Aase Katarina Bodin

Microporous bacterial cellulose as a potential scaffold for bone regeneration

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 Youngs 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.

Tissue-engineered conduit using urine-derived stem cells seeded bacterial cellulose polymer in urinary reconstruction and diversion.

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 Ia and AE1/AE3) and the SMC expressed SMC markers (α-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.

Bacterial cellulose as a potential vascular graft: Mechanical characterization and constitutive model development

Bacterial cellulose (BC) is a polysaccharide produced by Acetobacter Xylinum bacteria with interesting properties for arterial grafting and vascular tissue engineering including high-burst pressure, high-water content, high crystallinity, and an ultrafine highly pure fibrous structure similar to that of collagen. Given that compliance mismatch is one of the main factors contributing to the development of intimal hyperplasia in vascular replacement conduits, an in depth investigation of support mechanical properties of BC is required to further supporting its use in cardiovascular-grafting applications. The aim of this study was to mechanically characterize BC and also study its potential to accommodate vascular cells. To achieve these aims, inflation tests and uniaxial tensile tests were carried out on BC samples. In addition, dynamic compliance tests were conducted on BC tubes, and the results were compared to that of arteries, saphenous vein, expanded polytetrafluoroethylene, and Dacron grafts. BC tubes exhibited a compliance response similar to human saphenous vein with a mean compliance value of 4.27 × 10(-2) % per millimeter of mercury over the pressure range of 30-120 mmHg. In addition, bovine smooth muscle cells and endothelial cells were cultured on BC samples, and histology and fluorescent imaging analysis were carried out showing good adherence and biocompatibility. Finally, a method to predict the mechanical behavior of BC grafts in situ was established, whereby a constitutive model for BC was determined and used to model the BC tubes under inflation using finite element analysis.

Bacterial cellulose modified with xyloglucan bearing the adhesion peptide RGD promotes endothelial cell adhesion and metabolism—a promising modification for vascular grafts

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. Copyright

Small calibre biosynthetic bacterial cellulose blood vessels: 13-months patency in a sheep model

Abstract 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.

Visualization of the cellulose biosynthesis and cell integration into cellulose scaffolds.

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.

Surface-engineered bacterial cellulose as template for crystallization of calcium phosphate

Bacterial cellulose (BC), produced by Acetobacter xylinum, and cotton linters as reference were surface modified by ozone-induced graft polymerization of acrylic acid and used as a template for crystallization of calcium phosphate. The grafting was verified using attenuated total reflection-infrared radiation (ATR-IR) and electron spectroscopy for chemical analysis (ESCA). ATR-IR revealed an additional absorption band at 1700 cm−1, corresponding to the carbonyl group in polyacrylic acid. ESCA figures show, apart from the characteristic peaks for cellulose, additional peaks at 285 eV and 289 eV that correspond to groups in acrylic acid. The grafting yield is higher on cotton linters compared with BC, which most likely has to do with differences in crystallinity and reactivity of the different cellulose materials. No morphology difference directly caused by grafting could be seen with scanning electron microscopy (SEM), which might indicate that acrylic acid was grafted as a thin film on the surface of the cellulose micro fibrils. Calcium phosphate was formed on the surface-modified cellulose by first pre-soaking the materials in a saturated Ca(OH)2 and later in simulated body fluid (SBF). The atomic ratio of calcium phosphate was determined by ESCA to be about 1.5 for the different materials. Energy dispersive spectroscopy (EDS) was used to map and verify that the crystals were calcium phosphate. Secondary ion mass spectroscopy (SIMS) was also used to verify the presence of calcium phosphate complex onto BC. SEM images showed the difference in dimension, distribution and morphology of the crystals depending on the materials. Smaller and a greater number of crystals were obtained on the surface-modified BC and larger and fewer crystals on surface-modified cotton linters. Structural and grafting differences between the celluloses may lead to differences in nucleation sites and possibly differences in the morphology of the Ca-P crystals. The BC–calcium phosphate composite is expected to be useful as a scaffold for bone tissue regeneration.

Release of Antithrombotic Drugs from Alginate Gel Beads

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.

540 AUGMENTED MUSCLE REGENERATION AND INNERVATION AFTER IMPLANTATION OF URINE-DERIVED STEM CELLS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR

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 10 6

CARS and SHG microscopy for the characterization of bacterial cellulose

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.