Man-Il Huh

Gold nanoparticles coated with gadolinium-DTPA-bisamide conjugate of penicillamine (Au@GdL) as a T1-weighted blood pool contrast agent

The work is directed toward the synthesis and characterization of gold nanoparticles coated by Gd-chelate (Au@GdL), where L is a conjugate of diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and penicillamine. Au@GdL is obtained by the replacement of citrate from Au@citrate with gadolinium chelate (GdL). The average size of Au@GdL is 12 nm with the loading of GdL reaching up to 3.0 × 103 per particle, and they exhibit very high r1 relaxivity (∼107 mM−1 s−1) as well as X-ray attenuation. The r1 relaxivity per [Gd] is 20.1 mM−1 s−1. The small size of the present system also exhibits a USPIO-like behavior, such as contrast enhancement in the liver, the blood pool and the lymph node, as demonstrated by histological and TEM images, as well as CT and MR, rendering itself as a new class of T1 multimodal CT/MR contrast agent with potential application to the blood pool and the lymph node.

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowmans layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemets membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009.

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development†

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT‐PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related “a disintegrin and metalloproteinase“ (ADAM) family proteinases in chick corneal development. While MMP‐13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase‐1 (TIMP‐1), was expressed from ED 18 to 2 days post‐hatching (P2). Early MMP‐13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane‐bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad‐spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell‐derived mesenchymal cell migration. Finally, we identified high levels of active membrane‐type 3‐MMP (MT3‐MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3‐MMP takes part in MMP‐2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development. J. Cell. Biochem. 101:1222–1237, 2007.

Functional activity mapping of rat auditory pathway after intratympanic manganese administration.

In the present study, we report a new method of manganese enhanced magnetic resonance imaging (MEMRI) using intratympanic (IT) manganese administration. We explore Mn²⁺ uptake from the middle ear cavity into the cochlea through mechanically gated ion channels of the hair cell and also functional auditory tract tracing without the use of excessive auditory stimuli for a long time period outside the scanner. After manganese administration in animals with normal hearing and unilateral deafness, T1-weighted MR images were obtained for up to 48 h with a 3.0 T MR imager. In normal rats, the mean signal-to-noise ratio (SNR) at each region of interest on the auditory pathway was significantly higher in the IT injection group than in the intraperitoneal (IP) injection group (P<0.05). Furthermore, the cochlea showed Mn²⁺ signal enhancement only in the IT injection group. In unilateral deafness rats, the IT injection of Mn²⁺ into the deaf-side middle ear cavity demonstrated signal enhancement in the cochlea but not in other auditory structures without axonal transport of Mn²⁺ along the auditory pathway. On the other hand, the IT injection of Mn²⁺ into the normal-side middle ear cavity demonstrated that the mean SNRs at the cochlea, cochlear nucleus, superior olivary complex, lateral lemniscus and inferior colliculus were significantly higher in the ipsilateral auditory pathway than in the contralateral pathway (P<0.05). For the IP injection group, the mean SNRs at each auditory structure, except the cochlea, increased bilaterally. In conclusion, the present work demonstrated the potential advantages of a new IT MEMRI over conventional systemic injection strategies in that (i) the functional auditory tract tracing initiated by the hair cell function is possible and (ii) the axonal transport of Mn²⁺ ions by trans-synaptic activity is possible without auditory stimulation for a long time period outside MR scanner.

ADAM10 mediates N-cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina.

Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro‐form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10‐specific siRNA, the level of mature ADAM10 decreased. Phase‐contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X‐treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X‐treated cultures. N‐cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N‐cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N‐cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation. J. Cell. Biochem. 114: 942–954, 2013.

Constitutive collagenase-1 synthesis through MAPK pathways is mediated, in part, by endogenous IL-1α during fibrotic repair in corneal stroma

Collagenase‐1 is a protease expressed by active fibroblasts that is involved in remodeling of the extracellular matrix (ECM). In this study, we characterize the intracellular signaling mechanism of collagenase‐1 production by IL‐1α in subcultured normal fibroblasts (NF) from uninjured normal corneas, compared to that in repair wound fibroblasts (WF). In NF, collagenase‐1 was induced specifically after the exogenous addition of IL‐1α via activation of ERK and p38MAPK. Collagenase‐1 expression was strongly suppressed upon treatment with either a MEK or p38MAPK inhibitor. In contrast, repair WF constitutively synthesized both IL‐1α and collagenase‐1. Combined treatment with both mitogen‐activated protein kinase (MAPK) inhibitors dramatically reduced collagenase‐1 synthesis, while individual MEK1 or p38 inhibitors weakly modulated the collagenase‐1 level. The results indicate that both pathways are crucial in the regulation of collagenase‐1 synthesis. Furthermore, an IL‐1α receptor antagonist (IL‐1ra) could not abolish constitutive collagenase‐1 synthesis, even at high doses, suggesting that other cytokines/factors are additionally involved in this process. We propose that induction of collagenase‐1 by IL‐1α in both WF and NF depends on a unique combination of cell type‐specific signaling pathways. J. Cell. Biochem. 102: 453–462, 2007.

