Percutaneous tibial nerve stimulation (pTNS): success rate and the role of rectal capacity.

PURPOSE: Percutaneous tibial nerve stimulation (pTNS) was originally developed to treat urinary incontinence. Recently, some case series have also documented its success in the treatment of fecal incontinence. Nevertheless, the mechanism underlying this effect remains unknown but may be related to changes in rectal capacity. The aim of this study was to investigate the success of pTNS for the treatment of fecal urge incontinence and assess the influence of rectal capacity on treatment efficacy. METHODS: All patients undergoing pTNS for fecal incontinence between July 2009 and March 2014 were enrolled in a prospective, observational study consisting of a therapeutic regimen that lasted 9 months. Therapy success was defined as a reduction in the CCI (Cleveland Clinic incontinence) score of ≥50% and patient-reported success. Furthermore, quality of life (Rockwood's scale) and changes in anorectal physiology were recorded. RESULTS: Fifty-seven patients with fecal urge incontinence were eligible, nine of whom were excluded. The success rate was 72.5%. Incontinence events and urge symptoms were significantly reduced after 3 months and at the end of therapy. The median CCI score decreased from 12 to 4 (P < 0.0001), and the quality of life was significantly improved. However, rectal capacity was not significantly related to treatment success before or after therapy. No adverse events were observed. CONCLUSIONS: These results demonstrate that pTNS can improve the symptoms and quality of life of patients with fecal urge incontinence. However, the study fails to demonstrate a correlation between treatment success and changes in rectal capacity.

[Surgical treatment of fistula-in-ano--a journey between Skylla and Charybdis]. / Die Therapie der Analfistel - Wahl zwischen Skylla und Charybdis.

This review discusses the pathogenesis, symptomatology, diagnostic work-up, and treatment options for fistula-in-ano, which is a common condition that affects ~ 20 in 100,000 per year with a predominance for young males. Fistula-in-ano normally presents as an acute anorectal abscess that subsequently becomes established as a chronic discharging fistula. The illness is characterized by chronic perianal discharge and intermittent pain. The aim of surgical treatment is permanent cure of the fistula whilst maintaining patient continence. This principle forms the basis of surgical decision-making and means that treatment options often have to be individualized for each patient. Low, simple fistulae may be treated by fistulotomy because of the low risk of incontinence. In contrast, high fistulae that contain a greater proportion of sphincter muscle demand more complex operations. Traditional reconstructive techniques (transanal advancement flap, primary sphincter reconstruction) aim to eradicate the fistula whilst leaving the sphincter muscle intact or readapted, whilst newer techniques (biosynthetic plugs) provide a scaffold to encourage normal tissue ingrowth with fistula occlusion. The newer procedures preserve the sphincter ideally. On the other hand success rates of these techniques are somewhat disappointing.

Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells.

PURPOSE: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs). METHODS: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet's membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), natrium hydrogen carboanhydrase (Na(+)/HCO(3)(-)), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry. RESULTS: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet's membrane. A significant DNA reduction (mean before decelluraziation 365.3 ± 88.6 ng/mg, mean after decelluarization 23.2 ± 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 ± 179 cells/mm(2) on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na(+)/HCO(3)(-), and Na(+)/K(+)-ATPase. CONCLUSIONS: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.

Prospective randomized controlled trial of simulator-based versus traditional in-surgery laparoscopic camera navigation training.

