Wound healing in urology.

ADR-12720; No of Pages 13 Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

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Keywords: Urology Growth factors Stem cells siRNA Laser tissue welding Negative pressure wound therapy

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Wound healing is a dynamic and complex phenomenon of replacing devitalized tissues in the body. Urethral healing takes place in four phases namely inflammation, proliferation, maturation and remodelling, similar to dermal healing. However, the duration of each phase of wound healing in urology is extended for a longer period when compared to that of dermatology. An ideal wound dressing material removes exudate, creates a moist environment, offers protection from foreign substances and promotes tissue regeneration. A single wound dressing material shall not be sufficient to treat all kinds of wounds as each wound is distinct. This review includes the recent attempts to explore the hidden potential of growth factors, stem cells, siRNA, miRNA and drugs for promoting wound healing in urology. The review also discusses the different technologies used in hospitals to treat wounds in urology, which make use of innovative biomaterials synthesised in regenerative medicines like hydrogels, hydrocolloids, foams, films etc., incorporated with growth factors, drug molecules or nanoparticles. These include surgical zippers, laser tissue welding, negative pressure wound therapy, and hyperbaric oxygen treatment. © 2014 Published by Elsevier B.V.

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Université de Bretagne Sud, Laboratoire Ingénierie des Matériaux de Bretagne, BP 92116, 56321 Lorient Cedex, France Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Priyadarsini Hills PO, Kottayam 686 560, Kerala, India School of Chemical Sciences, Mahatma Gandhi University, Priyadarsini Hills PO, Kottayam 686 560, Kerala, India

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Neethu Ninan a,b,⁎, Sabu Thomas b,c, Yves Grohens a

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Wound healing in urology☆

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1. Introduction . . . . . . . . . . . . . . . . . . . 2. Wound healing in urology using growth factors . . . 3. Wound healing in urology using stem cells . . . . . . 4. Wound healing in urology using siRNA and miRNA . . 5. Wound healing in urology using drugs . . . . . . . . 6. Wound healing using laser tissue welding . . . . . . 7. Wound healing using surgical zippers . . . . . . . . 8. Wound healing using negative pressure wound therapy 9. Wound healing using hyperbaric oxygen therapy . . . 10. Conclusion . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

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Urology is the branch of science for diagnosing and curing diseases of the urinary tract, which includes the kidneys, urinary bladder,

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Regenerative Medicine Strategies in Urology” ⁎ Corresponding author at: Université de Bretagne-Sud, Laboratoire d'Ingénierie des MATériaux de Bretagne (LIMatB), Centre de Recherche Christiaan Huygens, Rue de St Maudé, BP 92116, 56321 Lorient Cedex, France. E-mail address: [email protected] (N. Ninan).

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urethra, penis, prostate and scrotum [1,2]. The disruption of the normal anatomical structure of the tissue is termed as injury or wound [3]. Injuries to the organs of the urinary tract may affect their regular functioning, and lead to further impediments or complications. Urinary tract wounds or injuries include blunt trauma, penetrating wounds or accidental wounds during surgery. These injuries are found to affect the surrounding abdominal organs and lead to continuous urine leakage, bleeding, infection. Urinary tract injuries are classified as kidney injuries, bladder injuries, urethral injuries and ureteral injuries [4,5]. Kidney injuries are caused due to extensive sports activities, motor vehicle accidents, unintentional falls, accidents during kidney biopsy or bullet

http://dx.doi.org/10.1016/j.addr.2014.12.002 0169-409X/© 2014 Published by Elsevier B.V.

Please cite this article as: N. Ninan, et al., Wound healing in urology, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.2014.12.002

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different phases of wound healing and the various methods adopted to accelerate the healing process, in the case of urology are discussed in the following paragraphs. Urethral healing takes place in four phases namely inflammation, proliferation, maturation and remodelling, similar to dermal healing. However, the duration of each phase of wound healing in urology is extended for a longer period when compared to that of the skin (Fig. 1) [22]. In normal tissue, bleeding occurs immediately after an injury. During haemostasis, blood vessels constrict and reduce the blood flow to the injury site. Platelets express sticky glycoproteins on their cell membranes that allow them to aggregate with each other. Fibrin and fibronectin connect together to form a plug that blocks the flow of blood from the wound and trap proteins and other particles [23]. Once the bleeding stops, an inflammatory reaction starts in the injured tissue (Fig. 2). Chemokines and cytokines are released to attract cells that phagocytize bacteria, damaged tissue, and debris. Various signalling molecules are produced to initiate the proliferation phase of wound healing. During this period, the intensity of inflammation is reduced and migration of fibroblasts is observed at the wounded site. Angiogenesis or neovascularisation occurs simultaneously with fibroblast proliferation. Endothelial stem cells move to the wounded area using pseudopodia to develop new blood vessels. Angiogenic factors and fibronectin attract these cells to the wound site [24]. During migration, endothelial cells degrade the clot and part of the extracellular matrix (ECM), using collagenases and plasminogen activator. Granulation tissue starts appearing, which mainly consists of blood vessels, fibroblasts, endothelial cells, inflammatory cells and components of the ECM. Fibroblasts help in collagen deposition (type III collagen) that increases the strength of the wound. Re-epithelialisation occurs in which epithelial cells migrate to form a barrier between the wound and the surrounding. The epithelialisation phase is moderated by basal keratinocytes from the wound edges [25]. Remodelling is the last stage of wound healing, which is characterised by collagen deposition and formation of scar

