Mechanical Properties of Mammalian and Fish Gelatins as a Function of the Contents of α-Chain, β-Chain,and Low and High Molecular Weight Fractions

Small-strain oscillatory measurements and size-exclusion chromatography coupled to multiangle laser light scattering were used to study the mechanical properties and the molecular weight distribution, respectively, of acid porcine skin gelatins (type A), lime bovine bone gelatins (type B), and cold water fish gelatins, while principal component analysis (PCA) and partial least squares regression were used to relate the mechanical properties with the molecular weight distribution. The present study suggests a linear relationship between the mechanical properties and the fractions of low molecular weight (LMW) molecules, α-chains, β-chains, and high molecular weight (HMW) molecules. The Bloom value for mammalian gelatin was positively correlated with the fractions of α-chains, β-chains, and HMW molecules and negatively correlated with the fraction of LMW molecules. The dynamic storage modulus for cold water fish gelatin was positively correlated with the fractions of β-chains and HMW molecules and negatively correlated with the fractions of LMW molecules and α-chains.     

Extraction and characterization of gelatin from camel skin (potential halal gelatin) and production of gelatin nanoparticles

Gelatin is used as an ingredient in both food and non-food industries as a gelling agent, stabilizer, thickener, emulsifier, and film former. Porcine skins, bovine hides, and cattle bones are the most common sources of gelatin. However, mammalian gelatins are rejected by some consumers due to social, cultural, religious, or health-related concerns. In the present study, gelatin was obtained from camel skin as an alternative source using a combination of processing steps. Central composite design combined with response surface methodology was used to achieve high gelatin yields under different extraction conditions: temperatures of 40, 60, and 80 °C; pH values of 1, 4, and 7; and extraction times of 0.5, 2.0, and 3.5 min. Maximum gelatin yield from camel skin (29.1%) was achieved at 71.87 °C and pH 5.26 after 2.58 min. The extracted gelatin samples were characterized for amino acid profile, foaming capacity, film formation, foam stability, and gel strength (Bloom value). Gelatin nanoparticles were produced, and their morphology and zeta potential were determined. Bloom value of the camel skin gelatin was 340 g. Amino acid analysis revealed that the extracted gelatin showed high glycine and proline contents. Analysis of camel skin gelatin nanoparticle and functional properties revealed high suitability for food and non-food applications, with potential use in the growing global halal food market.     

Comparative behaviour of L-929 fibroblastic and human endothelial cells onto crosslinked protein substrates

Studies were carried out to compare the behaviour of human umbilical vein endothelial cells (HUVEC) and L-929 fibroblastic cells towards proteins crosslinked by glutaraldehyde (GTA) or carbodiimide (CDI) proposed for coating of vascular prostheses. CDI crosslinking of bovine serum albumin used alone, or mixed with gelatin, allowed higher rates of cell growth and DNA synthesis than GTA crosslinking independent of cells. Assessment of the plating efficiency revealed a similar behaviour of both cells towards membranes and reference plastic surface in terms of percentages of bound cells. HUVEC proliferation onto CDI crosslinked gelatin and/or albumin membranes did not differ significantly whereas the growth of L-929 was enhanced onto gelatin albumin membranes in comparison with both gelatin membranes and the reference surface. The analysis of DNA synthesis corroborated the results of the growth curves and elicited a delay of the growth phases in HUVEC cultured onto CDI crosslinked membranes, unlike the L-929 fibroblast.     

Melatonin desensitizing effects on the in vitro responses to MCH,alpha-MSH,isoproterenol and melatonin in pigment cells of a fish (S. marmoratus), a toad (B. ictericus), a frog (R. pipiens), and a lizard (A. carolinensis), exposed to varying photoperiodic regimens

Melatonin is a weak dose-independent lightening agonist in fish skin, a moderate dose-dependent lightening agonist in toad skin and a potent lightening agent in frog and lizard skins (reversing in a dose-dependent manner the darkening caused by alpha-melanocyte-stimulating hormone). In frog skins, previous exposure to melatonin reduced further lightening actions of the indoleamine, and in toad skins, increasing concentrations of melatonin elicited decreasing lightening responses, suggesting an autodesensitizing action of the hormone. Various concentrations of melatonin diminished the responses to the lightening agonist melanin-concentrating hormone (MCH) in fish skins and to the darkening agonists alpha-MSH in toad, frog and lizard skins and isoproterenol in frog skins. In vitro inhibitory actions of melatonin are mimicked in the absence of the hormone in skin preparations from toads kept in continuous darkness for 48 hr. The lipophylic nature of the indoleamine associated with the results herein described suggests intracellular actions of melatonin on vertebrate pigment cells.     

