes biomarker levels; Repairs urinary epithelial tight junctions; None adverse symptoms [ 60 , 62–64 ] Bladder submucosal injection Derived from human blood Patients (clinical trial) Reduces pain; Increases bladder capacity [ 61 ] Intravesical injection Derived from human blood; BoNT-A Patients (clinical trial) Alleviates bladder symptoms [ 66 ] Medications Oral Prednisolone Patients (clinical trial) Improves pain and dysfunction; Reduces inflammation; Promotes epithelial regeneration [ 68 ] Oral Shionone Rats; CYP Reduces hemorrhage, edema, and cell pyroptosis; Decreases inflammation; Enhances urothelial cell viability [ 70 ] Oral Lactoferrin (Valpalf capsules) Patients (clinical trial) Modulates immune responses; Controls inflammation [ 72 ] Bladder instillation (22 patients); Intradetrusor injection (25 patients) Onabotulinumtoxin A Patients (clinical trial) Better patient satisfaction; Pain relief; Functional recovery [ 76 ] Combination of intravesical instillation and periurethral injection BoNT-A Patients (clinical trial) Improvements in urinary symptoms and pain [ 77 ] Molecular drugs Intravesical instillation PNAs; HIV TAT protein Rats; CYP Decreases immunoreactivity; Regenerates urethral epithelium [ 81 ] Nanomaterials Intraperitoneal injections Curcumin; CONPs Mice; CYP Eliminates ROS; Reducing inflammation; Restores bladder barrier function; Reestablishes tight arrangement and cell junctions of urethral epithelium [ 86 ] Intravesical infusion MSC-CM from human UC-MSCs; PLGA/CM-TMC NPs Rats; CYP Supports urinary epithelium regeneration; Reduces infiltration of inflammatory and mast cells; Restores bladder’s protective barrier [ 49 ] Intravesical injection PRP derived from human blood; Nanofat Patients (clinical trial) Improves bladder symptoms; Reduces pain; Alleviates inflammation-related and distress [ 67 ] Physical therapies Retrograde stimulating sacral nerve roots through electrical stimulating tibial nerve of the ankle PTNS Patients (clinical trial) Reduces nighttime urinary frequency and urgency symptoms [ 87 ] Paddle-shaped spinal cord stimulator Spinal cord stimulation Patients (clinical trial) Complete long-term relief from pain [ 93 ] Acupuncture combined with electrical stimulation Electroacupuncture Patients (clinical trial) Reduces pain; Decreases daytime and nighttime urine frequency; Improves mental well-being and sleep quality; None adverse effects over the subsequent year [ 98 ] Iintravesical instillation LESW; BoNT-A Patients (clinical trial) Enhances permeability of urinary tract epithelium; Improving drug delivery efficiency; Blocks bladder overactivity; Alleviates inflammation; Relieves pain; Improves bladder function [ 101 ] Diagnostic technologies Intravesical infusion NIR-II nanoprobe BTPE-NO 2 @F127; Acrolein; H 2 O 2 Mice; CYP H 2 O 2 triggers production of strong NIR-II fluorescence emission; Enabling clear optoacoustic imaging of bladder lesions [ 104 ] Intraperitoneal injection XM-TBS fluorescent probe; Superoxide anion radicals Mice; CYP Features unique two-photon excitation fluorescence bioimaging capabilities; Enables intracellular visualization of superoxide anion radicals [ 106 ] Abbreviations : HA, hyaluronic acid; NGF, nerve growth factor; CS, Chondroitin sulfate; α-CD, α-cyclodextrin; EGF, epidermal growth factor; SIS ECM, small intestinal submucosa extracellular matrix; E. coli, Escherichia coli; USCs, urine-derived stem cells; ADSCs, adipose tissue-derived stem cells; BMSCs, bone marrow-derived stem cells; AFSCs, amniotic fluid-derived stem cells; UCB-MSCs, umbilical cord blood derived mesenchymal stem cells; UC-MSCs, umbilical cord-derived mesenchymal stem cells; HCl, hydrochloric acid; Muse, multilineage-differentiating stress-enduring cells derived from BMSCs; DPSCs, dental pulp stem cells; μgelbot, microgelbot by embedding magnetic iron oxide nanochains into a poly(ethylene glycol) diacrylate microgel; ESCs, embryonic stem cells; NAC, N-acetylcysteine; EVs, extracellular vesicles; PLGA, poly(lactic-co-glycolic acid); TMC, N-trimethyl chitosan chloride; PLGA/CM-TMC NPs, PLGA/Conditioned medium-TMC nanoparticles; PPS, pentosan polysulfate sodium; SVF, stromal vascular fraction; PRP, platelet-rich plasma; BoNT-A, botulinum toxin A; PNAs, peptide nucleic acids; HIV, human immunodeficiency virus; TAT, transcriptional activation factor; CONPs, cerium oxide nanoparticles; ROS, reactive oxygen species; PTNS, percutaneous tibial nerve stimulation; LESW, low-energy shock wave; BTPE-NO 2 @F127, dual-mode near-infrared II (NIR-II) nanoprobe; H 2 O 2 , hydrogen peroxide. Table 2 Summarization of Advantages and Disadvantages of Stem Cells, EVs, Conditioned Media, and PRP Therapies in IC/BPS Management Biomaterials Advantages Disadvantages Ref. Stem cells Provide long-term symptom relief and reduce dependence on traditional drugs, especially suitable for difficult to treat cases. There is a lack of large-scale clinical trial data, and safety and efficacy need to be further validated, which may pose a risk of infection or immune rejection. [ 27 , 28 , 30–33 , 35–38 , 40 , 41 , 50 , 51 ] EVs It can be absorbed by bladder epithelial cells in a short period of time, thus directly reaching the lesion to exert therapeutic effects. It is simple to operate, minimally invasive, and easier to clinically translate. High stability, easy storage, mass production, excellent quality control. The individual differences in treatment effects are significant, and traditional medical methods cannot be completely replaced. Extraction and preservation techniques require high standards and need to maintain their biological activity. [ 45–47 ] Conditioned media, Avoiding live cell transplantation, with strong biological activity, standardized preparation, easy storage, no ethical restrictions, and avoiding risks such as allogeneic rejection, abnormal differentiation, and tumorigenicity. Suitable for patients who are not suitable for stem cell transplantation. The biological active ingredients are uncertain, their clinical effectiveness still needs to be verified in detail, large scale clinical validation data is also required. There are still unknown potential risks, insufficient regulation, and relatively high costs. [ 49 ] PRP Sourced from the patient’s own body, with no rejection, minimally invasive, high safety, and significant effects for difficult to treat cases. Multiple injections may be required. It may cause local redness, swelling or pain, and may lead to infection. The treatment effect still needs to be verified by a large number of clinical studies. [ 59–64 , 66 ] Summary of Biomaterials, Stem Cells, Nanomedicine, Namomaterials, Physical Therapies and Diagnostic Techniques for IC/BPS and Their Treatment Outcomes Abbreviations : HA, hyaluronic acid; NGF, nerve growth factor; CS, Chondroitin sulfate; α-CD, α-cyclodextrin; EGF, epidermal growth factor; SIS ECM, small intestinal submucosa extracellular matrix; E. coli, Escherichia coli; USCs, urine-derived stem cells; ADSCs, adipose tissue-derived stem cells; BMSCs, bone marrow-derived stem cells; AFSCs, amniotic fluid-derived stem cells; UCB-MSCs, umbilical cord blood derived mesenchymal stem cells; UC-MSCs, umbilical cord-derived mesenchymal stem cells; HCl, hydrochloric acid; Muse, multilineage-differentiating stress-enduring cells derived from BMSCs; DPSCs, dental pulp stem cells; μgelbot, microgelbot by embedding magnetic iron oxide nanochains into a poly(ethylene glycol) diacrylate microgel; ESCs, embryonic stem cells; NAC, N-acetylcysteine; EVs, extracellular vesicles; PLGA, poly(lactic-co-glycolic acid); TMC, N-trimethyl chitosan chloride; PLGA/CM-TMC NPs, PLGA/Conditioned medium-TMC nanoparticles; PPS, pentosan polysulfate sodium; SVF, stromal vascular fraction; PRP, platelet-rich plasma; BoNT-A, botulinum toxin A; PNAs, peptide nucleic acids; HIV, human immunodeficiency virus; TAT, transcriptional activation factor; CONPs, cerium oxide nanoparticles; ROS, reactive oxygen species; PTNS, percutaneous tibial nerve stimulation; LESW, low-energy shock wave; BTPE-NO 2 @F127, dual-mode near-infrared II (NIR-II) nanoprobe; H 2 O 2 , hydrogen peroxide. Summarization of Advantages and Disadvantages of Stem Cells, EVs, Conditioned Media, and PRP Therapies in IC/BPS Management There is currently no globally approved “biomaterial-based” standard treatment product for IC/BPS therapy. The core bottleneck of its clinical translation lies in the lack of Good Manufacturing Practice (GMP) scale production system and unclear regulatory classification, often classified as “tissue engineering products” or “advanced therapeutic pharmaceutical products”, but lacking unified classification. Firstly, there is a bottleneck in GMP production: biomaterials such as decellularized matrix, EVs, stem cell derivatives, etc. are often prepared in personalized or small batches, lacking standardized and scalable GMP processes. Secondly, regulatory classification faces difficulties in determining whether it should be classified as Class III medical devices or biological products, with unclear declaration pathways and inconsistent clinical evaluation requirements. Thirdly, there are obstacles to clinical translation, difficulties in designing clinical trials, and a lack of long-term safety data. Looking ahead to the future, the long-term safety and efficacy of innovative biomaterials, nanomedicine and stem cell therapy require continuous and rigorous scientific assessment in the clinical translation process. At present, stem cell therapy has not been widely recognized in clinical practice in China, especially in the field of bladder repair and regeneration. More reliable clinical validation data is still needed to support further clinical use. Future clinical research can also focus on developing more precise and customized biomaterials and cell therapy strategies suitable for individual patients and specific subtypes of IC/BPS symptoms. One possible research direction is to develop novel combination therapies that target multiple clinical symptoms or mechanisms, and even potential mechanisms that may involve stem cells, biomaterials, nanomaterials, molecular drugs, and combinations of nanomedicines. Finally, due to the complex pathogenesis of IC/BPS, we must seek to improve patient compliance and subjective perceptions of rehabilitation, and attempt to develop more standardized outcome evaluation criteria. This will help to scientifically and reasonably evaluate the effectiveness of biomaterials, nanomedicine and stem cell therapy in preclinical and clinical research.

