Moonsoo M. Jin

Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: Implications for cartilage tissue repair

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.

Cubic Mesoporous Graphitic Carbon(IV) Nitride: An All-in-One Chemosensor for Selective Optical Sensing of Metal Ions

Carbon nitride is an appealing class of material which can complement carbon in a variety of applications. At ambient conditions a graphitic carbon nitride (g-C3N4) is regarded to be the most stable allotrope. Kroke et al. proposed that gC3N4 consists of sheets of ordered tri-s-triazine moieties connected through planar tertiary amino groups stacked in a graphitic fashion. The bulk material synthesis of g-C3N4 was attempted by thermal condensation of nitrogen-rich precursors, even though most reactants do not proceed significantly past the polymeric form owing to incomplete condensation or polymerization in the bulk. A higher condensation degree was achieved by carrying out the condensation in molten salts. Graphitic carbon nitride has recently attracted great interests because of its semiconductor properties, which makes it suitable for photocatalytic applications. The electronic band structure and band gap of g-C3N4 depend on the degree of condensation of the material. It was also proposed that the band gap can be tuned to lower or higher values by protonation or synthesis of inclusion complex with metal cations such as Zn and Fe. g-C3N4 has been used as a photocatalyst for the production of hydrogen and oxygen from water. The introduction of porosity into g-C3N4 yields an increase in the accessible surface area of the material. It was also shown that the catalytic and photocatalytic activity of mpg-C3N4 (“mesoporous graphitic carbon nitride”) greatly improved over that of the bulk material. 10] Optical sensors are molecular receptors whose optical properties change upon binding to specific guests. Optical sensing systems have been intensively investigated for their capability of providing sensitivity and fast and easy detection, biocompatibility, and adaptability to a wide variety of assay conditions. One notable application of optical sensors is in the sensing of metal ions. Especially in industrial areas, large amounts of toxic and carcinogenic metals have been released into the environment, which has strongly raised interest in the biological and environmental monitoring of such compounds. Various optical sensors based on azo-coupled macrocycles, porphyrin, and phenanthroline derivatives have been described for the detection of a wide range of metal ions. The Lewis basic site on these molecular receptors provides strong coordination to metal ions while the net electron transfer from chromophore/fluorophore group in the receptor to the complexed metal ions leads to the qualitative and semiquantitative sensing of metal ions. Recent development in mesoporous materials has improved the performance of these sensors through immobilization of receptors, whereby the receptors are attached to a large and accessible surface area and well-defined pores, favorable for high adsorption capacity of chromogenic/fluorescent molecules and efficient transport of analytes and thus low detection limits of below tens of nanomolar concentrations. There is, however, still growing demand for more advanced optical sensing system with lower detection limit and faster kinetic response. Considering the properties of present chromogenic/fluorescent receptors, it seems that nanostructured g-C3N4 would be a promising alternative. As previously described, the electronic structure of g-C3N4 is adjustable by coupling events of protons or metals to the surface. The surface functionalities of g-C3N4, that is, NH2/ NH /=N , are well-characterized ligands exhibiting high adsorption capacity for metal ions through chelation or redox reaction. Finally, an additional supporting material is not necessary because it is possible to tailor its nanostructure by using any kind of silica hard template. In comparison with the systems in which the receptor is supported on a porous material, and thus constitute just a minor part, considering weight and volume fraction of the overall system, in mpgC3N4 the entire material would be composed of the functional, in this case sensing, material, which principally gives rise to very high sensitivity. Such a nanostructuring is also necessary for efficient transport of metal ions to the surface. Herein, we utilized g-C3N4 as an all-in-one chemosensor to detect trace amounts of metal ions in aqueous solutions. For this purpose 3D cubic (Ia 3d) mesoporous g-C3N4 (c-mpgC3N4) with high surface area was synthesized for the first time. The electronic properties as well as the surface functionalities of c-mpg-C3N4 should make it an efficient optical sensor. Structural characterization of c-mpg-C3N4 was carried out by smalland wide-angle X-ray scattering (SAXS and WAXS), transmission electron microscopy (TEM), N2 sorption, thermogravimetric analysis (TGA), Fourier transform infrared [*] E. Z. Lee, Dr. Y.-S. Jun, Prof. W. H. Hong Department of Chemical and Biomolecular Engineering, KAIST 335 Gwahak-ro, Yuseong-gu, Daejeon, 305-701 (Korea) Fax: (+82)42-350-3910 E-mail: st9750@kaist.ac.kr whhong@kaist.ac.kr Homepage: http://sep.kaist.ac.kr

