Bladder Regeneration Using a Polycaprolactone Scaffold with a Gradient Structure and Growth Factors in a Partially Cystectomized Rat Model.

BACKGROUND: Tissue engineering can be used for bladder augmentation. However, conventional scaffolds result in fibrosis and graft shrinkage. This study applied an alternative polycaprolactone (PCL)-based scaffold (diameter = 5 mm) with a noble gradient structure and growth factors (GFs) (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) to enhance bladder tissue regeneration in a rat model. METHODS: Partially excised urinary bladders of 5-week-old male Slc:SD rats were reconstructed with the scaffold (scaffold group) or the scaffold combined with GFs (GF group) and compared with sham-operated (control group) and untreated rats (partial cystectomy group). Evaluations of bladder volume, histology, immunohistochemistry (IHC), and molecular markers were performed at 4, 8, and 12 weeks after operation. RESULTS: The bladder volumes of the scaffold and GF group recovered to the normal range, and those of the GF group showed more enhanced augmentation. Histological evaluations revealed that the GF group showed more organized urothelial lining, dense extracellular matrix, frequent angiogenesis, and enhanced smooth muscle bundle regeneration than the scaffold group. IHC for α-smooth muscle actin, pan-cytokeratin, α-bungarotoxin, and CD8 revealed that the GF group showed high formation of smooth muscle, blood vessel, urothelium, neuromuscular junction and low immunogenicity. Concordantly, real-time polymerase chain reaction experiments revealed that the GF group showed a higher expression of transcripts associated with smooth muscle and urothelial differentiation. In a 6-month in vivo safety analysis, the GF group showed normal histology. CONCLUSION: This study showed that a PCL scaffold with a gradient structure incorporating GFs improved bladder regeneration functionally and histologically.

Activated carbon fiber filters could reduce the risk of surgical smoke exposure during laparoscopic surgery: application of volatile organic compounds.

BACKGROUND: Generation of smoke is inevitable during surgical procedures. Some volatile organic compounds (VOCs) in surgical smoke are known to be strong carcinogens. We used a prototype of a multi-layered complex filter in an attempt to eliminate VOCs. METHODS: From June 2015 to July 2015, 20 patients underwent transperitoneal laparoscopic nephrectomy for renal cell carcinoma. Smoke (pre-filter) was collected 20 min after the electrocautery device was first used during the surgery, by the direct collection method, with a 5-L Tedlar® gas-sampling bag. Twenty and 120 min after the filter was applied, smoke (post-filter) was again collected using the same method. The sample was analyzed by gas chromatography and mass spectrography. The cancer risk and hazard quotient were analyzed based on US Environmental Protection Agency guidelines. RESULTS: Twenty patients with a median age of 54.5 (30-80) years were enrolled in the study. Eighteen VOCs were detected using the Japanese indoor air standards mix analysis. The total elimination rate of the VOCs was 86.49 ± 2.83%. The post-filter (120 min) cancer risk (mean ± standard deviation) reduced to a negligible level for benzene, ethylbenzene, and styrene except 1,2-dichloroethane. The post-filter (120 min) hazard quotient for each compound decreased to levels posing a negligible risk for acetone, hexane, benzene, toluene, p-xylene, o-xylene, and styrene. CONCLUSION: Strong carcinogens, such as 1,2-dichloroethane, benzene, and ethylbenzene, were eliminated by more than 85% by using this activated carbon fiber filter and the risks from these compounds decreased to an almost negligible level. We suggest using every measure, including these filters, to protect the health of operating room personnel.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Human Urine-derived Stem Cells Seeded Surface Modified Composite Scaffold Grafts for Bladder Reconstruction in a Rat Model.

We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold(heparin-bFGF) group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 µL/cm H2O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 µL/cm H2O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 µL/cm H2O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 µL/cm H2O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 µL/cm H2O for the scaffold(heparin-bFGF) group, respectively). In histological and immunohistochemical analysis, the USC-scaffold(heparin-bFGF) group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold(heparin-bFGF) exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.

Local and systemic effects of a tissue engineered neobladder in a canine cystoplasty model.

