Novel electrospun polyurethane grafts for vascular access in rats.

OBJECTIVES: The purpose of this study is to develop a new and improved polyurethane (PU) graft using electrospinning and chemical modifications for hemodialysis patients, which will replace the current standard, polytetrafluoroethylene (PTFE) graft. The chemical modifications aim to improve hemocompatibility and reduce thrombogenicity and neointimal hyperplasia. METHOD: The study population was randomized and divided equally into four groups; one control group received a PTFE graft, and three treatment groups received three different types of polyurethane grafts. Two duplex measurements were performed directly on the graft on the same locations, followed by a histologic examination. RESULTS: In the first few days after the implantation animals lost some weight, it took a week to recover to pre-surgical weight. Throughout the 28 days, there was no significant difference between animals in wound, activity, and the general appearance. PTFE and PU A groups have lower compliance or reduced graft diameter due to neointimal hyperplasia development on Doppler interrogation. The histological analysis showed limited neointimal hyperplasia development and no excessive inflammatory response to any of the grafts. CONCLUSION: This study demonstrates that animals with polyurethane grafts show better blood flow because the developed NIH was inconspicuous, as indicated by the different velocity measure than controls on Duplex and minimal NIH development microscopically.

The Effect of Geometric Graft Modification on Arteriovenous Graft Patency in Haemodialysis Patients: A Systematic Review and Meta-Analysis.

OBJECTIVE: Arteriovenous grafts (AVGs) are the second best option for haemodialysis access when native arteriovenous fistulae placement is not possible, because they have a lower patency owing to neointimal hyperplasia at the venous anastomosis. This review aimed to evaluate the effect of geometric graft modification to the graft-vein interface on AVG patency. DATA SOURCES: The MEDLINE and Embase (OvidSP) databases were systematically searched for relevant studies analysing the effect of geometrically modified AVGs on graft patency and stenosis formation (last search July 2019). REVIEW METHODS: Data regarding AVG type, patency, and graft outlet stenosis was extracted for further evaluation. Data were pooled in a random effects model to estimate the relative risk of graft occlusion within one year. Follow up, number of patients, and relevant patient characteristics were extracted for the quality assessment of the included studies using Newcastle-Ottawa Scale and Cochrane Risk of Bias Tool. The quality of the evidence was determined according to the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) system. RESULTS: Search strategies produced 2772 hits, of which eight articles met predetermined inclusion criteria. Overall, the included articles had low to moderate risk of bias. In total, 414 expanded polytetrafluoroethylene AVGs (232 geometrically modified and 182 standard) were analysed, comprising two modified AVG types: a prosthetic cuff design (Venaflo®) and grafts with a Tyrell vein patch. Overall, modified grafts did not show a statistically significantly higher one year primary (relative risk [RR] 0.86, 95% confidence interval [CI] 95% 0.64-1.16; GRADE: "low to very low") or secondary patency (RR 0.57, 95% CI 0.32-1.02; GRADE: "low to very low") when compared with standard AVGs. Analysis of prosthetic cuffed grafts (112 patients) separately demonstrated a statistically significantly higher one year primary (RR 0.75, 95% CI 0.61-0.91) and one year secondary patency (RR 0.47, 95% CI 0.30-0.75) compared with standard grafts (92 patients). The results on stenosis formation were inconclusive and inadmissible to quantitative analyses. CONCLUSION: The meta-analysis showed that a prosthetic cuff design significantly improves AVG patency, while a venous cuff does not. Although the heterogeneity and low number of available studies limit the strength of the results, this review shows the potential of grafts with geometric modification to the graft-vein anastomosis and should stimulate further clinical and fundamental research on improving graft geometry to improve graft patency.

A salt-based method to adapt stiffness and biodegradability of porous collagen scaffolds.

