Biomaterials (v.26, #4)

Calendar (I).

Aiming at developing new reverse thermo-responsive polymers, poly(ethylene oxide)-poly(propylene oxide) multiblock copolymers were synthesized by covalently binding the two components using carbonyl chloride and diacyl chlorides as the coupling molecules. The appropriate selection of the various components allowed the generation of systems displaying much enhanced rheological properties. For example, 15 wt% aqueous solutions of an alternating poly(ether-carbonate) comprising PEO6000 and PPO3000 segments, achieved a viscosity of 140,000 Pa s, while the commercially available Pluronic F127 displayed 5,000 Pa s only. Furthermore, the structure of the chain extender played a key role in determining the sol–gel transition. While poly(ether-ester)s containing therephtaloyl (para) and isophtaloyl (metha) coupling units failed to gel at any concentration, a 15 wt% aqueous solution of the polymer chain-extended with phtaloyl chloride (ortho) gelled at 43°C. The water solutions were also studied by dynamic light scattering and a clear influence of the PEO/PPO ratio on the aggregate size was observed. By incorporating short aliphatic oligoesters into the backbone, prior to the chain extension stage, reverse thermal gelation-displaying biodegradable poly(ether-ester-carbonate)s, were generated.
Keywords: (PEO-PPO) multiblock copolymers; Poly(ether carbonate)s; Poly(ether ester)s; Reverse thermo-responsive systems; Viscosity vs. temperature profile;

Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern by Tatiana Segura; Brian C Anderson; Peter H Chung; Rebecca E Webber; Kenneth R Shull; Lonnie D Shea (359-371).
The physiological activity of hyaluronic acid (HA) polymers and oligomers makes it a promising material for a variety of applications. The development of HA–hydrogel scaffolds with improved mechanical stability against degradation and biochemical functionality may enhance their application to tissue engineering. In this report, a crosslinking strategy targeting the alcohol groups via a poly(ethylene glycol) diepoxide crosslinker was investigated for the generation of degradable HA hydrogels. To provide support for cell adhesion in vitro, collagen was incorporated into the HA solution prior to the crosslinking process. The hydrogels have a continuous exterior and a porous interior, with pore diameters ranging from 6 to 9 μm. HA and HA–collagen hydrogels degrade in the presence of hyaluronidase and collagenase enzymes, indicating that the chemical modification does not prevent biodegradation. Complete degradation of the hydrogels occurred within 14 days in hyaluronidase (100 U/ml) and 3 days in collagenase (66 U/ml). Pattern transfer was employed to introduce a surface topography onto the hydrogel, which was able to orient cell growth. Furthermore, the hydrogels could be functionalized with the biomolecule neutravidin by incorporation of biotin along the HA backbone. This biotinylation approach may allow attachment of bioactive molecules that are conjugated to avidin.
Keywords: Collagen; Biotin; Avidin; Orientation; Topographical control;

The influence of surface topography of ceramic abutments on the attachment and proliferation of human oral fibroblasts by Kamal Mustafa; Agneta Odén; Ann Wennerberg; Kjell Hultenby; Kristina Arvidson (373-381).
As different implant abutments are introduced to obtain a sufficient soft tissue barrier, the aim of this study was to determine the effect of three different surface modifications of densely sintered high-purity aluminium oxide on morphology, attachment and proliferation of human gingival fibroblasts. Fibroblasts were cultured on pressed aluminium oxide, milled, and then sintered to full density (1), on pressed, densely sintered (2), and on pressed, densely sintered and then polished surfaces (3). The different surfaces were analyzed using a confocal laser scanner, an atomic force microscope and a scanning electron microscope. The cell profile areas were measured using a semiautomatic interactive image analyzer and the figures were expressed as percent of attachment. The polished specimens had the smoothest surfaces and the roughest were the milled surfaces in terms of height deviation. No difference was found in the spacing between the peaks on the polished surfaces compared to the milled surfaces. Fibroblasts on the milled ceramic appeared to follow the direction of the fine irregularities on the surface. The analyses showed the polished surfaces had significantly higher percentages of initial cell attachment than the other surfaces (P<0.05). After 3 days of cell culture, significantly more cells were attached to the milled and sintered surfaces than to the polished one, possibly indicating higher proliferation capacity on those types of surfaces.
Keywords: Fibroblasts; Attachment; Impact abutments; Al2O3; Surface roughness;

