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Humoral factors that are secreted from skeletal muscles can regulate bone metabolism and contribute to muscle-bone relationships. Although extracellular vesicles (EVs) play important roles in physiological and pathophysiological processes, the roles of EVs that are secreted from skeletal muscles in bone repair have remained unclear. In the present study, we investigated the effects of the local administration of muscle cell-derived EVs on bone repair in control and streptozotocin-treated diabetic female mice. Muscle cell-derived EVs (Myo-EVs) were isolated from the conditioned medium from mouse muscle C2C12 cells by ultracentrifugation, after which Myo-EVs and gelatin hydrogel sheets were transplanted on femoral bone defect sites. The local administration of Myo-EVs significantly improved delayed bone repair that was induced by the diabetic state in mice 9 days after surgery. Moreover, this administration significantly enhanced the ratio of bone volume to tissue volume at the damaged sites 9 days after surgery in the control mice. Moreover, the local administration of Myo-EVs significantly blunted the number of Osterix-positive cells that were suppressed by the diabetic state at the damage sites after bone injury in mice. Additionally, Myo-EVs significantly blunted the mRNA levels of Osterix and alkaline phosphatase (ALP), and ALP activity was suppressed by advanced glycation end product 3 in ST2 cells that were treated with bone morphogenetic protein-2. In conclusion, we have shown for the first time that the local administration of Myo-EVs improves delayed bone repair that is induced by the diabetic state through an enhancement of osteoblastic differentiation in female mice.

OBJECTIVES Excessive and chronic inflammation after a myocardial infarction (MI) is associated with left ventricular remodelling and impaired cardiac function. Among inflammatory cells, macrophages play a critical role in polarizing proinflammatory M1 or the reparative M2 subtype. Pioglitazone (PGZ) is reported to regulate macrophage polarization to the M2 subtype. Our goal was to validate the therapeutic effects and the mechanisms of PGZ utilizing a drug delivery system. METHODS Poly L-lactic-co-glycolic acid microspheres (MS) incorporating PGZ were prepared. To validate the therapeutic potential of PGZ-MS, Sprague-Dawley rats were subjected to permanent left coronary artery ligation to induce an MI. Placebo-MS (100 μg) or PGZ-MS (100 μg) was injected to the infarct region just after induction. Cardiac function and size were assessed by echocardiography. At 28 days after surgery, the rats were sacrificed, and the excised hearts were evaluated histologically. RESULTS Sustained release of PGZ from the PGZ-MS was confirmed in vitro. PGZ-MS significantly rehabilitated cardiac dysfunction after an MI (fractional shortening: MI vs MI+placebo-MS vs MI+PGZ-MS, 24.4 ± 1.1 vs 24.3 ± 1.6 vs 32.2 ± 1.4%; P = 0.0035) with reverse remodelling. Immunohistochemical analyses revealed that PGZ-MS enhanced macrophage polarization (ratio of M2 subtype: 0.39 ± 0.03 vs 0.42 ± 0.02 vs 0.54 ± 0.02; P = 0.0004) and attenuated apoptosis of cardiomyocytes in the ischaemic border zone. CONCLUSIONS We confirmed macrophage polarization by sustained release of PGZ, which resulted in amelioration of adverse left ventricular remodelling and cardiac dysfunction. Drug delivery system-based macrophage polarization might serve as a promising strategy in cardiac regenerative therapy for ischaemic heart disease. (241 words)

Introduction Macrophages play an important role in regulating inflammation and tissue regeneration. It is known that anti-inflammatory macrophages play an important role for tissue regeneration. The objective of this study is to modify macrophages phenotypes for anti-inflammatory function by utilizing drug delivery technology. Method In this study, 4 types of poly (L-lactic-co-glycolic acid) (PLGA) microspheres incorporating pioglitazone of an anti-inflammatory modifier (pio-MS) with different sizes were prepared. In vitro release test of pio-MS was performed in phosphate buffered-saline solution (PBS) containing 1 wt% of sodium lauryl sulfate. The arginase activity and the secretion of interleukin (IL)−10 as anti-inflammatory macrophage markers of mouse bone marrow derived-macrophages (BMDM) cultured with the pio-MS were evaluated. Results The sustained release of pioglitazone was observed from all types of pio-MS in vitro. When BMDM were cultured with the pio-MS with an average diameter of 40 μm (pio-MS40), the arginase activity and the secretion of IL-10 increased to a significant extent compared with other pio-MS. Conclusions The pio-MS40 with an diameter of 40 μm had a potential to induce the anti-inflammatory modification of BMDM in this culture system. The sustained release of pioglitazone is promoting to modify the macrophage function., Highlights • Microspheres incorporating pioglitazone with different sizes were prepared. • Sustained release of pioglitazone from the microspheres were observed. • The effect of pioglitazone on macrophages was enhanced by the sustained release.

