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.

Grafting RGD containing peptides onto hydroxyapatite to promote osteoblastic cells adhesion.

Ceramics possess osteoconductive properties but exhibit no intrinsic osteoinductive capacity. Consequently, they are unable to induce new bone formation in extra osseous sites. In order to develop bone substitutes with osteogenic properties, one promising approach consists of creating hybrid materials by associating in vitro biomaterials with osteoprogenitor cells. With this aim, we have developed a novel strategy of biomimetic modification to enhance osseointegration of hydroxyapatite (HA) implants. RGD-containing peptides displaying different conformations (linear GRGDSPC and cyclo-DfKRG) were grafted onto HA surface by means of a three-step reaction procedure: silanisation with APTES, cross-linking with N-succinimidyl-3-maleimidopropionate and finally immobilisation of peptides thanks to thiol bonding. Whole process was performed in anhydrous conditions to ensure the reproducibility of the chemical functionalisation. The three-step reaction procedure was characterised by high resolution X-ray photoelectron spectroscopy. Efficiency of this biomimetic modification was finally demonstrated by measuring the adhesion of osteoprogenitor cells isolated from HBMSC onto HA surface.

Heterogeneous cell mechanical properties: an atomic force microscopy study.

Atomic force microscopy (AFM) is a non-invasive microscopy to explore living biological systems like cells in liquid environment. Thus AFM is an appropriate tool to investigate surface chemical modification and its influence on biological systems. In particular, control over biomaterial surface chemistry can result in a regulated cell response. This report investigates the influence of adhesive and non-adhesive surfaces on the cell morphology and the influence of the cytoskeleton structure on the local mechanical properties. In this study, the main work concerns a thorough investigation of the height images obtained with an AFM as therecorded images provide the evolution of the mechanical properties of the cell as function of its local structure. Information on the cell elasticity due to the cytoskeleton organization is deduced when comparing the AFM tip indentation depth versus the distance between the cytoskeleton bundles for the different samples.

Can magnetic resonance imaging be the key technique to visualize and investigate endovascular biomaterials?

OBJECTIVE: Magnetic resonance imaging (MRI) is an established modality in clinical use but may be potentially underutilized to visualize and investigate biomaterials. As its use is totally contraindicated only for ferromagnetic devices, it was employed to visualize deployment, biofonctionality, healing, and biodurability of a commercially available endovascular device, namely the Medtronic-AVE AneuRx. The quality of the observations coupled with the absence of ionizing radiations are likely to make this technique an attractive imaging modality in the future. METHOD: The potential benefits of the MRI technique were investigated in a GE Vectra-MR 0.5T MRI for the Medtronic-AVE AneuRx endovascular prosthesis, under different conditions: undeployed i.e., inserted in the delivery cartridge as received from the manufacturer (step 1), deployed in a mock glass-aneurysm tube (step 2), and as a pathological explant harvested at the autopsy of a patient (step 3). The device was submitted to X-rays for examination in addition to MRI. At step 3, the device was further investigated with light microscopy and scanning electron microscopy (SEM) together with X-ray diffraction. RESULTS: The device which was inserted and pleated in the delivery cartridge did not demonstrate any significant observation either in MRI or in X-rays. When it was deployed in the mock aneurysmal glass tube, light artefacts were associated with the T2 weighed FSE images around the Nitinol whereas X-rays gave images of indisputable interest. Similar results were noted using the explanted device. Very high contrasts were obtained with T1 whereas T2 images were almost defect free. The X-rays allowed to accurate imaging of the Nitinol skeleton but were poor to discriminate between the different tissues. Pathology observations using light microscopy were not really challenged, as the magnetic resonance imaging was performed using a 0.5T machine. DISCUSSION: The benefits of magnetic resonance imaging as a quality control technique to examine an endovascular device within its cartridge remains ill defined. Similarly, the role of conventional X-rays is unknown. The observation of devices fully deployed in a mock aneurysmal glass-tube under MRI are potentially useful but X-rays images allowed better definition. The MRI examination of the explanted device does permit observations related to the healing of the device that might be obtained in vivo and, thus offers new avenues for the follow-up of implanted devices. The pathological investigations brought additional informations about the tissues and the corrosion of the Nitinol. However, it is unlikely that MRI will permit detailed analysis of the biomaterials and in particular the corrosion process of the stents. CONCLUSION: These early observations of the follow-up of devices using MRI warrant further investigation. The absence of ionizing radiation with MRI makes this technique particularly attractive. As there is no emission of ionizing radiation associated with magnetic resonance, it is recommended that further investigation using this environment friendly technique for the follow-up of devices made of biomaterials that are MRI compatible.

[Pinctada margaritifera nacre (mother-of-pearl): physico-chemical and biomechanical properties, and in vitro cytocompatibility]. / La nacre Pinctada margaritifera: caractérisation physico-chimique, propriétés biomécaniques et cytocompatibilité in vitro.

