Biomaterials (v.29, #28)
3D imaging of tissue integration with porous biomaterials
by Robert E. Guldberg; Craig L. Duvall; Alexandra Peister; Megan E. Oest; Angela S.P. Lin; Ashley W. Palmer; Marc E. Levenston (pp. 3757-3761).
Porous biomaterials designed to support cellular infiltration and tissue formation play a critical role in implant fixation and engineered tissue repair. The purpose of this Leading Opinion Paper is to advocate the use of high resolution 3D imaging techniques as a tool to quantify extracellular matrix formation and vascular ingrowth within porous biomaterials and objectively compare different strategies for functional tissue regeneration. An initial over-reliance on qualitative evaluation methods may have contributed to the false perception that developing effective tissue engineering technologies would be relatively straightforward. Moreover, the lack of comparative studies with quantitative metrics in challenging pre-clinical models has made it difficult to determine which of the many available strategies to invest in or use clinically for companies and clinicians, respectively. This paper will specifically illustrate the use of microcomputed tomography (micro-CT) imaging with and without contrast agents to nondestructively quantify the formation of bone, cartilage, and vasculature within porous biomaterials.
Keywords: Imaging; Scaffolds; Micro-CT; Bone; Vascularization; Cartilage
Biodegradable injectable polyurethanes: Synthesis and evaluation for orthopaedic applications
by Raju Adhikari; Pathiraja A. Gunatillake; Ian Griffiths; Lisa Tatai; Malsha Wickramaratna; Shadi Houshyar; Tim Moore; Roshan T.M. Mayadunne; John Field; Margaret McGee; Tania Carbone (pp. 3762-3770).
Biodegradable polyurethanes offer advantages in the design of injectable or preformed scaffolds for tissue engineering and other medical implant applications. We have developed two-part injectable prepolymer systems (prepolymer A and B) consisting of lactic acid and glycolic acid based polyester star polyols, pentaerythritol (PE) and ethyl lysine diisocyanate (ELDI). This study reports on the formulation and properties of a series of cross linked polyurethanes specifically developed for orthopaedic applications. Prepolymer A was based on PE and ELDI. Polyester polyols (prepolymer B) were based on PE anddl-lactic acid (PEDLLA) or PE and glycolic acid (PEGA) with molecular weights 456 and 453, respectively. Several cross linked porous and non-porous polyurethanes were prepared by mixing and curing prepolymers A and B and their mechanical and thermal properties, in vitro (PBS/37°C/pH 7.4) and in vivo (sheep bi-lateral) degradation evaluated. The effect of incorporating β-tricalcium phosphate (β-TCP, 5microns, 10wt.%) was also investigated. The cured polymers exhibited high compressive strength (100–190MPa) and modulus (1600–2300MPa). β-TCP improved mechanical properties in PEDLLA based polyurethanes and retarded the onset of in vitro and in vivo degradation. Sheep study results demonstrated that the polymers in both injectable and precured forms did not cause any surgical difficulties or any adverse tissue response. Evidence of new bone growth and the gradual degradation of the polymers were observed with increased implant time up to 6months.
Keywords: Injectable polyurethanes; In vitro; degradation; In vivo; degradation; Biocompatibility; Mechanical properties; Orthopaedic
In vivo expression of osteogenic markers and bone mineral density at the surface of fluoride-modified titanium implants
by Marta Monjo; Sébastien F. Lamolle; S. Petter Lyngstadaas; H. Jacob Rønold; Jan Eirik Ellingsen (pp. 3771-3780).
The aim of the present study was to investigate the biological mechanisms of the functional attachment of fluoride-modified titanium implants to cortical bone by studying the association of the pull-out test results with gene expression of osteoblast (runx2, osteocalcin, collagen-I and IGF-I), osteoclast (TRAP, H+-ATPase and calcitonin receptor) and inflammation (TNF-α, IL-6 and IL-10) markers from peri-implant bone tissue using real-time RT–PCR, following a 4- and 8-week healing period. After implant detachment, wound fluid from the implant site was collected for LDH and ALP activity analysis. A new method to study volumetric bone mineral density (vBMD) of sub-implant cortical bone was developed using micro-computed tomography. Our results show lower LDH activity and TRAP mRNA levels in fluoride implants after 4weeks of healing, yet no differences were found either on the pull-out force or expression of bone formation marker genes. After 8weeks of healing, both pull-out, vBMD and osteocalcin, runx2 and collage type I gene expression were higher in fluoride implants. In conclusion, fluoride-modified implants seem to modulate both inflammation and bone resorption/formation events at the bone–implant interface, suggesting that these biological effects are an intrinsic part of the clinical performance of this surface.
