Scalable production of mechanically tunable block polymers from sugar.

Development of sustainable and biodegradable materials is essential for future growth of the chemical industry. For a renewable product to be commercially competitive, it must be economically viable on an industrial scale and possess properties akin or superior to existing petroleum-derived analogs. Few biobased polymers have met this formidable challenge. To address this challenge, we describe an efficient biobased route to the branched lactone, ß-methyl-δ-valerolactone (ßMδVL), which can be transformed into a rubbery (i.e., low glass transition temperature) polymer. We further demonstrate that block copolymerization of ßMδVL and lactide leads to a new class of high-performance polyesters with tunable mechanical properties. Key features of this work include the creation of a total biosynthetic route to produce ßMδVL, an efficient semisynthetic approach that employs high-yielding chemical reactions to transform mevalonate to ßMδVL, and the use of controlled polymerization techniques to produce well-defined PLA-PßMδVL-PLA triblock polymers, where PLA stands for poly(lactide). This comprehensive strategy offers an economically viable approach to sustainable plastics and elastomers for a broad range of applications.

Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission.

Collective migration of epithelial cells is an integral part of embryonic development, wound healing, tissue renewal and carcinoma invasion. While previous studies have focused on cell-extracellular matrix adhesion as a site of migration-driving, traction force-transmission, cadherin mediated cell-cell adhesion is also capable of force-transmission. Using a soft elastomer coated with purified N-cadherin as a substrate and a Hepatocyte Growth Factor-treated, transformed MDCK epithelial cell line as a model system, we quantified traction transmitted by N-cadherin-mediated contacts. On a substrate coated with purified extracellular domain of N-cadherin, cell surface N-cadherin proteins arranged into puncta. N-cadherin mutants (either the cytoplasmic deletion or actin-binding domain chimera), however, failed to assemble into puncta, suggesting the assembly of focal adhesion like puncta requires the cytoplasmic domain of N-cadherin. Furthermore, the cytoplasmic domain deleted N-cadherin expressing cells exerted lower traction stress than the full-length or the actin binding domain chimeric N-cadherin. Our data demonstrate that N-cadherin junctions exert significant traction stress that requires the cytoplasmic domain of N-cadherin, but the loss of the cytoplasmic domain does not completely eliminate traction force transmission.

Engineered bi-histidine metal chelation sites map the structure of the mechanical unfolding transition state of an elastomeric protein domain GB1.

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The φ(M2+)(U)-value is defined as ΔΔG(‡-N)/ΔΔG(U-N), which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG(‡-N)) relative to its thermodynamic stability (ΔG(U-N)) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify φ(M2+)(U)-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1's mechanical unfolding transition state, the interface between force-bearing ß-strands 1 and 4 is largely disrupted, and the first ß-hairpin is partially disordered while the second ß-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.

Photo-cross-linked poly(ethylene carbonate) elastomers: synthesis, in vivo degradation, and determination of in vivo degradation mechanism.

Low-molecular-weight poly(ethylene carbonate) diols of varying molecular weight were generated through catalyzed thermal degradation of high-molecular-weight poly(ethylene carbonate). These polymers were then end functionalized with acrylate groups. The resulting α,ω-diacrylates were effectively photo-cross-linked upon exposure to long-wave UV light in the presence of a photoinitiator to yield rubbery networks of low sol content. The degree of cross-linking effectively controlled the in vivo degradation rate of the networks by adherent macrophages; higher cross-link densities yielded slower degradation rates. The cross-link density did not affect the number of adherent macrophages at the elastomer/tissue interface, indicating that cross-linking affected the susceptibility of the elastomer to degradative species released by the macrophages. The reactive species likely responsible for in vivo degradation appears to be superoxide anion, as the in vivo results were in agreement with in vitro degradation via superoxide anion, while cholesterol esterase, known to degrade similar poly(alkylene carbonate)s, had no affect on elastomer degradation.

Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction.

