ABSTRACT
RATIONALE: Stable isotope analysis of bone provides insight into animal foraging and allows for ecological reconstructions over time, however pre-treatment is required to isolate collagen. Pre-treatments typically consist of demineralization to remove inorganic components and/or lipid extraction to remove fats, but these protocols can differentially affect stable carbon (δ13C) and nitrogen (δ15N) isotope values depending on the chemicals, tissues, and/or species involved. Species-specific methodologies create a standard for comparability across studies and enhance understanding of collagen isolation from modern cetacean bone. METHODS: Elemental analyzers coupled to isotope ratio mass spectrometers were used to measure the δ13C and δ15N values of powdered killer whale (Orcinus orca) bone that was intact (control) or subjected to one of three experimental conditions: demineralized, lipid-extracted, and both demineralized and lipid-extracted. Additionally, C:N ratios were evaluated as a proxy for collagen purity. Lastly, correlations were examined between control C:N ratios vs. historical age and control C:N ratios vs. sample characteristics. RESULTS: No significant differences in the δ15N values were observed for any of the experimental protocols. However, the δ13C values were significantly increased by all three experimental protocols: demineralization, lipid extraction, and both treatments combined. The most influential protocol was both demineralization and lipid extraction. Measures of the C:N ratios were also significantly lowered by demineralization and both treatments combined, indicating the material was closer to pure collagen after the treatments. Collagen purity as indicated via C:N ratio was not correlated with historical age nor sample characteristics. CONCLUSIONS: If only the δ15N values from killer whale bone are of interest for analysis, no pre-treatment seems necessary. If the δ13C values are of interest, samples should be both demineralized and lipid-extracted. As historical age and specimen characteristics are not correlated with sample contamination, all samples can be treated equally.
Subject(s)
Bone and Bones , Carbon Isotopes , Collagen , Mass Spectrometry , Nitrogen Isotopes , Whale, Killer , Animals , Carbon Isotopes/analysis , Nitrogen Isotopes/analysis , Bone and Bones/chemistry , Mass Spectrometry/methods , Collagen/analysis , Collagen/chemistry , Lipids/analysis , Lipids/chemistryABSTRACT
(+)4-cholesten-3-one has been proved to have potential wound healing effect in the process of wound regeneration. This study aimed to evaluate the effect of (+)4-cholesten-3-one/sodium alginate/gelatin on skin injury and reveal its potential molecular mechanism. First, we prepared sodium alginate/gelatin hydrogel (SA/Gel hydrogel) with different ratios and tested their characteristics. Based on these results, different concentrations of (+)4-cholesten-3-one were added into SA/Gel hydrogel. A full-thickness skin injury model was successfully established to evaluate wound healing activityin vivo. HE staining and Masson staining were used to evaluate the thickness of granulation tissue and collagen deposition level. Immunohistochemical staining and immunofluorescence staining were applied to detect the level of revascularization and proliferation in each group of wounds. Western blot, quantitative-PCR and immunofluorescence staining were used to detect the expression of proteins related to Wnt/ß-catenin signaling pathway in each group of wounds.In vitroresults showed that the hydrogel not only created a 3D structure for cell adhesion and growth, but also exhibited good swelling ability, excellent degradability and favorable bio-compatibility. Most importantly,in vivoexperiments further indicated that (+)4-cholesten-3-one/SA/Gel hydrogel effectively enhanced wound healing. The effectiveness is due to its superior abilities in accelerating healing process, granulation tissue regeneration, collagen deposition, promoting angiogenesis, tissue proliferation, as well as fibroblast activation and differentiation. The underlying mechanism was related to the Wnt/ß-catenin signaling pathway. This study highlighted that (+)4-cholesten-3-one/SA/Gel hydrogel holds promise as a wound healing dressing in future clinical applications.