Keywords: Titanium; Fluoride; Bone; In vivo test; Gene expression; Osseointegration
Synthesis and characterization of biodegradable elastomeric polyurethane scaffolds fabricated by the inkjet technique
by Changhong Zhang; Xuejun Wen; Naren R. Vyavahare; Thomas Boland (pp. 3781-3791).
Biodegradable polyurethanes (PUs) were synthesized from methylene di- p-phenyl-diisocyanate (MDI), polycaprolactone diol (PCL-diol) and N, N-bis (2-hydorxyethyl)-2-aminoethane-sulfonic acid (BES), serving as a hard segment, soft segment and chain extender, respectively. MDI was chosen due to its reactivity and wide application in synthesis of biomedical polyurethanes due to its reactivity; PCL-diol was chosen because of its biodegradability; and BES was chosen because it allowed the introduction sulfonic acid groups onto the polymer chains. We evaluated the polyurethanes' degradation rate, mechanical properties, hydrophilicity, antithrombogenecity, and ability to support fibroblast cell attachment and growth by comparing with polymers having a 2,2-(methylimino)diethanol (MIDE) chain extender. Mechanical testing demonstrated that the PU containing BES has tensile strengths of about 17MPa and elongations up to 400%, about three times the strength and four times the elongation than the MIDE based PUs. The polymers showed decreased in vitro degradation rates, lower glass transition temperature ( Tg) and hydrophilicity possibly due to enhanced microphase separation. Preliminary cytocompatibility studies showed that all the PUs are non-toxic, but PU containing BES exhibited much lower cell attachment and proliferation than the MIDE chain extended polymers. An in vitro platelet adhesion assay showed lower platelet attachment on BES containing PU. Additionally, due to the existence of sulfonic acid groups, the BES extended PU became water soluble in basic condition and insoluble in acidic condition, a phenomenon that is reversible at pH value of 8.7, making this a pH sensitive polymer attractive for bioprinting applications. By adding acetic acid into an inkjet cartridge and printing it onto a PU solution with pH above 8.7, precision fabricated scaffolds can be obtained, suggesting that BES based PUs are promising candidates as synthetic inks used for customizable fabrication of tissue engineering scaffolds.
Keywords: Biodegradation; Cell adhesion; Cell proliferation; Cell–material interaction; Degradation; Mechanical properties
The regulation of cell functions electrically using biodegradable polypyrrole–polylactide conductors
by Guixin Shi; Ze Zhang; Mahmoud Rouabhia (pp. 3792-3798).
Electricity has a long history of being used as an alternative clinical treatment and as an effective approach to modifying cellular behaviours in vitro. It has been difficult, however, to take advantage of this modality in tissue generation because of the lack of suitable conductive, biocompatible scaffolding materials. In this study, in order to electrically regulate cell activities, a largely biodegradable conductor made of 5% conductive polypyrrole and 95% biodegradable poly(l-lactide) (PPy/PLLA) was prepared. Human cutaneous fibroblasts were cultured on the conductors in the presence or absence of a direct current (DC) electrical field (EF) of 50mV/mm. The growth of the cells was characterized using fluorescent staining, SEM, and a MTT assay. The RNA expressions of interleukin-6 (IL-6) and interleukin-8 (IL-8) were assayed by RT-PCR. The amounts of IL-6 and IL-8 secreted by the fibroblasts were quantified by ELISA. The results showed that the PPy/PLLA conductors supported cell adhesion, spreading, and proliferation in both the presence and absence of the EF. Electrical stimulation (ES) applied through PPy/PLLA conductors dramatically enhanced cytokine secretion approximately 10-fold when compared to the non-ES controls. This effect lasted several days after the end of the ES. These findings highlight for the first time the possibility of a potent, effective approach to regulating tissue regeneration in conductive scaffolds through ES-modulated cytokine secretion, and to increasing cytokine productivity for biotechnological applications.
