Biomaterials (v.27, #28)

Calendar (I).

Chemistry and the biological response against immunoisolating alginate–polycation capsules of different composition by Sara Ponce; Gorka Orive; Rosa Hernández; Alicia R. Gascón; Jose Luis Pedraz; Bart J. de Haan; Marijke M. Faas; H.J. Mathieu; Paul de Vos (4831-4839).
Implantation of microencapsulated cells has been proposed as a therapy for a wide variety of diseases. An absolute requirement is that the applied microcapsules have an optimal biocompatibility. The alginate-poly-l-lysine system is the most commonly applied system but is still suffering from tissue responses provoked by the capsule materials. In the present study, we investigate the biocompatibility of microcapsules elaborated with two commonly applied alginates, i.e. an intermediate-G alginate and a high-G alginate. These alginates were coated with poly-l-lysine (PLL), poly-d-lysine (PDL) and poly-l-ornithine (PLO). The main objective of this study is to determine the interaction of each alginate matrix with the different polycations and the potential impact of these interactions in the modulation of the host's immune response. To address these issues the different types of microcapsules were implanted into the peritoneal cavity of rats for 1 month. After this period the microcapsules were recovered and they were evaluated by different techniques. Monochromatised X-ray photoelectron spectroscopy (XPS) was performance and the degree of capsular recovery, overgrowth on each capsule, and the cellular composition of the overgrowth were evaluated by histology. Our results illustrate that the different observed immune responses are the consequence of the variations in the interactions between the polycations and alginates rather than to the alginates themselves. Our results suggest that PLL is the best option available and that we should avoid using PLO and PDL in its present form since it is our goals to produce capsules that lack overgrowth and do not induce an immunological response as such.
Keywords: Alginate; Biocompatibility; Immune response; Microcapsule; Surface modification; X-ray photoelectron spectroscopy (XPS);

Practical application of a chromogenic FXIIa assay by Rui Zhuo; Erwin A. Vogler (4840-4845).
Autohydrolysis of blood factor XII ( FXII + FXIIa → 2 FXIIa ) is found to be a facile reaction in neat-buffer buffer solutions of FXII but an insignificant reaction in the presence of plasma proteins. Autohydrolysis causes a chromogenic assay for FXIIa in buffer solution to strongly deviate from the traditional plasma-coagulation assay. Autohydrolysis can be accommodated by performing chromogenic detection of FXIIa as a rate assay in swamping concentrations of FXII. Rate-assay results performed in this way are shown to be in analytical agreement with the plasma-coagulation assay. Autohydrolysis can be used as a means of amplifying FXIIa produced by contacting neat-buffer solutions of FXII with biomaterials, suggesting a route to highly sensitive measurement of biomaterial hemocompatibility.
Keywords: Blood coagulation; Factor XII; Hageman factor; Autoactivation; Autohydrolysis;

The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function by Coby C. Larsen; Faina Kligman; Kandice Kottke-Marchant; Roger E. Marchant (4846-4855).
We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120° to 6°, indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.
Keywords: Endothelial cells; Surface modification; ePTFE vascular grafts; RGD peptide;

The effect of silica nanoparticulate coatings on serum protein adsorption and cellular response by M.S. Lord; B.G. Cousins; P.J. Doherty; J.M. Whitelock; A. Simmons; R.L. Williams; B.K. Milthorpe (4856-4862).
Serum protein adsorption on colloidal silica surfaces was investigated using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The amount of serum proteins adsorbed on colloidal silica-coated surfaces was not significantly different from the control silica surfaces, with the exception of 21 nm colloidal silica which experienced significantly less ( P < 0.05 ) fibrinogen adsorption compared with control silica. The adhesion and proliferation of human endothelial cells (C11STH) on nano-scale colloidal silica surfaces were significantly reduced compared with control silica surfaces, suggesting that the conformation of adsorbed proteins on the colloidal silica surfaces plays a role in modulating the amount of cell binding. Fibronectin is one of the main extracellular matrix proteins involved in endothelial cell attachment to biomaterial surfaces. There was reduced binding of a monoclonal anti-fibronectin antibody, that reacted specifically with the cell-binding fragment, to fibronectin-coated colloidal silica surfaces compared with control silica surfaces. This suggests that the fibronectin adsorbed on the colloidal silica-coated surfaces was conformationally changed compared with control silica reducing the availability of the cell-binding domain of fibronectin.
Keywords: Surface topography; Nanostructures; Protein adsorption; Cellular response;

