Hyaluronic Acid Oligosaccharides Research Articles

Novel β1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides.

The efficiency of de novo synthesis of hyaluronic acid (HA) using Pasteurella multocida hyaluronate synthase (PmHAS) is limited by its low catalytic activity during the initial reaction steps when monosaccharides are the acceptor substrates. In this study, we identified and characterized a β-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant β1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-β-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, β1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (~ 12-fold) with GlcA-pNP as the acceptor, making it a better option for the initial step of de novo HA oligosaccharide synthesis. We then developed a biocatalytic approach for size-controlled HA oligosaccharide synthesis using the disaccharide produced by β1,4 EcGnT as a starting material, followed by stepwise PmHAS-catalyzed synthesis of longer oligosaccharides. Using this approach, we produced a series of HA chains of up to 10 sugar monomers. Overall, our study identifies a novel bacterial β1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides. KEY POINTS: • A novel β-1,4-N-acetylglucosaminyl-transferase (EcGnT) from E. coli O8:K48:H9. • EcGnT is superior to PmHAS for enabling de novo HA oligosaccharide synthesis. • Size-controlled HA oligosaccharide synthesis relay using EcGnT and PmHAS.

Hyaluronic acid oligosaccharide-modified zeolitic imidazolate framework-8 nanoparticles loaded with oxaliplatin as a targeted drug-delivery system for colorectal cancer therapy.

Aim: Exploring a nanoscale targeted drug-delivery system (DDS) for oxaliplatin (Oxa) to improve its therapeutic effect in colorectal cancer. Materials & methods: Nanoparticles were prepared using zeolitic imidazole framework-8 (ZIF-8) modified by hyaluronic acid oligosaccharide (oHA) as an Oxa carrier (oHA@ZIF-8@Oxa). After multiple characterizations, the therapeutic efficacy of the DDS was evaluated by cytotoxicity testing and a nude mouse tumor transplantation experiment in vivo. Results: The results of characterization showed the DDS was homogeneous in morphology and uniform in dispersion. The drug loading of Oxa was 11.82% and the encapsulation efficiency was 90.8%. The cytotoxicity test and in vivo experiments showed that oHA@ZIF-8@Oxa had a more significant anticolorectal cancer effect than free Oxa. Conclusion: This work offers a promising potential DDS for enhancing the anticolorectal cancer effect of Oxa.

Enhanced Tumor Accumulation of Low-Molecular-Weight Hyaluronic Acid/Chitosan Nanocomplexes for Photothermal Therapy

Targeted phototheranostic nanosystems involving both cancer-specific near-infrared (NIR) fluorescence imaging and NIR light-induced phototherapy have shown great potential to improve cancer detection and treatment. In this study, a bifunctional nanocomplex based on low-molecular-weight hyaluronic acid (LMW-HA) and chitosan oligosaccharide lactate (COL) conjugating a zwitterionic NIR dye (ZW800-1) was rationally designed and prepared, and it was simultaneously used to enhance tumor accumulation and photothermal therapy (PTT). When HA-COL-ZW nanocomplexes were intravenously injected into mice bearing NCI-H460 tumors, HA-COL-ZW revealed increased tumor accumulation with prolonged tumor retention. Moreover, the ZW800-1 incorporated in HA-COL-ZW nanocomplexes showed excellent capability to convert NIR light into heat energy at the tumor site, acting as a PTT agent. Therefore, the targeted phototherapeutic HA-COL-ZW nanocomplex is a biocompatible and effective photothermal nanoagent, which could be a good candidate for future clinical use.

A comparative study of the ameliorative effects of hyaluronic acid oligosaccharides and hyaluronic acid on DSS-induced colitis in mice and research on relevant mechanisms.