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wounds. The patients suffer from high blood pressure, kidney failure, infection, delayed bleeding, blood in urine and pain in the upper abdomen. Their treatment depends on the severity of the injury. Minor kidney injuries can be treated with bed rest and uptake of fluids, whereas major wounds are cured by surgery to repair the damaged tissue [6,7]. Bladder injuries are due to high-impact blows to the pelvis or accidents that occur during the caesarean section, hysterectomy or colectomy. Patients may experience lower abdominal pain, difficulty in urination, haematuria (presence of blood in urine) and urinary incontinence. Minor bladder injuries are treated using catheters whereas, surgical repairs are required in the case of major bladder injuries [8,9]. Ureteral injuries occur during ureteroscopy (diagnosis of ureter), pelvic surgeries, stabbed wounds or gunshots. Patients suffer from abdominal pain, infection, urine leakage, blood in urine, fistula formation, etc. Minor ureteral injuries are cured by placing stents in the ureter and major wounds require surgery to reconstruct the ureter [10–13]. Urethral injuries can arise during procedures such as cystoscopy, bladder catheterization or from pelvic fractures and straddle type falls that mainly affect the area between the legs. The main symptoms include permanent narrowing of the urethra, blood in urine, infection, urinary incontinence, blood discharge from the penis of the male or the urethral opening of the female and erectile dysfunction. Minor urethral injuries are cured by inserting a catheter into the urethra and major wounds are treated by surgery [14,15]. Wound healing is a dynamic and complex phenomenon of replacing devitalized tissues of the body [16–19]. Urethroplasty, hypospadias repairs and other surgical interventions that are used to treat defects or injuries of the urinary tract, rely on functional wound healing to be successful [20]. Impaired wound healing may result in the formation of fistula and strictures (scar formation) due to excessive fibrosis. Recurrent microtrauma caused by continuous displacement of damaged urethral tissue persuades a prolonged inflammatory response and increased metabolic activity during the wound healing process [21]. The

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N. Ninan et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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Fig. 1. Haematoxylin and eosin stained images that show different sections of skin.


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Fig. 2. Schematic diagram showing different phases of wound healing in urology namely, inflammation, proliferation, maturation and remodelling.

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tissue, which fills the wound bed. Fibroblasts differentiate into myofibroblasts, which assist in wound contraction [26]. Hofer et al. analysed the process of urethral wound healing in 36 male Sprague–Dawley rats after urethroplasty [27]. They counted the number of fibroblasts, neutrophils, macrophages and blood vessels

using immunostaining and picrosirius staining. They also evaluated the expression of growth factors like vascular endothelial growth factor (VEGF), tumour necrosis factors (TNFα, TNFβ), platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) using real time PCR. They found that the inflammatory phase was extended for four days


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Growth factors are naturally-occurring substances (proteins or steroid hormones) that stimulate the growth and proliferation of cells involved in wound healing [31]. They act as signalling molecules between cells. They monitor cell differentiation and maturation (Fig. 3). Growth factor therapy is employed in regenerative medicine for promoting tissue regeneration. To appreciate the functions of growth factors, the different stages of wound healing are discussed again. During haemostasis, sub-endothelial collagen is exposed to platelets. Thrombin, produced by exposing collagen activates platelets, which undergo degranulation, releasing an array of cytokines, vasoactive substances and growth factors like epidermal growth factor (EGF), transforming growth factor (TGF), platelet derived growth factor (PDGF), and platelet derived angiogenesis factors. During the inflammatory phase, these growth factors diffuse into tissues surrounding the wound and chemotactically draw inflammatory cells into the injured area [32]. During the proliferation phase, PDGF, EGF and FGF induce activation and proliferation of fibroblasts. TGF-β may either stimulate or reduce proliferation of fibroblasts, depending on their concentration in the wound. In response to the release of growth factors, by macrophages, fibroblasts produce collagen. Proliferation of fibroblasts is accompanied by angiogenesis, which is modulated by key growth factors like basic fibroblast growth factor (bFGF), released by damaged endothelial cells and macrophages along with VEGF, which is released by macrophages and keratinocytes. Endothelial cells produce bFGF, VEGF and PDGF whereas fibroblasts secrete bFGF, TGF-β, PDGF and keratinocyte growth factor (KGF). On the other hand, keratinocytes mainly release growth factors like TGF-β and TGF-α [33]. EGF is a protein with 53 amino acid residues and three intramolecular disulphide bonds. It is mainly secreted by platelets and macrophages. It promotes proliferation of fibroblasts and reduces the time for healing wounds [34]. KGF (also known as fibroblast growth factor, FGF-7) is released from the bladder stromal cells. It acts through the receptor located on the urothelium to promote proliferation of urothelial cells that support wound healing. It helps in the growth of keratinocytes, which secrete keratin [35]. TGF represents two classes of polypeptide growth

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and was characterised by neutrophil and macrophage predominance along with an increased level of expression of VEGF, TNFα, TNFβ, PDGF and interleukin (IL-10). The proliferation phase extended until the tenth day and was characterised by an increase of myofibroblasts and angiogenesis. They compared it to dermal wound healing whose proliferation phase extended until the sixth day. Maturation and remodelling started on the tenth day, characterised by decreased proliferation and angiogenesis, and increased formation of collagen I. The remodelling phase extended until the tenth day for rat dermis and until the twelfth day for rat urethra. The wound healing process was not limited to the site of injury, but involved the vast majority of periurethral tissue and corpus spongiosum, which constitute unique anatomical features of the urethra. A possible reason for the prolonged phase of urethral wound healing is due to urine extravasation through the urethra into the surrounding tissues. Unlike subcutaneous tissue, urethra is surrounded by spongiosum tissue, which reduces the migration rate of inflammatory cells to the site of injury [27]. In the following sections, the various developments in urological tissue engineering to promote wound healing is discussed. Tissue engineering is an emerging interdisciplinary science that encompasses potential strategies to reconstruct or regenerate living tissue for the replacement of damaged or diseased organs by a combination of cells, growth factors, biomaterials and bioactive molecules [28]. An ideal wound dressing material or scaffold will provide an immediate replacement of both lost dermis and epidermis with permanent wound coverage and can resist wound infection, prevent water loss, withstand shear forces, have a long shelf life and provide thermal insulation. It must be widely available, cheaper, durable, stable and lack antigenicity [29]. In urological tissue engineering, autologous bladder cells are harvested and seeded onto biodegradable scaffolds for regenerating wounded bladder tissue. Soon, urothelial cells, bladder smooth muscle cells and endothelial cells migrate into the implanted scaffold and proliferate to form organised bladder tissues [30]. The review also discusses the different technologies that are used in hospitals to treat wounds in urology, which make use of the recent advancements in the field of tissue engineering. These include surgical zippers, laser tissue welding, negative pressure wound therapy, and hyperbaric oxygen treatment.

