Triple Helix Structure Of Collagen Research Articles

Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers.The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 μm, 160 Å) using isocratic elution and MS-compatible mobile phase.The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Effects of Ultrasonic Power on the Structure and Rheological Properties of Skin Collagen from Albacore (Thunnus alalunga).

The effects of ultrasonic power (0, 150, 300, 450, and 600 W) on the extraction yield and the structure and rheological properties of pepsin-soluble collagen (PSC) from albacore skin were investigated. Compared with the conventional pepsin extraction method, ultrasonic treatment (UPSC) significantly increased the extraction yield of collagen from albacore skin, with a maximum increase of 8.56%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that peptides of low molecular weight were produced when the ultrasonic power exceeded 300 W. Meanwhile, secondary structure, tertiary structure, and X-ray diffraction analyses showed that the original triple helix structure of collagen was intact after the ultrasonic treatment. The collagen solutions extracted under different ultrasonic powers had significant effects on the dynamic frequency sweep, but a steady shear test suggested that the collagen extracted at 150 W had the best viscosity. These results indicate that an ultrasonic power between 150 and 300 W can improve not only the extraction yield of natural collagen, but also the rheological properties of the collagen solution without compromising the triple helix structure.

Evaluation of the Relationship Between Collagen Matrix Damage and Proteoglycan Activity Following Submaximal Fatigue Loading

Background and Hypothesis:Previous research indicated that fatigue loading of the anterior cruciate ligament (ACL) leads to disruption and unraveling of the collagen triple helix structure. Using a novel in vivo murine model, we have spectroscopically shown that collagen unraveling at the molecular level is associated with an increase in proteoglycan activity. Moreover, we found an increase in tissue compliance within these damaged tissue domains. We hypothesize that the increase in proteoglycan activity is largely responsible for this mechanical change. To investigate this, we first need to identify the proteoglycan(s) responsible for our biochemical spectra that indicatedan elevation in activity associated with unraveled collagen domains. For this pilot project, we histomorphometrically investigated the proteoglycan activity of two potential protein candidates (decorin, versican) following sub-maximal ACL fatigue loading.
 Experimental Design or Project Methods:The right knees of 20 mice underwent 5,000 cycles of moderate or strenuous fatigue loading. Left knees served as a control. Samples were collected 1 and 72 hours after loading. Immunohistochemistry was performed to detect decorin and versican activity in the ACLs.
 Results:A greater area of the ACL was stained with decorin relative to versican in ACLs that underwent moderate fatiguing after 1-hour rest (p = 0.02) and 72-hour rest (p < 0.01). Similar differences were found in ACLs after strenuous fatiguing and 72-hour rest (p < 0.01). No significant temporal differences were found in versican (p = 0.76) or decorin (p = 0.53) activity in fatigued ACLs relative to controls.
 Conclusion and Potential Impact:These preliminary findings suggest that versican nor decorin are the proteoglycans colocalizing with unraveled collagen. We are currently investigating aggrecan, lumican, and biglycan as other potential proteoglycan candidates to explain our spectroscopic data.

Characterization and application of photocrosslinkable collagen maleate as bioink in extrusion-based 3D bioprinting.

3D bioprinting, a significant advancement in biofabrication, is renowned for its precision in creating tissue constructs. Collagen, despite being a gold standard biomaterial, faces challenges in bioink formulations due to its unique physicochemical properties. This study introduces a novel, neutral-soluble, photocrosslinkable collagen maleate (ColME) that is ideal for 3D bioprinting. ColME was synthesized by chemically modifying bovine type I collagen with maleic anhydride, achieving a high substitution ratio that shifted the isoelectric point to enhance solubility in physiological pH environments. This modification was confirmed to preserve the collagen's triple-helix structure substantially. Bioprinting parameters for ColME were optimized, focusing on adjustments to the bioink concentration, extrusion pressure, nozzle speed, and temperature. Results demonstrated that lower temperatures and smaller nozzle sizes substantially improved the print quality of grid structures. Additionally, the application of intermittent photo-crosslinking facilitated the development of structurally robust 3D multilayered constructs, enabling the stable fabrication of complex tissues. Cell viability assays showed that encapsulated cells within the ColME matrix maintained high viability after printing. When compared to methacrylated gelatin, ColME exhibited superior mechanical strength, resistance to enzymatic digestion, and overall printability, positioning it as an outstanding bioink for the creation of durable, bioactive 3D tissues.

