Comparison of Compressive Stress-Relaxation Behavior in Osteoarthritic (ICRS Graded) Human Articular Cartilage.

Osteoarthritis (OA) is a common joint disorder found mostly in elderly people. The role of mechanical behavior in the progression of OA is complex and remains unclear. The stress-relaxation behavior of human articular cartilage in clinically defined osteoarthritic stages may have importance in diagnosis and prognosis of OA. In this study we investigated differences in the biomechanical responses among human cartilage of ICRS grades I, II and III using polymer dynamics theory. We collected 24 explants of human articular cartilage (eight each of ICRS grade I, II and III) and acquired stress-relaxation data applying a continuous load on the articular surface of each cartilage explant for 1180 s. We observed a significant decrease in Young's modulus, stress-relaxation time, and stretching exponent in advanced stages of OA (ICRS grade III). The stretch exponential model speculated that significant loss in hyaluronic acid polymer might be the reason for the loss of proteoglycan in advanced OA. This work encourages further biomechanical modelling of osteoarthritic cartilage utilizing these data as input parameters to enhance the fidelity of computational models aimed at revealing how mechanical behaviors play a role in pathogenesis of OA.

Non-linear optical microscopy of cartilage canals in the distal femur of young pigs may reveal the cause of articular osteochondrosis.

BACKGROUND: Articular osteochondrosis is a common cause of leg weakness in pigs and is defined as a focal delay in the endochondral ossification of the epiphysis. The first demonstrated steps in the pathogenesis consist of loss of blood supply and subsequent chondronecrosis in the epiphyseal growth cartilage. Blood vessels in cartilage are located in cartilage canals and become incorporated into the secondary ossification centre during growth. It has been hypothesized that vascular failure occurs during this incorporation process, but it is not known what predisposes a canal to fail. To obtain new information that may reveal the cause of vascular failure, the distal femur of 4 pigs aged 82-140 days was sampled and examined by non-linear optical microscopy. This novel technique was used for its ability to reveal information about collagen by second harmonic generation and cellular morphology by two-photon-excited fluorescence in thick sections without staining. The aims were to identify morphological variations between cartilage canal segments and to examine if failed cartilage canals could be followed back to the location where the blood supply ceased. RESULTS: The cartilage canals were shown to vary in their content of collagen fibres (112/412 segments), and the second harmonic and fluorescence signals indicated a variation in the bundling of collagen fibrils (245/412 segments) and in the calcification (30/412 segments) of the adjacent cartilage matrix. Failed cartilage canals associated with chondronecrosis were shown to enter the epiphyseal growth cartilage from not only the secondary ossification centre, but also the attachment site of the caudal cruciate ligament. CONCLUSION: The variations between cartilage canal segments could potentially explain why the blood supply fails at the osteochondral junction in only a subset of the canals. Proteins linked to these variations should be examined in future genomic studies. Although incorporation can still be a major cause, it could not account for all cases of vascular failure. The role of the caudal cruciate ligament in the cause of osteochondrosis should therefore be investigated further.

Second harmonic generation imaging reveals a distinct organization of collagen fibrils in locations associated with cartilage growth.

PURPOSE: The articular-epiphyseal cartilage complex (AECC) is responsible for the expansion of the bone ends and serves the function of the articular cartilage in juvenile mammals. Bundles of collagen fibrils surrounding cells were in the literature observed more frequently near the articular surface of the AECC. The articular surface, the perichondrium, and cartilage canals are interfaces where appositional growth of the AECC has been demonstrated. The current study aimed to evaluate the potential of second harmonic generation (SHG) to locate the collagen fibril bundles near the articular surface and to examine whether a comparable collagen fibril organization could be observed near the other interfaces of the AECC. MATERIALS AND METHODS: The study included the femoral condyle of four piglets aged 82-141 days. The forward and backward scattered SHG, and their ratio, was analyzed across the AECC using objectives with different numerical aperture. Two-photon-excited fluorescence was used to visualize cells. RESULTS: A similar pattern of collagen fibril organization was observed near the articular surface, around cartilage canals, and adjacent to the perichondrium. The pattern consisted of a higher ratio of forward to backward scattered SHG that increased relative to the surrounding matrix at lower numerical aperture. This was interpreted to reflect collagen fibril bundles in the territorial matrix of cells in these areas. CONCLUSIONS: The observed arrangement of collagen fibrils was suggested to be related to the presumed different growth activity in these areas and indicated that SHG may be used as an indirect and label-free marker for cartilage matrix growth.

