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A silk elastin-like protein (SELP) is an artificial compound with silk fibroin-like and elastin-like tandem repeats. The objective of this study is to evaluate the influence of SELP on the polarization of mouse bone marrow-derived macrophages. When the macrophages of inflammation-type (M1) were cultured with different concentrations of SELP solution, the secretion of a pro-inflammatory cytokine, tumor necrotizing factor (TNF)-α, was significantly suppressed at the higher concentrations. In addition, the secretion of an anti-inflammation cytokine, interleukin (IL)-10, was significantly enhanced from the macrophage of an original type (M0). By the incubation with soluble SELP, the morphology of M0- and M1-type macrophages changed to be of a round shape with a large size. Following incubation with the sponge of SELP, the M0-type macrophages secreted IL-10 with time. When injected into an air pouch of mice subcutis which had been prepared by the injection of air, the SELP sponge and 5 wt % of SELP solution induced IL-10 secretion to a significantly high extent compared with the saline injection. Cells isolated from the air pouch 24 h after the injection were stained by the CD206 of a M2 marker. It is concluded that the SELP itself in solution has an ability to induce the anti-inflammation M2-type macrophages.

Although choledochojejunostomy is the standard technique for biliary reconstruction, there are various associated problems that need to be solved such as reflux cholangitis. Interposition with an artificial bile duct (ABD) to replace the resected bile duct maintains a physiological conduit for bile and may solve this problem. This study investigated the usefulness of an ABD made of gelatin hydrogel nonwoven fabric (GHNF). GHNF was prepared by the solution blow spinning method. The migration and activity of murine fibroblast L929 cells were examined in GHNF sheets. L929 cells migrated into GHNF sheets, where they proliferated and synthesized collagen, suggesting GHNF is a promising scaffold for bile duct regeneration. ABDs made of GHNF were implanted in place of resected bile duct segments in rats. The rats were killed at 2, 6, and 12 weeks postimplantation. The implantation site was histologically evaluated for bile duct regeneration. At postoperative 2 weeks, migrating cells were observed in the ABD pores. The implanted ABD was mostly degraded and replaced by collagen fibers at 6 weeks. Ki67-positive bile duct epithelial cells appeared within the implanted ABD. These were most abundant within the central part of the ABD after 6 weeks. The percentages of Ki67-positive cells were 31.7 ± 9.1% in the experimental group and 0.8 ± 0.6% in the sham operation group at 6 weeks (p

Objective To improve lateral thyroarytenoid (TA) muscle atrophy after laryngeal paralysis, reconstruction of the vascular network of the atrophied muscle is necessary. We therefore evaluated whether the controlled release of basic fibroblast growth factor (bFGF) with autologous fascia implantation could affect vascular reconstruction in the lateral TA muscle. Study design Animal experiment. Setting Laboratory. Methods Unilateral laryngeal paralysis was induced in 20 rats. The rats were implanted with autologous fascia and a gelatin hydrogel sheet with or without 1 µg of bFGF (fascia and bFGF + fascia groups; n = 5 each) and with only a gelatin hydrogel sheet with bFGF (bFGF group: n = 5). Another group remained untreated (n = 5) at 4 months after paralysis. At 3 months since transplantation, intra- and intergroup comparisons of the muscle volumes and total area of blood vessels in the lateral TA muscle were performed. Results When compared with the untreated group, the bFGF + fascia group showed a significant increase in muscle volume (P =.0008) and vascular area (P =.0002) in the lateral TA muscle, whereas the other 2 treated groups demonstrated an insufficient effect. Conclusion bFGF + fascia implantation showed histologic improvement in severe laryngeal paralysis. We demonstrated that the decrease in lateral TA muscle mass after paralysis might be countered by the reconstruction of the vascular network. Our findings indicate that hypovascular and denervated areas of the laryngeal muscle can be regenerated by the implantation of growth factors and scaffolds with surgical stress. Level of evidence 5.

Introduction Postoperative dysphonia is mostly caused by vocal fold scarring, and careful management of vocal fold surgery has been reported to reduce the risk of scar formation. However, depending on the vocal fold injury, treatment of postoperative dysphonia can be challenging. Objective The goal of the current study was to develop a novel prophylactic regenerative approach for the treatment of injured vocal folds after surgery, using biodegradable gelatin hydrogel microspheres as a drug delivery system for basic fibroblast growth factor. Methods Videoendoscopic laryngeal surgery was performed to create vocal fold injury in 14 rabbits. Immediately following this procedure, biodegradable gelatin hydrogel microspheres with basic fibroblast growth factor were injected in the vocal fold. Two weeks after injection, larynges were excised for evaluation of vocal fold histology and mucosal movement. Results The presence of poor vibratory function was confirmed in the injured vocal folds. Histology and digital image analysis demonstrated that the injured vocal folds injected with gelatin hydrogel microspheres with basic fibroblast growth factor showed less scar formation, compared to the injured vocal folds injected with gelatin hydrogel microspheres only, or those without any injection. Conclusion A prophylactic injection of basic fibroblast growth factor -containing biodegradable gelatin hydrogel microspheres demonstrates a regenerative potential for injured vocal folds in a rabbit model. Resumo Introdução A disfonia pós-operatória é causada principalmente por cicatrizes nas pregas vocais. Tem sido relatado que o manejo cuidadoso da cirurgia das pregas vocais reduz o risco de formação de cicatriz. No entanto, a depender da lesão da prega vocal, o tratamento da disfonia pós-operatória pode ser desafiador. Objetivo Desenvolver uma nova abordagem regenerativa profilática para o tratamento de pregas vocais lesionadas após a cirurgia, com microesferas biodegradáveis de hidrogel de gelatina como sistema de administração de medicamentos para o Fator Básico de Crescimento de Fibroblastos (bFGF). Método A cirurgia laríngea videoendoscópica foi feita para criar lesão nas pregas vocais em 14 coelhos. Imediatamente após esse procedimento, microesferas biodegradáveis de hidrogel de gelatina com bFGF foram injetadas na prega vocal. Duas semanas após a injeção, as laringes foram excisadas para avaliação da histologia das pregas vocais e do movimento da mucosa. Resultados A presença de função vibratória deficiente foi confirmada nas pregas vocais lesionadas. A histologia e a análise de imagem digital demonstraram que as pregas vocais lesionadas injetadas com microesferas de hidrogel de gelatina com bFGF apresentaram menor formação de cicatriz, em comparação com as pregas vocais lesionadas injetadas apenas com microesferas de hidrogel de gelatina ou aquelas sem injeção. Conclusão Uma injeção profilática de microesferas biodegradáveis de hidrogel de gelatina com bFGF demonstra um potencial regenerativo para pregas vocais lesionadas em um modelo de coelho.

