Assessments of injectable alginate particle-embedded fibrin hydrogels for soft tissue reconstruction.

Soft tissue reconstruction is often needed after massive traumatic damage or cancer removal. In this study, we developed a novel hybrid hydrogel system consisting of alginate particles and a fibrin matrix that could maintain tissue volume long term. Alginate particles were fabricated by mixing 5% alginate with a 20 mM calcium solution. Cells and these alginate particles were then embedded in fibrin (alginate-fibrin) hydrogels using a dual syringe mixer. Cell-hydrogel constructs were evaluated in terms of cell survival and proliferation in the constructs in vitro. The results indicated that cellular viability, spreading and proliferation in the alginate-fibrin hydrogels were significantly higher compared to constructs fabricated with fibrin or alginate only. In vivo explants showed that cells contained within fibrin-only hydrogels did not contribute to neo-tissue formation, and the fibrin was fully degraded within a 12 week period. In the alginate-fibrin system, higher cellularity and vascular ingrowth were observed in vivo. This resulted in neo-tissue formation in the alginate-fibrin hydrogels. These results demonstrate that fibrin may enhance cell proliferation and accelerate the formation of extracellular matrix proteins in the alginate-fibrin system, while the alginate particles could contribute to volume retention. This injectable hybrid system composed of degradable and non-degradable hydrogels may be a preferable approach to the repair of soft tissue defects.

Embryoid body formation of human amniotic fluid stem cells depends on mTOR.

Human amniotic fluid stem cells (hAFSCs) harbor high proliferative capacity and high differentiation potential and do not raise the ethical concerns associated with human embryonic stem cells. The formation of three-dimensional aggregates known as embryoid bodies (EBs) is the principal step in the differentiation of pluripotent embryonic stem cells. Using c-Kit-positive hAFSC lines, we show here that these stem cells harbor the potential to form EBs. As part of the two kinase complexes, mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signaling pathway, which is involved in the regulation of cell proliferation, growth, tumor development and differentiation. Blocking intracellular mTOR activity through the inhibitor rapamycin or through specific small interfering RNA approaches revealed hAFSC EB formation to depend on mTORC1 and mTORC2. These findings demonstrate hAFSCs to be a new and powerful biological system to recapitulate the three-dimensional and tissue level contexts of in vivo development and identify the mTOR pathway to be essential for this process.

Expression-targeted gene therapy for the treatment of transitional cell carcinoma.

Targeted gene delivery for induced apoptosis of transitional cell carcinomas was carried out in vivo in mice via utilization of the murine cyclooxygenase type 2 (Cox-2) promoter (Tis10). MB49 cells, which constitutively overexpress Cox-2 like numerous other carcinomas, selectively expressed delivered genes that utilized this transcriptional control element. The products of the delivered genes were artificially inducible forms of caspases 3 and 9, which remained inactive until a chemical inducer of dimerization was later injected intraperitoneally. The genes were delivered intravesically as plasmids complexed with poly(ethylenimine). Significant improvements, in the form of reduced bladder mass, reduced tumor volume, anti-angiogenesis and inhibition of tumor growth were seen versus untreated or unactivated controls. In some instances, tumors were seen to go into complete remission. There were no apparent bystander effects associated with the treatments. This targeted gene therapy regimen could have wide applicability to numerous cancers due to constitutive overexpression of Cox-2.

Tunica repair with acellular bladder matrix maintains corporal tissue function.