PURPOSE OF THE STUDY: Pinctada maxima nacre (mother-of-pearl) has been recently proposed as a bone substitute. The purpose of this work was to assess the in vitro cytocompatibility of Pinctada margaritifera nacre and its physico-chemical and biomechanical properties. MATERIAL AND METHODS: The cytocompatiblity was assessed in contact with human osteoprogenetic cells. Attachment was measured at one hour and three hours by determining N-acetyl-beta-D-hexosaminidase activity. Proliferation was monitored by measuring metabolic activity with the MTT test. Cell morphology was studied under scanning electron microscopy and cell differentiation was assessed by immunocytochemistry monitoring of the synthesis of type I collagen and osteocalcin. Diffraction x-ray and scanning electron microscopy was used to study the physico-chemical structure. Two samples taken from the inner part of the shell and two other samples from the outer part of the shell were tested for resistance under compression and to calculate the Young module. RESULTS: The results showed that osteoprogenetic cells attached to the nacre (2/3 of the plastic control), proliferated according to a standard pattern (increased metabolic activity followed by a plateau then decreased activity), synthetized type I collagen and osteocalcin, and presented a morphology analogous to control cells cultured on the plastic culture wells. The diffraction spectrum of the crystalline structure corresponded to crystallized calcium carbonate in the form of calcite (CaCO(3)) for the outer part and in the form of aragonite for the inner part. The Young module was 46.1 Gpa and resistance to rupture was 185 Mpa. CONCLUSION: Pinctada margaritifera nacre is cytocompatible in vitro with mechanical properties very similar to cortical bone.

Characterization of dynamic cellular adhesion of osteoblasts using atomic force microscopy.

BACKGROUND: Atomic force microscopy (AFM) can be used to visualize the cell morphology in an aqueous environment and in real time. It also allows the investigation of mechanical properties such as cell compliance as a function of cell attachment. This study characterized and evaluated osteoblast adhesion by AFM. METHODS: Human bone marrow stromal cells were cultured on two types of surface to induce weak and strong cellular adhesions. RESULTS: Cells were considered as spreading if they had a flattened and lengthened shape and a cytoskeletal organization in the submembrane cytosolic region. Cell detachment demonstrated different adhesion states between adherent cells to be distinguished. The stability of the cytoskeletal fibers indicated that cells were adherent. The elastic modulus was estimated by two complementary approaches. The values deduced were between 3 x 10(2) and 2 x 10(5) Nm(-2) according to the state of cell adhesion and the approaches used to measure this elastic modulus. CONCLUSIONS: Although the results were qualitative, a relation may be deduced between the elasticity of living cells as demonstrated by cytoskeletal organization and the state of cell adhesion. The technique could be used to determine the adhesion state of an adherent osteoblast observed under AFM.

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 two grafting methods for obtaining a 3-aminopropyltriethoxysilane monolayer on silica surface.

In order to establish a 3-aminopropyltriethoxysilane (APTES) grafting procedure with limited number of APTESs noncovalently linked to the silica surface, two different methods of grafting (in acid-aqueous solution and in anhydrous solution) were compared. The grafted surface state was investigated by atomic force microscopy (AFM). The stability of the grafting was checked at different temperatures by AFM. Continuous and plane APTES grafted surfaces were successfully prepared using both grafting preparations. The grafting in an anhydrous solution behaves homogeneously and stably compared to the grafting in an acid-aqueous solution. Moreover, with anhydrous solution, results showed that a unique monolayer of APTES was grafted.

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.

[Can minimal arterial aggressions using non-penetrating mechanical clip suture prevent myo-intimal hyperplasia? Preliminary results]. / L'agression artérielle minimale lors de suture par clips mécaniques non pénétrants prévient-elle l'hyperplasie myo-intimale? Résultats préliminaires.

SUBJECT: Vascular anastomosis is still associated with a significant rate of early (stenosis, thrombosis) and delayed (intimal hyperplasia) complications. Even though suture closure remains the most widespread standard procedure, many mechanical systems have been developed mostly using non penetrating clips, aiming to make the suture easier, to reduce the operating time and to reduce the scarring process of the arterial wall. We investigated the usefulness of non penetrating titanium Vascular Closure Staple (VCS) developed for peripheral blood vessels anastomosis, in a study on 20 rabbits with the small VCS system. MATERIAL AND METHODS: On 20 rabbits, 9 of the aortic sutures were done with VCS clips and 11 were done by standard closure. RESULTS: We found a significant improvement in the operating time of the closure (9 +/- 2 minutes versus 14 +/- 4 minutes), early and delayed (10 weeks) patency and the respect of the aorta diameter (0.248 +/- 0.01 centimetres versus 0.246 +/- 0.039 centimetres) and loss of surface (40.3 +/- 5.59% versus 45.6 +/- 6.34%). The main improvement is the reduced intimal hyperplasia (0.128 +/- 0.05 millimetres versus 0.198 +/- 0.032 millimetres. P=0.012). CONCLUSION: Arterial closure can be performed more rapidly with VCS clips than with suture closure, and with a marked reduced reaction of intimal hyperplasia. With those elements it is necessary to continue the experimental studies and to evaluate the VCS sutures at mean and long term.

