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Biomaterials (v.26, #23)

Calendar (pp. i).

Viscoelastic and mechanical behavior of recombinant protein elastomers by Karthik Nagapudi; William T. Brinkman; Benjamin S. Thomas; Jung O. Park; Mohan Srinivasarao; Elizabeth Wright; Vince P. Conticello; Elliot L. Chaikof (pp. 4695-4706).
Recombinant DNA synthesis was employed to produce elastin–mimetic protein triblock copolymers containing chemically distinct midblocks. These materials displayed a broad range of mechanical and viscoelastic responses ranging from plastic to elastic when examined as hydrated gels and films. These properties could be related in a predictable fashion to polymer block size and structure. While these materials could be easily processed into films and gels, electrospinning proved a feasible strategy for creating protein fibers. All told, the range of properties exhibited by this new class of protein triblock copolymer in combination with their easy processability suggests potential utility in a variety of soft prosthetic and tissue engineering applications.

Keywords: Recombinant protein; Protein polymer; Triblock copolymer; Elastin–mimetic; Mechanical behavior; Viscoelastic properties

Biocatalytic synthesis of highly ordered degradable dextran-based hydrogels by Lino Ferreira; Maria H. Gil; Antonio M.S. Cabrita; Jonathan S. Dordick (pp. 4707-4716).
We have prepared unique macroporous and ordered dextran-based hydrogels using a single-step biocatalytic transesterification reaction between dextran and divinyladipate in neat dimethylsulfoxide. These hydrogels show a unimodal distribution of interconnected pores with average diameters from 0.4 to 2.0μm depending on the degree of substitution. In addition, the hydrogels show a higher elastic modulus for a given swelling ratio than chemically synthesized dextran-based hydrogels. In vivo studies in rats show that the hydrogel networks are degradable over a range of time scales from 5 to over 40 days, and possess good biocompatibility, as reflected in only a mild inflammatory reaction and minor fibrous capsule formation during the time-frame of subcutaneous implantation. These combined properties may offer competitive advantages in biomedical applications ranging from tissue engineering to controlled drug delivery.

Keywords: Enzyme; Dextran; Hydrogel; Cross-linking; Biodegradation

Effects of calcium ion implantation on human bone cell interaction with titanium by Saima N. Nayab; Frances H. Jones; Irwin Olsen (pp. 4717-4727).
The use of calcium ion (Ca) implantation of titanium (Ti), previously reported to encourage osseointegration in vivo, has been investigated using an in vitro model in order to understand the basic mechanisms involved in the response of target cells to such surfaces. Polished Ti discs were implanted with high, medium and low (1×1017, 1×1016, 1×1015ionscm−2) doses of Ca ions at 40keV. The effects of different levels of Ca implantation on morphology, attachment and spreading of MG-63 cells seeded on the surface of control (non-implanted) Ti and Ca–Ti discs were assessed. Further, to understand cell–material interactions at a molecular level, the expression ofβ1 andα5β1 integrins and the formation of vinculin-positive focal adhesion plaques were examined. In addition, the effects of pre-immersion of the Ca (high)–Ti in tissue culture medium on cell attachment were measured and correlated with specific chemical changes at the Ti surface. Our findings suggest that Ca implantation can affect the adhesion of MG-63 cells both qualitatively and quantitatively. However, this effect appears to depend on the level at which Ca ions are implanted. Results showed that although cell adhesion on Ca (high)–Ti was initially reduced, it nevertheless was not only restored but substantially increased with progressing culture times. In addition, a significantly enhanced cell spreading, formation of focal adhesion plaques and expression of integrins were measured on this particular surface. In contrast, no marked differences were observed in cell behaviour on Ca–Ti (low and medium). Pre-immersion studies indicated that the decrease in cell attachment to Ca (high)–Ti at early time periods may be linked to the presence of Ca- and P-rich particles on the surface. The absence of these particles at 24h was consistent with a significant increase in cell attachment.