Type I collagen scaffolds for tissue reconstruction often have impaired mechanical characteristics such as limited stiffness and lack of strength. In this study, a new technique is presented to fine-tune stiffness and biodegradability of collagen scaffolds by treatment with concentrated salt solutions. Collagen scaffolds were prepared by a casting, freezing and lyophilization process. Scaffolds were treated with 90% saturated salt solutions, the salts taken from the Hofmeister series, followed by chemical crosslinking. Treatment with salts consisting of a divalent cation in combination with a monovalent anion, e.g. CaCl2, resulted in fast shrinkage of the scaffolds up to approximately 10% of the original surface area. Effective salts were mostly at the chaotropic end of the Hofmeister series. Shrunken scaffolds were more than 10 times stiffer than non-shrunken control scaffolds, and displayed reduced pore sizes and swollen, less organized collagen fibrils. The effect could be pinpointed to the level of individual collagen molecules and indicates the shrinking effect to be driven by disruption of stabilizing hydrogen bonds within the triple helix. No calcium deposits remained in CaCl2 treated scaffolds. Subcutaneous implantation in rats showed similar biocompatibility compared to H2O and NaCl treated scaffolds, but reduced cellular influx and increased structural integrity without signs of major degradation after 3 months. In conclusion, high concentrations of chaotropic salts can be used to adjust the mechanical characteristics of collagen scaffolds without affecting biocompatibility. This technique may be used in regenerative medicine to stiffen collagen scaffolds to better comply with the surrounding tissues, but may also be applied for e.g. slow release drug delivery systems.

The Impact of γ-Irradiation and EtO Degassing on Tissue Remodeling of Collagen-based Hybrid Tubular Templates.

Clinical implementation of novel products for tissue engineering and regenerative medicine requires a validated sterilization method. In this study, we investigated the effect of γ-irradiation and EtO degassing on material characteristics in vitro and the effect on template remodeling of hybrid tubular constructs in a large animal model. Hybrid tubular templates were prepared from type I collagen and Vicryl polymers and sterilized by 25 kGray of γ-irradiation or EtO degassing. The in vitro characteristics were extensively studied, including tensile strength analysis and degradation studies. For in vivo evaluation, constructs were subcutaneously implanted in goats for 1 month to form vascularized neo-tissue. Macroscopic and microscopic appearances of the γ- and EtO-sterilized constructs slightly differed due to additional processing required for the COL-Vicryl-EtO constructs. Regardless of the sterilization method, incubation in urine resulted in fast degradation of the Vicryl polymer and decreased strength (<7 days). Incubation in SBF was less invasive, and strength was maintained for at least 14 days. The difference between the two sterilization methods was otherwise limited. In contrast, subcutaneous implantation showed that the effect of sterilization was considerable. A well-vascularized tube was formed in both cases, but the γ-irradiated construct showed an organized architecture of vasculature and was mechanically more comparable to the native ureter. Moreover, the γ-irradiated construct showed advanced tissue remodeling as shown by enhanced ECM production. This study shows that the effect of sterilization on tissue remodeling cannot be predicted by in vitro analyses alone. Thus, validated sterilization methods should be incorporated early in the development of tissue engineered products, and this requires both in vitro and in vivo analyses.

Ureteral Reconstruction in Goats Using Tissue-Engineered Templates and Subcutaneous Preimplantation.

Repair of long ureteral defects often requires long graft tissues and extensive surgery. This is associated with complications, including a lack of suitable tissue and graft site morbidity. Tissue engineering may provide an attractive alternative to the autologous graft tissues. In this study, ureteral repair using (preimplanted) tubular collagen-Vicryl templates was evaluated in a new goat model. Tubular templates were prepared from tubularized Vicryl meshes and 0.7% type-I collagen (length = 6 cm, inner diameter = 6 mm, wall thickness = 3 mm). In total, twelve goats were used and evaluated after 3 months. Eight goats were implanted with the collagen-Vicryl templates and in four goats the templates were first preimplanted in the subcutis and subsequently used as ureteral graft. Template implantation was successful in 92% of the goats(11/12). During follow-up, 82% of the animals (9/11) survived without signs of discomfort. Two animals were sacrificed prematurely due to kidney perforation by the stent and urine leakage. Two other animals presented with stenosis of the neoureter due to stent migration. After preimplantation, the templates were remodeled mostly to autologous tissue with similar mechanical characteristics as the native ureter. Goats grafted with preimplanted templates presented with predominantly healthy kidneys, whereas the goats grafted with the collagen-Vicryl templates presented with fibrotic and inflamed regions in the kidneys. The use of preimplanted tissue templates showed favorable results compared with direct functional implantation of the templates. Partial remodeling toward autologous tissue and similar mechanical characteristics likely improved the integration in the ureteral tissue. Preimplantation of tissue-engineered templates should therefore be considered when two-stage procedures using a nephrostomy catheter are indicated or when planning allows for additional time to treatment.

Clinical protocol levels are required in laboratory animal surgery when using medical devices: experiences with ureteral replacement surgery in goats.

