Biomaterials (v.27, #19)

Calendar (I).

Peripheral nerve regeneration presents a significant clinical challenge and the current state of the art using autografts to repair long peripheral nerve gaps is unsatisfactory. In this manuscript, the analytical framework that determines the fate of grafts (autografts or biomaterial-based grafts) is discussed. Also outlined are parameters and variables that might be manipulated to enhance the efficacy of scaffolds designed for peripheral nerve regeneration. The importance of using appropriate animal models and outcome measures in evaluating biomaterials-based scaffolds or other engineered constructs suitability for bridging peripheral nerve gaps is highlighted.
Keywords: Neutral tissue engineering; Hydrogels; Nanofibers;

Multifunctional Ti–(Ca,Zr)–(C,N,O,P) films for load-bearing implants by D.V. Shtansky; N.A. Gloushankova; I.A. Bashkova; M.A. Kharitonova; T.G. Moizhess; A.N. Sheveiko; F.V. Kiryukhantsev-Korneev; M.I. Petrzhik; E.A. Levashov (3519-3531).
Films of Ti–Ca–P–C–O–(N), Ti–Ca–C–O–(N) and Ti–Zr–C–O–(N) were deposited by DC magnetron sputtering or ion implantation-assisted magnetron sputtering of composite targets TiC0.5+10%Ca10(PO4)6(OH)2, TiC0.5+20%(CaO+TiO2) and TiC0.5+10%ZrO2 in an Ar atmosphere or reactively in a gaseous mixture of Ar+14%N2. The microstructure, elemental and phase composition of films were studied by means of X-ray diffraction, transmission electron microscopy, scanning force microscopy, X-ray photoelectron spectroscopy and energy-dispersive X-ray spectroscopy. The films were characterized in terms of their hardness, Young's modulus, elastic recovery, adhesion strength, and friction and wear both in air and under physiological solution. Particular attention was paid to the analysis of deformation and fracture for various film/substrate systems during scratch testing. The biocompatibility of the films was evaluated by both in vitro and in vivo experiments. In vitro studies involved the investigation of adhesion, spreading, and proliferation of MC3T3-E1 osteoblasts and IAR-2 epithelial cells, morphometric analysis, actin cytoskeleton, focal contacts staining, alkaline phosphatase activity and von Kossa staining of osteoblastic culture. Cell culture experiments demonstrated an increase of osteoblastic proliferation on Ca- and P-incorporated films. In vivo studies were fulfilled by subcutaneous implantation of Teflon plates coated with the tested films in mice and analysis of the population of adherent cells on their surfaces. The results obtained show that multicomponent nanostructured Ti–(Ca, Zr)–(C, N, O, P) films possess a combination of high hardness, wear resistance and adhesion strength, reduced Young's modulus, low friction coefficient and high biocompatibility.
Keywords: Multifunctional films; Composite targets; Magnetron sputtering; Physical, mechanical and tribological properties; Attachment, spreading and proliferation of cells; Alkaline phosphatase activity;

The significance of crystallographic texture of titanium alloy substrates on pre-osteoblast responses by Shahab Faghihi; Fereshteh Azari; Huolong Li; Mohammad R. Bateni; Jerzy A. Szpunar; Hojatollah Vali; Maryam Tabrizian (3532-3539).
The aim of this study is to investigate the effects of grain orientation in polycrystalline materials on cell-substrate interactions. Samples are prepared from rods and sheets of Ti–6Al–4V substrates with predominately two distinct crystallographic orientations. X-ray diffraction analysis indicates that 36% of the surfaces of rod samples consist of ( 1 0 1 ¯ 0 ) plane, while the predominant orientation in the surface of the sheet samples is ( 1 1 2 ¯ 0 ) plane (29%). Morphological studies and cell biological experiments including cell attachment, proliferation and differentiation are conducted using MC3T3 pre-osteoblast cells cultured on these two different samples. The number of attached cells on the rod Ti- ( 1 0 1 ¯ 0 ) samples (70% after 1 h and 50% after 2 h) is higher than on the sheet Ti- ( 1 1 2 ¯ 0 ) samples. Cell proliferation after 3 days is also significantly higher on the Ti- ( 1 0 1 ¯ 0 ) samples. Alkaline phosphatase activity, however, shows no significant difference between the two samples. Scanning electron microscopy (SEM) analysis of MC3T3 cells grown on samples with different crystallographic texture demonstrate significant differences in morphology with respect to attachment and growth pattern. This study shows that crystal orientation of the substrate can influence cell responses and, therefore, substrate engineering can be used to improve and control cell-substrate interactions.
Keywords: Titanium alloy; Crystallographic texture; Cell adhesion; Cell proliferation; Alkaline phosphatase; Cell morphology;

