Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) supports adhesion and migration of mesenchymal stem cells and tenocytes

AIM: To establish the potential of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) as a material for tendon repair. METHODS: The biocompatibility of PHBHHx with both rat tenocytes (rT) and human mesenchymal stem cells (hMSC) was explored by monitoring adhesive characteristics on films of varying weight/volume ratios coupled to a culture atmosphere of either 21% O2 (air) or 2% O2 (physiological normoxia). The diameter and stiffness of PHBHHx films was established using optical coherence tomography and mechanical testing, respectively. RESULTS: Film thickness correlated directly with weight/volume PHBHHx (r2 = 0.9473) ranging from 0.1 mm (0.8% weight/volume) to 0.19 mm (2.4% weight/volume). Film stiffness on the other hand displayed a biphasic response which increased rapidly at values > 1.6% weight/volume. Optimal cell attachment of rT required films of ≥ 1.6% and ≥ 2.0% weight/volume PHBHHx in 2% O2 and 21% O2 respectively. A qualitative adhesion increase was noted for hMSC in films ≥ 1.2% weight/volume, becoming significant at 2% weight/volume in 2% O2. An increase in cell adhesion was also noted with ≥ 2% weight/volume PHBHHx in 21% O2. Cell migration into films was not observed. CONCLUSION: This evaluation demonstrates that PHBHHx is a suitable polymer for future cell/polymer replacement strategies in tendon repair.

Silk fibroins modify the atmospheric low temperature plasma-treated poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) film for the application of cardiovascular tissue engineering

Tissue engineered scaffold is one of the hopeful therapies for the patients with organ or tissue damages. The key element for a tissue engineered scaffold material is high biocompatibility. Herein the poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) film was irradiated by the low temperature atmospheric plasma and then coated by the silk fibroins (SF). After plasma treatment, the surface of PHBHHx film became rougher and more hydrophilic than that of original film. The experiment of PHBHHx flushed by phosphate buffer solution (PBS) proves that the coated SF shows stronger immobilization on the plasma-treated film than that on the untreated film. The cell viability assay demonstrates that SF-coated PHBHHx films treated by the plasma significantly supports the proliferation and growth of the human smooth muscle cells (HSMCs). Furthermore, the scanning electron microscopy and hemotoylin and eosin (HE) staining show that HSMCs formed a cell sub-monolayer and secreted a large amount of extracellular matrix (ECM) on the films after one week's culture. The silk fibroins modify the plasma-treated PHBHHx film, providing a material potentially applicable in the cardiovascular tissue engi-neering.

Characterization of 3D Printed Poly(3-Hydroxybutyric-Co-3-Hydroxyvalerate) by Fused Granular Fabrication through Thermal and Mechanical Analyses

Poly[R-3-hydroxybutyrate-co-(R-3-hydroxyvalerate)] (PHBVs) copolymers are promising biopolymers, which could substitute petroleum-based plastics for various applications. PHB and PHBV pellets were processed on a customized 3D printer via Fused Granular Manufacturing (FGM) approach modified with a Mahor screw extruder. To anticipate the behaviour of PHBVs when transformed using conventional thermo-mechanical shaping processes, thermal and mechanical analyses were carried out in order to better understand the effect of annealing temperature on their crystallization behaviour and mechanical properties of PHB polymer and PHBV copolymer. The objectives of the present work were to propose an experimental strategy to study the melting and crystallization events, crystalline structure changes, and mechanical performances of both PHB homopolymer and PHBV copolymer according to identical thermal annealing treatments. A monitoring of 3D printed PHB and PHBV structures was achieved by coupling Differential Scanning Calorimetry (DSC) and tensile tests. .

BIOSYNTHESIS AND THERMAL PROPERTIES OF POLY(3-HYDROXYBUTYRATE-co-3-HYDROXYVALERATE)WITH LARGE VARIETY OF HYDROXYVALERATE CONTENTS BY BACILLUS CEREUS

Biosynthesis and thermal properties of poly（3-hydroxybutyrate-co-3-hydroxyvalerate） （PHBV） with different HV （hydrovalerate） content produced by a Bacillus cereus strain were investigated. A large variety of HV contents （up to about 90 mol%） of PHBV could be produced by this strain. Combined nitrogen sources containing both yeast extract and ammonium sulphate were better for cell growth and polyhydroxyalkanoates （PHA） production than either yeast extract or ammonium sulphate alone. Propionic acid is more favorable for the production of HV content than that of valeric acid. Finally, thermal properties of PHBV produced by this strain are found close to the results of other groups.

