Mineralization of regenerated cellulose hydrogels induced by human bone marrow stromal cells.

The proliferation of cultured human bone marrow stromal cells (HBMSC) on regenerated cellulose hydrogels was assessed. Regenerated cellulose hydrogels showed good rates of HBMSC proliferation, the cells exhibiting a flattened morphology, and after 22 days in culture, the cells had homogeneously colonized the surface of the materials. Moreover, since the early days in culture, between the surface of the materials and attached cells a continuous granulated hydroxyapatite layer was formed. It has been previously demonstrated in vitro, but without cells, that these materials did not mineralize. Hence, it seems that HBMSC promoted the mineralization of the surface.

Development of a resorbable macroporous cellulosic material used as hemostatic in an osseous environment.

The control of bleeding is a frequently encountered therapeutic problem, particularly during dental surgery. The most efficient substances used to resolve this problem are not risk-free because of their animal or human origins, so cellulosic materials are potentially of interest. The aim of this study was to develop a resorbable macroporous cellulosic material for use as a resorbable hemostatic agent in bone sites. The degradation and the cytocompatibility of the cellulosic material versus controls were evaluated and its behaviour in vivo was studied. An original process using calcium carbonate powder as inverse matrix was used to develop a macroporous material. In order to predegrade the cellulosic material for hemostatic use, oxidation was performed with periodate. A dialdehyde component unstable at physiological pH was thus obtained. The material was found to have cytotoxicity, biocompatibility, and resorption properties similar to control but its hemostatic power was higher.

Functional upregulation of granulocytes labeled with technetium-99m-HMPAO and indium-111-oxinate.

UNLABELLED: Indium-111-oxinate-labeled granulocytes have been used in vivo for several years for the detection of abscesses. Technetium-99-m-hexamethylpropyleneamine oxime (99mTc-HMPAO) labeling has more recently been described. METHODS: The influence of radiolabeling by both radiotracers on adhesion glycoprotein CD11b quantification was studied in quiescent and formyl-methionylleucylphenylalanine (fMLP)-activated neutrophils (PMN). Adhesion was assessed on human umbilical endothelial cells (HUVEC) as well as the repercussion of the granulocyte labeling on HUVEC viability (neutral red) and metabolic activity (MTT). Chemotaxis of PMN was evaluated by measuring migration under agarose with fMLP as chemoattractant. We also measured phagocytosis and the production of hydrogen peroxide induced by staphylococcus aureus. RESULTS: Whereas whole functional integrity is maintained after labeling, most of the functions (CD11b expression, adhesion, HUVEC metabolic activity) are up-regulated while chemotaxis is decreased in the presence of both radiotracers. Indium-111-oxinate induces larger alterations than 99mTc-HMPAO. CONCLUSION: These data were obtained in normal volunteers. In patients, alterations due to the in vitro labeling procedure, in addition to potential functional alterations caused by the underlying pathology, should be taken into account during image interpretation.

Haemocompatibility of Ti6A14V alloy.

Ti6A14V alloy has been mainly used as a biomaterial in the orthopaedic field. The present study describes the surface state of the Ti6A14V material and evaluates its in vitro haemocompatibility in terms of protein adsorption, platelet retention and haemolysis. The behaviour of the Ti6A14V alloy towards albumin and fibrinogen was compared to that of a reference medical-grade elastomer. The platelet retention test gave better results than those achieved with the elastomer. The haemolysis percentage of the alloy was almost zero. These results indicate that the Ti6A14V alloy is well tolerated by blood.

Biocompatibility of carbon-carbon materials: blood tolerability.

Carbon-carbon composites are well known in the field of aerospace technology. Such composites have been proposed to be used as biomaterials, particularly in contact with blood. To evaluate their haemocompatibility, samples were tested in vivo and in vitro, using radiotracers. In vivo study showed the accumulation of platelets on the exposed surface material with any surface morphology, whereas platelet concentration in blood remained constant. In vitro study allowed us to distinguish, among entrapped platelets, active adhering platelets from those mechanically retained and it appeared that the bulk structure of materials influenced the adhesion mechanism of platelets.

Blood haemolysis by ceramics.