Microenvironmental biophysical factors play a fundamental role in controlling cell behaviors including cell morphology, proliferation, adhesion and differentiation, and even determining the cell fate. Cells are able to actively sense the surrounding mechanical microenvironment and change their cellular morphology to adapt to it. Although cell morphological changes have been considered to be the first and most important step in the interaction between cells and their mechanical microenvironment, their regulatory network is not completely clear. In the current study, we generated silicon-based elastomer polydimethylsiloxane (PDMS) substrates with stiff (15:1, PDMS elastomer vs. curing agent) and soft (45:1) stiffnesses, which showed the Young's moduli of ~450 kPa and 46 kPa, respectively, and elucidated a new path in cytoskeleton re-organization in chondrocytes in response to changed substrate stiffnesses by characterizing the axis shift from the secreted extracellular protein laminin ß1, focal adhesion complex protein FAK to microfilament bundling. We first showed the cellular cytoskeleton changes in chondrocytes by characterizing the cell spreading area and cellular synapses. We then found the changes of secreted extracellular linkage protein, laminin ß1, and focal adhesion complex protein, FAK, in chondrocytes in response to different substrate stiffnesses. These two proteins were shown to be directly interacted by Co-IP and colocalization. We next showed that impact of FAK on the cytoskeleton organization by showing the changes of microfilament bundles and found the potential intermediate regulators. Taking together, this modulation axis of laminin ß1-FAK-microfilament could enlarge our understanding about the interdependence among mechanosensing, mechanotransduction, and cytoskeleton re-organization.

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.

Tunable mechanical stability and deformation response of a resilin-based elastomer.

Resilin, the highly elastomeric protein found in specialized compartments of most arthropods, possesses superior resilience and excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and produced a modular, recombinant resilin-like polypeptide bearing both mechanically active and biologically active domains to create novel biomaterial microenvironments for engineering mechanically active tissues such as blood vessels, cardiovascular tissues, and vocal folds. Preliminary studies revealed that these recombinant materials exhibit promising mechanical properties and support the adhesion of NIH 3T3 fibroblasts. In this Article, we detail the characterization of the dynamic mechanical properties of these materials, as assessed via dynamic oscillatory shear rheology at various protein concentrations and cross-linking ratios. Simply by varying the polypeptide concentration and cross-linker ratios, the storage modulus G' can be easily tuned within the range of 500 Pa to 10 kPa. Strain-stress cycles and resilience measurements were probed via standard tensile testing methods and indicated the excellent resilience (>90%) of these materials, even when the mechanically active domains are intercepted by nonmechanically active biological cassettes. Further evaluation, at high frequencies, of the mechanical properties of these materials were assessed by a custom-designed torsional wave apparatus (TWA) at frequencies close to human phonation, indicating elastic modulus values from 200 to 2500 Pa, which is within the range of experimental data collected on excised porcine and human vocal fold tissues. The results validate the outstanding mechanical properties of the engineered materials, which are highly comparable to the mechanical properties of targeted vocal fold tissues. The ease of production of these biologically active materials, coupled to their outstanding mechanical properties over a range of compositions, suggests their potential in tissue regeneration applications.

Biodegradation of polyether-polyol-based polyurethane elastomeric films: influence of partial replacement of polyether polyol by biopolymers of renewable origin.

In this work we investigated the degradation process ofpolyether-polyol-based polyurethane (PUR) elastomeric films in the presence of a mixed thermophilic culture as a model of a natural bacterial consortium. The presence of PUR material in cultivation medium resulted in delayed but intensive growth of the bacterial culture. The unusually long lag phase was caused by the release of unreacted polyether polyol and tin catalyst from the material. The lag phase was significantly shortened and the biodegradability of PUR materials was enhanced by partial replacement (10%) of polyether polyol with biopolymers (carboxymethyl cellulose, hydroxyethyl cellulose, acetyl cellulose and actylated starch). The process of material degradation consisted of two steps. First, the materials were mechanically disrupted and, second, the bacterial culture was able to utilize abiotic degradation products, which resulted in supported bacterial growth. Direct utilization of PUR by the bacterial culture was observed as well, but the bacterial culture contributed only slightly to the total mass losses. The only exception was PUR material modified by acetyl cellulose. In this case, direct biodegradation represented the major mechanism of material decomposition. Moreover, PUR material modified by acetyl cellulose did not tend to undergo abiotic degradation. In conclusion, the modification of PUR by proper biopolymers is a promising strategy for reducing potential negative effects of waste PUR materials on the environment and enhancing their biodegradability.