STUDY ON BIODEGRADABILITY OF POLY (3-HYDROXYBUTYRATE-co-3-HYDROXYVALERATE)/ORGANOPHILIC MONTMORILLONITE NANOCOMPOSITES

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/Organophilic montmorillonite (PHBV/OMMT) nanocomposites were prepared and the biodegradability of the PHBV/OMMT nanocomposites was studied by a cultivation degrading method in soil suspension. The relationship between structure and biodegradability of PHBV/OMMT nanocomposites was investigated. The results showed that the biodegradability of PHBV/OMMT nanocomposites decreased with increasing amount of OMMT and it was related to the number of PHBV degrading microorganisms in degradation environment, the anti-microbial property of OMMT and the degree of crystallinity of the nanocomposites.

嗜水气单胞菌胞内聚3-羟基丁酸-3-羟基己酸酯(PHBHHx)提取方法的研究

以嗜水气单胞菌利用月桂酸发酵生产PHBHHx,比较多种溶剂对提取率和产品质量的影响,建立了嗜水气单胞菌胞内提取PHBHHx的新方法。本方法使用湿细胞提取,节省干燥细胞的能耗,仲丁醇无异味、毒性小、价格低廉,而且可以循环多次利用、对相对分子质量影响小,解决了工业生产上能耗高,生产成本高,溶剂回收难,安全隐患多,提取率不高、产品质量低等问题。用仲丁醇作溶剂,预处理细胞提取率提高13%,相对分子质量达83万,提取率达78%,产品纯度达99%以上。

3-羟基丁酸-3-羟基己酸共聚酯纳米颗粒作为雷帕霉素缓释载体的应用研究

目的研究生物可降解高分子材料3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)作为疏水性药物雷帕霉素缓释载体的效果及其体外条件下对疏水性药物的缓释情况。方法超声乳化分散法制备PHBHHx载药纳米微球,颗粒尺度分析仪对颗粒粒径进行表征,透析袋法进行雷帕霉素体外缓释,高效液相色谱法检测雷帕霉素释放量。用包裹雷帕霉素的PHBHHx纳米颗粒处理体外培养的PC3细胞,MTT法检测细胞活力,Western blot检测mTOR底物p70S6K磷酸化情况。结果包裹雷帕霉素的PHBHHx纳米颗粒平均粒径为211.4nm,对雷帕霉素的包封率为92.5%,包裹雷帕霉素的PHBHHx纳米颗粒可以实现雷帕霉素的体外缓释,且能够抑制人前列腺癌细胞系PC3的体外增殖和胞内p70S6K的磷酸化。结论 PHBHHx是一种很有潜力的疏水性药物包封和递送载体材料。

3-羟基丁酸与3-羟基己酸共聚酯在心血管组织工程中的应用

为了研究可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯(3-hydroxybutyrate-co-3-hydroxyhexanoate,PHBHHx)的血管内生物相容性,采用脱细胞羊肺动脉为支架,以PHBHHx涂层,构建复合补片(Hybrid patch),植入New Zealand兔腹主动脉内(12只),以脱细胞未涂层羊肺动脉片(Uncoated patch)做为对照(12只)。分别于术后第1、4和12周处死动物,取出移植补片进行组织学、免疫荧光染色、扫描电镜和钙含量测定。结果表明:hybrid patch管腔面光滑无血栓,内膜增生适度,再细胞化完全:免疫荧光染色检测,新生内膜组织中类内皮细胞呈CD31阳性反应,单层连续排列,间质细胞呈现SMA阳性反应;钙含量测定,hybrid patch明显低于uncoated patch(P<0.05)。由此认为:PHBHHx的血管内生物相容性满意,是心血管组织工程较为理想的腔内涂层材料。

Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) Using Aeromonas hydrophila 4AK4 Grown in Mixed Carbon Source

Aeromonas hydrophila 4AK4 was grown on mixed substrates of soybean oil and lauric acid for the production of polyhydroxyalkanoate copolymer consisting of 3 hydroxybutyrate (3HB) and 3 hydroxyhexanoate (3HHx). A maximal poly(3 hydroxybutyrate co 3 hydroxyhexanoate) (PHBHHx) content of 49.13% in dry cells was obtained in a shake flask culture. PHBHHx of 6.26 g/L was produced in a fermentation experiment over 48 h on a sole carbon source containing 100 g/L soybean oil, while 12.40 g/L PHBHHx was produced on a mixed carbon source containing 80 g/L soybean and 20 g/L lauric acid over the same period of time, resulting in a polyhydroxyalkanoate (PHA) productivity of 0.25 g/(L·h). The results show that mixed carbon sources are suitable for industrialized production of PHBHHx from A. hydrophila 4AK4, as the mixed carbon sources also overcome the foaming problem that occurs when lauric acid is employed as a sole carbon source in PHBHHx production.