Optical investigation of osteoarthritic human cartilage (ICRS grade) by confocal Raman spectroscopy: a pilot study.

Biomolecular changes in the cartilage matrix during the early stage of osteoarthritis may be detected by Raman spectroscopy. The objective of this investigation was to determine vibrational spectral differences among different grades (grades I, II, and III) of osteoarthritis in human osteoarthritic cartilage, which was classified according to the International Cartilage Repair Society (ICRS) grading system. Degenerative articular cartilage samples were collected during total joint replacement surgery and were classified according to the ICRS grading system for osteoarthritis. Twelve cartilage sections (4 sections of each ICRS grades I, II, and III) were selected for Raman spectroscopic analysis. Safranin-O/Fast green was used for histological staining and assignment of the Osteoarthritis Research Society International (OARSI) grade. Multivariate principal component analysis (PCA) was used for data analysis. Spectral analysis indicates that the content of disordered coil collagen increases significantly during the early progression of osteoarthritis. However, the increase was not statistically significant during later stages of the disease. A decrease in the content of proteoglycan was observed only during advanced stages of osteoarthritis. Our investigation shows that Raman spectroscopy can classify the different stage of osteoarthritic cartilage and can provide details on biochemical changes. This proof-of-concept study encourages further investigation of fresh cartilage on a larger population using fiber-based miniaturized Raman probe for the development of in vivo Raman arthroscopy as a potential diagnostic tool for osteoarthritis.

Single cell confocal Raman spectroscopy of human osteoarthritic chondrocytes: a preliminary study.

A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA) especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS) grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes.

Characterization of cholesterol crystals in atherosclerotic plaques using stimulated Raman scattering and second-harmonic generation microscopy.

Cholesterol crystals (ChCs) have been identified as a major factor of plaque vulnerability and as a potential biomarker for atherosclerosis. Yet, due to the technical challenge of selectively detecting cholesterol in its native tissue environment, the physiochemical role of ChCs in atherosclerotic progression remains largely unknown. In this work, we demonstrate the utility of hyperspectral stimulated Raman scattering (SRS) microscopy combined with second-harmonic generation (SHG) microscopy to selectively detect ChC. We show that despite the polarization sensitivity of the ChC Raman spectrum, cholesterol monohydrate crystals can be reliably discriminated from aliphatic lipids, from structural proteins of the tissue matrix and from other condensed structures, including cholesteryl esters. We also show that ChCs exhibit a nonvanishing SHG signal, corroborating the noncentrosymmetry of the crystal lattice composed of chiral cholesterol molecules. However, combined hyperspectral SRS and SHG imaging reveals that not all SHG-active structures with solidlike morphologies can be assigned to ChCs. This study exemplifies the merit of combining SRS and SHG microscopy for an enhanced label-free chemical analysis of crystallized structures in diseased tissue.

Quantifying the hydroxyapatite orientation near the ossification front in a piglet femoral condyle using X-ray diffraction tensor tomography.

While a detailed knowledge of the hierarchical structure and morphology of the extracellular matrix is considered crucial for understanding the physiological and mechanical properties of bone and cartilage, the orientation of collagen fibres and carbonated hydroxyapatite (HA) crystallites remains a debated topic. Conventional microscopy techniques for orientational imaging require destructive sample sectioning, which both precludes further studies of the intact sample and potentially changes the microstructure. In this work, we use X-ray diffraction tensor tomography to image non-destructively in 3D the HA orientation in a medial femoral condyle of a piglet. By exploiting the anisotropic HA diffraction signal, 3D maps showing systematic local variations of the HA crystallite orientation in the growing subchondral bone and in the adjacent mineralized growth cartilage are obtained. Orientation maps of HA crystallites over a large field of view (~ 3 × 3 × 3 mm3) close to the ossification (bone-growth) front are compared with high-resolution X-ray propagation phase-contrast computed tomography images. The HA crystallites are found to predominantly orient with their crystallite c-axis directed towards the ossification front. Distinct patterns of HA preferred orientation are found in the vicinity of cartilage canals protruding from the subchondral bone. The demonstrated ability of retrieving 3D orientation maps of bone-cartilage structures is expected to give a better understanding of the physiological properties of bones, including their propensity for bone-cartilage diseases.

