Coupling of bone resorption and formation by RANKL reverse signalling.

Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis.

Extracellular vesicles secreted from mouse muscle cells improve delayed bone repair in diabetic mice.

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.

The Transfer of the Hepatocyte Growth Factor Gene by Macrophages Ameliorates the Progression of Peritoneal Fibrosis in Mice.

Growing evidence indicates that hepatocyte growth factor (HGF) possesses potent antifibrotic activity. Furthermore, macrophages migrate to inflamed sites and have been linked to the progression of fibrosis. In this study, we utilized macrophages as vehicles to express and deliver the HGF gene and investigated whether macrophages carrying the HGF expression vector (HGF-M) could suppress peritoneal fibrosis development in mice. We obtained macrophages from the peritoneal cavity of mice stimulated with 3% thioglycollate and used cationized gelatin microspheres (CGMs) to produce HGF expression vector-gelatin complexes. Macrophages phagocytosed these CGMs, and gene transfer into macrophages was confirmed in vitro. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) for three weeks; seven days after the first CG injection, HGF-M was administered intravenously. Transplantation of HGF-M significantly suppressed submesothelial thickening and reduced type III collagen expression. Moreover, in the HGF-M-treated group, the number of α-smooth muscle actin- and TGF-ß-positive cells were significantly lower in the peritoneum, and ultrafiltration was preserved. Our results indicated that the transplantation of HGF-M prevented the progression of peritoneal fibrosis and indicated that this novel gene therapy using macrophages may have potential for treating peritoneal fibrosis.

Classification of PD-L1 expression in various cancers and macrophages based on immunohistocytological analysis.

Programmed death (PD)-1/PD-ligand 1 (PD-L1) antibodies have shown an intense clinical effect in some patients with PD-L1+ tumors, and their applications have rapidly expanded to various cancer types with or without the application of new companion diagnostics (CDx) with a lower cutoff value and inclusion of macrophage evaluation. However, the pathological background explaining the difference in the cutoff value remains unknown. To address this, we evaluated tissue array samples from 231 patients with lung adenocarcinoma, 186 with lung squamous cell carcinoma, and 38 with renal cell carcinoma (RCC) who were not receiving PD-1/PD-L1 antibodies to investigate the relationship between PD-L1 expression on tumor cells and CD8+ T-cell infiltration in tumor tissues. PD-L1 expression in RCC was clearly lower than that in non-small-cell lung cancer (NSCLC) tissue, whereas CD8+ T-cell infiltration was low in all cancers. We next analyzed PD-L1 expression by interferon (α, ß, and γ) and LPS stimulation in both macrophages and 41 cancer cell lines derived from various organs and histological types. The PD-L1 expression patterns were classified into three types, which differed depending on each organ or tissue type. Interestingly, NSCLC cell lines showed highly diverse PD-L1 expression patterns compared with RCC cell lines. Conversely, PD-L1 expression was stronger and more prolonged in macrophages than in typical cell lines. Here, we revealed the diversity of the PD-L1 expression patterns in tumor cells and macrophages, demonstrating the pathological and cytological significance of the transition of cutoff values in PD-L1 CDx for PD-1/PD-L1 antibody administration.

Irisin improves delayed bone repair in diabetic female mice.

INTRODUCTION: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

In Vivo Multimodal Imaging of Stem Cells Using Nanohybrid Particles Incorporating Quantum Dots and Magnetic Nanoparticles.

The diagnosis of the dynamics, accumulation, and engraftment of transplanted stem cells in vivo is essential for ensuring the safety and the maximum therapeutic effect of regenerative medicine. However, in vivo imaging technologies for detecting transplanted stem cells are not sufficient at present. We developed nanohybrid particles composed of dendron-baring lipids having two unsaturated bonds (DLU2) molecules, quantum dots (QDs), and magnetic nanoparticles in order to diagnose the dynamics, accumulation, and engraftment of transplanted stem cells, and then addressed the labeling and in vivo fluorescence and magnetic resonance (MR) imaging of stem cells using the nanohybrid particles (DLU2-NPs). Five kinds of DLU2-NPs (DLU2-NPs-1-5) composed of different concentrations of DLU2 molecules, QDs525, QDs605, QDs705, and ATDM were prepared. Adipose tissue-derived stem cells (ASCs) were labeled with DLU2-NPs for 4 h incubation, no cytotoxicity or marked effect on the proliferation ability was observed in ASCs labeled with DLU2-NPs (640- or 320-fold diluted). ASCs labeled with DLU2-NPs (640-fold diluted) were transplanted subcutaneously onto the backs of mice, and the labeled ASCs could be imaged with good contrast using in vivo fluorescence and an MR imaging system. DLU2-NPs may be useful for in vivo multimodal imaging of transplanted stem cells.

