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Biomedical subjects

Anthony Atala

Publications and source records attributed to Anthony Atala.

At least 73 records · Page 4Linked to original sources
PubMed2003

Urethral stricture repair with an off-the-shelf collagen matrix.

PURPOSE: In select patients with urethral strictures in whom genital skin is insufficient alternative tissues are needed for urethral reconstruction. We explored the feasibility of using a bladder submucosa collagen based inert matrix as a free graft substitute for urethral stricture repair. MATERIALS AND METHODS: A total of 28 patients 22 to 61 years old with a diagnosis of urethral stricture underwent reconstructive surgery using a collagen based inert matrix for urethral repair. The inert collagen matrix was trimmed to size as needed for each patient and the neourethra was created by anastomosing the matrix in an onlay fashion to the urethral plate with continuous 6-zero absorbable sutures. The size of the created neourethra ranged from 1.5 to 16 cm. A voiding history, physical examination, retrograde urethrography, uroflowmetry and cystoscopic examinations were performed preoperatively and postoperatively. Random urethral biopsies were also performed. RESULTS: After a 36 to 48-month followup (mean 37) 24 of the 28 patients had a successful outcome. The remaining 4 patients had a slight caliber decrease at the anastomotic sites on urethrography. A subcoronal fistula developed in 1 patient which closed spontaneously 1 year after repair. Mean maximum urine flow rate increased from the preoperative value of 9 +/- 1.29 to 19.7 +/- 3.07 ml. per second postoperatively. Cystoscopic studies revealed adequate caliber conduits and normal appearing urethral tissues. Histological examination of the biopsy specimens showed the typical urethral stratified epithelium. CONCLUSIONS: Use of an off-the-shelf collagen inert matrix appears to be beneficial for patients with urethral strictures and obviates the need for obtaining an autologous graft, thus eliminating donor site morbidity.

Adult↗
PubMed2003

Tissue engineering, stem cells, and cloning for the regeneration of urologic organs.

Tissue engineering efforts are currently being undertaken for every type of tissue and organ within the urinary system. Most of the effort expended to engineer genitourinary tissues has occurred within the last decade. Tissue engineering techniques require a cell culture facility designed for human application. Personnel who have mastered the techniques of cell harvest, culture, and expansion as well as polymer design are essential for the successful application of this technology. Various engineered genitourinary tissues are at different stages of development, with some already being used clinically, a few in preclinical trials, and some in the discovery stage. Recent progress suggests that engineered urologic tissues may have an expanded clinical applicability in the future.

Biocompatible Materials↗
PubMed2003

Directed apoptosis in Cox-2-overexpressing cancer cells through expression-targeted gene delivery.

The principle of promoter-targeted gene delivery was used to direct the expression of reporter genes and inducible caspases to Cox-2-overexpressing cancer cells. The polycation poly(ethylenimine) was used in unmodified form to nonvirally deliver genes into cells, and targeting was achieved at the transcriptional level. Results demonstrated that reporter expression was reduced by an average of 89.8% in normal cells and cell lines not overexpressing Cox-2 when the strong cytomegalovirus promoter was replaced with the human Cox-2 promoter in delivered plasmids. Cocultures of normal and Cox-2-overexpressing cancer cells showed less than 0.5% reporter expression in normal fibroblast cells but over 35% reporter expression in PC3 prostate cancer cells. This targeting method was then used to direct the expression of inducible forms of caspases 3 and 9 to Cox-2-overexpressing cancer cells of the bladder and prostate. Following activation of the resulting caspase pro-forms, cells underwent apoptosis as evidenced by DNA fragmentation and cytoskeletal degradation. This result was also observed in cells resistant to apoptosis in terms of TNF-alpha initiation. Such directed apoptosis could eventually serve as a treatment for an entire class of Cox-2-overexpressing carcinomas.

Apoptosis↗
PubMed2003

Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices.