The Effects of Accelerated Photooxidation on Molecular Weight and Thermal and Mechanical Properties of PHBV/Cloisite 30B Bionanocomposites

The effects of accelerated photooxidation on the molecular weight and thermal and mechanical properties of Cast PHBV and PHBV/Cloisite 30B(3 wt%)bionanocomposites are investigated herein.Through size exclusion chromatography(SEC)analysis,a significant decrease in both weight and number average molecular weights was observed for all irradiated samples over time,resulting from the chain scission mechanism.Differential scanning calorimetry(DSC)data indicated a decrease in degree of crystallinity and melting temperature after UV exposure,with the appearance of double melting peaks related to the changes in the crystal structure of PHBV.Thermal stability,tensile and thermo-mechanical properties were also reduced consecutively in photooxidation,being more pronounced for Cast PHBV.This study shows that the incorporation of Cloisite 30B in PHBV provides a better resistance to photooxidation in comparison with the neat polymer.

Effects of Quenching Temperature and Time on Pore Diameter of Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) Porous Scaffolds and MC3T3-E1 Osteoblast Response to the Scaffolds

Poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） （PHBHHx） scaffolds were prepared by thermally inducing phase separation （TIPS） for bone reconstruction. Scanning electron microscopy and porosity measurements were used to analyze the structure and properties of the scaffolds. The pore diameter of the scaffolds could be easily controlled by changing the quenching temperature and time. The biocompatibility was assessed by examining the proliferation and morphology of MC 3T3-E1 osteoprogenitor cells seeded on the scaffolds. Cultures grown in the presence of a source of phosphate ions showed the formation of a mineralized extracellular matrix. The results indicate that PHBHHx scaffolds prepared using TIPS are a promising candidate for bone reconstruction.

The Effects of Gamma Irradiation on Molecular Weight, Morphology and Physical Properties of PHBV/Cloisite 30B Bionanocomposites

In this paper,the effects of gamma irradiation on Cast poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV)and PHBV/Cloisite 30B(C30B)(3 wt%)bionanocomposite prepared by melt compounding,were evaluated at various doses,i.e.,5,15,20,50 and 100 kGy at room temperature in air.Changes in molecular weight,morphology and physical properties were investigated.The study showed that the main degradation mechanism occurring in gamma irradiation in both Cast PHBV and C-PHBV/3C30B bionanocomposite is chain scission,responsible for the decrease of molecular weight.Differential scanning calorimetry(DSC)data indicated a regular decrease in crystallization temperature,melting temperature and crystallinity index for all irradiated samples with increasing the dose.Further,DSC thermograms of both Cast PHBV and PHBV bionanocomposite exhibited double melting peaks due probably to changes in the PHBV crystal structure.Tensile and DMA data showed a reduction in Young’s modulus,strength,elongation at break and storage modulus with the radiation dose;the decrease was however more pronounced for Cast PHBV.The morphological damages were much less pronounced for the PHBV bionanocomposite sample compared to Cast PHBV,for which some irregularities and defects were observed at 100 kGy.This study highlighted the ability of C30B to counterbalance the detrimental effect of radiolytic degradation on the functional properties of PHBV up to 100 kGy,thus acting as a potential anti-rad.

Evaluation of Biodegradation in Aqueous Medium of Poly(Hydroxybutyrate-Co-Hydroxyvalerate)/Carbon Nanotubes Films in Respirometric System

Biodegradable polymers have been increasingly used for scientific and commercial applications because they are similar to some conventional thermoplastics and exhibit the ability of self-degradation.Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV)nanocomposites films with 1 and 2 wt% of carbon nanotubes(CNT)were prepared by solution mixing,followed by solvent evaporation.In this work,PHBV/CNT nanocomposites were submitted to biodegradation in an aqueous medium for 34 days through a respirometric system.Then,the PHBV films were analyzed by the CO2 production and mineralization as a response of a microbial attack,which was monitored by back titration during all the experiment.The films were also characterized by measuring the weight loss;crystallinity was evaluated by differential scanning calorimetry(DSC)and the surface morphology by scanning electron microscopy(SEM).By analyzing the weight loss of the films,it was observed that adding CNT increases the resistance to biodegradation process.The obtained values of CO2 production and mineralization of the samples,as well as the values of weight loss,showed that the biodegradation of PHBV/CNT nanocomposites was minor in comparison to neat PHBV.The addition of CNT in PHBV matrix influences the surface morphology,causing the presence of cavities and an increase of roughness.