BACKGROUND: Surgical residents often use a laparoscopic camera in minimally invasive surgery for the first time in the operating room (OR) with no previous education or experience. Computer-based simulator training is increasingly used in residency programs. However, no randomized controlled study has compared the effect of simulator-based versus the traditional OR-based training of camera navigation skills. METHODS: This prospective randomized controlled study included 24 pregraduation medical students without any experience in camera navigation or simulators. After a baseline camera navigation test in the OR, participants were randomized to six structured simulator-based training sessions in the skills lab (SL group) or to the traditional training in the OR navigating the camera during six laparoscopic interventions (OR group). After training, the camera test was repeated. Videos of all tests (including of 14 experts) were rated by five blinded, independent experts according to a structured protocol. RESULTS: The groups were well randomized and comparable. Both training groups significantly improved their camera navigational skills in regard to time to completion of the camera test (SL P = 0.049; OR P = 0.02) and correct organ visualization (P = 0.04; P = 0.03). Horizon alignment improved without reaching statistical significance (P = 0.20; P = 0.09). Although both groups spent an equal amount of actual time on camera navigation training (217 vs. 272 min, P = 0.20), the SL group spent significantly less overall time in the skill lab than the OR group spent in the operating room (302 vs. 1002 min, P < 0.01). CONCLUSION: This is the first prospective randomized controlled study indicating that simulator-based training of camera navigation can be transferred to the OR using the traditional hands-on training as controls. In addition, simulator camera navigation training for laparoscopic surgery is as effective but more time efficient than traditional teaching.

Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts.

PURPOSE: To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering. METHODS: Corneal cells were isolated from human corneoscleral rims. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/ml) and 0.3% sodium dodecyl sulphate. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm removal of the corneal cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using DNA Purification Kit (Fermentas, St. Leon-Rot, Germany). Alcian blue staining was carried out to analyse the structure of the extracellular matrix (ECM). Corneal stromal cells were injected into the DPCs; limbal corneal epithelial cells and corneal endothelial cells were seeded onto the anterior and posterior surfaces of the DPCs, respectively. Evaluation was undertaken at days 14 and 30. The phenotypical properties of the cultivated corneal cells were investigated using Immunolocalization of type I collagen, keratocan, lumican, cytokeratin 3 (AE5) and type VIII collagen. RESULTS: Haematoxylin-eosin and DAPI staining showed efficient elimination of porcine corneal cells, whereas alcian blue confirmed gross preservation of the ECM. The quantitative analysis of the DNA content showed a significant reduction (mean before decellularization: 75.45 ± 13.71 ng/mg; mean after decellularization: 9.87 ± 2.04 ng/mg, p < 0.001). All three types of corneal cells were efficiently cultured and expanded on the DPCs. CONCLUSIONS: Decellularized porcine corneas might serve as a potential scaffold for tissue engineering of the cornea, possibly providing xenogenic substrate for corneal transplantation.

Reconstruction of corneal stroma with decellularized porcine xenografts in a rabbit model.

PURPOSE: To evaluate the potential use of decellularized porcine stromal matrix (PSM) for reconstruction of corneal stroma in a rabbit model. METHODS: Ten chinchilla bastard rabbit corneas were exposed to a circular half-thickness keratotomy with a 3.0 mm diameter at the central cornea. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 K IU/ml) and 0.3% sodium dodecyl sulphate (SDS). The 3.0 mm in diameter decellularized corneal stromal xenograft was inserted into the pocket, and the incision was closed with four 10.0 nylon sutures. Clinical photographs were taken at day 1, day 7, day 30 and on a monthly basis for up to 6 months after transplantation. Six months after surgery, the rabbits were killed and eyes were enucleated. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the complete removal of the corneal cells after decellularization of porcine corneas and repopulation with rabbit cells. Alcian blue staining was performed for analysing the structure of the extracellular matrix (ECM). RESULTS: Efficient elimination of porcine cells was achieved by our decellularization protocol and confirmed via HE and DAPI stainings. Moreover, the major histoarchitectural ECM structure had been maintained as visualized by the alcian blue stain. Finally, the PSM was biocompatible with the host's epithelium evidenced as a regrowth covering the exposed xenograft. CONCLUSIONS: This novel technique of tissue engineering may provide one of many solutions to addressing anterior corneal pathological conditions in the face of a shortage of human corneal material.

Laparoscopic versus open splenectomy for nontraumatic diseases.

