Sung Youn Jung

Plasma-treated silk fibroin nanofibers for skin regeneration

Silk fibroin (SF) nanofibers were prepared by electrospinning and treated with plasma in the presence of oxygen or methane gas to modify their surface characteristics. The surface characteristics of the SF nanofibers after plasma treatment were examined using contact angle measurements and XPS analysis. The hydrophilicity of the electrospun SF nanofibers decreased slightly by the CH(4) plasma treatment. On the other hand, the hydrophilicity of the SF nanofibers increased greatly by an O(2) plasma treatment. The O(2)-treated SF nanofibers showed higher cellular activities for both normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) than the untreated ones.

Osteoclast Precursors Display Dynamic Metabolic Shifts toward Accelerated Glucose Metabolism at an Early Stage of RANKL-Stimulated Osteoclast Differentiation

Mature osteoclasts have an increased citric acid cycle and mitochondrial respiration to generate high ATP production and ultimately lead to bone resorption. However, changes in metabolic pathways during osteoclast differentiation have not been fully illustrated. We report that glycolysis and oxidative phosphorylation characterized by glucose and oxygen consumption as well as lactate production were increased during receptor activator of nuclear factor-ĸB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 and bone marrow-derived macrophage cells. Cell proliferation and differentiation varied according to glucose concentrations (0 to 100 mM). Maximal cell growth occurred at 20 mM glucose concentration and differentiation occurred at 5 mM concentration. Despite the similar growth rates exhibited when cultured cells were exposed to either 5 mM or 40 mM glucose, their differentiation was markedly decreased in high glucose concentrations. This finding suggests the possibility that osteoclastogenesis could be regulated by changes in metabolic substrate concentrations. To further address the effect of metabolic shift on osteoclastogenesis, we exposed cultured cells to pyruvate, which is capable of promoting mitochondrial respiration. Treatment of pyruvate synergistically increased osteoclastogenesis through the activation of RANKL-stimulated signals (ERK and JNK). We also found that osteoclastogenesis was retarded by blocking ATP production with either the inhibitors of mitochondrial complexes, such as rotenone and antimycin A, or the inhibitor of ATP synthase, oligomycin. Taken together, these results indicate that glucose metabolism during osteoclast differentiation is accelerated and that a metabolic shift towards mitochondrial respiration allows high ATP production and induces enhanced osteoclast differentiation.

Epidermal cellular response to poly(vinyl alcohol) nanofibers containing silver nanoparticles.

A heat-treated PVA nanofibrous matrix containing silver (Ag) was prepared by electrospinning an aqueous 10 wt% PVA solution and followed by heat treatment at 150 degrees C for 10 min. The average diameter of the as-spun and heat-treated PVA nanofibers was 330 nm. The heat-treated PVA nanofibrous matrix containing Ag was irradiated with UV light to transform the Ag ions in the nanofibrous matrix into Ag nanoparticles. The in vitro cytotoxicity of the Ag ions and/or nanoparticles on normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) cultures was examined. The PVA nanofibrous matrix containing Ag showed slightly higher level of attachment and spreading in the early stage culture (1 h) than the PVA nanofibers without Ag (control). However, compared with the PVA nanofibers without Ag, the heat-treated and UV-irradiated PVA nanofibers, containing mainly Ag ions and nanoparticles, respectively, showed reduced cell attachment and spreading. This shows that both Ag ions and Ag nanoparticles are cytotoxic to NHEK and NHEF. There was no significant difference in cytotoxicity to NHEK and NHEF between Ag ions and Ag nanoparticles. NHEF appeared to be more sensitive to Ag ions or particles than NHEK. In addition, the residual nitrate ions (NO3(-)) in the PVA nanofibers had an adverse effect on the culture of both cells.

A Biologically Active Sequence of the Laminin α2 Large Globular 1 Domain Promotes Cell Adhesion through Syndecan-1 by Inducing Phosphorylation and Membrane Localization of Protein Kinase Cδ

Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 α2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin α2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cδ (PKCδ) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCδ activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 α2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCδ signaling pathway.

A biologically active sequence of the laminin α2 LG1 domain promotes cell adhesion through syndecan-1 by inducing phosphorylation and membrane localization of the PKCδ

Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 α2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin α2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cδ (PKCδ) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCδ activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 α2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCδ signaling pathway.

Titanium Surface Coating with a Laminin-Derived Functional Peptide Promotes Bone Cell Adhesion

Laminin-derived peptide coatings can enhance epithelial cell adhesion to implants, and the positive effect of these peptides on bone cell adhesion has been anticipated. The purpose of this study was to evaluate the improvement in bone cell attachment to and activity on titanium (Ti) scaffolds coated with a laminin-derived functional peptide, Ln2-P3 (the DLTIDDSYWYRI motif). Four Ti disc surfaces were prepared, and a human osteosarcoma (HOS) cell attachment test was performed to select two candidate surfaces for peptide coating. These two candidates were then coated with Ln2-P3 peptide, a scrambled peptide, or left uncoated to measure cell attachment to each surface, following which one surface was chosen to assess alkaline phosphatase (ALP) activity and osteogenic marker gene expression with quantitative real-time PCR. On the commercially pure Ti surface, the Ln2-P3 coating significantly increased cellular ALP activity and the expression levels of ALP and bone sialoprotein mRNA as compared with the scrambled peptide-coated and uncoated surfaces. In conclusion, although further in vivo studies are needed, the findings of this in vitro study indicate that the Ln2-P3-coated implant surface promotes bone cell adhesion, which has clinical implications for reducing the overall treatment time of dental implant therapy.

The potential of laminin-2-biomimetic short peptide to promote cell adhesion, spreading and migration by inducing membrane recruitment and phosphorylation of PKCδ.

Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand-receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3β1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3β1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/β inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3β1 integrin, and that binding of α3β1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation.

The potential of mouse skin-derived precursors to differentiate into mesenchymal and neural lineages and their application to osteogenic induction in vivo

Although previous studies indicate that skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells (NCSCs), a shared ability for multilineage differentiation toward neural crest lineages between SKPs and NCSCs has not been fully demonstrated. Here, we report the derivation of SKPs from adult mouse skin and their directed multilineage differentiation toward neural crest lineages. Under controlled in vitro conditions, mouse SKPs were propagated and directed toward peripheral nervous system lineages such as peripheral neurons and Schwann cells, and mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic, and smooth muscle cells. To ask if SKPs could generate these same lineages in vivo, a mixture of SKP-derived mesenchymal stem cells and hydroxyapatite/tricalcium phosphate was transplanted into the rat calvarial defects. Over the ensuing 4 weeks, we observed formation of osteogenic structure in the calvarial defect without any evidence of teratomas. These findings demonstrate the multipotency of adult mouse SKPs to differentiate into neural crest lineages. In addition, SKP-derived mesenchymal stem cells represent an accessible, potentially autologous source of precursor cells for tissue-engineered bone repair.

Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering.

Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678-2686; Ln2-LG3-P2-DN3), from the human laminin α2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering.

Protein kinase Cα/β inhibitor Gö6976 promotes PC12 cell adhesion and spreading through membrane recruitment and activation of protein kinase Cδ.

Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/β. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 μM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/β signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.