Co-reporter:Xiaoxuan Ma;Le Zhang;Wenjiao Xue;Chenhui Zhu
Journal of Materials Science 2017 Volume 52( Issue 7) pp:3771-3785
Publication Date(Web):2017 April
DOI:10.1007/s10853-016-0577-1
Injectable hydrogels have become a hot topic of research in tissue engineering field, with broad application prospects. In this paper, recombinant human-like collagen (HLC) and three different kinds of high molecular weight pullulan (P), crosslinked with 1,2,7,8-diepoxyoctane (DEO), were synthesized in a water bath. Pullulan/1,2,7,8-diepoxyoctane (PD) and pullulan human-like collagen/1,2,7,8-diepoxyoctane (PHD) hydrogels, with high crosslinking density, injectability, thermal stability, and biocompatibility were synthesized. According to hypothesis, the biocompatibility and biodegradability of PHD hydrogels was better than PD hydrogels and PHD hydrogels served as ideal filler biomaterials in tissue engineering. Physicochemical properties such as swelling ratio, elastic modulus, thermal stability, porosity, and rheological features of these hydrogels were studied. Results showed that an increase in the molecular weight of pullulan and the amount of added HLC enhanced its mechanical properties. However, it showed little influence on its thermal stability, since pullulan and HLC themselves were thermally stable. Acute toxicity test, hemolysis test, and L929 cell 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay (MTT) were employed for testing the toxicity and cytocompatibility. The cell attachment in vitro and biocompatibility in vivo were also investigated. The MTT and live/dead cell viability staining results demonstrated that PDH was less cytotoxic than PD due to the promotion of the L929 proliferation. In vitro degradation results indicated that the hydrogels had prolonged degradation times and excellent anti-enzymatic abilities. Subcutaneous injection of PDH hydrogel in rabbit showed a lower inflammatory response after 1, 2, and 8 weeks, and the hydrogels showed almost no obvious degradation in two months. Thus, the hydrogel had a great potential to be used as ideal filler biomaterial in tissue engineering.
Co-reporter:Gaoyu Chen;Xiaoyan Zheng;Chong Wang;Junfeng Hui;Xuexi Sheng;Xiangxing Xu;Jianchun Bao;Weijun Xiu;Lihui Yuwen
RSC Advances (2011-Present) 2017 vol. 7(Issue 89) pp:56537-56542
Publication Date(Web):2017/12/12
DOI:10.1039/C7RA10516A
Hydroxyapatite (HAp) is the main inorganic component of human bones and teeth. The doping of HAp nanocrystals plays an important role in tissue engineering, drug delivery, biomarkers and artificial bones. In this article, a postsynthetic metal ion exchange method was developed for doping hydroxyapatite (HAp) nanocrystals in organic solutions. It can be mono- or multi-ion doped with Mg2+, Sr2+, Zn2+, Mn2+, Fe3+ or Cu2+ etc., despite the significant radius variation of Ca2+ and doping ions. The doping ratio can be tuned in a wide range, e.g. Fe3+ of 0–20%, which is much higher than the ion exchange performed in aqueous solution. The structure and morphology of the HAp nanocrystals were preserved after postsynthetic doping, suggesting potential biological applications.
Co-reporter:Zhiguang Duan;Jianjun Deng;Yangfang Dong;Chenhui Zhu;Weina Li
Food & Function (2010-Present) 2017 vol. 8(Issue 10) pp:3723-3736
Publication Date(Web):2017/10/18
DOI:10.1039/C7FO00385D
Ginsenoside Rk3 (Rk3) is present in the roots of processed Panax notoginseng herbs and it exerts anti-platelet aggregation, pro-immunogenic and cardioprotective effects. However, little is known regarding the anticancer activities of this compound, especially in lung cancer. This study was designed to investigate the anticancer effects of Rk3 on non-small cell lung cancer (NSCLC) cells and in an H460 xenograft tumor model. Our results showed that Rk3 reduced cell viability, inhibited both cell proliferation and colony formation, and induced G1 phase cell cycle arrest by downregulating the expression of cyclin D1 and CDK4 and upregulating the expression of P21. Rk3 also induced apoptosis in a concentration-dependent manner in H460 and A549 cells by Annexin V/PI staining, TUNEL assay and JC-1 staining, resulting in a change in the nuclear morphology. Moreover, Rk3 induced the activation of caspase-8, -9, and -3, promoted changes in mitochondrial membrane potential, decreased the expression of Bcl-2, increased the expression of Bax, and caused the release of cytochrome c, which indicated that the apoptosis-inducing effects of Rk3 were triggered via death receptor-mediated mitochondria-dependent pathways. Furthermore, Rk3 significantly inhibited the growth of H460 xenograft tumors without an obvious effect on the body weight of the treated mice. Histological analysis indicated that Rk3 inhibited tumor growth by altering the proliferation and morphology of tumor cells. In addition, we confirmed that Rk3 inhibited angiogenesis via CD34 staining and chick embryo chorioallantoic membrane (CAM) assay in vivo. Taken together, our findings revealed not only the anticancer effect of Rk3 on NSCLC cells but also a new promising therapeutic agent for human NSCLC.
Co-reporter:Jingjing Shi;Xiaoxuan Ma;Yuan Gao;Chenhui Zhu;Yu Mi
The Protein Journal 2017 Volume 36( Issue 4) pp:322-331
Publication Date(Web):07 June 2017
DOI:10.1007/s10930-017-9723-0
High-level expression of recombinant collagen by genetic engineering is urgently required. Recombinant collagen is different from natural collagen in its hydroxyproline (Hyp) content and thermal stability. To obtain hydroxylated collagen for applications in biomedicine and biomaterials, the human collagen α1(III) chain was co-expressed with the viral prolyl 4-hydroxylase A085R in Escherichia coli. Unlike previous reports using human prolyl 4-hydroxylase, this study examined the hydroxylation of full-length human collagen α1(III) chain (COL3A1) by viral prolyl 4-hydroxylase. The genes encoding these two proteins were controlled by different promoters, Ptac and PRPL, on a recombinant pKK223-3 plasmid. The sequencing results verified that the target genes were successfully inserted into the recombinant vector. Based on quantitative PCR, SDS–PAGE, and western blotting, successful expression by E. coli BL21(DE3) was detected at the mRNA and protein levels for both loci. Liquid chromatography–mass spectrometry (LC–MS/MS) results suggested that the highest Hyp yield was obtained when the two proteins were induced with 0.5 mM IPTG and heat-shock treatment at 50 °C, corresponding to high enzyme expression and low human collagen α1(III) chain expression levels. A biological activity analysis indicated that the recombinant collagen with the highest hydroxylation level supported the growth of baby hamster kidney cells, similar to observations for native collagen. The production of hydroxylated collagen in this study establishes a new method for collagen hydroxylation and provides a basis for the application of recombinant collagen expressed in E. coli.