BACKGROUND: Laparoscopic splenectomy (LS) is the standard procedure for normal size or moderately enlarged spleens; open splenectomy (OS) is preferred in cases of splenomegaly. In this study, indications for and complications of open and laparoscopic splenectomy were analyzed, with the aim to identify patients who will benefit from either technique. METHOD: A consecutive series of 52 patients undergoing elective open or laparoscopic splenectomy between January 2001 and December 2006 was analyzed. Spleen volume was calculated as length x width x depth from the pathologist's measurements. RESULTS: LS was performed in 25 patients with a median age of 41 years (range = 24-65). OS was performed in 27 patients with a median age of 60 years (range = 24-86) (p < 0.001). Conversion to OS was necessary in two patients (8%). Operation time was significantly shorter in LS (p < 0.05). Spleen volume was significantly greater in patients who underwent open (median = 2520 ml, range = 150-16,800 ml) versus laparoscopic (median = 648 ml, range = 150-4860 ml) splenectomy (p = 0.001). In 36% of all laparoscopic procedures, spleen volume exceeded 1000 ml. The underlying disease was mainly immunothrombocytopenia in LS patients and lymphoma and osteomyelofibrosis in OS patients. Five patients died after OS. Significantly more patients were hospitalized longer than 7 days following OS than following LS (p < 0.05). Overall complication rate was higher after OS (LS, 8; OS, 13 patients; p < 0.05). CONCLUSIONS: LS was preferred in younger patients with moderate splenomegaly, while massive splenomegaly mostly led to OS. In view of the absence of technique-related differences, LS can primarily be attempted in all patients.

Routine stenting reduces urologic complications as compared with stenting "on demand" in adult kidney transplantation.

OBJECTIVES: To examine the impact of the chosen surgical technique and of systematic versus "on-demand" placement of a primary stent on the incidence of urologic complications in adult kidney transplantation. METHODS: Data of 497 consecutive patients undergoing kidney transplantation at a single center were retrospectively analyzed with respect to urologic complications. Three different surgical strategies for the ureteroneocystostomy were compared: (1) transvesical anastomosis with stenting "on demand," (2) extravesical anastomosis with stenting "on demand," and (3) extravesical anastomosis with routine stenting. Nine parameters were evaluated by logistic regression for a possible contribution to the development of urologic complications. RESULTS: Routine placement of a stent significantly reduced the number of urologic complications compared with both transvesical or extravesical anastomoses with stenting "on demand" (20.8% in transvesical "on demand," 17.9% in extravesical "on demand," and 5.8% in extravesical "routine"). Logistic regression analysis revealed that routine stenting versus stenting "on demand" (P = 0.001) and living donor transplantation (P = 0.038) are two independent factors associated with a significantly lower incidence of urologic complications. Although routine stenting was not associated with an increased incidence of urinary tract infections, female gender was the only independent factor associated with this complication (P = 0.001). CONCLUSIONS: Routine stenting of the ureteroneocystostomy is superior to stenting "on demand" in adult kidney transplantation, suggesting that the intraoperative decision of whether to stent is insufficient to avoid urologic complications.

Activation of human microvascular endothelial cells with TNF-alpha and hypoxia/reoxygenation enhances NK-cell adhesion, but not NK-Cytotoxicity.

BACKGROUND: Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-alpha and hypoxia/reoxygenation in an in vitro model of I/R. METHODS: MVEC were stimulated in vitro for 8 h with TNF-alpha, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using Cr-release assays. RESULTS: Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-alpha but unaffected by hypoxia/reoxygenation in the absence of TNF-alpha. ICAM-1 expression was further increased by a combination of TNF-alpha and hypoxia/reoxygenation, whereas TNF-alpha-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-alpha and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-alpha and slighty reduced by hypoxia/reoxygenation. CONCLUSIONS: Endothelial activation with TNF-alpha, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.

Chlamydia pneumoniae induces aponecrosis in human aortic smooth muscle cells.