Background Nanoparticle-loaded delivery systems have attracted much attention recently. Poly(lactic-co-glycolic acid) (PLGA) is one of the most successful biodegradable polymers for biomedical applications. There are only a few studies on the treatment of dermal fibrosis with sustained-release drugs. Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays an important role in endogenous anti-fibrotic defense mechanisms. Recent studies have suggested that pioglitazone, a synthetic PPAR-γ activator, has effects beyond reducing blood sugar and it can reduce fibrosis and inflammation when used systemically. Objective We aimed to assess the effects of local injections of pioglitazone-loaded PLGA nanoparticles (PGN-NP) on an experimental sclerosis and to demonstrate the in vivo pharmacokinetics of subcutaneously administered PLGA nanoparticles. Methods Locally injectable PGN-NP were prepared and subcutaneously administered to bleomycin (BLM)-induced scleroderma model mice. The effect of pioglitazone was also evaluated with cultured fibroblasts. Coumarin-6-loaded fluorescent PLGA nanoparticles (FL-NP) and silicon naphthalocyanine-loaded near-infrared PLGA nanoparticles (NIR-NP) were used to demonstrate in vitro cellular uptake by cultured fibroblasts and the in vivo pharmacokinetics of subcutaneously administered nanoparticles. Results Weekly subcutaneous injections of PGN-NP attenuated skin fibrosis in BLM-induced scleroderma model mice. Pioglitazone significantly suppressed migration ability and TGF-β-mediated myofibroblast differentiation in cultured fibroblasts. FL-NP were internalized into cultured fibroblasts within 60 min, and PGN-NP-primed fibroblasts expressed anti-fibrotic phenotypes. Subcutaneously injected NIR-NP remained in the vicinity of the injection site more than non-particulate silicon naphthalocyanine. Conclusion These results provide a basis for the development of new treatments for dermal fibrosis and a better understanding of the potential of PLGA nanoparticles in dermatology.

The objective of this study is to prepare cationized gelatin-molecular beacon (MB) complexes for the visualization of intracellular messenger RNA (mRNA). The complexes were prepared from cationized gelatins with different extents of cationization and different mixing ratios of MB to cationized gelatin. The apparent size of complexes was almost similar, while the zeta potential was different among the complexes. Irrespective of the preparation conditions, the complexes had a sequence specificity against the target oligonucleotides in hybridization. The cytotoxicity and the amount of complexes internalized into cells increased with an increase in the cationization extent and the concentration of cationized gelatin. After the incubation with complexes prepared from cationized gelatin with the highest extent of cationization and at mixing ratios of 10 and 20 pmole MB/μg cationized gelatin, a high fluorescent intensity was detected. On the other hand, the complex prepared with the mixing ratio at 20 pmole/μg did not show any cytotoxicity. The complex was the most effective to visualize the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA endogenously present. In addition, even for enhanced green fluorescent protein (EGFP) mRNA exogenously transfected, the complex permitted to effectively detect it as well. It is concluded that both the endogenous and exogenous mRNA can be visualized in living cells by use of cationized gelatin-MB complexes designed.

