Biomaterials (v.28, #31)
Influence of substratum surface chemistry/energy and topography on the human fetal osteoblastic cell line hFOB 1.19: Phenotypic and genotypic responses observed in vitro
by Xiaomei Liu; Jung Yul Lim; Henry J. Donahue; Ravi Dhurjati; Andrea M. Mastro; Erwin A. Vogler (pp. 4535-4550).
Time-dependent phenotypic response of a model osteoblast cell line (hFOB 1.19, ATCC, and CRL-11372) to substrata with varying surface chemistry and topography is reviewed within the context of extant cell-adhesion theory. Cell-attachment and proliferation kinetics are compared using morphology as a leading indicator of cell phenotype. Expression of ( α2, α3, α4, α5, αv, β1, and β3) integrins, vinculin, as well as secretion of osteopontin (OP) and type I collagen (Col I) supplement this visual assessment of hFOB growth. It is concluded that significant cell-adhesion events—contact, attachment, spreading, and proliferation—are similar on all surfaces, independent of substratum surface chemistry/energy. However, this sequence of events is significantly delayed and attenuated on hydrophobic (poorly water-wettable) surfaces exhibiting characteristically low-attachment efficiency and long induction periods before cells engage in an exponential-growth phase. Results suggest that a ‘time–cell–substratum–compatibility–superposition principle’ is at work wherein similar bioadhesive outcomes can be ultimately achieved on all surface types with varying hydrophilicity, but the time required to arrive at this outcome increases with decreasing cell–substratum–compatibility. Genomic and proteomic tools offer unprecedented opportunity to directly measure changes in the cellular machinery that lead to observed cell responses to different materials. But for the purpose of measuring structure–property relationships that can guide biomaterial development, genomic/proteomic tools should be applied early in the adhesion/spreading process before cells have an opportunity to significantly remodel the cell–substratum interface, effectively erasing cause and effect relationships between cell–substratum–compatibility and substratum properties.This review quantifies relationships among cell phenotype, substratum surface chemistry/energy, topography, and cell–substratum contact time for the model osteoblast cell line hFOB 1.19, revealing that genomic/proteomic tools are most useful in the pursuit of understanding cell adhesion if applied early in the adhesion/spreading process.
Keywords: Cell adhesion; Surface chemistry; Surface energy; Cell–substratum–compatibility; hFOB; Osteoblast
The potential impact of the preparation rich in growth factors (PRGF) in different medical fields
by Eduardo Anitua; Sanchez Mikel Sánchez; Gorka Orive; Andia Isabel Andía (pp. 4551-4560).
Platelet-rich preparations constitute a relatively new biotechnology for the stimulation and acceleration of tissue healing and bone regeneration. The versatility and biocompatibility of this approach has stimulated its therapeutic use in numerous medical and scientific fields including dentistry, oral implantology, orthopaedics, ulcer treatment, tissue engineering among others. Here we discuss the important progress that has been accomplished in the field of platelet-rich preparations in the last few years. Some of the most interesting therapeutic applications of this technology are discussed as are some of the limitations, future challenges and directions in the field.
Keywords: Growth factors; Platelets; Regenerative medicine; Tissue repair; Drug delivery; Biomaterials
The effect of gas plasma modification on platelet and contact phase activation processes
by Nicholas P. Rhodes; Darren J. Wilson; Rachel L. Williams (pp. 4561-4570).