Cellular Interaction of Human Skin Cells towards Natural Bioink via 3D-Bioprinting Technologies for Chronic Wound: A Comprehensive Review.

Skin substitutes can provide a temporary or permanent treatment option for chronic wounds. The selection of skin substitutes depends on several factors, including the type of wound and its severity. Full-thickness skin grafts (SGs) require a well-vascularised bed and sometimes will lead to contraction and scarring formation. Besides, donor sites for full-thickness skin grafts are very limited if the wound area is big, and it has been proven to have the lowest survival rate compared to thick- and thin-split thickness. Tissue engineering technology has introduced new advanced strategies since the last decades to fabricate the composite scaffold via the 3D-bioprinting approach as a tissue replacement strategy. Considering the current global donor shortage for autologous split-thickness skin graft (ASSG), skin 3D-bioprinting has emerged as a potential alternative to replace the ASSG treatment. The three-dimensional (3D)-bioprinting technique yields scaffold fabrication with the combination of biomaterials and cells to form bioinks. Thus, the essential key factor for success in 3D-bioprinting is selecting and developing suitable bioinks to maintain the mechanisms of cellular activity. This crucial stage is vital to mimic the native extracellular matrix (ECM) for the sustainability of cell viability before tissue regeneration. This comprehensive review outlined the application of the 3D-bioprinting technique to develop skin tissue regeneration. The cell viability of human skin cells, dermal fibroblasts (DFs), and keratinocytes (KCs) during in vitro testing has been further discussed prior to in vivo application. It is essential to ensure the printed tissue/organ constantly allows cellular activities, including cell proliferation rate and migration capacity. Therefore, 3D-bioprinting plays a vital role in developing a complex skin tissue structure for tissue replacement approach in future precision medicine.

TAT-dextran-mediated mitochondrial transfer enhances recovery from models of reperfusion injury in cultured cardiomyocytes.

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.

Retraction Note: In Vitro Transfection of Plasmid DNA by Amine Derivatives of Gelatin Accompanied with Ultrasound Irradiation.

The Editor-in-Chief has retracted this article [1] at the request of the corresponding author. Figure 1B appears to be identical to Figure 1D, despite being under different experimental conditions.

Improved viability of murine skin flaps using a gelatin hydrogel sheet impregnated with bFGF.

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.

Evaluation of dual release of stromal cell-derived factor-1 and basic fibroblast growth factor with nerve conduit for peripheral nerve regeneration: An experimental study in mice.

BACKGROUND: The development of drug delivery systems has enabled the release of multiple bioactive molecules. The efficacy of nerve conduits coated with dual controlled release of stromal cell-derived factor-1 (SDF-1) and basic fibroblast growth factor (bFGF) for peripheral nerve regeneration was investigated. MATERIALS AND METHODS: Sixty-two C57BL6 mice were used for peripheral nerve regeneration with a nerve conduit (inner diameter, 1 mm, and length, 7 mm) and an autograft. The mice were randomized into five groups based on the different repairs of nerve defects. In the group of repair with conduits alone (n = 9), a 5-mm sciatic nerve defect was repaired by the nerve conduit. In the group of repair with conduits coated with bFGF (n = 10), SDF-1 (n = 10), and SDF-1/bFGF (n = 10), it was repaired by the nerve conduit with bFGF gelatin, SDF-1 gelatin, and SDF-1/bFGF gelatin, respectively. In the group of repair with autografts (n = 10), it was repaired by the resected nerve itself. The functional recovery, nerve regeneration, angiogenesis, and TGF-ß1 gene expression were assessed. RESULTS: In the conduits coated with SDF-1/bFGF group, the mean sciatic functional index value (-88.68 ± 10.64, p = .034) and the axon number (218.8 ± 111.1, p = .049) were significantly higher than the conduit alone group, followed by the autograft group; in addition, numerous CD34-positive cells and micro vessels were observed. TGF-ß1 gene expression relative values in the conduits with SDF-1/bFGF group at 3 days (7.99 ± 5.14, p = .049) significantly increased more than the conduits alone group. CONCLUSION: Nerve conduits coated with dual controlled release of SDF-1 and bFGF promoted peripheral nerve regeneration.

