Shaanxi Provincial Institute of Microbiology

Xian, China

Shaanxi Provincial Institute of Microbiology

Xian, China
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Shi J.,Northwest University, China | Ma X.,Northwest University, China | Gao Y.,Northwest University, China | Fan D.,Northwest University, China | And 3 more authors.
Protein Journal | Year: 2017

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. © 2017 Springer Science+Business Media New York


Li X.,Northwest University, China | Xue W.,Shaanxi Provincial Institute of Microbiology | Liu Y.,Northwest University, China | Li W.,Northwest University, China | And 3 more authors.
Materials Science and Engineering C | Year: 2016

New locally injectable biomaterials that are suitable for use as soft tissue fillers are needed to address a significant unmet medical need. In this study, we used pullulan and human-like collagen (HLC) based hydrogels with various molecular weights (MWs) in combination therapy against tissue defects. Briefly, pullulan was crosslinked with NaIO4 to form a pullulan hydrogel and then may coupled with HLC using the reaction between the -NH2 end-group of HLC and the -CHO group present on the aldehyde pullulan to form the HLC/pullulan hydrogel, wherein the NaIO4 acted as the crosslinking and oxidizing agent. The good miscibility of pullulan and HLC in the hydrogels was confirmed via Fourier transform infrared spectroscopy, scanning electron microscopy, compression testing, enzyme degradation testing, cell adhesions, live/dead staining and subcutaneous filling assays. Here, pullulan hydrogels with various MWs were fabricated and physicochemically characterized. Limitations of the pullulan hydrogels included inflammation, poor mechanical strength, and degradation. By contrast, the properties of the HLC/pullulan hydrogels strongly enhanced. The efficacy of these hydrogels was evaluated both in vitro and in vivo. Our results indicate that HLC/pullulan hydrogels may have therapeutic value as efficient soft tissue fillers, with reduced inflammation, improved cell adhesion and delayed hydrogel degradation. © 2015 Published by Elsevier B.V.


Li X.,Northwest University, China | Fan D.-D.,Northwest University, China | Deng J.-J.,Northwest University, China | Hui J.-F.,Northwest University, China | And 3 more authors.
Asian Journal of Chemistry | Year: 2013

Injectable in situ forming biodegradable chitosan-human like collagen (CS-HLC) based hydrogels has proved to be a potential candidate as an injectable biomaterial for tissue engineering. With crosslinking agent of carbodiimide, the properties of the novel chitosan-humanlike collagen/b-sodium glycerophosphate-carbodiimide hydrogel were also examined. The gelation time, structure, equilibrium swelling and degradation in vitro and in vivo were dependent upon crosslinking and structure of composite hydrogels. The chitosan-human-like collagen/b-sodium glycerophosphate-carbodiimide hydrogel showed a desirable gelling time, swelling ratio, a smooth surface, regular porous networks and biodegradable. Therefore, the chitosan-human-like collagen/b-sodium glycerophosphate-carbodiimide hydrogels are excellent candidates for use in biomedical fields, such as in soft tissue defect filling and drug delivery.


Li X.,Northwest University, China | Xue W.,Shaanxi Provincial Institute of Microbiology | Zhu C.,Northwest University, China | Fan D.,Northwest University, China | And 2 more authors.
Materials Science and Engineering C | Year: 2015

Abstract Novel 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. © 2015 Elsevier B.V.


Li X.,Northwest University, China | Xue W.,Shaanxi Provincial Institute of Microbiology | Liu Y.,Northwest University, China | Fan D.,Northwest University, China | And 2 more authors.
Journal of Materials Chemistry B | Year: 2015

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. © The Royal Society of Chemistry 2015.