Design and evaluation of new wound dressings based on collagen-cellulose derivatives

The purpose of this research was to design and characterize new hydrogels, respectively, their lyophilized forms, spongious matrices, based on collagen and methylcellulose or hydroxyethylcellulose to be used as wound dressings. 1.1% collagen gel was blended with 3% cellulose ethers gels in different ratios. To design the new potential dressings, we evaluated the compatibility of biopolymers through rheological and spectroscopic analyses for hydrogels, respectively spectral, morphological, goniometric, enzymatic, and thermal stability. Rheological measurements indicated that all hydrogels were pseudoplastic, a required property for their manipulation and application. Spectroscopic and spectral analyses showed the conservation of the specific triple helix structure of collagen for samples with 30% cellulose ethers. The morphological evaluation demonstrated that matrices presented hydrophilic and porous structures with proper swelling ability. The thermal and biodegradation study illustrated the increase of matrices’ stability with the addition of cellulose ethers gels. Biocompatibility tests showed that none of the tested matrices had any cytotoxic effect and some compositions of collagen and methylcellulose or hydroxyethylcellulose release factors that might promote fibroblasts’ proliferation and had no inhibitor effect on fibroblasts migration ability; all these results guarantee the development of a promising wound dressing based on 70% collagen gel and 30% cellulose ethers gels.

Probing the impact of alkyl chain length of imidazolium ionic liquids on the conformational stability of collagen type-I from skin of Lutjanus erythropterus

Multispectroscopic techniques and molecular docking study were carried out to inspect the role of concentrations and cationic chain length of ionic liquids (ILs), on the secondary structure of collagen type-I. To attain this objective, a series of imidazolium ILs having same anion (Cl−) and a set of cations with varying chain length were used in this study. The ILs studied utilized are 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]), and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]). The effect of these ILs on collagen type-I was analyzed by using UV-vis, circular dichroism (CD), steady state fluorescence, time resolve fluorescence (TRF), synchronous fluorescence, dynamic light scattering (DLS), fourier transform infrared spectroscopy (FT-IR) and molecular docking approach. The results obtained shows the stability of the collagen was dependent on the alkyl chain length of cation as well as concentration of these ILs. The alteration in hydrogen bonding and hydrophobic interactions due to the different cations of ILs, with the amino acid residues, play a key role in unfolding/destabilization of collagen. Consequently, the destabilization propensity of the ILs toward the collagen increases with increasing chain length of ILs. In addition, when organized in the order of providing destability to collagen triple helix structure, the cations of the ILs obeyed the Hofmeister series. Molecular docking also confirms more hydrophobic interaction and reduced hydrogen bonding due to long alkyl chain length of cations in ILs. The docking results are in agreement with the spectroscopic results and provide the information about the ligand binding site on the protein.

A fluorescent sensing platform based on collagen peptides-protected Au/Ag nanoclusters and WS2 for determining collagen triple helix integrity.

The unique triple helix structure of collagen plays an important role in its biological properties, and the triple helix integrity is closely correlated with its molecular behavior and biological functions. Nevertheless, there is still a lack of convenient, accurate and practical methods for quantitatively determining collagen triple helix integrity. Herein, we first prepared bovine skin collagen peptide (BSCP)-protected Au/Ag nanoclusters (Au/AgNCs@BSCP) with excellent optical properties, high stability and good biocompatibility, which could adsorb on WS2 surface leading to fluorescence quenching. Upon the addition of collagen, the interaction of collagen and Au/AgNCs@BSCP led to the detachment of Au/AgNCs@BSCP from the WS2 surface, causing an increase in the fluorescence signal. Using the difference in the fluorescence recovery of the different samples, we achieved the quantitative determination of collagen triple helix integrity. This developed strategy exhibited excellent accuracy, selectivity, and practicality, thus showing promising potentials in biomedical applications.

