Chitosan-collagen biopolymer biofilm derived from cephalopod gladius; Evaluation of osteogenesis, angiogenesis and wound healing for tissue engineering application.

Chitosan (Ch) and acid-soluble collagen (ASC) from Doryteuthis singhalensis gladius were isolated to test their osteogenic, angiogenic, and wound healing capabilities in male Wistar rats. The results of the study showed that the ASC yield was 18.58 %, the total protein content was 86.43 % ± 0.18 %, and the amino acid composition was as follows: glycine, 15.68 %; proline, 13.84 %. A, B, I, II, and III show FT-IR amide regional bands at 3392, 2959, 1652, 1471, and 1237 cm-1 respectively. The electrophoretic pattern of ASC validated its molecular weights of 105 kDa and 96 kDa. The 1H NMR spectra showed pure singles at 1.99 ppm, and the UV-Vis spectrum showed a particular absorbance between 238 and 220 nm. The DSC showed two endothermic peaks: one with an To value of 119.72 °C and TP of 126.28 °C, and the other with 147.42 °C and 148.47 °C. Initially, we fabricated Ch and ASC biofilms at an 8.5:1.5 ratio for tissue engineering applications. A cellular-level study demonstrated good biocompatibility and enhanced osteoblastic differentiation of collagen chitosan films (CChF). Additionally, the biofilm exhibited increased angiogenic potency, as observed in the chick embryo chorioallantoic membrane (CAM) assay. The experimental animal model demonstrated that in wound healing, the CChF treated rats (95.75 ± 2.28 %) had a greater decrease in the diameter of the wound than the control rats (22.25 ± 2.45 %), followed by the CF (collagen film) treated rats (63.25 ± 2.08 %) and ChF (chitosan film) (52.67 ± 1.58 %). Rats treated with CChF had 48.82 ± 1.25 mg/g of hydroxyproline in NFGT and 75.25 ± 1.56 mg/g of overall protein. The higher hydroxyproline levels in the CChF-treated groups corroborated these histopathological findings. These results imply that by promoting the development of scars, inflammation, and proliferation, CChF accelerates the healing process.

Comparison of Physicochemical Characteristics and Fibril Formation Ability of Collagens Extracted from the Skin of Farmed River Puffer (Takifugu obscurus) and Tiger Puffer (Takifugu rubripes).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5-29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.

Physicochemical and Antioxidant Properties of Acid- and Pepsin-Soluble Collagens from the Scales of Miiuy Croaker (Miichthys Miiuy).

In this report, acid-soluble collagen (ASC-MC) and pepsin-soluble collagen (PSC-MC) were extracted from the scales of miiuy croaker (Miichthys miiuy) with yields of 0.64 ± 0.07% and 3.87 ± 0.15% of dry weight basis, respectively. ASC-MC and PSC-MC had glycine as the major amino acid with the contents of 341.8 ± 4.2 and 344.5 ± 3.2 residues/1000 residues, respectively. ASC-MC and PSC-MC had lower denaturation temperatures (32.2 °C and 29.0 °C for ASC-MC and PSC-MC, respectively) compared to mammalian collagen due to their low imino acid content (197.6 and 195.2 residues/1000 residues for ASC-MC and PSC-MC, respectively). ASC-MC and PSC-MC were mainly composed of type I collagen on the literatures and results of amino acid composition, SDS-PAGE pattern, ultraviolet (UV) and Fourier-transform infrared spectroscopy (FTIR) spectra. The maximum solubility of ASC-MC and PSC-MC was appeared at pH 1⁻3 and a sharp decrease in solubility was observed when the NaCl concentration was above 2%. Zeta potential studies indicated that ASC-MC and PSC-MC exhibited a net zero charge at pH 6.66 and 6.81, respectively. Furthermore, the scavenging capabilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, superoxide anion radical and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical of ASC-MC and PSC-MC were positively correlated with their tested concentration ranged from 0 to 5 mg/mL and PSC-MC showed significantly higher activity than that of ASC-MC at most tested concentrations (p < 0.05). In addition, the scavenging capability of PSC-MC on hydroxyl radical and superoxide anion radical was higher than those of DPPH radical and ABTS radical, which suggested that ASC-SC and PSC-SC might be served as hydroxyl radical and superoxide anion radical scavenger in cosmeceutical products for protecting skins from photoaging and ultraviolet damage.

Physicochemical properties of acid- and pepsin-soluble collagens from the cartilage of Siberian sturgeon.

