Biochemical and mechanical behavior of ostrich pericardium as a new biomaterial

We have performed a comparative analysis of glutaraldehyde-preserved ostrich pericardium, as a novel biomaterial, with bovine pericardium. The biochemical characteristics (histology, water content, amino acid composition, and collagen and elastin contents), mechanical properties, and in vivo calcification in a subcutaneous rat model were examined. Ostrich pericardium is slightly thinner and shows a higher water content (70+/-2% vs. 62+/-2%) than bovine pericardium. Additionally, ostrich pericardium presents 1.6-fold lower elastin content and a lower percentage of collagen in reference to the total protein content (68+/-2% vs. 76+/-2%). However, ostrich pericardium shows better mechanical properties, with higher tensile stress at rupture (32.4+/-7.5 vs. 11.5+/-4.6) than calf pericardium. In vivo calcification studies in a rat subcutaneous model show that ostrich pericardium is significantly less calcified than bovine pericardium (23.95+/-13.30 vs. 100.10+/-37.36 mg/g tissue) after 60 days of implantation. In conclusion, glutaraldehyde-stabilized ostrich pericardium tissue shows better mechanical properties than calf tissue. However, calcium accumulation in implanted ostrich tissue is still too high to consider it a much better alternative to bovine pericardium, and anticalcification treatments should be considered.     

The telescoping suture--Part II: A novel method to improve the mechanical behavior of a new biomaterial: ostrich pericardium

Ostrich pericardium, sutured using a telescoping or overlapping technique, was studied to determine its mechanical behavior. From each of 12 pericardial sacs, four contiguous strips were cut longitudinally, from root to apex, and another four contiguous strips were cut in transverse direction. One of the strips in each set of four was used as an unsutured control and the remaining three were sutured by overlapping 0.5 cm of the tissue and sewing with Gore-tex, Prolene or Pronova. These 96 samples were then subjected to tensile testing along their major axes until rupture. The tensile stresses recorded in the suture materials at the moment tears appeared in the pericardium ranged between 55.99 MPa and 70.23 MPa for Gore-tex in samples cut in the two directions. Shear stress became ostensible at 56 MPa, with clearly evident tears. However, microfracture of the collagen fibers must be produced at much lower stress levels. The comparison of the resistance in kilograms (machine-imposed), without taking into account the sections in which the load was applied, demonstrated only a slight loss of load when the telescoping suture was employed in ostrich pericardium samples. Ostrich pericardium may continue to be an alternative biological material for the construction of heart valve leaflets.     

Comparison of the mechanical behaviors of biological tissues subjected to uniaxial tensile testing: pig,calf and ostrich pericardium sutured with Gore-Tex

The purpose of this study was to compare the mechanical behavior of calf pericardium, pig pericardium and ostrich pericardium when subjected to tensile testing. Tensile stress was applied to 108 tissue samples, 36 of each type of tissue, until rupture. Groups of three adjacent strips measuring 12 x 2 cm(2) were cut longitudinally. Each group consisted of an unsutured center sample, or control, and the two contiguous samples, that on the right sutured with Gore-Tex at a 90 degrees angle with respect to the longitudinal axis and that on the left sewn with the same suture material at 45 degrees angle. The sutured samples showed a statistically significant loss of resistance (p<0.001) when compared with the corresponding unsutured tissue. The mean stresses at rupture for sutured ostrich pericardium were 21.81 and 20.81 MPa in the samples sewn at 45 degrees and 90 degrees, respectively, higher than those corresponding to unsutured calf and pig pericardium, 14.0 and 11.49 MPa, respectively, at rupture.The analysis of the stress/strain curve shows a smaller difference between sutured and unsutured ostrich pericardium than those observed in the other two biomaterials. These results demonstrate that, in addition to its greater resistance, ostrich pericardium also presents a less pronounced interaction with the suture material. Its capacity to absorb the shearing stress produced by the suture is greater. This report also confirms that the method of selection using paired samples ensures their homogeneity and makes it possible to predict the behavior of a sample by determining that of the other half of the pair.     

