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Poly(lactic-co-glycolic acid)(PLGA) microparticles represent an important class of materials used for drug delivery. Current synthesis frequently uses conventional emulsion, where dichloromethane(DCM) is used as the organic phase solvent. Due to the health and environmental toxicity of DCM and its slow degradation, this work replaces DCM with a greener solvent, dimethyl carbonate(DMC). To attain narrow distribution of PLGA particle size, microfluidic flow focusing was chosen over conventional emulsion. This new approach successfully produced PLGA microparticles encapsulated with flavopiridol, a kinase inhibitor. These particles exhibit sustained release profile more desirable than the conventional counterparts. The cytotoxicity and activity tests have demonstrated high biocompatibility and efficacy of these PLGA particles. The high sustainability is also evaluated using simple E-Factor(sEF) and complete E-Factor(cEF). The lower health and environmental toxicities of DMC than DCM are evidenced by approximately one order of magnitude higher in lethal dose, i. e., 50%(LD50) values in rat, 5-fold faster degradation rate, and 30% higher GlaxoSmithKline(GSK) combined greenness value. The approach reported in this work shall provide a new and green means for drug delivery in general. The products enable local sustained delivery of flavopiridol for prevention of post-traumatic osteoarthritis, and anti-cancer therapy.

This work reports the first direct observations of binding and complex formation between transforming growth factor beta 1 (TGF-β1) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM). Each COMP molecule consists of pentamers whose five identical monomeric units bundle at N-termini. From this central point, the five monomers’ flexible arms extend outward with C-terminal domains at the distal ends, forming a bouquet-like structure. In commonly used buffer solutions, TGF-β1 molecules typically form homodimers (majority), double dimers (minority), and aggregates (trace amount). Mixing of TGF-β1 and COMP leads to rapid binding and complex formation. The TGF-β1/COMP complexes contain one to three COMP and multiple TGF-β1 molecules. For complexes with one COMP, the structure is more compact and less flexible than that of COMP alone. For complexes with two or more COMP molecules, the conformation varies to a large degree from one complex to another. This is attributed to the presence of double dimers or aggregates of TGF-β1 molecules, whose size and multiple binding sites enable binding to more than one COMP. The number and location of individual TGF-β1 dimers are also clearly visible in all complexes. This molecular-level information provides new insight into the mechanism of chondrogenesis enhancement by TGF-β1/COMP complexes, i.e. simultaneous and multivalent presentation of growth factors. These presentations help explain the high efficacy in sustained activation of the signalling pathway to augment chondrogenesis.

Reactive oxygen species (ROS) generated by the NADPH oxidase (Nox) enzymes are important short-range signaling molecules. They have been extensively studied in the physiology and pathophysiology of the cardiovascular system, where they have important roles in vascular inflammation, angiogenesis, hypertension, cardiac injury, stroke, and aging. Increasing evidence demonstrates that ROS and Nox enzymes also affect bone homeostasis and osteoporosis, and more recent studies implicate ROS and Nox enzymes in both inflammatory arthritis and osteoarthritis. Mechanistically, this connection may be through the effects of ROS on signal transduction. ROS affect both transforming growth factor-β/Smad signaling, interleukin-1β/nuclear factor-kappa B signaling, and the resulting changes in matrix metalloproteinase expression. The purpose of this review is to describe the role of Nox enzymes in the physiology and pathobiology of bone and joints and to highlight the potential of therapeutically targeting the Nox enzymes.

© 2018 American Chemical Society. This work presents the first direct evidence of multivalent binding between bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM) imaging. AFM topographic images reveal the molecular morphology of COMP, a pentameric protein whose five identical monomer units bundle together at N-termini, extending out with flexible chains to C-termini. Upon addition of BMP-2, COMP molecules undergo conformational changes at the C-termini to enable binding with BMP-2 molecules. AFM enables local structural changes of COMP to be revealed upon binding various numbers, 1-5, of BMP-2 molecules. These BMP-2/COMP complexes exhibit very different morphologies from those of COMP: much more compact and thus less flexible. These molecular-level insights deepen current understanding of the mechanism of how the BMP-2/COMP complex enhances osteogenesis among osteoprogenitor cells, i.e., multivalent presentation of BMP-2 via the stable and relatively rigid BMP-2/COMP complex could form a lattice of interaction between multiple BMP-2 and BMP-2 receptors. These ligand-receptor clusters lead to fast initiation and sustained activation of the Smad signaling pathway, resulting in enhanced osteogenesis. This work is also of translational importance as the outcome may enable use of lower BMP-2 dosage for bone repair and regeneration.