Co-reporter:Leilei Zhao, Xian Li, Jiaqi Zhao, Saijian Ma, Xiaoxuan Ma, Daidi Fan, Chenhui Zhu, Yannan Liu
Materials Science and Engineering: C 2016 Volume 68() pp:317-326
Publication Date(Web):1 November 2016
DOI:10.1016/j.msec.2016.05.108
•Different concentrations of microbial transglutaminase are prepared for hydrogels.•HLC crosslinking with microbial transglutaminase (MTGase) producing novel smart hydrogels•Novel smart hydrogels formation in an enzymes/temperature sensitive manner•Novel smart hydrogels are exhibiting lower toxicity and mild inflammation.Various tissue scaffold materials are increasingly used to repair skin defects by cross-linking because of the ability to fill and implant in any form via operation. However, crosslinker residues cannot be easily removed from scaffold materials prepared by chemical crosslinking methods, limiting their use for skin tissue engineering. Here, microbial transglutaminase (MTGase), a nontoxic crosslinker with high specific activity and reaction rate under mild conditions, was employed crosslinks in human-like collagen (HLC) to yield novel smart MTGase crosslinked with human-like collagen (MTGH) hydrogels, which are sensitive to temperature and/or enzymes. Various ratios of MTGase/HLC were performed, and their physicochemical properties were characterized, including the swelling ratio, the elastic modulus, the morphology and the porosity. The degradation behavior and mechanism of MTGase in concentration-dependent manner involved in formation hydrogels were identifying in vitro. The cell attachment in vitro and biocompatibility in vivo were also investigated. The results demonstrated that the use of different concentrations of MTGase to crosslink HLC produced products with different degradation times and biocompatibilities. The 50 U/g MTGase-prepared MTGH hydrogels had a higher density of crosslinks, which made them more resistant to degradation by collagenase I and collagenase II. However, 40 U/g MTGase-prepared MTGH hydrogels were more suitable for cell attachment. In addition, compared with the Collagen Implant I® (SUM) used in animal experiments, the 40 U/g MTGase-prepared MTGH hydrogels had a lower toxicity and better biocompatibility. Therefore, 40 U/g MTGase crosslinked with HLC should be used to prepare MTGH hydrogels for potential application as soft materials for skin tissue engineering.
Co-reporter:Xian Li, Wenjiao Xue, Yannan Liu, Daidi Fan, Chenhui Zhu and Xiaoxuan Ma
Journal of Materials Chemistry A 2015 vol. 3(Issue 23) pp:4742-4755
Publication Date(Web):12 May 2015
DOI:10.1039/C5TB00408J
In this study, we designed multifunctionalized hydrogel scaffolds and injectable particles based on high-molecular-weight (MW) pullulan and human-like collagen (HLC) crosslinked with 1,4-butanediol diglycidyl ether (BDDE) for combination therapy tissue restoration. The properties of the pullulan/BDDE (PB) and pullulan/BDDE/human-like collagen (PBH) hydrogels were characterized via swelling ratio measurements, mechanical tests, and enzymatic degradation in vitro and via subcutaneous injections in vivo. The results demonstrate that the dry hydrogels completely returned to their original state in deionized water. The elastic modulus of the PBH53 dry hydrogels is higher than that of the other hydrogels after exposure to bending stress and compression stress with a maximum value of 7858.93 MPa. In addition, the in vitro live/dead staining and cell adhesion of the PBH hydrogels exhibited a superior fibroblast morphology without high levels of cell death, which were considerably better than those of PB hydrogels. In vivo, PB and PBH particles with good biocompatibility and anti-biodegradation were successfully prepared via the granulation of wet PB and PBH hydrogels for efficient subcutaneous injection in Kunming mice and New Zealand rabbits. Therefore, the PB and PBH hydrogels were found to be acceptable, safe, soft materials for use in skin restoration, cartilage treatment, and lacrimal dryness therapy.
Co-reporter:Xiaoyan Zheng, Meiying Liu, Junfeng Hui, Daidi Fan, Haixia Ma, Xiaoyong Zhang, Yaoyu Wang and Yen Wei
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 31) pp:20301-20307
Publication Date(Web):01 Jul 2015
DOI:10.1039/C5CP01845E
In this paper we report two different doping strategies to prepare a series of novel HAp:Ln3+ (Ln = Eu or Tb) nanocrystals with tunable aspect ratios via facile hydrothermal synthetic routes. Adopting a one-pot synthetic strategy, with increasing rare-earth doping dosage, the as-prepared nanocrystals have relatively weak fluorescence intensity, and change from nanorods with lengths of about 150 nm into nanowires with lengths of about 2 μm. Using the synthetic pure HAp nanorods as matrices, they are endowed with bright green or red luminescent properties by doping Tb3+ or Eu3+ ions via a second hydrothermal process, and simultaneously retain their original morphologies (diameter 8 nm, length 150 nm). The hydrophobic HAp:Ln3+ nanorods with strong optical properties are converted into hydrophilic particles with a surfactant (Pluronic F127) and successfully applied to live cell imaging.
Co-reporter:Junfeng Hui, Hui Li, Xiaoyan Zheng, Haixia Ma, Daidi Fan, Hang Liu, Yaoyu Wang
Ceramics International 2015 Volume 41(Issue 5) pp:6194-6202
Publication Date(Web):June 2015
DOI:10.1016/j.ceramint.2014.12.156
As members in the calcium apatite family, dicalcium phosphate dihydrate (DCPD), anhydrous dicalcium phosphate (DCPA), and hydroxyapatite (HAp) play important roles in many fields. Herein, by tuning the amount of oleic acid, octadecylamine, acetic acid and reaction conditions, hierarchically structured DCPD or DCPA nanoflowers, DCPD nanosheets, DCPA nanobelts or nanowires and HAp nanorods have been selectively synthesized via a simple, mild solution process. The phase conversion and morphological evolution of the three compounds were discussed in detail. The as-prepared samples were characterized by Fourier Transform Infrared Spectroscopy, X-ray Diffraction, Scanning Electron Microscope and Transmission Electron Microscope. Cytotoxicity assay showed that samples own good biocompatibility.
Co-reporter:Lin Liu, Ya Wang, Daidi Fan, Yu Mi
Materials Letters 2015 Volume 139() pp:245-248
Publication Date(Web):15 January 2015
DOI:10.1016/j.matlet.2014.10.047
•The degree of ionization of phenolphthalein is closely interrelated with the structure state of water.•Phenolphthalein is used as an indicator for monitoring the vacuum freeze-drying process.•During the three stages of freeze-drying process, the colour changes of phenolphthalein show a high sensitivity to the changes of water state, even the liquid-like state of water in desorption drying.To improve the quality of the product, an important subject in vacuum freeze-drying process is to accurately monitor the state of water at its freezing stage and that of the residual water at its drying stage. In this paper, an easy-to-perform method is shown to monitor the state of water during the whole freeze-drying process (i.e. the state of water in the freezing stage and that of the residual water in drying stage) based on colour changes of phenolphthalein. A sample prepared with phenolphthalein was sensitive to structural changes of water hydrogen bonding network and exhibited colour-changing effect. This makes it possible to accurately trace the dynamic freeze-drying process, thus providing a valuable tool for preparing high-quality products.
Co-reporter:Chenhui Zhu;Yanru Chen;Jianjun Deng;Wenjiao Xue;Xiaoxuan Ma;Junfeng Hui
Polymers for Advanced Technologies 2015 Volume 26( Issue 10) pp:1217-1225
Publication Date(Web):
DOI:10.1002/pat.3557
The aim of this study was to determine whether a phosphorylated human-like collagen calcium (PHLC–Ca) complex showed a higher bioavailability than other calcium supplements. To improve the calcium-binding ability of human-like collagen (HLC), modified HLC was prepared by introducing a reactive phosphate group using a dry-heating method, followed by the formation of PHLC–Ca in MOPS buffer. PHLC–Ca was characterized using atomic absorption spectrophotometry, Fourier transform infrared (FTIR), fluorescence quenching, circular dichroism (CD), and ultraviolet–visible absorption (UV–vis) spectroscopy. The calcium uptake test showed that the bioavailability of PHLC–Ca was higher than that of CaCl2 and G-Ca. Data from a mouse model of osteoporosis suggested that PHLC–Ca is superior for treating osteoporosis than CaCl2 or G-Ca. These results indicate that PHLC–Ca is a desirable calcium supplement for use in daily life and clinical application. Copyright © 2015 John Wiley & Sons, Ltd.