Subject(s)
Alginates , Gelatin , Hydrogels , Regeneration , Skin , Wound Healing , Wound Healing/drug effects , Alginates/chemistry , Animals , Gelatin/chemistry , Hydrogels/chemistry , Hydrogels/pharmacology , Skin/injuries , Skin/drug effects , Skin/metabolism , Regeneration/drug effects , Cell Proliferation/drug effects , Male , Mice , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Rats , Collagen/chemistry , Wnt Signaling Pathway/drug effects , HumansABSTRACT
Purpose: In this study, wound dressings were designed using zinc-modified marine collagen porous scaffold as host for wild bilberry (WB) leaves extract immobilized in functionalized mesoporous silica nanoparticles (MSN). These new composites were developed as an alternative to conventional wound dressings. In addition to the antibacterial activity of classic antibiotics, a polyphenolic extract could act as an antioxidant and/or an anti-inflammatory agent as well. Methods: Wild bilberry leaves extract was prepared by ultrasound-assisted extraction in ethanol and its properties were evaluated by UV-Vis spectroscopy (radical scavenging activity, total amount of polyphenols, flavonoids, anthocyanins, and condensed tannins). The extract components were identified by HPLC, and the antidiabetic properties of the extract were evaluated via α-glucosidase inhibitory activity. Spherical MSN were modified with propionic acid or proline moieties by post-synthesis method and used as carriers for the WB leaves extract. The textural and structural features of functionalized MSN were assessed by nitrogen adsorption/desorption isotherms, small-angle XRD, SEM, TEM, and FTIR spectroscopy. The composite porous scaffolds were prepared by freeze drying of the zinc-modified collagen suspension containing WB extract loaded silica nanoparticles. Results: The properties of the new composites demonstrated enhanced properties in terms of thermal stability of the zinc-collagen scaffold, without altering the protein conformation, and stimulation of NCTC fibroblasts mobility. The results of the scratch assay showed contributions of both zinc ions from collagen and the polyphenolic extract incorporated in functionalized silica in the wound healing process. The extract encapsulated in functionalized MSN proved enhanced biological activities compared to the extract alone: better inhibition of P. aeruginosa and S. aureus strains, higher biocompatibility on HaCaT keratinocytes, and anti-inflammatory potential demonstrated by reduced IL-1ß and TNF-α levels. Conclusion: The experimental data shows that the novel composites can be used for the development of effective wound dressings.
Subject(s)
Bandages , Collagen , Nanoparticles , Plant Extracts , Plant Leaves , Silicon Dioxide , Wound Healing , Zinc , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plant Leaves/chemistry , Silicon Dioxide/chemistry , Silicon Dioxide/pharmacology , Collagen/chemistry , Collagen/pharmacology , Zinc/chemistry , Zinc/pharmacology , Nanoparticles/chemistry , Wound Healing/drug effects , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Humans , Tissue Scaffolds/chemistry , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Cell Line , Porosity , Fibroblasts/drug effects , Antioxidants/pharmacology , Antioxidants/chemistryABSTRACT
Among all of the materials used in tissue engineering in order to develop bioequivalents, collagen shows to be the most promising due to its superb biocompatibility and biodegradability, thus becoming one of the most widely used materials for scaffold production. However, current imaging techniques of the cells within collagen scaffolds have several limitations, which lead to an urgent need for novel methods of visualization. In this work, we have obtained groups of collagen scaffolds and selected the contrasting agents in order to study pores and patterns of cell growth in a non-disruptive manner via X-ray computed microtomography (micro-CT). After the comparison of multiple contrast agents, a 3% aqueous phosphotungstic acid solution in distilled water was identified as the most effective amongst the media, requiring 24 h of incubation. The differences in intensity values between collagen fibers, pores, and masses of cells allow for the accurate segmentation needed for further analysis. Moreover, the presented protocol allows visualization of porous collagen scaffolds under aqueous conditions, which is crucial for the multimodal study of the native structure of samples.
Subject(s)
Collagen , Tissue Scaffolds , X-Ray Microtomography , Tissue Scaffolds/chemistry , X-Ray Microtomography/methods , Collagen/chemistry , Collagen/metabolism , Tissue Engineering/methods , Animals , Water/chemistry , Porosity , Cell Culture Techniques, Three Dimensional/methods , HumansABSTRACT
PURPOSE: Mitigating unwanted refractive errors is crucial for surgeons to ensure quality vision after penetrating keratoplasty (PK). The primary objective of the present study is to highlight the importance of microstructural matching of the host and the donor cornea during PK on the distribution of the corneal tissue while suturing. METHODS: For this purpose, the present study undertakes an in-vitro PK model to analyse the effect of suturing and host-donor misalignment on corneal birefringence. Five groups of experiments were performed using five corneoscleral buttons. In each group, N = 16 data points (corresponding to 16 simple interrupted sutures) were assessed before and after PK with five degrees of misalignments, 0°, 30°, 45°, 60° and 90° to detect the variations in corneal birefringence post-PK. The technique of digital photoelasticity is utilized to capture the corneal birefringence experimentally. RESULTS: The local and global features of corneal birefringence provided interesting insights into the nuances of corneal birefringence in PK. Statistical analysis was performed to study the effects of suturing on the birefringence around the suture bites. It was observed that the interaction of the suture tension and structural misalignment between the host and the donor cornea influences the corneal birefringence in PK. Conclusions The zero-degree structural misalignment of the host and the donor tissue is preferable to minimize the topographical irregularities and related astigmatism post-PK. The findings of the present study envisage an additional step of structurally aligning the donor tissue with the host before suturing to minimize topographical irregularities in PK.
