Biomaterials (v.28, #35)

Designing hydrogel adhesives for corneal wound repair by Mark W. Grinstaff (5205-5214).
Today, corneal wounds are repaired using nylon sutures. Yet there are a number of complications associated with suturing the cornea, and thus there is interest in an adhesive to replace or supplement sutures in the repair of corneal wounds. We are designing and evaluating corneal adhesives prepared from dendrimers—single molecular weight and highly branched polymers. We have explored two strategies to form these ocular adhesives. The first involves a photocrosslinking reaction and the second uses a peptide ligation reaction to couple the individual dendrimers together to form the adhesive. These adhesives were successfully used to repair corneal perforations, close the flap produced in a LASIK procedure, and secure a corneal transplant.
Keywords: Cornea; Wound healing; Hydrogel; Adhesive; Ophthalmology;

Covalent attachment of synthetic and biological molecules to tissue surfaces can be used to enhance local drug delivery, reduce adhesions after surgery, and attach reconstructive biomaterials and tissue-engineered matrices to tissues. We present here a mild approach to coupling polymers to tissue surfaces through an enzyme catalyzed reaction between peptide modified polymer and native protein components of the tissue extracellular matrix (ECM). Tissue transglutaminase (tTG), a Ca2+-dependent enzyme that catalyzes the reaction between lysine and glutamine residues to form a ε(γ-glutaminyl) lysine isopeptide bond, was incubated with cartilage in the presence of lysine (FKG-NH2) and glutamine (GQQQLG-NH2) peptides as well as peptide functionalized poly(ethylene glycol) (PEG). Immunohistochemistry was used to detect the presence of covalently bound PEG polymer at the tissue surface as well as to a depth of as much as 10 μm below the surface. Collagen II, fibronectin, osteopontin and osteonectin were found to react with the peptides and peptide modified PEG in the presence of tTG in solution, suggesting these cartilage ECM components as being substrates in the tissue reaction. The results illustrate the use of tTG as a simple, effective and biologically compatible method of coupling synthetic and biological molecules to cartilage and other tissues containing ECM proteins that are substrates of tTG.
Keywords: Transglutaminase; Cartilage; Peptide; Polymer; Conjugation; Surface;

Diffusion of vitamin E in ultra-high molecular weight polyethylene by Ebru Oral; Keith K. Wannomae; Shannon L. Rowell; Orhun K. Muratoglu (5225-5237).
Vitamin E-doped, radiation crosslinked ultra-high molecular weight polyethylene (UHMWPE) is developed as an alternate oxidation and wear resistant bearing surface in joint arthroplasty. We analyzed the diffusion behavior of vitamin E through UHMWPE and predicted penetration depth following doping with vitamin E and subsequent homogenization in inert gas used to penetrate implant components with vitamin E. Crosslinked UHMWPE (65- and 100-kGy irradiation) had higher activation energy and lower diffusion coefficients than uncrosslinked UHMWPE, but there were only slight differences in vitamin E profiles and penetration depth between the two doses. By using homogenization in inert gas below the melting point of the polymer following doping in pure vitamin E, the surface concentration of vitamin E was decreased and vitamin E stabilization was achieved throughout a desired thickness. We developed an analytical model based on Fickian theory that closely predicted vitamin E concentration as a function of depth following doping and homogenization.
Keywords: Vitamin E; Highly crosslinked polyethylene; Joint arthroplasty;