The interaction of long nanowisres and living cells is directly related to nanowires’ nanotoxicity and health impacts. Interactions of silver nanowires (AgNWs) and macrophage cell lines (NR8383) were investigated using laser scanning confocal microscopy and single cell compression (SCC). With high-resolution imaging and mechanics measurement of individual cells, AgNW-induced frustrated phagocytosis was clearly captured in conjunction with structural and property changes of cells. While frustrated phagocytosis is known for long microwires and long carbon nanotubes, this work reports first direct observations of frustrated phagocytosis of AgNWs among living cells in situ. In the case of partial penetration of AgNWs into NR8383 cells, confocal imaging revealed actin participation at the entry sites, whose behavior differs from microwire-induced frustrated phagocytosis. The impacts of frustrated phagocytosis on the cellular membrane and cytoskeleton were also quantified by measuring the mechanical properties using SCC. Taken collectively, this study reveals the structural and property characteristics of nanowire-induced frustrated phagocytosis, which deepens our understanding of nanowire–cell interactions and nanocytotoxicity.

Intervertebral disk degeneration (IVDD) is a spinal disk condition caused by an inflammatory response induced by various proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many diseases, especially in regulating the activation of primary inflammatory response genes. Our study investigated a highly selective CDK9 inhibitor, atuveciclib, which protects nucleus pulposus (NP) cells from proinflammatory stimuli-induced catabolism. The effects of CDK9 inhibition were determined in human and rat NP cells treated with IL-1β in the presence or absence of atuveciclib or small interfering RNA target CDK9. Inhibition of CDK9 led to the attenuation of inflammatory response. In addition, rat intervertebral disk (IVD) explants were used to determine the role of CDK9 inhibition in extracellular matrix degradation. The rat IVDD model also proved that CDK9 inhibition attenuated IVDD, as validated using magnetic resonance imaging and immunohistochemistry. Taken together, CDK9 is a potential therapeutic target to prevent IVDD.

While self-assembly of molecules is relatively well-known and frequently utilized in chemical synthesis and material science, controlled assembly of molecules represents a new concept and approach. The present work demonstrates the concept of controlled molecular assembly using a non-spherical biomolecule, heparosan tetrasaccharide (MW = 1.099 kD). The key to controlled assembly is the fact that ultra-small solution droplets exhibit different evaporation dynamics from those of larger ones. Using an independently controlled microfluidic probe in an atomic force microscope, sub-femtoliter aqueous droplets containing designed molecules produce well-defined features with dimensions as small as tens of nanometers. The initial shape of the droplet and the concentration of solute within the droplet dictate the final assembly of molecules due to the ultrafast evaporation rate and dynamic spatial confinement of the droplets. The level of control demonstrated in this work brings us closer to programmable synthesis for chemistry and materials science which can be used to develop vehicles for drug delivery three-dimensional nanoprinting in additive manufacturing.

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by hypertrophic differentiation of the engineered cartilaginous tissue caused by endochondral ossification. Since a healthy cartilage matrix contains high amounts of Aggrecan and COMP, we hypothesized that their supplementation in the biogel used in the generation of subperiosteal cartilage mimics the composition of the cartilage extracellular matrix environment, with beneficial properties for the engineered cartilage. Supplementation of COMP or Aggrecan was studied in vitro during chondrogenic differentiation of rabbit periosteum cells and periosteum-derived chondrocytes. Low melting agarose was supplemented with bovine Aggrecan, human recombinant COMP or vehicle and was injected between the bone and periosteum at the upper medial side of the tibia of New Zealand white rabbits. Generated subperiosteal cartilage tissue was analyzed for weight, GAG and DNA content and ALP activity. Key markers of different phases of endochondral ossification were measured by RT-qPCR. For the in vitro experiments, no significant differences in chondrogenic marker expression were detected following COMP or Aggrecan supplementation, while in vivo favorable chondrogenic marker expression was detected. Gene expression levels of hypertrophic markers as well as ALP activity were significantly decreased in the Aggrecan and COMP supplemented conditions compared to controls. The wet weight and GAG content of the in vivo generated subperiosteal cartilage tissue was not significantly different between groups. Data demonstrate the potential of Aggrecan and COMP to favorably influence the subperiosteal microenvironment for the in vivo generation of cartilage for the optimization of cartilage regenerative approaches.