Management of bone metastasis is becoming increasingly important. Thus, local and systemic treatment options have been developed for control. Although systemic administration of anticancer agents is effective for bone metastasis, it is often stopped because of poor general conditions or side effects. Therefore, it is highly desirable to develop a more effective and safer local treatment for bone metastasis. The purpose of the current study was to investigate the antitumor effects and safety of gelatin hydrogel microspheres incorporating cisplatin (GM-CDDP), which we developed as a sustained release system without harmful substances. First, we assessed GM-CDDP for its in vitro degradability and potential for sustained release. Second, in vivo antitumor and side effects were evaluated using a murine bone metastasis model of MDA-MB-231 human breast cancer cells incorporating GFP. In vitro, initial bursts were observed within 2 h and CDDP was released gradually with gelatin hydrogel degradation, which reached 100% at 48 h. In vivo, local administration of GM-CDDP (2 mg/kg) significantly suppressed tumor growth and bone osteolysis compared with the control, and local and systemic administration of free CDDP (2 mg/kg; p < 0.05). Local administration of GM-CDDP significantly reduced loss of body weight and elevation of blood urea nitrogen compared with the systemic administration of free CDDP (p < .05). The current study suggests that local administration of GM-CDDP achieves higher antitumor effects with a potential for lesser side effects compared with local or systemic administration of free CDDP.

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)

Duchenne muscular dystrophy (DMD) is an intractable genetic muscular disorder characterized by the loss of DYSTROPHIN. The restoration of DYSTROPHIN is expected to be a curative therapy for DMD. Because muscle stem cells (MuSCs) can regenerate damaged myofibers with full-length DYSTROPHIN in vivo, their transplantation is being explored as such a therapy. As for the transplanted cells, primary satellite cells have been considered, but donor shortage limits their clinical application. We previously developed a protocol that differentiates induced pluripotent stem cells (iPSCs) to MuSCs (iMuSCs). To ameliorate the respiratory function of DMD patients, cell transplantation to the diaphragm is necessary but difficult, because the diaphragm is thin and rapidly moves. In the present study, we explored the transplantation of iMuSCs into the diaphragm. First, we show direct cell injection into the diaphragm of mouse was feasible. Then, to enhance the engraftment of the transplanted cells in a rapidly moving diaphragm, we mixed polymer solutions of hyaluronic acid, alginate and gelatin to the cell suspension, finding a solution of 20% dissolved hyaluronic acid and 80% dissolved gelatin improved the engraftment. Thus, we established a method for cell transplantation into mouse diaphragm and show that an injectable hyaluronic acid-gelatin solution enables the engraftment of iMuSCs in the diaphragm., マウスの横隔膜にヒトiPS細胞から作った骨格筋幹細胞を移植する --デュシェンヌ型筋ジストロフィーの呼吸筋治療に向けた横隔膜移植方法の確立--. 京都大学プレスリリース. 2022-04-13.

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.

Introduction Excellent outcomes of arthroscopic rotator cuff repair for small and medium tears have been recently reported. However, re-tears after surgery have been a common complication after surgical repair of large and massive rotator cuff tears and often occur in early postoperative phase. It was previously reported that basic fibroblast growth factor and platelet-rich plasma enhanced rotator cuff tear healing. We hypothesized that this combined therapy could enhance rotator cuff healing after rotator cuff repair in a rat model. This study aimed to evaluate the efficacy of combined therapy of platelet-rich plasma and basic fibroblast growth factor with gelatin-hydrogel sheet. Methods To create a rotator cuff defect, the infraspinatus tendon of Sprague Dawley rat was resected from the greater tuberosity. The infraspinatus tendons were repaired and covered with gelatin-hydrogel sheet impregnated with PBS (control group), basic fibroblast growth factor (bFGF group), platelet-rich plasma (PRP group), or both basic fibroblast growth factor and platelet-rich plasma (combined group). Histological examinations were conducted using hematoxylin and eosin, safranin O, and immunofluorescence staining, such as Isolectin B4, type II collagen at 2 weeks postoperatively. For mechanical analysis, ultimate failure load of the tendon-humeral head complex was evaluated at 6 weeks postoperatively. Results In the hematoxylin and eosin staining, the tendon maturing score of the combined group was higher than that of the control group at postoperative 2 weeks. In the safranin O staining, stronger proteoglycan staining was observed in the combined group compared with the other groups at postoperative 2 weeks. Vascular staining with isolectin B4 in 3 treatment groups was significantly higher than that in the control group. Type II collagen expression in the combined group was significantly higher than those in the other groups. The ultimate failure load of the combined group was significantly higher than that of the control group. Conclusion Combined therapy of basic fibroblast growth factor and platelet-rich plasma promoted angiogenesis, tendon maturing and fibrocartilage regeneration at the enthesis, which could enhance the mechanical strength. It was suggested that combined basic fibroblast growth factor and platelet-rich plasma might enhance both tendon and bone–tendon junction healing, and basic fibroblast growth factor and platelet-rich plasma might be synergistic.