Medical-grade polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polyetherurethane (PEU) and ultrahigh molecular weight polyethylene (UHMWPE) were plasma treated with O2, Ar, N2 and NH3. Their surface properties were characterised using X-ray photoelectron spectroscopy (XPS), static secondary ion mass spectroscopy (SSIMS), atomic force microscopy (AFM) and dynamic contact angle (DCA) analysis. Platelet adhesion, aggregation, activation and release of microparticles were determined after contact with whole blood in a cone and plate viscometer. Activation of the coagulation system was quantified in a static environment using a partial thromboplastin time (PTT) assay. The chemical compositions of the untreated surfaces were found to be very similar to those of the bulk material except for PEU, whose surface was comprised almost entirely of soft ether segments. For all materials, the different plasma treatments resulted in moderate etching with the incorporation of functional groups and removal of side groups: defluorination, dehydrogenation, cleavage of methyl side groups and soft segments for PTFE, UHMWPE, PDMS and PEU, respectively. Consequently, plasma treatment resulted in increased wettability in all cases. Blood contact with the virgin materials resulted in activation of platelets and the clotting cascade. Plasma treatment resulted in a significant reduction in platelet adhesion for all materials and all treatments. In the case of PTFE and PEU, the activation status of these cells was also reduced. Plasma treatment of all materials reduced fluid-phase CD62P expression. Platelet aggregate size correlated well with degree of aggregate formation, but many treatments increased the degree of aggregation, as was the case for microparticle shedding. There was no correlation between CD62P expression, aggregate formation and platelet microparticle (PMP) shedding. It is concluded that despite incorporation of the same chemical groups, the pattern of response to blood in vitro is not the same across different polymers.
Keywords: Polymers; Haemocompatibility; Platelet; Microparticle; Coagulation
Reduced foreign body response at nitric oxide-releasing subcutaneous implants
by Evan M. Hetrick; Heather L. Prichard; Bruce Klitzman; Mark H. Schoenfisch (pp. 4571-4580).
The tissue response to nitric oxide (NO)-releasing subcutaneous implants is presented. Model implants were created by coating silicone elastomer with diazeniumdiolate-modified xerogel polymers capable of releasing NO. The host tissue response to such implants was evaluated at 1, 3, and 6 weeks and compared to that of uncoated silicone elastomer blanks and xerogel-coated controls incapable of releasing NO. Delivery of NO (∼1.35μmol/cm2 of implant surface area) reduced foreign body collagen capsule (“scar tissue”) thickness by >50% compared to uncoated silicone elastomer after 3 weeks. The chronic inflammatory response at the tissue/implant interface was also reduced by >30% at NO-releasing implants after 3 and 6 weeks. Additionally, CD-31 immunohistochemical staining revealed ∼77% more blood vessels in proximity to NO-releasing implants after 1 week compared to controls. These findings suggest that conferring NO release to subcutaneous implants may promote effective device integration into healthy vascularized tissue, diminish foreign body capsule formation, and improve the performance of indwelling medical devices that require constant mass transport of analytes (e.g., implantable sensors).
Keywords: Foreign body response; Angiogenesis; Inflammatory response; Nitric oxide; Xerogel; Wound healing
In vitro biological evaluation of high molecular weight hyperbranched polyglycerols
by Rajesh Kumar Kainthan; Samuel R. Hester; Elena Levin; Dana V. Devine; Donald Elliott Brooks (pp. 4581-4590).
Low molecular weight hyperbranched polyglycerols are highly water soluble and biocompatible polyether polyols, which can be synthesized in a controlled manner with narrow polydispersity. Recently we reported the synthesis and characterization of very high molecular weight ( Mn up to 700,000) and narrowly polydispersed polyglycerols which could be potentially used as alternatives to high generation dendrimers which are difficult to make. A detailed biocompatibility testing of these polymers conducted in vitro is reported here. The in vitro studies include hemocompatibility testing for effects on coagulation (prothrombin time (PT), activated partial thromboplastin time (APTT), plasma recalcification time (PRT), thrombelastograph parameters (TEG)), complement activation, platelet activation, red blood cell aggregation and cytotoxicity. Results from these studies show that these high molecular weight polyglycerols are highly biocompatible and are potential candidates for various applications in nanobiotechnology and in nanomedicine. Moreover these polymers are thermally and oxidatively stable.
