Collagen Chains Research Articles

High Performance of Covalently Grafting onto Collagen in The Presence of Graphene Oxide.

A collagen-based copolymer, ASC-g-Poly(methyl methacrylate-co-Ethyl Acrylate), was synthesized in the presence of Graphene Oxide (GO) via an in-situ polymerization. The presence of GO that increased the accessible surface area for initiated collagen chains allowed for an accelerated polymerization with highly improved grafting performance and efficiency. This was conducted from two polymerization systems with varied comonomer feed ratios, in which two distinguished GO loadings were used. The processability of the achieved nanocomposite was then evaluated through casting and electrospinning processing methods. Fourier Transform Infrared Spectroscopy (FT-IR), UV-Vis spectroscopy, Differential Scanning Calorimeter (DSC), Thermogravimetric analysis (TGA), Scanning Electron Microscope (SEM), Transmission electron microscopy (TEM), and tensile analysis were conducted to characterize the GO-ASC-g-P(MMA-co-EA). The nanocomposite films showed a unique morphology, multilayer nanostructure of the grafted GO monolayers that deposited simultaneously one on top of another. The morphology of the electrospun fibers was affected by the addition of GO loadings in which the increase in fiber diameter was observed while the surface of the nanofibers was decorated by the GO nanolayers. To modify the collagen, this research highlights the importance of introducing functional groups of GO and the substitution of GO loadings as an active nanostructure filler to highly monomer feed ratios improving the physiochemical properties of collagen. This easy-to-apply approach is suggested for applications intending the mechanical properties and deterred degradation of Collagen-based materials.

Anti–Glomerular Basement Membrane Disease

Anti-glomerular basement membrane (anti-GBM) disease is a rare autoimmune small vessel vasculitis characterized by autoreactivity to antigens in type IV collagen chains expressed in glomerular and alveolar basement membrane. The detection of circulating anti-GBM antibodies, which are shown to be directly pathogenic, is central to disease diagnosis. Clinically, anti-GBM disease usually presents with rapidly progressive glomerulonephritis with or without alveolar hemorrhage. Rapid diagnosis and early treatment are required to prevent mortality and to preserve renal function. Relapse in anti-GBM disease is uncommon. Variant and atypical forms of anti-GBM disease are increasingly recognised.

The effect of radio-frequency heating on vacuum-packed saury (Cololabis saira) in water.

We characterized vacuum-packed whole saury (Cololabis saira) treated using radio-frequency (RF) heating and compared it with that treated using conventional retort heating. RF heating is electrical heating based on dielectric heating. In this study, the effect of RF heating on softening and collagen in backbone was analyzed. RF heating heated the center of fish faster than water. The backbone was softened to a chewable level, and the heating time was shortened to one-third. The amount of crude protein and collagen in backbone decreased with decreasing elasticity, although that heated using RF (131°C) still contained a higher amount than conventional heating. However, β and γ collagen, and then α1 and α2 collagen chain in backbone disappeared with heating; therefore, collagen was degraded to collagen peptide. Results confirmed that RF heating provided wholly eatable fish containing low-molecular collagen peptide in a short heating time.

Comparison of covalently and physically cross-linked collagen hydrogels on mediating vascular network formation for engineering adipose tissue

Timely tissue vascularization and integration of engineered tissues into a patient plays an important role in the successful translation of engineered tissues into clinically relevant therapies. To decrease the time needed to vascularize an engineered adipose tissue, suitable local microenvironments provided by hydrogels to support cell-based functional vascular network formation have been investigated. Using the same biomolecule in solution, two types of hydrogels can be obtained: a “physical hydrogel” which is thermal-induced self-assemble fibril initiation and growth, due to amino and carboxyl telopeptides on collagen chains, and a “chemical hydrogel” which results from the covalently cross-linking of the side chains induced by one step enzyme mediation in aqueous solution. In this paper, we compare the capability of engineering vascular network and large-sized vascularized adipose tissue in vivo in different types of collagen hydrogels, physical and chemical crosslinking. The relationships between vascular network formation and hydrogel properties for the two types of hydrogels are discussed. Finally, we successfully engineered a vascularized adipose tissue construct (∼877.6 adipocytes/mm2; 94% area of a construct) in the absence of exogenous cytokines in chemical covalently crosslinking cell-laden hydrogel. These results show manipulating the polymerized methods of a hydrogel could not only modulate vascular network formation, but also regenerate adipose tissue in vivo.

