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Biomaterials (v.28, #11)

Editorial board (pp. co2).

Self-assembled molecular magnets on patterned silicon substrates: Bridging bio-molecules with nanoelectronics by C.-C. Chia-Ching Chang; Kien Wen Sun; S.-F. Shang-Fan Lee; L.-S. Lou-Sing Kan (pp. 1941-1947).
The paper reports the methods of preparing molecular magnets and patterning of the molecules on a semiconductor surface. A highly magnetically aligned metallothionein containing Mn and Cd (Mn,Cd-MT-2) is first synthesized, and the molecules are then placed into nanopores prepared on silicon (001) surfaces using electron beam lithography and reactive ion-etching techniques. We have observed the self-assemble growth of the MT molecules on the patterned Si surface such that the MT molecules have grown into rod or ring type three-dimensional nanostructures, depending on the patterned nanostructures on the surface. We also provide scanning electron microscopy, atomic force microscopy, and magnetic force microscope studies of the molecular nanostructures. This engineered molecule shows molecular magnetization and is biocompatible with conventional semiconductors. These features make Mn,Cd-MT-2 a good candidate for biological applications and sensing sources of new nanodevices. Using molecular self-assembly and topographical patterning of the semiconductor substrate, we can close the gap between bio-molecules and nanoelectronics built into the semiconductor chip.

Keywords: Silicon; Surface modification; Electron beam lithography; Molecular magnet

Antibacterial nitric oxide-releasing xerogels: Cell viability and parallel plate flow cell adhesion studies by Evan M. Hetrick; Mark H. Schoenfisch (pp. 1948-1956).
The ability of nitric oxide (NO)-releasing xerogels to reduce adhesion of Pseudomonas aeruginosa under flowing conditions was evaluated using a parallel plate flow chamber. At a controlled bacterial suspension flow rate of 0.2mL/min, the NO-releasing xerogels reduced bacterial adhesion in a flux-dependent fashion, with an NO flux of ∼21pmolcm−2s−1 reducing P. aeruginosa adhesion by ∼65% compared to controls. Fluorescent viability staining indicated that bacteria adhered to NO-releasing xerogels were killed within 7h. Quantitative cell-plating viability studies showed that the extent of bactericidal activity was dependent on the total amount of NO released, with 750nmolcm−2 killing >90% more adhered bacteria than xerogels releasing 25nmolcm−2. Thus, NO-releasing xerogels were shown to both inhibit P. aeruginosa adhesion and kill adhered bacteria cells, two important steps toward designing anti-infective biomaterial coatings.

Keywords: Nitric oxide; Xerogel; Bacterial adhesion; Cell viability; Implant infection

Functional and histological evaluation of transplanted pancreatic islets immunoprotected by PEGylation and cyclosporine for 1 year by Dong Yun Lee; Jong Hee Nam; Youngro Byun (pp. 1957-1966).
Pancreatic islet transplantation is one of the most promising strategies for patients suffering from type 1 diabetes mellitus, but several therapeutic immunosuppressive medications must be administered simultaneously to protect transplanted islets in the long-term, and these expose patients to the risk of serious complications. Thus, we developed chemically modified islets with a protective poly(ethylene glycol) (PEG) layer, which reduces immunogenicity by preventing cellular immune reactions. We report here that PEG-based chemical immunomodulation can provide a semi-permanent effective therapy that protects transplanted islets at least for 1 year when accompanied by cyclosporine. Moreover, this combinatorial approach appears to avoid the toxicities associated with immunosuppressive medications because of the reduced amounts of medication required. Also, the conjugated PEG molecules were found to be continuously present at the transplanted islets. However, unmodified islets (control) were completely eliminated within 2 weeks even when CsA was administered. These results strongly suggest that this new combinatorial therapy provides a semi-permanent, effective clinical means of attenuating transplanted islet immunogenicity for a long time, whilst avoiding the toxicities associated with therapeutic levels of immunosuppressants owing to the minimized immunosuppressant.

