Biomaterials (v.29, #12)

Influence of the degree of methacrylation on hyaluronic acid hydrogels properties by Sidi A. Bencherif; Abiraman Srinivasan; Ferenc Horkay; Jeffrey O. Hollinger; Krzysztof Matyjaszewski; Newell R. Washburn (1739-1749).
The properties of hyaluronic acid (HA) hydrogels having a broad range of methacrylation are presented. Increasing solubility of glycidyl methacrylate (GM) in a co-solvent mixture during the methacrylation of HA with GM was shown to produce photopolymerizable HAGM conjugates with various degree of methacrylation (DM) ranging from 14% up to 90%. Aqueous solutions of HAGM macromonomers were photocross-linked to yield hydrogels with nearly full vinyl group conversions after 10 min exposure under ultraviolet light (UV). Hydrogels were characterized by uniaxial compression and volumetric swelling measurements. Keeping the DM constant, the shear modulus was varied from 16 kPa up to 73 kPa by varying the macromonomer concentration. However, at a given macromonomer concentration while varying the DM, similarly the shear modulus varied from 22 kPa up to 65 kPa. Preliminary in-vitro cell culture studies showed that GRGDS modified HAGM hydrogels promoted similarly cell interaction at both low and high DMs, 32% and 60%, respectively. Densely cross-linked hydrogels with a high DM have been shown to be more mechanically robust while maintaining cytocompability and cell adhesion.
Keywords: Hyaluronic acid; Cross-linking; Hydrogels; Mechanical properties; Cell response;

Comparison of nanoscale and microscale bioactive glass on the properties of P(3HB)/Bioglass® composites by Superb K. Misra; Dirk Mohn; Tobias J. Brunner; Wendelin J. Stark; Sheryl E. Philip; Ipsita Roy; Vehid Salih; Jonathan C. Knowles; Aldo R. Boccaccini (1750-1761).
This study compares the effects of introducing micro (m-BG) and nanoscale (n-BG) bioactive glass particles on the various properties (thermal, mechanical and microstructural) of poly(3hydroxybutyrate) (P(3HB))/bioactive glass composite systems. P(3HB)/bioactive glass composite films with three different concentrations of m-BG and n-BG (10, 20 and 30 wt%, respectively) were prepared by a solvent casting technique. The addition of n-BG particles had a significant stiffening effect on the composites, modulus when compared with m-BG. However, there were no significant differences in the thermal properties of the composites due to the addition of n-BG and m-BG particles. The systematic addition of n-BG particles induced a nanostructured topography on the surface of the composites, which was not visible by SEM in m-BG composites. This surface effect induced by n-BG particles considerably improved the total protein adsorption on the n-BG composites compared to the unfilled polymer and the m-BG composites. A short term in vitro degradation (30 days) study in simulated body fluid (SBF) showed a high level of bioactivity as well as higher water absorption for the P(3HB)/n-BG composites. Furthermore, a cell proliferation study using MG-63 cells demonstrated the good biocompatibility of both types of P(3HB)/bioactive glass composite systems. The results of this investigation confirm that the addition of nanosized bioactive glass particles had a more significant effect on the mechanical and structural properties of a composite system in comparison with microparticles, as well as enhancing protein adsorption, two desirable effects for the application of the composites in tissue engineering.
Keywords: Poly(3hydroxybutyrate); MG-63 osteoblasts; Nanoparticles; Bioactive glass; Bioactive composites; Osteoblast;

Synthesis, mechanical properties, biocompatibility, and biodegradation of polyurethane networks from lysine polyisocyanates by Scott A. Guelcher; Abiraman Srinivasan; Jerald E. Dumas; Jonathan E. Didier; Sean McBride; Jeffrey O. Hollinger (1762-1775).
Bone defects, such as compressive fractures in the vertebral bodies, are frequently treated with acrylic bone cements (e.g., PMMA). Although these biomaterials have sufficient mechanical properties for fixing the fracture, they are non-degradable and do not remodel or integrate with host tissue. In an alternative approach, biodegradable polyurethane (PUR) networks have been synthesized that are designed to integrate with host tissue and degrade to non-cytotoxic decomposition products. PUR networks have been prepared by two-component reactive liquid molding of low-viscosity quasi-prepolymers derived from lysine polyisocyanates and poly(ɛ-caprolactone-co-dl-lactide-co-glycolide) triols. The composition, thermal transitions, and mechanical properties of the biomaterials were measured. The values of Young's modulus ranged from 1.20–1.43 GPa, and the compressive yield strength varied from 82 to 111 MPa, which is comparable to the strength of PMMA bone cements. In vitro, the materials underwent controlled biodegradation to non-cytotoxic decomposition products, and supported the attachment and proliferation of MC3T3 cells. When cultured in osteogenic medium on the PUR networks, MC3T3 cells deposited mineralized extracellular matrix, as evidenced by von Kossa staining and tetracycline labeling. Considering the favorable mechanical and biological properties, as well as the low-viscosity of the reactive intermediates used to prepare the PUR networks, these biomaterials are potentially useful as injectable, biodegradable bone cements for fracture healing.
Keywords: Polyurethane; Injectable; Biocompatibility; Biodegradation; Cell proliferation; Compression;

