Collagen Thin Films Research Articles

Formation of linear arrays of holes in self-assembled collagen films

Collagen is one of the main constituents of mammalian extracellular matrix and is used extensively as a coating for tissue culture dishes and medical implants to promote cell growth and proliferation. By modulating the topography of the collagen coating at the nanometer to micrometer length scales, it is possible to achieve spatial control over cell growth and morphology. In this work, we are exploring the self-assembly of a thin collagen film on a glass substrate as a way to create new nanoscale surface features. By controlling the collagen concentration and adding an oscillatory flow, we are able to enrich the collagen film surface with a localized pattern of ripples oriented perpendicular to the flow direction. We propose that these ripples are the result of dewetting of the collagen film that leads to the formation of adjacent holes. We observe that individual holes form with an anisotropic rim due to the microstructure of the deposited collagen fibril network. This intrinsic anisotropy and the oscillatory flow yield new holes being formed in the film next to existing rims. As holes keep growing deeper, the rims extend along the flow direction, and the holes appear rectangular in shape, which gives the linear array of holes the apparent morphology of a ripple. Overall, we are able to create localized ripples at the surface of collagen films that would be difficult to produce via standard nanofabrication techniques.

Fabrication of Porous Collagen Scaffolds Containing Embedded Channels with Collagen Membrane Linings.

Tissues and organs contain an extracellular matrix (ECM). In the case of blood vessels, endothelium cells are anchored to a specialized basement membrane (BM) embedded inside the interstitial matrix (IM). We introduce a multi-structural collagen-based scaffold with embedded microchannels that mimics in vivo structures within vessels. Our scaffold consists of two parts, each containing two collagen layers, i.e., a 3D porous collagen layer analogous to IM lined with a thin 2D collagen film resembling the BM. Enclosed microchannels were fabricated using contact microprinting. Microchannel test structures with different sizes ranging from 300 to 800 µm were examined for their fabrication reproducibility. The heights and perimeters of the fabricated microchannels were ~20% less than their corresponding values in the replication PDMS mold; however, microchannel widths were significantly closer to their replica dimensions. The stiffness, permeability, and pore size properties of the 2D and 3D collagen layers were measured. The permeability of the 2D collagen film was negligible, making it suitable for mimicking the BM of large blood vessels. A leakage test at various volumetric flow rates applied to the microchannels showed no discharge, thereby verifying the reliability of the proposed integrated 2D/3D collagen parts and the contact printing method used for bonding them in the scaffold. In the future, multi-cell culturing will be performed within the 3D porous collagen and against the 2D membrane inside the microchannel, hence preparing this scaffold for studying a variety of blood vessel-tissue interfaces. Also, thicker collagen scaffold tissues will be fabricated by stacking several layers of the proposed scaffold.

The Influence of Various Crosslinking Conditions of EDC/NHS on the Properties of Fish Collagen Film.

The process of crosslinking improves the physicochemical properties of biopolymer-based composites, making them valuable for biomedical applications. EDC/NHS-crosslinked collagen materials have a significant potential for tissue engineering applications, due to their enhanced properties and biocompatibility. Chemical crosslinking of samples can be carried out in several ways, which is crucial and has a direct effect on the final properties of the obtained material. In this study, the effect of crosslinking conditions on the properties of collagen films using EDC and NHS was investigated. Studies included FTIR spectroscopy, AFM, swelling and degradation tests, mechanical testing and contact angle measurements. Evaluation of prepared collagen films indicated that both crosslinking agents and crosslinking conditions influenced film properties. Notable alternations were observed in the infrared spectrum of the sample, to which EDC was added directly to the fish collagen solution. The same sample indicated the lowest Young modulus, tensile strength and breaking force parameters and the highest elongation at break. All samples reached the maximum swelling degree two hours after immersion in PBS solution; however, the immersion-crosslinked samples exhibited a significantly lower degree of swelling and were highly durable. The highest roughness was observed for the collagen film crosslinked with EDC, whereas the lowest was observed for the specimen crosslinked with EDC with NHS addition. The crosslinking agents increased the surface roughness of the collagen film, except for the sample modified with the addition of EDC and NHS mixture. All films were characterized by hydrophilic character. The films' modification resulted in a decrease in their hydrophilicity and wettability. Our research allows for a comparison of proposed EDC/NHS crosslinking conditions and their influence on the physicochemical properties of fish collagen thin films. EDC and NHS are promising crosslinking agents for the modification of fish collagen used in biomedical applications.