To look for the collagen alternatives of mammalian cartilages from aquatics and their by-products, acid-soluble collagen (ASC-SC) and pepsin-soluble collagen (PSC-SC) were extracted from cartilages of Siberian sturgeon (Acipenser baerii) with yields of 27.13 ± 1.15 and 14.69 ± 0.85% on dry weight basis. ASC-SC and PSC-SC had glycine as the major amino acid with the contents of 326.8 and 327.5 residues 1000 residues-1, and their contents of proline and hydroxyproline were 205.9 and 208.0 residues 1000 residues-1. ASC-SC and PSC-SC comprised type I collagen ([α1(I)]2α2(I)) and type II collagen ([α1(II)]3) on the literatures and results of amino acid composition, SDS-PAGE pattern, UV, and FTIR spectra. Meanwhile, FTIR spectra data indicated that there were more hydrogen bonds in ASC-SC and more intermolecular crosslinks in PSC-SC. The maximum transition temperature (Tmax) of the ASC (28.3 °C) and PSC (30.5 °C) was lower than those of collagens from mammalian cartilages (> 37 °C). ASC-SC and PSC-SC showed high solubility in the acidic pH ranges and the solubility decreased in the presence of NaCl at concentrations above 3%. Zeta potential studies indicated that both ASC-SC and PSC-SC exhibited a net zero charge at pH 6.30 and 6.32. SEM results indicated that ASC-SC and PSC-SC presented irregular dense sheet-like film linked by random-coiled filaments. Therefore, collagens from Siberian sturgeon cartilages might be the suitable alternatives of the collagens of mammal cartilages as functional ingredient to treat some diseases.

Preparation, Physicochemical and Antioxidant Properties of Acid- and Pepsin-Soluble Collagens from the Swim Bladders of Miiuy Croaker (Miichthys miiuy).

Collagen is one of the most useful biomaterials and widely applied in functional food and cosmetics. However, some consumers have paid close attention to the safety of mammalian collagens because of the outbreaks of bovine spongiform encephalopathy (BSE), foot-and-mouth disease (FMD), and other prion diseases. Therefore, there is a strong demand for developing alternative sources of collagen, with one promising source being from the process by-products of commercial fisheries. In this report, acid-soluble collagen (ASC-SB) and pepsin-soluble collagen (PSC-SB) from swim bladders of miiuy croaker (Miichthys miiuy) were isolated with yields of 1.33 ± 0.11% and 8.37 ± 0.24% of dry swim bladder weight. Glycine was the major amino acid present, with a content of 320.5 (ASC-SB) and 333.6 residues/1000 residues (PSC-SB). ASC-SB and PSC-SB had much lower denaturation temperatures compared to mammalian collagen, a consequence of low imino acid contents (196.7 and 199.5 residues/1000 residues for ASC-SB and PSC-SB, respectively). The data of amino acid composition, SDS-PAGE pattern, UV and FTIR spectra confirmed that ASC-SB and PSC-SB were mainly composed of type I collagen. FTIR spectra data indicated there were more hydrogen bonding and intermolecular crosslinks in ASC-SB. These collagens showed high solubility in the acidic pH ranges and low NaCl concentrations (less than 2%). The Zeta potential values of ASC-SB and PSC-SB were 6.74 and 6.85, respectively. ASC-SB and PSC-SB presented irregular, dense, sheet-like films linked by random-coiled filaments under scanning electron microscopy. In addition, ASC-SB and PSC-SB could scavenge DPPH radical, hydroxyl radical, superoxide anion radical, and ABTS radical in a dose-dependent manner. Overall, the results indicate that collagens from the swim bladders of miiuy croaker are a viable substitute for mammalian collagen, with potential functional food and cosmeceutical applications.

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna (Katsuwonus pelamis).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the spine (ASC-SP and PSC-SP) and skull (ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were (2.47 ± 0.39)%, (5.62 ± 0.82)%, (3.57 ± 0.40)%, and (6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly (330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH (1-5) and lost their solubilities when the NaCl concentration was above 2% (W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna (Katsuwonus pelamis) / 中国天然药物

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the spine (ASC-SP and PSC-SP) and skull (ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were (2.47 ± 0.39)%, (5.62 ± 0.82)%, (3.57 ± 0.40)%, and (6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly (330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH (1-5) and lost their solubilities when the NaCl concentration was above 2% (W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.