Surface energy effects on osteoblast spatial growth and mineralization by Jung Yul Lim; Michael C. Shaughnessy; Zhiyi Zhou; Hyeran Noh; Erwin A. Vogler; Henry J. Donahue (1776-1784).
While short-term surface energy effects on cell adhesion are relatively well known, little is revealed as regards its later stage effects on cell behavior. We examined surface energy effects on osteoblastic cell growth and mineralization by using human fetal osteoblastic (hFOB) cells cultured on plasma-treated quartz (contact angle, θ  = 0°) and octadecyltrichlorosilane (OTS)-treated quartz (θ  = 113°). hFOB cells formed a homogeneous cell layer on plasma-treated quartz, while those cultured on OTS-treated quartz produced randomly distributed clump-like structures that were filled with cells (confirmed by confocal microscopy). Mineral deposition by hFOB cells was spatially homogeneous when cultured on hydrophilic surfaces. Furthermore, cells on hydrophilic surfaces exhibited increased mineralized area as well as enhanced mineral-to-matrix ratio (assessed by Fourier transform infrared spectroscopy), relative to cells on hydrophobic surfaces. Experiments using other types of osteoblast-like cells (MC3T3-E1, MG63, and SAOS-2) revealed more or less similar effects in spatial growth morphology. It was concluded that hydrophilic surfaces induce homogeneous spatial osteoblastic cell growth and mineral deposition and enhance the quantity (e.g., area) and quality (e.g., mineral-to-matrix ratio) of mineralization relative to hydrophobic surfaces. Our data suggest that surface energy effects on osteoblastic cell differentiation, especially mineralization, may be correlated with surface energy dependent changes in spatial cell growth.
Keywords: Surface energy; Osteoblast; Hydrophilic; Hydrophobic; Mineralization;

Attenuation of protease activity in chronic wound fluid with bisphosphonate-functionalised hydrogels by Erin A. Rayment; Tim R. Dargaville; Gary K. Shooter; Graeme A. George; Zee Upton (1785-1795).
Chronic ulcers are an important and costly medical issue, imposing considerable pain, reduced mobility and decreased quality of life. The common pathology in these chronic wounds is excessive proteolytic activity, resulting in degradation of key factors critical to the ulcer's ability to heal. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), with many authors suggesting that they need to be inhibited for the ulcer to heal. The studies we report here show that the excessive MMP activity in CWF can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and tethered to a poly(2-hydroxy methacrylate) (PHEMA) hydrogel. Furthermore, these functionalised alendronate hydrogels appear to be biologically inert as assessed in a three-dimensional ex vivo human skin equivalent model. Together, these results highlight the potential use of a tethered MMP inhibitor to inhibit protease activity in wound fluid. This approach may improve wound healing as it still allows MMPs to remain active in the upper cellular layers of the ulcer bed where they perform vital roles in wound healing; thus may offer an attractive new device-orientated wound therapy.
Keywords: Wound healing; Wound dressing; Matrix metalloproteinase; PHEMA; Hydrogel; Biocompatibility;

Proliferation of aligned mammalian cells on laser-nanostructured polystyrene by Esther Rebollar; Irene Frischauf; Michael Olbrich; Thomas Peterbauer; Steffen Hering; Johannes Preiner; Peter Hinterdorfer; Christoph Romanin; Johannes Heitz (1796-1806).
Biomaterial surface chemistry and nanoscale topography are important for many potential applications in medicine and biotechnology as they strongly influence cell function, adhesion and proliferation. In this work, we present periodic surface structures generated by linearly polarized KrF laser light (248 nm) on polystyrene (PS) foils. These structures have a periodicity of 200–430 nm and a depth of 30–100 nm, depending on the angle of incidence of the laser beam. The changes in surface topography and chemistry were analysed by atomic force microscopy (AFM), advancing water contact-angle measurements, Fourier-transform infrared spectroscopy using an attenuated total reflection device (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). We show that the surface laser modification results in a significantly enhanced adhesion and proliferation of human embryonic kidney cells (HEK-293) compared to the unmodified polymer foil. Furthermore, we report on the alignment of HEK-293 cells, Chinese hamster ovary (CHO-K1) cells and skeletal myoblasts along the direction of the structures. The results indicate that the presence of nanostructures on the substrates can guide cell alignment along definite directions, and more importantly, in our opinion, that this alignment is only observed when the periodicity is above a critical periodicity value that is cell-type specific.
Keywords: Cell proliferation; Cell morphology; Laser manufacturing; Nanotopography; Polystyrene;

