Biomaterials (v.30, #9)
Inhibition of cytokine and surface antigen expression in LPS-stimulated murine macrophages by triethylene glycol dimethacrylate
by Alexander Eckhardt; Tolga Harorli; Jirakant Limtanyakul; Karl-Anton Hiller; Claudia Bosl; Carola Bolay; Franz-Xaver Reichl; Gottfried Schmalz; Helmut Schweikl (pp. 1665-1674).
Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) cause a shift in the cellular redox balance which influences redox-sensitive signaling pathways. The immediate response of the innate immune system to inflammatory challenges is controlled by related pathways. Therefore, the influence of TEGDMA on the expression of the pro- and anti-inflammatory cytokines TNF-α, IL-6, and IL-10 and surface antigens (CD14, CD40, CD80, CD86, CD54, MHC class I and II) was analyzed in RAW264.7 macrophages. No significant change in cytokine production or surface antigen expression was detected after the macrophages were treated with increasing TEGDMA concentrations for 6, 24, and 48h. However, co-stimulation with the bacterial endotoxin lipopolysaccharide (LPS) and TEGDMA resulted in a concentration-dependent inhibition of LPS-induced release of TNF-α, IL-6, and IL-10 by about 90% as detected by ELISA. Flow-cytometric analyses indicated an LPS-stimulated expression of all surface antigens. The LPS-induced expression of CD14 was inhibited by high TEGDMA concentrations. CD40 and CD80 expressions were down-regulated by TEGDMA in LPS-stimulated cells, and CD86 as well as MHC class I expression was inhibited to a lesser extent. The LPS-stimulated expression of CD54 (ICAM-1) was increased about twofold by increasing TEGDMA concentrations after a 24 and 48h exposure. Thus, the ability of macrophages to induce an appropriate immune response is inhibited by TEGDMA which reduces cytokine production and expression of surface antigens.
Keywords: Dental resin; TEGDMA; Lipopolysaccharide; Cytokine; Surface antigen
Covalent immobilisation of tropoelastin on a plasma deposited interface for enhancement of endothelialisation on metal surfaces
by Yongbai Yin; Steven G. Wise; Neil J. Nosworthy; Anna Waterhouse; Daniel V. Bax; Hani Youssef; Michael J. Byrom; Marcela M.M. Bilek; David R. McKenzie; Anthony S. Weiss; Martin K.C. Ng (pp. 1675-1681).
Currently available endovascular metallic implants such as stents exhibit suboptimal biocompatibility in that they re-endothelialise poorly leaving them susceptible to thrombosis. To improve the interaction of these implants with endothelial cells we developed a surface coating technology, enabling the covalent attachment of biomolecules to previously inert metal surfaces. Using horseradish peroxidase as a probe, we demonstrate that the polymerised surface can retain the presentation and activity of an immobilised protein. We further demonstrated the attachment of tropoelastin, an extracellular matrix protein critical to the correct arrangement and function of vasculature. Not only it is structurally important, but it plays a major role in supporting endothelial cell growth, while modulating smooth muscle cell infiltration. Tropoelastin was shown to bind to the surface in a covalent monolayer, supplemented with additional physisorbed multilayers on extended incubation. The physisorbed tropoelastin layers can be washed away in buffer or SDS while the first layer of tropoelastin remains tightly bound. The plasma coated stainless steel surface with immobilised tropoelastin was subsequently found to have improved biocompatibility by promoting endothelial cell attachment and proliferation relative to uncoated stainless steel controls. Tropoelastin coatings applied to otherwise inert substrates using this technology could thus have broad applications to a range of non-polymeric vascular devices.
Keywords: Stainless steel; Plasma polymerisation; Endothelialisation; Elastin
Reconstruction of calvarial defect using a tricalcium phosphate-oligomeric proanthocyanidins cross-linked gelatin composite
by Kuo-Yu Chen; Pei-Chi Shyu; Guo-Chung Dong; Yueh-Sheng Chen; Wei-Wen Kuo; Chun-Hsu Yao (pp. 1682-1688).