The objective of this study is to investigate the influence of shaking culture on the biological functions of cell aggregates incorporating gelatin hydrogel microspheres in terms of the microspheres/cells ratio. The mixture of MC3T3-E1 cells and the microspheres was cultured in the U-bottomed wells of 96-well plate pre-coated with poly (vinyl alcohol) (PVA) to form cell aggregates incorporating microspheres. When incubated in the static or shaking culture, the size of cell aggregates increased with amounts of gelatin hydrogel microspheres but was similar between the two cultures. At the smaller ratio of microspheres to cells, the viability of cell aggregates under the shaking culture was significantly higher than that of static culture. On the other hand, there was no significant difference in the viability between them at the higher ratio. Gelatin hydrogel microspheres enabled to enhance ATP and mitochondrial activities of cell aggregates under the shaking culture. The effect was high at the smaller microspheres/cells ratio. It is concluded that the shaking culture was promising to allow cells to enhance their activities.

Although surgical ventricular restoration for ischemic cardiomyopathy is expected as an alternative or bridge to heart transplantation, post-operative remodeling of left ventricle (LV) needs to be addressed. This study aimed to examine the effect of basic fibroblast growth factor (bFGF), which induces angiogenesis and tissue regeneration in ischemic myocardium, to prevent remodeling after surgical ventricular restoration (SVR) using a rat ischemic cardiomyopathy model. Four weeks after coronary artery ligation, rats were divided into two groups: rats treated with SVR alone (SVR; n = 21), and rats treated with SVR and local sustained release of bFGF using gelatin hydrogel sheet (SVR + bFGF; n = 22). Cardiac function was assessed by serial echocardiography and cardiac catheterization. Cardiac tissue sections were histologically examined for vascular density and fibrosis. Higher systolic function and lower LV end-diastolic pressure (LVEDP) were observed in rats treated with SVR + bFGF (SVR vs SVR + bFGF; Ees: 0.22 ± 0.11 vs 0.33 ± 0.22 mmHg/μL, p = 0.0328; LVEDP: 12.7 ± 7.0 vs 8.5 ± 4.3 mmHg, p = 0.0230). LV area tended to be lower in rats treated with SVR + bFGF compared to rats treated with SVR alone (left-ventricular end-diastolic area: 0.66 ± 0.07 vs 0.62 ± 0.07 cm2, p = 0.071). Vascular density tended to be higher in rats treated with SVR + bFGF than those without bFGF (23.3 ± 8.1 vs 28.8 ± 9.5/mm2, p = 0.0509). BFGF induced angiogenesis and attenuated remodeling after SVR which secured the efficacy of SVR in a rat ischemic cardiomyopathy model.

Clinical trials with autologous adipose-derived stem cell (AdSC) therapy for ischemic heart diseases (IHDs) are ongoing. However, little is known about combinational therapeutic effect of AdSCs and statin poly(lactic-co-glycolic) acid (PLGA) nanoparticles on the ischemic myocardium. We investigated the hypothesis that statins, which have pleiotropic effects, augment the therapeutic potential of AdSCs and that AdSCs also act as drug delivery tools. Simvastatin-conjugated nanoparticles (SimNPs) significantly promoted migration activity without changing proliferation activity and upregulated growth factor gene expression in vitro. A small number of intravenously administered SimNP-loaded AdSCs (10,000 cells per mouse) improved cardiac function following myocardial infarction, inducing endogenous cardiac regeneration in the infarcted myocardium. The de novo regenerated myocardium was thought to be derived from epicardial cells, which were positive for Wilms’ tumor protein 1 expression. These findings were attributed to the sustained, local simvastatin release from the recruited SimNP-loaded AdSCs in the infarcted myocardium rather than to the direct contribution of recruited AdSCs to tissue regeneration. SimNP-loaded AdSCs may lead to a novel somatic stem cell therapy for IHDs. Stem Cells Translational Medicine 2019;8:1055–1067

The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.

During embryonic development, cells differentiate in a coordinated manner, aligning their fate decisions and differentiation stages with those of surrounding cells. However, little is known about the mechanisms that regulate this synchrony. Here we show that cells in close proximity synchronize their differentiation stages and cellular phenotypes with each other via extracellular vesicle (EV)‐mediated cellular communication. We previously established a mouse embryonic stem cell (ESC) line harbouring an inducible constitutively active protein kinase A (CA‐PKA) gene and found that the ESCs rapidly differentiated into mesoderm after PKA activation. In the present study, we performed a co‐culture of Control‐ESCs and PKA‐ESCs, finding that both ESC types rapidly differentiated in synchrony even when PKA was activated only in PKA‐ESCs, a phenomenon we named ‘Phenotypic Synchrony of Cells (PSyC)’. We further demonstrated PSyC was mediated by EVs containing miR‐132. PKA‐ESC‐derived EVs and miR‐132‐containing artificial nano‐vesicles similarly enhanced mesoderm and cardiomyocyte differentiation in ESCs and ex vivo embryos, respectively. PSyC is a new form of cell‐cell communication mediated by the EV regulation of neighbouring cells and could be broadly involved in tissue development and homeostasis.

