Effect of different gamma-irradiation doses on cytotoxicity and material properties of porous polyether-urethane polymer.

Biomaterials respond to sterilization methods differently. Steam sterilization might decrease the performance of thermoplastic polyether-urethane (TPU); however, the effect of different gamma-radiation doses on this polymer is contradictory in present literature. The purpose of this study was to investigate the differences between irradiative doses in comparison with steam sterilization on a porous TPU scaffold produced by a new processing method. No significant differences in the surface chemical structure were found with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis when comparing with the sterilization methods. The molecular weight (M(w)) had a net increase from 11.5 +/- 0.039 to 13.2 +/- 0.072 kDa by gamma-sterilization from 10 to 60 kGy. The samples that were irradiated (>60 kGy) had also an increase in polydispersity index (PDI; 1.45 +/- 0.007) in comparison with the nonsterile ones (1.31 +/- 0.017), which indicate branching. Liquid chromatography/mass spectroscopy (LC/MS) analysis showed that there was a correlation between the concentration of the breakdown product, methyl dianiline, and cytotoxicity. The concentration of this compound was found to be four times higher in steam-sterilized sample (1.3 +/- 0.01 ppb) compared with that of the polymer sample gamma-sterilized at 10 kGy (0.3 +/- 0.01 ppb). The cytotoxicity of TPU was found to decrease with higher radiation doses, and was significantly higher for the steam-sterilized samples. It is recommended that TPU produced with the described foaming method should be sterilized by gamma-irradiation at 25 kGy or higher doses.

A novel processing method for injection-molded polyether-urethane scaffolds. Part 1: processing.

A large-scale scaffold processing method with injection molding has been successfully developed. Water was used as afoaming agent for the new technique. NaCl was used as a porogen to achieve an open-cell structure. Organic solvents, which are common foaming agents for polyurethane, where not used. Toxic remains in the polymer were therefore prevented. Pore size and porosity was adjustable through process parameters. A parameter study showed that an increase in injection pressure, plasticize speed, cylinder, and mold temperature raised the mean pore diameter. The porosity also could be mended by the cylinder and mold temperature, in addition to NaCl concentration. It was possible to produce scaffolds with a porosity of 64 +/- 3%, a pore size distribution from 30-450 microm, and a mean pore diameter of 270 +/- 90 microm. The interconnective pores were found to lie between 5 and 58 microm.

A novel processing method for injection-molded polyether-urethane scaffolds. Part 2: cellular interactions.

A large-scale scaffold processing method with injection molding has been successfully developed. Water was used as a foaming agent for the new technique. NaCl was used as a porogen to achieve an open-cell structure. Organic solvents, which are common foaming agents for polyurethane, where not used. Toxic remains in the polymer were therefore prevented. Biocompatibility tested gave a mean optical density of 81% from WST-1 proliferation assay. In comparison to the previously study processing method, hot pressing (Haugen H, Ried V, Brunner M, Will J, Wintermantel E. J Mater Sci: Mater Med2004;15:343-346), the current scaffolds had an increase of 20% of the mean optical density. Cell seeding showed that human fibroblasts adhered to the surface and proliferated. The spread of the adhered fibroblasts was uniform on the surface. A quantitative MTT analysis proved that there was a significant (p < 0.01) increase in the OD level after 7 and 14 days of incubation. This cell layer thickened with increased incubation time from 7 to 14 days (p < 0.05) and had typical fibroblast morphology.

Biostability of polyether-urethane scaffolds: a comparison of two novel processing methods and the effect of higher gamma-irradiation dose.

This article deals with enzyme-induced biodegradation behavior of thermoplastic polyether-urethane (TPU). Porous scaffolds were processed by a new foaming method applied in hot pressing and injection molding. The scaffolds were subsequently gamma sterilized. The samples were incubated with cholesterol esterase (CE) for 28 days to simulate an enzymatic degradation order to assess polymer biostability. The main focus of degradation products was the most toxic one: methylene dianiline (MDA). LC/MS was used to separate the breakdown products and to identify possible MDA amounts. The results showed that (a) the hot-pressed sample released an MDA amount almost twice as large (0.26 ng +/- 0.008) as that of the injection-molded samples (0.15 ng +/- 0.003) after incubation with enzyme activity in the physiological range, and (b) a tenfold increase in CE activity revealed considerably higher MDA amounts (7540.0 ng +/- 0.004). This enzyme concentration is physiologically unlikely, however, but may occur for extreme high inflammation behavior. Even for extremely high levels of CE enzyme, the scaffold will not discharge MDA above toxic levels. The injection-molded samples sterilized at 25 kGy seem to represent the most promising processing method. Therefore, the new injection-molding foaming process of polyether-urethane can be considered appropriate for use as a biomaterial.