Ceramics are more and more frequently under consideration for construction of blood-contacting devices, i.e. cardiac valves or cardiac assist devices. This study evaluated the haemolysis eventually initiated in vitro by ceramic powders (Al2O3, ZrO2/Y2O3, AlN, B4C, BN, SiC, Si3 N4, TiB2, TiN, TiC), graphite and diamond. The chemical composition of the powders was studied by X-ray microprobe and various other methods, and BET specific areas were determined. The haemolysis was almost zero for all powders, except AlN which showed slight haemolysis and TiB2 which had high haemolytic power.

Microencapsulation of isolated pituitary cells by polyacrylamide microlatex coagulation on agarose beads.

Microlatex beads of homogenous size were made by polymerization of a mixture of acrylamide/bisacrylamide dispersed in a microemulsion. The microlatex was aggregated by dilution of the microemulsion in acrylamide solutions. The aggregates were then coagulated by polymerization at the interfaces of agarose beads circulating in a capillary tube containing paraffin oil. Biocompatibility was tested on isolated pituitary cells microencapsulated by this procedure.

Beware of commercial human thrombins used to stimulate cultured endothelial cells.

In the field of in vitro biocompatibility testing, the investigation of cell response at the interface with a biomaterial is of great importance; there is a need for standard conditions and thus of well-defined and reliable sources of materials for an objective evaluation of cellular function. Thrombin is often used in vitro as a stimulating agent to check the specific functions of cultured endothelial cells. In the present work, and in order to select a thrombin of commercial origin, two criteria were borne in mind: purity towards the presence of the von Willebrand factor (vWF) and effectiveness towards vWF release by human umbilical venous endothelial cells (HUVEC) that have been submitted to four human commercial thrombins. We detected the presence of vWF in some thrombin solutions that have not yet been in contact with HUVEC. The different thrombins contained vWF antigen ranging from less than 0.1 mUnit per NIH unit of thrombin (from Diagnostica Stago and Sigma Chemical Co.) to 10-20 mUnit per NIH unit of Fibrindex thrombin (from Ortho Diagnostic Systems). Thus, if vWF is present in commercial thrombins, it contributes to and overestimates the vWF appearance in the media resulting from cell stimulation. Consequently, we fixed on thrombin from Diagnostica Stago for further studies involving HUVEC on biomaterials.

Evaluation of cell colonization on biomaterials: preventing cell attachment to plastic containers.

In biocompatibility evaluation involving cell culture models, we use samples of biomaterials of different forms and sizes. During cell seeding onto biomaterials of an inadequate size to cover the bottom of the culture wells completely, cells have the opportunity to attach to the plastic. As described in this report with two culture models and two biomaterials, we use an agarose gel sublayer to prevent this phenomenon.

In vitro cytocompatibility of radio-opacifiers used in ureteral endoprosthesis.

Ureteral endoprostheses are urinary catheters made of polymeric biomaterials made radio-opaque through the addition of X-ray absorbing additives such as barium, bismuth, tantale or tungsten. The aim of this work was to study the in vitro toxicity of solutions of these radio-opacifiers using two cell culture models. Primary-cultures of human urothelial cells (HUC) arising from normal adult urinary tract and permanent urothelial cell line were used. Solutions at different dilutions were placed into the wells containing monolayers of confluent cells. After 24 h incubation period, the solutions were removed and cell viability and cell metabolic activity tests were performed (Neutral Red assay and MTT assay). At a concentration lower than 1 mg l(-1) the different radio-opacifiers used showed no toxicity. From 1 to 3 mg l(-1) one can note a significant dose-dependent decrease of cell metabolic activity of solely HUC for barium chloride. At 3 mg l(-1) one can note a significant deleterious effect on HUC metabolic activity, with bismuth and tantale. For tungsten, there is no deleterious effect, but on the contrary a significant increase in HUC metabolic activity at a 0.5 mg l(-1) concentration. None of the solutions did provoke alterations in HUC viability for concentrations less than 3 mg l(-1). Interestingly, for permanent cell line one can note a solely significant decrease of cell viability at 3 mg l(-1) for tantale. All the other tested salts on permanent cell line were not significantly different from controls for cell viability as well as cell metabolic activity. HUC culture model may be of relevance for the screening of radio-opacifiers intended for ureteral endoprostheses.

TiN coating: surface characterization and haemocompatibility.