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.

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.

Mesenchymal stem cells (MSCs) are recognized as promising drug delivery vehicles. However, the limitation of drug loading capacity and safety considerations are two obstacles to the further application of MSCs. Here, we report MSC membrane-coated mesoporous silica nanoparticles (MSN@M) that maintain the active stealth and self-positioning drug delivery abilities of MSCs and resolve issues related to MSCs-mediated drug delivery. MSN@M was established through uniformly integrating MSC membrane onto a mesoporous silica nanoparticle (MSN) core by sonication. Reduced clearance of phagocytes mediated by CD47 marker on MSC membrane was observed in vitro, which explained the only ~ 25% clearance rate of MSN@M compared with MSN in vivo within 24 h. MSN@M also showed stronger tumor targeting and penetration ability compared with MSN in HepG2 tumor bearing mice. Simultaneously, MSN@M exhibited strong capacity for drug loading and sustained drug release ability of MSN when loaded with doxorubicin (DOX), the drug loading of MSN@M increased ~ 5 folds compared with MSC membrane. In HepG2 xenograft mice, DOX-loaded MSN@M effectively inhibited the growth of tumors and decreased the side effects of treatment by decreasing the exposure of other tissues to DOX. Consequently, our MSN@M may serve as alternative vehicles for MSCs and provide more options for antitumor treatment.

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

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.

Gelatin hydrogels are naturally derived scaffolds useful for tissue engineering because of their cytocompatibility and controllable degradability. However, they are brittle and inflexible when dry, which limits their use for in utero tissue engineering in large animal models. Therefore, in this study, we attempted to generate flexible gelatin sheets by adding various plasticizers with different molecular weights (MW). We systematically evaluated the flexibility, sustainability, and potential clinical utility of the resulting flexible gelatin sheets. Gelatin sheets with low-MW plasticizers, such as monosaccharides or sugar alcohols, showed a reduced tensile modulus in dynamic viscoelasticity, which reflected their actual flexibility. Wet gelatin sheets containing plasticizers showed higher tensile strength than the nonplasticizer control, although wet gelatin sheets under all conditions had a much lower tensile strength than dry gelatin sheets. In a functional study, gelatin sheets containing glycerol, which has the lowest MW among sugar alcohols, showed encouraging results, such as good fit to the curvature of the experimental animal, biocompatibility, and suitability for endoscopic approaches. The findings of this study should enable the expansion of future applications for flexible gelatin sheets.

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

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. 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. 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. GHG–CDDP is a promising treatment option for peritoneal metastases through the complete sustained-release of CDDP with less systemic toxicity.

Background Visceral obesity is one of the risk factors for clinically relevant pancreatic fistula after pancreatic resection. The objective of this study was to evaluate the impact of intraperitoneal lipolysis on postoperative pancreatic fistula. Methods The degree of intraperitoneal lipolysis was investigated by measuring the free fatty acid concentration in drain discharge in patients after pancreatic resection. An experimental pancreatic fistula model was prepared by pancreatic transection, and the impact of intraperitoneal lipolysis was evaluated by intraperitoneal administration of triolein (triglyceride) with, or without orlistat (lipase inhibitor). Results Thirty-three patients were included in the analysis. The free fatty acid concentration in drain discharge on postoperative day 1 was significantly associated with the development of a clinically relevant pancreatic fistula (P = 0·004). A higher free fatty acid concentration in drain discharge was associated with more visceral adipose tissue (P = 0·009). In the experimental model that included 98 rats, intraperitoneal lipolysis caused an increased amount of pancreatic juice leakage and multiple organ dysfunction. Intraperitoneal administration of a lipase inhibitor reduced lipolysis and prevented deterioration of the fistula. Conclusion Intraperitoneal lipolysis significantly exacerbates pancreatic fistula after pancreatic resection. Inhibition of lipolysis by intraperitoneal administration of a lipase inhibitor could be a promising therapy to reduce clinically relevant postoperative pancreatic fistula. Surgical relevance Clinically, there are two types of pancreatic fistula after pancreatic resections: harmless biochemical leak and harmful clinically relevant pancreatic fistula. Visceral obesity is one of the known risk factors for clinically relevant pancreatic fistula; however, the underlying mechanisms remained to be elucidated. Patients with clinically relevant pancreatic fistula had a higher free fatty acid concentration in the drain discharge, suggesting a relationship between intraperitoneal lipolysis and pancreatic fistula. The experimental model of pancreatic fistula demonstrated that intraperitoneal lipolysis caused deterioration in pancreatic fistula, suggesting that intraperitoneal lipolysis is one of the mechanisms that drives biochemical leakage to clinically relevant pancreatic fistula. Intraperitoneal administration of a lipase inhibitor prevented lipolysis as well as pancreatic fistula deterioration in the experimental model, suggesting a future clinical application for lipase inhibitors in prevention of clinically relevant pancreatic fistula.

