A solubilized liver-specific extracellular matrix (L-ECM) substratum was obtained by decellularization of porcine liver using Triton X-100 and pepsin treatments. The L-ECM was able to immobilize ...hepatocyte growth factor at a high efficiency of 87%. L-ECM gelled spontaneously in a physiologically neutral environment. Primary hepatocytes embedded in the L-ECM gel showed a high albumin synthesis activity and ethoxyresorufin-O-deethylase (EROD) activity even at 3 weeks in culture. In addition, the L-ECM gel-embedded hepatocytes implanted subcutaneously into partial hepatectomized rats showed a high survival rate (18%) and formed a large liver tissue-like structure. Their efficiencies of EROD activity and large liver tissue-like structure formation were about twice those of collagen gel-embedded hepatocytes. Based on these results, we clarified the effectiveness of L-ECM gel as a substrate for hepatocyte culture and transplantation.
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•Heparin functionalized PCL/gelatin co-spun nanofibrous dressings were synthesized.•Fabricated dressings showed improved mechanical and degradation properties.•Fabricated dressings ...showed exogenous and endogenous GFs sequestration ability.•In-vivo, synergistic effect of exogenous and endogenous GFs promoted tissue regeneration.
Growth factors (GFs) are signaling molecules that are principle mediators in tissue regeneration. Biomaterial scaffolds employed as wound dressings are often hampered by their limitations to deliver GFs exogenously due to their instability and low half-life. The key to overcome this challenge lies in the better organization and use of endogenous pro-regenerative GFs released at regenerative site, with an aim to minimize the sole dependency on exogenous factors. Considering such challenges, this research utilizes the exogenous and endogenous GFs sequestering capability of heparin functionalized PCL/gelatin co-spun nanofabrics to mediate synergistically driven tissue regeneration by utilizing combined therapeutic effect of exogenous and endogenous GFs, and thereby minimizing the sole dependency on exogenous GFs for tissue regeneration. Basic fibroblast growth factor (bFGF) was chosen as GF for exogenous loading whereas vascular endothelial growth factor (VEGF) was chosen as a representative example to demonstrate the endogenous pro-regenerative GF sequestration capability of fabricated nanofabrics. From our results, the fabricated nanofabrics showed loading efficiency of 80% for exogenous bFGF and can sequester 15-fold more amount of endogenous VEGF compared to non-heparin functionalized nanofibrous dressings. When applied as wound dressings, heparin functionalized nanofibers showed better therapeutic capability compared to control groups that were treated using patches without heparin functionalization, indicating endogenously driven tissue regeneration. This was indicated by significant higher number of newly formed skin appendages, lesser scarring and lower inflammatory levels in newly formed granulation. Additionally, further improvements in therapeutic effect was observed when exogenous bFGF was employed indicating effectiveness of synergistically mediated tissue regeneration.
Growth factors (GFs) are indispensable in regenerative medicine because of their high effectiveness. However, as GFs degenerate easily, the development of a suitable carrier with improved stability ...for GFs is necessary. In this study, we developed a gel-in-oil (G/O) emulsion technology for the transdermal delivery of growth factors. Nanogel particles prepared with heparin-immobilized gelatin that can bind growth factors were dispersed in isopropyl myristate. The particle size of the G/O emulsion could be controlled by changing the surfactant concentration, volume ratio of the water phase to the oil phase, and gelatin concentration. In vitro skin penetration studies showed better penetration through the stratum corneum of fluorescent proteins containing G/O emulsions than of the aqueous solution of GF. Similarly, an in vivo study showed an angiogenesis-inducing effect after transdermal application of GF-immobilized G/O emulsion. Angiogenesis in mice was confirmed owing to both an increased blood vessel network and higher hemoglobin content in the blood. Therefore, the G/O emulsion could be a promising carrier for GFs with better stability and can effectively deliver GFs at the target site.
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•Gel-in-oil (G/O) nanodispersion ensures effective delivery of functional molecules.•G/O could avoid degradation of bFGF at physiological temperature.•Nanogel droplets confirmed increased permeability through the skin.•Transdermal delivery of bFGF loaded G/O nanodispersion induces angiogenesis.
Organogels are semi-solid systems that can gel organic liquids at low concentrations. The use of organogels in drug delivery has grown rapidly in the last decade owing to their fibrous microstructure ...and suitability for different routes of administration. The current study is characterized by nanogel dispersion (NGD) development based on emulsion technology. The efficiency of this organogel based NGD as a carrier for anticancer drugs was assessed both in vitro and in vivo. 12-Hydroxystearic acid formed an organogel with lipiodol and encapsulated the anticancer drug paclitaxel. The gel-in-water (G/W) nanodispersion was prepared via ultrasonication and stabilized by a nonionic surfactant. The results showed that the organogel enabled sustained drug release from G/W nanodispersion over time, along with enhanced cellular uptake. The prepared G/W nanodispersion was found to be biocompatible with mouse hepatocytes and fibroblast cells in vitro, whereas paclitaxel-loaded G/W nanodispersion showed cytotoxicity (p <0.05) against lung cancer (A549) cell lines. Similarly, intravenous administration of paclitaxel-loaded G/W nanodispersion exerts an anticancer effect against lung cancer in vivo, with a significant decrease in tumor volume (p <0.05). Therefore, the proposed G/W nanodispersion could be a promising carrier for chemotherapy agents with sustained drug release and better therapeutic outcomes against cancer.