Sustainable biomass as green and efficient crosslinkers of collagen: Case of by-products from six pomegranate varieties with global commercial interest in Morocco

Chrome tanning is the most common method used to produce leather, but its environmental and health impacts have necessitated the search for alternatives that have proved to be challenging. The circular economy, which uses biomasses to reduce and recover waste, is seen as a potential solution. In Morocco, fruit waste produced by juice processing industries is an untapped source of tannins that could replace chrome tanning in a more sustainable manner. In this study, a new eco-friendly tanning method based on vegetable biomass has been developed to reduce chromium-related environmental pollution and enhance leather performance. Tanning activities of extracts from six Moroccan pomegranate rind (PR) varieties were investigated, by studying their crosslinking with the insoluble collagen (IC) extracted from a sheep hide, under industrial tanning conditions. Herein, molecular distribution of crosslinkers extracts were carried out using gel exclusion chromatography to follow polymerization degree and crosslinking kinetics during tanning process. Between linear and logarithmic profiles, tannins and phenolics behave differently during the crosslinking depending on their molecular weight. Fourier Transform Infrared Spectroscopy exhibits the existence of inter and intramolecular interaction between IC functional groups and extract. X-Ray Diffraction analysis shows the influence of crosslinkers extracts into the triple helix structure of collagen. Scanning Electron Microscopy observations shows the fibrial size variations of crosslinked collagenous structure by varying the extract variety. Differential Scanning Calorimetry and thermogravimetric measurements revealed that the thermal stabilities were affected by the extracts varieties origins. Theoretical studies using Gaussian was used as a complementally tool to compare the reactivity and the stability of the major molecules that contributes in the crosslinking process. Principal Component Analysis (PCA) was used to examine correlations between the crosslinked IC and PR extracts' analyzed properties. The data set's dimension was reduced to two components via PCA, which accounted for 69.79% of the observed variability.

Design of short peptides and peptide amphiphiles as collagen mimics and an investigation of their interactions with collagen using molecular dynamics simulations and docking studies.

Short peptide sequences and bolaamphiphiles derived from natural proteins are gaining importance due to their ability to form unique nanoscale architectures for a variety of biological applications. In this work, we have designed six short peptides (triplet or monomeric forms) and two peptide bolaamphiphiles that either incorporate the bioactive collagen motif (Gly-X-Y) or sequences where Gly, Pro, or hydroxyproline (Hyp) are replaced by Ala or His. For the bolaamphiphiles, a malate moiety was used as the aliphatic linker for connecting His with Hyp to create collagen mimics. Stability of the assemblies was assessed through molecular dynamics simulations and results indicated that (Pro-Ala-His)3 and (Ala-His-Hyp)3 formed the most stable structures, while the amphiphiles and the monomers showed some disintegration over the course of the 200ns simulation, though most regained structural integrity and formed fibrillar structures, and micelles by the end of the simulation, likely due to the formation of more thermodynamically stable conformations. Multiple replica simulations (REMD) were also conducted where the sequences were simulated at different temperatures. Our results showed excellent convergence in most cases compared to constant temperature molecular dynamics simulation. Furthermore, molecular docking and MD simulations of the sequences bound to collagen triple helix structure revealed that several of the sequences had a high binding affinity and formed stable complexes, particularly (Pro-Ala-His)3 and (Ala-His-Hyp)3. Thus, we have designed new hybrid-peptide-based sequences which may be developed for potential applications as biomaterials for tissue engineering or drug delivery.