Keywords: Hyperbranched polyglycerol; Biocompatibility; Blood compatibility; Thrombelastograph; Red cell aggregation; Coagulation
Long-term toxicity of holmium-loaded poly(l-lactic acid) microspheres in rats
by Sander W. Zielhuis; J. Frank W. Nijsen; Jan-Henry Seppenwoolde; Chris J.G. Bakker; Gerard C. Krijger; Hub F.J. Dullens; Bernard A. Zonnenberg; Peter P. van Rijk; Wim E. Hennink; Alfred D. van het Schip (pp. 4591-4599).
The aim of this study was to get insight into the toxic effects of holmium-166-loaded poly(l-lactic acid) microspheres (Ho-PLLA-MS) which have very interesting features for treatment of liver malignancies. Acute, mid- and long-term effects were studied in healthy Wistar rats by evaluating clinical, biochemical and tissue response. Rats were divided into four treatment groups: sham, decayed neutron-irradiated Ho-PLLA-MS, non-irradiated Ho-PLLA-MS and PLLA-MS. After implantation of the microspheres into the liver of the rats, the animals were monitored (body weight, temperature and liver enzymes) for a period of 14–18 months. Some of the rats that received previously neutron-irradiated Ho-PLLA-MS were periodically scanned with magnetic resonance imaging (MRI) to see if holmium was released from the microspheres. After sacrification, the liver tissue was histologically evaluated. Bone tissue was subjected to neutron-activation analysis in order to examine whether accumulation of released holmium in the bone had occurred. No measurable clinical and biochemical toxic effects were observed in any of the treatment groups. Furthermore, histological analyses of liver tissue samples only showed signs of a slight chronic inflammation and no significant differences in the tissue reaction between rats of the different treatment groups could be observed. The non-irradiated PLLA-MS and Ho-PLLA-MS stayed intact during the study. In contrast, 14 months after administration, the neutron-irradiated Ho-PLLA-MS was not completely spherical anymore, indicating that degradation had started. However, the holmium loading had not been released as was illustrated with MRI and affirmed by neutron-activation analysis of bone tissue. In conclusion, neutron-irradiated Ho-PLLA-MS does not provoke any toxic reaction and can be applied safely in vivo.
Keywords: Holmium; Microspheres; PLLA; Biodegradation; Irradiation; MRI
Protein adsorption and cellular uptake of cerium oxide nanoparticles as a function of zeta potential
by Swanand Patil; Amanda Sandberg; Eric Heckert; William Self; Sudipta Seal (pp. 4600-4607).
The surface chemistry of biomaterials can have a significant impact on their performance in biological applications. Our recent work suggests that cerium oxide nanoparticles are potent antioxidants in cell culture models and we have evaluated several therapeutic applications of these nanoparticles in different biological systems. Knowledge of protein adsorption and cellular uptake will be very useful in improving the beneficial effects of cerium oxide nanoparticles in biology. In the present study, we determined the effect of zeta potential of cerium oxide nanoparticles on adsorption of bovine serum albumin (BSA) and cellular uptake in adenocarcinoma lung cells (A549). The zeta potential of the nanoparticles was varied by dispersing them in various acidic and basic pH solutions. UV–visible spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) were used for the protein adsorption and cellular uptake studies, respectively. Nanoceria samples having positive zeta potential were found to adsorb more BSA while the samples with negative zeta potential showed little or no protein adsorption. The cellular uptake studies showed preferential uptake for the negatively charged nanoparticles. These results demonstrate that electrostatic interactions can play an important factor in protein adsorption and cellular uptake of nanoparticles.
Keywords: Cerium oxide nanoparticles; Protein adsorption; Cellular uptake; Zeta potential; Electrostatic interactions
Supramolecular crafting of cell adhesion
by Hannah Storrie; Mustafa O. Guler; Suha N. Abu-Amara; Tova Volberg; Mukti Rao; Benjamin Geiger; Samuel I. Stupp (pp. 4608-4618).
