Indentation-type Atomic Force Microscopy Research Articles

The Revolution in Breast Cancer Diagnostics: From Visual Inspection of Histopathology Slides to Using Desktop Tissue Analysers for Automated Nanomechanical Profiling of Tumours.

We aim to develop new portable desktop tissue analysers (DTAs) to provide fast, low-cost, and precise test results for fast nanomechanical profiling of tumours. This paper will explain the reasoning for choosing indentation-type atomic force microscopy (IT-AFM) to reveal the functional details of cancer. Determining the subtype, cancer stage, and prognosis will be possible, which aids in choosing the best treatment. DTAs are based on fast IT-AFM at the size of a small box that can be made for a low budget compared to other clinical imaging tools. The DTAs can work in remote areas and all parts of the world. There are a number of direct benefits: First, it is no longer needed to wait a week for the pathology report as the test will only take 10 min. Second, it avoids the complicated steps of making histopathology slides and saves costs of labour. Third, computers and robots are more consistent, more reliable, and more economical than human workers which may result in fewer diagnostic errors. Fourth, the IT-AFM analysis is capable of distinguishing between various cancer subtypes. Fifth, the IT-AFM analysis could reveal new insights about why immunotherapy fails. Sixth, IT-AFM may provide new insights into the neoadjuvant treatment response. Seventh, the healthcare system saves money by reducing diagnostic backlogs. Eighth, the results are stored on a central server and can be accessed to develop strategies to prevent cancer. To bring the IT-AFM technology from the bench to the operation theatre, a fast IT-AFM sensor needs to be developed and integrated into the DTAs.

Nano-Scale Mechanical Properties of the Articular Cartilage Zones in a Mouse Model of Post-Traumatic Osteoarthritis

Destabilization of the medial meniscus (DMM) surgery in mice is used to elucidate the mechanism of post-traumatic osteoarthritis (PT-OA). The study of cartilage biomechanics in PT-OA is important for understanding the pathophysiology of the condition. We used indentation-type atomic force microscopy (IT-AFM) to assess the nanostiffness of the interterritorial matrix of articular cartilage (AC) zones in the medial and the lateral tibia plateau (MTP and LTP) on native tissue sections 2 and 8 weeks after DMM or Sham surgery. At 2 weeks, pronounced stiffening of the DMM AC was observed compared to Sham, with the most marked changes occurring in the superficial zone and affecting the proteoglycan moiety rather than the collagen network. The LTP cartilage was obviously stiffer than the MTP in DMM, but not in Sham. At 8 weeks, only modest differences in nanostiffness were observed between DMM and Sham. The difference in stiffness between MTP and LTP was reduced, and the proteoglycan and collagen phases changed in a more similar manner. Interestingly, the deep zone was softer in the DMM compared to the Sham. Sham AC showed an increase in stiffness between 2 and 8 weeks, a trend that was counteracted in the DMM group. Collectively, our study demonstrates that nano-scale IT-AFM is a sensitive tool to monitor biomechanical changes during the course of PT-OA.

ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation.

Ageing or obesity are risk factors for protein aggregation in the endoplasmic reticulum (ER) of chondrocytes. This condition is called ER stress and leads to induction of the unfolded protein response (UPR), which, depending on the stress level, restores normal cell function or initiates apoptotic cell death. Here the role of ER stress in knee osteoarthritis (OA) was evaluated. It was first tested in vitro and in vivo whether a knockout (KO) of the protein disulfide isomerase ERp57 in chondrocytes induces sufficient ER stress for such analyses. ER stress in ERp57 KO chondrocytes was confirmed by immunofluorescence, immunohistochemistry, and transmission electron microscopy. Knee joints of wildtype (WT) and cartilage-specific ERp57 KO mice (ERp57 cKO) were analyzed by indentation-type atomic force microscopy (IT-AFM), toluidine blue, and immunofluorescence/-histochemical staining. Apoptotic cell death was investigated by a TUNEL assay. Additionally, OA was induced via forced exercise on a treadmill. ER stress in chondrocytes resulted in a reduced compressive stiffness of knee cartilage. With ER stress, 18-month-old mice developed osteoarthritic cartilage degeneration with osteophyte formation in knee joints. These degenerative changes were preceded by apoptotic death in articular chondrocytes. Young mice were not susceptible to OA, even when subjected to forced exercise. This study demonstrates that ER stress induces the development of age-related knee osteoarthritis owing to a decreased protective function of the UPR in chondrocytes with increasing age, while apoptosis increases. Therefore, inhibition of ER stress appears to be an attractive therapeutic target for OA.