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Fig. 3. Schematic diagram showing the role of growth factors in wound healing in urology. The growth factors played an integral role in signal cascade responsible for chemotaxis, cell migration and proliferation during wound healing.


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3. Wound healing in urology using stem cells In cell therapy, live cells are used for treatment purposes. The main objective of cell therapy is to rejuvenate, replace or restore biological function of damaged tissues or organs. Stem cells are undifferentiated cells that are capable of self-renewing and differentiating into precursor or progenitor cells of various cell types [45]. Among the different stem cells, adult stem cells are mostly used in tissue engineering as they are easy to obtain through in vitro culture, and they do not raise any ethical concerns compared to embryonic stem cells. However, the proliferation and the differentiating ability of adult stem cells are not so high. Bone marrow is the main source for collecting adult stem cells. Bone marrow consists of mesenchymal stem cells (MSCs), haematopoietic stem cells (HSCs), cellular precursor and endothelial progenitors. HSCs give rise to monocytes, erythrocytes, white blood cells, dendritic cells, etc. [46]. MSCs are found in mesodermal germinal layer in very small quantities, and they differentiate into chondrocytes, osteoblasts and adipocytes. Besides bone marrow, they are also found in the umbilical cord, skin and adipose layer [47]. At the time of injury, MSCs migrate from bone marrow to the wound site. During the inflammatory phase of wound healing, these cells regulate cell proliferation. They help in cell migration, angiogenesis and collagen deposition and promote the wound's ability to progress beyond the inflammatory phase by decreasing the production of pro-inflammatory cytokines and increasing the production of anti-inflammatory cytokines. They act as immune response modulators and help the transplanted cells to protect themselves from graft rejection [48]. Nakamura et al. investigated the ability of MSCs genetically engineered with stromal cell derived factor-1 (SCDF) to heal wounds. After gene transfection, MSCs secreted SCDF for seven days. SCDF engineered MSCs enhanced the migration of MSCs and fibroblasts. They also released VEGF, interleukin-6 (IL-6) and hepatocyte growth factor. They reduced the wound size significantly and increased the number of blood vessels. A cell tracing experiment was conducted with fluorescently labelled cells, which demonstrated that the percentage survival of SCDF–MSCs was higher when compared to MSCs. They concluded that SCDF genetic engineering was a promising way to promote wound healing ability of MSCs [23]. Tian et al. synthesised a highly porous poly L-lactic acid (PLLA) scaffold by the paraffin sphere leaching method. Onto these nanoporous PLLA matrices, growth factor induced bone marrow MSCs were seeded and then implanted subcutaneously into nude mice. They found that the PLLA scaffolds allowed cell–matrix penetration, myogenic differentiation and endorsed tissue remoulding with rich capillary formation (Fig. 4). The results demonstrated that the PLLA scaffold can be potentially used for cell based tissue engineering for patients suffering from bladder injuries [49]. De Coppi et al. have demonstrated that amniotic fluid and bone marrow derived MSCs could be converted to smooth muscle cells in cryo-injured rat bladder and could prevent compensatory hypertrophy of surviving smooth muscle cells. The differentiation of smooth muscles was evaluated by injecting

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bladder regeneration at the injury site. To fabricate this scaffold, the N terminal of bFGF was attached to the collagen binding domain. It promoted vascularisation and smooth muscle ingrowth, which was essential for wound healing [43]. Yang et al. have cultured human bladder smooth muscle cells (HMSC), human bladder urothelial cells (HBUC) and human umbilical vein endothelial cells (HUVEC) extracted from patients with neurogenic bladder and used them for comparative estimations of growth factors [44]. They used eight potential growth factors, including PDGF-BB, PDGF-CC, bFGF, VEGF, EGF, insulin-like growth factor (IGF-1), hepatocyte growth factor (HGF), and TGF-β1. Migration, proliferation and wound healing of HBUC and HUVEC were enhanced by bFGF, VEGF, EGF, IGF-1, HGF but were inhibited by TGF-β1. Among the different growth factors, PDGF-BB, EGF and VEGF were the most effective factors in stimulating the activities of HMSC, HBUC and HUVEC and helped in bladder regeneration, angiogenesis and wound healing [44].

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factors, like TGF-α and TGF-β. TGF-α stimulates the growth of keratinocytes and fibroblasts at the injured area. TGF-β1 and TGF-β2 promote angiogenesis and ensure blood supply to the wounded area. In 1997, Baskin et al. created a model of surgical bladder injury in rodents. After a defined period of time, they bisected the bladder and extracted total RNA from the wounded half. They found that the mRNA expressions for KGF and TGF-α were 6–8 times higher than control bladders after 12 h of injury. Urethral wound healing was greatly affected by exogenous KGF. Thus, KGF and TGF-α were potent mediators of early phases of bladder wound healing [36]. TGF-β2 and TGF-β3 come into play during the re-epithelialisation process and act as mediators for bladder wall remodelling. PDGF is a glycoprotein that has a significant role in blood vessel formation and cellular division of fibroblasts. It exists as three isomers namely, PDGF-AA, PDGF-BB and PDGF-AB. In monocytes, fibroblasts and macrophages, PDGF stimulates chemotaxis, gene expression and accelerate extracellular matrix and collagen formation. VEGF is a protein that assists in vasculogenesis and angiogenesis and restores oxygen supply to cells when blood circulation is inadequate. When tissue hypoxia occurs in the heart and neural tubes of the foetus, angiogenesis is initiated, by upregulating VEGF expression. Burgu et al. found that developing bladder was hypoxic in vivo, and oxygen tensions controlled VEGF expression in embryonic bladder [37]. They administered Pimonidazole, a hypoxia-marker, to pregnant mice during foetal days. Western blot and immunohistochemistry were used to detect CD31 (endothelial marker) and VEGF. Explanted bladders were cultured in 3% oxygen, to represent the hypoxic state. Endothelium (CD31) was visualised directly beneath the urothelium. Capillaries were prominent in detrusor layers and in lamina propria, at later embryonic stages. They found that developing bladder contained hypoxic regions rich in VEGF and close to nascent capillary beds. Fibrin sealants are widely used in urological surgery as topical agents for haemostasis and adhesive for tissue approximation. They are biodegradable and do not create inflammation or foreign body reaction [38]. They can be produced from pooled blood sources or a single blood donor. They consist of human fibrinogen, fibronectin, factor XIII and plasminogen [39]. They are reconstituted in a solution containing aprotinin, which is a bovine derived protease inhibitor. The vial also contains a catalyst solution consisting of bovine thrombin suspended in a calcium chloride solution. The effect of fibrin sealant mimics the final step of the coagulation cascade. The mechanism involves the conversion of fibrinogen (soluble blood component) into fibrin in the presence of thrombin. Factor XIII is activated by thrombin, which converts fibrin into a cross-linked network [40]. Kajbafzadeh et al. proved the efficacy of fibrin sealant for the repair of urethrocutaneous fistula after multiple failed hypospadias and epispadias surgeries. They conducted a survey on eleven boys with a history of hypospadias and at least two unsuccessful fistula repair surgeries, causing a recurrent urethrocutaneous fistula. A single donor fibrin glue either from the patients or parent was applied over the suture lines and under the skin. They also inserted a urethral catheter for 7–10 days. Nine patients showed remarkable recovery after surgery. The remaining two patients recuperated after six months with no further intervention. They did not report fistula recurrence during follow-up. Fibrin sealant improved the safety margin regarding the danger of disease transmission [41]. Platelet rich fibrin is an autologous source of growth factors and cytokines obtained from the sera of patients containing VEGF, PDGF, TGF-β, EGF, etc. Its tendency to polymerise slowly during centrifugation and the fibrin based structure make it a better healing material. Soyer et al. have estimated the use of platelet rich fibrin (PRF) for urethral repair. They included 18 Wistar albino rats in their study. Penile urethral incisions were made and repaired using the PRF. After urethral repair, penile urethras were sampled. They found that the levels of VEGF and TGF-β were increased in sub-epithelia of penile skin and urethra. Thus, PRF could be used as an alternative for successful urethral repair [42]. Chen et al. have fabricated collagen based scaffold containing basic fibroblast growth factor (bFGF) and proved that it was capable of