Expression of matrix metalloproteinase-13 (MMP-13) in the testes of growing and adult chicken.

Although several matrix metalloproteinases (MMPs) have been implicated in testis development, the presence of MMP-13 protein has not been directly substantiated in the male avian gonads. In this study, we examined the expression patterns of MMP-13 and MMP inhibitors, TIMP-1 and TIMP-2, in immature (4weeks), pre-pubertal (16weeks), and mature (1year) chicken testes. Using RT-PCR analysis, we observed that MMP-13 mRNA was expressed in immature testis. In Western blot analysis, the expression level of MMP-13 protein peaked in the immature testes during marked tissue remodeling, whereas it gradually decreased during testis maturation. High expression levels of TIMP-1 (34-kDa) and TIMP-2 (55-kDa) were detected only in immature and pre-pubertal testes and not in adult testis. Four different forms of TIMP-2 protein were differentially detected in the testes of growing and adult chicken. Using immunohistochemistry we localized both secreted and intracellular forms of MMP-13, TIMP-1, and TIMP-2 proteins. These proteins were temporally and spatially distributed in growing and adult testes, and all their expression levels were similar to the expression profile of Western blot results. These findings suggest that age-related changes of MMP-13 with balance of TIMPs act in concert to effect the controlled testicular remodeling and maturation.

Rapid expansion and auto-grafting efficiency of porcine full skin expanded by a skin bioreactor ex vivo

Full skin auto-grafts are required for reconstruction of skin burns and trauma scars. However, currently available clinical approaches such as sheet skin graft, mesh skin grafts, artificial skin graft, and in vivo skin expansion have limitations due to their potential danger for secondary damage and scar formation at the donor site, and discomfort during skin expansion. We developed an advanced bioreactor system and evaluated its function in skin expansion using porcine full skin. The reactor was designed as a pneumatic cylinder type, was programmed to adjust the pressure and the operating time. The system was composed of culture chamber unit, environmental control unit, and monitoring unit. Skins were expanded at 200 kPa pneumatic force and the expanded skins were analyzed by immunohistochemistry and histology. Furthermore we carried out auto-grafting experiment of the expanded skins in vivo using Yucatan pigs and skins were harvested and histologically analyzed after 8 weeks. The results showed that the bioreactor expanded skins to 160% in 4 hours. Histological analysis of the expanded skins revealed that epidermal cells and dermal fibroblasts were viable and remained integrity. The results of auto-grafting experiment indicated that fibrosis and scars were not detected in the grafted skins. This study demonstrates that the newly developed skin bioreactor enabled to obtain large sized full skin rapidly and successful grating.

Generation of Femtosecond Laser-Cut Decellularized Corneal Lenticule Using Hypotonic Trypsin-EDTA Solution for Corneal Tissue Engineering

Purpose To establish an optimized and standardized protocol for the development of optimal scaffold for bioengineering corneal substitutes, we used femtosecond laser to process human corneal tissue into stromal lenticules and studied to find the most efficient decellularization method among various reagents with different tonicities. Methods The decellularization efficacy of several agents (0.1%, 0.25%, and 0.5% of Triton X-100, SDS, and trypsin-EDTA (TE), resp.) with different tonicities was evaluated. Of all protocols, the decellularization methods, which efficiently removed nuclear materials examined as detected by immunofluorescent staining, were quantitatively tested for sample DNA and glycosaminoglycan (GAG) contents, recellularization efficacy, and biocompatibilities. Results 0.5% SDS in hypertonic and isotonic buffer, 0.25% TE in hypotonic buffer, and 0.5% TE in all tonicities completely decellularized the corneal lenticules. Of the protocols, decellularization with hypotonic 0.25 and 0.5% TE showed the lowest DNA contents, while the GAG content was the highest. Furthermore, the recellularization efficacy of the hypotonic TE method was better than that of the SDS-based method. Hypotonic TE-treated decellularized corneal lenticules (DCLs) were sufficiently transparent and biocompatible. Conclusion We generated an ideal protocol for DCLs using a novel method. Furthermore, it is possible to create a scaffold using a bioengineered corneal substitute.