The zonal evolution of collagen-network morphology quantified in early osteoarthritic grades of human cartilage.

Objective: We aimed to directly quantify the zone-specific evolution in morphology of collagen fibers and networks in human cartilage during the progression of early osteoarthritis. Collagen fibers exhibit depth-dependent orientations and diameters crucial to their mechanical roles. Cartilage degenerates in osteoarthritis, affecting the morphology of the collagen network and ultimately the intra-tissue mechanics. Design: We obtained specimens of human cartilage from healthy human knees ( n = 3 ) and from total knee arthroplasties ( n = 5 ). We utilized TEM and custom image analyses to visualize and quantify distributions in principal orientation, dispersion (about the principal orientation), and diameter of collagen fibers in the early grades of OA within each through-thickness zone. We then used histological and statistical analyses to probe for significant changes in the zone-specific evolution in collagen-network morphology as a function of Osteoarthritis Research Society International (OARSI) grade. Results: Dispersion in the alignment of collagen fibers increased with progression of early OA in both the superficial and deep zones, and decreased in the middle zone, while principal orientation did not change significantly. The non-normal and right-skewed distributions in fiber diameters did not evolve with the progression of OA. Conclusions: We provide the research community with quantitative data (1) on the through-thickness morphology of collagen in healthy cartilage and (2) on the evolution of through-thickness morphology of collagen with progressing early OA. Such quantitative data facilitate an improved mechanistic understanding of the progression of OA, and may facilitate identifying image-based biomarkers and treatment targets, and ultimately finding clinical interventions for OA.

Analyzing the feasibility of discriminating between collagen types I and II using polarization-resolved second harmonic generation.

According to previous studies, the nonlinear susceptibility tensor ratio χ33 /χ31 obtained from polarization-resolved second harmonic generation (P-SHG) under the assumption of cylindrical symmetry can be used to distinguish between fibrillar collagen types. Discriminating between collagen fibrils of types I and II is important in tissue engineering of cartilage. However, cartilage has a random organization of collagen fibrils, and the assumption of cylindrical symmetry may be incorrect. In this study, we simulated the P-SHG response from different collagen organizations and demonstrated a possible method to exclude areas where cylindrical symmetry is not fulfilled and where fibrils are located in the imaging plane. The χ33 /χ31 -ratio for collagen type I in tendon and collagen type II in cartilage was estimated to be 1.33 and 1.36, respectively, using this method. These ratios are now much closer than what has been reported previously in the literature, and the larger reported differences between collagen types can be explained by variation in the structural organization.

Characterization of cellular and matrix alterations in the early pathogenesis of osteochondritis dissecans in pigs using second harmonic generation and two-photon excitation fluorescence microscopy.

Osteochondritis dissecans is a joint disease that is observed in several species. The disease can develop as a cause of ischemic chondronecrosis in the epiphyseal growth cartilage. Some lesions of chondronecrosis undergo spontaneous resolution, but it is not possible to predict whether a lesion will resolve or progress and require intervention. Proliferation of cells into clusters occurs at the lesion margin, but it is unclear if the clusters have a repair function. The aims of the current study were to examine clusters and potential matrix changes in response to ischemic chondronecrosis in the distal femur of 10 pigs aged 70-180 days using advanced microscopy based on two-photon excitation fluorescence and second harmonic generation. These microscopy techniques can perform 3D imaging of cells and collagen without staining. The results indicated a lower collagen density in the chondronecrotic areas compared to the normal growth cartilage, and fissures and breaks in the matrix integrity were demonstrated that potentially can propagate and cause osteochondritis dissecans. A higher number of cells in clusters was correlated with reduction in collagen density in the lesions. Some of the cells in the clusters had a morphology similar to progenitor cells, suggesting a potential repair role of the clusters. The study has shed further light on the secondary responses after initial lesion formation, which information can be of potential use to create models that can predict lesion progression and that may hence avoid unnecessary interventions in the future. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes.

