Biomaterials (v.31, #4)

Some properties of keratin biomaterials: Kerateines by Paulina Hill; Helen Brantley; Mark Van Dyke (585-593).
Keratins are a family of structural proteins that can be isolated from a variety of tissues. “Soft” keratins are cytoskeletal elements found in epithelial tissues while protective tissues such as nails, hooves, and hair are composed of “hard” keratins. Hard keratins have been the subject of biomaterials investigations for more than three decades. Numerous methods exist for denaturing these proteins which are characterized by a high sulfur content and extensive disulfide bonding, under either oxidative or reductive conditions, extracting them from tissue and processing them into various physical states such as gels, films, coatings, and fibers. Kerateines or keratoses (oxidatively or reductively derived, respectively), alone or in combination with other biomaterials, have been tested in a small number of systems to demonstrate feasibility for medical applications such as wound healing, bone regeneration, hemostasis, and peripheral nerve repair. These investigations have shown generally good compatibility with cells and tissues, but the focus of prior investigations has been fairly narrow, and as a result there is relatively little published data on the general behavior of keratin biomaterials in biological systems beyond cell culture assays. The goal of this study was to produce a reduced form of keratin biomaterial, kerateine, using a typical and well-published technique, and characterize several aspects of its behavior that may have implications to its general use as a biomaterial. Kerateines were extracted from human hair, fabricated into gels and porous scaffolds, characterized, and placed into biological systems to determine their interactions with cells and tissue. Initially, the proteins were analyzed for molecular weight and amino acid content, as well as their ability to facilitate cell adhesion and proliferation. Crosslinked hydrogels were investigated for their hydrolytic stability in vitro; the micro-architecture and in vivo tissue response of lyophilized gels was also studied. These experiments both confirmed and expanded earlier findings that kerateines demonstrate excellent compatibility in biological systems.
Keywords: Keratin; Biomaterial; Protein; Hydrogel; Scaffold; Hair;

The host response to a biomaterial is characterized by both acute recruitment and attachment of cells as well as chronic encapsulating tissue reaction. The implantation procedure induces production of damage-associated molecular patterns (DAMPs) which may contribute to host recognition of the material. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that bind not only pathogen-associated molecular patterns (PAMPs) but also DAMPs. We sought to investigate whether TLR4/DAMP interactions were involved in the acute and chronic inflammatory response to an implanted biomaterial. When PET discs were implanted intraperitoneally for 16 h, no differences were found in the number of leukocytes recruited between TLR4+ (C57BL/10J) and TLR4 (C57BL/10ScNJ) mice. However, a significant shift in the leukocyte profile on the biomaterial surface was observed for TLR4 mice. While the total number of adherent cells was the same in both strains, TLR4+ mice had a profile with equivalent neutrophil and monocyte/macrophage presence on the material surface, and TLR4 mice had a profile of predominantly neutrophils with fewer monocyte/macrophages. When implants were placed subcutaneously for 2 weeks, the fibrous capsule thicknesses were not different between TLR4+ and TLR4 mouse strains. These findings illustrate that TLR4 may play a role in the initial recognition of a biomaterial by directing the adhesive cellular profile.
Keywords: Biomaterials; Toll-like receptors; Host response; Acute inflammation;

Altered calcium dynamics in cardiac cells grown on silane-modified surfaces by Melissa S. Ravenscroft-Chang; Jayna M. Stohlman; Peter Molnar; Anupama Natarajan; Heather E. Canavan; Maggie Teliska; Maria Stancescu; Victor Krauthamer; James J. Hickman (602-607).
Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca2+ because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca2+ imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca2+ concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca2+ changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca2+ dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell–surface interactions in cardiac systems.
Keywords: Biocompatibility; Calcium; Cardiomyocyte; Cell culture; Image analysis; Surface modification;