The in vivo tissue reactions and biodegradations of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(lactide) (PLA), poly(3-hydroxybutyrate) (PHB), blends of PHBHHx (X) and poly(ethylene glycol) (PEG) (E) with ratios of 1:1 (E1X1) and 1:5 (E1X5), respectively, were evaluated by subcutaneous implantation in rabbits. Results revealed that the degradation rate increased in the order of PHB<PHBHHx<PLA. During the implantation period, crystallinity of PHBHHx increased from 19% to 22% and then dropped to 14%. Gel permeation chromatography (GPC) displayed increasing polydispersity and typical bimodal distribution from 3 to 6 months. The above results suggested that rapid PHBHHx degradation occurred in amorphous region rather than in crystalline region. While the in vivo hydrolysis of PHB was found to start from a random chain scission both in amorphous and crystalline regions of the polymer matrix, as demonstrated by its hydrolysis process accompanied by a decrease in molecular weight with unimodal distribution and relatively narrow polydispersity. Compared to pure PHBHHx, PHBHHx–PEG blends showed accelerated weight loss of PHBHHx with weak molecular weight reduction. In general, PHBHHx elicited a very mild tissue response during implantation lasting 6 months compared with relative acute immunological reactions observed among PHB and PLA objects, respectively. Pronounced tissue responses were observed in the capsule surrounding E1X1 and E1X5 as characterized by the presence of lymphocytes, eosinophils and vascularization, which might be resulted from the continuous leaching of PEG.
Keywords: PHB; PHBHHx; In vivo biodegradation; Tissue response; In vivo biocompatibility;

The in vitro stability of air-filled polybutylcyanoacrylate microparticles by Carsten Olbrich; Peter Hauff; Frank Scholle; Wolfgang Schmidt; Udo Bakowsky; Andreas Briel; Michael Schirner (3549-3559).
Different methods of manufacturing permitted the production of air-filled PBCA microparticles (af-pbca-mp) with different physical properties such as size and wall thickness. These differences led to distinctions with respect to mechanical stability and, at the same time, to different levels of biochemical stability when incubated in biofluids. Microparticles, designed as they are to be mechanically more stable (composed of larger nanoparticles resulting in thicker shell wall, no surface hydrolysis), persist longer under in vitro conditions in biofluids such as serum, plasma and whole blood than do the more fragile ones. It was possible when using the measurement of ultrasound attenuation to characterize af-pbca-mp degradation with respect to the disappearance of the ultrasound properties of the particles and therefore to find out how long different formulations can be expected to be active as contrast agents under simulated in vivo conditions. The present examination showed that using either serum, plasma or whole blood leads to results with the same tendencies in terms of the stability and durability of af-pbca-mp in the media, mimicking in vivo conditions. It was thus possible to validate successfully the use of either serum or plasma as substitutes for whole blood. Further studies dealing with the in vitro in vivo correlation will be needed to find out if the situation in this in vitro assay corresponds to the situation in the body.
Keywords: Microcapsule; Biodegradation; Plasma; Blood; Polybutylcyanoacrylate;

The promotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels by Peter Prang; Rainer Müller; Ahmed Eljaouhari; Klaus Heckmann; Werner Kunz; Thomas Weber; Cornelius Faber; Maurice Vroemen; Ulrich Bogdahn; Norbert Weidner (3560-3569).
Appropriate target reinnervation and functional recovery after spinal cord injury depend on longitudinally directed regrowth of transected axons. To assess the capacity to promote directed axon regeneration, alginate-based highly anisotropic capillary hydrogels (ACH) were introduced into an axon outgrowth assay in vitro and adult rat spinal cord lesions in vivo. In an entorhino-hippocampal slice culture model, alginate-based scaffolds elicit highly oriented linear axon regrowth and appropriate target neuron reinnervation. Coating of alginate-based ACH with the extracellular matrix components collagen, fibronectin, poly l-ornithine and laminin did not alter the axon regrowth response as compared to uncoated alginate-based ACH. After implantation into acute cervical spinal cord lesions in adult rats, alginate-based ACH integrate into the spinal cord parenchyma without major inflammatory responses, maintain their anisotropic structure and in parallel to findings in vitro induce directed axon regeneration across the artificial scaffold. Furthermore, adult neural progenitor cells (NPC), which have been shown to promote cell-contact-mediated axon regeneration, can be seeded into alginate-based ACH as a prerequisite to further improve the regenerative capacity of these artificial growth supportive matrices. Thus, alginate-based ACH represent a promising strategy to induce directed nerve regrowth following spinal cord injury.
Keywords: Self-assembly; Stem cell; Nerve regeneration; Nerve tissue engineering; Hydrogel; Alginate;

Intraperitoneal stability of alginate–polyornithine microcapsules in rats: An FTIR and SEM analysis by Christopher G. Thanos; Briannan E. Bintz; William J. Bell; Haitao Qian; Patricia A. Schneider; Daniel H. MacArthur; Dwaine F. Emerich (3570-3579).
Alginate–polycation microcapsule systems have been used over decades as delivery vehicles for cell and protein therapy. These systems have been unpredictable across a range of indications with questions resulting around the inherent stability of the alginate polysaccharide and failure mode of the delivery system. The current study focuses on such a system using 5 different alginates, 2 of which are commercially purified, which are crosslinked by polyornithine. Capsules formed by frequency-generated droplet formation were studied in the peritoneal cavity of Long-Evans rats over the course of 3 months by morphometry, Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy of the surface. Individual capsule components were also investigated on FTIR and a relative stability index was generated by titration for comparison to explanted samples over time. Using these techniques, a distinct degradation pattern was noted and is compared between the 5 alginate sources.
Keywords: Alginate; Biodegradation; Cell encapsulation; FTIR; Hydrogel; SEM;

Viability and electrophysiology of neural cell structures generated by the inkjet printing method by Tao Xu; Cassie A. Gregory; Peter Molnar; Xiaofeng Cui; Sahil Jalota; Sarit B. Bhaduri; Thomas Boland (3580-3588).
Complex cellular patterns and structures were created by automated and direct inkjet printing of primary embryonic hippocampal and cortical neurons. Immunostaining analysis and whole-cell patch-clamp recordings showed that embryonic hippocampal and cortical neurons maintained basic cellular properties and functions, including normal, healthy neuronal phenotypes and electrophysiological characteristics, after being printed through thermal inkjet nozzles. In addition, in this study a new method was developed to create 3D cellular structures: sheets of neural cells were layered on each other (layer-by-layer process) by alternate inkjet printing of NT2 cells and fibrin gels. These results and findings, taken together, show that inkjet printing is rapidly evolving into a digital fabrication method to build functional neural structures that may eventually find applications in neural tissue engineering.
Keywords: Inkjet printing; Neural cell structure; Tissue engineering; 3D constructs;