The supramolecular design of bioactive artificial extracellular matrices to control cell behavior is of critical importance in cell therapies and cell assays. Most previous work in this area has focused on polymers or monolayers which preclude control of signal density and accessibility in the nanoscale filamentous environment of natural matrices. We have used here self-assembling supramolecular nanofibers that display the cell adhesion ligand RGDS at van der Waals density to cells. Signal accessibility at this very high density has been varied by changes in molecular architecture and therefore through the supramolecular packing of monomers that form the fibers. We found that branched architectures of the monomers and the consequent lower packing efficiency and additional space for epitope motion improves signaling for cell adhesion, spreading, and migration. The use of artificial matrices with nanoscale objects with extremely high epitope densities could facilitate receptor clustering for signaling and also maximize successful binding between ligands and receptors at mobile three-dimensional interfaces between matrices and cells. Supramolecular design of artificial bioactive extracellular matrices to tune cell response may prove to be a powerful strategy in regenerative medicine and to study biological processes.
Keywords: Cell adhesion; Cell signaling; Hydrogel; Self-assembly
Characterisation of the macroporosity of polycaprolactone-based biocomposites and release kinetics for drug delivery
by Yiwei Wang; H.-I. Hsin-I Chang; David F. Wertheim; Allan S. Jones; Chris Jackson; A.G.A. Allan G.A. Coombes (pp. 4619-4627).
Microporous, biocomposite matrices comprising a continuous phase of poly( ε-caprolactone) (PCL) and a dispersed phase of lactose or gelatin particles with defined size range (45–90, 90–125 and 125–250μm) were produced by precipitation casting from solutions of PCL in acetone. Scanning electron microscopy (SEM) analysis revealed a characteristic surface morphology of particulates interspersed amongst crystalline lamellae of the polymer phase. Rapid release of around 80% of the lactose content occurred in PBS at 37°C in 3 days, whereas biocomposites containing gelatin particles of size range 90–125 and 125–250μm, respectively, displayed gradual and highly efficient release of around 90% of the protein phase over 21 days. A highly porous structure was obtained on extraction of the water-soluble phase. Micro-computed tomography (Micro-CT) and image analysis enabled 3-D visualisation and quantification of the internal pore size distribution. A maximum fractional pore area of 10.5% was estimated for gelatin-loaded matrices. Micro-CT analysis confirmed the presence of an extensive system of macropores, sufficiently connected to permit protein diffusion, but an absence of high volume, inter-pore channels. Thus tissue integration would be confined to the matrix surface initially if the designs investigated were used as tissue-engineering scaffolds, with the core potentially providing a depot system for controlled delivery of growth factors.
Keywords: Biocomposite; Polycaprolactone; Protein delivery; Porosity; Scaffold; Micro-CT
Oxygen producing biomaterials for tissue regeneration
by Benjamin S. Harrison; Daniel Eberli; Sang Jin Lee; Anthony Atala; James J. Yoo (pp. 4628-4634).
A limiting factor in regenerating large organs and healing large wounds completely is the inability to provide oxygen to the affected areas for vascularization and healing to occur. An oxygen rich compound of sodium percarbonate was incorporated into films of Poly(d,l-lactide– co–glycolide) (PLGA) and used for in situ production of oxygen. Oxygen release could be observed from the film over a period of 24h. When the oxygen producing biomaterials were placed in contact with ischemic tissue in a mouse model, decreased tissue necrosis and cellular apoptosis was observed. This indicates that improved tissue viability could be maintained for several days using oxygen producing biomaterials.
Keywords: Oxygenation; Fibroblasts; Hypoxia; Controlled drug release; Tissue engineering
Combination of scaffold and adenovirus vectors expressing bone morphogenetic protein-7 for alveolar bone regeneration at dental implant defects
by Yufeng Zhang; Jianhua Song; Bin Shi; Yining Wang; Xiaohui Chen; Cui Huang; Xuecao Yang; Dongxuan Xu; Xiangrong Cheng; Xinwen Chen (pp. 4635-4642).
