The deterioration of calcified cartilage integrity reflects the severity of osteoarthritis-A structural, molecular, and biochemical analysis.

The calcified cartilage zone (CCZ) is a thin interlayer between the hyaline articular cartilage and the subchondral bone and plays an important role in maintaining the joint homeostasis by providing biological and mechanical support from unmineralized cartilage to the underlying mineralized subchondral bone. The hallmark of CCZ characteristics in osteoarthritis (OA) is less well known. The aim of our study is to evaluate the structural, molecular, and biochemical composition of CCZ in tissues affected by primary knee OA and its relationship with disease severity. We collected osteochondral tissue samples stratified according to disease severity, from 16 knee OA patients who underwent knee replacement surgery. We also used meniscectomy-induced rat samples to confirm the pathophysiologic changes of human samples. We defined the characteristics of the calcified cartilage layer using a combination of morphological, biochemical, proteomic analyses on laser micro-dissected tissue. Our results demonstrated that the Calcium/Phosphate ratio is unchanged during the OA progression, but the calcium-binding protein and cadherin binding protein, as well as carbohydrate metabolism-related proteins, undergo significant changes. These changes were further accompanied by thinning of the CCZ, loss of collagen and proteoglycan content, the occurrence of the endochondral ossification, neovasculature, loss of the elastic module, loss of the collagen direction, and increase of the tortuosity indicating an altered structural and mechanical properties of the CCZ in OA. In conclusion, our results suggest that the calcified cartilage changes can reflect the disease progression.

Influence of Pore Size and Surface Functionalization of Mesoporous Silica Nanoparticles on the Solubility and Antioxidant Activity of Confined Coenzyme Q10.

Coenzyme Q10 is a potent antioxidant that plays an important role in the maintenance of various biochemical pathways of the body and has a wide range of therapeutic applications. However, it has low aqueous solubility and oral bioavailability. Mesoporous silica nanoparticles (MCM-41 and SBA-15 types) exhibiting varying pore sizes and modified with phosphonate and amino groups were used to study the influence of pore structure and surface chemistry on the solubility, in vitro release profile, and intracellular ROS inhibition activity of coenzyme Q10. The particles were thoroughly characterized to confirm the morphology, size, pore profile, functionalization, and drug loading. Surface modification with phosphonate functional groups was found to have the strongest impact on the solubility enhancement of coenzyme Q10 when compared to that of pristine and amino-modified particles. Phosphonate-modified MCM-41 nanoparticles (i.e., MCM-41-PO3) induced significantly higher coenzyme Q10 solubility than the other particles studied. Furthermore, MCM-41-PO3 led to a twofold decrease in ROS generation in human chondrocyte cells (C28/I2), compared to the free drug in a DMSO/DMEM mixture. The results confirmed the significant contribution of small pore size and negative surface charge of MSNs that enable coenzyme Q10 confinement to allow enhanced drug solubility and antioxidant activity.

Inflammatory macrophages interrupt osteocyte maturation and mineralization via regulating the Notch signaling pathway.

BACKGROUND: It is well-known that both macrophages and osteocytes are critical regulators of osteogenesis and osteoclastogenesis, yet there is limited understanding of the macrophage-osteocyte interaction, and how their crosstalk could affect bone homeostasis and mineralization. This research therefore aims to investigate the effects of macrophage polarization on osteocyte maturation and mineralization process. METHODS: A macrophage-derived conditioned medium based osteocyte culture was set up to investigate the impact of macrophages on osteocyte maturation and terminal mineralization. Surgically induced osteoarthritis (OA) rat model was used to further investigate the macrophage-osteocyte interaction in inflammatory bone remodeling, as well as the involvement of the Notch signaling pathway in the mineralization process. RESULTS: Our results identified that osteocytes were confined in an immature stage after the M1 macrophage stimulation, showing a more rounded morphology, higher expression of early osteocyte marker E11, and significantly lower expression of mature osteocyte marker DMP1. Immature osteocytes were also found in inflammatory bone remodeling areas, showing altered morphology and mineralized structures similar to those observed under the stimulation of M1 macrophages in vitro, suggesting that M1 macrophages negatively affect osteocyte maturation, leading to abnormal mineralization. The Notch signaling pathway was found to be down regulated in M1 macrophage-stimulated osteocytes as well as osteocytes in inflammatory bone. Overexpression of the Notch signaling pathway in osteocytes showed a significant circumvention on the negative effects from M1 macrophage. CONCLUSION: Taken together, our findings provide valuable insights into the mechanisms involved in abnormal bone mineralization under inflammatory conditions.