Biosynthetic response and mechanical properties of articular cartilage after injurious compression

Traumatic joint injury is known to produce osteoarthritic degeneration of articular cartilage. To study the effects of injurious compression on the degradation and repair of cartilage in vitro, we developed a model that allows strain and strain rate‐controlled loading of cartilage explants. The influence of strain rate on both cartilage matrix biosynthesis and mechanical properties was assessed after single injurious compressions. Loading with a strain rate of 0.01 s−1 to a final strain of 50% resulted in no measured effect on the cells or on the extracellular matrix, although peak stresses reached levels of about 12 MPa. However, compression with strain rates of 0.1 and 1 s−1 caused peak stresses of approximately 18 and 24 MPa, respectively, and resulted in significant decreases in both proteoglycan and total protein biosynthesis. The mechanical properties of the explants (compressive and shear stiffness) were also reduced with increasing strain rate. Additionally, cell viability decreased with increasing strain rate, and the remaining viable cells lost their ability to exhibit an increase in biosynthesis in response to low‐amplitude dynamic mechanical stimulation. This latter decrease in reparative response was most dramatic in the tissue compressed at the highest strain rates. We conclude that strain rate (like peak stress or strain) is an important parameter in defining mechanical injury, and that cartilage injuriously compressed at high strain rates can lose its characteristic anabolic response to low‐amplitude cyclic mechanical loading.

A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants

We have developed an incubator housed, biaxial-tissue-loading device capable of applying axial deformations as small as 1 microm and sinusoidal rotations as small as 0.01 degrees. Axial resolution is 50 nm for applying sinewaves as low as 10 microm (or 1% based on a 1 mm thickness) or as large as 100 microm. Rotational resolution is 0.0005 degrees. The machine is small enough (30 cm high x 25 cm x 20 cm) to be placed in a standard incubator for long-term tissue culture loading studies. In metabolic studies described here, application of sinusoidal macroscopic shear deformation to articular cartilage explants resulted in a significant increase in the synthesis of proteoglycan and proteins (uptake of (35)S-sulfate and (3)H-proline) over controls held at the same static offset compression.

Shear and Compression Differentially Regulate Clusters of Functionally Related Temporal Transcription Patterns in Cartilage Tissue

Chondrocytes are subjected to a variety of biophysical forces and flows during physiological joint loading, including mechanical deformation, fluid flow, hydrostatic pressure, and streaming potentials; however, the role of these physical stimuli in regulating chondrocyte behavior is still being elucidated. To isolate the effects of these forces, we subjected intact cartilage explants to 1–24 h of continuous dynamic compression or dynamic shear loading at 0.1 Hz. We then measured the transcription levels of 25 genes known to be involved in cartilage homeostasis using real-time PCR and compared the gene expression profiles obtained from dynamic compression, dynamic shear, and our recent results on static compression amplitude and duration. Using clustering analysis, we determined that transcripts for proteins with similar function had correlated responses to loading. However, the temporal expression patterns were strongly dependent on the type of loading applied. Most matrix proteins were up-regulated by 24 h of dynamic compression or dynamic shear, but down-regulated by 24 h of 50% static compression, suggesting that cyclic matrix deformation is a key stimulator of matrix protein expression. Most matrix proteases were up-regulated by 24 h under all loading types. Transcription factors c-Fos and c-Jun maximally responded within 1 h to all loading types. Pre-incubating cartilage explants with either a chelator of intracellular calcium or an inhibitor of the cyclic AMP pathway demonstrated the involvement of both pathways in transcription induced by dynamic loading.

Shear-and Compression-induced Chondrocyte Transcription Requires MAPK Activation in Cartilage Explants