We explored the feasibility of developing corporal tissue, consisting of human cavernosal smooth muscle and endothelial cells in vivo, using three-dimensional acellular collagen matrices, which are similar in architecture to native corpora. Acellular collagen matrices were derived from processed donor rabbit corpora, using cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were seeded on the acellular matrices. A total of 80 matrices, 20 without cells and 60 with cells, were implanted subcutaneously in athymic mice. An additional 36 matrices seeded with cells were maintained in culture for up to 4 weeks. Hydroxyproline quantification, Western blot analysis, RT-PCR, and scanning electron microscopy of the matrices, with and without cells, were performed at various time points. Animals were killed 3 days and 1, 2, 3, 4, 6, and 8 weeks after implantation. Immunocytochemical and histological analyses were performed to confirm the muscle and endothelial phenotype. Organ bath studies were performed in order to determine the degree of tissue contraction. Western blot analysis detected alpha-actin, myosin, and tropomyosin proteins from human corporal smooth muscle cells. Expression of muscarinic acetylcholine receptor (mAChR) subtype m4 mRNA was demonstrated by RT-PCR from corporal muscle cells before and 8 weeks after seeding. The implanted matrices showed neovascularity into the sinusoidal spaces by 1 week after implantation. Increasing organization of smooth muscle and endothelial cells lining the sinusoidal walls was observed at 2 weeks and continued with time. The matrices were covered with the appropriate cell architecture 4 weeks after implantation. The matrices showed a stable collagen concentration over 8 weeks, as determined by hydroxyproline quantification. Immunocytochemical studies using alpha-actin and factor VIII antibodies confirmed the presence of corporal smooth muscle and endothelial cells, both in vitro and in vivo, at all time points. There was no evidence of cellular organization in the control matrices. Organ bath studies showed that the cell-seeded corporal tissue matrices responded to electrical field stimulation, whereas the unseeded implants failed to respond. This study demonstrates that human cavernosal smooth muscle and endothelial cells seeded on three-dimensional acellular collagen matrices derived from donor corpora are able to form well-vascularized corporal tissues in vivo.

Animals↗
PubMed2003

Engineering of vaginal tissue in vivo.

Congenital vaginal anomalies and cloacal malformations may require extensive surgical reconstruction. Surgical challenges are often encountered because of the limited amounts of native tissue available. We investigated the feasibility of using vaginal epithelial and smooth muscle cells for the engineering of vaginal tissues in vivo. Vaginal epithelial and smooth muscle cells of female rabbits were grown, expanded in culture, and characterized immunocytochemically. Vaginal epithelial and smooth muscle cells were seeded on polyglycolic acid (PGA) scaffolds at 10 x 10(6) and 20 x 10(6) cells/cm(3), respectively. The cell-seeded scaffolds were subcutaneously implanted into nude mice. The animals were killed 1, 4, and 6 weeks after implantation. Immunocytochemical and histochemical analyses were performed with pancytokeratins AE1/AE3 and with smooth muscle-specific alpha-actin antibodies to confirm the reconstituted tissue phenotype. Western blot analyses and electrical field stimulation studies were also performed to further characterize the tissue-engineered constructs. Vaginal epithelial cells were serially identified with anti-pancytokeratins AE1/AE3 at all culture stages. Smooth muscle cells in culture stained positively with alpha-smooth muscle actin antibodies. One week after implantation in vivo, the retrieved polymer scaffolds demonstrated multilayered tissue strips of both cell types, and penetrating native vasculature was also noted. Increased organization of the smooth muscle and epithelial tissue was evident by 4 weeks. There was no evidence of tissue formation in the controls. Immunocytochemical analyses using anti-pancytokeratins confirmed the presence of vaginal epithelial cells in each of the constructs. Anti-alpha-actin smooth muscle antibodies also confirmed the presence of multilayered smooth muscle fibers and tissue at each time point. Western blot analyses of the scaffolds confirmed the expression of cytokeratin and smooth muscle actin proteins when compared with controls. The contractile properties of the tissue-engineered vaginal constructs in response to electrical field stimulation were similar to those of normal vaginal tissue. Vaginal epithelial and smooth muscle cells can be easily cultured and expanded in vitro. Cell-seeded polymer scaffolds are able to form vascularized vaginal tissue in vivo that have phenotypic and functional properties similar to those of normal vaginal tissues. This is the first demonstration in tissue engineering wherein vaginal epithelial and smooth muscle cells are reconstituted in vivo into vaginal tissue. This technology may be pursued further experimentally in order to achieve the engineering of vaginal tissues for clinical applications.