Visualizing collagen proteolysis by peptide hybridization: From 3D cell culture to in vivo imaging

Degradation of the extracellular matrix (ECM) is one of the fundamental factors contributing to a variety of life-threatening or disabling pathological conditions. However, a thorough understanding of the degradation mechanism and development of new ECM-targeting diagnostics are severely hindered by a lack of technologies for direct interrogation of the ECM structures at the molecular level. Previously we demonstrated that the collagen hybridizing peptide [CHP, sequence: (GPO)9, O: hydroxyproline] can specifically recognize the degraded and unfolded collagen chains through triple helix formation. Here we show that fluorescently labeled CHP robustly visualizes the pericellular matrix turnover caused by proteolytic migration of cancer cells within 3D collagen culture, without the use of synthetic fluorogenic matrices or genetically modified cells. To facilitate in vivo imaging, we modified the CHP sequence by replacing each proline with a (2S,4S)-4-fluoroproline (f) residue which interferes with the peptide's inherent propensity to self-assemble into homo-triple helices. We show that the new CHP, (GfO)9, tagged with a near-infrared fluorophore, enables in vivo imaging and semi-quantitative assessment of osteolytic bone lesions in mouse models of multiple myeloma. Compared to conventional techniques (e.g., micro-CT), CHP-based imaging is simple and versatile in vitro and in vivo. Therefore, we envision CHP's applications in broad biomedical contexts ranging from studies of ECM biology and drug efficiency to development of clinical molecular imaging.

High pressure Raman study of type-I collagen.

The high pressure response of type-I collagen from bovine Achilles tendon is investigated with micro-Raman spectroscopy. Fluorinert™ and methanol-ethanol mixtures were used as pressure transmitting media (PTM) in a diamond anvil cell. The Raman spectrum of collagen is dominated by three bands centred at approximately 1450, 1660 and 2930cm-1 , attributed to C-H deformation, C=O stretching of the peptide bond (amide-I band) and C-H stretching modes respectively. Upon pressure increase, using Fluorinert™ as PTM, a shift towards higher frequencies of the C-H stretching and deformation peaks is observed. Contrary, the amide-I band peaks are shifted to lower frequencies with moderate pressure slopes. On the other hand, when using the alcohol mixture as PTM, the amide-I band exhibits more pronounced C=O bond softening, deduced from the shift to lower frequencies, suggesting a strengthening of the hydrogen bonds between glycine and proline residues of different collagen chains due to the presence of the polar alcohol molecules. Furthermore, some of the peaks exhibit abrupt changes in their pressure slopes at approximately 2 GPa, implying a variation in the compressibility of the collagen fibres. This could be attributed to a pitch change from 10/3 to 7/2, sliding of the tropocollagen molecules, twisting variation at the molecular level and/or elimination of the D-gaps induced by kink compression. All spectral changes are reversible upon pressure release, which indicates that denaturation has not taken place. Finally, a minor lipid phase contamination was detected in some sample spots. Its pressure response is also monitored.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.