In order to enhance the biocompatibility and cell affinity of metal nanoparticles for biosensing and drug delivering applications, we prepared the phospholipid derivatives containing disulfide groups to modify silver nanoparticle surfaces. By adding sodium borohydride to reduce both disulfide bonds of the derivatives and silver ions simultaneously, the generated thiol groups can be reacted with newborn silver atoms immediately to generate nanoclusters. The assemblies consisted of either phosphorylcholine (PC) or phosphorylethanolamine (PE) head groups, which made the silver clusters biocompatibile. Transmission electron microscope (TEM) and optical absorption spectra assisted in modulating reaction conditions, demonstrating that a surfactant/Ag ratio of 0.4 led to the formation of uniform, well-dispersed spherical particles about 3.8 nm in diameter. X-ray photoelectron spectra and infrared spectra also illustrated the elemental and molecular structures of nanoparticles. The insertion of rhodamine dye into the surfactant layer enabled the nanoparticles to be used as a fluorescent probe. In cell culture tests, the nanoparticles were internalized into platelet or fibroblast cells in a short period of incubation without harming the cells.
Keywords: Silver nanoparticle; Phosphorylcholine; Disulfide; Uptake; Rhodamine;

Polylactide/polyglycolide copolymer in bone defect healing in humans by Carlo Bertoldi; Davide Zaffe; Ugo Consolo (1817-1823).
This pilot study aims to evaluate the healing of a large defects in the human jawbone filled with a Poly-Lactide-co-Glycolide (PLG) polymer (Fisiograft®) by means of clinical, radiological and histological methods and to compare the results with those of platelet-rich plasma (PRP) clot or autologous bone (AB) fillings. Bone cysts, where previous non-surgical treatments failed to promote healing, underwent surgery. Nineteen consenting male patients were randomly split into three groups, packed with PRP, AB or PLG. A core biopsy was performed 4 and 6 months after surgery. All treated defects showed clinical, radiological and histological progresses over time. AB provided the best clinical and histological performance and PLG had overlapping outcomes; PRP filling was statistically different. Six months after surgery, bone activities were enhanced in sites treated with PLG and fairly good with PRP. Additionally, PLG showed some new lamellar formations. In conclusion, outcomes were best with AB graft, but suitable results were achieved using PLG to promote healing of severe bone defects. PLG shows only a delayed regenerative capability but does not require a secondary donor site.
Keywords: Autologous bone; Bone regeneration; Copolymer; In vivo test; Polyglycolic acid; Polylactic acid;

IgG deposition and activation of the classical complement pathway involvement in the activation of human granulocytes by decellularized porcine heart valve tissue by Fabienne Bastian; Marie-Elisabeth Stelzmüller; Klaus Kratochwill; Marie-Theres Kasimir; Paul Simon; Guenter Weigel (1824-1832).
Decellularization treatment of heart valves has been thought to eliminate tissue immunogenicity. Early failure of tissue-engineered xenogeneic heart valves was seen in children and has been a major drawback in this promising field of research. This study was designed to characterize the effects of acellular porcine heart valve tissue on immune activation in vitro. Incubation of decellularized porcine tissue with human plasma led to adsorption of IgG, activation of the classical complement pathway and adhesion of activated polymorphonuclear leukocytes (PMN). This inflammatory response was strongly inhibited by proteins extracted from native porcine tissue which might indicate that inhibitors of PMN activation present in the extracellular matrix (ECM) are lost during the decellularization process.
Keywords: Complement; Extracellular matrix; Inflammation; Leukocytes; Heart valve;

The role of electrosprayed apatite nanocrystals in guiding osteoblast behaviour by Eng San Thian; Zeeshan Ahmad; Jie Huang; Mohan J. Edirisinghe; Suwan N. Jayasinghe; Deborah C. Ireland; Roger A. Brooks; Neil Rushton; William Bonfield; Serena M. Best (1833-1843).
Apatite nanocrystals, which mimic the dimensions of natural bone mineral, were electrosprayed on glass substrates, as a suitable synthetic biomedical material for osteoblast outgrowth was explored. A variety of topographic patterns were deposited and the influence of these designs on osteoblast alignment and cell differentiation was investigated. Patterned cell growth and enhanced cell differentiation were seen. Osteoblasts were also cultured on apatite nanocrystals chemically modified with either carbonate or silicon ions. Enhanced cell proliferation and early formation of mineral nodules were observed on apatite nanocrystals with silicon addition. This work highlights the importance of the combined effects of surface topography and surface chemistry in the guidance of cell behaviour.
Keywords: Apatite; Carbonate; Electrospraying; Nanocrystals; Osteoblasts; Silicon;

