Integration of cellulases into bacterial cellulose: Toward bioabsorbable cellulose composites

Cellulose biodegradation resulting from enzymolysis generally occurs in nature rather than in the human body because of the absence of cellulose degrading enzymes. In order to achieve in-vivo degradation in human body for in-vivo tissue regeneration applications, we developed a bioaborbable bacterial cellulose (BBC) material, which integrates one or more cellulose degrading enzymes (cellulases), and demonstrated its degradability in vitro using buffers with pH values relevant to wound environments. We introduced a double lyophilizing process to retain the microstructure of the bacterial cellulose as well as the activity of embedded enzymes allowing for long-term storage of the material, which only requires hydration before use. Enzymes and their combinations have been examined to optimize the in-vitro degradation of the BBC material. In-vitro studies revealed that acidic cellulases from Trichoderma viride showed reasonable activity for pH values ranging from 4.5 to 6.0. A commercial cellulase (cellulase-5000) did not show good activity at pH 7.4, but its degrading ability increased when used in conjunction with a β-glucosidase from Bacillus subtilis or a β-glucosidase from Trichoderma sp. Given the harmless glucose product of the enzymatic degradation of cellulose, the BBC material may be ideal for many wound care and tissue engineering applications for the bioabsorbable purpose.     

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.     

In vitro and in vivo behavior of self-reinforced bioabsorbable polymer and self-reinforced bioabsorbable polymer/bioactive glass composites

The aim of this study was to investigate the in vitro and in vivo properties and degradation of (1) self-reinforced (SR) lactide copolymer, P(L/DL)LA 70:30, and (2) SR composites of the same polylactide and bioactive glass 13-93. The following three polymer and polymer-bioactive glass samples were studied: SR-PLA70, SR-PLA70 + BaG15s, and SR-PLA70 + BaG20c. In vitro behavior was studied in a phosphate-buffered saline for 87 weeks at 37 degrees +/- 1 degrees C and a pH of 7.4 +/- 0.2. In vivo behavior was studied by implanting the rods in the dorsal subcutaneous tissue of rats (SR-PLA70 + BaG20c) or rabbits (SR-PLA70 and SR-PLA70 + BaG15s) for 48 weeks. The degradation of the specimens was evaluated by measuring the changes in mechanical properties, crystallinity and molecular weight of polymer, water absorption, weight loss, and structural changes. Results showed that the addition of bioactive glass filler modified the degradation kinetics and material morphology.     

Mechanical properties and in vitro degradation of bioabsorbable self-expanding braided stents

The aim of this study was to characterize the mechanical and self-expansion properties of braided bioabsorbable stents. In total four different stents were manufactured from PLLA fibres using a braiding technique. The changes in radial pressure stiffness and diameter recovery of the stents were determined initially, and after insertion and release from a delivery device. The braided stents were compared to three commercially available metallic braided stents. The changes in physical and mechanical properties of the PLLA fibres and stents during in vitro degradation were investigated. After release from the delivery device, the PLLA stents did not fully recover to their original diameter. The radial pressure stiffness of the bioabsorbable stents was similar to that of the metallic stents. The in vitro degradation study showed that the stents would keep at least half of their initial radial pressure stiffness for more than 22 weeks.     

Mechanical properties and in vitro degradation of bioabsorbable self-expanding braided stents

The aim of this study was to characterize the mechanical and self-expansion properties of braided bioabsorbable stents. In total four different stents were manufactured from PLLA fibres using a braiding technique. The changes in radial pressure stiffness and diameter recovery of the stents were determined initially, and after insertion and release from a delivery device. The braided stents were compared to three commercially available metallic braided stents. The changes in physical and mechanical properties of the PLLA fibres and stents during in vitro degradation were investigated. After release from the delivery device, the PLLA stents did not fully recover to their original diameter. The radial pressure stiffness of the bioabsorbable stents was similar to that of the metallic stents. The in vitro degradation study showed that the stents would keep at least half of their initial radial pressure stiffness for more than 22 weeks.     

