Construction of Pedicled Smooth Muscle Tissues by Combining the Capsule Tissue and Cell Sheet Engineering.

The survival of engineered tissue requires the formation of its own capillary network, which can anastomose with the host vasculature after transplantation. Currently, while many strategies, such as modifying the scaffold material, adding endothelial cells, or angiogenic factors, have been researched, engineered tissue implanted in vivo cannot timely access to sufficient blood supply, leading to ischemic apoptosis or shrinkage. Constructing vascularized engineered tissue with its own axial vessels and subsequent pedicled transfer is promising to solve the problem of vascularization in tissue engineering. In this study, we used the tissue expander capsule as a novel platform for vascularizing autologous smooth muscle cell (SMC) sheets and fabricating vascularized engineered tissue with its own vascular pedicle. First, we verified which time point was the most effective for constructing an axial capsule vascular bed. Second, we compared the outcome of SMC sheet transplantation onto the expander capsule and classical dorsal subcutaneous tissue, which was widely used in other studies for vascularization. Finally, we transplanted multilayered SMC sheets onto the capsule bed twice to verify the feasibility of fabricating thick pedicled engineered smooth muscle tissues. The results indicated that the axial capsule tissue could be successfully induced, and the capsule tissue 1 week after full expansion was the most vascularized. Quantitative comparisons of thickness, vessel density, and apoptosis of cell sheet grafts onto two vascular beds proved that the axial capsule vascular bed was more favorable to the growth and vascularization of transplants than classical subcutaneous tissue. Furthermore, thick vascularized smooth muscle tissues with the vascular pedicle could be constructed by multi-transplanting cell sheets onto the capsule bed. The combination of axial capsule vascular bed and cell sheet engineering may provide an efficient strategy to overcome the problem of slow or insufficient vascularization in tissue engineering.

Harvesting prevascularized smooth muscle cell sheets from common polystyrene culture dishes.

Cell sheet engineering has recently emerged as a promising strategy for scaffold-free tissue engineering. However, the primary method of harvesting cell sheets using temperature-responsive dishes has potential limitations. Here we report a novel cell sheet technology based on a coculture system in which SMCs are cocultured with EPCs on common polystyrene dishes. We found that an intact and highly viable cell sheet could be harvested using mechanical methods when SMCs and EPCs were cocultured on common polystyrene dishes at a ratio of 6:1 for 5 to 6 days; the method is simple, cost-effective and highly repeatable. Moreover, the cocultured cell sheet contained capillary-like networks and could secrete a variety of angiogenic factors. Finally, in vivo studies proved that the cocultured cell sheets were more favorable for the fabrication of vascularized smooth muscle tissues compared to single SMC sheets. This study provides a promising avenue for smooth muscle tissue engineering.

Pharmacological Targeting of Plasminogen Activator Inhibitor-1 Decreases Vascular Smooth Muscle Cell Migration and Neointima Formation.

OBJECTIVE: Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that promotes and inhibits cell migration, plays a complex and important role in adverse vascular remodeling. Little is known about the effects of pharmacological PAI-1 inhibitors, an emerging drug class, on migration of vascular smooth muscle cells (SMCs) and endothelial cells (ECs), crucial mediators of vascular remodeling. We investigated the effects of PAI-039 (tiplaxtinin), a specific PAI-1 inhibitor, on SMC and EC migration in vitro and vascular remodeling in vivo. APPROACH AND RESULTS: PAI-039 inhibited SMC migration through collagen gels, including those supplemented with vitronectin and other extracellular matrix proteins, but did not inhibit migration of PAI-1-deficient SMCs, suggesting that its antimigratory effects were PAI-1-specific and physiologically relevant. However, PAI-039 did not inhibit EC migration. PAI-039 inhibited phosphorylation and nuclear translocation of signal transducers and activators of transcription-1 in SMCs, but had no discernable effect on signal transducer and activator of transcription-1 signaling in ECs. Expression of low-density lipoprotein receptor-related protein 1, a motogenic PAI-1 receptor that activates Janus kinase/signal transducers and activators of transcription-1 signaling, was markedly lower in ECs than in SMCs. Notably, PAI-039 significantly inhibited intimal hyperplasia and inflammation in murine models of adverse vascular remodeling, but did not adversely affect re-endothelialization after endothelium-denuding mechanical vascular injury. CONCLUSIONS: PAI-039 inhibits SMC migration and intimal hyperplasia, while having no inhibitory effect on ECs, which seems to be because of differences in PAI-1-dependent low-density lipoprotein receptor-related protein 1/Janus kinase/signal transducer and activator of transcription-1 signaling between SMCs and ECs. These findings suggest that PAI-1 may be an important therapeutic target in obstructive vascular diseases characterized by neointimal hyperplasia.

Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments for bladder augmentation: in vivo, long term, and functional pilot study.

BACKGROUND: Bladder augmentation technique has changed over the years and the current practice has significant adverse health effects and long-term sequelae. Previously, we reported a novel cell transfer technology for covering demucosalized colonic segments with bladder urothelium and smooth muscle cells through an aerosol spraying of these cells and a fibrin glue mixture. OBJECTIVE: To determine the long-term durability and functional characteristics of demucosalized segments of colon repopulated with urothelial cells in the bladder of swine for use in augmentation cystoplasty. STUDY DESIGN: Nine swine were divided into three groups. The first group (control) underwent standard colocystoplasty; the second group underwent colocystoplasty with colonic demucosalization and aerosol application of fibrin glue and urothelial cell mixture; in the third group detrusor cells were added to the mixture described in group two. The animals were kept for 6 months. Absorptive and secretory function was assessed. Bladders were harvested for histological and immunohistochemical evaluation. RESULTS: All animals but one in the experimental groups showed confluent urothelial coverage of the colonic segment in the bladder without any evidence of fibrosis, inflammation, or regrowth of colonic epithelial cells. Ten percent of the instilled water in the bladder was absorbed within an hour in the control group, but none in experimental groups(p = 0.02). The total urine sediment and protein contents were higher in the control group compared with experimental groups (p < 0.05). DISCUSSION: Both study groups developed a uniform urothelial lining. Histologically, the group with smooth muscle had an added layer of submucosal smooth muscle. Six months after bladder augmentation the new lining was durable. We were also able to demonstrate that the reconstituted augmented segments secrete and absorb significantly less than the control colocystoplasty group. We used a non-validated simple method to evaluate permeability of the new urothelial lining to water. To determine if the aerosol transfer of bladder cells would have behaved differently in the neurogenic bladder population, this experiment should have been performed in animals with neuropathic bladders. CONCLUSION: Aerosol spraying of single cell suspension of urothelial and muscular cells with fibrin glue resulted in coverage of the demucosalized intestinal segment with a uniform urothelial layer. This new lining segment was durable without regrowth of colonic mucosa after 6 months. The new reconstituted segment absorbs and secretes significantly less than control colocystoplasty.

Furlow double-opposing z-plasty.

First described in 1978 by Furlow for the repair of a cleft soft palate, the double-opposing z-plasty, also known as the Furlow palatoplasty, is an excellent procedure for repairing a submucous cleft. It is also useful in patients with touch closure who simply need lengthening of the soft palate and as an option for patients with anomalous carotid vasculature where pharyngeal flaps and sphincter pharyngoplasty are precarious. The primary aims of this chapter are to provide the clinician with indications for when to consider utilizing the Furlow palatoplasty and to give a stepwise description of how to perform the procedure.

Tissue engineering in the gut: developments in neuromusculature.

The complexity of the gastrointestinal (GI) tract lies in its anatomy as well as in its physiology. Several different cell types populate the GI tract, adding to the complexity of cell sourcing for regenerative medicine. Each cell layer has a specialized function in mediating digestion, absorption, secretion, motility, and excretion. Tissue engineering and regenerative medicine aim to regenerate the specific layers mimicking architecture and recapitulating function. Gastrointestinal motility is the underlying program that mediates the diverse functions of the intestines, as an organ. Hence, the first logical step in GI regenerative medicine is the reconstruction of the tubular smooth musculature along with the drivers of their input, the enteric nervous system. Recent advances in the field of GI tissue engineering have focused on the use of scaffolding biomaterials in combination with cells and bioactive factors. The ability to innervate the bioengineered muscle is a critical step to ensure proper functionality. Finally, in vivo studies are essential to evaluate implant integration with host tissue, survival, and functionality. In this review, we focus on the tubular structure of the GI tract, tools for innervation, and, finally, evaluation of in vivo strategies for GI replacements.