Penile conditions, such as Peyronie's disease or tumor resection may require surgical reconstruction of the tunica albuginea. Various materials have been proposed, as a biomaterial for tunica albuginea repair, however, little functional data are available. We examined the applicability and functional outcome of a collagen-based matrix derived from the bladder (acellular bladder matrix (ABM)), as a biomaterial for tunica repair. Biocompatibility testing was performed on the matrix, which included mitochondrial metabolic activity, cell viability and apoptosis. Approximately 50% of the dorsal penile tunica albuginea was replaced with the collagen-based matrix patch after surgical removal in 24 New Zealand White rabbits. Cavernosometry and cavernosography were performed. The animals were killed 1, 2 and 3 months after surgery for analyses. The matrix showed excellent biocompatibility. All animals implanted with the matrix survived without any noticeable untoward effects. There was no evidence of inflammation or infection at the time of retrieval. Cavernosometry of the implanted animals demonstrated normal intracavernosal pressures with visual erections. Cavernosography of the repaired corpora showed a normal anatomical configuration. Biomechanical analysis of the retrieved matrices demonstrated similar tensile strengths as native tunica. Histologically, there was only a minimal inflammatory response, which gradually decreased over time. These results show that ABM is biocompatible, durable and effective when used as a tunica substitute. The matrix may be useful as an off-the-shelf biomaterial for patients requiring tunica albuginea repair.

Alterations in angiogenic growth factors and neuronal nitric oxide synthase expression in chronic cavernosal ischemia.

Our aim was to study anatomical and molecular changes at varying time points after the induction of cavernosal ischemia (CI) in a rabbit model of arteriogenic erectile dysfunction. Tissue structure and the expression of angiogenic and neurogenic genes were examined using immunostaining and reverse transcription-polymerase chain reaction (RT-PCR) analyses. We found a progressive increase of erectile connective tissue together with a decrease in smooth muscle cell content as the duration of CI increased. Immunohistochemical staining showed an increase in vascular endothelial growth factor (VEGF) levels at the early stages and a decrease at the later stages of ischemia. RT-PCR analysis of VEGF and neuronal nitric oxide synthase (nNOS) confirmed these results and showed nearly a two-fold increase in VEGF and nNOS mRNA levels in the early stages of CI with a decrease at the later stages of CI. On the other hand, mRNA levels of VEGF receptor, KDR, decreased approximately by 50% over the course of CI. Our studies showed that the cellular and molecular responses of the erectile tissue to short-term ischemia are different than those seen after long-term ischemia. The dramatic reduction in KDR expression suggests that the cavernosal endothelium is very sensitive to ischemia. The similar changes in VEGF and nNOS expression over the course of CI suggest a tissue-defensive mechanism to CI via the VEGF and NO pathways. Taken together, this study suggests that supplementation of VEGF at earlier stages of ischemia may restore the damaged endothelial cells of the corpus cavernosum and support tissue perfusion.

[Tissue engineering in urology. Basic principles and application]. / Tissue engineering in der Urologie. Prinzipien und Anwendung.

Tissue engineering is a rather new field of science. Despite this fact, some experimental investigations have already been applied in clinical studies. Compared to other medical fields, tissue engineering in urology is well established. Tissue-engineered bulking agents and tissue-engineered bladder augments are being investigated in clinical trials. Even though the knowledge gained in recent years is promising, the results of cellular therapies need to be critically judged before being finally applied in patients. Genetic engineering and stem cell research (adult undifferentiated cells) have had major impact on the field of tissue engineering over the past 2 years. By using the technology of genetic engineering, biochemical and functional qualities of tissues may be modified. Adult stem cells may help to substitute lost tissue in an autologous fashion by isolating undifferentiated cells from the body and by differentiating them into a desired cell type. These cells may be used to form native functional tissue to replace a diseased organ or organ part.

Vascular endothelial growth factor-mediated autocrine stimulation of prostate tumor cells coincides with progression to a malignant phenotype.

