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1.
Adv Mater ; 30(13): e1706092, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29446165

ABSTRACT

Actin networks are adaptive materials enabling dynamic and static functions of living cells. A central element for tuning their underlying structural and mechanical properties is the ability to reversibly connect, i.e., transiently crosslink, filaments within the networks. Natural crosslinkers, however, vary across many parameters. Therefore, systematically studying the impact of their fundamental properties like size and binding strength is unfeasible since their structural parameters cannot be independently tuned. Herein, this problem is circumvented by employing a modular strategy to construct purely synthetic actin crosslinkers from DNA and peptides. These crosslinkers mimic both intuitive and noncanonical mechanical properties of their natural counterparts. By isolating binding affinity as the primary control parameter, effects on structural and dynamic behaviors of actin networks are characterized. A concentration-dependent triphasic behavior arises from both strong and weak crosslinkers due to emergent structural polymorphism. Beyond a certain threshold, strong binding leads to a nonmonotonic elastic pulse, which is a consequence of self-destruction of the mechanical structure of the underlying network. The modular design also facilitates an orthogonal regulatory mechanism based on enzymatic cleaving. This approach can be used to guide the rational design of further biomimetic components for programmable modulation of the properties of biomaterials and cells.

2.
J Vis Exp ; (128)2017 10 25.
Article in English | MEDLINE | ID: mdl-29155710

ABSTRACT

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.


Subject(s)
DNA/chemistry , Nanotubes/chemistry , Polymers/chemistry , Base Sequence , Microscopy, Atomic Force , Nucleic Acid Conformation , Nucleic Acid Hybridization
3.
Molecules ; 22(10)2017 Oct 24.
Article in English | MEDLINE | ID: mdl-29064446

ABSTRACT

Bundled actin structures play an essential role in the mechanical response of the actin cytoskeleton in eukaryotic cells. Although responsible for crucial cellular processes, they are rarely investigated in comparison to single filaments and isotropic networks. Presenting a highly anisotropic structure, the determination of the mechanical properties of individual bundles was previously achieved through passive approaches observing bending deformations induced by thermal fluctuations. We present a new method to determine the bending stiffness of individual bundles, by measuring the decay of an actively induced oscillation. This approach allows us to systematically test anisotropic, bundled structures. Our experiments revealed that thin, depletion force-induced bundles behave as semiflexible polymers and obey the theoretical predictions determined by the wormlike chain model. Thickening an individual bundle by merging it with other bundles enabled us to study effects that are solely based on the number of involved filaments. These thicker bundles showed a frequency-dependent bending stiffness, a behavior that is inconsistent with the predictions of the wormlike chain model. We attribute this effect to internal processes and give a possible explanation with regard to the wormlike bundle theory.


Subject(s)
Actin Cytoskeleton/chemistry , Actins/chemistry , Biomechanical Phenomena , Kinetics , Models, Chemical , Optical Tweezers , Protein Multimerization , Rheology , Stress, Mechanical
4.
Phys Rev Lett ; 117(19): 197801, 2016 Nov 04.
Article in English | MEDLINE | ID: mdl-27858441

ABSTRACT

The mechanics of complex soft matter often cannot be understood in the classical physical frame of flexible polymers or rigid rods. The underlying constituents are semiflexible polymers, whose finite bending stiffness (κ) leads to nontrivial mechanical responses. A natural model for such polymers is the protein actin. Experimental studies of actin networks, however, are limited since the persistence length (l_{p}∝κ) cannot be tuned. Here, we experimentally characterize this parameter for the first time in entangled networks formed by synthetically produced, structurally tunable DNA nanotubes. This material enabled the validation of characteristics inherent to semiflexible polymers and networks thereof, i.e., persistence length, inextensibility, reptation, and mesh size scaling. While the scaling of the elastic plateau modulus with concentration G_{0}∝c^{7/5} is consistent with previous measurements and established theories, the emerging persistence length scaling G_{0}∝l_{p} opposes predominant theoretical predictions.