Neo-vascularization and bone formation mediated by fetal mesenchymal stem cell tissue-engineered bone grafts in critical-size femoral defects by Zhi-Yong Zhang; Swee-Hin Teoh; Mark S.K. Chong; Eddy S.M. Lee; Lay-Geok Tan; Citra N. Mattar; Nicholas M. Fisk; Mahesh Choolani; Jerry Chan (608-620).
Tissue-engineered bone grafts (TEBG) require highly osteogenic cell sources for use in fracture repair applications. Compared to other sources of mesenchymal stem cells (MSC), human fetal MSC (hfMSC) have recently been shown to be more proliferative and osteogenic. We studied the functional performance of hfMSC-mediated TEBG in 7 mm rat femoral critical-sized bone defects (CSD). Dynamically-cultured and osteogenically-primed hfMSC seeded onto macroporous poly-ɛ-caprolactone tri-calcium phosphate scaffolds were transplanted into CSDs. After 12 weeks, hfMSC-mediated TEBG induced 2.1× more new bone formation (43.3 ± 10.5 vs. 21.0 ± 7.4 mm3, p  < 0.05), with greater compact and woven bone, and a 9.8× increase in stiffness (3.9 ± 1.7 vs. 0.4 ± 0.3 mNm/degree, p  < 0.05) compared to acellular scaffolds, such that only animals transplanted with TEBG underwent full fracture repair of the CSD. Although hfMSC survived for <4 weeks, by 4 weeks they were associated with a 3.9× larger vasculature network in the defect area (35.2 ± 11.1 vs. 6.5 ± 3.6 mm3 p  < 0.05), suggesting an important role for hfMSC in the promotion of neo-vasculogenesis. We speculate that hfMSC-mediated healing of the CSD by stimulating neo-vascularization through as yet undetermined mechanisms. This proof-of-principle study demonstrates the utility of primitive MSC for bone regeneration, and may be of relevance to vascularization in other areas of regenerative medicine.
Keywords: Fetal mesenchymal stem cells; Bone tissue engineering; Poly-caprolactone; Rat femur; Critical size defect;

Smooth muscle layer plays an important role in maintaining homeostasis of blood vessels, thus generating a functional smooth muscle layer is a prerequisite for successful construction of blood vessels via tissue-engineering approach. In this study, we investigated the feasibility of constructing an elastic vessel wall in small diameter (less than 6 mm) using smooth muscle cells (SMCs) differentiated from human adipose-derived stem cells (hASCs) under pulsatile stimulation in a bioreactor. With the induction of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-4 (BMP4) in combination for 7 days, hASCs were found to acquire an SMC phenotype characterized by the expression of SMC-related markers including smooth muscle alpha actin (α-SMA), calponin, and smooth muscle myosin heavy chain (SM-MHC). The SMCs derived from hASCs were seeded in polyglycolic acid (PGA) unwoven mesh and the cell–scaffold complex were subjected to pulsatile stimulation in a bioreactor for 8 weeks. The vessel walls engineered under the dynamic stimulation for 8 weeks showed a dense and well-organized structure similar to that of native vessels. The differentiated hASCs with dynamic loading were found to maintain their SMC phenotype within 3-dimensional PGA scaffold with a high level of collagen deposition close to that of native ones. Vessels constructed in the static condition showed a loose histological structure with less expression of contractile proteins. More importantly, the engineered vessel under pulsatile stimulation exhibited significant improvement in biomechanical properties over that generated from static conditions. Our results demonstrated that hASCs can serve as a new cell source for SMCs in blood vessel engineering, and an elastic small-diameter vessel wall could be engineered by in vitro culture of SMC-differentiated hASCs on the PGA scaffold with matchable biomechanical strength to that of normal blood vessels under pulsatile stimulation.
Keywords: Polyglycolic acid; Adipose-derived stem cells; Smooth muscle cells; Blood vessel tissue engineering;