Formulation and Evaluation of Fish Collagen Extract into Thin Films

Collagen is a macromolecule of connective tissues in animals and it is cosmopolitan in skin, bones, teeth, tendons, ligament, cartilage, eyes, blood vessels, gut and inter-vertebral discs. Collagen is attracting attention as a biomaterial with several properties such as inducing natural process of blood platelets, cell differentiation, and wound healing, through physical and chemical modifications. The present study was aimed to extract collagen from fish skin and formulate into collagen thin films. Fish collagen thin film was prepared using solvent casting method by using gelatin, sodium alginate, CMC, PEG and ferric oxide, and the formulations were named as F1 to F5. The prepared thin films were cut into strips of area 2X2 cm2 and evaluated for organoleptic evaluations, film weight, thickness, folding endurance, drug content of films, surface pH, disintegration time, in-vitro dissolution studies, moisture loss and uptake and stability studies. The results of the analysis parameters such as weight, thickness, percentage transparency, swelling property, surface pH and folding endurance depends on the concentration of sodium alginate and PEG used. Accelerated stability studies have not shown much remarkable changes in the reading of weight, disintegration time and surface pH, thus it indicates that temperature would not alter the properties of the thin film. From the results of percentage drug release collagen is released fast from F3 (60% in 10 min), and the total drug release was obtained in a span of 40 min, this formulation can be used for the preparation of thin films and F3 total drug release was obtained in 60 min

A Collagen Thin Film-Based Bioimpedance Sensor for Cell Proliferation Rate Assessment

Collagen is the main structural protein in the human body and plays an important role in different cellular behaviors like proliferation, migration, and differentiation. It is important to study different cellular characteristics on a natural substrate similar to the extracellular matrix (ECM) in the body. In this paper, a bioimpedance sensor was used to measure the impedance behavior of biological cells cultured on a scaffold of collagen thin films. The collagen thin films with different concentrations were created on a 1-hexadecanethiol modified surface of the interdigitated electrodes (IDEs). The mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were cultured on the collagen thin films. The interaction and the proliferation rate of these cells were investigated by the microscopic, and bioimpedance approaches. Impedance measurements of the cells cultured on the collagen thin film were performed by an impedance analyzer at the frequency band of 10 kHz to 1MHz at 10 mV. Differential impedances (ΔZ) were used as a quantitative criterion to evaluate cellular proliferation on collagen thin films. Results showed that collagen thin films with lower concentration lead to higher proliferation rate and vice versa.

Microstructured collagen films for 3D corneal stroma modelling

ABSTRACT Purpose/aim Corneal injury is a major cause of impaired vision around the globe. The fine structure of the corneal stroma plays a pivotal role in the phenotype and behavior of the embedded cells during homeostasis and healing after trauma or infection. In order to study healing processes in the cornea, it is important to create culture systems that functionally mimic the natural environment. Materials and methods Collagen solution was vitrified on top of a grated film to achieve thin collagen films with parallel microgrooves. Keratocytes (corneal stromal cells) were cultured on the films either as a single layer or as stacked layers of films and cells. SEM and F-actin staining were used to analyze the pattern transference onto the collagen and the cell orientation on the films. Cell viability was analyzed with MTS and live/dead staining. Keratocytes, fibroblasts, and myofibroblasts were cultured to study the pattern’s effect on phenotype. Results A microstructured collagen film-based culture system that guides keratocytes (stromal cells) to their native, layerwise perpendicular orientation in 3D and that can support fibroblasts and myofibroblasts was created. The films are thin and transparent enough to observe cells at least three layers deep. The cells maintain viability in 2D and 3D cultures and the films can support fibroblast and myofibroblast phenotypes. Conclusions The films provide an easily reproducible stroma model that maintains high cell viability and improves the preservation of the keratocyte phenotype in keratocytes that are differentiated to fibroblasts.