Tannerella forsythia‐produced methylglyoxal causes accumulation of advanced glycation endproducts to trigger cytokine secretion in human monocytes

The periodontal pathogen Tannerella forsythia has the unique ability to produce methylglyoxal (MGO), an electrophilic compound which can covalently modify amino acid side chains and generate inflammatory adducts known as advanced glycation endproducts (AGEs). In periodontitis, concentrations of MGO in gingival-crevicular fluid are increased and are correlated with the T. forsythia load. However, the source of MGO and the extent to which MGO may contribute to periodontal inflammation has not been fully explored. In this study we identified a functional homolog of the enzyme methylglyoxal synthase (MgsA) involved in the production of MGO in T. forsythia. While wild-type T.forsythia produced a significant amount of MGO in the medium, a mutant lacking this homolog produced little to no MGO. Furthermore, compared with the spent medium of the T. forsythia parental strain, the spent medium of the T. forsythia mgsA-deletion strain induced significantly lower nuclear factor-kappa B activity as well as proinflammogenic and pro-osteoclastogenic cytokines from THP-1 monocytes. The ability of T. forsythia to induce protein glycation endproducts via MGO was confirmed by an electrophoresis-based collagen chain mobility shift assay. Together these data demonstrated that T. forsythia produces MGO, which may contribute to inflammation via the generation of AGEs and thus act as a potential virulence factor of the bacterium.

A Shared Epitope of Collagen Type XI and Type II Is Recognized by Pathogenic Antibodies in Mice and Humans with Arthritis.

Collagen XI (CXI) is a heterotrimeric molecule with triple helical structure in which the α3(XI) chain is identical to the α1(II) chain of collagen II (CII), but with extensive posttranslational modifications. CXI molecules are intermingled in the cartilage collagen fibers, which are mainly composed of CII. One of the alpha chains in CXI is shared with CII and contains the immunodominant T cell epitope, but it is unclear whether there are shared B cell epitopes as the antibodies tend to recognize the triple helical structures. Mice expressing the susceptible immune response gene Aq were immunized with CII or CXI. Serum antibody responses were measured, monoclonal antibodies were isolated and analyzed for specificity to CII, CXI, and triple helical collagen peptides using bead-based multiplex immunoassays, enzyme-linked immunosorbent assays, and Western blots. Arthritogenicity of the antibodies was investigated by passive transfer experiments. Immunization with CII or CXI leads to a strong T and B cell response, including a cross-reactive response to both collagen types. Immunization with CII leads to severe arthritis in mice, with a response toward CXI at the chronic stage, whereas CXI immunization induces very mild arthritis only. A series of monoclonal antibodies to CXI were isolated and of these, the L10D9 antibody bound to both CXI and CII equally strong, with a specific binding for the D3 epitope region of α3(XI) or α1(II) chain. The L10D9 antibody binds cartilage in vivo and induced severe arthritis. In contrast, the L5F3 antibody only showed weak binding and L7D8 antibody has no binding to cartilage and did not induce arthritis. The arthritogenic L10D9 antibody bound to an epitope shared with CII, the triple helical D3 epitope. Antibody levels to the shared D3 epitope were elevated in the sera from mice with arthritis as well as in rheumatoid arthritis. CXI is immunologically not exposed in healthy cartilage but contains T and B cell epitopes cross-reactive with CII, which could be activated in both mouse and human arthritis and could evoke an arthritogenic response.

Soluble Activin Type IIB Receptor Decoy changes Gene Expression Profiles of Bone Cells in the OIM and not the G610C Mouse Model of Osteogenesis Imperfecta

