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Tissue engineering of functional trileaflet heart valves from human marrow stromal cells.

BACKGROUND: We previously demonstrated the successful tissue engineering and implantation of functioning autologous heart valves based on vascular-derived cells. Human marrow stromal cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically. The feasibility of creating tissue engineered heart valves (TEHV) from MSC as an alternative cell source, and the impact of a biomimetic in vitro environment on tissue differentiation was investigated. METHODS AND RESULTS: Human MSC were isolated, expanded in culture, and characterized by flow-cytometry and immunohistochemistry. Trileaflet heart valves fabricated from rapidly bioabsorbable polymers were seeded with MSC and grown in vitro in a pulsatile-flow-bioreactor. Morphological characterization included histology and electron microscopy (EM). Extracellular matrix (ECM)-formation was analyzed by immunohistochemistry, ECM protein content (collagen, glycosaminoglycan) and cell proliferation (DNA) were biochemically quantified. Biomechanical evaluation was performed using Instron(TM). In all valves synchronous opening and closing was observed in the bioreactor. Flow-cytometry of MSC pre-seeding was positive for ASMA, vimentin, negative for CD 31, LDL, CD 14. Histology of the TEHV-leaflets demonstrated viable tissue and ECM formation. EM demonstrated cell elements typical of viable, secretionally active myofibroblasts (actin/myosin filaments, collagen fibrils, elastin) and confluent, homogenous tissue surfaces. Collagen types I, III, ASMA, and vimentin were detected in the TEHV-leaflets. Mechanical properties of the TEHV-leaflets were comparable to native tissue. CONCLUSION: Generation of functional TEHV from human MSC was feasible utilizing a biomimetic in vitro environment. The neo-tissue showed morphological features and mechanical properties of human native-heart-valve tissue. The human MSC demonstrated characteristics of myofibroblast differentiation.

Absorbable Implants↗

[Mineralized collagen based composite for bone tissue engineering].

OBJECTIVE: To construct a mineralized collagen based composite by biomimetic synthesis for bone tissue engineering. METHODS: Using the molecular collagen as the template, the calcium phosphate is deposited on it to produce a mineralized collagen based composite, then is combined with minute amount of poly lactic acid (PLA), the three-dimensional scaffold composite is prepared by liquid phase separation. Using osteoblast culture technique, the biocompatibility of this biomaterial in vitro is detected by x-ray diffraction, SEM, TEM, fluoroscopy and CLSM. RESULTS: Both degree and the size of crystals in the composite are low, which are similar to that of nature bone. It possesses porous structure and the porosity of the composite is high. The typical fibrillar microstructure is self-assembled of the collagen and the nano-crystal hydroxyapatite (HA) in the composite, moreover, the x-ray diffraction graphic of HA crystal shows the [002]-oriented. CONCLUSIONS: The biomimetic three-dimensional composite can serve as one of the optimal scaffold material for bone tissue engineering both on structure and on property.

Animals↗

Physical-chemical and biological characterization of silk fibroin-coated porous membranes for medical applications.

Membranes in artificial organs and scaffolds for tissue engineering are often coated with biomimetic molecules (e.g., collagen) to improve their biocompatibility and promote primary cell adhesion and differentiation. However, animal proteins are expensive and may be contaminated with prions. Silk fibroin (SF) made by Bombyx Mori silk worms, used as a scaffold or grafted to other polymers, reportedly promotes the adhesion and growth of many human cell types. This paper describes how commercial porous membranes were physically coated with SF, and their physical-chemical properties were characterized by SEM, AFM, tensile stress analysis and dynamic contact angle measurements. The effect of the SF coating on membrane biocompatibility and resistance to bacterial colonization is also examined. The proposed technique yields SF coats of different thickness that strengthen the membranes and make their surface remarkably more wettable. The SF coat is not cytotoxic, and promotes the adhesion and proliferation of an immortalized fibroblast cell line. Similarly to collagen, SF-coated membranes also exhibit a much better resistance to the adhesion of S. epidermidis bacteria than uncoated membranes. These preliminary results suggest that SF is a feasible alternative to collagen as a biomimetic coating for 3D scaffolds for tissue engineering or bioartificial (as well as artificial) prosthesis.

Chemical Phenomena↗

Clinical comparison of Omafilcon A with four control materials.

