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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.

RNA interference (RNAi) is a gene silencing process by inhibiting a target messenger RNA (mRNA) in the sequence-specific manner in the cell cytoplasm. Small interfering RNA (siRNA) cleaves the target mRNA. However, siRNA is not generally internalized into cells in the native state. The objective of this study is to prepare cationized gelatin nanospheres (cGNS) incorporating small interfering RNA (siRNA) and to prolong the time period of gene expression suppression. The cGNS with different degradabilities were prepared to evaluate the effect on the suppression of gene expression. There was no difference in the apparent size and zeta potential of cGNS among the amounts of glutaraldehyde (GA) added for crosslinking. The degradation of cGNS tended to become slowly with an increase of GA amounts used in preparation. After MC3T3-E1 cells were incubated with cGNS incorporating siRNA, the gene expression of cells was evaluated by real-time polymerase chain reaction (PCR). The time period of gene suppression increased with an increased amount of siRNA incorporated in cGNS. Moreover, the significant gene suppression was extended over 4 days. It is concluded that the intracellular controlled release with the cGNS enabled siRNA to prolong the time period of gene expression suppression.

Composite scaffolds obtained by the combination of biodegradable porous scaffolds and hydroxyapatite with bone regeneration potential are feasible materials for bone tissue engineering. However, most composite scaffolds have been fabricated by complicated procedures or under thermally harsh conditions. We have previously demonstrated that hydroxyapatite coating onto various substrates under a thermally mild condition was achieved by erbium-doped yttrium aluminum garnet (Er: YAG) pulsed laser deposition (PLD). The purpose of this study was to prepare a polycaprolactone (PCL) porous scaffold coated with the hydroxyapatite by the Er: YAG-PLD method. Hydroxyapatite coating by the Er: YAG-PLD method was confirmed by morphology, crystallographic analysis, and surface chemical characterization studies. When cultured on PCL porous scaffold coated with hydroxyapatite, rat bone marrow-derived mesenchymal stem cells adhered, spread, and proliferated well. The micro-CT and staining analyses after the implantation of scaffold into the critical-sized calvaria bone defect in rats indicate that PCL porous scaffold coated with hydroxyapatite demonstrates accelerated and widespread bone formation. In conclusion, PCL porous scaffold coated with hydroxyapatite obtained by the Er: YAG-PLD method is a promising material in bone tissue engineering.

Basic fibroblast growth factor (bFGF) promotes epithelial cell proliferation and angiogenesis but its clinical applications are limited by its short half-life and low retention. Recently developed gelatin hydrogel sheets able to release physiologically active substances in a controlled manner have the potential to overcome these issues. In this study, the effects of gelatin hydrogel sheets impregnated with bFGF on flap survival and angiogenesis were examined in a murine skin flap model. A flap of 1 × 3 cm was generated on the backs of 60 C57BL/6 mice. The mice were divided into five groups (n = 12/group): Group I, untreated; Group II, treated with a gelatin hydrogel sheet impregnated with saline; Group III, treated with bFGF (50 µg) without sheets; Groups IV and V, treated with gelatin hydrogel sheets impregnated with 50 and 100 µg of bFGF, respectively. On the seventh day after surgery, the flap survival area and vascular network were examined and hematoxylin and eosin and von Willebrand factor staining were used for histological examinations. The flap survival areas were significantly larger in Groups IV and V than in other groups. The area of new vessels was significantly larger in Group IV than in the other groups. In the murine skin flap model, gelatin hydrogel sheets impregnated with bFGF promoted angiogenesis and improved flap survival. These findings support the use of bFGF-impregnated gelatin hydrogel sheets for improving ischemic flap survival in clinical settings.

Recently, many studies on the three-dimensional (3D) fabrication of cells have been performed. Under these circumstances, it is indispensable to develop the imaging technologies and methodologies for noninvasive visualization of 3D cells fabricated. The objective of this study is to develop the labeling method of human induced pluripotent stem (iPS) cells-derived 3D cartilage tissue with gelatin nanospheres coincorporating three kinds of quantum dots (QD) and iron oxide nanoparticles (IONP) (GNSQD+IONP). In this study, two labeling methods were performed. One is that a cartilage tissue was labeled directly by incubating with octaarginine (R8)-treated GNSQD+IONP (direct labeling method). The other one is a "dissociation and labeling method." First, the cartilage tissue was dissociated to cells in a single dispersed state. Then, the cells were incubated with R8-GNSQD+IONP in a monolayer culture. Finally, the cells labeled were fabricated to 3D pellets or cell sheets. By the direct labeling method, only cells residing in the surrounding site of cartilage tissue were labeled. On the contrary, the 3D cartilage pellets and the cell sheets were homogenously labeled and maintained fluorescently visualized over 4 weeks. In addition, the cartilage properties were histologically detected even after the process of dissociation and labeling. Homogenous labeling and visualization of human iPS cells-derived 3D cartilage tissue was achieved by the dissociation and labeling method with GNSQD+IONP. Impact statement The homogenous labeling and visualization of human iPS cells-derived three-dimensional (3D) cartilage tissue was achieved over 4 weeks by the dissociation and labeling method with gelatin nanospheres coincorporating quantum dots (QD) and iron oxide nanoparticles (IONP) (GNSQD+IONP). The cartilage properties of cells treated were maintained. It is concluded that the dissociation and labeling method with GNSQD+IONP is a promising to visualize the human iPS cells-derived 3D cartilage tissue.