Keywords: Electrical stimulation; Polypyrrole; Biodegradable polymer; Fibroblasts; Gene expression; Cytokine secretion
The morphology of anisotropic 3D-printed hydroxyapatite scaffolds
by Fabienne C. Fierz; Felix Beckmann; Marius Huser; Stephan H. Irsen; Barbara Leukers; Frank Witte; Özer Degistirici; Adrian Andronache; Michael Thie; Bert Müller (pp. 3799-3806).
Three-dimensional (3D) scaffolds with tailored pores ranging from the nanometer to millimeter scale can support the reconstruction of centimeter-sized osseous defects. Three-dimensional-printing processes permit the voxel-wise fabrication of scaffolds. The present study rests upon 3D-printing with nano-porous hydroxyapatite granulates. The cylindrical design refers to a hollow bone with higher density at the periphery. The millimeter-wide central channel follows the symmetry axis and connects the perpendicularly arranged micro-pores. Synchrotron radiation-based micro computed tomography has served for the non-destructive characterization of the scaffolds. The 3D data treatment is essential, since, for example, the two-dimensional distance maps overestimate the mean distances to the material by 33–50% with respect to the 3D analysis. The scaffolds contain 70% micrometer-wide pores that are interconnected. Using virtual spheres, which might be related to the cells migrating along the pores, the central channel remains accessible through the micro-pores for spheres with a diameter of up to (350±35)μm. Registering the tomograms with their 3D-printing matrices has yielded the almost isotropic shrinking of (27±2)% owing to the sintering process. This registration also allows comparing the design and tomographic data in a quantitative manner to extract the quality of the fabricated scaffolds. Histological analysis of the scaffolds seeded with osteogenic-stimulated progenitor cells has confirmed the suitability of the 3D-printed scaffolds for potential clinical applications.
Keywords: Hydroxyapatite; Image analysis; Porosity; Scaffold; Synchrotron radiation-based micro computed tomography; Three-dimensional-printing
Interactions between a poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyhexanoate) terpolyester and human keratinocytes
by Yan Ji; Xiao-Tao Li; Guo-Qiang Chen (pp. 3807-3814).
A new member of polyhydroxyalkanoates (PHA) family, namely, a terpolyester abbreviated as PHBVHHx consisting of 3-hydroxybutyrate (HB), 3-hydroxyvalerate (HV) and 3-hydroxyhexanoate (HHx) that can be produced by recombinant microorganisms, was found to have proper thermo- and mechanical properties for possible skin tissue engineering, as demonstrated by its strong ability to support the growth of human keratinocyte cell line HaCaT. In this study, HaCaT cells showed the strongest viability and the highest growth activity on PHBVHHx film compared with PLA, PHB, PHBV, PHBHHx and P3HB4HB, even the tissue culture plates were grown with less HaCaT cells compared with that on PHBVHHx. To understand its superior biocompatibility, PHBVHHx nanoparticles ranging from 200 to 350nm were prepared. It was found that the nanoparticles could increase the cellular activities by stimulating a rapid increase of cytosolic calcium influx in HaCaT cells, leading to enhanced cell growth. At the same time, 3-hydroxybutyrate (HB), a degradation product and the main component of PHBVHHx, was also shown to promote HaCaT proliferation. Morphologically, under the same preparation conditions, PHBVHHx film showed the most obvious surface roughness under atomic force microscopy (AFM), accompanied by the lowest surface energy compared with all other well studied biopolymers tested above. These results explained the superior ability for PHBVHHx to grow skin HaCaT cells. Therefore, PHBVHHx possesses the suitability to be developed into a skin tissue-engineered material.