Reconstructing full-length ureteral defects using a spiral bladder muscle flap with vascular pedicles.

INTRODUCTION: This study investigates the efficacy of ureteral reconstruction using a spiral bladder muscle flap with vascular pedicles (ie, the superior vesical arteries) to repair full-length ureteral defects and explores a surgical approach for repairing long ureteral defects (>20 cm) using a bladder muscle flap. TECHNICAL CONSIDERATIONS: The characteristics of the ureteral reconstruction surgery include the following: (1) Surgeons fully expose the bladder in the retroperitoneal space. (2) While dissecting the superior vesical arteries, the integrities of the blood vessel trunk and the primary branches are maintained as much as possible. (3) While preparing the bladder muscle flap, the surgeons make an S-shaped cut along the route of the superior vesical arteries along the bladder. In general, the basal width of the muscle flap is approximately 2-3 cm in length, and the total length is approximately 1-2 cm longer than the defective ureter. (4) During the surgery, kidney descent and fixation and psoas hitch are performed to reduce end-to-end anastomotic tension. (5) The addition of a submucosal tunnel to prevent postoperative ureteral reflux is unnecessary. (6) A pedicled greater omentum graft is transferred to cover the reconstructed ureter to enhance blood supply when necessary. CONCLUSION: Ureteroplasty using a spiral bladder muscle flap with vascular pedicles (ie, the superior vesical arteries) is an ideal treatment to repair full-length ureteral defects. Moreover, this technique is particularly useful for ureteral defects longer than 20 cm. This procedure should be strongly promoted.

A minimally invasive method to prevent postlaryngectomy major pharyngocutaneous fistula using infrahyoid myofascial flap.

INTRODUCTION: To prevent postoperative pharyngocutaneous fistula (PCF) after total (pharyngo)laryngectomy, simultaneous coverage of pharyngeal anastomosis with vascularised flaps such as pectoralis major muscle, anterolateral thigh or radial forearm, has been reported to be effective. As an alternative to the invasive methods using distant flaps, we used the infrahyoid myofascial flap (IHMFF), which was harvested from the same operation field of (pharyngo)laryngectomy, for covering the site of pharyngeal anastomosis. Herein, we describe the safety and effectiveness of our minimally invasive method for preventing PCF. METHODS: Eleven patients who were at a high risk of developing PCF due to previous chemoradiotherapy underwent simultaneous coverage of pharyngeal anastomosis with IHMFF after total (pharyngo)laryngectomy. The incidence of PCF and the rate of major fistula requiring surgical closure were determined, and the results were compared with the control group (23 patients without IHMFF cover after laryngectomy). RESULTS: PCF developed in 2 of the 11 patients (18.2%). The fistulae of these two patients were closed conservatively and did not require additional surgery. PCF developed in 6 of 23 patients (26.1%) in patients without IHMFF cover. All the six patients with fistula required additional closure surgery. The incidence of PCF did not differ in patients with or without IHMFF cover (Fisher's exact probability test; p=0.939, NS). However, the rate of major PCF requiring surgical closure was significantly lower in patients with IHMFF cover (Fisher's exact probability test; p=0.036<0.05). CONCLUSIONS: For (pharyngo)laryngectomy patients, IHMFF cover is a minimally invasive method that can prevent major PCF.