Keywords: Titanium; Calcium-implantation; Cell adhesion; MG-63 cells; Integrin; Vinculin

The influence of surface porosity on gap-healing around intra-articular implants in the presence of migrating particles by Ole Rahbek; Soren Kold; Berit Zippor; Soren Overgaard; Kjeld Soballe (pp. 4728-4736).
The aim of the present study was to compare the effect of two different porous coatings on bone ongrowth and on the peri-implant migration of polyethylene (PE) particles. Porous-coated cylindrical implants with an either plasma-sprayed closed-pore coating (Pl) or titanium fiber metal open-pore coating (Fi) were inserted intra-articular in exact fit or with a 0.75mm peri-implant gap. A weight-loaded implant device in the distal femur was used. We used a randomized paired design in eight dogs. PE particles were injected repeatedly intra-articular in the knee until the dogs were killed after 8 weeks.Fi implants had significantly more bone ongrowth 8 (0–21)% compared with Pl implants 0 (0–0)% in gap situations and reduced the number of peri-implant PE particles significantly. Among exact-fitted implants we found that peri-implant tissue around Pl implants consisted of significantly more fibrous tissue than around Fi implants. A sealing effect against the migration of PE particles was found for both Fi and Pl implants in exact fit.

Keywords: Animal model; Arthroplasty; Bone ingrowth; Polyethylene; Surface texture

Biocompatibility and stability of disulfide-crosslinked hyaluronan films by Yanchun Liu; Xiao Zheng Shu; Glenn D. Prestwich (pp. 4737-4746).
Hyaluronan (HA) can be chemically modified to engineer robust materials with pre-selected mechanical properties and resorption rates that can be dictated by the intended clinical use. Disulfide-crosslinked HA films were prepared by air oxidation of thiol-modified HA, followed by treatment with 0.3% hydrogen peroxide. The degradation of the disulfide-crosslinked films in vitro was very slow (<10% in 7 days) in buffer alone and shorter ( t1/2=3–5 days) in the presence of hyaluronidase (HAse). The cytocompatibility of the disulfide-crosslinked HA films was determined using two separate conditions: (i) in vitro culture of mouse fibroblasts in indirect contract with the films, and (ii) in vitro culture of fibroblasts directly on films coated with polyd-lysine. Excellent cytocompatibility was observed in murine fibroblasts that were cultured in indirect contact with thiolated HA films. Although cells were unable to attach and spread on thiolated HA films, pre-coating the thiolated HA films with poly D-lysine resulted in attachment and spreading equivalent to that observed on polystyrene. Rates of resorption in vivo were obtained by subcutaneous implantation of disulfide-crosslinked HA films into the backs of Wistar rats. Biocompatibility in vivo was determined in both subcutaneous flank and peritoneal cavity implantation of the films in Wistar rats. The disulfide-crosslinked HA films were less than 30% resorbed after 42 days in vivo, and histochemical and cytochemical analysis indicated that the films were well-tolerated with mild inflammatory response at both sites of implantation.

Keywords: Thiol modification; Hyaluronic acid; Films; Biodegradation; Fibroblast cytocompatibility; Peritoneal cavity; Subcutaneous implantation; Disulfide crosslinking; Hyaluronidase

Electrochemical corrosion and metal ion release from Co-Cr-Mo prosthesis with titanium plasma spray coating by Lucien Reclaru; Pierre-Yves Eschler; Reto Lerf; Andreas Blatter (pp. 4747-4756).
The corrosion behavior of CoCrMo implants with rough titanium coatings, applied by different suppliers by either sintering or vacuum plasma spraying, has been evaluated and compared with uncoated material. The open-circuit potential, corrosion current and polarization resistance were determined by electrochemical techniques. The Co, Cr and Ti ions released from the samples into the electrolyte during a potentiostatic extraction technique were analyzed using ICP-MS.The Ti coatings from the different suppliers showed a different porous morphology, and the implants exhibited a distinct corrosion activity, underlining the importance of the coating process parameters. Among the titanium coated samples, the one with the sintered overcoat turned out to be the most resistant. Yet, on an absolute scale, they all showed a corrosion resistance inferior to that of uncoated CoCrMo or wrought titanium.