Many new antioxidant, anti-inflammatory, and immunomodulatory drugs are gradually developed to treat IC/BPS, achieving good therapeutic effects. These drugs include the aforementioned anti-IC/BPS drugs, such as melatonin and PPS, as well as prednisolone and shionone. Strong evidence suggests that these drugs can significantly alleviate clinical symptoms of IC/BPS in different ways. In a clinical study, 31 patients (25 females and 6 males) took low-dose prednisolone orally (1.25–5.0 mg/day). 68 This immunosuppressive therapy significantly alleviated bladder pain and bladder urination disorders by controlling chronic inflammation and inducing bladder wall epithelium regeneration, responding to one of the most difficult clinical problems of IC/BPS. Shionone is a triterpenoid compound isolated from traditional Chinese Aster tataricus , which has potent anti-inflammatory activity. 69 Wang et al 70 administered shionone at a dose of 50 or 100 mg/kg body weight via gavage to CYP-induced IC/BPS rat models, significantly reducing bladder hemorrhage, edema, and cell pyroptosis. Further research has found that shionone reduced pro-inflammatory cytokines such as IL-1β and the N-terminal fragment of gasdermin D (GSDMD-N) by regulating the NF-κB/NLRP3/GSDMD-N signaling pathway, thereby modulating bladder inflammation and promote urethral epithelial viability. In addition, there is another drug called iron-binding milk glycoprotein lactoferrin, which is a key component of the innate immune system in mammals. Lactoferrin is abundant in breast milk and mucous secretions from many parts of the body, especially in human colostrum. Lactoferrin activates macrophages and natural killer cells, and plays an important role in immunoregulation. 71 A clinical trial led by Rosa showed that oral lactoferrin (Valpalf capsules) was not only effective in treating recurrent cystitis, but also had strong anti-inflammatory effects on IC/BPS patients with genetic thrombophilia. The treatment decreased the patient’s IL-6 levels by approximately 58%, and the immunomodulatory and anti-inflammatory effects could promote the repair of damaged epithelial layer. 72 , 73 Onabotulinumtoxin A is another widely used neurotoxin drug for medical treatment, which can block the release of acetylcholine in neuromuscular junction neurons and reduce bladder contractility. This helps to reduce the expression and release of harmful neurotransmitters, blocking peripheral and central sensitization. 74 The American Urological Association has recommended that intradetrusor injecting Onabotulinumtoxin A at an appropriate dosage (about 100 units) to avoid the risk of urinary tract infection and urinary retention. 75 Welch et al analyzed the differences in the treatment of IC/BPS between bladder instillation (22 patients) and intradetrusor injection (25 patients) of Onabotulinumtoxin A. They found that at 2 months, intradetrusor injection showed better patient satisfaction, pain and functional improvement. However, there was no significant difference between the two treatment methods during the 6-month or 9-month follow-up. 76 Chen’s group also conducted a parallel study comparing the clinical efficacy of single intravesical injection of BoNT-A and intravesical instillation plus periurethral injection for IC/BPS. 77 Periurethral injection was performed at 5 sites around the urethral meatus, with injection depths ranging from 1 to 1.5 cm. During the 12-month follow-up after treatment, both groups showed significant improvement in urinary symptoms and pain, with the combination of instillation and injection being more effective than the former. These results may indicate that combination therapy can effectively improve the defects of bladder and urethral mucosa in IC/BPS patients, with better effect. But both studies have major limitations: firstly, the sample size is small, and more research is needed to confirm that treatment may delay disease recurrence time. Secondly, the optimal injection dose for urethral injection or detrusor muscle injection is still unclear, and the required dose for combined injection may be greater than that of monotherapy, which may increase the risk of urethral injury. The research progress on decoding the molecular pathogenesis of IC/BPS provides a solid foundation for the development of molecular targeted therapy drugs and nanomedicine delivery systems. These therapies are based on the principle of delivering specific drugs to target cells or tissues to enhance therapeutic efficacy and reduce off-target side effects. Although the pathogenesis of IC/BPS is still partially unknown, it is widely believed that that bacterial triggering of the host immune system can lead to excessive inflammation, ultimately resulting in tissue injury. 78 Since the beginning of the 20th century, molecular therapies based on nucleic acid delivery and antisense nucleotides have attracted widespread attention. 79 Peptide nucleic acids (PNAs) are antisense nucleotides that are used to treat various diseases due to their ability to form double-stranded complexes with target mRNA and induce gene silencing through translation suppression. 80 Tyagi et al used a protein transduction domain (PTD, GGGGYGRKKRRQRRR) derived from the human immunodeficiency virus transcriptional activation factor (HIV TAT) protein to intravesically deliver PNA (corresponding sequence KTAACGATAGACACATGCC) into the bladder epithelium of rats, thereby suppressing overexpression of NGF in CYP-induced IC/BPS rats. 81 The colocalization of green fluorescence from nuclear counterstain with the red fluorescence from the rhodamine probe indicated that PNA covalently tethered to the 11-mer cell penetrating peptide could successfully penetrate into the cells of the urothelium, indicating TAT was required for successful delivery of PNA to the cells in the urothelium. Their results showed that compared with the control group, rats receiving antisense therapy has less bladder contraction per hour, and there was also less NGF immunoreaction in the bladder and regenerated urethral epithelium of rats. This confirmed the feasibility of using TAT-PNA complex for intravesical antisense therapy and in rat bladder sections treated with the TAT peptide-PNA conjugates, only the red fluorescence of rhodamine labeled on PNA was visible, indicating its stability. In addition, long noncoding RNAs (lncRNAs) regulate the expression of disease-related genes at different stages and play a role in various biological processes of the disease, such as cell proliferation, apoptosis, immune regulatory function, and other physiological and pathological processes. 82 For example, Wang et al established an IC/BPS rat model through intraperitoneal injection of CYP and intravesical instillation of fisetin and TNF-α. 83 They also established an in vitro model using rat bladder epithelial cells stimulated by TNF-α. Through in vivo and in vitro studies, they found that lncRNA Maternally Expressed Gene3 (lncRNA MEG3) was closely related to oxidative stress, inflammation, apoptosis and epithelial damage in bladder tissue. Downregulation of lncRNA MEG3 could alleviate inflammation and bladder tissue injury in IC/BPS rats by upregulating Nrf2 and inhibiting the p38/NF-kB pathway. These results will contribute to the design of novel nanotherapies targeting MEG63 expression to control oxidative stress and inflammation of IC/BPS. As mentioned above, chronic inflammation in IC/BPS continuously produces free radicals or reactive oxygen species (ROS). If these ROS cannot be effectively cleared by the endogenous antioxidant system, excessive ROS can cause oxidative stress, hinder the normal barrier function of the bladder, allow urine solutes to penetrate into the subepithelial layer, and exacerbate inflammatory responses. On the contrary, efficient clearing ROS in the early stages of inflammatory response is beneficial for early intervention of IC/BPS. 84 Cerium oxide is a well-known nanoenzyme with catalase- and superoxide dismutase-like antioxidant activity. It can reversibly convert Ce 3+ and Ce 4+ to form vacancies in the crystal structure to scavenge free radicals at the site of inflammation, prolong the clearance time of ROS, and effectively alleviate IC/BPS symptoms. 85 Therefore, cerium oxide nanoparticles (CONPs) have been used as carriers for antioxidant compound curcumin and obtained antioxidant cerium oxide nanoparticles (Cur-CONPs) for antioxidant intervention in CYP-induced IC/BPS mouse model ( Figure 4 ). 86 Curcumin is a natural polyphenol extracted from turmeric that has antioxidant and anti-inflammatory effects. When taken alone, it also has low bioavailability and poor stability in the gastrointestinal tract and physiological environment. When curcumin is loaded onto nanoparticle carriers, its antioxidant and anti-inflammatory effects are maximally enhanced, and its treatment effect on oxidative stress in IC/BPS is strengthened. Researchers found that Cur-CONPs effectively eliminated ROS, decreased the expression of pro-inflammation proteins, reduced the inflammation level in IC/BPS mice, restored the function of bladder barrier, and retained tight cell junctions in the urethral epithelium, demonstrating a powerful effect. Interestingly, Cur-CONPs exhibited a much stronger therapeutic effect on inflammation than CONPs themselves. Quantitative analysis revealed a Ce 3+ /Ce 4+ ratio of 0.518 for the CONPs and 0.564 for the Cur-CONPs. A higher Ce 3+ /Ce 4+ ratio indicates enhanced antioxidant properties, as a greater concentration of Ce 3+ signifies increased oxygen vacancies in the crystal structure, which facilitate oxygen exchange and redox reactions. This suggests that the complexation with curcumin elevates the antioxidant capacity of the CONPs, potentially augmenting therapeutic efficacy in individuals with IC. H&E staining images ( Figure 4B ) showed that severe denudation and thinner urothelium layers in the CYP group, suggesting bladder barrier dysfunction and a potential invasion of the urinary solutes into the underlying tissues. Conversely, images from the CONP and Cur-CONP groups displayed intact and thick urothelium layers, as well as mitigated inflammatory responses by Cur-CONPs in IC mice. In studies mentioned earlier, MSC-CM delivered by PLGA/CM-TMC NPs and PRP-assisted nanofat transplantation exhibited novel nanomaterial therapy strategy for IC/BPS. 49 , 67 Moreover, the cationic TMC coating on PLGA/CM-TMC NPs significantly increased the retention of nanoparticles in the urethral epithelium, greatly improving the epithelial barrier permeability during bladder therapy. 49 Autologous nanofat transplantation via PRP is also a new bladder therapy method, especially for refractory IC/BPS, which is both safe and effective. This remarkable therapeutic effect may be due to the tissue-engineering-based integration of multiple biological components in vivo, such as stem cells, growth factors, and ECM. 67 Figure 4 ( A ) Illustration of the designing and application of curcumin-loaded cerium oxide nanoparticles for IC/BPS treatment. Curcumin can be rapidly released after administration and serve as a short-term antioxidant for acute pain. CONPs serve as long-term antioxidants. Curcumin and CONPs synergically scavenge ROS and regulate bladder barrier dysfunction and inflammatory responses, thereby effectively improving symptoms of IC/BPS. ( B ) H&E-stained images reveal severe denudation, a thinner urothelium layer, and edema in the CYP group, while the CONP and Cur-CONP groups show intact and thick urothelium layers. U: Urothelium; LP: Lamina propria; M: Smooth muscle; L: Bladder lumen. Scale bar: 100 µm. Reproduced with permission from Yang-Chen Lin, et al. 86 Characterization and Therapeutic Potential of Curcumin-Loaded Cerium Oxide Nanoparticles for Interstitial Cystitis Management. Antioxidants 2024, 13, 826. Licensed under Creative Commons Attribution (CC BY) license/Adapted from original. Two-part scientific figure: Cur-CONP bladder schematic and 8 H and E micrographs for Control,CYP,CONP,Cur-CONP. The image A showing a scientific schematic with nanoparticles, arrows and labeled callouts around a human outline and a syringe aimed at the bladder area. Circular particles are labeled CONP and a molecular structure is labeled Curcumin. Text boxes read Long-term, Short-term, Scavenging, Scavenging, ROS, Inflammatory responses and Bladder barrier dysfunction. A side key lists Cur-CONP, CONP, Curcumin, Urothelium and Intermediate cell. The image B showing an 8-image grid of H and E stained tissue micrographs arranged as 4 columns by 2 rows. Column titles read Control, CYP, CONP and Cur-CONP. Row labels at left read 100x and 200x. Each micrograph contains letter labels U, LP, M and L. ( A ) Illustration of the designing and application of curcumin-loaded cerium oxide nanoparticles for IC/BPS treatment. Curcumin can be rapidly released after administration and serve as a short-term antioxidant for acute pain. CONPs serve as long-term antioxidants. Curcumin and CONPs synergically scavenge ROS and regulate bladder barrier dysfunction and