Co-reporter:Xian Li, Wenjiao Xue, Chenhui Zhu, Daidi Fan, Yannan Liu, XiaoxuanMa
Materials Science and Engineering: C 2015 Volume 57() pp:189-196
Publication Date(Web):1 December 2015
DOI:10.1016/j.msec.2015.07.059
•Carboxyl grafting onto pullulan forms the carboxyl pullulan prepared for hydrogels.•HLC crosslinking with carboxyl pullulan (PC) producing novel PCBH hydrogels•PC crosslinking with BDDE to prepare novel PCB hydrogels for tissue engineering•PC exhibiting high reactivity, superb selectivity and mild reaction conditionsNovel hydrogels based on carboxyl pullulan (PC) and human-like collagen (HLC) crosslinking with 1,4-butanediol diglycidyl ether (BDDE) are promising soft fillers for tissue engineering due to their highly tunable properties. Recent studies, however, have shown that incorporating hyaluronic acid and BDDE results in hydrogels with a microporous structure, a large pore size and high porosity, which reduce cell adhesion and enhance degradation in vivo. To improve biocompatibility and prevent biodegradation, the use of PC to replace hyaluronic acid in the fabrication of PC/BDDE (PCB) and PC/BDDE/HLC (PCBH) hydrogels was investigated. Preparation of gels with PC is a promising strategy due to the high reactivity, superb selectivity, and mild reaction conditions of PC. In particular, the Schiff base reaction of HLC and PC produces the novel functional group –RCONHR′ in PCBH hydrogels. Twenty-four weeks after subcutaneous injection of either PCB or PCBH hydrogel in mice, the surrounding tissue inflammation, enzymatic response and cell attachment were better compared to hyaluronic acid-based hydrogels. However, the biocompatibility, cytocompatibility and non-biodegradability of PCBH were milder than those of the PCB hydrogels both in vivo and in vitro. These results show that the proposed use of PC and HLC for the fabrication of hydrogels is a promising strategy for generating soft filler for tissue engineering.
Co-reporter:Xian Li, Daidi Fan, Chenhui Zhu and Xiaoxuan Ma
Journal of Materials Chemistry A 2014 vol. 2(Issue 9) pp:1234-1249
Publication Date(Web):19 Dec 2013
DOI:10.1039/C3TB21628D
CS–HLC–HA–β-GP (chitosan–human-like collagen–hyaluronic acid–β-sodium glycerophosphate) hydrogels were prepared based on the self-assembly of CS–HLC–HA (CCA) fibers. The effects of the fibers on the synthesis, characteristics and biomedical applications of CS–HLC–HA–β-GP (CCAG) hydrogels were studied for various HA contents. The synthesis mechanism of the novel CCAG hydrogel was explored using Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The hydrogels were characterized by a swelling test, gelling time and enzymatic treatments. The results indicated that a new amide bond (–CONH) and –NRH2+ were formed. The gelling time and swelling behaviors were dependent on the intertwining, overlap and adsorption of the polymer chains at various temperatures and pH. Furthermore, biomedical applications were evaluated by transmission electron microscopy (TEM), immunohistochemical analysis and haematoxylin and eosin (H&E) staining. The effect of the fibers on the histocompatibility of the hydrogels revealed that the fibers inside the hydrogel pores reduced the quantity of macrophages, decreased the degree of inflammation, and improved the anti-degradation of the modified hydrogels. This type of new hydrogel emerges as an interesting injectable filling biomaterial for tissue engineering.
Co-reporter:Xiaoxuan Ma, Jianjun Deng, Yuzhang Du, Xian Li, Daidi Fan, Chenhui Zhu, Junfeng Hui, Pei Ma and Wenjiao Xue
Journal of Materials Chemistry A 2014 vol. 2(Issue 18) pp:2749-2763
Publication Date(Web):21 Feb 2014
DOI:10.1039/C3TB21842B
Novel hydrogels (termed HCD hydrogels) were synthesized based on human-like collagen (HLC) and chitosan (CS) cross-linked with dialdehyde starch (DAS). The biological stability and biocompatibility of HCD hydrogels were determined through in vitro and in vivo tests. The mechanism of hydrogel formation was studied using Fourier transform infrared spectroscopy (FTIR), which showed that covalent bonds formed via acetalization and Schiff base reactions. Biological stability was evaluated in vitro by degrading HCD hydrogels with class I collagenase, class II collagenase, and both class I and class II collagenases and in vivo after subcutaneously injecting HCD into an animal model. The biological characteristics of HCD hydrogels was studied by two methods: (i) MTT and cytomorphology cytotoxicity and cytocompatibility and (ii) in vivo, whereby histomorphometry, transmission electron microscopy (TEM), and immunohistochemistry were used to compare different types of surgically introduced hydrogels, our HCD hydrogels, SunMax Collagen Implant hydrogels (SUM hydrogels), and OUTLINE&EVOLUTION Injectable Synthetic Gel hydrogels (EVL hydrogels). The in vivo analyses were performed at 1, 9, 12, and 28 weeks after surgery. The hydrogel biodegradation results showed that the normalized residual weight (WR) of HCD hydrogels varied with DAS content. In vitro, we found that the minimum WR of HCD hydrogels was 42.19% after 28 weeks when degraded by both types of class I and class II collagenase. The MTT assay indicated that the minimum relative growth rate (RGR) of cells was 93% after they were incubated with HCD hydrogels for 7 days, suggesting good cytocompatibility. In vivo histomorphometry results indicated that HCD hydrogels effectively filled tissue voids and did not cause redness, edema, festering, or color changes. In addition, a few vessels grew into the hydrogel and a thin fibrous capsule was eventually produced. TEM and immunohistochemistry studies suggested that HCD hydrogels produced less intense inflammatory responses than those produced by SUM hydrogels and EVL hydrogels. Overall, HCD hydrogels afford both enhanced biological stability and excellent biocompatibility, making them potentially promising for skin patch scaffolds, wrinkle treatments, and tissue cavity fillers.
