Comparison of acellular and cellular bioactivity of poly 3-hydroxybutyrate/hydroxyapatite nanocomposite and poly 3-hydroxybutyrate scaffolds

Nanocomposites have recently been identified as a useful scaffolding material in tissue engineering applications. Poly (3-hydroxybutyrate)/hydroxyapatite nanoparticles (P3HB)/(nHA) porous scaffolds were successfully fabricated through a solvent casting and particulate leaching technique. P3HB/nHA and P3HB scaffolds were prepared by the same technique for comparison. The structure of the nanocomposite and P3HB scaffolds was observed by SEM. The Energy Disperssive X-ray Analysis (EDXA, map of Ca) results indicated that HA nanoparticles were homogeneously dispersed in the P3HB matrix. X-ray diffraction (XRD) analysis showed that P3HB and HA coexist in the nanocomposite. Transmission electron microscopy (TEM) images also showed that the particle size of HA was 30 ～ 40 nm. The porosity of the scaffolds was 84%, and macropores and micropores coexisted and interconnected throughout the scaffolds. Acellular bioactivity experiments showed that more HA crystals formed on the surface of the nanocomposite scaffold than on the P3HB scaffold after 4 weeks immersion in Simulated Body Fluid (SBF). Cell culture experiments demonstrated that the P3HB/nHA nanocomposite scaffold had a better tendency of proliferation and Alkaline Phosphatase (ALP) activity to MG 63 cells than the pure P3HB scaffold. It was found that nHA addition can improve acellular and cellular bioactivity of the P3HB scaffold.     

Adsorption characteristics of P(3HB) depolymerase as evaluated by surface plasmon resonance and atomic force microscopy

Molecular recognition of poly[(R)-3-hydroxybutyrate] (P(3HB)) depolymerase from Ralstonia pickettii T1 to the surfaces of biodegradable aliphatic polyesters such as P(3HB) and poly(L-lactic acid) (PLLA) was examined from the viewpoints of kinetics and dynamics. To determine the kinetic parameters on the interaction between the substrate-binding domain (SBD) of P(3HB) depolymerase and various polymer substrates with different chemical structures, surface plasmon resonance (SPR) measurements were performed. On the other hand, using an atomic force microscopic (AFM) cantilever tip functionalized with the SBD of P(3HB) depolymerase, the mechanical parameters such as unbinding force to the polymer surfaces were measured. Both the SPR and AFM measurements showed that the SBD has a high affinity to P(3HB) and PLLA. From the results of kinetics and dynamics, the energy potential landscape of SBD-polymer interaction was disclosed on the basis of a phenomenological model, and the mechanism of the interaction was discussed.     

Hydroxyapatite surface modified by L-lactic acid and its subsequent grafting polymerization of L-lactide

A new method of surface modification of hydroxyapatite nanoparticles (n-HA) by surface grafting reaction of l-lactic acid and ring-opening polymerization of l-lactide (LLA) was developed. Two modified HA nanoparticles were obtained: HA modified by l-lactic acid (l-HA) and HA grafting with poly(l-lactide) (PLLA; p-HA). The modified surface of n-HA was attested by Fourier transformation infrared, (31)P MAS NMR, and thermal gravimetric analysis. The results showed that l-lactic acid could be easily grafted onto the n-HA surface by forming a Ca carboxylate bond and initiated by the hydroxyl group of the grafted l-lactic acid and LLA could be graft-polymerized onto the n-HA surface in the presence of stannous octanoate. The highest grafting amounts of l-lactic acid and PLLA were about 33 and 22 wt %, respectively. The modified HA/PLLA composites showed good mechanical properties and uniform microstructure. The tensile strength and modulus of the p-HA/PLLA composite containing 15 wt % of p-HA were 67 MPa and 2.1 GPa, respectively, while those of the n-HA/PLLA composites were 45 MPa and 1.7 GPa, respectively. The elongation at the break of the l-HA/PLLA composite containing 15 wt % l-HA could reach 44%, in comparison with 6.5% of the n-HA/PLLA composites containing 15 wt % n-HA.     