Control of focal adhesion dynamics by material surface characteristics by Annette Diener; Barbara Nebe; Frank Lüthen; Petra Becker; Ulrich Beck; Hans Georg Neumann; Joachim Rychly (383-392).
The mechanisms of cell adhesion to the extracellular matrix (ECM) which are of fundamental importance for function, survival, and growth of cells involve the formation of focal adhesions to facilitate integrin signaling. Recently, it became evident that focal adhesions are not stable but move to enable cell migration and ECM formation. We examined the number, size, and dynamic behavior of focal adhesions in living MG-63 osteoblastic cells, which were cultured on titanium surfaces with different roughnesses and on stainless steel (SS). As a marker for focal adhesions we used GFP-tagged vinculin, a cytoskeletal protein. Focal adhesions were smaller on titanium and on SS than on collagen-coated glass coverslips. The corundum-blasted rough surface of titanium induced the smallest adhesions. On all the surfaces that we have tested, we observed a mobility of focal adhesions. On collagen-coated coverslips focal adhesions moved with a speed of 60 nm/min. The speed was reduced on titanium and still more restricted on SS. The topography did not affect the mobility of focal adhesions. We conclude that on the material surfaces that we have studied a reduced mobility of focal adhesions may strengthen the linkages between cell and ECM but impair the ability to dynamically organize and remodel the ECM. The results may have a great impact in the functional evaluation of tailored biomaterial surfaces for the application in tissue engineering.
Keywords: Osteoblast; Titanium; Surface topography; Vinculin; Focal adhesion; Mobility;

Hydrolysis of tetracalcium phosphate under a near-constant-composition condition—effects of pH and particle size by Laurence C Chow; Milenko Markovic; Stanislav A Frukhtbeyn; Shozo Takagi (393-401).
Tetracalcium phosphate (TTCP) is a component of a number of calcium phosphate cements used clinically for bone defect repairs. The strength, phase composition, and solubility of the set cement are highly dependent on the reactions of the cement components during setting. This study investigated hydrolysis reactions of TTCP under solution compositions chosen to mimic the compositions of the cement liquid during setting. The study utilized a pseudo-constant-composition technique that allowed both the rate and stoichiometry of the reaction to be determined while the reaction proceeded under a specific, constantly held solution pH, thereby keeping a constant calcium-to-phosphate ratio in solution. The hydrolysis experiments were conducted using either a fine (median particle size 3.5 μm) or coarse (median particle size 13.2 μm) TTCP powder at pH 7, 8 and 10. Low crystalline calcium (Ca)-deficient hydroxyapatite (HA) was the product in all experiments. Both the solution pH and TTCP particle size produced significant effects on all aspects of the hydrolysis reaction. At a given pH, the fine TTCP produced a HA product with a greater Ca deficiency than did the coarse TTCP. For a given particle size, the Ca deficiency generally decreased with increasing pH. Hydrolysis reaction rate generally decreased with increasing pH or TTCP particle size. At pH 7 and 8, the solution was undersaturated with respect to TTCP and supersaturated with respect to HA, suggesting that the reaction rate was limited by TTCP dissolution. In contrast, at pH 10, the solution was approximately saturated with respect to TTCP and highly supersaturated with respect to HA, suggesting that HA formation was the rate-determining step of the reaction. The findings provided useful insights into the setting reaction mechanisms of TTCP-containing calcium phosphate cements.
Keywords: Hydrolysis; Tetracalcium phosphate; Hydroxyapatite; Calcium-deficient apatite; Constant-composition titration; Bone graft material;

Cytoprotection of PEG-modified adult porcine pancreatic islets for improved xenotransplantation by Dong Xie; Cheryl A Smyth; Christopher Eckstein; Guadalupe Bilbao; Jimmy Mays; Devin E Eckhoff; Juan L Contreras (403-412).
Functional poly(ethylene glycol) (PEG) derivatives, including monosuccinimidyl PEG (MSPEG) with molecular weight (MW) of 2000 (2 kDa) as well as 5 kDa and disuccinimidyl PEG (DSPEG) with MW of 3 and 6 kDa, were synthesized and characterized. They were used to modify the surface of adult porcine islets for cytoprotection. The islets were isolated, purified and modified with functional PEG. Untreated porcine islets were used as control. An in vitro human antibody/complement-mediated cytotoxicity test based on the release of intracellular lactate dehydrogenase was used to evaluate cytotoxicity of human serum to the modified islets. In vitro cell viability was assessed using membrane-integrity straining and islet metabolism in culture. In vitro islet functionality was evaluated by glucose-stimulated insulin release of islets in static incubation with human serum. In vivo islet functionality was evaluated by monitoring non-fasting blood glucose level in streptozotocin-induced diabetic (SCID) immunocompromized mice after intraportal transplantation of porcine islets. Results show that all the PEG derivatives used in the study showed significant in vitro and in vivo cytoprotections against cytotoxic effects elicited by human serum and diabetic SCID mice, respectively, to porcine islets. DSPEG derivatives combined with human albumin exhibited a better cytoprotection, as compared to MSPEG ones, due to the capacity of the succinimidyl groups to selectively react with amino groups of the albumin under physiological conditions. The effects of both MW and concentration of the PEG derivatives on cytoprotection were significant. It appears that this novel biotechnology will be an attractive approach for improved xenotransplantation of islets.
Keywords: PEG derivatives; Synthesis; Characterization; Xenotransplantation; Cytoprotection; Antibody/complement-mediated cytotoxicity; Cell viability; In vitron and in vivo islet functionality;