Early Detection of Cartilage Degeneration: A Comparison of Histology, Fiber Bragg Grating-Based Micro-Indentation, and Atomic Force Microscopy-Based Nano-Indentation.

We have determined the sensitivity and detection limit of a new fiber Bragg grating (FBG)-based optoelectronic micro-indenter for biomechanical testing of cartilage and compared the results to indentation-type atomic force microscopy (IT-AFM) and histological staining. As test samples, we used bovine articular cartilage, which was enzymatically degraded ex vivo for five minutes using different concentrations of collagenase (5, 50, 100 and 500 µg/mL) to mimic moderate extracellular matrix deterioration seen in early-stage osteoarthritis (OA). Picrosirius Red staining and polarization microscopy demonstrated gradual, concentration-dependent disorganization of the collagen fibrillar network in the superficial zone of the explants. Osteoarthritis Research Society International (OARSI) grading of histopathological changes did not discriminate between undigested and enzymatically degraded explants. IT-AFM was the most sensitive method for detecting minute changes in cartilage biomechanics induced by the lowest collagenase concentration, however, it did not distinguish different levels of cartilage degeneration for collagenase concentrations higher than 5 µg/mL. The FBG micro-indenter provided a better and more precise assessment of the level of cartilage degeneration than the OARSI histological grading system but it was less sensitive at detecting mechanical changes than IT-AFM. The FBG-sensor allowed us to observe differences in cartilage biomechanics for collagenase concentrations of 100 and 500 µg/mL. Our results confirm that the FBG sensor is capable of detecting small changes in articular cartilage stiffness, which may be associated with initial cartilage degeneration caused by early OA.

Sensory neuropeptides are required for bone and cartilage homeostasis in a murine destabilization-induced osteoarthritis model.

Numerous studies identified a role for the sensory neuropeptides substance P (SP) and alpha calcitonin gene-related peptide (αCGRP) in osteoarthritis (OA) pain behavior. Surprisingly, little attention has been paid on how their trophic effects on cartilage and bone cells might affect structural changes of bone and cartilage in OA pathology. Here, we sought to elucidate sensory neuropeptides influence on structural alterations of bone and cartilage during murine OA pathophysiology. OA was induced by destabilization of the medial meniscus (DMM) in the right knee joint of 12weeks old male C57Bl/6J wildtype (WT) mice and mice either deficient for SP (tachykinin 1 (Tac1)-/-) or αCGRP. By OARSI histopathological grading we observed significant cartilage matrix degradation after DMM surgery in αCGRP-deficient mice after 4weeks whereas Tac1-/- scores were not different to sham mice before 12weeks after surgery. Indentation-type atomic force microscopy (IT-AFM) identified a strong superficial zone (SZ) cartilage phenotype in Tac1-/- Sham mice. Opposed to WT and αCGRP-/- mice, SZ cartilage of Tac1-/- mice softened 2weeks after OA induction. In Tac1-/- DMM mice, bone volume to total volume ratio (BV/TV) increased significantly compared to the Tac1-/- Sham group, 2weeks after surgery. WT mice had reduced BV/TV compared to αCGRP-/- and Tac1-/- mice after 12weeks. Increased calcified cartilage thickness and medial condyle diameter were detected in the medial tibia of all groups 8weeks after OA induction by nanoCT analysis. Meniscal ossification occurred in all OA groups, but was significantly stronger in the absence of neuropeptides. Increased serum concentration of the respective non-deleted neuropeptide was observed in both neuropeptide-deficient mice strains. Both neuropeptides protect from age-related bone structural changes under physiological conditions and SP additionally demonstrates an anabolic effect on bone structure preservation in a pathophysiological situation. Both neuropeptide deficient mice display an intrinsic structural cartilage matrix phenotype that might alter progression of cartilage degeneration in OA.

The Protein Mat(ters)-Revealing the Biologically Relevant Mechanical Contribution of Collagen- and Fibronectin-Coated Micropatterns.

