Yu Mi

Encapsulation of probiotic Bifidobacterium longum BIOMA 5920 with alginate-human-like collagen and evaluation of survival in simulated gastrointestinal conditions.

Alginate (ALg)-human-like collagen (HLC) microspheres were prepared by the technology of electrostatic droplet generation in order to develop a biocompatible vehicle for probiotic bacteria. Microparticles were spherical with mean particle size of 400μm. The encapsulation efficiency (EE) of ALg-HLC microspheres could reach 92-99.2%. Water-soluble and fibrous human-like collagen is combined with sodium alginate through intermolecular hydrogen bonding and electrostatic force which were investigated by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC), thus the matrix of ALg-HLC was very stable. Bifidobacterium longum BIOMA 5920, as a kind of probiotic bacteria, was encapsulated with alginate-human-like collagen to survive and function in simulated gastrointestinal juice. Microparticles were very easy to degradation in simulated intestinal juices. After incubation in simulated gastric (pH 2.0, 2h), the encapsulated B. longum BIOMA 5920 numbers were 4.81 ± 0.38 log cfu/g.

Human-like collagen/nano-hydroxyapatite scaffolds for the culture of chondrocytes

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.

Protopanaxadiol and Protopanaxatriol-Type Saponins Ameliorate Glucose and Lipid Metabolism in Type 2 Diabetes Mellitus in High-Fat Diet/Streptozocin-Induced Mice

Ginsenoside is a major active component of ginseng, which exhibits various pharmacological properties such as hepatoprotection, tumor suppression and diabetes resistance. In this study, the anti-diabetic effects of protopanaxadiol (PPD) and protopanaxatriol (PPT)-type saponins were explored and compared in high-fat diet/streptozocin-induced type 2 diabetes mellitus (T2DM) mice. Our results showed that low or high dose (50 mg/kg bodyweight or 150 mg/kg bodyweight) PPD and PPT significantly reduced fasting blood glucose, improved glucose tolerance and insulin resistance in T2DM mice. PPD and PPT also regulated serum lipid-related markers such as reduced total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol in T2DM mice. In addition, PPD and PPT dramatically ameliorated the inflammatory responses by suppressing the secretion of pro-inflammatory cytokines like tumor necrosis factor-alpha and interleukin-6 in serum level and gene expression in liver level, and improved the antioxidant capacity by increasing the superoxide dismutase and decreasing malondialdehyde levels in the serum of T2DM mice. Moreover, the anti-diabetic effect of PPD and PPT appeared to be partially mediated by the suppression of hepatic metabolism genes expression such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase, as well as facilitating lipid metabolism genes expression such as microsomal TG transfer protein in the liver tissues of T2DM mice. Taken together, our results indicated that PPD and PPT might potentially act as natural anti-diabetic compounds to be used for preventing and treating the T2DM and its complications in the future.

Novel Scaffolds Fabricated Using Oleuropein for Bone Tissue Engineering

We investigated the feasibility of oleuropein as a cross-linking agent for fabricating three-dimensional (3D) porous composite scaffolds for bone tissue engineering. Human-like collagen (HLC) and nanohydroxyapatite (n-HAp) were used to fabricate the composite scaffold by way of cross-linking. The mechanical tests revealed superior properties for the cross-linked scaffolds compared to the uncross-linked scaffolds. The as-obtained composite scaffold had a 3D porous structure with pores ranging from 120 to 300 μm and a porosity of 73.6 ± 2.3%. The cross-linked scaffolds were seeded with MC3T3-E1 Subclone 14 mouse osteoblasts. Fluorescence staining, the Cell Counting Kit-8 (CCK-8) assay, and scanning electron microscopy (SEM) indicated that the scaffolds enhanced cell adhesion and proliferation. Our results indicate the potential of these scaffolds for bone tissue engineering.

Oxygen transfer rate control in the production of human‐like collagen by recombinant Escherichia coli

The effects of different methods for elevating the OTR (oxygen transfer rate) during foreign gene expression and the cell growth of recombinant Escherichia coli BL21 were investigated. Two strategies were introduced to control DO (dissolved oxygen) levels in the fermentation broth: (i) increasing fermentor pressure and (ii) supplying oxygen‐enriched air. These two methods were compared with the glucose feedback model, which acted as the control. By adopting a fed‐batch method of cultivation, the cell yield coefficient (YX/S), accumulation of acetic acid and volumetric product yield (Yp) were measured or estimated. Adoption of these two methods led to an improvement in the OTR. The cell density and volumetric product yield in the cultivation controlled by increasing the fermentor pressure reached 77 g·l−1 (dry cell weight) and 14 g·l−1 respectively, which were much higher than those obtained with the strategy of supplying oxygen‐enriched air (48 and 6 g·l−1 respectively) and in the control (46 and 7 g·l−1 respectively). The results indicate that increasing fermentor pressure is an effective way to enhance the OTR and recombinant protein (human‐like collagen) productivity.