inflammatory responses, thereby effectively improving symptoms of IC/BPS. ( B ) H&E-stained images reveal severe denudation, a thinner urothelium layer, and edema in the CYP group, while the CONP and Cur-CONP groups show intact and thick urothelium layers. U: Urothelium; LP: Lamina propria; M: Smooth muscle; L: Bladder lumen. Scale bar: 100 µm. Reproduced with permission from Yang-Chen Lin, et al. 86 Characterization and Therapeutic Potential of Curcumin-Loaded Cerium Oxide Nanoparticles for Interstitial Cystitis Management. Antioxidants 2024, 13, 826. Licensed under Creative Commons Attribution (CC BY) license/Adapted from original. Overall, nanomedicine and nanomaterial-based drug delivery system for the treatment of IC/BPS have great promise due to their unique advantages. Their sustained-release action prolongs drug action and reduces dosing frequency. Meanwhile, nanomedicine and nanomaterial-based drug delivery system can improve the stability and permeability of drugs, increase the penetration of drugs across the bladder mucosa barrier, and provide effective treatment for lesions in damaged GAG layers. These products can co-deliver different anti-inflammatory and antioxidant agent(s), enabling combination therapy to synergistically target the pathological processes of IC/BPS. However, there are still several disadvantages that need to be noted. Firstly, safety and biocompatibility, such as toxicity and persistent bladder irritation. Secondly, due to the complex preparation process, it is difficult to ensure uniformity and stability, which can lead to abnormal local aggregation or burst drug release may occur. Thus, caution should be exercised when translating nanomedicine and nanotechnologies for IC/BPS into clinical practice. Meanwhile, it is necessary to strengthen preclinical research and rigorously demonstrate its safety and effectiveness. Percutaneous tibial nerve stimulation (PTNS) is a Food and Drug Administration (FDA)-approved therapy for the treatment of overactive bladder. 87 It is electrical stimulation of the tibial nerve near the ankle, retrogradely activating sacral nerve roots, thereby affecting IC/BPS symptoms. 88 In a large single center study by Abdalla et al, 87 34 patients (9 men and 25 women) after 10 weekly PTNS sessions received between January 2010 and October 2021 were evaluated. After 12 weeks of PTNS induction therapy, no improvement was observed in daytime urinary frequency or bladder pain; however, significant improvement was observed in nocturnal urinary frequency and urgency. Although it is unclear how PTNS mediates symptom relief, one well received hypothesis is that electrical stimulation of the tibial nerve retrogradely influences sacral plexus activity, which modulates the pelvic floor, bladder, and urethral sphincter and thereby improves the bladder microenvironment and urothelial integrity. 89 , 90 Another neuromodulation approach is sacral nerve root stimulation, which is also promising in alleviating bladder pain and urinary symptoms in a minimally invasive manner. 91 In comparison with highly invasive operations such as bladder augmentation/reconstruction and urinary diversion, sacral nerve stimulation is a conservative alternative. The procedure involves the implantation of electrodes connected to an implantable pulse generator, through which electrical impulses are delivered to the sacral nerve roots via the intervertebral foramen to stimulate the tibial and pudendal nerves and spinal reflexes and the central nervous system. 92 This modality has a success rate of 60% to 98%; it is a safe and effective option to relieve pelvic pain and urinary symptoms. In patients who have no response or who are not eligible for a sacral nerve stimulation, the spinal cord stimulation can also relief symptoms. For example, Moufarrij et al reported a patient with IC/BPS and persistent back and lower extremity pain who had been implanted a paddle-type spinal cord stimulator. With the treatment, this patient had complete and permanent relief from IC/BPS-related pain. 93 In this modality, electrical currents from epidural electrodes alleviated afferent sensory input to the central nervous system and reduced nociceptive inputs and related neurochemical effect to modulate nociceptive reflexes. Also, this type of device has lower electricity consumption and lower incidence of electrode migration for clinical management. To our knowledge, this case study describes the first report of near-complete resolution of IC/BPS with spinal cord stimulation. Acupuncture is also a component of traditional Chinese medicine, and can be an effective treatment for IC/BPS. Acupuncture is the technique of needling through thin sterile needles in some characteristic acupoints along the meridian to have certain therapeutic effects. 94 Acupuncture has proven to remarkably increase the contents of enkephalin, endorphin, and serotonin in brain tissue and plasma to exert its therapy, which helps to provide sedation, analgesia, immune regulation, and motor dysfunction recovery, among others. 95 , 96 Besides, many studies have proved the effect of acupuncture for the treatment of refractory overactive bladder and pelvic pain of IC/BPS patients. 97 For instance, one clinical case from Guang’anmen Hospital, Chinese Academy of Medical Sciences, on a woman with IC/BPS, 67-year-old, has completed 4 weeks of acupuncture combined with electrical stimulation (electroacupuncture). 98 After treatment, her pain was substantially relieved, daily urinary frequency reduced from 10 times to 4‒5 times per day, nocturia decreased from once hourly to 1‒2 times a night. Her mental state and sleep quality improved markedly, with no adverse events found over the follow-up for 1 year. At present, the mechanism for electroacupuncture that takes care of the symptoms of IC/BPS are not clear, but it may probably mostly select effective acupoints of bladder and lumbosacral regions. These effective acupoints lie near the urethral and transverse muscle neurons in the sacral micturition center and are expected to probably show an action similar to sacral nerve stimulation and PTNS. 99 , 100 Another well-known physical therapy is low-energy shock wave (LESW) therapy. It takes the pain relief, anti-inflammation, and tissue regeneration action. It is applied successfully in treatment for overactive bladder, pelvic pain syndrome, chronic prostatitis, and IC/BPS. LESW relaxes pain, lowers the expression of NGF and pro-inflammatory factors and the abnormality of bladder overactivity, and also increases urothelial permeability, enhances urothelial intravesical delivery efficiency of BoNT-A, and maintains bladder function. 101 These therapeutic effects are thought to be due to angiogenesis, cell proliferation and differentiation induced by LESW, which facilitate the repair of tissue damage and improve the bladder condition of IC/BPS patients. 102 Although the above physical therapies clearly relieve patients’ pain and urinary symptoms of IC/BPS, the clinical evaluation system of these physical therapies is still relatively limited. More specifically, they cannot accurately and non-invasively monitor the repair and regeneration of the bladder mucosa, making it impossible to directly observe microstructure changes in bladder mucosa during treatment. This is mainly due to inherent limitations of routine clinical diagnoses, such as the low resolution of the routine clinical diagnosis instrument (cystoscopy), making it impossible to determine subtle improvement of the mucosa, including epithelial repair and neovascularization. Also, repeated tissue biopsies for pathological analysis is clinically inconvenient and patients barely tolerate it. Thus, treatment-induced improvement in the mucosal layer remains a “black box” and inferred only from recovery of functional rather than direct morphological evidence. Due to the lack of clinical biomarkers, it is difficult to accurately diagnose IC/BPS, so clinical diagnosis can often only be made by ruling out other diseases. Thereby, it is particularly important to develop practical and convenient detection methods and diagnostic probes. In CYP-induced IC/BPS, liver metabolism produces a toxic metabolite called acrolein. 103 It can damage the bladder transitional epithelium and produce a large amount of ROS, including hydrogen peroxide (H 2 O 2 ), in the metabolic process of the kidney, bladder, and urethra. Therefore, H 2 O 2 can serve as a distinctive in situ biomarker for the development of novel diagnostic and therapeutic tools for IC/BPS. For example, Chen et al designed and prepared an activatable dual-mode near-infrared II (NIR-II) nanoprobe called BTPE-NO 2 @F127, used for sensitive and non-invasive diagnosis of CYP-induced IC/BPS in mice using H 2 O 2 as an in situ biomarker ( Figure 5 ). 104 Briefly, benzothiadiazole was first partially conjugated with two tetraphenylethylene (TPE) moieties to construct its core structure, and then modified with two nitrophenyloxoacetamide groups to form the molecular probe BTPE-NO 2 . Finally, by encapsulating BTPE-NO 2 with Pluronic F127, biocompatible and water-soluble nanoprobes BTPE-NO 2 @F127 were obtained. After intravesically instillation into the injured bladder, pathological H 2 O 2 will cleave the two nitrophenoxyacetamide groups, activate BTPE-NH 2 chromophores, and show red shift absorption at 680‒850 nm and strong NIR-II fluorescence emission at 950‒1200 nm. Inert probes give out unvaried or “always-on” signals that constitute the added background noise, while the activatable probes in this study produce signals only if they encounter or react with the target biomarker, hence the detection or imaging using the activatable probes would have much higher sensitivity with negligible background noise and could effectively avoid giving false positive signals. The NIR-II fluorescence signal can be used to image bladder lesions (such as epithelial shedding, inflammation, and hemorrhage) through multispectral optoacoustic tomography, providing clear photoacoustic imaging of bladder lesions. Figure 5 ( A ) Illustration of fabricating and application of nanoprobe BTPE-NO 2 @F127 and H 2 O 2 biomarker-activated detection and imaging for mouse IC/BPS. To obtain the nanoprobe, a benzothiadiazole-based core is first synthesized by connecting benzothiadiazole with two TPE, which is then linked with two nitrophenyloxoacetamide groups at its both ends to afford the molecular probe BTPE-NO 2 . Without the presence of H 2 O 2 , the nanoprobe BTPE-NO 2 is almost non-fluorescent due to the existence of two fluorescence quenchers and the absorption is centering around 615 nm, whereas the H 2 O 2 at pathological level in the disease sites as indicated by the arrow (eg., in liver or bladder) cleaves the nitrophenyloxoacetamide and produces the activated chromophore (BTPE-NH 2 ), thereby red-shifting the absorption band to 680–850 nm and generating strong NIR-II fluorescent emission in the range of 950~1200 nm as shown in the Orange dotted box. Encapsulation of molecular probe BTPE-NO 2 by Pluronic F127 ensures necessary biocompatibility and water-dispersibility for biological applications. ( B and C ) Application of nanoprobe BTPE-NO 2 @F127 in IC mouse model via MSOT imaging and H&E analysis. ( B ) Representative cross-sectional MSOT images of the control mice (intraperitoneally injecting saline into healthy mice) and the IC model groups (24 h after intraperitoneal injecting 75 or 150 mg kg −1 CYP) at various time points post intravesical injection of BTPE-NO 2 @F127. Upper panel: overlay of the activated probe signal with the background (grayscale) signal. Lower panel: multispectrally resolved signal from the activated probe. Organ labeling: 1 artery. White dotted circle: bladder region. Color bar: L: 6.1×10 1 , H: 4.1×10 3 (arb. units). ( C ) H&E staining of various mice bladders. n=5. Scale bar: 50 μm. Reproduced with permission from from Junjie Chen, et al. 104 A H 2 O 2 -activatable nanoprobe for diagnosing interstitial cystitis and liver ischemia-reperfusion injury via multispectral optoacoustic tomography and NIR-II fluorescent imaging. Nature Communications, 2021, 12, 6870. Licensed under Creative Commons Attribution 4.0 International License/Adapted from original. Three-part scientific figure showing nanoprobe synthesis, MSOT imaging over time and bladder histology. Image A illustrates the synthesis of nanoprobe BTPE-NO@F127, where BTPE-NO reacts with hydrogen peroxide to form BTPE-NH, encapsulated in F127 for biocompatibility. The inset graph shows absorption shifting from 615 nm to 680-850 nm post-activation, with fluorescence emission at 950-1200 nm. A mouse model demonstrates applications in interstitial cystitis, trazodone-induced liver injury and liver ischemia-reperfusion injury. Image B presents multispectral optoacoustic tomography images across control, CYP 75 mg/kg and CYP 150 mg/kg groups at intervals of 0, 15, 30, 60, 90 and 120 minutes, showing increased probe signals in treated groups. Image C displays hematoxylin and eosin staining of mouse bladder tissue from control, 75 mg/kg and 150 mg/kg groups, with a 50 µm scale bar, indicating dose-related tissue differences. ( A ) Illustration of fabricating and application of nanoprobe BTPE-NO 2 @F127 and H 2 O 2 biomarker-activated detection and imaging for mouse IC/BPS. To obtain the nanoprobe, a benzothiadiazole-based core is first synthesized by connecting benzothiadiazole with two TPE, which is then linked with two nitrophenyloxoacetamide groups at its both ends to afford the molecular probe BTPE-NO 2 . Without the presence of H 2 O 2 , the nanoprobe BTPE-NO 2 is almost non-fluorescent due to the existence of two fluorescence quenchers and the absorption is centering around 615 nm, whereas the H 2 O 2 at pathological level in the disease sites as indicated by the arrow (eg., in liver or bladder) cleaves the nitrophenyloxoacetamide and produces the activated chromophore (BTPE-NH 2 ), thereby red-shifting the absorption band to 680–850 nm and generating strong NIR-II fluorescent emission in the range of 950~1200 nm as shown in the Orange dotted box. Encapsulation of molecular probe BTPE-NO 2 by Pluronic F127 ensures necessary biocompatibility and water-dispersibility for biological applications. ( B and C ) Application of nanoprobe BTPE-NO 2 @F127 in IC mouse model via MSOT imaging and H&E analysis. ( B ) Representative cross-sectional MSOT images of the control mice (intraperitoneally injecting saline into healthy mice) and the IC model groups (24 h after intraperitoneal injecting 75 or 150 mg kg −1 CYP) at various time points post intravesical injection of BTPE-NO 2 @F127. Upper panel: overlay of the activated probe signal with the background (grayscale) signal. Lower panel: multispectrally resolved signal from the activated probe. Organ labeling: 1 artery. White dotted circle: bladder region. Color bar: L: 6.1×10 1 , H: 4.1×10 3 (arb. units). ( C ) H&E staining of various mice bladders. n=5. Scale bar: 50 μm. Reproduced with permission from from Junjie Chen, et al. 104 A H 2 O 2 -activatable nanoprobe for diagnosing interstitial cystitis and liver ischemia-reperfusion injury via multispectral optoacoustic tomography and NIR-II fluorescent imaging. Nature Communications, 2021, 12, 6870. Licensed under Creative Commons Attribution 4.0 International License/Adapted from original. In addition to H 2 O 2 , superoxide anion, as the main precursor of most ROS, has also become another highly promising endogenous biomarker for IC/BPS due to its extremely high chemical reactivity. It plays an important role in the progression of many inflammatory diseases, exacerbating oxidative stress, tissue damage, and inflammation. 105 For example, Liu et al prepared a fluorescent probe XM-TBS based on merocyanine for detecting superoxide anions. 106 The probe exhibits high specificity at ultralow concentrations of superoxide anion as low as 49.3 nM, distinguishing superoxide anions from other potential interfering factors. This provides unique two-photon excited fluorescence bioimaging in the CYP-induced IC/BPS mouse models. In this system, bis(trifluoromethyl)benzenesulfonate derivatives are recognition elements that specifically react with superoxide radicals through addition reactions. The obtained product can be excited by 800 nm femtosecond laser and release green fluorescence at 557 nm. Another important feature of this probe is its ultrafast fluorescence response‒within only 10 seconds‒which gives it excellent responsiveness to currently available probes. Its imaging depth is up to millimeter level, and this satisfactory imaging depth is essentially due to the powerful tissue penetration ability excited by near-infrared light. Therefore, its high specificity, significant detection sensitivity, and excellent biological imaging performance make it a promising probe for reliable monitoring of elusive superoxide anions and their harmful effects on the inflammatory-infiltrated urethral epithelium in IC/BPS patients. Overall, IC patients still mainly rely on exclusive diagnosis, and the diagnostic principle should be based on detailed medical history collection, physical examination, and laboratory testing to record the patients’ clinical symptoms and signs, and exclude infections or other diseases that may cause the patients’ symptoms. Fortunately, not only have breakthroughs been made in the diagnostic and therapeutic technologies based on biological probes mentioned above, but magnetic resonance imaging (MRI), cystoscopy and hydrodistension have also gradually shown great potential in the differential diagnosis of IC patients.

Due to the unclear etiology of IC/BPS, its complicated pathological changes and numerous symptoms pose significant challenges to clinical practice. In this review, we summarized the latest research advances and clinical translation value of biomaterials, stem cells and their derivatives, nanomedicine, and nanomaterials in the treatment of IC/BPS. We discussed the treatment strategies of IC/BPS from multiple perspectives, including relieving common symptoms, pelvic pain and urinary dysfunction, regulating inflammation, and promoting bladder mucosal healing and regeneration. We also summarized some novel nucleic acid drugs, new combination therapies between tradition physical therapy and electrical stimulation, as well as the latest diagnostic methods for IC/BPS. The rapid development of biomaterials and nanomedicnes has brought new opportunities for controlling oxidative stress and excessive inflammation, as well as promoting the recovery of bladder mucosal barrier. These strategies are expected to partially or gradually overcome the inherent limitations of conventional therapies. The challenges in therapy standardization, large-scale production, and clinical regulatory approval will also inspire interdisciplinary integration between materials science, nanotechnology, biology, and medicine, and continue to develop efficient and innovative solutions for the treatment of IC/BPS. Successfully translating these promising therapeutic strategies into clinical practice requires close collaboration between clinicians, basic scientists and biomedical engineers, which will improve the quality of life for patients with this debilitating disease in the long run.

The GAG layer in the bladder urethral epithelium forms a protective barrier between urine contents and bladder tissue. The damage to this hydrophobic GAG layer allows urinary toxins to penetrate into bladder tissue, leading to bladder inflammation. 14 The urinary dilution effects and dynamic bladder emptying cycles will significantly reduce the retention and delivery efficiency of nanomedicine in the bladder, which is the core physiological barrier of drug delivery in IC/BPS therapy. The continuously generated urine will significantly dilute the infused nanomedicine and reduce the local effective drug concentration. At the same time, the flushing of urine flow will also accelerate the removal of nanomedicine and shorten the action time. During the dynamic urination cycle, the mechanical stress of bladder filling contraction and the periodic excretion of urine (usually every 3–6 hours) also physically remove the nanomedicine remaining in the bladder, affecting its targeted penetration. Therefore, nanomedicine design strategies should consider these factors, such as enhancing mucosal adhesion to enhance drug retention strength and time, or self-assembly of biomaterials to resist washing dilution. Therefore, the design and development of GAG “complementary” therapy have emerged, aiming to restore barrier protective function, improve the integrity and function of bladder mucosa, and provide effective treatment for IC/BPS patients who have no or poor response to existing therapies. Common GAG analogs used in GAG supplementation therapy for IC/BPS include hyaluronic acid (HA), chondroitin sulfate (CS), pentosan polysulfate (PPS), heparin (HEP), semi-synthetic glycosaminoglycan ethers (SAGE), and combinations of CS and HA. 15 For example, Kuo et al conducted a clinical study to evaluate the changes in urinary levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in IC/BPS patients after intravesical injection of HA. 16 They found that urinary NGF levels decreased after HA treatment, demonstrating its role in alleviating IC/BPS symptoms, and urinary NGF is potential biomarker for evaluating clinical response. However, BDNF levels did not significantly decrease, indicating that the role of HA as an anti-inflammatory factor is limited. Overall, the different changes in these two neurotrophic factors during HA treatment reflect the different roles of chronic inflammation in the pathogenesis of IC/BPS. In contrast, in another study, intravesical instillation of HA in cyclophosphamide (CYP)-induced IC/BPS rats significantly inhibited the secretion of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8. 17 Both in vitro and in vivo experiments showed enhanced release of sulfated GAG. In addition, an increase in epithelial permeability was found, directly indicating that the use of HA affected inflammatory response, GAG production, and epithelial barrier properties. To more accurately examine the position and function of HA in the bladder urothelium, researchers established a comprehensive, terminally differentiated urethral epithelial cell culture model with constant barrier properties. 18 These models are helpful in studying the physiological effects of HA on urinary tract epithelial inflammation and barrier repair. They found that HA cannot directly restore the GAG layer on the surface of urinary tract epithelium. On the contrary, it regulated the integrity of barrier by intervening in urinary tract epithelial cells. Further research is needed to reveal the mechanism of GAG layer reconstruction during HA treatment, as well as to elucidate the relationship between HA and inflammation in tissue repair. The main limitation of using GAG for complementary therapy in clinical practice is that GAG analogs such as HA and CS are linear polymers, making it difficult to achieve the impermeability of natural bladder GAGs. To solve this problem, chemical crosslinking or modification of natural GAGs were used to improve its clinical use status. For example, Heidebrecht et al adjusted the properties of the CS polymer by adding it in a base-initiated crosslinking reaction of diethyl sulfone ( Figure 1 ). 