A seeding device for tissue engineered tubular structures by Lorenzo Soletti; Alejandro Nieponice; Jianjun Guan; John J. Stankus; William R. Wagner; David A. Vorp (4863-4870).
One of the challenges in the tissue engineering of tubular tissues and organs is the efficient seeding of porous scaffolds with the desired cell type and density in a short period of time, without affecting cell viability. Though different seeding techniques have been investigated, a fast, reproducible, and efficient bulk seeding method with uniform cellular distribution has yet to be reported. In this paper, a novel seeding device utilizing the synergistic effects of vacuum, centrifugal force and flow has been developed and analyzed. The device allows porous tubular scaffolds to be uniformly bulk seeded as well as luminally surface-seeded with cells. Porous tubular polymer scaffolds were bulk and surface-seeded with cell suspensions, and cell viability and seeding efficiency were subsequently assessed. A rigorous quantitative analysis of shear stresses acting on the cells during the seeding process, and of cell location within the scaffolds following seeding was also performed. Our results showed that the scaffolds were uniformly seeded along the longitudinal and circumferential directions within the tube wall without affecting cell viability or exposing them to excessive shear stresses.
Keywords: Cell seeding; Computational fluid dynamics; Endothelialization; Tubular scaffold; Vascular grafts;

Transplantation of dopamine neurons is a promising approach to treat Parkinson's disease. Embryonic stem (ES) cells are expected to be a cell source of the dopaminergic neurons. Various difficulties, however, need to be overcome to realize cell therapy of Parkinson's disease using dopaminergic neurons from ES cells. For example, they are highly sensitive to enzymatic treatment and physical dissociation, and the patient's immune system may attack the transplanted cells. In this study, we attempted to induce dopaminergic neurons from mouse ES cells enclosed in hollow fibers using conditioning medium from PA6 cells, the stromal cells derived from skull bone marrow. β-tubulin type III positive cells and tyrosine hydroxylase positive cells were efficiently derived in hollow fibers after 16 days in culture, and dopamine release was observed when the hollow fibers containing cells were exposed to 56 mm KCl for 15 min to induce dopamine release through depolarization of the neurons. By our procedure, enclosure of dopaminergic neurons in hollow fibers was easily performed without loss of cells, and the hollow fiber membrane is expected to efficiently protect dopaminergic neurons from mechanical disturbances and attacks by the host immune system. Although there are many issues, especially related to immuno-isolation, that still remain to be addressed, we believe that differentiation of ES cells within hollow fibers is one of the crucial procedures so that cell therapy of Parkinson's disease can be realized.
Keywords: Embryonic stem cell; Hollow fiber; Dopaminergic neuron; PA6 cell; Parkinson's disease;

The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells by Shelly R. Peyton; Christopher B. Raub; Vic P. Keschrumrus; Andrew J. Putnam (4881-4893).
Hydrogels based on poly(ethylene glycol) (PEG) are of increasing interest for regenerative medicine applications and are ideal materials to direct cell function due to the ability to confer key functionalities of native extracellular matrix (ECM) on PEG's otherwise inert backbone. Given extensive recent evidence that ECM compliance influences a variety of cell functions, PEG-based hydrogels are also attractive due to the ease with which their mechanical properties can be controlled. In these studies, we exploited the chemical and mechanical tunability of PEG-based gels to study the impact of ECM chemistry and mechanics on smooth muscle cells (SMCs) in both 2-D and 3-D model systems. First, by controlling the extent of crosslinking and therefore the mechanical properties of PEG-based hydrogels (tensile moduli from 13.7 to 423.9 kPa), we report here that the assembly of F-actin stress fibers and focal adhesions, indicative of the state of actin contractility, were influenced by the compliance of 2-D PEG gels functionalized with either short adhesive peptides or full-length ECM proteins. Varying ECM ligand density and identity independent of gel compliance affected the physical properties of the focal adhesions, and also influenced SMC spreading in 2-D. Furthermore, SMCs proliferated to a greater extent as gel stiffness was increased. In contrast, the degree of SMC differentiation, which was qualitatively assessed by the extent of smooth muscle α-actin bundling and the association of calponin and caldesmon with the α-actin fibrils, was found to decrease with substrate stiffness in 2-D cultures. In 3-D, despite the fact that their viability and degree of spreading were greatly reduced, SMCs did express some contractile markers indicative of their differentiated phenotype when cultured within PEG–RGDS constructs. Combined, these data suggest that the mechanical and chemical properties of PEG hydrogels can be tuned to influence SMC phenotype in both 2-D and 3-D.
Keywords: ECM; Polyethylene; Smooth muscle cell; Hydrogel; Cell proliferation; Mechanical properties;