Keywords: CoCrMo implant; Titanium plasma spray coating; Electrochemical test; Cations extraction; Corrosion

Dynamic heterodimer-functionalized surfaces for endothelial cell adhesion by P. Jeanene Willcox; Reinhart-King Cynthia A. Reinhart-King; Steven J. Lahr; William F. DeGrado; Daniel A. Hammer (pp. 4757-4766).
The functionalization of hydrogels for receptor-mediated cell adhesion is one approach for targeted cell and tissue engineering applications. In this study, polyacrylamide gel surfaces were functionalized with specific cell adhesion ligands via the self-assembly of a peptide-based heterodimer. The system was comprised of a cysteine-terminated monomer, A (MW ∼ 5400), grafted to the polyacrylamide gels and a complementary ligand presenting monomer, BX (MW ∼ 5800) that was designed to heterodimerize with A. Two ligand presenting monomers were synthesized: one presenting the RGDS ligand, BD, for receptor-mediated cell adhesion, and the other, a control monomer presenting the nonadhesive RGES ligand, BE. Assembly of the peptide pair A–BX by association of the monomers into a coiled coil was verified by circular dichroism in solution. Binding studies were conducted to determine the dissociation constant of the pair A–BX, which was found to beKD∼10-8m. Polyacrylamide gels functionalized with A–BX heterodimers were evaluated for cell adhesion using bovine aortic endothelial cells (BAECs). Endothelial cells cultured on the A–BD functionalized surfaces demonstrated typical cell morphologies and expected spreading behavior as a function of the density of RGDS ligand, calculated as the amount of BD associated with grafted A on the surface of the gels. In contrast, A–BE linked surfaces supported no cell adhesion. The adhesion of the substrate was dynamically altered through the reassembly of A–BX dimers as BD molecules in the solution replaced BE molecules at the substrate. The molecular constructs described here demonstrate the potential to design a broad family of switchable peptides that impart the dynamic control of biofunctionality at an interface, which would be useful for precise manipulation of cell physiology.

Keywords: Cell adhesion; Endothelial cell; RGD peptide; Cell spreading; Integrin

Molecularly engineered p(HEMA)-based hydrogels for implant biochip biocompatibility by Sheena Abraham; Sean Brahim; Kazuhiko Ishihara; Anthony Guiseppi-Elie (pp. 4767-4778).
The strategy of phospholipid-based biomimicry has been used to molecularly engineer poly(2-hydroxyethyl methacrylate) [p(HEMA)]-based hydrogels for improved in vitro and potential in vivo biocompatibility. Two methacrylate-based monomers, poly(ethylene glycol) (200) monomethacrylate (PEGMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC), were incorporated at varying mole fractions of 0.0–0.5mol% PEGMA and 0–10mol% MPC respectively, into 3mol% tetraethyleneglycol diacrylate (TEGDA) cross-linked p(HEMA) networks. Upon hydration of these engineered hydrogels, a reduction in receding contact angle from 22±1.2° for p(HEMA) to 8±2.7° for p(HEMA) containing 0.5:10mol% PEGMA:MPC was observed, reflecting the significant increase in surface hydrophilicity with increasing PEGMA and MPC content upon prolonged hydration. Hydrogels containing MPC showed a temporal increase in hydrophilicity following continuous immersion in DI water over 5 days. Hydrogels containing 0.5mol% PEGMA and MPC in the range of 5–10mol% displayed reduced protein adsorption when incubated with the common extracellular matrix proteins; fibronectin, collagen or laminin, producing up to 64% less protein adsorption compared to p(HEMA). Compositional optima for cell viability and proliferation established from two-factor Central Composite design analysis of human muscle fibroblasts cultured on these hydrogels suggest that those containing PEGMA between 0.3 and 0.5mol% and MPC levels around 5–10mol% exhibit desirable characteristics for implant material coatings—high viability (>80%) with low proliferation (<40%), confirming a lack of cytotoxicity.