A biodegradable GTP composite which was composed of oligomeric proanthocyanidins (OPCs) cross-linked gelatin mixed with tricalcium phosphate was developed as a bone substitute. The subcutaneous implantation in rats was examined to determine the in vivo degradation and biocompatibility of the GTP composites with various cross-linking densities. Experimental results indicated that the rate of in vivo degradation was markedly attenuated as the concentration of OPCs increased above 5.0wt%. Furthermore, this study examined the biological response of rabbit calvarial bone to GTP composite to evaluate its potential for use as an osteoconductive bone substitute. Bone defects (10mm in diameter) in New Zealand white rabbits were filled with the GTP composite. The de-protenized bovine cancellous bone matrix was employed as the control material. The results of radiographic analyses demonstrated obviously greater new bone ingrowth in the GTP composite than in the de-proteinized bovine bone at the same implantation time. Progressive replacement of the GTP composite by new bone proceeded by a combination of osteoconduction and biodegradation. The biodegradable GTP composite thus has great potential for improving bone repair.
Keywords: Oligomeric proanthocyanidins; Cross-linking; Gelatin; Tricalcium phosphate; Osteoconductive; Bone repair
Cells and tissue interactions with glycated collagen and their relevance to delayed diabetic wound healing
by Huijuan Liao; Julia Zakhaleva; Weiliam Chen (pp. 1689-1696).
Dermal accumulation of advanced glycation end products (AGEs) has increasingly been implicated as the underlying cause of delayed diabetic wound healing. Devising an in vitro model to adequately mimic glycated tissues will facilitate investigation into the mechanism of glycation in conjunction with exploration of new approaches or improvement of current therapies for treating diabetic chronic wounds. Collagen matrices were artificially glycated and the presence of AGEs was demonstrated by immunostaining. Both the mechanical properties of the collagen matrices and their interactions with fibroblasts (morphology, attachment, proliferation, and migration) were altered after glycation, moreover, there was evidence of impairment on extracellular matrix (ECM) remodeling as well as inhibition of cell-induced material contraction. The actin cytoskeletons of the fibroblasts residing in the glycated collagen matrices were reorganized. In vivo mice full-thickness dermal wound models implanted with glycated collagen matrices showed delayed wound healing response. Thus, the glycated collagen matrix is an adequate in vitro model to mimic glycated tissues and could serve as a facile experimental tool to investigate the mechanism of glycation in conjunction with exploration of new approaches or improvement of current therapies for treating diabetic wounds.
Keywords: In vitro; model; Collagen matrix; Glycation; Fibroblast
The use of a shape-memory poly(ε-caprolactone)dimethacrylate network as a tissue engineering scaffold
by Sabine Neuss; Iris Blomenkamp; Rebekah Stainforth; Dagmar Boltersdorf; Marc Jansen; Nick Butz; Alberto Perez-Bouza; Ruth Knüchel (pp. 1697-1705).
Shape-memory polymers produced from many natural or synthetic raw polymers are able to undergo a shape transformation after exposure to a specific external stimulus. This feature enables their use in minimal-invasive surgery with a small, compact starting material switching over to a more voluminous structure in the body. The use of biomaterials in modern medicine calls for compatibility tests with cell types, encountering the biomaterial during a short-term or long-term in vivo application. We analysed the cell behaviour of L929 mouse fibroblasts, human mesenchymal stem cells, human mesothelial cells and rat mesothelial cells on a biodegradable shape-memory polymer network to assess its suitability for medical applications. Further, we investigated the differentiation capacity of mesenchymal stem cells into osteoblasts and adipocytes on the polymer and we analysed the influence of the shape-memory effect on adherent cells. The polymer was cytocompatible for all tested cell types, supporting cell viability and proliferation. The differentiation capacity of mesenchymal stem cells was supported by the polymer and shape-memory effect activation did not affect the majority of adherent cells.
Keywords: Shape memory; Mesenchymal stem cells; Cell adhesion; Cytotoxicity; Biocompatibility
The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells
by Gen-hong Mao; Gui-an Chen; Hai-yan Bai; Tian-ran Song; Yan-xia Wang (pp. 1706-1714).