Physicochemical Performance of Collagen Modified by Melissa officinalis Extract

Collagen-based materials are widely used as adhesives in medicine and cosmetology. However, for several applications, their properties require modification. In this work, the influence of Melissa officinalis on the properties of collagen films was studied. Collagen was extracted from Silver Carp skin. Thin collagen films were prepared by solvent evaporation. The structure of films was researched using infrared spectroscopy. The surface properties of films were investigated using Atomic Force Microscopy (AFM). Mechanical properties were measured as well. Antioxidant activity was determined by spectrophotometric methods using DPPH free radicals, FRAP, and CUPRAC methods. Total phenolic compounds were determined by the Folin–Ciocalteau method. It was found that the addition of Melissa officinalis modified the roughness of collagen films and their mechanical properties. Moreover, the obtained material has antioxidant properties. The parameters mentioned above are very important in potential applications of collagen films containing Melissa officinalis in cosmetics.

The Infuence of Salicin on Rheological and Film-Forming Properties of Collagen.

Collagen films are widely used as adhesives in medicine and cosmetology. However, its properties require modification. In this work, the influence of salicin on the properties of collagen solution and films was studied. Collagen was extracted from silver carp skin. The rheological properties of collagen solutions with and without salicin were characterized by steady shear tests. Thin collagen films were prepared by solvent evaporation. The structure of films was researched using infrared spectroscopy. The surface properties of films were investigated using Atomic Force Microscopy (AFM). Mechanical properties were measured as well. It was found that the addition of salicin modified the roughness of collagen films and their mechanical and rheological properties. The above-mentioned parameters are very important in potential applications of collagen films containing salicin.

Methods for the Assembly and Characterization of Polyelectrolyte Multilayers as Microenvironments to Modulate Human Mesenchymal Stromal Cell Response.

Thin films are of interest in materials design because they allow for the modification of surface properties of materials while the bulk properties of the material are largely unaffected. In this work, we outline methods for the assembly of thin films using a technique known as layer-by-layer (LbL). Furthermore, their interactions with human mesenchymal stromal cells (hMSCs) are discussed. hMSCs are a subject of growing interest because of their potential to treat or cure diseases, given their immunosuppressive properties, multipotent differentiation capabilities, and tissue regeneration capabilities. Numerous improvements and modifications have been suggested for the harvesting, treatment, and culture of hMSCs prior to their administration in human subjects. Here, we discuss methods to assess the interactions of hMSCs with thin LbL-assembled films of heparin and collagen. Three different methods are discussed. The first details the preparation of heparin/collagen multilayers on different surfaces and the seeding of cells on these multilayers. The second method details the characterization of multilayers, including techniques to assess the thickness, roughness, and surface charge of the multilayers, as well as in situ deposition of multilayers. The third method details the analysis of cell interactions with the multilayers, including techniques to assess proliferation, viability, real-time monitoring of hMSC behavior, analysis of hMSC-adhesive proteins on the multilayers, immunomodulatory factor expression of hMSCs, and cytokine expression on heparin/collagen multilayers. We propose that the methods described in this work will assist in the design and characterization of LbL-assembled thin films and the analysis of hMSCs cultured on these thin films.