Nanoreinforcement of poly(propylene fumarate)-based networks with surface modified alumoxane nanoparticles for bone tissue engineering

A novel composite material has been fabricated for bone tissue engineering scaffolds utilizing the biodegradable polymer poly(propylene fumarate)/poly(propylene fumarate)-diacrylate (PPF/PPF-DA) and surface-modified carboxylate alumoxane nanoparticles. Various surface-modified nanoparticles were added to the polymer including a surfactant alumoxane, an activated alumoxane, a mixed alumoxane containing both activated and surfactant groups, and a hybrid alumoxane containing both groups within the same substituent. These nanocomposites, as well as polymer resin and unmodified boehmite composites, underwent flexural and compressive mechanical testing and were examined using electron microscopy. Hybrid alumoxane nanoparticles dispersed in PPF/PPF-DA exhibited over a 3-fold increase in flexural modulus at 1 wt % loading compared to polymer resin alone. No significant loss of flexural or compressive strength was observed with increased loading of hybrid alumoxane nanoparticles. These dramatic improvements in flexural properties may be attributed to the fine dispersion of nanoparticles into the polymer and increased covalent interaction between polymer chains and surface modifications of nanoparticles.     

Laccase from Aspergillus niger: A novel tool to graft multifunctional materials of interests and their characterization

In the present study, we propose a green route to prepare poly(3-hydroxybutyrate) [(P(3HB)] grafted ethyl cellulose (EC) based green composites with novel characteristics through laccase-assisted grafting. P(3HB) was used as a side chain whereas, EC as a backbone material under ambient processing conditions. A novel laccase obtained from Aspergillus niger through its heterologous expression in Saccharomyces cerevisiae was used as a green catalyst for grafting purposes without the use of additional initiator and/or cross-linking agents. Subsequently, the resulting P(3HB)-g-EC composites were characterized using a range of analytical and imagining techniques. Fourier transform infrared spectroscopy (FT-IR) spectra showed an increase in the hydrogen-bonding type interactions between the side chains of P(3HB) and backbone material of EC. Evidently, X-ray diffraction (XRD) analysis revealed a decrease in the crystallinity of the P(3HB)-g-EC composites as compared to the pristine individual polymers. A homogeneous P(3HB) distribution was also achieved in case of the graft composite prepared in the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a mediator along with laccase as compared to the composite prepared using pure laccase alone. A substantial improvement in the thermal and mechanical characteristics was observed for grafted composites up to the different extent as compared to the pristine counterparts. The hydrophobic/hydrophilic properties of the grafted composites were better than those of the pristine counterparts.     

Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli

In order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB)s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB)s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers.     

Novel biobased nanocomposites from soybean oil and functionalized organoclay

Novel biobased nanocomposites have been prepared by the cationic polymerization of conjugated soybean oil (CSOY) or conjugated LoSatSoy oil (CLS) with styrene (ST) and divinylbenzene (DVB), and a reactive organomodified montmorillonite (VMMT) clay as a reinforcing phase. This filler has been prepared by the cationic exchange of sodium montmorillonite with (4-vinylbenzyl)triethylammonium chloride in aqueous solution. The nanostructures of the nanocomposites have been determined by using wide-angle X-ray diffraction (WAXD) and transmission electron microscopy (TEM), respectively. The results from WAXD and TEM indicate that a heterogeneous structure consisting of intercalation and partial exfoliation or an intercalation structure exists in the nanocomposites, depending on the amount of VMMT in the polymer matrix. The thermal, mechanical, and organic vapor barrier properties of the nanocomposites have been evaluated by dynamic thermal analysis, thermogravimetric analysis, mechanical testing, and toluene absorption. A significant improvement is observed in the thermal stability, the dynamic bending storage modulus, the compressive modulus, the compressive strength, the compressive strain at failure, and the vapor barrier performance for the CSOY-- and CLS-based nanocomposites with 1-2 wt % VMMT loading, where some individual exfoliated silicate platelets occur. For example, the CLS-based nanocomposite with 1-2 wt % VMMT exhibits increases of 100-128%, 86-92%, and 5-7% in compressive modulus, compressive strength, and compressive strain at failure, respectively. CLS with higher unsaturation and reactivity affords nanocomposites with higher thermal stability and higher mechanical properties than CSOY.     

Renewable resource-based green composites from recycled cellulose fiber and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) bioplastic

Novel "green" composites were successfully fabricated from recycled cellulose fibers (RCF) and a bacterial polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by melt mixing technique. Various weight contents (15%, 30%, and 40%) of the fibers were incorporated in the PHBV matrix. The effect of the fiber weight contents on the thermal, mechanical, and dynamic-mechanical thermal properties of PHBV was investigated and a comparative property analysis was performed with RCF-reinforced polypropylene (PP) composites. The tensile and storage moduli of the PHBV-based composites were improved by 220% and 190%, respectively, by reinforcement with 40 wt % RCF. Halpin-Tsai and Tsai-Pagano's equations were applied for the theoretical modeling of the tensile modulus of PHBV-based composites. The heat deflection temperature (HDT) of the PHBV-based composites was increased from 105 to 131 degrees C, while the coefficient of linear thermal expansion (CLTE) value was reduced by 70% upon reinforcement with 40 wt % RCF. The PHBV-based composites had also shown better tensile and storage moduli and lower CLTE values than PP-based composites. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) were used to study the melting behavior, thermal stability, and morphology of the composite systems, respectively.     

