Application of Autologous Human Bone Marrow-Derived Mesenchymal Stem Cells in Distraction Osteogenesis for the Treatment of Bilateral Mandibular Hypoplasia.

Distraction osteogenesis a surgical procedure conducted to improve craniofacial deformities. Compared with conventional operations, this technique has advantages such as the ability to lengthen the soft tissue and hard tissue. Therefore, this method is used to treat severe craniofacial abnormalities.The major disadvantage of distraction osteogenesis is the long treatment period. If the consolidation period is not sufficiently long after the distraction period, complications such as discontinuity or contraction of newly formed bone may occur. Recently, many researchers have attempted to develop methods for enhancing the ossification of newly formed bone, reducing shrinkage, and shortening the overall treatment period.The authors injected autologous bone marrow-derived mesenchymal stem cells during the consolidation phase after distraction osteogenesis surgery in a bilateral mandibular hypoplasia patient. Here, the authors report the treatment results, which were found to be favorable.

Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles.

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights, such as PEI25K (m.w. 25 kd) and PEI2K (m.w. 2 kd). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.

Biodistribution and tissue expression kinetics of plasmid DNA complexed with polyethylenimines of different molecular weight and structure.

Polyethylenimine (PEI) has been studied as an efficient and versatile in vitro and in vivo gene delivery agent. Here, we report the in vivo fate, tissue expression duration, and safety after the intravenous injection of plasmid DNA complexed to various PEIs under different conditions. Murine interleukin-2 plasmid DNA was complexed with branched PEI2Kd, 25Kd, or linear PEI25Kd at different N/P ratios. The mean residence time of plasmid DNA was found to be prolonged after delivery in PEI, evidencing the highest values in branched PEI25Kd. As compared to branched PEI25Kd, linear PEI25Kd at the same N/P ratio provided mRNA expression levels orders of magnitude higher in the lung over an 8-day period. In the branched PEI2Kd/DNA complexes, the N/P ratio of 80:1 evidenced higher gene expression efficiency in the kidney and spleen than the normal N/P ratio of 10:1. The generation of proinflammatory chemokine receptors was induced by branched PEI25Kd, but not by other PEIs. The complexes of DNA with linear 25Kd PEI or branched PEI2Kd exhibited no histological changes after repeated administrations. These results indicate that the structure, molecular weight, and N/P ratios of PEIs must be collectively considered and modulated for organ-targeted plasmid DNA delivery.

Mutational analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean Charcot-Marie-Tooth neuropathy patients.

We examined CMT1A duplication of 17p11.2-p12, mutations of PMP22, MPZ (P0), GJB1 (Cx32), EGR2 and NEFL genes in 57 Korean families with patients diagnosed as having Charcot-Marie-Tooth (CMT) disease. The CMT1A duplication was present in 53.6% of 28 CMT type 1 patients. In the 42 CMT families without CMT1A duplication, 10 pathogenic mutations were found in 9 families. The 10 mutations were not detected in 105 healthy controls. Seven mutations (c.318delT (p.Ala106fs) in PMP22, c.352G>A (p.Asp118Asn), c.449-1G>T (3'-splice site), c.706A>G (p.Lys236Glu) in MPZ, c.407T>C (p.Val136Ala)[corrected], c.502T>C (p.Cys168Arg) in GJB1, and c.1001T>C (p.Leu334Pro) in NEFL) were determined to be novel. The mutation frequencies of PMP22 and MPZ were similar to those found in several European populations, however, it appeared that mutations in GJB1 are less frequent in East Asian CMT patients than in Eur opean patients. We described the identified mutations and phenotype-genotype correlations based on nerve conduction studies.

Enzymatically degradable temperature-sensitive polypeptide as a new in-situ gelling biomaterial.

We are reporting a poly (ethylene glycol)-block-poly(alanine-co-phenyl alanine) (PEG-PAF) aqueous solution that undergoes sol-to-gel transition as the temperature increases. The sol-to-gel transition was observed at as low a concentration as 3.0-7.0 wt.%. Micellar aggregation accompanying small conformational changes of the peptide from random coils to beta-sheets is suggested as the sol-to-gel transition mechanism of the PEG-PAF aqueous solution. The PEG-PAF is stable in phosphate buffered saline, however, it degraded in the subcutaneous layer of rats. In vitro study showed that proteolytic enzymes such as cathepsin B, cathepsin C, and elastase that are present in the subcutaneous layer of the mammalian tissue might be responsible for the degradation of the polymer in rats. As a feasibility study of this material, a single shot of an aqueous insulin formulation (13.8 mg insulin/kg) showed a hypoglycemic effect over 18 days in rats. The current functional polypeptide may be very promising as an in-situ gelling system for tissue engineering, cell/stem cell therapy, and drug delivery.

Modulation of biodistribution and expression of plasmid DNA following mesenchymal progenitor cell-based delivery.

Although therapeutic applications of mesenchymal progenitor cells (MPCs) have been studied, the in vivo fate of genes delivered by the MPCs has received little attention. We report here the in vivo kinetics, tissue distribution, and duration of gene expression after systemic administration of plasmid DNA delivered by MPCs. Murine MPCs were isolated from bone marrow, cultured, and transfected with plasmid DNA using polyethylenimine. The gene-modified MPCs or naked plasmid DNA was administered intravenously to mice. Injected MPCs incorporating plasmid DNA yielded elevated serum concentrations when compared with the group treated with plasmid DNA alone, a 280-fold higher level measured at 5-min post-administration. Moreover, plasmid DNA delivered in MPCs was detected in several organs, lymph nodes, and bone marrow. The highest levels of distribution were observed in the liver, followed by lung and spleen at 4 days post-dose. Similar to the distribution of DNA, significant expression levels of the exogenous gene were observed only after delivery of the DNA in MPCs, demonstrating the sustained expression at the liver, lung, and kidney for 4 days after tail vein injection. This study provides perspectives regarding the in vivo fate and target tissue distribution of genes following MPC-based delivery.

Enhanced mucosal and systemic immunogenicity of human papillomavirus-like particles encapsidating interleukin-2 gene adjuvant.

Here, we report the enhanced mucosal and systemic immunogenicity of human papillomavirus type (HPV) 16 L1 virus-like particles (VLP) encapsidating a cytokine genetic adjuvant. Plasmid DNA expressing interleukin-2 (pIL2) was encapsidated in VLP using the reassembly property of VLP from disassembled L1 capsomeres. pIL2 in reassembled VLP showed stability against DNase I, indicating encapsidation. After intramuscular immunization into mice, the highest vaginal and salivary HPV16 L1-specific IgA titers were observed in pIL2-encapsidated VLP, followed by VLP plus pIL2 in separate plasmid, and VLP alone. Similar to mucosal responses, serum IgG, IgG1, and IgG2a antibody titers were the highest in the group treated with pIL2-encapsidated VLP. Moreover, the adjuvanticity of pIL2 encapsidated in VLP was stronger in IgG2a antibody relative to IgG1 antibody. Our results indicate that the encapsidation of a genetic cytokine adjuvant pIL2 would be beneficial for more effective induction of mucosal and systemic immune responses to VLP vaccines.