In this study, a comparison between structure, chemical composition and mechanical properties of collagen fibers at three regions within a human periodontium, has enabled us to define a novel tooth attachment mechanism. The three regions include, (1) the enthesis region: insertion site of periodontal ligament (PDL) fibers (collagen fibers) into cementum at the root surface, (2) bulk cementum, and (3) the cementum–dentin junction (CDJ). Structurally, continuity in collagen fibers was observed from the enthesis, through bulk cementum and CDJ. At the CDJ the collagen fibers split into individual collagen fibrils and intermingled with the extracellular matrix of mantle dentin. Under wet conditions, the collagen fibers at the three regions exhibited significant swelling suggesting a composition rich in polyanionic molecules such as glycosaminoglycans. Additionally, site-specific indentation illustrated a comparable elastic modulus between collagen fibers at the enthesis (1–3 GPa) and the CDJ (2–4 GPa). However, the elastic modulus of collagen fibers within bulk cementum was higher (4–7 GPa) suggesting presence of extrafibrillar mineral.It is known that the tooth forms a fibrous joint with the alveolar bone, which is termed a gomphosis. Although narrower in width than the PDL space, the hygroscopic CDJ can also be termed as a gomphosis; a fibrous joint between cementum and root dentin capable of accommodating functional loads similar to that between cementum and alveolar bone. From an engineering perspective, it is proposed that a tooth contains two fibrous joints that accommodate the masticatory cyclic loads. These joints are defined by the attachment of dissimilar materials via graded stiffness interfaces, such as: (1) alveolar bone attached to cementum with the PDL; and (2) cementum to root dentin with the CDJ. Thus, through variations in concentrations of basic constituents, distinct regions with characteristic structures and graded properties allow for attachment and the load bearing characteristics of a tooth.
Keywords: Atomic force microscopy (AFM); Interface; Collagen fibers; Periodontium; Nanoindentation;

Molecular design and characterization of the neuron–microelectrode array interface by Frauke Greve; Susanne Frerker; Anne Greet Bittermann; Claus Burkhardt; Andreas Hierlemann; Heike Hall (5246-5258).
Electrophysiological activities of neuronal networks can be recorded on microelectrode arrays (MEAs). This technique requires tight coupling between MEA–surfaces and cells. Therefore, this study investigated the interface between DRG neurons and MEA–surface materials after adsorption of neurite promoting proteins: laminin-111, fibronectin, L1Ig6 and poly-l-lysine. Moreover, substrate-induced effects on neuronal networks with time were analyzed. The thickness of adsorbed protein layers was found between ∼1 nm for poly-l-lysine and ∼80 nm for laminin-111 on platinum, gold and silicon nitride. The neuron-to-substrate interface was characterized by Scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and SEM after in situ focused-ion-beam milling demonstrating that the ventral cell membrane adhered inhomogeneously to laminin-111 or L1Ig6 surfaces. Tight areas of 20–30 nm and distant areas <1 μm alternated and even tightest areas did not correlate with the physical thickness of the protein layers. This study illustrates the difficulties to predict cell-to-material interfaces that contribute substantially to the success of in vitro or in vivo systems. Moreover, focused ion beam (FIB)/SEM is explored as a new technique to analyze such interfaces.
Keywords: Dorsal root ganglion neurons; CMOS-electronics; FIB/SEM; QCM-D; Neuron-to-substrate-interface;

In vitro degradation characteristics of photocrosslinked anhydride systems for bone augmentation applications by Ashley A. Weiner; Danielle M. Shuck; Jordan R. Bush; V. Prasad Shastri (5259-5270).
In the past decade, injectable biomaterials that are capable of in situ formation have garnered increased interest for use in restorative orthopedic procedures. In this study, the in vitro degradation of photocrosslinked polyanhydride matrices, derived from methacrylic anhydrides of 1,6-bis(p-carboxyphenoxy)hexane (MCPH) and sebacic acid (MSA) were evaluated over a 6-week period under physiological conditions. These matrices were augmented with two additives—the reactive diluent poly(ethylene glycol) diacrylate (PEGDA) and the buffering agent calcium carbonate (CaCO3). Disk shaped specimens were produced by crosslinking the components using both chemical and photoinitiators and exposure to visible light. The experimental variables studied included: MCPH:MSA ratio, PEGDA molecular weight and weight fraction, and incorporation of CaCO3. The effects of these variables on local pH, water uptake, mass loss, and mechanical properties were explored. Increasing the MCPH:MSA ratio decreased the mass loss and water uptake at predetermined endpoints, and decreased buffer acidity during degradation. Both PEGDA and CaCO3 were found to decrease acidity and to reduce water uptake during degradation. Incorporation of CaCO3 enabled maintenance of compressive modulus during degradation. These results demonstrate that incorporation of reactive diluents and nonreactive additives into networks of photocrosslinked anhydrides can improve system properties as a material for bone replacement.
Keywords: Injectable; Spinal fusion; Drug delivery; Photocurable; Bioerodible;