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green fluorescent protein labelled MSCs from rat amniotic fluid or bone marrow. At 30 days after transplantation, the majority of MSCs underwent differentiation into smooth muscle cells. Thus, they declared that stem cell transplantation could regulate post-injury bladder remodelling [50]. Asanuma et al. have studied the therapeutic potential of MSCs to repair kidney injury. They found that administering exogenous MSCs during acute and chronic kidney injuries can improve functional and structural recovery of glomerular, tubular and interstitial kidney compartments [51]. Kajbafzadeh et al. demonstrated the feasibility of adipose derived MSCs seeded on tissue engineering scaffolds for bladder wall regeneration. Rat adipose derived MSCs were cultured and seeded onto tissue engineered prepuce scaffold. An electrospun nanofibrous polyamide matrix was also prepared. Sprague–Dawley rats underwent bladder wall regeneration on an adipose derived MSC seeded scaffold and electropsun matrix. Immunohistochemistry staining showed higher density of CD34+ and CD31+ progenitor cells on scaffolds seeded with MSCs [52]. Adipose derived stem cells (ADSCs) are isolated through liposuction techniques from fat tissue. They are used to make muscle cells and new blood vessels. Unlike bone marrow stem cells, they are tremendously abundant and easily accessible. Jack et al. cultured human ADSCs in smooth muscle inductive media and seeded them onto synthetic bladder composites to regenerate bladder smooth cells [53]. The composites were made using poly(lactic-co-glycolic acid). The cell seeded bladders expressed proteins like actin, myosin, calponinin and caldesmon, which were confirmed by RT-PCR and immunofluorescence techniques. The scaffolds were implanted in rats and were successful in regenerating bladder tissue from ADSCs. Jianying et al. explored the

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Fig. 4. Schematic diagram showing the role of stem cells in wound healing in urology. Stem cells hasten wound closure, help in the migration of immune cells and endothelial progenitors and promote angiogenesis.

wound healing ability of ADSCs seeded on electrospun poly(L-lactideco-ε-caprolactone)/poloxamer scaffolds. The in vivo studies conducted on 12 Sprague–Dawley rats showed evidence that ADSCs were differentiated into epidermal like structures [54]. Zhao et al. demonstrated the differentiation potential of ADSCs in smooth muscle cells for ureteral tissue engineering [55]. Zhang et al. demonstrated that ADCSs could be differentiated into the urothelium cell phenotype when they were coimplanted with cells of the mature urothelium cell line, and the proportion of differentiated cells increased from 2–4 weeks [56]. Fu and coworkers stated that natural or synthetic polymers do not provide a satisfactory curative solution for long urethral defects. They successfully used ADSCs along with oral mucosal epithelial cells for producing tissue engineered urethras. Poly glycolic acid was chosen as a scaffold material to overcome pathogen infections [57]. Embryonic stem cells (ESCs) are pluripotent stem cells derived from the early stage of the embryo (blastocyst stage). The major limitations of ESCs are the ethical concerns regarding the use of human embryos for cell harvesting and allogenicity [58]. Lee et al. investigated the effect of ESCs on wound healing in 110 diabetes induced rats. ESCs were topically injected into full-thickness skin wounds. The mRNA levels of EGF, VEGF and fibronectin were markedly high and the wound sizes were greatly reduced [58]. Oottamasathien et al. demonstrated that mouse ESCs can be differentiated to form bladder tissue by specific interactions with foetal bladder mesenchyme. They could visualise the various stages in the differentiation of urothelium from ESCs, by the expression of specific endodermal transcription factors like Foxa1 and Foxa2. The final functional urothelium was characterised by verifying uroplakin expression. They proved that embryonic bladder mesenchyme can steer


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RNA interference or RNAi refers to knock down of genes to study the function of proteins [63]. Small interfering RNAs or siRNAs are double-stranded RNA molecules that have a prominent role in posttranscriptional gene silencing. A single siRNA consists of the 5′ phosphate group and 3′ hydroxyl group. It is produced from doublestranded RNA or hairpin looped RNA, which after entering a cell is cleaved by an RNase III enzyme called Dicer. It is then incorporated into a protein complex called RNAi induced silencing complex or RISC (Fig. 5). Once it finds mRNA, the siRNA unwinds and degrades the complementary strands of mRNA with the help of endonucleases and exonuclease enzymes [63]. They break down mRNA after transcription, resulting in no translation. They have gained a lot of importance in drug targeting and tissue engineering. They have several advantages that