A genetic algorithm for the optimization of the thermoinduction protocol for high-level production of recombinant human-like collagen from Escherichia coli

Production of recombinant human‐like collagen (RHLC) by thermoinduction of recombinant Escherichia coli BL 21 during high cell density cultivation was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon‐to‐nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermoinduction protocol for RHLC production was determined by using a model coupling genetic algorithm and artificial neural networks. The optimal operating conditions were as follows: maintenance of induction temperature at 42°C for 3 H and then at 39.4°C until the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, whereas the experimental value was 12.1 g/L under the optimal induction conditions.

A Novel Human-Like Collagen Hydrogel Scaffold with Porous Structure and Sponge-Like Properties

The aim of this research was to prepare a novel sponge-like porous hydrogel scaffold based on human-like collagen (HLC) that could be applied in cartilage tissue regeneration. In this study, bovine serum albumin (BSA) was used as a porogen to prepare the porous hydrogel, which had not been previously reported. Glutamine transaminase (TGase) was used as the cross-linker of the hydrogel, because it could catalyze the cross-linking of BSA. During the crosslinking process, BSA and HLC were mixed together, which affected the cross-linking of HLC. When the cross-linking was completed, the non-crosslinked section formed pores. The microstructure, porosity, swelling properties, and compressive properties of the hydrogel were studied. The results showed that the pore size of the hydrogel was between 100 and 300 μm, the porosity reached up to 93.43%, and the hydrogel had rapid water absorption and suitable mechanical properties. Finally, we applied the hydrogel to cartilage tissue engineering through in vitro and in vivo research. The in vitro cell experiments suggested that the hydrogel could promote the proliferation and adhesion of chondrocytes, and in vivo transplantation of the hydrogel could enhance the repair of cartilage. In general, the hydrogel is promising as a tissue engineering scaffold for cartilage.

Improving the production of human-like collagen by pulse-feeding glucose during the fed-batch culture of recombinant Escherichia coli

To increase the target protein production and reduce acetic acid accumulation during fed‐batch cultivation of recombinant Escherichia coli BL21 in a 30‐L bioreactor, 12 different models of pulse feeding were performed to evaluate the effect of pulse feeding at different cultivation phases and pulse frequency on cell growth, acetic acid accumulation, and human‐like collagen (HLC) synthesis. The results showed that the acetate concentration was kept at a low level (below 0.5 g/L) in all cases when pulse feeding was introduced before induction, whereas the pulse frequency affected cytoactivity significantly through cell growth rate, oxygen uptake rate, carbon dioxide evolution rate, and the synthesis of the target protein. The final biomass and HLC reached 75.46 and 7.26 g/L, respectively, in the model of 8‐Sec feedings per 188 Sec. After induction, the pulse frequency had a great effect on HLC synthesis after high‐temperature induction; low frequency was adverse to microorganisms. The model of 3‐Sec feeding per 27 Sec was best and resulted in the highest biomass and HLC production. Compared to the pseudo‐exponential feeding, pulse feeding reduced acetic acid accumulation effectively.

A novel thiolated human-like collage zinc complex as a promising zinc supplement: Physicochemical characteristics and biocompatibility

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 pH8.0. One mole of thiolated HLC-Zn (SHLC-Zn) complex possessed 24.3mol 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.

A Two-Step Protocol to Remove Endotoxins from Human-Like Collagen

A two-step protocol to remove endotoxins from recombinant human-like collagen was investigated. To meet the clinical need, Triton X-114 two-phase extraction system was applied as the first step to remove large amounts of endotoxins, while affinity chromatography was used as the second step to achieve further purification. Under the best experimental condition optimized through response surface methodology techniques, endotoxin level was 0.025˜0.25 EU/mg and protein recovery was 81.9%, respectively, which indicated that 99% of endotoxins were eliminated with high protein recovery. Therefore, the two-step protocol can be exploited as an efficient method for endotoxin elimination in biotechnology processes.