Comparative evaluation of physico-chemical characteristics of biopolyesters P（3HB） and P（3HB-co-3HV） produced by endophytic Bacillus cereus RCL 02

BACKGROUND： Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates （PHAs）. Bacillus cereus RCL 02 （MCC 3436）, a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly（3-hydroxybutyrate） [P（3HB）] of its cell dry biomass when grown in mineral salts （MS） medium. METHODS： The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P（3HB） isolated from RCL 02 in terms of thermal, mechanical and chemical properties. RESULTS： Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3- hydroxybutyrate （3HB） and 3-hydroxyvalerate （3HV） [P（3HB-co-3HV）] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction （XRD） analysis, Fourier transform infrared （FTIR） and nuclear magnetic resonance （NMR） spectroscopic studies. Thermogravimetric analysis （TGA） revealed that P（3HB） and P（3HB-co-3HV） films degraded at 278.66℃ and 273.49℃, respectively. The P（3HB-co-3HV） showed lower melting temperature （165.03℃） compared to P （3HB） （170.74℃） according to differential scanning calorimetry （DSC）. Incorporation of 3HV monomers decreased the tensile strength （21.52 MPa）, tensile modulus （0.93 GPa）, storage modulus （E＇） （0.99 GPa） and increased % elongation at break （12.2%） of the copolyester. However, P（3HB） showed better barrier properties with lower water vapor transmission rate （WVTR） of 0.55 g-mil/100 in2/24 h. CONCLUSION： These findings emphasized exploration of endophytic bacterial strain （RCL 02） to produce biodegradable polyesters which might have significant potential for industrial application.

Differentiation of smooth muscle progenitor cells in peripheral blood and its application in tissue engineered blood vessels

Background： A major shortcoming in tissue engineered blood vessels （TEBVs） is the lack of healthy and easily attainable smooth muscle cells （SMCs）. Smooth muscle progenitor cells （SPCs）, especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. Methods： SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV （EGM-2-MV） medium containing platelet-derived growth factoroBB （PDGF BB）. Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell （SOC） phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction （RT-PCR）. The cells were seeded onto the silk fibroin-modified poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） （SF-PHBHHx） scaflblds by 6× 10^4 cells/cm^2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-（4,5-dimethylthiazol-2-yl）-diphenyltetrazolium bromide （MTT） assay, scanning electron microscope （SEM）, and 4,6-diamidino-2-phenylindole （DAPI） staining. Results： SOCs displayed specific ＂hill and valley＂ morphology, expressed the specific markers of the SMC lineage： smooth muscle （SM） a-actin, calponin and smooth muscle myosin heavy chain （SM MHC） at protein and messenger ribonucleic acid （mRNA） levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22a （SM22a, a contractile protein, and extracellular matrix components elastin and matrix Gla protein （MGP）, as well as vascular endothelial growth factor （VEGF）. After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation. Conclusion： SPCs isolated from peripheral blood can be differentiated the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell sointo urce for constructing TEBVs.

Efficacy of nanofibrous conduits in repair of longsegment sciatic nerve defects

Our previous studies have histomorphologically confirmed that nanofibrous poly（3-hydroxybutyrate- co-3-hydroxyvalerate） conduit can be used to repair 30-mm-long sciatic nerve defects. However, the repair effects on rat behaviors remain poorly understood. In this study, we used nanofibrous poly（3-hydroxybutyrate-co-3-hydroxyvalerate） conduit and autologous sciatic nerve to bridge 30-ram-long rat sciatic nerve gaps. Within 4 months after surgery, rat sciatic nerve functional re- covery was evaluated per month by behavioral analyses, including toe out angle, toe spread anal- ysis, walking track analysis, extensor postural thrust, swimming test, open-field analysis and no- ciceptive function. Results showed that rat sciatic nerve functional recovery was similar after nanofibrous poly（3-hydroxybutyrate-co-3-hydroxyvalerate） conduit and autologous nerve grafting. These findings suggest that nanofibrous poly（3-hydroxybutyrate-co-3-hydroxyvalerate） conduit is suitable in use for repair of long-segment sciatic nerve defects.

