application-of-atomic-force-microscopy-to-detect-early-osteoarthritis

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

optical-sectioning-visualization-intervertebral-disc-from-embryonic

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

injection-porcine-adipose-tissue-derived-stroma-cells-via-waterjet

Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine ...

addressing-practical-issues-atomic-force-microscopy-based-micro

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

5-aminolevulinic-acid-mediated-photodynamic-therapy-on-primary-bone

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

University Hospital of Tübingen

University Hospital Tübingen

University Hospital Tübingen

A type VI collagen-rich pericellular matrix (PCM) encloses both intervertebral disk (IVD) and articular cartilage chondrocytes. In the latter, the PCM protects the chondrocytes from mechanical overload, whereas tissue degeneration is associated with PCM destruction. As little is known about the IVD PCM, we investigated chondrocyte survival after mechanical overload as well as PCM structural integrity as a function of clinical tissue degeneration. The hypothesis was that IVD degeneration may affect PCM integrity and overload-related chondrocyte survival. Cylindrical human IVD explants from patients undergoing surgical procedures for lumbar disk degeneration, disk prolapse, or spinal trauma were generated and scored. Mechanical overload was applied by single uniaxial 50% compression followed by immediate release, and the explants were live-dead stained (n = 20 explants). Type VI collagen, the major PCM component, was fluorescent stained and the extent was determined, in which individual cells were enclosed by a recognizable PCM; this was termed PCM fraction. More than 50% of chondrocytes in all degenerative IVD explants displayed <25% PCM fraction and a lower PCM fraction correlated with higher cell numbers (p < 0.001), suggesting a PCM structural impairment in IVD degeneration that is associated with chondrocyte clustering. Mechanical overload-induced significantly increased cell death (p = 0.005), and the PCM fraction was significantly lower in overload-induced cell death than in live cells (p = 0.042), suggesting that a fully present PCM has a protective role in mechanical overload. Collectively, human IVD degeneration is associated with a structural impairment of the PCM, which may promote cell death under mechanical overload.

Chondrocyte death after mechanically overloading degenerated human intervertebral disk explants is associated with a structurally impaired pericellular matrix.

During osteoarthritis (OA)-triggered cartilage degeneration, the chondrocytes spatially rearrange from single to double strings, and then to small and finally big clusters. Both the extracellular matrix (ECM) and the pericellular matrix (PCM) progressively degrade in osteoarthritis, changing the overall mechanical properties of the cartilage. We investigated the mechanical properties particularly elasticity of the ECM and PCM and their interconnection as a function of chondrocyte spatial organisation. Human articular cartilage samples from 30 patients were categorised according to their cellular pattern. Elasticity of the ECM and PCM was assessed by means of atomic force microscopy (AFM). Significant decreases were observed in the elasticity of both the ECM and the PCM with each change of cellular pattern, except from single to double strings in the ECM (p = 0.072). Spatial reorganisation strongly correlated with the elasticity of the ECM (r = -0.768, p < 0.001) and of the PCM (r = -0.729, p < 0.001). The ECM/PCM ratio remained unchanged (r = -0.099, p = 0.281). This study is the first to describe and quantify the differences in the elastic moduli of the ECM in relation to the PCM on the basis of chondrocyte spatial arrangement. This study shows that the elastic changes of the ECM and the PCM occur simultaneously, unidirectionally, and to a comparable degree.

Assessment of biomechanical properties of the extracellular and pericellular matrix and their interconnection throughout the course of osteoarthritis.

