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.

Preconditioning process for dermal tissue decellularization using electroporation with sonication.

Decellularization to produce bioscaffolds composed of the extracellular matrix (ECM) uses enzymatic, chemical and physical methods to remove antigens and cellular components from tissues. Effective decellularization methods depend on the characteristics of tissues, and in particular, tissues with dense, complex structure and abundant lipid content are difficult to completely decellularize. Our study enables future research on the development of methods and treatments for fabricating bioscaffolds via decellularization of complex and rigid skin tissues, which are not commonly considered for decellularization to date as their structural and functional characteristics could not be preserved after severe decellularization. In this study, decellularization of human dermal tissue was done by a combination of both chemical (0.05% trypsin-EDTA, 2% SDS and 1% Triton X-100) and physical methods (electroporation and sonication). After decellularization, the content of DNA remaining in the tissue was quantitatively confirmed, and the structural change of the tissue and the retention and distribution of ECM components were evaluated through histological and histochemical analysis, respectively. Conditions of the chemical pretreatment that increase the efficiency of physical stimulation as well as decellularization, and conditions for electroporation and sonication without the use of detergents, unlike the methods performed in previous studies, were established to enable the complete decellularization of the skin tissue. The combinatorial decellularization treatment formed micropores in the lipid bilayers of the skin tissues while removing all cell and cellular residues without affecting the ECM properties. Therefore, this procedure can be widely used to fabricate bioscaffolds by decellularizing biological tissues with dense and complex structures.

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.

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.

A collagen-AS/εPLL bilayered artificial substitute regulates anti-inflammation and infection for initial inflamed wound healing.

Despite the development of advanced tissue engineering substitutes, inflammation is still a significant problem that can arise from inflamed burn injuries, chronic wounds, or microbial diseases. Although topical wound dressing accelerates healing by minimizing or preventing the consequences of skin inflammation, there remains a need for the development of a novel substitute scaffold that can effectively eliminate immoderate inflammation and infection in the initial phase of the healing meachanism. In this study, an artificial skin substitute scaffold fabricated with asiaticoside (AS) and epsilon-poly-L-lysine (εPLL) was prepared. Upon the release of these bioactive compounds, they accelerate wound healing and inhibit any bacterial infection at the wound site. We determined whether AS and εPLL exhibit anti-inflammatory and bactericidal effects through different mechanisms. Collectively, the collagen-AS/εPLL artificial skin substitute could be a significant therapeutic agent for scar-less rapid wound healing (without infection and inflammation) of initially-inflamed full-thickness wounds.

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.

An effective method to generate controllable levels of ROS for the enhancement of HUVEC proliferation using a chlorin e6-immobilized PET film as a photo-functional biomaterial.

Reactive oxygen species (ROS) are byproducts of cellular metabolism; they play a significant role as secondary messengers in cell signaling. In cells, high concentrations of ROS induce apoptosis, senescence, and contact inhibition, while low concentrations of ROS result in angiogenesis, proliferation, and cytoskeleton remodeling. Thus, controlling ROS generation is an important factor in cell biology. We designed a chlorin e6 (Ce6)-immobilized polyethylene terephthalate (PET) film (Ce6-PET) to produce extracellular ROS under red-light irradiation. The application of Ce6-PET films can regulate the generation of ROS by altering the intensity of light-emitting diode sources. We confirmed that the Ce6-PET film could effectively promote cell growth under irradiation at 500 µW/cm2 for 30 min in human umbilical vein endothelial cells. We also found that the Ce6-PET film is more efficient in generating ROS than a Ce6-incorporated polyurethane film under the same conditions. Ce6-PET fabrication shows promise for improving the localized delivery of extracellular ROS and regulating ROS formation through the optimization of irradiation intensity.

Asiaticoside and polylysine-releasing collagen complex for effectively reducing initial inflammatory response using inflamed induced in vitro model.

Inflammation is a significant clinical problem that can arise from full-thickness wounds or burn injuries or microbial disease. Although topical wound healing substances could promote rapid wound healing by preventing or reducing the consequences of inflammation, there still remains a need for the development of novel substances that can effectively reduce infection and inflammation in initial wound healing phase. In this study, collagen was combined with asiaticoside (AS) and ε-poly-l-lysine (εPLL). This complex was then applied to in vitro models of infection and inflammation. Collagen-AS coatings inhibited the initial inflammatory response to LPS through a sustained release of AS, and a bilayer coating-εPLL showed a notable antimicrobial effect using microbial infection test. In this study, we determined whether asiaticoside and εPLL have anti-inflammatory and antibacterial effects through different mechanisms. Collectively, the collagen-AS/εPLL complex indicated great therapeutic potentials for accelerate wound healing and the complex may be considered as a artificial scaffold substitute product to full-thickness wound healing.

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.

Stem cell passage affects directional migration of stem cells in electrotaxis.