Keywords: Polyhydroxyalkanoates; PHB; Terpolyesters; 3-Hydroxybutyrate; 3-Hydroxyvalerate; 3-Hydroxyhexanoate
Induction of osteoblast differentiation phenotype on poly(l-lactic acid) nanofibrous matrix
by Jiang Hu; Xiaohua Liu; Peter X. Ma (pp. 3815-3821).
Thin sheets of nanofibrous (NF) poly(l-lactic acid) (PLLA) matrix were fabricated using a novel phase separation method, mimicking the structure of natural collagen fibers. In this study, the cell morphology, cytoskeleton and adhesion structure, proliferation and differentiation were investigated on NF PLLA matrix using an osteoblast cell line model. Scanning electron microscopy revealed that the MC3T3-E1 cells took a more rounded shape on NF matrix, with abundant interactions with nanofibers. There were no long dense stress fibers or typical focal adhesion structures formed on NF matrix. In consistence with the flat cell morphology and abundant focal adhesions, the cell proliferation was faster on flat PLLA films. With the addition of ascorbic acid (AA), cells were induced to differentiate both on NF matrix and flat films. Cells on NF matrix exhibited an enhanced osteoblast differentiation phenotype, with dramatically higher bone sialoprotein (BSP) gene expression (two orders of magnitude higher) and significantly higher alkaline phosphatase (ALP) activities. Strikingly, even without the addition of AA, thus no natural collagen fibers deposited into the matrix, the BSP gene expression was still highly up-regulated on NF matrix, showing a direct effect of PLLA nanofibers on BSP gene expression. Enhanced BSP gene expression was correlated with the down-regulation of the small GTPase RhoA activities. Inhibition of RhoA effector ROCK induced BSP gene expression of cells in AA-free medium on flat PLLA films. These results suggest that the nanofibers promote the differentiation of osteoblasts likely through RhoA-Rock signaling pathway.
Keywords: Polylactic acid; Osteoblast; Bone tissue engineering; Nanofiber; Differentiation
Advancing dental implant surface technology – From micron- to nanotopography
by Gustavo Mendonça; Daniela B.S. Mendonça; Francisco J.L. Aragão; Lyndon F. Cooper (pp. 3822-3835).
Current trends in clinical dental implant therapy include use of endosseous dental implant surfaces embellished with nanoscale topographies. The goal of this review is to consider the role of nanoscale topographic modification of titanium substrates for the purpose of improving osseointegration. Nanotechnology offers engineers and biologists new ways of interacting with relevant biological processes. Moreover, nanotechnology has provided means of understanding and achieving cell specific functions. The various techniques that can impart nanoscale topographic features to titanium endosseous implants are described. Existing data supporting the role of nanotopography suggest that critical steps in osseointegration can be modulated by nanoscale modification of the implant surface. Important distinctions between nanoscale and micron-scale modification of the implant surface are presently considered. The advantages and disadvantages of nanoscale modification of the dental implant surface are discussed. Finally, available data concerning the current dental implant surfaces that utilize nanotopography in clinical dentistry are described. Nanoscale modification of titanium endosseous implant surfaces can alter cellular and tissue responses that may benefit osseointegration and dental implant therapy.
Keywords: Nanotopography; Dental implant; Surface treatment; Sol–gel techniques; Bone regeneration; Cell signaling
The effect of silica nanoparticle-modified surfaces on cell morphology, cytoskeletal organization and function
by Anna M. Lipski; Christopher J. Pino; Frederick R. Haselton; I.-Wei Chen; V. Prasad Shastri (pp. 3836-3846).