BACKGROUND: The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis. RESULTS: Chlamydiae were found to be localized up to 72 h post infection in aortic smooth muscle cells either as single bacteria or inside of large inclusions. Quantification of host cell death by lactate dehydrogenase release assay revealed strictly dose and time dependent lysis for all tested isolates of Chlamydia pneumoniae. Phosphatidylserine exposure was detected by flow cytometry in Chlamydia pneumoniae infected cells. Ultrastructure of Chlamydia pneumoniae infected human aortic smooth muscle cells showed extensive membrane- and organelle damage, chromatin condensation but no nuclear fragmentation. DNA fragmentation as well as cell membrane permeability was analyzed by TUNEL and NHS-biotin staining and occurred exclusively in cells carrying Chlamydia pneumoniae spots but not in smooth muscle cells with inclusions. These morphological features of cell death were not accompanied by an activation of caspase-3 as revealed by analysis of enzyme activity but involved mitochondrial membrane depolarization as shown by TMRE uptake and release of cytochrome c from mitochondria. CONCLUSION: This study provides evidence that Chlamydia pneumoniae induce a spot like infection in human aortic smooth muscle cells, which results in a chimeric cell death with both apoptotic and necrotic characteristics. This aponecrotic cell death may assist chronic inflammation in atherosclerotic blood vessels.

Human umbilical cord cells for cardiovascular tissue engineering: a comparative study.

OBJECTIVE: Tissue engineering of viable, autologous cardiovascular replacements with the potential to grow, repair and remodel represents an attractive approach to overcome the shortcomings of available replacements for the repair of congenital cardiac defects. Currently, vascular myofibroblast cells represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments prior to scaffold seeding, a technique which may not be preferable, especially in pediatric patients. This study evaluates cells isolated from human umbilical cord artery, umbilical cord vein and whole cord as alternative autologous cell sources for cardiovascular tissue engineering. METHODS: Cells were isolated from human umbilical cord artery (UCA), umbilical cord vein (UCV), whole umbilical cord (UCC) and saphenous vein segments (VC), and were expanded in culture. All three expanded cell groups were seeded on bioabsorbable copolymer strips and grown in vitro for 28 days. Isolated cells were characterized by flow cytometry, histology, immunohistochemistry, proliferation assays and compared to VC. Morphological analysis of the seeded polymer strips included histology, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and uniaxial stress testing. RESULTS: UCA, UCV and UCC demonstrated excellent cell growth properties comparable to VC. Following isolation, all three cell groups showed myofibroblast-like morphology and characteristics by staining positive for alpha-smooth muscle actin (ASMA) and vimentin. Histology and immunohistochemistry of seeded polymers showed good tissue and extracellular matrix formation containing collagen I, III and elastin. TEM showed viable myofibroblasts and the deposition of collagen fibrils and progressive growing tissue formation, with a confluent surface, was observed in SEM. No difference was found among the mechanical properties of UCA, UCV, UCC and VC tissue engineered constructs. CONCLUSIONS: Tissue engineering of cardiovascular constructs by using UCA, UCV and UCC is feasible in an in vitro environment. Cell growth, morphology, characteristics and tissue formation were comparable between UCA, UCV, UCC and VC. UCC represent an attractive, readily available autologous cell source for cardiovascular tissue engineering offering the additional benefits of utilizing juvenile cells and avoiding the invasive harvesting of intact vascular structures.

Dextran sulfate acts as an endothelial cell protectant and inhibits human complement and natural killer cell-mediated cytotoxicity against porcine cells.

BACKGROUND: The innate immune system, including complement and natural killer (NK) cells, plays a critical role in activation and damage of endothelial cells (ECs) during xenograft rejection. The semisynthetic proteoglycan analog dextran sulfate (DXS, molecular weight 5,000) is known to inhibit the complement and coagulation cascades. We hypothesized that DXS may act as an "EC-protectant" preventing complement and NK lysis by functionally replacing heparan sulfate proteoglycans that are shed from the EC surface on activation of the endothelium. METHODS: Binding of DXS to ECs, deposition of human complement, cytotoxicity, and heparan sulfate expression after exposure to normal human serum were analyzed by flow cytometry. The efficacy of DXS to protect ECs from xenogeneic NK cell-mediated cytotoxicity was tested in standard 51Cr-release assays. RESULTS: DXS dose-dependently inhibited all three pathways of complement activation. Binding of DXS to porcine cells increased on treatment with human serum or heparinase I and correlated positively with the inhibition of human complement deposition. This cytoprotective effect of DXS was still present when the challenge with normal human serum was performed up to 48 hr after DXS treatment of the cells. DXS incubation of porcine ECs with and without prior tumor necrosis factor-alpha stimulation reduced xenogeneic cytotoxicity mediated by human NK cells by 47.3% and 25.3%, respectively. CONCLUSIONS: DXS binds to porcine cells and protects them from complement- and NK cell-mediated injury in vitro. It might therefore be used as a novel therapeutic strategy to prevent xenograft rejection and has potential for clinical application as an "EC protectant."

