A Novel Porous Butyryl Chitin-Animal Derived Hydroxyapatite Composite Scaffold for Cranial Bone Defect Repair.

Bone defects, a common orthopedic problem in clinical practice, are a serious threat to human health. As alternative materials to autologous bone grafts, synthetic cell-free functionalized scaffolds have been the focus of recent research in designing scaffolds for bone tissue engineering. Butyryl chitin (BC) is a derivative of chitin (CT) with improved solubility. It has good biocompatibility, but few studies have investigated its use in bone repair. In this study, BC was successfully synthesized with a degree of substitution of 2.1. BC films were prepared using the cast film method and showed strong tensile strength (47.8 ± 4.54 N) and hydrophobicity (86.4 ± 2.46°), which was favorable for mineral deposition. An in vitro cytological assay confirmed the excellent cell attachment and cytocompatibility of the BC film; meanwhile, in vivo degradation indicated the good biocompatibility of BC. Hydroxyapatite (HA), extracted from bovine cancellous bone, had good cytocompatibility and osteogenic induction activity for the mouse osteoblast cell line MC3T3-E1. With the aim of combining the advantages of BC and HA, a BC-HA composite scaffold, with a good pore structure and mechanical strength, was prepared by physical mixing. Administered into skull defects of rats, the scaffolds showed perfect bone-binding performance and effective structural support, and significantly promoted the regeneration of new bone. These results prove that the BC-HA porous scaffold is a successful bone tissue engineering scaffold and has strong potential to be further developed as a substitute for bone transplantation.

Nerve Regeneration Effect of a Composite Bioactive Carboxymethyl Chitosan-Based Nerve Conduit with a Radial Texture.

Chitosan (CTS) has been used as a nerve guidance conduit (NGC) material for bridging peripheral nerve defects due to its biocompatible, biodegradable, and non-toxic properties. However, the nerve regeneration effect of chitosan alone is restricted due to its inadequate biological activity. Herein, a composite, bioactive chitosan based nerve conduit, consisting of outer warp-knitted tube scaffold made from medical-grade chitosan fiber, and inner porous cross linked carboxymethyl chitosan (C-CM-CTS) sponge with radial texture was developed. The inner wall of the scaffold was coated with C-CM-CTS solution. CM-CTS provided favorable bioactivities in the composite chitosan-based nerve conduit. An in vitro study of CM-CTS revealed its satisfying biocompatibility with fibroblast and its inhibition of oxidative damage to Schwann cells. As the internal filler of the NGC, the lyophilized sponge of C-CM-CTS showed a longitudinal guidance effect for nerve reconstruction. After 10 mm defect in rat sciatic nerve was bridged with the composite bioactive chitosan-based nerve conduit, the nerve conduit was able to effectively promote axonal regeneration and played a positive role in inducing nerve regeneration and functional recovery. In addition to the functional advantages, which are equal to those of an autograft; the technology for the preparation of this conduit can be put into mass production.

Exosomal transfer of miR-769-5p promotes osteosarcoma proliferation and metastasis by targeting DUSP16.

BACKGROUND: Osteosarcoma (OS) is a malignant tumor originating from mesenchymal stem cells, and has an extremely high fatality rate and ability to metastasize. Although mounting evidence suggests that miR-769-5p is strongly associated with the malignant progression and poor prognosis of various tumors, the exact role of miR-769-5p in OS is still unclear. Therefore, this study aimed to explore the relationship between miR-769-5p and the malignant progression of OS, and its underlying mechanism of action. METHODS: miR-769-5p expression was analyzed in GSE28423 from the GEO database and measured in OS clinical specimens and cell lines. The effects of miR-769-5p on OS proliferation, migration and invasion were measured both in vivo and in vitro. In addition, bioinformatics analyses and luciferase reporter assays were used to explore the target genes of miR-769-5p. Rescue experiments were also conducted. Moreover, a co-culture model was used to test the cell interaction between bone mesenchymal stem cells (BMSC) and OS cells. RESULTS: We found that miR-769-5p is highly expressed in OS clinical specimens and cell lines. In vivo and in vitro experiments also showed that miR-769-5p significantly promoted the proliferation, migration and invasion of OS cells. Dual-specific phosphatase 16 (DUSP16) was negatively associated with miR-769-5p expression in OS cells and tissue samples and was validated as the downstream target by luciferase reporter assay and western blotting. Rescue experiments showed that DUSP16 reverses the effect of miR-769-5p on OS cells by negatively regulating the JNK/p38 MAPK signaling pathway. Additionally, the results of the co-culture of BMSCs and OS cells confirmed that miR-769-5p was transferred from BMSCs to OS cells through exosomes. CONCLUSIONS: In summary, this study demonstrates for the first time that BMSC-derived exosomal miR-769-5p promotes OS proliferation and metastasis by targeting DUSP16 and activating the JNK/p38 MAPK signaling pathway, which could provide rationale for a new therapeutic strategy for OS.

