New amniotic membrane based biocomposite for future application in reconstructive urology

OBJECTIVE Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model. MATERIAL AND METHODS Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-Ecaprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material. RESULTS Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson’s trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young’s elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process. CONCLUSIONS Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.http://www.plosone.orgam2016Internal Medicin

Ultrastructural Changes in Mitochondria in Patients with Dilated Cardiomyopathy and Parvovirus B19 Detected in Heart Tissue without Myocarditis

Understanding the meaning of parvovirus B19 (PB19V) in an etiology of dilated cardiomyopathy (DCM) is difficult. Viruses change the dynamics of the mitochondria by interfering with the mitochondrial process/function, causing the alteration of mitochondrial morphology. In this study, the ultrastructural changes in the mitochondria in endomyocardial biopsy (EMB) samples from patients with DCM and PB19V were determined. Methods: The PB19V evaluation was performed in EMB specimens by real-time PCR in 20 patients (age: 28 ± 6 years). The biopsy specimens were examined by histo- and immunohistochemistry to detect the inflammatory response. The ultrastructural features of the mitochondria were evaluated by electron microscopy. Results: The presence of PB19V in the heart tissue without the presence of inflammatory process, defined according to Dallas and immunohistochemical criteria, was associated with ultrastructural changes in the mitochondria. Distinctive ultrastructural pathologies were indicated, such as the presence of mitochondria in the vicinity of the expanded sarcoplasmic reticulum with amorphous material, blurred structure of mitochondria, interrupted outer mitochondrial membrane and mitophagy. Conclusions: Extending diagnostics with ultrastructural analysis of biopsy samples provides new knowledge of the changes associated with the presence of PB19V in the heart tissue. The observed changes can be a basis for searching for the damage mechanisms, as well as for new therapeutic solutions

Nano–bio interactions of upconversion nanoparticles at subcellular level: biodistribution and cytotoxicity

Background: Modern medicine requires intensive research to find new diagnostic and therapeutic solutions. Recently, upconverting nanoparticles (UCNPs) doped with lanthanide ions have attracted significant attention. Methods: The efficient internalization of UCNPs by cells was confirmed, and their precise cellular localization was determined by electron microscopy and confocal studies. Results: UCNPs colocalized only with specific organelles, such as early endosomes, late endosomes and lysosomes. Furthermore, experiments with chemical inhibitors confirmed the involvement of endocytosis in UCNPs internalization and helped select several mechanisms involved in internalization. Exposure to selected UCNPs concentrations did not show significant cytotoxicity, induction of oxidative stress or ultrastructural changes in cells. Conclusion: This study suggests that UCNPs offer new diagnostic options for biomedical infrared imaging.</p

Does the Mesenchymal Stem Cell Source Influence Smooth Muscle Regeneration in Tissue-Engineered Urinary Bladders?

A variety of tissue engineering techniques utilizing different cells and biomaterials are currently being explored to construct urinary bladder walls de novo, but so far no approach is clearly superior. The aim of this study was to determine whether mesenchymal stem cells (MSCs) isolated from different sources, (bone marrow [BM-MSCs] and adipose tissue [ADSCs]), differ in their potential to regenerate smooth muscles in tissue-engineered urinary bladders and to determine an optimal number of MSCs for urinary bladder smooth muscle regeneration. Forty-eight rats underwent hemicystectomy and bladder augmentation with approximately 0.8 cm graft. In the first and second groups, urinary bladders were reconstructed with small intestinal submucosa (SIS) seeded with 10 × 10 or 4 × 10 ADSCs/cm , respectively. In the third and fourth groups, urinary bladders were augmented with SIS seeded with 10 × 10 or 4 × 10 BM-MSCs/cm , respectively. In the fifth group, urinary bladders were augmented with SIS without cells. The sixth group (control) was left intact. Smooth muscle regeneration was evaluated by real-time polymerase chain reaction (RT-PCR) and histological examinations. Histologically, there were no significant differences between urinary bladders augmented with ADSCs and BM-MSCs, but there was a marked increase in smooth muscle formation in bladders augmented with grafts seeded with MSCs in higher density (10 × 10 /cm ) compared to lower density (4 × 10 /cm ). Molecular analysis revealed that bladders reconstructed with ADSC-seeded grafts expressed higher levels of smooth muscle myosin heavy chain, caldesmon, and vinculin. Bladders augmented with unseeded SIS were fibrotic and devoid of smooth muscles. ADSCs and BM-MSCs have comparable smooth muscle regenerative potential, but the number of MSCs used for graft preparation significantly affects the smooth muscle content in tissue-engineered urinary bladders