An ideal stent to treat cerebral aneurysms should have an antithrombotic effect on the inner stent blood-facing side and a tissue organization effect on the outer aneurysmal side of the stent. The objective of this study is to evaluate the feasibility of a drug containing stent in the in vivo treatment of cerebral aneurysms. Argatroban, an antithrombotic drug, is encapsulated in biodegradable poly (d,l-lactide-co-glycolide) (PLGA) microspheres for the controlled release with an in vitro study conducted to evaluate the drug release and anticoagulation behavior of released drug. Basic fibroblast growth factor (bFGF), an organization drug, is released from gelatin hydrogels. The stents are coated with gelatin hydrogels incorporating bFGF and PLGA microspheres containing argatroban, and applied to the carotid artery aneurysm of an elastase-induced rabbit model. Most of the aneurysm cavity is occupied by loose connective tissues in the group treated with drug-coated stents, whereas extensive massive hematomas are observed in the group treated with drug-free stents. The occurrence rate of in-stent thrombus is small in the drug-coated stents. The stent incorporating bFGF and PLGA microspheres containing argatroban is an effective device for cerebral aneurysm treatment. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2185-2194, 2019.

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.

Owing to the diversity and ease of preparation of nanomaterials, the rational nanocarriers with a rational design have become increasingly popular in medical researches. Although nanoparticle-based drug delivery exhibits great potential, there are some challenges facing like rapid plasma clearance, triggering or aggravation of immune response, etc. Herein, cell-based targeted drug delivery systems have drawn more and more attention owing to low immunogenicity and intrinsic mutation rate, and innate ability to allow targeted delivery. Mesenchymal stem cells (MSCs) have been used in gene and drug delivery. The use of MSCs is a promising approach for the development of gene transfer systems and drug loading strategies because of their intrinsic properties, including homing ability and tumor tropism. By combining the inherent cell properties and merits of synthetic nanoparticles (NPs), cell membrane coated NPs emerge as the time requires. Overall, we provide a comprehensive overview of the utility of MSCs in drug and gene delivery as well as MSC membrane coated nanoparticles for therapy and drug delivery, aiming to figure out the significant room for development and highlight the potential future directions.

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/β-tricalcium phosphate (β-TCP) sponges, which comprise 50 wt% gelatin and 50 wt% β-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/β-TCP sponges was significantly greater than that in the control group using acidic gelatin/β-TCP sponges alone in a ridge augmentation model in dogs. However, whether these results depend on controlled release by the gelatin/β-TCP sponges remains controversial. In this study, we evaluated the effects of controlled release by comparing acidic and basic gelatin/β-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/β-TCP sponges (IEP 5.0) soaked with 0.3% rhFGF-2 were applied to the defect in the acidic group, whereas basic gelatin/β-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.

Platelet-rich plasma (PRP) contains numerous growth factors and promotes bone fracture healing. The aim of this study was to evaluate the effectiveness of the controlled release of PRP from biodegradable gelatin hydrogel for promoting healing in a rabbit ischemic sternal model. PRP was prepared from the whole blood of a Japanese white rabbit. Sixteen rabbits were randomized into four groups (each n = 4) and all underwent median sternotomy and bilateral internal thoracic artery removal. Before the sternum was closed, the following solutions were applied between the sternum incisions in three of the groups: 30 mg of gelatin hydrogel incorporating 300 μL of phosphate-buffered saline, 300 μL of a solution form of PRP, or 30 mg of gelatin hydrogel incorporating 300 μL of PRP (PRP + Gel). The fourth group acted as a control. Sternal healing was evaluated by histology and microcomputed tomography 7 days after the intervention. The PRP + Gel group showed a significantly higher proportion of fibrosis within the fracture area (an indicator of sternal healing) than the other groups and a significantly higher mean intensity of osteocalcin. These results indicate that the controlled release of PRP from locally applied gelatin hydrogel was markedly effective in enhancing sternal healing in the early postoperative period. This novel therapy could potentially help prevent complications, such as deep sternal wound infection and could result in early postoperative ambulation after median sternotomy.

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

We investigated the reproducibility of creating a vascularized tissue flap in an in vivo tissue engineering chamber by incubating a vascular pedicle imbedded in a collagen sponge with activated platelet-rich plasma (aPRP) and basic fibroblast growth factor (bFGF).Collagen sponge soaked with saline (control group), bFGF (Group 1), aPRP (Group 2), and aPRP/controlled release bFGF (Group 3) was implanted with a saphenous arteriovenous pedicle into a tissue engineering chamber, located subcutaneously in the groin of rabbits. After 4 weeks of implantation, the contents in the chamber were harvested for volumetric and histological analyses.The total volume of generated tissue in Group 3 was the largest among the Groups (control group vs. Group 3, p 0.01). The volume of the pedicle vascular bundle/adipose tissue component was larger in Groups 1 and 3 than that in the control group (p 0.05 and p 0.01, respectively). The inflammatory tissue volume was larger in Groups 2 and 3 (control group vs. Group 3, p 0.05). In a smaller long-term study, inflammatory tissue at 4 weeks was gradually replaced by the adipose tissue within 8 weeks.PRP-induced inflammatory reactions were considered to be necessary to stimulate cell migration into the chamber, leading to more tissue regeneration with abundant cell components. We conclude that PRP contributes to the reproducibility of preparing vascularized flaps in an in vivo chamber.