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make them efficient for gene silencing. Researchers can carry out siRNA experiments quickly due to the availability of ready to use reagents. Their synthesis route can be scaled up. They can be easily labelled. The amount of siRNA can be strictly controlled when compared to vector driven approaches. Their stability can be increased by chemical modification [64]. However, clinical translation of siRNA has hindered numerous delivery barriers like endosomal membrane impermeability and nuclease degradation. Several attempts are added to improve the pharmaceutical properties of siRNA. The short life span of siRNA is a major challenge for rapidly growing cells where the maximum silencing activity will occur only after two days of infection. They are quite expensive for large-scale experiments. They may not work in cell lines as they are very difficult to transfect. They have no inducible expression and are not useful for long-term studies [65]. Several natural and synthetic polymers are used for their local delivery. Duvall et al. fabricated a novel scaffold that can deliver siRNA locally for several weeks. PHD2 gene was silenced using PHD2 siRNA in a mouse model. As a result, mice with PHD2 siRNA showed a threefold increase in blood vessel formation. Angiogenesis is a key factor in wound healing, and siRNA was thus found to promote the healing process in mice [66]. Nguyen et al. confirmed that wound healing can be improved by topical silencing of the p53 gene and is associated with augmented vasculogenic mediators. A cell cycle regulator gene like p53 is upregulated in diabetic wounds and has been shown to play regulatory roles in vasculogenic pathways. Full-thickness cutaneous wounds (circular in shape) were made on the shaved dorsal skin of diabetic (db/db) mice using a 4 mm punch biopsy tool. A donut shaped silicone splint was centred on the wound and affixed using a cyanoacrylate adhesive and then sutured. These stents prevented wound contraction and ensured wound healing by secondary intention. The wounds and stents were topically applied to an agarose matrix incorporated with p53 siRNA. Western blot analysis confirmed the breakdown of p53 in wounds treated with siRNA. The enzyme-linked immunosorbent assay

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ESCs towards developing endodermal derived urothelium [59]. To overcome the problem of graft rejection, induced pluripotent stem (iPS) cells are used, which are generated from the cells of patients and are reprogrammed to the stem-cell like state. However, great care needs to be taken, as these cells can be cancerous [60]. Li et al. used iPS for treating diabetic wounds. They reprogrammed somatic cells into iPS cells. They adopted direct lineage reprogramming, which had great potential to generate an enriched population of a particular subtype of cells [61]. Franck et al. have fabricated silk based biomaterials in combination with extracellular matrix coatings for bladder tissue engineering with primary and pluripotent cells. The scaffolds were prepared by the gel spinning process and were coated with collagen I, collagen IV and fibronectin. The RT-PCR results showed that fibronectin coated scaffolds facilitated ESC and induced pluripotent stem cell differentiation towards both urothelial and smooth muscle lineages [62]. More research can be conducted to utilise iPSs for accelerating wound healing in urology.

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Fig. 5. Schematic diagram depicting the role of siRNA in wound healing in urology. When the siRNA encapsulated delivery vector is injected into mice, it travels through the bloodstream and is taken up by cells. siRNA is then incorporated into the RNAi induced silencing complex or RISC. Once it finds the mRNA, the siRNA unwinds and degrades the complementary strands of mRNA and promotes cellular migration.


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The active components used in wound management include several pharmaceutical agents, including creams, ointments, powders and solutions that are applied to the wound site. The new generation of medicated dressings incorporates drug molecules that have therapeutic values which overcome the demerits of conventional pharmaceutics [76]. In the following paragraphs, we will discuss some of the drugs used in urology to promote wound healing. A normal male urethra contains pseudostratified columnar epithelium that is located on the basement membrane, a connective tissue layer

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exogenous miRNA-29B to modulate extracellular matrix remodelling following cutaneous injury. A release of miRNA from these scaffolds occurred by polymer degradation and vector diffusion [70]. The disadvantages of miRNA sequencing are high cost and time consuming, require a large amount of total RNA and involve extensive amplification. However, it is easy to obtain the sequences of miRNA and identify point mutations [71]. Wound angiogenesis is a prime factor in wound healing, denoted by migration of endothelial cells followed by capillary formation to support the reviving tissue in the wound bed. These capillaries are necessary to supply nutrients to the granulation tissue. Vascular endothelial growth factor (VEGF) is the main angiogenic factor for repairing skin. Research has proven that Dicer is required for embryonic angiogenesis, and its knockdown can seriously affect postnatal angiogenic response. MiRNAs are used for chronic wound healing as they can control VEGF by repression [72]. They are involved in the proliferation and remodelling phase of wound healing. During the proliferation phase, re-epithelialisation occurs, which includes proliferation and migration of keratinocytes at the wound edge. The migration of keratinocyte becomes faster when SH2-containing phosphoinositide-5-phosphatase (SHIP2) is silenced. MiRNA-205 can silence the SHIP2 gene, which in turn affect AKT signalling pathway and enable motility of keratinocytes. The up-regulation of miRNA-205 can modulate F-actin organisation and promote motility of keratinocytes [73]. MiRNA-210 is another type of miRNA, which is hypoxia sensitive and is used to treat ischaemic wounds [74]. MiRNA 29a directly regulates collagen deposition by using the remodelling phase of wound healing [75]. Most of the studies in miRNA are in vitro. So, research should be focussed on conducting more animal studies to prove their role in wound healing in urology.