As a dietary supplement, hyaluronic acid (HA) has exhibited appreciable immunomodulatory activity and an ameliorative effect on rodent colitis. However, its high viscosity is not only refractory to absorb through the gut, but also causes flatulence. In contrast to HA, hyaluronic acid oligosaccharides (o-HAs) can overcome the above-mentioned constraints, but their treatment effect still remains ill-defined contemporarily. Herein, the current study intends to compare the modulatory effects of HA and o-HA on colitis and assess the underlying molecular mechanism. We first showed that o-HA had a better preventive effect than HA in alleviating colitis symptoms, as evidenced by lower body weight loss, lower disease activity index scores, a lower inflammatory response (TNF-α, IL-6, IL-1β, p-NF-κB), and more intact colon epithelial integrity in vivo. The best efficiency was observed in the o-HA treated group with a dosage of 30 mg kg-1. In an in vitro barrier function assay, o-HA exerted a better protective effect on the transepithelial electrical resistance (TEER), FITC permeability, and wound healing and modulated the expression of tight junction (TJ) proteins (ZO-1, occludin) in lipopolysaccharide (LPS)-stimulated Caco-2 cells. In summary, both HA and o-HA showed the potential to reduce inflammation and ameliorate intestinal damage in DSS-induced colitis and LPS-induced inflammation, but o-HA had improved outcomes. The results also provided a glimpse of the latent mechanism by which HA and o-HA enhanced intestinal barrier function via MLCK/p-MLC signaling pathway suppression.

Cloning and Biochemical Characterization of a Hyaluronate Lyase from Bacillus sp. CQMU-D.

Hyaluronidase (HAase) can enhance drug diffusion and dissipate edema by degrading hyaluronic acid (HA) in the extracellular matrix into unsaturated HA oligosaccharides in mammalian tissues. Microorganisms are recognized as valuable sources of HAase. In this study, a new hyaluronate lyase (HAaseD) from Bacillus sp. CQMU-D was expressed in Escherichia coli BL21, purified, and characterized. The results showed that HAaseD belonged to the polysaccharide lyase (PL) 8 family and had a molecular weight of 123 kDa. HAaseD could degrade chondroitin sulfate (CS) -A, CS-B, CS-C, and HA, with the highest activity toward HA. The optimum temperature and pH value of HAaseD were 40°C and 7.0, respectively. In addition, HAaseD retained stability in an alkaline environment and displayed higher activity with appropriate concentrations of metal ions. Moreover, HAaseD was an endolytic hyaluronate lyase that could degrade HA to produce unsaturated HA oligosaccharides. Together, our findings indicate that HAaseD from Bacillus sp. CQMU-D is a new hyaluronate lyase and with excellent potential for application in industrial production.

Hyaluronic Acid Oligosaccharide Derivatives Alleviate Lipopolysaccharide-Induced Inflammation in ATDC5 Cells by Multiple Mechanisms.

High molecular weight hyaluronic acids (HMW-HAs) have been used for the palliative treatment of osteoarthritis (OA) for decades, but the pharmacological activity of HA fragments has not been fully explored due to the limited availability of structurally defined HA fragments. In this study, we synthesized a series glycosides of oligosaccharides of HA (o-HAs), hereinafter collectively referred to as o-HA derivatives. Their effects on OA progression were examined in a chondrocyte inflammatory model established by the lipopolysaccharide (LPS)-challenged ATDC5 cells. Cell Counting Kit-8 (CCK-8) assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed that o-HA derivatives (≤100 μg/mL) exhibited no cytotoxicity and pro-inflammatory effects. We found that the o-HA and o-HA derivatives alleviated LPS-induced inflammation, apoptosis, autophagy and proliferation-inhibition of ATDC5 cells, similar to the activities of HMW-HAs. Moreover, Western blot analysis showed that different HA derivatives selectively reversed the effects of LPS on the expression of extracellular matrix (ECM)-related proteins (MMP13, COL2A1 and Aggrecan) in ATDC5 cells. Our study suggested that o-HA derivatives may alleviate LPS-induced chondrocyte injury by reducing the inflammatory response, maintaining cell proliferation, inhibiting apoptosis and autophagy, and decreasing ECM degradation, supporting a potential oligosaccharides-mediated therapy for OA.