Islet-like cells derived from embryonic stem (ES) cells may be a promising therapeutic option for future diabetes treatment. Here, we demonstrated a five-stage protocol with adding exendin-4 instead of nicotinamide finally could generate islet-like cells from human embryonic stem (ES) cells. Immunofluorescence analysis revealed a high percentage of c-peptide positive cells in the derivation. However, in addition to insulin/c-peptide, most cells also coexpressed PDX-1 (pancreas duodenum homeobox-1), glucagon, somatostatin or pancreatic polypeptide. Insulin and other pancreatic beta-cell-specific genes were all present in the differentiated cells. Insulin secretion could be detected and increased significantly by adding KCL in high glucose concentration in vitro. Furthermore, subcutaneous transplantation of scaffolds seeded with the islet-like cells or cell transplantation under kidney capsules for further differentiation in vivo could improve 6h fasted blood glucose levels and diabetic phenotypes in streptozotocin-induced diabetic SCID mice. More interestingly, blood vessels of host origin, characterized by mouse CD31 immunostaining, invaded the cell–scaffold complexes. This work reveals a five-stage protocol with adding exendin-4 may be an effective protocol on the differentiation of human ES cells into islet-like cells, and suggests scaffolds can serve as vehicles for islet-like cell transplantation.
Keywords: Human embryonic stem cells; Islet; Scaffold; Transplantation
Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2- N, 6- O-sulfated chitosan in vitro and in vivo
by Huanjun Zhou; Jiangchao Qian; Jing Wang; Wantong Yao; Changsheng Liu; Jianguo Chen; Xuehua Cao (pp. 1715-1724).
Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6- O-sulfated chitosan (6SCS), while 2- N-sulfate was subsidiary group with less activation. Low dose of 2- N, 6- O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.
Keywords: BMP-2; Sulfated chitosan; Heparin; Bone tissue engineering
Tubular micro-scale multiwalled carbon nanotube-based scaffolds for tissue engineering
by Sharon L. Edwards; Jeffrey S. Church; Jerome A. Werkmeister; John A.M. Ramshaw (pp. 1725-1731).
In this study we have prepared a tubular knitted scaffold from a 9 ply multiwalled carbon nanotube (MWCNT) yarn and a composite scaffold, formed by electrospinning poly(lactic- co-glycolic acid) (PLGA) nanofibres onto the knitted scaffold. Both structures were assessed for in vitro biocompatibility with NR6 mouse fibroblast cells for up to 22 days and their suitability as tissue engineering scaffolds considered. The MWCNT yarn was found to support cell growth throughout the culture period, with fibroblasts attaching to, and proliferating on, the yarn surface. The knitted tubular scaffold contained large pores that inhibited cell spanning, leading to the formation of cell clusters on the yarn, and an uneven cell distribution on the scaffold surface. The smaller pores, created through electrospinning, were found to promote cell spanning, leading to a uniform distribution of cells on the composite scaffold surface. Evaluation of the electrical and mechanical properties of the knitted scaffold determined resistance levels of 0.9kΩ/cm, with a breaking load and extension to break approaching 0.7N and 8%, respectively. The PLGA/MWCNT composite scaffold presented in this work not only supports cell growth, but also has the potential to utilize the full range of electrical and mechanical properties that carbon nanotubes have to offer.
Keywords: Biocompatibility; Carbon nanotube; Fibroblasts; Scaffold
Long-term clinical outcomes following the use of synthetic hydroxyapatite and bone graft in impaction in revision hip arthroplasty
by Tajeshwar S. Aulakh; Narlaka Jayasekera; Jan-Herman Kuiper; James B. Richardson (pp. 1732-1738).