Actins↗
PubMed2002

In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo.

Recent findings implied that the progression of hematologic malignancies, like that of solid tumors, is dependent on neovascularization. Recent studies on patients with acute myeloid leukemia (AML) showed increased levels of leukocyte-associated vascular endothelial growth factor (VEGF) and neovascularization of the bone marrow. Murine (32D, M1) and human (HEL, U937, and UKE-1) leukemic cell lines and freshly isolated leukemic cells were analyzed for the expression of VEGF and VEGF receptor mRNA. The expression of VEGF and VEGF receptors KDR and neuropilin-1 (NRP-1) was detected in these cells. In a murine chloroma model, delivery of VEGF(165) using microencapsulation technology resulted in enhanced tumor growth and vascularization, whereas treatment with a VEGF antagonist soluble NRP-1 (sNRP-1) inhibited tumor angiogenesis and growth. In a systemic leukemia model, survival of mice injected with adenovirus (Ad) encoding for Fc-sNRP-1 (sNRP-1 dimer) was significantly prolonged as compared with mice injected with Ad-LacZ. Further analyses showed a reduction in circulating leukemic cells and infiltration of liver and spleen as well as bone marrow neovascularization and cellularity. Taken together, these results demonstrate that angiogenic factors such as VEGF promote AML progression in vivo. The use of VEGF antagonists as an antiangiogenesis approach offers a potential treatment for AML. Finally, our novel in vivo drug delivery model may be useful for testing the activities of other peptide antiangiogenic factors.

Acute Disease↗
PubMed2002

Generation of histocompatible tissues using nuclear transplantation.

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac "patches", nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.

Animals↗
PubMed2002

Spatial and temporal control of transgene expression through ultrasound-mediated induction of the heat shock protein 70B promoter in vivo.

Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) or Fas ligand (Adeno-HSP-FasL) under the control of the heat shock protein 70B (hsp70B) promoter. Cultures infected with Adeno-HSP-Luc transiently expressed high levels of luciferase after heat shock. When cultures infected with Adeno-HSP-FasL were maintained at 37 degrees C, no transgene expression was observed, but when cultures were exposed to heat stress, transgene expression resulted in apoptotic cell death. In vivo, transgene expression was induced by ultrasound-mediated heating of adenovirus-infected tissue. In mice or rats injected with the Adeno-HSP-Luc construct, high levels of localized expression of luciferase activity were observed in regions subjected to ultrasound-mediated irradiation. Adeno-HSP-FasL was administered systemically to mice via the tail vein to evaluate safety. Animals receiving Adeno-HSP-FasL in the absence of ultrasound treatment did not display liver toxicity, whereas animals receiving ultrasound treatment to induce the expression of Fas ligand from the hsp70B promoter had significant increases in serum levels of liver enzymes. These data demonstrate that combining the inducible hsp70B promoter with ultrasound induction allows safe local expression of cytotoxic genes with possible therapeutic utility.

Adenoviruses, Human↗
PubMed2002

Rate and predictors of spontaneous resolution of prenatally diagnosed primary nonrefluxing megaureter.