A technique for preparing undecalcified osteochondral fresh frozen sections for elemental mapping and understanding disease etiology.

The anatomy of the osteochondral junction is complex because several tissue components exist as a unit, including uncalcified cartilage (with superficial, middle, and deep layers), calcified cartilage, and subchondral bone. Furthermore, it is difficult to study because this region is made up of a variety of cell types and extracellular matrix compositions. Using X-ray fluorescence microscopy, we present a protocol for simultaneous elemental detection on fresh frozen samples. We transferred the osteochondral sample using a tape-assisted system and successfully tested it in synchrotron X-ray fluorescence. This protocol elucidates the distinct distribution of elements at the human knee's osteochondral junction, making it a useful tool for analyzing the co-distribution of various elements in both healthy and diseased states.

Application of Metabolomics to Osteoarthritis: from Basic Science to the Clinical Approach.

PURPOSE OF THE REVIEW: Osteoarthritis (OA) is a multifactorial and progressive disease affecting whole synovial joint. The extract pathogenic mechanisms and diagnostic biomarkers of OA remain unclear. In this article, we review the studies related to metabolomics of OA, discuss the biomarkers as a tool for early OA diagnosis. Furthermore, we examine the major studies on the application of metabolomics methodology in the complex context of OA and create a bridge from findings in basic science to their clinical utility. RECENT FINDINGS: Recently, the tissue metabolomics signature permits a view into transitional phases between the healthy and OA joint. Both nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry-based metabolomics approaches have been used to interrogate the metabolic alterations that may indicate the complex progression of OA. Specifically, studies on alterations pertaining to lipids, glucose, and amino acid metabolism have aided in the understanding of the complex pathogenesis of OA. The discovery of identified metabolites could be important for diagnosis and staging of OA, as well as for the assessment of efficacy of new drugs.

Mixed cell therapy of bone marrow-derived mesenchymal stem cells and articular cartilage chondrocytes ameliorates osteoarthritis development.

Of the many cell-based treatments that have been tested in an effort to regenerate osteoarthritic articular cartilage, none have ever produced cartilage that compare with native hyaline cartilage. Studies show that different cell types lead to inconsistent results and for cartilage regeneration to be considered successful, there must be an absence of fibrotic tissue. Here we report of a series of experiments in which bone marrow-derived stem cells (BMSCs) and articular cartilage chondrocytes (ACCs) were mixed in a 1:1 ratio and tested for their ability to enhance cartilage regeneration in three different conditions: (1) in an in vitro differentiation model; (2) in an ex vivo cartilage defect model implanted subcutaneously in mice; and (3) as an intra-articular injection in a meniscectomy-induced OA model in rats. The mixed cells were compared with monocultures of BMSCs and ACCs. In all three experimental models there was significantly enhanced cartilage regeneration and decreased fibrosis in the mixed BMSCs+ACCs group compared with the monocultures. Molecular analysis showed a reduction in vascularization and hypertrophy, coupled with higher chondrogenic gene expression resulting from the BMSCs+ACCs treatment. Together, our data suggest that mixed BMSCs+ACCs treatment is highly chondro-protective and is more effective in regenerating damaged cartilage in both the ex vivo cartilage defect and post-trauma OA disease models. The results from this approach could potentially be used for regeneration of cartilage in OA patients.

Protective effects of mitochondria-targeted antioxidants and statins on cholesterol-induced osteoarthritis.