MRI characterization of agarose gel micro-droplets at acute time-points within the rabbit lumbar muscle by Warren D. Foltz; Mark L. Ormiston; Duncan J. Stewart; David W. Courtman; Alexander J. Dick (1844-1852).
Agarose gel micro-droplets supplemented with provisional matrix proteins have been shown to enhance encapsulated cell survival for cell therapy applications. This study evaluated micro-droplet T 1 and T 2 relaxation on a 1.5 T clinical MRI scanner to guide the optimization of encapsulated cell delivery to intermediate-sized animals. Preliminary in vitro experiments using encapsulated human blood-derived endothelial progenitor cells (EPCs) documented a negligible impact of EPC encapsulation on agarose micro-droplet T 1 and T 2 relaxation, even following transient immersion in 2.3 mm Gd-DTPA. Furthermore, Gd-DTPA immersion did not adversely impact encapsulated cell viability. These results allowed for efficient pre-clinical methodological development using direct injections into the rabbit lumbar region of agarose droplets without cells (n  = 6). At time-points to 6 h, in vivo injection sites displayed elevated T 2 and T 1 (1.8%: ΔT 2  = 53 ± 28%, ΔT 1  = 50 ± 25%, n  = 13; 2.5%: ΔT 2  = 41 ± 10%, ΔT 1  = 41 ± 26%, n  = 11). Rapid imaging sequences displayed high conspicuity at sites of Gd-DTPA-immersed capsule injection, which persisted for less than 4 h. Therefore, basic differences of micro-droplet T 1 and T 2 when compared to tissue provide a platform for acute tracking of encapsulated cell fate. Transient Gd-DTPA encapsulation accentuates T 1 differences.
Keywords: MRI; Cell encapsulation; Microcapsule; Cell viability; Progenitor cell;

Partitioning microfluidic channels with hydrogel to construct tunable 3-D cellular microenvironments by Amy P. Wong; Raquel Perez-Castillejos; J. Christopher Love; George M. Whitesides (1853-1861).
Accurate modeling of the cellular microenvironment is important for improving studies of cell biology in vitro. Here, we demonstrate a flexible method for creating a cellular microenvironment in vitro that allows (i) controlled spatial distribution (patterning) of multiple types of cells within three-dimensional (3-D) matrices of a biologically derived, thermally curable hydrogel (Matrigel) and (ii) application of gradients of soluble factors, such as cytokines, across the hydrogel. The technique uses laminar flow to divide a microchannel into multiple subchannels separated by microslabs of hydrogel. It does not require the use of UV light or photoinitiators and is compatible with cell culture in the hydrogel. This technique makes it possible to design model systems to study cellular communication mediated by the diffusion of soluble factors within 3-D matrices. Such factors can originate either from secretions of neighboring cells patterned within the microchannel, or from an external source – e.g., a solution of growth factors injected into a subchannel. This method is particularly useful for studying cells such as those of the immune system, which are often weakly adherent and difficult to position precisely with standard systems for cell culture. We demonstrated this application by co-culturing two types of macrophage-like cells (BAC1.2F5 and LADMAC cell lines) within spatially separated regions of a slab of hydrogel. This pair of cell lines represents a simple model system for intercellular communication: the LADMAC cells produce colony-stimulating factor 1 (CSF-1), which is required by the BAC cells for survival.
Keywords: Cellular signaling; Co-culture; Hydrogel; Microenvironment modeling; Micropatterning; Macrophage;

Proliferation and differentiation of adipose-derived stem cells on naturally derived scaffolds by Lauren E. Flynn; Glenn D. Prestwich; John L. Semple; Kimberly A. Woodhouse (1862-1871).
A tissue-engineered substitute that facilitates large-volume regeneration of the subcutaneous adipose tissue layer is needed for reconstructive plastic surgery. Towards this goal, we describe the in vitro culture of primary human adipose-derived stem cells (ASC) seeded into placental decellular matrix (PDM) and cross-linked hyaluronan (XLHA) scaffolds. Specifically, we evaluated cellular proliferation and adipogenic differentiation in the PDM, XLHA, and PDM combined with XLHA scaffolds. Cellular proliferation, viability, and glucose consumption were determined prior to the induction of differentiation. Adipogenesis within each of the scaffolds was investigated through gene expression analysis using end point and real time reverse transcriptase polymerase chain reaction (RT-PCR). The results indicate that the cell-adhesive PDM scaffolds facilitated proliferation and viability, while differentiation was augmented when the cells were encapsulated in the non-adhesive XLHA gels.
Keywords: Adipose tissue engineering; Scaffold; Extracellular matrix; Hyaluronan; Stem cells;