Bone is a highly dynamic tissue which is continually remodeled by osteoblasts and osteoclasts, with regulation by osteocytes. In Osteogenesis Imperfecta (OI), a clinically heterogeneous disorder characterized by bone fragility, the functions of osteoblasts and osteoclasts are thought to be impaired. As a mechanosensitive tissue, bone responds to mechanical loads, some of the strongest of which are from skeletal muscles, by changing its shape.The inhibition of myostatin, a negative regulator of muscle growth and a member of the TGF‐β superfamily, has been shown to induce muscle growth via hypertrophy. In this study, we investigated the effect of pharmacologically inhibiting myostatin through treatment with the soluble activin receptor type IIB decoy molecule (sActRIIB‐mFc), focusing on the gene expression patterns of bone cells derived from two molecularly distinct mouse models of OI (osteogenesis imperfecta murine [OIM] vs. G610C). Because the molecular defects that give rise to the G610C and OIM models differ and muscle function is differentially impacted, we hypothesize that the two models may respond differently to sActRIIB‐mFc treatment. Homozygous mice (oim/oim) in the OIM model have a single nucleotide deletion in the COL1A2 gene that leads to functional null proα2 (I) collagen chains and the generation of homotrimeric type I collagen, whereas the G610C mouse, heterozygous for a missense mutation in the COL1A2 gene, has a glycine to cysteine substitution at the 610 position of the triple helical domain.At two months of age, WT, +/G610C, and oim/oim male mice were treated bi‐weekly either with vehicle (Tris‐buffered saline) or sActRIIB‐mFc for 8 weeks. Quantitative RT‐PCR analyses demonstrated that tibias from sActRIIB‐mFc treated oim/oim mice exhibited increased gene expression levels of the markers of osteoblast, osteoclast, and osteocyte function and differentiation analyzed. This contrasts with the significant changes seen in the femoral cortical bone microarchitecture and biomechanical integrity of sActRIIB‐mFc‐treated WT and +/G610C mice, which was not observed in the oim/oim mice.In conclusion, our findings indicate that sActRIIB‐mFc treatment has differential effects on the G610C and OIM mice, which may in part reflect differences in severity (severe in oim/oim vs. moderately severe in +/G610C), and/or their unique molecular defects.Understanding the molecular impact of therapeutics is critical in order to develop more effective and targeted treatment for OI.Support or Funding InformationLeda J. Sears TrustKansas City Area life Sciences Institute Patton Trust Research, Inc. University of Missouri Research Board GrantMarch of DimesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of congenic myostatin inhibition on a mouse model of osteogenesis imperfecta (OI) type I/IV

Bone is a dynamic organ in part due to its mechanosensitivity. Muscle mass and force are some of the largest physiological loads that bone experience during its life time, with muscle mass being directly proportional to bone mass. One of the factors regulating muscle mass is myostatin, a member of the TGF‐β superfamily, that acts as a negative regulator of muscle growth. Osteogenesis imperfecta (OI), commonly known as brittle bone disease, is a heritable connective tissue disorder characterized by skeletal fragility. A glycine to cysteine substitution in the proα2(I) collagen chain leads to reduced bone mineral density and compromised biomechanical integrity as modeled by the heterozygous G610C (+/G610C) mouse. +/G610C mice model both the genotype and phenotype of 64 affected individuals with mild to moderate type I/IV OI in an Old Order Amish kindred. We investigated the effects of myostatin deficiency on the +/G610C mouse muscle by breeding heterozygote myostatin deficient mice (+/mstn) with +/G610C mice to produce WT, +/mstn, +/G610C, and +/mstn +/G610C mice. After weaning at three weeks of age, mice were weighed weekly until 4 months of age when they were euthanized. After euthanization, 5 hindlimb muscles, the gastrocnemius, quadriceps, tibalis anterior, plantaris, and soleus, were harvested and weighed. Serum was separated from the blood and used to quantify myostatin levels. Serums from +/mstn and +/mstn +/G610C mice demonstrated decreased levels of myostatin as compared to WT and +/G610C mice. +/mstn mice showed increased body weights at four months of age when compared to WT littermates. +/mstn +/G610C mice also had increased body weights as compared to +/G610C littermates, although they did not reach significance. More importantly, however, +/mstn and +/mstn +/G610C mice exhibited significantly increased hindlimb skeletal muscle weights and relative muscle weights as compared to +/G610C mice for the gastrocnemius and quadriceps. These findings demonstrate that myostatin deficiency can lead to increased muscle mass in mice with OI which can potentially lead to increased bone mass and strength.Support or Funding InformationSupported by March of Dimes, National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (RO1 AR055907), the Leda J. Sears Trust, the Kansas City Area Life Sciences Institute Patton Trust Research, Inc., and the University of Missouri Research Board Grant. The project described was also supported by the IMSD EXPRESS Fellows Program (or simply the IMSD EXPRESS Program) via grant number R25GM056901 from the National Institute of General Medical Science (NIGMS), a component of the National Institutes of Health (NIH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potential biomarkers and targets in reversibility of pulmonary arterial hypertension secondary to congenital heart disease: an explorative study.