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170–200 μm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.
Keywords: Chitosan; Poly(lactic acid-glycolic acid); Microsphere; Scaffold; Bone tissue engineering;

Efficient cell attachment to biodegradable polymer scaffolds is a necessary prerequisite in tissue engineering. However, it is difficult to evenly cover scaffold surfaces with cells because scaffolds are generally highly porous, with complex three-dimensional (3D) surfaces. In this article, we demonstrate the efficiency of avidin–biotin binding systems (ABBS) for the initial attachment of biotinylated Hep G2 cells to avidin adsorbed flat, two-dimensional (2D) and highly porous 3D poly l-lactic acid (PLLA) surfaces. The potential toxicity of biotinylation and/or strong ABBS binding forces was also investigated. ABBS assisted Hep G2 cells to adhere to a flat PLLA surface within 10 min; the proliferation of these attached cells was comparable with control intact cells cultured on collagen. Hepatic functions of the attached cells, such as albumin secretion, induction of CYP1A1 and CYP1A2 genes, and metabolic capacity of CYP1A1/2 as measured by the ethoxyresorufin O-deethylase assay, were not significantly changed. Also, a stimulus of a cytokine: oncostatin M (OSM) phosphorylated an intracellular signaling molecule, extracellular signal-related kinase 1 (ERK1) via transmembrane receptor complex, at 24 h after inoculation by ABBS. In addition, efficient attachment of Hep G2 cells to a highly porous PLLA 3D scaffold was demonstrated. These results clearly show that ABBS is useful for rapidly trapping cells in both biodegradable, polymer-based, flat 2D surfaces, and in highly porous 3D scaffolds. Furthermore, binding hepatic cells by this technique has only small effects on liver-specific functions, or on signal transfer ability of transmembrane receptor complexes.
Keywords: Avidin–biotin binding system; Cell attachment; Poly l-lactic acid; Liver functions; Tissue engineering;

Electrospinning (ESP) has lately shown a great potential as a novel scaffold fabrication technique for tissue engineering. Scaffolds are produced by spinning a polymeric solution in fibers through a spinneret connected to a high-voltage electric field. The fibers are then collected on a support, where the scaffold is created. Scaffolds can be of different shapes, depending on the collector geometry, and have high porosity and high surface per volume ratio, since the deposited fibers vary from the microscale to the nanoscale range. Such fibers are quite effective in terms of tissue regeneration, as cells can bridge the scaffold pores and fibers, resulting in a fast and homogeneous tissue growth. Furthermore, fibers can display a nanoporous ultrastructure due to solvent evaporation. The aim of this study was to characterize electrospun scaffolds from poly(ethylene oxide terephthalate)–poly(butylene terephthalate) (PEOT/PBT) copolymers and to unravel the mechanism of pore formation on the fibers. The effect of different fiber diameters and of their surface nanotopology on cell seeding, attachment, and proliferation was studied. Smooth fibers with diameter of 10 μm were found to support an optimal cell seeding and attachment within the micrometer range analyzed. Moreover, a nanoporous surface significantly enhanced cell proliferation and cells spreading on the fibers. The fabrication of ESP scaffolds with incorporated dyes with different molecular dimensions is also reported and their release measured. These findings contribute to the field of cell–material interaction and lead to the fabrication of “smart” scaffolds which can direct cells morphology and proliferation, and eventually release biological signals to properly conduct tissue formation.
Keywords: Electrospinning; Nanoporosity; Cell attachment; Cell morphology; Drug release;