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proved upsurge in the VEGF expression, and the healing process was thus improved [67]. Researchers should now try to evaluate whether urothelial wound healing can be hastened by silencing the p53 gene. Li et al. tried to investigate the viability of replacing urinary epithelial cells with oral keratinocytes and TGF-β1 siRNA onto bladder acellular matrix grafts to reconstruct urethra. They found that these cells exhibited excellent cytocompatibility with the matrix grafts by the formation of stratified epithelial layer after six months [68]. siRNA can thus be used to silence the gene to promote angiogenesis, cell migration, proliferation and wound healing. MicroRNAs or miRNAs are a recently discovered class of non-coding RNA (containing ~ 22 nucleotides) that suppress gene expression by specifically cleaving targeted mRNA. Compared to siRNA, miRNA can take up to 100 target genes simultaneously, offering a more effective result. They are involved in several biological processes, including growth, differentiation and proliferation. Liu et al. analysed miRNA expression patterns in the process of embryogenesis to determine the mechanism of embryonic bladder development [69]. The miRNA expression patterns revealed that unique miRNA from submucosal and epithelial areas are responsible for mesenchymal stem cell differentiation into smooth muscle cells of the bladder. The mechanism of action of miRNA involves several steps. RNA nuclease II synthesises primary miRNA within the nucleus of a cell. RNA endonuclease (Drosha) along with its cofactors cleaves primary miRNA to produce smaller fragments of precursor miRNA containing ~70 nucleotides. These fragments are carried to cytosol using proteins like Exportin, where they are further cleaved by the RNA endonuclease called Dicer to form mature miRNA. One strand of miRNA duplex is incorporated within an RNA induced silencing complex or RISC that recognises target mRNA and suppresses their expression of post-transcriptional gene silencing (Fig. 6). Shilo et al. have conducted experiments on Dicer deficient mouse models [70]. It was found that such knockout mice were not able to convert miRNA to mature miRNA due to the absence of Dicer. As a result, targeted mRNA cannot be cleaved and protein expression cannot be suppressed. The knockout mice lost weight within 1–2 days. Their skin evaginated into the epidermis instead of folding downwards into the dermis. This also resulted in dehydration and apoptosis of cells. This experiment showed the critical role of miRNA in skin morphogenesis. Endothelial cell capillary sprouting, tubulogenesis and migration can be inhibited by knockdown of Drosha and Dicer. Monaghan et al. synthesised a collagen based scaffold to deliver

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Fig. 6. Schematic diagram depicting the synthesis of miRNA and their role in wound healing in urology. Endothelial cell capillary sprouting, tubulogenesis, migration and angiogenesis can be inhibited by knockdown of Drosha and Dicer.


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The main requirement for pleasing cosmetic results is wound healing without stress. Tension-free wound healing is achieved by providing sufficient mechanical support to maintain the wound edges that allow homogenous distribution of tension across the whole wound. Surgical wounds heal slowly compared to wounds treated with minimum tension. Risnes et al. conducted a prospective, randomized study to compare a non-invasive surgical zipper to intracutaneous suture closure with respect to post-operative wound infection rate and cosmetic results [88]. They studied 300 patients out of which 150 had their wounds closed with surgical zippers and 150 with intracutaneous suture. The end points were deep and superficial wound infections were within 6 weeks after operation. The superficial infection was 5.3% in the zipper group whilst it was 6% for patients treated with intracutaneous suture. The wound infection rate was the same for both groups; however, the cosmetic result was found to be better for patients treated with surgical zippers. Onuminya et al. conducted a randomized controlled prospective study to evaluate the outcome of using the surgical zipper

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Laser tissue welding (LTW) is a transformational technology that has proven its efficiency over current surgical procedures as they provide quick and accurate haemostasis and is lifesaving for patients suffering from blood clotting disorders. It is particularly useful for handling soft tissues of the urinary system. It enables doctors to join and seal the tissues without much thermal damage. It was implemented for the first

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time in the year 1966 for creating a vascular anastomosis using a neodymium laser and cyanoacrylate glue [81]. It is a ‘stitch-free’ surgical technique for joining ruptured tissues like blood vessels, cartilage, urinary tract, skin, nerve, etc. The basic principle involves absorption of laser light by tissues, which is converted to heat energy that ultimately results in deformation of tissue proteins and finally their fusion [82]. The photothermally altered tissue proteins connect each other through covalent or electrostatic interaction. LTW reduces the operation and recovery time, decreases blood transfusion, lessens healthcare costs and diminishes pain and scar formation. The disadvantages of LTW are peripheral tissue damage, insufficient strength and low depth of penetration of light [83]. The latest research is focussed on the development of innovative laser mediated instant haemostasis and sutureless surgical repair with the help of human albumin based bio-absorbable materials to join, repair and stop bleeding on any tissue surface [84]. Transparent bioabsorbable human albumin based wound dressing materials that are incorporated with growth factors and antibiotics are also fabricated. LTW contains a diode laser to coagulate human albumin dye solder along with human albumin scaffold that provide quick and accurate haemostasis. When the laser excites the photosensitive dye molecules, thermal energy that coagulates human serum albumin is produced, which undergoes a solid to liquid transformation. The thermal energy will be dissipated within the biomaterial, leaving the tissues unharmed. Lobik et al. proved the efficiency of LTW applied on urinary bladder in rats, which resulted in a higher survival rate and improved the quality of scar obtained from clinical and histological examination [85]. Poppas et al. used LTW in genitourinary reconstructive surgery and proved that it could decrease operative time, reduce postoperative fistula formation and improve healing when compared to conventional suture controls [86]. Huang et al. used a plasmonic nanocomposite extracted from an elastin like polypeptide as tissue solder [87]. The nanocomposite contained gold nanorods that were wrapped inside the polypeptide that made them elastic. This reduced the chance of seal splitting or rupturing. When chromophores were excited by laser, elastin protein solders got denatured and were incorporated within the weld site leading to an upsurge in the tensile strength of closure, minimised foreign body reaction and reduced peripheral tissue destruction. Deeper tissue penetration was achieved using near infrared spectroscopy (NIR). The mechanical properties of nanocomposites can be adjusted by varying the concentration of gold nanorods. They formed strong ‘liquid-tight’ but elastic seal that prevented harmful bacteria from leaking out. They tested the nanocomposite on pigs with intestinal injury and confirmed that they can replace stitches in surgery. Researchers should now try to explore the potential of plasmonic nanocomposites in promoting wound healing in urology.