PURPOSE: The purpose of this study was to assess the clinical performance of hydrogel contact lenses manufactured from a novel, biomimetic, 59% water content, hydrogel material (Omafilcon A) as compared with a representative range of control materials (Polymacon, Etafilcon A, Atlafilcon A, and Welcon CE). METHOD: Thirty subjects were enrolled in a 2-month, double-masked, randomized, comparative, daily wear study. Subjects wore two sets of lenses; each pair was worn for 1 month. In each part of the study, subjects wore a Proclear (Biocompatibles Ltd.) test lens (Omafilcon A) in one eye and a control lens in the other eye. In vivo lens dehydration was measured after 1 week of lens wear. Lenses were collected at the end of the study and subjected to in vitro spectrofluorimetric analysis to measure protein and lipid deposits. RESULTS: Omafilcon A showed significantly less lipid spoilation than all the controls and significantly less protein than the Etafilcon A and Atlafilcon A lenses. High levels of protein were found on the Group 4 lenses (Etafilcon A) and high levels of lipid were found on the Group 2 lenses (Weicon CE). The Omafilcon A lenses exhibited significantly less on-eye dehydration than the Etafilcon A lenses (P < 0.0001) and the Weicon CE lenses (P < 0.005). The majority of subjects expressed a preference for the Proclear lens, generally for reasons of comfort. Some minor differences in aspects of lens fit were noted between the test and control lenses, however, none of these were felt to be great enough to affect comfort. CONCLUSIONS: We hypothesize that the reduced spoliation seen with the Omafilcon A lens relates to the biomimetic nature of the material which incorporates phosphorylcholine.

Adsorption↗

Hydroxyapatite coating on stainless steel pre-coated with bovine serum albumin at ambient conditions.

A biomimetic process for coating of nanosized hydroxyapatite on stainless steel, which capitalises the dual nature of the protein bovine serum albumin in both metal binding and a strong affinity for calcium ions, has been developed. The novelty of the process lies in pre-conditioning the metallic surface using the above protein prior to its mineralization with hydroxyapatite at ambient conditions. The microporous morphology of these coatings may provide favourable solubility and resorbability as desired by many orthopaedic and orthodontic applications.

Animals↗

Functional incorporation of integrins into solid supported membranes on ultrathin films of cellulose: impact on adhesion.

Biomimetic models of cell surfaces were designed to study the physical basis of cell adhesion. Vesicles bearing reconstituted blood platelet integrin receptors alpha(IIb)beta(3) were spread on ultrathin films of cellulose, forming continuous supported membranes. One fraction of the integrin receptors, which were facing their extracellular domain toward the aqueous phase, were mobile, exhibiting a diffusion constant of 0.6 micro m(2) s(-1). The functionality of receptors on bare glass and on cellulose cushions was compared by measuring adhesion strength to giant vesicles. The vesicles contained lipid-coupled cyclic hexapeptides that are specifically recognized by integrin alpha(IIb)beta(3). To mimic the steric repulsion forces of the cell glycocalix, lipids with polyethylene glycol headgroups were incorporated into the vesicles. The free adhesion energy per unit area deltag(ad) was determined by micro-interferometric analysis of the vesicle's contour near the membrane surface in terms of the equilibrium of the elastic forces. By accounting for the reduction of the adhesion strength by the repellers and from measuring the density of receptors one could estimate the specific receptor ligand binding energy. We estimate the receptor-ligand binding energy to be 10 k(B)T under bioanalogue conditions.

Biomimetic Materials↗

Preparation of hydroxyapatite-gelatin nanocomposite.

A nanocomposite of gelatin[GEL]-hydroxyapatite[HAp] was prepared using the biomimetic process. The hydroxyapatite nanocrystals were precipitated in aqueous solution of gelatin at pH 8 and 38 degrees C. The chemical bonding between calcium ions of HAp and carboxyl ions of GEL molecules induced a red-shift of the 1339 cm(-1) band of GEL in FT-IR analysis. TEM images and electron diffraction patterns for the nanocomposite strongly indicate the self-organization of HAp nanocrystals along the GEL fibrils. Electron diffraction for the nanocomposites showed a strong preferred orientation of the (002) plane in HAp nanocrystals. The development of HAp nanocrystals in an aqueous GEL solution was highly influenced by the concentration ratio of GEL to HAp. A higher concentration of GEL induced the formation of tiny crystallites (4 nm x 9 nm size), while a lower concentration of GEL contributed to the development of bigger crystallites (30 nm x 70 nm size). From DT/TGA data, the HAp-GEL nanocomposite showed typically three exothermic temperatures. The increase in decomposition temperatures indicates the formation of a primary chemical bond between HAp and GEL. The higher concentration of GEL supplies abundant reaction sites containing groups such as carboxyl, which can bind with calcium ions. The abundant supply of reaction sites leads to a very large number of HAp nuclei. However, the formation of a large number of nuclei depletes the concentration of calcium ions that available for growth to the extent that the nuclei cannot grow very large. This in turn will lead to the creation of a large number of tiny nanocrystals at this higher GEL concentration.

