Cellulose is a biopolymer that has long been used as a biomedical material and is still used in a modified form in hemodialysis membranes and as a carrier material in drug release systems. The different sources of cellulose that have not yet been fully explored, for example, bacterial cellulose (BC), might possess properties that are needed for some very specific biomedical applications. BC differs considerably from other sources of cellulose and still requires a great deal of research to be understood completely. This chapter on BC as a biomaterial reports the findings and results of research carried out in the last decade. It discusses current understanding of the process and biochemistry of BC biosynthesis, and known techniques for the manipulation of its nanostructure and morphology. It reviews cell studies, BC׳s biocompatibility, and potential for use as a scaffold in tissue engineering. It concludes by looking forward to future improvements that will enhance the accessibility of BC commercially and in broader applications.

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology (II)1 Apr 2013540 AUGMENTED MUSCLE REGENERATION AND INNERVATION AFTER IMPLANTATION OF URINE-DERIVED STEM CELLS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR Guihua Liu, Aase Bodin, Shantaram Bharadwaj, Anthony Atala, and Yuanyuan Zhang Guihua LiuGuihua Liu Winston Salem, NC More articles by this author , Aase BodinAase Bodin Winston Salem, NC More articles by this author , Shantaram BharadwajShantaram Bharadwaj Winston Salem, NC More articles by this author , Anthony AtalaAnthony Atala Winston Salem, NC More articles by this author , and Yuanyuan ZhangYuanyuan Zhang Winston Salem, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1935AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Impairment of sphincter muscles or their neural and vascular support leads to stress urinary incontinence (SUI). Cell-based therapies with autologous stem cells have exhibited promising early clinical outcomes. We recently demonstrated that stem cells can be obtained from human urine via a non-invasive approach, and these urine-derived stem cells (USC) can give rise to mesodermal cell lineages, including myocytes. The aim of this study was to determine the effect of USC over-expressing vascular endothelial growth factor (VEGF) along with endothelial cells on angiogenesis, cell survival, growth, myogenic differentiation, and innervation following implantation in vivo. METHODS USC were obtained from 10 urine samples (five healthy donors; ages 3-27 years). USC were infected with adenovirus containing the human VEGF165 gene (USC/Ad-VEGF). The USC (5×106 cells) were suspended in 500 μl of collagen-I gel and subcutaneously implanted into 45 nude mice for 4 weeks in the following groups (G): G1: USC/Ad-VEGF plus endothelial cells (n=12 mice); G2: USC/Ad-VEGF (n=12); G3: USC/Ad-GFP (n=11); G4: human skeletal muscle cells as a positive control (n=5); and G5: cell-free as a negative control (n=5). Immunohistochemistry for human nuclear markers, endothelial markers (CD 31 and von Willebrand factor [vWF]), muscle markers (alpha-smooth muscle actin, desmin, and myosin), and nerve markers (S-100, GFAP, neurofilament) was also performed on the implanted graft tissues. RESULTS USC produced VEGF protein when cultured in vitro; VEGF levels in genetically modified USC (869.15±28.57 pg/ml) were 35 times higher than in USC on their own (30.73±1.89 pg/ml). Extensive vascularization was noted in VEGF-expressing USC implant groups compared to the non-VEGF groups in vivo. Real-time PCR and immunofluorescent staining revealed that in the VEGF-expressing groups, more cells displayed endothelial markers (CD 31 and vWF), and cells expressing a myogenic phenotype and biomarkers (myo-D, desmin and myosin) were abundant. The numbers of cells expressing human nuclear markers also significantly increased in these groups, and regenerated nerve fibers displaying neural markers (S-100, GFAP, and neurofilament) were found in the grafts composed of VEGF-expressing USC. CONCLUSIONS Improved angiogenesis by VEGF-expressing USC enhanced grafted cell survival, promoted myogenic differentiation of USC, and enhanced innervation. This approach has important clinical implications for the development of novel cell therapies to treat SUI. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e222 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guihua Liu Winston Salem, NC More articles by this author Aase Bodin Winston Salem, NC More articles by this author Shantaram Bharadwaj Winston Salem, NC More articles by this author Anthony Atala Winston Salem, NC More articles by this author Yuanyuan Zhang Winston Salem, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Objectives. Many patients in need of bypass surgery lack graft material and current synthetic alternatives have poor performance. A 4 mm vascular graft composed of bacterial cellulose (BC) was developed and tested in pilot study in a large animal model. Design. BC is a biopolymer made by the bacteria acetobacter xylinum. BC grafts (n = 16) with 4 cm length and 4 mm internal diameter were implanted bilaterally in the carotid arteries of eight sheep. No long-term antithrombotic therapy was administered. Patency was assessed with ultrasound. Histology, immunohistochemistry, and electron microscopy were performed after explantation. Results. Fifty percent of the grafts occluded within two weeks. One animal died with patent grafts after 14 days. In the three remaining animals 5/6 grafts were patent after nine months. Two animals were followed 13 months after implantation with 3/4 grafts patent at explantation. All patent grafts had confluent endothelial-like cells. Conclusions. Biosynthetic small calibre vascular grafts made from BC can be patent for up to 13 months in sheep carotid arteries. BC is a potential material for small calibre grafts but patency in animal models needs to be improved before clinical studies can be planned.