The contribution of metabolic factors on the severity of osteoarthritis (OA) is not fully appreciated. This study aimed to define the effects of hypercholesterolemia on the progression of OA. Apolipoprotein E-deficient (ApoE-/-) mice and rats with diet-induced hypercholesterolemia (DIHC) rats were used to explore the effects of hypercholesterolemia on the progression of OA. Both models exhibited OA-like changes, characterized primarily by a loss of proteoglycans, collagen and aggrecan degradation, osteophyte formation, changes to subchondral bone architecture, and cartilage degradation. Surgical destabilization of the knees resulted in a dramatic increase of degradative OA symptoms in animals fed a high-cholesterol diet compared with controls. Clinically relevant doses of free cholesterol resulted in mitochondrial dysfunction, overproduction of reactive oxygen species (ROS), and increased expression of degenerative and hypertrophic markers in chondrocytes and breakdown of the cartilage matrix. We showed that the severity of diet-induced OA changes could be attenuated by treatment with both atorvastatin and a mitochondrial targeting antioxidant. The protective effects of the mitochondrial targeting antioxidant were associated with suppression of oxidative damage to chondrocytes and restoration of extracellular matrix homeostasis of the articular chondrocytes. In summary, our data show that hypercholesterolemia precipitates OA progression by mitochondrial dysfunction in chondrocytes, in part by increasing ROS production and apoptosis. By addressing the mitochondrial dysfunction using antioxidants, we were able attenuate the OA progression in our animal models. This approach may form the basis for novel treatment options for this OA risk group in humans.-Farnaghi, S., Prasadam, I., Cai, G., Friis, T., Du, Z., Crawford, R., Mao, X., Xiao, Y. Protective effects of mitochondria-targeted antioxidants and statins on cholesterol-induced osteoarthritis.

Dietary Fats and Osteoarthritis: Insights, Evidences, and New Horizons.

Osteoarthritis (OA) is a progressive, age-related disease characterized by the degradation of the cartilage, abnormal bone remodeling, and joint pain eventually leading to disability. The occurrence of clinically diagnosed OA and the incidence of disability show geographic variations, which suggests that lifestyle and factors such as diet play a vital role in the formation and progression of OA. Obesity is associated with a state of low-grade inflammation and increased plasma concentrations of fatty acids such as the saturated fatty acids (SFA). Importantly, obesity is a major risk factor for the development of OA in both weight-bearing and non-weight-bearing joints. Further, obese individuals bear the full brunt of OA which poses a huge health, social and economic problem, and hence it is essential to increase our understanding of OA and obesity to improve patient care and decrease disease progression. Hence, the current state of knowledge on the relationship between obesity and OA is reviewed, especially the influence of different diets. In particular, we emphasize the role and mechanisms of SFA to cause or worsen OA. J. Cell. Biochem. 118: 453-463, 2017. © 2016 Wiley Periodicals, Inc.

Systematic Identification, Characterization and Target Gene Analysis of microRNAs Involved in Osteoarthritis Subchondral Bone Pathogenesis.

This study aimed to identify the microRNAs associated with sclerotic status of subchondral bone in the pathogenesis of osteoarthritis (OA). Total RNA was extracted from non-sclerotic and sclerotic OA subchondral bone from patients undergoing knee replacement surgeries. miRCURY™ LNA miRNA chip and qRT-PCR were used to profile and validate differential microRNA expression. In addition, we further confirmed profiles of altered miRNAs in an OA rat meniscectomy animal model and their putative targets of the miRNAs were predicted using ingenuity (IPA) software. Finally, five short-listed miRNAs were reactivated by transient in vitro overexpression (miRNA mimics) in subchondral bone osteoblasts and their phenotypes were assessed. Functional screening identified 30 differentiated miRNAs in sclerotic subchondral bone compared to non-sclerotic bone of OA patients. Data integration resulted in confirmation of the eight miRNAs, with aberrant expression in independent human OA bone sample set. In silico analysis (IPA) identified 732 mRNA transcripts as putative targets of the eight altered miRNAs, of which twenty genes were validated to be differentially expressed in sclerotic compared to non-sclerotic bone samples. Out of eight dysregulated miRNA's, five of them showed consistent time-dependent downregulation in a rat OA model. Furthermore, synthetic miR-199a-3p, miR-199a-5p, miR-590-5p, and miR-211-5p mimics rescued the abnormal osteoarthritic subchondral bone osteoblast gene expression and mineralization. We have identified four novel miRNAs that play important roles in subchondral bone pathogenesis in OA. Additional studies are required to develop these miRNAs into therapeutic modalities for OA.

Is Synovial Macrophage Activation the Inflammatory Link Between Obesity and Osteoarthritis?

Osteoarthritis (OA) is the most common musculoskeletal disease, affecting nearly 25 % of the world population (WHO reports), leading to pain and disability. There are as yet no clinically proven therapies to halt OA onset or progression; the development of such therapies is, therefore, a national as well as international research priority. Obesity-related metabolic syndrome has been identified as the most significant, but also an entirely preventable risk factor for OA; however, the mechanisms underlying this link remain unclear. We have examined the available literature linking OA and metabolic syndrome. The two conditions have a shared pathogenesis in which chronic low-grade inflammation of affected tissues is recognized as a major factor that is associated with systemic inflammation. In addition, the occurrence of metabolic syndrome appears to alter systemic and local pro-inflammatory cytokines that are also related to the development of OA-like pathologies. Recent findings highlight the importance not only of the elevated number of macrophage in inflamed synovium but also the activation and amplification of the inflammatory state and other pathological changes. The role of local inflammation on the synovium is now considered to be a pharmacological target against which to aim disease-modifying drugs. In this review, we evaluate evidence linking OA, synovitis and metabolic syndrome and discuss the merits of targeting macrophage activation as a valid treatment option for OA.