Analysis of computer-aided techniques for virtual planning in nasoalveolar moulding.

We compared two methods of planning virtual alveolar moulding as the first step in nasoalveolar moulding to provide the basis for an automated process to fabricate nasoalveolar moulding appliances by using computer-assisted design and computer-aided manufacturing (CAD/CAM). First, the initial intraoral casts taken from seven newborn babies with complete unilateral cleft lip and palate were digitised. This was repeated for the target models after conventional nasoalveolar moulding had been completed. The initial digital model for each patient was then virtually modified by two different modelling techniques to achieve the corresponding target model: parametric and freeform modelling with the software Geomagic(®). The digitally-remodelled casts were quantitatively compared with the actual target model for each patient, and the comparison between the two modified models and the target model showed that freeform modelling of the initial cast was successful (mean (SD) deviation n=7, +0.723 (0.148) to -0.694 (0.157)mm) but needed continuous orientation and was difficult to automate. The results from the parametric modelling (mean (SD) deviation, n=7, +1.168 (0.185) to -1.067 (0.221)mm) were not as good as those from freeform modelling. During parametric modelling, we found some irregularities on the surface, and transverse growth of the maxilla was not accounted for. However, this method seems to be the right one as far as automation is concerned. In addition, an external algorithm must be implemented because the function of the commercial software is limited.

Angiopolarity of cell carriers: directional angiogenesis in resorbable liver cell transplantation devices.

The purpose of this study was to obtain directional angiogenesis of small blood vessels and capillaries to an implant made from a resorbable polymer for hepatocyte transplantation. It was intended to mimic the native acinar structure of the liver in order to facilitate replication of the cells and organ growth. The implant device structure was designed for injection to minimize surgical trauma. Hollow microspheres with an open porous wall structure and one large central opening were made from poly(d,l-lactic-co-glycolic acid) (85:15 lactic:glycolic). This polymeric scaffold was seeded with hepatocytes and implanted into the abdominal wall muscle of syngeneic Fisher rats. Specimens explanted up to 56 days p.o. showed hepatocyte survival and the development of a directional blood supply. This phenomenon is coined "angiopolarity". The study should help in addressing the issue as to whether avascular cell implants with post-transplantation organ growth should be attempted. Processing options in applying heat to the polymer solution allow manufacturing of larger microspheres with different diameters of central openings. This would allow the use of the scaffold for other cell transplantations than hepatocytes.

Grooves affect primary bone marrow but not osteoblastic MC3T3-E1 cell cultures.

To elucidate the influence of microtextures on bone cell performance, primary adult rat bone marrow cells (RBMC) and osteoblastic MC3T3-E1 cells were cultured on tissue culture pretreated plates to which grooves at different density were applied. RBMC cells were found to be significantly affected by grooves in the substratum in contrast to osteoblastic MC3T3-E1 cells, taking culture morphology, total cell number, cell mass, and cell activity (MTT-dehydrogenase), parameter for differentiation of osteoblast progenitor cells into (pre-)osteoblasts (alkalinephosphatase activity, ALP) and tartrate-resistant acid phosphatase (TRAP) activity as indices. TRAP is located in lysosomes and secretory granules mainly although not solely in osteoclasts. By applying grooves to and/or by chemical treatment of unpretreated pure polysterene plates it could be concluded that the effects on RBMC cells were evoked not only by the presence of grooves but also by the surface chemistry of the grooved and ungrooved surface areas.

Protein adsorption and monocyte activation on germanium nanopyramids.