Integrin binding and focal adhesion assembly are critical to cellular responses to biomaterial surfaces in biomedical and biotechnological applications. While immunostaining techniques to study focal adhesion assembly are well established, a crucial need remains for quantitative methods for analyzing adhesive structures. We present simple yet robust approaches to quantify integrin binding and focal adhesion assembly on biomaterial surfaces. Integrin binding to fibronectin and a RGD-containing synthetic peptide was quantified by sequentially cross-linking integrin–ligand complexes via a water-soluble homo-bifunctional cross-linker, extracting bulk cellular components in detergent, and detecting bound integrins by ELISA. Focal adhesion components (vinculin, talin, α-actinin) localized to adhesion plaques were isolated from bulk cytoskeletal and cytoplasmic components by mechanical rupture at a plane close to the basal cell surface and quantified by Western blotting. These approaches represent simple and efficient methodologies to analyze structure–function relationships in cell–material interactions.
Keywords: Cell adhesion; Integrin; Focal adhesion; Vinculin; Talin;

Bacterial cellulose as a potential scaffold for tissue engineering of cartilage by A. Svensson; E. Nicklasson; T. Harrah; B. Panilaitis; D.L. Kaplan; M. Brittberg; P. Gatenholm (419-431).
Tissue constructs for cartilage with native mechanical properties have not been described to date. To address this need the bacterial cellulose (BC) secreted by Gluconacetobacter xylinus (=Acetobacter xylinum) was explored as a novel scaffold material due to its unusual material properties and degradability. Native and chemically modified BC materials were evaluated using bovine chondrocytes. The results indicate that unmodified BC supports chondrocyte proliferation at levels of approximately 50% of the collagen type II substrate while providing significant advantages in terms of mechanical properties. Compared to tissue culture plastic and calcium alginate, unmodified BC showed significantly higher levels of chondrocyte growth. Chemical sulfation and phosphorylation of the BC, performed to mimic the glucosaminoglycans of native cartilage, did not enhance chondrocyte growth while the porosity of the material did affect chondrocyte viability. The BC did not induce significant activation of proinflammatory cytokine production during in vitro macrophage screening. Hence, unmodified BC was further explored using human chondrocytes. TEM analysis and RNA expression of the collagen II from human chondrocytes indicated that unmodified BC supports proliferation of chondrocytes. In addition, ingrowth of chondrocytes into the scaffold was verified by TEM. The results suggest the potential for this biomaterial as a scaffold for tissue engineering of cartilage.
Keywords: Cellulose; Cartilage tissue engineering; Chondrocytes; Scaffold;

The effect of pore size on cell adhesion in collagen-GAG scaffolds by F.J. O’Brien; B.A. Harley; I.V. Yannas; L.J. Gibson (433-441).
The biological activity of scaffolds used in tissue engineering applications hypothetically depends on the density of available ligands, scaffold sites at which specific cell binding occurs. Ligand density is characterized by the composition of the scaffold, which defines the surface density of ligands, and by the specific surface area of the scaffold, which defines the total surface of the structure exposed to the cells. It has been previously shown that collagen–glycosaminoglycan (CG) scaffolds used for studies of skin regeneration were inactive when the mean pore size was either lower than 20 μm or higher than 120 μm (Proc. Natl. Acad. Sci., USA 86(3) (1989) 933). To study the relationship between cell attachment and viability in scaffolds and the scaffold structure, CG scaffolds with a constant composition and solid volume fraction (0.005), but with four different pore sizes corresponding to four levels of specific surface area were manufactured using a lyophilization technique. MC3T3-E1 mouse clonal osteogenic cells were seeded onto the four scaffold types and maintained in culture. At the experimental end point (24 or 48 h), the remaining viable cells were counted to determine the percent cell attachment. A significant difference in viable cell attachment was observed in scaffolds with different mean pore sizes after 24 and 48 h; however, there was no significant change in cell attachment between 24 and 48 h for any group. The fraction of viable cells attached to the CG scaffold decreased with increasing mean pore size, increasing linearly (R 2=0.95, 0.91 at 24 and 48 h, respectively) with the specific surface area of the scaffold. The strong correlation between the scaffold specific surface area and cell attachment indicates that cell attachment and viability are primarily influenced by scaffold specific surface area over this range (95.9–150.5 μm) of pore sizes for MC3T3 cells.
Keywords: Collagen; Scaffold; Microstructure; Cell adhesion;

