Marije Sloff

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.

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.

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.

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

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.

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.

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.

The influence of collagen density in cellular distribution

Adequate cellular in-growth into biomaterials is one of the fundamental requirements 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. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. 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 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...