Johan Sundberg

Mechanical evaluation of bacterial nanocellulose as an implant material for ear cartilage replacement

Bacterial nanocellulose (BNC) is a novel non-degradable biocompatible material that promotes chondrocyte adhesion and proliferation. In this work, its potential use in ear cartilage tissue engineering (TE) is investigated. Firstly, the mechanical properties of native ear cartilage are measured in order to set a preliminary benchmark for ear cartilage replacement materials. Secondly, the capacity of BNC to match these requirements is assessed. Finally, a biofabrication process to produce patient-specific BNC auricular implants is demonstrated. BNC samples (n=78) with varying cellulose content (2.5-15%) were compared using stress-relaxation indentation with human ear cartilage (n=17, from 4 males, aged 49-93 years old). Additionally, an auricle from a volunteer was scanned using a 3T MRI with a spoiled gradient-echo sequence. A negative ear mold was produced from the MRI data in order to investigate if an ear-shaped BNC prototype could be produced from this mold. The results show that the instantaneous modulus Ein, equilibrium modulus Eeq, and maximum stress σmax of the BNC samples are correlated to effective cellulose content. Despite significantly different relaxation kinetics, the Ein, Eeq and σmax of BNC at 14% effective cellulose content reached values equivalent to ear cartilage (for Eeq, BNC: 2.4±0.4MPa and ear cartilage: 3.3±1.3MPa). Additionally, this work shows that BNC can be fabricated into patient-specific auricular shapes. In conclusion, BNC has the capability to reach mechanical properties of relevance for ear cartilage replacement, and can be produced in patient-specific ear shapes.

In situ Imaging of Collagen Synthesis by Osteoprogenitor Cells in Microporous Bacterial Cellulose Scaffolds

Microscopy techniques based on laser-induced nonlinear optical processes allow for chemically specific imaging of unmodified samples at high spatial resolution in three dimensions and provide powerful tools for characterization of tissue-engineering constructs. This is highlighted by the simultaneous imaging of scaffold material, cells, and produced extracellular matrix collagen in samples consisting of osteoprogenitor MC3T3-E1 cells seeded on microporous bacterial cellulose (BC), a potential scaffold material for synthesis of osseous tissue. BC and collagen have been visualized by second harmonic generation (SHG) microscopy, and verification of collagen identification on cellulose scaffolds has been carried out on sectioned samples by comparison with the conventional histological staining technique. Both methods showed similar collagen distributions and a clear increase in the amount of collagen when comparing measurements from two time points during growth. For investigations of intact cellulose scaffolds seeded with cells, SHG was combined with simultaneous coherent anti-Stokes Raman scattering (CARS) microscopy for visualization of cell arrangement in three dimensions and to be correlated with the SHG data. Results showed that the osteoprogenitor cells were able to produce collagen already during the first days of growth. Further on, developed collagen fiber networks could be imaged inside compact regions of cells located in the cellulose micropores. Collagen production, the initial step of tissue mineralization, demonstrates the potential of BC as a scaffold material for bone tissue engineering. Furthermore, the noninvasive in situ monitoring of collagen inside compact tissue clearly manifests the benefits of nonlinear microscopy techniques, such as SHG and CARS, for use in tissue engineering.

Effect of xylan content on mechanical properties in regenerated cellulose/xylan blend films from ionic liquid

AbstractnWe report of cellulose and arabinoglucuronoxylan (AGX) blend films made from wood polymers extracted from one and the same tree. Blends were prepared by dissolution of wood polymers in 1-ethyl-3-methylimidazolium acetate (EmimAc). Films were produced by casting EmimAc solution followed by coagulation in ethanol. The films were optically transparent, fully amorphous as shown by wide angle X-ray scattering, and free from EmimAc residues as shown by Fourier transform infrared spectroscopy. Mechanical properties were analyzed as a function of water content. The plasticizing effect of water on the films was evidenced by both tensile and dynamical mechanical analysis measurements with humidity scans. Equilibrium moisture content (w/w) was measured at different relative humidities and the proportional water uptake was clearly related to the mechanical properties. We found good mechanical properties independent of the polysaccharide composition and an increased Young’s modulus at low humidities with a maximum at approximately 20xa0% AGX content. The strengthening effect was removed after leaching the AGX from the films. This study shows potential applications of biopolymer extracted from trees as future packaging.