Potential Analysis and Preparation of Chitosan Oligosaccharides as Oral Nutritional Supplements of Cancer Adjuvant Therapy.

Cancer is considered to have an adverse influence on health around the world. Chitosan, a linear polysaccharide that contains copolymers of ß -1-4 linked d-glucosamine and N-acetyl-d-glucosamine units, has been widely used in the field of biomedicine, owing to its nontoxicity, biocompatibility, biodegradability, and hemocompatibility. This study was aimed at preparing the chitosan oligosaccharides (COS) and examining its ability on suppressing lung cancer in vitro and in vivo. Human non-small-cell lung cancer A549 cells model and C57BL/6 mice bearing lung cancer model were adopted. COS showed inhibition on the viability and proliferation of lung carcinoma cells (A549) in time-dependent manners, but no cytotoxicity to human liver cell (HL-7702). Moreover, COS could significantly increase Bax expression of A549 cells while decreasing Bcl-2 expression. COS supplementation significantly inhibited the growth of Lewis tissues and promoted necrosis of tumor cells in vivo. After treatment with COS, significantly elevated concentrations of Bax and reduced expression of Bcl-2 in tumor tissues, as well as elevated levels of TNF- α , IL-2, Fas and Fas-L in mice serum were observed (p < 0.05). In conclusion, COS had certain anti-tumor effects and potential application as a synergic functional food ingredient to prevent cancer.

Dietary Natural N-Acetyl-d-Glucosamine Prevents Bone Loss in Ovariectomized Rat Model of Postmenopausal Osteoporosis.

Postmenopausal osteoporosis has seriously affected the life quality of elderly women. A natural polymer, chitin, obtained from shells of crab and shrimp, has been widely used in the biomedical field owing to its nontoxicity, biocompatibility, and biodegradability. In this study, natural N-acetyl-d-glucosamine (NAG) was prepared from liquefied chitin. The protective activities of NAG in postmenopausal osteoporosis were evaluated on Sprague Dawley rats and osteoblast-based models. Results showed that oral administration of NAG boosted trabecular bone volume and trabecular numbers. Additionally, the calcium content in the femur and tibia increased, and femoral biomechanical properties improved. Furthermore, NAG supplementation significantly lowered alkaline phosphatase levels and increased calcium content in the serum of ovariectomized rats. In vitro studies showed that NAG markedly promoted cell proliferation and stimulated osteoblast differentiation of mouse calvaria origin MC3T3-E1 cells with increased alkaline phosphatase activity in a concentration-dependent manner. Moreover, NAG effectively protected osteoblasts from oxidative damage induced by hydrogen peroxide. In conclusion, our data provide an additional foundation for dietary supplementation of NAG, which could protect and reverse osteopenia in postmenopausal women.

Evaluation of a photocrosslinkable hydroxyethyl chitosan hydrogel as a potential drug release system for glaucoma surgery.

Hydroxyethyl chitosan (HECTS) is a critical derivative of chitosan that has been widely used as biomedical materials due to great water-solubility and excellent biocompatibility. Here, photosensitive hydroxyethyl chitosan was synthesized by introducing azide group on NH2 of HECTS (HECTS-AZ), afterwards FTIR and 1H NMR spectra were detected to confirm the formation of HECTS-AZ. The solution of HECTS-AZ can achieve a sol-gel transition through UV irradiation for 30 s. The evaluation of biocompability and biodegradability in vivo was conducted in rats, visual and pathological examinations exhibited the HECTS-AZ has excellent biocompability and degradation time of the hydrogel is more than 14 weeks. Furthermore, HECTS-AZ hydrogel as an ocular drug delivery system loading heparin was prepared to implant under sclera of rabbit after glaucoma filtration surgery (GFS). The experimental results demonstrated the heparin loaded hydrogel can effectively maintain filtration bleb and lowing intraocular pressure (IOP) after GFS for prolonged time. Besides, obvious inflammatory reactions and side effects have not been observed in ocular during the experimental period. In conclusion, the HECTS-AZ hydrogel is a potential drug delivery device for the treatment of glaucoma and other ocular diseases.

Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01.

BACKGROUND: A detailed knowledge about the degradation mechanism of chitosanase hydrolysis is critical for the design of novel enzymes to produce well-defined chito-oligosaccharide products. METHODS: Through the combination of structural and biochemical analysis, we present new findings that provide novel insights into the degradation mechanism of chitosanase OU01. RESULTS: We have determined the crystal structure of Asp(43)/Ala mutant of OU01, and have trapped the hydrolyzed product of the reaction. This structure reveals the role of the general acid (Glu(25)) in catalysis. Two structural features about the mechanisms of the non-processive chitosanases are described for the first time. 1). Structural comparison reveals that the enzyme goes through an open-closed-open conformational transition upon substrate binding and product release; 2). polar residues constitute the substrate binding cleft. Additional site important for polymeric substrate recognition is identified and a three-step polymeric substrate recognition mechanism is proposed. CONCLUSIONS: Detailed substrate recognition mechanism is described for non-processive chitosanase for the first time. GENERAL SIGNIFICANCE: These findings provide new structural insights into the understanding of overall hydrolysis mechanism for non-processive chitosanase, and also will facilitate the design of new enzymes used for industrial purpose.

Structural insights into the substrate-binding mechanism for a novel chitosanase.

Chitosanase is able to specifically cleave ß-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase-substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion.

Tissue-engineered membrane based on chitosan for repair of mechanically damaged corneal epithelium.

In this study, hydroxyethyl chitosan (HECTS), a water-soluble derivative of chitosan, was used to create a blend membrane and its function and application as a scaffold in repair of mechanically damaged corneal epethelia were examined. The results showed that HECTS significantly promoted growth of corneal epithelial cells (CEpCs) in vitro and that CEpCs grew well on the HECTS-based blend membrane. Fluorescent imaging showed that CEpCs were interconnected and formed layers on the membrane. After transplanting the CEpCs-seeded membranes onto the damaged corneal epithelium, we found that the epithelium was repaired faster compared with control. The repaired corneal epithelium with the membrane had a more compact structure and a smoother surface than control when observed by histology and scanning electron microscope. These results demonstrate an ability of the tissue-engineered scaffold to speed up the repair of mechanically damaged corneal epithelium.

PeposX-Exhaust: A lightweight and efficient tool for identification of short peptides.

Short peptides have become the focus of recent research due to their variable bioactivities, good digestibility and wide existences in food-derived protein hydrolysates. However, due to the high complexity of the samples, identifying short peptides still remains a challenge. In this work, a tool, named PeposX-Exhaust, was developed for short peptide identification. Through validation with known peptides, PeposX-Exhaust identified all the submitted spectra and the accuracy rate reached 75.36%, and the adjusted accuracy rate further reached 98.55% when with top 5 candidates considered. Compared with other tools, the accuracy rate by PeposX-Exhaust was at least 70% higher than two database-search tools and 15% higher than the other two de novo-sequencing tools, respectively. For further application, the numbers of short peptides identified from soybean, walnut, collagen and bonito protein hydrolysates reached 1145, 628, 746 and 681, respectively. This fully demonstrated the superiority of the tool in short peptide identification.

Investigation of the skin repair and healing mechanism of N-carboxymethyl chitosan in second-degree burn wounds.

N-carboxymethyl chitosan (NCMC) was synthesized with the modification of chitosan; the substitution degree was measured by titration. The biocompatibility and degradability of the NCMC were studied in vivo and the results showed that the NCMC was nontoxic and biocompatible. The in vivo degradation rate of NCMC in musculature was faster than that in subcutaneous tissue due to the relatively high lysozyme concentration. The NCMC was used as biomaterial to heal deep second-degree burn wounds. The wound size reduction, histological examination, and the quantification of transforming growth factor-ß(1) , tumor necrosis factor-α and interleukin-8 protein levels, and Smad3 gene expression were measured to evaluate the healing effects. The results demonstrated that the NCMC was efficient in accelerating wound healing via activating transforming growth factor-ß(1) /Smad3 signaling pathway.