Manufacturing and morphology structure of polylactide-type microtubules orientation-structured scaffolds by Fei Yang; Xue Qu; Wenjin Cui; Jianzhong Bei; Fangyuan Yu; Shibi Lu; Shenguo Wang (4923-4933).
Tissue engineering using scaffold not only should have biodegradability and a certain 3D structure, but also its morphology structure should be mimetic to that of the repaired natural tissue. So to manufacture the scaffold with a biomimetic structure as the natural tissues is important. In this research, highly porous poly(l-lactic acid) (PLLA) and poly(l-lactic-co-glycolic acid) (PLGA) scaffolds with microtubules orientation structure were designed and fabricated by using dioxane as solvent and an improved thermal-induced phase separation (TIPS) technique. All the factors which will affect solvent crystallization and microtubules orientation structure of the scaffold, such as the type of the solvent and polymer, concentration of the polymer solution, and temperature-gradient of the system have been studied carefully. So the porosity, diameter, tubular morphology and orientation of the microtubules could be controlled by adjusting the concentration of the polymer solution and temperature-gradient of the system. The scaffold with diameter of microtubules from 40 to 240 μm and high porosity up to 96% could be obtained by adjusting temperature-gradient during the TIPS process. By increasing concentration of the polymer solution the regularity of the microtubular scaffold has been improved and the thickness of wall of the microtubules has been increased as well. In vitro cell culture results show that after the scaffolds have been improved by the ammonia plasma treatment and then collagen anchorage method, the human transparent cartilage cells H144, could be seeded deeply into the microtubules orientation-structured scaffolds and grew well there.
Keywords: Microtubules orientation-structured scaffold; Thermal-induced phase separation; Manufacturing; Polylactide; PLGA; Tissue engineering;

A new method to repair rib defects with biomaterials containing recombinant human bone morphogenetic protein-2 (rhBMP-2) is presented in this report. We had reported previously the successful regeneration of bony rib defects by placing a short chain of small beta-tricalcium phosphate (β-TCP) cylinders on the intact periosteum. The multi-cylinder implants were ineffective in promoting rib repair when the periosteum was absent. By adding rhBMP-2 to the β-TCP cylinders, we were able to promote rib bone regeneration in the presence or absence of the periosteum. The osteogenic capacity of the rhBMP-2/β-TCP composite implant and the time required to complete regeneration were evaluated in a canine model. An 8 cm long section of rib bone, including the periosteum, was removed and replaced with a chain of the rhBMP-2/β-TCP cylinders. At 6 weeks after implantation, the ribs were restored to their original configuration and mechanical strength. The multi-cylinder β-TCP implants were degraded and replaced by new bone in 12 weeks. This new degradable bone-inducing implant material has significant clinical potential for rib repair.
Keywords: Bone regeneration; Bone morphogenetic protein (BMP); Calcium phosphate; Biodegradation;

Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles by Dennis P. Link; Juliette van den Dolder; Wouter J.F.M. Jurgens; Joop G.C. Wolke; John A. Jansen (4941-4947).
In this study, the mechanical properties of an implanted calcium phosphate (CaP) cement incorporated with 20 wt% poly (dl-lactic-co-glycolic acid) (PLGA) microparticles were investigated in a rat cranial defect. After 2, 4 and 8 weeks of implantation, implants were evaluated mechanically (push-out test) and morphologically (Scanning Electron Microscopy (SEM) and histology). The results of the push-out test showed that after 2 weeks the shear strength of the implants was 0.44±0.44 MPa (average±sd), which increased to 1.34±1.05 MPa at 4 weeks and finally resulted in 2.60±2.78 MPa at 8 weeks. SEM examination showed a fracture plane at the bone–cement interface at 2 weeks, while the 4- and 8-week specimens created a fracture plane into the CaP/PLGA composites, indicating an increased strength of the bone–cement interface. Histological evaluation revealed that the two weeks implantation period resulted in minimal bone ingrowth, while at 4 weeks of implantation the peripheral PLGA microparticles were degraded and replaced by deposition of newly formed bone. Finally, after 8 weeks of implantation the degradation of the PLGA microparticles was almost completed, which was observed by the bone ingrowth throughout the CaP/PLGA composites.On basis of our results, we conclude that the shear strength of the bone–cement interface increased over time due to bone ingrowth into the CaP/PLGA composites. Although the bone–cement contact could be optimized with an injectable CaP cement to enhance bone ingrowth, still the mechanical properties of the composites after 8 weeks of implantation are insufficient for load-bearing purposes.
Keywords: Injectable CaP/PLGA cement; Mechanical properties; Bone ingrowth;