3D Culture of MSCs on a Gelatin Microsphere in a Dynamic Culture System Enhances Chondrogenesis.

Recent advancement in cartilage tissue engineering has explored the potential of 3D culture to mimic the in vivo environment of human cartilaginous tissue. Three-dimensional culture using microspheres was described to play a role in driving the differentiation of mesenchymal stem cells to chondrocyte lineage. However, factors such as mechanical agitation on cell chondrogenesis during culture on the microspheres has yet to be elucidated. In this study, we compared the 2D and 3D culture of bone-marrow-derived mesenchymal stem cells (BMSCs) on gelatin microspheres (GMs) in terms of MSC stemness properties, immune-phenotype, multilineage differentiation properties, and proliferation rate. Then, to study the effect of mechanical agitation on chondrogenic differentiation in 3D culture, we cultured BMSCs on GM (BMSCs-GM) in either static or dynamic bioreactor system with two different mediums, i.e., F12: DMEM (1:1) + 10% FBS (FD) and chondrogenic induction medium (CIM). Our results show that BMSCs attached to the GM surface and remained viable in 3D culture. BMSCs-GM proliferated faster and displayed higher stemness properties than BMSCs on a tissue culture plate (BMSCs-TCP). GMs also enhanced the efficiency of in-vitro chondrogenesis of BMSCs, especially in a dynamic culture with higher cell proliferation, RNA expression, and protein expression compared to that in a static culture. To conclude, our results indicate that the 3D culture of BMSCs on gelatin microsphere was superior to 2D culture on a standard tissue culture plate. Furthermore, culturing BMSCs on GM in dynamic culture conditions enhanced their chondrogenic differentiation.

Systematic chemical screening identifies disulfiram as a repurposed drug that enhances sensitivity to cisplatin in bladder cancer: a summary of preclinical studies.

BACKGROUND: Since the standard gemcitabine and cisplatin (GC) chemotherapy for advanced bladder cancer yields limited therapeutic effect due to chemoresistance, it is a clinical challenge to enhance sensitivity to GC. METHODS: We performed high-throughput screening by using a library of known chemicals and repositionable drugs. A total of 2098 compounds were administered alone or with GC to human bladder cancer cells, and chemicals that enhanced GC effects were screened. RESULTS: Disulfiram (DSF), an anti-alcoholism drug, was identified as a candidate showing synergistic effects with cisplatin but not with gemcitabine in multiple cell lines. Co-administration of DSF with GC affected cellular localisation of a cisplatin efflux transporter ATP7A, increased DNA-platinum adducts and promoted apoptosis. Micellar DSF nanoparticles (DSF-NP) that stabilised DSF in vivo, enhanced the inhibitory effect of cisplatin in patient-derived and cell-based xenograft models without severe adverse effects. A drug susceptibility evaluation system by using cancer tissue-originated spheroid culture showed promise in identifying cases who would benefit from DSF with cisplatin. CONCLUSIONS: The present study highlighted the advantage of drug repurposing to enhance the efficacy of anticancer chemotherapy. Repurposing of DSF to a chemotherapy sensitiser may provide additional efficacy with less expense by using an available drug with a well-characterised safety profile.

Efficacy of Gelatin Hydrogel Impregnated With Concentrated Platelet Lysate in Murine Wound Healing.