The left ventricular assist device under consideration is based on the principle of the Maillard-Wankel rotary pump. The construction materials must meet stringent requirements. Titanium nitride was chosen for its surface properties and graphite for its bulk characteristics. The purpose of this study was to characterize the chemical vapour deposition titanium nitride coating via morphology, roughness, crystallinity, chemical composition, to report and discuss the results of in vitro haemocompatibility tests (protein adsorption, platelet retention, haemolysis) and to discuss physico-chemical and biological results. This chemical vapour deposition titanium nitride coating is well tolerated by the blood despite its surface irregularities, and appears as a good candidate material after improvements.

Cellulose phosphates as biomaterials. In vivo biocompatibility studies.

Femoral implantation of regenerated cellulose hydrogels revealed their biocompatibility, but a complete osseointegration could not be observed. Phosphorylation was therefore envisaged as the means to enhance cellulose bioactivity. In vitro studies showed that regenerated cellulose hydrogels promote bone cells attachment and proliferation but do not mineralize in acellular simulated physiological conditions. On the contrary, phosphorylated cellulose has shown an opposite behavior, by inducing the formation of a calcium phosphate layer in simulated physiological conditions, but behaving as a poor substrate for bone cells attachment and proliferation. In order to investigate the in vivo behavior of these materials, and assess the influence of mineralization induction ability vs. bone cells compatibility, unmodified and phosphorylated cellulose hydrogels were implanted in rabbits for a maximum period of 6 months and bone regeneration was investigated. Despite the difficulties arising from the retraction of cellulose hydrogels upon dehydration during the preparation of retrieved implants, histological observations showed no inflammatory response after implantation, with bone intra-spongious regeneration of cells and the integration of the unmodified as well as the phosphorylated cellulose implants. After a maximum implantation period of 6 months, histological observations, histomorphometry and the measurement of the amount of 45Ca incorporated in the surrounding tissue indicated a slightly better osseointegration of phosphorylated cellulose, although no significant differences between the two materials were found.

Study of a (trimethylenecarbonate-co-epsilon-caprolactone) polymer--part 2: in vitro cytocompatibility analysis and in vivo ED1 cell response of a new nerve guide.

Future surgical strategies to restore neurological function in peripheral nerve loss may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. Random copolymers of trimethylene carbonate and epsilon caprolactone (P(epsilonCL-TMC), 50: 50) have been synthesized by ring opening polymerization using rare earth alkoxides as initiator. Their potential use as nerve guide repairs has been assessed through indirect and direct in vitro biocompatibility tests and in vivo soft tissue response to EDI subclass macrophages. In vitro, we exposed monolayers of human skin fibroblasts and an established continuous cell line (Hela) to liquid extracts (either pure or diluted in the culture medium) of epsilonCL-TMC copolymer including positive (phenol) and negative controls. Then, colorimetric assays (Neutral red and MTT) were performed. The extracts of epsilonCL-TMC induced no significant cytotoxic effect. We also exposed in vitro Schwann cells to pieces of P(epsilonCL-TMC) and P(LA-GA) copolymers. We evaluated cell attachment at 1 and 3 h by measuring the activity of the lysosomal enzyme (N-acetyl-beta-hexosaminidase) and cell proliferation at 1, 3, 6 and 9 days by measuring the cell metabolic activity (MTT assay). Values for attachment slightly decreased between 1 and 3 h but were significantly higher than on agars (negative control). Cells plated on epsilonCL-TMC showed a rate of proliferation comparable with that of normalized controls and higher than on PGA-PLA at day 9. Finally, we evaluated in vivo the soft tissue response after implantation of cylindrical tubes of P(epsilonCL-TMC) and P(LA-GA) copolymers with an immunohistochemistry staining procedure for the newly recruited ED1 macrophages. An image analysis system automatically measured the optical density of labelled positive ED1 cells at 9, 21 and 60 days after implantation. epsilonCL-TMC copolymer showed a mild soft tissue reaction with no adverse chronic inflammatory reaction. These data allowed us to consider this conduit as a potential effective substitute in nerve repair. El sevier Science Ltd. All rights reserved.

Radiosterilization of albuminated polyester prostheses.