Poly (ε-caprolactone-co-α-iodo-ε-caprolactone) was synthesized by binding iodine to PCL chain bearing carbanionic site on α-position of carbonyl groups using lithium diisopropylamide. Copolyesters containing from 10% to 25% of iodo-units were thus obtained. Viscoelastic properties (modulus, loss angle), thermal properties (T m, T c, ΔH m), crystallinity and in vitro degradability of this new type of copolymers were measured. Their opacity to X-rays was assessed, and appeared high enough to be of interest for biomedical applications.
Keywords: Polycaprolactone; Copolymer; Radio-opacity; Degradation;

Long-term biocompatibility of a corrodible peripheral iron stent in the porcine descending aorta by Matthias Peuster; Carola Hesse; Tirza Schloo; Christoph Fink; Philipp Beerbaum; Christian von Schnakenburg (4955-4962).
Currently there are no biodegradable stents available for treatment of vascular obstructions in patients with congenital heart defects. This study was performed to evaluate the safety of a corrodible stent produced from pure iron in a peripheral stent design (6–12 mm diameter) in a slotted tube design similar to a commercially available 316-L stent which served as control. Both stents were implanted into the descending aorta of 29 minipigs with an overstretch injury without technical problems. Two animals died after the implantation not related to the iron stent. The remaining 27 minipigs were followed for 1–360 days. Histomorphometry and quantitative angiography showed no difference with regard to the amount of neointimal proliferation between 316-L and iron stents. Histopathological examination of heart, lung, spleen, liver, kidney and para-aortic lymphatic nodes demonstrated no signs of iron overload or iron-related organ toxicity. Adjacent to the iron stent struts, there was no evidence for local toxicity due to corrosion products. We conclude that iron is a suitable metal for the production of a large-size degradable stent with no local or systemic toxicity. A faster degradation rate, however, is desirable and further studies have to focus on the modification of the composition and design of the stent to expedite the degradation process.
Keywords: Biodegradation; Corrosion; Degradation; Intimal hyperplasia; Stent;

The brain tissue response to biodegradable poly(methylidene malonate 2.1.2)-based microspheres in the rat by Elvire Fournier; Catherine Passirani; Nathalie Colin; Serge Sagodira; Philippe Menei; Jean-Pierre Benoit; Claudia N. Montero-Menei (4963-4974).
The aim of this study was to follow the in vivo biodegradation as well as to appreciate the brain tissue response to poly(methylidene malonate 2.1.2) (PMM 2.1.2)-based microspheres implanted into the rat brain. Ninety-three adult Sprague–Dawley female rats were engaged in the study in which 54 underwent stereotactic implantation of blank γ-sterilized PMM 2.1.2-based microspheres, prepared by an emulsion-extraction method. Twelve rats were implanted with the same 5-fluorouracil (5-FU)-loaded microspheres. Seventeen controls received the suspension medium alone (carboxymethylcellulose aqueous solution). The animals were sacrificed on post-operative days 1, 2, 8 and months 1, 2, 3, 6, 9, 12, 15 and 18. The brains were dissected, frozen, cut in a freezing microtome, and the slides were processed for immunohistological evaluation and scanning electron microscopy.During the first few days, the moderate inflammatory response to blank or loaded PMM 2.1.2 microspheres was largely a consequence of the mechanical trauma that occurs during surgery. The macrophagous-microglial reaction was similar to the one typically found following any damage in the CNS. There were also no differences in GFAP reactivity between the implanted animals and the controls. Blank microspheres began to degrade between 3 and 6 months, while 5-FU microspheres degraded between 8 days and 1 month. The polymer degradation generated in both cases a pronounced inflammatory and immunological reaction, leading to an important cell loss, a cerebral atrophy and to the death of several animals. PMM 2.1.2 was thus shown to be inadequate for intracerebral drug delivery.
Keywords: Poly(methylidene malonate 2.1.2); Biocompatibility; 5-fluorouracil; Biodegradation; Microspheres; Rat brain;