•Organogel nanodispersion effectively delivers drug through intravenous injection.•Diffusion-controlled drug release occurs from organogel.•Gel-in-water nanodispersion is biocompatible to proliferative cells in vitro.•Gel-in-water nanodispersion will be a promising carrier for anticancer drugs.
Cryopreservation is important for enabling long-term cell preservation. However, physical damage due to ice crystal formation and membrane permeation by dimethyl sulfoxide (DMSO) severely affects ...cryopreserved cell viability. To ensure cell survival and functional maintenance after cryopreservation, it is important to protect the cell membrane, the most vulnerable cell component, from freeze-thaw damage. This study aimed to create a glycolipid derivative having a positive interaction with the cell membrane and cytoprotective effects. As a result, we synthesized a novel trehalose derivative, oleyl-trehalose (Oleyl-Treh), composed of trehalose and oleyl groups. Its use led to increased viable cell counts when used with DMSO in a non-cytotoxic concentration range (1.6 nM–16 μM). Oleyl-Treh significantly improved viability and liver-specific functions of hepatocytes after cryopreservation, including albumin secretion, ethoxyresorufin-O-deethylase activity (an indicator of cytochrome P450 family 1 subfamily A member 1 activity), and ammonia metabolism. Oleyl-Treh could localize trehalose to the cell membrane; furthermore, the oleyl group affected cell membrane fluidity and exerted cryoprotective effects. This novel cryoprotective agent, which shows a positive interaction with the cell membrane, provides a unique approach toward cell protection during cryopreservation.
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Growth factors are crucial bioactive molecules in vitro and in vivo. Among them, basic fibroblast growth factor (bFGF) has been used widely for various applications such as cell culture and ...regenerative medicine. However, bFGF has extremely poor stability in aqueous solution; thus, it is difficult to maintain its high local concentration. Heparin-conjugated materials have been studied recently as promising scaffold-immobilizing growth factors for biological and medical applications. The previous studies have focused on the local concentration maintenance and sustained release of the growth factors from the scaffold.
In this paper, we focused on the biological stability of bFGF immobilized on the heparin-conjugated collagen (hep-col) scaffold. The stability of the immobilized bFGF was quantitatively evaluated at physiological temperature (37 °C) using cell culture and ELISA.
The immobilized bFGF had twice higher stability than the bFGF solution. Furthermore, the hep-col scaffold was able to immobilize not only bFGF but also other growth factors (i.e., vascular endothelial growth factor and hepatocyte growth factor) at high efficiency.
The hep-col scaffold can localize several kinds of growth factors as well as stabilize bFGF under physiological temperature and is a promising potent scaffold for regenerative medicine.
•Heparin-conjugated collagen scaffold immobilized bFGF, VEGF, and HGF with a high efficiency of 80–90% even at 100 ng/mL.•Cell proliferation of HUVECs was promoted depending on the bFGF amount on the scaffold, and slowed by pre-incubation at 37 °C.•Growth factor-immobilization on the scaffold stabilized bFGF and maintained its bioactivity longer than bFGF solution.
Liver transplantation plays an important role in the medical field. To improve the quality of a donor liver, there is a need to establish a preservation system to prevent damage and maintain liver ...function. In response to this demand, machine perfusion (MP) has been proposed as a new liver preservation method instead of the conventional static cold storage. There is controversy about the optimal MP temperature of the donor liver. Since the oxygen consumption of the liver differs depending on the temperature, construction of a system that satisfies the oxygen demand of the liver is crucial for optimizing the preservation temperature. In this study, an MP system, which satisfies the oxygen demand of liver at each temperature, was constructed using an index of oxygen supply; the overall volumetric oxygen transfer coefficient, the amount of oxygen retention of perfusate and oxygen saturation. Both subnormothermic MP (SNMP, 20–25 °C) and normothermic MP (NMP, 37 °C) could maintain liver viability at a high level (94%). However, lactate metabolism of the liver during NMP was more active than that during SNMP. Furthermore, the ammonia metabolism of liver after NMP was superior to that after SNMP. Hence, NMP, which maintains the metabolic activity of the liver, is more suitable for preservation of the donor liver than SNMP, which suppresses the metabolic activity. In summary, normothermia is the optimal temperature for liver preservation, and we succeeded in constructing an NMP system that could suppress liver damage and maintain function.