Chemical and morphological characteristics of a biomaterial surface are thought to play an important role in determining cellular differentiation and apoptosis. In this report, we investigate the effect of nanoparticle (NP) assemblies arranged on a flat substrate on cytoskeletal organization, proliferation and metabolic activity on two cell types, Bovine aortic endothelial cells (BAECs) and mouse calvarial preosteoblasts (MC3T3-E1). To vary roughness without altering chemistry, glass substrates were coated with monodispersed silica nanoparticles of 50, 100 and 300nm in diameter. The impact of surface roughness at the nanoscale on cell morphology was studied by quantifying cell spreading, shape, cytoskeletal F-actin alignment, and recruitment of focal adhesion complexes (FAC) using image analysis. Metabolic activity was followed using a thiazolyl blue tetrazolium bromide assay. In the two cell types tested, surface roughness introduced by nanoparticles had cell type specific effects on cell morphology and metabolism. While BAEC on NP-modified substrates exhibited smaller cell areas and fewer focal adhesion complexes compared to BAEC grown on glass, MC3T3-E1 cells in contrast exhibited larger cell areas on NP-modified surfaces and an increased number of FACs, in comparison to unmodified glass. However, both cell types on 50nm NP had the highest proliferation rates (comparable to glass control) whereas cells grown on 300nm NP exhibited inhibited proliferation. Interestingly, for both cell types surface roughness promoted the formation of long, thick F-actin fibers, which aligned with the long axis of each cell. These findings are consistent with our earlier result that osteogenic differentiation of human mesenchymal progenitor cells is enhanced on NP-modified surfaces. Our finding that nanoroughness, as imparted by nanoparticle assemblies, effects cellular processes in a cell specific manner, can have far reaching consequences on the development of “smart” biomaterials especially for directing stem cell differentiation.
Keywords: Focal adhesions; F-actin fibers; Nanoroughness; Endothelial cells; Preosteoblasts; Nano-mechanotransduction
Induced stepwise conformational change of human serum albumin on carbon nanotube surfaces
by Jia-Wei Shen; Tao Wu; Qi Wang; Yu Kang (pp. 3847-3855).
Non-covalent adsorption of proteins onto carbon nanotubes is important to understand the environmental and biological activity of carbon nanotubes as well as their potential applications in nanostructure fabrication. In this study, the adsorption dynamics and features of a model protein (the A sub-domain of human serum albumin) onto the surfaces of carbon nanotubes with different diameters were investigated out by molecular dynamics simulation. The adsorption behaviors were observed by both trajectory and quantitative analyses. During the adsorption process, the secondary structures of α-helices in the model protein were slightly affected. However, the random coils connecting these α-helices were strongly affected and this made the tertiary structure of protein change. The conformation and orientation selection of the protein were induced by the properties and the texture of surfaces indicated by the interaction curve. In addition, the stepwise adsorption dynamics of these processes are found. The mechanism of induced stepwise conformational change of protein on carbon nanotube surfaces would be helpful to better understand the protein–surface interaction at the molecular level.
Keywords: Human serum albumin; Carbon nanotube; Protein adsorption; Stepwise conformational change; Protein-surface interaction
Doxorubicin conjugated tod-α-tocopheryl polyethylene glycol 1000 succinate (TPGS): Conjugation chemistry, characterization, in vitro and in vivo evaluation
by Na Cao; Si-Shen Feng (pp. 3856-3865).
To develop a polymer–anticancer drug conjugate,d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was employed as a carrier of doxorubicin (DOX) to enhance its therapeutic effects and reduce its side effects. Doxorubicin was chemically conjugated to TPGS. The molecular structure, drug loading efficiency, drug release kinetics and stability of the conjugate were characterized. The cellular uptake, intracellular distribution, and cytotoxicity were accessed by using MCF-7 breast cancer cells and C6 glioma cells as in vitro cell model. The conjugate showed higher cellular uptake efficiency and broader distribution within the cells. Judged by IC50, the conjugate was found 31.8, 69.6, 84.1% more effective with MCF-7 cells and 43.9, 87.7, 42.2% more effective with C6 cells than the parent drug after 24, 48, 72h culture, respectively. The in vivo pharmacokinetics and biodistribution were investigated after an i.v. administration at 5mgDOX/kg body weight in rats. Promisingly, 4.5-fold increase in the half-life and 24-fold increase in the area-under-the-curve (AUC) of DOX were achieved for the TPGS–DOX conjugate compared with the free DOX. The drug level in heart, gastric and intestine was significantly reduced, which is an indication of reduced side effects. Our TPGS–DOX conjugate showed great potential to be a prodrug of higher therapeutic effects and fewer side effects than DOX itself.