Subject(s)
Collagen , Cornea , Keratoplasty, Penetrating , Tissue Donors , Keratoplasty, Penetrating/methods , Cornea/surgery , Collagen/chemistry , Sutures/adverse effects , Birefringence , Humans , Suture Techniques , Corneal Topography/methodsABSTRACT
The hierarchic assembly of fibrillar collagen into an extensive and ordered supramolecular protein fibril is critical for extracellular matrix function and tissue mechanics. Despite decades of study, we still know very little about the complex process of fibrillogenesis, particularly at the earliest stages where observation of rapidly forming, nanoscale intermediates challenges the spatial and temporal resolution of most existing microscopy methods. Using video rate scanning atomic force microscopy (VRS-AFM), we can observe details of the first few minutes of collagen fibril formation and growth on a mica surface in solution. A defining feature of fibrillar collagens is a 67-nm periodic banding along the fibril driven by the organized assembly of individual monomers over multiple length scales. VRS-AFM videos show the concurrent growth and maturation of small fibrils from an initial uniform height to structures that display the canonical banding within seconds. Fibrils grow in a primarily unidirectional manner, with frayed ends of the growing tip latching onto adjacent fibrils. We find that, even at extremely early time points, remodeling of growing fibrils proceeds through bird-caging intermediates and propose that these dynamics may provide a pathway to mature hierarchic assembly. VRS-AFM provides a unique glimpse into the early emergence of banding and pathways for remodeling of the supramolecular assembly of collagen during the inception of fibrillogenesis.
Subject(s)
Microscopy, Atomic Force , Single Molecule Imaging , Microscopy, Atomic Force/methods , Single Molecule Imaging/methods , Animals , Extracellular Matrix/metabolism , Fibrillar Collagens/metabolism , Fibrillar Collagens/chemistry , Collagen/metabolism , Collagen/chemistry , Aluminum SilicatesABSTRACT
Corneal defects can lead to stromal scarring and vision loss, which is currently only treatable with a cadaveric corneal transplant. Although in situ-forming hydrogels have been shown to foster regeneration of the cornea in the setting of stromal defects, the cross-linking, biomechanical, and compositional parameters that optimize healing have not yet been established. This, Corneal defects are also almost universally inflamed, and their rapid closure without fibrosis are critical to preserving vision. Here, an in situ forming, bioorthogonally cross-linked, nanocluster (NC)-reinforced collagen and hyaluronic acid hydrogel (NCColHA hydrogel) with enhanced structural integrity and both pro-regenerative and anti-inflammatory effects was developed and tested within a corneal defect model in vivo. The NCs serve as bioorthogonal nanocross-linkers, providing higher cross-linking density than polymer-based alternatives. The NCs also serve as delivery vehicles for prednisolone (PRD) and the hepatocyte growth factor (HGF). NCColHA hydrogels rapidly gel within a few minutes upon administration and exhibit robust rheological properties, excellent transparency, and negligible swelling/deswelling behavior. The hydrogel's biocompatibility and capacity to support cell growth were assessed using primary human corneal epithelial cells. Re-epithelialization on the NCColHA hydrogel was clearly observed in rabbit eyes, both ex vivo and in vivo, with expression of normal epithelial biomarkers, including CD44, CK12, CK14, α-SMA, Tuj-1, and ZO-1, and stratified, multilayered morphology. The applied hydrogel maintained its structural integrity for at least 14 days and remodeled into a transparent stroma by 56 days.