In vitro inflammatory and immune response to uncrosslinked hyaluronic acid (HA) and HA fillers

IntroductionDelayed-onset nodules are a complication associated with soft tissue filler products, including hyaluronic acid (HA) fillers; however, the etiology of these events remains unclear. The role of uncrosslinked HA and crosslinked HA fillers in the response of immune cells (dendritic cells, B cells, and T cells) was evaluated with and without concurrent bacterial stimulation. MethodsUncrosslinked HA of varying molecular weights and crosslinked HA fillers were tested in a series of in vitro assays to evaluate 1) human monocyte activation; 2) activation, maturation, and migration of human monocyte-derived dendritic cells (MoDC); 3) T-cell activation and associated skin damage; and 4) T-cell–mediated B-cell response. For the latter assay, the HA test articles were also evaluated following stimulation by bacteria. ResultsWhen treated with lipopolysaccharide (LPS) as a positive control, inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-10, IL-12p70, and tumor necrosis factor [TNF]-α) released by MoDC were significantly upregulated. Treatment of the MoDC with HA (0.776, 150, and 485 kDa) did not result in stimulation of IL-1β, IL-6, IL-10, and IL-12p70, although increased TNF-α was observed with all HA test samples. The TNF-α increase was significantly lower for HA samples (1.5–2.64 pg/mL) compared with LPS (966.96–4834.18 pg/mL). Similarly, treatment of MoDC with HA samples did not result in expression of surface markers indicative of dendritic cell maturation. MoDC treated with 0.776 and 150 kDa HA, but not with 485 kDa HA, had significantly increased migration toward fetal bovine serum, but not C–C motif chemokine ligand 21 (CCL21). In the T-cell activation and skin damage assay, VYC-15L, degraded VYC-15L, HYC-24L+, and oligosacharride HA samples were negative in the T-cell proliferation and interferon-γ assays at all tested concentrations. The degraded HYC-24L + sample was borderline at the highest concentration and negative at the middle and low concentrations. Skin damage was determined to be negative for all HA samples at all concentrations. In the human monocyte activation assay, all tested uncrosslinked HA samples (5, 150, and 731 kDa) were determined to be non-sensitizing (i.e., did not activate monocyte cells). In the T-cell–mediated B-cell activation assay, treatment with HA oligosaccharides, uncrosslinked HA, and crosslinked HA fillers did not result in any consistent stimulation of any cytokines measured. A molecular weight–dependent decrease in the secreted immunoglobulin G (IgG) was observed, and this effect was more pronounced with the crosslinked HA fillers. Addition of heat-inactivated Cutibacterium acnes bacteria (HIB) to uncrosslinked HA and crosslinked HA fillers resulted in increased cytokine production similar to when HIB was tested alone. The decrease in secreted IgG observed for crosslinked HA filler samples without HIB was maintained with addition of HIB to VYC-20, VYC-25, SGD-30XP, and HYC-24+ samples. The reduction of secreted IgG observed for RSQ and VYC-15 alone was no longer apparent when HIB was added to these cultures (i.e., returned to control levels but were not upregulated). ConclusionThe results from this study suggest that uncrosslinked HA and HA fillers themselves do not stimulate an inflammatory/immune response, regardless of molecular weight or crosslinking. However, the immune response following HIB stimulation may be impacted by the presence of HA fillers, suggesting a potential initiating role of contaminating factors such as bacteria and a modulating role of HA.