Silk fibroin microtubes for blood vessel engineering by Michael Lovett; Christopher Cannizzaro; Laurence Daheron; Brady Messmer; Gordana Vunjak-Novakovic; David L. Kaplan (5271-5279).
Currently available synthetic grafts demonstrate moderate success at the macrovascular level, but fail at the microvascular scale (<6 mm inner diameter). We report on the development of silk fibroin microtubes for blood vessel repair with several advantages over existing scaffold materials/designs. These microtubes were prepared by dipping straight lengths of stainless steel wire into aqueous silk fibroin, where the addition of poly(ethylene oxide) (PEO) enabled control of microtube porosity. The microtube properties were characterized in terms of pore size, burst strength, protein permeability, enzymatic degradation, and cell migration. Low porosity microtubes demonstrated superior mechanical properties in terms of higher burst pressures, but displayed poor protein permeability; whereas higher porosity tubes had lower burst strengths but increased permeability and enhanced protein transport. The microtubes also exhibited cellular barrier functions as low porosity tubes prevented outward migration of GFP-transduced HUVECs, while the high porosity microtubes allowed a few cells per tube to migrate outward during perfusion. When combined with the biocompatible and suturability features of silk fibroin, these results suggest that silk microtubes, either implanted directly or preseeded with cells, are an attractive biomaterial for microvascular grafts.
Keywords: Silk fibroin; Vascular grafts; Arterial tissue engineering; Porosity; Endothelial cells; Bioreactor;

Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose-derived mesenchymal stem cells with silk fibroin 3D scaffolds by Joshua R. Mauney; Trang Nguyen; Kelly Gillen; Carl Kirker-Head; Jeffrey M. Gimble; David L. Kaplan (5280-5290).
Biomaterials derived from silk fibroin prepared by aqueous (AB) and organic (HFIP) solvent-based processes, along with collagen (COL) and poly-lactic acid (PLA)-based scaffolds were studied in vitro and in vivo for their utility in adipose tissue engineering strategies. For in vitro studies, human bone marrow and adipose-derived mesenchymal stem cells (hMSCs and hASCs) were seeded on the various biomaterials and cultured for 21 days in the presence of adipogenic stimulants (AD) or maintained as noninduced controls. Alamar Blue analysis revealed each biomaterial supported initial attachment of hMSCs and hASCs to similar levels for all matrices except COL in which higher levels were observed. hASCs and hMSCs cultured on all biomaterials in the presence of AD showed significant upregulation of adipogenic mRNA transcript levels (LPL, GLUT4, FABP4, PPARγ, adipsin, ACS) to similar extents when compared to noninduced controls. Similarly Oil-Red O analysis of hASC or hMSC-seeded scaffolds displayed substantial amounts of lipid accumulating adipocytes following cultivation with AD. The data revealed AB and HFIP scaffolds supported similar extents of lipid accumulating cells while PLA and COL scaffolds qualitatively displayed lower and higher extents by comparison, respectively. Following a 4-week implantation period in a rat muscle pouch defect model, both AB and HFIP scaffolds supported in vivo adipogenesis either alone or seeded with hASCs or hMSCs as assessed by Oil-Red O analysis, however the presence of exogenous cell sources substantially increased the extent and frequency of adipogenesis observed. In contrast, COL and PLA scaffolds underwent rapid scaffold degradation and were irretrievable following the implantation period. The results suggest that macroporous 3D AB and HFIP silk fibroin scaffolds offer an important platform for cell-based adipose tissue engineering applications, and in particular, provide longer-term structural integrity to promote the maintenance of soft tissue in vivo.
Keywords: Adipose; Stem cells; Silk; Tissue;

