Incubation In Phosphate-buffered Saline Research Articles

In vivo bioactivity of rhBMP-2 delivered with novel polyelectrolyte complexation shells assembled on an alginate microbead core template

Electrostatic interactions between polycations and polyanions are being explored to fabricate polyelectrolyte complexes (PEC) that could entrap and regulate the release of a wide range of biomolecules. Here, we report the in vivo application of PEC shells fabricated from three different polycations: poly-l-ornithine (PLO), poly-l-arginine (PLA) and DEAE-dextran (DEAE-D) to condense heparin on the surface of alginate microbeads and further control the delivery of recombinant human bone morphogenetic protein 2 (rhBMP-2) in spinal fusion application. We observed large differences in the behavior of PEC shells fabricated from the cationic polyamino acids (PLO and PLA) when compared to the cationic polysaccharide, DEAE-D. Whereas DEAE-D-based PEC shells eroded and released rhBMP-2 over 2days in vitro, PLO- and PLA-based shells retained at least 60% of loaded rhBMP-2 after 3weeks of incubation in phosphate-buffered saline. In vivo implantation in a rat model of posterolateral spinal fusion revealed robust bone formation in the PLO- and PLA-based PEC shell groups. This resulted in a significantly enhanced mechanical stability of the fused segments. However, bone induction and biomechanical stability of spine segments implanted with DEAE-D-based carriers were significantly inferior to both PLO- and PLA-based PEC shell groups (p<0.01). From these results, we conclude that PEC shells incorporating native heparin could be used for growth factor delivery in functional bone tissue engineering application and that PLA- and PLO-based complexes could represent superior options to DEAE-D for loading and in vivo delivery of bioactive BMP-2 in this approach.

The application of layered double hydroxide clay (LDH)-poly(lactide-co-glycolic acid) (PLGA) film composites for the controlled release of antibiotics

Many sites of bacterial infection such as in-dwelling catheters and orthopedic surgical sites require local rather than systemic antibiotic administration. However, currently used controlled release vehicles, such as polymeric films, release water-soluble antibiotics too quickly, whereas nonporous bone cement, used in orthopedics, release very little drug. The purpose of this study was to investigate the use of nanoparticulates composed of layered double hydroxide clays to bind various antibiotics and release them in a controlled manner. Mg-Al (carbonate) layered double hydroxides were synthesized and characterized using established methods. These clay particles were suspended in solutions of the antibiotics tetracycline, doxorubicin (DOX), 5-fluorouracil, vancomycin (VAN), sodium fusidate (SF) and antisense oligonucleotides and binding was determined following centrifugation and quantitation of the unbound fraction by UV/Vis absorbance or HPLC analysis. Drug release from layered double hydroxide clay/drug complexes dispersed in polymeric films was measured by incubation in phosphate-buffered saline (pH 7.4) at 37°C using absorbance or HPLC analysis. Antimicrobial activity of drug released from film composites was determined using zonal inhibition studies against S. epidermidis. All drugs bound to the clay particles to various degrees. Generally, drugs released with a large burst phase of release (except DOX) with little further drug release after 4days. Dispersion of drug/clay complexes in poly(lactic-co-glycolic acid) films resulted in a reduced burst phase of release and a slow continuous release for many weeks with effective antimicrobial amounts of VAN and SF released at later time points. Layered double hydroxide clays may be useful for controlled release applications at sites requiring long-term antibiotic exposure as they maintain the drug in a non-degraded state and release effective amounts of drug over long time periods. LDH clay/drug complexes are amenable to homogenous dispersion in polymeric films where implant coating may be optimal or required.

Incorporation of salmon calcitonin-loaded poly(lactide-co-glycolide) (PLGA) microspheres into calcium phosphate bone cement and the biocompatibility evaluation in vitro

Slow tissue ingrowth is the major drawback for the use of calcium phosphate cements; to address the issue, salmon calcitonin–loaded biodegradable poly(lactide- co-glycolide) microspheres were incorporated into calcium phosphate cement in this study. The effects of poly(lactide- co-glycolide) weight ratio on the mechanical strength, self-setting properties, and salmon calcitonin release ability of calcium phosphate cement were systematically investigated. The in vitro degradation behavior and the cumulative mass loss (%) of the composite during incubation in phosphate-buffered saline were studied. The release of salmon calcitonin was sustained for at least 35 days, and the release rate can be tailored by adjusting the ratio of PLGA. The scanning electron microscopic images of the composites after incubation for 48 days indicated that the poly(lactide- co-glycolide) degraded completely and formed a porous structure in the calcium phosphate cement. An in vitro cell culture of the calcium phosphate cement/salmon calcitonin–poly(lactide- co-glycolide) cement provided more biocompatible than calcium phosphate cement. This composite possesses the basic performance for clinical needs, and it has potential use for treating osteoporosis and accelerating bone repair.