Co-reporter:Huizhi Zhang, Daidi Fan, Jianjun Deng, Chenghui Zhu, Junfeng Hui, Xiaoxuan Ma
Materials Science and Engineering: C 2014 Volume 42() pp:124-129
Publication Date(Web):1 September 2014
DOI:10.1016/j.msec.2014.05.015
•TA buffer is a mild buffer system for endotoxins removal of HLC.•TA buffer may facilitate endotoxins adsorbed on the resin efficiently.•TA buffer has high-efficiency endotoxin removal and high HLC recovery efficiency.Protein preparation, which has active ingredients designated for the use of biomaterials and therapeutical protein, is obtained by genetic engineering, but products of genetic engineering are often contaminated by endotoxins. Because endotoxin is a ubiquitous and potent proinflammatory agent, endotoxin removal or depletion from protein is essential for researching any biomaterials. In this study, we have used Tris-acetate (TA) buffer of neutral pH value to evaluate endotoxins absorbed on the Pierce high-capacity endotoxin removal resin. The effects of TA buffer on pH, ionic strength, incubation time as well as human-like collagen (HLC) concentration on eliminating endotoxins are investigated. In the present experiments, we design an optimal method for TA buffer to remove endotoxin from recombinant collagen and use a chromogenic tachypleus amebocyte lysate (TAL) test kit to measure the endotoxin level of HLC. The present results show that, the endotoxins of HLC is dropped to 8.3 EU/ml at 25 mM TA buffer (pH 7.8) with 150 mM NaCl when setting incubation time at 6 h, and HLC recovery is about 96%. Under this experimental condition, it is proved to exhibit high efficiencies of both endotoxin removal and collagen recovery. The structure of treated HLC was explored by Transmission Electron Microscopy (TEM), demonstrating that the property and structure of HLC treated by TA buffer are maintained. Compared to the most widely used endotoxin removal method, Triton X-114 extraction, using TA buffer can obtain the non-toxic HLC without extra treatment for removing the toxic substances in Triton X-114. In addition, the present study aims at establishing a foundation for further work in laboratory animal science and providing a foundation for medical grade biomaterials.The processes of endotoxins adsorbed from HLC.
Co-reporter:Chenhui Zhu, Xiaoxuan Ma, Yonghui Wang, Yu Mi, Daidi Fan, Jianjun Deng, Wenjiao Xue
Materials Science and Engineering: C 2014 Volume 44() pp:411-416
Publication Date(Web):1 November 2014
DOI:10.1016/j.msec.2014.08.010
•Human-like collagen was thiolated for improving its zinc binding ability.•pH and zinc concentration control the binding amount of zinc to thiolated HLC.•The physicochemical properties of thiolated HLC–Zn complex were investigated.•The biocompatibility of thiolated HLC–Zn complex was evaluated.To improve zinc binding ability to human-like collagen (HLC) and stability of metal complex, HLC was thiolated by mercaptosuccinylation reaction with S-acetylmercaptosuccinic anhydride (S-AMSA) at pH 8.0. One mole of thiolated HLC–Zn (SHLC–Zn) complex possessed 24.3 mol zinc ions when pH was 8.0 and zinc concentration was 15 mM. The physicochemical properties and biocompatibility of thiolated HLC–Zn (SHLC–Zn) complex were investigated by UV–vis, CD, electrophoresis analysis, differential scanning calorimetry (DSC) and cell viability assay, respectively. The results showed that SHLC–Zn complex(1) exhibited higher zinc ions than that of native HLC and still maintained the secondary structure of HLC though interaction occurred between SHLC and zinc ions, (2) increased the apparent molecular weight when compared with native HLC, (3) exhibited greater thermal stability than native HLC, and (4) presented toxicity free for BHK cells. This study suggests that the SHLC–Zn complex is a potential nutrition as well as zinc supplement in the medical application.
Co-reporter:Lina Wang;Jing He;Zhongcheng Lv
Biotechnology and Bioprocess Engineering 2014 Volume 19( Issue 5) pp:916-924
Publication Date(Web):2014 September
DOI:10.1007/s12257-014-0234-y
Recombinant human full-length mature collagen α1 (III) chain (rhCOL3A1) was secreted by Pichia pastoris GS115, using the Saccharmyces cerevisiae á-mating factor prepro signal, and the theoretical molecular weight of rhCOL3A1 was 95.344 kDa. The gene cloned from human placenta, was designed and cloned into expression vector pPIC9K under the control of a strong inducible promoter AOX1.The expression stage of rhCOL3A1 was sensitive to different carbon ratios through mixed fermentation. LCMS/ MS analysis and western blotting demonstrated that the recombinant human full-length mature collagen a1 (III) gene was successfully expressed in P. pastoris GS115 during the methanol induction stage. Furthermore, an effective strategy of mixed fermentation was established to express rhCOL3A1 in shake flash. Compared to single carbon induction, when induced with mixed carbon at the ration of 0.8 (glycerol/methanol), the time corresponding to the highest yield of rhCOL3A1 (1.27 g/L) was drastically reduced by 50%. The same conclusion was observed from RT-qPCR. Consequently, a new strategy which was more time-saving and effective was provided for the large-scale producing the full-length mature rhCOL3A1.
Co-reporter:Chenhui Zhu, Daidi Fan, Yaoyu Wang
Materials Science and Engineering: C 2014 Volume 34() pp:393-401
Publication Date(Web):1 January 2014
DOI:10.1016/j.msec.2013.09.044
•Human-like collagen/hyaluronic acid (HLC/HA) 3D scaffolds are fabricated at different mass ratios by freeze-drying.•Mass ratio (HLC to HA) controls the morphology, mechanical property, and degradation of scaffolds.•The scaffold with optimal mass ratio exhibits excellent biocompatibility.Following the previous work of media layer and adventitia layer construction for vascular scaffold, we developed a suitable intima layer scaffold for endothelialization using novel human-like collagen/hyaluronic acid composite at different mass ratios of 40/1, 20/1 and 10/1 (HLC to HA) by freeze-drying process. The structure, mechanical strength, degradation and biocompatibility of the vascular HLC/HA scaffold were evaluated. The results showed that the 10/1 HLC/HA composited an optimal scaffold with (1) an interconnected porous network with a pore diameter of 12 ± 2 μm and porosity of 89.3%, (2) better mechanical properties with higher stress of 321.7 ± 15 kPa and strain of 45.5 ± 0.2% than 40/1, 20/1 and pure HLC scaffolds, (3) only 9% degradation upon immersion in PBS for 45 days at 37 °C in vitro, and (4) excellent biocompatibility. This study suggests that the 10/1 HLC/HA composite has a broad prospect of application as luminal vascular scaffold in the tissue engineering.
Co-reporter:Jingjing Zhang, Xiaoxuan Ma, Daidi Fan, Chenhui Zhu, Jianjun Deng, Junfeng Hui, Pei Ma
Materials Science and Engineering: C 2014 Volume 43() pp:547-554
Publication Date(Web):1 October 2014
DOI:10.1016/j.msec.2014.07.058
•Human-like collagen was used with hyaluronic acid to prepare soft tissue filling meterials.•1,4-Butanediol diglycidyl ether (BDDE) was introduced to treat the hydrogels.•The addition of human-like collagen could improve the biological properties of hydrogels.Injectable hydrogel plays an important role in soft tissue filling and repair. We report an injectable hydrogel based on hyaluronic acid (HA) and human-like collagen (HLC), both with favorable biocompatibility and biodegradability. These two types of biomacromolecules were crosslinked with 1,4-butanediol diglycidyl ether to form a three-dimensional network. The redundant crosslinker was removed by dialysis and distillation. An HA-based hydrogel prepared by the same method was used as a control. The cytocompatibility was studied with a Cell Counting Kit-8 (CCK-8) test. Carbazole colorimetry was used to analyze the in vitro degradation rate. The histocompatibility was evaluated by hematoxylin and eosin (H&E) staining analysis and immunohistochemical analysis. The CCK-8 assay demonstrated that the HA/HLC hydrogel was less cytotoxic than the HA-based hydrogel and could promote baby hamster kidney cell (BHK) proliferation. The cell adhesion indicated that BHK could grow well on the surface of the materials and maintain good cell viability. The in vitro degradation test showed that the HA/HLC hydrogel had a longer degradation time and an excellent antienzyme ability. In vivo injection showed that there was little inflammatory response to HA/HLC after 1, 2, and 4 weeks. Therefore, the HA/HLC hydrogel is a promising biomaterial for soft tissue filling and repair.