Hypoxia and reoxygenation do not upregulate adhesion molecules and natural killer cell adhesion on human endothelial cells in vitro.

OBJECTIVES: Ischemia/reperfusion injury is characterized by endothelial cell activation leading to increased expression of adhesion molecules such as inter-cellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, endothelial- and platelet-selectin (E- and P-selectin), and to the subsequent recruitment of leukocytes. The aim of the present study was to investigate the respective effects of a proinflammatory cytokine (tumor necrosis factor alpha, TNF-alpha), hypoxia and/or reoxygenation on adhesion molecule expression and natural killer (NK) cell adhesion in an in vitro model of I/R. METHODS: Human aortic endothelial cells (HAEC) were stimulated in vitro for 8h with TNF-alpha (1000 U/ml) and exposed to hypoxia (1% O(2)), reoxygenation (21% O(2)) or different combinations thereof. Cell surface expression of ICAM-1, VCAM-1 and E-/P-selectin on HAEC was analyzed by flow cytometry, and culture supernatants were tested for soluble adhesion molecules by ELISA. Rolling adhesion of NK cells on HAEC was determined using a rotating assay. RESULTS: Untreated HAEC constitutively expressed ICAM-1 on their surface but neither expressed E-/P-selectin, VCAM-1, nor shedded soluble adhesion molecules. Exposure of HAEC to hypoxia or hypoxia and reoxygenation did not upregulate cell surface expression or shedding of adhesion molecules. In contrast, TNF-alpha significantly upregulated cell surface expression of ICAM-1, VCAM-1, and E-/P-selectin and led to the shedding of ICAM-1 and E-selectin. Combined treatment of HAEC with TNF-alpha, hypoxia and reoxygenation reduced E-/P-selectin surface expression and enhanced E-selectin shedding, but did not further influence ICAM-1 and VCAM-1. Soluble VCAM-1 was not detected. NK cell adhesion on HAEC increased 4-fold after TNF-alpha stimulation, but was not affected by hypoxia or hypoxia and reoxygenation. CONCLUSIONS: Both the expression of endothelial adhesion molecules and rolling NK cell adhesion was upregulated by TNF-alpha but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injury.

Human umbilical cord cells: a new cell source for cardiovascular tissue engineering.

BACKGROUND: Tissue engineering of viable, autologous cardiovascular constructs with the potential to grow, repair, and remodel represents a promising new concept for cardiac surgery, especially for pediatric patients. Currently, vascular myofibroblast cells (VC) represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments before scaffold seeding, a technique that may not be preferable, particularly in pediatric patients. In this study, we investigated the feasibility of using umbilical cord cells (UCC) as an alternative autologous cell source for cardiovascular tissue engineering. METHODS: Human UCC were isolated from umbilical cord segments and expanded in culture. The cells were sequentially seeded on bioabsorbable copolymer patches (n = 5) and grown in vitro in laminar flow for 14 days. The UCC were characterized by flow cytometry (FACS), histology, immunohistochemistry, and proliferation assays and were compared to saphenous vein-derived VC. Morphologic analysis of the UCC-seeded copolymer patches included histology and both transmission and scanning electron microscopy. Characterization of the extracellular matrix was performed by immunohistochemistry and quantitative extracellular matrix protein assays. The tissue-engineered UCC patches were biomechanically evaluated using uniaxial stress testing and were compared to native tissue. RESULTS: We found that isolated UCC show a fibroblast-like morphology and superior cell growth compared to VC. Phenotype analysis revealed positive signals for alpha-smooth muscle actin (ASMA), desmin, and vimentin. Histology and immunohistochemistry of seeded polymers showed layered tissue formation containing collagen I, III, and glycoaminoglycans. Transmission electron microscopy showed viable myofibroblasts and the deposition of collagen fibrils. A confluent tissue surface was observed during scanning electron microscopy. Glycoaminoglycan content did not reach values of native tissue, whereas cell content was increased. The biomechanical properties of the tissue-engineered constructs approached native tissue values. CONCLUSIONS: Tissue engineering of cardiovascular constructs using UCC is feasible in an in vitro environment. The UCC demonstrated excellent growth properties and tissue formation with mechanical properties approaching native tissue. It appears that UCC represent a promising alternative autologous cell source for cardiovascular tissue engineering, offering the additional benefits of using juvenile cells and avoiding the invasive harvesting of intact vascular structures.

