Design of an apoptotic cell-mimetic wound dressing using phosphoserine-chitosan hydrogels.

Inflammatory M1 macrophages create a hostile environment that impedes wound healing. Phosphoserine (PS) is a naturally occurring immunosuppressive molecule capable of polarizing macrophages from an inflammatory phenotype (M1) to an anti-inflammatory phenotype (M2). In this study, we designed, fabricated, and characterized PS-immobilized chitosan hydrogels as potential wound dressing materials. A PS group precursor was synthesized via a phosphoramidite reaction and subsequently immobilized onto the chitosan chain through an EDC/N-hydroxysuccinimide reaction using a crosslink moiety HPA. The PS/HPA-conjugated chitosan (CS-PS) was successfully synthesized by deprotecting the PS group in HCl. In addition, the hydrogels were prepared by the HRP/H2O2 enzyme-catalyzed reaction with different PS group contents (0, 7.27, 44.28 and 56.88 µmol g-1). The immobilization of the PS group improved the hydrophilicity of the hydrogels. Interestingly, CS-PS hydrogel treatment upregulated both pro-inflammatory and anti-inflammatory cytokines. This treatment also resulted in alterations in the macrophage cell morphology from the M1 to M2 phenotype. The CS-PS hydrogel significantly accelerated diabetic wound healing. Overall, this study provides insights into the potential of PS-immobilized hydrogel materials for improved inflammatory disease therapy.

Green tea catechin-grafted silk fibroin hydrogels with reactive oxygen species scavenging activity for wound healing applications.

BACKGROUND: Overproduction of reactive oxygen species (ROS) is known to delay wound healing by causing oxidative tissue damage and inflammation. The green tea catechin, (-)-Epigallocatechin-3-O-gallate (EGCG), has drawn a great deal of interest due to its strong ROS scavenging and anti-inflammatory activities. In this study, we developed EGCG-grafted silk fibroin hydrogels as a potential wound dressing material. METHODS: The introduction of EGCG to water-soluble silk fibroin (SF-WS) was accomplished by the nucleophilic addition reaction between lysine residues in silk proteins and EGCG quinone at mild basic pH. The resulting SF-EGCG conjugate was co-crosslinked with tyramine-substituted SF (SF-T) via horseradish peroxidase (HRP)/H2O2 mediated enzymatic reaction to form SF-T/SF-EGCG hydrogels with series of composition ratios. RESULTS: Interestingly, SF-T70/SF-EGCG30 hydrogels exhibited rapid in situ gelation (< 30 s), similar storage modulus to human skin (≈ 1000 Pa) and superior wound healing performance over SF-T hydrogels and a commercial DuoDERM® gel dressings in a rat model of full thickness skin defect. CONCLUSION: This study will provide useful insights into a rational design of ROS scavenging biomaterials for wound healing applications.

Bioabsorbable Carboxymethyl Starch-Calcium Ionic Assembly Powder as a Hemostatic Agent.

In contrast to hemostatic fabrics, foams, and gels, hemostatic spray powders may be conveniently applied on narrow and complex bleeding sites. However, powdered hemostatic agents are easily desorbed from the bleeding surface because of blood flow, which seriously decreases their hemostatic function. In this study, the hemostatic performance of a bioabsorbable powder with decreased desorption was investigated. The proposed hemostatic powder (OOZFIXTM) is an ionic assembly of carboxymethyl starch and calcium. The microstructure and chemical properties of the hemostatic powder were analyzed. The hemostatic performance (blood absorption, blood absorption rate, and coagulation time), thromboelastography (TEG), rheology, adhesion force, and C3a complement activation of the OOZFIXTM were evaluated and compared with those of the carboxymethyl starch-based commercial hemostatic powder (AristaTM AH). The in vivo rat hepatic hemorrhage model for hemostasis time and bioabsorption of the OOZFIXTM showed quick biodegradation (<3 weeks) and a significantly improved hemostasis rate (78 ± 17 s) compared to that of AristaTM AH (182 ± 11) because of the reduced desorption. The bioabsorbable hemostatic powder OOZFIXTM is expected to be a promising hemostatic agent for precise medical surgical treatments.