Whether pulmonary arterial hypertension (PAH) is reversible in congenital heart disease (CHD) is important for the operability of CHD. However, little is known about that. Our research was aimed at exploring novel biomarkers and targets in the reversibility of CHD-PAH. CHD-PAH patients diagnosed with right heart catheterization (RHC) were enrolled (n = 14). Lung biopsy was performed during the repair surgery. After one year follow-up, mean pulmonary arterial pressures (mPAP) were evaluated by RHC to determine the diagnosis of reversible (mPAP < 25 mmHg, n = 10) and irreversible (mPAP ≥ 25 mmHg, n = 4) PAH. Harvested normal lung tissues (n = 6) were included as the control group. Pulmonary arteriole lesions were identified by pathological grading in tissue staining. iTRAQ-labelled mass-spectrometry analysis followed by immunohistochemistry and western blot was used to explore the most meaningful differential proteins. For enrolled patients, the histopathological grading of pulmonary vascular lesions in reversible CHD-PAH patients was all at grades 0–II while grades III–IV were shown only in irreversible CHD-PAH patients. Proteomic analysis identified 85 upregulated and 75 downregulated proteins, including cytoskeletal proteins and collagen chains, mainly involved in cell adhesion, extracellular matrix, cytoskeleton, immune response, and complement pathways. Among them, caveolin-1, filamin A expression, and cathepsin D combined with macrophagocytes counts were significantly increased; glutathione S-transferase mu1 (GSTM1) expression was significantly decreased in the irreversible CHD-PAH group (all P < 0.05). Caveolin-1, filamin A, and cathepsin D expression showed a positive relation and GSTM1 showed a negative relation with pathological grading. Upregulated caveolin-1, filamin A, and cathepsin D combined with increased macrophagocytes and downregulated GSTM1 may be potential biomarkers and targets in the irreversibility CHD-PAH, and which may be useful in evaluating the operability and understanding the irreversibility of CHD-PAH. Expression of these pathological biomarkers combined with pathological changes in lung biopsy may have great value in predicting the irreversibility of PAH.

The Influence of Simple Phenols on Collagen Type I Fibrillogenesis in vitro

The influence of phenolic compounds with different numbers of hydroxy groups (phenol, pyrocatechol, resorcinol, and pyrogallol) on the kinetics of in vitro fibrillogenesis of collagen and on fibril structure has been studied. It has been shown that these phenols accelerate fibril formation mainly by shortening the lag phase, presumably facilitating the formation of collagen dimers and their subsequent association to linear aggregates. The accelerating activity of phenols is proportional to the number of hydroxy groups in the molecule. It increases in the series: phenol < resorcinol < pyrogallol. Therefore, the ability of phenols to accelerate fibril formation is likely to stem from the formation of hydrogen bonds with amino-acid residues in collagen chains. The hydrogen bonds may stabilize the structure of the intermediates, facilitating their interaction during fibrillogenesis.

Guide to electrospinning denatured whole chain collagen from hoki fish using benign solvents

Tissue engineering requires the design and manufacture of biomimetic scaffolds. Collagen-derived nanofibrous scaffolds have been intensively studied because collagen, in the form of fibrils, is one of the main components of the extra cellular matrix (ECM). Several collagen materials have been used in electrospinning studies including mammalian extracted Type I collagen and gelatin formulations. Denatured whole chain collagen (DWCC) can be prepared by heat denaturing acid-soluble collagen extracted from cold-water fish skin. This product provides a consistent source of collagen with a controlled molecular weight profile and intact alpha chains including telopeptides. In this work, we studied DWCC-water-acid systems in order to determine the effect of solution composition on nanofibre morphology. Whereas measurement of the classical physical properties of concentrated solutions failed to predict and only partially explained the electrospinning behavior of collagen derived polymers, hydrodynamic properties provided insight. All the samples are presented in ternary diagrams to map the electrospinnability of the systems. These “electrospinning maps” provide an informative resource to electrospinning collagen-derived product for biomedical or commercial applications and a practical alternative to complicated models developed for synthetic polymers.