Is the poly (L- lactide- co- caprolactone) nanofibrous membrane suitable for urinary bladder regeneration?

The purpose of this study was to compare: a new five-layered poly (L-lactide-co-caprolactone) (PLC) membrane and small intestinal submucosa (SIS) as a control in rat urinary bladder wall regeneration. The five-layered poly (L-lactide-co-caprolactone) membrane was prepared by an electrospinning process. Adipose tissue was harvested from five 8-week old male Wistar rats. Adipose derived stem cells (ADSCs) were seeded in a density of 3×10(6) cells/cm2 onto PLC membrane and SIS scaffolds, and cultured for 5-7 days in the stem cell culture medium. Twenty male Wistar rats were randomly divided into five equal groups. Augmentation cystoplasty was performed in a previously created dome defect. Groups: (I) PLC+ 3×10(6)ADSCs; (II) SIS+ 3×10(6)ADSCs; (III) PLC; (IV) SIS; (V) control. Cystography was performed after three months. The reconstructed urinary bladders were evaluated in H&E and Masson's trichrome staining. Regeneration of all components of the normal urinary bladder wall was observed in bladders augmented with cell-seeded SIS matrices. The urinary bladders augmented with SIS matrices without cells showed fibrosis and graft contraction. Bladder augmentation with the PLC membrane led to numerous undesirable events including: bladder wall perforation, fistula or diverticula formation, and incorporation of the reconstructed wall into the bladder lumen. The new five-layered poly (L-lactide-co-caprolactone) membrane possesses poorer potential for regenerating the urinary bladder wall compared with SIS scaffold

Preparation and structure of biocomposite.

<p>(A) The pieces of Am (black arrows) placed onto a sheet of PLCL nanofibers. A drum is used as a target during the nanofiber production process. SEM images are displayed in B-D. (B) A cross-section image of the biocomposite material. The biocomposite material is 389 um thick with an inner cavity containing the Am. (C) Visible drops of glycerin used for Am preservation are observed on surface of PLCL nanofibers (white arrows). (D) Two pieces of delaminated biocomposite material. The borders between consecutive sheets of nanofibers (*) are clearly visible with Am inside.</p

The mechanical evaluation of reconstructed bladder wall based on Young’s elastic modulus.

<p>Young’s modulus of intact and reconstructed bladder walls were compared to the digitally estimated content of smooth muscle content based on TRI staining average. Additionally, to reflect changes in the remodeling of the augmented bladder wall, Young’s modulus of Am and PLCL are presented. The presented values of smooth muscle content was rounded up.</p

Amniotic membrane extract cytotoxicity measurement using real-time cell analysis.

<p>Each result was presented as mean from 5 independent experiment with SD bars. No statistically significant differences in cell viability were observed between AME treated and control cells (p>0.05) after 72 hours.</p

Urinary bladder augmented with biocomposite.

<p>(A) Biocomposite material scaffold prepared for the suture procedure. (B) Urinary bladder after the augmentation procedure. Single fixing sutures are visible (black arrows). The optimal compliance of the biocomposite material scaffold allowed for bladder filling shortly after the surgical procedure. (C) Resected reconstructed bladder 3 months after augmentation. The regenerated bladder wall (blue and cyan line was well integrated with the native bladder wall (black line). The borderline between the intact part of the bladder and the reconstructed one was indistinct and without scar formation (blue line). The upper surface of regenerated bladder wall (cyan line) was covered with adipose tissue forming a vascular pedicle (red line) derived from the omentum majus (green line). The bladder neck (yellow line) can be observed with adjacent fragments of seminal vesicles (white line).</p