Xue W.-J.,Shaanxi Provincial Institute of Microbiology | Fan D.-D.,Northwest University, China
Huaxue Gongcheng/Chemical Engineering (China) | Year: 2011

To control the acetate production and achieve a high-level expression of the target protein in the different-scale bioreactors during fed-batch production of human-like collagen (HLC) with recombinant Escherichia coli. A probing feeding strategy using feed-up DO-transient control was employed in two different-scale bioreactors and the dissolved oxygen response signal was used to detect the acetate production in pulsed fed-batch cultivation of E. coli. The final dry cell weight (DCW) of 69.1 g/L and HLC mass concentration of 13.1 g/L were obtained on the laboratory scale and the results are similar to that optimized in the previous study. When the process was scaled up to pilot-scale (500 L), the final DCW decreased from 80.3 g/L to 54.1 g/L with non-induced cultivations while acetate mass concentration was similar on the both scales. The difference in DCW was mainly resulted from the difference in oxygen transfer capacity. The final DCW of 51.7 g/L and HLC mass concentration of 9.6 g/L were obtained on the pilot-scale upon optimization of induction timing, and the results are satisfied based on the HLC content. The feed-up DO-transient control strategy can be successfully employed to control the acetate production and achieve a high-level expression of the target protein during the fed-batch production of HLC with recombinant E. coli.


Li X.,Northwest University, China | Fan D.,Northwest University, China | Deng J.,Northwest University, China | Hui J.,Northwest University, China | And 3 more authors.
Journal of Pure and Applied Microbiology | Year: 2013

A novel hydrogel for biomedical applications was successfully fabricated by human-like collagen (HLC) and carboxymethylcellulose (CMC) with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and adipic acid dihydrazide (ADH) as crosslinkers. The interior morphologies of these hydrogels were also characterized before and after degradation by collagenase I and simulated body fluid (SBF). The results of in vitro degradability tests of these hydrogels that the CMC-HLC/EDC-ADH hydrogel in SBF indicated that possesses slow degradability than in collagenase I in vitro. Therefore, the results provided the possibility that CMC-HLC/EDC-ADH hydrogels are suitable for biomedical applications such as soft tissue augmentation for their good biocompatibility.


Ma X.,Northwest University, China | Deng J.,Northwest University, China | Du Y.,Northwest University, China | Li X.,Northwest University, China | And 5 more authors.
Journal of Materials Chemistry B | Year: 2014

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. This journal is © the Partner Organisations 2014.


PubMed | Northwest University, China and Shaanxi Provincial Institute of Microbiology
Type: | Journal: Materials science & engineering. C, Materials for biological applications | Year: 2015

New locally injectable biomaterials that are suitable for use as soft tissue fillers are needed to address a significant unmet medical need. In this study, we used pullulan and human-like collagen (HLC) based hydrogels with various molecular weights (MWs) in combination therapy against tissue defects. Briefly, pullulan was crosslinked with NaIO4 to form a pullulan hydrogel and then may coupled with HLC using the reaction between the -NH2 end-group of HLC and the -CHO group present on the aldehyde pullulan to form the HLC/pullulan hydrogel, wherein the NaIO4 acted as the crosslinking and oxidizing agent. The good miscibility of pullulan and HLC in the hydrogels was confirmed via Fourier transform infrared spectroscopy, scanning electron microscopy, compression testing, enzyme degradation testing, cell adhesions, live/dead staining and subcutaneous filling assays. Here, pullulan hydrogels with various MWs were fabricated and physicochemically characterized. Limitations of the pullulan hydrogels included inflammation, poor mechanical strength, and degradation. By contrast, the properties of the HLC/pullulan hydrogels strongly enhanced. The efficacy of these hydrogels was evaluated both in vitro and in vivo. Our results indicate that HLC/pullulan hydrogels may have therapeutic value as efficient soft tissue fillers, with reduced inflammation, improved cell adhesion and delayed hydrogel degradation.


PubMed | Northwest University, China and Shaanxi Provincial Institute of Microbiology
Type: | Journal: Materials science & engineering. C, Materials for biological applications | Year: 2015

Novel 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.

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