19 They found that the longer reaction times, the higher average molecular weights of the polymer, and the resulting product was named “SuperGAGs” (GLX-100, Figure 1A and B ). These crosslinked high-molecular-weight polymers could restore the persistent impermeability of lipopolysaccharide (LPS)-induced IC/BPS rat bladder, and prolong the adhesion time between the polymers and the bladder wall surface by forming a deep and stable layer of water molecules, which is very similar to the natural impermeability of the bladder ( Figure 1C–E ). On the 5th day, GLX-100 was more effective than CS in restoring bladder impermeability. The binding of GLX-100 to the surface of bladder lumen was characterized by biotin reporter, and the results showed that mice treated with GLX-100 exhibited excellent continued adherence on day 10. In contrast, CS showed a decrease in binding levels starting from day 5 and dissolved more on day 10. However, there is an urgent need to apply new formulation of biomaterials to clinical practice, which can increase the local drug concentration in the urothelium and reduce the frequency of intravesical instillation. In this regard, studies have found that GAG-based nanoplatelets with characteristic flat shapes could be fabricated through self-assembly processes. 20 The nanoplatelets were formed by the interaction between the cavities of α-cyclodextrin and the alkyl chains covalently attached to GAG molecules. In this study, three GAG were used including CS, HEP, and HA. The obtained hydrophobically modified CS, HEP and HA were denoted O-palmitoyl-chondroitin sulfate (CS-PA), O-palmitoyl-heparin (HEP-PA) and O-palmitoyl-hyaluronan (HA-PA). The sizes of the nanostructured GAG formulations with uniform size distribution are 333.3 nm for CS-PA, 187 nm HEP-PA, and xxx nm for HA-PA, respectively. The zeta potential are −47.1±2.0 mV for CS-PA, −46.7±3.3 mV for HEP-PA, and −37.3±1.9 mV for HA-PA, respectively. They found that HA nanoplatelets showed the strongest anti-inflammatory effects both in vitro and in vivo, reducing bladder inflammation and inducing bladder mucosal regeneration in LPS-induced rat IC/BPS models. These advantages are based on its flat morphology, which allows it to diffuse faster in diluted fluids and quickly adhere to the urethral epithelium at lower concentrations. Compared with natural GAGs, these nanostructured GAG materials have a longer retention time in the bladder, stronger anti-inflammatory and biological activities, and require lower dosing. Therefore, nanostructured GAG formulations are a promising new approach for developing GAG complementary therapies for the treatment of IC/BPS. Figure 1 ( A ) Illustration of synthesis for SuperGAG polymer (GLX-100) with controlled molecular weight. Longer crosslinking time results in higher molecular weight of GLX-100. ( B ) Schematic of capturing agents crosslinked GLX-100. ( C – E ) Adhesion of GLX-100 with bladder luminal wall. ( C ) Quantitation of biotinylated CS and biotinylated GLX-100 to the urothelium of LPS-induce URO-MCP1 mouse IC/BPS on days 1, 5, and 10. n=5 for each groups. Controls: treated with saline. Control vs ***p<0.001, ****p<0.0001. Bladder samples visualized via histology labeled by IHC with HRP: Sample images (40×) of biotinylated GLX-100 ( D ) and biotinylated CS ( E ) at days 1, 5 and 10. The luminal surface is oriented to the right side of all images, the signal is indicated with the arrows. Reproduced with permission from Richard W. Heidebrecht, Jr., et al. 19 Development of a unique crosslinked glycosaminoglycan for soft tissue repair: Treatment of interstitial cystitis/bladder pain syndrome. PLoSONE, 2025, 20(1): e0317790. Licensed under Creative Commons Attribution License/Adapted from original. Composite: GLX-100 synthesis, GAG polymer schematic, SA-HRP graph, histology images. The image A shows the synthesis stages of GLX-100. Starting Solution progresses through Stage 1: Formation of initial complex, Stage 2: Formation of larger soluble complex and Stage 3: Residual Vinyl Groups Conjugated. The key identifies Chondroitin Sulfate, Vinyl Sulfone, Linker and Capture Agent. The image B shows a schematic of GAG Polymer with Capture Agent attached, indicating continuation of polymer backbone. The image C shows a bar graph of SA-HRP Positivity comparing Control, CS and GLX-100 on Days 1, 5 and 10. Significant differences are marked with asterisks. The image D shows histology images of Biotinylated GLX-100 on Days 1, 5 and 10, with arrows indicating signal. Control (saline) is also shown. The image E shows histology images of Biotinylated CS on Days 1, 5 and 10, with arrows indicating signal. ( A ) Illustration of synthesis for SuperGAG polymer (GLX-100) with controlled molecular weight. Longer crosslinking time results in higher molecular weight of GLX-100. ( B ) Schematic of capturing agents crosslinked GLX-100. ( C – E ) Adhesion of GLX-100 with bladder luminal wall. ( C ) Quantitation of biotinylated CS and biotinylated GLX-100 to the urothelium of LPS-induce URO-MCP1 mouse IC/BPS on days 1, 5, and 10. n=5 for each groups. Controls: treated with saline. Control vs ***p<0.001, ****p<0.0001. Bladder samples visualized via histology labeled by IHC with HRP: Sample images (40×) of biotinylated GLX-100 ( D ) and biotinylated CS ( E ) at days 1, 5 and 10. The luminal surface is oriented to the right side of all images, the signal is indicated with the arrows. Reproduced with permission from Richard W. Heidebrecht, Jr., et al. 19 Development of a unique crosslinked glycosaminoglycan for soft tissue repair: Treatment of interstitial cystitis/bladder pain syndrome. PLoSONE, 2025, 20(1): e0317790. Licensed under Creative Commons Attribution License/Adapted from original. There are also studies that improve the biological function of GAG-based materials by combining them with other biomolecules, including growth factors and acellular matrices. Among numerous growth factors, epidermal growth factor (EGF) plays an important role in the regeneration of urethral epithelium. The bladder itself expresses a large number of EGF receptors, and the kidneys and urethra also secrete a large amount of EGF. 21 Therefore, researchers combine EGF with HA and instilled it into the bladder of LPS-induced rat IC/BPS model. 22 This combination effectively restored the epithelial lining and reduced inflammation. Compared with HA treatment alone, the HA/EGF group significantly decreased the number of urinary red blood cells, reduced inflammatory cell infiltration and relatively irregular re-epithelialization and fibrotic tissue. It also reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-6, and IL-1β, which regulated oxidative stress ultimately leading to an increase in bladder contraction interval and a significant improvement in urine output. However, a drawback of this study is that the reagent used, NewEpi, is a commercially available mixture with fixed concentrations of HA and EGF, making it difficult to determine the optimal therapeutic dosage ratio between the two. Moreover, this study did not separately evaluate the role of EGF, so more work is needed. Furthermore, to address the issues of short drug retention time and frequent intravesical administration, researchers have developed a hybrid material using rabbit small intestinal submucosa extracellular matrix (ECM) as a thermosensitive matrix and HA as a drug component. 23 This thermosensitive composite hydrogel is liquid at 15 to 37.5 °C, but will solidify in a gel at 37.5 to 50 °C. The transition within this temperature range makes it very suitable for intravesical infusion, as it solidifies within about 60 seconds after administration, greatly prolongs its residence time in the body. The G′ of HA-Gel is 87 Pa. The swelling rate of the hydrogel decreases with increasing temperature to the low critical temperature of approximately 37.5 °C. The retention time and drug release time of the composite hydrogel in vivo were more than 5 days, which was much higher than that of HA alone in the control group. In addition, the composite hydrogel had good antibacterial properties in the rat model of bacterial cystitis. 24 In the two studies, the GAG analogs was natural HA purchased directly without any modification. The former study did not characterize the retention time and degradation process of HA in the bladder, while the latter found that the thermosensitive gel extended the retention time of HA from 3 days to more than 5 days, and the retention concentration was also significantly higher. It helped to resist the erosion of urine and extend the release and action time of HA. Both have high efficiency in restoring the permeability of urotheliums. It is encouraging that innovative research on HA supplements continues to emerge. One of the most creative recent studies reported the use of probiotics for single-infusion and continuous in situ synthesis of HA. In this study, scientists developed an extremely biocompatible probiotic strain that can continuously secrete high-molecular-weight HA in situ under ultrasound stimulation as needed after perfusion into the bladder of CYP-induced rat IC/BPS model. 25 This strategy could specifically target and firmly adhere to damaged epithelium at the site of bladder inflammation, continuously and effectively produce HA, form a protective engineered material layer on the surface of bladder epithelium, accelerate mucosal healing, significantly upregulate the expression of tight junction proteins, and reduce the level of pro-inflammatory cytokines. The retention time of HA can significantly reach 21 days, and it completely degrades within 42 days, achieving complete restoration of the urothelium. This is more significant than the previous infusion of HA. These findings indicate that various GAG analog complementary therapies have shown promising therapeutic effects in treating IC/BPS. Although different forms of HA supplements have achieved significant therapeutic effects in treating IC/BPS, further and more detailed research is needed in the future to better improve them. Particularly, future research must develop well-designed, disease-specific, and controlled randomized clinical trials and provide sufficient samples to convincingly demonstrate the effectiveness of treatment. Only under rigorous research can the instillation of HA into the bladder be used as a effective method for the recurrence of IC/BPS and urinary tract infections. Research has shown that mesenchymal stem cells (MSCs) from tissues such as bone marrow, adipose tissue, and umbilical cord have significant therapeutic effects on IC/BPS. MSCs exert their functions through paracrine effects, immune regulation, specific differentiation to resist apoptosis, antioxidant and anti-inflammatory effects, reverse tissue fibrosis, induce angiogenesis and collagen synthesis, ultimately achieving the repair of bladder tissue. 26 The stem cell therapy of IC/BPS can be performed in different ways in vivo, such as intravenous injection and direct injection into tissues. For example, Kim et al compared the therapeutic effect of delivering stem cells (1×10 5 cells/rat for one time) via bladder submucosal injection, tail vein injection, and urethral instillationin a rat IC/BPS model induced by uroplakin II. 27 Their results showed that 10 days after direct injection into the submucosal layer of the bladder, a significant effect on reducing inflammatory cell infiltration and improving bladder function recovery was observed, presenting the best therapeutic effect. In addition, when they injected human urine-derived stem cells (USCs), human adipose tissue-derived stem cells (ADSCs), human bone marrow-derived stem cells (BMSCs), and human amniotic fluid-derived stem cells (AFSCs) into the submucosal layer of the bladder for IC/BPS treatment, there was no difference in treatment results. This study clearly elucidated the impact of stem cell therapy on IC/BPS, providing guidance on how to develop and optimize effective stem cell therapies for patients. Separately, Song et al investigated the therapeutic efficacy and molecular basis of human umbilical cord blood-derived MSCs (UCB-MSCs) by directly injecting them (1×10 6 cells/rat for one time) into the submucosal layer of the bladder of a rat IC/BPS model induced by hydrochloric acid (HCl) injection. 