The effect of gelatin incorporation into electrospun poly(l-lactide-co-ɛ-caprolactone) fibers on mechanical properties and cytocompatibility by Jongman Lee; Giyoong Tae; Young Ha Kim; In Su Park; Sang-Heon Kim; Soo Hyun Kim (1872-1879).
Very elastic poly(l-lactide-co-ɛ-caprolactone) (PLCL) (50:50) copolymer blended with gelatin was electrospun into microfibers from a hexafluoroisopropanol solution. PLCL fiber sheet exhibited the unique soft and flexible behavior while gelatin fiber was hard and brittle. As the gelatin content of PLCL/gelatin fibers increased, Young's modulus was increased, but the elongation was decreased compared to those of PLCL. However, fibers containing 10–30 wt% gelatin demonstrated an enhanced tensile strength with still high elongation to be beneficial for tissue engineering scaffolds. The cytocompatibility of electrospun fiber sheets was evaluated by fibroblasts (NIH-3T3) cell culture. The initial cell adhesion on various fibers after 5 h was somewhat similar, but in the order of PLCL > PLCL70/gelatin30 ≈ PLCL50/gelatin50 > PLCL90/gelatin10 ≈ gelatin > PLCL30/gelatin70. However, the cell proliferation exhibited a completely different and strong dependence on the fiber composition: a very high proliferation rate on PLCL90/gelatin10, followed by PLCL > gelatin > PLCL70/gelatin30. Such an enhanced effect of gelatin, especially at 10 wt% content, on strength and cytocompatibility of PLCL/gelatin fibers would be very preferable for tissue engineering scaffolds.
Keywords: PLCL-gelatin fibers; Cytocompatibility; Electrospinning; Fibroblasts;

The effect of modified polysialic acid based hydrogels on the adhesion and viability of primary neurons and glial cells by Yohannes Haile; Silke Berski; Gerald Dräger; Andrè Nobre; Katharina Stummeyer; Rita Gerardy-Schahn; Claudia Grothe (1880-1891).
In this study we present the enzymatic and biological analysis of polysialic acid (polySia) based hydrogel in terms of its degradation and cytocompatibility. PolySia based hydrogel is completely degradable by endosialidase enzyme which may avoid second surgery after tissue recovery. Viability assay showed that soluble components of polySia hydrogel did not cause any toxic effect on cultured Schwann cells. Moreover, green fluorescence protein transfected neonatal and adult Schwann cells, neural stem cells and dorsal root ganglionic cells (unlabelled) were seeded on polySia hydrogel modified with poly-l-lysine (Pll), poly-l-ornithine–laminin (porn–laminin) or collagen. Water soluble tetrazolium salt assay revealed that modification of the hydrogel significantly improved cell adhesion and viability. These results infer that polySia based scaffolds in combination with cell adhesion molecules and cells genetically modified to express growth factors would potentially be promising alternative in reconstructive therapeutic strategies.
Keywords: Polysialic acid hydrogel; Schwann cells; Dorsal root ganglia cells; Neural stem cells; Surface modification;

The effect of mesenchymal populations and vascular endothelial growth factor delivered from biodegradable polymer scaffolds on bone formation by Janos M. Kanczler; Patrick J. Ginty; John J.A. Barry; Nicholas M.P. Clarke; Steve M. Howdle; Kevin M. Shakesheff; Richard O.C. Oreffo (1892-1900).
The capacity to deliver, temporally, bioactive growth factors in combination with appropriate progenitor and stem cells to sites of tissue regeneration promoting angiogenesis and osteogenesis offers therapeutic opportunities in regenerative medicine. We have examined the bone regenerative potential of encapsulated vascular endothelial growth factor (VEGF165) biodegradable poly(dl-lactic acid) (PLA) scaffolds created using supercritical CO2 fluid technology to encapsulate and release solvent-sensitive and thermolabile growth factors in combination with human bone marrow stromal cells (HBMSC) implanted in a mouse femur segmental defect (5 mm) for 4 weeks. HBMSC seeded on VEGF encapsulated PLA scaffolds showed significant bone regeneration in the femur segmental defect compared to the scaffold alone and scaffold seeded with HBMSC as analysed by indices of increased bone volume (BV mm3), trabecular number (Tb.N/mm) and reduced trabecular separation (Tb.Sp. mm) in the defect region using micro-computed tomography. Histological examination confirmed significant new bone matrix in the HBMSC seeded VEGF encapsulated scaffold group as evidenced by Sirius red/alcian blue and Goldner's trichrome staining and type I collagen immunocytochemistry expression in comparison to the other groups. These studies demonstrate the ability to deliver, temporally, a combination of VEGF released from scaffolds with seeded HBMSC to sites of bone defects, results in enhanced regeneration of a bone defect.
Keywords: Bone tissue regeneration; Supercritical fluids; PLA scaffolds; VEGF; Segmental femur defect; Human bone marrow stromal cells;