Polarization-resolved second harmonic generation (P-SHG) microscopy has evolved as a promising technique to reveal subresolution information about the structure and orientation of ordered biological macromolecules. To extend the adoption of the technique, it should be easily integrated onto commercial laser scanning microscopes. Furthermore, procedures for easy calibration and assessment of measurement accuracy are essential, and measurements should be fully automated to allow for analysis of large quantities of samples. In this paper we present a setup for P-SHG which is readily incorporated on commercial multiphoton microscopes. The entire system is completely automated which allows for rapid calibration through the freely available software and for automated imaging for different polarization measurements, including linear and circular polarization of the excitation beam. The results show that calibration settings are highly system dependent. We also show that the accuracy of the polarization control is easily quantified and that it varies between systems. The accuracy can be tuned by iterative alignment of optics or a more fine-grained calibration procedure. Images of real samples show that the red accuracy of the results is easily visualized with the automated setup. Through this system we believe that P-SHG could develop a wider adoption in biomedical applications.

Reflection spectroscopy of atherosclerotic plaque.

Heart disease is the primary cause of death in the western world. Many of these deaths are caused by the rupture of vulnerable plaque. Vulnerable plaques are characterized by a large lipid core covered by a thin fibrous cap. One method for detecting these plaques is reflection spectroscopy. Several studies have investigated this method using statistical methods. A more analytic and quantitative study might yield more insight into the sensitivity of this detection modality. This is the approach taken in this work. Reflectance spectra in the spectral region from 400 to 1700 nm are collected from 77 measurement points from 23 human aortas. A measure of lipid content in a plaque based on reflection spectra is presented. The measure of lipid content is compared with the thickness of the lipid core, determined from histology. Defining vulnerable plaque as having a lipid core >500 microm and fibrous cap <500 microm, vulnerable plaques are detected with a sensitivity of 88% and a specificity of 94%. Although the method can detect lipid content, it is not very sensitive to the thickness of the fibrous cap. Another detection modality is necessary to detect this feature.

Novel imaging technologies for characterization of microbial extracellular polysaccharides.

Understanding of biology is underpinned by the ability to observe structures at various length scales. This is so in a historical context and is also valid today. Evolution of novel insight often emerges from technological advancement. Recent developments in imaging technologies that is relevant for characterization of extraceullar microbiological polysaccharides are summarized. Emphasis is on scanning probe and optical based techniques since these tools offers imaging capabilities under aqueous conditions more closely resembling the physiological state than other ultramicroscopy imaging techniques. Following the demonstration of the scanning probe microscopy principle, novel operation modes to increase data capture speed toward video rate, exploitation of several cantilever frequencies, and advancement of utilization of specimen mechanical properties as contrast, also including their mode of operation in liquid, have been developed on this platform. Combined with steps in advancing light microscopy with resolution beyond the far field diffraction limit, non-linear methods, and combinations of the various imaging modalities, the potential ultramicroscopy toolbox available for characterization of exopolysaccharides (EPS) are richer than ever. Examples of application of such ultramicroscopy strategies range from imaging of isolated microbial polysaccharides, structures being observed when they are involved in polyelectrolyte complexes, aspects of their enzymatic degradation, and cell surface localization of secreted polysaccharides. These, and other examples, illustrate that the advancement in imaging technologies relevant for EPS characterization supports characterization of structural aspects.

Nonlinear optical microscopy of early stage (ICRS Grade-I) osteoarthritic human cartilage.

In a synovial joint, the articular cartilage is directly affected during the progression of Osteoarthritis (OA). The characterization of early stage modification in extra-cellular matrix of cartilage is essential for detection as well as understanding the progression of disease. The objective of this study is to demonstrate the potential and capability of nonlinear optical microscopy for the morphological investigation of early stage osteoarthritic cartilage. ICRS Grade-I cartilage sections were obtained from the femoral condyle of the human knee. The surface of articular cartilage was imaged by second harmonic generation and two-photon excited fluorescence microscopy. Novel morphological features like microsplits and wrinkles were observed, which would otherwise not be visible in other clinical imaging modalities (e.g., CT, MRI, ultrasound and arthroscope. The presence of superficial layer with distinct collagen fibrils parallel to the articular surface in 4 specimens out of 14 specimens, indicates that different phases of OA within ICRS Grade-I can be detected by SHG microscopy. All together, the observed novel morphologies in early stage osteoarthritic cartilage indicates that SHG microscopy might be a significant tool for the assessment of cartilage disorder.

Combined imaging of oxidative stress and microscopic structure reveals new features in human atherosclerotic plaques.

