Enhancing effect of sodium butyrate on phosphatidylserine-liposome-induced macrophage polarization.

OBJECT: Phosphatidylserine-containing liposomes (PSLs) can mimic the immunomodulatory effects of apoptotic cells by binding to the phosphatidylserine receptors of macrophages. Sodium butyrate, an antiinflammatory short-chain fatty acid, is known to facilitate the M2 polarization of macrophages. This study aimed to investigate the effect of sodium butyrate on PSLs-induced macrophage polarization. METHODS: PSLs physical properties and cellular uptake tests, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry analysis were performed to assess the polarization-related indicators of M1/M2 macrophages. RESULTS: The results showed that sodium butyrate did not affect the size and cellular uptake of PSLs. For M1 macrophage polarization, sodium butyrate significantly intensified the antiinflammatory function of PSLs, inhibiting LPS-induced proinflammatory genes expression, cytokines and enzyme release (tumor necrosis factor-alpha, interleukin (IL)-1ß, IL-6, and inducible nitric oxide synthase), as well as CD86 (M1 marker) expression. In addition to the enhancing effect of antiinflammation, sodium butyrate also promoted PSL-induced M2 macrophages polarization, especially elevated thymus and activation-regulated chemokine (TARC) and arginase-1 (Arg-1) enzyme levels which are involved in tissue repair. CONCLUSION: Sodium butyrate enhanced antiinflammatory properties and M2-polarization inducing effect of PSLs. Therefore, sodium butyrate may represent a novel approach to enhance PSL-induced macrophage polarization.

Suppression of T24 human bladder cancer cells by ROS from locally delivered hematoporphyrin-containing polyurethane films.

Systemic injection of a photosensitizer is a general method in photodynamic therapy, but it has complications due to the unintended systemic distribution and remnants of photosensitizers. This study focused on the possibility of suppressing luminal proliferative cells by excessive reactive oxygen species from locally delivered photosensitizer with biocompatible polyurethane, instead of the systemic injection method. We used human bladder cancer cells, hematoporphyrin as the photosensitizer, and polyurethane film as the photosensitizer-delivering container. The light source was a self-made LED (510 nm, 5 mW cm-2) system. The cancer cells were cultured on different doses of hematoporphyrin-containing polyurethane film and irradiated with LED for 15 minutes and 30 minutes each. After irradiating with LED and incubating for 24 hours, cell viability analysis, cell cycle analysis, apoptosis assay, intracellular and extracellular ROS generation study and western blot were performed. The cancer cell suppression effects of different concentrations of the locally delivered hematoporphyrin with PDT were compared. Apoptosis dominant cancer cell suppressions were shown to be hematoporphyrin dose-dependent. However, after irradiation, intracellular ROS amounts were similar in all the groups having different doses of hematoporphyrin, but these values were definitely higher than those in the control group. Excessive extracellular ROS from the intended, locally delivered photosensitizer for photodynamic treatment application had an inhibitory effect on luminal proliferative cancer cells. This method can be another possibility for PDT application on contactable or attachable lesions.

Influence of Biomimetic Materials on Cell Migration.

In recent tissue engineering applications, the advance of biomaterials has focused on the devising of biomimetic materials that are directing new tissue formation and capable of causing specific cellular responses. These advances can be controlled by modifying the devising parameters of the materials. The biomimetic materials potentially mimic many roles of ECM in tissues. For the homogeneous distribution and biocompatibility of scaffolds by cell migration with biomimetic materials, cell migration is studied because it has a important role in physiological phenomenon and in pathologies; cancer metastasis, immune response or embryonic development. This review discusses the migration of cells with biomimetic materials for tissue engineering. It is also summarized that the recent advances of cell migration with biomimetic materials in 2-D and 3-D for tissue engineering.

Enhancement of human mesenchymal stem cell infiltration into the electrospun poly(lactic-co-glycolic acid) scaffold by fluid shear stress.