Recently, many studies on the three-dimensional (3D) fabrication of cells have been performed. Under these circumstances, it is indispensable to develop the imaging technologies and methodologies for noninvasive visualization of 3D cells fabricated. The objective of this study is to develop the labeling method of human induced pluripotent stem (iPS) cells-derived 3D cartilage tissue with gelatin nanospheres coincorporating three kinds of quantum dots (QD) and iron oxide nanoparticles (IONP) (GNSQD+IONP). In this study, two labeling methods were performed. One is that a cartilage tissue was labeled directly by incubating with octaarginine (R8)-treated GNSQD+IONP (direct labeling method). The other one is a "dissociation and labeling method." First, the cartilage tissue was dissociated to cells in a single dispersed state. Then, the cells were incubated with R8-GNSQD+IONP in a monolayer culture. Finally, the cells labeled were fabricated to 3D pellets or cell sheets. By the direct labeling method, only cells residing in the surrounding site of cartilage tissue were labeled. On the contrary, the 3D cartilage pellets and the cell sheets were homogenously labeled and maintained fluorescently visualized over 4 weeks. In addition, the cartilage properties were histologically detected even after the process of dissociation and labeling. Homogenous labeling and visualization of human iPS cells-derived 3D cartilage tissue was achieved by the dissociation and labeling method with GNSQD+IONP. Impact statement The homogenous labeling and visualization of human iPS cells-derived three-dimensional (3D) cartilage tissue was achieved over 4 weeks by the dissociation and labeling method with gelatin nanospheres coincorporating quantum dots (QD) and iron oxide nanoparticles (IONP) (GNSQD+IONP). The cartilage properties of cells treated were maintained. It is concluded that the dissociation and labeling method with GNSQD+IONP is a promising to visualize the human iPS cells-derived 3D cartilage tissue.

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)

Irisin is a proteolytic product of fibronectin type II domain-containing 5, which is related to the improvement in glucose metabolism. Numerous studies have suggested that irisin is a crucial myokine linking muscle to bone in physiological and pathophysiological states.We examined the effects of local irisin administration with gelatin hydrogel sheets and intraperitoneal injection of irisin on the delayed femoral bone repair caused by streptozotocin (STZ)-induced diabetes in female mice. We analyzed the femurs of mice using quantitative computed tomography and histological analyses and then measured the mRNA levels in the damaged mouse tissues.Local irisin administration significantly blunted the delayed bone repair induced by STZ 10 days after a femoral bone defect was generated. Local irisin administration significantly blunted the number of Osterix-positive cells that were suppressed by STZ at the damaged site 4 days after a femoral bone defect was generated, although it did not affect the mRNA levels of chondrogenic and adipogenic genes 4 days after bone injury in the presence or absence of diabetes. On the other hand, intraperitoneal injection of irisin did not affect delayed bone repair induced by STZ 10 days after bone injury. Irisin significantly blunted the decrease in Osterix mRNA levels induced by advanced glycation end products or high-glucose conditions in ST2 cells in the presence of bone morphogenetic protein-2.We first showed that local irisin administration with gelatin hydrogel sheets improves the delayed bone repair induced by diabetic state partially by enhancing osteoblastic differentiation.