Periurethral skeletal myofibre implantation in patients with urinary incontinence and intrinsic sphincter deficiency: a phase I clinical trial.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Cell therapy using muscle precursor cell (MPC) injections has shown promise for urinary incontinence due to intrinsic sphincter deficiency (ISD), but the cell-preparation process is complex and costly. Implantation of freshly isolated myofibres carrying MPCs, mainly satellite cells, was very efficient in repairing muscle damage in recent animal experiments. In a phase I clinical trial, we investigated whether periurethral myofibre implantation generated local myogenesis and improved continence in 10 patients (five men and five women) with ISD. We found that myofibre implantation increased intraurethral pressure and periurethral electromyographic activity in patients with ISD. There were no serious side-effects. OBJECTIVES: To assess the safety of periurethral myofibre implantation in patients with urinary incontinence due to intrinsic sphincter deficiency (ISD) To assess the resulting myogenic process and effects on urinary continence. PATIENTS AND METHODS: An open-label non-randomised phase I clinical trial was conducted in five men and five women with ISD (mean age, 62.5 years). A free muscle strip from the patient's gracilis muscle was implanted around the urethra as a means to deliver locally myofibres and muscle precursor cells (MPCs). Patients were assessed for collection formation and incomplete bladder emptying. The maximum urethral closure pressure (MUCP) and concomitant periurethral electromyographic (EMG) activity were recorded before surgery and 1 and 3 months after surgery. Continence was assessed using the 24-h pad test and self-completed questionnaires, for 12 months. RESULTS: There were no serious side-effects. Continence improved significantly during the 12-month follow-up in four of the five women, including two who recovered normal continence. In the women, MUCP increased two-fold and de novo EMG periurethral activity was recorded. In the men, MUCP and EMG recordings showed similar improvements but the effect on continence was moderate. The few patients enrolled could affect these results. CONCLUSIONS: This is the first report of a one-step procedure for transferring autologous MPCs via myofibre implantation in patients with ISD. EMG and urodynamic assessments showed improvement of periurethral muscle activity. Further work is needed to confirm and improve the therapeutic efficiency of this procedure.

Seromuscular grafts for bladder reconstruction: extra-luminal demucosalisation of the bowel.

OBJECTIVE: To develop a robust sterile, fully demucosalized and vascularized seromuscular patch for use as an adjunct to novel bioengineering techniques aimed at augmenting, reconstructing, or replacing the bladder because of endstage disease. To eliminate deep colonic epithelial crypts to prevent the possibility of colonocyte regrowth. To maintain sterility by excluding the possibility of contamination from the bowel contents. METHODS: Pilot studies were performed on euthanized pigs to optimize the technique, with tissue samples examined by immunohistochemistry. In vivo, vascularized seromuscular colonic flaps were created from the bowel exterior in 7 large white hybrid pigs. The dissection was facilitated by placing an inflated Foley catheter within the colonic lumen. The seromuscular ends were approximated with 5/0 Vicryl sutures and excess mucosa intussuscepted within the lumen. Demucosalized flaps were used to augment the bladder by composite cystoplasty and were examined immunohistochemically at 3 months. RESULTS: Pilot studies showed that the technique was successful in creating seromuscular segments with no epithelial remnants. When applied surgically, the seromuscular flaps survived and showed no evidence of colonocyte regrowth at 3 months. CONCLUSION: Extraluminal dissection creates robust seromuscular flaps and prevents both regrowth by colonic epithelial cells and contamination of the tissue by exposure to the bowel contents. This technique should find application in a range of bladder reconstruction techniques, including composite cystoplasty and autoaugmentation.

Both immediate and delayed intracavernous injection of autologous adipose-derived stromal vascular fraction enhances recovery of erectile function in a rat model of cavernous nerve injury.

BACKGROUND: Intracavernous injection of cultured adipose-derived stem cells (ADSCs) effectively restores erectile function in cavernous nerve (CN)-injured rats when administered at the time of injury. However, culturing exposes ADSCs to the risk of contamination and dedifferentiation. OBJECTIVE: Explore the effect of uncultured autologous adipose-derived stromal vascular fraction (SVF) on improving erectile function in a rat model of CN injury when administered at the time of injury or 4 wk after injury. DESIGN, SETTING, AND PARTICIPANTS: Eighty-nine male Sprague Dawley rats were randomly divided into four groups. CN injury or sham surgery was performed at the start of the study, and rats were treated with either SVF or vehicle. Functional testing and histologic analysis were performed 12 wk after CN crush or sham surgery. INTERVENTION: We used intracavernous injection of saline immediately after CN crush (n=23), intracavernous injection of SVF immediately after CN crush (n=17), intracavernous injection of SVF 4 wk after CN crush (n=23), or sham surgery (n=26). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We studied intracavernous pressure (ICP) response to CN electrostimulation and performed histologic examination of midpenile cross-sections. Data were analyzed using one-way analysis of variance followed by the Tukey-Kramer test. RESULTS AND LIMITATIONS: Both immediate and delayed treatment with SVF resulted in a significantly increased ICP-to-mean arterial pressure ratio compared with the vehicle-treated group. Both immediate and delayed treatment with SVF significantly increased expression of neuronal nitric oxide synthase and neurofilament in dorsal penile nerves compared to the vehicle group. Furthermore, the smooth muscle-to-collagen ratio within the corpus cavernosum was significantly improved in both of the SVF groups compared to vehicle-treated rats. The main limitation of the study is the lack of determination of the SVF components. CONCLUSIONS: Uncultured autologous SVF injected immediately or 4 wk after CN crush improved erectile function, promoted nerve regeneration, and prevented fibrosis of the corpus cavernosum following CN injury.