Characterization of nano-structural and nano-mechanical properties of osteoarthritic subchondral bone.

BACKGROUND: Although articular cartilage is the primary tissues affected by osteoarthritis (OA), the underlying subchondral bone also undergoes noticeable changes. Despite the growing body of research into the biophysical and mechanical properties of OA bone there are few studies that have analysed the structure of the subchondral sclerosis at the nanoscale. In this study, the composition and nano-structural changes of human osteoarthritis (OA) subchondral bone were investigated to better understand the site-specific changes. METHODS: OA bone samples were collected from patients undergoing total knee replacement surgery and graded according to disease severity (grade I: mild OA; grade IV: severe OA). Transmission electron microscopy (TEM), Electron Diffraction, and Elemental Analysis techniques were used to explore the cross-banding pattern, nature of mineral phase and orientation of the crystal lattice. Subchondral bone nano-hydroxyapatite powders were prepared and characterised using high resolution transmission electron microscopy (HR-TEM) and fourier transform infrared spectroscopy (FTIR). Subchondal bone mechanical properties were investigated using a nano-indentation method. RESULTS: In grade I subchondral bone samples, a regular periodic fibril banding pattern was observed and the c-axis orientation of the apatite crystals was parallel to the long axis of the fibrils. By contrast, in grade IV OA bone samples, the bulk of fibrils formed a random and undulated arrangement accompanied by a circular oriented pattern of apatite crystals. Fibrils in grade IV bone showed non-hierarchical intra-fibrillar mineralization and higher calcium (Ca) to phosphorous (P) (Ca/P) ratios. Grade IV OA bone showed higher crystallinity of the mineral content, increased modulus and hardness compared with grade I OA bone. CONCLUSIONS: The findings from this study suggest that OA subchondral sclerotic bone has an altered mineralization process which results in nano-structural changes of apatite crystals that is likely to account for the compromised mechanical properties of OA subchondral bones.

Gamma tocotrienol targets tyrosine phosphatase SHP2 in mammospheres resulting in cell death through RAS/ERK pathway.

BACKGROUND: There is increasing evidence supporting the concept of cancer stem cells (CSCs), which are responsible for the initiation, growth and metastasis of tumors. CSCs are thus considered the target for future cancer therapies. To achieve this goal, identifying potential therapeutic targets for CSCs is essential. METHODS: We used a natural product of vitamin E, gamma tocotrienol (gamma-T3), to treat mammospheres and spheres from colon and cervical cancers. Western blotting and real-time RT-PCR were employed to identify the gene and protein targets of gamma-T3 in mammospheres. RESULTS: We found that mammosphere growth was inhibited in a dose dependent manner, with total inhibition at high doses. Gamma-T3 also inhibited sphere growth in two other human epithelial cancers, colon and cervix. Our results suggested that both Src homology 2 domain-containing phosphatase 1 (SHP1) and 2 (SHP2) were affected by gamma-T3 which was accompanied by a decrease in K- and H-Ras gene expression and phosphorylated ERK protein levels in a dose dependent way. In contrast, expression of self-renewal genes TGF-beta and LIF, as well as ESR signal pathways were not affected by the treatment. These results suggest that gamma-T3 specifically targets SHP2 and the RAS/ERK signaling pathway. CONCLUSIONS: SHP1 and SHP2 are potential therapeutic targets for breast CSCs and gamma-T3 is a promising natural drug for future breast cancer therapy.

Role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis.

OBJECTIVE: The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA. METHODS: To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples. RESULTS: It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1ß, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression. CONCLUSION: Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA.

Osteocyte-induced angiogenesis via VEGF-MAPK-dependent pathways in endothelial cells.

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2-MAPK-ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK-ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.

Near infrared spectroscopy for rapid determination of Mankin score components: a potential tool for quantitative characterization of articular cartilage at surgery.