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus.

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6-10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10(3 )U (urokinase units) mg(-1) total fibrinolytic activity, which consisted of 692 U mg(-1) direct fibrinolytic activity and 769 U mg(-1) plasminogen-activator activity. Km and Vmax values for azocasein were 1 mg ml(-1) and 43 µg min(-1) ml(-1), respectively.

Multichannel nerve conduit based on chitosan derivates for peripheral nerve regeneration and Schwann cell survival.

In this study, a chitosan-based composite multichannel nerve conduit consisting of a warp-knitted chitosan scaffold and internally oriented N-succinyl-chitosan (NS-chitosan) fibers was applied to bridge a 10-mm nerve defect in rats. This study confirmed that an external pipeline with appropriate mechanical support was obtained by warp knitting techniques and that NS-chitosan fibers were not toxic to L-929 and PC-12 cells. These fibers degraded slowly for over 90 days and exerted sustained neuroprotective effects on peripheral nerves through their ability to drive cellular migration, promote survival, and block apoptosis of damaged Schwann cells through the Bcl-2/Bax/caspase-3 pathway. The multichannel chitosan/NS-chitosan conduit represented a histologically and functionally successful nerve reconstruction across a damaged 10-mm peripheral nerve model, showing regenerative efficacy equal to that of an autograft. The results demonstrated that the chitosan/NS-chitosan conduit with a warp-knitted tube construct and aligned inner fiber had good mechanical and bioactive properties for nerve repair.

A fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry and its application in food-derived protein hydrolysates.

Food-derived bioactive peptides have many outstanding features like high safety, easy absorption, etc. However, explorations of the peptides are suffering from the limited knowledge of sample composition and low efficiency of separation techniques. In this work, a fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS) was designed and constructed in-house. For chromatographic system optimization, the effects of column pairs and fraction transfer volumes on separation performance were studied. The pair of Protein BEH SEC and HSS T3 columns was found of high orthogonality. The peak capacity detected by the optimized 2DLC reached 1165 (for corn protein hydrolysates), indicating high resolving power. Moreover, the number of peptides identified from corn, soybean and casein protein hydrolysates reached as high as 8330, 8925 and 7215, respectively, demonstrating the high potential of the system. This would help reveal the peptide composition and facilitate the research on exploring bioactive peptides from food-derived protein hydrolysates.

Corrigendum: miR-766-3p targeting BCL9L suppressed tumorigenesis, epithelial-mesenchymal transition, and metastasis through the ß-catenin signaling pathway in osteosarcoma cells.

[This corrects the article DOI: 10.3389/fcell.2020.594135.].

Acute toxicity of high dosage carboxymethyl chitosan and its effect on the blood parameters in rats.

This experiment was aimed to study whether Carboxymethyl chitosan has acute toxicity and effects on the blood parameters of rats, which were treated with high dosage carboxymethyl chitosan (1350 mg/kg) through a laparotomy. Acute toxicity was first studied and then kinds of blood parameters were detected at different time points after the laparotomy, which contain coagulant parameters (thrombin time, prothrombin time, activated partial thromboplatin time and fibrinogen), anticoagulant parameter (antithrombin III), fibrinolytic parameters (plasminogen and fibrin degradation product) and hemorheology parameters (blood viscosity). Results showed that no acute toxicity was detected and no significant effects were found on the parameters of coagulation, anticoagulation, fibrinolysis or hemorheology of rats after the laparotomy, which indicated that carboxymethyl chitosan has no significant toxicity on the blood system of rats after being absorbed in the abdominal cavity and degraded gradually in the blood. And this study has provided experimental basis for carboxymethyl chitosan to be applied in the field of biomedical materials.

Effects of molecular weights on the absorption, distribution and urinary excretion of intraperitoneally administrated carboxymethyl chitosan in rats.