Vascular endothelial growth factor (VEGF), which is often produced at high levels by tumor cells, is a well-known mediator of tumor angiogenesis. VEGF receptor tyrosine kinases, KDR/Flk-1 and Flt-1, have been thought to be expressed exclusively by endothelial cells. In this study, we have used a prostate tumor progression series comprised of a differentiated rat prostate epithelial cell line, NbE-1, and its highly motile clonal derivative, FB2. Injection of NbE-1 cells into the inferior vena cava of syngeneic rats indicated that these cells are nontumorigenic. Using the same model, FB2 cells generated rapidly growing and well-vascularized tumors in the lungs. NbE-1 expressed marginal levels of VEGF, whereas high levels of VEGF protein were detected in FB2-conditioned medium and in FB2 tumors in vivo. Analysis of (125)I-VEGF(165) binding to NbE-1 and FB2 cells indicated that only motile FB2 cells expressed the VEGF receptor Flt-1. Consistent with this finding, physiological concentrations of VEGF induced chemotactic migration in FB2 but not in NbE-1 cells. This is the first documentation of a functional Flt-1 receptor in prostate tumor cells. Our results suggest two roles for VEGF in tumor progression: a paracrine role as an angiogenic factor and a previously undescribed role as an autocrine mediator of tumor cell motility.

In vitro biocompatibility assessment of naturally derived and synthetic biomaterials using normal human urothelial cells.

The reconstruction of urinary tissues often employs various types of biomaterials, and adequate material biocompatibility is essential for the successful reconstruction of urinary tissues. In this study we utilized a primary normal human urothelial cell culture system to evaluate the in vitro biocompatibility of a number of naturally derived biomaterials [i.e., bladder submucosa, small intestinal submucosa, collagen, and alginate] and polymeric biomaterials [i.e., poly(glycolic acid), poly(L-lactic acid), poly(lactic-co-glycolic acid), and silicone] that are either experimentally or clinically used in urinary reconstructive surgery. To determine the cytotoxic and bioactive effects of these biomaterials, the cell viability, metabolic activity, apoptotic properties, and DNA-synthesis activity were measured with four types of assays [Neutral Red, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide, apoptotic activity, and tritiated thymidine incorporation assays] using extract and direct contact methods. Most of the biomaterials tested did not induce significant cytotoxic effects and exhibited normal metabolic function and cell growth in vitro. This normal primary human urothelial cell culture model is suitable for in vitro biocompatibility assessments and is able to provide information on the cell-biomaterial interactions and the ability of biomaterials to support bioactive cell functions.

A noninvasive test for vesico-ureteric reflux in children.

OBJECTIVE: To report the development and testing of a device for the noninvasive diagnosis of vesico-ureteric reflux (VUR) which avoids the need for urethral catheterization (currently required to reliably determine the presence of VUR), and which thus avoids the anxiety of parents and patients that causes many families to refuse such evaluation. PATIENTS AND METHODS: Fifty-four children (49 girls and five boys, mean age 7.2 years, range 4-14) previously evaluated as having VUR volunteered to participate; no child was symptomatic at the time of the study. Refluxing units were known to be present by voiding cysto-urethrography (within 1 year, mean 7 months) in 45 and absent in 16. The device developed acquires electronically processed acoustic signals from the child during an observed urination. The signals are then analysed 'off-line' to determine the presence or absence of VUR. The initial preparation for the test included: (i) a full bladder [at least 0.80 x ((2 + age) x 30 mL)] measured by ultrasonography; and (ii) localization of the pelvi-ureteric junction by ultrasonography to accurately place the device's sensors on the child's back. The children were then positioned at a commode after placing the sensors; the recording was started and continued until voiding occurred. The children were tested with the recording and analysis team unaware of the presence and/or degree of VUR. The first 47 studies were single-kidney examinations and the remaining seven included simultaneous monitoring of both kidneys. RESULTS: Sixty-one renal units were assessed and interpretable signals were obtained from 54 (89%). There were seven episodes of 'system failure' when no interpretable data were obtained. One unit with no VUR had a 'reflux' signal; in four kidneys, spontaneous (two) and postsurgical (two) resolution of reflux was predicted by the testing and subsequently verified by cyclic radionuclide cystography. CONCLUSIONS: This noninvasive diagnostic technique detected VUR in 35 of 37 refluxing units and verified no reflux in 16 of 17 units without VUR. Further refinements may allow this technology to be used in all children with suspected VUR.