Viscoplastic fracture transition of a biopolymer gel.

Physical gels are swollen polymer networks consisting of transient crosslink junctions associated with hydrogen or ionic bonds. Unlike covalently crosslinked gels, these physical crosslinks are reversible thus enabling these materials to display highly tunable and dynamic mechanical properties. In this work, we study the polymer composition effects on the fracture behavior of a gelatin gel, which is a thermoreversible biopolymer gel consisting of denatured collagen chains bridging physical network junctions formed from triple helices. Below the critical volume fraction for chain entanglement, which we confirm via neutron scattering measurements, we find that the fracture behavior is consistent with a viscoplastic type process characterized by hydrodynamic friction of individual polymer chains through the polymer mesh to show that the enhancement in fracture scales inversely with the squared of the mesh size of the gelatin gel network. Above this critical volume fraction, the fracture process can be described by the Lake-Thomas theory that considers fracture as a chain scission process due to chain entanglements.

An antibacterial collagen membrane crosslinked by the inclusion complex of β-cyclodextrin dialdehyde and ofloxacin for bacterial keratitis.

Infectious keratitis is one of the leading causes of blindness in the world, especially in developing countries. Corneal transplant surgery is a feasible treatment for bacterial keratitis when drug therapy cannot be effective. However, the amount of corneal donors is far from requirements of clinical treatment and thus, the prognosis of bacterial keratitis is not satisfactory. In this study, we developed a novel antibacterial corneal repair material (β-CD-DA/OFLX-Col) for bacterial keratitis. The inclusion complex of β-cyclodextrin dialdehyde (β-CD-DA) with ofloxacin formed by host–guest interaction was used as both a drug vector and a crosslinker for further reaction with the amino groups on lysine of collagen chains. Physical properties, cytocompatibility, and antibacterial property of β-CD-DA/OFLX-Col film were characterized. The results indicated that the film was mainly transparent and possessed superior mechanical properties. Moreover, human corneal epithelial cells could adhere to the film and proliferate normally, indicating that the β-CD-DA/OFLX-Col film was non-cytotoxic and had good biocompatibility. Most importantly, the β-CD-DA/OFLX-Col film exhibited prominent antibacterial effects against E. coli and S. aureus in vitro, which could minimize the risks of infection. The prepared β-CD-DA/OFLX-Col film could greatly increase bioavailability of drugs and reduce toxic side effects, thus displaying great potential in bacterial keratitis treatment.

Potential of Electrospun Poly(3-hydroxybutyrate)/Collagen Blends for Tissue Engineering Applications.

In this work, tunable nonwoven mats based on poly(3-hydroxybutyrate) (PHB) and type I collagen (Coll) were successfully produced by electrospinning. The PHB/Coll weight ratio (fixed at 100/0, 70/30, and 50/50, resp.) was found to control the morphological, thermal, mechanical, and degradation properties of the mats. Increasing collagen amounts led to larger diameters of the fibers (in the approximate range 600–900 nm), while delaying their thermal decomposition (from 245°C to 262°C). Collagen also accelerated the hydrolytic degradation of the mats upon incubation in aqueous medium at 37°C for 23 days (with final weight losses of 1%, 15%, and 23% for 100/0, 70/30, and 50/50 samples, resp.), as a result of increased mat wettability and reduced PHB crystallinity. Interestingly, 70/30 meshes were the ones displaying the lowest stiffness (~116 MPa; p < 0.05 versus 100/0 and 50/50 meshes), while 50/50 samples had an elastic modulus comparable to that of 100/0 ones (~250 MPa), likely due to enhanced physical crosslinking of the collagen chains, at least at high protein amounts. All substrates were also found to allow for good viability and proliferation of murine fibroblasts, up to 6 days of culture. Collectively, the results evidenced the potential of as-spun PHB/Coll meshes for tissue engineering applications.