Germanium can form defect-free pyramidal islands on Si(1 0 0)-2 x 1 with a height of 15 nm and a width of 60 nm. Using chemical vapor deposition we have prepared substrates with different nanopyramid densities to study the impact on contact angles, protein adsorption and cell behavior. The advancing contact angle of a water droplet of millimeter size significantly raises with nanopyramid density. The dynamic contact angle measurements reveal that the substrate surface is highly hydrophilic. On such a surface the adsorption of hydrophilic proteins, i.e. albumin and globulin, is drastically increased by the presence of nanopyramids. More important, however, the globulin is inactive after adsorption on nanopyramid edges. This observation is supported by the cytokine release of IL-1beta and TNF-alpha of monocyte-like cell line U937. Consequently, the presence of nanopyramidal structures gives rise to less inflammatory reactions.

Future directions in biomaterials.

Biomaterials have made a great impact on medicine. However, numerous challenges remain. This paper discusses three representative areas involving important medical problems. First, drug delivery systems; major considerations include drug-polymer interactions, drug transformation, diffusion properties of drugs and, if degradation occurs, of polymer degradation products through polymer matrices developing a more complete understanding of matrix degradation in the case of erodible polymers and developing new engineered polymers designed for specific purposes such as vaccination or pulsatile release. Second, cell-polymer interactions, including the fate of inert polymers, the use of polymers as templates for tissue regeneration and the study of polymers which aid cell transplantation. Third, orthopaedic biomaterials, including basic research in the behaviour of chondrocytes, osteocytes and connective tissue-free interfaces and applied research involving computer-aided design of biomaterials and the creation of orthopaedic biomaterials.

Tissue engineering scaffolds using superstructures.

Here, scaffolds as cell and tissue carriers are approached from an engineering point of view, emphasizing material superstructuring in the design of supports. Superstructure engineering provides optimal spatial and nutritional conditions for cell maintenance by the arrangement of structural elements (e.g. pores or fibres) so as to vary the order of cell to cell contact. This approach is illustrated in the design of several scaffolds: knitted fabrics as three-dimensional superstructures for optimized osteosynthesis implants, a new injectable open porous implant system, an angiopolar non-degradable ceramic cell carrier, and an injectable or microsurgically implantable entangled carrier system. The implications for tissue engineering are discussed.

Titanium containing amorphous hydrogenated carbon films (a-C: H/Ti): surface analysis and evaluation of cellular reactions using bone marrow cell cultures in vitro.

Amorphous hydrogenated carbon (a-C : H) coatings, also called diamond-like carbon (DLC), have many properties required for a protective coating material in biomedical applications. The purpose of this study is to evaluate a new surface coating for bone-related implants by combining the hardness and inertness of a-C : H films with the biological acceptance of titanium. For this purpose, different amounts of titanium were incorporated into a-C : H films by a combined radio frequency (rf) and magnetron sputtering set-up. The X-ray photoelectron spectroscopy (XPS) of air-exposed a-C : H/titanium (a-C : H/Ti) films revealed that the films were composed of TiO2 and TiC embedded in and connected to an a-C : H matrix. Cell culture tests using primary adult rat bone marrow cell cultures (BMC) were performed to determine effects on cell number and on osteoblast and osteoclast differentiation. By adding titanium to the carbon matrix, cellular reactions such as increased proliferation and reduced osteoclast-like cell activity could be obtained, while these reactions were not seen on pure a-C : H films and on glass control samples. In summary, a-C : H/Ti could be a valuable coating for bone implants, by supporting bone cell proliferation while reducing osteoclast-like cell activation.

Titanium dioxide ceramics control the differentiated phenotype of cardiac muscle cells in culture.

A new approach, the cultivation of heart muscle cells on biocompatible scaffolds made from titanium dioxide ceramics was established to provide a mechanism for in vitro engineering of a vital heart tissue. Terminally differentiated ventricular myocytes isolated from hearts of adult rats were kept in primary culture for long periods of time and used as an experimental model. The microenvironmental properties of titanium dioxide ceramics helped to maintain the tissue-like structural organisation of the cardiac cells in vitro. Coating of the cell substrata with fine-grained titanium dioxide ceramics imitating cell surface topography favoured the formation of focal adhesion complexes in the ventral plasma membrane of cardiomyocytes. It also promoted the cellular expression of vinculin, a protein that connects the ECM integrin receptors to the network of cytoplasmic filaments, which define cell shape. This topographical reinforcement of cell-material interactions led to stabilisation of the molecular linkage between the extracellular contacts and the intracellular cytoskeleton and thus assisted the preservation and maintenance of the heart muscle cell differentiated phenotype in long-term primary culture. The results of this work demonstrate a promising pathway for the regulation of cellular organisation in vitro by local geometric control.