28 Their results showed that a single injection of UCB-MSCs could significantly alleviate urinary irregularity and shortened intervals in affected rats at 1 week after stem cell injection. The transplanted UCB-MSCs could engrafted to the epithelium and stroma. Molecular studies have found that UCB-MSCs activated the wingless-related integration site (Wnt) pathway, stimulated epithelial regeneration and functional recovery. 29 Similarly, another study injected MSCs into the bladder wall of HCl-induced IC/BPS rats, resulting in positive therapeutic effects. 30 In this study, researchers found that injecting rat ADSCs into the bladder wall could notably reduce inflammation and fibrosis in a rat model, including inhibiting the invasion of mast cells, suppressing the expression of inflammatory factors such as TNF-α and transforming growth factor-β (TGF-β), and eliminating the excessive deposition of collagen fibers. The pathological manifestations of bladder, such as detachment of bladder mucosa, excessive activity, and bladder pain, have been significantly alleviated. In addition, other types of stem cells, such as multilineage-differentiating stress-enduring (Muse) cells prepared from human BMSCs, have also been used to treat HCl-induced rat IC/BPS by injecting cells (1×10 4 cells/rat for one time) into the anterior and posterior walls of the bladder. 31 After 4 weeks of implantation, histological results showed Muse cells had a greater degree of retention in the submucosal and lamina propria of the bladder and urethral epithelium, increasing the recovery of bladder epithelium, eliminating inflammation, and restoring function. On the other hand, intravenous infusion of MSCs is another treatment method applied to IC/BPS. 32 , 33 For example, Tabata et al 32 reported that intravenous injection of MSCs (1×10 6 cells/rat for one time) significantly inhibited mucosal bleeding, leukocyte infiltration, and mucosal edema in the Hunner-type IC/BPS rat model induced by loxoribine. After 72 h of treatment, MSCs efficiently infiltrated the submucosal and mucosal layers of inflamed bladder, exhibiting excellent mucosal repair effects. The molecular analysis results also indicated that MSCs enhanced their therapeutic efficacy in IC/BPS rats through anti-inflammatory effects associated with Toll-like receptor-7. 34 Another research team examined the effect of intravenous injection of MSCs (5×10 6 cells/rat for three injections) on chronic radiation-induced cystitis in rats. 33 After 8–12 months of cell therapy, the damage to blood vessels and bladder epithelium caused by radiation was obviously reduced. The reduction in vascular lesions and correction of the urinary tract epithelial barrier were due to stem cells promoting the regeneration of the urethral epithelium, suggesting that rat MSCs helped prevent the progression of radiation cystitis. These results indicate that MSC therapy may not only be suitable for traditional IC/BPS patients, but also for patients with radiation cystitis. Also, intravenous injection of UC-MSCs showed good therapeutic effects on IC/BPS. Xu et al found that the injection of human UC-MSCs (1×10 6 cells/rat for one time) could greatly restore urinary function and shorten bladder urination time in CYP-induced IC/BPS rats after 1 week. 35 In addition, UC-MSCs markedly reduced bladder tissue inflammation and prevented the formation of mast cell granulation, highlighting the enormous potential of UC-MSCs in the treatment of IC/BPS. Researchers also injected stem cells directly into IC/BPS bladder through indwelling catheters, and monitored the retention time of stem cells on the bladder urethral epithelium. 36 In this study, Hirose et al chose human dental pulp stem cells (DPSCs) as the source of MSCs. Firstly, they prepared a rat model induced by HCl, and then injected DPSCs (2×10 6 cells/rat for one time) into the rat bladder using an indwelling catheter. Compared with PBS injection, DPSCs injection greatly improved the tissue morphology of inflammation, and the damaged epithelium fully healed on the 6 th day of injection. In contrast, the urethral epithelium of the PBS treatment group still displayed dense fibrosis and mast cell infiltration. The alleviation of bladder inflammation in the DPSC group was closely related to the decrease in levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, suggesting that the healing of damaged bladder epithelium would benefit from downregulation of these cytokines. The mucous layer in the bladder and urination make it extremely difficult for stem cell therapy to efficiently and persistently act on the bladder, which leads to poor delivery of stem cells to the target bladder site and poor effective retention of stem cells. To solve this problem, researchers encapsulated magnetic iron oxide nanochains with controllable shapes and sizes in poly(ethylene glycol) diacrylate microgel to prepare soft microgelbot (μgelbot). 37 Given the shear-thinning property of the bladder mucous layer (meaning that shear forces make it easier for stem cells to move through the mucous layer), the design of μgelbot was intended to help achieve penetrating the mucus layer. The shape of μgelbot was firstly optimized for enhancing shear force to the mucus layer, including triangle, quadrangle, pentagon, and star. In the reconstructed mucus extracted from the intestine of pig, triangle shaped μgelbots were not able to rotate under rotating magnetic field (RMF). The other shapes of μgelbots could rotate with above 70% of efficiency. Triangle shaped μgelbots hardly penetrated through the reconstructed mucus, whereas the other μgelbots could penetrate efficiently. In particular, quadrangle-shaped μgelbots could penetrate through the reconstructed mucus with minimal “unmovable” μgelbots. After that, the authors generated the torque map depending on the shape of the μgelbot. The torque was concentrated at the vertices of the μgelbot because the torque was proportional to the distance from the axis of rotation. Quadrangle shaped μgelbots showed the better penetration in the mucus layer. The quadrangle-shaped μgelbot much more efficiently penetrated through the reconstructed mucus with MG and RMF than with only MG. That’s to say, compared with other different μgelbot shapes tested (triangle, pentagon and star), the quadrilateral shape was more efficient in transmitting shear force to the mucus layer under the drive of a RMF, resulting in greater penetration and retention of MSCs (1×10 5 cells/rat for three injections) in CYP-induced IC/BPS mouse bladder walls. Moreover, the gelatin coating on the surface of μgelbot facilitated to load MSCs. The effective delivery and paracrine effect of stem cells prevented the invasion of mast cells, deposition of collagen in the bladder, and apoptosis of bladder cells, thereby reconstructing the mucus layer after 9 days of injection. Prior to in vivo treatment evaluation, the authors first assessed the tissue toxicity of μgelbot on the bladder wall after intravesical injection. There was no significant toxicity of μgelbot on the bladder wall. To investigate the long-term in vivo behavior of MSCs@μgelbots, they evaluated the clearance of GFP-MSC@μgelbots from the bladder after IC treatment. The in vivo imaging system clearly showed that MSCs@μgelbots were gradually cleared from the bladder within 7 days. In addition, after 7 days, there was no difference in the main organ tissue pathology and blood biochemical analysis between PBS treatment group and MSCs@μgelbots treatment group, reflecting the long-term safety of MSCs@μgelbots. To our knowledge, this is the first study using μgelbots to treat bladder diseases. The delivery of stem cells through the mucous layer by μgelbot is expected to bring bright prospects for diseases such as IC/BPS. When evaluating the efficacy of stem cell therapy, it is necessary to consider the safety of treatment, especially the risk of tumorigenicity and uncontrolled differentiation. The efficacy and tumor risk of stem cells depend on their dosage of use. The time and cost of preparing stem cells will also rapidly increase with the increase of dosage. To reduce the quantity of stem cells used for IC/BPS therapy, Shin et al investigated the synergistic therapeutic effect of anti-fibrotic agent N-acetylcysteine (NAC) and human embryonic stem cells (ESCs, 2.5 or 5×10 4 cells/rat for one time) injected through the anterior wall of the bladder. 38 IC/BPS rats induced by LPS exhibited irregular urination, short bladder contraction time, and low urine output. Both individual stem cell therapy and the combination therapy of stem cells and NAC significantly prolonged the interval between bladder contract events, increased urine volume, and lowered residual urine output after 2 weeks of treatment. It is worth noting that the use of NAC greatly reduced the number of ESCs used, significantly restored urinary function, repaired damaged urethral epithelium, and effectively alleviated tissue inflammation. This can be explained by the known antioxidant properties of NAC, which can directly scavenge oxygen free radicals and inhibit fibrosis. 39 Although the effect of using NAC alone is similar to stem cell therapy, its contribution to reducing the required stem cell dose is crucial. However, the above study still has limitations as it only tested precise therapeutic effects on bladder tissue repair and functional recovery, without exploring its mechanism of action. Of course, this is closely related to the vague pathophysiology of IC/BPS, which may have multiple factors and require further in-depth and detailed research. Monitoring and accurately analyzing the basic behavior of implanted stem cells in the physiological and pathological environments of human body is crucial for the biosafety of stem cell therapy, as it will address concerns about the efficacy and appropriate use of MSCs. To investigate the relationship and molecular mechanisms between MSC implantation and microenvironment at the whole-genome level, Yu et al 40 developed a two-photon in vivo microscopy technology combined with single-cell transcriptome analysis. They jointly applied these two techniques to study the in vivo behavior of MSCs derived from human ESCs in an acute IC/BPS animal model (injecting 1×10 6 cells/rat for one time). The two-photon in vivo imaging directly witnessed the dynamic correlation between ESCs and the bladder vascular system over a period of 28 days, and tracked the conformation of the partially integrated ESCs in the perivascular structure. Single-cell transcriptome analysis enabled researchers to identify genes involved in the homing, migration, and anti-inflammatory effects of ESCs, including Fos proto-oncogene (FOS, AP-1 transcription factor subunit) and cyclin dependent kinase-1 (CDK1). This in vivo tracking method tracks the behavior of a single implanted ESCs in the body, providing direct answer in vivo, thereby enhancing the understanding of the safety outcomes of ESC therapy in living animals. Surprisingly, clinical research on MSC therapy has also made significant progress. For example, MR-MC-01 is an MSC drug derived from human ESCs, which has shown good safety and efficacy in IC/BPS patients, especially in Hunner’s lesion patients with poor PPS response. 