The current rapid progression in tissue engineering and local gene delivery systems has enhanced applications of osseointegration in dental implants. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with an adenoviral vector encoding human bone morphogenetic proteins (BMP7). These scaffolds were evaluated in vitro by scanning electron microscopy (SEM), and human periodontal ligament cells (HPLCs) were seeded in this scaffold. We used Reverse transcription-polymerase chain reaction (RT-PCR) to determine the expression levels of osteopontin and bone sialoprotein. Alkaline phosphatase (ALP) activity was also determined. Then these scaffolds were implanted into defects on both sides of the mandible. Three months later, the animals were sacrificed and non-decalcified sections were evaluated histologically. Histomorphometric analyses were performed at the bone–implant interface using the image obtained by confocal laser scanning microscopy. Results indicated that the scaffold containing Ad-BMP7 exhibited the higher ALP activity, and the expression of osteopontin and bone sialoprotein were up-regulated. After implanting in defects around implant, the bone formation in Ad-BMP7 scaffolds was greater than that in other scaffolds at 4 or 8 weeks. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-BMP7 as a good substrate candidate in bone tissue engineering.
Keywords: Bone morphogenetic protein; Bone; Chitosan; Implant; Tissue engineering
Construction and characterization of fragmented mesenchymal-stem-cell sheets for intramuscular injection
by C.-H. Chun-Hung Chen; Yen Chang; C.-C. Chung-Chi Wang; C.-H. Chih-Hao Huang; C.-C. Chieh-Cheng Huang; Y.-C. Yi-Chun Yeh; S.-M. Shiaw-Min Hwang; H.-W. Hsing-Wen Sung (pp. 4643-4651).
Cell transplantation via local intramuscular injection is a promising therapy. However, the cells used are usually expanded in vitro and dissociated by trypsinization, which may be harmful to the cells. In the study, a novel method for the construction of fragmented sheets of mesenchymal stem cells (MSCs) with a uniform size, without treating with any enzymes, was reported. The obtained MSC sheets preserved the intercellular junctions and endogenous extracellular matrix and kept their cell phenotype. After injection through a needle, the fragmented MSC sheets maintained intact and retained their activity upon transferring to another growth surface, while the complete cell sheets were torn into pieces. Transplantation of fragmented MSC sheets in the skeletal muscle of a syngeneic rat model via local injection was evaluated. The transplanted MSC sheets were mainly localized at the site of injection, while the dissociated MSCs were scattered around. Additionally, there were significantly more MSCs retained in the local skeletal muscle for the group injected with fragmented MSC sheets than that injected with dissociated MSCs. These results indicated that the fragmented cell sheets might be used as a novel therapeutic cell-carrier for intramuscular administration.
Keywords: Temperature-responsive hydrogel; Mesenchymal stem cell; Cell sheet; Cell transplantation; Intramuscular injection
Self-assembled collagen–human mesenchymal stem cell microspheres for regenerative medicine
by Barbara P. Chan; T.Y. Hui; C.W. Yeung; J. Li; I. Mo; G.C.F. Chan (pp. 4652-4666).
Mesenchymal stem cells (MSCs)-based therapy is a promising approach in regenerative medicine and tissue engineering. However, the outcomes of existing treatments have not been satisfactory owing to suboptimal localization to implantation site, poor viability, low engraftment efficacy and lack of functional remodeling of the delivered cells. Therefore, adopting an effective cell delivery modality is among the biggest technological challenges for successful clinical applications of MSC-based therapy. We developed a novel microencapsulation technique producing self-assembled collagen–MSC microspheres and demonstrated that these microspheres could serve as excellent cell delivery devices as they were stable, injectable and able to provide a protective, growth- and migration-supporting matrix to MSCs. We also showed that MSCs could preserve their stem cell nature upon microencapsulation and easily be localized with retained viability upon in vivo implantation. These microspheres present novel cell delivery devices with optimal biological and functional profile that may facilitate clinical applications of MSC-based therapy.
Keywords: Collagen; Microencapsulation; Mesenchymal stem cell; Cell delivery; Microspheres
The effect of TiO2 nanotubes in the enhancement of blood clotting for the control of hemorrhage
by Somnath C. Roy; Maggie Paulose; Craig A. Grimes (pp. 4667-4672).
