Martin Stolz

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.

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.

Sliding motion modulates stiffness and friction coefficient at the surface of tissue engineered cartilage

OBJECTIVEnFunctional cartilage tissue engineering aims to generate grafts with a functional surface, similar to that of authentic cartilage. Bioreactors that stimulate cell-scaffold constructs by simulating natural joint movements hold great potential to generate cartilage with adequate surface properties. In this study two methods based on atomic force microscopy (AFM) were applied to obtain information about the quality of engineered graft surfaces. For better understanding of the molecule-function relationships, AFM was complemented with immunohistochemistry.nnnMETHODSnBovine chondrocytes were seeded into polyurethane scaffolds and subjected to dynamic compression, applied by a ceramic ball, for 1h daily [loading group 1 (LG1)]. In loading group 2 (LG2), the ball additionally oscillated over the scaffold, generating sliding surface motion. After 3 weeks, the surfaces of the engineered constructs were analyzed by friction force and indentation-type AFM (IT-AFM). Results were complemented and compared to immunohistochemical analyses.nnnRESULTSnThe loading type significantly influenced the mechanical and histological outcomes. Constructs of LG2 exhibited lowest friction coefficient and highest micro- and nanostiffness. Collagen type II and aggrecan staining were readily observed in all constructs and appeared to reach deeper areas in loaded (LG1, LG2) compared to unloaded scaffolds. Lubricin was specifically detected at the top surface of LG2.nnnCONCLUSIONSnThis study proposes a quantitative AFM-based functional analysis at the micrometer- and nanometer scale to evaluate the quality of cartilage surfaces. Mechanical testing (load-bearing) combined with friction analysis (gliding) can provide important information. Notably, sliding-type biomechanical stimuli may favor (re-)generation and maintenance of functional articular surfaces and support the development of mechanically competent engineered cartilage.

Nanofibrous poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) scaffolds provide a functional microenvironment for cartilage repair

Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a ‘guiding’ structure to support the body’s self-healing process.

Nanotechnology in Medicine: Moving from the Bench to the Bedside

While living matter is composed of a large number of biological nanomachines, it has been recog- nized early in the history of nanotechnology that medicine could be a prime field for application. Now that nanotechnology has gone beyond its infancy, its mature arsenal of tools, methods and materials is ready for applications outside physics. While true clinical applications of nanotechnology are still practically non-exis- tent at the current time, a significant number of promising medical projects is at an advanced experimental stage. Tools based on the atomic force microscope will not only allow improved imaging of living matter but can also serve as functional probes and will even serve as sensitive sensors for a broad range of molecules of medical interest. New immunological tests based on microcontact printing and microfluidics will signifi- cantly improve medical laboratory diagnosis. New materials, including nanotubes and fullerenes, nanocon- tainers and other self-assembled structures may improve mechanical properties and biocompatibility of im- plants and will allow new approaches in drug targeting.

Supramolecular Organization of Collagen Fibrils in Healthy and Osteoarthritic Human Knee and Hip Joint Cartilage.

Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.

Towards monitoring transport of single cargos across individual nuclear pore complexes by time-lapse atomic force microscopy

A new preparation procedure was developed for the stable adsorption of either the cytoplasmic or the nuclear face of native (i.e. in physiological buffer without detergent extraction and in the absence of chemical fixatives) Xenopus oocyte nuclear envelopes (NEs) onto silicon (Si) surfaces. This yields optimal structural preservation of the nuclear pore complexes (NPCs) without compromising their functional properties. The functional viability of thus prepared NPCs was documented by time-lapse atomic force microscopy (AFM) of the reversible calcium-mediated opening (i.e. +Ca(2+)) and closing (i.e. -Ca(2+)) of the iris diaphragm-like distal ring topping the NPCs nuclear baskets. Moreover, site-specific single colloidal gold particle detection was documented by AFM imaging one and the same NPC before and after immuno-gold labeling the sample with a nucleoporin-specific antibody. With this new preparation protocol at hand, we should eventually be able to follow by time-lapse AFM transport of single gold-conjugated cargos across individual NPCs.

Calibration of colloidal probes with atomic force microscopy for micromechanical assessment

Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by testing agarose samples with indentation moduli ranging over three orders of magnitude via AFM and compared these results with bulk compression tests. Our results show that quantitative measurements of indentation modulus is achieved over three orders of magnitude ranging from 1 kPa to 1000 kPa via AFM cantilever-based microindentation experiments. Therefore, our approach could be used for quantitative micromechanical measurements without the need to perform further validation via bulk compression experiments.

