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Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis.

Acute myocardial infarction is a leading cause of death among single organ diseases. Despite successful reperfusion therapy, ischaemia reperfusion injury (IRI) can induce oxidative stress (OS), cardiomyocyte apoptosis, autophagy and release of inflammatory cytokines, resulting in increased infarct size. In IRI, mitochondrial dysfunction is a key factor, which involves the production of reactive oxygen species, activation of inflammatory signalling cascades or innate immune responses, and apoptosis. Therefore, intercellular mitochondrial transfer could be considered as a promising treatment strategy for ischaemic heart disease. However, low transfer efficiency is a challenge in clinical settings. We previously reported uptake of isolated exogenous mitochondria into cultured cells through co-incubation, mediated by macropinocytosis. Here, we report the use of transactivator of transcription dextran complexes (TAT-dextran) to enhance cellular uptake of exogenous mitochondria and improve the protective effect of mitochondrial replenishment in neonatal rat cardiomyocytes (NRCMs) against OS. TAT-dextran-modified mitochondria (TAT-Mito) showed a significantly higher level of cellular uptake. Mitochondrial transfer into NRCMs resulted in anti-apoptotic capability and prevented the suppression of oxidative phosphorylation in mitochondria after OS. Furthermore, TAT-Mito significantly reduced the apoptotic rates of cardiomyocytes after OS, compared to simple mitochondrial transfer. These results indicate the potential of mitochondrial replenishment therapy in OS-induced myocardial IRI.

[Introduction]Inflammatory response plays an important role in the disease progress or therapeutic effect. In this context, it is highly required to develop a technology to visualize the inflammatory response. In this study, macrophages and their microRNA (miRNA) which are involved in the inflammatory response, were focused while a system of molecular beacon (MB) to detect the miRNA of macrophages was designed and prepared. [Methods]Gelatin nanospheres were prepared by the conventional coacervation method. An antibody with an affinity for the surface receptor of macrophages was immobilized onto the gelatin nanospheres by several methods. A nucleic acid-based MB for a pro-inflammatory miRNA 155–5p was designed and incorporated into the antibody-immobilized gelatin nanospheres (MB-gelatin NS). Macrophages before and after the polarization into pro-inflammatory or anti-inflammatory phenotypes were cultured with the MB-gelatin NS and change in the intracellular fluorescence was observed. [Results]The antibody-immobilized gelatin nanospheres prepared by a coupling between the amino groups of gelatin and the sugar chains of antibody with NaIO4 showed the highest affinity for cellular receptor. MB complexed with the cell-penetrating (CP) peptide was successfully incorporated into the antibody-immobilized gelatin nanospheres. When cultured with pro-inflammatory macrophages, MB-gelatin NS efficiently detected the miRNA 155–5p to emit fluorescence. [Conclusions]By the NaIO₄ method, the antibody was immobilized onto gelatin nanospheres with a high affinity remaining while the MB was incorporated into the antibody-immobilized gelatin nanospheres. The MB incorporated allowed mRNA to visualize the pro-inflammatory nature of macrophages.

Regenerative medicine is a new and promising medical method aiming at treating patients with defective or dysfunctional tissues by maintaining or enhancing the biological activity of cells. The development of biomaterial-based technologies, such as cell scaffolds and carriers for drug delivery system, are highly required to promote the regenerative research and regenerative therapy. Nucleic acids are one of the most feasible factors to efficiently modify the biological activity of cells. The effective and stable delivery of nucleic acids into cells is highly required to succeed in the modification. Biomaterials-based non-viral carriers or biological carriers, like exosomes, play an important role in the efficient delivery of nucleic acids. This review introduces the examples of regenerative research and regenerative therapy based on the delivery of nucleic acids with biomaterials technologies and emphasizes their importance to accomplish regenerative medicine.

Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1⁺ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche.