Subject(s)
Hydrogels , Hydrogels/chemistry , Hydrogels/pharmacology , Animals , Rabbits , Hyaluronic Acid/chemistry , Hyaluronic Acid/pharmacology , Cornea/drug effects , Regeneration/drug effects , Humans , Cross-Linking Reagents/chemistry , Collagen/chemistry , Hepatocyte Growth Factor/pharmacology , Hepatocyte Growth Factor/metabolism , Hepatocyte Growth Factor/chemistryABSTRACT
Periodontitis is a bacteria-induced chronic inflammatory disease characterized by degradation of the supporting tissue and bone in the oral cavity. Treatment modalities seek to facilitate periodontal rehabilitation while simultaneously preventing further gingival tissue recession and potentially bone atrophy. The aim of this study was to compare two differently sourced membranes, a resorbable piscine collagen membrane and a porcine-derived collagen membrane, in the repair of soft tissue defects utilizing a preclinical canine model. This in vivo component consisted of 10 beagles which were subjected to bilateral maxillary canine mucogingival flap defects, as well as bilateral soft tissue defects (or pouches) with no periodontal ligament damage in the mandibular canines. Defects received either a piscine-derived dermal membrane, (Kerecis® Oral, Ísafjörður, Iceland) or porcine-derived dermal membrane (Geistlich Mucograft®, Wolhusen, Switzerland) in a randomized fashion (to avoid site bias) and were allowed to heal for 30, 60, or 90 days. Statistical evaluation of tissue thickness was performed using general linear mixed model analysis of variance and least significant difference (LSD) post hoc analyses with fixed factors of time and membrane. Semi-quantitative analysis employed for inflammation assessment was evaluated using a chi-squared test along with a heteroscedastic t-test and values were reported as mean and corresponding 95% confidence intervals. In both the mucogingival flap defects and soft tissue gingival pouches, no appreciable qualitative differences were observed in tissue healing between the membranes. Furthermore, no statistical differences were observed in the thickness measurements between piscine- and porcine-derived membranes in the mucogingival flap defects (1.05 mm [±0.17] and 1.29 mm [±0.17], respectively [p = .06]) or soft tissue pouches (1.36 mm [±0.14] and 1.47 mm [±0.14], respectively [p = .27]), collapsed over time. Independent of membrane source (i.e., piscine or porcine), similar inflammatory responses were observed in both the maxilla and mandible at the three time points (p = .88 and p = .79, respectively). Histologic and histomorphometric evaluation results indicated that both membranes yielded equivalent tissue responses, remodeling dynamics and healing patterns for the mucogingival flap as well as the soft tissue gingival pouch defect models.
Subject(s)
Collagen , Wound Healing , Animals , Dogs , Swine , Collagen/chemistry , Collagen/pharmacology , Membranes, Artificial , Gingiva/pathologyABSTRACT
Collagen from paleontological bones is an important organic material for isotopic measurement, radiocarbon analysis, and paleoproteomic analysis to provide information on diet, dating, taxonomy, and phylogeny. Current paleoproteomic methods are destructive and require from a few milligrams to several tens of milligrams of bone for analysis. In many cultures, bones are raw materials for artifacts that are conserved in museums, which hampers damage to these precious objects during sampling. Here, we describe a low-invasive sampling method that identifies collagen, taxonomy, and post-translational modifications from Holocene and Upper Pleistocene bones dated to 130,000 and 150 BC using dermatological skin tape discs for sampling. The sampled bone micropowders were digested following our highly optimized enhanced filter-aided sample preparation protocol and then analyzed by MALDI FTICR MS and LC-MS/MS for identifying the genus taxa of the bones. We show that this low-invasive sampling does not deteriorate the bones and achieves results similar to those obtained by more destructive sampling. Moreover, this sampling method can be carried out at archeological sites or in museums.
Subject(s)
Bone and Bones , Collagen , Fossils , Paleontology , Proteomics , Bone and Bones/chemistry , Proteomics/methods , Paleontology/methods , Animals , Collagen/chemistry , Collagen/analysis , Archaeology/methods , Specimen Handling/methods , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Protein Processing, Post-Translational , HumansABSTRACT
Selective scleral crosslinking has been proposed as a novel treatment to increase scleral stiffness to counteract biomechanical changes associated with glaucoma and high myopia. Scleral stiffening has been shown by transpupillary peripapillary scleral photocrosslinking in rats, where the photosensitizer, methylene blue (MB), was injected retrobulbarly and red light initiated crosslinking reactions with collagen. Here, we adapted a computational model previously developed to model this treatment in rat eyes to additionally model MB photocrosslinking in minipigs and humans. Increased tissue length and subsequent diffusion and light penetration limitations were found to be barriers to achieving the same extent of crosslinking as in rats. Per cent inspired O2, injected MB concentration and laser fluence were simultaneously varied to overcome these limitations and used to determine optimal combinations of treatment parameters in rats, minipigs and humans. Increasing these three treatment parameters simultaneously resulted in maximum crosslinking, except in rats, where the highest MB concentrations decreased crosslinking. Additionally, the kinetics and diffusion of photocrosslinking reaction intermediates and unproductive side products were modelled across space and time. The model provides a mechanistic understanding of MB photocrosslinking in scleral tissue and a basis for adapting and screening treatment parameters in larger animal models and, eventually, human eyes.