Keywords: Diabetes; Islet; Cyclosporine; Immunomodulation; PEGylation; Transplantation

The effect of nanofiber alignment on the maturation of engineered meniscus constructs by Brendon M. Baker; Robert L. Mauck (pp. 1967-1977).
The fibrocartilaginous menisci are load-bearing tissues vital to the normal functioning of the knee. Removal of damaged regions of the meniscus subsequent to injury impairs knee function and predisposes patients to osteoarthritis. In this study, we employed biodegradable nanofibrous scaffolds for the tissue engineering of the meniscus. Non-aligned (NA) or fiber-aligned (AL) nanofibrous scaffolds were seeded with meniscal fibrochondrocytes (MFCs) or mesenchymal stem cells (MSCs) to test the hypothesis that fiber-alignment would augment matrix content and organization, resulting in improved mechanical properties. Additionally, we proposed that MSCs could serve as an alternative to MFCs. With time in culture, MSC- and MFC-seeded NA and AL constructs increased in cellularity and extracellular matrix (ECM) content. Counter our initial hypothesis, NA and AL constructs contained comparable amounts of ECM, although a significantly larger increase in mechanical properties was observed for AL compared to NA constructs seeded with either cell type. Cell-seeded NA constructs increased in modulus by ∼1MPa over 10 weeks while cell-seeded AL construct increased by >7MPa. Additionally, MSC-constructs yielded greater amounts of ECM and demonstrated comparable increases in mechanical properties, thereby confirming the utility of MSCs for meniscus tissue engineering. These results demonstrate that cell-seeded fiber-aligned nanofibrous scaffolds may serve as an instructive micro-pattern for directed tissue growth, reconstituting both the form and function of the native tissue.

Keywords: Tissue engineering; Mechanical properties; Electrospinning; Anisotropy; Fibrochondrocytes; Mesenchymal stem cells

Pharmacologically active microcarriers releasing glial cell line – derived neurotrophic factor: Survival and differentiation of embryonic dopaminergic neurons after grafting in hemiparkinsonian rats by V.M. Valérie M. Tatard; Laurence Sindji; Jennifer (Godbee) Branton; Aubert-Pouëssel Anne Aubert-Pouëssel; Jacqueline Colleau; J.-P. Jean-Pierre Benoit; Montero-Menei Claudia N. Montero-Menei (pp. 1978-1988).
To improve the outcome of foetal dopaminergic cell transplantation for the treatment of Parkinson's disease, pharmacologically active microcarriers (PAM) were developed. PAM are able to convey cells on their surface and release a growth factor to improve cell survival, differentiation and integration after brain implantation. Lysozyme-releasing PAM were first produced and characterized. They served as a model system for the development of glial cell line-derived neurotrophic factor (GDNF)-releasing PAM conveying foetal ventral mesencephalic (FVM) cells. The effects of the intrastriatal implantation of this system were studied in hemiparkinsonian rats during a 6-week period. This study reports on the degradation of coated and non-coated PAM and the release of lysozyme and of biologically active GDNF for 42 days. Unloaded and GDNF-loaded PAM conveying FVM cells allowed a high improvement of the grafted cell survival and of fibre outgrowth, when compared to the cells transplanted alone. The animals receiving the PAM showed an earlier improvement in amphetamine-induced rotational behaviour compared to animals receiving FVM cells only; behaviour that appears to be more regular and stable with the GDNF-releasing PAM. The use of PAM to convey foetal cells is thus an efficient strategy for cell therapy in neurodegenerative diseases, as it allows improvement of cell survival and fibre outgrowth inducing a rapid recovery of behaviour using only low amounts of cells.