Bone resorption and homeostasis is carried out by osteoclasts, whose differentiation and activity are regulated by the RANK/RANKL axis. Our previous studies using a mouse model of joint injury show that joint trauma induces local inflammation followed by bone remodeling. The transcription factor cyclin-dependent kinase 9 (CDK9) is the major regulator of inflammation, as CDK9 inhibitor flavopiridol effectively suppress injury-induced inflammatory response. The objective of this study was to investigate the underlying mechanism through which flavopiridol regulates bone resorption. The effects of CDK9 inhibition, by the specific-inhibitor flavopiridol, on bone resorption were determined in vivo using two distinct and clinically relevant bone remodeling models. The first model involved titanium particle-induced acute osteolysis, and the second model was ovariectomy-induced chronic osteoporosis. The effects and mechanism of CDK9 inhibition on osteoclastogenesis were examined using in vitro culture of bone marrow macrophages (BMMs). Our results indicated that flavopiridol potently suppressed bone resorption in both in vivo bone-remodeling models. In addition, CDK9 inhibition suppressed in vitro osteoclastogenesis of BMM and reduced their expression of osteoclast-specific genes. Finally, we determined that flavopiridol suppressed RANKL signaling pathway via inhibition of p65 phosphorylation and nuclear translocation of NF-κB. Summary, CDK9 is a potential therapeutic target to prevent osteolysis and osteoporosis by flavopiridol treatment.

© 2015, AO Research Institute. All rights Reserved. Joint injury often leads to post-traumatic osteoarthritis (PTOA). Acute injury responses to trauma induce production of pro-inflammatory cytokines and catabolic enzymes, which promote chondrocyte apoptosis and degrade cartilage to potentiate PTOA development. Recent studies show that the rate-limiting step for transcriptional activation of injury response genes is controlled by cyclin-dependent kinase 9 (CDK9), and thus it is an attractive target for limiting the injury response. Here, we determined the effects of CDK9 inhibition in suppressing the injury response in mechanically-injured cartilage explants. Bovine cartilage explants were injured by a single compressive load of 30% strain at 100%/s, and then treated with the CDK9 inhibitor Flavopiridol. To assess acute injury responses, we measured the mRNA expression of pro-inflammatory cytokines, catabolic enzymes, and apoptotic genes by RT-PCR, and chondrocyte viability and apoptosis by TUNEL staining. For long-term outcome, cartilage matrix degradation was assessed by soluble glycosaminoglycan release, and by determining the mechanical properties with instantaneous and relaxation moduli. Our data showed CDK9 inhibitor markedly reduced injury-induced inflammatory cytokine and catabolic gene expression. CDK9 inhibitor also attenuated chondrocyte apoptosis and reduced cartilage matrix degradation. Lastly, the mechanical properties of the injured explants were preserved by CDK9 inhibitor. Our results provide a temporal profile connecting the chain of events from mechanical impact, acute injury responses, to the subsequent induction of chondrocyte apoptosis and cartilage matrix deterioration. Thus, CDK9 is a potential disease-modifying agent for injury response after knee trauma to prevent or delay PTOA development.

Cartilage oligomeric matrix protein (COMP) is an important non-collagenous cartilage protein that is essential for the structural integrity of the cartilage extracellular matrix. The repeated modular structure of COMP allows it to "bridge" and assemble multiple cartilage extracellular matrix components such as collagens, matrilins, and proteoglycans. With its modular structure, COMP also has the potential to act as a scaffold for growth factors, thereby affecting how and when the growth factors are presented to cell-surface receptors. However, it is not known whether COMP binds growth factors. We studied the binding interaction between COMP and TGF-β1 in vitro and determined the effect of COMP on TGF-β1-induced signal transduction in reporter cell lines and primary cells. Our results demonstrate that mature COMP protein binds to multiple TGF-β1 molecules and that the peak binding occurs at slightly acidic pH. These interactions were confirmed by dual polarization interferometry and visualized by rotary shadow electron microscopy. There is cation-independent binding of TGF-β1 to the C-terminal domain of COMP. In the presence of manganese, an additional TGF-β-binding site is present in the TSP3 repeats of COMP. Finally, we show that COMP-bound TGF-β1 causes increased TGF-β1-dependent transcription. We conclude that TGF-β1 binds to COMP and that TGF-β1 bound to COMP has enhanced bioactivity.