Stem cells can differentiate into various body tissues and organs and thus are considered as promising tools for cell therapy and tissue engineering. Early passage stem cells have high differentiation ability compared to late passage stem cells. Thus, it is important to use early passage stem cells in cell therapy. Here, we investigated whether cell migration could be used to compare young and senescent cells. We used 'electrotaxis' where cells under electric treatment move towards the anode or cathode. Without an electric stimulus, stem cells moved randomly. However, under a direct electric current, the cells moved with directionality. Under stimulation with a direct electric current, early passage stem cells moved towards the anode; when the cells became senescent with increasing passages, the percentage of cells migrating to the anode decreased. These results suggest that the behavior of stem cells under the influence of a direct electric current is also related to their passage number. Therefore, electrotaxis migration analysis can be used to distinguish between young cell and senescent cells.

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.

Author Correction: Homogeneity evaluation of mesenchymal stem cells based on electrotaxis analysis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

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.

Recombinant batroxobin-coated nonwoven chitosan as hemostatic dressing for initial hemorrhage control.

The choice of hemostat is determined by the situation and the degree of hemorrhage. One common hemostat, the nonwoven dressing, is easy to handled and controls severe bleeding on wider wounds. In this study, chitosan-based nonwoven dressings with recombinant batroxobin (rBat) were used as efficacious hemostatic dressing agents. Hemostatic agents need to absorb blood quickly in the early stages of blood coagulation cascade to rapidly and effectively control of excessive hemorrhages. To date, most studies of hemostatic agents focused on a single material and hemostats composed of multiple materials have not been studied sufficiently. Thus, we made a chitosan dressing coated with rBat and investigated the microstructure, mechanical properties, hemostatic efficacy, and clotting properties of the coated dressing. Our results showed that the rBat had a synergetic effect on chitosan that improved blood coagulation. Furthermore, the dressing had excellent bleeding control in an Sprague-Dawley (SD) rat femoral artery hemorrhage model. In conclusion, hemostasis can be improved by combining a chitosan-based nonwoven dressing with other agents, and rBat-coated chitosan-based nonwoven dressings have enormous potential to improve blood coagulation.

Exogenous ROS-induced cell sheet transfer based on hematoporphyrin-polyketone film via a one-step process.

To date, most of invasive cell sheet harvesting methods have used culture surface property variations, such as wettability, pH, electricity, and magnetism, to induce cell detachment. These methods that rely on surface property changes are effective when cell detachment prior to application is necessary, but of limited use when used for cell sheet transfer to target regions. The study reports a new reactive oxygen species (ROS)-induced strategy based on hematoporphyrin-incorporated polyketone film (Hp-PK film) to transfer cell sheets directly to target areas without an intermediate harvesting process. After green LED (510 nm) irradiation, production of exogenous ROS from the Hp-PK films induces cell sheet detachment and transfer. The study suggests that ROS-induced cell detachment property of the Hp-PK film is closely related to conformational changes of extracellular matrix (ECM) proteins. Also, this strategy with the Hp-PK film can be applied by regulating production rate of exogenous ROS in various types of cells, including fibroblasts, mesenchymal stem cells and keratinocytes. In conclusion, ROS-induced method using the Hp-PK film can be used for one-step cell sheet transplantation and has potential in biomedical applications.

Microneedle Vascular Couplers with Heparin-Immobilized Surface Improve Suture-Free Anastomosis Performance.

To make up for the shortcomings of the suture-based approach and current coupler devices including long suturing time, exhaustive training, additional mechanical setting, and narrow working windows for size and type of diverse vessel types, a new, suture-free microneedle coupler was developed in this study. The needle shape for improved anastomosis performance and the condition for antithrombotic surface immobilization were determined. In particular, the polymer materials help to maintain healthy phenotypes of main vascular cell types. The performance in rabbit and porcine models of end-to-end vascular anastomosis indicate that this device can serve as a potent alternative to the current approaches.

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.

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.

Homogeneity evaluation of mesenchymal stem cells based on electrotaxis analysis.

Stem cell therapy that can restore function to damaged tissue, avoid host rejection and reduce inflammation throughout body without use of immunosuppressive drugs. The established methods were used to identify and to isolate specific stem cell markers by FACS or by immunomagnetic cell separation. The procedures for distinguishing population of stem cells took a time and needed many preparations. Here we suggest an electrotaxis analysis as a new method to evaluate the homogeneity of mesenchymal stem cells which can observe the stem cell population in culture condition and wide use to various types of stem cells. Human mesenchymal stem cell, adipose derived stem cell, tonsil derived stem cell and osteogenic differentiated cells migrated toward anode but the migration speed of differentiated cells was significantly decreased versus that of stem cells. In mixture of stem cells and differentiated cells condition, we identified that the ratio of stem cell versus differentiated cell was matched with the homogeneity evaluation data of stem cells based on electrotaxis analysis. As a result, our evaluation tool has the possibility of the wide use to stem cell homogeneity evaluation and might be used as the stem cell quality control during stem cell culture without any additional antibodies.