Enhancement of chondrogenesis of human adipose derived stem cells in a hyaluronan-enriched microenvironment by Shun-Cheng Wu; Je-Ken Chang; Chih-Kuang Wang; Gwo-Jaw Wang; Mei-Ling Ho (631-640).
Microenvironment plays a critical role in guiding stem cell differentiation. We investigated the enhancing effect of a hyaluronan (HA)-enriched microenvironment on human adipose derived stem cell (hADSC) chondrogenesis for articular cartilage tissue engineering. The hADSCs were obtained from patients undergoing hip replacement. HA-coated wells and HA-modified poly-(lactic-co-glycolic acid) (HA/PLGA) scaffolds were used as the HA-enriched microenvironment. The mRNA expressions of chondrogenic (SOX-9, aggrecan and collagen type II), fibrocartilage (collagen type I), and hypertrophic (collagen type X) marker genes were quantified by real-time polymerase chain reaction. Sulfated glycosaminoglycan (sGAG) deposition was detected by Alcian blue, safranin-O staining, and dimethylmethylene blue (DMMB) assays. Localized collagen type II was detected by immunohistochemistry. The hADSCs cultured in HA-coated wells (0.005–0.5 mg/cm2) showed enhanced aggregation and mRNA expressions (SOX-9, collagen type II, and aggrecan) after 24 h, and sGAG content was also significantly increased after 9 days of culture. The HA-modified PLGA did not change the cell adherence and viability of hADSCs. The mRNA expressions of chondrogenic marker genes were significantly enhanced in hADSCs cultured in HA/PLGA rather than those cultured in the PLGA scaffold after 1, 3, and 5 days of culture. The hADSCs cultured in HA/PLGA produced higher levels of sGAG and collagen type II, compared to those in the PLGA scaffold after 4 weeks of cultures. Our results suggest that HA-enriched microenvironment induces chondrogenesis in hADSCs, which may be beneficial in articular cartilage tissue engineering.
Keywords: Human adipose derived stem cells (hADSCs); Hyaluronic acid/hyaluronan (HA)-enriched microenvironment; Poly-(lactic-co-glycolic acid) (PLGA) scaffold; Chondrogenesis; Cell aggregation; Articular cartilage tissue engineering;

Hierarchical scaffolds via combined macro- and micro-phase separation by Peter A. George; Katie Quinn; Justin J. Cooper-White (641-647).
Recent advances in biomaterial surface engineering have shown that surface biomechanical, spatial and topographical properties can elicit control over fundamental biological processes such as cell shape, proliferation, differentiation and apoptosis. Along these lines, we have very recently shown that the self-assembly of block copolymers into thin films can be used as an extremely labile method to precisely position cellular adhesion molecules, at nanometre lateral spacings, to effect control over cell attachment and morphology. Here, we extend our work in 2-dimensional block copolymer films into the production of 3-dimensional porous block copolymer scaffolds. The reported method combines macro-scale temperature induced phase separation and micro-phase separation of block copolymers to produce highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available scaffold engineering technologies. The phase behaviour of these polymer–solvent systems is described and potential mechanisms leading to the observed structure formation are presented. The nano-domains have thereafter been functionalised with CGRGDS peptides throughout the scaffold and shown to effect changes in cell attachment and spreading, in agreement with previous 2-dimensional studies. These multi-scale, functional scaffolds are easy to manufacture and scaleable, making them ideal candidates for tissue engineering applications.
Keywords: Nanotechnology; Integrin clustering; Self assembly; Surface modification; Block copolymer;

Endothelial cell recovery, acute thrombogenicity, and monocyte adhesion and activation on fluorinated copolymer and phosphorylcholine polymer stent coatings by Shawn L. Chin-Quee; Steve H. Hsu; Kim L. Nguyen-Ehrenreich; Julie T. Tai; George M. Abraham; Stephen D. Pacetti; Yen F. Chan; Gaku Nakazawa; Frank D. Kolodgie; Renu Virmani; Nadine N. Ding; Leslie A. Coleman (648-657).
This study compares the effects of two polymers currently being marketed on commercially available drug-eluting stents, PVDF-HFP fluorinated copolymer (FP) and phosphorylcholine polymer (PC), on re-endothelialization, acute thrombogenicity, and monocyte adhesion and activity. Rabbit iliac arteries were implanted with cobalt–chromium stents coated with FP or PC polymer (without drug) and assessed for endothelialization at 14 days by confocal and scanning electron microscopy (SEM). Endothelialization was equivalent and near complete for FP and PC polymer-coated stents (>80% by SEM). Acute thrombogenicity was assessed in a Chandler loop model using porcine blood. Thrombus adherence was similar for both polymers as assessed by clot weight, thrombin–antithrombin III complex, and lactate dehydrogenase expression. In vitro cell adhesion assays were performed on FP and PC polymer-coated glass coupon surfaces using HUVECs, HCAECs, and THP-1 monocytes. The number of ECs adhered to FP and control surfaces were equivalent and significantly greater than on PC surfaces (p  < 0.05). There were no differences in THP-1 monocyte adhesion and cytokine (MCP-1, RANTES, IL-6, MIP-1α, MIP-1β, G-CSF) expression. The data suggests that biological responses to both FP and PC polymer are similar, with no mechanistic indication that these polymers would be causative factors for delayed vessel healing in an acute timeframe.
Keywords: Biocompatibility; Endothelialization; Fluorinated copolymer; Phosphorylcholine; Stent; Thrombogenicity;