Cells have various applications in biomedical research. Cryopreservation is a cell-preservation technique that provides cells for such applications. After cryopreservation, sensitive cells, such as ...primary hepatocytes, suffer from low viability due to the physical damage caused by ice crystals, highlighting the need for better methods of cryopreservation to improve cell viability. Given the importance of effectively suppressing ice crystal formation to protect cellular structure, trehalose has attracted attention as cryoprotectant based on its ability to inhibit ice crystal formation; however, trehalose induces osmotic stress. Therefore, to establish a cell-cryopreservation technique, it is necessary to provide an optimal balance between the protective and damaging effects of trehalose.
In this study, we evaluated the effects of osmotic stress and ice crystal formation on the viability and function of primary rat hepatocytes at wide range of trehalose concentration.
There was no osmotic stress at very low concentrations (2.6 μM) of trehalose, and 2.6 μM trehalose drives the formation of finer ice crystals, which are less damaging to the cell membrane. Furthermore, we found that the number of viable hepatocytes after cryopreservation were 70% higher under the 2.6 μM trehalose-supplemented conditions than under the dimethyl sulfoxide-supplemented conditions. Moreover, non-cryopreserved cells and cells cryopreserved with trehalose showed comparable intracellular dehydrogenase activity.
We showed that trehalose at very low concentrations (2.6 μM) improved dramatically viability and liver function of hepatocyte after cryopreservation.
•Very low concentration of trehalose could suppress ice crystal formation and protect cell structure.•There was a correlation between osmotic pressure of trehalose and hepatocytes viability.•Very low concentration of trehalose improved viability and liver function of hepatocyte after cryopreservation.
Immune checkpoint inhibitors (ICI) are promising therapeutic agents for relapsed or refractory classical Hodgkin’s lymphoma (RRcHL). This retrospective study evaluated patients with RRcHL registered ...in the clinical research program Tohoku-Hematology-Forum-26, between 2016 and 2020, and treated with ICI in 14 centers in Northeast Japan. We analyzed the usage, efficacy, and safety of ICI therapy (ICIT). Among a total of 27 patients with RRcHL, 21 and nine were treated with nivolumab and/or pembrolizumab, respectively. The best response was complete response (CR), partial response (PR), stable disease (SD), and progressive disease in 11 (40.8%), seven (25.9%), eight (29.6%), and one (3.7%) patient, respectively. In all patients undergoing ICIT, the 2-year progression-free survival and 2-year overall survival (OS) were 48.6% and 87.4%, respectively. The 2-year OS for patients with CR, PR, and SD were 100%, 68.6%, and 87.5%, respectively. A total of 36 events of immune-related adverse events (irAEs) or immune-related like adverse events (irlAEs) were observed in 19 of the 27 patients (70.4%). Two thirds of these irAEs or irlAEs were grade 1–2 and controllable. During the observation period, ICIT was discontinued in 22 of 27 (81.4%) patients due to CR, inadequate response, irAE and patient circumstances in five (22.7%), seven (31.8%), eight (36.4%) and two patients (9.1%), respectively. Therapy-related mortality-associated irAE were observed in only one patient during ICIT. These results suggest that ICIT for RRcHL is effective and safe in real-world settings. The optimal timing of induction and duration of ICIT remains to be established.
Abnormal activation of hypoxia-inducible factor-1 (HIF-1), one of the most important transcription factors for the adaptation
of cells to hypoxia, is frequently observed in numerous types of solid ...tumors. Dysregulation of HIF-1 induces tumor angiogenesis
and enhances the expression of anti-apoptotic proteins and glycolysis-associated enzymes in cancer cells, which in turn leads
to the promotion of tumor growth. In the present study, we examined the pathophysiologic role of HIF-1 in multiple myeloma.
Furthermore, we explored the possibility that HIF-1 may be a molecular target for myeloma therapy. We identified constitutive
expression of the hypoxia-inducible factor-1 α (HIF-1α)-subunit in established myeloma cell lines and in primary myeloma cells.
Treatment with insulin-like growth factor-1 (IGF-1) significantly increased HIF-1α expression through activation of the AKT
and mitogen-activated protein kinase signaling pathways. Inhibition of HIF-1 function either by echinomycin, a specific HIF-1
inhibitor, or a siRNA against HIF-1α resulted in enhanced sensitivity to melphalan in myeloma cells. This inhibition of HIF-1
also reversed the protective effect of IGF-1 on melphalan-induced apoptosis. Inhibition of HIF-1 drastically reduced both
basal and IGF-1–induced expression of survivin, one of the most important anti-apoptotic proteins in myeloma cells. We conclude
that HIF-1 inhibition may be an attractive therapeutic strategy for multiple myeloma. Mol Cancer Ther 2009;8(8):2329–38
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