Keywords: p(HEMA) hydrogels; Biocompatibility; Phosphorylcholine; Polyethylene glycol; Protein absorption; Human muscle fibroblasts; Wettability

Bone tissue induction, using a COLLOSS-filled titanium fibre mesh-scaffolding material by X. Frank Walboomers; John A. Jansen (pp. 4779-4785).
Scaffold materials for bone tissue engineering often are supplemented with bone morphogenetic proteins (BMPs). In the current study we aimed to investigate COLLOSS, a bovine extracellular matrix product containing native BMPs. Hollow cylindrical implants were made, with a length of 10mm, a 3mm inner diameter, and a 5mm outer diameter, from titanium fibre mesh. The central space of the tube was filled with 20mg COLLOSS. Subsequently, these implants, as well as non-loaded controls, were implanted subcutaneously into the back of Wistar rats, withn=6 for all study groups. After implantation periods of 2, 8, and 12 weeks, tissue-covered implants were retrieved, and sections were made, perpendicular to the long axis of the tube. Histology showed, that all implants were surrounded by a thin fibrous tissue capsule. After 2 weeks of implantation, the COLLOSS material was reduced in size inside the loaded implants, but no bone-like tissue formation was evident. After 8 weeks, in two out of six loaded specimens, new-formed bone- and bone marrow-like tissues could be observed. After 12 weeks, this had increased to five out of six COLLOSS-loaded samples. The amount of bone-like tissue did not differ between 8 and 12 weeks, and on average occupied 15% of the central space of the tube. In the non-loaded control samples, only connective tissue ingrowth was observed. In conclusion, we can say that COLLOSS material loaded in a titanium fibre mesh tube, showed bone-inducing properties. The final efficacy of these osteo-inductive properties has to be confirmed in future large animal studies.

Keywords: Bone tissue; Tissue engineering; Titanium fibre mesh; COLLOSS; ; Animal study

Development of mammalian cell-enclosing subsieve-size agarose capsules (<100μm) for cell therapy by Shinji Sakai; Kenji Kawabata; Tsutomu Ono; Hiroyuki Ijima; Koei Kawakami (pp. 4786-4792).
Agarose capsules were prepared using a droplet breakup method in a coflowing stream. Subsieve-size capsules 76±9μm in diameter were obtained by extruding 4wt% agarose solution from a needle (300μm inner diameter) at a velocity of 1.2cm/s into an ambient liquid paraffin flow of 20.8cm/s. Increasing the flow rate of the liquid paraffin and decreasing that of the agarose solution resulted in a decreased resultant capsule diameter. Reduction in diameter from several hundred micrometers to subsieve-size (<100μm) enhanced molecular exchange and mechanical stability. Measurements based on the percentage of intact mitochondria in the cells demonstrated that the viability of the enclosed cells was independent of capsule diameter. No significant difference was observed between the viabilities of cells enclosed in capsules with diameters of 79±8 and 351±41μm (p=0.43). Compared with cells seeded in a tissue culture dish, the cells enclosed in the subsieve-size capsules showed 89.2% viability.

Keywords: Microcapsule; Cell encapsulation; Microencapsulation; Transplantation; Polysaccharide; Cell viability

The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polycaprolactone by Giovanni Marletta; Gabriela Ciapetti; Cristina Satriano; Stefania Pagani; Nicola Baldini (pp. 4793-4804).
Using techniques of tissue engineering, synthetic substitutes can be applied for the repair and regeneration of damaged bone.It has been found that material surface properties are crucial for cell adhesion and spreading, i.e. cell activities that are related directly to the ability of osteoblasts to proliferate.This fact has promoted the strategy of creating an ECM-like layer onto materials, so as to influence the cell response. In this study human bone-derived osteoblasts have been used to test the effects of surface modification by low energy ion beams of a polyε-caprolactone (PCL) substrate and subsequent RGD adsorption.Osteoblasts were seeded and grown onto untreated and irradiated polyε-caprolactone films, with or without RGD-adsorption step, and viability, morphology, and spreading of the osteoblasts were studied at different time endpoints. Differences were observed in the organization of cytoskeleton within cells: stress fibers were more evident in irradiated samples vs. untreated and total cell adhesion was higher. Surface characterization by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy, and surface free energy measurements showed that the polar character of PCL, i.e., the acid–base term, was increased following irradiation treatment. Moreover the irradiated PCL had a nano-sized topography, which also could improve osteoblasts adhesion.We found that the treatment of the surface with ion beam is per se improving osteoblasts adhesion and spreading onto PCL. Furthermore, also if a significant RGD adsorption was obtained for irradiated PCL surfaces, it was found that in the investigated conditions it seems to have only a minor effect on the cell response.This study suggests that new strategies involving irradiation-based treatments can be adopted to promote the initial steps of bone deposition onto synthetic surfaces, exploiting the surface-induced reorganization of the ECM matrix.

