A. U. Daniels

Dynamic Elastic Modulus of Porcine Articular Cartilage Determined at Two Different Levels of Tissue Organization by Indentation-Type Atomic Force Microscopy

Cartilage stiffness was measured ex vivo at the micrometer and nanometer scales to explore structure-mechanical property relationships at smaller scales than has been done previously. A method was developed to measure the dynamic elastic modulus, |E(*)|, in compression by indentation-type atomic force microscopy (IT AFM). Spherical indenter tips (radius = approximately 2.5 microm) and sharp pyramidal tips (radius = approximately 20 nm) were employed to probe micrometer-scale and nanometer-scale response, respectively. |E(*)| values were obtained at 3 Hz from 1024 unloading response curves recorded at a given location on subsurface cartilage from porcine femoral condyles. With the microsphere tips, the average modulus was approximately 2.6 MPa, in agreement with available millimeter-scale data, whereas with the sharp pyramidal tips, it was typically 100-fold lower. In contrast to cartilage, measurements made on agarose gels, a much more molecularly amorphous biomaterial, resulted in the same average modulus for both indentation tips. From results of AFM imaging of cartilage, the micrometer-scale spherical tips resolved no fine structure except some chondrocytes, whereas the nanometer-scale pyramidal tips resolved individual collagen fibers and their 67-nm axial repeat distance. These results suggest that the spherical AFM tip is large enough to measure the aggregate dynamic elastic modulus of cartilage, whereas the sharp AFM tip depicts the elastic properties of its fine structure. Additional measurements of cartilage stiffness following enzyme action revealed that elastase digestion of the collagen moiety lowered the modulus at the micrometer scale. In contrast, digestion of the proteoglycans moiety by cathepsin D had little effect on |E(*)| at the micrometer scale, but yielded a clear stiffening at the nanometer scale. Thus, cartilage compressive stiffness is different at the nanometer scale compared to the overall structural stiffness measured at the micrometer and larger scales because of the fine nanometer-scale structure, and enzyme-induced structural changes can affect this scale-dependent stiffness differently.

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy

The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

Use of isothermal microcalorimetry to monitor microbial activities

Isothermal calorimetry measures the heat flow of biological processes, which is proportional to the rate at which a given chemical or physical process takes place. Modern isothermal microcalorimeters make measurements of less than a microwatt of heat flow possible. As a result, as few as 10 000-100 000 active bacterial cells in culture are sufficient to produce a real-time signal dynamically related to the number of cells present and their activity. Specimens containing bacteria need little preparation, and isothermal microcalorimetry (IMC) is a nondestructive method. After IMC measurements, the undisturbed samples can be evaluated by any other means desired. In this review, we present a basic description of microcalorimetry and examples of microbiological applications of IMC for medical and environmental microbiology. In both fields, IMC has been used to quantify microbial activity over periods of hours or even days. Finally, the recent development of highly parallel instruments (up to 48 channels) and the constantly decreasing costs of equipment have made IMC increasingly attractive for microbiology. Miniaturization of isothermal calorimeters provides an even wider range of possibilities.

Micro- and Nanomechanical Analysis of Articular Cartilage by Indentation-Type Atomic Force Microscopy: Validation with a Gel-Microfiber Composite

As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilages soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composites water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilages microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilages lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage.

Effect of Fibular Plate Fixation on Rotational Stability of Simulated Distal Tibial Fractures Treated with Intramedullary Nailing

Background: The effect of an intact fibula on rotational stability after a distal tibial fracture has, to the best of our knowledge, not been clearly defined. We designed a cadaver study to clarify our clinical impression that fixation of the fibula with a plate increases rotational stability of distal tibial fractures fixed with a Russell-Taylor intramedullary nail.Methods: Seven matched pairs of embalmed human cadaveric legs and sixteen fresh-frozen human cadaveric legs, including one matched pair, were tested. To simulate fractures, 5-mm transverse segmental defects were created at the same level in the tibia and fibula, 7 cm proximal to the ankle joint in each bone. The tibia was stabilized with a 9-mm Russell-Taylor intramedullary nail that was statically locked with two proximal and two distal screws. Each specimen was tested without fibular fixation as well as with fibular fixation with a six-hole semitubular plate. A biaxial mechanical testing machine was used in torque control mode with an initial axial load of 53 to 71 N applied to the tibial condyle. Angular displacement was measured in 0.56-N-m torque increments to a maximal torque of 4.52 N-m (40 in-lb).Results: Initially, significantly less displacement (p ⩽ 0.05) was produced in the specimens with fibular plate fixation than in those without fibular plate fixation. The difference in angular displacement between the specimens treated with and without plate fixation was established at the first torque data point measured but did not increase as the torque was increased. No significant difference in the rotational stiffness was found between the specimens treated with and without plate fixation after measurement of the second torque data point (between 1.68 and 4.48 N-m).Conclusions: Fibular plate fixation increased the initial rotational stability after distal tibial fracture compared with that provided by tibial intramedullary nailing alone. However, there was no difference in rotational structural stiffness between the specimens treated with and without plate fixation as applied torque was increased.Clinical Relevance: In patients with ipsilateral distal tibial and fibular fractures who are treated with Russell-Taylor intramedullary nailing of the tibia, rotational stability of the tibial fracture can be increased by plate-and-screw fixation of the fibula, which may reduce the risk of valgus malunion.