Histerocystoplasty: a novel surgical procedure in the rat.

BACKGROUND: Enterocystoplasties are associated to complications. To avoid them, different types of tissue templates have been used to augment the bladder and induce native bladder regeneration. MATERIALS AND METHODS: A novel surgical technique for bladder reconstruction using autologous uterine tissue was evaluated in a rat model. Forty-two female Wistar rats were randomly allocated into three groups: sham-operation hysterocystorrhaphy (n = 12), hysterocystoplasty (n = 18), and control (n = 12). Two weeks after surgery, ultrasound examination of the bladder was performed. At 2, 4, or 6 mo after surgery, the rats were anesthetized and blood and urine samples were taken. They were then euthanized and post-mortem and histologic examination were performed. Ultrasound examination, analytical parameters and weight control, as well as gross and histologic examination were performed in all the operated animals. The statistical analysis was performed using Kruskal-Wallis and the extension of Fisher's exact tests. Significance was set at 5% (P < 0.05). RESULTS: Serum chemistry, blood count and peripheral blood smears, electrolytes, and urinary parameters were all within the normal range for the rat. Histologic sections of the surgically augmented zone between the bladder and uterine horn demonstrated urothelial epithelization, providing adequate coverage of the transition area in 72.22% of the rats that underwent hysterocystoplasty. CONCLUSIONS: The hysterocystoplasty was technically viable in all the cases and proved to be an easy and safe surgical model for bladder reconstruction. All animals were healthy after surgery and all systemic parameters analyzed were within normal physiologic range for the rat.

Successful implantation of bioengineered, intrinsically innervated, human internal anal sphincter.

BACKGROUND & AIMS: To restore fecal continence, the weakened pressure of the internal anal sphincter (IAS) must be increased. We bioengineered intrinsically innervated human IAS to emulate sphincteric physiology in vitro. METHODS: We cocultured human IAS circular smooth muscle with immortomouse fetal enteric neurons. We investigated the ability of bioengineered innervated human IAS, implanted in RAG1-/- mice, to undergo neovascularization and preserve the physiology of the constituent myogenic and neuronal components. RESULTS: The implanted IAS was neovascularized in vivo; numerous blood vessels were observed with no signs of inflammation or infection. Real-time force acquisition from implanted and preimplant IAS showed distinct characteristics of IAS physiology. Features included the development of spontaneous myogenic basal tone; relaxation of 100% of basal tone in response to inhibitory neurotransmitter vasoactive intestinal peptide (VIP) and direct electrical field stimulation of the intrinsic innervation; inhibition of nitrergic and VIPergic electrical field-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction); contraction in response to cholinergic stimulation with acetylcholine; and intact electromechanical coupling (evidenced by direct response to potassium chloride). Implanted, intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiology of myogenic and neuronal components. CONCLUSIONS: Intrinsically innervated human IAS bioengineered tissue can be successfully implanted in mice. This approach might be used to treat patients with fecal incontinence.

Re-innervation of smooth muscle that is transplanted to provide urethral sphincter augmentation.