Measuring and assessment of impedance spectrum of collagen thin films in the presence of deionized water

Collagen is the major structural protein in the human body, which plays a significant role in retaining the structural and biological integrity of the extracellular matrix. In this study, the electrical impedance of collagen thin films on gold electrodes on an FR4 substrate was measured in dry and humid states. The magnitude and phase of the measured electrical impedances showed a significant difference (P < 0.05) in the dry and wet conditions. By adding deionized water to the dry collagen thin films, the magnitude of the electrical impedance decreased, indicating an increase in conductance. It can be reasonably inferred that chemical and electrical interactions between the water molecules and the functional groups present in the collagen molecules such as the hydroxyl group are responsible for the increase in the conductance. After adding a certain amount of water, the impedance reduction trend is reversed and an increase is observed. This could be due to the saturation of chemical interactions between the collagen and water molecules: by adding more deionized water to the medium, there is a greater number of free water molecules in the medium, reducing the conductance of the medium and the impedance increases by decreasing concentrations of the ions which are produced from interactions between water and collagen molecules. The quantitative study of this finding has been performed with the help of electrical impedance spectroscopy. Finally, an electrical model was fitted to the measured data which describes the impedance behavior of collagen thin films in the vicinity of water molecules.

Deposition of Cell Culture Coatings Using a Cold Plasma Deposition Method

Collagen coatings were applied onto polystyrene microplates using a cold atmospheric pressure plasma process. The coatings were compared to standard wet chemical collagen thin films using microscopy, surface energy, infra-red spectroscopy, electrophoresis, and cell culture techniques. Thin films were also deposited on gold electrodes using both coating methods and their structural and barrier properties probed using cyclic voltammetry. While the wet chemical technique produced a thicker deposit, both films appear equivalent in terms of coverage, porosity, structure, and chemistry. Significantly, the cold plasma method preserves both the primary and secondary structure of the protein and this results in high biocompatibility and cell activity that is at least equivalent to the standard wet chemical technique. The significance of these results is discussed in relation to the benefits of a single step plasma coating in comparison to the traditional multi-step aseptic coating technique.

Modification of Collagen Properties with Ferulic Acid.

Collagen materials are widely used in biomedicine and in cosmetics. However, their properties require improvement for several reasons. In this work, collagen solution as well as collagen films were modified by the addition of ferulic acid (FA). Thin collagen films containing FA were obtained by solvent evaporation. The properties of collagen solution have been studied by steady shear tests. The structure and surface properties of collagen thin films were studied. It was found that for collagen solution with 5% addition of FA, the apparent viscosity was the highest, whereas the collagen solutions with other additions of FA (1%, 2%, and 10%), no significant difference in the apparent viscosity was observed. Thin films prepared from collagen with 1 and 2% FA addition were homogeneous, whereas films with 5% and 10% FA showed irregularity in the surface properties. Mechanical properties, such as maximum tensile strength and elongation at break, were significantly higher for films with 10% FA than for films with smaller amount of FA. Young modulus was similar for films with 1% and 10% FA addition, but bigger than for 2% and 5% of FA in collagen films. The cross-linking of collagen with ferulic acid meant that prepared thin films were elastic with better mechanical properties than collagen films.

Investigation of process to form collagen thin film with aligned collagen fiber.

Investigation of process to form collagen thin film with aligned collagen fiber.

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve.

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

Development of Young’s modulus for collagen thin films reinforced with ZnO nanorods probed by Atomic force microscopy

Zinc Oxide (ZnO) nanorods have been prepared and proposed for collagen Type I reinforcement. The insertion of ZnO has been confirmed by an x-ray Diffraction pattern. A nano-indentation technique implemented through an Atomic Force Microscopy has been effectively handled to probe a mechanical behavior of soft tissue like collagen. Measurements at different temperatures for collagen thin films filled with different amounts of ZnO reveals that ZnO significantly develops Young’s modulus from 9 GPa to 18 GPa. Without ZnO, the temperature increment leads to the dehydration of pure collagen while the reinforced ones were still elastic for cell growth. ZnO-reinforced collagen has, therefore, been improved both inherent rigidity and its stability against ambient temperature. The finding in this work suggests that nanoscale ZnO is good candidate for collagen reinforcement which is crucial for tissue engineering technology.

Artificial sensory organs: latest progress.