X-ray imaging optimization of 3D tissue engineering scaffolds via combinatorial fabrication methods by Yanyin Yang; Shauna M. Dorsey; Matthew L. Becker; Sheng Lin-Gibson; Gary E. Schumacher; Glenn M. Flaim; Joachim Kohn; Carl G. Simon (1901-1911).
We have developed a combinatorial method for determining optimum tissue scaffold composition for several X-ray imaging techniques. X-ray radiography and X-ray microcomputed tomography enable non-invasive imaging of implants in vivo and in vitro. However, highly porous polymeric scaffolds do not always possess sufficient X-ray contrast and are therefore difficult to image with X-ray-based techniques. Incorporation of high radiocontrast atoms, such as iodine, into the polymer structure improves X-ray radiopacity but also affects physicochemical properties and material performance. Thus, we have developed a combinatorial library approach to efficiently determine the minimum amount of contrast agent necessary for X-ray-based imaging. The combinatorial approach is demonstrated in a polymer blend scaffold system where X-ray imaging of poly(desaminotyrosyl-tyrosine ethyl ester carbonate) (pDTEc) scaffolds is improved through a controlled composition variation with an iodinated-pDTEc analog (pI2DTEc). The results show that pDTEc scaffolds must include at least 9%, 16%, 38% or 46% pI2DTEc (by mass) to enable effective imaging by microradiography, dental radiography, dental radiography through 0.75 cm of muscle tissue or microcomputed tomography, respectively. Only two scaffold libraries were required to determine these minimum pI2DTEc percentages required for X-ray imaging, which demonstrates the efficiency of this new combinatorial approach for optimizing scaffold formulations.
Keywords: Combinatorial library; Polycarbonate; Scaffolds; Radiopacity; X-ray microcomputed tomography; X-ray radiography;

Particle size-dependent organ distribution of gold nanoparticles after intravenous administration by Wim H. De Jong; Werner I. Hagens; Petra Krystek; Marina C. Burger; Adriënne J.A.M. Sips; Robert E. Geertsma (1912-1919).
A kinetic study was performed to determine the influence of particle size on the in vivo tissue distribution of spherical-shaped gold nanoparticles in the rat. Gold nanoparticles were chosen as model substances as they are used in several medical applications. In addition, the detection of the presence of gold is feasible with no background levels in the body in the normal situation. Rats were intravenously injected in the tail vein with gold nanoparticles with a diameter of 10, 50, 100 and 250 nm, respectively. After 24 h, the rats were sacrificed and blood and various organs were collected for gold determination. The presence of gold was measured quantitatively with inductively coupled plasma mass spectrometry (ICP-MS).For all gold nanoparticle sizes the majority of the gold was demonstrated to be present in liver and spleen. A clear difference was observed between the distribution of the 10 nm particles and the larger particles. The 10 nm particles were present in various organ systems including blood, liver, spleen, kidney, testis, thymus, heart, lung and brain, whereas the larger particles were only detected in blood, liver and spleen. The results demonstrate that tissue distribution of gold nanoparticles is size-dependent with the smallest 10 nm nanoparticles showing the most widespread organ distribution.
Keywords: Nanoparticles; Biodistribution; Biocompatibility; Toxicokinetics;