Mineralization of regenerated cellulose hydrogels.

Due to their high water swelling, regenerated cellulose hydrogels and sponges were pre-incubated in a Ca-containing solution, and their mineralization was investigated. Results obtained demonstrate that a simple pre-incubation treatment in a Ca containing solution can induce mineralization in materials with limited or no tendency to mineralize. The minerals formed had an apatitic carbonated and poorly crystalline structure, resembling carbonated hydroxyapatite found in bone mineral. The apatitic layer formed showed a relatively accelerated growth using this technique, exhibiting nodules in their macroscopic structure, which seem to indicate lateral growth. The porous structure of regenerated cellulose sponges was also homogeneously mineralized using this technique.

[New polymers, surface treatments, bioactive materials: value of vascular access devices]. / Nouveaux polymères, traitements de surface, matériaux bioactifs: intérêts pour les dispositifs d'accès vasculaire.

Complications associated with hemodialysis vascular access are mainly due to a relative lack of biocompatibility and hemocompatibility of the materials used for the making of these devices, and to infection phenomena which may be induced by these devices. New compositions which can be processed into polymers, and a functionalisation of the latter aimed at endowing them with a specific ability to interact with molecular and cellular actors involved in the already mentioned lack of fiability on the one hand, and with infectious pathogens on the other, open the way to some improvement of the situation. Let us hope that device manufacturers will make sufficient efforts in order to see the technology transfers undertaken and completed.

Cytotoxicity assessment of latex urinary catheters on cultured human urothelial cells.

PURPOSE: To study the toxicity of latex urinary catheters on cultured human urothelial cells (HUC). MATERIALS AND METHODS: We exposed monolayers of HUC (well characterized for their proliferation, qualitative evaluation and quantitative measurement of cytokeratins) to either pure or diluted liquid latex extracts, obtained under standard conditions or by direct contact with materials. RESULTS: The latex urinary catheter appears to be highly toxic since cell viability and metabolic activity were about 10% of those of negative controls for original extracts. Concerning direct contact, latex reduced cell viability, metabolic activity and cell proliferation of HUC on days 1, 3 and 8. CONCLUSION: The high toxicity of latex on HUC is confirmed for extracts and direct contact. Therefore, it should no longer be used for urinary catheters.

Development of "heparin-like" polymers using swift heavy ion and gamma radiation. I. Preparation and characterization of the materials.

The development of ideal antithrombogenic polymers, a major problem in biomaterials sciences, is a primary objective in the fields of cardiovascular prostheses, artificial hearts, and other devices. To decrease their thrombogenicity, which remains the major obstacle, we have developed polymeric materials endowed with a specific affinity for antithrombin III (ATIII) and thus able, like heparin, to catalyze the inhibition of thrombin by ATIII. Sulfonate and sulfonamide groups are introduced onto phenyl rings belonging to styrene residues, which are radiation grafted (using swift heavy ion and gamma radiation) onto poly(vinylidene difluoride) (PVDF) and also onto poly(vinylidene fluoride/hexafluoropropylene) [P(VDF-HFP)]. In contrast to gamma radiation, which leads to a homogeneous modification, the advantage of swift heavy ion grafting is that only small regions are modified; thus, the surface may present hydrophilic (corresponding to the modified areas) and hydrophobic microdomains (corresponding to the unmodified areas) of different sizes, depending on the absorbed dose and grafting yield. Surface topography and composition are characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Sulfur, sodium, fluorine, and carbon are determined by scanning electron microscopy combined with energy-dispersive X-ray analysis (SEM-EDXA). The amount of fluorine decreases as polystyrene (PS) is grafted, whatever the kind of radiation and polymer. When the polymers are functionalized, the amount of fluorine also decreases while sodium and sulfur appear. Functionalization seems to increase the roughness of the surface, and its area.

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.

Polymorphonuclear cell apoptosis in exudates generated by polymers.

