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Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N -acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9- Galns ) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9- Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients. Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disorder causing severe skeletal and non-skeletal alterations in patients. Here, the authors generate a MPSIVA rat model that mimics the disabling human pathology and develop an AAV9-Galns gene therapy to treat the disease.

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.

Osteoarthritis (OA) is a common degenerative musculoskeletal disease highly prevalent in aging societies worldwide. Traditionally, knee OA is diagnosed using conventional radiography. However, structural changes of articular cartilage or menisci cannot be directly evaluated using this method. On the other hand, ultrasound is a promising tool able to provide direct information on soft tissue degeneration. The aim of our study was to systematically determine the site-specific diagnostic performance of semi-quantitative ultrasound grading of knee femoral articular cartilage, osteophytes and meniscal extrusion, and of radiographic assessment of joint space narrowing and osteophytes, using MRI as a reference standard. Eighty asymptomatic and 79 symptomatic subjects with mean age of 57.7 years were included in the study. Ultrasound performed best in the assessment of femoral medial and lateral osteophytes, and medial meniscal extrusion. In comparison to radiography, ultrasound performed better or at least equally well in identification of tibio-femoral osteophytes, medial meniscal extrusion and medial femoral cartilage morphological degeneration. Ultrasound provides relevant additional diagnostic information on tissue-specific morphological changes not depicted by conventional radiography. Consequently, the use of ultrasound as a complementary imaging tool along with radiography may enable more accurate and cost-effective diagnostics of knee osteoarthritis at the primary healthcare level.

Articular cartilage (AC) lacks the ability to self-repair and cell-based approaches, primarily based on using chondrocytes and mesenchymal stem cells (MSCs), are emerging as effective technology to restore cartilage functionality, because cells synergic functionality may support the maintenance of chondrogenic phenotype and promote extracellular matrix regeneration. This work aims to develop a more physiologically representative co-culture system to investigate the influence of MSCs on the activity of chondrocytes. A thermo-sensitive chitosan-based hydrogel, ionically crosslinked with β–glycerophosphate, is optimised to obtain sol/gel transition at physiological conditions within 5 minutes, high porosity with pores diameter <30 µm, and in vitro mechanical integrity with compressive and equilibrium Young’s moduli of 37 kPa and 17 kPa, respectively. Live/dead staining showed that after 1 and 3 days in culture, the encapsulated MSCs into the hydrogels are viable and characterised by round-like morphology. Furthermore chondrocyte spheroids, seeded on top of gels that contained either MSCs or no cells, show that the encapsulated MSCs stimulate chondrocyte activity within a gel co-culture, both in terms of maintaining the coherence of chondrocyte spheroids, leading to a larger quantity of CD44 (by immunofluorescence) and a higher production of collagen and glycosaminoglycans (by histology) compared with the mono-culture.

Background The surface microstructures of cartilage are closely related to its functions. Accurate observation of cartilage surface is key to researching cartilage-related diseases. Traditional observation methods have limitations: conventional optical microscopes have a narrow depth of field and cannot clearly image the uneven cartilage surface; other methods either have low resolution or damage the sample. Ultra depth of field microscopy(UDFM)is a new high-precision method for cartilage surface research. It can non-invasively show clear 3D microstructural details of the cartilage surface without damaging the sample. At present, its application in cartilage surface research is still in the exploration stage, and it is of great significance to use this new method for related research. Methods Fresh porcine knee joints and discarded cartilage tissue from human total knee arthroplasty were utilized to prepare a range of cartilage damage models and sectioned specimens. UDFM was employed to examine the cartilage surface, cross-sections, and lateral profiles at various magnifications (10–2000×), generating both two-dimensional and three-dimensional topographic images. The integrated UDFM software was used for qualitative assessment of surface morphology, cellular architecture, and damage characteristics, as well as for quantitative measurements of height differences, surface roughness, fissure length, and stained area. Results UDFM provides non-destructive and direct visualization of the authentic three-dimensional morphology and subtle surface damage of articular cartilage, with 3D topographic mapping precisely delineating the depth and extent of lesions. This technology enables continuous, high-resolution observation of the macroscopic appearance, surface microarchitecture, and cellular morphology of cartilage across a broad range of magnifications. In fresh specimens, it reliably reveals in situ paired or clustered chondrocytes. The OPt-SEM special lighting mode further enhances the detection sensitivity for subtle surface damage. UDFM delivers rapid and highly accurate quantitative measurements of surface height differences, roughness, fissure length, and stained area, all with excellent reproducibility. In comparison to conventional techniques, UDFM is straightforward to operate, eliminates the need for complex sample preparation, and preserves the native structure of cartilage, thereby effectively addressing the inherent limitations of stereomicroscopy and scanning electron microscopy. Conclusion Ultra depth of field microscopy is an efficient, non-destructive, and precise tool for three-dimensional observation of cartilage surfaces, offering high resolution, large depth of field, convenient operation, and strong quantitative capabilities. This technology can comprehensively and authentically reflect the microstructure and damage characteristics of cartilage surfaces, providing a new technical approach and objective quantitative method for basic and clinical cartilage research, early disease diagnosis, and evaluation of repair efficacy, with broad application prospects.