Pivotal Role of ADP-ribosylation Factor 6 in Toll-like Receptor 9-mediated Immune Signaling

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.

Surface mineralization of Ti6Al4V substrates with calcium apatites for the retention and local delivery of recombinant human bone morphogenetic protein-2

Titanium alloys are prevalently used as orthopedic prosthetics. Inadequate bone–implant interactions can lead to premature prosthetic loosening and implant failure. Local delivery of osteogenic therapeutics promoting osteointegration of the implant is an attractive strategy to address this clinical challenge. Given the affinity of calcium apatites for bone matrix proteins we hypothesize that titanium alloys surface mineralized with calcium apatites should be explored for the retention and local delivery of osteogenic recombinant human bone morphogenetic protein-2 (rhBMP-2). Using a heterogeneous surface nucleation and growth process driven by the gradual pH elevation of an acidic solution of hydroxyapatite via thermal decomposition of urea, Ti6Al4V substrates were surface mineralized with calcium apatite domains exhibiting good affinity for the substrate. The microstructures, size and surface coverage of the mineral domains as a function of the in vitro mineralization conditions were examined by light and scanning electron microscopy and the surface calcium ion content quantified. An optimal mineralization condition was identified to rapidly (<10 h) achieve surface mineral coverage far superior to those accomplished by week long incubation in simulated body fluids. In vitro retention–release profiles of rhBMP-2 from the mineralized and unmineralized Ti6Al4V, determined by an enzyme-linked immunosorbent assay, supported a higher degree of retention of rhBMP-2 on the mineralized substrate. The rhBMP-2 retained on the mineralized substrate after 24 h incubation in phosphate-buffered saline remained bioactive, as indicated by its ability to induce osteogenic transdifferentiation of C2C12 myoblasts attached to the substrate. This mineralization technique could also be applied to the surface mineralization of calcium apatites on dense tantalum and titanium and porous titanium substrates.

Biological hydrogel synthesized from hyaluronic acid, gelatin and chondroitin sulfate by click chemistry

In order to mimic the natural cartilage extracellular matrix, which is composed of core proteins and glycosaminoglycans, a biological hydrogel was synthesized from the biopolymers hyaluronic acid (HA), chondroitin sulfate (CS) and gelatin via click chemistry. HA and CS were modified with 11-azido-3,6,9-trioxaundecan-1-amine (AA) and gelatin was modified with propiolic acid (PA). The molecular structures were verified by 1H nuclear magnetic resonance, infrared spectroscopy and elemental analysis, giving substitution degrees of 29%, 89% and 44% for HA–AA, CS–AA and gelatin–PA (G–PA), respectively. The –N3 groups of HA–AA and CS–AA were reacted with the acetylene groups of G–PA, catalyzed by Cu(I), to form triazole rings, thereby forming a cross-linked hydrogel. The gelation time was decreased monotonically with increasing Cu(I) concentration up to 0.95mgml−1. The hydrogel obtained was in a highly swollen state and showed the characteristics of an elastomer. Incubation in phosphate-buffered saline for 4weeks resulted in a weight loss of up to 45%. Moreover, about 20% gelatin and 10% CS were released from the hydrogel in 2weeks. In vitro cell culture showed that the hydrogel could support the adhesion and proliferation of chondrocytes.