Co-reporter:Liping Jia, Zhiguang Duan, Daidi Fan, Yu Mi, Junfeng Hui, Le Chang
Materials Science and Engineering: C 2013 Volume 33(Issue 2) pp:727-734
Publication Date(Web):1 March 2013
DOI:10.1016/j.msec.2012.10.025
Three dimensional (3D) biodegradable porous scaffolds play a key role in cartilage tissue repair. Freeze-drying and cross-linking techniques were used to fabricate a 3D composite scaffold that combined the excellent biological characteristics of human-like collagen (HLC) and the outstanding mechanical properties of nano-hydroxyapatite (nHA). The scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and compression tests, using Relive® Artificial Bone (RAB) scaffolds as a control. HLC/nHA scaffolds displayed homogeneous interconnected macroporous structure and could withstand a compression stress of 2.67 ± 0.37 MPa, which was higher than that of the control group. Rabbit chondrocytes were seeded on the composite porous scaffolds and cultured for 21 days. Cell/scaffold constructs were examined using SEM, histological procedures, and biochemical assays for cell proliferation and the production of glycosaminoglycans (GAGs). The results indicated that HLC/nHA porous scaffolds were capable of encouraging cell adhesion, homogeneous distribution and abundant GAG synthesis, and maintaining natural chondrocyte morphology compared to RAB scaffolds. In conclusion, the presented data warrants the further exploration of HLC/nHA scaffolds as a potential biomimetic platform for chondrocytes in cartilage tissue engineering.Highlights► Human-like collagen was first used to prepare cartilage tissue engineering scaffold. ► Genipin, a natural biological cross-linking agent, was introduced to treat scaffold. ► We chose market product as a control.
Co-reporter:Yu Mi, Ran Su, Dai-Di Fan, Xiao-Li Zhu, Wen-Ni Zhang
Materials Science and Engineering: C 2013 Volume 33(Issue 5) pp:3047-3053
Publication Date(Web):1 July 2013
DOI:10.1016/j.msec.2013.03.035
•Synthesised N,O-carboxymethyl chitosan (NOCs) coated alginate (ALg) microspheres.•Their effect on intestinal microflora was investigated in simulated gastric juices.•NOCs A coated ALg microspheres improved Bifidobacterium longum survival in SGJ.•The modified chitosan layer improved the pH-response of alginate microspheres.•NOCs A coated microspheres could be used to deliver oral bioactive compounds.In order to greatly improve vitality of probiotic bacteria within the application, a novel biocompatible vehicle, N,O-carboxymethyl chitosan (NOCs) with appropriate degrees of substitution coat alginate (ALg) microparticles, was prepared by electrostatic droplet generation. The amount of chitosan (Cs) and N,O-carboxymethyl chitosan (NOCs) coated on the ALg microparticles was determined by differential scanning calorimetry. The surface morphology of ALg microparticles, Cs coated ALg microparticles and NOCs coated ALg microparticles was determined using scanning electron microscopy. The coating thickness of Cs coated ALg microparticles and that of NOCs coated ALg microparticles was directly observed with confocal laser scanning microscopy. In order to assess pH sensitivity of microparticles, the bovine serum albumin release from the microspheres was tested in acid solution (pH 2.0) for 2 h and subsequently in alkaline solution (pH 7.0) for 2 h. The survival of Bifidobacterium longum BIOMA 5920 loaded in NOCs coated with ALg microparticle was improved in simulated gastric juice (pH 2.0, for 2 h) compared to that of B. longum BIOMA 5920 loaded in ALg microparticles and Cs coated ALg microparticles. After incubation in simulated intestinal juices (pH 7.0, 2 h), the release of microencapsulated B. longum BIOMA 5920 was investigated.
Co-reporter:Chenhui Zhu, Yan Sun, Yaoyu Wang, Yane Luo, Daidi Fan
Materials Science and Engineering: C 2013 Volume 33(Issue 5) pp:2611-2619
Publication Date(Web):1 July 2013
DOI:10.1016/j.msec.2013.02.028
In order to develop the nutritional trace elements which could be absorbed and utilized effectively, protein chelates were adopted. Calcium, copper and manganese were considered based on their physiological functions, and the new chelates of HLC-Ca, HLC-Cu and HLC-Mn were formed in MOPS or MES buffer and purified by gel chromatography, and then freeze-dried. And they were detected and analyzed by atomic absorption spectrophotometry, ultraviolet–visible absorption (UV–vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, fluorescence quenching method, circular dichroism (CD) and differential scanning calorimetry (DSC). The results showed that some chemical reactions happened between HLC and the three metal ions to form new chemical compounds. The thermodynamic parameters, ∆H, ∆G and ∆S, showed that the chelation process between HLC and metal ions was performed spontaneously. Fluorescence quenching spectra of HLC indicated that the quenching mechanism was static in nature. According to the data of DSC, the new chelates were more stable than the free HLC. And HLC-metal complex was non-toxic to the BHK21 cell through MTT assay.Highlights► HLC-Ca, HLC-Cu and HLC-Mn were new chemical compounds and different to free HLC. ► Possible sites for Ca2 +, Cu2 + and Mn2 + to bind with HLC were presented. ► The chelation process between HLC and metal ions was performed spontaneously. ► The thermodynamic stability of the new chelates was higher than that of free HLC.
Co-reporter:Xian Li, Xiaoxuan Ma, Daidi Fan and Chenhui Zhu
Soft Matter 2012 vol. 8(Issue 14) pp:3781-3790
Publication Date(Web):22 Feb 2012
DOI:10.1039/C2SM06994F
The ability of four gel-forming copolymers to act as in situ dermal fillers in plastic and reconstruction surgery was studied. The four hydrogels, which were based on chitosan (CS), included CS/β-GP (β-sodium glycerophosphate), CS–HLC (human-like collagen)/β-GP, CS–AC (animal-derived collagen)/β-GP and CS–Gelatin/β-GP. The potential of the hydrogels as tissue engineering scaffolds was explored by an MTT test and an in vitro degradation assay. The interior morphologies of these hydrogels were also characterized before and after degradation. Histocompatibility in vivo was evaluated by hematoxylin and eosin (H&E) staining, immunohistochemical analysis and transmission electron microscopy (TEM). An MTT assay showed that the CS–HLC/β-GP hydrogel was less cytotoxic and could promote marrow stromal cell (MSC) proliferation better than other hydrogels. Data from an in vitro degradation test showed that the CS–HLC/β-GP hydrogel had a longer degradation time and led to a lower weight loss than the other hydrogels. Furthermore, the CS–HLC/β-GP hydrogel rapidly formed a stable gel that maintained its integrity even after 6 weeks in vivo. From TEM, there were found to be a large quantity of macrophages, fibroblasts, fibrocytes, lymphocytes and capillaries in the CS–AC/β-GP and CS–Gelatin/β-GP hydrogels, whilst levels in the CS–HLC/β-GP hydrogel were small. Furthermore, abnormalities of cell morphology were observed in the CS–AC/β-GP and CS–Gelatin/β-GP hydrogels whereas cell morphology in the CS–HLC/β-GP hydrogel was regular. Therefore, the CS–HLC/β-GP hydrogel, with good cytocompatibility and histocompatibility, is suitable for soft tissue defect filling, such as skin patches, wrinkles, and tissue cavities formed by surgery.