During osteoarthritis, chondrocytes change their spatial arrangement from single to double strings, then to small and big clusters. This change in pattern has recently been established as an image-based biomarker for osteoarthritis. The pericellular matrix (PCM) appears to degrade together alongside cellular reorganization. The aim of this study was to characterize this PCM-degradation based on different cellular patterns. We additionally wanted to identify the earliest time point of PCM-breakdown in this physiopathological model. To this end, cartilage samples were selected according to their predominant cellular pattern. Qualitative analysis of PCM degradation was performed immunohistochemically by analysing five main PCM components: collagen type VI, perlecan, collagen type III, biglycan, and fibrillin-1 (n = 6 patients). Their protein content was quantified by enzyme-linked immunosorbent assay (127 patients). Accompanying spatial cellular rearrangement, the PCM is progressively destroyed, with a pericellular signal loss in fluorescence microscopy for collagen type VI, perlecan, and biglycan. This loss in protein signal is accompanied by a reduction in total protein content from single strings to big clusters (P < .001 for collagen type VI, P = .003 for perlecan, and P < .001 for biglycan). As a result of an increase in the number of cells from single strings to big clusters, the amount of protein available per cell also decreases for collagen type III and fibrillin-1, where total protein levels remain constant. Biochemical changes of the PCM and cellular rearrangement are thus highly interconnected hallmarks of osteoarthritis. Interestingly, the earliest point in time for a relevant PCM impairment appears to be at the transition to small clusters.

Biochemical changes of the pericellular matrix and spatial chondrocyte organization-Two highly interconnected hallmarks of osteoarthritis.

Previously, we developed a novel, needle-free waterjet (WJ) technology capable of injecting viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we aimed to investigate the effect of WJ technology on cell viability, surface markers, differentiation and attachment capabilities, and biomechanical features. Porcine adipose tissue-derived stromal cells (pADSCs) were isolated, expanded, and injected by WJ technology. Cell attachment assays were employed to investigate cell-matrix interactions. Cell surface molecules were analyzed by flow cytometry. Cells injected by Williams Needle (WN), normal cannula, or not injected cells served as controls. Biomechanical properties were assessed by atomic force microscopy (AFM). pADSCs injected by the WJ were viable (85.9%), proliferated well, and maintained their in vitro adipogenic and osteogenic differentiation capacities. The attachment of pADSCs was not affected by WJ injection and no major changes were noted for cell surface markers. AFM measurements yielded a significant reduction of cellular stiffness after WJ injections ( < 0.001). WJ cell delivery satisfies several key considerations required in a clinical context, including the fast, simple, and reproducible delivery of viable cells. However, the optimization of the WJ device may be necessary to further reduce the effects on the biomechanical properties of cells.

Injection of Porcine Adipose Tissue-Derived Stromal Cells by a Novel Waterjet Technology.

Using two-dimensional top-down view microscopy, researchers have recently described chondrocytes as being spatially arranged in distinct patterns such as strings, double strings, and small and large clusters. Because of the seeming association of these changes with tissue degeneration, they have been proposed as an image-based biomarker for early osteoarthritis (OA) staging. The aim of our study was to investigate the spatial arrangement of chondrocytes in human articular cartilage in a 3D fashion and to evaluate the 3D changes of these patterns in the context of local tissue destruction. Decalcified femoral condyle resections from the load-bearing area were analysed in 3D for their spatial chondrocyte organisation by means of fluorescence microscopy and synchrotron-radiation micro-computed tomography (SR-µCT). In intact cartilage chondrocyte strings can be found in the superficial, transitional and deep zones. The proposed pattern changes accompanying tissue destruction could be located not just along the surface but also through all layers of cartilage. Each spatial pattern was characterised by a different cellular density (the only exception being between single and double strings with p = 0.062), with cellular density significantly increasing alongside the increase in local tissue degeneration as defined by the chondrocyte patterns. We can thus corroborate that the proposed cellular spatial changes are a three-dimensional function of local tissue degeneration, underlining their relevance as an image-based biomarker for the early diagnosis and description of OA.Clinical trial registration number: Project number of the ethics committee of the University of Tübingen:171/2014BO2.

Exploration of changes in spatial chondrocyte organisation in human osteoarthritic cartilage by means of 3D imaging.

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.