Keywords: Surface; Poly; ε; -caprolactone; Irradiation; RGD; Osteoblasts

Anterior cruciate ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies by Helen H. Lu; James A. Cooper Jr.; Sharron Manuel; Joseph W. Freeman; Mohammed A. Attawia; Frank K. Ko; Cato T. Laurencin (pp. 4805-4816).
The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee, and limitations in existing reconstruction grafts have prompted an interest in tissue engineered solutions. Previously, we reported on a tissue-engineered ACL scaffold fabricated using a novel, three-dimensional braiding technology. A critical factor in determining cellular response to such a graft is material selection. The objective of this in vitro study was to optimize the braided scaffold, focusing on material composition and the identification of an appropriate polymer. The selection criteria are based on cellular response, construct degradation, and the associated mechanical properties. Three compositions of poly- α-hydroxyester fibers, namely polyglycolic acid (PGA), poly-L-lactic acid (PLLA), and polylactic- co-glycolic acid 82:18 (PLAGA) were examined. The effects of polymer composition on scaffold mechanical properties and degradation were evaluated in physiologically relevant solutions. Prior to culturing with primary rabbit ACL cells, scaffolds were pre-coated with fibronectin (Fn, PGA-Fn, PLAGA-Fn, PLLA-Fn), an important protein which is upregulated during ligament healing. Cell attachment and growth were examined as a function of time and polymer composition. While PGA scaffolds measured the highest tensile strength followed by PLLA and PLAGA, its rapid degradation in vitro resulted in matrix disruption and cell death over time. PLLA-based scaffolds maintained their structural integrity and exhibited superior mechanical properties over time. The response of ACL cells was found to be dependent on polymer composition, with the highest cell number measured on PLLA-Fn scaffolds. Surface modification of polymer scaffolds with Fn improved cell attachment efficiency and effected the long-term matrix production by ACL cells on PLLA and PLAGA scaffolds. Therefore based on the overall cellular response and its temporal mechanical and degradation properties in vitro, the PLLA braided scaffold pre-coated with Fn was found to be the most suitable substrate for ACL tissue engineering.

Keywords: Polymer composition; Anterior cruciate ligament (ACL); Poly-; α; -hydroxyester; Degradation; Fibronectin; Ligament tissue engineering

Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering by Jessica M. Williams; Adebisi Adewunmi; Rachel M. Schek; Colleen L. Flanagan; Paul H. Krebsbach; Stephen E. Feinberg; Scott J. Hollister; Suman Das (pp. 4817-4827).
Polycaprolactone (PCL) is a bioresorbable polymer with potential applications for bone and cartilage repair. In this work, porous PCL scaffolds were computationally designed and then fabricated via selective laser sintering (SLS), a rapid prototyping technique. The microstructure and mechanical properties of the fabricated scaffolds were assessed and compared to the designed porous architectures and computationally predicted properties. Scaffolds were then seeded with bone morphogenetic protein-7 (BMP-7) transduced fibroblasts and implanted subcutaneously to evaluate biological properties and to demonstrate tissue in-growth. The work done illustrates the ability to design and fabricate PCL scaffolds with porous architecture that have sufficient mechanical properties for bone tissue engineering applications using SLS. Compressive modulus and yield strength values ranged from 52 to 67MPa and 2.0 to 3.2Mpa, respectively, lying within the lower range of properties reported for human trabecular bone. Finite element analysis (FEA) results showed that mechanical properties of scaffold designs and of fabricated scaffolds can be computationally predicted. Histological evaluation and micro-computed tomography (μCT) analysis of implanted scaffolds showed that bone can be generated in vivo. Finally, to demonstrate the clinical application of this technology, we designed and fabricated a prototype mandibular condyle scaffold based on an actual pig condyle. The integration of scaffold computational design and free-form fabrication techniques presented here could prove highly useful for the construction of scaffolds that have anatomy specific exterior architecture derived from patient CT or MRI data and an interior porous architecture derived from computational design optimization.