Trastuzumab-modified nanoparticles: Optimisation of preparation and uptake in cancer cells by Isabel Steinhauser; Birgit Spänkuch; Klaus Strebhardt; Klaus Langer (4975-4983).
Nanoparticles consisting of human serum albumin (HSA) represent a promising strategy for targeted drug delivery to tumour cells. The coupling of the antibody trastuzumab to HSA nanoparticles takes advantage of the capability of HER2-positive cells to incorporate substances binding to HER2. In our present study, we developed nanoparticles based on HSA which were covalently modified on their surface with thiolated trastuzumab. A special focus was on the optimisation of the thiolation procedure of the antibody under the aspect of an effective binding to particle surfaces. Different thiolation conditions were evaluated and the degree of antibody dimerisation was determined. We analysed the thiolated antibody by size exclusion chromatography (SEC) and identified the best thiolation procedure for the preparation of trastuzumab-conjugated nanoparticles. Over a storage period of 6 weeks the resulting particles were stable and physico-chemical properties such as size and zetapotential did not show any changes. Biological activity was confirmed under cell culture conditions: antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis. These data provide the basis for the development of stable and biological active carrier systems for directed targeting of tumour cells using trastuzumab-conjugated HSA nanoparticles.
Keywords: Albumin; Drug delivery; Flow cytometry; Nanoparticle; Surface modification; Thiol;

A study of thermoresponsive poly(N-isopropylacrylamide)/polyarginine bioconjugate non-viral transgene vectors by Nan Cheng; Wenguang Liu; Zhiqiang Cao; Weihang Ji; Dongchun Liang; Gang Guo; Jingyu Zhang (4984-4992).
A thermoresponsive poly(N-isopropylacrylamide)/poly(l-arginine)bioconjugate (PNIPArg) was prepared by radical polymerization and EDC-activated coupling. The lower critical solution temperature (LCST) of PNIPArg aqueous solution determined by turbidimetry was around 35.2 °C. The transmission electron microscope (TEM) showed that the PNIPArg/DNA complexes appeared uniform nanospheres with size about 50–120 nm. Variable temperature circular dichroism (CD) and gel electrophoresis results revealed that the association and dissociation of PNIPArg/DNA complexes could be tuned by varying temperature; polyarginine (PArg) showed no temperature-controllable change of DNA condensate. Incorporation of PNIPAAm considerably decreased the cytotoxicity of PArg. The transfection level of PNIPArg and PArg was evaluated with COS-1 cells using two different reporter genes, pGL3-Control encoding luciferase and pEGP-C1 encoding green fluorescent protein (GFP). The transfection efficiency of PNIPArg incubated at 37 °C for 22 h, 20 °C for 2 h and 37 °C for 24 h was enhanced to a different extent depending on PNIPArg/DNA ratios compared to that incubated at 37 °C for 48 h. Encouragingly, at PNIPArg/DNA mass ratio of 3/1, the transfection efficiency of PNIPArg obtained with variable temperature route was equivalent to that of LipofectamineTM 2000.
Keywords: Poly(N-isopropylacrylamide); Poly(l-arginine); Thermoresponsive; Non-viral vector; Gene transfection;

Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: Comparison of adenovirus mediated gene transfer and protein delivery of BMP-2 by Lorenz Meinel; Sandra Hofmann; Oliver Betz; Robert Fajardo; Hans P. Merkle; Robert Langer; Christopher H. Evans; Gordana Vunjak-Novakovic; David L. Kaplan (4993-5002).
Bone tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs) and silk fibroin biomaterials were combined to study the impact of viral transfection on MSC osteogenic performance in vitro. MSCs were transduced with adenovirus containing a human BMP-2 (Ad-BMP-2) gene at clinically reasonable viral concentrations and cultured for 4 weeks. Controls with nontransfected MSCs, but exposed to exogenous BMP-2 concentrations on an analogous time profile as that secreted by the Ad-BMP-2 group, were compared. Both the Ad-BMP-2 MSC group and the exogenous protein BMP-2 group strongly expressed osteopontin and bone sialoprotein. Cells secreted a matrix that underwent mineralization on the silk fibroin scaffolds, forming clusters of osseous material, as determined by micro-computed tomography. The expression of osteogenic marker proteins and alkaline phosphatase was significantly higher in the Ad-BMP-2 MSC group than in the exogenous protein BMP-2 group, and no significant differences in mineralization were observed in two of the three MSC sources tested. The results demonstrate that transfection resulted in higher levels of expression of osteogenic marker genes, no change in proliferation rate and did not impact the capacity of the cells to calcify tissues on these protein scaffolds. These findings suggest additional options to control differentiation where exogenous additions of growth factors or morphogens can be replaced with transfected MSCs.
Keywords: Mesenchymal stem cells; bone morphogenetic protein (BMP-2); Silk; Tissue engineering; Adenoviral gene transfer; Micro-computed tomography;