It is common to test medical devices in large animal studies that are or could also be used in humans. In this short report we describe the use of a ureteral J-stent for the evaluation of biodegradable tubular constructs for tissue reconstruction, and the regeneration of ureters in Saanen goats. Similarly to a previous study in pigs, the ureteral J-stent was blindly inserted until some resistance was met. During evaluation of the goats after three months, perforation of the renal cortex by the stent was observed in four out of seven animals. These results indicated that blind stent placement was not possible in goats. In four new goats, clinical protocols were followed using X-ray and iodinated contrast fluids to visualize the kidney and stent during stent placement. With this adaptation the stents were successfully placed in the kidneys of these four new goats with minimal additional effort. It is likely that other groups in other fields ran into similar problems that could have been avoided by following clinical protocols. Therefore, we would like to stress the importance of following clinical protocols when using medical devices in animals to prevent unnecessary suffering and to reduce the number of animals needed.

Novel tubular constructs for urinary diversion: a biocompatibility study in pigs.

The use of bowel tissue for urinary diversion can be associated with severe complications, and regenerative medicine may circumvent this by providing an engineered conduit. In this study, a novel tubular construct was identified for this purpose. Three constructs (diameter 15 mm) were prepared from type I collagen and either (a) a semi-biodegradable Vypro II polymer (COL-Vypro), (b) a rapidly biodegradable Vicryl polymer (COL-Vicryl) or (c) an additional collagenous layer (COL-DUAL). After freezing, lyophilization and crosslinking, all constructs showed a porous structure with a two-fold higher strength for the polymer-containing constructs. These constructs were connected to full bladder defects of 11 female pigs and evaluated after 1 (n = 4) or 3 months (n = 5). With respect to surgical handling, the polymer-containing constructs were superior. All pigs voided normally without leakage and the survival rate was 82%. For the implanted COL-Vypro constructs (8/9), stone formation was observed. COL-DUAL and COL-Vicryl showed better biocompatibility and only small remnants were found 1 month post-implantation. Histological and immunohistochemical analysis showed the best regeneration for COL-Vicryl with respect to urothelium; muscle pedicles and elastin formation were best developed in the COL-Vicryl constructs. In this study, COL-Vicryl constructs were superior in both biocompatibility and bladder tissue regeneration and have high potential for artificial urinary diversions. Copyright © 2016 John Wiley & Sons, Ltd.

Tubular Constructs as Artificial Urinary Conduits.

PURPOSE: A readily available artificial urinary conduit might be substituted for autologous bowel in standard urinary diversions and minimize bowel associated complications. However, the use of large constructs remains challenging as host cellular ingrowth and/or vascularization is limited. We investigated large, reinforced, collagen based tubular constructs in a urinary diversion porcine model and compared subcutaneously pre-implanted constructs to cell seeded and basic constructs. MATERIALS AND METHODS: Reinforced tubular constructs were prepared from type I collagen and biodegradable Vicryl® meshes through standard freezing, lyophilization and cross-linking techniques. Artificial urinary conduits were created in 17 female Landrace pigs, including 7 with a basic untreated construct, 5 with a construct seeded with autologous urothelial and smooth muscle cells, and 5 with a free graft formed by subcutaneous pre-implantation of a basic construct. All pigs were evaluated after 1 month. RESULTS: The survival rate was 94%. At evaluation 1 basic and 1 cell seeded conduit were occluded. Urinary flow was maintained in all conduits created with pre-implanted constructs. Pre-implantation of the basic construct resulted in a vascularized tissue tube, which could be used as a free graft to create an artificial conduit. The outcome was favorable compared to that of the other conduits. Urinary drainage was better, hydroureteronephrosis was limited and tissue regeneration was improved. CONCLUSIONS: Subcutaneous pre-implantation of a basic reinforced tubular construct resulted in a vascularized autologous tube, which may potentially replace bowel in standard urinary diversions. To our knowledge we introduce a straightforward 2-step procedure to create artificial urinary conduits in a large animal model.

New technique for needle-less implantation of eukaryotic cells.