Artificial feeding of northern fowl mites, Ornithonyssus sylviarum (Canestrini and Fanzago) (Acari: Macronyssidae), through membranes

A new device and technique are described for the in vitro feeding of northern fowl mites, Ornithonyssus sylviarum (Canestrini and Fanzago). The device consisted of a glass cylinder 25 mm in length and in outside diameter, capped with a chick skin membrane at one end and a snap-cap with a wire cloth window at the other end. Maximum feeding by northern fowl mites on warmed heparinized chicken blood occurred after 60 min and at a blood temperature range of 36-42 C. Skin membranes prepared from 1-wk-old chicks gave significantly higher feeding rates than those from 4-wk-old chicks, but unfrozen skins and skins frozen up to 4 wk were equally effective. Also, mites fed equally well through white leghorn and broiler chick skin membranes. About 80% of northern fowl mites fed. The in vitro technique described simplifies the approach to studies of northern fowl mite biology and physiology.     

壳聚糖基角膜细胞载体的制备及其细胞相容性

为探讨羟丙基壳聚糖基共混膜作为组织工程技术中角膜细胞培养载体的可行性, 分别制备了羟丙基壳聚糖/硫酸软骨素、羟丙基壳聚糖/明胶/硫酸软骨素以及羟丙基壳聚糖/氧化透明质酸/硫酸软骨素三种共混膜。测定其透光率、含水量和蛋白吸附性能; 在共混膜上培养兔角膜上皮细胞, 通过观察角膜上皮细胞在不同载体膜上的生长状态、贴附情况, 测定细胞活性以及上清液中乳酸脱氢酶的活性, 研究三种壳聚糖基载体膜片与角膜上皮细胞的相容性。膜片理化性质测定结果表明三种共混膜片具有良好的透明度, 适宜的含水量和较强的蛋白吸附性能; 细胞相容性实验结果表明羟丙基壳聚糖/明胶/硫酸软骨素共混膜对细胞的损伤最小, 有利于细胞在膜上的贴附和生长, 表现出良好的细胞相容性, 有望作为角膜细胞载体体外构建组织工程化角膜。     

Characterization of Fish Gelatin at Nanoscale Using Atomic Force Microscopy

Atomic force microscopy (AFM) was used as a meaningful tool to characterize the nanostructure of gelatin from catfish (Ictalurus punctatus) skin. The gelatins extracted with pretreatments including acid pretreatment, alkaline pretreatment, and alkaline followed by acid pretreatment (optimized extraction conditions). The resulting gelatins were imaged using AFM and their nanostructure was studied. The AFM images showed that gelatin extracted with acid pretreatment had a coacervate structure while with alkaline pretreatment there were separate aggregates. Spherical aggregates and annular pores were observed in AFM images of gelatin with the optimized extraction conditions. AFM imaging of gelatin with a relative high concentration (0.5%) was successfully done and the results help researchers to understand gelatin structures at the nanoscale.     

Physical and chemical properties of gelatin from the skin of cultured Amur sturgeon (Acipenser schrenckii)