The objective of the present study was to investigate the effect of platelet-rich plasma (PRP) combined with gelatin β-tricalcium phosphate (β-TCP) sponge on bone generation in a lumbar vertebral body defect of ovariectomized rat. After creating critical-size defects in the center of the anterior vertebral body, the defects were filled with the following materials: (1) no material (control group), (2) gelatin β-TCP sponge with PRP (PRP sponge group), and (3) gelatin β-TCP sponge with phosphate-buffered saline (PBS sponge group). Microcomputed tomography and histological evaluation were performed immediately after surgery and at 4, 8, and 12 weeks to assess bone regeneration. Biomechanical test was also performed at postoperative week 12. In the PRP sponge group, both imaging and histological examination showed that visible osteogenesis was first induced and additional growth of bone tissue was observed in the transplanted sponge, compared with the PBS sponge group. There was no negative effect of either PRP sponge or PBS sponge transplantation on bone tissue generation around the periphery of the defect. Biomechanical test showed increased stiffness of the affected vertebral bodies in the PRP sponge group. These results indicate that PRP-impregnated gelatin β-TCP sponge is effective for facilitating bone regeneration in lumbar vertebral bone defect under osteoporotic condition. PRP combined with gelatin β-TCP sponges could be potentially useful for developing a new approach to vertebroplasty for osteoporotic vertebral fracture.

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.

© 2022 by the authors. Licensee MDPI, Basel, Switzerland.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 splitthickness 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.

The objective of this study is to design the manner of ephrinB2 immobilized onto polyacrylamide (PAAm) hydrogels with varied elasticity and evaluate the effect of hydrogels elasticity and the immobilized manner of ephrinB2 on the Runx2 expression of human mesenchymal stem cells (hMSC). The PAAm hydrogels were prepared by the radical polymerization of acrylamide (AAm), and N , N′ -methylenebisacrylamide (BIS). By changing the BIS concentration, the elasticity of PAAm hydrogels changed from 1 to 70 kPa. For the bio-specific immobilization of ephrinB2, a chimeric protein of ephrinB2 and Fc domain was immobilized onto protein A-conjugated PAAm hydrogels by making use of the bio-specific interaction between the Fc domain and protein A. When hMSC were cultured on the ephrinB2-immobilized PAAm hydrogels with varied elasticity, the morphology of hMSC was of cuboidal shape on the PAAm hydrogels immobilized with ephrinB2 compared with non-conjugated ones, irrespective of the hydrogels elasticity. The bio-specific immobilization of ephrinB2 enhanced the level of Runx2 expression. The expression level was significantly high for the hydrogels of 3.6 and 5.9 kPa elasticity with bio-specific immobilization of ephrinB2 compared with other hydrogels with the same elasticity. The hydrogels showed a significantly down-regulated RhoA activity. It is concluded that the Runx2 expression of hMSC is synergistically influenced by the hydrogels elasticity and their immobilized manner of ephrinB2 immobilized. Statement of Significance Differentiation fate of mesenchymal stem cells (MSC) is modified by biochemical and biophysical factors, such as elasticity and signal proteins. However, there are few experiments about combinations of them. In this study, to evaluate the synergistic effect of them on cell properties of MSC, we established to design the manner of Eph signal ligand, ephrinB2, immobilized onto polyacrylamide hydrogels with varied elasticity. The gene expression level of an osteogenic maker, Runx2, was enhanced by the immobilized manner, and significantly enhanced for the hydrogels of around 4 kPa elasticity with bio-specific immobilization of ephrinB2. This is the novel report describing to demonstrate that the Runx2 expression of MSC is synergistically influenced by the hydrogels elasticity and their manner of ephrinB2 immobilized.

The objective of this study is to investigate the utility of gelatin hydrogel-fragmented fibers (GHFF) as a material to suppress the shrinkage of cell sheets, which often happens upon detaching from a culture plate. The GHFF were fabricated by cutting gelatin hydrogel nonwoven fabrics. MC3T3-E1 cells were simply mixed with different amounts of GHFF, followed by culturing to formulate the cell sheet homogeneously incorporating GHFF. When detached from the culture plate, the cell sheet formulated without GHFF shrunk while the area became about 23% of the original one before detachment. On the contrary, the cell sheet formulated with GHFF hardly shrunk. The lactate/glucose ratio of a metabolic activity was significantly lower and the adenosine triphosphate (ATP) production was higher for the cell sheet formulated with the GHFF than that obtained without the GHFF. An osteogenic activity was high for the cell sheet formulated with the GHFF compared with that obtained without the GHFF. The GHFF addition was a simple and promising method to fabricate active cell sheets without size change. Impact Statement This study introduces the utility of gelatin hydrogel-fragmented fibers (GHFF) for cell sheet engineering. Upon detaching from the culture plate, the cell sheet formulated without GHFF shrunk, while the area became about 23% of the original one before detachment. On the contrary, the cell sheet formulated with GHFF hardly shrunk. The GHFF allowed cell sheets to enhance the metabolic and osteogenic activities. The GHFF addition was a simple and promising method to fabricate active cell sheets without size change.

The objective of this study is to design hydrogel microspheres of a cell scaffold, which not only function as a scaffold to form cell aggregates of three-dimensional culture but also can disappear to release growth factors in the well-controlled manner by noncytotoxic stimulation in any timing. The hydrogel microspheres were prepared by a water-in-oil emulsion method from m-aminophenylboronic acid (APBA)-introduced gelatin (APBA-gelatin) with or without poly(vinyl alcohol) (PVA) mixing. Irrespective of the PVA concentration, the microspheres with the same diameter were prepared. The microspheres were water solubilized only by adding sorbitol of a sugar although the solubilization extent depended on the PVA concentration. When cocultured with the microspheres, mesenchymal stem cells formed cell aggregates homogeneously incorporating the microspheres. Upon adding sorbitol in the culture medium, mixed APBA-gelatin-PVA hydrogel microspheres disappeared with time in the cell aggregates. The microspheres containing basic fibroblast growth factor or bone morphogenetic protein-2 released the respective growth factor accompanied with the microspheres disappearance. It is concluded that the present microspheres of sugar-responsive water solubilization are promising scaffold of cell aggregates and have an ability to allow growth factors to be released in the cell aggregates when it is required.