The effect of electrospun fibre alignment on the behaviour of rat periodontal ligament cells

It is envisioned that for the regeneration of highly organized structures, like tendon and ligaments, only aligned fibrous scaffolds can provide adequate topographic guidance to cells. In this study, a novel method to electrospin an aligned scaffold is presented. Electrospun fibres were deposited into a water bath and then the fibres were drawn to a rotating mandrel in a controlled manner. In this way, parallel and cross-aligned fibrous poly (lactide-co-glycolide) (PLGA) scaffolds were fabricated, which were subsequently used to study their effect on the growth behaviour of rat periodontal ligament (PDL) cells. First, the scaffolds were characterized regarding mechanical properties, scaffold stability and degradation in vitro. Then, rat PDL cells were seeded and cultured on these scaffolds for up to 7 days. Randomly oriented PLGA and solvent cast plain PLGA films served as controls. Results showed that the alignment of fibres resulted in a higher tensile stress and Young's modulus. Aligned scaffolds maintained their structural stability better compared to the controls after incubation in phosphate-buffered saline for 6 weeks. Further, cells were observed to elongate along the fibre after 3 days of culture. Proliferation and migration of PDL cells was significantly more prevalent on the aligned fibres compared to the controls. It was concluded that aligned scaffolds seem to be able to promote the organized regeneration of periodontal tissue.

Cationic vesicles from novel bolaamphiphilic compounds

Effective targeted drug delivery by cationic liposomes is difficult to achieve because of their rapid clearance from the blood circulation. Bolaamphiphiles that form monolayer membrane may provide vesicles with improved stability, as shown for archaeosomes. We investigated a series of bolaamphiphiles with acetylcholine head groups and systematic structural changes in their hydrophobic domain for their ability to form stable nanovesicles. Bolaamphiphiles with two aliphatic chains separated by a short amide midsection produced spherical nanovesicles ranging in diameter from 80 to 120 nm. These vesicles lost their encapsulated material within 24 hours of incubation in phosphate-buffered saline (PBS). Similar bolaamphiphiles with a longer midsection produced a mixture of fibers and more stable nanovesicles. Bolaamphiphiles with ester amide midsection produced only spherical nanovesicles that were stable during incubation in PBS for several days. Vesicles made from bolaamphiphiles with acetylcholine head groups conjugated to the aliphatic chain via the amine were less stable than vesicles made from bolaamphiphiles with head groups conjugated to the aliphatic chain via the acetyl group. Vesicles that were stable in vitro showed good stability in the blood circulation after intravenous administration to mice. These results help in elucidating the bolaamphiphile structures needed to form stable cationic vesicles for targeted drug delivery.

An improved synthesis and biological evaluation of a new cage-like bifunctional chelator, 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1-ylamino)methyl)benzoic acid, for 64Cu radiopharmaceuticals

Stable attachment of (64)Cu(2+) to a targeting molecule usually requires the use of a bifunctional chelator (BFC). Sarcophagine (Sar) ligands rapidly coordinate (64)Cu(2+) within the multiple macrocyclic rings comprising the cage structure under mild conditions, providing high stability in vivo. Previously, we have designed a new versatile cage-like BFC Sar ligand, 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1-ylamino)methyl)benzoic acid (AmBaSar), for (64)Cu radiopharmaceuticals. Here we report the improved synthesis of AmBaSar, (64)Cu(2+) labeling conditions and its biological evaluation compared with the known BFC 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). The AmBaSar was synthesized in four steps starting from (1,8-diamine-Sar) cobalt(III) pentachloride ([Co(DiAmSar)]Cl(5)) using an improved synthetic method. The AmBaSar was labeled with (64)Cu(2+) in pH 5.0 ammonium acetate buffer solution at room temperature, followed by analysis and purification with HPLC. The in vitro stability of (64)Cu-AmBaSar complex was evaluated in phosphate buffered saline (PBS), fetal bovine serum and mouse blood. The microPET imaging and biodistribution studies of (64)Cu-AmBaSar were performed in Balb/c mice, and the results were compared with (64)Cu-DOTA. The AmBaSar was readily prepared and characterized by MS and (1)H NMR. The radiochemical yield of (64)Cu-AmBaSar was >or=98% after 30 min of incubation at 25 degrees C. The (64)Cu-AmBaSar complex was analyzed and purified by HPLC with a retention time of 17.9 min. The radiochemical purity of (64)Cu-AmBaSar was more than 97% after 26 h of incubation in PBS or serum. The biological evaluation of (64)Cu-AmBaSar in normal mouse demonstrated renal clearance as the primary mode of excretion, with improved stability in vivo compared to (64)Cu-DOTA. The new cage-like BFC AmBaSar was prepared using a simplified synthetic method. The (64)Cu-AmBaSar complex could be obtained rapidly with high radiochemical yield (>/=98%) under mild conditions. In vitro and in vivo evaluation of AmBaSar demonstrated its promising potential for preparation of (64)Cu radiopharmaceuticals.