Biomimetic Materials↗

Advance and prospect of bionanomaterials.

Over the past few years, bionanomaterial science has emerged as a new exciting field in which theoretical and experimental studies of structure and function of bionanomaterials have become a focus, and the importance of DNA, RNA, and peptides as bionanomaterials to the fundamental development in biology and nanomaterials has begun to be recognized. In particular, biochemistry, biophysics, biomechanics, thermodynamics, and electronic properties of DNA, RNA, and peptides, as well as intelligent composite biological materials, have become a new interdisciplinary frontier in life science and material science. There is an increasing need for a more systematic study of the basic issues involved in bionanomaterials and a more active participation of researchers in the application domain of such novel materials. Great advances have been and are being made in nanobiochip materials, nanoscale biomimetic materials, nanomotors, nanocomposite materials, interface biomaterials, and nanobiosensor and nano drug delivery systems, with enormous prospect in industrial, defense, and clinical medicine applications. Here we review some of the main advances in this field over the past few years, explore the application prospects, and discuss the concepts, issues, approaches, and challenges, with the aim of stimulating a broader interest in developing bionanomaterials technology.

Biocompatible Materials↗

Some got away, but others didn't.

A history of research projects undertaken in the group-ranging from mechanistic investigations to biomimetic chemistry to molecular devices and synthesis-is presented.

Biological Factors↗

On the role(s) of additives in bioinspired silicification.

Biological organisms are able to direct the formation of patterned and hierarchical biomineral structures. Extractable organic materials have been found entrapped in diatom, sponge and plant biosilica, some of which have been isolated by selective chemical dissolution methods and their composition and structure studied. Information gained from the bioextracts has inspired materials chemists to design biomimetic analogues and develop bioinspired synthetic schemes for silica formation. The results obtained from bioinspired silicification investigations are hypothesised to arise from specific modes of action of the organic additives, which are described in this review. Specifically, additives in bioinspired silicification act either as catalysts, aggregation promoting agents or structure-directing agents or more typically, exhibit a combination of these behaviours.

Biomimetic Materials↗

Phytol-modified heme in mesoporous silica: conjugates as models of hemoproteins.

A ferriprotoporphyrin, hemin (Fe(3+)), modified with 3,7,11,15-tetramethyl-2-hexadecen-1-ol, phytol, was adsorbed in nano-spaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material) forming a phytol-modified hemin (Fe(3+))-FSM nano-conjugate. The properties and the structure of the conjugate were studied by UV-visible light absorption, IR absorption spectroscopy, and a nitrogen adsorption isotherm. Although the hemin without phytol could not be adsorbed to the mesoporous silica, modification with phytol imparted preferential adsorption properties. The conjugate was not only stable but also had a peroxidase-like activity in a 0.1% hydrogen peroxide solution, while free hemin in the solution was easily destroyed. The hemin (Fe(3+)) in the FSM was reduced to heme (Fe(2+)) by hydrazine. The phytol-modified heme (Fe(2+))-FSM conjugate formed an O(2)-heme complex with a superoxide type structure, resembling oxyhemoglobin or oxymyoglobin, which has not been previously observed for free heme in solution. The addition of carbon monoxide or nitrogen monoxide to the phytol-modified heme (Fe(2+))-FSM conjugate caused the formation of CO- or NO-heme complex in the nano-spaces of the FSM. These properties are attributed not only to the Fe-complex but also to the cooperative functions of the heme with mesoporous silica, resembling properties of a natural heme-protein conjugate; hemoglobin or peroxidase. These results are an elegant example of biomimetic nano-technology.

Adsorption↗

The reaction of hydrogen atoms with methionine residues: A model of reductive radical stress causing tandem protein-lipid damage.

The occurrence of tandem damage, due to reductive radical stress involving proteins and lipids, is shown by using a biomimetic model. It is made of unsaturated lipid vesicle suspensions in phosphate buffer in the presence of methionine, either as a single amino acid or as part of a protein such as RNase A, which contains four methionine residues. The radical process starts with the formation of H(.) atoms by reaction of solvated electrons with dihydrogen phosphate anions, which selectively attack the thioether function of methionine. The modification of methionine to alpha-aminobutyric acid is accompanied by the formation of thiyl radicals, which in turn cause the isomerization of the cis fatty acid residues to the trans isomers. The relationship between methionine modification and lipid damage and some details of the reductive radical stress obtained by proteomic analysis of irradiated RNase A are presented.

Biomimetics↗

Antibiotic resistance: mono- and dinuclear zinc complexes as metallo-beta-lactamase mimics.