Self-assembled glycol chitosan nanoparticles for the sustained and prolonged delivery of antiangiogenic small peptide drugs in cancer therapy by Jong-Ho Kim; Yoo-Shin Kim; Kyeongsoon Park; Eunah Kang; Seulki Lee; Hae Yun Nam; Kwangmeyung Kim; Jae Hyung Park; Dae Yoon Chi; Rang-Woon Park; In-San Kim; Kuiwon Choi; Ick Chan Kwon (1920-1930).
Antiangiogenic peptide drugs have received much attention in the fields of tumor therapy and tumor imaging because they show promise in the targeting of integrins such as αvβ3 on angiogenic endothelial cells. However, systemic antiangiogenic peptide drugs have short half-lives in vivo, resulting in fast serum clearance via the kidney, and thus the therapeutic effects of such drugs remain modest. In this study, we prepared self-assembled glycol chitosan nanoparticles and explored whether this construct might function as a prolonged and sustained drug delivery system for RGD peptide, used as an antiangiogenic model drug in cancer therapy. Glycol chitosan hydrophobically modified with 5β-cholanic acid (HGC) formed nanoparticles with a diameter of 230 nm, and RGD peptide was easily encapsulated into HGC nanoparticles (yielding RGD-HGC nanoparticles) with a high loading efficiency (>85%). In vitro work demonstrated that RGD-HGC showed prolonged and sustained release of RGD, lasting for 1 week. RGD-HGC also inhibited HUVEC adhesion to a βig-h3 protein-coated surface, indicating an antiangiogenic effect of the RGD peptide in the HGC nanoparticles. In an in vivo study, the antiangiogenic peptide drug formulation of RGD-HGC markedly inhibited bFGF-induced angiogenesis and decreased hemoglobin content in Matrigel plugs. Intratumoral administration of RGD-HGC significantly decreased tumor growth and microvessel density compared to native RGD peptide injected either intravenously or intratumorally, because the RGD-HGC formulation strongly enhanced the antiangiogenic and antitumoral efficacy of RGD peptide by affording prolonged and sustained RGD peptide delivery locally and regionally in solid tumors.
Keywords: Glycol chitosan nanoparticles; Drug delivery system; Antiangiogenic peptide drugs; Cancer therapy;

The potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization by Hu-Lin Jiang; Mi Lan Kang; Ji-Shan Quan; Sang Gyun Kang; Toshihiro Akaike; Han Sang Yoo; Chong-Su Cho (1931-1939).
A vaccine delivery system based on mannosylated chitosan microspheres (MCMs) was studied in vitro and in vivo. Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) were loaded in MCMs or chitosan microspheres (CMs). Fluorescence confocal microscopy indicated that BBD-loaded MCMs (BBD–MCMs) bound with mannose receptors on murine macrophages (RAW264.7 cells). In vitro experiments using macrophages demonstrated that BBD–MCMs had more effective immune-stimulating activity than BBD-loaded CMs (BBD–CMs). Mice intranasally immunized with BBD–MCMs showed significantly higher BBD-specific IgA antibody responses in saliva and serum than mice immunized with BBD–CMs (p  < 0.05). After challenge with B. bronchiseptica via the nasal cavity, groups treated with BBD–MCMs or BBD–CMs showed similar patterns with a high survival rate even though there was no significant difference between those groups. These results suggested that mannose moieties in the MCMs enhanced immune-stimulating activities through mucosal delivery due to a specific interaction between mannose groups in the MCMs and mannose receptors on the macrophages.
Keywords: Mannosylated chitosan microspheres; Vaccine delivery system; Nasal vaccination; Macrophage mannose receptors;

The effect of local simvastatin delivery strategies on mandibular bone formation in vivo by Yeonju Lee; Marian J. Schmid; David B. Marx; Mark W. Beatty; Diane M. Cullen; Melissa E. Collins; Richard A. Reinhardt (1940-1949).
Systemic simvastatin is known to reduce cholesterol and stimulate modest bone formation, but local surgical placement in polylactic acid domes causes robust bone formation and local swelling. A less invasive and more flexible injection protocol was studied to evaluate the bone-inducing effects compared to surgical implantation. Bone formation rate, short- and long-term bone augmentation histology, and mechanical properties were evaluated to characterize the new bone in a rat bilateral mandible model (test and control sides in same animal). Results demonstrated that multiple (3) injections of 0.5 mg simvastatin effectively reduced soft tissue swelling while preserving bone growth (60% increase of bone width at 24 days) compared to simvastatin dome placement (43% increase at 24 days). Compared to controls, bone formation rate was significantly higher on the simvastatin side, especially in the dome. Three-point bending tests revealed higher maximum force to fracture and stiffness at 24 days with simvastatin injections. Long-term evaluation showed that 55% of maximum new bone formed 24 days post-injection was retained at 90 days.
Keywords: Simvastatin; Local delivery; Bone formation; In vivo; Long-term; Mechanical tests;

pH-Sensitive poly(N,N-dimethylaminoethyl methacrylate (DMAEMA)/2-hydroxyethyl methacrylate (HEMA)) nanoparticles were prepared for the triggered release of paclitaxel within a tumor microenvironment. Tumors exhibit a lower extracellular pH than normal tissues. We show that paclitaxel release from DMAEMA/HEMA particles can be actively triggered by small, physiological changes in pH (within 0.2–0.6 pH units). Monodispersed nanoparticles were synthesized by forming an O/W emulsion followed by photopolymerization. Particles were characterized by transmission electron microscopy, dynamic light scattering, electrophoresis, and cytotoxicity. High release rates and swelling ratios are achieved at low pH, low crosslinking density, and high content of DMAEMA. Paclitaxel release is limited to 9% of the payload at pH 7.4 after a 2-h incubation at 37 °C. After adjusting to pH 6.8, 25% of the payload is released within 2 h. Cell viability studies indicate that pH-sensitive DMAEMA/HEMA nanoparticles are not cytotoxic and may be used as an efficient, feedback-regulated drug delivery carrier.
Keywords: pH-Sensitive nanoparticle; DMAEMA; HEMA; Paclitaxel; Controlled release; Cancer therapeutic;