Subject(s)
Sclera , Swine, Miniature , Animals , Rats , Sclera/metabolism , Swine , Humans , Models, Biological , Methylene Blue/chemistry , Collagen/metabolism , Collagen/chemistry , Cross-Linking Reagents , Computer Simulation , Photosensitizing Agents/pharmacologyABSTRACT
Despite the fact that several rheumatoid arthritis treatments have been utilized, none of them achieved complete joint healing and has been accompanied by several side effects that compromise patient compliance. This study aims to provide an effective safe RA treatment with minimum side effects through the encapsulation of melatonin (MEL) in hyalurosomes and loading these hyalurosomes in collagen thermos-sensitive poloxamer 407 (PCO) hydrogels, followed by their intra-articular administration in AIA model rats. In vitro characterization of MEL-hyalurosomes and PCO hydrogel along with in vivo evaluation of the selected formulation were conducted. Particle size, PDI and EE % of the selected formulation were 71.5 nm, 0.09 and 90 %. TEM micrographs demonstrated that the particles had spherical shape with no aggregation signs. Loading PCO hydrogels with MEL-hyalurosomes did not cause significant changes in pH although it increased its viscosity and injection time. FTIR analysis showed that no interactions were noted among the delivery system components. In vivo results revealed the superior effect of MEL-hyalurosomes PCO hydrogel over MEL-PCO hydrogel and blank PCO hydrogels in improving joint healing, cartilage repair, pannus formation and cell infiltrations. Also, MEL-hyalurosomes PCO hydrogel group showed comparable levels of TNF-α, IL1, MDA, NRF2 and HO-1 with the negative control group. These findings highlight the MEL encapsulation role in augmenting its pharmacological effects along with the synergistic effect of hyaluronic acid in hyalurosomes and collagen in PCO hydrogel in promoting joint healing.
Subject(s)
Arthritis, Rheumatoid , Collagen , Hydrogels , Melatonin , Poloxamer , Animals , Melatonin/administration & dosage , Melatonin/chemistry , Melatonin/pharmacology , Hydrogels/chemistry , Hydrogels/administration & dosage , Collagen/chemistry , Arthritis, Rheumatoid/drug therapy , Poloxamer/chemistry , Male , Injections, Intra-Articular , Rats , Arthritis, Experimental/drug therapy , Rats, Wistar , Temperature , Particle Size , Drug Carriers/chemistryABSTRACT
The protein-polysaccharide nanofibers have attracted intensive attention in promoting wound healing, due to their components and nanoscale fibrous structure that mimics the native extracellular matrix (ECM). For the full-thickness wounds, in addition to promoting healing, hemostatic property and antibacterial activity are also of critical importance. However, currently, protein-polysaccharide-based nanofiber membranes exhibit poor mechanical properties, lack inherent hemostatic and antibacterial capabilities, as well as the ability to promote tissue repair. In this study, we developed composited membranes, which were composed of collagen (Col) and chitosan (Chs), through solvent alteration and post-processing, the membranes showed enhanced stability under physiological conditions, proper hydrophilic performance and improved mechanical property. Appropriated porosity and water vapor transmission rate, which benefit to wound healing, were detected among all the membranes except for Col membrane. Aimed at wound dressing, hemocompatibility, antibacterial activity and cell proliferation of the electrospun membranes were evaluated. The results indicated that the Col/Chs composited membranes exhibited superior blood clotting capacity, and the membranes with Chs exceeding 60% possessed sufficient antibacterial activity. Moreover, compared with Chs nanofibers, significant increase in cell grow was detected in Col/Chs (1:3) membrane. Taken together, the electrospun membrane with multiple properties favorable to wound healing, superior blood coagulation, sufficient antibacterial performance and promoting cell proliferation property make it favorable candidate for full-thickness skin wound healing.
Subject(s)
Anti-Bacterial Agents , Biocompatible Materials , Cell Proliferation , Chitosan , Collagen , Materials Testing , Membranes, Artificial , Nanofibers , Wound Healing , Chitosan/chemistry , Wound Healing/drug effects , Nanofibers/chemistry , Collagen/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cell Proliferation/drug effects , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Humans , Bandages , Animals , Porosity , Blood Coagulation/drug effects , Mice , Extracellular Matrix/metabolismABSTRACT
Lipid-based nanocarriers have been extensively utilized for the solubilization and cutaneous delivery of water-insoluble active ingredients in skincare formulations. However, their practical application is often limited by structural instability, leading to premature release and degradation of actives. Here we present highly robust multilamellar nanovesicles, prepared by the polyionic self-assembly of unilamellar vesicles with hydrolyzed collagen peptides, to stabilize all-trans-retinol and enhance its cutaneous delivery. Our results reveal that the reinforced multilayer structure substantially enhances dispersion stability under extremely harsh conditions, like freeze-thaw cycles, and stabilizes the encapsulated retinol. Interestingly, these multilamellar vesicles exhibit significantly lower cytotoxicity to human dermal fibroblasts than their unilamellar counterparts, likely due to their smaller particle number per weight, minimizing potential disruptions to cellular membranes. In artificial skin models, retinol-loaded multilamellar vesicles effectively upregulate collagen-related gene expression while suppressing the synthesis of metalloproteinases. These findings suggest that the robust multilamellar vesicles can serve as effective nanocarriers for the efficient delivery and stabilization of bioactive compounds in cutaneous applications.