Design, Synthesis, and Photo-Responsive Properties of a Collagen Model Peptide Bearing an Azobenzene

Collagen is a vital component of the extracellular matrix in animals. Collagen forms a characteristic triple helical structure and plays a key role in supporting connective tissues and cell adhesion. The ability to control the collagen triple helix structure is useful for medical and conformational studies because the physicochemical properties of the collagen rely on its conformation. Although some photo-controllable collagen model peptides (CMPs) have been reported, satisfactory photo-control has not yet been achieved. To achieve this objective, detailed investigation of the isomerization behavior of the azobenzene moiety in CMPs is required. Herein, two CMPs were attached via an azobenzene linker to control collagen triple helix formation by light irradiation. Azo-(PPG)10 with two (Pro-Pro-Gly)10 CMPs linked via a photo-responsive azobenzene moiety was designed and synthesized. Conformational changes were evaluated by circular dichroism and the cis-to-trans isomerization rate calculated from the absorption of the azobenzene moiety indicated that the collagen triple helix structure was partially disrupted by isomerization of the internal azobenzene.

S2616 Melena: An Uncommon Presentation of Scurvy

Introduction: Scurvy is the clinical manifestation of vitamin C deficiency and presents with hemorrhage (petechiae, ecchymoses, bleeding mucosa), follicular hyperkeratosis, hemolytic anemia, hypochondriasis, hysteria, depression, and fatigue. Scurvy is a rare diagnosis in industrialized countries; however, it can be seen in alcoholic, elderly, and institutionalized patients or those following fad diets or suffering from malabsorptive disorders. The association of scurvy and GI pathology is rare, but it exists. There have been reported cases of scurvy presenting as GI bleed and even as the initial manifestation of Whipple’s disease. Case Description/Methods: A 71-year-old female with past medical history of systemic lupus erythematosus, carcinoid tumor of the small bowel and abnormal bruising presents to the hospital with worsening purpura and intermittent polyarthralgia affecting her feet, ankles, knees, hips, shoulders, and elbows. Labs were significant for anemia of 8.25 and mildly elevated CRP and ESR of 5.9 and 30 respectively. During hospitalization, the patient had multiple black bowel movements and her hemoglobin dropped to 6.8. Iron studies were significant for iron deficiency. She was transfused and underwent EGD and colonoscopy. EGD showed characteristic findings of gastric antral vascular ectasia (Figure 1) which was treated with APC and colonoscopy revealed erythematous mucosa throughout the entire colon (Figure 1). Biopsy of the colon mucosa was unremarkable. The patient was discharged with dermatology follow up for biopsy of purpuric skin lesions. After discharge, patient's labs resulted and showed an undetectable level of Vitamin C. She was seen at follow up by her PCP and treatment was started. Discussion: The water-soluble vitamin C plays a crucial component in collagen synthesis by activating hydrolases that form stabilizing components of the collagen triple-helix structure. When deficient, small vessels in the skin and mucosa become fragile resulting in submucosal hemorrhage in the stomach, duodenum and colon that presents as hematochezia or melena. Treatment consists of dietary modification and intravenous vitamin C replacement with resolution of lesions in only a few days. Vitamin C is replaced daily and generally normalizes in 3-4 weeks. This case demonstrates an unusual presentation of scurvy in the form of GI bleed. It is important for gastroenterologists to consider scurvy in the differential diagnosis of patients presenting with GI bleed, purpura and polyarthralgia.Figure 1.: (A) EGD images of mucosal hemorrhage seen in scurvy (B) Colonoscopy images of mucosal hemorrhage seen in scurvy.

Studying on effects of boiling on texture, microstructure and physiochemical properties of sea cucumber body wall and its mechanism using second harmonic generation (SHG) microscopy

The structural foundation of texture changes in sea cucumber body wall (SCBW) during boiling was investigated using second harmonic generation (SHG) microscopy for the first time. The results from SHG signal imaging, light microscopy, scanning electron microscopy and transmission electron microscopy indicated the hierarchical structures of collagen in SCBW underwent progressive destruction with the prolongation of boiling time, including the depolymerization of collagen fibres, the uncoiling and disaggregation of collagen fibrils, the destruction of collagen microfibrils, the loosing of triple helix structure of collagen, and the degradation and gelatinization of collagen, which contributed to the progressive decline in texture indicators including shear force and hardness. SHG analysis also indicated that although collagen macromolecules such as collagen fibres, collagen fibrils and collagen microfibrils could be observed in 0.5 h-boiled and 2 h-boiled SCBW, monomeric collagen, the basic structural components of those macromolecules, has been already damaged.