Preparation of electrospun poly( l-lactide- co-caprolactone- co-glycolide)/phospholipid polymer/rapamycin blended fibers for vascular application

Nanopores generated from the gap among electrospun nanofibers can provide vascular tissues with oxygen, nutrients, and growth factors. Here, we report the first fabrication of poly( l-lactide- co-caprolactone- co-glycolide) (PLCG) electrospun nanofibers. A water-soluble amphiphilic copolymer bearing phosphorylcholine groups (PMB30W) and an antiproliferative (rapamycin) were incorporated into PLCG by solvent blending and subsequent electrospinning. The nanostructure of the electrospun PLCG/PMB30W/rapamycin blended fibers was stable during incubation in phosphate-buffered saline without addition of chemical crosslinkers. Amphiphilic characteristics of PMB30W and hydrophobic nature of rapamycin could make the electrospun PLCG/PMB30W/rapamycin blended fibers stable under a physiologic condition. The electrospun PLCG/PMB30W/rapamycin blended fibers may be preferable for vascular prostheses and tissue-engineered vascular grafts.

Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms byPseudomonas aeruginosaandStaphylococcus aureus

Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.

Collaborative Cell-Resistant Properties of Polyelectrolyte Multilayer Films and Surface PEGylation on Reducing Cell Adhesion to Cytophilic Surfaces

Both poly(ethylene glycol) (PEG) grafting and layer-by-layer polyelectrolyte multilayer (PEM) deposition for surface modification of biomaterials have been shown to decrease cell adhesion. The aim of this study was to investigate the synergic efficacy of PEGylated PEM films on reducing cell adhesion. PEG grafted to poly(ethylene imine) (PEI) was deposited onto the top of PEI/PAA (poly(acrylic acid)) multilayer films which were deposited onto cytophilic substrates, including tissue culture polystyrene and collagen-based substrate. The efficacy of the PEGylated PEM films in blocking adhesion of L929 cells was investigated by varying the amount of conjugated PEG and the layer numbers of PEM films. We found that cell adhesion was reduced on the swollen PEM films and further decreased by deposition of PEI-g-PEG as the topmost layer. The ability in cell resistance was enhanced with increasing PEG contents of PEGylated PEM films. PEGylated PEM films were stable for long-term incubation in phosphate-buffered saline. We demonstrated that cell affinity of cytophilic surfaces could be depressed by deposition of PEGylated PEM films.

Decreased Ceramide Transport Protein (CERT) Function Alters Sphingomyelin Production following UVB Irradiation

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

In vitro biocompatibility and degradation of terpolyester 3HB-co-4HB-co-3HHx, consisting of 3-hydroxybutyrate, 4-hydroxybutyrate and 3-hydroxyhexanoate

A terpolyester consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyhexanoate (3HHx), abbreviated as P(3HB-co-4HB-co-3HHx), was studied for possible application as an implant biomaterial. L929 mouse fibroblasts, MC3T3-E1 murine osteoblasts and a human cell line of immortalized human keratinocyte (HaCat cells) were used to study the biocompatibility of P(3HB-co-4HB-co-3HHx). Cell morphology and cell activity were studied using scanning electron microscopy (SEM) and the MTT assay, respectively. All three cell types showed higher activities when grown on films of P(3HB-co-4HB-co-3HHx) compared with their growth on poly(lactic acid) (PLA), co-polyester PHBHHx films and on polylysine-coated plates (blank), respectively. The three cell types grown on the terpolyester also demonstrated a well-spread cell shape and large number of pseudopods due to strong cell–cell and cell–material interactions. It was clearly observed that P(3HB-co-4HB-co-3HHx) had a much faster degradation rate than PHBHHx after 15 weeks of incubation in phosphate-buffered saline under dynamic conditions. The results proved that the terpolyester had favorable biocompatibility and biodegradability compared with the well-studied polyesters PLA and PHBHHx.