Tubularized incised plate urethroplasty: expanded use in primary and repeat surgery for hypospadias.

PURPOSE: We evaluated the impact of tubularized incised plate urethroplasty on primary and repeat hypospadias repair. MATERIALS AND METHODS: We retrospectively reviewed the medical records of all boys who underwent hypospadias repair at our institution during a recent 3-year period. The level of the hypospadias defect, technique of repair, primary repair versus reoperation, age at surgery and complications were recorded. RESULTS: A total of 520 hypospadias repairs were done from May 1996 through June 1999. We began to perform tubularized incised plate urethroplasty in November 1996. During the ensuing consecutive 32 months 181 primary and 25 repeat hypospadias repairs were done using this technique. Mean patient age at surgery was 22 months (range 3 months to 30 years). During the 6 months immediately before we began to use this method the Mathieu flip-flap procedure was the most commonly performed technique, accounting for 38% of all hypospadias repairs. In contrast, during the last 6 months reviewed tubularized incised plate urethroplasty accounted for 63% of all repairs, including 41 of 65 primary operations (63%) and 4 of 6 reoperations (67%), while no Mathieu procedures were performed. Postoperative followup was 6 to 38 months for tubularized incised plate repair. Overall meatal stenosis and a urethrocutaneous fistula developed in 1 and 14 boys, respectively (7% complication rate). CONCLUSIONS: Tubularized incised plate urethroplasty has become the preferred technique of primary and repeat hypospadias repair at our institution. The technique has few complications as well as proved success and versatility that continues to expand its applicability and popularity.

Continuous release of endostatin from microencapsulated engineered cells for tumor therapy.

Research studies suggest that tumor-related angiogenesis contributes to the phenotype of malignant gliomas. We assessed the effect of local delivery of the angiogenesis inhibitor endostatin on human glioma cell line (U-87MG) xenografts. Baby hamster kidney (BHK) cells were stably transfected with a human endostatin (hES) expression vector and were encapsulated in alginate-poly L-lysine (PLL) microcapsules for long-term delivery of hES. The release of biologically active endostatin was confirmed using assays of bovine capillary endothelial (BCE) proliferation and of tube formation. Human endostatin released from the microcapsules brought about a 67. 2% inhibition of BCE proliferation. Furthermore, secreted hES was able to inhibit tube formation in KDR/PAE cells (porcine aortic endothelial cells stably transfected with KDR, a tyrosine kinase) treated with conditioned U-87MG medium. A single local injection of encapsulated endostatin-secreting cells in a nude mouse model resulted in a 72.3% reduction in subcutaneous U87 xenografts' weight 21 days post treatment. This inhibition was achieved by only 150.8 ng/ml human endostatin secreted from 2 x 10(5) encapsulated cells. Encapsulated endostatin-secreting cells are effective for the treatment of human glioblastoma xenografts. Continuous local delivery of endostatin may offer an effective therapeutic approach to the treatment of a variety of tumor types.

Tissue engineering in urology.

Congenital abnormalities, cancer, trauma, infection, inflammation, iatrogenic injuries, and other conditions may lead to genitourinary organ damage or loss, requiring eventual reconstruction. Tissue engineering follows the principles of cell transplantation, materials science, and engineering toward the development of biological substitutes that would restore and maintain normal function. Tissue engineering may involve matrices alone, wherein the body's natural ability to regenerate is used to orient or direct new tissue growth, or the use of matrices with cells. Both synthetic (polyglycolic acid polymer scaffolds alone and with co-polymers of poly-1-lactic acid and poly-DL-lactide-coglycolide) and natural biodegradable materials (processed collagen derived from allogeneic donor bladder submucosa and intestinal submucosa) have been used, either alone or as cell delivery vehicles. Tissue engineering has been applied experimentally for the reconstitution of several urologic tissues and organs, including bladder, ureter, urethra, kidney, testis, and genitalia. Fetal applications have also been explored. Recently, several tissue engineering technologies have been used clinically, including the use of cells as bulking agents for the treatment of vesicoureteral reflux and incontinence, urethral replacement, and bladder reconstruction. Recent progress suggests that engineered urologic tissues may have clinical applicability in the future.