The main biological purpose of blood coagulation is formation of an obstacle to prevent blood loss of hydraulic strength sufficient to withstand the blood pressure. The ability to rapidly stem hemorrhage in trauma patients significantly impacts their chances of survival, and hence is a subject of ongoing interest in the medical community. Herein, we report on the effect of biocompatible TiO2 nanotubes on the clotting kinetics of whole blood. TiO2 nanotubes 10μm long were prepared by anodization of titanium in an electrolyte comprised of dimethyl sulfoxide and HF, then dispersed by sonication. Compared to pure blood, blood containing dispersed TiO2 nanotubes and blood in contact with gauze pads surface-decorated with nanotubes demonstrated significantly stronger clot formation at reduced clotting times. Similar experiments using nanocrystalline TiO2 nanoparticles showed comparatively weaker clot strengths and increased clotting times. The TiO2 nanotubes appear to act as a scaffold, facilitating fibrin formation. Our results suggest that application of a TiO2 nanotube functionalized bandage could be used to help stem or stop hemorrhage.
Keywords: TiO; 2; nanotube; Blood clotting; Coagulation; Hemorrhage; Magnetoelastic sensor; Fibrin
The drug loading, cytotoxicty and tumor vascular targeting characteristics of magnetite in magnetic drug targeting
by Suman Dandamudi; Robert B. Campbell (pp. 4673-4683).
Chemotherapy is a popular treatment approach against cancer but significant uptake of drugs by normal tissues is still a major limitation. Magnetic drug targeting (MDT) has been used to improve localized drug delivery to interstitial tumor targets. MDT is now being developed to improve drug delivery to tumor vessels. We thus seek to understand the role of magnetite (MAG-C) in drug loading, influence on cytotoxicity and vascular targeting characteristics. The inclusion of MAG-C at lower concentrations (0.5mg/ml) in cationic liposomes did not alter the efficiency of loading etoposide, but at higher concentrations (2.5mg/ml) incorporation decreased from 80±3.4% to 44±4.26%. MAG-C reduced the incorporation of dacarbazine. The incorporation was significantly lower compared to liposomal etoposide, both in the presence and absence of MAG-C. The incorporation efficiency of vinblastine sulfate in cationic liposomes was similar for low and relatively high MAG-C content; values for incorporation were 21±0.11 and 23±2, respectively. Polyethylene-glycol improved the efficiency of loading chemotherapeutic agents regardless of drug type. Additionally, cytotoxicity and tumor vascular targeting characteristics of liposome therapeutics were not influenced by MAG-C. The components used to prepare magnetic liposomes for MDT should be optimized for maximum therapeutic benefit.
Keywords: Cancer; Chemotherapy; Endothelial cell; Tumor vasculature; Magnetic liposomes; HPLCAbbreviations; DMPC; 1; 2-dimyristoyl-phosphatidylcholine; DMTAP; 1; 2-dimyristoyl-trimethyl-ammonium propane; DMPE-PEG; 5000; 1; 2-dimyristoyl-phosphatidylethanolamine-polyethylene glycol; CHOL; cholesterol; MAG-C; fluid MAG-CS (magnetite and citric acid matrix); DMEM; Dulbecco's modified eagle medium; EBM-2; endothelial cell basal medium-2; HMEC-1; human dermal microvascular endothelial cells; B16-F10; murine melanoma; CL; cationic liposomes; PCL; PEGylated cationic liposomes; SRB; sulforhodamine B; H; 2; O; 2; hydrogen peroxide; DPH; 1; 6-diphenyl-1; 3; 5-hexatriene; SQUID; superconducting quantum interference device
Enhance immune response to DNA vaccine based on a novel multicomponent supramolecular assembly
by Xianfeng Zhou; Bin Liu; Xianghui Yu; Xiao Zha; Xizhen Zhang; Xueyun Wang; Yu Chen; Yan Chen; Yue Chen; Yaming Shan; Yinghua Jin; Yongge Wu; Junqiu Liu; Wei Kong; Jiacong Shen (pp. 4684-4692).