Magnetic Resonance Imaging (MRI) contrast agents with rapid renal excretion that do not penetrate the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCFB) are preferred for safer and low-risk diagnosis. Gadolinium (Gd)-conjugated nanoparticles have been proposed for use as contrast agents; however, the particle size must range between 1 to 7 nm to ensure rapid renal excretion. In this study, three types of gelatin, dissolved in water at varying concentrations of 0.1-2 wt.%, were irradiated with 5 kGy γ-rays at 25°C under aerated conditions to produce ultra-small gelatin nanogels having an average particle size ranging between 6 ± 2 to 21 ± 4 nm. Ultra-small Gd-coordinated gelatin nanogels (GdGN) suitable for use as MRI contrast agents were produced using 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS) and DOTA-butylamine as Gd ligand derivatives. Non-cytotoxicity and effective relaxivity of GdGN as a positive MRI contrast agent were verified using in vivo experiments. Rapid renal excretion of GdGN was observed in mice within 1 h with no accumulation in the liver. GdGN did not migrate across the BCFB in normal mice, thus emphasizing its safety as an MRI contrast agent. STATEMENT OF SIGNIFICANCE: The authors developed ultra-small sized gelatin nanogels as blood-brain-barrier impermeable contrast agents for magnetic resonance imaging (MRI). The authors used radiation crosslinking technique to ensure better integrity of the amino acids present in the gelatin nanogels while conjugating with gadolinium (Gd) to form gadolinium-coordinated gelatin nanogels (GdGN). The safety and efficacy of GdGN, as MRI contrast agents, were verified by in vivo studies. GdGN exhibited rapid renal excretion within 90 minutes and no passage across the barriers in the brain.

A promising strategy for augmenting bone formation involves the local delivery of multiple osteoinductive and vasculogenic growth factors. However, success depends on sustained growth factor release and its appropriate combination to induce stem cells and osteogenic cells at the bony site. Herein, we have developed a nanocomposite fibrous scaffold loaded with fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) and its ability to promote vascularisation and bone regeneration in critical sized calvarial defect was compared to the scaffold with VEGF + BMP2 and FGF2 + BMP2. Simple loading of growth factors on the scaffold could provide a differential release pattern, both in vitro and in vivo (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks). Among all the groups, dual growth factor loaded scaffold (VEGF + BMP2 & FGF2 + BMP2) enhanced vascularisation and new bone formation, but there was no difference between FGF2 and VEGF loaded scaffolds although its release pattern was different. FGF2 mainly promoted stem cell migration, whereas VEGF augmented new blood vessel formation at the defect site. This study suggests that biomimetic nanocomposite scaffold is a promising growth factor delivery vehicle to improve bone regeneration in critical sized bone defects. Statement of Significance Many studies have shown the effect of growth factors like VEGF-BMP2 or FGF2-BMP2 in enhancing bone formation in critical sized defects, but there are no reports that demonstrate the direct comparison of VEGF-BMP2 and FGF2-BMP2. In this study, we have developed a nanocomposite fibrous scaffold that could differentially release growth factors like VEGF, BMP2 and FGF2 (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks), which in turn promoted neovascularisation and new bone formation in critical sized defects. There was no difference in vascularisation and bone formation induced by VEGF + BMP2 or FGF2 + BMP2. The growth factor was loaded in a simple manner, which would ensure ease of use for the end-user, especially for the surgeons treating a patient in an operating room.

Chronic myocardial infarction (CMI) tends to be resistant to treatments possibly due to extensive solid fibrotic scar, hypoxia mediated by poorly vascularized environment, and/or inflammation and apoptosis. Here we aimed to testify the therapeutic effects of sustained release of basic fibroblast growth factor (bFGF) using gelatin hydrogel (GH) in a rat chronic MI model and to elucidate the therapeutic mechanism including the alteration of extracellular matrix component. CMI model rats are prepared by the permanent ligation of proximal left anterior descending coronary artery. After 4 weeks, GH sheets (GHSs) with bFGF (100 µg) (bFGF group) or with phosphate-buffered saline (Vehicle group) were implanted to the CMI models to evaluate the effect of bFGF–GHS on chronic scar tissue. Sham operation group was also prepared (n = 5 for each). 4 weeks after implantation, bFGF–GHS significantly improved cardiac contractile function (fractional shortening: 21.8 ± 1.1 vs 21.5 ± 1.3 vs 29.7 ± 1.8%; P

Summary 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., Graphical Abstract, Highlights • FGF2 and GDNF induce morphologically distinct spermatogonial clusters in vivo • FGF2-expanded undifferentiated spermatogonia are prone to be RARG+ in vivo • FGF2 is more permissive than GDNF for Rarg expression in cultured spermatogonia • FGF2 suppresses GDNF production and retinoic acid metabolism in the germline niche, Takashima and colleagues demonstrate the novel functions of FGF2 in the germline niche. Although FGF2 induces self-renewal of spermatogonial stem cells in vitro, this molecule expands “differentiation-prone” GFRA1+RARG+ spermatogonia and facilitates retinoic acid actions required for differentiation in vivo. The present study suggests that FGF2 contributes to spermatogonial differentiation, indicating reconsideration of the role of FGF2 in the germline niche.

