Faculty Bibliography

A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP.DNA(8:1).FG lipoproteoplex complex demonstrated enhanced transfection of beta-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat.FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP.DNA(8:1).FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.

A combination of modified HIV-1 Tat (mTat) peptide and cationic lipids, FuGENE HD (FH), dramatically enhanced transfection efficiency across a range of cell lines when compared to mTat or FH alone (Biomaterials 35:1705-1715 2014). The efficiency of this Tat peptide combination was significantly higher than many commercial non-viral vectors. In this present study, we tested the feasibility of this non-viral vector, mTat/FH, in vivo using plasmid DNA encoding a luciferase gene. The results of the in vivo studies showed that animals administered mTat/FH/DNA intramuscularly had significantly higher and longer luciferase expression ( approximately 7 months) than those with mTat/DNA, FH/DNA, or DNA alone. Histological evaluation showed little immune response in the muscles, livers, and kidneys of mice administered with the mTat/FH. The combination of mTat with FH could significantly improve transfection efficiency, expanding the potential use of non-viral gene vectors in vivo.

Regenerative procedures using barrier membrane technology are presently well established in periodontal/endodontic surgery. The objective of this study was to compare the subsequent effects of the released platelet-derived growth factor (PDGF) and growth/differentiation factor 5 (GDF-5) from collagen membranes (CMs) on bone regeneration in vitro and in vivo. In vitro studies were conducted using MC3T3-E1 mouse preosteoblasts cultured with or without factors. Cell viability, cell proliferation, alkaline phosphatase (ALP) activity and bone marker gene expression were then measured. In vivo studies were conducted by placing CMs with low or high dose PDGF or GDF-5 in rat mandibular defects. At 4 weeks after surgery new bone formation was measured using muCT and histological analysis. The results of in vitro studies showed that CM/GDF-5 significantly increased ALP and cell proliferation activities without cytotoxicity in MC3T3-E1 cells when compared to CM/PDGF or CM alone. Gene expression analysis revealed that Runx2 and Osteocalcin were significantly increased in CM/GDF-5 compared to CM/PDGF or control. Quantitative and qualitative muCT and histological analysis for new bone formation revealed that although CM/PDGF significantly enhanced bone regeneration compared to CM alone or control, CM/GDF-5 significantly accelerated bone regeneration to an even greater extent than CM/PDGF. The results also showed that GDF-5 induced new bone formation in a dose-dependent manner. These results suggest that this strategy, using a CM carrying GDF-5, might lead to an improvement in the current clinical treatment of bone defects for periodontal and implant therapy.

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement ( approximately 5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression ( approximately 7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.

The mucosa of alimentary tract heals more rapidly than cutaneous wounds. The underlying mechanisms of this enhanced healing have not been completely elucidated. Constant exposure to salivary growth factors has been shown to play a critical role in mucosal homeostasis and tissue repair. Angiogenesis also has an essential role in successful wound repair. One of the main angiogenic growth factors, vascular endothelial growth factor (VEGF), has a pleiotropic role in tissue repair via neovascularization, reepithelialization, and regulation of extracellular matrix. We have previously reported a critical role for salivary VEGF in bowel adaptation after small bowel resection. We hypothesize that salivary VEGF is an essential stimulus for oral mucosal tissue repair, and use the murine palatal wound model to test our hypothesis. In a loss-of-function experiment, we removed the primary source of VEGF production through selective submandibular gland (SMG) sialoadenectomy in a murine model and observed the effects on wound closure and neovascularization. We then performed a selective loss-of-function experiment using the protein VEGF-Trap to inhibit salivary VEGF. In a gain-of-function experiment, we supplemented oral VEGF following SMG sialoadenectomy. After SMG sialoadenectomy, there was significant reduction in salivary VEGF level, wound closure, and vessel density. Lower levels of salivary VEGF were correlated with impaired neovascularization and reepithelialization. The selective blockade of VEGF using VEGF-Trap resulted in a similar impairment in wound healing and neovascularization. The sole supplementation of oral VEGF after SMG sialoadenectomy rescued the impaired wound healing phenotype and restored neovascularization to normal levels. These data show a novel role for salivary-VEGF in mucosal wound healing, and provide a basis for the development of novel therapeutics aimed at augmenting wound repair of the oral mucosa, as well as wounds at other sites in the alimentary tract.