Albumin-derived nanocarriers: Substrates for enhanced cell adhesive ligand display and cell motility by Ram I. Sharma; Marian Pereira; Jean E. Schwarzbauer; Prabhas V. Moghe (3589-3598).
Cell-adhesive ligands organized on nanoscale synthetic biomaterials can potentially recapitulate the nanoscale organization of extracellular matrix and the consequent effects of cell dynamics. In this study, 100 nm albumin nanocarriers (ANC) were fabricated to serve as nanoscale organizational units for a well-defined ligand, the recombinant fragment from fibronectin comprised of the RGD-containing module 10 and the synergy-region-containing module 9. Conventional protein conjugation chemistry was employed to fabricate nanocarriers with increasing levels of displayed ligand. Presentation of ligand-functionalized ANCs adsorbed onto substrates was found to enhance keratinocyte attachment when compared to equivalent levels of adsorbed ligands, supported by ELISA data that the display of ligand on ANCs essentially increased the accessibility of the cell-binding domain and AFM data that the ligand was likely exposed due to ligand–ANC repulsion. The ligand presentation from ANCs converted the cellular morphology from a stationary phenotype to a motile phenotype, with the expression of filopodia-like microextensions, and a decrease in focal adhesions, indicating decreased cell adhesion strength. Consequently, cell motility was found to be significantly elevated on ligand–ANC substrates relative to substrates with equivalent levels of ligand. Overall, the ligand-functionalized albumin nanocarriers offer a unique model platform with two distinct properties: enhanced ligand exposure for enhancement of cell attachment to ligands at low concentrations; and enhanced cell detachment, motile phenotype, and migration kinetics.
Keywords: Albumin nanocarriers; Fibronectin; Cell adhesion; Cell migration; Nanobiotechnology;

Characterisation of the nanoporous structure of collagen-glycosaminoglycan hydrogels by freezing-out of bulk and bound water by Lyuba I. Mikhalovska; Vlad M. Gun’ko; Vlad V. Turov; Vlad I. Zarko; Stuart L. James; Pankaj Vadgama; Paul E. Tomlins; Sergey Victorovich Mikhalovsky (3599-3607).
The nanoporous structure of collagen-glycosaminoglycan (CG) hydrogels was studied using 1H NMR spectroscopy and thermally stimulated depolarisation (TSD) current with layer-by-layer freezing-out of bulk and interfacial water. The depression of the freezing point of water is related to the size of the nanopore, to which it is confined. Changes in the Gibbs free energy of the unfrozen interfacial water are related to the amount of bound water in the hydrogel matrix and to the re-arrangement of the 3D network structure of the biopolymer. Analysis of the thermodynamic properties of bulk and interfacial water using the layer-by-layer freezing-out technique combined with NMR and TSDC provides valuable information about the structural features of CG hydrogels that can be used for characterisation of different types of hydrogels and soft tissue scaffolds, artificial skin substitutes and other biomaterials.
Keywords: Collagen; Hydrogel; Porosity; Nanopore structure; NMR;

A new compliant scaffold suitable for small-diameter vascular grafts has been developed that promotes strong attachment of endothelial cells. Composite scaffolds were produced by wet spinning polycaprolactone (PCL) fibres which form the luminal surface, then electrospinning porous polyurethane (PU) onto the back of the PCL fibres to form the vessel wall substitute. Human endothelial cells demonstrated strong attachment to the composite PCL–PU scaffold, and proliferated to form a monolayer with strong PECAM-1 expression and cobblestone morphology. Attached cells demonstrated abundant release of von Willebrand factor, nitric oxide and ICAM-1 under physiological stimuli, and exhibited an immune response to lipopolysaccharide. The composite scaffold may also deliver bioactive molecules. Active trypsin, used as a test molecule, had a defined 48 h pattern of release from luminal PCL fibres. These data confirm the potential of this novel composite scaffold in vascular tissue engineering.
Keywords: Polycaprolactone; Polyurethane; Vascular tissue engineering; Endothelial cells;