Keywords: Transplantation; Microspheres; Drug release; Growth factor; Nerve tissue engineering; Progenitor cell

A fusion protein of hepatocyte growth factor for immobilization to collagen by Takashi Kitajima; Hiroshi Terai; Yoshihiro Ito (pp. 1989-1997).
We describe here a fusion protein consisting of hepatocyte growth factor (HGF; an angiogenic factor) and a collagen-binding domain (CBD) polypeptide of fibronectin (FN). This fusion protein (CBD-HGF), produced by a baculovirus expression system, exhibited much stronger collagen binding activity than native HGF in the range of 0.4–6.4μg/ml. Its binding at the lowest concentration exceeded that of HGF at the highest concentration. In addition, the collagen-bound CBD-HGF promoted growth of endothelial cells (ECs) to a greater degree at least 4 days longer than HGF added to the culture medium; about 5-fold greater increase in cell number after 10 days. These findings suggest that the fused CBD moiety not only helped immobilize HGF on collagen but also helped stabilize the fusion molecule, resulting in prolonged activity. The angiogenic activity of CBD-HGF in animal tissues was examined by subcutaneously implanting collagen sponges containing bound CBD-HGF. Blood vessel formation in the sponges after 7 days was 4–6-fold extensive as compared to the control sponges without sample. Implanted sponges with native HGF did not show significant difference from control. These results indicate that CBD-HGF is suitable for in vitro culture of ECs, and that this fusion protein can be used to confer HGF activity on biomaterials for use in tissue engineering.

Keywords: Growth factors; HGF; Immobilization; Collagen; Endothelial cells; Angiogenesis

One-step fabrication of porous micropatterned scaffolds to control cell behavior by Bernke J. Papenburg; Laura Vogelaar; Lydia A.M. Bolhuis-Versteeg; Rob G.H. Lammertink; Dimitrios Stamatialis; Matthias Wessling (pp. 1998-2009).
This paper reports a one-step method to fabricate highly porous micropatterned 2-D scaffold sheets. The scaffold sheets have high glucose diffusion, indicating that the porosity and pore morphology of the scaffolds are viable with respect to nutrient transport, and a micropattern for cell alignment. HUVEC culturing proved that the scaffold sheets are suitable for cell culturing. More extensive culturing experiments with mouse myoblasts, C2C12, and mouse osteoblasts, MC3T3, showed that tissue organization can be controlled; the micropattern design affects the extent of cell alignment and tissue formation. Cells are favorably settled in the micropattern and even at higher confluence levels, when the cells start to overgrow the ridges of the micropattern, these cells align themselves in the direction of the micropattern. Preliminary multi-layer stacking experiments indicate that the 2-D scaffold sheets are very promising as basis for building 3-D scaffolds.

Keywords: Scaffold; Tissue engineering; Phase separation; Micropatterning; Porosity; Cell alignment

Three-dimensional culture of annulus fibrosus cells within PDLLA/Bioglass® composite foam scaffolds: Assessment of cell attachment, proliferation and extracellular matrix production by Wilda Helen; Catherine L.R. Merry; Jonny J. Blaker; Julie E. Gough (pp. 2010-2020).
The objective of the present study was to assess cell attachment, proliferation and extracellular matrix (ECM) production by bovine annulus fibrosus (BAF) cells cultured in vitro in PDLLA/Bioglass® composite foams. PDLLA foams incorporated with different percentages (0, 5 and 30wt%) of Bioglass® particles were prepared by thermally induced phase separation (TIPS) process and characterized by scanning electron microscopy (SEM). BAF cell morphology and attachment within the PDLLA/Bioglass® foams were analysed using SEM. An assessment of cell proliferation was conducted using the WST-1 assay. The amount of sulphated glycosaminoglycans (sGAG) were quantified using the 1,9-dimethylmethylene blue (DMMB) assay after 4 weeks in culture. Furthermore, the amount of collagen synthesis was determined using a hydroxyproline assay, and the presence of collagen types I and II was investigated using Western blotting. Our results reveal that PDLLA/Bioglass® foam scaffolds can provide an appropriate microenvironment for BAF cell culture which enhances cell proliferation and promotes the production of sGAG, collagen type I and collagen type II. These findings provide preliminary evidence for the use of PDLLA/Bioglass® composite scaffolds as cell-carrier materials for future treatments of intervertebral discs with damaged AF regions.