Arabinoglucuronoxylan was extracted from Norway spruce and films prepared by casting from aqueous solution. The sugar analysis and NMR confirmed that the spruce xylan was composed of arabinose, 4-O-methyl-glucuronic acid and xylose in a ratio of 1:2:11 respectively. Substitutions of 4-O-methyl-alpha-D-GlcpA at O-2 and of alpha-L-Araf at O-3 on the xylose backbone were found by NOE analysis. NOE cross-peaks indicated as well that there is at least one free xylose on the main chain present between two substitutions. Whether the distribution of side chains was random or in blocks was uncertain. The average molecular weight of the sample was determined by size exclusion chromatography to be 12,780 g/mol. Arabinoglucoronoxylan casting yielded transparent flexible films with an average stress at break of 55 MPa, strain at break of 2.7% and a Young's Modulus 2735 MPa. Wide-angle X-ray scattering analysis showed that the arabinoglucuronoxylan films were totally amorphous. Addition of sorbitol as plasticizer resulted in less strong but more flexible films (strain at break of 5%). Peaks of crystallinity could be seen in X-ray which corresponds to sorbitol crystallizing in distinct phases. The dynamic mechanical analysis showed that the arabinoglucuronoxylan film softened at a later relative humidity (80% RH) in comparison with plasticized films (60% RH). The films showed low oxygen permeability and thus have a potential application in food packaging. (C) 2011 Elsevier Ltd. All rights reserved.

Nanoporous cellulose biosynthesized by bacteria is an attractive biomaterial scaffold for tissue engineering due to its biocompatibility and good mechanical properties. However, for bone applications a microscopic pore structure is needed to facilitate osteoblast ingrowth and formation of a mineralized tissue. Therefore, in this study microporous bacterial cellulose (BC) scaffolds were prepared by incorporating 300-500 microm paraffin wax microspheres into the fermentation process. The paraffin wax microspheres were subsequently removed, and scanning electron microscopy confirmed a microporous surface of the scaffolds while Fourier transform infrared spectroscopy verified the elimination of paraffin and tensile measurements showed a Young's modulus of approximately 1.6 MPa. Microporous BC and nanoporous (control) BC scaffolds were seeded with MC3T3-E1 osteoprogenitor cells, and examined by confocal microscopy and histology for cell distribution and mineral deposition. Cells clustered within the pores of microporous BC, and formed denser mineral deposits than cells grown on control BC surfaces. This work shows that microporous BC is a promising biomaterial for bone tissue engineering applications.

Today, biomaterials such as polytetrafluorethylene (ePTFE) are used clinically as prosthetic grafts for vascular surgery of large vessels (>5 mm). In small diameter vessels, however, their performance is poor due to early thrombosis. Bacterial-derived cellulose (BC) is a new promising material as a replacement for blood vessels. This material is highly biocompatible in vivo but shows poor cell adhesion. In the native blood vessel, the endothelium creates a smooth non-thrombogenic surface. In order to sustain cell adhesion, BC has to be modified. With a novel xyloglucan (XG) glycoconjugate method, it is possible to introduce the cell adhesion peptide RGD (Arg-Gly-Asp) onto bacterial cellulose. The advantage of the XG-technique is that it is an easy one-step procedure carried out in water and it does not weaken or alter the fiber structure of the hydrogel. In this study, BC was modified with XG and XGRGD to asses primary human vascular endothelial cell adhesion, proliferation, and metabolism as compared with unmodified BC. This XG-RGD-modification significantly increased cell adhesion and the metabolism of seeded primary endothelial cells as compared with unmodified BC whereas the proliferation rate was affected only to some extent. The introduction of an RGD-peptide to the BC surface further resulted in enhanced cell spreading with more pronounced stress fiber formation and mature phenotype. This makes BC together with the XG-method a promising material for synthetic grafts in vascular surgery and cardiovascular research.