In vitro and in vivo degradation of bioabsorbable PLLA spinal fusion cages

The in vitro and in vivo degradation of poly-L-lactic acid cages used as an adjunct to spinal arthrodesis was investigated. In the in vitro experiments cages were subjected to aging up to 73 weeks in phosphate-buffered solution (pH 7.4) at 37 degrees C. Inherent viscosity, crystallinity, and mechanical strength were determined at different time points. In the in vivo study, the poly-L-lactic acid cages were packed with bone graft and implanted in the L3-L4 spinal motion segment of 18 Dutch milk goats. At 12, 26, and 52 weeks, the motion segments were isolated and poly-L-lactic acid samples retrieved. On evaluation, the in vivo implanted cages showed an advanced decline in inherent viscosity compared to the cages subjected to in vitro degradation experiments. At 6 months of implantation, the geometrical shape and original height of 10 mm was maintained during 6 months of follow up. This finding fits well with the observation that mechanical strength was maintained for a period of 6 months in vitro. At 12 months, the poly-L-lactic acid cage had been disintegrated into multiple fragments with signs of absorption. Despite the high-load-bearing conditions, the poly-L-lactic acid cage allowed interbody fusion to occur without collapse of the cage.     

In vitro and in vivo testing of bioabsorbable antibiotic containing bone filler for osteomyelitis treatment

The use of local antibiotics from a biodegradable implant is appealing concept for treatment of chronic osteomyelitis. Our aim was to develop a new drug delivery system based on controlled ciprofloxacin release from poly(D/L-lactide). Cylindrical composite pellets (1.0 x 0.9 mm) were manufactured from bioabsorbable poly(D/L-lactide) matrix and ciprofloxacin (7.4 wt %). In vitro studies were carried out to delineate the release profile of the antibiotic and to verify its antimicrobial activity by means of MIC testing. A long-term study in rabbits was performed to validate the release of ciprofloxacin from the composite in vivo. Therapeutic level of ciprofloxacin (>2 microg/mL) was maintained between 60 and 300 days and the concentration remained below the potentially detrimental level of 20 microg/mL in vitro. The released ciprofloxacin had retained its antimicrobial properties against common pathogens. In an exploratory long-term in vivo study with three rabbits, ciprofloxacin could not be detected from the serum after moderate filling (160 mg) of the tibia (follow-up 168 days), whereas after high dosing (a total dose of 1,000 mg in both tibias) ciprofloxacin was found temporarily at low serum concentrations (14-34 ng/mL) during the follow-up of 300 days. The bone concentrations of ciprofloxacin could be measured in all samples at 168 and 300 days. The tested copolylactide matrix seems to be a promising option in selection of resorbable carriers for sustained release of antibiotics, but the composite needs modifications to promote ciprofloxacin release during the first 60 days of implantation.     

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 vitro mechanical properties comparsion of four room temperature curing denture base resin:

The room temperature curing denture base resin has low mechanical properties,so itwas limited in clinical application.Ithad previously disscused thatreinfored ma-chinical properties with metal fiber,glass fiber,plastic fiberand by adding the frac-ture resistance substances in powder　　The aim of this study was to determined the mechanical properitiesof fourroomtemperature curing denture base resin which had been modified performance withhigh boil pointmethacrylate.MADTERIALSAND METHOD…     

In vitro and in vivo studies on bioabsorbable ultra-high-strength poly(L-lactide) rods.

Ultra-high-strength poly(L-lactide) (PLLA) rods were fabricated using a drawing technique. Rods with a diameter of 3.2 mm and a draw ratio of 2.5:1 showed initial bending strength and modulus values of 240 MPa and 13 GPa, respectively. The purpose of this study was to investigate the in vitro and in vivo degradation of PLLA rods with a draw ratio of 2.5:1. The greater the rod diameter, the longer the bending strength was maintained in phosphate buffered saline at 37 degrees C. The bending strength retention of rods (diam. 3.2 mm) implanted in the subcutis of rabbits was almost equal to that of rods in the in vitro study, while those rods implanted in the medullary cavity of rabbit femora showed a slightly lower bending strength retention. Molecular weight was reduced to the greatest extent in the medullary cavity, followed by in the subcutis and in vitro. The weight of PLLA rods in the medullary cavity was reduced by 22% at 52 weeks and by 70% at 78 weeks after implantation. Histologically, no inflammatory or foreign body reaction was observed in the medullary cavity for 52 weeks. The drawn PLLA rods maintained a bending strength exceeding that of human cortical bone in the medullary canal for a period of 8 weeks, suggesting that the drawn PLLA rods may be useful in the repair of fractured human bones.     