Eicosapentanoic acid (EPA) is an antioxidant and omega-3 polyunsaturated fatty acid that reduces inflammatory cytokine production. Gelatin hydrogel can be used as a carrier of a physiologically active substance that release it gradually for an average of ~3 weeks. Therefore, this study aimed to clarify the effect of EPA-incorporating gelatin hydrogels on osteoarthritis (OA) progression in vivo. Ten-week-old male C57BL/6J mice were randomly divided into six groups (n = 6): Sham, destabilization of the medial meniscus (DMM), Corn: DMM + 2 µL corn oil, EPA injection alone (EPA-I): DMM + 2 µL corn oil + 125 μg/μL EPA, Gel: DMM + gelatin hydrogels, and EPA-G: DMM + 125 μg/μL EPA-incorporating gelatin hydrogels. The mice were euthanized at 8 weeks after DMM or Sham surgery, and subjected to histological evaluation. Matrix-metalloproteinases-3 (MMP-3), MMP-13, interleukin-1β (IL-1β), p-IKK α/β, CD86, and CD163 protein expression in the synovial cartilage was detected by immunohistochemical staining. F4/80 expression was also assessed using the F4/80 score of macrophage. Histological score was significantly lower in EPA-G than in EPA-I. MMP-3-, MMP-13-, IL-1β-, and p-IKK α/β-positive cell ratio was significantly lower in EPA-G than in EPA-I. However, CD86- and CD163-positive cell ratio was not significantly different between EPA-I and EPA-G. The average-sum F4/80 score of macrophage in EPA-G was significantly lower than that in EPA-I. EPA-incorporating gelatin hydrogels were shown to prevent OA progression in vivo more effectively than EPA injection alone. Our results suggested that intra-articular administration of controlled-release EPA can be a new therapeutic approach for treating OA.

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.

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.

Spinal cord injury (SCI) is one of the most debilitating injuries, and transplantation of stem cells in a scaffold is a promising strategy for treatment. However, stem cell treatment of SCI has been severely impaired by the increased generation of reactive oxygen species in the lesion microenvironment, which can lead to a high level of stem cell death and dysfunction. Herein, a MnO

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.

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. Osteoclasts secrete small extracellular vesicles that stimulate osteoblasts, promoting bone formation via receptor activator of nuclear factor-kappa B ligand (RANKL), thereby linking bone formation and resorption.

The objective of this study is to design bioabsorbable injectable hydrogels based on the physico-chemical interaction between biocompatible polymers and ferric ions, and evaluate the survival, proliferation, and osteogenic differentiation of cells encapsulated in the hydrogels. The injectable hydrogels were prepared by simply mixing mixed alginate/gelatin solution at various ratios and FeCl3 solution. The hydrogels prepared disappeared within a few days in the phosphate buffered-saline solution (PBS) with containing collagenase although the disappearance rate increased with an increase of the gelatin ratio in the hydrogel. For the hydrogel of alginate/gelatin low ratio, the survival and proliferation of cells in the hydrogel-encapsulated condition were significantly high compared with those of hydrogel at the higher ratios. The cells collected 3 days after cultured in the hydrogel also proliferated to a significantly higher extent than those collected from other hydrogels. The proliferation ability of cells was similar that of cells cultured on the standard tissue culture polystyrene (TCPS) dish. When evaluated to compare with cells cultured on the TCPS dish, the expression of runt-related transcription factor-2 (RUNX2) gene, the alkaline phosphatase (ALP) activity, and the calcium precipitation were significantly high. The cells were encapsulated by the mixed alginate/gelatin and FeCl3 hydrogel and injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the injectable hydrogel prepared by simple mixing mixed alginate/gelatin solution and FeCl3 solution is a promising material for the cell transplantation. STATEMENT OF SIGNIFICANCE: Injectable hydrogels prepared by simple mixing mixed alginate/gelatin solution at various ratios and FeCl3 solution. For the hydrogel of alginate/gelatin low ratio, the survival, the proliferation, and the differentiate properties of cells in the hydrogel-encapsulated condition were similar those of cells cultured on the TCPS dish. When the cells encapsulated hydrogels were injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the present injectable hydrogel is a promising material for the cell transplantation.

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-β1 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-β1 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.