Co-reporter:Y.S. Ren, X.S. Zhu, D.D. Fan, P. Ma, L.H. Liang
Energy Procedia 2012 Volume 16(Part A) pp:211-216
Publication Date(Web):2012
DOI:10.1016/j.egypro.2012.01.035
An efficient agar plate mutagenesis and screening technique for improving mutation frequency was established by using dielectric barrier discharge (DBD) plasma. Enterobacter agglomerans is well known as a phosphate-solubilizing plant-associated bacterium. In this study, DBD plasma was conducted toE. agglomerans mutation for improving the phosphate-soubilizing activity. The results showed that the phosphate-soubilizing activity of mutants increased compared with original strain, and the phosphate-soubilizing activity of the best mutants is 1.49-fold of original strain. It demonstrated that DBD plasma treatment has a high-efficient quality, and it will be a useful method of mutation.
Co-reporter:Yuanyuan Yu
Biological Trace Element Research 2012 Volume 145( Issue 1) pp:33-38
Publication Date(Web):2012 January
DOI:10.1007/s12011-011-9167-x
The calcium complex of human-like collagen (HLC) was generated in aqueous solution, purified by dialysis or ultrafiltration, and isolated by freeze-drying. Evidence for interactions between HLC and calcium(II) was obtained through measurements of the complex's fluorescence and UV-visible spectra, the interpretation of its Fourier transform infrared spectroscopy and circular dichroism spectra, thermogravimetric (TG) analysis, and differential scanning calorimetry (DSC). The HLC–Ca complex is considered a new chemical compound in which HLC retains its helical structure and the calcium ion is linked to the protein via C=O and N–H groups. Additionally, TG and DSC measurements indicate that the HLC–Ca complex is thermodynamically more stable than free HLC. The results obtained provide important evidence for the further study of the interactions of HLC with Ca(II).
Co-reporter:Lan Chen;Chenhui Zhu;Bowen Liu;Xiaoxuan Ma;Zhiguang Duan;Yang Zhou
Journal of Biomedical Materials Research Part A 2011 Volume 99A( Issue 3) pp:395-409
Publication Date(Web):
DOI:10.1002/jbm.a.33202
Abstract
Novel human-like collagen (HLC)/chitosan blended with poly(ethylene oxide) (PEO) nanofibrous meshes of different ratios were fabricated by electrospinning from aqueous solutions. Through studying the effects of the three composition on the solution rheological properties and the morphology of electrospun meshes, the mechanism of electrospinning was explored at the molecular level, and the ratio of PEO/(HLC & chitosan) (w/w) should be controlled below 1/4 as a plasticizer and HLC/chitosan maintained 4/3 w/w. Obtained meshes were treated by 0.2% glutaraldehyde solution (95% ethanol) for crosslinking and 0.2 M glycine solution for blocking unreacted aldehyde groups and became insoluble with fiber diameters of 151 ± 33 to 278 ± 46 nm, PEO was leached out after crosslinking and rinsing. HLC/chitosan scaffolds (4/3, w/w) could mimic native ECM in both chemical component and structure and support cellular in-growth in vivo while exhibited proper degradation rate in vivo. Bone marrow stromal cells adopted a flattened shape with filopodia- and lamellipodia-like extensions in the scaffolds and grew as a confluent layer after 7 days of culture in vitro. This study indicated the feasibility of electrospun nanofabrious HLC/chitosan scaffold from aqueous solution for tissue engineering application. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
Co-reporter:Daidi Fan;Jianyu Xing;Wenjiao Xue;Chenhui Zhu;Xiaoxuan Ma;Rong Ma
Chinese Journal of Chemistry 2011 Volume 29( Issue 9) pp:1811-1816
Publication Date(Web):
DOI:10.1002/cjoc.201180318
Abstract
Effects of temperature on self-interaction of human-like collagen (HLC) were investigated by hydrophobic interaction chromatography, calorimetric measurement, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Results show that three types of interaction roles may exist between HLC molecules at 3–50°C, which were divided into three narrower temperature ranges. In temperature range from 3–22°C, hydrogen bonding plays a key role in the formation of a gelatinous aggregate. In the range of 22–38°C, hydrophobic bonds accompanied by hydrogen bonds are involved in the formation compact aggregates. When temperature is above 38°C the hydrophobic effect formed in the HLC monomer results in the loss of its ability to self-interact.
Co-reporter:Yuan-Yuan Yu, Dai-Di Fan
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2011 Volume 81(Issue 1) pp:412-416
Publication Date(Web):15 October 2011
DOI:10.1016/j.saa.2011.06.030
In the present investigation, the complex of recombinant human-like collagen (r-HLC) with zinc (II) has been synthesized in aqueous solution and was analyzed by UV–vis spectroscopy, FTIR spectroscopy, circular dichroism (CD) spectroscopy, thermo gravimetric (TG) and differential scanning calorimetry (DSC) analysis. It can be concluded from UV–vis spectra that there exists interaction between r-HLC and zinc, and the complex is a new chemical compound different from pure r-HLC. In the complex of Zn, recombinant human-like collagen acts as ligand, linking the zinc ion via both groups of CO and N–H. Besides, the results of TG and DSC confirm that the complex was significantly different from ligand, and the former is more thermally stable in comparison with the latter. The results obtained from the current investigation are of crucial importance to understand the r-HLC–Zn complex and provide theoretical evidence for the further study.Graphical abstractHighlights• The zinc complex of r-HLC was synthesized and investigated. • r-HLC could coordinate with Zn to form a new chemical compound. • The coordination between r-HLC and Zn is by both groups of CO and N–H. • The thermodynamic property of the complex is more stable in comparison with r-HLC.
Co-reporter:Chenhui Zhu;Zhiguang Duan;Wenjiao Xue;Longan Shang;Fulin Chen;Yane Luo
Journal of Biomedical Materials Research Part A 2009 Volume 89A( Issue 3) pp:829-840
Publication Date(Web):
DOI:10.1002/jbm.a.32256
Abstract
With the increasing occurrence of vascular diseases and poor long-term patency rates of current small diameter vascular grafts, it becomes urgent to pursuit biomaterial as scaffold to mimic blood vessel morphologically and mechanically. In this study, novel human-like collagen (HLC, produced by recombinant E. coli)/chitosan tubular scaffolds were fabricated by cross-linking and freeze-drying process. The scaffolds were characterized by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and tensile test, respectively. Human venous fibroblasts were expanded and seeded onto the scaffolds in the density of 1 × 105 cells/cm2. After a 15-day culture under static conditions, the cell–polymer constructs were observed using SEM, confocal laser scanning microscopy (CLSM), histological examination, and biochemical assays for cell proliferation and extracellular matrix production (collagen and glycosaminoglycans). Furthermore, the scaffolds were implanted into rabbits' livers to evaluate their biocompatibility. The results indicated that HLC/chitosan tubular scaffolds (1) exhibited interconnected porous structure; (2) achieved the desirable levels of pliability (elastic up to 30% strain) and stress of 300 ± 16 kPa; (3) were capable of enhancing cell adhesion and proliferation and ECM secretion; (4) showed superior biocompatibility. This study suggested the feasibility of HLC/chitosan composite as a promising candidate scaffold for blood vessel tissue engineering. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res 2009
Co-reporter:Xiao-Jun Wang;Dai-Di Fan;Yan-E Luo
Applied Biochemistry and Biotechnology 2009 Volume 158( Issue 2) pp:262-276
Publication Date(Web):2009 August
DOI:10.1007/s12010-008-8351-8
The adsorption of recombinant human-like collagen by metal chelate media was investigated in a batch reactor and in a fixed-bed column. The adsorption equilibrium and kinetics had been studied by batch adsorption experiments. Equilibrium parameters and protein diffusivities were estimated by matching the models with the experimental data. Using the parameters of equilibrium and kinetics, various models, such as axial diffusion model, linear driving force model, and constant pattern model, were used to simulate the breakthrough curves on the columns. As a result, the most suitable isotherm was the Langmuir–Freundlich model, and the ionic strength had no effect on the adsorption capacity of chelate media. In addition, the pore diffusion model fitted very well to the kinetic data. The pore diffusivities decreased with increasing the initial protein concentration, however had little change with the ionic strength. The results also indicated that the models predict breakthrough curves reasonably well to the experimental data, especially at low initial protein concentration (0.3 mg ml−1) and low flow rate (34 cm h−1). By the results, we optimized the experimental conditions of a chromatographic process using immobilized metal affinity chromatography to purify recombinant human-like collagen.