Keywords: Bone tissue engineering; Finite element analysis; Laser manufacturing; Mechanical properties; Micro-computed tomography (μCT); Polycaprolactone; Porosity; Rapid prototyping; Solid free-form fabrication

Influence of stirring-induced mixing on cell proliferation and extracellular matrix deposition in meniscal cartilage constructs based on polyethylene terephthalate scaffolds by A.A. Andr A. Neves; Nick Medcalf; Kevin M. Brindle (pp. 4828-4836).
The response of engineered meniscal cartilage constructs to stirring-induced mixing in spinner flasks was investigated. Polyethylene terephthalate scaffolds were seeded with meniscal fibrochondrocytes from 6 month-old sheep and cultured under a variety of stirring regimes for 28 days. Stirring-induced mixing increased up to 7-fold the deposition of glucosaminoglycans and up to 3-fold the deposition of collagen, when compared to static cultures. High and medium intensity stirring induced rapid cell proliferation, with maximal cell densities achieved within the first seven days of cultivation. Under these conditions, collagen and glucosaminoglycan deposition occurred predominantly in association with cell proliferation, the specific deposition rate of these biopolymers decreasing markedly after 7 days of cultivation, when the cell number reached a plateau. Constructs exposed to the highest intensity stirring had the highest levels of collagen and glucosaminoglycans and a more homogeneous cell distribution. As the success of the integration at a repair site in the knee of a meniscal construct is likely to be dependent on the cellular activity of the construct, these studies suggest that cultivation of meniscal cartilage constructs, under these conditions, should not extend for more than 7 days.

Keywords: Meniscus; Cartilage; Mixing; ECM; PET

Self-assembling short oligopeptides and the promotion of angiogenesis by Daria A. Narmoneva; Olumuyiwa Oni; Alisha L. Sieminski; Shugang Zhang; Jonathan P. Gertler; Roger D. Kamm; Richard T. Lee (pp. 4837-4846).
Because an adequate blood supply to and within tissues is an essential factor for successful tissue regeneration, promoting a functional microvasculature is a crucial factor for biomaterials. In this study, we demonstrate that short self-assembling peptides form scaffolds that provide an angiogenic environment promoting long-term cell survival and capillary-like network formation in three-dimensional cultures of human microvascular endothelial cells. Our data show that, in contrast to collagen type I, the peptide scaffold inhibits endothelial cell apoptosis in the absence of added angiogenic factors, accompanied by enhanced gene expression of the angiogenic factor VEGF. In addition, our results suggest that the process of capillary-like network formation and the size and spatial organization of cell networks may be controlled through manipulation of the scaffold properties, with a more rigid scaffold promoting extended structures with a larger inter-structure distance, as compared with more dense structures of smaller size observed in a more compliant scaffold. These findings indicate that self-assembling peptide scaffolds have potential for engineering vascularized tissues with control over angiogenic processes. Since these peptides can be modified in many ways, they may be uniquely valuable in regeneration of vascularized tissues.