Contributions of polystyrene to the mechanical properties of blended mixture of old newspaper and wood pulp

Composites from recycled newspaper would result in the effective use of the waste product which is currently burned or land-filled, as well as potential reduction in the cost of manufactured composite. In this work, old newspaper (ONP) together with yellowish wood pulp and waste polystyrene from packaging were used to produce composite. The technique studied in this work is an alternative to the conventional melt compounding and was expected to provide efficient wetting of fibers by the polymer. Polystyrene was grafted with acrylonitrile, ethylmethacrylate and butylmethacrylate, respectively, using benzoyl peroxide as an initiator. The amount of polystyrene to monomer is 1:0.75 and to initiator is 1:1. The grafted copolymers were characterized using thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC). Different ratios of waste polystyrene or grafted waste polystyrene were mixed with a blend of old newsprint and wood pulp to form composites. The mechanical properties of these composites as well as water uptake were studied. The tensile properties of the prepared composites did not show essential improvement, except for the modulus of elasticity. Scanning electron microscopy indicate that composites with grafted polystyrene showed more homogeneity than the composite with polystyrene and also than blank, so the grafted polymer is distributed very well improving the mechanical properties of the composites. Strong adhesion between the fiber and grafted polymer was found.     

Production of copolymer poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) through a one-step cultivation process

A one-step cultivation process for the production of biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by Cupriavidus sp. USMAA2-4 was carried out using various carbon sources. It was found that Cupriavidus sp. USMAA2-4 could produce approximately 44 wt.% copolymer of P(3HB-co-4HB) with 27 mol% 4HB composition when the combination of oleic acid and 1,4-butanediol are used as carbon sources in 60 h cultivation. The manipulation of carbon-to-nitrogen ratio (C/N) resulted in the increase of dry cell weight, PHA content as well as 4HB composition. A new strategy of introducing oleic acid and 1,4-butanediol together and separately at different concentration demonstrated different yield in PHA content ranging from 47 to 58 wt.%. The molecular weight obtained was 234 kDa (by adding 1,4-butanediol and oleic acid together) and 212 kDa (by adding 1,4-butanediol separately). The copolymer of P(3HB-co-4HB) produced by Cupriavidus sp. USMAA2-4 was detected statistically as a random copolymer when analysed by nuclear magnetic resonance (NMR) spectroscopy.     

Poly(3-hydroxubutyrate-co-4-hydroxubutyrate)/bacterial cellulose composite porous scaffold: Preparation, characterization and biocompatibility evaluation

Bio-composite scaffolds were prepared by freeze-drying using poly(3-hydroxubutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) and bacterial cellulose (BC) as raw materials and trifluoroacetic acid (TFA) as co-solvent. The characteristics of the composite scaffold were investigated by field emission scanning electron microscopy (FESEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), water contact angle measurement and tensile testing. Preliminary biodegradation test was performed for P(3HB-co-4HB) and P(3HB-co-4HB)/BC composite scaffold in buffer solution and enzyme solution. The biocompatibility of the composite scaffold was preliminarily evaluated by cell adhesion studies using Chinese Hamster Lung (CHL) fibroblast cells. The cells incubated with composite scaffold for 48 h were capable of forming cell adhesion and proliferation, which showed better biocompatibility than pure P(3HB-co-4HB) scaffold. Thus, the prepared P(3HB-co-4HB)/BC composite scaffold was bioactive and may be suitable for cell adhesion/attachment suggesting that these scaffolds can be used for wound dressing or tissue-engineering scaffolds.     

Simultaneously Enhancing the Thermal Stability,Mechanical Modulus,and Electrochemical Performance of Solid Polymer Electrolytes by Incorporating 2D Sheets

Solid state electrolytes are the key components for high energy density lithium ion batteries and especially for lithium metal batteries where lithium dendrite growth is an inevitable obstacle in liquid electrolytes. Solid polymer electrolytes based on a complex of polymers and lithium salts are intrinsically advantageous over inorganic electrolytes in terms of processability and film‐forming properties. But other properties such as ionic conductivity, thermal stability, mechanical modulus, and electrochemical stability need to be improved. Herein, for the first time, 2D additives using few‐layer vermiculite clay sheets as an example to comprehensively upgrade poly(ethylene oxide)‐based solid polymer electrolyte are introduced. With clay sheet additives, the polymer electrolyte exhibits improved thermal stability, mechanical modulus, ionic conductivity, and electrochemical stability along with reduced flammability and interface resistance. The composite polymer electrolyte can suppress the formation and growth of lithium dendrites in lithium metal batteries. It is anticipated that the clay sheets upgraded solid polymer electrolyte can be integrated to construct high performance solid state lithium ion and lithium metal batteries with higher energy and safety.     