Modifications on collagen structures promoted by 1,4-dioxane improve thermal and biological properties of bovine pericardium as a biomaterial

Collagen is a widely used raw material for biomaterial manufacture, which generally depends on chemical modifications of this fibrillar protein with cross-linking agents to improve biocompatibility and mechanical properties. However, cross-linking reduces the natural properties of collagen, such as low immune response, low toxicity as well as the ability to promote cellular growth and attachment. In this work, the modifications promoted by 1,4-dioxane solvent on the collagen present in native bovine pericardium (NBP) matrix routinely used in bioprosthesis manufacture, with or without subsequent cross-linking by glutaraldehyde, has been studied. The structural changes of NBP evaluated by scanning electron microscopy show that 1,4-dioxane induces a more homogeneous material by increasing aggregation of collagen fibers, while transmission scanning electron microscopy shows that natural collagen fibril arrangement, integrity, and the D-periodicity pattern are maintained by solvent treatments. Measurements of thermal stability and resistance to collagenase enzymatic digestion of NBP matrices treated with 1,4-dioxane show an increase in melting temperature and decrease in biodegradability, as compared to native pericardium. Cross-linking with glutaraldehyde improves all the analyzed NBP properties, which are not impaired by previous treatment with 1,4-dioxane. Histological evaluation of NBP submitted to 1,4-dioxane treatment shows lower lipid and cell contents and improvement in other morphologic characteristics compared to native pericardium. Altogether, these results suggest the use of 1,4-dioxane organic solvent as an alternative non-cross-linking treatment for direct utilization on rich collagen matrices, resulting in materials with improved biocompatibility and physicochemical properties suitable for tissue engineering.     

Mechanical evaluation and design of a multilayered collagenous repair biomaterial

One method of fabricating implantable biomaterials is to utilize biologically derived, chemically modified tissues to form constructs that are both biocompatible and remodelable. Rigorous mechanical characterization is a necessary component in material evaluation to ensure that the constructs will withstand in vivo loading. In this study we performed an in-depth biaxial mechanical and quantitative structural analysis of GraftPatch (GP), a biomaterial constructed by assembling chemically treated layers of porcine small intestinal submucosa (SIS). The mechanical behavior of GP was compared to both native SIS and to glutaraldehyde-treated bovine pericardium (GLBP) as a reference biomaterial. Under biaxial loading, GP was found to be stiffer than native SIS and mechanically anisotropic, with the preferred fiber direction demonstrating greater stiffness. Quantitative structural analysis using small-angle light scattering indicated a uniform fiber structure similar to GLBP and SIS. To enable test-protocol-independent quantitative comparisons, the biaxial mechanical data were fit to an orthotropic constitutive model, which indicated a similar degree of mechanical anisotropy between the three groups. We also demonstrate how the constitutive model can be used to design layered biocomposite materials that can undergo large deformations.     

Mechanical and corrosion resistance of a new nanostructured Ti-Zr-Ta-Nb alloy

In this work, a multi-elementary Ti–10Zr–5Nb–5Ta alloy, with non-toxic alloying elements, was used to develop an accumulative roll bonding, ARB-type procedure in order to improve its structural and mechanical properties. The alloy was obtained by cold crucible semi-levitation melting technique and then was ARB deformed following a special route. After three ARB cycles, the total deformation degree per layer is about 86%; the calculated medium layer thickness is about 13 μm. The ARB processed alloy has a low Young’s modulus of 46 GPa, a value very close to the value of the natural cortical bone (about 20 GPa). Data concerning ultimate tensile strength obtained for ARB processed alloy is rather high, suitable to be used as a material for bone substitute. Hardness of the ARB processed alloy is higher than that of the as-cast alloy, ensuring a better behaviour as a implant material. The tensile curve for the as-cast alloy shows an elastoplastic behaviour with a quite linear elastic behaviour and the tensile curve for the ARB processed alloy is quite similar with a strain-hardening elastoplastic body. Corrosion behaviour of the studied alloy revealed the improvement of the main electrochemical parameters, as a result of the positive influence of ARB processing. Lower corrosion and ion release rates for the ARB processed alloy than for the as-cast alloy, due to the favourable effect of ARB thermo-mechanical processing were obtained.     