Photodynamic Activity of Protoporphyrin IX-Immobilized Cellulose Monolith for Nerve Tissue Regeneration.

The development of nerve conduits with a three-dimensional porous structure has attracted great attention as they closely mimic the major features of the natural extracellular matrix of the nerve tissue. As low levels of reactive oxygen species (ROS) function as signaling molecules to promote cell proliferation and growth, this study aimed to fabricate protoporphyrin IX (PpIX)-immobilized cellulose (CEPP) monoliths as a means to both guide and stimulate nerve regeneration. CEPP monoliths can be fabricated via a simple thermally induced phase separation method and surface modification. The improved nerve tissue regeneration of CEPP monoliths was achieved by the activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinases (ERKs). The resulting CEPP monoliths exhibited interconnected microporous structures and uniform morphology. The results of in vitro bioactivity assays demonstrated that the CEPP monoliths with under 0.54 ± 0.07 µmol/g PpIX exhibited enhanced photodynamic activity on Schwann cells via the generation of low levels of ROS. This photodynamic activation of the CEPP monoliths is a cell-safe process to stimulate cell proliferation without cytotoxic side effects. In addition, the protein expression of phospho-ERK increased considerably after the laser irradiation on the CEPP monoliths with low content of PpIX. Therefore, the CEPP monoliths have a potential application in nerve tissue regeneration as new nerve conduits.

Shape-dependent antimicrobial activities of silver nanoparticles.

Purpose: An important application of silver nanoparticles (Ag NPs) is their use as an antimicrobial and wound dressing material. The aim of this study is to investigate the morphological dependence on the antimicrobial activity and cellular response of Ag NPs. Materials and methods: Ag NPs of various shapes were synthesized in an aqueous solution using a simple method. The morphology of the synthesized Ag NPs was observed via TEM imaging. The antimicrobial activity of the Ag NPs with different morphologies was evaluated against various microorganisms (Escherichia coli [E. coli], Staphylococcus aureus [S. aureus], Pseudomonas aeruginosa [P. aeruginosa]). The antimicrobial activity of the Ag NPs was also examined according to the concentration in terms of the growth rate of E. coli. Results: The TEM images indicated that the Ag NPs with different morphologies (sphere, disk and triangular plate) had been successfully synthesized. The antimicrobial activity obtained from the inhibition zone was in the order of spherical Ag NPs > disk Ag NPs > triangular plate Ag NPs. In contrast, fibroblast cells grew well in all types of Ag NPs when the cell viability was evaluated via an MTT assay. An inductively coupled plasma mass assay showed that the difference in the antimicrobial activities of the Ag NPs was closely associated with the difference in the release rate of the Ag ions due to the difference in the surface area of the Ag NPs. Conclusion: The morphological dependence of the antimicrobial activity of the Ag NPs can be explained by the difference in the Ag ion release depending on the shape. Therefore, it will be possible to control the antimicrobial activity by controlling the shape and size of the Ag NPs.

Polypeptide Thermogel-Filled Silk Tube as a Bioactive Nerve Conduit.

To repair nerve damages, nerve conduits are clinically used. However, when the gap distance is greater than 3 cm, nerve damages remain difficult to repair using current empty nerve conduits. To improve the nerve conduit, a silk nerve conduit (diameter = 5 mm and length = 3 cm) filled with a hydrogel matrix was prepared by injecting a thermogelling aqueous poly(ethylene glycol)-poly(l-alanine) (PEG-PA) solution containing stem cells and neurotropic factors into a silk tube at 37 °C. Successful differentiation of the incorporated stem cells into neuronal cells is a key factor for this system to be used as a nerve conduit. Therefore, neuronal differentiation of the incorporated stem cells in the thermogel-filled conduit system was investigated as a bioactive nerve conduit. The study found that the stem cells exhibited 20 to 60-fold increases in mRNA expression for the neuronal biomarkers of NFM and the glial biomarker of GFAP, in the PEG-PA thermogel-filled silk nerve conduit systems containing neurotropic factors of NGF and BDNF compared to control experiments. This article proved the potential of thermogel as an effective filler of a soft nerve conduit. The PEG-PA thermogel-filled silk conduit system incorporating stem cells and neurotropic factors can be promising as a bioactive nerve conduit system.