Comparison of nitrate-removal efficiency and bacterial properties using PCL and PHBV polymers as a carbon source to treat aquaculture water

Nitrate(NO_(3)^(−))accumulation in recirculating aquaculture systems(RASs)with high stocking densities presents a problem for reared animals and the environment.The use of a biodegradable polymer as organic carbon for heterotrophic denitrification exhibits good performance for NO_(3)^(−)removal from wastewater.A comparison of NO_(3)^(−)–N removal efficiency and bacterial properties using polycaprolactone(PCL)and poly(3-hydroxybutyrateco-3-hydroxyvalerate)(PHBV)as carbon sources to treat aquaculture water was conducted for a 102-day period.The results indicated that the NO_(3)^(−)–N removal rates of 0.27±0.07 and 0.19±0.05 g/L per day,respectively,could be achieved with influent concentrations ranging from 81.1 to 132.75 mg/L and a flow rate of 1 L/h.The removal of NO_(3)^(−)–N versus consumed PCL(1:1 w/w)was significantly higher than that versus consumed PHBV(0.3:1 w/w)(P<0.05).The concentrations of effluent nitrite-nitrogen and total ammonium nitrogen were maintained at an acceptable level.The bacterial community structures between the two types of reactors varied significantly.Acidovorax and Denitratisoma were the top two genera of the bacterial community in the biofilm in the PCL beads with a dominance of 26.83%and 6.67%,respectively.In the PHBV beads,Acidovorax at 17.95%and Bdellovibrio at 6.37%were the top two genera.The PCL-denitrification reactor developed in this study showed better potential than the PHBV-denitrification reactor in removing NO_(3)^(−)from aquaculture water.

小鼠诱导多能干细胞与PHBHHx膜体外共培养的实验研究

目的探讨小鼠诱导多能干细胞(mi PSCs)与聚3-羟基丁酸-3-羟基己酸共聚物(PHBHHx)膜材料的体外相容性。方法复苏mi PSCs,培养传代后,分为两组,一组与PHBHHx膜材料在体外共培养,通过DAPI染色方法观察mi PSCs在膜上的粘附情况,细胞计数试剂盒-8(CCK-8)测定mi PSCs在PHBHHx膜上的增殖,扫描电镜观察细胞与膜材料的复合情况;另一组用传统的培养皿培养mi PSCs,CCK-8测定细胞活力。结果小鼠诱导多能干细胞能够与PHBHHx膜材料良好的粘附和增殖,CCK-8实验测定细胞活力OD值结果显示PHBHHx膜显著大于传统的细胞培养皿培养(0.617±0.019 vs.0.312±0.004,P<0.05)。结论 PHBHHx与mi PSCs体外共培养制成的细胞补片表面上有细胞粘附及增殖,与传统的细胞培养皿培养相比,PHBHHx膜培养在细胞的粘附和增殖中具有很大的优势,PHBHHx可作为干细胞治疗多种疾病的支架材料之一。

聚羟基脂肪酸酯的亲水性改性及其与人脐静脉内皮细胞相容性的研究

用丝素蛋白(SF)对微生物合成的高分子聚合物聚(3-羟基丁酸酯-co-3-羟基己酸酯)(PHBHHx)进行亲水改性,以提高材料的生物相容性.水接触角测定和表面自由能分析表明,丝素蛋白在支架表面吸附,使PHBHHx材料表面的水接触角从90°降至51°,表面自由能从37.9 mJ/m2增至57.4 mJ/m2,因而增加了材料的亲水性.进一步对亲水性改性前后PHBHHx多孔支架与人脐静脉内皮细胞(HUVECs)的相容性进行了比较.MTT法细胞活力分析表明,细胞在支架上培养3,5,7天后,其在SF改性PHBHHx多孔支架上的活力显著高于在未改性的PHBHHx支架上的活力;扫描电镜观察细胞生长形貌表明,细胞在改性后多孔支架上黏附及生长5天后,形成了连续细胞单层,其生长状态优于在未改性的PHBHHx支架上的生长状态;胶原含量测定表明细胞在改性后支架上比在未改性支架上有更好的胶原分泌能力,即改性后支架更利于诱导HUVECs分泌细胞外基质(ECM)从而构建类似体内的生长环境.