Lubricity and stability of poly(2-methacryloyloxyethyl phosphorylcholine) polymer layer on Co–Cr–Mo surface for hemi-arthroplasty to prevent degeneration of articular cartilage by Masayuki Kyomoto; Toru Moro; Ken-ichi Saiga; Fumiaki Miyaji; Hiroshi Kawaguchi; Yoshio Takatori; Kozo Nakamura; Kazuhiko Ishihara (658-668).
Migration of the artificial femoral head to the inside of the pelvis due to the degeneration of acetabular cartilage has emerged as a serious issue in resurfacing or bipolar hemi-arthroplasty. Surface modification of cobalt–chromium–molybdenum alloy (Co–Cr–Mo) is one of the promising means of improving lubrication for preventing the migration of the artificial femoral head. In this study, we systematically investigated the surface properties, such as lubricity, biocompatibility, and stability of the various modification layers formed on the Co–Cr–Mo with the biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer by dip coating or grafting. The cartilage/poly(MPC) (PMPC)-grafted Co–Cr–Mo interface, which mimicked a natural joint, showed an extremely low friction coefficient of <0.01, as low as that of a natural cartilage interface. Moreover, the long-term stability in water was confirmed for the PMPC-grafted layer; no hydrolysis of the siloxane bond was observed throughout soaking in phosphate-buffered saline for 12 weeks. The PMPC-grafted Co–Cr–Mo femoral head for hemi-arthroplasty is a promising option for preserving acetabular cartilage and extending the duration before total hip arthroplasty.
Keywords: Phosphorylcholine; Cobalt alloy; Hip replacement prosthesis; Surface modificaiton; Cartilage; Friction;

The use of chitosan to damage Cryptococcus neoformans biofilms by Luis R. Martinez; Mircea Radu Mihu; George Han; Susana Frases; Radames J.B. Cordero; Arturo Casadevall; Adam J. Friedman; Joel M. Friedman; Joshua D. Nosanchuk (669-679).
The use of indwelling medical devices (e.g. pacemakers, prosthetic joints, catheters, etc) continues to increase, yet these devices are all too often complicated by infections with biofilm-forming microbes with increased resistance to antimicrobial agents and host defense mechanisms. We investigated the ability of chitosan, a polymer isolated from crustacean exoskeletons, to damage biofilms formed by the pathogenic fungus Cryptococcus neoformans. Using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay and CFU determinations, we showed that chitosan significantly reduced both the metabolic activity of the biofilms and cell viability, respectively. We further demonstrated that chitosan penetrated biofilms and damaged fungal cells using confocal and scanning electron microscopy. Notably, melanization, an important virulence determinant of C. neoformans, did not protect cryptococcal biofilms against chitosan. The chitosan concentrations used in this study to evaluate fungal biofilm susceptibility were not toxic to human endothelial cells. Our results indicate that cryptococcal biofilms are susceptible to treatment with chitosan, suggesting an option for the prevention or treatment of fungal biofilms on indwelling medical devices.
Keywords: Cryptococcus neoformans; Fungi; Biofilms; Chitosan; Planktonic; Melanin;