Introduction Nitric oxide (NO) is an intracellular and intercellular messenger that plays an important role in cellular events in physiological and pathophysiological processes. NO is one of the inflammation markers and macrophages of an inflammatory cell produce a large amount of NO compared with other cells. Non-invasive detection system of NO is highly required to realize an early therapeutic treatment considering the process of pathophysiological changes. The objective of this study is to develop an imaging agent of nitric oxide (NO). Methods A water-insoluble DAR-4M of fluorescent dye for NO was solubilized in water through the micelle formation with gelatin grafted with l-α-phosphatidylethanolamine distearoyl (DAR-4M micelles). Physicochemical and biological properties of DAR-4M micelles were investigated by using cultured cells and animals. Results The DAR-4M micelles responded to NO secreted from a NO donors, in contrast to the same concentration of free DAR-4M. When RAW264.7 of a macrophage cell line was stimulated by lipopolysaccharide (LPS) to allow them to generate NO, the DAR-4M micelles could detect NO of the cells to a significant great extent compared with free DAR-4M. After the intravenous injection of DAR-4M micelles or free DAR-4M to a mouse model of aristolochic acid (AA) induced acute interstitial nephritis, the DAR-4M micelles enhanced the fluorescence intensity from the kidneys to a significant great extent compared with the free DAR-4M injection. In case of DAR-4M micelles injection into normal mice, such an enhanced kidney fluorescence was not observed. A body distribution experiment demonstrated that the kidney accumulation of DAR-4M micelles was not modified by the AA-induced inflammation. After the AA injection, the number of CD11b-positive cells increased with time, indicating the increased number of inflammatory macrophages. Conclusion DAR-4M micelles are effective in imaging NO generated from macrophages accompanied with inflammation., Highlights • DAR-4M has been limited to use in vivo due to the lack of water-solubility. • DAR-4M was water-solubilized by the micelle formation with a hydrophobidized gelatin. • DAR-4M micelles were effective to visualize NO-producing macrophages in vivo.

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., Highlights • Antibody could be immobilized onto gelatin nanospheres with the affinity remaining. • MB for a pro-inflammatory miRNA was incorporated into gelatin nanospheres. • MB incorporated emitted the fluorescence in the pro-inflammatory macrophages.

Osteonecrosis of the jaw induced by administration of bisphosphonates (BPs), BP-related osteonecrosis (BRONJ), typically develops after tooth extraction and is medically challenging. As BPs inhibit oral mucosal cell growth, we hypothesized that suppression of the wound healing-inhibiting effects could prevent BRONJ onset after tooth extraction. Since basic fibroblast growth factor (bFGF) promotes wound healing, but has a short half-life, we examined whether the initiation of BRONJ could be prevented by applying a bFGF-containing gelatin hydrogel over the extraction sockets of BRONJ model rats. Forty-three rats, received two intravenous injections of zoledronic acid 60 μg/kg, once per week for a period of 2 weeks, underwent extraction of a unilateral lower first molar. The rats here were randomly assigned to the bFGF group (n = 15 rats, gelatin hydrogel sheets with incorporated bFGF applied over the sockets); the phosphate-buffered saline (PBS) group (n = 14 rats, gelatin hydrogel sheets without bFGF applied over the sockets); or the control group (n = 14 rats, nothing applied over the sockets). One rat in the bFGF group was sacrificed immediately after tooth extraction. Twenty-one rats were sacrificed at 3 weeks, and the remaining 21 rats were sacrificed at 8 weeks after tooth extractions. The harvested mandibles were analyzed using micro-computed tomography and sections were evaluated qualitatively for mucosal disruption and osteonecrosis. The incidence of osteonecrosis at 8 weeks after tooth extraction was 0% in the bFGF group, 100% in the PBS group, and 85.7% in the control group. The frequency of complete coverage of the extraction socket by mucosal tissue was significantly greater in the bFGF group than in the other groups. These results suggest that application of bFGF in the extraction socket promoted socket healing, which prevented BRONJ development. The growth-stimulating effects of bFGF may have offset the inhibition of wound healing by BP.