Subject(s)
Administration, Cutaneous , Collagen , Fibroblasts , Lipids , Nanoparticles , Vitamin A , Vitamin A/administration & dosage , Vitamin A/chemistry , Humans , Collagen/chemistry , Lipids/chemistry , Nanoparticles/chemistry , Fibroblasts/drug effects , Drug Stability , Skin/metabolism , Drug Carriers/chemistry , Cell Survival/drug effects , Skin Absorption , Skin, ArtificialABSTRACT
Many bones experience bending, placing one side in net compression and the other in net tension. Because bone mechanical properties are relatively reduced in tension compared with compression, adaptations are needed to reduce fracture risk. Several toughening mechanisms exist in bone, yet little is known of the influences of secondary osteon collagen/lamellar 'morphotypes' and potential interplay with intermolecular collagen cross-links (CCLs) in prevalent/predominant tension- and compression-loaded regions. Paired third metacarpals (MC3s) from 10 adult horses were prepared for mechanical testing. From one MC3/pair, 5â mm cubes were tested in compression at several mid-shaft locations. From contralateral bones, dumbbell-shaped specimens were tested in tension. Hence, habitual/natural tension- and compression-loaded regions were tested in both modes. Data included: elastic modulus, yield and ultimate strength, and energy absorption (toughness). Fragments of tested specimens were examined for predominant collagen fiber orientation (CFO; representing osteonal and non-osteonal bone), osteon morphotype score (MTS, representing osteonal CFO), mineralization, porosity and other histological characteristics. As a consequence of insufficient material from tension-tested specimens, CCLs were only examined in compression-tested specimens (HP, hydroxylysylpyridinoline; LP, lysylpyridinoline; PE, pentosidine). Among CCLs, only LP and HP/LP correlated significantly with mechanical parameters: LP with energy absorption, HP/LP with elastic modulus (both r=0.4). HP/LP showed a trend with energy absorption (r=-0.3, P=0.08). HP/LP more strongly correlated with osteon density and mineralization than CFO or MTS. Predominant CFO more strongly correlated with energy absorption than MTS in both testing modes. In general, CFO was found to be relatively prominent in affecting regional toughness in these equine MC3s in compression and tension.
Subject(s)
Collagen , Haversian System , Metacarpal Bones , Animals , Horses/physiology , Collagen/chemistry , Collagen/metabolism , Metacarpal Bones/physiology , Metacarpal Bones/anatomy & histology , Metacarpal Bones/chemistry , Haversian System/physiology , Biomechanical Phenomena , Compressive Strength , Stress, Mechanical , Elastic ModulusABSTRACT
Objective: Collagen, a widely used natural biomaterial polymer in skin tissue engineering, can be innovatively processed into nanocollagen through cryogenic milling to potentially enhance skin tissue healing. Although various methods for fabricating nanocollagen have been documented, there is no existing study on the fabrication of nanocollagen via cryogenic milling, specifically employing graphene oxide as separators to prevent agglomeration. Methods: In this study, three research groups were created using cryogenic milling: pure nanocollagen (Pure NC), nanocollagen with 0.005% graphene oxide (NC + 0.005% GO), and nanocollagen with 0.01% graphene oxide (NC+0.01% GO). Characterization analyses included transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, x-ray diffraction (XRD), zeta potential (ZP), and polydispersity index (PDI). Results: TEM and SEM analysis revealed that nanocollagen groups alone exhibited particle sizes of less than 100 nm. FTIR spectroscopic investigations indicated the presence of amide A, B, and I, II, and III (1800 to 800 cm-1) in all nanocollagen study groups, with the characteristic C-O-C stretching suggesting the incorporation of graphene oxide (GO). XRD data exhibited broadening of the major peak as the proportion of GO increased from pure NC to the nanocollagen groups with GO. Zeta potential measurements indicated electrostatic attraction of the samples to negatively charged surfaces, accompanied by sample instability. PDI results depicted size diameters ranging from 800 to 1800 nm, indicating strong polydispersity with multiple size populations. Conclusion: This research demonstrated that collagen can be successfully fabricated into nanoparticles with sizes smaller than 100 nm.