Computer-aided tissue-engineering approach was used to develop a novel precision extrusion deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/hydroxyapatite (PCL–HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL–HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0°/90° pattern. Two groups of scaffolds having 60% and 70% porosity and with pore sizes of 450 and 750 μm, respectively, were evaluated for their morphology and compressive properties using scanning electron microscopy (SEM) and mechanical testing. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell-scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue-engineering application. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL–HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL–HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.
Keywords: Bone tissue engineering; Polycaprolactone; Hydroxyapatite; Composite; Free form fabrication; Precision extrusion deposition;

A fibrinogen-based precision microporous scaffold for tissue engineering by Michael P. Linnes; Buddy D. Ratner; Cecilia M. Giachelli (5298-5306).
Fibrin has been long used as an effective scaffolding material to grow a variety of cells and tissue constructs. It has been utilized mainly as a hydrogel in varying concentrations to provide an environment in which suspended cells work to rearrange the fibers and lay down their own extracellular matrix. For these fibrin hydrogels to be useful in many tissue-engineering applications, the gels must be cultured for long periods of time in order to increase their mechanical strength to the levels of native tissues. High concentrations of fibrinogen increase the mechanical strength of fibrin hydrogels, but at the same time reduce the ability of cells within the scaffold to spread and survive. We present a method to create a microporous, nanofibriliar fibrin scaffold that has controllable pore size, porosity, and microstructure for applications in tissue engineering. Fibrin has numerous advantages as a scaffolding material as it is normally used by the body as temporary scaffolding for tissue regeneration and healing, and can be autologously sourced. We present here a scaffolding process which enhances the mechanical properties of the fibrin hydrogel by forming it surrounding poly(methyl-methacrylate) beads, then removing the beads with acetone to form an interconnected microporous network. The acetone serves the dual purpose of precipitating and fixing the fibrinogen-based scaffolds as well as adding strength to the network during polymer bead removal. Effects of fibrinogen concentration and time in acetone were examined as well as polymerization with thrombin. A natural crosslinker, genipin, was also used to add strength to the scaffolds, producing a Young's modulus of up to 184±5 kPa after 36 h of reaction. Using these methods we were able to produce microporous fibrin scaffolds that support cell growth and have mechanical properties similar to many native tissues.
Keywords: Fibrin; Fibrinogen; Scaffold; Micropatterning; Hydrogel; Cross-linking;

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell–matrix biology to regulate cell attachment and function.
Keywords: Endothelial cells; Extracellular matrix; Shear stress; Adhesion molecules; Smooth muscle cells;

Epithelialization of hydrogels achieved by amine functionalization and co-culture with stromal cells by Stephen Rimmer; Claire Johnson; Bojun Zhao; Joyleen Collier; Louisa Gilmore; Subodh Sabnis; Paul Wyman; Christopher Sammon; Nigel J. Fullwood; Sheila MacNeil (5319-5331).
The aim of this study was to develop a hydrogel which would be suitable for corneal cell re-epithelialization when used as a corneal implant. To achieve this, a series of hydrogels were functionalized with primary amines by post-polymerization reactions between amine compounds and glycidyl ether groups attached to the hydrogels. We report a strong correlation between the structure of the amine and the viability of stromal cells and epithelial cells cultured on these hydrogels. Subsequent co-culture of epithelial and stromal cells on the amine modified hydrogels allowed successful expansion of epithelial cells on surfaces functionalized with alkyl αω diamines with carbon chain lengths of between 3 and 6. Analysis of variance showed that corneal epithelial cells had a strong preference for surfaces functionalized by the reaction of excess 1,3 diaminopropane with units of glycidyl methacrylate compared to the reaction products of other amines (ammonia; 1,2-diaminoethane; 1,4-diaminobutane or 1,6-diaminohexane). We suggest this approach of amine functionalization combined with stromal/epithelial co-culture offers a promising new approach to achieving a secure corneal epithelium.
Keywords: Hydrogels; Amines; Cornea; Epithelial cells;