Structural properties of gelatin extracted from croaker fish (Johnius sp) skin waste

ABSTRACTIn the present study, the gelatin from fresh croaker fish (Johnius sp) skin was obtained by rapid extraction and its properties were assessed. The gelling and melting temperature of croaker skin gelatin was 17.4 and 23.8°C as revealed by dynamic visco-elastic analysis using Controlled Stress Rheometer. Croaker skin gelatin (CSG) had the bloom value of 193.4 g. Scanning electron microscopic analysis of freeze dried gelatin showed the presence of voids surrounded by a loose network imitating a sponge or coral like structure. Texture profile parameters such as hardness, springiness, cohesiveness, gumminess, and chewiness of croaker fish skin gelatin gel were relatively lower than the porcine gelatin gel. The amino acid composition of gelatin revealed a higher proportion of glycine, alanine, and hydroxyl proline (30.25, 23.4, and 11.82 g/100 g protein, respectively) content. The electrophoretic analysis showed the presence of two peptide chains with the approximate molecular weight of 200 and 116 kDa corresponds to β and α peptide chain of collagen. The Fourier Transform Infrared (FT-IR) spectra of gelatin showed peaks correspond to the structures like α helix, β sheet, and random coil in the gelatin preparations.

Case of epidermolysis bullosa acquisita with concomitant anti‐laminin‐332 antibodies

Subepidermal autoimmune blistering disease including bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid, anti-laminin-γ1 pemphigoid, linear immunoglobulin A bullous disease and epidermolysis bullosa acquisita (EBA), are all characterized by direct immunofluorescence microscopy or immunoglobulin deposition on the basement membrane zone. Among them, EBA is a rare acquired subepidermal autoimmune blistering disease of the skin and mucous membranes reactive with type VII collagen, a major component of the epidermal basement membrane zone. Anti-laminin-332-type mucous membrane pemphigoid has pathogenic autoantibodies against laminin-332, which is a basement membrane heterotrimeric protein composed of α3, β3 and γ2 laminin chains. We describe a 73-year-old Japanese man presenting with multiple, annular, tense blisters on the lower legs and oral lesions. Despite the severe clinical manifestations, the disease was successfully controlled by combination therapy of oral prednisolone and mizoribine. This case was confirmed to have autoantibodies to both type VII collagen and laminin-332 α3 chain by indirect immunofluorescence of 1 mol NaCl-split normal human skin, various immunoblot analyses and enzyme-linked immunosorbent assays. This case was a rare case of EBA with concomitant anti-laminin-332 antibodies.

Rose Bengal Binding to Collagen and Tissue Photobonding.

We investigated two critical aspects of rose Bengal (RB) photosensitized protein cross-linking that may underlie recently developed medical applications. Our studies focused on the binding of RB to collagen by physical interaction and the effect of this binding and certain amino acids on RB photochemistry. Molecular dynamics simulations and free-energy calculation techniques, complemented with isothermal titration calorimetry, provided insight into the binding between RB and a collagen-like peptide (CLP) at the atomic level. Electrostatic interactions dominated, which is consistent with the finding that RB bound equally well to triple helical and single chain collagen. The binding free energy ranged from −5.7 to −3 kcal/mol and was strongest near the positively charged amino groups at the N-terminus and on lysine side chains. At high RB concentration, a maximum of 16 ± 3 bound dye molecules per peptide was found, which is consistent with spectroscopic evidence for aggregated RB bound to collagen or the CLP. Within a tissue-mimetic collagen matrix, RB photobleached rapidly, probably due to electron transfer to certain protein amino acids, as was demonstrated in solutions of free RB and arginine. In the presence of arginine and low oxygen concentrations, a product absorbing at 510 nm formed, presumably due to dehalogenation after electron transfer to RB. In the collagen matrix without arginine, the dye generated singlet oxygen as well as the 510 nm product. These results provide the first evidence of the effects of a tissue-like environment on the photochemical mechanisms of rose Bengal.