Gene expression profiling of human articular cartilage grafts generated by tissue engineering by Christian Kaps; Simone Frauenschuh; Michaela Endres; Jochen Ringe; Andreas Haisch; Jörg Lauber; Jan Buer; Veit Krenn; Thomas Häupl; Gerd-Rüdiger Burmester; Michael Sittinger (3617-3630).
Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. In this study autologous human cartilage tissue engineering grafts based on bioresorbable polyglactin/polydioxanone scaffolds were analyzed on the broad molecular level. RNA from freshly isolated, primary and expanded adult articular chondrocytes and from three-dimensional cartilage grafts were used for gene expression profiling using oligonucleotide microarrays.The capacity of cartilage grafts to form cartilage matrix was evaluated after subcutaneous transplantation into nude mice. Gene expression profiling showed reproducibly the regulation of 905 genes and documented that chondrocytes undergo fundamental changes during cartilage tissue engineering regarding chondrocyte metabolism, growth, and differentiation. Three-dimensional assembly of expanded, dedifferentiated chondrocytes initiated the re-differentiation of cells that was accompanied by the reversal of the expression profile of multiple players of the transforming growth factor (TGF) signaling pathway including growth and differentiation factor-5 and inhibitor of differentiation-1 as well as by the induction of typical cartilage-related matrix genes such as type II collagen and cartilage oligomeric matrix protein. Cartilage grafts formed a cartilaginous matrix after transplantation into nude mice. Three-dimensional tissue culture of expanded articular chondrocytes initiates chondrocyte re-differentiation in vitro and leads to the maturation of cartilage grafts towards hyaline cartilage in vivo.
Keywords: Cartilage tissue engineering; Cartilage regeneration; Chondrocyte differentiation; Gene expression profiling; Microarray; Polyglactin/polydioxanone scaffold;

Design and analysis of tissue engineering scaffolds that mimic soft tissue mechanical anisotropy by Todd Courtney; Michael S. Sacks; John Stankus; Jianjun Guan; William R. Wagner (3631-3638).
Tissue engineered constructs must exhibit tissue-like functional properties, including mechanical behavior comparable to the native tissues they are intended to replace. Moreover, the ability to reversibly undergo large strains can help to promote and guide tissue growth. Electrospun poly (ester urethane) ureas (ES-PEUU) are elastomeric and allow for the control of fiber diameter, porosity, and degradation rate. ES-PEUU scaffolds can be fabricated to have a well-aligned fiber network, which is important for applications involving mechanically anisotropic soft tissues. We have developed ES-PEUU scaffolds under variable speed conditions and modeled the effects of fiber orientation on the macro-mechanical properties of the scaffold. To illustrate the ability to simulate native tissue mechanical behavior, we demonstrated that the high velocity spun scaffolds exhibited highly anisotropic mechanical properties closely resembling the native pulmonary heart valve leaflet. Moreover, use of the present fiber-level structural constitutive model allows for the determination of electrospinning conditions to tailor ES-PEUU scaffolds for specific soft tissue applications. The results of this study will help to provide the basis for rationally designed mechanically anisotropic soft tissue engineered implants.
Keywords: Cardiac tissue engineering; Elastomer; Constitutive modeling; Polyurethane; Scaffold design; Biomechanics;

Transplantable retinal pigment epithelial cell sheets for tissue engineering by Akira Kubota; Kohji Nishida; Masayuki Yamato; Joseph Yang; Akihiko Kikuchi; Teruo Okano; Yasuo Tano (3639-3644).
The native retinal pigment epithelium (RPE) exists as a monolayer structure and is critically involved in the maintenance of photoreceptors. Damage or destruction of the RPE due to a variety of diseases therefore often results in loss of vision. With regenerative purposes in mind, we have examined various culture conditions such as the initial cell density and the addition of various supplements in an effort to produce transplantable RPE cell sheets that can be harvested without defects. We demonstrate that the cell density in cultured RPE sheets increased linearly with the number of seeded cells and that RPE sheets were harvested without defects and limited contraction due to cytoskeletal reorganization, when TGF-β2 was added to the growth medium. Results from histological analysis and the measurement of trans-epithelial resistance also demonstrates that these RPE cell sheets exist as monolayer structure, similar to the native RPE, with intact cell-to-cell junctions. Therefore, these methods provide significant insight into the fabrication of transplantable RPE cell sheets that can be applied to RPE regenerative therapies to restore lost vision.
Keywords: Retina; Thermally responsive material; Epithelium; TGF(transforming growth factor); ECM(extracellular matrix); Ophthalmology;