Surfaces modified with nanometer-thick silver-impregnated polymeric films that kill bacteria but support growth of mammalian cells by Ankit Agarwal; Tahlia L. Weis; Michael J. Schurr; Nancy G. Faith; Charles J. Czuprynski; Jonathan F. McAnulty; Christopher J. Murphy; Nicholas L. Abbott (680-690).
Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (∼100 μg/cm2) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with ∼0.4 μg/cm2 to ∼23.6 μg/cm2 of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as ∼0.4 μg/cm2 of silver in the polymeric films caused a reduction of 6 log10  CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing ∼0.4 μg/cm2 of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.
Keywords: Silver nanoparticles; Wound healing; Wound-bed engineering; Nanostructured polymer films; Antibacterial activity; Cytotoxicity;

The in vivo performance of magnetic particle-loaded injectable, in situ gelling, carriers for the delivery of local hyperthermia by Pol-Edern Le Renard; Olivier Jordan; Antonin Faes; Alke Petri-Fink; Heinrich Hofmann; Daniel Rüfenacht; Frederik Bosman; Franz Buchegger; Eric Doelker (691-705).
We investigated the use of in situ implant formation that incorporates superparamagnetic iron oxide nanoparticles (SPIONs) as a form of minimally invasive treatment of cancer lesions by magnetically induced local hyperthermia. We developed injectable formulations that form gels entrapping magnetic particles into a tumor. We used SPIONs embedded in silica microparticles to favor syringeability and incorporated the highest proportion possible to allow large heating capacities. Hydrogel, single-solvent organogel and cosolvent (low-toxicity hydrophilic solvent) organogel formulations were injected into human cancer tumors xenografted in mice. The thermoreversible hydrogels (poloxamer, chitosan), which accommodated 20% w/v of the magnetic microparticles, proved to be inadequate. Alginate hydrogels, however, incorporated 10% w/v of the magnetic microparticles, and the external gelation led to strong implants localizing to the tumor periphery, whereas internal gelation failed in situ. The organogel formulations, which consisted of precipitating polymers dissolved in single organic solvents, displayed various microstructures. A 8% poly(ethylene-vinyl alcohol) in DMSO containing 40% w/v of magnetic microparticles formed the most suitable implants in terms of tumor casting and heat delivery. Importantly, it is of great clinical interest to develop cosolvent formulations with up to 20% w/v of magnetic microparticles that show reduced toxicity and centered tumor implantation.
Keywords: In situ forming implant; Intratumoral injection; Physical gels; Superparamagnetic nanoparticles; Silica composite microparticles; Magnetic induced hyperthermia;

The relationship between the nanostructure of titanium surfaces and bacterial attachment by Sabrina D. Puckett; Erik Taylor; Theresa Raimondo; Thomas J. Webster (706-713).
Infection of an orthopedic prosthesis is undesirable and causes a decrease in the success rate of an implant. Reducing the adhesion of a broad range of bacteria could be an attractive means to decrease infection and allow for subsequent appropriate tissue integration with the biomaterial surface. In this in vitro study, nanometer sized topographical features of titanium (Ti) surfaces, which have been previously shown to enhance select protein adsorption and subsequent osteoblast (bone-forming cell) functions, were investigated as a means to also reduce bacteria adhesion. This study examined the adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa on conventional Ti, nanorough Ti produced by electron beam evaporation, and nanotubular and nanotextured Ti produced by two different anodization processes. This study found that compared to conventional (nano-smooth) Ti, the nanorough Ti surfaces produced by electron beam evaporation decreased the adherence of all of the aforementioned bacteria the most. The conventional and nanorough Ti surfaces were found to have crystalline TiO2 while the nanotubular and nanotextured Ti surfaces were found to be amorphous. The surface chemistries were similar for the conventional and nanorough Ti while the anodized Ti surfaces contained fluorine. Therefore, the results of this study in vitro study demonstrated that certain nanometer sized Ti topographies may be useful for reducing bacteria adhesion while promoting bone tissue formation and, thus, should be further studied for improving the efficacy of Ti-based orthopedic implants.
Keywords: Titanium; Nanotopography; Bacteria; Adhesion; Fibronectin;