Background Evaluations for the tumorigenicity of transplantation of stem cell products is mandatory for clinical application. It is of importance to establish a system to accurately quantify contaminated tumorigenic cells regardless of the format of stem cell product. In the present report, we aimed to examine the accuracy of the quantification of tumorigenic cell numbers with commonly used 2 methods, quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM) using experimental models of stem cell products spiked with tumorigenic cells. Methods Human mesenchymal stem cells (hMSCs) and melanoma Mewo-Luc cells constitutively expressing luciferase were used. We stained Mewo-Luc cells with a cell linker then spiked onto hMSC suspensions and hMSC sheets. We validated the accuracy of 10-fold serial dilution technique for Mewo-Luc cell suspension using a Coulter counter. The samples spiked with Mewo-Luc cells were subjected to qPCR and FCM analyses, respectively for the quantification of Mewo-Luc cells. Results Ten-fold serial dilutions of Mewo-Luc cells were performed accurately with small deviation. In samples spiked with or less than 100 cells in hMSC suspensions, and samples spiked with or less than 1,000 cells in hMSC sheets showed significantly higher cell numbers in calculations by FCM, respectively (suspensions; qPCR vs FCM: 100 cells: 59 ± 25 vs 232 ± 35 cells, p = 0.022/10 cells: 21 ± 7 vs 114 ± 27 cells, p = 0.030, sheets; qPCR vs FCM: 1,000 cells: 1723 ± 258 vs 5810 ± 878 cells, p = 0.012/100 cells: 110 ± 18 vs 973 ± 232 cells, p = 0.012/10 cells: 20 ± 6 vs 141 ± 36 cells, p = 0.030). Conclusion Differences in accuracy between quantification methods should be considered in designing a tumorigenicity study model., Biochemistry; Cell biology; Tissue engineering; Cell culture; Stem cells research; Biomedical engineering; Regenerative medicine; Stem cell therapy; Tumorigenicity; Pre-clinical safety tests; Methodology.

The objective of this study is to prepare cationized gelatin nanospheres (cGNS) incorporating a molecular beacon (MB), and visualize cellular apoptosis. Two types of MB to detect the messenger RNA (mRNA) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GAP MB), and caspase-3 (casp3 MB) were incorporated in cGNS, respectively. MB incorporated in cGNS showed the DNA sequence specificity in hybridization. The cGNS incorporation enabled MB to enhance the stability against nuclease to a significantly great extent compared with free MB. The cGNS incorporating GAP MB were internalized into the KUM6 of a mouse bone marrow-derived stem cell by an endocytotic pathway. The cGNS were not distributed at the lysosomes. After the incubation with cGNS, the cell apoptosis was induced at different concentrations of camptothecin. No change in the intracellular fluorescence was observed for cGNSGAPMB. On the other hand, for the cGNScasp3MB, the fluorescent intensity significantly enhanced by the apoptosis induction of cells. It is concluded that cGNS incorporating MB is a promising system for the visualization of cellular apoptosis.

Macrophages play an important role in regulating inflammation and tissue regeneration. In the present study, uniform fibrin hydrogel scaffolds were engineered in millimeters. These scaffolds induced anti-inflammatory macrophages to digest and infiltrate the scaffold. The culture conditions of the fibrin hydrogels decreased the secretion of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, and increased the secretion of interleukin-10 (IL-10), an anti-inflammatory cytokine, in mouse bone marrow-derived macrophages. Similar results were also observed in human monocyte-derived macrophages (HMDMs). In addition, most of cells that infiltrated the fibrin hydrogels were macrophages expressing CD163, CD204, and CD206, which are anti-inflammatory macrophages markers, both in mice and in human cells. Therefore, to induce increased macrophage infiltration, we attempted to combine fibrin hydrogels with SEW2871, a monocyte/macrophage recruitment agent that is known to be a sphingosine-1 phosphate receptor 1 agonist, solubilized in water by micelle formation with a cholesterol-grafted gelatin. However, the fibrin hydrogels alone retained the same monocyte migration activity as the hydrogels with SEW2871-incorporated micelles in the hydrogel-bearing mouse model. These findings indicate that fibrin hydrogels have a strong promoting effect on the recruitment of anti-inflammatory macrophages. Therefore, fibrin hydrogels may be an optimal biomaterial in the design of medicines for macrophage-induced regenerative therapies. STATEMENT OF SIGNIFICANCE: The immune response to tissue injury is important for determining the speed and the result of the regeneration. Alternatively activated macrophages (M2 macrophages) resolve inflammatory response and promote tissue repair by producing anti-inflammatory factors. Promoting the recruitment of macrophages is a hopeful strategy in the design of biomaterials for tissue regeneration. In the present study, we combined the fibrin hydrogel, which promotes anti-inflammatory polarization, with a macrophage recruitment agent. We revealed that the fibrin hydrogel significantly promoted anti-inflammatory polarization in mouse in vivo and human in vitro. Moreover, macrophages significantly infiltrated into the fibrin hydrogel regardless of the agent combination. Fibrin hydrogels may become a reliable biomaterial for tissue regeneration, and the present study is believed to provide information for many researchers.