Initial experience with the transurethral self-detachable balloon system for urinary incontinence in pediatric patients.

PURPOSE: A new endoscopic technique to treat urinary incontinence in children using a self-detachable balloon device was studied. MATERIALS AND METHODS: The study includes 11 patients with a mean age of 14.6 years and all of whom had intrinsic sphincter deficiency due to myelomeningocele in 9, spinal artery bleed in 1 and cloacal exstrophy in 1. All patients were on clean intermittent catheterization preoperatively and postoperatively. Endoscopic balloon treatment was performed on an outpatient basis. A mean of 5 balloons (range 2 to 8) were placed per patient. All patients underwent formal urodynamic study preoperatively and at 6 weeks and 6 months following balloon placement. RESULTS: Of the 9 patients without prior bladder neck surgery 7 had improvement in urodynamic parameters, including urethral pressure profile in all 7 and functional bladder capacity in 6, 4 were markedly improved clinically and 2 were dry. Two patients with prior bladder neck surgery were clinically unchanged following balloon placement, although 1 had urodynamic improvement. CONCLUSIONS: Our initial experience with the transurethral self-detachable balloon system as a minimally invasive outpatient procedure to treat urinary incontinence in children has been encouraging. To date this procedure appears most applicable to the patient who has not undergone surgery and has a neurogenic etiology for urinary incontinence.

Defunctionalized bladders: effects before and after refunctionalization in an animal model.

PURPOSE: Bladder behavior after refunctionalization is usually unpredictable. We comparatively analyze various aspects of bladder defunctionalization and subsequent refunctionalization using an animal model. MATERIALS AND METHODS: A total of 18 rabbits were divided equally into 3 groups. Animals in group 1 underwent 2 successive surgical procedures, including bladder division and reattachment. Bladder division was performed by hemisecting the bladder from dome to trigone into a functioning and nonfunctioning chamber. Bladder reattachment was achieved by reanastomosing both hemibladders. Group 2 animals underwent sham operations, and group 3 animals were age matched normal controls. Serial urodynamic studies and fluoroscopic cystograms were performed in all animals. Gross, histochemical (hematoxylin and eosin, Masson's trichrome and Sirius red) and immunocytochemical (alpha-actin, collagen I and III) analyses, collagen content determination and organ bath studies were performed. RESULTS: The defunctionalized hemibladders demonstrated lower wet weight, capacity and compliance compared to the functional contralateral and normal control bladders. Refunctionalization of the bladders resulted in a progressive recovery of capacity and compliance with time. The bladder contractile response and connective tissue-to-muscle ratio were abnormal in the defunctionalized segments but normalized after bladder refunctionalization. CONCLUSIONS: Defunctionalization results in remarkable alterations in bladder growth, capacity, compliance and distribution of connective tissue. However, these bladders demonstrate an innate capacity to recover from these alterations following refunctionalization.

Ketorolac suppresses postoperative bladder spasms after pediatric ureteral reimplantation.