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that comprises vascular sinusoids of the corpus spongiosum and smooth muscle fibres. Urethral lesions have a complex structure and are difficult to be treated using endoscopy. Tensile distracting forces arising at these lesions due to muscle pull can further widen the scars within the tissue. A new technique to minimise the tension at the edge of lesions or wounds is a temporary paralysis of a muscle underlying a wound. Botulin toxin-A (BTX) is an effective drug that can cause temporary paralysis of smooth muscles by hindering acetylcholine release at the presynaptic cholinergic junction and thereby blocking neural transmission when injected locally [77]. In urology, BTX is used for treating motor and sensory urges, chronic prostatic pain, neurogenic detrusor overactivity, etc. Sahinkanat et al. proved that BTX can improve urethral wound healing by inhibiting urethral muscle contraction [21]. They made urethral injuries in 30 male albino rats by cutting urethra transversely using scissors and then sutured them. The rats were injected with BTX, and the urethras were harvested for histopathology examination, which produced significant results of tissue regeneration. Research has proven that anaesthetics play a role in wound healing. Infiltration of surgical wounds in urology with long-acting local anaesthetic is used to lessen postoperative incisional pain and increase rate of wound healing [78]. Hanci et al. compared the effect of infiltration of local anaesthetics like bupivacaine, lidocaine and tramadol, on wound healing in 32 male Wistar rats. Surgical procedures were carried out using intraperitoneal injection of ketamine. The wounded area of the test groups was subcutaneously infiltrated with the study drugs (bupivacaine, lidocaine or tramadol). The control rats were infiltrated with normal saline. After drug infiltration, surgical incisions (including cutaneous and subcutaneous connective tissues) were done with a scalpel, and tissues were joined using sutures. The rats were euthanized, and the skin was excised for analysis. In their study, they found that bupivacaine and lidocaine reduced collagen production and wound breaking strength and created high cores for edema, inflammation and vascularity when compared to control rats not treated with the drugs. To the contrary, there was no significant difference between control group and rats treated with tramadol. The results confirmed that tramadol can be used for wound infiltration anaesthesia without adverse effect on the surgical healing process [79]. Rebamipide is an amino acid derivative of quinolinone used for healing gastroduodenal ulcers and for providing mucosal protection. The clinically effective concentration for rebamipide is 1–100 μm. To achieve this concentration in the target organ, it needs to be administered topically because orally administered rebamipide is not absorbed in the digestive tract. The potential healing of rebamipide on damaged urothelium was evaluated by Funahashi et al. [80]. They injected the bladders of female Sprague–Dawley rats with hydrochloride to induce cystitis. They were then injected with rebamipide and kept for 1 h. They found that the intravesical application of rebamipide accelerated the repair of damaged urothelium, protected its barrier function and suppressed bladder overactivity. Research should now be focussed on preparing nanoformulations of these drugs to reduce the concentration induced toxicity. Such drug loaded nanoparticles can be incorporated within wound dressing agents to promote urothelial wound healing. In the following sections, the different technologies used in hospitals to accelerate wound healing in urology are discussed. They make use of innovative biomaterials synthesised in regenerative medicines like hydrogels, hydrocolloids, foams, films, etc. incorporated with growth factors or drug molecules or nanoparticles.

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Negative pressure wound therapy (NPT) is a technique which employs a vacuum dressing to accelerate healing in acute or chronic wounds and enhance healing of first and second-degree burns. NPT involves the monitored application of sub-atmospheric pressure to the local wound environment by a sealed wound dressing coupled to a vacuum pump [91]. The vacuum draws out fluid from the wound and increases the blood flow to that area. The vacuum can be applied continuously or, occasionally, depending on the type of wound being treated. Open-cell foam dressing, layers of non-woven polyester and gauze sealed with an occlusive dressing are used as wound dressing materials for this technique [92]. Occlusive dressings include transparent film, hydrogels, hydrocolloids and alginates. Transparent films are polyurethane membranes with an acrylic adhesive that are waterproof, breathable, and transparent but are non-absorptive. Hydrogels are

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carbohydrate based polymers that can hold 80% water, but have poor exudate absorption. Saline and antibiotic fluids are used in the NPT to irrigate the wound. The dressing or filler material will be connected to the contours of the wound, and the overlying foam is sealed with a clear film [93]. The dressing is coupled to the drainage tube through an opening of the transparent film. The NPT was thus found to endorse wound healing by removing wound exudate, increasing blood perfusion and promoting growth of granulation tissue. Hydrocolloids are gel forming agents available as adhesive wafers and pastes that can absorb exudates and limit moisture loss but may leave a residue in the wound. Alginates are composed of cellulose fibres, derived from seaweed that are ideal for severe wounds, but suffer from foreign body reaction [94]. The applications of the NPT for healing wounds in urology are discussed in the following paragraphs. Artificial urinary sphincters (AuS) are used to treat masculine stress urinary incontinence that occurs after male prostatectomy. Although they have proven to be useful, some patients have reported infectious complications due to their usage. In urology, NPT is often used to remove infected foreign materials like abdominal mesh or orthopaedic implants and accelerate wound healing. Yokoyama et al. successfully treated surgical site infection due to AuS implantation, using NPT and antibiotic therapy [95]. Their patient was a 65-year-old man who underwent an unsuccessful AuS implantation and suffered serious infectious complications. They treated the patient using NPT by applying a pressure of − 125 mm Hg, and the patient was continent for one year without any complications (Fig. 8). Pasquier et al. applied NPT for the management of battlefield scrotum trauma and found that it could increase the rate of wound healing. The NPT is a therapeutic alternative that complements surgical and medical involvement in patients with complex scrotal wounds, particularly suitable for austere settings [96]. Fournier's gangrene is a life-threatening disease that affects the perineum (the region between scrotum and anus). It is a urological emergency condition that requires intravenous antibiotics and surgical removal of necrotic tissue. The wound remains open for a prolonged period of time and requires multiple regular wound dressings. Ozturk and coworkers investigated the use of vacuum assisted closure (VAC) for treating Fournier's gangrene after initial debridement [97]. VAC is a

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technique in closing 50 clean surgical wounds. The age, gender and wound distribution were similar for the surgical zipper study group, and the conventional nylon suture control group. The outcome of the scar was good in 86% of the study group when compared with 42% of the conventional control group. They proved that medical zippers are superior techniques to treat surgical wounds [89]. Bastian et al. used Medizip, a non-invasive skin closure system for urological procedures [90]. Medizip is a surgical zipper used for healing open wounds. It is a combination of hypoallergenic, microporous polyester coated with acrylate adhesive and a zipper, with a size ranging from 6–50 cm. After radial prostatectomy, twelve patients underwent non-invasive skin closure using Medizip. Once the operation was over, the wound was inspected for six weeks. They were satisfied with the cosmetic results of the wound healing progress (Fig. 7). Medizip has proven to be a safe alternative to conventional suture material for skin closure. However, its use was limited for diabetic wounds and wounds with substantial curves of more than 20°. It has several advantages as it can be opened for wound inspection and is comfortable for patients. It also decreases the cost and the time for skin closure in the operating room by more than 50% [90].

