Instytut Podstawowych Problemów Techniki

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BACKGROUND: Electrospun nanofibers have widespread putative applications in the field of regenerative medicine and tissue engineering. When compared to naturally occurring collagen matrices, electrospun nanofiber scaffolds have two distinct advantages: they do not induce a foreign body reaction and they are not at risk for biological contamination. However, the exact substrate, structure, and production methods have yet to be defined. MATERIAL AND METHODS: In the current study, tubular-shaped poly(L-lactide-co-caprolactone) (PLCL) constructs produced using electrospinning technology were evaluated for their potential application in the field of tissue regeneration in two separate anatomic locations: the skin and the abdomen. The constructs were designed to have an internal diameter of 3 mm and thickness of 200 μm. Using a rodent model, 20 PLCL tubular constructs were surgically implanted in the abdominal cavity and subcutaneously. The constructs were then evaluated histologically using electron microscopy at 6 weeks post-implantation. RESULTS: Histological evaluation and analysis using scanning electron microscopy showed that pure scaffolds by themselves were able to induce angiogenesis after implantation in the rat model. Vascularization was observed in both tested groups; however, better results were obtained after intraperitoneal implantation. Formation of more and larger vessels that migrated inside the scaffold was observed after implantation into the peritoneum. In this group no evidence of inflammation and better integration of scaffold with host tissue were noticed. Subcutaneous implantation resulted in more fibrotic reaction, and differences in cell morphology were also observed between the two tested groups. CONCLUSIONS: This study provides a standardized evaluation of a PLCL conduit structure in two different anatomic locations, demonstrating the excellent ability of the structure to achieve vascularization. Functional, histological, and mechanical data clearly indicate prospective clinical utilization of PLCL in critical size defect regeneration.

Słowa kluczowe:

Polymers, Regenerative medicine, Tissue Engineering, Tissue Scaffolds, Urinary Diversion

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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-E-caprolactone) (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.

Słowa kluczowe:

Bladder, Smooth muscles, Muscle regeneration, Bionanotechnology, Renal system, Urothelium, Urology, Nanomaterials

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Streszczenie:

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 3×106 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+ 3×106ADSCs; (II) SIS+ 3×106ADSCs; (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.

Słowa kluczowe:

urinary bladder regeneration, electrospinning

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Streszczenie:

The aim of this study was to compare two different acellular scaffolds: natural and synthetic, for urinary conduit construction and ureter segment reconstruction. Acellular aortic arch (AAM) and poly(L-lactide-co-caprolactone) (PLCL) were used in 24 rats for ureter reconstruction in both tested groups. Follow-up period was 4 weeks. Intravenous pyelography, histological and immunohistochemical analysis were performed. All animals survived surgical procedures. Patent uretero-conduit junction was observed only in one case using PLCL. In case of ureter segment reconstruction ureters were patent in one case using AAM and in four cases using PLCL scaffolds. Regeneration of urothelium layer and focal regeneration of smooth muscle layer was observed on both tested scaffolds. Obtained results indicates that synthetic acellular PLCL scaffolds showed better properties for ureter reconstruction than naturally derived acellular aortic arch.

Słowa kluczowe:

Ureter, Muscle regeneration, Kidneys, Collagens, Urine, Surgical and invasive medical procedures, Smooth muscles, Inflammation

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Streszczenie:

Large ureter damages are difficult to reconstruct. Current techniques are complicated, difficult to perform, and often associated with failures. The ureter has never been regenerated thus far. Therefore the use of tissue engineering techniques for ureter reconstruction and regeneration seems to be a promising way to resolve these problems. For proper ureter regeneration the following problems must be considered: the physiological aspects of the tissue, the type and shape of the scaffold, the type of cells, and the specific environment (urine). 
This review presents tissue engineering achievements in the field of ureter regeneration focusing on the scaffold, the cells, and ureter healing.

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Introduction.
Tissue engineering and biomaterials science currently offer the technology needed to replace the urinary tract wall. This review addresses current achievements and barriers for the regeneration of the urinary bladder based on tissue engineering methods. Materials and methods. Medline was search for urinary bladder tissue engineering regenerative medicine and stem cells.

Results.
Numerous studies to develop a substitute for the native urinary bladder wall using the tissue engineering approach are ongoing. Stem cells combined with biomaterials open new treatment methods, including even de novo urinary bladder construction. However, there are still many issues before advances in tissue engineering can be introduced for clinical application.

Conclusions.
Before tissue engineering techniques could be recognize as effective and safe for patients, more research studies performed on large animal models and with long follow–up are needed to carry on in the future.

Słowa kluczowe:

stem cells, bladder regeneration, tissue engineering

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Słowa kluczowe:

electrospinning, microfibers, nanofibers, in vivo, animal model

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Streszczenie:

We used the principles of electrospinning to produce materials for applications in regenerative medicine of urinary bladder wall, ureter, wound dressing and potential applications in cancer therapy. Our research is based on biodegradable polymers produced by ring-opening polymerization. Scaffolds of poly(L-lactide-co-caprolactone) (PLCL) gradually degrade leaving no artificial material behind to be replaced by natural extracellular collagen matrix. We formed flat membranes of micro- and nanofibers to carry out regeneration of urinary bladder wall as animal model of cancer treatment. Grafts were tested for biocompatibility and aimed for guided cell growth, yet we were unsuccessful in mechanical compliance of nanomaterial and reconstructed tissue. We tested tubular scaffolds made of nanofibers aimed for ureter tissue engineering. We found stem cells seeding unnecessary. The results of nanomaterial implantation on animal model were better than for collagen matrices. Animal model was also tested for use of nanofibers of human serum albumin as wound dressing. The native structure of the protein was retained to maintain its anti-adhesive properties, despite poor mechanical characteristics. Nanomaterial caused no inflammation and was resorbed during 16 days. Last application of presented materials was targeted drug delivery system made of PLCL nanofibers. Release of anticancer drug complexed with nanoparticles is to be triggered by tumor cells. Such nanomaterial is potential drug delivery system. Acknowledgements: The authors wishes to thank for the cooperation: T. Chmielewski, P. Nakielski, K. Zembrzycki, G. Mikulowski and prof. T. A. Kowalewski from IPPT PAN. The project was partially supported by the National Centre for Research and Development. Grant No. STRATEGMED1/235368/8/NCBR/2014.

Słowa kluczowe:

electrospinning, nanofibers, regenerative medicine, wound dressing, urology, biodegradable polymers, animal model.

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