Keywords: Intervertebral disc; Tissue engineering; Polylactic acid; Bioactive glass

Limits to the durability of arterial elastic tissue by M.A. Lillie; J.M. Gosline (pp. 2021-2031).
To engineer a better blood vessel, we must identify which structural, mechanical, and biological features of the native vessel must be replicated to ensure long-term survival of the implant. In this study, we tested autoclave-purified elastic tissue from along the pig thoracic aorta under long-term static and cyclic loading to identify factors that affected its durability. Samples were tested in water or in sucrose, which enhances viscoelasticity. Samples failed between 50% and 80% extension, which is lower than the failure extension in shorter, quasi-static tensile tests. Cyclic loading had a small effect on the durability of samples tested in water. Samples from the distal thoracic aorta and samples pre-treated in 70% ethanol showed enhanced durability. Failure between 50% and 80% extension appears associated with structural features of the individual fibre, and indirect evidence suggests it may be due to failure of the microfibrils, not the elastin. Cross-linked elastin may be necessary but insufficient to prevent failure. Durability appears also affected by regional differences in tissue structure, possibly the three-dimensional fibre organization. These results suggest ensuring normal fibre synthesis and organization may be crucial to the design of a successful vascular implant.

Keywords: Elastin; Microfibril; Arterial tissue engineering; Fatigue; Mechanical test

Biofilm formation on surface characterized micro-implants for skeletal anchorage in orthodontics by Mervyn Y.H. Chin; Andrew Sandham; Joop de Vries; Henny C. van der Mei; Henk J. Busscher (pp. 2032-2040).
Micro-implants are increasingly popular in clinical orthodontics to effect skeletal anchorage. However, biofilm formation on their surfaces and subsequent infection of peri-implant tissues can result in either exfoliation or surgical removal of these devices. The present study aimed to assess biofilm formation on five commercially available, surface characterized micro-implant systems in vitro. The elemental surface compositions of as-received and autoclave-sterilized micro-implants were characterized by X-ray photoelectron spectroscopy. High carbon contamination was detected on the oxide surfaces, along with traces of inorganic elements (Ca, Cu, Cr, Pb, Zn, and P) which disappeared after Ar+ ion sputtering. The mean surface roughnesses ( Ra) were around 182nm for titanium micro-implants, and 69nm for stainless steel micro-implants, as measured by atomic force microscopy. Scanning electron microscopy revealed different surface topographies between manufacturers, varying from typical machined grooves to structural defects like pores and pits. Overnight biofilms were grown on micro-implant surfaces by immersion in pooled human whole saliva. Biofilms on micro-implants treated with chlorhexidine and fluoride mouthrinses contained comparable numbers of viable organisms, but significantly less than did untreated micro-implants. Comparison of different implant systems using multiple linear regression analysis indicated that biofilm formation was governed by roughness of the implant surface and the prevalence of carbon- and oxygen-rich components.

Keywords: Biofilm; Titanium; Stainless steel; Orthodontic micro-implants; Chlorhexidine; Fluoride