Folate receptor targeted, rare-earth oxide nanocrystals for bi-modal fluorescence and magnetic imaging of cancer cells by Sonali Setua; Deepthy Menon; Adersh Asok; Shantikumar Nair; Manzoor Koyakutty (714-729).
Targeted cancer imaging using rare-earth oxide nanocrystals, free from heavy metals (Cd, Se, Te, Hg and Pb), showing bright red-fluorescence and magnetic resonance imaging (MRI) is presented. Y2O3 nanocrystals (YO NC) doped in situ with fluorescent (Eu3+) and paramagnetic (Gd3+) impurities and conjugated with a potential cancer targeting ligand, folic acid (FA), were prepared using an all-aqueous wet-chemical process. Structural, optical and magnetic properties of these multifunctional nanocrystals were investigated by X-ray diffraction, electron microscopy, photoluminescence and magnetization studies. Highly monodisperse nanocrystals of size ∼20 nm with cubic bixbyite crystal structure showed bright red-fluorescence when doped with Eu3+. Co-doping with Gd3+ and mild air drying resulted significantly enhanced fluorescence quantum efficiency of ∼60% together with paramagnetic functionality, enabling T 1-weighted MR contrast with ∼5 times higher spin-lattice relaxivity compared to the clinically used Gd3+ contrast agent. Cytotoxicity and reactive oxygen stress studies show no toxicity by YO NC in both normal and cancer cells up to higher doses of 500 μm and longer incubation time, 48 h. Cancer targeting capability of FA conjugated NCs was demonstrated on folate receptor positive (FR+) human nasopharyngeal carcinoma cells (KB) with FR depressed KB (FRd) and FR negative (FR-) lung cancer cells A549 as controls. Fluorescence microscopy and flow-cytometry data show highly specific binding and cellular uptake of large concentration of FA conjugated NCs on FR+ve cells compared to the controls. Thus, the present study reveals, unique bi-modal contrast imaging capability, non-toxicity and cancer targeting capability of multiple impurities doped rare-earth oxide nanocrystals that can find promising application in molecular imaging.

Size-mediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells by Yuan Yuan; Changsheng Liu; Jiangchao Qian; Jing Wang; Yuan Zhang (730-740).
Hydroxyapatite nanoparticles (HAPN) have been discovered to exert cytotoxicity and apoptosis-induction in some cancer cells. But it is still not clear how tumor cells interact with HAPNs with various sizes. In this study, we investigated the effect of the particle size of the HAPN on the anti-tumor activity, apoptosis-induction and the levels of the apoptotic signaling proteins in human hepatoma HepG2 model cells. HAPNs within 20–180 nm size range were synthesized by a modified sol–gel method. The cellular internalization and biolocalization of the FITC-labeled HAPNs were also identified. The results showed that in HepG2 cells, the anti-tumor activity and HAPN-induced apoptosis strongly depended on the size of HAPNs, and the efficacies all decreased in the order of 45-nm > 26-nm > 78-nm > 175-nm. HAPNs, ranging from 20 nm to 80 nm, were found to effectively activate caspase-3 and -9, decrease the Bcl-2 protein level, and increase the levels of Bax, Bid and the release of cytochrome c from mitochondria into cytoplasm, with the best efficiency from 45-nm HAPN. Correlating the cellular response with the cellular internalization, it can be inferred that the size of HAPN and thereby the cellular localization had predominant effect on the HAPN-induced cytotoxicity, apoptotis, and the levels of the apoptotic proteins in HepG2 cells. The findings presented here could provide new means to modulate the cellular behaviors of HAPN and to guide the design of HAPN-based delivery and therapeutic systems.
Keywords: Hydroxyapatite nanoparticles; Cytotoxicity; Apoptosis-induction; Human hepatoma HepG2 cells; Size-mediated; Mitochondrial-dependent pathway;

Enhancement of incisional wound healing by thrombin conjugated iron oxide nanoparticles by Ofra Ziv-Polat; Moris Topaz; Tamar Brosh; Shlomo Margel (741-747).
Thrombin has been clinically used for topical hemostasis and wound management for more than six decades. The half-life of thrombin in human plasma is shorter than 15 s due to close control by inhibitors. In order to stabilize the thrombin, it was bound to maghemite (γ-Fe2O3) nanoparticles, as demonstrated in previous work. The aim of the present study was to examine the efficiency of the bound thrombin for wound healing applications compared to the free thrombin. For this purpose incisional wounds on rat skin were treated with a mixture of fibrinogen, CaCl2 solution and free or bound thrombin. The wounds' edges were then approximated by skin staples. The control incisional wounds were closed with staples only. In the course of 28 days of healing the highest values of skin tensile strength were observed following treatment with the bound thrombin. Significantly lower values of tensile strength were observed following treatment with the free thrombin, and the lowest values were obtained following treatment with staples only. The histological findings correlate with the mechanical strength measurements, which demonstrate the most advanced stages of healing following treatment with the bound thrombin.
Keywords: Iron oxide nanoparticles; Immobilized thrombin; Wound healing; Fibrin; Tensile strength; Haemostasis;