Guided Regeneration of Rabbit Calvarial Defects Using Silk Fibroin Nanofiber-Poly(glycolic acid) Hybrid Scaffolds.

Bone tissue engineering aims to regenerate defected bones by combining cells, scaffolds, and growth factors. In general, defected bone tissues are treated with barrier membranes or guiding scaffolds to achieve bone restoration. However, the growth rate of bone tissue is slower than that of adjacent soft tissue. Therefore, we propose patient-customizable guided bone regeneration (GBR) and membrane-guided tissue regeneration (GTR) scaffold hybrid constructs for precise bone tissue restoration without dimensional collapse beyond the critical bone defect size. Silk fibroin (SF) nanofiber membranes and poly(glycolic acid) (PGA) scaffolds were fabricated using electrospinning and hot-melt additive manufacturing methods based on a computer-generated scaffold design. Their manipulation parameters, microstructures, compressive moduli, and biodegradability were investigated. The initial attachment and proliferation of preosteoblasts on a PGA scaffold were analyzed based on seeding efficiency and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The regenerated bone volumes of control and SF-PGA hybrid scaffolds were 14.8 and 21.4%, respectively, after 8 weeks of in vivo rabbit calvarial defect regeneration. The SF-PGA hybrid scaffold group exhibits greater regeneration of bone tissue than the control and PGA scaffold groups, indicating that this is a promising material combination as a GBR-GTR agent.

Surface modification of PHBV nanofiber mats for rapid cell cultivation and harvesting.

To maintain the original function of a specific tissue for therapeutic tissue engineering, an advanced cell culture surface for repeat cell proliferation is necessary. We designed a novel cell proliferation and rapid harvesting surface by combining nonwoven nanofiber mat and a thermo-responsive polymer. Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) mats were fabricated by the electrospinning technique. A poly(N-isopropylacrylamide) (PNIPAM) thermo-responsive layer was grafted on the PHBV nanofiber mat by electron beam irradiation. The average diameter of the PNIPAM-grafted PHBV nanofibers was determined by SEM. ATR-FTIR and ESCA were used to confirm the grafting of PNIPAM onto the PHBV nanofiber surface. Water contact angles on the mats were measured in response to temperature changes. Human adipose-derived stem cells (ADSCs) were cultured on the PNIPAM-grafted PHBV nanofiber mat to investigate cell proliferation, harvesting, and functionality during repeat subculture. Detached ADSCs from each surface by low temperature treatment and trypsin-EDTA were compared by a fluorescence-activated cell sorter (FACS) using expression of stem cell membrane-specific markers such as CD-13 PE, CD-29 PE, and CD-90 FITC. The mass cultivation and intact harvesting of stem cells by low temperature treatment using a thermo-responsive PHBV nanofiber mat is a promising technique for use in regenerative medicine and stem cell therapy.

Modification and optimization of electrospun gelatin sheets by electron beam irradiation for soft tissue engineering.

BACKGROUND: Crosslinked gelatin nanofibers are one of the widely used scaffolds for soft tissue engineering. However, modifying the biodegradation rate of chemically crosslinked gelatin is necessary to facilitate cell migration and tissue regeneration. Here, we investigated the optimal electron beam (e-beam) irradiation doses with biodegradation behavior on changes in the molecular weight, morphology, pore structure, and cell proliferation profiles of electrospun nanofibrous gelatin sheets. METHODS: The molecular weights of uncrosslinked gelatin nanofibers were measured using gel permeation chromatography. The morphology and pore structure of the gelatin scaffolds were analyzed by scanning electron microscopy and a porosimeter. Biodegradation tests were performed in phosphate-buffered saline solutions for 4 weeks. Cell proliferation and tissue regeneration profiles were examined in fibroblasts using WST-1 assays and hematoxylin and eosin staining. RESULTS: Crosslinked gelatin nanofiber sheets exposed to e-beam irradiation over 300 kGy showed approximately 50% weight loss in 2 weeks. Gelatin scaffolds exposed to e-beam irradiation at 100-200 kGy showed significantly increased cell proliferation after 7 days of incubation. CONCLUSIONS: These findings suggested that the biodegradation and cell proliferation rates of gelatin nanofiber scaffolds could be optimized by varying e-beam irradiation doses for soft tissue engineering.