The objective of this study is to investigate the effect of lipopolysaccharide (LPS) addition on the gene transfection of human mesenchymal stem cells (hMSC). hMSC were treated with the LPS at different concentrations and the complex of spermine-introduced pullulan and luciferase plasmid DNA for 3 h. The maximum level of gene expression was observed for hMSC treated with a certain concentration range of LPS. In addition, the cytotoxicity, cellular internalization of complexes, and cell cycle after LPS treatment were investigated. The cytotoxicity increased with an increase in the LPS concentration treated. On the other hand, the cellular internalization of complexes increased with the increased LPS concentration, although the internalization was sharply reduced at the high concentration. The LPS treatment increased the actin polymerization of cells to allow to spread more. The enhanced cells spreading would enhance the cellular internalization of complexes. In addition, the LPS treatment increased the rate of cell cycle. It is possible that the balance of cytotoxicity, cellular internalization, and cell cycle caused by the LPS addition results in the enhanced gene transfection at a certain LPS concentration. It is concluded that LPS treatment positively modified the cellular internalization and the cell cycle, resulting in the enhanced gene transfection.

The objective of this study is to visualize cell apoptosis in three-dimensional (3D) cell aggregates based on molecular beacons (MB). Two types of MB for messenger RNA were used: caspase-3 MB as a target for apoptosis and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, caspase-3 and GAPDH MB were incorporated in cationized gelatin nanospheres (cGNS), respectively (cGNScasp3 MB and cGNSGAP MB). In addition, cGNS co-incorporating caspase-3 and GAPDH MB (cGNSdual MB) were prepared to perform the dual-color imaging for the same cell aggregate. The cGNSMB were incubated with mouse mesenchymal stem cells to label with MB in the two-dimensional culture. The cell apoptosis mediated by the addition of antibody for a death receptor Fas was ratiometrically detected by the cGNSdual MB to the same extent as single MB. The cell aggregates were prepared from MB-labeled cells, and the MB fluorescence was detected from almost all the cells even in the 3D aggregates to show the homogenous distribution. In addition to the Fas-mediated apoptosis, the aggregates were treated with camptothecin of a low-molecular weight apoptosis inducer. The fluorescence of caspase-3 MB was mainly distributed at the surface surrounding site of Fas-mediated apoptotic aggregates rather than the center site, while that of GAPDH MB was detected even in the interior site. On the other hand, in the camptothecin-induced apoptotic aggregates, both caspae-3 and GAPDH MB fluorescence were detected from the interior site of aggregates as well as the surrounding site. It is likely that the MB fluorescence reflected the localization of apoptotic position caused by the different molecular sizes of apoptosis inducer and the consequent penetration into the aggregates. It is concluded that the cGMSMB are a promising system to visualize cell apoptosis in 3D cell aggregates without the destruction of aggregates.

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.

The objective of this study is to visualize the ability of cell proliferation based on molecular beacons (MB). Two types of MB to detect messenger RNA (mRNA) were used. One is a Ki67 MB of a target for cell proliferation ability. The other one is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, the MB were incorporated into cationized gelatin nanospheres (cGNS). There was no difference in the physicochemical properties and the cell internalization between the cGNS

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.

We developed a simple method to prepare PEG-based soft nanoparticles that encapsulate dual imaging probes. Because the probes could be encapsulated by either chemical or physical means, a variety of probe molecules were encapsulated within the nanoparticles simultaneously. The nanoparticles were administrated to mice and the pharmacokinetics of the nanoparticles was analyzed by means of MRI, fluorescence spectroscopy, and transmission electron microscopy. The soft nanoparticles were excreted by the mice rapidly through the urine without collapse of the nanoparticles and without leaking of the probe molecules, and no accumulation of the nanoparticles in the body was observed. The pharmacokinetics of the nanoparticles was not changed by the encapsulated molecules and acute toxicity to mice was negligible. It was expect that these PEG-based soft nanoparticles will be applicable for use as a safe diagnostic agent.

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.

The objective of this study is to visualize the ability of cell proliferation based on molecular beacons (MB). Two types of MB to detect messenger RNA (mRNA) were used. One is a Ki67 MB of a target for cell proliferation ability. The other one is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, the MB were incorporated into cationized gelatin nanospheres (cGNS). There was no difference in the physicochemical properties and the cell internalization between the cGNSKi67 MB and cGNSGAP MB. When basic fibroblast growth factor (bFGF) was added to KUM6 cells of a mouse bone marrow-derived mesenchymal stem cell line, the expression of Ki67 and the cell proliferation increased with the bFGF concentration. After the incubation for the cell internalization of cGNSMB, the cells were further incubated for 24 hr with or without different concentrations of bFGF. The fluorescence of cGNSKi67 MB significantly increased with the increase of bFGF concentration, whereas that of cGNSGAP MB was constant, irrespective of the bFGF concentration. A timelapse imaging assay revealed that a fast enhancement of cGNSKi67 MB fluorescence after the bFGF addition compared with no bFGF addition. On the other hand, for cGNSGAP MB, a constant fluorescence was observed even at any time point after the bFGF addition. It is concluded that the cGNSMB system is a promising for the chronological visualization of proliferation ability in living cells.