Keywords: Cancer chemotherapy; Chemotherapeutic engineering; Drug modification; Prodrug; Polymer–drug conjugation
Design and evaluation of polypseudorotaxanes of pegylated insulin with cyclodextrins as sustained release system
by Taishi Higashi; Fumitoshi Hirayama; Shogo Misumi; Hidetoshi Arima; Kaneto Uekama (pp. 3866-3871).
Supramolecular assemblies have attracted a great attention, due to their intriguing topologies and their application in various fields such as nanodevices, sensors, molecular switches, and drug delivery systems. In this study, we prepared the monosubstituted insulin with poly(ethylene glycol) (PEG, MW about 2200) and its cyclodextrin (CyD) polypseudorotaxanes. The pegylated insulin formed polypseudorotaxanes with α- and γ-CyDs, by inserting one PEG chain in the α-CyD cavity and two PEG chains in the γ-CyD cavity. The pegylated insulin/α- and γ-CyD polypseudorotaxanes were less soluble in water and the release rate of the drug decreased in the order of drug alone>the γ-CyD polypseudorotaxane>the α-CyD polypseudorotaxane. The plasma levels of the pegylated insulin after subcutaneous administration of the γ-CyD polypseudorotaxane to rats were significantly prolonged, accompanying an increase in the area under plasma concentration-time curve, which was clearly reflected in the prolonged hypoglycemic effect. The results indicated that the pegylated insulin/CyD polypseudorotaxanes can work as a sustained drug release system, and the polypseudorotaxane formation with CyDs may be useful as a sustained drug delivery technique for other pegylated proteins and peptides.
Keywords: Cyclodextrin; Pegylated insulin; Polypseudorotaxane; Sustained release; Drug delivery system
Acid-degradable cationic methacrylamide polymerized in the presence of plasmid DNA as tunable non-viral gene carrier
by In Kap Ko; Assem Ziady; Shiwei Lu; Young Jik Kwon (pp. 3872-3881).
New acid-degradable cationic nanoparticles were synthesized using a monomer-to-polymer approach, which enabled highly flexible nanoparticle fabrication to obtain controlled properties such as size and conjugation with additional functionalities. The nanoparticles were designed to cause swelling and osmotic destabilization of the endosome, while cationic branches holding anionic DNA are cleaved from the polymeric backbone of the nanoparticles and make plasmid DNA accessible for efficient gene expression. Efficient release of plasmid DNA upon hydrolysis of the nanoparticles at the endosomal pH 5.0 and transportation of the released DNA to the nucleus of a cell were shown. In vitro studies showed significantly higher transfection efficiency by the degradable nanoparticles than polyethylenimine (PEI) polyplexes at very low concentrations (i.e., ng/mL). Size-dependent selective transfection of phagocytic cells (e.g., RAW 309 macrophages) and non-phagocytic cells (e.g., NIH 3T3 fibroblasts) was also achieved by using nanoparticles of two different sizes (240nm and 680nm in diameter), which implies feasibility of tunable gene therapy and DNA vaccination using the nanoparticle system. Preliminary pulmonary transfection of mice using the degradable nanoparticles demonstrated a remarkably higher expression of firefly luciferase at 70% lower concentration than using naked DNA alone. Implications and further improvement of the nanoparticles to be used in gene therapy are also discussed.
Keywords: Controlled drug release; Drug delivery; Gene transfer; Nanoparticles
The effect of surface demineralization of cortical bone allograft on the properties of recombinant adeno-associated virus coatings
by Cemal Yazici; Laura Yanoso; Chao Xie; David G. Reynolds; R. Jude Samulski; Jade Samulski; Judith Yannariello-Brown; Arthur A. Gertzman; Xinping Zhang; Hani A. Awad; Edward M. Schwarz (pp. 3882-3887).