Preventing postoperative tissue adhesion using injectable carboxymethyl cellulose-pullulan hydrogels.

An injectable adhesive hydrogel composed of carboxymethyl cellulose (CMC) and pullulan is developed and evaluated as a postoperative anti-adhesion barrier. CMC was modified with tyramine to introduce crosslinking site via an EDC-NHS reaction. The in situ hydrogel was prepared by an enzyme-mediated reaction of tyramine-immobilized CMC with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Pullulan was added to the hydrogel solution to improve adhesiveness to the wound area and accelerate biodegradation. The modified CMC was confirmed by ATR-FTIR spectroscopy. The gelation time, storage modulus (G'), and weight loss of the hydrogels were measured as functions of the amounts of HRP and H2O2. The hydrogel group showed negligible cell proliferation and cytotoxicity, compared to that shown by the control group. The in vivo animal test demonstrated that significant decrease of postoperative tissue adhesion by applying the hydrogels. The CMC-pullulan hydrogel could be a useful treatment as an injectable in situ anti-adhesive agent.

Silk fibroin/hydroxyapatite composite hydrogel induced by gamma-ray irradiation for bone tissue engineering.

BACKGROUND: In this study, silk fibroin (SF) composite hydrogels containing hydroxyapatite (HAP) nanoparticles (NPs) for bone tissue engineering were fabricated using gamma-ray (γ-ray) irradiation treatment. During the irradiation, the HAP dispersed SF solution was changed to the chemically crosslinked SF hydrogel. METHODS: Distribution of HAP NPs in the SF hydrogel was examined by SEM imagery and energy dispersive X-ray spectrophotometry, and the crystalline structure of SF composite hydrogels was also confirmed by X-ray diffractometry. An optimum preparation condition of the SF/HAP composite hydrogels was determined with various HAP contents. For evaluation of the osteogenic differentiation of human mesenchymal stem cells (hMSCs), alkaline phosphatase activity (ALP), HAP nucleation in SBF and in vitro calcium accumulation were measured. RESULTS: The results revealed that compared with the pure SF hydrogels, the SF/HAP composite hydrogels improved osteogenic differentiation. CONCLUSION: This paper demonstrates the great potential of the SF/HAP composite hydrogels in terms of the production of the bone tissue engineering scaffolds for which osteogenesis is required.

Nanofiber mats composed of a chitosan-poly(d,l-lactic-co-glycolic acid)-poly(ethylene oxide) blend as a postoperative anti-adhesion agent.

Postoperative tissue adhesion causes serious complications and suffering in 90% of patients after peritoneum surgery, while commercial anti-adhesion agents cannot completely prevent postoperative peritoneal adhesions. This study demonstrates electrospining of a blended solution of chitosan, poly(d,l-lactic-co-glycolic acid) (PLGA), and poly(ethylene oxide) (PEO) to fabricate a chitosan-based nanofibrous mat as a postoperative anti-adhesion agent. Rheological studies combined with scanning electron microscopy reveal that the spinnability of the chitosan-PLGA solution could be controlled by adjusting the blend ratio and concentration with average fiber diameter from 634 to 913 nm. Biodegradation of the nanofiber specimens showed accelerated hydrolysis by chitosan. Proliferation of fibroblasts and antimicrobial activity of nanofibers containing chitosan was analyzed. Abdominal defects with cecum adhesion in rats demonstrated that the blend nanofiber mats were effective in preventing tissue adhesion as a barrier (4 weeks after abdominal surgery) by coverage of exfoliated peritoneum and insufficient wound sites at the beginning of the wound healing process. Chitosan-PLGA-PEO blend nanofiber mats will provide a promising key as a postoperative anti-adhesion agent. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1906-1915, 2017.