Understanding cell-material interactions requires accurate characterization of the substrate mechanics, which are generally measured by indentation-type atomic force microscopy. To facilitate cell-substrate interaction, model extracellular matrix coatings are used although their tensile mechanical properties are generally unknown. In this study, beyond standard compressive stiffness estimation, we performed a novel tensile mechanical characterization of collagen- and fibronectin-micropatterned polyacrylamide hydrogels. We further demonstrate the impact of the protein mat on the tensile stiffness characterization of adherent cells. To our knowledge, our study is the first to uncover direction-dependent mechanical behavior of the protein coatings and to demonstrate that it affects cellular response relative to substrate mechanics.

Effects of non-enzymatic glycation on the micro- and nano-mechanics of articular cartilage.

The mechanical properties of articular cartilage depend on the quality of its matrix, which consists of collagens and glycosaminoglycans (GAGs). The accumulation of advanced glycation end products (AGEs) can greatly affect the mechanics of cartilage. In the current study, we simulated the accumulation of AGEs by using L-threose to cross-link collagen molecules in the cartilage matrix (in vitro). The resulting changes in the mechanical properties (stiffness) of cartilage are then measured both at the micrometer-scale (using micro-indenter) and nanometer-scale (using indentation-type atomic force microscopy). Non-enzymatic cross-linking within the cartilage matrix was confirmed by the browning of L-threose-treated samples. We observed > 3 times increase in the micro-scale stiffness and up to 12-fold increase in the nano-scale stiffness of the glycated cartilage in the peak pertaining to the collagen fibers, which is caused by cartilage network embrittlement. At the molecular level, we found that besides the collagen component, the glycation process also influenced the GAG macromolecules. Comparing cartilage samples before and after L-threose treatment revealed that artificially induced-AGEs also decelerate in vitro degradation (likely via matrix metalloproteinases), observed at both micro- and nano-scales. The combined observations suggest that non-enzymatic glycation may play multiple roles in mechanochemical functioning of articular cartilage.

Effect of calcium ions on the nanostiffness of articular cartilage

Calcium is one of the major elements in human body, and critical in the biological and physiological functions. Soft tissue hardening due to calcification has been reported in various diseases. However, the quantitative relationship between the mechanical properties of soft tissues and calcification has not yet been fully understood. In this study, using human articular cartilage as prototype material, the relationship between the ex vivo calcium concentration and the nanostiffness of soft tissues was investigated by indentation-type atomic force microscopy. It is found that the nanostiffness of tissue shows a significant dependence on concentration of calcium ions, when it is higher than the normal serum calcium concentration. Structural observation of collagen fibrils indicated that the increase of nanostiffness should be the result of the increase of crosslinking with the introduction of calcium among collagen molecules.

A mechanical characterization of polymer scaffolds and films at the macroscale and nanoscale

Biomaterials should be mechanically tested at both the nanoscale and macroscale under conditions simulating their working state, either in vitro or in vivo, to confirm their applicability in tissue engineering applications. In this article, polyester-urethane-based films and porous scaffolds produced by hot pressing and thermally induced phase separation respectively, were mechanically characterized at both the macroscale and nanoscale by tensile tests and indentation-type atomic force microscopy. All tests were conducted in wet state with the final aim of simulating scaffold real operating conditions. The films showed two distinct Young Moduli populations, which can be ascribed to polyurethane hard and soft segments. In the scaffold, the application of a thermal cooling gradient during phase separation was responsible for a nanoscale polymer chain organization in a preferred direction. At the macroscale, the porous matrices showed a Young Modulus of about 1.5 MPa in dry condition and 0.3 MPa in wet state. The combination of nanoscale and macroscale values as well as the aligned structure are in accordance with stiffness and structure required for scaffolds used for the regeneration of soft tissues such as muscles.

The elastic modulus of articular cartilage at nano-scale and micro-scale measured using indentation type atomic force microscopy

The elastic modulus of articular cartilage at nano-scale and micro-scale measured using indentation type atomic force microscopy

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering.

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects.