Fabrication, characterization, and biological assessment of multilayered DNA-coatings for biomaterial purposes

This study describes the fabrication of two types of multilayered coatings onto titanium by electrostatic self-assembly (ESA), using deoxyribosenucleic acid (DNA) as the anionic polyelectrolyte and poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Both coatings were characterized using UV-vis spectrophotometry, atomic force microscopy (AFM), X-ray photospectroscopy (XPS), contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and for the amount of DNA immobilized. The mutagenicity of the constituents of the coatings was assessed. Titanium substrates with or without multilayered DNA-coatings were used in cell culture experiments to study cell proliferation, viability, and morphology. Results of UV-vis spectrophotometry, AFM, and contact angle measurements clearly indicated the progressive build-up of the multilayered coatings. Furthermore, AFM and XPS data showed a more uniform build-up and morphology of [PDL/DNA]-coatings compared to [PAH/DNA]-coatings. DNA-immobilization into both coatings was linear, and approximated 3microg/cm(2) into each double-layer. The surface morphology of both types of multilayered DNA-coatings showed elevations in the nanoscale range. No mutagenic effects of DNA, PDL, or PAH were detected, and cell viability and morphology were not affected by the presence of either type of multilayered DNA-coating. Still, the results of the proliferation assay revealed an increased proliferation of primary rat dermal fibroblasts on both types of multilayered DNA-coatings compared to non-coated controls. The biocompatibility and functionalization of the coatings produced here, will be assessed in subsequent cell culture and animal-implantation studies.     

Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues,whole-animals,insects and plants

A method for preparing thin fresh-frozen sections from large samples and hard tissues is described and the applications are shown. A new adhesive film is introduced to produce the frozen sections. The sample is frozen in a cooled hexane or liquid nitrogen, and then freeze-embedded with 4-5% carboxymethyl cellulose (CMC) in the coolant. A specially prepared adhesive film is fastened to the cut surface of the sample in order to support the section and cut slowly with a disposable tungsten carbide blade. The adhesive film is made of a thin plastic film and an adhesive before use. This method produces 2-microm thick fresh-frozen sections from a large sample, bone or tooth. The "film-section" i.e. the section attached to the adhesive film, can be used for many types of studies such as histology, general histochemistry, enzyme histochemistry, immunohistochemistry, in situ hybridization, elemental analysis, and autoradiography for water-soluble materials. Immunohistochemistry and in situ hybridization can be carried out with nonfixed and undecalcified sections. The section on the adhesive film can be transferred to a glass slide and mounted under a cover slip, and stained sections can be examined with an optical microscope at high magnification. This method is also useful for preparing frozen sections from samples of fish, insects, and plants. Furthermore, samples of particular areas can be collected from the film-section by means of a laser microdissection technique. The multiple possible applications of the adhesive film render it highly useful for studies in biological and medico-dental fields.     

Ostrich pericardium, a biomaterial for the construction of valve leaflets for cardiac bioprostheses: mechanical behaviour, selection and interaction with suture materials.