Preparation of Hyaluronic Acid Oligosaccharides by Enzymatic Method and Its Transdermal Absorption Activity

Preparation of Hyaluronic Acid Oligosaccharides by Enzymatic Method and Its Transdermal Absorption Activity

The anti-inflammatory activity of specific-sized hyaluronic acid oligosaccharides

Severe acute inflammatory conditions may cause tissue damage, sepsis, and death. As a critical component of the extracellular matrix, hyaluronic acid (HA) has been reported to possess pro- and anti-inflammatory properties via Toll-like receptors (TLRs). In this study, we prepared different sizes and structures of HA oligosaccharides and derivatives and investigated the effects on inflammation in vitro and in vivo. Our results showed that HA tetra-saccharide was the minimum fragment to enhance inflammation, whereas HA disaccharide competitively blocked TLR4-dependent inflammation. The enzymatic HA disaccharide (ΔHA2) inhibited lipopolysaccharide (LPS)-induced inflammation. Based on structure-activity relationship analysis, we observed that anti-inflammatory activity depended on HAs polymerization degree, acetyl group, and configuration. In addition, we demonstrated that ΔHA2 reduced LPS-induced pro-inflammatory cytokines production in vivo. ΔHA2, a native metabolite of HA polysaccharides, may have a potential role against LPS-mediated inflammatory diseases.

Hyaluronic acid oligosaccharides modified mineralized collagen and chitosan with enhanced osteoinductive properties for bone tissue engineering

In this study, we prepared a biomimetic hyaluronic acid oligosaccharides (oHAs)-based composite scaffold to develop a bone tissue-engineered scaffold for stimulating osteogenesis and endothelialization. The functional oHAs products were firstly synthesized, namely collagen/hyaluronic acid oligosaccharides/hydroxyapatite (Col/oHAs/HAP), chitosan/hyaluronic acid oligosaccharides (CTS/oHAs), and then uniformly distributed in poly (lactic-co-glycolic acid) (PLGA) solution followed by freeze-drying to obtain three-dimensional interconnected scaffolds as temporary templates for bone regeneration. The morphology, physicochemical properties, compressive strength, and degradation behavior of the fabricated scaffolds, as well as in vitro cell responses seeded on these scaffolds and in vivo biocompatibility, were investigated to evaluate the potential for bone tissue engineering. The results indicated that the oHAs-based scaffolds can promote the attachment of endothelial cells, facilitate the osteogenic differentiation of MC3T3-E1 and BMSCs, and have ideal biocompatibility and tissue regenerative capacity, suggesting their potential to serve as alternative candidates for bone tissue engineering applications.

Anti-HA antibody does not detect hyaluronan.

It is generally known that hyaluronic acid (HA) is a biocompatible and biodegradable glycosaminoglycan distributed widely throughout epithelial, connective and neural tissues. HA is one of the chief components of the extracellular matrix. Lack of immunogenicity is one of the biggest advantages of the therapeutic use of HA, but it also prevents the production of specific anti-HA antibodies. Contrary to this, there are still several studies performing HA detection by immunohistochemical or immunohistofluorescent method using an anti-HA antibody. Therefore, this short study discusses whether the anti-HA antibody is specific for HA. To verify the specificity of the HA staining the hyaluronidase treatment of histological samples was performed and the ability of anti-HA antibody and biotinylated HA binding protein (bHABP)-based probe to bind to their targets was evaluated. Additionally, the competitive binding assay with short HA oligosaccharides and subsequent histological staining was performed. Both assays showed that the anti-HA antibody is not sufficiently specific for HA and that the bHABP probe is a reliable method for HA detection in histological samples. The conclusion made by previous investigators based on using HA antibodies should be reevaluated and future use of anti-HA antibody should be avoided.