To expand the usefulness of cultured Amur sturgeon, Acipenser schrenckii, its skin was used to explore the production of gelatin. After acetic acid pre‐treatment (0.05 m for 3 h), gelatin was extracted at temperatures of 50 or 70°C for 1 or 6 h. Gelatin yield ranged from 9.42 to 12.47% (wet weight basis) (P < 0.05). With increasing extraction time and temperature, the content of imino acids (proline + hydroxyproline), gel strength and L*‐value (lightness) decreased, while the a*‐value (redness) and b*‐value (yellowness) of gelatin gel increased (P < 0.05). Electrophoretic analysis revealed that α‐chains and β‐chains were predominant components in all extracted gelatins. Higher molecular weight proteins (γ‐chain) were also observed. Gelling and melting temperatures of gelatin were 13.6–14.6°C and 20.3–22.6°C, respectively. Circular dichroism (CD) spectra and Fourier transform infrared (FTIR) spectroscopy revealed the triple helix loss in gelatin (A₁₂₃₅ (AIII)/A₁₄₅₁ < 1). Extraction conditions caused secondary structure changes in the gelatin. More likely due to the differences in the culture water temperature, gelatin exhibited gelling and melting temperatures intermediate between those of cold‐ and warm‐water fish gelatins. The obtained gelatin can be used in food products or in the production of bioactive compounds.     

Isolation of 2-Pyruvoylaminobenzamide as an Antiauxin from Colletotrichum lagenarium

To learn of possible contributions of amide side-groups of asparagine and glutamine residues to viscoelastic properties of food proteins, lime-processed gelatin, which has lost these amide side-groups, was amidated by reaction with ammonia and the water soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC). Depending on the quantity of EDC used, 13 to 40.3% of carboxyl side-groups of gelatin was amidated without intra- and inter-molecular cross-linkages, as judged from analyses of free-amino groups, gel-filtration chromatography, and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis. The amidated gelatins had higher isoionic points as expected, but their regeneration of the triple helical structure of collagen was prevented as judged from analyses of circular dichroism. Gels made from amidated gelatins showed fairly high viscosity but low gel strength.     

Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris

A custom-designed, highly hydrophilic gelatin was produced in Pichia pastoris. Secreted production levels in single-copy transformants were in the range 3-6 g/l of clarified broth and purification to near homogeneity could be accomplished by differential ammonium sulfate precipitation. Despite the fact that gelatins are highly susceptible to proteolysis because of their unfolded structure, the recombinant protein was shown to be fully intact by SDS-PAGE, N-terminal sequencing, gel filtration chromatography and mass spectrometry. Owing to its highly hydrophilic nature, the migration of the synthetic gelatin in SDS-PAGE was severely delayed. Esterification of the carboxylic amino acid side chains resulted in normal migration. The high polarity of the synthetic gelatin also accounts for its negligible surface activity in water at concentrations up to 5% (w/v), as determined by tensiometry. Circular dichroism spectrometry showed that the non-hydroxylated gelatin did not form triple helices at 4 degrees C. The spectrum was even more representative of the random coil conformation than the spectrum of natural non-hydroxylated gelatins.     

Efficacy of mechanically modified electrospun poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactide-co-<Emphasis Type="Italic">ε</Emphasis>-caprolactone)/gelatin membrane on full-thickness wound healing in rats

Bioengineered skin substitute offers new opportunities for treating various skin ailments. To compensate the structural integrity problems of scaffolds prepared from natural components, we mechanically developed highly modified electrospun nanofibrous membranes, incorporating poly(l-lactide-co-ε-caprolactone) (PLCL) into gelatin [poly(l-lactide-co-ε-caprolactone)/ gelatin membrane, (P/G (3/7)]. Subsequent to our previous in vitro study, our goal was to evaluate the in vivo performance of PLCL, gelatin, and P/G (3/7) membranes, and investigate the feasibility of the newly developed P/G (3/7) membrane for wound healing. Histological analysis using the mathematical model of wound healing and contraction, revealed the association between stiffness of skin substitute with cytokeratin production and wound contraction rate, and the defect site covered with the stiffer membrane showed lower cytokeratin production, and inversely, higher wound contraction rate. Overall, the P/G (3/7) membrane induced a satisfactory wound healing outcome. However, lower cytokeratin production rate with the mechanically modified P/G membrane involves the importance of the conditional blending of PLCL. Conversely, the condition of PLCL showed some incompatibility and hindrance of skin regeneration, consistent with previous in vitro results. With proper mechanical strength and cell viability, the P/G (3/7) membrane could successfully be used as a suitable skin substitute scaffold.     

Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications

Chitin is a natural biopolymer have been used for several biomedical applications due to its biodegradability and biocompatibility. By using the calcium solvent system, chitin regenerated hydrogel (RG) was prepared by using -chitin. And also, the swelling hydrogel (SG) was prepared by using β-chitin with water. Then, both RG and SG were mixed with gelatin and N-acetyl-d-(+)-glucosamine (GlcNAc) at 120 °C for 2 h. The chitin/gelatin membranes with GlcNAc were also prepared by using RG and SG with GlcNAc. The prepared chitin/gelatin membranes with or without GlcNAc were characterized by mechanical, swelling, enzymatic degradation, thermal and growth of NIH/3T3 fibroblast cell studies. The stress and elongation of chitin/gelatin membrane with GlcNAc prepared from RG was showed higher than the chitin/gelatin membranes without GlcNAc. But, the chitin/gelatin membranes prepared from SG with GlcNAc was showed higher stress and elongation than the chitin/gelatin membranes without GlcNAc. It is due to the crosslinking effect of GlcNAc. The chitin/gelatin membranes prepared from SG showed higher swelling than the chitin/gelatin membranes prepared from RG. In contrast, the chitin/gelatin membranes prepared from RG showed higher degradation than the chitin/gelatin membranes prepared from SG. And also, these chitin/gelatin membranes are showing good growth of NIH/3T3 fibroblast cell. So these novel chitin/gelatin membranes are useful for tissue engineering applications.     

Fabrication of gelatin/calcium phosphate composite nanofibrous membranes by biomimetic mineralization

Based on the principles of biomimetic mineralization, biocomposite nanofibrous membranes were fabricated by the growth of CaP crystals on electrospun gelatin nanofibers to mimic both the physical architecture and chemical composition of natural bone ECM. Plenty more CaP crystals formed on the nanofibrous membrane containing Ca(2+) ion precursors, in which these crystals were also observed on the inner side of membrane. The release rate of Ca(2+) ion precursors from the nanofibrous membrane was slower than that of PO(4)(3-) ion precursors, suggesting the existence of more strong intermolecular interaction between gelatin and Ca(2+) ions. ATR-FTIR and XRD results clearly revealed the formation of CaP crystals mixed with apatite and CaCO(3), or apatite and TCP on the membranes. The Ca/P molar ratio of crystals obtained from the XPS data was 2.03 and 1.60, which depended on the mineralization conditions. Higher amount of CaP crystals significantly accelerated the deposit rate of bone-like apatite on the surface of composite membrane, meaning to the improved in vivo bone bioactivity.     

Evaluation of Fibroblasts Adhesion and Proliferation on Alginate-Gelatin Crosslinked Hydrogel

Due to the relatively poor cell-material interaction of alginate hydrogel, alginate-gelatin crosslinked (ADA-GEL) hydrogel was synthesized through covalent crosslinking of alginate di-aldehyde (ADA) with gelatin that supported cell attachment, spreading and proliferation. This study highlights the evaluation of the physico-chemical properties of synthesized ADA-GEL hydrogels of different compositions compared to alginate in the form of films. Moreover, in vitro cell-material interaction on ADA-GEL hydrogels of different compositions compared to alginate was investigated by using normal human dermal fibroblasts. Viability, attachment, spreading and proliferation of fibroblasts were significantly increased on ADA-GEL hydrogels compared to alginate. Moreover, in vitro cytocompatibility of ADA-GEL hydrogels was found to be increased with increasing gelatin content. These findings indicate that ADA-GEL hydrogel is a promising material for the biomedical applications in tissue-engineering and regeneration.     

Reconstruction of a tissue-engineered skin containing melanocytes

The objective of this study was to establish a new method for reconstruction of a tissue-engineered skin containing melanocytes by employing tissue engineering. The keratinocytes, melanocytes and dermal fibroblasts were isolated and purified from human foreskin biopsies. Then the cells were used to construct a tissue-engineered skin containing melanocytes. The localization of melanocytes in the tissue-engineered skin was detected by DOPA staining, S-100 immunohistochemical staining and transmission electron microscope (TEM). The results showed that the melanocytes could be detected in the basal layer of the constructed skin and the melanocytes showed dendritic morphology. Moreover the constructed skins were used to repair the athymic mice skin defects. Animal experiment results indicated that the skin equivalents could successfully repair full thickness skin defects in athymic mice and generated black skins by 6weeks after grafting. Melanocytes located in the basal layer of the athymic mice skin could also be detected by using the S-100 immunohistochemical staining. Our established method is useful to repair the full-thickness skin defects.     