Co-reporter:Wen J. Xue;Dai D. Fan;Longan Shang;Chen H. Zhu;Xiao X. Ma
Biotechnology Letters 2009 Volume 31( Issue 2) pp:221-226
Publication Date(Web):2009 February
DOI:10.1007/s10529-008-9852-9
The evolution of CO2 in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O2-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO2 concentration of 12.7% in the effluent gas was detected. The CO2 pulse injection experiments showed that: (1) a 20% CO2 pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases slightly stimulated growth; and (2) CO2 inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20% CO2 pulse. The higher the CO2 concentration and/or the longer the duration of the CO2 pulse, the stronger the stimulatory or inhibitory effects.
Co-reporter:Yan E. Luo;Dai D. Fan;Long A. Shang;Hui J. Shi;Xiao X. Ma
Biotechnology Letters 2008 Volume 30( Issue 4) pp:637-643
Publication Date(Web):2008 April
DOI:10.1007/s10529-007-9593-1
Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation.
Co-reporter:Li-Heng Wang;Dai-Di Fan;Long-An Shang;Hui-Juan Shi;Xiao-Xuan Ma;Yu Mi;Li-Feng Gu;Kang-Zhen Xu
Chinese Journal of Chemistry 2007 Volume 25(Issue 1) pp:
Publication Date(Web):8 JAN 2007
DOI:10.1002/cjoc.200790011
A study of the effect of drug, kanamycin, on the growth metabolism of recombinant Escherichia coli B1 was carried out by microcalorimeter monitoring of the metabolic activity of treated cells. Power-time curves of growing recombinant Escherichia coli cell suspensions, treated with different kanamycin doses, were recorded. The extent of the effect was evaluated by changes in the slopes of the microcalorimetric curves and the kinetics of the drug action was interpreted from the time at which these changes reached their maximum values and maintained their maximum values. Experimental dose-effect relationships conform to the median-effect principle of the mass-action law: fa/(1−fa)=(D/D50)m. A plot of y=lg[(fa)1−1]−1 versus x=lg D gives the slope m, D50 and R∞. The experimental results revealed that high concentration of kanamycin had an inhibitory effect on the growth of recombinant Escherichia coli B1 in the lg phase, and had a promoting effect in the stationary period. Moreover, it was demonstrated that microcalorimetry was a reliable method for the detection of modulatory effects in biology.
Co-reporter:Jiaqing GUO, Yan'e LUO, Daidi FAN, Pengfei GAO, Xiaoxuan MA, Chenhui ZHU
Chinese Journal of Chemical Engineering (October 2010) Volume 18(Issue 5) pp:830-836
Publication Date(Web):1 October 2010
DOI:10.1016/S1004-9541(09)60135-X
Recombinant Escherichia coli BL21 is used to produce human-like collagen. The key constituents of media are optimized using response surface methodology (RSM). Before thermal induction, the highest biomass production and the lowest production of some hazardous by-products, especially acetic acid, were obtained in the media containing 0.085 mol·L−1 glucose and 0.019 mol·L−1 nitrogen (carbon-nitrogen ratio, 4.47:1). After thermal induction, when the concentrations of glucose and nitrogen in the media were 0.065 mol·L−1 and 0.017 mol·L−1, respectively (carbon-nitrogen ratio, 3.82:1), the productivity of human-like collagen per cell was the highest while that of acetic acid was the lowest. The extended analysis showed that the production of lactic acid and propionic acid increased while that of some intermediate acids of the tricarboxylic acid cycle decreased if the dose of glucose increased.
Co-reporter:Huoqing CHEN, Xiaoxuan MA, Daidi FAN, Yan'e LUO, Pengfei GAO, Chanyuan YANG
Chinese Journal of Chemical Engineering (August 2010) Volume 18(Issue 4) pp:681-686
Publication Date(Web):1 August 2010
DOI:10.1016/S1004-9541(10)60275-3
The effects of L-cysteine concentration on biohydrogen production by Enterobacterium Bacterium M580 were investigated in batch cultivation. The experimental results showed that L-cysteine could enhance the cell growth, hydrogen production rate and hydrogen yield when its concentration was less than 500 mg·L−1, while it had negative effects when its concentration was higher than 500 mg·L−1. The hydrogen production was the highest [1.29 mol·mol−1 (H2/glucose)] when 300 mg·L−1 L-cysteine was added into the culture, and the yield was 9.4% higher than that in the control. The oxidation-reduction potential (ORP), which was influenced by L-cysteine, also affected hydrogen production. The ORP values were in the range (300 mV to (150 mV when the L-cysteine concentration was higher than 500 mg·L−1. Although the ORP in this range was favorable for hydrogen production, it was not suitable for the biomass growth. Hence, less hydrogen was produced. When the L-cysteine concentration was lower than 500 mg·L−1, the ORP was more suitable for both biomass growth and hydrogen production. In addition, at least 91% glucose was consumed when L-cysteine was added to the culture media, compared to the 97.37% consumption without L-cysteine added.
Co-reporter:Xiaoyan Zheng, Junfeng Hui, Hui Li, Chenhui Zhu, Xiufu Hua, Haixia Ma, Daidi Fan
Materials Science and Engineering: C (1 June 2017) Volume 75() pp:
Publication Date(Web):1 June 2017
DOI:10.1016/j.msec.2017.02.103
•A simple and effective hydrothermal method was developed for preparing uniform HAp nanorods with amphiphilic surfaces.•The synthesized amphiphilic HAp nanorods could be dispersed in water, ethyl alcohol or cyclohexane.•The prepared HLC/HAp porous scaffolds had good mechanical properties, biocompatibility and osteoconductive etc.This paper describes a new synthetic strategy and biological application for novel amphiphilic hydroxyapatite (HAp) nanorods. The prepared HAp nanorods were able to be dispersed in water, ethyl alcohol and cyclohexane. The co-anchoring of the multidentate ligands of PEG 20000 and hydrophobic oleic acid (OA) on the rods' surfaces endowed them with excellent amphibious properties. Utilizing amphiphilic HAp nanorods with excellent biocompatibility as the inorganic phase, human-like collagen (HLC) as the organic phase and natural genipin as the cross-linker, optimal HLC/HAp porous scaffolds (HLC: HAp = 1:4, w/w) were fabricated. The compression stress and three-point bending strength of the scaffolds with pore diameters of 150 to 200 μm reached approximately 3.4 MPa and 5.4 MPa, respectively, and their porosity was 77.35 ± 3.75%. Cytological tests showed that HLC/HAp scaffolds could contribute to cell proliferation and differentiation. The results indicated that these novel amphiphilic HAp nanorods can be expected to become recognized as an excellent inorganic material for the porous scaffolds used in repairing bone and related applications.