Actin and tubulin cytoskeletal components are studied extensively in chondrocytes, but less is known about vimentin intermediate filaments. In other cell types, vimentin is a determinant of cell stiffness and disruption of vimentin networks weakens the mechanical integrity of cells. Changes in vimentin organization correlate with osteoarthritis progression, but the functional consequences of these changes remain undetermined in chondrocytes. The objective of this study was to compare the contribution of vimentin to the mechanical stiffness of primary human chondrocytes isolated from normal versus osteoarthritic cartilage. Chondrocytes were embedded in alginate and vimentin networks disrupted with acrylamide. Constructs were imaged while subjected to 20% nominal strain on a confocal microscope stage, and the aspect ratios of approximately 1,900 cells were measured. Cytosolic stiffness was estimated with a finite element model, and live-cell imaging of GFP-vimentin was used to further analyze the nature of vimentin disruption. Vimentin in normal chondrocytes formed an inner cage-like network that was substantially stiffer than the rest of the cytosol and contributed significantly to overall cellular stiffness. Disruption of vimentin reduced stiffness approximately 2.8-fold in normal chondrocytes. In contrast, osteoarthritic chondrocytes were less stiff and less affected by vimentin disruption. This 3D experimental system revealed contributions of vimentin to chondrocyte stiffness previously not apparent, and correlated changes in vimentin-based chondrocyte stiffness with osteoarthritis.

Objective Mechanical stimulation of cartilage affects tissue homeostasis and chondrocyte function. The chondrocyte phenotype is dependent on cell shape, which is largely determined by the actin cytoskeleton. Reorganization of the actin cytoskeleton results from Rho GTPase activation. The purpose of this study was to examine the roles of both actin and Rho in mechanotransduction in chondrocytes. Methods We embedded human articular chondrocytes in 2 × 6–mm agarose discs at 5 × 106 cells/ml and subjected the discs to unconfined dynamic compression at 0.5 Hz. By comparing samples with and without dynamic compression, we identified Rho activation according to the GTP-bound active RhoA measured in cell lysates. We identified rearrangements in filamentous actin structures using fluorescence-labeled phalloidin and confocal microscopy of fixed samples. We identified altered gene expression using TaqMan quantitative reverse transcription–polymerase chain reaction analysis. We tested for a requirement for Rho signaling by performing the dynamic compression in the presence of Rho kinase inhibitors. Results RhoA activation occurred within 5–10 minutes of dynamic compression. Rho kinase–dependent actin reorganization occurred within 20 minutes after application of dynamic compression and was apparent as “punctate” F-actin structures that were visible under confocal microscopy. We identified early-phase mechanoresponsive genes (CCL20 and inducible nitric oxide synthase) that were highly up-regulated within 1 hour of dynamic compression in a Rho kinase–dependent and actin-dependent manner. Conclusion Together, these results are the first demonstration that the Rho–Rho kinase pathway and actin cytoskeletal reorganization are required for changes in the expression of genes involved in human chondrocyte mechanotransduction.

Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a ‘lattice’ to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions.

Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.

Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15-2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level.

Link protein (LP) is an abundant protein of cartilage which stabilizes the interaction of aggrecan with hyaluronic acid (HA). In this study we report that LP is also present in a large number of embryonic non-cartilaginous tissues. We demonstrate, using RNase protection experiments, that the coding region of the LP mRNAs isolated from these tissues is identical to that present in cartilage. Furthermore, we show that the LP mRNAs are translated in non-cartilaginous tissues by the identification of LP polypeptides with monoclonal antibody 4B6/A5 in Western blots. LP is localized in the extracellular matrix of the mesoderm along the entire digestive tract and in the dermis of the embryonic skin as revealed by immunofluorescence analysis. Investigations on the interactions between LP and proteoglycans from skin and proventriculus demonstrate that LP can enhance the binding of proteoglycans from these tissues to HA. In addition, we find that the same proteoglycans bound to HA in the presence of LP are always more resistant to competition by soluble HA than in the absence of LP. Our results suggest that LP is involved in the stabilization of extracellular matrices of a wide variety of non-cartilaginous tissues.