BACKGROUND: The efficacy of a gelatin hydrogel (GH) sheet impregnated with platelet-rich plasma in full-thickness wound healing has been reported. Human platelet lysate is another potential natural product for use in wound healing. The present study examined the effects of a GH sheet impregnated with concentrated freeze-dried platelet lysate on wound healing after storage for 9 mo. MATERIALS AND METHODS: Platelet concentrates were subjected to three freeze-thaw cycles and freeze-dried then preserved at 4°C. Reconstitution with saline was then performed to produce 1-fold (hPL1), 2-fold (hPL2), and 3-fold (hPL3) concentrations of preserved platelet lysate. Full-thickness wounds were made on the back of male C57Bl6J/Jcl mice. Wounds were treated with saline, hPL1, or a GH sheet impregnated with saline, hPL1, hPL2, or hPL3. Histologic examinations using hematoxylin-eosin, Azan, and anti-CD31 staining were performed on days 4, 7, and 14 to assess neoepithelialization and granulation tissue and capillary formation. RESULTS: This study showed that the GH sheet itself or the simple administration of hPL1 did not accelerate the healing process. However, the GH sheet impregnated with hPL1 accelerated the granulation tissue formation to some extent, and the GH sheet impregnated with hPL2 or hPL3 clearly accelerated the capillary formation and the granulation tissue formation. In addition, the GH sheet impregnated with hPL3 had the longest epithelium formation. CONCLUSIONS: A GH sheet impregnated with long-term preserved 2-fold or 3-fold concentrated platelet lysate enhances the wound healing process.

Comparison between different isoelectric points of biodegradable gelatin sponges incorporating ß-tricalcium phosphate and recombinant human fibroblast growth factor-2 for ridge augmentation: A preclinical study of saddle-type defects in dogs.

BACKGROUND AND OBJECTIVE: It is well known that recombinant human fibroblast growth factor-2 (rhFGF-2) signaling plays an important role in tissue repair and regeneration. rhFGF-2 strongly binds to acidic gelatin via ionic linkages and is gradually released upon gelatin decomposition. On the other hand, the linkage between rhFGF-2 and basic gelatin is so weak that most rhFGF-2 is rapidly released from basic gelatin by simple desorption. Gelatin/ß-tricalcium phosphate (ß-TCP) sponges, which comprise 50 wt% gelatin and 50 wt% ß-TCP in a cross-linked structure, can release rhFGF-2 gradually owing to their electrical features. In a previous study, we reported that new bone height in the test group using rhFGF-2 with acidic gelatin/ß-TCP sponges was significantly greater than that in the control group using acidic gelatin/ß-TCP sponges alone in a ridge augmentation model in dogs. However, whether these results depend on controlled release by the gelatin/ß-TCP sponges remains controversial. In this study, we evaluated the effects of controlled release by comparing acidic and basic gelatin/ß-TCP sponges with different isoelectric points (IEP) on ridge augmentation in dogs. MATERIALS AND METHODS: Twelve weeks after extraction of the maxillary second and third incisors of six dogs, critically sized saddle-type defects (8 mm length × 4 mm depth) were surgically created bilaterally 2 mm from the mesial side of the canine. Acidic gelatin/ß-TCP sponges (IEP 5.0) soaked with 0.3% rhFGF-2 were applied to the defect in the acidic group, whereas basic gelatin/ß-TCP sponges (IEP 9.0) soaked with 0.3% rhFGF-2 were applied to the defect in the basic group. Twelve weeks after surgery, biopsy specimens were obtained and subjected to microcomputed tomography (micro-CT) and histological analyses. RESULTS: New bone area detected by micro-CT analysis was significantly smaller in the basic group than in the acidic group. New bone height calculated by histologic sections was significantly lower in the basic group than in the acidic group. The total tissue height was lower in the basic group than in the acidic group. However, the differences between both sites were not significant. CONCLUSIONS: These findings suggest that in ridge augmentation of saddle-type defects, controlled release of rhFGF-2 induces notably more alveolar bone formation than does short-term application of rhFGF-2.

Intraperitoneal chemotherapy for peritoneal metastases using sustained release formula of cisplatin-incorporated gelatin hydrogel granules.

PURPOSE: We previously reported the effectiveness of gelatin microspheres incorporating cisplatin in a mouse model of peritoneal metastases. In this study, we report our new complete sustained-release formula of gelatin hydrogel granules incorporating cisplatin (GHG-CDDP), which exerted a good anti-tumor effect with less toxicity. METHODS: GHG-CDDP was prepared without organic solvents to enable its future clinical use. The pharmaceutical characterization of GHG-CDDP was performed, and its in vivo degradability was evaluated. The anti-tumor effect was evaluated using a murine peritoneal metastasis model of the human gastric cancer MKN45-Luc cell line. RESULTS: Our new manufacturing process dramatically reduced the initial burst of CDDP release to approximately 2% (wt), while the previous product had a 25-30% initial burst. In intraperitoneal degradation tests, approximately 30% of GHG-CDDP remained in the murine abdominal cavity 7 days after intraperitoneal injection and disappeared within 3 weeks. GHG-CDDP significantly suppressed the in vivo tumor growth (p = 0.02) and prolonged the survival time (p = 0.0012) compared with the control. In contrast, free CDDP did not show a significant therapeutic effect at any dose. Weight loss and hematological toxicity were also significantly ameliorated. CONCLUSIONS: GHG-CDDP is a promising treatment option for peritoneal metastases through the complete sustained-release of CDDP with less systemic toxicity.