PURPOSE: The purpose of this study was to demonstrate the potential of near infrared (NIR) spectroscopy for characterizing the health and degenerative state of articular cartilage based on the components of the Mankin score. METHODS: Three models of osteoarthritic degeneration induced in laboratory rats by anterior cruciate ligament (ACL) transection, meniscectomy (MSX), and intra-articular injection of monoiodoacetate (1 mg) (MIA) were used in this study. Degeneration was induced in the right knee joint; each model group consisted of 12 rats (N = 36). After 8 weeks, the animals were euthanized and knee joints were collected. A custom-made diffuse reflectance NIR probe of 5-mm diameter was placed on the tibial and femoral surfaces, and spectral data were acquired from each specimen in the wave number range of 4,000 to 12,500 cm(-1). After spectral data acquisition, the specimens were fixed and safranin O staining (SOS) was performed to assess disease severity based on the Mankin scoring system. Using multivariate statistical analysis, with spectral preprocessing and wavelength selection technique, the spectral data were then correlated to the structural integrity (SI), cellularity (CEL), and matrix staining (SOS) components of the Mankin score for all the samples tested. RESULTS: ACL models showed mild cartilage degeneration, MSX models had moderate degeneration, and MIA models showed severe cartilage degenerative changes both morphologically and histologically. Our results reveal significant linear correlations between the NIR absorption spectra and SI (R(2) = 94.78%), CEL (R(2) = 88.03%), and SOS (R(2) = 96.39%) parameters of all samples in the models. In addition, clustering of the samples according to their level of degeneration, with respect to the Mankin components, was also observed. CONCLUSIONS: NIR spectroscopic probing of articular cartilage can potentially provide critical information about the health of articular cartilage matrix in early and advanced stages of osteoarthritis (OA). CLINICAL RELEVANCE: This rapid nondestructive method can facilitate clinical appraisal of articular cartilage integrity during arthroscopic surgery.

3D Printed Microneedles for the Transdermal Delivery of NAD+ Precursor: Toward Personalization of Skin Delivery.

3D printing of microneedles (µNDs) for transdermal therapy has the potential to enable patient personalization based on the target disease, site of application, and dosage requirements. To convert this concept to reality, it is necessary that the 3D printing technology can deliver high resolution, an affordable cost, and large print volumes. With the introduction of benchtop 4K and 8K 3D printers, it is now possible to manufacture medical devices like µNDs at sufficient resolution and low cost. In this research, we systematically optimized the 3D printing design parameters such as resin viscosity, print angle, layer height, and curing time to generate customizable µNDs. We have also developed an innovative 3D coating microtank device to optimize the coating method. We have applied this to the development of novel µNDs to deliver an established NAD+ precursor molecule, nicotinamide mononucleotide (NMN). A methacrylate-based polymer photoresin (eSun resin) was diluted with methanol to adjust the resin viscosity. The 3D print layer height of 25 µm yielded a smooth surface, thus reducing edge-ridge mismatches. Printing µNDs at 90° to the print platform yielded 84.28 ± 2.158% (n = 5) of the input height thus increasing the tip sharpness (48.52 ± 10.43 µm, n = 5). The formulation containing fluorescein (model molecule), sucrose (viscosity modifier), and Tween-20 (surface tension modifier) was coated on the µNDs using the custom designed microtank setup, and the amount deposited was determined fluorescently. The dye-coated µND arrays inserted into human skin (in vitro) showed a fluorescence signal at a depth of 150 µm (n = 3) into the skin. After optimization of the 3D printing parameters and coating protocol using fluorescein, NMN was coated onto the µNDs, and its diffusion was assessed in full-thickness human skin in vitro using a Franz diffusion setup. Approximately 189 ± 34.5 µg (5× dipped coated µNDs) of NMN permeated through the skin and 41.2 ± 7.53 µg was left in the skin after 24 h. Multiphoton microscopy imaging of NMN-coated µND treated mouse ear skin ex vivo demonstrated significantly (p < 0.05) increased free-unbound NADPH and reduced fluorescence lifetime of NADPH, both of which are indicative of cellular metabolic rates. Our study demonstrates that low-cost benchtop 3D printers can be used to print high-fidelity µNDs with the ability to rapidly coat and release NMN which consequently caused changes in intracellular NAD+ levels.

Spatial analysis of the osteoarthritis microenvironment: techniques, insights, and applications.