Spatial mapping of the biomechanical properties of the pericellular matrix of articular cartilage measured in situ via atomic force microscopy.

In articular cartilage, chondrocytes are surrounded by a narrow region called the pericellular matrix (PCM), which is biochemically, structurally, and mechanically distinct from the bulk extracellular matrix (ECM). Although multiple techniques have been used to measure the mechanical properties of the PCM using isolated chondrons (the PCM with enclosed cells), few studies have measured the biomechanical properties of the PCM in situ. The objective of this study was to quantify the in situ mechanical properties of the PCM and ECM of human, porcine, and murine articular cartilage using atomic force microscopy (AFM). Microscale elastic moduli were quantitatively measured for a region of interest using stiffness mapping, or force-volume mapping, via AFM. This technique was first validated by means of elastomeric models (polyacrylamide or polydimethylsiloxane) of a soft inclusion surrounded by a stiff medium. The elastic properties of the PCM were evaluated for regions surrounding cell voids in the middle/deep zone of sectioned articular cartilage samples. ECM elastic properties were evaluated in regions visually devoid of PCM. Stiffness mapping successfully depicted the spatial arrangement of moduli in both model and cartilage surfaces. The modulus of the PCM was significantly lower than that of the ECM in human, porcine, and murine articular cartilage, with a ratio of PCM to ECM properties of approximately 0.35 for all species. These findings are consistent with previous studies of mechanically isolated chondrons, and suggest that stiffness mapping via AFM can provide a means of determining microscale inhomogeneities in the mechanical properties of articular cartilage in situ.

1,3-Butadiene: I. Review of metabolism and the implications to human health risk assessment.

1,3-Butadiene (BD) is a multisite carcinogen in laboratory rodents following lifetime exposure, with mice demonstrating greater sensitivity than rats. In epidemiology studies of men in the styrene-butadiene rubber industry, leukemia mortality is associated with butadiene exposure, and this association is most pronounced for high-intensity BD exposures. Metabolism is an important determinant of BD carcinogenicity. BD is metabolized to several electrophilic intermediates, including epoxybutene (EB), diepoxybutane (DEB), and epoxybutane diol (EBD), which differ considerably in their genotoxic potency (DEB >> EB > EBD). Important species differences exist with respect to the formation of reactive metabolites and their subsequent detoxification, which underlie observed species differences in sensitivity to the carcinogenic effects of BD. The modes of action for human leukemia and for the observed solid tumors in rodents are both likely related to the genotoxic potencies for one or more of these metabolites. A number of factors related to metabolism can also contribute to nonlinearity in the dose-response relationship, including enzyme induction and inhibition, depletion of tissue glutathione, and saturation of oxidative metabolism. A quantitative risk assessment of BD needs to reflect these species differences and sources of nonlinearity if it is to reflect the current understanding of the disposition of BD.

Degradation of ozonized tire rubber by aniline - Degrading Candida methanosorbosa BP6 strain.

Aniline-degrading yeast strain - Candida methanosorbosa BP-6 was tested for its ability to degrade ground tire rubber, treated and non-treated with ozone. The protein content, respiratory activity, critical oxygen concentration (COC) and emulsifying activity of the yeast strain were monitored during 21 day degradation process. The effects of biodegradation were evaluated using aldehyde detection, Scanning Electrone Microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR) analysis. Pre-treatment of ground tire rubber with ozone resulted in lower microbial growth. However, metabolic condition of the C. methanosorbosa BP-6 yeast strain was higher in sample with ozonized tire rubber. Furthermore, the COC values in the last days of the process were about 30% lower regarding non-ozonized polymer. Also, the ozonization of tire rubber resulted in higher biosurfactant production of the yeast strain. The roughness and visible gaps in rubber matrix (SEM analysis) confirmed the ability of Candida methanosorbosa BP-6 yeast strain for tire rubber biodegradation.

Development of metformin chain extended polyurethane elastomers as bone regenerative films.