Subject(s)
Collagen , Graphite , Particle Size , Graphite/chemistry , Collagen/chemistry , Spectroscopy, Fourier Transform Infrared , Biocompatible Materials/chemistry , X-Ray Diffraction , Tissue Engineering/methods , Microscopy, Electron, Scanning , Microscopy, Electron, TransmissionABSTRACT
BACKGROUND: A strong seal of soft-tissue around dental implants is essential to block pathogens from entering the peri-implant interface and prevent infections. Therefore, the integration of soft-tissue poses a challenge in implant-prosthetic procedures, prompting a focus on the interface between peri-implant soft-tissues and the transmucosal component. The aim of this study was to analyse the effects of sandblasted roughness levels on in vitro soft-tissue healing around dental implant abutments. In parallel, proteomic techniques were applied to study the interaction of these surfaces with human serum proteins to evaluate their potential to promote soft-tissue regeneration. RESULTS: Grade-5 machined titanium discs (MC) underwent sandblasting with alumina particles of two sizes (4 and 8 µm), resulting in two different surface types: MC04 and MC08. Surface morphology and roughness were characterised employing scanning electron microscopy and optical profilometry. Cell adhesion and collagen synthesis, as well as immune responses, were assessed using human gingival fibroblasts (hGF) and macrophages (THP-1), respectively. The profiles of protein adsorption to the surfaces were characterised using proteomics; samples were incubated with human serum, and the adsorbed proteins analysed employing nLC-MS/MS. hGFs exposed to MC04 showed decreased cell area compared to MC, while no differences were found for MC08. hGF collagen synthesis increased after 7 days for MC08. THP-1 macrophages cultured on MC04 and MC08 showed a reduced TNF-α and increased IL-4 secretion. Thus, the sandblasted topography led a reduction in the immune/inflammatory response. One hundred seventy-six distinct proteins adsorbed on the surfaces were identified. Differentially adsorbed proteins were associated with immune response, blood coagulation, angiogenesis, fibrinolysis and tissue regeneration. CONCLUSIONS: Increased roughness through MC08 treatment resulted in increased collagen synthesis in hGF and resulted in a reduction in the surface immune response in human macrophages. These results correlate with the changes in protein adsorption on the surfaces observed through proteomics.
Subject(s)
Fibroblasts , Macrophages , Surface Properties , Humans , Fibroblasts/metabolism , Fibroblasts/cytology , Macrophages/metabolism , Macrophages/cytology , Dental Abutments , Titanium/chemistry , Gingiva/cytology , Gingiva/metabolism , Proteomics , Cell Adhesion , Collagen/metabolism , Collagen/chemistry , AdsorptionABSTRACT
Contrary to the initial belief that myofibroblasts are terminally differentiated cells, myofibroblasts have now been widely recognized as an activation state that is reversible. Therefore, strategies targeting myofibroblast to be a quiescent state may be an effective way for antihypertrophic scar therapy. Graphene quantum dots (GQDs), a novel zero-dimensional and carbon-based nanomaterial, have recently garnered significant interest in nanobiomedicine, owing to their excellent biocompatibility, tunable photoluminescence, and superior physiological stability. Although multiple nanoparticles have been used to alleviate hypertrophic scars, a GQD-based therapy has not been reported. Our in vivo studies showed that GQDs exhibited significant antiscar efficacy, with scar appearance improvement, collagen reduction and rearrangement, and inhibition of myofibroblast overproliferation. Further in vitro experiments revealed that GQDs inhibited α-SMA expression, collagen synthesis, and cell proliferation and migration, inducing myofibroblasts to become quiescent fibroblasts. Mechanistic studies have demonstrated that the effect of GQDs on myofibroblast proliferation blocked cell cycle progression by disrupting the cyclin-CDK-E2F axis. This study suggests that GQDs, which promote myofibroblast-to-fibroblast transition, could be a novel antiscar nanomedicine for the treatment of hypertrophic scars and other types of pathological fibrosis.