The objective of this study is to design a new multimodal imaging system for the evaluation of bone regeneration process. Pamidronate (PA) of bisphosphonates with a high affinity for hydroxyapatite, was introduced to pullulan with different molecular weights (PA-pullulan). Then, two probes for fluorescence and magnetic resonance (MR) imagings were introduced into the PA-pullulan to prepare the PA-pullulan conjugates containing both the imaging probes (PA-pullulan-F/M). The PA-pullulan-F/M conjugates had an affinity for hydroxyapatite. A gelatin hydrogel incorporating bone morphogentic protein (BMP)-2 was prepared and implanted subcutaneously into mice to obtain an animal model of bone regeneration. When intravenously injected into mice with the bone tissue ectopically formed by the BMP-2-incorporated hydrogel to fluorescently evaluate their body distribution, the PA-pullulan-F/M conjugates were accumulated in the bone tissue regenerated. The time profile of fluorescent intensity well corresponded with that of calcium amount in the bone tissue newly formed. In addition, the PA-pullulan-F/M conjugates showed an MR ability similar to Gd-DTPA (gadopentetate dimeglumine) of a MR imaging agent clinically used. The MR signal around the bone tissue newly formed was enhanced for mice injected with PA-pullulan-F/M prepared from pullulan with the molecular weight of 6000. It is concluded that the PA-pullulan-F/M conjugate is a useful multimodal agent of polymeric delivery system to evaluate the process of bone regeneration.

A simple modification of a DNA aptamer with poly(ethylene glycol) (PEG) based on metal coordination was developed. N, N-bis(carboxymethyl)-L-lysine (NTA) of a metal chelate residue was chemically introduced to one terminus of PEG. The NTA-introduced PEG (PEG-NTA) chelated Cu(2+) ions form a Cu(2+)-chelated PEG (PEG-Cu). When PEG-Cu was mixed with a DNA aptamer of anti-tumor activity (AS1411) in aqueous solution, a complex of PEG-Cu and AS1411 based on metal coordination was formed. The complex inhibited in vitro tumor growth in a dose-dependent manner. A body distribution study with tumor-bearing mice revealed that PEG-Cu-AS1411 complexes injected intravenously had a significant longer lifetime in the blood circulation and 1.5-2.0-fold higher accumulation in the tumor tissue than free AS1411. Intravenous injection of complexes suppressed the in vivo growth of tumor mass to a significantly greater extent compared with that of free AS1411. The Cu(2+)-coordinated PEG modification is a simple and promising method to enhance accumulation of the aptamer in the tumor, resulting in the augmented anti-tumor effect.

Miho Watanabe, Hiaying Li, Jessica Roybal, Matthew Santore, Antonetta Radu, Jun-Ichiro Jo, Michio Kaneko, Yasuhiko Tabata, and Alan Flake. Tissue Engineering Part A.Apr 2011.1099-1110. http://doi.org/10.1089/ten.tea.2010.0390, Myelomeningocele (MMC) is a common and devastating malformation. As an alternative to fetal surgical repair, tissue engineering has the potential to provide a less invasive approach for tissue coverage applicable at an earlier stage of gestation. We have previously evaluated the use of gelatin hydrogel composites composed of gelatin sponges and sheets as a platform for tissue coverage of the MMC defect in the retinoic acid induced fetal rat model of MMC. In the current study, we compare our previous composite with gelatin microspheres as a scaffold for tissue ingrowth and cellular adhesion within the amniotic fluid environment. We also examine the relative efficacy of various bioactive protein coatings on the adhesion of amniotic fluid cells to the construct within the amniotic cavity. We conclude from this study that gelatin microspheres are as effective as gelatin sponges as a scaffold for cellular ingrowth and amniotic fluid cell adhesion and that collagen type I and fibronectin coatings enhance amniotic fluid cell adhesion to the gelatin-based scaffolds. These findings support the potential for the development of a tissue-engineered injectable scaffold that could be applied by ultrasound-guided injection, much earlier and less invasively than sponge or sheet-based composites. © 2011 Mary Ann Liebert, Inc.

The objective of this study was to genetically engineer dendritic cells (DC) for biological activation and evaluate their anti-tumor activity in a tumor-bearing mouse model. Mouse DC were incubated on the surface of culture dishes which had been coated with the complexes of a cationized dextran and luciferase plasmid DNA complexes plus a cell adhesion protein, Pronectin, for gene transfection (reverse transfection). When compared with the conventional transfection where DC were transfected in the medium containing the complexes, the level of gene expression by the reverse method was significantly higher and the time period of gene expression was prolonged. Following the reverse transfection of DC by a plasmid DNA of mouse interleukin-12 (mIL-12) complexed with the cationized dextran, the mIL-12 protein was secreted at higher amounts for a longer time period. When injected intratumorally into mice carrying a mass of B16 tumor cells, the DC genetically activated showed significant anti-tumor activity.