In this study, novel elastomeric biodegradable bone regenerative films were developed from metformin (Met) and polyurethane (PU). Metformin was selected due to its osteogenic properties and proper chemical structure to react with PU prepolymer. Metformin was integrated into PU macromolecular structure as chain extender after the synthesis of PU prepolymer via condensation polymerization of polycaprolactone diol and hexamethylene diisocyanate. Chemical, thermal, viscoelastic properties of PU-Met films where characterized and discussed in terms of structure-property relationships. PU-Met films had Tg value around -45 °C and showed superior viscoelastic properties under 1 Hz and 10 Hz tensile oscillation frequencies during dynamic mechanical analysis. On the 21st day of biodegradation studies, PU-Met films degraded 2.3 ±â€¯0.1% and 37.8 ±â€¯4.2% in oxidative and enzymatic media, respectively. Cell-material interactions of elastomeric films were investigated by proliferation (MTT assay), alkaline phosphatase activity (ALP), calcium depositions (Alizarin Red Quantification) and morphological evaluations (SEM). Presence of metformin in PU formulation increased MC3T3-E1 attachment, proliferation and calcium deposition.

Synthesis and characterization of biodegradable elastomeric polyurethane scaffolds fabricated by the inkjet technique.

Biodegradable polyurethanes (PUs) were synthesized from methylene di-p-phenyl-diisocyanate (MDI), polycaprolactone diol (PCL-diol) and N,N-bis (2-hydorxyethyl)-2-aminoethane-sulfonic acid (BES), serving as a hard segment, soft segment and chain extender, respectively. MDI was chosen due to its reactivity and wide application in synthesis of biomedical polyurethanes due to its reactivity; PCL-diol was chosen because of its biodegradability; and BES was chosen because it allowed the introduction sulfonic acid groups onto the polymer chains. We evaluated the polyurethanes' degradation rate, mechanical properties, hydrophilicity, antithrombogenecity, and ability to support fibroblast cell attachment and growth by comparing with polymers having a 2,2-(methylimino)diethanol (MIDE) chain extender. Mechanical testing demonstrated that the PU containing BES has tensile strengths of about 17 MPa and elongations up to 400%, about three times the strength and four times the elongation than the MIDE based PUs. The polymers showed decreased in vitro degradation rates, lower glass transition temperature (T(g)) and hydrophilicity possibly due to enhanced microphase separation. Preliminary cytocompatibility studies showed that all the PUs are non-toxic, but PU containing BES exhibited much lower cell attachment and proliferation than the MIDE chain extended polymers. An in vitro platelet adhesion assay showed lower platelet attachment on BES containing PU. Additionally, due to the existence of sulfonic acid groups, the BES extended PU became water soluble in basic condition and insoluble in acidic condition, a phenomenon that is reversible at pH value of 8.7, making this a pH sensitive polymer attractive for bioprinting applications. By adding acetic acid into an inkjet cartridge and printing it onto a PU solution with pH above 8.7, precision fabricated scaffolds can be obtained, suggesting that BES based PUs are promising candidates as synthetic inks used for customizable fabrication of tissue engineering scaffolds.

Amino alcohol-based degradable poly(ester amide) elastomers.

Currently available synthetic biodegradable elastomers are primarily composed of crosslinked aliphatic polyesters, which suffer from deficiencies including (1) high crosslink densities, which results in exceedingly high stiffness, (2) rapid degradation upon implantation, or (3) limited chemical moieties for chemical modification. Herein, we have developed poly(1,3-diamino-2-hydroxypropane-co-polyol sebacate)s, a new class of synthetic, biodegradable elastomeric poly(ester amide)s composed of crosslinked networks based on an amino alcohol. These crosslinked networks feature tensile Young's modulus on the order of 1MPa and reversable elongations up to 92%. These polymers exhibit in vitro and in vivo biocompatibility. These polymers have projected degradation half-lives up to 20 months in vivo.

Transfer of CO2, N2O and CH4 to butyl rubber (polyisobutylene) septa during storage.