Expansion of human bone marrow stromal cells on poly-(dl-lactide-co-glycolide) (PDLLGA) hollow fibres designed for use in skeletal tissue engineering by Suzanne M. Morgan; Simon Tilley; Semali Perera; Marianne J. Ellis; Janos Kanczler; Julian B. Chaudhuri; Richard O.C. Oreffo (5332-5343).
Strategies to expand human bone marrow stromal cells (HBMSC) for bone tissue engineering are a key to revolutionising the processes involved in three-dimensional skeletal tissue reconstruction. To facilitate this process we believe the use of biodegradable porous poly(dl-lactide-co-glycolide) (PDLLGA) hollow fibres as a scaffold used in combination with HBMSC to initiate natural bone repair and regeneration offers a potential solution. In this study, the biocompatibility of 75:25 PDLLGA fibres with HBMSC and the capacity of a PDLLGA fibre-associated HBMSC-monolayer to establish an osteogenic phenotype in vivo was examined. A high proportion of HBMSC survived when expanded on PDLLGA fibres for 6 days, with only 10% of the propidium iodide (pI)-labelled population represented in the sub-G1 DNA peak on analysis by flow cytometry. Tracking carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labelled HBMSC by flow cytometry indicated that HBMSC attachment to the PDLLGA fibres does not interfere with their rate of proliferation. Furthermore, in response to osteogenic stimuli, HBMSC expanded on PDLLGA fibres can differentiate, as expected, along the osteogenic lineage with associated alkaline phosphatase activity. Following implantation into SCID mice, osteogenic-conditioned PDLLGA fibre–HBMSC graft resulted in type I collagen deposition and associated bone mineralisation and osteoid formation, as evidenced by immunohistochemistry and histology. These studies provide evidence that porous PDLLGA hollow fibre–HBMSC graft is an innovative biomaterial that offers new approaches to mesenchymal cell expansion, which could be utilised as a scaffold for skeletal tissue generation.
Keywords: Bone tissue engineering; Poly-(dl-lactide-co-glycolide); Hollow fibre; Human bone marrow stromal cells; Osteogenesis; Wet-spin phase-inversion technique and biodegradable scaffolds;

Size of the polymeric particulate antigen delivery system and its interactions with antigen-presenting cells (APCs) influence the immune response both qualitatively and quantitatively. In this paper, we report that antigen-loaded polymeric microparticles elicit antibody titers without being phagocytosed by macrophages; and size of the antigen-loaded particles modulates immune response from single-point immunization. Antibody titers varied significantly from single-point immunization with different sized polylactide (PLA) particles entrapping hepatitis B surface antigen. Nanoparticles (200–600 nm) were efficiently taken up by macrophages and elicited lower antibody titers in comparison to microparticles (2–8 μm). PLA microparticles that elicited highest and long-lasting antibody titers from single-point immunization were not taken up by the macrophages and found attached to the surface of the macrophages. Immunization with nanoparticles (200–600 nm) was associated with higher levels of IFN-γ production, upregulation of MHC class I molecules along with antibody isotypes favoring Th1-type immune response. Immunization with microparticles (2–8 μm size) promoted IL-4 secretion, upregulated MHC class II molecules and favored Th2-type immune response. Western blot analysis showed that release of HBsAg from surface-attached microparticles into macrophages increased with time, but was more or less constant in case of nanoparticles. Our results suggest that continuous release of high concentration of antigen from cell surface-attached PLA microparticles into APCs results in improved antibody response from single-point immunization. It also offers an exciting possibility of designing size-based polymer particle delivery system to modulate immune response.
Keywords: Polylactide; Microparticles; Nanoparticles; Macrophage; Cellular interaction; Immune response;