骨髓间充质干细胞与聚3-羟基丁酸酯-co-4-羟基丁酸酯生物材料共培养细胞补片的初步研究

目的:研究聚3-羟基丁酸酯-co-4-羟基丁酸酯[P(3HB-co-4HB)]这种新型高分子材料与骨髓间充质干细胞(BMSCs)共培养,观察材料对干细胞的存活及增殖的影响,形成细胞补片的效果;从而找到一种适合BMSCs生长、增殖的高分子生物材料,作为治疗心肌梗死,软骨损伤等多种组织损伤疾病的修复方法之一。方法:取清洁级雄性健康BSL-C57小鼠作为实验对象,通过分离培养获得小鼠BMSCs,并进行流式细胞仪鉴定表面标志物。BMSCs培养至5代后,将BMSCs与P(3HB-co-4HB)制成的生物材料薄膜共培养,24h后固定进行电镜扫描,并用DAPI荧光染料染色处理,在荧光显微镜下观察并进行细胞计数,并描绘生长曲线。结果:BMSCs流式细胞术鉴定:CD34、CD45阴性,CD90弱阳性,CD73阳性。扫描电镜下,P(3HB-co-4HB)材料与BMSCs共培养形成的细胞补片,其表面细胞数量多,细胞状态正常。荧光显微镜下,对其细胞补片表面的细胞进行计数,并绘制生长曲线,显示表面细胞有逐渐增多的趋势。结论:P(3HB-co-4HB)材料与BMSCs共培养制成的细胞补片表面有细胞存活及增殖,由于P(3HB-co-4HB)材料本身具有良好的生物组织相容性及可降解等性质,所以该新型高分子材料可以作为干细胞治疗多种疾病的支架材料之一。     

Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers

In support of programs to identify polyhydroxyalkanoates with improved materials properties, we report on our efforts to characterize the mechanical and thermal properties of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). The copolyesters, having molar fraction of 3HHx ranging from 2.5 to 35 mol % and average molecular weights ranging from 1.15 x 10(5) to 6.65 x 10(5), were produced by fermentation using Aeromonas hydrophila and a recombinant strain of Pseudomonas putida GPp104. The polymers were chloroform extracted and characterized by solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy and a variety of mechanical and thermal tests. Solution-state (1)H NMR data were used to determine polymer composition-of-matter, while solution-state (13)C NMR data provided polymer-sequence information. Solvent fractionation and NMR spectroscopic characterization of these polymers showed that polymers containing up to 9.5 mol % 3HHx had a Bernoullian compositional distribution. By contrast, polymers containing more than 9.5 mol % 3HHx had a bimodal polymer composition. Solvent fractionation of these 3HHx-rich polyesters produced two polymer fractions, each of which was again consistent with Bernoullian polymerization statistics. Solid-state NMR relaxation experiments provided insight into aging in poly(3HB-co-3HHx) copolymers, demonstrating increased polymer-chain motion with increasing 3HHx content. The elongation-to-break ratio in the polyesters increased with increasing molar fraction of 3HHx monomers. Aging properties of the poly(3HB-co-3HHx) copolymers were very similar to copolymers of 3HB and 3-hydroxyvalerate (3HV). However, poly(3HB-co-3HHx) exhibited increased activation energy to thermal degradation with increasing 3HHx content.     

Biodegradability and mechanical properties of poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate)-starch blends.

Wheat starch granules and poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate) [P(HB-co-HV), (19.1 mol% HV)] were blended at 160 degrees C. Increasing the starch content from 0 to 50% (wt/wt) decreased the tensile strength of P(HB-co-HV) from 18 MPa to 8 MPa and diminished flexibility as Young's modulus increased from 1,525 MPa to 2,498 MPa, but overall mechanical properties of the polymer remained in a useful range. A mixed microbial culture required more than 20 days to degrade 150-microns-thick samples of 100% P(HB-co-HV), whereas samples containing 50% (wt/wt) starch disappeared in fewer than 8 days. Starch granules degraded before P(HB-co-HV) did. Aerobic degradation proceeded more rapidly than anaerobic degradation.     