Preparation and Characterization of Tilapia Collagen-Thermoplastic Polyurethane Composite Nanofiber Membranes.

Marine collagen is an ideal material for tissue engineering due to its excellent biological properties. However, the limited mechanical properties and poor stability of marine collagen limit its application in tissue engineering. Here, collagen was extracted from the skin of tilapia (Oreochromis nilotica). Collagen-thermoplastic polyurethane (Col-TPU) fibrous membranes were prepared using tilapia collagen as a foundational material, and their physicochemical and biocompatibility were investigated. Fourier transform infrared spectroscopy results showed that thermoplastic polyurethane was successfully combined with collagen, and the triple helix structure of collagen was retained. X-ray diffraction and differential scanning calorimetry results showed relatively good compatibility between collagen and TPU.SEM results showed that the average diameter of the composite nanofiber membrane decreased with increasing thermoplastic polyurethane proportion. The mechanical evaluation and thermogravimetric analysis showed that the thermal stability and tensile properties of Col-TPU fibrous membranes were significantly improved with increasing TPU. Cytotoxicity experiments confirmed that fibrous membranes with different ratios of thermoplastic polyurethane content showed no significant toxicity to fibroblasts; Col-TPU fibrous membranes were conducive to the migration and adhesion of cells. Thus, these Col-TPU composite nanofiber membranes might be used as a potential biomaterial in tissue regeneration.

Integrity of the ECM Influences the Bone Regenerative Property of ECM/Dicalcium Phosphate Composite Scaffolds.

Due to the limitation of clinical autologous bone supply and other issues, the development of bone regeneration materials is still a hot topic. Natural tissue-derived bone repair materials have good biocompatibility and degradability, but their structure and properties are likely to be adversely affected during terminal sterilization. In this study, a composite scaffold consisting of the acellular extracellular matrix and dicalcium phosphate (ECM/DCP) was fabricated and terminally sterilized by γ-ray irradiation. In addition, the ECM/DCP scaffold was saturated with water and was also sterilized by γ-ray irradiation (RX-ECM/DCP). Results showed that the triple helix structure of collagen was better maintained in RX-ECM/DCP than in ECM/DCP. The thermal stability of RX-DCP/ECM was much better than that of ECP/ECM. The in vitro and in vivo performances of both types of scaffolds were also evaluated. The RX-ECM/DCP scaffold exhibited better in vitro bioactivity than that of the ECM/DCP scaffold as evidenced by more mineral formation in the simulated body fluid. In addition, RX-ECM/DCP also induced more effective bone regeneration than the ECM/DCP scaffold did in a rat calvarial defect model. Results sufficiently demonstrated that the addition of water to the scaffold could protect the structure of the ECM/DCP scaffold from being damaged by γ-ray irradiation during the terminal sterilization process. In summary, this study demonstrated a means to protect the ECM structure, which in turn led to the improvement of bone regenerative properties of the materials during γ-ray irradiation of ECM-based bone repair materials.