Subject(s)
Cell Proliferation , Cicatrix, Hypertrophic , Graphite , Myofibroblasts , Quantum Dots , Quantum Dots/chemistry , Myofibroblasts/drug effects , Myofibroblasts/pathology , Myofibroblasts/metabolism , Graphite/chemistry , Graphite/pharmacology , Cicatrix, Hypertrophic/drug therapy , Cicatrix, Hypertrophic/pathology , Cell Proliferation/drug effects , Animals , Humans , Mice , Collagen/chemistry , Cell Movement/drug effectsABSTRACT
Skin aging, characterized by reduced regeneration, chronic inflammation, and heightened skin cancer risk, poses a significant challenge. Collagen fillers have emerged as a potential solution for skin rejuvenation by stimulating collagen regeneration. However, their clinical efficacy is limited by inherent instability and vulnerability toin vivodegradation by collagenase. Chemical cross-linking presents a promising approach to enhance stability, but it carries risks such as cytotoxicity, calcification, and discoloration. Here, we introduce a highly durable 1,4-butanediol diglycidyl ether (BDDE) cross-linked collagen filler for skin rejuvenation. BDDE effectively cross-links collagen, resulting in fillers with exceptional mechanical strength and injectability. These fillers demonstrate favorable stability and durability, promoting proliferation, adhesion, and spreading of human foreskin fibroblast-1 cellsin vitro. In vivostudies confirm enhanced collagen regeneration without inducing calcification. BDDE cross-linked collagen fillers offer promising prospects for medical cosmetology and tissue regeneration.
Subject(s)
Butylene Glycols , Cell Proliferation , Collagen , Cross-Linking Reagents , Fibroblasts , Rejuvenation , Skin Aging , Skin , Humans , Collagen/chemistry , Butylene Glycols/chemistry , Cross-Linking Reagents/chemistry , Fibroblasts/metabolism , Skin Aging/drug effects , Animals , Cell Proliferation/drug effects , Skin/metabolism , Dermal Fillers/chemistry , Biocompatible Materials/chemistry , Materials Testing , Regeneration , Epoxy Compounds/chemistry , Male , Cell Adhesion , Tissue Engineering/methods , MiceABSTRACT
Liver fibrosis, a major global health issue, is marked by excessive collagen deposition that impairs liver function. Noninvasive methods for the direct visualization of collagen content are crucial for the early detection and monitoring of fibrosis progression. This study investigates the potential of spectral photoacoustic imaging (sPAI) to monitor collagen development in liver fibrosis. Utilizing a novel data-driven superpixel photoacoustic unmixing (SPAX) framework, we aimed to distinguish collagen presence and evaluate its correlation with fibrosis progression. We employed an established diethylnitrosamine (DEN) model in rats to study liver fibrosis over various time points. Our results revealed a significant correlation between increased collagen photoacoustic signal intensity and advanced fibrosis stages. Collagen abundance maps displayed dynamic changes throughout fibrosis progression. These findings underscore the potential of sPAI for the noninvasive monitoring of collagen dynamics and fibrosis severity assessment. This research advances the development of noninvasive diagnostic tools and personalized management strategies for liver fibrosis.
Subject(s)
Collagen , Liver Cirrhosis , Photoacoustic Techniques , Photoacoustic Techniques/methods , Animals , Liver Cirrhosis/diagnostic imaging , Liver Cirrhosis/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/metabolism , Collagen/metabolism , Collagen/chemistry , Rats , Liver/diagnostic imaging , Liver/pathology , Liver/metabolism , Male , Diethylnitrosamine/toxicity , Disease Models, AnimalABSTRACT
Cancer metastasis starts from early local invasion, during which tumor cells detach from the primary tumor, penetrate the extracellular matrix (ECM), and then invade neighboring tissues. However, the cellular mechanics in the detaching and penetrating processes have not been fully understood, and the underlying mechanisms that influence cell polarization and migration in the 3D matrix during tumor invasion remain largely unknown. In this study, we employed a dual tumor-spheroid model to investigate the cellular mechanisms of the tumor invasion. Our results revealed that the tensional force field developed by the active contraction of cells and tissues played a pivotal role in tumor invasion, acting as the driving force for remodeling the collagen fibers during the invasion process. The remodeled collagen fibers promoted cell polarization and migration because of the stiffening of the fiber matrix. The aligned fibers facilitated tumor cell invasion and directed migration from one spheroid to the other. Inhibiting/shielding the cellular contractility abolished matrix remodeling and re-alignment and significantly decreased tumor cell invasion. By developing a coarse-grained cell model that considers the mutual interaction between cells and fibers, we predicted the tensional force field in the fiber network and the associated cell polarization and cell-matrix interaction during cell invasion, which revealed a mechano-chemical coupling mechanism at the cellular level of the tumor invasion process. Our study highlights the roles of cellular mechanics at the early stage of tumor metastasis and may provide new therapeutic strategies for cancer therapy.