Spruce xylan/HEMA-SBA15 hybrid hydrogels as a potential scaffold for fibroblast growth and attachment.

A hybrid hydrogel (GHC-SBA15) based on spruce xylan (HC), 2-hydroxyethyl methacrylate (HEMA), and mesoporous silica (SBA15) was prepared with the intended use of fibroblast attachment and growth. Xylan was functionalized with acryloyl chloride to introduce vinyl groups and was crosslinked by radical polymerization with HEMA in presence of SBA15. Infrared spectroscopy and nuclear magnetic resonance confirmed the copolymerization of HEMA with xylan. Up to 20 wt.% addition, SBA15 was homogenously incorporated in the structured hydrogel network as observed by SEM. Moreover, nitrogen adsorption-desorption, small angle X-ray scattering and transmission electron microscopy indicated that the mesoporous SBA15 framework was maintained and that the hybrid hydrogel was a physical mixture of SBA15 with the copolymer HC/HEMA. Rheological analysis revealed that addition of 20% w/w SBA15 into hydrogel enhanced significantly the mechanical properties. In addition, we demonstrate that fibroblast L929 cells grew and spread on GHC-SBA15. Cell viability was within the expected range.

Increased lipid accumulation and adipogenic gene expression of adipocytes in 3D bioprinted nanocellulose scaffolds.

Compared to standard 2D culture systems, new methods for 3D cell culture of adipocytes could provide more physiologically accurate data and a deeper understanding of metabolic diseases such as diabetes. By resuspending living cells in a bioink of nanocellulose and hyaluronic acid, we were able to print 3D scaffolds with uniform cell distribution. After one week in culture, cell viability was 95%, and after two weeks the cells displayed a more mature phenotype with larger lipid droplets than standard 2D cultured cells. Unlike cells in 2D culture, the 3D bioprinted cells did not detach upon lipid accumulation. After two weeks, the gene expression of the adipogenic marker genes PPARγ and FABP4 was increased 2.0- and 2.2-fold, respectively, for cells in 3D bioprinted constructs compared with 2D cultured cells. Our 3D bioprinted culture system produces better adipogenic differentiation of mesenchymal stem cells and a more mature cell phenotype than conventional 2D culture systems.

Lipases efficiently stearate and cutinases acetylate the surface of arabinoxylan films.

This is the first report on successful enzyme catalyzed surface esterification of hemicellulose films. Enzyme catalyzed surface acetylation with vinyl acetate and stearation with vinyl stearate were studied on rye arabinoxylan (AX) films. Different surface analytical techniques (FT-IR, TOF-SIMS, ESCA, CA) show that lipases from Mucor javanicus, Rhizopus oryzae and Candida rugosa successfully surface stearate AX films and that a cutinase from Fusarium solani pisi surface acetylates these films. The specificities of cutinase and lipases were also compared, and higher activity was observed for lipases utilizing long alkyl chain substrates while higher activity was observed for cutinase utilizing shorter alkyl chain substrates. The contact angle analysis showed films with increased initial hydrophobicity on the surfaces.

Description of a novel approach to engineer cartilage with porous bacterial nanocellulose for reconstruction of a human auricle.

In this study, we investigated the effects of human primary chondrocytes, derived from routine septorhino- and otoplasties on a novel nondegradable biomaterial. This biomaterial, porous bacterial nanocellulose, is produced by Gluconacetobacter xylinus. Porosity is generated by paraffin beads embedded during the fermentation process. Human primary chondrocytes were able to adhere to bacterial nanocellulose and produce cartilaginous matrix proteins such as aggrecan (after 14 days) and collagen type II (after 21 days) in the presence of differentiation medium. Cells were located within the pores and in a dense cell layer covering the surface of the biomaterial. Cells were able to re-differentiate, as cell shape and extra cellular matrix gene expression showed a chondrogenic phenotype in three-dimensional bacterial nanocellulose culture. Collagen type I and versican expression decreased during three-dimensional culture. Variations in pore sizes of 150-300 µm and 300-500 µm did not influence cartilaginous extra cellular matrix synthesis. Varying seeding densities from 9.95 × 10(2) to 1.99 × 10(3) cells/mm(2) and 3.98 × 10(3) cells/mm(2) did not result in differences in quality of extra cellular matrix neo-synthesis. Our results demonstrated that both nasal and auricular chondrocytes are equally suitable to synthesize new extra cellular matrix on bacterial nanocellulose. Therefore, we propose both cell sources in combination with bacterial nanocellulose as promising candidates for the special needs of auricular reconstruction.

Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly(urethane urea) scaffolds.

The chemical and physical properties of scaffolds affect cellular behaviour, which ultimately determines the performance and outcome of tissue-engineered cartilage constructs. The objective of this study was to assess whether a degradable porous poly(urethane urea) scaffold could be a suitable material for cartilage tissue engineering. We also investigated whether the post-expansion redifferentiation and cartilage tissue formation of in vitro expanded adult human chondrocytes could be regulated by controlled modifications of the scaffold architecture. Scaffolds with different pore sizes, < 150 µm, 150-300 µm and 300-500 µm, were seeded with chondrocytes and subjected to chondrogenic and osteogenic induction in vitro. The poly(urethane urea) scaffold with the smaller pore size enhanced the hyaline-like extracellular matrix and thus neocartilage formation. Conversely, the chondrocytes differentiated to a greater extent into the osteogenic pathway in the scaffold with the larger pore size. In conclusion, our results demonstrate that poly(urethane urea) may be useful as a scaffold material in cartilage tissue engineering. Furthermore, the chondrogenic and the osteogenic differentiation capacity of in vitro expanded human articular chondrocytes can be influenced by the scaffold architecture. By tailoring the pore sizes, the performance of the tissue-engineered cartilage constructs might be influenced and thus also the clinical outcome in the long run.

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.

Bioreactors for tissue engineering of cartilage.

The cartilage regenerative medicine field has evolved during the last decades. The first-generation technology, autologous chondrocyte transplantation (ACT) involved the transplantation of in vitro expanded chondrocytes to cartilage defects. The second generation involves the seeding of chondrocytes in a three-dimensional scaffold. The technique has several potential advantages such as the ability of arthroscopic implantation, in vitro pre-differentiation of cells and implant stability among others (Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L, N Engl J Med 331(14):889-895, 1994; Henderson I, Francisco R, Oakes B, Cameron J, Knee 12(3):209-216, 2005; Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E, Lindahl A, Clin Orthop (374):212-234, 2000; Nagel-Heyer S, Goepfert C, Feyerabend F, Petersen JP, Adamietz P, Meenen NM, et al. Bioprocess Biosyst Eng 27(4):273-280, 2005; Portner R, Nagel-Heyer S, Goepfert C, Adamietz P, Meenen NM, J Biosci Bioeng 100(3):235-245, 2005; Nagel-Heyer S, Goepfert C, Adamietz P, Meenen NM, Portner R, J Biotechnol 121(4):486-497, 2006; Heyland J, Wiegandt K, Goepfert C, Nagel-Heyer S, Ilinich E, Schumacher U, et al. Biotechnol Lett 28(20):1641-1648, 2006). The nutritional requirements of cells that are synthesizing extra-cellular matrix increase along the differentiation process. The mass transfer must be increased according to the tissue properties. Bioreactors represent an attractive tool to accelerate the biochemical and mechanical properties of the engineered tissues providing adequate mass transfer and physical stimuli. Different reactor systems have been [5] developed during the last decades based on different physical stimulation concepts. Static and dynamic compression, confined and nonconfined compression-based reactors have been described in this review. Perfusion systems represent an attractive way of culturing constructs under dynamic conditions. Several groups showed increased matrix production using confined and unconfirmed systems. Development of automatic culture systems and noninvasive monitoring of matrix production will take place during the next few years in order to improve the cost affectivity of tissue-engineered products.

Bacterial cellulose as a potential scaffold for tissue engineering of cartilage.

Tissue constructs for cartilage with native mechanical properties have not been described to date. To address this need the bacterial cellulose (BC) secreted by Gluconacetobacter xylinus (= Acetobacter xylinum) was explored as a novel scaffold material due to its unusual material properties and degradability. Native and chemically modified BC materials were evaluated using bovine chondrocytes. The results indicate that unmodified BC supports chondrocyte proliferation at levels of approximately 50% of the collagen type II substrate while providing significant advantages in terms of mechanical properties. Compared to tissue culture plastic and calcium alginate, unmodified BC showed significantly higher levels of chondrocyte growth. Chemical sulfation and phosphorylation of the BC, performed to mimic the glucosaminoglycans of native cartilage, did not enhance chondrocyte growth while the porosity of the material did affect chondrocyte viability. The BC did not induce significant activation of proinflammatory cytokine production during in vitro macrophage screening. Hence, unmodified BC was further explored using human chondrocytes. TEM analysis and RNA expression of the collagen II from human chondrocytes indicated that unmodified BC supports proliferation of chondrocytes. In addition, ingrowth of chondrocytes into the scaffold was verified by TEM. The results suggest the potential for this biomaterial as a scaffold for tissue engineering of cartilage.