Functionalization of polysulfide nanoparticles and their performance as circulating carriers by Annemie Rehor; Hugo Schmoekel; Nicola Tirelli; Jeffrey A. Hubbell (1958-1966).
We here present an evaluation of the carrier performance of nanoparticles that are biofunctional, i.e. derivatized to provide a controlled biological activity, and environmentally responsive, since they respond to the presence of oxidants. In particular, we focus on the possibilities (a) to make the nanoparticles detectable and (b) to control their uptake in phagocytic cells, which determines their lifetime in vivo. We first describe techniques for labeling selectively the nanoparticle surface or bulk with imaging moieties (fluorophores or gold). We then show how surface composition and size, which are both controlled through the use of PEG derivatives, influence uptake by macrophages in vitro and blood circulation in vivo: for example, in vitro uptake is negligible for small (40 nm) particles but not for larger (100 nm) ones and, correspondingly, in vivo blood circulation half-life time decreases from 6.0 to 2.9 h. However, upon decoration with RGD peptides also the small particles can be significantly internalized.
Keywords: Nanoparticles; Biofunctionalization; Labeling; Phagocytosis; Macrophages;

Spatial distribution and acute anti-inflammatory effects of Methylprednisolone after sustained local delivery to the contused spinal cord by Stacie A. Chvatal; Young-Tae Kim; Andres M. Bratt-Leal; Hyunjung Lee; Ravi V. Bellamkonda (1967-1975).
Methylprednisolone (MP) has been shown to reduce acute inflammation resulting from a secondary damage cascade initiated by the primary physical injury to the spinal cord. The current clinical practice for delivering systemic MP is inefficient, and high doses are required, resulting in adverse, undesired, dose-related side effects in patients. Here, we report a novel, minimally invasive, localized drug delivery system for delivering MP to the contused adult rat spinal cord that potentially side-steps the deleterious consequences of systemic cortico-steroid therapy. MP was encapsulated in biodegradable PLGA based nanoparticles (NP), and these nanoparticles were embedded in an agarose hydrogel for localization to the site of contusion injury. To visualize and quantify its spatial distribution within the injured spinal cord, MP was conjugated to Texas-red cadaverine prior to encapsulation in nanoparticles. When delivered via the hydrogel-nanoparticle system, MP entered the injured spinal cord and diffused up to 1.5 mm deep and up to 3 mm laterally into the injured spinal cord within 2 days. Furthermore, topically delivered MP significantly decreased early inflammation inside the contusion injured spinal cord as evidenced by a significant reduction in the number of ED-1+ macrophages/activated microglia. This decreased early inflammation was accompanied by a significantly diminished expression of pro-inflammatory proteins including Calpain and iNOS. Additionally, topically delivered MP significantly reduced lesion volume 7 days after contusion injury. The minimally invasive MP delivery system reported in this study has the potential to enhance the effectiveness of MP therapy after contusion injury to the spinal cord and avoid the side effects arising from high dose cortico-steroid therapy.
Keywords: Drug delivery; Nanoparticle; Hydrogel;

Dynamic imaging of arginine-rich heart-targeted vehicles in a mouse model by Hua Zhang; Jiro Kusunose; Azadeh Kheirolomoom; Jai W. Seo; Jinyi Qi; Katherine D. Watson; Heather A. Lindfors; Erkki Ruoslahti; Julie L. Sutcliffe; Katherine W. Ferrara (1976-1988).
Efficacious delivery of drugs and genes to the heart is an important goal. Here, a radiolabeled peptide-targeted liposome was engineered to bind to the heart, and the biodistribution and pharmacokinetics were determined by dynamic positron emission tomography in the FVB mouse. Efficient targeting occurred only with an exposed ligand and a dense concentration of peptide (6000 peptides/particles). Liposomes targeted with CRPPR or other arginine-rich peptides with an exposed guanidine moiety bound within 100 s after intravenous injection and remained stably bound. With CRPPR-targeted particles, the radioisotope density in the heart averaged 44 ± 9% injected dose/gram of tissue, more than 30-fold higher than in skeletal muscle. The rapid and efficient targeting of these particles can be exploited in drug and gene delivery systems and with dynamic positron emission tomography provides a model system to optimize targeting of engineered particles.
Keywords: Endothelium; Heart; Liposome; Peptides; Positron emission tomography (PET); Molecular imaging;