The infiltration of the cells into the scaffolds is important phenomenon to give them good biocompatibility and even biodegradability. Fluid shear stress is one of the candidates for the infiltration of cells into scaffolds. Here we investigated the directional migration of human mesenchymal stem cells and infiltration into PLGA scaffold by fluid shear stress. The human mesenchymal stem cells showed directional migrations following the direction of the flow (8, 16 dyne/cm(2)). In the scaffold models, the fluid shear stress (8 dyne/cm(2)) enhanced the infiltration of cells but did not influence on the infiltration of Poly(lactic-co-glycolic acid) particles.

Effects of vehicles on the physical properties and biocompatibility of premixed calcium silicate cements.

Premixed calcium silicate cements (pCSCs) contain vehicles which endow fluidity and viscosity to CSCs. This study aimed to investigate the effects of three vehicles, namely, polyethylene glycol (PEG), propylene glycol (PG), and dimethyl sulfoxide (DMSO), on the physicochemical properties and biocompatibility of pCSCs. The setting time, solubility, expansion rate, and mechanical strength of the pCSCs were evaluated, and the formation of calcium phosphate precipitates was assessed in phosphate-buffered saline (PBS). The effects of pCSC extracts on the osteogenic differentiation of mesenchymal stem cells (MSCs) were investigated. Finally, the tissue compatibility of pCSCs in rat femurs was observed. CSC containing PEG (CSC-PEG) exhibited higher solubility and setting time, and CSC-DMSO showed the highest expansion rate and mechanical strength. All pCSCs generated calcium phosphate precipitates. The extract of CSC-PG induced the highest expressions of osteogenic markers along with the greatest calcium deposites. When implanted in rat femurs, CSC-PEG was absorbed considerably, whereas CSC-PG remained relatively unaltered inside the femur.

Synergistic effects of arginine-glycine-aspartic acid and phosphatidylserine on the surface immunomodulation and osseointegration of titanium implants.

The control of macrophage polarization is important in bone tissue regeneration such as osseointegration. In this study, a coating method was developed to improve the osseointegration of titanium (Ti) implants by generating an immunomodulatory effect. The surface of the Ti discs was coated with a poly(lactide-co-glycolide)(PLGA) polymer, phosphatidylserine (PS), and arginine-glycine-aspartic acid (RGD) peptide conjugated phospholipid. In in vitro assay using mouse bone marrow-derived macrophages (BMDMs), the most significant expression of the M2 marker genes (Arg-1, YM-1, FIZZ1) and CD206, an M2 surface marker, was obtained with coatings containing 6 mol% RGD conjugates and phospholipids consisting of 50 mol% PS. The M2-inducing effect of RGD and PS was also verified in rat femurs where coated Ti rods were implanted. The RGD and PS coating significantly enhanced the osseointegration of the Ti implants. Moreover, a biomechanical push-out test showed that the RGD and PS coating increased the interfacial binding force between the bone and implants. These results indicate that PS and RGD can be applied to the solid surface of implantable biomedical devices to improve immunomodulation and tissue regeneration.

Sterilization of sealed PVDF pouches containing decellularized scaffold by electrical stimulation.

A terminal sterilization process for tissue engineering products, such as allografts and biomaterials is necessary to ensure complete removal of pathogenic microorganisms such as the bacteria, fungi, and viruses. However, it can be difficult to sterilize allografts and artificial tissue models packaged in wet conditions without deformation. In this study, we investigated the sterilization effects of electrical stimulation (ES) and assessed its suitability by evaluating sterility assurance levels in pouches at a constant current. Stability of polyvinylidene fluoride pouches was determined by a sterility test performed after exposure to five microorganisms (Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans) for 5 days; the sterility test was also performed with decellularized human dermal tissues inoculated with the five microorganisms. Sterilization using ES inactivated microorganisms both inside and outside of sealed pouches and caused no damage to the packaged tissue. Our results support the development of a novel system that involves ES sterilization for packaging of implantable biomaterials and human derived materials.

Effects of RGD-grafted phosphatidylserine-containing liposomes on the polarization of macrophages and bone tissue regeneration.