Biaxial mechanical properties of muscle-derived cell seeded small intestinal submucosa for bladder wall reconstitution by Shing-Hwa Lu; Michael S Sacks; Steve Y Chung; D.Claire Gloeckner; Ryan Pruchnic; Johnny Huard; William C de Groat; Michael B Chancellor (443-449).
Bladder wall replacement remains a challenging problem for urological surgery due to leakage, infection, stone formation, and extensive time needed for tissue regeneration. To explore the feasibility of producing a more functional biomaterial for bladder reconstitution, we incorporated muscle-derived cells (MDC) into small intestinal submucosa (SIS) scaffolds. MDC were harvested from mice hindleg muscle, transfected with a plasmid encoding for β-galactosidase, and placed into single-layer SIS cell culture inserts. Twenty-five MDC and/or SIS specimens were incubated at 37°C for either 10 or 20 days. After harvesting, mechanical properties were characterized using biaxial testing, and the areal strain under 1 MPa peak stress used to quantify tissue compliance. Histological results indicated that MDC migrated throughout the SIS after 20 days. The mean (±SE) areal strain of the 0 day control group was 0.182±0.027 (n=5). After 10 days incubation, the mean (±SE) areal strain in MDC/SIS was 0.247±0.014 (n=5) compared to 10 day control SIS 0.200±0.024 (n=6). After 20 days incubation, the mean areal strain of MDC/SIS was 0.255±0.019 (n=5) compared to control SIS 0.170±0.025 (n=5). Both 10 and 20 days seeded groups were significantly different (p=0.027) than that of incubated SIS alone, but were not different from each other. These results suggest that MDC growth was supported by SIS and that initial remodeling of the SIS ECM had occurred within the first 10 days of incubation, but may have slowed once the MDC had grown to confluence within the SIS.
Keywords: Bladder tissue engineering; Stem cell; Scaffold; Mechanical properties; Soft tissue biomechanics;

Locally delivered nanoencapsulated tyrphostin (AGL-2043) reduces neointima formation in balloon-injured rat carotid and stented porcine coronary arteries by Shmuel Banai; Michael Chorny; S.David Gertz; Ilia Fishbein; Jianchuan Gao; Louise Perez; Galila Lazarovichi; Aviv Gazit; Alexander Levitzki; Gershon Golomb (451-461).
Local delivery of antiproliferative drugs encapsulated in biodegradable nanoparticles (NP) has shown promise as an experimental strategy for preventing restenosis development. A novel PDGFRβ-specific tyrphostin, AGL-2043, was formulated in polylactide-based nanoparticles and was administered intraluminally to the wall of balloon-injured rat carotid and stented pig coronary arteries. The disposition and elimination kinetics within the vessel wall, as well as the antirestenotic potential of the novel drug and delivery system, were evaluated. The efficacy and the local drug elimination kinetics were affected by the size of the NP and the drug-carrier binding mode. Despite similar arterial drug levels 90 min after delivery in rats, small NP were more efficacious in comparison to large NP (90 and 160 nm, respectively). AGL-2043 selectively inhibited vascular SMC in a dose-dependent manner. The antiproliferative effect of nanoencapsulated tyrphostin was considerably higher than that of surface-adsorbed drug. In the pig model, intramural delivery of AGL-2043 resulted in reduced in-stent neointima formation in the coronary arteries over control despite similar degrees of wall injury. The results of this study suggest that locally delivered tyrphostin AGL-2043 formulated in biodegradable NP may be applicable for antirestenotic therapy independent of stent design or type of injury.
Keywords: Controlled drug release; Drug delivery; In-stent restenosis; Intimal hyperplasia; Local delivery; Nanoparticles; Neointima; Protein tyrosine kinase blocker; Restenosis; Smooth muscle cell; Stent; Tyrphostin;