A number of methods to augment the resistance of the outlet of the urinary bladder and to improve continence have been developed, including the artificial urinary sphincter and the placement of skeletal muscle around the urethra. It has been recently shown in a rabbit model that transplantation of smooth muscle around the proximal urethra reduces incontinence caused by internal sphincter deficiency. In the present work we have investigated the re-innervation of a peri-urethral smooth muscle transplant, and whether re-innervating axons have an appropriate effect when they are stimulated. Detrusor muscle from the dome of the bladder was transplanted to encircle the proximal urethras of rats. Rats tolerated the surgery and transplantation without any signs of compromised health. At 8 weeks the new sphincter was intact and easily recognised. The transplant contracted in response to transmural stimulation (1-5Hz for up to 5min) in a similar way to freshly removed detrusor strips. Contractions were graded with stimulus frequency, they peaked at about 10s and faded to a lower tension that was maintained. The amplitudes of sustained contractions of the transplants were reduced to about 10% by hyoscine and were almost abolished by tetrodotoxin. Histological examination revealed healthy, vascularised smooth muscle in the transplants, similar in appearance to freshly dissected detrusor. Re-innervation was confirmed immunohistochemically for transplanted detrusor muscle and transplants of dartos muscle. We conclude that smooth muscle transplanted to form a new sphincter around the urethra becomes functionally re-innervated and has potential to be used for sphincter augmentation.

Perineal colostomy with spiral smooth muscle graft for neosphincter reconstruction following abdominoperineal resection of very low rectal cancer: long-term outcome.

BACKGROUND: To avoid abdominal colostomy and improve quality of life, several types of anorectal reconstruction following abdominoperineal resection have been proposed. The aim of this study was to assess functional results and the quality of life of patients with very low rectal cancer after abdominoperineal resection and neosphincter reconstruction by perineal colostomy with a colonic muscular cuff. PATIENTS AND METHODS: Twenty-seven patients who had undergone neosphincter reconstruction with a perineal spiral cuff plasty after abdominoperineal resection were included in a retrospective study to evaluate long-term outcome. The functional results were analyzed using anal manometry and the continence score. The quality of life was measured with the global and disease-specific questionnaires European Organization for Research and Treatment of Cancer QLQ-C30 and C38. RESULTS: Median follow-up time was 105 months (range, 18-185 mo). The median Holschneider continence score of the study sample was 13 (continent), with a range of 10 (partially continent) to 16 (continent), thus demonstrating satisfactory functional results. The functional assessment was completed by neosphincter manometry which revealed a median resting vs compression pressure of 40 vs 96 cmH2O with a range of 5 to 81 cmH2O vs 49 to 364 cmH2O. The quality-of-life analyses showed an above-average score for both global health and disease-specific status. CONCLUSION: Spiral cuff colostomy with reconstruction after abdominoperineal resection of very low distal rectal cancer offers a surgical option for a selective group of patients with reasonable functional long-term results and an improved quality of life.

Stimulated smooth muscle neosphincter in male intrinsic sphincter deficiency: Proof of principle studies in a rabbit model.

AIMS: Intrinsic sphincter deficiency (ISD) causes significant disability and impairment of quality of life despite a range of treatment options. We investigated a novel method to improve sphincter function that does not appear to have been previously attempted, that is, transplantation to create a smooth muscle cuff, that subsequently becomes innervated, around the urethra. METHODS: Bladder pressure and passage of urine were measured in conscious, sedated rabbits of three groups: 6 control (unoperated) rabbits, 8 rabbits rendered incontinent by incision of their urethral wall, and 12 lesioned rabbits treated by transplantation of a circumferential strip of autologous dartos muscle whose innervation was later stimulated electrically. Effects of stimulation were tested up to 6 months after surgery. RESULTS: Lesions of the proximal urethra caused the bladder to leak at filling volumes that previously caused no leak. The volume added to cause first leak was less than half the volume added to cause a voiding reflex in unoperated rabbits. Transplantation of dartos to the lesioned bladder neck did not affect urodynamic parameters. However, electrical stimulation of the innervation of the transplant increased the bladder volume necessary to cause voiding and restored voiding pressures and filling volumes towards normal. These effects were maintained for 6 months and were not related to spontaneous healing. CONCLUSIONS: Free transplants of smooth muscle that become innervated offer promise as a treatment for ISD that is unlikely to cause urethral erosion and will not require a pump to restore continence.