Analysis of behavioural rejection of micro-textured surfaces and implications for recruitment by the barnacle Balanus improvisus.

Experiments performed in the field and in the laboratory show that the barnacle, Balanus improvisus, preferentially settles on smooth surfaces. Settlement and recruitment of B. improvisus was evaluated on micro-textured surfaces with scales of surface texture ranging from 1 to 100 µm in profile heights. Surface texture with profile heights within a topographic range of 30-45 µm reduced settlement and recruitment by 92% as compared to smooth surfaces. The reduction in recruitment on micro-textured surfaces is best explained by behavioural responses to surface topography. Behavioural experiments show that cyprids have a higher propensity for smooth surfaces than for micro-textured surfaces. Cyprids spend more time exploring smooth surfaces and more time swimming when exposed to micro-textured surfaces. Micro-textured surfaces are more often rejected by cyprids after exploration than smooth surfaces. It is suggested that some scales of surface texture could be exploited to improve future anti-fouling techniques in geographical areas where Balanus improvisus is a severe fouling problem.

Design and microstructuring of PDMS surfaces for improved marine biofouling resistance.

In this study room temperature vulcanized (RTV) silicone surfaces with designed surface microstructure and well-defined surface chemistry were prepared. Their resistance to marine macrofouling by barnacles Balanus improvisus was tested in field experiments for deducing optimal surface topography dimensions together with a better understanding of macrofouling mechanisms. Polydimethylsiloxane (PDMS) surfaces were microstructured by casting the PDMS pre-polymer on microfabricated molds. The master molds were made by utilizing photolithography and anisotropic etching of monocrystalline silicon wafers. Several iterative casting steps of PDMS and epoxy were used to produce large quantities of microstructured PDMS samples for field studies. The microstructured PDMS surface consisted of arrays of pyramids or riblets creating a surface arithmetic mean roughness ranging from 5 to 17 microm for different microstructure sizes and geometries, as determined by scanning electron microscopy. Chemophysical properties of the microstructured films were investigated by electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectroscopy and dynamic contact angle measurements. Films were chemically homogeneous down to the submicron level. Hydrophobicity and contact angle hysteresis increased with increased surface roughness. Field tests on the west coast of Sweden revealed that the microstructure containing the largest riblets (profile height 69 microm) reduced the settling of barnacles by 67%, whereas the smallest pyramids had no significant influence on settling compared to smooth PDMS surfaces. The effect of dimensions and geometry of the surface microstructures on the B. improvisus larvae settling is discussed.

Surface Science of a Filled Polydimethylsiloxane-Based Alkoxysilane-Cured Elastomer: RTV11.

Characterization of a filled polydimethylsiloxane (PDMS)-based elastomer, RTV11, is reported. Included in this work is resin characterization, kinetics of cure as a function of catalyst concentration, and surface properties of pristine and water-aged films. X-ray spectroscopy for chemical analysis (ESCA), dynamic contact angle (DCA) analysis, and ATR-IR were used to characterize cured films. ESCA reveals C and Si peaks in ratios expected for PDMS, but CaCO3 which comprises 32% of the bulk is not detected. The surface of cured RTV11 films is thus predominantly a PDMS network crosslinked by a siliceous domain, the latter comprising about 1.3% by weight. The presence of Ca was confirmed by energy dispersive X-ray analysis (EDX) which probes at micron depth. Stability of films in water was evaluated by tapping mode atomic force microscopy (TM-AFM), mass loss, changes in contact angles, ESCA, and optical microscopy. TM-AFM images of films aged in water for three months show an increase in surface roughness due to the formation of micro-pits which occupy about 4% of the surface. Gravimetric analysis showed fully cured films lose mass at a rate of about 0.09%/wk over a three month period in water. The mass loss associated with pitting/surface roughening comprises only 0.85% of that measured gravimetrically and by analysis of immersion water. Analysis of Si and Ca in the storage water was performed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) in order to quantify the products of surface erosion. An unexpected finding was the surface depletion-limited loss of CaCO3 during the first two months of immersion. The results of surface analytical studies are discussed in the context of the use of alkoxysilane cured PDMS resins for nontoxic fouling release applications. Copyright 1999 Academic Press.