Effects of low dose X‐ray irradiation on porcine articular cartilage explants

Ionizing radiation therapy is a crucial treatment for cancer, but can damage surrounding normal tissues. Damage to articular cartilage leading to arthropathy can occur at irradiated sites. It is unclear whether this response is due to damaging surrounding skeletal structures or direct effects on cartilage. In this study, we showed that irradiation with 2 Gy of X-rays causes a significant reduction in the stiffness of porcine explants 1 week post-irradiation. By using both microindentation and indentation-type atomic force microscopy, ionizing radiation reduces stiffness in both the superficial zone, and throughout the entire thickness of the tissue. Young's modulus values were 75% and 60% lower in 2 Gy irradiated samples when compared with controls using microindentation and nanoindentation, respectively. Glycosaminoglycans (GAGs) released into the culture media of irradiated samples was nearly 100% greater at 24 h after exposure. While collagen content in the tissue is similar between groups, GAG content is 55% lower in irradiated explants compared with controls 7 days after exposure. Therefore, the irradiated explants are unable to recover from the initial loss of GAGs by 1 week. This acute loss of GAGs is a likely contributor to the reduction in modulus seen after exposure to ionizing radiation.

Collagen fibril stiffening in osteoarthritic cartilage of human beings revealed by atomic force microscopy

This study aimed to characterize the in-situ mechanical property and morphology of individual collagen fibril in osteoarthritic cartilage using indentation-type atomic force microscopy (IT-AFM). The specimens with intact articular cartilage (AC), mild to severe degenerated cartilage from osteoarthritis (OA) were collected with informed consent from the postmenopausal women who underwent hip or knee arthroplasty. The fresh specimens were cryo-sectioned by layers with 50μm thick for each from the articular surface to calcified cartilage, and then processed for AFM imaging and nanoindentation test. For each layer, a total of 20 collagen fibrils were randomly selected for testing. AFM tips with the nominal radius less than 10nm were employed for probing the individual collagen fibril, and the obtained cantilever deflection signal and displacement were recorded for calculating its elastic modulus. An intact AC exhibited a gradation in elastic modulus of collagen fibrils from articular surface (2.65 ± 0.31 GPa) to the cartilage-bone interface (3.70 ± 0.44 GPa). It was noted in mildly degenerated OA cartilage that the coefficient of variation for mechanical properties of collagen fibers, ranging from 25% to 48%, significantly increased as compared with intact one (12%). The stiffened collagen fibrils occurred at either articular surface (3.11 ± 0.91 GPa) or the cartilage-bone interface (5.64 ± 1.10 GPa), accompanied by loosely organized meshwork with advancement of OA cartilage degeneration. It was echoed by histological findings of OA cartilage, including fibrotic changes of surface region and tidemark irregularities. The stiffened collagen fibrils in AC occurred with OA onset and progression, not only at articular surface but also the cartilage-bone interface.

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

Functional 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. Bovine 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. The 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. This 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.

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 cartilage's 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 composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's 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 cartilage's 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.

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.

The measurement of biomechanical properties of porcine articular cartilage using atomic force microscopy

We have recently demonstrated that indentation-type atomic force microscopy (IT-AFM) is capable of detecting early onset osteoarthritis (OA) (Stolz, 2009). This study was based on biopsies, using a desk-top commercial atomic force microscope (AFM). However, cartilage analysis in the knee joints needs to be non-destructive to avoid new seeding points for OA by the taking of biopsies. This requires bringing the probe tip in contact with the articular cartilage (AC) surface inside the joint. Here we present our recent progress towards a medical instrument for performing such IT-AFM measurements for in-vivo knee diagnostics. The scanning force arthroscope (SFA) integrates a miniaturized AFM into a standard arthroscopic sleeve, and is used for direct, quantitative, in situ inspection of AC (Imer et al., 2006). The stabilization and the positioning of the instrument relative to the surface under investigation were performed by means of eight inflatable balloons. An integrated three-dimensional, piezoelectric scanner allowed raster scanning and probing of a small area of cartilage around the point of insertion. An AFM probe with an integrated deflection sensor was mounted at the distal end of the instrument. Using this instrument, several measurements were performed on agarose gel and on porcine cartilage samples. The load-displacement curves obtained were analyzed and the dynamic elastic moduli | E(*) | were calculated. A good correlation between these values and those published in the scientific literature was found. Therefore, we concluded that the SFA can provide quantitative measurements to detect early pathological changes in OA.

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 = ∼2.5 μm) and sharp pyramidal tips (radius = ∼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 ∼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.