Keywords: Angiogenesis; Endothelial cell; Peptide; Scaffold

The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture by Shinichi Maeno; Yasuo Niki; Hideo Matsumoto; Hideo Morioka; Taku Yatabe; Atsushi Funayama; Yoshiaki Toyama; Tetsushi Taguchi; Junzo Tanaka (pp. 4847-4855).
Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca2+ and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca2+ concentration were examined in two- and three-dimensional cultures. The present results indicate that 2–4mM Ca2+ is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6–8mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10mM) are cytotoxic. Purely from the perspective of calcium deposition, higher concentrations lead to increased accumulation of Ca2+.Culturing cells in phosphate-containing gel in media with Ca2+ also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca2+ concentrations around 5mM is useful for both HAp deposition and osteoblast behavior.

Keywords: Calcification; Medium calcium; Osteoblast; Viability; Type II collagen gel; Tissue engineering

Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and β-tricalcium phosphate by Yoshitake Takahashi; Masaya Yamamoto; Yasuhiko Tabata (pp. 4856-4865).
Biodegradable gelatin sponges at different contents of β-tricalcium phosphate ( β-TCP) were fabricated to allow bone morphogenetic protein (BMP)-2 to incorporate into them. The in vivo osteoinduction activity of the sponges incorporating BMP-2 was investigated, while their in vivo profile of BMP-2 release was evaluated. The sponges prepared had an interconnected pore structure with an average pore size of 200μm, irrespective of the β-TCP content. The in vivo release test revealed that BMP-2 was released in vivo at a similar time profile, irrespective of the β-TCP content. The in vivo time period of BMP-2 retention was longer than 28 days. When the osteoinduction activity of gelatin or gelatin– β-TCP sponges incorporating BMP-2 was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges, although the extent of bone formation was higher in the sponges with the lower contents of β-TCP. On the other hand, the level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges decreased with an increase in the content of β-TCP. The gelatin sponge exhibited significantly higher osteoinduction activity than that of any gelatin– β-TCP sponge, although every sponge with or without β-TCP showed a similar in vivo profile of BMP-2 release. In addition, the in vitro collagenase digestion experiments revealed that the gelatin– β-TCP sponge collapsed easier than the gelatin sponge without β-TCP incorporation. These results suggest that the maintenance of the intrasponge space necessary for the osteoinduction is one factor contributing to the osteoinduction extent of BMP-2-incorporating sponges.

Keywords: Bone morphogenetic protein; Gelatin–; β; -TCP sponge; Controlled release; Osteoinduction

PEG-appended β-(1→3)-d-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation by Ryouji Karinaga; Kazuya Koumoto; Masami Mizu; Takahisa Anada; Seiji Shinkai; Kazuo Sakurai (pp. 4866-4873).
Schizophyllan is a natural β-(1→3)-d-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG–SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004–1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG–SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier.

Keywords: Antisense; Polysaccharide; Gene transfer; RGD peptideAbbreviations; s-SPG; single chain of schizophyllan; AS ODN; antisense oligonucleotide; AS-c-myb; 5′-GTGCCGGGGTCTTCGGGC-(dA); 40; -3′ phosphorothioate; antisense ODN for; c-myb; mRNA; Sc-c-myb; 5′-TGC TGC GCG TGG TCG GCG-(dA); 40; -3′ phosphorothioate; scramble sequence of AS-c-myb; s-SPG/AS-c-myb; complex made from s-SPG and AS-c-myb with the molar ratio of 1.5:1.0; RGD(0.68)–SPG/AS-c-myb; complex made from RGD(0.68)–SPG and AS-c-myb with the molar ratio of 1.5:1.0; RGD(5.0)–SPG/AS-c-myb; complex made from RGD(5.0)–SPG and AS-c-myb with the molar ratio of 1.5:1.0.; PEG 5; K(10.1)–SPG/AS-c-myb; complex made from PEG 5; K(10.1)–SPG and AS-c-myb with the molar ratio of 1.5:1.0.; s-SPG+PEG; mixture of s-SPG and PEG.