Freeze-dried recombinant adeno-associated virus (rAAV) coated structural allografts have emerged as an approach to engender necrotic cortical bone with host factors that will persist for weeks following surgery to facilitate revascularization, osteointegration, and remodeling. However, one major limitation is the nonporous cortical surface that prohibits uniform distribution of the rAAV coating prior to freeze-drying. To overcome this we have developed a demineralization method to increase surface absorbance while retaining the structural integrity of the allograft. Demineralized bone wafers (DBW) made from human femoral allograft rings demonstrated a significant 21.1% (73.6±3.9% versus 52.5±2.6%; p<0.001) increase in percent surface area coating versus mineralized controls. Co-incubation of rAAV-luciferase (rAAV-Luc) coated DBW with a monolayer of C3H10T1/2 cells in culture led to peak luciferase levels that were not significantly different from soluble rAAV-Luc controls ( p>0.05), although the peaks occurred at 60h and 12h, respectively. To assess the transduction efficiency of rAAV-Luc coated DBW in vivo, we first performed a dose response with allografts containing 107, 109 or 1010 particles that were surgically implanted into the quadriceps of mice, and assayed by in vivo bioluminescence imaging (BLI) on days 1, 3, 5, 7, 10, 14, and 21. The results demonstrated a dose response in which the DBW coated with 1010 rAAV-Luc particles achieved peak gene expression levels on day 3, which persisted until day 21, and was significantly greater than the 107 dose throughout this time period ( p<0.01). A direct comparison of mineralized versus DBW coated with 1010 rAAV-Luc particles failed to demonstrate any significant differences in transduction kinetics or efficiency in vivo. Thus, surface demineralization of human cortical bone allograft increases its absorbance for uniform rAAV coating, without affecting vector transduction efficiency.
Keywords: Allograft; Bone; Recombinant adeno-associated virus (rAAV); Freeze-dried; Bioluminescence imaging (BLI)
Aligned fibrillar collagen matrices obtained by shear flow deposition
by Babette Lanfer; Uwe Freudenberg; Ralf Zimmermann; Dimitar Stamov; Vincent Körber; Carsten Werner (pp. 3888-3895).
Here we present a new technique to generate surface-bound collagen I fibril matrices with differing structural characteristics. Aligned collagen fibrils were deposited on planar substrates from collagen solutions streaming through a microfluidic channel system. Collagen solution concentration, degree of gelation, shear rate and pre-coating of the substrate were demonstrated to determine the orientation and density of the immobilized fibrils. The obtained matrices were imaged using confocal reflection microscopy and atomic force microscopy. Image analysis techniques were applied to evaluate collagen fibril orientation and coverage. As expected, the degree of collagen fibril orientation increased with increasing flow rates of the solution while the matrix density increased at higher collagen solution concentrations and on hydrophobic polymer pre-coatings. Additionally, length of the immobilized collagen fibrils increased with increasing solution concentration and gelation time.
Keywords: Collagen fibrils; Alignment; Microfluidics; Self-assembly; Material properties
Full-field deformation of bovine cornea under constrained inflation conditions
by Brad L. Boyce; J. Mark Grazier; Reese E. Jones; Thao D. Nguyen (pp. 3896-3904).
The viscoelastic response of bovine corneas was characterized using in vitro inflation (bulge) experiments combined with spatially-resolved deformation mapping via digital image correlation. A complex fixture conforming to the limbal annulus was developed to hold the attached sclera rigid while allowing deformation only in the cornea. A statistical set of experiments was performed for a pressure range of 3.6–8kPa (27–60mmHg), representing nominal bovine intraocular pressure (IOP) to acute glaucoma conditions. A broader pressure range of 0–32kPa (0–240mmHg) was also examined to characterize the nonlinear finite deformation behavior of the tissue. Results showed that for pressures near and above IOP, the majority of the deformation was localized in the limbus and peripheral regions, which left the central cornea largely undeformed. This observation was consistent with the known preferred circumferential alignment of collagen fibrils outside of the central cornea. In general, the inflation experiments observed viscoelastic behavior in the form of rate-dependent hysteresis in the pressure–deformation response of the apex of the cornea, creep in the apex deformation at a constant inflation pressure, and relaxation in the pressure response at a constant inflation volume. The 3.6–8kPa (27–60mmHg) pressure range produced small viscoelastic deformations and a nearly linear pressure–deformation response, which suggests that for physiological pressure ranges, the cornea can be approximated as a linear viscoelastic or linear pseudo-elastic material.
Keywords: Cornea; Elasticity; Viscoelasticity; Creep