Johannes H. Kindt

Molecular mechanistic origin of the toughness of natural adhesives, fibres and composites

Natural materials are renowned for their strength and toughness,,,,. Spider dragline silk has a breakage energy per unit weight two orders of magnitude greater than high tensile steel,, and is representative of many other strong natural fibres,,. The abalone shell, a composite of calcium carbonate plates sandwiched between organic material, is 3,000 times more fracture resistant than a single crystal of the pure mineral,. The organic component, comprising just a few per cent of the composite by weight, is thought to hold the key to nacres fracture toughness,. Ceramics laminated with organic material are more fracture resistant than non-laminated ceramics,, but synthetic materials made of interlocking ceramic tablets bound by a few weight per cent of ordinary adhesives do not have a toughness comparable to nacre. We believe that the key to nacres fracture resistance resides in the polymer adhesive, and here we reveal the properties of this adhesive by using the atomic force microscope to stretch the organic molecules exposed on the surface of freshly cleaved nacre. The adhesive fibres elongate in a stepwise manner as folded domains or loops are pulled open. The elongation events occur for forces of a few hundred piconewtons, which are smaller than the forces of over a nanonewton required to break the polymer backbone in the threads. We suggest that this ‘modular’ elongation mechanism might prove to be quite general for conveying toughness to natural fibres and adhesives, and we predict that it might be found also in dragline silk.

Bone indentation recovery time correlates with bond reforming time

Despite centuries of work, dating back to Galileo, the molecular basis of bones toughness and strength remains largely a mystery. A great deal is known about bone microsctructure and the microcracks that are precursors to its fracture, but little is known about the basic mechanism for dissipating the energy of an impact to keep the bone from fracturing. Bone is a nanocomposite of hydroxyapatite crystals and an organic matrix. Because rigid crystals such as the hydroxyapatite crystals cannot dissipate much energy, the organic matrix, which is mainly collagen, must be involved. A reduction in the number of collagen cross links has been associated with reduced bone strength and collagen is molecularly elongated (‘pulled’) when bovine tendon is strained. Using an atomic force microscope, a molecular mechanistic origin for the remarkable toughness of another biocomposite material, abalone nacre, has been found. Here we report that bone, like abalone nacre, contains polymers with ‘sacrificial bonds’ that both protect the polymer backbone and dissipate energy. The time needed for these sacrificial bonds to reform after pulling correlates with the time needed for bone to recover its toughness as measured by atomic force microscope indentation testing. We suggest that the sacrificial bonds found within or between collagen molecules may be partially responsible for the toughness of bone.

Probing protein-protein interactions in real time.

We have used a prototype small cantilever atomic force microscope to observe, in real time, the interactions between individual protein molecules. In particular, we have observed individual molecules of the chaperonin protein GroES binding to and then dissociating from individual GroEL proteins, which were immobilized on a mica support. This work suggests that the small cantilever atomic force microscope is a useful tool for studying protein dynamics at the single molecule level.

Direct Observation of the Transition from Calcite to Aragonite Growth as Induced by Abalone Shell Proteins

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.

Evidence that Collagen Fibrils in Tendons Are Inhomogeneously Structured in a Tubelike Manner

The standard model for the structure of collagen in tendon is an ascending hierarchy of bundling. Collagen triple helices bundle into microfibrils, microfibrils bundle into subfibrils, and subfibrils bundle into fibrils, the basic structural unit of tendon. This model, developed primarily on the basis of x-ray diffraction results, is necessarily vague about the cross-sectional organization of fibrils and has led to the widespread assumption of laterally homogeneous closepacking. This assumption is inconsistent with data presented here. Using atomic force microscopy and micromanipulation, we observe how collagen fibrils from tendons behave mechanically as tubes. We conclude that the collagen fibril is an inhomogeneous structure composed of a relatively hard shell and a softer, less dense core.

Force Spectroscopy of Collagen Fibers to Investigate Their Mechanical Properties and Structural Organization

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length <20 microm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of <7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.

Data acquisition system for high speed atomic force microscopy

With the development of atomic force microscopes that allow higher scan speeds, the need for data acquisition systems (DAQ) that are capable of handling the increased amounts of data in real time arises. We have developed a low cost data acquisition and scan control system around a commercially available DAQ board in a WINDOWS environment. By minimizing the involvement of the processor in the data transfer using direct memory access, and generation of the scan signals synchronously with the data acquisition, we were able to record 30 frames per second with a pixel resolution of 150×150pixels and 14bit per channel.

Atomic force microscopy study of living diatoms in ambient conditions

We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain‐forming, benthic freshwater species –Eunotia sudetica, Navicula seminulum and a yet unidentified species – are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long‐term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man‐made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh‐ and in seawater environments. Our phase‐imaging and force‐pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man‐made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge.

Investigations into the polymorphism of rat tail tendon fibrils using atomic force microscopy

Collagen type I displays a typical banding periodicity of 67 nm when visualized by atomic force or transmission electron microscopy imaging. We have investigated collagen fibers extracted from rat tail tendons using atomic force microscopy, under different ionic and pH conditions. The majority of the fibers reproduce the typical wavy structure with 67 nm spacing and a height difference between the peak and the grooves of at least 5 nm. However, we were also able to individuate two other banding patterns with 23+/-2 nm and 210+/-15 nm periodicities. The small pattern showed height differences of about 2 nm, whereas the large pattern seems to be a superposition of the 67 nm periodicity showing height differences of about 20 nm. Furthermore, we could show that at pH values of 3 and below the fibril structure gets dissolved whereas high concentrations of NaCl and CaCl(2) could prevent this effect.

Atomic Force Microscopy-Based Screening of Drug-Excipient Miscibility and Stability of Solid Dispersions

ABSTRACTPurposeDevelopment of a method to assess the drug/polymer miscibility and stability of solid dispersions using a melt-based mixing method.MethodsAmorphous fractured films are prepared and characterized with Raman Microscopy in combination with Atomic Force Microscopy to discriminate between homogenously and heterogeneously mixed drug/polymer combinations. The homogenous combinations are analyzed further for physical stability under stress conditions, such as increased humidity or temperature.ResultsCombinations that have the potential to form a molecular disperse mixture are identified. Their potential to phase separate is determined through imaging at molecular length scales, which results in short observation time. De-mixing is quantified by phase separation analysis, and the drug/polymer combinations are ranked to identify the most stable combinations.ConclusionsThe presented results demonstrate that drug/polymer miscibility and stability of solid dispersions, with many mechanistic details, can be analyzed with Atomic Force Microscopy. The assay allows to identify well-miscible and stable combinations within hours or a few days.