Injectable pullulan hydrogel for the prevention of postoperative tissue adhesion.

Methods for reducing and preventing postoperative abdominal adhesions have been researched for decades; however, despite these efforts, the formation of postoperative peritoneal adhesions is continuously reported. Adhesions cause serious complications such as postoperative pain, intestinal obstruction, and infertility. Tissue adhesion barriers have been developed as films, membranes, knits, sprays, and hydrogels. Hydrogels have several advantages when used as adhesion barriers, including flexibility, low tissue adhesiveness, biodegradability, and non-toxic degraded products. Furthermore, compared with preformed hydrogels, injectable hydrogels can fill and cover spaces of any shape and do not require a surgical procedure for implantation. In this study, pullulan was modified through reaction with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to introduce carboxyl and phenyl groups as crosslinking sites. The grafting of tyramine on pullulan allows crosslinking branches on pullulan backbone. We successfully fabricated pullulan hydrogel with an enzymatic reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The chemical structure of modified pullulan was analyzed with ATR-FTIR and (1)H NMR spectroscopies. Rheological properties were tested by measuring storage modulus with varying H2O2, HRP, polymer solution concentrations and tyramine substitution rates. Cell viability and animal tests were performed. The modified pullulan hydrogel is an invaluable advance in anti-adhesion agents.

Growth behavior of endothelial cells according to electrospun poly(D,L-lactic-co-glycolic acid) fiber diameter as a tissue engineering scaffold.

Investigating the effect of electrospun fiber diameter on endothelial cell proliferation provides an important guidance for the design of a fabric scaffold. In this study, we prepared biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) fibrous nonwoven mats with different fiber diameters ranged from 200 nm to 5 µm using the electrospinning technique. To control the fiber diameters of PLGA mats, 4 mixture solvents [hexafluoro-2-propanol, 2,2,2,-trifluoroethanol:dimethylformamide (9:1), 2,2,2,-trifluoroethanol:hexafluoro-2-propanol (9:1), chloroform] were used. Average diameters were 200 nm, 600 nm, 1.5 µm, and 5.0 µm, respectively. Stereoscopic structure and spatial characterization of fibrous PLGA mats were analyzed using atomic force microscopy and a porosimeter. The mechanical properties of PLGA mats were analyzed using a universal testing machine. The spreading behavior and infiltration of endothelial cells on PLGA mats were visualized by field emission scanning electron microscopy and hematoxylin and eosin staining. Cell proliferation on different PLGA fibers with different diameters was quantified using the MTT assay. Cells on 200 nm diameter PLGA mats showed rapid attachment and spreading. However, the cells did not penetrate the PLGA mat. Cells cultured on 600 nm and 1.5 µm diameter fibers could infiltrate the pores and cell proliferation was dramatically increased after 14 days. Secreted prostacyclin from endothelial cells on each mat was measured to examine the ability to inhibit platelet activation. This basic study on cell proliferation and fiber diameter with physical characterization provides a foundation for studies examining nonwoven fibrous PLGA mats as a tissue engineering scaffold.

Antimicrobial Silver Chloride Nanoparticles Stabilized with Chitosan Oligomer for the Healing of Burns.

Recently, numerous compounds have been studied in order to develop antibacterial agents, which can prevent colonized wounds from infection, and assist the wound healing. For this purpose, novel silver chloride nanoparticles stabilized with chitosan oligomer (CHI-AgCl NPs) were synthesized to investigate the influence of antibacterial chitosan oligomer (CHI) exerted by the silver chloride nanoparticles (AgCl NPs) on burn wound healing in a rat model. The CHI-AgCl NPs had a spherical morphology with a mean diameter of 42 ± 15 nm. The burn wound healing of CHI-AgCl NPs ointment was compared with untreated group, Vaseline ointment, and chitosan ointment group. The burn wound treated with CHI-AgCl NPs ointment was completely healed by 14 treatment days, and was similar to normal skin. Particularly, the regenerated collagen density became the highest in the CHI-AgCl NPs ointment group. The CHI-AgCl NPs ointment is considered a suitable healing agent for burn wounds, due to dual antibacterial activity of the AgCl NPs and CHI.