Greenhouse gases are more sampled than ever because of environmental interests. Gas samples are often inserted into vials with gas tight butyl rubber septa before concentration analysis. Little is known on the global transfer property of butyl rubber septa for CO2, N2O and CH4. Sorption kinetics were measured by injecting CO2, N2O or CH4 into glass vials with either one of four butyl rubber septa types and stored during 90 days. CO2 and N2O concentrations decreased during storage depending upon septa type and initial concentration, with the highest linear rate being 0.023 for CO2 and 0.0015 mg L(-3) day(-1) for N2O. When a low concentration was injected, CH4 concentration changes over time were small and did not differ between septa types. Sorption isotherms were measured using nine concentrations and stored during 45 days. CO2 sorption isotherms ranged from 0 to 3.7 x 10(-3) m(3) m(-2) and N2O from 0.3 to 1.4 x 10(-3) m(3) m(-2). Examples of errors associated with the use of these butyl rubber septa are given.

Star-shaped polyester-based elastomers as an implantable delivery system for insulin: Development, pharmacokinetics, pharmacodynamics, and biocompatibility.

Elastomers are largely developed for biomedical applications; however, little is reported on them although they are an effective and controllable delivery system for proteins. In the present study, we investigated the pharmacokinetics, biosecurity, and hypoglycemic effect of an insulin-loaded elastomer formulation in diabetic rats. Cylindrical insulin-loaded elastomers were fabricated using a UV cross-linking process based on methyl-acrylic-star-poly(ε-caprolactone-co-D,L-lactide) cyclic ester and methyl-bi-acrylic-poly(ε-caprolactone-b-polyethylene glycol-b-ε-caprolactone) (CLPEGCLMA). The encapsulated insulin was well protected during the formulation. An in vitro pharmacokinetic study revealed that the rate of insulin release from the elastomers was affected by the hydrophilicity/hydrophobicity of the system and controlled by the CLPEGCLMA (hydrophilic prepolymer) composition. It was observed that insulin release followed the Higuchi model. In addition, the more hydrophilic elastomers showed higher degradation rates in vivo. Furthermore, in the pharmacodynamic study, all the elastomers, except those that contained star-poly(ε-caprolactone-co-D,L-lactide) (number-average molecular weight, Mn), polyethylene glycol (PEG) (kMn), ε-caprolactone/PEG (mol/mol), and CLPEGCLMA (weight, %) at a ratio of 3432:10:20:30, respectively, decreased blood glucose concentration and maintained it at a stable level. It was observed that the hypoglycemic effect of the drug-loaded elastomers was directly proportional to the rate of in vitro insulin release; however, emaciation was not observed. Moreover, elastomers play a positive role in biosecurity. Therefore, the elastomers might be effective carriers for the delivery of peptide drugs in the form of implants.

Cell adhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering.

Cardiac tissue engineering is focused on obtaining functional cardiomyocyte constructs to provide an alternative to cellular cardiomyoplasty. Mechanical stimuli have been shown to stimulate protein expression and the differentiation of mammalian cells from contractile tissues. Our aim was to obtain a flexible scaffold which could be used to apply mechanical forces during tissue regeneration. Poly(1,8-octanediol-co-citric acid) (POC) is an elastomer that can be processed into scaffolds for tissue engineering. We investigated the effect of modifying the porosity on the mechanical properties of the POC scaffolds. In addition, the effects of the storage method and strain rate on material integrity were assessed. The maximum elongation of POC porous films varied from 60% to 160% of their original length. A decrease in the porosity caused a rise in this elastic modulus. The attachment of HL-1 cardiomyocytes to POC was assessed on films coated with fibronectin, collagen and laminin. These extracellular matrix proteins promoted cell adhesion in a protein-type- and concentration-dependent manner. Therefore, POC scaffolds can be optimised to meet the mechanical and biological parameters needed for cardiac culture. This porous material has the potential to be used for cardiac tissue engineering as well as for other soft tissue applications.