A therapeutic angiogenesis of sustained release of basic fibroblast growth factor using biodegradable gelatin hydrogel sheets in a canine chronic myocardial infarction model.

This study investigated the safety and efficacy of a sustained release of basic fibroblast growth factor (bFGF) with biodegradable gelatin hydrogel sheets as therapeutic angiogenesis in canine chronic myocardial infarction (MI) models. Canine chronic MI model was induced by ligating the left anterior descending coronary artery and its diagonal branches. At 4 week post-induction, we applied either saline (Control group, n = 5) or 200 µg of bFGF (Treatment group, n = 6) soaked gelatin hydrogel sheets on the ischemic area of the left ventricular (LV) wall. At 6 weeks after the procedure, we evaluated the efficacy by echocardiography and immunohistochemical study. There were no procedure-related adverse events or deaths. The serum bFGF level was under detectable levels in all animals at any sampling points. In terms of efficacy, echocardiographic evaluation demonstrated that fractional shortening was significantly improved in the treatment group. In addition, immunohistochemical study showed that the capillary density in the border zone of the MI area, as well as the MI area, significantly increased in the treatment group. Therapeutic angiogenesis by bFGF using biodegradable gelatin hydrogel sheets was safe, increased the capillary density, and improved LV function in canine chronic MI models.

Peptide drugs accelerate BMP-2-induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling.

Both W9 and OP3-4 were known to bind the receptor activator of NF-κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide-induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3-4 accelerated BMP-2-induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL-binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP-2-induced bone regeneration by the RANKL-binding peptides.

Effect of sustained release of basic fibroblast growth factor using biodegradable gelatin hydrogels on frozen-thawed human ovarian tissue in a xenograft model.

AIM: Ovarian tissue cryopreservation before cancer treatment is the only option to preserve fertility under some circumstances. However, tissue ischemia after transplantation while awaiting angiogenesis induces dysfunctional folliculogenesis and reduces ovarian reserve and is one of the disadvantages of frozen-thawed ovarian tissue transplantation. Basic fibroblast growth factor (bFGF) is a major regulator of angiogenesis. However, bFGF rapidly loses biological activity when its free form is injected in vivo. This study investigated whether administration of active bFGF helps establish a nurturing environment for follicular survival. METHODS: A sheet form of a sustained release drug delivery system for bFGF was developed using biodegradable acidic gelatin hydrogel (bFGF sheet). The bFGF sheets or phosphate-buffered saline sheets, as a negative control, were transplanted with frozen-thawed human ovarian tissues subcutaneously into the backs of severe combined immunodeficient mice. Neovascularization, cell proliferation, fibrosis and follicular survival of ovarian grafts were analyzed at 6 weeks after xenografting. RESULTS: The bFGF sheets were optimized to release bFGF for at least 10 days. The transplantation of bFGF sheets with frozen-thawed ovarian tissues significantly increased human and mouse CD31-positive areas and stromal and endothelial cell proliferations. The administration of bFGF also significantly decreased the percentage of the fibrotic area in the graft, resulting in a significant increase in primordial and primary follicular density. CONCLUSION: Local administration of a sustained release of biologically active bFGF induced neovascularization in frozen-thawed ovarian tissue grafts, which could establish the nurturing environment required for follicular survival in heterotopic xenografts.

4D printing of polymeric materials for tissue and organ regeneration.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex, stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applications. Although the 4D concept was first highlighted in 2013, extensive research has rapidly developed, along with more-in-depth understanding and assertions regarding the definition of 4D. In this review, we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in the field. Both transformation-preprogrammed 4D printing and 4D printing of shape memory polymers are intensively surveyed. Afterwards we will explore and discuss the applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissues and implantable scaffolds, as well as future perspectives and conclusions.