BACKGROUND AIMS: On review of the use of stem cells in the literature, promissory outcomes for functional organ recovery in many subspecialties in medicine underscore its therapeutic potential. The application of stem cells through the use of a needle can result in additional scar formation, which is undesired for delicate organs. The present work describes the use of a needle-less stem cell injector with the Immediate Drop on Demand Technology (I-DOT) for cell injection in vitro. METHODS: Mesenchymal stromal cells from human bone marrow were labeled with ethynyl-deoxyuridine (EdU) for 2 days and then were re-suspended. With the use of I-DOT, the cells were applied to type 1 collagen matrices or pig bladder tissue specimens with or without mucosa at different levels of energy. The collagen matrices were analyzed after 4 h and 5 days; bladder tissue specimens were analyzed 4 h after cell implantation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT) assay was performed immediately after cell application to the collagen matrices. Histological analysis with the use of frozen sections and immunofluorescence was used to localize EdU-labeled cells. RESULTS: A considerable number of cells were detected by use of the MTT assay for collagen matrices. In the collagen matrix, the mean measured depth immediately after application ranged between 210 µm and 489 µm, 220 µm and 270 µm for entire bladder specimens, and 230 µm and 370 µm for bladder without mucosa. Cells survived for up to 5 days in the collagen matrix in both bladder specimens. CONCLUSIONS: Cells can survive during I-DOT application, which suggests that the I-DOT device may be a potentially suitable technology for needle-less cell application onto tissues.

Tissue engineering in animal models for urinary diversion: a systematic review.

Tissue engineering and regenerative medicine (TERM) approaches may provide alternatives for gastrointestinal tissue in urinary diversion. To continue to clinically translatable studies, TERM alternatives need to be evaluated in (large) controlled and standardized animal studies. Here, we investigated all evidence for the efficacy of tissue engineered constructs in animal models for urinary diversion. Studies investigating this subject were identified through a systematic search of three different databases (PubMed, Embase and Web of Science). From each study, animal characteristics, study characteristics and experimental outcomes for meta-analyses were tabulated. Furthermore, the reporting of items vital for study replication was assessed. The retrieved studies (8 in total) showed extreme heterogeneity in study design, including animal models, biomaterials and type of urinary diversion. All studies were feasibility studies, indicating the novelty of this field. None of the studies included appropriate control groups, i.e. a comparison with the classical treatment using GI tissue. The meta-analysis showed a trend towards successful experimentation in larger animals although no specific animal species could be identified as the most suitable model. Larger animals appear to allow a better translation to the human situation, with respect to anatomy and surgical approaches. It was unclear whether the use of cells benefits the formation of a neo urinary conduit. The reporting of the methodology and data according to standardized guidelines was insufficient and should be improved to increase the value of such publications. In conclusion, animal models in the field of TERM for urinary diversion have probably been chosen for reasons other than their predictive value. Controlled and comparative long term animal studies, with adequate methodological reporting are needed to proceed to clinical translatable studies. This will aid in good quality research with the reduction in the use of animals and an increase in empirical evidence of biomedical research.

Tissue engineering of the bladder--reality or myth? A systematic review.

PURPOSE: We systematically reviewed preclinical studies in the literature to evaluate the potential of tissue engineering of the bladder. Study outcomes were compared to the available clinical evidence to assess the feasibility of tissue engineering for future clinical use. MATERIALS AND METHODS: Preclinical studies of tissue engineering for bladder augmentation were identified through a systematic search of PubMed and Embase™ from January 1, 1980 to January 1, 2014. Primary studies in English were included if bladder reconstruction after partial cystectomy was performed using a tissue engineered biomaterial in any animal species, with cystometric bladder capacity as an outcome measure. Outcomes were compared to clinical studies available at http://www.clinicaltrials.gov and published clinical studies. RESULTS: A total of 28 preclinical studies are included, demonstrating remarkable heterogeneity in study characteristics and design. Studies in which preoperative bladder volumes were compared to postoperative volumes were considered the most clinically relevant (18 studies). Bladder augmentation through tissue engineering resulted in a normal bladder volume in healthy animals, with the influence of a cellular component being negligible. Furthermore, experiments in large animal models (pigs and dogs) approximated the desired bladder volume more accurately than in smaller species. The initial clinical experience was based on seemingly predictive healthy animal models with a promising outcome. Unfortunately these results were not substantiated in all clinical trials, revealing dissimilar outcomes in different clinical/disease backgrounds. Thus, the translational predictability of a model using healthy animals might be questioned. CONCLUSIONS: Through this systematic approach we present an unbiased overview of all published preclinical studies investigating the effect of bladder tissue engineering on cystometric bladder capacity. Preclinical research in healthy animals appears to show the feasibility of bladder augmentation by tissue engineering. However, in view of the disappointing clinical results based on healthy animal models new approaches should also be evaluated in preclinical models using dysfunctional/diseased bladders. This endeavor may aid in the development of clinically applicable tissue engineered bladder augmentation with satisfactory long-term outcome.