Signalling pathways involved in the activation of dendritic cells by layered double hydroxide nanoparticles by Ang Li; Lili Qin; Di Zhu; Rongrong Zhu; Jing Sun; Shilong Wang (748-756).
Layered double hydroxide (LDH) nanoparticles are attractive as potential drug vectors for the targeting not only of tissues, but also of intracellular organelles, and particularly the acidic endolysosomes created after cell endocytosis. The purpose of this study was to investigate the ability of LDH nanoparticles designed as vectors to activate dendritic cells (DCs), as measured by various cellular functions. The study also explored the possible signaling pathway through which the LDH nanoparticles exerted their effects on the cellular functions of DCs. First, LDH nanoparticles with different ratios of Mg(OH)2 to Al(OH)3 (1:1, 2:1 and 3:1, called R1, R2 and R3 respectively) were optimized and had a hydrodynamic diameter of 57 nm with a zeta potential of +35 mV. Then, the efficient endocytosis of the optimized LDH nanoparticles by bone marrow-derived dendritic cells (MDDCs) was monitored by fluorescence-activated cell sorting. The effect of R1, R2 and R3 on the expression of the pro- and anti-inflammatory cytokines (TNF-α, IL-6, and IL-12) and the co-stimulatory molecules (CD40, CD80, CD86, and MHC class II) in MDDCs was examined. The exposure of R1 caused a dose-dependent increase in the expression of TNF-α, IL-12, CD86 and CD40, while R2 and R3 did not up-regulate these cytokines and co-stimulatory molecules. Migration assays showed that R1 could increase the migration capacity of DCs to CCL21 and up-regulate the expression of CCR7. Furthermore, we found that R1 significantly increased the NF-κB expression in the nucleus (in a dose-dependent manner) and promoted the degradation of total IκBα levels, indicating that the NF-κB signaling pathway might involve in an R1-induced DC activation. Our results suggested that LDH nanoparticles, in the future, may function as a useful vector for ex vivo engineering to promote vaccine delivery in immune cells.
Keywords: Layered double hydroxides; Nanoparticles; Vaccine; Dendritic cell;

The purpose of this study was to develop polymeric nano-carriers of doxorubicin (DOX) that can increase the therapeutic efficacy of DOX for sensitive and resistant cancers. Towards this goal, two polymeric DOX nano-conjugates were developed, for which the design was based on the use of multi-functionalized poly(ethylene oxide)-block-poly(ɛ-caprolactone) (PEO-b-PCL) micelles decorated with αvβ3 integrin-targeting ligand (i.e. RGD4C) on the micellar surface. In the first formulation, DOX was conjugated to the degradable PEO-b-PCL core using the pH-sensitive hydrazone bonds, namely RGD4C-PEO-b-P(CL-Hyd-DOX). In the second formulation, DOX was conjugated to the core using the more stable amide bonds, namely RGD4C-PEO-b-P(CL-Ami-DOX). The pH-triggered drug release, cellular uptake, intracellular distribution, and cytotoxicity against MDA-435/LCC6WT (a DOX-sensitive cancer cell line) and MDA-435/LCC6MDR (a DOX-resistant clone expressing a high level of P-glycoprotein) were evaluated. Following earlier in vitro results, SCID mice bearing MDA-435/LCC6WT and MDA-435/LCC6MDR tumors were treated with RGD4C-PEO-b-P(CL-Hyd-DOX) and RGD4C-PEO-b-P(CL-Ami-DOX), respectively. In both formulations, surface decoration with RGD4C significantly increased the cellular uptake of DOX in MDA-435/LCC6WT and MDA-435/LCC6MDR cells. In MDA-435/LCC6WT, the best cytotoxic response was achieved using RGD4C-PEO-b-P(CL-Hyd-DOX), that correlated with the highest cellular uptake and preferential nuclear accumulation of DOX. In MDA-435/LCC6MDR, RGD4C-PEO-b-P(CL-Ami-DOX) was the most cytotoxic, and this effect correlated with the accumulation of DOX in the mitochondria. Studies using a xenograft mouse model yielded results parallel to those of the in vitro studies. Our study showed that RGD4C-decorated PEO-b-P(CL-Hyd-DOX) and PEO-b-P(CL-Ami-DOX) can effectively improve the therapeutic efficacy of DOX in human MDA-435/LCC6 sensitive and resistant cancer, respectively, pointing to the potential of these polymeric micelles as the custom-designed drug carriers for clinical cancer therapy.
Keywords: Drug targeting; Chemotherapy; Polymeric micelles; Multidrug resistance; Doxorubicin;