Preparation and in vivo testing of porous alumina ceramics for cell carrier applications.

Microporous alumina was used to develop implantable cell carriers shaped as a hollow-sphere with a central opening to allow ingrowth of vascularised tissues. The carriers were produced by suspending the ceramic raw materials in water, homogenising and dropping the resulting slurry onto a heated plate (hot plate moulding, HPM). Morphological characteristics of the cell carriers were investigated by SEM and optical microscopy. Produced carriers had an average diameter of 4.9 mm. The material was highly porous (56 +/- 8%). For in vivo testing the cell carriers were implanted into abdominal wall of Zur: SIV rats for up to 50 weeks and investigated by light microscopy, SEM and TEM. The surface of the hollow carriers was in close contact with unirritated muscle tissue; no inflammation or capsule formation was observed. Loose connective tissue had grown into the hollow cell carrier, and after prolonged implantation >20 weeks adipocytes were observed. The absence of scar tissue formation around the implant and the vitality within the cavity of the hollow carriers indicate that porous alumina may be used for cell transplantation devices.

Degradation of poly(D,L)lactide implants with or without addition of calciumphosphates in vivo.

The study was aimed at examining the in vivo degradation of pure poly(D,L)lactide (PDLLA) or PDLLA with an admixture of calciumphosphates. One rod (20 x 3 x 2 mm) and one cube (3 x 2 x 2 mm) of pure PDLLA, PDLLA with tricalciumphosphate (PDLLA + TCP) or PDLLA with calciumhydrogenphosphate (PDLLA + CHP), respectively, were implanted into the dorsal muscles of 50 male Wistar Albino rats. After definite intervals (from 2nd to 72nd week), pH measurements were performed in the environment of the implants. Afterwards, the cubes with their surrounding tissues were excised for histological examinations, measurements of the outer dimensions and mechanical analyses of the explanted rods were performed. No drop of more than 0.1 pH units was detectable in the tissue surrounding any type of implants. No advantageous effect of the calciumphosphates could be proved. A mild foreign body reaction could be observed around PDLLA implants. After 72 weeks, pure PDLLA had been totally resorbed from the extracellular space, the degradation of calciumphosphate-enriched PDLLA was still in progress. A large amount of inflammations occurred in the tissues surrounding PDLLA with an admixture of slowly degrading TCP or CHP, leading to two abscesses and four fistulas at PDLLA + TCP, and two abscesses and three fistulas at PDLLA + CHP implantation site. Bending strength of pure PDLLA was constant up to the 4th week post-implantation and reduced to 60% of the initial value up to the 12th week. No traces of crystallinity could be observed during the degradation of PDLLA. As a conclusion of the study, complete resorption from the extracellular space and tissue tolerance of pure PDLLA is proved. An admixture of small calciumphosphate particles is not suitable to improve the biocompatibility of PDLLA but leads to a decrease in the mechanical characteristics.

The stiffness of bone marrow cell-knit composites is increased during mechanical load.

A novel device for mechanical stimulation of primary adult rat bone marrow cells cultured on three-dimensional knitted textiles has been prototyped. A method has been developed ensuring a well-defined, high-density, and reproducible cell seeding on the knitted fabric. After culturing for 18-52 days the cell-knit composites were subjected to uniaxial 2% stretching and relaxation. The frequency was altered between 0.1 Hz (196 min, loading phase) and 0.01 Hz (360 min, resting phase). Identically treated knits without cells exhibited a slight stiffness reduction, whereas the stiffness of knits with cells increased from cycle to cycle. The stiffness increase was found to depend on the duration of the culture period before mechanical loading. Our data suggest that the extracellular matrix deposited by the cells on the knit and intact microtubuli of living cells cause the observed stiffness increase. In comparison to the unstrained static cell-knit composites cell proliferation and bone cell differentiation were reduced by the mechanical load.