The advent of nanoparticle DDSs (drug delivery systems, nano-DDSs) is opening new pathways to understanding physiology and pathophysiology at the nanometer scale. A nano-DDS can be used to deliver higher local concentrations of drugs to a target region and magnify therapeutic effects. However, interstitial cells or fibrosis in intractable tumors, as occurs in pancreatic or scirrhous stomach cancer, tend to impede nanoparticle delivery. Thus, it is critical to optimize the type and size of nanoparticles to reach the target. High-resolution 3D imaging provides a means of “seeing” the nanoparticle distribution and therapeutic effects. We introduce the concept of “nano-pathophysiological imaging” as a strategy for theranostics. The strategy consists of selecting an appropriate nano-DDS and rapidly evaluating drug effects in vivo to guide the next round of therapy. In this article we classify nano-DDSs by component carrier materials and present an overview of the significance of nano-pathophysiological MRI.

The objective of this study was to design and prepare a new contrast agent of magnetic resonance (MR) imaging for the evaluation of therapeutic angiogenesis. Diethylenetriaminepentaacetic acid (DTPA) residue of a chelator was chemically introduced to dextran with a molecular weight of 74,000 (dextran-DTPA). Cyclic peptide containing an arginine-glycine-aspartic acid (RGD) sequence (cyclic RGD) with an inherent affinity for the α(v)β(3) integrin was then introduced to dextran-DTPA (Cyclic RGD-dextran-DTPA). Gd(3+) was added to cyclic RGD-dextran-DTPA to prepare a dextran-based MR contrast agent (Cyclic RGD-dextran-DTPA-Gd). Cyclic RGD-dextran-DTPA-Gd had affinity for cells expressing the α(v)β(3) integrin and showed a higher longitudinal relaxivity compared with DTPA-Gd of an MR contrast agent clinically used. Right femoral, external iliac, and deep femoral and circumflex arteries and veins were surgically ligated to prepare a mouse model of hindlimb ischemia. A laser Doppler analysis and histological evaluation confirmed that hindlimb ischemia healed naturally and was accompanied by angiogenesis, while α(v)β(3) integrin was expressed in the ischemic-angiogenic region without any treatment. Mice at 7 days after vascular ligation were used as an angiogenesis model. When intravenously injected into mice with hindlimb ischemia, cyclic RGD-dextran-DTPA accumulated in the ischemic-angiogenic region and showed the MR ability to detect the ischemic-angiogenic region. It is concluded that cyclic RGD-dextran-DTPA-Gd is a promising material for evaluation of therapeutic angiogenesis.

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 design three-dimensional scaffolds of bone marrow-derived stem cells capable of their gene transfection and evaluate the transfection extent. Three-dimensional scaffolds of gelatin and β-tricalcium phosphate (β-TCP) were prepared. Spermine-introduced pullulan (spermine-pullulan) was prepared as the non-viral carrier of gene transfection. The spermine-pullulan was mixed with a luciferase plasmid DNA to prepare their polyion complex. The scaffolds were treated with succinylated gelatin (suc-gel) at different concentrations, treated in different methods of freeze-drying and dehydrothermal treatment, and placed in the aqueous of complexes to prepare various scaffolds containing the complex. When the stem cells were seeded into the scaffolds to evaluate the gene transfection of cells, the level of plasmid DNA transfection depended on the method of complex containing scaffolds preparation. The complexes were released with time from the scaffolds although the release profile depended on the type of scaffolds. The order of gene transfection for the stem cells in the scaffolds was in good accordance with that of plasmid DNA released. It is possible that cells were transfected with the complexes released from the scaffolds.

