Xiaoxuan Ma

Effects of Chitosan on Properties of Novel Human-like Collagen/Chitosan Hybrid Vascular Scaffold

Novel human-like collagen (HLC)/chitosan hybrid scaffolds were fabricated at blend ratios of 0%, 0.02%, 0.2% by crosslinking and freeze-drying process. The properties of the scaffolds were investigated, including morphology, mechanical strength, degradability, and cell biocompatibility. When the blend ratio was 0.02%, the morphology of the scaffolds was highly homogeneous with interconnected porous structure 46 ± 9 μm in size (SEM). The X-ray photoelectron spectroscopy analysis indicated intermolecular crosslinks between HLC and chitosan. The strain and stress of the scaffolds were 37.9 ± 3.3% and 309.7 ± 19.7 KPa, respectively. Human venous fibroblasts were expanded and seeded into the scaffolds in the density of 1 × 10 5 cells/cm3 under static conditions. The cell morphology and proliferation were investigated using SEM, H&E, and MTT assay, which showed that the optimal content of the chitosan was signifcantly enhanced the cells adhesion, proliferation, and viability, compared to pure HLC, pure chitosan, and 0.2% chitosan/HLC scaffolds. These hybrid scaffolds appear to have favorable characteristics for vascular tissue engineering application.

A human-like collagen/chitosan electrospun nanofibrous scaffold from aqueous solution: electrospun mechanism and biocompatibility.

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.

New suitable for tissue reconstruction injectable chitosan/collagen-based hydrogels

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.

A Novel Injectable pH/Temperature Sensitive CS-HLC/β-GP Hydrogel: The Gelation Mechanism and Its Properties

The chitosan-human-like collagen/β-sodium glycerophosphate (CS-HLC/β-GP) hydrogel, is sensitive to pH and temperature changes and has proved to be a potential candidate as an injectable filling biomaterial in plastic and reconstruction surgery. The formation and pH/temperature-sensitivity mechanism of the novel CS-HLC/β-GP hydrogel were explored by Fourier transform infrared spectroscopy (FTIR) and swelling tests. Our results showed that an amide bond (-CONH) is formed between the carbonyl (CO) of HLC and the amino of CS and -NRH2 + is formed between the amide bond and β-GP; the temperature sensitivity depended on the hydrophobic-hydrophilic interaction of molecular chains, and the pH sensitivity depended on the electrostatic interaction of ionic groups (-NRH2 + and -OPO3 2-). The properties of the novel CS-HLC/β-GP hydrogel were also examined using the tube inverting method, the swelling test, the liquid displacement method, and mechanical testing. The gelling time before and after addition of HLC is 30 min and 8 min, respectively, at 37°C. Compared with conventional CS/β-GP hydrogels, such as CS-AC (animal-derived collagen)/β-GP hydrogel, the CS-HLC/β-GP hydrogel showed a desirable porosity level, swelling ratio, pH/temperature sensitivity, a smooth surface, regular porous networks, and adequate mechanical strength and cross-linking densities. Therefore, the CS–HLC/β-GP hydrogels are excellent candidates for use in biomedical fields, such as in soft tissue defect filling and drug delivery.

Synthesis and characterization of hyaluronic acid/human-like collagen 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.

Medium optimization based on the metabolic‐flux spectrum of recombinant Escherichia coli for high expression of human‐like collagen II

Recombinant Escherichia coli BL21 was used to produce human‐like collagen II in fed‐batch cultivation. By performing MFA (metabolic‐flux analysis), the carbon/nitrogen molar ratios in both the batch and feeding media were optimized for high‐level production of human‐like collagen II. Three carbon/nitrogen molar ratios in both the batch and feeding media were used in the present study, and the MFA results showed that the optimal carbon/nitrogen molar ratios for the batch and feeding media were 2.36:1 and 5.12:1 respectively, yielding the highest dry‐cell density (67.2 g/l dry cell weight) and human‐like collagen production (10.8 g/l).

A novel smart injectable hydrogel prepared by microbial transglutaminase and human-like collagen: Its characterization and biocompatibility.

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 50U/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, 40U/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 40U/g MTGase-prepared MTGH hydrogels had a lower toxicity and better biocompatibility. Therefore, 40U/g MTGase crosslinked with HLC should be used to prepare MTGH hydrogels for potential application as soft materials for skin tissue engineering.

Effects of acetic acid and its assimilation in fed-batch cultures of recombinant Escherichia coli containing human-like collagen cDNA

The primary processing problem in recombinant Escherichia coli fermentation is the production of acetic acid, which can inhibit both cell growth and recombinant protein production. The ability of E. coli to assimilate acetate permits it to solve this problem in a rather creative manner. In this study, the effects of acetic acid assimilation through a glucose starvation period at different cell growth phases were investigated in fed-batch cultures of recombinant E. coli. Experimental results showed that the human-like collagen (HLC) production could be improved by introducing glucose starvation at the end of batch culture and pre-induction phase, while the glucose starvation at the induction phase resulted in a poor HLC productivity. The acetic acid assimilation was observed during all the glucose starvation periods. In addition, a systematic study for evaluating the effects of acetic acid was carried out by adding acetate into culture media at different cell growth phases and then employing a glucose starvation after several hours. It was found that obvious acetate inhibition on cell growth occurred in the batch culture phases while its inhibitory effect on HLC expression occurred only in the post-induction phase. The longer the elevated acetic acid concentration maintained, the stronger the inhibitory effects were. These results are of significance for optimizing and scaling-up fermentation processes.

A novel chitosan–collagen-based hydrogel for use as a dermal filler: initial in vitro and in vivo investigations

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.

Effects of self-assembled fibers on the synthesis, characteristics and biomedical applications of CCAG hydrogels

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.