Actin: From cell biology to atomic detail

Over the past 2 decades our knowledge about actin filaments has evolved from a rigid pearls on a string model to that of a complex, highly dynamic protein polymer which can now be analyzed at atomic detail. To achieve this, exploring actins oligomerization, polymerization, polymorphism, and dynamic behavior has been crucial to understanding in detail how this abundant and ubiquitous protein can fulfill its various functions within living cells. In this review, a correlative view of a number of distinct aspects of actin is presented, and the functional implications of recent structural, biochemical, and mechanical data are critically evaluated. Rational analysis of these various experimental data is achieved using an integrated structural approach which combines intermediate-resolution electron microscopy-based 3-D reconstructions of entire actin filaments with atomic resolution X-ray data of monomeric and polymeric actin.

Early osteoarthritis were only detected at the nanometer scale but not at the micrometer or millimeter scale

1. On page 3096 the authors referring to two of my own papers (Stolz et al., 2004, 2009): ‘‘In these studies, the outermost 1 mm of the cartilage surface was removed via microtoming, and the mechanical properties measured by AFM were therefore those of articular cartilage surface 1 mm beneath the native surface.’’ We removed the outer zone of the articular cartilage only at the time when we established the indentation-type atomic force microscopy (IT-AFM) method, which was then presented in the Biophysical Journal in 2004 (Stolz et al., 2004). Importantly, all measurements provided in the Stolz et al.’s Nature Nanotechnology paper (Stolz et al., 2009) and the accompanying Biophysical Journal paper by Loparic et al. (2010) are on original cartilage surfaces. Therefore, the statement by Desrocher et al. is misleading. Respectfully I would suggest the authors submit a corrigendum. 2. What Desrocher et al. call ‘‘early osteoarthritis’’ is already intermediate to late stage osteoarthritis. As documented in the Stolz et al.’s Nature Nanotechnology paper (Stolz et al., 2009), we found a strong correlation between visual grading using the Outerbridge scale and the corresponding values of nanostiffness by IT-AFM. When using sharp nanometer-sized tips, IT-AFM measured structure/functional changes of the proteoglycan moiety in grade 0 cartilage, but scanning electron microscopy (SEM) and IT-AFM using micrometer-sized tips on grade 0 samples did not reveal changes in the collagen meshwork. In addition, SEM revealed significant structural changes in Grade 1 osteoarthritis cartilage and increasing damage of the collagen meshwork in the higher grades. Obviously, changes in the proteoglycan moiety start a long time before damages of the collagen structures are visible. Visible changes in the collagen meshwork correspond to late stage osteoarthritis. Therefore, the term ‘‘early osteoarthritis’’ should only be used when monitoring alterations of the proteoglycan moiety in an intact collagen meshwork (grade 0 cartilage). Treating cartilage in ‘‘early osteoarthritis’’ at a time when the collagen meshwork is still intact – which is in grade 0 cartilage only – would certainly increase the chances of stopping or even reversing osteoarthritis. We have started collaborating with pharmaceutical industry to use the IT-AFM method to measure nanometer structure/functional changes in the small joints of rats and mice to facilitate the development of drugs against osteoarthritis. Additionally, for early detection of osteoarthritis in patient’s knee and hip joints, we are currently developing a hand-held clinical tool, the arthroscopic AFM. Following an evidence-based medicine approach, the arthroscopic AFM will allow the orthopedic surgeon to base his decision on ‘hard numbers’ and to compare his results to a reference database (Aigner et al., 2009; Imer et al., 2006, 2009; Stolz et al., 2009). 3. The authors state in their Discussion that ‘‘cartilage is a material’’ (Desrochers et al., 2010). As stated by Lukkassen and Meidell (2003): ‘‘Material sciences defines a composite as a material even though it consists of two or more components, but a honeycomb core built up of two different components is a structure’’. Articular cartilage exhibits a zonal architecture with irregular imbedded chondrocytes and should not be referred to as a material. This has the following implications: As has been demonstrated by Stolz et al. (2009), an early detection of osteoarthritis was only possible at the nanometer scale, but not at the micrometer scale or above. However, changes at the nanometer scale may be also reflected at microto centimeter scales as concerted events where cartilage structural elements are acting together. The nanostiffness calculated as the arithmetic average from cartilage biopsies from seven osteoarthritis patients changed from roughly about Enano1⁄450 kPa (1⁄40.050 MPa) for grade 0 to Enano1⁄420 kPa (1⁄40.020 MPa) for grade 1 osteoarthritis cartilage [cf. Table 1 in Stolz et al. (2009)]. In first approximation this change in proteoglycan moiety stiffness should result into a change of bulk mechanical property and be measurable as a change of microstiffness: Emicro1⁄41.350 MPa (grade 0) to 1.320 MPa (grade 1), corresponding to a 2% change when measured at the level of microstiffness. From our experience, it is not possible to measure those changes when using 2 mm diameter spherical tips on mouse femoral heads, or 5 mm diameter spherical tips for the measurements on human articular cartilage. It is therefore not a lack of sensitivity on the part of the AFM, but the structural inhomogeneity of cartilage that prevents the measurement of changes in the proteoglycan moiety. It seems that any technique that addresses an early detection of osteoarthritis must be able to resolve the collagen fibrils and proteoglycan moiety directly.