The aim of the present work was to evaluate alginate hydrogels in the form of spherical beads as carrier for antithrombotic drugs for future use in artificial grafts. The ionotropic gelation technique was employed to prepare beads from the L. hyperborea stipe of alginate with two different alginate concentrations and two different guluronic to manuronic acid ratios. The beads were loaded, via soaking, with three different types of low molecular weight model molecules representing drugs with antithrombotic action and their release characteristics were subsequently evaluated. The entire release process of the negatively charged model drugs under study (Salicylic acid and Hirudin), was found to be governed by diffusion, while additional electrostatic interactions between drug molecule and alginate matrix was indicated to influence the release rate of the analyzed positively charged drug molecule (Dipyridamole). It was found that the alginate hydrogel matrix imposed a decrease of the drug diffusion rate on the molecules under study as compared to the corresponding diffusion rates in water. All diffusion coefficients decreased slightly with increasing concentration of alginate and with increasing guluronic to manuronic acid ratio. The results show on the potential use of alginate gel beads when developing vehicles for release of low molecular weight antithrombotic drugs.

The objective of this study was to generate bacterial cellulose (BC) scaffolds seeded with human urine-derived stem cells (USC) to form a tissue-engineered conduit for use in urinary diversion. Microporous BC scaffolds were synthesized and USC were induced to differentiate into urothelial and smooth muscle cells (SMC). Induced USC (10(6) cells/cm(2)) were seeded onto BC under static and 3D dynamic (10 or 40 RPM) conditions and cultured for 2 weeks. The urothelial cells and SMC derived from USC formed multilayers on the BC scaffold surface, and some cells infiltrated into the scaffold. The urothelium derived from USC differentiation expressed urothelial markers (uroplakin la and AE1/AE3) and the SMC expressed SMC markers (a-smooth muscle actin and desmin). In addition, USC/BC scaffold constructs were implanted into athymic mice, and the cells were tracked using immunohistochemical staining for human nuclear antigen. In vivo, the cells appeared to differentiate and express urothelial and SMC markers. In conclusion, porous BC scaffolds allow 3 dimensional growth of USC, leading to formation of a multilayered urothelium and cell matrix infiltration. Thus, cell-seeded BC scaffolds hold promise for use in tissue-engineered urinary conduits for urinary reconstruction. (C) 2010 Elsevier Ltd. All rights reserved.

By controlling the microarchitecture of bioengineered scaffolds for artificial tissues, their material and cell-interaction properties can be designed to mimic native correspondents. Current understanding of this relationship is sparse and based on microscopy requiring harsh sample preparation and labeling, leaving it open to which extent the natural morphology is studied. This work introduces multimodal nonlinear microscopy for label-free imaging of tissue scaffolds, exemplified by bacterial cellulose. Unique three-dimensional images visualizing the formation of nanofiber networks throughout the biosynthesis, revealing that supra-structures (layered structures, cavities) are formed. Cell integration in compact scaffolds was visualized and compared with porous scaffolds. While the former showed distinct boundaries to the native tissue, gradual cell integration was observed for the porous material. Thus, the degree of cell integration can be controlled through scaffold supra-structures. This illustrates the potential of nonlinear microscopy for noninvasive imaging of the intriguing interaction mechanisms between scaffolds and cells.

We have developed a protocol employing dual-mode non-linear microscopy for the monitoring of the biosynthesis of bacterial cellulose at a single-fiber level, with the fundamental aim to achieve a product with material properties similar to those of human blood vessels. Grown in a tubular geometry it could then be used as a natural and biocompatible source of replacement tissue in conjunction with cardiovascular surgery. The bacteria (Acetobacter xylinum) were selectively visualized based on the CH2 vibration of its organic macromolecular contents by the Coherent Anti-Stokes Raman Scattering (CARS) process and, simultaneously, the non-centrosymmetrically ordered, birefringent cellulose fibers were depicted by the Second Harmonic Generation (SHG) process. This dual-channel detection approach allows the monitoring of cellulose-fiber formation in vivo and to determine the influence of e. g. different growth conditions on fiber thickness and orientation, their assembling into higher-order structures and overall network density. The bacterial and fiber distributions were monitored in a simple microscope cultivation chamber, as well as in samples harvested during the actual fermentation process of tubular cellulose grafts. The CARS and SHG co-localization images reveal that highest bacterial population densities can be observed in the surface regions of the cellulose tissue, where the primary growth presumably takes place. The cellulose network morphology was also compared with that of human arteries and veins, from which we conclude that the cellulose matrix is comparatively homogeneous in contrast to the wavy band-like supra-formations of collagen in the native tissue. This prompts for sophisticated fermentation methods by which tunnels and pores of appropriate sizes and shapes can be introduced in the cellulose network in a controllable way. With this protocol we hope to contribute to the fundamental knowledge required for optimal production of bioengineered cellulose tissues, eventually being available for clinical use.