The self-assembly of biodegradable cationic polymer micelles as vectors for gene transfection by Yong Wang; Chyan-Ying Ke; Cyrus Weijie Beh; Shao-Qiong Liu; Suat-Hong Goh; Yi-Yan Yang (5358-5368).
Cationic micelles self-assembled from a biodegradable amphiphilic copolymer, poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate} (P(MDS-co-CES)) have recently been reported for efficient gene delivery and co-delivery of drug and nucleic acid. In this study, poly(ethylene glycol) (PEG) of various molecular weights (M n=550, 1100 and 2000) was conjugated to P(MDS-co-CES) having different cholesterol grafting degrees to improve the stability of micelle/DNA complexes in the blood for systemic in vivo gene delivery. DNA binding ability, gene transfection efficiency and cytotoxicity of P(MDS-co-CES), PMDS, PEGylated PMDS and PEGylated P(MDS-co-CES) micelles were studied and compared. As with P(MDS-co-CES), PEG–P(MDS-co-CES) polymers could also self-assemble into stable micelles of small size. However, PMDS and PEG–PMDS without cholesterol could not form stable micelles but formed large particles. PEGylation of polymers significantly decreased their gene transfection efficiency in HEK293, HepG2, HeLa, MDA-MB-231 and 4T1 cells. However, increasing N/P ratio promoted gene transfection. An increased cholesterol grafting degree led to greater gene expression level possibly because of the more stable core–shell structure of the micelles. PEG550–P(MDS-co-CES) micelles induced high gene transfection level, comparable to that provided by P(MDS-co-CES) micelles. PEGylated polymers were much less cytotoxic than P(MDS-co-CES). PEGylated P(MDS-co-CES) micelles may provide a promising non-viral vector for systemic in vivo gene delivery.
Keywords: Cationic polymer micelles; PEGylation; Self-assembly; Gene transfection;

A 3D collagen microsphere culture system for GDNF-secreting HEK293 cells with enhanced protein productivity by Hoi-Ling Wong; Ming-Xi Wang; Pik-To Cheung; Kwok-Ming Yao; Barbara Pui Chan (5369-5380).
Mammalian cell culture technology has been used for decades in mass production of therapeutic proteins. However, unrestricted cell proliferation usually results in low-protein productivity. Controlled proliferation technologies such as metabolism intervention and genetic manipulation are therefore applied to enhance the productivity. Nevertheless, these strategies induced growth arrest with reduced viability and increased apoptosis. In this study, we report a new controlled proliferation technology by encapsulating human embryonic kidney (HEK) 293 cells over-expressing glial-derived neurotrophic factor (GDNF) in 3D collagen microspheres for extended culture. We investigated the viability, proliferation, cell cycle and GDNF productivity of HEK293 cells in microspheres as compared to monolayer culture. This system provides a physiologically relevant tissue-like environment for cells to grow and exerts proliferation control throughout the culture period without compromising the viability. A significant increase in the production rate of GDNF was found in the 3D microsphere system comparing with the monolayer culture. GDNF productivity was also significantly affected by the initial cell number and the serum concentration. The secreted GDNF was still bioactive as it induced neurite extension in PC12 cells. In summary, the 3D collagen microsphere system presents a cost-effective controlled growth technology for protein production in pharmaceutical manufacturing.
Keywords: 3D culture; Biopharmaceutical manufacturing; GDNF-secreting HEK293 cells; Microsphere; Collagen; Microencapsulation;