Carboxymethyl chitosan (CM-chitosan) is one of water-soluble derivatives of chitosan. Numerous studies have been focused on its applications as pharmaceutical excipient, bioactive reagent and nontoxic drug carrier. Like other polysaccharides, CM-chitosan is inhomogenous in molecular weight. Originations and preparation procedures considerably influence its molecular weight and molecular weight distributions. Understanding the molecular weight related biological behaviour of this inhomogenous glycopolymer in vivo were crucial for the quality control and clinical applications of chitosan and chitosan based medical devices. In this study, we investigated the effects of molecular weights on the absorption, distribution, degradation and urinary excretion of the fluorescein isothiocyanate-labeled CM-chitosan in rats. The results indicated that molecular weight significantly influenced the uptake of CM-chitosan from the lumen of abdomen and blood vessels to peripheral tissues, the distribution of this chemical and urinary excretion after intraperitoneal administration. These findings provided an important reference for the clinical applications of this versatile derivative of chitosan as postsurgical and other biomedical materials and important clues for the exploitation of CM-chitosan based drug targeting and delivery systems.

Preparation, Characterization, and Biological Evaluation of Transparent Thin Carboxymethyl-Chitosan/Oxidized Carboxymethyl Cellulose Films as New Wound Dressings.

Full thickness burns in which the damage penetrates deep into the skin layers and reaches underneath the muscle, compel the need for more effective cure. Herein, cross-linked carboxymethyl-chitosan (CM-chitosan) films, prepared by Schiff base association with oxidized carboxymethyl cellulose (OCMC), are investigated regarding the wound healing capacity on full thickness burn injuries in vivo. Transparent thin CM-chitosan/OCMC films are obtained with tensile strength reaching 6.11 MPa, elongation at break above 27%, and water absorption more than 800%, which operates in favor of absorbing excess exudate and monitoring the wound status. Furthermore, the nonadherent CM-chitosan/OCMC films, with satisfactory biodegradability, cell, and tissue compatibility, are readily used to the wound sites and easily removed following therapy on scalded tissue so as to alleviate the suffering from burn. The films efficiently promote epithelial and dermal regeneration compared to the control, achieving 75.9% and 94.4% wound closure, respectively, after 14 and 27 days. More importantly, CM-chitosan/OCMC films accelerate wound healing with natural mechanisms which include controlling inflammatory response, reducing apoptosis, promoting fibroblast cell proliferation, and collagen formation. In conclusion, the CM-chitosan/OCMC films elevate the repair ratio of burn injuries and have great potential for facilitating the healing process on full-thickness exuding wounds.

Multifunctional effects of wound dressing based on chitosan-coordinated argentum with resistant bacterial penetration.

Third-degree scald, causing serious tissue destruction with continuous pain, easily leads to microbial infections and delayed wound healing. Therefore, a multifunctional treatment is attractive for seriously damaged tissue. Herein, carboxymethyl chitosan-coordinated argentum (Ag-CMC) was synthesized via a complexation method, and then the Ag+ release, antibacterial activity, biocompatibility, pain relief and wound healing properties of Ag-CMC were investigated in vitro and in vivo. The results revealed that Ag+ had interacted with carboxymethyl chitosan, containing approximately 1.2% of silver. The Ag-CMC (50-200 µg/mL) with Ag+ sustained release exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, drug-resistant E. coli, PA, MRSA and good biocompatibility with L929 cells. Furthermore, antibacterial and wound healing experiments demonstrated that Ag-CMC achieved an effective contraction rate of 90% after 28 days by accelerating re-epithelialization, regulating inflammation response, relieving pain and infections. Therefore, Ag-CMC is a safe multifunctional treatment for wound healing and infections.

Study on repair of abdominal wall defect rats with hernia mesh coated with chitosan-based photosensitive hydrogel.

Herein, hydroxypropyl chitosan azide (AZ-HPCTS) was synthesized and prepared as a hydrogel coating applied to a polypropylene mesh (PPM) through UV irradiation. This study confirmed the hypothesis that hydrogels with porous three-dimensional network structures exhibited excellent biocompatibility and biodegradability and adhered well to PPM. During the 180-day follow-up period, the AZ-HPCTS-coated PPM (AH-PPM) promoted wound healing by promoting the secretion of transforming growth factor-beta1 (TGF-beta1) in the acute reaction stage, which was reduced to a lower level at 30 d. The PPM exhibited a lower fibrin lysozyme activity based on the expression of tissue plasminogen activator (tPA) compared with that of AH-PPM (P < 0.05). The intraperitoneal adhesion score of AH-PPM decreased to 2.4 at 180 d in contrast with PPM (P < 0.01), which remained at a high level throughout the study. In conclusion, the AZ-HPCTS hydrogel is a potential coating for hernia patches that deserves further study in the biomaterial field.