Impaction grafting using morsellised allograft bone restores bone stock, but carries the potential for transmission of infection. Synthetic bone graft substitutes can eliminate this risk but may, however, influence outcome. In this study we tested the hypothesis that a 50/50 mix of hydroxyapatite and allograft does not affect long-term function, survival or radiological outcome. Sixty-five patients had revision hip arthroplasty using impaction grafting with either pure allograft (42 patients) or a 50/50 mixture of allograft and solid particulate hydroxyapatite. Harris hip scores were assessed pre-operatively and annual intervals thereafter. Function was analyzed using multilevel modeling, the Kaplan–Meier method used for survival analysis and graft incorporation was assessed radiologically. The hip score improved in both groups but showed a small annual decline (average 1.2/year, p<0.01). This decline was higher for females (average 3.4, p=0.025) and significantly related to pre-op scores ( p<0.001). After adjusting for these, allograft patients had marginally higher scores (difference=3.1, p=0.3). The majority of revisions were for aseptic loosening. At 13 years survival in the allograft group was 84%, and 82% in the mixture group ( p=0.96, log rank test). Radiologically the graft incorporation was similar in both groups ( p=0.62). We conclude that long-term prosthesis survival and function following revision arthroplasty with a 50/50 mixture of allograft and hydroxyapatite are comparable to allograft alone.
Keywords: Allograft; Hip arthroplasty; Hydroxyapatite; Impaction grafting; Revision
A DNA nanomachine induced by single-walled carbon nanotubes on gold surface
by Chao Zhao; Yujun Song; Jinsong Ren; Xiaogang Qu (pp. 1739-1745).
Single-walled carbon nanotubes (SWNTs) can selectively induce human telomeric i-motif DNA formation at pH 7.0. Based on this property, we design a DNA nanomachine induced by SWNTs on gold surface. The motor DNA is human telomeric G-quadruplex DNA. The reversible hybridization between the motor DNA and its complementary human telomeric i-motif DNA can be modulated by SWNTs without changing solution pH. Up to now, to our knowledge, there is no report to show that a DNA nanomachine is induced by SWNTs or a DNA nanomachine can detect i-motif formation at pH 7.0. Our work may provide a new concept for designing an SWNT-induced DNA nanomachine and for the detection of i-motif DNA structure at pH 7.0. DNA hybridization, conformational transition and i-motif formation have been characterized on surface or in solution by fluorescence confocal microscopy, circular dichroism, DNA melting and gel electrophoresis. The folding and unfolding kinetics of the DNA nanomachine on gold surface were studied by Fourier transform-surface plasmon resonance (FT-SPR). All these results indicate that SWNTs can induce the DNA nanomachine to work efficiently and reversibly. Therefore our work will provide new insights into the design and application of SWNT-induced DNA nanodevice under physiological conditions.
Keywords: Single-walled carbon nanotube; Human telomere DNA; DNA detection; i-Motif DNA; DNA conformation; Nanomachine
Delivery of sodium borocaptate to glioma cells using immunoliposome conjugated with anti-EGFR antibodies by ZZ-His
by Bin Feng; Kazuhito Tomizawa; Hiroyuki Michiue; Shin-ichi Miyatake; Xiao-Jian Han; Atsushi Fujimura; Masaharu Seno; Mitsunori Kirihata; Hideki Matsui (pp. 1746-1755).
Nanoparticles are effective of delivering cargo into cells. Here, sodium borocaptate (BSH) was encapsulated in liposomes composed of nickel lipid, and anti-epidermal growth factor receptor (EGFR) antibodies were conjugated to the liposomes using the antibody affinity motif of protein A (ZZ) as an adaptor (immunoliposomes). The immunoliposomes were used to deliver BSH into EGFR-overexpressing glioma cells. Immunohistochemical analysis using an anti-BSH monoclonal antibody revealed that BSH was delivered effectively into the cells but not into EGFR-deficient glioma or primary astrocytes. In an animal model of brain tumors, both the liposomes and the BSH were only observed in the tumor. Moreover, the efficiency of10B's delivery into glioma cells was confirmed by inductively coupled plasma-atomic emission spectrometry (ICP-AES) both in vitro and in vivo. The results suggest that this system utilizing immunoliposomes provides an effective means of delivering10B into glioma cells in boron neutron capture therapy (BNCT).
Keywords: Glioma cells; BNCT; EGFR; Immunoliposome; Drug delivery
Inhibition of titanium particle-induced osteoclastogenesis through inactivation of NFATc1 by VIVIT peptide
by Fengxiang Liu; Zhenan Zhu; Yuanqing Mao; Ming Liu; Tingting Tang; Shijing Qiu (pp. 1756-1762).