The mechanical behavior of ostrich pericardium was studied for the purpose of assessing its utility in the construction of bioprosthetic cardiac valve leaflets. The tissue was tested biaxially using a hydraulic simulator that subjected it to increasing stress until rupture. One hundred eighty trials were performed, 36 with unsutured pericardium and four series of 36 trials each with pericardium sutured with silk, Prolene, nylon or Gore-Tex. The samples were tested in pairs from three different pericardial regions. One sample from each pair (the predictive specimen) was assessed according to morphological and mechanical criteria, while the other (the predicted or selectable specimen) was subjected only to morphological analysis. The findings show that ostrich pericardium treated with glutaraldehyde according to standard methods has an excellent resistance to rupture in biaxial testing, withstanding stresses of up to 100 MPa, and never lower than 30 MPa. Its resistance to rupture is lowered by suturing, a loss that is less pronounced when silk sutures are used. The results with Gore-Tex are very homogeneous and the elastic behavior of the pericardium/suture unit appears to be similar to that of unsutured tissue, suggesting that the interaction between the two biomaterials is minor. Similar results were observed in the series sutured with Prolene and nylon. The use of paired samples makes it possible to closely estimate the mechanical behavior of the tissue in a given zone by determining that of its mate. The statistical study shows that this estimation is not conditioned by the suture employed, thus validating this approach and providing more precise criteria for tissue selection.     

A physical vapor deposition method for controlled evaluation of biological response to biomaterial chemistry and topography

The purpose of this study was to characterize a technique to effectively mask surface chemistry without modifying surface topography. A thin layer of titanium was deposited by physical vapor deposition (PVD) onto different biomaterial surfaces. Commercially pure titanium disks were equally divided into three groups. Disks were either polished to a mirror finish, grit blasted with alumina particles, or grit blasted and subsequently plasma sprayed with a commercial grade of hydroxyapatite (HA). A subgroup of each of these treatment types was further treated by masking the entire disk surface with a thin layer of commercially pure titanium deposited by PVD. A comparison of surface topography and chemical composition was carried out between disks within each treatment group. Canine marrow cells were seeded on all disk surfaces to determine the stability of the PVD Ti mask under culture conditions. The PVD process did not significantly alter the surface topography of any samples. The thin titanium layer completely masked the underlying chemistry of the plasma sprayed HA surface and the chemistry of the plasma vapor deposited titanium layer did not differ from that of the commercially pure titanium disks. Aliquots obtained from the media during culture did not indicate any significant differences in Ti concentration amongst the Ti and Ti-masked surfaces. The PVD application of a Ti layer on HA coatings formed a stable, durable, and homogenous layer that effectively masked the underlying surface chemistry without altering the surface topography.     

Resistance and stability of a new method for bonding biological materials using sutures and biological adhesives

The valve leaflets of cardiac bioprostheses are secured and shaped by sutures which, given their high degree of resistance and poor elasticity, have been implicated in the generation of stresses within the leaflets, contributing to the failure of the bioprostheses. Bioadhesives are bonding materials that have begun to be utilized in surgery, although there is a lack of experience in their use with inert tissues or bioprostheses.Tensile testing is performed until rupture in samples of calf pericardium, a biomaterial employed in the manufacture of bioprosthetic heart valve leaflets.One hundred and thirty-two trials are carried out in three types of samples: intact or control tissue (n = 12); samples transected and glued in an overlapping manner with a cyanoacrylate (n = 60); and samples transected, sewn with a commercially available suture material and reinforced at the suture holes with the same cyanoacrylate (n = 60). Seven days after their preparation, 12 samples from each group, including the controls, are subjected to tensile testing until rupture and the findings are compared.In the stability study, groups of 12 each of the remaining 48 glued and 48 sutured and glued samples underwent tensile testing until rupture on days 30, 60, 90, and 120, after their preparation.The results show that bonding with the adhesive provided a resistance ranging between 1.04 and 1.87 kg, probably insufficient for use in valve leaflets, but also afforded a high degree of elasticity. After 120 days, both the glued and the sutured and glued series show excellent elastic behavior, with no rigidity or hardening of the pericardium. These samples present reversible elongation, or strain, when they surpass their elastic limit at rupture. This finding may be due to a load concentration that is damaging to the pericardium, to the behavior of the tissue as an amorphous material, or perhaps to both circumstances.These results need to be confirmed in future studies as they may be of value in the design and manufacture of cardiac bioprostheses.     

Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.     