Osteoarthritis (OA) is a debilitating degenerative disease affecting multiple joint tissues, including cartilage, bone, synovium, and adipose tissues. OA presents diverse clinical phenotypes and distinct molecular endotypes, including inflammatory, metabolic, mechanical, genetic, and synovial variants. Consequently, innovative technologies are needed to support the development of effective diagnostic and precision therapeutic approaches. Traditional analysis of bulk OA tissue extracts has limitations due to technical constraints, causing challenges in the differentiation between various physiological and pathological phenotypes in joint tissues. This issue has led to standardization difficulties and hindered the success of clinical trials. Gaining insights into the spatial variations of the cellular and molecular structures in OA tissues, encompassing DNA, RNA, metabolites, and proteins, as well as their chemical properties, elemental composition, and mechanical attributes, can contribute to a more comprehensive understanding of the disease subtypes. Spatially resolved biology enables biologists to investigate cells within the context of their tissue microenvironment, providing a more holistic view of cellular function. Recent advances in innovative spatial biology techniques now allow intact tissue sections to be examined using various -omics lenses, such as genomics, transcriptomics, proteomics, and metabolomics, with spatial data. This fusion of approaches provides researchers with critical insights into the molecular composition and functions of the cells and tissues at precise spatial coordinates. Furthermore, advanced imaging techniques, including high-resolution microscopy, hyperspectral imaging, and mass spectrometry imaging, enable the visualization and analysis of the spatial distribution of biomolecules, cells, and tissues. Linking these molecular imaging outputs to conventional tissue histology can facilitate a more comprehensive characterization of disease phenotypes. This review summarizes the recent advancements in the molecular imaging modalities and methodologies for in-depth spatial analysis. It explores their applications, challenges, and potential opportunities in the field of OA. Additionally, this review provides a perspective on the potential research directions for these contemporary approaches that can meet the requirements of clinical diagnoses and the establishment of therapeutic targets for OA.

Polymer nanocarrier system for endosome escape and timed release of siRNA with complete gene silencing and cell death in cancer cells.

An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U-2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 µg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 µg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA.

Controlling Microenvironments with Organs-on-Chips for Osteoarthritis Modelling.

Osteoarthritis (OA) remains a prevalent disease affecting more than 20% of the global population, resulting in morbidity and lower quality of life for patients. The study of OA pathophysiology remains predominantly in animal models due to the complexities of mimicking the physiological environment surrounding the joint tissue. Recent development in microfluidic organ-on-chip (OoC) systems have demonstrated various techniques to mimic and modulate tissue physiological environments. Adaptations of these techniques have demonstrated success in capturing a joint tissue's tissue physiology for studying the mechanism of OA. Adapting these techniques and strategies can help create human-specific in vitro models that recapitulate the cellular processes involved in OA. This review aims to comprehensively summarise various demonstrations of microfluidic platforms in mimicking joint microenvironments for future platform design iterations.

Inhibition of Leukotriene A4 Hydrolase Suppressed Cartilage Degradation and Synovial Inflammation in a Mouse Model of Experimental Osteoarthritis.

OBJECTIVE: Chronic inflammation plays an important role in the osteoarthritis (OA) pathology but how this influence OA disease progression is unclear. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator generated from arachidonic acid through the sequential activities of 5-lipoxygenase, 5-lipoxygenase-activating protein, Leukotriene A4 hydrolase (LTA4H) and its downstream product LTB4. The aim of this study is to investigate the involvement and the potential therapeutic target of the LTB4 pathway in OA disease progression. DESIGN: Both clinical human cartilage samples (n = 7) and mice experimental OA models (n = 6) were used. The levels of LTA4H and leukotriene B4 receptor 1 were first examined using immunostaining in human OA/non-OA cartilage and mice experimental OA models. We also determined whether the LTA4H pathway was associated with cartilage degeneration and synovitis inflammation in OA mice models and human articular chondrocytes. RESULTS: We found that both LTA4H and LTB4 receptor (BLT1) were highly expressed in human and mice OA cartilage. Inhibition of LTA4H suppressed cartilage degeneration and synovitis in OA mice model. Furthermore, inhibition of LTA4H promoted cartilage regeneration by upregulating chondrogenic genes expression such as aggrecan (ACAN), collagen 2A1 (COL2A1), and SRY-Box transcription factor 9 (SOX9). CONCLUSIONS: Our results indicate that the LTA4H pathway is a crucial regulator of OA pathogenesis and suggest that LTA4H could be a therapeutic target in combat OA.