Osteoclastogenesis induced by particulate wear debris is a major pathological factor contributing to periprosthetic osteolysis. Although the nuclear factor of activated T cells c1 (NFATc1) is known to be involved in osteoclast differentiation, its effect on osteoclastogenesis in response to wear particles remains unclear. In the present study, we investigated the role of NFATc1 in the regulation of osteoclast differentiation from bone marrow macrophages (BMMs) stimulated with titanium (Ti) particles. The results showed that Ti particles could stimulate BMMs to produce proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) and differentiate into multinucleated osteoclasts in the presence of receptor activator of nuclear factor-κB ligand (RANKL). NFATc1 was expressed in BMMs and multinucleated cells cultured with Ti particles and RANKL. Inactivation of NFATc1 by 11R-VIVIT peptide potently impeded the Ti particle-induced osteoclastogenesis. 11R-VIVIT peptide does not have toxic effect on BMMs. Based on these data, we conclude that inactivation of NFATc1 by VIVIT peptide would provide a promising therapeutic target for the treatment of periprosthetic osteolysis.
Keywords: Titanium particles; Osteoclast; NFATc1; Cytokines; Osteolysis
Bone healing induced by local delivery of an engineered parathyroid hormone prodrug
by Isabelle Arrighi; Silke Mark; Monica Alvisi; Brigitte von Rechenberg; Jeffrey A. Hubbell; Jason C. Schense (pp. 1763-1771).
Regenerative medicine requires innovative therapeutic designs to accommodate high morphogen concentrations in local depots, provide their sustained presence, and enhance cellular invasion and directed differentiation. Here we present an example for inducing local bone regeneration with a matrix-bound engineered active fragment of human parathyroid hormone (PTH1–34), linked to a transglutaminase substrate for binding to fibrin as a delivery and cell-invasion matrix with an intervening plasmin-sensitive link (TGplPTH1–34). The precursor form displays very little activity and signaling to osteoblasts, whereas the plasmin cleavage product, as it would be induced under the enzymatic influence of cells remodeling the matrix, was highly active. In vivo animal bone-defect experiments showed dose-dependent bone formation using the PTH–fibrin matrix, with evidence of both osteoconductive and osteoinductive bone-healing mechanisms. Results showed that this PTH-derivatized matrix may have potential utility in humans as a replacement for bone grafts or to repair bone defects.
Keywords: Bone healing; Bone graft; Fibrin; Peptide; Osteoblast
Dexamethasone-containing PLGA superparamagnetic microparticles as carriers for the local treatment of arthritis
by Nicoleta Butoescu; Christian A. Seemayer; Michelangelo Foti; Olivier Jordan; Eric Doelker (pp. 1772-1780).
Superparamagnetic iron oxide nanoparticles (SPIONs) are attractive materials that have been widely used in medicine for diagnostic imaging and therapeutic applications. In our study, SPIONs and the corticosteroid dexamethasone acetate (DXM) are co-encapsulated into PLGA microparticles for the aim of locally treating inflammatory conditions such as arthritis. The magnetic properties conferred by the SPIONs could help to maintain the microparticles in the joint with an external magnet. The aim of this study was to investigate the interaction between magnetic microparticles and human synovial fibroblasts in terms of microparticle uptake (FACS, confocal and optical microscopy), internalization mechanism (Prussian Blue staining, TEM, immunofluorescence), cell toxicity (MTT) and tissue reaction after intra-articular injection (histology). The results show that the microparticles have an excellent biocompatibility with synoviocytes and that they are internalized through a phagocytic process, as demonstrated by fluorescence-activated cell sorting and morphological analyses of cells exposed to microparticles. Histological analysis showed that the prepared microparticles did not induce any inflammatory reaction in the joint. This type of carrier could represent a suitable magnetically retainable intra-articular drug delivery system for treating joint diseases such as arthritis or osteoarthritis.
Keywords: Synoviocyte; Intra-articular injection; Microparticles; PLGA; SPIONs; Phagocytosis
The covalent attachment of adhesion molecules to silicone membranes for cell stretching applications
by Pierre-Jean Wipff; Hicham Majd; Chitrangada Acharya; Lara Buscemi; Jean-Jacques Meister; Boris Hinz (pp. 1781-1789).