5.
Graefes Arch Clin Exp Ophthalmol ; 254(8): 1567-1577, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27270346

ABSTRACT

PURPOSE: We aimed to determine the ultrastructural changes of collagen fibrils and cells in the rabbit sclera after scleral crosslinking using riboflavin and blue light of different intensities. Scleral crosslinking is known to increase scleral stiffness and may inhibit the axial elongation of progressive myopic eyes. METHODS: The equatorial parts of the sclera of one eye of six adult albino rabbits were treated with topical riboflavin solution (0.5 %) followed by irradiation with blue light (200, 400, 650 mW/cm(2)) for 20 min. After 3 weeks, the ultrastructure of scleral cells and the abundance of small- (10-100 nm) and large-diameter (>100 nm) collagen fibrils in fibril bundles of different scleral layers were examined with electron microscopy. RESULTS: In the scleral stroma of control eyes, the thickness of collagen fibrils showed a bimodal distribution. The abundance of small-diameter collagen fibrils decreased from the inner towards the outer sclera, while the amount of large-diameter fibrils and the scleral collagen content did not differ between different stroma layers. Treatment with riboflavin and blue light at 200 mW/cm(2) did not induce ultrastructural changes of cells and collagen fibrils in the scleral stroma. Treatment with blue light of higher intensities induced scleral cell activation in a scleral layer-dependent manner. In addition, outer scleral layers contained phagocytes that engulfed collagen fibrils and erythrocytes. Blue light of the highest intensity induced a reduction of the scleral collagen content, a decreased abundance of large-diameter collagen fibrils, and an increased amount of small-diameter fibrils in the whole scleral stroma. CONCLUSIONS: The data indicate that in rabbits, scleral crosslinking with riboflavin and blue light of 200 mW/cm(2) for 20 min is relatively safe and does not induce ultrastructural alterations of scleral cells and of the collagen composition of the scleral stroma. Irradiation with blue light of intensities between 200 and 400 mW/cm(2) induces scleral cell activation, which may contribute to scleral scarring and stiffening. Higher intensities cause scleritis.


Subject(s)
Collagen/pharmacology , Cross-Linking Reagents/pharmacology , Light , Myopia/therapy , Riboflavin/pharmacology , Sclera/ultrastructure , Animals , Biomechanical Phenomena , Disease Models, Animal , Microscopy, Electron , Myopia/physiopathology , Photosensitizing Agents/pharmacology , Rabbits , Sclera/drug effects , Sclera/physiopathology
6.
Phys Rev Lett ; 116(10): 108102, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-27015510

ABSTRACT

Attractive depletion forces between rodlike particles in highly crowded environments have been shown through recent modeling and experimental approaches to induce different structural and dynamic signatures depending on relative orientation between rods. For example, it has been demonstrated that the axial attraction between two parallel rods yields a linear energy potential corresponding to a constant contractile force of 0.1 pN. Here, we extend pairwise, depletion-induced interactions to a multifilament level with actin bundles, and find contractile forces up to 3 pN. Forces generated due to bundle relaxation were not constant, but displayed a harmonic potential and decayed exponentially with a mean decay time of 3.4 s. Through an analytical model, we explain these different fundamental dynamics as an emergent, collective phenomenon stemming from the additive, pairwise interactions of filaments within a bundle.


Subject(s)
Actin Cytoskeleton/physiology , Actins/physiology , Models, Biological , Muscle Contraction/physiology , Optical Tweezers , Linear Models
7.
Exp Eye Res ; 139: 37-47, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26208440

ABSTRACT

Several scleral cross-linking (SXL) methods were suggested to increase the biomechanical stiffness of scleral tissue and therefore, to inhibit axial eye elongation in progressive myopia. In addition to scleral cross-linking and biomechanical effects caused by riboflavin and light irradiation such a treatment might induce tissue damage, dependent on the light intensity used. Therefore, we characterized the damage threshold and mechanical stiffening effect in rabbit eyes after application of riboflavin combined with various blue light intensities. Adult pigmented and albino rabbits were treated with riboflavin (0.5 %) and varying blue light (450 ± 50 nm) dosages from 18 to 780 J/cm(2) (15 to 650 mW/cm(2) for 20 min). Scleral, choroidal and retinal tissue alterations were detected by means of light microscopy, electron microscopy and immunohistochemistry. Biomechanical changes were measured by shear rheology. Blue light dosages of 480 J/cm(2) (400 mW/cm(2)) and beyond induced pathological changes in ocular tissues; the damage threshold was defined by the light intensities which induced cellular degeneration and/or massive collagen structure changes. At such high dosages, we observed alterations of the collagen structure in scleral tissue, as well as pigment aggregation, internal hemorrhages, and collapsed blood vessels. Additionally, photoreceptor degenerations associated with microglia activation and macroglia cell reactivity in the retina were detected. These pathological alterations were locally restricted to the treated areas. Pigmentation of rabbit eyes did not change the damage threshold after a treatment with riboflavin and blue light but seems to influence the vulnerability for blue light irradiations. Increased biomechanical stiffness of scleral tissue could be achieved with blue light intensities below the characterized damage threshold. We conclude that riboflavin and blue light application increased the biomechanical stiffness of scleral tissue at blue light energy levels below the damage threshold. Therefore, applied blue light intensities below the characterized damage threshold might define a therapeutic blue light tolerance range.