Phosphatidylserine-containing liposomes (PSLs) can mimic the anti-inflammatory effects of apoptotic cells by binding to the phosphatidylserine receptors of macrophages. MGF-E8, a bridge molecule between phosphatidylserine and macrophages, can promote M2 polarization by activating macrophage integrin with its arginine-glycine-aspartic acid (RGD) motif. In this study, to mimic MGF-E8, PSLs presenting RGD peptide (RGD-PSLs) were prepared, and their immunomodulatory effects on macrophages and the bone tissue regeneration of rat calvarial defects were investigated. RGD peptides enhanced the phagocytosis of PSLs by macrophages, especially when the PSLs contained 3% RGD. RGD-PSLs were also more effective than PSLs for the suppression of lipopolysaccharide-induced gene expression of proinflammatory cytokines (i.e., IL-1ß, IL-6, and TNF-α) as well as CD86 (M1 marker) expression. Furthermore, RGD promoted PSL-induced M2 polarization: 3%-RGD-PSLs significantly enhanced the mRNA expression of Arg-1, FIZZ1, and YM-1, as well as CD206 (M2 marker) expression. In a calvarial defect model, a significant increase in M2 with a decrease in M1 macrophages was observed with 3%-RGD-PSL treatment compared with the effects of PSLs alone. Finally, new bone formation was also accelerated by 3%-RGD-PSLs. Thus, these results suggest that the intensive immunomodulatory effect of RGD-PSLs led to the enhancement of bone tissue regeneration.

Development of a Shape-Memory Tube to Prevent Vascular Stenosis.

Inserting a graft into vessels with different diameters frequently causes severe damage to the host vessels. Poor flow patency is an unresolved issue in grafts, particularly those with diameters less than 6 mm, because of vessel occlusion caused by disturbed blood flow following fast clotting. Herein, successful patency in the deployment of an ≈2 mm diameter graft into a porcine vessel is reported. A new library of property-tunable shape-memory polymers that prevent vessel damage by expanding the graft diameter circumferentially upon implantation is presented. The polymers undergo seven consecutive cycles of strain energy-preserved shape programming. Moreover, the new graft tube, which features a diffuser shape, minimizes disturbed flow formation and prevents thrombosis because its surface is coated with nitric-oxide-releasing peptides. Improved patency in a porcine vessel for 18 d is demonstrated while occlusive vascular remodeling occurs. These insights will help advance vascular graft design.

Zwitterionic sulfobetaine polymer-immobilized surface by simple tyrosinase-mediated grafting for enhanced antifouling property.

Introducing antifouling property to biomaterial surfaces has been considered an effective method for preventing the failure of implanted devices. In order to achieve this, the immobilization of zwitterions on biomaterial surfaces has been proven to be an excellent way of improving anti-adhesive potency. In this study, poly(sulfobetaine-co-tyramine), a tyramine-conjugated sulfobetaine polymer, was synthesized and simply grafted onto the surface of polyurethane via a tyrosinase-mediated reaction. Surface characterization by water contact angle measurements, X-ray photoelectron spectroscopy and atomic force microscopy demonstrated that the zwitterionic polymer was successfully introduced onto the surface of polyurethane and remained stable for 7days. In vitro studies revealed that poly(sulfobetaine-co-tyramine)-coated surfaces dramatically reduced the adhesion of fibrinogen, platelets, fibroblasts, and S. aureus by over 90% in comparison with bare surfaces. These results proved that polyurethane surfaces grafted with poly(sulfobetaine-co-tyramine) via a tyrosinase-catalyzed reaction could be promising candidates for an implantable medical device with excellent bioinert abilities. STATEMENT OF SIGNIFICANCE: Antifouling surface modification is one of the key strategy to prevent the thrombus formation or infection which occurs on the surface of biomaterial after transplantation. Although there are many methods to modify the surface have been reported, necessity of simple modification technique still exists to apply for practical applications. The purpose of this study is to modify the biomaterial's surface by simply immobilizing antifouling zwitterion polymer via enzyme tyrosinase-mediated reaction which could modify versatile substrates in mild aqueous condition within fast time period. After modification, pSBTA grafted surface becomes resistant to various biological factors including proteins, cells, and bacterias. This approach appears to be a promising method to impart antifouling property on biomaterial surfaces.

Heparin-functionalized polymer graft surface eluting MK2 inhibitory peptide to improve hemocompatibility and anti-neointimal activity.