Nonhydrolytic fragmentation of a poly[(R)-3-hydroxybutyrate] single crystal revealed by use of a mutant of polyhydroxybutyrate depolymerase

This paper reports the initial process of the enzymatic degradation of solution-grown lamellar single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1. We used a hydrolytic-activity-disrupted mutant of the PHB depolymerase in order to avoid the influence of hydrolytic reaction in the system. The effect of addition of the mutant enzyme upon the P(3HB) single crystals was investigated by turbidimetric assay, high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Suspension turbidity of the P(3HB) single crystals increased after addition of the mutant enzyme having no hydrolytic activity. No soluble product from the P(3HB) single crystals with the mutant enzyme was detected by HPLC. AFM observation of the P(3HB) single crystals adsorbed on highly ordered pyrolytic graphite revealed that the mutant enzyme yielded a lot of lengthwise crystal fragments from the P(3HB) single crystals. On the basis of these results, we concluded that the mutant enzyme disturbs the molecular packing of the P(3HB) polymer chain around the loose chain packing region in the single crystal, resulting in the fragmentation. Therefore, it is suggested that the enzymatic degradation of P(3HB) single crystals with a wild-type PHB depolymerase progresses via three steps: (1) adsorption of the enzyme onto the surface of the single crystal; (2) disturbance of the molecular packing of P(3HB) polymer chain in the single crystal by the adsorbed enzyme; and (3) hydrolysis of the disturbed polymer chain by the adsorbed enzyme.     

Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters

Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_Ps) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_Ps that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436–2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_Ps was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_Ps mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.     

Intracellular degradation of poly(3-hydroxybutyric acid) accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [P(3HB)] accounting 69% of cell dry weight (CDW) from glucose during growth in nitrogen-free Stockdale medium. Degradation of the accumulated polymer by the organism was studied under carbon-free medium following two-step cultivation method. P(3HB) content of cells decreased rapidly from 69% to 4.8% of CDW after 35 h under carbon-deprived condition. Autodigestion of P(3HB) was evident from the estimation of intracellular P(3HB) depolymerase (i-depolymerase) activity in cell-free extract using artificial P(3HB) granules as substrate. Polymer content decreased rapidly along with the increase in i-depolymerase activity and rate of polymer degradation when medium was supplemented with (NH4)2SO4 at 0.1% (w/v) level. However, the effects were reverse when organic nitrogenous substrate, beef extract at similar concentration was present in the medium. The optimum temperature and pH for i-depolymerase activity were 35 degrees C and 7.7 respectively. The oxygen-limiting condition (culture volume per flask volume, 50%) decreased 10.7% activity of i-depolymerase over control resulting a slow P(3HB) degradation. The presence of NaCl (6 x 10(3) microg/ml) showed a positive effect on i-depolymerase whereas EDTA (40 microg/ml) resulted in 20% less activity. Furthermore, the intracellular degradation of P(3HB) decreased the intrinsic viscosity, molecular weight and tensile strength of the accumulated polymer.     

Injectable nanocomposites of single-walled carbon nanotubes and biodegradable polymers for bone tissue engineering

We have investigated the dispersion of single-walled carbon nanotubes (SWNTs) and functionalized SWNTs (F-SWNTs) in the unsaturated, biodegradable polymer poly(propylene fumarate) (PPF) and examined the rheological properties of un-cross-linked nanocomposite formulations as well as the electrical and mechanical properties of cross-linked nanocomposites. F-SWNTs were produced from individual SWNTs by a diazonium-based method and dispersed better than unmodified SWNTs in both un-cross-linked and cross-linked PPF matrix. Cross-linked nanocomposites with F-SWNTs were superior to those with unmodified SWNTs in terms of their mechanical properties. Specifically, nanocomposites with 0.1 wt % F-SWNTs loading resulted in a 3-fold increase in both compressive modulus and flexural modulus and a 2-fold increase in both compressive offset yield strength and flexural strength when compared to pure PPF networks, whereas the use of 0.1 wt % SWNTs gained less than 37% mechanical reinforcement. These extraordinary mechanical enhancements considered together with Raman scattering and sol fraction measurements indicate strong SWNT-PPF interactions and increased cross-linking densities resulting in effective load transfer. With enhanced mechanical properties and capabilities of in situ injection and cross-linking, these SWNT/polymer nanocomposites hold significant implications for the fabrication of bone tissue engineering scaffolds.     

Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava

A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.