Controlled formation of magnetite crystal by partial oxidation of ferrous hydroxide in the presence of recombinant magnetotactic bacterial protein Mms6 by Yosuke Amemiya; Atsushi Arakaki; Sarah S. Staniland; Tsuyoshi Tanaka; Tadashi Matsunaga (5381-5389).
Mms6 is a small acidic protein that is tightly associated with bacterial magnetite in Magnetospirillum magneticum AMB-1. This protein has previously shown iron binding activity, allowing it to generate uniform magnetic crystals by co-precipitation of ferrous and ferric ions. Here, magnetite crystals were formed by the partial oxidation of ferrous hydroxide in the presence and absence of Mms6. The crystals synthesised were systematically characterised according to their sizes and morphologies using high-resolution transmission electron microscopy. Mms6-mediated synthesis of magnetite by this methods produced crystals of a uniform size and narrow size distribution with a cubo-octahedral morphology, similar to bacterial magnetite observed in M. magneticum AMB-1. The crystals formed in the absence of Mms6 were octahedral, larger with an increased size distribution. Protein quantification analysis of Mms6 in the synthesised particles indicated tight association of this protein onto the crystal. Furthermore, high affinities to iron ions and a highly charged electrostatic quality suggest that the protein acts as a template for the nucleus formation and/or acts as a growth regulator by recognising crystal faces. The method introduced in this study presents an alternative route for controlling the size and shape of magnetite crystals without the use of organic solvent and high temperatures.
Keywords: Magnetite; Nanoparticle; Crystal growth; Biomimetic material; Biomineralisation; Magnetotactic bacteria;

In vivo biomechanical assessment of triglycidylamine crosslinked pericardium by Michael S. Sacks; Hirotsugu Hamamoto; Jeanne M. Connolly; Robert C. Gorman; Joseph H. Gorman; Robert J. Levy (5390-5398).
While glutaraldehyde crosslinking is most often used to fabricate bioprosthetic heart valves (BHV) using heterograft tissues, it predisposes BHV to calcification and dramatically stiffens the heterograft tissues. Our group previously reported the synthesis and characterization of a novel epoxy-crosslinker, triglycidylamine (TGA). TGA pretreatment of BHV tissues compared to glutaraldehyde results in both calcification resistance in subdermal implants and improved leaflet compliance. In these prior studies, optimal calcification inhibition was noted with the combined use of TGA with mercapto-aminobisphosphonate (MABP). In the present study, we investigated the hypothesis that bovine pericardium cross-linked with TGA-MABP retains these beneficial biomechanical properties in vivo using a novel mitral valve anterior leaflet (MVAL) ovine valvuloplasty model. Bovine pericardial specimens were crosslinked with either glutaraldehyde or TGA-MABP, from which 1 cm2 sections were implanted in the ovine MVAL after removal of the original tissue of the same size. An array of four sonomicrometry transducers were implanted on the corners and used to compute the complete in-surface strain tensor cardiac cycle over the cardiac cycle at 0 and 4 weeks. Following explant samples were fixed in formalin for histology studies. At 4 weeks both treatment groups experienced no dimensional changes in the unloaded state, indicating no shrinkage. When fully loaded during peak systolic ejection, TGA-MABP valvuloplasty patches were significantly more compliant, which did not change at 4 weeks. In contrast, the glutaraldehyde areal strain increased significantly by 4 weeks. Estimated implant stresses for both treatment groups, based on previously measured biomechanical properties [Connolly JM, Alferiev I, Clark-Gruel JN, Eidelman N, Sacks M, Palmatory E, et al. Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms. Am J Pathol 2005;166(1):1–13], were 40 and 250 kPa in the circumferential and radial directions, respectively, which are comparable to predicted BHV peak stress levels. We conclude that TGA-MABP crosslinked bovine pericardium, when subjected to in vivo BHV stress levels in a blood-contacting environment, maintains stable functionality.
Keywords: Bioprosthetic heart valves; Heterograft biomaterials; Biomechanics; Biocompatibility;