Novel hyaluronic acid oligosaccharide-loaded and CD44v6-targeting oxaliplatin nanoparticles for the treatment of colorectal cancer

Oxaliplatin resistance is one of the main causes of failed colorectal cancer treatment, followed by recurrence and metastasis. In this study, we found that colorectal cancer cells secrete a high level of hyaluronic acid (HA), which interacts with its receptor CD44v6 to mediate colorectal cancer resistance to chemotherapy. HA oligosaccharide (oHA) is a degradation product of HA. We found that it competitively binds to CD44v6, reversing the HA–CD44v6-mediated effect of HA on oxaliplatin resistance. In addition, oHA showed no toxicity or immunogenicity but exhibited good biocompatibility and tumor-targeting capability. Therefore, we synthesized oHA-loaded oxaliplatin liposome nanoparticles (oHA-Lipid-Oxa) using a thin-film hydration method. The cytotoxicity of oHA-Lipid-Oxa was assessed in vitro using flow cytometry, which revealed greater lethality than oxaliplatin alone. Finally, we established a tumor-bearing nude mouse model and separately injected oHA-Lipid-Oxa, Lipid-Oxa, Oxa, oHA, and phosphate-buffered saline (PBS) into the tail vein to observe the antitumor effects of nanoparticles in vivo. The oHA-Lipid-Oxa group exhibited the highest tumor suppression rate, but the weight loss was not obvious. Hematoxylin and eosin staining showed greatest lymphocyte and macrophage infiltration in the oHA-Lipid-Oxa group. Moreover, oHA-Lipid-Oxa induced tumor cell apoptosis and necrosis most robustly compared with the other groups. We showed that oHA-Lipid-Oxa has excellent histocompatibility and CD44v6-targeting capabilities, thus greatly increasing the sensitivity to oxaliplatin and reducing adverse reactions. Accordingly, oHA-Lipid-Oxa has a broad potential for therapeutic application.

Biochemical characterization of a thermophilic hyaluronate lyase TcHly8C from Thermasporomyces composti DSM22891

Hyaluronic acid (HA) is an anionic linear polysaccharide abundantly distributed in the extracellular matrix of mammalian connective, growing, and tumor tissues. Hyaluronidase is used as an important drug diffusion promoter and a tool enzyme to produce HA oligosaccharides. However, there is no thermostable hyaluronidase suitable for application to date. In this study, a thermophilic hyaluronate lyase, TcHly8C, from Thermasporomyces composti DSM22891 was expressed in Escherichia coli. The recombinant TcHly8C was most active at 70 °C, and it retained about 30% of initial activity after incubation at 60 °C for 28 days. The half-lives of TcHly8C at 60 °C and 70 °C were 16.1 d and 2.3 h, respectively. The optimum pH of TcHly8C is 5.93, and it was stable at pH 6.15–10.90. The presence of Mg2+ could enhance its enzymatic activity significantly. Km, kcat, and kcat/Km of TcHly8C towards HA were 3.69 mg∙ml−1, 17.82 s−1, and 4.82 ml∙mg−1∙s−1, respectively. TcHly8C degraded HA in an exolytic mode, and the end product was unsaturated HA disaccharide (ΔUA-GlcNAc). Overall, our results show that TcHly8C is the first reported PL8 exo-type hyaluronate lyase with high thermostability, which provides a potential enzyme used in medicine and production of HA oligosaccharides.

Synthesis of defined oligohyaluronates-decorated liposomes and interaction with lung cancer cells

In this work hyaluronic acid (HA) oligosaccharides with degree of polymerization (DP) 4, 6 and 8, obtained by enzymatic depolymerization of HA, were conjugated to a PEG-phospholipid moiety. The products (HA-DP4, HA-DP6 and HA-DP8) were used to prepare decorated liposomes. The cellular uptake of HA-DP4, HA-DP6 and HA-DP8-decorated fluorescently labelled liposomes was significantly higher (12 to 14-fold) in lung cancer cell lines with high CD44 expression than in those with low CD44 expression, suggesting a receptor-mediated entry of HA-conjugated formulations. Competition assays showed that the uptake followed this rank order: HA-DP8>HA-DP6>HA-DP4 liposomes. Moreover, they are capable of a faster interaction with CD44, followed by phagocytosis, than HA liposomes obtained from HA of higher molecular weight (4800 and 14800 Da). HA-DP4, HA-DP6 and HA-DP8-liposomes did not show cytotoxicity or inflammatory effects. Overall, we propose our new HA-DP oligosaccharides as biocompatible and effective tools for a potential drug delivery to CD44-positive cells.