Surface characteristics and biological studies of hydroxyapatite coating by a new method

Hydroxyapatite was coated on the Ti implants by a new coating method called low temperature high speed collision (LTHSC). Higher roughness values were estimated in the formed thick coating on the implant surface. While the roughness value was lower than plasma sprayed HA coatings, LTHSC HA-coated surface showed higher hydrophilicity. The cell proliferation of oseteoblastic cells evaluated by MTT assay showed that HA-coated surface had significantly higher cell viability than the control. The HA-coated surface showed higher mean total protein production even though there was no statistical difference between two surfaces. Osteoblast differentiation, ALP activity and expression of differentiation marker genes such as osteopontin and osteocalcin were elevated in the cells cultured on HA-coated surface and this surface also showed higher collagen protein expression than titanium control. Moreover, in experimental group, BIC values were significantly increased after 12 weeks of implantation and about 30-50% cortical bone-implant contact was observed.     

Physiological and cell biological aspects of perfusion culture technique employed to generate differentiated tissues for long term biomaterial testing and tissue engineering

Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.     

Physiological and cell biological aspects of perfusion culture technique employed to generate differentiated tissues for long term biomaterial testing and tissue engineering

Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.     

Simple and reliable method of doxycycline determination in human plasma and biological tissues

Over recent years there has been a resurgence in the use of doxycycline in clinical practice, which does not depend on its antibacterial properties. This paper describes a method of determination of doxycycline in human plasma and atheromatous tissue using high-performance liquid chromatography (HPLC), and a cheap commercially available extraction system. Doxycycline is extracted in the mobile phase and injected directly into the HPLC system, avoiding time consuming drying up steps. A limit of detection of 0.125 microg/ml of plasma, and a relative standard deviation of 3% was achieved, making the method very reliable and useful for assays within the usual therapeutic range. The method has also been applied to the extraction of a mixture of tetracyclines from plasma and atherma with equal efficacy, making it useful for assays of this class of drugs in veterinary practice and assays of food contaminants.     

Long-term stability of bone tissues induced by an osteoinductive biomaterial, recombinant human bone morphogenetic protein-2 and a biodegradable carrier

The long-term stability of bone tissues induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) and poly[L-lactide-co-glycolide] copolymer-coated gelatin sponge (PGS) was examined. In 16 dogs, 2.5 cm unilateral bone defects were created in the left tibial diaphyses. Tibia was fixed with metal plate, and PGS impregnated with (0.4 mg/cm(3)) or without rhBMP-2 was implanted into 15 or one defects, respectively. The metal plates of rhBMP-2-treated limbs were removed 16 weeks after the implantation. The bilateral tibiae of five animals each of the rhBMP-2-treated group were harvested at 32, 52 or 104 weeks, and served for biomechanical testing and histology. Although the defect that received PGS alone resulted in nonunion at 16 weeks, all defects treated with rhBMP-2 achieved radiographic bony union by 8 weeks. Biomechanical properties of the regenerated bones restored to the levels of intact tibiae at 32 weeks, but torsional stiffness was significantly higher. No statistical significances were detected in all parameters between regenerated and intact tibiae at 104 weeks. No radiographic and histological findings suggesting enhanced resorption to the regenerated bones were observed. These results suggest the long-term stability of the bone tissues induced by rhBMP-2, and the usefulness of rhBMP-2-impregnated PGS as a biomaterial for long bone defect filling.     

A new method to determine a fractal dimension of non-stationary biological time-serial data

We devised a new analysis using quartile deviation of integrated and subtracted fluctuation, termed QIS-A, to determine a fractal dimension of non-stationary fluctuation. In the algorithm, computations of the quartile deviation, Q(n), of all integrated and subtracted fluctuations are repeated over all scales (n). The fractal scaling exponent is determined as a slope of the line relating log Q(n) to log n. Comparison of the QIS-A and a spectral analysis using 20 computer-simulated fractional Brownian motions demonstrates robustness of the QIS-A to non-stationary fluctuations.