Background and Objective Tissue engineering by using recombinant human (rh) growth factor technology may offer a promising therapeutic approach for treatment of gingival recession. Fibroblast growth factor-2 (FGF-2) has shown the ability to promote periodontal regeneration. Gelatin/beta-tricalcium phosphate (gelatin/β-TCP) sponges have been developed to control the release of growth factors. The present study evaluated the periodontal regenerative efficacy of rhFGF-2 by comparing gelatin/β-TCP sponges incorporated with rhFGF-2 to the scaffolds alone in artificially created recession-type defects in dogs. Material and methods Critically sized buccal gingival recession defects were surgically created on maxillary canine teeth of five dogs. In each animal, defects were randomized to receive either a gelatin/β-TCP sponge soaked with rhFGF-2 (gelatin/β-TCP/rhFGF-2) or phosphate-buffered saline (gelatin/β-TCP). Eight weeks after surgery, biopsy specimens were obtained and subjected to microcomputed tomography and histological analyses. Results Complete root coverage was achieved in both groups. Microcomputed tomography revealed significantly greater new bone volume in the gelatin/β-TCP/rhFGF-2 group. Histologically, both groups achieved periodontal regeneration; however, gelatin/β-TCP/rhFGF-2 sites exhibited more tissue regeneration, characterized by significantly larger amounts of new cementum and new bone. Gelatin/β-TCP sites featured increased long junctional epithelium and connective tissue attachment. In the gelatin/β-TCP/rhFGF-2 sites, new bone exhibited many haversian canals and circumferential lamellae as well as remarkably thick periosteum with blood vascularization and hypercellularity. Conclusion Within the limitations of this study, rhFGF-2 in gelatin/β-TCP sponges exhibits an increased potential to support periodontal wound healing/regeneration in canine recession-type defects.

Cartilage tissue is characterized by its poor regenerative properties, and the clinical performance of cartilage grafts to replace cartilage defects has been unsatisfactory. Recently, cartilage regeneration with mature chondrocytes and stem cells has been developed and applied in clinical settings. However, there are challenges with the use of mature chondrocytes and stem cells for tissue regeneration, including the high costs associated with the standard stem cell isolation methods and the decreased cell viability due to cell manipulation. Previous studies demonstrated that cartilage can be regenerated from chondrocyte clusters that contain stem cells. Based upon some of the existing techniques, the goal of this study was to develop a novel and practical method to induce cartilage regeneration. A microslicer device was developed to process cartilage tissues into micron-size cartilage (microcartilage) in a minimally invasive manner. We evaluated microcartilage sizes and demonstrated 100-400 µm as optimal for generating a high cell yield with collagenase digestion. In addition, autologous intrafascial implantation of the composites of microcartilage and an absorbable scaffold with a slow-release system of basic fibroblast growth factor (bFGF) was carried out to induce cartilage regeneration. Our results demonstrated that the extent of bFGF diffusion depends on the size of microcartilage, and that cartilage regeneration was induced most effectively with 100 µm of microcartilage via SOX5 upregulation. These findings suggest that cartilage regeneration is possible with microcartilage as a source of cells without ex vivo cell expansion.

Li Wan,1,* Xinglei Yao,1–3,* Francesco Faiola,3 Bojun Liu,4 Tianyuan Zhang,2 Yasuhiko Tabata,5 Hiroyuki Mizuguchi,6 Shinsaku Nakagawa,7 Jian-Qing Gao,2 Robert Chunhua Zhao1 1Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Peking Union Medical College Hospital, Center of Excellence in Tissue Engineering Chinese Academy of Medical Sciences, Beijing, 2Institute of Pharmaceutics, Zhejiang University, Hangzhou, 3State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 4YouAn Hospital, Capital Medical University, Beijing, People’s Republic of China; 5Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 6Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 7Department of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan *These authors contributed equally to this work Abstract: Mesenchymal stem cells (MSCs) are adult stem cells with multilineage potential, which makes them attractive tools for regenerative medicine applications. Efficient gene transfer into MSCs is essential not only for basic research in developmental biology but also for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors, but not into MSCs, which are deficient in coxsackievirus and adenovirus receptors expression. To overcome this problem, we developed an Adv coated with a spermine-pullulan (SP) cationic polymer and investigated its physicochemical properties and internalization mechanisms. We demonstrated that the SP coating could enhance adenoviral transduction of MSCs without detectable cytotoxicity or effects on differentiation. Our results argue in favor of the potentiality of the SP-coated Adv as a prototype vector for efficient and safe transduction of MSCs. Keywords: mesenchymal stem cells, adenovirus vectors, spermine-pullulan, polymer, gene transduction

Knowledge of the microenvironment of articular cartilage in health and disease is the key to accomplishing fundamental disease-modifying treatments for osteoarthritis. The proteins comprising the CCN Family are matricellular proteins with a remarkable relevance within the context of cartilage metabolism. CCN2 displays a great capability for regenerating articular cartilage, and CCN3 has been shown to activate the expression of genes related to articular chondrocytes and to repress genes related to endochondral ossification in epiphyseal chondrocytes. Moreover, mice lacking CCN3 protein have been shown to display ostearthritic changes in their knee articular cartilage. In this study, we employed a monoiodoacetic acid (MIA)-induced osteoarthritic model to investigate whether osteoarthritic changes in the cartilage are reciprocally accompanied by CCN3 down-regulation and an inducible overexpression system to evaluate the effects of CCN3 on articular chondrocytes in vitro. Finally, we also investigated the effects of exogenous CCN3 in vivo during the early stages of MIA-induced osteoarthritis. We discovered that CCN3 is expressed by articular chondrocytes in normal rat knees, whereas it is rapidly down-regulated in osteoarthritic knees. In vitro, we also discovered that CCN3 increases the proteoglycan accumulation, the gene expression of type II collagen, tenascin-C and lubricin, as well as the protein production of tenascin-C and lubricin in articular chondrocytes. In vivo, it was discovered that exogenous CCN3 increased tidemark integrity and produced an increased production of lubricin protein. The potential utility of CCN3 as a future therapeutic agent and possible strategies to improve its therapeutic functions are also discussed.