Biomimetic systems containing one or two zinc(II) ions supported by phenolate ligands were developed as functional mimics of metallo-beta-lactamase. These complexes were shown to catalytically hydrolyze beta-lactam substrates, such as oxacillin and penicillin G. The dinuclear zinc complex 1, which has a coordinated water molecule, exhibits high beta-lactamase activity, whereas the dinuclear zinc complex 2, which has no water molecules, but labile chloride ligands, shows a much lower activity. The high beta-lactamase activity of complex 1 can be ascribed to the presence of a zinc-bound water molecule that is activated by being hydrogen bonded to acetate substituents. The kinetics of the hydrolysis of oxacillin by complex 1 and the effect of pH on the reaction rates are reported in detail. In addition, the kinetic parameters obtained for the synthetic analogues are compared with those of the natural metallo-beta-lactamase from Bacillus cereus (BcII). To understand the role of the second metal ion in hydrolysis, the syntheses and catalytic activities of two mononuclear complexes (3 and 4) that include coordinated water molecules are described. Interestingly, the mononuclear zinc complexes 3 and 4 also exhibit high activity, supporting the assumption that the second zinc ion is not crucial for the beta-lactamase activity.

Bacillus cereus↗

Determination of specific interactions between glucose ligand carrying polymer and glucose transporter type-1 (GLUT-1) using different cell types.

In order to develop a biomimetic polymer for cell recognition, poly [3-O-(4'-vinylbenzyl)-D-glucose] (PVG) and different types of glucose transport (GLUT)-carrying cells, namely, HepG2 cells (GLUT-1), 3T3-L1 fibroblast cells (GLUT-1 and GLUT-4), and MIN6 cells (GLUT-2), were tested for specific interaction. To clarify the nature of interaction between PVG and the different cell types, rhodamine-B isothiocyanate (RITC)-labeled polymers were used to prove and visualize the interactions. In this study, we found that labeled polymer strongly binds to HepG2 cells. The fluorescence intensity of PVG with in the presence of HepG2 cells was found to be stronger than that of 3T3-L1 fibroblast cells (0.11 +/- 0.05) and MIN6 cells, which carry GLUT-2 (0.028 +/- 0.01); a confocal laser microscopic study confirmed this interaction. To confirm the specificity of the interaction mediated by GLUT, the cells were pretreated with phloretin, an inhibitor of GLUT-1, before adding RITC-labeled PVG polymer to the cell culture medium. This treatment suppressed the interaction of PVG with HepG2 cells and partially suppressed its interaction with 3T3-L1 fibroblast cells.

Animals↗

Osteogenic effects of bioactive glass on bone marrow stromal cells.

Bioactive glass (BG) is an effective synthetic bone graft material. BG granules of narrow size range (300-355 mum) have the ability to form new bone tissue inside excavations produced by in vivo resorption. Previously, we demonstrated that BG stimulates the differentiation of cultured osteoblast precursors if the glass surface was biomimetically modified by the formation of bone-like apatite and adsorption of serum proteins. We now report that modified BG can also increase the rate at which multipotential rat bone marrow stromal cells (rMSC) will undergo osteogenesis. BG promoted rMSC osteogenesis both when cells were plated in contact with BG and when cells were not directly in contact with the BG. Alkaline phosphatase activity, a marker of bone cell differentiation, was used as an indicator for osteogenesis. Alkaline phosphatase activity of rMSCs exposed to osteoinducers such as ascorbate, dexamethasone, and BMP-2 was enhanced in the presence of BG. The stimulatory effect of BG was more pronounced in rMSC cultures with low basal alkaline phosphatase activity than in those with higher activity. The enhanced differentiation of rMSCs was associated with both a change in rMSC morphology and altered chemical composition of the cell culture media. rMSCs cultured on BG in the presence of BMP or dexamethasone exhibited a more rounded osteoblast-like appearance as compared with cells grown on tissue culture plastic. In the presence of BG, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period, suggesting a continuous dissolution of surface-modified BG and resulting release of BG dissolution products. The data suggest that both surface- and solution-mediated events play a role in the osteogenic effect of BG.

Alkaline Phosphatase↗

Co-electrospun poly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds.

In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic acid), PLGA, 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and alpha-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting PLGA-gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 +/- 80 nm, which was considerably smaller than fibers made under identical conditions from the starting materials (PLGA, 780 +/- 200 nm; gelatin, 447 +/- 123 nm; elastin, 1060 +/- 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds. Histological characterization of the PGE constructs indicated that BMSCs penetrated into the center of scaffolds and began proliferating shortly after seeding. Our results suggest that fibrous scaffolds made of PGE and similar biomimetic blends of natural and synthetic polymers may be useful for engineering soft tissues, such as heart, lung, and blood vessels.

Animals↗

Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

Basement Membrane↗