Flow cytometry was used to quantify apoptotic and necrotic polymorphonuclear (PMN) cells in an exudate generated by biomaterials, and the results were compared with determinations of spontaneous apoptosis and necrosis in PMN cells from the bloodstream. The exudate formed inside cylindrical tubes subcutaneously implanted in the dorsal region of rats was collected over a 1-week period. A rapid and simple staining procedure based on the spectral properties of the bisbenzemide Hoechst 33342 was used to identify apoptotic PMN cells. Quantification of permeabilized PMN cells stained by propidium iodide was possible in the same unfixed specimens. The percentages of apoptotic and permeabilized PMN cells in peripheral rat blood were low (1.8 +/-0 0.5% and 1.7 +/- 0.7%, respectively), similar to results found in humans. In exudates generated by polyvinyl chloride (PVC), the percentages of apoptotic and permeabilized PMN cells were higher than in the blood. The percentage of PMN cells undergoing apoptosis progressively increased with time and reached a maximum at day 2 (27% +/- 6%). The percentage of permeabilized cells progressively increased with time and was much higher than the percentage of apoptotic cells on days 4 and 8. Apoptosis and necrosis of PMN cells at day 2 were inhibited when tubes were filled with 10% serum. Selective inhibition of apoptosis with a caspase inhibitor in vivo indicated that apoptosis and necrosis are two separate pathways leading to the death of PMN cells in the exudate. At day 2, polyurethane (PU) was associated with a lower rate of apoptosis than PVC or a random copolymer of trimethylene carbonate (TMC) and epsiloncaprolactone (ECL). Apoptosis was interpreted as an organized cell removal process that limits inflammation. Apoptosis was the natural route of PMN cell death at the early stage of inflammation.

Development of RGD peptides grafted onto silica surfaces: XPS characterization and human endothelial cell interactions.

The attachment of human umbilical vein endothelial cells (HUVECs) on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) was investigated. This approach was used to provide substrates that are adhesive to cells even in the absence of serum proteins and to cells that have had no prior treatment of the surface with proteins that promote cell adhesion. We wanted to improve control of cellular interactions with cell-adhesive materials by providing fixedly bound adhesion ligands. Silica was examined as a model surface. The peptides were grafted using three different steps: grafting of aminosilane molecules; reaction with a maleimide molecule; and immobilization of cell-binding peptides containing the RGD sequence. The RGD-grafted surface was characterized by X-ray photoelectron spectroscopy (XPS) and contact-angle measurements.

Xenogeneic ossicular implants: an experimental study of heterotopic, demineralized, lyophilized, porcine implants in the guinea-pig.

This study was done to compare the outcome of porcine ossicular implants in the middle ear and the subcutaneous dorsal region of the guinea-pig to those of allo-implants implanted in parallel in the dorsal region. The implants were heteropic, xenogeneic, demineralized (HCl), lyophilized and sterilized. The evaluation was histological (light microscopy and scanning electron microscopy) and immunological (immunofluorescence staining). Fifty-four guinea-pigs were implanted in the middle ear and 14 of them were also implanted subcutaneously in the dorsal region with xeno-implants and allo-implants. The middle ear implants were found to be constantly reossified and coated with normal mucosa with only a minimal immune reaction. In contrast, the dorsal xeno-implants were found to be the target of mononucleic infiltration, fibrous encapsulation and an influx of immunoglobulins resulting in segregation. The corresponding allo-implants were found to be partially reoccupied and reossified. These findings highlight the value of HCl demineralization in the induction of non-species-specific Bone Morphogenetic Protein and the failure of attempts at immuno-despecification. It appears that the fate of the implant depends less on its antigenic load than on the site of implantation. In this regard the middle ear is apparently very advantageous. The very good short-term tolerance and recovery observed in the middle ear xeno-implant suggest that these implants offer sufficiently good results to warrant clinical testing.

Expansion and differentiation of haemopoietic progenitor cells on endothelialized hydroxyapatite under static conditions.

We have analysed the potential of an endothelialized hydroxyapatite matrix (HAP), a synthetic bone substitute, as a cellularized support for the expansion of haemopoietic progenitor cells. Scanning electron microscopy (SEM) showed that the endothelial cells (EC) tended to form a monolayer fitting closely to the matrix, and that the progenitors adhered to the EC layer. Endothelialized HAP supported the proliferation and differentiation of the progenitors with the addition of the exogenous cytokines IL-1, and IL-3. The expanded cell population essentially belonged to the myeloid and monocytic lineages, with a smaller percentage of the megakaryocyte lineage. In comparative experiments CD34+ progenitors were expanded on endothelialized tissue culture flasks, and a significant higher viability of the expanded cells was found with the endothelialized HAP. A high percentage (approx. 40%) of mature granulocytes was generated in accordance with the presence of differentiating cytokines such as G-CSF and GM-CSF at high concentrations in the coculture medium. Other cytokines that could be detected were IL-6, M-CSF, SCF, flt3-ligand. More than 50% of the expanded cell population was able to phagocytose bacteria and to generate an oxydative burst. These data indicate that cellularized HAP may be a useful matrix for stromal cell-based expansion systems.