PURPOSE: We assessed clinical and ultrasound predictors of the spontaneous resolution of prenatally diagnosed, primary nonrefluxing megaureter. MATERIALS AND METHODS: Primary nonrefluxing megaureter was prenatally diagnosed in 54 neonates from 1993 to 1998, including 15 in whom it was bilateral (69 megaureters). Median followup period from birth to the last radiology examination was 25.8 months (range 2.3 to 72). RESULTS: Of the patients 10 underwent surgical repair for severe hydroureteronephrosis, including decreasing renal function on diuretic renography in 1, and an episode of bilateral obstruction and acute renal failure in 1. Hydroureteronephrosis resolved in 39 cases (72%) and persisted in 5 at 30 to 72 months of followup. Laterality, gender and retrovesical ureteral diameter had no significant effect on the resolution rate (p >0.05). Mean initial ureteral diameter in patients with resolution was less than in those without resolution and in those who underwent surgery (0.8 versus 1.15 and 1.32 cm., respectively). Presenting hydronephrosis grade was a significant predictor of the resolution rate (p = 0.03). Grades 1 to 3 hydronephrosis resolved at a median age of 12.9, 23.9 and 34.6 months, respectively (range 4.1 to 66). In patients in whom grades 4 and 5 hydronephrosis resolved the median age at resolution was 48.5 months. CONCLUSIONS: Most cases of prenatally diagnosed, primary nonrefluxing megaureter resolve spontaneously. Grades 1 to 3 hydronephrosis tend to resolve between ages 12 and 36 months. For these grades followup intervals may be progressively elongated after a pattern of improving hydronephrosis has been established. In children with grade 4 or 5 hydronephrosis, or a retrovesical ureteral diameter of greater than 1 cm. the condition may resolve slowly and require surgery.

Child↗
PubMed2002

Proximal hypospadias.

Approximately 20% of hypospadiac urethras are located proximally, anywhere from the penoscrotal to the perineal region. Repair of proximal defects is still one of the most challenging surgical procedures facing the hypospadiologist. Modifications of current techniques for the correction of proximal hypospadias as well as new innovative techniques continue to be proposed.

Humans↗
PubMed2002

Complications of hypospadias repair.

Hypospadias is one of the most frequently encountered congenital malformations of the genitourinary system. The incidence in studies of large populations has been reported to be from 1 to 8 per 1000 male births. For centuries the imagination and creativity of surgeons has been challenged to create a phallus that is both functional and cosmetic. Over the last 30 years numerous new operations and techniques have been developed with the objective of achieving improved cosmetic results with minimal complications.

Balanitis↗
PubMed2002

Experimental and clinical experience with tissue engineering techniques for urethral reconstruction.

Tissue engineering has been proposed as a strategy for urethral reconstruction. This 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. Acellular collagen matrices derived from donor bladder submucosa have been used both experimentally and clinically for onlay urethral replacement with good success at our center. If a tubularized urethral repair is needed, the use of cells on the collagen matrix is essential for adequate tissue formation. Tissue engineering techniques are useful for urethral reconstruction.

Biocompatible Materials↗
PubMed2002

Principals of neovascularization for tissue engineering.

The goals in tissue engineering include the replacement of damaged, injured or missing body tissues with biological compatible substitutes such as bioengineered tissues. However, due to an initial mass loss after implantation, improved vascularization of the regenerated tissue is essential. Recent advances in understanding the process of blood vessel growth has offered significant tools for therapeutic neovascularization. Several angiogenic growth factors including vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) were used for vascularization of ischemic tissues. Three approaches have been used for vascularization of bioengineered tissue: incorporation of angiogenic factors in the bioengineered tissue, seeding endothelial cells with other cell types and prevascularization of matrices prior to cell seeding. This paper reviews the process of blood vessel growth and tissue vascularization, and discuss strategies for efficient vascularization of engineered tissues.

Animals↗
PubMed2002

Pancreatic tumor growth is regulated by the balance between positive and negative modulators of angiogenesis.