PURPOSE: Tissue engineered bladders are emerging as a potential treatment option in urological surgery. Although successful neobladders can be engineered with autologous cells on a biodegradable polymer scaffold, studies of the local and systemic effects on host tissue have not been extensively pursued. We examined such effects at predetermined time points after implantation of tissue engineered neobladders in a canine cystoplasty model. MATERIALS AND METHODS: Eight dogs underwent trigone sparing cystectomies. Six dogs (experimental group) received bladder augmentation with tissue engineered constructs produced from autologous urothelial and smooth muscle cells on a prefabricated polyglycolic acid polymer scaffold. Two beagles (control group) received bladder augmentation with the polyglycolic acid scaffold alone. Serial urodynamic studies, cystograms, peripheral blood smears, urinalysis, serum chemistry, complete blood count and electrolytes were done at predetermined time points postoperatively. The bladder, and local and distant organs were retrieved 6 months after surgery for analysis. RESULTS: Capacity and compliance of the engineered bladders reached normal levels by 6 months. Engineered bladders showed tissue composition similar to that of normal bladders. Infiltration of inflammatory cells was minimal and subsided with time. An increase in the total systemic leukocyte count and in bacteriuria was evident initially at 1 week but they returned to normal levels by 1 month postoperatively. Other systemic parameters remained within normal levels at all time points. There was no evidence of abnormal findings in local or distant organs. CONCLUSIONS: Implantation of polymer molds seeded with autologous bladder cells did not show significant local or systemic toxicity in a canine model. This study suggests that such engineered neobladders are safe and effective for reconstructive surgery.

Modified Magnesium Hydroxide Nanoparticles Inhibit the Inflammatory Response to Biodegradable Poly(lactide- co-glycolide) Implants.

Biodegradable polymers have been extensively used in biomedical applications, ranging from regenerative medicine to medical devices. However, the acidic byproducts resulting from degradation can generate vigorous inflammatory reactions, often leading to clinical failure. We present an approach to prevent acid-induced inflammatory responses associated with biodegradable polymers, here poly(lactide- co-glycolide), by using oligo(lactide)-grafted magnesium hydroxide (Mg(OH)2) nanoparticles, which neutralize the acidic environment. In particular, we demonstrated that incorporating the modified Mg(OH)2 nanoparticles within degradable coatings on drug-eluting arterial stents efficiently attenuates the inflammatory response and in-stent intimal thickening by more than 97 and 60%, respectively, in the porcine coronary artery, compared with that of drug-eluting stent control. We also observed that decreased inflammation allows better reconstruction of mouse renal glomeruli in a kidney tissue regeneration model. Such modified Mg(OH)2 nanoparticles may be useful to extend the applicability and improve clinical success of biodegradable devices used in various biomedical fields.

Hypoxia Enhances Cell Properties of Human Mesenchymal Stem Cells.

Atmospheric (in vitro) oxygen pressure is around 150 mm Hg (20% O2), whereas physiologic (in vivo) oxygen pressure ranges between 5 and 50 mm Hg (0.7-7% O2). The normoxic environment in cell culture does not refer to a physiological stem cell niche. The aim of this study is to investigate the effect of oxygen concentration on cell properties of human mesenchymal stem cells (MSCs). We analyzed cell proliferation rate, senescence, immunophenotype, stemness gene expression and differentiation potency with human urine stem cells (USCs), dental pulp stem cells (DPSCs), amniotic fluid stem cells (AFSCs), and bone marrow stromal cells (BMSCs). USCs, DPSCs, AFSCs and BMSCs were cultured under either 5% O2 hypoxic or 20% O2 normoxic conditions for 5 days. MSCs cultured under hypoxia showed significantly increased proliferation rate and high percentage of S-phase cells, compared to normoxic condition. In real-time PCR assay, the cells cultured under hypoxia expressed higher level of Oct4, C-Myc, Nanog, Nestin and HIF-1α. In immunophenotype analysis, MSCs cultured under hypoxia maintained higher level of the MSC surface markers, and lower hematopoietic markers. Senescence was inhibited under hypoxia. Hypoxia enhances osteogenic differentiation efficiency compared to normoxia. Hypoxia showed enhanced cell proliferation rate, retention of stem cell properties, inhibition of senescence, and increased differentiation ability compared to normoxia.

Biomimetic Porous PLGA Scaffolds Incorporating Decellularized Extracellular Matrix for Kidney Tissue Regeneration.