Subject(s)
Cross-Linking Reagents/pharmacology , Riboflavin/pharmacology , Sclera/drug effects , Animals , Biomechanical Phenomena , Disease Models, Animal , Light , Microscopy, Electron , Photosensitizing Agents/pharmacology , Rabbits , Sclera/radiation effects , Sclera/ultrastructure
8.
Acta Ophthalmol ; 93(5): e328-e336, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25516112

ABSTRACT

PURPOSE: To determine the visco-elastic properties of isolated rabbit scleral tissue and dose-dependent biomechanical and morphological changes after collagen cross-linking by riboflavin/blue light treatment. MATERIAL: Scleral patches from 87 adult albino rabbit eyes were examined by dynamic shear rheology. Scleral patches were treated by riboflavin and different intensities of blue light (450 nm), and the impact on the visco-elastic properties was determined by various rheological test regimes. The relative elastic modulus was calculated from non-treated and corresponding treated scleral patches, and treatments with different blue light intensities were compared. RESULTS: Shear rheology enables us to study the material properties of scleral tissue within physiological relevant parameters. Cross-linking treatment increased the viscous as well as the elastic modulus and changed the ratio of the elastic versus viscous proportion in scleral tissue. Constant riboflavin application combined with different blue light intensities from 12 mW/cm(2) up to 100 mW/cm(2) increased the relative elastic modulus of scleral tissue by factors up to 1.8. Further enhancement of the applied light intensity caused a decline of the relative elastic modulus. This might be due to destructive changes of the collagen bundle structure at larger light intensities, as observed by histological examination. CONCLUSION: Collagen cross-linking by riboflavin/blue light application increases the biomechanical stiffness of the sclera in a dose-dependent manner up to certain light intensities. Therefore, this treatment might be a suitable therapeutic approach to stabilize the biomechanical properties of scleral tissue in cases of pathological eye expansion.


Subject(s)
Collagen/metabolism , Cross-Linking Reagents , Elastic Modulus/physiology , Light , Riboflavin/pharmacology , Sclera/drug effects , Sclera/metabolism , Animals , Biomechanical Phenomena , Dose-Response Relationship, Radiation , Photochemotherapy , Photosensitizing Agents/pharmacology , Rabbits , Rheology
9.
J Mater Chem B ; 2(27): 4297-4309, 2014 Jul 21.
Article in English | MEDLINE | ID: mdl-32261568

ABSTRACT

Natural hydrogels such as gelatin are highly desirable biomaterials for application in drug delivery, biosensors, bioactuators and extracellular matrix components due to strong biocompatibility and biodegradability. Typically, chemical crosslinkers are used to optimize material properties, often introducing toxic byproducts into the material. In this present work, electron irradiation is employed as a reagent-free crosslinking technique to precisely tailor the viscoelasticity, swelling behavior, thermal stability and structure of gelatin. With increasing electron dose, changes in swelling behavior and rheology indicate increasing amounts of random coils and dangling ends as opposed to helical content, a result confirmed through Fourier transform infrared spectroscopy. Gel fraction, rheology and swelling measurements at 37 °C were used to verify thermal stability in biological conditions. Scanning electron microscopy images of dried gelatin samples support these conclusions by revealing a loss of free volume and apparent order in the fracture patterns. The degree of crosslinking and mesh size are quantified by rubber elasticity theory and the Flory-Rehner equation. Overall, precise control of material properties is demonstrated through the interplay of concentration and irradiation dose, while providing an extensive parameter-property database suitable for optimized synthesis.

10.
Article in English | MEDLINE | ID: mdl-24229308

ABSTRACT

We studied the influence of fluorescent polystyrene beads on both entangled and cross-linked actin networks. Thermal bead fluctuations were observed via video particle tracking and analyzed with one-point microrheology. Illumination of fluorescent beads with their appropriate excitation wavelength leads to a drastic softening of actin gels. Other wavelengths and bright field microscopy do not increase thermal bead fluctuations. This effect cannot be significantly reduced by adding common oxygen scavengers. We conclude that the usage of fluorescent beads impairs results when studying the microrheology of actin networks.


Subject(s)
Actins/chemistry , Fluorescent Dyes/chemistry , Microspheres , Actins/drug effects , Fluorescent Dyes/pharmacology , Glycerol/chemistry , Microscopy, Fluorescence , Oxygen/chemistry , Polystyrenes/chemistry , Polystyrenes/pharmacology , Rheology , Temperature
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