Bletilla striata polysaccharide modified collagen fiber composite sponge with rapid hemostasis function

Emergencies often result in uncontrollable bleeding, which is thought to be the leading cause of death at the scene of the injured. Among various hemostasis scenarios, collagen fiber (CF) is gradually replacing traditional hemostatic materials due to its superior properties and ease of sourcing from animals. Herein, we use CF and the natural herbaceous Bletilla striata as raw materials to prepare a collagen fiber-oxidized Bletilla striata composite hemostatic sponge (CFOB). During the cross-linking process, the triple helix structure of collagen stays intact, and its porous three-dimensional network structure brings excellent bulkiness and water absorption properties. Experiments show that the optimal amount of sponge CFOB-10, namely oxidized Bletilla striata polysaccharide 0.5 mg/mL and CF 5 mg/mL, only needed 25 ± 4.06 s for hemostasis time in the rat liver hemorrhage model. In addition, CFOB meets the safety performance requirements of cytotoxicity classification standard 0. Therefore, the optimal amount of CFOB is an excellent new hemostatic material with application potential.Graphical

Impact of Collagen Triple Helix Structure on Melanoma Cell Invadopodia Formation and Matrix Degradation upon BRAF Inhibitor Treatment.

A collagen-rich tumor microenvironment (TME) is associated with worse outcomes in cancer patients and contributes to drug resistance in many cancer types. In melanoma, stiff and fibrillar collagen-abundant tissue is observed after failure of therapeutic treatments with BRAF inhibitors. Increased collagen in the TME can affect properties of the extracellular matrix (ECM), including stiffness, adhesiveness, and interaction of integrins with triple helix forming nanostructures. Decoupling these biochemical and biophysical properties of the ECM can lead to a better understanding of how each of these individual properties affect melanoma cancer behavior and drug efficacy. In addition, as drug treatment can induce cancer cell phenotypic switch, cancer cell responsiveness to the TME can be dynamically changed during therapeutic treatments. To investigate cancer cell phenotype changes and the role of the cancer TME, poly(ethylene glycol) (PEG) hydrogels functionalized with collagen mimetic peptides (CMPs) is utilized, or an interpenetrating network (IPN) of type І collagen within the PEG system to culture various melanoma cell lines in the presence or absence of Vemurafenib (PLX4032) drug treatment is prepared. Additionally, the potential of using CMP functionalized PEG hydrogels, which can provide better tunability is explored, to replace the existing invadopodia assay platform based on fluorescent gelatin.

Mineralization of Phosphorylated Fish Skin Collagen/Mangosteen Scaffolds as Potential Materials for Bone Tissue Regeneration.

In this study, a potential hard tissue substitute was mimicked using collagen/mangosteen porous scaffolds. Collagen was extracted from Tilapia fish skin and mangosteen from the waste peel of the respective fruit. Sodium trimetaphosphate was used for the phosphorylation of these scaffolds to improve the nucleation sites for the mineralization process. Phosphate groups were incorporated in the collagen structure as confirmed by their attenuated total reflection Fourier transform infrared (ATR-FTIR) bands. The phosphorylation and mangosteen addition increased the thermal stability of the collagen triple helix structure, as demonstrated by differential scanning calorimetry (DSC) and thermogravimetry (TGA) characterizations. Mineralization was successfully achieved, and the presence of calcium phosphate was visualized by scanning electron microscopy (SEM). Nevertheless, the porous structure was maintained, which is an essential characteristic for the desired application. The deposited mineral was amorphous calcium phosphate, as confirmed by energy dispersive X-ray spectroscopy (EDX) results.

Fabrication and Properties of Electrospun Collagen Tubular Scaffold Crosslinked by Physical and Chemical Treatments.

Tissue engineered scaffold was regarded as a promising approach instead of the autograft. In this study, small diameter electrospun collagen tubular scaffold with random continuous smooth nanofibers was successfully fabricated. However, the dissolution of collagen in concentrated aqueous (conc. aq.) acetic acid caused to the serious denaturation of collagen. A novel method ammonia treatment here was adopted which recovered the collagen triple helix structure according to the analysis of IR spectra. Further dehydrothermal (DHT) and glutaraldehyde (GTA) treatments were applied to introduce the crosslinks to improve the properties of collagen tube. The nanofibrous structure of collagen tube in a wet state was preserved by the crosslinking treatments. Swelling ratio and weight loss decreased by at least two times compared to those of the untreated collagen tube. Moreover, tensile strength was significantly enhanced by DHT treatment (about 0.0076 cN/dTex) and by GTA treatment (about 0.075 cN/dTex). In addition, the surface of crosslinked collagen tube kept the hydrophilic property. These results suggest that DHT and GTA treatments can be utilized to improve the properties of electrospun collagen tube which could become a suitable candidate for tissue engineered scaffold.

