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

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.

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.

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.

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.

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.

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 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.

Modification of nanocellulose with a xyloglucan-RGD conjugate enhances adhesion and proliferation of endothelial cells: implications for tissue engineering.

This paper describes a novel method for introducing the RGD cell adhesion peptide to enhance cell adhesion onto bacterial cellulose (BC). BC and cotton linters as reference were modified with xyloglucan (XG) and xyloglugan bearing a GRGDS pentapeptide. The adsorptions followed Langmuir adsorption behavior, where both XGs probably decorate the cellulose surfaces as a monolayer. The adsorption maximum of the XGs reached around 180 mg/g on BC and only about three times as much on cotton linters. The adsorption was verified with colorimetric methods. The specific surface area of BC measured with XG and XG-GRGDS was about 200 m (2)/g and was almost three times less for cotton linters, 60 m (2)/g. The difference in the amounts of XGs adsorbed might be explained by the swollen network of bacterial cellulose and a more exposed and accessible bulk as compared to cotton linters. The nanocellulose material was modified homogeneously throughout the material, as seen by the z-scan in confocal microscopy. Moreover, the modification in the water phase, in comparison with organic solvents, was clearly advantageous for preserving the morphology, as observed with SEM. The modification slightly increased the wettability, which might explain the decrease in or undetectable adsorption of adhesive protein shown by QCM-D. Initial cell studies showed that adhesion of human endothelial cells is enhanced when the BC hydrogel is modified with XG-GRGDS. QCM-D studies further revealed that the cell enhancement is due to the presence of the RGD epitope on XG and not to a nonspecific adsorption of fibronectin from cell culture medium. Optimization and proliferation studies of human endothelial cells onto bacterial cellulose modified with XG-GRGDS are currently being carried out at the Vascular Engineering Center, Sahlgrenska University Hospital, Gothenburg.

Bacterial cellulose as a potential meniscus implant.

Traumatic or degenerative meniscal lesions are a frequent problem. The meniscus cannot regenerate after resection. These lesions often progress and lead to osteoarthritis. Collagen meniscal implants have been used in clinical practice to regenerate meniscal tissue after partial meniscectomy. The mechanical properties of bacterial cellulose (BC) gel were compared with a collagen material and the pig meniscus. BC was grown statically in corn steep liquid medium, as described elsewhere. Pig meniscus was harvested from pigs. The collagen implant was packed in sterile conditions until use. The different materials were evaluated under tensile and compression load, using an Instron 5542 with a 500 N load cell. The feasibility for implantation was explored using a pig model. The Young's modulus of bacterial cellulose was measured to be 1 MPa, 100 times less for the collagen material, 0.01 MPa in tensile load. The Young's modulus of bacterial cellulose and meniscus are similar in magnitude under a compression load of 2 kPa and with five times better mechanical properties than the collagen material. At higher compression strain, however, the pig meniscus is clearly stronger. These differences are clearly due to a more ordered and arranged structure of the collagen fibrils in the meniscus. The combination of the facts that BC is inexpensive, can be produced in a meniscus shape, and promotes cell migration makes it an attractive material for consideration as a meniscus implant.

Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes.

Bacterial cellulose (BC) was deposited in tubular form by fermenting Acetobacter xylinum on top of silicone tubes as an oxygenated support and by blowing different concentrations of oxygen, that is, 21% (air), 35%, 50%, and 100%. Mechanical properties such as burst pressure and tensile properties were evaluated for all tubes. The burst pressure of the tubes increased with an increase in oxygen ratio and reached a top value of 880 mmHg at 100% oxygen. The Young's modulus was approximately 5 MPa for all tubes, irrespective of the oxygen ratio. The elongation to break decreased from 30% to 10-20% when the oxygen ratio was increased. The morphology of the tubes was characterized by Scanning Electron Microscopy (SEM). All tubes had an even inner side and a more porous outer side. The cross section indicated that the tubes are composed of layers and that the amount of layers and the yield of cellulose increased with an increase in oxygen ratio. We propose that an internal vessel wall with high density is required for the tube to sustain a certain pressure. An increase in wall thickness by an increase in oxygen ratio might explain the increasing burst pressure with increasing oxygen ratio. The fermentation method used renders it possible to produce branched tubes, tubes with unlimited length and inner diameters. Endothelial cells (ECs) were grown onto the lumen of the tubes. The cells formed a confluent layer after 7 days. The tubes potential as a vascular graft is currently under investigation in a large animal model at the Centre of Vascular Engineering, Sahlgrenska University

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.

Mechanical properties of bacterial cellulose and interactions with smooth muscle cells.

Tissue engineered blood vessels (TEBV) represent an attractive approach for overcoming reconstructive problems associated with vascular diseases by providing small calibre vascular grafts. The aim of this study has been to evaluate a novel biomaterial, bacterial cellulose (BC), as a potential scaffold for TEBV. The morphology of the BC pellicle grown in static culture was investigated with SEM. Mechanical properties of BC were measured in Krebs solution and compared with the properties of porcine carotid arteries and ePTFE grafts. Attachment, proliferation and ingrowth of human smooth muscle cells (SMC) on the BC were analysed in vitro. The BC pellicle had an asymmetric structure composed of a fine network of nanofibrils similar to a collagen network. The shape of the stress-strain response of BC is reminiscent of the stress-strain response of the carotid artery, most probably due to the similarity in architecture of the nanofibrill networks. SMC adhered to and proliferated on the BC pellicle; an ingrowth of up to 40 microm was seen after 2 weeks of culture. BC exhibit attractive properties for use in future TEBV.

In vivo biocompatibility of bacterial cellulose.

The biocompatibility of a scaffold for tissue engineered constructs is essential for the outcome. Bacterial cellulose (BC) consists of completely pure cellulose nanofibrils synthesized by Acetobacter xylinum. BC has high mechanical strength and can be shaped into three-dimensional structures. Cellulose-based materials induce negligible foreign body and inflammatory responses and are considered as biocompatible. The in vivo biocompatibility of BC has never been evaluated systematically. Thus, in the development of tissue engineered constructs with a BC scaffold, it is necessary to evaluate the in vivo biocompatibility. BC was implanted subcutaneously in rats for 1, 4, and 12 weeks. The implants were evaluated in aspects of chronic inflammation, foreign body responses, cell ingrowth, and angiogenesis, using histology, immunohistochemistry, and electron microscopy. There were no macroscopic signs of inflammation around the implants. There were no microscopic signs of inflammation either (i.e., a high number of small cells around the implants or the blood vessels). No fibrotic capsule or giant cells were present. Fibroblasts infiltrated BC, which was well integrated into the host tissue, and did not elicit any chronic inflammatory reactions. The biocompatibility of BC is good and the material has potential to be used as a scaffold in tissue engineering.