Intravascular Application of Labelled Cell Spheroids: An Approach for Ischemic Peripheral Artery Disease.

Bone is a frequent site of metastases, being typically associated with a short-term prognosis in affected patients. Photodynamic therapy (PDT) emerges as a promising alternative treatment for controlling malignant disease that can directly target interstitial metastatic lesions. The aim of this study was to assess the effect induced by PDT treatment on both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma). After 24 h post light delivery (blue light-wavelength 436 nm) with 5-aminolevulinic acid, the effect on cellular migration, viability, apoptosis, and senescence were assessed. Our results showed that bone metastasis derived from breast cancer reacted with an inhibition of cell migration coupled with reduced viability and signs of apoptosis such as nuclei fragmentation following PDT exposure. A limited effect in terms of cellular viability inhibition was observed for the cells of giant cell bone tumors. In contrast, bone metastasis derived from renal carcinoma followed a different fate-cells were characterized by senescent features, without a notable effect on cell migration or viability. Collectively, our study illustrates that PDT could act as a successful therapy concept for local tumor control in some entities of bone metastases.

Assessment of 5-Aminolevulinic Acid-Mediated Photodynamic Therapy on Bone Metastases: An in Vitro Study.

Osteoarthritis (OA) is a degenerative joint disease currently affecting half of all women and one-third of all men aged over 65 and it is predicted to even increase in the next decades. In the variety of causes leading to OA, the first common denominator are changes in the extracellular matrix of the cartilage. In later stages, OA affects the whole joint spreading to higher levels of tissue architecture causing irreversible functional and structural damage. To date, the diagnosis of OA is only formulated in the late stages of the disease. This is also, where most present therapies apply. Since a precise diagnosis is a prerequisite for targeted therapy, tools to diagnose early OA, monitor its progression, and accurately stage the disease are wanted. This review article focuses on recent advances in indentation technologies to diagnose early OA through describing biomechanical cartilage characteristics. We provide an overview of microindentation instruments, indentation-type Atomic Force Microscopy, ultrasound, and water-jet ultrasound indentation, Optical Coherence Tomography-based air-jet indentation, as well as fiber Bragg grating.

Detecting early osteoarthritis through changes in biomechanical properties - A review of recent advances in indentation technologies in a clinical arthroscopic setup.

ArticleTotal : 5	Year
Publication title Cited by 5	2020
Publication title	2021
Publication title	2021
Publication title Cited by 1	2022
Publication title Cited by 1	2022

Article	Year
Chondrocyte death after mechanically overloading degenerated human intervertebral disk explants is associated with a structurally impaired pericellular matrix. Journal of tissue engineering and regenerative medicine\| PubMed ID: 30053767	2018
Assessment of biomechanical properties of the extracellular and pericellular matrix and their interconnection throughout the course of osteoarthritis. Journal of biomechanics\| PubMed ID: 31629545	2019
Biochemical changes of the pericellular matrix and spatial chondrocyte organization-Two highly interconnected hallmarks of osteoarthritis. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 32301522	2020
Injection of Porcine Adipose Tissue-Derived Stromal Cells by a Novel Waterjet Technology. International journal of molecular sciences\| PubMed ID: 33921246	2021
Exploration of changes in spatial chondrocyte organisation in human osteoarthritic cartilage by means of 3D imaging. Scientific reports\| PubMed ID: 33963289	2021
Intravascular Application of Labelled Cell Spheroids: An Approach for Ischemic Peripheral Artery Disease. International journal of molecular sciences\| PubMed ID: 34202056	2021
Assessment of 5-Aminolevulinic Acid-Mediated Photodynamic Therapy on Bone Metastases: An in Vitro Study. Biology\| PubMed ID: 34681119	2021
Detecting early osteoarthritis through changes in biomechanical properties - A review of recent advances in indentation technologies in a clinical arthroscopic setup. Journal of biomechanics\| PubMed ID: 35042088	2022

Marina Danalache

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