41 In a prospective, randomized, double-blind, placebo-controlled Phase I/IIa clinical trial, 22 patients were recruited (6 in Phase I injecting 2.5×10 7 cells as low dose and 16 in phase IIa injecting 5×10 7 cells as high dose). MSC treatment has achieved good results, improving patient’s symptoms and quality of life. After 6 months of treatment, most patients’ lesions have disappeared or significantly reduced. This is due to the structural regeneration caused by the differentiation of stem cells into epithelial cells and endothelial cells, which also reduces nocturia. The above three studies related to ESC research have been approved by relevant Institutional Animal Care and Use Committee or the clinical protocol has been approved by the Institutional Review Board of Asan Medical Center and registered at ClinicalTrials.gov. 38 , 40 , 41 All cell experiments and animal studies strictly follow relevant regulations. No ethical or regulatory issues have arisen. Although this study showed positive clinical outcomes, it is impossible to achieve statistical significance of all the results due to the small number of patients. No significant difference was found between the treatment group and the placebo group. Therefore, more research is needed to further confirm this results using larger and more representative sample populations, which is also necessary for the universal applicability of stem cell clinical therapy. Overall, injecting stem cells into submucosal layer of bladder wall is more easier to achieve local high concentration of stem cells compared to intravenous injection and catheter infusion, so that it can avoid the problem of fewer stem cells reaching the bladder and exerting their effects as much as possible. It may be a more ideal treatment method for clinical translation and application. Increasing evidence indicates that the paracrine effect of MSCs is largely achieved through the release of extracellular vesicles (EVs), also known as exosomes. MSC-derived vesicles (MSC-EVs) are approximately 50‒1000 nm in size and carry various cellular components such as nucleic acids, proteins, and other bioactive molecules. 42 MSC-EVs provide a novel cell-free biological therapy approach that is highly stable and can avoid risks such as tumor formation and immune rejection, making them more advantageous than MSCs. 43 , 44 For example, two independent studies evaluated the therapeutic effect and mechanism of MSC-EVs and its nucleic acid contents in reducing neuroinflammation and bladder pain during the treatment of IC/BPS. 45 , 46 Liu et al 45 found that NLRP3 inflammasomes‒belonging to the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3‒in neurons of the spinal dorsal horn (SDH) were activated in IC/BPS rats. This leads to the activation of glial cells and spinal neuroinflammation, as well as mechanical pain and frequent urination in the pubic region. Intrathecal injection of MSC-EVs derived from human UC-MSCs could significantly alleviate pain and urinary frequency in rats, inhibit glial cell activation and neuroinflammation in SDH. Further results confirmed that this was due to significant inhibition of the NLRP3 inflammasome activation and TLR4/NF-κB signaling pathway. These findings verified the hypothesis that MSC-EVs alleviated neuroinflammation and mechanical allodynia in IC by inhibiting the activation of NLRP3 inflammasome. In addition, MSC-EVs inhibited the activation of microglia and astrocytes in the SDH, contributing to the relief of spinal neuroinflammation in IC rats. The activation of transcription factor NF-κB is an essential event for NLRP3 inflammasome activation, it promotes the synthesis of NLRP3, pro-IL-1β and pro-IL-18. And TLR4 is an important NF-κB-activating receptor. The authors assessed the activity of TLR4/NF-κB signal pathway in the SDH of IC rats and found that the expression of TLR4 was upregulated, and phosphorylation ratio of NF-κB (p65) was also increased which represented a higher transcriptional regulatory activity. While after MSC-EV treatment, expression level of TLR4 and phosphorylation ratio of NF-κB (p65) showed significant decline. It indicated that MSC-EVs downregulated the activity of TLR4/NF-κB signal pathway, which might be the regulatory mechanism for MSC-EVs to inhibit activation of neuron-derived NLRP3 inflammasome. Despite significant functional improvements, this study still has limitations as MSC-EVs contain biological active non-coding RNAs and proteins with different compositions, and the active component in MSC-EVs that inhibit the TLR4/NF-κB signaling pathway and NLRP3 inflammasome activation are still unclear. In another study, Cui et al aimed to prepare miR-9-rich EVs from human UC-MSCs and further investigate their roles and mechanisms in combating neuroinflammation ( Figure 2 ). 46 Images showed isolated EVs as circular double-layered vesicles with the common exosomal markers (Hsp70 and CD63). Nano-flow cytometry results showed that the accurate size of vesicles were approximate 60–120 nm in diameter. And miR-9 expression was increased in miR-9 enriched EVs by approximately 6.20-fold compared with normal EVs. The low expression of miR-9 in glial cells triggered TLR4 activation and acute pain in protamine-induced IC/BPS mice. Their results showed that miR-9 could directly target and suppress TLR4 function. Intrathecal injection of miR-9-rich EVs could block the TLR4/NF-κb signaling pathway in SDH of IC/BPS mice, penetrate the blood-brain barrier into spinal cord tissue, alleviate bladder pain and urinary frequency, reduce glial cell activation, decrease TLR4-mediated systemic and central inflammation, and reduce NLRP3 inflammasome activity. Ultimately, EVs purified from miR-9-modified MSCs alleviated neuroinflammation and bladder pain in IC/BPS mice by blocking the TLR4/NLRP3 pathway. Therefore, these MSCs-derived EVs, rich in bioactive molecules, possess promise for new regenerative medicine treatments. In this study, EVs derived from miR-9-enriched MSCs can freely cross the blood-brain barrier into the spinal cord tissue. However, one shortcoming is that the authors did not characterize the pharmacokinetics and biodistribution of pharmacokinetics and biodistribution. And there is little evidence of systemic off-target immune modulation. The possibility of deviating from the target may exist, although TLR4/NLRP3 is widely expressed in myeloid/endothelial cells, there is currently no reliable data indicating systemic immune suppression. The impact seems to be limited to the sites of EV accumulation or local damage, as well as systemic risks after intravenous injection, which may have some impact on peripheral blood. These situations deserve further attention in future research. The above therapy is highly similar to another treatment that enhances the therapeutic effect of MSC-EVs using exogenous TGF-β3 under advanced three-dimensional culture conditions, namely T-a3D-EV. 47 Compared with traditional MSC therapy, injecting T-a3D-EV into the submucosal layer of chronic IC/BPS animal models markedly improved wound healing and anti-inflammatory effects, restored bladder function, and repaired the defective urethral epithelium. Researchers have confirmed that the anti-inflammatory function of T-a3D-EV treatment is induced by a greatly increase in TGF-β1 levels, which helps enhance immune regulation and urinary tract epithelium regeneration. This strategy is expected to be applied clinically in the treatment of bladder diseases. Figure 2 ( A – D ) Characterization of MSC-EVs. ( A ) TEM image of EVs. ( B ) Western blot analysis of EV enriched protein markers (Hsp70 and CD63). ( C ) Nano-flow cytometry (NanoFCM) of HUC-MSCs-EV ranging from 60 to 120 nm. ( D ) QRT-PCR analysis of miR-9 expression in the EVs. **p<0.01, n=3. ( E and F ) MiR-9 enriched EVs suppress NLRP3 inflammasome via TLR4/NF‑κb signal pathway in SDH of IC/BPS mice. Intrathecal injecting miR-9 enriched EVs increases the expression level of TLR4, phosphorylation ratio of NF-κB (p65), NLRP3, caspase-1, IL-1β and IL-18, while decreases the expression level of all proteins in SDH of IC/BPS mice. Compared with the Cystitis+i.t. vehicle group. **p<0.01; ***p<0.005, n=6. ( G ) EVs derived from miR-9-modified MSCs alleviate neuroinflammation and cystitis-induced bladder pain via inhibiting TLR4/NLRP3 pathway in IC/BPS mice. Reproduced with permission from Xiangrong Cui, et al. 46 MiR-9-enriched mesenchymal stem cells derived exosomes prevent cystitis-induced bladder pain via suppressing TLR4/NLRP3 pathway in interstitial cystitis mice. Immun Inflamm Dis., 2024, 12, e1140. Licensed under Creative Commons Attribution License/Adapted from original. Image: MSC-EVs, miR-9, TLR4/NLRP3 effects in IC/BPS mice. Image A displays a TEM image of extracellular vesicles (EVs). Image B presents a Western blot of EV protein markers Hsp70, CD63 and β-actin. Image C shows a nano-flow cytometry graph of HUC-MSCs-EV size distribution (60-120 nm). Image D compares relative miR-9 expression in miR-9 enriched EVs versus normal EVs. Image E provides Western blot results for TLR4, p65 NF-κB, p-p65 NF-κB and GAPDH, with graphs showing TLR4/GAPDH and p-p65/p65 NF-κB ratios under different conditions: normal, cystitis+i.t. vehicle, cystitis+i.t. miR-9 enriched EVs and cystitis+i.t. control EVs. Image F shows Western blot results for NLRP3, caspase-1, IL-1β, IL-18 and GAPDH, with graphs of NLRP3/GAPDH, caspase-1/GAPDH, IL-1β/GAPDH and IL-18/GAPDH ratios across the same conditions. Image G illustrates how miR-9 enriched EVs suppress the NLRP3 inflammasome via the TLR4/NF-κB pathway in IC/BPS mice, highlighting interactions between neuroglia, exosomes and pain response pathways. ( A – D ) Characterization of MSC-EVs. ( A ) TEM image of EVs. ( B ) Western blot analysis of EV enriched protein markers (Hsp70 and CD63). ( C ) Nano-flow cytometry (NanoFCM) of HUC-MSCs-EV ranging from 60 to 120 nm. ( D ) QRT-PCR analysis of miR-9 expression in the EVs. **p<0.01, n=3. ( E and F ) MiR-9 enriched EVs suppress NLRP3 inflammasome via TLR4/NF‑κb signal pathway in SDH of IC/BPS mice. Intrathecal injecting miR-9 enriched EVs increases the expression level of TLR4, phosphorylation ratio of NF-κB (p65), NLRP3, caspase-1, IL-1β and IL-18, while decreases the expression level of all proteins in SDH of IC/BPS mice. Compared with the Cystitis+i.t. vehicle group. **p<0.01; ***p<0.005, n=6. ( G ) EVs derived from miR-9-modified MSCs alleviate neuroinflammation and cystitis-induced bladder pain via inhibiting TLR4/NLRP3 pathway in IC/BPS mice. Reproduced with permission from Xiangrong Cui, et al. 46 MiR-9-enriched mesenchymal stem cells derived exosomes prevent cystitis-induced bladder pain via suppressing TLR4/NLRP3 pathway in interstitial cystitis mice. Immun Inflamm Dis., 2024, 12, e1140. Licensed under Creative Commons Attribution License/Adapted from original. Since the therapeutic effect of MSCs is mainly established by the large number of bioactive molecules and EVs secreted by MSCs, the conditioned medium of MSC culture (MSC-CM) has also received widespread attention from researchers and is considered a promising potential treatment method for IC/BPS. 48 In a study, Lin et al 49 developed a biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticle platform for delivering MSC-CM in the bladder to prolong the duration of treatment and improve the permeability of MSC-CM in CYP-induced IC/BPS rats. These nanoparticles could effectively encapsulate MSC-CM from human UC-MSCs, and their surface coating of cationic N-trimethyl chitosan chloride (PLGA/CM-TMC NPs) enhanced the adhesion and penetration of the nanoparticles at epithelium level, improving the effectiveness of bladder therapy. Therefore, the PLGA/CM-TMC NPs loaded with MSC-CM significantly accumulated in the urethral epithelium, facilitating sustained release and penetration of these nanoparticles into the submucosal lamina propria, supporting the regeneration of urinary epithelium, reducing the infiltration of inflammatory and mast cells, and restoring the barrier of the urethral epithelium to harmful substances. Ultimately, this approach remarkably improved symptoms such as mechanical allodynia and urinary function in damaged bladder, providing a new pathway for nanoparticle-functional, cell-free therapy in IC/BPS, thereby improving health conditions more quickly. Other studies have gone beyond the use of stem cells alone or their own paracrine effects, exploring the combination therapy of stem cells with drugs such as melatonin and PPS to protect oxidative stress and inflammation of IC/BPS, enhance immune regulation or other therapeutic effects of stem cells. 