The modulation of angiogenesis in the foreign body response by the poxviral protein M-T7 by Jasper M. Boomker; Danil T. Luttikhuizen; Henrike Veninga; Lou F.M.H. de Leij; T. Hauw The; Aalzen de Haan; Marja J.A. van Luyn; Martin C. Harmsen (pp. 4874-4881).
The foreign body response is characterized by enhanced recruitment of inflammatory cells. As the directional movement of cells is controlled by chemokines, disruption of the chemokine network would be an attractive approach to improve biocompatibility of an implanted material. The sequestration of chemokines by cell surface-expressed glycosaminoglycans (GAGs) is vital for in vivo chemokine activity. The myxoma virus encodes a soluble protein, M-T7, that interacts with conserved GAG-binding domains of chemokines to block chemokine-mediated leukocyte recruitment. We hypothesized that M-T7 might also affect the function of other inflammation-associated proteins in addition to chemokines that bind to GAG. In our studies, we focussed on the modulation of the GAG-binding molecules macrophage chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor-164 (VEGF164) in the inflammatory reaction against subcutaneously implanted degradable cross-linked dermal sheep collagen discs in AO rats. Genetic delivery of M-T7 delays the influx of macrophages into the collagen discs. In addition, angiogenesis around the implanted material was reduced. The discs revealed reduced levels of rat MCP-1 and rat VEGF164. This was not due to down regulation of transcription of the genes that encode MCP-1 and VEGF164. Our in vivo observations suggest that, in addition to chemokines such as MCP-1, M-T7 neutralizes VEGF164.

Keywords: Gene therapy; Glycosaminoglycan; Immunomodulation; Macrophage; Chemotaxis

Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays by In Kap Ko; Koichi Kato; Hiroo Iwata (pp. 4882-4891).
Neural stem cells are the attractive cell source for functional regeneration of damaged central nervous tissues by means of cell transplantation or in situ induction of differentiated neural cells. Such stem cell therapies require the prospective identification and isolation of neural stem cells. However they are difficult due to limited information on surface markers. This study aimed at developing an antibody microarray that permits parallel analysis of multiple surface antigens expressed on neural stem cells present in a neurosphere-forming cell population. A microarray was prepared by micro-spotting antibodies directed to surface antigens and ligands for membrane-associated receptors onto the patterned monolayer of alkanethiols self-assembled on a gold-evaporated glass plate. Neurosphere-forming cells were subjected to a cell-binding assay on the microarray followed by immunofluorescent staining of nestin, an intracellular marker of neural stem cells. It was demonstrated that such a cell based assay facilitated to examine the specificity of surface antigens for nestin-positive neural stem cells. Furthermore, the microarray could also be used to assess proliferation capability of cells bound to individual spots. These results suggest that the microarray-based strategy will provide a useful tool for the parallel analysis of surface markers expressed on a specific cell type in a heterogeneous population.

Keywords: Antibody microarray; Neural stem cell; Cell-based high-throughput screening; Self-assembled monolayer; Micropatterning; Immunocytochemical staining; Proliferation assay

Biomaterial microarrays: rapid, microscale screening of polymer–cell interaction by Daniel G. Anderson; David Putnam; Erin B. Lavik; Tahir A. Mahmood; Robert Langer (pp. 4892-4897).
The identification of biomaterials that induce optimal gene expression patterns and allow for appropriate levels of cellular attachment is of central importance in tissue engineering and cell therapy. Herein, we describe the creation of cell-compatible, biomaterial microarrays, that allow rapid, microscale testing of biomaterial interactions with cells. As proof of principle, we simultaneously characterized over 3456 human mesenchymal stem cell (hMSC)–biomaterial composite interactions, and describe preliminary studies on the utility of these arrays with a neural stem cell line (NSC), and primary articular chondrocytes.

Keywords: Microarray; Biomaterials; PLGA

Erratum to ‘Locally delivered nanoencapsulated tyrphostin (AGL-2043) reduces neointima formation in balloon-injured rat carotid and stented porcine coronary arteries’ [Biomaterials 26 (2005) 451–461] by Shmuel Banai; Michael Chorny; S. David Gertz; Ilia Fishbein; Jianchuan Gao; Louise Perez; Galila Lazarovich; Aviv Gazit; Alexander Levitzki; Gershon Golomb (pp. 4898-4901).
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