Silver coordination compounds as light-stable, nano-structured and anti-bacterial coatings for dental implant and restorative materials

Silver coordination polymer chains were deposited on Au(111) as a model surface, as well as on gold alloy and titanium as dental implant and restorative materials. The topography of the surface was analysed on the model substrate and it was found to be a nano-structured crystalline material. In vitro investigations in a flow chamber imitating the oral environment prove the anti-bacterial properties of the silver compound.

Isothermal micro calorimetry – a new method for MIC determinations: results for 12 antibiotics and reference strains of E. coli and S. aureus

BackgroundAntimicrobial susceptibility testing of microorganisms is performed by either disc diffusion or broth dilution tests. In clinical use, the tests are often still performed manually although automated systems exist. Most systems, however, are based on turbidometric methods which have well-known drawbacks.ResultsIn this study we evaluated isothermal micro calorimetry (IMC) for the determination of minimal inhibitory concentrations (MICs) of 12 antibiotics for five micro-organisms. Here we present the data for the 12 antibiotics and two representative microorganisms E. coli (a Gram-) and S. aureus (a Gram+). IMC was able to determine the MICs correctly according to CLSI values. Since MICs require 24 hours, time was not reduced. However, IMC provided new additional data – a continuous record of heat-producing bacterial activity (e.g. growth) in calorimetry ampoules at subinhibitory antibiotic concentrations. Key features of the heatflow (P) and aggregate heat (Q) vs. time curves were identified (tdelayand ΔQ/Δt respectively). Antibiotics with similar modes of action proved to have similar effects on tdelayand/or ΔQ/Δt.ConclusionIMC can be a powerful tool for determining the effects of antibiotics on microorganisms in vitro. It easily provides accurate MICs – plus a potential means for analyzing and comparing the modes of action of antibiotics at subinhibitory concentrations. Also IMC is completely passive, so after evaluation, ampoule contents (media, bacteria, etc.) can be analyzed by any other method desired.

Proteoglycans and Glycosaminoglycans Improve Toughness of Biocompatible Double Network Hydrogels

Based on the molecular stent concept, a series of tough double-network hydrogels (St-DN gels) made from the components of proteoglycan aggregates - chondroitin sulfate proteoglycans (1), chondroitin sulfate (2), and sodium hyaluronate (3) - are successfully developed in combination with a neutral biocompatible polymer. This work demonstrates a promising method to create biopolymer-based tough hydrogels for biomedical applications.

Of Chains and Rings: Synthetic Strategies and Theoretical Investigations for Tuning the Structure of Silver Coordination Compounds and Their Applications

Varying the polyethyleneglycol spacer between two (iso)-nicotinic groups of the ligand systems, a large structural variety of silver coordination compounds was obtained, starting with zero-dimensional ring systems, via one-dimensional chains, helices and double-helices to two-dimensional polycatenanes. Theoretical calculations help to understand their formation and allow predictions in some cases. These structures can be tuned by careful design of the ligand, the use of solvent and the counter ions, influencing also other important properties such as light stability and solubility. The latter is important in the context of biomedical applications, using silver compounds as antimicrobial agents.

Engineering human cell-based, functionally integrated osteochondral grafts by biological bonding of engineered cartilage tissues to bony scaffolds

In this study, we aimed at developing and validating a technique for the engineering of osteochondral grafts based on the biological bonding of a chondral layer with a bony scaffold by cell-laid extracellular matrix. Osteochondral composites were generated by combining collagen-based matrices (Chondro-Gide) containing human chondrocytes with devitalized spongiosa cylinders (Tutobone) using a fibrin gel (Tisseel). We demonstrate that separate pre-culture of the chondral layer for 3 days prior to the generation of the composite allows for (i) more efficient cartilaginous matrix accumulation than no pre-culture, as assessed histologically and biochemically, and (ii) superior biological bonding to the bony scaffold than 14 days of pre-culture, as assessed using a peel-off mechanical test, developed to measure integration of bilayered materials. The presence of the bony scaffold induced an upregulation in the infiltrated cells of the osteoblast-related gene bone sialoprotein, indicative of the establishment of a gradient of cell phenotypes, but did not affect per se the quality of the cartilaginous matrix in the chondral layer. The described strategy to generate osteochondral plugs is simple to be implemented and--since it is based on clinically compliant cells and materials--is amenable to be readily tested in the clinic.