Chronic kidney disease is now recognized as a major health problem, but current therapies including dialysis and renal replacement have many limitations. Consequently, biodegradable scaffolds to help repairing injured tissue are emerging as a promising approach in the field of kidney tissue engineering. Poly(lactic-co-glycolic acid) (PLGA) is a useful biomedical material, but its insufficient biocompatibility caused a reduction in cell behavior and function. In this work, we developed the kidney-derived extracellular matrix (ECM) incorporated PLGA scaffolds as a cell supporting material for kidney tissue regeneration. Biomimetic PLGA scaffolds (PLGA/ECM) with different ECM concentrations were prepared by an ice particle leaching method, and their physicochemical and mechanical properties were characterized through various analyses. The proliferation of renal cortical epithelial cells on the PLGA/ECM scaffolds increased with an increase in ECM concentrations (0.2, 1, 5, and 10%) in scaffolds. The PLGA scaffold containing 10% of ECM has been shown to be an effective matrix for the repair and reconstitution of glomerulus and blood vessels in partially nephrectomized mice in vivo, compared with only PLGA control. These results suggest that not only can the tissue-engineering techniques be an effective alternative method for treatment of kidney diseases, but also the ECM incorporated PLGA scaffolds could be promising materials for biomedical applications including tissue engineered scaffolds and biodegradable implants.

Development of a porcine renal extracellular matrix scaffold as a platform for kidney regeneration.

Acellular scaffolds, possessing an intact three-dimensional extracellular matrix (ECM) architecture and biochemical components, are promising for regeneration of complex organs, such as the kidney. We have successfully developed a porcine renal acellular scaffold and analyzed its physical/biochemical characteristics, biocompatibility, and kidney reconstructive potential. Segmented porcine kidney cortexes were treated with either 1% (v/v) Triton X-100 (Triton) or sodium dodecyl sulfate (SDS). Scanning electron microscopy showed both treatments preserved native tissue architecture, including porosity and composition. Swelling behavior was higher in the Triton-treated compared with the SDS-treated scaffold. Maximum compressive strength was lower in the Triton-treated compared with the SDS-treated scaffold. Attenuated total reflective-infrared spectroscopy showed the presence of amide II (-NH) in both scaffolds. Furthermore, richer ECM protein and growth factor contents were observed in the Triton-treated compared with SDS-treated scaffold. Primary human kidney cell adherence, viability, and proliferation were enhanced on the Triton-treated scaffold compared with SDS-treated scaffold. Following murine in vivo implantation, tumorigenecity was absent for both scaffolds after 8 weeks and in the Triton-treated scaffold only, glomeruli-like structure formation and neovascularity were observed. We identified 1% Triton X-100 as a more suitable decellularizing agent for porcine renal ECM scaffolds prior to kidney regeneration.

Characterization of a novel composite scaffold consisting of acellular bladder submucosa matrix, polycaprolactone and Pluronic F127 as a substance for bladder reconstruction.

The bladder is an organ susceptible to a variety of congenital anomalies, injuries and disorders. To address the clinical limitations of existing scaffolds, we fabricated a novel scaffold that can be applied to morphological and functional bladder reconstruction. As a first step to prove the benefit of the scaffold, intensive in vitro and in vivo analyses were conducted. The novel composite scaffold was fabricated using polycaprolactone/Pluronic F127 (PCL/F127) and variable proportions (1, 3, 5 and 10wt.%) of porcine acellular bladder submucosa matrix (BSM). Physicochemical properties and biocompatibilities of the scaffolds were characterized. For cell-mediated analysis, upper-urinary-tract-derived urine stem cells were used. Observations of tensile strength, modulus, porosity, cell adhesion, viability and proliferation characteristics of scaffolds indicated that the optimum proportion of BSM in the composite scaffolds was 3 or 5 wt.%. Based on comparison of 3 and 5 wt.% BSM/PCL/F127 scaffolds with respect to degradability, hydrophilicity, surface properties and functional group presence, the 3 wt.% BSM was chosen for in vivo studies. 8 weeks after kidney-subcapsular implantation of the 3 wt.% BSM/PCL/F127 scaffold, cells remained attached to the surface and there was no evidence of teratomas. A BSM content of 3 wt.% was the optimum proportion for fabrication of the neo scaffold. We predict that the 3 wt.% BSM/PCL/F127 composite scaffold could act as an ideal matrix after cystectomy based on its favorable physicochemical properties and biocompatibilities.