The leading cause of synthetic graft failure includes thrombotic occlusion and intimal hyperplasia at the site of vascular anastomosis. Herein, we report a co-immobilization strategy of heparin and potent anti-neointimal drug (Mitogen Activated Protein Kinase II inhibitory peptide; MK2i) by using a tyrosinase-catalyzed oxidative reaction for preventing thrombotic occlusion and neointimal formation of synthetic vascular grafts. The binding of heparin-tyramine polymer (HT) onto the polycarprolactone (PCL) surface enhanced blood compatibility with significantly reduced protein absorption (64.7% decrease) and platelet adhesion (85.6% decrease) compared to bare PCL surface. When loading MK2i, 1) the HT depot surface gained high MK2i-loading efficiency through charge-charge interaction, and 2) this depot platform enabled long-term, controlled release over 4weeks (92-272µg/mL of MK2i). The released MK2i showed significant inhibitory effects on VSMC migration through down-regulated phosphorylation of target proteins (HSP27 and CREB) associated with intimal hyperplasia. In addition, it was found that the released MK2i infiltrated into the tissue with a cumulative manner in ex vivo human saphenous vein (HSV) model. This present study demonstrates that enzymatically HT-coated surface modification is an effective strategy to induce long-term MK2i release as well as hemocompatibility, thereby improving anti-neointimal activity of synthetic vascular grafts.

Controlled Delivery of Extracellular ROS Based on Hematoporphyrin-Incorporated Polyurethane Film for Enhanced Proliferation of Endothelial Cells.

The principle of photodynamic treatment (PDT) involves the administration of photosensitizer (PS) at diseased tissues, followed by light irradiation to produce reactive oxygen species (ROS). In cells, a moderate increase in ROS plays an important role as signaling molecule to promote cell proliferation, whereas a severe increase of ROS causes cell damage. Previous studies have shown that low levels of ROS stimulate cell growth through PS drugs-treating PDT and nonthermal plasma treatment. However, these methods have side effects which are associated with low tissue selectivity and remaining of PS residues. To overcome such shortcomings, we designed hematoporphyrin-incorporated polyurethane (PU) film induced generation of extracellular ROS with singlet oxygen and free radicals. The film can easily control ROS production rate by regulating several parameters including light dose, PS dose. Also, its use facilitates targeted delivery of ROS to the specific lesion. Our study demonstrated that extracellular ROS could induce the formation of intracellular ROS. In vascular endothelial cells, a moderated increase in intracellular ROS also stimulated cell proliferation and cell cycle progression by accurate control of optimum levels of ROS with hematoporphyrin-incorporated polymer films. This modulation of cellular growth is expected to be an effective strategy for the design of next-generation PDT.

Exovascular application of epigallocatechin-3-O-gallate-releasing electrospun poly(L-lactide glycolic acid) fiber sheets to reduce intimal hyperplasia in injured abdominal aorta.

Intimal hyperplasia is an excessive ingrowth of tissue resulting in chronic structural lesions commonly found at sites of atherosclerotic lesions, arterial angioplasty, vascular graft anastomoses, and other vascular abnormalities. Epigallocatechin-3-O-gallate (EGCG) was shown to elicit antioxidant, anti-proliferative, and anti-thrombogenic effects. In this study, we used an electrospinning technique to synthesize EGCG-eluting biodegradable poly(L-lactide glycolic acid) (PLGA) fiber sheets for local delivery of EGCG and investigated the effect of their exovascular application on intimal hyperplasia following balloon-induced abdominal aorta injury in a rabbit experimental model. The morphology of the composite sheets was characterized using scanning electron microscopy and Fourier transform-infrared spectroscopy. EGCG was loaded and dispersed into the PLGA-based electrospun fibers. The EGCG-loaded PLGA sheets exhibited sustained EGCG release following the initial 24 h of burst release in phosphate-buffered saline. In vivo studies demonstrated significant inhibition of intimal hyperplasia following the application of the EGCG-eluting electrospun PLGA fiber sheets, compared with vehicle PLGA controls. In conclusion, our results show that exovascular application of EGCG-eluting PLGA electrospun fiber sheets may provide a useful system for the effective local delivery of drugs for the prevention of intimal hyperplasia.