Tailored doxorubicin-hyaluronan conjugate as a potent anticancer glyco-drug: an alternative to prodrug approach.

Releasibility of doxorubicin from drug-conjugates is believed to be a prerequisite for its anti-cancer activity. Here, a new glyco-drug approach that circumvents the releasibility restriction is reported, opening a new possibility to design efficient, target specific drug delivery system. It is discovered that stable amide coupling of doxorubicin (DOX) tohyaluronan (HA) shows dose dependent cytotoxicity to CD44 positive human coloncancer cells (HCT116) as compared to human breast cancer cells(MCF-7) and mouse fibroblast cells (NIH-3T3), which express less CD44 receptor. This direct conjugation approach is an easy scalable strategy that could be adopted to design innocuous anti-tumor nanoparticle formulations.

Ureteral tissue engineering: where are we and how to proceed?

In the field of regenerative medicine, various types of biodegradable and nonbiodegradable scaffolds have been developed for urinary tract tissue-engineering applications. Naturally derived or synthetic materials have been tested to determine their properties and their effectiveness. However, the majority of the current literature focuses on the reconstruction of the urethra, urinary diversion, and urinary bladder, while limited data have been published regarding the use of biomaterials in ureteral reconstruction. Tissue engineering might offer alternative and less invasive therapeutic options for long ureteral defects compared with the current surgical reconstructive techniques and their potential complications. In this article, we aimed to review the literature regarding ureteral tissue engineering through a Medline search and describe new potential options for future clinical applications. We concluded that the available literature is inconclusive since the superiority of a specific scaffold has not been demonstrated and the latest developments of regenerative medicine have not been evaluated in ureteral tissue engineering yet.

Tissue engineered tubular construct for urinary diversion in a preclinical porcine model.

PURPOSE: The ileal conduit has been considered the gold standard urinary diversion for patients with bladder cancer and pediatric patients. Complications are mainly related to the use of gastrointestinal tissue. Tissue engineering may be the technical platform on which to develop alternatives to gastrointestinal tissue. We developed a collagen-polymer conduit and evaluated its applicability for urinary diversion in pigs. MATERIALS AND METHODS: Tubular constructs 12 cm long and 15 mm in diameter were prepared from bovine type I collagen and Vypro® II synthetic polymer mesh. Characterized tubes were sterilized, seeded with and without primary porcine bladder urothelial cells, and implanted as an incontinent urostomy using the right ureter in 10 female Landrace pigs. At 1 month the newly formed tissue structure was functionally and microscopically evaluated by loopogram and immunohistochemistry, respectively. RESULTS: The survival rate was 80% with 1 related and 1 unrelated death. By 1 month the collagen was resorbed and a retroperitoneal tunnel had formed that withstood 40 cm H(2)O water pressure. In 5 cases the tunnel functioned as a urostomy. Histological analysis revealed a moderate immune response, neovascularization and urothelial cells in the construct lumen. The polymer mesh provoked fibroblast deposition and tissue contraction. No major differences were observed between cellular and acellular constructs. CONCLUSIONS: After implanting the tubular constructs a retroperitoneal tunnel was formed that functioned as a urinary conduit in most cases. Improved large tubular scaffolds may generate alternatives to gastrointestinal tissue for urinary diversion.

Improving the cell distribution in collagen-coated poly-caprolactone knittings.

Adequate cellular in-growth into biomaterials is one of the fundamental requirements of scaffolds used in regenerative medicine. Type I collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support can be produced. However, in general, adequate cell in-growth and cell seeding has been suboptimal. In this study we prepared collagen scaffolds of different collagen densities and investigated the cellular distribution. We also prepared a hybrid polymer-collagen scaffold to achieve an optimal cellular distribution as well as sufficient mechanical strength. Collagen scaffolds [ranging from 0.3% to 0.8% (w/v)] with and without a mechanically stable polymer knitting [poly-caprolactone (PCL)] were prepared. The porous structure of collagen scaffolds was characterized using scanning electron microscopy and hematoxylin-eosin staining. The mechanical strength of hybrid scaffolds (collagen with or without PCL) was determined using tensile strength analysis. Cellular in-growth and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and human urothelium seeding. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a hybrid scaffold prepared from 0.4% collagen strengthened with knitting achieved the best cellular distribution.