Fabrication and characterization of thermoresponsive polystyrene nanofibrous mats for cultured cell recovery.

Rapid cell growth and rapid recovery of intact cultured cells are an invaluable technique to maintain the biological functions and viability of cells. To achieve this goal, thermoresponsive polystyrene (PS) nanofibrous mat was fabricated by electrospinning of PS solution, followed by the graft polymerization of thermoresponsive poly(N-isopropylacrylamide)(PIPAAm) on PS nanofibrous mats. Image analysis of the PS nanofiber revealed a unimodal distribution pattern with 400 nm average fiber diameter. Graft polymerization of PIPAAm on PS nanofibrous mats was confirmed by spectroscopic methods such as ATR-FTIR, ESCA, and AFM. Human fibroblasts were cultured on four different surfaces, PIPAAm-grafted and ungrafted PS dishes and PIPAAm-grafted and ungrafted PS nanofibrous mats, respectively. Cells on PIPAAm-grafted PS nanofibrous mats were well attached, spread, and proliferated significantly much more than those on other surfaces. Cultured cells were easily detached from the PIPAAm-grafted surfaces by decreasing culture temperature to 20 °C, while negligible cells were detached from ungrafted surfaces. Moreover, cells on PIPAAm-grafted PS nanofibrous mats were detached more rapidly than those on PIPAAm-grafted PS dishes. These results suggest that thermoresponsive nanofibrous mats are attractive cell culture substrates which enable rapid cell growth and recovery from the culture surface for application to tissue engineering and regenerative medicine.

Effect of methylcellulose on the formation and drug release behavior of silk fibroin hydrogel.

In this study, methylcellulose (MC) was used to control the gelation time of silk fibroin (SF) aqueous solution. The gelation time was measured using a Vibro Viscometer at 50 °C. Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and a texture meter were used to investigate the effect of MC on the hydrogelation of SF solution. SF/MC hydrogels could be formed by the addition of MC, although their gelation time was increased with MC content. To examine the conformational change of SF/MC hydrogels, time-resolved FT-IR spectra were obtained at constant temperature using a custom-made IR chamber. From FT-IR spectra focused on the amide I peak position, the transition of SF molecules in SF/MC solution from a random coil to a ß-sheet structure was inhibited in the presence of MC molecules. In addition, the drug release of SF/MC hydrogels loaded with 5-aminosalicylic acid was studied in 2-dimensional (2-D) and 3-dimensional (3-D) conditions in vitro. The drug release behavior of SF or SF/MC hydrogels was measured using UV-Vis spectroscopy. The release rate of 5-aminosalicylic acid in SF/MC hydrogel was lower than that of SF hydrogel, which may be closely associated with the hydrophilic interaction between MC and 5-aminosalicylic acid. This approach to controlling the sol-gel transition and the drug release of SF hydrogels by the addition of MC will be useful in the design and tailoring of novel materials for biomedical applications.

Efficient formation of cell spheroids using polymer nanofibers.

Spheroid culture has been used for suspension cultures of anchorage-dependent cells. In this study, we developed a new method for the suspension cultures of anchorage-dependent animal cells using polymer nanofibers. Poly(lactic-co-glycolic acid) nanofibers (785 nm in average fiber-diameter, 88 µm in average fiber-length) fabricated by the electrospinning method were added to each suspension culture of human embryonic kidney 293 cells and human dermal fibroblasts. As compared to no addition of nanofibers to the suspension cultures, nanofibers enhanced cell spheroid formation, thereby reducing cell death resulting from a lack of cell adhesion. Efficient formation of spheroids in the presence of polymer nanofibers may be useful for the suspension cultures of anchorage-dependent cells.