Objective This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Methods Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Results Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. Conclusions This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell–cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.

The extracellular matrix (ECM) of articular cartilage is structurally and mechanically inhomogeneous and anisotropic, exhibiting variations in composition, collagen fiber architecture, and pericellular matrix (PCM) morphology among the different zones (superficial, middle, and deep). Joint loading exposes chondrocytes to a complex biomechanical environment, as the microscale mechanical environment of the chondrocyte depends on the relative properties of its PCM and local ECM. ECM anisotropy and chondrocyte deformation are influenced by the split-line direction, the preferred collagen fiber orientation parallel to the articular surface. While previous studies have demonstrated that cartilage macroscale properties vary with depth and the direction of loading relative to the split-line direction, the potential anisotropic behavior of the ECM and PCM at the microscale has yet to be examined. The goal of this study was to characterize the depth and directional dependence of the microscale biomechanical properties of porcine cartilage ECM and PCM in situ. Cartilage was cryosectioned to generate samples oriented parallel and perpendicular to the split-line direction and normal to the articular surface. Atomic force microscopy (AFM)-based stiffness mapping was utilized to measure ECM and PCM microscale elastic properties in all three directions within each zone. Distinct anisotropy in ECM elastic moduli was observed in the superficial and deep zones, while the middle zone exhibited subtle anisotropy. PCM elastic moduli exhibited zonal uniformity with depth and directional dependence when pooled across the zones. These findings provide new evidence for mechanical inhomogeneity and anisotropy at the microscale in articular cartilage.

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 . Atomic force microscopy can be used to detect the early onset of osteoarthritis in cartilage samples obtained from mice and patients, well before conventional diagnosis methods. This work could lead to a minimally invasive tool for the early detection of osteoarthritis and the development of more effective therapies for treating this disease.

Ozone (O3) is an oxidizing natural gas widely applied as adjunctive therapeutic treatment for a variety of pathological conditions. Currently, O3-based therapies rely on the low-dose concept i.e., the administration of low O3 concentrations able to induce a mild oxidative stress stimulating antioxidant and anti-inflammatory response without causing cell damage. In addition, low O3 concentrations are thought to activate cellular and molecular mechanisms responsible for analgesic and regenerative effects. Due to these properties, in the last decade interest has arisen in the fields of orthopedics and regenerative medicine on the potential of O3 to counteract joint diseases involving cartilage degeneration. In this pilot study, we have explored the anti-degenerative potential of O3 on knee articular cartilage explanted from a healthy adult rabbit and maintained in vitro. Light and transmission electron microscopy were used to monitor chondrocyte and extracellular matrix features of cartilage samples undergoing O3 treatment every three days for two weeks. Results demonstrated that low O3 concentrations act on chondrocytes and the molecular components of the extracellular matrix of articular cartilage explants, significantly improving their preservation under in vitro conditions, likely by promoting both protective and pro-regenerative pathways. This opens promising perspectives for further investigations on the therapeutic potential of O3 for the treatment of cartilage degeneration not only as painkilling and anti-inflammatory agent but also as a cartilage regenerative agent.

Atomic force microscopy (AFM) is an easy-to-use, powerful, high-resolution microscope that allows the user to image any surface and under any aqueous condition. AFM has been used in the investigation of the structural and mechanical properties of a wide range of biological matters including biomolecules, biomaterials, cells, and tissues. It provides the capacity to acquire high-resolution images of biosamples at the nanoscale and allows at readily carrying out mechanical characterization. The capacity of AFM to image and interact with surfaces, under physiologically relevant conditions, is of great importance for realistic and accurate medical and pharmaceutical applications. The aim of this paper is to review recent trends of the use of AFM on biological materials related to health and sickness. First, we present AFM components and its different imaging modes and we continue with combined imaging and coupled AFM systems. Then, we discuss the use of AFM to nanocharacterize collagen, the major fibrous protein of the human body, which has been correlated with many pathological conditions. In the next section, AFM nanolevel surface characterization as a tool to detect possible pathological conditions such as osteoarthritis and cancer is presented. Finally, we demonstrate the use of AFM for studying other pathological conditions, such as Alzheimer’s disease and human immunodeficiency virus (HIV), through the investigation of amyloid fibrils and viruses, respectively. Consequently, AFM stands out as the ideal research instrument for exploring the detection of pathological conditions even at very early stages, making it very attractive in the area of bio- and nanomedicine.