DNA vaccination has tremendous potential for treating or preventing numerous diseases for which traditional vaccines are ineffective but the technique can be limited by low immunogenicity. Current synthetic DNA delivery systems are versatile and safe, but substantially less efficient than viruses. Here, a novel multicomponent supramolecular system involving the preparation of mannose-bearing chitosan oligomers microspheres with entrapping complexes of DNA vaccine and polyethylenimine was developed to mimic many of the beneficial properties of the viruses. After delivery by intramuscular immunization in BALB/c mice, the microspheres induced an enhanced serum antibody responses two orders of magnitude greater than naked DNA vaccine. Additionally, in contrast to naked DNA, the microspheres induced potent cytotoxic T lymphocyte responses at a low dose. Consequently, formulation of DNA vaccines into multicomponent vectors is a powerful means of increasing vaccine potency.
Keywords: Mannose-bearing Chitosan oligomers microspheres; Supramolecular assembly; DNA vaccine; Artificial virus; ImmunogenicityAbbreviation; MBCO microspheres; mannose-bearing chitosan oligomers microspheres; PEI; polyethylenimine; CTL; cytotoxic T lymphocyte; i.m.; intramuscular; TA; anterior tibialis; APCs; antigen presenting cells; DCs; dendritic cells; HSPGs; heparin sulfate proteoglycans; DS; degree of substitution; LDH; lactate dehydrogenase; DLS; dynamic light scattering.
A comparative evaluation of poly-l-lysine-palmitic acid and Lipofectamine ™ 2000 for plasmid delivery to bone marrow stromal cells
by B.A. Başak Açan Clements; Vanessa Incani; Cezary Kucharski; Afsaneh Lavasanifar; Bruce Ritchie; Uludag Hasan Uludağ (pp. 4693-4704).
The current study compared the effectiveness of an amphiphilic biomaterial poly(l-lysine)-palmitic acid (PLL–PA), and the lipid-based transfection agent Lipofectamine™ 2000 for plasmid delivery to bone marrow stromal cells (BMSC). We investigated the utility of the carriers to deliver a plasmid containing enhanced green fluorescent protein (pEGFP) to BMSC in vitro. Confocal microscopy was used to investigate the intracellular trafficking of pEGFP/carrier complexes. pEGFP delivery and EGFP expression were assessed by flow cytometry. PLL–PA formed condensed structures with pEGFP and successfully delivered the plasmid into the nucleus within 5h of incubation with the cells. PLL–PA delivered the pEGFP to ∼80% of the cells, achieving a maximum transfection efficiency of ∼22%. This was significantly higher than Lipofectamine™ 2000-mediated transfection, which was 11% under most optimal conditions. Dosing the BMSC two or three times during the 24h period increased the transfection efficiency by 2–3 folds, without compromising cell viability. When chloroquine was employed as an ensomolytic agent, 100μm of the drug increased the transfection efficiency while reducing cell viability, but lower concentrations (1–10μm) were not beneficial for transfection. Combining PLL–PA with Lipofectamine™ 2000 created an additive effect, increasing the transfection efficiency of PLL–PA. Long-term evaluation of gene expression with pEGFP/PLL–PA yielded ∼17% transfection on day 1, which gradually decreased over a 12-day period. We conclude that PLL–PA is an effective biomaterial carrier and a promising candidate for non-viral gene delivery to BMSC.
Keywords: Gene transfer; Bone marrow; Mesenchymal stem cell; Polyamino acid; Poly(; l; -lysine); Palmitic acidAbbreviations; AF546: Alexa Fluor 546 carboxylic acid succinimidyl ester; AdGFP: adenovirus expressing green fluorescent protein; BMSC: bone marrow stromal cells; EGFP: enhanced green fluorescent protein; pEGFP: plasmid containing the enhanced green fluorescent protein gene; PA: palmitic acid; PLL: poly(; l; -lysine)
Delivery of non-viral gene carriers from sphere-templated fibrin scaffolds for sustained transgene expression
by Justin M. Saul; Michael P. Linnes; Buddy D. Ratner; Cecilia M. Giachelli; Suzie H. Pun (pp. 4705-4716).