Human atherosclerotic samples collected by carotid endarterectomy were investigated using electronic paramagnetic resonance imaging (EPRI) for visualization of reactive oxygen species, and nonlinear optical microscopy (NLOM) to study structural features. Regions of strong EPRI signal, indicating a higher concentration of reactive oxygen species and increased inflammation, were found to colocalize with regions dense in cholesterol crystals as revealed by NLOM.

Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics.

Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p-SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p-SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p-SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second-order χ tensor elements. These values may be used as a bio-marker to detect any structural alterations in pathological tissue for diagnostic purposes. The SHG intensity image (red) in (a) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution (b) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.

Alterations in collagen fibre patterns in breast cancer. A premise for tumour invasiveness?

Stromal tissue in the breast plays a key role in cancer invasiveness due to molecular and cellular changes. Collagen is the main component of the stroma. The purposes of this study were to investigate differences in collagen fibre patterns between tumour-induced stromal tissue and normal stroma, and between high-grade and low-grade breast cancer stroma, using second harmonic generation microscopy. Thirty-seven ductal carcinomas were examined: Twenty-one Luminal A phenotype and sixteen HER2 or Basal-like phenotype. Three regions were examined in each case: intratumoral, juxtatumoral and extratumoral. Two images were captured in each region. Two characteristics of collagen fibres were examined: the degree of straightness, and the degree of alignment. Collagen fibres were visually classified as curly, intermediate or straight, and as parallel or not parallel. The results of angle measurement and visual analysis showed that collagen fibres were straightest in the intratumoral region and curliest in the extratumoral region. Collagen fibres were more parallel in the juxtatumoral region compared to the two other regions. There were no significant differences between high-grade and low-grade tumours. As a breast tumour progresses, collagen fibres appear to straighten and align at the tumour boundary. This could facilitate invasion of the tumour into the surrounding stroma.

Biochemical and structural characterization of neocartilage formed by mesenchymal stem cells in alginate hydrogels.

A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM). Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC) immobilized in alginate hydrogels, and used immunohistochemistry (IHC) and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG) microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml) showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide meaningful insight into ECM assembly of neocartilage, which will lead to better understanding of cartilage formation and development of new tissue engineering strategies.

Ultrasound-enhanced drug delivery in prostate cancer xenografts by nanoparticles stabilizing microbubbles.

The delivery of nanoparticles to solid tumors is often ineffective due to the lack of specificity towards tumor tissue, limited transportation of the nanoparticles across the vascular wall and poor penetration through the extracellular matrix of the tumor. Ultrasound is a promising tool that can potentially improve several of the transportation steps, and the interaction between sound waves and microbubbles generates biological effects that can be beneficial for the successful delivery of nanocarriers and their contents. In this study, a novel platform consisting of nanoparticle-stabilized microbubbles has been investigated for its potential for ultrasound-enhanced delivery to tumor xenografts. Confocal laser scanning microscopy was used to study the supply of nanoparticles from the vasculature and to evaluate the effect of different ultrasound parameters at a microscopic level. The results demonstrated that although the delivery is heterogeneous within tumors, there is a significant improvement in the delivery and the microscopic distribution of both nanoparticles and a released model drug when the nanoparticles are combined with microbubbles and ultrasound. The mechanisms that underlie the improved delivery are discussed.

Structural analysis of articular cartilage using multiphoton microscopy: input for biomechanical modeling.

The 3-D morphology of chicken articular cartilage was quantified using multiphoton microscopy (MPM) for use in continuum-mechanical modeling. To motivate this morphological study we propose aspects of a new, 3-D finite strain constitutive model for articular cartilage focusing on the essential load-bearing morphology: an inhomogeneous, poro-(visco)elastic solid matrix reinforced by an anisotropic, (visco)elastic dispersed fiber fabric which is saturated by an incompressible fluid residing in strain-dependent pores. Samples of fresh chicken cartilage were sectioned in three orthogonal planes and imaged using MPM, specifically imaging the collagen fibers using second harmonic generation. Employing image analysis techniques based on Fourier analysis, we derived the principal directionality and dispersion of the collagen fiber fabric in the superficial layer. In the middle layer, objective thresholding techniques were used to extract the volume fraction occupied by extracellular collagen matrix. In conjunction with information available in the literature, or additional experimental testing, we show how this data can be used to derive a 3-D map of the initial solid volume fraction and Darcy permeability.