50 , 51 Melatonin is a natural free radical scavenger and anti-inflammatory agent, serving as a stabilizer for cell membrane oxidative damage and an inhibitor of inflammation. PPS is a common oral IC drug. 52 , 53 Therefore, studying its combined effect with stem cell therapy is very valuable. For example, the combination of intraperitoneal injection of melatonin with intravenous injection of ADSCs significantly improved collagen deposition in the submucosa and muscles, inhibited inflammatory responses and oxidative stress, and reduced proteinuria and urinary output. 50 On the other hand, in the treatment of uroplakin3A-induced IC/BPS rats, the combination of oral PPS and bladder submucosal injection of human ADSCs significantly improved urethral epithelial regeneration, reduced fibrosis, controlled mast cell infiltration, and downregulated inflammatory marker genes (TNF-α, IFN-γ, IL-6, and TLR2). These results clearly showed that the combination of drugs and stem cell therapy could produce synergistic therapeutic effects and has enormous clinical application potential. In addition, there is a relatively unique stromal vascular fraction (SVF) containing abundant stem cells, which has been used in some clinical trails for patients who have failed various drug treatments and surgical interventions and have poor response. SVF is obtained from the fat collected during liposuction, and then digested with collagenase to separate the stromal and vascular frictions from the adipocyte fraction. One study involved 18 centers and 109 patients (91 females and 18 males), making it the first and largest human trial using autologous stem cell therapy for the treatment of IC/BPS. 54 Half of the extracted SVF was intravenously infused and the other half was injected into the pelvic floor target area. After receiving SVF therapy, 71.5% of patients showed improvement in symptoms and overall pain relief. This is because the processed SVF still contains MSCs, hematopoietic stem cells (HSCs), and cytokines with regenerative, immunomodulatory and anti-inflammatory effects. When these stem cells reach the target area, they have the same function as autologous stem cells in the damaged tissue, starting to repair the damaged epithelium. Therefore, this autologous adipose tissue SVF, which includes adult stem cells, has several ideal characteristics: easy collection, higher stem cell content, sufficient supply due to human fat sources, easy storage and proliferation, and no ethical issues with the source. These results are consistent with the efficacy and safety shown by other studies of SVF derived from human perirenal adipose tissue or heterologous porcine urothelial cells in IC/BPS animal models, as well as longer contraction intervals, complete protection and restoration of bladder epithelial integrity, and recovery of basement membrane and lamina propria. 55 , 56 Overall, these treatments have great potential as safe and widely applicable new regenerative therapies. Platelet-rich plasma (PRP) therapy is one of the recently emerging biological treatment methods, along with platelet poor plasma (PPP) and platelet rich fibrin (PRF). PRP therapy uses the patient’s own platelet-rich plasma, which is rich in various growth factors such as platelet-derived growth factor (PDGF), EGF, and TGF-β. These bioactive factors can reduce inflammation, reconstruct the integrity of bladder epithelial cells, and ultimately promote the regeneration of bladder tissue. 57 The preparation method of PRP involves obtaining a patient’s blood sample, concentrating platelets through certain procedures, and injecting the resultant PRP into the bladder wall to take effect. 58 In an animal model study, Chen et al investigated the effect of intravesical infusion of PRP on acute CYP-induced injury in IC/PBS rats. 59 They found that PRP enhanced the proliferation of hum562, an fibroblasts in vitro, reduced IL-6 expression and promoted urothelial repair in vivo, thereby correcting urinary frequency in treated rats. These results suggest the enormous potential of PRP in repairing urinary tract epithelium. Compared with the basic research related to PRP, clinical trials on PRP therapy have also made remarkable progress. In the past 5 years, many clinical trials using PRP alone or in combination with biopharmaceuticals have reported good results. 60 For example, Jiang et al 61 injected 10 mL of PRP into the suburothelium at the posterior and lateral walls of the bladder of 40 IC/BPS patients who were unable to be successfully treated with conventional therapy, and repeated the injections for 3 times ( Figure 3 ). Researchers evaluated urinary biomarkers (cytokines and functional proteins), pain scores, daytime and nighttime urinary frequency, bladder capacity, and other biomarkers. After treatment, all symptoms and scores improved obviously, and relevant biomarkers also presented favorable changes. In another clinical trial conducted by Jhang et al, 62 researchers injected PRP into the bladder of 19 patients through 4 submucosal injections (once a month). After treatment, the patients’ pain decreased, bladder capacity increased, and biomarkers of bladder barrier function such as E-cadherin and TGF-β also increased significantly. This confirmed that PRP injection substantially improved the urinary tract epithelial barrier function and epithelial proliferation in IC/BPS patients, and this epithelial improvement promoted symptom relief. In addition, three other clinical studies on PRP treatment for IC/BPS have also reported favorable results. Researchers have successfully repaired the defect of tight junctions in the urinary epithelium. All patients had no adverse reactions, such as urinary retention, urinary tract infection or difficulty in urination. 61 , 63 , 64 The significant therapeutic effect of PRP is due to its high content of bioactive substances, which can promote the proliferation of urinary tract epithelial cells, modulate inflammation, promote vascularization, and possibly regulate the bladder nerves. 65 Figure 3 ( A – G ) Preparation of PRP. ( A ) Withdrawing 100 mL of whole blood; ( B ) First round of centrifugation (190×g, 20 min, <20 °C); ( C ) Collecting the platelet-contained supernatant plasma; ( D ) Transferring to another sterile tube; ( E ) Second round of centrifugation (2000×g, 20 min, <20 °C); ( F ) Forming platelet pellets at tube bottom; ( G ) Obtaining PRP (the precipitate) and platelet-poor plasma (PPP, the upper part). PRP can be injected by adding appropriate volume of PPP or normal saline. ( H ) Changong of IC symptom index (ICSI) and problem index (ICPI), visual analog scale (VAS), global response assessment (GRA) after 1 or 4 PRP injection. Reproduced with permission from Yuan-Hong Jiang, et al. 61 Therapeutic Efficacy of Intravesical Platelet-Rich Plasma Injections for Interstitial Cystitis/Bladder Pain Syndrome—A Comparative Study of Different Injection Number, Additives and Concentrations. Front. Pharmacol., 2022, 13, 853776. Licensed under Creative Commons Attribution License/Adapted from original. Composite image showing PRP preparation steps and graphs of ICSI, ICPI, VAS and GRA scores over time. The image A shows blood samples in tubes. The image B shows a centrifuge with tubes inside and a control panel displaying 190 times g, 15 and 16. The image C shows tubes with separated plasma. The image D shows a person handling a tube with separated components. The image E shows a centrifuge with tubes and a control panel displaying 2000 times g, 20 and 20. The image F shows two tubes with separated plasma. The image G shows tubes in a rack. The image H shows four graphs: ICSI, ICPI, VAS and GRA. The ICSI graph shows scores from 12.1 to 5.5 over 6 months. The ICPI graph shows scores from 12.3 to 4.8 over 6 months. The VAS graph shows scores from 5.5 to 1.4 over 6 months. The GRA graph shows scores from 0 to 1.8 over 6 months. Each graph compares PRP times 1 and PRP times 4 treatments at baseline, 1 month, 3 months and 6 months. ( A – G ) Preparation of PRP. ( A ) Withdrawing 100 mL of whole blood; ( B ) First round of centrifugation (190×g, 20 min, <20 °C); ( C ) Collecting the platelet-contained supernatant plasma; ( D ) Transferring to another sterile tube; ( E ) Second round of centrifugation (2000×g, 20 min, <20 °C); ( F ) Forming platelet pellets at tube bottom; ( G ) Obtaining PRP (the precipitate) and platelet-poor plasma (PPP, the upper part). PRP can be injected by adding appropriate volume of PPP or normal saline. ( H ) Changong of IC symptom index (ICSI) and problem index (ICPI), visual analog scale (VAS), global response assessment (GRA) after 1 or 4 PRP injection. Reproduced with permission from Yuan-Hong Jiang, et al. 61 Therapeutic Efficacy of Intravesical Platelet-Rich Plasma Injections for Interstitial Cystitis/Bladder Pain Syndrome—A Comparative Study of Different Injection Number, Additives and Concentrations. Front. Pharmacol., 2022, 13, 853776. Licensed under Creative Commons Attribution License/Adapted from original. There are studies comparing or combining PRP perfusion with other treatments. A study assessed the clinical efficacy and complications of intravesical injection of PRP and botulinum toxin A (BoNT-A) in the treatment of IC/BPS. 66 The injection site was approximately 1 mm in depth into the suburothelium equally distributed at posterior and lateral bladder walls. During a 6-month follow-up period, intravesical injection of PRP and BoNT-A was equally effective in alleviating the symptoms of IC/BPS. Patients who received BoNT-A injection experienced more difficulty in urinating and a higher incidence of urinary tract infections compared to those who received PRP treatment. Hence, BoNT-A has a higher incidence of urinary tract infections. In another study, Hung et al combined patients’ PRP with autologous emulsified nanofat prepared during surgery for bladder instillation. After 6 months, all 6 patients showed significant improvement in bladder symptoms, such as relief of bladder pain, inflammation symptoms, and pain. After treatment, cystoscopy revealed that the structure of the bladder mucosa tended to recover to normal. 67 The combination of PRP and autologous nanofat significantly restored the morphology of bladder mucosa, indicating that this therapy (combined with autologous biomaterials for tissue repair and regeneration) is efficient. The mixed transplants contain a large amount of active ingredients, such as stem cells, growth factors, and ECM components. However, due to the small sample size and lack of controlled studies, further research on a larger scale and longer observation and follow-up periods are needed to evaluate the morphological benefits of this treatment. Although PRP therapy has made promising progress, there are also challenges, including difficulty in assessing the penetration and delivery of PRP in the submucosal space of the bladder, and the lack of a placebo control group due to the absence of standard treatment controls. In addition, repeated blood draws can have an impact on patients, and the PRP preparation protocols also needs to be more standardized. It is believed that the choices and timing of PRP administration for IC/BPS will soon be overcome.