In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres

Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-l-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications.     

Biogenesis of bacterial cellulose

Cellulose is the most abundant biological polymer on Earth. It is found in wood and cotton, and forms the basic structural foundation of the cell wall of almost all eukaryotic plants. Bacteria are known to secrete cellulose as part of their metabolism of glucose and other sugars. The focus of this review is upon bacterial cellulose synthesis. We emphasize recent literature directed primarily upon Acetobacter xylinum, which has been most widely studied. Our review covers the following topics relating to cellulose synthesis: genetics, biochemistry, ultrastructure, growth conditions, and ecological considerations as they relate to the diversity of microbes capable of synthesizing this abundant, unique polymer--cellulose.     

In vivo mechanical properties of thoracic aortic aneurysmal wall estimated from in vitro biaxial tensile test

To investigate the mechanism of aneurysm rupture, it is necessary to examine the mechanical properties of aneurysm tissues in vivo. A new approach to evaluate in vivo mechanical properties of aortic aneurysmal tissues has been proposed in this study. The shape of the aneurysm was modeled as a sphere, and equi-biaxial stress in the in vivo state was estimated from the diameter and the wall thickness of each aneurysm and mean blood pressure of each patient. The mechanical properties of the aneurysm at the in vivo stress were estimated from its in vitro biaxial tensile properties. There were no significant correlations among maximum diameter D, wall thickness t, and mean infinitesimal strain in the in vivo state epsilon(m). This indicates the wall deformation during aneurysm development was not elastic but plastic. The mean incremental elastic modulus H(m), an index of tissue stiffness, had a significant positive correlation with elastic modulus anisotropy index K(H). This indicates the aneurysmal wall got more anisotropic in vivo as it becomes stiffer.     

In situ hybridization of carbon nanotubes with bacterial cellulose for three-dimensional hybrid bioscaffolds

Carbon nanotubes (CNTs) have shown great potential in biomedical fields. However, in vivo applications of CNTs for regenerative medicine have been hampered by difficulties associated with the fabrication of three-dimensional (3D) scaffolds of CNTs due to CNTs' nano-scale nature. In this study, we devised a new method for biosynthesis of CNT-based 3D scaffold by in situ hybridizing CNTs with bacterial cellulose (BC), which has a structure ideal for tissue-engineering scaffolds. This was achieved simply by culturing Gluconacetobacter xylinus, BC-synthesizing bacteria, in medium containing CNTs. However, pristine CNTs aggregated in medium, which hampers homogeneous hybridization of CNTs with BC scaffolds, and the binding energy between hydrophobic pristine CNTs and hydrophilic BC was too small for the hybridization to occur. To overcome these problems, an amphiphilic comb-like polymer (APCLP) was adsorbed on CNTs. Unlike CNT-coated BC scaffolds (CNT-BC-Imm) formed by immersing 3D BC scaffolds in CNT solution, the APCLP-adsorbed CNT-BC hybrid scaffold (CNT-BC-Syn) showed homogeneously distributed CNTs throughout the 3D microporous structure of BC. Importantly, in contrast to CNT-BC-Imm scaffolds, CNT-BC-Syn scaffolds showed excellent osteoconductivity and osteoinductivity that led to high bone regeneration efficacy. This strategy may open a new avenue for development of 3D biofunctional scaffolds for regenerative medicine.     

Characteristics and anticancer properties of bacterial cellulose films containing ethanolic extract of mangosteen peel

Bacterial cellulose (BC) films containing an ethanolic extract of mangosteen peel were prepared and their physical, chemical, and anticancer properties were characterized. The cumulative absorption and release profiles of bioactive compounds in the films were determined based on total phenolic and α-mangostin content. The BC films were filled with total phenolic compounds expressed as gallic acid equivalent varying from 4.72 to 275.91?mg/cm³ dried film, and α-mangostin varying from 2.06 to 248.20?mg/cm³ dried film. A Fourier transform infrared spectroscopy evaluation showed that there were weak interactions between the functional groups of the extract and the BC. Decreases in the water absorption capacity and water vapor transmission rate of the modified films were detected. Release studies were performed using Franz diffusion cells. In a non-transdermal system, the release of bioactive compounds from the films depended on concentration, immersion time, and the pH of the dissolution medium. A transdermal diffusion study showed that 59–62% of total phenolic compounds that were initially loaded were released from the films and more than 95% of bioactive compounds released from the films were adsorbed into pig skin. Only very small amount of the bioactive compounds penetrated through pig skin and into phosphate and acetate buffers. In studies of anticancer abilities, the release of 2.0?μg/ml α-mangostin from the BC films could suppress the growth of B16F10 melanoma (approximately 31% survival). With the release of α-mangostin at greater than 17.4–18.4?μg/ml, less than 15 and 5% survival of B16F10 melanoma and MCF-7 breast cancer cells, respectively, was observed.     