Synthesis, characterization and stability of a luteinizing hormone-releasing hormone (LHRH)-functionalized poly(amidoamine) dendrimer conjugate

Cancer targeting is crucial for cancer detection, therapy and targeted drug delivery. A dendrimer-peptide conjugate has been synthesized based on poly(amidoamine) dendrimer generation 5 (PAMAM G5) as a platform and a luteinizing hormone-releasing hormone (LHRH) peptide as a targeting moiety. The synthesized conjugate was fully characterized using nuclear magnetic resonance (NMR), UV-Vis spectrometry, reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Further stability experiments showed that the synthesized conjugate was stable after 72-h incubation in phosphate-buffered saline (PBS) buffer (pH 7.4) at 37°C. The synthesized conjugate may find applications in biomedical targeting, gene delivery and imaging.

Native Umbilical Cord Matrix Stem Cells Express Hepatic Markers and Differentiate Into Hepatocyte-like Cells

Umbilical cord matrix stem cells (UCMSCs) are able to differentiate into mesodermal and ectodermal lineages. The present study investigates the differentiation potential of human UCMSCs into hepatic lineage. We isolated human UCMSCs and characterized them in vitro by measuring their expansion potential, by assessing expression of mesenchymal stem cell (MSC) markers, and by evaluating their ability to differentiate into adipocytes and osteocytes. UCMSCs were thereafter subjected to a hepatogenic differentiation protocol. Expression of hepatic and MSC markers in differentiated cells was analyzed by reverse-transcription polymerase chain reaction, flow cytometry, and immunocytochemical assays and compared with undifferentiated UCMSCs and freshly isolated liver cells. UCMSCs were transplanted into livers of hepatectomized-SCID mice, and engraftment capacity was investigated by detection of human nucleus and mitochondria and human hepatic-specific proteins. In vitro expanded UCMSCs constitutively expressed markers of hepatic lineage, including albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. In vitro-differentiated UCMSCs exhibited hepatocyte-like morphology, up-regulated several hepatic markers, stored glycogen, produced urea, and exhibited an inducible CYP 3A4 activity. However, absence of some hepatic markers in differentiated UCMSCs, as HepPar1 or hepatocyte nuclear factor 4 (HNF-4), implied that their differentiation did not reach the level of mature hepatocytes. We also noticed that differentiated UCMSCs partially preserved MSC markers. Engraftment capacity of UCMSCs was observed, and expression of human albumin and alpha-fetoprotein was detected 2, 4, and 6 weeks after transplantation in mice livers, while cytokeratin 19 was completely down-regulated. We conclude that UCMSCs, with a newly demonstrated endodermic differentiation potential, might be an alternative source for liver-directed cell therapies.

Synthesis and Evaluation of Novel Biodegradable Hydrogels Based on Poly(ethylene glycol) and Sebacic Acid as Tissue Engineering Scaffolds

Novel biodegradable poly(ethylene glycol) (PEG) based hydrogels, namely, PEG sebacate diacrylate (PEGSDA) were synthesized, and their properties were evaluated. Chemical structures of these polymers were confirmed by Fourier transform infrared and proton nuclear magnetic resonance (1H NMR) spectroscopy. After photopolymerization, the dynamic shear modulus of the hydrogels was up to 0.2 MPa for 50% PEGSDA hydrogel, significantly higher than conventional hydrogels such as PEG diacrylate (PEGDA). The swelling ratios of these macromers were significantly lower than PEGDA. The in vitro degradation study demonstrated that these hydrogels were biodegradable with weight losses about 66% and 32% for 25% and 50% PEGSDA after 8 weeks of incubation in phosphate-buffered saline at 37 degrees C. In vitro biocompatibility was assessed using cultured rat bone marrow stromal cells (MSCs) in the presence of unreacted monomers or degradation products. Unlike conventional PEGDA hydrogels, PEGSDA hydrogel without RGD peptide modification induced MSC cell adhesion similar to tissue culture polystyrene. Finally, complex three-dimensional structures of PEGSDA hydrogels using solid free form technique were fabricated and their structure integrity was better maintained than PEGDA hydrogels. These hydrogels may find use as scaffolds for tissue engineering applications.