Strain devices with expandable polydimethylsiloxane (PDMS) culture membranes are frequently used to stretch cells in vitro, mimicking mechanically dynamic tissue environments. To immobilize cell-adhesive molecules to the otherwise non-adhesive PDMS substrate, hydrophobic, electrostatic and covalent surface coating procedures have been developed. The efficacy of different coating strategies to transmit stretches to cells however is poorly documented and has not been compared. We describe a novel and simple procedure to covalently bind extracellular matrix proteins to the surface of stretchable PDMS membranes. The method comprises PDMS oxygenation, silanization, and covalent protein cross-linking to the silane. We demonstrate improved attachment (∼2-fold), spreading (∼2.5-fold) and proliferation (∼1.2-fold) of fibroblasts to our new coating over established coating procedures. Further, we compared the efficiency of different PDMS coating techniques to transmit stretches. After 15% stretch, the number of maximally (15 ± 5%) stretched cells on our PDMS surface coating was ∼7-fold higher compared with alternative coating protocols. Hence, covalent linkage of adhesive molecules is superior to non-covalent methods in providing a coating that resists large deformations and that fully transmit this stretch to cultured cells.
Keywords: PDMS (silicone); Mechanical stretch; Cell adhesion; Collagen
The effect of surface immobilized bisphosphonates on the fixation of hydroxyapatite-coated titanium implants in ovariectomized rats
by Ying Gao; Shujuan Zou; Xiaoguang Liu; Chongyun Bao; Jing Hu (pp. 1790-1796).
Immobilized bisphosphonates (BPs) have been introduced to improve implant fixation, however, no information could be found about the efficiency of this approach in osteoporotic bone. This study was designed to evaluate the bone response to surface immobilized BPs on implants inserted in tibiae of ovariectomized (OVX) rats. Three months after bilateral ovariectomy, 40 rats were randomly assigned into four groups for implantation of hydroxyapatite-coated titanium implants with or without immobilized BPs: (1) control group (without BP treatments); (2) pamidronate (PAM) group (1mg/ml of PAM immersing); (3) ibandronate group (1mg/ml of ibandronate immersing); and (4) zoledronic acid (ZOL) group (1mg/ml of ZOL immersing). After implantation periods of 3 months, the peri-implant–bone density, trabecular microstructure, bone–implant interface and mechanical fixation of implants were evaluated by dual energy X-ray absorptiometry, micro-computed tomography, histology and push-out test. We found that three BPs triggered pronounced bone–implant integration and early bone formation around implants in OVX rats, with a rank order of ZOL>ibandronate>PAM. These results provide new evidence that immobilized BPs have positive effects on implant fixation in osteoporotic bone, in addition to their well-documented potency to inhibit implant loosening in normal bone.
Keywords: Bisphosphonate; Immobilize; Hydroxyapatite; Implant; Osteoporosis
Effects of incorporation of poly(γ-glutamic acid) in chitosan/DNA complex nanoparticles on cellular uptake and transfection efficiency
by Shu-Fen Peng; Mei-Ju Yang; Chun-Jen Su; Hsin-Lung Chen; Po-Wei Lee; Ming-Cheng Wei; Hsing-Wen Sung (pp. 1797-1808).
Chitosan (CS)/DNA complex nanoparticles (NPs) have been considered as a vector for gene delivery. Although advantageous for DNA packing and protection, CS-based complexes may lead to difficulties in DNA release once arriving at the site of action. In this study, an approach through modifying their internal structure by incorporating a negatively charged poly(γ-glutamic acid) (γ-PGA) in CS/DNA complexes (CS/DNA/γ-PGA NPs) is reported. The analysis of small angle X-ray scattering results revealed that DNA and γ-PGA formed complexes with CS separately to yield two types of domains, leading to the formation of “compounded NPs”. With this internal structure, the compounded NPs might disintegrate into a number of even smaller sub-particles after cellular internalization, thus improving the dissociation capacity of CS and DNA. Consequently, after incorporating γ-PGA in CS/DNA complexes, a significant increase in their transfection efficiency was found. Interestingly, in addition to improving the release of DNA intracellularly, the incorporation of γ-PGA in CS/DNA complexes significantly enhanced their cellular uptake. We further demonstrated that besides a non-specific charged-mediated binding to cell membranes, there were specific trypsin-cleavable proteins involved in the internalization of CS/DNA/γ-PGA NPs. The aforementioned results indicated that γ-PGA played multiple important roles in enhancing the cellular uptake and transfection efficiency of CS/DNA/γ-PGA NPs.