Electrospun nanofibrous cellulose scaffolds with controlled microarchitecture

Introducing porosity in electrospun scaffolds is critical to improve cell penetration and nutrient diffusion for tissue engineering. Nanofibrous cellulose scaffolds were prepared by electrospinning cellulose acetate (CA) followed by saponification to regenerate cellulose. Using a computer-assisted design approach, scaffolds underwent laser ablation resulting in pores with diameters between 50 and 300 μm without damaging or modifying the surrounding scaffold area. A new mineralization method was employed in conjunction with microablation using commercial phosphate buffered saline (PBS) to soak carboxymethylcellulose surface-modified electrospun scaffolds. The resulting crystals within the scaffold on the interior of the pore had a calcium to phosphate ratio of 1.56, similar to hydroxyapatite. It was observed that porosity of the cellulose scaffolds enhanced osteoblast cell attachment at the edge of the pores, while mineralization enhanced overall cell density.

3D Culturing and differentiation of SH-SY5Y neuroblastoma cells on bacterial nanocellulose scaffolds

Abstract A new in vitro model, mimicking the complexity of nerve tissue, was developed based on a bacterial nanocellulose (BNC) scaffold that supports 3D culturing of neuronal cells. BNC is extracellularly excreted by Gluconacetobacter xylinus (G. xylinus) in the shape of long non-aggregated nanofibrils. The cellulose network created by G. xylinus has good mechanical properties, 99% water content, and the ability to be shaped into 3D structures by culturing in different molds. Surface modification with trimethyl ammonium beta-hydroxypropyl (TMAHP) to induce a positive surface charge, followed by collagen I coating, has been used to improve cell adhesion, growth, and differentiation on the scaffold. In the present study, we used SH-SY5Y neuroblastoma cells as a neuronal model. These cells attached and proliferated well on the BNC scaffold, as demonstrated by scanning electron microscopy (SEM) and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Following neuronal differentiation, we demonstrated functional action potentials (APs) by electrophysiological recordings, indicating the presence of mature neurons on the scaffolds. In conclusion, we have demonstrated for the first time that neurons can attach, proliferate, and differentiate on BNC. This 3D model based on BNC scaffolds could possibly be used for developing in vitro disease models, when combined with human induced pluripotent stem (iPS) cells (derived from diseased patients) for detailed investigations of neurodegenerative disease mechanisms and in the search for new therapeutics.

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 150–300u2009µm and 300–500u2009µm did not influence cartilaginous extra cellular matrix synthesis. Varying seeding densities from 9.95u2009×u2009102 to 1.99u2009×u2009103u2009cells/mm2 and 3.98u2009×u2009103u2009cells/mm2 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.

13C NMR assignments of regenerated cellulose from solid-state 2D NMR spectroscopy

From the assignment of the solid-state (13)C NMR signals in the C4 region, distinct types of crystalline cellulose, cellulose at crystalline surfaces, and disordered cellulose can be identified and quantified. For regenerated cellulose, complete (13)C assignments of the other carbon regions have not previously been attainable, due to signal overlap. In this study, two-dimensional (2D) NMR correlation methods were used to resolve and assign (13)C signals for all carbon atoms in regenerated cellulose. (13)C-enriched bacterial nanocellulose was biosynthesized, dissolved, and coagulated as highly crystalline cellulose II. Specifically, four distinct (13)C signals were observed corresponding to conformationally different anhydroglucose units: two signals assigned to crystalline moieties and two signals assigned to non-crystalline species. The C1, C4 and C6 regions for cellulose II were fully examined by global spectral deconvolution, which yielded qualitative trends of the relative populations of the different cellulose moieties, as a function of wetting and drying treatments.

Biosynthesis and in vitro evaluation of macroporous mineralized bacterial nanocellulose scaffolds for bone tissue engineering

Macroporous bacterial nanocellulose (BNC) scaffolds with calcium phosphate coated surfaces is a candidate for future bone tissue engineering applications. The mineralization of the macroporous BNC scaffolds was achieved by a biomimetic process, resulting in an environment resembling native bone tissues mineralized extra cellular matrix both topographically and chemically. The deposited crystals were analyzed with electron spectroscopy for chemical analysis (ESCA), energy-dispersive X-ray spectroscopy (EDX) and X-ray crystallography (XRD). MSCs were cultured in osteogeneic medium for 21 days on the scaffolds. The results of this study show that macroporous BNC can be mineralized with hydroxyapatite and that MSCs retain their ability to proliferate and differentiate towards an osteoblastic phenotype within the mineralized BNC, showing the promise of this material in bone tissue engineering applications.