Biodegradable polycation and plasmid DNA multilayer film for prolonged gene delivery to mouse osteoblasts

Sustained release of functional plasmid DNA from the surfaces of materials which support cell adhesion for tissue formation could have a significant impact on gene therapy and tissue engineering. We report here layer-by-layer assembled multilayer film from a degradable cationic poly(2-aminoethyl propylene phosphate) and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) for mouse osteoblast cell adhesion and prolonged gene delivery. Multilayer film growth was monitored by UV spectrophotometry and intensity of absorbance at 260 nm related to incorporated DNA increased in an exponential manner with increase the number of deposited polymer and plasmid layers. It degraded upon incubation in phosphate-buffered saline (PBS) at 37 °C and sustained the release of bioactive plasmid DNA up to 2 months. The multilayer film facilitated initial mouse osteoblast cell adhesion onto the surface and enhanced cellular alkaline phosphatase activity and calcium accumulation. It sustained delivering transcriptional active DNA to mouse osteoblast cells cultured on the film, and directly prolonged gene expression in the presence of serum without any exogenous transfection agent. This biodegradable multilayer assembly is promising for the local and sustained delivery of plasmid DNA and such a layer-by-layer system suggests an alternative method for plasmid DNA incorporation which may be useful for surface modification of implanted materials or scaffold for gene therapy and tissue regeneration.

Apparent Dewetting of Ultrathin Multilayered Polyelectrolyte Films Incubated in Aqueous Environments

We have investigated and characterized changes in film morphology and surface structure that occur when ultrathin multilayered polyelectrolyte films fabricated from linear poly(ethylene imine) (LPEI), sodium poly(styrene sulfonate) (SPS), and two hydrolytically degradable polyamines (polymers 1 and 2) are incubated in physiologically relevant environments. Characterization of the physical erosion profiles of films having the structure (LPEI/SPS)10(1/SPS)4(2/SPS)4 (approximately 80 nm thick) by atomic force microscopy (AFM), reflective optical microscopy, and scanning electron microscopy (SEM) demonstrated that these materials undergo large-scale changes in surface structure and morphology upon incubation in phosphate-buffered saline (PBS) at 37 degrees C. The patterns and structures generated during this transformation (e.g., nucleation and growth of holes, coalescence of holes, formation of cell-type structures, and the subsequent breakup of these features into droplets) are similar in many ways to those observed for the dewetting of thin films of conventional polymers, such as polystyrene, on nonwetting surfaces. The processes reported here are sufficiently slow (they occur over approximately 100 h) and occur under sufficiently mild conditions (e.g., incubation in PBS at 37 degrees C) to permit characterization and quantification of the structures and features that arise during the course of these transformations. The apparent dewetting of these ultrathin films upon exposure to aqueous environments creates future opportunities to investigate and characterize processes of mass transport in this class of ionically cross-linked assemblies.

Covalently Attached Vancomycin Provides a Nanoscale Antibacterial Surface

Despite improved strategies for treating periprosthetic infection, current antibiotic delivery approaches are imperfect and can result in bacterial resistance and recalcitrant bio-films. To address the issues, we developed a covalently linked vancomycin-titanium implant interface that prevents and possibly eliminates bacterial colonization. We determined the amount of vancomycin immobilized on the titanium surface and assessed vancomycin stability and activity over time. When incubated with Staphylococcus aureus, the vancomycin-titanium surface showed an almost complete absence of adherent bacteria. To determine if continual exposure to vancomycin-titanium would cause decreased susceptibility to the antibiotic, S. aureus was incubated with vancomycin-titanium for 1 week or 4 weeks; these bacteria did not show an increased minimum inhibitory concentration for vancomycin. We tested the long-term stability of the vancomycin-titanium surface by incubation in phosphate-buffered saline for 11 months and then challenging the surface with S. aureus. Fluorescent staining for bacteria indicated the vancomycin-titanium retained its bactericidal activity. Finally, osteoblasts seeded on the vancomycin-titanium surface exhibited no change in viability, indicating the surface supports bone cell adhesion. Based on these observations, covalent modification of the titanium surface with an antibiotic may be viewed as a potential new tool in preventing or eliminating periprosthetic infection.