Keywords: Chitosan; Poly(γ-glutamic acid); Transfection efficiency; Cellular uptake
Direct cell entry of gold/iron-oxide magnetic nanoparticles in adenovirus mediated gene delivery
by Kazumasa Kamei; Yohei Mukai; Hiroki Kojima; Tomoaki Yoshikawa; Mai Yoshikawa; George Kiyohara; Takao A. Yamamoto; Yasuo Yoshioka; Naoki Okada; Satoshi Seino; Shinsaku Nakagawa (pp. 1809-1814).
Gold/iron-oxide MAgnetic Nanoparticles (GoldMAN) imparts useful magnetic properties to various biomolecules. Gold nanoparticles immobilized on the surface of magnetic nanoparticles allow for the conjugation of biomolecules via an Au–S bond. Here, we present a practical application by utilizing GoldMAN and a magnetic field to induce intracellular transduction. This method has great potential for application of the adenovirus gene delivery vector (Ad), widely used for in vitro/ in vivo gene transfer, to Ad-resistant cells. We demonstrated that Ad was easily immobilized on GoldMAN and the Ad/GoldMAN complex was introduced into the cell by the magnetic field, which increased gene expression over 1000 times that of Ad alone. The GoldMAN penetrated the plasma membrane directly, independent of the cell-surface virus receptors and endocytosis pathway. This mechanism will contribute to improve the gene expression efficiency of Ad. This technology is a useful tool for extending Ad tropism and enhancing transduction efficiency. GoldMAN also makes possible the effective use of various biomolecules within the cell because of its interesting cell-entry mechanism.
Keywords: Adenovirus; Gene expression; Gold; Magnetism; Nanoparticle
Neuronal gene delivery by negatively charged pullulan–spermine/DNA anioplexes
by Devang K. Thakor; Yang D. Teng; Yasuhiko Tabata (pp. 1815-1826).
Nonviral gene transfer to neurons remains unreliable due to a lack of effective and nontoxic vectors. Here, we achieved effective neuronal gene delivery through salt-free complexation of plasmid DNA and pullulan–spermine, a conjugate prepared from a naturally derived polysaccharide and polyamine. Specifically, at low spermine nitrogen:DNA phosphate (N:P) ratios, complexes formed with ζ-potential and diameter of approximately−40mV and 350nm, respectively. Higher N:P ratios increased the ζ-potential to approximately +10mV. All complexes were stable for at least 1 week and protected DNA from degradation. In vitro transfection of rat sensory neurons occurred at all N:P ratios, but uniquely, efficiency was highest for anionic complexes (anioplexes). Subsequent analyses revealed the inhibition of reporter gene expression by asialofetuin (1mg/ml) and methyl-beta-cyclodextrin (5mm), indicating utilization of glycoprotein-specific interactions and lipid rafts for uptake and intracellular trafficking. In marked contrast to a commercial cationic lipid reagent, anioplexes did not exhibit measurable cytotoxicity at up to 20μg/ml DNA. Additionally, transfection efficiency was maintained in the presence of serum and antibiotics. Based on these favorable properties, we successfully established two transfection methods for cultured adult sensory neurons and tissue explants. Collectively, these data suggest that negatively charged pullulan–spermine/DNA anioplexes could represent an effective gene delivery technology, particularly for neurons.
Keywords: Gene therapy; Gene transfer; Nerve regeneration; Nerve; Neuron; Sensory
Stimuli-responsive interfaces and systems for the control of protein–surface and cell–surface interactions
by Martin A. Cole; Nicolas H. Voelcker; Helmut Thissen; Hans J. Griesser (pp. 1827-1850).