In vitro interactions of biomedical polyurethanes with macrophages and bacterial cells

Three commercial medical-grade polyurethanes (PUs), a poly-ether-urethane (Pellethane), and two poly-carbonate-urethanes, the one aromatic (Bionate) and the other aliphatic (Chronoflex), were tested for macrophages and bacterial cells adhesion, in the presence or absence of adhesive plasma proteins. All the experiments were carried out on PUs films obtained by solvent casting. The wettability of these films was analysed by measuring static contact angles against water. The ability of the selected PUs to adsorb human fibronectin (Fn) and fibrinogen (Fbg) was checked by ELISA with biotin-labelled proteins. All PUs were able to adsorb Fn and Fbg (Fn > Fbg). Fn adsorption was in the order: Pellethane > Chronoflex > Bionate, the highest Fbg adsorption being detected onto Bionate (Bionate > Chronoflex > Pellethane). The human macrophagic line J111, and the two main bacterial strains responsible for infection in humans (Staphylococcus aureus Newman and Staphylococcus epidermidis 14852) were incubated in turn with the three PUs, uncoated or coated with plasma proteins. No macrophage or bacterial adhesion was observed onto uncoated PUs. PUs coated with plasma, Fn or Fbg promoted bacterial adhesion (S. aureus > S. epidermidis), whereas macrophage adhered more onto PUs coated with Fn or plasma. The coating with Fbg did not promote cell adhesion. Pellethane showed the highest macrophage activation (i.e. spreading), followed, in the order, by Bionate and Chronoflex.     

In vivo biocompatibility and mechanical properties of porous zein scaffolds

In our previous study, a three-dimensional zein porous scaffold with a compressive Young's modulus of up to 86.6+/-19.9 MPa and a compressive strength of up to 11.8+/-1.7 MPa was prepared, and was suitable for culture of mesenchymal stem cells (MSCs) in vitro. In this study, we examined its tissue compatibility in a rabbit subcutaneous implantation model; histological analysis revealed a good tissue response and degradability. To improve its mechanical property (especially the brittleness), the scaffolds were prepared using the club-shaped mannitol as the porogen, and stearic acid or oleic acid was added. The scaffolds obtained had an interconnected tubular pore structure, 100-380 microm in pore size, and about 80% porosity. The maximum values of the compressive strength and modulus, the tensile strength and modulus, and the flexural strength and modulus were obtained at the lowest porosity, reaching 51.81+/-8.70 and 563.8+/-23.4 MPa; 3.91+/-0.86 and 751.63+/-58.85 MPa; and 17.71+/-3.02 and 514.39+/-19.02 MPa, respectively. Addition of 15% stearic acid or 20% oleic acid did not affect the proliferation and osteogenic differentiation of MSCs, and a successful improvement of mechanical properties, especially the brittleness of the zein scaffold could be achieved.     

In vitro technique in estimation of passive mechanical properties of bovine heart part I. Experimental techniques and data

Myocardium generally demonstrates viscoelastic behavior. Since the stress-stain relationships of tissues are pseudo-elastic, their mechanical behavior can be defined as hyperelastic. In this work, mechanical properties of bovine heart were studied. In this study, the experimental technique for testing myocardium is explained and the experimental data are presented. First, the heart was perfused and the specimens were cut from different regions of the heart. Second, the materials preferred direction was identified. Then, a series of uniaxial, biaxial and equibiaxial test were performed on specimens taken from: left ventricle free wall (LVFW), right ventricle free wall (RVFW), left ventricle mid-wall (LVMW) and apex. Test specimens were preconditioned by applying cyclic load to reduce the viscoelastic effect. After preconditioning, the samples were tested at various stretch rates and loading conditions. Finally, a conclusion is made on the experimental data.