Drug delivery system of dual-responsive PF127 hydrogel with polysaccharide-based nano-conjugate for textile-based transdermal therapy

Pluronic F-127 based dual-responsive (pH/temperature) hydrogel drug delivery system was developed involving polysaccharide-based nano-conjugate of hyaluronic acid and chitosan oligosaccharide lactate and applied for loading of gallic acid which is the principal component of traditional Chinese medicine Cortex Moutan recommended in the treatment of atopic dermatitis. The polysaccharide-based nano-conjugate was used as pH-responsive compound in the formulation and its amphiphilic character was determined colorimetrically. Microstructure analysis by SEM and TEM indicated highly porous hydrogel network and well-dispersed micellar structures, respectively, after modification with the nano-conjugate, and so, release property of the hydrogel for drug was significantly improved. Different pH-conditions were applied here to see pH-responsiveness of the formulation and increase in acidity of external environment gradually diminished mechanical stability of the hydrogel and that was reflected on the drug release property. Rheology was performed to observe sol-gel transition of the formulation and showed better rheological properties after modification with nano-conjugate. In this study, the cytotoxicity results of PF127 based formulations loaded with/without gallic acid showed cell viability of > 80.0 % for human HaCaT keratinocytes in the concentration range of 0.0–20.0 μg/ml.

Synthesis of hyaluronic acid oligosaccharides with a GlcNAc-GlcA repeating pattern and their binding affinity with CD44.

Hyaluronic acid (HA) is a ubiquitous glycosaminoglycan in the extracellular matrix and a ligand of CD44, a transmembrane glycoprotein that is important in cell migration. Crystal and NMR studies found a hexasaccharide of the pattern (GlcA-GlcNAc)3 as the shortest HA that could bind to CD44, but molecular dynamics simulations indicated that a tetrasaccharide of the pattern (GlcNAc-GlcA)2 is the key structure interacting with CD44. Access to oligomers with such a repeat pattern is crucial in binding studies with CD44. Here we developed a synthetic procedure to afford the HA oligosaccharides with the GlcNAc-GlcA repeating unit and measured the binding interaction between these sugars and human CD44 by isothermal titration calorimetry (ITC). During the chemical synthesis, we successfully generated the β-glycosidic bond in the absence of neighbouring group participation and overcome the issues in the oxidation step. In addition, ammonia-free dissolving metal reduction for debenzylation and azido reduction has been applied in carbohydrate synthesis for the first time. ITC analysis revealed that the HA tetrasaccharide (GlcNAc-GlcA)2 could indeed interact and bind to the human CD44.

Preparation, Characterization, and Inhibition of Hyaluronic Acid Oligosaccharides in Triple-Negative Breast Cancer.

Hyaluronic acid (hyaluronan, HA) is a critical component of the extracellular matrix and plays an important biological function of interacting with different molecules and receptors. In this study, both odd- and even-numbered HA oligosaccharides (HAOs) with specific degrees of polymerization (DP) were prepared by different hydrochloric acid hydrolyses, and their structures were characterized by means of HPLC, ESI-MS, and NMR. The data show that the odd-numbered HAOs (DP3-11) have a glucuronic acid reducing end, while the even-numbered HAOs (DP2-10) have an N-acetylglucosamine reducing end. Biological evaluations indicated that all HAOs significantly inhibited the growth and migration of triple-negative breast cancer (TNBC) MDA-MB-231 cells. Among these oligosaccharides, the HA tetrasaccharide (DP4) was confirmed to be the minimum fragment necessary to inhibit MDA-MB-231 cells. Our data suggest that HAOs have potential value in the treatment of TNBC.