Miho Watanabe, Jun-ichiro Jo, Antonetta Radu, Michio Kaneko, Yasuhiko Tabata, and Alan W. Flake. Tissue Engineering Part A.May 2010.1645-1655. http://doi.org/10.1089/ten.tea.2009.0532., Myelomeningocele (MMC) is a common and devastating malformation. Although fetal surgical closure may improve outcome, a less invasive approach that can be applied earlier in gestation is desirable. The objective of this study was to evaluate the therapeutic feasibility of a tissue engineering approach for prenatal coverage of MMC. A gelatin hydrogel composite combining a gelatin sheet and gelatin sponge was prepared with or without basic fibroblast growth factor incorporation, and applied prenatally to retinoic-acid-induced fetal MMC in the rat model. Most of the composites were adherent to the MMC within the amniotic fluid environment with the help of cyanoacrylate adhesive. Histological examination revealed cells layered over the composites with associated extracellular matrix as well as cellular ingrowth into the sponges. The layer over the composite was composed of mixed nonepithelial and epithelial cells with the extracellular matrix consisting of collagen type I and hyaluronic acid. The tissue inside the sponge consisted of nonepithelial cells and hyaluronic acid. Epidermal ingrowth underneath the sponges and neovascularization into the sponges occurred and were significantly increased by the incorporation of basic fibroblast growth factor. Although further development is needed, this study supports the therapeutic potential of a tissue engineering approach for prenatal coverage of MMC. © 2010 Mary Ann Liebert, Inc.

The objective of this study is to prepare a non-viral carrier of gene transfection from various polysaccharides and evaluate the feasibility in gene expression for mesenchymal stem cells (MSCs). Various amounts of spermine were chemically introduced into pullulan, dextran and mannan with a molecular weight of around 40 000 or pullulan with different molecular weights to prepare cationized polysaccharides with different extents of spermine introduced (spermine-polysaccharide). Each cationized polysaccharide was complexed with a plasmid DNA at various ratios and in vitro gene transfection was investigated for rat bone marrow-derived MSCs. The level of gene expression depended on the type of cationized polysaccharide. The highest level was observed for the complex of spermine-pullulan and plasmid DNA. Additionally, the level also depended on the molecular weight of pullulan and the extent of spermine introduced to pullulan. Suppression of gene expression with chlorpromazine and methyl-beta-cyclodextrin of endocytosis inhibitors demonstrated that the cellular uptake of spermine-pullulan-plasmid DNA complexes was mediated by clathrin- and raft/caveolae-dependent endocytic pathways. The cationized pullulan is a promising non-viral carrier of plasmid DNA for MSCs.

Purpose To assess whether Gd-DTPA-Gel-Cis, a conjugate of gadolinium (Gd), cis diamminedichloroplatinum (Cis), diethylenetriaminepentaacetic acid (DTPA)-dianhydride, and bovine gelatin (Gel) can be used as an intravascular contrast agent at MRI and as an antitumor cell proliferation agent in vitro. Materials and Methods We injected Gd-DTPA-Gel-Cis (200 mg/mL) into the caudal vein of female HER-2/neu transgenic mice with spontaneous mammary tumors. The tumor signal intensity was measured with a 0.3 Tesla MRI scanner. HER-2/neu-expressing NT cells were treated with Gd-DTPA-Gel-Cis (5 μM cisplatin, 200 mg/mL Gel), Cis alone (5 μM cisplatin), or Gel alone (200 mg/mL gelatin). Differences of P < 0.05 were considered to be statistically significant. Results On T1-weighted MRI scans of mice injected with Gd-DTPA-Gel-Cis we observed a 23% increase in signal intensity. The survival rates of cells exposed to Gd-DTPA-Gel-Cis or Cis were 70.9% and 58.3%, respectively, of the survival rates observed after treatment with Gel alone. Gd-DTPA-Gel-Cis showed significant toxicity (P < 0.05). Conclusion Gd-DTPA-Gel-Cis shows promise for use as an MRI contrast medium and as an antitumor agent. J. Magn. Reson. Imaging 2009;30:907–910. © 2009 Wiley-Liss, Inc.