Strong inflammation is essential for expression of articular cartilage-specific citrullinated antigens

To investigate the expression of citrullinated epitopes in articular cartilage protein and whether its expression is sufficient to induce anti-citrullinated protein antibody (ACPA) response in mice. The experimental group was treated with different concentrations of lipopolysaccharide (LPS), heat-inactivated bacteria (Escherichia coli and Staphylococcus aureus) or specific monoclonal antibody against type Ⅱ collagen to induce citrullination of articular cartilage protein, with PBS as the control. Immunohistochemistry with the monoclonal antibody ACC4 (IgG1) that specifically binds to the citrullinated epitope of cartilage protein was performed for detecting the expression of citrullinated protein, with ACC1 (IgG2a) as a positive control antibody and L243 (IgG2a) and Hy2.15 (IgG1) as the negative isotype control. In the in vivo experiment, SD rats were subjected to injection of different doses of LPS in the right knee (with PBS as the controls in the left knee), and 3 days later frozen sections were prepared for immunohistochemical detection of the expression of citrullinated protein. Models of collagen-induced arthritis (CIA) established in different mouse strains were observed for incidence and severity of CIA. Serum samples collected from these models and the sera from rheumatoid arthritis patients were examined for anti-citrullinated protein antibody, and immunohistochemistry was performed to detect the expression of citrullinated protein in the cartilage of the mouse. The citrullinated CII epitope-specific antibody ACC4 did not bind to articular cartilage tissues with different treatments as compared with the positive control antibody ACC1. The ACC4 antibody and the antibodies from patients with rheumatoid arthritis with high titers of anti-citrullinated protein antibody were capable of binding to the synovial tissue around the articular cartilage of the CIA. Luminex analysis showed that the anti-citrullinated protein antibody was lowly expressed in mouse serum, but the anti-type Ⅱ collagen triple helix structure peptide antibody exhibited strong reactivity. Mild acute inflammatory response is not enough to cause citrullination of articular cartilage protein, and the expression of specific epitope requires a high-intensity inflammatory response. Inflammatory articular cartilage protein can express citrullinated epitopes in type Ⅱ collagen-induced arthritis in mice, but the expression of citrullinated epitopes is not sufficient to induce an immune response to anti-citrullinated antibodies. Stronger stimulation signals are required to induce an immune response for producing anti-citrullinated protein antibodies.

New Nanofibers Based on Protein By-Products with Bioactive Potential for Tissue Engineering.

Concentrated collagen hydrolysate (HC10CC), rabbit collagen glue (RCG), and keratin hydrolysate (KH) were investigated in terms of their extraction from mammalian by-products and processing by electrospinning. The electrospun nanofibers were characterized by scanning electron microscopy coupled with the energy dispersive X-ray spectroscopy (SEM/EDS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), and indentation tests. The cytotoxicity of the electrospun nanofibers was conducted on L929 fibroblast cells using MTT and LDH assays and cell morphology observations. The electrospun RCG and KH nanofibers morphology showed an average size of nanofibers ranging between 44 and 410 nm, while the electrospun HC10CC nanofibers exhibited higher sizes. The ATR-FTIR spectra performed both on extracted proteins and electrospun nanofibers showed that the triple helix structure of collagen is partially preserved. The results were in agreement with the circular dichroism analysis for protein extracts. Furthermore, the viscoelastic properties of electrospun KH nanofibers were superior to those of electrospun RCG nanofibers. Based on both in vitro quantitative and qualitative analysis, the electrospun nanofibers were not cytotoxic, inducing a healthy cellular response. The results of new electrospun protein-based nanofibers may be useful for further research on bioactive properties of these nanofibers for tissue engineering.