UNLABELLED: We evaluated the efficacy of ketorolac in suppressing postoperative bladder spasms after ureteroneocystostomy (ureteral reimplantation). Twenty-four pediatric patients undergoing intravesical ureteroneocystostomy were enrolled prospectively to receive either ketorolac or placebo via double-blinded randomization. Twelve patients in each group shared similar preoperative characteristics. All were maintained on an epidural infusion of bupivacaine (0.1%) with fentanyl (2 microg/mL) throughout the study. Patients were given either ketorolac (0.5 mg. kg(-1). dose(-1)) or placebo (equivalent volume saline) IV after surgery and every 6 h thereafter for 48 h. Parents were instructed to record bladder spasm episodes prospectively by using a standardized time-flow diary. Three patients (25%) in the ketorolac group experienced bladder spasms, compared with 10 patients (83%) in the placebo group (two-sided P < 0.05). The median severity score for the ketorolac group was 1.2 (mild = 1.0, severe = 3.0), compared with 2.6 for the placebo group (P = 0.003). We conclude that IV ketorolac reduces the frequency and severity of postoperative bladder spasms after intravesical ureteroneocystostomy. IMPLICATIONS: We studied the efficacy of ketorolac, a prostaglandin synthesis inhibitor, in the treatment of bladder spasm after ureteroneocystostomy (antireflux operation). Patients were randomized in a double-blinded manner to receive either ketorolac or placebo after the surgery. We demonstrate that ketorolac reduces the frequency and severity of postoperative bladder spasm.

Systems for therapeutic angiogenesis in tissue engineering.

The goals in tissue engineering include the replacement of damaged, injured, or missing body tissues with biologically compatible substitutes. To overcome initial tissue-mass loss, improved vascularization of the regenerated tissue is essential. Two pathways of tissue neovascularization are known: vasculogenesis, the in situ assembly of capillaries from undifferentiated endothelial cells (EC), and angiogenesis, the sprouting of capillaries from preexisting blood vessels. Recent advances in our understanding of the process of bloodvessel growth have provided significant tools for the neovascularization of bioengineered tissues. Several growth factors serve as stimuli for EC proliferation and migration as well as the formation of new blood vessels. They convey their effects via specific receptors expressed on the surface of EC. Vascular epithelial growth factor (VEGF) is a major regulator of neovascularization. VEGF plays a major role in the early development of blood-cell progenitors. Basic fibroblast growth factor (bFGF) was identified as the first angiogenic factor. It is a potent inducer of EC proliferation and blood-vessel growth in vitro and in vivo. VEGF and bFGF have been injected into undervascularized ischemic tissues, resulting in new blood-vessel formation and tissue perfusion. Gene-therapy approaches using VEGF cDNA injection into ischemic tissues have augmented the formation of collateral vessels. Angiogenic factors such as VEGF and bFGF have also been incorporated into bioengineered tissues and have facilitated blood-vessel growth. Other approaches such as prevascularization of the matrix prior to cell seeding and incorporation of EC into the bioengineered tissues have produced encouraging results. This article reviews the process of blood-vessel growth and tissue vascularization, placing emphasis on strategies that can be employed for efficient vascularization of engineered tissues in vitro and in vivo.

Tissue engineering of the bladder.

When gastrointestinal tissue is used for bladder augmentation or replacement, multiple complications may ensue, such as infection, metabolic disturbances, urolithiasis, perforation, increased mucous production, and malignancy. Therefore, alternative methods are being sought for cystoplasty. There has been a resurgence of interest in the use of acellular collagen-based matrices as scaffolds for bladder regeneration. Experimental work involving several collagen matrices, such as allogenic bladder and intestinal tissues, is currently being conducted in several academic centers. Recently, functional bladder tissue has been engineered using selective cell transplantation. The approach that has been followed for bioengineering of bladder tissue involves the use of autologous cells, thus avoiding rejection, whereby a biopsy of tissue is obtained from the host, after which the cells are dissociated and expanded in vitro, reattached to a matrix, and implanted into the same host.

Tissue-engineering applications for phallic reconstruction.

Pathologic penile conditions often require reconstructive surgery. Due to the limited amount of autologous tissues available for reconstruction, other tissue substitutes have been used. Phallic reconstruction using engineered autologous genital tissue, i.e., tissue derived from the patient's own cells, may be preferable. In this article we describe tissue-engineering approaches that may be applicable to genital reconstruction.