BACKGROUND: Healing of tooth extraction sockets in poorly controlled diabetic patients is often delayed and accompanied by severe infection. The exact cellular and molecular mechanisms underlying the pathogenesis of this complication are still not fully understood. OBJECTIVES: The purpose of this study was to investigate molecular changes associated with delayed oral wound healing in diabetes. MATERIALS AND METHODS: Six to eight weeks old male type 2 diabetes and age matched control inbred mice were used and maxillary molar tooth extractions were performed. At 4 and 7 days after tooth extraction, the edentulous mucosa of the mice were harvested, and analyzed for histology and gene expression of key wound healing factors. RESULTS: In the diabetic model, histological analysis showed that epithelial tissue migration for wound closure was delayed after tooth extraction compared to the control. Quantitative real-time PCR revealed that expression of the TGF-beta1, TGF-beta2, TGF-beta3, TGFbetaRII and TGFbetaRIII genes was significantly downregulated in the diabetic model at 4 and 7 days after tooth extraction. CONCLUSION: These results suggest that delayed wound healing of oral mucosa in diabetes may be associated with decreased expression levels of these regulatory genes which play important roles in controlling epithelial wound closure.

PURPOSE: The aim of this study was to investigate the cellular response of human gingival fibroblasts (HGFs) cultured on smooth or rough zirconia (Zr) or titanium (Ti) disks. MATERIALS AND METHODS: Disks fabricated from four different materials--smooth Zr (Zr-S), rough Zr (Zr-R), smooth Ti (Ti-S), and rough Ti (Ti-R)--were used, and surface roughness was analyzed by atomic force microscopy. After HGFs were cultured on these disks, cell morphology was examined by scanning electron microscopy, cell proliferation activity was evaluated by a monotetrazolium assay, and gene expression levels of various collagens and integrins were measured by real-time polymerase chain reaction. RESULTS: The Ti-R disks were the roughest, followed by Zr-R, Ti-S, and Zr-S, in that order. The cells cultured on the Zr-S and Ti-S disks appeared to be more aligned with the fine irregularities at later time points, whereas the cells cultured on the Zr-S showed the weakest spreading compared to the other surfaces after 3 hours of culture. With respect to proliferation, cells proliferated significantly faster on the Zr-S surface than on the other surfaces. Gene expression of integrin alpha2 at 3 hours and integrin alpha5 and type I collagen at 48 hours on Zr-S was significantly up-regulated compared to Ti. Conversely, the expression of integrins beta1 and beta3 and type III collagen was significantly decreased on Zr-S at 1 hour compared to the other materials. CONCLUSION: These data indicate that different surface materials and topographies may induce a distinct HGF morphology, proliferation, and gene expression.

Objectives: The purpose of the present study was to investigate the early peri-implant soft tissue healing on different titanium surface topographies. Material and methods: Titanium implants with smooth or rough surfaces were placed in the extraction site at 1 month after the maxillary first molar extractions of 15 male Sprague-Dawley rats. At 4 and 7 days after implant surgery, the peri-implant oral mucosa was randomly harvested and analyzed for collagen fiber orientation, and expression of extracellular matrix genes. Descriptive and paired t-tests were performed where appropriate (alpha=0.05). Results: The laser scanning microscopic analysis of the Sirius red stained peri-implant connective tissue revealed a parallel and uniform collagen fiber orientation along the smooth implant at both 4 and 7 days. In contrast, the collagen fibers of the comparative peri-implant tissue from rough surface implants at 7 days were not arranged in parallel orientation but in an irregular a 'cotton-ball-like.' The levels of mRNA of types III and XII collagen and transforming growth factor-beta1 significantly increased in the smooth implant group compared with the rough implant group at both 4 and 7 days or either one of days. Conclusion: The results of this study suggest that implant surface characteristics may affect early events of soft tissue healing by influencing collagen fiber orientation and expression of key genes for initial healing. To cite this article: Yamano S, Al-Sowygh ZH, Gallucci GO, Wada K, Weber H-P, Sukotjo C. Early peri-implant tissue reactions on different titanium surface topographies. Clin. Oral Impl. Res. xx, 2010; 000-000

The HIV-1 Tat peptide has been successfully used for intracellular gene delivery. Likewise, various lipid-based methods have shown increased endocytosis and can influence endosomal escape. This study combines the favorable properties of Tat peptide with that of lipid systems for DNA delivery. We combined the lipid FuGENE HD (FH) with the Tat peptide sequence modified with histidine and cysteine residues (mTat). mTat/FH transfection was evaluated by luciferase expression plasmid in five cell types. mTat/FH produced significant improvement in transfection efficiency of all cell lines when compared to FH or mTat. Treatment with chloroquine, associated with energy-dependent endocytosis, significantly increased transfection efficiency with mTat/FH while incubation at low temperature decreased it. The zeta potential of mTat/FH/DNA was significantly higher compared to FH, mTat, or their DNA combination in the presence of serum, and it was correlated with transfection efficiency. The particle size of the FH/DNA complex was significantly reduced by addition of mTat. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/FH transfection, but transfection was increased by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. These findings demonstrated the feasibility of using a combination of mTat with lipids, utilizing temperature-dependent and caveolae-mediated endocytosis, as a potentially attractive non-viral gene vector