The study reported here is concerned with the radio-sterilization of Dacron vascular prostheses coated with crosslinked albumin. gamma-Radiations have no effect on the mechanical properties of the polyester fibres or on their crystallinity, whether irradiated in a dry state or immersed in saline. Special attention has been paid to the release of the albumin, or protein fragments from the reticulum using 125I-labelled albumin as a radiotracer. The albumin leakage depends upon the type of Dacron fabrics considered but the values derived from radioactivity measurements are always greater than those directly measured, which indicates a radio-induced break of the bond between iodine and albumin; this has nothing to do with the break of the association between albumin and Dacron. Moreover no cytotoxicity of the irradiated immersion medium has been observed using a test based on organotypic culture in liquid medium. Thus radio-sterilization of an albuminated polyester vascular prosthesis immersed in saline appears to be a suitable procedure.

Biocompatibility of carbon-carbon materials: in vivo study of their erosion using 14carbon labelled samples.

Several uncertainties have to be resolved concerning the in vivo erosion of carbon-carbon composite materials and the outcome of the resulting particles. We studied these phenomena with implants superficially doped with 14carbon, specially prepared using the usual production processes of these materials. The samples implanted in rats presented changes in their measured radioactivity which proved erosion. Autoradiographies of the whole animal as well as pathological studies of peri-implanted tissues with histoautoradiographies of the related sections revealed the presence of carbon at a distance from the implant. However, the majority of the eroded particles were retained in the fibrous capsule surrounding the implant. The methods of electron-diffraction, associated with electron microscopy, seem to be a tool suitable to characterize the nature (fibrous or pyrolytic) of the carbon particles present in the capsule.

Cultured differentiated human urothelial cells in the biomaterials field.

To review the use of normal cultured differentiated human urothelial cells in the biomaterials field, we checked the literature for human urothelial cells in culture (HUC) both for their use in biocompatibility assessment and as bioartificial devices. The in vitro culture of differentiated human urothelium is now a simple and reliable procedure. These techniques provide new tools for biocompatibility assessment of urinary biomaterials, because for the rational design of a testing procedure, it is preferable that the particular cell culture models selected should be closely related to the end-use application. The emerging use of HUC culture should lead to the development of bioartificial tissue for urinary tract reconstruction. Tissue engineering techniques require urothelial cells and cell delivery matrices. The cytocompatibility of novel artificial delivery matrices should be assessed in vitro before implantation using cultured HUC to find the best material available.

Experimental evaluation of a gelatin-coated polyester graft used as an arterial substitute.

Protein coating and endothelial cell preseeding have been proposed and studied as improvements to arterial prostheses. In this paper, an impervious polyester vascular graft which had been coated with cross-linked gelatin was compared to a porous one over a period of up to 8 months in dogs. This evaluation involved in vivo methods using radio tracers to study patency and thrombogenicity and in vitro controls of the healing processes. The main advantages offered by coated grafts over uncoated include the absence of preclotting and better biointegration.

New artificial connective matrix-like structure: thrombogenicity and use as endothelial cell culture support.

The recently described artificial connective matrix made of elastin solubilized peptides, type I+III collagens and connective proteins is shown to have structural and biological properties very close to the natural arterial subendothelium: the capacity to promote endothelial cell cultures maintaining their phenotype expression and its non-thrombogenicity. This new bioactive composite material could be used to replace arteries.

Ex vivo leucocyte adhesion and protein adsorption on TiN.

Titanium nitride (TiN) is regarded as a potential biomaterial for blood-contact applications. Its in vitro haemocompatibility has been evaluated already and gave promising results. The purpose of this study was to continue studying its 'biological' behaviour through an ex vivo evaluation. The material was a physical vapour deposition elaborated TiN coating and the phenomena observed were leucocyte adhesion and albumin and fibrinogen adsorption. These ex vivo results were compared with in vitro results obtained previously. Two reference medical grade silicone elastomer and three TiN arterio-arterial extra-corporeal circuits were tested. No leucocyte was retained by TiN, as in in vitro experiments; the ex vivo fibrinogen adsorbed quantity was higher and albumin adsorption was about the same in in vitro and in ex vivo situations. TiN can be considered as a suitable blood-contacting material.

Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces.

Heparin-like materials, characterized by a defined superficial density of functional groups which activate antithrombin III (AT III), when in contact with blood specifically inhibit thrombin as soon as it appears. This paper describes an isotopic method to estimate this density and to visualize the distribution of the affinity sites concerned, both directly with AT III labelled with 125Iodine and indirectly with an anti AT III monoclonal antibody labelled with 111Indium.