Gene transfer using self-assembled ternary complexes of cationic magnetic nanoparticles, plasmid DNA and cell-penetrating Tat peptide by Hai Peng Song; Jing Ye Yang; Seong Loong Lo; Yi Wang; Wei Min Fan; Xiao Sheng Tang; Jun Min Xue; Shu Wang (769-778).
Nonviral magnetofection facilitates gene transfer by using a magnetic field to concentrate magnetic nanoparticle-associated plasmid delivery vectors onto target cells. In light of the well-established effects of the Tat peptide, a cationic cell-penetrating peptide, that enhances the cytoplasmic delivery of a variety of cargos, we tested whether the combined use of magnetofection and Tat-mediated intracellular delivery would improve transfection efficiency. Through electrostatic interaction, gene transfer complexes were generated by mixing polyethylenimine-coated cationic magnetic iron beads with plasmid DNA, followed by addition of a bis(cysteinyl) histidine-rich Tat peptide. These ternary magnetofection complexes provided a 4-fold improvement in transgene expression at a dose of 1 μg of plasmid DNA per 20,000 cells over the binary complexes without the Tat peptide and transfected up to 60% of cells in vitro. The enhanced transfection efficiency was also observed in vivo in the rat spinal cord after lumbar intrathecal injection. Moreover, the injected ternary magnetofection complexes in the cerebrospinal fluid responded to a moving magnetic filed by shifting away from the injection site and mediating transgene expression in a remote region. Thus, our approach could potentially be useful for effective gene therapy treatments of localized diseases.
Keywords: Gene transfer; Peptide; Nanoparticle; Self assembly;

Long-term biostability of self-assembling protein polymers in the absence of covalent crosslinking by Rory E. Sallach; Wanxing Cui; Fanor Balderrama; Adam W. Martinez; Jing Wen; Carolyn A. Haller; Jeannette V. Taylor; Elizabeth R. Wright; Robert C. Long; Elliot L. Chaikof (779-791).
Unless chemically crosslinked, matrix proteins, such as collagen or silk, display a limited lifetime in vivo with significant degradation observed over a period of weeks. Likewise, amphiphilic peptides, lipopeptides, or glycolipids that self-assemble through hydrophobic interactions to form thin films, fiber networks, or vesicles do not demonstrate in vivo biostability beyond a few days. We report herein that a self-assembling, recombinant elastin-mimetic triblock copolymer elicited minimal inflammatory response and displayed robust in vivo stability for periods exceeding 1 year, in the absence of either chemical or ionic crosslinking. Specifically, neither a significant inflammatory response nor calcification was observed upon implantation of test materials into the peritoneal cavity or subcutaneous space of a mouse model. Moreover, serial quantitative magnetic resonance imaging, evaluation of pre- and post-explant ultrastructure by cryo-high resolution scanning electron microscopy, and an examination of implant mechanical responses revealed substantial preservation of form, material architecture, and biomechanical properties, providing convincing evidence of a non-chemically or ionically crosslinked protein polymer system that exhibits long-term stability in vivo.
Keywords: Recombinant protein polymer; Elastin-mimetic; Biocompatibility; Biostability; Magnetic resonance imaging;