Structural requirements for stabilization of vascular elastin by polyphenolic tannins by Jason C. Isenburg; Nishant V. Karamchandani; Dan T. Simionescu; Narendra R. Vyavahare (3645-3651).
Elastin-associated degeneration and calcification are potential causes of long-term failure of glutaraldehyde (Glut) fixed tissue bioprostheses used in cardiovascular surgery. This vulnerability may be attributed to the inability of Glut to cross-link and adequately protect vascular elastin from enzymatic attack. Tannic acid (TA), a poly galloyl glucose (Glc), is compatible with Glut fixation, binds to vascular elastin, improves resistance to degradation and reduces in vivo calcification. While these results provided evidence of a beneficial interaction between elastin and TA, the nature and mechanisms of these interactions are unclear; moreover, TA–elastin binding exhibits a partial instability after long-term interaction with vascular elastin which could contribute to issues of implant toxicity. In present studies, we used resistance to elastase, mechanical properties, and cell viability assays to evaluate the elastin-stabilizing potential and cytotoxicity of TA derivatives and individual TA components such as acetylated TA (AcTA), pentagalloylglucose (PGG), free gallic acid (Gall) and Glc. Our comparative study demonstrates that polyphenolic hydroxyl groups are the main structural groups essential to the interaction between TA and elastin. Furthermore, we show that PGG, the core structure of TA, possesses the same unique elastin-stabilizing qualities of TA, yet it is much less cytotoxic than TA and thus could be potentially useful as an elastin-stabilizing agent for cardiovascular bioprostheses.
Keywords: Glutaraldehyde; Pentagalloylglucose; Elastase; Tannic acid; Aorta; Cross linking;

A new mathematical model was developed and an exact analytical solution without approximations of previous work was derived for the description of the kinetics and equilibrium characteristics of drug loading from a finite external solution onto ion-exchange microspheres. The influence of important parameters pertinent to material properties and loading conditions on the kinetics, efficiency, and equilibrium of drug loading was analyzed using the developed model and equations. The numerical results showed that the rate of drug loading increased with increasing initial drug concentration in the solution or with the relative volume of the external solution and the microsphere. The maximum binding capacity of the micrsophere and the association rate constant had positive effects on the loading rate and the equilibrium loading. A decrease in microsphere radius or an increase in drug diffusion coefficient accelerated the loading process but did not influence the equilibrium drug loading. The model prediction agreed with experimental results of verapamil hydrochloride loading onto sulfopropyl dextran microspheres. The usefulness of the model in the design of loading experiments for desired drug loading efficiency and equilibrium loading was demonstrated by numerical analysis.
Keywords: Ion-exchange microspheres; Mathematical modeling; Exact analytical solution; Drug loading kinetics; Equilibrium loading and efficiency; Experimental verification;