This study introduces the latest progress on the study of artificial sensory organs, with a special emphasis on the clinical results of artificial nerves and the concept of in situ tissue engineering. Peripheral nerves have a strong potential for regeneration. An artificial nerve uses this potential to recover a damaged peripheral nerve. The polyglycolic acid collagen tube (PGA-C tube) is a bio-absorbable tube stuffed with collagen of multi-chamber structure that consists of thin collagen films. The clinical application of the PGA-C tube began in 2002 in Japan. The number of PGA-C tubes used is now beyond 300, and satisfactory results have been reported on peripheral nerve repairs. This PGA-C tube is also effective for patients suffering from neuropathic pain.

Atomic Force Microscopy for Collagen-Based Nanobiomaterials

Novel nanobiomaterials are increasingly gaining ground in bioengineering research. Among the numerous biomaterials, collagen-nanobiomaterials, such as collagen thin films, are of great interest since they present a wide range of applications in the fields of biomaterials, tissue engineering, and biomedicine. Collagen type I is the most abundant protein within extracellular matrix and, due to its unique characteristics, is widely used as biomaterial. A thorough characterization of the structure and properties of nanomaterials can be achieved by Atomic Force Microscopy (AFM). AFM is a very powerful tool which can be used to obtain qualitative or quantitative information without destroying the collagen fibrillar structure. This mini review covers issues related to the use of AFM for studying the structure and mechanical properties of collagen-based nanobiomaterials, collagen-substrate interactions during the formation of collagen thin films, collagen-cells interactions, and the collagen-optical radiation interactions.

The Human Cornea as a Model Tissue for Additive Biomanufacturing: A Review

The recent development and clinical success of molded recombinant collagen corneal substitutes offers a tantalizing prospect for the application of additive biomanufacturing in corneal tissue engineering and regenerative medicine (TERM). The cornea has a number of advantages over other tissues for additive biomanufacturing in that it is relatively homogeneous and completely avascular and has low metabolic demand. Additive biomanufacturing could bring a number of advantages over molding to corneal TERM, including increased visual acuity and mechanical strength. We have developed a method for producing thin collagen films, which are suitable for the culturing of corneal mesenchymal stem cells (MSCs), using additive manufacturing techniques, and we hope to use this technique to produce functional corneal substitutes.

Saliva Sensor on Collagen Based Film to Detect Blood Sugar Level

Saliva is been increasingly observed as an attractive diagnostic fluid. This paper demonstrates one of the application of saliva in detecting blood glucose level. Though many different methods had been identified to measure blood glucose level this method particularly concentrated on salivary resistance of both diabetes and healthy person (Before and after fasting). A biological sensing element which is saliva based collagen from tannery solid waste is characterized for many bio sensing applications such as blood sugar level, harmone level (cortisol), cancer biomarkers……etc. The collagen thin film sheet was prepared from chrome shavings which is used as a substrate for identification of salivary resistance. Saliva(1μl) had been placed in contact with multimeter electrode on the collagen substrate where the transformation of biological response to an electrical signal displays the value on multimer digital screen. The value on the digital display has been related to find blood sugar level of every individual. After measuring under wet condition it's been air dried to measure under dry condition. Likewise procedure is been repeated under different concentration of oral fluid henceforth its resistance value has been recorded.

Investigation of the mechanical properties of collagen fibrils under the influence of low power red laser irradiation

The low level red laser has been increasingly used for medical applications over the last decade. Despite the recorded positive effects of low level red laser in various medical treatments, the exact collagen—red light interaction mechanism still remains unknown. Hence, in this paper, thin collagen films were irradiated using red low power laser in order to investigate the effects of red light on collagen nanoscale features and mechanical properties. The results demonstrated that the red light did not induce statistically significant changes on nanoscale collagen topography features. The topography of the alternating sequence of overlapping and gap regions (D-band periodicity) and the surface roughness were not affected. However, significant changes on the mechanical properties of the collagen fibrils were recorded. In particular, the red light induced major changes in the mechanical heterogeneity of the fibrils due to the D-band periodicity. The above results provide new information about the red light-collagen interaction mechanism and are a step forward towards to the clarification of the impact of red light on collagen nanoscale features.