Delivery of safe and controlled levels of biomimetic cues to govern host response and reorganization is a fundamental component in the design of tissue engineering scaffolds. Non-viral gene delivery is an approach that exploits the cell machinery to produce proteins while avoiding genomic DNA incorporation. We describe a method to integrate polymeric non-viral gene carriers (polyplexes) within a novel three-dimensional, sphere-templated fibrin scaffold suitable for soft tissue engineering applications. After seeding the scaffolds with NIH-3T3 fibroblasts, different transgene expression profiles were achieved based on the spatial distribution of polyplexes within the scaffold. Scaffolds with polyplexes coated onto the surface of inter-connected pores showed peak transfection at day 5 and linear expression through 15 days. Scaffolds with polyplexes embedded within the fibrils of the biopolymer showed peak expression at 7–9 days and showed linear expression for 21–29 days, depending on the polymer:DNA ratio. Surface-coated polyplexes achieved one order of magnitude greater expression than polyplexes embedded within the scaffold. The integrated material formulations developed in this work provide a useful technology for tissue engineering applications by demonstrating the ability to provide long-term biomimetic cues through non-viral gene delivery.
Keywords: Tissue engineering; Scaffold; Controlled release; Gene delivery; Transfection
Biological applications of quantum dots
by Timothy Jamieson; Raheleh Bakhshi; Daniela Petrova; Rachael Pocock; Mo Imani; Alexander M. Seifalian (pp. 4717-4732).
Quantum dots (QDs) are a novel class of inorganic fluorophore which are gaining widespread recognition as a result of their exceptional photophysical properties. They are rapidly being applied to existing and emerging technologies, and could have an important role in many areas. Significant challenges remain, however, which must be understood and more fully defined before they can be widely validated.This review provides on overview of QD technology, covering QD characteristics, synthesis methods, and the applications in which they have been put to use. The influence of synthesis methods on QD characteristics and their subsequent suitability to different applications is discussed, and a broad outline of the technologies into which they have been incorporated is presented, and the relative merits and weaknesses of their incorporation are evaluated. The potential for further development, and inclusion in other technologies is also discussed, and barriers restricting further progress specified, particularly with regard to the poorly understood surface chemistry of QDs, the potential for alteration of function of biological molecules when complexed with QDs, and on a larger scale the significant potential for cytotoxicity both in vitro and in vivo.
Keywords: Quantum dot; Nanocrystal; Fluorescence imaging; Gene technology; Tumor imaging; Polymer and NanomaterialAbbreviation; CdSe; cadmium selenide; DHLA; dihydrolipoic acid; FRET; fluorescence resonance energy transfer; GFP; green fluorescent protein; MPA; mercaptoacetic acid; PEG; polyethylene glycol; SLN; sentinel lymph node; QD; quantum dot; SiO; 2; silica; ZnS; zinc sulfide
Shear modulus of porcine coronary artery in reference to a new strain measure
by Wei Zhang; Xiao Lu; Ghassan S. Kassab (pp. 4733-4738).
To simplify the stress–strain relationship of blood vessels, we define a logarithmic–exponential (log–exp) strain measure to absorb the nonlinearity. As a result, the constitutive relation between the second Piola–Kirchhoff stress and the log–exp strain can be written as a generalized Hooke's law. In this work, the shear modulus of porcine coronary arteries is determined from the experimental data in inflation–stretch–torsion tests. It is found that the shear modulus with respect to the log–exp strain can be viewed as a material constant in the full range of elasticity, and the incremental shear modulus for Cauchy shear stress and small shear strain at various loading levels can be predicted by the proposed Hooke's law. This result further validates the linear constitutive relation for blood vessels when shear deformation is involved.
Keywords: Blood vessel; Constitutive relation; Strain measure; Nonlinearity
Wiley Announces the 10th edition of the Wiley Registry of Mass Spectral Data
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