There is increasing evidence for the implication of tumor-derived angiogenic and anti-angiogenic factors in controlling tumor growth in vivo. In this study, we documented the production of inhibitors of angiogenesis by pancreatic cancer cells and examined how changes in the balance between pro- and anti-angiogenic factors regulate tumor growth in vivo. The human pancreatic cancer cell line Hs-776T (HS-W) produces slow-growing tumors in SCID mice. Cells of a variant form (HS-R) of Hs-776T produced faster-growing tumors compared to HS-W. Characterization of HS-W and HS-R cells in vitro showed similar proliferation rates and production of the angiogenic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Analyzes of anti-angiogenic factors showed comparable levels of angiostatin and thrombospondin 1 and 2, but endostatin was only detected in conditioned media of HS-W cells and was absent in HS-R. Cell proliferation was similar in both tumor types in vivo, whereas HS-W tumors demonstrated increased apoptosis with a high percentage of apoptotic endothelial cells (EC). Subsequently, VEGF was over-expressed in Hs-776T cells (HS-VF), resulting in rapidly growing tumors and lowering tumor and EC apoptosis. Collectively, our study confirms that tumor growth is dependent on its ability to increase the angiogenic stimulus or to reduce the amounts of endogenous anti-angiogenic factors.

Animals↗
PubMed2002

Future trends in bladder reconstructive surgery.

The incorporation of bowel into the urinary tract is associated with significant long-term complications. Therefore, considerable efforts are being made to avoid the use of enteric epithelium in bladder reconstruction. The simplest of these entail the use of native urothelium that is already available, with techniques such as auto-augmentation, auto-augmentation de-epithelialized enterocystoplasty, and ureterocystoplasty. Unfortunately, in many patients, the bladder is too small, or dilated ureters are not available, and these techniques cannot be applied. Recently, experimental techniques are examining the use of tissue expansion to the ureter and bladder to increase the volume of tissue available. Tissue engineering techniques are being applied to bladder regeneration, and considerable advances have already been made leading to in vivo animal experimentation, the results of which are very encouraging. The details of these most recent advances will be discussed in detail in this report.

Animals↗
PubMed2002

Reconstitution of human corpus cavernosum smooth muscle in vitro and in vivo.

A large number of congenital and acquired abnormalities of the genitalia would benefit from the availability of transplantable, autologous corpus cavernosum tissue for use in reconstructive procedures. We describe the results of preliminary experiments designed to determine the feasibility of using cultured human corporal smooth muscle cells seeded onto biodegradable polymer scaffolds for the formation of corpus cavernosum smooth muscle in vitro and in vivo. Primary cultures of human corpus cavernosum smooth muscle cells were derived from operative biopsies obtained during penile prosthesis implantation. Cells were characterized in vitro and seeded as a contiguous multilayered sheet onto polymers of non-woven polyglycolic acid. The seeded polymer constructs were then implanted subcutaneously in athymic mice. Animals were killed 7, 14, and 24 days after surgery and implants were examined via histology, immunocytochemistry, and Western blot analyses. Cultured cell multilayers were identified as smooth muscle before implantation via phase-contrast microscopy, immunocytochemistry and Western blot analyses. Retrieved implants from all time points demonstrated corporal smooth muscle tissue grossly, and histologically, at the time of sacrifice. Intact smooth muscle cell multilayers were observed growing along the surface of the polymers. There was evidence of early vascular ingrowth at the periphery of the implants by 7 days. By 24 days, there was evidence of polymer degradation. Maintenance of the smooth muscle phenotype in vivo was confirmed immunocytochemically and by Western blot analyses with antibodies to alpha-smooth muscle actin. This study provides evidence that cultured human corporal smooth muscle cells may be used in conjunction with biodegradable polymer scaffolds to create corpus cavernosum smooth muscle tissue in vitro and in vivo.

Animals↗