Pilot scale production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) biopolymers with high molecular weight and elastomeric properties.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [(P(3HB-co-4HB)] copolymer receives attention as next generation biomaterial in medical application. However, the exploitation of the copolymer is still constrained since such copolymer has not yet successfully been performed in industrial scale production. In this work, we intended to establish pilot production system of the copolymer retaining the copolymer quality which has recently discovered to have novel characteristic from lab scale fermentation. An increase of agitation speed has significantly improved the copolymer accumulation efficiency by minimizing the utilization of substrates towards cell growth components. This is evidenced by a drastic increase of PHA content from 28 wt% to 63 wt% and PHA concentration from 3.1 g/L to 6.5 g/L but accompanied by the reduction of residual biomass from 8.0 g/L to 3.8 g/L. Besides, fermentations at lower agitation and aeration have resulted in reduced molecular weight and mechanical strength of the copolymer, suggesting the role of sufficient oxygen supply efficiency in improving the properties of the resulting copolymers. The KLa-based scale-up fermentation was performed successfully in maintaining the yield and the quality of the copolymers produced without a drastic fluctuation. This suggests that the scale-up based on the KLa values supported the fermentation system of P(3HB-co-4HB) copolymer production in single-stage using mixed-substrate cultivation strategy.

Microbial-based synthesis of highly elastomeric biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thermoplastic.

This study reports the production of P(3HB-co-4HB) [Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)] in possession of high molecular weight and elastomeric properties by Cupriavidus sp. USMAA1020 in single-stage mixed-substrate cultivation system. 1,4-butanediol and 1,6-hexanediol are found to be efficient substrate mixture that has resulted in high copolymer yield, occupying a maximum of 70wt% of the total biomass and producing higher 4HB monomer composition ranging from 31mol% to 41mol%. In substrate mixtures involving 1,6-hexanediol, cleavage of the 6-hydroxyhexanoyl-CoA produces Acetyl-CoA and 4-hydroxybutyryl-CoA. Acetyl-CoA is instrumental in initiating the cell growth in the single-stage fermentation system, preventing 4-hydroxybutyryl-CoA from being utilized via ß-oxidation and retained the 4HB monomer at higher ratios. Macroscopic kinetic models of the bioprocesses have revealed that the P(3HB-co-4HB) formation appears to be in the nature of mixed-growth associated with higher formation rate during exponential growth phase; evidenced by higher growth associated constants, α, from 0.0690g/g to 0.4615g/g compared to non-growth associated constants, ß, from 0.0092g/g/h to 0.0459g/g/h. The P(3HB-co-31mol% 4HB) produced from the substrate mixture exhibited high weight-average molecular weight, Mw of 927kDa approaching a million Dalton, and possessed elongation at break of 1637% upon cultivation at 0.56wt% C. This is the first report on such properties for the P(3HB-co-4HB) copolymer. The copolymer is highly resistant to polymer deformation after being stretched.

Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction / 国际口腔科学杂志·英文版

Microenvironmental biophysical factors play a fundamental role in controlling cell behaviors including cell morphology, proliferation, adhesion and differentiation, and even determining the cell fate. Cells are able to actively sense the surrounding mechanical microenvironment and change their cellular morphology to adapt to it. Although cell morphological changes have been considered to be the first and most important step in the interaction between cells and their mechanical microenvironment, their regulatory network is not completely clear. In the current study, we generated silicon-based elastomer polydimethylsiloxane (PDMS) substrates with stiff (15:1, PDMS elastomer vs. curing agent) and soft (45:1) stiffnesses, which showed the Young's moduli of ~450 kPa and 46 kPa, respectively, and elucidated a new path in cytoskeleton re-organization in chondrocytes in response to changed substrate stiffnesses by characterizing the axis shift from the secreted extracellular protein laminin β1, focal adhesion complex protein FAK to microfilament bundling. We first showed the cellular cytoskeleton changes in chondrocytes by characterizing the cell spreading area and cellular synapses. We then found the changes of secreted extracellular linkage protein, laminin β1, and focal adhesion complex protein, FAK, in chondrocytes in response to different substrate stiffnesses. These two proteins were shown to be directly interacted by Co-IP and colocalization. We next showed that impact of FAK on the cytoskeleton organization by showing the changes of microfilament bundles and found the potential intermediate regulators. Taking together, this modulation axis of laminin β1-FAK-microfilament could enlarge our understanding about the interdependence among mechanosensing, mechanotransduction, and cytoskeleton re-organization.