Hyaluronic acid oligosaccharide-modified collagen nanofibers as vascular tissue-engineered scaffold for promoting endothelial cell proliferation

Hyaluronic acid oligosaccharides (oHAs) have shown promising results in promoting vascular endothelial cell (EC) proliferation and endothelialization. To engineered develop tissue scaffold for promoting EC proliferation and vessel endothelialization, different sizes of oHAs were prepared and grafted onto collagen to improve the biological properties of the synthesized materials, especially in angiogenesis. Firstly, oHAs were successfully prepared and conjugated with collagen to construct glycosylated collagens by reductive amination. The glycosylated collagens were then electrospun into various nanofibrous structures and their morphology and hemocompatibility, as well as EC responses on these nanofibrous scaffolds, were studied to evaluate the potential for vascular tissue engineering. The results showed that the nanofibrous scaffolds grafted by oHAs promoted EC proliferation whereas high molecular weight HA inhibited proliferation. The scaffolds had no detectable degree of hemolysis and coagulation, suggesting their promise as engineered vascular tissue scaffolds.

Improving in vitro biocompatibility on biomimetic mineralized collagen bone materials modified with hyaluronic acid oligosaccharide.

Hyaluronic acid (HA) has great potential in bone tissue engineering due to its favorable bioactivity and biocompatibility, especially hyaluronic acid oligosaccharides (oHAs) shows a promising result in endothelialization of blood vessel. To improve endothelialized effect and osteogenic performance of bone scaffold, we have created a biomimetic nanofiber network based on collagen modified with hyaluronic acid oligosaccharides (Col/oHAs) and its mineralized product. Biomimetically mineralized Col/oHAs based composite (Col/oHAs/HAP) was prepared via self-assembly at room temperature. The resultant composites were characterized by fourier transform infrared spectroscopy (FT-IR), X-Ray diffractometry (XRD), thermogravimetric analysis (TGA), scanning electron microscope (SEM) and transmission electron microscopy (TEM). They show some characteristics of natural bone both in composition and microstructure. The nanofiber was fabricated as a hybrid network which bionics extracellular matrix (ECM) and was prepared to culture artery endothelial cell (PIEC) and the mouse parietal bone cell (MC3T3-E1). Cells attached tightly to the nanofibers and infiltrated into the materials, forming an interconnected cell community. Moreover, the as-prepared nanofiber was found to noticeably enhance cells adhesion and proliferation and upregulate alkaline phosphatase activity (ALP) and osteocalcin (OCN) expression suggesting positive cellular responses. These results indicated that the Col/oHAs/HAP composite has a promising capacity to direct the osteogenic differentiation by providing an adaptable environment and can be expected as an excellent candidate for bone tissue engineering approaches with improved performance of promoting PIEC proliferation.

Construction of engineered Streptococcus zooepidemicus for the production of hyaluronic acid ligosaccharide

Hyaluronic acid (HA) is widely used in many fields, such as medicine, cosmetics and food. The bioactivity of HA depends on its molecular weight (Mw). Owing to the important physiological activities and special physiological functions, HA oligosaccharides have important application prospects in medicine fields. Streptococcus zooepidemicus has wide applications in commercial production of HA, due to its short fermentation cycle and strong production intensity. In order to efficiently synthesize HA oligosaccharides and solve the dissolved oxygen in the fermentation process, in this study, we overexpressed HA synthase (HasA) and introduced and optimized the leech hyaluronidase LHAase in Streptococcus zooepidemicus WSH-24. As a result, HA oligosaccharides were efficiently produced with improved dissolved oxygen. After 24 h, HA oligosaccharides production intensity reached to 294.2 mg/(L·h), and the concentration accumulated to 0.97 g/L in flask cultures, which was 1.82 times of the wild strain. Impressively, HA oligosaccharides were increased to 7.06 g/L in 3 L fermentor. The constructed Streptococcus zooepidemicus strain for producing HA oligosaccharides would have broad application prospects.