Optical resolution and mechanism using enantioselective cellulose,sodium alginate and hydroxypropyl‐β‐cyclodextrin membranes

Chiral solid membranes of cellulose, sodium alginate, and hydroxypropyl‐β‐cyclodextrin were prepared for chiral dialysis separations. After optimizing the membrane material concentrations, the membrane preparation conditions and the feed concentrations, enantiomeric excesses of 89.1%, 42.6%, and 59.1% were obtained for mandelic acid on the cellulose membrane, p ‐hydroxy phenylglycine on the sodium alginate membrane, and p ‐hydroxy phenylglycine on the hydroxypropyl‐β‐cyclodextrin membrane, respectively. To study the optical resolution mechanism, chiral discrimination by membrane adsorption, solid phase extraction, membrane chromatography, high‐pressure liquid chromatography ultrafiltration were performed. All of the experimental results showed that the first adsorbed enantiomer was not the enantiomer that first permeated the membrane. The crystal structures of mandelic acid and p ‐hydroxy phenylglycine are the racematic compounds. We suggest that the chiral separation mechanism of the solid membrane is “adsorption – association – diffusion,” which is able to explain the optical resolution of the enantioselective membrane. This is also the first report in which solid membranes of sodium alginate and hydroxypropyl‐β‐cyclodextrin were used in the chiral separation of p ‐hydroxy phenylglycine.     

Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin

Microelectrodes were used to investigate the effect of 0.5 mM mucosal lanthanum (La3+) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potential Vsco (mean value = -65.4 +/- 3.2 mV, inside negative) hyperpolarized to -108.7 +/- 2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potential Vo or the inner membrane potential Vi. The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La3+ to the outer medium increased the short-circuit current to 190% of the control value. Vsco concomitantly changed to -28 +/- 3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La3+ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La3+ on short-circuit current has been observed. It is ascribed to the penetration of La3+ into the sodium channels.     

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na⁺,K⁺)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na⁺,K⁺)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na⁺,K⁺)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na⁺,K⁺)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change. Accepted: 21 January 1997     

Electric organ morphology of Sternopygus macrurus, a wave-type, weakly electric fish with a sexually dimorphic EOD.

In several species of electric fish with a sex difference in their pulse-type electric organ discharge (EOD), the action potential-generating cells of the electric organ (electrocytes) of males are larger and more invaginated compared to females. Androgen treatment of females and juveniles produces a longer-duration EOD pulse that mimics the mature male EOD, with a concurrent increase in electrocyte size and/or membrane infolding. In Sternopygus macrurus, which generates a wave-type EOD, androgen also increases EOD pulse duration. To investigate possible morphological correlates of hormone-dependent changes in EOD in Sternopygus, we examined electric organs from both fish collected in the field, and untreated and androgen-treated specimens in the laboratory. The electrocytes are cigar shaped, with prominent papillae on the posterior, innervated end. Electrocytes of field-caught specimens were significantly larger in all parameters than were electrocytes of specimens maintained in the laboratory. EOD pulse duration and frequency were highly correlated, and were significantly different between the sexes in sexually mature fish. Nevertheless, no sex difference in electrocyte morphology was observed, nor did any parameters of electrocyte morphology correlate with EOD pulse duration or frequency. Further, whereas androgen treatment significantly lowered EOD frequency and broadened EOD pulse duration, there was no difference in electrocyte morphology between hormone-treated and control groups. Thus, in contrast to results from studies on both mormyrid and gymnotiform pulse fish, electrocyte morphology is not correlated with EOD waveform characteristics in the gymnotiform wave-type fish Sternopygus. The data, therefore, suggest that sex differences in EOD are dependent on changes in active electrical properties of electrocyte membranes.