Real-time control over and reversibility of biomolecule–surface interactions at interfaces is an increasingly important goal for a range of scientific fields and applications. The field of stimuli-responsive, smart or switchable systems has generated much research interest due to its potential to attain unprecedented levels of control over biomolecule adsorption processes and interactions at engineered interfaces, including the control over reversibility of adsorption. Advances in this field are particularly relevant to applications in the areas of biosensing, chromatography, drug delivery and regenerative medicine. The control over biomolecule adsorption and desorption processes at interfaces is often used to control subsequent events such as cell–surface interactions. Considerable research interest has been directed at systems that can be reversibly switched between interacting and non-interacting states and used thus for switching, on and off, bio-interfacial interactions such as protein adsorption. Such switchable coatings often incorporate features such as temporal resolution, spatial resolution and reversibility. Here we review recent literature on switchable coatings that employ stimuli such as light, temperature, electric potential, pH and ionic strength to control protein adsorption/desorption and cell attachment/detachment en route to the development of next-generation smart bio-interfaces.
Keywords: Surface modification; Cell culture; Protein adsorption; Thermally responsive polymer; pH sensitive; Stimuli-responsive polymersAbbreviations; AA; acrylic acid; ATR; attenuated total reflection; ATRP; atom transfer radical polymerisation; BCAm; benzo-18-crown-6-acrylamide; BIS; N; ,; N; -methylene-bis-acrylamide; BMA; butylmethacrylate; BSA; bovine serum albumin; CA; contact angle; CP-AFM; colloid probe atomic force microscopy; DP; degree of polymerisation; ECM; extracellular matrix; EG-12A; endoglucanase 12A; ELP; elastin-like polypeptide; FN; fibronectin; FT-IR; Fourier transform infrared; HRP; horse-radish peroxidase; LCST; lower critical solution temperature; NIPAM; N; -isopropylacrylamide; NRs; nanorods; OEG; oligo ethylene glycol; PAA; poly(acrylic acid); PBS; phosphate buffered saline; pDEAM; poly(; N; ,; N; -diethylacrylamide); PDI; polydispersity index; PEG; poly(ethylene glycol); pI; isoelectric point; PLGA; poly(; dl; lactic acid-; co; -glycolic acid); pNIPAM; poly(; N; -isopropylacrylamide); ppNIPAM; plasma polymerised NIPAM; P2VP; poly(2-vinylpyridine); QCM; quartz crystal microbalance; RGDS; arginine–glycine-aspartic acid–serine; RGD-Cp; arginine–glycine–aspartic acid–cyclopentadiene; SCF; self-consistent field; SFA; surface force apparatus; SPR; surface plasmon resonance; TCPS; tissue culture polystyrene
Characterization of type I collagen gels modified by glycation
by Megan E. Francis-Sedlak; Shiri Uriel; Jeffery C. Larson; Howard P. Greisler; David C. Venerus; Eric M. Brey (pp. 1851-1856).
Chronic exposure to reducing sugars due to diabetes, aging, and diet can permanently modify extracellular matrix (ECM) proteins. This non-enzymatic glycosylation, or glycation, can lead to the formation of advanced glycation end products (AGE) and crosslinking of the ECM. This study investigates the effects of glycation on the properties of type I collagen gels. Incubation with glucose-6-phopshate (G6P), a reducing sugar that exhibits similar but more rapid glycation than glucose, modified the biological and mechanical properties of collagen gels. Measures of AGE formation that correlate with increased complications in people with diabetes, including collagen autofluorescence, crosslinking, and resistance to proteolytic degradation, increased with G6P concentration. Rheology studies showed that AGE crosslinking increased the shear storage and loss moduli of type I collagen gels. Fibroblasts cultured on glycated collagen gels proliferated more rapidly than on unmodified gels, but glycated collagen decreased fibroblast invasion. These results show that incubation of type I collagen gels with G6P increases clinically relevant measures of AGE formation and that these changes altered cellular interactions. These gels could be used as in vitro models to study ECM changes that occur in diabetes and aging.
Keywords: Collagen; Diabetes; Wound healing; Mechanical properties; Confocal microscopy; Glycation