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Fig. 7. Intraoperative wound appearance (a) before applying surgical zipper, (b) after applying surgical zipper, (c) zipper closure and (d) wound after surgical zipper. Postoperative wound inspection after (e) 8 days and (f) 6 weeks. Reprinted from [90], Copyright 2003, with permission from Elsevier.


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The process of wound healing involves a complex interaction of several factors to replace damaged tissues. Human beings are in the quest for finding better solutions for wound closure. Urethral lesions have complex structure and are difficult to be treated using endoscopy. So, several studies have been conducted in the field of regenerative medicine to promote wound healing using growth factors, drug molecules, siRNA, miRNA, stem cells, etc. Growth factor therapy is employed in tissue engineering for monitoring cell differentiation, maturation and for promoting tissue regeneration. Stem cells hasten wound closure, help in the migration of immune cells and endothelial progenitors and promote angiogenesis. Gene silencing can be used to deactivate a particular gene to promote angiogenesis, cell migration, and proliferation. The new generation of medicated dressings incorporates drug molecules that have therapeutic values, which overcome the demerits of conventional pharmaceutical agents. Several technologies are used in hospitals, which make use of innovative materials synthesised in tissue engineering. Negative pressure wound therapy involves the monitored application of subatmospheric pressure to the local wound environment by a sealed wound dressing coupled to a vacuum pump. Laser tissue welding is a transformational technology that has proven its efficiency over current surgical procedures as they provide quick and accurate haemostasis and is lifesaving for patients suffering from blood clotting disorders. The surgical zipper is the combination of a hypoallergenic, microporous polyester coated with acrylate adhesive and a zipper, for tension-free wound healing. The main principle behind HBOT is to increase dissolved oxygen in the blood when it is provided at a pressure higher than atmospheric pressure and can be used to promote vasculogenesis and tissue regeneration. In the future, research can be conducted to fabricate novel materials that can heal urothelial wounds in a few days and with minimum pain.

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Acknowledgement

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The main principle behind hyperbaric oxygen therapy (HBOT) is to upsurge dissolved oxygen in the blood when it is provided at a pressure higher than atmospheric pressure [98]. It mainly requires an equipment that consists of a pressure chamber with the provision for supplying 100% oxygen. Trained persons are allowed to carry out this therapy [99]. Hyper-oxygenation of the tissues is achieved by distribution of oxygen across the pressure gradient. This improves the anti-inflammatory and pain killing effects, increases bacterial permeability to antibiotics, enhances the function of lymphocytes and macrophages, boosts testosterone production and promotes wound healing [100]. Thus, hyper oxygenation can be used in urology for treating scrotal–perineal fascitis, urgency–frequency syndrome (interstitial cystitis), chronic pelvic pain, Fournier's gangrene and radiation-induced cystitis [101]. Radiation cystitis is characterised by inflammation of the inner lining of small blood vessels that lead to acute and incurable ischaemia of the bladder wall and finally to muscle fibrosis because of cellular hypoxia. HBOT can increase oxygen tension in bladder tissue and promotes neovascularisation of normal tissue. Due to an increase in oxygen gradient, tissue macrophages stimulate angiogenesis. HBOT also initiates vasoconstriction and promotes wound healing. Following HBOT, the tissue oxygen of patients with radiation cystitis remained at normal levels for many years [99]. Interstitial cystitis (IC) is a chronic bladder syndrome that leads to pelvic pain [102]. The main symptoms include urinary urgency and frequent need to urinate along with severe bladder pain. Most of the patients develop reduction in bladder capacity secondary to fibrosis of the bladder wall. Research has shown that HBOT can significantly reduce pelvic pain, haematuria, urinary urgency and irritative voiding symptoms in patients with radiation cystitis. The promising results of HBOT for radiation cystitis prompted researchers to conduct a prospective pilot study of HBOT for patients with IC. The urothelium becomes a hypoxic, hypo vascular and the bladder wall shows chronic ischemia, in patients with late-stage IC. van Ophoven and colleagues conducted 30 sessions of HBOT on six patients in a hyperbaric chamber and followed up for 15 months [103]. They measured pain, and urgency and evaluated symptom severity. Four patients reported that HBOT was highly efficient in reducing pain whilst two patients showed short term amelioration of their symptoms. Thus, they stated that HBOT resulted in a sustained decrease of pelvic pain and urgency and was found to be effective in treating patients with IC. Around 26% of patients who underwent penile brachytherapy or radiotherapy of penile cancer have reported soft-tissue necrosis. They are usually treated using local irrigation, antibiotics, analgesics, antiinflammatory medication and wound debridement. HBOT has a welldefined role in treating such patients with tissue necrosis after penile

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brachytherapy. Gomez-Iturriaga et al. conducted HBOT on seven patients with soft-tissue necrosis of penis and found that all of them experienced excellent response with the healing of necrosis and resolution of symptoms [104]. Pedersen et al. has used HBOT in tissue engineering to promote vascularisation and regeneration of tissues. Scaffolds were made from poly(L-lactide-co-1,5-dioxepan-2-one) and were implanted into calvarial defects in Wistar rats. The animals received five sessions of HBOT for 90 min, for up to four weeks. The results confirmed that HBOT was effective in bone healing. Researchers should now conduct research to understand the efficiency of HBOT for tissue engineering in urology [105].

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type of the NPT that reduces oedema and promotes wound healing. They found that VAC is an effective and economical way to manage Fournier's gangrene. It is very cost-effective and can be used for a long period of time.

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promoting granulation tissue formation. Reprinted from [95], Copyright 2013, with permission from Wiley.

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