Chondrocytes regulate the composition of cartilage extracellular matrix in response to mechanical signals, but the intracellular pathways involved in mechanotransduction are still being defined. Mitogen-activated protein kinase (MAPK) pathways are activated by static and dynamic compression of cartilage, which simultaneously induce intratissue fluid flow, pressure gradients, cell, and matrix deformation. First, to determine whether cell and matrix deformation alone could induce MAPK activation, we applied dynamic shear to bovine cartilage explants. Using Western blotting, we measured ERK1/2 and p38 activation at multiple time points over 24 h. Distinct activation time courses were observed for different MAPKs: a sustained 50% increase for ERK1/2 and a delayed increase in p38 of 180%. We then investigated the role of MAPK activation in mechano-induced chondrocyte gene expression. Cartilage explants were preincubated with inhibitors of ERK1/2 and p38 activation before application of 1–24 h of three distinct mechanical stimuli relevant to in vivo loading (50% static compression, 3% dynamic compression at 0.1 Hz, or 3% dynamic shear at 0.1 Hz). mRNA levels of selected genes involved in matrix homeostasis were measured using real-time PCR and analyzed by k-means clustering to characterize the time- and load-dependent effects of the inhibitors. Most genes examined required ERK1/2 and p38 activation to be regulated by these loading regimens, including matrix proteins aggrecan and type II collagen, matrix metalloproteinases MMP13, and ADAMTS5, and transcription factors downstream of the MAPK pathway, c-Fos, and c-Jun. Thus, we demonstrated that the MAPK pathway is a central conduit for transducing mechanical forces into biological responses in cartilage.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5‐phosphatase domain followed by an ASH and a RhoGAP‐like domain. Their divergent NH2‐terminal portions remain uncharacterized. We show that the NH2‐terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin‐coated pits, was earlier shown to contain another binding site for clathrin in its COOH‐terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2‐terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin‐binding site in OCRL maps to an unusual clathrin‐box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin‐dependent membrane trafficking.

Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases.

Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (K(D) near 1 nM) and include potent inhibitors of BoNT/A protease activity (K(i) near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication.

Combined effects of dynamic tissue shear deformation and insulin-like growth factor I on chondrocyte biosynthesis in cartilage explants.

Biophysical forces and biochemical factors play crucial roles in the maintenance of the integrity of articular cartilage. In this study, we explored the effect of dynamic tissue shear deformation and insulin-like growth factor I (IGF-I) on matrix synthesis by chondrocytes within native cartilage explants. Dynamic tissue shear in the range of 0.5-6% strain amplitude at 0.1 Hz was applied to cartilage explants cultured in serum-free medium. Dynamic tissue shear above 1.5% strain amplitude significantly stimulated protein and proteoglycan synthesis, by maximum values of 35 and 25%, respectively, over statically held control specimens. In the absence of tissue shear, IGF-I augmented protein and proteoglycan synthesis up to twofold at IGF-I concentrations in the range of 100-300 ng/ml. When tissue shear and IGF-I stimuli were combined, matrix biosynthesis levels were significantly higher than the maximal effect caused by either stimulus alone. However, there was no significant interaction between tissue shear and IGF-I as determined by two-way ANOVA. We then quantified the effect of dynamic tissue shear on the transport of IGF-I into and within cartilage explants. [125I]IGF-I was added to the medium, and the levels of intratissue [125I]IGF-I were directly measured as a function of time over 48 h in the presence and absence of continuous dynamic shear strain. Dynamic shear did not alter the rate of uptake of [125I]IGF-I into the explants, suggesting that convective diffusion of [125I]IGF-I is negligible under the shear strain conditions used. This is in marked contrast to the enhancement of transport reported in response to uniaxial dynamic compression. Taken together, these data suggest that (1) the stimulatory effect of tissue shear is via mechanotransduction pathways and not by facilitated transport of biochemical factors and (2) chondrocytes may possess complementary signal transduction pathways for biophysical and biochemical factors leading to changes in metabolic activity.

Reducing the object orientation dependence of susceptibility effects in gradient echo MRI through quantitative susceptibility mapping.

This study demonstrates the dependence of non‐local susceptibility effects on object orientation in gradient echo MRI and the reduction of non‐local effects by deconvolution using quantitative susceptibility mapping. Imaging experiments were performed on a 3T MRI system using a spoiled 3D multi‐echo GRE sequence on phantoms of known susceptibilities, and on human brains of healthy subjects and patients with intracerebral hemorrhages. Magnetic field measurements were determined from multiple echo phase data. To determine the quantitative susceptibility mapping, these field measurements were deconvolved through a dipole inversion kernel under a constraint of consistency with the magnitude images. Phantom and human data demonstrated that the hypointense region in GRE magnitude image corresponding to a susceptibility source increased in volume with TE and varied with the source orientation. The induced magnetic field extended beyond the susceptibility source and varied with its orientation. In quantitative susceptibility mapping, these blooming artifacts, including their dependence on object orientation, were reduced, and the material susceptibilities were quantified. Magn Reson Med, 2012.