Background— In-stent thrombosis is mainly triggered by adenosine diphosphate (ADP)-dependent platelet aggregation after percutanous coronary stent implantation. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) rapidly hydrolyzes ADP to adenosine monophosphate, inhibiting platelet aggregation. We tested the hypothesis that local delivery of human placental E-NTPDase (pE-NTPDase) gene into injured arteries via gene-eluting stent could prevent subacute in-stent thrombosis. Methods and Results— We generated gene-eluting stents by coating bare metal stents with cationic gelatin hydrogel containing pE-NTPDase cDNA (pE-NTPDase stent), and implanted the stents into rabbit femoral arteries (FA) prone to production of platelet-rich thrombi due to repeated balloon injury at 4-week intervals. After the second injury, E-NTPDase gene expression was severely decreased; however, the implantation of pE-NTPDase stent increased E-NTPDase mRNA levels and NTPDase activity to higher level than normal FA. The FAs with pE-NTPDase stents maintained patency in all rabbits ( P Conclusions— Human pE-NTPDase gene transfer via cationic gelatin-coated stents inhibited subacute in-stent thrombosis and suppressed neointimal hyperplasia and inflammation without antiplatelet drugs.

Autoimmune alopecia (alopecia areata) is considered to be triggered by a collapse of immune privilege in hair follicles. Here we confirmed that infiltrating CD4 T lymphocytes around hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells, suggesting a dominant role of Th1 cells in the alopecic lesion. Given this finding, we sought to elucidate the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant interleukin-4 and neutralizing anti-interferon-gamma antibody. We found that local injections of both interleukin-4 and neutralizing anti-interferon-gamma antibody effectively treated alopecia in C3H/HeJ mice. Results from immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction demonstrated that intralesional injection of interleukin-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon-gamma mRNA expression in the affected alopecic skin. Furthermore, Th1 transcription factor T-box21 small interfering RNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. Taken together, the use of gelatin-small interfering RNA conjugates promises to be a novel, efficient, and safe tool as an alternative gene therapy for the treatment of various human diseases. To our knowledge, this is the first report of effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease.

次世代DDS型治療システムとは，従来のDDS治療効果をさらに向上させるために，外部エネルギーをDDS技術と融合させる新しい技術・方法論である．この新治療システムによって，体内の深部にある病気を治療できるであろう．本稿では，磁場とDDSとを組み合わせた，磁気誘導DDSによる次世代治療システムを実現させるために必要となる技術要素を概説するとともに，その治療ポテンシャルについて述べる．

The feasibility and mechanism of gene delivery by pullulan–spermine, a recently developed cationic polysaccharide, were investigated. Pullulan–spermine-mediated transfection of plasmid DNA resulted in greatly reduced cytotoxicity and a 10-fold increase in the level of gene expression when compared to Lipofectamine 2000, a commercially available cationic lipid. Additionally, after transfection of p53-expressing plasmid DNA by pullulan–spermine but not Lipofectamine 2000, the in vitro proliferation of T24 cells was significantly reduced. Pullulan–spermine-mediated gene expression was inhibited by both chlorpromazine of clathrin-mediated endocytosis inhibitor and methyl-β-cyclodextrin and filipin of raft/caveolae inhibitors. We conclude that pullulan–spermine is a promising carrier for gene transfection, and that cellular uptake of pullulan–spermine–plasmid DNA complexes is mediated by clathrin- and raft/caveolae-dependent endocytotic pathways.

The human noncoding RNA gene RGM249 has been shown to regulate the degree of cancer cell differentiation. In this study, we investigated the effects of 3 microRNA-like molecules digested from RGM249 on the loss of malignant properties in cancer cells in immunodeficient KSN/Slc mice. We utilized small interfering RNAs (siRNAs) alone or in combination with a cationized drug delivery system (DDS) consisting of atelocollagen or gelatin hydrogel microspheres. The results demonstrated growth inhibition and apoptosis and the inhibition of both neovascularization and metastasis, indicating that the DDSs effectively infiltrated the majority of tumor cells in vivo. Systemic administration of the 3 siRNAs inhibited the metastatic ability of malignant cells. Cotransfection of these siRNAs exerted a regulatory effect upon the genes involved in differentiation, pluripotency, and proliferation in cancer cells. These results suggest that RGM249-derived oligonucleotides may be involved in the regulation of metastasis, proliferation, and differentiation in vivo, and that the tested siRNAs may therefore represent a new anticancer therapeutic approach.