Nanoparticles of poly(lactide)—tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers for protein drug delivery by Sie Huey Lee; Zhiping Zhang; Si-Shen Feng (pp. 2041-2050).
Nanoparticles (NPs) of poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers with various PLA:TPGS component ratios were prepared by the double emulsion technique for protein drug formulation with bovine serum albumin (BSA) as a model protein. Influence of the PLA:TPGS component ratio and the BSA loading level on the drug encapsulation efficiency (EE) and in vitro drug release behavior was investigated. The PLA-TPGS NPs achieved 16.7% protein drug loading and 75.6% EE, which exhibited a biphasic pattern of controlled protein release with higher initial burst for those NPs of more TPGS content. Furthermore, the released proteins retained good structural integrity for at least 35 days at 37°C as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy. Compared with other biodegradable polymeric NPs such as poly(d,l-lactide-co-glycolide) (PLGA) NPs, PLA-TPGS NPs could provide the encapsulated proteins a milder environment. Confocal laser scanning microscopy (CLSM) observation demonstrated the intracellular uptake of the PLA-TPGS NPs by NIH-3T3 fibroblast cells and Caco-2 cancer cells. This research suggests that PLA-TPGS NPs could be of great potential for clinical formulation of proteins and peptides.

Keywords: Biodegradable polymers; Cancer nanotechnology; Bovine serum albumin; Nanomedicine; Protein drug delivery

Design and evaluation of biodegradable, biosensitive in situ gelling system for pulsatile delivery of insulin by N. Kashyap; B. Viswanad; G. Sharma; V. Bhardwaj; P. Ramarao; M.N.V. Ravi Kumar (pp. 2051-2060).
Biodegradable glucose-sensitive in situ gelling system based on chitosan for pulsatile delivery of insulin was developed. The sols/gels were thoroughly characterized for swelling properties, rheology, texture analysis and water content. The developed glucose-sensitive gels responded to varied glucose concentrations in vitro indicating their ability to function as environment-sensitive systems. Insulin load onto the gels was optimized and was found to affect the rheological behavior of these gels, the final preparation used for in vitro contained 1IU/200μl of the sol. These gels released the entrapped insulin in a pulsatile manner in response to the glucose concentration in vitro. Furthermore, the formulations when evaluated for their in vivo efficacy in streptozotocin-induced diabetic rats at a dose of 3IU/kg, demonstrated their ability to release insulin in response to glucose concentration and were preferred much better against subcutaneously given plain insulin formulation used as the control. Together, these preliminary results indicate that biosensitive chitosan in situ gelling systems have substantial potential as pulsatile delivery systems for insulin.

Keywords: Biosensitive; Biodegradable; Chitosan; Hydrogels; Pulsatile delivery

Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier by T.-i. Tae-il Kim; J.-u. Jung-un Baek; Cheng Zhe Bai; J.-s. Jong-sang Park (pp. 2061-2067).
We synthesized arginine-conjugated polypropylenimine dendrimer G2 (DAB-8), PPI2-R for gene delivery systems. Synthesized PPI2-R could retard plasmid DNA at a weight ratio of 4 completely and PPI2-R polyplexes showed a fluorescence of less than 10% over a charge ratio of 2 by PicoGreen reagent assay, suggesting its good DNA condensing ability. The size of PPI2-R polyplex was measured to about 200nm at a charge ratio of 150. PPI2-R displayed 80–90% cell viability at even a 150μg/mL concentration. Transfection efficiency of PPI2-R was found to be high comparable to that of PEI25kD and to be 8–214 times higher than that of unmodified PPI2 on HeLa and 293 cells. Moreover, PPI2-R showed 4 times higher transfection efficiency than PEI25kD, treating with 10μg pDNA because of its low cytotoxicity on HeLa cells. Finally, PPI2-R showed a transfection efficiency 2–3 times higher than PEI25kD on HUVECs, showing its potency as a gene delivery carrier for primary cells. These results demonstrate that arginine-conjugation of PPI2 is successful in developing a low toxic and highly transfection efficient gene delivery carrier.

Keywords: Gene delivery; Polypropylenimine dendrimer; Arginine-conjugation; Low cytotoxicity; High transfection

Erratum to “A citric acid-based hydroxyapatite composite for orthopedic implants” by Hongjin Qiu; Jian Yang; Pradeep Kodali; Jason Koh; Guillermo A. Ameer (pp. 2068-2068).
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