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 such 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 5.5 ± 1.9 to 21 ± 3.7 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.

Recent studies on bone regeneration demonstrate the use of low cost and stable small molecules, which avoid the adverse effect and high cost of growth factors. Herein, we investigate the chemotactic, angiogenic and osteoinductive potential of a prostacyclin analogue, ONO-1301, when delivered through a biomimetic nanocomposite scaffold (nanohydroxyapatite-gelatin matrix reinforced with fibers) for bone tissue regeneration. The small molecule was loaded onto the scaffold in three different concentrations. There was burst release from all the groups of scaffolds within 24 h followed by a sustained release up to 14 days, but the concentration was dependent on loading percentage. ONO-1301 loaded scaffolds augmented the migration, proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs), but increasing the concentration beyond a certain dose did not show any effect. The osteoinduction was mediated through the prostaglandin I2 receptor and cyclic AMP (cAMP) signaling pathway. They also promoted new bone formation in large sized calvarial defects in rats compared to the scaffold alone, but did not show any impact on angiogenesis. Hence, this study suggests the chemotactic and osteoinductive capability of ONO-1301 for the repair and regeneration of critical sized bone defects.

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.

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.

The objective of this study is to prepare cationized gelatin nanospheres incorporating molecular beacon (MB) (cGNSMB) and a cationized gelatin-molecular beacon complex, and evaluate the time period of messenger RNA (mRNA) visualization. The cGNS with different degradabilities were prepared to incorporate MB. There was no difference in the apparent size and zeta potential between the cGNSMB and the complex, while the MB release from the complex was faster than that from the cGNSMB. When mouse mesenchymal stem cells were incubated with the complex and cGNSMB, the amount of MB internalized into cells and cytotoxicity of complex were higher compared with cGNSMB. However, in the amount range of noncytotoxicity, the amount of MB internalized was at a similar level among them. The intracellular fluorescence of cGNSMB was observed over 14 days, whereas that of complex disappeared within 5 days. Moreover, the time period of cGNSMB remaining in the cells prolonged with the increase of glutaraldehyde amount used in cGNSMB preparation. As the result, it is likely that the intracellular fluorescence was retained at a high level for longer time periods. It is concluded that the intracellular controlled release of MB is promising, to achieve long-term and continuous visualization of mRNA. Impact Statement Molecular beacon (MB) is a versatile activatable imaging probe to detect messenger RNA (mRNA). Cationized gelatin nanospheres incorporating MB (cGNSMB) with different degradabilities and a cationized gelatin-MB complex were prepared. The intracellular fluorescence of cGNSMB was observed over 14 days, whereas that of complex disappeared within 5 days. This is because the intracellular decrease of cGNSMB was slower than that of complex. It is concluded that the intracellular controlled release of MB is promising, to achieve long-term and continuous visualization of mRNA.

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.

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.

We previously have developed the photoresponsive tetra-gel and nanoparticles for controlling the function of the encapsulated substance by UV irradiation. However, the penetration ability of the UV is not high enough. Here, we developed a radiation-responsive tetra-gel and nanoparticle based on γ-ray-responsive X-shaped polyethylene glycol (PEG) linker with a disulfide bond. The nanoparticle could retain small molecules and biomacromolecules. γ-Rays were used as a trigger signal because of their higher penetrating ability. This allowed a spatiotemporal release and control of the encapsulated substances from the nanoparticle in the deeper region, which is impossible by using light exposure (ultraviolet, visible, and near-infrared).

Many types of gene delivery systems have been developed to enhance the level of gene expression. Controlled release technology is a feasible gene delivery system which enables genes to extend the expression duration by maintaining and releasing them at the injection site in a controlled manner. This technology can reduce the adverse effects by the bolus dose administration and avoid the repeated administration. Biodegradable biomaterials are useful as materials for the controlled release-based gene delivery technology and various biodegradable biomaterials have been developed.Controlled release-based gene delivery plays a critical role in a conventional gene therapy and genetic engineering. In the gene therapy, the therapeutic gene is released from biodegradable biomaterial matrices around the tissue to be treated. On the other hand, the intracellular controlled release of gene from the sub-micro-sized matrices is required for genetic engineering. Genetic engineering is feasible for cell transplantation as well as research of stem cells biology and medicine.DNA hydrogel containing a sequence of therapeutic gene and the exosome including the individual specific nucleic acids may become candidates for controlled release carriers. Technologies to deliver genes to cell aggregates will play an important role in the promotion of regenerative research and therapy.

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.