Living, autologous pulmonary artery conduits tissue engineered from human umbilical cord cells.

BACKGROUND: Tissue engineering represents a promising approach to in vitro creation of living, autologous replacements with the potential to grow, repair, and remodel. Particularly in a congenital operation, there is a substantial need for such implantation materials. We previously demonstrated fabrication of completely autologous, functional heart valves on the basis of peripheral vascular cells. Presently the feasibility of creating pulmonary artery conduits from human umbilical cord cells was investigated. METHODS: Human umbilical cord cells were harvested and expanded in culture. Pulmonary conduits fabricated from rapidly bioabsorbable polymers were seeded with human umbilical cord cells and grown in vitro in a pulse duplicator bioreactor. Morphologic characterization of the generated neo-tissues included histology, transmission, and scanning electron microscopy. Characterization of extracellular matrix was comprised of immunohistochemistry. Extracellular matrix protein content and cell proliferation were quantified by biochemical assays. Biomechanical testing was performed using stress-strain and burst-stress tests. RESULTS: Histology of the conduits revealed viable, layered tissue and extracellular matrix formation with glycosaminoglycans and collagens I and III. Cells stained positive for vimentin and alpha-smooth muscle actin. Scanning electron microscopy showed confluent, homogenous tissue surfaces. Transmission electron microscopy demonstrated elements typical of viable myofibroblasts, such as collagen, fibrils, and elastin. Extracellular matrix proteins were significantly lower compared with native tissue; the cell content was increased. The mechanical strength of the pulsed constructs was comparable with native tissue; the static controls were significantly weaker. CONCLUSIONS: In vitro fabrication of tissue-engineered human pulmonary conduits was feasible utilizing human umbilical cord cells and a biomimetic culture environment. Morphologic and mechanical features approximated human pulmonary artery. Human umbilical cord cells demonstrated excellent growth properties representing a new, readily available cell source for tissue engineering without necessitating the sacrifice of intact vascular donor structures.

A new source for cardiovascular tissue engineering: human bone marrow stromal cells.

OBJECTIVE: Vascular-derived cells represent an established cell source for tissue engineering of cardiovascular constructs. Previously, cell isolation was performed by harvesting of vascular structures prior to scaffold seeding. Marrow stromal cells (MSC) demonstrate the ability to differentiate into multiple mesenchymal cell lineages and would offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. We studied the feasibility of using MSC as an alternative cell source for cardiovascular tissue engineering. METHODS: Human MSC were isolated from bone marrow and expanded in culture. Subsequently MSC were seeded on bioabsorbable polymers and grown in vitro. Cultivated cells and seeded polymers were studied for cell characterization and tissue formation including extracellular matrix production. Applied methods comprised flow cytometry, histology, immunohistochemistry, transmission (TEM) and scanning electron microscopy (SEM), and biochemical assays. RESULTS: Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for alpha-smooth muscle actin and vimentin. Histology and SEM of seeded polymers showed layered tissue formation. TEM demonstrated formation of extracellular matrix with deposition of collagen fibrils. Matrix protein analysis showed production of collagen I and III. In comparison to vascular-derived cell constructs quantitative analysis demonstrated comparable amounts of extracellular matrix proteins in the tissue engineered constructs. CONCLUSIONS: Isolated MSC demonstrated myofibroblast-like characteristics. Tissue formation on bioabsorbable scaffolds was feasible with extracellular matrix production comparable to vascular-cell derived tissue engineered constructs. It appears that MSC represent a promising cell source for cardiovascular tissue engineering.