Promotion of opsonization by antibodies and phagocytosis of Gram-positive bacteria by a bifunctional polyacrylamide by Vijay M. Krishnamurthy; Lee J. Quinton; Lara A. Estroff; Steven J. Metallo; Jessica M. Isaacs; Joseph P. Mizgerd; George M. Whitesides (3663-3674).
This paper describes the application of a bifunctional polyacrylamide (pA–V–F) presenting both vancomycin and fluorescein groups, to modify the surfaces of multiple species of Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis) to control molecular recognition of these surfaces. The vancomycin groups allowed the specific recognition of a structural component of the bacterial cell wall: peptides terminated in d–Ala–d–Ala. The fluorescein groups allowed the imaging of binding of polymer to the surfaces of bacteria by fluorescence, and are representative, low molecular weight haptens; their recognition by anti-fluorescein antibodies provides proof-of-principle that bifunctional polymers can be used to introduce haptens onto the surface of the bacteria. Flow cytometry revealed that polymer-labeled S. aureus and S. pneumoniae were opsonized by anti-fluorescein antibodies ∼20-fold more than were untreated bacteria; nearly all (∼92%) polymer-labeled S. aureus, and a large (76%) fraction of polymer-labeled S. pneumoniae were opsonized. The bound antibodies then promoted phagocytosis of the bacteria by cultured J774 macrophage-like cells. Flow cytometry revealed that macrophages ingested S. aureus decorated with the polymer-antibody complexes ∼2-fold more efficiently than S. aureus in control groups, in spite of the high background (caused by efficient antibody-independent ingestion of S. aureus by macrophages). This paper, thus, demonstrates the ability of a bifunctional polymer to carry out three distinct functions based on polyvalent molecular recognition: (i) recognition of the surface of Gram-positive bacteria, (ii) modification of this surface to generate specific binding sites recognized by an antibody, and (iii) promotion of phagocytosis of the opsonized bacteria.
Keywords: Antibacterial agent; Bifunctional polyacrylamide; Specific immunostimulation; Antibody; Macrophage; Flow cytometry;

Decellularization of tissues and organs by Thomas W. Gilbert; Tiffany L. Sellaro; Stephen F. Badylak (3675-3683).
Decellularized tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications, and the decellularization methods used vary as widely as the tissues and organs of interest. The efficiency of cell removal from a tissue is dependent on the origin of the tissue and the specific physical, chemical, and enzymatic methods that are used. Each of these treatments affect the biochemical composition, tissue ultrastructure, and mechanical behavior of the remaining extracellular matrix (ECM) scaffold, which in turn, affect the host response to the material. Herein, the most commonly used decellularization methods are described, and consideration give to the effects of these methods upon the biologic scaffold material.
Keywords: Extracellular matrix; Decellularization; Tissue engineering; Scaffolds;

Crosslinking of decellularized porcine heart valve matrix by procyanidins by Wanyin Zhai; Jiang Chang; Kaili Lin; Junyin Wang; Qiang Zhao; Xiaoning Sun (3684-3690).
Heart valve diseases have a significant high mortality, and the valve replacement using glutaraldehyde crosslinked porcine heart valves is one of the main curing techniques. But its application is limited due to poor durability, calcification of the valves and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of procyanidins on porcine heart valve matrix. After crosslinking of the decellularized porcine aortic heart valves by procyanidins, the tensile strength, the in vitro enzymatic degradation resistance, procyanidins release from the crosslinked materials and the cytotoxicity of procyanidins to heart valvular interstitial cells were examined. The results showed that the tensile strength of procyanidins crosslinked valve matrix was higher than that of glutaraldehyde crosslinked valve matrix. Valve matrix crosslinked by 10 mg/ml procyanidins could be stored in D-Hanks solution for at least 45 days without any decline in ultimate tensile strength and maintained the elasticity as the fresh valves. Furthermore, procyanidins was found to release when the crosslinked tissue stored in D-Hanks solution. The release rate was high during the first 4 days and then dramatically decreased thereafter. During releasing phase, the concentration of procyanidins was no toxicity to heart valve interstitial cells. In vitro enzymatic degradation revealed that crosslinked matrix could resist the enzymatic hydrolysis, and the resistant capacity was approximately the same as glutaraldehyde crosslinked valve matrix. This study shows that procyanidins can crosslink porcine heart valves effectively without toxicity. Our results suggested that this method might be a useful approach for preparation of bioprosthetic heart valve.
Keywords: Procyanidins; Crosslinking; Heart valve matrix; Biological valve material;