Co-reporter:Xiaoyan Zheng, Meiying Liu, Junfeng Hui, Daidi Fan, Haixia Ma, Xiaoyong Zhang, Yaoyu Wang and Yen Wei
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 31) pp:NaN20307-20307
Publication Date(Web):2015/07/01
DOI:10.1039/C5CP01845E
In this paper we report two different doping strategies to prepare a series of novel HAp:Ln3+ (Ln = Eu or Tb) nanocrystals with tunable aspect ratios via facile hydrothermal synthetic routes. Adopting a one-pot synthetic strategy, with increasing rare-earth doping dosage, the as-prepared nanocrystals have relatively weak fluorescence intensity, and change from nanorods with lengths of about 150 nm into nanowires with lengths of about 2 μm. Using the synthetic pure HAp nanorods as matrices, they are endowed with bright green or red luminescent properties by doping Tb3+ or Eu3+ ions via a second hydrothermal process, and simultaneously retain their original morphologies (diameter 8 nm, length 150 nm). The hydrophobic HAp:Ln3+ nanorods with strong optical properties are converted into hydrophilic particles with a surfactant (Pluronic F127) and successfully applied to live cell imaging.
Co-reporter:Xiaoxuan Ma, Jianjun Deng, Yuzhang Du, Xian Li, Daidi Fan, Chenhui Zhu, Junfeng Hui, Pei Ma and Wenjiao Xue
Journal of Materials Chemistry A 2014 - vol. 2(Issue 18) pp:NaN2763-2763
Publication Date(Web):2014/02/21
DOI:10.1039/C3TB21842B
Novel hydrogels (termed HCD hydrogels) were synthesized based on human-like collagen (HLC) and chitosan (CS) cross-linked with dialdehyde starch (DAS). The biological stability and biocompatibility of HCD hydrogels were determined through in vitro and in vivo tests. The mechanism of hydrogel formation was studied using Fourier transform infrared spectroscopy (FTIR), which showed that covalent bonds formed via acetalization and Schiff base reactions. Biological stability was evaluated in vitro by degrading HCD hydrogels with class I collagenase, class II collagenase, and both class I and class II collagenases and in vivo after subcutaneously injecting HCD into an animal model. The biological characteristics of HCD hydrogels was studied by two methods: (i) MTT and cytomorphology cytotoxicity and cytocompatibility and (ii) in vivo, whereby histomorphometry, transmission electron microscopy (TEM), and immunohistochemistry were used to compare different types of surgically introduced hydrogels, our HCD hydrogels, SunMax Collagen Implant hydrogels (SUM hydrogels), and OUTLINE&EVOLUTION Injectable Synthetic Gel hydrogels (EVL hydrogels). The in vivo analyses were performed at 1, 9, 12, and 28 weeks after surgery. The hydrogel biodegradation results showed that the normalized residual weight (WR) of HCD hydrogels varied with DAS content. In vitro, we found that the minimum WR of HCD hydrogels was 42.19% after 28 weeks when degraded by both types of class I and class II collagenase. The MTT assay indicated that the minimum relative growth rate (RGR) of cells was 93% after they were incubated with HCD hydrogels for 7 days, suggesting good cytocompatibility. In vivo histomorphometry results indicated that HCD hydrogels effectively filled tissue voids and did not cause redness, edema, festering, or color changes. In addition, a few vessels grew into the hydrogel and a thin fibrous capsule was eventually produced. TEM and immunohistochemistry studies suggested that HCD hydrogels produced less intense inflammatory responses than those produced by SUM hydrogels and EVL hydrogels. Overall, HCD hydrogels afford both enhanced biological stability and excellent biocompatibility, making them potentially promising for skin patch scaffolds, wrinkle treatments, and tissue cavity fillers.
Co-reporter:Xian Li, Daidi Fan, Chenhui Zhu and Xiaoxuan Ma
Journal of Materials Chemistry A 2014 - vol. 2(Issue 9) pp:NaN1249-1249
Publication Date(Web):2013/12/19
DOI:10.1039/C3TB21628D
CS–HLC–HA–β-GP (chitosan–human-like collagen–hyaluronic acid–β-sodium glycerophosphate) hydrogels were prepared based on the self-assembly of CS–HLC–HA (CCA) fibers. The effects of the fibers on the synthesis, characteristics and biomedical applications of CS–HLC–HA–β-GP (CCAG) hydrogels were studied for various HA contents. The synthesis mechanism of the novel CCAG hydrogel was explored using Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The hydrogels were characterized by a swelling test, gelling time and enzymatic treatments. The results indicated that a new amide bond (–CONH) and –NRH2+ were formed. The gelling time and swelling behaviors were dependent on the intertwining, overlap and adsorption of the polymer chains at various temperatures and pH. Furthermore, biomedical applications were evaluated by transmission electron microscopy (TEM), immunohistochemical analysis and haematoxylin and eosin (H&E) staining. The effect of the fibers on the histocompatibility of the hydrogels revealed that the fibers inside the hydrogel pores reduced the quantity of macrophages, decreased the degree of inflammation, and improved the anti-degradation of the modified hydrogels. This type of new hydrogel emerges as an interesting injectable filling biomaterial for tissue engineering.
Co-reporter:Xian Li, Wenjiao Xue, Yannan Liu, Daidi Fan, Chenhui Zhu and Xiaoxuan Ma
Journal of Materials Chemistry A 2015 - vol. 3(Issue 23) pp:NaN4755-4755
Publication Date(Web):2015/05/12
DOI:10.1039/C5TB00408J
In this study, we designed multifunctionalized hydrogel scaffolds and injectable particles based on high-molecular-weight (MW) pullulan and human-like collagen (HLC) crosslinked with 1,4-butanediol diglycidyl ether (BDDE) for combination therapy tissue restoration. The properties of the pullulan/BDDE (PB) and pullulan/BDDE/human-like collagen (PBH) hydrogels were characterized via swelling ratio measurements, mechanical tests, and enzymatic degradation in vitro and via subcutaneous injections in vivo. The results demonstrate that the dry hydrogels completely returned to their original state in deionized water. The elastic modulus of the PBH53 dry hydrogels is higher than that of the other hydrogels after exposure to bending stress and compression stress with a maximum value of 7858.93 MPa. In addition, the in vitro live/dead staining and cell adhesion of the PBH hydrogels exhibited a superior fibroblast morphology without high levels of cell death, which were considerably better than those of PB hydrogels. In vivo, PB and PBH particles with good biocompatibility and anti-biodegradation were successfully prepared via the granulation of wet PB and PBH hydrogels for efficient subcutaneous injection in Kunming mice and New Zealand rabbits. Therefore, the PB and PBH hydrogels were found to be acceptable, safe, soft materials for use in skin restoration, cartilage treatment, and lacrimal dryness therapy.