Matrices for tissue engineering-scaffold structure for a bioartificial liver support system.

This study proposes a new composite scaffold system. A woven polyethylenterephtalate (PET) fabric was coated on one side with a biodegradable PLGA film, in order to obtain a geometrically polarized scaffold structure for an bioartificial liver support system. The composite structure ensures the stability of the membrane during degradation of the membrane polymer. The mesh size of the composite does not significantly influence the degradation behavior. Hepatocyte culturing studies reveal that the formation of aggregates depends on the mesh size and on the pretreatment: The largest aggregates could be observed after 48 h when PVLA coating, large mesh size and EGF were combined. Thus, the combination of a geometrically structured, partially degradable scaffold with receptor-mediated cell attachment sites offers promising possibilities in liver tissue engineering.

[Biomaterials, human tolerance and integration]. / Biomaterialien--humane Toleranz und Integration.

Biomaterials and related process engineering in order to obtain optimal surface and structural biocompatibility of implants and devices are presented. Vital-avital composites for tissue engineering, cell culture models, porous ceramics and degradable polymers are introduced as examples. Emphasis is laid on the conversion of basic research results into clinical applications and on the exchange of technologies from the non-medical into the medical field and vice versa.

The effect of an addition of sodium hydrogenphosphate to poly(D,L)lactide--results of in vitro examinations.

Aim of the study was to examine the influence of sodium hydrogenphosphate (NaP) on the pH value and the mechanical characteristics of degrading poly(D,L)lactide (PDLLA). Test rods of PDLLA with or without NaP amounting to 1, 10, 25 or 50 mol per 100 mol lactate, the degradation product of PDLLA, were produced by injection molding. Molecular weight and bending strength of the rods were measured before and after an accelerated in vitro-test (55 degrees C, Ringer's solution (RS)). For a long-time degradation test PDLLA-rods with or without 1 mol% NaP were placed in RS or Soerensen buffer solution (SB, pH 7.4) at 37 +/- 1 degrees C. Measurements of pH and determination of length, volume and weight of the samples were done in 2-4 week intervals up to the 52nd week after incubation. A pH-drop was measured in RS or SB containing pure PDLLA after 28 or 36 weeks respectively. Stabilization of the pH value due to admixed NaP delayed the degradation related pH drop for 8 weeks in RS or SB. A strong increase of length, volume and weight was measured in PDLLA + NaP-rods. In conclusion minimal stabilization of pH but also an increase of outer dimensions of the samples was found due to the admixture of NaP to PDLLA. Thus, an addition of substantially higher amounts than 1 mol% NaP in PDLLA can not be recommended, regardless of the positive effects on pH stabilization.

The effect of an admixture of sodium hydrogen phosphate or heparin-coating to poly(D,L)lactide--results of an animal study.

The study was aimed at investigating the effect of an admixture of sodium hydrogen phosphate (NaP) on the pH value around degrading poly(D,L)lactide (PDLLA) and the possible improvement of PDLLA biocompatibility by coating its surface with heparin. PDLLA +/- NaP was injection-molded to form rods (20 x 3 x 2 (mm)) and cubes (3 x 2 x 2 (mm)). Half of the pure PDLLA samples were surface-coated using heparin. One rod and cube each of PDLLA, PDLLA + NaP and PDLLA/Hep were implanted into the dorsal muscles of 42 rats. From the 2nd to 52nd week after operation, pH measurements were performed in the environment around the implants. The samples were then harvested for histological and mechanical analyses. No significant decrease in pH-values was observed in the tissue around the implants. Pure PDLLA and PDLLA/Hep samples were macroscopically resorbed after 52 weeks, while the degradation of PDLLA + NaP was still in progress. Approximately 80% of the initial bending strength of PDLLA or PDLLA/Hep rods was present after six weeks, while the bending strength of PDLLA + Nap was reduced to 50% after 4 weeks. Heparin-coating of PDLLA did not improve its biocompatibility but did increase its resorption. While no significant effect of NaP on pH value was found, its admixture did reduce the mechanical characteristics of the implants.